Conjugated polymer nanoparticles for bioimaging applications

.. .CONJUGATED POLYMER NANOPARTICLES FOR BIOIMAGING APPLICATIONS GENG JUNLONG (M.S University of Science and Technology of China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR... PUBLICATIONS 151 vi SUMMARY Conjugated polymer nanoparticles (CP NPs) have emerged as an attractive fluorescent probe in bioimaging as well as other biological applications due to their large... Jablonski diagram IC stands for internal conversion, ISC stands for intersystemm crossing, S0 stands for ground singlet state, S1 stands for first excited singlet state, Sn stands for higher excited singlet

CONJUGATED POLYMER NANOPARTICLES FOR BIOIMAGING APPLICATIONS GENG JUNLONG NATIONAL UNIVERSITY OF SINGAPORE 2014 CONJUGATED POLYMER NANOPARTICLES FOR BIOIMAGING APPLICATIONS GENG JUNLONG (M.S. University of Science and Technology of China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Geng Junlong 30 December 2014 i ACKNOWLEDGEMENTS The person I would like to thank firstly is my supervisor, Prof, Liu Bin, for offering me the opportunity to join her group and start my postgraduate study at NUS. I appreciate her kindly support, valuable guidance and inspiration when I pursue the Ph.D. degree here. Her profound knowledge, research enthusiasm and vigorous methodology guided me to finish my Ph.D. projects successfully. I sincerely thank my labmates, Dr. Li Kai and Dr. Ding Dan, for guiding in cell culture and animal experiments; Dr. Liu Jie, Dr. Pu Kanyi, Dr. Zhou Li and Dr. Zhan Ruoyu for providing polymer materials, Dr. Cai Liping, Dr. Shi Haibin, Dr. Wang Guan, Dr. Wang Yanyan, Dr. Wang Yusong, Dr. Hu Qinglian, Dr. Yuan Youyong, Mr. Feng Guangxue, Ms. Liang Jing and Ms. Zhang Ruoyu for kind help. I also appreciate other collaborators for their great help in my experiment. I am grateful to Mr. Boey Kok Hong, Ms. Lee Chai Keng, Mr. Liu Zhicheng, Ms. Lim Kwee Mei and other technicians in ChBE for their assistance and support. The scholarship from National University of Singapore, the award for outstanding self-financed students abroad in 2012 from Chinese government and the facilities from Department of Chemical and Biomolecular Engineering and National University of Singapore are also greatly acknowledged. Finally, I would love to thank my parents, parents in law, relatives, friends and especially my beloved wife, Ms. Kang Jun, for their unconditional love, supporting and encouragement in my everyday life. ii TABLE OF CONTENTS DECLARATION .............................................................................................. i ACKNOWLEDGEMENTS ............................................................................. ii TABLE OF CONTENTS ................................................................................iii SUMMARY ...................................................................................................vii LIST OF TABLES ........................................................................................viii LIST OF FIGURES ........................................................................................ ix LIST OF SCHEMES.....................................................................................xiii LIST OF SYMBOLS .................................................................................... xiv CHAPTER 1 INTRODUCTION ..................................................................... 1 1.1 Background ............................................................................................ 1 1.2 Properties of CPs .................................................................................... 3 1.3 Synthesis of CP NPs............................................................................... 6 1.3.1 Emulsion .......................................................................................... 7 1.3.2 Precipitation ................................................................................... 11 1.3.3 Cross Linking ................................................................................ 15 1.4 Biological Application of CP NPs ....................................................... 15 1.4.1 Bioimaging .................................................................................... 16 1.4.2 Biological Detection ...................................................................... 23 1.4.3 Therapy .......................................................................................... 27 1.5 Research Objectives ............................................................................. 34 1.6 Thesis Outline ...................................................................................... 36 CHAPTER 2 FACILE SYNTHESIS OF STABLE MULTIHYDROXY CONJUGATED POLYMER NANOPARTICLES BY DENTRITIC CROSSLINKING ....................................................................................................... 38 2.1 Introduction .......................................................................................... 38 2.2 Experimental Section ........................................................................... 40 2.2.1 Materials ........................................................................................ 40 2.2.2 Characterization ............................................................................. 41 2.2.3 Synthesis of PFBT-N3 ................................................................... 41 iii 2.2.4 Synthesis of HPG .......................................................................... 42 2.2.5 Synthesis of HPG-Alk ................................................................... 43 2.2.6 Preparation of CP-HPG NPs ......................................................... 43 2.2.7 Cell Imaging .................................................................................. 44 2.2.8 Cytotoxicity Evaluation ................................................................. 44 2.3 Results and Discussion ......................................................................... 45 2.3.1 Synthesis and Characterization of PFBT-N3 and HPG-Alk .......... 45 2.3.2 Synthesis and Characterization of CP-HPG NPs .......................... 47 2.3.3 Stability Characterization of CP-HPG1......................................... 52 2.3.4 Cellular Imaging of CP-HPG1 ...................................................... 54 2.4 Conclusion............................................................................................ 56 CHAPTER 3 A GENERAL APPROACH TO PREPARE CONJUGATED POLYMER DOT EMBEDDED SILICA NANOPARTICLES WITH A SIO2@CP@SIO2 STRUCTURE FOR TARGETED HER2-POSITIVE CELLULAR IMAGING ................................................................................ 57 3.1 Introduction .......................................................................................... 57 3.2 Experimental Section ........................................................................... 60 3.2.1 Materials ........................................................................................ 60 3.2.2 Characterization ............................................................................. 61 3.2.3 Preparation of SiO2@CP@SiO2 NPs ............................................ 61 3.2.4 Synthesis, Purification, and Characterization of Peptide .............. 62 3.2.5 Preparation of SiO2@PFBT@SiO2-Pep NPs ................................ 63 3.2.6 Preparation of PFBT NPs .............................................................. 63 3.27 Cell Culture .................................................................................... 64 3.2.8 Cellular Imaging ............................................................................ 64 3.2.9 Flow Cytometry Study .................................................................. 65 3.2.10 Cytotoxicity of SiO2@PFBT@SiO2-pep NPs ............................. 65 3.2.11 Photostability of SiO2@PFBT@SiO2-pep NPs ........................... 66 3.3 Results and Discussion ......................................................................... 67 iv 3.3.1 Preparation and Characterization of SiO2@CP@SiO2 NPs .......... 67 3.3.2 Surface Functionalization with HER2 Targeting Peptide ............. 76 3.3.3 Targeted Cellular Imaging ............................................................. 77 3.3.4 Cytotoxicity and Photostability ..................................................... 80 3.4 Conclusion............................................................................................ 81 CHAPTER 4 MICELLE/SILICA CO-PROTECTED CONJUGATED POLYMER NANOPARTICLES FOR TWO-PHOTON EXCITED BRAIN VASCULAR IMAGING ............................................................................... 83 4.1 Introduction .......................................................................................... 83 4.2 Experimental Section ........................................................................... 86 4.2.1 Materials ........................................................................................ 86 4.2.2 Characterization ............................................................................. 86 4.2.3 TPA Measurements ....................................................................... 87 4.2.4 Wide-Field Microscopy Imaging .................................................. 88 4.2.5 Synthesis of PFBT-F127-SiO2 NPs ............................................... 89 4.2.6 Synthesis of PFBT-DSPE NPs ...................................................... 90 4.2.7 Synthesis of PFBT-F127 NPs ........................................................ 90 4.2.8 Fluorescence Stability of PFBT-F127-SiO2 NPs .......................... 90 4.2.9 Photostability of PFBT-F127-SiO2 NPs ........................................ 91 4.2.10 Cytotoxicity of PFBT-F127-SiO2 NPs ........................................ 91 4.2.11 Real-Time Two-Photon Intravital Blood Vascular Imaging ....... 92 4.3 Results and Discussion ......................................................................... 93 4.3.1 Synthesis and Characterization of PFBT-F127-SiO2 NPs............. 93 4.3.2 Single NP Imaging of PFBT-F127-SiO2 NPs ............................... 99 4.3.3 Fluorescence Stability and Cytotoxicity of PFBT-F127-SiO2 NPs .............................................................................................................. 100 4.3.4 TPA Spectra of PFBT-F127-SiO2 NPs........................................ 101 4.3.5 Intravital TPFI of PFBT-F127-SiO2 NPs ................................... 102 4.4 Conclusion.......................................................................................... 104 v CHAPTER 5 ORGANIC NANOPARTICLES WITH PROCESSABLE CONJUGATED POLYMER FOR PHOTOACOUSTIC VASCULAR IMAGING .................................................................................................... 105 5.1 Introduction ........................................................................................ 105 5.2 Experimental Section ......................................................................... 108 5.2.1 Materials ...................................................................................... 108 5.2.2 Characterization ........................................................................... 108 5.2.3 PA Measurement .......................................................................... 109 5.2.4 Synthesis of Monomers and PFTTQ ........................................... 110 5.2.5 Synthesis of PFTTQ NPs............................................................. 115 5.2.6 Synthesis of Au NRs .................................................................... 116 5.2.7 Cytotoxicity of PFTTQ NPs ........................................................ 116 5.2.8 Photostability of PFTTQ NPs and Au NRs ................................. 117 5.2.9 In Vivo PA Brain Vascular Imaging ............................................. 117 5.3 Results and Discussion ....................................................................... 119 5.3.1 Synthesis and Characterization of PFTTQ .................................. 119 5.3.2 Preparation of PFTTQ NPs ......................................................... 121 5.3.3 Photostability Investigation of PFTTQ NPs ................................ 125 5.3.4 In Vivo Vasculature Imaging ........................................................ 127 5.4 Conclusion.......................................................................................... 129 CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS ................ 130 6.1 Conclusions ........................................................................................ 130 6.2 Recommendations .............................................................................. 133 LIST OF PUBLICATIONS ......................................................................... 151 vi SUMMARY Conjugated polymer nanoparticles (CP NPs) have emerged as an attractive fluorescent probe in bioimaging as well as other biological applications due to their large absorption coefficient, high brightness, good photostability and low cytotoxicity, which are even superior to the conventional fluorescent probes (e.g. organic dyes and quantum dots). Currently, CP NPs have been mainly fabricated through emulsion method and precipitation approach. Unfortunately, CP NPs prepared by these strategies are mainly built up through the hydrophobic or π-interaction between CP chains or CP/matrices, which could lose their structural stability in aqueous media. In this thesis, several strategies have been developed to improve the structural and mechanical stability of CP NPs to facilitate their bioimaging applications. Firstly, CP NPs with covalent cross-linked surfaces have been synthesized via click reaction between CPs and hyperbranched polyglycerol in miniemulsion. Secondly, CP embedded silica NPs with a SiO2@CP@SiO2 structure have been fabricated by combination of a precipitation approach and a modified Stöber method. Thirdly, a micelle/silica co-protection strategy has been developed to yield CP loaded NPs with a high fluorescence quantum yield and a large two-photon action cross section in aqueous medium. The obtained CP NPs show good performance in both cellular and vascular imaging. On the other hand, CP NPs with strong near-infrared absorbance and high non-radiative fluorescence quantum yield have also been designed for photoacoustic imaging, broadening the applications of CP NPs in bioimaging. vii LIST OF TABLES Table 2.1 Reaction Parameters and the Optical Properties of the WaterDispersible CP-HPG NPs.[a] ............................................................................ 47 Table 3.1 Characterization of SiO2@CP@SiO2 NPs. ..................................... 68 Table 4.1 Emission decay components of PFBT-F127-SiO2 NPs and PFBTDSPE NPs. ....................................................................................................... 97 viii LIST OF FIGURES Figure 1.1 FE-TEM image of poly(para-phenylene) NPs prepared with SDS as the emulsifier (A)[58] and CP NPs prepared with PVA as the emulsifier (B).[59] Copyright 2002 and 2009 John Wiley and Sons. ................................... 8 Figure 1.2 (A) SEM image of poly(phenylene ethynylene) NPs.[70] (B) AFM images of MEH-PPV NPs.[71] Copyright 2002 Royal Society of Chemistry and 2006 American Chemical Society. .................................................................. 12 Figure 1.3 Fluorescence images of live (A) and fixed (B) cells. A) BALB/C 3T3 cells were incubated sequentially with PPE NPs (green) and Hoechst dye (blue). B) Live BALB/C 3T3 cells were incubated with PPE NPs and fixed for confocal microscopic study.[72] Copyright 2007 John Wiley and Sons. .......... 17 Figure 1.4 Confocal images of MCF-7 (A) and NIH/3T3 cells (B) incubated with folic acid-functionalized CP NPs.[59] Copyright 2009 John Wiley and Sons. ................................................................................................................. 18 Figure 1.5 Confocal images of live MCF-7 cells incubated sequentially with anti-EpCAM primary antibody and PFBT NP-IgG conjugates (A), and PFBT NP-IgG conjugates only (B).[89] Copyright 2010 American Chemical Society. .......................................................................................................................... 21 Figure 1.6 (A) Ex vivo fluorescence imaging of healthy brains in wild-type mice and medullo blastoma tumors in ND2:SmoA1 mice. Each mouse was injected with either PFBT/ PFDBT NP (top), or PFBT/PFDBT-CTX NP (middle). Control: no injection (bottom). (B) Biophotonic images of resected livers, spleens, and kidneys from wild-type (middle) and ND2:SmoA1 (bottom) mice receiving PFBT/PFDBT-CTX NP injection. Control: no injection (top).[84] Copyright 2011 John Wiley and Sons. .............................................. 22 Figure 1.7 Effects of various ions on the fluorescence intensity of solution containing PFBT NP and PFVT/PFTBT NP.[86] Copyright 2011 American Chemical Society ............................................................................................. 25 Figure 1.8 (A) The schematic illustration of pH sensitive CP-FITC NPs. (B) Fluorescence spectra of CP-FITC NPs at different pH.[82] .............................. 25 Figure 1.9 (A) SEM images of PFO/doxorubicin NPs. (B) A schematic illustration of the component of NPs and uptake of NP by cells.[108] Copyright 2010 American Chemical Society. .................................................................. 28 Figure 1.10 (A) Chemical structures of CP and photosensitizer TPP. (B) A schematic illustration of NP formulation. (C) AFM image of CP/TPP coencapsulated NPs. (D) Normalized UV-vis and PL spectra of PDHF NPs doped with 10% of TPP as well as that of pure PHDF NPs.[112] Copyright 2011 Royal Society of Chemistry. ............................................................................ 30 ix Figure 1.11 (A) FE-TEM images of PPy NPs. (B) The temperature evolution of PPy NPs at different concentrations.[120] Copyright 2012 Royal Society of Chemistry. ........................................................................................................ 33 Figure 2.1 FT-IR spectra of HPG, HPG-Alk, PFBT-N3 and CP-HPG-1. ....... 46 Figure 2.2 (A) Hydrodynamic radius distribution and (B) FE-TEM image of CP−HPG-1. UV−vis absorption (dashed line) and emission (solid line) spectra (C) of PFBT−N3 in dichloromethane and CP−HPG−1 in aqueous solution (λex = 450 nm). The inset shows the aqueous solution of CP−HPG-1 NPs under daylight (left) and 365 nm UV light illumination (right). ................................ 48 Figure 2.3 Hydrodynamic radius distribution of (A) CP–HPG–2, (B) CP– HPG–3, (C) CP–HPG–4 and (D) CP–HPG–5. ................................................ 48 Figure 2.4 Photograph of HPG–Alk (left), PFBT–N3 (middle), and CP–HPG NPs (right) in the mixture of water and chloroform. ....................................... 50 Figure 2.5 FE-TEM image of CP–HPG–6 NPs. ............................................. 51 Figure 2.6 pH (A), NaCl concentration (B), BSA and γ-globulin concentration (C) dependent fluorescence intensity ratio (I/I0), where I0 is the emission intensity of CP-HPG-1 in aqueous solution at pH 6.8 without addition of NaCl, BSA and γ-globulin, and I is the emission intensity of CP-HPG-1 in aqueous solution at different (A) pH or (B) different concentrations of NaCl, (C) BSA and γ-globulin. (D) Radiation time dependent fluorescence intensity ratio (I/I0) of CP-HPG, Alexafluo 488, and fluorescein, the radiation was provided by confocal laser, where I0 and I are the emission intensity of fluorescent probe without and with radiation for different time, respectively. ............................ 53 Figure 2.7 (A) Cell viability of MCF-7 cells after incubation with CP−HPG-1 at different concentrations for 24 and 48 h, respectively. Confocal fluorescence image of MCF-7 cells upon incubation (B) with and (D) without CP−HPG-1 ([RU] = 1 μM) for 2 h. (C) 3-D confocal image of cell line MCF-7 incubated with CP-HPG-1 for 2 h. ................................................................... 55 Figure 3.1 (A) Normalized UV-vis absorption (dashed line) and PL spectra (solid line) of SiO2@CP@SiO2 NPs in water. (B) Photographs of SiO2@CP@SiO2 NP suspensioin in water under a hand held UV lamp (excited at 365 nm). ....................................................................................................... 68 Figure 3.2 FE-TEM images of SiO2@PFBT@SiO2 NPs taken at different reaction times after adding TEOS (A-E) as well as APTES (F). All images share the same scale bar as that in F. ............................................................... 70 Figure 3.3 FE-TEM images of PFBT dots in ethanol/water mixture (v/v = 9:1) upon sonication. ............................................................................................... 72 Figure 3.4 FE-TEM images of the mixture of SiO2 NPs and CP dots before APTES addition (A) and further reaction for 12 h in the presence of APTES (B). ................................................................................................................... 72 x Figure 3.5 FE-TEM images of SiO2@PFBT@SiO2 NPs with the addition of APTES (upper row; A, B) or TEOS (bottom row; C, D) for further 12 h reaction followed by centrifuging once (A, C) and five times (B, D). The insets show the respective photographs of SiO2@PFBT@SiO2 NPs after centrifugation. All images share the same scale bar as that in D. .................... 73 Figure 3.6 Confocal fluorescence images of SKBR-3 breast cancer cells after 2 h incubation with (A) SiO2@PFBT@SiO2-pep and (B) SiO2@PFBT@SiO2COOH NP suspensions at 100 μg·mL-1 NPs at 37 °C. Confocal fluorescence images of NIH/3T3 fibroblast cells after 2 h incubation with (C) SiO2@PFBT@SiO2-pep and (D) SiO2@PFBT@SiO2-COOH NP suspensions at 100 μg mL-1 NPs at 37 °C. All images have the same scale bar as that in A. .......................................................................................................................... 78 Figure 3.7 (A) CLSM fluorescence image of SKBR-3 breast cancer cells without incubation with SiO2@PFBT@SiO2 NPs. (B) 3D CLSM fluorescence image of SKBR-3 breast cancer cells incubated with SiO2@PFBT@SiO2-pep NPs. .................................................................................................................. 79 Figure 3.8 Flow cytometry histograms of pure SKBR-3 breast cancer cells without NP incubation (black) and SKBR-3 breast cancer cells after 2 h incubation with SiO2@PFBT@SiO2-Pep NP (red) and SiO2@PFBT@SiO2COOH NP (blue) suspensions at 100 μg·mL-1 NPs......................................... 79 Figure 3.9 (A) Metabolic viability of SKBR-3 breast cancer cells after incubation with SiO2@PFBT@SiO2-pep at various NP concentrations for 24 h (gray) and 48 h (shadow). (B) Photostability of SiO2@PFBT@SiO2-pep NPs, PFBT NPs and fluorescein in SKBR-3 breast cancer cells upon continuous laser excitation at 488 nm for 10 min. I0 is the initial fluorescence intensity and I is the fluorescence intensity of the sample at different time points after illumination. ..................................................................................................... 80 Figure 4.1 (A) UV-vis (dash-dotted line) and PL (solid line) spectra of PFBTF127-SiO2 NPs (red) and PFBT-DSPE NPs (black) at 25 µg/mL of PFBT. (B) Fluorescence decay curves of PFBT-F127-SiO2 NPs (red) and PFBT-DSPE NPs (black). Instrument response (IRF) (blue) is also indicated. FE-TEM images of PFBT-F127-SiO2 NPs (C) and PFBT-DSPE NPs (D). ................... 94 Figure 4.2 The DLS size evolution of PFBT-F127-SiO2 NPs in 10 days. ...... 97 Figure 4.3 (A) UV-vis (dashed) and PL (solid) spectra of PFBT loaded F127 NPs with (red) and without (black) silica layers, respectively. (B) Photostability of PFBT loaded F127 NPs with and without the protection silica layer upon continuous laser excitation at both 405 nm and 488 nm with 100% light confluence, where I0 is the initial fluorescence intensity and I is the fluorescence intensity of the sample at different time points after illumination. .......................................................................................................................... 97 Figure 4.4 Histograms of the total number of photons collected for (A) PFBTF127-SiO2 NPs, (B) PFBT-DSPE NPs and (C) QD655. Note the different binning and scales for A, B and C, λex = 488 nm for all samples. ................... 99 xi Figure 4.5 (A) PL intensity evolution of PFBT-F127-SiO2 NPs upon incubation with 1 × PBS at 37 °C for different times, where I0 is the fluorescence intensity at 545 nm for the fresh NP suspension and I is that for NPs after incubation for different time, respectively. The inset shows the PL spectra of freshly prepared PFBT-F127-SiO2 NPs (black) and after 10 days incubation with 1 × PBS at 37 °C (red). (B) Metabolic viability of NIH/3T3 fibroblast cells after incubation with PFBT-F127-SiO2 NP suspensions at various NP concentrations for 24 h and 48 h, respectively. ........................... 100 Figure 4.6 TPA spectra of PFBT-F127-SiO2 NPs (based on CP chain concentration) and Evans blue in water. ........................................................ 102 Figure 4.7 Intravital TPFI of PFBT-F127-SiO2 NPs stained blood vessels of mice brain at depth of 0 µm (A), 50 µm (B), 100 µm (C), 200 µm (D), 300 µm (E), 400 µm (F) and 500 µm (G), and the respective Z-projected image (H) as well as 3D image (I). All the images share the same scale bar of 50 µm. ..... 103 Figure 4.8 Images of intravital TPFI of brain blood vessels in mouse without injection of PFBT-F127-SiO2 NPs. The scale bar is 50 µm……………… 104 Figure 5.1 (A) UV-vis absorption spectra and (B) PA imaging as well as the PA intensity of PFTTQ NPs and Au NRs with same mass, the scale bar is 100 µm. (C) The PA intensity of PFTTQ NPs at concentrations from 0.05 mg/mL to 0.5 mg/mL. ................................................................................................. 122 Figure 5.2 (A) UV-vis absorption spectra and (B) PA imaging as well as the PA intensity of PFTTQ NPs and Au NRs, the scale bar is 100 µm. .............. 124 Figure 5.3 (A) Absorbance intensity evolution of PFTTQ NPs and Au NRs upon continuous pulse laser irradiation with a power of 15 mJ/cm2 for different times, where I0 is the absorbance intensity at 800 nm for the fresh NP suspensions and I is that for NPs after irradiation for different time, respectively. (B) UV-vis absorption spectra of PFTTQ NPs and Au NRs before and after laser irradiation with a power density of 15 mJ/cm2 for 6 mins. FE-TEM images of PFTTQ NPs (C) and Au NRs (D) before (1) and after (2) laser irradiation for 6 mins. All images share the scale bar of 100 nm.......... 126 Figure 5.4 Metabolic viability of NIH/3T3 fibroblast cells after incubation with PFTTQ NP suspensions at various NP concentrations for 24 h. ........... 127 Figure 5.5 (A) Schematic illustration of PA imaging for rat brain vasculature. (B) PA rat cortical vasculature C-scan images before and after 10 mins injection of PFTTQ NPs. (C) The evolution of the integrated PA intensity at the red line spot as a function of time. ........................................................... 128 xii LIST OF SCHEMES Scheme 1.1 Chemical structures of representitive CPs used in devices and luminescence. ..................................................................................................... 4 Scheme 1.2 Schematic illustration of Jablonski diagram. IC stands for internal conversion, ISC stands for intersystemm crossing, S0 stands for ground singlet state, S1 stands for first excited singlet state, Sn stands for higher excited singlet state, T1 stands for excited triplet state. .................................................. 5 Scheme 1.3 Schematic illustration of CP NPs prepared with emulsion method (A), precipitation approach (B) and cross linking approach (C). ...................... 8 Scheme 2.1 Schematic illustration of the preparation of CP-HPG NPs. ......... 45 Scheme 3.1 Chemical structures of four CPs used in this study. .................... 67 Scheme 3.2 Schematic illustration of the synthesis of SiO2@PFBT@SiO2 NPs with surface functionalized targeting peptide. (A) Sonicatioin of a THF solution of CPs in ethanol/water mixture (v/v = 9:1) affords single CP chain dots. (B) Addition of TEOS and ammonia into the CP dots dispersed solution leads to the formation of CP dots-punctuated silica NP pattern after reaction. (C) Further addition of APTES into the mixture results in CP dots embedded silica NPs with surface functionalized amine groups. (D1) Surface carboxylation with maleic anhydride in the presence of Et3N. (D2) Peptide conjugation via EDC/NHS reaction. ................................................................ 75 Scheme 3.3 The chemical structure of peptide, GGHAHFG. ......................... 76 Scheme 4.1 (A) Chemical structure of F127 and PFBT. (B) Schematic illustration of the fabrication of PFBT-F127-SiO2 NPs. .................................. 93 Scheme 5.1 Synthetic route and chemical structure of PFTTQ. Reagents and conditions: I) KOH, 1-bromohexane, H2O, 100 oC, 1 h; II) CuI, Pd(PPh3)2Cl2, trimethylsilylaceylene, i-Pr2NH/THF, room temperature, overnight; III) KOH, THF/MeOH/H2O, room temperature, 1 h; IV) CuI, Pd(PPh3)2Cl2, 1, iPr2NH/THF, room temperature, overnight; V) KMnO4, NaHCO3, TBAB, CH2Cl2, room temperature, 2 days; VI) H2SO4, HNO3, 100 oC, overnight; VII) Pd(PPh3)2Cl2, 2-(tributylstannyl)thiophene, THF, 80 oC, overnight; VIII) NBS, DMF, 60 oC, 3 h; IX) i) Iron, acetic acid; ii) acetic acid, 135 oC, 24 h; X) Pd(OAc)2, Cy3P, Et4NOH, toluene, 18 h. ...................................................... 119 Scheme 5.2 Schematic illustration of the fabrication of PFTTQ NPs using DSPE-PEG2000 as the matrix through a precipitation method. ...................... 121 Scheme 5.3 Experimental setup of the PAM system. The pulled tubing was filling with the venous samples in the focusing depth. The laser was pulsed with a pulse repetition rate of 10 Hz and coupled by a lens to an optical fiber to illuminate samples. PA waves were detected with a 50-MHz transducer and then through the A/D card to the PC for further data analysis. ...................... 122 xiii LIST OF SYMBOLS APTES 3-aminopropyl triethoxysilane Au NRs gold nanorods BSA bovine serum albumin CLSM confocal laser-scanning microscopy CMC critical micelle concentration CP conjugated polymer CPEs conjugated polyelectrolytes CTAB cetyltrimethylammonium bromide DAPI 4',6-diamidino-2-phenylindole DCC N,N’-dicyclohexylcarbodiimide DCM Dichloromethane DLS dynamic light scattering DMDES dimethyldimethoxysilane DMEM Dulbecco’s Modified Eagle Medium DMF dimethylformamide DMSO dimethyl sulfoxide DSPE-PEG2000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000] EPR enhanced permeability and retention FBS fetal bovine serum F127 poly(ethylene oxide)-block-poly(propylene block-poly(ethylene oxide) FE-TEM field emission transmission electron microscopy FITC fluorescein isothiocyanate FRET förster resonance energy transfer FT-IR fourier transform infrared xiv oxide)- GPC gel permeation chromatography HA 5-hexynoic acid HPG hyperbranched polyglycerol IC inter conversion ICG Indocyanine green LLS laser light scattering MRI magnetic resonance imaging NMR nuclear magnetic resonance NIR near infrared NPs Nanoparticles PA Photoacoustic PAM photoacoustic microscopy PAE photoacoustic endoscopy PAT photoacoustic computed tomography PBS phosphate buffer saline PDI polydispersity index PEG polyethylene glycol PET positron emission tomography PL photoluminescence PLGA poly(lactide-co-glycolide) PVA poly (vinyl alcohol) QDs quantum dots QY quantum yield RU repeat unit S0 ground state S1 first singlet excited states SDS sodium dodecyl sulphate xv T1 first triplet excited states TEOS Tetraethsilane THF Tetrahydrofuran TPA two-photon absorption TPFI two-photon fluorescence imaging  radiative decay rate knr non-radiative decay rate  quantum yield  lifetime xvi Chapter 1 CHAPTER 1 INTRODUCTION 1.1 Background Biological imaging is one of the most important techniques to study the fundamental issues in life science and solve the practical problems in clinical diagnosis and therapy.[1-4] It helps researchers to observe the objects and investigate fundamental biological processes from the macroscopic level to microscopic range and further to the sub-cellular scope. So far, various imaging modalities including magnetic resonance imaging (MRI),[5-7] positron emission tomography (PET),[8,9] ultrasound,[10,11] photoacoustic (PA)[12-14] and fluorescence imaging[15-17] have been widely utilized to achieve reliable and accurate biodetection and bioimaging. Among these strategies, the fluorescence imaging technique has emerged as an essential strategy in biosensing as well as bioimaging due to their non-invasive, high temporalspatial resolution and real time properties.[18-22] The precise and accurate analysis of the targets from bimolecular level to cellular and tissue stages usually depends on the performance of exogenous fluorescent reporters in biological system. As a result, fluorescent probes with high fluorescence QY, good photostability, low cytotoxicity and easily functionalized groups in aqueous or biological environment are highly desirable for biological scientists. Organic dyes, the conventional versatile fluorophores, have been widely utilized in bioimaging and biodetection.[16,23] Advances in the progress 1 Chapter 1 of organic chemistry and material science have facilitated the synthesis and design of numerous organic fluorophores, including rhodamine, alexa, BODIPY and cynanine dyes. Unfortunately, these organic fluorophores usually show poor photostability and narrow Stokes shift, which hamper their applications in bioimaging with high sensitivity and resolution. Although encapsulation of organic fluorophores into matrices could improve their stability, the intrinsic narrow Stokes shift could not be solved. In this respect, inorganic quantum dots (QDs) have shown large Stokes shift, good photostability and high brightness.[20,24] However, their intrinsic toxicities from heavy metal components also limit their practical applications in biological and clinical studies.[15] The inherent limitations of conventional fluorescent materials are incentives to explore alternative fluorescent probes with improved performance. More recently, fluorescent conjugated polymer (CP) based materials have attracted great research interest in biological applications.[25-29] CPs are macromolecules with delocalized π-conjugated backbones and fantastic lightharvesting property. Fluorescent CP encapsulated nanoparticles (CP NPs) show large absorption coefficients, large Stokes shifts, good photostabilities, low cytotoxicity and high brightness, which are even superior to the conventional fluorescent reporters (e.g. organic dyes, QDs).[30-34] The NP formulation could not only help to improve the fluorescence and colloidal stability of CPs in biological environment but also provide surfacefunctionalized groups, allowing to further conjugation for specific detection or targeted imaging.[35,36] Furthermore, fine-tuning the size and surface property of CP NPs would facilitate the enhanced permeability and retention (EPR) 2 Chapter 1 effect of NPs in tumor microvasculature through passive targeting role to improve their performance in targeted tumor imaging.[37-39] In view of the obvious advantages of NP formulation for CPs, various approaches have been developed to synthesize CP NPs and their applications in biological detection, imaging and therapy have been well investigated.[30,35,40] In this chapter, the developed approaches for CP NP synthesis have been summarized firstly. Secondly, the applications of CP NPs in specific biological applications including imaging, detection and therapy have been reviewed. Finally, the objectives and outline of this thesis have been illustrated. 1.2 Properties of CPs CPs are macromolecules with unsaturated backbones. Due to the highly delocalized π-electrons, CPs show distinguished conductivity or photoluminescence, which have been widely applied in light-emitting diodes, field effect transistors, photovoltaic devices[41-45] as well as chemical/biological detection and bioimaging.[25,26,33,46] Noteworthy is that both conductive and optical properties of CPs can be easily tuned by choosing suitable monomers as well as proper conjugation approaches.[47-49] Specifically, polyacetylene (PA), polyaniline (PAN), polypyrrole (PPy) and their derivatives are famous for their intrinsic conductivity, while polyfluorene (PF), polyphenylene (PPE), poly(phenylene vinylene) (PPV), and polythiophene (PT) are prominent for their electro- and photoluminescence (PL). The backbone structures of these commonly used CPs are shown in Scheme 1.1. 3 Chapter 1 Scheme 1.1 Chemical structures of representitive CPs used in devices and luminescence. The electron transfer process of CPs could be depicted with the classical Jablonski diagram. As shown in Scheme 1.2, the energy level of CP is divided into the ground state (S0), singlet excited states (S1, Sn) and triplet excited states (T1). Upon energy absorption, the electrons in the ground state are excited into higher energy levels. When electrons return to the ground state from the excited states, the energy could be relaxed through non-radiative and radiative pathways. The non-radiative pathway is divided into internal conversion and intersystem crossing, which is generally related with heat generation; while the radiative pathway involves the production of fluorescence or phosphorescence. In detail, the electrons in CPs are excited from the lowest energy level in the ground state (S0) to the higher excited states (S1 to Sn). The excited electrons in higher energy level could release part of energy and relax to the lowest energy level with singlet excited state (S 1) or to ground state through inter conversion (IC) or change their spin to the lowest energy level with triplet excited state (T1). The fluorescence is produced from the radiation when electrons transfer from the lowest excited energy level with singlet state (S1) to the ground state (S0). Alternatively, phosphorescence is induced when electrons relax from the lowest excited energy level with triple 4 Chapter 1 state (T1) to the ground state (S0). It is noted that the triple state energy could transfer to nearby molecules (e.g. oxygen) to induce singlet species. Both fluorescence and phosphorescence signals could be applied for detection and imaging in biological system. On the contrary, the excited electrons could release their energy through the non-radiative decay pathway, which is accompanied with the heat generation. This could be utilized in photothermal therapy. Meanwhile, the excited electrons could also induce the production of singlet species, which may be utilized in photodynamic therapy for disease treatment. Scheme 1.2 Schematic illustration of Jablonski diagram. IC stands for internal conversion, ISC stands for intersystemm crossing, S0 stands for ground singlet state, S1 stands for first excited singlet state, Sn stands for higher excited singlet state, T1 stands for excited triplet state. The photophysical properties of CPs (e.g. absorption and emission) are highly dependent on their chemical structures as well as conformations.[25,26,50,51] Specifically, the backbone structure, the effective 5 Chapter 1 conjugation length, the side chain arrangement and the packing or conformation of CP chains could influence the absorption, radiative and nonradiative processes of CPs. For instance, when the interchain interaction of a specific CP backbone increases, the excited electrons could find low-energy level to relax, resulting in obvious red-shifts of the emission spectra of CP aggregates as compared with separated CP chains.[26] This also means that the photophysical properties of CPs could be simply adjusted by chemical modification of their backbone and side chain structures, controlling their conjugation length, as well as adjusting their packing states or morphologies for different applications. 1.3 Synthesis of CP NPs The pioneer work of preparation of CP NPs can be traced back to 1980s, in which CP NPs have been synthesized by direct polymerization. The first generation of CP NPs include polyacetylene,[52] polyaniline[53] and polypyrrole.[54] They have been mainly utilized in device applications. Although the direct polymerization method has been extended to fluorescent CPs, it requires either oxidative monomers or water compatible metal catalysts, which is hard to be widely explored into other CP systems. In contrast, the postpolymerization approach mainly utilizes commercially available polymers or the synthesized CPs to fabrication of CP NPs, which is facile and convenient to be applied to various types of CPs. Since this thesis is mainly focused on bioimaging applications of CP NPs, the discussion is limited to CP NPs with water solubility or dispersibility. It is noted to worth that there is another formulation of water soluble CPs, conjugated polyelectrolytes (CPEs). CPEs are macromolecules with 6 Chapter 1 hydrophobic backbones and hydrophilic side chains, which own the property of polyelectrolytes.[33,50,51,55-57] The synthesis of CPEs usually starts with sophisticated design of monomers for both backbone conjugation and side chain modification to render them water solubility requirement, which is tedious, cost, time consuming and more importantly not generic.[36] On the other hand, the NP strategy is generally applied to various hydrophobic CPs. To make our discussion more concise, the NPs discussed in this thesis is confined to water soluble or dispersible CP NPs prepared with the postpolymerization approaches. In general, the process of CP NP fabrication involves the dissolving of hydrophobic CPs in the organic solvent, mixing the organic solvent with water, and removing the organic solvent from the mixture. Depending on miscibility of the organic phase and the water phase, the methods of CP NP fabrication are generally classified into emulsion and nanoprecipitation. In addition, there is another apporach, named cross-linking, which is aimed to improve the stability of CP NPs. 1.3.1 Emulsion A typical procedure for emulsion method is shown in Scheme 1.3A. CPs are firstly dissolved in the organic solvent which is immiscible with water, while emulsifier is dissolved in the aqueous phase. These two solutions are mixed together, followed by rapid mixing or sonication to disperse the organic phase into aqueous phase. The small emulsion droplets are obtained simultaneously and protected by the surfactants absorbed at the interface of organic/water droplets. The organic solvent is subsequently evaporated to yield water dispersible CP NPs. 7 Chapter 1 A CP Solvent evaporation Rapidly mixing Emulsion Emulsifier in Water B CP Rapidly mixing Solvent evaporation Precipitation C CP Rapidly mixing Solvent evaporation Cross-linking Precipitation Scheme 1.3 Schematic illustration of CP NPs prepared with emulsion method (A), precipitation approach (B) and cross linking approach (C). A B 500 nm 500 nm Figure 1.1 FE-TEM image of poly(para-phenylene) NPs prepared with SDS as the emulsifier (A)[58] and CP NPs prepared with PVA as the emulsifier (B).[59] Copyright 2002 and 2009 John Wiley and Sons. The early work of CP NPs prepared with emulsion method is reported 8 Chapter 1 by Landfester and co-workers in 2003.[58] In detail, they chose poly(paraphenylene), polyfluorene and polycyclopentadithiophenes and utilized sodium dodecyl sulphate (SDS) as the surfactant. Upon ultrasonication of chloroform solution containing CPs in the aqueous solution with SDS as the emulsifier, stable miniemulsion droplets have been formed. The water dispersible CP NPs has been obtained after chloroform evaporation. The size of CP NPs could be controlled from 30 to 500 nm by adjusting the initial CP concentration. A typical field emission transmission electron microscopy (FE-TEM) image of poly(para-phenylene) NPs is shown in Figure 1.1A. This approach has been further applied to synthesize other CP NPs, including polyfluorene, poly(2methoxy-5-(2-ethylhexyl)oxy)-1,4-phenyl-ene)vinylene (MEH-PPV) and poly(cyclopentadithiophene).[60] In addition, two types of CP co-encapsulated NPs have also been fabricated using SDS as the surfactant, which shows phase separation of two different polymers due to their different hydrophobicities.[61] Moreover, this approach has been utilized to encapsulated the förster resonance energy transfer (FRET) pair of two different CPs, poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly(2,5-dioctyl-1,4-phenylenevinylene) (POPPV). The emission of CP NPs could be manipulated by adjusting their respective percentages in NPs.[62] In 2009, Beneventi and co authors utilized the cationic surfactant, tetradecyltrimethylammonium bromide (TTAB) as an emulsifier and 2,7poly(9,9-dialkylfluorene-co-fluorenone) as a typical CP to study the influence of cationic surfactant in the CP NPs. Their study suggested that large amounts of TTAB segregated at the NP surface to reduce the interface tension between polymers and water, yielding stable CP NPs.[63] Although the emulsifiers of 9 Chapter 1 SDS and TTAB could help to synthesize water dispersible CP NPs, they could destroy cellular membranes due to their detergent effect, which hamper their applications in biological system. In 2009, Li et al. has developed a generic strategy to fabricate CP loaded poly(lactide-co-glycolide) (PLGA) NPs through a modified solvent extraction single emulsion method by sonication of the mixture of dichloromethane (DCM) solution containing CPs as well as PLGA matrices using poly (vinyl alcohol) (PVA) as the emulsifier.[59] The emission of PLGA NP suspensions could be easily manipulated by choosing suitable CPs. The hydrodynamic diameters of the CP NPs in water were measured to be in the range of 240 to 270 nm by DLS. The FE-TEM images illustrate that the obtained CP NPs are spherical in shape with a size ~150 nm as shown in Figure 1.1B. The smaller CP NP size is resulted from the shrink of polymeric NPs in vacuum. Although low CP concentration was presented in NP formulation, the obtained CP NPs showed high brightness and good photostability. Due to good biocompatibility of PLGA matrices and PVA emulsifiers, the obtained CP NPs have been successfully applied in cellular imaging. In addition, Li and co-workers also developed a modified emulsion approach using a mixture of PLGA and poly((lactic-co-glycoclic)-bamine(polyethylene glycol)) (PLGA-b-PEG-NH2) as co-encapsulation matrix to facilitate conjugation with antibodies for targeted cellular imaging. [64] Through conjugation of trastuzumab (Herceptin) at NP surfaces, efficient targeted cellular imaging for SKBR-3 breast cancer cells has been achieved. More recently, this strategy has been extended to encapsulate both CPs and inorganic NPs (e.g. magnetic NPs and gold NPs) to realize multifunctional 10 Chapter 1 biological imaging.[65,66] At the same time, Green’s group reported a facile strategy to prepare CP NPs using polyethylene glycol (PEG) as both emulsifier and encapsulation matrices.[67] Thanks to the highly diluted CPs in DCM, the attained CP NPs only show a small size of ~5 nm. Later, both block copolymer 1,2-diacyl-snglycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)] (lipidPEG) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) have been utilized as co-emulsifier to prepare CP NPs.[68] The hydrophobic lipid parts were embedded into CP to form a core and PEG segments oriented into water phase to stabilize the formed CP NPs. The obtained CP NPs had an average diameter of 80-100 nm in water. They further used this strategy to synthesize magnetic NPs and CP co-loaded DSPE-PEG NPs for both fluorescent and magnetic resonance imaging.[69] 1.3.2 Precipitation A typical scheme of the precipitation approach, also named reprecipitation or nanoprecipitation by different researchers, is shown in Scheme 1.3B. Generally, a hydrophobic CP is dissolved in a benign organic solvent, followed with rapid injection into a large amount of water. Upon vigorous stirring or sonication, CP chains rapidly collapse from organic phase to water phase. Subsequent evaporation of water yields the CP NPs. Unlike the above emulsion method, emulsifiers are not used in the precipitation approach. The CP NPs are mainly built up through the hydrophobic interaction between CP chains. When the organic solution is mixed with water, hydrophobic CP chains fold into spherical morphology to minimize their surface tension to form CP NPs. 11 Chapter 1 A B 1.5 µm 100 nm Figure 1.2 (A) SEM image of poly(phenylene ethynylene) NPs.[70] (B) AFM images of MEH-PPV NPs.[71] Copyright 2002 Royal Society of Chemistry and 2006 American Chemical Society. In 2003, Moon et al. reported the synthesis of poly(phenylene ethynylene) (PPE) NPs by mixing dimethyl sulfoxide (DMSO) solution containing (PPE) with aqueous medium.[32] The obtained NPs show a diameter of 500-800 nm as shown in Figure 1.2A. Later, Moon and co-worker optimized the CP structures by conjugation of PPE with amine groups and obtained CP NPs with an average size of 97 nm with a polydispersity index of 0.13.[72] Using the same strategy, CP NPs with pentiptycene-containing poly(p-phenylene ethynylene) have also been synthesized.[73] Due to the purification with a membrane filter, the size of CP NP was decreased to ~28 nm characterized with laser light scattering (LLS). By grafting the side chain PPE with amine functionalized tartaric acid, the CP NPs have been optimized with a size of ~8 nm.[74] In addition, the synthesized CP NPs showed good stability, which did not show obvious size change in 3 months. In 2005, McNeill and co-workers developed a novel strategy to synthesize CP NPs by ultrasonication of CP dissolved tetrahydrofuran (THF) solution in aqueous medium.[75] Noteworthy is that no need to modify CP side 12 Chapter 1 chains with cationic or water soluble groups and the CP NPs could be formed through hydrophobic interaction between CP chains. Furthermore, the NP size could be fine-tuned by changing the starting CP concentration.[71,76] The AFM image of MEH-PPV NPs prepared with this approach is shown in Figure 1.2B, which shows a size around 15 nm.[71] The surfaces of CP NPs prepared with this approach could be functionalized with reactive groups by using poly(styrene-g-ethylene oxide) (PS-PEG-COOH) or poly(styrene-co-maleic anhydride) (PSMA) as co-matrices to conjugate with biological reagents for targeted detection or imaging, which have been widely developed by Chiu’s group.[34,77-93] Increasing the concentration of CPs in THF solution resulted in the enlarged sizes of CP NPs. The comparison of the photophysical properties of CP NPs with that of Qdot 565 and Alexa 488 demonstrated that CP NPs show better photostability and high brightness than the other two, facilitating their biological applications.[89] In addition to single CP encapsulated NPs, a donor CP, poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-thiadiazole)] (PFBT), and an acceptor CP, poly[(9,9-dihexylfluorene)-co-4,7-di(thiophen-2yl)-2,1,3-benzothiadiazole] (PFDBT), have been co-encapsulated into PSMA matrix to afford FRET based NPs.[84] The obtained CP NPs show an emission maximum at ~650 nm with a higher QY of 0.56 (measured with 4(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran in methanol as the standard). The surface functionalized carboxyl groups could be conjugation with chlorotoxin (a tumour targeting peptide). In combination with the high brightness of near infrared (NIR) emission and low cytotoxicity, the FRET based CP NPs have been successfully applied in targeted brain tumour imaging. This FRET strategy has also been utilized to endow CP NPs 13 Chapter 1 with strong NIR emission by taking silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775)[84] and QDs[88] as the acceptors, which open a new avenue for CP NPs in NIR biological imaging. Another type of precipitation approach is to synthesize amphiphilic polymers, which are composed of hydrophobic backbones and hydrophilic side chains. By adding a large amount of water into the THF solution of amphiphilic CPs, NPs with hydrophobic CP backbones as the core and hydrophilic side chains as the shell would be obtained. Controlling the hydrophobic/hydrophilic ratio of polymer, fluorescent CP NPs with various sizes from 85 nm to 178 nm have been synthesized as reported by Chen and co-workers.[94] In addition, poly(fluorenylene-alt-phenylene) modified with lipid groups and alkylammonium groups have also been reported to form CP NPs with a size about 50 nm in water.[95] The same strategy has been utilized by Liu’s group to synthesize various types of CP NPs with strong far-red/near infrared fluorescence[96,97] and high fluorescence QY.[98] The CP NPs prepared from both emulsion and precipitation approaches are mainly built up through the hydrophobic interaction between CPs or CPs/matrices. From the long term application perspective, these NPs are deficiency in stability due to the weak hydrophobic interaction. In addition, these CP NPs are mechanically instabilbe and cannot be redispersed in water after drying. To further improve the stability of CP NPs, a cross-linking approach has been developed. 14 Chapter 1 1.3.3 Cross Linking A typical scheme for the cross linking approach is shown in Scheme 1.3C. The cross linking method mainly involves grafting the hydrophobic backbones with hydrophilic side chains as well as cross linking groups. As the existence of functional groups at the side chains, the cross link reaction can occur to form a stable shell to improve the stability of CP NPs. Park, et al. synthesized CP backbones with azide groups at side chains.[99] Followed by precipitation, CP chains entangled to form CP NPs in water. The formed CP NPs solution was further irridated with UV light, which initiated the cross linking reaction between azide groups. The obtained CP NPs show obvious improved mechanical and photophysical stabilities as compared to that prepared with precipitation method, which is due to the protective crosslinking shell. Later, the same group utilized similar strategy to prepare CP NPs with cross linked shell using copper (І) or cucurbit[6]uril as catalysts, which could be potentially applied in cellular imaging.[100] Although these approaches could greatly enhance the stability of CP NPs, the density of functional groups at NP surfaces is hard to control, which may hamper their further biological applications. 1.4 Biological Application of CP NPs Accompanied by the extensive development of CP NPs, their biological applications have also been widely investigated. So far, their applications in biological system have also been mainly explored in imaging, detection and therapy. 15 Chapter 1 1.4.1 Bioimaging CP NPs usually show large absorption coefficient, high fluorescence brightness, good colloidal stability and photostability, low toxicity and functional surfaces, which meet the harsh requirement of fluorescent probes for biological imaging. The bioimaging application of CP NPs could be divided into non-specific cellular imaging, targeted cellular imaging and in vivo imaging. The early application of CP NPs in cellular imaging was mainly conducted by nonspecific imaging with bare hydrophobic NPs. For example, in 2007, Moon and co-workers prepared PPE NPs with an average size of 97 nm and a QY of 0.17 in aqueous medium.[72] The obtained PPE NPs could help to stain both living and fixed cell lines through overnight incubation. CP NPs were found exclusively around the perinuclear region as shown in Figure 1.3. In addition, the location of CP NPs could not overlap with the LysoTracker, and the uptake mechanism of these NPs by cells has not been investigated in this study. Another typical of CP NPs developed by Wu et al. has also been studied in nonspecific cellular imaging of J774A1 macrophages cells. The developed CP NPs without any encapsulation layer were found to be efficient labelling probes for cellular imaging. The high fluorescence signal of CP NPs could help to clearly observe the cell morphologies under confocal microscopy. Moreover, the CP NPs showed good overlap with LysoTracker, illustrating that they entered the cells through endocytosis process.[101] The detailed mechanism has been further examined by Fernando and coworkers.[102] As the endocytosis process is highly dependent on temperature, the authors studied the NP uptake at different temperature. Only weak 16 Chapter 1 fluorescence could be observed after long incubation time at 0 °C, which is much lower than that at 37 °C, illustrating that endocytosis plays an important role in NP uptake. In addition, PFBT NPs showed good overlap with the dextran tracker (a specific endocytosis tracker) which further confirmed the endosytosis mechanism. Moreover, the endocytosis involves several possible pathways, including clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis. To better understand the pathway, the authors further investigated the NP uptake by cells treated with various inhibitors, which could influence the definite pathways. The authors concluded that CP NPs with a size around 18 nm enter the cells mainly through macropinocytosis pathway.[102] A B Figure 1.3 Fluorescence images of live (A) and fixed (B) cells. A) BALB/C 3T3 cells were incubated sequentially with PPE NPs (green) and Hoechst dye (blue). B) Live BALB/C 3T3 cells were incubated with PPE NPs and fixed for confocal microscopic study.[72] Copyright 2007 John Wiley and Sons. Another non-specific staining example using CP NPs have been reported by Green’s group.[67] The PEG capped CP NPs could stain HEp-2 cells after 1 hour incubation and more efficient staining could be achieved by increasing incubation time. Strong fluorescence signal could be detected in the 17 Chapter 1 cytoplasm of cells after 24 h incubation. Noteworthy is that the fluorescence intensity of this CP NP did not decrease obviously upon laser irradiation for 5 minutes at a power density of 12.5 mW/cm2. The same group also investigated non-specific cellular imaging of lipid-PEG encapsulated CP NPs,[68] which also showed low cytotoxicity and good staining effect. Besides nonspecific cellular imaging, CP NPs could be endowed with targeting capacity by surface conjugation with targeting molecules, such as proteins, or peptides. One of the earliest examples of targeted cellular imaging for CP NPs has been demonstrated with folic acid conjugated PPE polymer chains.[103] As the side chains of CPs have numerous ionic carboxyl groups, CP NPs could be well dispersed in water. In addition, the carboxyl groups have been further conjugated with folic acid molecules via an amide coupling reaction. The folic acid conjugated CPs could enter KB cells more efficiently than those without folic acid conjugation. A B Figure 1.4 Confocal images of MCF-7 (A) and NIH/3T3 cells (B) incubated with folic acid-functionalized CP NPs.[59] Copyright 2009 John Wiley and Sons. Another example of targeted cellular imaging has been reported by Li and co-workers. They demonstrated a general strategy to prepare CP 18 Chapter 1 encapsulated PLGA NPs, and the CP NP surfaces could be functionalized with folic acid due to the existence of carboxyl acid groups at NP surfaces.[32] Taking PFVP as an example, folic acid conjugated PLGA NPs capped PFVP NPs (FPPFVP NPs) have been synthesized. After incubation of MCF-7 cancer cells with FPPFVP NP suspension for 2.5 h, bright fluorescence signal from NPs were detected from cell cytoplasm (Figure 1.4A). On the contrary, only week fluorescence was observed from NIH/3T3 fibroblast cells upon staining with FPPFVP NPs since lower folate groups at cell surfaces (Figure 1.4B). In addition, FPPFVP NPs showed almost no cytotoxicity with a concentration up to 2 mg/mL of NPs (700 nM of PFVP based on polymer chain), indicating that FPPFVP NPs have low cytotoxicity. As a result, FPPFVP NPs were able to act as an efficient targeting probe to discriminate folate receptors over expressed cancer cell lines. In 2010, Wu et al. reported the application of PS-PEG-COOH encapsulated PFBT NPs for visualizing cell surface marker (EpCAM) in MCF-7 cancer cell membrane.[89] Thanks to the carboxyl groups at NP surfaces, biomolecules, such as IgG, could be grafted at NP surfaces. Taking a specific cellular target of EpCAM as an example, the authors demonstrate the specific targeting ability of CP NPs. The PFBT NPs were conjugated with IgG via the EDC catalyzed coupling reaction. On the other hand, the surfaces of MCF-7 breast cancer cells were labelled with EpCAM receptors. In the presence of both PFBT-IgG probes and the detection antibody, EpCAM, MCF-7 cancer cells could be labelled with fluorescent probe as shown in Figure 1.5A; while in the absence of EpCAM, PFBT-IgG probe could not label MCF-7 cell surfaces (Figure 1.5B). In addition, they compared the 19 Chapter 1 labelling effect of their probes with a commercially available probe, Qdot-IgG. The quantitative analysis of the flow cytometry data suggested that the average intensity of PFBT NP-labeled cells was ~25 times brighter than the Qdot-IgG-labeled cells and ~18 times brighter than Alexa-IgG-labeled ones. As a result, the CP NPs provide a significantly higher signal-to-noise ratio than that of Qdots and Alexa at low excitation conditions. Besides immunofluorescent labelling, Wu and co-workers have also utilized the CP NPs for bioorthogonal labelling of targeted cells through click chemistry.[85] Using biosynthetic approaches, the targeted biomolecules could be endowed with the bioorthogonal groups, which is ready for highly specific targeted labelling. Wu et al. utilized the amino acids of azidohomoalanine (AHA) and homopropargylglycine to synthesize fresh proteins in the cells, which possess the functional groups for click reaction. The counterpart groups were conjugated at the surfaces of CP NPs. Bright fluorescence have been detected at the AHA incubation cells after reaction with alkyne modified CP NPs under cobber (І) catalysts. In contrast, no cell labelling was observed for the cells without eating AHA amino acids. However, the existence of copper ion is toxic to cells, which may hinder their applications in live cellular imaging. In addition to the applications of CP NPs in in vitro cellular targeting, the application of such fluorescent probes in in vivo imaging has also been investigated. For in vivo imaging, NIR fluorescent probes are highly desirable due to their high penetration depth, low autofluorescence background and weak photodamage to biological species. The first example of CP NPs applied in in vivo imaging has been demonstrated by Kim and co-workers in 2010.[104] 20 Chapter 1 They synthesized CP NPs by in suit Knoevenagel polymerization in the micelles formed by Tween 80. Through choosing different monomers, CP NPs could show fluorescence from blue color to red color. Among these CP NPs, the NIR-cvPDs showed an intense NIR fluorescence with an emission peak at 693 nm and a QY of 21%. The NIR-cvPDs NPs have been further applied into sentinel lymph node imaging by intradermally injected into the forepaw pad of mice. Their data showed that the NP could efficiently be trapped in the sentinel lymph nodes of the mice. A B Figure 1.5 Confocal images of live MCF-7 cells incubated sequentially with anti-EpCAM primary antibody and PFBT NP-IgG conjugates (A), and PFBT NP-IgG conjugates only (B).[89] Copyright 2010 American Chemical Society. Another example of in vivo imaging has been demonstrated using both 21 Chapter 1 PFBT and PFDBT co-encapsulated PSMA NPs. The obtained NPs showed an NIR emission with a peak at 655 nm and a high QY of 56%. The NP surfaces were conjugated to a peptide with targeting effect, chlorotoxin (CTX), for targeted tumor imaging and PEG to increase the circulation time of NP in in vivo condition. The capability of PFBT/PFDBT-CTX conjugates to traverse the blood-brain barrier and specifically target a tumor was evaluated in a transgenic mouse model (ND2:SmoA1), which was counter-illustrated with wide-type mouse. It was observed that PFBT/PFDBT-CTX conjugates accumulated at the brain tumor regions of the ND2:SmoA1 mice after NP injection, confirming their targeting ability to malignant brain tumors (Figure 1.6A). On the contrary, PFBT/PFDBT NPs without CTX conjugation did not show obvious tumor site accumulation in brain. Real-time investigation of the circulation profile of the PFBT/PFDBT NP-CTX conjugates indicated that the accumulation of conjugates in the brain tumor has been accomplished within 24 h and the signal intensity remained steady for 48 h during the 72 h analysis. However, the conjugates also showed obvious non-specific accumulation in liver and kidney during the tested period (Figure 1.6B). A Wide type ND2:SmoA1 B Liver Spleen Kidney Figure 1.6 (A) Ex vivo fluorescence imaging of healthy brains in wild-type mice and medullo blastoma tumors in ND2:SmoA1 mice. Each mouse was injected with either PFBT/ PFDBT NP (top), or PFBT/PFDBT-CTX NP (middle). Control: no injection (bottom). (B) Biophotonic images of resected 22 Chapter 1 livers, spleens, and kidneys from wild-type (middle) and ND2:SmoA1 (bottom) mice receiving PFBT/PFDBT-CTX NP injection. Control: no injection (top).[84] Copyright 2011 John Wiley and Sons. Besides the fluorescence imaging, the application of CP NPs in PA imaging have also been reported recently. PA imaging is built up through measurement of ultrasonic waves generated by the targets upon pulse laser absorption.[12-14] Benefiting from ballistic light, PA imaging could observe high deeper biological targets than other optical imaging modalities. In general, the PA signal is highly dependent on the absorption coefficient and non-radiative QY of the contrast agent. The first report of CP NP based PA contrast agent has been reported by Zha et al.[12] The synthesized PPy NPs showed high PA intensity due to their large absorption coefficient and high non-radiative QY. The application of PPy NPs as PA contrast agents have been further demonstrated in mice vascular imaging. 1.4.2 Biological Detection The applications of CP NPs have also been extended to the detection of physiologically important species. Most of the CP NP based fluorescent biosensor is utilizing the FRET between CPs and another sensitive reporter. Generally, the CP NPs with high stable fluorescence have been synthesized, and small fluorophores which are sensitive to the targeted molecules or environments have been doped or grafted at the surface of CP NPs, leading to efficient energy transfer between CPs and sensitive fluorophores. In the presence of the detection molecules or environment variation, the fluorescence property of fluorescent reporter could be influenced, resulting in the change of energy transfer between CP and organic dyes. This could further lead to a big 23 Chapter 1 ratiomatric change of donor and acceptor intensity for highly sensitive detection. The early example of CP NP based bisosensors has been developed by Wu et al. in 2009.[105] The sensor was consisted of a polymer (PDHF) and an oxygen sensitive platinum octaethylporphyrin (PtOEB). The fluorescence of PDHF was greatly quenched by PtOEB in the presence of nitrogen due to efficient energy transfer between them, and strong red fluorescence has been detected upon excitation of PDHT. However, in the presence of oxygen, the fluorescence of PtOEB was quenched, resulting in less energy transfer between PDHF and PtOEB, and strong blue emission was observed upon excitation of PDHF. Another example of CP NP based singlet oxygen sensor has been reported by Zhang and co-workers.[106] A singlet sensitive oxygen molecule (diaryltetracene) has been conjugated at the side chain of poly(phenlyene-co-fluorene). Due to energy transfer from CPs to the side diaryltetracene pendants, their conjugates showed a yellow emission from the diaryltetracene pendants upon excitation of the CPs. Upon mixing with the singlet oxygen molecules, energy transfer between them was blocked and the fluorescence of CP backbones was restored, resulting in an observable strong blue fluorescence from CPs. A large ratiometric contrast of 200-fold from the “off” state to the “on” state for singlet oxygen has been observed in this system. 24 Chapter 1 Figure 1.7 Effects of various ions on the fluorescence intensity of solution containing PFBT NP and PFVT/PFTBT NP.[86] Copyright 2011 Royal Society of Chemistry. A B Figure 1.8 (A) The schematic illustration of pH sensitive CP-FITC NPs. (B) Fluorescence spectra of CP-FITC NPs at different pH.[82] Copyright 2011 American Chemical Society. A ratiometric fluorescent sensor for metal ion detection has been constructed by Chiu’s group using a mixture of bare PFDBT NPs and PSMA encapsulated PFBT NPs.[86] The mixture showed two emission peaks at 540 nm (from PFBT NPs) and 623 nm (from PFDBT NPs), respectively. In the presence of Cu2+ or Fe2+, the fluorescence intensity at 540 nm was quenched while the fluorescence at 623 nm was stable, leading to an efficient ratiometic changing. The linear correlation between the ratio of I540 nm/I623 nm and ion concentration allowed the detection of Cu2+ and Fe2+ in 1-30 μM and 10-25 μM ranges, respectively (Figure 1.7). As the NP fluorescence could be 25 Chapter 1 recovered by addition of EDTA into NP/Cu2+ aggregates, which is not achievable in NP/Fe2+ aggregates, the NPs have been used to quantify Cu2+ and Fe2+ concentration in Dulbecco’s Modified Eagle Medium (DMEM). In addition to metal ions, intracellular pH is also of high importance to understand the physiological processes in subcellular organelles.[82] Chan developed an intracellular pH sensing platform, which was consisted of pHinsensitive PFVP and pH-responsive fluorescein isothiocyanate (FITC). Two emission peaks at 440 nm and 513 nm have been observed upon excitation at 390 nm. Gradual changing pH from 5.0 to 8.0 increased the emission peak of FITC while the fluorescence intensity of PFVP kept constant as shown in Figure 1.8. The ratio of fluorescence intensities (I513nm/I440nm) was found to change linearly as a function of pH in the range of 5.0-8.0 with good sensitivity and reversibility. The ability of the NPs in intracellular pH monitoring was further carried out. Quantitative study of the confocal images of Hela cells after internalization of the NPs via endocytosis suggested that the average pH value of Hela cells was ~5.0, which was consistent with the reported pH range for the acidic organelles involved in endocytosis (4.5-5.0 in lysosomes and ~6.5 in early endosomes). This strategy has also been utilized for temperature monitoring through conjugation a temperature sensitive dye with CP NPs.[90] Besides small molecules and environment monitoring, the FRET based strategy has also been extended to biomolecular detection. The detection of proteases, an important species involved in cancer and other diseases, has been achieved by Cordovilla and co-workers.[107] The CP was consisted of a highly emissive PPE backbone grafted with perylene unit for red emission and a 26 Chapter 1 hydrophilic chain with a terminal functional succinimide groups. The amphiphilic polymers could self assemble into NPs. Due to the aggregation induced fluorescence quenching, CP NPs showed weak fluorescence after cross-linking the succinimide groups with a proteases sensitive peptide. In the presence of proteases, the sensitive peptide broke and the aggregation induced CP fluorescence quenching became less dominant, resulting in the enhancement of CP fluorescence. The platform shows a 15-fold enhancement of the fluorescence intensity upon incubation with protease under physiological condition. 1.4.3 Therapy Both imaging and detection applications utilize the fluorescent properties of CP NPs. More recently, other properties of CP NPs have been explored in therapy applications. In view of the polymer nature of CP NPs, several researchers investigated their applications as drug and gene carriers. In addition, CPs could produce singlet oxygen, which has been used in photodynamic therapy. Moreover, fine-tuning CP structure could increase their non-radiative QY, which could be utilized as photothermal therapy reagents. In 2010, Wang’s group reported the CP NP based drug delivery system.[108] The NPs were synthesized by electrostatic assembly of cationic PFO and anionic poly(L-glutamic acid) grafted with doxorubicin. The CP NPs showed a size of 50 nm with good photostability and low cytotoxicity as show in Figure 1.9A. The advantageous feature of these NPs is due to their capacity to trace the drug delivery and release process. As shown in Figure 1.9B, the 27 Chapter 1 fluorescence of PFO was quenched due to energy transfer between PFO and doxorubicin. After drug release, the quenching effect would greatly decrease and the fluorescence of PFO would recover. As a result, the PFO could be used an indicator of drug delivery and release process in targeted cancer cells. A B Figure 1.9 (A) SEM images of PFO/doxorubicin NPs. (B) A schematic illustration of the component of NPs and uptake of NP by cells.[108] Copyright 2010 American Chemical Society. The CP NPs could be functionalized with cationic groups to be used as a platform for gene delivery. Wang’s group graft lipid at the side chain of cationic poly(fluorenylene phenylene), which has formed CP NPs in water due to its amphiphilic property.[95] The plasmid (pCX-EGFP) has been successfully loaded in NPs and delivered into A549 lung cancer cells for transcription and translation. The fluorescence from CP could help researchers to track the entrance of plasmids into cells. The expression of GFP in A549 cancer cells has been observed, illustrating the successfully plasmid delivery with the help of CP NPs. Moon and co-workers synthesized amine groups functionalized PPE polymer, which were precipitation to form CP NPs with a size about 60-80 nm.[109] Thanks to the existence of positively amine groups at NP surfaces, negatively charged gene could be easily loaded in CP NPs. The results showed that CP NPs could efficiently deliver siRNA into cell. In 28 Chapter 1 addition, CP NPs have also been used as fluorescent probes to monitor the uptake mechanism of NPs by cells. The photodynamic therapy of CP has been discovered by Whitten and co-workers in 2005. They found that a cationic PPE could efficiently kill bacterial through produce singlet oxygen or other reactive oxygen species upon white light irradiation.[110] Whitten’s group synthesized various types of CPs for photodynamic therapy. One typical example is that through absorbing positively charged PPE at the surface of negatively charged silica particles, efficient biocidal reagents have been constructed.[111] They chose silica particles with a size of 5 µm or 30 µm, respectively, and PPE with positively charged chains were adsorbed on silica particles surfaces. The fluorescence of PPE could be clearly observed under fluorescent microscopy. Light induced biocidal activity has been successfully demonstrated on Cobetia marina and Pseudomonas aeruginosa treatment. Besides the capacity to generate singlet oxygen by CPs, they also help to increase the singlet oxygen generation ability of other photosensitizer. Since CPs could help to collect excitation light more efficiently due to their large extinction coefficient, and the absorbed light energy could transfer from CP to photosensitizer, resulting in great enhancement of singlet oxygen generation amount. One example of this concept has been demonstrated by Wang’s group in 2009.[113] The authors selected an anionic polythiophene (PTP) as the donor and a cationic porphyrin (TPPN) as an acceptor to form complexes through electrostatic interactions in aqueous. Due to efficient energy transfer between them, the singlet oxygen generated from TPPN has been greatly improved by PTP as compared with bare TPPN. Under the irradiation with white light with 29 Chapter 1 a fluence of 90 mW/cm2 for only 5 minutes, 70% reduction of bacterial viability has been realized. A C B D Figure 1.10 (A) Chemical structures of CP and photosensitizer TPP. (B) A schematic illustration of NP formulation. (C) AFM image of CP/TPP coencapsulated NPs. (D) Normalized UV-vis and PL spectra of PDHF NPs doped with 10% of TPP as well as that of pure PHDF NPs.[112] Copyright 2011 Royal Society of Chemistry. In 2011, Shen et al. synthesized photosensitizer doped CP NPs for photodynamic therapy, which can be utilized upon both one-photon and twophoton excitation.[114,115] Meanwhile, McNeil’s group also developed CP based photodynamic therapy reagents under one- and two-photon excitation.[112] They chose poly(9,9-dihexylfluorene) (PDHF) and poly(9,9dioctylfluorenyl-2,7-diyl) (PFO) as the donor and tetraphenylporphyrin (TPP) as the photosensitizer, which were used to form CP and photosensitizer co- 30 Chapter 1 encapsulated NPs. The chemical structures of the used reagents and the corresponding CP NPs with a size of ~15 nm are shown in Figure 1.10A and 1.10B, respectively. Efficient energy transfer occurs between CPs and TPP due to the good overlap between the PFO emission and the TPP absorption (Figure 1.10D). In addition, the NPs are suitable for two-photon excitation, which shows deeper penetration depth as compared with one-photon excitation. In 2011, Wang’s group further conjugated the photosensitizer to the side chain of PTP, which forms CP NPs by self-assembly.[116] In addition, targeted molecules could be grafted at the surface of CP NPs for targeted photodynamic therapy. In addition to the biocidal capacity, they can distinguish malignant and normal cells. Taking folic acid as an example target molecule, the formed CP NPs could specifically kill more folate positive KB cells (80% damage) than that of folate negative NIH-3T3 cells (30% damage). Very recently, the same group grafted a photosensitizer (a protein inactivation drug) as well as targeted molecules (folic acid) with CP backbone for targeted cancer therapy.[117] Upon light irradiation, the CP could induce the drug to release singlet oxygen species to inactivate the targeted protein at specific cancer cells, leading to the inhibition of tumor growth. More recently, CP NPs with strong NIR absorbance and high nonradiative decay QY start to serve as photothermal reagents. Photothermal therapy mainly utilizes the heat generation of materials under light absorption. For CPs, the heat is mainly produced when the excited electrons are transferred to the ground state through non-radiative pathway. In this regard, 31 Chapter 1 conductive CPs should have great potential to generate heat due to the high non-radiative QY. The first report of CP based photothermal agents is polyaniline. Polyamiline is easily to be doped with strong aid, alkali ion or transition metal to generate an interband gap between valence and conduction bands, facilitating the decrease of excitation-energy level and the transfer of electrons. The doping process could transfer the NP state from emeralidine base (EB) to emeralidine salt (ES), which is accompanied by the red-shift of its absorption peak to NIR region. In view of multi-components exist in cellular microenvironment, polyaniline could be potentially doped by protons, alkali ions or other oxidative species inside cytosol to change its status from EB state to ES state with a strong NIR absorbance. Yang et al. synthesized polyaniline NPs with EB state with good water solubility, which could be uptaken by cancer cells.[118] In the intracellular environment, EB polyaniline is changed to ES state. The temperature could be increased from 27 °C to 68 °C upon laser irradiation (2.45 W/cm2) of 0.5 mg/mL of polyaniline NPs. Upon laser irradiation, cancer cells could be effectively killed. Taking A431 cells as an example, both in vitro and tissue experiments illustrated that polyaniline was an efficient photothermal material for cancer cell therapy. In addition, Liu’s group ethylenedioxythiophene):poly(4-styrenesulfonate) developed poly-(3,4- (PEDOT:PSS) based organic photothermal agents with strong NIR absorbance in 2011.[119] The synthesized NPs showed an NIR absorbance with an average diameter of 8090 nm based on scanning electron microscope characterization. PEGs were absorbed at NP surfaces to render NPs with stealth property, resulting in their 32 Chapter 1 efficient accumulation in mice tumor due to EPR effect after 48 h intravenous injection of NPs. To monitor the therapy effect of NPs, an infrared thermal mapping instrument was applied to check the temperature evolution in the tumor upon NIR laser irradiation. The temperature in tumor was increased to 51 °C after 5 minutes laser irradiation with a power density of 0.5 W/cm2, while no obvious temperature increase was observed for tumor without NP injection. The tumor was eliminated after 15 days treatment, demonstrating the good therapy effect of the synthesized NPs as a photothermal agent. A B Figure 1.11 (A) FE-TEM images of PPy NPs. (B) The temperature evolution of PPy NPs at different concentrations.[120] Copyright 2012 Royal Society of Chemistry. In addition, polypyrrole (PPy) has also been extensively utilized as a photothermal agent.[120-124] PPy NPs could be simply fabricated through emulsion polymerization using FeCl3 as oxidation agent and PVA as an emulsifier. Zheng’s group firstly reported the photothermal application of the PVA stabilized PPy NPs in 2012.[120] The synthesized NPs show good water dispersibility and excellent photostability with a size around 50 nm as shown in Figure 1.11A. The temperature of PPy NP suspension in water (with a concentration of 20 ppm) could be increased from 20 °C to 48 °C in 10 33 Chapter 1 minutes upon 808 nm laser irradiation at a density of 1 W/cm2 as shown in Figure 1.11B. In view of the high heat generation efficiency and good biocompatibility of PPy NPs, they have been further used in in vitro and in vivo photothermal experiment. The tumour temperature reached 60 ℃ after only 5 minutes laser irradiation with the power density of 1 W/cm2, and the treated 4T1 tumors disappeared quickly without noticeable side effects on normal tissues. Later, both Liu’s group[121] and Dai’s group[124] reported the application of PPy NPs in photothermal therapy. In a recent work, MacNeill’s group synthesized CPs through a donaracceptor (D-A) concept to fine-tune their absorption properties.[125] Conjugation of 2-ethylhexyl cyclopentadithiophene with 2,1,3- benzothiadiazole or 2,1,3-benzoselenadiazole, two different polymers, PCPDBO and PCPDTBSe were synthesized. They respective polymeric NPs have been synthesized via the conventional precipitation apporach. The obtained PCPDTBSe NPs showed an obvious NIR absorbance peak, benefiting energy conversion to heat. In addition, these CP NPs were low cytotoxicity in MTT experiment using RKO and HCT116 colorectal cancer cells as examples. However, most cancer cells were killed by PCPDTBSe NPs at above 125 μg/mL concentration upon 808-nm NIR laser irradiation at a power density of 0.6 W/cm2. 1.5 Research Objectives In view of the obvious advantages of CP NPs in fluorescence imaging as compared with the conventional fluorescent materials, various strategies have been developed to synthesize CP NPs. Generally, the obtained CP NPs show large absorption coefficient, bright fluorescence, large Stokes shifts, 34 Chapter 1 good photostabilities and benign biocompatibility in their respective bioimaging applications. However, most CP NPs are synthesized by taking advantage of the weak hydrophobic interaction of CP backbones, which may not be stable in long term study. Considering the above issues, it is necessary to design and develop new approaches to synthesize CP NPs and improve their colloid and structure stabilities. In addition, the fluorescence performance of CP NPs (e.g. brightness) is essentially important for their fluorescent imaging. However, less work is focused on the optimization of their performance by controlling the internal structures. Furthermore, although some CP NPs show high fluorescence in imaging, there are also many CP NPs do not emit high fluorescence, which means they have strong high non-radiative QYs. Since the PA imaging performance of contrasts is highly related to non-radiative QY, we also investigated whether CPs with high non-radiative QYs could be applied as PA imaging contrast agents, which broadens the imaging application of CPs based materials. As such, this Ph.D. thesis is focused on developing new strategy to synthesize CP NPs with high structure stabilities, high fluorescence as well as exploring their applications in PA imaging. The specific objectives are listed as following. 1. To develop a facial approach to synthesize CP NPs with cross-linked shell to increase their stability as well as surface functional groups for specific applications. 2. To develop a general method to fabricate CP loaded silica NPs to increase both physical stability and photostability and explore their applications in targeted cellular imaging. 35 Chapter 1 3. To enhance fluorescent performance (e.g. fluorescence QY) of CP NPs as well as their application in in vivo fluorescence imaging. 4. To design CP NPs with high non-radiative QYs and study their applications in PA imaging. Through this Ph.D. project, it is anticipated that both stability and the fluorescence performance of CP NPs would be improved through our material and formulation design. In addition, the application of CP NPs in PA imaging modality would also be exploited. 1.6 Thesis Outline This thesis consists of six chapters. Chapter 1 introduces the general research background, the progress of CP NPs in literatures as well as the objectives of this Ph.D. project. In Chapter 2, a facile strategy to synthesize stable CP NPs with multihydroxy by dendritic cross-linking is illustrated. The detailed influencing factors on the size and other property of CP NPs are discussed and their application in cellular imaging is also investigated. In Chapter 3, a general approach to prepare CP embedded silica NPs without any tedious chemical modification of both CP chains and silica precursors is developed. The formation mechanism of CP doped silica NPs is studied followed by the examination of their application in targeted cellular imaging. . In view of the protection of both polymer and silica matrices, we explored the polymer/silica co-protection strategy to improve both stability and fluorescence QY of CP NPs in Chapter 4. In addition, their two-photo absorption property is investigated. Their application as two-photon fluorescent probes is further studied in two-photon excited vascular imaging. 36 Chapter 1 Chapter 5 describes the synthesis of CP NPs for PA imaging, which involves the design of CPs with a high non-radiative QY, formulation of CP NPs and their application as a PA probe in biological imaging. Chapter 6 summarizes the conclusions of this thesis and future research directions are recommended. 37 Chapter 2 CHAPTER 2 FACILE SYNTHESIS OF STABLE MULTIHYDROXY CONJUGATED POLYMER NANOPARTICLES BY DENTRITIC CROSS-LINKING 2.1 Introduction The development of fluorescent bioimaging reagents has gained significant attention in recent years. An ideal bioimaging reagent generally requires high fluorescence QY, good optical stability, biocompatibility, and surface functionality.[126,127] As a promising candidate, CPs have shown advantages over small fluorophores, fluorescent proteins, and even semiconductor QDs, which include large absorption cross sections, bright fluorescence, and favourable biocompatibility.[26,35,36,74,85] However, CPs with inherent hydrophobic backbones are insoluble in water. As a consequence, it is highly desirable to endow CPs with good water dispersibility to facilitate their biological applications. Several strategies have been developed to transform organic soluble CPs into aqueous media, which include emulsion technique, precipitation technique, design and synthesis of conjugated polyelectrolytes (CPEs) and direct polymerization in heterophase system.[31,51,128-133] Among them, synthesis of CPEs and formation of CP NPs) via precipitation technique are the two most widely used strategies. However, the synthesis of CPEs generally requires laborious synthetic steps and the modification has to be specifically designed for each CP. In addition, the charged side chains of CPEs often lead 38 Chapter 2 to nonspecific interactions with biomolecules.[134,135] The other commonly employed strategy is to form CP NPs by precipitation technique in the absence or presence of surfactants.[99,136,137] It involves the dissolution of CPs in organic solvents, followed by injection of the CP solutions into water with ultrasonication. CP NPs are formed when hydrophobic CP chains fold into spherical shape to release interface energy. A variety of water-dispersible CP NPs have been synthesized via this approach, however, these CP NPs with hydrophobic surface are not inherently stable, and their surface possesses no functional groups for further functionalization.[32,138-140] Recently, embedding CPs into polymer matrix such as carboxylic polyethylene glycol and poly(DLlactide-co-glycolide) afforded CP NPs with surface functional groups.[59,141] The poor structural stability resulted from noncovalent interaction may cause microphase separation or CP leakage, which is problematic for practical applications.[59,142] As the polymer encapsulated CP NPs generally have COOH or -NH2 functional groups, they could also lead to undesired association with cell membrane and other charged biomolecules via hydrophobic and electrostatic interactions.[59,100,141-143] Despite the progress on CP NPs in recent years, the preparation of CP NPs with inherent hydrophilic surface, multiple neutral functional groups, and high structural stability is still in its infancy. In this chapter, we report a new strategy toward water dispersible CP NPs via copper-free click chemistry in an oil-in water miniemulsion system, as shown in Scheme 2.1. Hyperbranched polyglycerol (HPG) was selected as the cross-linker because it is a well-known biocompatible dendritic polymer with good water solubility and numerous reactive hydroxyl groups.[144-146] In 39 Chapter 2 addition, HPG can be easily synthesized by one step polymerization as compared to dendrimers.[147] As one of the most widely used CPs for CP NP formation, PFBT was chosen as a representative CP to react with HPG. Detailed studies reveal that the cross-linked CP-HPG NPs. with multiple surface hydroxyl groups exhibit good structural stability and water dispersibility, high fluorescence QY, stable optical properties, and low cytotoxicity. In addition, CP-HPG NPs show tunable size in the range of 40210 nm with narrow size distribution, simply by adjusting the oil phase and surfactant used for miniemulsion. This work opens a new opportunity to synthesize functional water-dispersible fluorescent CP NPs. 2.2 Experimental Section 2.2.1 Materials Glycidol, potassium methylate solution in methanol (CH3OK, 25%), N,N’-dicyclohexylcarbodiimide (DCC), 5-hexynoic acid (HA), 4- (dimethylamino) pyridine (DMAP), MTT, bovine serum albumin (BSA), γglobulins from bovine blood, sodium dodecyl sulfate (SDS), and sodium azide were purchased from Sigma-Aldrich. Glycidol (96%) was purified by vacuum distillation and stored in a refrigerator (-4 oC) before use. Dioxane and methanol was distilled before use. NMR solvents were purchased from Cambridge Isotope Laboratories, Inc. Poly[9,9’-bis(6’’-bromohexyl)fluoreneco-4,7-(2,1,3-benzothiadiazole)] (PFBT-Br, Mn 15000, PDI 1.87) was synthesized according to our previous report.[148] All other chemicals were analytical grade and used as received without further purification. Milli-Q water (18.2 MΩ) was used for all experiments. 40 Chapter 2 2.2.2 Characterization Fourier transform infrared (FT-IR) spectra were recorded using a PE Paragon 1000 spectrometer (KBr disk). NMR spectra were collected on Bruker Avance 500 (DRX 500, 500 MHz). UV-vis spectra were recorded on a Shimadzu UV-1700 spectrometer. PL measurements were carried out on a Perkin-Elmer LS-55 equipped with a xenon lamp excitation source and a Hamamatsu (Japan) 928 PMT, using 90 degree angle detection for solution sample. PL QYs were measured using quinine sulphate as the standard, with a QY of 55% in H2SO4 (0.1 M). DLS measurements were performed using Brookhaven Instrument Corporation (BIC) 90 Plus with λ = 659 nm. FE-TEM studies were performed on a JEOL JEM-2010 electron microscopy with an accelerating voltage of 200 kV. Gel permeation chromatography (GPC) analysis was conducted with a Waters 2690 liquid chromatography system equipped with Waters 996 photodiode detector, using polystyrene as the standard and LiBr/DMF (0.01 mol/L) (for HPG and HPG-Alk) or THF (for PFBT-N3) as the eluent at a flow rate of 1 mL/min. Confocal laser-scanning microscopy (CLSM) images was recorded on a Zeiss LSM 410 (Jena, Germany) with imaging software (Fluoview FV500). 2.2.3 Synthesis of PFBT-N3 PFBT-Br (624 mg, 1 mmol of PFBT repeat unit) was dissolved in 100 mL of THF. Sodium azide (NaN3, 260 mg, 4 mmol) was dissolved in DMF (50 mL). Then the PFBT-Br and NaN3 solutions were mixed in a 250 mL flask equipped with a magnetic stirring bar. The mixture was heated to reflux for 2 days, filtrated, concentrated, and then precipitated in methanol. After removal of the solvent, 527 mg of PFBT-N3 as an orange powder was obtained (Yield: 41 Chapter 2 ca. 96%). 1H NMR (500 MHz, CDCl3, ppm) δ = 8.15-7.97 (m, 6H), 7.96-7.81 (m, 2H), 3.26-3.06 (m, 6H), 2.26-2.04 (m, 4H), 178-1.42 (m, 4H), 1.39-1.04 (m, 8H) 0.95-0.85 (m, 4H). FT-IR (KBr, cm-1): ν = 2926 and 2862 (-CH2, CH3), 2098 (-N3). Element analysis calculated (%): C 67.88, H 5.48, N 20.43. Found: C 67.88, H 5.48, N 20.43. GPC (THF as eluent): number-average molecular weight (Mn) = 14200, polydispersity index (PDI) = 1.87. 2.2.4 Synthesis of HPG HPGs with molecular weight 15.5 KDa was prepared according to the literature.[149,150] Typically, in a three-neck flask, trimethylolpropane (TMP) (20.1 mg, 0.15 mmol) was added to the flask under argon atmosphere followed by 100 μL of potassium methylate solution in methanol (25 wt %) and 2 mL methanol. The mixture was stirred for 30 min at 45 oC, and then excess methanol was removed in a vacuum. Anhydrous dioxane (15 mL) was added. The flask was kept in an oil bath at 95 oC, and 20 mL of glycidol (0.3 mol) was added dropwise over a period of 24 h. After completion of monomer addition the mixture was stirred for an additional 5 h. The product was dissolved in methanol then was precipitated in acetone, and this process was repeated at least three times. 1H NMR (500 MHz, DMSO, ppm) δ = 4.83-4.39 (-OH), 3.82-3.25 (-CH, -CH2). 13C NMR (500 MHz, DMSO, ppm) δ: 80.555.6. FT-IR (KBr, cm-1): ν = 3440 (-OH), 2926 and 2862 (-CH2, ν-CH3), 1120 (C-O-C). GPC (DMF as eluent): Mn = 15500 (210 -OH groups per HPG macromolecule), PDI = 1.23. 42 Chapter 2 2.2.5 Synthesis of HPG-Alk Typically, HPG (740 mg, 10.0 mmol -OH) was mixed with 25 mL of DMF solution containing the DCC (60 mg, 0.29 mmol) and DMAP (30 mg, 0.25 mmol). Then 340 mg of 5-hexynoic acid (28 mg, 0.25 mmol) was added to the mixture at room temperature under magnetic stirring for 24 h. After removing the insoluble dicyclohexylurea (DCU) by filtration and centrifugation, the centrifugal liquid was precipitated in diethyl ether and then in acetone. The final obtained HPG-Alk straw yellow viscous liquid can be dissolved in organic solvents such as chloroform and dichloromethane but cannot be dissolved in water anymore. 1H NMR (500 MHz, DMSO, ppm) δ = 4.77-4.37 (-OH), 4.25-4.01 (HPG conjugated HA unit, -CH, -CH2), 3.85-3.14 (HPG backbone, -CH, -CH2), 2.48 (-CH2CO), 2.27 (-CH2C≡CH), 2. 2.0 (C≡CH), 1.85 (-CH2CH2C≡CH). FT-IR (KBr, cm-1): ν = 3440 (-OH), 2926 and 2862 (-CH2, -CH3), 2105 (-C≡CH), 1724 (-C=O) 1120 (C-O-C). GPC (DMF as eluent): Mn = 19200 (about 20% of –OH groups were transformed into C≡CH groups), PDI = 1.32. 2.2.6 Preparation of CP-HPG NPs Typically, HPG-Alk (11.8 mg, 0.025 mmol of -C≡CH) and PFBT-N3 (7.0 mg, 0.025 mmol of -N3) were dissolved in chloroform (1 mL) and stirred at room temperature for 0.5 h before pouring into the SDS (65 mg, 0.23 mmol) containing water (25 mL). Then the mixture was ultrasonicated for 10 min in a water-ice bath to obtain miniemulsion. After stir at room temperature for 1 h, the reaction temperature was rise to 85 oC and stirred for additional 24 h to ensure the complete of click reaction. After reaction, the formed CP-HPG NPs were freeze-dried and washed by hexane repeatedly. Subsequently, the CP43 Chapter 2 HPG NPs were dispersed in water again and dialysed against Milli-Q water for 5 days using a cut off 3500 membrane. 2.2.7 Cell Imaging MCF-7 breast cancer cells were cultured in folate-free RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a humidified environment containing 5% CO2. Before experiment, the cells were precultured until confluence was reached. MCF-7 breast cancer cells were cultured in the chambers at 37 oC. After 80% confluence, the medium was removed and the adherent cells were washed twice with 1 × PBS buffer. The CP-HPG NP suspension (1 µM of PFBT repeat unit, 0.4 mL) was then added to the chamber. After incubation for 2 h, cells were washed three times with 1 × PBS buffer and then fixed by 75% ethanol for 20 min, which was further washed twice with 1 × PBS buffer and imaged by CLSM (Zeiss LSM 410, Jena, Germany) with imaging software (Fluoview FV1000) . 2.2.8 Cytotoxicity Evaluation The cytotoxicities of CP-HPG-1 NPs were examined using MTT assay. MCF-7 breast cancer cells were seeded in 96-well plates (Costar, IL, USA) at a density of 4 × 104 cells mL-1. After 24 h incubation, the medium was replaced by the CP-HPG NP suspension at PFBT repeat unit concentrations of 10, 5, and 2 µM, and the cells were then incubated for 24, and 48 h, respectively. After the designated time intervals, the wells were washed twice with 1 × PBS buffer, and 100 µL of freshly prepared MTT (0.5 mg/mL) solution in culture medium was added to each well. The MTT medium solution was carefully removed after 3 h incubation. DMSO (100 µL) was 44 Chapter 2 then added into each well, and the plate was gently shaken for 10 min at room temperature to dissolve all precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan). Cell viability was expressed by the ratio of absorbance of the cells incubated with CP-HPG-1 to that of the cells incubated with culture medium only. 2.3 Results and Discussion 2.3.1 Synthesis and Characterization of PFBT-N3 and HPG-Alk HO O OH HO OH HO CH3OK HO O O O O O O O HO O HO OH O O OH HO O O O OH HO OH O O S N O HO OH HPG-ALK N S N Click n n PFBT-N3 PFBT-Br Br N3 PFBT OH O OH OH NaN3 Br O O O HO OH O O O O OH OH O OH O HPG N O O O OH OH O O O O HO OH O OH O O O DCC, DMAP OH O O O HO HO OH OH O O HO HO HO O O O O OH OH O O O O OH OH O O O OH HO O O HO OH OH HO OH HO HO OH HO HO OH O N3 HPG-ALK CP-HPG nanoparticle Scheme 2.1 Schematic illustration of the preparation of CP-HPG NPs. The synthetic route to water-dispersible CP NPs is depicted in Scheme 2.1. Poly[9,9′-bis(6″-bromohexyl)fluorene-co-4,7-(2,1,3-benzothiadiazole)] (PFBT-Br, Mn = 15000, polydispersity (PDI) = 1.75) was synthesized according to our previous report.[148] The -Br groups were then transformed to -N3 groups by reaction between PFBT-Br and sodium azide to afford poly[9,9′-bis(6″-azidohexyl)fluorene-co-4,7-(2,1,3-benzothiadiazole)] (PFBTN3) in 95% yield. After the reaction, the resonance peaks at around 3.30 ppm for -CH2Br disappears and new peaks appear at 3.18 ppm for -CH2N3 in the 1H NMR spectrum for PFBT-N3, indicating the successful transformation of -Br 45 Chapter 2 to -N3. This is further verified by the appearance of an obvious peak at 2104 cm-1 corresponding to -N3 groups in the FT-IR spectrum of PFBT-N3 (Figure 2.1). Absorbance HPG HPG-Alk PFBT-N3 CP-HPG-1 4000 3000 2000 1000 -1 Wavenumber (cm ) Figure 2.1 FT-IR spectra of HPG, HPG-Alk, PFBT-N3 and CP-HPG-1. HPG (Mn = 15500, PDI = 1.23) was synthesized by anionic ringopening polymerization according to the literature,[149-151] which has excellent water solubility. The structure of HPG was characterized by FT-IR and NMR spectra (Figure 2.1 and experimental section). The degree of branching (DB) for the HPG calculated from the results of inverse-gated 13C NMR spectrum is about 0.53.[149,152] The surface hydroxyl groups of HPG were subsequently esterified with 5-hexynoic acid (HA) in the presence of DCC to afford alkynemodified HPG (HPG-Alk). As compared to the FT-IR spectrum of HPG, two new peaks at 2105 and 1724 cm-1 corresponding to -C≡CH and -C=O groups appear on the FT-IR spectrum for HPG-Alk. In addition, the resonance peaks at 2.27 ppm (-CH2C≡CH), 2.0 ppm (-C≡CH), and 1.85 ppm (-CH2CH2C≡CH) in the 1H NMR spectrum of HPG-Alk also suggest its right structure. The conversion efficiency of the hydroxyl groups to alkyne groups calculated from 46 Chapter 2 the 1H NMR spectra of HPG and HPG-Alk is ∼20%, which is further confirmed by the GPC result of HPG-Alk (Mn = 19200, PDI = 1.32) relative to HPG. It should be noted that the HPG-Alk still possesses numerous hydroxyl groups (∼168 -OH groups per HPG-Alk macromolecule calculated from the GPC and NMR results). The HPG-Alk shows good solubility in organic solvents such as dichloromethane and chloroform but is not dispersible in water. The detailed synthesis and characterization are shown in the experimental section. Similar to HPG-Alk, HPG-Alk2 (Mn = 35600, PDI = 1.20) with over 80% surface alkyne groups was also synthesized simply by increasing the feed ratio of HA to HPG. 2.3.2 Synthesis and Characterization of CP-HPG NPs Table 2.1 Reaction Parameters and the Optical Properties of the WaterDispersible CP-HPG NPs.[a] Product Voil phase (mL) Diameter[b] PDI[b] (nm) CSDS (mM) QY (%) CP-HPG-1 1 9.5 83 0.046 0.24 CP-HPG-2 2 9.5 114 0.053 0.23 CP-HPG-3 3 9.5 152 0.048 0.21 CP-HPG-4 1 12.5 44 0.042 0.25 CP-HPG-5 1 8.0 210 0.068 0.21 CP-HPG-6 1 0 135 0.724 0.17 [a] Volume of the water phase is 20 mL. The feed amounts of PFBT-N3 and HPG-Alk are 11.8 and 7 mg, respectively; [azide]/[alkyne] = 1:1. [b] Determined by DLS. 47 Chapter 2 A 100 B 60 40 20 300 nm 0 100 200 300 Diameter (nm) CP-HPG-1 PL Intensity (a.u.) 80 Intensity PFBT-N3 C Absorbance (a.u.) CP-HPG-1 300 400 500 600 700 800 Wavelength (nm) Figure 2.2 (A) Hydrodynamic radius distribution and (B) FE-TEM image of CP−HPG-1. UV−vis absorption (dashed line) and emission (solid line) spectra (C) of PFBT−N3 in dichloromethane and CP−HPG−1 in aqueous solution (λex = 450 nm). The inset shows the aqueous solution of CP−HPG-1 NPs under daylight (left) and 365 nm UV light illumination (right). 100 A 100 CP-HPG-2 Intensity Intensity 60 40 75 100 C 100 125 150 Diameter (nm) 40 0 140 175 CP-HPG-4 100 80 D 150 160 Diameter (nm) 170 CP-HPG-5 80 Intensity Intensity (a.u.) 60 20 20 60 40 20 0 CP-HPG-3 80 80 0 B 60 40 20 40 60 Diameter (nm) 80 0 100 200 300 Diameter (nm) 400 Figure 2.3 Hydrodynamic radius distribution of (A) CP–HPG–2, (B) CP– HPG–3, (C) CP–HPG–4 and (D) CP–HPG–5. As copper ions are toxic to biosubstrates, biocompatible copper-free click coupling was adopted to synthesize CP-HPG NPs using thermal 48 Chapter 2 initiation in oil-in-water miniemulsion.[153,154] PFBT-N3 and HPG-Alk with 1:1 azide/alkyne ratio were first dissolved in chloroform (oil phase) and then the mixture was miniemulsified with water in the presences of SDS surfactant. As the temperature increased to 85 °C, click reaction between HPG-Alk and PFBT-N3 occurred. After reaction, transparent orange aqueous solution was obtained, indicating good water dispersibility of the CP-HPG NPs. The disappearance of the peak at about 2100 cm-1 corresponding to alkyne or azide groups in the FT-IR spectrum of CP-HPG confirms the nearly complete “click” cross-linking and efficient thermal initiation (Figure 2.1). Meanwhile, the peak at 3400 cm-1 remains strong, indicating that the CP-HPG NPs possess numerous hydroxyl groups. To probe the effect of miniemulsion conditions on the size of CP-HPG NPs, we conducted a series of experiments by changing the feed ratio of oil phase or surfactant to water (Table 2.1). The sizes of the obtained CP-HPG NPs were investigated by DLS and the respective results are shown in Figure 2.2A and Figure 2.3. As the critical micelle concentration (CMC) for SDS in water is about 8.0 mM, we first fixed the concentration of SDS (CSDS) at 9.5 mM. The size of CP-HPG NPs increased from 83 to 152 nm with increased oil phase from 1 to 3 mL. This is due to the fact that more oil phase could result in the formation of bigger micelles at the same CSDS in an oil-in-water system.[155] On the other hand, it is found that at a constant oil-to-water ratio, the size of CP-HPG increased from 44 to 210 nm when the CSDS was varied from 12.5 to 8.0 mM (Table 2.1). This is consistent with previous reports on using miniemulsion to synthesize polystyrene and poly(N-isopropylacrylamide) NPs.[156] These results indicate that the size of CP-HPG NPs could be fine- 49 Chapter 2 tuned simply by adjusting the oil phase and surfactant used in the miniemulsion. All the CP-HPG NPs synthesized with SDS as the surfactant showed narrow size distribution (PDI < 0.07) and good water dispersibility. FE-TEM further verified the well-defined nanostructure and uniform size. As shown in Figure 2.2B, the CP-HPG-1 NPs exhibit spherical shape with an average size of 78 nm, which is slightly smaller than that for DLS data (Table 2.1). Figure 2.4 Photograph of HPG–Alk (left), PFBT–N3 (middle), and CP–HPG NPs (right) in the mixture of water and chloroform. It is worth noting that both the HPG-Alk and the PFBT-N3 have good solubility in chloroform. Interestingly, after click reaction, the products show good dispersibility in water phase as shown in Figure 2.4. The SDS surfactant should be removed by repeated washing with hexane, followed by dialysis against Milli-Q water for five days. We deduce that the good water dispersibility of the CP-HPG NPs is attributed to the presence of numerous hydrophilic hydroxyl groups on the surface of CP-HPG NPs while the hydrophobic domain is encapsulated as the core after the click reaction. To prove this conjecture, first, a control experiment for CP-HPG-6 in the absence of SDS was conducted (Table 2.1). The resulted CP-HPG-6 presents good 50 Chapter 2 dispersibility in the upper aqueous layer of a water/chloroform mixture, which indicates that SDS is not directly related to water dispersibility of the CP-HPG NPs, although it significantly affects the nanoparticle size and morphology (Figure 2.5). In addition, HPG-Alk2 was also reacted with PFBT-N3 (1:1 alkyne/azide) in miniemulsion and the product showed good dispersibility in water. However, when HPG-Alk2 with 4:1 alkyne/azide was used, the resulting CP-HPG NPs exhibited amphibious dispersibility that can be dispersed both in water and in chloroform phase as a result of the presence of hydroxyl groups and un-cross-linked HA chains on their surface. Based on these results, the good water dispersibility of the CP-HPG NPs is attributed to the presence of numerous hydrophilic neutral hydroxyl groups on the surface of CP-HPG NPs. These results also suggest that the surface functionality of CP-HPGs can be easily fine-tuned by choosing suitable cross-linkers. 2 µm Figure 2.5 FE-TEM image of CP–HPG–6 NPs. The absorption and emission spectra of CP-HPG-1 NPs in aqueous solution are shown in Figure 2.2C. Compared with the PFBT-N3 in dichloromethane, no obvious shift is observed for the absorption and emission 51 Chapter 2 peaks of the CP-HPG-1. All the CP-HPG NPs prepared using SDS as surfactant exhibit strong fluorescence with QYs higher than 20% (Table 2.1), which compare favorably to other reported CP NPs based on PFBT with QYs less than 10%.[101] Such a high fluorescence QY of CP-HPG NPs could be attributed to the minimized self-quenching of PFBT in the CP NPs format. As compared to the CP NPs prepared by the precipitation approach, the introduction of HPG as a spherical scaffold to the polymer side chain can effectively reduce the chain tangling and π-π stacking of CPs, leading to reduced polymer fluorescence self-quenching. Therefore, using HPG as crosslinker can not only endow CPs with hydrophilic neutral hydroxyl groups associated with good water dispersibility but also can retain strong fluorescence of the CPs. 2.3.3 Stability Characterization of CP-HPG1 Considering the complex physiological environment for bioimaging applications, the physical stability of CP-HPG NPs in aqueous solution was examined under different conditions. Because all the CP-HPG NPs show similar optical properties, CP-HPG-1 was chosen as a representative for the following studies. The concentration of CP-HPG-1 solution was calculated based on the concentration of PFBT repeat unit (RU). The fluorescence change of CP-HPG-1 at [RU] = 20 μM and different pH is shown in Figure 2.6A. Less than 5% variation in emission intensity is observed when the pH is changed from 3 to 11. In addition, there is very little fluorescence change when the solution ionic strength is increased from 0 to 1 M (Figure 2.6B). This compares favourably to CPEs and other CP NPs with a charged surface that show obvious fluorescence quenching in solutions with relatively high ionic 52 Chapter 2 strength.[157,158] In addition, the CP-HPG-1 possesses high colloidal stability in water and no obvious precipitate is observed even after being stored at room temperature for three months. A 1.3 B pH 1.2 1.2 NaCl 1.1 1.1 1.0 I/I0 I/I0 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 4 5 6 7 8 9 10 11 0 10 20 30 40 50 Concentration (mM) pH 1.2 C D BSA 1.1 CP-HPG-1 Alexa fluo 488 Fluorescein 1.25  - Globulin 1.00 I/I0 I/I0 1.0 0.9 0.50 0.8 0.7 0.75 0.25 0 20 40 60 80 100 0.00 120 0 Concentration (mg/L) 2 4 6 8 10 Time (min) Figure 2.6 pH (A), NaCl concentration (B), BSA and γ-globulin concentration (C) dependent fluorescence intensity ratio (I/I0), where I0 is the emission intensity of CP-HPG-1 in aqueous solution at pH 6.8 without addition of NaCl, BSA and γ-globulin, and I is the emission intensity of CP-HPG-1 in aqueous solution at different (A) pH or (B) different concentrations of NaCl, (C) BSA and γ-globulin. (D) Radiation time dependent fluorescence intensity ratio (I/I0) of CP-HPG, Alexafluo 488, and fluorescein, the radiation was provided by confocal laser, where I0 and I are the emission intensity of fluorescent probe without and with radiation for different time, respectively. As proteins largely exist in biological media, BSA and γ-globulin (a kind of immunoglobulin in blood) were chosen as the representative examples to study their interactions with CP-HPG-1. As shown in Figure 2.6C, the CPHPG-1 fluorescence remains unchanged when the protein concentrations are increased from 0 to 300 μg/mL. DLS measurements further confirm that there is no significant difference in size and size distribution of CP-HPG-1 in 53 Chapter 2 aqueous solution before and after the addition of BSA and γ-globulin, suggesting good antiprotein adsorption property of CP-HPG-1. In addition, the photostability of CP-HPG-1 was studied by continuousimaging using CLSM for 10 min. For comparison, commercially available fluorescent dyes such as Alexafluo 488 and fluorescein were also investigated under the same condition. As shown in Figure 2.6D, the emission intensities decrease by 11%, 28%, and 90% at 10 min for CP-HPG-1, Alexafluo 488, and fluorescein, respectively. These studies indicate that the CP-HPG-1 NPs possess good physical and photo stabilities, which make them good candidates for biological applications. 2.3.4 Cellular Imaging of CP-HPG1 The cytotoxicity of CP-HPG-1 was evaluated for MCF-7 breast cancer cells using MTT cell-viability assay. Figure 2.7A summarizes the in vitro MCF-7 cell viability after being cultured for 24 or 48 h. It is noteworthy that the concentrations of the CP-HPG-1 solution are much higher than that used for cell imaging ([RU] = 1 μM). The cell viabilities are close to 100% at [RU] = 2 and 5 μM and close to 90% at [RU] = 10 μM within the tested period, suggesting the low cytotoxicity of CP-HPG-1. This result is consistent with the previous studies of CP NPs prepared by precipitation technique.[59] The good biocompatibility of CP-HPG-1 should also benefit from the highly biocompatible HPG. To demonstrate the CP-HPG NPs in cell imaging, MCF7 cells were incubated with CP-HPG-1 solution at [RU] = 1 54 Chapter 2 Figure 2.7 (A) Cell viability of MCF-7 cells after incubation with CP−HPG-1 at different concentrations for 24 and 48 h, respectively. Confocal fluorescence image of MCF-7 cells upon incubation (B) with and (D) without CP−HPG-1 ([RU] = 1 μM) for 2 h. (C) 3-D confocal image of cell line MCF-7 incubated with CP-HPG-1 for 2 h. μM. After 2 h incubation, the cells were fixed for fluorescence imaging by CLSM. The excitation wavelength was fixed at 488 nm, and the fluorescent signals were collected above 540 nm. The CLSM image of CP-HPG-1 stained MCF-7 cells is displayed in Figure 2.7B. Strong fluorescence from the cellular cytoplasm is observed for MCF-7 cells, implicating that CP-HPG-1 is efficiently internalized by MCF-7 cells and accumulated in the cytoplasm, which is further confirmed by the 3D CLSM image (Figure 2.7C). In contrast, no fluorescence can be observed for the cells without incubation of CP-HPG-1 55 Chapter 2 (Figure 2.7D). These data suggest that CP-HPG-1 can be used as an effective fluorescent stain for cell imaging with good fluorescence contrast. 2.4 Conclusion In this chapter, we have successfully demonstrated a simple and effective approach to fabricate robust CP-HPG NPs via copper-free click reaction in oil-in-water miniemulsion. The CP-HPG NPs with tunable size are born with multiple surface neutral hydroxyl groups, which have shown good water dispersibility, bright fluorescence, good optical stability, and low cytotoxicity. As the first example to combine dendritic polymer and CP by covalent cross-linking, this study not only exploits a new type of CP NP with desired properties, but also provides new opportunities and fundamental guidelines to design advanced CP NPs for biological applications. 56 Chapter 3 CHAPTER 3 A GENERAL APPROACH TO PREPARE CONJUGATED POLYMER DOT EMBEDDED SILICA NANOPARTICLES WITH A SIO2@CP@SIO2 STRUCTURE FOR TARGETED HER2-POSITIVE CELLULAR IMAGING 3.1 Introduction Fluorescence microscopy technique has attracted great interest in biological research and cancer diagnosis due to its noninvasive, real time and high resolution characteristics.[159-163] It has been recognized that fluorescent probes with high brightness, good photostability, benign biocompatibility and easy surface functionalization capabilities are beneficial for achieving optimal physiological performance in biological studies. The traditional small fluorophores and fluorescent proteins show small Stokes shift and poor photostability,[164,165] while semiconductor QDs have the notorious cytotoxicity,[166-168] all hindering their practical biological applications. Recently, CPs have emerged as effective fluorescent materials due to their distinguished advantages.[33,36,169] CP based materials show large absorption cross section, strong fluorescence, high photostability and favourable biocompatibility, which meet the requirements of fluorescent probes for biological applications.[25,26,35,51,170,171] As the inherent hydrophobic backbones hinder CPs to soluble in aqueous environment, strategies to endow CPs with good water solubility or dispersibility are highly desirable.[33,35,36] So far, three main strategies have been developed to render CPs with 57 Chapter 3 water solubility or dispersibility. One strategy is to graft ionic side chains to CP backbones to yield CPEs, which can further self-assemble in aqueous media.[36,98] Unfortunately, the generated CPEs often create problems of nonspecific interaction with biosubstrates because of their charged side chains. Another strategy is to prepare cross-linked CP NPs with high stability by click chemistry,[99,100,172] which involves multiple-step monomer modification. The third strategy is to prepare CP NPs through precipitation or emulsion method,[59,65,68,69,101,138,173] which requires mixing of organic phase with aqueous phase under sonication or magnetic stirring. The CP NPs are formed based on the weak hydrophobic interaction between CPs and polymer matrix, which could result in low stability in aqueous media for long term studies. In addition, CP NPs prepared via nanoprecipitation or emulsion method could easily lose their physical stability and may not be redispersed in water after drying.[99,172] As a result, development of a facile and effective approach for fabricating CP NPs that simultaneously possess good water dispersibility, excellent structural and optical stability and surface functional groups remains challenging. The pioneer work by Stöber has stimulated the development of silica NPs for a variety of applications in biomedicine and photonics.[174-180] Organic dye doped silica NPs have shown great promise in various biological applications due to their good photostability, excellent brightness and benign biocompatibility.[178,180] In view of the notable advantages of silica matrix, it holds great potential to improve CP NP performance in stability and biocompatibility through encapsulate CPs into silica matrix. As the highly hydrophobic CP chains are immiscible with the hydrophilic silica matrix, the 58 Chapter 3 encapsulation of CPs in silica NPs generally requires the assistance of block copolymers[181] or tedious modification of CP chain and silanes.[178,180] Although mesoporous silica matrices have also been used to directly encapsulate untreated CPs, the porous structure is unable to protect CPs from the invasion of solvents or oxygen molecules, leading to poor stability for long term biological applications.[171,182-184] As a consequence, a more general and straightforward approach to prepare CP embedded silica NPs is still in demand. In this chapter, we report a general, simple and effective strategy to synthesize CP embedded silica NPs with a unique SiO2@CP@SiO2 structure (SiO2@CP@SiO2 NPs) by integrating a precipitation method and a modified Stöber approach. This approach allows the encapsulation of CPs inside the silica NPs without sophisticated CP modification or tedious silane monomer conjugation. The obtained SiO2@CP@SiO2 NPs not only show high stability but also have surface functionalized amine groups that enable bioconjugation for further biological applications. The formation mechanism of SiO2@CP@SiO2 NPs is investigated by FE-TEM and their detailed optical properties are also studied. The application of SiO2@CP@SiO2 NPs as an effective fluorescent probe is demonstrated by functionalization of NP surfaces with a HER2 specific peptide (GGHAHFG) for targeted cellular imaging using HER2-overexperssed SKBR-3 breast cancer cells as an example. In conjugation of its simple synthetic procedure, high fluorescence QY, excellent physical stability and good photostability, the developed SiO2@CP@SiO2 NPs can serve as an efficient fluorescent nanoprobe for biological applications. 59 Chapter 3 3.2 Experimental Section 3.2.1 Materials Poly(9,9′-dihexylfluorene-alt-1,4-phenylene) didehexylfluorenyl divinylene-alt-1,4-phenylene) dihexylfluorene-alt-2,1,3-benzothiadiazole) (PFBT) (PFP), poly(9,9′- (PFVP), poly(9,9- and poly[(9,9- dihexylfluorenyl-co-2,1,3,-benzothiadiazole)-co-4,7-di(thiophen-2-yl)-2,1,3benzothadiazole] (PFBTDBT) were synthesized according to the previous reports.[185-188] Tetraethsilane (TEOS), 3-aminopropyl triethoxysilane (APTES), 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDAC), N-hydroxysulfosuccinimide (Sulfo-NHS), trifluoroacetic acid (TFA), N,N-diisopropylethylamine (DIEA), acetic anhydride (Ac2O), triisopropylsilane (TIS), 4',6-diamidino-2-phenylindole (DAPI), quinine sulphate, fluorescein, 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran, MTT, penicillin-streptomycin solution and DMSO were purchased from Sigma-Aldrich. DMEM, fetal bovine serum (FBS) and trypsin-EDTA solution were purchased from Gibco (Lige Technologies, Ag, Switzerland). Hexane, DCM, dimethylformamide (DMF), ethanol, methanol, THF, concentrated sulphuric acid and ammonia solution were obtained from Merck (Germany). Rink-amide resin, O-benzotriazole-N,N,N’,N’-tetramethyluronium-hexafluoro-phosphate (HBTU), N-hydroxybenzotriazole (HOBt) and Fmoc-protective amino acids were purchased from GL Biochem Ltd. Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, Breford, USA). SKBR-3 breast cancer cells and NIH/3T3 fibroblast cells were provided by American Type Culture Collection. 60 Chapter 3 3.2.2 Characterization The UV-vis absorption spectra of CP solutions and their corresponding NPs were measured using a Shimadzu UV-1700 spectrophotometer. Their fluorescence spectra were measured using a fluorometer (LS-55, Perkin Elmer, USA). The average particle sizes and size distributions were determined by laser light scattering with a particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90° at room temperature. The surface morphologies of SiO2@CP@SiO2-NPs were studied with FE-TEM (JEM-2010F, JEOL, Japan). Zeta potential was measured using a zeta potential analyzer (ZetaPlus, Brookhaven Instruments Co. USA) at room temperature. 3.2.3 Preparation of SiO2@CP@SiO2 NPs The SiO2@CP@SiO2 NPs were prepared by the combination of a precipitation method and a modified Stöber approach. Typically, a THF solution (0.3 mL) containing CP (0.15 mg) was poured into a mixture of ethanol and water (v/v = 9:1, 10 mL) under sonication using a microtip probe sonicator (XL2000, Misonix Incorporated, NY) for 2 min. The solution was then stirred at room temperature for 2 h to evaporate THF. Subsequently, 100 μL of TEOS was added into the mixture followed by 300 μL of ~30% ammonium solution. The solution was further stirred for 12 h at room temperature. To investigate the evolution mechanism of the formed NPs, aliquots were taken from the solution at different time points to conduct FETEM measurements. Followed, 40 μL of APTES was added into the above reaction mixture and stirred for another 12 h. The formed SiO2@CP@SiO2NPs were then centrifuged for 10 min at 7500 rpm and washed with ethanol five times to remove ammonia and impurities. In between the washing steps, 61 Chapter 3 SiO2@CP@SiO2 NPs were redispersed by sonication. 3.2.4 Synthesis, Purification, and Characterization of Peptide GGHAHFG was synthesized using a standard Fmoc strategy with rink amide resin as the solid support.[189] Standard HOBT/HBTU/DIEA coupling method was used throughout the whole process. The resin (100 mg, loading ∼0.5 mmol·g-1) was swelled in HPLC-grade DMF for 1 h at room temperature. Subsequently, Fmoc group was deprotected in piperidine/DMF (v/v = 1:4) for 2 h at room temperature. Following piperidine removal, the resin was washed extensively with DMF and DCM and dried thoroughly under high vacuum. Next, glycine was dissolved in dry DMF (1.5 mL) together with HBTU (4 equiv), HOBt (4 equiv), and DIEA (8 equiv). The dry resin was then added and the resulting mixture was shaken at room temperature. After overnight reaction, the resin was filtered and washed thoroughly with DMF (3×), DCM (3×) and DMF (3×) until the filtrate became colorless. After drying thoroughly under high vacuum, the resin was deprotected again with 20% piperidine in DMF for the next coupling cycle. The above cycle was repeated until the last amino acid has been coupled. Finally, the resin was capped with a solution of Ac2O (10 eq) and DIEA (20 eq) in DCM (200 mL), and the mixture was allowed to react for 2 h at room temperature. After the whole coupling process, the resin was washed thoroughly with DMF and dried under high vacuum for 2 h at room temperature. The peptide was then cleaved in a mixture of 95% TFA, 2.5% triisopropylsilane (TIS) and 2.5% H2O for 4 h at room temperature. Following prolonged concentration in vacuum until >80% of cleavage cocktail was removed. Cold ether (chilled to 20 °C) was added to the liquid residue to precipitate the peptide. The ether layer was then decanted and the precipitates 62 Chapter 3 were dried thoroughly in vacuum. The resulting peptide was further purified by prep-HPLC and characterized by LC-MS. IT-TOF m/z calcd: 681.70, found 681.32. The HPLC condition is: 5-20% B for 10 min, then 20-40% B for 2 min, 5% B for 2 min (Solvent A: 100% H2O with 0.1% TFA; Solvent B: 100% CH3CN with 0.1% TFA). 3.2.5 Preparation of SiO2@PFBT@SiO2-Pep NPs The amine groups functionalized SiO2@PFBT@SiO2 NPs were firstly modified with maleic anhydride to render carboxyl acid groups functionalized SiO2 NPs. Typically, maleic anhydride (10 mg) in 10 mL of ethanol and 0.5 mL of triethylamine were added into 10 mL of SiO2@PFBT@SiO2 NP suspensions and stirred overnight. Then 20 mL of H2O was added into the mixture, followed with washing with ethanol and H2O three times, respectively. Subsequently, 3.2 mg of EDAC and 3.6 Sulfo-NHS were added into NP suspension to activate carboxyl groups for 4 h at room temperature. The activated SiO2 NPs were centrifuged to remove excess ligands and redispersed into 5 mL H2O. 5 mL of 0.2 M borate buffer was then added to the suspension, which was followed with 0.1 mL of 0.1 M peptide (GGHAHFG). The mixture was stirred at room temperature overnight and then washed with water as well as DMSO to eliminate the excess peptide. 3.2.6 Preparation of PFBT NPs PFBT NPs were prepared using a precipitation method.[35] Briefly, a THF solution (1 mL) containing a mixture of PFBT (0. 5 mg) was poured into 10 mL of MilliQ water under sonication. THF was then evaporated by magnetic stirring in fume hood. PFBT NPs were obtained by filtration through 63 Chapter 3 a 0.2 μm filter. The QY of PFBT- NPs was estimated to be ~20% using 4(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran in methanol as a standard. 3.27 Cell Culture SKBR-3 breast cancer cells and NIH/3T3 fibroblast cells were cultured in folate-free DMEM medium containing 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in a humidified environment containing 5% CO2. Before experiment, the cells were precultured until confluence was reached. 3.2.8 Cellular Imaging SKBR-3 breast cancer cells were cultured in chamber (LAB-TEK, Chambered Coverglass System) at 37 °C. After 80% confluence, the medium was removed and the adherent cells were washed three times with 1× phosphate buffer saline (PBS) buffer. SiO2@PFBT@SiO2-pep NP and SiO2@PFBT@SiO2-COOH NP suspensions at 100 μg·mL-1 NPs in FBS free DMEM medium were then added to the chamber. After incubation for 2 h, cells were washed three times with 1× PBS buffer and then fixed by 75% ethanol for 10 min. Subsequently, the cells were incubated with DAPI for 15 min to stain the cell nuclei. The cell monolayer was further washed three times with 1× PBS buffer and imaged by CLSM (Zeiss LSM 410, Jena, Germany) with imaging software (Fluoview FV1000). The confocal images of NIH/3T3 cells treated with SiO2@PFBT@SiO2-pep SiO2@PFBT@SiO2-COOH NPs under the same condition were also studied. 64 Chapter 3 3.2.9 Flow Cytometry Study Three groups of SKBR-3 breast cancer cells were cultured in a 6-well culture plate to achieve the desired confluence. One group was used as the control without treatment. The other two groups were treated with SiO2@PFBT@SiO2-pep NP and SiO2@PFBT@SiO2-COOH NP suspensions at 100 μg·mL-1 NPs in FBS free DMEM medium, respectively. After incubation for 2 h at 37 °C, the control and sample groups were washed with 1× PBS buffer three times and then treated with 1× PBS trypsin, followed by washing with DMEM medium through centrifugation. MCF-7 breast cancer cells with a density of ~1.5 × 105 cells·mL-1 were dispersed in FBS free DMEM medium (2 mL) for both control and sample groups. Flow cytometry measurements were conducted using Cyan-LX (DakoCytomation). The mean fluorescence was determined by counting 10,000 events (λex = 488 nm, 680/40 nm bandpass filter). 3.2.10 Cytotoxicity of SiO2@PFBT@SiO2-pep NPs MTT assays were performed to assess the metabolic activity of NIH/3T3 fibroblast cells. NIH/3T3 fibroblast cells were seeded in 96-well plates (Costar, IL, USA) with a density of 4 × 104 cells·mL-1. After 24 h incubation, the medium was replaced by SiO2@PFBT@SiO2-pep NP suspensions with various NP concentrations (100, 500 and 1000 μg·mL-1), and the cells were further incubated for 24 and 48 h, respectively. After the designated time intervals, the wells were washed three times with 1× PBS buffer, and freshly prepared MTT (0.5 mg·mL-1) solution (100 µL) in culture medium was added to each well. The MTT medium solution was carefully removed after 3 h incubation in the incubator. DMSO (100 µL) was then 65 Chapter 3 added into each well and the plate was gently shaken for 10 min at room temperature to dissolve all precipitates. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan) after subtracting the absorbance of the corresponding control cells incubated with SiO2@PFBT@SiO2-pep NPs at the same concentration but without the addition of MTT to eliminate the absorbance interference from PFBT. Cell viability was expressed by the ratio of absolute absorbance of the cells incubated with NP suspensions to that of the cells incubated with culture medium only. 3.2.11 Photostability of SiO2@PFBT@SiO2-pep NPs To compare the photostability of SiO2@PFBT@SiO2 NPs with that of fluorescein and PFBT NPs, the photostablility of SiO2@PFBT@SiO2-pep, PFBT NPs and fluorescein was investigated. SKBR-3 breast cancer cells incubated with SiO2@PFBT@SiO2-pep NPs, PFBT NPs and fluorescein were prepared according to the procedures described above. The CLSM images of the sample were recorded at 1 min interval under continuous laser scanning at excitation wavelength of 488 nm with a laser power of 4.75 mW. The fluorescence intensity of each image was analyzed by Image Pro Plus software. Their photostability was expressed by the ratio of the fluorescence intensity after excitation for a designated time interval to its initial value as a function of the exposure time. 66 Chapter 3 3.3 Results and Discussion 3.3.1 Preparation and Characterization of SiO2@CP@SiO2 NPs Scheme 3.1 Chemical structures of four CPs used in this study. To synthesize SiO2@CP@SiO2 NPs, the THF solution of CP was first poured into an ethanol/water mixture (v/v = 9:1) under sonication. Upon magnetic stirring, THF gradually evaporated and CPs formed small CP dots. The resulting solution was subsequently added with silica precursor, TEOS, as well as ammonia catalyst. A sol-gel silica reaction took place in the presence of the CP dots. After 12h, APTES was then added to modify silica NPs with amine functional groups for further bioconjugation. To the best of our knowledge, this is the first report on the synthesis of SiO2@CP@SiO2 NPs at room temperature without specific CP or silane modification. To demonstrate the versatility of this method for preparing CP encapsulated silica NPs, four different polymers with various emission colours were chosen in this study. These CPs are PFP, PFVP, PFBT and PFBTDBT, respectively, which were synthesized according to our previous reports [185187,190] . The fluorescence emission wavelengths of the CPs are fine-tuned from 400 nm to 850 nm and their chemical structures are shown in Scheme 3.1. 67 Chapter 3 Norm. Absorbance 300 400 500 600 700 800 B Norm. PL Intensity PFP PFVP PFBT PFBTDBT A 900 Wavelength (nm) Figure 3.1 (A) Normalized UV-vis absorption (dashed line) and PL spectra (solid line) of SiO2@CP@SiO2 NPs in water. (B) Photographs of SiO2@CP@SiO2 NP suspensioin in water under a hand held UV lamp (excited at 365 nm). Table 3.1 Characterization of SiO2@CP@SiO2 NPs. Loaded CP Particle size (nm) Zeta potential (mV) Encapsulation efficiency (%) QY (%)a) PFP 65 +26 100 54 PFVP 62 +23 100 35 PFBT PFBTDBT 68 72 +25 +25 100 100 27 20 [a] The QYs of SiO2@PFP@SiO2, SiO2@PFVP@SiO2, SiO2@PFBT@SiO2 and SiO2@PFBTDBT@SiO2 NPs were measured with quinine sulfate (0.1 M H2SO4; QY = 0.54), fluorescein (0.1 M NaOH; QY = 0.95), 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (methanol; QY = 0.43) as the standard, respectively. Figure 3.1A shows the UV-vis absorption and fluorescence spectra of the prepared SiO2@CP@SiO2 NP aqueous suspensions. The increased baselines of the absorption spectra at the short wavelength are due to the strong scattering of silica matrix.[178] The emission maxima of PFP-, PFVP-, PFBT- and PFBTDBT encapsulated silica NP suspensions are 421 nm, 482 nm, 554 nm and 699 nm, respectively. It is noted that the emission maxima of PFP-, PFVP-, PFBT- and PFBTDBT-SiO2 NP suspensions are red-shifted by 8 nm, 12 nm, 11nm and 41 nm, respectively, as compared to those of pure CPs in THF. The red shifts of the emission maxima of these NPs are mainly 68 Chapter 3 derived from the aggregation of CP chains in silica NP formulation, which is consistent with the previous reports of CP NPs prepared through nanoprecipitation and emulsion methods.[59,68,181] In addition, the aqueous suspensions of SiO2@CP@SiO2 NPs show bright and vivid colours under the excitation of a hand held UV lamp as shown in Figure 1b. These results illustrate that the photophysical properties of SiO2@CP@SiO2 NPs are tunable by choosing different CPs. Table 3.1 shows that the SiO2@CP@SiO2 NPs have an average diameter from ~60 to ~70 nm determined by laser light scattering. It has been reported that NPs with a size from 50 nm to 200 nm show good cellular uptake efficiency.[191,192] The zeta potential of SiO2@CP@SiO2 NPs was measured to be around +25 mV in neutral aqueous environment, which is mainly derived from the grafted amine groups at NP surfaces. The positively charged surfaces of SiO2@CP@SiO2 NPs guarantee the electrostatic repulsion among particles, leading to a relatively good colloidal stability in water. The encapsulation efficiencies of the four CPs in silica matrix are calculated from the absorption difference of the CP solution in ethanol/water mixture prior to encapsulation and that of the supernatant after NP centrifugation, which are almost 100% under our experimental conditions. The QY of each SiO2@CP@SiO2 NP was measured using commercial fluorophores as the standard. The relatively high QYs (> 20%) for these four SiO2@CP@SiO2 NPs are comparable with the previously reported water dispersible CP NPs.[35,68,138,181] The fluorescence stability of the obtained SiO2@CP@SiO2 NPs was also investigated by monitoring the fluorescence intensity change of SiO2@PFBT@SiO2 NPs upon incubation in PBS at 37 °C as an example. The fluorescence intensity of 69 Chapter 3 SiO2@PFBT@SiO2 NP suspension remains ~92% of its initial value after 5 day incubation in 1 × PBS. The excellent physical stability of CP NPs is ascribed to the protection of PFBT from the silica matrix.[178-180] Moreover, the dried SiO2@CP@SiO2 NPs can be redispersed easily in ethanol or water due to the robust silica matrix. The high fluorescence QY and outstanding stability of SiO2@CP@SiO2 NPs benefit their biological applications. A 20 min B 40 min C D 3h E 12 h F 60 min +APTES 12h 50 nm Figure 3.2 FE-TEM images of SiO2@PFBT@SiO2 NPs taken at different reaction times after adding TEOS (A-E) as well as APTES (F). All images share the same scale bar as that in F. It is generally observed that silica condensation in the presence of seed particles leads to core/shell morphologies.[193,194] In the case of CPs and silica hybrid system, it is of interest to investigate the evolution of their structures at various reaction times. Taking PFBT as an example, the morphology and structure changes of CP-silica hybrid composites were studied with FE-TEM. In these experiments, PFBT dots were firstly obtained by precipitation method combining ultrasonication and solvent evaporation. Upon addition of TEOS, a sol-gel silica reaction proceeded in the suspension of PFBT dots. During the process of silica NP formation, aliquots were taken from the reaction solution 70 Chapter 3 at the time interval of 20 min, 40 min, 60 min, 3 h and 12 h after TEOS addition. The FE-TEM samples were prepared by directly dropping the reaction solution onto copper grid with subsequent drying in vacuum oven to minimize further TEOS precursor cross-linking. As shown in Figure 3.2, at the initial 20 min, small black PFBT dots are adsorbed on the silica NP surfaces. The sizes of PFBT dots and silica NPs are estimated to be ~3 nm and ~27 nm, respectively, from Figure 3.2A. The 3 nm PFBT dot should only contain a single polymer chain according to the estimation method described in the SI. Each PFBT dot is adsorbed at silica NP surface to form a PFBT dotspunctuated silica NP structure. This interesting pattern may result from the phase separation between the immiscible PFBT and silica matrix components. The sizes of silica NPs increase to ~32 nm at 40 min (Figure 3.2B) and further grow to be ~35 nm (Figure 3.2C) and ~42 nm (Figure 3.2D) at the time intervals of 1h and 3h, respectively. There is little difference in silica NP size between samples prepared at 3h and 12h. During the time interval from 20 min to 12 h, the small black PFBT dots with a size ~3 nm can be clearly observed, further illustrating that hydrophobic PFBT dots cannot be encapsulated into hydrophilic silica matrix. It is worth noting that PFBT dots are not stable in the absence of silica matrix. The FE-TEM image of PFBT dots in ethanol/water mixture without TEOS addition is shown in Figure 3.3. No regular PFBT aggregates are observed in Figure 3.3, indicating small PFBT dots are not stable and aggregate easily even during the sample preparation. However, the silica substrate provides a robust support for PFBT dots, leading to an observable PFBT dots-punctuated silica NP structure. 71 Chapter 3 100 nm Figure 3.3 FE-TEM images of PFBT dots in ethanol/water mixture (v/v = 9:1) upon sonication. B A 50 nm 50 nm Figure 3.4 FE-TEM images of the mixture of SiO2 NPs and CP dots before APTES addition (A) and further reaction for 12 h in the presence of APTES (B). Interestingly, if SiO2 NPs and CP dots were synthesized separately, mixed and followed by 12 h stirring, the PFBT dots-punctuated SiO2 NPs could also be observed (Fig 3.4), which further confirm that CP dots tend to be absorbed at the SiO2 NP surfaces rather than encapsulated inside of SiO2 cores. After reaction for 12 h, APTES was further added into the reaction mixture, which was followed by another 12 h magnetic stirring. The FE-TEM image of the PFBT-silica mixture after 12 h reaction with APTES is shown in Figure 3.4B. An interesting surface morphology evolution from PFBT dotspunctuated silica NP patterns (Figure 3.2A-3.2E) to smooth silica NP surfaces 72 Chapter 3 (Figure 3.2F) is observed. The disappearance of small black PFBT dots after APTES addition illustrates that PFBT dots are more likely encapsulated into the silica matrix upon APTES hydrolysis. It is noted that the size of SiO2@PFBT@SiO2 NPs can be easily controlled by changing the amount of TEOS precursor in the reaction. Doubling the TEOS concentration at the feed will lead to the size of SiO2@PFBT@SiO2 NPs increase from ~40 to ~65 nm. A B C D 50 nm Figure 3.5 FE-TEM images of SiO2@PFBT@SiO2 NPs with the addition of APTES (upper row; A, B) or TEOS (bottom row; C, D) for further 12 h reaction followed by centrifuging once (A, C) and five times (B, D). The insets show the respective photographs of SiO2@PFBT@SiO2 NPs after centrifugation. All images share the same scale bar as that in D. The successful embedment of PFBT dots into silica layers was further confirmed by checking the colours of the precipitations and supernatants after centrifugation. The yellow precipitation and a clear supernatant are obtained by centrifugation of the reaction solution upon addition of APTES after 12 h reaction, which is shown in the inset of Figure 3.5A. After washing for five 73 Chapter 3 times, the yellow colour is still present in the precipitate (Figure 3.5B inset), indicating that PFBT is stably embedded in silica NPs upon APTES crosslinking. However, addition of TEOS into the PFBT dots-punctuated silica NP suspension followed by 12 h reaction cannot encapsulate PFBT dots in silica matrix and the supernatant remains yellow (Figure 3.5C inset). In fact, black PFBT dots are separated from silica NPs upon sonication/centrifugation washing as shown in Figure 3c. Further washing will only lead to clean silica NPs as white precipitates (the inset of Figure 3.5D). These data not only illustrate that CP dots cannot be embedded by further addition of TEOS reagents, but also indicate that CP dots adsorbed at silica surfaces are not stable and can be easily removed by sonication/centrifugation treatment. The other three CPs including PFP, PFVP and PFBTDBT can also be embedded into silica NPs with the assistance of APTES reagents (data not shown). Based on FE-TEM images from Figure 3.2 and Figure 3.4, a possible mechanism of the formation of SiO2@CP@SiO2 NPs has been proposed. Scheme 3.2 illustrates our proposed mechanism for the formation of SiO2@CP@SiO2 NPs. CP dots are firstly obtained in ethanol/water mixture (v/v = 9:1) through unltrasonication and THF evaporation (Scheme 3.2A). Due to the low solubility of hydrophobic backbones in polar ethanol/water 74 Chapter 3 A B C D1 D2 Scheme 3.2 Schematic illustration of the synthesis of SiO2@PFBT@SiO2 NPs with surface functionalized targeting peptide. (A) Sonicatioin of a THF solution of CPs in ethanol/water mixture (v/v = 9:1) affords single CP chain dots. (B) Addition of TEOS and ammonia into the CP dots dispersed solution leads to the formation of CP dots-punctuated silica NP pattern after reaction. (C) Further addition of APTES into the mixture results in CP dots embedded silica NPs with surface functionalized amine groups. (D1) Surface carboxylation with maleic anhydride in the presence of Et3N. (D2) Peptide conjugation via EDC/NHS reaction. mixture, CP dots are obtained upon THF evaporation. After adding TEOS, a sol-gel procedure is performed which may take the present CP dots as nucleation centers.[194] However, the hydrophilic silica component cannot grow homogeneously around hydrophobic CP dots due to their different hydrophilicities. On the other hand, the hydrophobic CP dots are not stable in the polar ethanol/water mixtures. To minimize the surface energy, CP dots tend to be adsorbed at the surfaces of the formed silica NPs rather than being encapsulated inside SiO2 core (Scheme 3.2B). As the aminopropyl groups from APTES can entangle with the hydrophobic CP dots, a self-assembled structure with CP dots as the core and triethoxy silane groups pointing 75 Chapter 3 outwards is obtained upon APTES addition. The silane groups can further react with other monomers to form a cross-linked network, leading to the encapsulation of CP dots in silica matrix (Scheme 3.2C). Moreover, APTES monomers can render the surface of SiO2@CP@SiO2 NPs with functional amine groups, which facilitate further conjugation with peptides for targeted cellular imaging (Scheme 3.2D). 3.3.2 Surface Functionalization with HER2 Targeting Peptide Scheme 3.3 The chemical structure of peptide, GGHAHFG. To demonstrate the potential of SiO2@CP@SiO2 NPs in biological application, SiO2@PFBT@SiO2 NPs were chosen as an example for targeted cellular imaging. As the positively charged amine groups tend to stick to the negatively charged cell membranes,[162,163,192] we first modified NP surfaces with carboxyl groups using maleic anhydride to minimize the non-specific interaction between NPs and cells. The successful grafting of carboxyl groups at silica NP surfaces was confirmed by the zeta potential measurement. The zeta potential changed from +25 mV for SiO2@PFBT@SiO2 NPs with surface functionalized amine groups to -34 mV for SiO2@PFBT@SiO2 NPs with surface functionalized carboxyl acid groups (SiO2@PFBT@SiO2-COOH NPs). The peptide, GGHAHFG, which can selectively bind to HER2 overexpressed SKBR-3 breast cancer cells was chosen to endow NPs with targeting ability.[195] The amine-bearing GGHAHFG peptide was synthesized by 76 Chapter 3 standard solid-phase fluorenylmethoxy carbonyl peptide chemistry.[195] The detailed synthetic procedure is illustrated in the experiment section and its structure is show in Scheme 3.3. The peptide was further purified by HPLC and confirmed by LC-MS characterization. Subsequent coupling between SiO2@PFBT@SiO2-COOH NPs and the peptide via the carbodiimide chemistry yielded fluorescent NPs (SiO2@PFBT@SiO2-Pep NPs) with targeting ability to HER2 positive cancer cell lines. The zeta potential of SiO2@PFBT@SiO2-Pep NPs was further changed to -11 mV, demonstrating the successful coupling between carboxyl groups at NP surface and amine groups of the peptides. 3.3.3 Targeted Cellular Imaging To examine targeting ability of SiO2@PFBT@SiO2-Pep NPs, SiO2@PFBT@SiO2-COOH NPs were used as a control in cellular imaging by CLSM. In these experiments, SKBR-3 breast cancer cells were incubated with both NP suspensions in culture medium for 2 h at 100 μg·mL-1 NPs. The CLSM images of SKBR-3 breast cancer cells after incubation with both NPs are shown in Figures 3.6A and 3.6B, respectively. The higher fluorescence intensity in Figure 3.6A as compared to that in 3.6B clearly indicates that the peptide promotes cellular uptake due to the over-expressed HER2 on SKBR-3 breast cancer cell membranes. The low fluorescence intensity from Figure 3.6B also illustrates that the carboxyl groups grafted at NP surfaces reduce non-specific interaction between NPs and breast cancer cells.[196-198] It is noted that there is no autofluorescence from the control cells without incubation of NPs under the same experimental conditions as shown in Figure 3.7A. The 3D image of SKBR-3 cells incubated with SiO2@PFBT@SiO2-Pep indicates that 77 Chapter 3 NPs are mainly located in cell cytoplasm as shown in Figure 3.7B. As a control, NIH/3T3 fibroblast cells with low HER2 receptors were also incubated with both NP suspensions. The fluorescence intensity from NIH/3T3 cells upon treatment with SiO2@PFBT@SiO2-Pep NPs (Figure 3.6C) is similar to that from cells upon treatment with SiO2@PFBT@SiO2-COOH NPs (Figure 3.6D). In addition, the fluorescence intensities from Figures 3.6C and 3.6D are lower than that from Figure 3.6A. These data indicate the receptor mediated uptake of SiO2@PFBT@SiO2-Pep NPs by SKBR-3 breast cancer cells.[59,161,199] A B 40 μm C D Figure 3.6 Confocal fluorescence images of SKBR-3 breast cancer cells after 2 h incubation with (A) SiO2@PFBT@SiO2-pep and (B) SiO2@PFBT@SiO2COOH NP suspensions at 100 μg·mL-1 NPs at 37 °C. Confocal fluorescence 78 Chapter 3 images of NIH/3T3 fibroblast cells after 2 h incubation with (C) SiO2@PFBT@SiO2-pep and (D) SiO2@PFBT@SiO2-COOH NP suspensions at 100 μg mL-1 NPs at 37 °C. All images have the same scale bar as that in A. B A 40 μm 40 μm Figure 3.7 (A) CLSM fluorescence image of SKBR-3 breast cancer cells without incubation with SiO2@PFBT@SiO2 NPs. (B) 3D CLSM fluorescence image of SKBR-3 breast cancer cells incubated with SiO2@PFBT@SiO2-pep NPs. 800 Control SiO2@PFBT@SiO2-pep SiO2@PFBT@SiO2-COOH Counts 600 400 200 0 0 10 1 10 2 10 3 10 4 Fluorescence Intensity (a.u.) Figure 3.8 Flow cytometry histograms of pure SKBR-3 breast cancer cells without NP incubation (black) and SKBR-3 breast cancer cells after 2 h incubation with SiO2@PFBT@SiO2-Pep NP (red) and SiO2@PFBT@SiO2COOH NP (blue) suspensions at 100 μg·mL-1 NPs. To obtain quantitative analysis of the targeting effect of the peptides on NP surfaces, flow cytometry was used to evaluate the intracellular fluorescence for SKBR-3 breast cancer cells upon incubation with both NPs. The mean fluorescence intensity of SKBR-3 cells incubated with 79 Chapter 3 SiO2@PFBT@SiO2-Pep NPs is ~2-fold higher as compared to that upon incubation with SiO2@PFBT@SiO2-COOH NPs as shown in Figure 3.8. These data further prove that peptide functionalized NPs favour their cellular uptake by SKBR-3 breast cancer cells due to receptor mediated endocytosis. 3.3.4 Cytotoxicity and Photostability 24 h 48 h A 100 B SiO2@PFBT@SiO2-pep NPs 120 PFBT NPs fluorescein I/I0 (%) Cell Viability (%) 100 80 60 40 60 40 20 0 80 20 1000 500 0 100 0 -1 SiO2 Concentration (gmL ) 2 4 6 8 10 Time (min) Figure 3.9 (A) Metabolic viability of SKBR-3 breast cancer cells after incubation with SiO2@PFBT@SiO2-pep at various NP concentrations for 24 h (gray) and 48 h (shadow). (B) Photostability of SiO2@PFBT@SiO2-pep NPs, PFBT NPs and fluorescein in SKBR-3 breast cancer cells upon continuous laser excitation at 488 nm for 10 min. I0 is the initial fluorescence intensity and I is the fluorescence intensity of the sample at different time points after illumination. The cytotoxicity of the SiO2@PFBT@SiO2-Pep NPs was evaluated by metabolic viability of NIH/3T3 fibroblast cells after incubation with NP suspensions at 100, 500 and 1000 g·mL-1 NPs for 24 h and 48 h, respectively. As shown in Figure 3.9A, the cell viabilities remain ~90% upon incubation with SiO2@PFBT@SiO2-pep NP suspension for 48 h at 1000 µg·mL-1 NP, a concentration that is 10-fold higher than that used for imaging. These results indicate the low cytotoxicity of the probe, which is ideal for targeted cellular imaging. The photostability of SiO2@CP@SiO2-Pep NPs in cellular environment was also investigated using SiO2@PFBT@SiO2-Pep NPs as an example under continuous laser scanning upon excitation at 488 nm. And the 80 Chapter 3 results were compared with those of fluorescein and PFBT NPs prepared with a typical precipitation method. The photostability results for SiO2@PFBT@SiO2-pep NPs, PFBT NPs and fluorescein are shown in Figure 3.9B. After 10 min continuous laser illumination, the fluorescence intensity from SKBR-3 breast cells incubated with SiO2@PFBT@SiO2-Pep NPs decreases ~9%, which is obviously better than that for PFBT NPs. Under the same condition, the fluorescence intensity of fluorescein is almost completely quenched by laser illumination. The improved photostability for PFBT-SiO2Pep NPs as compared to PFBT NPs is expected to result from the robust silica matrix protection, which is beneficial for long term bioimaging studies. 3.4 Conclusion In this chapter, we developed a simple yet effective strategy to prepare CP embedded NPs with a unique SiO2@PFBT@SiO2 structure via an integration of the precipitation method and a modified Stöber approach. Through FE-TEM investigation of the sample aliquots at various reaction times, the morphology change from CP dots-punctuated silica NPs to CP embedded silica NPs with smooth surfaces was observed. The successful encapsulation of CPs into silica matrix is resulting from the entanglement between hydrophobic CP chains and APTES reagents. The as-prepared NPs exhibit high QY, excellent photostability, low cytotoxicity and surface available functional groups, benefiting their conjugation with targeting ligands. By conjugation with a specific targeting peptide, the obtained NPs also allow targeted imaging of SKBR-3 breast cancer cells in a high contrast manner. Based on the developed strategy for simple synthesis of SiO2@CP@SiO2 NPs, further encapsulation of metal NPs into silica core can 81 Chapter 3 easily lead to nanocomposites with metal enhanced fluorescence.[200,201] Furthermore, the incorporation of magnetic NPs could afford multimodal nanocomposites with imaging and therapeutic functions,[69,202] which may further broaden CP based material in theranostics applications. 82 Chapter 4 CHAPTER 4 MICELLE/SILICA CO-PROTECTED CONJUGATED POLYMER NANOPARTICLES FOR TWO-PHOTON EXCITED BRAIN VASCULAR IMAGING 4.1 Introduction Two-photon fluorescence imaging (TPFI) has recently attracted great research interest as a non-invasive technique to investigate complex biological issues.[203-206] As TPFI is usually excited by NIR lasers (700-1000), it shows obvious advantages in deeper penetration depth, lower autofluorescence, less scattering and minimal phototoxicity as compared with the conventional onephoton excited technique.[207-210] To design a two-photon absorption (TPA) material, the two-photon action cross section (defined as ηδ, where η is the fluorescence QY and δ is the TPA cross section) is a paramount parameter to be considered. Large ηδ could allow spatial resolution go down to nanometer level or require less excitation power to reach the same resolution, which would further minimize photodamage to fluorescent materials as well as biological targets.[211-215] Although a number of organic TPA materials show high ηδ values in organic solvents, their ηδ values, especially QY, decrease significantly due to severe aggregation caused fluorescence quenching after being transferred to aqueous media upon either ionic side chain modification or NP formulation.[216-226] As a result, development two-photon excited probes with large cross section and high fluorescence QY in aqueous media is highly desirable. 83 Chapter 4 CPs are macromolecules consisting of a delocalized π-electron system along the polymer backbone.[26-28,33] Due to their large absorption coefficient and high fluorescence efficiency, CPs have been widely applied in optoelectronic devices.[25,28] Advances in the progress of material and chemistry science have facilitated fine-tuning of CP structures and their photophysical properties, which have further broadened their applications to biological fields.[27,28,33,34,65,101,128,138,140] As hydrophobic CPs cannot be dispersed in aqueous media, approaches to render CPs with good water dispersibility have been developed. So far, CPs have been mainly endowed with water solubility or dispersibility through ionic side chain modification,[36,227] emulsion[59,139] and precipitation.[68,101,138,140,188,228] These types of CP NPs are generally formed with the hydrophobic CP chains in the core and hydrophilic side chains or matrices in the shell, resulting in water dispersible CP NPs. The reported CP NPs have shown large absorption cross section, moderate to high fluorescence QY and low toxicity upon one-photon excitation.[68,101,138,140,188,228] However, the conventional issue of aggregation caused polymer fluorescence self-quenching is disregarded in these reported CP NPs. When CPs are in the solid or aggregated states, excitons could migrate along both interchains and intrachains to find the low energy traps, leading to non-radiative decay to result in severe fluorescence quenching.[25,26] Although the previously reported CP NPs have shown reasonable brightness, little research has been conducted to optimize their QYs.[183,229-234] Improving fluorescence QYs of CP NPs would not only improve their brightness under one-photon excitation, but also increase two-photon action cross sections (ηδ), 84 Chapter 4 both facilitating their biological applications. These benefits inspire us to further explore strategies that could increase the fluorescence QY of CP NPs. Previous studies have shown that blending CPs with other inert polymers or surfactants could reduce CP self-aggregation, leading to improved fluorescence QYs in the film state.[229,230,234] In addition, stretching CP chains in the solid film of polystyrene could also optimize their photophysical properties.[233] On the other hand, encapsulation of CPs in mesoporous silica matrices could also enhance their fluorescence QYs by blocking oxygen attacking to CP backbones.[183,231] These studies imply that controlling the morphology or formation of CP domains in NP formulations may help to optimize their QYs. Recently, Tian et. al. reported that the fluorescence QY of PFBT NPs could be enhanced to 75% by decreasing CP concentration in NPs via a nanoprecipitation method.[91] However, these CP NPs are built on weak hydrophobic interactions between CP backbones and amphiphilic matrices, which are not redispersable in water after drying.[68,80,99,172,235 ] In this contribution, we report a simple and efficient approach to improve the QY as well as TPA cross section of CP NPs through the micelle/silica co-protection strategy. By isolating CP chains in NP formulation and blocking oxygen and water molecule attacking to CPs, the QYs of the obtained CP NPs have been significantly improved. Taking PFBT as an example, CP NPs were encapsulated into the micelles formed by a triblock polymer, poly(ethylene oxide)-block-poly(propylene oxide)-block- poly(ethylene oxide) (F127), followed by silica cross-linking. The obtained CP NPs show a QY of 75%, among the highest for PFBT based NPs. [34,101,236] In addition, PFBT loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- 85 Chapter 4 N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) NPs with a QY of 40% were prepared according to the conventional precipitation method.[173,228,237] Both static and dynamic photoluminescence comparison between the two types of PFBT NPs were conducted to understand the QY enhancement of NPs prepared with the micelle/silica approach. Moreover, single NP imaging was also conducted to compare their brightness using a commercial QD as reference. In conjugation with their large TPA cross section, excellent stability and benign biocompatibility, the obtained PFBT NPs have been successfully utilized in real-time two-photon brain vascular imaging. 4.2 Experimental Section 4.2.1 Materials PFBT was synthesized according to the previous report.[235] DSPEPEG2000 was purchased from Avanti Polar Lipids, Inc. QD655 was purchased from Invitrogen (Eugene, OR, USA). F127, TEOS, dimethyldimethoxysilane (DMDMS), 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4Hpyran, MTT, penicillin-streptomycin solution, FBS, trypsin-EDTA solution, Evans blue, methanol and THF were purchased from Sigma-Aldrich. Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, Breford, USA). NIH/3T3 fibroblast cells were provided by American Type Culture Collection. 4.2.2 Characterization The UV-vis absorption spectra of CP NPs were measured using a Shimadzu UV-1700 spectrophotometer. The fluorescence spectra were 86 Chapter 4 measured using a fluorometer (LS-55, Perkin Elmer, USA). The surface morphologies of PFBT-F127-SiO2 NPs were studied with FE-TEM (JEM2010F, JEOL, Japan). Average particle size and size distribution of the NPs were determined by DLS with particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90° at room temperature. Fluorescence lifetime measurements were performed on a FluoTime 200 TCSPC fluorescence platform from Picoquant GmbH (Berlin, Germany). A Titanium-sapphire 100 fs laser (Chameleon, Coherent) with second- and thirdharmonic generation was used as the excitation source with an excitation wavelength of 460 nm. In time correlated single photon counting (TCSPC) apparatus, the detector is a microchannel plate (MCP) PMT system HAMR3809U-50 (Hamamatsu) and has a spectral sensitivity from 160 nm to 850 nm and instrument response function of 30 ps. Fluorescence lifetimes were extracted from the decay curves using commercially available fluorescence lifetime analysis software (FluoFit Pro. PicoQuant GmbH). Fluorescence decay curves were fitted using a two or three exponential mode. 4.2.3 TPA Measurements TPA spectra were measured using two-photon induced fluorescence spectroscopy. The samples were excited with laser pulses of 100 fs produced by the mode-locked Ti:Sapphire laser (Spectraphysics Tsunami) with a repetition rate of 82 MHz, and a femtosecond optical parametric amplifier (OPA) was used within the spectral range 800-920 nm. The emission from PFBT aqueous suspensions was collected at a 90o angle by a high numerical aperture lens and directed to a spectrometer’s entrance slit. Rhodamine 6G in 87 Chapter 4 methanol was used as a reference. TPA cross section was calculated from the following equation:[238] 2 1  F21c1n1 F12c2n2 Where δ1 and δ2 are the TPA cross sections, F1 and F2 are the TPIF intensities, η1 and η2 are the fluorescence QYs, c1 and c2 are the concentrations, n1 and n2 are the refractive indexes of solvents (1 corresponds to Rhodamine 6G, 2 is PFBT-F127-SiO2 NPs or Evans blue). The concentration of PFBT-F127-SiO2 NP suspensions is calculated based on PFBT chains. 4.2.4 Wide-Field Microscopy Imaging Fluorescence imaging of individual PFBT-F127-SiO2 NP, PFBTDSPE NPs and QD655 was performed with a Wide-Field Microscope (WFM) based on a Nikon ECLIPSE Ti-U inverted microscope frame. Light from a CW multi-line Ar ion laser (Melles Griot, CA, USA) emitting at 488 nm (0.55 mW, measured above the glass cover slide) was fiber-coupled to a Nikon TIRF attachment and focused on the back aperture of a high NA objective (Nikon TIRF Apo 100x, NA = 1.49, oil immersion) after passing through an excitation filter (z488/10x ). Immersion oil (nD = 1.4790, Cargille, USA) was added between the high NA objective and the cover slip for index matching. The luminescence was collected by the same objective and after passing through the dichroic mirror (Z514RDC) and the emission filter (HQ530 LP ) it was directed onto an iXonEM+897 EMCCD camera (512 x 512 pixels, 150 nm per pixel resolution, Andor Technology, Northern Ireland) connected to the side port of the microscope. The camera was connected to a computer furbished with camera-dedicated software to control the imaging parameters, 88 Chapter 4 and for data acquisition. The samples were prepared by depositing a droplet of nanoparticle solutions on a glass cover slide, waiting for 30s for particles adsorption to the substrate, removing excess of liquid and drying under N2. Fluorescence intensity time-traces were obtained by acquiring 1000 consecutive frames at a rate of 10 frames per second (100 ms exposure time, 100 s time-traces) and extracting the number of counts per particle at each frame. The images were analysed using Andor Solis (ver. 4.14.30001.0, Andor Technology, Northern Ireland) and NIS Elements Ar 4.10.00 (Nikon, Japan) software. Statistical analysis of the data was performed using a custom analysis software (based on LabView, National Instruments) and OriginPro8 software (OriginLab Corporation, USA). 4.2.5 Synthesis of PFBT-F127-SiO2 NPs Micelle/Silica co-protection NPs (PFBT-F127-SiO2 NPs) were synthesized by modifying a previously reported experiment.[239] Firstly, F127 (100 mg) and PFBT (0.5 mg) were dissolved in 1.5 mL THF in a 20 mL glass vial and stirred for 3 h at room temperature to obtain a homogeneous solution. The THF solvent was then evaporated with a gentle nitrogen flow to yield a solid residue. Subsequently, 1.6 mL of 0.85 M hydrochloride solution was added into the solid F127/PFBT mixture, which was followed by 10 min sonication to form a homogeneous suspension. Then, 180 µL of TEOS were added into the mixture and the solution was further stirred for 2 h at room temperature. 30 µL of DMDMS were subsequently added to terminate the silica shell growth at NP surfaces. The mixture was kept stirring for another 24 h at room temperature. Finally, the solution was dialyzed with a cut-off molecular weight of 14 000 membrane against Milli-Q water for three days to 89 Chapter 4 remove hydrochloride as well as unreacted reagents. The suspension was then purified with a 0.2 µm syringe filter to attain PFBT-F127-SiO2 NPs. 4.2.6 Synthesis of PFBT-DSPE NPs A THF solution (1 mL) containing DSPE-PEG2000 (20 mg) and PFBT (0.1 mg) was poured into water (10 mL) upon sonication. The sonication lasted for 60 seconds at 12 W output using a microtip probe sonicator (XL2000, Misonix Incorporated, NY). The mixture was then stirred at room temperature overnight to evaporate THF. The NP suspension was further purified with a 0.2 µm syringe filter to obtain PFBT-DSPE NPs. 4.2.7 Synthesis of PFBT-F127 NPs A THF solution (1 mL) containing F127 (20 mg) and PFBT (0.1 mg) was poured into water (10 mL) upon sonication. The sonication lasted for 60 seconds at 12 W output using a microtip probe sonicator (XL2000, Misonix Incorporated, NY). The mixture was then stirred at room temperature overnight to evaporate THF. The NP suspension was further purified with a 0.2 µm syringe filter to obtain PFBT-F127 NPs. 4.2.8 Fluorescence Stability of PFBT-F127-SiO2 NPs The fluorescence stability of PFBT-F127-SiO2 NPs in 1 × PBS at 37 °C was monitored by a fluorometer. At designed time interval, the fluorescence intensity of the incubated NP suspension was measured with the fluorometer, which was further expressed by I/I0, where I0 is the fluorescence intensity at 545 nm of fresh prepared PFBT-F127-SiO2 NP suspension and I is that of NPs after incubation at designed time, respectively. 90 Chapter 4 4.2.9 Photostability of PFBT-F127-SiO2 NPs To compare the photostability of PFBT loaded F127 NPs with and without protection silica layer, 20 µL of both NPs at same PFBT concentrations were put in 96 well and irradiated at excitation wavelength of both 405 nm and 488 nm with 100% confluence. The fluorescence intensity at the designed time interval was measured by the fluorometer. The photostability was expressed by the ratio of the fluorescence intensity after excitation for a designated time interval to its initial value as a function of the exposure time. 4.2.10 Cytotoxicity of PFBT-F127-SiO2 NPs NIH/3T3 fibroblast cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin streptomycin at 37 °C in a humidified environment containing 5% CO2. The metabolic activity of NIH/3T3 fibroblast cells was evaluated using MTT assays. NIH/3T3 cells were seeded in 96-well plates (Costar, IL, USA) at an intensity of 4 × 104 cells·mL-1. After 24 h incubation, the medium was replaced by PFBT-F127-SiO2 NP suspensions at different NP concentrations, and the cells were then incubated for 24 and 48 h, respectively. After the designated time intervals, the wells were washed twice with 1 × PBS and 100 µL of freshly prepared MTT (0.5 mg·mL-1) solution in culture medium was added into each well. The MTT medium solution was carefully removed after 3 h incubation in an incubator. DMSO (100 µL) was then added into each well and the plate was gently shaken for 10 minutes at room temperature to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan). Cell viability was expressed by the ratio of absorbance 91 Chapter 4 of the cells incubated with NP suspensions to that of the cells incubated with culture medium only. 4.2.11 Real-Time Two-Photon Intravital Blood Vascular Imaging The experimental setup for two-photon intravital blood vascular imaging is described elsewhere.[240] First, a skin incision was made to expose the skull. The head was then immobilized on a custom-built intravital imaging stage.[241] A a small 2 mm circular piece of parietal bone was excised using a dental drill, exposing the meninges and the brain of the immobilized mouse. For TPFI experiments, mice were anesthetized (150 mg/kg ketamine and 10 mg/kg xylazine) and placed on a heating pad to maintain a core body temperature of 37 °C throughout each imaging procedure. 200 µL of PFBTF127-SiO2 NPs at the PFBT concentration of 2 µM were administered via retro-orbital injection prior to imaging. As a control, a group of mice without PFBT-F127-SiO2 were also imaged under the same experimental conditions. All procedures were performed under the guidelines of Institutional Animal Care and Use Committee. A TriM Scope II single-beam two-photon microscope (LaVision BioTec) with a tunable 680-1080 nm laser (Coherent) was used to acquire the images. PFBT-F127-SiO2 NPs and second harmonic generation were excited at 810 nm, and the emitted light was split by 560 nm and 495 nm long pass mirrors and detected through 542/27 nm and 390/40 nm filters, respectively. 92 Chapter 4 4.3 Results and Discussion 4.3.1 Synthesis and Characterization of PFBT-F127-SiO2 NPs A B F127 1) micellization + 2) silica growth PFBT Scheme 4.1 (A) Chemical structure of F127 and PFBT. (B) Schematic illustration of the fabrication of PFBT-F127-SiO2 NPs. The PFBT loaded fluorescent NPs were prepared via a one-pot reaction using the commercially available triblock polymer F127 as the template. [239] The chemical structures of PFBT and F127 are shown in Scheme 4.1A. PFBT was synthesized according to our previous report.[235] F127 is a biocompatible triblock copolymer composed of a central hydrophobic chain of poly(propylene oxide) (PPO) connected with two hydrophilic chains of poly(ethylene oxide) (PEO). To prepare the NPs, the THF solution of F127 and PFBT was dried with nitrogen blow. Upon sonication in hydrochloride solution, micelles were formed with the entangled hydrophobic PPO segments and PFBT chains in the core and the hydrophilic PEO segments extending into aqueous phase, which could help to prevent nonspecific interaction with biomolecules. The hydrophilic silica components further formed in the region 93 Chapter 4 of hydrophilic PEO shell through TEOS hydrolysis under acidic condition.[239] Adding DEDMS could terminate the silicate cross-linking and provide micelles with thin silica shells (Scheme 4.1B). The mixture was further dialyzed to remove excess hydrochloride and excess reactants. The silica cross linked PFBT doped F127 NPs (PFBT-F127-SiO2 NPs) were finally obtained through filtration with 0.2 µm membrane filter. 0.08 600 500 400 0.06 300 0.04 200 0.02 400 500 600 B F127-SiO2fitting 100 0.00 300 IRF F127-SiO2 10000 0 700 Intensity (counts) DSPE 700 PL Intensity (a.u.) Absorbance (a.u.) 0.10 F127-SiO2 A DSPE DSPE fitting 1000 100 10 2 4 6 8 10 12 Time (ns) Wavelength (nm) C D 100 nm 100 nm Figure 4.1 (A) UV-vis (dash-dotted line) and PL (solid line) spectra of PFBTF127-SiO2 NPs (red) and PFBT-DSPE NPs (black) at 25 µg/mL of PFBT. (B) Fluorescence decay curves of PFBT-F127-SiO2 NPs (red) and PFBT-DSPE NPs (black). Instrument response (IRF) (blue) is also indicated. FE-TEM images of PFBT-F127-SiO2 NPs (C) and PFBT-DSPE NPs (D). FE-TEM shows that PFBT-F127-SiO2 NPs have a relative uniform size of 12 nm as shown in Figure 4.1C. No obvious size variation of PFBTF127-SiO2 NPs was observed in 10 days by laser light scattering measurement 94 Chapter 4 (Figure 4.2), indicating their excellent colloidal stability. In addition, PFBT loaded DSPE-PEG2000 NPs (PFBT-DSPE NPs) were also synthesized through the conventional reported precipitation methods.[173,228] The detailed preparation procedure is illustrated in the experimental section. The size of PFBT-DSPE NPs was estimated to be 30 ± 10 nm using FE-TEM as shown in Figure 4.1D. The UV-vis absorption and PL spectra of PFBT-F127-SiO2 NP and PFBT-DSPE NP suspensions are shown in Figure 4.1A. Both NP suspensions have similar absorption peaks around 320 nm and 460 nm, which are ascribed to the fluorene and benzothiadiazole units, respectively. Although both NPs exhibit similar absorbance, PFBT-F127-SiO2 NPs show much higher fluorescence intensity than that of PFBT-DSPE NPs. The QYs of PFBT-F127SiO2 NPs and PFBT-DSPE NPs were measured to be 75% and 40% using 4(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran in methanol as the standard. To the best of knowledge, the 40% QY of PFBTDSPE NPs is higher than most reported PFBT NPs, while the 75% QY of PFBT-F127-SiO2 NP is the highest among PFBT NPs.[34,80,101,232] To understand the high QY of PFBT-F127-SiO2 NPs as compared to PFBT-DSPE NPs as well as other reported PFBT based NPs, the different condition of PFBT chains in their respective NP formulations should be clarified. One of the most important different environments between PFBTF127-SiO2 NPs and PFBT-DSPE NPs is that there is a protection silica shell at the surface of PFBT-F127-SiO2 NPs. As well known, the silica matrices or silica layers at NP surfaces could help to prevent the attack of organic fluorophores and CPs by water or oxygen molecules, which facilitate the 95 Chapter 4 increase of NP QYs.[175,180,230,231,242] To verify this hypothesis, PFBT-F127 NPs without silica shell were also synthesized according to the procedure listed in the experimental section. However, PFBT encapsulated F127 NPs without silica protection layers (PFBT-F127 NPs) also shows a higher QY of 70%, which is only a little lower that that with silica layer (PFBT-F127-SiO2 NPs) as shown in Figure 4.3A. This comparison demonstrates that the F127 matrices could already help prevent the attack of PFBT chains by water or oxygen molecules and the protection effect of the silica layer in avoiding quencher attack is not the dominant factor to greatly enhance the fluorescence QY. One noteworthy function of the silica layer is that it could help to improve the photostability of PFBT-F127-SiO2 NPs as compared to that without silica layers (PFBT-F127 NPs) upon laser irradiation (Figure 4.3B). Carefully comparing the F127 matrix with the DSPE-PEG2000 matrix, we find that F127 chain have a much longer hydrophobic PPO segments (201 hydrophobic carbon atoms) than that of DSPE-PEG2000 matrix (34 hydrophobic carbon atoms). The longer hydrophobic PPO segments in F127 chains could entangle with PFBT backbones more efficiently than that in DSPE-PEG2000 matrices, leading to successful isolation of the CPs in the PFBT-F127 NPs. In theory, the isolation of CP chains could help to reduce CP interchain interaction, benefiting their fluorescence QY enhancement.229,230,234 As a result, the high fluorescence QY of PFBT-F127-SiO2 NPs should be mainly ascribed to the sufficient isolation and protection of PFBT chains by the longer hydrophobic PPO segments from F127 matrices and less protection of the silica layers. 96 Chapter 4 Table 4.1 Emission decay components of PFBT-F127-SiO2 NPs and PFBTDSPE NPs. sample 1 (ns) A1 (%) 2 (ns) A2 (%) 3 (ns) A3 (%) av 2 F127 0.16 39.44 0.71 39.55 2.96 21.01 0.97 1.04 DSPE 0.18 48.14 0.52 49.90 2.15 1.96 0.38 1.03 , where  is the decay time and A is the amplitude; 2 is the ∑ value of fit. 40 Size (nm) 30 20 10 0 0 2 4 6 8 10 Time (day) Figure 4.2 The DLS size evolution of PFBT-F127-SiO2 NPs in 10 days. 120 700 F127-SiO2 F127 0.08 500 0.06 400 300 0.04 200 0.02 0.00 300 F127-SiO2 B 600 F127 100 I/I0 (%) A PL intensity (a.u.) Absorbance (a.u.) 0.10 80 60 100 400 500 600 40 0 700 0 2 4 6 8 10 Time (min) Wavelength (nm) Figure 4.3 (A) UV-vis (dashed) and PL (solid) spectra of PFBT loaded F127 NPs with (red) and without (black) silica layers, respectively. (B) Photostability of PFBT loaded F127 NPs with and without the protection silica layer upon continuous laser excitation at both 405 nm and 488 nm with 100% light confluence, where I0 is the initial fluorescence intensity and I is the fluorescence intensity of the sample at different time points after illumination. 97 Chapter 4 To better understand the enhanced QY of PFBT-F127-SiO2 NPs as compared to that of PFBT-DSPE NPs, the fluorescence lifetimes for both NPs were investigated with the FluoTime 200-Fluorescence system. The QY () and fluorescence lifetime () of a fluorophore is given by  =  / ( + knr) and  = ( + knr)-1,[243] where  is the radiative decay rate, and knr is the nonradiative decay rate. In general, the radiative lifetime () of a fluorophore is its intrinsic property, which is considered to be a constant. As such, the changes in QY () and lifetime () are mainly due to the change in non-radiative decay rate (knr), and the values of QY () and lifetime () of a fluorophore should change in the same direction, either both increasing or both decreasing.[243] As shown in Figure 4.2, the average fluorescence lifetime of PFBT-F127-SiO2 NPs (0.97 ns) is much longer than that of PFBT-DSPE NPs (0.38 ns). The elongated lifetime of PFBT-F127-SiO2 NPs should result from the blocking of non-radiative decay pathways of PFBT chains through minimizing their selfaggregation and environment attack, resulting in the observed higher QY of PFBT-F127-SiO2 NPs. In addition, the respective emission decay components of PFBT-F127-SiO2 NPs and PFBT-DSPE NPs are shown in Table 4.1. Generally, the longer lifetime component (2.96 ns for PFBT-F127-SiO2 NPs and 2.15 ns for PFBT-DSPE NPs) represents fluorescence decay from the isolated polymer chains.[230,234] The other two short decay components are related to quenching process induced by self-aggregation or attacking from molecular quenchers (e.g. oxygen and water).[234] Clearly, the data in Table 4.1 validate that the micelle/silica approach is able to increase the percentage of the isolated single chain and decrease the environment quenching influence on CP fluorescence. 98 Chapter 4 4.3.2 Single NP Imaging of PFBT-F127-SiO2 NPs To further investigate the behavior of individual PFBT-F127-SiO2 NP and PFBT-DSPE NP, single NP fluorescence imaging was conducted upon excitation at 488 nm, using a commercial QD655 as the benchmark. The intensity time-traces of PFBT-F127-SiO2 NPs, PFBT-DSPE NPs and QD655 were collected by integrating the emission from individual NPs over 100 s A F127-SiO2 20 Occurrence 20 30 10 30 20 10 10 0 0.0 QD655 C 40 Occurrence Occurrence DSPE B 40 2.0x10 6 4.0x10 6 6.0x10 Total counts in 100 s 6 0 0.0 2.0x10 6 4.0x10 6 6.0x10 Total counts in 100 s 6 0 0.0 5.0x10 5 1.0x10 6 Total counts in 100 s Figure 4.4 Histograms of the total number of photons collected for (A) PFBTF127-SiO2 NPs, (B) PFBT-DSPE NPs and (C) QD655. Note the different binning and scales for A, B and C, λex = 488 nm for all samples. for 1000 consecutive frames. The histograms of the total number of photon emitted by each type of NPs in 100 s are shown in Figure 4.4A to 4.4C. The average number of the total photons emitted by single PFBT-F127-SiO2 NP (9.4105 counts) is similar with that of PFBT-DSPE NP (9.6105 counts), and much higher than that QD655 (2.8105 counts). Considering the smaller size of PFBT-F127-SiO2 NPs (~12 nm) than that of PFBT-DSPE NPs (~30 nm), on average, each PFBT-F127-SiO2 NPs should has less CP chains than PFBTDSPE NPs. The similar counts for both NPs further support the higher fluorescence QY of PFBT-F127-SiO2 NPs. 99 Chapter 4 4.3.3 Fluorescence Stability and Cytotoxicity of PFBT-F127-SiO2 NPs B 1.0 I/I0 0.6 0.4 0.2 120 24 h 48 h 100 Day 0 Day 10 PL intensity (a.u.) 0.8 Cell viability (%) A 80 60 40 20 Wavelength (nm) 0.0 0 2 4 6 8 0 10 1000 500 100 NP Concentration (g/mL) Time (day) Figure 4.5 (A) PL intensity evolution of PFBT-F127-SiO2 NPs upon incubation with 1 × PBS at 37 °C for different times, where I0 is the fluorescence intensity at 545 nm for the fresh NP suspension and I is that for NPs after incubation for different time, respectively. The inset shows the PL spectra of freshly prepared PFBT-F127-SiO2 NPs (black) and after 10 days incubation with 1 × PBS at 37 °C (red). (B) Metabolic viability of NIH/3T3 fibroblast cells after incubation with PFBT-F127-SiO2 NP suspensions at various NP concentrations for 24 h and 48 h, respectively. Excellent fluorescence stability in aqueous media is essential for biological applications, especially in long-term studies. The fluorescence intensity evolution of PFBT-F127-SiO2 NPs was investigated by monitoring their fluorescence changes upon incubation with PBS at 37 °C for different time. As shown in Figure 4.5A and its inset, no obvious fluorescence decrease is observed after 10 days incubation of the PFBT-F127-SiO2 NPs with 1 × PBS at 37 °C, suggesting outstanding stability of the prepared NPs. To evaluate the cytotoxicity of the PFBT-F127-SiO2 NPs, the metabolic viability of NIH/3T3 fibroblast cells after incubation with NPs was investigated at different NP concentrations. Figure 4.5B shows the cytotoxicity results of the PFBT-F127-SiO2 NPs upon incubation with suspensions at 100 to 1000 g/mL NPs for 24 h and 48 h, respectively. The cell viabilities remain ~90% upon incubation with PFBT-F127-SiO2 NPs for 48 h at 1000 µg/mL 100 Chapter 4 concentration, indicating the low cytotoxicity of the probe, which is ideal for biological imaging. 4.3.4 TPA Spectra of PFBT-F127-SiO2 NPs In vivo visualizing blood vessels is essential to investigate biological processes, such as angiogenesis, vascular leakage, and leukocyte extravasation.[240,244,245] Materials with large TPA cross section is beneficial to achieve a good imaging resolution. The TPA spectra of PFBT-F127-SiO2 NP aqueous suspensions were measured with a TPIF microscope with a tunable Ti:Sapphire laser. As a comparison, the TPA property of Evans blue, a commonly used blood vascular imaging contrast,[240] was also investigated. The relative TPFI intensities of PFBT-F127-SiO2 NPs and Evans blue were measured using Rhodamine 6G in methanol as the standard. The emission signals were collected from an excitation spectra ranging from 800 nm to 920 nm at 10 nm intervals. Details of the measurement and calculation method are provided in the experimental section. The TPA cross section of PFBT-F127SiO2 NPs was calculated based on CP chain concentrations and the obtained TPA spectra of PFBT-F127-SiO2 NPs as well as Evans blue are shown in Figure 4.6 The maximum δ is ~1085 GM at 810 nm, which gives a ηδ value of 814 GM, which is much larger than that of Evans blue. This larger TPA active cross-section of PFBT-F127-SiO2 NPs in aqueous media is highly desirable to ensure a high signal-to-noise ratio in bioimaging experiment. 101 Chapter 4 1200 PFBT-F127-SiO2 TPA Cross Section (GM) Evans Blue 900 600 300 0 760 800 840 880 920 Wavelength (nm) Figure 4.6 TPA spectra of PFBT-F127-SiO2 NPs (based on CP chain concentration) and Evans blue in water. 4.3.5 Intravital TPFI of PFBT-F127-SiO2 NPs After investigate the TPA properties of PFBT-F127-SiO2 NPs, their biological application was demonstrated by labeling and visualizing the brain vasculature of an anaesthetized mouse using two-photon microscopy. The PFBT-F127SiO2 NPs were administered intravenously prior to imaging and the twophoton excited fluorescence was collected at 542  27 nm upon excitation at 800 nm.[240] Figures 4.7A-4.7G show representative images of the blood vessels at various depth in the brain imaged through a cranial window over 20 minutes. The major blood vessels as well as the smaller capillaries in the pia mater could be visualized with the help of PFBT-F127-SiO2 NPs. In addition, no large aggregates are observed in brain blood vessels, owing to the PEO segments on the surface of NPs blocking nonspecific binding. More importantly, microvasculature deep in the brain that lies beyond the pia matter (500 µm) could still be detected with high resolution (Figure 4.7G). As a control, no fluorescence was detected from the brain blood vessels before PFBT-F127-SiO2 NPs was administered (Figure 4.8). The 3D reconstruction 102 Chapter 4 (Figure 4.7H) and Z-projected (Figure 4.7I) images of the brain vasculature illustrate that our probe can efficiently label the blood vasculature in in vivo condition. A 0 µm B 50 µm C 100 µm D 200 µm E 300 µm F 400 µm G 500 µm H Z-projection I 3D Figure 4.7 Intravital TPFI of PFBT-F127-SiO2 NPs stained blood vessels of mice brain at depth of 0 µm (A), 50 µm (B), 100 µm (C), 200 µm (D), 300 µm (E), 400 µm (F) and 500 µm (G), and the respective Z-projected image (H) as well as 3D image (I). All the images share the same scale bar of 50 µm. 103 Chapter 4 Figure 4.8 Images of intravital TPFI of brain blood vessels in mouse without injection of PFBT-F127-SiO2 NPs. The scale bar is 50 µm. 4.4 Conclusion In conclusion, we developed a simple and effective strategy to increase the QY as well as tow-photon action cross section of PFBT NPs using F127 as the matrices and silica shells as the protection layer. The obtained PFBT-F127SiO2 NPs show a high QY of 0.75 by reducing CP self-aggregation and environment quenching, which was evidenced by fluorescence lifetime measurements. In addition, single NP study illustrates that the bright PFBTF127-SiO2 NPs exhibit good photostability as compared to PFBT-DSPE NPs. Moreover, PFBT-F127-SiO2 NPs show a large two-photon action cross section and non-toxic biocompatibility, benefiting their biological applications as TPFI probes. Real-time in vivo TPFI reveals that PFBT-F127-SiO2 NPs could be utilized as an effective TPA probe for in vivo blood vascular imaging with a deep depth of 500 µm and high contrast. The strategy developed in this study should provide a new avenue for CP NPs in both one-photon and two-photon excited biological imaging. 104 Chapter 5 CHAPTER 5 ORGANIC NANOPARTICLES WITH PROCESSABLE CONJUGATED POLYMER FOR PHOTOACOUSTIC VASCULAR IMAGING 5.1 Introduction Photoacoustic (PA) imaging is an emerging technique in biological imaging due to its noninvasive, high resolution and deep penetration characteristics.[13,246-248] Currently, PA imaging technique has three major implementations: dark-field photoacoustic microscopy (PAM), photoacoustic computed tomography (PAT) and photoacoustic endoscopy (PAE). Whereas PAM and PAE usually aim to image millimeter deep at micrometer-scale resolution, PAT can be implemented for deeper imaging ability with few hundred micrometer-scale resolution. PA imaging is based on the measurement of ultrasonic waves generated by the targets upon pulse laser absorption.[12,249] Utilizing the intrinsic optical contrast molecules in biological systems, PA imaging has been successfully applied in imaging blood vessels,[250] studying brain hemodynamic changes,[14,251-253] and visualizing tumor angionenesis[254]. Unfortunately, most intrinsic optical contrast agents, such as hemoglobin and deoxy-hemoglobin, absorb light in the visible spectral region, where exists the overwhelming light scattering in biological tissues, resulting in limited sensitivity and resolution.[246,247] In addition, many biological objects (e.g. disease signal molecules) do not absorb light in any region, which hampers the utilization of PA technique for specific detection 105 Chapter 5 and imaging.[246] Exogenous contrast agents can not only enhance the sensitivity of PA imaging by enabling the absorption region in NIR region to reduce the scattering interference but also target the specific biological objects to generate sufficient PA signals for accurate analysis.[250,255-258] To increase the detection sensitivity and resolution, several optically absorptive contrast agents have been developed for biological applications.[258261] So far, the most widely used PA contrast agents are gold nanomaterials including nanorods, nanocages and nanoshells.[12,262-267] In addition, several reports have also utilized carbonous nanomaterials, such as carbon nanotubes and polyhydroxy fullerenes.[268-271] The recent research interest in the development of PA probes has been extended to NIR dyes and organic NPs due to their benign biocompatibility.[250,254,256,272,273] For example, Indocyanine green (ICG), a nontoxic and water soluble dye, can help to visualize more detailed vasculature structures in sentinel lymph nodes brain as compared to the intrinsic signals.[250,256,273] In addition, organic dye loaded polymer NPs and porphysome organic NPs have also been utilized as PA contrast agents for targeted tumor imaging.[256,274,275] These successful examples have motivated researchers to look for organic biocompatible materials with large absorption coefficients for further improvement of PA contrast. CPs are macromolecules characterized with π-conjugated backbone and readily modified electrical and optical properties, which have been widely applied in optoelectronic devices and biomedical field.[25-28,51] The extensive bioimaging studies based on CPs have revealed that they exhibit good photostability and benign biocompatibility.[33,35,36,98,128,188] In view of their conjugated backbones, CPs generally show much larger absorption coefficient 106 Chapter 5 as compared to their small molecular counterparts, which provide the new opportunity for them to serve as organic exogenous contrast agents for PA imaging. Recently, polypyrrole-based NPs, prepared through oxidative polymerization of pyrrole monomer using PVA as stabilizing agent and FeCl 3 as catalyst, have been successfully applied as PA contrast agent in deep tissue imaging.[12] The NPs show strong absorption in the NIR region with high PA signals. However, polypyrrole is not soluble in almost any organic solvent and the chemical processability of this polymer is poor. In addition, the NPs generated from in-suit polymerization can be hardly modified with functional groups for further modification. Nonetheless, the successful application of polypyrrole for PA imaging has opened new opportunities for the development of CP based PA reagents. The well-developed nanoparticle encapsulation strategy requires the encapsulated polymers to be soluble in organic solvents. An effective strategy to obtain CPs with NIR absorption is the combination of electron-rich and electron-deficient moieties in an alternating fashion to form alternating donoracceptor (D‒A) backbone structure. In this regard, thiadiazoloquinoxaline derivatives are good electron acceptors due to their high electron-deficiency, which are ideal for achieving small band gap CPs with long wavelength absorption. In this chapter, we synthesize poly[9,9-bis(4-(2- ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4-(hexyloxy)phenyl)-4,9di(thiophen-2-yl)thiadiazolo-quinoxaline] (PFTTQ) and its NP formation to yield a potential PA probe. Using DSPE-PEG2000 as the encapsulation matrix,[173,228] the obtained PFTTQ loaded DSPE-PEG2000 NPs (PFTTQ NPs) show strong absorbance in NIR range with high non-radiative QY. To evaluate 107 Chapter 5 the PA signal from the PFTTQ NPs, Au NRs with a strong NIR extinction coefficient have also been synthesized. The PA intensities of both PFTTQ NPs and Au NRs with the same absorbance have been studied using a 50-MHz dark field confocal PAM system. In addition, the photostability and biocompatibility of the PFTTQ NPs have also been investigated. The potential of PFTTQ NPs as a PA probe was further demonstrated using brain vascular imaging as an example. 5.2 Experimental Section 5.2.1 Materials DSPE-PEG2000 was purchased from Avanti Polar Lipids, Inc. MTT, penicillin-streptomycin solution, FBS, trypsin-EDTA solution, methanol and THF, 4-iodophenol, 1-bromohexane, trimethylsilylaceylene, copper iodide, 2(tributylstannyl)thiophene, N-bromosuccinimide (NBS), palladium(II) acetate (Pd(AcO)2), tricyclohexylphosphine (Cy3P) and tetraethylammonium hydroxide (Et4NOH) solution (35 wt% in H2O) were purchased from SigmaAldrich and used as received. Toluene used for Suzuki polycondensation was pretreated with sulfuric acid followed by distillation. All other chemical reagents were used as received. Compounds 6,[277] 7,[277] 8[278] and 10[279] were prepared according to previous reports. Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, Breford, USA). NIH/3T3 fibroblast cells were provided by American Type Culture Collection. 5.2.2 Characterization Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500 NMR spectrometer (500 MHz for 1H, referenced to TMS at δ = 108 Chapter 5 0.00 ppm and 125 MHz for 13 C, referenced to CDCl3 at 77.0 ppm). Average particle size and size distribution of the NPs were determined by DLS with particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90° at room temperature. FE-TEM studies were performed on a JEOL JEM-2010 electron microscope with an accelerating voltage of 200 kV. UV‒vis spectra were collected on a Shimadzu UV-1700 spectrometer. All UV-vis spectra were collected at 24 ± 1 oC. MilliQ water (18.2 MQ) was used for all the experiments. 5.2.3 PA Measurement Polyethylene tubing (~20 cm) was filled with various designed samples. Afterwards, the tubing was positioned at a depth of the transducer’s focus, i.e., the depth of 9 mm with respect to the transducer in water tank. The system was maintained in a 25°C water bath throughout the experiment. The contrast changes of the developed probes were imaged using a 50-MHz dark field confocal PAM system with 32 × 61-m resolution (Scheme 5.3). An optical parametric oscillator pumped by a frequency-tripled Nd:YAG Qswitched laser was employed to provide ~4-ns laser pulses at a pulse repetition rate of 10 Hz. Laser energy was delivered by a 1-mm multimode fiber. The fiber tip was coaxially aligned with a convex lens, an axicon, a plexiglass mirror, and an ultrasonic transducer on an optical bench, forming dark field illumination that is confocal with the focal point of the ultrasonic transducer. Laser pulses at two visible wavelengths, 800 (λ480) was used for PA wave excitation. A large numerical-aperture, wideband 50-MHz ultrasonic transducer was employed to allow for the efficient collection of PA signals. The scanning step size was 20 m for each B-scan. 109 Chapter 5 The PA signals received by the ultrasonic transducer were preamplified by a low-noise amplifier (noise figure 1.2 dB, gain 55 dB, AU-3A0110, Miteq, USA), cascaded to an ultrasonic receiver (5073 PR, Olympus, USA), then digitized and sampled by a computer-based 14-bit analog to digital (A/D) card (CompuScope 14200, GaGe, USA) at a 200-MHz sampling rate for data storage. Fluctuations in the laser energy were monitored by a photodiode (DET36A/M, Thorlabs, USA). Recorded photodiode signals were applied to compensate for PA signal variations caused by laser energy instability before any further signal processing. No signal averaging was performed in this study. Note that the amplitude of the envelope-detected PA signal was used in the imaging analysis. 5.2.4 Synthesis of Monomers and PFTTQ 1-Hexyloxy-4-iodobenzene (1). To a solution of tetrabutylammonium bromide (100 mg, 0.31 mmol) and 4-iodophenol (5 g, 22.7 mmol) in aqueous possium hydroxide (50 mL, 50wt%) at 100 oC was added 1-bromohexane (4.37 mL, 31.1 mmol) in one portion. The reaction was kept for 1 h at this temperature. After stopping the reaction, the mixture was extracted with dichloromethane, washed with water and dried over MgSO4. After solvent removal, the residue was purified by silica gel column chromatography using hexane as eluent to provide 1-hexyloxy-4-iodobenzene as colorless liquid (6.3 g, yield: 91%). 1H NMR (500 MHz, CDCl3, ppm) δ: 7.53 (d, J = 8.5 Hz, 2 H), 6.67 (d, J = 8.5 Hz, 2 H), 3.91 (t, J = 6.5 Hz, 2 H), 1.75 (m, 2 H), 1.45 (m, 2 H), 1.33 (m, 4 H), 0.91 (t, J = 7 Hz, 3 H). 13C NMR (125 MHz, CDCl3, ppm) δ: 159.06, 138.16, 116.97, 82.40, 68.16, 31.57, 29.14, 25.69, 22.60, 14.04. 110 Chapter 5 ((4-(Hexyloxy)phenyl)ethynyl)trimethylsilane (2). To a solution of 1-hexyloxy-4-iodobenzene (3.25 g, 10.69 mmol), copper iodide (104 mg, 0.54 mmol) and Pd(PPh3)2Cl2 (150 mg, 0.21 mmol) in diisopropylamine/tetrahydrofuran (30/10 mL) at room temperature under argon atmosphere was added trimethylsilylaceylene (2.45 g, 25 mmol) via syringe. The reaction was performed at room temperature overnight. The mixture was diluted with dichloromethane, filtered through a celite pad, concentrated under reduced pressure and purified by silica gel column chromatography using hexane as eluent to afford ((4- (hexyloxy)phenyl)ethynyl)trimethylsilane as colorless liquid (2.8 g, yield: 95%). 1H NMR (500 MHz, CDCl3, ppm) δ: 7.38 (d, J = 8.5 Hz, 2 H), 6.79 (d, J = 8.5 Hz, 2 H), 3.94 (t, J = 6.5 Hz, 2 H), 1.75 (m, 2 H), 1.45 (m, 2 H), 1.33 (m, 4 H), 0.89 (t, J = 7 Hz, 3 H), 0.29 (s, 9 H). 13 C NMR (125 MHz, CDCl3, ppm) δ: 159.37, 133.44, 115.02, 114.36, 105.34, 92.27, 68.07, 31.57, 29.15, 25.68, 22.58, 14.00, 0.08. 1-Ethynyl-4-(hexyloxy)benzene (3). A round bottle flask was charged with ((4-(hexyloxy)phenyl)ethynyl)trimethylsilane (2.74 g, 10 mmol), potassium hydroxide (5.6 g, 100 mmol), THF (50 mL), methanol (25 mL) and water (18 mL). The mixture was stirred at room temperature under argon atmosphere for 1 h. After solvent removal, the residue was subsequently redissolved in dichloromethane, washed with water and dried over MgSO4. The crude product was purified by silica gel column chromatography using hexane as eluent to yield 1-ethynyl-4-(hexyloxy)benzene as colorless liquid (1.9 g, yield: 94%). 1H NMR (500 MHz, CDCl3, ppm) δ: 7.42 (d, J = 8.5 Hz, 2 H), 6.83 (d, J = 8.5 Hz, 2 H), 3.95 (t, J = 6.5 Hz, 2 H), 3.00 (s, 1 H), 1.77 (m, 2 111 Chapter 5 H), 1.46 (m, 2 H), 1.35 (m, 4 H), 0.93 (t, J = 7 Hz, 3 H). 13C NMR (125 MHz, CDCl3, ppm) δ: 159.59, 133.57, 114.49, 113.95, 83.80, 75.67, 68.08, 31.59, 29.17, 25.71, 22.61, 14.03. 1,2-Bis(4-(hexyloxy)phenyl)ethyne (4). A solution of 1-ethynyl-4(hexyloxy)benzene (2.02 g, 10 mmol), 1-hexyloxy-4-iodobenzene (3.19 g, 10.5 mmol), copper iodide (95 mg, 0.50 mmol) and Pd(PPh3)2Cl2 (140 mg, 0.20 mmol) in diisopropylamine (30 mL) was stirred for 20 h at room temperature under argon atmosphere. After the removal of solvent, the residue was subsequently redissolved with dichloromethane, washed with water and dried over MgSO4. The crude product was purified by silica gel column chromatography (hexane/dichloromethane = 9/1) to afford compound 1,2bis(4-(hexyloxy)phenyl)ethyne as white solid (2.6 g, yield: 69%). 1H NMR (500 MHz, CDCl3, ppm) δ: 7.42 (d, J = 8.5 Hz, 4 H), 6.84 (d, J = 8.5 Hz, 4 H), 3.97 (t, J = 6.5 Hz, 4 H), 1.78 (m, 4 H), 1.46 (m, 4 H), 1.35 (m, 4 H), 0.91 (t, J = 7 Hz, 6H). 13 C NMR (125 MHz, CDCl3, ppm) δ: 158.99, 132.83, 115.54, 114.52, 87.96, 68.09, 31.59, 29.19, 25.71, 22.59, 14.02. 1,2-Bis(4-(hexyloxy)phenyl)ethane-1,2-dione (5). To a solution of 1,2-bis(4-(hexyloxy)phenyl)ethyne (2.5 g, 6.6 mmol) in dichloromethane (20 mL) was added tetrabutylammonium bromide (100 mg, 0.31 mmol), NaHCO3 (1.2 g, 14.3 mmol), KMnO4 (3.3 g, 20.9 mmol) and water (40 mL). The mixture was vigorously stirred at room temperature for 2 days, then sodium bisulfite (8 g) and hydrochloric acid (10 mL) were subsequently added. The mixture was extracted with dichloromethane and washed with water, and the organic phase was dried over MgSO4. After solvent removal, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 19/1) to 112 Chapter 5 afford white solid (2.5 g, yield: 92%). 1H NMR (500 MHz, CDCl3, ppm) δ: 7.92 (d, J = 8.5 Hz, 4 H), 6.94 (d, J = 8.5 Hz, 4 H), 4.03 (t, J = 7 Hz, 4 H), 1.80 (m, 4 H), 1.46 (m, 4 H), 1.34 (m, 4 H), 0.91 (t, J = 7 Hz, 6 H). 13C NMR (125 MHz, CDCl3, ppm) δ: 193.56, 164.50, 132.35, 126.13, 114.72, 68.48, 31.50, 28.98, 25.60, 22.56, 13.99. 4,7-Dibromo-5,6-dinitrobenzo[1,2,5]thiodiazole (6). To a mixture of sulphuric acid (10 mL) and nitric acid (70%, 10 mL) at 0 oC was added 4,7dibromo-2,1,3-benzothiodiazole (2 g, 6.8 mmol). After stirring at 100 oC overnight, the mixture was cooled down to room temperature and poured into ice-water (100 mL), followed by addition of sodium hydroxide solution to neutralize the excess acid. The precipitate was filtered and washed with water. The crude product was purified by silica gel column chromatography (hexane/dichloromethane = 8/2) to afford white solid (522 mg, yield: 20%). MS: m/z = 383.9. 5,6-Dinitro-4,7-di(thiophen-2-yl)benzo[1,2,5]thiadiazole (7). This compound was prepared with modified procedures according to previous reports.1 In brief, a solution of 4,7-dibromo-5,6-dinitro-benzothiadiazole (250 mg, 0.65 mmol), 2-(tributylstannyl)thiophene (971 mg, 2.6 mmol) and dichlorobis(triphenylphosphine)palladium (45 mg, 0.65 mmol) in anhydrous THF was heated to 80 oC under argon atmosphere. The mixture was allowed at 80 oC overnight. After the solvent removal, the residue was purified by silica gel column chromatography (hexane followed by hexane/dichloromethane = 7:3) to afford 5,6-dinitro-4,7-di(thiophen-2-yl)benzo[1,2,5]thiadiazole as orange solid (230 mg, yield : 90.7%). 1H NMR (500 MHz, CDCl3, ppm) δ: 7.78 (d, J = 4 Hz, 2 H), 7.55 (d, J = 4 Hz, 2 H), 7.27 (t, J = 4 Hz, 2 H). 113 Chapter 5 4,7-Bis(5-bromothiophen-2-yl)-5,6-dinitrobenzo[1,2,5]thiadiazole (8). This compound was prepared with modified procedures according to previous reports.2 In brief, a solution of 5,6-dinitro-4,7-di(thiophen-2yl)benzo[1,2,5]thiadiazole (230 mg, 0.59 mmol) in DMF (20 mL) was heated to 60 oC. The mixture was added N-bromosuccinimide (215 mg, 1.21 mmol) in one portion. Another portion of NBS (215 mg, 1.21 mmol) was added after 1 h. 2 h later, TLC indicates all the starting material was completely consumed, and only one new spot appeared. The reaction mixture was cooled down to room temperature and poured into water. The collected precipitation was washed with methanol twice to afford 4,7-bis(5-bromothiophen-2-yl)-5,6dinitrobenzo[1,2,5]thiadiazole as orange solid (274 mg, yield: 85%). 1H NMR (500 MHz, CDCl3, ppm) δ: 7.29 (d, J = 4 Hz, 2 H), 7.22 (d, J = 4 Hz, 2 H). 4,9-Bis(5-bromothiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-[1,2,5] thiadiazolo[3,4-g]quinoxaline (9). A mixture of 4,7-bis(5-bromothiophen-2yl)-5,6-dinitrobenzo[1,2,5]thiadiazole (300 mg, 0.55 mmol) and fine iron powder (368 mg, 6.5 mmol) in acetic acid (20 mL) was heated to 80 oC. After 6 h, the mixture was cooled down to room temperature and filtered off. The filtration was mixed with 1,2-bis(4-(hexyloxy)phenyl)ethane-1,2-dione (225 mg, 0.55 mmol) and stirred at 135 oC under argon atmosphere for 24 h. After removing the solvent, the residue was washed with methanol. The precipitation was purified by silica gel column chromatography (hexane/dichloromethane = 7/3) to afford compound M3 as black-green solid (241 mg, yield: 51%). 1H NMR (500 MHz, CDCl3, ppm) δ: 8.83 (d, J = 4 Hz, 2 H), 7.70 (d, J = 8.5 Hz, 4 H), 7.21 (d, J = 4 Hz, 2 H), 6.96 (d, J = 8.5 Hz, 4 H), 4.05 (t, J = 6.5 Hz, 4 H), 1.85 (m, 4 H), 1.54 (m, 4 H), 1.38 (m, 8 H), 0.93 114 Chapter 5 (t, J = 7 Hz, 6 H). 13C NMR (125 MHz, CDCl3, ppm) δ: 160.63, 153.01, 150.67, 137.32, 133.79, 133.05, 132.50, 129.94, 129.40, 120.21, 119.46, 114.11, 68.19, 31.68, 29.30, 25.81, 22.66, 14.09. Poly[9,9-bis(4-(2-ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4(hexyl-oxy)phenyl)-4,9-di(thiophen-2-yl)thiadiazoloquinoxaline] (PFTTQ). A Schlenk tube was charged with 4,9-bis(5-bromothiophen-2-yl)-6,7-bis(4(hexyloxy)phenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline (100.0 mg, 0.116 mmol), 2,7-bis(4,4,5,5-tetramethyl-1,3,3-dioxaboralan-2-yl)-9,9-bis(4-(2- ethylhexyloxy)phenyl)fluorene (95.8 mg, 0.116 mmol), palladium acetate (3 mg) and tricyclohexylphosphine (7 mg) in toluene (10 mL) before it was sealed with a rubber septum. The Schlenk tube was degassed with three freeze-pump-thaw cycles to remove air. After the mixture was heated to 80 oC, an aqueous Et4NOH solution (20 wt%, 1.5 mL) was added to initiate the reaction. After 18 h, the reaction was stopped and cooled down to room temperature. The mixture was dropped slowly into methanol (100 mL) to precipitate the crude polymer followed by centrifugation. The crude polymer was subsequently redissolved in chloroform (200 mL), washed with water 3 times, and dried over MgSO4. After solvent removal, the polymer (45 mg, yield: 30%) was obtained as a black-green solid by precipitation in methanol. 1 H NMR (500 MHz, CDCl3, ppm) δ: 9.00 (br, 4 H), 7.81‒7.14 (br, 14 H), 6.80 (br, 8 H), 3.98 (br, 4 H), 3.78 (br, 4 H), 1.83 (br, 4 H), 1.67 (br, 2 H), 1.50‒1.26 (br, 28 H), 0.93‒0.85 (br, 18 H). 5.2.5 Synthesis of PFTTQ NPs A THF solution (1 mL) containing DSPE-PEG2000 (1.5 mg), and PFTTQ (1 mg) was poured into water (10 mL). This was followed by 115 Chapter 5 sonicating the mixture for 2 minutes at 12 W output using a microtip probe sonicator (XL2000, Misonix Incorporated, NY). The mixture was then stirred at room temperature overnight to evaporate the organic solvent. The NP suspension was further purified with a 0.2 µm syringe filter to obtain PFTTQ NPs. 5.2.6 Synthesis of Au NRs Au NRs was synthesized by a seed-mediated growth approach following the previous reports.[280] Briefly, Au NP seed was prepared by rapidly injecting NaBH4 (0.01 M, 0.6 mL) in a mixture of HAuCl4 (0.01 M, 0.25 mL) and cetyltrimethylammonium bromide (CTAB, 0.1 M, 9.75 mL). After stirring for 5 mins, the obtained brownish solution was kept for at least 2 h at room temperature before being used as the seed. To grow Au NRs, 2 mL of HAuCl4 and 400 mL of AgNO3 were added into 40 mL of 0.1 M CTAB under gentle stirring. Subsequently, 0.8 mL of 1.0 mL HCL was added, following with 0.32 mL of 0.1 M ascorbic acid. Next, 8 μL of the seed solution were injected in the mixture and gentle stirred for another 5 mins. The reaction mixture was then left undisturbed overnight to get Au NRs. 5.2.7 Cytotoxicity of PFTTQ NPs NIH/3T3 fibroblast cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin streptomycin at 37 °C in a humidified environment containing 5% CO2. The metabolic activity of NIH/3T3 fibroblast cells was evaluated using MTT assays. NIH/3T3 cells were seeded in 96-well plates (Costar, IL, USA) at an intensity of 4 × 104 cells·mL-1. After 24 h incubation, the medium was replaced by PFTTQ NP suspensions at 116 Chapter 5 different mass concentrations, and the cells were then incubated for another 24 h. After the designated time interval, the wells were washed twice with 1 × PBS and 100 µL of freshly prepared MTT (0.5 mg·mL-1) solution in culture medium was added into each well. The MTT medium solution was carefully removed after 3 h incubation in an incubator. DMSO (100 µL) was then added into each well and the plate was gently shaken for 10 minutes at room temperature to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan) after subtracting the absorbance of the corresponding control cells incubated with PFTTQ NPs at the same concentration but without the addition of MTT to eliminate the absorbance interference from PFTTQ. Cell viability was expressed by the ratio of absolute absorbance of the cells incubated with NP suspensions to that of the cells incubated with culture medium only. 5.2.8 Photostability of PFTTQ NPs and Au NRs The photostability of PFTTQ NPs as well as Au NRs was investigated by monitoring their respective UV-vis absorption intensity changes after pulse laser irradiation for different time intervals. At designed time interval, the UVvis absorption intensity of the NP suspension was measured with the spectrometer, which was further expressed by I/I0, where I0 is the absorption intensity at 800 nm of fresh NP suspension and I is that of NPs after irradiation, respectively. 5.2.9 In Vivo PA Brain Vascular Imaging The experimental setup for brain vasculatur imaging using dark-field strong-focusing PAM is described elsewhere in detailed.[251,281] Male Sprague 117 Chapter 5 Dawley rats weighing 250 to 300 g each were used for PA imaging. The animals were housed at a constant temperature and humidity with free access to food and water. Before the imaging experiment, the rats were fasted for 24 hours but given water ad libitum. All animal experiments were conducted in accordance with the guidelines of the Animal Research Committee of National University of Singapore. The animals were initially anesthetized with 3% isoflurane. Supplemental α-chloralose anesthesia (70 mg∕kg) was injected intraperitoneally as needed. The anesthetized rats were mounted on a custom built acrylic stereotaxic head holder, and the skin and muscle were cut off to expose the bregma landmark. The anteroposterior distance between the bregma and the interaural line was surveyed directly. Furthermore, the craniotomy was performed for each animal and the bilateral cranial window with approximately 6 (horizontal) × 3 (vertical) mm size was fashioned with a high-speed drill. The interaural and bregma references were then used to position the heads in the fPAM system without additional surgery in following experiments. Quantitative data were expressed as mean ± SD. Statistical comparisons were made by ANOVA analysis and Student’s t-test. P value < 0.05 was considered statistically significant. 118 Chapter 5 5.3 Results and Discussion 5.3.1 Synthesis and Characterization of PFTTQ Scheme 5.1 Synthetic route and chemical structure of PFTTQ. Reagents and conditions: I) KOH, 1-bromohexane, H2O, 100 oC, 1 h; II) CuI, Pd(PPh3)2Cl2, trimethylsilylaceylene, i-Pr2NH/THF, room temperature, overnight; III) KOH, THF/MeOH/H2O, room temperature, 1 h; IV) CuI, Pd(PPh3)2Cl2, 1, iPr2NH/THF, room temperature, overnight; V) KMnO4, NaHCO3, TBAB, CH2Cl2, room temperature, 2 days; VI) H2SO4, HNO3, 100 oC, overnight; VII) Pd(PPh3)2Cl2, 2-(tributylstannyl)thiophene, THF, 80 oC, overnight; VIII) NBS, DMF, 60 oC, 3 h; IX) i) Iron, acetic acid; ii) acetic acid, 135 oC, 24 h; X) Pd(OAc)2, Cy3P, Et4NOH, toluene, 18 h. PFTTQ was synthesized by Suzuki polymerization between 4,9-bis(5bromothiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazolo [3,4- g]quinoxaline 9 and 2,7-bis(4,4,5,5-tetramethyl-1,3,3-dioxaboralan-2-yl)-9,9- 119 Chapter 5 bis(4-(2-ethylhexyloxy)-phenyl)fluorene 10 in 57% yield. The synthetic route toward the monomer 9 is illustrated in Scheme 5.1. 1-Hexyloxy-4iodobenzene 1 was synthesized in 91% yield through alkylation of 4iodophenol in 50% KOH aqueous solution. Sonogashira reaction between 1 and trimethylsilylaceylene was performed in the presence of CuI/Pd(PPh 3)2Cl2 to yield ((4-(hexyloxy)phenyl)-ethynyl)trimethylsilane 2 in 95% yield, which was followed by hydrolysis under alkaline conditions to afford 1-ethynyl-4(hexyloxy)benzene 3 in 95% yield. The Sonogashira reaction between 1 and 3 yielded 1,2-bis(4-(hexyloxy)phenyl)ethyne 4 as a white solid in 69% yield, which was oxidized in the presence of KMnO4 to afford 1,2-bis(4(hexyloxy)phenyl)ethane-1,2-dione 5 in 92% dinitrobenzo[1,2,5]thiodiazole yl)benzo[1,2,5]thiadiazole 7, 6, and yield. 4,7-Dibromo-5,6- 5,6-dinitro-4,7-di(thiophen-24,7-bis(5-bromothiophen-2-yl)-5,6- dinitrobenzo[1,2,5]thiadiazole 8 were prepared according to previous reports.[282] 5 and 8 were treated in reflux acetic acid to afford 9 in 51% yield. The chemical structures of the intermediates and PFTTQ were verified by 1H NMR. The correct structure of 1 was confirmed by the appearance of a triplet at 3.91 ppm for ‒OCH2(CH2)4CH3. The strong singlet at 0.29 ppm for 2 is attributed to ‒CCSi(CH3)3, and the integrated area ratio between peaks at 0.29 ppm and 3.94 ppm (‒OCH2(CH2)4CH3) is calculated to be 4.5, which suggests the correct structure of 2. After hydrolysis, the ethynyl proton of 3 is observed at 3.00 ppm, which disappears in the 1H NMR spectrum of 4 due to the successful coupling reaction between 3 and 1. Compared to 4, the aryl protons of 5 are shifted toward downfield due to the presence of dione, which reduces electron density. As compared to the 1H NMR spectra of 9 and 10, the 120 Chapter 5 broaden signals for PFTTQ are another evidence of successful polymerization. GPC results reveal that PFTTQ has a number-average molecular weight (Mn) of 12900. It is found that PFTTQ can be easily soluble in nonpolar organic solvent, such as toluene, tetrahydrofuran, dichloromehtane, etc. 5.3.2 Preparation of PFTTQ NPs Scheme 5.2 Schematic illustration of the fabrication of PFTTQ NPs using DSPE-PEG2000 as the matrix through a precipitation method. PFTTQ NPs were synthesized through a modified precipitation method using DSPE-PEG2000 as the matrix (Scheme 5.2).[173,228] A THF solution containing PFTTQ as well as the matrix of DSPE-PEG2000 was poured into water under sonication. The hydrophobic DSPE segments tend to entangle with PFTTQ chains and the hydrophilic PEG chains extend into aqueous phase under sonication. The NP suspension was obtained after THF evaporation under stirring overnight. The NP suspension was further purified with a 0.2 µm syringe filter to obtain PFTTQ NPs. 121 Chapter 5 Optical Bench Tunable Laser System Fiber Lens Lens Fixed Sync Out Sampler Photodiode R Mode LNA Axicon T/R Mode Ultrasonic Pulser/Receiver RF Out Sync Out Ultrasonic Motor 1 Micro-Meter Ultrasonic Motor 2 Plexiglass Y X Z PC: Control, Data Acquisition, Storage, and Display Probe Water Tank TCP/IP X-Direction Sample Y-Direction Motor Driver Y X Z B-scan images C-scan images Scheme 5.3 Experimental setup of the PAM system. The pulled tubing was filling with the venous samples in the focusing depth. The laser was pulsed with a pulse repetition rate of 10 Hz and coupled by a lens to an optical fiber to illuminate samples. PA waves were detected with a 50-MHz transducer and then through the A/D card to the PC for further data analysis. Figure 5.1 (A) UV-vis absorption spectra and (B) PA imaging as well as the PA intensity of PFTTQ NPs and Au NRs with same mass, the scale bar is 100 µm. (C) The PA intensity of PFTTQ NPs at concentrations from 0.05 mg/mL to 0.5 mg/mL. 122 Chapter 5 Figure 5.1 shows the UV-vis absorption spectrum of PFTTQ NP suspensions in water. The absorption band at 425 nm should result from π‒π* transition of the conjugated backbone, while the broad absorption spectrum from 700 to 850 nm arises from charge transfer between the fluorene (donor) units and the thiadiazolo-quinoxaline (acceptor) units. As we know, Au nanorods (NRs) show high extinction coefficient in NIR range, which have been widely applied to PA imaging with relatively high PA intensities.[266] To evaluate the photophysical and PA signals of PFTTQ NPs, Au NRs with an extinction maximum at ~805 nm have been synthesized according to the seedmediated approach.[280] The absorption coefficient of PFTTQ NPs is calculated to be 3.6 L/g·cm based on mass concentration, which is around 26% of that of Au NRs (13.9 L/g·cm).[283] The PA intensities of PFTTQ NPs and Au NRs under the same mass are evaluated using a non-absorbing polyethylene tube and a 50-MHz dark field confocal PAM system to collect the images (Scheme 5.3). To eliminate the absorbance influence of PFTTQ NPs and Au NRs on their PA generation capacity, their concentrations were adjusted so that they have the same absorbance at 800 nm (Figure 5.1A). Under this condition, PFTTQ NPs show a higher PA intensity as compare to that of Au NRs as shown in Figure 1b. Although the absorption intensity of PFTTQ NPs is only one quarter of the same mass amount of Au NRs, PFTTQ NPs still shows higher PA intensity in comparison with Au NRs as shown in Figure 5.1B. 123 Chapter 5 Figure 5.2 (A) UV-vis absorption spectra and (B) PA imaging as well as the PA intensity of PFTTQ NPs and Au NRs, the scale bar is 100 µm. To eliminate the absorbance influence of PFTTQ NPs and Au NRs on their PA generation capacity, their concentrations are adjusted so that they have the same absorbance at 800 nm (Figure 5.2A). Under this condition, PFTTQ NPs show a higher PA intensity as compare to that of Au NRs as shown in Figure 5.2B. This result further demonstrates that the PFTTQ NPs have stronger capacity to generate PA signal as compare to Au NRs. It is worth noting that almost no NIR fluorescence from PFTTQ NPs are detected upon excitation at 800 nm, illustrating that most of the excited excitons return to the ground state via the non-radiative decay pathway. It is generally believed that large intrinsic extinction coefficient and high non-radiative QY (which is obtained by 1 – QY) are the two determining factors for organic materials to facilitate their PA signal generation.[246,261] The strong PA signals from PFTTQ NPs should result from their large absorption coefficient and high non-radiative QY.[246] Moreover, the PA intensities of PFTTQ NPs increase linearly with NP concentrations in aqueous medium as shown in Figure 5.1C. PFTTQ NPs show an average size of 50 ± 10 nm observed from 124 Chapter 5 FE-TEM. The dark contrast of NPs results from the high electron density of PFTTQ chains (Figure 5.3C1). On the other hand, they show a hydrodynamic average effective diameter of 80 ± 20 nm, which should result from the extension of hydrophilic PEG chains at NP surfaces. 5.3.3 Photostability Investigation of PFTTQ NPs Excellent NP stability in aqueous media is essentially important for biological applications, particularly in long-term studies. As such, the photostability of PFTTQ NPs as well as the conventional PA contrast, Au NRs, were investigated by monitoring their absorbance changes upon continuous laser irradiation at a power density of 15 mJ/cm2 for different time. As shown in Figure 5.3A, the absorbance intensity of PFTTQ NPs decrease around 17% after 10 mins pulse laser irradiation. On the contrary, Au NRs decrease more than 80% of their NIR absorption intensity at the same condition. Figure 5.3B shows that no obvious damage for the absorption spectrum of PFTTQ NPs; whereas the NIR absorption profile of Au NPs greatly decrease and the absorption intensity at ~520 nm increase upon laser irradiation for only 6 mins. Their respective morphology changes before and after laser irradiation were also studied by FE-TEM. As shown in Figure 5.3C, the PFTTQ NPs are kept well in sphere morphologies after laser irradiation. However, Au NRs are degraded and most of Au NRs are melted into Au NPs by pulse laser (Figure 5.3D2). This result illustrates the excellent photostability of PFTTQ NPs as compared to Au NRs, benefiting long-term tracing of the obtained organic contrasts in biomedical studies. In addition, no obvious precipitation from the prepared PFTTQ NP suspensions was observed after being stored at 4 °C for 3 months, demonstrating their excellent colloidal stability. 125 Chapter 5 Figure 5.3 (A) Absorbance intensity evolution of PFTTQ NPs and Au NRs upon continuous pulse laser irradiation with a power of 15 mJ/cm2 for different times, where I0 is the absorbance intensity at 800 nm for the fresh NP suspensions and I is that for NPs after irradiation for different time, respectively. (B) UV-vis absorption spectra of PFTTQ NPs and Au NRs before and after laser irradiation with a power density of 15 mJ/cm2 for 6 mins. FE-TEM images of PFTTQ NPs (C) and Au NRs (D) before (1) and after (2) laser irradiation for 6 mins. All images share the scale bar of 100 nm. 126 Chapter 5 Cell viability (%) 100 80 60 40 20 0 500 200 100 50 NP Concentration (g/mL) Figure 5.4 Metabolic viability of NIH/3T3 fibroblast cells after incubation with PFTTQ NP suspensions at various NP concentrations for 24 h. In addition to good photostability and physical stability, benign biocompatibility is also an important virtue to be considered for materials in biological applications. To evaluate the cytotoxicity of the PFTTQ NPs, the metabolic viability of NIH/3T3 fibroblast cells after incubation with NPs was investigated at different NP concentrations. Figure 5.4 shows the cytotoxicity results of the PFTTQ NPs upon incubation with NP suspensions at 50 to 500 g/mL concentrations for 24 h. The cell viabilities remain ~90% upon incubation with the NP suspension at 500 µg/mL for 24 h, indicating their low cytotoxicity, which is ideal for biological studies. 5.3.4 In Vivo Vasculature Imaging The application of PFTTQ NPs as PA contrast reagents in biological applications was demonstrated in conrtical vasculature imaging in living rat using PA microscopy at a 31 × 62 µm spatial resolution. As shown in Figure 5.5A, the rat was mounted on a stereotaxic head holder, and the skin and 127 Chapter 5 Figure 5.5 (A) Schematic illustration of PA imaging for rat brain vasculature. (B) PA rat cortical vasculature C-scan images before and after 10 mins injection of PFTTQ NPs. (C) The evolution of the integrated PA intensity at the red line spot as a function of time. muscle were cut off from the skull to expose the bregma. The region of interest (indicated by the red line in Figure 5.5B), the superior sinus, was imaged at 800 nm before and after PFTTQ NP injection through retro-orbital. The images before PFTTQ NP injection as well as that after 10 mins administration of NPs are shown in Figure 5.5B. Only weak PA signals are collected from pure brain vessels, which may due to low absorption coefficient from hemoglobin at 800 nm. In contrary, the PA signal of blood vessels is greatly enhanced from circulating PFTTQ NPs upon NPs administration. The PA intensity is improved by ~3-fold after NP injection as compared to that before NP injection. The obvious differentiation of blood vessels and the brain parenchyma demonstrates the strong PA generation capacity of PFTTQ NPs, which should result from the large absorption coefficient and high non- 128 Chapter 5 radiative decay rate of PFTTQ NPs. Moreover, PA imaging of blood vessels remain similar resolution after 30 mins NP injection (Figure 5.5C), which may resulting from the suitable size as well as the PEG surface of the circulating PFTTQ NPs. 5.4 Conclusion In this chapter, we have synthesized a CP as well as CP loaded NPs, PFTTQ NPs, and demonstrated their feasibility as an effective PA imaging probe in brain vasculature imaging. Due to the large conjugation system of PFTTQ chain, the obtained CP NPs show a large absorption coefficient in NIR range (700-850 nm). In addition, PFTTQ NPs shows weak fluorescence, in other word, high non-radiative QY, as a result of their charge transfer characteristic in their backbones. Both the large absorption coefficient and high non-radiative QY benefit the strong PA generation of PFTTQ in NP formulation. As a result, PFTTQ NPs even show higher PA intensity as compared to Au NR under the same absorbance condition. More importantly, PFTTQ NPs exhibit good photostability upon continuously pulse laser irradiation. In addition to their benign biocompatibility, PFTTQ NPs have been successfully applied in brain vasculature imaging with high PA contrast. Further conjugation PFTTQ NPs with specific targeting ligands could further broaden our probes in targeted biological imaging.[267,284,285] Moreover, in view of their strong absorption coefficient and high non-radiative QY, PFTTQ NPs could also be applied in photothermal therapy,[119,125,286,287] which may achieve theranositic functionality in biomedical applications. 129 Chapter 6 CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS 6.1 Conclusions CP NPs are emerging as attractive fluorescent probes in bioimaging due to the large extinction coefficient, strong luminescence signal, excellent photostability and benign biocompatibility. In these respects, CP NPs shows better performance as compared with the conventional organic dyes and QDs. Currently, CP NPs have been mainly fabricated through the emulsion method and the precipitation strategy. Unfortunately, CP NPs prepared by these approaches are mainly built up through weak hydrophobic interaction, resulting in their low structural stability in aqueous. In this thesis, we aimed to improve the performance of CP NPs in stability and fluorescent property as well as explore their bioimaging applications. Firstly, a new strategy has been developed to synthesize CP NPs with numerous surface hydroxyl groups via click chemistry between a CP (PFBT) and hyperbranched polyglycerol (HPG) in miniemulsion. DLS and FE-TEM characterizations indicate that the synthesized CP NPs have spherical shapes with uniform sizes tunable in the range of 40-210 nm simply by adjusting the feed amount of the oil phase or surfactant in the miniemulsion. The obtained CP NPs have good water dispersibility and orange emission with high fluorescence QYs of 23 %. Detailed spectroscopy studies reveal that the CP NPs have shown stable fluorescence against pH change, ionic strength variation, or protein disturbance. In addition, they have good photostability 130 Chapter 6 and low cytotoxicity, which make them an ideal fluorescent moiety for cellular imaging. This study provides important fundamental understanding of crosslinking modification on CP to form CP NPs. Secondly, we developed a one-step synthesis of CP embedded silica NPs with a SiO2@CP@SiO2 structure by combination of a precipitation approach and a modified Stöber method. Four types of CPs have been employed to demonstrate the versatility of the developed strategy, yielding fluorescent silica NPs with emission across the visible spectrum. The FE-TEM investigation reveal that the entanglement between hydrophobic CPs and the aminopropyl groups of 3-aminopropyl triethoxysilane contributes to the successful encapsulation of CPs into silica matrix. The synthesized NPs exhibit excellent physical stability and good photostability. In addition, they have amine groups on surfaces, which benefit further conjugation for biological applications. Through reaction with a peptide (GGHAHFG) that is specific to HER2 receptor, the synthesized NPs have been successfully applied for targeted cellular imaging of HER2-overexpressed SKBR-3 breast cancer cells. Along with its high QY and benign biocompatibility, the developed CP embedded silica NPs have a great potential for applications in biological imaging. Thirdly, taking PFBT as an example, we developed a one-step approach to synthesize PFBT loaded NPs with high fluorescence QY and large two-photon action cross section in aqueous medium through both micelle and silica co-protection strategy. PFBT NPs show a two photon absorption maximum of 814 GM at 810 nm and an emission maximum at 545 nm with a high fluorescence QY of 75%. The fluorescence lifetime investigation reveals 131 Chapter 6 that the high fluorescence QY is mainly due to reduced polymer aggregation and minimized environment effect on CP fluorescence quenching. The synthesized PFBT NPs have shown good colloid stability and photostability as well as benign biocompatibility, which have been further applied to visualize the mouse brain vasculature through intravital two-photon excited brain vascular imaging with high contrast. The developed micelle/silica coprotection strategy should be generally applicable to other CP NPs with improved brightness and stability for various biological applications. In addition to improve the performance of CP NPs in fluorescent imaging, we also explored their application in PA imaging. PA imaging is an emerging technique in biological imaging, which is able to provide high resolution and deep penetration depth. Exogenous contrast agents with strong NIR absorbance are highly desirable for PA imaging. In the fourth project, we synthesized PFTTQ by Suzuki polymerization, which shows strong absorption in the NIR range. Thanks to the processability of PFTTQ, it was further encapsulated into DSPE-PEG2000 matrix to yield PFTTQ loaded DSPEPEG2000 NPs (PFTTQ NPs). At the same mass or with the same absorbance at 800 nm, PFTTQ NPs show stronger PA signal as compared to gold nanorods (Au NRs), one of the most widely used PA agent. In addition, PFTTQ NPs show much higher photostability in comparison with Au NRs upon laser irradiation. The obtained PFTTQ NPs has been successfully applied in rat brain vascular PA imaging. The developed strategies in this thesis efficiently improve the structural and mechanical stability of CP NPs. The detailed strategies are defined as HPG cross-linking, silica coating, silica and micelle co-protection. 132 Chapter 6 In addition, they have been successfully applied in fluorescent biological imaging (e.g. cellular imaging, targeted cellular imaging and vascular imaging) with good performance. Moreover, these strategies could be generally explored to other CPs to improve their fluorescent performance and optimize their biological applications. Furthermore, to broaden the imaging modality of CP NPs, CP with strong NIR absorption and low fluorescence QY has also been synthesized followed by NP formulation. Thanks to the high non-radiative QY, the obtained CP NPs have shown strong PA signal in rat vasculature imaging. From the customized viewpoint, the CP NPs with desired optical properties and specific imaging modalities could be fine-tuned by choosing the suitable CPs. This project thus provides a new avenue to synthesize CP NPs for biological applications. 6.2 Recommendations Based on the obtained results in this thesis, some future investigations are recommended to further optimize and broaden the application of CP NPs. Stable CP NPs have been successfully fabricated and applied in biological imaging in this thesis. However, the current application of CP NPs is limited in in vitro or in vivo subcutaneous model (tumour, blood vessel). This is due to the intrinsic drawback of fluorescence imaging, in which both absorption and emission lights could be largely absorbed and scattered by biological tissues. One approach to overcome this limitation is to fabricate multifunctional nanocomposites, which could integrate fluorescence imaging with other modalities to provide multiple signals. As well known, magnetic NP or gadolinium (Gd) based magnetic resonance imaging (MRI) can provide high tissue penetration depth but with low resolution in microscopic 133 Chapter 6 targets.[7,66] On the other hand, as mentioned above, CP NP based fluorescence imaging could offer good resolution in in vitro subjects but poor penetration depth. Integration CP NPs with magnetic NPs or Gd based agents can simultaneously combine the advantages of both fluorescence imaging and MRI. For example, the cross-linked CP NPs (Chapter 2) have many free hydroxyl groups at surface, which could further react with diethylenetriaminepentaacetic acid for chelating with Gd to afford CP-Gd nanocomposites. In addition, magnetic NPs could be firstly covered with silica shell and further encapsulated CP dots at silica surface utilizing SiO2-CP-SiO2 formulation (Chapter 3) to build Magnetic NP-SiO2-CP-SiO2 nanocomposites for multifunctional imaging. These multifunctional probes should facilitate both in vitro detection and deep in vivo cancer diagnosis. Another approach to conquer the restriction of low penetration depth of CP NP based optical imaging is to explore new imaging modalities by synthesizing CPs with unique properties. As an emerging non-invasive imaging modality, PA imaging has attracted great research interest in view of the deeper penetration depth in in vivo condition. As low absorption coefficient of most biological targets in the NIR range, only weak PA signal could be detected.[253,265] As such, exogenous contrast agents with high PA generation capacity and good selectivity to targets are highly desirable. In view of large absorption coefficient and excellent stability, CP NPs have great potential to be applied as PA contrast agents. Our preliminary result (Chapter 5) shows that CP NP based exogenous contrasts could provide high PA signal in in vivo condition. However, the relationship between CP structure and PA signal generation needs to be further investigated. In addition to the 134 Chapter 6 development of CP based PA contrast agents, designing CP NPs with both fluorescence and PA signals may provide an advantageous nanoplatform for dual-modality imaging. Since fluorescence signal could provide high resolution and PA signal could endow deep penetration depth, complementary information from biological system could be obtained utilizing the designed multifunctional nanoplatform. The third important aspect deserved to be investigated is the toxicity and clearance of CP NPs in in vivo condition. So far, CP NPs have shown good biocompatibility in both in vitro and in vivo conditions based the fluorescence imaging modality. Although no obvious toxicity for CP NPs has been observed on H&E staining in many reports, their long term toxicities in in vivo condition have been less studied.[288] In addition, the clearance time of CP NPs is hard to obtained only based on the fluorescence imaging. The PA signal from CP or integration CP NPs with other functional materials may provide new opportunities to study the clearance of CP NPs. For example, integration CP NPs with Gd could help to determine the residue amount of Gd in organs based on the ICP technique. This will no doubt help to determine the fate of CP based materials in in vivo condition. Finally, synthesizing CP based theranostic NPs is also an interesting topic.[123,258,263] The materials with high extinction coefficient and low fluorescence QY could produce heat upon laser irradiation since most excited electrons return to ground state through non-radiative decay pathway. Several reports have shown that CP NPs could be utilized in PTT.[121,124] On the other hand, the similar materials with large absorbance and low QY have been utilized in PA imaging.[12] As such, there is a great potential to achieve both 135 Chapter 6 imaging and therapeutic function with the same CP NPs. For example, PFTTQ NPs have shown high PA intensities in in vivo condition. In addition, they could also produce heat upon NIR laser irradiation. Conjugation of CP NPs with specific targeting ligands could endow PFTTQ NPs with good targeted capacity to specific cancer cell or tumor. The PA signal may help to track the location of PFTTQ NPs. When NPs arrived at tumor, therapy treatment would be conducted to kill cancer cell or tumor with the help of PA signal. 136 References References (1) Weissleder, R.; Pittet, M. J., Nature 2008, 452, 580. (2) Gusnard, D. A.; Raichle, M. E., Nat. Rev. Neurosci. 2001, 2, 685. (3) Massoud, T. F.; Gambhir, S. S., Genes Dev. 2003, 17, 545. 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Rep. 2013, 3, 1150. 152 [...]... mJ/cm2 for different times, where I0 is the absorbance intensity at 800 nm for the fresh NP suspensions and I is that for NPs after irradiation for different time, respectively (B) UV-vis absorption spectra of PFTTQ NPs and Au NRs before and after laser irradiation with a power density of 15 mJ/cm2 for 6 mins FE-TEM images of PFTTQ NPs (C) and Au NRs (D) before (1) and after (2) laser irradiation for. .. collected for (A) PFBTF127-SiO2 NPs, (B) PFBT-DSPE NPs and (C) QD655 Note the different binning and scales for A, B and C, λex = 488 nm for all samples 99 xi Figure 4.5 (A) PL intensity evolution of PFBT-F127-SiO2 NPs upon incubation with 1 × PBS at 37 °C for different times, where I0 is the fluorescence intensity at 545 nm for the fresh NP suspension and I is that for NPs after incubation for different... Jablonski diagram IC stands for internal conversion, ISC stands for intersystemm crossing, S0 stands for ground singlet state, S1 stands for first excited singlet state, Sn stands for higher excited singlet state, T1 stands for excited triplet state The photophysical properties of CPs (e.g absorption and emission) are highly dependent on their chemical structures as well as conformations.[25,26,50,51]... practical applications in biological and clinical studies.[15] The inherent limitations of conventional fluorescent materials are incentives to explore alternative fluorescent probes with improved performance More recently, fluorescent conjugated polymer (CP) based materials have attracted great research interest in biological applications. [25-29] CPs are macromolecules with delocalized π -conjugated. .. devices and luminescence 4 Scheme 1.2 Schematic illustration of Jablonski diagram IC stands for internal conversion, ISC stands for intersystemm crossing, S0 stands for ground singlet state, S1 stands for first excited singlet state, Sn stands for higher excited singlet state, T1 stands for excited triplet state 5 Scheme 1.3 Schematic illustration of CP NPs prepared with emulsion method... states or morphologies for different applications 1.3 Synthesis of CP NPs The pioneer work of preparation of CP NPs can be traced back to 1980s, in which CP NPs have been synthesized by direct polymerization The first generation of CP NPs include polyacetylene,[52] polyaniline[53] and polypyrrole.[54] They have been mainly utilized in device applications Although the direct polymerization method has... contrast, the postpolymerization approach mainly utilizes commercially available polymers or the synthesized CPs to fabrication of CP NPs, which is facile and convenient to be applied to various types of CPs Since this thesis is mainly focused on bioimaging applications of CP NPs, the discussion is limited to CP NPs with water solubility or dispersibility It is noted to worth that there is another formulation... earliest examples of targeted cellular imaging for CP NPs has been demonstrated with folic acid conjugated PPE polymer chains.[103] As the side chains of CPs have numerous ionic carboxyl groups, CP NPs could be well dispersed in water In addition, the carboxyl groups have been further conjugated with folic acid molecules via an amide coupling reaction The folic acid conjugated CPs could enter KB cells more... waves were detected with a 50-MHz transducer and then through the A/D card to the PC for further data analysis 122 xiii LIST OF SYMBOLS APTES 3-aminopropyl triethoxysilane Au NRs gold nanorods BSA bovine serum albumin CLSM confocal laser-scanning microscopy CMC critical micelle concentration CP conjugated polymer CPEs conjugated polyelectrolytes CTAB cetyltrimethylammonium bromide DAPI 4',6-diamidino-2-phenylindole... without and with radiation for different time, respectively 53 Figure 2.7 (A) Cell viability of MCF-7 cells after incubation with CP−HPG-1 at different concentrations for 24 and 48 h, respectively Confocal fluorescence image of MCF-7 cells upon incubation (B) with and (D) without CP−HPG-1 ([RU] = 1 μM) for 2 h (C) 3-D confocal image of cell line MCF-7 incubated with CP-HPG-1 for 2 h 55 Figure