Nanoencapsulation of quantum dot nanoparticles in biological labeling

SYNTHESIS OF QUANTUM DOT-POLYMER NANOPARTICLES FOR BIOLOGICAL LABELING HUANG NING (B.Eng) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements The successful completion of this thesis would not have been possible without the help of a handful of people whom I would like to thank here First of all, I have been indebted in many ways to my thesis supervisor, Dr Zhang Yong, whose patience and kindness have been invaluable to me I would also like to thank Dr Yang Xiaotun, research fellow of our lab This project has benefited tremendously from their diligent guidance and sound advice I am also grateful to the many laboratory officers whose assistance has been indispensable; in particular, Miss Satinderpal Kaur of the Biochemistry Lab and Miss Xu Xiaojing of the TEM My fun-loving friends in the group have also made my stint in the lab a truly enjoyable and memorable one Lastly, I would like to acknowledge the immense support as well as strong encouragement of my family and close friends that accompanied me along the way ii Summary Quantum Dots (QDs) have attracted considerable interest in luminescence tagging due to their unique optical and electronic properties The ideal optical properties of QDs offer the possibility of using them as fluorescent probes in biological staining and diagnostics But some problems associated with QDs, for example, poor water solubility, poor biocompatibility and chemical stability in physiological media, limit their use in biomedical applications Though some efforts have been made to prepare QD-tagged microbeads, the sizes of the beads are above 100 nm, which make them not suitable for labeling of subcellular components In this work, fluorescent QDs were incorporated into polystyrene nanoparticles using emulsion polymerization method and particle sizes were controlled The nanoparticles have carboxyl groups on the surfaces that can be used for further attachment of biomolecules After surface modification with folic acid, the intracellular delivery of the nanoparticles into NIH3T3 and HT-29 cells was investigated using confocal microscope The QD encoded nanoparticles are suitable for staining or labeling of subcellular components or intracellular measurements due to their very small size Keywords: Quantum dots; Nanoparticles; Polystyrene; Surface modification iii Table of Contents Page Acknowledgements ii Summary iii List of figure vii Chapter Introduction 1.1 Introduction 1.2 Aims and Objectives Chapter Literature Review 2.1 Introduction to Fluorescent Labeling 2.2 Fluorescent Materials 2.2.1 Flurescent Proteins 2.2.2 Organic Fluorophores 2.2.3 Lanthanide Chelates 12 2.2.4 Nanoparticles 15 2.3 2.2.4.1 Optically Active Metal Nanoparticels 15 2.2.4.2 Quantum Dots 16 2.2.4.3 Latex Nanospheres 22 Reference 23 iv Chapter Materials and Methods 32 3.1 Chemicals 32 3.2 Synthesis of CdSe 33 3.3 Synthesis of CdSe/ZnS 34 3.4 Synthesis of Polystyrene Encapsulated CdSe/ZnS Nanoparticles 35 3.4.1 35 3.4.2 Synthesis of Polystyrene Encapsulated CdSe/ZnS with COOH Group on the Surface Surface Modification with Folic Acid 36 3.5 Cell Culture 37 3.6 Characterization 38 3.6.1 Fluorescent Microscopy 38 3.6.2 Confocal Laser Scanning Microscopy 38 3.6.3 Transmission Electron Microscopy (TEM) 38 3.6.3 Fourier Transform Infrared (FT-IR) 39 3.6.4 UV Spectrometry 39 3.6.5 Fluorescent Spectrometry 39 3.7 Reference 41 Chapter Experimental Results and Discussions 42 4.1 Synthesis of CdSe 42 4.2 Synthesis of CdSe/ZnS 47 4.3 Synthesis of Polystyrene Particles 50 4.4 Surface Modification of the Nanoparticles with Folic Acid 55 4.5 Intercellular Delivery of Nanoparticles 58 v 4.6 Reference 63 Chapter Conclusions 69 Appendix 71 vi List of Figures Page Fig 2.1 Wild type GFP chromophore, consisting of a cyclized tripeptide made of Ser65, Tyr66, and Gly67 Fig 2.2 The structure of dicarbocyanines dyes Fig 2.3 Structure of thiazole orange (TO) 10 Fig 2.4 Structure of oxazole yellow (YO) 10 Fig 2.5 Structure of Cy bis functional dye 11 Fig 2.6 Structure of Cy monofunctional dye 11 Fig 2.7 Structure of representative chelates 14 Fig 3.1 The schematic of the synthesis of CdSe 33 Fig 3.2 Diagram of synthesis of polystyrene/quantum dot nanocomposites conjugated with carboxyl 35 Fig 4.1 TEM image of CdSe 43 Fig 4.2 The absorbance spectrum of CdSe 43 Fig 4.3 The emission spectrum of CdSe 44 Fig 4.4 The compared emission spectra of samples collected at different reaction time 45 Fig 4.5 Comparison of emission spectra of the quantum dots at different coating stage 48 Fig 4.6 TEM image of CdSe/ZnS 49 Fig 4.7 TEM images of PS@CdSe/ZnS micro/nano particles of different sizes 52 Fig 4.8 The optical (a) and fluorescence (b) images of PS@CdSe/ZnS micro-sized particles 52 vii Fig 4.9 TEM image of nano-sized PS@CdSe/ZnS 53 Fig 4.10 FT-IR spectra of (a) PS@CdSe/ZnS with COOH on the surface and (b) PS@CdSe/ZnS without surface modification 54 Fig 4.11 FTIR spectra of (a) PS@CdSe/ZnS nanoparticles, (b) pure folic acid and (c) folic acid modified PS@CdSe/ZnS nanoparticles 57 Fig 4.12 Confocal images of NIH-3T3 cells after cultured with (a) unmodified and (b) folic acid modified nanoparticles and(c, d) their corresponding bright field images 58 Fig 4.13 Confocal images of HT-29 cells after cultured with folic acid modified nanoparticles for (b) hours and (c) hours, and(a) the bright field images 59 Fig 4.14 Bright field and confocal images of HT-29 cells after cultured with folic acid modified nanoparticles The bright field image was taken after culture for 30 mins and the confocal images were taken every 30 mins 61 Fig 4.15 Bright field and confocal images of HT-29 cells after cultured with unmodified nanoparticles The bright field image was taken after culture for 30 mins and the confocal images were taken every 30 mins 61 viii Chapter Introduction Chapter Introduction 1.1 Introduction Semiconductor quantum dots (QDs) have good potential for use as fluorescent probes in biological staining and diagnostics Compared with conventional organic fluorophores, QDs have a strong fluorescence emission and narrow, symmetric emission spectrum, and are photochemically stable QDs also exhibit a wide range of size-tunable colors and a series of different-colored dots can be activated using a single laser The ideal optical properties of QDs offer the possibility of using them to tag biomolecules in ultra-sensitive biological detection based on optical coding technology Some techniques have been developed to incorporate QDs into polymer beads, to solve the problems relating to QDs’ surface chemistry such as water solubility, biocompatibility, chemical stability in physiological media, etc., or to pack different combinations of QDs and produce QD encoded polymer beads; for example, create QD bar codes and the use of six colors and 10 intensity levels can theoretically encode one million biomolecules However, the QD encoded polymer beads that have been reported so far are above 100 nm, therefore they are very useful for multiplexed bioassays, but not suitable for staining or labeling of subcellular components or intracellular measurements due to the relatively big size of the beads In addition, it is difficult to produce uniform beads and to control the number of QDs incorporated These drawbacks severely limit their applications in biological labeling Chapter Introduction In this work, luminescent CdSe/ZnS QDs were incorporated into polystyrene (PS) nanoparticles grafted with carboxyl groups using emulsion polymerization method and separation of nanoscale QD encoded PS particles (30 nm) were performed through centrifugation at high speed in viscous solution The nanoparticles were further surface modified with folic acid and their intracellular delivery into NIH3T3 and HT-29 cell lines was investigated using confocal laser scanning microscope The longevity of QDs allows us to track the intracellular delivery of the nanoparticles over a certain time period Chapter Results and Discussion folic acid modified nanoparticles [75] Moreover, after surface modification of the nanoparticles with folic acid, the absorption peak corresponding to C-N stretching vibration between 1000 cm-1 and 1300 cm-1 becomes prominent, indicating the formation of amide bond between folic acid and carboxyl groups of the nanoparticles Figure 4.11 FTIR spectra of (a) PS@CdSe/ZnS nanoparticles, (b) pure folic acid and (c) folic acid modified PS@CdSe/ZnS nanoparticles Both folic acid modified and unmodified particles are water soluble and stable in solution After several months, the particle solution (in PBS) remains clear and the fluorescence can still be observed under fluorescence microscope 57 Chapter Results and Discussion 4.5 Intercellular Delivery of Nanoparticles The intracellular uptake of the nanoparticles to both NIH-3T3 and HT-29 cells was studied [76, 77] The confocal images of NIH-3T3 cells cultivated with both unmodified and folic acid modified nanoparticles are given in figure 4.12 For unmodified nanoparticles, fluorescence was observed in the cells but not in the nuclei of most cells, indicating that the nanoparticles could not go into the nuclei easily After surface modification of the nanoparticles with folic acid, fluorescence appeared in the nuclei of some cells This suggested that the intracellular uptake of the nanoparticles may be affected by the surface modification, and the folic acid attached may help the nanoparticles to go into the cell nuclei, because naturally folic acid Figure 4.12 Confocal images of NIH-3T3 cells after cultured with (a) unmodified and (b) folic acid modified nanoparticles and(c, d) their corresponding bright field images 58 Chapter Results and Discussion participates in the synthesis of DNA and RNA Confocal images of HT-29 cells after culture for different time periods with the surface modified nanoparticles are shown in figure 4.13 The nanoparticles were only attached to the surface of the cells after hour of culture, while they spread over the cytoplasm of cells after hours of culture Figure 4.13 Confocal images of HT-29 cells after cultured with folic acid modified nanoparticles for (b) hours and (c) hours, and(a) the bright field images The above confocal results suggested that the QD encoded nanoparticles can be used to study the intracellular uptake of nanoparticles after attaching suitable biomolecules to the surface However, the same results can be obtained using conventional fluorescent dyes An important advantage of QDs over organic dyes is their longevity, which makes it possible to track the intracellular trafficking of 59 Chapter Results and Discussion nanoparticles continuously Therefore the experiments were designed to record the fluorescence images of the same cells over a time period of hours at 30 minutes intervals, using a confocal microscope The confocal images of HT-29 cells growing with folic acid modified and unmodified nanoparticles were given in figure 4.14 and figure 4.15 respectively For folic acid modified nanoparticles, they first attached to the folate receptors expressed on the cell membrane [76] It was demonstrated in the image of cells after 0.5 hour of culture with the nanoparticles, as fluorescence was only observed on the cell membrane After hour, more fluorescence was present on the cell membrane and some fluorescence spots were seen in the cytoplasm, indicating the migration of the nanoparticles into cells For the cells growing with the nanoparticles for more than 1.5 hours, more fluorescence was observed in the cells, suggesting that more nanoparticles were detached from the cell membrane and spread over the cytoplasm Furthermore, during the 3-hour time period, fluorescence was seen all the time on the cell membrane After the nanoparticles on the cell membrane moved into the cells, the binding sites on the cell membrane were filled up with some other nanoparticles from the media This further confirmed that the intracellular uptake of the folic acid modified nanoparticles is based on receptor-mediated endocytosis However, after cells were growing with unmodified nanoparticles for 0.5 hour, fluorescence was observed in the cells and not much change was seen as culture time was prolonged This could be explained as that, the uptake of the unmodified nanoparticles into HT29 cells was based on non-specific endocytosis because no specific ligands were 60 Chapter Results and Discussion present on the surface Although similar results for intracellular uptake of nanoparticles have been reported using conventional dyes as fluorescent labels, it is only possible to use QDs that have long fluorescence lifetime, to visualize the intracellular trafficking of nanoparticles via imaging techniques Figure 4.14 Bright field and confocal images of HT-29 cells after cultured with folic acid modified nanoparticles The bright field image was taken after culture for 30 mins and the confocal images were taken every 30 mins Figure 4.15 Bright field and confocal images of HT-29 cells after cultured with unmodified nanoparticles The bright field image was taken after culture for 30 mins and the confocal images were taken every 30 mins 61 Chapter Results and Discussion From the above experiments, we could make the conclusion that the PS@CdSe/ZnS nanoparticles are suitable for intracellular study because of their very small sizes Their long-term cell biocompatibility will be studied in future Compared with single QDs, the QD encoded nanoparticles have more advantages, for examples, multi-colored QDs can be packed into the nanoparticles for multiplexed analysis, the nanoparticles are more chemically stable and can be made biocompatible, and their sizes can be adjusted to that of polymer nanoparticles for drug/gene delivery and the same surface modification protocols can be used to control their intracellular delivery 62 Chapter Results and 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synthesized by one-pot reaction using CdO as precursor These QDs emited green fluorescent light at 570 nm Luminescent CdSe/ZnS QDs were incorporated into polystyrene (PS) particles grafted with carboxyl groups using emulsion polymerization method By studying the micro-sized PS particles under fluorescent microscopy, strong green fluorescent light was observed with a thin film of PS encapsulating the QDs Separation of nanoscale QD encoded PS particles were performed through centrifugation at high speed in viscous solution TEM image gave us a clear image of the nano-sized PS particles which were about 30 to 40 nm These nanoparticles were much smaller than that reported on the previous similar studies which are so far above 100 nm These small nanoparticles made it possible to be used in intracellular study The nanoparticles were further surface-modified with folic acid and their intracellular delivery into NIH-3T3 and HT-29 cell lines was investigated using confocal microscope Since folic acid receptor is expressed on most of the cancer cells, the nanoparticles modified with folic acid on their surface may specifically target to the cancer cells Furthermore, the longevity of QDs allows us to track the intracellular delivery of the nanoparticles over a certain time period 69 Chapter Conclusion From the confocal images, we concluded that surface modification with folic acid helped the nanoparticles go further into the nuclei of cell Their long life-time against photobleaching also gave us the privilege to track the intracellular uptake of these nanoparticles By using both modified and unmodified luminescent nanoparticles, we did observe better for the modified nanoparticles as compared with the unmodified ones From this work, we believe that the QD encoded nanoparticles are suitable for staining or labeling of subcellular components or intracellular measurements 70 Appendix Appendix Publication list “Intracellular uptake of CdSe-ZnS/Polystyrene nanobeads” Journal of Biomedical Materials Research: Part B (In press) “Encapsulation of luminescent quantum nanodots in polystyrene nanocapsules by microemulsion polymerization” Journal of Metastable and Nanocrystalline Materials 23 (2005) 19 “Preparation of quantum dot based fluorescent labels” Nanotech2004, Singapore “Synthesis and luminescent properties of biocompatible silica-coated polystyrenequantum dots capsules” 7th World Biomaterials Congress, Sydney, Australia “Synthesis of polystyrene/cdse-zns nanocomposites modified with PLL-PEI-PEGFA” 1st Nano-Engineering and Nano-Science Congress, Singapore “Use of Quantum Dot Nanocomposites as Fluorescent Labels” Fourth Asian International Symposium on Biomaterials (AISB4), Japan 71 [...]... by gene transfer into cells The resulting chimera often remains parent-protein targeting and function when expressed in cells [14] The fluorescent proteins have been used as tools in numerous applications, including as markers to track and quantify individual or multiple protein species [16], as probes to monitor protein-protein interactions [14, 17, 18], and as biosensors to describe biological events... instance chlorobenzene, in the conjugating chain of the fluorophore 10 Chapter 2 Literature Review Polymethine dyes are highly fluorescent and they absorb and emit light mostly in the visible region of the optical spectrum, as a function of the chain length and of the terminating moieties It is known that, at first, elongation of the polymethine chain increases the fluorescence quantum yields, but, as... Proteins The most popular member of fluorescent proteins family is Green fluorescent protein (GFP) which was originally isolated from the light-emitting organ of the jellyfish Aequorea Victoria by Shimomura et al in 1962 [11] as a companion protein to aequorin, the famous chemiluminescent protein from Aequorea jellyfish In a footnote to their account of aequorin purification, it is said that “a protein... decrease of the efficiency [44] Polymethine dyes like Cy3 and Cy5 have been widely and routinely used in the labeling of biological compounds such as antibodies, nucleic acids, lipids and other amino groups-containing materials [45, 46] and a popular choice of fluorescent probes in microarray technology [47] The Cy 3 dye is an orange fluorescing cyanine that produces a signal easily detected using a fluorescein... and enter “off” states for periods of time (blinking) [94, 110, 111] Despite their potential and their success so far in biological applications, quantum dots also have limitations associated with their use Owing to their size and chemical nature, quantum dots cannot diffuse through the cell membrane To use QDs for labeling and imaging cytoplasmic proteins, the QDs must be delivered by invasive approaches... surface of the nanoparticles This capping leads to a stabilization and prevents uncontrolled growth and aggregation of the nanoparticles In the case of a labile capping layer such as citrate, biomolecules can be linked directly with the nanoparticles The nanoparticles are usually highly sensitive which makes single-molecule detection (SMD) possible Three types of nanoparticles are potentially useful as singlemolecule... salicylic acid and its derivatives in biological systems, and in fluorimetric immunoassay [51-53] On the other hand, they are a kind of potential luminescent materials for further application There has been a growing interest in the study of the luminescence property of such chelates The specific physical and chemical properties of lanthanides which make them so useful in studies of biochemical systems are... replaced by the nanoparticles [118-120] Medcalf et al have developed an immunoturbidimetric assay for urine albumin and indicator of kidney problems [118] Agglutination in the presence of urine albumin was detected by measuring the change in absorbance caused by light scattering at 340 nm [118] In 2000, Nie and his colleagues used 20 nm fluorescent latex particles that are conjugated to proteins through... application of GFP was to detect gene expression in vivo [15] The most successful application is using GFP as a genetic fusion partner to host proteins By genetic engineering, GFP can be fused as a tag to the protein of interest, often without altering the function of the protein As GFP is spontaneously fluorescent, chimeric GFP fusions give the great advantages that they can be expressed in situ by... potentially useful as singlemolecule probes, in particular, latex nanospheres, optically active metal nanoparticles and luminescent quantum dots Using nanoparticles as probes in bioanalysis offers several potential advantages First, suspensions of nanoparticles do not appreciably scatter light Second, the low background results in low detection limits In addition, nanoparticles form more stable suspensions ... assay for urine albumin and indicator of kidney problems [118] Agglutination in the presence of urine albumin was detected by measuring the change in absorbance caused by light scattering at 340... routinely used in the labeling of biological compounds such as antibodies, nucleic acids, lipids and other amino groups-containing materials [45, 46] and a popular choice of fluorescent probes in. .. the number of QDs incorporated These drawbacks severely limit their applications in biological labeling Chapter Introduction In this work, luminescent CdSe/ZnS QDs were incorporated into polystyrene