In situ forming supramolecules and chitosan based hydrogels for regenerative medicine

A dissertation for the degree of doctor of philosophy Stimuli responsive PEGylated nano-assemblies for cancertargeted drug delivery Department of Molecular Science and Technology The Graduate School of Ajou University Dai Hai Nguyen Stimuli responsive PEGylated nano-assemblies for cancertargeted drug delivery Supervisor: Professor Ki Dong Park A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy June 2013 Department of Molecular Science and Technology The Graduate School of Ajou University Dai Hai Nguyen Acknowledgement I wish to express in this part my gratitude to the scientists, technicians and other people who were directly and indirectly involved in this work, without the help of whom the findings of this thesis surely could not have been done First and foremost, I would like to extend immeasurable gratitude to Professor Ki Dong Park, for giving me the opportunity to my PhD thesis under his supervision I greatly appreciated his supervision for teaching, advising and supporting me throughout my work I am very grateful for his extreme patience and encouragement during the most stressful time when my results were not good He is a respectable mentor who has kindly supported me in the name of family It was an honor to work under his supervisor I am grateful to my thesis committee members, Professor Sung-Hwa Yoon, Professor Won-Hee Suh at Ajou University, Professor Ji Hoon Jeong at Sungkyunkwan University, Dr In Kwon Jung at Genoss Company for their numerous suggestions and helpful advice This is a good opportunity to express my gratitude to Professors at Ajou University whose teaching and advice helped me to complete my PhD coursework I would especially like to thank Dr Yoon Ki Joung who has supported for me for about three years He kindly and friendly guided me from laboratory studies to routine life in Korea I also have deep gratitude towards Dr Jin Woo Bae for being a great mentor His scientific comments are always useful in doing experiments, preparing presentation, and writing a scientific paper I would like to thank my Vietnamese Professors Thi Phuong Thoa Nguyen, Thi Kieu Xuan Huynh, and Huu Khanh Hung Nguyen for giving this opportunity to me, who taught me fundamental knowledge of chemistry at University of Science-HCMC I especially appreciate all supports of my past and current members in Biomaterial and Tissue Engineering Laboratory: Dr Kyoung Soo Jee, Dr Jin Woo Bae, Dr Dong Hyun Go, Dr Jung Seok Lee, Dr Kyung Min Park, Dr Se Jin Son, Dr Ngoc Quyen Tran, Dr Eugene Lih, Jong Hoon Choi, Yeo Jin Jun, In Kyu Hwang, Bae Young Kim, Ji Ho Heo, Seung Soo You, Ki Seong Ko, Ji Hye Oh, Seung Mee Hyun, Dong Hwan Oh, Joo Young Son, Yun Ki Lee, Ji Ho Kim, Min Yong Eom, Thi Thai Thanh Hoang, Thi Phuong Le I hope all members in BT Lab will obtain the outstanding achievement in your dream and get the happiness in their life I appreciate all help of my Vietnamese best friends in Korea, Minh Dung Truong, Van Thinh Nguyen, Dinh Chuong Pham, Ngoc Hoi Nguyen, Thanh Quy Nguyen, Hung Cuong Dinh, Thi Hiep Nguyen, Chan Khon Huynh, who helped in several experiments such as XRD, AFM, DLS, Confocal, FACS, cell culture, and animal studies Without them this thesis surely would not have been so multifaceted and prolific I also would like to be thankful to Korean friends in School of Engineering, Medicine School for your help and support me during my stay here Good luck to all of them Korean life could be some times stressful and tough, with all the competitiveness and perfectionism Luckily, I have had extensive care, support, and help from my family and friends, who shared with me many wonderful and unforgettable moments throughout my time here I would like to devote this thesis to them with my sincere gratitude I would like to thank many of my best friends, Hoang Duy Nguyen, Minh Triet Thieu, Hoang Chuong Nguyen, Nhat Nguyen Nguyen, Xuan Huong Ho… With them I shared the first journey to Korea, as well as the sadness of leaving our lovely home and country All this would not be possible without my loving immediate family For good or for bad, they are the ones who always stand behind me, and let me know that I am not alone Finally, deeply from my heart, I would like to thank my parents who believe and support me at all time My best regards to all, Dai Hai Nguyen Abstract Cancer is one of the leading causes of death worldwide and chemotherapy is a major therapeutic approach for the treatment which may be used alone or combined with other forms of therapy However, conventional chemotherapy has the potential to harm healthy cells in addition to tumor cells Using targeted nanoparticles to deliver chemotherapeutic agents in cancer therapy offers many advantages to improve drug delivery and to overcome many problems associated with conventional chemotherapy This work covers the general areas of responsive nanocarriers and encompassed methods of fabricating nanocarrier-based drug delivery systems for controlled and targeted therapeutic application Chapter provides general information of cancer and cancer treatment strategies The recently cancer treatment based on nanocarrier were introduced In addition, the special features as well as requirements of nanoparticles for targeted drug delivery were presented This chapter describes overall objectives of this study with the current status of stimuli-responsive self-assembled nanocarriers for cancer chemotherapy In chapter 2, self-assembled nanogels based on reducible heparin-Pluronic copolymer was developed for intracellular protein delivery Heparin was conjugated with cystamine and the terminal hydroxyl groups of Pluronic were activated with the VS group, followed by coupling of VS groups of Pluronic with cystamine of heparin The chemical structure, heparin content and VS group content of the resulting product were determined by 1H i NMR, FT-IR, toluidine blue assay and Ellman's method The HP conjugate showed a critical micelle concentration of approximately 129.35 mg L−1, a spherical shape and the mean diameter of 115.7 nm, which were measured by AFM and DLS The release test demonstrated that HP nanogels were rapidly degraded when treated with glutathione Cytotoxicity results showed a higher viability of drug-free HP nanogel than that of drugloaded one Cyclo(Arg–Gly–Asp–D-Phe–Cys) (cRGDfC) peptide was efficiently conjugated to VS groups of HP nanogels and exhibited higher cellular uptake than unmodified nanogels In chapter 3, stimuli–responsive Pluronic micelles is developed for targeting cancer chemotherapy In particularly, the role of crosslinking disulfide bond and hydrazone bond in arrangement of environmental stimuli including redox and pH were discussed Specifically, acrylic acid was grafted onto PPO blocks of Pluronic by dispersion/emulsion polymerization and used to introduce thiol groups as well as hydrazine groups DOX was conjugated to the hydrazone groups to achieve the pHtriggered release The micelles were crosslinked by the formation of disulfide bonds due to the presence of thiol groups on the polymer backbones The physico-chemical properties of the micelles were characterized In vitro release studies were performed to investigate pH-dependent release of DOX from the Pluronic micelles FA was conjugated to the Pluronic polymer for targeting cancer cell FA conjugated micelles were compared with the micelles without FA using confocal laser scanning microscopy (CLSM) and flow cytometry The Pluronic micelles functionalized with FA targeting ligand on the surface showed the enhanced cellular uptake In chapter 4, self-assembled magnetic nanoparticles ii (SAMNs) were fabricated from β-cyclodextrins functionalized superparamagnetic iron oxide (SPIO@CD), paclitaxel (PTX), adamantylamine-poly(ethylene glycol)-vinyl sulfone (ADA-PEG-VS), and c(RGDfC) peptide for integrated cancer cell-targeted drug delivery In this approach, PTX and ADA-PEG-VS enabled the host-guest inclusion with SPIO@CD to form PEG-ADA:SPIO@CD:PTX SAMNs Furthermore, cyclo(Arg-GlyAsp-d-Phe-Cys) (c(RGDfC)) peptide, a targeting ligand, could conjugate onto the VS groups of the PEG arms of SAMNs The architecture of SAMNs were characterized FTIR, TEM, and thermo gravimetric analysis (TGA), which confirmed that PEG, CD have been effectively functionalized on the surface of SPIO nanoparticles SAMNs were enabling to be controlled over the sizes, surface chemistry, payloads of supramolecular nanoparticle vector The sizes, drug entrapment efficiency (DEE), drug loading efficiency (DLE), and SIPO encapsulation of SAMNs could turn by changing its components In vitro PTX release profile from SAMNs was highly ADA response Cumulative releases of PTX from SAMNs were 44.1% and 9.6% with and without ADA treatment after 120 h Most importantly, the analyses of vibration sample magnetometer (VSM) verified that the magnetic property of SAMNs was increased under the external magnetic field c(RGDfC)-conjugated SPIO nanocarriers exhibited a higher level of cellular uptake than unmodified ones in vitro according to flow cytometry and confocal laser scanning microscopy (CLSM) iii Table of Contents Abstract i Table of Contents iv List of Figures viii List of Tables xv Chapter General introduction 1 Cancer and strategy treatment .2 Nanocarrier strategies in cancer chemotherapy Self-assembled nanocarrier for drug delivery .6 PEGylated nanocarriers for systemic deliver Targeted drug delivery systems for cancer therapy 13 5.1 Passive targeting strategies and recent developments 15 5.2 Active targeting strategies and stimuli-triggered ligand presentation 16 Stimuli-response for controlled drug delivery 18 6.1 Concepts for designing stimuli-responsive nanoparticles 18 6.2 Previous studies of stimuli-response for controlled drug delivery 26 Overall objectives .28 References 30 iv Chapter Self-assembled nanogels based on reducible heparin-Pluronic copolymer for targeted protein delivery .35 Introduction 36 Materials and methods 40 2.1 Materials 40 2.2 Synthesis of copolymers and preparation of drug loaded nanogels 40 2.3 Polymer characterizations 44 2.4 In vitro release test .47 2.5 Cytotoxicity assay 47 2.6 Intracellular uptake study 47 2.7 Statistical analysis .48 Results and Discussion .50 3.1 Characterization of polymers and nanogels .50 3.2 CMC and size distribution of nanogels 51 3.3 In vitro release profiles of RNase A and heparin .55 3.4 Cytotoxicity of RNase A-loaded nanogels 55 3.5 Cellular uptake of HP−RGD nanogels .58 Conclusions 61 References 62 Chapter pH- and redox-stimuli sensitive Pluronic micelle for targeted v In vitro PTX release test The in vitro PTX release experiments were performed in triplicate using dialysis method mL of PEG-ADA:SPIO@CD:PTX SAMNs suspended in PBS (0.01 M, pH 7.4; PTX content, 0.3 mg/mL) were transferred to a dialysis bag (MWCO 12 kDa) and immersed into 14 mL of PBS at 37 °C The vials then were placed in an orbital shaker bath, which was maintained at 37 °C and shaken horizontally at 100 rpm At specific time intervals, 14 mL of the release medium was collected and an equal volume of fresh media was added After lyophilization of the collected media, the released amounts of PTX were determined using a HPLC To investigate the effect host-guest interaction of PTX and SAMN systems, a release evaluation of PEG-ADA:SPIO@CD:PTX SAMNs suspended in PBS content of ADA at the final concentration of 50 µM In vitro intracellular uptake study HeLa cells purchased from American Type Culture Collection Cells were grown in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% FBS (fetal bovine serum) The cultures were maintained at 37 °C under a humidified atmosphere containing 5% CO2 The cellular uptake behavior and the intracellular distribution of the SAMNs were analyzed using both confocal laser scanning microscopy (CLSM) and fluorescenceactivated cell-sorting (FACS) For CLSM studies, HeLa cells were seeded on a coverslip in 24-well plate (5 × 104 cells/well) and cultured overnight For blocking studies, cells were pretreated for 45 with an 10 µM solution of a c(RGDfC) peptide that was previously identified to bind tightly to the integrin αvβ3 receptor The cells and pretreated cell ware then treated with c(RGDfC)-conjugated PEG-ADA:SPIO@CD:PTX/Rho for 106 h (SAMNs concentration 50 μg/mL) Cells were then washed times with PBS, fixed with paraformaldehyde for 10 min, treated with DAPI for 15 for nuclei staining, and then washed times with PBS The coverslips were mounted in Vectashield anti-fade mounting medium (Vector Labs) and imaged using a confocal laser scanning microscope (CLSM, Zeiss LSM 510 laser) Images were analyzed using image software (Carl Zeiss LSM) For FACS, HeLa cells seeded into a 6-well plate (5 × 106 cells/well) and cultured overnight Cells were also pretreated for 45 with an 10 µM solution of a c(RGDfC) peptide The cells and pretreated cell were then treated with c(RGDfC)-conjugated PEGADA:SPIO@CD:PTX/Rho for h (SAMNs concentration 50 μg/mL) Thereafter, Cells were then washed times with PBS, trypsinized at 37 °C, centrifuged (1500 rpm, min), and resuspended in PBS (pH 7.4, 1% BSA) Fluorescence histograms were recorded using a BD FACS Calibur (Beckton Dickinson) flow cytometer and analyzed using the Cell Quest software supplied by the manufacturer Each histogram was generated by analyzing at least 10,000 cells Results and discussion 3.1 Characterization General routes employed for the synthesis of ADA-PEG-VS and DOPA-CD was shown in Fig Its chemical structure was verified by1H NMR and FT-IR characterization The binding of CDs on the surface of SPIO is verified by FT-IR spectroscopy Figure 4.4 shows FT-IR spectra of SPIO, SPIO@CD and PEG-ADA:SPIO@CD:PTX It is shown that the characteristic absorption band of the oleic acid ligands (stretching vibration –CH2–) from surface of bare magnetic nanocarriers is 2877 cm-1 In the spectra 107 of SPIO@CD, the appearance of the characteristic peaks of the 1027 cm-1 (stretching vibration of O–H), 1630 cm-1 (stretching vibration of N–H), 2877 cm-1 (stretching vibration of C–H), and the broad band at 3300–3450 cm-1 (stretching vibration of –OH) indicated the DOPA-CD deposition on the SPIO In the spectra of PEGADA:SPIO@CD:PTX, the appearance of the characteristic peaks of the 1247 cm-1 (stretching vibration of C–O–C) and 2785cm-1 (stretching vibration of C–H) indicated the PEG-ADA deposition on the SPIO@CD The spectrums of DOPA-CD and PEG-ADA are quite alike, with all the characteristic peaks of DOPA-CD at 1027, 1630, 2877 and 3300-3450 cm-1 and the characteristic peaks of PEG-ADA at 1247, 1630, and 2785 cm-1 Furthermore, it is clear that the spectrum of the PEG-ADA:SPIO@CD:PTX and SPIO@CD show a smart change as compared to that of the SPIO Thus, on the basis of all the above results, it could be concluded that DOPA-CD has been grafted on the SPIO and PEG-ADA attached on SPIO@CD successfully 108 a) b) c) d) e) 3500 3000 2500 2000 1500 Wavelength (cm-1) 1000 Figure 4.4 FT-IR spectra of (a) bare Fe3O4 NPs; (b) DOPA-CD; (c) ADA-PEG-VS, (d) SPIO@CD, (e) VS-PEG-ADA:SPIO@CD:PTX SAMNS 12 10 b c 25 10 Intensity (%) a Intensity (%) Intensity (%) 12 15 10 0 Diameter (nm) d 20 Diameter (nm) Diameter (nm) e f Figure 4.5 The particles size distribution of SPIO, SPIO@CD, and VS-PEGADA:SPIO@CD:PTX by DLS (a, b, and c) and by TEM (d, e, and f) 109 a b Figure 4.6 In vitro characterization: (A) Magnetization curve of SIPO (white) and SAMNs (black) at 298 oK measured by SQUID exhibiting magnetic saturation, (B) Photograph of magnetic separation of SAMNs by a magnet 110 The particles size distribution of SPIO, SPIO@CD, and VS-PEG- ADA:SPIO@CD:PTX by DLS are shown in Figure 4.5a, b, and c, respectively The mean diameter of SPIO nanocarriers is about nm, which is consistent with information of manufacture The average size of SPIO@CD is around 10 nm The average size of VSPEG-ADA:SPIO@CD:PTX SAMNs is 91.7 nm The size and shape of nanocarriers are deduced from the TEM images As seen in Figure 4.5f, SAMNs have well shaped spherical The dimensions of SAMNs determined by TEM reasonably agree with that determined by dynamic DLS The core structure can be distinguished and the darker magnetite cores are around 85 nm The TEM images can be considered as the direct evidence that PEG-ADA:SPIO@CD:PTX is fabricated successfully The magnetization properties of bare SPIO and PEG-ADA:SPIO@CD:PTX are investigated at room temperature by vibration sample magnetometer (VSM) At the same field, the saturation magnetization value (Ms) for SPIO is found to be 34.5 emu/g, which is less than its bulk counterpart (90 emu/g 2626) For PEG-ADA:SPIO@CD:PTX, the magnetization obtained is 45.0 emu/g The increased saturation magnetization of the SAMNs is generally believed to be due to preparation of SAMNs under magnetic field, resulting in increased magnetic isotropy, in agreement with literature.18 As seen Figure 4.6a, it shows that there is no hysteresis in the hysteresis loop and remanence and coercivity are zero, illustrating that PEG-ADA:SPIO@CD:PTX are superparamagnetic Superparamagnetism is the responsiveness to an applied magnetic field without retaining any magnetism after removal of the applied magnetic field As saturation magnetization of 16.3 emu/g is enough for magnetic separation from solution with a magnet,19 the large saturation magnetization of MNPs could be easily separated from solution by applying an 111 external magnetic field Figure 4.6b shows that when the CD-MNPs solution is placed adjacent to a magnet it became limpid within a few seconds This simple magnetic separation experiment affirmed that PEG-ADA:SPIO@CD:PTX are magnetic and can be removed from aqueous solutions easily 3.2 In vitro release test Drug loading efficacy played an important role in formulation of the drug delivery system and directly affects the therapeutic effect of the system The drug entrapment efficiency (DEE) and loading efficiency (DLE) of SAMNs can be changed by adjusting the ratio of PEG-ADA: SPIO@CD: PTX The DEE and DLE were from 4.9% to 11.4% and from 42.5% to 77.8%, respectively This adjustment also led to the change of SPIO weight ratio in SAMNs from 57.6% to 72.4% Achieving drug controlled release in cancer cells was played a very important role for not only improving anticancer efficacy but also reducing side effect It has been wellestablished that SPIONs vibrate in the presence of an external magnetic field, and this characteristic has been previously utilized for the magnetic nanocarriers in external triggered release features.20 In recently, Kim and his et al have demonstrated the use of synthesis host- guest chemistry to provide triggered activation of a therapeutic system via competitive inclusion Fabrication of the SAMNs was based on host-guest interaction, and it was disintegrable in presence of guest (ADA) Thus, we could expect that after intracellular uptake of SAMNs, PTX would be released through the use of competitive interactions of orthogonally presented guest molecules To further verify that the in vitro drug release characteristics from SAMNs were further examined in PBS buffer with and 112 without ADA at 37 °C and results were shown in Figure 4.7 Without ADA, only 9.6% cumulative PTX release was observed over the period of 120 h, which is in accordance with the fact that SAMNs were relatively stable at neutral conditions In the contrast to the results obtained from the PEG-ADA:SIPO@CD:PTX SAMNs in PBS buffer with ADA, the drug release rate from the SAMNs is impeded significantly with 44.1% drug cumulative release over the same period The host-guest interactions between PTX and CD could effectively preserve the structural integrity of SAMNs, which may lead to a slower drug release rate The higher rate in PTX release in presence of ADA may due to the competitive interactions between the free ADA and PTX with CD These results demonstrate that SAMNs can be used as ADA-disintegrable and intracellular triggerable controlled release nanocarriers 3.3 In vitro cellular uptake Flow cytometry and CLSM were used to evaluate the effect of c(RGDfC) on the cellular uptake behavior of the SAMNs against αvβ3 and αvβ5 positive HeLa cells (derived from human cervical cancer) Fig.2.8.(A) shows the flow cytometry histograms of Rho fluorescence from HeLa cells and c(RGDfC) pretreated HeLa cells incubated with c(RGDfC)-PEG-ADA:SPIO@CD:PTX/Rho SAMNs at the same equivalent Rho concentration of μg/ml for h Cells without any treatment were used as a negative control to detect auto-fluorescence The flow cytometry analysis clearly demonstrated that c(RGDfC) targeting ligand can significantly enhance the cellular uptake of the SAMNs after h incubation CLSM was used to further characterize the cell uptake behavior and to investigate the intracellular distribution of the Rho in the HeLa cells after 113 h incubation with c(RGDfC)-PEG-ADA:SPIO@CD:PTX/Rho SAMNs As shown in Fig 2.8B, significantly higher intracellular Rho fluorescence intensity was observed in the HeLa cells incubated c(RGDfC)-PEG-ADA:SPIO@CD: PTX/Rho SAMNs cooperation to pretreated one, which was consistent with the cellular uptake behavior observed by flow cytometry and was attributed to the receptor-mediated endocytosis process 114 Cumulative PTX release (%) 50 40 30 20 10 0 20 40 60 Time (h) 80 100 120 Figure 4.7 In vitro PTX release profiles in PBS (pH 7.4) with (●) and without (■) ADA from SAMNs Figure 4.8 Confocal microscopic images (A) and fluorescence intensity (B) of HeLa cells and c(RGDfC) pretreated HeLa celles incubated with c(RGDfC)-PEGADA:SPIO@βCD:PTX SAMNs at 37 oC for h 115 Conclusions VS-PEG-ADA:SPIO@CD:PTX SAMNs synthesized and characterized successfully by host guest interaction for targeted drug delivery The prepared SAMNs had a spherical sharp with a narrow size distribution It have a mean diameter of 91.7 nm, and an encapsulation efficiency > 50% The VS-PEG-ADA:SPIO@CD:PTX SAMNs has large saturation magnetization so it could be applied in magnetic targeted drug delivery c(RGDfC) was covalently coupled to the SAMNs surface via a chemical reaction between thiol and a vinyl sulfone functional group The cellular uptake of c(RGDfC)conjugated PEG-ADA:SPIO@CD:PTX SAMNs vesicles against HeLa cell line was greater than the pretreatment cells Furthermore, a competitive binding experiment indicated that integrin receptor-mediated endocytosis had been involved in the cell uptake c(RGDfC)-PEG-ADA:SPIO@CD:PTX SAMNs can be disassembled intracellular by the orthogonal guest molecule ADA, thereby triggering drug release The results of the present study indicate that c(RGDfC)-PEG-ADA:SPIO@CD:PTX SAMNs could be used as a potential targeted drug delivery system 116 Reference Shen JM, Gao FY, Yin T, Zhang HX, Ma M, et al 2013 Pharmacol Res 70: 10215 Xu Y, Karmakar A, Heberlein WE, Mustafa T, Biris AR, Biris AS 2012 Adv Healthc Mater 1: 493-501 Wang SY, Liu MC, Kang KA 2013 Adv Exp Med Biol 765: 315-21 Singh A, Dilnawaz F, Mewar S, Sharma U, Jagannathan NR, Sahoo SK 2011 ACS Appl Mater Interfaces 3: 842-56 Alexiou C, Tietze R, Schreiber E, Lyer S 2010 Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 53: 839-45 Kong SD, Lee J, Ramachandran S, Eliceiri BP, Shubayev VI, et al 2012 J Control Release 164: 49-57 Gautier J, Munnier E, Paillard A, Herve K, Douziech-Eyrolles L, et al 2012 Int J Pharm 423: 16-25 Kayal S, Ramanujan RV 2010 J Nanosci Nanotechnol 10: 5527-39 Chertok B, David AE, Yang VC 2010 Biomaterials 31: 6317-24 10 Alexiou C, Schmid RJ, Jurgons R, Kremer M, Wanner G, et al 2006 Eur Biophys J 35: 446-50 11 Basti H, Ben Tahar L, Smiri LS, Herbst F, Vaulay MJ, et al 2010 J Colloid Interface Sci 341: 248-54 12 Shenoy D, Fu W, Li J, Crasto C, Jones G, et al 2006 Int J Nanomedicine 1: 51-7 117 13 Petter RC, Salek JS, Sikorski CT, Kumaravel G, Lin FT 1990 J Am Chem Soc 112: 3860-8 14 GH J, J B, J M, RC D 1989 Vogel ’s text book of quantitative chemical analysis 15 Nguyen DH, Joung YK, Choi JH, Moon HT, Park KD 2011 Biomed Mater 6: 055004 16 Nguyen DH, Choi JH, Joung YK, Park KD 2011 Bioactive and Compatible Polymers 26: 17 Jagetia GC, Rao SK 2006 Evid Based Complement Alternat Med 3: 267-72 18 Lu Y, Zhao Y, Yu L, Dong L, Shi C, et al 2010 Adv Mater 22: 1407-11 19 Ma ZY, Guan YP, Liu XQ, Liu HZ 2005 Langmuir 21: 6987-94 20 Azagarsamy MA, Alge DL, Radhakrishnan SJ, Tibbitt MW, Anseth KS 2012 Biomacromolecules 13: 2219-24 118 Overall conclusion Research activity aimed towards achieving specific and targeted delivery of anticancer agents has expanded tremendously in the last years or so with new avenues of directing drugs to tumors as well as new types of drugs In this dissertation, we presented how nanoparticles took advantage of these special features and how nanoparticles could act as a vehicle to specifically deliver cancer-fighting drugs to tumors We have developed three differ drug delivery systems using PEG and its block copolymer for targeted drug delivery The presence of PEG outer shell helps nanoscale carriers to bypass the RES clearance, thereby prolonging the circulation time in the blood stream Another advantage that could be taken from the stability of PEG-coated nanospheres is the possibility of attaching antibodies or a fragment of them to the surface of the particles, without destabilizing them, in order to achieve site-specific drug delivery, a major challenge for drug administration Ideally, these “magic missiles” would accumulate at the diseased tissue and locally liberate the necessary amount of drug The drugs can be released at the desired sites of actions by designing environment-sensitive linkers in side structure of nanoparticles where the linkers respond to the extra/intracellular microenvironment or external stimuli The design of these types of nanoparticles remains a very interesting research area Controlled release of drug at the site of action will enhance the efficacy and reduce the side effect of drug The combination of the use of stimuli-responsive material and targeting moieties will lead to nanoparticles which can be targeted to the side of action and which will deliver the drug These approach should provide the creative treatment methods have made it to the clinic and hopefully are well on their way to improving the length and quality of life for cancer patients However, it should be noted that extensive preclinical evaluations are required for these types of nanoparticles before they can be considered to use in patients Subjects which have to be evaluated are the pharmacokinetics of drug loaded/conjugated nanoparticles, effect of the surface-located targeting molecules on the opsonization process and blood circulation times as well as the efficacy and toxicity of the nanoparticles in particlular after repeated administration Mechanistic studies of the intracellular drug release from the nanoparticles are also required to further unravel the kinetics of intracellular nanoparticle destabilization and intracellular drug release [...]... various diseases.13 Interest in this concept has increased significantly in recent decades with the innovations of nanomedicine Cancer nanomedicines have the ability to improve the therapeutic index of drugs by preferential localization at target sites, lower distribution in healthy tissues, delivery of hydrophobic drugs and extended release rate Progress in the development of nanomedicines for targeted drug... substances are under investigation for drug delivery and more specifically for cancer therapy are used in the clinic Interestingly pharmaceutical sciences are using nanocarriers to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient The kind of hazards that are introduced by using nanocarriers for drug delivery are... and free DOX (e, f) were incubated with HeLa cells for 1 h (a, c, e) and 4 h (b, d, f) xii Flow cytometry histrogram (g) and fluorescence intensity (h) of various DOX formulations internalized into HeLa cells for 4h Figure 3.8 Dose-dependent cytotoxicity of (a) FA-Pluronic-C/H micelles and (b) FA-Pluronic-C/H-DOX against HeLa cells after 48 h incubation (n = 4) Free DOX (×), Pluronic-C/H-DOX (○), and. .. are coated with opsonin proteins (a2) and associate with macrophages (a3) for transit to the liver (a4) Macrophages stationary in the liver, known as Kupffer cells, also participate in nanoparticle scavenging (b) Nanocarriers coated with PEG coating (b1) prevents this opsonization (b2), resulting in decreased liver accumulation (b3) and increased availability of the NP for imaging or therapy Figure... nanocarriers for systemic delivery Clearly, particles with longer circulation times have superior ability to reach the tumor site through passive targeting As opsonization is an integral step in the removal of foreign macromolecules by the RES, many efforts for increasing serum stability and extending circulation time have focused on blocking absorption of opsonins onto the nanoparticle surface.5 For passive... are coated with opsonin proteins (a2) and associate with macrophages (a3) for transit to the liver (a4) Macrophages stationary in the liver, known as Kupffer cells, also participate in nanoparticle scavenging (b) Nanocarriers coated with PEG coating (b1) prevents this opsonization (b2), resulting in decreased liver accumulation (b3) and increased availability of the NP for imaging or therapy.12 12... (b) and the AFM x image of heparin−Cya−Pluronic−VS (c) Figure 2.7 Release profiles of heparin from heparin−Cya−Pluronic−VS nanogel (A) and RNase A from RNase A-loaded heparin−Cya−Pluronic−VS nanogel (B) Release studies were performed primarily in PBS media ((pH 7.4) and then treated with GSH at final concentration of 5 mM at 37oC Experiments were performed three times and the data indicate mean ± standard... of NIH3T3 cells incubated with heparin−Cya−Pluronic −cRGDfC (♦) and RNase A-loaded heparin−Cya−Pluronic−cRGDfC (■) nanogels for 48 h at 37 oC The cell viability was determined by MTT assay and plotted against the polymer concentration in DMEM at 37 oC Experiments were performed four times and significant differences between the treatment means and control values at respective times are indicated by *... polyethylene oxide (PEO)), which was used both for chemical modification of various drugs (peptide and protein, first of all) to make them more stable and long-circulating and for the decoration of pharmaceutical carriers to improve their pharmacokinetic properties Figure 1.6 illustrates how opsonin proteins associate with foreign bodies and coat its surface As bacteria and viruses have the same negative surface... microenvironments, promoting targeted delivery and controlled release of therapeutic drugs and imaging agents Figure 2.1 Schematic illustration showing the formation and redox-sensitive intracellular delivery of a protein-loaded HP nanogel Figure 2.2 Synthetic route of heparin−SS−Pluronic−VS conjugate Figure 2.3 1 H NMR (D2O) spectra of Pluronic−DVS (A), heparin−Cya (B), and heparin−Cya−Pluronic−VS (C)