PNIPAAM MODIFIED PCL MATRIX FOR IN VITRO CELL CULTURE STUDY LUO XUAN NATIONAL UNIVERSITY OF SINGAPORE 2007 PNIPAAM MODIFIED PCL MATRIX FOR IN VITRO CELL CULTURE STUDY LUO XUAN B.Sci., Zhejiang University A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAMME IN BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE ACKNOWLEDGEMENTS This thesis grew out of a series of trials and experiments for the idea of an interdisciplinary project, from which I benefited and learned quite a lot I would like to gratefully acknowledge Prof Teoh Swee Hin’s efforts during this work and the precious opportunity provided for me to propose and execute this project I would like to thank Professor Ding Jeak Ling and Associate Professor Ho Bo very much for the insightful thoughts, constant guidance, and detailed instruction My sincere thanks also go to Doctor Li Jun, who has been kindly offering experiment equipments and technical support, and Doctor Tong Yen Wah for the PNIPAAm polymer provided I am grateful to all my classmates and friends from different labs at my study, without whose help this thesis wouldn’t have appeared Finally, I am forever indebted to my parents It would be unimaginable for me to overcome all the difficulties when one of them has been suffering from the serious disease, if it were not their understanding, support and encouragement TABLE OF CONTENTS Summary List of Tables List of Figures Introduction 1.1 Poly(ε-caprolactone) 1.1.1 Background 1.1.2 Physical and chemical properties 1.1.3 Major biomedical applications of PCL 1.1.3.1 Drug delivery system 1.1.3.2 Internal fixation 1.1.3.3 Membrane implantation 1.1.3.4 Skin coverings for wounds 1.1.4 PCL scaffolds manufactured by fused deposition modelling (FDM) 1.2 Poly(N-isopropyl acrylamide) 1.2.1 History 1.2.2 Physical and chemical properties 1.2.3 Major biomedical applications of PNIPAAm 1.2.3.1 Phase separation and “molecule gate” 1.2.3.2 Two dimensional cell sheet technology 1.2.3.3 Polymeric carriers Materials and methods 2.1 Physical coating of PNIPAAm onto PCL scaffolds 2.1.1 Materials 2.1.2 Methods 2.2 Chemical grafting of PNIPAAm 2.2.1 3D grafting of PNIPAAm onto PCL scaffolds 2.2.1.1.Materials 2.2.1.2.Preparation of purified NIPAAm and polymerization solution 2.2.1.3.Plasma treatment polymerization 2.2.2 2D grafting of PNIPAAm onto PCL films 2.2.2.1.Materials 2.2.2.2.Methods 2.2.2.2.1 Preparation of purified NIPAAm and polymerization solution 2.2.2.2.2 Preparation of PCL films 2.1.1.1.1 Plasma treatment 2.1.1.1.2 U.V polymerization 2.1.1.1.3 Water contact angle measurements 2.1.1.1.4 Cell seeding and culture 2.1.1.1.5 MTS assay 2.1.1.1.6 Confocal laser scanning microscopy (CLSM) 2.1.1.1.7 Scanning electron microscopy (SEM) Results, discussion 3.1.1 Physical coating of PNIPAAm onto PCL scaffolds 3.1.2 Chemical grafting of PNIPAAm 3.1.3 3D grafting of PNIPAAm onto PCL scaffolds 3.1.4 2D grafting of PNIPAAm onto PCL films Conclusion Bibliography SUMMARY 2D cell sheet technology with the aid of PNIPAAm temperature responsive changes on the hydrophobicity was already invented and widely applied as reviewed earlier PNIPAAm caught much attention in recent years mainly because of its LCST of 32℃ , which is close to human body and cell culture temperature, and moreover, adjustable by adding in functional groups bearing different hydrophobicity during the polymerization procedure The idea, development of 2D cell sheet technology into 3D in vivo cell culture instrument came from the high volume/space ratio 3D structure, especially PCL scaffolds, provided In the research, the 3D exploration did not achieve as expected, partially due to the low crystallization point of PCL and limits in 3D grafting PCL scaffolds did not withstand the last heat treatment step of the whole PNIPAAm chemical grafting process However, the change of morphology and N% on 3D physically coated PCL scaffolds still indicates how different that a PNIPAAm layer may assist in cell culture, in vitro Further investigations about 3D cell harvest can by done by selecting other scaffolds that may undergo the temperature required for 3D chemical grafting Moreover, verified by the drop in water contact angle experiment, MTS assay, SEM result and confocal microscopy images, the PNIPAAm chemical grafting onto 2D PCL I films was well targeted and established The 2D PNIPAAm grafted matrix greatly supported cell culture with the non-cytotoxic character PCL exhibited II LIST OF TABLES Table 1.1 Fact sheet of microsphere based PCL or PCL blended Table 1.2 Fact sheet of microsphere based PCL co-polymers Table 1.3 Fact sheet of nanosphere based PCL or PCL blended Table 1.4 Fact sheet of nanosphere based PCL co-polymers III LIST OF FIGURES Figure 1.1 Structure of monomer ε-caprolactone and polymer PCL Figure 1.2 Brief illustration of the basic FDM process Figure 1.3 (a) Lay-down pattern of 0º/90º/0º forming square honeycomb scaffolds Its frontal (b) and lateral view (c) under electronic microscope Figure 1.4 (a) Lay-down pattern of 0º/60º/120º forming square honeycomb scaffolds Its frontal (b) and lateral view (c) under electronic microscope Figure 1.5 Structure of monomer N-isopropylacrylamide and polymer PNIPAAm Figure 1.6 Schematic illustration of the variety of natural or synthetic biomolecules which may be conjugated to a smart polymer In some cases, only one molecule may be conjugated, such as a recognition protein, which may be linked to the protein at a reactive terminal group of the polymer, or it may be linked at a reactive pendant group along the polymer backbone In other cases more than one molecule may be onjugated along the polymer backbone, such as a targeting ligand along with many drug molecules Figure 1.7 Copolymerization of a thermally sensitive polymer, PNIPAAm, with a more hydrophilic comonomer, AAm, raises the LCST of the copolymer, while copolymerization with a more hydrophobic comonomer, N-test butylacrylamide (N-tBAAm), lowers the LCST Figure 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Nanostructured designs of biomedical materials: applications of cell sheet engineering to functional regenerative tissues and organs Akihiko Kikuchi, Teruo Okano Journal of Controlled Release 101 (2005) 69–84 108 Kwon OH, Kikuchi A, Yamato M, Sakurai Y, Okano T Rapid cell sheet detachment from poly(N-isopropyl acrylamide)-grafted porous cell culture membranes J Biomed Mater Res 2000 Apr;50(1):82-9 109 Hyeong Kwon O, Kikuchi A, Yamato M, Okano T Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes Biomaterials 2003 Mar;24(7):1223-32 110 H.G Schild Poly(N-isopropyl acrylamide): experiment, theory, and application Prog Polym Sci., 1992, Vol 17, 163-249 54 [...]... PEAD /PCL single emulsion In vitro Atkins29 PCL/ poly(ethylene oxide)– poly(propylene oxide) solvent evaporation In vitro Huatan et al.30 PCL solvent evaporation In vitro Lin and Yu31; Lin et al.32 PCL In vitro Lin and Huang33 In vitro Ethyl salicylate PCL emulsion-solvent evaporation solvent evaporation Nerve growth factor PCL; PCL/ PLGA solvent evaporation In vitro Indomethacin PCL melt-dispersion In vitro. .. PCL/ PLA; PCL/ cellulose acetate butyrate solvent evaporation In vitro Chang et al.21,22,23 Cyclosporine PCL solvent evaporation In vitro Aberturas et al.24 Cisplatin PCL/ PLA solvent evaporation In vitro Chandy et al.25 E- and P-selectin Ribozymes PCL PCL In vitro In vitro Dickerson et al.26 Jackson et al27 Bovine serum albumin PCL single emulsion Injection of polymeric paste solvent evaporation In vitro. .. drugs PCL o/w or w/o/w emulsion In vitro Perez et al.16 Taxol PCL In vitro Dordunoo et al.17 Gentamicin PCL In vitro Sondhof et al.18 Ketoprofen PCL; PCL/ hydroxypropylmethylcellulose phthalate PCL; PCL/ PLA solvent evaporation double emulsion spray-drying in vitro Giunchedi et al.19 Entrapping; PEG coated In vitro Das et al.20 Antigen Colchicine Chlorpromazine; chlorpromazine–HCL and progesterone PCL/ PLA;... Nitrofurantoin Insulin PCL PCL solvent evaporation solvent evaporation In vitro In vitro Gadzinowski et al.34 Cao and Shoichet35 Bodmeier and Chen,36 Dubernet et al.37 Shenoy et al.38 3,4-diaminopyridine PCL In vitro Gibaud et al.39 Heparin PCL/ poly(d,l-lactic-co-glycolic acid)/Eudragit RS and RL by different methods for double emulsification; evaporation process In vitro Jiao et al.40 Injectable bone... caprolactone PCL/ ethylene oxide ε-caprolactone/l-lactide PCL/ poly(ε-block d,llactide) PCL/ poly(ether ester amide) PCL/ lactic acid PCL/ 2,2-bis(2oxazoline) Techniques Vitro/ Vivo Researchers In vitro In vitro Pitt et al.42; Buntner et al.43,44 Martini et al.45 Guerra et al.46 both Limin et al.47 In vitro Barbato et al.48 In vitro Cha and Pitt49 In vitro Tarvainen et al.50 Solvent evaporation hot melt solvent... glycol) /PCL Taxol Methoxy poly(ethylene glycol) /PCL Bovine serum poly(ethylene albumin Oxide /PCL Clonazepam poly(ethylene glycol) /PCL 1.1.3.2 Techniques Vitro/ Vivo Researchers dialysis In vitro Kim et al.75,76 dialysis In vitro Kim and Lee77 w/o/w double emulsion dialysis In vitro Lu et al.78 In vitro Ryu et al.79 Internal fixation Early in the 1980’s, PCL had been studied as the replacement of stainless... 1.4 5 Table 1.3 Fact sheet of nanosphere based PCL or PCL blended Drug Components delivered Indomethacin PCL Techniques three submicron systems Vitro/ Vivo Researchers both Calvo et al.51,52 Cartelol PCL In vivo Flurbiprofen PCL Solvent diplacement In vitro Primidone PCL In vitro Aceclofenac PCL both Alonso et al.57 Diclofenac PCL interfacial deposition Interfacial precipitation Spray dry MarchalHeussler... especially helpful in harvesting cardiac cells beating at the same rate and endothelial cell sheets for repair of vessels 19 Sandwich cell culture layers can be formed when a PNIPAAm layer is inserted in between the tissue culture grade polystyrene (TCP) and another fibronectin (FN) cell adhesive layer (Fig 1.15) Figure 1.14 Schematic diagrams for possible interactions of materials surfaces with cells The “deadhesion”... been studied, by blending PCL with other polymers, or using PCL alone, as listed in Table 1.1 3 Tabble 1.1 Fact sheet of microsphere based PCL or PCL blended Drug Delivered Component(s) Techniques Vitro/ Vivo Researcher(s) PCL double emulsion both Jameela et al.12 PCL melt encapsulation both Jameela et al.13 PCL in vitro Youan et al.14 PLGA w/o/w solvent evaporation Spray-drying in vitro Murillo et al.15... carried out by Schwope et al, both in vitro and in vivo, with three types of materials formed by PCL: 1) a freeze-dried collagen /PCL film laminate, 2) a freeze-dried PCL foam /PCL film laminate, and 3) a heat-dried collagen /PCL film laminate Results showed that one graft was extremely adherent to wound (burned rat model) and most successful in promoting tissue bed formation.82 In the year of 1995, Jurgens’ ... PCL/ PLGA solvent evaporation In vitro Indomethacin PCL melt-dispersion In vitro Nitrofurantoin Insulin PCL PCL solvent evaporation solvent evaporation In vitro In vitro Gadzinowski et al.34 Cao and... Tamoxifen PCL Metipranolol PCL Isradipine PCL Bovine serum albumin PCL In vitro In vitro In vivo interfacial deposition nanoprecipitation both double emulsion pressure homogenization In vitro both... solvent evaporation In vitro Chandy et al.25 E- and P-selectin Ribozymes PCL PCL In vitro In vitro Dickerson et al.26 Jackson et al27 Bovine serum albumin PCL single emulsion Injection of polymeric