function ali zed polymer nano containers for targeted drug delivery

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Functionalized Polymer Nanocontainers for Targeted Drug Delivery

Inaugurationaldissertation zur Erlangung der Wrde einesDoktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultt der Universitt Basel

von

Samantha Mariela Benitoaus Buenos Aires, Argentina

Basel, 2006

Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultt Auf Antrag der Herren: Prof. Dr. Wolfgang Meier Dr. Andreas Taubert Prof. J. P. Maier

Basel, den 28. September 2004 Prof. M Tanner Dekan

Table of Contents

Table of Contents1. Introduction ____________________________________________________ 1 1.1.General introduction _____________________________________________________ 1 1.2.Cells, membranes, and proteins ____________________________________________ 2 1.3.Amphiphiles, lipids, and self-assembly _______________________________________ 3 1.4.Polymers and block-copolymers ____________________________________________ 5 1.5.Aggregation of amphiphilic block-copolymers in aqueous media___________________ 71.5.1.Micelles____________________________________________________________________ 8 1.5.2.Vesicles___________________________________________________________________ 10

1.6.Polymer nanocontainers _________________________________________________ 10 1.7.General concept of encapsulation __________________________________________ 12 1.8.General concept of drug delivery __________________________________________ 121.8.1.Drug delivery based on liposomes ______________________________________________ 13 1.8.2.Polymer based drug delivery systems ___________________________________________ 13 1.8.3.Controlled-release systems ___________________________________________________ 14 1.8.4.Site specific or Selective targeting______________________________________________ 15

1.9.General concept of surface immobilization ___________________________________ 16 2. Scope of this thesis ______________________________________________ 19 3. Results and Discussions __________________________________________ 21 3.1.Polymer synthesis, functionalization and characterization _______________________ 213.1.1.Synthesis of ABA triblock-copolymers ___________________________________________ 213.1.1.1. 3.1.1.2. 3.1.1.3. Introduction to block-copolymer synthesis__________________________________________ 21 Considerations regarding the polymer selection _____________________________________ 22 Results and discussion _________________________________________________________ 23

3.1.2.End group functionalization of ABA triblock-copolymers_____________________________ 263.1.2.1. 3.1.2.2. 3.1.2.3. Introduction and theoretical considerations _________________________________________ 26 Biotinylated ABA triblock-copolymers ______________________________________________ 27 Fluorescently labeled ABA triblock-copolymers ______________________________________ 28

3.1.3.Characterization techniques and assays _________________________________________ 293.1.3.1. 3.1.3.2. 3.1.3.3. 3.1.3.4. 3.1.3.5. Polymer characterization by 1H-NMR, GPC, and IR ___________________________________ 29 Characterization and quantification of biotinylated ABA triblock-copolymer ________________ 36 Fluorescently modified ABA triblock-copolymer ______________________________________ 38 Block-copolymer monolayer isotherms _____________________________________________ 40 Interaction of biotinylated block-copolymer monolayer with streptavidin __________________ 43

3.1.4.Conclusions and Summary____________________________________________________ 46

3.2.Vesicle preparation and characterization ____________________________________ 473.2.1.Introduction _______________________________________________________________ 47 3.2.2.Results and discussion _______________________________________________________ 483.2.2.1. Vesicle characterization by DLS, SEC, and TEM ______________________________________ 49

Table of contents

3.2.2.2. 3.2.2.3. 3.2.2.4. 3.2.2.5.

Fluorescent Giant Unilamellar Vesicles ____________________________________________ 53 Trapped volume of vesicles _____________________________________________________ 53 FCS studies of vesicles_________________________________________________________ 56 Density determination and Sedimentation velocity studies_____________________________ 59

3.2.3.Conclusions and Summary ___________________________________________________ 60

3.3.Biotinylated Nanocontainers for selective targeting of cells ______________________ 613.3.1.Introduction to Biotin-Avidin technology_________________________________________ 61 3.3.2.Criteria for material selection and targeting strategy _______________________________ 61 3.3.3.Results and discussion_______________________________________________________ 633.3.3.1. 3.3.3.2. 3.3.3.3. 3.3.3.4. 3.3.3.5. Chromatographic separation ____________________________________________________ 63 UV-vis characterization of biotin-streptavidin conjugation _____________________________ 64 TEM and SEM studies on biotinylated nanocontainers ________________________________ 67 Binding and Uptake analyzed by Fluorescence Microscopy ____________________________ 68 Cytotoxicity studies ___________________________________________________________ 73

3.3.4.Conclusions and Summary ___________________________________________________ 74

3.4.Gold encapsulation______________________________________________________ 753.4.1.Introduction _______________________________________________________________ 75 3.4.2.Results and discussions ______________________________________________________ 753.4.2.1. 3.4.2.2. Encapsulation of pre-formed gold nanoparticles _____________________________________ 75 In situ gold encapsulation ______________________________________________________ 77

3.4.3.Conclusions _______________________________________________________________ 81

3.5.Vesicle immobilization onto surfaces ________________________________________ 833.5.1.Introduction _______________________________________________________________ 83 3.5.2.Results and discussions ______________________________________________________ 843.5.2.1. Quartz crystal microbalance with dissipation experiments _____________________________ 84

3.5.3.Conclusions _______________________________________________________________ 91

4. Materials and Methods___________________________________________ 93 4.1.Materials______________________________________________________________ 93 4.2.Synthesis of ABA triblock-copolymers _______________________________________ 94 4.3.End-group functionalization of ABA triblock-copolymers ________________________ 95 4.4.Spectroscopy techniques - 1H-NMR and IR __________________________________ 96 4.5.Gel Permeation Chromatography __________________________________________ 964.5.1.Theoretical description ______________________________________________________ 96 4.5.2.Experimental part __________________________________________________________ 97

4.6.Quantification of biotinylation of ABA triblock-copolymer________________________ 98 4.7.Langmuir film experiments _______________________________________________ 984.7.1.Theoretical description ______________________________________________________ 98 4.7.2.Experimental part _________________________________________________________ 104

4.8.Methods of vesicle preparation ___________________________________________ 1054.8.1.Standard (solvent-injection-extrusion) or Ethanol method__________________________ 105

Table of Contents

4.8.2.Direct dispersion method ____________________________________________________ 105 4.8.3.Electroformation (GUV) _____________________________________________________ 106

4.9.Methods of proteo-vesicle preparation _____________________________________ 1064.9.1.Standard (solvent-injection-extrusion) or Ethanol method __________________________ 106 4.9.2.Direct dispersion/Detergent and Biobeads method________________________________ 106

4.10.Size Exclusion Chromatography _________________________________________ 1074.10.1.Preparative Chromatography ________________________________________________ 108

4.11.UV-Vis measurements _________________________________________________ 108 4.12.Fluorescence measurements____________________________________________ 1094.12.1.Theoretical description_____________________________________________________ 109 4.12.2.Experimental part_________________________________________________________ 110

4.13.Light Scattering ______________________________________________________ 1114.13.1.Theoretical description_____________________________________________________ 111 4.13.2.Experimental part_________________________________________________________ 114

4.14.Transmission and scanning electron microscopy ____________________________ 1154.14.1.Theoretical description_____________________________________________________ 115 4.14.2.Experimental part_________________________________________________________ 116

4.15.FCS experiments _____________________________________________________ 1164.15.1.Technique description _____________________________________________________ 116 4.15.2.Experimental part_________________________________________________________ 118

4.16.Density determinations ________________________________________________ 1194.16.1.Theoretical description_____________________________________________________ 119 4.16.2.Experimental part_________________________________________________________ 121

4.17.Selective cell targeting with biotinylated Nanocontainers _____________________ 122 4.18.Gold encapsulation ___________________________________________________ 1254.18.1.Pre-formed gold Nanoparticles encapsulation ___________________________________ 125 4.18.2.In situ gold formation