Injectable gelatin-peg hydrogels for long-term delivery of dexamethasone or avastin to treat choroidal neovascularization
Injectable gelatin-peg hydrogels for long-term delivery of dexamethasone or avastin to treat choroidal neovascularization
초록/요약
Abstract Injectable hydrogel systems have received much attention due to their versatile and tunable characteristics based on a minimal invasive technique. In a free flow state, hydrogel precursor solutions can fill up a tissue defect or target site while containing bioactive molecules, and then act as a localized therapeutic depot after physical or chemical cross-linking. A horseradish peroxidase (HRP)-catalyzed cross-linking reaction has recently received much attention as a promising approach to developing in situ forming hydrogels. For the reaction, HRP and hydrogen peroxide (H2O2) are both considered as essential prerequisites for controlling the degree and rate of cross-linking. Choroidal neovascularization (CNV) resulted by various retina and posterior segment disease particularly age‐related macular degeneration is among the main causes of blindness worldwide and in light of ongoing population ageing its prevalence is increasing specially in developed countries. As a major health concern, these ocular diseases have detrimental effects on patients’ quality of life and society economy. Considering the key role of vascular endothelium growth factor (VEGF) in the pathogenesis of CNV, Although the pathophysiology of macular edema is not yet fully understood, VEGF has been recognized as a major contributor. VEGF inhibitors have changed the paradigm of therapy and have become the standard of care during the last decade. Dexamethasone has potentially many more pathogenic targets and thus much more potential to address a therapeutic response on chronic/recalcitrant, longstanding, or severe macular edema. Avastin is a full‐length humanized antibody against all isoforms of VEGF. Avastin is widely used intravitreally in the treatment of CNV as an off‐labeled drug. Dexamethasone and Avastin have demonstrated promising effect on treatment of the disease, repetitive intravitreal injection is required due to short half‐life of the drug in vitreous. The frequency of intravitreal injection would be a leading cause in reducing patient compliance besides deleterious adverse effects including retinal detachment, subconjunctival hemorrhage and endophthalmitis. Hence, developing extended release drug delivery systems seems to be necessary to decrease the number of administrations. The main objective of this dissertation is to develop and evaluate injectable gelatin hydrogels for long term drug delivery of dexamethasone or avastin to treat choroidal neovascularization. The first work of the thesis was to prepare and characterize an injectable gelatin hydrogel via HRP catalyzed cross-linking. The gelatin hydrogels were prepared from a gelatin solution above 3 wt% in the presence of their mechanical properties such as gelation time, elastic modulus and degradation time were evaluated at different HRP, H2O2 concentration. Drug release behaviors from hydrogel were evaluated HPLC (dexamethasone) and ELISA (avastin). Through circular dichroism analysis, the structural stability of released avastin from the gelatin hydrogel was confirmed. In the cell study, the drug loaded hydrogels showed no apparent cytotoxicity. We intravitreal injected the drug loaded hydrogels into the eyes of SD rats and rabbits. The results of in vivo study demonstrated successful injection of the drug loaded hydrogel at the injected site and excellent therapeutic efficacy and pharmacokinetics.
more목차
I. General introduction ............................................................................................ 1
1. Choroidal vascular diseases ................................................................................. 1
2. Hydrogels for opthalmology ................................................................................ 2
3. Injectable hydrogels ............................................................................................. 3
3.1. Cross-linking strategies for injectable hydrogelation ..................................... 4
3.1.1. Physically cross-linked hydrogels .............................................................. 5
3.1.2. Chemically cross-linked hydrogels ............................................................ 6
4. Enzyme-catalyzed cross-linking system .............................................................. 7
4.1. Horseradish peroxidase-catalyzed cross-linking system................................. 9
5. Injectable hydrogels for drug delivery ............................................................... 12
6. Overall objectives ............................................................................................. .13
II. Intravitreal injectable dexamethasone loaded gelatin hydrogels for macular edema treatment ..................................................................................................... 14
1. Introduction ........................................................................................................ 14
1.1. Macular edema ................................................................................................ 14
2. Materials and Methods ....................................................................................... 17
2.1. Materials........................................................................................................ 17
2.2. Synthesis of gelatin-peg-tyramine (GPT) ................................................... 17
2.3. Characterizations of GPT .............................................................................. 19
2.4. Hydrogel preparation and gelation time measurement of GPT hydrogel ..... 20
2.5. Rheological experiment .............................................................................. 21
2.6. In vitro proteolytic degradation of hydrogels ............................................... 21
2.7. Preparation of dexamethasone loaded hydrogel systems ............................ 22
2.8. Quantification of dexamethasone released from hydrogel systems .............. 22
2.9. In vitro two-dimensional cell study .............................................................. 23
2.10. In vivo Intravitreal injection of dexamethasone loaded GPT hydrogel ...... 23
2.10.1. ME induction ........................................................................................ 23
2.10.2. Intravitreal injection of dexamethasone loaded GPT hydrogel ............. 24
2.10.3 Fluorescence imaging techniques for flat retinal preparations ............... 24
3. Results and discussion ....................................................................................... 25
3.1. Synthesis and characterizations of GPT ...................................................... 25
3.2. Hydrogel formation and gelation time .......................................................... 27
3.3. Effect of hydrogen peroxide concentration on mechanical strength ............. 29
3.4. In vitro proteolytic degradation of hydrogels ............................................... 30
3.5. In vitro drug release behaviors ...................................................................... 31
3.6. In vitro hDFB cell viability on dexamethasone loaded hydrogels ................ 32
3.7. Efficacy of macular edema treatment using dexamethasone loaded GPT hydrogels 3.3. Preparation of Ava/HPs loaded hydrogel systems ........................... 34
4. Conclusions ........................................................................................................ 35
III. Injectable heparin nanogel/gelatin hydrogel composite for avastin long term delivery to treat age-related macular degeneration ............................................ 37
1. Introduction ........................................................................................................ 37
1.1. Age-related macular degeneration ................................................................ 37
1.2. Anti-VEGF proteins ..................................................................................... 38
2. Materials and Methods ....................................................................................... 43
2.1. Materials........................................................................................................ 43
2.2. Synthesis of GPT and heparin-pluronic (HP) ............................................... 43
2.3. Preparation of avastin loaded HP nanogels (Ava/HPs) ................................ 44
2.4. Preparation of Ava/HPs loaded hydrogel systems ........................................ 45
2.5. Quantification of avastin release behaviors from hydrogel systems ............. 46
2.6. In vitro two-dimensional cell study .............................................................. 47
3. Results and discussion ....................................................................................... 47
3.1. Characterization of HP copolymer ................................................................ 47
3.2 Preparation of Ava/HP nanogels .................................................................... 48
3.3. Preparation of Ava/HP loaded GPT hydrogel ............................................. 48
3.4. Evaluation of in vitro release kinetics ........................................................... 49
3.5. In vitro RPE cell viability analysis ............................................................... 50
4. Conclusion ......................................................................................................... 51
IV. References......................................................................................................... 53
