Injectable Hyaluronan Microgel-embedded Gelatin Hydrogel and Gelatin Hydrogel Rod with Controlled Protein Delivery for Neovascular Retinal Therapy
- 주제(키워드) Retinal neovascularization , intravitreal controlled anti-VEGF delivery , formulation design of hydrogels , microgels , hydrogel rods , minimally invasiveness , inhibited drug clearance , extended intravitreal drug maintenance , biodegradability
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 박기동
- 발행년도 2022
- 학위수여년월 2022. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032078
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Globally, the development of treatment strategies for the neovascularization in the posterior segment of eye is inevitable. There have been numerous studies for retinal neovascularization treatment including laser therapies and intraocular anti-vascular endothelial growth factor (anti-VEGF) delivery systems. However, the laser therapies temporarily stabilize the existing leaky blood vessels, not prevent the neovascularization which cause disease progression. While intravitreal drug delivery systems enhance the therapeutic efficacy to prevent the retinal neovascularization compared to conventional drug administration, they still have limitations in requirement of surgery, fast clearance, and non-biodegradability in the eyes, causing frequent administration, additional surgery, and adverse effects. Thus, the development of injectable drug delivery systems with long-term drug efficacy, and degradability is required. The objectives of this dissertation are to develop the hydrogel-based drug delivery carriers with minimally invasiveness, intravitreal controlled anti-VEGF delivery for long-term efficacy, and biodegradability for improved clinical performance and less adverse effects by reducing intravitreal injection frequency, especially in treatment of retinal neovascularization, through formulation design of hydrogels in particle and rod shapes. In chapter 2, injectable microgel/hydrogel composites were developed to achieve long-term and sustained ranibizumab delivery for the treatment of neovascular retinal diseases. Hyaluronic acid (HA) microgels were fabricated through thiol-ene reaction and emulsification, showing adjustable ranibizumab loading efficiency (43.3 ~ 97.6%) depending on size of microgels. The microgel/hydrogel composites were prepared by incorporating HA microgels into enzyme-mediated in situ crosslinkable gelatin-PEG-tyramine (GPT) hydrogels as a secondary barrier matrix, exhibiting reduced initial burst release and prolonged release of ranibizumab over 90 days compared to HA microgels. The released ranibizumab revealed biological activity by neutralizing VEGF in vitro. Then, in vivo pharmacokinetics (PK) were evaluated via rabbit intravitreal injection of bolus ranibizumab, ranibizumab-loaded microgel, and ranibizumab-loaded microgel/hydrogel composites, resulting in significantly reduced burst release and maintenance of ranibizumab concentration for 120 days in the gel-implant groups compared to bolus ranibizumab. Moreover, all eyes showed no inflammatory signs or abnormal apoptosis by microscopic and histologic analyses. These results indicated that both HA microgels and microgel/hydrogel composites are expected to serve as promising carriers independently to overcome current challenges of intravitreal anti-VEGF therapy by sustaining anti-VEGF release and extending the injection intervals. Chapter 3 described a novel intravitreally injectable pre-crosslinked hydrogel rod for controlling the bevacizumab release profiles and extend the intravitreal maintenance of bevacizumab concentration for several months to contribute the treatment of posterior segment eye disease. As prescribed, enzyme-mediated in situ cross-linkable gelatin-PEG-tyramine (GPT) hydrogels were utilized for fabricating the hydrogel rod to achieve a potentially sustainable and controllable protein delivery. The GPT hydrogel rod loaded with bevacizumab was prepared using easily removable thermo-responsible gelatin mold. The hydrogel rods were injectable through 21-gauge needle using precisely designed rod injector. By adjusting the crosslinking degree of hydrogel rods, precise control of initial release amount and long-term release of bevacizumab for several months were achieved. Moreover, the in vitro protein release profile of pre-crosslinked hydrogel rods was significantly controlled in comparison to in situ forming hydrogels at the same crosslinking degree. The therapeutic effects of anti-VEGF and cytocompatibility were proven via in vitro cellular assays using Human Umbilical Vein Endothelial Cells (HUVECs). Then, in vivo PK were evaluated via rabbit intravitreal injection of bolus bevacizumab, bevacizumab-loaded injectable hydrogels, and bevacizumab-loaded hydrogel rods, resulting in significantly inhibited bevacizumab clearance and prolonged maintenance of bevacizumab over effective concentration for 120 days in the hydrogel rod groups compared with bolus bevacizumab and injectable hydrogel groups. The structural stability, safety, and biodegradability of hydrogel rods were assessed by ultrasound images. Moreover, the non-inflammatory response of eyes was evaluated via enzyme-linked immunosorbent assay (ELISA) of rabbit TNF-α and rabbit VEGF. These results indicated that this novel hydrogel rods have a potential for drug delivery in a controlled manner, contributing to the clinical application to treat retinal neovascular diseases as well as various diseases that need potential sustained local drug delivery.
more목차
Chapter 1. General introduction 1
1. Retinal neovascularization (RNV) 2
2. Conventional clinical treatment for RNV 3
2.1. Laser therapy 3
2.1.1. Thermal laser photocoagulation (TLP) 3
2.1.2. Photodynamic therapy (PDT) 4
2.1.3. Limitation of laser therapies 5
2.2. Anti-vascular endothelial growth factor (anti-VEGF) administration 6
2.2.1. Anti-VEGF therapy with various routes of administration 7
2.2.2. Intravitreal anti-VEGF administration 9
2.2.3. Requirement of intravitreal controlled anti-VEGF delivery 9
3. Current strategies for intravitreal controlled drug delivery 11
3.1. Invasive surgical drug delivery system 11
3.1.1. Port delivery system (PDS) 11
3.1.2. Current challenges in invasive drug delivery system 12
3.2. Minimally invasive non-surgical drug delivery systems 12
3.2.1. Micro-, nano- particles 13
3.2.1.1. Liposome 13
3.2.1.2. Micelle 14
3.2.1.3. Polymeric spheres 15
3.2.2. In situ forming hydrogels 18
3.2.2.1. In situ forming particle/hydrogel composite 20
3.2.3. Injectable rod-type implants 21
3.2.4. Current challenges in minimally invasive drug delivery systems 22
4. Overall objectives 24
5. References 26
Chapter 2. Injectable Hyaluronan Microgel-embedded Gelatin Hydrogel for Controlled Ranibizumab Delivery 41
1. Introduction and objective 42
2. Experimental section 44
2.1. Materials 44
2.2. Synthesis of HA microgel and GPT hydrogel precursors 45
2.3. Fabrication of microgels and microgel/hydrogel composites 47
2.4. Preparation of ranibizumab-loaded microgels 48
2.5. Evaluation of ranibizumab release profiles 49
2.6. Assessment of in vitro anti-VEGF efficacy 50
2.7. Investigation of in vitro degradation 50
2.8. Procedures of animal experiments 51
2.9. Evaluation of intraocular toxicity 53
2.10. Statistical analysis 53
3. Results and discussion 54
3.1. Synthesis and chemical structure of polymer precursors 54
3.2. Quantitative analysis of conjugated functional groups 56
3.3. Morphology analysis of microgels and microgel/hydrogel composites 56
3.4. Controllable protein loading and release capacity of microgels 58
3.5. Long-term controlled ranibizumab release profiles 59
3.6. In vitro VEGF neutralization efficiency 61
3.7. In vitro proteolytic degradability 62
3.8. In vivo immunoassay and PK analyses 64
3.9. In vivo toxicity of microgels and microgel/hydrogel composites 71
4. Conclusions 73
5. References 74
Chapter 3. Injectable Gelatin Hydrogel Rod for Controlled Bevacizumab Delivery and Long-term Stability 81
1. Introduction and objective 82
2. Experimental section 85
2.1. Materials 85
2.2. Synthesis of GPT hydrogel precursors 86
2.3. Preparation and rheological analysis of GPT hydrogel 87
2.4. Fabrication of GPT hydrogel rod and rod injector 87
2.5. Determination of Swelling ratio 88
2.6. Evaluation of bevacizumab release profiles 89
2.6.1. Release from hydrogel rods depending on the crosslinking degree 89
2.6.2. Release from hydrogel rods vs. in situ forming hydrogels 90
2.7. Assessment of in vitro biological activity and toxicity 90
2.8. Investigation of in vitro degradation 91
2.9. Procedures of animal experiment, immunoassay, and PK analyses 92
2.10. Evaluation of intraocular stability and safety 93
2.11. Statistical analysis 94
3. Results and discussion 95
3.1. Chemical analysis of GPT polymer 95
3.2. Preparation and mechanical strength of GPT hydrogel with tunable crosslinking degree 96
3.3. Fabrication of GPT hydrogel rods with controlled crosslinking degree 97
3.4. Controlled swelling ratio of hydrogel rods 98
3.5. Controllable bevacizumab release profiles 99
3.5.1. Release from hydrogel rods depending on the crosslinking degree 99
3.5.2. Release from hydrogel rods vs. in situ forming hydrogels 101
3.6. Long-term inhibited proliferation and viability of endothelial cells 103
3.7. In vitro proteolytic degradability of hydrogel rods 105
3.8. In vivo immunoassay and PK analyses 106
3.9. Intraocular ultrasound images and pro-inflammatory analysis 112
4. Conclusions 114
5. References 116
Chapter 4. Overall conclusions 121
