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Title Page
Abstract
Contents
CHAPTER 1. General Introduction 24
1.1. Drug delivery systems 25
1.2. Hydrogels 27
1.3. Biomaterials 27
1.4. Natural materials 28
1.5. Synthetic materials 29
1.6. Strategy of this work 29
CHAPTER 2. Preparation and in vivo evaluation of an injectable crosslinked cartilage acellular matrix-PEG hydrogel scaffold derived from porcine cartilage 32
2.1. Introduction 33
2.2. Experimental section 37
2.2.1. Materials 37
2.2.2. Preparation of a CAM powder 38
2.2.3. Determination of Double Strand DNA of CAM Before and After Decellularization 39
2.2.4. Preparation of a Near Infrared (NIR) tagged CAM powder 40
2.2.5. Synthesis of COOH-PEG-400-COOH 40
2.2.6. Synthesis of NHS-PEG-400-NHS (PEG crosslinker) 41
2.2.7. Preparation of crosslinked CAM using PEG cross-linker 42
2.2.8. Preparation of CAM suspensions 43
2.2.9. Hydrophilicity test of CAM films and CAM hydrogels 44
2.2.10. Rheological properties of CAM hydrogels 45
2.2.11. Injectability test of CAM hydrogels 45
2.2.12. Animal experiment 46
2.2.13. In vivo biodegradation and biocompatibility experiments of NIR-labeled CAM, 0.6-CAM-GA, 0.6-CAM-PEG, 1-CAM-PEG, 3-CAM-PEG and 5-CAM-PEG hydrogels 47
2.2.14. In vivo implantation of CAM, 0.6-CAM-GA, 0.6-CAM-PEG 47
2.2.15. Histological assay of CAM, 0.6-CAM-GA, 0.6-CAM-PEG hydrogels 48
2.2.16. Statistical analysis 50
2.3. Results and Discussion 51
2.3.1. Preparation of CAM powder 51
2.3.2. Synthesis of COOH-PEG-400-COOH and NHS-PEG-400-NHS. 54
2.3.3. Preparation of CAM, 0.6-CAM-GA, 0,6-CAM-PEG films. 56
2.3.4. Preparation and characterization of CAM-PEG Powders 61
2.3.5. Hydrophilicity test of CAM, 0.6-CAM-GA, 0.6-CAM-PEG, 1-CAM-PEG, 3-CAM-PEG, and 5-CAM-PEG films 64
2.3.6. Rheological properties of CAM hydrogels 69
2.3.7. Injectability test of CAM hydrogels 72
2.3.8. In vivo biodegradation of NIR-labeled CAM, 0.6-CAM-GA, 0.6-CAM-PEG, 1-CAM-PEG, 3-CAM-PEG and 5-CAM-PEG hydrogels 75
2.3.9. In vivo biodegradation of CAM, 0.6-CAM-GA, 0.6-CAM-PEG hydrogels 79
2.3.10. In vivo biocompatibility of CAM, 0.6-CAM-GA, 0.6-CAM-PEG hydrogels 81
2.4. Conclusion 85
CHAPTER 3. Anti-cancer activity of intratumorally injectable in-situ forming hyaluronic acid hydrogel 86
3.1. Introduction 87
3.2. Experimental section 92
3.2.1. Materials 92
3.2.2. Preparation of HA-Tet and HA-TCO hydrogels 92
3.2.3. Preparation of near-infrared (NIR) fluorescence-labeled HA-Tet (HA-Tet-NIR), HA-TCO (HA-TCO-NIR) and HA (HA-NIR) hydrogels 93
3.2.4. Preparation of HA-Dox Cx-HA-Dox, HA-NIR-Dox and Cx-HA-NIR-Dox formulation 94
3.2.5. Zeta potential of HA, HA-TCO, HA-Tet, Dox, HA-Dox, HA-TCO-Dox and HA-Tet-Dox 95
3.2.6. Rheological properties of hydrogels 95
3.2.7. Injectability test of hydrogels 96
3.2.8. In vitro release of Dox from HA and Cx-HA hydrogels. 97
3.2.9. In vitro degradation of NIR tagged HA and Cx-HA hydrogels and Dox release from HA and Cx-HA hydrogels 98
3.2.10. In vitro anti-tumor activity 99
3.2.11. Inhibitiory effects. 100
3.2.12. Animal study 100
3.2.13. Ex vivo fluorescent images of remained Dox and NIR labeled Cx-HA hydrogel within tumors and organs. 101
3.2.14. Distribution of Dox in tumors after intratumoral injection 102
3.2.15. Histological assay 102
3.2.16. Statistical analysis 105
3.3. Result and discussion 106
3.3.1. Preparation and charaterization of Cx-HA hydrogel 106
3.3.2. Zeta potential of HA hydrogel formulations 109
3.3.3. Rheological properties and injectability of Cx-HA-Dox hydrogel formulation 113
3.3.4. In vitro Dox release and in vitro degradation of HA and Cx-HA hydrogels. 118
3.3.5. In vivo Dox release and in vivo degradation of HA and Cx-HA hydrogels. 122
3.3.6. In vivo anti-tumor effect of Dox of formulations. 125
3.3.7. In vivo fluorescent images of remained Dox and NIR labelled Cx-HA hydrogel. 128
3.3.8. Distribution of Dox in tumors after intra-tumoral injection 132
3.3.9. Histological assay 134
3.4. Discussion 140
3.5. Conclusion 144
CHAPTER 4. Overall conclusion 145
References 147
List of Publications 151
List of Patents 154
List of Book chapter 155
List of Presentations 155
List of Awards 158
Abstract (In Korean) 160
CHAPTER 1. 14
Figure 1.1. Drug release profile and drug concentration of (a) conventional drug administration and (b) drug delivery system 26
Figure 1.2. Natural materials and synthetic materials for use of DDS 28
CHAPTER 2. 14
Figure 2.1. Overall scheme of preparation of CAM hydrogel via PEG crosslinker. 36
Figure 2.2. Images of (a) porcine cartilage, (b) Decellularized porcine cartilage (CAM), (c) dried CAM, (d) CAM powder after freeze-milling, (e) dsDNA contents of native tissue and CAM (*p 〈0.01). 53
Figure S1. ¹H NMR spectra of (a) COOH-PEG-400-COOH and (b) NHS-PEG-400-NHS cross-linker. 55
Figure 2.3. Heat of fusion and temperature in a) DSC at 0-300°C, b) enlarged DSC at 0-175°C, and c) enlarged DSC at 150-300°C of CAM, 0.6-CAM-GA, 0.6-CAM-PEG, 1-CAM-PEG, 3-CAM-PEG, and 5-CAM-PEG. 58
Figure S2. In vitro biodegradation test of CAM, 0.6-CAM-GA, and 0.6-CAM-GA films by 10 CDU collagenase/mL in 1X PBS. 60
Figure 2.4. Particle sizes of (a) CAM, (b) 0.6-CAM-GA, (c) 0.6-CAM-PEG, (d) 1-CAM-PEG, (e) 3-CAM-PEG, and (f) 5-CAM-PEG measured by DLS. 62
Figure 2.5. FT-IR spectra of (a) CAM, (b) 0.6-CAM-GA, (c) 0.6-CAM-PEG, (d) 1-CAM-PEG, (e) 3-CAM-PEG, and (f) 5-CAM-PEG. 63
Figure 2.6. (a) Image of a dried CAM film and (b) images of water contact angle and water contact angle degrees of CAM, 0.6-CAM-GA, 0.6-CAM-PEG, 1-CAM-PEG, 3-CAM-PEG, and 5-CAM-PEG. 66
Figure 2.7. (a) Images of DW absorbed by CAM powders loaded into Pastuer pipettes, (b) plots of CAM, 0.6-CAM-PEG, 1-CAM-PEG, 3-CAM-PEG, and 5-... 68
Figure 2.8. (a) Images of 0.6-CAM-PEG hydrogel, (b) complex viscosities, (c) complex moduli, (d) shear stress and (e) viscosity versus strain graphs of 0.6-CAM-GA, CAM, 0.6-CAM-PEG, 1-CAM-PEG, 3-CAM-PEG and 5-CAM-PEG... 71
Figure 2.9. (a) Image of 0.6-CAM-PEG hydrogel loaded in 3 mL Norm-Ject Luer Lock tip syringe, image of injectability test and injectability test of CAM, 0.6-... 74
Figure S3. Synthesis of ¹H NMR spectra of (a) IR-783 and (b) IR-783-COOH. 77
Figure 2.10. (a) Bright and NIR fluorescent images of NIR conjugated CAM hydrogel in a syringe, (b) in vivo injection and (c) in vivo NIR fluorescent image of... 78
Figure 2.11. (a) In vivo subcutaneous injection image of CAM hydrogel, (b) image of CAM hydrogel formed as a depot and (c) gross images of harvested CAM, 0.6-CAM-GA, 0.6-CAM-PEG on week 1, 2 and 4. 80
Figure 2.12. H&E staining images of CAM, 0.6-CAM-PEG, and 0.6-CAM-GA hydrogel scaffold section in weeks 1, 2, and 4 (scale bar: 200 μm, red scale bar: 20 μm). 83
Figure 2.13. (a) ED1 staining images, (b) CD4 staining images, (c) ED1 positive cells/hematoxylin positive cells and (d) CD4 positive cells/hematoxylin positive cells of CAM, 0.6-CAM-GA, and 0.6-CAM-PEG hydrogels section on weeks 1,... 84
CHAPTER 3. 18
Figure 3.1. Schematic images of preparation of doxorubicin containing drug depot and tumor suppression via intratumoral injections and sustained release of Dox 91
Figure S1. ¹H NMR spectra of free HA, HA-TCO, HA-Tet and Cx-HA 107
Figure 3.2. (a) Zeta potential of HA, HA-Dox, HA-TCO, HA-TCO-Dox HA-Tet, HA-Tet-Dox, Cx-HA, Cx-HA-Dox, and Dox, (b) Storage and loss moduli, (c) tan δ, (d) storage moduli and (e) complex viscosities of HA, HA-Dox, Cx-HA,... 111
Figure S2. Particle sizes of HA, HA-Dox, HA-TCO, HA-TCO-Dox, HA-Tet, HA-Tet-Dox, Cx-HA, and Cx-HA-Dox (*p 〈0.01) 112
Figure 3.3. (A) Images of the formulations of (a1) HA, and (a2) HA-Dox in a single barrel syringe, (a3) HA-Tet, and HA-TCO, (a4) HA-Tet-Dox, and HA-TCO-Dox at room temperature, (B) injected hydrogel depot of (b1)HA, (b2) HA-... 116
Figure S3. Injection and remained hydrogel images of HA-Dox and Cx-HA-Dox at each time point 117
Figure 3.4. (a) In vitro accumulated Dox, (b) Dox release from HA, Cx-HA-Dox hydrogel, and (c) enlarged in vitro Dox release, (d) Tₘₐₓ, Cₘₐₓ, Dox conc. at Cₘₐₓ, and AUC₀₋ₜ of HA-Dox and Cx-HA-Dox, (e) in vitro degradation of NIR... 120
Figure S4. In vitro fluorescent images Dox in red color and NIR tagged Cx-HA hydrogel (NIR-Cx-HA-Dox) in green color remaining after injected in 1X PBS 121
Figure 3.5. (a) Optical and fluorescent images showing the morphology of B16F10 cells treated with PBS, Dox single, Dox repeat, HA-Dox, and Cx-HA-Dox after 1 d, 2 d, and 3 d (scale bar: 50 μm), (b) in vitro anticancer activity of Dox... 124
Figure 3.6. Inhibitory effects of (a) full tumor volume, (b) plots of tumor growth rate constant, and (c) tumor volume doubling time, tumor growth rate, and tumor... 126
Figure S5. Body weight after injection of Control, Dox single, Dox repeat, HA-Dox, and Cx-HA-Dox. Each solution was injected into xenograft-bearing mice... 127
Figure 3.7. (a) In vivo fluorescent images of remained Dox (red) and NIR labelled Cx-HA hydrogel (green) into harvested tumors on 1, 4, 6, 12, and 18 days.... 130
Figure S6. In vivo fluorescent images of remained Dox (red) and NIR labelled Cx-HA hydrogel (green) into harvested tumors and tissues on 1, 6, 12, and 18 days 131
Figure 3.8. Distribution of (a) Dox in tumors after intra-tumoral injection of Dox single, Dox repeat, HA-Dox and Cx-HA-Dox as well as Dox distribution in all organs after intra-tumoral injection of (b) Dox single, (c) Dox repeat, (d) HA-... 133
Figure 3.9. (a) H&E-stained histological sections of tumors on 1, 6, 12, and 18 days after intratumoral injection of xenograft-bearing mice with saline, Dox single,... 137
Figure 3.10. (a) DAPI staining (blue, nuclei), BCL-2 staining (green, BCL-2 positive cells) of tumors on 1, 6, 12, and 18 days after intra-tumoral injection of... 138
초록보기
약물전달시스템(DDS)은 필요한 양의 약물을 원하는 신체 부위에 효율적으로 전달하고 부작용을 최소화할 수 있는 제형을 만들어 약물 치료를 최적화하는 기술이다. 지난 수십 년 동안 DDS 는 기존 제형의 문제점을 해결하기 위해 다양한 방식으로 연구되었습니다. 기존의 약물 전달 시스템은 빠른 초기 방출, 생체 내 세포 독성 및 면역 거부와 같은 미해결 문제를 가지고 있으며 생체에 이식하여 사용하기에는 적합하지 않습니다. 따라서, 약물 농도의 혈장 농도를 약효가 발휘되는 수준으로 유지하고, 함유된 약물을 원하는 부위에 방출하고, 세포 독성 및 면역 거부 반응을 일으키지 않는 첨단 약물 전달 시스템이 필요하다. 많은 DDS 제제 중 주사형 하이드로겔은 수술 없이 원하는 부위에 약물을 효과적으로 전달하고 약물의 서방형 방출을 유도할 수 있다. 본 연구에서는 천연 생체재료를 이용하여 약효를 증가시키기 위한 주사형 하이드로겔을 제조하였다.
1 장에서는 연구 배경, 전반적인 약물 전달 시스템 및 천연 유래 물질을 사용한 주사 가능한 하이드로겔 제조에 대한 일반적인 소개를 설명합니다.
2 장에서는 천연 생체재료인 돼지 연골 유래 세포 외 기질을 이용한 생체적합성이 우수한 주사형 하이드로겔의 제조에 대한 연구를 설명하였다. 돼지연골 유래 소재는 친수성이 낮아 주사용 하이드로겔 제조에 한계가 있으며, 매우 빠르게 생분해 된다는 한계가 있다. 따라서 이러한 단점을 보완하기 위해 생체적합성과 친수성이 높은 PEG 를 이용하여 돼지 연골 유래 조직을 가교하였다. PEG 를 이용하여 가교된 하이드로겔은 in vivo 실험에서 우수한 생체적합성을 보였으며, PEG 의 가교 농도에 따라 물성 및 주입성이 조절됨을 확인하였다.
3 장에서는 생체적합성이 우수한 생체재료인 히알루론산(HA)을 이용하여 주사 가능한 하이드로겔을 제조하였다. 히알루론산은 생체적합성이 뛰어나다는 장점이 있지만 생분해가 매우 빠르다는 단점이 있다. 이러한 단점으로 인해 HA 를 약물 전달 시스템으로 사용할 경우 HA 에 담지된 약물의 초기 폭발을 억제하기 어렵고 약물 효과의 지속 시간도 짧다. 따라서 이러한 단점을 해결하기 위해 HA 에 Tetrazine 과 trans cyclooctene 을 각각 도입한 후 Diels Alder click reaction 을 통해 cross-linkin 을 유도하고 종양에 직접 주입 시 in-situ forming 하이드로겔을 형성할 수 있도록 하였다. HA 하이드로겔은 가교반응을 통해 항암제인 독소루비신의 서방출을 유도하여 항암효과를 높였다.
결론적으로 본 연구의 결과는 천연 생체재료를 이용한 주사형 하이드로겔의 제조방법을 제시하였다.MARC 보기
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