repository
효소 촉매 반응 효소 고정화 방법을 이용한 In situ 형성 하이드로젤의 제조 및 특성 평가
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 박기동 | - |
| dc.contributor.author | Kim Bae Young | - |
| dc.date.accessioned | 2018-11-08T08:11:15Z | - |
| dc.date.available | 2018-11-08T08:11:15Z | - |
| dc.date.issued | 2015-02 | - |
| dc.identifier.other | 19378 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/11375 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :분자과학기술학과,2015. 2 | - |
| dc.description.tableofcontents | I. Introduction 1 1. In situ forming hydrogels 1 1.1. Cross-linking strategies for in situ hydrogelation 2 1.1.1. Physically cross-linked hydrogels 5 1.1.2. Chemically cross-linked hydrogels 5 2. Enzyme-catalyzed cross-linking system 6 2.1. Transglutaminase-catalyzed cross-linking system 9 2.2. Tyrosinase-catalyzed cross-linking system 12 2.3. Phosphatase-catalyzed cross-linking system 14 2.4. Horseradish peroxidase-catalyzed cross-linking system 15 2.5. Glucose oxidase-catalyzed cross-linking system 20 3. Ideal cross-linking method for clinical translation of hydrogels 22 4. Overall objectives 23 II. Materials and Methods 24 1. Materials 24 2. In situ forming gelatin hydrogels via HRP- and GOx-mediated reaction 25 2.1. Synthesis of GPT conjugates 25 2.2. Preparation of GPT hydrogels and gelation time measurement 27 2.3. Rheological experiment 28 2.4. Compressive test 29 2.5. In vitro cytotoxicity study 29 2.6. In vitro three-dimensional cell study 30 2.7. In vitro proteolytic degradation test 31 3. In situ forming enzyme-free hydrogels via ferromagnetic microbead-assisted enzymatic cross-linking 31 3.1. Preparation and characterizations of enzyme immobilized beads 31 3.1.1. Preparation of iron-containing polymeric beads 31 3.1.2. Immobilization of enzymes onto beads 32 3.1.3. Characterizations of enzyme-beads 33 3.2. Preparation and characterizations of enzyme-free hydrogels 35 3.3. Fluorescent detection of enzymes in hydrogels 36 3.4. In vitro three-dimensional culture of hDFBs 37 III. Results and Discussion 39 1. In situ forming gelatin hydrogels via HRP- and GOx-mediated reaction 39 1.1. Synthesis and characterizations of GPT conjugates 39 1.2. Hydrogel formation and gelation time 41 1.3. Glucoseoxidaseandglucosedependentelasticmodulus 43 1.4. Additional cross-linking of hydrogels in culture medium 46 1.5. In vitro cytotoxicity of incorporated enzymes in hydrogels 47 1.6. In vitro hDFB cell proliferation in hydrogels 49 1.7. In vitro proteolytic degradation of hydrogels 49 2. In situ forming HRP-free gelatin hydrogels via ferromagnetic microbead-assisted enzymatic cross-linking 53 2.1. Characterizations of iron-containing poly(GMAMMA) beads 53 2.2. Characterizations of HRP immobilized ferromagnetic microbeads 54 2.3. Iron content changes of HRP-bead in aqueous solution 57 2.4. Catalytic activity of HRP-beads 59 2.5. Gelation time of gelatin hydrogels 61 2.6. Elastic modulus of gelatin hydrogels 62 2.7. Fluorescent detection of enzyme in gelatin hydrogels 64 2.8. In vitro proliferation study of hDFBs in hydrogels 67 3. In situ forming catalyst-free gelatin hydrogels via dual-enzyme immobilized beads 68 3.1. Characterizations of GOx immobilized ferromagnetic microbeads 68 3.2. Catalytic activity of enzyme-beads 71 3.3. Gelation time and elastic modulus of gelatin hydrogels 72 3.4. Reusability and storage stability of enzyme-beads 75 3.5. Fluorescent detection of enzyme in gelatin hydrogels 77 3.6. In vitro proliferation study of hDFBs in hydrogels 77 IV. Conclusion 81 V. References 83 Abstract in Korean 93 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 효소 촉매 반응 효소 고정화 방법을 이용한 In situ 형성 하이드로젤의 제조 및 특성 평가 | - |
| dc.title.alternative | Bae Young Kim | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Bae Young Kim | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2015. 2 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 695819 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000019378 | - |
| dc.subject.keyword | 하이드로젤 | - |
| dc.description.alternativeAbstract | 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. However, the inevitable incorporation of HRP and H2O2 during cross-linking may limit more extensive use of the system. Although HRP appears to be non-toxic, it may cause immune response resulted from its plant-derived origin. In addition, H2O2 is known to cause DNA damage and cell death at a high concentration. Therefore, there is an increasing demand for an alternative to the conventional HRP-catalyzed cross- linking method. The main objective of this dissertation is to develop and evaluate in situ forming enzymatically cross-linked hydrogels for injectable biomedical applications, which is based on enzyme immobilization and H2O2-generating enzyme. The first work of the thesis was to prepare and characterize an in situ forming gelatin hydrogel via HRP- and GOx-catalyzed cross-linking. The gelatin hydrogels were prepared from a gelatin solution above 5 wt% in the presence of HRP, GOx and Glucose. Their mechanical properties such as gelation time, swelling ratio and degradation time were evaluated at different HRP, GOx and glucose concentrations. In addition, it was clearly observed that gelatin hydrogels prepared via HRP- and GOx-mediated reaction were not cytotoxic under all given conditions, suggesting that the GOx-triggered cross-linking system enables us not only to minimize cellular damage by excess H2O2, but also maintain the catalytic activity of HRP. To improve unavoidable incorporation of enzymes into hydrogels, in situ forming enzyme-free hydrogels via ferromagnetic microbead- assisted enzymatic cross-linking were developed and characterized. We showed that the iron content in microbeads contributes greatly to the HRP- and GOx- catalyzed gelation process. The hydrogel was formed rapidly using HRP- and GOx immobilized beads with polymer solution containing glucose. The mechanical properties could be controlled from 400 Pa to over 7500 Pa by changing the bead-contact time. Furthermore, an in vitro 3D cell studies revealed that the enzyme-free GPT hydrogels could serve as a bioactive injectable matrix for cell delivery. Therefore, we expect that the interfacial in situ enzymatic cross- linking system using enzyme immobilized beads can be used as a certain alternative to the conventional method that has been widely used to prepare enzymatically cross-linked hydrogels. | - |
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
- Files in This Item:
- There are no files associated with this item.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.
-
- License
- STATISTICS
- Total Visit :3,747,772
- Total Download :1,818
- Today View :25,430
