HPRT1 Gene Amplification via In-Gel PCR and pH-Responsive Drug Release from DOX@ZIF-8-Loaded Photo-Crosslinked GelMA Hydrogels

다국어 초록 (Multilingual Abstract)

This thesis investigates photo-crosslinkable gelatin methacrylate (GelMA) hydrogels as an adaptable hydrogel matrix for two independently evaluated biomedical applications: HPRT1 gene amplification via in-gel PCR and pH-responsive drug release from DOX@ZIF-8-loaded GelMA composite hydrogels.

In the first study, thermal PCR amplification of the HPRT1 housekeeping gene was performed within UV-crosslinked GelMA hydrogels. Among the tested formulations containing 0.25–0.75% (w/v) LAP, GEL-LAP5 showed the strongest apparent amplification signal under the tested conditions. PCR amplification was inhibited when PCR reagents were exposed to UV crosslinking, likely due to radical-induced damage to DNA templates and polymerase. In contrast, loading PCR reagents into pre-crosslinked GelMA hydrogels restored amplification. Gel electrophoresis and supplementary real-time PCR results supported the feasibility of pre-crosslinked GelMA hydrogels as matrices for in-gel PCR amplification.
In the second study, doxorubicin-loaded ZIF-8 (DOX@ZIF-8) nanoparticles were synthesized by a one-pot aqueous method and incorporated into GelMA hydrogels. Structural and physicochemical characterization supported the formation of DOX@ZIF-8 nanoparticles and their incorporation into the GelMA matrix. Based on indirect analysis of residual DOX in the supernatant and wash solutions, the encapsulation efficiency was estimated to be approximately 99%. The GelMA/DOX@ZIF-8 composite hydrogels exhibited pH-dependent DOX release, with cumulative release of approximately 64% at pH 5.0 and 14% at pH 7.4 over 72 h. In vitro MTT assays showed dose-dependent cytotoxicity in MCF-7 cells, reducing cell viability to approximately 43% at 4 μM, while NIH 3T3 cells retained approximately 64% viability under the same DOX-equivalent concentration.

Collectively, these findings indicate that photo-crosslinked GelMA hydrogels can be adapted for HPRT1 in-gel PCR amplification and pH-responsive DOX release. Because these two functions were evaluated independently using different GelMA-based formulations, this study provides proof-of-concept evidence for separate GelMA-based in-gel PCR and drug release applications, and may serve as a basis for future integrated GelMA-based biomedical systems.

국문 초록 (Abstract)

본 학위논문에서는 광가교 젤라틴 메타크릴레이트(gelatin methacrylate, GelMA) 하이드로젤을 생의학적 응용을 위한 하이드로젤 매트릭스로 활용하여, HPRT1 유전자의 겔 내 PCR 증폭과 DOX@ZIF-8 담지 GelMA 복합 하이드로젤의 pH 반응성 약물 방출 가능성을 각각 독립적으로 평가하였다.

첫 번째 연구에서는 UV로 가교된 GelMA 하이드로젤 내에서 HPRT1 housekeeping gene의 thermal PCR 증폭을 수행하였다. 0.25–0.75% (w/v) LAP 농도를 포함하는 GelMA 제형 중 GEL-LAP5가 본 실험 조건에서 가장 강한 증폭 신호를 나타냈다. PCR 시약이 UV 가교 과정 중 함께 존재할 경우 증폭이 억제되었으며, 이는 LAP 광분해 과정에서 생성된 라디칼에 의한 DNA template와 polymerase 손상 때문으로 판단된다. 반면, GelMA 하이드로젤을 먼저 가교한 후 PCR 시약을 도입한 경우 증폭이 회복되었다. Agarose gel electrophoresis와 보조적인 real-time PCR 결과는 pre-crosslinked GelMA 하이드로젤이 겔 내 PCR 증폭을 위한 매트릭스로 활용될 수 있음을 보여주었다.

두 번째 연구에서는 doxorubicin이 담지된 ZIF-8(DOX@ZIF-8) 나노입자를 one-pot 수계 합성법으로 제조하고, 이를 GelMA 하이드로젤에 도입하여 GelMA/DOX@ZIF-8 복합 하이드로젤을 제작하였다. 구조적 및 물리화학적 분석을 통해 DOX@ZIF-8 나노입자의 형성과 GelMA 매트릭스 내 도입을 확인하였다. 상등액과 세척액에 잔류한 DOX를 기반으로 간접 정량한 결과, 봉입효율은 약 99%로 추정되었다. GelMA/DOX@ZIF-8 복합 하이드로젤은 pH 의존적 DOX 방출 거동을 보였으며, 72시간 동안 pH 5.0 조건에서는 약 64%, pH 7.4 조건에서는 약 14%의 누적 방출률을 나타냈다. In vitro MTT assay 결과, DOX 등가 농도 4 μM에서 MCF-7 유방암 세포 생존율은 약 43%까지 감소하였으며, NIH 3T3 섬유아세포는 약 64%의 생존율을 유지하였다. 이는 본 실험 조건에서 NIH 3T3 세포보다 MCF-7 세포에서 더 높은 세포독성 반응이 나타났음을 의미한다.

종합적으로, 본 연구는 광가교 GelMA 하이드로젤이 HPRT1 겔 내 PCR 증폭과 pH 반응성 DOX 방출에 각각 적용될 수 있음을 보여준다. 두 기능은 각 응용 목적에 따라 선택된 서로 다른 GelMA 기반 제형에서 독립적으로 평가되었으므로, 본 연구는 GelMA 기반 하이드로젤의 겔 내 PCR 및 약물 방출 응용 가능성을 확인한 기초 가능성 평가 연구로 해석될 수 있다. 이러한 결과는 향후 통합형 GelMA 기반 생의학 시스템 개발을 위한 기초 자료로 활용될 수 있다.

목차 (Table of Contents)

• CHAPTER 1. Introduction 1
• 1.1 Research Background 1
• 1.2 Gelatin Methacrylate (GelMA) Hydrogels 4
• 1.2.1 Synthesis and Photo-Crosslinking Mechanism 4
• 1.2.2 Physicochemical Properties and Tunability 7
• 1.2.3 Biomedical Applications of GelMA 9
• 1.3 Hydrogel-Based Molecular Diagnostics 11
• 1.3.1 Principles and Limitations of Polymerase Chain Reaction (PCR) 11
• 1.3.2 In-Gel PCR: Concept and Advantages 13
• 1.3.3 HPRT1 as a Housekeeping Gene for PCR Validation 16
• 1.4 MOF-Based Drug Delivery Systems 18
• 1.4.1 Structure and Properties of ZIF-8 18
• 1.4.2 pH-Responsive Degradation Mechanism of ZIF-8 20
• 1.4.3 Doxorubicin (DOX) as a Model Anticancer Drug 22
• 1.5 GelMA as a Common Matrix for In-Gel PCR and pH-Responsive DOX Release 24
• 1.5.1 Rationale for Combining MOF Nanoparticles with Hydrogels 24
• 1.5.2 Tumor Microenvironment and Localized Drug Delivery 27
• 1.6 Research Motivation and Objectives 29
• CHAPTER 2. GelMA Hydrogel as a Platform for In-Gel PCR Amplification 32
• 2.1 Introduction 32
• 2.2 Experimental 35
• 2.2.1 Materials 35
• 2.2.2 Synthesis of GelMA 35
• 2.2.3 Preparation of Photo-Crosslinked GelMA Hydrogels 36
• 2.2.4 Characterization Methods 38
• 2.2.5 PCR Amplification of HPRT1 Gene 40
• (1) PCR Reaction Setup and Thermal Cycling Conditions 40
• (2) Experimental Groups and Controls 42
• (3) Agarose Gel Electrophoresis and Real-Time PCR 42
• (4) Band Intensity Analysis (ImageJ) 43
• 2.3 Results and Discussion 44
• 2.3.1 Morphological and Elemental Analysis (SEM–EDX) 44
• 2.3.2 Confirmation of Methacrylation (¹H NMR) 46
• 2.3.3 Chemical Structure Analysis (FTIR) 48
• 2.3.4 Crystallinity Analysis (XRD) 51
• 2.3.5 Optical Properties and LAP Incorporation (UV–Vis) 53
• 2.3.6 Viscoelastic Properties and Crosslinking Behavior (Rheology) 56
• 2.3.7 Thermal Stability (TGA) 59
• 2.3.8 PCR Amplification Performance 61
• (1) Effect of GelMA and LAP Concentration 62
• (2) Effect of UV Crosslinking Strategy 64
• (3) Quantitative PCR Analysis and Validation 66
• 2.4 Summary 69
• CHAPTER 3. GelMA/DOX@ZIF-8 Composite Hydrogel for pH-Responsive Drug Release 71
• 3.1 Introduction 71
• 3.2 Experimental 73
• 3.2.1 Materials 73
• 3.2.2 Synthesis of DOX@ZIF-8 Nanoparticles 73
• 3.2.3 Fabrication of GelMA/DOX@ZIF-8 Composite Hydrogels 75
• 3.2.4 Characterization Methods 77
• 3.2.5 In Vitro Drug Release Study (pH 7.4 and pH 5.0) 79
• 3.2.6 In Vitro Cytotoxicity Evaluation 80
• (1) Cell Culture (MCF-7 and NIH 3T3) 80
• (2) Sample Preparation 81
• (3) MTT Assay Protocol 82
• 3.3 Results and Discussion 84
• 3.3.1 Structural Confirmation of ZIF-8 and DOX@ZIF-8 (PXRD) 84
• 3.3.2 Chemical Interaction Analysis (FT-IR) 87
• 3.3.3 Morphological Analysis (FE-SEM) 90
• 3.3.4 Particle Size and Distribution (DLS) 93
• 3.3.5 Drug Loading and Encapsulation Efficiency (UV–Vis) 96
• 3.3.6 Thermal Analysis (TGA) 101
• 3.3.7 pH-Responsive Drug Release Behavior 104
• 3.3.8 Cytotoxicity Against MCF-7 Cancer Cells 109
• 3.3.9 Biocompatibility with NIH 3T3 Normal Cells 113
• 3.3.10 Comparative Cytotoxicity and Therapeutic Implications 117
• 3.4 Summary 121
• CHAPTER 4. Conclusions and Outlook for GelMA-Based Biomedical Hydrogel Systems 123
• 4.1 Summary of In-Gel PCR and Drug Release Applications 123
• 4.2 Limitations of This Study 125
• 4.3 Suggestions for Future Research 127
• References 129
• 국문초록 135