Development of Stimuli-responsive Hydrogels based on Carboxymethyl-Cellulose for Drug Delivery System
- Alternative Title
- 약물 전달 시스템을 위한 카르복시메틸 셀룰로오스 기반 자극 반응성 하이드로겔의 개발
- Abstract
- 스티뮬러스 반응성 하이드로겔은 특정 부위에서 화학요법 분자의 원격 조절된 방출 덕분에 의약품 전달 시스템에서 널리 연구되고 있다. 조기 약물 방출은 치료 분자의 효율성을 감소시키고 원하지 않는 부작용을 유발할 수 있다. 최소한의 부작용으로 약물의 효능을 향상시키기 위해, 스튜뮬러스 반응성 하이드로겔은 외부 또는/그리고 내부 특정 트리거(예: 온도, pH, 산화환원, 빛, 자기장 또는 전기장, 효소, 반응산소종 (ROS) 등) 하에서 약물 방출을 조절할 수 있는 유망한 소재로 나타날 수 있다. 생체 적합성과 생분해성의 고유 속성으로 인해 탄수화물 기반 자연 고분자인 카르복시메틸 셀룰로오스(CMC)를 크로스링크된 디그레이더블 크로스링커로 사용하여 하이드로겔을 합성하고 특성화하였다. 크로스링커 및 폴리머 백본은 바이오-오소고널 "클릭 화합" 반응인 역전자 전자 수요 딜스-알더(IEDDA)와 같은 반응을 통해 공유결합되어 하이드로겔 시스템의 물리화학적 특성을 조절하는 데 기여했다. 이 반응은 생리학적 조건 하에서 간단하고 용이한 절차로 빠르게 진행되는 특성으로 인해 선호되었다.
논문의 후반부는 디셀레나이드 (Se-Se) 결합을 포함하는 환원 반응성 하이드로겔을 합성하는 데 전념되었다. 약물의 반응적 방출 행동, 즉 디셀레나이드 결합의 클리브리지 및 이후 환원 환경에서 하이드로겔 네트워크의 분해가 조사되었다. 나중에는 반응산소종 (ROS) 클리브러블 티오키탈 크로스링커를 사용하여 근적외선 (NIR) 반응성 약물 전달 시스템을 개발하였다. NIR-조사에 노출됨에 따라 ICG에서 생성된 ROS와 상호 작용하여 약물의 나노 지역적 방출 패턴이 조사되었다. NIR-조사는 무해하고 비침습적인 전략으로 알려져 있다.
이 논문에서 사용된 하이드로겔은 1H NMR, FE-SEM, UV-VIS 분광, FTIR 및 레오메트리와 같은 여러 특성화 기술을 사용하여 자세히 연구되었다. 크로스링커 농도를 변화시켜 젤레이션 시간, 팽만 행동, 기공도 및 레오로지적 특성을 조사했다. 하이드로겔의 세포 독성은 정상 세포 라인을 사용하여 분석되었으며, 무균 환경에서 암 세포 라인에 대한 하이드로겔의 체외 항암 효과가 NIR 및 환원 환경에서 결정되었다.
이 연구는 크로스링커의 중요한 역할을 강조하며 바이오 폴리머 기반 스튜뮬러스 반응성 약물 전달 물질의 개발과 특성화에 관여함을 강조한다. 디셀레나이드 기반 크로스링커인 DSe-DPEG-DTz를 사용하여 개발된 환원 반응성 및 생체 적합성 소프트 하이드로겔은 높은 팽만률(>35 배), 튜네이블 젤레이션 시간(1-5분) 및 우수한 독소률(>85%)을 보여주었다. 이 하이드로겔은 글루타치온 10 mmol의 존재하에서 PBS 솔루션 (38%)에 비해 글루타치온이 존재할 때 독소(DOX)의 유동 응답 및 빠른 방출(12시간 후 99%)을 보여주었다. NIR 반응성 하이드로겔 개발에서는 ROS-클리블 티오키탈 크로스링커를 사용하였다. NIR-조사에 노출 됨에 따라 하이드로겔은 ICG에서 생성된 ROS와 상호 작용하여 NIR-조사에 노출되면 특이 및 지속적인 약물 방출 패턴을 보여주었다. 체외 세포 독성 결과는 하이드로겔이 높은.|Stimuli-responsive hydrogels have been widely studied in drug delivery system owing to the remotely controlled release of chemotherapeutic molecules at the targeted site. The premature drug release reduces the efficiency of therapeutic molecules and can induce unwanted adverse impacts. Whether to improve drug efficacy with minimal side effects, stimuli-responsive hydrogel could be a promising material enabled to control release of the drug under external or/and internal specific triggers (e.g. temperature, pH, redox, light, magnetic or electric field, enzyme, reactive oxygen species (ROS) etc.). The controlled dynamic behavior of the materials in transporting the therapeutic substance in physiological conditions could be regulated by carefully crafting the chemical structure of the crosslinkers.
The main objective of this dissertation is to develop and design stimuli-responsive hydrogels for on-demand drug delivery system by employing a novel crosslinkers which could be decomposed ideally under specific stimulus. Responsiveness is introduced by incorporating the cleavable moieties into the structure of crosslinkers. The hydrogels composed of carbohydrate based natural polymer i.e., carboxymethyl cellulose (CMC) crosslinked with degradable crosslinkers were synthesized and characterized. CMC was used in hydrogel system owing to the inherent properties of biocompatibility and biodegradability. The crosslinker and polymer backbone were covalently bonded through bio-orthogonal “click chemistry” reaction such as inverse electron demand Dields Alder (IEDDA) contributed to tailor the physiochemical properties of hydrogel system. This reaction was preferred since it occurred rapidly under physiological conditions by simple and facile procedure. The second part of this thesis is devoted to synthesize reduction-responsive hydrogels containing di-selenide bond. The responsive release behavior of the drug was investigated i.e., the cleavage of di-selenide bond and subsequently decomposed the hydrogel network under reducing environment. In the later part, NIR-responsive drug delivery system was developed by employing a reactive oxygen species (ROS)-cleavable thioketal crosslinker. A NIR-dye (FDA approved), indocyanine green (ICG) was encapsulated into the hydrogel network to generate ROS-stimulus upon exposure of NIR-irradiation. The sustained and spatiotemporal release pattern of drug from its carrier was investigated by employing NIR-light known as an innocuous and non-invasive strategy.
Hydrogels were further studied in this dissertation using several characterization techniques such as, proton nuclear magnetic resonance (1H NMR), field emission scanning electron microscopy (FE-SEM), ultraviolet-visible (UV-VIS) spectroscopy, and Fourier transform infrared spectroscopy (FTIR). The physiochemical properties were measured by using a rheometer. The gelation times, swelling behavior, porosity, and rheological properties were investigated by varying the concentration of cross-linker. The cytotoxicity of hydrogels was analyzed by using normal cells lines. Moreover, in vitro anti-tumor efficacy of the hydrogels against cancer cell lines were determined under NIR and reducing environment.
This study highlights the essential role of crosslinker involves in development and characterization of biopolymer-based stimuli-responsive drug delivery material. Reduction-responsive and biocompatible soft hydrogels derived from CMC were successfully prepared by employing diselenide (Se-Se) based crosslinker, DSe-DPEG-DTz. The hydrogels showed high swelling ratios (>35 times), tunable gelation times (1–5 min), and excellent doxorubicin (DOX) loading efficiencies (>85 %). The hydrogels demonstrated stimuli-responsive and fast release of DOX (99 %, after 12 h) in the presence of 10 mmol of glutathione as compared to the normal PBS solution (38 %). In developing of NIR-responsive hydrogels, the reactive oxygen species (ROS)-cleavable thioketal cross-linkers was used. Upon NIR-irradiation, the hydrogels showed spatiotemporal release of encapsulated DOX (>99%) owing to the cleavage of thioketal bonds by interacting with ROS generated from ICG. The in vitro cytotoxicity results revealed that the hydrogels were highly cytocompatible and did not induce any toxic effect on the HEK-293 cells. In contrast, the DOX+ICG-encapsulated hydrogels enhanced the chemotherapeutic effect and effectively inhibited the proliferation of Hela cancer cells when irradiated with NIR-light.
- Issued Date
- 2024
- Awarded Date
- 2024-02
- Type
- Dissertation
- Publisher
- 국립부경대학교 대학원
- Affiliation
- 국립부경대학교 대학원
- Department
- 대학원 스마트그린기술융합공학과
- Advisor
- Kwon Taek Lim
- Table Of Contents
- 1. Introduction 1
1.1 Smart hydrogels in drug delivery system 1
1.2 Natural polymers 6
1.2.1 Cellulose 9
1.2.1.1 Carboxymethyl-cellulose . 10
1.3 Fabrication of hydrogel 12
1.3.1 Cross-linking of hydrogels 12
1.3.2 Bio-orthogonal Click chemistry reactions 13
1.3.3 On-demand cleavage of cross-linkers . 16
2. Reduction-responsive and bio-orthogonal carboxymethyl cellulose based soft hydrogels cross-linked via IEDDA click chemistry for cancer therapy application 18
2.1 Introduction 18
2.2 Experimental and Characterization 24
2.2.1 Materials 24
2.2.2 Synthesis of water-soluble DSe-DPEG-DTz cross-linker 28
2.2.3 Synthesis of CMC-Nb . 29
2.3 Preparation of hydrogels . 30
2.4 Characterization of materials 33
2.4.1 Rheological analysis 33
2.4.2 Swelling study . 34
2.4.3 Drug loading 35
2.4.4 Drug release analysis 36
2.4.5 Hemolysis assay . 36
2.4.6 In vitro degradation study of hydrogels . 37
2.4.7 Cell culture and cytotoxicity evaluation 38
2.4.8 Live/ Dead assay 39
2.5 Results and discussion 40
2.5.1 Synthesis of CMC-Nb . 40
2.5.2 Preparation and characterization of hydrogels 43
2.5.3 Drug loading and in vitro reduction-triggered release of DOX from hydrogels 49
2.5.4 Cytotoxicity studies . 53
2.5.5 Live/dead analysis . 56
2.5.6 Hemolytic activity of hydrogels . 58
2.5.7 Biodegradation study 60
2.6 Conclusions 62
3. NIR-responsive carboxymethyl-cellulose hydrogels containing thioketal-linkages for on-demand drug delivery system . 64
3.1 Introduction 64
3.2 Materials and methods . 72
3.2.1 Materials 72
3.2.2 Methods 73
3.2.2.1 Synthesis and characterization of precursors . 73
3.2.2.1.1 Synthesis of ROS-cleavable small molecule thioketal-di-tetrazine cross-linker (TK-DTz) 73
3.2.2.1.1.1 Synthesis of thioketal diacid (TKDA) 73
3.2.2.1.2 Synthesis of Amino-Functionalized Tetrazine (Tz-NH2) . 73
3.2.2.1.2.1 Synthesis of Tert-butyl 4-cyanobenzylcarbamate (TBC) 73
3.2.2.1.2.2 Synthesis of Tert-butyl 4-(1,2,4,5-tetrazin-3-yl)benzylcarbamate (Tz-NH-Boc) . 74
3.2.2.1.2.3 De-protection of BOC-group to obtain Tz-NH2 75
3.2.2.1.2.4 Synthesis of TK-DTz 76
3.2.3 Functionalization of CMC with Nb . 77
3.3 Formulation of hydrogels 77
3.4 Characterization 80
3.4.1 Measurements and instruments 80
3.4.2 Drug loading and release assessment 80
3.4.3 In vitro cytotoxicity analysis of precursors and fabricated hydrogels 82
3.4.4 In vitro anti-tumor activity of DOX+ICG-encapsulated hydrogels 83
3.4.5 Hemolysis assay . 84
3.4.6 In vitro evaluation of ROS production 85
3.4.7 Statistical analysis . 85
3.5 Results and discussion 86
3.5.1 Synthesis of TK-DTz 86
3.5.2 Synthesis of CMC-Nb . 90
3.5.3 Fabrication and characterization of the hydrogels 93
3.5.4 Gelation times and viscoelastic properties of CMC hydrogels 93
3.5.5 Swelling and water retention properties. 96
3.5.6 Morphological analysis 98
3.5.7 Drug loading and release studies 100
3.5.8 In vitro cytocompatibility of precursors, blank hydrogels and DOX/ICG-encapsulated hydrogels 105
3.5.9 Hemolytic activity of hydrogels 108
3.5.10 The anti-cancer activity of formulated hydrogels 110
3.5.11 In vitro ROS production 115
3.6 Conclusions . 117
4. Dissertation Summary and future perspectives . 119
Bibliography 123
- Degree
- Doctor
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- 대학원 > 스마트그린기술융합공학과
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