Cellulose Nanofibril-Based Functional Hydrogels for Drug Release Application : 셀룰로오스 나노피브릴을 이용한 약물방출용 기능성 수화젤

국문 초록 (Abstract)

셀룰로오스는 자연에서 가장 풍부한 천연고분자로 재생가능성, 친환경성 및 인체 친화성으로 인해 다양한 기능성재료를 만드는 많은 관심을 받고 있다. 더욱이 최근 나노기술과 나노재료의 발달은 다양한 나노세룰로오스를 이용에 많은 관심을 일으켰다. 본 연구의 목적은 셀룰로오스 C6의 수산기를 카르복실기로 전환시키는 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) 산화반응으로 단리한 셀룰로오스 나노 피브릴(CNF: cellulose nanofibril)을 이용하여 약물 방출용 기능성 수화젤(hydrogel)을 개발하고자 하는 것이다. 본 연구의 목적을 달성하고자 본 연구는 네 부분, 즉 CNF의 단리, CNF 특성분석, CNF기반 수화젤 제조 및 특성분석, 그리고 기능성 CNF/고분자 복합수화젤 제조를 중점적으로 연구하였다.
첫 번째 부분의 연구는 셀룰로오스 원료종류, 초음파처리 시간, 및 산화제인 NaCl의 첨가량에 따라TEMO산화반응 후CNF 또는 셀룰로오스 마이크로 피브릴(CMF: cellulose micro fibril) 표면의 카르복실기 특성을 분석하였다. 전도도 적정(conduct metric titration)과 적외선 분광기(FT-IR)를 이용하여 cellulose의 표면 carboxylate 함량을 측정하였다. 산화반응 후 초음파 처리는 표면 carboxylate 함량을 증가시켰다. 또한 셀룰로오스의 원료 종류에 따라 표면 carboxylate 함량이 다르게 나타났다. 또 산화제 첨가량을 조절하면 CNF와CMF의 표면 carboxylate 함량을 조절할 수 있는 것으로 나타났다. 따라서 적절한 원료 선택과 산화제 투입량으로 CNF 또는 CMF의 표면 carboxylate 함량을 제어 할 수 있었다.
두 번째 부분의 연구는 CNF 또는 CMF의 현탁액에 양이온 즉 Al3+, Ca2+ 및H+을 첨가하여 셀룰로오스표면의 음이온성 표면 carboxylate와 이온성 가교결합을 유도하여 수화젤인 nanogel과 microgel을 제조하고자 하였다. 먼저 본 연구는 Al3+를 이용하여 microgel를 제조하는데 초음파 처리시간이 미치는 영향을 연구하였다. 초음파 처리 시간은 CMF 현탁액의CNF 함량에 영향을 미쳤으며 이는 수화젤의 내부 구조를 영향을 미쳤다. 긴 초음파 처리시간은 수화젤의 저장탄성계수(G’), 압축강도, 표면적을 증가시켰다. 이 같은 결과는 수화젤의 내부구조는 수화제의 팽윤 및 약물방출 거동에 큰 영향을 미치며 초음파 처리로 내부구조를 제어 할 수 있다는 것을 보여주었다. 또한 삼가(tri-valent) 양이온으로 만든 수화젤이 이가(di-valent) 및 수소이온(H+)으로 제조한 수화제로 보다 더 강한 피브릴간 또는 피브릴 내 상호작용을 나타냈다. 두 종류의 수화젤의 기계적 물성과 팽윤거동은 약물방출 과 반대의 상관관계를 나타내었다. 단단하고 기공이 많은 모든 수화젤은 낮은 약물방출을 나타내었다. Microgel과 nanogel의 약물방출 거동은 Higuchi model에 가장 잘 적합하였다. 이들 연구결과는 수화젤의 구조-물성 간의 밀접한 관련이 있다는 것을 나타내었다. 즉 수화젤의 내부구조는 약물방출에 많은 영향을 주었으며 내부구조 제어를 통한 약물방출 거동을 제어할 수 있음을 시사하였다.
세 번째 부분의 연구는 다른 수준의 표면전하를 갖는 CNF와 저임계용액온도(LCST: low critical solution temperature)에서 온도반응성 고분자인 poly(N-isoproylacrylamide) (PNIPAAm)를 이용하여 온도에 반응하는 수화젤를 개발하기 위한 실험을 실시하였다. 순수한 PNIPAAm와 CNF/PNIAAm 복합수화젤은 CNF와 단량체를 혼합하여 용기 내에서 합성하였다. Carboxylate 함량이 증가할수록, CNF 현탁액의 전단점도와 전단응력이 감소하는 반면, 저장탄성계수 및 손실탄성계수는 유동학적 측정에서 각 주파수에 더 의존하게 된다. 결과적으로, CNF/PNIPAAm 복합수화젤의 압축강도는 보다 낮은 CNF 표면전하 수준에서 더 커지며, 이것은 그들의 긴밀한 상호 가교결합에 기인할 수 있다. CNF 표면전하 수준은 CNF/PNIPAAm 수화젤의 특성에 큰 영향을 미치며, 이는 CNF 표면 전하의 적절한 수준의 선택이 온도반응성 수화젤에 중요하다는 것을 나타낸다.
이 연구의 마지막 부분에서는 pH와 온도에 반응하는 CNF/PNIPAAm 복합수화젤을 제조하고자 PNIPAAm의 2가지 다른 중합온도 즉, -20℃ 및 11℃와 CNF농도의 영향을 연구하였다. CNF/PNIPAAm 복합수화젤은 11℃와 낮은 CNF농도(8% 이하)에서 제조되었으며, CNF의 첨가 최소량이 10% 일 떄 나타나는 표면층(skin layer) 형성효과에 의해 기포가 형성되었다. CNF/PNIPAAm 복합수화젤은 순수한 PNIPAAm 수화젤보다 더 큰 팽윤을 나타내었고, -20℃에서 제조된 복합수화젤의 팽윤비율이 11℃에서 제조된 것보다 우수했다. CNF 첨가와 동결 중합 온도 사이의 상승 효과는 빠른 수분보유(water retention)값으로 높은 압축강도를 나타냈다. CNF/PNIPAAm 복합수화젤의 팽창거동 및 박동 온도거동은 pH와 온도에 대해 이중 반응성을 보였다. 그러나 11℃에서 제조된 수화젤은 박동 pH에 대하여 매우 낮은 반응성을 보였다. 예상한 바와 같이 보다 높은 표면 전하의 레벨의 CNF로 제조된 복합수화젤은 낮은 표면 전하를 사용하여 제조된 것보다 더 큰 pH반응성을 나타냈다. -20℃에서 준비된 복합수화젤에서 미세 기공 구조로 인하여 온도에 따른 약물모델로서의 methylene blue 의 더 큰 방출이 발견되었다. 그러나 pH에 대한 반응에서는 반대였다.
전반적으로, 이 연구는 TEMPO-산화반응에 의해 분리된 carboxylated CNF가 순수 CNF기반 수화젤 및 CNF/PNIPAAm 복합수화젤을 포함하여 CNF 기반 기능성 수화젤을 약물 방출 용도로 성공적으로 개발한다는 것을 밝혀냈다. 그리고 이 연구는 CNF의 특성과 수화젤의 제조방법이 수화젤 네트워크 매개 변수, 즉 내부 구조의 소형화, 상호 작용의 정도, 기공 크기 및 생성된 수화젤의 특성, 약물 방출 거동, 압축 강도에 큰 영향을 준다는 것을 발견했다.

다국어 초록 (Multilingual Abstract)

Cellulose is the most abundant natural polymer, and attracting a great amount of attention in developing numerous cellulose-based functional materials because it is renewable, biodegradable, environmentally friendly and biocompatible in a broad range of applications. Furthermore, recent advances in nanotechnologies and nanomaterials also increasingly brought a lot of attention on the utilization of a variety of nanocellulose materials. Thus, the goal of this study was to develop functional hydrogels for drug release application, using cellulose nanofibril (CNF) isolated by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation which had converted the primary hydroxyl groups at C6 to carboxylate groups. In order to achieve the goal, this study focused on four main parts, including the isolation and characterization of CNFs, CNF-based hydrogels, and functional CNF/polymer composite hydrogels.
In the first part of this research, different carboxyl group contents on the surface of CNFs or cellulose microfibrils (CMFs) after TEMPO-oxidation were characterized, depending on the source of cellulose raw materials, sonication treatments, and the addition level of NaClO as an oxidant. Both conductometric titration and infrared spectroscopy were employed to quantify the amount of carboxyl content. It was found that a post sonication treatment of the oxidized cellulose had no influence on the level of the surface carboxylate content of CMFs. However, the carboxylate content of CNF increased after the sonication treatment. The results also showed that the carboxyl content of CNFs was different from those of their corresponding bulk oxidized cellulose. The carboxyl content of CNFs or CMFs could be optimized by controlling the addition level of NaClO for the oxidation and by selecting the source of cellulose raw materials. These findings revealed ways of controlling the level of carboxylate groups on CNFs for further functionalization of CNFs.
The second part of this study emphasized on the development of CNF- or CMF-based hydrogels by inducing ionic cross-linking with cationic ions such as Al3+, Ca2+ and H+. Both CNFs and CMFs were used to prepare nanogels and microgels, respectively. In particular, this study investigated the effect of sonication time to characteristics of microgels that were prepared by combing CMFs with the addition of Al3+. Sonication treatment time influenced the proportion of CNFs in the CMF suspension, which in turn influenced the internal structure of the obtained cation rendered-hydrogels. A longer sonication time resulted in an increase of the storage modulus, compression strength, and surface area of the microgels. This result suggests that the internal structure of the hydrogel has a significant impact on the swelling and drug release behavior of the hydrogels, and the internal structure of the microgels be controlled by the sonication time to manipulate the release of drugs from the microgels. It was found that the tri-valent ion showed stronger intra- and inter-fibrillar interactions than those of di-valent or mono-valent ions. Freeze-dried hydrogels were composed of interconnected porous networks. Mechanical properties and swelling ratios of both hydrogels were inversely proportional to their cumulative drug release profiles. All hydrogels with compact and highly porous structures resulted in slow and low drug release. Drug release kinetics of microgels and nanogels were best fitted to the Higuchi model. These results of this study demonstrate that there is close structure-property relationships for all hydrogels, and suggest that characteristics and internal structure of hydrogel had a great impact on its properties and drug-release behavior.
The third part of this study was conducted to develop thermally responsive hydrogels using CNF with different levels of the surface charge and poly (N-isoproylacrylamide) (PNIPAAm), a thermally responsive polymer at the low critical solution temperature (LCST). Pure PNIPAAm and CNF/PNIAAm composite hydrogels were prepared in situ polymerization and by incorporating CNF into the polymer. As the carboxyl content increased, the shear viscosity and shear stress of the CNF suspension decreased, while the storage and loss modulus become more dependent on the angular frequency in the rheological measurements. Consequently, the compression strength of the CNF/PNIPAAm composite hydrogels was greater at lower CNF surface charge level, which could be ascribed to their tight interconnections structure. It was found that the CNF surface charge level had a great impact on the characteristics of the CNF/PNIPAAm hydrogels, indicating that the selection of a proper level of CNF surface charge was important for temperature responsive hydrogels.
In the last part of this study, CNF/PNIPAAm composite hydrogels responsive to pH and temperature were developed by combining CNFs with thermal responsive PNIPAAm at two different temperatures for in situ polymerization, i.e., -20 oC and 11 oC as well as at different CNF addition levels. The CNF/PNIPAAm composite hydrogels prepared at 11 oC and lower CNF levels (below 8%) showed the formation of bubbles by a skin layer effect, indicating that the minimum amount of CNF loading was 10% w/w. CNF/PNIPAAm composite hydrogels displayed greater swelling than those of pure PNIPAAm hydrogels, and the swelling ratio for the composite hydrogels prepared at 11 oC was superior compared to those prepared at -20 oC. Synergistic effect between CNF loading and freezing polymerization temperature resulted in high compression strength with a fast water retention value. Swelling behavior and pulsatile temperature behavior of CNF/PNIPAAm composite hydrogels showed dual responsiveness to pH and temperature. However, those hydrogels prepared at 11 oC had a very low responsiveness to the pulsatile pH. As expected, the composite hydrogels prepared with CNFs of higher surface charge level resulted in greater pH responsiveness than those prepared with low surface charge. A greater release of methylene blue as a model drug in the response to temperature was found at the composite hydrogels prepared at -20 oC, owing to their micro porous structures. However, it was opposite in the response to pH.
Overall, this study revealed that the carboxylated CNFs isolated by TEMPO-mediated oxidation were successfully used to develop CNF-based functional hydrogels, including pure CNF-based hydrogels and CNF/PNIPAAm composite hydrogels for drug release application. And this study also found that characteristics of CNFs and preparation methods of hydrogels had a great impact to properties and drug release behavior of the resultant hydrogels in term of the hydrogel network parameters such as compact internal structure, the degree of interaction, pore size, and compression strength

목차 (Table of Contents)

• Acknowledgments i
• Table of Contents iii
• List of Tables xi
• List of Figures xii
• List of Abbreviatons and Symbols xix
• Chapter 1. Introduction 1
• 1.1. Background 1
• 1.2. Objectives 6
• 1.3. Outline of the dissertation 7
• Chapter 2. Literature Review 10
• 2.1. Introduction 10
• 2.1.1. Why cellulose? 10
• 2.1.2. Mass production of nanocellulose 12
• 2.1.3. Nanocellulose composite hydogels 13
• 2.2. Nanocellulose 16
• 2.2.1. Cellulose nanowhisker 17
• 2.2.2. Cellulose nanofibril 18
• 2.3. Surface charged CNF 21
• 2.3.1. Surface pre-treatment of CNF 21
• 2.3.2. TEMPO-mediated cellulose nanofibril (TEMPO-CNF) 23
• 2.4. TEMPO-CNF applications 26
• 2.4.1. CNF-based films 26
• 2.4.2. CNF-based nanocomposites 27
• 2.4.3. Nanoparticles deposition substrate 27
• 2.5. CNF-based hydrogel 28
• 2.6. Cellulose-based polymer hydrogels 30
• 2.7. Stimuli responsive hydrogels 30
• Chapter 3. Materials and Methods 34
• 3.1. Materials 34
• 3.2. TEMPO-mediated oxidation of cellulose 34
• 3.2.1. Simultaneous sonication oxidation reaction of cellulose 35
• 3.3. CNF preparation 36
• 3.3.1. Post-sonication of the oxidation treatment 36
• 3.3.2. Isolation of TEMPO-CNF 36
• 3.4. Conductometric titration 37
• 3.5. Hydrogel preparations 38
• 3.5.1. CNF-based hydrogel preparation 38
• 3.5.1.1. Microgels prepared from CMFs rendered by cations 38
• 3.5.1.2. Nanogels prepared from CNFs rendered by different cations 39
• 3.5.2. Functional CNF/polymer composite hydrogels 40
• 3.5.2.1. CNF/PNIPAAm hydrogels with different carboxyl contents 40
• 3.5.2.2. Dual temperature and pH responsive hydrogels polymerized at different temperatures 41
• 3.6. Characterizations 42
• 3.6.1. Viscosity 42
• 3.6.2. CNF transparency 42
• 3.6.3. Rheological behavior and compression strength 42
• 3.6.3.1. CNF suspensions 42
• 3.6.3.2. Hydrogels 43
• 3.6.4. Infra-red spectroscopy 43
• 3.6.5. Aerogel preparation 44
• 3.6.6. FE-SEM analysis 44
• 3.6.7. Specific surface and pore size analysis 44
• 3.6.8. XRD analysis 45
• 3.6.9. DSC analysis 46
• 3.6.10. Swelling ratio of hydrogels 46
• 3.6.11. Water retention of hydrogels 47
• 3.6.12. Temperature dependence of hydrogels 47
• 3.6.13. Pulsatile temperature dependence of hydrogels 48
• 3.6.14. Pulsatile pH dependence of hydrogels 48
• 3.7. Drug release behavior of CNF-based hydrogels 49
• 3.7.1. Drug loading 49
• 3.7.1.1. CNF-based hydrogels rendered by cations 49
• 3.7.1.2. CNF/PNIPAAm hydrogel 50
• 3.7.2. In vitro drug release 50
• 3.7.2.1. CNF-based hydrogels rendered by cations 50
• 3.7.2.2. CNF/PNIPAAm hydrogels 51
• Chapter 4. Results and Discussion 53
• 4.1. Cellulose carboxylation by TEMPO-mediated oxidation 53
• 4.1.1. Effect of different sources of raw materials 54
• 4.1.2. Effect of simultaneous ultrasonication during TEMPO-mediated oxidation 57
• 4.1.3. Effect of post-sonication time after the TEMPO-oxidation 60
• 4.1.4. Effect of different amounts of sodium hyprochlorite 63
• 4.1.5. ATR-FTIR spectroscopy analysis 65
• 4.1.6. Conclusion 68
• 4.2. Sonication treatment on CMF-based microgels rendered by ionic interaction 70
• 4.2.1. Characterization of CMFs 71
• 4.2.1.1. Effect of sonication time on carboxylate content 71
• 4.2.1.2. Morphology of CMFs 72
• 4.2.1.3. Viscosity and transparency of CMFs 74
• 4.2.2. Microgel properties 75
• 4.2.2.1. Mechanical properties of microgels 75
• 4.2.2.2. Chemical groups of microgels 76
• 4.2.2.3. Cellulose crystallinity of microgels 78
• 4.2.2.4. Morphology of microgels 80
• 4.2.2.5. Surface area of microgels 82
• 4.2.2.6. Swelling behavior of hydrogels 83
• 4.2.2.7. Drug release behavior of microgels 85
• 4.2.3. Conclusion 86
• 4.3. CNF-based nanogels rendered by different cationic ions 88
• 4.3.1. Mechanical properties of nanogels 90
• 4.3.2. Chemical groups of hydrogels 95
• 4.3.3. Morphology of hydrogels 96
• 4.3.4. Surface area and pore size of nanogels 98
• 4.3.5. Cellulose crystallinity of hydrogels 101
• 4.3.6. Swelling behavior of hydrogels 104
• 4.3.7. Drug release behavior of hydrogels 106
• 4.3.8. Drug release kinetics 108
• 4.3.9. Conclusion 110
• 4.4. CNF/PNIPAAm hydrogels at different carboxyl contents at low cellulose concentration 112
• 4.4.1. Characterizations of CNFs 113
• 4.4.2. Characterization of CNF/PNIPAAm hydrogels 119
• 4.4.2.1. Morphology of CNF/PNIPAAm hydrogels 119
• 4.4.2.2. Composite strength of CNF/PNIPAAm hydrogels 121
• 4.4.2.3. ATR-FTIR spectroscopy of CNF/PNIPAAm hydrogels 122
• 4.4.2.4. Lower critical solution temperature of CNF/PNIPAAm hydrogels 124
• 4.4.2.5. Microscopic morphology of CNF/PNIPAAm hydrogels 125
• 4.4.2.6. Swelling behavior of CNF/PNIPAAm hydrogels 126
• 4.4.2.7. Water retention of CNF/PNIPAAm hydrogels 128
• 4.4.3. Conclusion 129
• 4.5. Dual responsive CNF/PNIPAAm hydrogel at different polymerization temperatures 131
• 4.5.1. Characterizations of CNF/PNIPAAm hydrogel 132
• 4.5.1.1. Mechanical properties of CNF/PNIPAAm hydrogels 135
• 4.5.1.2. Microscopic morphology of CNF/PNIPAAm hydrogels 137
• 4.5.1.3. ATR-FTIR and DSC analysis of CNF/PNIPAAm hydrogels 138
• 4.5.1.4. Temperature dependence of CNF/PNIPAAm hydrogels 140
• 4.5.1.5. Swelling behavior of CNF/PNIPAAm hydrogels 141
• 4.5.1.6. Pulsatile swelling behavior of CNF/PNIPAAm at different temperature and pH 143
• 4.5.2. Drug release study at different temperatures and pH levels 146
• 4.5.3. Conclusion 150
• 4.6. CNF/PNIPAAm hydrogels at different carboxyl contents prepared at low temperature (-20 oC). 152
• 4.6.1. Characterization of CNF/PNIPAAm hydrogels 152
• 4.6.1.1. Mechanical properties of CNF/PNIPAAm hydrogels 152
• 4.6.1.2. Microscopic morphology of CNF/PNIPAAm 155
• 4.6.1.3. Water retention and temperature dependence of CNF/PNIPAAm hydrogels 156
• 4.6.1.4. ATR-FTIR and DSC analysis of CNF/PNIPAAm hydrogels 158
• 4.6.2. Drug release study at different pH levels 160
• 4.6.3. Drug release kinetics 161
• 4.6.4. Conclusion 164
• Chapter 5. Conclusions 165
• REFERENCES 168
• Appendix 1 List of publications 189
• a. Peer reviewed papers 189
• b. Publication in progress 189
• c. Publication in preparation 189
• d. Conferences 190
• Abstract 193