本研究為溫感型複合水凝膠薄膜之開發，藉由改變羧甲基幾丁聚醣之比例以及交聯程度，來觀察對薄膜溫感性及物化性質的影響，希望能夠應用在傷口敷料，並且可應用於藥物控制釋放。本研究分為三部分：第一部分為合成P(NI-co-AA)共聚物，利用起始劑過硫酸鉀(KPS)將氮-異丙基丙烯醯胺(NIPAAm)及丙烯酸(AA)進行自由基聚合，當AA添加量為0.1、0.125、0.15 g並反應兩小時後，可以得到AA單元在鏈段上所佔比例分別為1.0%、5.7%、8.0%的P(NI-co-AA)共聚物。在低於PAA的pKa值環境下，隨著AA的比例改變，低臨界溶液溫度(LCST)並沒有明顯變化；在高於PAA的pKa值環境下，隨著AA的比例增加，LCST會隨之上升。第二部分為合成羧甲基幾丁聚醣(CMCS)，在鹼性環境下加入氯乙酸與幾丁聚醣反應，利用FTIR及1H-NMR進行結構鑑定，發現添加不同量的氫氧化鈉以及改變反應溫度，會得到不同取代度的CMCS，當氫氧化鈉的濃度提高，取代度也會隨之上升；增加反應溫度，亦可以得到較高取代度的CMCS。將CMCS做溶解度測試，發現CMCS可以溶在鹼性環境下，隨著取代度越高，在酸性的溶解度越差。第三部分為利用氯化鋁(AlCl3)交聯明膠(Gelatin)、CMCS及P(NI-co-AA)，並倒入鐵氟龍盤中室溫乾燥成水膠膜，利用FTIR進行結構鑑定後確認有成功交聯出水膠薄膜；另外進行SEM、LCST、薄膜含水率、膨潤性、水氣透濕性、抗菌性、生物相容性等測試，透過SEM可得知合成出之水膠薄膜為一緻密薄膜、透過UV及DSC測試，LCST數值大約都在32 oC附近、另外可以發現交聯程度的改變會對薄膜含水率、膨潤率及水氣透濕性有影響；抗菌性則隨著CMCS的含量添加，不論是大腸桿菌還是金黃色葡萄球菌，抗菌能力都有上升；利用L929及HaCaT進行細胞相容性測試，可以顯示水膠複合薄膜具有生物相容性。

In this study, thermo-responsive hydrogel membranes were prepared to serve as wound dressing materials. We researched into different prescriptions and degree of crosslink were adopted to change the properties of hydrogel membrane. Furthermore, by adding thermal responsive polymer to hydrogel, the complex hydrogel could be sensitive to temperature.
To begin with, free radical polymerization of N-isopropylacrylamide (NIPAAm) and acrylic acid (AA) was used to synthesize P(NI-co-AA) copolymers. Structure of P(NI-co-AA) was characterized by FTIR and 1H-NMR. AA accounts for 1.0%, 5.7% and 8.0% of segemts. Adjusting the pH to lower than pKa of PAA, the lower critical solution temperature (LCST) of the copolymers decrease as AA proportion increase, and vice versa. Then, carboxymethyl chitosan (CMCS) was prepared and characterized by FTIR and 1H-NMR. The results showed that the degree of carboxymethyl substitution depended on the degree of reaction temperature, and it was also relevant to NaOH concentration. It could be increased by raising the degree of reaction temperature and NaOH concentration. The solubility experiment showed that CMCS could dissolved at high pHs, the insoluble-region moving to lower pHs when the degree of carboxymethyl substitution increased. Finally, we prepared thermo-responsive composite hydrogel membranes, and evaluated chemical properties and physical properties of the membranes. After testing LCST by UV-Visible Spectrophotometer (UV) and Differential Scanning Calorimeter (DSC), the result showed that its degree of membranes around 32 oC. Besides, degree of crosslink affected moisture content, swelling properties and water vapor transmission rates. An increase in CMCS content would improve the antibacterial activity. In vitro studies showed that membranes could promote cell growth on HaCaT and L929 cells.

目錄
中文摘要	I
 Abstract III
 圖目錄 VIII
 表目錄 XI
 第一章 緒論 1
1.1 前言 1
1.2 研究動機 1
 第二章 文獻回顧 3
2.1 幾丁聚醣 3
2.1.1 幾丁聚醣簡介 3
2.1.2 幾丁聚醣應用 5
2.1.3 幾丁聚醣的抗菌機制 7
2.2 化學修飾幾丁聚醣 9
2.2.1 羧甲基幾丁聚醣 9
2.3 環境敏感型高分子 12
2.3.1 溫度敏感型高分子 12
2.3.2 酸鹼敏感型高分子 15
2.4 明膠 17
2.5 水膠簡介 19
2.6 傷口敷料 20
2.6.1 皮膚構造 20
2.6.2 傷口癒合機制 22
2.6.3 傷口敷料需求 24
第三章 實驗方法 25
3.1 實驗流程與架構 25
3.2 實驗藥品 27
3.3 實驗儀器 31
3.4 實驗步驟 35
3.4.1 NIPAAm單體純化 35
3.4.2 合成P(NI-co-AA)高分子 35
3.4.3 合成羧甲基幾丁聚醣(Carboxymethyl chitosan，CMCS) 37
3.4.4 水膠薄膜製備 39
3.5 結構分析與性質測試 41
3.5.1 化學結構鑑定 41
3.5.2 形態分析(SEM) 41
3.5.3 性質測試 42
 第四章 結果與討論 47
4.1 P(NI-co-AA)結構與性質分析 47
4.1.1 結構分析(FTIR) 47
4.1.2 高分子鏈結構組成(NMR) 49
4.1.3 不同pH水溶液對高分子相轉變溫度(LCST)的影響 51
4.2 幾丁聚醣結構鑑定 56
4.2.1 結構分析(FTIR) 56
4.2.2 去乙醯度計算(NMR) 58
4.3 羧甲基幾丁聚醣(CMCS)結構與性質分析 60
4.3.1 結構分析(FTIR) 60
4.3.2 羧甲基取代度計算(NMR) 62
4.3.3 CMCS在不同pH水溶液中之溶解性測試 67
4.4 水膠薄膜性質與分析 68
4.4.1 結構分析(FTIR-ATR) 68
4.4.2 薄膜含水率計算 70
4.4.3 膨潤度測試	70
4.4.4 水氣透濕性	72
4.4.5 薄膜型態觀察(SEM) 73
4.4.6 水膠薄膜相轉移溫度(LCST)測試 75
4.4.7 抗菌性測試	77
4.4.8 細胞相容性測試 80
 第五章 結論 85
 第六章 參考文獻 87
 第七章 附錄 95
7.1 調控不同酸鹼環境對大腸桿菌的抗菌能力影響之測試 95
7.2 調控不同酸鹼環境對大腸桿菌的抗菌能力之影響 96


圖目錄
圖 2-1 幾丁質之化學結構 4
圖 2-2 幾丁聚醣之化學結構 (n > m)	4
圖 2-3 幾丁質去乙醯反應 4
圖 2-4 水膠結構(a)幾丁聚醣自行交聯(b)共聚物網狀結構(c)半互穿網狀結構 6
圖 2-5幾丁聚醣寡聚物對B. cereus和E. coli的抗菌反應模擬機制[16] 8
圖 2-6 不同反應溫度CMCS的溶解範圍[20] 10
圖 2-7 (a) N-羧甲基幾丁聚醣；(b) O-羧甲基幾丁聚醣；(c) N,O-羧甲基幾丁聚醣 11
圖 2-8 (a) (Poly(N-isopropylacrylamide，PNIPAAm) (b) (Poly(N,N'-diethylacrylamide)，PDEAAm) 結構式 13
圖 2-9 不同結構PNIPAAm水膠升溫後之應答示意圖[31] 13
圖 2-10 引入不同比例親疏水性單體對LCST值之影響；AAm為Acrylamide，N-tBAAm為N-tert butylacrylamide [32] 14
圖 2-11 (a) Poly(methacrylic acid)；(b) Poly(methacrylic acid)；(c) Poly(2-ethyl acrylic acid)；(d) Poly(2-propyl acrylic acid)結構式 15
圖 2-12 (a) Poly(N,N'-dimethyl aminoethyl methacrylate)；(b) Poly(N,N'-diethyl aminoethyl methacrylate)；(c) Poly(4 or 2-vinylpyridine)；(d) Poly(vinyl imidazole)結構式 16
圖 2-13 明膠結構[40] 17
圖 2-14 皮膚構造示意圖 21
圖 2-15 表皮組織示意圖 21
圖 2-16 真皮組織示意圖 22
圖 2-17 傷口修復機制與時間 23
圖 3-1 實驗流程架構圖 26
圖 3-2 P(NI-co-AA) 合成反應流程圖 35
圖 3-3 P(NI-co-AA)反應裝置圖 36
圖 3-4 Carboxymethyl chitosan合成反應流程圖 37
圖 3-5 CMCS反應裝置圖 38
圖 3-6 水膠薄膜合成反應流程圖 39
圖 3-7 Gelatin/CMCS/P(NI-co-AA)水膠薄膜成品 39
圖 3-8 四區畫線塗盤示意圖 45
圖 4-1 (a) PNIPAAm；(b) PAA；(c) P(NI-co-AA1.0) 的紅外線吸收光譜圖 48
圖 4-2 P(NI-co-AA) 結構式 49
圖 4-3利用KPS起始劑聚合不同AA添加量，在75 oC反應兩小時後(a) P(NI-co-AA1.0)；(b) P(NI-co-AA5.7)；(c) P(NI-co-AA8.0) 1H-NMR光譜圖 50
圖 4-4 P(NI-co-AA1.0)共聚物在不同pH值水溶液下穿透率與溫度之關係圖 51
圖 4-5 P(NI-co-AA5.7)共聚物在不同pH值水溶液下穿透率與溫度之關係圖 52
圖 4-6 P(NI-co-AA8.0)共聚物在不同pH值水溶液下穿透率與溫度之關係圖 52
圖 4-7 不同P(NI-co-AA)共聚物在pH=4水溶液下穿透率與溫度之關係圖 53
圖 4-8 不同P(NI-co-AA)共聚物在pH=5.5水溶液下穿透率與溫度之關係圖 54
圖 4-9 不同P(NI-co-AA)共聚物在pH=7.4水溶液下穿透率與溫度之關係圖 55
圖 4-10 幾丁聚醣的紅外線吸收光譜圖(DDA= 94%) 56
圖 4-11 CS之1H-NMR光譜圖 58
圖 4-12 (a) CS；(b) CMCS 的紅外線吸收光譜圖 60
圖 4-13 Carboxymethyl chitosan 結構式 64
圖 4-14 (a) 30N5；(b) 30N20 1H-NMR光譜圖 65
圖 4-15 水膠薄膜交聯示意圖 69
圖 4-16 (a) Gelatin；(b) CMCS；(c) P(NI-co-AA)；(d) Membrane的紅外線吸收光譜圖 69
圖 4-17 PAA在不同環境下的紅外線吸收光譜圖 (a) PAA in base；(b) PAA in acid；(c) PAA crosslinked by AlCl3 69
圖 4-18 不同交聯程度對G5C0.5P0.5複合薄膜水氣透濕之影響 72
圖 4-19 G5C0.5P0.5A0.1薄膜的掃瞄式電子顯微鏡圖 73
圖 4-20 G5C0.5P0.5A0.05薄膜的掃瞄式電子顯微鏡圖 74
圖 4-21 G5C0.5P0.5A0.02薄膜的掃瞄式電子顯微鏡圖 74
圖 4-22 不同交聯程度水膠的光穿透率和溫度之關係曲線圖 75
圖 4-23 不同交聯程度水膠的DSC升溫曲線圖 76
圖 4-24 不同CMCS含量的Gelatin/CMCS/P(NI-co-AA)薄膜對大腸桿菌的抑菌性	78
圖 4-25 不同CMCS含量的Gelatin/CMCS/P(NI-co-AA)薄膜對金黃色葡萄球菌的抑菌性 79
圖 4-26 不同CMCS含量的Gelatin/CMCS/P(NI-co-AA)水膠薄膜對HaCaT細胞存活率之影響 81
圖 4-27 不同AlCl3含量的Gelatin/CMCS/P(NI-co-AA)水膠薄膜對HaCaT細胞存活率之影響 82
圖 4-28 不同CMCS含量的Gelatin/CMCS/P(NI-co-AA)水膠薄膜對L929細胞存活率之影響 83
圖 4-29 不同AlCl3含量的Gelatin/CMCS/P(NI-co-AA)水膠薄膜對L929細胞存活率之影響 84


表目錄

表目錄
表 2-1 明膠組合成分 18
表 3-1 水膠薄膜實驗配方 40
表 4-1 P(NI-co-AA)官能基紅外線光譜吸收峰的位置[55] 48
表 4-2 不同P(NI-co-AA)共聚物在不同pH環境下的LCST值 55
表 4-3 CS官能基紅外線光譜吸收峰的位置[56,57] 57
表 4-4 CS 1H-NMR吸收峰的位置[58] 59
表 4-5 CMCS官能基紅外線光譜吸收峰的位置[59,60] 61
表 4-6 CMCS 1H-NMR吸收峰的位置[61,62] 64
表 4-7 Carboxymethyl chitosan取代度(substituted)整理表 64
表 4-8 在不同反應溫度下所合成出的CMCS在不同pH水溶液下溶解度測試 67
表 4-9 G5C0.5P0.5不同交聯程度含水率 70
表 4-10 G5C0.5P0.5A0.02 膨潤度測試 71
表 4-11 G5C0.5P0.5A0.05 膨潤度測試 71
表 4-12 G5C0.5P0.5A0.1 膨潤度測試 71
表 4-13 不同交聯程度對LCST之影響 76
表 4-14 不同樣品對大腸桿菌抗菌活性 78
表 4-15 不同樣品對金黃色葡萄球菌抗菌活性 79
表 4-16 不同CMCS含量的水膠薄膜對HaCaT細胞存活率之影響 81
表 4-17 不同AlCl3含量的水膠薄膜對HaCaT細胞存活率之影響 82
表 4-18 不同CMCS含量的水膠薄膜對L929細胞存活率之影響 83
表 4-19 不同AlCl3含量的水膠薄膜對L929細胞存活率之影響 84
表 7-1 不同酸鹼環境對大腸桿菌抗菌活性之影響 96

[1] Kast CE, Frick W, Losert U, Bernkop-Schnürch A. Chitosan-thioglycolic acid conjugate: a new scaffold material for tissue engineering? Int J Pharm 2003;256:183-9.
[2] Muzzarelli RAA, Barontini G, Rocchetti R. Immobilized enzymes on chitosan columns: a Chymotrypsin and acid phosphatase. Biotechnol Bioeng 1976;18:1445-54.
[3] Rinaudo M, Pavlov G, Desbrières J. Influence of acetic acid concentration on the solubilization of chitosan. Polymer 1999;40:7029-32.
[4] Struszczyk MH. Chitin and chitosan Part II. Applications of chitosan. Polimery 2002;47:396-403.
[5] Berger J, Reist M, Mayer JM, Felt O, Peppas NA, Gurny R. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics 2004;57:19-34.
[6] Crini G. Recent developments in polysaccharide-based materials used as adsorbents in wastewater treatment. Progress in Polymer Science 2005;30:38-70.
[7] Rinaudo M. Chitin and chitosan: Properties and applications. Progress in Polymer Science 2006;31:603-32.
[8] Boucard N, Viton C, Agay D, Mari E, Roger T, Chancerelle Y et al. The use of physical hydrogels of chitosan for skin regeneration following third-degree burns. Biomaterials 2007;28:3478-88.
[9] Kossovich LY, Salkovskiy Y, Kirillova IV. Electrospun chitosan nanofiber materials as burn dressing. IFMBE Proc 2010;31 IFMBE:1212-4.
[10] Thomas V, Yallapu MM, Sreedhar B, Bajpai SK. Fabrication, characterization of chitosan/nanosilver film and its potential antibacterial application. J Biomater Sci Polym Ed 2009;20:2129-44.
[11] Denkbas EB, Öztürk E, Özdemir N, Keçeci K, Agalar C. Norfloxacin-loaded chitosan sponges as wound dressing material. J Biomater Appl 2004;18:291-303.
[12] Berger J, Reist M, Mayer JM, Felt O, Peppas NA, Gurny R. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics 2004;57:19-34.
[13] Tsai G-, Su W-, Chen H-, Pan C-. Antimicrobial activity of shrimp chitin and chitosan from different treatments and applications of fish preservation. Fish Sci 2002;68:170-7.
[14] Liu H, Du Y, Wang X, Sun L. Chitosan kills bacteria through cell membrane damage. Int J Food Microbiol 2004;95:147-55.
[15] Helander IM, Nurmiaho-Lassila E-, Ahvenainen R, Rhoades J, Roller S. Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. Int J Food Microbiol 2001;71:235-44.
[16] Vishu Kumar AB, Varadaraj MC, Gowda LR, Tharanathan RN. Characterization of chito-oligosaccharides prepared by chitosanolysis with the aid of papain and Pronase, and their bactericidal action against Bacillus cereus and Escherichia coli. Biochem J 2005;391:167-75.
[17] Shahidi F, Arachchi JKV, Jeon Y-. Food applications of chitin and chitosans. Trends Food Sci Technol 1999;10:37-51.
[18] Zheng L-, Zhu J-. Study on antimicrobial activity of chitosan with different molecular weights. Carbohydr Polym 2003;54:527-30.
[19] Chung Y-, Su Y-, Chen C-, Jia G, Wang H-, Wu JCG et al. Relationship between antibacterial activity of chitosan and surface characteristics of cell wall. Acta Pharmacol Sin 2004;25:932-6.
[20] Chen X, Park H. Chemical characteristics of O-carboxymethyl chitosans related to the preparation conditions. Carbohydr Polym 2003;53:355-9.
[21] Nishimura S-, Ikeuchi Y, Tokura S. The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin. Carbohydr Res 1984;134:305-12.
[22] Muzzarelli RAA, Ilari P, Petrarulo M. Solubility and structure of N-carboxymethylchitosan. Int J Biol Macromol 1994;16:177-80.
[23] Liu Y, Zhao D, Wang J. Preparation of O-Carboxymethyl chitosan by schiff base and antibacterial activity. Adv Mater Res 2013;647:794-7.
[24] Sun T, Xu P, Liu Q, Xue J, Xie W. Graft copolymerization of methacrylic acid onto carboxymethyl chitosan. European Polymer Journal 2003;39:189-92.
[25] Chen S, Wu Y, Mi F, Lin Y, Yu L, Sung H. A novel pH-sensitive hydrogel composed of N,O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. J Controlled Release 2004;96:285-300.
[26] Gil ES, Hudson SM. Stimuli-reponsive polymers and their bioconjugates. Prog Polym Sci (Oxford) 2004;29:1173-222.
[27] Kubota K, Hamano K, Kuwahara N, Fujishige S, Ando I. Characterization of poly(N-isopropylmethacrylamide) in water. Polym J 1990;22:1051-7.
[28] Boutris C, Chatzi EG, Kiparissides C. Characterization of the LCST behaviour of aqueous poly(N-isopropylacrylamide) solutions by thermal and cloud point techniques. Polymer 1997;38:2567-70.
[29] Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Adv Drug Deliv Rev 2001;53:321-39.
[30] Wu XS, Hoffman AS, Yager P. Synthesis and characterization of thermally reversible macroporous poly(N-isopropylacrylamide) hydrogels. J Polym Sci Part A 1992;30:2121-9.
[31] Yoshida R, Uchida K, Kaneko Y, Sakai K, Kikuchi A, Sakurai Y et al. Comb-type grafted hydrogels with rapid deswelling response to temperature changes. Nature 1995;374:240-2.
[32] Hoffman AS, Stayton PS, Bulmus V, Chen G, Chen J, Cheung C et al. Really smart bioconjugates of smart polymers and receptor proteins. J Biomed Mater Res 2000;52:577-86.
[33] Philippova OE, Hourdet D, Audebert R, Khokhlov AR. pH-responsive gels of hydrophobically modified poly(acrylic acid). Macromolecules 1997;30:8278-85.
[34] Torres-Lugo M, Peppas NA. Molecular design and in vitro studies of novel pH-sensitive hydrogels for the oral delivery of calcitonin. Macromolecules 1999;32:6646-51.
[35] Tonge SR, Tighe BJ. Responsive hydrophobically associating polymers: A review of structure and properties. Adv Drug Deliv Rev 2001;53:109-22.
[36] Murthy N, Robichaud JR, Tirrell DA, Stayton PS, Hoffman AS. The design and synthesis of polymers for eukaryotic membrane disruption. J Control Release 1999;61:137-43.
[37] Lee AS, Bütün V, Vamvakaki M, Armes SP, Pople JA, Gast AP. Structure of pH-dependent block copolymer micelles: Charge and ionic strength dependence. Macromolecules 2002;35:8540-51.
[38] Okubo M, Ahmad H, Suzuki T. Synthesis of temperature-sensitive micron-sized monodispersed composite polymer particles and its application as a carrier for biomolecules. Colloid Polym Sci 1998;276:470-5.
[39] Tabata Y, Ikada Y. Protein release from gelatin matrices. Adv Drug Deliv Rev 1998;31:287-301.
[40] Choi YS, Hong SR, Lee YM, Song KW, Park MH, Nam YS. Studies on gelatin-containing artificial skin: II. Preparation and characterization of cross-linked gelatin-hyaluronate sponge. J Biomed Mater Res 1999;48:631-9.
[41] Ponticiello MS, Schinagl RM, Kadiyala S, Barry FP. Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J Biomed Mater Res 2000;52:246-55.
[42] Rathna GVN, Chatterji PR. Controlled drug release from gelatin-sodium carboxymethylcellulose interpenetrating polymer networks. J Macromol Sci Pure Appl Chem 2003;40 A:629-39.
[43] Lysaght MJ, Reyes J. The growth of tissue engineering. Tissue Eng 2001;7:485-93.
[44] Kim S, Nimni ME, Yang Z, Han B. Chitosan/gelatin-based films crosslinked by proanthocyanidin. J Biomed Mater Res Part B Appl Biomater 2005;75:442-50.
[45] Chen Y-, Chang J-, Cheng C-, Tsai F-, Yao C-, Liu B-. An in vivo evaluation of a biodegradable genipin-cross-linked gelatin peripheral nerve guide conduit material. Biomaterials 2005;26:3911-8.
[46] Chen Y-, Chiou S-, Wong T-, Ku H-, Lin H-, Chung C- et al. Using Gelatin Scaffold With Coated Basic Fibroblast Growth Factor as a Transfer System for Transplantation of Human Neural Stem Cells. Transplant Proc 2006;38:1616-7.
[47] Wichterle O, Lím D. Hydrophilic Gels for Biological Use. Nature 1960;185:117-8.
[48] Yannas IV, Lee E, Orgill DP, Skrabut EM, Murphy GF. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin. Proc Natl Acad Sci U S A 1989;86:933-7.
[49] Hamidi M, Azadi A, Rafiei P. Hydrogel nanoparticles in drug delivery. Adv Drug Deliv Rev 2008;60:1638-49.
[50] He H, Cao X, Lee LJ. Design of a novel hydrogel-based intelligent system for controlled drug release. J Controlled Release 2004;95:391-402.
[51] 黃世偉. 高分子材料與醫療器材. 科學發展 2010:14-19.
[52] 林怡伶. 化學修飾雙性幾丁聚醣衍生物及其持水特性研究. 2005.
[53] 李承明. 明膠在創傷敷料中的應用. The Science and Technology of Gelatin 2003:57-62.
[54] Huang X, Zhang Y, Zhang X, Xu L, Chen X, Wei S. Influence of radiation crosslinked carboxymethyl-chitosan/gelatin hydrogel on cutaneous wound healing. Materials Science and Engineering: C 2013;33:4816-24.
[55] Schilli CM, Zhang M, Rizzardo E, Thang SH, Chong YK, Edwards K et al. A new double-responsive block copolymer synthesized via RAFT polymerization: Poly(N-isopropylacrylamide)-block-poly(acrylic acid). Macromolecules 2004;37:7861-6.
[56] Shigemasa Y, Matsuura H, Sashiwa H, Saimoto H. Evaluation of different absorbance ratios from infrared spectroscopy for analyzing the degree of deacetylation in chitin. Int J Biol Macromol 1996;18:237-42.
[57] Brugnerotto J, Lizardi J, Goycoolea FM, Argüelles-Monal W, Desbrières J, Rinaudo M. An infrared investigation in relation with chitin and chitosan characterization. Polymer 2001;42:3569-80.
[58] Lavertu M, Xia Z, Serreqi AN, Berrada M, Rodrigues A, Wang D et al. A validated 1H NMR method for the determination of the degree of deacetylation of chitosan. J Pharm Biomed Anal 2003;32:1149-58.
[59] Chen S-, Wu Y-, Mi F-, Lin Y-, Yu L-, Sung H-. A novel pH-sensitive hydrogel composed of N,O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. J Control Release 2004;96:285-300.
[60] Wang L, Wang A. Adsorption properties of congo red from aqueous solution onto N,O-carboxymethyl-chitosan. Bioresour Technol 2008;99:1403-8.
[61] Chen X-, Park H-. Chemical characteristics of O-carboxymethyl chitosans related to the preparation conditions. Carbohydr Polym 2003;53:355-9.
[62] Cai Z-, Yang C-, Zhu X-. Preparation of quaternized carboxymethyl chitosan and its capacity to flocculate COD from printing wastewater. J Appl Polym Sci 2010;118:299-305.
[63] Lamke L-, Nilsson GE, Reithner HL. The evaporative water loss from burns and the water-vapour permeability of grafts and artificial membranes used in the treatment of burns. Burns 1977;3:159-65.
[64] Kim IY, Yoo MK, Seo JH, Park SS, Na HS, Lee HC et al. Evaluation of semi-interpenetrating polymer networks composed of chitosan and poloxamer for wound dressing application. Int J Pharm 2007;341:35-43.