羟丁基壳聚糖的制备及其水凝胶敏感性（温度/pH）与生物相容性研究
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摘要
壳聚糖是广泛存在于自然界的一种氨基多糖，是甲壳素经脱乙酰基后的产物。壳聚糖具有许多独特的性质如可生物降解、无毒性、生物相容性以及抗菌性等，在生物医学领域被广泛用作药物释放载体、组织工程支架、伤口敷料等。但是由于分子链上分布着大量的氨基和羟基，形成分子内和分子间氢键，壳聚糖的结晶度较高、溶解性差，这极大地限制了它的开发和应用。通过化学改性制备水溶性壳聚糖衍生物是改善壳聚糖性能、拓宽其应用范围的重要途径之一。本文通过醚化改性方法在壳聚糖分子链上引入羟丁基基团，制备出水溶性良好的壳聚糖衍生物—羟丁基壳聚糖，研究了改性后产物的理化性质、生物活性、水凝胶敏感性、生物相容性以及作为药物缓释载体的可行性，对壳聚糖类产品的潜在应用价值的开发具有重要意义。
本实验以1,2-环氧丁烷在碱性条件下的开环产物为醚化剂，对壳聚糖进行改性反应，制得羟丁基壳聚糖。探讨了主要反应条件如反应介质、1,2-环氧丁烷用量、反应温度和时间对产物取代度、特性粘度及水溶性的影响。实验结果表明制备羟丁基壳聚糖的最佳反应条件为壳聚糖1g，10mL50%的NaOH水溶液，N2保护下碱化24h，挤出多余碱液，异丙醇水溶液中分散(异丙醇：水(v:v)=1:1)，1,2-环氧丁烷20mL，60℃反应24h。元素分析法测定此条件下产物的取代度为1.91。采用红外光谱、固体13C核磁共振对产物的分子结构进行了表征，证明在壳聚糖分子上成功引入了亲水性的羟丁基基团，取代位置为C6-OH和C2-NH2。通过多种方法测定合成产物的理化性质，与原料壳聚糖相比，羟丁基壳聚糖具有水溶性好、pH适应范围广、吸湿保湿性强等优点。
研究了羟丁基壳聚糖抑制细胞迁移、免疫活性及抑菌活性。采用划痕法研究了羟丁基壳聚糖对L929细胞迁移的影响作用，实验由预处理阶段和细胞迁移阶段组成，两个阶段都影响羟丁基壳聚糖对L929细胞迁移的作用。预处理24h时，羟丁基壳聚糖表现出了较强的抑制L929细胞迁移的作用；细胞迁移阶段中，羟丁基壳聚糖对L929细胞迁移的抑制作用在6h时最强，作用时间和羟丁基壳聚糖对L929细胞的相对迁移率没有相关性。另外，羟丁基壳聚糖的浓度也是一个重要影响因素，浓度越高对细胞迁移的抑制作用越强，与对照组相比，羟丁基壳聚糖（0.25mg/mL）对细胞迁移速度的最大抑制率可达到48%。在免疫活性方面，考察了羟丁基壳聚糖对小鼠巨噬细胞吞噬功能和正常小鼠淋巴细胞增殖作用的影响。羟丁基壳聚糖具有免疫调节活性：①羟丁基壳聚糖可增强小鼠单核巨噬细胞和腹腔巨噬细胞的吞噬功能，并且随着剂量的提高，吞噬功能增强；②羟丁基壳聚糖能够刺激小鼠脾淋巴细胞增殖，也可同时促进ConA、LPS诱导的T、B淋巴细胞的增殖作用。采用浊度法测定了羟丁基壳聚糖的抑菌活性，在浓度﹥0.01%时，羟丁基壳聚糖表现出了对大肠杆菌和金黄色葡萄球菌生长的抑制作用，随着浓度的增加对细菌生长的抑制作用增强。
通过分子内和分子间氢键的交联作用，在未加入任何交联剂的情况下制备了羟丁基壳聚糖水凝胶。水凝胶体系具有温度敏感性，在冰浴中为澄清透明溶液，温度升高至室温则发生凝胶化。2.5wt%的羟丁基壳聚糖水凝胶半透明，有一定的强度，可用镊子夹起。通过流变学手段系统地考察了影响水凝胶性能的主要因素，结论如下：1.随着羟丁基壳聚糖浓度和温度的提高，成胶时间缩短，不同情况下羟丁基壳聚糖的成胶时间都﹤90s，属于快速响应型水凝胶。2.羟丁基壳聚糖浓度不同、溶剂的pH不同和溶剂中的离子种类都对成胶温度造成影响：随着羟丁基壳聚糖浓度的提高，成胶温度降低；不同pH溶剂制备的羟丁基壳聚糖水溶液，成胶温度也不同，5.0wt%羟丁基壳聚糖以蒸馏水为溶剂的成胶温度为16.5℃，pH7.4的PBS缓冲液中为15.5℃，pH4的HCl溶液中为17.5℃，pH10的NaOH溶液中则不成胶，证明溶剂的pH值影响羟丁基壳聚糖的成胶温度；溶剂中的离子种类也影响成胶温度，体系中加入0.1mol/L的CaCl2、MgCl2使羟丁基壳聚糖在0℃时已表现出凝胶性质，加入0.1mol的Na+，羟丁基壳聚糖的成胶温度降至3.5℃。3.羟丁基壳聚糖属于热可逆性水凝胶，可随着体系温度的变化不断地发生sol-gel-sol之间的转换。羟丁基壳聚糖的这些性质提示其具有温度和pH敏感性。采用SEM对凝胶的内部形态进行观察，羟丁基壳聚糖水凝胶内部为多孔网络结构，孔壁光滑致密，水凝胶内部孔径随着羟丁基壳聚糖含量的增加而减小。溶胀度测试显示羟丁基壳聚糖凝胶60min内就达到了最大溶胀平衡，属于快速溶胀型水凝胶。
依据中国卫生部《生物材料和医疗器材生物学评价技术要求》，以羟丁基壳聚糖为材料，进行细胞毒性实验、溶血实验、急性毒性试验、肌肉植入和皮下注射实验，对羟丁基壳聚糖的体内、体外生物相容性进行综合评价。选用胎鼠成纤维细胞为靶细胞，采用细胞增殖度法检测羟丁基壳聚糖溶液及其水凝胶浸提液的细胞相容性，结果表明：水溶液组，当羟丁基壳聚糖浓度﹥0.5mg/mL时表现出微弱的细胞毒性；而浸提液组不同稀释倍数及不同作用时间内，胎鼠成纤维细胞的相对增殖率都高于75%，全部无细胞毒性作用，材料的毒性评级为Ⅰ级。采用直接接触法检测壳聚糖和羟丁基壳聚糖的血液相容性，浓度为1mg/mL的壳聚糖的溶血率极高，达到15.33%，羟丁基壳聚糖具有改善壳聚糖溶血性能的作用，壳聚糖和羟丁基壳聚糖浓度为1mg/mL，m(壳聚糖):m(羟丁基壳聚糖)≦1:1时，在1h无溶血和血细胞凝聚现象发生，溶血率﹤5%，满足生物材料对血液相容性的要求。同时，不同溶剂的羟丁基壳聚糖的溶血率也不同，以0.9%生理盐水为溶剂的溶血率低于以2%醋酸为溶剂的溶血率。急性毒性实验结果表明，不同稀释倍数的羟丁基壳聚糖水凝胶浸提液都没有表现出对小鼠的毒性作用，各实验组小鼠体重增加正常，无急性不良反应，处死后解剖观察小鼠主要脏器无异常。羟丁基壳聚糖溶胶埋植于大鼠背部肌肉及注射到皮下组织都可原位形成凝胶结构，在植入和注射后的不同时期处死动物进行相关检测后发现，肌肉植入和皮下注射后，凝胶周围组织没有明显的炎症反应，未发现组织变性和坏死。在植入的初期可见水凝胶周围有纤维薄膜产生，随着时间的延长，包膜变薄直至消失。羟丁基壳聚糖水凝胶可在肌肉组织和皮下组织内缓慢降解。以上结果证明，羟丁基壳聚糖是一种无细胞毒、不致急性溶血、无急性体内毒性、并且具有良好组织相容性的生物材料。
以羟丁基壳聚糖为载体材料，BSA为模型药物，制备负载BSA的羟丁基壳聚糖水凝胶控释体系，并对其体外释放行为进行了研究。羟丁基壳聚糖水凝胶对BSA的释放效果受羟丁基壳聚糖浓度、释放环境温度及释放环境pH值的影响。随着水凝胶内羟丁基壳聚糖浓度的增加，BSA的释放速度减慢，释放量减少；与25℃下的释放行为相比，37℃条件下BSA的释放速率较快；释放介质的pH也影响BSA的释放行为，在pH=6.0时，羟丁基壳聚糖发生溶蚀，1h内已将BSA全部释放出来，释放介质pH=7.4比pH=8.0时，药物释放速度快。实验结果提示羟丁基壳聚糖水凝胶体系是一种理想的缓释载体，可以通过调节羟丁基壳聚糖浓度、释放环境的温度和释放环境的pH控制药物的释放行为。
Chitosan (CS) is a widespread amino polysaccharide in nature, and is the product ofchitin after deacetylation. CS has many unique properties, such as biodegradability,non-toxicity, good biocompatibility, and antibacterial activity. In the biomedical field, CS iswidely used for drug delivery carriers, tissue engineering scaffolds, wound dressings, and soon. However, there are many amino and hydroxyl groups in the macromolecular chain of theCS, and those groups can form intramolecular and intermolecularhydrogen bonds. Thisreason led to be higher crystallinity and poor soluble of CS, which greatly limits itsdevelopment and application. Preparation for water soluble CS derivatives by chemicalmodification will be one important way of improving the performance and broadening itsapplication range. In this thesis we prepare a good water-soluble CS derivative–hydroxybutyl chitosan (HBCS) by etherifying modification method, which introducehydroxybutyl groups into the CS molecular chain. Then for this modified product we researchthe physical and chemical properties, gelation properties, bioactivities and biocompatibilities,and all of these will have great significance for the development of the CS products on thepotential values.
In this study, we use the ring-opening product of1,2-epoxy butane in the alkalinecondition as the etherification agent to modify the CS. We research the main reactionconditions, such as the reaction medium, the dose of1,2-epoxy butane, the temperature andtime, all of which would have an influence on the substitution of product, the intrinsicviscosity and water-solubility. The results show that the optimum condition for thepreparation of the HBCS is as follows: CS1g,10ml50%NaOH solution, under N2protection, reacting for24h, then squeezing out the excess lye, and dispersing in the mixtureof water and isopropanol (isopropanol: water (v: v)=1:1), then adding1,2-epoxy butane20ml, and at60℃reacting for24h. By the elemental analysis, we know the substitution of theproduct under this condition is1.91. By FTIR and solid-state13C NMR, we character themolecular structure of the product, and demonstrate the successful introduction of thehydroxybutyl groups into the CS molecular, confirm the substituted position is C6-OH andC2-NH2. We further determine the physical and chemical properties of the synthesis product, and find HBCS has good water-solubility, pH wide adaptation, strongly hygroscopic andmoisture capacity and other advantages, comparing with the raw material of CS.
We research the inhibition of cell migration, immune activity and antibacterial activity ofHBCS, respectively. Using Scratch Test, we study HBCS play the role in L929cell migration.There were two phases in this experiment, the pretreatment and cell migration, and both of thephases affect the role of HBCS impacting on the cell migration. After pretreatment for24h,HBCS show the strong inhibition of L929cell migration. During the cell migration phase, weuse HBCS (0.25mg/mL) to stimulate cells, after24h scratching, and respectively at differenttimes observe the situation of cell migration. The results show that for L929cells thestrongest inhibiting effect of HBCS is after6h, and the relative mobility is not relevant to thecell migration time. In addition, we also examine different concentration of HBCS in the roleof cell migration, the higher concentration bring the stronger inhibition of cell migration.Compared with the control group, when HBCS is0.25mg/mL, the maximum inhibiting cellmigration speed could reach48%. The results of the effects of HBCS on the phagocytosis ofmacrophage and the proliferation of lymphocyte in normal mice show that HBCS canenhance the phagocytosis function of monocyte and peritoneal macrophages, and as the doseincrease, the phagocytosis capacity enhance. Similarly, HBCS can stimulate the proliferationof lymphocyte in mice spleen, and also promote ConA, LPS-induced T, B lymphocyteproliferation. By turbidity method, we determine the antibacterial activity of the HBCS, at theconcentration>0.01%, HBCS show inhibiting the growth of Escherichia coli andStaphylococcus aureus, and this inhibiting capacity has the dependence of the concentrationof the HBCS, increasing with the concentration.
The HBCS hydrogel is prepared through intramolecular and intermolecular hydrogenbonds without any cross-linking agent. The HBCS hydrogel is temperature-sensitive, it isclear and transparent solution in an ice bath and become to be turbidity when the temperatureincrease.2.5wt%of the HBCS hydrogel is translucent and has a certain intensity, which canbe clamped. The main factors affect the gel process are investigated by systematic rheologicalmethod, and the results are as follows:1. The gelation time decrease with increasing CSconcentration and temperature, the gelation time is <90s under different circumstances,belonging to a rapidly responsive hydrogel.2. The concentration, pH and solvent types ofHBCS all affect the gelation temperature. The gelation temperature is decrease with increasing the HBCS concentration. The solvent pH also affect the gelation temperature of theHBCS hydrogel.5wt%HBCS solution in distilled water has a gelation temperature of16.5℃, in PBS7.4is15.5℃, in pH4HCl is17.5℃and in pH10NaOH can not be a gel. Theions in the solvent also affect the gelation temperature, when the CaCl2, MgCl2is added to thesystem, the HBCS show gel characteristics at0℃. The Na+, when the concentration is0.1mol/L, the gelation temperature is3.5℃.3. HBCS hydrogel is heat reversible exchange withsurroundings temperature and can occur sol-gel-sol transformation continuously with theconversion of ambient temperature. The internal morphology of the hydrogel was observed bySEM and the results show that it is porous network structure, with smooth and dense porewalls. The pore size of the hydrogel decreased with the increasing content of HBCS. Theresults of the swelling ratio test certify the hydrogel could reach swelling equilibrium within60min, belonging to a fast swelling hydrogel.
To study the biocompatibilities of the HBCS hydrogel, the cytotoxicity test, hemolysistest, acute systemic toxicity test, muscle and subcutaneous implantation experiments areconducted according to the "Chinese Ministry of Health, Biological evaluation of biomaterialsand medical devices". The cytotoxicity test is performed using fetal fibroblast cells as targetcells and the cell proliferation degree is detected. The result show that in the group of HBCSsolution, the HBCS demonstrate weak cytotoxicity when its concentration>0.5mg/mL.While in the extract group, the fetal mouse fibroblast cells relative growth rate is higher than75%, independent of the concentration and the stimulus time, indicating the goodcyto-compatibility. The hemolysis test of CS and HBCS is detected by direct contact methodand the results show that the1mg/mL of CS solution has a higher hemolytic rate with15.33%. HBCS could improve the hemolytic properties of CS, when the m (CS): m (HBCS)≦1:1and the concentration is1mg/ml, the hemolysis rate is <5%. The hemolysis rates are alsodifferent when the solvents of the HBCS are different, the solution with a0.9%NaCl assolvent has a2.24%hemolysis rate, while solution with a2%acetic acid as solvent has a2.67%hemolysis rate. The results of the acute systemic toxicity show that the HBCShydrogel extracts has not toxic effects in mice. The weight gain of the mice is normal, noadverse reactions are found, and sections of the main organs in mice are normal too. When theHBCS solution is embedded in the back muscles and subcutaneous tissue of mice, it can forma gel structure in situ, the tests are conducted at different time after implantation. There are no significant surrounding tissue inflammation, tissue degeneration and necrosis afterimplantation. In the early stages after implantation, the fibrous capsule can be seen around thehydrogel. With the time extend, the fibrous capsule become thinner until disappearance. TheHBCS hydrogel could be slowly depredated within the muscle tissue and subcutaneous tissue.All results prove that the HBCS hydrogel has no cytotoxicity in vitro, no acute hemolytic, noacute toxicity in vivo, and good tissue compatibility.
We use HBCS as the carrier material and BSA as a model drug to prepare HBCShydrogel delivery systems which is loaded BSA. The release behaivor in vitro is studied. Therelease of BSA resulted from HBCS hydrogel is affected by the concentration of HBCS,environmental temperature and pH values. As the increase of the HBCS concentration, theBSA release speed becomes slower and the release ratio decrease. Compared with the releasebehavior at25℃, the BSA release speed becomes faster at37℃. The pH also affects theBSA release behavior, at pH=6.0, HBCS begins to be dissolute and all of the BSA is releasedout in1h. Compared with at pH=9.0, the BSA release is faster at pH=6.0. These resultssuggest that HBCS hydrogel is an ideal delivery system, which can be adjusted by theconcentration of HBCS, the release of environmental including the release temperature andpH to control the release behavior.
本实验以1,2-环氧丁烷在碱性条件下的开环产物为醚化剂，对壳聚糖进行改性反应，制得羟丁基壳聚糖。探讨了主要反应条件如反应介质、1,2-环氧丁烷用量、反应温度和时间对产物取代度、特性粘度及水溶性的影响。实验结果表明制备羟丁基壳聚糖的最佳反应条件为壳聚糖1g，10mL50%的NaOH水溶液，N2保护下碱化24h，挤出多余碱液，异丙醇水溶液中分散(异丙醇：水(v:v)=1:1)，1,2-环氧丁烷20mL，60℃反应24h。元素分析法测定此条件下产物的取代度为1.91。采用红外光谱、固体13C核磁共振对产物的分子结构进行了表征，证明在壳聚糖分子上成功引入了亲水性的羟丁基基团，取代位置为C6-OH和C2-NH2。通过多种方法测定合成产物的理化性质，与原料壳聚糖相比，羟丁基壳聚糖具有水溶性好、pH适应范围广、吸湿保湿性强等优点。
研究了羟丁基壳聚糖抑制细胞迁移、免疫活性及抑菌活性。采用划痕法研究了羟丁基壳聚糖对L929细胞迁移的影响作用，实验由预处理阶段和细胞迁移阶段组成，两个阶段都影响羟丁基壳聚糖对L929细胞迁移的作用。预处理24h时，羟丁基壳聚糖表现出了较强的抑制L929细胞迁移的作用；细胞迁移阶段中，羟丁基壳聚糖对L929细胞迁移的抑制作用在6h时最强，作用时间和羟丁基壳聚糖对L929细胞的相对迁移率没有相关性。另外，羟丁基壳聚糖的浓度也是一个重要影响因素，浓度越高对细胞迁移的抑制作用越强，与对照组相比，羟丁基壳聚糖（0.25mg/mL）对细胞迁移速度的最大抑制率可达到48%。在免疫活性方面，考察了羟丁基壳聚糖对小鼠巨噬细胞吞噬功能和正常小鼠淋巴细胞增殖作用的影响。羟丁基壳聚糖具有免疫调节活性：①羟丁基壳聚糖可增强小鼠单核巨噬细胞和腹腔巨噬细胞的吞噬功能，并且随着剂量的提高，吞噬功能增强；②羟丁基壳聚糖能够刺激小鼠脾淋巴细胞增殖，也可同时促进ConA、LPS诱导的T、B淋巴细胞的增殖作用。采用浊度法测定了羟丁基壳聚糖的抑菌活性，在浓度﹥0.01%时，羟丁基壳聚糖表现出了对大肠杆菌和金黄色葡萄球菌生长的抑制作用，随着浓度的增加对细菌生长的抑制作用增强。
通过分子内和分子间氢键的交联作用，在未加入任何交联剂的情况下制备了羟丁基壳聚糖水凝胶。水凝胶体系具有温度敏感性，在冰浴中为澄清透明溶液，温度升高至室温则发生凝胶化。2.5wt%的羟丁基壳聚糖水凝胶半透明，有一定的强度，可用镊子夹起。通过流变学手段系统地考察了影响水凝胶性能的主要因素，结论如下：1.随着羟丁基壳聚糖浓度和温度的提高，成胶时间缩短，不同情况下羟丁基壳聚糖的成胶时间都﹤90s，属于快速响应型水凝胶。2.羟丁基壳聚糖浓度不同、溶剂的pH不同和溶剂中的离子种类都对成胶温度造成影响：随着羟丁基壳聚糖浓度的提高，成胶温度降低；不同pH溶剂制备的羟丁基壳聚糖水溶液，成胶温度也不同，5.0wt%羟丁基壳聚糖以蒸馏水为溶剂的成胶温度为16.5℃，pH7.4的PBS缓冲液中为15.5℃，pH4的HCl溶液中为17.5℃，pH10的NaOH溶液中则不成胶，证明溶剂的pH值影响羟丁基壳聚糖的成胶温度；溶剂中的离子种类也影响成胶温度，体系中加入0.1mol/L的CaCl2、MgCl2使羟丁基壳聚糖在0℃时已表现出凝胶性质，加入0.1mol的Na+，羟丁基壳聚糖的成胶温度降至3.5℃。3.羟丁基壳聚糖属于热可逆性水凝胶，可随着体系温度的变化不断地发生sol-gel-sol之间的转换。羟丁基壳聚糖的这些性质提示其具有温度和pH敏感性。采用SEM对凝胶的内部形态进行观察，羟丁基壳聚糖水凝胶内部为多孔网络结构，孔壁光滑致密，水凝胶内部孔径随着羟丁基壳聚糖含量的增加而减小。溶胀度测试显示羟丁基壳聚糖凝胶60min内就达到了最大溶胀平衡，属于快速溶胀型水凝胶。
依据中国卫生部《生物材料和医疗器材生物学评价技术要求》，以羟丁基壳聚糖为材料，进行细胞毒性实验、溶血实验、急性毒性试验、肌肉植入和皮下注射实验，对羟丁基壳聚糖的体内、体外生物相容性进行综合评价。选用胎鼠成纤维细胞为靶细胞，采用细胞增殖度法检测羟丁基壳聚糖溶液及其水凝胶浸提液的细胞相容性，结果表明：水溶液组，当羟丁基壳聚糖浓度﹥0.5mg/mL时表现出微弱的细胞毒性；而浸提液组不同稀释倍数及不同作用时间内，胎鼠成纤维细胞的相对增殖率都高于75%，全部无细胞毒性作用，材料的毒性评级为Ⅰ级。采用直接接触法检测壳聚糖和羟丁基壳聚糖的血液相容性，浓度为1mg/mL的壳聚糖的溶血率极高，达到15.33%，羟丁基壳聚糖具有改善壳聚糖溶血性能的作用，壳聚糖和羟丁基壳聚糖浓度为1mg/mL，m(壳聚糖):m(羟丁基壳聚糖)≦1:1时，在1h无溶血和血细胞凝聚现象发生，溶血率﹤5%，满足生物材料对血液相容性的要求。同时，不同溶剂的羟丁基壳聚糖的溶血率也不同，以0.9%生理盐水为溶剂的溶血率低于以2%醋酸为溶剂的溶血率。急性毒性实验结果表明，不同稀释倍数的羟丁基壳聚糖水凝胶浸提液都没有表现出对小鼠的毒性作用，各实验组小鼠体重增加正常，无急性不良反应，处死后解剖观察小鼠主要脏器无异常。羟丁基壳聚糖溶胶埋植于大鼠背部肌肉及注射到皮下组织都可原位形成凝胶结构，在植入和注射后的不同时期处死动物进行相关检测后发现，肌肉植入和皮下注射后，凝胶周围组织没有明显的炎症反应，未发现组织变性和坏死。在植入的初期可见水凝胶周围有纤维薄膜产生，随着时间的延长，包膜变薄直至消失。羟丁基壳聚糖水凝胶可在肌肉组织和皮下组织内缓慢降解。以上结果证明，羟丁基壳聚糖是一种无细胞毒、不致急性溶血、无急性体内毒性、并且具有良好组织相容性的生物材料。
以羟丁基壳聚糖为载体材料，BSA为模型药物，制备负载BSA的羟丁基壳聚糖水凝胶控释体系，并对其体外释放行为进行了研究。羟丁基壳聚糖水凝胶对BSA的释放效果受羟丁基壳聚糖浓度、释放环境温度及释放环境pH值的影响。随着水凝胶内羟丁基壳聚糖浓度的增加，BSA的释放速度减慢，释放量减少；与25℃下的释放行为相比，37℃条件下BSA的释放速率较快；释放介质的pH也影响BSA的释放行为，在pH=6.0时，羟丁基壳聚糖发生溶蚀，1h内已将BSA全部释放出来，释放介质pH=7.4比pH=8.0时，药物释放速度快。实验结果提示羟丁基壳聚糖水凝胶体系是一种理想的缓释载体，可以通过调节羟丁基壳聚糖浓度、释放环境的温度和释放环境的pH控制药物的释放行为。
Chitosan (CS) is a widespread amino polysaccharide in nature, and is the product ofchitin after deacetylation. CS has many unique properties, such as biodegradability,non-toxicity, good biocompatibility, and antibacterial activity. In the biomedical field, CS iswidely used for drug delivery carriers, tissue engineering scaffolds, wound dressings, and soon. However, there are many amino and hydroxyl groups in the macromolecular chain of theCS, and those groups can form intramolecular and intermolecularhydrogen bonds. Thisreason led to be higher crystallinity and poor soluble of CS, which greatly limits itsdevelopment and application. Preparation for water soluble CS derivatives by chemicalmodification will be one important way of improving the performance and broadening itsapplication range. In this thesis we prepare a good water-soluble CS derivative–hydroxybutyl chitosan (HBCS) by etherifying modification method, which introducehydroxybutyl groups into the CS molecular chain. Then for this modified product we researchthe physical and chemical properties, gelation properties, bioactivities and biocompatibilities,and all of these will have great significance for the development of the CS products on thepotential values.
In this study, we use the ring-opening product of1,2-epoxy butane in the alkalinecondition as the etherification agent to modify the CS. We research the main reactionconditions, such as the reaction medium, the dose of1,2-epoxy butane, the temperature andtime, all of which would have an influence on the substitution of product, the intrinsicviscosity and water-solubility. The results show that the optimum condition for thepreparation of the HBCS is as follows: CS1g,10ml50%NaOH solution, under N2protection, reacting for24h, then squeezing out the excess lye, and dispersing in the mixtureof water and isopropanol (isopropanol: water (v: v)=1:1), then adding1,2-epoxy butane20ml, and at60℃reacting for24h. By the elemental analysis, we know the substitution of theproduct under this condition is1.91. By FTIR and solid-state13C NMR, we character themolecular structure of the product, and demonstrate the successful introduction of thehydroxybutyl groups into the CS molecular, confirm the substituted position is C6-OH andC2-NH2. We further determine the physical and chemical properties of the synthesis product, and find HBCS has good water-solubility, pH wide adaptation, strongly hygroscopic andmoisture capacity and other advantages, comparing with the raw material of CS.
We research the inhibition of cell migration, immune activity and antibacterial activity ofHBCS, respectively. Using Scratch Test, we study HBCS play the role in L929cell migration.There were two phases in this experiment, the pretreatment and cell migration, and both of thephases affect the role of HBCS impacting on the cell migration. After pretreatment for24h,HBCS show the strong inhibition of L929cell migration. During the cell migration phase, weuse HBCS (0.25mg/mL) to stimulate cells, after24h scratching, and respectively at differenttimes observe the situation of cell migration. The results show that for L929cells thestrongest inhibiting effect of HBCS is after6h, and the relative mobility is not relevant to thecell migration time. In addition, we also examine different concentration of HBCS in the roleof cell migration, the higher concentration bring the stronger inhibition of cell migration.Compared with the control group, when HBCS is0.25mg/mL, the maximum inhibiting cellmigration speed could reach48%. The results of the effects of HBCS on the phagocytosis ofmacrophage and the proliferation of lymphocyte in normal mice show that HBCS canenhance the phagocytosis function of monocyte and peritoneal macrophages, and as the doseincrease, the phagocytosis capacity enhance. Similarly, HBCS can stimulate the proliferationof lymphocyte in mice spleen, and also promote ConA, LPS-induced T, B lymphocyteproliferation. By turbidity method, we determine the antibacterial activity of the HBCS, at theconcentration>0.01%, HBCS show inhibiting the growth of Escherichia coli andStaphylococcus aureus, and this inhibiting capacity has the dependence of the concentrationof the HBCS, increasing with the concentration.
The HBCS hydrogel is prepared through intramolecular and intermolecular hydrogenbonds without any cross-linking agent. The HBCS hydrogel is temperature-sensitive, it isclear and transparent solution in an ice bath and become to be turbidity when the temperatureincrease.2.5wt%of the HBCS hydrogel is translucent and has a certain intensity, which canbe clamped. The main factors affect the gel process are investigated by systematic rheologicalmethod, and the results are as follows:1. The gelation time decrease with increasing CSconcentration and temperature, the gelation time is <90s under different circumstances,belonging to a rapidly responsive hydrogel.2. The concentration, pH and solvent types ofHBCS all affect the gelation temperature. The gelation temperature is decrease with increasing the HBCS concentration. The solvent pH also affect the gelation temperature of theHBCS hydrogel.5wt%HBCS solution in distilled water has a gelation temperature of16.5℃, in PBS7.4is15.5℃, in pH4HCl is17.5℃and in pH10NaOH can not be a gel. Theions in the solvent also affect the gelation temperature, when the CaCl2, MgCl2is added to thesystem, the HBCS show gel characteristics at0℃. The Na+, when the concentration is0.1mol/L, the gelation temperature is3.5℃.3. HBCS hydrogel is heat reversible exchange withsurroundings temperature and can occur sol-gel-sol transformation continuously with theconversion of ambient temperature. The internal morphology of the hydrogel was observed bySEM and the results show that it is porous network structure, with smooth and dense porewalls. The pore size of the hydrogel decreased with the increasing content of HBCS. Theresults of the swelling ratio test certify the hydrogel could reach swelling equilibrium within60min, belonging to a fast swelling hydrogel.
To study the biocompatibilities of the HBCS hydrogel, the cytotoxicity test, hemolysistest, acute systemic toxicity test, muscle and subcutaneous implantation experiments areconducted according to the "Chinese Ministry of Health, Biological evaluation of biomaterialsand medical devices". The cytotoxicity test is performed using fetal fibroblast cells as targetcells and the cell proliferation degree is detected. The result show that in the group of HBCSsolution, the HBCS demonstrate weak cytotoxicity when its concentration>0.5mg/mL.While in the extract group, the fetal mouse fibroblast cells relative growth rate is higher than75%, independent of the concentration and the stimulus time, indicating the goodcyto-compatibility. The hemolysis test of CS and HBCS is detected by direct contact methodand the results show that the1mg/mL of CS solution has a higher hemolytic rate with15.33%. HBCS could improve the hemolytic properties of CS, when the m (CS): m (HBCS)≦1:1and the concentration is1mg/ml, the hemolysis rate is <5%. The hemolysis rates are alsodifferent when the solvents of the HBCS are different, the solution with a0.9%NaCl assolvent has a2.24%hemolysis rate, while solution with a2%acetic acid as solvent has a2.67%hemolysis rate. The results of the acute systemic toxicity show that the HBCShydrogel extracts has not toxic effects in mice. The weight gain of the mice is normal, noadverse reactions are found, and sections of the main organs in mice are normal too. When theHBCS solution is embedded in the back muscles and subcutaneous tissue of mice, it can forma gel structure in situ, the tests are conducted at different time after implantation. There are no significant surrounding tissue inflammation, tissue degeneration and necrosis afterimplantation. In the early stages after implantation, the fibrous capsule can be seen around thehydrogel. With the time extend, the fibrous capsule become thinner until disappearance. TheHBCS hydrogel could be slowly depredated within the muscle tissue and subcutaneous tissue.All results prove that the HBCS hydrogel has no cytotoxicity in vitro, no acute hemolytic, noacute toxicity in vivo, and good tissue compatibility.
We use HBCS as the carrier material and BSA as a model drug to prepare HBCShydrogel delivery systems which is loaded BSA. The release behaivor in vitro is studied. Therelease of BSA resulted from HBCS hydrogel is affected by the concentration of HBCS,environmental temperature and pH values. As the increase of the HBCS concentration, theBSA release speed becomes slower and the release ratio decrease. Compared with the releasebehavior at25℃, the BSA release speed becomes faster at37℃. The pH also affects theBSA release behavior, at pH=6.0, HBCS begins to be dissolute and all of the BSA is releasedout in1h. Compared with at pH=9.0, the BSA release is faster at pH=6.0. These resultssuggest that HBCS hydrogel is an ideal delivery system, which can be adjusted by theconcentration of HBCS, the release of environmental including the release temperature andpH to control the release behavior.
引文
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