摘要
温敏可注射水凝胶为组织工程的一类软支架材料,由于能够在温度的调控下发生溶胶-凝胶转变,可通过注射的方法将复合有细胞或药物的聚合物溶液注射到所需部位,原位形成凝胶,避免了创伤性的外科手术,达到微创伤目的。壳聚糖/甘油磷酸钠(CS/GP)温敏水凝胶具有良好的相容性、可注射等优点,但也存在一些问题,如温度响应慢,机理不够明确等。
本研究以CS和不同醛为原料制备了一系列的烷基化壳聚糖(ACS) (ACS-4、ACS-6、ACS-8、ACS-4i),通过调节原料投料比,分别得到不同取代度(DS)的ACS。利用FITR、1H-NMR等对产物的结构和烷基取代度进行了表征。结果显示所得ACS的DS介于5%-54.8%之间。且ACS均具有良好的水溶性。
利用试管倒置法和流变仪分别对CS/GP和ACS/GP的溶胶-凝胶转变过程进行了研究。研究表明,随着GP浓度的增加,CS/GP和ACS/GP的凝胶温度(Tgel)均表现为下降趋势。对于ACS体系,Tgel随烷基取代度增加先升高后降低。当取代度过大时,体系趋于形成沉淀而不是凝胶。DS约5%时,不同烷基链对ACS/GP的Tgel影响不大。此外,发现当取代度较大时,ACS/GP凝胶在降温过程中可发生凝胶-溶胶的回复,而CS/GP凝胶不能完全回复。进一步推测体系的凝胶机理,得出:低温时,GP分子与CS分子相互作用在CS周围形成水保护层;达到凝胶温度时,水保护层遭到破坏,原本难以接近的CS分子得以相互作用,包括氢键、疏水缔合、分子链缠结等,其中急剧增多的CS之间的氢键是导致凝胶形成的主要作用力。ACS/GP凝胶体系的凝胶过程与其相似,但随取代度的增加疏水缔合作用对凝胶形成的贡献增加,甚至可成为凝胶的主要驱动力。
通过SEM、称重法等研究了凝胶的性能,结果表明干凝胶的形貌为不规则的多孔结构。干凝胶最大溶胀度可达到118.9%-310%;约334min溶胀度达平衡。14d后凝胶湿重仍能保持90-95%,凝胶能够在较长时间内保持完整的凝胶形态。凝胶的牛血清蛋白(BSA)释放曲线表明,CS/GP、ACS-4-1/GP、ACS-6-1/GP凝胶的释放行为相似,在第一阶段即最初的24h出现了较严重的突释,约43.5%-49.4%,第二阶段2d之后,BSA释放逐渐变得缓慢,累计释放量稳定上升。ACS/GP凝胶的控释性能略好于CS/GP凝胶。
As a kind of soft scaffolds in tissue engineering, thermosensitive and injectable hydrogels which show sol-gel transition in response to temperature have gained great interest because the cell or drug can be easily loaded into them and can be injected into the needed place to form in-situ gel. This method can avoid the surgery and relieve the suffering of patients. Chitosan/sodium-β-glycerophosphate (CS/GP) hydrogel has some advantages such as biocompatibility and injectability. However, there are some problems for CS/GP hydrogel, for example the low rate of the response to temperature, the ambiguous mechanism and so on.
In this research, alkylated chitosan (ACS-4, ACS-6, ACS-8, ACS-4i) was prepared by using CS and different kinds of aldehyde. The degree of substitution of the derivative (DS) was mainly controlled by CS/aldehyde ratio. The structure and DS of ACS was investigated by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance. Results showed that the obtained DS ranged from 5% to54.8%. All of the obtained alkylated chitosan was water soluble.
The sol-gel transition of CS/GP and ACS/GP were studied by the test tube inverted method and rheometer. The gelation temperature (Tgel) decreased as the concentration of GP increased for both CS/GP and ACS/GP. As for ACS/GP, Tgel firstly increased and then decreased when DS increased. However, if SD was too large, ACS/GP could not gel but precipitate. When DS was about 5%, the alkyl chain length had little effect on Tgel. During cooling process, the gel-sol transition could occur for ACS/GP when DS was large enough, whereas CS/GP hydrogel was only partially thermoreversible. The mechanism of sol-gel transition was speculated as follows. As for CS/GP, CS was stabilized by the formation of the shield of water around CS at the lower temperature, which was promoted by the interaction between CS and GP. At Tgel, the water shield was destroyed and thus all kinds of the interaction between CS began forming, including the entanglement, hydrogen bond, hydrophobic interaction and so on. Hydrogen bond played an important role. As for ACS/GP, hydrophobic forces played more and more important role on sol-gel transition as the amount of hydrophobic group increased.
Properties of the hydrogel were investigated by SEM and gravimetric method. It was found that xerogel showed irregular porous structure. The largest swelling degree (SD) of xerogel was about 118.9%-310% and reached equilibrium after about 334 minutes. The wet weight remaining of the hydrogels in PBS were 90%-95% after 14 days, indicating the stability of these gels. The released profile of bovine serum albumin (BSA) form these in-situ hydrogels showed that CS/GP, ACS-4-1/GP, ACS-6-1/GP had the similar release behavior. The burst release occurred in the first day, about 43.5%-49.4%. After that, the release behaviors of all gels were much smoother. BSA was released relatively more slowly from ACS/GP hydrogel than from CS/GP hydrogel.
引文
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