多功能聚羧酸-NHS酯改性溶液态胶原及其构效机理

Cross-linking modification is a method which is widely used in the improvement of collagen stability. However, most of the relevant studies focus on the modification of collagen which is in solid state, mainly because there are two problems in the cross-linking modification of collagen in liquid state: the first problem is that collagen has been endured by acylated modification in order to obtain a good water-solubility under the physiological conditions, therefore there is no enough amino-group in collagen molecules for cross-linking reaction, since amino-group has been used up by acylated modification. The second problem is that collagen solution is liable to gelation in the process of cross-linking modification. From the point of view of solving these problems in an integrated manner，in the present project we are planning to synthetize polycarboxylic acid-NHS esters as a novel modification agent for collagen in liquid state. The stability, water-solubility and flowability of collagen solutions could be modified and controlled synchronously by the use of this novel modifier. By building the model of denaturation kinetics of collagen in the process of heating, the effect of modification on the stability of collagen will be investigated. By exploring the dissolution behavior under physiological conditions，the effect of modification on the water-solubility of collagen will be studied. By examining the rheological properties of modified collagen, by building the corresponding rheological models, and by revealing the mechanism of collagen gelation caused by cross-linking, the effect of modification on the water-solubility of collagen will be explored. On the basic of these results, by combining the influencing mechanism of modification on the molecular structure and the state of aggregation, finally the relationship of structure-activity of collagen subjected to multi-modification will be revealed.In the present project, we tried to break through the traditional train of thought on collagen cross-linking, that is, to research and develop a kind of muti-functional modifier for collagen, which is of great significance for brodening the range of application of collagen solution and for facilitating the progress of the high-value ultization of animal biomass.

交联改性是当今广泛采用的用于改善胶原稳定性的方法。但目前研究基本集中于固态胶原的改性，这是由于液态胶原的交联改性尚存在两个问题：一是胶原需经酰化改性以改善生理条件下水溶性，无剩余氨基供交联反应使用，二是交联改性过程中胶原易凝胶化。本项目从统筹解决的角度出发，拟合成聚羧酸-NHS酯作为新型胶原改性剂，实现胶原稳定性、水溶性和流动性的同步改性调控。通过构建胶原热变性动力学模型，研究改性对稳定性的影响；通过考察胶原在生理条件下的溶解行为，研究改性对水溶性的影响；通过考察胶原的流变性能、构建相关流变学模型，并揭示交联致胶原凝胶化的机理，研究改性对流动性的影响并实现凝胶化行为有效调控。在此基础上，结合改性对胶原分子结构、聚集态结构的影响机制，最终阐明多功能化改性液态胶原的构效机理。本项目力图突破传统改性思路，研发一类多功能胶原改性剂，对于拓宽液态胶原应用范围、推动动物生物质高值利用进程具有重要意义。

传统交联改性的特点决定了其与酰化改性制备水溶性胶原无法同时实现，且具有易使胶原发生凝胶化的缺陷。本课题从统筹解决的角度出发，选用具有“梳型”结构的天然生物大分子聚谷氨酸（γ-PGA）进行功能化改造，制备聚谷氨酸-NHS 活性酯作为多功能胶原改性剂，同步实现对胶原进行酰化改性与交联改性。首先对合成的γ-PGA-NHS 酯进行表征，测定其基本结构；再将其用于胶原改性，采用多种方法和技术研究γ-PGA-NHS 酯对胶原分子结构与基础性能的影响；在此基础上，研究了胶原溶解性、热稳定性对γ-PGA-NHS酯结构特征与用量的复杂依赖性；揭示了γ-PGA-NHS 酯改性对胶原流变学性质的影响，同时探明改性对高浓胶原体系聚集态结构的影响；进一步通过荧光法研究了改性对低浓胶原体系聚集态结构的影响；综合分析γ-PGA-NHS 酯改性胶原的上述影响机制，明确了γ-PGA-NHS 酯改性胶原溶液体系内同时存在γ-PGA大分子与胶原大分子之间的交联作用、氢键作用、静电作用及链段缠结作用。在不同的γ-PGA-NHS 酯酯化度、不同的γ-PGA-NHS 酯用量下及不同的胶原浓度体系中，占据主导地位的作用并完全不相同。这些复杂作用共同构建了胶原稳定性、水溶性和流动性的制约关系，是多功能化改性液态胶原构效机理的核心。此外，细胞毒性实验表明γ-PGA-NHS酯不仅无细胞毒性，反而还可提高细胞活力。总之，本项目通过研制一种同步调控型多功能胶原改性剂，制备出一种新型高热稳定性（≥44.8°C）的液态水溶胶原，对于拓宽胶原应用范围，推动动物生物质在美容护肤、生物医学材料等领域的高值化利用具有重要意义。通过项目资助发表SCI论文7篇，申请专利1项，培养硕士生1名。