磷酸胆碱化壳聚糖衍生物的合成、表征及生物学评价
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摘要
本研究从细胞膜仿生的角度出发，设计合成一种新的壳聚糖磷酰化衍生物，以磷酰胺键的方式将细胞膜结构单元磷酸胆碱基团偶联到壳聚糖骨架上，研究其合成方法、生物学性能及其纳米化方法。
为了合成磷酸胆碱化壳聚糖衍生物，我们研究比较了三种壳聚糖的均相磷酸胆碱化方法：(A)采用新型高极性六氟异丙醇作反应介质，基于Antherton-Todd反应实现壳聚糖的直接磷酰化；(B)利用N-邻苯二甲酰化壳聚糖为中间体，以二氯磷酰胆碱为磷酰化试剂；(C)利用6-O-三苯基甲醚化壳聚糖为中间体，基于Antherton-Todd反应实现磷酰化。研究表明合成路线(C)适合用来均相合成磷酸胆碱化壳聚糖衍生物。
应用合成路线(C)，通过改变投料比，基于Antherton-Todd反应合成了三种不同取代度的水溶性磷酸胆碱化壳聚糖衍生物。NMR和FTIR谱图上对应-N+(CH3)3基团吸收峰的出现表明磷酸胆碱基团成功偶联到壳聚糖骨架的氨基上，根据1H NMR谱图的峰强度比计算出三种壳聚糖衍生物的取代度分别为16%、27%和42%。GPC数据显示，与壳聚糖相比，磷酸胆碱化壳聚糖衍生物的分子量有所降低，分子量分布有所拓宽；XRD、TGA、DSC和水溶性实验表明，磷酸胆碱化壳聚糖衍生物的结晶性能和热稳定性均有不同程度地下降，但是其在水中的溶解性能得到了很大地提升，三种取代度的衍生物均可溶于pH=1-12的水溶液中。
细胞毒性实验表明，3T3细胞与磷酸胆碱化壳聚糖衍生物共培养的相对增殖率在80%-110%之间，细胞毒性为0级或1级，属于无细胞毒性范畴；血液相容性实验表明，引入磷酸胆碱基团可以延缓衍生物的凝血时间，而且可以有效抑制血小板在其上的黏附与激活；与牛血清白蛋白(BSA)的相互作用表明，磷酸胆碱基团的引入可以有效抑制壳聚糖衍生物与BSA之间的相互作用，减小蛋白质的构象改变，这对避免激活因蛋白质构象改变而导致的不良生物反应具有重要意义。
磷酸胆碱化壳聚糖衍生物可自组装形成纳米粒子。研究表明，磷酸胆碱化壳聚糖衍生物仍可与三聚磷酸钠进行离子交联形成纳米粒子，这些纳米粒子呈现规则的球形结构，粒径在60-120nm之间，Zeta电位介于18-28mV；同时，磷酸胆碱化壳聚糖衍生物具有两亲性，可在中性水溶液中自组装成具有疏水核亲水壳的纳米胶束，由低到高，三种取代度衍生物的临界胶束浓度分别为0.129mg/mL，0.201mg/mL，0.256mg/mL。所形成的纳米胶束粒径范围在70-110nm之间，Zeta电位接近于0，介于0-4mV之间。这两类纳米粒子有望应用于药物/基因载体。
Through constructing a cell outer membrane mimetic structure, a novel phosphorylatedchitosan derivatives were designed to couple phosphorylcholine(PC) with bioactivity ontochitosan with a phosphamide binding, which can be used in biomedical materials.
Three phosphorylated methods of chitosan were investigated, including (A)1,1,1,3,3,3-Hexafluoro-2-propanol used as reaction medium to synthesize phosphorylatedchitosan directly based on Antherton-Todd reaction,(B) using Cs-NPTh as the intermediate andphosphorylcholine dichloride as phosphorylated agent,(C) Cs-Tr as the intermediate tosynthesize phosphorylated chitosan based on Antherton-Todd reaction. It was found thatphosphorylated chitosan can be synthesized using method (C).
Using Antherton-Todd reaction, phosphorylated chitosan derivatives with different DSwere synthesized. The new peaks in NMR and FTIR spectra indicated that the PC moiety hadbeen conjugated to the amino group of the chitosan. The DS of PC moiety was calculated by theamount ratio of H of-N+(CH3)3(from PC) to H1(from glucosamine units of GlcN andGlcN-PC) based on the1H NMR spectra. The DS values ranged from16to42mol%. The GPCanalysis found that PCCs appeared lower Mw values but higher Mw/Mn values compared withthe starting chitosan. All these PCCs with decreased crystallization showed excellent solubilityin the aqueous solutions within a wide pH range (1-12). TGA and DSC results revealed that thethermal stability of PCCs decresed with the increase of DS value.
The in vitro cycotoxicity of PCCs copolymers was evaluated using a MTT assayperformed with NIH/3T3cells. The cells showed almost100%viability in the presence ofPCCs with different DS value, which suggests that all the PCCs with low toxicity are safety forbiomedical application. The blood compatibility were evaluated by means of blood-clotting andplatelet adhesion assay. The blood-clotting assay indicated that PCCs could prolong theblood-clotting process. Platelet adhesion assay showed that PCCs could effectively inhibit theplatelet adhesion and activation. Using bovine serum albumin (BSA) as a model protein, UVadsorption spectra and fluorescence spectra revealed that the non-specific interactions betweenPCCs and BSA were effectively suppressed and the conformation of BSA was almostunchanged with the addition of PCCs.
Further, PCCs nanoparticles could be still formed in a spherical shape similar to chitosannanoparticles with zeta potential between18-28mV by ionically crosslinking withtripolyphosphate (TPP), the sizes were in the ranges of60-120nm. In addition, the amphiphilicPCCs copolymers could self-assemble to form spherical nano-aggregates. The CMC values ofPCCs were in the range of0.129-0.256mg/mL. The sizes of PCCs self-aggregates with zetapotential between0-4mV were in the ranges of30-60nm. These PCCs nanoparticles could beused as promising delivery systems for drug or gene delivery applications.
为了合成磷酸胆碱化壳聚糖衍生物，我们研究比较了三种壳聚糖的均相磷酸胆碱化方法：(A)采用新型高极性六氟异丙醇作反应介质，基于Antherton-Todd反应实现壳聚糖的直接磷酰化；(B)利用N-邻苯二甲酰化壳聚糖为中间体，以二氯磷酰胆碱为磷酰化试剂；(C)利用6-O-三苯基甲醚化壳聚糖为中间体，基于Antherton-Todd反应实现磷酰化。研究表明合成路线(C)适合用来均相合成磷酸胆碱化壳聚糖衍生物。
应用合成路线(C)，通过改变投料比，基于Antherton-Todd反应合成了三种不同取代度的水溶性磷酸胆碱化壳聚糖衍生物。NMR和FTIR谱图上对应-N+(CH3)3基团吸收峰的出现表明磷酸胆碱基团成功偶联到壳聚糖骨架的氨基上，根据1H NMR谱图的峰强度比计算出三种壳聚糖衍生物的取代度分别为16%、27%和42%。GPC数据显示，与壳聚糖相比，磷酸胆碱化壳聚糖衍生物的分子量有所降低，分子量分布有所拓宽；XRD、TGA、DSC和水溶性实验表明，磷酸胆碱化壳聚糖衍生物的结晶性能和热稳定性均有不同程度地下降，但是其在水中的溶解性能得到了很大地提升，三种取代度的衍生物均可溶于pH=1-12的水溶液中。
细胞毒性实验表明，3T3细胞与磷酸胆碱化壳聚糖衍生物共培养的相对增殖率在80%-110%之间，细胞毒性为0级或1级，属于无细胞毒性范畴；血液相容性实验表明，引入磷酸胆碱基团可以延缓衍生物的凝血时间，而且可以有效抑制血小板在其上的黏附与激活；与牛血清白蛋白(BSA)的相互作用表明，磷酸胆碱基团的引入可以有效抑制壳聚糖衍生物与BSA之间的相互作用，减小蛋白质的构象改变，这对避免激活因蛋白质构象改变而导致的不良生物反应具有重要意义。
磷酸胆碱化壳聚糖衍生物可自组装形成纳米粒子。研究表明，磷酸胆碱化壳聚糖衍生物仍可与三聚磷酸钠进行离子交联形成纳米粒子，这些纳米粒子呈现规则的球形结构，粒径在60-120nm之间，Zeta电位介于18-28mV；同时，磷酸胆碱化壳聚糖衍生物具有两亲性，可在中性水溶液中自组装成具有疏水核亲水壳的纳米胶束，由低到高，三种取代度衍生物的临界胶束浓度分别为0.129mg/mL，0.201mg/mL，0.256mg/mL。所形成的纳米胶束粒径范围在70-110nm之间，Zeta电位接近于0，介于0-4mV之间。这两类纳米粒子有望应用于药物/基因载体。
Through constructing a cell outer membrane mimetic structure, a novel phosphorylatedchitosan derivatives were designed to couple phosphorylcholine(PC) with bioactivity ontochitosan with a phosphamide binding, which can be used in biomedical materials.
Three phosphorylated methods of chitosan were investigated, including (A)1,1,1,3,3,3-Hexafluoro-2-propanol used as reaction medium to synthesize phosphorylatedchitosan directly based on Antherton-Todd reaction,(B) using Cs-NPTh as the intermediate andphosphorylcholine dichloride as phosphorylated agent,(C) Cs-Tr as the intermediate tosynthesize phosphorylated chitosan based on Antherton-Todd reaction. It was found thatphosphorylated chitosan can be synthesized using method (C).
Using Antherton-Todd reaction, phosphorylated chitosan derivatives with different DSwere synthesized. The new peaks in NMR and FTIR spectra indicated that the PC moiety hadbeen conjugated to the amino group of the chitosan. The DS of PC moiety was calculated by theamount ratio of H of-N+(CH3)3(from PC) to H1(from glucosamine units of GlcN andGlcN-PC) based on the1H NMR spectra. The DS values ranged from16to42mol%. The GPCanalysis found that PCCs appeared lower Mw values but higher Mw/Mn values compared withthe starting chitosan. All these PCCs with decreased crystallization showed excellent solubilityin the aqueous solutions within a wide pH range (1-12). TGA and DSC results revealed that thethermal stability of PCCs decresed with the increase of DS value.
The in vitro cycotoxicity of PCCs copolymers was evaluated using a MTT assayperformed with NIH/3T3cells. The cells showed almost100%viability in the presence ofPCCs with different DS value, which suggests that all the PCCs with low toxicity are safety forbiomedical application. The blood compatibility were evaluated by means of blood-clotting andplatelet adhesion assay. The blood-clotting assay indicated that PCCs could prolong theblood-clotting process. Platelet adhesion assay showed that PCCs could effectively inhibit theplatelet adhesion and activation. Using bovine serum albumin (BSA) as a model protein, UVadsorption spectra and fluorescence spectra revealed that the non-specific interactions betweenPCCs and BSA were effectively suppressed and the conformation of BSA was almostunchanged with the addition of PCCs.
Further, PCCs nanoparticles could be still formed in a spherical shape similar to chitosannanoparticles with zeta potential between18-28mV by ionically crosslinking withtripolyphosphate (TPP), the sizes were in the ranges of60-120nm. In addition, the amphiphilicPCCs copolymers could self-assemble to form spherical nano-aggregates. The CMC values ofPCCs were in the range of0.129-0.256mg/mL. The sizes of PCCs self-aggregates with zetapotential between0-4mV were in the ranges of30-60nm. These PCCs nanoparticles could beused as promising delivery systems for drug or gene delivery applications.
引文
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