叶酸偶联壳聚糖纳米粒的制备及其载药性能研究
摘要
叶酸受体和其配体的结合具有特异性、选择性。利用叶酸受体在某些肿瘤部位过度表达而在正常组织低水平表达的特性,选择价廉易得、天然无毒、生物相容性好,并具有一定抗肿瘤活性的生物大分子——壳聚糖作为药物载体,将叶酸偶联至壳聚糖后再与抗肿瘤药物相结合,通过叶酸受体的介导作用,增加药物在病灶局部的浓度、提高疗效、降低毒副作用,达到靶向给药的目的,在肿瘤治疗领域具有重要的实际意义。
本课题首次采用新的均相合成法制备叶酸偶联壳聚糖。通过红外光谱和紫外光谱对产物的结构进行了表征。运用正交实验设计的方法,分别对非均相和均相两种合成法的制备工艺进行了优化,结果表明：叶酸活性酯与壳聚糖的质量比对偶联比的影响最大,其次是反应时间,反应温度的影响最小；当叶酸活性酯与壳聚糖的质量比为2：1、反应时间为48 h、反应温度为45℃时,两种方法制备的产物的偶联比都达到最大,分别为0.029和0.088。
通过离子交联法制备壳聚糖纳米粒,研究了影响纳米粒粒径的主要因素,结果表明：壳聚糖和TPP浓度分别为5.0 mg/mL和2.0 mg/mL、二者质量比为4：1；室温下反应20 min时,壳聚糖纳米粒的粒径最小,为91.3 nm。在上述条件下,以相同方法制备出叶酸偶联壳聚糖纳米粒,经激光粒度仪测得其平均粒径为122 nm, Zeta电位为+47.1 mV；扫描电镜照片显示纳米粒的形状规则,基本呈球形,大小分布均匀,平均粒径约为150 nm。
以阿霉素和5-氟尿嘧啶为模型药物,制备了载阿霉素的叶酸偶联壳聚糖纳米粒和载5-氟尿嘧啶的叶酸偶联壳聚糖纳米粒。激光粒度仪测得二者的平均粒径分别为190 nm和164 nm；两种载药纳米粒的稳定性均良好,放置25天后平均粒径基本无变化。经检测,在90%置信度下,3批载阿霉素的叶酸偶联壳聚糖纳米粒的平均包封率为(37.89±0.70)%,平均载药量为(16.22±0.17)%；载5-氟尿嘧啶的壳聚糖叶酸偶联壳聚糖纳米粒的平均包封率为(44.73±0.83)%,平均载药量为(24.91±0.08)%。
The binding between folate receptor and its ligand is specific and selective. Based on the fact that folate receptor can be overexpressed on the surface of some kinds of tumor cells but scarecely expressed on normal cells, chosing chitosan as the material of drug carrier which is an inexpensive, nontoxic, biocompatiable macromolecule, folate-conjugated chitosan that coupled with anticancer drug can increase the concentration of drugs, enhance the curative effect and reduce side effect through folate receptor-mediated delivery, which makes practically significant sense in the field of tumor therapy.
New method of preparing in homogeneous phase was brought forward in this research for the first time. The folate-conjugated chitosan was prepared and the structure of product was characterized by FT-IR and UV-Vis spectra. The optimum condition of synthesis was obtained by orthogonal experimental design and the results were as follows:The mass ratio between folate and chitosan was the most important factor, the next one was the reacting time, the third one was the reacting temperature; When the mass ratio between folate and chitosan was 2:1 with reacting for 48 hours at the temperature of 45℃, the conjugated scale arrived at the maximum of 0.088 in homogeneous phase and 0.029 in unhomogeneous phase.
Chitosan nanoparticles with average particle size of 91.3 nm were prepared by ionically cross-linking method. The optimum technologies were obtained by researching the major factors in preparation and the results were as follows:the concentration of chitosan and folate were 5.0 mg/mL and 2.0 mg/mL respectively, the mass ratio was 4:1 with reacting for 20 minutes at room temperature. Under this condition, folate-conjugated chitosan nanoparticles were prepared by the same method. The average particle size and the Zeta potential were 122 nm and +47.1 mV respectively. Photo given by SEM showed an spheric shape and the average particle size was about 150 nm.
Doxorubicin-loaded-folate-conjugated chitosan nanoparticles and 5-Fluorouracil-loaded-folate-conjugated chitosan nanoparticles were prepared in this research. The nanoparticles were stable and had no change in size during 25 days. The average particle size of the former was 190 nm with the average embedding ratio and the average drug loading were (37.89±0.70)% and (16.22±0.17)% respectively on condition that the confidence coefficient was 90%. For the latter, the average particle size was 164nm with the average embedding ratio and the average drug loading were (44.73±0.83)% and (24.91±0.08)% respectively on condition that the confidence coefficient was 90%.
本课题首次采用新的均相合成法制备叶酸偶联壳聚糖。通过红外光谱和紫外光谱对产物的结构进行了表征。运用正交实验设计的方法,分别对非均相和均相两种合成法的制备工艺进行了优化,结果表明：叶酸活性酯与壳聚糖的质量比对偶联比的影响最大,其次是反应时间,反应温度的影响最小；当叶酸活性酯与壳聚糖的质量比为2：1、反应时间为48 h、反应温度为45℃时,两种方法制备的产物的偶联比都达到最大,分别为0.029和0.088。
通过离子交联法制备壳聚糖纳米粒,研究了影响纳米粒粒径的主要因素,结果表明：壳聚糖和TPP浓度分别为5.0 mg/mL和2.0 mg/mL、二者质量比为4：1；室温下反应20 min时,壳聚糖纳米粒的粒径最小,为91.3 nm。在上述条件下,以相同方法制备出叶酸偶联壳聚糖纳米粒,经激光粒度仪测得其平均粒径为122 nm, Zeta电位为+47.1 mV；扫描电镜照片显示纳米粒的形状规则,基本呈球形,大小分布均匀,平均粒径约为150 nm。
以阿霉素和5-氟尿嘧啶为模型药物,制备了载阿霉素的叶酸偶联壳聚糖纳米粒和载5-氟尿嘧啶的叶酸偶联壳聚糖纳米粒。激光粒度仪测得二者的平均粒径分别为190 nm和164 nm；两种载药纳米粒的稳定性均良好,放置25天后平均粒径基本无变化。经检测,在90%置信度下,3批载阿霉素的叶酸偶联壳聚糖纳米粒的平均包封率为(37.89±0.70)%,平均载药量为(16.22±0.17)%；载5-氟尿嘧啶的壳聚糖叶酸偶联壳聚糖纳米粒的平均包封率为(44.73±0.83)%,平均载药量为(24.91±0.08)%。
The binding between folate receptor and its ligand is specific and selective. Based on the fact that folate receptor can be overexpressed on the surface of some kinds of tumor cells but scarecely expressed on normal cells, chosing chitosan as the material of drug carrier which is an inexpensive, nontoxic, biocompatiable macromolecule, folate-conjugated chitosan that coupled with anticancer drug can increase the concentration of drugs, enhance the curative effect and reduce side effect through folate receptor-mediated delivery, which makes practically significant sense in the field of tumor therapy.
New method of preparing in homogeneous phase was brought forward in this research for the first time. The folate-conjugated chitosan was prepared and the structure of product was characterized by FT-IR and UV-Vis spectra. The optimum condition of synthesis was obtained by orthogonal experimental design and the results were as follows:The mass ratio between folate and chitosan was the most important factor, the next one was the reacting time, the third one was the reacting temperature; When the mass ratio between folate and chitosan was 2:1 with reacting for 48 hours at the temperature of 45℃, the conjugated scale arrived at the maximum of 0.088 in homogeneous phase and 0.029 in unhomogeneous phase.
Chitosan nanoparticles with average particle size of 91.3 nm were prepared by ionically cross-linking method. The optimum technologies were obtained by researching the major factors in preparation and the results were as follows:the concentration of chitosan and folate were 5.0 mg/mL and 2.0 mg/mL respectively, the mass ratio was 4:1 with reacting for 20 minutes at room temperature. Under this condition, folate-conjugated chitosan nanoparticles were prepared by the same method. The average particle size and the Zeta potential were 122 nm and +47.1 mV respectively. Photo given by SEM showed an spheric shape and the average particle size was about 150 nm.
Doxorubicin-loaded-folate-conjugated chitosan nanoparticles and 5-Fluorouracil-loaded-folate-conjugated chitosan nanoparticles were prepared in this research. The nanoparticles were stable and had no change in size during 25 days. The average particle size of the former was 190 nm with the average embedding ratio and the average drug loading were (37.89±0.70)% and (16.22±0.17)% respectively on condition that the confidence coefficient was 90%. For the latter, the average particle size was 164nm with the average embedding ratio and the average drug loading were (44.73±0.83)% and (24.91±0.08)% respectively on condition that the confidence coefficient was 90%.
引文
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