细粒棘球绦虫多肽⁃壳聚糖季铵盐纳米颗粒的制备

Preparation of Echinococcus granulosus peptide embedded in chitosan quaternary ammonium salt nanoparticles

目的以羟丙基三甲基氯化铵壳聚糖(HACC)和三聚磷酸钠(TPP)为载体,制备载有细粒棘球绦虫表位多肽FL46的纳米颗粒(NPs),筛选最优制备条件,对包封效果及形态表征进行评价。方法采用离子交联法,将FL46溶液缓慢滴加到HACC溶液中,以800 r/min转速搅拌并逐滴加入TPP溶液,制备FL46⁃HACC⁃NPs混悬液。使用纳米粒度电位仪检测平均粒径、多分散系数(PDI)和表明电位(Zeta),透射电子显微镜下观察其微观形态,二辛可宁酸(BCA)蛋白浓度测定试剂盒计算包封率。以HACC浓度(0.25、0.50、0.75、1.00 mg/ml)、TPP浓度(0.25、0.50、0.75、1.00 mg/ml)、HACC与TPP质量比(8∶1、9∶1、10∶1、11∶1)进行三因素四水平正交实验,单因素法考察对FL46⁃HACC⁃NPs表征的影响。结果不同的HACC和TPP质量浓度及质量比下,FL46⁃HACC⁃NPs溶液可呈澄清、乳光、沉淀状态。经单因素实验,以粒径小、包封率高为筛选条件,筛选出的最优制备条件为HACC浓度0.25 mg/ml,TPP浓度0.50 mg/ml,HACC与TPP的质量比9∶1。最优条件下可制备类球形或多边形FL46⁃HACC⁃NPs,粒径为(307.37±2.17)nm,PDI为(0.228±0.008),Zeta电位为(29.77±0.46)mV,包封率为(75.95±1.60)%。透射电镜下显示,FL46⁃HACC⁃NPs形态结构大小均匀,呈类球形或多边形。结论离子交联法可有效制备出优化的FL46⁃HACC⁃NPs,为细粒棘球绦虫多肽疫苗保护效果的研究提供基础。

Objective To prepare nanoparticles(NPs)containing the epitope peptide FL46 of Echinococcus granulosus using hydroxypropyltrimethyl ammonium chloride chitosan(HACC)and sodium tripolyphosphate(TPP)as carriers,to screen the optimal preparation conditions,and evaluate the encapsulation effect and morphological charac⁃terisation.Methods The FL46⁃HACC⁃NPs suspension was prepared by slowly adding FL46 solution dropwise to HACC solution using the ionic cross⁃linking method,stirring at 800 r/min and adding TPP solution dropwise.The average particle size,polydispersity index(PDI)and surface potential(Zeta)were measured using a nanoparticle size potentiometer,the microscopic morphology was observed under the transmission electron microscope and the encapsu⁃lation rate was calculated by BCA protein concentration measurement assay kit.A three⁃factor,four⁃level orthogonal experiment was conducted with HACC concentration(0.25,0.50,0.75,1.00 mg/ml),TPP concentration(0.25,0.50,0.75,1.00 mg/ml),and HACC to TPP mass ratio(8∶1,9∶1,10∶1,11∶1),with single factor method to examine the effect on the characterisation of FL46⁃HACC⁃NPs.Results FL46⁃HACC⁃NPs solution can be clarified,milky and precipi⁃tated at different mass concentrations and mass ratios of HACC and TPP.After single⁃factor experiments,the opti⁃mal preparation conditions were screened for small particle size and high encapsulation rate.The optimal prepara⁃tion conditions were HACC concentration of 0.25 mg/ml,TPP concentration of 0.50 mg/ml,and the mass ratio of HACC to TPP 9∶1.Under the optimal conditions,spherical or polygonal FL46⁃HACC⁃NPs with the particle size of(307.37±2.17)nm,PDI of(0.228±0.008),Zeta potential of(29.77±0.46)mV and encapsulation rate of(75.95±1.60)%can be prepared.Transmission electron microscopy shows that the FL46⁃HACC⁃NPs have a homogeneous morphological structure and are sphere⁃like or polygonal in shape.Conclusion Optimised FL46⁃HACC⁃NPs can be effectively prepared by the ion cross⁃linking method,which can provide basis for the study of the protective effect of E.granulosus polypeptide vaccine.