细粒棘球绦虫多肽-壳聚糖季铵盐纳米颗粒的制备

doi:10.12140/j.issn.1000-7423.2023.03.006

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (3): 300-305.doi: 10.12140/j.issn.1000-7423.2023.03.006

细粒棘球绦虫多肽-壳聚糖季铵盐纳米颗粒的制备

吴晓莹(), 胡媛^*(), 曹建平

中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心），国家卫生健康委员会寄生虫病原与媒介生物学重点实验室，世界卫生组织热带病合作中心，国家热带病国际联合研究中心，上海 200025

收稿日期:2022-11-10 修回日期:2023-03-26 出版日期:2023-06-30 发布日期:2023-05-30
通讯作者: *胡媛（1976-），女，博士，研究员，从事寄生虫疫苗的研制。E-mail: huyuan@nipd.chinacdc.cn
作者简介:吴晓莹（1996-），女，硕士研究生，从事寄生虫疫苗的研制。E-mail: 2369528046@qq.com
基金资助:
上海市自然科学基金(19ZR1462600);国家自然科学基金面上项目(81971969);国家自然科学基金面上项目(82272369)

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

WU Xiaoying(), HU Yuan^*(), CAO Jianping

National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research; Key Laboratory of Parasite and Vector Biology; National Health Commissionof People’s Republic of China; World Health Organization Collaborating Centre for Tropical Diseases, Shanghai 200025, China

Received:2022-11-10 Revised:2023-03-26 Online:2023-06-30 Published:2023-05-30
Contact: *E-mail: huyuan@nipd.chinacdc.cn
Supported by:
Shanghai Natural Science Foundation(19ZR1462600);National Nature Science Foundation of China(81971969);National Nature Science Foundation of China(82272369)

摘要/Abstract

摘要：

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。

关键词: 细粒棘球绦虫, 壳聚糖季铵盐, 多肽-纳米颗粒, 离子交联法

Abstract:

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 characterisation. 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 encapsulation 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 precipitated at different mass concentrations and mass ratios of HACC and TPP. After single-factor experiments, the optimal preparation conditions were screened for small particle size and high encapsulation rate. The optimal preparation 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.

Key words: Echinococcus granulosus, Quaternary ammonium salt of chitosan, Peptide-nanoparticles, Ion cross-linking method

中图分类号:

R383.3

吴晓莹, 胡媛, 曹建平. 细粒棘球绦虫多肽-壳聚糖季铵盐纳米颗粒的制备[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(3): 300-305.

WU Xiaoying, HU Yuan, CAO Jianping. Preparation of Echinococcus granulosus peptide embedded in chitosan quaternary ammonium salt nanoparticles[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(3): 300-305.

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分组 Group	质量比（HACC∶TPP） Mass ratio (HACC∶ TPP)	粒径/nm Particle size/nm	多分散系数 PDI	Zeta 电位/mV Zeta potential/mV	包封率/% Entrapment efficiency/%
1	8∶1	310.50 ± 4.20	0.23 ± 0.01	28.27 ± 0.63	58.56 ± 0.91
2	9∶1	296.20 ± 1.59^a	0.25 ± 0.01	29.63 ± 0.49	53.44 ± 2.24^b
3	10∶1	325.77 ± 1.76^c	0.25 ± 0.01	30.97 ± 0.66	53.01 ± 0.61
4	11∶1	333.37 ± 3.06	0.26 ± 0.01	31.97 ± 0.21	48.24 ± 1.75

分组 Group	TPP浓度/mg·ml^-1 Concentration of TPP/mg·ml^-1	粒径/nm Particle size/nm	多分散系数 PDI	Zeta 电位/mV Zeta potential/mV	包封率/% Entrapment efficiency/%
1	0.25	339.80 ± 1.61	0.26 ± 0.01	29.03 ± 0.12	65.83 ± 0.93
2	0.50	296.20 ± 1.59^a	0.25 ± 0.01	29.63 ± 0.49	53.44 ± 2.24^b
3	0.75	313.97 ± 2.92^c	0.27 ± 0.02	29.63 ± 0.40	52.55 ± 0.71
4	1.00	319.10 ± 1.39	0.24 ± 0.01	28.83 ± 0.39	55.72 ± 3.87

分组 Group	HACC浓度/mg·ml^-1 Concentration of HACC/mg·ml^-1	粒径/nm Particle size/nm	多分散系数 PDI	Zeta 电位/mV Zeta potential/mV	包封率/% Entrapment efficiency/%
1	0.25	307.37 ± 2.17	0.23 ± 0.01	29.77 ± 0.46	75.95 ± 1.60
2	0.50	296.20 ± 1.59^a	0.25 ± 0.01	29.63 ± 0.49	53.44 ± 2.24^b
3	0.75	280.93 ± 1.47^c	0.27 ± 0.01	29.47 ± 0.25	35.99 ± 2.53^d
4	1.00	-	-	-	-

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

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