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

doi:10.12140/j.issn.1000-7423.2023.03.006

Abstract

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

CLC Number:

R383.3

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	-	-	-	-