Preparation Of Latexin Protein-loaded Chitosan Nanoparticles And Its Role In Tumor Immune Regulation

The activity of traditional drug dosage forms in vivo is limited by the physical and chemical properties of drugs,and has low bioavailability,high side effects and low compliance.Therefore,it is very necessary to find appropriate vectors to effectively control drug release and successfully target the treatment site,so as to improve the pharmacokinetic effect,stability and regulation of metabolic time of drugs.Nanoparticles can penetrate the cell and tissue space to reach the liver,spleen,lung,spinal cord,lymph and other target organs.Due to the biodegradability,p H value,ion or temperature sensitivity of the material,they can show controlled release characteristics,which can improve the effectiveness of drugs and reduce the toxic side effects.Chitosan is a polymer basic polysaccharide obtained from the deacetylation of chitin.It has biodegradability,biocompatibility,non-toxicity,antibacterial properties,cell affinity and unique adhesion.Chitosan nanoparticles are widely used in drug delivery systems because they can prolong the retention time of drugs during absorption,improve drug stability and utilization.Latexin(LXN),a carboxyl peptidase inhibitor with a total length of 222 amino acids,was first identified in the lateral neocortex of the rat brain as a marker of neurons in the lateral neocortex of the developing rat brain.Due to its protease inhibitor properties,it is thought that LXN may be involved in protein degradation and metabolism.In addition,LXN has also been shown to be involved in inflammation as it is expressed in macrophages and mast cells.Previous studies have shown that LXN is secreted from macrophages through exosome pathway,and a large amount of LXN in the form of exosome is distributed in tissue lesions.Macrophage exosomes containing LXN can promote the activation of Jurkat cells and inhibit the differentiation of CD4+T cells into Treg cells.Therefore,in this study,chitosan nanoparticles loaded with LXN protein were used to explore the mechanism of inhibiting CD4+T cell differentiation into Treg cells by LXN,and the effect of chitosan nanoparticles loaded with LXN protein on tumor growth.First of all,this experiment successfully prepared blank chitosan nanoparticles by ion gel method to prepare for LXN protein loading.The focus is to explore the biological characteristics of chitosan nanoparticles and the optimal conditions suitable for LXN protein loading.Chitosan nanoparticles were characterized by scanning electron microscopy,transmission electron microscopy,Fourier transform infrared spectroscopy,nanoparticle potentiometer and other means,and the blank chitosan nanoparticles were explored by time stability experiment,p H stability experiment,encapsulation rate test experiment,and cytotoxicity test experiment.The experimental results showed that the size of blank chitosan nanoparticles was 196.6 nm,the potential was 26.5 mv,and the PDI was 0.237.The nanoparticles were spheroidal particles with uniform shape,ideal shape and good dispersion.The blank nanoparticles showed good stability in a short time or in a month.The smaller size and corrected potential can be maintained at p H 4-7,and the encapsulation rate of the prepared chitosan nanoparticles for BSA increases with the increase of protein concentration.In addition,the positive charge of the prepared nanoparticles is more conducive to the loading of protein.At the same time,MTT assay showed that the synthesized nanoparticles had no obvious toxicity to cells.Secondly,we explored the effect of LXN-loaded nanoparticles on T cell activation and immunomodulatory performance in tumors.Firstly,the nanoparticles loaded with LXN protein were synthesized.The nanoparticles loaded with LXN protein were characterized by morphology characterization,immunocolloidal gold staining,particle size and distribution coefficient determination,time stability and p H stability.The immunocolloidal gold staining experiment proved that chitosan nanoparticles successfully loaded LXN protein.The measurement of particle size and distribution coefficient indicated that the prepared LXN-loaded nanoparticles presented regular spherical shape.The average particle size was 204.4 nm and the potential was 27.1 mv.The stability test indicated that the nanoparticles loaded with LXN protein had good properties within 8 hours and p H between 2 and 7.After the phagocytosis of LXN-loaded nanoparticles was detected by flow cytometry,we investigated the activation of Jurkat cells by LXN-loaded nanoparticles in mouse lymphocytes.The expression levels of CD44 and IFN-γ m RNA of Jurkat cells loaded with LXN proteomic cells increased compared with the activated group.The m RNA expression levels of CD44 and IFN-γ in Jurkat cells without LXN-loaded nanoparticles did not change significantly,indicating that the activation ability of Jurkat cells ingested with LXN-loaded nanoparticles was enhanced.After the phagocytosis of LXN-loaded nanoparticles was detected by flow cytometry,we investigated the activation of Jurkat cells by LXN-loaded nanoparticles in mouse lymphocytes.The expression levels of CD44 and IFN-γ m RNA of Jurkat cells loaded with LXN proteomic cells increased compared with the activated group.The m RNA expression levels of CD44 and IFN-γ in Jurkat cells without LXN-loaded nanoparticles did not change significantly,indicating that the activation ability of Jurkat cells ingested with LXN-loaded nanoparticles was enhanced.According to the results of cell experiments,we constructed a mouse tumor bearing model,which further confirmed the immunomodulatory effect of LXN protein supported by nanoparticles.The results showed that,compared with CTL treatment group,tumor growth was slower in the nanoparticle loaded LXN protein treatment group,and there were more CD4+T cells in spleen T lymphocytes.The proportion of CD4+T cells and Treg cells in the tumor was measured.Compared with CTL treatment group,there were more CD4+T cells and fewer Treg cells in the nanoparticle loaded LXN protein treatment group.The results of in vivo experiments indicated that the LXN protein supported by nanoparticles inhibited the differentiation of CD4+T cells into Treg cells in tumors.In summary,this paper focuses on the formation of chitosan nanoparticles and the promotion of tumor immunity as LXN protein carrier.