Application Of Exosome-like Nano-vesicles Derived From Tea-leaves In The Prevention And Treatment Of Colon Diseases Effect

Inflammatory bowel disease(IBD)is one of the modern refractory diseases with chronic recurrent episodes of unknown etiology.With the continuous improvement of national living standard in China,the incidence of ulcerative colitis increases year by year,which has seriously threatened the healthy life of residents in China.At present,the treatment for IBD is mainly through anti-inflammatory drugs and immunosuppressive agents.Although these drugs have a certain therapeutic effect,they still have the disadvantages of low drug utilization and large toxic and side effects,which cannot meet the needs of clinical treatment at present.There is an urgent need to develop new treatments for IBD.Complete eradication of IBD can be achieved through antiinflammation,anti-oxidation,mucosal repair and the reconstruction of a dynamic balance of intestinal microorganisms.As a new therapeutic strategy,nano-drug carriers can not only change the drug solubility,but also target the corresponding target cells through modification to exert the maximum drug effect and the minimum toxic and side effects.In recent years,researchers have prepared different nano-drug carriers using a variety of materials,such as liposomes,proteins,polymer micelles,graphite,inorganic silicon nanoparticles,and some nano-drugs have been applied to the diagnosis and treatment of the corresponding diseases.And with the development of technology,nano drug delivery system has attracted more and more attention from researchers,not only because the nano drug carrier has a good biocompatibility but also has the characteristics of no degradation and no immunogenicity.In addition,the special properties of natural biological materials also deserve further study and exploration.As a natural nano-drug factory in plants,the natural nano-drug contains a large amount of lipids,some functional proteins and many bioactive small molecules,which has good biocompatibility and strong potential as a drug delivery carrier material.Therefore,in this study,we successfully extracted the natural targeting nano-drugs from three natural tea leaves and evaluated their therapeutic effects by in vitro and in vivo experiments.Through in vitro characterization,we found that the three nanocapsules were spherical nanoparticles with the uniform size and particle size of about 140 nm.At the same time,in vitro cell experiments and in vivo animal experiments prove that the three lipid nano-vesicles have good anti-inflammation,anti-oxidation performance and biosafety performance;and after the IBD mouse model is orally administrated,the three lipid nano-vesicles can be gathered to the inflammatory lesion site and specifically swallowed by target cells to achieve the effects of anti-inflammation,anti-oxidation and mucosal repair and achieve the effect of relieving inflammation at one time.The main research contents and conclusions of this paper are divided into the following three parts.Part I: Extraction and characterization of three kinds of natural lipid nanovesicles.Nanovesicles were identified from three tea leaves through the steps of juicing,differential speed centrifugation,ultracentrifugation,gradient ultracentrifugation and cleaning;The extracted lipid nanovesicles were characterized by dynamic light scattering(DLS),transmission electron microscope(TEM)and atomic force microscope(AFM).Organic small molecules contained in the particles were detected by HPLC,lipid components of nanoparticles were detected by quadrupole mass spectrometer,and proteins were identified and quantified by LC-MS/MS and orbital rap mass spectrometry.Results: Uniform spherical nanoparticles with average diameter of 140 nm and surface potential of-14.5 m V.Nanovesicles contain a variety of flavonoids and polyphenols,proteins related to inflammation regulation.The three lipid nanovesicles in the simulated fluid of gastrointestinal tract have good stability.Part II: In vitro cell experiment and biological safety detection of natural lipid nanocapsules.The toxicity of the three lipid nanocapsules to colitis-associated cells(CT-26,Raw264.7)was determined by MTT;the phagocytosis of three nanocapsules by Raw264.7 was quantified and characterized by flow cytometry(FCM)and laser confocal microscopy(CLSM).The secretion of inflammatory factors in Raw264.7 macrophages after treatment with three nanocapsules was detected by ELISA.The antioxidant capacity of Raw264.7 macrophage treated with that three nano-vesicle was detected by using a fluorescent probe DCFH-DA.The in vivo biological safety of the drug was evaluated by administering it to healthy mice for 7 days.RESULTS: By means of MTT assay and in vivo animal experiment,we found that the three nano-vesicles all had good biocompatibility,and would not cause harm to the body even after long-term oral administration to mice.The quantitative experiment of cell phagocytosis further proved that the three lipid nano-vesicles had natural targeting.After treating the macrophages,the three nanoparticles could effectively inhibit the production of pro-inflammatory factors and promote the production of anti-inflammatory cytokines,and at the same time,had the ability to remove oxygen free radicals,showing a time-dependent characteristic,which indicated that the three nano-vesicles could effectively exert the therapeutic effects to resist inflammatory diseases for a long time.Part III: Animal model experiments of three kinds of lipid nanocapsules in vivo.After the oral administration of three lipid nano-vesicles,the treatment differences of different nano-drugs on IBD and inflammation-caused in situ colon cancer were evaluated through DSS-induced mouse IBD model and AOM-induced in situ colon cancer model.Results: After oral administration,the three nanoparticles showed different therapeutic effects in different animal models.Evaluation of intestinal microbial remodeling induced by three lipid nanocapsules.The feces of mice in each experimental group in the IBD model treatment experiment were collected.By extracting the total RNA of the flora,and applying the second-generation sequencing technology to sequence the 16 S r RNA and OUT clustering,and by means of α diversity analysis and β diversity analysis,the reconstruction of intestinal microorganisms was analyzed.Results: There were significant differences in the abundance,diversity and microflora composition of intestinal microorganisms between the healthy group and the DSS control group.The flora structure of the nano-drug treatment group was different from that of the healthy control group and the DSS control group,but the general rule was that the better the drug treatment effect,the closer the intestinal microbial richness,diversity and flora structure were to those of the healthy group.