Study On The Oral Anti-hepatocellular Carcinoma Effect Of Tyroserupide/PLGA Nanoparticles Mediated By Intestinal Targeting Penetrating Peptide

Background Cancer is one of the most common chronic diseases in the world,characterized by uncontrolled proliferation and spread of cells.According to data released by the International Agency for Research on Cancer,cancer has become the deadliest disease in both China and the United States,and its incidence is expected to continue to rise in the next decade.Hepatocellular carcinoma(HCC)is the sixth most common cancer in the world,and the third leading cause of cancer-related death.It is a common malignant tumor that seriously threatens human life and health.The treatment of HCC is incredibly tricky.The side effects of chemotherapy limit its application in liver cancer,especially in thin patients.Therefore,it is necessary to search for drugs with low toxicity and side effects in the treatment of liver cancer.Tyroserleutide(YSL)is composed of L-tyrosine,L-serine,and L-leucine.It can inhibit the invasion and adhesion of mouse melanoma cell lines,and the more encouraging result is its anti-HCC effect.YSL was mainly distributed in the cytoplasm and co-located within the mitochondria of tumor cells.Moreover,YSL directly acts on the mitochondria of tumor cells,reduces the mitochondrial membrane potential,affects the permeability of the membrane and causes mitochondrial swelling.YSL has been shown to significantly prolong the survival of mice transplanted with H22 hepatoma cells and inhibit the growth of human BEL-7402 tumor in nude mice At the same time,YSL is a polypeptide with little side effects.Chemotherapy is often used to treat cancer clinically,i.e.intravenous drip is used to make drugs with cancer cell killing effect enter the systemic circulation directly.This mode of administration causes a rapid increase in the blood concentration of anti-cancer drugs,which exceeds the maximum tolerable concentration(MTC)of the drug in humans.YSL,as a polypeptide,is cleared or degraded quickly after intravenous injection.Therefore,it is necessary to give the drugs frequently,which brings more inconvenience to patients.In contrast,oral chemotherapy is administered in the form of oral anti-cancer drugs,which gradually enter the systemic circulation through intestinal absorption.This method can maintain the appropriate drug concentration in the blood,avoid blood drug concentration exceeding human MTC and prolong the action time of cancer cells and drugs.Thus,the side effects of drugs are reduced while improving the therapeutic effect.Also,oral chemotherapy is convenient to use,and patients can take their own medicine at home,which significantly saves the time and treatment cost of patients and the patient's compliance is high.However,how to improve the oral bioavailability of YSL is a major problem.Aim The aim of this study is to develop a new penetrating peptide targeting intestinal epithelial cells,and further modify YSL-PLGA NPs,which can not only be orally administered for YSL,but also enhance the inhibitory effect of YSL on hepatocellular carcinoma tumors.Specifically,the penetrating peptide(R6)was combined with the polypeptide of LRVG(targeting intestinal epithelial cells)to form intestinal targeting penetrating peptide(R6LRVG).We prepared R6LRVG-modified YSL-PLGA NPs(YSL-PLGA/R6 LRVG NPs)to enhance the absorption of YSL.First,YSL-PLGA NPs were prepared by double emulsion volatilization(W/O/W)giving the nano platform high drug-carrying capacity,then R6 LRVG was modified on its surface using the principle of electrostatic adsorption.After that,the NPs were characterized by transmission electron microscopy(TEM),Fourier transform infrared(FTIR)spectrum study and zeta-potential.Furthermore,the cell uptake,in vitro bioactivity and in vivo anti-tumor activity of the NPs were investigated.In addition,the mechanism was further studied,including internalization modes and the respiration rate of mitochondria.Methods1 Preparation and characterization of nanoparticles: YSL-PLGA NPs and PLGA NPs were prepared according to the double emulsion solvent evaporation method(W/O/W),and YSL-PLGA/R6 LRVG NPs and PLGA/R6 LRVG NPs were prepared by electrostatic adsorption.The morphological structure,size distribution,and degree of dispersion of the four nanoparticles were determined by TEM and the particle size potential.The drug loading of nanoparticles was characterized by elemental analysis,FTIR spectra analysis,and elemental analysis,and the entrapment efficiency and drug loading efficiency was examined by UV.Elemental analysis and FTIR spectra analysis were used to examine whether R6 LRVG peptide was successfully adsorbed.2 Nanoparticle stability and release in vitro: UV was used to detect the drug release from the drug loaded nanoparticles at different times under simulated gastrointestinal environment in vitro.Particle size to examine the stability of nanoparticles in simulated gastrointestinal solution.3 Cytoxicity and antitumor effect in vitro: The Cytotoxicity of PLGA NPs,PLGA/R6 LRVG NPs,YSL-PLGA NPs and YSL-PLGA/R6 LRVG NPs on Caco-2cells was examined using CCK-8 kit.Antitumor effect studies of drug loaded nanoparticles(YSL-PLGA NPs,YSL-PLGA/R6 LRVG NPs)were performed using BEL-7402 cells.4 Evaluation of cellular uptake and targeting: Instead of the stock drug,FITC was used to observe the fluorescence intensity profiles of FITC-PLGA NPs,FITC-PLGA/R6 LRVG NPs,FITC-PLGA/R6 NPs,and FITC-PLGA/LRVG NPs in Caco-2 cells or BEL-7402 cells by laser confocal microscopy,to evaluate the cellular uptake ability and targeting effect of the penetrating peptide,and to quantify the cellular uptake results of the two nanoparticles by flow cytometry experiments.Using the Caco-2 cell monolayer model,the transmembrane transport rates of the two nanoparticles at different incubation times were evaluated separately to illustrate the penetration versus targeting effect of the prepared nanoparticles.5 Endocytic pathway exploration: Several common cytostatics were chosen to treat Caco-2 cells incubated with FITC-PLGA NPs and FITC-PLGA/R6 LRVG NPs with temperature conditions to comparatively analyze the possible endocytic pathways.6 Cellular Respiration Rate: Basal respiration values,ATP synthesis oxygen consumption,maximal oxygen consumption,and mitochondrial inhibition reserve maximal respiration values of BEL-7402 cells mitochondria treated with YSL-PLGA/R6 LRVG NPs at different times versus different concentrations were measured by using XF cell Mito stress test kit and analyzed comparatively.7 Antitumor activity and safety in vivo: The in vivo antitumor activity of nanoparticles against HCC was investigated in a tumor bearing BALB/c mouse model induced by BEL-7402 cells.The body weight of the mice was measured and the major organs(heart,liver,spleen,lung,and kidney)of the mice were collected for H& E staining for histological analysis.Result1 TEM and size potential,showing that all four kinds of nanoparticles have a good globular morphology structure with a size around 200 nm and a uniform distribution,and a good degree of dispersion is not easy to aggregate.The positively charged nanoparticles modified with R6 LRVG peptide are favorable for oral route absorption.The results confirmed successful drug loading and good entrapment efficiency of the nanoparticles as characterized by elemental analysis,FTIR spectra analysis,and thermal analysis.Elemental analysis and FTIR spectra analysis confirmed successful electrostatic adsorption of R6 LRVG peptide.2 Through particle size and ultraviolet detection,the nanoparticles have good drug loading controlled release characteristics in gastrointestinal tract,high stability in simulated intestinal fluid,but not good stability in simulated gastric fluid.By oral gavage with 3% sodium bicarbonate solution,the nanoparticles can effectively avoid a large loss in the acidic gastric environment.3 The cytotoxicity assay results showed that the nanoparticles all exhibited nontoxicity with good cytocompatibility.Antitumor effect studies results showed that YSL-PLGA/R6 LRVG NPs significantly increased the cytotoxicity against BEL-7402 cells with a more pronounced antitumor effect.4 The results of laser confocal experiment and flow cytometry experiment indicated that the cell targeting effect of LRVG peptide modification contributed to the cellular uptake in Caco-2 cells.However,R6 LRVG modified nanoparticles showed targeting function to intestinal epithelial cells and could significantly enhance their absorption.More intuitively,the results of Caco-2 cell monolayer transport assay illustrated that the modification of R6 LRVG significantly improved the intestinal epithelial penetration ability of nanoparticles.5 Endocytic pathway showed that reticulin and caveolae/lipid rafts mediated endocytosis played an important role in the cellular uptake of FITC-PLGA NPs and FITC-PLGA/R6 LRVG NPs.6 In the examination of the cellular respiration rate,YSL-PLGA/R6 LRVG NPs reduced the basal respiration value,mitochondrial oxygen consumption and maximal respiration rate more effectively than YSL pure drug.Demonstrated that R6 LRVG modified nanoparticles enhanced mitochondrial targeting so that YSL could eventually be more enriched into mitochondria.7 Animal pharmacodynamics results showed that the oral administration of YSL-PLGA/R6 LRVG group achieved better tumor inhibition than YSL-PLGA NPs group,with better anticancer efficacy in vivo and without causing significant general side effects in normal tissues.Conclusion In conclusion,we have successfully prepared and evaluated R6LRVG-modified YSL-PLGA nanoparticles(YSL-PLGA/R6 LRVG NPs)for oral anti-tumor therapy of hepatocellular carcinoma.The results showed that with the modification of R6 LRVG,a novel multifunctional nanocarrier with good biocompatibility,high permeability and excellent tumor therapeutic effect was designed.R6 LRVG peptide modification plays a vital role in improving gastrointestinal absorption,cellular internalization and tumor therapeutic efficacy.Further studies on the mechanism showed that cell uptake of YSL-PLGA/R6 LRVG NPs could be related to multiple pathways mediated by endocytosis of reticulin and caveolae/lipid rafts.In addition,the YSL-PLGA/R6 LRVG NPs could interfere with the function of mitochondria.In particular,the YSL-PLGA/R6 LRVG NPs could decrease the basal respiratory value,mitochondrial oxygen consumption and maximal respiratory rate.Most importantly,oral experiments in vivo have achieved significant anti-tumor effects.H&E analysis did not show any histological changes in the major organs.Taken together,YSL-PLGA/R6 LRVG NPs are an effective oral drug delivery system for cancer treatment,which provides a promising prospect for intestinal drug absorption and has potential application value in oral cancer treatment.