Synthesis Of Targeted Transmembrane Drug Delivery Materials Of R6RGD-modified Cyclodextrin And Its Antitumor Mediated Tyroserupide

BackgroundThe clinical treatment of cancer is mainly based on injection,which is limited by poor patient compliance,intensive cell killing and large side effects.Oral administration has the advantages of simplicity,low side effects and good patient compliance,which can be better used in cancer treatment,but its absorption is limited by the enzyme barrier and gastrointestinal barrier.Nanotechnology has a good application prospect in tumor oral drug delivery system because of its good stability,large specific surface area and surface specific modification.In recent years,peptide protein anticancer drugs have been widely studied for their advantages of high efficiency,low toxicity and high specificity,but their bioavailability is low because they can not enter tumor cells stably.Peptides with the ability to penetrate the membrane can specifically recognize tumor cells and effectively mediate drug transmembrane absorption.At present,oral delivery combining targeted membrane peptides and nanoparticles(NPs)for polypeptide protein anticancer drugs not only improves the stability,specificity and bioavailability of drugs,but also serves as a better alternative for injection delivery,which has great practical significance in the tumor delivery system.Hexaarginine(R6)is a transmembrane peptide containing six arginines.RGD(arginine glycine aspartate)is an integrin that can specifically recognize the high expression on the surface of tumor cells?_v?₃.The combination of them(R6RGD)with anticancer drugs can effectively target tumor cells,promote drug absorption and play a specific killing role.Carboxymethyl-?-cyclodextrin(CM?CD)has good water solubility and high safety,and carboxymethyl on the surface can provide good specific modification sites.Based on the good biological characteristics of CM?CD and the effective tumor targeting property of R6RGD,combined with the advantages of nanodrug delivery system,CM?CD nanoparticles modified with targeted penetrating peptide can be used as an effective tumor targeting nanooral drug delivery system to achieve good drug delivery applications.AimIn this project,we designed a kind of composite nanoparticles(YSL/R6RGD-CM?CD NPs)to achieve oral targeted delivery of biomacromolecule anticancer drug tyroserleutide(YSL).The physical and chemical characterization and stability of NPs were studied,and the safety and absorption promoting effect of NPs were explored at the cellular level.In addition,the targeted absorption effect and tumor target of nanoparticles in tumor cells were further verified.At the same time,through the study of anti-tumor effect in vitro and in vivo and the preliminary study of anti-tumor mechanism,it is proved that the nanoparticles drug delivery system has good targeted drug delivery effect and great application prospect.Methods1 Preparation and characterization of packaging materials:R6RGD modified carboxymethyl-?-cyclodextrin drug carrier R6RGD-CM?CD was prepared with amide bond by dehydration and condensation.Fourier transform infrared spectroscopy(FTIR)and matrix assisted laser desorption time of flight mass spectrometry(MALDI-TOF-MS)were used to analyze whether R6RGD was successfully bonded to CM?CD.The bonding amount of the packaging material was determined by ¹H-NMR and protein content detection,and then the thermal stability of the successfully synthesized packaging material was explored by differential scanning calorimetry(DSC).2 Preparation and characterization of nanoparticles:R6RGD-CM?CD and YSL were used to form stable nanoparticles by saturated solution method under the action of electrostatic interaction and hydrogen bonding.The morphology and distribution of nanoparticles were observed by transmission electron microscope and particle size analyzer.The encapsulation efficiency and drug loading rate of YSL nanoparticles were determined by FTIR.3 Stability and in vitro release of nanoparticles:The stability and in vitro drug release of the nanoparticles were evaluated by simulating the gastrointestinal environment and detecting the content of YSL under specific conditions by UV spectrophotometer.The release of YSL from nanoparticles was evaluated at different p H to analyze the possible release of YSL from nanoparticles in the gastrointestinal tract;and the feasibility of oral antineoplastic drug delivery was evaluated by measuring the activity of casein loaded R6RGD-CM?CD NPs in the gastrointestinal enzyme environment.4 Cytotoxicity and uptake mechanism:CCK8 method was used to evaluate the cytotoxicity of CM?CD NPs,YSL/CM?CD NPs,R6RGD-CM?CD NPs and YSL/R6RGD-CM?CD NPs on Caco-2 cells.CM?CD NPs and R6RGD-CM?CD NPs were prepared by using FITC.Caco-2 cells were treated with inhibitors of different absorption ways,and then two kinds of fluorescent labeled NPs were applied to the pretreated cells.The possible absorption ways of nanoparticles were explored by comparing the absorption ways of different treatment groups.5 Cell uptake promoting and tumor cell targeting:Laser confocal microscopy was used to observe the uptake of FITC labeled CM?CD NPs and R6RGD-CM?CD NPs by Caco-2 cells to evaluate the absorption promoting ability of targeted transmembrane peptide in cells.By comparing the uptake of tumor cells and normal cells,and RGD competitive uptake within the group,the qualitative and quantitative analysis was carried out by fluorescence microscope and flow cytometry to further evaluate the tumor cell targeting effect and targeting sites of NPs.6 Pharmacodynamics and mechanism of nanoparticles in vivo and in vitro:CCK8method was used to detect the killing effect of YSL/CM?CD NPs and YSL/R6RGD-CM?CD NPs on BEL-7402 cells,and to explore the anti-tumor effect of drug loaded nanoparticles in vitro.Then,the antitumor effects of YSL/CM?CD NPs and YSL/R6RGD-CM?CD NPs in vivo were detected in mice bearing BEL-7402hepatoma,and the toxicity of drug loaded nanoparticles was observed by organ tissue sections.At the same time,in order to further explore its mechanism of tumor inhibition,XF96 cell mitotic stress test kit(MA)was used to measure the respiratory rate of BEL-7402 cells.Concentration-dependent and time-dependent groups were set up to explore the inhibition of drug loaded nanoparticles on tumor respiration under different time and concentration.Results1 R6RGD-CM?CD was successfully formed by R6RGD and CM?CD by amide bond in the ratio of 1:1.R6RGD-CM?CD nanoparticles with stable structure and reasonable size were prepared by using R6RGD-CM?CD and YSL nanoparticles.The particle size of the nanoparticles was 165 nm,with high drug loading rate,negative charge and good dispersion.2 R6RGD modified nanoparticles had good stability in simulated gastrointestinal environment.The release curve in p H environment showed that R6RGD modified nanoparticles had good tumor microenvironment release effect.In vitro cytotoxicity experiments showed that nanoparticles were a non-toxic drug delivery carrier.3 The results of Caco-2 cell uptake assay showed that R6RGD modified drug loaded nanoparticles had better uptake effect than unmodified drug loaded nanoparticles,and the cellular uptake mechanism was related to reticulin mediated endocytosis and giant cell mediated endocytosis.At the same time,targeted uptake and absorption experiments on normal and tumor cells showed that R6RGD modified nanoparticles had better tumor targeting uptake effect,and the main target was integrin?_V?₃overexpressed in tumor cells.4 The in vivo and in vitro cytotoxicity of R6RGD loaded nanoparticles showed that both R6RGD modified and unmodified drug loaded nanoparticles had certain tumor inhibition effect,and R6RGD modified drug loaded nanoparticles showed stronger tumor inhibition rate.We found that R6RGD modified drug loaded nanoparticles could inhibit mitochondrial respiration.ConclusionTherefore,a cyclodextrin derivative(R6RGD-CM?CD)with tumor targeting and cell penetrating ability was successfully synthesized in this project,and YSL was loaded on the derivative to prepare excellent nanoparticles(YSL/R6RGD-CM?CD NPs)with good drug loading efficiency.The synthesized nanoparticles had uniform particle size,good dispersion and high biological safety.R6RGD modification promoted the uptake of nanoparticles by Caco-2 cells,and the cellular uptake mechanism was related to the endocytosis and macropinocytosis mediated by trellin.At the same time,YSL/R6RGD-CM?CD NPs could over express integrin on tumor surface?_v?_3,which allowed the nanoparticles to aggregate on the surface of tumor cells.In addition,YSL could enter tumor cells more easily to achieve killing effect under the action of transmembrane peptide.Therefore,the killing effect of YSL/R6RGD-CM?CD NPs on tumor cells in vitro and in vivo was significantly higher than that of YSL/CM?CD NPs.Meanwhile,further study on its anti-tumor mechanism showed that nanoparticles could inhibit mitochondrial respiration,which may be related to YSL directly acting on mitochondria,changing mitochondrial membrane permeability,reducing mitochondrial membrane potential and accelerating mitochondrial dissolution.To sum up,R6RGD-CM?CD could be used as a kind of nano drug material with good tumor targeting and mediate YSL to form an effective anti-cancer system,which had a good application prospect in oral nano drug delivery of anti-tumor.