Research On The Molecular Design Of PH-sensitive-amphiphilic White And Polysaccharide Nanoparticles And Their Application In Tumor Targeted Drug Delivery

Bletilla Striata Polysaccharide(BSP)is a natural plant polysaccharide which extracted from Bletilla striata of traditional Chinese medicine.BSP has proper biosafety and biodegradability.BSP's backbone is ?-glucosidic bond linked mannose and glucose.Therefore,there are more reactive groups(such as hydroxyl groups)in the main chain.BSP's structural properties can be modified.In this paper,a pH-sensitive amphiphilic BSP derivative will prepare by structural modification using BSP as the original matrix.When it is in the aqueous phase solvent system,this BSP derivative will Self-assembly of nanoparticles(NPs)by amphiphilic interaction,simultaneously pH sensitivity to weakly acidic conditions.The pH environment in tumor tissue is weakly acidic.Furthermore,the anti-tumor hydrophobic drug(Dox)can be encapsulated by a hydrophobic core in the NPs structure.They are thereby forming an intelligent nano drug delivery system that targets tumor tissue.Subsequently,the properties of NPs explored from the perspective of material structure.Cytotoxicity,tumor cell inhibition rate,cellular uptake,and drug distribution in cells evaluated at the cellular level.At the individual level,tumor-bearing mice used as models to investigate the antitumor activity,distribution in vivo and pathological analysis of major organs and tumor sites.First,extract BSP from Chinese medicine Bletilla striata.Purification of BSP by depigmentation,deproteinization,exchange cellulose column chromatography and Sephadex column chromatography.Through structural analysis,BSP is mainly composing of ?-mannose and ?-glucose,both of the relative molar ratio of 4.8: 1.After detection by HPGPC,BSP showed a symmetrical peak shape with a molecular weight of 178 kDa.It characterized by Fourier transform-infrared spectroscopy(FT-IR),which showed the characteristic peak of the polysaccharide.The structure of BSP confirmed by nuclear magnetic resonance(NMR);BSP is a polysaccharide compound with concentrated molecular weight,uniform composition,and high purity.On this basis,the BSP structurally modified in three steps.Since histidine is protonated in an acidic environment and protonated to produce pH sensitivity,histidine(His)will act as a pH-sensitive group.The first step is to carboxymethylated BSP,the second step is to modify the His group to the C-BSP structure,and the third step is to introduce the hydrophobic group stearic acid(SA)onto the His-BSP structure.Product His-SA-BSP.FT-IR and NMR separately characterized the product structures of each step,and it confirmed that the modification was successful.Subsequently,the NPs prepared by using His-SA-BSP in the aqueous phase system by ultrasonic dispersion method.The related properties tested.It was found that the NPs structure expanded when the pH of the environment decreased,and the NPs decreased with the decrease of pH.The zeta potential increases and the phenomenon of charge reversal occurs.With the rise of the substitution degree of His group,the critical micelle concentration(CMC)value of His-SA-BSP NPs shows a downward trend;finally,the NPs detected.The acid dissociation value(pHd)indicates that the structure dissociated under the condition of pH=5.0.Doxorubicin(Dox)was used as a model drug and loaded into NPs.X-ray diffractometry(XRD)and FT-IR were used to detect the characteristic peaks of Dox in drug-loaded NPs,indicating that Dox successfully loaded.Subsequently,the relevant properties of the drug-loaded NPs tested.When the pH lowered from 7.4 to 5.0,the particle size of NPs increased sharply from 120 nm to 530 nm,and the same swelling phenomenon observed by transmission electron microscopy(TEM).The environment decreased from 7.4 to 4.0,the zeta potential of NPs increased from-13 mV to 19 mV,and a negative to positive charge reversal occurred at a pH of 6.5.Further preparation of Dox-SA-BSP NPs as a control was carried out in vitro.At pH=5.0,70% of Dox in Dox-His-SA-BSP NPs released within 36 hours.Under the same conditions,Dox-SABSP NPs released 40% of Dox,verifying that the pH sensitivity of His-SA-BSP derived from the His group in the polymer.At the cellular level,the CCK-8 method confirmed that NPs constructed with BSP as the primary substrate had extremely low cytotoxicity.After carrying the drug,it showed that Dox-His-SA-BSP NPs had the best antitumor activity.Subsequently,the uptake of Dox by MCF-7 cells examined.The results showed that the Dox-His-SA-BSP NPs group had stronger fluorescence intensity at the same acquisition time point.The distribution of Dox-NPs in tumor cells observed by laser confocal microscopy.The results showed that His-SA-BSP NPs first formed in lysosomes after being internalized by cells,under acidic conditions of lysosomes(pH 4~5)Swelling of NPs accelerates the dissociation of NPs,causing Dox to released into the nucleus.Finally,the mechanism of internalization of His-SA-BSP NPs was studied.The results showed that His-SA-BSP NPs enter the tumor cells through multiple pathways.The minicell growth pathway is the primary pathway for NPs internalization.BALB/C nude mice with murine breast cancer used as animal models,and the situation of His-SA-BSP NPs in small animals was also analyzed.On the one hand,the DIR probe was used to image living animals through small animals.The system explores the targeting and distribution of NPs.In contrast,His-SA-BSP delivers drugs to the tumor site more effectively,significantly increasing the delivery efficiency of the drug to the tumor site,and increasing the drug at the tumor site.Savings.On the other hand,mice were treated with anti-tumor by injection of Dox,Dox-SA-BSP NPs and Dox-HisSA-BSP.During the treatment period,the body weight of the Dox group and the saline group decreased.This NPs was not the case in the BSP NPs group,indicating that BSP NPs have good biocompatibility;after treatment,the Dox-His-SA-BSP group had the best therapeutic effect in the four experimental groups.Pathological studies revealed that no significant organ tissue lesions observed in the BSP NPs group,and severe apoptosis found in the His-SA-BSP group,indicating that the His-SA-BSP group has Excellent tumor targeting with good biocompatibility.