Polysaccharide-based Core/shell Nanoparticles With Breast Tumor Microenvironment-responsive Drug Delivery

Malignant breast tumor is a common cancer among women with the highest mobidity and morality in the worldwide.The patients in the early stage of breast cancer are commonly treated by surgery,while those in the terminal state mostly rely on chemotherapy.The anti-cancer drugs will kill the normal tissue cells while killoing the cancer cells,leading to a significant side-effect to the human body.With the development of nanotechnology,drug nanocarriers,taking the advantages of materials science,biomedicine,and pharmacology,provide new ideas for cancer therapy.Many properties of drug nanocarriers,such as the stability in blood circulation,active targeting to cancer cells,and rapid intercellular drug delivery,can be improved by the functional modifications.Based on the features of breast cancer cell(such as high concentrations of ATP and Hyaluronidase),a novel quaternary ammonium chitosan(HTCC)/hyaluronic acid(HA)-based composite nanoparticle with good stability in the bloodstream,active targeting to breaset cancer cells,and rapid intercellular drug devliery,is developed in this work.The core is the self-assembled nanoparticle from the amphiphilic phenylboronic acid-conjugated quaternary ammonium chitosan(HTCC-FPBA)molecule,and the outer shell is the hyaluronic acid derivative(mHA-PEG)modified by polyethylene glycol(PEG)and methacrylate(MA),which combines with the core by electrostatic interaction followed by ultraviolet photo-crosslinking.The PEG group,cross-linked structure,and negative potential of the outer shell are in favor of the enhancement of stability in the bloodstream.After uptake by breast cancer cells,the outer shell of nanoparticles is degraded by hyaluronidase(Hyals)(Hyals-responsive).The phenylboronic acid group in the core combined with the ATP in the cytoplasm,leading to the change from hydrophobicity to hydrophilicity and disassembly of the core,and the subsequent fast drug delivery(ATP-responsive).The research comments and results of this work are as follows:(1)Preparation of drug-loaded self-assembled nanoparticles SNX@HTCC-FPBA NPs as the core layer.First,chitosan(Cs)was modified by the quaternary ammonium salt to synthesize a quaternary ammonium salt chitosan(HTCC)with good water solubility.Then,an amphiphilic molecule(HTCC-FPBA)was synthesized by amidation reaction between HTCC and 3-fluoro-4-carboxyphenylboronic acid(FPBA).The ~1H-NMR results showed that both quaternary ammonium salt and phenylboronic acid were successfully bonded to chitosan with a substitution degree of about 63%and 34%respectively.The critical micelle concentration(CMC)value of HTCC-FPBA self-assembled nanoparticles was measured to be 0.082 mg/mL by pyrene fluorescent spectrometry.The drug-loaded nanoparticles(SNX@HTCC-FPBA NPs)were obtained by a rotary evaporation method and the drug loading capacity was determined to be about 18.1%by UV-Vis.The particle size and zeta potential of SNX@HTCC-FPBA NPs were measured to be about 296.9 nm and+25.7mV respectively by DLS.The TEM observation showed that the drug-loaded nanoparticles were spherical with a particle size of about 150 nm.(2)Synthesis of mHA-PEG polymer as the component of shell layer.First,HA-PEG was synthesized from HA and PEG by amidation reaction,and then mHA-PEG polymer was obtained by the reaction between HA-PEG and methacrylic anhydride(MA).The ~1H-NMR results showed that both PEG and MA were successfully bonded to HA with a substitution degree of about 16.8%and 12.0%respectively.(3)Preparation of core/shell drug-loaded nanoparticles(SNX@HTCC-FPBA/mHA-PEG NPs).SNX@HTCC-FPBA NPs(core)and mHA-PEG(shell)were combined by electrostatic interaction and cross-linked by ultraviolet radiation to obtain the stablized core/shell SNX@HTCC-FPBA/mHA-PEG NPs.The particle size and zeta potential of SNX@HTCC-FPBA/mHA-PEG NPs were measured to be about 233 nm and-10.8 mV respectively by DLS.The TEM observation showed that the nanoparticles were spherical with a particle size of about140 nm.The stability test showed that in the conditions of mimicking bloodstream,the particle size of SNX@HTCC-FPBA/mHA-PEG NPs was little affected by the ionic strength of sodium chloride solution,fetal bovine serum,neutral or weak acid(pH7.4 or pH6.8?pH 5.0),and low ATP concentration(0.4 mM),indicative of their good stability in the bloodstream.In vitro drug release experiment showed that the drug delivery of SNX@HTCC-FPBA/mHA-PEG NPs was ATP/Hyals-responsive.By dual stimuli of ATP(4.0 mM)and Hyals(150 unit/mL)for 48 h,the cumulative drug delivery percentage was close to 90%,meanwhile the nanoparticles were observed to be swollen and partially degraded by TEM.The blood coagulation,hemolysis and BSA adsorption experiments showed that the nanoparticles had good blood compatibility.The cell viability by MTT assay showed that the drug-unloaded HTCC-FPBA/m HA-PEG NPs had no cytotoxicity on human epidermal fibroblasts(HSF)and breast cancer cells(MCF-7 and MDA-MB-453),however the drug-loaded SNX@HTCC-FPBA/mHA-PEG NPs presented a similar killing effect to pure SNX2112 for two breast cancer cells,i.e.MCF-7 and MDA-MB-453,overexpressing CD44 receptor.The CLSM observation showed that much more SNX@HTCC-FPBA/mHA-PEG NPs were swallowed by MCF-7 and MDA-MB-453 cells in comparison to HSF cells lowly expressing CD44 receptor,indicating that the endocytosis of SNX@HTCC-FPBA/mHA-PEG NPs by MCF-7 and MDA-MB-453 cells was mediated by CD44 receptor,that is to say,these nanoparticles were of active targeting to MCF-7 breast cancer cells.In summary,SNX@HTCC-FPBA/mHA-PEG NPs synthesized above show good stability in the bloodsream,active targeting to breast cancer cell overexpressed CD44 receptor,and ATP/Hyals dual stimuli responsive drug delivery after endocytosis by breast cancer cells.This work provides a good idea for the design of drug nanocarriers in the therapy of breast cancer.