Construction Of Oxidized Hyaluronic Acid-doxorubicin Prodrug Delivery System For Breast Cancer Therapy

Breast cancer has become the leading cause of cancer morbidity and mortality among women worldwide.Currently,chemotherapy is one of the most common methods in breast cancer therapy.Nonetheless,such therapy still elicits severe side effects and frequently leads to low survival owing to indiscriminate drug distribution between cancerous and normal tissues.With the rapid development of nanomedicine,nanoparticles(NPs)-based drug delivery systems have attracted extensive attention from many aspects.Using chemical synthesis and self-assembled nanotechnology,the prepared NPs have considerable distribution coefficient and therapeutic efficacy for breast cancer.Herein,a CD44-targeted acid-sensitive nanovehicle for the delivery of doxorubicin(DOX)was designed to simultaneously achieve active-targeting of breast cancer and intracellular release of drugs.We partially oxidized HA with sodium periodate to obtain oxidized hyaluronic acid(OHA).The OHA could be conjugated with the DOX via a Schiff base condensation reaction,followed by the selfassembly of an amphiphilic nanosystem with a hydrophilic OHA shell and a hydrophobic DOX core(OHA-DOX NPs).The pH-dependent nanosystem exhibited excellent stability in the blood circulation but rapidly triggered the drug release upon reaching tumor cells,thereby improving the systemic toxicity and anti-cancer efficacy of the DOX.In this paper,the physicochemical properties,biological stability,active-targeting ability,as well as anti-breast cancer effects in vitro and in vivo of the synthesized NPs were evaluated comprehensively.The synthesis of OHA-DOX NPs was driven by Schiff base reaction and self-assembly.The oxidation degree of OHA was 24.3% ± 1.6% and the drug loading content was 9.86% ± 0.52% by ultraviolet-visible spectrophotometry.The successful synthesis of OHA-DOX was characterized by ultraviolet-visible absorption spectroscopy,infrared spectroscopy and hydrogen nuclear magnetic resonance spectroscopy.The critical micelle concentration of OHA-DOX was 4.5 μg/ml by fluorescence spectroscopy.The average particle size of OHA-DOX was 129.2 ± 2.1 nm and the Zeta potential was-21.9 ± 1.2 m V using dynamic light scattering via a Zetasizer nanoparticle analyzer.The OHA-DOX had good stability,and there was no significant change in particle size in normal saline for 7consecutive days.The drug release of OHA-DOX at pH 5.0 was significantly higher than the normal physiological pH by dynamic dialysis.The hemolysis test and cytotoxicity test on MCF-10 A,HUVEC and H9C2 confirmed that OHA-DOX had good biocompatibility compared with DOX.The targeting ability and anti-breast cancer effect of OHA-DOX were discussed from the cellular level.Firstly,the targeting ability of OHADOX towards MCF-10 A cells(CD44 in resting state),MCF-7 cells(CD44with normal expression)and MDA-MB-231 cells(CD44 with overexpression)was studied.OHA-DOX showed the highest fluorescence intensity in MDA-MB-231 cells via both confocal laser scanning microscopy and flow cytometry,which demonstrated the effective accumulation of OHA-DOX in CD44-overexpressed MDA-MB-231 cells.Secondly,HA-competitive inhibition assay showed that compared with the groups without HA pretreatment,the uptake efficiency of NPs was remarkably diminished in all cells pretreated with HA,indicating CD44-mediated selective endocytosis of OHA-DOX.Finally,the OHA-DOX showed excellent anti-proliferative activity on breast cancer cells MCF-7and MDA-MB-231 by a MTT assay,which gradually equaled or even exceeded the cytotoxicity of free DOX.The targeting ability and anti-breast cancer effect of OHA-DOX were further discussed from the animal level.The CD44-overexpressed MDAMB-231 cells were selected to establish a tumor-bearing nude mouse model.Firstly,ex vivo DOX fluorescence imaging showed that OHADOX obviously accumulated on tumor tissues instead of internal organs after 24 h post-injection.Next,the anti-breast cancer effect of OHA-DOX on MDA-MB-231 tumor-bearing nude mice was evaluated.Compared with DOX,OHA-DOX exhibited the strongest anti-tumor proliferation activity and almost no weight loss in nude mice after administration.The H&E staining of tumors and major organs confirmed the low toxicity of OHA-DOX towards normal tissues and the largest necrosis area for breast cancer.Finally,the CD44 antibody incubation and staining of tumors verified the breast cancer targeting of OHA-DOX mediated by the CD44.In this study,DOX and CD44-targeting nanocarrier OHA were chemically coupled through pH-sensitive imine bonds,and then successfully self-assembled into an OHA-DOX nanosystem in an aqueous environment.The nanosystem had a simple preparation process,good particle size stability,low CMC value,pH-responsive drug release behavior and excellent biocompatibility.Based on CD44-mediated specific breast cancer targeting,the OHA-DOX nanosystem had excellent antiproliferation effects of breast cancer in vivo and in vitro.