Tumor Microenvironment PH-responsive PEG-detachment Hyaluronic Acid-cyclodextrin Nanoplatform For Cancer Chemothrapy

Hyaluronic acid(HA)has been extensively used as drug carrier and natural active targeted ligand of CD44 receptor overexpressed on numerous tumor cells,and it possesses superior physiological and physicochemical properties of stability,water-soluble,biocompatibility,biodegradable,non-toxic,and non-immunogenic.However,HA is also a ligand of other HA receptors usually expressed in the reticuloendothelial system(RES),which results in unexpected off-target biodistribution of HA-based nanoparticles in RES and rapid clearance from the body.It was an effective strategy to modify the HA-based nanoparticles with PEG,which could effectively reduce RES capture,prolong the circulation time and enhance the accumulation of tumor site after intravenous administration.Unfortunately,the PEGylation remarkably inhibits tumor cellular uptake and endosomal escapement and significantly compromising the in vivo antitumor efficacy,that is "PEG dilemma".Here,we develop a tumor microenvironment pH-responsive PEG-detachment HA and cyclodextrin supramolecular nanoplatform to overcome this dilemma for improving the efficiency of active targeting and the anti-tumor efficacy.At first,three different molecular weight HAs(MW=7,63,102 kDa)were covalently modified with hydroxypropyl-β-cyclodextrin(HPCD)by the esterification between the carboxyl of HA and the hydroxy of HPCD,and the structures were characterized by 1H-NMR and FT-IR method.The tumor microenvironment pH-responsive adamantane-PEG conjugate(AD-B-PEG)with the benzoic imine linkage was synthesized,and the PEGylation HA and HPCD inclusion complex was prepared by the interaction of adamantane and cyclodextrin.The pH-responsive characteristic of nanocarriers was investigated,and the results indicated that the prepared inclusion complex could hydrolyze rapidly to detach the PEG at tumor microenvironment acid condition.The non-pH sensitive adamantane-PEG conjugate(AD-O-PEG)with an ester linkage was synthesized as the negative control.The Dox-loaded nanoparticles(Dox/HCVs)with three different HA MWs nanocarriers were prepared by the emulsion solvent evaporation method with the help of hydrophobic molecular vitamin E succinate(VES)served as a nanobridge.The physiochemical and pharmaceutical characteristics of Dox/HCVs were similar,and they possessed a spherical shape morphology,uniform size distribution,similar particle size(～140 nm)and zeta potential(～-30 mv),high drug encapsulation efficiency and excellent colloidal stability.Their similar in vitro release behaviors were pH-dependent.The cytotoxicity,cellular uptake and endocytosis mechanism of Dox/HCVs were carried out on CD44 overexpressed cells(Hela and H460 cells)and CD44 underexpressed cells(MCF-7 and A549 cells).The results indicated that the cellular internalization of Dox/HCVs was energy-consuming,CD44-dependent and primarily mediated by clathrin-mediated endocytosis.And the Dox/HCV-63 had the best active targeting efficiency.The effects of Dox/HCVs on affinity to CD44 receptor and CD44 clustering were investigated.And it was interesting to found that both the HA-CD44 binding affinity and induced CD44 clustering were HA MW-dependent,the two of which determine the apparent targeting efficiency of Dox/HCVs in the conflicting directions.The PEGylation Dox-loaded nanoparticles(Dox/HCVPs)were prepared with 63 kDa HA nanocarrier.They had similar physiochemical and pharmaceutical characteristics with Dox/HCVs,a slightly higher Zeta potential and pH-dependent release behavior.The cytotoxicity and cellular uptake of Dox/HCVPs were evaluated on CD44 overexpressed Hela and H460 cells.The Dox/HCVBP had a better cellular uptake capacity and cytotoxicity effect when compared with Dox/HCVOP.The investigation of cellular uptake mechanisms suggested that the Dox/HCVBP nanoparticles could improve the CD44 receptor-mediated cellular endocytosis for better cytotoxicity by detaching the PEG shell at the tumor microenvironment acidic condition.Furthermore,the endosomal escape ability was observed by immunofluorescence method.The results demonstrated that the Dox/HCVBP group had plenty of nanoparticles escaped from endo/lysosomes at 3 h and nearly completely escaped at 5 h,but there still were numerous nanoparticles trapped in endo/lysosomes in Dox/HCVOP group at 5 h.The in vivo pharmacokinetic of Dox solution,Dox/HCVOP and Dox/HCVBP were investigated in SD rats by UPLC-MS/MS method.The Dox/HCVOP and Dox/HCVBP had a similar pharmacokinetic profile,and PEGylation remarkably prolonged t1/2(～3 fold)and significantly increased the AUC0-t(～4 fold)in comparison with Dox solution.DiR-loaded HCVPs NPs biodistribution also was observed in 4T1 tumor-bearing mice by ex vivo imaging of major organs and tumor tissue.The two of Dox/HCVOP and Dox/HCVBP nanoparticles could effective enhanced the tumor accumulation,and there have no significant difference between their average fluorescence intensity of DiR in tumor tissues at 24 h post-administration.These results further demonstrated that benzoic imine was stable enough in blood circulation for helping guarantee the PEGyaltion of Dox/HCBP.In vivo anticancer effects of Dox solution,Dox/HCVOP and Dox/HCVBP were studied in the 4T1 tumor xenograft model.The results indicated that the Dox/HCVBP had a best antitumor effect,and TUNEL assay illustrated a high apoptosis level in tumor tissue of Dox/HCVBP group.Comparing with Dox solution,Dox/HCVPs had little influence on body weight owing to the decreased systemic toxicity.Those results suggested that tumor microenvironment pH-responsive PEG-detachment was an effective strategy to enhance the tumor targeting ability of nanoparticles by improving biodistribution and active targeting ability.Our findings pave the way to rationally design the natural ligand-targeting NPs by balancing two conflicting processes of tumor cellular uptake and in vivo nonspecific biodistribution.