Studies On Folate And CD44 Receptors Dual Targeting Hyaluronic Acid Polymeric Micelles

In our study,hyaluronic acid(HA)polymer was selected as polysaccharide main chain for hydrophobized modification due to its specific targeting to tumor cells via CD44 receptor.We developed a series of novel self-assembled hyaluronic acid-octadecyl(HA-C18)copolymer by chemical conjugation of hydrophobic octadecyl moiety and further to synthesis folate-conjugated HA-octadecyl(FA-HA-C18)copolymer for active dual targeting.Novel polymeric micellar systems were developed by the self-assembly of the synthesized HA-C18 and FA-HA-C18 graft copolymers using paclitaxel(PTX)as model drug.The in vitro and in vivo characteristics of the self-assembled CD44 receptor single-targeting HA-C18 micelles and folate and CD44 receptors dual-targeting FA-HA-C18 micelles were investigated systematically including pharmaceutical properties,cytotoxicity,cellular uptake,endocytosis mechanisms,pharmacokinetic study and tissue distribution behaviors.Hemolysis study showed that HA-C18 and FA-HA-C18 polymers could be used as an injectable pharmaceutical adjuvant for PTX.The chemical structure of the synthesized HA-C18 and FA-HA-C18 copolymers were characterized by 1H NMR and FTIR.The degree of substitution(DS)of octadecyl group in a series of HA-C18 copolymers ranged from 12.7%to 19.3%,the critical aggregation concentration of which decreased from 37.3 to 10.0 ?g/mL.The DS of folic acid in HA-C18 copolymer was 6.8%,the critical aggregation concentration of which was 11.2 ?g/mL.PTX was successfully encapsulated into the hydrophobic cores of the HA-C18 and FA-HA-C18 micelles by ultrasonic method,with encapsulation efficiency as high as 97.3%.The physicochemical properties of the polymeric micelles were investigated.The average sizes of PTX-loaded HA-C18 of different DS of octadecyl group and FA-HA-C18 micelles with different drug loading amounts was in the range of 184.8-379.7 nm.PTX-loaded HA-C18 and FA-HA-C18 micelles presented spherical micellar nanoparticle with uniform size.Differential scanning calorimetry(DSC)and X-ray diffraction(XRD)analysis suggested that PTX was converted from the crystalline state to the amorphous state after loading into polymeric micelles.Moreover,in vitro release behavior of PTX was investigated by dialysis bag method and PTX was released from micelles in a near zero-order sustained manner.The cytotoxicity,cellular uptake and endocytosis mechanisms studies of HA-C18 and FA-HA-C18 micelles were evaluated in folate and CD44 receptors overexpressing MCF-7 cell line and CD44 receptor overexpressing,folate receptor deficient cell lines.In vitro antitumor activity tests suggested PTX-loaded HA-C18 and FA-HA-C18 micelles exhibited significantly higher cytotoxic activity against MCF-7 and A549 cells compared to Taxol at a lower PTX concentration.The cellular inhibition of FA-HA-C18 micelles was significantly greater than that by HA-C18 micelles for folate receptor-positive MCF-7 cells.The cellular uptake experiments were conducted by quantitative assay of PTX cellular accumulation and confocal laser scanning microscopy and fluorescence microscopy imaging of coumarin-6 labeled HA-C18 and FA-HA-C18 micelles.The quantitative and qualitative cellular uptake experiments showed that HA micellar systems possessed highed cellular uptake capacity,and the cellular uptake behavior of micelles was concentration-,time-and particle size-dependent.The folate and CD44 receptors dual-targeting of FA-HA-C18 micelles exhibited higher cellular uptake amount compared to the CD44 receptor single-targeting HA-C18 micelles.Folate and CD44 receptors competitive inhibition studies performed by fluorescence microscopy imaging suggested intracellular delivery of HA-C18 and FA-HA-C18 micelles was efficiently taken up via CD44 receptor-mediated endocytosis.The folate receptor-mediated endocytosis further enhanced internalized amounts of FA-HA-C18 micelles in MCF-7 cells,as compared with HA-C18 micelles.The internalization pathways of PTX-loaded HA-C18 and FA-HA-C18 micelles included clathrin-mediated endocytosis,caveolae-mediated endocytosis and macropinocytosis.The studies on cytotoxicity and cellular uptake were conducted to investigate the multidrug resistance(MDR)of HA-C18 and FA-HA-C18 micelles in MCF-7/Adr cell by comparison with MCF-7 cell.The investigations indicated that HA-C18 and FA-HA-C18 micelles could overcome PTX resistance by enhancing cellular accumulation of PTX in resistance cells.A sensitive and selective UPLC-MS/MS method was developed for the determination of PTX in biological specimen.The pharmacokinetic study of PTX-loaded HA-C18 and FA-HA-C18 micelles were evaluated.The results demonstrated HA-C18 and FA-HA-C18 micelles of PTX possessed much longer circulation and moderately larger AUC than Taxol.Tissue biodistribution behavior was also investigated in normal and MCF-7-bearing mice respectively.The results showed PTX-loaded HA-C18 and FA-HA-C19 micelles were widely and rapidly distributed into most tissues and the nanomicelles significantly altered the distribution performances of PTX in mice.PTX in HA-C18 and FA-HA-C18 micelleswere more distributed into liver,spleen and lung,while decreased the distribution of PTX in heart and kidney than Taxol.In addition,more PTX accumulated in solid tumor in MCF-7-bearing mice.The folate and CD44 receptors dual-targeting of FA-HA-C18 micelles exhibited higher tumor targeting efficiency compared to the CD44 receptor single-targeting HA-C18 micelles.