| The novel enoxaparin sodium-PLGA hybrid nanoparticles(EPNs)was successfully developed for oral and intravenous delivery of water-soluble cytotoxic drug doxorubicin hydrochloride to improve oral bioavailability and overcome the multidrug resistance(MDR),and thereby enhance antitumor activity.HUVEC cells proliferation experiment was performed by MTT experiment.ES exhibited an anti-proliferation effect in a dose-and time-dependent manner in HUVEC cells.The prepared DOX-EPNs by emulsion-solvent evaporation method exhibited excellent performances of spherical and round shape,narrow particle distribution with small size(158.6nm),and zeta potentials was-14.5mV.By incorporation of negative polymer enoxaparin sodium(ES)to form electrostatic complex,the encapsulation efficiency(93.78%)of DOX was significantly improved.DSC and XRD experiments showed that DOX was in an amorphous form in the core of nanoparticles(NPs).In vitro release showed the pronounced sustained-release characteristics of DOX-loaded EPNs(DOX-EPNs).The cytotoxicity,cellular uptake and uptake mechanism were studied in DOX sensitive cells MCF-7 and resistant cells MCF-7/Adr.EPNs showed higher cytotoxicity and cellular uptake in comparison with DOX solution(DOX-Sol)in MCF-7/Adr cells,suggesting EPNs could reverse the MDR.The internalization of DOX-EPNs might be energy-dependent endocytosis processes involving caveolin-dependent endocytosis,clathrin-dependent endocytosis,macropinocytosis,and caveolae-/clathrin-independent endocytosis.UPLC-MS/MS method was developed to investigate DOX-Sol and DOX-EPNs in rat pharmacokinetics after intravenous injection.The results showed that EPNs enhanced the AUC(0-t)and t1/2 of DOX.Tissue biodistribution experiment indicated the hydration shell of pegylated-PLGA nanoparticles might prevent from the identification of reticuloendothelial system,and resulted in an improved plasma concentration of drug.In situ intestinal perfusion indicated the improved membrane permeability in all intestine segments(duodenum,jejunum and ileum)of EPNs than that of DOX-Sol,and the main absorption site for DOX-EPNs was ileum.Intestinal absorption mechanism showed that clathrin-dependent pathway and caveolin-dependent pathway were mainly involved in the endocytosis processes of DOX-EPNs.Confocal laser scanning microscopy(CLSM)and in vivo imaging system showed better permeability and intestinal wall bioadhesion of NPs.Compared with DOX-Sol,higher DOX level was observed in liver in DOX-EPNs treated group,suggesting EPNs could prolong circulation time of DOX in body.MDCK cells were used to study the cellular biological mechanism of DOX-EPNs.EPNs could improve the cellular accumulation due to endocytosis.Furthermore,EPNs could enhance the permeation of DOX from AP side to BL side.Multiple transcytosis pathways were involved in DOX-EPNs transporting the MDCK cellular monolayers mainly including clathrin-dependent endocytosis,energy-dependent endocytosis,and caveolin-dependent endocytosis pathways.UPLC-MS/MS method was developed to investigate the pharmacokinetics of DOX-Sol and DOX-EPNs.The assay was conducted by oral administration.Compared with DOX-Sol treated group,t1/2 and AUC(0-t)of the DOX-EPNs treated group were increased to 2.47-fold and 3.63-fold,respectively.Hence,DOX-EPNs were verified higher oral bioavailability and longer retention time in systemic circulation.Toxicity results showed that oral DOX-EPNs can significantly decrease the cardiotoxicity and renal toxicity of DOX. |
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