| The new CD44 receptors-targeted,pH-triggered degradable and multifunctional hyaluronic acid micelles were prepared to overcome the shortcomings of doxorubicin(DOX),such as the poor water solubility,low bioavailability,toxic side effects,multidrug resistance(MDR)and so on.Hyaluronic acid(HA),which targeted to the CD44 receptor,was used as the hydrophilic chain.Hyaluronic acid-octadecylamine(HOA)conjugates with different degrees of substitution were synthesized to verify whether the DS was the main factor in cellular uptake.A new acid degradable compound of 2-(octadecyloxy)-1,3-dioxan-5-amine(OD)with an ortho ester group was synthesized,and then conjugated to HA to develop pH-responsive and tumor-targeted hyaluronic acid-2-(octadecyloxy)-1,3-dioxan-5-amine(HOD)conjugates for intracellular delivery of DOX.The pH sensitive characteristic and drug release behaviour of the micelles were studied in detail.In vitro cytotoxicity,cell uptake,internalization mechanism and intracellular drug release of the HOD micelles were investigated.In vivo biodistribution and tumor-targeting characteristics of the micelles were also observed.Furthermore,DOX and VES were conjugated by hydrazone bond to get DOX-NN-VES prodrug,which was loaded by HOD to prepare DNVM drug delivery system with reversing MDR function.The mechanisms of reversal MDR and in vivo pharmacodynamics were also researched.Hyaluronic acid-octadecylamine(HOA)conjugates with 12%~51% degrees of substitution(DS)were synthesized.And a series of DOX loaded HOA micelles(DOX/HOA)were prepared and characterized.With the increase of DS,the size of DOX/HOA decreased obviously.The micelles had a good fetal bovine serum stability and presented a controlled-release manner.The in vitro cytotoxicity and cellular uptake studies showed that DOX/HOA23,based on appropriate DS and proper particle size,presented enhanced anticancer activity and efficient internalization to achieve the highest intracellular drug concentration.These results indicate that both the carboxyl groups and the particle size play important roles in the cellular uptake of HA-based micelles.2-(octadecyloxy)-1,3-dioxan-5-amine(OD)with an acid degradable ortho ester group was synthesized and conjugated to hyaluronic acid(HA)backbone to prepare pH-responsive and tumor-targeted hyaluronic acid-2-(octadecyloxy)-1,3-dioxan-5-amine(HOD)conjugates.1H NMR was used to confirm the structures of the OD and HOD.DOX/HOD micelles,with a narrow size distribution,were stable under physiological conditions,but the drug was released quickly in the tumor acidic microenvironment.In vitro cell tests proved that the pH-sensitive DOX/HOD micelles had enhanced cytotoxicity,effective internalization and quick pH-triggered release compared to DOX/HOA micelles.Furthermore,endocytosis inhibition studies presented that DOX/HOD micelles were internalized into cells mainly via the caveo-lae-mediated route.In vivo study of micelles in tumor-bearing mice indicateed that HOD micelles were effectively accumulated into the tumor tissue than HOA micelles.The DOX-NN-VES prodrug was constructed by acid-sensitive hydrazone bond and encapsulated into HOD micelles to prepare DNVM multifunctional drug delivery system.DVSM encapsulating DOX-VES(via amide bond)and DOXM encapsulating DOX were prepared as controls.MCF-7/ADR cells were adopted to study the capacity and mechanism of reversal MDR.The cytotoxicity and cell uptake efficiency of DNVM were significantly higher than DVSM and DOXM,indicating DNVM with a better capacity of reversal MDR.Reversal mechanism of MDR experimental results showed that compared with DVSM and DOXM,DNVM could more effectively inhibit drug efflux and the expression of P-gp and make the cells produce more reactive oxygen species(ROS)to reverse tumor MDR.In vivo pharmacodynamics results showed that DNVM significantly inhibited the tumor growth and no significant change in body weight during the experiment,indicating that DNVM can improve the treatment effect of anti-tumor drug and reduce the side effects. |
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