The Construction And Evaluation Of Drug Delivery System Based On Redox-responsive PEG-sheddable Nanomicelle

PEGydration micelles can’t guarantee the rapid release of drug within tumor cells and then limits its antitumor efficacy.The problem of PEG hydration shell have been concerned about by researchers.We develop a redox-responsive PEG-sheddable copolymer of disulfide-linked polyethylene glycol 5000-lysine-di-tocopherol succinate(P5kSSLV),which can self-assemble into nanomicelles in aqueous condition and trigger the rapid release of encapsulated drugs within tumor cells.Star-shape redox-responsive PEG-sheddable DOX-loaded disulfide-linked polyethylene glycol 5000-lysine-di-tocopherol succinate(P5kSSLV-DOX)was prepared with P5kSSLV as the nanocarrier and DOX as the poorly soluble drug.The synthesis and characteristic of P5kSSLV were investigated.In addition,we further studied P5kSSLV-DOX nanomicelle including formulation characteristics,reduction response,cytotoxicity,cellular uptake mechanisms,apoptosis,in vivo pharmacokinetics and in vivo pharmacodynamic behavior.The structure of P5kSSLV copolymer was verified by]H-NMR,and the critical micelle concentration was 1.97 μg-mL-1.Hemolytic experiment showed that P5kSSLV had a lower hemolytic rate compared with Tween-80 and cremoohor EL,which suggested P5kSSLV could be intravenously injected.Reducing-responsive experimental showed that the disulfide bond in P5kSSLV could cleave in reducing condition,which led to the separation between hydrophilic group and hydrophobic group.With DOX as a model drug,redox-responsive P5kSSLV-DOX nanomicelle was prepared by film hydration method.The particle size,drug loading,encapsulation efficiency and Zeta potential of P5kSSLV-DOX nanomicelle were 26.64 nm,4.07%,92.37%and-0.84 mV,respectively.The nanomicelle was spherical with uniform size.DSC results showed that DOX in amorphous state was encapsulated in P5kSSLV-DOX nanomicelle.The size change,turbidity change and in vitro release of P5kSSLV-DOX nanomicelle in reducing condition revealed that P5kSSLV-DOX nanomicelle was reducing-sensitive.Under non-reducing condition,P5kSSLV-DOX had following behaviors:(1)The particle size remained at about 30 nm;(2)nanomicelle solution was clear and transparent red solution;(3)DOX was released about 30%within 24 h;(4)The micelle diameter remained at about 30 nm after 24 h release.Under reducing condition,P5kSSLV-DOX nanomicelle had following changes:(1)The particle size became smaller firstly and then larger;(2)Nanomicelle solution became cloudy;(3)DOX was released more than 50%within 24 h;(4)There was no nanomicelle in solution after 24 h release.The above results indicated that P5kSSLV-DOX was stable in the outside of tumor cell and was disassembled within tumor cells.The cytotoxicity,cellular uptake and mechanism and apoptosis of P5kSSLV-DOX nanomicelle were investigated in MCF-7 cells.Redox-insensitive P5kLV-DOX nanomicelle was chosen as control.Cytotoxicity and mitochondrial membrane potential test showed the blank P5kSSLV nanomicelle had the antitumor activity.P5kSSLV-DOX had a higher cytotoxicity compared with P5kLV-DOX.The cellular uptake of P5kSSLV-DOX was conducted by confocal laser scanning microscopy and flow cytometry.The uptake of P5kSSLV-DOX was time-dependent and concentration-dependent.P5kSSLV-DOX prompted more DOX to come into nucleus compared with P5kLV-DOX.Annexin V-FITC/PI staining was chosen to investigate the apoptosis of DOX-Sol,P5kSSLV-DOX and P5kLV-DOX.P5kSSLV-DOX had a higher apoptosis than P5kLV-DOX.Endocytosis inhibition test showed the uptake of P5kSSLV-DOX was energy-dependent caveolae-mediated endocytosis and macropinocytosis.Overcoming multidrug resistance of P5kSSLV-DOX was preliminarily investigated in MCF-7/Adr cells.In comparison with DOX-Sol and P5kLV-DOX,P5kSSLV-DOX had the highest cytotoxicity.In addition,P5kSSLV-DOX could improve the uptake of DOX in MCF-7/Adr cells.UPLC/MS/MS method was developed to determine DOX-Sol and P5kSSLV-DOX in rat pharmacokinetics after intravenous injection.Results showed P5kSSLV-DOX increased the AUC and t1/2 of DOX,which prolonged the circulation time of DOX significantly.Tissue biodistribution behaviors of fluorochrome Cy7 and P5kSSLV-Cy7 were conducted in Balb/C mice bearing 4T1 breast transplanted tumor.At predetermined time points,ex vivo fluorescent intensity of Cy7 in different tissues were measured.12 h or 24 h after injection,P5kSSLV-Cy7 had higher signal intensity of Cy7 than free Cy7,which suggested P5kSSLV-Cy7 had the advantages of long circulation and tumor targeting.In vivo anticancer efficacy test showed P5kLV-DOX and P5kSSLV-DOX could significantly inhibit the growth of tumor and decrease the toxicity in comparison with DOX-Sol,and redox-responsive P5kSSLV-DOX had the highest inhibitory effect.