Preparation Of K5-DOX Conjugate And In Vitro Evaluation Of The Drug Delivery Properties

K5polysaccharide is the capsular polysaccharide of E. Coli K5. As the structure of K5polysaccharide is similar to heparin’s backbone, it is widely used for the chemoenzymicsynthesis of heparin and heparan sulfate. Since K5polysaccharide has water solubilty,non-immunogenity and long half-life, it can be used as drug carrier. In this study, K5polysaccharide-doxorubicin conjugate (K5-DOX) was prepared and the drug’s physical andchemical properties, in vitro drug release, anti-tumor activity and cellular uptake mechanismswere determined.The main contents and results are as follows:(1) K5polysaccharide was extracted and purified from the fermentation of E. coli K5strain, by the method of high-density fermentation in inorganic medium. K5polysaccharidewas oxidation by periodic acid to obtain aldehyde K5polysaccharide. Doxorubicin waschemically conjugated with aldehyde K5polysaccharide through “Schiff” base to obtainpH-sensitive K5-DOX. The chemical structure of K5-DOX was characterized by1H NMRspectrum, the drug content of K5-DOX is determined to be17.4%.(2) As K5-DOX is amphiphilic, it could self-assemble into nanoparticles in aqueoussolution. Transmission electron microscopy (TEM) observation demonstrated that K5-DOXnanoparticles were roughly spherical in shape. The average size of K5-DOX nanoparticles inPBS (pH7.4) is125nm (PDI=0.164). The average size of K5-DOX nanoparticles in RPMI1640medium containing10%FBS shows no significant variation, indicating serum resistantactivity.(3) In vitro drug release profiles were obtained by a dynamic dialysis method at variouspH conditions. The release rate of DOX from K5-DOX at pH5.0was much faster than that atpH7.4, revealing a pH-triggered release manner. K5-DOX nanoparticles were instablility atpH5.0, its size increases with time in pH5.0PBS.(4) MTT assay indicated that K5polysaccharide has good biocompatibility. Thecytotoxicity assay and flow cytometry analysis demonstrated the cellular uptake of K5-DOXwas faster than that of free DOX, showing improved efficacy. In vitro cellular uptake showedthat HeLa and A549cells could uptake K5-DOX in a short period after incubation. A549cellsuptake more K5-DOX as compared with HeLa cells. The cellular uptake mechanism indicatedthat K5-DOX were mainly internalized via clathrin-mediated endocytosis andmacropinocytosis pathways.In summary, K5polysaccharide has good biocompatibility, high blood stability, cellularuptake ability, has a wide range of applications in drug delivery system.