Preparation And Property Study Of Layer-by-layer Heparin/Chitosan Microcapsule

Layer-by-layer (LbL) assembly microcapsule (MC) possesses many advantages such asregulatory structure, easy to be modified, controllable drug release, which is widely used inthe field of drug delivery. However, LbL-MCs made of synthetic polymers have major draw-back of low biocompatibility, while LbL-MCs from natural polymers have bottleneckproblem of stability. Thus it is very essential to explore new natural polymers to prepareLbL-MCs with improved biocompatibility as well as stability.Heparin (HEP) is the most negative charged polysaccharide in nature, while Chitosan(CHI) is the only naturally occurred positive charged polysaccharide. HEP and CHI couldform stable complex via electrostatic interaction in aqueous solution. In addition, both HEPand CHI have excellent biocompatibility and are widely used as biomedical materials. Thiswork aimed to prepare a LbL-MC (HEP/CHI)5based on HEP and CHI and study itsmorphology, size, surface charge, biocompatibility, stability, drug loading property as well asdrug releasing behavior. Our results indicated that the microcapsule have good stability,biocompatibility and excellent drug loading capacity and pH sensitive drug releasing behavior.The study contents are as follows:(1) Preparation and characterization of (HEP/CHI)5Firstly (HEP/CHI)5microcapsules was prepared, and the preparation process wasoptimized. The optimal concentration of Heparin (HEP) and Chitosan (CHI) for (HEP/CHI)5preparation was measured as2g/L. ζ-potential values on MC surface would appearednegative or positive alternatively, suggesting that HEP and CHI has been adsorbed onto thesurface of cores step by step, and pH change did not affect their electrostatic adsorptionsignificantly. The optimal layer number of MC is5, under which MCs are well separatedwithout aggregation and have good mechanical strength. Morphology characterizationshowed that (HEP/CHI)5had spherical structure with loose porous membrane-like shell. MCshave an averaged size about4.2μm in dry state and5.2μm in wet state. The HEP content in(HEP/CHI)5was measured as (37.5±0.3) wt%, its release rates in pH5.0,7.4and10.0PBSsolution were all less than30%after24h, suggesting it was stable under different pHenviroments. MTT assay indicated it had no obvious toxicity against ECs, HeLa and A549. Insummary,(HEP/CHI)5microcapsule is an excellent drug carrier candidate.(2)(HEP/CHI)5-DOX microcapsules for anticancer therapyDoxorubicin (DOX) was loaded in (HEP/CHI)5to prepare (HEP/CHI)5-DOXmicrocapsules. Confocal laser scanning microscope (CLSM) images and drug loading testresults indicated that both HEP and DOX are loaded in the microcapsules, i.e., HEP mainlyexisted in microcapsule wall and its content was (48.0±0.5) wt%, while DOX located both inwall and cavity, and its loading rate is (7.3±0.3) wt%. DOX released faster at pH5.0thanneutral and alkaline pH, indicating a pH sensitive drug release behavior. After24h, theaccumulated release rate reached92.1%at pH5.0. Heparanase are over-expressed in tumor microenvironment, it may digest heparin and thus corrode the microcapsules. In vitroheparanase digestion experiment found the outer of CHI layer of MCs could protect heparinfrom heparanase degradation and maintain MC stability. CLSM observation found that MCscould achieve intracellular co-delivery of both doxorubicin and heparin. It is mainly becausethe outer of microcapsules were chitosan, which could help drug transport across cellmembranes. MTT assay results also presented that intracellular heparin and doxorubicinmight show synergistic anticancer effect, and this effect is more obvious in A549cell line.