Synthesis, Surface Immobilization And Biological Evaluation Of Bio-inspired Double-positively Charged Phosphodicholine-chitosan

Based on the biomimetic molecular design, novel biomimetic chitosan derivatives, double-positively charged phosphodicholine-chitosan conjugates(PdCCs) with excellent antibacterial and biocompatible properties were synthesized based on the Atherton-Todd reaction for coupling double-positively charged phosphodicholine(PdC) to chitosan, then immobilized onto PCL membrane surfaces to develop an approach for constructing an integrated antibacterial and biocompatible surface, which can be widely used in biomedical and health fields.PdC composed of a phosphate bonded to double-positively charged choline groups was coupled to chitosan via Atherton-Todd reaction, and its degree of substitution(DS, mol%) can be adjusted by changing the feed ratio of related reactants. The chemical structure of PdCCs was confirmed by 1H and 31 P NMR. When the feed ratio of 6-O-trityl chitosan and double choline chloride phosphite was 1:3 and 1:5, the DS value was 32% and 42% respectively determined by their 1H NMR spectra, which was well consistent with the results of potentiometric titration and elemental analysis. As a control, single-positively charged quaternarized chitosan(HTCC) was also synthesized using 2, 3-epoxypropyl trimethyl ammonium chloride(GTMAC). Both of them had similar degree of polymerization(DP) by GPC measurement. The associated water structure in PdCCs with different water content(Wc) was investigated by DSC, and the appearance of cold crystallization peak proved the existence of freezing bound water in the Wc value ranged from 0.5 to 2.5, implying that PdCCs may show better biocompatibility than HTCC.Bacterial inhibition experiments, including MIC and bacterial content release measurements exhibited that both PdCCs and HTCC had excellent antibacterial properties against Gram-positive bacteria(S. aureus) and Gram-negative bacteria(E. coli). In vitro cytotoxicity assay revealed that the IC50 values of PdCCs against NIH/3T3 were above 2 mg/m L, comparable to 0.5 mg/mL for the IC50 of HTCC64. Blood compatibility suggested that all the hemolysis values of PdCCs and HTCC were below 2%; but unlike HTCC, PdCCs did not induce the morphology change and shorten the clotting time. And PdC can suppress the conformational change of BSA contacting with PdCCs. It was suggested that PdCCs showed better biocompatibility than HTCC, possibly due to the fact that the water barrier restrained by the local branched charge distribution can suppress the non-specific interaction with proteins and biomembranes.In order to build an excellent antibacterial and biocompatible surface, PdCCs with DS of 42% was used for further investigation. ATR-FTIR results showed that PdCCs has been successfully grafted onto the electrospun PCL membrane surface. SEM showed that the morphology of PCL membrane surface changed obviously after the immobilization. Contact angle measurements, antibacterial tests and protein adsorption experiments revealed that surface immobilization of biomimetic PdCCs can enhance the hydrophilicity, antibacterial properties and protein resistance capacity.In summary, the double-positively charged phosphodicholine-chitosan conjugate(PdCCs) with excellent antibacterial and biocompatibility properties was synthesized, and it can be grafted onto the surface to effectively integrate antibacterial and biocompatible properties, which is expected to be widely used in biomedical and health fields.