Preparation Of Maleic Anhydride Functional Copolymers And Construction Of Antibacterial Surface

Polymer materials are widely used in the field of medical devices due to their low density,ease of processing,good flexibility and biological inertness.However,since most of commonly used polymer medical device substrates lack antibacterial activity,the infection caused by implants/interventions has become one of the common clinical diseases,which not only seriously affects the quality of medical care,but also aggravates the pain and economic burden of injured patients.By modulating the surface/interface properties of the substrate,it is possible to impart functions such as antimicrobial activity to the materials without altering its intrinsic properties,which is an effective way to develop new anti-infective medical devices.However,at present,polymeric materials for medical devices generally suffer from surface chemical inertness and the commonly used antimicrobial agents still show high cytotoxicity.How to construct a stable,efficient and low-toxicity antibacterial coating on its surface by simple strategies is still an urgent problem to be solved in this field.Based on the above background,in this dissertation,a strategy was proposed to construct antibacterial surface by introducing functional maleic anhydride copolymer coating on the surface of polymeric substrates.By modulating the chemical structure and surface morphology of the antimicrobial coating,an antimicrobial surface with both high bactericidal efficiency and low cytotoxicity has been developed.The main results are as follows:（1）A cross-linked antibacterial coating based on maleic anhydride（MAH）-2-ethylhexyl acrylate（2-EHA）copolymer conjugated with polyhexamethylene biguanide（PHMB）was constructed.Firstly,2-EHA-MAH copolymer with different composition was synthesized by free radical polymerization.Proton nuclear magnetic resonance（¹H NMR）,attenuated total reflection flourier transformed infrared spectroscopy（ATR-FTIR）and gel permeation chromatography（GPC）were used to confirm the structure and composition of copolymer,and its molecular weight ranged from 1.2 to 1.5×10⁴Da.The copolymerization kinetics was monitored by gas chromatography,and the reactivity ratios of the monomers were calculated by Kelen-Tüd（?）s method as r_（2-EHA）=0.6653 and r_（MAH）=0.0403,respectively.The copolymer was coated on the surface of the silicone rubber modified by（3-aminopropyl）triethoxysilane by dip coating method.The effect of polymer coatings with different copolymer components was compared by SEM observation and it was found that the coating with complete structure and high flexibility could be obtained when the composition ratio of copolymer was[2-EHA]/[MAH]=6:4.By adjusting the concentration of copolymer solution,the coating thickness can be controlled in the range of 1.5-7.5μm.Poly（hexamethylene biguanide）（PHMB）was covalently coupled to the coating by the reaction of amino groups with anhydride groups,with a fixed density of up to 3 mg/cm².In order to improve the durability of antibacterial coating,gas-solid crosslinking reaction was carried out using gaseous organic diamine.The effect of ethylenediamine,1,2-diaminopropane and 1,4-butanediamine on the toughness of the cross-linked coatings was investigated and it was found that cross-linking with 1,4-butanediamine enhanced the durability of the coatings while maintaining good flexibility,with no cracking or peeling after 12 days immersion in water and no cracks observed after500 times bending.In vitro antibacterial test results showed that the modified substrate showed excellent antibacterial activity,with 99.9%and 95.1%inhibition against S.aureus and E.coli respectively.After 12days of continuous culture in simulated urine environment,the antibacterial activity against S.aureus remained above 96.2%,indicating that the coating had good long-term antibacterial stability.（2）A maleic anhydride copolymer coating with self-condensing cross-linking properties,high antimicrobial activity and low cytotoxicity has been developed without the addition of conventional antimicrobial reagents.Firstly,a terpolymer（PPTM）of maleic anhydride（MAH）,poly（ethyleneglycol）dimethacrylate（PEGMA）andγ-（methacryloyloxy）propyltrimethoxysilane（TMSPMA）was synthesized by free radical polymerization.The effects of different monomer content and PEGDA molecular weight on copolymer behavior and copolymer composition were studied.The molecular weight of copolymer was determined to be in the range of 1.2～1.9×10⁴Da by GPC characterization.The polymerization kinetics was monitored by gas chromatography,and it was found that TMSPMA had the highest polymerization rate of the three monomers,the MAH unit content in the copolymer was less than the feed amount and the MAH unit content in the copolymer increased with increasing PEGDA feed amount.Then,PPTM was dipped onto the surfaceofsiliconerubber（SPM）modifiedby（3-aminopropyl）triethoxysilane,and the effect of PPTM coating with different monomer ratios was compared.The coating thickness varied from 0.5-30μm when the polymer solution concentration increased from5%to 50%.The self-condensed cross-linking of the coating was achieved by adding 2.4 vol%water to the terpolymer solution and heating at125℃for 12 h.The transformation of Si-OH groups and formation of Si-O-Si in the self-condensed cross-linking process was confirmed by Fourier transform attenuation total reflectance infrared spectroscopy.It was found that the coating with complete coverage,long-term water resistance and good flexibility can be obtained when the molecular weight of PEGMA was 950 g/mo L and the copolymerization composition was[PEGMA]/[TMSPMA]/[MAH]=3/2/5.The cross-linked coating was significantly more flexible and without showing any cracks after 500times bending.The sliding friction of the modified catheter is reduced to1/2 of the initial value.The antibacterial results showed that SPM killed almost 100%of S.aureus and E.coli on the surface,and the surface bacterial coverage was reduced to less than 20%.The results of cycle antibacterial test showed that SPM could show effective antibacterial activity within 5 cycles,and the antibacterial activity within the first 3cycles was almost 100%.In addition,CCK-8 assay was used to evaluate the cytotoxicity of the coating on mouse fibroblasts（L929）in vitro.The results indicated that after 48 h of co-culture,the cell activity of SPM coating group could be maintained at 73%,which was higher than that of SPM coating coupled with antimicrobial peptide E6（54%）,showing lower cytotoxicity.（3）A hierarchical pattern-modified polyvinyl chloride（PVC）surface was constructed by surface living photografting technique,which could endow the surface with high bactericidal efficiency as well as low cytotoxicity and.Firstly,polyethylene glycol gel layer（PEGMA/PEGDA）was introduced into PVC surface as antifouling layer（PIG）based on the visible-light-induced controlled/living grafting polymerization,and the grafting density of gel layer was 0.18±0.01 mg/cm².By utilizing the residual dormant group on the surface of hydrogel layer,a secondary graft polymerization of quaternary ammonium polymer brush（PDMATDM）and maleic anhydride cross-linked copolymer（MAH-PEGDA）components was performed（noted as PIGQ and PIGM respectively）.SEM observation revealed that the hierarchical grafting system reduced bacterial coverage by more than 89%compared to the PVC surface directly grafted with the antimicrobial component,confirming that the introduction of a hydrophilic layer significantly resisted bacterial adhesion.On this basis,DMATDM or MAH-PEGDA strips with different width were grafted onto PIG surface by using photomasks to form a hierarchical graft patterned structure（PIGQ-stripe width（μm）/interval distance（μm）and PIGM-stripe width（μm）/interval distance（μm））.The morphology of the graft stripe was uniform with high fidelity.The height of the strips ranged from 0.05～0.5μm and 0.2～2μm,respectively,and increased with the increase of the interval distance.Although patterned PIGQ and patterned PIGM surfaces had obvious hydrophilic differences（100°/50°）,the anti-bacterial adhesion effects of the two patterned surfaces showed none direct correlation with hydrophilic differences,but are more affected by band spacing and height.Combined with the statistics of surface bacterial adhesion and antibacterial tests,it can be concluded that PIGQ-2/2,PIGM-1/2 and PIGM-2/2 have excellent anti-bacterial adhesion effect and nearly 100%bacterial inhibition rate against E.coli.Bacterial adhesion test showed that the surface of PIGQ-2/2 had good anti-biofilm effect within 7 days,but the stripe was easily damaged,and the surface was covered with a large number of bacteria after 14 days.On the contrary,the stripe on the surface of PIGM-1/2 was not broken after prolonged immersion,and showed good anti-biofilm effect within 14 days.In addition,cytotoxicity test showed that the layered graft patterned structure had good cytocompatibility.In addition,the cytotoxicity test showed that the layered graft patterned structure had good cytocompatibility.After co-culture with non-patterned PIGQ and PIGM surface for 48 h,the viability of L929 cells was only about 70%,while PIGQ-2/2 and PIGM-1/2 could ensure more than 95%,and the cytotoxicity was significantly reduced.