Study On The Stabilization Modification Of Liposomes And Their In-Situ Formable Hydrogels

In this paper,chitosan(CS)with good biocompatibility was used as the raw material,and the reactive functional group of double bond and thiol groups were connected to the CS side chain by the chemical modification of maleic anhydride and cysteine.Then these functional groups were further used to react to form hydrogels.Liposomes were prepared by thin-film hydration method using phosphatidylcholine and cholesterol as raw materials,which were coated with modified CS to improve their stability,and then reacted with the functional groups of CS to form hydrogels to prepare liposome hydrogels.The hydrogels are expected to be used as tissue engineering scaffolds for drug loading and tissue repair.CSMA was prepared by the modification of CS with maleic anhydride.The results showed that the CS was successfully modified by maleic anhydride and the double bond was attached to the side chain of CS.The degree of substitution of double bond and the amount of vinyl groups of CSMA under different conditions were discussed.The concentration of CS was 1 wt % and the reaction time was 24 hours was chosen.The molar ratio of chitosan repeating unit to maleic anhydride was 1:1 for the next experimental conditions.The degree of substitution of double bond was 72.5% and the amount of vinyl groups was 1780.46 ± 32.65 μmol/g.Modified CS with cysteine,under the action of catalyst(EDAC/NHS),the amide bond is formed by condensation reaction between the amino group of CS and the carboxyl group of cysteine to obtain thiolated chitosan(CSSH).The results of FTIR and 1H NMR analysis showed that CS was successfully modified by cysteine.The degree of substitution of thiolated chitosan,total thiol groups,free thiols content and disulfide bond content of CSSH under different conditions were discussed.Finally,the concentration of CS was chosen to be 1 wt %,and the molar ratio of CS repeats to cysteine was 1:1 for the next experimental conditions.The total thiol groups content of the product was 420.36 ± 0.14 μmol/g,the free thiol content was 200.84 ± 16.16 μmol/g,the degree of substitution of thiolated chitosan was 6.0% by 1H NMR and 5.4% by Ellman’s method.Liposomes(Lip)were prepared with phosphatidylcholine and cholesterol.To improve the stability of blank liposomes,CSSH and CSMA were coated on liposomes to obtain Lip-CSSH and Lip-CSMA,liposomes can load curcumin.The liposomes,coated liposomes and curcumin liposomes were analyzed by transmission electron microscopy,nano laser particle size analyzer,differential scanning calorimetry(DSC),rheology and stability.Transmission electron microscopy showed that the liposomes were spherical,and the particle size of the blank liposomes were 115.1±9.6 nm,negatively charged at-56.8±1.47 mV.After coating,the modified CS and Lip interacted,the liposomes particle size increased,the potential changed from negative to positive and the stability has improved.Through the encapsulation of curcumin in liposomes,the entrapment efficiency and drug loading were improved by coating CSSH and CSMA.Lip-CSSH improved the physical stability and storage stability of liposomes significantly.In vitro release experiments showed that CSSH-coated liposomes could delay the release of curcumin and had sustained release effect.The results showed that free curcumin had a significant inhibitory effect on MCF-7 at 100 μM,while Lip-CSSH contained curcumin with concentration of 100 μM,its inhibitory effect was not obvious.When its concentration and time were further increased,the growth of MCF-7 were inhibited obviously after treatment of 200 μM,72 h.The results show that curcumin released from Lip-CSSH over time,which facilitated the delivery and released of curcumin with long-cycle therapy.The CSSH/CSMA hydrogels and CSSH/CSMA/Lip liposomes hydrogels were prepared and the mechanism of hydrogels formation were analyzed.The rheological analysis showed that with the increase of CSMA,the gel time were reduced and their mechanical properties were improved.The Scanning electron microscopy showed that the hydrogels had porous structure,which were conducive to the growth of nutrients and waste transport.Tensile experiments showed that the tensile strength of the dry and wet freeze-drying hydrogels were increased after the addition of liposomes.Polarizing microscopic observation showed that the dried gel films had a crystalline property and had a crystal structure under a polarizing microscope.Cell experiments showed that the hydrogels had good biocompatibility and are expected to be used for tissue repair.