| Collagen is a natural polymer with good biocompatibility,low antigenicity and biodegradability.Collagen is widely used in cosmetics,cosmetic skin care,drug delivery and tissue engineering and other fields.However,collagen has some shortcomings,such as poor water solubility,poor thermal stability,and weak resistance to enzyme degradation,which limit the further application of collagen.A kind of natural polymer poly(γ-glutamic acid)(γ-PGA)with large amount of carboxyl group was used as raw material to the synthesis of its esterified derivativeγ-PGA-NHS esters,as a novel non-toxic multifunctional collagen modifier for the fabrication of modified collagen solutions with excellent performances in respect of water solubility,thermal stability and flowability.Firstly,γ-PGA-NHS esters were synthesized at room temperature using γ-PGA and NHS as raw materials,with carbodiimide as catalyst and dimethyl sulfoxide as solvent.Fourier infrared spectroscopy(FT-IR)displayed the ester peak at 1817 cm-1 and the C-O stretching vibration peak at 1110 cm-1 and 1035 cm-1,indicating that the synthetic product was γ-PGA-NHS ester.The results of nuclear magnetic resonance(NMR)showed that with the molar ratio of NHS to carboxyl group on γ-PGA side chains(NHS/COOH)increased from 1/20 to 5/1,the esterification degree ofγ-PGA-NHS ester increased from 2.8%to 89.0%.In addition,with the increase of temperature and pH,the hydrolysis of γ-PGA-NHS ester was accelerated.While with the increase of concentration,the hydrolysis rate of γ-PGA-NHS ester decreased.Secondly,collagen solutions were modified by the reaction between γ-PGA-NHS esters and natural collagen.The infrared absorption peak of the modified collagen was similar to that of native collagen,and the intensity ratio of amide Ⅲ to 1450 cm-1(AⅢ/A1450)was higher than that of native collagen,suggesting that the triple-helix structure of the modified collagen was not destroyed.The absorption peak position of the modified collagen in the circular dichroism(CD)was basically the same as that of native collagen,and the absolute value of positive and negative peak intensity ratio Rpn of the modified collagen was higher than that of the latter,further confirming that the modified collagen still maintains an integrated triple helix structure.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)showed that the fraction of low molecular weight of the modified collagen was decreased,accompanied by the increase of the fraction of the high molecular weight.The results of trinitro-benzene-sulfonic acid(TNBS)showed that with increasing either the esterification degree or the dosage of γ-PGA-NHS ester,the crosslinking degree of the modified collagen would be increased.It could be found from field emission scanning electron microscopy(FESEM)images that the porosity and average pore size of the modified collagen sponge were larger than those of the native collagen,which suggested a superior permeability for the modified collagen sponge.The results of resistance to enzymolysis test revealed that the modified collagen possessed a stronger resistance to enzymatic hydrolysis than the native one.MTT assay and DAPI method were used to evaluate the cytotoxicity of modifier.The results reflected that both of native collagen and the modified collagen exhibited excellent cell activity,and the cell activity of the modified one was even better.Finally,the effects of γ-PGA-NHS ester modification on the thermal stability,water solubility,rheological properties,as well as the aggregation structure of collagen in solutions were studied.The results of differential scanning calorimetry(DSC)showed that the thermal denaturation temperatures(45.0-46.9℃)of all the modified collagens were higher than that of native collagen(38.8℃).The results of isoelectric point(PI)test displayed that the PI values(3.61-5.31)of all the modified collagens were lower than that of native collagen(6.91),which indicated that the PI of collagen could be effectively reduced via modification using γ-PGA-NHS esters,resulting in the improvement on its water solubility.The rheological properties of modified collagen were quite different from those measured from native collagen.The values of complex viscosity of all the modified collagens were higher than those of native collagen;the values of loss factor of the modified one were lower than the native one,but still greater than 1,indicating that the modified collagen retained a good fluidity.Hence,different from compared with glutaraldehyde,γ-PGA could inhibit the gelation of collagen.The modified collagens also had a stronger resistance to deformation and a higher creep recovery rate.Additionally,the fluorescence spectra showed that the aggregation tendency of collagen molecules in solution was inhibited by γ-PGA in the low concentration collagen solutions(≤ 0.3 mg/mL),while in the collagen solutions with higher concentration(≥ 0.5 mg/mL),γ-PGA would promote the aggregation of collagen molecules.In the low concentration collagen solution,the electrostatic repulsion of the side chains of γ-PGA in the modified collagen molecules played a dominating role.Whereas,in the high concentration collagen solution,the hydrophobic interaction,hydrogen bonding,and entanglement effect might become primary,facilitating the aggregation of collagen molecules in solution. |
