Optimization Of The Preparation Process Of Gelatin-Stearic Acid Composite Gel With Response Surface Methodology And Its Properties

Gelatin is an amphoteric polymer substance.Its structure contains a large number of hydroxyl,carboxyl,and amino groups.It has strong hydrophilicity,and it is easy to produce hydrogen bonds and Van der Waals forces between molecules.Because of its unique gelation,gelatin has become an important material for research and preparation of gels.Gelatin gel is widely used in biomedicine and other fields due to its excellent biocompatibility,cell adhesion,biodegradability,and bioactive factor loading capacity.However,due to the poor rigidity of the gelatin molecular chain,the single-component gel has poor mechanical properties and water retention,is not easy to mold,has a fast degradation rate,and cannot meet the needs of medical tissues.In order to overcome the above drawbacks,the hydrophobic substance stearic acid was used to modify it,and citric acid,sodium citrate and triethyl citrate were selected as crosslinking agents to obtain gelatin-citric acid-stearic acid complex Gelatin-Citric Acid-Stearic Acid（GA-CA-SA）,Gelatin-Sodium Citrate-Stearic Acid（GA-SC-SA）and Gelatin-Triethyl Citrate-Stearic Acid（GA-TEC-SA）.The effects of stearic acid quality,crosslinker quality and temperature on the compressive strength of the prepared composite gels were compared,and the compressive strength of single-component gelatin gels was compared.Response surface methodology was used to prepare the composite gel.Optimized to determine the best process conditions.Scanning electron microscope,infrared spectrometer and thermogravimetric analyzer were used to analyze its micromorphology,chemical structure and thermal stability,and the deconvolution curve of infrared spectrum was used to analyze the intermolecular hydrogen bonding.According to the detection method of composite gel extracts,composite gel powders of different qualities were dissolved in serum-free medium by ultrasonic vibration,and placed in 6-well plates under sterile conditions to prepare composite gel culture solutions of different concentrations.As a blank control group,only serum-free medium was added.After being placed in a 37℃incubator,extracts of composite gel samples and blank extracts of different concentrations were extracted after 24 hours.The cytotoxicity was evaluated by measuring the absorbance of the samples and comparing the relative cell proliferation to explore the cytotoxicity of each composite gel.In order to investigate the drug release behavior of the prepared composite gel,curcumin（Cur）was used as a model drug to prepare different composite gels containing curcumin,and the composite gel phases and single-component gelatin gels were studied.Effect of drug release.This study provides a new approach for the preparation of high mechanical strength,degradable and non-toxic composite gel materials,and also provides the necessary theoretical support for the application of gelatin-based composite gels in the field of biomedical drug loading.The main experimental results are as follows:（1）The optimal preparation conditions of GA-CA-SA composite gel optimized by response surface method are:Stearic acid mass is 0.1261 g,Citric acid is 0.1545 g,and Temperature is 69℃.The optimal preparation conditions of GA-SC-SA composite gel are:Stearic acid mass 0.1382 g,Sodium citrate0.2015 g,Temperature 69℃.The optimal preparation conditions of GA-TEC-SA composite gel are:Stearic acid mass is 0.1261 g,Triethyl citrate is0.0909 g,and Temperature is 70℃.Under the best conditions,the compressive strength of the prepared composite gel was increased by 117%,220.59%,and93.25%compared with the gelatin gel.The increase rate of compressive strength is GA-SC-SA>GA-CA-SA>GA-TEC-SA,which may be due to the more positively charged sodium ions in sodium citrate and negatively charged with stearic acid The electrostatic effect formed by the carboxyl group and the hydrogen bonds and van der Waals forces between other molecules are stronger than the hydrogen bonds and van der Waals forces after crosslinking by the other two cross-linking agents.The effect of improving the compressive strength after crosslinking with triethyl citrate is not as good as citric acid and sodium citrate,probably because triethyl citrate is a lipid substance,and the active groups in the gel solution are relative to citric acid and lemon It is caused by less sodium.（2）Infrared spectroscopy studies show that in the composite gel,the protonated amino group on the gelatin chain interacts with the anion provided by the citric acid and sodium citrate crosslinking agent,and there is hydrogen bonding between the active groups of the molecule The active group of the cross-linking agent interacts with the amino group and amide group of the gelatin molecule.The strengthening and contracting vibration of the C-C bond indicates that stearic acid was successfully introduced into the composite gel.The curve fitting of the unwinding integral peak shows that the secondary structure of gelatin changes significantly after compounding.In the GA-CA-SA composite gel,the random curl structure and theβangle change are relatively large,which is attributed to the hydrogen bonding effect and electrostatic effect formed between the citric acid active group and gelatin than the other two composite gels.The force is strong.Various interactions between the composite gel molecules,especially hydrogen bonding,lead to changes in the secondary structure of gelatin.（3）Fluorescence analysis showed that the GA-CA-SA composite gel had a binding constant K_Λis 3.606×10³L mo L^-1,the number of binding sites n is1.038,and citric acid and gelatin produced a molar ratio of approximately 1:1.GA-SC-SA composite gel,the binding constant K_Λis 4.819×10⁷L mo L^-1,the number of binding sites n is 2.119,the interaction of sodium citrate and gelatin produces a conjugate with a molar ratio approximately 1:2.In the process of cross-linking gelatin and stearic acid,sodium citrate has a larger binding constant and doubles the binding sites than citric acid,and the cross-linking degree is better.It may be due to the combination of citric acid,sodium citrate and gelatin molecules and stearic acid molecules through hydrogen bonding,van der Waals force and electrostatic interaction,and sodium citrate is also rich in positively charged sodium ions and anions to form an electrostatic effect.（4）Thermogravimetric analysis shows that the decomposition temperature of the prepared GA-CA-SA composite gel,GA-SC-SA composite gel,and GA-TEC-SA composite gel decreases,the weight loss rate of the thermogravimetric curve becomes smaller,and heat resistance Performance has also been enhanced.It may be caused by the crosslinking agent crosslinking with gelatin and stearic acid through interactions such as hydrogen bonding and electrostatic interaction.As the temperature rises,the increase of molecular kinetic energy overcomes the limitation of the weak force of hydrogen bonding,and the composite gel oxidizes and decomposes at a lower temperature.The composite gel after crosslinking at high temperature has a faster weight loss rate.SEM observation showed that the inside of the composite gel showed a loose void structure.（5）Cytotoxicity experiments showed that GA-CA-SA composite gel,GA-SC-SA composite gel,and GA-TEC-SA composite gel were qualified for cytotoxicity and had good biocompatibility.Drug sustained-release studies have shown that GA-CA-SA composite gel,GA-SC-SA composite gel,GA-TEC-SA composite gel has a slow release effect and a high drug release rate,the highest drug release rate respectively up to 98.6%,97.2%,97.3%,can be applied to the field of medicine to improve the practical value of composite gel.