Preparation,Characterization And Preliminary Pharmacodynamic Evaluation Of Sinomenine Hydrochloride-loaded Liposomes-in-hyaluronic Acid Hydrogel In Acute Eczema

Objective:The main objective of this project is to design a formulation containing pentoxifylline hydrochloride for the treatment of eczema that can be used for dermal administration.Firstly,through pre-prescription research,the basic physical and chemical characteristics of sinomenine hydrochloride were discussed,and the preparation methods of sinomenine hydrochloride liposomes were preliminarily determined by screening the preparation methods of liposomes.Then the formulation process of sinomenine hydrochloride-loaded liposomes was optimized,and sinomenine hydrochloride-loaded liposomes-in-hydrogels was prepared by using this liposome as an intermediate.The characterization,release rate and antioxidant activity of sinomenine hydrochloride-loaded liposomes-in-hydrogels were studied,and the pharmacodynamics of eczema were further studied,which provided a theoretical basis and technology platform for the development of sinomenine hydrochloride-loaded liposomes-in-hydrogels.Methods:（1）To find out the basic physical and chemical characteristics of sinomenine hydrochloride（SH）by consulting the literature,establish the ultraviolet content determination method of SH,and preliminarily screen the preparation method of sinomenine hydrochloride-loaded liposomes（SH-L）,which lays the groundwork for the subsequent preparation of sinomenine-loaded liposomes-in-hydrogels（SH-L-H）.（2）To determine the final prescription process of SH-L and evaluate the quality of SH-L.（3）To determine the final prescription process for SH-L-H and to establish a methodology for the determination of the ultraviolet content of SH-L-H.Suitable hyaluronic acid matrix concentrations were obtained by viscosity screening,and a methodology for ultraviolet content determination was established for them.The quality evaluation,in vitro release and ex vivo transdermal release of the SH-L-H were performed to investigate the performance and quality of this liposome hydrogel.（4）The in vitro antioxidant ability of SH-L-H was researched by the scavenging ability of 1,1-diphenyl-2-picrylhydrazyl（DPPH）,H₂O₂and the scavenging ability of malondialdehyde（MDA）in mouse organs,which laid the foundation for further in vivo drug efficacy experiments in mice.（5）To judge the skin irritation of SH-L-H by single and multiple times of mouse skin administration,and to do a good job of safety investigation for further in vivo pharmacodynamic experiments in mice.（6）A mouse acute eczema model was established,and the inhibitory effect of SH-L-H on mouse acute eczema was investigated by macroscopic indexes including visual observation and scoring of mouse skin condition,biochemical indexes including organ index,MDA content in skin and microscopic indexes of H&E staining.Results:（1）According to the literature review and preliminary experimental operation,SH has a stable property,and it was suitable to be determined by ultraviolet content determination method.The precision,stability and repeatability all met the standard.（2）Thin film dispersion method was chosen as the preparation method of SH-L.The best preparation formula of SH-L was:（1）Weighed 100 mg of SH powder into 50m L of PBS solution（p H=7.4）and dissolved well to obtain 2 mg/m L of SH solution,and set aside.（2）Weighed 0.3 g of phospholipids and 0.1 g of cholesterols in an eggplant bottle,added a small amount of absolute ethanol and sonicated at 70°C for 10 min to dissolve the phospholipids and cholesterols.The lipid solution was evacuated on a rotary evaporator（vacuum:70 k Pa,temperature:25°C,speed:100 r/min）until a thin film of lipid was formed on the inner wall of the flask.（3）Took 10 m L of the prepared solution（1）and put it in the lipid-like film formed in the above step（2）,shook it properly,added a stirrer,then put it on a rotary evaporator,and hydrated it at 70℃and100 r/min for 30 min to obtain a milky white suspension.（4）Ultrasonic treatment of the suspension obtained in the above step（3）for 10 min,and filtration with 0.22μm microporous membrane for 3 times to obtain SH-L.According to the observation of the cryo-electron microscope,the prepared liposomes was orbiculate with evenly scatter,the particle size was 135.7±11.4 nm,and the Zeta potential was-38.5±3.2 m V.The stability experiment showed that the prepared liposome has good stability when it is placed at 4℃.（3）SH-L-H prepared with 1%hyaluronic acid was used in the experiment.The quality evaluation of the prepared liposome hydrogel showed that the preparation was milky white with a certain viscosity,p H=7.32±0.01 and had the good stability and was also non-irritating to mouse skin.The release evaluation of dialysis membrane in vitro showed that the liposome hydrogel had a good release performance in vitro and a certain sustained release effect,and the release law was in accordance with weibull CDF model.Ex vivo release evaluation showed that the cumulative release rate of SH-L-H reached 44%at 84 h,and the release law was in accordance with weibull CDF model,which provided a basis for transdermal drug administration.（4）Antioxidant experiments showed that both SH and its preparations have good antioxidant capacity.The scavenging capacity of SH for DPPH radicals and H₂O₂both increased with increasing concentration,with IC₅₀of 0.084 mg/m L and 0.3771 mg/m L,respectively;SH-L-H also has the ability to scavenge DPPH radicals and H₂O₂,with0.125 mg/m L SH-L-H scavenging approximately 60%of DPPH radicals and 0.02mg/m L SH-L-H scavenging approximately 10%of H₂O₂.SH and SH-L-H can inhibit MDA in liver and kidney of mice,with the inhibition rates of 70%and 20%in liver and40%and 10%in kidney respectively.（5）Single and repeated mouse skin administration showed that SH-L-H was a safe preparation without skin irritation,which provided a basis for animal experiments.（6）A mouse model of acute eczema induced by 1-chloro-2,4-dinitrobenzene（DNCB）was established.After applying SH-L-H to it,it was found that SH-L-H has a good therapeutic effect on acute eczema.Compared with the blank group,the weight of mice in the drug group remained almost constant during the experiment,the skin condition recovered well,the organ index and MDA content in the skin decreased compared with the model group.H&E staining showed that the skin keratinization degree and inflammatory cell infiltration were reduced compared with the model group,which indicated that SH-L-H had a certain therapeutic effect on acute eczema.Conclusion:In this study,the fact that SH can be prepared into liposome hydrogel was elucidates:SH-L-H prepared by combining liposomes prepared by film dispersion method with hyaluronic acid hydrogel had good characterization results,and the diffusion test results in vitro and ex vivo proved that SH-L-H has a certain sustained release effect and is suitable for skin administration;antioxidant experiments had shown that SH-L-H has a good antioxidant effect and has great potential for the treatment of diseases associated with oxidative stress;preliminary pharmacodynamic experiments show that the preparation of SH-L-H has a good therapeutic effect on the acute eczema of mice caused by DNCB,and provides a new idea for the further development of this preparation.