Preliminary Investigation On Preparation Of Drug-loaded Oral Film Based On Natural Spider Silk Protein And Its Drug Release Properties

Objectives: The efficacy of Kangfuxin Liquid,an insect-originated drug developed from Periplaneta americana Linn.,has been clinically verified in the treatment of oral mucositis caused by radiotherapy and chemotherapy in cancer patients,inosine is a drug that increases leukocyte,as one of the active ingredients in Kangfuxin Liquid,also plays a certain role in the treatment of oral mucositis.However,the current treatment method of Kangfuxin liquid often adopts administration with gargle,which has a short residence time at the lesion site and low efficacy,and the use of sustained-release oral film can greatly improve the drug utilization rate.Spider silk in nature is a natural biomaterial with good biocompatibility,excellent mechanical properties,and biodegradability.Therefore,this thesis is proposed for the first time to use Chilobrachy jingzhao of natural spider silk as the carrier of film agent,design and prepare spider silk protein matrix membrane by the solvent-casting method,this arachnid protein matrix membrane was utilized to investigate the possibility of loading the lyophilized powder of P.americana Linn（semi-finished product of Kangfuxin liquid）as well as inosine material drug on the film,and to examine the sustained-release characteristics of the loaded drugs,in order to achieve the effect of sustained-release drug on oral mucositis.Methods: In this thesis,preliminary investigation of film forming was firstly carried out.Chilobrachy jingzhao spider silk was respectively dissolved by hexafluoroisopropanol,trifluoroacetic acid,formic acid,to compare the film formation and releasetion of the spider silk after dissolution.The stability of the two drugs in the above three solvents was detected by TLC and HPLC;Then,the solvent casting method was used to prepare the drug-loaded spider silk protein film,and the preliminary screening process was carried out.Evaluation of film agent appearance,thickness uniformity,surface p H value,mechanical properties and adhesion properties,structure verification（infrared spectrum scanning,X-ray diffraction,scanning electron microscopy,water contact Angle）.The degradation of film agent in artificial saliva and stimulated gastric fluid,ans the in vitro simulated release effect of drug-loaded film in artificial saliva was investigated.Moreover,in vitro cytotoxicity test,cell and membrane co-culture examination,dermal irritation and skin sensitization,mouse acute toxicity test,as well as the oral compliance test were used to determine the cytocompatibility and biosafety.Results:（1）The ability of dissolving spider silk was trifluoroacetic acid > hexafluoroisopropanol >> formic acid,and the HFIP film was easy to return to soft unpeel after film formation.The stability of loaded drugs on the film in 2 hours was verified by HPLC and TLC;（2）Through preliminary screening,the spider silk protein solution was heated and dissolved at 58℃ for 8h,and then concentrated to 80% of the original volume as spider silk protein membrane forming solution,with drug loading of more than 15%;（3）The appearance meet the requirements of the Chinese Pharmacopoeia（2020 edition）;the average thickness is in the range of 0.150～0.170 mm;the water contact angle indicated a certain degree of hydrophilicity.The surface p H value was between 6.70 and 6.90,showing no irritation to the oral mucosa.Adhesive force,adhesive time,flexability,breaking strength,elongation at break show that the film agent has enough adhension,strength and flexibility;（4）A series of structural characterizations showed that the drug existed in the membrane pore of spider silk protein film.Furthermore,the weight loss rate of the film agent was only 5% within 7 days in artificial saliva,but more than 65% in artificial gastric fluid after 72 hours co-existence;（5）The cumulative release rate of both two drug films was more than 80% in vitro,the release time was more than 4h.It was concluded both of them belonged to the diffusion drug release mechanism;（6）MTT method examination show that the film agents possess no cytotoxicity to L-929 cells whereas the cells could coexist with the film for at least 5 days,meeting the requirements of three routine tests（cytotoxicity,stimulation and sensitization）in GB/T16886.1-2017 biological evaluation test.The film agent had no acute toxicity to mice,and exhibited good oral compliance.Conclusions: In this paper,the oral film agent containing lyophilized powder of P.americana Linn and inosine was successfully prepared using natural spider silk/hexafluoroisopropanol solution as film forming material by solvent casting method.Its appearance,mechanical and adhensive properties meet the need of oral film agent,and has satisfactory release in vitro.It has good biocompatibility and biosafty.It is expected to be developed into a treatment method with good effect for oral mucositis in patients with radiotherapy and chemotherapy in the future and applied in the study of more advanced oral film agent.