Preparation And Preliminary Evaluation Of Insulin-loaded Bletilla Striata Polysaccharide Dissolving Microneedles With Flexible Patches

In recent years,the new transdermal drug delivery form-microneedles(MN)has gradually become a research hotspot.Painless,minimally invasive self-management and non-invasive applications are advantages of MN.Among them,dissolving microneedles(DMNs)are prepared from a rapidly dissolving matrix,such as polymers or sugars,that can be inserted and dissolved in the skin to release encapsulating drugs.Natural polymer materials have attracted much attention in recent years because of their good biocompatibility,biodegradability,film-forming property and low cost.In addition,compared with some synthetic polymer materials,most natural materials can play a special pharmacokinetic role,and the preparation process does not need high temperature treatment,thus avoiding the damage to the loaded drugs.Therefore,the research on the preparation of soluble microneedles with natural polymer materials has been increasing year by year.Bletilla striata polysaccharide(BSP)s as a natural polymer dextran material,BSP is considered as an ideal biomedical material due to its good stability,biocompatibility and biodegradability.It is widely used in the manufacture of adhesives,wound dressings and gels.In addition,BSP has also been shown to have extremely high medicinal properties,promoting wound healing and hemostasis,and has anti-inflammatory,antioxidant and antimicrobial activities.These pharmacological activities of BSP may be helpful in repairing micro-damage caused by DMNs insertion,such as preventing bacterial infection and reducing inflammation.In our previous work,we have successfully prepared BSP dissolving microneedles(BMNs),which are easy to manufacture and can penetrate the skin to achieve transdermal drug delivery.However,BMNs lacks the desired flexibility to adhere tightly to the skin.In fact,due to the elasticity of the skin,the tips of microneedles need enough mechanical strength to penetrate the stratum corneum,and the backing needs to be flexible enough to adhere tightly to the skin.As a result,a single matrix material often fails to meet both this particular need.Based on these findings,our research group prepared a compound soluble microneedle patch with BSP as the needle material and polyvinyl alcohol(PVA)with excellent flexibility as the back material on the basis of previous research,in order to provide a new technology for the development of soluble microneedle transdermal insulin delivery system.Abstract:Objective This study attempted to prepare INS loaded soluble microneedle flexible patches(INS-DMNs)with BSP and PVA as the needle material and the backing material,respectively,to provide a new technology for the transdermal delivery of INS.The physicochemical properties and structure of BSP were preliminarily analyzed.The preparation process of microneedle was screened and optimized to investigate the properties of related preparations of microneedle.The material safety of BSP was tested.The pharmacodynamics of INS DMNs in vivo was investigated.Methods1.The purified BSP was obtained by the extraction and purification process published in our research group,and the BSP was characterized by UV spectroscopic scanning,phenol-sulfuric acid method,Coomassie bright blue method,high performance liquid chromatography,dextran gel chromatography and infrared chromatography.2.Rabbit erythrocyte hemolysis and CCK-8 assay were used to investigate the blood compatibility and cytotoxicity of BSP as the matrix material for microneedles.3.Polydimethylsiloxane(PDMS)molds were prepared by wet etching method.4.INS-DMNs was prepared by micro-molding method.The concentration of BSP and PVA were investigated with the hardness of the needle body,the flexibility of the backing and the formability as the indexes.5.The morphological characteristics of INS-DMNs were observed by scanning electron microscope.Draize method was used to evaluate the skin irritation of rats with INS-DMNs.The mechanical strength of INS-DMNs was evaluated by texture analyzer.The stability of Ins loaded in the Bletilla striata polysaccharide dissolving microneedles with flexible patches was determined by circular dichromatography.Confocal microscopy was used to evaluate the transdermal release and diffusion of INS after the application of INS-DMNs to the skin of rats.The in vitro transdermal properties of INS-DMNS were investigated by Franz diffusion cell.6.The drug loading of INS-DMNs was determined by insulin kit.The administration time was determined according to the dissolution of microneedles over time.To evaluate in vivo pharmacodynamics the hypoglycemic effect of INS-DMNS on diabetic rats.Results1.There was no obvious absorption peak at 260 nm and 280 nm after UV scanning,and the determination of protein content and total polysaccharide content showed that the total polysaccharide content in BSP was 67.36%,and no protein was tested.The relative molecular weights of BSP were 2.36×10~5 g/mol and 6.63×10~4 g/mol,respectively.The monosaccharide composition and molar ratio of BSP were:mannose:glucose=3.13:1.2.The hemolysis test results showed that the hemolysis rate of BSP matrix solution with the concentration range of 0.625-10 mg/m L was all lower than 2%required by ISO 10993-4,indicating that BSP had no hemolysis effect on rabbit erythrocytes and was a blood compatible material.The results of cytotoxicity showed that the concentration of microneedle matrix solution ranged from 0.3125-10 mg/m L,which had no inhibition on the growth and reproduction of L929 cells and had no toxic effect on cells.3.The PDMS mold prepared by wet etching method is milky white,strong toughness,uniform pinhole and no incomplete,and can be reused for many times.4.Based on the hardness of the needle body,the flexibility and formability of the backlining,the preparation process of the needle body(BSP)and the backlining(PVA)were optimized.The concentration of the needle body(BSP)and the backlining(PVA)were 15%and 15%respectively.5.INS-DMNs showed smooth pyramidal shape,uniform needle body with good mechanical strength,flexible and smooth back,little skin irritation and quick recovery.Ins coated in white and polysaccharide soluble micro-needle flexible patches had good stability at room temperature,and the application of INS-DMNs could make the INS reach 220μm inside the skin.The results of skin penetration in vitro showed that the cumulative permeability of INS in microneedles reached(89.63±6.00)%after 12 h of applying INS-DMNs to rat skin,and the cumulative permeability of INS in the INS solution group was(2.77±1.34)%,which proved that INS-DMNS could effectively improve the transdermal penetration of INS.6.The kit test results showed that INS-DMNs contained(0.25±0.02)IU per tablet and could be dissolved within 2 h.In vivo pharmacodynamics results showed that INS-DMNs showed significant hypoglycemic effect on diabetic rats.Conclusion1.The physicochemical properties and structural characteristics of BSP were characterized and analyzed,and the biocompatibility of BSP was good.2.INS-DMNs prepared according to the optimized process of the prescription showed that the needle body prepared by BSP had good mechanical properties and could Pierce the skin to release drugs.Meanwhile,the back lining prepared by PVA was flexible and flexible,providing good skin adhesion.INS-DMNs showed less skin irritation.INS loaded in Bletilla striata polysaccharide dissolving microneedles with flexible patches showed good stability,and the application of INS-DMNs could reach subcutaneous 220μm.3.INS-DMNS can effectively reduce the blood glucose level of diabetic rats.Due to the sustained release effect of Bletilla striata polysaccharide dissolving microneedles with flexible patches,the administration of INS-DMNs is more continuous than that of subcutaneous injection of INS.This study shows that INS-DMNS is expected to provide a new idea and reference for the transdermal delivery of INS.