Preparation Of Tanshinone?A Self-dissolving Microneedles And Its Inhibition Of HSF Proliferation

Research background:Hypertrophic scars?HS?is a type of tissue fibrosis disease that is specific to human skin.It has pathological features that are difficult to cure and high recurrence rate.It is a long-term research hot spot in dermatology.At present,clinical methods of surgery and drug treatment are often used in the clinic,but the high cost of treatment and the long treatment cycle can easily cause patients with both economic and physical and mental burdens.Modern understanding of HS mainly focuses on inflammatory response,cytokines,etc.Among them,fibroblasts are an indispensable type of functional cells in the process of wound repair.Their activation,excessive division,and the synthesis of a large amount of extracellular matrix can directly cause the occurrence of hypertrophic scars.Tanshinone IIA?TSA?has pharmacological effects such as anti-inflammatory,anti-infection,and immune regulation.Studies show that TSA can dose-dependently reduce the proliferation index of abnormally activated fibroblasts in HS,block its entry from G1 to S phase,and Cells stagnate in G1 phase,which further induces apoptosis,and can also enhance collagenase bioactivity in HS,and accelerate the reabsorption and degradation of collagen fibers in scar sites.This project intends to prepare TSA self-dissolving microneedles to meet the requirements of the drug to efficiently penetrate the skin barrier and more effectively enter the dermis to play a therapeutic role,breaking through the difficulty of significantly thickening epidermal connective tissue and stratum corneum at the HS site.Further exert the effect of suppressing scars,and provide a new idea for the clinical use of HS.Objective:Tanshinone IIA was prepared as TSA-PVP SD and characterized;TSA-PVP SD was further prepared as TSA-MN,the optimal preparation process parameters were optimized,and the quality of TSA-MN was investigated;the in vitro transdermal performance and skin transport of TSA-MN Characteristics and skin irritation;To investigate the effects of TSA-MN on the cell proliferation,cell migration ability,TGF-?₁ and Smad 7 expression levels of HSF-induced cell proliferation induced by TGF-?₁in vitro,and to comprehensively evaluate the design of TSA-MN The rationality and safety of the method provide research methods and theoretical basis for its prevention and control of HS.Method:?1?Preparation and characterization of TSA-PVP SD.The solvent method was used to prepare TSA-PVP SD.A method for determination of TSA by high-performance liquid chromatography?HPLC?was established.The phase identification of TSA-PVP SD was performed using microscopic observation,solubility experiments,and in vitro dissolution tests.X-ray powder diffraction was used.?XRD?,differential scanning calorimetry?DSC?,and infrared spectroscopy?FTIR?for structural identification.?2?Study on the preparation process of TSA-MN.The microneedle matrix,injection molding and curing methods are optimized.The TSA-MN is prepared by negative casting method,and its 24h dermal retention is measured by in vitro transdermal absorption method.The dermal retention is used as an evaluation index,and the box-Behnken effect surface method is used to optimize the micro Needle prescription process.?3?Quality evaluation of TSA-MN.The physical and chemical properties of tsa-mn,such as morphology,pH value,drug loading and hygroscopicity,were investigated;Dynamic dialysis was used to investigate the drug release of^-TSA-MN in vitro;use the modified Franz diffusion cell method to investigate the transdermal performance of microneedles in vitro,Draw the in vitro permeability curve and fit the pharmacokinetic equation;use Coumarin-6?coumarin-6?as a fluorescent probe to investigate its skin transport characteristics;investigate the skin irritation of TSA-MN,observe the skin state comprehensive score,and remove The skin of the administration site was subjected to tissue sectioning and HE staining to evaluate the skin medication safety of the transdermal preparation.?4?The effect of TSA-MN on the biological characteristics of HSF.Establish HSF in vitro culture method;use CCK8 method to screen the optimal time and concentration of TSA for HSF;use CCK8 method to investigate the effect of micro-targeting on the proliferation of HSF cells induced by TGF-?₁;use scratch method to investigate the micro-targeting of HSF cell migration ability Effects;TSA-MN was used to detect the expression of TGF-?₁ and Smad 7 protein in HSF by Western blot.Result:?1?TSA-PVP SD can significantly enhance the solubility and dissolution of TSA in vitro,and the cumulative dissolution rate in vitro can reach more than 90%at 80 minutes.Interaction with PVP.TSA-PVP SD can be used in the next microneedle preparation experiment.?2?The optimal preparation process of TSA-MN is as follows:take the prescribed amount of TSA-PVP SD,chondroitin sulfate?CS??2:1,w/w?,add 0.5 times the amount of purified water?w/w?,and stir well.Allow to stand until swelling is complete,cast on a 0.5mm polytetrafluoroethylene micro-hole array mold,ultrasonically oscillate?power 270W,15min?to fully fill the solution in the micro-holes to obtain the needle-loaded drug layer,remove the mold and place in a vacuum dryer Dry for 12h.Take the matrix solution?PVP and CS are mixed 1:1and fully swell?and pour it over the drug-carrying layer.Evacuate to make the solution evenly spread without bubbles between the drug-carrying layer and the microneedle backing layer.Continue the dryer.After drying for 12 hours,take it out of the mold.TSA-MN patch.Three batches of TSA autolysed microneedle samples were prepared according to the optimal process.The dermal retention was?30.4813±0.7181??g·cm^-2.The results showed that the preparation process was stable and reliable.?3?Each needle of TSA-MN is in a regular conical shape,the needle is in good condition without broken needles,the pH value is between6.1-7.8,the drug content is?10.06±0.15?mg/tablet,and it is easy to work under high humidity Hygroscopicity,the moisture absorption rate can reach 23.2%±0.6%in 24 hours,it should be stored in a vacuum dryer;the cumulative release rate in vitro for 24 hours is 55.68%,no sudden release effect,with obvious sustained release effect,in line with Hixon-crowell Equation model;the cumulative amount of skin in vitro for24 h is?3.5867±0.172??g?cm^-2,which conforms to the zero-order equation and the Hixon-crowell equation model.Using coumarin-6,TSA-MN can be found at the initial stage of administration.The released drug reaches the dermis layer of the lesion and stays in the dermis layer to continue to exert its efficacy.The left and right parity comparison method has verified that TSA-MN is almost non-irritating to the skin.?4?After different concentrations of TSA-MN acted on HSF at different times,they all showed a certain proliferation inhibition effect,and within a certain range,the effect was time-and dose-dependent,and at the same time significantly inhibited cell migration ability;Western blot experiments It was shown that TSA-MN significantly reduced the TGF-?₁secretion of HSF and increased the expression of Smad7.Conclusion:PVP is a good carrier to enhance the dissolution of TSA.TSA-PVP SD is prepared as auto-soluble microneedles,which has a stable and good dermal retention effect after process optimization.The TSA-MN prepared in this subject is of good quality and has almost no skin irritation.Its in vitro release and transdermal properties are better than those of TSA APIs and ordinary dosage forms.Migration ability plays a significant role in down-regulating the secretion of TGF-?₁ in HSF and increasing the expression level of Smad7.The study of this project laid a foundation for the use of TSA-MN in the prevention and treatment of HS in the later period.