Preparation Of Cell Penetrating Peptide TAT-modified Liposome Loaded With Salvianolic Acid B And Its Effect On Proliferation Of HSF

Research background:Hypertrophic scars(HS)are a common skin fibrosis disorder,the effective treatment of which is still inaccessible.Current HS treatment focuses on surgery and drug therapy.However,surgical treatment leads to pain and high rate of recurrence,whilst hormonal drugs are expensive and cause adverse-effects.Therefore,the development of safe and effective drugs for the prevention and treatment of HS is a critical subject in this field.Human Skin Fibroblasts(HSF)are key effector cells of HS and appear at the site of tissue injury,during the ending of the inflammatory phase,and at the early phase of wound healing.Studies have shown that HSF activation and differentiation into myofibroblasts are key processes in the pathophysiology of HS.As such,HSF are important drug targets for HS treatment at the cytological level.Salvianolic acid B(SAB)is the most active ingredient in salvia miltiorrhiza and displays anti-fibrotic effects on the lung,kidney and myocardium.SAB can resist free-radical-mediated peroxidative damage,adjust humoral and cellular immunity,inhibit fibroblasts proliferation and collagen synthesis,and inhibit multiple signaling pathways.Therefore,SAB plays a comprehensive inhibitory role in the pathogenesis of fibrosis.Thecell penetratingpeptide TAT(TAT,Cys-AYGRKKRRQRRR)possesses high cell penetrating ability and can be used to enhance drug transport.Drugs can be up to 20 times larger than the molecular weight of TAT,and their transmembrane transport can be enhanced without loss of biological activity.As a new adjuvant for drug delivery,TAT has unparalleled advantages in the conventional transdermal transport of macromolecules and hydrophilic drugs.Based on the above assumptions,in order to establish a topical transdermal delivery system for the prevention and treatment of HS in proliferative phase,we developed the cell penetrating peptide TAT-modified liposome loaded with salvianolic acid B(SAB-TAT-LIP),aiming to enhance the retention of the drug for a high local effect in the dermis,prolong the drug release,in addition to the expected high stability on storage.Objective:To optimize the preparation techniques,lipid composition,and the determinant factors of SAB-TAT-LIP by means of Box-Behnken design to achieve the desirable physicochemical parameters and therapeutic effect;to investigate its in vitro transdermal properties,retention effect in dermis layer,skin irritation and allergies;to investigate its effect on the proliferation,migration,cell cycle,apoptosis and TGF-?1 expression of HSF,and to preliminarily evaluate its effect on the prevention and treatment of HS.Method:(1)The conjugation of DSPE-PEG1000-TAT micelles.The DSPE-PEG1000-TAT was synthesized by DSPE-PEG1000-MAL and TAT by means Michael addition reaction,was used as liposome materials to enhance transdermal delivery.The molecular weight was verified by MALDI-TOF-MS system,and the molecular structure determined by1H-NMR and FT-IR.(2)The preparation of SAB-TAT-LIP.The content of SAB was determined by HPLC.SAB-TAT-LIP was prepared by p H gradient reverse-phase evaporation method.Ultrafiltration method was employed to determine the encapsulation efficiency that was used as the evaluation index.Single factor test was used to select the significant factors.The Box-Behnken response surface method was adopted to optimize the preparation of the liposomes.(3)The characterization of SAB-TAT-LIP.The properties of SAB-TAT-LIP including entrapment efficiency,morphology,mean diameter,Zeta potential,polydispersity index,preliminary stability and freeze-drying stability were studied.In vitro SAB-release from SAB-TAT-LIP was performed using the equilibrium dialysis method.Franz diffusion cell method was employed to determine the dermal retention and transdermal rate of SAB-TAT-LIP,and the in vitro penetration curves were drawed.Skin irritation and allergies studies were conducted to evaluate the safety of SAB-TAT-LIP.(4)The effect of SAB-TAT-LIP on biological behavior of HSF.HSF were cultured in vitro.The stability of SAB-TAT-LIP in 50% Fetal bovine serum was studied.Cell proliferation was examined by MTT assay.Cell migration was assessed by Transwell chamber method and Scratch method.Cell cycle was detected by Flow cytometry.Cell apoptosis rate was analyzed by Annexin V-FITC / PI method.The level of TGF-?1was measured by ELISA method.Result:(1)MALDI-TOF-MS,FT-IR and 1H-NMR spectra of the reactant and final product were compared,the results of which showed that the final product was the desired compound.The DSPE-PEG1000-TAT phospholipid material was successfully synthesized.(2)The optimal preparative conditions were as follows: The p H gradient reverse-phase evaporation technique was used to fabricate the SAB-TAT-LIP.Firstly,Lipid,Chol,DSPE-PEG1000,DSPE-PEG2000 and DSPE-PEG1000-TAT were dissolved in chloroform: methanol(2:1 v/v)in a round-bottomed flask at a ratio of 100: 33.33: 2: 1: 1(mol/mol).SAB(SAB:lipid=0.094:1,w/w)was dissolved in 1% glycine-hydrochloric acid buffered saline solution(p H 3.28).The mixture was evaporated to form a thin film under reduced pressure at 50 ?,60 rpm.The film was purged with nitrogen gas and incubated overnight to eliminate traces of the solvents.Next,phoshate buffer solution was added to adjust the p H to 6.0.The thin film was hydrated for 30 min,and then sonicated for 6 min(300W for 1 s,interval 1 s).The resultant liposomes were further extruded 15 times using a LF-50 liposome mini-extruder assembled with a membrane filter of 400 nm pore size to adjust the diameter of the SAB-TAT-LIP.The encapsulation efficiency of SAB-TAT-LIP was(86.70±0.85)%.(3)The SAB-TAT-LIP displayed unimodal size-distribution with mean diameter of(219.90±5.09)nm,polydispersity index of 0.190±0.013,and Zeta potential of(-9.25±0.92)m V.The investigation of SAB-TAT-LIP in vitro 24 h cumulative release was 62.49% of the total drug with no burst effect,and showed significant sustained release effect that fit to Hixon-crowell equation model.The in vitro 32 h total accumulative transdermal rate of SAB-TAT-LIP was 17.21%,the steady penetration rate of SAB-TAT-LIP was(28.33±4.9)?g·cm-2·h-1,the transdermal absorption equation fit to Zero-order and Hixon-crowell equation model;and the dermal retention was(44.39±6.87)?g·cm-2,which were significantly higher than ordinary liposomes.SAB-TAT-LIP showed non-irritation and non-sensitization to mouse skin.(4)SAB-TAT-LIP showed high levels of stability in 50%Fetal bovine serum.The in vitro cell studies showed that blank liposome had no toxic effect on HSF.Different concentrations SAB-TAT-LIP of all inhibited proliferation in varying degrees after intervention HSF at different time.And in a linear concentration range,enhanced inhibitory effect on the proliferation of HSF was in dose and time dependent manner.SAB-TAT-LIP significantly inhibited the migration and invasion of HSF.At the same time,SAB-TAT-LIP significantly increased the apoptosis rate,increased the cell cycle G0/G1 phase and decreased the level of TGF-?1after intervention of 48 h,compared with the blank control group,P<0.01.Conclusion:Box-Behnken effect surface optimization of SAB-TAT-LIP preparation process is feasible.SAB-TAT-LIP showed high encapsulation efficiency,small size-distribution,and its in vitro release behavior and transdermal absorption properties are suitable for topical transdermal administration formulation.SAB-TAT-LIP in vitro can significantly inhibit HSF proliferation,migration and invasion,induce cell apoptosis,block the cell cycle G0/G1 phase,decreased the level of TGF-?1.Conclusively,our experimental data quantitatively demonstrate that SAB-TAT-LIP may offer a promising therapeutic strategy for transdermal delivery in prevention and treatment of HS.