The Anti-Hypertrophic Scars Agent Liposome Of Paeonol Modified With Anti-VEGF Antibody Transfersomes:Preparation And Pharmacodynamics Evaluation

Research background The clinical treatments of Hypertrophic scars mainly include surgical resection and drug treatment.Surgical treatment usually cost is high and higher recurrence rate.Currently clinical inhibition drugs for treatment of HS have great side effects and high recurrence rate.Therefore,it is the top priority to develop effective drugs for treatment of HS.Bevacizumab?BEV?can specifically block the biological effects of VEGF,and inhibit hypertrophic scar hypervascular hyperplasia,as well as delay fibroblast growth and metastasis,so as to achieve the purpose of inhibiting HS.Paeonol can inhibit TGF-?₁,TNF-?and intracellular oxygen free radicals,which can effectively inhibit the excessive proliferation of microvessels.We intend to connect BEV with phospholipid via polyethylene glycol to prepare antibody-modified liposomes to entrap paeonol,which can enhance the retention effect of the drug in the dermis layer,to achieve the purpose of preventing and treating HS.Objective To prepare liposomes of paeonol modified with anti-VEGF antibody transfersomes;to optimize prescription and preparation process,investigate its in vitro transdermal properties and dermal hysteresis;and to study the skin irritation and the effect of inhibiting scar by liposomes of paeonol modified with anti-VEGF antibody transfersomes.Method?1?The conjugation of BEV-PEG₃₀₀₀-DOPE antibody.Pnp-PEG₃₀₀₀-DOPE conjugation and structure identification using MS;coupling rate determined by Sephadex G-150 gel column method;molecular weight determined by GPC andstructure identification using IR.?2?The preparation of liposomes of paeonol modified with anti-VEGF antibody,and study its quality.The liposomes were prepared by membrane dispersion-ultrasonic method.The entrapment efficiency 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.The morphology of liposomes was observed by transmission electron microscopy and the particle size and Zeta potential were measured by particle size analyzer.?3?The preparation of liposomes of paeonol modified with anti-VEGF antibody transfersomes.To prepare liposomes of paeonol modified with anti-VEGF antibody;to examine the quality of liposomes of paeonol modified with anti-VEGF antibody,including traits,viscosity,pH value,centrifuge test and determination of content.To determine the dermal retention and transdermal rate of liposomes of paeonol modified with anti-VEGF antibody transfersomes,paeonol liposome gel and paeonol gel;to draw the in vitro penetration curves.To investigate the skin irritation with contrast method;to observe the erythema and edema of skin;to determine the cell of skin by HE statining.?4?Hypertrophic scar models were established in rabbits,which were randomly divided into 5 groups:the model group,asiaticoside group,paeonol group,liposomes of paeonol group,and liposomes of paeonol modified with anti-VEGF antibody transfersomes group.And the control group was not given any treatment.And the rate of hypertrophic scar and SEI were measured.At the meantime,the morphology of hypertrophic scarwere observed by HE staining and Masson staining.The expression of VEGF,TGF-?₁ and TNF-?were measured by ELISA.Result?1?Pnp-PEG₃₀₀₀-DOPE was successfully synthesized with a molecular weight of4090;BEV-PEG₃₀₀₀-DOPE was successfully synthesized with a coupling ratio of63.72%and a molecular weight of 226570.IR successfully measured the desired functional goups.?2?The optimal preparative conditions were as follows:ratio of lecithin cholesterol:Paeonol:Pnp-PEG₃₀₀₀-DOPE:BVE-PEG₃₀₀₀-DOPE?14:5:4:0.28:0.05?;phosp-holipid concentration of 7.36 mg·mL^-1;film forming temperature of 41?;stripping using PH 7.5 sodium dihydrogen phosphate buffer;ultrasound 3 min?ultrasound time2 s,interval 3 s,power 300 W?.Prepare 3 batches using optimal preparation,which showed paeonol encapsulation efficiency of?73.61±2.36?%;particle size of?235.7±4.67?nm;and Zeta potential of-?5.13±0.25?mV.Preparation of liposomes of paeonol modified with anti-VEGF antibody transfersomes.?3?Liposomes of paeonol modified with anti-VEGF antibody transfersomes with viscosity between 6.6⁷.4 Pa·S,pH value of 7.0⁷.5,the content of?2.193±0.3107?mg·g^-1.Liposomes of paeonol modified with anti-VEGF antibody transfersomes with slow body transdermal rate and significant effect of dermal retention.Liposomes of paeonol modified with anti-VEGF antibody transfersomes is non-irritating.?4?The rate of hypertrophic scar in liposomes of paeonol modified with anti-VEGF antibody transfersomes group was 47.92%,which was lower than other groups.Compared with model group,SEI?1.34±0.51?and the expression of VEGF(30.90±3.57 ng·L^-1),TGF-?₁(733.2±43.19 ng·L^-1)and TNF-?(66.76±2.98 ng·L^-1)significantly reduced in liposomes of paeonol modified with anti-VEGF antibody transfersomes group?P<0.01?,where collagen fibers and muscles fibers was decreased and arranged sparsely,but cartilage cells were orderly pattern,which cannot observed obvious calcification and inflammatory cells.Conclusion:Ethanol injection preparation of BEV-PEG₃₀₀₀-DOPE if feasible;Box-Behnken effect surface optimization of liposomes of paeonol modified with anti-VEGF antibody preparation process is feasible;liposomes of paeonol modified with anti-VEGF antibody transfersomes has a sustained release effect and obvious retention effect in dermis layer.Liposomes of paeonol modified with anti-VEGF antibody transfersomes is non-irritating to rabbit skin and rabbit ear hyperplastic scar have a good therapeutic effect.