Transdermal Delivery Of Vascular Endothelial Growth Factor With Surface Engineered Gold Nanoparticles

Background and ObjectTransderal drug delivery systems?TDDS?refers to a kind of drug delivery route that is administered directly to the surface of the skin by coating,allowing the drugs to be absorbed into the skin,resulting in systemic or topical therapeutic effects.TDDS is a noninvasive route of administration,and compared with the traditional route of drug delivery?oral and injected?,it has obvious advantages as follows : ?To avoid drugs being hydrolyzed in gastrointestinal duct;?To avoid first pass effect in hepatic;?Instantly withdraw if necessary.How to cure skin injury was caused by burn or radiation is still a serious concern in the clinical therapy.The large areas of skin is deep damaged that is hard to heal.The healthy skin has to be grafted onto the wounds.Due to the destruction of subcutaneous vascular,the wounds after grafting can not get enough blood supply,and it is difficult to heal.The angiogenesis is necessary for restoring blood supply to repair damaged skin.Vascular endothelial growth factor?VEGF?is the most effective factors on promoting angiogenesis and preventing injury progression,which triggers a cascade reaction to induce endothelial cell activation,assembly of new vascular structures,mural cell recruitment,and vessel stabilization.However,the traditional method fails to delivery VEGF to the epidermis or dermis efficiently and accurately.Thus,how to find a way that facilitates VEGF to cross the skin barrier directly to promote angiogenesis may become the key point to treat skin injuries.Recently,the nanoparticles?NPs?-mediated drug delivery into or through the epidermis and dermis has attracted much attention.However,there is few reports to study the efficacy of AuNPs-assisted drug penetrate the skin.Here,we applied gold nanoparticles with different surface charges to penetrate the skin and evaluate their percutaneous efficiency.We found AuNPs with a negative surface charge had higher percutaneous efficiency.VEGF-conjugated surface engineered gold nanoparticles still had surface negative charges,which enhanced VEGF penitriting skin and inducing angiogenesis.Our findings may provide new directions for the treatment of cutaneous injury.Methods1.Synthesis of PEGylated AuNPsThe gold nanoparticles were synthesized by a reduction of citrate buffer solution.Then,SH-PEG-COOH,SH-PEG-NH2 and PEG-SH prepared freshly were added to solution of Au NPs respectively.After chemical reaction,we got gold nanoparticles with different surface functional groups?citrate-AuNP,AuNP-PEG-OCH₃,AuNP-PEG-COOH,and AuNP-PEG-NH₂?.2.In Vivo Percutaneous Absorption of AuNPsBalbc/c mice?female,6⁷ weeks old?were chose to establish animal model and the mice's back skin were shaved.The gauzes which were soaked with different gold nanoparticles?citrate-AuNP,AuNP-PEG-OCH₃,AuNP-PEG-COOH,and AuNP-PEG-NH₂?suspensions(2×10¹⁰ /mL,1.44 ?M)covered the exposed area for 24 h.Through TEM of AuNP-treated skin to study whether the surface engineered gold nanoparticles can penetrate the skin.The silver-enhanced staining was used to evaluate the the efficacy of gold nanoparticles.3.MTT AssayBalbc/c 3T3 cells were chose to evaluate the cytotoxicity of AuNP-PEG-COOH.4.Bioconjugation of AuNP-PEG-COOH with VEGFA certain amount of AuNP-PEG-COOH was bioconjugated with VEGF by EDC/sulfo-NHS.5.In vivo Study of the Co-delivery of AuNP-PEG-COOH and VEGFThe animal model was established in the same manner as described above.The gauzes which were soaked with AuNP-CONH-VEGF suspensions covered the exposed skin for 24 h.After 24 h,immunohistochemical staining was used to detect whether VEGF penetrated the skin.After 7 days,to further investigate whether VEGF can promote angiogenesis after penetrating the skin via AuNP-PEG-COOH,immunohistochemistry and H&E staining were used to detect the skin after the transdermal treatment.Results1 The diameter of citrate-capped AuNPs was about 11.2±0.1 nm with spherical geometry and very low polydispersity.After modified with the thiol-PEG molecules,the hydrodynamic diameter of AuNPs was increased by approximately 8-10 nm and different Zeta potential was obtained.2.TEM images proveed that all gold nanoparticles with different surface charge can pass the skin.In the silver-enhanced staining images,the number of silver-stained particles in the AuNP-PEG-COOH group was the most compared with the groups.3.The safety grade was 1 when the gold nanoparticles' concentration was within the range of 10 ?M-50 ?M,which meant they had low cytotoxicity.4.After conjugated with VEGF,the surface charge of the compound?AuNP-CONHVEGF?was still negative,which was maintained consistent with AuNP-PEG-COOH.5.After transdermal treatment on the skin of mice,we detected VEGF?His-tagged?in the experimental group which applied AuNP-CONH-VEGF by immunohistochemical staining,while none in the control group.7 days later,using the immunohistochemical staining and H&E staining,we found that the subcutaneous vascular density is significantly higher in the skin applied with AuNP-CONH–VEGF than the control group.Conclusion1.AuNP-PEG-COOH,which's surface charge was negative,had the highest transdemal efficiency.2.After conjugated with VEGF,the surface charge of gold nanoparticles?AuNPCONH-VEGF?was still negative.3.AuNP-PEG-COOH connected with VEGF by using EDC/sulfo-NHS could permeate through the skin barrier and induce angiogenesis.