Pharmacodynamics And Mechanism Based On Anti-biofilm Activities Of Hexyl-aminolevulinate Ethosomes In The Treatment Of Acne

BackgroundAcne is an inflammatory skin disease involving the hair follicle and sebaceous glands,which has a high incidence rate,affects the appearance of the patients,and imposes a physiological and psychological burden on them.The pathogenesis of acne is complicated and it is easy to relapse,which makes treatment difficult.As a multifactorial disease,the pathogenesis of acne is mainly related to microbial infection and proliferation,inflammation,elevated androgen levels,abnormal keratinization of hair follicle,and increased secretion of sebaceous glands.Among the skin microorganisms,the quantity of bacteria is the maximum,most of which can form biofilm(BF).BF is a kind of extracellular polymer covering the surface of the fixation secreted by the bacteria,enveloping the bacteria to evade the host’s immune response and antibiotic killing effect,especially the propionibacterium and staphylococcus related to acne.Treating acne can be difficult as a consequence of biofilm formed by bacteria such as Propionibacterium acnes and Staphylococcus epidermidis.In general,the treatment of acne includes systemic and topical treatment,such as vitamin A acid drugs,antibiotics,hormones and so on.Nevertheless,isotretinoin has teratogenic effects;long-term use of antibiotics can easily cause drug resistance;hormones can inhibit the body’s immune function and increase the risk of infection;laser can cause pigmentation.In contrast,photodynamic therapy(PDT)has great advantages.Killing microorganisms such as Propionibacterium acnes and Staphylococcus epidermidis is one of the important means of treating acne.However,the efficacy of antibiotics is reduced by the protective effect of bacterial biofilm on microorganisms.Worse still,the proliferation of surviving Propionibacterium acnes leads to prolonged acne inflammation.PDT has a good antibacterial effect without drug resistance,becoming an important method for the treatment of acne.With high targeting and few side effects,PDT uses a certain wavelength of excitation light to excite photosensitizers in cells,producing reactive oxygen free radicals,which induces cell apoptosis and necrosis.Currently,the most widely used photosensitizer 5-aminolevulinic acid(ALA)is poor in fat solubility and transdermal permeability.ALA ester derivative 5-hexyl-aminolevulinate(h-ALA)has higher stability,higher fat solubility and better transdermal permeability.Studies have shown that h-ALA at a relatively lower concentration can produce the same fluorescence intensity as ALA does.Nowadays,the third-generation photosensitizer is the original photosensitizer coated with a drug delivery carrier or antibody to achieve better penetration of tissue or targeting purposes.Ethosomes are lipid bimolecular vesicles with good stability,high encapsulation efficiency,good transdermal properties,non-toxicity and low skin irritation.It proves to be an excellent transdermal drug carrier.The ALA liposomes and h-ALA liposomes reported have good pharmacological effects.H-ALA ethosomes had been synthesized in our previous work.The characteristics of high encapsulation efficiency,good stability,and excellent percutaneous permeability had been verified,as well as its good therapeutic effect in the psoriasis mouse model.This study will explore the therapeutic effect of h-ALA ethosomes on acne and its mechanism of anti-biofilm effect on Staphylococcus epidermidis and P.acnes biofilms.ObjectiveThe objective of this study is to analyze the pharmacodynamics of h-ALA ethosomes for the treatment of acne and to explore the mechanism from the perspective of anti-biofilm effect.MethodsH-ALA ethosomes were prepared.A model of acne inflammation in rats was established by injection of P.acnes into the auricle.The acne models were treated with topical application of 0.5%h-ALA ethosomes,blank ethosomes,0.5%h-ALA,0.5%ALA,5%h-ALA and 5%ALA,followed by red light irradiation.The effect on acne inflammation was evaluated by the appearance of the auricle,the pathological changes of HE staining,the immunohistochemical expression of IL-1β and the counting number of cultured P.acnes colonies from tissue homogenate.On the other hand,S.epidermidis and P.acnes biofilms were established by 96-well plate method,incubated with 0.5%h-ALA ethosomes,blank ethosomes,5%h-ALA and 5%ALA an then irradiated by red light.The antibacterial effect was evaluated by XTT colorimetric method and scanning electron microscopy(SEM)observation of biofilm changes.Results1.Therapeutic effect of acne inflammation model in ratsThe appearance of the auricle:the acne model untreated group still had obvious inflammation such as erythema,swelling and telangiectasia;the 0.5%h-ALA ethosomes group,the 0.5%h-ALA group and the 0.5%ALA group still had mild inflammation performance;the 5%h-ALA group and the 5%ALA group had little erythema in the auricle;in the blank ethosomes group,the auricle showed erythema and telangiectasia.P.acnes colony count:the count was significantly reduced in the 5%h-ALA group and there was no significant difference between the other groups.HE staining:the 0.5%h-ALA ethosomes group had a few inflammatory cells in the dermis;erythema and swelling along with diffuse infiltration of inflammatory cells were observed in the acne model untreated group;the blank ethosomes group showed a large number of inflammatory cells;abscesses and necrosis were observed in the stratum corneum of the 0.5%h-ALA group and the 0.5%ALA group,scattered in inflammatory cells;the 5%h-ALA group and the 5%ALA group had the least inflammatory cells.Immunohistochemical expression of IL-1β:the 0.5%h-ALA ethosomes group was lower than the 0.5%h-ALA group and the 0.5%ALA group,but higher than the 5%h-ALA group and the 5%ALA group;there was no significant difference between the 0.5%h-ALA group,the 0.5%ALA group,and the blank ethosomes group;the 5%h-ALA group was lower than the 5%ALA group.2.Antibacterial effects of Staphylococcus aureus and P.acnes biofilmsFor S.epidermidis biofilm,the 0.5%h-ALA ethosomes group was lower in quantification of survival bacteria than the blank ethosomes group and the 5%ALA group.There was no significant difference between the 0.5%h-ALA ethosomes group and the 5%h-ALA group.The quantification of survival bacteria in the 5%h-ALA group was lower than the 5%ALA group.Surprisingly,the blank ethosomes group was found to be less quantitative than the 5%ALA group.For P.acnes biofilm,the 0.5%h-ALA ethosomes group was lower in quantification of survival bacteria than the blank ethosomes group,5%h-ALA group and 5%ALA group.The quantification of survival bacteria in the 5%h-ALA group was lower than the 5%ALA group.Different from S.epidermidis biofilm,the 5%ALA group was less quantitative than the blank ethosomes group.Conlusion1.The experimental model of acne inflammation in rats showed that the therapeutic effect of 0.5%h-ALA ethosomes was better than that of 0.5%h-ALA.The therapeutic effect of 5%h-ALA was better than 5%ALA.2.The antibacterial ability of 5%h-ALA against S.epidermidis and P.acnes biofilm was stronger than 5%ALA.The 0.5%h-ALA ethosomes’ antibacterial ability was improved compared with 0.5%h-ALA.Blank ethosomes could be antibacterial.