Application Of Novel Transdermal Drug Delivery In Inflammatory Dermatosis

First part【Background and Objective】Fractional laser and microneedles are two controllable auxiliary transdermal drug delivery systems,which can reversibly ablate or destroy the skin to make the drug penetrate deeper through the skin,thereby increasing the transdermal absorption efficiency of the drug.Fractional laser and microneedle-assisted drug transdermal technology has been widely used in disease detection,disease treatment,vaccination,medical cosmetology and other fields in recent years.In this study,we combined the fractional Er:YAG laser or solid microneedles and cyclosporin nanoparticles on the skin lesions to alleviate the skin damage in the psoriasis-like mouse model induced by imiquimod,and then discussed the corresponding mechanism.【Method】1.We synthesize cyclosporin nanoparticles by chemical methods,characterize their physical and chemical properties,and evaluate their stability.Detection of the toxicity of cyclosporin nanoparticles to cells: Cyclosporin nanoparticles of different concentrations were used to interfere with human keratinocytes(Ha Ca T cells)and fibroblasts for 48 hours,and the CCK8 experiment was used to detect them.Detected the auxiliary transdermal effect of fractional laser and solid microneedle: use the fractional Er:YAG laser and solid microneedle to treat the isolated pig skin,and use confocal fluorescence microscope to observe the permeation effect of liposoluble fluorescence on the pig skin.2.Applied 5% imiquimod(IMQ)to the right ear of the mouse for 5 days to induce the formation of a psoriasis-like mouse model.Starting from the 6th day,used the fractional Er: YAG laser treat the skin of the right ear and pierced the skin of the right ear with solid microneedle for three consecutive days,and applied Cyclosporine nanoparticles of different concentrations(0.4 mg/mL,2 mg/mL,10 mg/mL)on the corresponding area at the same time.On the 6th day,the right ear of the mouse was taken for pathological section to evaluate the micropores in the skin after the fractional laser and microneedle treatment,and to measure the changes in epidermal thickness.On the 9th day,the mouse serum was taken to evaluate the safety of the drug by detecting the liver and kidney function of the mouse.3.The lesions of IMQ-treated mice were administrated with fractional Er: YAG laser and solid microneedles,and then externally treated with 2 mg/mL cyclosporin nanoparticles.On the fourth day after 3 days of treatment,the serum was taken to measure the liver and kidney function,and the liver and kidney were taken for pathological section to evaluate the safety.We used the following methods to evaluate the therapeutic effects of various treatments:(1)skin thickness and epidermal thickness;(2)PASI score;(3)the levels of PCNA,iNOS,VEGF,ki67,CD3 in the skin of right ear(immunohistochemistry);(4)the concentration of IL-2,IL-17,IL-22,IL-23,TNF-α in serum(ELISA);(5)the m RNA expression of IL-17 A,IL-17 C,IL-22,IL-23,IL-17 F in the lesions(RT-PCR);(6)the protein expression of JAK3,p-JAK3,STAT3,p-STAT3,IFN-α,SOCS3 in the right ear of mice(Western-blot).【Results】1.We successfully synthesized cyclosporin nanoparticles with a particle size of187±12.6 nm and a concentration of(0.4 mg/mL,2 mg/mL,10 mg/mL).Among them,the stability of the nanoparticles in the low concentration group(0.4 mg/mL and 2mg/mL)was good,and the stability of the nanoparticles in the high concentration group(10 mg/mL)was poor.When the drug concentration was less than 1 μg/mL,the cyclosporin nanoparticles had no obvious toxicity to the two kinds of cells;when the drug concentration reached more than 10 μg/mL,the proliferation of the two kinds of cells was significantly inhibited.We observed micro-channels formed by microneedles and fractional lasers on the isolated pig skin through a confocal fluorescence microscope,and liposoluble fluorescent agents accumulated around the channels.2.Compared with the negative control group,the fractional laser or solid microneedle combined with cyclosporine nanoparticles(2 mg/mL,10 mg/mL)can significantly reduce the thickness of the epidermis of the psoriasis-like mouse model.The liver and kidney functions of mice were all normal.After the right ears of normal mice and IMQ treated mice were processed by fractional laser and solid microneedles,microchannels can be observed.3.Compared with the negative control group(IMQ treatment group,microneedle group,fractional laser group,cyclosporin nanoparticle group),fractional laser or microneedle combined with cyclosporin nanoparticles can significantly down-regulate IMQ-induced psoriatic lesions,including:(1)the thickness of the right ear of the mice model was significantly thinner;(2)the PASI score was lower;(3)PCNA,iNOS,VEGF,ki67,and CD3 expression levels in local lesions were significantly reduced;(4)the m RNA expression levels of IL-17 A,IL-17 C,IL-22,IL-23 and IL-17 F in the skin lesions were significantly reduced;(5)IFN-γ expression in the skin lesions was down-regulated;(6)the phosphorylation of JAK3 and STAT3 decreased remarkable;(7)the concentration of IL-2,IL-17,IL-22,IL-23,and TNF-α in serum decreased significantly.【Conclusion】Firstly,we successfully synthesized cyclosporin nanoparticles and verified their stability and safety.Secondly,through the combined treatment of solid microneedles or fractional laser and cyclosporin nanoparticles,we found that solid microneedles and fractional Er:YAG laser can not only effectively but also safely and controllably deliver cyclosporin nanoparticles into the skin.Finally,we found that compared with solid microneedles or fractional laser or cyclosporin nanoparticles alone,the combination of solid microneedles or fractional laser and cyclosporin nanoparticles can significantly alleviate IMQ-induced skin lesions in mice,for example,the alleviation of skin thickness,down-regulation of PASI score,reduction of local immune cell infiltration in skin lesions,and down-regulation of the expression of pro-inflammatory cytokines.In this study,cyclosporine,a systemic drug for moderate to severe psoriasis in dermatology department,was combined with transdermal technology to establish a successful local topical treatment fot IMQ-induced skin lesions in mice,which provided a new theoretical and practical basis for the topical treatment of inflammatory skin diseases.Second part【Background and Objective】Lupus band test is a direct immunofluorescence method to detect the deposition of immunoglobulin and complement in the skin of patients with lupus erythematosus(LE).It is one of the important auxiliary examinations in the diagnosis of lupus erythematosus.However,LBT is often limited due to its invasive skin biopsy nature in clinics,which is due to that the skin lesions in LE patients usually occur in the exposed areas(mostly on face).In this study,we chose Er: YAG laser as a transdermal drug delivery system with small trauma,fast repair and good controllability,assisted by water-soluble antibody(TRITC-conjugated Goat Anti-Mouse IgG,molecular weight about 150 kda).Previous studies had successfully verified the effectiveness,safety and controllability of this transdermal drug delivery system.On the basis of previous studies,we aim to obtain a broad insight in the dynamics of TRITC-conjugated Goat Anti-Mouse IgG(T-IgG)uptake in fractional Er:YAG laser pretreated skin and provide a new,non-invasive option for detecting lupus erythematosus(LE)in mice.【Method】1.H&E staining was used to observe whether MRL/lpr mice had similar pathological characteristics with lupus erythematosus.The normal skin around the lesion was taken from the 14 week old female MRL/lpr mice.The slices were incubated with T-IgG or FITC-Goat Anti-Mouse IgG as the antibody for 30 mins,in order to validate the presence of lupus band.Then,the slices were stained with H&E to determine the position of lupus band.2.14 week female MRL/lpr mice with obvious skin lesions were selected as the experimental group,and 14 week female MRL/MPJ mice were selected as the negative control group.The skins of MRL/lpr and MRL/Mp J mice were treated by fractional Er:YAG laser at a power density of 25 J/cm~2 and 210 pores/cm~2.After 24 hours of T-IgG application,the normal skin around the lesions was taken.Frozen section of half specimen was used to observe whether there was band fluorescence near the basement membrane band by fluorescence microscope.Further more,H&E staining was used to determine the location of the fluorescence band.Another part of MRL/lpr mice skin was scanned with confocal microscope to observe the intensity and depth of fluorescence distribution.【Results】1.The pathological manifestations of dorsal skin lesions of MRL/lpr mice showed colloid bodies,keratinous plugs blocking the pores,and signifificantly thickened basement membrane band at DEJ.Lupus band test was taken on the skin of MRL/lpr mice,as the positive control test.Obvious banded fluorescence was observed at the DEJ,indicating that the presence of IgG deposition at basement membrane zone,and can be combined with T-IgG,which verified that MRL/lpr lupus mice can be used as an ideal lupus band test model.2.In the lupus band test in vivo,Red fluorescence could be observed at basement membrane bands in MRL/lpr murine skin,indicating positive LBT.Bright field and H&E staining confirmed that the fluorescence signal was located at DEJ.In the skin lesion of MRL/lpr mice without laser treatment,no significant fluorescence signal was detected within the skin.The MRL/Mp J mice control group showed only a few weak hair follicle-shaped fluorescence signals in the skin and LBT was negative.Confocal microscopy showed that spatially diffusion fluorescence signals were the strongest when the scanning depth was 60–80 mm in the MRL/lpr murine skin,corresponding to the basement membrane band.【Conclusion】Our study successfully verified that MRL/lpr lupus mice is an ideal lupus band test model.We innovatively used Er: YAG laser as a transdermal drug delivery system to accomplish lupus band test in vivo.This study provides a new alternative to an invasive pathological examination for the diagnosis of LE.Also,this study provides theoretical basis and direction for other diagnosis and therapy in the future.Our study successfully verified that fractional Er:YAG laser and solid microneedle are two successful novel transdermal drug delivery systems,which increase the transdermal efficiency of macromolecular drugs,antibodies and other substances,and achieve the effect of transdermal drug therapy and transdermal immunity.They provide a new idea and theoretical basis for the diagnosis and treatment of inflammatory skin diseases such as lupus erythematosus and psoriasis.