| Part Ⅰ The fabrication and basic property characterization of methotrexate-loaded dissolving microneedle patchBackground: Methotrexate(MTX)is one of the first-line drugs for moderate to severe psoriasis.However,the adverse effects caused by systemic administration of MTX greatly restrict its clinical application.Transdermal drug delivery has many advantages over the conventional approaches,but the performances of currently available skin permeation enhancement techniques are not so satisfactory.Drug-loaded microneedle(MN)patches that dissolve upon insertion and release the drug simultaneously may be a desirable strategy to administer MTX for psoriasis patients.Objective: To fabricate the MTX-loaded dissolving MN patches and characterize their basic properties.Methods: The MTX-loaded dissolving MN patches with different doses were fabricated by a two-step micromolding process.Their morphology was observed using a dermoscope and the determination of the drug content in the needles of the patch was assisted by an ultraviolet-visible spectrophotometer.The mechanical strength of MN patches was measured by a nanoindenter.MN-based drug permeation into the skin was visualized using a confocal laser scanning microscope(CLSM).We employed Franz diffusion cell to perform in vitro transdermal delivery of MTX.In vivo dissolution of the MTX-loaded MN patches was observed by an optical microscope and a scanning electron microscope(SEM).Psoriasis-like lesion was induced by imiquimod(IMQ)and skin tissue sections were investigated to evaluate in vivo skin insertion capability after the application of the MN patch.The recovery process was recorded by a dermoscope to test acute skin irritation of the MTX-loaded MN patch.The inhibitory effect on an immortalized human keratinocyte cell line(Ha Ca T)was assessed using Cell Counting Kit-8(CCK-8)to examine the pharmaceutical activity of MTX encapsulated in the MN patch.Results: Each MN patch consisted of 100(10×10)pyramid-like shaped needles with a height of 650 ± 19 μm,a base width of 220 ± 7 μm,and a center space of adjacent needles of 500 ± 12 μm on a baseplate.The amounts of MTX in the needles of the patches fabricated by solutions containing 6.25,12.5,25,and 50 mg/m L MTX were 6.5 ± 1.3,13.8 ± 1.8,30.6 ± 2.3,and 65.3 ± 2.9 μg,respectively.The mechanical strength of MTX-loaded MN patches decreased with the increase of the drug content in the needles.The depth of fluorescent dye diffusion was deeper than 150 μm after the rhodamine 6G-loaded dissolving MN patch inserted into the porcine cadaver skin.The accumulated release percentage was 52% within 10 min and it kept almost unchanged as ～90% after 1.5 h.The MN patch successfully penetrated the thickened epidermis and completely dissolved within 10 min.No obvious irritative reaction was observed and the microholes on the skin became nearly invisible 1 h later.There was no significant difference between the proliferation of Ha Ca T cells inhibited by MTX-loaded MN patches and MTX solutions(p>0.05).Conclusion: We developed the MTX-loaded dissolving MN patch with excellent water solubility,biocompatibility,biodegradability,and mechanical properties and controllability of drug loading.The MTX-loaded MN patch successfully penetrated IMQ-induced thickened epidermis in mice,dissolved rapidly and delivered the drug intralesionally.Meanwhile,the pharmaceutical activity of MTX remained almost unaffected during the fabrication of MN patch.Part Ⅱ The effects of MTX-loaded dissolving MN patches with various doses on IMQ-induced psoriasis-like skin lesionBackground: Recent studies have demonstrated that solid MN patches could enhance the skin permeability of MTX in vitro.These MN patches reported before were merely used to create microchannels in skin prior to the application of a topical drug formulation.The drug absorbation into the skin was limited due to the receding micropores,probably reducing the therapeutic efficacy.Therefore,exploration of in vivo effects is of great significance.In the first part of our study,we have developed the MTX-loaded dissolving MN patches with various doses and proved that they effectively promote the transdermal delivery of MTX in vitro.We further investigate the effects of MTX-loaded dissolving MN patches on a mouse model of psoriasis-like skin inflammation induced by IMQ.Objective: To study the therapeutic effects and systemic impacts of various doses of MTXloaded dissolving MN patches on IMQ-induced psoriasis-like skin lesion in vivo.Methods: The BALB/c mice were randomly divided into 6 groups.The mice given no IMQ served as the control group,while the other mice received topical application of IMQ cream from day 1 to day 6 on their left ears and were differently treated(days 2~6):(1)model group(no treatment),(2)HA MN group,(3)low-dose(LD)MTX MN group(～6.5 μg MTX/ patch),(4)medium-dose(MD)MTX MN group(～13.8 μg MTX/ patch),and(5)high-dose(HD)of MTX MN group(～30.6 μg MTX/ patch).1 MN patch was applied to the mouse ear once daily.The ear swelling was recorded to evaluate the severity of skin inflammation.Mice left ear skin was further analyzed by hematoxylin-eosin(HE)staining and immunohistochemistry(IHC)staining for Ki67,IL-23 and IL-17 A.Furthermore,their blood cell counts,serum levels of alanine transaminase(ALT),aspartate transaminase(AST),blood urea nitrogen(BUN),and creatinine(CREA)were determined.Results: The application of IMQ to the ear skin of mice caused typical manifestations of erythema,thickening,and scaling.Skin inflammation was alleviated after the treatment with MTX-loaded MN patches,especially in the MD and HD groups.Whereas the lesions of the HA MN group were as severe as that of the model group on day 7.The ear thickness of mice treated with the LD,MD,and HD of MTX-loaded MN patches was significantly reduced from 500.8 ± 29.8 μm to 421.8 ± 28.7(p<0.01),339.6 ± 23.2(p<0.001),and 269.2 ± 21.1 μm(p<0.001),respectively.Meanwhile,their left ear epidermal thickness was repressed from 108.1 ± 9.4 μm to 90.3 ± 9.0(p<0.05),62.3 ± 8.9(p<0.001),and 34.6 ± 6.6 μm(p<0.001).The IMQ-induced upregulated expressions of Ki67,IL-23,and IL-17 A were markedly inhibited by the MTX-loaded MN patches in a dose-dependent manner.However,there was no significant difference between the model group and the HA MN group in the ear swelling,epidermal thickening and expressions of Ki67,IL-23,and IL-17A(p>0.05).IMQ substantially elevated white blood cell(WBC)(p<0.001)and neutrophil(p<0.001)counts of mice.The HA MN,LD,and MD MTX MN groups showed no pronounced difference compared with the model group in blood cell counts(p>0.05).By contrast,the high dose of MTX-loaded MN patches dramatically reversed the boost of WBC(p<0.001)and neutrophil(p<0.001)counts.No distinguishable change in the serum levels of ALT,AST,BUN,and CREA was observed after all treatments(p>0.05).Conclusion: The MTX-loaded dissolving MN patches could attenuate the IMQ-induced psoriatic skin inflammation in mice and the improvement was positively correlated with the dose of MTX.The MN patches within the dosage range of this study hardly affected the hepatic and renal functions of mice.Nevertheless,the high dose of MTX-loaded MN patches showed inhibitory effects on sensitized systemic immunity and the hematopoietic system.Part Ⅲ The comparison of effect and safety between MTX-loaded MN patch and oral administration of MTX in the treatment of IMQ-induced psoriasis-like skin lesionBackground: Oral administration is the most common route in the therapy of psoriasis with MTX.However,the drug is partly degraded in the gastrointestinal tract and eliminated by the liver,resulting in limited therapeutic effect and adverse events of digestive system.The MTX-loaded MN patch rapidly dissolved after insertion into skin and delivered the drug directly into lesions,which is likely to increase the bioavailability,improve treatment efficacy and reduce systemic toxicity.We thus compared the therapeutic effect and safety of MTXloaded MN patch with oral MTX in vivo.Objective: To compare the therapeutic efficacy,systemic effect and toxicity of MTX-loaded MN patch with oral administration of MTX in a mouse model of psoriasiform dermatitis.Methods: The BALB/c mice were randomly divided into 5 groups.Psoriasis-like lesions were induced by IMQ on left ears of the mice except the control group.In the MTX MN group,the MTX-loaded dissolving MN patches with medium dose(～13.8 μg MTX/ patch)were applied to mice 1 patch/day from day 2 to day 6.For comparison,the mice were given intragastric administration of 13.8 and 27.6 μg of MTX once daily in the oral MTX group and the 2× oral MTX group,respectively.The severity of skin inflammation was evaluated by daily measurement of ear thickness,and the left ears of mice were observed on day 7.All skin tissue sections were assessed by HE staining as well as IHC staining for Ki67.Blood cell counts,serum levels of ALT,AST,BUN and CREA were analyzed to examine the systemic impacts,hepatotoxicity and nephrotoxicity of the MTX-loaded dissolving MN patch and oral administration of MTX.Results: IMQ-induced skin erythema,thickening and scaling were attenuated after different MTX treatments.The mice treated with the same dose(13.8 μg)of MTX-loaded MN patches and a double dose(27.6 μg)of oral MTX showed much milder psoriasiform dermatitis than the mice in the oral MTX group.When given the same dose(13.8 μg)of MTX,the reductions of ear swelling(p<0.01),epidermal hyperplasia(p<0.01),and expression level of Ki67(p<0.05)by the MN patches were more significant than oral administration.Notably,there was no significant difference in therapeutic effect between the MTX MN group(13.8 μg)and the 2× oral MTX group(27.6 μg)(p>0.05).The WBC(p<0.001),neutrophil(p<0.01)and lymphocyte(p<0.05)counts of the mice were remarkably reduced by 27.6 μg of oral MTX rather than the other treatments.In addition,taking 27.6 μg of MTX orally also significantly elevated serum levels of ALT(p<0.001)and AST(p<0.05).Conclusion: Compared with oral administration,the MTX-loaded dissolving MN patch showed enhanced efficacy to ameliorate the IMQ-induced psoriasiform skin inflammation and achieved a comparable therapeutic effect at a reduced dosage,which could compromise the systemic effect and hepatotoxicity.Part Ⅳ The effects of MTX-loaded dissolving MN patch on IMQ-induced secondary psoriasis-like skin lesion at a distant site.Background: Psoriasis is a chronic,recurrent inflammatory skin disease.It has been reported that the IL-17A+Vγ4+ γδT memory-like cells from the draining lymph nodes(d LNs)of one psoriasiform skin lesion can home to the initial site of skin inflammation,and they will also travel to other parts of the body.Thus,the recurrence of psoriasis-like skin inflammation will be exacerbated when being rechallenged with IMQ.The MTX-loaded MN patch was demonstrated to be more efficient and safe than oral administration in previous parts of our study.It is of practical significance to explore whether MTX-loaded MN patch has the potential prevent the relapse of psoriasis while attenuating local skin inflammation and to elucidate the possible therapeutic mechanism.Objective: To investigate the effects of MTX-loaded MN patch on the secondary induction of skin inflammation at a distant site and the underlying mechanism.Methods: We conducted 2 animal experiments in this part.First,3 groups(the control group,the model group and the MTX MN group)of mice were employed to evaluate the effects of MTX-loaded MN patches on IL-17A+Vγ4+ γδT cells in the IMQ-induced psoriasis-like ear skin lesion and the d LNs.IMQ and MTX-loaded MN patches were applied to mice left ears from day 1 to day 5.Flow cytometric analysis of left ear dermis and its draining lymph nodes was peformed on day 6.In the other experiment,mice were randomly divided into 4 groups(the control group,the IMQ group,the IMQ + MTX MN group and the MTX MN group)according to different treatments their left ears received from day 1 to day 5.After an interval of 5 days(days 6~10)without any treatment,psoriasiform skin lesion was elicited on the right ears of all mice by IMQ for consecutive 4 days(days 11~14).The right ear thickness was measured from day 11 to day 15 to assess the severity of inflammation,and all skin tissue sections on day 15 were further analyzed by HE staining as well as Ki67 IHC staining.The accumulation of IL-17A+Vγ4+ γδT cells in the right ear dermis was also determined by flow cytometric analysis.Results: In the first experiment,the accumulation of IL-17AVγ4 γδT cells in left ear dermis(p<0.001)and the draining lymph nodes(p<0.001)of mice treated with MTX-loaded MN patches were both significantly inhibited in comparison with the model group.In the other experiment,the secondary generation(days 11~14)of psoriasiform phenotype on mice right ears was markedly restrained due to the treatment with MTX-loaded MN patches(days 1~5)on their left ear lesions induced by IMQ.Specifically,the ear thickening(p<0.001),epidermal hyperplasia(p<0.05)and the expression level of Ki67(p<0.01)of mice right ears were all obviously inhibited compared with the IMQ group.Importantly,IL-17A+Vγ4+ γδT cells in right ear dermis was substantially lower than that of the IMQ group(p<0.001).However,there was no significant difference between the MTX MN group and the control group in all aspects(p>0.05).Conclusion: The MTX-loaded dissolving MN patch could not only ameliorate local psoriasis-like skin inflammation induced by IMQ but also show a concomitant antipsoriatic effect against secondary IMQ sensitization at a distant site by impairing the expansion of IL-17A+Vγ4+ γδT cells in the dermis of MN patch-treated skin and the d LNs. |
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