The Study On Efifcacy And Immunological Mechanisms Of Treatment SKH-1Mouse Skin Squamous Cell Carcinoma With ALA PLGA Nps Photodynamic Therapy Combined With Microneedles

Background Cutaneous squamous cell carcinoma (SCC) is one of the most commonhuman skin tumors. The efficacy of traditional treatments is less than ideal,especially when lesion is large and the cosmetic result is required.5-aminolevulinic acid photodynamic therapy (ALA-PDT) is a photodynamictherapy using5-aminolevulinic acid (ALA) as photosensitizers. It is noveltherapy techniques which damage tumor tissue and hyperplastic cells byphotodynamic reaction. Its advantages include approving effect, no pain,no scar formation, free from the restrictions of the number and locationof the lesions and the ability to be repeatedly at the same site. But thedisadvantage of ALA-PDT is the limited depth of treatment, the efficacyfor deep skin SCC of is still not ideal. This is mainly because of the lowconcentration after administration of ALA in the organization, SCC, unevendistribution, the efficiency of the photodynamic reaction is limited. Inorder to further improve the ALA-PDT treatment the skin SCC efficacy, weenvisaged to enhance the photodynamic effect by the administration ofnanotechnology and microneedles. In this study, pre-research groupprepared5-aminolevulinic acid PLGA nanoparticles (ALA PLGA NPs), combinedwith microneeds, photodynamic therapy of cutaneous squamous cell carcinomain mice by enhance the stability of the photosensitizer EPR effect (enhanced permeability and retention effect, EPR effect) and SCC tissue targetingselective。PLGA combined with the microneedles administration to improvethe permeability of the NPs, thereby improving the ALA-PDT effect. Andimmunological mechanisms is an exploratory study.Objective Explore the new ALA PLGA NPs PDT combined microneedles treatmentSKH-1mice squamous cell carcinoma of the efficacy and immunologic effectsin mice.(1) ALA PLGA NPs PDT combined with microneedles treatment SKH-1skin squamous cell carcinoma into a mold mice drug pharmacokinetics;(2)ALA PLGA NPs PDT combined with microneedles treatment of SKH-1mousesquamous cell carcinoma of the efficacy studies;(3) ALA PLGA NPS PDTcombined with microneedles of the immunological effects after treatmentof SKH-1mice squamous cell carcinoma.Methods (1) ALA PLGA NPs PDT combined with microneedles treatment SKH-1squamous cell carcinoma of the skin into a mold mice pharmacokinetic studies:the establishment of SKH-1hairless mice with multiple skin cancer, tumordiameter, length to1to5mm after select24tumor diameter of about1to5mm, biopsy confirmed SCC tumor-bearing mice were randomly divided intoA, B, C, D4groups, Group A (6) ALA PLGA NPs combined with microneedlesgroup, Group B (6)ALA PLGA NPs group,Group C(6) ALGroup A,Group D(6) ALAcombined with microneedles group. Each group were randomly selected threetumor-bearing mice, conducted ALA/PpⅨ drug pharmacokinetic study,4groups of mice were given different treatment.Group A were treated withmicroneedles before outer coating ALA PLGA NPS cream (content equal to0.8%ALA cream), Group B were treated with ALA PLGA NPs cream(content equal to0.8%ALA), Group C were treated with8%ALA cream, Group D were treated with microneedles before outer coating8%ALA cream. The curalux spectrumanalyzer detects the component mode mouse tumor and the surroundingfluorescence intensity changes over time, and draw the fluorescence curve,ALA/PpⅨ drug pharmacokinetic studies;3tumor-bearing mice in each groupwere randomly selected, the camera shooting fluorescence images of miceand observed by fluorescence microscopy PpⅨ fluorescence distribution inthe tumor tissue; ALA PLGA NPs treatment group using transmission electronmicroscopy PDT combined with microneedles after administration of ALA PLGANPs in the tumor tissue distribution.(2) ALA PLGA NPs PDT combined withmicroneedles treatment of SKH-1mouse skin squamous cell carcinoma of theefficacy studies: Select50tumor diameter of about1to5mm biopsyconfirmed SCC tumor-bearing mice were randomly divided A, B, C, D, E5groups,Group A (10) ALA PLGA NPs PDT combined with microneedles treatment, GroupB (10) ALA PLGA NPs PDT treatment, Group C (10) ALA-PDT treatment, D(10)ALA-PDT combined with microneedles treatment group. E(10) was thecontrol group, not any treatment. A, B, C, D of each group of treatmentonce a week after the end of the4consecutive treatment efficacy.(3) TheALA PLGA NPs PDT combined with microneedles treatment SKH-1mice squamouscell carcinoma of the immunologic effects of the mouse: Select35tumordiameter of about1to5mm, the biopsy confirmed SCC tumor-bearing smallmice were randomly divided into A, B, C, D, E5groups, Group A (7) usingthe ALA PLGA NPs PDT combined with microneedles treatment, Group B (7) ALAPLGA NPs PDT treatment, Group C(7) the use of red light therapy alone, GroupD (7) ALA-PDT treatment, Group E(7) was the control group, not any treatment.Before treatment and after treatment1d,3d,7d drawn usingimmunohistochemical method of tumor tissue or the appearance of normaltissue local CD4~+T and CD8~+T distribution study; flow cytometry mouse CD4~+T cells (CD3~+CD4~+), CD8~+T cells (CD3~+CD8~+); flow cytometry mouse cell factorTNF-α, IL-6, IL-10the expression level.Results (1) ALA PLGA NPs PDT combined with microneedles treatment SKH-1squamous cell carcinoma of the skin into a mold mice pharmacokinetic studyresults: Curalux spectrum analyzer detection ALA PLGA NPs PDTcombined withmicroneedles administration group SCC cells PpⅨ fluorescence intensitygenerated within6hours after dressing reached a peak after slowly reduced.And detecting SCC vicinity0.5cm,1cm at the fluorescent display ALA PLGANPs after administration of the microneedles, the fluorescence confinedto the tumor site, tumor vicinity fluorescence intensity is low. ALA PLGANPs group of PpⅨ fluorescence intensity is also six hours and reached apeak, and then slowly lower, but the fluorescence intensity is lower thanALA PLGA NPs combined with microneedles administration group, thefluorescence is confined to the tumor site, tumor surrounding lowfluorescence intensity. ALA PLGA NPs combined with microneedlesadministration group and ALA PLGA NPS group fluorescence intensity werelower than ALA and ALA combined with microneedles group. PpⅨ fluorescenceintensity generated in the the SCC cells treated reached a peak after slowlyreduced. And detection SCC surrounding0.5cm,1cm at the fluorescentdisplay strong fluorescence at the tumor site and around the tumorfluorescence intensity. ALA combined with microneedles group PpⅨfluorescence intensity also reached the peak at5hours, and then slowlyreduced, but the fluorescence intensity is lower than ALA. And detectionSCC surrounding0.5cm,1cm at the fluorescent display fluorescence at thetumor site and tumor surrounding fluorescence intensity is lower than theALA. The fluorescence images of mice topical ALA combined with microneedlesand simple topical ALA treatment comparison. ALA system to absorb and spread to the mouse body, as well as throughout the whole body fluorescence inmice. microneedles treated of topical ALA PLGA NPs with simple topical ALAPLGA NPs group compared fluorescence images show strong fluorescence, butshowed good targeting, compared with simple topical ALA and ALA combinedwith microneedles treatment groups, fluorescent confined to the tumor site,tumor fluorescence intensity around, did not spread throughout the body,visible ALA PLGA NPs targeting better. Fluorescence microscopemicroneedles treatment before topical ALA compare topical ALA,fluorescence microscopy shows strong fluorescence intensity, ALA PLGA NPsgroup plus microneedles, help to increase the permeability of the ALA PLGANPs, fluorescence microscopy showed strong fluorescence intensity. Thetreatment group deal with microneedles, topical ALA PLGA NPs cream for3h electron microscopy results show a large number of spherical ALA PLGANPs visible within the cytoplasm, most of the ALA PLGA NPs form remainsintact.(2) ALA PLGA NPs PDT combined with micro needles to drug groupcomplete response rate was84.06%, the diameter of <1~4mm, thickness2.5mm cancer treatment effect is good, can be completely subsided;>4mm indiameter, thickness2.5mm after the treatment for tumor inhibition. Endof treatment2weeks after complete remission at the end of the tumors hadhistopathological lesion disappeared, structure back to normal. ALA PLGANPs PDT group complete response rate was62.75%, the treatment effect islower than the ALA PLGA NPs PDT combined with microneedles to drug group,may be related to medication after ALA PLGA NPs into squamous carcinomatissues of fewer small and release of ALA, photodynamic effect is poor.ALA-PDT complete response rate was88.89%, ALA-PDT combined microneedlesto drug group complete response rate was90.38%, the diameter of <1~4mm,thickness2.5mm cancer treatment effect is good, can be completely subsided; >4mm in diameter, thickness2.5mm part can fade after treatment. End oftreatment2weeks after complete remission at the end of the tumors hadhistopathological lesion disappeared, structure back to normal. Tumor inblank control group, each mouse tumors increased to more than10pieces,part of tumors in mice back plate shaped or dense carpet distribution, sometumors merging into larger tumor, central necrosis, ulceration, totalnumber of tumors in mice up to hundred. Histopathological tumor hyperplasiaof height, a large number of cell atypia breakthrough basement membranezone diffuses whole layer, visible pathological karyokinesis and keratinpearl.(3) immunological studies. ALA PLGA NPs PDT combined withmicroneedles group and ALA-PDT group the day after the end of the treatment,3d,7d by immunohistochemical detection of CD4~+T, CD8~+T expressiongradually increased, compared with before treatment. After treatment,3dpositive cells was significantly increased (P<0.05), no significantincrease in other groups. AlA PLGA NPs PDT combined with microneedles groupcompared with ALA-PDT group,the difference was not statisticallysignificant (P>0.05), compared with the other groups was statisticallysignificant (P<0.05), CD4~+T/CD8~+T ratio change significantly. ALA PLGA NPsPDT combined with microneedles treatment SCC mice, can enhance the localimmunological effects. ALA PLGA NPs PDT combined with microneedles groupand ALA-PDT group the day after treatment,3d,7d by flow cytometrydetection of CD4~+T, CD8~+T increase, peak at day3, followed by a slow decline.TNF-α, IL-6, IL-10expression with time gradually increase, peak at day3, followed by a slow decline, days after treatment, TNF-α, IL-6, IL-10significantly increased (P<0.05). The other group had no significantchange. ALA PLGA NPs PDT combined with microneedles group compared withALA-PDT group no significant difference (P>0.05), compared with the other groups were statistically significant (P<0.05). Visible combined withmicroneedles ALA PLGA NPs PDT drug treatment SCC mice, but also can enhancethe system immunological effects.Conclusion (1) AlA PLGA NPs combined with microneedles enhances thestability of the photosensitizer ALA for the selective targeting of skinSCC tissue, improves the efficiency of the SCC tissue absorption of ALAPLGA NPs and improve the generation of PpⅨ amount;(2) The efficacy oftreatment SKH-1mouse skin squamous cell carcinoma with AlA PLGA NPsphotodynamic therapy combined with microneedles is safe and effective,adverse reactions;(3) ALA PLGA NPs PDT combined with microneedlestreatment can improve the SCC miceCD4~+, CD8~+percentage of significantimprovement in the serum IL-10, IL-6, TNF-α level, it is possible toenhance the immunological effects of the treatment of SKH-1mice squamouscell carcinoma.