Establishment And Evaluation Of Phototoxic Microneedles Targeting Skin Squamous Cell Carcinoma

Docetaxel?DTX?is a taxane antitumor drug.Its mechanism of action is to form a stable non-functional microtubule bundle by strengthening tubulin polymerization and inhibiting microtubule depolymerization,thereby it destroys the mitosis of tumor cells and achieves anti-tumor effect.Temoporfin?m-THPC?is a second-generation photosensitizer,which can be used for the photodynamic treatment of squamous cell carcinoma of the head and neck with high selectivity for tumor.After selective uptake by tumor tissue,m-THPC concentration can reach fourteen times of that of normal tissue.After light irradiation with 652 nm wavelength in the tumor tissue,a large amount of reactive oxygen species?such as singlet oxygen,anion and anion?are produced in the surrounding medium,which cause mitochondrial damage,and then make tumor cells die.At present,skin squamous cell carcinoma has the characteristics of refractory,recurrent,and invasive,and it is necessary to use preparations to improve the treatment effect of skin squamous cell carcinoma.In order to increase the concentration of the tumor site,local administration was used.In order to improve the penetration ability of the cuticle,a phototoxic microneedle was prepared,which can puncture the cuticle and improve the drug penetration effect.The combination of microneedle and drug nanoparticles can improve the slow release and permeability of drug in tumor site.Nanoparticles connect with the target base of EGF receptor,which can enhance the targeting of nanoparticles in the tumor site,reduce it into the systemic circulation,and comprehensively improve the therapeutic effect of skin squamous cell carcinoma.Therefore,according to the characteristics of skin squamous cell carcinoma,this study aims to design a drug delivery system to improve the treatment effect of skin squamous cell carcinoma.The specific structure of phototoxic microneedle is to construct a local delivery system,in which DTX and photosensitizer m-THPC are wrapped in nanoparticles,and then nanoparticles are encapsulated in microneedles.The local drug delivery of skin is carried out by the puncture of microneedles.Part one Pre-prescription studies and preparation and characterization of DTX-Nps/m-THPC-NpsObjectives:Established an in vitro content analysis method for DTX and m-THPC.Determined the solubility of DTX and m-THPC in water and the oil-water partition coefficient,and determined whether it was suitable for transdermal administration.Synthesized modified PLGA carrier materials with end-group modification.Characterized and optimized the nanoparticles prepared by DTX modified PLGA and m-THPC modified PLGA respectively.Methods:Determined the content of DTX and m-THPC by high-performance liquid chromatography,and investigated the specificity,precision,stability and recovery rate of the chromatographic conditions.Measured the solubility of DTX and m-THPC in water and the oil-water partition coefficient in noctanol-water system.Synthesized end-group modified PLGA by ring-opening polymerization method,and confirmed the structure by nuclear magnetic and gel chromatography?GPC?.Prepared drug-loading and contained coumarin 6 nanoparticles by emulsion-solvent evaporation method.Combining with particle size,encapsulation efficiency and other factors,optimized the formulation of drug loaded nanoparticles by Box-behnken method.Results:The established condition of high-performance liquid chromatography was specificity,and its precision,repeatability and recovery rate all met the requirements of the methodology.The oil-water partition coefficient and water solubility of DTX and m-THPC were Log P 2.41 and Log P 3.16 and 2.820?g/ml and 0.0042?g/ml,respectively,which were suitable for transdermal application.The synthesis products were verified by hydrogen spectrum,which proved that m PEG-PLGA,COOH-PEG-PLGA,and Mal-PEG-PLGA carrier materials were synthesized,and their molecular weights were 16274,9877,9356,respectively.Optimize formulation is drug carrier ratio of 0.5:10,oil-water volume ratio of 1:10 and poloxamer concentration of 0.1%.DTX-PLGA-Nps encapsulation rate was 52.75±3.59%and drug loading was 0.95±0.02%.m-THPC-PLGA-Nps encapsulation rate was 63.96±0.02%and drug loading was 2.49±0.01%.The prepared two kinds of nanoparticle solutions were uniform with the particle size less than 200 nm,and the polydispersity index?PDI?less than0.2.Conclusion:The established DTX and m-THPC content determination methods were easy to operate and had good reproducibility,all of which met the methodological requirements.End-group modified PLGA was synthesized by ring-opening polymerization method.DTX and m-THPC and coumarin 6?Cou-6-EGF-Nps?nanoparticles were prepared by emulsion-solvent evaporation method.Part two Cell uptake and pharmacodynamic test of DTX-EGF-Nps and m-THPC-EGF-NpsObjectives:To evaluate the in vitro activity of the prepared DTX-EGF-Nps and m-THPC-EGF-Nps.Methods:A431?cutaneous squamous carcinoma?cells and Ha Cat?normal keratinocyte?cells were used to evaluate the prepared DTX-Nps and m-THPC-Nps.Different nanoparticle concentrations were set in the experiment.The survival rate of A431 and Ha Cat cells after administration was measured by MTT method.The uptake of Cou-6-EGF-Nps by the cells was observed by an inverted fluorescence microscope.the cellular uptake mechanisms were studied too.Pharmacodynamic test was carried out by irradiating the cells with He-Ne laser for 60 s,and observeed the survival rate of the cells.Results:The blank nanoparticles were less toxic to two kinds of cells.It could be seen that the synthesized carrier material had less toxicity.The survival rate of DTX-Nps on A431 and Ha Cat cells decreases with the increase of the concentration of administration;when the m-THPC-Nps was at a high concentration,the survival rate of A431 cells was low.With the increase of drug concentration,the toxicity of drug to cells gradually increased.The IC₅₀ values of Blank-Nps,DTX,m THPC,DTX-Nps and m-THPC-Nps were calculated separately.For A431 and Ha Cat cells,the IC₅₀ values of Blank-Nps were similar,but the IC₅₀values of DTX-Nps and m-THPC-Nps were higher than that of DTX and m-THPC respectively.Compared with the control group,the groups of chlorpromazine,sodium azide,and colchicine had an inhibitory effect on the uptake of nanopaticles in A431 cells,however,those inhibitors did not inhibit the uptake of Ha Cat cells.DTX+m-THPC-EGF-Nps,m-THPC-EGF-Nps and m-THPC of 3concentrations?low,medium and high?acted on Ha Cat cells respectively.After laser irradiation,the cell survival rates were lower than that of non irradiation cells.DTX+m-THPC-EGF-Nps,m-THPC-EGF-Nps and m-THPC of 3 concentrations?low,medium and high?acted on A431 cells respectively.After laser irradiation,the cell survival rates of m-THPC-EGF-Nps and m-THPC were lower than that of non irradiation cells,while only at high concentration,the cell survival rate of DTX+m-THPC-EGF-Nps was lower than that of non irradiated cells.Compared with Ha Cat cells,A431 was more toxic to Ha Cat cells.Conclusion:In this section,the MTT method,inverted fluorescence microscope,fluorescence microplate reader,and LJL40-HA He-Ne laser treatment instrument were used to evaluate the in vitro activity of DTX-EGF-Nps and m-THPC-EGF-Nps.It was known that the prepared nanoparticles could be used in subsequent experiments.Part three Preparation and in vitro evaluation of microneedles cont-aining nanoparticelsObjectives:To prepare fast-dissolving microneedles containing DTX-EGF-NPs and m-THPC-EGF-NPs,and to pave the way for subsequent experiments.Methods:Blank microneedles were prepared by inverted mold casting method,and suitable carrier materials were screened.The optimal carrier material was used to prepare drug-loaded nanoparticle microneedles,and their appearance,puncture rate,drug loading,confocal microscopy,and histology were evaluated in vitro.Results:In this paper,hyaluronic acid?HA?was selected as a carrier material for the preparation of microneedles,and nanoparticle-encapsulated microneedles were successfully prepared.The formulation of microneedles was 40%HA as the tip concentration and 50%HA as the backing layer.The prepared microneedles had a height of 598.69±0.65?m and a substrate width of 203.43±1.71?m.The needles contained DTX-EGF-NPs and m-THPC-EGF-NPs,and the drug loading was 10.07±1.58?g?n=10?and0.50±0.13?g?n=10?,respectively.Histological results showed that microneedles could puncture the mouse skin to form holes.Confocal microscopy results showed that microneedle contained coumarin-6nanoparticles could puncture into rat skin with a depth of 220?m and could be used to treat skin squamous cell carcinoma within 0.5 cm in diameter.Conclusion:The method of preparing microneedles by inverted mold casting method was simple and reproducible.The microneedles were successfully prepared and could be puncture into rat skin to treat skin squamous cell carcinoma within 0.5 cm in diameter,but the drug loading in microneedles was low.