| Objective:The skin with an area of about 2 m~2 is the largest organ in the human body,which is a very important drug administration site.Currently,there are many problems with traditional transdermal preparations,such as slow absorption,low blood concentration,and delayed effects.Although permeability enhancer and various physical methods such as magnetic field introduction are used to solve above problems,these methods are costly,inconvenient and stimulating.Dissolving microneedles have sharp needles,and the short length of the needles will not touch the nerve tissue,which can not only overcome the pain of patients and reduce infection,but also improve the efficiency of transdermal transmission.In this paper,Estradiol was used as the model drug.A convenient,microemulsion-loaded,organic solvent-free,sustained-release transdermal microneedle system with high mechanical strength,fast transdermal speed was proposed,which is expected to solve the existing problems of transdermal drug delivery.Methods:1.A HPLC method was established for the determination of estradiol in microemulsions and microneedles.2.The single factor method was used to screen the water drop oil process for the preparation of microemulsion.The most suitable oil phase was selected by the determination of estradiol solubility.The optimal emulsifier,co-emulsifier and Km were selected based on microemulsion area as the index.And the microemulsion prescription was further optimized by D-optimal mixture design.3.The appearance of the microemulsion was observed.The type of the microemulsion was identified by staining method.The particle size,transparency,p H and drug content of the microemulsion was determined by HPLC.The centrifugal stability and short-term stability of the microemulsion were also investigated.4.The single factor method was used to screen freeze-thaw methods for the preparation of estradiol-microemulsion-loaded microneedle.Four factors were selected,including PVA concentration,PVP concentration,PVA to PVP gel ratio,and the ratio of mixed gel to estradiol microemulsion.The Box-Behnken response surface method was used to optimize the prescription of estradiol-microemulsion-loaded microneedle.5.The optimized estradiol microemulsion microneedles were evaluated in vitro,including morphological examination,drug content determination,FTIR spectroscopy,release experiment,skin puncture experiment,skin permeability experiment,skin retention experiment,guinea pig skin irritation experiment and stability experiment.6.The LC-MS method was established to determine the content of estradiol in rat plasma using losartan as the internal standard.7.The pharmacokinetics of estradiol-microemulsion-loaded microneedle in rats were investigated.Results:1.A HPLC method with high specificity,high precision,and good reproducibility was established for the determination of estradiol content in microemulsions and microneedles.2.The preparation temperature,stirring speed,dropping speed,stirring time and emulsification time of HS-ME were 40℃,800 rpm/min,1 m L/min,15 min and 8 h,respectively.The prescription of HS-ME optimized by the D-optimal mixture design was 0.20%estradiol,19.46%Kolliphor HS15,6.49%PEG 400,7.98%isopropyl myristate and 65.87%distilled water.HS-ME was an O/W microemulsion with a particle size of 70.87±1.60 nm,transparency of 68.64±0.28%,p H of6.42±0.07,and drug content of 2.01±0.05 mg/g.It had good centrifugal stability and was stabile when stored at room temperature and 40℃for 7 days.3.The preparation temperature,stirring speed,dropping speed,stirring time,emulsification time and ultrasonic time of ALF-ME were 40℃,1000 rpm/min,0.5 m L/min,20 min,8 h and 30 min,respectively.The prescription of ALF-ME optimized by the D-optimal mixture design was 0.83%estradiol,40.91%Labrasol ALF,13.64%PEG 400,11.57%isopropyl myristate and 33.05%distilled water.ALF-ME was an O/W microemulsion with a particle size of 395.50±4.37 nm,transparency of 67.44±0.37%,p H of6.89±0.09 and drug content of 8.83±0.09 mg/g.It had good centrifugal stability and was stabile when stored at 4℃for 7 days.4.The freeze-thaw conditions for preparation of estradiol microemulsion microneedles were as follows:freezing time 1 h,thawing time 0.5 h,freeze-thaw cycle 2 times.Drying conditions:electric blast drying oven drying(40℃,48 h).Prescription of HS-ME-MN:PVA(20%):PVP(40%)=0.75,mixed gel:HS-ME=3.The length of HS-ME-MN was600μm,the area was 0.81 cm~2,and the content of estradiol in each piece of HS-ME-MN was177.12±0.72μg.Prescription of ALF-ME-MN:PVA(20%):PVP(30%)=0.75,mixed gel:ALF-ME=4,the length of the needle body of ALF-ME-MN was 600μm,the area was 0.81 cm~2,and the content of estradiol in each piece of ALF-ME-MN was 671.10±13.19μg.5.Estradiol was successfully encapsulated into the microneedle and there was no chemical interaction between the drug and the excipients.Estradiol-microemulsion-loaded microneedle could release the drug for a long time and had sufficient mechanical strength to pierce the skin for faster delivery of the drug to the receiving fluid.Estradiol-microemulsion-loaded microneedle was safe and non-irritating to the skin.Estradiol-microemulsion-loaded microneedle should be stored away from light and humid environments.6.An LC-MS method with high precision,good stability and specificity was established for the determination of estradiol in rats plasma.In vivo pharmacokinetic experiments of rats,Estradiol-microemulsion-loaded microneedle could not only deliver drugs to the body to reach effective blood concentration quickly through the puncture of the skin in the needle body part and the transport of microemulsion in the microneedles,but also maintain the blood concentration through the continuous release of drugs in the backing part.Conclusion:In this paper,a microemulsion-loaded,organic solvent-free,sustained-release transdermal microneedles system for the delivery of lipophilic drugs was designed.1.The HPLC method with good linearity,high precision,high accuracy,strong specificity and good reproducibility for the determination of estradiol in microemulsion and microneedles and the LC-MS method for the determination of estradiol in plasma were established.2.Two O/W microemulsions of estradiol with high transparency and good stability were obtained.3.Two kinds of estradiol-microemulsion-loaded microneedles were obtained.Estradiol-microemulsion-loaded microneedles are simple in preparation,reliable in quality,sharp in tip with good mechanical strength,uniform in drug distribution,no reaction with excipients,excellent in drug release performance,low drug retention in skin,high in safety,and can quickly reach and maintain effective blood drug concentration.Therefore,the microemulsion-loaded,organic solvent-free,sustained-release transdermal microneedles system in this paper will be a progress in transdermal drug delivery of lipophilic drugs and provide a new idea for transdermal delivery of lipophilic drugs. 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