Design And Evaluation Of Silk Fibroin Double-Layer Composite Microneedles Loaded With Triproline

Triptorelin,a widely used agonist of gonadotropin-releasing hormone,was approved for the treatment of sex steroid-dependent carcinomas of the prostate gland,and is also commonly used in assisted reproductive technology in China.Currently,triptorelin is marketed as a subcutaneous injection（0.1 mg）and a sustained-release formulation with poly（lactic-co-glycolic acid）（PLGA）as the polymer.The disadvantages of formulations（reconstitution before use,professional manipulation,and injection administration）result in a low quality of life for patients.Subcutaneous injections requiring daily administration result in low bioavailability.Moreover,incomplete release and inflammation caused by the acid-degradation products of synthetic polymers and activity loss of peptide/protein structures are the main challenges hindering its wide clinical application.In the study,we developed double-layer silk fibroin microneedles for transdermal delivery of triptorelin.Sustained-release microneedles and nanoparticle microneedles were prepared respectively according to different therapeutic applications.Triptorelin was mixed with silk fibroin to form a microneedle tip supported on a backing made of dissolving polyacrylic acid.Upon contact with the tissue fluid in skin,the polyacrylic acid backing dissolves rapidly and is separated from the microneedle tip.Triptorelin encapsulated in the microneedle tip is implanted intracutaneously and released through drug diffusion and degradation of silk fibroin.Microneedles were characterized（morphological structure,mechanical properties,insertion properties,degradation properties,skin permeability,pharmacokinetics and pharmacodynamics studies）to assess the therapeutic effect.1.Preparation and performance evaluation of silk fibroin blank microneedlesThe micromolding method was employed to prepare silk fibroin microneedles without triptorelin.The secondary structure of silk fibroin was changed by methanol vapor treatment to improve the mechanical properties of the microneedles.Each microneedle was fabricated into a cone of height～600μm,basic width of～300μm and tip radius of～10μm.The β-sheet content was increased with the treatment time of methanol vapor,and the mechanical strength of MNs was positively correlated with theβ-sheets content.Methanol-treated microneedles were successfully inserted into porcine skin and showed good biodegradability.2.Preparation and performance evaluation of triptorelin-loaded sustained-release microneedlesA low shear mixing method was performed for the preparation of triptorelin microcrystals.The well-dispersed microcrystals size was ～1 μm.The micromolding method was employed to prepare double-layer composite triptorelin microneedles,and methanol vapor treatment was used to change the conformation of the microneedles.Triptorelin microneedles were composed of a 10×10 array with a center-to-center spacing of ～600μm over an area of 9×9 mm².Each microneedle was fabricated into a cone of height～600μm,basic width of～300μm and tip radius of～10μm.The β-sheet content of the microneedles was proportional to the methanol vapor treatment time within 24 h,and inversely proportional to the treatment time in 48 h～96 h.The β-sheet content of triptorelin microneedles prepared by 8%silk fibroin concentration in annealing using methanol vapor for 24 h was the highest（～42%）,and the failure force reached～330 mN/MN.Triptorelin microneedles were evaluated to have good insertion properties,matrix separation performance,biodegradability,biocompatibility,and safety.3.In vitro release and skin penetration of sustained-release microneedlesThe triptorelin encapsulation of the microneedle array was determined to be 45.62±1.56μg.A microneedle array with a design size of 9 cm² would be sufficient to provide a therapeutic dose of triptorelin for 1 week.Triptorelin microneedles were incubated in phosphate-buffered saline（PBS;pH 7.4）to determine the release behavior of drug in vitro.The cumulative drug release decreased with the increase ofβ-sheet content in the microneedles.Triptorelin microneedles prepared by 8%silk fibroin concentration in annealing using methanol vapor for 24 h showed a more obvious long-term sustained-release effect.Release data were analyzed and fitted to establish the relationship between β-sheet content and diffusion coefficient.The higher the β-sheet content in microneedles,the slower the diffusion rate of triptorelin.Our results indicated that the release behavior of triptorelin from microneedles could be predicted and adjusted by controlling the β-sheet content.Transdermal release of triptorelin from microneedles was monitored with microneedles piercing into porcine skin.Approximately 30%of triptorelin was released into PBS,indicating that the triptorelin microneedles could improve the skin permeability compared to membranes.4.Pharmacokinetic and pharmacodynamic evaluation of sustained-release microneedlesA rapid,sensitive,and selective method for simultaneous determination of triptorelin and testosterone in rat plasma by liquid chromatography-tandem mass spectrometry（LC-MS/MS）was developed using leuprolide and testosterone-¹³C as internal standards.Endogenous testosterone was determined by reference to a calibration curve prepared using testosterone-d₃ as a surrogate analyte.ESI source was operated in positive ionization mode and nitrogen was used as nebulization and desolvation gas.Curtain gas,gas 1,and gas 2 flows were 40,55,and 55 arbitrary units,respectively.The ionspray voltage was 5500 V and source temperature was kept at 550℃.Protein precipitation was used for sample preparation,and an Shimadzu Shim-pack XR-ODSⅢcolumn（1.6μm,75×2.0 mm）was applied for chromatographic separation.The mobile phase was a water-methanol mixture consisting of 0.01%formic acid,with a flow rate of 0.4 m L/min.The method was fully validated in terms of selectivity,linearity,precision,accuracy,extraction recovery,and stability according to the US Food and Drug Administration guidelines for the determination of biological samples.According to the guiding principles of GnRH agonist clinical trial design,healthy rats were employed as animal models for pharmacokinetic evaluation of sustained-release microneedles,testosterone levels were selected as pharmacodynamic indicators for the treatment of prostate cancer,and the levels of luteinizing hormone（LH）and estradiol（E2）were selected as pharmacodynamic indicators for the treatment of central precocious puberty and gynecological diseases.Triptorelin microneedles extended the half-life of triptorelin in male rats compared with that obtained by subcutaneous injection.Triptorelin was released continuously from microneedles into the plasma until 8 d,indicating that the triptorelin microneedles have a long-lasting sustained-release effect.In the pharmacodynamic study,triptorelin microneedles exhibited long-term testosterone inhibition and maintained castration levels for≥7 d.The levels of LH and E2 in the plasma of microneedles-treated rats at 7 d were significantly lower than normal physiological levels,indicating that microneedles have a down-regulation effect on sex hormones.5.Preparation of nanoparticle microneedles and evaluation of drug release performance in vivo and in vitroTriptorelin nanoparticles were prepared using triptorelin incorporation to increase the β-sheet content of silk fibroin.The well-dispersed nanoparticles size was～200 nm and the surface of nanoparticles showed a slightly negative charge（～-4.82 mV）.Measurement of the amount of free drug in supernatants revealed triptorelin to be encapsulated in nanoparticles with a high encapsulation efficiency of～95%.The nanoparticles exhibited sustained release in vitro and protected the drug from degradation by peptidases in skin interstitial fluid.The micromolding method was employed to prepare nanoparticles microneedles（NPs-MNs）.Each microneedle was fabricated into a cone of height～600μm and basic width of～300μm.The β-sheet content in the secondary structure of NPs-MNs increased compared with blank microneedles（BK-MNs）.The increased β-sheet content in the conformation ensured that NPs-MNs had good mechanical properties to pierce the stratum corneum.Transdermal release of triptorelin was increased to～65%indicating that the NPs-MNs could improve the skin permeability of triptorelin.Triptorelin was released continuously into the plasma of NPs-MNs-treated rats until 48 h and the relative bioavailability of NPs-MNs was increased to 180%.Administration of NPs-MNs increased the secretion of LH and E2 in the plasma of female rats initially,followed by prolonged inhibition of secretion of LH and E2,indicating that NPs-MNs have a therapeutic effect.Two types of silk fibroin-based bilayer composite microneedles,sustained-release microneedles and nanoparticle microneedles,were developed for transdermal delivery of triptorelin.Triptorelin was wrapped in sustained-release microneedles with a high drug loading.Moreover,due to the sustained-release effect of silk fibroin treated with methanol vapor,sustained-release microneedles can meet the needs of long-term medication for the treatment of hormone-dependent diseases.Triptorelin was encapsulated in nanoparticles to control the release and avoid the degradation of triptorelin by enzymes in the skin.By improving the relative bioavailability of drugs and reducing the dosage,nanoparticle microneedles can provide safe and convenient drug administration to pregnant women using assisted reproductive technology.The favorable mechanical properties,insertion properties,degradation properties,skin permeability,and therapeutic effects in vivo render the microneedle-mediated triptorelin delivery system as a potential strategy for the industrial scale-up and broad application.