Preparation Of Magnetic Silk Fibroin Nanoparticles In A Supercritical Process And Combination With Magnetophoresis For Transdermal Drug Delivery System

Transdermal drug delivery system has some advantages in avoiding the hepatic first-pass metabolism, maintaining a constant and prolonged drug level, providing a local target and convenience for administration, which can reduce adverse reaction and improve the therapeutic index and patient compliance. However, the stratum corneum, as one of the main skin barrier, limits the percutaneous penetration of a number of molecules. In order to disrupt the barrier function of the stratum corneum, using nanoparticles?NPs? as a drug carrier, has become an effective way to promote drug percutaneous penetration. Meanwhile, magnetophoresis, as an active penetration enhancement technique can improve the drug permeation effectively. In this study, methotrexate?MTX? was selected as a drug model and silk fibrin?SF? was used as a drug carrier, the MTX-SF NPs and MTX-Fe₃O₄-SF NPs were prepared by suspension-enhanced dispersion by supercritical CO2?Sp EDS? to study their effects in promoting drug percutaneous penetration, and then the MTX-Fe₃O₄-SF NPs and magnetic field were combined to investigate the effect of magnetic field to drug permeation across the skin. The mechanism of penetration enhancement of magnetic field was discussed. The cytotoxicity and skin irritation of the drug carrier were also preliminarily investigated.?1? The SF NPs and Fe₃O₄-SF NPs were prepared by Sp EDS, and their morphology, particle size and distribution were characterized. The results showed that when the ratio of SF/Fe₃O₄ was 20:3, nanoparticles had a good sphericity and uniform particle size. The resulting MTX-SF NPs and MTX-Fe₃O₄-SF NPs had a small particle size?<100 nm? and a narrow particle size distribution. FTIR spectra measurement indicated a successful loading of MTX into SF NPs. The nanoparticles had a high drug load and encapsulation efficiency. And the encapsulation efficiency tended to become lower when the theoretical drug load was increased. Compared with the raw drugs, the drug-loaded nanoparticles eshibited a better sustained-release profile.?2? In vitro permeation data showed that after administration for 24 h, the MTX-SF NPs formulation significantly promoted the percutaneous permeation amount of MTX?89.20±1.86 ?g/cm2?, which was 3.33 times of MTX formulation?26.76±2.92 ?g/cm2?. The confocal microscope images indicated that the fluorescence was detected in dermis by applying nanoparticles while the fluorescence signal was only observed on the surface of skin at the control.?3? The percutaneous permeation effect of stationary magnetic field combined with MTX-Fe₃O₄-SF NPs was studied. The results showed that MTX-Fe₃O₄-SF NPs formulation in the magnetic field obviously promoted the permeation amount of MTX?91.35±2.01 ?g/cm2?, compared to MTX-Fe₃O₄-SF NPs formulation?67.87±3.08 ?g/cm2? without the magnetic field. The permeation study by pretreatment of skin and ATR-FTIR analysis demonstrated that the mechnism of stationary magnetic field is magnetokinesis but not effecting the structure of SC or inducing the pore of skin.?4? The percutaneous permeation effect of alternating magnetic field combined with MTX-Fe₃O₄-SF NPs was investigated. The results clearly demonstrated the capability of the magnetic field to enhance the percutaneous delivery of MTX. The application of different kinds of magnetic fields all obtained a higher cumulative amount of MTX penetration than the passive administration. The cumulative amount of MTX penetrating the skin to the receptor compartment was 11.37±0.55 ?g/cm2 for the passive transdermal drug delivery while the permeation amount was found to increase gradually when employing a stationary magnetic field?13.83±0.85 ?g/cm2?, an alternating magnetic field?19.08±2.67 ?g/cm2?, and a stationary/alternating magnetic field?27.39±1.70 ?g/cm2?. The alternating magnetic field enhanced the permeation of MTX across the skin much more effectively than the stationary magnetic field. This reveals that the driving force in the horizontal direction for the drug-loaded magnetic nanoparticles is more favorable for the permeation of MTX across the skin than the driving force in the vertical direction. Importantly, the combination of stationary and alternating magnetic fields, which can generate a massage-like driving force in both horizontal and vertical directions, achieved the best enhancement for the permeation of MTX across the skin. The combination of stationary and alternating magnetic fields achieves a synergistic effect to enhance the drug permeation via the simulated massage of the skin.The result of CLSM showed that the stationary magnetic field generated a stronger fluorescence signal than the control group without any magnetic field, which means that more of the drug penetrated across the skin. In the case of the alternating magnetic field or stationary/alternating magnetic field, a fluorescence signal with a bigger area was detected in deeper regions of the skin. It is suggested that the alternating magnetic field can drive the movement of magnetic nanoparticles on the surface of the skin, thus enhancing the follicular penetration.The result of ATR-FTIR revealed that the skin subjected to a stationary magnetic field shows a similar spectrum to the control. However, after exposure to an alternating magnetic field or stationary/alternating magnetic fields, the symmetric and asymmetric methylene stretching bands were shifted to lower values. This result indicates that there was a lipid order-disorder transition from the gel to the lipid-crystalline state. This transition can increase the fluidity of the SC lipids, which could increase the skin penetration.?5? The cytotoxicity test was carried out by co-culturing HES cells with extract of SF NPs and Fe₃O₄-SF NPs. Cytotoxic activity measured by Alamar Blue method concluded that SF NPs and Fe₃O₄-SF NPs possessed a good biocompatibility and no cellular toxicity. The irritation test demonstrated that the SF NPs and Fe₃O₄-SF NPs had a mild irritation. The results indicated that SF NPs and Fe₃O₄-SF NPs have potential for transdermal drug delivery system.