森部 久仁一

This study aimed to assess the applicability of solution-state 1H NMR for molecular-level characterization of siRNA-loaded lipid nanoparticles (LNP). Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, MC3) was used as an ionizable lipid, and siRNA-loaded LNPs were prepared by pre-mixing and post-mixing methods. The pre-mixing method involved mixing an acidic solution containing siRNA with an ethanolic lipid solution using a microfluidic mixer. The pre-mixed LNP was prepared by dialyzing the mixed solution into the phosphate buffered saline (PBS, pH 7.4). The post-mixed LNP was prepared by mixing the siRNA solution with empty LNP in an acidic condition with and without ethanol, resulting in post-mixed LNP (A) and (B), respectively. Both pre-mixed and post-mixed LNPs formed LNP particles with an average diameter of approximately 50 nm. Moreover, the ratio of encapsulated siRNA to lipid content in each LNP particle remained constant regardless of the preparation method. However, small-angle X-ray scattering measurements indicated structural variations in the siRNA-MC3 stacked bilayer structure formed in the LNPs, depending on the preparation method. Solution-state 1H NMR analysis suggested that the siRNA was incorporated uniformly into the LNP core for pre-mixed LNP compared to post-mixed LNPs. In contrast, the post-mixed LNPs contained siRNA-empty regions with local enrichment of siRNA in the LNP core. This heterogeneity was more pronounced in post-mixed LNP (B) than in post-mixed LNP (A), suggesting that ethanol facilitated the homogeneous mixing of siRNA with LNP lipids. The silencing effect of each siRNA-loaded LNP was reduced in the order of pre-mixed LNP, post-mixed LNP (A), and post-mixed LNP (B). This suggested that the heterogeneity of the siRNA-loaded LNP could cause a reduction in the silencing effect of the incorporated siRNA inside LNPs. The present study highlighted that NMR-based characterization of siRNA-loaded LNP can reveal the molecular-level heterogeneity of siRNA-loaded LNP, which helps to optimize the preparation conditions of siRNA-loaded LNP formulations.

Drug-rich droplets formed through liquid-liquid phase separation (LLPS) have the potential to enhance the oral absorption of drugs. This can be attributed to the diffusion of these droplets into the unstirred water layer (UWL) of the gastrointestinal tract and their reservoir effects on maintaining drug supersaturation. However, a quantitative understanding of the effect of drug-rich droplets on intestinal drug absorption is still lacking. In this study, the enhancement of intestinal drug absorption through the formation of drug-rich droplets was quantitatively evaluated on a mechanistic basis. To obtain fenofibrate (FFB)-rich droplets, an amorphous solid dispersion (ASD) of FFB/hypromellose (HPMC) was dispersed in an aqueous medium. Physicochemical characterization confirmed the presence of nanosized FFB-rich droplets in the supercooled liquid state within the FFB/HPMC ASD dispersion. An in situ single-pass intestinal perfusion (SPIP) assay in rats demonstrated that increased quantities of FFB-rich nanodroplets enhanced the intestinal absorption of FFB. The effective diffusion of FFB-rich nanodroplets through UWL would partially contribute to the improved FFB absorption. Additionally, confocal laser scanning microscopy (CLSM) of cross sections of the rat intestine after the administration of fluorescently labeled FFB-rich nanodroplets showed that these nanodroplets were directly taken up by small intestinal epithelial cells. Therefore, the direct uptake of drug-rich nanodroplets by the small intestine is a potential mechanism for improving FFB absorption in the intestine. To quantitatively evaluate the impact of FFB-rich droplets on the FFB absorption enhancement, we determined the apparent permeabilities of the FFB-rich nanodroplets and dissolved FFB based on the SPIP results. The apparent permeability of the FFB-rich nanodroplets was 110-130 times lower than that of dissolved FFB. However, when the FFB-rich nanodroplet concentration was several hundred times higher than that of dissolved FFB, the FFB-rich nanodroplets contributed significantly to FFB absorption improvement. The present study highlights that drug-rich nanodroplets play a direct role in enhancing drug absorption in the gastrointestinal tract, indicating their potential for further improvement of oral absorption from ASD formulations.

In this study, we investigated the mechanism of curcumin (CUR) release from poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) nanoparticles (NPs) by evaluating the temperature-dependent CUR release. NPs were prepared by the nanoprecipitation method using various PLGA/PLA polymers with different lactic:glycolic ratios (L:G ratios) and molecular weights. Increasing the polymer molecular weight resulted in a decrease in the particle size of NPs. The wet glass transition temperature (Tg) of PLGA/PLA NPs was lower than the intrinsic polymer Tg, which can be derived from the water absorption and nanosizing of the polymer. The reduction in Tg was more significant for the PLGA/PLA NPs with lower polymer L:G ratios and lower polymer molecular weight. The greater decrease of Tg in the lower polymer L:G ratios was possibly caused by the higher water absorption due to the more hydrophilic nature of the glycolic acid segment than that of the lactic acid segment. The efficient water absorption in PLGA/PLA NPs with lower molecular weight could cause a significant reduction of Tg as it has lower hydrophobicity. CUR release tests from the PLGA/PLA NPs exhibited enhanced CUR release with increasing temperatures, irrespective of polymer species. By fitting the CUR release profiles into mathematical models, the CUR release process was well described by an initial burst release followed by a diffusion-controlled release. The wet Tg and particle size of the PLGA/PLA NPs affected the amount and temperature dependence of the initial burst release of CUR. Above the wet Tg of NPs, the initial burst release of CUR increased sharply. Smaller particle sizes of PLGA/PLA NPs led to a higher fraction of initial CUR burst release, which was more pronounced above the wet Tg of NPs. The wet Tg and particle sizes of the PLGA/PLA NPs also influenced the diffusion-controlled CUR release. The diffusion rate of CUR in the NPs increased as the wet Tg values of the NPs decreased. The diffusion path length of CUR was affected by the particle size, with larger particle size resulting in a prolonged diffusion-controlled release of CUR. This study highlighted that for the formulation development of PLGA/PLA NPs, suitable PLGA/PLA polymers should be selected considering the physicochemical properties of PLGA/PLA NPs and their correlation with the release behavior of encapsulated drugs at the application temperature.