RESUMEN

Poloxamer 407 (P407) is used as a safety-guaranteed, invaluable pharmaceutical nanocarrier. The aqueous solution of P407 exhibits sol-to-gel and gel-to-sol transitions, specifically during a temperature rise. Here, we develop a method to determine the pair potential between colloidal particles based primarily on experimental small-angle scattering data. Using this approach, the pair potential between the P407 micelles in the sol-gel-sol transition state is revealed without prelimiting any type of interaction forces. The results indicate that the increase in the attractive interaction contributes to enhancing the volume fraction, which is the decisive parameter for the gelation in terms of the Alder transition theory, i.e., the fluid-to-crystal phase transition of the hard-sphere model in the micelle system. This study demonstrates that one of the key mechanisms of the gel-to-sol transition upon heating is the enhancement of the structural fluctuation due to widening of the potential well in the intermicellar pair potential.

RESUMEN

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.

Asunto(s)

RESUMEN

Poloxamer hydrogel possesses thermosensitive sol-gel transition characteristics and is widely used as a drug-controlled-release carrier for topical or injectable formulations. In this study, the effect of loading of a drug, acetaminophen (ACE), on the physical and structural properties of poloxamer 407 (P407) micelles and hydrogels was investigated. Differential scanning calorimetry measurements revealed that ACE reduced the critical micelle temperature and enthalpy of micellization of P407 solutions. The P407 micellization was promoted by ACE incorporation. Rheometry showed that ACE increased the sol-gel transition temperature and reduced the gel strength of P407. In situ small-angle X-ray scattering (SAXS) using synchrotron radiation revealed that ACE altered the structure of P407 micelles and their packing in the P407 gels. As ACE concentration increased, the P407 micelle packing changed from a face-centered cubic phase to a body-centered cubic phase. Furthermore, ACE disordered the micelle packing structure and induced the formation of an amorphous phase. Structural analysis of the P407 micelle packing indicated that ACE reduced the aggregation number (Nagg) of P407 micelles in the gels. The SAXS study for diluted P407 solutions revealed that ACE reduced the P407 micelle size and its uniformity. The structural changes in P407 micelles by ACE loading (e.g., the reduction of Nagg, size, and size uniformity) would alter the micelle packing structure. It was found that these structural changes of micelle packing, especially the formation of an amorphous phase, could destabilize the P407 gel. As a result, the physical properties of P407 gels, such as gelation temperature and gel strength, were changed. This relationship between the structure and physical property of drug-loaded P407 gels was well-explained by correlating the micelle and gel structures. The mechanistic understanding of the change in the physical properties of P407 gels by drug loading is essential for the effective development of poloxamer gel formulations.

RESUMEN

This study aimed to investigate the impact of amorphous solubility and colloidal drug-rich droplets on drug absorption. The amorphous solubility of cilnidipine (CND) in AS-HF grade of hypromellose acetate succinate (HPMC-AS) solution was significantly reduced compared to that in non-polymer solution due to AS-HF partitioning into the CND-rich phase. In contrast, AS-LF grade of HPMC-AS has minimal effect on the amorphous solubility. The size of colloidal CND-rich droplets formed in the CND-supersaturated solution was less than 100 nm in the presence of AS-HF, while 200-450 nm in the presence of AS-LF. When the CND concentrations were near the amorphous solubility, CND membrane flux was reduced in the presence of AS-HF due to the decrease in the amorphous solubility of CND. However, the CND flux increased with the increase in CND-rich droplets, especially in the AS-HF solution. The size reduction of the CND-rich droplets led to their effective diffusion into the unstirred water layer, enhancing CND flux. In higher CND concentration regions, the CND flux became higher in the AS-HF solution than in the AS-LF solution. Thus, it is essential to elucidate the drug concentration-dependent impact of the colloidal drug-rich droplets on the drug absorption performance to optimize supersaturating formulations.

RESUMEN

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.

Asunto(s)

RESUMEN

RNA vaccines are applicable to the treatment of various infectious diseases via the inducement of robust immune responses against target antigens by expressing antigen proteins in the human body. The delivery of messenger RNA by lipid nanoparticles (LNPs) has become a versatile drug delivery system used in the administration of RNA vaccines. LNPs are widely considered to possess adjuvant activity that induces a strong immune response. However, the properties of LNPs that contribute to their adjuvant activity continue to require clarification. To characterize the relationships between the lipid composition, particle morphology, and adjuvant activity of LNPs, the nanostructures of LNPs and their antibody production were evaluated. To simply compare the adjuvant activity of LNPs, empty LNPs were subcutaneously injected with recombinant proteins. Consistent with previous research, the presence of ionizable lipids was one of the determinant factors. Adjuvant activity was induced when a tiny cholesterol assembly (cholesterol-induced phase, ChiP) was formed according to the amount of cholesterol present. Moreover, adjuvant activity was diminished when the content of cholesterol was excessive. Thus, it is plausible that an intermediate structure of cholesterol (not in a crystalline-like state) in an intra-particle space could be closely related to the immunogenicity of LNPs.

RESUMEN

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.

Asunto(s)

RESUMEN

Encapsulation of active pharmaceutical ingredients (APIs) in confined spaces has been extensively explored as it dramatically alters the molecular dynamics and physical properties of the API. Herein, we explored the effect of encapsulation on the molecular dynamics and physical stability of a guest drug, salicylic acid (SA), confined in the intermolecular spaces of γ-cyclodextrin (γ-CD) and poly(ethylene glycol) (PEG)-based polypseudorotaxane (PPRX) structure. The sublimation tendency of SA encapsulated in three polymorphic forms of the γ-CD/PEG-based PPRX complex, monoclinic columnar (MC), hexagonal columnar (HC), and tetragonal columnar (TC), was investigated. The SA sublimation rate was decreased by 3.0-6.6-fold and varied in the order of MC form > HC form > TC form complex. The 13C and 1H magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectra and 13C spin-lattice relaxation time (T1) indicated that the encapsulated SA molecules existed as the monomeric form, and its molecular mobility increased in the order of MC form > HC form > TC form complex. In the complexes, a rapid chemical exchange between two dynamic states of SA (free and bound) was suggested, with varying adsorption/desorption rates accounting for its distinct molecular mobility. This adsorption/desorption process was influenced by proton exchange at the interaction site and interaction strength of SA in the complexes, as evidenced by 1H MAS spectra and temperature dependency of the 13C carbonyl chemical shift. A positive correlation between the molecular mobility of SA and its sublimation rate was established. Moreover, the molecular mobility of γ-CD and PEG in the complexes coincided with that of SA, which can be explained by fast guest-driven dynamics. This is the first report on the stability improvement of an API through complexation in polymorphic supramolecular host structures. The relationship between the molecular dynamics and physical properties of encapsulated API will aid in the rational design of drug delivery systems.

Asunto(s)

RESUMEN

Flavonoids often exhibit broad bioactivity but low solubility and bioavailability, limiting their practical applications. The transglycosylated materials α-glucosyl rutin (Rutin-G) and α-glucosyl hesperidin (Hsp-G) are known to enhance the dissolution of hydrophobic compounds, such as flavonoids and other polyphenols. In this study, the effects of these materials on flavone solubilization were investigated by probing their interactions with flavone in aqueous solutions. Rutin-G and Hsp-G prepared via solvent evaporation and spray-drying methods were evaluated for their ability to dissolve flavones. Rutin-G had a stronger flavone-solubilizing effect than Hsp-G in both types of composite particles. The origin of this difference in behavior was elucidated by small-angle X-ray scattering (SAXS) and nuclear magnetic resonance analyses. The different self-association structures of Rutin-G and Hsp-G were supported by SAXS analysis, which proved that Rutin-G formed polydisperse aggregates, whereas Hsp-G formed core-shell micelles. The observation of nuclear Overhauser effects (NOEs) between flavone and α-glucosyl materials suggested the existence of intermolecular hydrophobic interactions. However, flavone interacted with different regions of Rutin-G and Hsp-G. In particular, NOE correlations were observed between the protons of flavone and the α-glucosyl protons of Rutin-G. The different molecular association states of Rutin-G or Hsp-G could be responsible for their different effects on the solubility of flavone. A better understanding of the mechanism of flavone solubility enhancement via association with α-glucosyl materials would permit the application of α-glucosyl materials to the solubilization of other hydrophobic compounds including polyphenols such as flavonoids.

Asunto(s)

RESUMEN

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

Asunto(s)

RESUMEN

Coamorphous formulation is a useful approach for enhancing the solubility of poorly water-soluble drugs via intermolecular interactions. In this study, a hydrogen-bonding-based coamorphous system was developed to improve drug solubility, but it barely changed the apparent permeability (Papp) of the drug. This study aimed to design a novel coamorphous salt using ionic interactions to improve drug permeability and absorption. Telmisartan (TMS), with an acidic group, was used to form a coamorphous salt with basic amlodipine (AML). Evaluation of the physicochemical properties confirmed the formation of a coamorphous salt via ionic interactions between the amine group of AML and the carboxyl group of TMS at a molar ratio of 1:1. The coamorphous salt of TMS/AML enhanced the partitioning of both drugs into octanol, indicating increased lipophilicity owing to the interaction between TMS and AML. The coamorphous salt dramatically enhanced TMS solubility (99.8 times that of untreated TMS) and decreased AML solubility owing to the interaction between TMS and AML. Although the coamorphous salt showed a decreased Papp in the permeation study in the presence of a thicker unstirred water layer (UWL) without stirring, Papp increased in the presence of a thinner UWL with stirring. The oral absorption of TMS from the coamorphous salt increased by up to 4.1 times compared to that of untreated TMS, whereas that of AML remained unchanged. Although the coamorphous salt with increased lipophilicity has a disadvantage in terms of diffusion through the UWL, the UWL is thin in human/animal bodies owing to the peristaltic action of the digestive tract. Dissociation of the coamorphous salt on the membrane surface could contribute to the partitioning of the neutral form of drugs to the membrane cells compared with untreated drugs. As a result, coamorphous salt formation has the advantage of improving the membrane permeation and oral absorption of TMS, owing to the enhanced solubility and supply of membrane-permeable free TMS on the surface of the membrane.

Asunto(s)

RESUMEN

In our previous reports, ternary amorphous solid dispersions (ASDs) of probucol (PBC)/polymer/surfactant were prepared by spray-drying and cryo-grinding, and colloidal dispersions of amorphous PBC nanoparticles were obtained by dispersing the ternary ASD into water. In this study, hot-melt extrusion, which is a practical method for preparing ASD formulations, was utilized to obtain ternary ASDs and colloidal dispersions of amorphous PBC nanoparticles. Polyvinylpyrrolidone K12 (PVP) with a relatively low Tg (below 100 °C) was used as a polymer, while poloxamer P407 (P407), which is chemically stable during the hot-melt extrusion process, was utilized as a surfactant. Ternary ASDs were successfully produced with high weight ratios of PVP and P407. A hydrogen bond between the PBC hydroxyl proton and PVP carbonyl oxygen in the ternary ASD was detected using solid-state NMR spectroscopy, suggesting that amorphous PBC was stabilized mainly by PVP. Stable colloidal dispersions of amorphous PBC nanoparticles were obtained from the PBC/PVP/P407 ASD at a weight ratio of 1:4:2. The mean particle size was below 200 nm and the amorphous state of PBC remained stable upon storage at 25 °C for 14 d. Solution-state 1H NMR and zeta-potential measurements suggested that P407 mainly stabilized the colloidal dispersion of amorphous PBC nanoparticles by steric hindrance at the solid/liquid interface. The findings of this study demonstrate that hot-melt extrusion can form practical ternary ASDs that provide colloidal dispersion of amorphous drug nanoparticles. Thus, this study advocates for the use of hot-melt extrusion in the design of an amorphous formulation for a variety of poorly water-soluble drugs.

Asunto(s)

RESUMEN

This study utilized temperature-variable nuclear magnetic resonance (NMR) spectroscopy to investigate the effects of a solubilizing agent on the ketoprofen (KTP) supersaturation region. Quantitative NMR analysis showed that the solubilizing agent cetyltrimethylammonium bromide (CTAB) increased both the crystalline and amorphous solubilities of KTP, shifting the KTP supersaturation region to a higher KTP concentration range. The amorphous solubility of KTP was found to be independent of the enantiomeric composition of KTP, even in the presence of CTAB. However, the supersaturation region of the S-enantiomer of KTP (s-KTP) in CTAB solutions was smaller than that of the racemic form of KTP (rac-KTP), likely because of the higher crystalline solubility of s-KTP. When KTP formed a KTP-rich phase via liquid-liquid phase separation from KTP-supersaturated solutions, CTAB was observed to be distributed into the KTP-rich phase, decreasing the chemical potential of KTP and the maximum thermodynamic activity of KTP in the aqueous phase. Additionally, the incorporation of CTAB into the KTP-rich phase diminished the solubilization effect of CTAB micelles in the aqueous phase, narrowing the KTP supersaturation region to a greater extent at higher KTP dose concentrations. Furthermore, the upper-temperature limit of the supersaturated dissolvable region of KTP was lowered in the presence of CTAB, which was rationalized by the melting point depression of the KTP crystal upon mixing with CTAB. The findings of this study highlight the importance of considering the molecular-level impact of solubilizing agents on the drug supersaturation region to fully exploit the potential benefits of supersaturated formulations.

Asunto(s)

RESUMEN

We examined the effects of the polymer-additive and drug chiralities on the ketoprofen (KTP) supersaturation region using temperature-variable nuclear magnetic resonance (NMR). Quantitative NMR analysis revealed that the racemic KTP and corresponding S-enantiomer (rac- and s-KTP) exhibited similar amorphous solubilities in a buffer, while the crystalline solubility of s-KTP was higher than that of rac-KTP. Therefore, rac-KTP exhibited a larger supersaturation region than s-KTP. In contrast, polyvinylpyrrolidone (PVP) reduced the amorphous solubility of both rac- and s-KTP, whereas the crystalline solubility of KTP remained unchanged. Partitioning PVP into the KTP-rich phase reduced the chemical potential of KTP in the KTP-rich phase and the amorphous solubility of KTP. At higher temperatures, the distribution of PVP into the KTP-rich phase became more significant, which considerably reduced the amorphous solubility. Because the upper limit of the KTP supersaturation decreased, PVP narrowed the KTP supersaturation region. The maximum KTP supersaturation ratio decreased with increasing temperature, and the supersaturated dissolvable area of KTP finally disappeared. The maximum temperature at which KTP can form the supersaturation was lowered by replacing rac- with s-KTP and the addition of PVP. The maximum supersaturation temperature was dominated by the melting behavior of crystalline KTP in an aqueous solution. The present study highlighted that a quantitative understanding of the supersaturation region is essential to determine whether supersaturated formulations are beneficial for improving the oral absorption of poorly water-soluble drugs.

Asunto(s)

RESUMEN

Cyclodextrin (CD) is used to solubilize poorly water-soluble drugs by inclusion complex formation. In this study, we investigated the effect of CD derivatives on stabilizing the supersaturation by inhibiting the crystallization of two poorly water-soluble drugs, carvedilol (CVD) and chlorthalidone (CLT). The phase solubility test showed that ß-CD and γ-CD derivatives enhanced the solubility of CVD to a greater extent, whereas the solubility of CLT was enhanced more by ß-CD derivatives. The solubilization efficacy of CD derivatives was dependent on the size fitness between the drug molecule and the CD cavity. In the drug crystallization induction time measurement, the same initial drug supersaturation ratio (S) was employed in all the CD solutions, and the methylated CD derivatives greatly outperformed unmethylated CD derivatives in stabilizing the supersaturation of both CVD and CLT. The crystallization inhibition strength of CD derivatives was strongly affected by the CD derivative substituent. Moreover, the calculated logarithm of octanol/water partition coefficients (log P) of CD derivatives showed a good correlation with drug crystallization inhibition ability. Thus, the high hydrophobicity of methylated CD plays an essential role in inhibiting crystallization. These findings can provide a valuable guide for selecting appropriate stabilizing agents for drug-supersaturation formulations.

Asunto(s)

RESUMEN

Based on the clinical success of an in vitro transcribed mRNA (IVT-mRNA) that is encapsulated in lipid nanoparticles (mRNA-LNPs), there is a growing demand by researchers to test whether their own biological findings might be applicable for use in mRNA-based therapeutics. However, the equipment and/or know-how required for manufacturing such nanoparticles is often inaccessible. To encourage more innovation in mRNA therapeutics, a simple method for preparing mRNA-LNPs is prerequisite. In this study, we report on a method for encapsulating IVT-mRNA into LNPs by rehydrating a Ready-to-Use empty freeze-dried LNP (LNPs(RtoU)) formulation with IVT-mRNA solution followed by heating. The resulting mRNA-LNPs(RtoU) had a similar intraparticle structure compared to the mRNA-LNPs prepared by conventional microfluidic mixing. In vivo genome editing, a promising application of these types of mRNA-LNPs, was accomplished using the LNPs(RtoU) containing co-encapsulated Cas9-mRNA and a small guide RNA.

Asunto(s)

RESUMEN

We previously established a nanoparticle-based drug delivery system (DDS) for high-dose ascorbic acid therapy by self-assembly of a lipid-modified ascorbic acid derivative, L-ascorbyl 2,6-dipalmitate (ASC-DP). The particles' morphology should be modified for effective DDSs. Here, we modulated the morphology of self-assembled ASC-DP nanoparticles using two different PEGylated lipids, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and cholesterol-polyethylene glycol (Chol-PEG), with various PEG molecular weights. At the preparation molar ratio of 10 : 1 (ASC-DP/PEGylated lipid), rod-like nanoparticles emerged in the ASC-DP/DSPE-PEG system, whereas the ASC-DP/Chol-PEG system yielded tube-like nanoparticles. The internal structures of both rod-like ASC-DP/DSPE-PEG and tube-like ASC-DP/Chol-PEG nanoparticles were similar to that of repeated ASC-DP bilayers. The particles' surfaces featured PEGylated lipids, which stabilized the structure and dispersion of the nanoparticles. For both systems, the particle size increased slightly with increasing the PEGylated lipid's PEG molecular weight. Increasing the PEG molecular weight decreased the inner tunnel size of tube-like ASC-DP/Chol-PEG nanoparticles. A mechanism has been proposed for the rod-to-tube transformation. Surface-layer free-energy changes owing to the mixing of multiple lipids and PEG chain repulsion are thought to underlie the inner tunnels' formation. The rod-to-tube morphology of self-assembled ASC-DP nanoparticles can be modulated by controlling the PEGylated lipids' structure, including the lipid species and the PEG chain length.

Asunto(s)

RESUMEN

A mixture of poly(benzyl methacrylate) (PBnMA) and 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)amide ([C2mim][NTf2]) exhibits lower-critical-solution-temperature (LCST)-type phase separation. An investigation combining magic-angle spinning NMR spectroscopy and small-angle scattering was performed to gain new insights into the interaction between PBnMA and the ionic liquid. The molecular mobility and the solute-solvent interaction in the system were investigated using 1H high-resolution magic-angle spinning NMR. Applying a magic-angle spinning frequency of 2 kHz allowed identifying the PBnMA peaks, which were not observed by conventional solution-state NMR. The peaks of [C2mim]+ almost coincided in the presence and absence of PBnMA, indicating the decoupling of the bulk solvent and polymer. The conformational state of PBnMA in [C2mim][NTf2] was investigated using small-angle X-ray scattering (SAXS). The pair distribution functions of PBnMA chains calculated from SAXS profiles suggest that PBnMA adopts a random coil conformation upon dissolution in [C2mim][NTf2]. The combined study clarifies the decoupled low mobility of polymers with a random coil conformation. It is considered that the specific decoupled low mobility is one of the origins of the decoupling conductivity of [C2mim][NTf2] in a matrix polymer. In addition, an increase in temperature induced a downfield shift and broadening of the [C2mim]+ peaks, suggesting that a larger amount of [C2mim]+ was bound to the PBnMA chains even at temperatures approaching the LCST.

Asunto(s)

RESUMEN

Myofibroblast-like activated hepatic stellate cells (aHSCs), which produce collagen, a major cause of liver fibrosis, are specific target cells for antifibrotic treatment. Recently, several reports have indicated that extracellular vesicles (EVs) play important roles in cell-to-cell communication through their tropism for specific cells or organs. Therefore, the present study aimed to identify aHSC-directed EVs by focusing on cell-to-cell interactions in the liver under pathological conditions. EVs were derived from the hepatocyte cell line AML12 treated with or without palmitic acid (PA) and evaluated for their physical properties and uptake by the aHSC cell line LX-2. AML12-derived EVs had a mean particle diameter of 110-130 nm, negative charge, and expressed the exosomal makers CD9 and CD63. PA-treated AML12 cells released larger EVs with higher protein levels than those without PA treatment. The intracellular uptake efficacy of EVs derived from PA-treated AML12 cells into activated LX-2 cells was significantly higher than those without PA treatment. Our study revealed that PA treatment induces hepatocytes to release EVs with aHSC-tropism. These findings may contribute to the development of an EV-based drug delivery system (DDS) for aHSC-targeted therapy and provide new insights into the role of steatotic hepatocyte-derived EVs in physiological or pathophysiological functions.

RESUMEN

Herein, we investigated the effect of the solubilizers, cetyltrimethylammonium bromide (CTAB) and amino methacrylate copolymer (Eudragit E PO, EUD-E), on the apparent amorphous solubility of ketoprofen (KTP) and free KTP concentrations in an aqueous phase when a KTP-rich phase was generated by liquid-liquid phase separation. Quantitative analysis by solution nuclear magnetic resonance (NMR) revealed that the apparent amorphous solubility of KTP increased with increasing EUD-E concentrations by the solubilization of KTP into the EUD-E micelles; this was reminiscent of the improvement in the apparent crystalline solubility of KTP observed when EUD-E was added. In contrast, the apparent amorphous solubility of KTP decreased with increasing CTAB concentrations, although the solubilizing ability of CTAB was stronger than that of EUD-E when the KTP-rich phase was absent. NMR analysis revealed that CTAB was distributed into the KTP-rich phase to a relatively large extent. This resulted in a significant reduction of the chemical potential of KTP in the KTP-rich phase in the CTAB solution. Thus, the maximum free KTP concentration in the aqueous phase was reduced more significantly in the CTAB solution than in the EUD-E solution. Moreover, the solubilization effect of KTP by the CTAB micelles in the aqueous phase was drastically diminished due to the distribution of CTAB into the KTP-rich phase. As a result, the apparent amorphous solubility of KTP reached a minimum at a CTAB concentration of 200 µg/mL. A further increase in the CTAB concentration resulted in an improvement in the apparent amorphous solubility of KTP due to the solubilization effect of CTAB remaining in the aqueous phase. The present study highlights the impact of solubilizer selection on the apparent amorphous solubility and attainable supersaturation of the drug, which should be considered during the development of supersaturating formulations to obtain preferable oral absorption.

Asunto(s)