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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.
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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.
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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.
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ROS are involved in the pathogenesis of bullous pemphigoid (BP), but this involvement has not been fully elucidated. In this study, to further elucidate the pathogenic role of ROS in BP, we examined the results of the diacron-reactive oxygen metabolite test and the biological antioxidant potential test for 16 patients with BP who visited our hospital before being treated with systemic corticosteroids. In the patients with BP, the average diacron-reactive oxygen metabolite levels, expressed in Carratelli units, were significantly reduced at 1 month of treatment (from 335.6 ± 40.3 Carratelli units to 224.7 ± 61.6 Carratelli units, P < .001). Bullous Pemphigoid Disease Area Index (erosions/blisters) scores correlated with diacron-reactive oxygen metabolite levels (r = 0.51), suggesting that those levels reflect the disease severity. We also performed staining of 3,5-dibromotyrosine in skin tissues. The 3,5-dibromotyrosine is expected to be a marker of tissue damage related to inflammation and allergies. The 3,5-dibromotyrosine was stained in infiltrated cells around the dermis, throughout the blister fluid, and at the basement membrane within the blister. It is considered that tissue destruction caused by the myeloperoxidase released from neutrophils and by eosinophil peroxidase released from eosinophils is involved in blister formation. The results suggest that ROS play a role in BP.
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Bullous pemphigoid (BP), an autoimmune subepidermal blistering disease, shows tense blisters associated with urticarial erythema. Tissue-bound Immunoglobulin G (IgG) at the basement membrane zone (BMZ) detected by direct immunofluorescence (DIF) is strong evidence for a diagnosis of BP. The sensitivity of DIF is higher in complement component 3 (C3) than in IgG, but the reason for this different sensitivity is not fully understood. In this study, we performed several ex vivo studies to investigate the possible mechanism of IgG negativity and C3 positivity at the BMZ by DIF in some BP cases. First, sera from BP patients showing IgG negativity by DIF were found to clearly react to the BMZ in their own DIF skin samples. Next, indirect immunofluorescence (IIF) was performed using sera diluted with different pH phosphate-buffered saline (PBS), pH 7.4, 6.0, and 3.0. Patients' sera diluted with pH 7.4 PBS showed linear staining at the BMZ, but sera diluted with pH 6.0 PBS and pH 3.0 PBS showed lower fluorescence intensities. Finally, sections of skin from BP patients were pre-incubated with different pH PBS (pH 3.0, 6.0, and 7.4), followed by staining with anti-human IgG and C3. The fluorescence intensities were notably lower for IgG and C3 that had been pre-incubated with pH 3.0 PBS and pH 6.0 PBS than for IgG and C3 that had been pre-incubated with pH 7.4 PBS. These results suggest that a low pH condition hinders the binding of autoantibodies to the BMZ, that is, the drop in tissue pH induced by inflammation inhibits autoantibodies from depositing at the BMZ. Furthermore, the drop in tissue pH causes tissue-bound autoantibodies to detach from the BMZ. Complement fragments are activated not only on IgG but also on the cell surface of cells close to IgG during complement activation. IgG may detach from the BMZ under low pH condition induced by inflammation, but some complement fragments remain at the BMZ. These phenomena may help to explain why C3 is more sensitive than IgG when DIF is used to diagnose BP.
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Membrana Basal , Complemento C3 , Inmunoglobulina G , Penfigoide Ampolloso , Humanos , Membrana Basal/inmunología , Membrana Basal/metabolismo , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/metabolismo , Concentración de Iones de Hidrógeno , Penfigoide Ampolloso/inmunología , Penfigoide Ampolloso/diagnóstico , Penfigoide Ampolloso/patología , Complemento C3/inmunología , Complemento C3/metabolismo , Masculino , Femenino , Anciano , Autoanticuerpos/inmunología , Autoanticuerpos/sangre , Técnica del Anticuerpo Fluorescente Directa , Piel/inmunología , Piel/patología , Técnica del Anticuerpo Fluorescente Indirecta , Anciano de 80 o más Años , Persona de Mediana EdadRESUMEN
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.
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Fenofibrato , Separación de Fases , Ratas , Animales , Preparaciones Farmacéuticas , Fenofibrato/química , Absorción Intestinal , Intestinos , SolubilidadRESUMEN
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.
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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.
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Curcumina , Nanopartículas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Curcumina/química , Ácido Poliglicólico/química , Temperatura , Preparaciones de Acción Retardada , Glicoles , Poliésteres , Tamaño de la Partícula , Nanopartículas/química , AguaRESUMEN
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.
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Ciclodextrinas , Simulación de Dinámica Molecular , Poloxámero , Rotaxanos , Preparaciones Farmacéuticas , Ciclodextrinas/química , Espectroscopía de Resonancia Magnética , Ácido Salicílico/químicaRESUMEN
OBJECTIVE: To determine how caregivers describe dystonia in people with cerebral palsy (CP). METHODS: In this prospective cohort study, paper surveys were administered to caregivers between September 7, 2021 and October 28, 2021 during CP Center visits at a large tertiary care center. Caregivers were asked to describe involuntary movements triggered by voluntary movement or triggered by tactile stimulation in the people with CP they cared for. Their CP Center medical provider separately assessed people with CP for dystonia. Movement features described exclusively by caregivers of people with CP and dystonia were determined using conventional content analysis. RESULTS: 113 caregivers responded on behalf of 56 people with and 57 people without dystonia. If caregivers noted that both voluntary movement and tactile stimulation triggered involuntary movements, that had a 92% positive predictive value for a dystonia diagnosis. Movement features exclusively described in people with CP and dystonia included: (1) stiffening, tensing, or tightening (15% of respondents); (2) involvement of the head (10%), torso (5%), or feet (5%); and (3) triggers of stretching (12.5%), excitement (5%), or transfers (5%). INTERPRETATION: In addition to a thorough exam, asking caregivers of people with CP to describe involuntary movements triggered by voluntary movement or tactile stimulation may inform clinical dystonia diagnosis.
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Parálisis Cerebral , Distonía , Trastornos Distónicos , Humanos , Parálisis Cerebral/complicaciones , Distonía/diagnóstico , Distonía/etiología , Cuidadores , Estudios Prospectivos , Trastornos Distónicos/diagnósticoRESUMEN
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.
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Flavonas , Hesperidina , Hesperidina/química , Rutina/química , Difracción de Rayos X , Protones , Dispersión del Ángulo Pequeño , Flavonoides , Espectroscopía de Resonancia Magnética , SolubilidadRESUMEN
OBJECTIVE: : To examine activities of daily living (ADL) and physical activity in older adults with heart failure admitted to a rehabilitation ward for subacute musculoskeletal disease. METHODS: : This study included patients with musculoskeletal disease (aged ≥75 years) who were admitted to the rehabilitation ward. Data on age, ADL, and time for physical activity (metabolic equivalents [METs]) were collected. Patients were divided into groups with or without heart failure, and the differences were compared using Mann-Whitney U-test. RESULTS: : This study included 84 musculoskeletal patients, including 25 with heart failure. The heart-failure group had similar levels of ADL independence compared to the without-heart-failure group (p=0.28) but had shorter duration of continuous and sustained physical activities and less total time (p<0.01) of light-intensity physical activity or higher. CONCLUSION: : Older adults with subacute musculoskeletal disease with heart failure do not necessarily require a large amount of physical activity to maintain ADL at the time of discharge. But very low physical activity may increase the risk for developing hospitalization-associated disability. Physical activity in older adults with subacute musculoskeletal disease with heart failure should be monitored separately from ADL.
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Background and Objectives: Dystonia in cerebral palsy (CP) is debilitating and common, but underdiagnosed, especially when coexistent with spasticity. With dedicated research-based assessment, dystonia is found in most people with spastic CP but is only clinically diagnosed in the minority. To begin addressing the high rates of dystonia underdiagnosis in this population, we determined the key feature experts use to assess upper extremity dystonia in people with spastic CP. Methods: In this prospective cohort study, 3 pediatric movement disorder specialists assessed upper extremity dystonia in neurologic examination videos of people with spastic CP and isolated periventricular leukomalacia (PVL) on brain MRI (i.e., those with a brain injury pattern typical for spastic CP). Dystonia severity was rated using the 10-point Global Dystonia Severity Rating Scale, first by each expert independently and then again after consensus-building discussion. Conventional content analysis of these discussions revealed salient features ("codes") that experts used to assess upper extremity dystonia. Code frequency distributions were compared between dystonia severity categories using χ2 tests. Results: We identified 96 people with spastic CP with isolated PVL on brain MRI seen in the St. Louis Children's Hospital CP Center between 2005 and 2018. Of them, 26 people were able and willing to be recorded while doing a standardized set of upper extremity examination maneuvers (age 4-25 years; 28% nonambulatory, 77% White). When assessing their videos, experts cited the "hand" less often and "shoulder" more often with increasing dystonia severity (p < 0.005, χ2 test). "Mirror movements" and the "hand open/close" examination maneuver were cited significantly more frequently in videos when experts were attempting to distinguish between no dystonia and mild dystonia (p < 0.005). Discussion: Expert clinicians use distinct movement features to assess upper extremity dystonia in people with spastic CP and PVL. Attention to involuntary shoulder (vs hand) movements can help gauge dystonia severity. Differentiation between mirror movements and dystonia, particularly during the hand open/close examination maneuver, may help identify mild dystonia. These results can help guide upper extremity dystonia assessment in people with spastic CP, thus potentially helping mitigate dystonia underdiagnosis.
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This is the ninth yearly article in the Tourette Syndrome Research Highlights series, summarizing selected research reports from 2022 relevant to Tourette syndrome. The authors briefly summarize reports they consider most important or interesting.
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Síndrome de Tourette , HumanosRESUMEN
BACKGROUND: Dystonia in cerebral palsy (CP) is classically associated with deep gray matter injury at term gestation, but the patterns of injury associated with dystonia following premature birth are unclear. We examined whether there were brain regional size differences associated with dystonia in people with CP born premature. METHODS: In this retrospective cohort study, we identified subjects with CP born premature (<37 weeks gestational age) seen at a tertiary care CP center between February 1, 2017, to February 1, 2021, who had T1-weighted brain magnetic resonance imaging (MRI) done between ages one and five years available in the clinical record. We measured the following on these brain MRI images per the 2013 Kidokoro criteria: interhemispheric distance, biparietal width, lateral ventricle diameter, transcerebellar diameter, deep gray matter area, and corpus callosum thickness. We then compared the sizes of these structures between those with and without dystonia correcting for gestational age at birth and gross motor functional ability (univariate general linear models). RESULTS: Fifty-five subjects met the inclusion and exclusion criteria. Interhemispheric distance was significantly greater in those with dystonia, suggesting decreased cortical volume (P = 0.005). There was no significant difference in the other measured structures between those with and without dystonia, including deep gray matter area. CONCLUSIONS: Increased interhemispheric distance, not measures of deep gray matter size, correlate with the presence of dystonia in people with CP born premature.
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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.
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Imagen por Resonancia Magnética , Protones , ARN Interferente Pequeño , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
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.