RESUMEN
A phospholipid bilayer is a typical structure that serves crucial functions in various cells and organelles. However, it is not unusual for it to take part in pathological processes. The cell membrane may be a binding target for amyloid-forming proteins, becoming a factor modulating the oligomerization process leading to amyloid deposition-a hallmark of amyloidogenic diseases-e.g., Alzheimer's disease. The information on the mechanisms governing the oligomerization influenced by the protein-membrane interactions is scarce. Therefore, our study aims to describe the interactions between DPPA, a cell membrane mimetic, and amyloidogenic protein human cystatin C. Circular dichroism spectroscopy and differential scanning calorimetry were used to monitor (i) the secondary structure of the human cystatin C and (ii) the phase transition temperature of the DPPA, during the protein-membrane interactions. NMR techniques were used to determine the protein fragments responsible for the interactions, and molecular dynamics simulations were applied to provide a molecular structure representing the interaction. The obtained data indicate that the protein interacts with DPPA, submerging itself into the bilayer via the AS region. Additionally, the interaction increases the content of α-helix within the protein's secondary structure and stabilizes the whole molecule against denaturation.
Asunto(s)
Membrana Celular , Cistatina C , Unión Proteica , Cistatina C/química , Cistatina C/metabolismo , Humanos , Membrana Celular/metabolismo , Membrana Celular/química , Simulación de Dinámica Molecular , Dicroismo Circular , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/metabolismo , Estructura Secundaria de Proteína , Rastreo Diferencial de CalorimetríaRESUMEN
Chondroitin sulfates (CSs) are important components of the extracellular matrix and side chains of membrane proteoglycans. These polysaccharides are, therefore, likely to interact with plasma membranes and play a significant role in modulating cellular functions. So far, the details of the processes occurring at the interface between the extracellular matrix and cellular membranes are not fully understood. In this study, we used experimental methods and atomic-scale molecular dynamics (MD) simulations to reveal the molecular picture of the interactions between CS and phosphocholine (PC) membranes, used as a simplified model of cell membranes. MD simulations reveal that the polysaccharide associates to the PC bilayer as a result of electrostatic interactions between the positively charged quaternary ammonium groups of choline and the negatively charged sulfate groups of CS. Compared to an aqueous medium, the adsorbed polysaccharide chains adopt more elongated conformations, which facilitates the electrostatic interactions with the membrane, and have a high degree of freedom to change their conformations and to adhere to and detach from the membrane surface. Penetrating slightly between the polar groups of the bilayer, they form a loosely anchored layer, but do not intrude into the hydrophobic region of the PC bilayer. The CS adsorption spread the PC headgroups apart, which is manifested by an increase in the value of the area pre lipid. The expansion of the lipid polar groups weakens the dispersion interactions between the lipid acyl chains. As a result, the lipid membrane in the membrane-polysaccharide contact areas becomes more fluid. Our outcomes may help to understand in detail the interaction of chondroitin sulfate with zwitterionic membranes at the molecular level, which is of biological interest since many biological processes depend on lipid-CS interactions.
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Sulfatos de Condroitina , Membrana Dobles de Lípidos , Simulación de Dinámica Molecular , Electricidad Estática , Sulfatos de Condroitina/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Fosfatidilcolinas/químicaRESUMEN
Oil-core nanocapsules (NCs, also known as nanoemulsions) are of great interest due to their application as efficient carriers of various lipophilic bioactives, such as drugs. Here, we reported for the first time the preparation and characterization of NCs consisting of chondroitin sulfate (CS)-based shells and liquid oil cores. For this purpose, two amphiphilic CS derivatives (AmCSs) were obtained by grafting the polysaccharide chain with octadecyl or oleyl groups. AmCS-based NCs were prepared by an ultrasound-assisted emulsification of an oil phase consisting of a mixture of triglyceride oil and vitamin E in a dispersion of AmCSs. Dynamic light scattering and cryo-transmission electron microscopy showed that the as-prepared core-shell NCs have typical diameters in the range of 30-250 nm and spherical morphology. Since CS is a strong polyanion, these particles have a very low surface potential, which promotes their stabilization. The cytotoxicity of the CS derivatives and CS-based NCs and their impact on cell proliferation were analyzed using human keratinocytes (HaCaTs) and primary human skin fibroblasts (HSFs). In vitro studies showed that AmCSs dispersed in an aqueous medium, exhibiting mild cytotoxicity against HaCaTs, while for HSFs, the harmful effect was observed only for the CS derivative with octadecyl side groups. However, the nanocapsules coated with AmCSs, especially those filled with vitamin E, show high biocompatibility with human skin cells. Due to their stability under physiological conditions, the high encapsulation efficiency of their hydrophobic compounds, and biocompatibility, AmCS-based NCs are promising carriers for the topical delivery of lipophilic bioactive compounds.
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Sulfatos de Condroitina , Portadores de Fármacos , Nanocápsulas , Nanocápsulas/química , Humanos , Sulfatos de Condroitina/química , Portadores de Fármacos/química , Suplementos Dietéticos , Fibroblastos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Emulsiones/química , Tamaño de la Partícula , Vitamina E/química , Supervivencia Celular/efectos de los fármacos , Línea Celular , Células HaCaTRESUMEN
The study carried out systematic research on the influence of selected oxysterols on cells viability, phospholipidosis and the level of secreted extracellular vesicles. Three oxidized cholesterol derivatives, namely 7α-hydroxycholesterol (7α-OH), 7- ketocholesterol (7-K) and 24(S)-hydroxycholesterol (24(S)-OH) were tested in three different concentrations: 50 µM, 100 µM and 200 µM for 24 h incubation with A549 lung cancer cell line. All the studied oxysterols were found to alter cells viability. The lowest survival rate of the cells was observed after 24 h of 7-K treatment, slightly better for 7α-OH while cells incubated with 24(S)-OH had the best survival rate among the oxysterols used. 7-K increased phospholipids accumulation in cells, however, most noticeable effect was noticed for 24(S)-OH. Changes in the level of extracellular vesicles secreted in cells culture after the treatment with oxysterols were also observed. It was found that all oxysterols used increased the level of secreted vesicles, both exosomes and ectosomes. The strongest effect was noticed for 24(S)-OH. Taken together, these results suggest that 7-K is the most potent inducer of cancer cell death, while 7α-OH is slightly less potent in this respect. The lower cytotoxic effect of 24(S)-OH correlates with greater phospholipids accumulation, extracellular vesicles production and better cells survival.
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Supervivencia Celular , Vesículas Extracelulares , Hidroxicolesteroles , Neoplasias Pulmonares , Oxiesteroles , Fosfolípidos , Humanos , Supervivencia Celular/efectos de los fármacos , Fosfolípidos/farmacología , Fosfolípidos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Vesículas Extracelulares/metabolismo , Oxiesteroles/metabolismo , Células A549 , Hidroxicolesteroles/farmacología , Hidroxicolesteroles/metabolismo , Cetocolesteroles/farmacología , Cetocolesteroles/metabolismoRESUMEN
BACKGROUND & AIMS: Butyric (one of the short-chain fatty acids), a major byproduct of the fermentation of non-digestible carbohydrates (e.g. fiber), is supposed to have anti-obesity and anti-inflammatory properties. However, butyrate's potential and mechanism in preventing obesity and the efficient form of administration remain to be clarified. METHODS: Hence, we studied the effect of oral supplementation with 5% (w/w) sodium butyrate and 4% (w/w) ß-glucan (fiber) on young male mice (C57BL/6J) with high-fat diet-induced obesity (HFD: 60 kcal% of fat + 1% of cholesterol). Six weeks old mice were fed diets based on HFD or control (AIN-93G) diet with/without supplements for 4 weeks. The unique, interdisciplinary approach combining several Raman-based techniques (including Raman microscopy and fiber optic Raman spectroscopy) and next-generation sequencing was used to ex vivo analyze various depots of the adipose tissue (white, brown, perivascular) and gut microbiome, respectively. RESULTS: The findings demonstrate that sodium butyrate more effectively prevent the pathological increase in body weight caused by elevated saturated fatty acids influx linked to a HFD in comparison to ß-glucan, thereby entirely inhibiting diet-induced obesity. Moreover, butyrate significantly affects the white adipose tissue (WAT) reducing the epididymal WAT mass in comparison to HFD without supplements, and decreasing lipid saturation in the epididymal WAT and perivascular adipose tissue of the thoracic aorta. Contrarily, ß-glucan significantly changes the composition and diversity of the gut microbiome, reversing the HFD effect, but shows no effect on the epididymal WAT mass and therefore the weight gain inhibition is not as effective as with sodium butyrate. CONCLUSIONS: Here, oral supplementation with sodium butyrate and ß-glucan (fiber) has been proven to have an anti-obesity effect through two different targets. Administration-dependent effects that butyrate imposes on the adipose tissue (oral administration) and microbiome (fiber-derived) make it a promising candidate for the personalized treatment of obesity.
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Obesidad , beta-Glucanos , Masculino , Animales , Ratones , Ratones Endogámicos C57BL , Ácido Butírico , Obesidad/tratamiento farmacológico , Obesidad/prevención & control , Suplementos Dietéticos , beta-Glucanos/farmacologíaRESUMEN
Phosphatidylserine (PS) exposure on the plasma membrane is crucial for many cellular processes including apoptotic cell recognition, blood clotting regulation, cellular signaling, and intercellular interactions. In this study, we investigated the arrangement of PS headgroups in mixed PS/phosphatidylcholine (PC) bilayers, serving as a simplified model of the outer leaflets of mammalian cell plasma membranes. Combining atomistic-scale molecular dynamics (MD) simulations with Langmuir monolayer experiments, we unraveled the mutual miscibility of POPC and POPS lipids and the intricate intermolecular interactions inherent to these membranes as well as the disparities in position and orientation of PC and PS headgroups. Our experiments revealed micrometer-scale miscibility at all mole fractions of POPC and POPS, marked by modest deviations from ideal mixing with no apparent microscale phase separation. The MD simulations, meanwhile, demonstrated that these deviations were due to strong electrostatic interactions between like-lipid pairs (POPC-POPC and POPS-POPS), culminating in lateral segregation and nanoscale clustering. Notably, PS headgroups profoundly affect the ordering of the lipid acyl chains, leading to lipid elongation and subtle PS protrusion above the zwitterionic membrane. In addition, PC headgroups are more tilted with respect to the membrane normal, while PS headgroups align at a smaller angle, making them more exposed to the surface of the mixed PC/PS membranes. These findings provide a detailed molecular-level account of the organization of mixed PC/PS membranes, corroborated by experimental data. The insights gained here extend our comprehension of the physiological role of PSs.
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Membrana Dobles de Lípidos , Fosfatidilcolinas , Membrana Dobles de Lípidos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Membranas Artificiales , Membrana Celular/metabolismoRESUMEN
Cartilage loss is a common clinical problem, which leads to significant pain, dysfunction, and even disability. As a result, there is growing interest in using small, non-protein molecules to protect or repair cartilage. Kartogenin (KGN), a small hydrophobic molecule, shows chondroprotective and chondrogenic properties. In this study, we embedded KGN in liposomes, and the whole system was stabilized by covering it with n-octadecylated (at two different substitution degrees) chondroitin sulfate (CS) derivatives. We investigated the interactions of empty liposomes and KGN-loaded liposomes with both CS derivatives using various physicochemical techniques, which revealed that hydrophobically modified CSs can interact with both neutral lipid membrane and negatively charged loaded-KGN lipid membrane. The cytotoxicity and chondrogenic properties of the polysaccharides and liposome-CS formulations of KGN were analyzed towards mesenchymal stem cells (MSCs). The results showed that the alkylated CS exhibited cytotoxic properties. The higher substituted CS self-assembles into stable nanoaggregates that can form a corona on the surface of liposomes, eliminating the overall cytotoxicity of this polymer. However, all tested chondrogenic markers' expression levels are enhanced for KGN-loaded liposomes and coated by lower substituted CS. Furthermore, the undesirable hypertrophy effect for this formulation significantly decreased compared to pure polymeric derivative.
RESUMEN
Considering the potential of nanostructured titanium dioxide layers as drug delivery systems, it is advisable to indicate the possibility of creating a functional drug delivery system based on anodic TiO2 for celecoxib as an alternative anti-inflammatory drug and its inclusion complex with ß-cyclodextrin. First, the optimal composition of celecoxib-ß-cyclodextrin complexes was synthesized and determined. The effectiveness of the complexation was quantified using isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), infrared spectroscopy (FT-IR) nuclear magnetic resonance (1H NMR), and scanning electron microscopy (SEM). Then, nanostructured titanium dioxide layers (TiO2) were synthesized using the electrochemical oxidation technique. The TiO2 layers with pore diameters of 60 nm and layer thickness of 1.60 µm were used as drug delivery systems. The samples were modified with pure celecoxib and the ß-cyclodextrin-celecoxib complex. The release profiles shown effective drug release from such layers during 24 h. After the initial burst release, the drug was continuously released from the pores. The presented results confirm that the use of nanostructured TiO2 as a drug delivery system can be effectively used in more complicated systems composed of ß-cyclodextrin-celecoxib complexes, making such drugs available for pain treatment, e.g., for orthopedic surgeries.
RESUMEN
The miscibility of phospholipids in a hydrated bilayer is an issue of fundamental importance for understanding the organization of biological membranes. Despite research on lipid miscibility, its molecular basis remains poorly understood. In this study, all-atom MD simulations complemented by Langmuir monolayer and DSC experiments have been performed to investigate the molecular organization and properties of lipid bilayers composed of phosphatidylcholines with saturated (palmitoyl, DPPC) and unsaturated (oleoyl, DOPC) acyl chains. The experimental results showed that the DOPC/DPPC bilayers are systems exhibiting a very limited miscibility (strongly positive values of excess free energy of mixing) at temperatures below the DPPC phase transition. The excess free energy of mixing is divided into an entropic component, related to the ordering of the acyl chains, and an enthalpic component, resulting from the mainly electrostatic interactions between the headgroups of lipids. MD simulations showed that the electrostatic interactions for lipid like-pairs are much stronger than that for mixed pairs and temperature has only a slight influence on these interactions. On the contrary, the entropic component increases strongly with increasing temperature, due to the freeing of rotation of acyl chains. Therefore, the miscibility of phospholipids with different saturations of acyl chains is an entropy-driven process.
RESUMEN
Hydrophobically modified chondroitin sulfate (CS) is widely used in the preparation of nano-sized drug delivery systems. Herein, the behavior of amphiphilic CSs in aqueous media and the drug accumulation inside the formed micelle-like structures were studied using experimental methods and molecular dynamics simulations. In particular, we focused on the impact of the degree of substitution (DS) with hydrophobic groups and the presence of drug on the morphology of the nanostructures and their molecular organization. Our results show that with increasing DS, the morphology of the amphiphilic CS nanostructures changes from irregular, loosely packed nanogels to cylindrical micelles with a core-shell architecture. These structures can efficiently accumulate hydrophobic drugs. However, the drug molecules preferentially locate at the interface between the hydrophobic part and the hydrophilic corona formed by the CS chains. Our work provides detailed information that may be relevant to the development of amphiphilic polysaccharide-based drug delivery systems.
Asunto(s)
Sulfatos de Condroitina , Micelas , Sulfatos de Condroitina/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Interacciones Hidrofóbicas e Hidrofílicas , Nanogeles , AguaRESUMEN
Endothelial inflammation is recognized as a critical condition in the development of cardiovascular diseases. TNF-induced inflammation of endothelial cells is linked to the formation of lipid droplets, augmented cortical stiffness, and nanostructural endothelial plasma membrane remodelling, but the insight into the mechanism linking these responses is missing. In the present work, we determined the formation of lipid droplets (LDs), nanomechanical, and nanostructural responses in the model of TNF-activated vascular inflammation in the isolated murine aorta using Raman spectroscopy, fluorescence imaging, atomic force microscopy (AFM), and scanning electron microscopy (SEM). We analysed the possible role of Rac1, a major regulator of cytoskeletal organization, in TNF-induced vascular inflammation. We demonstrated that the formation of LDs, polymerization of F-actin, alterations in cortical stiffness, and nanostructural protuberances in endothelial plasma membrane were mediated by the Rac1. In particular, we revealed a significant role for Rac1 in the regulation of the formation of highly unsaturated LDs formed in response to TNF. Inhibition of Rac1 also downregulated the overexpression of ICAM-1 induced by TNF, supporting the role of Rac1 in vascular inflammation. Altogether, our results demonstrate that LDs formation, an integral component of vascular inflammation, is activated by Rac1 that also regulates nanomechanical and nanostructural alterations linked to vascular inflammation.
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Células Endoteliales , Endotelio Vascular , Animales , Aorta , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Inflamación/metabolismo , Gotas Lipídicas/metabolismo , RatonesRESUMEN
BACKGROUND: The sealer penetration into the dentinal tubules might be beneficial, especially in necrotic endodontic cases, as it provides the obstruction of the contaminated tubules. OBJECTIVES: To determine the effect of 3 final irrigants (sodium hypochlorite (NaOCl), alcohol and chlorhexidine (CHX)) on the penetration of an epoxy sealer into the dentinal tubules. MATERIAL AND METHODS: The study was carried out on 60 single-canal human teeth with straight roots. The root canals were prepared to the ISO 40/04 size, using the Reciproc® instruments. The teeth were divided into 4 groups (n = 15). The canals in each group were irrigated according to the following scheme: group 1 (control) - 5.25% NaOCl; group 2 - smear layer removal (40% citric acid (CA) and 5.25% NaOCl) and 5.25% NaOCl; group 3 - smear layer removal (as in group 2), and 40% CA, water and 98% isopropyl alcohol; and group 4 - smear layer removal (as in group 2), and 40% CA, water and 2% CHX. The root canals were filled using the vertical condensation technique with gutta-percha and the porphyrin-labeled AH Plus™ sealer. After 3 days, 1-milimeter-thick cross-section slices were cut from the roots at a distance of 2 mm, 5 mm and 8 mm from the apex. The sections were imaged under a confocal microscope and the sealant penetration depth into the dentinal tubules was measured. RESULTS: The longest resin tags in all parts of the roots were found in group 4 (CHX), and the shortest in group 1 (control). The mean depth of the sealer penetration (in micrometers) was as follows: 21, 22 and 23 (group 1); 201, 231 and 374 (group 2); 170, 232 and 280 (group 3); and 330, 408 and 638 (group 4) in the apical, middle and coronal parts, respectively. CONCLUSIONS: The final irrigation with CHX resulted in the deepest penetration of the epoxy sealer into the tubules. Isopropyl alcohol had the most negative impact on the sealer penetration into the tubules.
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Materiales de Obturación del Conducto Radicular , Hipoclorito de Sodio , 2-Propanol , Clorhexidina , Ácido Edético , Humanos , Microscopía Confocal/métodos , Irrigantes del Conducto Radicular , Preparación del Conducto RadicularRESUMEN
Excessive lipid accumulation is a serious problem in obesity leading to adipose tissue (AT) overgrowth, chronic inflammation, endothelial dysfunction, and elevated risk of cardiovascular complications. In this work, Raman techniques coupled with fluorescence imaging were applied to characterize the effects of short-term (2 weeks) and extended (up to 8 weeks) high-fat diet (HFD) feeding on various depots of the adipose tissue of young and mature mice. Our results proved the synergistic effect of age and HFD-induced obesity manifested by changes in the morphology of adipocytes and the chemical composition of lipids. After 2 weeks of HFD feeding of young animals, substantial hypertrophy of adipocytes but only for the periaortic adipose tissue was detected with a significant decrease in lipid unsaturation degree solely in the epididymal white adipose tissue. The periaortic AT did not altered chemically due to short-term HFD feeding, however, it changed with age and with prolonged exposure to harmful factors. For older animals only brown AT remains resistant on HFD underlying its protective role and highlighting its potential as a target in obesity therapies.
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Adipocitos/patología , Dieta Alta en Grasa/efectos adversos , Hipertrofia/patología , Inflamación/patología , Obesidad/patología , Animales , Hipertrofia/etiología , Inflamación/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/etiologíaRESUMEN
The aim was to assess the impact of irrigation protocols ended with ethanol or chlorhexidine on AH Plus penetration into dentinal tubules. 45 root canals were prepared to ISO 40/04, divided into three groups and irrigated with three protocols: Group 1 (control): 5.25% NaOCl; Group 2: 40% CA (citric acid), 5.25% NaOCl, 40% CA, water, ethanol; Group 3: 40% CA, 5.25% NaOCl, 40% CA, water, 2% chlorhexidine. Canals were filled using vertical condensation technique with gutta-percha and fluorescein-stained AH Plus sealer. After 72 h, 1 mm thick cross-sections were cut at 2, 5, 8 mm from the apex. Confocal laser microscope was used to measure the sealer penetration into dentinal tubules. Mean depth of sealer penetration (in micrometres) was 107, 131, 170 (Group 1); 146, 233, 317 (Group 2); 185, 301, 542 (Group 3); in apical, middle and coronal parts, respectively. Irrigation protocol ended with chlorhexidine resulted in the deeper sealer penetration compared with alcohol.
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Materiales de Obturación del Conducto Radicular , Irrigantes del Conducto Radicular , Clorhexidina , Resinas Epoxi , Etanol , Microscopía Confocal/métodos , Preparación del Conducto Radicular/métodos , Hipoclorito de Sodio , AguaRESUMEN
Temporal partitioning of daily food intake is crucial for survival and involves the integration of internal circadian states and external influences such as the light-dark cycle and dietary composition. These intrinsic and extrinsic factors are interdependent with misalignment of circadian rhythms promoting body weight gain, while consumption of a calorie-dense diet elevates the risk of obesity and blunts circadian rhythms. Recently, we defined the circadian properties of the dorsal vagal complex of the brainstem, a structure implicated in the control of food intake and autonomic tone, but whether and how 24 h rhythms in this area are influenced by diet remains unresolved. Here we focused on a key structure of this complex, the nucleus of the solitary tract (NTS). We used a combination of immunohistochemical and electrophysiological approaches together with daily monitoring of body weight and food intake to interrogate how the neuronal rhythms of the NTS are affected by a high-fat diet. We report that short-term consumption of a high-fat diet increases food intake during the day and blunts NTS daily rhythms in neuronal discharge. Additionally, we found that a high-fat diet dampens NTS responsiveness to metabolic neuropeptides, and decreases orexin immunoreactive fibres in this structure. These alterations occur without prominent body weight gain, suggesting that a high-fat diet acts initially to reduce activity in the NTS to disinhibit mechanisms that suppress daytime feeding. KEY POINTS: The dorsal vagal complex of the rodent hindbrain possesses intrinsic circadian timekeeping mechanisms In particular, the nucleus of the solitary tract (NTS) is a robust circadian oscillator, independent of the master suprachiasmatic clock Here, we reveal that rat NTS neurons display timed daily rhythms in their neuronal activity and responsiveness to ingestive cues These daily rhythms are blunted or eliminated by a short-term high-fat diet, together with increased consumption of calories during the behaviourally quiescent day Our results help us better understand the circadian control of satiety by the brainstem and its malfunctioning under a high-fat diet.
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Dieta Alta en Grasa , Núcleo Solitario , Animales , Ritmo Circadiano/fisiología , Ingestión de Alimentos/fisiología , Neuronas/metabolismo , Ratas , Núcleo Solitario/metabolismoRESUMEN
The orexinergic system delivers excitation for multiple brain centers to facilitate behavioral arousal, with its malfunction resulting in narcolepsy, somnolence, and notably, visual hallucinations. Since the circadian clock underlies the daily arousal, a timed coordination is expected between the orexin system and its target subcortical visual system, including the superior colliculus (SC). Here, we use a combination of electrophysiological, immunohistochemical, and molecular approaches across 24 h, together with the neuronal tract-tracing methods to investigate the daily coordination between the orexin system and the rodent SC. Higher orexinergic input was found to occur nocturnally in the superficial layers of the SC, in time for nocturnal silencing of spontaneous firing in this visual brain area. We identify autonomous daily and circadian expression of clock genes in the SC, which may underlie these day-night changes. Additionally, we establish the lateral hypothalamic origin of the orexin innervation to the SC and that the SC neurons robustly respond to orexin A via OX2 receptor in both excitatory and GABAA receptor-dependent inhibitory manners. Together, our evidence elucidates the combination of intrinsic and extrinsic clock mechanisms that shape the daily function of the visual layers of the SC.
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Relojes Circadianos , Orexinas/metabolismo , Colículos Superiores/metabolismo , Visión Ocular/fisiología , Animales , Relojes Circadianos/genética , Relojes Circadianos/fisiología , Oscuridad , Área Hipotalámica Lateral/metabolismo , Masculino , Ratones , Neuronas/metabolismo , Receptores de Orexina/metabolismo , Ratas , Ratas Sprague-Dawley , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Nanoparticles made of amphiphilic block copolymers are commonly used in the preparation of nano-sized drug delivery systems. Poly(styrene)-block -poly(acrylic acid) (PS-PAA) copolymers have been proposed for drug delivery purposes; however, the drug loading capacity and cytotoxicity of PS-PAA nanoparticles are still not fully recognized. Herein, we investigated the accumulation of a model hydrophobic drug, curcumin, and its spatial distribution inside the PS-PAA nanoparticles. Experimental methods and atomistic molecular dynamics simulations were used to understand the molecular structure of the PS core and how curcumin molecules interact and organize within the PS matrix. The hydrophobic core of the PS-PAA nanoparticles consists of adhering individually coiled polymeric chains and is compact enough to prevent post-incorporation of curcumin. However, the drug has a good affinity for the PS matrix and can be efficiently enclosed in the PS-PAA nanoparticles at the formation stage. At low concentrations, curcumin is evenly distributed in the PS core, while its aggregates were observed above ca. 2 wt %. The nanoparticles were found to have relatively low cytotoxicity to human skin fibroblasts, and the presence of curcumin further increased their biocompatibility. Our work provides a detailed description of the interactions between a hydrophobic drug and PS-PAA nanoparticles and information on the biocompatibility of these anionic nanostructures which may be relevant to the development of amphiphilic copolymer-based drug delivery systems.
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KEY POINTS: Rhythmic processes in living organisms are controlled by biological clocks. The orexinergic system of the lateral hypothalamus carries circadian information to provide arousal for the brain during the active phase. Here, we show that orexins exert an excitatory action in three parts of the lateral geniculate nucleus (LGN), in particular upon directly retinorecipient neurons in the non-image forming visual structures. We provide evidence for the high nocturnal levels of orexins with stable circadian expression of predominant orexin receptor 2 in the LGN. Our data additionally establish the convergence of orexinergic and pituitary adenylate cyclase (PAC)-activating peptide/PAC1 receptor systems (used by melanopsin-expressing retinal ganglion cells), which directly regulates responses to the retinal input. These results help us better understand circadian orexinergic control over the non-image forming subcortical visual system, forming the animal's preparedness for the behaviourally active night. ABSTRACT: The orexinergic system of the lateral hypothalamus is tightly interlinked with the master circadian clock and displays daily variation in activity to provide arousal-related excitation for the plethora of brain structures in a circadian manner. Here, using a combination of electrophysiological, optogenetic, histological, molecular and neuronal tracing methods, we explore a particular link between orexinergic and visual systems in rat. The results of the present study demonstrate that orexinergic fibre density at the area of subcortical visual system exerts a clear day to night variability, reaching a maximum at behaviourally active night. We also show pronounced electrophysiological activations of neurons in the lateral geniculate nucleus by orexin A through 24 h, via identified distinct orexin receptors, with the ventrolateral geniculate displaying a daily cycle of responsiveness. In addition, for the first time, we provide a direct evidence for orexins to act on retinorecipient neurons with a high convergence of orexinergic and putatively retinal pituitary adenylate cyclase (PAC)-activating peptide/PAC1 receptor systems. Altogether, the present study ties orexins to non-image forming visual structures with implications for circadian orexinergic modulation of neurons, which process information on ambient light levels.
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Cuerpos Geniculados , Neuronas , Animales , Ritmo Circadiano , Área Hipotalámica Lateral/metabolismo , Neuronas/metabolismo , Receptores de Orexina/metabolismo , Orexinas/metabolismo , RatasRESUMEN
Micro- and nanostructures prepared from biodegradable homopolymers and amphiphilic block copolymers (AmBCs) have found application as drug-delivery systems (DDSs). The ability to accumulate a drug is a very important parameter characterizing a given DDS. This work focuses on the impact of DDS size, the packing of polymer chains in the DDS, and drug - polymer matrix compatibility on the hydrophobic drug - loading capacity (DLC) of nano/microcarriers prepared from a biodegradable polymer or its copolymer. Using experimental measurements in combination with atomistic molecular dynamics simulations, an analysis of curcumin encapsulation in microspheres (MSs) from polylactide (PLA) homopolymer and nanoparticles (NPs) from PLA-block-poly(2-methacryloyloxyethylphosphorylcholine) AmBC was performed. The results show that curcumin has good affinity for the PLA matrix due to its hydrophobic nature. However, the DLC value is limited by the fact that curcumin only accumulates in the peripheral part of these structures. Such uneven drug distribution in the PLA matrix results from the non-homogeneous density of MSs (non-uniform packing of the polymer chains in the coil). The results also indicate that the MSs can retain a greater amount of hydrophobic drug compared to the NPs, which is associated with the formation of drug aggregates inside the PLA microparticles.