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1.
Molecules ; 26(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34443296

RESUMO

In recent decades liposomes have been used in different field thanks to their ability to act as a vehicle for a wide range of biomolecules, their great versatility and their easy production. The aim of this study was to evaluate liposomes as a vehicle for the actives present in the HelixComplex (HC) snail mucus for topical delivery. Liposomes composed of a mixture of phosphatidylcholine, cholesterol and octadecylamine were prepared with and without HC (empty liposomes) and their biological efficacy was tested by evaluating cell viability and migration. HC-loaded liposomes (LHC) were stable throughout 60 days of observation, and showed interesting effects on wound healing reconstitution. In particular, we observed that 25 µg/mL LHC were already able to induce a higher cell monolayer reconstitution in comparison to the untreated samples and HC treated samples after only 4 h (28% versus 10% and 7%, p = 0.03 and p= 0.003, respectively). The effect was more evident at 24 h in comparison with the untreated control (54% versus 21.2% and 41.6%, p = 0.006 and p = NS, respectively). These results represent a preliminary, but promising, novelty in the delivery strategy of the actives present in the HelixComplex mucus.


Assuntos
Muco/química , Caramujos/química , Animais , Morte Celular , Linhagem Celular , Fibroblastos/citologia , Técnica de Fratura por Congelamento , Humanos , Lipídeos/análise , Lipossomos/ultraestrutura , Espectrofotometria Infravermelho , Cicatrização/efeitos dos fármacos
2.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105

RESUMO

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
3.
Molecules ; 26(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805693

RESUMO

Lecithins of different origins and compositions were used for the liposomal encapsulation of carvacrol within the framework of the development of active films for food packaging. Liposomes were incorporated into aqueous polymeric solutions from fully (F) and partially (P) hydrolysed Poly (vinyl alcohol) (PVA) to obtain the films by casting. The particle size distribution and ζ-potential of the liposomal suspensions, as well as their stability over time, were evaluated. Liposomal stability during film formation was analysed through the carvacrol retention in the dried film and the film microstructure. Subtle variations in the size distributions of liposomes from different lecithins were observed. However, the absolute values of the ζ-potential were higher (-52, -57 mV) for soy lecithin (SL) liposomes, followed by those of soy lecithin enriched with phosphatidylcholine (SL-PC) (-43, -50 mV) and sunflower lecithin (SFL) (-33, -38 mV). No significant changes in the liposomal properties were observed during the study period. Lyotropic mesomorphism of lipid associations and carvacrol leakage occurred to differing extents during the film drying step, depending on the membrane lipid composition and surface charge. Liposomes obtained with SL-PC were the most effective at maintaining the stability of carvacrol emulsion during film formation, which led to the greatest carvacrol retention in the films, whereas SFL gave rise to the least stable system and the highest carvacrol losses. P-PVA was less sensitive to the emulsion destabilisation due to its greater bonding capacity with carvacrol. Therefore, P-PVA with carvacrol-loaded SL-PC liposomes has great potential to produce active films for food packaging applications.


Assuntos
Cimenos/administração & dosagem , Embalagem de Alimentos/métodos , Lipossomos/química , Álcool de Polivinil/química , Anti-Infecciosos/administração & dosagem , Antioxidantes/administração & dosagem , Plásticos Biodegradáveis/química , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Emulsões , Conservação de Alimentos/métodos , Humanos , Lecitinas/química , Lipossomos/ultraestrutura , Microscopia Eletrônica de Varredura , Tamanho da Partícula
4.
Int J Biol Macromol ; 167: 834-844, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33181211

RESUMO

A polylactide composite fracture fixator loaded with vancomycin cationic liposome (PLA@VL) was prepared by reverse evaporation method. The method of cationic liposome encapsulating vancomycin could effectively improve antibacterial property and achieve drug sustained release effect, so as to reduce toxicity of antibiotics in vivo. Scanning electron microscope (SEM) was used to observe morphology and Fourier transform infrared spectroscopy (FTIR) was used to detect the composition of the internal fixator. In vitro drug release model, in vitro degradation model and body fluid osteogenesis model were designed in this study. On the other hand, the experiments of inhibition zone and MC3T3-E1 osteoblasts in mice were conducted to explore antibacterial property, cell activity and adhesion of the PLA@VL composite internal fixator. Alkaline phosphatase (ALP) staining method and alizarin red assay were used to detect the osteogenic induction ability of the composite internal fixator. Finally, mice fracture models were established to verify osteogenic and anti-infection abilities of the composite internal fixator in vivo. The results showed that MC3T3-E1 cells had better adhesion and proliferation abilities on the PLA@VL composite internal fixator than on the PLA fixator, which indicated that the PLA@VL composite internal fixator possessed excellent osteogenic and anti-infection abilities both in vivo and in vitro. Therefore, the above experiments showed that the fracture internal fixator combined with vancomycin cationic liposome had better biocompatibility, antibacterial ability and osteogenic ability, which provides a promising anti-infection material for the clinical field of fracture.


Assuntos
Antibacterianos/administração & dosagem , Fixadores Internos , Lipossomos/química , Poliésteres/análise , Vancomicina/administração & dosagem , Fosfatase Alcalina/metabolismo , Animais , Antibacterianos/química , Materiais Biocompatíveis/química , Biomarcadores , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Liberação Controlada de Fármacos , Lipossomos/ultraestrutura , Camundongos , Testes de Sensibilidade Microbiana , Células NIH 3T3 , Osteogênese/efeitos dos fármacos , Solubilidade , Análise Espectral , Engenharia Tecidual , Tecidos Suporte/química , Vancomicina/química
6.
Proc Natl Acad Sci U S A ; 117(50): 31871-31881, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33257567

RESUMO

TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.


Assuntos
Morte Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Proteínas Ativadoras de GTPase/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cricetulus , Proteínas Ativadoras de GTPase/uso terapêutico , Células HeLa , Humanos , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Microscopia Eletrônica , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/uso terapêutico
7.
Int J Nanomedicine ; 15: 7627-7650, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33116492

RESUMO

The skin is the largest organ in the human body, providing a barrier to the external environment. It is composed of three layers: epidermis, dermis and hypodermis. The most external epidermis is exposed to stress factors that may lead to skin conditions such as photo-aging and skin cancer. Some treatments for skin disease utilize the incorporation of drugs or bioactive compounds into nanocarriers known as liposomes. Liposomes are membranes whose sizes range from nano to micrometers and are composed mostly of phospholipids and cholesterol, forming similar structures to cell membranes. Thus, skin treatments with liposomes have lower toxicity in comparison to traditional treatment routes such as parenteral and oral. Furthermore, addition of edge activators to the liposomes decreases the rigidity of the bilayer structure making it deformable, thereby improving skin permeability. Liposomes are composed of an aqueous core and a lipidic bilayer, which confers their amphiphilic property. Thus, they can carry hydrophobic and hydrophilic compounds, even simultaneously. Current applications of these nanocarriers are mainly in the cosmetic and pharmaceutic industries. Nevertheless, new research has revealed promising results regarding the effectiveness of liposomes for transporting bioactive compounds through the skin. Liposomes have been well studied; however, additional research is needed on the efficacy of liposomes loaded with bioactive peptides for skin delivery. The objective of this review is to provide an up-to-date description of existing techniques for the development of liposomes and their use as transporters of bioactive compounds in skin conditions such as melanoma and skin inflammation. Furthermore, to gain an understanding of the behavior of liposomes during the process of skin delivery of bioactive compounds into skin cells.


Assuntos
Inflamação/patologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Pele/patologia , Transporte Biológico , Humanos , Lipossomos/ultraestrutura , Absorção Cutânea
8.
Nat Commun ; 11(1): 4502, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908136

RESUMO

Biological tissues, such as muscle, can increase their mechanical strength after swelling due to the existence of many biological membrane barriers that can regulate the transmembrane transport of water molecules and ions. Oppositely, typical synthetic materials show a swelling-weakening behavior, which always suffers from a sharp decline in mechanical strength after swelling, because of the dilution of the network. Here, we describe a swelling-strengthening phenomenon of polymer materials achieved by a bioinspired strategy. Liposomal membrane nanobarriers are covalently embedded in a crosslinked network to regulate transmembrane transport. After swelling, the stretched network deforms the liposomes and subsequently initiates the transmembrane diffusion of the encapsulated molecules that can trigger the formation of a new network from the preloaded precursor. Thanks to the tough nature of the double-network structure, the swelling-strengthening phenomenon is achieved to polymer hydrogels successfully. Swelling-triggered self-strengthening enables the development of various dynamic materials.


Assuntos
Materiais Biomiméticos/química , Hidrogéis/química , Lipossomos/química , Nanoestruturas/química , Força Compressiva , Reagentes para Ligações Cruzadas/química , Lipossomos/ultraestrutura , Teste de Materiais , Microscopia Eletrônica de Transmissão , Nanoestruturas/ultraestrutura , Resistência à Tração
9.
Proc Natl Acad Sci U S A ; 117(31): 18497-18503, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32680969

RESUMO

Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipossomos/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Lipossomos/metabolismo , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/ultraestrutura , Conformação Proteica
10.
Int J Mol Sci ; 21(15)2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32707876

RESUMO

In this work, we aimed to develop liposomal nanocomposites containing citric-acid-coated iron oxide magnetic nanoparticles (CMNPs) for dual magneto-photothermal cancer therapy induced by alternating magnetic field (AMF) and near-infrared (NIR) lasers. Toward this end, CMNPs were encapsulated in cationic liposomes to form nano-sized magnetic liposomes (MLs) for simultaneous magnetic hyperthermia (MH) in the presence of AMF and photothermia (PT) induced by NIR laser exposure, which amplified the heating efficiency for dual-mode cancer cell killing and tumor therapy. Since the heating capability is directly related to the amount of entrapped CMNPs in MLs, while the liposome size is important to allow internalization by cancer cells, response surface methodology was utilized to optimize the preparation of MLs by simultaneously maximizing the encapsulation efficiency (EE) of CMNPs in MLs and minimizing the size of MLs. The experimental design was performed based on the central composite rotatable design. The accuracy of the model was verified from the validation experiments, providing a simple and effective method for fabricating the best MLs, with an EE of 87% and liposome size of 121 nm. The CMNPs and the optimized MLs were fully characterized from chemical and physical perspectives. In the presence of dual AMF and NIR laser treatment, a suspension of MLs demonstrated amplified heat generation from dual hyperthermia (MH)-photothermia (PT) in comparison with single MH or PT. In vitro cell culture experiments confirmed the efficient cellular uptake of the MLs from confocal laser scanning microscopy due to passive accumulation in human glioblastoma U87 cells originated from the cationic nature of MLs. The inducible thermal effects mediated by MLs after endocytosis also led to enhanced cytotoxicity and cumulative cell death of cancer cells in the presence of AMF-NIR lasers. This functional nanocomposite will be a potential candidate for bimodal MH-PT dual magneto-photothermal cancer therapy.


Assuntos
Glioblastoma/tratamento farmacológico , Hipertermia Induzida/métodos , Lipossomos/química , Nanopartículas de Magnetita/química , Nanocompostos/química , Fototerapia/métodos , Células 3T3 , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ácido Cítrico/química , Endocitose/efeitos dos fármacos , Glioblastoma/radioterapia , Humanos , Hipertermia , Hipertermia Induzida/instrumentação , Lasers , Lipossomos/síntese química , Lipossomos/ultraestrutura , Campos Magnéticos , Nanopartículas de Magnetita/efeitos da radiação , Nanopartículas de Magnetita/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Nanocompostos/efeitos da radiação , Tamanho da Partícula
11.
Biochem Biophys Res Commun ; 529(2): 362-365, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32703436

RESUMO

Army Liposome Formulations (ALF) are potent adjuvants, of which there are two primary forms, lyophilized ALF (ALFlyo) containing monophosphoryl lipid A (MPLA) and ALF containing MPLA and QS21 (ALFQ). ALFlyo and ALFQ adjuvants are essential constituents of candidate vaccines for bacterial, viral, and parasitic diseases. They have been widely used in preclinical immunogenicity studies in small animals and non-human primates and are progressing to phase I/IIa clinical trials. ALFQ was prepared by adding saponin QS21 to small unilamellar liposome vesicles (SUVs) of ALF55 that contain 55 mol% cholesterol, whereas ALFlyo was created by reconstituting lyophilized SUVs of ALF43, consisting of 43 mol% cholesterol, in aqueous buffer solution. These formulations display heterogenous particle size distribution. Since biophysical characteristics of liposomes may impact their adjuvant potential, we characterized the particle size distribution and lamellarity of the individual liposome particles in ALFlyo and ALFQ formulations using cryo-electron microscopy and a newly developed MANTA technology. ALFlyo and ALFQ exhibited similar particle size distributions with liposomes ranging from 50 nm to several µm. However, fundamental differences were observed in the lamellar structures of the liposomes. ALFlyo displayed a greater number of multilamellar and multivesicular liposome particles, as compared to that in ALFQ, which was predominately unilamellar.


Assuntos
Adjuvantes Imunológicos/química , Lipídeo A/análogos & derivados , Lipossomos/química , Saponinas/química , Colesterol/química , Microscopia Crioeletrônica , Liofilização , Lipídeo A/química , Lipossomos/ultraestrutura , Tamanho da Partícula
12.
Int J Nanomedicine ; 15: 4079-4090, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606665

RESUMO

Purpose: The aim of this study is to develop efficient localized therapy of sertaconazole nitrate for the treatment of vaginal candidiasis. Methods: Sertaconazole nitrate-loaded cationic liposomes were prepared by thin-film hydration method and coated with different concentrations of pectin (0.05%, 0.1% and 0.2%) to develop mucoadhesive liposomes. The formulated mucoadhesive vesicles were characterized in terms of morphology, entrapment efficiency, particle size, zeta value, mucoadhesive properties and drug release. The selected formula was incorporated into a gel base and further characterized by an ex vivo permeation study in comparison with conventional sertaconazole gel. Also, the in vivo study was performed to assess the efficacy of sertaconazole mucoadhesive liposomal gel in treating rats with vaginal candidiasis. Results: The mucoadhesive liposomes were spherical. Coating liposomes with pectin results in increased entrapment efficiency and particle size compared with uncoated vesicles. On the contrary, zeta values were reduced upon coating liposomes with pectin indicating efficient coating of liposomes with pectin. Mucoadhesive liposomes showed a more prolonged and sustained drug release compared with uncoated liposomes. Ex vivo study results showed that mucoadhesive liposomal gel increased sertaconazole tissue retention and reduced drug tissue penetration. In the invivo study, the mucoadhesive liposomal gel showed a significant reduction in the microbial count with a subsequent reduction in inflammatory responses with the lowest histopathological change compared with conventional gel. Conclusion: The study confirmed the potentiality of employing mucoadhesive liposomes as a successful carrier for the vaginal delivery of antifungal drugs.


Assuntos
Antifúngicos/uso terapêutico , Candidíase Vulvovaginal/tratamento farmacológico , Imidazóis/uso terapêutico , Muco/química , Tiofenos/uso terapêutico , Adesividade , Animais , Anti-Infecciosos/farmacologia , Biomarcadores/metabolismo , Preparações de Ação Retardada , Liberação Controlada de Fármacos , Feminino , Géis , Humanos , Imidazóis/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Mediadores da Inflamação/metabolismo , Lipossomos/ultraestrutura , Mucinas/metabolismo , Tamanho da Partícula , Ratos Sprague-Dawley , Ovinos , Eletricidade Estática , Tiofenos/farmacologia , Vagina/patologia , beta-Glucanas/metabolismo
13.
J Am Chem Soc ; 142(25): 10989-10995, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32476414

RESUMO

Polymer hexosomes are block copolymer solution morphologies that adopt an internal structure composed of an inverse hexagonal (HII) phase. To date, most polymer hexosomes are reportedly rotationally symmetric solid structures that possess a common feature where hexagonally ordered inverted cylinders rotate along a central axis of symmetry to form circular hoops. Here, we report on the formation of polymer hexosomes whose inverted cylinders orient in an unusual manner, forming hoops that are noncircular. For topological reasons, this led to the generation of four defects in the resulting hexosome structure. We find that these defect-bearing hexosomes are hollow, thereby resembling polymer vesicles or polymersomes with an inverse hexagonal cylindrical morphology in the shell. The topological defects of these so-called "vesicular hexosomes" are enticing as they could serve as a platform to spatially anchor targeting ligands or biomolecules on the surface, while the hollow cylindrical shell and the vesicular lumen could spatially accommodate cargoes within the different domains. We propose that these vesicular hexosomes do not form via a conventional nucleation-growth self-assembly pathway, but rather via a two-step process involving first liquid-liquid phase separation followed by polymer microphase separation.


Assuntos
Resinas Acrílicas/química , Lipossomos/química , Polivinil/química , Ouro/química , Lipossomos/síntese química , Lipossomos/ultraestrutura , Nanopartículas Metálicas/química
14.
Int J Nanomedicine ; 15: 3771-3790, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32547027

RESUMO

Introduction: Rapamycin has been considered as a potential treatment for osteoarthritis (OA). Drug carriers fabricated from liposomes can prolong the effects of drugs and reduce side effects of drugs. Low-intensity pulsed ultrasound (LIPUS) has been found to possess anti-OA effects. Materials and Methods: The anti-osteoarthritic effects of liposome-encapsulated rapamycin (L-rapa) combined with LIPUS were examined by culture of normal and OA chondrocytes in alginate beads and further validated in OA prone Dunkin-Hartley guinea pigs. Results: L-rapa with LIPUS largely up-regulated aggrecan and type II collagen mRNA in human OA chondrocytes (HOACs). L-rapa with LIPUS caused significant enhancement in proteoglycan and type II collagen production in HOACs. Large decreases in both MMP-13 and IL-6 proteins were found in the HOACs exposed to L-rapa with LIPUS. Intra-articular injection of 40 µL L-rapa at both 5 µM and 50 µM twice a week combined with LIPUS thrice a week for 8 weeks significantly increased GAGs and type II collagen in the cartilage of knee. Results on OARSI score showed that intra-articular injection of 5 µM L-rapa with LIPUS displayed the greatest anti-OA effects. Immunohistochemistry revealed that L-rapa with or without LIPUS predominantly reduced MMP-13 in vivo. The values of complete blood count and serum biochemical examinations remained in the normal ranges after the injections with or without LIPUS. These data indicated that intra-articular injection of L-rapa collaborated with LIPUS is not only effective against OA but a safe OA therapy. Conclusion: Taken together, L-rapa combined with LIPUS possessed the most consistently and effectively anabolic and anti-catabolic effects in HOACs and the spontaneous OA guinea pigs. This study evidently revealed that liposome-encapsulation collaborated with LIPUS is able to reduce the effective dose and administration frequency of rapamycin and further stably reinforce its therapeutic actions against OA.


Assuntos
Osteoartrite/terapia , Sirolimo/uso terapêutico , Ondas Ultrassônicas , Animais , Peso Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Condrócitos/efeitos da radiação , Colágeno Tipo II/metabolismo , Liberação Controlada de Fármacos , Cobaias , Humanos , Injeções Intra-Articulares , Interleucina-6/metabolismo , Lipossomos/ultraestrutura , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Pessoa de Meia-Idade , Osteoartrite/sangue , Osteoartrite/patologia , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirolimo/administração & dosagem , Sirolimo/farmacologia
15.
Nat Commun ; 11(1): 1516, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471995

RESUMO

ESCRT-III proteins assemble into ubiquitous membrane-remodeling polymers during many cellular processes. Here we describe the structure of helical membrane tubes that are scaffolded by bundled ESCRT-III filaments. Cryo-ET reveals how the shape of the helical membrane tube arises from the assembly of two distinct bundles of helical filaments that have the same helical path but bind the membrane with different interfaces. Higher-resolution cryo-EM of filaments bound to helical bicelles confirms that ESCRT-III filaments can interact with the membrane through a previously undescribed interface. Mathematical modeling demonstrates that the interface described above is key to the mechanical stability of helical membrane tubes and helps infer the rigidity of the described protein filaments. Altogether, our results suggest that the interactions between ESCRT-III filaments and the membrane could proceed through multiple interfaces, to provide assembly on membranes with various shapes, or adapt the orientation of the filaments towards the membrane during membrane remodeling.


Assuntos
Membrana Celular/química , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Anisotropia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Lipossomos/ultraestrutura , Modelos Biológicos , Polímeros/química , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo
16.
Sci Rep ; 10(1): 6938, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332809

RESUMO

Neurodegenerative diseases (Alzheimer's, Parkinson's etc.) causes brain cell damage leading to dementia. The major restriction remains in delivering drug to the central nervous system is blood brain barrier (BBB). The aim of this study was to develop a liposomal drug delivery system of Aphanamixis polystachya leaf extract for the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. In this study GC-MS analysis is used to determine major constituents of Aphanamixis polystachya leaf extract. Liposomal batches of Aphanamixis polystachya leaf extract was prepared using design of experiment (DoE) and characterized using Malvern zetasizer, transmission electron microscopy (TEM), and FT-IR. Stability study of blank and leaf extract loaded liposome were performed in gastric media. In-vivo neurobehavioral and anti-inflammatory studies were performed on mice and rat model respectively. GC-MS data showed that major constituents of Aphanamixis polystachya leaf extract are 2-Pentanone, different acids (Octadec-9-enoic acid, 5-Hydroxypipeloic acid etc.), and Beta-Elemene etc. Malvern Zetasizer and TEM data showed that liposome batches of Aphanamixis polystachya leaf extract were in the range of 120 - 180 nm. Interactions between process parameters and material attributes found to have more impact on the average particle size and polydispersity of liposome batches compared to the impact of each parameter in isolation. Stability studies data suggest that blank and leaf extract loaded liposomes were stable at gastric conditions after 4 hours. In-vivo neurobehavioural study data indicated that significant improvement in the memory function, locomotor activity and ambulatory performance of dementia induced mice was observed for the liposomal batches compared to merely A. polystachya leaf extract.


Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/fisiologia , Sistemas de Liberação de Medicamentos , Meliaceae/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Animais , Anti-Inflamatórios/farmacologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Lipossomos/ultraestrutura , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Modelos Animais , Tamanho da Partícula , Ratos Long-Evans , Espectroscopia de Infravermelho com Transformada de Fourier
17.
Elife ; 92020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286222

RESUMO

Cation-chloride-cotransporters (CCCs) catalyze transport of Cl- with K+ and/or Na+across cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, regulation of blood pressure, and renal function. CCCs are targets of clinically important drugs including loop diuretics and their disruption has been implicated in pathophysiology including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the structure of a CCC, the Mus musculus K+-Cl- cotransporter (KCC) KCC4, in lipid nanodiscs determined by cryo-EM. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We identify binding sites for substrate K+ and Cl- ions, demonstrate the importance of key coordinating residues for transporter activity, and provide a structural explanation for varied substrate specificity and ion transport ratio among CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.


Assuntos
Relação Estrutura-Atividade , Simportadores/ultraestrutura , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Lipossomos/química , Lipossomos/ultraestrutura , Camundongos , Nanopartículas/química , Nanopartículas/ultraestrutura , Conformação Proteica , Simportadores/química
18.
Biochim Biophys Acta Biomembr ; 1862(8): 183241, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126227

RESUMO

The presence of an asymmetric distribution of lipids in biological membranes was first described ca. 50 years ago. While various studies had reported the role of loss of lipid asymmetry on signaling processes, its effect on membrane physical properties and membrane-protein interactions lacks further understanding. The recent description of new technologies for the preparation of asymmetric model membranes has helped to fill part of this gap. However, the major effort so far has been on plasma membrane models. Here we describe the preparation of liposomes mimicking the mitochondria outer membrane (MOM) in regard to its lipid composition and asymmetry. By employing the methyl-ß-cyclodextrin-catalyzed lipid exchange technology and accurate quantification of lipid asymmetry with head group-specific probes we showed the successful preparation of a MOM model bearing a physiologically relevant lipid composition and asymmetry. In addition, by a direct comparison with its lipid symmetrical counterpart it is shown that asymmetric models were more resistant to tBid-promoted Bax-permeabilization, suggesting a role played by MOM lipid asymmetry on the mitochondria pathway of apoptosis. The barrier imposed by lipid asymmetry on membrane permeabilization was in part due to a decrease in the concentration of membrane-bound proteins, which was likely a consequence of the two mutually-dependent properties; i.e., the lower electrostatic surface potential and the higher molecular packing imposed by lipid asymmetry. It is proposed that MOM lipid asymmetry imparts different physical properties on the membrane and might add an additional component of regulation in intricate mitochondrial processes.


Assuntos
Lipídeos/química , Mitocôndrias/genética , Membranas Mitocondriais/química , Proteína X Associada a bcl-2/genética , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Lipossomos/química , Lipossomos/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/ultraestrutura , Fosfolipídeos/química , Fosfolipídeos/genética
19.
Int J Pharm ; 579: 119178, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32105722

RESUMO

OBJECTIVE: The dimeric artesunate phospholipid conjugate (Di-ART-GPC) is a novel amphipathic artemisinin derivative, which can be assembled into liposomes. Di-ART-GPC liposomes were prepared and evaluated as potential anti-inflammatory agents for rheumatic arthritis (RA). METHODS: Di-ART-GPC was assembled into liposomes utilizing thin film dispersion-high pressure homogenization. Dynamic light scattering (DLS), transmission electron microscopy (TEM), and electron cryo microscopy (cryo-EM) were employed to characterize the liposomal size and morphology. The in vitro cytotoxicity of the Di-ART-GPC liposomes was assessed using Cell Counting Kit-8 (CCK8). The anti-inflammatory effects were studied utilizing the inflammatory cell model. Finally, the in vivo efficacy of the Di-ART-GPC-conjugated liposomes was investigated using the arthritis rat model. RESULTS: The particle size of the Di-ART-GPC liposomes decreased to a narrow range of approximately 70 nm following high-pressure homogenization. The in vitro studies revealed low cytotoxicity and good anti-inflammatory effects of the Di-ART-GPC liposomes, which exhibited significantly higher inhibition of the cell secretion of pro-inflammatory cytokines than ART. The in vivo evaluation confirmed that treatment with Di-ART-GPC resulted in a decline in the ankle swelling rate and a low inflammatory response compared with the model control and ART. CONCLUSION: Di-ART-GPC liposomes demonstrate remarkable potential as novel ART-based anti-inflammatory agents for RA.


Assuntos
Anti-Inflamatórios/farmacologia , Artesunato/química , Artrite Reumatoide/prevenção & controle , Lipossomos/ultraestrutura , Fosfolipídeos/química , Pró-Fármacos/uso terapêutico , Animais , Anti-Inflamatórios/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Tamanho da Partícula , Pró-Fármacos/química , Ratos
20.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952316

RESUMO

This study aims to evaluate the potency of cisplatin (Cispt)-loaded liposome (LCispt) and PEGylated liposome (PLCispt) as therapeutic nanoformulations in the treatment of bladder cancer (BC). Cispt was loaded into liposomes using reverse-phase evaporation method, and the formulations were characterized using dynamic light scattering, scanning electron microscopy, dialysis membrane, and Fourier-transform infrared spectroscopy (FTIR) methods. The results showed that the particles were formed in spherical monodispersed shapes with a nanoscale size (221-274 nm) and controlled drug release profile. The cytotoxicity effects of LCispt and PLCispt were assessed in an in vitro environment, and the results demonstrated that PLCispt caused a 2.4- and 1.9-fold increase in the cytotoxicity effects of Cispt after 24 and 48 h, respectively. The therapeutic and toxicity effects of the formulations were also assessed on BC-bearing rats. The results showed that PLCispt caused a 4.8-fold increase in the drug efficacy (tumor volume of 11 ± 0.5 and 2.3 ± 0.1 mm3 in Cispt and PLCispt receiver rats, respectively) and a 3.3-fold decrease in the toxicity effects of the drug (bodyweight gains of 3% and 10% in Cispt and PLCispt receiver rats, respectively). The results of toxicity were also confirmed by histopathological studies. Overall, this study suggests that the PEGylation of LCispt is a promising approach to achieve a nanoformulation with enhanced anticancer effects and reduced toxicity compared to Cispt for the treatment of BC.


Assuntos
Cisplatino/farmacologia , Lipossomos/química , Polietilenoglicóis/química , Neoplasias da Bexiga Urinária/prevenção & controle , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Butilidroxibutilnitrosamina , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/química , Cisplatino/farmacocinética , Liberação Controlada de Fármacos , Feminino , Humanos , Lipossomos/ultraestrutura , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Ratos Wistar , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Neoplasias da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/metabolismo
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