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1.
Biomolecules ; 11(8)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34439800

RESUMO

The eye is at the forefront of developing therapies for genetic diseases. With the FDA approval of the first gene-therapy drug for a form of congenital blindness, numerous studies have been initiated to develop gene therapies for other forms of eye diseases. These examinations have revealed new information about the benefits as well as restrictions to using drug-delivery routes to the different parts of the eye. In this article, we will discuss a brief history of gene therapy and its importance to the eye and ocular delivery landscape that is currently being investigated, and provide insights into their advantages and disadvantages. Efficient delivery routes and vehicle are crucial for an effective, safe, and longer-lasting therapy.


Assuntos
Oftalmopatias/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Animais , Efusões Coroides , DNA/genética , DNA/metabolismo , DNA/uso terapêutico , Olho/metabolismo , Olho/patologia , Oftalmopatias/genética , Oftalmopatias/metabolismo , Oftalmopatias/patologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , Humanos , Injeções Intravítreas , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/uso terapêutico , Nanopartículas/administração & dosagem , Nanopartículas/química , Nanopartículas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Líquido Sub-Retiniano , Vírus/genética , Vírus/metabolismo , Corpo Vítreo
2.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361779

RESUMO

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.


Assuntos
Cavéolas/efeitos dos fármacos , Colesterol/química , Células Endoteliais/efeitos dos fármacos , Lipossomos/química , Microdomínios da Membrana/efeitos dos fármacos , Transfecção/métodos , Animais , Cavéolas/química , Cavéolas/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Clatrina/metabolismo , DNA/química , DNA/metabolismo , Endocitose/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Filipina/química , Filipina/farmacologia , Expressão Gênica , Lipossomos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Nistatina/química , Nistatina/farmacologia , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacologia , Pinocitose/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos
3.
Nat Commun ; 12(1): 4972, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404795

RESUMO

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells.


Assuntos
Fusão de Membrana/fisiologia , Membranas/metabolismo , Polímeros/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Animais , Microscopia Crioeletrônica , Lipossomos/metabolismo , Proteínas do Tecido Nervoso , Ligação Proteica , Proteínas R-SNARE , Ratos , Proteína 25 Associada a Sinaptossoma , Sintaxina 1 , Proteína 2 Associada à Membrana da Vesícula
4.
Nat Nanotechnol ; 16(9): 1039-1044, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34294909

RESUMO

Plasma SARS-CoV-2 RNA may represent a viable diagnostic alternative to respiratory RNA levels, which rapidly decline after infection. Quantitative PCR with reverse transcription (RT-qPCR) reference assays exhibit poor performance with plasma, probably reflecting the dilution and degradation of viral RNA released into the circulation, but these issues could be addressed by analysing viral RNA packaged into extracellular vesicles. Here we describe an assay approach in which extracellular vesicles directly captured from plasma are fused with reagent-loaded liposomes to sensitively amplify and detect a SARS-CoV-2 gene target. This approach accurately identified patients with COVID-19, including challenging cases missed by RT-qPCR. SARS-CoV-2-positive extracellular vesicles were detected at day 1 post-infection, and plateaued from day 6 to the day 28 endpoint in a non-human primate model, while signal durations for 20-60 days were observed in young children. This nanotechnology approach uses a non-infectious sample and extends virus detection windows, offering a tool to support COVID-19 diagnosis in patients without SARS-CoV-2 RNA detectable in the respiratory tract.


Assuntos
COVID-19/diagnóstico , Vesículas Extracelulares/metabolismo , Lipossomos/uso terapêutico , RNA Viral/sangue , SARS-CoV-2/isolamento & purificação , Animais , Técnicas Biossensoriais , COVID-19/sangue , Teste de Ácido Nucleico para COVID-19 , Chlorocebus aethiops , Modelos Animais de Doenças , Células HEK293 , Humanos , Cinética , Lipossomos/metabolismo , RNA Viral/genética , SARS-CoV-2/genética , Tetraspanina 28/imunologia , Tetraspanina 28/metabolismo
5.
Sci Rep ; 11(1): 14748, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285303

RESUMO

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (Km and Vmax) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose-response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.


Assuntos
Candida albicans/enzimologia , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Lipossomos/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Cinética , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200063

RESUMO

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.


Assuntos
Archaea/fisiologia , Membrana Celular/fisiologia , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fluidez de Membrana , Esqualeno/análogos & derivados , Archaea/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Esqualeno/farmacologia , Temperatura
7.
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
8.
Nat Commun ; 12(1): 4121, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226541

RESUMO

Effective treatments of neurodegenerative diseases require drugs to be actively transported across the blood-brain barrier (BBB). However, nanoparticle drug carriers explored for this purpose show negligible brain uptake, and the lack of basic understanding of nanoparticle-BBB interactions underlies many translational failures. Here, using two-photon microscopy in mice, we characterize the receptor-mediated transcytosis of nanoparticles at all steps of delivery to the brain in vivo. We show that transferrin receptor-targeted liposome nanoparticles are sequestered by the endothelium at capillaries and venules, but not at arterioles. The nanoparticles move unobstructed within endothelium, but transcytosis-mediated brain entry occurs mainly at post-capillary venules, and is negligible in capillaries. The vascular location of nanoparticle brain entry corresponds to the presence of perivascular space, which facilitates nanoparticle movement after transcytosis. Thus, post-capillary venules are the point-of-least resistance at the BBB, and compared to capillaries, provide a more feasible route for nanoparticle drug carriers into the brain.


Assuntos
Encéfalo/metabolismo , Capilares/metabolismo , Portadores de Fármacos , Nanopartículas/uso terapêutico , Transcitose/fisiologia , Vênulas/metabolismo , Animais , Arteríolas , Transporte Biológico , Barreira Hematoencefálica , Capilares/patologia , Endotélio/diagnóstico por imagem , Endotélio/patologia , Cinética , Lipossomos/metabolismo , Camundongos , Receptores da Transferrina/metabolismo , Vênulas/patologia
9.
Chemphyschem ; 22(15): 1547-1565, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34086399

RESUMO

The aggregation and deposition of amyloid ß (Aß) peptide onto neuronal cells, with consequent cellular membrane perturbation, are central to the pathogenesis of Alzheimer's disease (AD). Substantial evidence reveals that biological membranes play a key role in this process. Thus, elucidating the mechanisms by which Aß interacts with biomembranes and becomes neurotoxic is fundamental to developing effective therapies for this devastating progressive disease. However, the structural basis behind such interactions is not fully understood, largely due to the complexity of natural membranes. In this context, lipid biomembrane models provide a simplified way to mimic the characteristics and composition of membranes. Aß-biomembrane interactions have been extensively investigated applying artificial membrane models to elucidate the molecular mechanisms underlying the AD pathogenesis. This review summarizes the latest findings on this field using liposomes as biomembrane model, as they are considered the most promising 3D model. The current challenges and future directions are discussed.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Lipossomos/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Membrana Celular/química , Membrana Celular/patologia , Permeabilidade da Membrana Celular , Humanos , Lipossomos/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Estresse Oxidativo
10.
FEBS Lett ; 595(14): 1914-1919, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34080704

RESUMO

Biological structures with highly curved membranes, such as caveolae and transport vesicles, are essential for signal transduction and membrane trafficking. Although membrane proteins in these structures are subjected to physical stress due to the curvature of the lipid bilayers, the effect of this membrane curvature on protein structure and function remains unclear. In this study, we established an experimental procedure to evaluate membrane curvature-induced structural changes in the prototypical potassium channel KcsA. The effect of a large membrane curvature was estimated using fluorescently labeled KcsA by incorporating it into liposomes with a small diameter (< 30 nm). We found that a large membrane curvature significantly affects the activation gate conformation of the KcsA channel.


Assuntos
Proteínas de Bactérias/química , Lipossomos/química , Fosfatidilcolinas/química , Canais de Potássio/química , Potássio/química , Coloração e Rotulagem/métodos , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quimotripsina/química , Corantes Fluorescentes/química , Expressão Gênica , Transporte de Íons , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fosfatidilcolinas/metabolismo , Potássio/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Rodaminas/química , Streptomyces coelicolor/genética
11.
Theranostics ; 11(14): 6860-6872, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093858

RESUMO

Background: Immunotherapy has profoundly changed the landscape of cancer management and represented the most significant breakthrough. Yet, it is a formidable challenge that the majority of cancers - the so-called "cold" tumors - poorly respond to immunotherapy. To find a general immunoregulatory modality that can be applied to a broad spectrum of cancers is an urgent need. Methods: Magnetic hyperthermia (MHT) possesses promise in cancer therapy. We develop a safe and effective therapeutic strategy by using magnetism-mediated targeting MHT-immunotherapy in "cold" colon cancer. A magnetic liposomal system modified with cell-penetrating TAT peptide was developed for targeted delivery of a CSF1R inhibitor (BLZ945), which can block the CSF1-CSF1R pathway and reduce M2 macrophages. The targeted delivery strategy is characterized by its magnetic navigation and TAT-promoting intratumoral penetration. Results: The liposomes (termed TAT-BLZmlips) can induce ICD and cause excessive CRT exposure on the cell surface, which transmits an "eat-me" signal to DCs to elicit immunity. The combination of MHT and BLZ945 can repolarize M2 macrophages in the tumor microenvironment to relieve immunosuppression, normalize the tumor blood vessels, and promote T-lymphocyte infiltration. The antitumor effector CD8+ T cells were increased after treatment. Conclusion: This work demonstrated that TAT-BLZmlips with magnetic navigation and MHT can remodel tumor microenvironment and activate immune responses and memory, thus inhibiting tumor growth and recurrence.


Assuntos
Neoplasias do Colo/terapia , Terapia Combinada/métodos , Hipertermia , Imunoterapia/métodos , Lipossomos/química , Terapia de Campo Magnético/métodos , Nanopartículas de Magnetita/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Benzotiazóis/farmacocinética , Benzotiazóis/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/imunologia , Feminino , Humanos , Lipossomos/metabolismo , Lipossomos/efeitos da radiação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/terapia , Ácidos Picolínicos/farmacocinética , Ácidos Picolínicos/farmacologia , Ratos , Microambiente Tumoral/efeitos dos fármacos , Macrófagos Associados a Tumor/efeitos dos fármacos , Macrófagos Associados a Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Theranostics ; 11(14): 7110-7125, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093874

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fatal interstitial lung disease characterized by abnormal transition and proliferation of fibroblasts. The uncontrolled transition of fibroblasts, commonly known as myofibroblasts, are the principal source of the enormous extracellular matrix (ECM) depositing in lung parenchyma, leading to gradual failure of gas exchange and mortality of the patients. However, up to now, rare effective therapeutic strategies have been developed to blockade fibroblast-to-myofibroblast transition (FMT) in IPF. Method: We illustrated that the lungs originated from IPF patients and mice with pulmonary fibrosis are characterized by the overexpression of sushi-repeat-containing protein, X-linked 2 (SRPX2). Further functionality studies identified the pivotal role of SRPX2 in FMT. Mechanistically, SRPX2 was involved in a TGFßR1/SMAD3/SRPX2/AP1/SMAD7 positive feedback loop. Specifically, SRPX2 was upregulated by TGF-ß1 in a TGFßR1/SMAD3-dependent manner, after which SRPX2 in turn repressed the expression of AP1, subsequently minimized SMAD7 expression, through which it reduced the formation of inhibitory complex with TGFßR1 and enhanced SMAD signaling pathway to promote FMT and exacerbate pulmonary fibrosis. Notably, intratracheal administration of siRNA-loaded liposomes could effectively suppress the expression of Srpx2 in the lung and remarkably protect mice against BLM-induced pulmonary fibrosis, concomitant with a significant reduction of FMT. Results: Accordingly, these data indicate that Srpx2 plays an essential role in the pathogenesis of pulmonary fibrosis and suggests the strategy aiming at silencing Srpx2 could be a promising therapeutic approach against pulmonary fibrosis in clinical settings.


Assuntos
Proliferação de Células/genética , Fibroblastos/metabolismo , Terapia Genética/métodos , Lipossomos/administração & dosagem , Proteínas de Membrana/metabolismo , Mioblastos/metabolismo , Proteínas de Neoplasias/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/terapia , Idoso , Animais , Movimento Celular/genética , Retroalimentação Fisiológica , Feminino , Fibroblastos/patologia , Inativação Gênica , Humanos , Lipossomos/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mioblastos/patologia , Proteínas de Neoplasias/genética , Fibrose Pulmonar/genética , RNA Interferente Pequeno , RNA-Seq , Proteína Smad3/metabolismo , Proteína Smad7/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima
13.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069905

RESUMO

Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and initiate lipid exchange. These isolated fragments, known as fusion peptides (HAfp), already possess own fusogenic activity towards liposomes. Although they have long been studied with the hope to uncover the details of HA-mediated fusion, their actual mechanism of action remains elusive. Here, we use extensive molecular dynamics simulations combined with experimental studies of three HAfp variants to fully characterize their free energy landscape and interaction with lipid bilayer. In addition to customary assumed peptides localization at lipid-water interface, we characterize membrane-spanning configurations, which turn out to be metastable for active HAfps and unstable for the fusion inactive W14A mutant. We show that, while the degree of membrane perturbation by surface peptide configurations is relatively low and does not show any mutation-related differences, the effect of deeply inserted configurations is significant and correlates with insertion depth of the N-terminal amino group which is the highest for the wild type HAfp. Finally, we demonstrate the feasibility of spontaneous peptide transition to intramembrane location and the critical role of strictly conserved tryptofan residue 14 in this process.


Assuntos
Orthomyxoviridae/metabolismo , Proteínas Virais de Fusão/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Bicamadas Lipídicas/química , Lipossomos/metabolismo , Fusão de Membrana , Membranas/metabolismo , Modelos Teóricos , Simulação de Dinâmica Molecular , Orthomyxoviridae/patogenicidade , Proteínas Virais de Fusão/química
14.
J Agric Food Chem ; 69(27): 7593-7602, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34190554

RESUMO

The present study describes the development of a novel liposome nanocarrier system. The liposome was coated with Lactobacillus acidophilus CICC 6074 S-layer protein (SLP) to improve the intestinal absorption of the cholesterol-lowering peptide Leu-Gln-Pro-Glu (LQPE). The SLP-coated liposomes were prepared and characterized with morphology, particle size, zeta potential, membrane stability, Fourier transform infrared spectroscopy, and dual-channel surface plasma resonance. The results showed that SLP could successfully self-assemble on liposomes. Then, LQPE liposomes and SLP-coated LQPE liposomes (SLP-L-LQPE) were prepared. SLP-L-LQPE not only showed better sustained release properties and gastrointestinal tolerance in vitro but also increased the retention time in mice intestine. Transepithelial transport experiment indicates that the transshipment of LQPE increased significantly after being embedded by liposomes and coated with SLP. The research provides a theoretical basis for the study of SLP-coated liposomes and a potential drug delivery system for improving the intestinal absorption of peptides.


Assuntos
Absorção Intestinal , Lactobacillus acidophilus , Lipossomos , Peptídeos/metabolismo , Animais , Mucosa Intestinal/metabolismo , Lipossomos/metabolismo , Camundongos , Tamanho da Partícula
15.
Nat Commun ; 12(1): 3310, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083531

RESUMO

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Fenômenos Biomecânicos , Divisão Celular/fisiologia , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Hidrólise , Lipossomos/metabolismo , Proteínas Luminescentes/metabolismo , Membranas/metabolismo , Modelos Biológicos , Pinças Ópticas , Proteínas Recombinantes de Fusão/metabolismo , Torção Mecânica
16.
PLoS Genet ; 17(5): e1009137, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33999937

RESUMO

Polarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified a F-BAR domain protein (ArF-BAR) in the necrotrophic fungus Ascochyta rabiei and demonstrate its involvement in endosome-dependent fungal virulence on the host plant Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is necessary for virulence. The loss-of-function of ArF-BAR gene results in delayed formation of apical septum in fungal cells near growing hyphal tip that is crucial for host penetration, and impaired secretion of a candidate effector having secretory signal peptide for translocation across the endoplasmic reticulum membrane. The mRNA transcripts of ArF-BAR were induced in response to oxidative stress and infection. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formation in vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1), as key transcriptional regulator of ArF-BAR expression. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate A. rabiei virulence through a mechanism that involves endocytosis, effector secretion, and actin cytoskeleton regulation.


Assuntos
Actinas/metabolismo , Ascomicetos/citologia , Ascomicetos/patogenicidade , Cicer/microbiologia , Endocitose , Proteínas Fúngicas/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Endocitose/genética , Endossomos/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Lipossomos/metabolismo , Mutação , Estresse Oxidativo , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas/genética , Virulência/genética
17.
Chem Biol Interact ; 345: 109511, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-33989593

RESUMO

Methylglyoxal is a dicarbonyl compound recruited as a potential cytotoxic marker, initially presents in cells and considered as a metabolite of the glycolytic pathway. Our aim is to demonstrate the inhibitory effect of 3, 3'-[3-(5-chloro-2-hydroxyphenyl)-3-oxopropane-1, 1-diyl] Bis (4-hydroxycoumarin) on the glyoxalase system, and indirectly its anticancer activity. The docking of OT-55 was conducted by using Flexible docking protocol, ChiFlex and libdock tools inside the active site of Glo-I indicated that both hydrogen bonding and hydrophobic interactions contributed significantly in establishing potent binding with the active site which is selected as a strong inhibitor with high scoring values and maximum Gibbs free energy. Coumarin-liposome formulation was characterized and evaluated in vivo against chemically induced hepatocarcinoma in Wistar rats. After Diethylnitrosamine (DEN) induction, microscopic assessment was realized; precancerous lesions were developed showing an increase of both tumor-associated lymphocyte and multiple tumor acini supported by the blood investigation. Our finding also suggested a preferential uptake of liposomes respectively in liver, kidney, lung, brain and spleen in the DEN-treated animals. OT-55 has also been shown to inhibit the activity of Glo-I in vitro as well as in DEN-treated rats. An abnormal high level of MGO of up to 50% was recorded followed by a reduction in glucose consumption and lactate dehydrogenase production validated in the positive control. MGO generates apoptosis as depicted by focal hepatic lesions. Also, no deleterious effects in the control group were observed after testing our coumarin but rather a vascular reorganization leading to nodular regenerative hyperplasia. Involved in the detoxification process, liver GSH is restored in intoxicated rats, while no changes are seen between controls. At the endothelial cell, OT-55 appears to modulate the release of NO only in the DEN-treated group. OT-55 would behave both as an anticancer agent but also as an angiogenic factor regarding results obtained.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Espaço Intracelular/efeitos dos fármacos , Lactoilglutationa Liase/antagonistas & inibidores , Neoplasias Hepáticas/patologia , Modelos Moleculares , Aldeído Pirúvico/metabolismo , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transporte Biológico , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lactoilglutationa Liase/química , Lactoilglutationa Liase/metabolismo , Lipossomos/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Conformação Proteica , Ratos , Ratos Wistar , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Int J Nanomedicine ; 16: 3073-3089, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953556

RESUMO

Introduction: Hyperoside (HYP), a flavonol glycoside compound, has been shown to significantly inhibit the proliferation of malignant tumors. Mitochondria serve as both "energy factories" and "suicide weapon stores" of cells. Targeted delivery of cytotoxic drugs to the mitochondria of tumor cells and tumor vascular cells is a promising strategy to improve the efficacy of chemotherapy. Objective: We report a novel dual-functional liposome system possessing both extracellular charge reversal and mitochondrial targeting properties to enhance drug accumulation in mitochondria and trigger apoptosis of cancer cells. Methods: L-lysine was used as a linker to connect 2,3-dimethylmaleic anhydride (DMA) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) to yield a new compound, DSPE-Lys-DMA (DLD). Then, DLD was mixed with other commercially available lipids to form charge reversed and mitochondria-targeted liposomes (DLD-Lip). The size, morphology, zeta potential, serum stability, and protein adsorption of the HYP loaded DLD-Lip (HYP/DLD-Lip) were measured. The release profile, cellular uptake, in vitro and in vivo toxicity, and anticancer activity of HYP/DLD-Lip were investigated. Results: The results showed that the mean diameter of the liposomes was less than 200 nm. The zeta potential of the liposomes was negative at pH 7.4. However, the zeta potential was positive at weak acidic pH values with the cleavage of the DMA amide. The charge reversion of HYP/DLD-Lip facilitated the cellular internalization and mitochondrial accumulation for enhanced antitumor effect. The strongest tumor growth inhibition (TGI 88.79%) without systemic toxicity was observed in DLD/HYP-Lips-treated CBRH-7919 tumor xenograft BALB/C mice. Conclusion: The charge reversed and mitochondria-targeted liposomes represented a promising anticancer drug delivery system for enhanced anticancer therapeutic efficacy.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Lipossomos/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Quercetina/análogos & derivados , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidiletanolaminas/química , Quercetina/administração & dosagem , Quercetina/química , Quercetina/farmacologia
19.
AAPS PharmSciTech ; 22(5): 164, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34041632

RESUMO

Psoriasis is a life-threatening autoimmune inflammatory skin disease, triggered by T lymphocyte. Recently, the drugs most commonly used for the treatment of psoriasis include methotrexate (MTX), cyclosporine (CsA), acitretin, dexamethasone, and salicylic acid. However, conventional formulations due to poor absorptive capacity, inconsistent drug release characteristics, poor capability of selective targeting, poor retention of drug molecules in target tissue, and unintended skin reactions restrict the clinical efficacy of drugs. Advances in topical nanocarriers allow the development of prominent drug delivery platforms can be employed to address the critical issues associated with conventional formulations. Advances in nanocarriers design, nano-dimensional configuration, and surface functionalization allow formulation scientists to develop formulations for a more effective treatment of psoriasis. Moreover, interventions in the size distribution, shape, agglomeration/aggregation potential, and surface chemistry are the significant aspects need to be critically evaluated for better therapeutic results. This review attempted to explore the opportunities and challenges of current revelations in the nano carrier-based topical drug delivery approach used for the treatment of psoriasis.


Assuntos
Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/tendências , Nanocápsulas/administração & dosagem , Psoríase/tratamento farmacológico , Administração Cutânea , Animais , Ciclosporina/administração & dosagem , Ciclosporina/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos/efeitos dos fármacos , Liberação Controlada de Fármacos/fisiologia , Humanos , Lipossomos/administração & dosagem , Lipossomos/metabolismo , Metotrexato/administração & dosagem , Metotrexato/metabolismo , Psoríase/metabolismo , Ácido Salicílico/administração & dosagem , Ácido Salicílico/metabolismo
20.
Methods Mol Biol ; 2256: 75-87, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34014517

RESUMO

Surface plasmon resonance (SPR)/BIAcore technology enables the characterization of molecular interactions, including determination of affinities and kinetics. In BIAcore, one of the interaction partners (the ligand) is immobilized on a chip and the other (the analyte) is provided in solution. BIAcore allows to study association and dissociation rates in real time without the use of labeling. BIAcore can be applied to molecular interactions involving small compounds and biological macromolecules such as proteins, lipids, nucleic acids, or carbohydrates. Here we describe protocols for the measurements of PDZ domain-peptide (oriented biotinylated peptides), PDZ domain-liposomes (lipid membranes), and PDZ-lipid-peptide tripartite interactions.


Assuntos
Técnicas Biossensoriais/métodos , Lipossomos/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Domínios PDZ , Fragmentos de Peptídeos/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Humanos , Cinética , Ligantes , Ligação Proteica
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