RESUMO
Lipid-based nanoparticles and lipoplexes containing ionizable lipids are among the most successful nanocarriers for mRNA-based therapies. The molecular structure of these assemblies is still not fully understood, as well as the role played by the ionizable lipids. SAXS experiments have shown that lipoplexes including the ionizable lipid 2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), under specific conditions, have a lamellar structure, where lipid bilayers are separated by mRNA-rich layers, with an overall spacing between 6.5 and 8.0 nm and a complex pH-dependence. Here, the structure and dynamics of these lipoplexes are investigated at varying pH and mRNA concentration using multiscale molecular dynamics simulations. It is observed that the interaction between DODMA and RNA is slightly attractive only at low pH levels, while it becomes effectively repulsive at high and intermediate pH. This results into a pH-dependent relocation of the RNA inside the multilayers, from the lipid head groups at low pH to a more uniform distribution inside the hydrophilic slabs of the multilayers at high pH. It is also observed that at high pH, DODMA lipids shift toward the hydrophobic part of the bilayer, consequently increasing their leaflet-flipping rate, a phenomenon which may ultimately affect the fusion process of the lipoplex with the endosomal membrane.
Assuntos
Lipídeos , Simulação de Dinâmica Molecular , Cátions/química , Concentração de Íons de Hidrogênio , Lipídeos/química , Lipossomos/química , RNA Mensageiro , Espalhamento a Baixo Ângulo , Transfecção , Difração de Raios XRESUMO
Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.
Assuntos
Portadores de Fármacos/química , Composição de Medicamentos/métodos , RNA Mensageiro/administração & dosagem , Animais , Cátions/química , Linhagem Celular , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Bicamadas Lipídicas/química , Lipossomos , Luciferases/genética , Camundongos , Modelos Moleculares , Mioblastos , RNA Mensageiro/síntese química , Espalhamento a Baixo Ângulo , Transfecção/métodos , Difração de Raios XRESUMO
Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.
Assuntos
Nanopartículas/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Animais , Cromatografia Líquida , Lipossomos/química , Espectrometria de Massas , Camundongos , Tamanho da PartículaRESUMO
We have used pulsed field gradient (PFG)-NMR diffusion experiments, also known as DOSY, in combination with small angle X-ray scattering measurements to investigate structure and molecular exchange dynamics between pharmaceutical lipid nanoparticles and the bulk phase. Using liposomes and lipoplexes formed after complexation of the liposomes with messenger mRNA as test systems, information on dynamics of encapsulated water molecules, lipids and excipients was obtained. The encapsulated fraction, having a diffusivity similar to that of the liposomes, could be clearly identified and quantified by the NMR diffusion measurements. The unilamellar liposome membranes allowed a fast exchange of water molecules, while sucrose, used as an osmolyte and model solute, showed very slow exchange. Upon interactions with mRNA a topological transition from a vesicular to a lamellar organization took place, where the mRNA was inserted in repeating lipid bilayer stacks. In the lipoplexes, a small fraction of tightly bound water molecules was present, with a diffusivity that was influenced by the additional presence of sucrose. This extended information on dynamic coherencies inside pharmaceutical nanoparticle products, provided by the combined application of SAXS and PFG-NMR diffusion measurements, can be valuable for evaluation of quality and comparability of nanoscaled pharmaceuticals.
Assuntos
Lipossomos , Espectroscopia de Ressonância Magnética , Nanopartículas , RNA Mensageiro , Espalhamento a Baixo Ângulo , Difração de Raios X , Nanopartículas/química , Espectroscopia de Ressonância Magnética/métodos , Difusão , Cinética , Difração de Raios X/métodos , Sacarose/química , Lipídeos/química , Água/química , Excipientes/química , Bicamadas Lipídicas/químicaRESUMO
mRNA based infectious disease vaccines have opened the venue for development of novel nucleic acids-based therapeutics. For all mRNA therapeutics dedicated delivery systems are required, where different functionalities and targeting abilities need to be optimized for the respective applications. One option for advanced formulations with tailored properties are lipid-polymer hybrid nanoparticles with complex nanostructure, which allow to combine features of several already well described nucleic acid delivery systems. Here, we explored hyaluronic acid (HA) as coating of liposome-mRNA complexes (LRCs) to investigate effects of the coating on surface charge, physicochemical characteristics and biological activity. HA was electrostatically attached to positively charged complexes, forming hybrid LRCs (HLRCs). At different N/P ratios, physico-chemical characterization of the two sets of particles showed similarity in size (around 200 nm) and mRNA binding abilities, while the presence of the HA shell conferred a negative surface charge to otherwise positive complexes. High transfection efficiency of LRCs and HLRCs in vitro has been obtained in THP-1 and human monocytes derived from PBMC, an interesting target cell population for cancer and immune related pathologies. In mice, quantitative biodistribution of radiolabeled LRC and HLRC particles, coupled with bioluminescence studies to detect the protein translation sites, hinted towards both particles' accumulation in the hepatic reticuloendothelial system (RES). mRNA translated proteins though was found mainly in the spleen, a major source for immune cells, with preference for expression in macrophages. The results showed that surface modifications of liposome-mRNA complexes can be used to fine-tune nanoparticle physico-chemical characteristics. This provides a tool for assembly of stable and optimized nanoparticles, which are prerequisite for future therapeutic interventions using mRNA-based nanomedicines.
Assuntos
Nanopartículas , Ácidos Nucleicos , Camundongos , Humanos , Animais , Lipossomos/química , Distribuição Tecidual , Leucócitos Mononucleares , Polímeros/química , Nanopartículas/química , TransfecçãoRESUMO
Liposomal formulations are used to improve the safety and cellular absorption of conventional drugs by limiting their interaction with phagocytes. The uptake behaviour of these nanocarriers is affected by the blood composition, and accordingly the presence of an anticoagulant in the blood could have a critical impact on the efficiency of nanomedicines. For the negatively charged liposomes, such as AmBisome®, no significant change in the uptake could be observed when co-incubated with heparin and primary phagocytes. Yet, we observed that a peak of the uptake extent of cationic liposomes was reached at a clinically relevant concentration of heparin for phagocytes and cancer cells. Hence, we recommend avoiding treatment of a heparinized patient with cationic nanomedicines because unexpectedly high uptake can occur in phagocytes.
Assuntos
Heparina , Nanomedicina , Anticoagulantes , Cátions , Humanos , LipossomosRESUMO
Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.
Assuntos
Biopolímeros/química , Lipídeos/química , Nanopartículas/química , RNA Mensageiro/metabolismo , Transfecção/métodos , Animais , Linhagem Celular , Ácidos Graxos Monoinsaturados/química , Feminino , Heparina/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Imagem Óptica , Tamanho da Partícula , Compostos de Amônio Quaternário/química , RNA Mensageiro/químicaRESUMO
We studied the incorporation of the hydrophobic anticancer drug paclitaxel (PXL), into a variety of lipid matrices by X-ray diffraction (XRD) measurements. Liposome suspensions from cationic and zwitterionic lipids, containing different molar fractions of paclitaxel were made and deposited on planar glass substrates. After drying at controlled relative humidity, aligned multilayer stacks were obtained. The structure perpendicular to the substrate plane was investigated by X-ray diffraction measurements. Bragg peaks to several orders were detected, indicative of well-ordered multilamellar lipid layers. The drug induced a modification of the bilayer spacing, which was the characteristic for a given type of lipid matrix. With an excess of the drug, Bragg peaks of drug crystals could be observed. The results provide insight into the solubility of paclitaxel in the different lipid membranes. A structural model of the organization of the drug in the membrane was discussed.
Assuntos
Lipossomos/química , Modelos Biológicos , Paclitaxel/química , Difração de Raios X , Interações Hidrofóbicas e Hidrofílicas , Íons , Bicamadas Lipídicas , SolubilidadeRESUMO
Neutron reflectivity measurements were used to investigate the thermal stability of layer-by-layer (LBL) films of poly(o-methoxyaniline) (POMA), which was probed by increasing the temperature up to 80 degrees C of a D2O solution in contact with the LBL films. The study was made possible by adsorbing POMA layers on a PEI/(PSS/PAH)5/PSS LBL film template, leading to less rough POMA layers in comparison with the POMA/poly(vinylsulfonic acid sodium salt) (PVS) LBL films adsorbed directly on glass and silicon substrates. While the latter yielded almost fringeless neutron reflectivity curves due to the large roughness, the fitting of the data for POMA films adsorbed onto the template film and UV-vis measurements indicated that the topmost layer is affected for films heated in solution up to 80 degrees C. This is essentially the same thermal stability of LBL films from the template films made with conventional polyelectrolytes. A decrease in thickness of approximately 10 A was inferred when the solution temperature increased from 25 degrees C to 80 degrees C, which was maintained when the sample was cooled back to 25 degrees C. This decrease, observed for solutions of pH 3 and pH 8, is consistent with thermally-stimulated desorption and was corroborated by UV-VIS absorption experiments. The unexpected stability of the POMA layer at pH 8 is attributed to the layer-by-layer structure of the films that allows POMA to remain doped, in its salt emeraldine form, even at high pH.
Assuntos
Compostos de Anilina/análise , Compostos de Anilina/química , Teste de Materiais/métodos , Membranas Artificiais , Nanoestruturas/química , Refratometria/métodos , Espectrofotometria Ultravioleta/métodos , Fluidez de Membrana , Conformação Molecular , Nanoestruturas/análise , Nanotecnologia/métodos , TemperaturaRESUMO
The development of nucleic acid based vaccines against cancer has gained considerable momentum through the advancement of modern sequencing technologies and on novel RNA-based synthetic drug formats, which can be readily adapted following identification of every patient's tumor-specific mutations. Furthermore, affordable and individual 'on demand' production of molecularly optimized vaccines should allow their application in large groups of patients. This has resulted in the therapeutic concept of an active personalized cancer vaccine, which has been brought into clinical testing. Successful trials have been performed by intranodal administration of sterile isotonic solutions of synthetic RNA vaccines. The second generation of RNA vaccines which is currently being developed encompasses intravenously injectable RNA nanoparticle formulations (lipoplexes), made up from lipid excipients, denoted RNA(LIP). A first product that has made its way from bench to bedside is a therapeutic vaccine for intravenous administration based on a fixed set of four RNA lipoplex drug products, each encoding for one shared tumor antigen (Lipoplex Melanoma RNA Immunotherapy, 'Lipo-MERIT'). This article describes the steps for translating these novel RNA nanomedicines into clinical trials.
Assuntos
Vacinas Anticâncer , Lipossomos/química , Melanoma/terapia , Nanopartículas/química , RNA Mensageiro/química , RNA/química , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Ensaios Clínicos como Assunto , Excipientes , Humanos , Imunoterapia/métodos , Melanoma/imunologia , Nanomedicina , Nanopartículas/uso terapêutico , Medicina de Precisão , RNA/administração & dosagem , RNA/imunologia , RNA Mensageiro/administração & dosagem , RNA Mensageiro/farmacologia , RNA Mensageiro/uso terapêuticoRESUMO
Asymmetrical flow field-flow fractionation (AsFlFFF)/multi-angle light scattering (MALS) was employed for studying filter-extruded liposomes in carrier solutions with different ionic strength and osmolarity. By dilution of preformed liposome suspensions with different media, only the ionic strength in the external free aqueous phase was changed. Under such conditions the liposomes were found to elute at almost identical elution times, which is in contrast to earlier studies. This may be explained by two opposing effects: (a) modulation of inter-particulate and particle-wall-repulsion effects and (b) osmotic stress-induced changes in vesicle size. The latter effect was demonstrated when analysing liposomes upon dilution in media of constant ionic strength, but varying osmotic pressure (with or without 150mmolL(-1) sucrose supplement). The osmotic stress-induced change in liposome size was found to be size dependent. Larger liposomes appeared to both shrink and swell when exposed to hyper- or hypoosmotic media, respectively. Smaller liposomes appeared to shrink but not to swell. The potential causes of this effect are discussed.
Assuntos
Fracionamento por Campo e Fluxo/métodos , Luz , Lipossomos/análise , Lipossomos/química , Concentração Osmolar , Pressão Osmótica , Tamanho da Partícula , Espalhamento de RadiaçãoRESUMO
The binding selectivity of charged liposomes to the spinal cord of rats affected by experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, was investigated. Positively and negatively charged liposomes were injected into the tail vein of rats, and blood/brain barrier (BBB) targeting was determined by confocal microscopy as a function of the temporal evolution of the inflammatory response. Accumulation in spinal cord endoneural vessels was observed for cationic, but not for anionic, liposomes, and only in EAE but not in healthy rats. The overall binding efficacy paralleled the severity of the clinical score, but targeting was observed already before clinical manifestation of inflammation. Preferential binding of positively charged liposomes in the course of acute EAE can be ascribed to subtle changes of BBB morphology and charge distribution in a similar way as for the binding of cationic particles to proliferating vasculature in chronic inflammation and angiogenesis. Our findings suggest that vascular changes related to increased binding affinity for cationic particles are very early events within the inflammatory reaction in acute EAE. Investigation of cationic vascular targeting can help to shed further light on these occurrences, and, potentially, new diagnostic and therapeutic options may become available. In neuroinflammatory diseases, cationic colloidal carrier particles may enable intervention at affected BBB by an approach which is independent from permeability increase.