RESUMO
mRNA based gene therapies hold the potential to treat multiple diseases with significant advantages over DNA based therapies, including rapid protein expression and minimized risk of mutagenesis. However, successful delivery of mRNA remains challenging, and clinical translation of mRNA therapeutics has been limited. This study investigated the use of a lipid/polymer hybrid (LPH) nanocarrier for mRNA, designed to address key delivery challenges and shuttle mRNA to targeted tissues. LPH nanocarriers were synthesized using a scalable microfluidic process with a variety of material compositions and mRNA loading strategies. Results show that a combination of permanently ionized and transiently, pH-dependent ionizable cationic lipids had a synergistic effect upon on mRNA gene translation, when compared to each lipid independently. Upon intravenous administration, particles with adsorbed mRNA outperformed particles with encapsulated mRNA for protein expression in the lungs and the spleen despite significant LPH nanoparticle localization to the liver. In contrast, encapsulated particles had higher localized expression when injected intramuscularly with protein expression detectable out to 12 days post injection. Intramuscular administration of particles with OVA mRNA resulted in robust humoral immune response with encapsulated outperforming adsorbed particles in terms of antibody titers at 28 days. These results demonstrate LPH nanocarriers have great potential as a vehicle for mRNA delivery and expression in tissues and that tissue expression and longevity can be influenced by LPH composition and route of administration.
Assuntos
Nanopartículas , Polímeros , Cátions , Microfluídica , RNA MensageiroRESUMO
Lipid nanoparticles (LNPs) containing mRNA can deliver genetic material to cells for use as vaccines or protein replacement therapies. We characterized the effect of solution pH on cationic LNPs containing green fluorescent protein (EGFP) mRNA and their transfection efficiency. We compared the structural and colloidal properties of mRNA LNPs with LNPs not containing mRNA and mRNA free in solution. We used a combination of biophysical technique to build a picture of the structure of the lipids and mRNA across pH and temperature in the form of an empirical phase diagram (EPD). A combination of Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry was used to investigate lipid phase behavior. The mRNA-LNPs transition from an inverse hexagonal phase at pH values below the pKa of the cationic lipid to a lamellar phase above the pKa. At higher temperatures the mRNA-LNPs also transitioned from an inverse hexagonal phase to a lamellar phase indicating the inverse hexagonal phase is more thermodynamically favorable. Based on circular dichroism, the mRNA within the LNP has more A form structure at pH values below the lipid pKa than above it. Optical density, zeta potential and dynamic light scattering measurements were used to probe the colloidal stability of the mRNA-LNPs. The particles were larger and more prone to aggregation below the pKa. A stability study was performed to relate the biophysical characteristics to the storage of the particles in solution at 4 and 25 °C. mRNA-LNPs had the highest transfection efficiency and stability at pH values below the pKa. However, there was a trade-off between the stability and aggregation propensity since at very low pH the particles were most prone to aggregation. We performed kinetic experiments to show that the time scale of the pH-dependent phase behavior is slow (6 hour transition) and the transition from lamellar to inverse hexagonal phases is irreversible. This suggests that the lamellar phase is less stable and kinetically trapped. Our findings deepen our structural understanding of mRNA-LNPs and will aid the development of related formulations.
Assuntos
Lipídeos , Nanopartículas , Cátions , Concentração de Íons de Hidrogênio , Lipídeos/química , Lipossomos , Nanopartículas/química , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
Nanoparticle-based mRNA therapeutics hold great promise, but cellular internalization and endosomal escape remain key barriers for cytosolic delivery. We developed a dual nanoparticle uptake and endosomal disruption assay using high-throughput and high-content image-based screening. Using a genetically encoded Galectin 8 fluorescent fusion protein sensor, endosomal disruption could be detected via sensor clustering on damaged endosomal membranes. Simultaneously, nucleic acid endocytosis was quantified using fluorescently tagged mRNA. We used an array of biodegradable poly(beta-amino ester)s as well as Lipofectamine and PEI to demonstrate that this assay has higher predictive capacity for mRNA delivery compared to conventional polymer and nanoparticle physiochemical characteristics. Top nanoparticle formulations enabled safe and efficacious mRNA expression in multiple tissues following intravenous injection, demonstrating that the in vitro screening method is also predictive of in vivo performance. Efficacious nonviral systemic delivery of mRNA with biodegradable particles opens up new avenues for genetic medicine and human health.
RESUMO
Controlling the biodistribution of nanoparticles upon intravenous injection is the key to achieving target specificity. One of the impediments in nanoparticle-based tumor targeting is the inability to limit the trafficking of nanoparticles to liver and other organs leading to smaller accumulated amounts in tumor tissues, particularly via passive targeting. Here we overcome both these challenges by designing nanoparticles that combine the specificity of antibodies with favorable particle biodistribution profiles, while not exceeding the threshold for renal filtration as a combined vehicle. To that end, ultrasmall silica nanoparticles are functionalized with anti-human epidermal growth factor receptor 2 (HER2) single-chain variable fragments to exhibit high tumor-targeting efficiency and efficient renal clearance. This ultrasmall targeted nanotheranostics/nanotherapeutic platform has broad utility, both for imaging a variety of tumor tissues by suitably adopting the targeting fragment and as a potentially useful drug delivery vehicle.
Assuntos
Neoplasias da Mama/metabolismo , Nanopartículas/química , Receptor ErbB-2/metabolismo , Anticorpos de Cadeia Única/química , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/prevenção & controle , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Feminino , Humanos , Camundongos , Nanopartículas/administração & dosagem , Tamanho da Partícula , Tomografia por Emissão de Pósitrons , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Dióxido de Silício/química , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Theranostic nanoparticles offer the potential for mixing and matching disparate diagnostic and therapeutic functionalities within a single nanoparticle for the personalized treatment of diseases. In this article, we present composite iron oxide-gadolinium-containing Prussian blue nanoparticles (Fe3O4@GdPB) as a novel theranostic agent for T1-weighted magnetic resonance imaging (MRI) and photothermal therapy (PTT) of tumors. These particles combine the well-described properties and safety profiles of the constituent Fe3O4 nanoparticles and gadolinium-containing Prussian blue nanoparticles. The Fe3O4@GdPB nanoparticles function both as effective MRI contrast agents and PTT agents as determined by characterizing studies performed in vitro and retain their properties in the presence of cells. Importantly, the Fe3O4@GdPB nanoparticles function as effective MRI contrast agents in vivo by increasing signal:noise ratios in T1-weighted scans of tumors and as effective PTT agents in vivo by decreasing tumor growth rates and increasing survival in an animal model of neuroblastoma. These findings demonstrate the potential of the Fe3O4@GdPB nanoparticles to function as effective theranostic agents.
Assuntos
Meios de Contraste/química , Ferrocianetos/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Fototerapia/métodos , Animais , Meios de Contraste/uso terapêutico , Feminino , Compostos Férricos/química , Gadolínio/química , Humanos , Magnetismo , Camundongos Endogâmicos , Nanopartículas/uso terapêutico , Neuroblastoma/diagnóstico por imagem , Neuroblastoma/tratamento farmacológico , Fototerapia/instrumentação , Razão Sinal-Ruído , Nanomedicina Teranóstica/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: This work evaluated the feasibility of combining insulin PEGylation with pH responsive hydrogels for oral insulin delivery. METHODS: A mono-substituted PEG-insulin conjugate was synthesized and purified. The site of conjugation was determined by MALDI-TOF MS. Uptake and release of PEGylated insulin was performed in complexation hydrogels to simulate oral dosing. The bioactivity of the conjugate and PK/PD profile was measured in vivo in rats. RESULTS: PEGylation was confirmed to be specifically located at the amino terminus of the B-chain of insulin. Higher loading efficiency was achieved with PEGylated insulin than regular human insulin in pH responsive hydrogels. The release of PEGylated insulin was lower than that of human insulin at all pH levels considered. Full retention of bioactivity of the PEG-insulin conjugate was confirmed by intravenous dosing while subcutaneous dosing exhibited a relative hypoglycemic effect 127.8% that of human insulin. CONCLUSIONS: Polyethylene glycol conjugated specifically to the amino terminus of the B-chain of insulin maintained the bioactivity of the protein and significantly extended the duration of the hypoglycemic effect. Used in combination with pH responsive hydrogels, PEGylated insulin has significant potential for oral delivery.
Assuntos
Portadores de Fármacos/síntese química , Hidrogéis/síntese química , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Polietilenoglicóis/química , Administração Oral , Animais , Portadores de Fármacos/química , Humanos , Hidrogéis/química , Hipoglicemiantes/química , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Insulina/química , Insulina/farmacocinética , Insulina/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Hydrogels comprised of poly(methacrylic acid) grafted with poly(ethylene glycol) (P(MAA-g-EG)) were characterized and examined for their potential as oral insulin carriers. Insulin loaded polymer (ILP) samples were made using two different polymer formulations. The values for the effective molecular weight between crosslinks, M _e , and the network mesh size, xi, were characterized and increased with increasing pH levels for both formulations. Insulin uptake studies indicated a high insulin loading efficiency for all samples tested, however release was dependent on the amount of insulin loaded. The effect of total polymer dosing was investigated by in situ administration in isolated ileal segments in rats. All ILP samples induced a hypoglycemic effect and an increase in insulin levels, proving that insulin was still biologically active. Insulin dosing amounts were varied by (i) maintaining a constant insulin fraction within an ILP sample while changing the amount of ILP and (ii) by varying the insulin fraction while dosing with the same amount of ILP. The total insulin absorption was dependent on both the amount of the polymer present and the concentration of insulin within an ILP sample, with a maximum relative bioavailability of 8.0%.
Assuntos
Portadores de Fármacos/química , Hidrogéis/química , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Polietilenoglicóis/química , Ácidos Polimetacrílicos/química , Administração Oral , Animais , Disponibilidade Biológica , Cromatografia em Gel , Preparações de Ação Retardada , Portadores de Fármacos/síntese química , Composição de Medicamentos , Estabilidade de Medicamentos , Elasticidade , Humanos , Hidrogéis/síntese química , Hipoglicemiantes/farmacocinética , Insulina/farmacocinética , Absorção Intestinal , Masculino , Tamanho da Partícula , Polietilenoglicóis/síntese química , Ácidos Polimetacrílicos/síntese química , Ratos , Ratos Sprague-DawleyRESUMO
The insulin-transport enhancing effects of a pH-sensitive poly((methacrylic acid)-grafted-poly(ethylene glycol)) hydrogel system were studied using Caco-2 monolayers as an in vitro model of intestinal transport. Further, the ability of the hydrogel system to protect entrapped proteins through the upper gastrointestinal tract via digestion in simulated gastric and simulated intestinal fluids with digestive enzymes was confirmed. Caco-2 cell monolayers were exposed to a series of formulations including insulin alone, the polymer in insulin solution, insulin-loaded polymer (ILP) and ILP previously subjected to simulated digestive fluids with enzymes. These studies demonstrated greatly increased insulin transport for the ILP samples when compared with insulin alone and insulin in the presence of polymer, P(app)=12.7 x 10(-8) cm/s and 6.61 x 10(-8) cm/s versus 0.07 x 10(-8) cm/s and 0.06 x 10(-8) cm/s, respectively. While enhanced transport with the ILP was observed, the largest changes in TEER values did not coincide with the highest amounts of insulin transport, this suggests that the paracellular route may not be the sole mechanism of transport. Further, as the Caco-2 cell line has been demonstrated to possess the insulin receptor, active transport or a mixed mechanism cannot be ruled out.