RESUMO
mRNA vaccines have recently received significant attention due to their role in combating the SARS-CoV-2 pandemic. As a platform, mRNA vaccines have been shown to elicit strong humoral and cellular immune responses with acceptable safety profiles for prophylactic use. Despite their potential, industrial challenges have limited realization of the vaccine platform on a global scale. Critical among these challenges are supply chain considerations, including mRNA production, cost of goods, and vaccine frozen-chain distribution. Here, we assess the delivery of lipid nanoparticle-encapsulated mRNA (mRNA/LNP) vaccines using a split-dose immunization regimen as an approach to develop mRNA dose-sparing vaccine regimens with potential to mitigate mRNA supply chain challenges. Our data demonstrate that immunization by a mRNA/LNP vaccine encoding respiratory syncytial virus pre-F (RSV pre-F) over a 9 day period elicits comparable or superior magnitude of antibodies when compared to traditional bolus immunization of the vaccine. The split-dose immunization regimens evaluated in our studies were designed to mimic reported drug or antigen release profiles from microneedle patches, highlighting the potential benefit of pairing mRNA vaccines with patch-based delivery technologies to enable sustained release and solid-state stabilization. Overall, our findings provide a proof of concept to support further investigations into the development of sustained delivery approaches for mRNA/LNP vaccines.
Assuntos
COVID-19 , Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Humanos , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Anticorpos Antivirais , Vacinas contra Vírus Sincicial Respiratório/genética , SARS-CoV-2/genética , COVID-19/prevenção & controle , Imunidade , RNA Mensageiro/genética , Anticorpos NeutralizantesRESUMO
Antimicrobial preservatives are used as functional excipients in multidose formulations of biological therapeutics to destroy or inhibit the growth of microbial contaminants, which may be introduced by repeatedly administering doses. Antimicrobial agents can also induce the biophysical instability of proteins and peptides, which presents a challenge in optimizing the drug product formulation. Elucidating the structural basis for aggregation aids in understanding the underlying mechanism and can offer valuable knowledge and rationale for designing drug substances and drug products; however, this remains largely unexplored due to the lack of high-resolution characterization. In this work, we utilize solution nuclear magnetic resonance (NMR) as an advanced biophysical tool to study an acylated 31-residue peptide, acyl-peptide A, and its interaction with commonly used antimicrobial agents, benzyl alcohol and m-cresol. Our results suggest that acyl-peptide A forms soluble octamers in the aqueous solution, which tumble slowly due to an increased molecular weight as measured by diffusion ordered spectroscopy and 1H relaxation measurement. The addition of benzyl alcohol does not induce aggregation of acyl-peptide A and has no chemical shift perturbation in 1H-1H NOESY spectra, suggesting no detectable interaction with the peptide. In contrast, the addition of 1% (w/v) m-cresol results in insoluble aggregates composed of 25% (w/w) peptides after a 24-hour incubation at room temperature as quantified by 1H NMR. Interestingly, 1H-13C heteronuclear single-quantum coherence and 1H-1H total correlation experiment spectroscopy have identified m-cresol and peptide interactions at specific residues, including Met, Lys, Glu, and Gln, suggesting that there may be a combination of hydrophobic, hydrogen bonding, and electrostatic interactions with m-cresol driving this phenomenon. These site-specific interactions have promoted the formation of higher-order oligomerization such as dimers and trimers of octamers, eventually resulting in insoluble aggregates. Our study has elucidated a structural basis of m-cresol-induced self-association that can inform the optimized design of drug substances and products. Moreover, it has demonstrated solution NMR as a high-resolution tool to investigate the structure and dynamics of biological drug products and provide an understanding of excipient-induced peptide and protein aggregation.
Assuntos
Anti-Infecciosos , Excipientes , Antibacterianos , Anti-Infecciosos/química , Álcool Benzílico/química , Excipientes/química , Peptídeos , Conservantes Farmacêuticos/químicaRESUMO
Regimen adherence remains a major hurdle to the success of daily oral drug regimens for the treatment and prevention of human immunodeficiency virus (HIV) infection. Long-acting drug formulations requiring less-frequent dosing offer an opportunity to improve adherence and allow for more forgiving options with regard to missed doses. The administration of long-acting formulations in a clinical setting enables health care providers to directly track adherence. MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine [EFdA]) is an investigational nucleoside reverse transcriptase translocation inhibitor (NRTTI) drug candidate under investigation as part of a regimen for HIV treatment, with potential utility as a single agent for preexposure prophylaxis (PrEP). The active triphosphate of MK-8591 (MK-8591-TP) exhibits protracted intracellular persistence and, together with the potency of MK-8591, supports its consideration for extended-duration dosing. Toward this end, drug-eluting implant devices were designed to provide prolonged MK-8591 release in vitro and in vivo Implants, administered subcutaneously, were studied in rodents and nonhuman primates to establish MK-8591 pharmacokinetics and intracellular levels of MK-8591-TP. These data were evaluated against pharmacokinetic and pharmacodynamic models, as well as data generated in phase 1a (Ph1a) and Ph1b clinical studies with once-weekly oral administration of MK-8591. After a single administration in animals, MK-8591 implants achieved clinically relevant drug exposures and sustained drug release, with plasma levels maintained for greater than 6 months that correspond to efficacious MK-8591-TP levels, resulting in a 1.6-log reduction in viral load. Additional studies of MK-8591 implants for HIV treatment and prevention are warranted.
Assuntos
Desoxiadenosinas/uso terapêutico , Portadores de Fármacos/química , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Inibidores da Transcriptase Reversa/uso terapêutico , Animais , Fármacos Anti-HIV , Desoxiadenosinas/química , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Macaca mulatta , Masculino , Polímeros/química , Ratos , Ratos Wistar , Inibidores da Transcriptase Reversa/químicaRESUMO
Lipid nanoparticles (LNPs) represent the most clinically advanced technology for the systemic delivery of therapeutic siRNA in vivo. Toward this end, a novel class of LNPs comprising low molecular weight (MW) ionizable amino lipids having asymmetric architecture was recently reported.1 LNPs of these amino lipids, termed asymmetric LNPs, were shown to be highly efficacious and well tolerated in vivo; advances were enabled by improved endosomal escape, coupled with enhanced amino lipid metabolism and clearance. In this work, we show that, in contrast to their desirable pharmacological performance, asymmetric LNPs present a significant pharmaceutical developability challenge, namely physical instability limiting extended shelf life. Using orthogonal characterization methods, we identify the mechanism of LNP instability as Ostwald ripening and establish it to be driven predominantly by the asymmetric amino lipid component. Through rational optimization of LNP physical and macromolecular properties, we are able to significantly attenuate or entirely eliminate the Ostwald ripening instability. Modulation of LNP size, for example, effectively halts particle growth. Similarly, optimization of LNP macromolecular packing through deliberate selection of structurally matched colipids significantly diminishes the rate of ripening. This later experimental observation is substantiated by molecular dynamics simulations of LNP self-assembly, which establish a quantitative dependence of LNP macromolecular order on colipid structure. In totality, the experimental and molecular dynamics outcomes of this work support the rational design of LNP physical and chemical properties leading to effective Ostwald ripening stabilization and enable the advance of asymmetric LNPs as a clinic-ready platform for siRNA therapeutics.
Assuntos
Aminoácidos/química , Apolipoproteínas B/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Lipídeos/química , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Animais , Apolipoproteínas B/genética , Cromatografia em Gel , Feminino , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Peso Molecular , Tamanho da Partícula , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Propriedades de SuperfícieRESUMO
Lipid nanoparticles (LNPs) are a leading platform for therapeutic delivery of small interfering RNAs (siRNAs). Optimization of LNPs as therapeutic products is enabled by the development of structure-activity relationships (SAR) linking LNP physiochemical and macromolecular properties to bioperformance. Methods by which LNP properties can be rationally manipulated are thus critical enablers of this fundamental knowledge build. In this work, we present a mechanistic study of LNP self-assembly via a rapid antisolvent precipitation process and identify critical physiochemical and kinetic parameters governing the evolution of LNP three-dimensional macromolecular structure as a biorelevant SAR feature. Using small-angle X-ray scattering, LNPs are shown to undergo a temporal evolution in macromolecular structure during self-assembly, rearranging from initially disordered phases after precipitation into well-ordered structures following a necessary annealing stage of the assembly sequence. The ability of LNPs to undergo structural reorganization is shown to be effected by the chemical nature of the aqueous antisolvent used for precipitation. Antisolvents of varying buffering species differentially influence LNP macromolecular features, revealing a new participatory role of buffer ions in LNP self-assembly. Furthermore, the formation of macromolecular structure in LNPs is shown to improve the efficiency of siRNA encapsulation, thereby offering a simple, nonchemical route for preparation of high-payload LNPs that minimize the dose of lipid excipients. The developed LNP precipitation process and mechanistic understanding of self-assembly are shown to be generalizable, enabling the production of LNPs with a tunable range of macromolecular features, as evidenced by the cubic, hexagonal, and oligo-lamellar phase LNPs exemplarily generated.
Assuntos
Lipídeos/química , Nanopartículas/química , RNA Interferente Pequeno/química , Relação Estrutura-AtividadeRESUMO
PURPOSE: To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs. METHODS: siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics. RESULTS: Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2 weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing. CONCLUSIONS: Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Técnicas de Silenciamento de Genes/métodos , RNA Interferente Pequeno/farmacologia , Animais , Química Farmacêutica , Diclofenaco/metabolismo , Técnicas In Vitro , Masculino , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Midazolam/metabolismo , Nanopartículas , Ligação Proteica , RatosRESUMO
In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.
Assuntos
Imunidade Celular , Imunidade Humoral , Vacinas contra Influenza , Infecções por Orthomyxoviridae , Vacinas de mRNA , Animais , Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza/imunologia , Camundongos , Nucleoproteínas/genética , Infecções por Orthomyxoviridae/prevenção & controle , Primatas , Vacinas Sintéticas , Vacinas de mRNA/imunologiaRESUMO
New classes of therapeutic peptides are being developed to prosecute biological targets which have been inaccessible to other modalities. Higher potency and longer half-life peptides have given rise to multiuse injectable formulations that enable convenient, low volume, and self-administered dosing; however, inclusion of antimicrobial preservatives to meet bactericidal requirements can impact other attributes of peptide formulations. Peptide-preservative interactions influencing solution-phase self-association of a non-insulin, linear, palmitoylated 31 amino acid peptide and two structurally similar peptides were assessed via turbidity, intrinsic fluorescence shifts and quenching, isothermal titration calorimetry, and 1H NMR. Meta-cresol and phenol specifically interact with the peptide, result in increased hydrophobicity near the tryptophan residue, and induce conformational changes, while benzyl alcohol does not impact tryptophan fluorescence, demonstrate any interaction enthalpy, or induce conformational changes. These same trends did not hold true for the other palmitoylated peptides evaluated, reinforcing the impacts of unique peptide sequences. Importantly, the presence of benzyl alcohol does increase the physical stability and solubility of the linear, 31 amino acid peptide under salt stress. We report new insights into the physical interactions of peptides with antimicrobial excipients, demonstrating a reversible association phenomenon and highlighting practical implications for formulation design and excipient selection.
Assuntos
Anti-Infecciosos , Excipientes , Peptídeos , Conservantes Farmacêuticos , SolubilidadeRESUMO
The RSV Fusion (F) protein is a target for neutralizing antibody responses and is a focus for vaccine discovery; however, the process of RSV entry requires F to adopt a metastable prefusion form and transition to a more stable postfusion form, which displays less potent neutralizing epitopes. mRNA vaccines encode antigens that are translated by host cells following vaccination, which may allow conformational transitions similar to those observed during natural infection to occur. Here we evaluate a panel of chemically modified mRNA vaccines expressing different forms of the RSV F protein, including secreted, membrane associated, prefusion-stabilized, and non-stabilized structures, for conformation, immunogenicity, protection, and safety in rodent models. Vaccination with mRNA encoding native RSV F elicited antibody responses to both prefusion- and postfusion-specific epitopes, suggesting that this antigen may adopt both conformations in vivo. Incorporating prefusion stabilizing mutations further shifts the immune response toward prefusion-specific epitopes, but does not impact neutralizing antibody titer. mRNA vaccine candidates expressing either prefusion stabilized or native forms of RSV F protein elicit robust neutralizing antibody responses in both mice and cotton rats, similar to levels observed with a comparable dose of adjuvanted prefusion stabilized RSV F protein. In contrast to the protein subunit vaccine, mRNA-based vaccines elicited robust CD4+ and CD8+ T-cell responses in mice, highlighting a potential advantage of the technology for vaccines requiring a cellular immune response for efficacy.
RESUMO
We are interested in developing a vaccine that prevents genital herpes. Adjuvants have a major impact on vaccine immunogenicity. We compared two adjuvants, an experimental Merck Sharp & Dohme lipid nanoparticle (LNP) adjuvant, LNP-2, with CpG oligonucleotide combined with alum for immunogenicity in mice when administered with herpes simplex virus type 2 (HSV-2) glycoproteins C, D and E (gC2, gD2, gE2). The immunogens are intended to produce neutralizing antibodies to gC2 and gD2, antibodies to gD2 and gE2 that block cell-to-cell spread, and antibodies to gE2 and gC2 that block immune evasion from antibody and complement, respectively. Overall, CpG/alum was better at producing serum and vaginal IgG binding antibodies, neutralizing antibodies, antibodies that block virus spread from cell-to-cell, and antibodies that block immune evasion domains on gC2. We used a novel high throughput biosensor assay to further assess differences in immunogenicity by mapping antibody responses to seven crucial epitopes on gD2 involved in virus entry or cell-to-cell spread. We found striking differences between CpG/alum and LNP-2. Mice immunized with gD2 CpG/alum produced higher titers of antibodies than LNP-2 to six of seven crucial epitopes and produced antibodies to more crucial epitopes than LNP-2. Measuring epitope-specific antibodies helped to define mechanisms by which CpG/alum outperformed LNP-2 and is a valuable technique to compare adjuvants.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Formação de Anticorpos , Epitopos/imunologia , Herpes Genital/prevenção & controle , Proteínas do Envelope Viral/imunologia , Compostos de Alúmen/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Técnicas Biossensoriais , Feminino , Herpes Genital/imunologia , Vacinas contra Herpesvirus/imunologia , Evasão da Resposta Imune , Imunogenicidade da Vacina , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Proteínas do Envelope Viral/administração & dosagem , Internalização do VírusRESUMO
Alternative formulations of entecavir, a once daily oral hepatitis B antiretroviral, may improve treatment adherence by patients. We explored the use of biocompatible polymers to control entecavir dissolution in two formats suitable for subcutaneous implantation. Hot melt extrudates were prepared by extruding entecavir-polymer blends at specified weight ratios. Dip-coated tablets were prepared by compressing entecavir in a multi-tip tooling. Tablets were dip-coated in solutions of polymer and dried. In rodents, entecavir-poly(caprolactone) extrudates demonstrated >180â¯days of continuous drug release, although below the estimated efficacious target input rate. Drug pharmacokinetic profiles were tunable by varying the polymer employed and implant format. The rank order trends of drug input rates observed in vitro were observed in vivo in the detected plasma concentrations of entecavir. In all dose groups entecavir was not tolerated locally at the site of administration where adverse event severity correlated with drug input rate. These polymer-based implantable formats have applicability to long-acting formulations of high solubility compounds beyond entecavir.
Assuntos
Antivirais/química , Antivirais/farmacologia , Guanina/análogos & derivados , Hepatite B/tratamento farmacológico , Animais , Química Farmacêutica/métodos , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos/efeitos dos fármacos , Excipientes/química , Feminino , Guanina/química , Guanina/farmacologia , Masculino , Polímeros/química , Ratos , Ratos Wistar , Solubilidade/efeitos dos fármacos , Comprimidos/química , Comprimidos/farmacologiaRESUMO
Maleimide-functional poly(ethylene glycol)-b-poly(epsilon-caprolactone) nanoparticles (NPs) were prepared via the Flash NanoPrecipitation technique. Subsequent reaction with a model ligand, bovine serum albumin (BSA), was conducted using thiol-maleimide conjugation. Reaction of up to 22% of NP surface maleimide-PEG tethers was obtained, with the percent conversion being essentially independent of the ratio of maleimide-PEG to methyl-PEG over the range 30-100%, respectively. At the highest surface coverage, BSA is calculated to essentially cover the NP surface area. Reaction parameters (reaction order and docking constant) describing the extent of ligand conjugation were determined. The reaction order is applicable to the conjugation of ligands presenting free thiol functionalities, while the value of the docking constant is ligand-dependent and accounts for physical and dynamic properties of the ligand-PEG interaction. Jointly, the particle formation process, using block copolymer-directed kinetically controlled assembly and surface functionalization represent a versatile new platform for the preparation of bioconjugated NPs with accurate control of ligand density and minimal processing steps.
Assuntos
Nanopartículas/química , Polietilenoglicóis/química , Animais , Bovinos , Ligantes , Luz , Maleimidas/química , Conformação Molecular , Nanotecnologia/métodos , Tamanho da Partícula , Polímeros/química , Espalhamento de Radiação , Soroalbumina Bovina/química , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.
Assuntos
Vacinas contra Dengue , Vírus da Dengue/imunologia , Dengue , Imunização Secundária , Lipídeos , Proteínas do Envelope Viral , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Dengue/imunologia , Dengue/prevenção & controle , Vacinas contra Dengue/química , Vacinas contra Dengue/imunologia , Vacinas contra Dengue/farmacologia , Feminino , Cobaias , Humanos , Lipídeos/química , Lipídeos/imunologia , Lipídeos/farmacologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/uso terapêutico , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/farmacologiaRESUMO
Magnetic resonance imaging (MRI)- and near-infrared (NIR)-active, multimodal composite nanocarriers (CNCs) are prepared using a simple one-step process, flash nanoprecipitation (FNP). The FNP process allows for the independent control of the hydrodynamic diameter, co-core excipient and NIR dye loading, and iron oxide-based nanocrystal (IONC) content of the CNCs. In the controlled precipitation process, 10 nm IONCs are encapsulated into poly(ethylene glycol) (PEG) stabilized CNCs to make biocompatible T2 contrast agents. By adjusting the formulation, CNC size is tuned between 80 and 360 nm. Holding the CNC size constant at an intensity weighted average diameter of 99 ± 3 nm (PDI width 28 nm), the particle relaxivity varies linearly with encapsulated IONC content ranging from 66 to 533 × 10(-3) m(-1) s(-1) for CNCs formulated with 4-16 wt% IONC. To demonstrate the use of CNCs as in vivo MRI contrast agents, CNCs are surface functionalized with liver-targeting hydroxyl groups. The CNCs enable the detection of 0.8 mm(3) non-small cell lung cancer metastases in mice livers via MRI. Incorporating the hydrophobic, NIR dye tris-(porphyrinato)zinc(II) into CNCs enables complementary visualization with long-wavelength fluorescence at 800 nm. In vivo imaging demonstrates the ability of CNCs to act both as MRI and fluorescent imaging agents.
Assuntos
Meios de Contraste , Portadores de Fármacos , Compostos Férricos , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Imagem Óptica/métodos , Animais , Meios de Contraste/química , Meios de Contraste/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Compostos Férricos/química , Compostos Férricos/farmacologia , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Camundongos , Tamanho da Partícula , Polietilenoglicóis/química , Polietilenoglicóis/farmacologiaRESUMO
The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA.
RESUMO
INTRODUCTION: Harnessing RNA interference as a therapeutic approach has the potential to significantly expand the druggable target space, offering new hope for treatment of diseases that cannot be addressed with existing classes of drugs. A number of siRNA therapeutics have already progressed into preclinical and clinical development. Of these, lipid-based systems have emerged as one of the most mature classes of systemic delivery technologies. Despite tremendous advances in development, a number of significant challenges must still be addressed to enable commercialization of a lipid-based siRNA pharmaceutical product. AREAS COVERED: This review addresses specific challenges inherent to the pharmaceutical development of lipid-based siRNA therapeutics. Focus is placed on the development of a robust manufacturing process, the setting of appropriate product specifications and controls, development of strategies to assess and ensure product stability, and the evaluation of product comparability throughout development. EXPERT OPINION: Discovering and developing a lipid-based siRNA therapeutic that can be commercialized requires engineering a particle that selectively and efficiently delivers the cargo to the target tissue and cellular compartment. The particle assembly must be strictly controlled and physical properties thoroughly characterized to successfully develop an understanding of particle attributes that influence in vivo pharmaceutical properties. Correlation of particle physio-chemical properties to product performance is the foundation for advancements in discovery and assuring quality in a commercial drug product. Although difficult, we believe these development challenges can be addressed with appropriate scientific resources and that the industry will continue to progress siRNA therapeutic candidates through clinical development.
Assuntos
Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Química Farmacêutica/métodos , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Estabilidade de Medicamentos , Humanos , Lipídeos/administração & dosagem , Lipídeos/química , Nanopartículas/administração & dosagem , Tecnologia FarmacêuticaRESUMO
The application of nanoparticles for the delivery and targeting of pharmaceutical, therapeutic and diagnostic agents in cancer therapy has received significant attention in recent years. Nanoparticles may be constructed from a wide range of materials and used to encapsulate or solubilize chemotherapeutic agents for improved delivery in vivo or to provide unique optical, magnetic and electrical properties for imaging and therapy. Several functional nanoparticles have already been demonstrated, including some clinically approved liposome drug formulations and metallic imaging agents. The next generation of nanoparticle-based research is directed at the consolidation of functions into strategically engineered multifunctional systems, which may ultimately facilitate the realization of individual therapy. These multiplexed nanoparticles may be capable of identifying malignant cells by means of molecular detection, visualizing their location in the body by providing enhanced contrast in medical imaging techniques, killing diseased cells with minimal side effects through selective drug targeting, and monitoring treatment in real time. This article highlights recent progress in the design and engineering of multifunctional systems, as well as discusses the development of a new, scalable and economic method for the modular preparation of multiplex nanoparticles where functional properties can be precisely and simply tailored.
Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos , Nanopartículas , Neoplasias , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Diagnóstico por Imagem , Humanos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológicoRESUMO
Nanomaterials have been demonstrated as useful tools for molecular imaging, molecular diagnosis and targeted therapy in biomedical research. The main advantages of such nanomaterials are improved circulation times, precise targeting, enhancement of dissolution rates and enhanced contrast. A challenge and opportunity for nanotechnological strategies is that multiple functionalities, such as therapeutics, targeting, imaging and stimuli responsiveness can be achieved within one nanoparticle. Multifunctional nanoparticles are now actively under investigation and are imminent as the next generation of nanoparticles for providing custom and tailored treatment. This review considers contemporary approaches and possible future directions in the emerging area of multifunctional nanoparticles with a special focus on targeted drug delivery.
RESUMO
We describe the preparation and characterization of hybrid block copolymer nanoparticles (NPs) for use as multimodal carriers for drugs and imaging agents. Stable, water-soluble, biocompatible poly(ethylene glycol)-block-poly(epsilon-caprolactone) NPs simultaneously co-encapsulating hydrophobic organic actives (beta-carotene) and inorganic imaging nanostructures (Au) are prepared using the flash nanoprecipitation process in a multi-inlet vortex mixer. These composite nanoparticles (CNPs) are produced with tunable sizes between 75 nm and 275 nm, narrow particle size distributions, high encapsulation efficiencies, specified component compositions, and long-term stability. The process is tunable and flexible because it relies on the control of mixing and aggregation timescales. It is anticipated that the technique can be applied to a variety of hydrophobic active compounds, fluorescent dyes, and inorganic nanostructures, yielding CNPs for combined therapy and multimodal imaging applications.
Assuntos
Ouro/química , Nanopartículas/química , Polímeros/química , beta Caroteno/química , Caproatos/química , Interações Hidrofóbicas e Hidrofílicas , Lactonas/química , Modelos Químicos , Tamanho da Partícula , Polietilenoglicóis/química , Solubilidade , Espectrofotometria Ultravioleta , Água/químicaRESUMO
The solution phase behavior of short, strictly alternating multiblock copolymers of type (A(n)B(n))(m) was studied using lattice Monte Carlo simulations. The polymer molecules were modeled as flexible chains in a monomeric solvent selective for block type A. The degree of block polymerization n and the number of diblock units per chain m were treated as variables. We show that within the regime of parameters accessible to our study, the thermodynamic phase transition type is dependent on the ratio of m / n. The simulations show microscopic phase separation into roughly spherical aggregates for m / n ratios less than a critical value and first-order macroscopic precipitation otherwise. In general, increasing m at fixed n, or n at fixed m, promotes the tendency toward macroscopic phase precipitation. The enthalpic driving force of phase change is found to universally scale with chain length for all multiblock systems considered and is independent of the existence of a true phase transition. For aggregate forming systems at low amphiphile concentrations, multiblock chains are shown to self-assemble into intramolecular, multichain clusters. Predictions for microstructural dimensions, including critical micelle concentration, equilibrium size, shape, aggregation parameters, and density distributions, are provided. At increasing amphiphile density, interaggregate bridging is shown to result in the formation of networked structures, leading to an eventual solution-gel transition. The gel is swollen and consists of highly interconnected aggregates of approximately spherical morphology. Qualitative agreement is found between experimentally observed physical property changes and phase transitions predicted by simulations. Thus, a potential application of the simulations is the design of multiblock copolymer systems which can be optimized with regard to solution phase behavior and ultimately physical and mechanical properties.