RESUMO
PURPOSE: Traditional polymeric nanoparticle formulations for prolonged local action during inhalation therapy are highly susceptible to muco-ciliary clearance. In addition, polymeric carriers are typically administered in high doses due to finite drug loading. For toxicological reasons, these carriers and their degradation byproducts are undesirable for inhalation therapy, particularly for chronic use, due to potential lung accumulation. METHODS: We synthesized a novel, insoluble prodrug (MRPD) of a time-dependent ß-lactam, meropenem, and formulated MRPD into mucus-penetrating crystals (MRPD-MPCs). After characterizing their mucus mobility (in vitro) and stability, we evaluated the lung pharmacokinetics of intratracheally-instilled MRPD-MPCs and a meropenem solution in guinea pigs. RESULTS: Meropenem levels rapidly declined in the lungs of guinea pigs receiving meropenem solution compared to those given MRPD-MPCs. At 9 h after dosing, drug levels in the lungs of animals that received meropenem solution dropped to 12 ng/mL, whereas those that received MRPD-MPCs maintained an average drug level of ≥1,065 ng/mL over a 12-h period. CONCLUSIONS: This work demonstrated that the combination of prodrug chemistry and mucus-penetrating platform created nanoparticles that produced sustained levels of meropenem in guinea pig lungs. This strategy represents a novel approach for sustained local drug delivery to the lung without using encapsulating matrices.
Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/química , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Portadores de Fármacos/química , Pulmão/metabolismo , Água/química , Animais , Química Farmacêutica/métodos , Sistemas de Liberação de Medicamentos/métodos , Cobaias , Humanos , Masculino , Meropeném , Muco/metabolismo , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Solubilidade , Soluções/administração & dosagem , Soluções/química , Tienamicinas/administração & dosagem , Tienamicinas/químicaRESUMO
Self-amplifying messenger RNA (mRNA) of positive-strand RNA viruses are effective vectors for in situ expression of vaccine antigens and have potential as a new vaccine technology platform well suited for global health applications. The SAM vaccine platform is based on a synthetic, self-amplifying mRNA delivered by a nonviral delivery system. The safety and immunogenicity of an HIV SAM vaccine encoding a clade C envelope glycoprotein formulated with a cationic nanoemulsion (CNE) delivery system was evaluated in rhesus macaques. The HIV SAM vaccine induced potent cellular immune responses that were greater in magnitude than those induced by self-amplifying mRNA packaged in a viral replicon particle (VRP) or by a recombinant HIV envelope protein formulated with MF59 adjuvant, anti-envelope binding (including anti-V1V2), and neutralizing antibody responses that exceeded those induced by the VRP vaccine. These studies provide the first evidence in nonhuman primates that HIV vaccination with a relatively low dose (50 µg) of formulated self-amplifying mRNA is safe and immunogenic.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , RNA Viral/imunologia , Vacinas contra a AIDS/administração & dosagem , Imunidade Adaptativa , Animais , Animais não Endogâmicos , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Cátions , Células Cultivadas , Emulsões , Infecções por HIV/imunologia , Imunidade Celular , Macaca mulatta , Masculino , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Nucleic acid-based vaccines such as viral vectors, plasmid DNA (pDNA), and mRNA are being developed as a means to address limitations of both live-attenuated and subunit vaccines. DNA vaccines have been shown to be potent in a wide variety of animal species and several products are now licensed for commercial veterinary but not human use. Electroporation delivery technologies have been shown to improve the generation of T and B cell responses from synthetic DNA vaccines in many animal species and now in humans. However, parallel RNA approaches have lagged due to potential issues of potency and production. Many of the obstacles to mRNA vaccine development have recently been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These studies demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable to those induced by EP delivery of pDNA.
RESUMO
Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/administração & dosagem , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Alphavirus/genética , Análise de Variância , Animais , Eletroforese em Gel de Ágar , Escherichia coli , Feminino , Imunofluorescência , Humanos , Lipídeos/química , Nanopartículas/química , RNA Interferente Pequeno/química , Ratos , Estatísticas não ParamétricasRESUMO
Intravaginal (ivag) delivery, which is a proven way to confer local protection against STDs contracted via the reproductive tract, is complicated by the mucus gel barrier, the hormone cycle, and the harsh mucosal environment that leads to low residence-time for administered agents. Polymer delivery vehicles may be useful in overcoming these barriers. In this study, we explored the fate of nanoparticles (NP) made from poly(lactide-co-glycolide) (PLGA) in the mouse reproductive tract after ivag delivery. The nanoparticles were modified to display avidin (Avid-NP) or 2 kDa PEG (PEG-NP) on their surface. Vaginal retention fractions for both muco-adhesive Avid-NP and stealthy PEG-NP were 5× higher than unmodified PLGA particles (NP). The amount of particles associated with mucus differed across formulations (Avid-NP>NP>PEG-NP). PEG-NP was found at higher concentration in the tissue than Avid-NP and NP up to 6h after delivery, and particles were found within epithelial cells, the underlying submucosal stromal and fibroblast cells of the vaginal tissue. Our results demonstrate that surface properties of nanoparticles can impact their fates following ivag delivery. Moreover, we show that the muco-evasive PEG-modified nanoparticles are the most effective among the delivery vehicles tested for this application.
Assuntos
Avidina/química , Portadores de Fármacos/química , Nanopartículas/química , Polietilenoglicóis/química , Poliglactina 910/química , Vagina/metabolismo , Administração Intravaginal , Animais , Cumarínicos/administração & dosagem , Portadores de Fármacos/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Polietilenoglicóis/metabolismo , Poliglactina 910/metabolismo , Tiazóis/administração & dosagem , Vagina/ultraestruturaRESUMO
Design of easily administered vaccines to protect the female reproductive tract against STIs such as HIV, HPV and HSV is a major step in improving world health standards. However, the effect of immunization routes and regimens (prime/boost) on immune response is not well-understood. Here, we present a systematic study of vaccine delivery by different routes and prime/boosting regimens to produce a robust humoral immune response in the reproductive tract. A model antigen, ovalbumin (OVA), was delivered orally or intranasally via polymer particles, and intravaginally via polymer disks to female mice. Repeated prime/boost at a single site result in high OVA-specific antibody levels in the serum for mice immunized orally (IgA) and invaginally (IgA and IgG) after 3 months. Vaginal antibody titers were the highest for mice immunized by intravaginal routes. Vaginal boosting following intranasal or oral priming did not appear to offer similar advantages to those primed intravaginally. Systemic immunization with OVA in Freund's adjuvant produced robust serum IgG levels, but little serum IgA or antibodies in the vaginal washings. All immunization schemes produced a significant level of IgG in the intestinal mucosa, with the exception of nasal priming followed by intravaginal boost with slow-releasing disks. In contrast, only immunization by nasal priming and intravaginal boost with fast-releasing disks was able to achieve significantly high intestinal IgA titers.
Assuntos
Vacinas/administração & dosagem , Administração Intravaginal , Administração Oral , Animais , Especificidade de Anticorpos , Sistemas de Liberação de Medicamentos , Feminino , Imunidade nas Mucosas , Imunização Secundária/métodos , Imunoglobulina A/sangue , Imunoglobulina A/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Ácido Láctico , Camundongos , Camundongos Endogâmicos BALB C , Microesferas , Modelos Imunológicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Vagina/imunologiaRESUMO
DNA delivery to cells can be improved by using particle carriers made from biodegradable polymers such as poly(lactic-co-glycolic)acid (PLGA). It is speculated that addition of targeting moieties to the particle surface to facilitate uptake can further enhance gene expression in specific cells or tissues. Taking advantage of well-known receptor/ligand interactions in intestinal and renal epithelial cells, we formulated PLGA particles with high density of surface-bound bovine serum albumin (BSA; approximately 768 molecules/particle). BSA-coated particles exhibited significantly higher uptake by cells expressing the albumin receptor, megalin, and resisted degradation in low pH. However, gene expression from BSA-coated particles was 3- to 10-fold lower than that from unmodified particles; this reduction in transfection efficiency was probably due to the slower DNA release rate from modified particles. In this setting, addition of a targeting feature to particles reduced their effectiveness. Our study highlights the importance of the interplay between cell uptake and payload release in the design of polymer drug carriers. FROM THE CLINICAL EDITOR: DNA delivery to cells can be improved by using particle carriers such as PLGA. Taking advantage of known receptor/ligand interactions in intestinal and renal epithelial cells, PLGA particles with high density surface-bound BSA were formulated. BSA-coated particles exhibited significantly higher uptake; however, gene expression was 3 to 10-fold lower. Unexpectedly, the addition of a targeting feature to these particles reduced their overall effectiveness.
Assuntos
DNA/administração & dosagem , DNA/farmacocinética , Portadores de Fármacos/química , Rim/química , Rim/fisiologia , Transfecção/métodos , Animais , Células Cultivadas , Ligantes , GambásRESUMO
Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protection against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternative approach using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing, we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA to the vaginal mucosa.
Assuntos
Materiais Biocompatíveis , Inativação Gênica , Ácido Láctico/administração & dosagem , Nanopartículas , Ácido Poliglicólico/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Vagina , Animais , Linhagem Celular , Feminino , Fluorescência , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos ICR , Mucosa , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
The rate of molecular transport through the mucus gel can be an important determinant of efficacy for therapeutic agents delivered by oral, intranasal, intravaginal/rectal, and intraocular routes. Transport through mucus can be described by mathematical models based on principles of physical chemistry and known characteristics of the mucus gel, its constituents, and of the drug itself. In this paper, we review mathematical models of molecular diffusion in mucus, as well as the techniques commonly used to measure diffusion of solutes in the mucus gel, mucus gel mimics, and mucosal epithelia.
Assuntos
Modelos Teóricos , Muco/fisiologia , Animais , Fenômenos Químicos , Difusão , Humanos , Substâncias Macromoleculares/farmacocinética , Modelos Biológicos , Muco/química , Muco/metabolismo , ViscosidadeRESUMO
Drug delivery to mucosal epithelia is severely limited by the mucus gel, which is a physical diffusion barrier as well as an enzymatic barrier in some sites. Loading of drug into polymer particles can protect drugs from degradation and enhance their stability. To improve efficacy of nanoparticulate drug carriers, it has been speculated that polymers such as poly(ethylene)glycol (PEG) incorporated on the particle surface will enhance transport in mucus. In the present study, we demonstrate the direct influence of PEG on surface properties of poly(lactic-co-glycolic)acid (PLGA) nanoparticles (d = 170 +/- 57 nm). PEG of various molecular weights (MW = 2, 5, 10 kDa) were incorporated at a range of densities from 5-100% on the particle surface. Our results indicate PEG addition improves dispersion, neutralize charge, and enhance particle diffusion in cervical mucus in a manner strongly dependent on polymer MW and density. Diffusion of PEGylated particles was 3-10x higher than that of unmodified PLGA particles. These findings improve the understanding of, and confirm a possible direction for, the rational design of effective carriers for mucosal drug/vaccine delivery.
Assuntos
Muco do Colo Uterino/metabolismo , Ácido Láctico/química , Ácido Láctico/metabolismo , Nanopartículas/química , Polietilenoglicóis/química , Ácido Poliglicólico/química , Ácido Poliglicólico/metabolismo , Difusão , Humanos , Microscopia Eletrônica de Varredura , Nanopartículas/ultraestrutura , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Propriedades de Superfície , ÁguaRESUMO
DNA can be used as a generic delivery vector in addition to its genetic role as a antigen expression vector. This is inspired in part by the fact that DNA molecules are true polymers. Surprisingly, DNA molecules have not been used as a delivery vector material. This is probably due to the fact that almost all DNA have only two shapes: linear or circular. This chapter details our efforts in fabricating highly branched dendrimer-like DNA (DL-DNA) that may serve as a multivalent DNA delivery vector. Just like chemical dendrimers, DL-DNA is multi-valent and monodisperse. However, unlike traditional chemical dendrimers, DL-DNA is much larger (~100 nm, generation 4) and can be designed to be nonsymmetric as well. Most importantly, DL-DNA possesses two unique properties: anisotropicity and biodegradability, making multiple, specific conjugations of viral peptides possible. Our method suggests that viral-pep-tide conjugated DL-DNA vectors can deliver genes into cells without any other transfection reagents. This viral-nonviral hybrid system can be further tailored to specific cells by conjugating specific ligands. We believe that such a DL-DNA-based, viral, and nonviral hybrid assembly will provide a new platform for drug delivery in general and gene delivery in particular.
Assuntos
Dendrímeros/química , Técnicas de Transferência de Genes , Vetores Genéticos/química , Peptídeos/química , Vacinas de DNA/química , Proteínas Virais/químicaRESUMO
Rapid, multiplexed, sensitive and specific molecular detection is of great demand in gene profiling, drug screening, clinical diagnostics and environmental analysis. One of the major challenges in multiplexed analysis is to identify each specific reaction with a distinct label or 'code'. Two encoding strategies are currently used: positional encoding, in which every potential reaction is preassigned a particular position on a solid-phase support such as a DNA microarray, and reaction encoding, where every possible reaction is uniquely tagged with a code that is most often optical or particle based. The micrometer size, polydispersity, complex fabrication process and nonbiocompatibility of current codes limit their usability. Here we demonstrate the synthesis of dendrimer-like DNA-based, fluorescence-intensity-coded nanobarcodes, which contain a built-in code and a probe for molecular recognition. Their application to multiplexed detection of the DNA of several pathogens is first shown using fluorescence microscopy and dot blotting, and further demonstrated using flow cytometry that resulted in detection that was sensitive (attomole) and rapid.