RESUMEN
Acute bacterial skin and skin structure infections (ABSSSIs) confer a substantial burden on the healthcare system. Local antibiotic delivery systems can provide controlled drug release directly to the site of infection to maximize efficacy and minimize systemic toxicity. The purpose of this study was to examine the antibacterial activity of antibiotic-loaded glutathione-conjugated poly(ethylene glycol) hydrogels (GSH-PEG) against ABSSSIs utilizing an ex vivo porcine dermal explant model. Vancomycin- or meropenem-loaded GSH-PEG hydrogels at 3 different dose levels were loaded over 1 h. Drug release was monitored in vitro under submerged conditions, by the Franz cell diffusion method, and ex vivo utilizing a porcine dermis model. Antibacterial activity was assessed ex vivo on porcine dermis explants inoculated with Staphylococcus aureus or Pseudomonas aeruginosa isolates treated with vancomycin- or meropenem-loaded GSH-PEG hydrogels, respectively. Histological assessment of the explants was conducted to evaluate tissue integrity and viability in the context of the experimental conditions. A dose-dependent release was observed from vancomycin and meropenem hydrogels, with in vitro Franz cell diffusion data closely representing ex vivo vancomycin release, but not high dose meropenem release. High dose vancomycin-loaded hydrogels resulted in a >3 log10 clearance against all S. aureus isolates at 48 h. High dose meropenem-loaded hydrogels achieved 6.5, 4, and 2 log10 reductions in CFU/ml against susceptible, intermediate, and resistant P. aeruginosa isolates, respectively. Our findings demonstrate the potential application of GSH-PEG hydrogels for flexible, local antibiotic delivery against bacterial skin infections.
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Antibacterianos , Vancomicina , Animales , Porcinos , Antibacterianos/farmacología , Antibacterianos/química , Hidrogeles/química , Staphylococcus aureus , Meropenem , Materiales BiocompatiblesRESUMEN
Block copolymer micelles have demonstrated great promise in the solubilization of hydrophobic drugs, but an understanding of the blood stability of the drug-laden micelles is needed for therapeutic advancement of micelle technologies. Following intravenous administration, mPEG-CL and mPEG-LA micelles have demonstrated quick release of their cargo and disassembly in blood, but the prevailing mechanisms of micelle disruption and key biomacromolecules driving this disruption have yet to be elucidated. Although protein interactions with solid polymeric nanoparticles have been characterized, not much is known regarding protein interactions with dynamic block copolymer micelles. Herein, we characterize the interaction of bovine and human serum albumins (BSA and HSA) with polymeric micelles, mPEG-CL and mPEG-LA, using protein fluorescence, isothermal titration calorimetry (ITC), and circular dichroism (CD) spectroscopy. We find that BSA and HSA have interactions with mPEG-CL, while only HSA is observed to weakly interact with mPEG-LA. Protein fluorescence suggests that binding of HSA to mPEG-CL and mPEG-LA is driven by electrostatic interactions. ITC suggests an interaction between serum albumin and mPEG-CL block copolymers driven by hydrogen bonding and electrostatic interactions in physiological MOPS-buffered saline, while mPEG-LA has no measurable interaction with either of the serum albumins. CD spectroscopy demonstrates that the protein secondary structure is intact in both proteins in the presence of mPEG-CL and mPEG-LA. Overall, BSA is not always predictive of polymeric interactions with HSA. Understanding of interactions between serum proteins and block copolymer micelles and the exact mechanisms of destabilization will direct the rational design of block copolymer systems for improving blood stability.
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Micelas , Nanopartículas , Animales , Bovinos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Polímeros , Albúmina SéricaRESUMEN
PURPOSE: Skin and soft tissue infections are increasingly prevalent and often complicated by potentially fatal therapeutic hurdles, such as poor drug perfusion and antibiotic resistance. Delivery vehicles capable of versatile loading may improve local bioavailability and minimize systemic toxicities yet such vehicles are not clinically available. Therefore, we aimed to expand upon the use of glutathione-conjugated poly(ethylene glycol) GSH-PEG hydrogels beyond protein delivery and evaluate the ability to deliver traditional therapeutic molecules. METHODS: PEG and GSH-PEG hydrogels were prepared using ultraviolet light (UV)-polymerization. Hydrogel loading and release of selected drug candidates was examined using UV-visible spectrometry. Therapeutic molecules and GST-fusion protein loading was examined using UV-visible and fluorescent spectrometry. Efficacy of released meropenem was assessed against meropenem-sensitive and -resistant P. aeruginosa in an agar diffusion bioassay. RESULTS: For all tested agents, GSH-PEG hydrogels demonstrated time-dependent loading whereas PEG hydrogels did not. GSH-PEG hydrogels released meropenem over 24 h. Co-loading of biologic and traditional therapeutics into a single vehicle was successfully demonstrated. Meropenem-loaded GSH-PEG hydrogels inhibited the growth of meropenem-sensitive and resistant P. aeruginosa isolates. CONCLUSION: GSH ligands within GSH-PEG hydrogels allow loading and effective delivery of charged therapeutic agents, in addition to biologic therapeutics.
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Antibacterianos/administración & dosificación , Productos Biológicos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Hidrogeles/química , Infecciones por Pseudomonas/tratamiento farmacológico , Antibacterianos/farmacocinética , Disponibilidad Biológica , Productos Biológicos/farmacocinética , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/farmacocinética , Liberación de Fármacos , Farmacorresistencia Bacteriana , Quimioterapia Combinada , Glutatión/química , Humanos , Meropenem/administración & dosificación , Meropenem/farmacocinética , Pruebas de Sensibilidad Microbiana , Polietilenglicoles/química , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Enfermedades Cutáneas BacterianasRESUMEN
Micelles, as a class of drug delivery systems, are underrepresented among United States Food and Drug Administration approved drugs. A lack of clinical translation of these systems may be due to, in part, to a lack of understanding of micelle interactions with biologic fluids following injection. Despite the limited clinical translation, micelles remain an active area of research focus and pre-clinical development. The goal of the present study was to examine the stability of amphiphilic block copolymer micelles in biologic fluids to identify the properties and components of biologic fluids that influence micelle stability. Micelle stability, measured via Förster resonance energy transfer-based fluorescent spectrometry, was complemented with density ultracentrifugation to reveal the colocalized, or dissociated, state of the dye cargo after exposure to human biologic fluids. Polymeric micelles composed of poly(ethylene glycol-block-caprolactone) (mPEG-CL) and poly(ethylene glycol-block-lactide) (mPEG-LA) were unstable in fetal bovine serum, human serum and synovial fluid, with varying levels of instability observed in ascites and pleural fluid. All polymeric micelles exhibited stability in cerebrospinal fluid, highlighting the potential for local cerebro-spinal administration of micelles. Interestingly, mPEG2.2k-CL3.1k and mPEG2k-LA2.7k micelles favored dissolution whereas mPEG5.4k-LA28.5k micelles favored stability. Taken together, our data offers both quantitative and qualitative evidence for micelle stability within human biologic fluids and offers evidence of polymer micelle instability in biologic fluids that is not explained by either total protein content or total unsaturated lipid content. The results help to identify potential sites for local delivery where stability is maintained.
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Micelas , Polímeros , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Humanos , Poliésteres , Polietilenglicoles , UltracentrifugaciónRESUMEN
Natural Deep Eutectic Solvent (NADES) species can exhibit unexpected solubilizing power for lipophilic molecules despite their simple composition: hydrophilic organic molecules and water. In the present study, the unique properties of NADES species were applied in combination with a model polymer system: a hydrophilic chitosan/alginate hydrogel. Briefly, NADES species (e.g., mannose-dimethylurea-water, 2:5:5, mole/mole) formed matrices to 1) dissolve lipophilic molecules (e.g., curcumin), 2) load lipophilic molecule(s) into the hydrogel, and 3) spontaneously vacate from the system. NADES species ubiquitously occur in natural sources, and a crude extract is a mixture of the NADES species and bioactive metabolites. Based on these ideas, we hypothesized that the crude extract may also allow the loading of natural bioactive molecules from a natural NADES species into (bio)hydrogel systems. To evaluate this hypothesis in vitro, Schisandra chinensis fruit extract was chosen as a representative mixture of lipophilic botanical molecules and hydrophilic NADES species. The results showed that the NADES matrix of S. chinensis was capable of loading at least three bioactive lignans (i.e., gomisin A, gomisin J, and angeloylgomisin H) into the polymer system. The lipophilic metabolites can subsequently be released from the hydrogel. The outcomes suggest that a unique drug delivery mechanism may exist in nature, thereby potentially improving the bioavailability of lipophilic metabolites through physicochemical interactions with the NADES.
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Lignanos/química , Fitoquímicos/química , Schisandra/química , Solventes/química , Disponibilidad Biológica , Frutas/química , Hidrogeles/química , Extractos Vegetales/químicaRESUMEN
Macrophages are the initial biologic responders to biomaterials. These highly plastic immune sentinels control and modulate responses to materials, foreign or natural. The responses may vary from immune stimulatory to immune suppressive. Several parameters have been identified that influence macrophage response to biomaterials, specifically size, geometry, surface topography, hydrophobicity, surface chemistry, material mechanics, and protein adsorption. In this review, the influence of these parameters is supported with examples of both synthetic and naturally derived materials and illustrates that a combination of these parameters ultimately influences macrophage responses to the biomaterial. Having an understanding of these properties may lead to highly efficient design of biomaterials with desirable biologic response properties.
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Materiales Biocompatibles/farmacología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Animales , HumanosRESUMEN
Glioblastoma multiforme (GBM) and other central nervous system (CNS) cancers have poor long-term prognosis, and there is a significant need for improved treatments. GBM initiation and progression are mediated, in part, by microRNA (miRNA), which are endogenous posttranscriptional gene regulators. Misregulation of miRNAs is a potential target for therapeutic intervention in GBM. In this work, a micelle-like nanoparticle delivery system based upon the block copolymer poly(ethylene glycol-b-lactide-b-arginine) was designed with and without a reducible linkage between the lactide and RNA-binding peptide, R15, to assess the ability of the micelle-like particles to disassemble. Using confocal live cell imaging, intracellular dissociation was pronounced for the reducible micelleplexes. This dissociation was also supported by higher efficiency in a dual luciferase assay specific for the miRNA of interest, miR-21. Notably, micelleplexes were found to have significantly better stability and higher anti-miRNA activity in cerebrospinal fluid than in human plasma, suggesting an advantage for applying micelleplexes to CNS diseases and in vivo CNS therapeutics. The reducible delivery system was determined to be a promising delivery platform for the treatment of CNS diseases with miRNA therapy.
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Antineoplásicos/farmacología , Líquido Cefalorraquídeo/metabolismo , MicroARNs/metabolismo , Arginina/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Glioblastoma/tratamiento farmacológico , Humanos , Micelas , Nanopartículas/administración & dosificación , Poliésteres/administración & dosificación , Polietilenglicoles/administración & dosificación , Polímeros/administración & dosificaciónRESUMEN
PURPOSE: To prolong the release of a heparan sulfate binding peptide, G2-C, using a commercially available contact lens as a delivery vehicle and to demonstrate the ability of the released peptide to block herpes simplex virus-1 (HSV-1) infection using in vitro, ex vivo, and in vivo models of corneal HSV-1 infection. METHODS: Commercially available contact lenses were immersed in peptide solution for 5 days prior to determining the release of the peptide at various time points. Cytotoxicity of the released samples was determined by MTT and cell cycle analysis, and the functional activity of the released samples were assessed by viral entry, and viral spread assay using human corneal epithelial cells (HCE). The ability to suppress infection in human and pig cornea ex vivo and mouse in vivo models were also assessed. RESULTS: Peptide G2-C was released through the contact lens. Following release for 3 days, the peptide showed significant activity by inhibiting HSV-1 viral entry and spread in HCE cells. Significant suppression of infection was also observed in the ex vivo and in vivo experiments involving corneas. CONCLUSIONS: Extended release of an anti-HS peptide through a commercially available contact lens can generate significant anti-HSV-1 activity and provides a new and effective way to control corneal herpes.
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Lentes de Contacto , Córnea/virología , Infecciones Virales del Ojo/tratamiento farmacológico , Heparitina Sulfato/farmacología , Herpesvirus Humano 1 , Queratitis Herpética/tratamiento farmacológico , Animales , Córnea/metabolismo , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Infecciones Virales del Ojo/metabolismo , Infecciones Virales del Ojo/virología , Femenino , Humanos , Queratitis Herpética/metabolismo , Queratitis Herpética/virología , Masculino , Ratones , Ratones Endogámicos BALB C , PorcinosRESUMEN
Macrophages exhibit phenotypic diversity permitting wide-ranging roles in maintaining physiologic homeostasis. Hyaluronic acid, a major glycosaminoglycan of the extracellular matrix, has been shown to have differential signaling based on its molecular weight. With this in mind, the main objective of this study was to elucidate the role of hyaluronic acid molecular weight on macrophage activation and reprogramming. Changes in macrophage activation were assessed by activation state selective marker measurement, specifically quantitative real time polymerase chain reaction, and cytokine enzyme-linked immunoassays, after macrophage treatment with differing molecular weights of hyaluronic acid under four conditions: the resting state, concurrent with classical activation, and following inflammation involving either classically or alternatively activated macrophages. Regardless of initial polarization state, low molecular weight hyaluronic acid induced a classically activated-like state, confirmed by up-regulation of pro-inflammatory genes, including nos2, tnf, il12b, and cd80, and enhanced secretion of nitric oxide and TNF-α. High molecular weight hyaluronic acid promoted an alternatively activated-like state, confirmed by up regulation of pro-resolving gene transcription, including arg1, il10, and mrc1, and enhanced arginase activity. Overall, our observations suggest that macrophages undergo phenotypic changes dependent on molecular weight of hyaluronan that correspond to either (1) pro-inflammatory response for low molecular weight HA or (2) pro-resolving response for high molecular weight HA. These observations bring significant further understanding of the influence of extracellular matrix polymers, hyaluronic acid in particular, on regulating the inflammatory response of macrophages. This knowledge can be used to guide the design of HA-containing biomaterials to better utilize the natural response to HAs.
RESUMEN
Nanocrystals are carrier-free solid drug particles that are sized in the nanometer range and have crystalline characteristics. Due to high drug loading (as high as 100%) - free of organic solvents or solubilizing chemicals - nanocrystals have become attractive in the field of drug delivery for cancer treatment. Top-down and bottom-up approaches have been developed for preparing anticancer nanocrystals. In this review, preparation methods and in vivo performance of anticancer nanocrystals are discussed first, followed by an introduction of hybrid nanocrystals in cancer theranostics.
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Antineoplásicos/uso terapéutico , Nanopartículas , Neoplasias/terapia , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Humanos , Propiedades de SuperficieRESUMEN
Micelleplexes are a class of nucleic acid carriers that have gained acceptance due to their size, stability, and ability to synergistically carry small molecules. MicroRNAs (miRNAs) are small non-coding RNA gene regulator that is consists of 19-22 nucleotides. Altered expression of miRNAs plays an important role in many human diseases. Using a model 22-nucleotide miRNA sequence, we investigated the interaction between charged groups on the micelle surface and miRNA. The model micelle system was formed from methoxy-poly(ethylene glycol)-b-poly(lactide) (mPEG-PLA) mixed with methoxy-poly(ethylene glycol)-b-poly(lactide)-b-oligoarginine (mPEG-PLA-Rx, x = 8 or 15). Surface properties of the micelles were varied by controlling the oligoarginine block length and conjugation density. Micelles were observed to have a core-shell conformation in the aqueous environment where the PLA block constituted the hydrophobic core, mPEG and oligoarginine formed a hydrophilic corona. Significantly different thermodynamic behaviors were observed during the interaction of single stranded miRNA with micelles of different surface properties, and the resulting micelleplexes mediated substantial cellular association. Depending upon the oligoarginine length and density, micelles exhibited miRNA loading capacity directly related to the presentation of charged groups on the surface. The effect of charged group accessibility of cationic micelle on micelleplex properties provides guidance on future miRNA delivery system design.
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Portadores de Fármacos/química , Micelas , MicroARNs/química , Poliésteres/química , Polietilenglicoles/química , Línea Celular Tumoral , Portadores de Fármacos/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Propiedades de Superficie , TermodinámicaRESUMEN
Although potent, proteins often require chemical modification for therapeutic use. Immunogenicity, difficult synthesis, and scale-up of these modifications are all engineering obstacles that stand in the way of expanding the use of these therapeutics. Melittin, a peptide derived from bee venom, has been shown to modulate inflammation. Although potentially therapeutic, the native peptide causes cell lysis and toxicity significantly hindering therapeutic application. Based upon the knowledge of the pore formation mechanism, we examined the toxicity and therapeutic effect of a melittin fusion protein with glutathione-S-transferase. The fusion of melittin and glutathione S-transferase results in diminished toxicity of the peptide and retained anti-inflammatory properties at doses that exceed toxic concentration of native melittin. Our results suggest that fusion proteins, particularly those of glutathione-S-transferase, may be facile modifications to control protein activity.
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Antiinflamatorios/farmacología , Glutatión Transferasa/farmacología , Meliteno/farmacología , Proteínas Recombinantes/farmacología , Animales , Venenos de Abeja/farmacología , Células Cultivadas , Inflamación/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Ratones , Péptidos/farmacologíaRESUMEN
MicroRNAs (miRNAs) are a class of gene regulators originating from non-coding endogenous RNAs. Altered expression, both up- and down-regulation, of miRNAs plays important roles in many human diseases. Correcting miRNA dysregulation by either inhibiting or restoring miRNA function may provide therapeutic benefit. However, efficient, nontoxic miRNA delivery systems are in need. Cell penetrating peptides (CPPs) have been widely exploited for protein, DNA, and RNA delivery. Few have examined CPP transfection efficiency with single stranded anti-miRNA. The R8 peptide condensed both siRNA and anti-miRNA. Greater than 50% of cells had anti-miRNA/R8 complexes associated and in these cells 68% of anti-miRNA escapes the endosome/lysosome. Single-stranded antisense miR-21 inhibitor (anti-miR-21) administered using the R8 peptide elicited efficient downstream gene upregulation. Glioblastoma cell migration was inhibited by 25% compared to the negative control group. To our knowledge, this is the first demonstration of miRNA modulation with anti-miR-21/R8 complexes, which has laid the groundwork for further exploring octaarginine as intracellular anti-miRNAs carrier.
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Movimiento Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Glioblastoma/metabolismo , MicroARNs/antagonistas & inhibidores , ARN sin Sentido/farmacología , ARN Neoplásico/antagonistas & inhibidores , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Glioblastoma/genética , Glioblastoma/patología , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN sin Sentido/química , ARN sin Sentido/genética , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Transfección/métodosRESUMEN
Heterogeneous toroidal-spiral particles (TSPs) were generated by polymer droplet sedimentation, interaction, and cross-linking. TSPs provide a platform for encapsulation and release of multiple compounds of different sizes and physicochemical properties. As a model system, we demonstrate the encapsulation and independently controlled release of an anti-VEGFR-2 antibody and irinotecan for the treatment of glioblastoma multiforme. The anti-VEGFR-2 antibody was released from the TS channels and its binding to HUVECs was confirmed by confocal microscopy and flow cytometry, suggesting active antibody encapsulation and release. Irinotecan, a small molecule drug, was released from the dense polymer matrix of poly(ethylene glycol) diacrylate (MW ~ 700 g/mol; PEGDA 700). Released irinotecan inhibited the proliferation of U251 malignant glioma cells. Since the therapeutic compounds are released through different pathways, specifically diffusion through the polymer matrix versus TS channels, the release rate can be controlled independently through the design of the structure and material of particle components.
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Anticuerpos Antiidiotipos/administración & dosificación , Sistemas de Liberación de Medicamentos , Glioblastoma/tratamiento farmacológico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Anticuerpos Antiidiotipos/química , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Glioblastoma/patología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Irinotecán , Tamaño de la Partícula , Polímeros/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/administración & dosificaciónRESUMEN
PURPOSE: To develop novel hybrid paclitaxel (PTX) nanocrystals, in which bioactivatable (MMPSense® 750 FAST) and near infrared (Flamma Fluor® FPR-648) fluorophores are physically incorporated, and to evaluate their anticancer efficacy and diagnostic properties in breast cancer xenograft murine model. METHODS: The pure and hybrid paclitaxel nanocrystals were prepared by an anti-solvent method, and their physical properties were characterized. The tumor volume change and body weight change were evaluated to assess the treatment efficacy and toxicity. Bioimaging of treated mice was obtained non-invasively in vivo. RESULTS: The released MMPSense molecules from the hybrid nanocrystals were activated by matrix metalloproteinases (MMPs) in vivo, similarly to the free MMPSense, demonstrating its ability to monitor cancer progression. Concurrently, the entrapped FPR-648 was imaged at a different wavelength. Furthermore, when administered at 20 mg/kg, the nanocrystal formulations exerted comparable efficacy as Taxol®, but with decreased toxicity. CONCLUSIONS: Hybrid nanocrystals that physically integrated two fluorophores were successfully prepared from solution. Hybrid nanocrystals were shown not only exerting antitumor activity, but also demonstrating the potential of multi-modular bioimaging for diagnostics.
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Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Paclitaxel/administración & dosificación , Paclitaxel/uso terapéutico , Animales , Peso Corporal/efectos de los fármacos , Línea Celular Tumoral , Química Farmacéutica , Femenino , Colorantes Fluorescentes , Humanos , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Desnudos , Nanopartículas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer metastasis is difficult to treat, and its outcome becomes dreadful for a patient. Lung is a major metastatic site for many types of cancers, and the need for finding effective treatment for lung metastasis cannot be overemphasized. In a previous study, we showed that camptothecin nanocrystals demonstrated greater anticancer efficacy and achieved significantly higher concentration in lungs than a conventional, solution-based formulation. In this study, we further determined the pharmacokinetics of camptothecin nanocrystals in rats and investigated treatment efficacy in mice against metastatic lung tumors. The results show that camptothecin nanocrystals were capable of eliciting greater antimetastatic efficacy and achieve a longer survival time in the murine model compared with camptothecin salt solution. The study suggests that using engineered, solid nanoparticles may be a feasible approach in the treatment of lung cancer and lung metastatic cancer.
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Antineoplásicos Fitogénicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Camptotecina/farmacocinética , Neoplasias Pulmonares/tratamiento farmacológico , Nanopartículas/química , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Camptotecina/administración & dosificación , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Ratas , Tasa de Supervivencia , Distribución Tisular , Resultado del Tratamiento , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MicroRNAs (miRNAs) are non-coding endogenous RNAs that direct post-transcriptional regulation of gene expression by several mechanisms. Activity is primarily through binding to the 3' untranslated regions (UTRs) of messenger RNAs (mRNA) resulting in degradation and translation repression. Unlike other small-RNAs, miRNAs do not require perfect base pairing, and thus, can regulate a network of broad, yet specific, genes. Although we have only just begun to gain insights into the full range of biologic functions of miRNA, their involvement in the onset and progression of disease has generated significant interest for therapeutic development. Mounting evidence suggests that miRNA-based therapies, either restoring or repressing miRNAs expression and activity, hold great promise. However, despite the early promise and exciting potential, critical hurdles often involving delivery of miRNA-targeting agents remain to be overcome before transition to clinical applications. Limitations that may be overcome by delivery include, but are not limited to, poor in vivo stability, inappropriate biodistribution, disruption and saturation of endogenous RNA machinery, and untoward side effects. Both viral vectors and nonviral delivery systems can be developed to circumvent these challenges. Viral vectors are efficient delivery agents but toxicity and immunogenicity limit their clinical usage. Herein, we review the recent advances in the mechanisms and strategies of nonviral miRNA delivery systems and provide a perspective on the future of miRNA-based therapeutics.
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Sistemas de Liberación de Medicamentos , Técnicas de Transferencia de Gen , MicroARNs/administración & dosificación , MicroARNs/uso terapéutico , Animales , Dendrímeros/química , Sistemas de Liberación de Medicamentos/métodos , Regulación de la Expresión Génica , Humanos , Ácido Láctico/química , Lípidos/química , MicroARNs/genética , MicroARNs/farmacocinética , Polietileneimina/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Distribución TisularRESUMEN
The stem cell differentiation paradigm is based on the progression of cells through generations of daughter cells that eventually become restricted and committed to one lineage resulting in fully differentiated cells. Herein, we report on the differentiation of adult human mesenchymal stem cells (hMSCs) towards adipogenic and osteogenic lineages using established protocols. Lineage specific geneswere evaluated by quantitative real-time PCR relative to two reference genes. The expression of osteoblast-associated genes (alkaline phosphatase, osteopontin, and osteocalcin)was detected in hMSCs that underwent adipogenesis. When normalized, the expression of adipocyte marker genes (adiponectin, fatty acid binding protein P4, and leptin) increasedin a time-dependent manner during adipogenic induction. Adiponectin and leptin were also detected in osteoblast-induced cells. Lipid vacuoles that represent the adipocyte phenotype were only present in the adipogenic induction group. Conforming to the heterogeneous nature of hMSCs and the known plasticity between osteogenic and adipogenic lineages, these data indicatea marker overlap between MSC-derived adipocytes and osteoblasts. Weproposea careful consideration of experimental conditions such as investigated timepoints, selected housekeeping genesand the evidence indicating lack of differentiation into other lineageswhen evaluating hMSC differentiation.
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Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of â¼ 0.5-1 mg/L of E. coli culture.
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Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Meliteno/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Antibacterianos/aislamiento & purificación , Antineoplásicos/aislamiento & purificación , Línea Celular Tumoral , Clonación Molecular , Escherichia coli/metabolismo , Humanos , Meliteno/genética , Proteínas Recombinantes/genética , Streptococcus pyogenes/efectos de los fármacosRESUMEN
Paclitaxel (PTX) nanocrystals (200 nm) were produced by crystallization from a solution. Antitumor efficacy and toxicity were examined through a survival study in a human HT-29 colon cancer xenograft murine model. The antitumor activity of the nanocrystal treatments was comparable with that by the conventional solubilization formulation (Taxol®), but yielded less toxicity as indicated by the result of a survival study. Tritium-labeled PTX nanocrystals were further produced with a near infrared (NIR) fluorescent dye physically integrated in the crystal lattice. Biodistribution and tumor accumulation of the tritium-labeled PTX nanocrystals were determined immediately after intravenous administration and up to 48 h by scintillation counting. Whole-body optical imaging of animals was concurrently carried out; fluorescent intensities were also measured from excised tumors and major organs of euthanized animals. It was found that drug accumulation in the tumor was less than 1% of 20mg/kg intravenous dose. Qualitatively correlation was identified between the biodistribution determined by using tritium-labeled particles and that using optical imaging, but quantitative divergence existed. The divergent results suggest possible ways to improve the design of hybrid nanocrystals for cancer therapy and diagnosis. The study also raises questions of the general role of the enhanced permeability and retention (EPR) effect in tumor targeting and the effectiveness of bioimaging, specifically for theranostics, in tracking drug distribution and pharmacokinetics.