RESUMEN

Transfusion of healthy red blood cells (RBCs) is a lifesaving process. However, upon storing RBCs, a wide range of damage-associate molecular patterns (DAMPs), such as cell-free DNA, nucleosomes, free-hemoglobin, and poly-unsaturated-fatty-acids are generated. DAMPs can further damage RBCs; thus, the quality of stored RBCs declines during the storage and limits their shelf-life. Since these DAMPs consist of either positive or negative charged species, we developed taurine and acridine containing electrospun-nanofibrous-sheets (Tau-AcrNFS), featuring anionic, cationic charges and an DNA intercalating group on their surfaces. We show that Tau-AcrNFS are efficient in scavenging DAMPs from stored human and mice RBCs ex vivo. We find that intermittent scavenging of DAMPs by Tau-AcrNFS during the storage reduces the loss of RBC membrane integrity and reduces discocytes-to-spheroechinocytes transformation in stored-old-RBCs. We perform RBC-transfusion studies in mice to reveal that intermittent removal of DAMPs enhances the quality of stored-old-RBCs equivalent to freshly collected RBCs, and increases their shelf-life by ~22%. Such prophylactic technology may lead to the development of novel blood bags or medical device, and may therefore impact healthcare by reducing transfusion-related adverse effects.

Asunto(s)

Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Nanofibras , Humanos , Animales , Ratones , Eritrocitos , Acridinas , Investigadores

RESUMEN

Lifelong systemic immunosuppression remains the biggest challenge in vascularized composite allotransplantation (VCA) due to the adverse effects it causes. Since VCA is a life-enhancing procedure as compared with solid organ transplant which is life-saving; one needs to weigh the benefits and risks carefully. Thus, there is a huge unmet clinical need to design biomaterial-based vehicles that can deliver drugs more efficiently, topically and locally to eliminate adverse effects of systemic immune suppression. This review discusses several biomaterial-based systems that have been carefully designed, conceived and attempted to make VCA a more patient compliant approach. Variety of promising preclinical studies has shown the feasibility of the approaches, and clinical trials are required to bridge the gap. Several challenges for the future and new approaches have been discussed.

Asunto(s)

Materiales Biocompatibles/uso terapéutico , Terapia de Inmunosupresión , Procedimientos de Cirugía Plástica/métodos , Alotrasplante Compuesto Vascularizado/efectos adversos , Materiales Biocompatibles/química , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Trasplantes/efectos de los fármacos

RESUMEN

Organophosphate-based pesticides inhibit acetylcholinesterase (AChE), which plays a pivotal role in neuromuscular function. While spraying in the field, farmworkers get exposed to pesticides through the dermal route. Internalized pesticide inhibits AChE, which leads to neurotoxicity, cardiotoxicity, cognitive dysfunction, loss of endurance, and death in severe cases. Here, we present a nucleophilic pyridine-2-aldoxime-functionalized chitosan-based topical gel (poly-Oxime gel) that rapidly deactivates organophosphates, methyl parathion (MPT), on the skin of rats, which leads to reduced AChE inhibition in the blood and tissues. Testing the robustness of poly-Oxime gel, we report reduction in AChE inhibition following repeated dermal administration of MPT in the presence of poly-Oxime gel. Furthermore, poly-Oxime gel prevented MPT-induced neuromuscular dysfunction, loss of endurance, and locomotor coordination. We observe a 100% survival in rats following topical MPT administration in the presence of poly-Oxime gel. This prophylactic gel may therefore help farmworkers by limiting pesticide-induced toxicity and mortality.

Asunto(s)

Metil Paratión/toxicidad , Actividad Motora/efectos de los fármacos , Síndromes de Neurotoxicidad/prevención & control , Intoxicación por Organofosfatos/prevención & control , Oximas/administración & dosificación , Administración Tópica , Animales , Insecticidas/toxicidad , Masculino , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/mortalidad , Intoxicación por Organofosfatos/etiología , Intoxicación por Organofosfatos/mortalidad , Ratas , Ratas Sprague-Dawley , Tasa de Supervivencia

RESUMEN

In the recent studies, it has been demonstrated that incorporation of unnatural amino acid, α,ß-dehydrophenylalanine, in small peptides results in stable self-assembled nanostructures with different sizes and shapes. Here, we have replaced the natural amino acid, phenylalanine, from our earlier reported work on self-assembled peptide, Boc-Pro-Phe-Gly-OMe, with a constrained dehydro amino acid, α,ß-dehydrophenylalanine, to study its influence on self-assembled nanostructures. Dehydrotripeptide, Boc-Pro-ΔPhe-Gly-OMe, self-assembled into nanostructures in aqueous solutions and formed hydrophobic matrix with improved encapsulation efficiency of hydrophobic molecules. The hydrodynamic size of peptide nanostructures from DLS study was found to be ∼257nm. The morphology and size of the loaded nanoparticles were also determined by TEM. To improve aqueous dispersibility the projected nanostructures for efficient use in drug delivery, self-assembled dehydropeptide nano carriers were further stabilized with Vitamin-E-TPGS. The final complex drug nanoparticles provided controlled drug release. These findings demonstrated that incorporation of constrained dehydro amino acids in peptides have the potential to construct stable nanostructures for development of nano materials with controlled drug release.

Asunto(s)

Curcumina/química , Preparaciones de Acción Retardada , Nanopartículas/química , Oligopéptidos/química , Ornidazol/química , Fenilalanina/análogos & derivados , Composición de Medicamentos , Liberación de Fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Nanopartículas/ultraestructura , Tamaño de la Partícula , Fenilalanina/química , Vitamina E/química

RESUMEN

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.

Asunto(s)

Antibacterianos/farmacología , Biguanidas/farmacología , Cromosomas Bacterianos/genética , Animales , Antibacterianos/metabolismo , Bacillus megaterium/efectos de los fármacos , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Biguanidas/metabolismo , Células CHO , Bovinos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Estructuras Cromosómicas/efectos de los fármacos , Cricetinae , Cricetulus , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Células HeLa , Caballos , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Estrés Fisiológico/efectos de los fármacos

RESUMEN

Here, we have designed and synthesized a novel cationic amphiphilic stimuli-responsive azobenzene-aminoglycoside (a small molecule) conjugate, Azo-AG 5, and characterized it by UV and FTIR. Light responsive nature of Azo-AG 5 was assessed under UV-vis light. Self- assembly of Azo-AG 5 in aqueous solutions into nanostructures and their ability to act as drug carrier were also investigated. The nanostructures of Azo-AG 5 showed average hydrodynamic diameter of ∼ 255 nm with aminoglycoside moiety (neomycin) and 4-dimethylaminoazobenzene forming hydrophilic shell and hydrophobic core, respectively. In the hydrophobic core, eosin and aspirin were successfully encapsulated. Dynamic light scattering (DLS) measurements demonstrated that the nanoassemblies showed expansion and contraction on successive UV and visible light irradiations exhibiting reversible on-off switch for controlling the drug release behavior. Similar behavior was observed when these nanostructures were subjected to pH-change. In vitro drug release studies showed a difference in UV and visible light-mediated release pattern. It was observed that the release rate under UV irradiation was comparatively higher than that observed under visible light. Further, azoreductase-mediated cleavage of the azo moiety in Azo-AG 5 nanoassemblies resulted in the dismantling of the structures into aggregated microstructures. Azo-AG 5 nanostructures having positive surface charge (+9.74 mV) successfully interacted with pDNA and retarded its mobility on agarose gel. Stimuli responsiveness of nanostructures and their on-off switch like behavior ensure the great potential as controlled drug delivery systems and in other biomedical applications such as colon-specific delivery and gene delivery.

Asunto(s)

Aminoglicósidos/química , Compuestos Azo/química , Nanoestructuras/química , Aminoglicósidos/toxicidad , Compuestos Azo/toxicidad , Supervivencia Celular , ADN/química , Preparaciones de Acción Retardada , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , NADH NADPH Oxidorreductasas/química , Nanoestructuras/toxicidad , Nitrorreductasas , Procesos Fotoquímicos

RESUMEN

Self-assembled peptides and specifically small peptide based nanostructures have been the focus of research in past decade due to their potential biological applications. In this study, we prepared a protected peptide, Boc-Pro-Phe-Gly-OMe, which self-assembled in aqueous solutions leading to the formation of nanostructures and ability to act as a drug carrier. Dynamic light scattering (DLS) measurements showed nanostructures with average size of 119.6 nm containing hydrophobic core, wherein hydrophobic drugs, viz, eosin, aspirin and curcumin, were successfully encapsulated. These encapsulated nanostructures, were further stabilized with Vitamin E-TPGS. In-vitro drug release studies revealed the release of drugs in controlled fashion from the nanostructures. The results advocate the potential of the proposed peptide nanostructures as controlled drug delivery systems and could be used in other biomedical applications.

Asunto(s)

Portadores de Fármacos/química , Nanoestructuras/química , Oligopéptidos/química , Vitamina E/análogos & derivados , Aspirina/metabolismo , Aspirina/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Curcumina/metabolismo , Curcumina/farmacología , Preparaciones de Acción Retardada , Composición de Medicamentos , Liberación de Fármacos , Eosina Amarillenta-(YS)/metabolismo , Eosina Amarillenta-(YS)/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Células MCF-7 , Nanoestructuras/ultraestructura , Tamaño de la Partícula , Polietilenglicoles/química , Vitamina E/química

RESUMEN

Previously, we demonstrated that 6-(N,N,N',N'-tetramethylguanidinium chloride)-hexanoyl-polyethylenimine (THP) polymers exhibited significantly enhanced transfection efficiency and cell viability. Here, in the present study, we have synthesized a series of N,N,N',N'-tetramethylguanidinium-polyethylenimine (TP1-TP5) polymers via a single-step reaction involving peripheral primary amines of bPEI and varying amounts of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). These polymers were found to interact efficiently with negatively charged pDNA and formed stable complexes in the size range of ~240-450 nm. Acid-base titration profiles revealed improved buffering capacity of TP polymers as compared to bPEI. Transfection and cytotoxicity assays performed with TP/pDNA complexes on HEK293, CHO, and HeLa cells showed significantly higher transfection efficiency and cell viability with one of the complexes, TP2/pDNA complex, exhibited the highest transfection efficiency (~1.4-2.3-fold) outcompeting native bPEI and the commercially available transfection reagent, Lipofectamine 2000. Compared to previously reported THP polymers, the transfection efficiency of TP/pDNA complexes was found to be lower, as examined by flow cytometry. These results highlight the importance of the hydrophobic C-6 linker in THP polymers in forming compact nanostructures with pDNA, which might lead to efficient uptake and internalization of the complexes; however, the projected TP polymers offer an advantage of their rapid and economical one-step synthesis.

Asunto(s)

Técnicas de Transferencia de Gen , Guanidina/química , Guanidina/síntesis química , Guanidinas/química , Guanidinas/síntesis química , Polietileneimina/análogos & derivados , Polietileneimina/química , Polietileneimina/síntesis química , Animales , Tampones (Química) , Línea Celular , Supervivencia Celular , Fenómenos Químicos , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Espacio Intracelular/metabolismo , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Plásmidos/metabolismo , Electricidad Estática , Transfección

RESUMEN

Recently, polyethylenimines (PEIs) have emerged as efficient vectors for nucleic acids delivery. However, inherent cytotoxicity has limited their in vivo applications. To address this concern as well as to incorporate hydrophobic domains for improving interactions with the lipid bilayers in the cell membranes, we have tethered varying amounts of amphiphilic pyridoxyl moieties onto bPEI to generate a small series of pyridoxyl-PEI (PyP) polymers. Spectroscopic characterization confirms the formation of PyP polymers, which subsequently form stable complexes with pDNA in nanometric range with positive surface charge. The projected modification not only accounts for a decrease in the density of 1° amines but also allows formation of relatively loose complexes with pDNA (cf. bPEI). Alleviation of the cytotoxicity, efficient interaction with cell membranes and easy disassembly of the pDNA complexes have led to the remarkable enhancement in the transfection efficiency of PyP/pDNA complexes in mammalian cells with one of the formulations, PyP-3/pDNA complex, showing transfection in ∼68% cells compared to ∼16% cells by Lipofectamine/pDNA complex. Further, the efficacy of PyP-3 vector has been established by delivering GFP-specific siRNA resulting in ∼88% suppression of the target gene expression. These results demonstrate the efficacy of the projected carriers that can be used in future gene therapy applications.

Asunto(s)

Aminas/química , Materiales Biocompatibles/farmacología , Técnicas de Transferencia de Gen , Polietileneimina/farmacología , Tampones (Química) , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Hemólisis/efectos de los fármacos , Heparina/metabolismo , Humanos , Ligandos , Lípidos/química , Células MCF-7 , Ensayos de Protección de Nucleasas , Tamaño de la Partícula , Plásmidos/metabolismo , Polietileneimina/síntesis química , Polietileneimina/química , ARN Interferente Pequeño/metabolismo , Electricidad Estática , Volumetría

RESUMEN

In this study, a modified dehydropeptide, Boc-FΔF-εAhx-OH, was conjugated with an aminoglycoside antibiotic, neomycin, to construct a multifunctional conjugate, Pep-Neo. The amphiphilic conjugate (Pep-Neo) was able to self-assemble into cationic nanostructures in an aqueous solution at low concentrations. Nanostructure formation was evidenced by TEM and dynamic light scattering analyses. The average hydrodynamic diameter of the self-assembled Pep-Neo nanostructures was found to be ∼279 nm with a zeta potential of +28 mV. The formation of nanostructures with a hydrophobic core and cationic hydrophilic shell resulted in an increased local concentration of cationic charge (ca. in 50% aqueous methanol, i.e. disassembled structure, zeta potential decreased to +17.6 mV), leading to efficient interactions with negatively charged plasmid DNA (pDNA). The size and zeta potential of the resulting Pep-Neo/pDNA complex were found to be ∼154 nm and +19.4 mV, respectively. Having been characterized by physicochemical techniques, the complex was evaluated for its toxicity and ability to deliver nucleic acid therapeutics. The flow cytometry results on MCF-7 cells revealed that Pep-Neo/pDNA complex transfected ∼27% cells at a w/w ratio of 66.6 while the standard transfection reagent, Lipofectamine, could transfect only ∼15% cells. MTT and hemolysis assays showed the non-toxic nature of the projected conjugate at various concentrations. Further, these nanostructures were shown to encapsulate hydrophobic drugs in the core. Finally, Pep-Neo nanostructures showed efficient antibacterial activity against different strains of Gram-positive and -negative bacteria. Interestingly, unlike neomycin, which is highly effective against Gram-negative bacteria, these nanostructures showed considerably high efficiency against Gram-positive strains, highlighting the promising potential of these nanostructures for various biomedical applications.

RESUMEN

Recent advancements in polymeric gene delivery have raised the potential of gene therapy as treatment for various acquired and inherited diseases. Here, we report on the synthesis and characterization of N-ethyl-N'-(3-dimethylaminopropyl)-guanidinyl-polyethylenimine (sGP) polymers and investigation of their capability to carry DNA and siRNA in vitro. Zinc triflate-mediated activation of primary amines of branched polyethylenimine (bPEI) followed by reaction with varying amounts of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDAC) resulted in the generation of a small series of trisubstituted guanidinyl-modified polyethylenimine polymers. Determination of primary amines on modified polymers by TNBS assay revealed 62-84% of the attempted conjugation of EDAC onto bPEI. These modified polymers were shown to condense plasmid DNA and retard its mobility on 0.8% agarose gel. Further, these polymers were evaluated for their capability to carry pDNA into the cells by performing transfection assay on various mammalian cells. All the modified polymer/pDNA complexes exhibited significantly higher levels of gene expression with one of the complexes, sGP3/pDNA complex, displayed ~1.45 to 3.0 orders of magnitude higher transfection efficiency than that observed in the native bPEI and the commercial transfection reagent, Lipofectamine™. The efficacy of sGP3 polymer was further assessed by siRNA delivery, which resulted in ~81% suppression of the target gene. In conclusion, these studies demonstrate the potential of these substituted guanidinyl-modified PEIs as efficient gene delivery vectors.

Asunto(s)

ADN/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/síntesis química , Guanidinas/química , Guanidinas/síntesis química , Polietileneimina/análogos & derivados , Polímeros/química , Polímeros/síntesis química , ARN Interferente Pequeño/metabolismo , Animales , Células CHO , Supervivencia Celular , Cricetulus , ADN/administración & dosificación , Portadores de Fármacos/metabolismo , Células HEK293 , Células HeLa , Humanos , Polietileneimina/síntesis química , Polietileneimina/química , ARN Interferente Pequeño/administración & dosificación

RESUMEN

Recently, non-viral gene delivery vectors have shown promising results in the treatment of inherited as well as infectious diseases. Among various cationic polymers, branched polyethylenimine (bPEI, 25 kDa) has been the most widely used vector and is known as a gold standard in gene delivery. However, in vivo applications of PEI have been hampered by its charge-associated toxicity. Here, in order to address the toxicity concern as well as to enhance the transfection efficacy of bPEI, it has been modified by tethering a hydrophobic and positively charged 6-(N,N,N',N'-tetramethylguanidinium chloride)-hexanoic acid (TH) linker using water soluble coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC). A small series of 6-(N,N,N',N'-tetramethylguanidinium chloride)-hexanoyl-polyethylenimine (THP) polymers was synthesized by varying the amounts of TH-linker . These modified polymers were subsequently characterized by spectroscopic techniques and evaluated for their buffering capacity, transfection efficiency and cytotoxicity on HeLa, HEK293 and CHO cells and the results were compared with those obtained with the standard transfection reagent, Lipofectamine™. From these studies, it was observed that one of the formulations, the THP2-DNA complex, exhibited significantly higher transfection efficiency (∼1.5-3.6 folds) and cell viability, outcompeting native bPEI and Lipofectamine™ in the absence and presence of serum. Further, to establish the superiority of THP2, it was examined for its ability to bind and deliver plasmid DNAs (pDNAs) of different molecular weight, provide protection against nucleases present in the cellular milieu and also deliver siRNAs (GFP- and GAPDH-specific). Taken together, the results show the potential of THP polymers as promising carriers of nucleic acids for in vivo applications.

Asunto(s)

ADN/química , Técnicas de Transferencia de Gen , Polietileneimina/química , ARN Interferente Pequeño/química , Tensoactivos/química , Animales , Células CHO , Supervivencia Celular , Cricetinae , ADN/metabolismo , Células HEK293 , Células HeLa , Humanos , Tamaño de la Partícula , Plásmidos , Polietileneimina/metabolismo , ARN Interferente Pequeño/metabolismo , Tensoactivos/metabolismo , Transfección

RESUMEN

Self-assembled peptide-based nanostructures have been the focus of research in the past decade because of their potential applications in various biological systems. Normally, small self-assembled peptide nanostructures contain hydrophobic moieties, therefore, their solubility in aqueous systems poses the important challenge in the field of molecular self-assembly in order to make effective use of these in a wide variety of applications. To improve their aqueous solubility, the self-assembled amphiphilic α,ß-dehydrophenylalanine containing small glyco-dehydropeptides, Boc-Phe-ΔPhe-εAhx-GA (I) and H-Phe-ΔPhe-εAhx-GA (II) with glucosamine (GA) attached at the C-terminal through a 6-aminocaproic acid linker, were synthesized, demonstrating the formation of nanostructures in aqueous media, which were characterized by DLS, AFM and TEM. Further, nanostructure II reduced auric chloride to gold nanoparticles and formed a peptide-gold conjugate (VII). The feasibility of using the nanostructures I and II as nanovectors for drug delivery was demonstrated by loading hydrophobic molecules, eosin and N-fluoresceinyl-2-aminoethanol (FAE) dyes. Besides, these peptides displayed antimicrobial activity against Micrococcus flavus, Bacillus subtilis and Pseudomonas aeruginosa. All these results advocate the potential of these nanostructures as efficient vectors for drug delivery applications.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/química , Glicopéptidos/química , Nanoestructuras/química , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Glicopéptidos/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía de Fuerza Atómica , Solubilidad