RESUMEN
Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs' technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro-in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics.
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Vesículas Extracelulares , Pez Cebra , Animales , Humanos , Antibacterianos/farmacología , Distribución Tisular , Vesículas Extracelulares/metabolismo , Línea Celular , MamíferosRESUMEN
Gene knockdown by siRNA offers an unrestricted choice of targets and specificity based on the principle of complementary Watson-Crick base pairing with mRNA. However, the negative charge, large molecular size, and susceptibility to enzymatic degradation of siRNA impede its successful transfection, hence limiting its potential for therapeutic use. The development of efficient and safe siRNA transfection agents is, therefore, critical for siRNA-based therapy. Herein, we developed a protein-based biodynamic polymer (biodynamer) that showed potential as a siRNA transfection vector, owing to its excellent biocompatibility, easy tunability, and dynamic polymerization under acidic environments. The positively charged biodynamers formed stable dynamic nanocomplexes (XL-DPs, hydrodynamic diameter of approximately 104 nm) with siRNA via electrostatic interactions and chemical cross-linking. As a proof of concept, the optimized XL-DPs were stable in physiological conditions with serum proteins and demonstrated significant pH-dependent size change and degradability, as well as siRNA release capability. The minimal cytotoxicity and excellent cellular uptake of XL-DPs effectively supported the intracellular delivery of siRNA. Our study demonstrated that the XL-DPs in survivin siRNA delivery enabled potent knockdown of survivin mRNA and induced notable apoptosis of carcinoma cells (2.2 times higher than a lipid-based transfection agent, Lipofectamine 2000). These findings suggested that our XL-DPs hold immense potential as a promising platform for siRNA delivery and can be considered strong candidates in the advancement of next-generation transfection agents.
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Apoptosis , Survivin/genética , ARN Interferente Pequeño , Transfección , Concentración de Iones de Hidrógeno , ARN Mensajero , Línea Celular TumoralRESUMEN
The analysis of nanoparticle (NP) dynamics in live cell studies by video tracking provides detailed information on their interactions and trafficking in the cells. Although the video analysis is not yet routinely used in NP studies, the equipment suitable for the experiments is already available in most laboratories. Here, we compare trajectory patterns, diffusion coefficients, and particle velocities of NPs in A549 cells with a rather simple experimental setup consisting of a fluorescence microscope and openly available trajectory analysis software. The studied NPs include commercial fluorescent polymeric particles and two subpopulations of PC-3 cell-derived extracellular vesicles (EVs). As bioderived natural nanoparticles, the fluorescence intensities of the EVs limited the recording speed. Therefore, we studied the effect of the recording frame rate and analysis parameters to the trajectory results with bright fluorescent commercial NPs. We show that the trajectory classification and the apparent particle velocities are affected by the recording frame rate, while the diffusion constants stay comparable. The NP trajectory patterns were similar for all NP types and resembled intracellular vesicular transport. Interestingly, the EV movements were faster than the commercial NPs, which contrasts with their physical sizes and may indicate a greater role of the motor proteins in their intracellular transports.
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Vesículas Extracelulares , Nanopartículas , Humanos , Células A549 , Microscopía Fluorescente , Vesículas Extracelulares/metabolismo , Colorantes Fluorescentes/metabolismoRESUMEN
BACKGROUND: Pulmonary infections associated with Pseudomonas aeruginosa can be life-threatening for patients suffering from chronic lung diseases such as cystic fibrosis. In this scenario, the formation of biofilms embedded in a mucus layer can limit the permeation and the activity of anti-infectives. OBJECTIVES: Native human pulmonary mucus can be isolated from endotracheal tubes, but this source is limited for large-scale testing. This study, therefore, aimed to evaluate a modified artificial sputum medium (ASMmod) with mucus-like viscoelastic properties as a surrogate for testing anti-infectives against P. aeruginosa biofilms. METHODS: Bacterial growth in conventional broth cultures was compared with that in ASMmod, and PAO1-GFP biofilms were imaged by confocal microscopy. Transport kinetics of three antibiotics, tobramycin, colistin, and ciprofloxacin, through native mucus and ASMmod were studied, and their activity against PAO1 biofilms grown in different media was assessed by determination of metabolic activity and cfu. RESULTS: PAO1(-GFP) cultured in human pulmonary mucus or ASMmod showed similarities in bacterial growth and biofilm morphology. A limited permeation of antibiotics through ASMmod was observed, indicating its strong barrier properties, which are comparable to those of native human mucus. Reduced susceptibility of PAO1 biofilms was observed in ASMmod compared with LB medium for tobramycin and colistin, but less for ciprofloxacin. CONCLUSIONS: These findings underline the importance of mucus as a biological barrier to antibiotics. ASMmod appears to be a valuable surrogate for studying mucus permeation of anti-infectives and their efficacy against PAO1 biofilms.
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Infecciones por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacología , Biopelículas , Humanos , Moco , Tobramicina/farmacologíaRESUMEN
PURPOSE: The aim of this work was to formulate and characterize surfactant-free glibenclamide nanoparticles using Eudragit RLPO and polyethylene glycol as sole stabilizer. METHODS: Glibenclamide nanoparticles were obtained by nanoprecipitation and evaluated in terms of drug content, encapsulation efficiency, apparent saturation solubility, drug release profile, solid state and storage stability. The influence of different stirring speed on the particle size, size distribution and zeta potential of the nanoparticles was investigated. The nanoparticle biocompatibility and permeability were analyzed in vitro on Caco-2 cell line (clone HTB-37) and its interaction with mucin was also investigated. RESULTS: It was found that increasing the molecular weight of polyethylene glycol from 400 to 6000 decreased drug encapsulation, whereas the aqueous solubility and dissolution rate of the drug increased. Particle size of the nanoformulations, with and without polyethylene glycol, were between 140 and 460 nm. Stability studies confirmed that glibenclamide nanoparticles were stable, in terms of particle size, after 120 days at 4°C. In vitro studies indicated minimal interactions of glibenclamide nanoparticles and mucin glycoproteins suggesting favorable properties to address the intestinal mucus barrier. Cell viability studies confirmed the safety profile of these nanoparticles and showed an increased permeation through epithelial cells. CONCLUSION: Taking into consideration these findings, polyethylene glycol is a useful polymer for stabilizing these surfactant-free glibenclamide nanoparticles and represent a promising alternative to improve the treatment of non-insulin dependent diabetes.
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Composición de Medicamentos/métodos , Gliburida/metabolismo , Hipoglucemiantes/metabolismo , Mucosa Intestinal/metabolismo , Nanopartículas/metabolismo , Tensoactivos , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Evaluación Preclínica de Medicamentos/métodos , Gliburida/administración & dosificación , Gliburida/química , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/química , Mucosa Intestinal/efectos de los fármacos , Nanopartículas/administración & dosificación , Nanopartículas/química , Tamaño de la Partícula , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Polietilenglicoles/metabolismo , Polímeros/administración & dosificación , Polímeros/química , Polímeros/metabolismoRESUMEN
Elimination of pulmonary Pseudomonas aeruginosa (PA) infections is challenging to accomplish with antibiotic therapies, mainly due to resistance mechanisms. Quorum sensing inhibitors (QSIs) interfering with biofilm formation can thus complement antibiotics. For simultaneous and improved delivery of both active agents to the infection sites, self-assembling nanoparticles of a newly synthesized squalenyl hydrogen sulfate (SqNPs) were prepared. These nanocarriers allowed for remarkably high loading capacities of hydrophilic antibiotic tobramycin (Tob) and a novel lipophilic QSI at 30 % and circa 10 %, respectively. The drug-loaded SqNPs showed improved biofilm penetration and enhanced efficacy in relevant biological barriers (mucin/human tracheal mucus, biofilm), leading to complete eradication of PA biofilms at circa 16-fold lower Tob concentration than Tob alone. This study offers a viable therapy optimization and invigorates the research and development of QSIs for clinical use.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Nanopartículas/química , Pseudomonas aeruginosa/efectos de los fármacos , Percepción de Quorum/efectos de los fármacos , Tobramicina/farmacología , Animales , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/toxicidad , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Moco/microbiología , Nanopartículas/toxicidad , Pseudomonas aeruginosa/fisiología , Quinolonas/farmacología , Escualeno/análogos & derivados , Escualeno/toxicidad , Ésteres del Ácido Sulfúrico/química , Ésteres del Ácido Sulfúrico/toxicidad , Pez CebraRESUMEN
Mucus is a complex hydrogel that acts as a protective barrier in various parts of the human body. Both composition and structural properties play a crucial role in maintaining barrier properties while dictating diffusion of molecules and (nano)materials. In this study, we compare previously described mucus surrogates with the native human airway and pig intestinal mucus. Oscillatory shear rheology was applied to characterize mucus on the bulk macrorheological level, revealing that the artificial airway surrogate deviates from the elastic-dominant behavior of native mucus samples. We circumvented this limitation through the addition of a cross-linking polymer to the surrogate, adjusting the rheological properties closer to those of native mucus. Applying particle tracking microrheology, we further demonstrated that the mechanical properties at the microscale differ significantly between artificial and native mucus. We conclude that proper characterization of mucus and its surrogates is vital for a reliable investigation of nanoparticle-based mucosal drug delivery.
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Sistemas de Liberación de Medicamentos , Mucosa Intestinal/química , Moco/química , Nanopartículas/química , Animales , Difusión , Humanos , Hidrogeles/química , Intestinos/química , Reología , Porcinos , ViscosidadRESUMEN
Biodegradable polymeric nanoparticles (NP) made from poly (lactid-co-glycolide) acid (PLGA) and chitosan (CS) hold promise as innovative formulations for targeted delivery. Since interactions of such NP with primary human immune cells have not been characterized, yet, here we assessed the effect of PLGA or CS-PLGA NP treatment on human peripheral blood mononuclear cells (PBMC), as well as on monocyte-derived DC (moDC). Amongst PBMC, antigen presenting cells (APC) showed higher uptake of both NP preparations than lymphocytes. Furthermore, moDC internalized CS-PLGA NP more efficiently than PLGA NP, presumably because of receptor-mediated endocytosis. Consequently, CS-PLGA NP were delivered mostly to endosomal compartments, whereas PLGA NP primarily ended up in lysosomes. Thus, CS-PLGA NP confer enhanced delivery to endosomal compartments of APC, offering new therapeutic options to either induce or modulate APC function and to inhibit pathogens that preferentially infect APC.
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Células Presentadoras de Antígenos/metabolismo , Quitosano , Endosomas/metabolismo , Leucocitos Mononucleares/metabolismo , Nanopartículas/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Células Presentadoras de Antígenos/citología , Quitosano/química , Quitosano/farmacocinética , Quitosano/farmacología , Humanos , Leucocitos Mononucleares/citología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacologíaRESUMEN
Aberrant immune activation mediated by T effector cell populations is pivotal in the onset of autoimmunity in type 1 diabetes (T1D). T follicular helper (TFH) cells are essential in the induction of high-affinity antibodies, and their precursor memory compartment circulates in the blood. The role of TFH precursors in the onset of islet autoimmunity and signaling pathways regulating their differentiation is incompletely understood. Here, we provide direct evidence that during onset of islet autoimmunity, the insulin-specific target T-cell population is enriched with a C-X-C chemokine receptor type 5 (CXCR5)+CD4+ TFH precursor phenotype. During onset of islet autoimmunity, the frequency of TFH precursors was controlled by high expression of microRNA92a (miRNA92a). miRNA92a-mediated TFH precursor induction was regulated by phosphatase and tension homolog (PTEN) - phosphoinositol-3-kinase (PI3K) signaling involving PTEN and forkhead box protein O1 (Foxo1), supporting autoantibody generation and triggering the onset of islet autoimmunity. Moreover, we identify Krueppel-like factor 2 (KLF2) as a target of miRNA92a in regulating human TFH precursor induction. Importantly, a miRNA92a antagomir completely blocked induction of human TFH precursors in vitro. More importantly, in vivo application of a miRNA92a antagomir to nonobese diabetic (NOD) mice with ongoing islet autoimmunity resulted in a significant reduction of TFH precursors in peripheral blood and pancreatic lymph nodes. Moreover, miRNA92a antagomir application reduced immune infiltration and activation in pancreata of NOD mice as well as humanized NOD Scid IL2 receptor gamma chain knockout (NSG) human leucocyte antigen (HLA)-DQ8 transgenic animals. We therefore propose that miRNA92a and the PTEN-PI3K-KLF2 signaling network could function as targets for innovative precision medicines to reduce T1D islet autoimmunity.
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Autoinmunidad , Diabetes Mellitus Tipo 1/inmunología , Factores de Transcripción de Tipo Kruppel/inmunología , MicroARNs/inmunología , Fosfohidrolasa PTEN/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adolescente , Animales , Antagomirs/genética , Antagomirs/inmunología , Autoanticuerpos/biosíntesis , Niño , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Femenino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/inmunología , Regulación de la Expresión Génica , Humanos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/patología , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Cultivo Primario de Células , Receptores CXCR5/genética , Receptores CXCR5/inmunología , Transducción de Señal , Linfocitos T Colaboradores-Inductores/patologíaRESUMEN
The majority of the currently used and developed anti-infectives are poorly water-soluble molecules. The poor solubility might lead to limited bioavailability and pharmacological action of the drug. Novel pharmaceutical materials have thus been designed to solve those problems and improve drug delivery. In this study, we propose a facile method to produce submicrocarriers (sMCs) by electrostatic gelation of anionic ß-cyclodextrin (aß-CD) and chitosan. The average hydrodynamic size ranged from 400 to 900 nm by carefully adjusting polymer concentrations and N/C ratio. The distinct host-guest reaction of cyclodextrin derivative is considered as a good approach to enhance solubility, and prevent drug recrystallization, and thus was used to develop sMC to improve the controlled release profile of a poorly soluble and clinically relevant anti-infective ciprofloxacin. The optimal molar ratio of ciprofloxacin to aß-CD was found to be 1:1, which helped maximize encapsulation efficiency (â¼90%) and loading capacity (â¼9%) of ciprofloxacin loaded sMCs. Furthermore, to recommend the future application of the developed sMCs, the dependence of cell uptake on sMCs size (500, 700, and 900 nm) was investigated in vitro on dTHP-1 by both flow cytometry and confocal microscopy. The results demonstrate that, regardless of their size, an only comparatively small fraction of the sMCs were taken up by the macrophage-like cells, while most of the carriers were merely adsorbed to the cell surface after 2 h incubation. After continuing the incubation to reach 24 h, the majority of the sMCs were found intracellularly. However, the sMCs had been designed to release sufficient amount of drug within 24 h, and the subsequent phagocytosis of the carrier may be considered as an efficient pathway for its safe degradation and elimination. In summary, the developed sMC is a suitable system with promising perspectives recommended for pulmonary extracellular infection therapeutics.
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Antibacterianos/farmacocinética , Ciprofloxacina/farmacocinética , Portadores de Fármacos/química , Composición de Medicamentos/métodos , Pulmón/metabolismo , Antibacterianos/administración & dosificación , Disponibilidad Biológica , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ciprofloxacina/administración & dosificación , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/farmacocinética , Liberación de Fármacos , Humanos , Pulmón/efectos de los fármacos , Nanopartículas/química , Tamaño de la Partícula , Neumonía/tratamiento farmacológico , Polisacáridos/química , Distribución Tisular , Agua/químicaRESUMEN
Amphiphilic polymer-based drug delivery systems hold potential in enhancing pharmacokinetics and therapeutic efficacy due to their ability to simultaneously codeliver different drugs in a controlled manner. We propose here a facile method for synthesizing a new amphiphilic polymer, farnesylated glycol chitosan (FGC), which self-assembles into nanoparticles upon being dispersed in aqueous media. The characteristics of FGC nanoparticles, in particular the size, could be tuned in a range from 200 to 500 nm by modulating the degree of farnesylation and the pH and polymer concentration during particle preparation. Carrier capacity, release kinetics, and surface modification of the established system were investigated using different model compounds. The colloids were biocompatible and stable at biologically relevant pH values. The interactions between the carriers and human mucus were examined by multiple particle tracking, which revealed that â¼80% of the particles remain immobilized within the mucus matrix. These results postulate FGC as a versatile drug delivery platform.
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Quitosano/análogos & derivados , Nanopartículas/química , Mucosa Respiratoria/efectos de los fármacos , Línea Celular Tumoral , Glicoles/química , Humanos , Nanopartículas/efectos adversos , Prenilación , Mucosa Respiratoria/metabolismoRESUMEN
BACKGROUND: Messenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. A variety of biodegradable nanoparticles (NPs) has been developed including lipid-based and polymer-based systems for mRNA delivery. However, both systems still lack in achieving an efficient transfection rate and a detailed understanding of the mRNA transgene expression kinetics. Therefore, quantitative analysis of the time-dependent translation behavior would provide a better understanding of mRNA's transient nature and further aid the enhancement of appropriate carriers with the perspective to generate future precision nanomedicines with quick response to treat various diseases. RESULTS: A lipid-polymer hybrid system complexed with mRNA was evaluated regarding its efficiency to transfect dendritic cells (DCs) by simultaneous live cell video imaging of both particle uptake and reporter gene expression. We prepared and optimized NPs consisting of poly (lactid-co-glycolid) (PLGA) coated with the cationic lipid 1, 2-di-O-octadecenyl-3-trimethylammonium propane abbreviated as LPNs. An earlier developed polymer-based delivery system (chitosan-PLGA NPs) served for comparison. Both NPs types were complexed with mRNA-mCherry at various ratios. While cellular uptake and toxicity of either NPs was comparable, LPNs showed a significantly higher transfection efficiency of ~ 80% while chitosan-PLGA NPs revealed only ~ 5%. Further kinetic analysis elicited a start of protein translation after 1 h, with a maximum after 4 h and drop of transgene expression after 48 h post-transfection, in agreement with the transient nature of mRNA. CONCLUSIONS: Charge-mediated complexation of mRNA to NPs enables efficient and fast cellular delivery and subsequent protein translation. While cellular uptake of both NP types was comparable, mRNA transgene expression was superior to polymer-based NPs when delivered by lipid-polymer NPs.
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Quitosano/análogos & derivados , Células Dendríticas/metabolismo , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Biosíntesis de Proteínas , ARN Mensajero/administración & dosificación , Transfección/métodos , Células A549 , Humanos , Cinética , Nanopartículas/ultraestructura , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , ARN Mensajero/genética , Ácidos Esteáricos/químicaRESUMEN
Nucleotide analogues are a therapeutic class that is very promising and currently used in clinics, notably against viral infectious diseases and cancer. However, their therapeutic potential is often restricted by a poor stability in vivo, the induction of severe side effects, and limited passive intracellular diffusion due to their hydrophilicity. Polysaccharide-based polymers (e.g., starch) have considerable advantages, including a lack of toxicity and the absence of antigenicity. The aim of this study was to develop new cationic starches able to form complexes with nucleotide analogues, thus protecting them and increasing their cell uptake. At the same time, the material should demonstrate good biocompatibility and low cytotoxicity. Different polyamines, (TREN, TEPA, and spermine) were grafted to starch to evaluate the impact of side-chain properties. The resulting cationic starch derivatives were characterized (e.g., degree of modification) and compared in their ability to form polyplexes with ATP as a model nucleotide. Among the tested candidates, the formulation of starch-TEPA and ATP with an N/P ratio of 2 led to nanoparticles with a size of 429 nm, a PdI of 0.054, and a ζ potential of -9 mV. MTT and LDH assays on A549 cell line showed low toxicity for this polymer. Confocal microscopy study proved that the cell internalization was an incubation-time- and energy-dependent process. Most important, starch-TEPA complexed with ddGTP showed significant biological activity on A549 cancer cells compared to that of plain ddGTP at the same concentration.
RESUMEN
Acute respiratory distress syndrome is linked to inflammatory processes in the human lung. The aim of this study was to mimic in vitro the treatment of lung inflammation by using a cell-based human autologous co-culture model. As a potential trial medication, we developed a pulmonary dry powder formulation loaded with interleukin-10 (IL-10), a potent anti-inflammatory cytokine. The inflammatory immune response was stimulated by lipopolysaccharide. The co-culture was combined with the Pharmaceutical Aerosol Deposition Device on Cell Cultures )PADDOCC), to deposit the IL-10-loaded microparticles on the inflamed co-culture model at the air-liquid interface. This treatment significantly reduced the secretion of interleukin-6 and tumour necrosis factor, as compared to the deposition of placebo (unloaded) particles. Our results show that the alveolar co-culture model, in combination with a deposition device such as the PADDOCC, may serve as a powerful tool for testing the safety and efficacy of dry powder formulations for pulmonary drug delivery.
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Aerosoles/farmacología , Células Epiteliales/fisiología , Interleucina-10/farmacología , Macrófagos Alveolares/fisiología , Pruebas de Toxicidad/métodos , Alternativas a las Pruebas en Animales , Técnicas de Cocultivo , Citocinas/genética , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/tratamiento farmacológico , Enfermedades Pulmonares/tratamiento farmacológico , Macrófagos Alveolares/efectos de los fármacos , Microscopía Electrónica de Rastreo , NanopartículasRESUMEN
The present study describes the synthesis of chitosan-squalene (chitosan-SQ), a unique amphiphilic chitosan derivative, which enables the efficient formation of nanoparticles in acetate buffer by self-assembly. The influence of different parameters on the nanoparticle size such as percentage of substitution, pH of the acetate buffer, concentration in chitosan-SQ, and time of stirring was studied. It could be demonstrated that this new polymer was nontoxic to cells, biodegradable, and preserved the anti-infective properties of the initial chitosan.1 Moreover, chitosan-SQ showed good carrier properties by allowing the encapsulation of both hydrophilic and hydrophobic model drug compounds.
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Antiinfecciosos/química , Quitosano/química , Portadores de Fármacos/química , Nanopartículas/química , Escualeno/químicaRESUMEN
Starch and starch derivatives are widely utilized pharmaceutical excipients. The concept of this study was to make use of starch as a biodegradable backbone and to modify it with low-toxic, but poor transfecting low molecular weight polyethylenimine (PEI) in order to achieve better transfection efficacy while maintaining enzymatic degradability. A sufficiently controllable conjugation could be achieved via a water-soluble intermediate of oxidized starch and an optimized reaction protocol. Systematic variation of MW fraction of the starch backbone and the amount of cationic side chains (0.8 kDa bPEI) yielded a series of starch-graft-PEI copolymers. Following purification and chemical characterization, nanoscale complexes with plasmid DNA were generated and studied regarding cytotoxicity and transfection efficacy. The optimal starch-graft-PEI polymers consisted of >100 kDa MW starch and contained 30% (wt) of PEI, showing similar transfection levels as 25 kDa bPEI, and being less cytotoxic and enzymatically biodegradable.
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Materiales Biocompatibles/síntesis química , Diseño de Fármacos , Polietileneimina/química , Almidón/química , Transfección/métodos , Materiales Biocompatibles/química , Materiales Biocompatibles/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN/química , Humanos , Estructura Molecular , Nanoestructuras/química , Nanoestructuras/toxicidad , Tamaño de la Partícula , Plásmidos/química , Polietileneimina/toxicidad , Solubilidad , Almidón/toxicidad , Propiedades de SuperficieRESUMEN
An inhalation-based Biopharmaceutics Classification System for pulmonary drugs (iBCS) holds the perspective to allow for scientifically sound prediction of differences in the in vivo performance of orally inhaled drug products (OIDPs). A set of nine drug substances were selected, that are administered via both the oral and pulmonary routes. Their solubility was determined in media representative for the oral (Fasted State Simulated Intestinal Fluid (FaSSIF)) and pulmonary (Alveofact medium and Simulated Lung Fluid (SLF)) routes of administration to confirm the need for a novel approach for inhaled drugs. The complexity of these media was then stepwise reduced with the purpose of understanding the contribution of their components to the solubilizing capacity of the media. A second reason for varying the complexity was to identify a medium that would allow robust but accurate dissolution testing. Hence, Hank's balanced salt solution (HBSS) as a medium used in many in vitro biological tests, non-buffered saline solution, and water were included. For some drug substances (salbutamol sulfate, tobramycin, isoniazid, and tiotropium bromide), no significant differences were observed between the solubility in the media used. For other drugs, however, we observed either just small (rifampicin, budesonide, salmeterol) or unexpectedly large differences (beclomethasone dipropionate). Based on the minimum theoretical solubility required for their common pulmonary dose in 10 ml of lung lining fluid, drug solubility was classified as either high or low. Two high solubility and two low solubility compounds were then selected for refined solubility testing in pulmonary relevant media by varying their content of phospholipids, surfactant proteins and other proteins. The solubility of drug substances in simulated lung lining fluids was found to be dependent on the physicochemical properties of the drug substance and the composition of the media. While a pulmonary dissolution medium that would fit all drugs could not be established, our approach may provide guidance for finding the most suitable dissolution medium for a given drug substance and better designing in vitro tests for predicting the in vivo performance of inhalable drug products.
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Biofarmacia , Intestinos , Preparaciones Farmacéuticas/química , Solubilidad , Administración por InhalaciónRESUMEN
Co-delivery of different protein-encoding polynucleotide species with varying expression kinetics of their therapeutic product will become a prominent requirement in the realm of combined nucleic acid(NA)-based therapies in the upcoming years. The current study explores the capacity for time-staggered expression of encoded proteins by simultaneous delivery of plasmid DNA (pDNA) in the core and mRNA on the shell of the same nanocarrier. The core is based on a Gelatin Type A-pDNA coacervate, thermally stabilized to form an irreversible nanogel stable enough for the deposition of cationic coats namely, protamine sulfate or LNP-related lipid mixtures. Only the protamine-coated nanocarriers remained colloidally stable following mRNA loading and could successfully co-transfect murine dendritic cell line DC2.4 with fluorescent reporter mRNA(mCherry) and pDNA (pAmCyan1). Further investigation of the protamine-coated nanosystem only, the transfection efficiency (percentage of transfected cells) and level of protein expression (mean fluorescence intensity, MFI) of mRNA and pDNA, simultaneously delivered by the same nanocarrier, were compared and kinetically assessed over 48 h in DC2.4 using flow cytometry. The onset of transfection for both nucleotides was initially delayed, with levels < 5% at 6 h. Thereafter, mRNA transfection reached 90% after 24 h and continued to slightly increase until 48 h. In contrast, pDNA transfection was clearly slower, reaching approximately 40% after 24 h, but continuing to increase to reach 94% at 48 h. The time course of protein expression (represented by MFI) for both NAs essentially followed that of transfection. Model-independent as well as model-dependent kinetic parameters applied to the data further confirmed such time-staggered expression of the two NA's where mRNA's rate of transfection and protein expression initially exceeded those of pDNA in the first 24 h of the experiment whereas the opposite was true during the second 24 h of the experiment where pDNA displayed the higher response rates. We expect that innovative nanocarriers capable of time-staggered co-delivery of different nucleotides could open new perspectives for multi-dosing, pulsatile or sustained expression of nucleic acid-based therapeutics in protein replacement, vaccination, and CRISPR-mediated gene editing scenarios.
RESUMEN
Treatment of microbial infections is becoming daunting because of widespread antimicrobial resistance. The treatment challenge is further exacerbated by the fact that certain infectious bacteria invade and localize within host cells, protecting the bacteria from antimicrobial treatments and the host's immune response. To survive in the intracellular niche, such bacteria deploy surface receptors similar to host cell receptors to sequester iron, an essential nutrient for their virulence, from host iron-binding proteins, in particular lactoferrin and transferrin. In this context, we aimed to target lactoferrin receptors expressed by macrophages and bacteria; as such, we prepared and characterized lactoferrin nanoparticles (Lf-NPs) loaded with a dual drug combination of antimicrobial natural alkaloids, berberine or sanguinarine, with vancomycin or imipenem. We observed increased uptake of drug-loaded Lf-NPs by differentiated THP-1 cells with up to 90% proportion of fluorescent cells, which decreased to about 60% in the presence of free lactoferrin, demonstrating the targeting ability of Lf-NPs. The encapsulated antibiotic drug cocktail efficiently cleared intracellular Staphylococcus aureus (Newman strain) compared to the free drug combinations. However, the encapsulated drugs and the free drugs alike exhibited a bacteriostatic effect against the hard-to-treat Mycobacterium abscessus (smooth variant). In conclusion, the results of this study demonstrate the potential of lactoferrin nanoparticles for the targeted delivery of antibiotic drug cocktails for the treatment of intracellular bacteria.
Asunto(s)
Antibacterianos , Lactoferrina , Nanopartículas , Staphylococcus aureus , Lactoferrina/química , Lactoferrina/farmacología , Humanos , Nanopartículas/química , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/química , Células THP-1 , Macrófagos/efectos de los fármacos , Vancomicina/farmacología , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Pruebas de Sensibilidad MicrobianaRESUMEN
Antimicrobial resistance is becoming more prominent day after day due to a number of mechanisms by microbes, especially the sophisticated biological barriers of bacteria, especially in Gram-negatives. There, the lipopolysaccharides (LPS) layer is a unique component of the outer leaflet of the outer membrane which is highly impermeable and prevents antibiotics from passing passively into the intracellular compartments. Biodynamers, a novel class of dynamically bio-responsive polymers, may open new perspectives to overcome this particular barrier by accommodating various secondary structures and form supramolecular structures in such bacterial microenvironments. Generally, bio-responsive polymers are not only candidates as bio-active molecules against bacteria but also carriers via their interactions with the cargo. Based on their dynamicity, design flexibility, biodegradability, biocompatibility, and pH-responsiveness, we investigated the potential of two peptide-based biodynamers for improving antimicrobial drug delivery. By a range of experimental methods, we discovered a greater affinity of Arg-biodynamers for bacterial membranes than for mammalian membranes as well as an enhanced LPS targeting on the bacterial membrane, opening perspectives for enhancing the delivery of antimicrobials across the Gram-negative bacterial cell envelope. This could be explained by the change of the secondary structure of Arg-biodynamers into a predominant ß-sheet character in the LPS microenvironment, by contrast to the α-helical structure typically observed for most lipid membrane-permeabilizing peptides. In comparison to poly-L-arginine, the intrinsic antibacterial activity of Arg-biodynamers was nearly unchanged, but its toxicity against mammalian cells was >128-fold reduced. When used in bacterio as an antibiotic potentiator, however, Arg-biodynamers improved the minimum inhibitory concentration (MIC) against Escherichia coli by 32 times compared to colistin alone. Similar effect has also been observed in two stains of Pseudomonas aeruginosa. Arg-biodynamers may therefore represent an interesting option as an adjuvant for antibiotics against Gram-negative bacteria and to overcome antimicrobial resistance.