RESUMEN
Microcontact printing (µCP) of polyelectrolytes is a facile and powerful method for surface micro/nanopatterning and functionalization. Poly(4-aminostyrene) (PAS) is a polyelectrolyte that can be converted to aryldiazonium salt and exhibits pH-dependent hydrophobicity. Here we demonstrate µCP of PAS and the expansion of this technique in various directions. First, the microcontact-printed PAS can be diazotized to micropattern biomolecules including DNA and protein and nanomaterials including single-walled carbon nanotubes and gold nanoparticles. Second, the diazotized PAS enables µCP of a metallic structure on a carbon surface. Third, the hydrophobic nature of PAS at the neutral pH allows the microcontact-printed PAS-based polyelectrolyte multilayer to be used as masks for wet etching. Lastly, this technique allows facile fabrication of highly engineered microparticles with a unique structure. Overall, this work has established a novel µCP platform with various potential applications.
Asunto(s)
Poliestirenos/química , Impresión , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Biomaterial properties have recently been shown to modulate extracellular vesicle (EV) secretion and cargo; however, the effects of substrate composition on EV production remain underexplored. This study investigates the impacts of surface coatings composed of collagen I (COLI), fibronectin (FN), and poly l-lysine (PLL) on EV secretion for applications in therapeutic EV production and to further understanding of how changes in the extracellular matrix microenvironment affect EVs. EV secretion from primary bone marrow-derived mesenchymal stromal cells (BMSCs), primary adipose-derived stem cells (ASCs), HEK293 cells, NIH3T3 cells, and RAW264.7 cells was characterized on the different coatings. Expression of EV biogenesis genes and cellular adhesion genes was also analyzed. COLI coatings significantly decreased EV secretion in RAW264.7 cells, with associated decreases in cell viability and changes in EV biogenesis-related and cell adhesion genes at day 4. FN coatings increased EV secretion in NIH3T3 cells, while PLL coatings increased EV secretion in ASCs. Surface coatings had significant effects on the capacity of EVs derived from RAW264.7 and NIH3T3 cells to impact in vitro macrophage proliferation. Overall, surface coatings had different cell-specific effects on EV secretion and in vitro functional capacity, thus highlighting the potential of substrate coatings to further the development of clinical EV production systems.
Asunto(s)
Vesículas Extracelulares , Fibronectinas , Células Madre Mesenquimatosas , Ratones , Animales , Humanos , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Células 3T3 NIH , Células RAW 264.7 , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Fibronectinas/química , Fibronectinas/metabolismo , Propiedades de Superficie , Polilisina/química , Polilisina/farmacología , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Células HEK293 , Proliferación Celular/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/química , Colágeno Tipo I/genéticaRESUMEN
Phagocytosis performed by a macrophage involves complex membrane trafficking and reorganization among various membranous cellular structures including phagosomes and vesicles derived from the phagosomes known as phagosome-derived vesicles. The present work reports on development of a technique that allows to specifically label the phagosome-derived vesicles in macrophages with a membrane dye. The technique is based on the use of microfabricated microparticles that are made of a thermosensitive nonbiodegradable polymer poly(N-isopropylacrylamide) (PNIPAM) or its derivative and contain a membrane dye 1,1'-dialkyl-3,3,3',3'-tetramethylindodicarbocyanine (DiI). The microparticles can be phagocytosed by RAW264.7 macrophages into their phagosomes, resulting in formation of intracellular DiI-positive vesicles derived from the phagosomes. The DiI-positive vesicles are motile and acidic; can be stained by fluorescently labelled dextran added in the culture medium; and can accumulate around new phagosomes, indicating that they possess properties of lysosomes. This technique is also applicable to another membrane dye 3,3'-dioctadecyloxacarbocyanine (DiO) and holds great potential to be useful for advancing our understanding of phagocytosis. STATEMENT OF SIGNIFICANCE: Phagocytosis performed by macrophages is a cellular process of great importance to various applications of biomaterials such as drug delivery and medical implantation. This work reports on a technique for characterizing phagocytosis based on the use of poly(N-isopropylacrylamide), which is a major biomaterial with numerous applications. This technique is the first of its kind and has generated an original finding about phagocytosis. In addition to drug delivery and medical implantation, phagocytosis plays critical roles in diseases, injuries and vaccination. This work could thus attract immediate and widespread interests in the field of biomaterials science and engineering.
Asunto(s)
Fagocitosis , Fagosomas , Materiales Biocompatibles , Lisosomas , MacrófagosRESUMEN
Existing clinical cell therapies, which rely on the use of biological functionalities of living cells, can be further enhanced by conjugating functional particles to the cells to form cell-particle complexes. Disk-shaped microparticles produced by the top-down microfabrication approach possess unique advantages for this application. However, none of the current mechanisms for conjugating the microfabricated microparticles to the cells are principally applicable to all types of cells with therapeutic potentials. On the other hand, membrane intercalation is a well-established mechanism for attaching fluorescent molecules to living cells or for immobilizing cells on a solid surface. This paper reports a study on conjugating disk-shaped microparticles, referred to as micropatches, to living cells through membrane intercalation for the first time. The procedure for producing the cell-micropatch complexes features an unprecedented integration of microcontact printing of micropatches, end-grafting of linear molecules of octadecyl chain and poly(ethylene glycol) to the printed micropatches, and use of gelatin as a temperature-sensitive sacrificial layer to allow the formation and subsequent release of the cell-micropatch complexes. Complexes composed of mouse neuroblastoma cells were found to be stable in vitro, and the micropatch-bound cells were viable, proliferative, and differentiable. Moreover, complexes composed of four other types of cells were produced. The membrane-intercalation mechanism and the corresponding fabrication technique developed in this study are potentially applicable to a wide range of therapeutic cells and thus promise to be useful for developing new cell therapies enhanced by the disk-shaped microparticles.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos , Micropartículas Derivadas de Células , Humanos , Microtecnología , Tamaño de la Partícula , Polietilenglicoles/química , TemperaturaRESUMEN
BACKGROUND: Ethanol injection is widely used in liposome preparation. However, the parameters determining particle size distribution of the liposomal preparation has not been fully defined. MATERIALS AND METHODS: A syringe pump-driven microfluidic injection device was used to produce liposomes under different conditions. RESULTS: Particle size of the liposomes was decreased with decrease in needle diameter (or increase in hydrodynamic pressure), decrease in lipid concentration in the alcohol solution, decrease in phase transition temperature (T(m)) of the lipid bilayer and the absence of cholesterol (or decrease in, membrane rigidity). CONCLUSION: The device used is simple to adopt and can be used for affordable production of liposomes with tunable particle size.
Asunto(s)
Etanol/farmacología , Liposomas , Tecnología Farmacéutica/métodos , Tamaño de la Partícula , PresiónRESUMEN
Soft lithographic techniques were used to fabricate polymeric microparticles for drug delivery applications. The microparticles were made of thermoplastics and thermosets from different types of precursors including reactive resin and polymer solutions in organic solvents or water. The microparticles produced using these methods were made of widely used polymers for drug delivery with highly uniform sizes, plate-like morphology, and well-defined lateral sizes and shapes, making them potentially useful for drug delivery applications and as platform for the construction of multi-functional drug delivery devices.
Asunto(s)
Materiales Biocompatibles/química , Preparaciones de Acción Retardada/química , Portadores de Fármacos/química , Polímeros/química , Microesferas , Tamaño de la Partícula , Fotograbar/métodos , Propiedades de SuperficieRESUMEN
A self-folding miniature device has been developed to provide enhanced mucoadhesion, drug protection, and targeted unidirectional delivery. The main part of the device is a finger like bilayered structure composed of two bonded layers. One is a pH-sensitive hydrogel based on crosslinked poly(methyacrylic acid) (PMAA) that swells significantly when in contact with body fluids, while the other is a non-swelling layer based on poly(hydroxyethyl methacrylate) (PHEMA). A mucoadhesive drug layer is attached on the bilayer. Thus, the self-folding device first attaches to the mucus and then curls into the mucus due to the different swelling of the bilayered structure, leading to enhanced mucoadhesion. The non-swelling PHEMA layer can also serve as a diffusion barrier, minimizing any drug leakage in the intestine. The resulting unidirectional release provides improved drug transport through the mucosal epithelium. The functionality of this device is successfully demonstrated in vitro using a porcine small intestine.
Asunto(s)
Sistemas de Liberación de Medicamentos , Hidrogeles , Adhesivos , Administración Oral , Animales , Disponibilidad Biológica , Diseño de Equipo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Membrana Dobles de Lípidos , Mucosa Bucal/metabolismo , Polihidroxietil Metacrilato , Ácidos Polimetacrílicos , Albúmina Sérica Bovina/administración & dosificación , Albúmina Sérica Bovina/metabolismo , PorcinosRESUMEN
Use of live cells as carriers for drug-laden particulate structures possesses unique advantages for drug delivery. In this work, we report on the development of a novel type of particulate structures called microdevices for cell-borne drug delivery. The microdevices were fabricated by soft lithography with a disklike shape. Each microdevice was composed of a layer of biodegradable thermoplastic such as poly(lactic-co-glycolic acid). One face of the thermoplastic layer was covalently grafted with a cell-adhesive polyelectrolyte such as poly-l-lysine. This asymmetric structure allowed the microdevices to bind to live cells through bulk mixing without causing cell aggregation. Moreover, the cell-microdevice complexes were largely stable, and the viability and proliferation ability of the cells were not affected by the microdevices over a week. In addition, sustained release of a mock drug from the microdevices was demonstrated. This type of microdevice promises to be clinically useful for sustained intravascular drug delivery.
Asunto(s)
Implantes Absorbibles , Trasplante de Células/instrumentación , Preparaciones de Acción Retardada/administración & dosificación , Implantes de Medicamentos/administración & dosificación , Ácido Láctico/química , Ácido Poliglicólico/química , Andamios del Tejido , Preparaciones de Acción Retardada/química , Diseño de Equipo , Análisis de Falla de Equipo , Ensayo de Materiales , Miniaturización , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
A challenge of X-ray radiation therapy is that high dose X-rays at therapeutic conditions damage normal cells. This paper describes the use of gold nanoparticle-loaded multilayer microdisks to enhance X-ray radiation therapy, where each microdisk contains over 10(5) radiosensitizing nanoparticles. The microdisks are attached on cell membranes through electrostatic interaction. Upon X-ray irradiation, more photoelectrons and Auger electrons are generated in the vicinity of the nanoparticles, which cause water ionization and lead to the formation of free radicals that damage the DNA of adjacent cancer cells. By attaching a large amount of gold nanoparticles on cancer cells, the total X-ray dose required for DNA damage and cell killing can be reduced. Due to their controllable structure and composition, multilayer microdisks can be a viable choice for enhanced radiation therapy with nanoparticles.
Asunto(s)
Oro/química , Nanopartículas del Metal/química , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Fluoresceína-5-Isotiocianato/química , Humanos , Nanopartículas del Metal/toxicidad , Microscopía Fluorescente , Polímeros/química , Radiación IonizanteRESUMEN
The functionalization and assembly of live cells with microfabricated polymeric biomaterials have attracted considerable interest in recent years, but the conventional methods suffer from high cost, high complexity, long processing time or inadequate capability. The present study reports on the development of a novel method for functionalizing and assembling live cells by integrating microcontact printing of polymeric biomaterials with a temperature-sensitive sacrificial layer prepared by spin-coating. This method has been used not only to functionalize live cells with microscopic polyelectrolyte and thermoplastic structures of various sizes and shapes, but also to assemble the cells into macroscopic stripes and sheets. The method is applicable to multiple types of cells, including human leukemic cells, mouse embryonic stem cells and human mesenchymal stem cells in the forms of single cells and cell aggregates. In addition, the microcontact-printed structures can be prepared using biodegradable and biocompatible polyelectrolytes and thermoplastic. The unique combination of low cost, ease of use and high versatility renders this method potentially useful for diverse biomedical applications, including drug delivery, cell tracking and tissue engineering.
Asunto(s)
Plásticos Biodegradables/química , Ensayo de Materiales , Animales , Adhesión Celular , Humanos , Células K562 , RatonesRESUMEN
Micro/nanoparticles containing densely packed gold nanoparticles (AuNPs) possess unique properties potentially useful for various biomedical applications. The micro/nanoparticles are conventionally produced by the bottom-up methods, which have limited capability for controlling the particle size, shape and structure. This article reports development of a top-down method that integrates layer-by-layer assembly and microcontact printing to fabricate disk-shaped microparticles named microdisks composed of densely packed AuNPs. This method allows precise control of not only the size, shape and structure of the microdisks but also the amount of the AuNPs in the microdisks. The microdisks can be loaded with different Raman reporters to generate characteristic surface-enhanced Raman scattering spectra under the near infrared excitation over a centimetre-scale lens-sample distance. Moreover, the microdisks can be attached to single live cells. This microdisk platform holds potential for multiplex Raman labelling of therapeutic cells for in vivo tracking of the cells.
Asunto(s)
Oro/química , Nanopartículas del Metal/química , Nanotecnología/métodos , Vidrio/química , Humanos , Células K562 , Microscopía de Fuerza Atómica , Microesferas , Tamaño de la Partícula , Alcohol Polivinílico/química , Silicio/química , Espectrometría Raman , Propiedades de SuperficieAsunto(s)
Cápsulas/síntesis química , Cristalización/métodos , Dimetilpolisiloxanos/química , Sistemas de Liberación de Medicamentos/métodos , Nanoestructuras/química , Nanoestructuras/ultraestructura , Polietilenglicoles/química , Ácidos Polimetacrílicos/química , Composición de Medicamentos/métodos , Sustancias Macromoleculares/química , Ensayo de Materiales , Conformación Molecular , Nanotecnología/métodos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
A multi-inlet microfluidic hydrodynamic focusing (MF) system to prepare lipopolyplex (LP) containing Bcl-2 antisense deoxyoligonucleotide (ODN) was developed and evaluated. The lipopolyplex nanoparticles consist of ODN:protamine:lipids (1:0.3:12.5wt/wt ratio) and the lipids included DC-Chol:egg PC:PEG-DSPE (40:58:2mol/mol%). Using K562 human erythroleukemia cells, which contain an abundance of Bcl-2 and overexpression of transferrin receptors (TfR), and G3139 (oblimerson sodium or Genasense(TM)) as a model cell line and drug, respectively, the Bcl-2 down-regulation at the mRNA and protein levels as well as cellular uptake and apoptosis was compared between the conventional bulk mixing (BM) method and the MF method. The lipopolyplex size and surface charge were characterized by dynamic light scattering (DLS) and zeta potential (zeta) measurement, respectively, while the ODN encapsulation efficiency was determined by gel electrophoresis. Cryogenic transmission electron microscopy (Cryo-TEM) was used to determine the morphology of LPs. Our results demonstrated that MF produced LP nanoparticles had similar structures but smaller size and size distribution compared to BM LP nanoparticles. MF LP nanoparticles had higher level of Bcl-2 antisense uptake and showed more efficient down-regulation of Bcl-2 protein level than BM LP nanoparticles.
Asunto(s)
Portadores de Fármacos/química , Microfluídica/métodos , Nanopartículas/química , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Apoptosis/efectos de los fármacos , Western Blotting , Microscopía por Crioelectrón , Regulación hacia Abajo , Fluoresceína-5-Isotiocianato , Humanos , Células K562 , Liposomas , Oligodesoxirribonucleótidos Antisentido/química , Oligodesoxirribonucleótidos Antisentido/genética , Tamaño de la Partícula , Receptores de Transferrina/biosíntesis , Propiedades de SuperficieRESUMEN
Polyethylenimine (PEI) and plasmid DNA (pDNA) complexes (PEI/pDNA) are nonviral vectors for gene delivery. The conventional method for producing these complexes involves bulk mixing (BM) of PEI and DNA followed by vortexing which at low N/P ratios results in large particle size distribution, low cytotoxicity, and poor gene transfection, while at high N/P ratios it results in small particle size and better gene transfection but high cytotoxicity. To improve size control, gene transfection efficiency, and cytotoxicity, in this study, we used a microfluidic hydrodynamic focusing (MF) device to prepare PEI/pDNA complexes at N/P = 3.3 and 6.7. We used bulk mixing as control, mouse NIH 3T3 fibroblast cells and mouse embryonic stem (mES) cells as model cell lines, plasmid encoding green fluorescent protein (pGFP) and secreted alkaline phosphatase (pSEAP) as the reporter gene, and commercially available Lipofectamine 2,000 as a positive control. The complexes were characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), and zeta potential (zeta) measurement. Confocal laser scanning microscopy (CLSM) and fluorescent labeling techniques were used to visualize the complex size distribution, complexation uniformity, and cellular distribution. The results showed that MF produced complexes were smaller and more uniformly complexed and had higher cell viability and improved exogenous gene expression.