RESUMO
Cationic nanogels with site-selected functionality were designed for the delivery of nucleic acid payloads targeting numerous therapeutic applications. Functional cationic nanogels containing quaternized 2-(dimethylamino)ethyl methacrylate and a cross-linker with reducible disulfide moieties (qNG) were prepared by activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) in an inverse miniemulsion. Polyplex formation between the qNG and nucleic acid exemplified by plasmid DNA (pDNA) and short interfering RNA (siRNA duplexes) were evaluated. The delivery of polyplexes was optimized for the delivery of pDNA and siRNA to the Drosophila Schneider 2 (S2) cell-line. The qNG/nucleic acid (i.e., siRNA and pDNA) polyplexes were found to be highly effective in their capabilities to deliver their respective payloads.
Assuntos
Técnicas de Transferência de Genes , Ácidos Nucleicos/química , Polietilenoglicóis , Polietilenoimina , Animais , Cátions , Linhagem Celular , DNA/genética , Drosophila/genética , Metacrilatos/química , Nanogéis , Plasmídeos/genética , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
Star polymers with poly(ethylene glycol) (PEG) arms and a degradable cationic core were synthesized by the atom transfer radical copolymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate macromonomer (PEGMA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), and a disulfide dimethacrylate (cross-linker, SS) via an "arm-first" approach. The star polymers had a diameter ~15 nm and were degraded under redox conditions by glutathione treatment into individual polymeric chains due to cleavage of the disulfide cross-linker, as confirmed by dynamic light scattering. The star polymers were cultured with mouse calvarial preosteoblast-like cells, embryonic day 1, subclone 4 (MC3T3-E1.4) to determine biocompatibility. Data suggest star polymers were biocompatible, with ≥ 80% cell viability after 48 h of incubation even at high concentration (800 µg/mL). Zeta potential values varied with N/P ratio confirming complexation with siRNA. Successful cellular uptake of the star polymers in MC3T3-E1.4 cells was observed by confocal microscopy and flow cytometry after 24 h of incubation.
Assuntos
Materiais Biocompatíveis/síntese química , Portadores de Fármacos/síntese química , Metacrilatos/química , Polietilenoglicóis/química , Animais , Materiais Biocompatíveis/análise , Materiais Biocompatíveis/farmacologia , Cátions/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Portadores de Fármacos/análise , Portadores de Fármacos/farmacologia , Endocitose , Citometria de Fluxo , Glutationa/metabolismo , Luz , Metacrilatos/metabolismo , Camundongos , Osteoblastos/efeitos dos fármacos , Tamanho da Partícula , Polietilenoglicóis/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Espalhamento de RadiaçãoRESUMO
Star polymers with thermoresponsive arms, consisting of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate with ~4 ethylene oxide units (OEOMA300, M(n) = 300), were synthesized via atom transfer radical polymerization (ATRP). 25% of the arms contained benzophenone chain-end functionality at the star periphery. A mixture of linear poly(MEO2MA-co-OEOMA300)-Br macroinitiators without and with benzophenone end-group macroinitiators were (MI and Bzp-MI, respectively) cross-linked with ethylene glycol dimethacrylate to form star polymers. Formation of star polymers was monitored by GPC, and the presence of benzophenone functionality in the stars was confirmed by ¹H NMR. The UV-vis spectroscopy revealed that the star polymers exhibit the low critical solution temperature (LCST) at 27 °C, slightly lower than LCST of either MI or Bzp-MI. Commercially available tissue culture grade polystyrene surface was modified by depositing a thin film of functionalized stars and UV cross-linking (λ = 365 nm). The star polymers covalently attached onto surfaces allowed a control of cell shrinkage and attachment in response to temperature changes.
Assuntos
Reagentes de Ligações Cruzadas/química , Polimerização , Polímeros/síntese química , Animais , Benzofenonas/química , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Óxido de Etileno/química , Espectroscopia de Ressonância Magnética , Metacrilatos/química , Camundongos , Microscopia de Contraste de Fase , Estrutura Molecular , Células NIH 3T3 , Processos Fotoquímicos , Polímeros/química , Polímeros/farmacologia , Poliestirenos/química , Propriedades de Superfície , Temperatura , Raios UltravioletaRESUMO
Poly(ethylene glycol) (PEG) star polymers containing GRGDS (Gly-Arg-Gly-Asp-Ser) peptide sequences on the star periphery were synthesized by atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMA), GRGDS modified poly(ethylene glycol) acrylate (GRGDS-PEG-Acryl), fluorescein o-methacrylate (FMA), and ethylene glycol dimethacrylate (EGDMA) via an "arm-first" method. Star polymers were approximately 20 nm in diameter, as measured by dynamic light scattering and atomic force microscopy. Conjugation of FMA to the stars was confirmed by fluorescence microscopy, and successful attachment of GRGDS segments to the star periphery was confirmed by (1)H NMR spectroscopy. Both fluorescent PEG star polymers with and without peripheral GRGDS peptide segments were cultured with MC3T3-E1.4 cells. These star polymers were biocompatible with ≥ 90% cell viability after 24 h of incubation. Cellular uptake of PEG star polymers in MC3T3-E1.4 cells was observed by confocal microscopy. Rapid uptake of PEG star polymers with GRGDS peptides (â¼ 100% of FITC-positive cells in 15 min measured by flow cytometry) was observed, suggesting enhanced delivery potential of these functional star polymers.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/síntese química , Proliferação de Células/efeitos dos fármacos , Polímeros/química , Polímeros/síntese química , Animais , Materiais Biocompatíveis/farmacologia , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Células Cultivadas , Citometria de Fluxo , Espectroscopia de Ressonância Magnética , Camundongos , Oligopeptídeos/química , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Fragmentos de Peptídeos/química , Polietilenoglicóis/química , Polímeros/farmacologia , Polimetil Metacrilato/químicaRESUMO
This study presents the synthesis and evaluation of cell adhesive poly(ethylene oxide) (PEO) star polymers for potential biomedical applications. Star polymers with a size of approximately 20 nm and with relatively low polydispersities (M(w)/M(n) ≤ 1.6), containing GRGDS (Gly-Arg-Gly-Asp-Ser) segments, were prepared by atom transfer radical copolymerization of PEO methyl ether methacrylate macromonomer (MM), telechelic GRGDS-PEO-acrylate MM, and ethylene glycol dimethacrylate (EGDMA). Results from (1)H NMR spectroscopy confirmed the covalent incorporation of the peptide into the star periphery. In vitro cytotoxicity experiments showed star polymers to be cytocompatible (≥95% cell viability) and GRGDS-star hybrid hydrogels supported the attachment of MC3T3.E1 (subclone 4) cells. Hybrid hydrogels were prepared by free radical photopolymerization based on 10% (wt/v) PEO dimethacrylates M(n) = 4000 g/mol with 1% (wt/v) GRGDS-star polymers having different peptide content. Cell adhesiveness was also determined from thin film coatings prepared with GRGDS-containing star polymers on nonadherent plastic plates. After 24 h incubation, phase contrast microscopy and scanning electron microscopy (SEM) images showed uniform cell adhesion and distribution over the film containing cell-adhesive star polymers. These results confirm that incorporation of RGD ligand-binding motifs into PEO-based star polymers is required to influence substrate-cell interactions.
Assuntos
Adesão Celular , Hidrogéis/química , Oligopeptídeos/química , Polietilenoglicóis/química , Células 3T3 , Motivos de Aminoácidos , Animais , Materiais Biocompatíveis/síntese química , Hidrogéis/síntese química , Camundongos , Oligopeptídeos/metabolismo , PorosidadeRESUMO
Atom transfer radical polymerization (ATRP) was used to produce a versatile drug delivery system capable of encapsulating a range of molecules. Inverse miniemulsion ATRP permitted the synthesis of biocompatible and uniformly cross-linked poly(ethylene oxide)-based nanogels entrapping gold nanoparticles, bovine serum albumin, rhodamine B isothiocyanate-dextran, or fluoresceine isothiocyanate-dextran. These moieties were entrapped to validate several biological outcomes and to model delivery of range of molecules. Cellular uptake of nanogels was verified by transmission electron microscopy, gel electrophoresis, Western blotting, confocal microscopy, and flow cytometry. Fluorescent colocalization of nanogels with a fluorophore-conjugated antibody for clathrin indicated clathrin-mediated endocytosis. Furthermore, internalization of nanogels either with or without GRGDS cell attachment-mediating peptides was quantified using flow cytometry. After 45 min of incubation, the uptake of unmodified FITC-Dx-loaded nanogels was 62%, whereas cellular uptake increased to >95% with the same concentration of GRGDS-modified FITC-Dx nanogels. In addition, a spheroidal coculture of human umbilical vascular endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) validated cell endocytosis. Application of ATRP enabled the synthesis of a functionalized drug delivery system with a uniform network that is capable of encapsulating and delivering inorganic, organic, and biological molecules.
Assuntos
Carboidratos/química , Endocitose , Ouro/química , Nanopartículas , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoimina/química , Polietilenoimina/farmacocinética , Proteínas/metabolismo , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Nanogéis , Crânio/citologia , Crânio/metabolismo , Esferoides Celulares , Células-Tronco/metabolismo , Distribuição Tecidual , Veias Umbilicais/citologia , Veias Umbilicais/metabolismoRESUMO
The properties of hyaluronic acid (HA) hydrogels having a broad range of methacrylation are presented. Increasing solubility of glycidyl methacrylate (GM) in a co-solvent mixture during the methacrylation of HA with GM was shown to produce photopolymerizable HAGM conjugates with various degree of methacrylation (DM) ranging from 14% up to 90%. Aqueous solutions of HAGM macromonomers were photocross-linked to yield hydrogels with nearly full vinyl group conversions after 10 min exposure under ultraviolet light (UV). Hydrogels were characterized by uniaxial compression and volumetric swelling measurements. Keeping the DM constant, the shear modulus was varied from 16 kPa up to 73 kPa by varying the macromonomer concentration. However, at a given macromonomer concentration while varying the DM, similarly the shear modulus varied from 22 kPa up to 65 kPa. Preliminary in-vitro cell culture studies showed that GRGDS modified HAGM hydrogels promoted similarly cell interaction at both low and high DMs, 32% and 60%, respectively. Densely cross-linked hydrogels with a high DM have been shown to be more mechanically robust while maintaining cytocompability and cell adhesion.
Assuntos
Materiais Biocompatíveis/química , Ácido Hialurônico/química , Hidrogéis/química , Teste de Materiais , Metacrilatos/química , Fibras Musculares Esqueléticas/fisiologia , Animais , Materiais Biocompatíveis/administração & dosagem , Adesão Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Elasticidade , Dureza , Ácido Hialurônico/administração & dosagem , Hidrogéis/administração & dosagem , Metacrilatos/administração & dosagem , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Solubilidade , Soluções/químicaRESUMO
Bone defects, such as compressive fractures in the vertebral bodies, are frequently treated with acrylic bone cements (e.g., PMMA). Although these biomaterials have sufficient mechanical properties for fixing the fracture, they are non-degradable and do not remodel or integrate with host tissue. In an alternative approach, biodegradable polyurethane (PUR) networks have been synthesized that are designed to integrate with host tissue and degrade to non-cytotoxic decomposition products. PUR networks have been prepared by two-component reactive liquid molding of low-viscosity quasi-prepolymers derived from lysine polyisocyanates and poly(epsilon-caprolactone-co-DL-lactide-co-glycolide) triols. The composition, thermal transitions, and mechanical properties of the biomaterials were measured. The values of Young's modulus ranged from 1.20-1.43 GPa, and the compressive yield strength varied from 82 to 111 MPa, which is comparable to the strength of PMMA bone cements. In vitro, the materials underwent controlled biodegradation to non-cytotoxic decomposition products, and supported the attachment and proliferation of MC3T3 cells. When cultured in osteogenic medium on the PUR networks, MC3T3 cells deposited mineralized extracellular matrix, as evidenced by von Kossa staining and tetracycline labeling. Considering the favorable mechanical and biological properties, as well as the low-viscosity of the reactive intermediates used to prepare the PUR networks, these biomaterials are potentially useful as injectable, biodegradable bone cements for fracture healing.
Assuntos
Implantes Absorvíveis , Materiais Biocompatíveis/química , Substitutos Ósseos/administração & dosagem , Substitutos Ósseos/química , Adesão Celular/efeitos dos fármacos , Isocianatos/administração & dosagem , Isocianatos/química , Células 3T3 , Animais , Linhagem Celular , Força Compressiva , Elasticidade , Dureza , Lisina/química , Teste de Materiais , Camundongos , Estresse MecânicoRESUMO
The development of minimally invasive therapeutics for orthopedic clinical conditions has substantial benefits, especially for osteoporotic fragility fractures and vertebral compression fractures. Poly(ester urethane)urea (PEUUR) foams are potentially useful for addressing these conditions because they cure in situ upon injection to form porous scaffolds. In this study, the effects of water concentration and polyester triol composition on the physicochemical, mechanical, and biological properties of PEUUR foams were investigated. A liquid resin (lysine diisocyanate) and hardener (poly(epsilon-caprolactone-co-glycolide-co-DL-lactide) triol, tertiary amine catalyst, anionic stabilizer, and fatty acid-derived pore opener) were mixed, and the resulting reactive liquid mixture was injected into a mold to harden. By varying the water content over the range of 0.5 to 2.75 parts per hundred parts polyol, materials with porosities ranging from 89.1 to 95.8 vol-% were prepared. Cells permeated the PEUUR foams after 21 days post-seeding, implying that the pores are open and interconnected. In vitro, the materials yielded non-cytotoxic decomposition products, and differences in the half-life of the polyester triol component translated to differences in the PEUUR foam degradation rates. We anticipate that PEUUR foams will present compelling opportunities for the design of new tissue-engineered scaffolds and delivery systems because of their favorable biological and physical properties.
Assuntos
Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/metabolismo , Poliésteres/síntese química , Poliésteres/metabolismo , Polímeros , Água , Células 3T3 , Animais , Materiais Biocompatíveis/química , Camundongos , Poliésteres/química , Polímeros/síntese química , Polímeros/químicaRESUMO
Motion-based therapies have been applied to promote healing of arthritic joints. The goal of the current study was to determine the early molecular events that are responsible for the beneficial actions of motion-based therapies on meniscal fibrocartilage. Rabbit knees with Antigen-Induced-Arthritis (AIA) were exposed to continuous passive motion (CPM) for 24 or 48 h and compared to immobilized knees. The menisci were harvested and glycosaminoglycans (GAG), interleukin-1beta (IL-1beta), matrix metalloproteinase-1 (MMP-1), cyclooxygenase-2 (COX-2), and interleukin-10 (IL-10) were determined by histochemical analysis. Within 24 h, immobilized knees exhibited marked GAG degradation. The expression of proinflammatory mediators MMP-1, COX-2, and IL-1beta was notably increased within 24 h and continued to increase during the next 24 h in immobilized knees. Knees subjected to CPM revealed a rapid and sustained decrease in GAG degradation and the expression of all proinflammatory mediators during the entire period of CPM treatment. More importantly, CPM induced synthesis of the anti-inflammatory cytokine IL-10. The results demonstrate that mechanical signals generated by CPM exert potent anti-inflammatory signals on meniscal fibrochondrocytes. Furthermore, these studies explain the molecular basis of the beneficial effects of CPM observed on articular cartilage and suggest that CPM suppresses the inflammatory process of arthritis more efficiently than immobilization.
Assuntos
Artrite Experimental/terapia , Cartilagem/metabolismo , Meniscos Tibiais/metabolismo , Terapia Passiva Contínua de Movimento , Animais , Artrite Experimental/metabolismo , Ciclo-Oxigenase 2 , Glicosaminoglicanos/análise , Interleucina-1/análise , Interleucina-10/biossíntese , Masculino , Metaloproteinase 1 da Matriz/análise , Prostaglandina-Endoperóxido Sintases/biossíntese , CoelhosRESUMO
A new method to prepare nanostructured hybrid hydrogels by incorporating well-defined poly(oligo (ethylene oxide) monomethyl ether methacrylate) (POEO(300)MA) nanogels of sizes 110-120 nm into a larger three-dimensional (3D) matrix was developed for drug delivery scaffolds for tissue engineering applications. Rhodamine B isothiocyanate-labeled dextran (RITC-Dx) or fluorescein isothiocyanate-labeled dextran (FITC-Dx)-loaded POEO(300)MA nanogels with pendant hydroxyl groups were prepared by activators generated electron transfer atom transfer radical polymerization (AGET ATRP) in cyclohexane inverse miniemulsion. Hydroxyl-containing nanogels were functionalized with methacrylated groups to generate photoreactive nanospheres. (1)H NMR spectroscopy confirmed that polymerizable nanogels were successfully incorporated covalently into 3D hyaluronic acid-glycidyl methacrylate (HAGM) hydrogels after free radical photopolymerization (FRP). The introduction of disulfide moieties into the polymerizable groups resulted in a controlled release of nanogels from cross-linked HAGM hydrogels under a reducing environment. The effect of gel hybridization on the macroscopic properties (swelling and mechanics) was studied. It is shown that swelling and nanogel content are independent of scaffold mechanics. In-vitro assays showed the nanostructured hybrid hydrogels were cytocompatible and the GRGDS (Gly-Arg-Gly-Asp-Ser) contained in the nanogel structure promoted cell-substrate interactions within 4 days of incubation. These nanostructured hydrogels have potential as an artificial extracellular matrix (ECM) impermeable to low molecular weight biomolecules and with controlled pharmaceutical release capability. Moreover, the nanogels can control drug or biomolecule delivery, while hyaluronic acid based-hydrogels can act as a macroscopic scaffold for tissue regeneration and regulator for nanogel release.
Assuntos
Materiais Biocompatíveis/química , Materiais Biomiméticos/química , Matriz Extracelular/química , Hidrogéis/química , Nanoestruturas/química , Polímeros/química , Animais , Materiais Biomiméticos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Força Compressiva , Módulo de Elasticidade , Radicais Livres/química , Hidrogéis/farmacologia , Teste de Materiais , Camundongos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Nanoestruturas/administração & dosagem , Nanoestruturas/ultraestrutura , Polímeros/farmacologia , PorosidadeRESUMO
A series of resorbable poly(ethylene glycol)-co-poly(glycolic acid) (PEG-co-PGA, 4KG5) macromonomers have been synthesized with the chemistries from three different photopolymerizable end-groups (acrylates, methacrylates and urethane methacrylates). The aim of the study is to examine the effects of the chemistry of the cross-linker group on the properties of photocross-linked hydrogels. 4KG5 hydrogels were prepared by photopolymerization with high vinyl group conversion as confirmed by (1)H nuclear magnetic resonance spectrometry using a 1D diffusion-ordered spectrometry pulse sequence. Our study reveals that the nature of end-groups in a moderately amphiphilic polymer can adjust the distribution and size of the micellar configuration in water, leading to changes in the macroscopic structure of hydrogels. By varying the chemistry of the cross-linker group (diacrylates (DA), dimethacrylates (DM) and urethane dimethacrylates (UDM)), we determined that the hydrophobicity of a single core polymer consisting of poly(glycolic acid) could be fine-tuned, leading to significant variations in the mechanical, swelling and degradation properties of the gels. In addition, the effects of cross-linker chemistry on cytotoxicity and proliferation were examined. Cytotoxicity assays showed that the three types of hydrogels (4KG5 DA, DM and UDM) were biocompatible and the introduction of RGD ligand enhanced cell adhesion. However, differences in gel properties and stability differentially affected the spreading and proliferation of myoblast C2C12 cells.
Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Mioblastos/fisiologia , Polietilenoglicóis/química , Ácido Poliglicólico/química , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Teste de Materiais , Camundongos , Mioblastos/citologia , Propriedades de SuperfícieRESUMO
Poly(N-isopropylacrylamide-co-5,6-benzo-2-methylene-1,3-dioxepane) (poly(NIPAAm-co-BMDO)) was synthesized by atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) polymerization. Using UV-vis spectroscopy, the lower critical solution temperature (LCST) of poly(NIPAAm) and poly(NIPAAm-co-BMDO) copolymers were measured, varying with respect to the amount of incorporated BMDO. This material is degradable and possesses a LCST above room temperature and below body temperature, making it a potential candidate for use as an injectable tissue engineering scaffold to enhance fracture repair. ATRP and RAFT enabled preparation of polymers with control over molecular weight up to M(n) = 50,000 g/mol and M(w)/M(n) < 1.2. Degradation studies were performed in basic solution and in complete Dulbecco's modified Eagle medium. The cytotoxicity of the material and its degradation products were analyzed by in vitro cell culture analyses, including cytotoxicity live/dead and CyQUANT cell proliferation assays. Crosslinked scaffolds with degradable units within the polymer backbone and at the crosslinking sites were prepared using an ester-containing diacrylate crosslinker. Furthermore, incorporation of a GRGDS peptide sequence improved cell attachment to the gels. Controlled/living radical polymerization techniques allow for precise control over macromolecular structure and are poised to become powerful tools for tissue engineering scaffold synthesis.