RESUMO
The steady-state shear and linear viscoelastic deformations of semidilute suspensions of rod-shaped nanocrystalline cellulose (NCC) particles in 1.0% hydroxyethyl cellulose and carboxymethyl cellulose solutions were investigated. Addition of NCC at the onset of semidilute suspension concentration significantly altered the rheological and linear viscoelastic properties of semidilute polymer solutions. The low-shear viscosity values of polymers solutions were increased 20-490 times (depending on polymer molecular weight and functional groups) by the presence of NCC. NCC suspensions in polymer solutions exhibited yield stresses up to 7.12 Pa. Viscoelasticity measurements also showed that NCC suspended polymer solutions had higher linear elastic moduli than the loss moduli. All of those results revealed the gel formation of NCC particles and presence of internal structures. The formation of a weak gel structure was due to the nonadsorbing macromolecules which caused the depletion-induced interaction among NCC particles. A simple interaction energy model was used to show successfully the flocculation of NCC particles in the presence of nonadsorbing polymers. The model is based on the incorporation of the depletion interaction term between two parallel plates into the DLVO theory for cubic prismatic rod shaped NCC particles.
Assuntos
Carboximetilcelulose Sódica/química , Celulose/análogos & derivados , Nanopartículas/química , Água/química , Celulose/química , Coloides , Elasticidade , Soluções , ViscosidadeRESUMO
Enabling gene expression in skin fibroblasts using safe, nonviral gene delivery has the potential to stimulate wound healing and aid in skin tissue engineering efforts. In this study, several lipid-substituted poly(L-Lysines) (PLL) were investigated for their ability to deliver a plasmid DNA (pEGFP) to human skin fibroblasts. While native and lipid-substituted PLLs showed complete complexation with pEGFP, polymers with higher lipid substitution were more resilient to dissociation after heparin treatment. All polymers showed good protection of pEGFP against DNase I and DNase II digestion in vitro. DNA delivery studies using fluorescently labeled pEGFP showed that native PLL lacked the ability to deliver pEGFP into cells, whereas most of the lipid-substituted PLLs gave efficient pEGFP delivery into the cells. Extent of lipid substitution was an important factor in DNA delivery efficiency. The intracellular pEGFP was intact after delivery with lipid-substituted polymers up to 7 days. An RT-PCR methodology indicated successful transcription of the reporter GFP gene, which was not the case when the cells were transfected with a blank plasmid containing no functional GFP gene. Further studies with flow cytometry showed that successful protein expression was obtained with PLLs substituted with myristic and stearic acid, the latter displaying a relatively lower toxicity. We conclude that substituting lipids on PLL results in effective gene carriers and the extent of substitution, rather than the individual lipid, appeared to be critical for effective plasmid delivery.
Assuntos
Fibroblastos/metabolismo , Lipídeos/química , Polilisina/química , Pele/metabolismo , Transfecção , Fragmentação do DNA , Desoxirribonucleases/metabolismo , Eletroforese em Gel de Ágar , Citometria de Fluxo , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Fluorescência , Ácido Mirístico/química , Plasmídeos/metabolismo , Polilisina/síntese química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácidos Esteáricos/químicaRESUMO
The current study compared the effectiveness of an amphiphilic biomaterial poly(L-lysine)-palmitic acid (PLL-PA), and the lipid-based transfection agent Lipofectamine 2000 for plasmid delivery to bone marrow stromal cells (BMSC). We investigated the utility of the carriers to deliver a plasmid containing enhanced green fluorescent protein (pEGFP) to BMSC in vitro. Confocal microscopy was used to investigate the intracellular trafficking of pEGFP/carrier complexes. pEGFP delivery and EGFP expression were assessed by flow cytometry. PLL-PA formed condensed structures with pEGFP and successfully delivered the plasmid into the nucleus within 5 h of incubation with the cells. PLL-PA delivered the pEGFP to approximately 80% of the cells, achieving a maximum transfection efficiency of approximately 22%. This was significantly higher than Lipofectamine 2000-mediated transfection, which was 11% under most optimal conditions. Dosing the BMSC two or three times during the 24 h period increased the transfection efficiency by 2-3 folds, without compromising cell viability. When chloroquine was employed as an ensomolytic agent, 100 microM of the drug increased the transfection efficiency while reducing cell viability, but lower concentrations (1-10 microM) were not beneficial for transfection. Combining PLL-PA with Lipofectamine 2000 created an additive effect, increasing the transfection efficiency of PLL-PA. Long-term evaluation of gene expression with pEGFP/PLL-PA yielded approximately 17% transfection on day 1, which gradually decreased over a 12-day period. We conclude that PLL-PA is an effective biomaterial carrier and a promising candidate for non-viral gene delivery to BMSC.
Assuntos
Portadores de Fármacos/química , Lipídeos/química , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Ácido Palmítico/química , Plasmídeos/administração & dosagem , Polilisina/química , Transfecção/métodos , Animais , Células Cultivadas , Feminino , Teste de Materiais , Ratos , Ratos Sprague-DawleyRESUMO
Nonviral gene carriers are actively explored in gene therapy due to safety concerns of the viral carriers. To design effective gene carriers for modification of bone marrow stromal cells (BMSC), an important cell phenotype for clinical application of gene therapy, cationic polymers polyethyleneimine (PEI), and poly-L-Lysine (PLL) were substituted with palmitic acid (PA) via amide linkages. Depending on the reaction conditions, PEI and PLL was substituted with 2.2-5.2 and 13.4-16.2 PA per polymer chain. The PA substituted polymers displayed slightly lower binding efficiency towards a plasmid containing Enhanced Green Fluorescent Protein (pEGFP) in an agarose gel binding assay. The cell binding of PLL-PA, but not PEI-PA, was particularly enhanced, resulting in higher percentage of the cells displaying a significant polymer uptake. pEGFP delivery into the BMSC was also significantly increased with the PLL-PA (vs. PLL), but not PEI-PA (vs. PEI). The transfection efficiency of PLL-PA was significantly higher ( approximately fivefold) than the unmodified polymer. We conclude that PA substitution on PLL provides an effective carrier for transfection of primary cells derived from the bone marrow.
Assuntos
Materiais Biocompatíveis/química , Células da Medula Óssea/metabolismo , Plasmídeos/administração & dosagem , Polímeros/química , Animais , Cátions/química , Sistemas de Liberação de Medicamentos , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Terapia Genética , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Teste de Materiais , Ácido Palmítico/química , Plasmídeos/genética , Polietilenoimina/química , Polilisina/química , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Células Estromais/metabolismo , TransfecçãoRESUMO
This work explores the unique antibiofilm activity of pentasilver hexaoxoiodate (Ag(5)IO(6)). To test this activity, wound dressings were impregnated with Ag(5)IO(6) and compared with various commercially available silver-containing dressings, as well as dressings containing chlorhexidine, iodine and polyhexamethylene biguanide (PHMB). The materials were tested against Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans for their ability to prevent micro-organism adherence, eliminate planktonic micro-organisms and disrupt/eliminate mature biofilms generated using the MBEC™ assay within 24 h of microbial exposure. Only the Ag(5)IO(6)-containing dressings were able to prevent adherence and eliminate surrounding planktonic micro-organisms for all species tested for ≥28 days of elution with log reductions >4. Two other silver dressings succeeded against P. aeruginosa only after 28 elution days, whilst the PHMB dressing succeeded after 28 days of elution against C. albicans only. Ag(5)IO(6)-containing dressings were able to generate >4 log reductions against all biofilms tested. The only commercial dressings able to generate >4 log reductions against biofilms were iodine against P. aeruginosa and S. aureus, and PHMB against S. aureus. The Ag(5)IO(6) dressings demonstrated complete kill (>4 log reduction) in a standard 30-min planktonic log reduction assay against all species. These results demonstrate that Ag(5)IO(6) has superior activity to a number of antimicrobials, with broad-spectrum efficacy that includes long-term prevention of microbial adherence, rapid kill of planktonic micro-organisms, and the ability to disrupt and eliminate mature biofilms. Thus, Ag(5)IO(6) may be a valuable antimicrobial agent for use in a number of medical device applications, including wound dressings, various catheters or implants.
Assuntos
Anti-Infecciosos Locais/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Desinfetantes/farmacologia , Equipamentos e Provisões/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Candida albicans/fisiologia , Contagem de Colônia Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/fisiologia , Fatores de TempoRESUMO
A conjugate of distearoylphosphoethanolamine-polyethylene glycol with 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was synthesized and incorporated into micelles and liposomes to create mineral-binding nanocarriers for therapeutic agents. The micelles and liposomes were used to encapsulate the anticancer drug doxorubicin (DOX) and a model protein lysozyme (LYZ) by using lipid film hydration (LFH) and reverse-phase evaporation vesicle (REV) methods. The results indicated that the micelles and LFH-derived liposomes were better at DOX loading than the REV-derived liposomes, while the REV method was preferable for encapsulating LYZ. The affinity of the micellar and liposomal formulations to hydroxyapatite (HA) was assessed in vitro, and the results indicated that all the thiolBP-incorporated nanocarriers had stronger HA affinity than their counterparts without thiolBP. The thiolBP-decorated liposomes also displayed a strong binding to a collagen/HA composite scaffold in vitro. More importantly, thiolBP-decorated liposomes gave increased retention in the collagen/HA scaffolds after subcutaneously implantation in rats. The designed liposomes were able to entrap the bone morphogenetic protein-2 in a bioactive form, indicating that the proposed nanocarriers could deliver bioactive factors locally in mineralized scaffolds for bone tissue engineering.
Assuntos
Conservadores da Densidade Óssea , Doenças Ósseas/tratamento farmacológico , Difosfonatos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Lipídeos/química , Nanopartículas/química , Animais , Materiais Biocompatíveis/química , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/uso terapêutico , Osso e Ossos/metabolismo , Difosfonatos/química , Difosfonatos/uso terapêutico , Feminino , Lipossomos/química , Teste de Materiais , Micelas , Estrutura Molecular , Tamanho da Partícula , Ratos , Ratos Sprague-DawleyRESUMO
Poly(L-lysine) (PLL) is a commonly used polymer for nonviral gene delivery. However, the polymer exhibits significant toxicity and is not very effective for transgene expression. To enhance the gene delivery efficiency of the polymer, we imparted an amphiphilic property to PLL by substituting approximately 10% of epsilon-NH2 with several endogenous lipids of variable chain lengths (lipid carbon chain ranging from 8 to 18). Lipid-modified PLL with high molecular weight (approximately 25 vs 4 kDa) was found to be more effective in delivering plasmid DNA intracellularly in clinically relevant bone marrow stromal cells (BMSC). For lipid-substituted 25 kDa PLL, a correlation between the extent of lipid substitution and the plasmid DNA delivery efficiency was obtained. Additionally, transgene expression by BMSC significantly increased (20-25%) when amphiphilic PLLs were used for plasmid delivery as compared to native PLL and the commercial transfection agent Lipofectamine-2000. The transfection efficiency of the polymers was positively correlated with the extent of lipid substitution. The amphiphilic polymers were able to modify the cells up to 7 days after transfection, after which the expression was decreased to background levels within 1 week. We conclude that lipid-substituted PLL can be used effectively as a nonviral carrier for DNA, and the extent of lipid substitution was an important determinant of gene delivery.
Assuntos
DNA/química , DNA/genética , Portadores de Fármacos/química , Lipídeos/química , Células-Tronco Mesenquimais/fisiologia , Polilisina/química , Transfecção/métodos , Animais , Células Cultivadas , Composição de Medicamentos/métodos , Teste de Materiais , Ratos , Ratos Sprague-DawleyRESUMO
This study was conducted to formulate a nonviral delivery system for the delivery of small interfering RNA (siRNA) to B16 melanoma cells in vitro. For this purpose, oleic and stearic acid modified derivatives of branched polyethylenimine (PEI) were prepared and evaluated. The hydrophobically modified polymers increased siRNA condensation up to 3 folds as compared to the parent PEI. The modified PEIs exhibited up to 3-fold higher siRNA protection from degradation in fetal bovine serum as compared to the parent PEI. The formulated complexes were shown to enter B16 cells in a time-dependent fashion, reaching over 90% of the cells after 24 h, as compared to only 5% of the cells displaying siRNA uptake in the absence of any carrier. A proportional reduction in siRNA cell uptake was observed with reduced polymeric content in the formulations. When used to deliver various doses of siRNA to B16 cells, the modified PEIs were superior or comparable to some of the commercially available transfection agents; the hydrophobically modified polymers gave 3-fold increased siRNA delivery than the parent PEI, approximately 5-fold higher delivery than jetPEI and Metafectene, a comparable delivery to Lipofectamine 2000, but a 1.6-fold decreased delivery compared to INTERFERin, which was the most efficient reagent in our hands. Using an siRNA specific for integrin alpha(v), a dose-dependent decrease in integrin alpha(v) levels was demonstrated in B16 cells by flow cytometry, revealing a more pronounced reduction of integrin alpha(v) levels for oleic- and stearic-acid modified PEIs. The overall results suggested that the hydrophobically modified PEIs provide a promising delivery strategy for siRNA therapeutic applications.
Assuntos
Ácido Oleico/química , Polietilenoimina/química , RNA Interferente Pequeno/química , Ácidos Esteáricos/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Camundongos , Estrutura Molecular , Ácido Oleico/toxicidade , Polietilenoimina/toxicidade , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/toxicidade , Soro/metabolismo , Ácidos Esteáricos/toxicidadeRESUMO
Palmitic acid conjugates of poly-L-lysine (PLL-PA) were prepared, and their ability to deliver plasmid DNA into human skin fibroblasts was evaluated in vitro. The conjugates were capable of condensing a 4.7 kb plasmid DNA into 50-200 nm particles (mean +/- SD = 112 +/- 34 nm), which were slightly smaller than the particles formed by PLL (mean +/- SD = 126 +/- 51 nm). Both PLL and PLL-PA were readily taken up by the cells, but PLL-PA delivered the plasmid DNA into a higher proportion of cells. DNA delivery was found to be reduced by endocytosis inhibitor Brefeldin A, suggesting an active mechanism of particle uptake. Using enhanced green fluorescent protein (EGFP) as a reporter gene, PLL-PA was found to give the highest number of EGFP-positive cells among several carriers tested, including polyethyleneimine, Lipofectamine-2000, and an adenovirus. Although some carriers gave a higher percentage of EGFP-positive cells than PLL-PA, they were also associated with higher toxicities. We conclude that PLL-PA is a promising gene carrier for non-viral modification of human fibroblasts.