RESUMO
Efficient tools are still being searched for to substitute the viral vectors in nucleic acid delivery applications. One of the most severe constraints in producing them is related to the strict reproducibility of their molecular characteristics, which is ensured through the synthesis. In this work, we report an original route to obtain polycationic nanoentities with low variability, which are able to act as cooperating carriers for dsDNA complexation and transport. The carriers are synthesized by rigorous conjugation of ß-cyclodextrin (ß-CD) with precise ratios of 2 kDa branched poly(ethyleneimine) (b-PEI) and 0.75 kDa poly(ethylene glycol) (PEG). Low cytotoxicity was the key parameter of the carrier design, besides the highest possible transfection ability, and both of these features were proven by HeLa cell culture assays. A reporter gene which induces the expression of green fluorescent protein (GFP), inserted in a plasmid, was used to perform the necessary quantitative measurements. In silico molecular modelling guided the carrier design and confirmed the functional mimicry of histones in the tight and compact nucleosome-like spiral packaging of dsDNA. The carrier molecules, synthesized with high reproducibility, are expected to be feasible for application in gene transfection.
RESUMO
Carboxymethyl guar gum (CMGG) synthesized from commercially available polysaccharide was formulated into nanoparticles via ionic gelation using trisodium trimetaphosphate (STMP) as cross-linking agent. Characterisation using a range of analytical techniques (FTIR, NMR, GPC, TGA and DLS) confirmed the CMGG structure and revealed the effect of the CMGG and STMP concentration on the main characteristics of the obtained nanoformulations. The average nanoparticle diameter was found to be around 208 nm, as determined by dynamic light scattering (DLS) and confirmed by scanning electron microscopy (SEM) and nanoparticle tracking analysis (NTA). Experiments using simulated gastric and intestinal fluids evidenced significant pH-dependent drug release behaviour of the nanoformulations loaded with Rhodamine B (RhB) as a model drug (loading capacity in excess of 83%), as monitored by UV-Vis. While dose-dependent cytotoxicity was observed, the nanoformulations appeared completely non-toxic at concentrations below 0.3 mg/mL. Results obtained so far suggest that carboxymethylated guar gum nanoparticles formulated with STMP warrant further investigations as polysaccharide based biocompatible drug nanocarriers.
Assuntos
Portadores de Fármacos/química , Galactanos/química , Mananas/química , Nanopartículas/química , Gomas Vegetais/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Liberação Controlada de Fármacos , Difusão Dinâmica da Luz , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Rodaminas/química , Rodaminas/metabolismo , Rodaminas/toxicidade , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
UNLABELLED: Bone marrow mesenchymal stem cells are important for both research and clinical purpose. A number of culture methods for these cells are available on the market, many of them consisting of specialized growing media in combination with growth factors. Our goal was to optimize a less expensive culture method for bone marrow mesenchymal cells. MATERIAL AND METHODS: Eight samples of bone marrow aspirates from patients were used. Out these 8 samples 2 were from healthy people, 3 from chronic granulocytic leukemia patients, 2 from multiple myeloma patients and 2 from patients with myelodysplastic syndrome. Bone aspirates from healthy people were used to optimize the culture method and the rest were used for testing the optimized method. Two methods were tried: 1. Cell culture starting from whole bone marrow, 2) cell culture after bone marrow separation in density gradient with Histopaque. RESULTS: Cell culture starting from whole bone marrow gives better yields for mesenchymal stem cells than methods which include gradient density separation of mononuclear cells with Ficoll-Histopaque. CONCLUSIONS: We have optimised a less expensive cell culture method for bone marrow mesenchymal cells.
Assuntos
Técnicas de Cultura de Células , Meios de Cultura/química , Células-Tronco Mesenquimais/citologia , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/economia , Diferenciação Celular , Proliferação de Células , Separação Celular/métodos , Centrifugação com Gradiente de Concentração/economia , Meios de Contraste/farmacologia , Meios de Cultura/economia , Diatrizoato/farmacologia , Ficoll/farmacologia , Citometria de Fluxo/métodos , Humanos , Romênia , Transplante de Células-Tronco/economiaRESUMO
UNLABELLED: Single nucleotide polymorphisms (SNPs) of the CYP19A1 gene have shown the ability to modify its activity, but no association has been established with aromatase inhibitor (AI) efficiency in hormone receptor positive breast cancer (BC). MATERIAL AND METHODS: The study included blood samples from 53 patients (p) with BC and 1 control (male DNA); 22p. (investigational group) were administered an Al and followed up. RESULTS: Homozygous (hh) -CC and TT -, and heterozygous (hz) - TC - genotypes of the rs10046 SNP were balanced. Response to treatment or progression were not affected (p=0.630) in patients with T allele 1; local relapse occurred significantly more rarely and overall survival (OS) was superior (p=0.046). For rs4646, genotypes were mainly hhGG (57%), with no implication to study parameters (p>0.05). For rs727479 SNP, major genotypes were hhTT (40%) and hzTG (45%). Therapeutic response was better in patients with T allele 2 (p=0.040), T allele 1 was associated with reduced local recurrence (p=0.047) and TT genotype with better OS (p = 0.008). In rs700518 SNP, genotype was mostly hzGA (49%). Local recurrence rates were reduced in the presence of G allele 1 (p=0.047) and GG homozygosity. CONCLUSIONS: At this stage, the study was purely exploratory and hypothesis generating. Genotyping of at least three of the four CYP19A1 SNPs evaluated (except rs4646) may have an impact in clinical practice, providing better criteria for patient selection, prognosis and therapeutic decision in BC.