ABSTRACT
The thrombin-induced platelet aggregation was studied in the absence and presence of magnesium sulfate, acetylsalicylic acid and emoxypine. It was found that all the preparations studied were able separately to decrease platelet aggregation. In contrast, their joint action was not able to affect the aggregation of platelets. The data obtained can be used to choose the treatment strategy for patients with ischemic stroke.
Subject(s)
Aspirin/pharmacology , Blood Platelets/metabolism , Magnesium/pharmacology , Picolines/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Brain Ischemia/drug therapy , Brain Ischemia/metabolism , Female , Humans , Male , Stroke/drug therapy , Stroke/metabolismABSTRACT
Genetic engineering of stem cells and their derivatives has the potential to enhance their regenerative capabilities. Here, dendrimer- and lipofection-based approaches were used for non-viral neurotrophin-3 (NT-3) over-expression in Schwann cells differentiated from skin precursors (SKP-SCs). A variety of dendrimers were first tested for transfection efficiency on HEK 293T cells, with PAMAMNH2 G4 found most effective and used subsequently for SKP-SCs transfection. Plasmid-based expression resulted in increased NT-3 release from SKP-SCs in both adherent and microcarrier-based culture. In a proof-of-concept study, the microcarrier/SKP-SCs were implanted into the injured nerve, and transfected cells were shown to detach, integrate into the nerve tissue and associate with regenerating axons. Virus-free systems for transient neurotrophin expression are a feasible and biologically safe option to increase the therapeutic value of stem cells and stem cell-derived cells in nerve repair strategies. Further work to develop bioprocesses for expansion of SKP-SCs on microcarriers in bioreactors is still needed.
Subject(s)
Neurotrophin 3/metabolism , Schwann Cells/metabolism , Transfection/methods , Animals , Cells, Cultured , Dendrimers , Female , HEK293 Cells , Humans , Nerve Regeneration , Polypropylenes , Rats , Rats, Inbred Lew , Sciatic Nerve/injuries , Sciatic Nerve/physiology , Skin/cytology , Stem Cell TransplantationABSTRACT
Expression of transgenes in neurons and stromal/mesenchymal stem cells (MSC) can greatly enhance their therapeutic potential. In transfection experiments, we studied properties of linear and branched (dendrimers) polycations as transgene delivery vehicles. Linear polyethyleneimine transfected neurons, but was ineffective in MSC. Polyamidoamine dendrimers showed greater transfection efficiency and mean GFP fluorescence intensity compared to phosphorus dendrimers of the same (4th) generation. Expression of neurotrophic factor BDNF in MSC transfected with polyamidoamine dendrimers was also by more than 10 times higher.
Subject(s)
Dendrimers/chemistry , Mesenchymal Stem Cells/metabolism , Neurons/metabolism , Polyamines/chemistry , Transfection/methods , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , Gene Expression , Genes, Reporter , Green Fluorescent Proteins , Humans , Mesenchymal Stem Cells/cytology , Neurons/cytology , Plasmids/genetics , Polyelectrolytes , Polyethyleneimine/chemistry , TransgenesABSTRACT
Research concerning new targeting delivery systems for pharmacologically active molecules and genetic material is of great importance. The aim of the present study was to investigate the potential of fourth generation (P4) cationic phosphorus-containing dendrimers to bind fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS), anti-neoplastic drug cisplatin, anti-HIV siRNA siP24 and its capability to deliver green fluorescent protein gene (pGFP) into cells. The interaction between P4 and ANS (as the model drug) was investigated. The binding constant and the number of binding centers per one molecule of P4 were determined. In addition, the dendriplex between P4 and anti-HIV siRNA siP24 was characterized using circular dichroism, fluorescence polarization and zeta-potential methods; the average hydrodynamic diameter of the dendriplex was calculated using zeta-size measurements. The efficiency of transfection of pGFP using P4 was determined in HEK293 cells and human mesenchymal stem cells, and the cytotoxicity of the P4-pGFP dendriplex was studied. Furthermore, enhancement of the toxic action of the anti-neoplastic drug cisplatin by P4 dendrimers was estimated. Based on the results, the fourth generation cationic phosphorus-containing dendrimers seem to be a good drug and gene delivery carrier candidate.
ABSTRACT
Dendrimers are a new class of nanocomposite materials. They are branching polymers whose structure is formed by monomeric subunit branches diverging to all sides from a central nucleus. The type of nucleus, attached monomers, and functional groups can be chosen during synthesis, which produces dendrimers of definite size, shape, density, polarity, branch mobility, and solubility. This review deals with problems of dendrimer molecular structures and capability of in vitro, in vivo, ex vivo, and in situ transfection of genetic material. Advantages and shortcomings of different types of dendrimers in this respect are discussed.
Subject(s)
Dendrimers/chemistry , Drug Carriers , Drug Delivery Systems , Solubility , Transfection , Dendrimers/administration & dosage , Drug Compounding , Drug Design , Genetic Vectors , Molecular Structure , Nanostructures , Nanotechnology , Particle Size , Structure-Activity Relationship , Technology, PharmaceuticalABSTRACT
The analysis of binding between cationic PAMAM G5 dendrimer and anionic fluorescent probe using fluorescence and equilibrium dialysis has been made. It was found that at low concentrations of ANS the double fluorimetric titration technique can be successfully used for quantitative analysis of binding of ANS to dendrimer. Based on fluorescence and dialysis data the constants of binding and the number of binding centers were calculated for binding of ANS to PAMAM G5 dendrimer: K(b) is approx. (0.5-1)x10(5)M(-1) and n is (0.5-0.7).
Subject(s)
Anilino Naphthalenesulfonates/chemistry , Fluorescent Dyes/chemistry , Polyamines/chemistry , Algorithms , Dendrimers , Dialysis , Fluorescence , Spectrometry, FluorescenceABSTRACT
A series of new, low molecular mass, lysine-based peptide dendrimers with varying distribution of cationic and aromatic groups in the structure were synthesized. They expressed antimicrobial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria as well as against fungal pathogens (Candida albicans). Their cytotoxic, haematotoxic, and genotoxic effects were studied. It appears that degree of branching and steric distribution and types of hydrophobic (aromatic) groups and cationic centres are important components of dendrimeric structure and influence both antimicrobial potency and toxicity. Such 3D structure of our dendrimers mimics that of the natural antimicrobial peptides and can be achieved by application of dendrimer chemistry.
Subject(s)
Anti-Infective Agents/pharmacology , Dendrimers/pharmacology , Oligopeptides/pharmacology , Animals , Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/toxicity , Candida albicans/drug effects , Candida albicans/growth & development , Cell Line , Cell Survival/drug effects , Cricetinae , DNA/chemistry , Dendrimers/chemical synthesis , Dendrimers/toxicity , Escherichia coli/drug effects , Escherichia coli/growth & development , Hemolysis/drug effects , Humans , Inhibitory Concentration 50 , Lysine/chemistry , Microbial Sensitivity Tests , Molecular Weight , Oligopeptides/chemical synthesis , Oligopeptides/toxicity , Protein Conformation , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & developmentABSTRACT
The effect of polyamidoamine (PAMAM) dendrimers on activity and fluorescence of pure acetylcholinesterase (EC 3.1.1.7.) was studied. It has been shown that all dendrimers studied decreased the enzymatic activity of acetylcholinesterase. This effect depended on the type of dendrimers. The data on the intrinsic fluorescence have shown that the dendrimers changed acetylcholinesterase conformation and the strongest effect was induced by PAMAM G3.5 dendrimer.
Subject(s)
Acetylcholinesterase/chemistry , Acetylcholinesterase/metabolism , Dendrimers/chemistry , Dendrimers/pharmacology , Protein Conformation/drug effects , Spectrometry, FluorescenceABSTRACT
The effect of PAMAM G3.5, PAMAM G4 and PAMAM-OH G4 dendrimers on human and bovine serum albumins has been studied by fluorescence spectroscopy at different pH and ionic strength. It has been shown that the interactions between dendrimers and proteins depend on pH and the efficiency of interactions can be regulated by changing pH. The maximal pH dependence was observed for interactions between albumins and PAMAM G4 dendrimer. At physiological pH all dendrimers affect proteins in the maximum degree. Dendrimers had no effect on N-F and N-B transitions of albumins. The effect of dendrimers on HSA was smaller than for BSA. The increase of NaCl concentration led to a decrease of interactions between dendrimers and proteins.
Subject(s)
Polyamines/pharmacology , Serum Albumin/drug effects , Animals , Cattle , Dendrimers , Humans , Hydrogen-Ion Concentration , Osmolar Concentration , Polyamines/chemistry , Protein Conformation/drug effects , Serum Albumin/chemistry , Sodium Chloride , Spectrometry, FluorescenceABSTRACT
The interactions between three types of polyamidoamine dendrimers (with anionic, cationic, and neutral charge on a surface) and fluorescent dye 1-anilinonaphthalene-8-sulfonate (ANS) were studied. Double fluorimetric titration method was employed to estimate a binding constant and the number of binding centers. As fluorescent probes can serve as models of toxin molecules, dendrimers, and human serum albumin (HSA) abilities to bind ANS were compared. In the presence of HSA and dendrimers, ANS located both in HSA and in dendrimers, but the interactions between ANS and HSA were stronger.
Subject(s)
Fluorescent Dyes/chemistry , Polyamines/chemistry , Serum Albumin/chemistry , Anilino Naphthalenesulfonates/chemistry , Binding Sites , Dendrimers , Humans , In Vitro Techniques , Spectrometry, FluorescenceABSTRACT
Room temperature tryptophan phosphorescence (RTTP) of suspensions of human platelets was studied. RTTP spectra and decay kinetics of both intact platelets and those after short-term incubation with low concentrations of thrombin or trypsin (0.3-50 microg/ml) were investigated. Protease-induced changes in the RTTP lifetime of platelets were observed, and interpreted in terms of the slow internal dynamics of membrane protein modification. The functional role of membrane protein internal dynamics is discussed in the context of platelet aggregation and signal transduction processes.
