ABSTRACT
INTRODUCTION: Authors of numerous publications have proved the therapeutic effect of laser irradiation on biological material, but the mechanisms at cellular and subcellular level are not yet well understood. OBJECTIVE: The aim of this study was to assess the effect of laser radiation emitted by the MLS M1 system (Multiwave Locked System) at two wavelengths (808 nm continuous and 905 nm pulsed) on the stability and fluidity of liposomes with a lipid composition similar to that of human erythrocyte membrane or made of phosphatidylocholine. MATERIAL AND METHODS: Liposomes were exposed to low-energy laser radiation at surface densities 195 mW/cm2 (frequency 1,000 Hz) and 230 mW/cm2 (frequency 2,000 Hz). Different doses of radiation energy in the range 0-15 J were applied. The surface energy density was within the range 0.46 - 4.9 J/cm 2. RESULTS: The fluidity and stability of liposomes subjected to such irradiation changed depending on the parameters of radiation used. CONCLUSIONS: Since MLS M1 laser radiation, depending on the parameters used, affects fluidity and stability of liposomes with the lipid content similar to erythrocyte membrane, it may also cause structural and functional changes in cell membranes.
Subject(s)
Cell Membrane/radiation effects , Membrane Lipids/radiation effects , Erythrocytes/radiation effects , Humans , Lasers , Liposomes/chemistry , Liposomes/radiation effects , Low-Level Light Therapy/instrumentation , Membrane Fluidity/radiation effectsABSTRACT
We have studied the effect of polyamidoamine (PAMAM) dendrimers of various generations on the thermal stability and fibrillation of human insulin. Thermostability of human insulin used differential scanning calorimetry (DSC), which showed two phase-transitions for insulin at 60 and 82°C. After adding dendrimers at 0.6 µmol/l, the first peaks disappeared and the second peaks were higher. We posited that, in the presence of dendrimers, the dimers in the solution were transformed into hexamers. The effect of dendrimers on insulin fibrillation was monitored by measuring ThT fluorescence, and visualization of insulin fibrils by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The effect of PAMAM dendrimers on insulin fibrillation was strongly dependent on the dendrimers generation and dendrimer:protein ratio.
Subject(s)
Amyloid/chemistry , Dendrimers/chemistry , Insulin/chemistry , Protein Aggregates , Amyloid/ultrastructure , Calorimetry, Differential Scanning , Humans , Microscopy, Atomic Force , Polymers/chemistry , Protein Binding , Protein StabilityABSTRACT
This paper examines a perspective on the use of newly engineered nanomaterials as effective and safe carriers of genes for the therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus and carbosilane) were complexed with anticancer siRNA and their biophysical properties of the dendriplexes analyzed. The potential of the dendrimers as nanocarriers for anticancer siBcl-xl, siBcl-2, siMcl-1 siRNAs and a siScrambled sequence was explored. Dendrimer/siRNA complexes were characterized by methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. Some of the experiments were done with heparin to check if siRNA can be easily disassociated from the complexes, and whether released siRNA maintains its structure after interaction with the dendrimer. The results indicate that siRNAs form complexes with all the dendrimers tested. Oligoribonucleotide duplexes can be released from dendriplexes after heparin treatment and the structure of siRNA is maintained in the case of PAMAM or carbosilane dendrimers. The dendrimers were also effective in protecting siRNA from RNase A activity. The selection of the best siRNA carrier will be made based on cell culture studies (Part B).
Subject(s)
Dendrimers/chemistry , Phosphorus/chemistry , RNA, Small Interfering/chemistry , RNAi Therapeutics/methods , Silanes/chemistry , Transfection , Circular Dichroism , Electrophoresis, Agar Gel , Gene Expression Regulation, Neoplastic , Heparin/chemistry , Humans , Light , Microscopy, Electron, Transmission , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/therapy , Nucleic Acid Conformation , Particle Size , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Scattering, Radiation , Spectrometry, Fluorescence , Transfection/methods , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
Dendrimers' action on proteins and peptides has a dual and controversial character. On one hand, they dissolve prion protein and amyloid fibrils aggregates, which are otherwise only soluble in solvents containing both detergents and high denaturant concentrations. On the other hand they are able to destabilize proteins in generation dependent manner. In present work we estimated the influence of small concentrations (up to 1.4 µg/ml) of cationic, neutral and anionic poly(amidoamine) dendrimers of 3rd and 4th generations on dithiotreitol induced aggregation of insulin. It was found that cationic dendrimers decreased the insulin aggregation, while anionic and neutral ones did not. At the same time, destabilizing effect of dendrimers on insulin structure was not observed. The conclusion was made that small concentrations of dendrimers can be applied to prevent or decrease the formation of misfolded structures of protein.
Subject(s)
Dendrimers/chemistry , Insulin/chemistry , Animals , Cattle , Models, MolecularABSTRACT
The biostimulating activity of low level laser radiation of various wavelengths and energy doses is widely documented in the literature, but the mechanisms of the intracellular reactions involved are not precisely known. The aim of this paper is to evaluate the influence of low level laser radiation from an multiwave locked system (MLS) of two wavelengths (wavelength = 808 nm in continuous emission and 905 nm in pulsed emission) on the human erythrocyte membrane and on the secondary structure of human serum albumin (HSA). Human erythrocytes membranes and HSA were irradiated with laser light of low intensity with surface energy density ranging from 0.46 to 4.9 J cm(-2) and surface energy power density 195 mW cm(-2) (1,000 Hz) and 230 mW cm(-2) (2,000 Hz). Structural and functional changes in the erythrocyte membrane were characterized by its fluidity, while changes in the protein were monitored by its secondary structure. Dose-dependent changes in erythrocyte membrane fluidity were induced by near-infrared laser radiation. Slight changes in the secondary structure of HSA were also noted. MLS laser radiation influences the structure and function of the human erythrocyte membrane resulting in a change in fluidity.
Subject(s)
Erythrocyte Membrane/radiation effects , Membrane Fluidity/radiation effects , Protein Structure, Secondary/radiation effects , Serum Albumin/radiation effects , Dose-Response Relationship, Radiation , Humans , Lasers , Light , Membrane Proteins/radiation effects , Serum Albumin/ultrastructureABSTRACT
OBJECTIVE: In this study the mechanism of interactions between polyamidoamine (PAMAM) dendrimers and bovine insulin was examined. The insulin is a 51 amino acid peptide-hormone involved in the homeostasis of blood glucose levels. This molecule consists of two chains - A and B - linked by two disulphide bridges. As insulin contains four tyrosine residues it was possible to evaluate dendrimers effect on protein conformation by measuring changes in the fluorescence spectra of insulin after addition of dendrimers or classical quenchers. METHODS: PAMAM dendrimers are based on ethylenediamine core and branched units which are built from methyl acrylate and ethylenediamine. PAMAM dendrimers with different surface groups (-COOH, -NH2, -OH) were used. Their size is comparable to insulin. RESULTS: The experiments show that interactions exist between PAMAM dendrimers and insulin. It was found that these interactions depend on a kind of dendrimer surface groups. CONCLUSIONS: It is very likely that interactions are of electrostatic nature and cause insulin conformational changes.
Subject(s)
Dendrimers/chemistry , Dendrimers/metabolism , Insulin/chemistry , Insulin/metabolism , Animals , Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Cattle , Drug Interactions , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/metabolism , Protein Binding , Protein Conformation , Spectrometry, Fluorescence , Tyrosine/chemistry , Tyrosine/metabolismABSTRACT
A new mechanism of gene expression inhibition has been discovered as RNA interference, in which the ability of double-stranded RNA to stimulate specific degradation of an mRNA target with a complementary sequence to one of the double-stranded RNA strands. Water-soluble carbosilane dendrimers containing ammonium or amine groups at their periphery are biocompatible molecules that may be good candidates as non-viral carriers of small interfering RNA. In studying the formation of complex between anti-HIV siRNA siGAG1 and carbosilane dendrimers NN8 and NN16 by circular dichroism, fluorescence, and zeta-potential, the size of nanoparticles formed has been estimated by dynamic light scattering. At a charge ratio of 1:3-4 (siGAG1:dendrimer), the dendriplexes formed were in the size range of 250-350 nm.
