RESUMEN

Amphiphilic dendrons represent a relatively novel class of molecules which may show many unique properties suitable for applications in a field of molecular biology and nanomedicine. They were frequently studied as platforms suitable for drug delivery systems as were, e.g. polymersomes or hybrid lipid-polymer nanoparticles. Recently, natural extracellular lipid vesicles (EVs), called exosomes (EXs), were shown to be a promising candidate in drug delivery applications. Formation of hybrid exosome-dendron nanovesicles could bring benefits in their simple conjugation with selective targeting moieties. Unfortunately, the complex architecture of biological membranes, EXs included, makes obstacles in elucidating the important parameters and mechanisms of interaction with the artificial amphiphilic structures. The aim of the presented work was to study the interaction of two types of amphiphilic carbosilane dendritic structures (denoted as DDN-1 and DDN-2) suitable for further modification with streptavidin (DDN-1) or using click-chemistry approach (DDN-2), with selected neutral and negatively charged lipid model membranes, partially mimicking the basic properties of natural EXs biomembranes. To meet the goal, a number of biophysical methods were used for determination of the degree and mechanisms of the interaction. The results showed that the strength of interactions of amphiphilic dendrons with liposomes was related with surface charge of liposomes. Several steps of interactions were disclosed. The initialization step was mainly coupled with amphiphilic dendrons - liposomes surface interaction resulting in destabilization of large self-assembled amphiphilic dendrons structures. Such destabilization was more significant with liposomes of higher negative charge. With increasing concentration of amphiphilic dendrons in a solution the interactions were taking place also in the hydrophobic part of bilayer. Further increase of nanoparticle concentration resulted in a gradual dendritic cluster formation in a lipid bilayer structure. Due to high affinity of amphiphilic dendrons to model lipid bilayers the conclusion can be drawn that they represent promising platforms also for decoration of exosomes or other kinds of natural lipid vehicles. Such organized hybrid dendron-lipid biomembranes may be advantageous for their subsequent post-functionalization with small molecules, large biomacromolecules or polymers suitable for targeted drug-delivery or theranostic applications.

Asunto(s)

Dendrímeros , Liposomas , Silanos , Dendrímeros/síntesis química , Dendrímeros/química , Silanos/química , Liposomas/química , Potenciales de la Membrana , Anisotropía , Calorimetría , Nanopartículas/química

RESUMEN

Supramolecular structures, such as micelles, liposomes, polymerosomes or dendrimerosomes, are widely studied and used as drug delivery systems. The behavior of amphiphilic building blocks strongly depends on their spatial distribution and shape of polar and nonpolar component. This report is focused on the development of new versatile synthetic protocols for amphiphilic carbosilane dendrons (amp-CS-DDNs) capable of self-assembly to regular micelles and other supramolecular objects. The presented strategy enables the fine modification of amphiphilic structure in several ways and also enables the facile connection of a desired functionality. DLS experiments demonstrated correlations between structural parameters of amp-CS-DDNs and the size of formed nanoparticles. For detailed information about the organization and spatial distribution of amp-CS-DDNs assemblies, computer simulation models were studied by using molecular dynamics in explicit water.

Asunto(s)

Antracenos/química , Simulación por Computador , Interacciones Hidrofóbicas e Hidrofílicas , Micelas , Nanopartículas/química , Silanos/química , Agua/química

RESUMEN

The complexity of drug delivery mechanisms calls for the development of new transport system designs. Here, we report a robust synthetic procedure toward stable glycodendrimer (glyco-DDM) series bearing glucose, galactose, and oligo(ethylene glycol)-modified galactose peripheral units. In vitro cytotoxicity assays showed exceptional biocompatibility of the glyco-DDMs. To demonstrate applicability in drug delivery, the anticancer agent doxorubicin (DOX) was encapsulated in the glyco-DDM structure. The anticancer activity of the resulting glyco-DDM/DOX complexes was evaluated on the noncancerous (BJ) and cancerous (MCF-7 and A2780) cell lines, revealing their promising generation- and concentration-dependent effect. The glyco-DDM/DOX complexes show gradual and pH-dependent DOX release profiles. Fluorescence spectra elucidated the encapsulation process. Confocal fluorescence microscopy demonstrated preferential cancer cell internalization of the glyco-DDM/DOX complexes. The conclusions were supported by computer modeling. Overall, our results are consistent with the assumption that novel glyco-DDMs and their drug complexes are very promising in drug delivery and related applications.

Asunto(s)

Antineoplásicos , Neoplasias Ováricas , Antineoplásicos/farmacología , Línea Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacología , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Liberación de Fármacos , Femenino , Humanos , Polietilenglicoles/química , Silanos

RESUMEN

Glycodendrimers are a novel group of dendrimers (DDMs) characterized by surface modifications with various types of glycosides. It has been shown previously that such modifications significantly decrease the cytotoxicity of DDMs. Here, we present an investigation of glucose-modified carbosilane DDMs (first-third-generation, DDM1-3Glu) interactions with two models of biological structures: lipid membranes (liposomes) and serum protein (human serum albumin, HSA). The changes in lipid membrane fluidity with increasing concentration of DDMs was monitored by spectrofluorimetry and calorimetry methods. The influence of glycodendrimers on serum protein was investigated by monitoring changes in protein fluorescence intensity (fluorescence quenching) and as protein secondary structure alterations by circular dichroism spectrometry. Generally, all generations of DDMGlu induced a decrease of membrane fluidity and interacted weakly with HSA. Interestingly, in contrast to other dendritic type polymers, the extent of the DDM interaction with both biological models was not related to DDM generation. The most significant interaction with protein was shown in the case of DDM2Glu, whereas DDM1Glu induced the highest number of changes in membrane fluidity. In conclusion, our results suggest that the flexibility of a DDM molecule, as well as its typical structure (hydrophobic interior and hydrophilic surface) along with the formation of larger aggregates of DDM2-3Glu, significantly affect the type and extent of interaction with biological structures.

Asunto(s)

Dendrímeros/farmacología , Portadores de Fármacos/farmacología , Glucosa/farmacología , Albúmina Sérica Humana/metabolismo , Silanos/farmacología , Antineoplásicos/administración & dosificación , Dicroismo Circular , Dendrímeros/química , Portadores de Fármacos/química , Glucosa/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas , Fluidez de la Membrana/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Silanos/química , Espectrometría de Fluorescencia

RESUMEN

Non-viral gene delivery vectors studied in the gene therapy applications are often designed with the cationic nitrogen containing groups necessary for binding and cell release of nucleic acids. Disadvantage is a relatively high toxicity which restricts the in vivo use of such nanoparticles. Here we show, that the 3rd generation carbosilane dendrimers possessing (trimethyl)phosphonium (PMe3) groups on their periphery were able to effectively deliver the functional siRNA into the cells (B14, Cricetulus griseus), release it into the cytosol and finally to achieve up to 40% gene silencing of targeted gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) with the comparable or, in some cases, even better effectivity as their ammonium counterparts. Moreover, such cationic dendrimers show relatively low in vivo toxicity as compared to their ammonium analogues when analyzed by standard fish embryo test (FET) on Danio rerio in vivo model, with LD50 = 6.26 µM after 48 h of incubation. This is more than 10-fold improvement as compared to published values for various other types of cationic dendrimers. We discuss the potential of further increase of the transfection efficiency, endosomal escape and decrease of toxicity of such non-viral vectors, based on the systematic screening of different types of substituents on central phosphonium atom.

Asunto(s)

Dendrímeros/toxicidad , Compuestos Organofosforados/toxicidad , ARN Interferente Pequeño/administración & dosificación , Silanos/toxicidad , Transfección/métodos , Animales , Línea Celular , Cricetulus , Dendrímeros/administración & dosificación , Embrión no Mamífero , Silenciador del Gen , Dosificación Letal Mediana , Compuestos Organofosforados/administración & dosificación , Silanos/administración & dosificación , Pez Cebra

RESUMEN

Glycodendrimers (Glyco-DDMs) represent a rapidly growing class of nanoparticles with promising properties for biomedical applications but concerns regarding the impact on human health and environment are still justified. Here we report, for the first time, the comparative study of in vivo developmental toxicity of carbosilane Glyco-DDMs and their cytotoxicity in vitro. Carbosilane Glyco-DDMs (generation 1-3) containing 4, 8, and 16 ß-d-glucopyranosyl units at the periphery (DDM1Glu, DDM2Glu, and DDM3Glu) were synthesized and characterized by 1H, 13C and 29Si NMR, mass spectrometry, dynamic light scattering, atomic force microscopy (AFM), and computer modeling. In vitro cytotoxicity assay (MTT) of DDM1-3Glu was performed on three different rodent cell lines (Cricetulus griseus) - B14 (ATCC, CCL-14.1), BRL 3A (ATCC, CRL-1442), and NRK 52E (ATCC, CRL-1571). Overall, very low cytotoxicity was observed with calculated IC50 in mM range with slight difference between each cell line and DDM generation investigated. Modified fish embryo test (FET) was further used for DDM3Glu developmental toxicity testing on zebrafish (Danio rerio) embryos. While seemingly harmless to intact embryos, adverse effects of DDMs on the embryonic development become evident after chorion removal (LD50=2.78 µM at 96 hpe). We summarized that the modified FET test showed a two to three orders of magnitude difference between the in vitro cytotoxicity and in vivo developmental toxicity of DDM3Glu. While, in general, the Glyco-DDMs show great promises as efficient vectors in targeted drug delivery or as therapeutic molecules itself, we suggest that their developmental toxicity should be thoroughly investigated to exclude safety risks associated with their potential biomedical use.

Asunto(s)

Dendrímeros/toxicidad , Embrión no Mamífero/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Silanos/toxicidad , Teratógenos/toxicidad , Pez Cebra , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetulus , Dendrímeros/química , Glucosa/química , Humanos , Dosificación Letal Mediana , Modelos Moleculares , Silanos/química , Propiedades de Superficie , Teratógenos/química , Pruebas de Toxicidad , Pez Cebra/embriología

RESUMEN

The influence of three generations of five different phosphonium carbosilane dendrimers and one ammonium carbosilane dendrimer as a reference (PMe3, PBu3, P(Et2)2(CH2)3OH, PPh3, P(MeOPh)3 and NMe3, peripheral functional groups) on dimyristoylphosphatidylcholine (DMPC) or a lipid mixture dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) of liposomes was studied by fluorescence polarization measurements and differential scanning calorimetry. All types of dendrimers interacted with neutral as well as negatively charged liposomes, but the strength and observed influence were different. Concentration, type of peripheral functional group modification and dendrimer generation were the main factors influencing the interaction. Generally, weak interactions as well as destabilization of the lipid membranes at low concentrations, regardless of liposome type, were observed in the case of DmPMe3, DmNMe3, DmPBu3 and DmP(Et2)2(CH2)3OH. Dendrimers with PPh3 and P(MeOPh)3 peripheral functional groups interacted much more strongly and increased the rigidity of liposomes. Electrostatic interactions, the hydrophobicity of substituents and charge shielding on the peripheral phosphonium group are important factors in the interaction. We suggest that, among the other types of dendrimers, the dendrimer with the P(MeOPh)3 peripheral functional group is a highly promising candidate for the design of a drug delivery system due to its positive charge, efficient interaction with lipidic membranes and low cytotoxicity.

Asunto(s)

Dendrímeros/química , Glicerofosfolípidos/química , Membrana Dobles de Lípidos/química , Liposomas/química , Silanos/química , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Tamaño de la Partícula , Electricidad Estática , Relación Estructura-Actividad , Propiedades de Superficie

RESUMEN

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.

Asunto(s)

Membrana Celular/efectos de la radiación , Lípidos de la Membrana/efectos de la radiación , Eritrocitos/efectos de la radiación , Humanos , Rayos Láser , Liposomas/química , Liposomas/efectos de la radiación , Terapia por Luz de Baja Intensidad/instrumentación , Fluidez de la Membrana/efectos de la radiación

RESUMEN

Regardless of the route of administration, some or all of a therapeutic agent will appear in the blood stream, where it can act on blood cells and other components of the plasma. Recently we have shown that poly(ethylene imines) (PEIs) which interact with plasma proteins are taken up into erythrocyte membranes. These observations led us to investigate the interactions between maltose functionalized hyperbranched PEIs (PEI-Mal) and plasma proteins. Two model proteins were chosen - human serum albumin (HSA) (albumins constitute ∼60% of all plasma proteins), and lysozyme. HSA is a negatively charged 66kDa protein at neutral pH, whereas lysozyme is a positively charged 14kDa protein. Fluorescence quenching and changes in the conformation of the amino acid tryptophan, diameter and zeta potential of proteins were investigated to evaluate the interaction of PEI-Mal with proteins. PEI-Mal interacts with both types of proteins. The strength of dendritic glycopolymer interactions was generally weak, especially with lysozyme. Greater changes were found with HSA, mainly triggered by hydrogen bonds and the electrostatic interaction properties of dendritic glycopolymers. Moreover, the structure and the size of PEI-Mal macromolecules affected these interactions; larger macromolecules with more sugar groups (95% maltose units) interacted more strongly with proteins than smaller ones with lower sugar modification (33% maltose units). Due to (i) the proven overall low toxicity of sugar-modified PEIs and, (ii) their ability to interact preferentially through hydrogen bonds with proteins of human plasma or possibly with other interesting protein targets, PEI-Mal is a good candidate for creating therapeutic nanoparticles in the fast developing field of nanomedicine.

Asunto(s)

Proteínas Sanguíneas/química , Iminas/química , Maltosa/química , Muramidasa/química , Polietilenos/química , Polímeros/química , Albúmina Sérica/química , Dendrímeros/química , Fluorescencia , Humanos

RESUMEN

For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures.

Asunto(s)

Dendrímeros/síntesis química , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Albúmina Sérica/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biotina/química , Biotinilación , Línea Celular , Dendrímeros/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Ligandos , Modelos Moleculares , Nanopartículas/ultraestructura , Poliaminas/química , Polietilenglicoles/química , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Estreptavidina/química , Streptomyces/genética , Streptomyces/metabolismo

RESUMEN

Interactions between maltose functionalized hyperbranched poly(ethylene imine)s (95% maltose decoration denoted as Mal-PEI A; 33% maltose decoration denoted as Mal-PEI B) and red blood cells (RBCs) and between red blood cell membranes were investigated. We monitored the degree of hemolysis, the change in cell shape, the influence of polymers on the fluidity of the cell membrane and some cell membrane enzymes to determine their possible cytotoxic impact on them. To observe the extent of hemolysis, the RBCs were incubated with different concentrations of Mal-PEIs. The first significant lysis of RBCs was observed after 6h of incubation. Prolongation of the incubation time increased the number of ruptured cells. Moreover, we observed that Mal-PEI B was more hemolytic than Mal-PEI A in buffer solution. In contrast, an incubation of RBCs with Mal-PEIs in human plasma significantly decreased the hemolytic process and showed higher hemolytic property of Mal-PEI A compared to Mal-PEI B. Also several changes in the shape of the RBCs occurred after incubation with Mal-PEIs. Some of the erythrocytes shrank (echinocytes), but their morphology generally remained unchanged during the incubation. As shown by fluorescence experiments, both polymers induced the increase of fluidity of RBCs membranes. In summary, both types of hyperbranched poly(ethylene imine)s were practically non-hemolytic even at high polymer concentrations. Mal-PEI B was slightly more noxious than the Mal-PEI A in a buffer solution, while in blood plasma, the situation was opposite. Decrease of Na+/K+ ATPase and total ATPase enzymes activity was related with molecule size and number of maltose groups on the surface of molecule. The low hemolytic properties only observed at higher concentration (100µM and 400µM) indicated that Mal-PEIs are promising macromolecules in the area of drug delivery systems.

Asunto(s)

Membrana Eritrocítica/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Iminas/farmacología , Maltosa/farmacología , Polietilenos/farmacología , Adenosina Trifosfatasas/metabolismo , Humanos , Iminas/química , Maltosa/química , Nanopartículas , Polietilenos/química , Espectrometría de Fluorescencia

RESUMEN

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers - OS) or completely (dense shell glycodendrimers - DS) modified with maltose residues. As a model membrane, two types of 100nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D2O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.

Asunto(s)

Dendrímeros/química , Membrana Dobles de Lípidos/química , Maltosa/química , Lípidos de la Membrana/química , Polipropilenos/química , Rastreo Diferencial de Calorimetría , Dendrímeros/metabolismo , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Difenilhexatrieno/química , Polarización de Fluorescencia , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/metabolismo , Liposomas/química , Liposomas/metabolismo , Espectroscopía de Resonancia Magnética , Maltosa/metabolismo , Fluidez de la Membrana , Lípidos de la Membrana/metabolismo , Fosfatidilgliceroles/química , Fosfatidilgliceroles/metabolismo , Polipropilenos/metabolismo , Electricidad Estática

RESUMEN

Polymers with functionalized surfaces have attracted a lot of attention in the last few years. Due to the progress in the techniques of polymer micro-patterning, miniaturized bioanalytical assays and biocompatible devices can be developed. In the presented work, we performed surface modification of polyethylene naphthalate (PEN) foil by an excimer laser beam through a photolithographic contact mask. The aim was to fabricate micro-patterned areas with surface functional groups available for localized covalent immobilization of biotin. It was found out that depending on the properties of the laser scans, a polymer surface exhibits different degrees of modification and as a consequence, different degrees of surface biotinylation can be achieved. Several affinity tests with optical detection of fluorescently labeled streptavidin were successfully performed on biotinylated micro-patterns of a PEN foil. The polymer surface properties were also evaluated by electrokinetic analysis, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The results have shown that PEN foils can be considered suitable substrates for construction of micro-patterned bioanalytical affinity assays.

Asunto(s)

Biotina/química , Naftalenos/química , Polietilenos/química , Estreptavidina/química , Biotinilación , Dispositivos Laboratorio en un Chip , Láseres de Excímeros , Microtecnología , Naftalenos/efectos de la radiación , Procesos Fotoquímicos , Polietilenos/efectos de la radiación , Propiedades de Superficie

RESUMEN

The therapeutic effects of low-power laser radiation of different wavelengths and light doses are well known, but the biochemical mechanism of the interaction of laser light with living cells is not fully understood. We have investigated the effect of MLS (Multiwave Locked System) laser near-infrared irradiation on cell membrane structure, functional properties, and free radical generation using human red blood cells and breast cancer MCF-4 cells. The cells were irradiated with low-intensity MLS near-infrared (simultaneously 808 nm, continuous emission and 905 nm, pulse emission, pulse-wave frequency, 1,000 or 2,000 Hz) laser light at light doses from 0 to 15 J (average power density 212.5 mW/cm(2), spot size was 3.18 cm(2)) at 22 °C, the activity membrane bound acetylcholinesterase, cell stability, anti-oxidative activity, and free radical generation were the parameters used in characterizing the structural and functional changes of the cell. Near-infrared low-intensity laser radiation changed the acetylcholinesterase activity of the red blood cell membrane in a dose-dependent manner: There was a considerable increase of maximal enzymatic rate and Michaelis constant due to changes in the membrane structure. Integral parameters such as erythrocyte stability, membrane lipid peroxidation, or methemoglobin levels remained unchanged. Anti-oxidative capacity of the red blood cells increased after MLS laser irradiation. This irradiation induced a time-dependent increase in free radical generation in MCF-4 cells. Low-intensity near-infrared MLS laser radiation induces free radical generation and changes enzymatic and anti-oxidative activities of cellular components. Free radical generation may be the mechanism of the biomodulative effect of laser radiation.

Asunto(s)

Estructuras de la Membrana Celular/efectos de la radiación , Radicales Libres/metabolismo , Rayos Infrarrojos , Rayos Láser , Acetilcolinesterasa/metabolismo , Línea Celular Tumoral , Eritrocitos/metabolismo , Eritrocitos/efectos de la radiación , Glutatión/metabolismo , Hemólisis/efectos de la radiación , Humanos , Metahemoglobina/metabolismo , Oxidación-Reducción/efectos de la radiación , Estrés Oxidativo/efectos de la radiación , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo

RESUMEN

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.

Asunto(s)

Membrana Eritrocítica/efectos de la radiación , Fluidez de la Membrana/efectos de la radiación , Estructura Secundaria de Proteína/efectos de la radiación , Albúmina Sérica/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Rayos Láser , Luz , Proteínas de la Membrana/efectos de la radiación , Albúmina Sérica/ultraestructura

RESUMEN

We have investigated the interactions between cationic NN16 and BDBR0011 carbosilane dendrimers with red blood cells or their cell membranes. The carbosilane dendrimers used possess 16 cationic functional groups. Both the dendrimers are made of water-stable carbon-silicon bonds, but NN16 possesses some oxygen-silicon bonds that are unstable in water. The nucleic acid used in the experiments was targeted against GAG-1 gene from the human immunodeficiency virus, HIV-1. By binding to the outer leaflet of the membrane, carbosilane dendrimers decreased the fluidity of the hydrophilic part of the membrane but increased the fluidity of the hydrophobic interior. They induced hemolysis, but did not change the morphology of the cells. Increasing concentrations of dendrimers induced erythrocyte aggregation. Binding of short interfering ribonucleic acid (siRNA) to a dendrimer molecule decreased the availability of cationic groups and diminished their cytotoxicity. siRNA-dendrimer complexes changed neither the fluidity of biological membranes nor caused cell hemolysis. Addition of dendriplexes to red blood cell suspension induced echinocyte formation.

Asunto(s)

Dendrímeros/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Hemólisis/efectos de los fármacos , Silanos/química , Cationes/química , Proliferación Celular , Dendrímeros/farmacología , Eritrocitos/efectos de los fármacos , Humanos , ARN Interferente Pequeño/genética , Silanos/farmacología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo

RESUMEN

Modification of the surface groups of dendrimers is one of the methods to improve their biocompatibility. This article presents results of experiments related to the toxicity of a modified polyamidoamine (PAMAM) dendrimer of the fourth generation with 4-carbomethoxypyrrolidone surface groups (PAMAM-pyrrolidone dendrimer). The cytotoxic activity of the dendrimer was tested on Chinese hamster fibroblasts (B14), embryonic mouse hippocampal cells (mHippoE-18) and rat liver derived cells (BRL-3A). The same cell lines were used to investigate the influence of pyrrolidone dendrimer on the mitochondrial membrane potential, intracellular ROS level and its ability to induce apoptosis or necrosis. The analyzed dendrimer showed only minor toxicity and no ability to induce apoptosis. The most important finding is the lack of influence of the PAMAM-pyrrolidone dendrimer on intracellular ROS level and mitochondrial membrane potential. FROM THE CLINICAL EDITOR: The authors demonstrate that pyrrolidone-functionalized PAMAM dendrimers have very low toxicity in the tested cell lines, as evidenced by no alteration of mitochondrial membrane potential and no increase of ROS production.

Asunto(s)

Dendrímeros/toxicidad , Pirrolidinonas/toxicidad , Animales , Línea Celular , Cricetinae , Cricetulus , Ratones , Ratas

RESUMEN

One of the major limitations in gene therapy is an inability of naked siRNA to passively diffuse through negatively charged cell membranes. Therefore, the siRNA transport into a cell requires efficient carriers. In this work we analyzed the charge-dependent interaction of the complexes of cationic carbosilane dendrimers (CBD) and anti-HIV siRNA (dendriplexes) with the model membranes - large unilamellar vesicles (LUV). We used the second generation of branched with CBD carbon-silicon bonds (CBD-CS) which are water-stable and that of oxygen-silicon bonds (CBD-OS) which are slowly hydrolyzed in aqueous solutions. The LUVs were composed of zwitterionic dimyristoylphosphatidylcholine (DMPC), negatively charged dipalmitoylphosphatidylglycerol (DPPG) and their mixture (DMPC/DPPG, molar ratio 7:3). The interaction of dendriplexes with LUVs affected both zeta potential and size of the vesicles. The changes of these values were larger for the negatively charged LUV. CBD-CS resulted in the decrease of zeta potential values to more negative ones, whereas an opposite effect took place for CBD-OS suggesting a different kind of interaction between LUVs and the dendriplexes. The results indicate that both CBD-CS and CBD-OS can be used for transport of siRNA into the cells. However, CBD-CS are preferred due to a better stability in water and improved bioavailability of siRNA on their surface.

Asunto(s)

Fosfolípidos/química , ARN Interferente Pequeño/metabolismo , Silanos/química , Fármacos Anti-VIH/química , Biofisica/métodos , Carbono/química , Dendrímeros/química , Dimiristoilfosfatidilcolina/química , Relación Dosis-Respuesta a Droga , Terapia Genética/métodos , Hidrólisis , Lípidos/química , Liposomas/química , Modelos Químicos , Oxígeno/química , Tamaño de la Partícula , Fosfatidilgliceroles/química , Unión Proteica , Silicio/química , Espectrometría de Fluorescencia/métodos

RESUMEN

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.

Asunto(s)

Dendrímeros/química , Membrana Dobles de Lípidos/química , Silanos/química , Cationes/química , Interacciones Hidrofóbicas e Hidrofílicas , Fluidez de la Membrana , Electricidad Estática

RESUMEN

Large unilamellar liposomes and multilamellar vesicles consisting of DMPC interacted with cationic phosphorus-containing dendrimers CPDs G3 and G4. DSC and ζ-potential measurements have shown that liposomal-dendrimeric molecular recognition probably occurs due to the interaction between the complementary surface groups. Calorimetric studies indicate that the enthalpy of the transition of the lipids that interact with CPDs is dependent on the dendrimers generation. These results can be used in order to rationally design mixed modulatory liposomal locked-in dendrimeric, drug delivery nano systems.

Asunto(s)

Dendrímeros/química , Dimiristoilfosfatidilcolina/química , Liposomas/química , Fósforo/química , Rastreo Diferencial de Calorimetría , Cationes/química , Portadores de Fármacos/química , Tamaño de la Partícula , Termodinámica