Dominika Wrobel

Modified PAMAM dendrimer with 4-carbomethoxypyrrolidone surface groups reveals negligible toxicity against three rodent cell-lines

UNLABELLED 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.

Interactions of phosphorus-containing dendrimers with liposomes.

The influence of cationic phosphorus-containing dendrimers generation 3 and 4 on model DMPC or DPPC lipid membranes was studied. Measurements of fluorescence anisotropy and differential scanning calorimetry (DSC) were applied to assess changes in lipid bilayer parameters, including fluidity, anisotropy, and phase-transition temperature. Interaction with both hydrophobic and hydrophilic regions of the bilayer was followed by these methods. Dendrimers of both generations influence lipid bilayers by decreasing membrane fluidity. The results suggest that dendrimers can interact both with the hydrophobic part and the polar head-group region of the phospholipid bilayer. Higher generation dendrimers interact more strongly with model membranes, and the concentration, as well as the generation, is of similar importance.

INTERACTION OF CATIONIC PHOSPHORUS DENDRIMERS (CPD) WITH CHARGED AND NEUTRAL LIPID MEMBRANES

Despite the rapid development of modern pharmaceutics, delivery of drugs to sites of action is not always effective. The research on new targeting delivery systems of pharmacologically active molecules is of great importance. Surface properties such as surface charge of drug delivery particles frequently define their pharmacokinetic profile; hence the efficiency of drugs can be increased by application of nanoparticles having appropriate surface properties. The aim of the present work was to study the interactions of cationic phosphorus-containing dendrimers (CPD) with model lipid membranes with no charge or bearing surface charge. The interactions of two generations of phosphorus dendrimers on the thermotropic behavior of model lipid membranes composed of DMPC (uncharged) or DMPC/DPPG (negatively charged) were studied using differential scanning calorimetry (DSC). The results of this study showed that CPDs can alter the thermotropic behaviour of the bilayer by reducing the cooperativity of phospholipids and this effect strongly depends on membrane surface charge. The information resulting from this study may be applied to the rational design of new drug carriers combining liposomal and dendrimeric technology.

siRNA carriers based on carbosilane dendrimers affect zeta potential and size of phospholipid vesicles.

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.

Effect of phosphorus dendrimers on DMPC lipid membranes

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.

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes.

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.

Direct immobilization of biotin on the micro-patterned PEN foil treated by excimer laser

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.

Biocompatible Size-Defined Dendrimer–Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications

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.

Interaction of cationic carbosilane dendrimers and their complexes with siRNA with erythrocytes and red blood cell ghosts.

Abstract 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.

Phosphonium carbosilane dendrimers for biomedical applications – synthesis, characterization and cytotoxicity evaluation

We report the synthesis and cytotoxicity evaluation of a completely new class of cationic carbosilane dendrimers functionalized with several different phosphonium peripheral groups and an ammonium functionalised one as a reference. The carbosilane dendrimers with NMe3, PMe3, P(Et2)2(CH2)3OH, PBu3, P(C6H4-OMe)3 and P(Ph)3 peripheral substituents were synthesized, thoroughly characterized and modelled by computer simulations. The cytotoxicities of the dendrimers were investigated in vitro on three model cell lines (B14, BRL and NRK cells) by MTT and CV assay methods. Generally, the cytotoxicities of PMe3 carbosilane dendrimers were similar or slightly lower when compared with NMe3 dendrimers. The substitution of methyl groups in PMe3 carbosilane dendrimers with more hydrophobic and bulky alkyl substituents (PBu3 and P(Et2)2(CH2)3OH dendrimers) resulted in an increase of cytotoxicity. The P(C6H4-OMe)3 dendrimer showed exceptionally low cytotoxicity across all cell lines or assay methods used. Generally, phosphonium carbosilane dendrimers could represent a valuable alternative to ammonium ones in gene therapy applications due to comparable or lower cytotoxicities, the presence of positive charge for nucleic acid electrostatic binding and in the cases of P(C6H4-OMe)3 and P(Ph)3 dendrimers high potential of mitochondrial targeting.