Barbara Klajnert

Fluorescence studies on PAMAM dendrimers interactions with bovine serum albumin

Polyamidoamine (PAMAM) dendrimers (generation 3.5 and 4) interaction with bovine serum albumin (BSA) was studied. The intensity of intrinsic fluorescence of two tryptophan residues and a shift in wavelength of their emission maxima were chosen as indicators of protein conformational changes. It is shown that the generation 4 has a greater impact on spectral properties of serum albumin than generation 3.5.

Interactions between PAMAM dendrimers and bovine serum albumin.

Dendrimers are a new class of polymeric materials. They are globular, highly branched, monodisperse macromolecules. Due to their structure, dendrimers promise to be new, effective biomedical materials as oligonucleotide transfection agents and drug carriers. More information about biological properties of dendrimers is crucial for further investigation of dendrimers in therapeutic applications. In this study the mechanism of interactions between polyamidoamine (PAMAM) dendrimers and bovine serum albumin (BSA) was examined. PAMAM dendrimers are based on an ethylenediamine core and branched units are constructed from both methyl acrylate and ethylenediamine. We used three types of PAMAM dendrimers with different surface groups (-COOH, -NH(2), -OH). As BSA contains two tryptophan residues we were able to evaluate dendrimers influence on protein molecular conformation by measuring the changes in the fluorescence of BSA in the presence of dendrimers. Additionally experiments with a fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS) were carried out. The differential scanning calorimetry (DSC) was chosen to investigate impact on protein thermal stability upon the dendrimers. Our experiments showed that the extent of the interactions between BSA and dendrimers strongly depends on their surface groups and is the biggest for amino-terminated dendrimers.

The Influence of Densely Organized Maltose Shells on the Biological Properties of Poly(propylene imine) Dendrimers: New Effects Dependent on Hydrogen Bonding

Maltose-modified poly(propylene imine) (PPI) dendrimers were synthesized by reductive amination of unmodified second- to fifth-generation PPI dendrimers in the presence of excess maltose. The dendrimers were characterized by using (1)H NMR, (13)C NMR, and IR spectroscopies; laser-induced liquid beam ionization/desorption mass spectrometry; dynamic light scattering analyses; and polyelectrolyte titration. Their scaffolds have enhanced molecular rigidity and their outer spheres, at which two maltose units are bonded to the former primary amino groups on the surface, have hydrogen-bond-forming properties. Furthermore, the structural features reveal the presence of a dense shell. Experiments involving encapsulation (1-anilinonaphthalene-8-sulfonic acid) and biological properties (hemolysis and interactions with human serum albumin (HSA) and prion peptide 185-208) were performed to compare the modified with the unmodified dendrimers. These experiments gave the following results: 1) The modified dendrimers entrapped a low-molecular-weight fluorescent dye by means of a dendritic box effect, in contrast to the interfacial uptake characteristic of the unmodified PPI dendrimers. 2) Both low- and high-generation dendrimers containing maltose units showed markedly reduced toxicity. 3) The desirable features of bio-interactions depended on the generation of the dendrimer; they were retained after maltose substitution, but were now mainly governed by nonspecific hydrogen-bonding interactions involving the maltose units. The modified dendrimers interacted with HSA as strongly as the parent compounds and appeared to have potential use as antiprion agents. These improvements will initiate the development of the next platform of glycodendrimers in which apparently contrary properties can be combined, and this will enable, for example, therapeutic products such as more efficient and less toxic antiamyloid agents to be synthesized.

In vivo toxicity of poly(propyleneimine) dendrimers

Dendrimers are highly branched macromolecules with the potential to be used for biomedical applications. Several dendrimers are toxic owing to their positively charged surfaces. However, this toxicity can be reduced by coating these peripheral cationic groups with carbohydrate residues. In this study, the toxicity of three types of 4th generation poly (propyleneimine) dendrimers were investigated in vivo; uncoated (PPI-g4) dendrimers, and dendrimers in which 25% or 100% of surface amino groups were coated with maltotriose (PPI-g4-25%m or PPI-g4-100%m), were administered to Wistar rats. Body weight, food and water consumption, and urine excretion were monitored daily. Blood was collected to investigate biochemical and hematological parameters, and the general condition and behavior of the animals were analyzed. Unmodified PPI dendrimers caused changes in the behavior of rats, a decrease in food and water consumption, and lower body weight gain. In the case of PPI-g4 and PPI-g4-25%m dendrimers, disturbances in urine and hematological and biochemical profiles returned to normal during the recovery period. PPI-g4-100%m was harmless to rats. The PPI dendrimers demonstrated dose- and sugar-modification-degree dependent toxicity. A higher dose of uncoated PPI dendrimers caused toxicity, but surface modification almost completely abolished this toxic effect.

Dendrimers in gene transfection

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.

Influence of surface functionality of poly(propylene imine) dendrimers on protease resistance and propagation of the scrapie prion protein

Accumulation of PrP(Sc), an insoluble and protease-resistant pathogenic isoform of the cellular prion protein (PrP(C)), is a hallmark in prion diseases. Branched polyamines, including PPI (poly(propylene imine)) dendrimers, are able to remove protease resistant PrP(Sc) and abolish infectivity, offering possible applications for therapy. These dendrimer types are thought to act through their positively charged amino surface groups. In the present study, the molecular basis of the antiprion activity of dendrimers was further investigated, employing modified PPI dendrimers in which the positively charged amino surface groups were substituted with neutral carbohydrate units of maltose (mPPI) or maltotriose (m3PPI). Modification of surface groups greatly reduced the toxicity associated with unmodified PPI but did not abolish its antiprion activity, suggesting that the presence of cationic surface groups is not essential for dendrimer action. PPI and mPPI dendrimers of generation 5 were equally effective in reducing levels of protease-resistant PrP(Sc) (PrP(res)) in a dose- and time-dependent manner in ScN2a cells and in pre-existing aggregates in homogenates from infected brain. Solubility assays revealed that total levels of PrP(Sc) in scrapie-infected mouse neuroblastoma (ScN2a) cells were reduced by mPPI. Coupled with the known ability of polyamino dendrimers to render protease-resistant PrP(Sc) in pre-existing aggregates of PrP(Sc) susceptible to proteolysis, these findings strongly suggest that within infected cells dendrimers reduce total amounts of PrP(Sc) by mediating its denaturation and subsequent elimination.

Biological properties of new viologen-phosphorus dendrimers.

Some biological properties of eight dendrimers incorporating both phosphorus linkages and viologen units within their cascade structure or at the periphery were investigated for the first time. In particular cytotoxicity, hemotoxicity, and antimicrobial and antifungal activity of these new macromolecules were examined. Even if for example all these species exhibited good antimicrobial properties, it was demonstrated that their behavior strongly depends on several parameters as their size and molecular weight, the number of viologen units and the nature of the terminal groups.

Dendrimer interactions with hydrophobic fluorescent probes and human serum albumin.

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.

Phosphorus dendrimers affect Alzheimer's (Aβ1-28) peptide and MAP-Tau protein aggregation.

Alzheimers disease (AD) is characterized by pathological aggregation of β-amyloid peptides and MAP-Tau protein. β-Amyloid (Aβ) is a peptide responsible for extracellular Alzheimers plaque formation. Intracellular MAP-Tau aggregates appear as a result of hyperphosphorylation of this cytoskeletal protein. Small, oligomeric forms of Aβ are intermediate products that appear before the amyloid plaques are formed. These forms are believed to be most neurotoxic. Dendrimers are highly branched polymers, which may find an application in regulation of amyloid fibril formation. Several biophysical and biochemical methods, like circular dichroism (CD), fluorescence intensity of thioflavin T and thioflavin S, transmission electron microscopy, spectrofluorimetry (measuring quenching of intrinsic peptide fluorescence) and MTT-cytotoxicity assay, were applied to characterize interactions of cationic phosphorus-containing dendrimers of generation 3 and generation 4 (CPDG3, CPDG4) with the fragment of amyloid peptide (Aβ(1-28)) and MAP-Tau protein. We have demonstrated that CPDs are able to affect β-amyloid and MAP-Tau aggregation processes. A neuro-2a cell line (N2a) was used to test cytotoxicity of formed fibrils and intermediate products during the Aβ(1-28) aggregation. It has been shown that CPDs might have a beneficial effect by reducing the system toxicity. Presented results suggest that phosphorus dendrimers may be used in the future as agents regulating the fibrilization processes in Alzheimers disease.

Water-soluble carbosilane dendrimers protect phosphorothioate oligonucleotides from binding to serum proteins

Treatment of dendriplexes formed between water-soluble carbosilane dendrimers and phosphorothioate oligodeoxynucleotides (ODN) with the anionic detergent sodium dodecyl sulfate disrupted the complexes indicating that the nature of the union in such dendriplexes is merely electrostatic. However, dendriplexes were not dissociated by serum proteins like bovine or human serum albumins, as assessed by gel electrophoresis and fluorescence experiments. This would imply a dendrimer-mediated protective effect able to prevent ODN interactions with serum proteins and additionally could translate into a reduction of the ODN doses needed to achieve the biological effects. The employment of carbosilane dendrimers as carriers may solve the problem of ODN kidnapping by plasmatic proteins as a key drawback for therapeutics involving ODNs. As examples, transfection processes on normal primary peripheral blood cells and diagnosis of HIV infection in the presence of serum have been assayed.