Dendrimers in Biomedical Applications: Rsc - Hardcover

Hardcover

ISBN 10: 1849736111 ISBN 13: 9781849736114

Publisher: Royal Society of Chemistry, 2013

Synopsis

This book will provide comprehensive coverage of dendrimer applications and clear indications for future research. It will appeal to chemists, biologists and materials scientists, working in both academia and industry.

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Dendrimers are important molecules that are currently undergoing investigation for use in a variety of different biomedical applications. This book explores the use of dendrimers for a variety of potential functions, including antiamyloidogenic agents, drug delivery systems, nucleic acid and RNA delivery vectors and to produce hybrid fibre platforms for nantechnology. Following the work of COST action TD0802, the main objective of which is to improve existing therapies and find new drugs based on dendrimers, the book will provide comprehensive coverage of dendrimer applications. Coverage includes modelling and molecular dynamic studies of dendrimers and dendrons, anionic dendrimer polymers, cationic carbosilane dendrimers and self-assembled multivalent dendrimers. Providing clear indications for future research and applications, this text will appeal to chemists, biologists and materials scientists, working in both academia and industry.

Excerpt. © Reprinted by permission. All rights reserved.

Dendrimers in Biomedical Applications

By Barbara Klajnert, Ling Peng, Valentín Ceña

The Royal Society of Chemistry

Copyright © 2013 COST Office
All rights reserved.

ISBN: 978-1-84973-611-4

Contents

DENDRIMERS AS ANTIAMYLOIDOGENIC AGENTS. DENDRIMER-AMYLOID AGGREGATES MORPHOLOGY AND CELL TOXICITY D. Appelhans, N. Benseny, 0. Klementiveva, M Bryszewska, B. Klajnert, and J. Cladera, 1, 
DENDRIMER-BASED HYBRID FIBERS AS POTENTIAL PLATFORM FOR 1D-OBJECTS IN NANOTECHNOLOGY A. Fahrni, D. Appelhans, A. Danani, G. M Pavan, and B. Voit, 14, 
NATURAL AND SYNTHETIC BIOMATERIALS AS COMPOSITES OF ADVANCED DRUG DELIVERY NANO SYSTEMS (ADDNSS). BIOMEDICAL APPLICATIONS K. Gardikis, E. A. Mourelatou, M Jonov, A. Aserin, D. Libster, B. Klajnert, M Bryszewska, N Garti, J. P. Majoral, K. Dimas, and C. Demetzos, 30, 
CATIONIC CARBOSILANE DENDRIMERS AS NON-VIRAL VECTORS OF NUCLEIC ACIDS (OLIGONUCLEOTIDE OR siRNA) FOR GENE THERAPY PURPOSES R. Gomez, F. J. de la Mata, J. L. Jimenez-Fuentes, P. Ortega, B. Klajnert, E. Pedziwiatr-Werbicka, D. Shcharbin, M Bryszewska, M Maly, J. Maly, M J. Serramia, R. Lorente, and M A. Munoz-Fernandez, 40, 
ANIONIC DENDRITIC POLYMERS FOR BIOMEDICAL APPLICATIONS D. Gröpger, A. Sousa-Herves, M Calderón, E. Fernandez-Megia, and R. Haag, 56, 
POLY(AMIDOAMINE) DENDRIMERS AS NON-VIRAL VECTORS FOR THE DELIVERY OF RNA THERAPEUTICS X Liu, P. Posocco, C. Liu, T Yu, Q. Wang, V Dal Col, C. Chen, Y Wang, P. Rocchi, S. Friel, and L. Peng, 73, 
DENDRIMERIC ANTIGENS. NEW APPROACHES TOWARDS DETECTION OF IgE-MEDIATED DRUG ALLERGY REACTIONS M.I. Montanez, C. Mayorga, M.J. Torres, A.J. Ruiz-Sanchez, M. Malkoch, A. Hult, M. Blanca, and E. Perez-Inestrosa, 84, 
MOLECULAR DYNAMICS OF LYSINE DENDRIMERS. COMPUTER SIMULATION AND NMR I. Neelov, S. Falkovich, D. Markelov, E. Paci, A. Darinskii, and H. Tenhu, 99, 
CHARACTERIZATION OF DENDRIMERS AND THEIR INTERACTIONS WITH BIOMOLECULES FOR MEDICAL USE BY MEANS OF ELECTRON MAGNETIC RESONANCE M. F. Ottaviani, D. Appelhans, F. Javier de la Mata, S. García-Gallego, R. Mazzeo, M. Cangiotti, L. Fiorani, J. P. Majoral, A. M. Caminade, M Bryszewska, and B. Klajnert, 115, 
DENDRIMERS AS VECTORS FOR SMALL INTERFERING RNA TRANSFECTION IN THE NERVOUS SYSTEM F. C. Pérez-Martínez, A. V. Ocaña, G. M. Pavan, A. Danani, and V. Ceña, 134, 
MULTISCALE MODELING OF DENDRIMERS AND DENDRONS FOR DRUG AND NUCLEIC ACID DELIVERY P. Posocco, E. Laurini, V Dal Col, D. Marson, L. Peng, D.K. Smith, B. Klajnert, M. Bryszewska, A.-M. Caminade, J.P. Majoral, M. Fermeglia, K. Karatasos and S. Pricl, 148, 
POLY(AMINOESTER) DENDRIMERS: DESIGN, SYNTHESIS AND CHARACTERIZATION G. Quéléver, C. Bouillon, P. Moreno, A. Tintaru, L. Charles, S. Pricl, and L. Peng, 167, 
FROM MULTIVALENT DENDRONS TO SELF-ASSSEMBLED MULTIVALENT DENDRIMERS: A COMBINED EXPERIMENTAL AND THEORETICAL APPROACH D.K. Smith, and S. Pricl, 179, 
SUBJECT INDEX, 200, 

CHAPTER 1

DENDRIMERS AS ANTIAMYLOIDOGENIC AGENTS. DENDRIMER-AMYLOID AGGREGATES MORPHOLOGY AND CELL TOXICITY

D. Appelhans, N. Bensen, O. Klementiveva, M. Bryszewska, B. Klajnert and J. Cladera K.

1 SUMMARY

Dendrimers are branched polymeric structures that have been shown to have a promising antiamyloidogenic potential by interfering with the polymerization process leading to the formation of the amyloid aggregates related to conformational diseases, such as Alzheimer's and prion diseases. It has been established that there is a relationship between the morphology of the amyloid aggregates and the amyloid peptides or proteins toxicity: fibrillar structures present low or no toxicity, whereas oligomeric species and amorphous aggregates, the so called granular non-fibrillar aggregates (GNAs), are toxic to cells. When interacting with the amyloid peptide associated to the onset and development of Alzheimer's disease, dendrimers can either accelerate the formation of fibrillar structures or inhibit it. Inhibition however may mean promoting the formation of amorphous aggregates. We summarize in the present chapter the experimental evidence showing that when used in a way that favors the formation and clumping of fibrils, dendrimers (glycodendrimers in particular) can reduce amyloid toxicity. However the same glycodendrimers used under different conditions can generate toxic GNAs, an aggregated form that could represent a general morphological signature for amyloid toxicity.

2 AMYLOID AGGREGATION AND ALZHEIMER'S DISEASE.

Alzheimer's disease is one of the so called 'conformational diseases', characterized by the accumulation in the organism of a misfolded variant of a peptide or protein in the form of an amyloid deposit, usually associated to tissue regions where cell deterioration is observed. In Alzheimer's disease, a pathological condition of the Central Nervous System (CNS) which evolution implies the degeneration of cognitive functions, amyloid plaques are typically observed in histological preparations from the affected brains. Such plaques, observed under the electron microscope are made of very thin (approximately 10 nm in diameter) and long (micrometers) amyloid fibrils (Fig. 1A). The main component of amyloid fibrils is the amyloid peptide, a 40-42 residues long peptide (Fig. 1B) which is the proteolytic product of the Amyloid Precursor Protein (APP). APP is a membrane protein which function in the CNS is yet not well established and that can be processed by three different secretases: when cleaved by the α and β secretases a non-amyloidogenic peptide is generated; however when processed by the β and γ secreatases, α mixture of 40 and 42 residues long amyloid peptides, with a high tendency to aggregate is produced into the extracellular space of the CNS.

2.1 Amyloid Peptide Aggregation and Cell Toxicity.

The formation of amyloid fibrils has been thoroughly studied in vitro. The amyloid peptide is structured in the fibril in the form of a cross β-sheet and fibril formation follows a nucleation-dependent polymerization mechanism (Fig. 2). During the lag phase of the typically sigmoid-shaped kinetics different forms of non-fibrillar, low and high molecular weight intermediates are formed. There is a mounting amount of evidence pointing to some of these non-fibrillar species that form during the nucleation phase as the amyloid species that may cause cytotoxicity, whereas mature fibrils would have very low toxicity.

In the search for compounds that would inhibit cell deterioration in Alzheimer's disease, there is a marked interest in finding compounds which are able to inhibit the formation of amyloid deposits either by promoting the removal of the amyloid peptide from the CNS or by inhibiting the formation of the toxic amyloid species or blocking their action. Given the non-toxic character of amyloid fibrils it has to be considered that one way of avoiding the amyloid peptide toxicity could be via its rapid association into fibrils.

Many studies can be found in the literature dedicated to study the effects of a very diverse number of compounds on the amyloid peptide aggregation kinetics. In many cases, it is considered that the power of a compound to inhibit fibril formation represents the possibility that such a compound could be useful to inhibit the peptide's cell toxicity.

However, when evaluating the results of such studies, one has to consider t he possibility that the fact of inhibiting the formation of fibrils, may implicate the accumulation of non-fibrillar toxic species, high and low molecular weight intermediates, or the accumulation of the so-called (toxic) 'Granular Non-fibrillar Aggregates' (GNAs).

2.2 Granular Non-Fibrillar Aggregates (GNAs).

When we consider a protein folding energy landscape diagram like the one depicted in Fig. 3, it can be seen that amyloid fibrils represent a misfolded form of a peptide or protein which can arise sometimes, given the right conditions, from some of the many intermediate (high energy) forms present at the beginning of the process, between which the structure of the peptide or protein fluctuates. The misfolded, fibrillar form, represents a minimum of energy which is even lower than that of the native form. However, fibrils are not the only misfolded, stable form. Peptides and proteins can aggregate as well in the form of amorphous, non-fibrillar aggregates.

It has been mentioned above that it is believed nowadays that the toxic forms of the amyloid peptide, have to be looked for, in Alzheimer's disease, among those species formed during the nucleation phase of the fibril formation process. In a recent work carried out by the group of Josep Cladera in Barcelona (Spain) in collaboration with Jan Maly's group in Usti nad Labem (Czech Republic) we have described, in the case of Alzheimer's disease, the formation of a kind of amorphous, non-fibrillar aggregates, which have been named 'Granular Non-fibrillar Aggregates' (GNAs) (Fig. 4), that could have an important role in cell toxicity. GNAs have been proposed as an off-pathway mechanism in amyloid aggregation. Its formation is related to relevant physiological parameters in Alzheimer's disease, such as an acid medium (pH between 5 and 6.5) or the interaction of the peptide with negatively charged biological membranes. Formation of amorphous toxic aggregates has been as well described as a consequence of the interaction of the amyloid peptide with Cu2+, another significant factor related to the development of the pathology.

3 INTERACTION OF DENDRIMERS WITH ALZHEIMER'S AMYLOID PEPTIDES. AGGREGATE MORPHOLOGY AND TOXICITY.

In the past seven years, in a series of collaborations between the labs of Maria Bryszewska and Barbara Klajnert in Lodz (Poland), Jean Pierre Majoral in Toulouse (France), Francesca Ottaviani in Urbino (Italy), Dietmar Appelhans in Dresden (Germany) and Josep Cladera in Barcelona (Spain), dendrimers have been shown to be capable of interfering in vitro with the formation of the amyloid aggregated structures typically related to the onset and development of Alzheimer's disease and prion diseases. This makes dendrimers potentially useful as compounds that could prevent or inhibit the action of the cytotoxic amyloid species. Such a possibility has come out in the first place from observations showing that dendrimers (PAMAM and phosphorus dendrimers) could interfere with the aggregation kinetics of two amyloid model peptides: the hydrophilic Aβ(1-28) (28 residues long) bit of the 40-42 residues long amyloid peptide (Fig. 1) found in Alzheimer's plaques and a segment of the human prion protein (the Prpl85-206 peptide). In summary it has been found that PAMAM and phosphorus dendrimers can interfere with amyloid fibril formation by either accelerating fibril formation or by slowing down and/or inhibiting its formation.

3.1 Interaction of PAMAM Dendrimers with the Aβ(1-40) Amyloid Peptide.

Fig. 5, shows the influence of PAMAM dendrimers on the aggregation process of the Alzheimer's whole amyloid Aβ(1-40) peptide. Amyloid fibril formation can be easily monitored using the fluorescence dye Thioflavin T (ThT). The dye becomes only fluorescent when interacting with the well-ordered β-sheet structures present in amyloid fibrils, so it is a good method to detect the appearance of fibrils. Fig. 5 reveals the sygmoid-shaped curve corresponding to the fibril formation kinetics of Aβ (1-40), where the typical lag (nucleation) phase followed by the elongation phase are clearly observed. Similarly to what had been observed for Aβ(1-28), PAMAM dendrimers may, in the case of Aβ(1-40), either accelerate fibril formation (this happens at low dendrimer/peptide ratios) or inhibit it (at higher dendrimer/peptide ratios). According to the ThT fluorescent measurements, when the peptide interacts with PAMAM dendrimers at a low dendrimer/peptide ratio there is a clear shortening of the nucleation phase, the presence of the dendrimer accelerates nuclei formation, but, as in the case of the amyloid peptide alone, ThT fluorescence increases until its value reaches a plateau. This is already indicative of the fact that under such conditions the peptide will still form fibrils at the end of the process. However, what is the morphology of the peptide-dendrimer aggregates at the end of the process when they interact at high dendrimer/peptide ratios and no ThT fluorescence increase is observed? This can be checked using electron microscopy. Three electron micrographs, corresponding to the end of three of the kinetics shown in Fig. 5, are shown in Fig. 6. As expected, Aβ(1-40) forms the typical amyloid fibrils when let to aggregate alone or in the presence of PAMAM dendrimers at low dendrimer/peptide ratios. However, when the dendrimer is present at high dendrimer/peptide ratios, no fibrillar structures are present in the electron micrograph. Only amorphous aggregates, similar in shape to those (toxic ones) described for the peptide at low pH or in the presence of Cu2+ ions to which we have referred above (GNAs), can be observed. In the case of the dendrimer/Aβ(1-40) complexes though, are these structures toxic to cells? When using PAMAM dendrimers, their intrinsic cell toxicity makes it impossible to answer the question. In order to measure cell toxicity, the use of biocompatible dendrimers having similar effects to those of PAMAM on amyloid aggregation is necessary. This, as we shall see in the next section, can be achieved using maltose-decorated dendrimers.

3.2 Interaction of Glycodendrimers with the Aβ(1-40) Amyloid Peptide.

The intrinsic cell toxicity of PAMAM dendrimers, with high superficial electrical charge density, is thought to be related to their capacity of establishing very strong interactions of electrostatic nature with biological structures. Glycodendrimers, dendrimers in which the surface is decorated with polysaccharides, have been recently shown in different works to be useful compounds to overcome cell toxicity. In the case of glycodendrimers, the interaction with biological macromolecules would proceed via the establishment of hydrogen bonds. Glycodendrimers have already been shown to have remarkably low or zero toxicity towards different cell lines and experiments on their properties when interacting with biological systems (hemolytic power, interaction with HSA and PrP185–208) have been reported. Fig. 7 illustrates how PPI-maltose glycodendrimers, generations 4 and 5, are not toxic to PC12 and SH-SY5Y cells up to 50 µM dendrimer concentration and have a low toxic effect between 50 and 100 µM. These two cell lines have been routinely used as neuronal models to test cell toxicity.

As shown in a recent work by the labs of Josep Cladera in Barcelona (Spain) and Dietmar Appelhans in Dresden (Germany) the interference capacity of glycodendrimers on the nucleation-dependent aggregation of Aβ(1–40), as illustrated for PPI-maltose generation 4 in Fig. 8, turns out to be very similar to the effect of PAMAM dendrimers (shown in Fig. 5): at low dendrimer-peptide ratios there is a slight acceleration of fibril formation; as the dendrimer/peptide ratio is increased the rate and the amount of fibrils at the end of the process decrease. In the case of PPI-maltose generation 5 glycodendrimers, a complete inhibitory effect of the ThT fluorescence increase (fibril formation) has been observed at any dendrimer/peptide ratio. When considering the similar effect of PAMAM and gycodendrimers on amyloid aggregation we have to consider the fact that in both cases the observed effect depends on the dendrimer-peptide ratio. At low ratios, dendrimers are not able to hamper fibril formation, that is, they cannot inhibit the elongation phase that takes place by combination of amyloid nuclei. And as a matter of fact, at low dendrimer-peptide ratios dendrimers become (although the molecular mechanism is unknown) as amyloid nucleation accelerators, shortening the nucleation phase and accelerating the formation of fibrils. It is reasonable to think that in the case of PAMAM dendrimers this nucleation effect could take place mainly through the establishment of electrostatic interactions between the amyloid oligomers that constitute the nuclei and the dendrimer, whereas in the case of glycodendrimers the effect would come through the formation of hydrogen bonds between the amyloid dendrimers and the surface of the dendrimers. At high dendrimer-peptide ratios, the dendrimer concentration would be enough to hamper the combination of the amyloid nuclei that results in the formation of fibrils. Again the interaction between PAMAM dendrimers and amyloid nuclei would be of electrostatic nature and the interaction between glycodendrimers and the nuclei would take place through the formation of hydrogen bonds.

The electron microscope (Fig. 9) reveals that fibril formation inhibition causes the formation of globular non-fibrillar aggregates (GNAs, generated by incubating dendrimer and peptide at high dendrimer/peptide ratios), whereas fibril formation enhancement favors fibril clumping (fibril clumps generated by incubating G4 maltose glycodendrimers with Ab(1–40) at low dendrimer/peptide ratios, compared to normal fibrils). As it has been hypothesized by Klementieva et al. fibril clumping could be due to the fact that at low dendrimer-peptide ratio, the dendrimers cannot inhibit the formation of fibrils, whereas once formed the same dendrimers may act as a 'glue', bringing the fibrils together in the form of clumps, through the establishment of hydrogen bonds.

It seems therefore that the effect of dendrimers on amyloid aggregation would be greatly due to the properties of the dendrimer surface. Klementieva et al. have estimated that the differential effect of G4 and G5 glycondendrimers is most likely due to the difference in the maltose surface density. An effect due to the electric charge density in model lipid membranes has been as well described by Benseny-Cases et al. Beyond dendrimers and lipid vesicles, they may be other globular polymeric structures with surfaces able to interact with amyloids, that could have similar effects and that may be worth investigating. The intrinsic biocompatibility of glycodendrimers permits to evaluate the toxic capacity of both fibrillar (clumped fibrils) and amorphous glycodendrimer/Aβ(1-40) complexes. When PC12 cell viability is assessed it can be observed that fibril clumping, achieved by using G4 at low dendrimer peptide ratios (G4:AB 0.05 and G4:AB 1 in Fig. 10), renders amyloid fibrils non-toxic. However, fibril formation inhibition, using G5 at any dendrimer/peptide ratio (G5:AB 0.1 and G5:AB 1 in Fig. 10) or G4 at high dendrimer/peptide ratios (G4:AB 10, not sown in fig. 10), generates toxic GNAs (Fig. 10).

(Continues...)

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Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2013
• Language: English
• ISBN 10: 1849736111
• ISBN 13: 9781849736114
• Binding: Hardcover
• Number of pages: 216
• Editor: Barbara Klajnert