Polycationic Dendrimers Research Articles

Intermolecular forces between low generation PAMAM dendrimer condensed DNA helices: role of cation architecture

In recent years, dendriplexes, complexes of cationic dendrimers with DNA, have become attractive DNA delivery vehicles due to their well-defined chemistries. To better understand the nature of the forces condensing dendriplexes, we studied low generation poly(amidoamine) (PAMAM) dendrimer-DNA complexes and compared them to comparably charged linear arginine peptides. Using osmotic stress coupled with X-ray scattering, we have investigated the effect of molecular chain architecture on DNA-DNA intermolecular forces that determine the net attraction and equilibrium interhelical distance within these polycation condensed DNA arrays. In order to compact DNA, linear cations are believed to bind in DNA grooves and to interact with the phosphate backbone of apposing helices. We have previously shown a length dependent attraction resulting in higher packaging densities with increasing charge for linear cations. Hyperbranched polycations, such as polycationic dendrimers, presumably would not be able to bind to DNA and correlate their charges in the same manner as linear cations. We show that attractive and repulsive force amplitudes in PAMAM-DNA assemblies display significantly different trends than comparably charged linear arginines resulting in lower DNA packaging densities with increasing PAMAM generation. The salt and pH dependencies of packaging in PAMAM dendrimer-DNA and linear arginine-DNA complexes were also investigated. Significant differences in the force curve behaviour and salt and pH sensitivities suggest that different binding modes may be present in DNA condensed by dendrimers when compared to linear polycations.

Interaction between viologen-phosphorus dendrimers and α-synuclein

In this study the interaction between viologen-phosphorus dendrimers and α-synuclein (ASN) was examined. Polycationic viologen-phosphorus dendrimers (two positive charges per viologen unit) are novel compounds with relatively unknown properties. The influence of these viologen dendrimers on ASN was tested using fluorimetric and circular dichroism methods. ASN contains four tyrosine residues; therefore, the influence of dendrimers on protein molecular conformation by measuring the changes in the ASN fluorescence in the presence of dendrimers was evaluated. The interaction of dendrimers with free l-tyrosine was also monitored. Results show that viologen-phosphorus dendrimers interact with ASN; they quenched the fluorescence of ASN as well as free tyrosine by dynamic and static ways. However, the quenching was not accompanied by modifications in the ASN secondary structure.

The Use of Solid Supports to Generate Nucleic Acid Carriers

Nucleic acids are the foundation stone of all cellular processes. Consequently, the use of DNA or RNA to treat genetic and acquired disorders (so called gene therapy) offers enormous potential benefits. The restitution of defective genes or the suppression of malignant genes could target a range of diseases, including cancers, inherited diseases (cystic fibrosis, muscular dystrophy, etc.), and viral infections. However, this strategy has a major barrier: the size and charge of nucleic acids largely restricts their transit into eukaryotic cells. Potential strategies to solve this problem include the use of a variety of natural and synthetic nucleic acid carriers. Driven by the aim and ambition of translating this promising therapeutic approach into the clinic, researchers have been actively developing advanced delivery systems for nucleic acids for more than 20 years. A decade ago we began our investigations of solid-phase techniques to construct families of novel nucleic acid carriers for transfection. We envisaged that the solid-phase synthesis of polycationic dendrimers and derivatized polyamimes would offer distinct advantages over solution phase techniques. Notably in solid phase synthesis we could take advantage of mass action and streamlined purification procedures, while simplifying the handling of compounds with high polarities and plurality of functional groups. Parallel synthesis methods would also allow rapid access to libraries of compounds with improved purities and yields over comparable solution methodologies and facilitate the development of structure activity relationships. We also twisted the concept of the solid-phase support on its head: we devised miniaturized solid supports that provided an innovative cell delivery vehicle in their own right, carrying covalently conjugated cargos (biomolecules) into cells. In this Account, we summarize the main outcomes of this series of chemically related projects.

Photo-physical and structural interactions between viologen phosphorus-based dendrimers and human serum albumin

This work deals with photo-physical and structural interactions between viologen phosphorus dendrimers and human serum albumin (HSA). Viologens are derivatives of 4,4′-bipyridinium salts. Aiming to rationalize the parameters governing such interactions eight types of these polycationic dendrimers in which the generation, the number of charges, the nature of the core and of the terminal groups vary from one to another, were designed and used. The influence of viologen-based dendrimers' on human serum albumin has been investigated. The photo-physical interactions of the two systems have been monitored by fluorescence quenching of free l-tryptophan and of HSA tryptophan residue. Additionally, using circular dichroism (CD) the effect of dendrimers on the secondary structure of albumin was measured. The obtained results show that viologen dendrimers interact with human serum albumin quenching its fluorescence either by collisional (dynamic) way or by forming complexes in a ground state (static quenching). In some cases the quenching is accompanied by changes of the secondary structure of HSA.

Simple enzyme immobilization inside glass tubes for enzymatic cascade reactions

The two enzymes Aspergillus nigerglucose oxidase (GOD) and horseradish peroxidase (HRP) were immobilized with a simple and quick procedure inside micropipette glass tubes with the help of the avidin–biotin system and of a biotinylated second generation polycationic dendronized polymer (de-PG2). De-PG2 strongly adheres to SiO2 surfaces and served as soft organic layer for non-covalently gluing the biotinylated enzyme–avidin complexes to the solid glass surface. The immobilized enzymes remained highly active for a period of several weeks. Furthermore, after connecting a glass tube containing immobilized GOD (first tube) with a glass tube containing immobilized HRP (second tube), this system was applied as a simple flow reactor for the spectrophotometric quantification of D-glucose in aqueous solution via a cascade reaction catalysed by the two immobilized enzymes. Moreover, preliminary measurements showed that the general procedure developed for the immobilization of HRP and GOD inside the glass tubes can also be applied to E. coli β-galactosidase, allowing the spectrophotometric determination of lactosevia a three-enzyme cascade reaction in a system composed of three sequentially connected tubes. The success of the work is based on two factors: (i) on the use of the dendronized polymerde-PG2 which combines properties of polycationic dendrimers as well as polycationic polymers in one and the same molecule, leading to efficient and reproducible binding to SiO2 surfaces; and (ii) on the monitoring of the immobilization on SiO2 with the transmission interferometric adsorption sensor (TInAS) for elaborating the optimal experimental conditions for the immobilization of the enzymes inside the tubes.

Combinatorial Discovery of Peptide Dendrimer Enzyme Models Hydrolyzing Isobutyryl Fluorescein

Two 6750-membered one-bead-one-compound peptide dendrimer combinatorial libraries L (X(4))(8)(LysX(3))(4)(LysX(2))(2)LysX(1) (X(1-4) = 14 different amino acids or deletion, Lys = branching lysine residue) and AcL (with N-terminal acetylation) were prepared by split-and-mix solid phase peptide synthesis. Screening toward fluorogenic substrates for esterase and aldolase activities using the in silica off-bead assay (N. Maillard et al., J. Comb. Chem. 2009, 11, 667-675) and bead decoding by amino acid analysis revealed histidine containing sequences active against fluorescein diacetate. Isobutyryl fluorescein, a related hydrophobic fluorogenic substrate, was preferentially hydrolyzed by dendrimers from library AcL containing hydrophobic residues such as AcH3 (AcHis)(8)(LysLeu)(4)(LysVal)(2)LysLysOH, compared to simple oligohistidine peptides as reference catalysts. Polycationic dendrimers from library L with multiple free N-termini such as H8 (His)(8)(LysβAla)(4)(LysThr)(2)LysaProNH(2) (aPro = (2S,4S)-4-aminoproline) showed stronger reactivity toward 8-acetoxypyrene-1,3,6-trisulfonate with partial acylation of N-termini. These experiments highlight the role of noncatalytic amino acids to determine substrate selectivity in peptide dendrimer esterase models.

Trimethylenedipyridinium Dendrimers: Synthesis and Sequential Complexation of Anthraquinone Disulfonate in Molecular Shells

The synthesis of a new class of polycationic dendrimers of generation 0, 1, and 2 (G0 (1.07 nm), G1 (1.63 nm), and G2 (3.03 nm)) is described. Core and 2-fold branching units consist of interconnected 1,3,5-tris(methylene)benzene and 4,4′-trimethylenedipyridinium units. 4-tert-Butylbenzyl has been used as the peripheral end group because best solubility of the dendrimers was achieved. The dendrimers act as a host for anthraquinone-2,6-disulfonate (AQDS) and can be stoichiometrically titrated and even overcharged as shown by 1H NMR, DOSY and cyclic voltammetry. Upon loading them with AQDS, the dendrimers undergo first a contraction, they reach a minimum hydrodynamic radius for complete charge compensation and they reopen when overcharging takes place. The contraction is supported by MM+ calculations. Upon stepwise loading of G2 (42 positive charges) with AQDS (2 negative charges), the first 3 mol equiv (6 negative charges) occupy the innermost dendrimer shell (consisting of 6 positive charges), the next 6 equiv (12 negative charges) occupy the middle shell (12 positive charges), and the last 12 equiv AQDS (24 negative charges) occupy the outermost shell of the dendrimer (24 positive charges), as supported by 1H NMR titrations yielding the magic equivalent numbers of 3, 9 = 3 + 6, and 21= 3 + 6 + 12.

The Detection of DNA Hybridization on Phosphorus Dendrimer Multilayer Films by Surface Plasmon Field Enhanced-Fluorescence Spectroscopy

Dendrimer multilayers on gold substrates prepared via layer-by-layer (LbL) assembly technique were characterized and used as substrates for DNA immobilization/hybridization. The multilayers, built using alternately polycationic and polyanionic phosphorus dendrimers of generation 4, were studied by surface plasmon resonance (SPR) spectroscopy. By varying the concentration of NaCl, the optimized optical thickness of a single dendrimer layer (about 4.5 nm) was achieved. Using the multilayers as the substrate, a high loading of DNA probes was obtained through covalent coupling of probe DNA on dendrimer multilayer film. The following hybridization of Cy5-dye labeled complementary target DNA with immobilized probe DNA was detected by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The limit of detection of target DNA upon hybridization reached 50 pM and 30 pM on 1 bilayer and 4 bilayers, respectively. The phosphorus dendrimer multilayer films display high stability during repeated regeneration and hybridization cycles. The sensitive platforms based on dendrimer multilayers deposited in the presence of NaCl make them attractive candidates for application in DNA sensing.

Polycationic phosphorus dendrimers: synthesis, characterization, study of cytotoxicity, complexation of DNA, and transfection experiments

Four series of phosphorus dendrimers (generations 1 and 4) having various types of amine terminal groups (pyrrolidine, morpholine, methyl piperazine, or phenyl piperazine) are synthesized. After protonation, the fourth generations of three of them are found water-soluble, and used for several biological experiments. First, the cytotoxicity of these polycationic dendrimers towards three cell strains (one healthy: HUVEC, and two cancerous: HEK 293 and HeLa) is assayed and found low. Second, their ability to interact with DNA is tested by electrophoresis: the dendrimer terminated by pyrrolidinium groups is found efficient to form dendriplexes. Finally, the polycationic dendrimers are used as transfection agents to deliver single- and double-stranded DNA into the three above-mentioned cell strains. Here also the dendrimer having pyrrolidinium groups is found the most efficient.

Polycationic triazine-based dendrimers: effect of peripheral groups on transfection efficiency.

A panel of eight, second generation triazine dendrimers differing in the number of amines, guanidines, hydroxyls and aliphatic groups on the periphery was synthesized and assayed for gene transfer in an attempt to correlate the effects of surface functionality on transfection efficiency. The physicochemical and biological properties of the dendrimers and dendriplexes, such as condensation of DNA, size, surface charge and morphology of dendriplexes, toxicity and ultimately transfection efficiency in MeWo cells, were analyzed. The results from an ethidium bromide exclusion assay showed that the complexation efficiency of the dendrimers with DNA is moderately affected by surface groups. Increasing the number of surface amines, reducing the number of surface hydroxyl groups, or replacing the amine moiety with guanidines all help strengthen the complex formed. Results from dynamic light scattering and zeta potential analyses indicate that the smallest particles correlate with complexes that exhibit the highest zeta potentials. Cytotoxicity was low for all compounds, particularly for the G2-5 dendrimer containing alkyl groups on the periphery, indicating the benefit of incorporating such neutral functionality onto the surface of the triazine dendrimers. Within this panel, the highest transfection efficiency was observed for the dendrimers that formed the smallest complexes, suggesting that this physicochemical property is an accurate predictor for determining which dendrimers will show high transfection efficiency.

Enhanced specificity and efficiency of polymerase chain reactions using poly(amidoamine) dendrimers and derivatives

The polymerase chain reaction (PCR) has become a fundamental technique in molecular biology. Improvements to enhance the specificity and efficiency of PCR are always required and remain a great challenge. Here we introduce generation 4 (G4) and 5 (G5) poly(amidoamine) (PAMAM) dendrimers and their derivatives with different terminal groups as a novel class of enhancers to improve the specificity and efficiency of PCR. We show that the dendrimers are effective in optimizing error-prone two-round PCR and non-specific PCR in specificity and efficiency. With the increase of the number and density of terminal amine groups, the optimum concentration of polycationic dendrimers could be decreased to as low as 1.35 nM, which is four orders of magnitude smaller than that of fully acetylated dendrimers and carboxyl-terminated dendrimers. The present study underlines a fact that dendrimers could be used as a powerful additive to enhance the specificity and efficiency of PCR.

Polyviologen Dendrimers as Hosts and Charge‐Storing Devices

Two dendrimers were designed and synthesized that contain a 1,3,5-trisubstituted benzenoid core and incorporate 9 and 21 viologen (4,4'-bipyridinium) units in their branches in addition to hydrophilic (aryloxy) terminal groups. For comparison purposes, model compounds containing one and two viologen units were also studied. These polycationic dendrimers form strong host-guest complexes with the dianionic form of the red dye eosin in dilute CH(2)Cl(2) solutions. Titration experiments, based on fluorescence measurements, showed that each viologen unit in the dendritic structures becomes associated with an eosin dianion. Electrochemical (in MeCN) and photosensitization (in CH(2)Cl(2)) experiments revealed that only a fraction of the viologen units present in the dendritic structures can be reduced. This fraction corresponds to the number of viologen units present in the outer shells of the dendrimers. The reasons for incomplete charge pooling are discussed. Comparison with the behavior of polyviologen dendrimers that are terminated with bulky tetraarylmethane groups and were studied previously enabled the role played by the terminal groups in the redox and hosting properties to be elucidated.

The characterization of a novel dendritic system for gene delivery by isothermal titration calorimetry

Understanding the nature of binding of polycationic dendrimers to DNA provides useful information on their role in gene delivery. In the present study, we have characterized the interaction of several peptide-based polycationic dendrimers with salmon sperm DNA using isothermal titration calorimetry. The dendrimers consisted of the cell penetrating peptide TAT, a nuclear localization signal peptide and dendritic polylysine. The binding affinity and thermodynamic parameters were found to increase as the number of positive charges on the dendrimer increased, indicating that ionic interactions were the major binding forces between the two molecules. The effect of acidic pH (3.2) compared to a more neutral pH (7.2) was also examined. The binding affinity was stronger at the lower pH but precipitation of the complex was more prominent at pH 7.2 which was shown by large enthalpies. The results indicate that our dendrimers are forming stable complexes with DNA.

The Advance of Dendrimers - A Versatile Targeting Platform for Gene/Drug Delivery

The quest towards achieving a better understanding of underlying mechanisms by which genetic factors contribute to human disease has gathered considerable momentum, most notably due to the drafting of the complete human genome sequence. This has in turn accelerated research into identifying genes responsible for a plethora of genetic, infectious and metabolic diseases with the vision that therapies can then be developed. Although achieving a therapeutic intervention by gene delivery is perfectly feasible, the practical approach to achieving such a goal, at least in vivo, has proved far more challenging. Employing viruses as gene vectors has to-date proven to be the most effective method of delivery however concerns have emerged about both the short and long-term risks they pose. These fears being confirmed by incidents which led to the tragic deaths of subjects believed to have been triggered by adeno- & retroviral vectors used in clinical trials. This prompted many in the field to turn their research focus towards developing non-viral vectors deemed not only to be safer (non-immunogenic) than their viral counterparts but with a greater gene loading capacity. Polycationic dendrimers (PCDs) as vectors for this purpose have attracted significant interest due to their ease of synthesis, versatility and tolerability. This review will explore the physicochemical parameters crucial to PCD-mediated gene delivery and highlight some innovative strategies designed to maximise transfection efficacy and facilitate tissue-targeting of these elaborate macromolecules.

Use of poly(vinyl alcohol)‐coated capillaries for separation of amino‐terminated polyamidoamine dendrimers

Characterization of amino-terminated polyamidoamine dendrimers by CE suffers from a lack of resolution for higher generations and poor between-day reproducibility of retention times. Under optimal conditions of temperature, voltage, and sample amount, 0-5 generations of dendrimers could be resolved with a bare fused-silica capillary. However, reproducibility was poor due to potential interactions of the polycationic dendrimers with the uncoated quartz capillary wall. Use of a poly(vinyl alcohol)-coated capillary significantly decreased the migration times of the nanomolecules without compromising resolution. Dendrimer mixtures containing generations 0-5 are separated as discrete, nonoverlapping peaks in about 15 min. In addition, the between-day precision of retention times was dramatically improved without the need for internal standards or data normalization. Dendrimers of various generations and cores run on different days showed an RSD of retention times of less than 4%. The poly(vinyl alcohol) coating was very stable as shown by the excellent precision of migration times obtained on a capillary used for a month with more than 100 injections. Similar to PAGE, separation of polyamidoamine dendrimers on a bare fused-silica and poly(vinyl alcohol)-coated capillary showed an exponential relationship between migration times and calculated charge density of the nanomolecules.

Influence of cationic phosphorus dendrimers on the surfactant-induced synthesis of mesostructured nanoporous silica

The concomitant use of polycationic dendrimers and cationic surfactants for the elaboration of periodic mesoporous silica of type MCM-41 is described. The hexagonal structure is preserved up to about 26% in weight of dendrimer included in the silica. The cationic surfactant can be selectively removed to liberate the pores, while preserving the non-covalently incorporated dendrimers. These dendrimers included in the mesoporous silica are fully accessible through the mesoporous volume to small molecules such as HCl and tetrahydrofuran.

The Interaction between Polycationic Poly-Lysine Dendrimers and Charged and Neutral Fluorescent Probes

The interactions between polycationic poly-lysine dendrimers and hydrophobic fluorescent probes (anionic ANS and neutral Prodan) were studied. R121 and R131 dendrimers were not able to interact with anionic and neutral hydrophobic groups. R124 was able to interact with neutral and anionic hydrophobic fluorescent probes, however mainly through hydrophobic forces. Dendrimers R155 and R169 showed the maximal effects. The strongest interactions observed for R169 can be explained by intramolecular folding (stacking) of its two L-proline residues. Using double fluorescence titration technique for ANS probe allowed to receive such constant of binding and the number of binding centers: for R121, 1.8 x 10(3) (mol/l)(-1) and 1.07; for R124, 12.1 x 10(3) (mol/l)(-1) and 0.48; for R131, 4.7 x 10(3) (mol/l)(-1) and 0.48; for R155, 9.2 x 10(3) (mol/l)(-1) and 1.36; for R169, 39.6 x 10(3) (mol/l)(-1) and 0.97. Thus, neutral and anionic hydrophobic probes can be used for the fast preliminary screening of binding properties of newly synthesized polycationic dendrimers.

Investigations on the toxicological profile of functionalized fifth-generation poly(propylene imine) dendrimer

Dendrimers have generated tremendous interest in the field of drug delivery. Despite indications of their utility as drug carriers, the inherent cytotoxicity associated with polycationic dendrimers acts as a limiting factor to their clinical applications. Many functionalization strategies have been adopted to mask peripheral amines in order to overcome this limitation. The object of the present investigation was to evaluate the effect of functionalization on the toxicological profile of fifth-generation poly(propylene imine) dendrimer (PPI-5.0G). Four forms of functionalized dendrimers, including protected glycine and phenylalanine, and mannose and lactose functionalized poly(propylene imine) (PPI) dendrimer, were synthesized as prospective drug carriers. These dendrimeric systems were evaluated for haemolytic toxicity, cytotoxicity, immunogenicity and haematological parameters. PPI-5.0G demonstrated a positive charge-based time- and concentration-dependent toxicity profile. Functionalization greatly improved the toxicity profile of the parent dendrimer. Hence it is proposed that these functionalized forms of PPI dendrimer have great potential as bio-compatible drug vehicles.

Synthesis of a library of polycationic lipid core dendrimers and their evaluation in the delivery of an oligonucleotide with hVEGF inhibition

This article follows on from our previous work in the area of non-viral gene delivery using polycationic dendrimers (PCDs). Herein we report on the synthesis and efficacy of a new library of lipid core PCDs in the delivery of the anti-angiogenic oligonucleotide (ODN-1) to retinal pigment epithelial cells. ELISA was used to monitor hVEGF levels in cells transfected with dendriplexes, Cytofectin GSV™ and control (non-transfected). At 48 h, hVEGF titres had returned to that of the untransfected control for Cytofectin GSV™ however, a number of dendriplexes continued to exhibit a marked reduction in hVEGF titres.

The importance of valency in enhancing the import and cell routing potential of protein transduction domain-containing molecules

Protein transduction domains (PTDs) are peptides that afford the internalization of cargo macromolecules (including plasmid DNA, proteins, liposomes, and nanoparticles). In the case of polycationic peptides, the efficiency of PTDs to promote cellular uptake is directly related to their molecular mass or their polyvalent presentation. Similarly, the efficiency of routing to the nucleus increases with the number of nuclear localization signals (NLS) associated with a cargo. The quantitative enhancement, however, depends on the identity of the PTD sequence as well as the targeted cell type. Thus the choice and multivalent presentation of PTD and NLS sequences are important criteria guiding the design of macromolecules intended for specific intracellular localization. This review outlines synthetic and recombinant strategies whereby PTDs and signal sequences can be assembled into multivalent peptide dendrimers and promote the uptake and routing of their cargoes. In particular, the tetramerization domain of the tumour suppressor p53 (p53 tet) is emerging as a useful scaffold to present multiple routing and targeting moieties. Short cationic peptides fused to the 31-residue long p53 tet sequence resulted in tetramers displaying a significant enhancement (up to 1000 fold) in terms of their ability to be imported into cells and delivered to the cell nucleus in relation to their monomeric analogues. The design of future polycationic peptide dendrimers as effective delivering vehicles will need to incorporate selective cell targeting functions and provide solutions to the issue of endosomal entrapment.