Use of poly(amido)amine dendrimers in prevention of early non-enzymatic modifications of biomacromolecules

Hyperglycaemia triggers the formation of both ‘early’ and advanced glycation end products, which are considered the major factors responsible for the complications of diabetes. Poly(amido)amine (PAMAM) dendrimers are relatively new class of materials with unique molecular structure predisposing them for the use as anti-glycation agents. The ability of poly(amido)amine (PAMAM) dendrimers G2 (MW 3256, 120 μmol/l) and G4 (MW 14215, 30 μmol/l) to inhibit the modification of proteins by high glucose (30 mmol/l, 37 °C, 72 h) was investigated using radiometric and spectrofluorometric assays. We monitored (a) non-enzymatic modifications of primary amino groups in BSA and polyamine compounds, and (b) the impact of anti-glycation agents on BSA conformation. Both PAMAM dendrimers and poly(l-lysine) (MW 70 kDa) effectively reduced BSA glycation, while undergoing the time-dependent modification themselves. Such a modification was a function of a number of available free amino groups per molecule, however, both dendrimers and poly(l-lysine) were equally effective in glucose scavenging. PAMAMs neither affected BSA conformation nor formed stable complexes with a protein, while non-glycated poly(l-lysine) significantly quenched BSA fluorescence. Our results encourage raising the hypothesis that PAMAM dendrimers may be considered effective and safe chemical competitors for non-enzymatic modification by glucose, thus confirming the earlier in vivo study showing the inhibition of protein modification in experimental diabetes in the presence of PAMAM dendrimers.     

Polyamidoamine dendrimers with different surface charge as carriers in anticancer drug delivery

Second-generation (G2) polyamidoamine (PAMAM) dendrimers are branched polymers containing 16 surface primary amine groups. Due to their structural properties, these polymers can be used as universal carriers in various drug delivery systems. Amine-terminated PAMAM dendrimers are characterized by a high positive surface charge, leading to effective but nonspecific interactions with negatively charged cell plasmatic membranes. To reduce the nonspecific internalization of PAMAM dendrimers, their primary amine groups are often modified by acetic or succinic anhydrides, polyethylene glycol derivatives and other compounds. In this work, the role of primary amine groups, which are localized on the surface of doxorubicin-conjugated (Dox) dendrimers, was studied with regard to their intracellular distribution and internalization rates using SKOV3 human ovarian adenocarcinoma cells. It was demonstrated that all Dox-labeled G2-derivatives containing different numbers of acetamide groups synthesized in this work show high rates of cellular uptake at 37°С. As expected, the conjugate carrying the maximum number of primary amine groups demonstrated the highest rates of binding and endocytosis. At the same time, the G2-Dox conjugate containing the maximum number of acetamide groups showed colocalization with LAMP2, a marker of lysosomes and late endosomes, as well as the highest level of cytotoxic activity against SKOV3 cells. We conclude that second-generation PAMAM dendrimers are characterized by varied pathways of internalization and intracellular distribution due to the number of primary amine groups on their surface and, as a consequence, a different surface charge.     

Evaluation of poly(amidoamine) dendrimers as potential carriers of iminodiacetic derivatives using solubility studies and 2D-NOESY NMR spectroscopy

The interactions between dendrimers and different types of drugs are nowadays one of the most actively investigated areas of the pharmaceutical sciences. The interactions between dendrimers and drugs can be divided into: internal encapsulation, external electrostatic interaction, and covalent conjugation. In the present study, we investigated the potential of poly(amidoamine) (PAMAM) dendrimers for solubility of four iminodiacetic acid derivatives. We reported that PAMAM dendrimers contribute to significant solubility enhancement of iminodiacetic acid analogues. The nature of the dendrimer–drug complexes was investigated by ¹H NMR and 2D-NOESY spectroscopy. The ¹H NMR analysis proved that the water-soluble supramolecular structure of the complex was formed on the basis of ionic interactions between terminal amine groups of dendrimers and carboxyl groups of drug molecules, as well as internal encapsulation. The 2D-NOESY analysis revealed interactions between the primary amine groups of PAMAM dendrimers and the analogues of iminodiacetic acid. The results of solubility studies together with ¹H NMR and 2D-NOESY experiments suggest that the interactions between PAMAM dendrimers of generation 1–4 and derivatives of iminodiacetic acid are based on electrostatic interactions and internal encapsulation.     

Effects of solute-solute interactions on protein stability studied using various counterions and dendrimers

Much work has been performed on understanding the effects of additives on protein thermodynamics and degradation kinetics, in particular addressing the Hofmeister series and other broad empirical phenomena. Little attention, however, has been paid to the effect of additive-additive interactions on proteins. Our group and others have recently shown that such interactions can actually govern protein events, such as aggregation. Here we use dendrimers, which have the advantage that both size and surface chemical groups can be changed and therein studied independently. Dendrimers are a relatively new and broad class of materials which have been demonstrated useful in biological and therapeutic applications, such as drug delivery, perturbing amyloid formation, etc. Guanidinium modified dendrimers pose an interesting case given that guanidinium can form multiple attractive hydrogen bonds with either a protein surface or other components in solution, such as hydrogen bond accepting counterions. Here we present a study which shows that the behavior of such macromolecule species (modified PAMAM dendrimers) is governed by intra-solvent interactions. Attractive guanidinium-anion interactions seem to cause clustering in solution, which inhibits cooperative binding to the protein surface but at the same time, significantly suppresses nonnative aggregation.     

Effect of size, surface charge, and hydrophobicity of poly(amidoamine) dendrimers on their skin penetration

The barrier functions of the stratum corneum and the epidermal layers present a tremendous challenge in achieving effective transdermal delivery of drug molecules. Although a few reports have shown that poly(amidoamine) (PAMAM) dendrimers are effective skin-penetration enhancers, little is known regarding the fundamental mechanisms behind the dendrimer-skin interactions. In this Article, we have performed a systematic study to better elucidate how dendrimers interact with skin layers depending on their size and surface groups. Franz diffusion cells and confocal microscopy were employed to observe dendrimer interactions with full-thickness porcine skin samples. We have found that smaller PAMAM dendrimers (generation 2 (G2)) penetrate the skin layers more efficiently than the larger ones (G4). We have also found that G2 PAMAM dendrimers that are surface-modified by either acetylation or carboxylation exhibit increased skin permeation and likely diffuse through an extracellular pathway. In contrast, amine-terminated dendrimers show enhanced cell internalization and skin retention but reduced skin permeation. In addition, conjugation of oleic acid to G2 dendrimers increases their 1-octanol/PBS partition coefficient, resulting in increased skin absorption and retention. Here we report that size, surface charge, and hydrophobicity directly dictate the permeation route and efficiency of dendrimer translocation across the skin layers, providing a design guideline for engineering PAMAM dendrimers as a potential transdermal delivery vector.     

Biomimetics: From Bioinformatics to Rational Design of Dendrimers as Gene Carriers

Biomimetics, or the use of principles of Nature for developing new materials, is a paradigm that could help Nanomedicine tremendously. One of the current challenges in Nanomedicine is the rational design of new efficient and safer gene carriers. Poly(amidoamine) (PAMAM) dendrimers are a well-known class of nanoparticles, extensively used as non-viral nucleic acid carriers, due to their positively charged end-groups. Yet, there are still several aspects that can be improved for their successful application in in vitro and in vivo systems, including their affinity for nucleic acids as well as lowering their cytotoxicity. In the search of new functional groups that could be used as new dendrimer-reactive groups, we followed a biomimetic approach to determine the amino acids with highest prevalence in protein-DNA interactions. Then we introduced them individually as terminal groups of dendrimers, generating a new class of nanoparticles. Molecular dynamics studies of two systems: PAMAM-Arg and PAMAM-Lys were also performed in order to describe the formation of complexes with DNA. Results confirmed that the introduction of amino acids as terminal groups in a dendrimer increases their affinity for DNA and the interactions in the complexes were characterized at atomic level. We end up by briefly discussing additional modifications that can be made to PAMAM dendrimers to turned them into promising new gene carriers.     

Low‐generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA

Conventional dendrimers are spherical symmetrically branched polymers ending with active surface functional groups. Polyamidoamine (PAMAM) dendrimers have been widely studied as gene delivery vectors and have proven effective at delivering DNA to cells in vitro. However, higher‐generation (G4‐G8) PAMAM dendrimers exhibit toxicity due to their high cationic charge density and this has limited their application in vitro and in vivo. Another limitation arises when attempts are made to functionalize spherical dendrimers as targeting moieties cannot be site‐specifically attached. Therefore, we propose that lower‐generation asymmetric dendrimers, which are likely devoid of toxicity and to which site‐specific attachment of targeting ligands can be achieved, would be a viable alternative to currently available dendrimers. We synthesized and characterized a series of peptide‐based asymmetric dendrimers and compared their toxicity profile and ability to condense DNA to spherical PAMAM G1 dendrimers. We show that asymmetric dendrimers are minimally toxic and condense DNA into stable toroids which have been reported necessary for efficient cell transfection. This paves the way for these systems to be conjugated with targeting ligands for gene delivery in vitro and in vivo. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Surface acetylation of polyamidoamine (PAMAM) dendrimers decreases cytotoxicity while maintaining membrane permeability

Improving the oral bioavailability of therapeutic compounds remains a challenging area of research. Polyamidoamine (PAMAM) dendrimers are promising candidates for oral drug delivery due to their well-defined compact structure, versatility of surface functionalities, low polydispersity, and ability to enhance transepithelial transport. However, potential cytotoxicity has hampered the development of PAMAM dendrimers for in vivo applications. In this article, we have systematically modified the surface groups of amine-terminated PAMAM dendrimers with acetyl groups. The effect of this modification on cytotoxicity, permeability, and cellular uptake was investigated on Caco-2 cell monolayers. Cytotoxicity was reduced by more than 10-fold as the number of surface acetyl groups increased while maintaining permeability across the cell monolayers. Furthermore, a decrease in nonspecific binding was evident for surface-modified dendrimers compared to their unmodified counterparts. These studies point to novel strategies for minimizing PAMAM dendrimer toxicity while maximizing their transepithelial permeability.     

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers.

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.     

PAMAM dendrimers — diverse biomedical applications. Facts and unresolved questions

In this mini-review a number of novel outcomes, originating from studies in the field of PAMAM dendrimers, are presented and discussed. Owing to the multi-disciplinary nature of dendrimer chemistry it seems important to focus on the relevant topical research of PAMAM dendrimers, including their function, toxicity, surface modifications, and also possible new applications of these spherical polymers. We also consider the possibilities of specific functionalisation of PAMAM dendrimers — both novel ideas and those that have already been reported; as well as their cell-mediated effects (toxic and non-toxic). Then the reactivity of dendrimers’ terminal groups, and their anticipated protective role against modifications of biomacromolecules, are discussed with regard to future developments in biomedical research.     

Serum albumins have five sites for binding of cationic dendrimers

The detailed analysis of the interaction between PAMAM G4 dendrimer and serum albumins was performed using circular dichroism, isothermal titration calorimetry, capillary electrophoresis, zeta-potential and fluorescence polarization. It was shown that serum albumins and PAMAM G4 dendrimer form the complex with stoichiometry of 4-6:1 for G4:HSA and 4-5:1 for G4:BSA molar ratio. The possible sites of PAMAM G4 dendrimers binding to protein surface were discussed. Also, it has been proposed that dendrimer does not significantly affect the protein secondary structure studied by circular dichroism.     

The influence of PAMAM dendrimers surface groups on their interaction with porcine pepsin

In this study the ability of three polyamidoamine (PAMAM) dendrimers with different surface charge (positive, neutral and negative) to interact with a negatively charged protein (porcine pepsin) was examined. It was shown that the dendrimer with a positively charged surface (G4 PAMAM-NH₂), as well as the dendrimer with a neutral surface (G4 PAMAM-OH), were able to inhibit enzymatic activity of pepsin. It was also found that these dendrimers act as mixed partially non-competitive pepsin inhibitors. The negatively charged dendrimer (G3.5 PAMAM-COOH) was not able to inhibit the enzymatic activity of pepsin, probably due to the electrostatic repulsion between this dendrimer and the protein. No correlation between changes in enzymatic activity of pepsin and alterations in CD spectrum of the protein was observed. It indicates that the interactions between dendrimers and porcine pepsin are complex, multidirectional and not dependent only on disturbances of the secondary structure.     

Glycopeptide dendrimers. Part I.

Glycopeptide dendrimers are branched structures containing both carbohydrates and peptides. Various classes of these compounds differing in composition and structure are mentioned, together with their practical use spanning from catalysis, transport vehicles to synthetic vaccines. The main stress is given to glycopeptide dendrimers, namely multiple antigen glycopeptides (MAGs). In MAGs, the core, branches or both are composed of amino acids or peptides. Other classes of glycodendrimers (PAMAM, polypropylene imine, cyclodextrin, calixarene, etc.) are mentioned too, but to a smaller extent. Their syntheses, physicochemical properties and biological activities are given with many examples. Glycopeptide dendrimers can be used as inhibitors of cell surface protein-carbohydrate interactions, intervention with bacterial adhesion, for studying of recognition processes, diagnostics, imaging and contrast agents, mimetics, for complexation of different cationts, as site-specific molecular delivery systems, for therapeutic purposes, as immunodiagnostics and in drug design. Biomedical applications of glycopeptide dendrimers as drug and gene delivery systems are also given.     

The breakdown of bilayer lipid membranes by dendrimers

The BLM-system for studying the electrophysical properties of bilayer lipid membranes (BLM) was applied to investigate interactions between polyamidoamine (PAMAM) dendrimers and lipid bilayers. The cationic PAMAM G5 dendrimer effectively disrupted planar phosphatidylcholine membranes, while the hydroxyl PAMAM-OH G5 and carboxyl PAMAM G4.5 dendrimers had no significant effect on them.     

Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport

We have investigated poly(amidoamine) (PAMAM) dendrimer interactions with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and KB and Rat2 cell membranes using atomic force microscopy (AFM), enzyme assays, flow cell cytometry, and fluorescence microscopy. Amine-terminated generation 7 (G7) PAMAM dendrimers (10-100 nM) were observed to form holes of 15-40 nm in diameter in aqueous, supported lipid bilayers. G5 amine-terminated dendrimers did not initiate hole formation but expanded holes at existing defects. Acetamide-terminated G5 PAMAM dendrimers did not cause hole formation in this concentration range. The interactions between PAMAM dendrimers and cell membranes were studied in vitro using KB and Rat 2 cell lines. Neither G5 amine- nor acetamide-terminated PAMAM dendrimers were cytotoxic up to a 500 nM concentration. However, the dose dependent release of the cytoplasmic proteins lactate dehydrogenase (LDH) and luciferase (Luc) indicated that the presence of the amine-terminated G5 PAMAM dendrimer decreased the integrity of the cell membrane. In contrast, the presence of acetamide-terminated G5 PAMAM dendrimer had little effect on membrane integrity up to a 500 nM concentration. The induction of permeability caused by the amine-terminated dendrimers was not permanent, and leaking of cytosolic enzymes returned to normal levels upon removal of the dendrimers. The mechanism of how PAMAM dendrimers altered cells was investigated using fluorescence microscopy, LDH and Luc assays, and flow cytometry. This study revealed that (1) a hole formation mechanism is consistent with the observations of dendrimer internalization, (2) cytosolic proteins can diffuse out of the cell via these holes, and (3) dye molecules can be detected diffusing into the cell or out of the cell through the same membrane holes. Diffusion of dendrimers through holes is sufficient to explain the uptake of G5 amine-terminated PAMAM dendrimers into cells and is consistent with the lack of uptake of G5 acetamide-terminated PAMAM dendrimers.     

Paramagnetic NMR Investigation of Dendrimer-Based Host-Guest Interactions

In this study, the host-guest behavior of poly(amidoamine) (PAMAM) dendrimers bearing amine, hydroxyl, or carboxylate surface functionalities were investigated by paramagnetic NMR studies. 2,2,6,6-Tetramethylpiperidinyloxy (TEMPO) derivatives were used as paramagnetic guest molecules. The results showed that TEMPO-COOH significantly broaden the ¹H NMR peaks of amine- and hydroxyl-terminated PAMAM dendrimers. In comparison, no paramagnetic relaxation enhancement (PRE) was observed between TEMPO-NH₂, TEMPO-OH and the three types of PAMAM dendrimers. The PRE phenomenon observed is correlated with the encapsulation of TEMPO-COOH within dendrimer pockets. Protonation of the tertiary amine groups within PAMAM dendrimers plays an important role during this process. Interestingly, the absence of TEMPO-COOH encapsulation within carboxylate-terminated PAMAM dendrimer is observed due to the repulsion of TEMPO-COO- anion and anionic dendrimer surface. The combination of paramagnetic probes and ¹H NMR linewidth analysis can be used as a powerful tool in the analysis of dendrimer-based host-guest systems.     

Influence of dendrimer surface chemistry and pH on the binding and release pattern of chalcone studied by molecular dynamics simulations

Poly(amidoamine) (PAMAM) dendrimers are promising nanocarriers that can enhance the solubility of hydrophobic drugs. The surface chemistry of dendrimers is of great relevance as end groups of these nanocarriers can be easily modified to improve the bioavailability and sustained release of the cargo. Therefore, a molecular‐level understanding of the host‐guest interactions that can give both qualitative and quantitative information is particularly desirable. In this work, fully atomistic molecular dynamics simulations were used to study the association of a bioactive natural product, ie, chalcone, with amine‐, acetyl‐, and carboxyl‐terminated PAMAM dendrimers at physiological and acidic pH environments. Amine‐ and carboxyl‐terminated PAMAM dendrimers have an open microstructure at low pH that is not able to hold the ligand tightly, resulting in an unfavorable encapsulation of the chalcone molecule. In the case of acetyl‐terminated dendrimer, chalcone molecule diffuses out of the dendritic cavities a few times during the simulation time and prefers to locate close to the surface of dendrimer. Average center of mass distance values at neutral pH showed that the chalcone molecule bounds firmly in the internal pockets of amine‐, acetyl‐, and carboxyl‐terminated dendrimers and forms stable complexes with these nanovectors. The potential of mean force calculations showed that the release of the ligand from the dendrimers occurs at a controlled rate in the body.     

Systematic investigation of polyamidoamine dendrimers surface-modified with poly(ethylene glycol) for drug delivery applications: synthesis, characterization, and evaluation of cytotoxicity

Surface modification of amine-terminated polyamidoamine (PAMAM) dendrimers by poly(ethylene glycol) (PEG) groups generally enhances water-solubility and biocompatibility for drug delivery applications. In order to provide guidelines for designing appropriate dendritic scaffolds, a series of G3 PAMAM-PEG dendrimer conjugates was synthesized by varying the number of PEG attachments and chain length (shorter PEG 550 and PEG 750 and longer PEG 2000). Each conjugate was purified by size exclusion chromatography (SEC) and the molecular weight (MW) was determined by (1)H NMR integration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). NOESY experiments performed in D 2O on selected structures suggested no penetration of PEG chains to the central PAMAM domain, regardless of chain length and degree of substitution. CHO cell cultures exposed to PAMAM-PEG derivatives (< or =1 microM) showed a relatively high cell viability. Generally, increasing the degree of PEG substitution reduced cytotoxicity. Moreover, compared to G3 PAMAM dendrimers that were N-acetylated to varying degrees, a lower degree of surface substitution with PEG was needed for a similar cell viability. Interestingly, when longer PEG 2000 was fully incorporated on the surface, cell viability was reduced at higher concentrations (32 muM), suggesting increased toxicity potentially by forming intermolecular aggregates. A similar observation was made for anionic carboxylate G5.5 PAMAM dendrimer at the same dendrimer concentration. Our findings suggest that a lower degree of peripheral substitution with shorter PEG chains may suffice for these PAMAM-PEG conjugates to serve as efficient universal scaffolds for drug delivery, particularly valuable in relation to targeting or other ligand-receptor interactions.     

Comparative biodistribution of PAMAM dendrimers and HPMA copolymers in ovarian-tumor-bearing mice

The biodistribution profile of a series of linear N-(2-hydroxylpropyl)methacrylamide (HPMA) copolymers was compared with that of branched poly(amido amine) dendrimers containing surface hydroxyl groups (PAMAM-OH) in orthotopic ovarian-tumor-bearing mice. Below an average molecular weight (MW) of 29 kDa, the HPMA copolymers were smaller than the PAMAM-OH dendrimers of comparable molecular weight. In addition to molecular weight, hydrodynamic size and polymer architecture affected the biodistribution of these constructs. Biodistribution studies were performed by dosing mice with (125)iodine-labeled polymers and collecting all major organ systems, carcass, and excreta at defined time points. Radiolabeled polymers were detected in organ systems by measuring gamma emission of the (125)iodine radiolabel. The hyperbranched PAMAM dendrimer, hydroxyl-terminated, generation 5 (G5.0-OH), was retained in the kidney over 1 week, whereas the linear HPMA copolymer of comparable molecular weight was excreted into the urine and did not show persistent renal accumulation. PAMAM dendrimer, hydroxyl-terminated, generation 6.0 (G6.0-OH), was taken up by the liver to a higher extent, whereas the HPMA copolymer of comparable molecular weight was observed to have a plasma exposure three times that of this dendrimer. Tumor accumulation and plasma exposure were correlated with the hydrodynamic sizes of the polymers. PAMAM dendrimer, hydroxyl-terminated, generation 7.0 (G7.0-OH), showed extended plasma circulation, enhanced tumor accumulation, and prolonged retention with the highest tumor/blood ratio for the polymers under study. Head-to-head comparative study of HPMA copolymers and PAMAM dendrimers can guide the rational design and development of carriers based on these systems for the delivery of bioactive and imaging agents.     

Surface modified dendrimers: synthesis and characterization for cancer targeted drug delivery

Dendrimers represents a highly branched three-dimensional structure that provides a high degree of surface functionality and versatility. PAMAM dendrimers are used as well-defined nanocontainers to conjugate, complex or encapsulate therapeutic drugs or imaging moieties. Star-burst [PAMAM] dendrimers represent a superior carrier platform for drug delivery. The present study was aimed at synthesis of a surface modified dendrimer for cancer targeted drug delivery system. For this 4.0 G PAMAM dendrimer was conjugated with Gallic acid [GA] and characterized through UV, IR, 1H NMR and mass spectroscopy. Cytotoxicity study of dendrimer conjugate was carried out against MCF-7 cell line using MTT assay. The study revealed that the conjugate is active against MCF-7 cell line and might act synergistically with anti-cancer drug and gallic acid-dendrimer conjugate might be a promising nano-platform for cancer targeting and cancer diagnosis.