Enzymatic activation of second-generation dendritic prodrugs: Conjugation of self-immolative dendrimers with poly(ethylene glycol) via click chemistry

Single-triggered disassemble dendrimers were recently developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units through a self-immolative chain fragmentation initiated by a single cleavage at the dendrimer's core. There are several examples for the bioactivation of first-generation self-immolative dendritic prodrugs. However, enzymatic activation failed for second-generation self-immolative dendrimers. The hydrophobic large molecular structure of the dendritic prodrugs results in aggregation under aqueous conditions and prevented the enzyme from reaching the triggering substrate. Here we show a simple solution for the enzymatic activation of second-generation self-immolative dendrimers. Poly(ethylene glycol) (PEG) was conjugated to the dendritic platform via click chemistry. The poly(ethylene glycol) tails significantly decreased the hydrophobic properties of the dendrimers and thereby prevented aggregate formation. We designed and synthesized a dendritic prodrug with four molecules of the anticancer agent camptothecin and a trigger that can be activated by penicillin-G-amidase. The PEG5000-conjugated, self-immolative dendritic prodrug was effectively activated by penicillin-G-amidase under physiological conditions and free camptothecin was released to the reaction media. Cell-growth inhibition assays demonstrated increased toxicity of the dendritic prodrug upon incubation with the enzyme.     

Enzymatic activation of hydrophobic self-immolative dendrimers: The effect of reporters with ionizable functional groups

Self-immolative dendrimers are uniquely structured molecules that release multiple tail units through a chain fragmentation initiated by a single cleavage at the dendrimer’s core. Although bioactivation of self-immolative dendritic molecules with only two reporter groups was demonstrated, enzymatic activation failed for self-immolative dendrimers with more reporters. These large and hydrophobic dendrimers aggregated under aqueous conditions and enzyme did not efficiently trigger chain fragmentation. Here we demonstrate a simple solution to the problem of enzymatic activation of hydrophobic self-immolative dendrimers. The reporter units on the dendritic platform were equipped with ionizable functional group. Polar interactions with water significantly decreased hydrophobicity of the dendrimers and prevented aggregate formation. Consequently, hydrophobic self-immolative dendrons were effectively activated.     

Synthesis and characterization of nanoscale dendritic RGD clusters for potential applications in tissue engineering and drug delivery

Spatial control over the distribution and the aggregation of arginine-glycine-aspartate (RGD) peptides at the nanoscale significantly affects cell responses. For example, nanoscale clustering of RGD peptides can induce integrins to cluster, thus triggering complete cell signaling. Dendrimers have a unique, highly branched, nearly spherical and symmetrical structure with low polydispersity, nanoscale size, and high functionality. Therefore, dendrimers are a class of ideal scaffold for construction of nanoscale dendritic RGD clusters in which RGD loading degree and cluster size can be finely adjusted. This new type of nanoscale dendritic RGD cluster will aid us to better understand the impact of spatial arrangement of RGD on cellular responses and to engineer RGD to trigger more favorable cellular responses. In this study, nanoscale dendritic RGD clusters were synthesized based on Starburst anionic G3.5 and cationic G4.0 polyamidoamine (PAMAM) dendrimers. The multiple terminal functional groups on the outermost layer of the dendrimer were coupled with RGD tripeptides. Biofunctionalized dendrimer structures were found to be highly dependent on the generation and the extent of peptide modification (ie, number of peptides per PAMAM dendrimer). Fluorescein isothiocyanate (FITC)-conjugated PAMAM dendrimers were utilized to monitor cellular internalization of dendrimers by adherent fibroblasts. Anionic G3.5-based dendritic RGD clusters have been shown to have no negative effect on fibroblast viability and a concentration-dependent effect on lowering cell adhesion on tissue culture polystyrene (TCPS) as that of free RGD. A similar concentration-dependent effect in cell viability and adhesion was also observed for cationic G4.0-based dendritic RGD clusters at lower but not at high concentrations. The results imply that the synthesized nanoscale dendritic RGD clusters have great potential for tissue engineering and drug delivery applications.     

Core and periphery functionalized dendrimers for transition metal catalysis; a covalent and a non-covalent approach

Dendrimers are well-defined hyperbranched macromolecules with characteristic globular structures for the larger systems. The recent impressive strides in synthetic procedures increased the accessibility of functionalized dendrimers at a practicable scale, resulting in a rapid development of dendrimer chemistry. Dendrimers have inspired many chemists to develop new materials and several applications have been explored, catalysis being one of them. The position of the catalytic site(s) as well as the spatial separation of the catalysts within the dendritic framework is of crucial importance. Dendrimers that are functionalized with transition metals in the core can potentially mimic properties of enzymes, their efficient natural counterparts, whereas the surface-functionalized systems have been proposed to fill the gap between homogeneous and heterogeneous catalysis. We prepared both core- and periphery-functionalized dendritic catalysts that are sufficiently large to enable separation by modern nanofiltration techniques. Here we review our recent findings using these promising novel transition metal-functionalized dendrimers as catalysts in several reactions. We will discuss some of the consequences of the architecturally different systems that have been studied and will elaborate on a novel non-covalent strategy of dendrimer functionalization.     

Potent antioxidant dendrimers lacking pro-oxidant activity

It is well known that antioxidants have protective effects against oxidative stress. Unfortunately, in the presence of transition metals, antioxidants, including polyphenols with potent antioxidant activities, may also exhibit pro-oxidant effects, which may irreversibly damage DNA. Therefore, antioxidants with strong free radical-scavenging abilities and devoid of pro-oxidant effects would be of immense biological importance. We report two antioxidant dendrimers with a surface rich in multiple phenolic hydroxyl groups, benzylic hydrogens, and electron-donating ring substituents that contribute to their potent free radical-quenching properties. To minimize their pro-oxidant effects, the dendrimers were designed with a metal-chelating tris(2-aminoethyl)amine (TREN) core. The dendritic antioxidants were prepared by attachment of six syringaldehyde or vanillin molecules to TREN by reductive amination. They exhibited potent radical-scavenging properties: 5 times stronger than quercetin and 15 times more potent than Trolox according to the 1,1-diphenyl-2-picrylhydrazyl assay. The antioxidant dendrimers also protected low-density lipoprotein, lysozyme, and DNA against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced free radical damage. More importantly, unlike quercetin and Trolox, the two TREN antioxidant dendrimers did not damage DNA via their pro-oxidant effects when incubated with physiological amounts of copper ions. The dendrimers also showed no cytotoxicity toward Chinese hamster ovary cells.     

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.     

Polyphenylene dendrimers as scaffolds for shape-persistent multiple peptide conjugates

The present work describes synthetic concepts for the coupling of peptides to polyphenylene dendrimers (PPDs). Novel functionalized cyclopentadienones have been synthesized whose Diels-Alder cycloaddition with various core molecules leads to polyphenylene dendrimers possessing (protected) amino or carboxyl groups. In addition, the resulting functionalized molecules exhibit the characteristic shape-persistence and monodispersity of PPDs. Their functions have been used for the attachment of polylysine to the dendritic scaffold. Three different methods for the decoration of dendrimers with polypeptides are presented. First, polylysine segments are grafted from the surface of the dendrimers employing alpha-amino acid N-carboxyanhydride (NCA) polymerization. Second, the C-terminal carboxyl groups of protected polypeptides are activated and then coupled to the amino groups on the surface of the PPD. Finally, cysteine terminated, unprotected peptide sequences are attached to polyphenylene dendrimers utilizing the addition of the sulfhydryl group of a cysteine to the maleimide functions on the dendrimer surface. Moreover, Diels-Alder cycloaddition of suitably functionalized cyclopentadienons to a desymmetized core molecule allows the design of a dendritic scaffold with a specific number of different anchor groups on its periphery. These approaches are important for the tailoring of new, shape-persistent, polyfunctional multiple antigen conjugates.     

PEGylated dendrimers with core functionality for biological applications

The synthesis of a variety of core functionalized PEGylated polyester dendrimers and their in vitro and in vivo properties are described in this report. These water-soluble dendrimers have been designed to carry eight functional groups on their dendritic core for a variety of biological applications such as drug delivery and in vivo imaging as well as eight solubilizing groups. Using a common symmetrical aliphatic ester dendritic core and trifunctional amino acid moieties, a library of dendrimers with phenols, alkyl alcohols, alkynes, ketones, and carboxylic acid functionalities has been synthesized without the need for column chromatography. The amines were PEGylated, leaving the other functionality of the amino acid available for further manipulation such as the attachment of drugs and/or labels. Radiolabeling experiments with the PEGylated dendrimers showed that they had a long circulation half-life in mice, confirming the potential of this class of dendrimers for therapeutic and/or diagnostic applications. A carboxylic acid functionalized dendrimer was elaborated to carry doxorubicin bound via a hydrazone bond. The drug-loaded carrier accumulated more in tumors and less in healthy organs than the clinically used PEGylated liposomal formulation Doxil. The efficient synthesis, high versatility, and favorable biological properties make these PEGylated polyester dendrimers promising structures for therapeutic and/or imaging applications.     

Design of dendritic macromolecules containing folate or methotrexate residues

Polyether dendritic compounds bearing folate residues on their surface were prepared as model drug carriers with potential tumor cell specificity. Starting from ester-terminated polyether dendrimers, hydrazide groups were easily introduced to the surface of the dendrimers by reaction with hydrazine. Folate residues were then conjugated to the hydrazide chain ends of the dendrimers by direct condensation with folic acid in the presence of a condensing agent or by reaction with an active ester derivative of folic acid. Essentially complete functionalization of the terminal hydrazide groups was achieved for both the first and the second generation dendrimers with four and eight hydrazide groups. For the G-2 dendrimer with 16 hydrazide groups, an average number of only 12.6 folate residues were attached to each dendrimer. The conjugates are soluble in aqueous medium above pH 7.4. In addition, a similar conjugation of the antitumor drug methotrexate to the dendrimer was also investigated. Once optimized, these molecules may form the basis for a novel family of multivalent drug carriers.     

Three generations of alpha,gamma-diaminobutyric acid modified poly(propyleneimine) dendrimers and their cisplatin-type platinum complexes

Three generations of alpha,gamma-diaminobutyric acid modified poly(propyleneimine) dendrimers [DAB(AM)n, n = 4, 8, 16] containing 4, 8, 16 free amino groups were coupled with Boc-protected alpha,gamma-diaminobutyric acid (DABA) moieties in high yields. These modified dendrimers were deprotected and the chiral dendritic amines with 8, 16 and 32 amino groups on the surface were isolated in excellent yields. Dendrimers with cisplatin moieties at the periphery were obtained in the reaction of the free amine dendrimers and potassium tetrachloroplatinate(II). The highly insoluble complexes were isolated as hydrates and characterized by means of IR, TGA and elemental analysis.     

Poly(propylene imine) dendrimers can bind to PEGylated albumin at PEG and albumin surface: Biophysical examination of a PEGylated platform to transport cationic dendritic nanoparticles

Cationic dendrimers are considered one of the best drug transporters in the body. However, in order to improve their biocompatibility, modification of them is required to reduce toxicity. In this way, many dendrimers may lose their original properties, for example, anticancer. To improve biocompatibility of dendrimers, it is possible to complex them with albumin, as is done very often in drug delivery. However, the interaction of dendrimers with albumin can lead to protein structure disruption or no complexation at all. Therefore, the investigation of the interaction between cationic poly-(propylene imine) dendrimers and polyethylene glycol (PEG)-albumin by fluorescence, circular dichroism, small angle X-ray scattering (SAXS), and transmission electron microscopy was carried out. Results show that cationic dendrimers bind to PEGylated albumin at PEG and albumin surfaces. The obtained results for 5k-PEG indicate a preferential binding of the dendrimers to PEG. For 20k-PEG binding of dendrimers to PEG and protein could induce a collapse of the PEG chain onto the protein surface. This opens up new possibilities to the use of PEGylated albumin as a platform to carry dendrimers without changing the albumin structure and improve the pharmacokinetic properties of dendrimers without further modification.     

Combinatorial synthesis, selection, and properties of esterase peptide dendrimers

A 65,536-member combinatorial library of peptide dendrimers was prepared by split-and-mix synthesis and screened on solid support for esterolytic activity in aqueous buffer using 8-butyryloxypyrene-1,3,6-trisulfonate (2) as a fluorogenic substrate. Active sequences were identified by analysis of fluorescent beads. The corresponding dendrimers were resynthesized by solid-phase synthesis, cleaved from the resin, and purified by preparative reverse-phase HPLC. The dendrimers showed the expected catalytic activity in aqueous buffer. Catalysis was studied against a pannel of fluorogenic 8-acyloxypyrene-1,3,6-trisulfonate substrates. The catalytic peptide dendrimers display enzyme-like kinetics in aqueous buffer with substrate binding in the range K(M) approximately 0.1 mM, catalytic rate constants k(cat) approximately 0.1 min(-1), and specific rate accelerations over background up to k(cat)/k(uncat) = 10,000.     

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.     

Genotoxicity of poly(propylene imine) dendrimers

Dendrimers are highly branched macromolecules with the potential in biomedical applications. Due to positively charged surfaces, several dendrimers reveal toxicity. Coating peripheral cationic groups with carbohydrate residues can reduce it. In this study, the cytotoxicity and genotoxicity of three types of 4th generation poly(propylene imine) dendrimers were investigated. Peripheral blood mononuclear cells (PBMCs) were treated with uncoated (PPI-g4) dendrimers, and dendrimers in which approximately 40% or 90% of peripheral amino groups were coated with maltotriose (PPI-g4-OS or PPI-g4-DS) at concentration of 0.05, 0.5, 5 mg/ml. Abbreviations OS and DS stand for open shell and dense shell respectively, that describes the structure of carbohydrate modified dendrimers. After 1 h of cell incubation at 37°C, the MTT and comet assays were performed. PPI dendrimers demonstrated surface-modification-degree dependent toxicity, although genotoxicity of PPI-g4 and PPI-g4-OS measured by the comet assay was concentration dependent up to 0.5 mg/ml and at 5 mg/ml the amount of DNA that left comet's head decreased. Results may suggest a strong interaction between dendrimers and DNA, and furthermore, that coating PPI dendrimers by maltoriose is an efficient method to reduce their genotoxicity what opens the possibilities to use them as therapeutic agents or drug carriers.     

Enhanced porphyrin accumulation using dendritic derivatives of 5-aminolaevulinic acid for photodynamic therapy: an in vitro study

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid has been widely used in photodynamic therapy for a range of malignant and certain non-malignant lesions. However, cellular uptake of 5-aminolaevulinic acid is limited by its hydrophilic nature and improved means of delivery are therefore being sought. Highly branched polymeric drug carriers known as dendrimers are a promising new approach to drug delivery. The aim of this study was to investigate the efficacy of dendrimers conjugated with 5-aminolaevulinic acid for porphyrin production in the transformed PAM 212 keratinocyte cell line and skin explants. Each dendritic derivative incorporated three 5-aminolaevulinic acid residues which were conjugated as esters via methyl or propyl linkers to a central tertiary carbon whose remaining terminal bore an amino, aminobenzyloxycarbonyl or nitro group. In the cell line, all compounds were more efficient at low concentrations compared to equimolar 5-aminolaevulinic acid for porphyrin production, with the most efficient incorporating the longer propyl linker. This compound was also the most lipophilic according to partition coefficient measurements. The intracellular porphyrin fluorescence levels showed good correlation with cellular phototoxicity following light exposure for all the compounds, together with minimal dark toxicity. Our findings indicate that the key factors influencing the efficacy of the dendritic derivatives are lipophilicity and steric hindrance within the dendritic structure which could restrict access to intracellular esterases for liberation of 5-aminolaevulinic acid. These findings should be taken into account in the design of larger dendrimers of 5-aminolaevulinic acid.     

Light Harvesting Dendrimers

Tree-like dendrimers with decreasing number of chromophores from periphery to core is an attractive candidate for light-harvesting applications. Numerous dendritic designs with different kinds of light-collecting chromophores at periphery and an energy-sink at the core have been demonstrated with high energy transfer efficiency. These building blocks are now being developed for several applications such as light-emitting diodes, frequency converters and other photonic devices. This review outlines the efforts that are based on both conjugated and non-conjugated dendrimers.     

Guanidino- and urea-modified dendrimers as potent solubilizers of misfolded prion protein aggregates under non-cytotoxic conditions. dependence on dendrimer generation and surface charge

Amino-terminated dendrimers are well-defined synthetic hyperbranched polymers and have previously been shown to destabilize aggregates of the misfolded, pathogenic, and partially protease-resistant form of the prion protein (PrPSc), transforming it into a partially dissociated, protease-sensitive form with strongly reduced infectivity. The mechanism behind this is not known, but a low pH, creating multiple positively charged primary amines on the dendrimer surface, increases the efficiency of the reaction. In the present study, surface amines of the dendrimers were modified to yield either guanidino surface groups (being positively charged at neutral pH) or urea groups (uncharged). The ability of several generations of modified dendrimers and unmodified amino-terminated dendrimers to deplete PrPSc from persistently PrPSc-infected cells in culture (SMB cells) was studied. It was found that destabilization correlated with both the generation number of the dendrimer, with higher generations being more efficient, and the charge density of the surface groups. Urea-decorated dendrimers having an uncharged surface were less efficient than positively charged unmodified- (amino) and guanidino-modified dendrimers. The most efficient dendrimers (generation 4 (G4) and G5-unmodified and guanidino dendrimers) cleared PrPSc completely by incubation for 4 days at less than 50 nM. In contrast to both unmodified and guanidine-modified dendrimers, the uncharged urea dendrimers showed much lower cytotoxicity toward noninfected SMB cells. Therapeutic uses of modified dendrimers are indicated by the low concentrations of dendrimers needed.     

SiRNA delivery systems based on neutral cross-linked dendrimers

A neutral cross-linked dendritic system is described for use of delivering small interfering RNA (siRNA) for targeted gene silencing. By replacing the terminal amines with hydrazide groups and N-acetylgalactosamine (GalNAc) ligands, cationic polyamidoamine (PAMAM) dendrimers were transformed into neutral glycosylated carriers for siRNA delivery. Mainly owing to the pH sensitivity and the proton-absorption capability of the tertiary amines within the interior branches, these PAMAM derivatives showing neutrality under physiological conditions (pH 7.4) formed complexes with siRNA through electrostatic interactions at pH 5. Cross-linking procedures via reactions with glutaraldehyde were established, and cytocompatible cross-linked systems loaded with siRNA obtained, with the particulate properties variable with the cross-linking condition and the GalNAc level in the dendritic carrier. In vitro cellular uptake and RNAi experiments showed that the cross-linked dendritic systems with an appropriate level of GalNAc composition effectively interacted with HepG2 cells and inhibited the expression of a luciferase reporter gene. Neutral cross-linked dendritic systems provide a new paradigm for designing siRNA delivery systems with biocompatibility and targeting capability.     

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.     

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.