PAMAM dendrimers for the delivery of the antibacterial Triclosan

Many oral care products incorporate an antibacterial compound to prevent the formation of dental plaque which predisposes teeth to dental caries or periodontal disease. Triclosan (TCN) is a commonly used antiplaque agent in toothpastes. Strategies to increase the delivery efficiency of antibacterials using formulation aids such as polyamidoamine (PAMAM) dendrimers are of interest. Solubilisation studies over the pH range 5-12 demonstrated an increase in the level of TCN solubilised with increasing dendrimer concentration (1 mM-5 mM). However, the dendrimer was unable to enhance TCN solubility at lower pH values and the solubilising effect observed was attributed to the ionization of TCN (pKa 8.14) resulting from dendrimer induced pH changes. End group modification of G3 PAMAM dendrimer with phenylalanine in order to promote solubility through pi-pi stacking between TCN and the amino acid has been carried out. Phenylalanine:G3 PAMAM conjugates of different ratios (32:1, 21:1, 16:1) were synthesized. The fully conjugated dendrimer (32:1) had poor aqueous solubility, whereas the 21:1 and 16:1 dendrimer conjugates were water soluble. The 21:1 conjugate was tested for its ability to solubilise TCN, however, again there was no increase over control buffer solutions of the same pH. An alternative approach under investigation is to directly conjugate TCN to PAMAM dendrimers via a hydrolysable linkage.     

PAMAM dendrimers for the delivery of the antibacterial Triclosan

Many oral care products incorporate an antibacterial compound to prevent the formation of dental plaque which predisposes teeth to dental caries or periodontal disease []. Triclosan (TCN) is a commonly used antiplaque agent in toothpastes []. Strategies to increase the delivery efficiency of antibacterials using formulation aids such as polyamidoamine (PAMAM) dendrimers are of interest.

Solubilisation studies over the pH range 5-12 demonstrated an increase in the level of TCN solubilised with increasing dendrimer concentration (1 mM–5 mM). However, the dendrimer was unable to enhance TCN solubility at lower pH values and the solubilising effect observed was attributed to the ionization of TCN (pKa 8.14) resulting from dendrimer induced pH changes.

End group modification of G3 PAMAM dendrimer with phenylalanine in order to promote solubility through π–π stacking between TCN and the amino acid has been carried out. Phenylalanine:G3 PAMAM conjugates of different ratios (32:1, 21:1, 16:1) were synthesized. The fully conjugated dendrimer (32:1) had poor aqueous solubility, whereas the 21:1 and 16:1 dendrimer conjugates were water soluble. The 21:1 conjugate was tested for its ability to solubilise TCN, however, again there was no increase over control buffer solutions of the same pH. An alternative approach under investigation is to directly conjugate TCN to PAMAM dendrimers via a hydrolysable linkage.     

Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers

PAMAM dendrimers are cationic polymers that have been used for the delivery of genes and oligonucleotides to cells. However, little is known about the behavior of dendrimer–nucleic acid complexes once they reach the cell interior. To pursue this issue, we prepared dendrimers conjugated with the fluorescent dye Oregon green 488. These were used in conjunction with oligonucleotides labeled with a red (TAMRA) fluorophore in order to visualize the sub-cellular distribution of the dendrimer–oligonucleotide complex and of its components by two-color digital fluorescence microscopy. The 2′-O-methyl antisense oligonucleotide sequence used in these studies was designed to correct splicing at an aberrant intron inserted into a luciferase reporter gene; thus effective delivery of the antisense agent results in the expression of the reporter gene product. The dendrimer–oligonucleotide complex remained associated during the process of uptake into vesicular compartments and eventual entry into the nucleus. Since the pharmacological activity of the antisense compound was manifest under these conditions, it suggests that the dendrimer–oligonucleotide complex is functionally active. A surprising result of these studies was that the Oregon green 488-conjugated dendrimer was a much better delivery agent for antisense compounds than unmodified dendrimer. This suggests that coupling of relatively hydrophobic small molecules to PAMAM dendrimers may provide a useful means of enhancing their capabilities as delivery agents for nucleic acids.     

Targeted delivery of doxorubicin: Drug delivery system based on PAMAM dendrimers

Polyamidoamine (PAMAM) dendrimers of the second generation (G2) are branched polymers containing 16 surface amino groups that allow them to be used as universal carriers on creating systems for drug delivery. G2 labeled with fluorescein isothiocyanate (FITC) efficiently bound with the surface of tumor cells at 4°C and was absorbed by the cells at 37°C. The covalent binding to G2-FITC of a vector protein, a recombinant fragment of the human alpha-fetoprotein receptor-binding domain (rAFP3D), increased the binding and endocytosis efficiency more than threefold. Covalent conjugates of G2 with doxorubicin (Dox) obtained by acid-labile linking of cis-aconitic anhydride (CAA) without the vector protein (G2-Dox) and with the vector protein rAFP3D (rAFP3D-G2-Dox) were accumulated by the tumor cells with high efficiency. However, a selective effect was observed only in rAFP3D-G2-Dox, which also demonstrated high cytotoxic activity against the human ovarian adenocarcinoma SKOV3 cells and low cytotoxicity against human peripheral blood lymphocytes. Based on these results, rAFP3D-G2 conjugate is promising for selective delivery of antitumor drugs.     

Design of riboflavin-presenting PAMAM dendrimers as a new nanoplatform for cancer-targeted delivery

This communication describes the synthesis and in vitro biological evaluation of novel generation 5 PAMAM dendrimers conjugated with riboflavin as a targeting ligand. Cell-based experiments demonstrated that a dendrimer conjugated with riboflavin is able to undergo cellular binding and uptake in KB cells, and when the dendrimer is also conjugated with methotrexate, the riboflavin dendrimer conjugate can potently inhibit cell growth.     

Surface-modified and internally cationic polyamidoamine dendrimers for efficient siRNA delivery

A novel internally quaternized and surface-acetylated poly(amidoamine) generation four dendrimer (QPAMAM-NHAc) was synthesized and evaluated for intracellular delivery of siRNA. The proposed dendrimer as a nanocarrier possesses the following advantages: (1) modified neutral surface of the dendrimer for low cytotoxicity and enhanced cellular internalization; (2) existence of cationic charges inside the dendrimer (not on the outer surface) resulting in highly organized compact nanoparticles, which can potentially protect nucleic acids from degradation. The properties of this dendrimer were compared with PAMAM-NH 2 dendrimer, possessing surface charges, and with an internally quaternized charged and hydroxyl-terminated QPAMAM-OH dendrimer. Atomic force microscopy studies revealed that internally charged and surface neutral dendrimers, QPAMAM-OH and QPAMAM-NHAc, formed well-condensed, spherical particles (polyplexes) with siRNA, while PAMAM-NH 2 resulted in the formation of nanofibers. The modification of surface amine groups to amide significantly reduced cytotoxicity of dendrimers with QPAMAM-NHAc dendrimer showing the lowest toxicity. Confocal microscopy demonstrated enhanced cellular uptake and homogeneous intracellular distribution of siRNA delivered by the proposed QPAMAM-NHAc nanocarrier. The results clearly demonstrated distinct advantages of developed QPAMAM-NHAc/siRNA polyplexes over the existing nucleic acid dendrimeric carriers.     

Anionic PAMAM dendrimers as drug delivery vehicles for transition metal-based anticancer drugs

The use of anionic half-generation poly(amidoamine) dendrimers as drug delivery vehicles for [Pt(S,S-dach)(5,6-Me₂phen)]²⁺ (56MESS) (where S,S-dach = 1S,2S-diaminocyclohexane; 5,6-Me₂phen = 5,6-dimethyl-1,10-phenanthroline) and [{Δ,Δ-Ru(phen)₂}₂(μ-bb7)]⁴⁺ (Rubb₇) (where phen = 1,10-phenanthroline; bb7 = 1,7-bis[4-(4′-methyl-2,2′-bipyridyl)heptane]) has been studied by nuclear magnetic resonance spectroscopy. From one- and two-dimensional ¹H NMR spectra both 56MESS and Rubb₇ were found to bind to the surface of generation 3.5, 4.5, 5.5 and 6.5 dendrimers through electrostatic interactions. The higher charge and larger size of Rubb₇ resulted in stronger binding to all dendrimer generations (K_b ? 2 × 10⁵ M⁻¹) compared with 56MESS (K_b ? 1 × 10⁴ M⁻¹). Interestingly, there appeared to be no observable trend between dendrimer size and binding constant strength. The size of the free and 56MESS-bound dendrimers were examined using pulsed-gradient spin-echo NMR. The dendrimers ranged in hydrodynamic diameter from 11 to 20 nm and in all cases were larger than their corresponding full-generation dendrimer. Upon the addition of 56MESS the diameter of the dendrimers increased, consistent with surface binding.     

Aptamer-targeted delivery of Bcl-xL shRNA using alkyl modified PAMAM dendrimers into lung cancer cells

RNAi-based gene therapy has been recently considered as a promising approach against cancer. Targeted delivery of drug, gene or therapeutic RNAi-based systems to tumor cells is one of the important issues in order to reduce side effects on normal cells. Several strategies have been developed to improve the safety and selectivity of cancer treatments including antibodies, peptides and recently aptamers with various attractive characteristics including higher target specificity, affinity and reduced toxicity. Here we described a novel targeted delivery platform comprising modified PAMAM with 10-bromodecanoic acid (10C) and 10C-PEG for improvement of transfection efficiency, AS1411 aptamer for targeting nucleolin ligand on target cancer cells and shRNA plasmid for specific knockdown of Bcl-xL protein. Modified vector could significantly improve the transfection efficiency even after covalent or non-covalent aptamer binding compared to the non-targeted vector in A549 cells. The results of gene silencing and apoptosis assay indicated that our targeted shRNA delivery system could efficiently down-regulate the Bcl-xL expression up to 25% and induce 14% late apoptosis in target cancer cells with strong cell selectivity. This study proposed a novel targeted non-viral system for shRNA-mediated gene-silencing in cancer cells.     

Interactions between PAMAM dendrimers and bovine serum albumin

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

Polyamidoamine (Yet Not PAMAM) dendrimers as bioinspired materials for drug delivery: structure-activity relationships by molecular simulations

In this paper, we report the results obtained from a comprehensive characterization of newly synthesized dendrimeric molecules in a solvated environment, by computer-aided simulations. The evidences allowed us to formulate some structure-activity relationships (SARs) between the experimentally verified cytotoxicity/noncytotoxicity of these compounds and some molecular features such as, for instance, radius of gyration, molecular shape, and dimensions. In particular, all noncytotoxic dendrimers were characterized by a more dense and globular shape and by a smoother surface pattern, as quantified by their fractal dimension D.     

The interaction of tryptophan and ANS with PAMAM dendrimers

Dendrimers are globular, hyperbranched polymers possessing a high concentration of surface functional groups and internal cavities. These unique features make them very useful in many biomedical applications, especially as carrier molecules. In this study, the interaction of tryptophan and 1-anilinonaphthalene-8-sulfonic acid with three types of polyamidoamine dendrimers was examined. It was observed that the type of dendrimer surface group has a strong impact on the interactions between the dendrimers and fluorescent molecules.     

Synthesis of symmetrical and unsymmetrical PAMAM dendrimers by fusion between azide- and alkyne-functionalized PAMAM dendrons

A new convergent synthetic method for the synthesis of PAMAM dendrimers has been developed. The fusion between propargyl-functionalized PAMAM dendrons and azido-functionalized PAMAM dendrons via the Cu(I)-catalyzed Huisgen [2 + 3] dipolar cycloaddition reaction (click chemistry) of an alkyne and an azide leads to the formation of symmetric PAMAM dendrimers in high yields. Furthermore, the coupling reactions between the different generation dendrons afford the size-differentiated unsymmetrical PAMAM dendrimers.     

Electrostatic theory of the assembly of PAMAM dendrimers and DNA

The electrostatic interactions mediated by counterions between a cationic PAMAM dendrimer, modelized as a sphere of radius and cationic surface charge highly increasing with generation, and a DNA, modelized as an anionic elastic line, are analytically calculated in the framework of condensation theory. Under these interactions the DNA is wrapped around the sphere. For excess phosphates relative to dendrimer primary amines, the free energy of the DNA‐dendrimer complex displays an absolute minimum when the complex is weakly negatively overcharged. This overcharging opposes gene delivery. For a highly positive dendrimer and a DNA fixed by experimental conditions to a number of phosphates less than the number of dendrimer primary amines, excess amine charges, the dendrimer may at the same time bind stably DNA and interact with negative cell membranes to activate cell transfection in fair agreement with molecular simulations and experiments. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 276–286, 2016.     

Synthesis of new polymer-bound adenine nucleotides using starburst PAMAM dendrimers

Two types of new polymer-bound adenine nucleotides were synthesized by coupling adenine nucleotides (ATP and ADP) with starburst polyamidoamine (PAMAM) dendrimers. The first type was obtained by coupling native adenine nucleotides directly with a carboxy-terminated PAMAM dendrimer. In the second type, the nucleotides were modified by introducing a spacer arm containing a carboxylic end group (N(6)-R-ATP and N(6)-R-ADP) and coupled with an amine-terminated PAMAM dendrimer. Both types of the dendrimers were coupled with native or the modified nucleotides using the well-known carbodiimide activation technique. The optimum coupling pH and temperature were 4 and 30 degrees C, respectively, for preparing the carboxy-terminated PAMAM-bound ATP or ADP, and were 9 and 50 degrees C, respectively, for preparing the amine-terminated PAMAM-bound N(6)-R-ATP or N(6)-R-ADP. The ATP or ADP contents in the synthesized polymers were found to be 4 mol of ATP or of ADP/mol of carboxy-terminated PAMAM-bound ATP or ADP and 25 mol of ATP or of ADP/mol of amine-terminated PAMAM-bound N(6)-R-ATP or N(6)-R-ADP. The coenzymatic activities relative to the native ATP of the carboxy-terminated PAMAM-bound ATP against glucokinase and hexokinase were 16 and 7%, respectively, and those of the amine-terminated PAMAM-bound N(6)-R-ATP 2 and 1%, respectively. The coenzymatic activities relative to the native ADP of the carboxy-terminated PAMAM-bound ADP and the amine-terminated PAMAM-bound N(6)-R-ADP against acetate kinase were 24 and 3.5%, respectively.     

Dicer-labile PEG conjugates for siRNA delivery

Poly(ethylene glycol) (PEG) conjugates of Dicer-substrate small interfering RNA (DsiRNA) have been prepared to investigate a new siRNA release strategy. 3'-sense or 5'-antisense thiol-modified, blunt-ended DsiRNAs, inhibiting enhanced green fluorescent protein (eGFP) expression, were covalently conjugated to PEG with varying molecular weights (2, 10, and 20 kg/mol) through a stable thioether bond using a Michael addition reaction. The DsiRNA conjugates with 2 kg/mol PEG (both 3'-sense or 5'-antisense strand conjugated) and the 10 kg/mol PEG conjugated to the 3'-sense strand of DsiRNA were efficiently cleaved by recombinant human Dicer to 21-mer siRNA, as determined by gel electrophoresis. Importantly, 2 and 10 kg/mol PEG conjugated to the 3'-sense strand of DsiRNA showed potent gene silencing activity in human neuroblastoma (SH-EP) cells, stably expressing eGFP, at both the mRNA and protein levels. Moreover, the 10 kg/mol PEG conjugates of the 3'-sense strand of DsiRNA were less immunogenic when compared with the unmodified DsiRNA, determined via an immune stimulation assay on human peripheral blood mononuclear cells.     

siRNA delivery technologies for mammalian systems

Inhibition of gene expression using the RNA interference (RNAi) pathway is rapidly becoming the method of choice for studying gene function in mammalian cells. However, successful knockdown of the target gene requires efficient delivery of short interfering RNAs (siRNAs). Several technologies have been developed that enable effective delivery of siRNAs to both cells in culture and whole animals. These technologies will allow the use of RNAi to study gene function in mammalian model systems in which classical methods are often limited and costly.     

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.     

Structural studies of biologically active glycosylated polyamidoamine (PAMAM) dendrimers

The partial modification of carboxylic acid terminated polyamidoamine (PAMAM) dendrimers with glucosamine has been reported to give dendrimer glucosamine conjugates novel immuno-modulatory and anti-angiogenic properties. Experimental analysis of these glycosylated dendrimers showed that, on average, eight glucosamine molecules were covalently bound to each dendrimer. In order to better understand the surface loading and distribution of these glucosamine molecules, molecular reactivity was determined by evaluation of electronic properties using frontier molecular orbital theory (FMOT) and molecular dynamics simulations. It was shown that the surface loading and distribution of zero length amide bond-conjugated glucosamine molecules was determined by both electronic effects and by the different dynamic conformations adopted by the modified dendrimer during the incremental addition of glucosamine. Importantly, the structural features and the dynamic behavior of the partially glycosylated generation 3.5 PAMAM dendrimer showed that its flexibility and polarity changed with the incremental addition of glucosamine. These peripheral glucosamine molecules remained available on the dendrimer’s surface for interaction with the biological target.