Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs

Polyamidoamine dendrimers having poly(ethylene glycol) grafts were designed as a novel drug carrier which possesses an interior for the encapsulation of drugs and a biocompatible surface. Poly(ethylene glycol) monomethyl ether with the average molecular weight of 550 or 2000 was combined to essentially every chain end of the dendrimer of the third or fourth generation via urethane bond. The poly(ethylene glycol)-attached dendrimers encapsulating anticancer drugs, adriamycin and methotrexate, were prepared by extraction with chloroform from mixtures of the poly(ethylene glycol)-attached dendrimers and varying amounts of the drugs. Their ability to encapsulate these drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts. Among the poly(ethylene glycol)-attached dendrimers prepared, the highest ability was achieved by the dendrimer of the fourth generation having the poly(ethylene glycol) grafts with the average molecular weight of 2000, which could retain 6.5 adriamycin molecules or 26 methotrexate molecules/dendrimer molecule. The methotrexate-loaded poly(ethylene glycol)-attached dendrimers released the drug slowly in an aqueous solution of low ionic strength. However, in isotonic solutions, methotrexate and adriamycin were readily released from the poly(ethylene glycol)-attached dendrimers.     

Remnant cationic dendrimers block RNA migration in electrophoresis after monophasic lysis

Cationic dendrimers such as poly(amidoamine) (PAMAM) and poly(propyleneimine) (PPI) have attractive characteristics for the delivery of nucleic acid and various biomedical applications. Most studies have focused on cationic dendrimer-based intracellular delivery, and very few studies have focused on the non-specific interaction of remnant cationic dendrimers with total RNA after isolation directly from cells in vitro. We examined RNA isolation using the common method of monophasic lysis from human macrophage-like cells (U937) and mouse fibroblast cells (NIH/3T3) that had been exposed to dendrimers and DNA/dendrimer complexes using gel electrophoresis. We found that PAMAM and PPI dendrimers strongly altered the mobility of RNA in the gels. In addition, the extent of dendrimer-induced alteration in RNA mobility was directly dendrimer-generation-dependent: the alteration was greater with higher-generation dendrimers. We also found that DNA/dendrimer complexes at higher dendrimer to DNA ratios interacted with RNA after isolation while gene expression was maintained. The interactions between RNA and remnant dendrimers after isolation were caused by electrostatic bindings, and we recovered total RNA using high ionic strength solvents (2M NaCl solution) to disrupt the electrostatic forces binding dendrimers to RNA. Because RNA isolation is routinely used for biological applications, such dendrimer-induced alteration in RNA mobility should be accounted for in the further processing of RNA-related applications.     

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.     

Direct plant gene delivery with a poly(amidoamine) dendrimer

Plant gene delivery is challenging due to the presence of plant cell walls. Conventional means such as Agrobacterium infection, biolistic particle bombardment, electroporation, or polyethylene glycol attachment are often characterized by high cost, labor extensiveness, and a significant perturbation to the growth of cells. We have succeeded in delivering GFP-encoding plasmid DNA to turfgrass cells using poly(amidoamine) dendrimers. Our new scheme utilizes the physiochemical properties as well as the nanosize of the poly(amidoamine) dendrimer for direct and noninvasive gene delivery. The GFP gene was expressed in the plant cells as observed by confocal fluorescence microscopy. The transfection efficiency may be further improved by optimizing the pH of the cell culture medium and the molar ratio of the dendrimer to DNA. The use of the current delivery system can be extended to virtually all plant species having successful regeneration systems in place.     

Dendrimers destabilize proteins in a generation-dependent manner involving electrostatic interactions

Dendrimers are well-defined chemical polymers with a characteristic branching pattern that gives rise to attractive features such as antibacterial and antitumor activities as well as drug delivery properties. In addition, dendrimers can solubilize prion protein aggregates at very low concentrations, but their mode of action is unclear. We show that poly(propylene imine) dendrimers based on di-aminobutane (DAB) and modified with guanidinium surface groups reduce insulin thermostability and solubility considerably at microgram per microliter concentrations, while urea-modified groups have hardly any effect. Destabilization is markedly generation-dependent and is most pronounced for generation 3, which is also the most efficient at precipitating insulin. This suggests that proteins can interact with both dendrimer surface and interior. The pH-dependence reveals that interactions are mainly mediated by electrostatics, confirmed by studies on four other proteins. Ability to precipitate and destabilize are positively correlated, in contrast to conventional small-molecule denaturants and stabilizers, indicating that surface immobilization of denaturing groups profoundly affects its interactions with proteins.     

Using ethidium to probe nonequilibrium states of DNA condensed for gene delivery

Here we explore the use of ethidium to determine relative affinities of different gene delivery vectors for DNA and describe an improved method for studying the interaction. Specifically, we investigate the binding of poly(amidoamine) dendrimers and show that the DNA-dendrimer-ethidium system is far from thermodynamic equilibrium. Moreover, dendrimer surface modification through PEGylation appears to make the interaction with DNA more reversible, which is favorable from the perspective of vector unpacking. Probing the nonequilibrium state of DNA during condensation processes is thus important for developing novel vectors, and further, it could also be useful in the study of chromatin folding.     

In vitro gene delivery using polyamidoamine dendrimers with a trimesyl core

Polyamidoamine (PAMAM) dendrimer represents one of the most efficient polymeric gene carriers. To investigate the effect of the core structure and generation of dendrimers on the complex formation and transfection efficiency, a series of PAMAM dendrimers with a trimesyl core (DT) at different generations (DT4 to DT8) were developed as gene carriers and compared with the PAMAM dendrimers derived from pentaerythritol (DP) and inositol (DI). The minimal generation number of DTs at which the dendrimer has enough amino group density to effectively condense DNA was higher (generation 6) than those of DPs and DIs (generation 5). DTs of generation 6 or higher condensed DNA into complexes with an average diameter ranging from 100 to 300 nm, but the 4th and 5th generations of DT (DT4 and DT5) formed only a severe aggregate with DNA. Interestingly, the DT6/pDNA complex was determined to be much smaller (100-300 nm) than those prepared with DP5 or DI5 (>600 nm) at N/P ratios higher than 15. The optimal generation numbers at which the dendrimers showed the highest transgene expression in COS-7 cells were 5 for DPs and DIs but 6 for DTs. The DT6/pDNAcomplex with smaller size mediated higher transgene expression in COS-7 cells than those prepared with DP5 or DI5. The in vitro transfection efficiency of the DT dendrimers as evaluated in HeLa cells, COS-7 cells, and primary hepatocytes decreased in the order of DT6 > DT7 > DT8 > DT5 > DT4. The transfection mediated by DT6 was significantly inhibited by bafilomycin A1. The acid-base titration curve for DT6 showed high buffer capacity in the pH range from 5.5 to 6.4 (pK(a) approximately 6). This permits dendrimers to buffer the pH change in the endosomal compartment. However, the transfection efficiency mediated by DT6 decreased significantly in the presence of serum in both HeLa cells and COS-7 cells. The cytotoxicity of DTs evaluated in HeLa cells using the 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide assay showed a trend of increasing toxicity with the polymer generations. The LD50 values of DT4 through DT8 were 628, 236, 79, 82, and 77 microg/mL, respectively, which were higher than that of poly(ethyleneimide) (18 microg/mL) and poly(L-lysine) (28 microg/mL) in the same assay. With a lower cytotoxicity and versatility for chemical conjugation, these PAMAM dendrimers with a DT core warrant further investigation for nonviral gene delivery.     

Enhancement of gene expression by polyamidoamine dendrimer conjugates with alpha-, beta-, and gamma-cyclodextrins.

To improve the transfection efficiency of nonviral vector, we synthesized the starburst polyamidoamine dendrimer conjugates with alpha-, beta-, and gamma-cyclodextrins (CDE conjugates), expecting the synergistic effect of dendrimer and cyclodextrins (CyDs). The (1)H NMR spectroscopic data indicated that alpha-, beta-, and gamma-CyDs are covalently bound to dendrimer in a molar ratio of 1:1. The agarose gel electrophoretic studies revealed that CDE conjugates formed the complexes with plasmid DNA (pDNA) and protected the degradation of pDNA by DNase I in the same manner as dendrimer. CDE conjugates showed a potent luciferase gene expression, especially in the dendrimer conjugate with alpha-CyD (alpha-CDE conjugate) which provided the greatest transfection activity (approximately 100 times higher than those of dendrimer alone and of the physical mixture of dendrimer and alpha-CyD) in NIH3T3 and RAW264.7 cells. In addition, the gene transfer activity of alpha-CDE conjugate was superior to that of Lipofectin. The enhancing gene transfer effect of alpha-CDE conjugate may be attributable to not only increasing the cellular association, but also changing the intracellular trafficking of pDNA. These findings suggest that alpha-CDE conjugate could be a new preferable nonviral vector of pDNA.     

Acid- and salt-triggered multifunctional poly(propylene imine) dendrimer as a prospective drug delivery system

A novel dendrimeric compound is designed with the objective of simultaneously addressing issues commonly encountered in drug delivery, i.e., stability in biological milieu as well as targeting. For this purpose, a multifunctional dendrimeric system derived from diaminobutane poly(propylene imine) dendrimers (DAB) is prepared bearing at its external surface poly(ethylene glycol) chains and guanidinium moieties. For these moieties, it has been established that they exhibit protective and targeting properties, respectively. The release of encapsulated compounds is triggered by titration with acids followed by the addition of sodium chloride solution. Specifically for pyrene, the solubilization site of which can be clearly traced, protonation leads to a distribution between the core and the poly(ethylene glycol) chains in the periphery of the dendrimer while it is released to the aqueous bulk solution by the addition of sodium chloride. The release of betamethasone valerate is also triggered by the addition of sodium chloride solution.     

Lysine dendrimers as vectors for delivery of genetic constructs to eukaryotic cells

Asymmetrical lysine dendrimers are promising as vectors for delivering gene expression constructs into mammalian cells. The condensing, protective, and transfection properties were studied for pentaspherical lysine dendrimer D5 and its analog D5C10, modified with capric acid residues at the outer sphere; in addition, the transfection activity was assayed for complexes DNA-dendrimer-endosomolytic peptide JTS-1. Fatty acid residues incorporated in lysine dendrimers proved to improve their ability to bind DNA, to protect DNA from nuclease degradation, and to ensure its transfer into the nucleus. Peptide JTS-1 introduced in DNA-dendrimer complexes significantly increased their transfection activity. The potentiating effect of JTS-1 was especially high with the DNA-D5C10 complex. An excess of JTS-1 changed the structure of the complexes and reduced their transfection activity. It was assumed that dendrimers D5 and D5C10 are promising vectors for DNA delivery to eukaryotic cells and provide a basis for constructing more refined nonviral module carriers.     

Synthesis of a folate functionalized PEGylated poly(propylene imine) dendrimer as prospective targeted drug delivery system

Based on fourth generation diaminobutane poly(propylene imine) dendrimer, a novel targeted drug nanocarrier was prepared, bearing protective PEG chains and a folate targeting ligand. As a control a PEGylated derivative without folate was also synthesized. The encapsulation and release properties of these PEGylated derivatives were investigated employing etoposide, an anticancer hydrophobic drug. Enhanced solubility of etoposide was achieved inside the dendrimeric scaffold which was subsequently released in a controlled manner. These properties coupled with specificity towards the folate receptor and the low toxicity render folate functionalized PEGylated poly(propylene imine) dendrimer promising candidate for targeted drug delivery.     

Transfection activity of polyamidoamine dendrimers having hydrophobic amino acid residues in the periphery

We designed poly(amidoamine) dendrimers with phenylalanine or leucine residues at their chain ends. Thereby, we achieved efficient gene transfection of cells through synergy of the proton sponge effect, which is induced by the internal tertiary amines of the dendrimer, and hydrophobic interaction by the hydrophobic amino acid residues in the dendrimer periphery. Dendrimers having 16, 29, 46, and 64 terminal phenylalanine residues were prepared by the reaction of the amine-terminated poly(amidoamine) G4 dendrimer and L-phenylalanine using condensing reagent 1,3-dicyclohexylcarbodiimide. Transfection activity of these phenylalanine-modified dendrimers for CV1 cells, an African green monkey kidney cell line, increased concomitant with the increasing number of the terminal phenylalanine residues, except for the dendrimer with 64 phenylalanine residues, which showed poor water solubility and hardly formed a complex with DNA at neutral pH. However, under weakly acidic conditions, the dendrimer with 64 phenylalanine residues formed a complex with DNA, thereby achieving highly efficient transfection. In contrast, the attachment of L-leucine residues was unable to improve the transfection activity of the parent dendrimer, probably because of the relatively lower hydrophobicity of this amino acid. The phenylalanine-modified dendrimer exhibited a higher transfection activity and a lower cytotoxicity than some widely used transfection reagents. For that reason, the phenylalanine-modified dendrimers are considered to be promising gene carriers.     

Cytotoxicity of polypropylenimine dendrimer conjugates on cultured endothelial cells

The cytotoxicity and time-dependent membrane disruption by polypropylenimine dendrimer conjugates on cultured human umbilical vein endothelial cells (HUVEC) is reported. Fluorescently labeled derivatives of generation 5 polypropylenimine dendrimers were prepared via conversion of amines to acetamides or through the covalent attachment of high molecular weight poly(ethylene glycol) (PEG) chains. Direct interactions between the fluorescent dendrimer conjugates and HUVEC were monitored using confocal fluorescence microscopy to track dendrimer movement across the plasma membrane and the fluorescent staining of cell nuclei. Propidium iodide and lactate dehydrogenase cytotoxicity assays confirmed that chemical modification of the surface amines of the parental dendrimer to neutral acetamide or PEG functionalities eliminated their acute cytotoxicity. Cationic primary-amine-containing dendrimers demonstrated drastic time-dependent changes in the plasma membrane permeability and prominent cytotoxicity. However, complete removal of the primary amines or masking of the cationic surface via PEGylation decreased dendrimer cytotoxicity. Thus, preventing electrostatic interactions of dendrimers with cellular membranes apparently is a necessary step toward minimizing the toxicity of delivery vehicles to the endothelium.     

Direct real-time molecular scale visualisation of the degradation of condensed DNA complexes exposed to DNase I

The need to protect DNA from in vivo degradation is one of the basic tenets of therapeutic gene delivery and a standard test for any proposed delivery vector. The currently employed in vitro tests, however, presently provide no direct link between the molecular structure of the vector complexes and their success in this role, thus hindering the rational design of successful gene delivery agents. Here we apply atomic force microscopy (AFM) in liquid to visualise at the molecular scale and in real time, the effect of DNase I on generation 4 polyamidoamine dendrimers (G4) complexed with DNA. These complexes are revealed to be dynamic in nature showing a degree of mobility, in some cases revealing the addition and loss of dendrimers to individual complexes. The formation of the G4–DNA complexes is observed to provide a degree of protection to the DNA. This protection is related to the structural morphology of the formed complex, which is itself shown to be dependent on the dendrimer loading and the time allowed for complex formation.     

In vitro and in vivo gene transfer by an optimized alpha-cyclodextrin conjugate with polyamidoamine dendrimer

The purpose of the present study is to optimize the structure of the polyamidoamine starburst dendrimer (dendrimer) conjugate with alpha-cyclodextrin (alpha-CDE conjugate) as a nonviral vector. alpha-CDE conjugates of dendrimer (generation 3, G3) with various average degrees of substitution (DS) of alpha-CyD of 1.1, 2.4, and 5.4 were prepared. alpha-CDE conjugates formed the complexes with pDNA, resulting in a change of the particle sizes of pDNA complexes, but the distinction of physicochemical properties among their vector/pDNA complexes was only very slight. The membrane-disruptive ability of alpha-CDE conjugates on liposomes encapsulating calcein and their cytotoxicity to NIH3T3 and HepG2 increased with an increase in the DS value of alpha-CyD. In vitro gene transfer activity of alpha-CDE conjugates in both NIH3T3 and HepG2 cells augmented as the charge ratio (vector/pDNA) increased, and the activity of alpha-CDE conjugate (DS 2.4) was the highest at higher charge ratios among dendrimer (G3), the three alpha-CDE conjugates, and TransFast. After intravenous administration of pDNA complexes in mice, alpha-CDE conjugate (DS 2.4) delivered pDNA more efficiently in spleen, liver, and kidney, compared with dendrimer and other alpha-CDE conjugates (DS 1.1 and 5.4). The potential use of alpha-CDE conjugate (G3, DS 2.4) could be expected as a nonviral vector in vitro and in vivo, and these data may be useful for design of alpha-CyD conjugates with other nonviral vectors.     

Coarse-grained modelling of urea-adamantyl functionalised poly(propylene imine) dendrimers

To investigate the behaviour of poly(propylene imine) dendrimers – and urea–adamantyl functionalised ones – in solution using molecular dynamics simulations, we developed a coarse-grained model to tackle the relatively large system sizes and time scales needed. Harmonic bond and angle potentials were derived from atomistic simulations using an iterative Boltzmann inversion scheme, modified to incorporate Gaussian fits of the bond and angle distributions. With the coarse-grained model and accompanying force field simulations of generations 1–7 of both dendrimer types in water were performed. They compare favourably with atomistic simulations and experimental results on the basis of size, shape, monomer density, spacer back-folding and atomic form factor measurements. These results show that the structural dynamics of these dendrimers originate from flexible chains constrained by configurational and spatial requirements. Large dendrimers are more rigid and spherical, while small ones are flexible, alternatively rod-like and globular.     

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.     

Gene delivery into mesenchymal stem cells: a biomimetic approach using RGD nanoclusters based on poly(amidoamine) dendrimers

Poly(amidoamine) dendrimers (generations 5 and 6) with amine termini were conjugated with peptides containing the arginine-glycine-aspartic acid (RGD) sequence having in view their application as gene delivery vectors. The idea behind the work was to take advantage of the cationic nature of dendrimers and of the integrin targeting capabilities of the RGD motif to improve gene delivery. Dendrimers were used as scaffolds for RGD clustering and, by controlling the number of peptides (4, 8, and 16) linked to each dendrimer, it was possible to evaluate the effect of RGD density on the gene delivery process. The new vectors were characterized in respect to their ability to neutralize and compact plasmid DNA (pDNA). The complexes formed by the vectors and pDNA were studied concerning their size, zeta potential, capacity of being internalized by cells and ability of transferring genes. Transfection efficiency was analyzed, first, by using a pDNA encoding for Enhanced Green Fluorescent Protein and Firefly Luciferase and, second, by using a pDNA encoding for Bone Morphogenetic Protein-2. Gene expression in mesenchymal stem cells was enhanced using the new vectors in comparison to native dendrimers and was shown to be dependent on the electrostatic interaction established between the dendrimer moiety and the cell surface, as well as on the RGD density of nanoclusters. The use of dendrimer scaffolds for RGD cluster formation is a new approach that can be extended beyond gene delivery applications, whenever RGD clustering is important for modulating cellular responses.     

Structurally flexible triethanolamine core PAMAM dendrimers are effective nanovectors for DNA transfection in vitro and in vivo to the mouse thymus

With the aim of developing dendrimer nanovectors with a precisely controlled architecture and flexible structure for DNA transfection, we designed PAMAM dendrimers bearing a triethanolamine (TEA) core, with branching units pointing away from the center to create void spaces, reduce steric congestion, and increase water accessibility for the benefit of DNA delivery. These dendrimers are shown to form stable nanoparticles with DNA, promote cell uptake mainly via macropinocytosis, and act as effective nanovectors for DNA transfection in vitro on epithelial and fibroblast cells and, most importantly, in vivo in the mouse thymus, an exceedingly challenging organ for immune gene therapy. Collectively, these results validate our rational design approach of structurally flexible dendrimers with a chemically defined structure as effective nanovectors for gene delivery, and demonstrate the potential of these dendrimers in intrathymus gene delivery for future applications in immune gene therapy.     

Preparation of poly(ethylene glycol)-attached dendrimers encapsulating photosensitizers for application to photodynamic therapy

Photodynamic therapy (PDT) is a noninvasive treatment of some diseases including cancer. We have developed poly(ethylene glycol) (PEG)-attached dendrimers as a drug-carrier candidate. In this study, we prepared nanocapsules of photosensitizers using PEG-attached dendrimers for application to PDT. Two PEG-attached dendrimers derived from poly(amido amine) (PAMAM) and poly(propylene imine) (PPI) dendrimers (PEG-PAMAM and PEG-PPI) were synthesized, and rose bengal (RB) and protoporphyrin IX (PpIX) were used as photosensitizers. Results showed that fewer PpIX molecules were encapsulated by both PEG-attached dendrimers than RB, but the complexes were more stable under physiological conditions. Furthermore, we demonstrated that PEG-PPI held photosensitizers in a more stable manner than PEG-PAMAM because of their inner hydrophobicity. We described the cytotoxicity of the complexes of photosensitizers induced by light irradiation in vitro. The complex of PpIX with PEG-PPI exhibited efficient cytotoxicity, compared with free PpIX. It was suggested that the cytotoxicity was caused by the high level of singlet oxygen production and the efficient delivery to mitochondria. Our results suggest that these PEG-attached dendrimers are a promising vehicle for PDT.