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.     

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.     

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.     

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.     

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.     

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.     

Activity of dendrimer-methotrexate conjugates on methotrexate-sensitive and -resistant cell lines

Dendritic nanostructures can play a key role in drug delivery, due to the high density and variety of surface functional groups that can facilitate and modulate the delivery process. We have investigated the effect of dendrimer end-functionality on the activity of polyamido amine (PAMAM) dendrimer-methotrexate (MTX) conjugates in MTX-sensitive and MTX-resistant human acute lymphoblastoid leukemia (CCRF-CEM) and Chinese hamster ovary (CHO) cell lines. Two amide-bonded PAMAM dendrimer-MTX conjugates were prepared using a dicyclohexylcarbodiimide (DCC) coupling reaction: one between a carboxylic acid-terminated G2.5 dendrimer and the amine groups of the MTX (conjugate A) and another between an amine-terminated G3 dendrimer and the carboxylic acid group of the MTX (conjugate B). Our studies suggest that conjugate A showed an increased drug activity compared to an equimolar amount of free MTX toward both sensitive and resistant cell lines, whereas conjugate B did not show significant activity on any of the cell lines. Despite substantially impaired MTX transport by MTX-resistant CEM/MTX and RII cells, conjugate A showed sensitivity increases of approximately 8- and 24-fold (based on IC50 values), respectively, compared to free MTX. Co-incubation of the cells with adenosine and thymidine along with either conjugate A or MTX resulted in almost complete protection, suggesting that the conjugate achieves its effect on dihyrofolate reductase (DHFR) enzyme through the same mechanism as that of MTX. The differences in cytotoxicity of these amide-bonded conjugates may be indicative of differences in the intracellular drug release from the cationic dendrimer (conjugate B) versus the anionic dendrimer (conjugate A), perhaps due to the differences in lysosomal residence times dictated by the surface functionality. These findings demonstrate the feasibility of using dendrimers as drug delivery vehicles for achieving higher therapeutic effects in chemotherapy, especially in drug-resistant cells.     

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.     

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.     

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.     

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.     

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.     

Polyplexes assembled with internally quaternized PAMAM-OH dendrimer and plasmid DNA have a neutral surface and gene delivery potency

Interior tertiary amine groups of PAMAM-OH dendrimers (hydroxyl-terminated polyamidoamine, PAMAM) were modified by methylation to make these polymers have a more cationic character, which enabled electrostatic interaction between PAMAM-OH and plasmid DNA. A methylation reaction was dose-dependent, producing internally quaternized PAMAM-OH (QPAMAM-OH), thereby making tertiary amine/quaternary amine ratio adjustment possible. More highly condensed particles of plasmid DNA were formed as the degree of quaternization increased, whereas unmodified polymer (PAMAM-OH) could not. The location of positive charges in the internal position of QPAMAM-OH resulted in the formation of neutral polyplexes in which zeta potential leveled off near the zero value even at high charge ratios (+/-) of 10. A light scattering experiment showed that the polyplex formed by QPAMAM-OH was very small with the size of 53.3 nm at the optimum condition. QPAMAM-OH/DNA polyplexes were round-shaped with the more compact and small particles formed as the charge ratio increased. QPAMAM-OH showed much reduced cytotoxicity compared with starburst PAMAM and branched polyethyleneimine (PEI) in which shielding of interior positive charges by surface hydroxyls might be the reason for this favorable result. These results suggest that QPAMAM-OH could be a promising tool as a nonviral vector both by itself and in conjugated form with targeting ligands.     

PAMAM dendrimer-based multifunctional conjugate for cancer therapy: synthesis, characterization, and functionality

Poly(amidoamine) (PAMAM) dendrimer-based multifunctional cancer therapeutic conjugates have been designed and synthesized. The primary amino groups on the surface of the generation 5 (G5) PAMAM dendrimer were neutralized through partial acetylation, providing enhanced solubility of the dendrimer (in conjugation of FITC (fluorescein isothiocyanate)) and preventing nonspecific targeting interactions (in vitro and in vivo) during delivery. The functional molecules fluorescein isothiocyanate (FITC, an imaging agent), folic acid (FA, targets overexpressed folate receptors on specific cancer cells), and paclitaxel (taxol, a chemotherapeutic drug) were conjugated to the remaining nonacetylated primary amino groups. The appropriate control dendrimer conjugates have been synthesized as well. Characterization of the G5 PAMAM dendrimer and its nanosize conjugates, including the molecular weight and number of primary amine groups, has been determined by multiple analytical methods such as gel permeation chromatography (GPC), nuclear magnetic resonance spectroscopy (NMR), potentiometric titration, high-performance liquid chromatography (HPLC), and UV spectroscopy. These multifunctional dendrimer conjugates have been tested in vitro for targeted delivery of chemotherapeutic and imaging agents to specific cancer cells. We present here the synthesis, characterization, and functionality of these dendrimer conjugates.     

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.     

Tumor cell imaging using the intrinsic emission from PAMAM dendrimer: a case study with HeLa cells

HeLa 229 cells were treated with methotrexate (MTX) and doxorubicin (DOX), utilizing fourth generation (G4), amine terminated poly(amidoamine) {PAMAM} dendrimer as the drug carrier. In vitro kinetic studies of the release of both MTX and DOX in presence and absence of G4, amine terminated PAMAM dendrimers suggest that controlled drug release can be achieved in presence of the dendrimers. The cytotoxicity studies indicated improved cell death by dendrimer-drug combination, compared to the control experiments with dendrimer or drug alone at identical experimental conditions. Furthermore, HeLa 229 cells were imaged for the first time utilizing the intrinsic emission from the PAMAM dendrimers and drugs, without incorporating any conventional fluorophores. Experimental results collectively suggest that the decreased rate of drug efflux in presence of relatively large sized PAMAM dendrimers generates high local concentration of the dendrimer-drug combination inside the cell, which renders an easy way to image cell lines utilizing the intrinsic emission properties of PAMAM dendrimer and encapsulated drug molecule.     

Effect of poly(amido)amine (PAMAM) G4 dendrimer on heart and liver mitochondria in an animal model of diabetes

Diabetes‐induced injury related to hyperglycaemia is associated with impaired function of mitochondria. Regardless of their cytotoxicity, PAMAM [poly(amido)amine] G4 dendrimers lower plasma glucose and suppress long‐term markers of diabetic hyperglycaemia in experimental diabetes. In the present study, we aimed at verifying whether such modulatory effects of PAMAM G4 (0.5 μmol/kg of body weight daily for 60 days) may contribute to improved respiration in heart and liver mitochondria from streptozotocin‐diabetic rats. PAMAM G4 alleviated long‐term markers of hyperglycaemia and reduced blood and tissue lipophilic antioxidants in diabetic animals, but did not restore mitochondrial function. In hearts, but not livers, dendrimers further reduced respiratory function and oxidative phosphorylation. Thus ameliorating effects of PAMAM G4 on glycation and glycoxidation in experimental diabetes are not sufficient to restore the impaired mitochondrial function in diabetes.     

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.     

GPCR ligand dendrimer (GLiDe) conjugates: adenosine receptor interactions of a series of multivalent xanthine antagonists

Previously, G protein-coupled receptor (GPCR) agonists were tethered from polyamidoamine (PAMAM) dendrimers to provide high receptor affinity and selectivity. Here, we prepared GPCR ligand--dendrimer (GLiDe) conjugates from a potent adenosine receptor (AR) antagonist; such agents are of interest for treating Parkinson's disease, asthma, and other conditions. Xanthine amine congener (XAC) was appended with an alkyne group on an extended C8 substituent for coupling by Cu(I)-catalyzed click chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. These conjugates also contained triazole-linked PEG groups (8 or 22 moieties per 64 terminal positions) for increasing water-solubility and optionally prosthetic groups for spectroscopic characterization and affinity labeling. Human AR binding affinity increased progressively with the degree of xanthine substitution to reach K(i) values in the nanomolar range. The order of affinity of each conjugate was hA(2A)AR > hA(3)AR > hA(1)AR, while the corresponding monomer was ranked hA(2A)AR > hA(1)AR ≥ hA(3)AR. The antagonist activity of the most potent conjugate 14 (34 xanthines per dendrimer) was examined at the G(i)-coupled A(1)AR. Conjugate 14 at 100 nM right-shifted the AR agonist concentration--response curve in a cyclic AMP functional assay in a parallel manner, but at 10 nM (lower than its K(i) value), it significantly suppressed the maximal agonist effect in calcium mobilization. This is the first systematic probing of a potent AR antagonist tethered on a dendrimer and its activity as a function of variable loading.     

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.