Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane.

Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40-50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.

G5-PEG PAMAM dendrimer incorporating nanostructured lipid carriers enhance oral bioavailability and plasma lipid-lowering effect of probucol.

This work aimed to improve the oral bioavailability and plasma lipid-lowering effect of probucol (PB) by constructing a combined drug delivery system (CDDS) composed of nanostructured lipid carrier (NLC) and PEGylated poly(amidoamine) dendrimer (PEG-PAMAM). PEG-PAMAM with dendrimer generations of 5 (G5-PEG) or 7 (G7-PEG) were incorporated in PB-NLCs to form PB-CDDSs, PB-NLCs/G5-PEG and PB-NLCs/G7-PEG. The resultant two kinds of PB-CDDSs were characterized by particle size, zeta potential, drug encapsulation efficacy, PB release rates, and physical stability. Formulation effects of NLC and CDDS on the cellular uptake of hydrophobic drug were explored in Caco-2 cells by fluorescent Cy5 dye as a hydrophobic drug model. Furthermore, in vivo pharmacokinetics of the PB-CDDS composed of G5-PEG and PB-NLCs were investigated in a low density lipoprotein receptor knockout (LDLr-/-) mouse model, including plateau plasma PB concentrations after oral administration of multiple doses, and bioavailability after oral administration of a single dose of different PB formulations. In addition, lipid-lowering effect of PB-NLCs/G5-PEG was studied. The results indicate that both G5-PEG and G7-PEG significantly improved aqueous solubility of PB. The two PB-CDDSs exhibited similar particle size (around 150nm) as PB-NLCs, but slower PB burst release rate, higher total PB release amount, and better particle morphology and storage stability than PB-NLCs. In comparison with traditional NLC, CDDS dramatically enhanced cellular uptake of Cy5 into Caco-2 cells. In vivo results demonstrate that PB-NLCs/G5-PEG had the highest plateau plasma PB concentration and oral bioavailability, and the greatest cholesterol-lowering effect in comparison with PB suspensions and PB-NLCs. Therefore, G5-PEG incorporating NLC can be exploited as a promising drug delivery system to improve oral bioavailability and lipid-lowering effect of PB.

G5 PAMAM dendrimer versus liposome: a comparison study on the in vitro transepithelial transport and in vivo oral absorption of simvastatin.

This study compared formulation effects of a dendrimer and a liposome preparation on the water solubility, transepithelial transport, and oral bioavailability of simvastatin (SMV). Amine-terminated G5 PAMAM dendrimer (G5-NH2) was chosen to form SMV/G5-NH2 molecular complexes, and SMV-liposomes were prepared by using a thin film dispersion method. The effects of these preparations on the transepithelial transport were investigated in vitro using Caco-2 cell monolayers. Results indicated that the solubility and transepithelial transport of SMV were significantly improved by both formulations. Pharmacokinetic studies in rats also revealed that both the SMV/G5-NH2 molecular complexes and the SMV-liposomes significantly improved the oral bioavailability of SMV with the liposomes being more effective than the G5-NH2. The overall better oral absorption of SMV-liposomes as compared to SMV/G5-NH2 molecular complexes appeared to arise from better liposomal solubilization and encapsulation of SMV and more efficient intracellular SMV delivery. FROM THE CLINICAL EDITOR: Various carrier systems have been designed to enhance drug delivery via the oral route. In this study, the authors compared G5 PAMAM dendrimers to liposome preparations in terms of solubility, transepithelial transport, and oral bioavailability of this poorly water-soluble drug. This understanding has improved our knowledge in the further development of drug carrier systems.

Oral absorption enhancement of probucol by PEGylated G5 PAMAM dendrimer modified nanoliposomes.

Probucol (PB), an antioxidant drug, is commonly used as a lipid concentration lowering drug to reduce blood plasma cholesterol levels in the clinic. However, the therapeutic effects of this drug are negatively impacted by its poor water solubility and low oral absorption efficiency. In this study, a PEGylated G5 PAMAM dendrimer (G5-PEG) modified nanoliposome was employed to increase water solubility, transepithelial transport, and oral absorption of PB. The uptake mechanism was explored in vitro in Caco-2 cells with the results suggesting that the absorption improvement of G5-PEG modified PB-liposome (PB-liposome/G5-PEG) was related to P-glycoprotein (P-gp) efflux pump but was independent of caveolae endocytosis pathways. Additionally, plasma lipid concentration lowering effects of PB-liposome/G5-PEG were evaluated in vivo in a LDLR-/- hyperlipidemia mouse model. Compared with saline treated group, treatment with PB-liposome/G5-PEG significantly inhibited the increase of plasma total cholesterol (TC) and triglyceride (TG) of mice induced by a high fat diet. Moreover, its lipid concentration lowering effects and plasma drug concentration were greater than PB alone or commercial PB tablets. Our results demonstrated that PB-liposome/G5-PEG significantly increased the oral absorption of PB and therefore significantly improved its pharmacodynamic effects.

Poly(amidoamine) dendrimer-methotrexate conjugates: the mechanism of interaction with folate binding protein.

Generation 5 poly(amidoamine) (G5 PAMAM) methotrexate (MTX) conjugates employing two small molecular linkers, G5-(COG-MTX)n, G5-(MFCO-MTX)n were prepared along with the conjugates of the G5-G5 (D) dimer, D-(COG-MTX)n, D-(MFCO-MTX)n. The monomer G5-(COG-MTX)n conjugates exhibited only a weak, rapidly reversible binding to folate binding protein (FBP) consistent with monovalent MTX binding. The D-(COG-MTX)n conjugates exhibited a slow onset, tight-binding mechanism in which the MTX first binds to the FBP, inducing protein structural rearrangement, followed by polymer-protein van der Waals interactions leading to tight-binding. The extent of irreversible binding is dependent on total MTX concentration and no evidence of multivalent MTX binding was observed.

Multivalent polymers for drug delivery and imaging: the challenges of conjugation.

Multivalent polymers offer a powerful opportunity to develop theranostic materials on the size scale of proteins that can provide targeting, imaging, and therapeutic functionality. Achieving this goal requires the presence of multiple targeting molecules, dyes, and/or drugs on the polymer scaffold. This critical review examines the synthetic, analytical, and functional challenges associated with the heterogeneity introduced by conjugation reactions as well as polymer scaffold design. First, approaches to making multivalent polymer conjugations are discussed followed by an analysis of materials that have shown particular promise biologically. Challenges in characterizing the mixed ligand distributions and the impact of these distributions on biological applications are then discussed. Where possible, molecular-level interpretations are provided for the structures that give rise to the functional ligand and molecular weight distributions present in the polymer scaffolds. Lastly, recent strategies employed for overcoming or minimizing the presence of ligand distributions are discussed. This review focuses on multivalent polymer scaffolds where average stoichiometry and/or the distribution of products have been characterized by at least one experimental technique. Key illustrative examples are provided for scaffolds that have been carried forward to in vitro and in vivo testing with significant biological results.

The role of caveolin-1 and syndecan-4 in the internalization of PEGylated PAMAM dendrimer polyplexes into myoblast and hepatic cells.

To improve gene delivery efficiency of PEGylated poly(amidoamine) dendrimers in livers and muscles, the roles of syndecan-4 receptor and caveolin-1 protein in the endocytosis of PEGylated generation 5 (G5-PEG) or 7 (G7-PEG) dendrimers and plasmid DNA polyplexes were explored in C2C12 and HepG2 cells. Expression levels of syndecan-4 for both cell lines were downregulated by transfection of the cells with syndecan-4 specific siRNA. Caveolin-1 was upregulated by infecting the cells with adenovirus vector expressed caveolin-1 (Ad-CAV-1). The impact of syndecan-4 and caveolin-1 on endocytosis of G5-PEG/DNA or G7-PEG/DNA polyplexes was then measured by flow cytometry. Our results demonstrate that downregulation of syndecan-4 and upregulation of caveolin-1 significantly improved internalization of PEG-PAMAM dendrimer polyplexes in HepG2 cells; however, in C2C12 cells, downregulation of syndecan-4 decreased the internalization of the polyplexes while upregulation of caveolin-1 had no effect on internalization. Gene expression results for G5-PEG/pGFP on the two cell lines exhibited the same trends for syndecan-4 and caveolin-1 as was observed for endocytosis of the polyplexes. This study gives a clue how to take strategies by up- or down-regulation of the expressions of syndecan-4 and caveolin-1 to improve in vivo gene delivery efficiency of the PEG-PAMAM dendrimers in clinical transgenic therapy.

Diffusion NMR study of generation-five PAMAM dendrimer materials.

Commercial generation-five poly(amidoamine) dendrimer material (G5c) was fractionated into its major structural components. Monomeric G5 (G5m; 21--30 kDa) was isolated to compare its functional properties to the G5c material. Diffusion-ordered nuclear magnetic resonance spectroscopy was employed to measure the self-diffusion coefficients and corresponding hydrodynamic radii of G5m and other G5c components as a function of dendrimer size (i.e., molecular weight) and tertiary structure (i.e., generational or oligomeric nature). It was found that the hydrodynamic radius (R(H)) scales with approximate numbers of atoms in the trailing generations, G5m, and oligomeric material at a rate of R(H)∝N(0.35), in good agreement with previous reports of RH scaling for PAMAM dendrimer with generation. G5c materials can be thought of as a heterogeneous mixture of dendrimers ranging in size from trailing generation two to tetramers of G5, approximately the same in size as a G7 dendrimer, with G5m comprising ∼65% of the material. The radius of hydration for G5m was measured to be 3.1 ± 0.1 nm at pH 7.4. The 10% swelling in response to a drop in pH observed for the G5c material was not observed for isolated G5m; however, the isolated G5--G5 dimers had an increase of 44% in R(H), indicating that the G5c pH response results from the increase in R(H) of the oligomeric fraction upon protonation. Finally, the data allow for an experimental test of the "slip" and "stick" boundary models of the Stokes--Einstein equation for PAMAM dendrimer in water.

Avidity mechanism of dendrimer-folic acid conjugates.

Multivalent conjugation of folic acid has been employed to target cells overexpressing folate receptors. Such polymer conjugates have been previously demonstrated to have high avidity to folate binding protein. However, the lack of a monovalent folic acid-polymer material has prevented a full binding analysis of these conjugates, as multivalent binding mechanisms and polymer-mass mechanisms are convoluted in samples with broad distributions of folic acid-to-dendrimer ratios. In this work, the synthesis of a monovalent folic acid-dendrimer conjugate allowed the elucidation of the mechanism for increased binding between the folic acid-polymer conjugate and a folate binding protein surface. The increased avidity is due to a folate-keyed interaction between the dendrimer and protein surfaces that fits into the general framework of slow-onset, tight-binding mechanisms of ligand/protein interactions.

Isolation and characterization of precise dye/dendrimer ratios.

Fluorescent dyes are commonly conjugated to nanomaterials for imaging applications using stochastic synthesis conditions that result in a Poisson distribution of dye/particle ratios and therefore a broad range of photophysical and biodistribution properties. We report the isolation and characterization of generation 5 poly(amidoamine) (G5 PAMAM) dendrimer samples containing 1, 2, 3, and 4 fluorescein (FC) or 6-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dyes per polymer particle. For the fluorescein case, this was achieved by stochastically functionalizing dendrimer with a cyclooctyne "click" ligand, separation into sample containing precisely defined "click" ligand/particle ratios using reverse-phase high performance liquid chromatography (RP-HPLC), followed by reaction with excess azide-functionalized fluorescein dye. For the TAMRA samples, stochastically functionalized dendrimer was directly separated into precise dye/particle ratios using RP-HPLC. These materials were characterized using (1)H and (19)F NMR spectroscopy, RP-HPLC, UV/Vis and fluorescence spectroscopy, lifetime measurements, and MALDI.

PAMAM Dendrimers as Quantized Building Blocks for Novel Nanostructures.

The desire to synthesize soft supramolecular structures with size scales similar to biological systems has led to work in assembly of polymeric nanomaterials. Recent advances in the isolation of generationally homogenous poly(amidoamine) (PAMAM) dendrimer enables their use as quantized building blocks. Here, we report their assembly into precise nanoclusters. In this work, click-functional ligands are stochastically conjugated to monomeric generation 5 PAMAM dendrimer and separated via reverse-phase HPLC to isolate dendrimers with precise numbers of click ligands per dendrimer particle. The click-ligand/dendrimer conjugates are then employed as modular building blocks for the synthesis of defined nanostructures. Complimentary click chemistry employing dendrimers with 1, 2, 3, or 4 ring-strained cyclooctyne ligands and dendrimers with 1 azide ligand were utilized to prepare megamer structures containing 2 to 5 ~30,000 kDa monomer units as characterized by mass spectrometry, size exclusion chromatography, and reverse-phase liquid chromatography. The resulting structures are flexible with masses ranging from 60,000 to 150,000 kDa, and are soluble in water, methanol, and dimethylsulfoxide.

Quantitative analysis of generation and branch defects in G5 poly(amidoamine) dendrimer.

Although methods have been developed to synthesize and isolate generation 5 (G5) PAMAM dendrimers containing precise numbers of ligands per polymer particle, the presence of skeletal and generational defects in this material can substantially hamper the process. Here we provide a quantitative analysis of G5 PAMAM dendrimer defects via high performance liquid chromatography, potentiometric titration, mass spectrometry, size exclusion chromatography, and nuclear magnetic resonance. We identified, isolated, and characterized the major structural defects of G5 dendrimer, trailing generations, and dimer, trimer, and tetramer species. We determine that the G5 material present in the as-received mixture contains 93 arms on average. We have developed two model systems capable of generating the experimentally observed mass range and polydispersity at defect rates of 8-15%.

Avidity modulation of folate-targeted multivalent dendrimers for evaluating biophysical models of cancer targeting nanoparticles.

We investigated two types of generation 5 polyamidoamine (PAMAM) dendrimers, each conjugated stochastically with a mean number of 5 or 10 methotrexate (MTX) ligands per dendrimer (G5-MTX5, G5-MTX10), for their binding to surface-immobilized folate binding protein (FBP) as a function of receptor density. The binding study was performed under flow by surface plasmon resonance spectroscopy. Two multivalent models were examined to simulate binding of the dendrimer to the receptor surface, showing that at relatively high receptor density, both dendrimer conjugates exhibit high avidity. However, upon reducing the receptor density by a factor of 3 and 13 relative to the high density level, the avidity of the lower-valent G5-MTX5 decreases by up to several orders of magnitude (KD = nM to µM), whereas the avidity of G5-MTX10 remains largely unaffected regardless of the density variation. Notably, on the 13-fold reduced FBP surface, G5-MTX5 displays binding kinetics similar to that of monovalent methotrexate, which is patently different from the still tight binding of the higher-valent G5-MTX10. Thus, the binding analysis demonstrates that avidity displayed by multivalent MTX conjugates varies in response to the receptor density and can be modulated for achieving tighter, more specific binding to the higher receptor density by modulation of ligand valency. We believe this study provides experimental evidence supportive of the mechanistic hypothesis of multivalent NP uptake to a cancer cell over a healthy cell where the diseased cell expresses the folate receptor at higher density.

Best practices for purification and characterization of PAMAM dendrimer.

Poly(amidoamine) (PAMAM) dendrimer are branched polymers with low degrees of heterogeneity. Current synthesis methods, however, result in substantial batch variability. We present our optimized procedure for post-synthesis (and post-market) purification of a generation 5 PAMAM dendrimer by membrane dialysis and demonstrate its effectiveness and limitations using a representative lot of biomedical grade dendrimer. This method successfully removes trailing generation defect structures, thereby reducing the heterogeneity of the material (PDI reduced from 1.04 to 1.02). Optimized analytical techniques to characterize the unpurified and purified dendrimer are also detailed. The efficiency of the purification method is successfully monitored by these analytics and dendrimer parameters that are critical for subsequent modification reactions and biological evaluation (M(n), M(w), PDI, average number of end groups) obtained. To provide better definition of the variability that should be expected between lots of synthesized material, HPLC traces for three additional commercial lots of dendrimer are also presented.