Functional nanoparticles often contain ligands including targeting molecules, fluorophores, and/or active moieties such as drugs. Characterizing the number of these ligands bound to each particle, and the distribution of nanoparticle-ligand species, is important for understanding the nanomaterial’s function. In this study, the amide coupling methods commonly used to conjugate ligands to poly(amidoamine) (PAMAM) dendrimers were examined. A skewed Poisson distribution was observed and quantified using HPLC for two sets of dendrimer-ligand samples prepared using the amine terminated form of the PAMAM dendrimer and a partially acetylated form of the PAMAM dendrimer that has been used for targeted in vivo drug delivery. The prepared samples had an average number of ligands per dendrimer ranging from 0.4 to 13. Distributions identified by HPLC are in excellent agreement with the mean ligand/dendrimer ratio, measured by 1H NMR, gel permeation chromatography (GPC), and potentiometric titration. These results provide insight into the heterogeneity of distributions that are obtained for many classes of nanomaterials to which ligands are conjugated and belie the use of simple cartoon models that present the “average” number of ligands bound as a physically meaningful representation for the material.

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper we show that non-cytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A and KB cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm 2 in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1 -100 ms, while membrane resealing may occur over tens of seconds. Patchclamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for AMO-3, a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making ~3 nm holes in living cell membranes.

CONSPECTUS Nanoparticles conjugated with functional ligands are expected to have a major impact in medicine, photonics, sensing, and nanoarchitecture design. One major obstacle to realizing the promise of these materials, however, is the difficulty in controlling the ligand/nanoparticle ratio. This obstacle can be segmented into three key areas: First, many system designs have failed to account for the true heterogeneity of ligand/nanoparticle ratios that compose each material. Second, the field's accepted level of characterization of ligand-nanoparticle materials is deficient as it does not provide an accurate definition of material composition that is necessary to both understand the material-property relationships as well as to monitor the consistency of the material. In particular, many characterization assays only determine the mean ligand/nanoparticle ratio and do not provide the number and relative amount of the different ligand/nanoparticle ratios that compose a material. Third, some synthetic approaches that are presently in use may not produce consistent material as they are sensitive to reaction kinetics, and the synthetic history of the nanoparticle. In this account we describe the recent advances that we have made in understanding the material composition of ligand-nanoparticle systems. Our work has been enabled by a model system using poly(amidoamine) dendrimers and two small molecule ligands. Using reverse phase high-pressure liquid chromatography (HPLC) we have successfully resolved and quantified the relative amount of each ligand/dendrimer ratio present. This type of information is rare for the entire field of ligand-nanoparticle materials because most analytical techniques have been unable to identify the components in the distribution. Our experimental data indicates that the actual distribution of ligand-nanoparticle components is much more heterogeneous than commonly assumed. The mean ligand/nanoparticle ratio that is so often the only information known about a material is insufficient. This is because the mean does not provide information on the diversity of components in the material and often does not describe the most common component (the mode). Additionally, our experimental data has provided examples of material batches with the same mean ligand/nanoparticle ratio and very different distributions. This discrepancy indicates that the mean cannot be used as the sole metric to assess the reproducibility of a system. We further found that distribution profiles can be highly sensitive to the synthetic history of the starting material as well as slight changes in reaction conditions. We have incorporated the lessons from our experimental data into new systems designs to provide improved control over the ligand/nanoparticle ratios.

The energetics, stoichiometry, and structure of poly(amidoamine) (PAMAM) dendrimer-phospholipid interactions were measured with isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), and molecular dynamics (MD) simulations. Dendrimers of sixth-generation and smaller interacted with the lipids at an average stoichiometry and enthalpy proportional to the number of primary amines per dendrimers (4.5 ± 0.1 lipids/primary amine and 6.3 ± 0.3 kJ/mol of primary amines, respectively). Larger dendrimers, however, demonstrated a decreased number of bound lipids and heat release per primary amine, presumably due to the steric restriction of dendrimer deformation on the lipid bilayer. For example, eighth-generation PAMAM dendrimers bound to 44% fewer lipids per primary amine and released 63% less heat per primary amine as compared to the smaller dendrimers. These differences in binding stoichiometry support generation-dependent models for dendrimer-lipid complexation, which are consistent with previously observed generation-dependent differences in dendrimer-induced membrane disruption. Dendrimers of seventh-generation and larger bound to lipids with an average stoichiometry consistent with each dendrimer having been wrapped by a bilayer of lipids, where as smaller dendrimers did not.

Stochastic synthesis of a ligand coupled to a nanoparticle results in a distribution of populations with different numbers of ligands per nanoparticle. This distribution was resolved and quantified using HPLC, and is in excellent agreement with the ligand/nanoparticle average measured by 1H NMR, GPC, and potentiometric titration, yet significantly more disperse than commonly held perceptions of monodispersity. Two statistical models were employed to confirm that the observed heterogeneity is consistent with theoretical expectations.

Generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers with amine, acetamide, and carboxylate end groups were prepared to investigate polymer/cell membrane interactions in vitro. G7 PAMAM dendrimers were used in this study because higher generation of dendrimers are more effective in permeabilization of cell plasma membranes and in the formation of nanoscale holes in supported lipid bilayers than smaller, lower generation dendrimers. Dendrimer-based conjugates were characterized by 1H NMR, UV/Vis spectroscopy, GPC, HPLC, and CE. Positively charged amine-terminated G7 dendrimers (G7-NH2) were observed to internalize into KB, Rat2 and C6 cells at a 200 nM concentration. By way of contrast, neither negatively charged G7 carboxylate-terminated dendrimers (G7-COOH) nor neutral acetamide-terminated G7 dendrimers (G7-Ac) associated with the cell plasma membrane or internalized under similar conditions. A series of in vitro experiments employing endocytic markers cholera toxin subunit B (CTB), transferrin, and GM1-pyrene were performed to further investigate mechanisms of dendrimer internalization into cells. G7-NH2 dendrimers co-localized with CTB, however, experiments with C6 cells indicated that internalization of G7-NH2 was not ganglioside GM1 dependent. The G7/CTB co-localization was thus ascribed to an artifact of direct interaction between the two species. The presence of GM1 in the membrane also had no effect upon XTT assays of cell viability or lactate dehydrogenase (LDH) assays of membrane permeability.

Poly(amidoamine) (PAMAM) dendrimers carrying different amounts of surface amino groups were synthesized and tested for their effects on cellular cytotoxicity, lysosomal pH, and mitochondriadependent apoptosis. In KB cells, the PAMAM dendrimers were taken up into the lysosomal compartment, and they increased the lysosomal pH and cytotoxicity as a function of the number of surface amino groups on the dendrimer. PAMAM dendrimers that were surface-neutralized by acetylation of >80% of the surface amino groups failed to show any cytotoxicity. The positively charged, amine-terminated PAMAM dendrimer induced cellular apoptosis, as demonstrated by mitochondrial membrane potential changes and caspase activity measurements. These results suggest that PAMAM dendrimers are endocytosed into the KB cells through a lysosomal pathway, leading to lysosomal alkalinization and induction of mitochondria-mediated apoptosis.

Poly(amidoamine) (PAMAM) dendrimer nanobiotechnology shows great promise in targeted drug delivery and gene therapy. Because of the involvement of cell membrane lipids with the pharmacological activity of dendrimer nanomedicines, the interactions between dendrimers and lipids are of particular relevance to the pharmaceutical applications of dendrimers. In this study, solid-state NMR was used to obtain a molecular image of the complex of generation 5 PAMAM dendrimer with the lipid bilayer. Using 1H radio frequency driven dipolar recoupling (RFDR) and 1H magic angle spinning (MAS) nuclear Overhauser effect spectroscopy (NOESY) techniques, we show that dendrimers are thermodynamically stable when inserted into zwitterionic lipid bilayers. 14N and 31P NMR experiments on static samples and measurements of the mobility of C–H bonds using a 2D proton detected local field protocol under MAS corroborate these results. The localization of dendrimers in the hydrophobic core of lipid bilayers restricts the motion of bilayer lipid tails, with the smaller G5 dendrimer having more of an effect than the larger G7 dendrimer. Fragmentation of the membrane does not occur at low dendrimer concentrations in zwitterionic membranes. Because these results show that the amphipathic dendrimer molecule can be stably incorporated in the interior of the bilayer (as opposed to electrostatic binding at the surface), they are expected to be useful in the design of dendrimer-based nanobiotechnologies.