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.