Bradford G. Orr

Wide varieties of cationic nanoparticles induce defects in supported lipid bilayers

Nanoparticles with widely varying physical properties and origins (spherical versus irregular, synthetic versus biological, organic versus inorganic, flexible versus rigid, small versus large) have been previously noted to translocate across the cell plasma membrane. We have employed atomic force microscopy to determine if the physical disruption of lipid membranes, formation of holes and/or thinned regions, is a common mechanism of interaction between these nanoparticles and lipids. It was found that a wide variety of nanoparticles, including a cell penetrating peptide (MSI-78), a protein (TAT), polycationic polymers (PAMAM dendrimers, pentanol-core PAMAM dendrons, polyethyleneimine, and diethylaminoethyl-dextran), and two inorganic particles (Au-NH2, SiO2-NH2), can induce disruption, including the formation of holes, membrane thinning, and/or membrane erosion, in supported lipid bilayers.

Cationic Nanoparticles Induce Nanoscale Disruption in Living Cell Plasma Membranes

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 noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) 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. Patch-clamp 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 an amphiphilic phenylene ethynylene antimicrobial oligomer (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 approximately 3 nm holes in living cell membranes.

A Model for Strain-Induced Roughening and Coherent Island Growth

We have investigated the morphological evolution of strained films during growth. Novel Monte Carlo studies, which incorporate linear elasticity, have been performed to simulate film growth with misfit. These studies demonstrate the onset of islanding for sufficiently large misfit. We present an analytic calculation which shows that from the onset of deposition the films are energetically unstable to large-scale islanding. We argue that the kinetics ultimately determines the surface morphology. Dislocations are not necessary for surface lattice relaxation. Support for this picture is inferred from experimental results on a number of strained growth systems.

Synthesis, characterization, and in vitro testing of superparamagnetic iron oxide nanoparticles targeted using folic Acid-conjugated dendrimers.

Organic-coated superparamagnetic iron oxide nanoparticles (OC-SPIONs) were synthesized and characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. OC-SPIONs were transferred from organic media into water using poly(amidoamine) dendrimers modified with 6-TAMRA fluorescent dye and folic acid molecules. The saturation magnetization of the resulting dendrimer-coated SPIONs (DC-SPIONs) was determined, using a superconducting quantum interference device, to be 60 emu/g Fe versus 90 emu/g Fe for bulk magnetite. Selective targeting of the DC-SPIONs to KB cancer cells in vitro was demonstrated and quantified using two distinct and complementary imaging modalities: UV-visible and X-ray fluorescence; confocal microscopy confirmed internalization. The results were consistent between the uptake distribution quantified by flow cytometry using 6-TAMRA UV-visible fluorescence intensity and the cellular iron content determined using X-ray fluorescence microscopy.

Large scale surface structure formed during GaAs (001) homoepitaxy

Atomic force microscopy studies have been performed on GaAs (001) homoepitaxy films grown by molecular beam epitaxy. Multilayered features are seen to evolve when the growth conditions favor island nucleation. As the epilayer thickness is increased these features grow in all dimensions but the angle of inclination remains approximately constant at 1°. The mounding does not occur on surfaces grown in step flow. We propose that the multilayered features are an unstable growth mode which relies on island nucleation and the presence of a step edge barrier.

Dendrimer-Based Multivalent Vancomycin Nanoplatform for Targeting the Drug-Resistant Bacterial Surface

Vancomycin represents the preferred ligand for bacteria-targeting nanosystems. However, it is inefficient for emerging vancomycin-resistant species because of its poor affinity to the reprogrammed cell wall structure. This study demonstrates the use of a multivalent strategy as an effective way for overcoming such an affinity limitation in bacteria targeting. We designed a series of fifth generation (G5) poly(amidoamine) (PAMAM) dendrimers tethered with vancomycin at the C-terminus at different valencies. We performed surface plasmon resonance (SPR) studies to determine their binding avidity to two cell wall models, each made with either a vancomycin-susceptible (D)-Ala-(D)-Ala or vancomycin-resistant (D)-Ala-(D)-Lac cell wall precursor. These conjugates showed remarkable enhancement in avidity in the cell wall models tested, including the vancomycin-resistant model, which had an increase in avidity of four to five orders of magnitude greater than free vancomycin. The tight adsorption of the conjugate to the model surface corresponded with its ability to bind vancomycin-susceptible Staphylococcus aureus bacterial cells in vitro as imaged by confocal fluorescent microscopy. This vancomycin platform was then used to fabricate the surface of iron oxide nanoparticles by coating them with the dendrimer conjugates, and the resulting dendrimer-covered magnetic nanoparticles were demonstrated to rapidly sequester bacterial cells. In summary, this article investigates the biophysical basis of the tight, multivalent association of dendrimer-based vancomycin conjugates to the bacterial cell wall, and proposes a potential new use of this nanoplatform in targeting Gram-positive bacteria.

Poly(amidoamine) dendrimers on lipid bilayers II: Effects of bilayer phase and dendrimer termination.

The molecular structures and enthalpy release of poly(amidoamine) (PAMAM) dendrimers binding to 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) bilayers were explored through atomistic molecular dynamics. Three PAMAM dendrimer terminations were examined: protonated primary amine, neutral acetamide, and deprotonated carboxylic acid. Fluid and gel lipid phases were examined to extract the effects of lipid tail mobility on the binding of generation-3 dendrimers, which are directly relevant to the nanoparticle interactions involving lipid rafts, endocytosis, lipid removal, and/or membrane pores. Upon binding to gel phase lipids, dendrimers remained spherical, had a constant radius of gyration, and approximately one-quarter of the terminal groups were in close proximity to the lipids. In contrast, upon binding to fluid phase bilayers, dendrimers flattened out with a large increase in their asphericity and radii of gyration. Although over twice as many dendrimer-lipid contacts were formed on fluid versus gel phase lipids, the dendrimer-lipid interaction energy was only 20% stronger. The greatest enthalpy release upon binding was between the charged dendrimers and the lipid bilayer. However, the stronger binding to fluid versus gel phase lipids was driven by the hydrophobic interactions between the inner dendrimer and lipid tails.

Stoichiometry and Structure of Poly(amidoamine) Dendrimer−Lipid Complexes

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, whereas smaller dendrimers did not.

Interaction of Dendrimers (Artificial Proteins) with Biological Hydroxyapatite Crystals

This investigation sets out to mimic protein-crystal interaction during biomineralization with the use of artificial proteins (dendrimers). It is hypothesized that these interactions depend on the surface charge of hydroxyapatite crystals. This was investigated with the use of dendrimers with capped surfaces of different charges to probe the surface. We used AFM images of crystal-bound dendrimers to determine the distribution of the surface charge, and its magnitude was correlated to the binding capacity of the dendrimers to the surface. The binding capacity of the dendrimers in ascending order at pH 7.4 was: acetamide-capped, -NHC(O)CH3, neutral charge; carboxylic-acid-capped, -COOH, negative charge; and amine-capped, -NH2, positive charge. AFM images of the crystals showed dendrimers spaced equally along the crystal. The results suggest that the crystal surface has alternating bands of positive and negative charge or a differential charge array, i.e., alternating bands of either more or less positive or negative charge.

Nanoscale morphology of Type I collagen is altered in the Brtl mouse model of Osteogenesis Imperfecta.

Bone has a complex hierarchical structure that has evolved to serve structural and metabolic roles in the body. Due to the complexity of bone structure and the number of diseases which affect the ultrastructural constituents of bone, it is important to develop quantitative methods to assess bone nanoscale properties. Autosomal dominant Osteogenesis Imperfecta results predominantly from glycine substitutions (80%) and splice site mutations (20%) in the genes encoding the α1 or α2 chains of Type I collagen. Genotype-phenotype correlations using over 830 collagen mutations have revealed that lethal mutations are located in regions crucial for collagen-ligand binding in the matrix. However, few of these correlations have been extended to collagen structure in bone. Here, an atomic force microscopy-based approach was used to image and quantitatively analyze the D-periodic spacing of Type I collagen fibrils in femora from heterozygous (Brtl/+) mice (α1(I)G349C), compared to wild type (WT) littermates. This disease system has a well-defined change in the col1α1 allele, leading to a well characterized alteration in collagen protein structure, which are directly related to altered Type I collagen nanoscale morphology, as measured by the D-periodic spacing. In Brtl/+ bone, the D-periodic spacing shows significantly greater variability on average and along the length of the bone compared to WT, although the average spacing was unchanged. Brtl/+ bone also had a significant difference in the population distribution of collagen D-period spacings. These changes may be due to the mutant collagen structure, or to the heterogeneity of collagen monomers in the Brtl/+ matrix. These observations at the nanoscale level provide insight into the structural basis for changes present in bone composition, geometry and mechanical integrity in Brtl/+ bones. Further studies are necessary to link these morphological observations to nanoscale mechanical integrity.