Nanostructured materials designed for cell binding and transduction.

The surface-functionalization of shell cross-linked (SCK) nanoparticles with the oligomeric peptide sequence YGRKKRRQRRR, the protein transduction domain (PTD) from the human immunodeficiency virus TAT protein, is described, and the cell binding interactions these nanobioconjugates exhibit are demonstrated. A convergent synthetic strategy was employed, whereby the SCK nanoparticles and the PTD were prepared independently and then coupled together during immobilization of the PTD component on a solid support. The SCK nanoparticles were prepared by the micellization of amphiphilic block copolymers of poly(epsilon-caprolactone-b-acrylic acid), followed by amidation-based cross-linking of the acrylic acid residues located within the micellar corona. The PTD sequence was constructed upon a solid support, from C-terminus to N-terminus, followed by extension with four glycine residues, leaving the amino chain end for subsequent coupling with remaining acrylic acid functionalities present on the surface of the SCK. Finally, cleavage from the solid support was performed, which also facilitated deprotection of the peptide side chain functionalities as well as hydrolysis of the poly(epsilon-caprolactone) segments composing the SCK core domain, to yield PTD-derivatized nanocage structures (PTD-nanocage). Covalent labeling of the SCK precursor with fluorescein-5-thiosemicarbazide provided fluorescently tagged PTD-nanocage nanobioconjugates to allow for their detection by fluorescence microscopy. The fluorescent PTD-nanocage bioconjugates were found to interact with CHO cells and HeLa cells, whereas the analogous structure lacking the PTD component did not. CHO cells bound with fluorescent PTD-nanocage bioconjugates were analyzed using flow cytometry and fluorescence activated cell sorting (FACS). Fluorescence confocal microscopy of isolated bioconjugate-bound CHO cells indicated that the bioconjugated nanoparticles were primarily located near the cell periphery; however, transduction of the nanoparticle into the cells also occurred.

Modulation of the binding affinity of myelopoietins for the interleukin-3 receptor by the granulocyte colony-stimulating factor receptor agonist.

Myelopoietins (MPOs) are a family of recombinant chimeric proteins that are both interleukin-3 (IL-3) receptor and granulocyte colony-stimulating factor (G-CSF) receptor agonists. In this study, MPO molecules containing one of three different IL-3 receptor agonists linked with a common G-CSF receptor agonist have been examined for their IL-3 receptor binding characteristics. Binding to the alpha-subunit of the IL-3 receptor revealed that the affinity of the MPO molecules was 1.7-3.4-fold less potent than those of their individual cognate IL-3 receptor agonists. The affinity decrease was reflected in the MPO chimeras having approximately 2-fold slower dissociation rates and 2.7-5.5-fold slower association rates than the corresponding specific IL-3 receptor agonists alone. The affinity of binding of the MPO molecules to the heteromultimeric alphabeta IL-3 receptor expressed on TF-1 cells was either 3-, 10-, or 42-fold less potent than that of the individual cognate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonance, near-UV circular dichroism, dynamic light scattering, analytical ultracentrifugation, and size exclusion chromatography experiments determined that there were significant tertiary structural differences between the MPO molecules. These structural differences suggested that the IL-3 and G-CSF receptor agonist domains within the MPO chimera may perturb one another to varying degrees. Thus, the differential modulation of affinity observed in IL-3 receptor binding may be a direct result of the magnitude of these interdomain interactions.

Large-scale purification of myeloperoxidase from HL60 promyelocytic cells: characterization and comparison to human neutrophil myeloperoxidase.

A large-scale purification procedure was developed for the isolation of myeloperoxidase from HL60 promyelocytic cells in culture. Initial studies showed the bulk of peroxidase-positive myeloperoxidase activity to be located in the cetyltrimethylammonium bromide solubilized particulate fraction of cell homogenates. The myeloperoxidase was then chromatographically purified using concanavalin A followed by gel filtration. SDS-PAGE analysis of the final preparation showed the presence of only two proteins with molecular masses of approximately 55 and 15 kDa, corresponding to the large and small subunits of myeloperoxidase. These data, along with Reinheit Zahl (RZ) values (A(430)/A(280)) of greater than or equal to 0.72, indicate that the myeloperoxidase prepared by this method is apparently homogeneous. Preparations routinely yielded 12-20 mg of pure myeloperoxidase per 10 ml of cell pellet. The HL60 myeloperoxidase was shown to be indistinguishable from purified human neutrophil myeloperoxidase by size exclusion chromatography, analytical ultracentrifugation, SDS-PAGE, Western blot, and NH(2)-terminal sequence analysis. The activities of the two myeloperoxidase samples, as measured using either the tetramethylbenzidine or the taurine chloramine assay, were indistinguishable. Finally, both enzymes responded identically to dapsone and aminobenzoic acid hydrazide, known inhibitors of myeloperoxidase. A protocol is presented here for the rapid, large-scale purification of myeloperoxidase from cultured HL60 cells, as well as evidence for the interchangeability of this myeloperoxidase and that purified from human neutrophils.

Packaging of DNA by shell crosslinked nanoparticles.

We demonstrate compaction of DNA with nanoscale biomimetic constructs which are robust synthetic analogs of globular proteins. These constructs are approximately 15 nm in diameter, shell crosslinked knedel-like (SCKs) nanoparticles, which are prepared by covalent stabilization of amphiphilic di-block co-polymer micelles, self-assembled in an aqueous solution. This synthetic approach yields size-controlled nanoparticles of persistent shape and containing positively charged functional groups at and near the particle surface. Such properties allow SCKs to bind with DNA through electrostatic interactions and facilitate reduction of the DNA hydrodynamic diameter through reversible compaction. Compaction of DNA by SCKs was evident in dynamic light scattering experiments and was directly observed by in situ atomic force microscopy. Moreover, enzymatic digestion of the DNA plasmid (pBR322, 4361 bp) by Eco RI was inhibited at low SCK:DNA ratios and prevented when [le]60 DNA bp were bound per SCK. Digestion by Msp I in the presence of SCKs resulted in longer DNA fragments, indicating that not all enzyme cleavage sites were accessible within the DNA/SCK aggregates. These results have implications for the development of vehicles for successful gene therapy applications.

Physical and kinetic characterization of recombinant human cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) mediates the exchange of triglycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) between lipoproteins in the plasma. In order to better understand the lipid transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity chromatography. Purified recombinant CETP had a weight-average relative molecular mass (MW) of 69561, determined by sedimentation equilibrium, and a specific absorption coefficient of 0.83 litre.g-1.cm-1. The corresponding hydrodynamic diameter (Dh) of the protein, determined by dynamic light scattering, was 14 nm, which is nearly twice the expected value for a spheroidal protein of this molecular mass. These data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% alpha-helix, 35% beta-sheet, 17% turn and 16% random coil. Like the natural protein from plasma, the recombinant protein consisted of several glycoforms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesterified cholesterol (UC), PL, TG and fatty acids (FA) were associated with the pure protein. Quantitative analyses verified that each mol of CETP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments, the transfer rates for CE or TG from HDL to LDL were found to be proportional to the initial concentrations of the respective ligands in the donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a Km of 700 nM for LDL particles and of 2000 nM for HDL particles, and a kcat of 2 s-1. The Km values were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified independently for CE, TG, PL and UC in 'half-reaction' experiments.

The interaction of calmodulin and polylysine as studied by 1H NMR spectroscopy and sedimentation equilibrium centrifugation.

The effects of polylysine on calmodulin were assessed using 1H NMR and sedimentation equilibrium centrifugation. Sedimentation equilibrium centrifugation measurements demonstrated that calmodulin associates with polylysine at calmodulin-polylysine molar ratios ranging from 10:1 to 2.5:1 and when polylysine is increased above the molar ratio of 1:1 a precipitate is formed. At a 1:2.5 calmodulin:polylysine molar ratio, 75% of the calmodulin precipitates from the solution and virtually no polylysine is present in the precipitate. 1H NMR studies of the aromatic region of calmodulin identified chemical shifts of three peaks at a calmodulin:polylysine molar ratio of 1:1. These studies suggest that polylysine associates with calmodulin in aqueous solution and can alter the structure of calmodulin to cause calmodulin self-aggregation.

Physicochemical properties of amaranthin, the lectin from Amaranthus caudatus seeds.

Amaranthin is the lectin present in the seeds of Amaranthus caudatus, which specifically binds the T-disaccharide (Gal beta 1,3GalNAc alpha-O-). The lectin is composed of a single type of subunit with Mr = 33,000-36,000 (Rinderle et al., 1989). Equilibrium sedimentation (Mr = 62,900) and low-angle laser light scattering (Mr = 61,400) methods have been used to unambiguously establish the native multimeric structure of amaranthin as a homodimer. These absolute molecular weight methods and the calculated Stokes radius (27.2 A) indicate that the amaranthin dimer is highly compact relative to typical globular proteins, and thus, anomalous molecular weight values are obtained when simple size exclusion chromatography is used to determine the molecular weight of amaranthin. Studies with a homobifunctional cross-linking reagent and amaranthin further support the existence of a lectin homodimer. The stoichiometry of carbohydrate binding was determined to be one T-disaccharide-binding site per amaranthin subunit (Ka = 3.6 X 10(5) M-1). Amaranthin exhibits hydrophobic-binding properties as indicated by binding of 8-anilino-1-naphthalene-sulfonate (Ka = 3.6 X 10(3) M-1) and 6-toluidinyl-2-naphthalenesulfonate (Ka = 2 X 10(4) M-1). Serological studies suggest that amaranthin does not appear to be present in the stems or leaves of the A. caudatus plant, nor were there any indications for the presence of cross-reactive material.