Tumor-specific gene transfer with receptor-mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers.

Synthetic nanoparticle formulations have the potential for tumor-targeted gene delivery. Receptor-targeted nanocomplex (RTN) formulations comprise mixtures of cationic liposomes and targeting peptides that self-assemble on mixing with nucleic acids. RTN formulations were prepared containing different polyethylene glycol (PEG)ylated lipids with esterase-cleavable linkers (e.g., ME42) to promote intracellular PEG detachment and nanoparticle disassembly. In addition, integrin-targeting peptides (peptide ME27) were tested with endosomal furin- and cathepsin B-cleavable peptide linkers located between the integrin-binding ligand and the K(16) nucleic acid-binding domain to promote intracellular disengagement from the receptor. ME42/ME27 RTNs formed stable particles of <200 nm in isotonic salt buffers, compared with 4-microm particles formed by un-PEGylated RTNs. Transfection efficiency by PEG-modified, cleavable RTNs improved approximately 2-fold in 4 different cell lines, with 80% efficiency in murine neuroblastoma cells. In an in vivo model of neuroblastoma, ME42/ME27 RTNs delivering luciferase genes were tumor specific, with little expression in other organs tested. PEGylation of the RTNs enhanced luciferase transfection 5-fold over non-PEG formulations, whereas the cleavability of the peptide ME27 enhanced transfection 4-fold over that of RTNs with noncleavable peptides. Cleavability of the lipid for in vivo transfections had no effect. PEGylated, cleavable RTN formulations offer prospects for tumor-specific therapeutic gene transfer.

Stabilized integrin-targeting ternary LPD (lipopolyplex) vectors for gene delivery designed to disassemble within the target cell.

Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM.

A receptor-targeted nanocomplex vector system optimized for respiratory gene transfer.

Synthetic vectors for cystic fibrosis (CF) gene therapy are required that efficiently and safely transfect airway epithelial cells, rather than alveolar epithelial cells or macrophages, and that are nonimmunogenic, thus allowing for repeated delivery. We have compared several vector systems against these criteria including GL67, polyethylenimine (PEI) 22 and 25 kd and two new, synthetic vector formulations, comprising a cationic, receptor-targeting peptide K(16)GACSERSMNFCG (E), and the cationic liposomes (L) DHDTMA/DOPE or DOSEP3/DOPE. The lipid and peptide formulations self assemble into receptor-targeted nanocomplexes (RTNs) LED-1 and LED-2, respectively, on mixing with plasmid (D). LED-1 transfected airway epithelium efficiently, while LED-2 and GL67 preferentially transfected alveolar cells. PEI transfected airway epithelial cells with high efficiency, but was more toxic to the mice than the other formulations. On repeat dosing, LED-1 was equally as effective as the single dose, while GL67 was 30% less effective and PEI 22 kd displayed a 90% reduction of efficiency on repeated delivery. LED-1 thus was the only formulation that fulfilled the criteria for a CF gene therapy vector while GL67 and LED-2 may be appropriate for other respiratory diseases. Opportunities for PEI depend on a solution to its toxicity problems. LED-1 formulations were stable to nebulization, the most appropriate delivery method for CF.

Multivalence and spot heterogeneity in microarray-based measurement of binding constants.

Microarray technology is increasingly used for a miniaturised and parallel measurement of binding constants. In microarray experiments heterogeneous functionalization of surfaces with capture molecules is a problem commonly encountered. For multivalent ligands, especially, however, binding is strongly affected by receptor density. Here we show that high-resolution imaging of microarrays followed by image segmentation and separate analysis of bright and dark parts provides valuable information about ligand binding. Binding titrations were conducted with monovalent and bivalent fluorescent ligand peptides for the model receptor vancomycin. Microarrays were scanned with a confocal microscope and inhomogeneous spots were evaluated either as a whole or after segmentation into bright and dark areas. Whereas the binding constant for the monovalent ligand was hardly affected by spot heterogeneity, for the bivalent ligand affinity was higher for the parts of the spots with a greater density of receptors. This information was lost if the spots were analysed as a whole. These results reveal that imaging resolution may be a key factor in miniaturised binding assays, emphasising the importance of high-resolution images and image segmentation for new techniques, for example SPR imaging.

Peptide microarrays for the detection of molecular interactions in cellular signal transduction.

The formation of protein complexes is a hallmark of cellular signal transduction. Here, we show that peptide microarrays provide a robust and quantitative means to detect signalling-dependent changes of molecular interactions. Recruitment of a protein into a complex upon stimulation of a cell leads to the masking of an otherwise exposed binding site. In cell lysates this masking can be detected by reduced binding to a microarray carrying a peptide that corresponds to the binding motif of the respective interaction domain. The method is exemplified for the lymphocyte-specific tyrosine kinase 70 kDa zeta-associated protein binding to a bis-phosphotyrosine-motif of the activated T-cell receptor via its tandem SH2 domain. Compared to established techniques, the method provides a significant shortcut to the detection of molecular interactions.

Determination of binding constants on microarrays with confocal fluorescence detection.

Confocal laser scanning microscopy was employed for the determination of binding constants of receptor-ligand interactions in a microarray format. Protocols for a localized immobilization of amine containing substances on glass via GOPTS (3-glycidyloxypropyl)trimethoxysilane) were optimized with respect to the detection of ligand binding by fluorescence. Compatibility with miniaturization by nanopipetting devices was ensured during all steps. The interaction of the tripeptide L-Lys-D-Ala-D-Ala with vancomycin immobilized on glass served as a model. To minimize consumption of ligand, binding constants were determined by stepwise titration of binding sites. The binding constant of the unlabeled ligand was determined by competitive titration with a fluorescently labeled analogue. The determined binding constants agreed well with those determined by other techniques, previously. Labeled ligand bound stronger than the unlabeled one. This difference was dye-dependent. Still, binding was specific for the tripeptide moiety confirming that ligand and fluorescent analogue competed for the same binding sites these results validate the determination of binding constants by competitive titration. The protocols established for confocal fluorescence detection are applicable to axially resolved detection modalities and screening for unlabeled ligands by competitive titration in general.

Modulation of neuronal activity by the endogenous pentapeptide QYNAD.

Inflammation and demyelination both contribute to the neurological deficits characteristic of multiple sclerosis. Neurological dysfunctions are attributable to inflammatory demyelination and, in addition, to soluble factors such as nitric oxide, cytokines and antibodies. QYNAD, an endogenous pentapeptide identified in the cerebrospinal fluid of patients with demyelinating disorders, has been proposed to promote axonal dysfunction by blocking sodium channels. The present study aimed at characterizing the properties of QYNAD in acutely isolated thalamic neurons in vitro. QYNAD, but not a scrambled peptide (NYDQA), blocked sodium channels in neurons by shifting the steady-state inactivation to more negative potentials. Blocking properties followed a dose-response curve with a maximum effect at 10 microm. A fluorescently labelled QYNAD analogue with retained biological activity specifically stained thalamic neurons, positive for type II sodium channels, thus demonstrating the specificity of QYNAD binding. Our study confirms and extends previous observations describing QYNAD as a potent sodium channel-blocking agent. These data as well as our preliminary observations in in vivo experiments in an animal model of inflammatory CNS demyelination warrant further in vivo studies in order to clarify the exact pathogenetic role of QYNAD in inflammatory neurological diseases.