Best Practices for Aggregate Quantitation of Antibody Therapeutics by Sedimentation Velocity Analytical Ultracentrifugation.

Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations.

Supramolecular Delivery Systems for Non-Platinum Metal-Based Anticancer Drugs.

Stimulated by the enormous success of the inorganic complex cisplatin in tumor treatment, interest in metal complexes has recently grown. Within cells, metal complexes can participate in reactions that are not possible with conventional organic substances, and most of them have promising efficacy as anticancer drugs. However, to be effective in vivo metal complexes need adequate delivery systems able to increase their water solubility, the in vivo bioavailability, and the safe delivery to target organs.

Liposomes derivatized with multimeric copies of KCCYSL peptide as targeting agents for HER-2-overexpressing tumor cells.

Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]DTPA[Gd]) have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide) sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems toward cancer cells overexpressing human epidermal growth factor receptor-2 (HER-2) receptors.

Diolein Based Nanostructures as Targeted Theranostics.

Diolein based non-targeted theranostic nanoparticles (DO-NPs) containing 10%wt of the amphiphilic Gadolinium complex (C18)2DTPA(Gd), and targeted NPs, obtained by introducing growing amounts (3% wt, 6% wt or 10% wt) of (C18)2-Peg3000- FA in the sample composition, have been studied for their in vitro and in vivo properties. Cellular binding was studied by lCP-MS analysis of the Gadolinium content and by Surface Plasmon Resonance (SPR) assays. The best formulation in terms of selectivity towards IGROV-1 cells with respect to non-targeted DO-NPs, was that containing 3% (C18)2Peg3000- FA (P < 0.

The influence of liposomal formulation on the incorporation and retention of PNA oligomers.

Liposomal formulations composed of phospholipids with different unsaturation degrees, head groups and at different cholesterol content have been tested for the encapsulation of Peptide Nucleic Acid (PNA) oligomers. The best loading capability (177μg, ER%=87.2) was obtained for pure liposomes of phosphatidylglycerol (DOPG) with negatively charged head group.

Pre-clinical evaluation of eight DOTA coupled gastrin-releasing peptide receptor (GRP-R) ligands for in vivo targeting of receptor-expressing tumors.

Background: Overexpression of the gastrin-releasing peptide receptor (GRP-R) has been documented in several human neoplasms such as breast, prostate, and ovarian cancer. There is growing interest in developing radiolabeled peptide-based ligands toward these receptors for the purpose of in vivo imaging and radionuclide therapy of GRP-R-overexpressing tumors. A number of different peptide sequences, isotopes, and labeling methods have been proposed for this purpose.

Effect of cisplatin containing liposomes formulated by unsaturated chain-containing lipids on gynecological tumor cells.

Gynecological tumors are major therapeutic areas of platinum-based anticancer drugs. Here, we report the characterization and in vitro biological assays of cisplatin-containing Egg L-α-phosphatidylcholine liposomes with different amounts of cholesterol. Dynamic light scattering estimated sizes of all obtained liposomes in the 100 nm range that are suitable for in vivo use.

Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery.

The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells.

Liposomal doxorubicin doubly functionalized with CCK8 and R8 peptide sequences for selective intracellular drug delivery.

A new dual-ligand liposomal doxorubicin delivery system, which couples targeting to enhanced cellular uptake and may lead to a more efficient drug delivery system, is here designed and synthetized. Liposomes based on the composition 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Peg2000-R8/(C18)2-L5-SS-CCK8 (87/8/5 mol/mol/mol) were prepared and loaded with doxorubicin. Presence of the two peptides on the external surface is demonstrated by fluorescence resonance energy transfer assay.

Target selective micelles for bombesin receptors incorporating Au(III)-dithiocarbamato complexes.

Pure sterically stabilized micelles (SSM) of DSPE-PEG2000, and sterically stabilized mixed micelles (SSMM) containing PC or DOPC phospholipids (5, 10 or 20% mol/mol with respect to DSPE-PEG2000) are developed as delivery systems for the gold based cytotoxic drug Au(III)-dithiocarbamato complex AuL12. In particular, SSMM containing 5% of PC at 5mM of lipid concentration encapsulates 61.0 μg of AuL12 with a DL% of 1.

Self-assembled or mixed peptide amphiphile micelles from Herpes simplex virus glycoproteins as potential immunomodulatory treatment.

The use of micelle aggregates formed from peptide amphiphiles (PAs) as potential synthetic self-adjuvant vaccines to treat Herpes simplex virus (HSV) infection are reported here. The PAs were based on epitopes gB409-505 and gD301-309, selected from HSV envelope glycoprotein B (gB) and glycoprotein D (gD), that had their N-terminus modified with hydrophobic moieties containing two C18 hydrocarbon chains. Pure and mixed micelles of gB and/or gD peptide epitopes were easily prepared after starting with the synthesis of corresponding PAs by solid phase methods.

Influence of PEG length on conformational and binding properties of CCK peptides exposed by supramolecular aggregates.

Five novel peptide amphiphiles (PAs), with common formula (C18)2-PEGx-CCK8 in which the CCK8 peptide and the (C18)2-hydrophobic moiety are spaced by polyethylene linkers of different length (PEG moieties with molecular weights of 700, 1000, 1500, 2000, and 3000 Daltons) are described. They act as potential target-selective nanocarriers towards tumor cells overexpressing cholecistokynin receptors. PAs self-assemble in supramolecular aggregates, with hydrodynamic radius ranging between 63 and 104 nm, as indicated by DLS measurements.

Peptides targeting chemokine receptor CXCR4: structural behavior and biological binding studies.

CXCR4 is a G-protein-coupled receptor involved in a number of physiological processes in the hematopoietic and immune systems. CXCL12/CXCR4 axis plays a central role in diseases, such as HIV, cancer, WHIM syndrome, rheumatoid arthritis, pulmonary fibrosis, and lupus and, hence, indicated as putative therapeutic target. Although multiple CXCR4 antagonists have been developed, there is only one marketed drug, plerixafor, indicated for stem cell mobilization in poor mobilizer patients.

A heme-peptide metalloenzyme mimetic with natural peroxidase-like activity.

Mimicking enzymes with alternative molecules represents an important objective in synthetic biology, aimed to obtain new chemical entities for specific applications. This objective is hampered by the large size and complexity of enzymes. The manipulation of their structures often leads to a reduction of enzyme activity.

Redox and electrocatalytic properties of mimochrome VI, a synthetic heme peptide adsorbed on gold.

Mimochrome VI (MC-VI) is a synthetic heme peptide containing a helix-heme-helix sandwich motif designed to reproduce the catalytic activity of heme oxidases. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of the electron-transfer process for MC-VI immobilized through hydrophobic interactions on a gold electrode coated with a nonpolar SAM of decane-1-thiol have been determined through cyclic voltammetry. Immobilization slightly affects the reduction potential of MC-VI, which under these conditions electrocatalytically turns over molecular oxygen.

Molecular strategies for protein stabilization: the case of a trehalose/maltose-binding protein from Thermus thermophilus.

The trehalose/maltose-binding protein (MalE1) is one component of trehalose and maltose uptake system in the thermophilic organism Thermus thermophilus. MalE1 is a monomeric 48 kDa protein predominantly organized in alpha-helix conformation with a minor content of beta-structure. In this work, we used Fourier-infrared spectroscopy and in silico methodologies for investigating the structural stability properties of MalE1.

D-galactose/D-glucose-binding Protein from Escherichia coli as Probe for a Non-consuming Glucose Implantable Fluorescence Biosensor.

D-Galactose/D-glucose-binding protein from E. coli (GGBP) is a monomer thatbinds glucose with high affinity. The protein structure of GGBP is organized in twoprincipal domains linked by a hinge region that form the sugar-binding site.