Application of DSC and Imaging Techniques on the Development of Innovative Chimeric/Mixed Nanosystems

The aim of this study was to rationally design, develop and investigate chimeric/mixed liposomes, comprising the lipid L-a-phosphatidylcholine, hydrogenated (Soy) (HSPC) and two pH-sensitive amphiphilic diblock copolymers poly(2-(dimethylamino)ethyl methacrylate)-b-poly(lauryl methacrylate) (PDMAEMA-b-PLMA), at various molar ratios to be proposed as new drug nanocarriers. Initially, chimeric bilayers of phospholipid and polymer were prepared and characterized by differential scanning calorimetry (DSC) in order to assess the thermotropic behavior in physiological and acidic environment. Based on the results, chimeric liposomes were developed by thin-film hydration and their physicochemical properties, as well as colloidal physical stability, were investigated with dynamic, electrophoretic and static light scattering (DLS, ELS and SLS). In addition, their size and morphology were evaluated through atomic force microscopy (AFM) and cryogenic transmission electron microscopy (cryo-TEM). An in vitro screening confirmed the low toxicity of these bioinspired and biocompatible nanosystems, which are composed of non-toxic biomaterials as building blocks. Finally, based on the above set of results, the most promising for in vivo applications chimeric liposomes were optimized. Classic and micro-DSC techniques were employed to highlight the thermodynamic phenomena that drive the self-assembly of these mixed nanosystems and that contribute to the membrane properties (transition cooperativity, fluidity, phase separation, etc.), also quantifying the pH-responsive character of these nanosystems. Complementary information as regard the morphological aspects emerged by imaging techniques and the influence of the concentration and hydrophilic-to-hydrophobic balance of the copolymer was assessed. Drug molecule entrapment/incorporation and release studies will be the next step of this work and require a multidisciplinary approach. In this integrated frame, the calorimetric methods are of great relevance as regards both the characterization and the design of these nanosystems

The 4C5 Cell-Impermeable Anti-HSP90 Antibody with Anti-Cancer Activity, Is Composed of a Single Light Chain Dimer

MAb 4C5 is a cell impermeable, anti-HSP90 murine monoclonal antibody, originally produced using hybridoma technology. We have previously shown that mAb 4C5 specifically recognizes both the α- and to a lesser extent the β-isoform of HSP90. Additionally, in vitro and in vivo studies revealed that by selectively inhibiting the function of cell-surface HSP90, mAb 4C5 significantly impairs cancer cell invasion and metastasis. Here we describe the reconstitution of mAb 4C5 into a mouse-human chimera. More importantly we report that mAb 4C5 and consequently its chimeric counterpart are completely devoid of heavy chain and consist only of a functional kappa light chain dimer. The chimeric antibody is shown to retain the original antibody's specificity and functional properties. Thus it is capable of inhibiting the function of surface HSP90, leading to reduced cancer cell invasion in vitro. Finally, we present in vivo evidence showing that the chimeric 4C5 significantly inhibits the metastatic deposit formation of MDA-MB-453 cells into the lungs of SCID mice. These data suggest that a chimeric kappa light chain antibody could be potentially used as an anti-cancer agent, thereby introducing a novel type of antibody fragment, with reduced possible adverse immunogenic effects, into cancer therapeutics

A Novel Extracellular Hsp90 Mediated Co-Receptor Function for LRP1 Regulates EphA2 Dependent Glioblastoma Cell Invasion

Extracellular Hsp90 protein (eHsp90) potentiates cancer cell motility and invasion through a poorly understood mechanism involving ligand mediated function with its cognate receptor LRP1. Glioblastoma multiforme (GBM) represents one of the most aggressive and lethal brain cancers. The receptor tyrosine kinase EphA2 is overexpressed in the majority of GBM specimens and is a critical mediator of GBM invasiveness through its AKT dependent activation of EphA2 at S897 (P-EphA2(S897)). We explored whether eHsp90 may confer invasive properties to GBM via regulation of EphA2 mediated signaling.We find that eHsp90 signaling is essential for sustaining AKT activation, P-EphA2(S897), lamellipodia formation, and concomitant GBM cell motility and invasion. Furthermore, eHsp90 promotes the recruitment of LRP1 to EphA2 in an AKT dependent manner. A finding supported by biochemical methodology and the dual expression of LRP1 and P-EphA2(S897) in primary and recurrent GBM tumor specimens. Moreover, hypoxia mediated facilitation of GBM motility and invasion is dependent upon eHsp90-LRP1 signaling. Hypoxia dramatically elevated surface expression of both eHsp90 and LRP1, concomitant with eHsp90 dependent activation of src, AKT, and EphA2.We herein demonstrate a novel crosstalk mechanism involving eHsp90-LRP1 dependent regulation of EphA2 function. We highlight a dual role for eHsp90 in transducing signaling via LRP1, and in facilitating LRP1 co-receptor function for EphA2. Taken together, our results demonstrate activation of the eHsp90-LRP1 signaling axis as an obligate step in the initiation and maintenance of AKT signaling and EphA2 activation, thereby implicating this pathway as an integral component contributing to the aggressive nature of GBM

A Novel Intracellular Isoform of Matrix Metalloproteinase-2 Induced by Oxidative Stress Activates Innate Immunity

Experimental and clinical evidence has pinpointed a critical role for matrix metalloproteinase-2 (MMP-2) in ischemic ventricular remodeling and systolic heart failure. Prior studies have demonstrated that transgenic expression of the full-length, 68 kDa, secreted form of MMP-2 induces severe systolic failure. These mice also had unexpected and severe mitochondrial structural abnormalities and dysfunction. We hypothesized that an additional intracellular isoform of MMP-2, which affects mitochondrial function is induced under conditions of systolic failure-associated oxidative stress.Western blots of cardiac mitochondria from the full length MMP-2 transgenics, ageing mice and a model of accelerated atherogenesis revealed a smaller 65 kDa MMP-2 isoform. Cultured cardiomyoblasts subjected to transient oxidative stress generated the 65 kDa MMP-2 isoform. The 65 kDa MMP-2 isoform was also induced by hypoxic culture of cardiomyoblasts. Genomic database analysis of the MMP-2 gene mapped transcriptional start sites and RNA transcripts induced by hypoxia or epigenetic modifiers within the first intron of the MMP-2 gene. Translation of these transcripts yields a 65 kDa N-terminal truncated isoform beginning at M(77), thereby deleting the signal sequence and inhibitory prodomain. Cellular trafficking studies demonstrated that the 65 kDa MMP-2 isoform is not secreted and is present in cytosolic and mitochondrial fractions, while the full length 68 kDa isoform was found only in the extracellular space. Expression of the 65 kDa MMP-2 isoform induced mitochondrial-nuclear stress signaling with activation of the pro-inflammatory NF-κB, NFAT and IRF transcriptional pathways. By microarray, the 65 kDa MMP-2 induces an innate immunity transcriptome, including viral stress response genes, innate immunity transcription factor IRF7, chemokines and pro-apoptosis genes.A novel N-terminal truncated intracellular isoform of MMP-2 is induced by oxidative stress. This isoform initiates a primary innate immune response that may contribute to progressive cardiac dysfunction in the setting of ischemia and systolic failure

Hsp90 binds directly to fibronectin (FN) and inhibition reduces the extracellular fibronectin matrix in breast cancer cells:

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells

Position-sensorless control of a submersible PMSM fed over a long cable and two transformers

This paper proposes the combination of two sensorless control algorithms for the secure start-up and the efficient operation of a remotely-controlled PMSM. V / f control is applied during the start-up of the motor and vector control is used for operation at higher speeds. The investigated system consists of a long step-out configuration with a cable and two transformers between the PMSM and its VSD. The system is tested with simulations and with experiments on a lab model. The results demonstrate that V / f control can safely start the PMSM. The effect of the inertia of the rotating assembly, the load torque and the initial rotor position on the start-up performance of the system is investigated and the successful transition from V / f control to vector control is demonstrated