Preclinical evaluation of dual PI3K-mTOR inhibitors and histone deacetylase inhibitors in head and neck squamous cell carcinoma.

BACKGROUND: We examine the potential value of a series of clinically relevant PI3K-mTOR inhibitors alone, or in combination with histone deacetylase inhibitors, in a model of head and neck squamous cell carcinoma (HNSCC). METHODS: Head and neck squamous cell carcinoma cell lines, human keratinocyte and HNSCC xenograft models were treated with histone deacetylase inhibitors (HDACIs) and new generation PI3K and dual PI3K-mTOR inhibitors either alone or in combination. Cell and tumour tissue viability and proliferation were then determined in vitro and in vivo. RESULTS: Phosphatidylinositol-3-phosphate kinase, AKT and dual PI3K-mTOR inhibitors caused marked in vitro enhancement of cytotoxicity induced by HDACIs in HNSCC cancer cells. This effect correlates with AKT inhibition and is attenuated by expression of constitutively active AKT. Histone deacetylase inhibitor and phosphatidylinositol-3-phosphate kinase inhibitors (PI3KIs) inhibited tumour growth in xenograft models of HNSCC. Importantly, we observed intratumoural HDAC inhibition and PI3K inhibition as assessed by histone H3 acetylation status and phospho-AKT staining, respectively. However, we saw no evidence of improved efficacy with an HDACI/PI3KI combination. INTERPRETATION: That PI3K and dual PI3K-mTOR inhibitors possess antitumour effect against HNSCC in vivo.

Spin torque, tunnel-current spin polarization, and magnetoresistance in MgO magnetic tunnel junctions.

We employ the spin-torque response of magnetic tunnel junctions with ultrathin MgO tunnel barrier layers to investigate the relationship between spin transfer and tunnel magnetoresistance (TMR) under finite bias, and find that the spin torque per unit current exerted on the free layer decreases by < 10% over a bias range where the TMR decreases by > 40%. This is inconsistent with free-electron-like spin-polarized tunneling and reduced-surface-magnetism models of the TMR bias dependence, but is consistent with magnetic-state-dependent decay lengths in the tunnel barrier.

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.

Silyl ether-coupled poly(epsilon-caprolactone)s with stepwise hydrolytic degradation profiles.

Silyl ether-coupled poly(epsilon-caprolactone)s (PCLs) with stepwise degradation profiles were synthesized via the cross-dehydrocoupling polymerizations between 1,4-bis(dimethylsilyl)benzene (BDSB) and telechelic, diol-terminated PCL macromonomers. With the presence of 10 wt % palladium on activated carbon as the catalyst, the condensations between BDSB and diol-terminated PCL macromonomers having molecular weights of 1200, 2010, and 5500 g/mol were performed in toluene at 100 degrees C under argon. Hydrogen was eliminated as the condensate upon the formation of silyl ether bonds linking the PCL blocks, yielding within 24 h, silyl ether-coupled PCLs of molecular mass 7590, 29,900, and 29,500 g/mol, respectively. The characterization of each polymer included (1)H NMR, (13)C NMR, and (29)Si NMR spectroscopies, size exclusion chromatography (SEC), and differential scanning calorimetry. The hydrolytic degradation properties of the polymers in solution were studied, and the molecular weight reductions over time were monitored by SEC. The silyl ether linkages of the polymers underwent hydrolysis in the presence of mineral acids, whereas the PCL segments released from the cleavage of the labile silyl ether coupling unit did not undergo detectable molecular weight reduction over 15 days. In the presence of acetic acid, the silyl ether functionalities were cleaved with a half-life of 3 days; however, the PCL chain required reaction with trifluoroacetic acid to give a number-average molecular weight loss half-life of 4 days. The silyl ether-coupled PCLs underwent degradation in a gradient fashion, therefore, by a protocol that involved the addition of acetic acid for cleavage of the silyl ether functionalities, followed by further addition of trifluoroacetic acid to bring the hydrolysis of the silyl ether functionalities to completion and to trigger the degradation of PCL segments.

Novel polymers: molecular to nanoscale order in three dimensions.

The assembly of polymer chains in solution is a powerful method that is leading to the preparation of interesting and unique macromolecular-based synthetic nanostructures. Specific control over the intramolecular and intermolecular physical interactions dictates either the folding of single chains or the aggregation and ordering of multiple chains. This control is provided through the selective placement of functional groups along the polymer backbone and the relative strengths of their attractive and repulsive interactions.

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.

Location of fluorotryptophan sequestered in an amphiphilic nanoparticle by rotational-echo double-resonance NMR.

Rotational-echo double-resonance (REDOR) 13C NMR spectra (with 19F dephasing) have been obtained of 6-fluorotryptophan complexed by a polymeric amphiphilic nanosphere consisting of a polystyrene core covalently attached to a poly(acrylic acid)-polyacrylamide shell. The REDOR spectra show that aromatic carbons from the polystyrene core and oxygenated carbons in the poly(acrylic acid)-polyacrylamide shell are both proximate to the 19F of 6-fluorotryptophan. Molecular modeling restrained by distances inferred from the REDOR spectra suggests that all of the 6-fluorotryptophans are in the shell but within 10 A of the core-shell interface.

Knowledge and skill retention of emergency care attendants, EMT-As, and EMT-Ps.

The retention of knowledge and skill proficiency was evaluated for emergency care attendants (ECAs), emergency medical technicians (EMT-As) and paramedics (EMT-Ps). The sample represented 4.1% of the total number of individuals trained and certified in these positions in Texas. The average loss of didactic knowledge did not exceed 10% over a two-year period. After two years the ECAs had lost approximately 55%, EMT-As 50%, and EMT-Ps 61% of the their basic skills proficiency. Retention of knowledge and skill appears to be directly related to frequency of use. Participants in continuing education programs experienced an 11% better retention average for skill than did nonparticipants. The employees of privately operated ambulance services retained their basic skills better than did members of other types of services. The rate of knowledge deterioration for the sample evaluated was not correlated (R = 0.08) to the original written score.