We find that conjugation and chemical composition can alter fundamental aspects of aptamer residence in circulation and distribution to tissues. Though the primary effect of PEGylation was on aptamer clearance, the prolonged systemic exposure afforded by presence of the 20 kDa moiety appeared to facilitate distribution of aptamer to tissues, particularly those of highly perfused organs.

Aptamers (protein binding oligonucleotides) have potential as a new class of targeted therapeutics. For applications requiring chronic systemic administration, aptamers must achieve high-affinity target binding while simultaneously retaining high in vivo stability, tolerability, and ease of chemical synthesis. To this end, we describe a method for generating aptamers composed entirely of 2'-O-methyl nucleotides (mRmY). We present conditions under which 2'-O-methyl transcripts can be generated directly and use these conditions to select a fully 2'-O-methyl aptamer from a library of 3 x 10(15) unique 2'-O-methyl transcripts. This aptamer, ARC245, is 23 nucleotides in length, binds to vascular endothelial growth factor (VEGF) with a Kd of 2 nM, and inhibits VEGF activity in cellular assays. Notably, ARC245 is so stable that degradation cannot be detected after 96 hr in plasma at 37 degrees C or after autoclaving at 125 degrees C. We believe ARC245 has considerable potential as an antiangiogenesis therapeutic.

No abstract

Inhibition of platelet derived growth factor (PDGF) can increase the efficacy of other cancer therapeutics, but the cellular mechanism is incompletely understood. We examined the cellular effects on tumor vasculature of a novel DNA oligonucleotide aptamer (AX102) that selectively binds PDGF-B. Treatment with AX102 led to progressive reduction of pericytes, identified by PDGF receptor B, NG2, desmin, or A-smooth muscle actin immunoreactivity, in Lewis lung carcinomas. The decrease ranged from 35% at 2 days, 63% at 7 days, to 85% at 28 days. Most tumor vessels that lacked pericytes at 7 days subsequently regressed. Overall tumor vascularity decreased 79% over 28 days, without a corresponding decrease in tumor size. Regression of pericytes and endothelial cells led to empty basement membrane sleeves, which were visible at 7 days, but only 54% remained at 28 days. PDGF-B inhibition had a less pronounced effect on pancreatic islet tumors in RIP-Tag2 transgenic mice, where pericytes decreased 47%, vascularity decreased 38%, and basement membrane sleeves decreased 21% over 28 days. Taken together, these findings show that inhibition of PDGF-B signaling can lead to regression of tumor vessels, but the magnitude is tumor specific and does not necessarily retard tumor growth. Loss of pericytes in tumors is an expected direct consequence of PDGF-B blockade, but reduced tumor vascularity is likely to be secondary to pericyte regression. [Cancer Res 2007;67(15):7358-67]

The transition from microcrystalline to nanocrystalline diamond films grown from Ar/H 2 /CH 4 microwave plasmas has been investigated. Both the cross-section and plan-view micrographs of scanning electron microscopy reveal that the surface morphology, the grain size, and the growth mechanism of the diamond films depend strongly on the ratio of Ar to H 2 in the reactant gases. Microcrystalline grain size and columnar growth have been observed from films produced from Ar/H 2 /CH 4 microwave discharges with low concentrations of Ar in the reactant gases. By contrast, the films grown from Ar/H 2 /CH 4 microwave plasmas with a high concentration of Ar in the reactant gases consist of phase pure nanocrystalline diamond, which has been characterized by transmission electron microscopy, selected area electron diffraction, and electron energy loss spectroscopy. X-ray diffraction and Raman spectroscopy reveal that the width of the diffraction peaks and the Raman bands of the as-grown films depends on the ratio of Ar to H 2 in the plasmas and are attributed to the transition from micron to nanometer size crystallites. It has been demonstrated that the microstructure of diamond films deposited from Ar/H 2 /CH 4 plasmas can be controlled by varying the ratio of Ar to H 2 in the reactant gas. The transition becomes pronounced at an Ar/H 2 volume ratio of 4, and the microcrystalline diamond films are totally transformed to nanocrystalline diamond at an Ar/H 2 volume ratio of 9. The transition in microstructure is presumably due to a change in growth mechanism from CH 3 • in high hydrogen content to C 2 as a growth species in low hydrogen content plasmas.

No abstract

Nanocrystalline diamond thin films have been synthesized in an ArCH 4 microwave discharge, without the addition of molecular hydrogen. X-ray diffraction, transmission electron microscopy, and electron energy loss spectroscopy characterizations show that the films consist of a pure crystalline diamond phase with very small grain sizes ranging from 3 to 20 nm. Atomic force microscopy analysis demonstrates that the surfaces of the nanocrystalline diamond films remain smooth independent of the film thicknesses. Furthermore, the reactant gas pressure, which strongly affects the concentration of C 2 dimer in the ArCH 4 plasma as well as the growth rate of the films, has been found to be a key parameter for the nanocrystalline diamond thin film depositions.

We have investigated the effect of substrate temperature on the growth rate and properties of nanocrystalline diamond thin films prepared by microwave plasma-assisted chemical vapor deposition on (100) Si from a 1% methane (CH4) precursor in argon (Ar). In previous work we have shown that the carbon dimer C2 is the dominant growth species for this CH4/Ar system without the addition of molecular hydrogen. In the present work, the apparent activation energy for this growth process from C2 was determined from a standard Arrhenius-type analysis of the growth rate data for substrate temperatures between 500 and 900 °C. The measured value of 5.85±0.438 kcal/mol (0.254±0.019 eV/atom) is shown to be in close agreement with the results of recent modeling studies of the energetics of C2 addition to the diamond (110)–(1×1):H surface. These results have important implications for low-temperature diamond coating of nonrefractory materials such as glasses.