8 research outputs found
Acidification reduces and alkalization augments cancer cell glucose uptake.
<p>(A) Reduced and augmented FDG uptake in breast cancer and colon cancer cells after 10 min incubation in buffer pH 6.2 and 7.8, respectively, compared to buffer pH 7.2. (B) Linear increase of FDG uptake in T47D breast cancer cells after 10 min incubation in buffers with graded pH increments. Bars are mean ± SD of uptake (n = 3) relative to cells in pH 7.2 (A) or 7.4 (B). *, p <0.05; **, p <0.01; †, p <0.005; ‡, p <0.001; n.s., not significant.</p
Time course of changes in cellular pH following alkaline and acidic exposure.
<p>(A) Close linear relationship between fluorescent emission ratios from BCECF-AM by excitation at 485 and 430 nm and the pH of standards applied to cell interior. (B) Linear relationship between applied buffer pH and intracellular pH of live T47D cells after 30 min. (C), Time course of extracellular pH (open circles) and intracellular pH (closed circles) over 3 h following incubation in pH 7.8 (left) or 6.2 (right). Data points are mean ± SD of triplicate samples.</p
Effect of pH on HK association with mitochondria.
<p>(A) Immunoblots (top) and quantified band intensities (bottom) of free HK-1 and HK-2 protein dissociated from isolated mitochondria into the supernatant following exposure to pH 6.2 or 7.8. (B) Immunoblots and quantified band intensities of HK-1 and HK-2 protein remaining in the mitochondrial pellets following pH exposure as above. Band intensities of HK from the mitochondrial pellet were normalized by COX4 bands. In supernatants, negative COX-4 bands confirmed the absence of mitochondria in the samples. Bars are mean ± SD of band intensities (n = 2) relative to that at pH 6.2 (A) or pH 7.8 (B).</p
Subcellular localization of hexokinase (HK) by acidification and alkalization.
<p>(A,B) Immunoblots of HK-1 and HK-2 protein (top) and quantified band intensities (bottom) in mitochondrial (A) and cytosolic fractions (B) of T47D cells following 50 min incubation in pH 7.8 or 6.2. HK band intensities of mitochondrial and cytosolic fractions were normalized for loading using COX4 and β-actin bands, respectively. Bars are mean ± SD of band intensities (n = 3) relative to cells in pH 6.2.</p
Effects of pH on co-localization of HK-1 and HK-2 with VDAC.
<p>(A,B) Confocal microscopy images of T47D cells double stained for VDAC and HK-1 (A) or VDAC and HK-2 (B) following incubation at pH 6.2 or 7.8.</p
Effect of pH on HK-VDAC binding.
<p>(A, B) Immunoblots (top) and quantified band intensities (bottom) of recombinant HK-1 (A) and HK-2 (B) co-immunoprecipitated with purified VDAC protein. Recombinant HK and purified VDAC protein were pre-incubated in pH 6.2 or 7.8 for 50 min. Bars are mean ± SD (n = 3) of band intensities normalized by VDAC bands relative to that at pH 6.2.</p
Effects of HK-1, HK-2 and VDAC1 siRNA on pH-modulated glucose uptake.
<p>Relative FDG uptake of T47D cells after transfection with specific siRNA against HK-1, HK-2, or VDAC1. Bars are mean ± SD of six samples from two separate experiments. †, p <0.0005; ‡, p <0.0001, compared to cells transfected with respective siRNA and incubated in pH 6.2.</p
Facile Method To Radiolabel Glycol Chitosan Nanoparticles with <sup>64</sup>Cu via Copper-Free Click Chemistry for MicroPET Imaging
An efficient and straightforward
method for radiolabeling nanoparticles
is urgently needed to understand the <i>in vivo</i> biodistribution
of nanoparticles. Herein, we investigated a facile and highly efficient
strategy to prepare radiolabeled glycol chitosan nanoparticles with <sup>64</sup>Cu via a strain-promoted azide–alkyne cycloaddition
strategy, which is often referred to as click chemistry. First, the
azide (N<sub>3</sub>) group, which allows for the preparation of radiolabeled
nanoparticles by copper-free click chemistry, was incorporated to
glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne
derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTA) chelator, was synthesized for preparing the preradiolabeled
alkyne complex with <sup>64</sup>Cu radionuclide. Following incubation
with the <sup>64</sup>Cu-radiolabeled DBCO complex (DBCO-PEG<sub>4</sub>-Lys-DOTA-<sup>64</sup>Cu with high specific activity, 18.5 GBq/μmol),
the azide-functionalized CNPs were radiolabeled successfully with <sup>64</sup>Cu, with a high radiolabeling efficiency and a high radiolabeling
yield (>98%). Importantly, the radiolabeling of CNPs by copper-free
click chemistry was accomplished within 30 min, with great efficiency
in aqueous conditions. In addition, we found that the <sup>64</sup>Cu-radiolabeled CNPs (<sup>64</sup>Cu-CNPs) did not show any significant
effect on the physicochemical properties, such as size, zeta potential,
or spherical morphology. After <sup>64</sup>Cu-CNPs were intravenously
administered to tumor-bearing mice, the real-time, <i>in vivo</i> biodistribution and tumor-targeting ability of <sup>64</sup>Cu-CNPs
were quantitatively evaluated by microPET images of tumor-bearing
mice. These results demonstrate the benefit of copper-free click chemistry
as a facile, preradiolabeling approach to conveniently radiolabel
nanoparticles for evaluating the real-time <i>in vivo</i> biodistribution of nanoparticles