We synthesized macromolecular ligands for CdSe-ZnS core-shell quantum dots incorporating multiple thiol groups, poly(ethylene glycol) chains, and either carboxylic acids or primary amines along a common poly(methacrylate) backbone. The thiol groups encourage the adsorption of these macromolecular constructs on the ZnS shell of the nanoparticles, and the poly(ethylene glycol) chains impose hydrophilic character on the resulting assemblies. Indeed, the coated quantum dots are readily soluble in water and are stable under these conditions for months over a broad pH range (4.0-12.0) and even in the presence of large salt concentrations. In addition, these nanoparticles have relatively small hydrodynamic diameters (17-30 nm) and good quantum yields (0.3-0.4). Furthermore, the pendant carboxylic acids or primary amines of the macromolecular ligands can be exploited to modify the quantum dots after the adsorption of the polymers on their surface. For example, boron dipyrromethene dyes can be connected to the hydrophilic quantum dots on the basis of amide bond formation to encourage the transfer of energy from the luminescent CdSe core to the organic dyes. Our hydrophilic nanoparticles can also cross the membrane of Chinese hamster ovarian cells and accumulate in the cytosol with limited nuclear localization. Moreover, the internalized quantum dots are not cytotoxic and have essentially no influence on cell viability. Thus, our strategy for the preparation of biocompatible quantum dots can evolve into the development of valuable luminescent probes with nanoscaled dimensions and optimal photophysical properties for a diversity of biomedical applications.

We designed four polymeric ligands for semiconductor quantum dots and synthesized these macromolecular constructs in four steps, starting from commercial precursors. These ligands have a poly(methacrylate) backbone with pendant thiol groups and poly(ethylene glycol) chains. The thiol groups anchor these ligands on the surface of preformed CdSe-ZnS core-shell quantum dots, and the poly(ethylene glycol) chains impose hydrophilic character on the resulting assemblies. Indeed, three of the four sets of quantum dots are soluble in aqueous environments and are stable under these conditions for days over a wide pH range (5.0-9.0). Furthermore, the polymeric coatings wrapped around the inorganic nanoparticles preserve the photophysical properties of the CdSe core and ensure relatively compact dimensions. Specifically, the luminescence quantum yield is ca. 0.4 and the hydrodynamic diameter ranges from 15 to 29 nm with the nature of the polymeric ligand. Model studies with human umbilical vein endothelial cells demonstrated that these hydrophilic quantum dots cross the cell membrane and localize either in the cytosol or in the nucleus. The length of the poly(ethylene glycol) chains appears to guide the intracellular localization of these luminescent probes. In addition, these studies indicated that these particular nanoparticles are not cytotoxic. In fact, their cellular internalization has essentially no influence on cell growth. In summary, we developed novel polymeric ligands able to impose hydrophilic character and biocompatibility on CdSe-ZnS core-shell nanoparticles. Thus, our results can lead to a new family of valuable luminescent probes for cellular imaging, based on the unique photophysical properties of semiconductor quantum dots.

1 The effect of the Cl À channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4 0 -diisothiocyanatostilbene-2,2 0 -disulfonic acid (DIDS), and anthracene-9-carboxylic acid (A-9-C), on Ca 2 þ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca 2 þ concentration ([Ca 2 þ ] i ) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca 2 þ from the sarcoplasmic reticulum (SR). 2 NFA and NPPB significantly increased basal [Ca 2 þ ] i and attenuated the caffeine-induced increase in [Ca 2 þ ] i . These Cl À channel blockers also increased the half-time (t 1/2 ) to peak for the caffeineinduced [Ca 2 þ ] i transient, and slowed the removal of Ca 2 þ from the cytosol following application of caffeine. Since DIDS and A-9-C were found to adversely affect fura-2 fluorescence, fluo-4 was used to monitor intracellular Ca 2 þ in studies involving these Cl À channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca 2 þ , and while DIDS had no significant effect on the t 1/2 to peak for the caffeine-induced Ca 2 þ transient, it was significantly increased by A-9-C. 3 In the absence of extracellular Ca 2 þ , NFA significantly increased basal [Ca 2 þ ] i , suggesting that the release of Ca 2 þ from an intracellular store was responsible for the observed effect. 4 Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca 2 þ ] i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca 2 þ ] i induced by NFA. 5 These data show that Cl À channel blockers have marked effects on Ca 2 þ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca 2 þ ] i indicates that it is likely due to Ca 2 þ release from an intracellular store, most probably the SR.

No abstract

Hypoxia contracts the pulmonary vein, but the underlying cellular effectors remain unclear. Utilizing contractile studies and whole cell patch-clamp electrophysiology, we report for the first time a hypoxia-sensitive K(+) current in porcine pulmonary vein smooth muscle cells (PVSMC). Hypoxia induced a transient contractile response that was 56 ± 7% of the control response (80 mM KCl). This contraction required extracellular Ca(2+) and was sensitive to Ca(2+) channel blockade. Blockade of K(+) channels by tetraethylammonium chloride (TEA) or 4-aminopyridine (4-AP) reversibly inhibited the hypoxia-mediated contraction. Single-isolated PVSMC (typically 159.1 ± 2.3 μm long) had mean resting membrane potentials (RMP) of -36 ± 4 mV with a mean membrane capacitance of 108 ± 3.5 pF. Whole cell patch-clamp recordings identified a rapidly activating, partially inactivating K(+) current (I(KH)) that was hypoxia, TEA, and 4-AP sensitive. I(KH) was insensitive to Penitrem A or glyburide in PVSMC and had a time to peak of 14.4 ± 3.3 ms and recovered in 67 ms following inactivation at +80 mV. Peak window current was -32 mV, suggesting that I(KH) may contribute to PVSMC RMP. The molecular identity of the potassium channel is not clear. However, RT-PCR, using porcine pulmonary artery and vein samples, identified Kv(1.5), Kv(2.1), and BK, with all three being more abundant in the PV. Both artery and vein expressed STREX, a highly conserved and hypoxia-sensitive BK channel variant. Taken together, our data support the hypothesis that hypoxic inhibition of I(KH) would contribute to hypoxic-induced contraction in PVSMC.