Hypoxia-induced resistance to cisplatin-mediated apoptosis in osteosarcoma cells is reversed by gambogic acid independently of HIF-1α.

In vitro evidence of hypoxia-induced resistance to cisplatin (CDDP)-mediated apoptosis exists in human osteosarcoma (OS). Gambogic acid (GA) is a promising chemotherapeutic compound that could increase the chemotherapeutic effectiveness of CDDP in human OS cells by inducing cell cycle arrest and promoting apoptosis. This study examined whether GA could overcome OS cell resistance to CDDP. Hypoxia significantly reduced levels of CDDP-induced apoptosis in the OS cell lines MG63 and HOS. However, combined treatment with GA and CDDP revealed a strong synergistic action between these drugs, and higher protein levels of the apoptosis-related factor Fas, cleaved caspase-8 and cleaved caspase-3 and lower expression of hypoxia-inducible factor (HIF)-1α are detected in both cell lines. Meanwhile, drug resistance was not reversed by exposure to the HIF-1α inhibitor 2-methoxyestradiol. These findings strongly suggest that hypoxia-induced resistance to CDDP is reversed by GA in OS cells independently of HIF-1α. Furthermore, in vivo studies using xenograft mouse models revealed that combination therapy with CDDP and GA exerted increased antitumor effects by inducing apoptosis. Taken together, our results demonstrate that GA may be a new potent therapeutic agent useful for targeting human OS cells.

Viability inhibition effect of gambogic acid combined with cisplatin on osteosarcoma cells via mitochondria-independent apoptotic pathway.

We previously demonstrated that gambogic acid (GA) is a promising chemotherapeutic compound for human osteosarcoma treatment. The aim of this study was to detect whether the combination of lower-dose GA (0.3 mg/L) and cisplatin (CDDP) (1 mg/L) could perform a synergistic effect on inhibiting tumor in four osteosarcoma cell lines. Our results showed that the combination between GA at lower dose and CDDP significantly exerts a synergistic effect on inhibiting the cellular viability in MG63, HOS, and U2OS cells. In contrast, an antagonistic character was detected in SAOS2 cells exposed to the combined use of lower-dose GA (0.3 mg/L) and CDDP (1 mg/L). Then, analysis of cell cycle showed the combination of both drugs significantly induced the G2/M phase arrest, without any difference relative to GA treatment alone, in MG63 cells. Flow-cytometric analysis of cell apoptosis displayed that the apoptotic rate in the combination group is higher than that in GA treatment alone in MG63, HOS, and U2OS cells. The combined use of both drugs had no effect on mitochondrial membrane potential, but promoted the apoptosis-inducing function through triggering of CDDP in the three cell lines. By measurement of mitochondrial membrane potential, the activity of caspase-3 and the expressions of caspase-8 and caspase-9, it was showed that the apoptosis-promoting effect of the combined use of both drugs could be dependent on the death receptor apoptosis pathway, not dependent on the mitochondria apoptosis mechanism. This research, for the first time, demonstrates that GA could increase the chemotherapeutic effect of CDDP in human osteosarcoma treatment through inducing the cell cycle arrest and promoting cell apoptosis.

Enzyme-amplified array sensing of proteins in solution and in biofluids.

We have developed an enzyme-nanoparticle sensor array where the sensitivity is amplified through enzymatic catalysis. In this approach cationic gold nanoparticles are electrostatically bound to an enzyme (beta-galactosidase, beta-Gal), inhibiting enzyme activity. Analyte proteins release the beta-Gal, restoring activity and providing an amplified readout of the binding event. Using this strategy we have been able to identify proteins in buffer at a concentration of 1 nM, substantially lower than current strategies for array-based protein sensing. Moreover, we have obtained identical sensitivity in studies where the proteins are spiked into the complex protein matrix provided by desalted human urine ( approximately 1.5 muM total protein; spiked protein concentrations were 0.067% of the overall protein concentration), demonstrating the potential of the method for diagnostic applications.

Nanoparticles for detection and diagnosis.

Nanoparticle based platforms for identification of chemical and biological agents offer substantial benefits to biomedical and environmental science. These platforms benefit from the availability of a wide variety of core materials as well as the unique physical and chemical properties of these nanoscale materials. This review surveys some of the emerging approaches in the field of nanoparticle based detection systems, highlighting the nanoparticle based screening methods for metal ions, proteins, nucleic acids, and biologically relevant small molecules.

Photoregulated release of caged anticancer drugs from gold nanoparticles.

An anticancer drug (5-fluorouracil) was conjugated to the surface of gold nanoparticles through a photocleavable o-nitrobenzyl linkage. In this system, the particle serves as both cage and carrier for the therapeutic, providing a nontoxic conjugate that effectively releases the payload upon long wavelength UV irradiation.

Photoinduced energy and electron-transfer processes in porphyrin-perylene bisimide symmetric triads.

The photophysics of two symmetric triads, (ZnP)2PBI and (H2P)2PBI, made of two zinc or free-base porphyrins covalently attached to a central perylene bisimide unit has been investigated in dichloromethane and in toluene. The solvent has been shown to affect not only quantitatively but also qualitatively the photophysical behavior. A variety of intercomponent processes (singlet energy transfer, triplet energy transfer, photoinduced charge separation, and recombination) have been time-resolved using a combination of emission spectroscopy and femtosecond and nanosecond time-resolved absorption techniques yielding a very detailed picture of the photophysics of these systems. The singlet excited state of the lowest energy chromophore (perylene bisimide in the case of (ZnP)2PBI, porphyrin in the case of (H2P)2PBI) is always quantitatively populated, besides by direct light absorption, by ultrafast singlet energy transfer (few picosecond time constant) from the higher energy chromophore. In dichloromethane, the lowest excited singlet state is efficiently quenched by electron transfer leading to a charge-separated state where the porphyrin is oxidized and the perylene bisimide is reduced. The systems then go back to the ground state by charge recombination. The four charge separation and recombination processes observed for (ZnP)2PBI and (H2P)2PBI in dichloromethane take place in the sub-nanosecond time scale. They obey standard free-energy correlations with charge separation lying in the normal regime and charge recombination in the Marcus inverted region. In less polar solvents, such as toluene, the energy of the charge-separated states is substantially lifted leading to sharp changes in photophysical mechanism. With (ZnP)2PBI, the electron-transfer quenching is still fast, but charge recombination takes place now in the nanosecond time scale and to triplet state products rather than to the ground state. Triplet-triplet energy transfer from the porphyrin to the perylene bisimide is also involved in the subsequent deactivation of the triplet manifold to the ground state. With (H2P)2PBI, on the other hand, the driving force for charge separation is too small for electron-transfer quenching, and the deactivation of the porphyrin excited singlet takes place via intersystem crossing to the triplet followed by triplet energy transfer to the perylene bisimide and final decay to the ground state.

Controlled nanoparticleassembly through protein conformational changes.

Selective surface recognition by proteins provides programmed bottom-up assembly of synthetic nanomaterials. We have investigated the controlled self-assembly of functionalized gold nanoparticles (Au-TAsp) with cytochrome c (Cyt c) and apoCyt c through complementary electrostatic interactions. Au-TAsp formed discrete, water-soluble adducts with native Cyt c, whereas unfolded apoCyt c induced nanocomposite formation at high Cyt c : Au-TAsp ratios. The binding of random-coil apoCyt c to Au-TAsp at low ratios induced α-helix formation in soluble nanocomposites, but at elevated ratios insoluble micron-scale aggregates were formed. The local structure of the assemblies was critically dependent on the Cyt c : Au-TAsp ratio. The dispersibility of apoCyt c-Au-TAsp was pH dependent, providing rapid and reversible control over nanocomposite assembly. The apoCyt c-Au-TAsp aggregates could likewise be disassembled through proteolytic cleavage of apoCyt c, demonstrating the ability to selectively remodel these hybrid materials.

Isomeric control of protein recognition with amino acid- and dipeptide-functionalized gold nanoparticles.

Amino acid and dipeptide-functionalized gold nanoparticles (NPs) possessing L/D-leucine and/or L/D-phenylalanine residues have been constructed in order to target the surfaces of alpha-chymotrypsin (ChT) and cytochrome c (CytC). Isothermal titration calorimetry (ITC) was conducted to evaluate the binding thermodynamics and selectivity of these NP-protein interactions. The chirality of the NP end-groups substantially affects the resultant complex stability, with up to 20-fold differences seen between particles of identical hydrophobicity, demonstrating that structural information from the ligands can be used to control protein recognition.

Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study.

Gold nanoparticles (NPs) functionalized with L-amino acid-terminated monolayers provide an effective platform for the recognition of protein surfaces. Isothermal titration calorimetry (ITC) was used to quantify the binding thermodynamics of these functional NPs with alpha-chymotrypsin (ChT), histone, and cytochrome c (CytC). The enthalpy and entropy changes for the complex formation depend upon the nanoparticle structure and the surface characteristics of the proteins, e.g., distributions of charged and hydrophobic residues on the surface. Enthalpy-entropy compensation studies on these NP-protein systems indicate an excellent linear correlation between DeltaH and TDeltaS with a slope (alpha) of 1.07 and an intercept (TDeltaS0) of 35.2 kJ mol(-1). This behavior is closer to those of native protein-protein systems (alpha = 0.92 and TDeltaS0 = 41.1 kJ mol(-1)) than other protein-ligand and synthetic host-guest systems.

Detection and identification of proteins using nanoparticle-fluorescent polymer 'chemical nose' sensors.

A sensor array containing six non-covalent gold nanoparticle-fluorescent polymer conjugates has been created to detect, identify and quantify protein targets. The polymer fluorescence is quenched by gold nanoparticles; the presence of proteins disrupts the nanoparticle-polymer interaction, producing distinct fluorescence response patterns. These patterns are highly repeatable and are characteristic for individual proteins at nanomolar concentrations, and can be quantitatively differentiated by linear discriminant analysis (LDA). Based on a training matrix generated at protein concentrations of an identical ultraviolet absorbance at 280 nm (A280 = 0.005), LDA, combined with ultraviolet measurements, has been successfully used to identify 52 unknown protein samples (seven different proteins) with an accuracy of 94.2%. This work demonstrates the construction of novel nanomaterial-based protein detector arrays with potential applications in medical diagnostics.

Modulation of the catalytic behavior of alpha-chymotrypsin at monolayer-protected nanoparticle surfaces.

Amino-acid-functionalized gold clusters modulate the catalytic behavior of alpha-chymotrypsin (ChT) toward cationic, neutral, and anionic substrates. Kinetic studies reveal that the substrate specificity (k(cat)/K(M)) of ChT-nanoparticle complexes increases by approximately 3-fold for the cationic substrate but decreases by 95% for the anionic substrate as compared with that of free ChT, providing enhanced substrate selectivity. Concurrently, the catalytic constants (k(cat)) of ChT show slight augmentation for the cationic substrate and significant attenuation for the anionic substrate in the presence of amino-acid-functionalized nanoparticles. The amino acid monolayer on the nanoparticle is proposed to control both the capture of substrate by the active site and release of product through electrostatic interactions, leading to the observed substrate specificities and catalytic constants.

Regulation of alpha-chymotrypsin activity on the surface of substrate-functionalized gold nanoparticles.

A gold nanoparticle functionalized with substrates for alpha-chymotrypsin was fabricated to afford an enzyme modulator that exhibited enzyme-specific activation coupled with general inhibition of other proteases.

Light-harvesting metallosupramolecular squares composed of perylene bisimide walls and fluorescent antenna dyes.

The fluorescent dye 4-dimethylamino-1,8-naphthalimide was incorporated at the bay area of N,N'-bispyridyl perylene bisimide to afford a fourfold-functionalized perylene bisimide ligand. Through self-assembly directed by metal-ion coordination, a multichromophore supramolecular entity composed of sixteen dimethylaminonaphthalimide antennas and a perylene bisimide-walled square core was subsequently constructed from this linear ditopic ligand and 90 degrees metal corner [Pd(dppp)](OTf)2 (dppp=1,3-bis(diphenylphosphino)propane; OTf=trifluoromethanesulfonate) in good yield. The isolated metallosupramolecular square was characterized by elemental analysis and 1H, 13C, and 31P{1H} NMR and UV/Vis spectroscopy. Furthermore, by means of 1H NMR diffusion-ordered spectroscopy (DOSY) the dimension of this assembly was evaluated by employing a previously reported perylene bisimide ligand and its square assembly as references. The results obtained confirm the square framework of the current assembly. The optical properties of this multichromophore dye assembly were investigated by UV/Vis and steady-state and time-resolved fluorescence spectroscopy. It was revealed that light captured by dimethylaminonaphthalimide antennas could be efficiently transported to the perylene bisimide core by a fluorescence resonance mechanism (energy-transfer efficiency E=95%), and this resulted in almost exclusive detection of intense perylene bisimide emission, irrespective of the excitation wavelength applied. The present square scaffold containing aminonaphthalimide antenna dyes exhibits more than seven times higher fluorescence quantum yield (Phifl=0.37) than a previously reported pyrene-bearing perylene bisimide-walled square (Phifl=0.05). Thus, this multichromophore square assembly with aminonaphthalimide antenna dyes is an artificial model for the cyclic light-harvesting complexes in purple bacteria.

Engineering the nanoparticle-biomacromolecule interface.

Monolayer-protected nanoparticles feature tunable size, surface functionality and core material, providing scaffolds for targeting biomacromolecules. This review highlights recent advances in nanoparticle-biomacromolecule interactions, focusing on two key areas: (1) The modulation of structure and function of biomacromolecules through engineered interactions with nanoparticle surfaces; (2) The use of biomacromolecules as building blocks for nanostructured materials.

Contrasting effects of exterior and interior hydrophobic moieties in the complexation of amino acid functionalized gold clusters with alpha-chymotrypsin.

[chemical structure: see text]. A series of L-amino acid functionalized gold nanoparticles with oligo(ethylene glycol) (OEG) tethers of varying length are prepared. These studies show that the hydrophobic side chains of amino acids facilitate the structural retention of alpha-chymotrypsin (ChT) but the interior alkyl chains promote its denaturation. An 80-fold range of denaturation rate constants were obtained for ChT in the presence of various nanoparticles. Thus, the tunable denaturation of protein could be achieved by rational combination of amino acid side chains and OEG tethers.