Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma.

The telomerase-specific oncolytic adenovirus Telomelysin and the histone deacetylase inhibitor AR42 have demonstrated anticancer effects in preclinical models of human hepatocellular carcinoma (HCC). However, the clinical development of Telomelysin may be hindered by human antiviral immunity and tumor resistance. Combining oncolytic and epigenetic therapies is a viable approach for treating various cancers. This study investigated the potential synergism of Telomelysin and AR42 and the relevant underlying mechanisms. Telomelysin and AR42 exhibited synergistic antiproliferative effects in human HCC models in vitro and in vivo. Apoptosis induced by Telomelysin was significantly enhanced by AR42 in both PLC5 and Hep3B HCC cells. AR42 treatment unexpectedly attenuated the expression of the coxsackievirus and adenovirus receptor and the mRNA levels of human telomerase reverse transcriptase, which may be positively associated with the cytotoxicity of Telomelysin. Meanwhile, the cellular antiviral interferon response was not altered by AR42 treatment. Further, we found that Telomelysin enhanced Akt phosphorylation in HCC cells. AR42 reduced Telomelysin-induced phospho-Akt activation and enhanced Telomelysin-induced apoptosis. The correlation of Akt phosphorylation with drug-induced apoptosis was validated in HCC cells with upregulated or downregulated Akt signaling. Combination therapy with Telomelysin and AR42 demonstrated synergistic anti-HCC efficacy. Clinical trials investigating this new combination regimen are warranted.

Thiolated Mesoporous Silica Nanoparticles as an Immunoadjuvant to Enhance Efficacy of Intravesical Chemotherapy for Bladder Cancer.

The characteristics of global prevalence and high recurrence of bladder cancer has led numerous efforts to develop new treatments. The spontaneous voiding and degradation of the chemodrug hamper the efficacy and effectiveness of intravesical chemotherapy following tumor resection. Herein, the externally thiolated hollow mesoporous silica nanoparticles (MSN-SH(E)) is fabricated to serve as a platform for improved bladder intravesical therapy. Enhanced mucoadhesive effect of the thiolated nanovector is confirmed with porcine bladder. The permeation-enhancing effect is also verified, and a fragmented distribution pattern of a tight junction protein, claudin-4, indicates the opening of tight junction. Moreover, MSN-SH(E)-associated reprogramming of M2 macrophages to M1-like phenotype is observed in vitro. The antitumor activity of the mitomycin C (MMC)-loaded nanovector (MMC@MSN-SH(E)) is more effective than that of MMC alone in both in vitro and in vivo. In addition, IHC staining is used to analyze IFN-γ, TGF-ß1, and TNF-α. These observations substantiated the significance of MMC@MSN-SH(E) in promoting anticancer activity, holding the great potential for being used in intravesical therapy for non-muscle invasive bladder cancer (NMIBC) due to its mucoadhesivity, enhanced permeation, immunomodulation, and prolonged and very efficient drug exposure.

Comprehensive Thione-Derived Perylene Diimides and Their Bio-Conjugation for Simultaneous Imaging, Tracking, and Targeted Photodynamic Therapy.

In this study, the chromophore 3,4,9,10-perylenetetracarboxylic diimide (PDI) is anchored with phenyl substituents at the imide N site, followed by thionation, yielding a series of thione products 1S-PDI-D, 2S-cis-PDI-D, 2S-trans-PDI-D, 3S-PDI-D, and 4S-PDI-D, respectively, with n = 1, 2, 3, and 4 thione. The photophysical properties are dependent on the number of anchored thiones, where the observed prominent lower-lying absorption is assigned to the S0 → S2(ππ*) transition and is red-shifted upon increasing the number of thiones; the lowest-lying excited state is ascribed to a transition-forbidden S1(nπ*) configuration. All nS-PDIs are non-emissive in solution but reveal an excellent two-photon absorption cross-section of >800 GM. Supported by the femtosecond transient absorption study, the S1(nπ*) → T1(ππ*) intersystem crossing (ISC) rate is > 1012 s-1, resulting in ∼100% triplet population. The lowest-lying T1(ππ*) energy is calculated to be in the order of 1S-PDI-D > 2S-cis-PDI-D ∼ 2S-trans-PDI-D > 3S-PDI-D > 4S-PDI-D, where the T1 energy of 1S-PDI-D (1.10 eV) is higher than that (0.97 eV) of the 1O2 1Δg state. 1S-PDI-D is further modified by either conjugation with peptide FC131 on the two terminal sides, forming 1S-FC131, or linkage with peptide FC131 and cyanine5 dye on each terminal, yielding Cy5-1S-FC131. In vitro experiments show power of 1S-FC131 and Cy5-1S-FC131 in recognizing A549 cells out of other three lung normal cells and effective photodynamic therapy. In vivo, both molecular composites demonstrate outstanding antitumor ability in A549 xenografted tumor mice, where Cy5-1S-FC131 shows superiority of simultaneous fluorescence tracking and targeted photodynamic therapy.

Noninvasive assessment of liver function reserve with fluorescent dosimetry of indocyanine green.

Using in vivo multiphoton fluorescent dosimetry, we demonstrate that the clearance dynamics of Indocyanine Green (ICG) in the blood can quickly reveal liver function reserve. In normal rats, the ICG retention rate was below 10% at the 15-minute post-administration; While in the rat with severe hepatocellular carcinoma (HCC), the 15-minute retention rate is over 40% due to poor liver metabolism. With a 785 nm CW laser, the fluorescence dosimeter can evaluate the liver function reserve at a 1/10 clinical dosage of ICG without any blood sampling. In the future, this low-dosage ICG 15-minute retention dosimetry can be applied for the preoperative assessment of hepatectomy or timely perioperative examination.

Imaging of Cancer Cells and Dictated Cytotoxicity Using Aptamer-Guided Hybridization Chain Reaction (HCR)-Generated G-Quadruplex Chains.

DNA nanotechnology provides powerful tools for developing cancer theranostics. Here we introduce the autonomous surface-nucleolin-guided HCR that leads to the polymerization of G-quadruplex polymer chains, in which the ZnII -protoporphyrin IX is intercalated. We demonstrate that MDA-MB-231 (Triple Negative Breast Cancer cells, TNBC) with overexpressed surface nucleolin were able to induce HCR leading to the formation of the ZnII PPIX-loaded G-quadruplex polymer chains, while the M10 epithelial breast cells served as control. The ZnII PPIX-loaded nanowires allow the selective imaging of TNBC, and their permeation into the TNBC leads to selective cytotoxicity and guided photodynamic therapy toward the cancer cells due to structural perturbation of the membranes. The aptamer-guided HCR-generated G-quadruplex polymer chains may serve as a versatile tool to target TNBC featuring poor prognosis and high pathological risk of recurrence, thus offering a promising theranostic platform.

Targeting triple-negative breast cancer with an aptamer-functionalized nanoformulation: a synergistic treatment that combines photodynamic and bioreductive therapies.

BACKGROUND: Areas of hypoxia are often found in triple-negative breast cancer (TNBC), it is thus more difficult to treat than other types of breast cancer, and may require combination therapies. A new strategy that combined bioreductive therapy with photodynamic therapy (PDT) was developed herein to improve the efficacy of cancer treatment. Our design utilized the characteristics of protoporphyrin IX (PpIX) molecules that reacted and consumed O2 at the tumor site, which led to the production of cytotoxic reactive oxygen species (ROS). The low microenvironmental oxygen levels enabled activation of a bioreductive prodrug, tirapazamine (TPZ), to become a toxic radical. The TPZ radical not only eradicated hypoxic tumor cells, but it also promoted therapeutic efficacy of PDT. RESULTS: To achieve the co-delivery of PpIX and TPZ for advanced breast cancer therapy, thin-shell hollow mesoporous Ia3d silica nanoparticles, designated as MMT-2, was employed herein. This nanocarrier designed to target the human breast cancer cell MDA-MB-231 was functionalized with PpIX and DNA aptamer (LXL-1), and loaded with TPZ, resulting in the formation of TPZ@LXL-1-PpIX-MMT-2 nanoVector. A series of studies confirmed that our nanoVectors (TPZ@LXL-1-PpIX-MMT-2) facilitated in vitro and in vivo targeting, and significantly reduced tumor volume in a xenograft mouse model. Histological analysis also revealed that this nanoVector killed tumor cells in hypoxic regions efficiently. CONCLUSIONS: Taken together, the synergism and efficacy of this new therapeutic design was confirmed. Therefore, we concluded that this new therapeutic strategy, which exploited a complementary combination of PpIX and TPZ, functioned well in both normoxia and hypoxia, and is a promising medical procedure for effective treatment of TNBC.

Simple aminophenol-based electrochemical probes for non-enzymatic, dual amperometric detection of NADH and hydrogen peroxide.

Non enzymatic detection of NADH and H2O2 is of practical significance for both environmental and biological prospective. However, there is no simple, straight forward electrochemical sensor available for sensing of them in real samples. Addressing this challenge, we report a simple stimuli responsive aminophenol, pre-anodized screen printed carbon electrode (SPCE*/AP) based electrochemical probes for dual detection of NADH and H2O2. Aminophenol prepared and adsorbed on the electrode from aminophenylboronic acid via boronic acid deprotection with H2O2. The SPCE*/AP fabricated with this process was characterized by cyclic voltammetry (CV), scanning electron microscope (SEM), Raman spectroscopy, UV-visible spectroscopy, and X-ray photoelectron spectroscopy (XPS). Amperometric detection results showed that SPCE*/AP electrodes exhibited linearity from 50 µM to 500 µM and from 200 µM to 2 mM with a detection limit (S/N = 3) of 4.2 µM and 28.9 µM for NADH and H2O2, respectively. Excellent reproducibility and selectivity for NADH and H2O2 were observed for this electrochemical platform. In addition, the matrix effect was investigated further using the same technique to analyze NADH and H2O2 in human urine samples, human serum samples, cell culture medium (containing 10% fetal bovine serum, FBS), and environmental water samples (tap water and rain water). Also, the present sensor demonstrated promising outcomes with living cells (normal cells and cancer cells).

Diagnosing the RGS11 Lung Cancer Biomarker: The Integration of Competitive Immunoassay and Isothermal Nucleic Acid Exponential Amplification Reaction.

Lung cancer is the primary cause of cancer-associated mortality worldwide, which makes the identification of reliable lung cancer biomarkers a pressing need for early diagnosis and prognosis. RGS11, which is a regulator of G-protein signaling and also a lung cancer biomarker, plays an important role in cancer-related metastasis. However, trace levels of RGS11 (in the range of pg/mL) in serum samples make it difficult to quantify using currently available enzyme-linked immunosorbent assay (ELISA) kits and, therefore, this hinders progress in the discovery of new approaches for treating lung cancer. The aim of this study is to develop a rapid, sensitive, and reliable platform for the detection of RGS11 lung cancer biomarker based on a suspension immunoassay coupled with an isothermal exponential amplification strategy. Our study was initiated by the functionalization of magnetic beads with anti-RGS11 antibodies (Ab-MB) by EDC (1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide)/NHS ( N-hydroxysulfosuccinimide) activation. Ab-MB served as a sensing probe for the competitive immunorecognitions between known concentrations of His-tag RGS11 and unknown concentrations of target RGS11 in serum. The reporter anti-His antibodies, which were modified with primers that induced an isothermal exponential amplification reaction, were subsequently introduced to the reaction mixture that resulted in the formation of immunosandwich complexes. The exponentially amplified DNA duplex that was intercalated with SYBR Green was designated as a signal reporter for the assessment of RGS11 in an inversely proportional relationship. The sensing platform was excellent for the determination of RGS11 with an exceptional detection limit of 148 fg/mL and a linear dynamic range of 0.1-10 pg/mL using a minimal sample volume (20 µL) and with a reaction time of 1.5 h. In addition, we challenged the sensing platform with RGS11-spiked samples (in 2× diluted serum), and an acceptable recovery rate (>90%) was observed. Finally, 24 clinical samples acquired from patients with advanced lung cancer (C), inflammation (I), and heart failure (H) were analyzed by this newly developed sensing platform and a commercial ELISA kit for validation. This sensing platform has potential in biomedical applications for clinically diagnosing liquid biopsy samples for patients with lung cancer. Moreover, the universal design of our proposed system is easily adapted to detect any other protein if a His-tag recombinant protein is available.

Chemopreventive Potential of Ethanolic Extracts of Luobuma Leaves (Apocynum venetum L.) in Androgen Insensitive Prostate Cancer.

Luobuma (Apocynum venetum L. (AVL)) is a popular beverage in Asia and has been reportedly to be associated with the bioactivities such as cardiotonic, diuretic, antioxidative, and antihypertensive. However, its biofunction as chemoprevention activity is seldom addressed. Herein, we aimed to characterize the anti-androgen-insensitive-prostate-cancer (anti-AIPC) bioactive compounds of Luobuma, and to investigate the associated molecular mechanisms. Activity-guided-fractionation (antioxidative activity and cell survivability) of Luobuma ethanolic extracts was performed to isolate and characterize the major bioactive compounds using Ultra Performance Liquid Chromatography (UPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and Nuclear Magnetic Resonance (NMR). Plant sterols (lupeol, stigamasterol and ß-sitosterol) and polyphenolics (isorhamnetin, kaempferol, and quercetin) were identified. Lupeol, a triterpene found in the fraction (F8) eluted by 10% ethyl acetate/90% hexane and accounted for 19.3% (w/w) of F8, inhibited the proliferation of PC3 cells. Both lupeol and F8 induced G2/M arrest, inhibition of ß-catenin signaling, regulation of apoptotic signal molecules (cytochrome c, Bcl-2, P53, and caspase 3 and 8), and suppression DNA repair enzyme expression (Uracil-DNA glycosylase (UNG)). To our knowledge, our study is the first report that lupeol inhibited the expression of UNG to elicit the cytotoxicity against androgen-insensitive-prostate-cancer cells. Collectively, Luobuma, which contains several antitumor bioactive compounds, holds the potential to be a dietary chemopreventive agent for prostate cancer.

Resuscitation Using Liposomal Vasopressin in an Animal Model of Uncontrolled Hemorrhagic Shock.

BACKGROUND: Current research suggests that administration of vasopressin to patients with uncontrolled hemorrhagic shock (UHS) can avoid the detrimental effects associated with aggressive fluid resuscitation. However, vasopressin has a short half-life of 10~35 minutes in in vivo use and precludes its use in the pre-hospital setting. To increase the half-life of vasopressin, we proposed to synthesize liposome-encapsulated vasopressin and test it in a rat model of UHS. METHODS: The film hydration method was used to prepare liposomal vasopressin consisting of: Dipalmitoylphosphatidylcholine, cholesterol, and dipalmitoyl phosphatidylethanolamine (20:20:1 mole ratio). 42 rats were subjected to UHS and randomly received 5 different treatments (vasopressin, liposomal vasopressin, lactate ringer (LR), liposome only and sham). Outcome of UHS were measured using 4 common prognostic tests: mean arterial pressure (MAP), serum lactate level, inflammatory profile and pulmonary edema. RESULTS: The dynamic light scattering results confirmed that we had prepared a successful liposomal vasopressin complex. Comparing the serum vasopressin concentration of liposomal vasopressin and vasopressin treated animals by ELISA, we found that the concentration of vasopressin for the liposomal vasopressin treated group is higher at 60 minutes. However, there was no significant difference between the MAP profile of rats treated with vasopressin and liposomal vasopressin in UHS. We also observed that animals treated with liposomal vasopressin performed indifferently to vasopressin treated rats in serum lactate level, inflammatory profile and edema profile. For most of our assays, the liposome only control behaves similarly to LR resuscitation in UHS rats. CONCLUSION: We have synthesized a liposomal vasopressin complex that can prolong the serum concentration of vasopressin in a rat model of UHS. Although UHS rats treated with either liposomal vasopressin or vasopressin showed no statistical differences, it would be worthwhile to repeat the experiments with different liposomal compositions.

Gallic acid induces necroptosis via TNF-α signaling pathway in activated hepatic stellate cells.

Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA), a natural phenolic acid widely found in gallnuts, tea leaves and various fruits, possesses several bioactivities against inflammation, oxidation, and carcinogenicity. The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property. However, the cytotoxicity of GA autoxidation causing cell death has also been reported. Herein, we postulated that GA might target activated hepatic stellate cells (aHSCs), the cell type responsible for hepatofibrosis, to mitigate the process of fibrosis. The molecular cytotoxic mechanisms that GA exerted on aHSCs were then analyzed. The results indicated that GA elicited aHSC programmed cell death through TNF-α-mediated necroptosis. GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH). Elevated oxidative stress triggered the production of TNF-α facilitating the undergoing of necroptosis through the up-regulation of key necroptotic regulatory proteins TRADD and receptor-interacting protein 3 (RIP3), and the inactivation of caspase-8. Calmodulin and calpain-1 activation were engaged, which promoted subsequent lysosomal membrane permeabilization (LMP). The TNF-α antagonist (SPD-304) and the RIP1 inhibitor (necrostatin-1, Nec-1) confirmed GA-induced TNFR1-mediated necroptosis. The inhibition of RIP1 by Nec-1 diverted the cell death from necroptosis to apoptosis, as the activation of caspase 3 and the increase of cytochrome c. Collectively, this is the first report indicating that GA induces TNF signaling-triggered necroptosis in aHSCs, which may offer an alternative strategy for the amelioration of liver fibrosis.

Synergism through combination of chemotherapy and oxidative stress-induced autophagy in A549 lung cancer cells using redox-responsive nanohybrids: a new strategy for cancer therapy.

A combination of various therapeutic approaches has emerged as a promising strategy for cancer treatment. A safe and competent nano-delivery system is thus in urgent demand to facilitate the simultaneous transport of various therapeutic agents to cancer cells and a tumor region to achieve synergistic effect. Gold nanoparticles (GNPs) and mesoporous silica nanoparticle (MSNs) were fabricated herein as potential candidates for drug delivery. Serving as gatekeepers, GNPs (5 nm in diameter) were attached onto the amino-functionalized MSNs (denoted as NMSNs) via a relatively weak gold-nitrogen bonding. The resulting nanohybrids (denoted as GCMSNs) were uptaken by cells, and the detachment of GNPs and subsequent intracellular drug release from NMSNs were achieved by competitive binding of intracellular glutathione to GNPs. In addition to the function of gatekeeping, GNPs also play another role as the oxidative stress elicitor. Our in vitro studies revealed that GCMSNs induced higher oxidative stress in lung cancer cells (A549) than in normal cells (3T3-L1). This growth inhibitory effect found in the cancer cells was likely induced by mitochondria dysfunction originated from the GCMSN-induced, oxidative stress-triggered mitochondria-mediated autophagy. The redox-responsive nanohybrids were further loaded with camptothecin and the intensified synergistic therapeutic effects were observed associated with combined chemotherapy and oxidative stress strategy. The results clearly demonstrate that such unique nanohybrids hold great promise for selective and effective cancer treatments.

Diagnosing the miR-141 prostate cancer biomarker using nucleic acid-functionalized CdSe/ZnS QDs and telomerase.

The microRNA, miR-141, is a promising biomarker for prostate cancer. We implement here a two-step sensing platform for the sensitive detection of miR-141. The first step involves the use of semiconductor CdSe/ZnS quantum dots (QDs) modified by FRET quencher-functionalized nucleic acids, that include the recognition sequence for miR-141 and a telomerase primer sequence for the second step of the analytical platform. Subjecting the probe-modified QDs to miR-141, in the presence of duplex specific nuclease, DSN, leads to the formation of a miR-141/probe duplex and to its DSN-mediated cleavage, while regenerating the miR-141. The DSN-induced cleavage of the quencher units leads to the activation of the fluorescence of the QDs, thus allowing the optical detection of miR-141 with a sensitivity corresponding to 1.0 × 10-12 M. The nucleic acid residues associated with the QDs after cleavage of the probe nucleic acids by DSN act as primers for telomerase. The subsequent telomerase/dNTPs-stimulated elongation of the primer units forms G-quadruplex telomer chains. Incorporation of hemin in the resulting G-quadruplex telomer chains yields horseradish peroxidase-mimicking DNAzyme units, that catalyze the generation of chemiluminescence in the presence of luminol/H2O2. The resulting chemiluminescence intensities provide a readout signal for miR-141, DL = 2.8 × 10-13 M. The first step of the sensing platform is non-selective toward miR-141 and the resulting fluorescence may be considered only as an indicator for the existence of miR-141. The second step in the sensing protocol, involving telomerase, provides a selective chemiluminescence signal for the existence of miR-141. The two-step sensing platform is implemented for the analysis of miR-141 in serum samples from healthy individuals and prostate cancer carriers. Impressive discrimination between healthy individuals and prostate cancer carriers is demonstrated.

Facile preparation of high-quantum-yield gold nanoclusters: application to probing mercuric ions and biothiols.

This paper describes an eco-friendly, one-pot strategy for the synthesis of water-soluble, high-quantum-yield gold nanoclusters (AuNCs) stabilized with 11-mercaptoundecanoic acid (MUA) on their surfaces. The as-prepared ultrasmall MUA-AuNCs (1.9 nm) exhibited a quantum yield (QY) of 13%, higher than those of most previously described thiol-protected AuNCs. We applied these MUA-AuNCs as a versatile probe to develop a fluorescence "turn-off" assay for sensing Hg(2+) ions as well as a fluorescence "turn-on" assay for sensing biothiols. The former assay operated through aggregation-induced fluorescence quenching upon interaction of the MUA-AuNCs with Hg(2+) ions in a buffer containing 2,6-pyridinedicarboxylic acid (PDCA); this probe provided high sensitivity and remarkable selectivity over other selected metal ions with a limit of detection (LOD) for Hg(2+) ions of 450 pM and linearity from 2 to 50 nM. In the latter assay for biothiols [i.e., cysteine (Cys), homocysteine (Hcy), glutathione (GSH)], the fluorescence of the Hg(2+)-MUA-AuNCs complexes was turned on because the affinity of Hg(2+) ions toward the SH group of the biothiols was greater than that toward the COOH groups of the MUA units on the surface of the AuNCs. This assay provided good linearity for the tested biothiols, ranging from 10 to 100 nM for Cys, from 10 to 100 nM for Hcy, and from 5 to 75 nM for GSH, with LODs of 5.4, 4.2, and 2.1 nM, respectively. In addition, these environmentally and biologically friendly AuNC probes tested satisfactorily against interference from a range of amino acids.

Phospholipid-functionalized mesoporous silica nanocarriers for selective photodynamic therapy of cancer.

This paper describes the fabrication of a highly efficient, non-cytotoxic drug delivery platform designed for photodynamic therapy (PDT): phospholipid-capped, protoporphyrin IX-loaded and FITC-sensitized mesoporous silica nanocarriers (Lipo-FMSNs/PpIX). After derivatization with folate on the phospholipid-capped FMSNs (denoted fa-Lipo-FMSNs/PpIX, the so-called nanoPDT system), we confirmed the nanoPDT systems' selective targeting of and entry into the folic acid receptor-overexpressed HeLa cells by means of cell viability assessment and confocal microscopic analysis. The decrease in the unfavorable dark toxicity of fa-Lipo-FMSNs/PpIX enabled the delivery of high concentrations of PpIX into cells. Moreover, the cellular uptake of the nanoPDT systems was greater than that of free PpIX. Upon irradiation with visible light, the nanoPDT system generated singlet oxygen efficaciously in aqueous environments-a decisive factor affecting its therapeutic applicability in PDT, demonstrating enhanced in vitro photocytotoxicity. Furthermore, an in vivo study of subcutaneous melanoma in nude mice inoculated with B16F10 cells revealed the capability for the nanoPDT system to mitigate nearly 65% of tumor growth.

Monitoring the subcellular localization of doxorubicin in CHO-K1 using MEKC-LIF: liposomal carrier for enhanced drug delivery.

Doxorubicin (DOX) is an extensively used anthracycline that has proven to be effective against a variety of human malignant tumors, such as ovarian or breast cancer. While DOX was administered into cultured cancer cell targets (such as CHO-K1) in either free drug form or in drug carrier-associated form (i.e., DOX encapsulated in the drug delivery carrier), various action of mechanisms for DOX were initiated, among which, it has been long believed that DOX enters the nucleus, interacts with DNA in numerous ways, and finally halts cell proliferation. Aside from its therapeutic effect, regrettably DOX treatment may be accompanied by the occurrence of cardiac and liver toxicity and drug resistance that are attributed from cellular processes involving the parent drug or its metabolites. Liposomal formulation of DOX, known to be capable of attenuating direct uptake of reticuloendothelial system (RES) and prolonging the circulation time in blood, demonstrated reduced toxic side-effects. We herein report the development of a modified MEKC-LIF (Micellar electrokinetic chromatography-Laser-induced fluorescence) method suitable for analyzing DOX in biological samples. The MEKC migration buffer, consisting of 10 mM borate, 100 mM sodium dodecyl sulfate (SDS) (pH 9.3), was found to provide an efficient and stable electrophoretic separation and analysis for DOX. Responses were linear in the range of 11.3-725 ng/mL; the limit of quantitation (LOQ) was found to be 43.1 ng/mL (S/N=10) (equivalent to 74.3 nM) and limit of detection (LOD) was calculated as 6.36 ng/mL (S/N=3) (equivalent to 11.0 nM). This approach was subsequently employed to compare the intracellular accumulation in three subcellular fractions of DOX-treated CHO-K1 cells. These fractions form a pellet at <1400 g, 1400-14000 g, and >14000 g and are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosole components, respectively, resulting from treatment of CHO-K1 cells with 25 µM (equivalent to 14.5 µg/mL) of two DOX formats (in free drug form or liposomal form synthesized in current study) for different periods of time. Our results indicated that the most abundant DOX was found in the nuclear-enriched fraction of cells treated for 12 h and 6 h with free and liposomal DOX, respectively, providing direct evidence to confirm the enhanced efficiency of liposomal carriers in delivering DOX into the nucleus. The observations presented herein suggest that subcellular fractionation followed by liquid-liquid extraction and MEKC-LIF could be a powerful diagnostic tool for monitoring intracellular DOX distribution, which is highly relevant to cytotoxicity studies of anthracycline-type anticancer drugs.

Optical aptasensors for the analysis of the vascular endothelial growth factor (VEGF).

The vascular endothelial growth factor, VEGF, is an important biomarker for different diseases and clinical disorders. We present a series of optical aptasensor-based sensing platforms for VEGF that include the following: (i) A FRET-based sensor that involves the VEGF-induced separation of aptamer-functionalized quantum dots blocked by a quencher nucleic acid (detection limit 1 nM). (ii) A FRET-based sensor based on the VEGF-induced assembly of the aptamer subunits functionalized with QDs and a dye acceptor (Cy5), respectively (detection limit 12 nM). (iii) A chemiluminescence aptasensor based on VEGF-induced assembly of a hemin/G-quadruplex catalyst (detection limit 18 nM). (iv) A chemiluminescence aptasensor based on the VEGF-stimulated assembly of two aptamer subunits into the hemin/G-quadruplex catalyst (detection limit 2.6 nM). (v) A chemiluminescence resonance energy transfer (CRET) aptasensor based on the VEGF-induced assembly of a semiconductor QDs-hemin/G-quadruplex supramolecular structure (detection limit 875 pM). Furthermore, an amplified optical aptasensor system based on the Exonuclease III (Exo III) recycling of the VEGF analyte was developed. In this system, one aptamer subunit is modified at its 5' and 3' ends with QDs and a black hole quencher, respectively. The VEGF-induced self-assembly of the aptamer subunits result in the digestion of the quencher units and the autonomous recycling of the analyte, while triggering-on the luminescence of the QDs (detection limit 5 pM). The system was implemented to analyze VEGF in human sera samples.

Synthesis, characterization, and electrochemical applications of carbon nanoparticles derived from castor oil soot.

A simple procedure for the modification of carbon nanoparticles (CNPs) from castor oil soot using acid treatment was described herein. Characterization studies revealed the presence of edge plane sites and surface carbon-oxygen functionalities at the surface of the CNP material. Voltammetric studies revealed the increased electrochemical activity of the CNP-modified electrode toward various biologically important molecules, including dopamine, uric acid, dihydronicotinamide adenine dinucleotide, tyrosine, and serotonin, relative to those obtained using the unmodified electrode. The improved electro-oxidation potentials for these compounds-and, thereby, the enhanced sensitivity of related sensors-was due directly to the presence of surface C(δ+)O(δ-) functional groups and the greater number of edge plane sites developed after acid treatment of the soot sample.

Binding kinetics of biomolecule interaction at ultralow concentrations based on gold nanoparticle enhancement.

Measuring the kinetic constants of protein-protein interactions at ultralow concentrations becomes critical in characterizing biospecific affinity, and exploring the feasibility of clinical diagnosis with respect to detection sensitivity, efficiency and accuracy. In this study, we propose a method that can calculate the binding constants of protein-protein interactions in sandwich assays at ultralow concentrations at the pg/mL level, using a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB). We discuss a two-compartment model to achieve reaction-limited kinetics under the stagnant conditions of the reaction chamber. The association rate constant, dissociation rate constant, and the equilibrium dissociation constant, that is, k(a), k(d), K(D), respectively, of the kinetics of binding between total prostate-specific antigen (t-PSA) and anti-t-PSA at concentrations from 0.1 pg/mL to 1 ng/mL, were measured either in PBS or in human serum. This is the first time that k(a), k(d), and K(D) have been measured at such a low concentration range in a complex sample such as human serum.