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.

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.

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.

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.

Antioxidant, anti-inflammatory and anti-browning activities of hot water extracts of oriental herbal teas.

Traditionally, antioxidants are used to scavenge reactive oxygen species (ROS), which are harmful by-products of aerobic metabolism. Inulae Flos, Horsetail, Chinese Leucas, Broomweed and Indian Wikstroemia are five herbal teas commonly consumed by Asians. Our aim was to investigate the hot water extracts of these five herbal teas for their total phenolics/flavonoid contents and antioxidant capacities. Furthermore, with inflammation and hyper-pigmentation considered as two biological processes associated with elevated cellular oxidative stress, Inulae Flos water extract was chosen for further evaluation of its inhibitory effects on the production of LPS-induced inflammatory mediators (such as, TNF-α, IL-6, IL-1ß) in RAW 264.7 cells and its anti-tyrosinase activity. Our findings suggest that Inulae Flos might be an alternative source as a potential antioxidant, and a noteworthy inhibitor of production of pro-inflammatory cytokines in a dose-dependent manner. Moreover, it could also serve as a potential natural food additive to prevent browning.

Anti-inflammatory effect of spilanthol from Spilanthes acmella on murine macrophage by down-regulating LPS-induced inflammatory mediators.

Spilanthes acmella (Paracress), a common spice, has been administered as a traditional folk medicine for years to cure toothaches, stammering, and stomatitis. Previous studies have demonstrated its diuretic, antibacterial, and anti-inflammatory activities. However, the active compounds contributing to the anti-inflammatory effect have seldom been addressed. This study isolates the active compound, spilanthol, by a bioactivity-guided approach and indicates significant anti-inflammatory activity on lipopolysaccharide-activated murine macrophage model, RAW 264.7. The anti-inflammatory mechanism of paracress is also investigated. Extracts of S. acmella are obtained by extraction with 85% ethanol, followed by liquid partition against hexane, chloroform, ethyl acetate, and butanol. The ethyl acetate extract exhibits a stronger free radical scavenging capacity than other fractions do, as determined by DPPH and ABTS radical scavenging assays. The chloroform extract significantly inhibits nitric oxide production ( p < 0.01) and is selected for further fractionation to yield the active compound, spilanthol. The diminished levels of LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) mRNA and protein expression support the postulation that spilanthol inhibits proinflammatory mediator production at the transcriptional and translational levels. Additionally, the LPS-stimulated IL-1beta, IL-6, and TNF-alpha productions are dose-dependently reduced by spilanthol. The LPS-induced phosphorylation of cytoplasmic inhibitor-kappaB and the nuclear NF-kappaB DNA binding activity are both restrained by spilanthol. Results of this study suggest that spilanthol, isolated from S. acmella, attenuates the LPS-induced inflammatory responses in murine RAW 264.7 macrophages partly due to the inactivation of NF-kappaB, which negatively regulates the production of proinflammatory mediators.

Development of a long-life capillary enzyme bioreactor for the determination of blood glucose.

A long-life capillary enzyme bioreactor was developed that determines glucose concentrations with high sensitivity and better stability than previous systems. The bioreactor was constructed by immobilizing glucose oxidase (GOx) onto the inner surface of a 0.53mm i.d. fused-silica capillary that was part of a continuous-flow system. In the presence of oxygen, GOx converts glucose to gluconic acid and hydrogen peroxide (H(2)O(2)). Hydrogen peroxide detection was accomplished using an amperometric electrochemical detector. The integration of this capillary reactor into a flow-injection (FIA) system offered a larger surface-to-volume ratio, reduced band-broadening effects, and reduced reagent consumption compared to packed column in FIA or other settings. To obtain operational (at ambient temp) and storage (at 4 degrees C) stability for 20 weeks, the glucose biosensing system was prepared using an optimal GOx concentration (200mg/mL). This exhibited an FIA peak response of 7min and a detection limit of 10muM (S/N=3) with excellent reproducibility (coefficient of variation, CV<0.75%). It also had a linear working range from 10(1) to 10(4)muM. The enzyme activity in this proposed capillary enzyme reactor was well maintained for 20 weeks. Furthermore, 20 serum samples were analyzed using this system, and these correlated favorably (correlation coefficient, r(2)=0.935) with results for the same samples obtained using a routine clinical method. The resulting biosensing system exhibited characteristics that make it suitable for in vivo application.