Inhibition of Melanoma Cells A375 by Carotenoid Extract and Nanoemulsion Prepared from Pomelo Leaves.

This study aims to determine carotenoids in pomelo leaves (Citrus grandis Osbeck), a rich source of nutrients and phytochemicals, by high-performance liquid chromatography-mass spectrometry and prepare carotenoid nanoemulsions for the study of its inhibitory mechanism on melanoma cells A375. Fourteen carotenoids were separated within 27 min by using a YMC-C30 column and a gradient mobile phase of methanol-acetonitrile-water (84:14:2, v/v/v) and methylene chloride with a flow rate of 1 mL/min and detection wavelength of 450 nm. All-trans-lutein plus its cis-isomers were present in the largest amount (3012.97 µg/g), followed by all-trans-neoxanthin (309.2 µg/g), all-trans-violaxanthin (208.5 µg/g), all-trans-ß-carotene plus its cis-isomers (203.17 µg/g), all-trans-α-carotene plus its cis-isomers (152.5 µg/g), all-trans-zeaxanthin (54.67 µg/g), and all-trans-ß-cryptoxanthin plus its cis-isomers (24.56 µg/g). A stable carotenoid nanoemulsion was prepared with a mean particle size of 13.3 nm, zeta-potential of -66.6 mV, a polydispersity index of 0.132 and an encapsulation efficiency of 99%. Both the carotenoid extract and nanoemulsion could upregulate p53, p21, cyclin B and cyclin A expressions in melanoma A375 cells and downregulate CDK1 and CDK2 in a concentration-dependent manner. Also, they could upregulate Bax and cytochrome-C and downregulate Bcl-2, leading to cell apoptosis through activation of caspase-9, caspase-8 and caspase-3. Compared to extract, carotenoid nanoemulsion was shown to be more effective in inhibiting the growth of melanoma cells A375. This finding further demonstrated that a carotenoid nanoemulsion prepared from pomelo leaves possessed a great potential to be developed into functional foods or even botanic drugs.

Preparation of Chlorophyll Nanoemulsion from Pomelo Leaves and Its Inhibition Effect on Melanoma Cells A375.

Pomelo (Citrus grandis), an important fruit crop grown in tropical and subtropical areas, is cultivated mainly in Asian countries. The dominant pigment in pomelo leaves, chlorophyll, has been reported to possess many biological activities such as antioxidant, anti-inflammation and anticancer. The objectives of this study were to determine chlorophylls in Pomelo leaves by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and to encapsulate the isolated chlorophylls from preparative column chromatography into a nanoemulsion system for elucidating the inhibition mechanism on the growth of melanoma cells A375. The results showed that chlorophyll a and chlorophyll b could be separated within 25 min by using a C18 column and a gradient ternary mobile phase of acetone, acetonitrile and methanol. Pomelo leaves mainly contained chlorophyll a (2278.3 µg/g) and chlorophyll b (785.8 µg/g). A highly stable chlorophyll nanoemulsion was prepared with the mean particle size being 13.2 nm as determined by a dynamic light scattering (DLS) method. The encapsulation efficiency of chlorophyll nanoemulsion was 99%, while the zeta potential was -64.4 mV. In addition, the chlorophyll nanoemulsion possessed high thermal stability up to 100 °C and remained stable over a 90-day storage period at 4 °C. Western blot analysis revealed that chlorophyll nanoemulsion and extract could upregulate p53, p21, cyclin B and cyclin A as well as downregulate CDK1 and CDK2 in a concentration-dependent manner for inhibition of melanoma cells A375. Furthermore, chlorophyll nanoemulsion and extract could upregulate Bax and cytochrome C and downregulate Bcl-2, leading to activation of caspase-9, caspase-8 and caspase-3 for the induction of cell apoptosis. Compared to chlorophyll extract, chlorophyll nanoemulsion was more effective in inhibiting the growth of melanoma cells A375.

[Wound healing effects of silk fibroin-bone morphogenetic protein-2 scaffolds on inflammatory pulp in rats].

OBJECTIVE: To identify the healing effect of electrospun silk fibroin-BMP-2 as a biologic pulp capping agent to inflammatory pulp in rat caused by lipopolysaccharide (LPS). METHODS: A total of 30 healthy adult male Wistar rats were randomly divided into five groups:(1) normal control group without operation; (2) blank control group without capping agents; (3) calcium hydroxide capping group; (4) electrospun silk fibroin capping group; (5) electrospun silk fibroin-BMP-2 capping group. Bilateral upper first molars of each rat in group 2-5 were drilled to expose the pulp to LPS which was used to establish a model of inflammatory pulp. The exposed pulp was capped with different capping agents or without capping agents. Then the hole was sealed. The animals were sacrificed on days 3, 7, and 14 post-operation and histological analysis was carried out, including HE stain and CD14 immunohistochemical stain. RESULTS: On day 7 and 14, the lowest inflammatory reaction score in HE stain among pulp capping groups was that of silk fibroin-BMP-2 group. The next were calcium hydroxide group and silk fibroin group. That of blank control group was the highest. The ranking of reparative dentine scores of those groups was just reversed. The D values of immunohistochemical stain of CD14 were not significantly different in groups applied pulp capping agents but significantly lower than blank control group on days 3 and 7. However, the D value of silk fibroin-BMP-2 group (0.145±0.011) was significantly lower than blank control group (0.287±0.019), calcium hydroxide group (0.170±0.017) and silk fibroin group (0.175±0.018) on day 14. CONCLUSION: Electrospun silk fibroin compounded with BMP-2 promoted wound healing of exposed pulp and had better potential to stimulate formation of reparative dentine to establish a suitable environment for pulp recovery.

Dielectrophoretic capture of E. coli cells at micropatterned nanoelectrode arrays.

This paper reports capture and detection of pathogenic bacteria based on AC dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS) employing an embedded vertically aligned carbon nanofiber (VACNF) nanoelectrode array (NEA) versus a macroscopic indium-tin-oxide (ITO) transparent electrode in "points-and-lid" configuration. The nano-DEP device was fabricated using photolithography processes to define an exposed active region on a randomly distributed NEA and a microfluidic channel on ITO to guide the flow of labeled Escherichia coli cells, respectively, and then bond them into a fluidic chip. A high-frequency (100 kHz) AC field was applied to generate positive DEP at the tips of exposed CNFs. Enhanced electric field gradient was achieved due to reduction in electrode size down to nanometer scale which helped to overcome the large hydrodynamic drag force experienced by E. coli cells at high flow velocities (up to 1.6 mm/s). This DEP device was able to effectively capture a significant number of E. coli cells. Significant decrease in the absolute impedance at the NEA was observed in EIS experiments. The results obtained in this study suggest the possibility of integration of a fully functional electronic device for rapid, reversible and label-free capture and detection of pathogenic bacteria.

An intrinsically fluorescent recognition ligand scaffold based on chaperonin protein and semiconductor quantum-dot conjugates.

Genetic engineering of a novel protein-nanoparticle hybrid system with great potential for biosensing applications and for patterning of various types of nanoparticles is described. The hybrid system is based on a genetically modified chaperonin protein from the hyperthermophilic archaeon Sulfolobus shibatae. This chaperonin is an 18-subunit double ring, which self-assembles in the presence of Mg ions and ATP. Described here is a mutant chaperonin (His-beta-loopless, HBLL) with increased access to the central cavity and His-tags on each subunit extending into the central cavity. This mutant binds water-soluble semiconductor quantum dots, creating a protein-encapsulated fluorescent nanoparticle. The new bioconjugate has high affinity, in the order of strong antibody-antigen interactions, a one-to-one protein-nanoparticle stoichiometry, and high stability. By adding selective binding sites to the solvent-exposed regions of the chaperonin, this protein-nanoparticle bioconjugate becomes a sensor for specific targets.

A self-assembling protein template for constrained synthesis and patterning of nanoparticle arrays.

Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here, we introduce a general technique for patterning nanoparticle arrays using two-dimensional crystals of genetically modified hollow protein structures called chaperonins. Constrained chemical synthesis of transition metal nanoparticles is initiated using templates functionalized with polyhistidine sequences. These nanoparticles are ordered into arrays because the template-driven synthesis is constrained by the nanoscale structure of the crystallized protein. We anticipate that this system may be used to pattern different classes of nanoparticles based on the growing library of sequences shown to specifically bind or direct the growth of materials.

Comparison of plasma N-brain natriuretic peptide, peak oxygen consumption, and left ventricular ejection fraction for severity of chronic heart failure.

We investigated whether a simple blood test of plasma N-brain natriuretic peptide (N-BNP), compared with echocardiographic left ventricular ejection fraction (LVEF), both measured at rest, correlated well with aerobic exercise capacity (peak oxygen consumption [VO(2)]) in patients with chronic heart failure. Plasma N-BNP was found to significantly correlate with peak VO(2) (p <0.001) and exercise duration (p = 0.001), whereas LVEF showed very poor correlations with peak VO(2) and exercise duration (both p >0.3). The results suggest that N-BNP actually reflects functional cardiac impairment better than LVEF.