A rapid and universal method for isolating starch granules in plant tissues.

Starch is the major form of carbohydrate storage in plants and exists as discrete starch granules (SGs). Isolation of high-quality SGs in different plant tissues is a prerequisite for studying the roles of SGs during plant growth, development, and responses to abiotic stress. However, it is difficult to isolate transitory SGs from leaves and storage SGs from pollen grains due to their small sizes and low quantities. Herein, we develop a novel method for isolating SGs by using the aqueous two-phase system (ATS) of ethanol/NaH2 PO4 . The ATS method efficiently separated SGs from contaminants based on their differences in density, solubility, and polarity. Using this method, we first isolated and purified three kinds of SGs from maize seeds, pollen, and leaves. The biochemical, microscopic, and proteomic analyses demonstrated the high purity of the isolated SGs. Proteomic analysis revealed distinct differences in SG-bound proteins between seed SGs and pollen SGs. As a simple, rapid, and low-cost method, the ATS-based method exhibits highly universal and reproducible results for starch-containing tissues in various plant species.

Water-soluble, membrane-permeable organic fluorescent nanoparticles with large tunability in emission wavelengths and Stokes shifts.

Entrapment within surface-crosslinked micelles (SCMs) enhanced the emission of conventional hydrophobic fluorescent dyes, endowed them with excellent water solubility and membrane permeability, and greatly expanded the Stokes shifts without any covalent structural modification of the dyes.

Characterization and optimization of mTHPP nanoparticles for photodynamic therapy of head and neck cancer.

OBJECTIVES: To characterize the properties of polymeric micelles containing different loading percentages of mTHPP, a photosensitizer for photodynamic therapy (PDT), with respect to fluorescence, singlet oxygen ((1)O(2)) yield, and in vitro cytotoxicity in head and neck cancer cells. STUDY DESIGN: Laboratory study. SETTING: Polymer chemistry laboratory. SUBJECTS AND METHODS: Absorption and emission spectroscopy was used to characterize the mTHPP-loaded micelles. The (1)O(2) yield was measured to determine the efficiency of reactive oxygen species (ROS) generation. In vitro studies were conducted using the HN5 cells and confirmed with H2009 cells to determine the photodynamic efficacy. DNA assay and confocal microscopy was used to measure intracellular fluorescence. RESULTS: The mTHPP micelles demonstrated the highest fluorescence intensity at 0.5% loading. The (1)O(2) generation of the micelles in solution peaked at 2% loading. Phototoxicity and dark toxicity experiments in HN5 and H2009 cells demonstrated that the best therapeutic index was achieved with the 2% loaded micelles with 100% cell cytotoxicity at a micelle concentration of 10 µg/mL and less than 10% dark cytotoxicity. In comparison, 10% loaded micelles demonstrated 100% cell cytotoxicity at a concentration of 20 µg/mL under both light and dark conditions. Confocal microscopy demonstrated increasing intracellular fluorescence with higher loading. CONCLUSIONS: The 2% mTHPP-loaded micelles generated greater (1)O(2), and 0.5% loading led to the most efficient generation of fluorescence in solution. Higher mTHPP loading density led to increased cellular fluorescence and dark cytotoxicity. Overall, 2% mTHPP-loaded micelles provided the optimal composition for photodynamic therapy with the largest therapeutic window.

Determination of neonicotinoid pesticides residues in agricultural samples by solid-phase extraction combined with liquid chromatography-tandem mass spectrometry.

This work reports a new sensitive multi-residue liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection, confirmation and quantification of six neonicotinoid pesticides (dinotefuran, thiamethoxam, clothiandin, imidacloprid, acetamiprid and thiacloprid) in agricultural samples (chestnut, shallot, ginger and tea). Activated carbon and HLB solid-phase extraction cartridges were used for cleaning up the extracts. Analysis is performed by LC-MS/MS operated in the multiple reaction monitoring (MRM) mode, acquiring two specific precursor-product ion transitions per target compound. Quantification was carried by the internal standard method with D(4)-labeled imidacloprid. The method showed excellent linearity (R(2)≥0.9991) and precision (relative standard deviation, RSD≤8.6%) for all compounds. Limits of quantification (LOQs) were 0.01 mg kg(-1) for chestnut, shallot, ginger sample and 0.02 mg kg(-1) for tea sample. The average recoveries, measured at three concentrations levels (0.01 mg kg(-1), 0.02 mg kg(-1) and 0.1 mg kg(-1) for chestnut, shallot, ginger sample, 0.02 mg kg(-1), 0.04 mg kg(-1) and 0.2 mg kg(-1) for tea sample), were in the range 82.1-108.5%. The method was satisfactorily validated for the analysis of 150 agricultural samples (chestnut, shallot, ginger and tea). Imidacloprid and acetamiprid were detected at concentration levels ranging from 0.05 to 3.6 mg kg(-1).

Nanoscopic micelle delivery improves the photophysical properties and efficacy of photodynamic therapy of protoporphyrin IX.

Nanodelivery systems have shown considerable promise in increasing the solubility and delivery efficiency of hydrophobic photosensitizers for photodynamic therapy (PDT) applications. In this study, we report the preparation and characterization of polymeric micelles that incorporate protoporphyrin IX (PpIX), a potent photosensitizer, using non-covalent encapsulation and covalent conjugation methods. Depending on the incorporation method and PpIX loading percentage, PpIX existed as a monomer, dimer or aggregate in the micelle core. The PpIX state directly affected the fluorescence intensity and (1)O(2) generation efficiency of the resulting micelles in aqueous solution. Micelles with lower PpIX loading density (e.g. 0.2%) showed brighter fluorescence and higher (1)O(2) yield than those with higher PpIX loading density (e.g. 4%) in solution. However, PDT efficacy in H2009 lung cancer cells showed an opposite trend. In particular, 4% PpIX-conjugated micelles demonstrated the largest PDT therapeutic window, as indicated by the highest phototoxicity and relatively low dark toxicity. Results from this study contribute to the fundamental understanding of nanoscopic structure-property relationships of micelle-delivered PpIX and establish a viable micelle formulation (i.e. 4% PpIX-conjugated micelles) for in vivo evaluation of antitumor efficacy.

Photoactivation switch from type II to type I reactions by electron-rich micelles for improved photodynamic therapy of cancer cells under hypoxia.

Photodynamic therapy (PDT) is an emerging clinical modality for the treatment of a variety of diseases. Most photosensitizers are hydrophobic and poorly soluble in water. Many new nanoplatforms have been successfully established to improve the delivery efficiency of PS drugs. However, few reported studies have investigated how the carrier microenvironment may affect the photophysical properties of photosensitizer (PS) drugs and subsequently, their biological efficacy in killing malignant cells. In this study, we describe the modulation of type I and II photoactivation processes of the photosensitizer, 5,10,15,20-tetrakis(meso-hydroxyphenyl)porphyrin (mTHPP), by the micelle core environment. Electron-rich poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) micelles increased photoactivations from type II to type I mechanisms, which significantly increased the generation of O(2)(-) through the electron transfer pathway over (1)O(2) production through energy transfer process. The PDPA micelles led to enhanced phototoxicity over the electron-deficient poly(D,L-lactide) control in multiple cancer cell lines under argon-saturated conditions. These data suggest that micelle carriers may not only improve the bioavailability of photosensitizer drugs, but also modulate photophysical properties for improved PDT efficacy.

Determination of Gibberellin A3 residue in fruit samples by liquid chromatography-tandem mass spectrometry.

A fast, simple, low cost, and high throughput method has been developed for the determination of Gibberellin A3 residue in fruit samples (apple, orange, peach, pear and grape). Analysis is performed by LC-MS/MS operated in the multiple reaction monitoring (MRM) mode, acquiring two specific precursor-product ion transitions per target compound. The method has been validated showing good linearity and selectivity. Limits of quantification (LOQs) were 10µgkg(-1) for apple, orange, peach, pear and grape samples. The average recoveries, measured at three concentration levels (10, 20 and 200µgkg(-1)) were in the range 77.8-96.2% for the compound tested with relative standard deviations below 13.7%. The proposed method is rapid, simple and could be utilised for the routine analysis of Gibberellin A3 in fruit samples.

Fluorescence monitoring of a photosensitizer and prediction of the therapeutic effect of photodynamic therapy for port wine stains.

In this study, a fluorescence method was established to obtain the local concentration of a photosensitizer (PS) based on the realtime fluorescence measurement of skin with port wine stain (PWS) during photodynamic therapy (PDT). This algorithm corrected for the distortions of PS fluorescence spectra imposed by the absorption of melanin and hemoglobin in skin and other factors, which yields a semi-quantitative measurement of PS concentration. Based on this information, a therapeutic effect correlation index (TECI) was proposed as the area under the PS concentration-time curve during PDT. The correlation between TECI and PDT treatment outcome was analyzed from 31 PWS patients. The measured PS fluorescence spectra showed that under the same PS dose, there were clear variations in the concentrations of the PS during PDT. Statistical analysis showed that TECI has a positive correlation with PDT outcome. Patients with a higher TECI value had a better treatment outcome. These results suggest that fluorescence spectroscopy can be used in situ to monitor skin PS concentration during PDT and to provide a valuable diagnostic tool to predict PDT outcome.

Polymeric micelle nanoparticles for photodynamic treatment of head and neck cancer cells.

OBJECTIVE: To encapsulate 5,10,15,20-tetrakis(meso-hydroxyphenyl)porphyrin (mTHPP), a photosensitizer, into polymeric micelles; characterize the micelles; and test in vitro photodynamic therapy efficacy against human head and neck cancer cells. STUDY DESIGN: A nanoparticle design, fabrication, and in vitro testing study. SETTING: Polymer chemistry laboratory. SUBJECTS AND METHODS: Micelles encapsulating mTHPP were produced, and micellar size was measured. Ultraviolet visible spectra and fluorescence spectroscopy were used to characterize the mTHPP-loaded micelles. In vitro cell culture using HSC-3 and HN-5 cancer cells was performed to test the photodynamic therapy efficacy of the micelles using confocal microscopy and method of transcriptional and translational (MTT) assay. RESULTS: mTHPP was encapsulated with high loading efficiency (> 85%) and density (up to 17%) into micelles. Micelle size was 30.6 +/- 3.3 nm by transmission electron microscopy and 30.8 +/- 0.6 nm by dynamic light scattering. The absorption maximum for each sample was 418 nm, and fluorescent spectroscopy revealed quenching with maximal fluorescence at five percent loading. Significant cytotoxicity was observed with confocal microscopy when HSC-3 cells were treated with 10 percent mTHPP micelles, with 100 percent cytotoxicity within the zone of laser light exposure at 420 nm. Phototoxicity and dark toxicity against HSC-3 and HN-5 cells measured using the MTT assay with five and 10 percent loaded mTHPP micelles demonstrated greater than 90 percent cytotoxicity with photodynamic therapy and less than 10 percent dark toxicity at a micelle concentration of 25 microg/mL for both cell lines. CONCLUSION: Micelles were able to encapsulate and solubilize mTHPP at high loading densities with uniform size distribution. These micelles exhibit fluorescence and photodynamic therapy mediated cytotoxicity against head and neck cancer cells in vitro.

[Determination of five macrolide antibiotic residues in royal jelly samples by using high performance liquid chromatography tandem mass spectrometry].

The macrolides are lipophilic molecules having a central lactone ring bearing 12 to 20 atoms to which several amino and/or neutral sugars are bound. They are broad spectrum antibiotics active against Gram-positive bacteria and mycoplasmas, as well as some Gram-negative organisms and members of the chlamydia group. Macrolides are a group of antibacterial compounds that have been widely used in medical and veterinary practices. A method of high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was developed for the confirmation of five macrolide antibiotic residues (spiramycin, oleandomycin, tylosin, roxithromycin, josamycin) in royal jelly samples. Trichloroacetic acid solution was used to precipitate the protein in the sample. The upper layer solution was extracted with acetonitrile. Then it was cleaned up with a C18 column. The one precursor/two product ion transitions for each macrolide antibiotics were monitored. The results show that the working curves for five macrolide antibiotics were linear in the range of 0.002 - 0.05 mg/L by HPLC-MS/MS in selective ion monitoring model. The limits of quantitation of the antibiotics in royal jelly were all 20 microg/kg. The recoveries were between 73.0% -90.2% at three spiked levels (20, 100 and 200 microg/kg for each macrolide antibiotic), and the relative standard deviations were between 5.6% - 10.5%.

[Determination of imidacloprid residues in vegetable and tea samples using liquid chromatography-mass spectrometry/mass spectrometry].

A liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method was developed for the determination of imidacloprid in vegetable and tea samples. The sample was first extracted with acetonitrile and then cleaned up with Florisil and active charcoal column. The precursor ion/product ion transitions m/z 256.0/209.3 and m/z 256.0/175.2 were monitored, and m/z 209.3 was used for quantification. The good linearity was obtained in the range of 0.01 - 0.5 mg/L with the correlation coefficients (nu2) more than 0.997. The limit of quantification was 0.01 mg/kg. The recoveries were 76% - 90% at the spiked levels of 0.01, 0.05 and 0.2 mg/kg with the relative standard deviations of 7.4% - 11.0%. The method is rapid, sensitive and specific for imidacloprid analysis.