Molecular and biochemical characterization of two 4-coumarate: CoA ligase genes in tea plant (Camellia sinensis).

KEY MESSAGE: Two 4-coumarate: CoA ligase genes in tea plant involved in phenylpropanoids biosynthesis and response to environmental stresses. Tea plant is rich in flavonoids benefiting human health. Lignin is essential for tea plant growth. Both flavonoids and lignin defend plants from stresses. The biosynthesis of lignin and flavonoids shares a key intermediate, 4-coumaroyl-CoA, which is formed from 4-coumaric acid catalyzed by 4-coumaric acid: CoA ligase (4CL). Herein, we report two 4CL paralogs from tea plant, Cs4CL1 and Cs4CL2, which are a member of class I and II of this gene family, respectively. Cs4CL1 was mainly expressed in roots and stems, while Cs4CL2 was mainly expressed in leaves. The promoter of Cs4CL1 had AC, nine types of light sensitive (LSE), four types of stress-inducible (SIE), and two types of meristem-specific elements (MSE). The promoter of Cs4CL2 also had AC and nine types of LSEs, but only had two types of SIEs and did not have MSEs. In addition, the LSEs varied in the two promoters. Based on the different features of regulatory elements, three stress treatments were tested to understand their expression responses to different conditions. The resulting data indicated that the expression of Cs4CL1 was sensitive to mechanical wounding, while the expression of Cs4CL2 was UV-B-inducible. Enzymatic assays showed that both recombinant Cs4CL1 and Cs4CL2 transformed 4-coumaric acid (CM), ferulic acid (FR), and caffeic acid (CF) to their corresponding CoA ethers. Kinetic analysis indicated that the recombinant Cs4CL1 preferred to catalyze CF, while the recombinant Cs4CL2 favored to catalyze CM. The overexpression of both Cs4CL1 and Cs4CL2 increased the levels of chlorogenic acid and total lignin in transgenic tobacco seedlings. In addition, the overexpression of Cs4CL2 consistently increased the levels of three flavonoid compounds. These findings indicate the differences of Cs4CL1 and Cs4CL2 in the phenylpropanoid metabolism.

An Electrospun Scaffold Loaded with an Enteromorpha Polysaccharide for Accelerated Wound Healing in Diabetic Mice.

The design and development of innovative multifunctional wound dressing materials in engineered biomaterials is essential for promoting tissue repair. In this study, nanofibrous wound dressing materials loaded with anti-inflammatory ingredients were manufactured by a promising electrospinning strategy, and their capability for treating diabetic wounds was also investigated. A scaffold blend consisting of an Enteromorpha polysaccharide and polyvinyl alcohol (PVA) was fabricated. The in vitro and in vivo studies confirmed the efficacy of PVA/EPP1 fiber. We found that PVA/EPP1 fiber accelerated the repair of a full-thickness skin wound in diabetic mice. The results suggest that this scaffold could effectively shorten the wound healing time by inhibiting inflammatory activity, which makes it a promising candidate for the treatment of hard-to-heal wounds caused by diabetes.

Preparation, optimization and cellular uptake study of tanshinone I nanoemulsion modified with lactoferrin for brain drug delivery.

Tanshinone I (TSI) is one of the bioactive compound obtained from the root of Salvia miltiorrhiza which is a well-known traditional Chinese medicine (TCM) used for the treatment of various diseases. Although TSI possesses several pharmacological effects, it has poor water solubility, blood-brain barrier (BBB) permeability and brain bioavailability. Therefore, in the present study, we developed TSI nanoemulsion (TSI-NE) modified with a brain targeting ligand (Lactoferrin (Lf)) to improve the BBB permeability. Pseudo-ternary phase diagrams were used to optimize the formulation. The optimal TSI-NE and TSI-Lf-NE were prepared and characterized. Finally, the uptake of TSI-Lf-NE by mouse brain microvascular endothelial cell line (bEnd.3 cells) was assessed using Coumarin-6 as a fluorescent probe. The results of the study showed that the stable optimal formulation of O/W nanoemulsion was successfully developed and modified with Lf. The cellular uptake study has shown that the fluorescence intensity (FI) increased with time over the incubation period. The FI at all time intervals increased in the following order: Coumarin-6-Solution＜Coumarin-6-NE＜Coumarin-6-Lf-NE. The results suggest that the BBB permeability of Coumarin-6-Lf-NE was better than those of Coumarin-6-NE and Coumarin-6 solution. Lf modified nanoemulsion has great potential for improving the brain delivery of TSI.

Mixed polyethylene glycol-modified breviscapine-loaded solid lipid nanoparticles for improved brain bioavailability: preparation, characterization, and in vivo cerebral microdialysis evaluation in adult Sprague Dawley rats.

Breviscapine is used in the treatment of ischemic cerebrovascular diseases, but it has a low bioavailability in the brain due to its poor physicochemical properties and the activity of P-glycoprotein efflux pumps located at the blood-brain barrier. In the present study, breviscapine-loaded solid lipid nanoparticles (SLN) coated with polyethylene glycol (PEG) derivatives were formulated and evaluated for their ability to enhance brain bioavailability. The SLNs were either coated with polyethylene glycol (40) (PEG-40) stearate alone (Bre-GBSLN-PS) or a mixture of PEG-40 stearate and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE-PEG2000) (Bre-GBSLN-PS-DSPE) and were characterized both in vitro and in vivo. The mean particle size, polydispersity index, and entrapment efficiency for Bre-GBSLN-PS and Bre-GBSLN-PS-DSPE were 21.60 ± 0.10 and 22.60 ± 0.70 nm, 0.27 ± 0.01 and 0.26 ± 0.04, and 46.89 ± 0.73% and 47.62 ± 1.86%, respectively. The brain pharmacokinetic parameters revealed that the brain bioavailability of breviscapine from the Bre-GBSLN-PS and Bre-GBSLN-PS-DSPE was significantly enhanced (p < 0.01) with the area under concentration-time curve (AUC) of 1.59 ± 0.39 and 1.42 ± 0.58 µg h/mL of breviscapine, respectively, in comparison to 0.11 ± 0.02 µg h/mL from the commercial breviscapine injection. The ratios of the brain AUC for scutellarin in comparison with the plasma scutellarin AUC for commercial breviscapine injection, Bre-GBSLN-PS, and Bre-GBSLN-PS-DSPE were 0.66%, 2.82%, and 4.51%, respectively. These results showed that though both SLN formulations increased brain uptake of breviscapine, Bre-GBSLN-PS-DSPE which was coated with a binary combination of PEG-40 stearate and DSPE-PEG2000 had a better brain bioavailability than Bre-GBSLN-PS. Thus, the coating of SLNs with the appropriate PEG derivative combination could improve brain bioavailability of breviscapine and can be a promising tool for brain drug delivery.

[Experimental study of repairing femoral bone defects with nHA/RHLC/PLA scaffold composite with endothelial cells and osteoblasts in canines].

OBJECTIVE: To explore whether a tissue-engineered construct composed of autogenous endothelial cells, osteoblasts and a new bioresorbable nano-hydroxyapatite/recombinant human-like collagen/polylactic acid (nHA/RHLC/PLA) would enhance bone regeneration and repair femoral head defects in canine models. METHODS: The bone marrow stem cells (BMSCs) were isolated from bone marrow of canine ilium and cultured in Dulbecco's modified eagle medium:nutrient mixture F-12 culture media for 1 week and the second-generation BMSCs were further induced by osteogenic medium (1×10(-8) mol/L dexamethasone, 10 mmol/L B-sodium glycerophosphate and 50 µg/ml vitamin C) and by endothelial cell grow medium (vascular endothelial growth factor and basic fibroblast growth factor) for 14 days in vitro. Thus BMSCs were induced into ECs and OBs. After the second passage, cells were digested and collected.And cell density was adjusted to 1.0×10(6)/ml.The cells and nHA/RHLC/PLA scaffold were co-cultured for 2-4 hours then nHA/RHLC/PLA scaffold composites prepared. Cavity defects of 8 mm in diameter and 10 mm in height were made in femoral heads.The nHA/RHLC/PLA scaffold composited with ECs and osteoblasts (OBs) (group A) and composited with OBs (group B) were inserted into different defects while cell-free nHA/RHLC/PLA scaffold served as controls (group C). New bone formation and defect repair were evaluated at 3 and 6 months by radiographic examination, histology and bone histomorphometry. RESULTS: New bone formation was evident as early as 3 months in groups A, B and C.At 6 months, abundant bone tissue within defects was observed in group A. The control animals with cell-free scaffold showed less bone formation at both timepoints.The scaffold of nHA/RHLC/PLA was degraded and absorbed gradually with the formation of new bone tissues.Histology and bone histomorphometry further revealed significantly increased trabecular bones in group A compared with groups B and C at 6 months postimplantation (P < 0.01). CONCLUSION: More abundant new bone tissue may be found in the bone defect areas implanted with osteoblast-endotheliocyte composite than osteoblasts composite and scaffold materials only.ECs and osteoblasts derived from BMSC are ideal seed cells for repairing femoral head defects.

Nucleotide-Based Lanthanide Coordination Polymer Nano-Probe for Turn-On Fluorescence Sensing of Zn2+ in Serum.

BACKGROUND: Water-dispersed lanthanide coordination polymers (LCPs) have attracted considerable attention owing to their superiority in bioanalysis. However, so far, most of the reported LCPs, due to the employment of water-insoluble and toxic organic molecules as ligands, are only competent in organic solution or the gaseous phase. Therefore, the construction of a water-dispersed, LCP-based, especially LCP nanoparticle (LCPNP)-based, sensor is still lacking and challenging. OBJECTIVE: The aim was to obtain a novel and effective LCPNP-based sensor for Zn2+ by simple self-assembly, utilizing water-soluble guanosine monophosphate (GMP) as ligand and Eu3+ as luminescence center, . METHODS: In aqueous solutions, Eu-GMP NPs were formed via self-assembly reaction between Eu3+ and GMP, and displayed very weak fluorescence due to low energy transfer from GMP to Eu3+ and the rate constant of nonradiactive deactivation of the excited states caused by the O-H vibration of coordinated water molecules. After the introduce of Zn2+, forming Eu-GMP/Zn, very interestingly, an 8-fold fluorescence enhancement was observed due to the removal of coordination water molecules and fluorescence sensitization of Zn2+. RESULTS: The fluorescence intensity of Eu-GMP NPs at 614 nm showed a linear relationship with the concentration of Zn2+ from 4 to 240 µM with a detection limit of 4 µM. Due to possessing long fluorescence, Eu-GMP showed prominent achievment for application in serum Zn2+ determination. CONCLUSION: The LCPNP probe exhibited excellent performance for the determination of Zn2+ in serum. HIGHLIGHTS: For the first time, we developed and designed a kind of water-dispersed, LCPNP-based turn-on fluorescence assay for Zn2+ in serum. High sensitivity and good recoveries were achieved due to long fluorescence life, good water-dispersed behavior, and the turn-on fluorescence response of the LCPNP probe.

Biodegradable JDBM coating stent has potential to be used in the treatment of benign biliary strictures.

In our previous study, to find out the optimal alloy suitable for biliary surgery, magnesium alloy Jiao Da Bio-magnesium (denoted as JDBM) alloy, Zn-3Cu alloys, and their respective coating (MgF2-PDLLA) products were produced for our research. We found that JDBM seems to be a potential material for clinical biliary stent application due to its uniform degradation and good compatibility. In order to apply the JDBM alloy to treat benign bile duct stricture, our group prepared the bare JDBM and its coating product into finished stents by mesh weaving carving technology and conducted the mechanical property tests, degradation tests and biocompatibility tests. During the mechanical property tests, we found the bare JDBM stent was more suitable than titanium alloy stent when applies to the bile duct, and the coating of the JDBM coating stent has no effect on its mechanical properties. Our in vitro and in vivo experiments revealed that the degradation rate of the JDBM coating stent is lower than that of the JDBM stent, and both stents were biosafe. Thus, there is promise for JDBM coating stents for the treatment of benign biliary strictures.

A novel molecularly imprinted sensor based on PtCu bimetallic nanoparticle deposited on PSS functionalized graphene with peroxidase-like activity for selective determination of puerarin.

In this work, PtCu bimetallic nanoparticle was deposited on poly (styrene sulfonate) (PSS) functionalized graphene (Gr) to form a nanocomposite PtCu/PSS-Gr and its enzyme-like activity was investigated. Benefiting from the synergistic effect from Pt and Cu monometal as well as the superior properties of PSS-Gr, such as large surface area, good dispersity, strong adsorption of substrate and additional peroxidase-like activity, the PtCu/PSS-Gr nanocomposite was demonstrated as an excellent peroxidase mimic to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Combined with molecularly imprinted polymer (MIP), a new colorimetric approach for puerarin detection was proposed with the linear range of 2 × 10-5-6 × 10-4 mol L-1 and LOD of 1 × 10-5 mol L-1. The combination of MIP with PtCu/PSS-Gr nanocomposite not only endowed the determination of puerarin with high selectivity, but also realized the detection of small molecules which are neither substrate of the nanozyme nor substances with strong oxidizing or reducing activity by using peroxidase-like catalytic activity of nanozyme, expanding the application of nanozyme.

[Impact of different traffic crossroads exhaust pollution on immune function of the school-age children in Taiyuan City].

OBJECTIVE: to discuss the impact of different traffic crossroad exhaust pollution on the immune function of school-age children. METHODS: A school in the heavy traffic polluted area and B school in the less traffic polluted area were selected. Total 142 students from five to six grades in both areas were sampled. The level of peripheral blood cells (WBC, lymphocytes, neutral cells, red blood cells, platelets) and the T lymphocyte subpopulations (the percentages of CD3+, CD4+, CD8+ and the ratio of CD4+/CD8+), saliva lysozyme contents and immunoglobulin content were determined. RESULTS: Except for neutral cells, the peripheral blood cells level (WBC, lymphocytes, red blood cells, platelets) of children in the A school were higher than those in the B school. The contents of saliva lysozyme, immunoglobulin (IgG), T lymphocyte subpopulations (the percentages of CD3+, CD4+ and the ratio of CD4+/CD8+) in the A school were lower than those in the B school (P < 0.05). CONCLUSION: Exhaust pollution of different traffic crossroads could inhibit nonspecific immunity and cell immunity, and impact the level of blood cells and humoral immunity.

Drug-eluting fully covered self-expanding metal stent for dissolution of bile duct stones in vitro.

BACKGROUND: The treatment of difficult common bile duct stones (CBDS) remains a big challenge around the world. Biliary stenting is a widely accepted rescue method in patients with failed stone extraction under endoscopic retrograde cholangiopancreatography. Fully covered self-expanding metal stent (FCSEMS) has gained increasing attention in the management of difficult CBDS. AIM: To manufacture a drug-eluting FCSEMS, which can achieve controlled release of stone-dissolving agents and speed up the dissolution of CBDS. METHODS: Customized covered nitinol stents were adopted. Sodium cholate (SC) and disodium ethylene diamine tetraacetic acid (EDTA disodium, EDTA for short) were used as stone-dissolving agents. Three different types of drug-eluting stents were manufactured by dip coating (Stent I), coaxial electrospinning (Stent II), and dip coating combined with electrospinning (Stent III), respectively. The drug-release behavior and stone-dissolving efficacy of these stents were evaluated in vitro to sort out the best manufacturing method. And the selected stone-dissolving stents were further put into porcine CBD to evaluate their biosecurity. RESULTS: Stent I and Stent II had obvious burst release of drugs in the first 5 d while Stent III presented controlled and sustainable drug release for 30 d. In still buffer, the final stone mass-loss rate of each group was 5.19% ± 0.69% for naked FCSEMS, 20.37% ± 2.13% for Stent I, 24.57% ± 1.45% for Stent II, and 33.72% ± 0.67% for Stent III. In flowing bile, the final stone mass-loss rate of each group was 5.87% ± 0.25% for naked FCSEMS, 6.36% ± 0.48% for Stent I, 6.38% ± 0.37% for Stent II, and 8.15% ± 0.27% for Stent III. Stent III caused the most stone mass-loss no matter in still buffer or in flowing bile, which was significantly higher than those of other groups (P < 0.05). In vivo, Stent III made no difference from naked FCSEMS in serological analysis (P > 0.05) and histopathological examination (P > 0.05). CONCLUSION: The novel SC and EDTA-eluting FCSEMS is efficient in diminishing CBDS in vitro. When conventional endoscopic techniques fail to remove difficult CBDS, SC and EDTA-eluting FCSEMS implantation may be considered a promising alternative.

Facile construction of luminescent supramolecular assemblies with aggregation-induced emission feature through supramolecular polymerization and their biological imaging.

Supramolecular polymerization is a novel method for the fabrication of multifunctional polymeric composites that mainly relied on the non-covalent interactions between different components. In this work, a novel and facile strategy has been developed for the construction of fluorescent organic nanoparticles (FONs) with aggregation-induced emission (AIE) characteristic based on the host-guest interaction between ß cyclodextrin terminating polyethylene glycol (ß-CD-PEG) and adamantine (Ad) containing AIE dye (Ph-Ad). Through the host-guest interaction, the fluorescent amphiphiles can be facilely obtained. The characterization results suggested we have successfully prepared the AIE-active FONs through the supramolecular polymerization. The Ph-Ad/ß-CD-PEG FONs possess many advantages such small size, high water dispersibility, desirable fluorescence properties, low cytotoxicity and efficient cell dyeing performance. All of the above results implied that these AIE-active supramolecular assemblies should be promising luminescent probes with great potential for different biomedical applications.

Novel Silicone-Coated 125I Seeds for the Treatment of Extrahepatic Cholangiocarcinoma.

125I seeds coated with titanium are considered a safe and effective interstitial brachytherapy for tumors, while the cost of 125I seeds is a major problem for the patients implanting lots of seeds. The aim of this paper was to develop a novel silicone coating for 125I seeds with a lower cost. In order to show the radionuclide utilization ratio, the silicone was coated onto the seeds using the electro-spinning method and the radioactivity was evaluated, then the anti-tumor efficacy of silicone 125I seeds was compared with titanium 125I seeds. The seeds were divided into four groups: A (control), B (pure silicone), C (silicone 125I), D (titanium 125I) at 2 Gy or 4 Gy. Their anti-tumour activity and mechanism were assessed in vitro and in vivo using a human extrahepatic cholangiocarcinoma cell line FRH-0201 and tumor-bearing BALB/c nude mice. The silicone 125I seeds showed higher radioactivity; the rate of cell apoptosis in vitro and the histopathology in vivo demonstrated that the silicone 125I seeds shared similar anti-tumor efficacy with the titanium 125I seeds for the treatment of extrahepatic cholangiocarcinoma, while they have a much lower cost.

In vitro comparison of antimicrobial effectiveness of QMix and other final irrigants in human root canals.

Final root canal irrigation stands as an effective strategy for eliminating the dentin infection. This study aimed to investigate and compare the antibacterial efficacy of QMix and other four final irrigation regimens in reducing Enterococcus faecalis within human root canals. Single-canal human teeth contaminated with E. faecalis for 4 weeks were prepared chemomechanically with sodium hypochlorite (NaOCl). Then, the teeth were randomly assigned into six groups according to the final irrigation protocols: (1) EDTA/NaOCl, 17% EDTA followed by 5.25% NaOCl; (2) EDTA/chlorhexidine (CHX), 17% EDTA followed by 2% CHX; (3) EDTA/cetrimide (CTR), 17% EDTA followed by 2% CTR; (4) MTAD; (5) QMix; and (6) control, 0.9% saline. Bacterial samples collected before instrumentation and after final irrigation were cultured and the colony-forming units (CFUs) were counted. The CFUs in the QMix, EDTA/CHX, and EDTA/CTR groups were significantly lower than those in the EDTA/NaOCl group. No significant differences were observed between the QMix, EDTA/CHX, and EDTA/CTR groups. MTAD showed weaker ability than QMix and EDTA/CHX to eliminate E. faecalis, but it caused a greater reduction in CFU than EDTA/NaOCl. Hence, the antimicrobial activity of QMix was comparable to that of EDTA/CHX and EDTA/CTR and more effective than that of EDTA/NaOCl against intracanal E. faecalis.

Effect of Baicalin-loaded PEGylated cationic solid lipid nanoparticles modified by OX26 antibody on regulating the levels of baicalin and amino acids during cerebral ischemia-reperfusion in rats.

Baicalin has many pharmacological activities, including neuroprotective function against ischemia and neurodegeneration. In our previous study, we found that Baicalin-loaded PEGylated cationic solid lipid nanoparticles modified by OX26 antibody (OX26-PEG-CSLN) might be a promising carrier to deliver drugs across the blood-brain barrier for the treatment of brain diseases. So, the aim of this present study was to further elucidate the mechanisms of OX26-PEG-CSLN cerebral ischemia protection by monitoring the changes of extracellular amino acids. In addition, we investigated the effect of OX26-PEG-CSLN on the excitotoxic neuronal injury as well as the pharmacokinetic profiles of baicalin in cerebrospinal fluid during ischemia-reperfusion period. The cerebrospinal fluid was collected by a microdialysis technique and divided into two parts - one part for pharmacokinetic study of baicalin using LC-MS/MS method and the other for pharmacodynamic study which was done by pre- column derivatization of the amino acids and analysis using a high-performance liquid chromatography with fluorescence detector (HPLC-FLD). The pharmacokinetic study showed that the AUC value of OX26-PEG-CSLN was 5.69-fold higher than that of the baicalin solution (Sol) (P<0.05) and the Cmax value of OX26-PEG-CSLN was 6.84-fold higher than that of the Sol (P<0.05). Moreover, the extracellular levels of glutamate (Glu), aspartic acid (Asp), glycine (Gly), taurine (Tau) and γ-aminobutyric acid (GABA) were measured for monitoring the imbalance of amino acids caused by ischemia and reperfusion. The excitotoxic index (EI) was also calculated for evaluating the imbalance between excitatory amino acid and inhibitory amino acid. The pharmacodynamic study showed that OX26-PEG-CSLN had stronger effect than Sol in reducing the content of aspartic, glutamic acid and increasing the concentrations of glycine, taurine and γ-aminobutyric acid during ischemia-reperfusion period. In conclusion, OX26-PEG-CSLN improved uptake of baicalin across the BBB into the brain, and elevated bioavailability of baicalin in cerebral spinal fluid of rats under the cerebral ischemia-reperfusion injury. OX26-PEG-CSLN had much higher protection effect against cerebral ischemia injury than Sol by relieving the excitotoxic neuronal injury via regulating amino acid levels in cerebral spinal fluid.

A novel magnetic nanoparticle drug carrier for enhanced cancer chemotherapy.

BACKGROUND: Magnetic nanoparticles (NPs) loaded with antitumor drugs in combination with an external magnetic field (EMF)-guided delivery can improve the efficacy of treatment and may decrease serious side effects. The purpose of this study was 1) to investigate application of PEG modified GMNPs (PGMNPs) as a drug carrier of the chemotherapy compound doxorubicin (DOX) in vitro; 2) to evaluate the therapeutic efficiency of DOX-conjugated PGMNPs (DOX-PGMNPs) using an EMF-guided delivery in vivo. METHODS: First, DOX-PGMNPs were synthesized and the cytotoxicity of DOX-PGMNPs was assessed in vitro. Second, upon intravenous administration of DOX-PMGPNs to H22 hepatoma cell tumor-bearing mice, the DOX biodistribution in different organs (tissues) was measured. The antitumor activity was evaluated using different treatment strategies such as DOX-PMGPNs or DOX-PMGPNs with an EMF-guided delivery (DOX-PGMNPs-M). RESULTS: The relative tumor volumes in DOX-PGMNPs-M, DOX-PGMNPs, and DOX groups were 5.46±1.48, 9.22±1.51, and 14.8±1.64, respectively (each p<0.05), following treatment for 33 days. The life span of tumor-bearing mice treated with DOX-PGMNPs-M, DOX-PGMNPs, and DOX were 74.8±9.95, 66.1±13.5, and 31.3±3.31 days, respectively (each p<0.05). CONCLUSION: This simple and adaptive nanoparticle design may accommodate chemotherapy for drug delivery optimization and in vivo drug-target definition in system biology profiling, increasing the margin of safety in treatment of cancers in the near future.

PEG-modified GoldMag nanoparticles (PGMNs) combined with the magnetic field for local drug delivery.

Polyethylene glycol-modified GoldMag nanoparticles (PGMNs) were synthesized and characterized by several analysis including transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy, and vibrating sample magnetometer. Here, we showed that the composite nanoparticles have a saturated magnetization of 34 emu/g with an average diameter of 50 nm. The kinetics analysis showed the maximum doxorubicin (DOX) loading capacity of PGMNs is 107.78 mg/g. The PGMNs loaded with DOX (DOX-loaded PGMNs) exhibited a controlled drug release pattern within the first 4 h and then a slowly, sustained release pattern for 4 days. The biodistribution of DOX-loaded PGMNs in vivo indicated that the concentrations of DOX in liver exposed to magnetic field group (60.7 ± 8.14, 53.6 ± 4.89, 44.8 ± 6.41, and 38.4 ± 2.58 ng/g) were significantly higher than those of no applied magnetic field group (40.8 ± 9.96, 31.9 ± 7.01, 28 ± 6.11, and 20.7 ± 5.78 ng/g) at 0.5, 1, 2, and 4 h (P < 0.05), respectively. The amount of Fe in liver exposed magnetic field showed a similar trend. Histological studies revealed an enhanced particle aggregation in the targeted liver area. Under the external magnetic field, the biodistribution of DOX-loaded PGMNs was changed compared to absence of magnet field, and the PGMNs were capable of carrying drug for targeting therapeutic purposes.

Effect of membrane mimicking environment on the conformation of a pore-forming (xSxG)6 peptide.

The mechanism of membrane interaction by beta-sheet peptides is important to understand fundamental principles of folding of beta-barrel proteins and various beta-amyloid proteins. Here, we examined the conformational characteristics of a porin-like channel forming (xSxG)(6) peptide in solution and membrane-mimicking environments (CD and ATR-IR) to understand the structural changes of the peptide during membrane association and channel formation. A comparison of the peptide conformations in different microenvironments showed that beta-sheet formation is enhanced in membrane-mimicking liposomes and SDS-micelles. The lipid-induced beta-sheet formation was confirmed by the formation of a characteristic beta-sheet structure on mixing a methanolic solution of the peptide (partially folded) with preformed liposomes. The amphipathicity of the peptide; increased hydrogen bonding, hydrophilicity, and reduction in dimensionality of the membrane surface; membrane-peptide interaction-forces; and presence of flexible glycines might facilitate beta-sheet formation in membranes. Though the CD spectra of both the peptide-bound and peptide-incorporated lipids are reminiscent of a beta-sheet structure, a significant variation in the peak positions of the two beta-sheet structures was noticed. The channel characteristics of (xSxG)(6) in the presence of low ionic strength solutions of NEt(3)BzCl and glucosammonium chloride are comparable to those reported under high ionic strength solutions. Altogether the data suggest that the channel formation by (xSxG)(6) proceeds via beta-sheet aggregate formation at the membrane surface, beta-sheet insertion, and rearrangement into a beta-barrel-like structure. The beta-barrel-like channel formation most likely arises from a sequence similarity to beta-barrel porins whereas the lipid-induced beta-sheet formation is governed by the above-mentioned factors.

Aggregation and porin-like channel activity of a beta sheet peptide.

Beta sheet peptides (e.g., amyloid beta) are known to form ion channels in lipid bilayers possibly through aggregation, though the channel structure is not clear. We have recently reported that a short beta sheet peptide, (xSxG)(6), forms porin-like voltage-gated channels in lipid bilayers [Thundimadathil et al. (2005) Biochem. Biophys. Res. Commun. 330, 585-590]. To account for the porin-like activity, oligomerization of the peptide into a beta barrel-like structure was proposed. In this work, peptide aggregation in aqueous and membrane environments and a detailed study of channel properties were performed to gain insight into the mechanism of channel formation. The complex nature of the channel was revealed by kinetic analysis and the occurrence of interconverting multiple conductance states. Ion channels were inhibited by Congo red, suggesting that the peptide aggregates are the active channel species. Peptide aggregation and fibril formation in water were confirmed by electron microscopy (EM) and Congo red binding studies. Furthermore, oligomeric structures in association with lipid bilayers were detected. Circular dichroism of peptide-incorporated liposomes and peptide-lipid binding studies using EM suggest a lipid-induced beta sheet aggregation. Gel electrophoresis of peptide-incorporated liposomes showed dimeric and multimeric structures. Taken together, this work indicates insertion of (xSxG)(6) as oligomers into the lipid bilayer, followed by rearrangement into a beta barrel-like pore structure. A large peptide pore comprising several individual beta sheets or smaller beta sheet aggregates is expected to have a complex behavior in membranes. A dyad repeat sequence and the presence of glycine, serine, and hydrophobic residues in a repeated pattern in this peptide may be providing a favorable condition for the formation of a beta barrel-like structure in lipid bilayers.

Pteroyl-gamma-glutamate-cysteine synthesis and its application in folate receptor-mediated cancer cell targeting using folate-tethered liposomes.

Cell membrane-associated folate receptors are selectively overexpressed in certain human tumors. The high affinity of folic acid for folate receptors provides a unique opportunity to use folic acid as a targeting ligand to deliver chemotherapeutic agents to cancer cells. Folate-tethered liposomes bearing pteroyl-gamma-glutamate-cysteine-polyethylene glycol (PEG)-distearoylphosphatidylethanolamine (DSPE) as the targeting component are under investigation as mediators of drug and gene delivery to cancer cells that overexpress folate receptors. Pteroyl-gamma-glutamate-cysteine synthesis is one of the crucial starting steps in the preparation of pteroyl-gamma-glutamate-cysteine-PEG-DSPE. However, published methods for the synthesis of pteroyl-gamma-glutamate-cysteine provide low yields and are not easily reproducible. Therefore, we developed a modified synthetic method for the removal of the N(10)-trifluoroacetyl group after cleavage/deprotection that is reliable, is easily reproducible, and has high yield (38%) compared with an unreliable yield of 3-20% with the earlier methods. Folate-tethered liposomes containing calcein or doxorubicin were prepared using pteroyl-gamma-glutamate-cysteine-PEG-DSPE as the targeting component along with nontargeted liposomes with PEG-DSPE. The results of the uptake of calcein and cytotoxicity of doxorubicin in human cervical cancer HeLa-IU(1) cells and human colon cancer Caco-2 cells demonstrated that folate-tethered liposomes were efficient in selective delivery to cancer cells overexpressing folate receptors. The improvement in yield of the targeting component can significantly facilitate "scale up" of folate receptor-mediated liposomal cancer therapy to the preclinical and clinical levels of investigations.