A study on the aflatoxin M1 rate and seasonal variation in pasteurized cow milk from northwestern Iran.

Present study aims to assess aflatoxin M1 (AFM1) contamination in 100 samples of pasteurized milk which were conventionally gathered during spring, summer, autumn, and winter from supermarkets located in Maragheh city of northwestern Iran. Samples were evaluated for AFM1 with a high-performance liquid chromatography (HPLC) method and with fluorimetric detection. The results showed that approximately 44% (11.25) of samples in winter, 32% (8.25) of samples in spring, 24% (6.25) of samples in summer, and 20% (5.25) of samples in autumn had AFM1 concentrations that exceeded the limit (0.05 µg/l) set by the European, Codex Alimentarius Commission and Iran standards. According to the statistical analysis of the data, there was no significant variation between the mean content of AFM1 during different seasons (P = 0.076). The results of our study suggest a high level of contamination of AFM1 in pasteurized milk in all seasons which may be due to the fact that milk supply for dairy factories is provided from dairy farms that are low in livestock feed quality. In Iran, pasteurized milk is consumed more than other milk products by all age groups. The total daily aflatoxin intake from contaminated milk and possibly other food products will be a significant risk to public health.

Reducible poly(amido ethylenimine)-based gene delivery system for improved nucleus trafficking of plasmid DNA.

In a nonviral gene delivery system, localization of a plasmid DNA in the nucleus is a prerequisite for expression of a desired therapeutic protein encoded in the plasmid DNA. In this study, a reducible polymer-based gene delivery system for improved intracellular trafficking and nuclear translocation of plasmid DNA is introduced. The system is consisted of two components, a plasmid DNA having repeated binding sequence for a karyophilic protein, NFkappaB, and a reducible polymer. A reducible poly(amido ethylenimine), poly(TETA-CBA), was synthesized by a Michael-type addition polymerization between cystamine bisacrylamide and triethyl tetramine. The polymer forming tight complexes with plasmid DNA could be degraded in the reductive cytosol to release the plasmid DNA. The triggered release mechanism in the cytosol could facilitate the interaction between cytosolic NFkappaB and the plasmid DNA having repeated NFkappaB biding motif. Upon activation of NFkappaB by interleukin-1beta (IL-1beta), most of the plasmid distributed in the cytoplasm was localized within the nucleus, resulting in significantly higher gene transfection efficiency than controls with nondegradable PEI. The current study suggests an alternative way of improving transfection efficiency by taking advantage of endogenous transport machinery for intracellular trafficking and nuclear translocation of a plasmid DNA.

Reducible poly(amido ethylenimine) directed to enhance RNA interference.

Designing synthetic macromolecular vehicles with high transfection efficiency and low cytotoxicity has been a major interest in the development of non-viral gene carriers. A reducible poly(amido ethylenimine) (SS-PAEI) synthesized by addition copolymerization of triethylenetetramine and cystamine bis-acrylamide (poly(TETA/CBA)) was used as a carrier for small interference RNA (siRNA). Poly(TETA/CBA) could efficiently condense siRNA to form stable complexes under physiological conditions and perform complete release of siRNA in a reductive environment. When formulated with VEGF-directed siRNA, poly(TETA/CBA) demonstrated significantly higher suppression of VEGF than linear-polyethylenimine (PEI) (L-PEI, 25kDa) in human prostate cancer cells (PC-3). After 5h of transfection, substantial dissociation and intracellular distribution of siRNA was observed in the poly(TETA/CBA) formulation, but not in the L-PEI formulation. The triggered release of siRNA by reductive degradation of poly(TETA/CBA) in the cytoplasm may affect the RNAi activity by increasing cytoplasmic availability of siRNA. These results suggest that the rational design of non-viral carriers should involve considerations for intracellular dissociation and trafficking of a nucleic acid drug to maximize its effect, in conjunction with formation of stable complexes under physiological conditions.

Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle.

Naked plasmid DNA (pDNA)-based gene therapy has low delivery efficiency, and consequently, low therapeutic effect. We present a biodegradable nonionic triblock copolymer, PEG(13)-PLGA(10)-PEG(13), to enhance gene delivery efficiency in skeletal muscle. Effects of PEG(13)-PLGA(10)-PEG(13) on physicochemical properties of pDNA were evaluated by atomic force microscopy (AFM) imaging, gel electrophoresis and zeta-potential analysis. AFM imaging suggested a slightly compacted structure of pDNA when it was mixed with the polymer, while zeta-potential measurement indicated an increased surface potential of negatively charged pDNA. PEG(13)-PLGA(10)-PEG(13) showed a relatively lower toxicity compared to Pluronic P85 in a skeletal muscle cell line. The luciferase expression of pDNA delivered in 0.25% polymer solution was up to three orders of magnitude more than branched polyethylenimine (bPEI(25 k))/pDNA and three times more than that of naked pDNA five days after intramuscular administration. This in vivo gene delivery enhancement was also observed displaying a two-fold higher expression of human vascular endothelial growth factor (VEGF). Based on fluorescence labeled pDNA distribution, it is speculated that the greater diffusivity of PEG(13)-PLGA(10)-PEG(13)/pDNA compared to bPEI(25 k)/pDNA accounts for better transfection efficiency in vivo. To summarize, combining PEG(13)-PLGA(10)-PEG(13) with pDNA possesses the potential to improve gene delivery efficiency in skeletal muscle.

Reducible poly(amido ethylenediamine) for hypoxia-inducible VEGF delivery.

Delivery of the hypoxia-inducible vascular endothelial growth factor (RTP-VEGF) plasmid using a novel reducible disulfide poly(amido ethylenediamine) (SS-PAED) polymer carrier was studied in vitro and in vivo. In vitro transfection of primary rat cardiomyoblasts (H9C2) showed SS-PAED at a weighted ratio of 12:1 (polymer/DNA) mediates 16 fold higher expression of luciferase compared to an optimized bPEI control. FACS analysis revealed up to 57+/-2% GFP positive H9C2s. The efficiency of plasmid delivery to H9C2 using SS-PAED was found to depend upon glutathione (GSH) levels inside the cell. SS-PAED mediated delivery of RTP-VEGF plasmid produced significantly higher levels of VEGF expression (up to 76 fold) under hypoxic conditions compared to normoxic conditions in both H9C2 and rat aortic smooth muscle cells (A7R5). Using SS-PAED, delivery of RTP-VEGF was investigated in a rabbit myocardial infarct model using 100 mug RTP-VEGF. Results showed up to 4 fold increase in VEGF protein expression in the region of the infarct compared to injections of SS-PAED/RTP-Luc. In conclusion, SS-PAED mediated therapeutic delivery improves the efficacy of ischemia-inducible VEGF gene therapy both in vitro and in vivo and therefore, has potential for the promotion of neo-vascular formation and improvement of tissue function in ischemic myocardium.

Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-g-PEG-RGD/pCMV-sFlt-1 complexes in tumor-bearing mice.

Vascular endothelial growth factor (VEGF) is an endogenous mediator of tumor angiogenesis. Blocking associations of the VEGF with its corresponding receptors (Flt-1, KDR/flk-1) have become critical for anti-tumor angiogenesis therapy. Previously, we synthesized PEI-g-PEG-RGD conjugate and evaluated as an angiogenic endothelial polymeric gene carrier. In this study, PEI-g-PEG-RGD/pCMV-sFlt-1 complexes are evaluated in terms of tumor growth inhibition in vivo. Complexes were repeatedly injected systemically via tail vein into subcutaneous tumor-bearing mice. As a result, tumor growth was inhibited in the PEI-g-PEG-RGD/pCMV-sFlt-1 injected group. However, this effect was not identified in PEI-g-PEG/pCMV-sFlt-1 or PEI-g-PEG-RGD/pCMV-GFP control groups. Moreover, the survival rate increased in the PEI-g-PEG-RGD/pCMV-sFlt-1 group compared with the controls group. These results suggest that delivery of pCMV-sFlt-1 using PEG-g-PEG-RGD may be effective for anti-angiogenic gene therapy.

Efficient expression of vascular endothelial growth factor using minicircle DNA for angiogenic gene therapy.

The application of plasmid DNA (pDNA)-based gene therapy is limited by its inefficient transgene expression. In this study, minicircle DNA was evaluated for efficient vascular endothelial growth factor (VEGF) expression in skeletal muscle cells. Production of minicircle DNA encoding VEGF was studied by a semi-quantitative electrophoresis method leading to optimized bacterial culture conditions and producing high purity (86.6%) minicircle DNA. The VEGF minicircle DNA under control of the SV40 promoter (pMini-SV-VEGF) showed an increased amount of VEGF mRNA and up to 8 times more VEGF expression than a conventional plasmid (pSV-VEGF) in two different skeletal muscle cell lines (C2C12 and L8). Minicircle DNA with different promoters, including the SV40, CMV and chicken beta-actin, was tested for VEGF expression in a C2C12 skeletal muscle cell line. The high VEGF expression generated by minicircle DNA stimulated efficient endothelial cell growth in vitro. Furthermore, minicircle DNA expressed higher VEGF compared to conventional plasmid in the tibialis anterior (TA) muscle of mice. Taken together, the results suggest that minicircle DNA is an efficacious gene vector for angiogenic VEGF expression in skeletal muscle and may be useful for treating peripheral arterial disease (PAD).

Pharmacokinetics and pharmacodynamics of propofol microemulsion and lipid emulsion after an intravenous bolus and variable rate infusion.

BACKGROUND: The aim of this trial was to evaluate the induction and recovery characteristics of microemulsion propofol (Aquafol; Daewon Pharmaceutical Co., Ltd., Seoul, Korea). Pharmacokinetics, pharmacodynamics, and safety profile were investigated. Lipid emulsion propofol (Diprivan; AstraZeneca, London, United Kingdom) was used as a comparator. METHODS: Thirty-one healthy volunteers aged 20-79 yr were given an intravenous bolus of propofol 2 mg/kg, followed by variable rate infusion for 60 min. Each volunteer was studied twice with different formulations at an interval of 1 week. Arterial concentrations of propofol were measured, and Bispectral Index was used as a surrogate measure of propofol effect. The induction and recovery characteristics including bioequivalence were evaluated by noncompartmental analysis. The pharmacokinetics and pharmacodynamics were investigated using a population approach with mixed effects modeling. The rate, severity, and causal relation of adverse events were analyzed. RESULTS: Both formulations were bioequivalent. The observed time to peak effect after a bolus of both formulations was 1.5 min. Plasma concentration of propofol at loss of consciousness, time to loss of consciousness after a bolus, and time to recovery of consciousness after discontinuation of infusion did not show significant differences. The population pharmacokinetics and pharmacodynamics revealed a variety of differences between two formulations. Aquafol showed similar safety profile to Diprivan. CONCLUSIONS: The efficacy and safety of Aquafol were not different from those of Diprivan within the dose range in this study.

Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma.

Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic growth factor that is a primary stimulant of the development and maintenance of a vascular network in the vascularization of solid tumors. It has been reported that a blockade of VEGF-mediated angiogenesis is a powerful method for tumor regression. RNA interference represents a naturally occurring biological strategy for inhibition of gene expression. In mammalian systems, however, the in vivo application of small interfering RNA (siRNA) is severely limited by the instability and poor bioavailability of unmodified siRNA molecules. In this study, we tested the hypothesis that a hydrophobically modified protein transduction domain, cholesteryl oligo-d-arginine (Chol-R9), may stabilize and enhance tumor regression efficacy of the VEGF-targeting siRNA. The noncovalent complexation of a synthetic siRNA with Chol-R9 efficiently delivered siRNA into cells in vitro. Moreover, in a mouse model bearing a subcutaneous tumor, the local administration of complexed VEGF-targeting siRNA, but not of scrambled siRNA, led to the regression of the tumor. Hence, we propose a novel and simple system for the local in vivo application of siRNA through Chol-R9 for cancer therapy.

Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery.

Poly(amido ethylenimine) polymers, a new type of peptidomimetic polymer, containing multiple disulfide bonds (SS-PAEIs) designed to degrade after delivery of plasmid DNA (pDNA) into the cell were synthesized and investigated as new carriers for triggered intracellular gene delivery. More specifically, three SS-PAEIs were synthesized from Michael addition reactions between cystamine bisacrylamide (CBA) and three different ethylene amine monomers, i.e., ethylenediamine (EDA), diethylenetriamine (DETA), or triethylenetetramine (TETA). Complete addition reactions were confirmed by (1)H NMR. The molecular weight, buffer capacity, and relative degree of branching for each SS-PAEI was determined by gel permeation chromatography (GPC), acid-base titration, and liquid chromatography-mass spectroscopy (LC-MS), respectively. Physicochemical characteristics of polymer/pDNA complexes (polyplexes) were analyzed by gel electrophoresis, particle size, and zeta-potential measurements. All three SS-PAEIs effectively complex pDNA to form nanoparticles with diameters less than 200 nm and positive surface charges of approximately 32 mV. The in vitro gene transfer properties of SS-PAEIs were evaluated using mouse embryonic fibroblast cell (NIH3T3), primary bovine aortic endothelial cell (BAEC), and rat aortic smooth muscle cell (A7R5) lines. Interestingly, polyplexes based on all three SS-PAEIs exhibited remarkably high levels of reporter gene expression with nearly 20x higher transfection efficiency than polyethylenimine 25k. The high transfection efficiency was maintained in the presence of 10% serum in the transfection medium. Furthermore, confocal microscopy experiments using labeled pDNA indicated that polyplexes of SS-PAEI displayed greater intracellular distribution of pDNA as compared to PEI, most likely due to environmentally triggered release. Therefore, SS-PAEIs are a new class of transfection agents that facilitate high gene expression while maintaining a low level of toxicity.

Low molecular weight linear polyethylenimine-b-poly(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties.

Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.