Characterization of cannabinoid plasma concentration, maternal health, and cytokine levels in a rat model of prenatal Cannabis smoke exposure.

Cannabis sativa has gained popularity as a "natural substance", leading many to falsely assume that it is not harmful. This assumption has been documented amongst pregnant mothers, many of whom consider Cannabis use during pregnancy as benign. The purpose of this study was to validate a Cannabis smoke exposure model in pregnant rats by determining the plasma levels of cannabinoids and associated metabolites in the dams after exposure to either Cannabis smoke or injected cannabinoids. Maternal and fetal cytokine and chemokine profiles were also assessed after exposure. Pregnant Sprague-Dawley rats were treated daily from gestational day 6-20 with either room air, i.p. vehicle, inhaled high-Δ9-tetrahydrocannabinol (THC) (18% THC, 0.1% cannabidiol [CBD]) smoke, inhaled high-CBD (0.7% THC, 13% CBD) smoke, 3 mg/kg i.p. THC, or 10 mg/kg i.p. CBD. Our data reveal that THC and CBD, but not their metabolites, accumulate in maternal plasma after repeated exposures. Injection of THC or CBD was associated with fewer offspring and increased uterine reabsorption events. For cytokines and chemokines, injection of THC or CBD up-regulated several pro-inflammatory cytokines compared to control or high-THC smoke or high-CBD smoke in placental and fetal brain tissue, whereas smoke exposure was generally associated with reduced cytokine and chemokine concentrations in placental and fetal brain tissue compared to controls. These results support existing, but limited, knowledge on how different routes of administration contribute to inconsistent manifestations of cannabinoid-mediated effects on pregnancy. Smoked Cannabis is still the most common means of human consumption, and more preclinical investigation is needed to determine the effects of smoke inhalation on developmental and behavioural trajectories.

Design of Smart Nanodiamonds: Introducing pH Sensitivity to Improve Nucleic Acid Carrier Efficiency of Diamoplexes.

The mechanism of cellular uptake and intracellular fate of nanodiamond/nucleic acid complexes (diamoplexes) are major determinants of its performance as a gene carrier. Our group designed lysine-nanodiamonds (K-NDs) as vectors for nucleic acid delivery. In this work, we modified the surface of K-NDs with histidine to overcome endo-lysosomal entrapment diamoplexes, the major rate limiting step in gene transfer. Histidine is conjugated onto the NDs in two configurations: lysyl-histidine-NDs (HK-NDs) where histidine is loaded on 100% of the lysine moieties and lysine/lysyl-histidine-NDs (H50K50-NDs) where histidine is loaded on 50% of the lysine moieties. Both HK-NDs and H50K50-NDs maintained the optimum size distribution (i.e., <200 nm) and a cationic surface (zeta potential > 20 mV), similar to K-NDs. HK-NDs binds plasmid deoxyribonucleic acid (pDNA) and small interfering ribonucleic acid (siRNA) forming diamoplexes at mass ratios of 10:1 and 60:1, respectively. H50K50-NDs significantly improved nucleic acid binding, forming diamoplexes at a 2:1 mass ratio with pDNA and a 30:1 mass ratio with siRNA, which are at values similar to the K-NDs. The amount of histidine on the surface also impacted the interactions with mammalian cells. The HK-NDs reduced the cell viability by 30% at therapeutic concentrations, while H50K50-NDs maintained more than 90% cell viability, even at the highest concentrations. H50K50-NDs also showed highest cellular uptake within 24 h, followed by K-NDs and HK-NDs. Most functionalized NDs show cellular exit after 5 days, leaving less than 10% of cells with internalized diamonds. The addition of histidine to the ND resulted in higher transfection of anti-green fluorescent protein siRNA (anti-GFP siRNA) with the fraction of GFP knockdown being 0.8 vs. 0.6 for K-NDs at a mass ratio of 50:1. H50K50-NDs further improved transfection by achieving a similar fraction of GFP knockdown (0.8) at a lower mass ratio of 30:1. Overall, this study provides evidence that the addition of histidine, a pH-modulating entity in the functionalization design at an optimized ratio, renders high efficiency to the diamoplexes. Further studies will elucidate the uptake mechanism and intracellular fate to build the relationship between physicochemical characteristics and biological efficacy and create a platform for solid-core nanoparticle-based gene delivery.

Megakaryocytes form linear podosomes devoid of digestive properties to remodel medullar matrix.

Bone marrow megakaryocytes (MKs) undergo a maturation involving contacts with the microenvironment before extending proplatelets through sinusoids to deliver platelets in the bloodstream. We demonstrated that MKs assemble linear F-actin-enriched podosomes on collagen I fibers. Microscopy analysis evidenced an inverse correlation between the number of dot-like versus linear podosomes over time. Confocal videomicroscopy confirmed that they derived from each-other. This dynamics was dependent on myosin IIA. Importantly, MKs progenitors expressed the Tks4/5 adaptors, displayed a strong gelatinolytic ability and did not form linear podosomes. While maturing, MKs lost Tks expression together with digestive ability. However, those MKs were still able to remodel the matrix by exerting traction on collagen I fibers through a collaboration between GPVI, ß1 integrin and linear podosomes. Our data demonstrated that a change in structure and composition of podosomes accounted for the shift of function during megakaryopoiesis. These data highlight the fact that members of the invadosome family could correspond to different maturation status of the same entity, to adapt to functional responses required by differentiation stages of the cell that bears them.

Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids.

BACKGROUND: Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production. OBJECTIVE: The aim of this study was to investigate how megakaryocytes interact and progress through the endothelial cells of mouse bone marrow sinusoids. METHODS: We used a combination of fluorescence, electron, and three-dimensional microscopy to characterize the cellular events between megakaryocytes and endothelial cells. RESULTS: We identified protrusive, F-actin-based podosome-like structures, called in vivo-MK podosomes, which initiate the formation of pores through endothelial cells. These structures present a collective and spatial organization through their interconnection via a contractile network of actomyosin, essential to regulate the endothelial openings. This ensures proper passage of megakaryocyte-derived processes into the blood circulation to promote thrombopoiesis. CONCLUSION: This study provides novel insight into the in vivo function of podosomes of megakaryocytes with critical importance to platelet production.

Liquid chromatography-tandem mass spectrometry bioanalytical method for the determination of kavain in mice plasma: Application to a pharmacokinetic study.

A simple and fast bioanalytical method for the quantification of kavain in mice plasma was developed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A full method validation was performed, according to regulatory guidelines, employing isotopically labeled kavain as the internal standard (racemic-kavain-d3). For the quantification, [M+H]+ was formed using an electrospray ionization (ESI) source in the positive ion mode and multiple reaction monitoring (MRM) was employed using a quadrupole-linear ion trap (4000 QTRAP®) instrument. The monitored MRM transitions were 231.0 â†’ 115.1 and 231.0 â†’ 152.8 for kavain; and 234.2 â†’ 199.2 for the internal standard. A linear response was obtained at the concentration range of 10 to 200 ng/mL with intra- and inter-day variations within the acceptable criteria for all quality control samples. After validation, the method was successfully applied for the quantification of kavain in mice plasma after oral administration of the kavain standard and Kava-kava extract. The plasma concentration over time results were applied for a pharmacokinetics study. The obtained pharmacokinetic parameters indicated a considerably higher bioavailability for kavain when Kava-kava extract was administered due to a pharmacokinetic synergism between the analyte and the other compounds present in the extract.

A 89Zr-labeled lipoplex nanosystem for image-guided gene delivery: design, evaluation of stability and in vivo behavior.

BACKGROUND: With the advances in radiopharmaceutical research, the development of image-guided therapy has become a major interest. While the development of theranostic nanotherapeutics is frequently associated with cancer chemotherapy, phototherapy and radiotherapy, there is little information available on the in vivo monitoring of gene delivery systems and the application of image-guided approach in gene therapy. The goal of this work was to determine the in vivo behavior of DNA delivery nanosystems - based on cationic gemini surfactants - designed for image-guided gene therapy. We tested the feasibility of monitoring tumor accumulation of gene delivery nanoparticles by positron emission tomography. METHODS: To be able to conjugate radiotracers to the nanoparticles, a deferoxamine-modified gemini surfactant was synthesized, DNA-containing lipoplex nanoparticles were formulated, and radiolabeled with Zirconium-89 (89Zr). The pharmacokinetics and biodistribution of 89Zr labeled surfactant and 89Zr labeled nanoparticles were monitored in mice by microPET/CT imaging and ex vivo gamma counting. RESULTS: Modification of the nanoparticles with deferoxamine did not alter their physicochemical properties. The radiolabeled nanoparticles (labeling efficiency of 95±3%) were stable in PBS and serum. The biological half-life of the 89Zr labeled nanoparticles was significantly higher compared to 89Zr labeled surfactant. As expected, the nanoparticles had significantly higher liver accumulation than the radiolabeled surfactant alone and lower kidney accumulation. Tumor uptake was detected at 2 hours post injection and decreased throughout the 3-day monitoring. CONCLUSION: We propose that radiolabeling DNA delivery lipoplex nanosystems is a promising approach for the design and optimization of image-guided nanomedicines, especially in the context of cancer gene therapy.

Molecular Engineering as an Approach To Modulate Gene Delivery Efficiency of Peptide-Modified Gemini Surfactants.

The unique molecular structure confers the diquaternary ammonium gemini surfactants with enhanced nucleic acid complexation ability, bottom-up design flexibility, and relatively low cytotoxicity. To capitalize on their potential as gene delivery vectors, novel structural modifications should be explored. In this work, 22 novel peptide-modified gemini surfactants with various alkyl tails and peptide spacer modifications were evaluated. This work represents the first report of dendrimer-like gemini surfactants and first evaluation of the impact of incorporating a hydrocarbon linker into the peptide chain. Our aim was to establish a structure activity relationship of the peptide-modified gemini surfactants and to identify the fundamental architectural requirements needed for the ultimate gene delivery systems. In vitro assessment revealed that the highest transfection efficiency and lowest cytotoxicity were associated with the glycyl-lysine modified gemini surfactants having the hexadecyl tail, 16-7N(G-K)-16. In fact, it showed an 8-fold increase in secreted protein with 20% increase in cell viability relative to the first-generation unsubstituted gemini surfactants. Further increase in the size of the attached peptides resulted in a decrease in the transfection efficiency and cell viability. Whereas the incorporation of a hydrocarbon linker into the peptide chain decreased the transfection efficiency of compounds with dipeptides, it increased the transfection efficiency of compounds with larger peptide chains. Such an increase was more prominent with the incorporation of a longer hydrocarbon linker. We conclude that a balance between the hydrophilic and hydrophobic characteristics of the compound is necessary since it results in physicochemical parameters conducive to the gene delivery process.

The development of simple flow injection analysis tandem mass spectrometric methods for the cutaneous determination of peptide-modified cationic gemini surfactants used as gene delivery vectors.

Diquaternary ammonium gemini surfactants are a class of non-viral gene delivery vectors, primarily studied for their dermal applications. However, their biological fate has rarely been investigated. In this work, we developed simple flow injection analysis tandem mass spectrometric methods, (FIA)-MS/MS, to understand the fate and biodistribution of topically applied gemini surfactant-based therapeutics in an ex-vivo skin model. Three peptide-modified gemini surfactants with varied structures and transfection efficiencies were evaluated. For each compound, two methods were developed to quantify their presence in skin tissue and in phosphate buffered saline (PBS). The methods were developed using single-point calibration mode. Skin penetration was assessed on CD1 mice dorsal skin tissue mounted in a Franz diffusion cell after extraction. Amongst the five evaluated liquid-liquid extraction protocols, the Folch method provides the highest extraction efficiency for all compounds. Weak cationic exchange solid phase extraction was also used to further isolate gemini surfactants from endogenous skin lipids. FIA-MS/MS analysis of the skin revealed that all compounds were detected in the skin with minimal partition into the PBS compartment, which represents circulation. Interestingly, the detected amounts of gemini lipids in the skin were correlated with their transfection efficiencies.

HPLC-MS/MS analysis of anthocyanins in human plasma and urine using protein precipitation and dilute-and-shoot sample preparation methods, respectively.

A high-performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS) method has been developed to analyze anthocyanins in urine and plasma to further understand their absorption, distribution, metabolism and excretion. The method employed a Synergi RP-Max column (250 × 4.6 mm, 4 µm) and an API 4000 mass spectrometer. A gradient elution system consisted of mobile phase A (water-1% formic acid) and mobile phase B (acetonitrile) with a flow rate of 0.60 mL/min. The gradient was initiated at 5% B, increased to 21% B at 20 min, and then increased to 40% B at 35 min. The analysis of anthocyanins presents a challenge because of the poor stability of anthocyanins during sample preparation, especially during solvent evaporation. In this method, the degradation of anthocyanins was minimized using protein precipitation and dilute-and-shoot and sample preparation methods for plasma and urine, respectively. No interferences were observed from endogenous compounds. The method has been used to analyze anthocyanin concentrations in urine and plasma samples from volunteers administered saskatoon berries. Cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside, cyanidin-3-xyloside and quercetin-3-galactoside, the five major flavonoid components in saskatoon berries, were identified in plasma and urine samples.

Rapid and simple flow injection analysis tandem mass spectrometric method for the quantification of melphalan in a lipid-based drug delivery system.

RATIONALE: The use of the anticancer drug melphalan is limited due to its poor water solubility. To address this limitation, it is incorporated within a novel delivery system using ß-cyclodextrin-gemini surfactants (18:1ßCDg). METHODS: Herein, two fast and simple flow injection analysis/tandem mass spectrometric (FIA-MS/MS) methods are developed for the quantification of melphalan (Mel) within the drug delivery system so that the solubilization efficiency of the system can be assessed. FIA-MS/MS methods are developed using a triple quadrupole linear ion trap mass spectrometer, equipped with electrospray ionization (ESI) in the positive ion mode. A deuterated form of melphalan (melphalan-d8) was used as an internal standard (IS). The methods were validated according to the FDA guidance. RESULTS: A linearity in the range of 2-100 ng/mL and accuracy and precision below 15% were observed for all standard points and quality control samples. The intra- and inter-day variations and freeze-thaw stability were within the acceptable range according to the criteria set by regulatory guidelines. On the other hand, other stability measures, such as room temperature stability and long-term stability, did not meet the required guidelines in some cases, indicating the need for quick sample analysis upon preparation. Such a fact could have been overlooked if full method validation had not been performed. CONCLUSIONS: The developed methods were applied to determine the encapsulation/solubilization of the [18:1ßCDg/Mel] delivery system. 18:1ßCDg enhances the aqueous solubility of melphalan without the need for co-solvent. The highest melphalan solubility was observed at a melphalan18:1ßCDg/Mel complex molar ratio of 2:1. This study demonstrated that a fast analysis for the purpose of quantifying a chemically unstable drug, such as melphalan, is feasible and important for the development of commercial dosage forms.

Evaluation of ß-cyclodextrin-modified gemini surfactant-based delivery systems in melanoma models.

Novel drug delivery systems are developed to improve the biological behavior of poorly soluble drugs and to improve therapeutic outcomes. In melanoma therapy, the goal is efficient drug delivery and mitigation of drug resistance. Melphalan (Mel), a currently used therapeutic agent for melanoma, requires solvent system for solubilization, leading to poor chemical stability. Moreover, drug resistance often renders the drug inefficient in clinical setting. A novel ß-cyclodextrin-modified gemini surfactant (CDgemini) delivery system was developed to incorporate Mel in order to improve its physicochemical and biological behavior. Melphalan nanoparticles (Mel-NP) showed optimal particle size in the 200-250 nm range for endocytosis and induced significantly higher cell death compared with Mel (50% of inhibitory concentration [IC50] of 36 µM for the complexes vs 82 µM for Mel). The CDgemini delivery system did not alter the pathway of the cellular death triggered by Mel and caused no intrinsic toxicity to the cells. The Mel-NP complexes induced significant cell death in melanoma cells that were rendered resistant to Mel. These findings demonstrate in principle the applicability of the CDgemini delivery system as safe and efficient alternative to the current melanoma therapy, especially in chemoresistant cases.

Evaluating the cytotoxic effects of the water extracts of four anticancer herbs against human malignant melanoma cells.

Malignant melanoma (MM) is the most dangerous type of skin cancer, killing more than 1,100 people each year in Canada. Prognosis for late stage and recurrent MM is extremely poor due to insensitivity to chemotherapy drugs, and thus many patients seek complementary and alternative medicines. In this study, we examined four commonly used anticancer herbs in traditional Chinese medicine, Hedyotis diffusa, Scutellaria barbata, Lobelia chinensis, and Solanum nigrum, for their in vitro antitumor effects toward human MM cell line A-375. The crude water extract of S. nigrum (1 g of dry herb in 100 mL water) and its 2-fold dilution caused 52.8%±13.0% and 17.3%±2.7% cytotoxicity in A-375 cells, respectively (P<0.01). The crude water extract of H. diffusa caused 11.1%±12.4% cytotoxicity in A-375 cells with no statistical significance (P>0.05). Higher concentrated formulation might be needed for H. diffusa to exert its cytotoxic effect against A-375 cells. No cytotoxicity was observed in A-375 cells treated with crude water extract of S. barbata and L. chinensis. Further high performance liquid chromatography-tandem mass spectroscopy analysis of the herbal extracts implicated that S. nigrum and H. diffusa might have adopted the same bioactive components for their cytotoxic effects in spite of belonging to two different plant families. We also showed that the crude water extract of S. nigrum reduced intracellular reactive oxygen species generation in A-375 cells, which may lead to a cytostatic effect. Furthermore, synergistic effect was achieved when crude water extract of S. nigrum was coadministered with temozolomide, a chemotherapy drug for skin cancer.

Effect of creatine supplementation and drop-set resistance training in untrained aging adults.

OBJECTIVE: To investigate the effects of creatine supplementation and drop-set resistance training in untrained aging adults. Participants were randomized to one of two groups: Creatine (CR: n=14, 7 females, 7 males; 58.0±3.0yrs, 0.1g/kg/day of creatine+0.1g/kg/day of maltodextrin) or Placebo (PLA: n=17, 7 females, 10 males; age: 57.6±5.0yrs, 0.2g/kg/day of maltodextrin) during 12weeks of drop-set resistance training (3days/week; 2 sets of leg press, chest press, hack squat and lat pull-down exercises performed to muscle fatigue at 80% baseline 1-repetition maximum [1-RM] immediately followed by repetitions to muscle fatigue at 30% baseline 1-RM). METHODS: Prior to and following training and supplementation, assessments were made for body composition, muscle strength, muscle endurance, tasks of functionality, muscle protein catabolism and diet. RESULTS: Drop-set resistance training improved muscle mass, muscle strength, muscle endurance and tasks of functionality (p<0.05). The addition of creatine to drop-set resistance training significantly increased body mass (p=0.002) and muscle mass (p=0.007) compared to placebo. Males on creatine increased muscle strength (lat pull-down only) to a greater extent than females on creatine (p=0.005). Creatine enabled males to resistance train at a greater capacity over time compared to males on placebo (p=0.049) and females on creatine (p=0.012). Males on creatine (p=0.019) and females on placebo (p=0.014) decreased 3-MH compared to females on creatine. CONCLUSIONS: The addition of creatine to drop-set resistance training augments the gains in muscle mass from resistance training alone. Creatine is more effective in untrained aging males compared to untrained aging females.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry quantitative method for the cellular analysis of varying structures of gemini surfactants designed as nanomaterial drug carriers.

Diquaternary gemini surfactants have successfully been used to form lipid-based nanoparticles that are able to compact, protect, and deliver genetic materials into cells. However, what happens to the gemini surfactants after they have released their therapeutic cargo is unknown. Such knowledge is critical to assess the quality, safety, and efficacy of gemini surfactant nanoparticles. We have developed a simple and rapid liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative determination of various structures of gemini surfactants in cells. Hydrophilic interaction liquid chromatography (HILIC) was employed allowing for a short simple isocratic run of only 4min. The lower limit of detection (LLOD) was 3ng/mL. The method was valid to 18 structures of gemini surfactants belonging to two different structural families. A full method validation was performed for two lead compounds according to USFDA guidelines. The HILIC-MS/MS method was compatible with the physicochemical properties of gemini surfactants that bear a permanent positive charge with both hydrophilic and hydrophobic elements within their molecular structure. In addition, an effective liquid-liquid extraction method (98% recovery) was employed surpassing previously used extraction methods. The analysis of nanoparticle-treated cells showed an initial rise in the analyte intracellular concentration followed by a maximum and a somewhat more gradual decrease of the intracellular concentration. The observed intracellular depletion of the gemini surfactants may be attributable to their bio-transformation into metabolites and exocytosis from the host cells. Obtained cellular data showed a pattern that grants additional investigations, evaluating metabolite formation and assessing the subcellular distribution of tested compounds.

Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application.

PURPOSE: Nanodiamonds (NDs) are emerging as an attractive tool for gene therapeutics. To reach their full potential for biological application, NDs should maintain their colloidal stability in biological milieu. This study describes the behavior of lysine-functionalized ND (lys-ND) in various dispersion media, with an aim to limit aggregation and improve the colloidal stability of ND-gene complexes called diamoplexes. Furthermore, cellular and macromolecular interactions of lys-NDs are also analyzed in vitro to establish the understanding of ND-mediated gene transfer in cells. METHODS: lys-NDs were synthesized earlier through covalent conjugation of lysine amino acid to carboxylated NDs surface generated through re-oxidation in strong oxidizing acids. In this study, dispersions of lys-NDs were prepared in various media, and the degree of sedimentation was monitored for 72 hours. Particle size distributions and zeta potential measurements were performed for a period of 25 days to characterize the physicochemical stability of lys-NDs in the medium. The interaction profile of lys-NDs with fetal bovine serum showed formation of a protein corona, which was evaluated by size and charge distribution measurements. Uptake of lys-NDs in cervical cancer cells was analyzed by scanning transmission X-ray microscopy, flow cytometry, and confocal microscopy. Cellular uptake of diamoplexes (complex of lys-NDs with small interfering RNA) was also analyzed using flow cytometry. RESULTS: Aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Size distributions showed good stability, remaining under 100 nm throughout the testing period. A positive zeta potential of >+20 mV indicated a preservation of surface charges. Size distribution and zeta potential changed for lys-NDs after incubation with blood serum, suggesting an interaction with biomolecules, mainly proteins, and a possible formation of a protein corona. Cellular internalization of lys-NDs was confirmed by various techniques such as confocal microscopy, soft X-ray spectroscopy, and flow cytometry. CONCLUSION: This study establishes that dispersion of lys-NDs in aqueous medium maintains long-term stability and also provides evidence that lysine functionalization enables NDs to interact effectively with the biological system to be used for RNAi therapeutics.

Development of amino acid substituted gemini surfactant-based mucoadhesive gene delivery systems for potential use as noninvasive vaginal genetic vaccination.

AIM: Recently, we synthesized amino acid- and peptide-substituted gemini surfactants, 'biolipids' that exhibited high transfection efficiency in vitro. In this study, we developed these plasmid DNA and gemini surfactant lipid particles for noninvasive administration in vaginal cavity. MATERIAL & METHODS: Novel formulations of these gene delivery systems were prepared with poloxamer 407 to induce in situ gelling of the formulation and diethylene glycol monoethyl ether to improve their penetration across mucosal tissue. RESULTS: Poloxamer at 16% w/v concentration in diethylene glycol monoethyl ether aqueous solution produced dispersions that gelled near body temperature and had a high yield value, preventing leakage of the formulation from the vaginal cavity. Intravaginal administration in rabbits showed that the glycyl-lysine-substituted gemini surfactant led to a higher gene expression compared with the parent unsubstituted gemini surfactant. CONCLUSION: This provides a proof-of-concept that amino acid substituted gemini surfactants can be used as noninvasive mucosal (vaginal) gene delivery systems to treat diseases associated with mucosal epithelia.

Characterization of the host-guest complex of a curcumin analog with ß-cyclodextrin and ß-cyclodextrin-gemini surfactant and evaluation of its anticancer activity.

BACKGROUND: Curcumin analogs, including the novel compound NC 2067, are potent cytotoxic agents that suffer from poor solubility, and hence, low bioavailability. Cyclodextrin-based carriers can be used to encapsulate such agents. In order to understand the interaction between the two molecules, the physicochemical properties of the host-guest complexes of NC 2067 with ß-cyclodextrin (CD) or ß-cyclodextrin-gemini surfactant (CDgemini surfactant) were investigated for the first time. Moreover, possible supramolecular structures were examined in order to aid the development of new drug delivery systems. Furthermore, the in vitro anticancer activity of the complex of NC 2067 with CDgemini surfactant nanoparticles was demonstrated in the A375 melanoma cell line. METHODS: Physicochemical properties of the complexes formed of NC 2067 with CD or CDgemini surfactant were investigated by synchrotron-based powder X-ray diffraction, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. Synchrotron-based small- and wide-angle X-ray scattering and size measurements were employed to assess the supramolecular morphology of the complex formed by NC 2067 with CDgemini surfactant. Lastly, the in vitro cell toxicity of the formulations toward A375 melanoma cells at various drug-to-carrier mole ratios were measured by cell viability assay. RESULTS: Physical mixtures of NC 2067 and CD or CDgemini surfactant showed characteristics of the individual components, whereas the complex of NC 2067 and CD or CDgemini surfactant presented new structural features, supporting the formation of the host-guest complexes. Complexes of NC 2067 with CDgemini surfactants formed nanoparticles having sizes of 100-200 nm. NC 2067 retained its anticancer activity in the complex with CDgemini surfactant for different drug-to-carrier mole ratios, with an IC50 (half-maximal inhibitory concentration) value comparable to that for NC 2067 without the carrier. CONCLUSION: The formation of host-guest complexes of NC 2067 with CD or CDgemini surfactant has been confirmed and hence the CDgemini surfactant shows good potential to be used as a delivery system for anticancer agents.

Development and validation of fast and simple flow injection analysis-tandem mass spectrometry (FIA-MS/MS) for the determination of metformin in dog serum.

A simple, fast and sensitive quantification method for the drug metformin in dog serum was developed using flow injection analysis (FIA)-tandem mass spectrometry (MS/MS). The method was fully validated according to industry standards. It is the first time that FIA-MS/MS for metformin was developed surpassing all existing methods in terms of time of analysis. The quantification method was dependent on the formation of [M+H](+) using electrospray ionization (ESI) and employing multiple reaction monitoring (MRM) using quadrupole-linear ion trap (4000 QTRAP(®)) instrument. A deuterated internal standard (IS) of metformin bearing six deuterium atoms was used to compensate for matrix effects and for variation in ion current within the ESI source. The ion transitions that were monitored were m/z 130.1→m/z 71.0 and m/z 130.1→m/z 60.1 for metformin and m/z 136.0→m/z 77.0 for the internal standard. A linear response (r=0.9966) was established for a range of concentrations of 5-2340 ng/mL. The inter- and intra-day variations were within the acceptable criteria for all quality control samples. The method was successfully applied for measurement of serum metformin concentration in dogs after intravenous injection.

Cytotoxic 1,5-diaryl-3-oxo-1,5-pentadienes: an assessment and comparison of membrane permeability using Caco-2 and MDCK monolayers.

A number of cytotoxic conjugated unsaturated ketones were screened for their membrane permeability characteristics using Caco-2 and MDCK cells with the view of finding promising leads for in vivo evaluations. 3b-e and 4a-b demonstrated high permeability characteristics. In particular, 4a emerged as a promising lead which showed excellent apparent permeability (P(app): 54.70) and efflux ratio (ER: 0.15) values. In general, the relative apparent permeabilities of these enones are similar in both bioassays.

Optimization of the reactional medium and a food impact study for a colorimetric in situ Salmonella spp. detection method.

Foodborne pathogens are still a major concern for public health authorities. In this paper, we describe the optimization of a previously reported method which combines a highly specific capture of targeted food pathogens with an intracellular staining method. The reaction medium was optimized to simultaneously allow specific enrichment of Salmonella and maximize the staining of the target pathogen. This in situ colorimetric concept was evaluated with a broad range of food samples artificially contaminated with low levels of stressed Salmonella to mimic natural contamination conditions. This direct detection method compared favorably to a commercially available immunoassay system (Vidas® UP Salmonella), for cooked meat, dry milk powder and egg products. Globally 88% agreement was obtained between the two methods with a sensitivity of 80% and a specificity of 100% for the tested method. Main discordances were obtained with food matrices having high levels of competitive Gram negative microflora. These observations show that the design of an adapted culture medium is necessary to enhance the specific in situ capture and revelation system.