Enhancing Methotrexate Delivery in the Brain by Mesoporous Silica Nanoparticles Functionalized with Cell-Penetrating Peptide using in Vivo and ex Vivo Monitoring.

The blood-brain barrier (BBB) acts as a physical/biochemical barrier that protects brain parenchyma from potential hazards exerted by different xenobiotics found in the systemic circulation. This barrier is created by "a lipophilic gate" as well as a series of highly organized influx/efflux mechanisms. The BBB bottleneck adversely affects the efficacy of chemotherapeutic agents in treating different CNS malignancies such as glioblastoma, an aggressive type of cancer affecting the brain. In the present study, mesoporous silica nanoparticles (MSNs) were conjugated with the transactivator of transcription (TAT) peptide, a cell-penetrating peptide, to produce MSN-NH-TAT with the aim of improving methotrexate (MTX) penetration into the brain. The TAT-modified nanosystem was characterized by Fourier transform infrared spectrometry (FTIR), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), and N2 adsorption-desorption analysis. In vitro hemolysis and cell viability studies confirmed the biocompatibility of the MSN-based nanocarriers. In addition, in vivo studies showed that the MTX-loaded MSN-NH-TAT improved brain-to-plasma concentration ratio, brain uptake clearance, and the drug's blood terminal half-life, compared with the use of free MTX. Taken together, the results of the present study indicate that MSN functionalization with TAT is crucial for delivery of MTX into the brain. The present nanosystem represents a promising alternative drug carrier to deliver MTX into the brain via overcoming the BBB.

Curcumin mediated down-regulation of αV ß3 integrin and up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) in Erlotinib resistant SW480 colon cancer cells.

Erlotinib is a potent, selective, and orally active inhibitor of the epidermal growth factor receptor, but the development of erlotinib resistance during chemotherapy can lead to treatment failure. To shed light on the erlotinib-resistant pathway, this study investigated the effect of combination therapy using curcumin- and erlotinib-loaded nanoparticles on the expression of αv ß3 integrin and pyruvate dehydrogenase kinase 4 (PDK4) in an erlotinib-resistant SW480 colon cancer cell line. An erlotinib-resistant SW480 colon cancer cell line was produced by long-term exposure to erlotinib. Curcumin-loaded Methoxy poly ethylene glycol Poly caprolactone (cur/mPEG-PCL) and erlotinib-loaded mPEG-PCL (erl/mPEG-PCL) micelles were provided using a single step nanoprecipitation method and used as combination therapy of resistant SW480 cancer cells. After that, gene expression levels of PDK4, αv, and ß3 mRNA were determined by the semiquantitative reverse transcription-polymerase chain reaction. Protein levels of whole αv ß3 integrin were evaluated using the enzyme-linked immunosorbent assay method. In SW480 cell line, the IC50 of nonresistant and resistant cells was 87.6 ± 1.2 nM and 19.1 ± 0.14 µM, for erlotinib and it was about 21.8 and 30 µM for curcumin, respectively. Although PDK4 expression was not significantly different in resistant and nonresistant cells, its expression was up regulated (1.4 fold) in resistant cells by a combination therapy of cur/mPEG-PCL at a dose of 3 µM and erl/mPEG-PCL at a dose of 5 µM. ß3 mRNA and the protein level of whole αv ß3 integrin was significantly higher in resistant SW480 cells as compared with those in nonresistant cells. In terms of treatment, a combination of 6-µM cur/mPEG-PCL and 5-µM erl/mPEG-PCL down regulated ß3 gene expression 6.6-fold in resistant cells as compared with nonresistant cells. At the protein level, a combination of 3-µM-cur/mPEG-PCL and 10-µM erl/mPEG-PCL reduced αv ß3 protein in resistant cells. The results indicated that combination therapy using cur/mPEG-PCL and erl/mPEG-PCL could decrease αv ß3 integrin expression and increase PDK4 gene expression in resistant colon cancer cells, which may have effects on drug resistance signaling pathways.

The effect of benzo[α]pyrene on expression and signaling cross talk of aryl hydrocarbon receptor and NFATc1 in mouse lung tissue.

OBJECTIVE: Polycyclic aromatic hydrocarbons (PAHs) are potent environmental pollutants. Benzo[α]pyrene (B[α]P) is the major compound of PAHs that acts by activating aryl hydrocarbon receptor (AhR) in cells. B[α]P is a known carcinogen and an immunotoxicant; however, its role with regard to nuclear factor of activated T cell (NFAT) pathway is unclear. AhR and NFAT signaling pathways have common roles in pathological functions in immunotoxicity and lung cancer. In this study, the effect of AhR activation on expression and signaling cross talk of AhR and NFATc1 pathways in mouse lung tissue has been investigated. METHODS: Swiss albino mice were randomly allocated to five groups and administered with cyclosporin A (CsA) and B[α]P for seven constitutive days. Animals were then killed, and lung tissues were obtained after washing the whole blood. Paraffin-embedded blocks were prepared, and 5 µm sections were cut for histopathological and immunohistochemical assessments. The results were scored by observer and digitally analyzed using ImageJ software. RESULTS: Our data showed that CsA administration resulted in a significant reduction of AhR expression. This effect was partly blocked in mice coadministrated with B[α]P and CsA. NFATc1 expression was also reduced in CsA-treated animals. Furthermore, CsA inhibited the pathological effects of B[α]P in mouse lung tissue. CONCLUSION: AhR expression is dependent on NFATc1 activation, and NFATc1 inhibition remarkably decreases AhR expression. However, it seems that total expression of NFATc1 is not dependent on AhR expression or activation. Moreover, CsA can prevent B[α]P-induced lung tissue damage, and it remarkably decreases NFATc1 expression. The results from this study point toward the molecular interactions of AhR and NFATc1 activation in lung tissue and the benefit of CsA treatment in B[α]P-induced lung damage.

Design, synthesis and biological evaluation of novel anti-cytokine 1,2,4-triazine derivatives.

A series of 16 novel 1,2,4-triazine derivatives bearing hydrazone moiety (7a-7p) have been designed, synthesized and evaluated for their activity to inhibit IL-1ß and TNF-α production. All compounds are reported for the first time. The chemical structures of all compounds were confirmed by spectroscopic methods and elemental analyzes. Most of the synthesized compounds were proved to have potent anti-cytokine activity and low toxicity on PBMC and MCF-7 cell lines. Compounds 7f, 7k, 7l and 7j presented simultaneously good levels of inhibition of both cytokines. Moreover, compound 7l exhibited good anti-inflammatory effect in carrageenan-induced rat paw edema. The results of Western blotting demonstrated that the anti-cytokine potential of compound 7l is mainly mediated through the inhibition of p38 MAPK signaling pathway. Molecular docking was performed to position compound 7l into p38α binding site in order to explore the potential target. The information of this work might be helpful for the design and synthesis of novel scaffold toward the development of new therapeutic agent to fight against inflammatory diseases.

Evaluation of anti-invasion effect of cannabinoids on human hepatocarcinoma cells.

CONTEXT: Cancer is a disease characterized by abnormal growth of cells. One of the most common types of liver cancers is called hepatocellular carcinoma (HCC) which is highly metastatic. As most of cannabinoids have shown anticancer effect against different cell lines in a number of reports, a biological investigation of two cannabinoids, CB65 (CB2 receptor agonist) and ACEA (CB1 receptor agonist) was carried out in this study. OBJECTIVE: In an attempt to find natural products as a new solution of cancer, this study was designed to investigate the potential antitumoral and anti-invasive activity of cannabinoids on HepG2 cells and the possible roles of matrix metalloproteinase-2 (MMP-2) and MMP-9 in its action. MATERIALS AND METHODS: The researchers examined the effect of various concentrations of CB65 (CB2 receptor agonist) and ACEA (CB1 receptor agonist), on the cell proliferation, viability, and invasion as well as expression of MMP-2 and MMP-9 in HepG2 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, matrigel invasion assay, and western blotting method. RESULTS: The results revealed that both cannabinoids reduce cell viability, cell invasion as well as MMP-2 and MMP-9 expression in higher dose of 20 nM. Furthermore, higher concentrations of examined cannabinoids were more effective. DISCUSSION: These data suggest ACEA and CB65 as an option for novel treatment of hepatocellular cancer. CONCLUSION: Our findings may contribute to design of new therapeutic strategies for the management of HCC.

Fabrication of ciprofloxacin-loaded chitosan/polyethylene oxide/silica nanofibers for wound dressing application: In vitro and in vivo evaluations.

Silica plays an effective role in collagen creation; hence, the degradation products of silica-based materials accelerate wound healing. In this regard, chitosan/polyethylene oxide/silica hybrid nanofibers were prepared by the combining the sol-gel method with electrospinning technique to accelerate the wound healing process. Ciprofloxacin, as an antibacterial drug, was then added to the electrospinning mixture. The nanofibers were characterized by SEM, EDX, X-ray mapping, TEM, TGA, FTIR, and XRD analysis. The degradation, swelling ratio, and release of ciprofloxacin were investigated in PBS. The prepared nanofiber could absorb water, maintain its morphological integrity during the degradation process, and gradually release ciprofloxacin. The nanofibers revealed an efficient antibacterial activity against Escherichia coli and Staphylococcus aureus. Cell viability assays showed that the nanofibers had no cytotoxicity against L929 mouse fibroblast and HFFF2 human foreskin fibroblast cell lines. The potential of the chitosan/polyethylene oxide/silica/ciprofloxacin nanofiber for healing full-thickness wound was assessed by applying the scaffold in the dorsal cutaneous wounds of the Balb/C mice. The white blood cell counts of the animals indicated the nanofiber-treated mice compared with the untreated ones had less infection and inflammation. According to the histopathologic data, the prepared nanofiber accelerated and enhanced tissue regeneration by increasing fibroblast cells and angiogenesis as well as decreasing the inflammation phase. The findings suggest that the prepared antibacterial scaffold with drug delivery properties could be an appropriate candidate for many medical and hygienic applications, especially as a bio-compatible and bio-degradable wound dressing.

Enhancement of the brain delivery of methotrexate with administration of mid-chain ester prodrugs: In vitro and in vivo studies.

In the present study, with the aim of improving the permeability of methotrexate (MTX) to the brain, the lipophilic MTX prodrugs containing the ester functional moiety were synthesized. The chemical structure of synthesized prodrugs was characterized and confirmed by FT-IR, NMR and mass spectral studies. Based on the results of in vitro cytotoxic studies, all of the synthesized prodrugs led to decrease in the IC50 in 72 h on U87 cancer cell line and the best result was observed for dihexyl methotrexate (MTX-DH) in comparison with free MTX, which led to decrease the IC50 amount up to 6 folds. In addition, in vivo toxicity on Artemia salina (A. salina) showed that the lipophilic MTX prodrugs have been able to partially mask the toxic profile of free MTX, at the same concentrations. These findings were also in compliance with hemolysis assay results, which confirm that the conjugates has not made the drug more toxic. Furthermore, in vivo study in rat model, was employed to determine the simultaneous drug concentration in brain and plasma. According to the obtained results, the brain-to-plasma concentration ratios (Kp values) of MTX-DH and dioctyl methotrexate (MTX-DO) groups were significantly higher compared with free MTX. Moreover, the uptake clearance of MTX by brain parenchyma increased significantly (3.85 and 9.08-time increased for MTX-DH and MTX-DO prodrugs, respectively). These findings indicate that the synthesized lipophilic MTX prodrugs are non-toxic and able to enhance brain penetration of MTX.

Magnetic nanoparticles decorated with PEGylated curcumin as dual targeted drug delivery: Synthesis, toxicity and biocompatibility study.

The problems associated with hydrophobic anticancer drugs are among the most important challenges to achieve efficient therapeutics for cancer treatment. In this study, PEGylated curcumin was used as the surface modification of magnetic nanoparticles (MNP@PEG-Cur) in order to simultaneously take advantage of magnetic targeting characteristic of nanoparticles and PEG conjugated drug. Curcumin was conjugated through EDC/NHS chemistry to the PEG hydroxyl functional groups, and then physically decorated on the surface of magnetic nanoparticles (MNP). The analysis of the conjugate and nanoparticles by FT-IR, 1HNMR, FE-SEM, TEM, EDX, TGA and VSM confirmed the successful synthesis and proper physicochemical properties of MNP@PEG-Cur nanoparticles. The carrier showed pH dependent drug release profile with higher drug release at acidic media (pH = 5.4) compared to neural condition (pH = 7.4). In addition, LD50 and hemolysis assay confirmed the biocompatibility of MNP@PEG-Cur. The cell viability assay also revealed that neither carrier, nor curcumin-loaded nanoparticles are cytotoxic at physiologic pH (7.4).

Naproxen conjugated mPEG-PCL micelles for dual triggered drug delivery.

A conjugate of the NSAIDs drug, naproxen, with diblock methoxy poly(ethylene glycol)-poly(ε-caprolactone) (mPEG-PCL) copolymer was synthesized by the reaction of copolymer with naproxen in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. The naproxen conjugated copolymers were characterized with different techniques including (1)HNMR, FTIR, and DSC. The naproxen conjugated mPEG-PCL copolymers were self-assembled into micelles in aqueous solution. The TEM analysis revealed that the micelles had the average size of about 80 nm. The release behavior of conjugated copolymer was investigated in two different media with the pH values of 7.4 and 5.2. In vitro release study showed that the drug release rate was dependant on pH as it was higher at lower pH compared to neutral pH. Another feature of the conjugated micelles was a more sustained release profile compared to the conjugated copolymer. The kinetic of the drug release from naproxen conjugated micelles under different values of pH was also investigated by different kinetic models such as first-order, Makoid-Banakar, Weibull, Logistic, and Gompertz.

Non-addictive opium alkaloids selectively induce apoptosis in cancer cells compared to normal cells.

BACKGROUND: Cytotoxic effects of some of the members of papaveraceae family have been reported in Iranian folk medicine. Recent reports has indicated that alkaloids fraction of opium may be responsible for its cytotoxic effect; however, the mechanism of this effect is not fully understood. This study has been designed to investigate the selective cytotoxic, genotoxic and also apoptosis induction effects of noscapine, papaverine and narceine, three non-addictable opium alkaloids, on HT29, T47D and HT1080 cancer cell lines. Mouse NIH3T3 cell line was chosen to present non-cancerous cells and Doxorubicin was selected as the positive control. METHODS: Cells were treated by different concentrations of Noscapine, Papaverine, Narceine and doxorubicin; viability was assessed by MTT assay. The genotoxicity and apoptosis induction were tested with comet assay and Annexin-V affinity when the concentration of each these drugs is less than its IC50. In addition, the DNA damage and caspase activity of the T47D cells were examined and the results were compared. RESULTS: This study noted the cytotoxicity and genotoxicity of noscapine and papaverine, specifically on cancerous cell lines. Furthermore, papaverine induces apoptosis in all studied cancer cell lines and noscapine showed this effect in T47D and HT29 cells but not in NIH-3 T3 cells as noncancerous cell line. narceine also showed genototoxicity in the studied cell lines at its IC50 concentration. CONCLUSIONS: This experiment suggests that noscapine and papaverine may be of use in cancer treatment due to their specific cytotoxicity and genotoxicity. However, further in vivo studies are needed to confirm its usefulness in cancer treatment.

Pomegranate juice (punica granatum): a new storage medium for avulsed teeth.

OBJECTIVE: There is evidence indicating that pomegranate juice contains many of the essential properties necessary to retain cell viability and cell proliferation. These properties indicate that pomegranate juice is a suitable storage medium for avulsed teeth. However, this idea has not yet been tested. In this study, the capacity of pomegranate juice (PJ) as a storage medium for retaining avulsed teeth was evaluated. MATERIALS AND METHODS: PDL fibroblasts were obtained from healthy human premolars and cultured in Dulbecco's Modified Eagle's Medium (DMEM). Cultured cells were subjected to different concentrations of pomegranate juice (PJ), 1% Hank's balanced salt solution (HBSS) and tap water for 1, 3, 6 and 24 hours. PDL cell viability was assessed by the neutral red uptake assay. RESULTS: The results indicated that 7.5% PJ was the most effective solution for maintaining PDL cell viability amongst all the experimental solution's and time intervals (P<0.05). The results also showed that 1% PJ was as effective as HBSS for maintaining PDL cell viability. The amount of cell viability increased with increasing concentration of PJ at all time intervals (P<0.001). This effect is suggestive of the proliferative potential of PJ solution. CONCLUSION: In conclusion, PJ can be recommended as a suitable transport medium for avulsed teeth.

Neuroprotective Effect of Total and Sequential Extract of Scrophularia striata Boiss. in Rat Cerebellar Granule Neurons Following Glutamate- Induced Neurotoxicity: An In-vitro Study.

Neuroprotective effect of the extract from aerial parts of Scrophularia striata Boiss (Scrophulariaceae) was investigated against glutamate-induced neurotoxicity on cultured rat pups Cerebellar Granule Neurons (CGNs). CGNs from 8 days old Sprague-Dawley rat were prepared and cultured. The experiments were performed after 8 days in culture. The plant was collected from the northeastern part (Ruin region) of Iran and air-dried at room temperature. The total extract was prepared with maceration of prepared powder in ethanol 80% for three times. SEQUENTIAL EXTRACTS WERE OBTAINED USING DRIED AND POWDERED AERIAL PARTS WITH INCREASINGLY POLAR SOLVENTS: petroleum ether, chloroform, ethyl acetate and methanol 80% solution. Cultured cells were exposed to 125 µM of glutamate for 12 h following a 24 h of incubation with test fractions at concentration of 10 mcg/mL. Morphological assay was performed using invert light microscope after fixation and staining with haematoxylin. Neuronal viability was measured using MTT assay. Statistical analysis was done using SPSS software. One way analysis of variance (ANOVA) was performed by Tukey post-hoc test. Values were considered statistically significant when p-value ≤ 0.05. Results of this study showed a significant neuroprotective activity of high polarity methanolic fraction of aerial parts of Scrophularia striata against glutamate-induced neurotoxicity in a dosedependent manner. Treatment with 10 mcg/mL of the fractions showed the best result.

Evaluation of Enamel Matrix Derivative (EMD) Teratogenicity on the Rat Embryo Neural Crest Culture.

Enamel matrix derivative Emdogain (EMD) is widely used in periodontal treatment in spite of the fact that its effect on the developing embryo has not been elucidated. The aim of this study was to investigate the teratogenic effect of EMD on the rat embryo neural crest cells. The neural crest is a unique population of cells that migrates from the dorsal neural tube along defined pathways and produces various cell types including the melanocytes, neuronal and glial cells of the sensory, autonomic and enteric nervous system as well as the chromaffin cells of the adrenal gland. These cells have been used extensively for in-vitro studies of neurogenesis. Cultured cells by micromass culture method derived from midbrain of six embryos (13 day postcoitum; 34-36 smites) and exposed to various concentrations of EMD for 5 days at 37°C and differentiated foci were counted. Retinoic Acid (20 µg/mL) was used as standard positive control. These cells were stained using Mayer's hematoxylin which is specific for staining differentiated cell nucleus. Neutral red staining determines cell viability rather than related cell differentiation but is used for normalization of Mayer's hematoxylin results. At the concentration as low as 8 µg/mL of EMD, no toxic effect on fetal cells was observed and it is suggested that EMD has no teratogenic effect at studied concentrations.