Search results for: cytotoxicity.

Authors: Yung-Chih Kuo, I-Hsin Wang

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Catanionic solid lipid nanoparticles (CASLNs) with surface wheat germ agglutinin (WGA) and lactoferrin (Lf) were formulated for entrapping and releasing etoposide (ETP), crossing the blood–brain barrier (BBB), and inhibiting the growth of glioblastoma multiforme (GBM). Microemulsified ETP-CASLNs were modified with WGA and Lf for permeating a cultured monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes and for treating malignant U87MG cells. Experimental evidence revealed that an increase in the concentration of catanionic surfactant from 5 μM to 7.5 μM reduced the particle size. When the concentration of catanionic surfactant increased from 7.5 μM to 12.5 μM, the particle size increased, yielding a minimal diameter of WGA-Lf-ETP-CASLNs at 7.5 μM of catanionic surfactant. An increase in the weight percentage of BW from 25% to 75% enlarged WGA-Lf-ETP-CASLNs. In addition, an increase in the concentration of catanionic surfactant from 5 to 15 μM increased the absolute value of zeta potential of WGA-Lf-ETP-CASLNs. It was intriguing that the increment of the charge as a function of the concentration of catanionic surfactant was approximately linear. WGA-Lf-ETP-CASLNs revealed an integral structure with smooth particle contour, displayed a lighter exterior layer of catanionic surfactant, WGA, and Lf and showed a rigid interior region of solid lipids. A variation in the concentration of catanionic surfactant between 5 μM and 15 μM yielded a maximal encapsulation efficiency of ETP ata 7.5 μM of catanionic surfactant. An increase in the concentration of Lf/WGA decreased the grafting efficiency of Lf/WGA. Also, an increase in the weight percentage of ETP decreased its encapsulation efficiency. Moreover, the release rate of ETP from WGA-Lf-ETP-CASLNs reduced with increasing concentration of catanionic surfactant, and WGA-Lf-ETP-CASLNs at 12.5 μM of catanionic surfactant exhibited a feature of sustained release. The order in the viability of HBMECs was ETP-CASLNs ≅ Lf-ETP-CASLNs ≅ WGA-Lf-ETP-CASLNs > ETP. The variation in the transendothelial electrical resistance (TEER) and permeability of propidium iodide (PI) was negligible when the concentration of Lf increased. Furthermore, an increase in the concentration of WGA from 0.2 to 0.6 mg/mL insignificantly altered the TEER and permeability of PI. When the concentration of Lf increased from 2.5 to 7.5 μg/mL and the concentration of WGA increased from 2.5 to 5 μg/mL, the enhancement in the permeability of ETP was minor. However, 10 μg/mL of Lf promoted the permeability of ETP using Lf-ETP-CASLNs, and 5 and 10 μg/mL of WGA could considerably improve the permeability of ETP using WGA-Lf-ETP-CASLNs. The order in the efficacy of inhibiting U87MG cells was WGA-Lf-ETP-CASLNs > Lf-ETP-CASLNs > ETP-CASLNs > ETP. As a result, WGA-Lf-ETP-CASLNs reduced the TEER, enhanced the permeability of PI, induced a minor cytotoxicity to HBMECs, increased the permeability of ETP across the BBB, and improved the antiproliferative efficacy of U87MG cells. The grafting of WGA and Lf is crucial to control the medicinal property of ETP-CASLNs and WGA-Lf-ETP-CASLNs can be promising colloidal carriers in GBM management.

Keywords: catanionic solid lipid nanoparticle, etoposide, glioblastoma multiforme, lactoferrin, wheat germ agglutinin

Procedia PDF Downloads 237

Authors: Francisco J. Cedano, Laura M. Pinzón, Camila I. Castro, Felipe Salcedo, Juan P. Casas, Juan C. Briceño

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Complex fractures located in articular surfaces are challenging to treat and their reduction with conventional treatments could compromise the functionality of the affected limb. An adhesive material to treat those fractures is desirable for orthopedic surgeons. This adhesive must be biocompatible and have a high adhesion to bone surface in an aqueous environment. The proposed adhesive is based on chitosan, given its adhesive and biocompatibility properties. Chitosan is mixed with calcium carbonate and hydroxyapatite, which contribute to structural support and a gel like behavior, and glutaraldehyde is used as a cross-linking agent to keep the adhesive mechanical performance in aqueous environment. This work aims to evaluate the rheological, adhesion strength and biocompatibility properties of the proposed adhesive using in vitro tests. The gelification process of the adhesive was monitored by oscillatory rheometry in an ARG-2 TA Instruments rheometer, using a parallel plate geometry of 22 mm and a gap of 1 mm. Time sweep experiments were conducted at 1 Hz frequency, 1% strain and 37°C from 0 to 2400 s. Adhesion strength is measured using a butt joint test with bovine cancellous bone fragments as substrates. The test is conducted at 5 min, 20min and 24 hours after curing the adhesive under water at 37°C. Biocompatibility is evaluated by a cytotoxicity test in a fibroblast cell culture using MTT assay and SEM. Rheological results concluded that the average gelification time of the adhesive is 820±107 s, also it reaches storage modulus magnitudes up to 106 Pa; The adhesive show solid-like behavior. Butt joint test showed 28.6 ± 9.2 kPa of tensile bond strength for the adhesive cured for 24 hours. Also there was no significant difference in adhesion strength between 20 minutes and 24 hours. MTT showed 70 ± 23 % of active cells at sixth day of culture, this percentage is estimated respect to a positive control (only cells with culture medium and bovine serum). High vacuum SEM observation permitted to localize and study the morphology of fibroblasts presented in the adhesive. All captured fibroblasts presented in SEM typical flatted structure with filopodia growth attached to adhesive surface. This project reports an adhesive based on chitosan that is biocompatible due to high active cells presented in MTT test and these results were correlated using SEM. Also, it has adhesion properties in conditions that model the clinical application, and the adhesion strength do not decrease between 5 minutes and 24 hours.

Keywords: bioadhesive, bone adhesive, calcium carbonate, chitosan, hydroxyapatite, glutaraldehyde

Procedia PDF Downloads 321

Authors: S. E. Mohmad-Saberi, W. Song, N. Oliver, M. Adrian, T.C. Hsu, A. Darbyshire

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Biodegradable polyurethane (PU) has emerged as a potential material to promote repair and regeneration of damaged/diseased tissues in heart valve regeneration due to its excellent biomechanical profile. Understanding the effects of sterilization on their properties is vital since they are more sensitive and more critical of porous structures compared to bulk ones. In this study, the effects of low concentration of hydrogen peroxide (H₂O₂) solution sterilization has been investigated to determine whether the procedure would be efficient and non-destructive to porous three-dimensional (3D) elastomeric nanocomposite, polyhedral oligomeric silsesquioxane-terminated poly (ethylene-diethylene glycol succinate-sebacate) urea-urethane (POSS-EDSS-PU) scaffold. All the samples were tested for sterility following sterilization using phosphate buffer saline (PBS) as control and 5 % v/v H₂O₂ solution. The samples were incubated in tryptic soy broth for the cultivation of microorganisms under agitation at 37˚C for 72 hours. The effects of the 5 % v/v H₂O₂ solution sterilization were evaluated in terms of morphology, chemical and mechanical properties using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and tensile tester apparatus. Toxicity effects of the 5 % v/v H₂O₂ solution decontamination were studied by in vitro cytotoxicity test, where the cellular responses of human dermal fibroblast (HDF) were examined. A clear, uncontaminated broth using 5 % v/v H₂O₂ solution method indicated efficient sterilization after 3 days, while the non-sterilized control shows clouding broth indicated contamination. The morphology of 3D POSS-EDSS-PU scaffold appeared to have similar morphology after sterilization with 5 % v/v H₂O₂ solution regarding of pore size and surface. FTIR results show that the sterilized samples and non-sterilized control share the same spectra pattern, confirming no significant alterations over the surface chemistry. For the mechanical properties of the H₂O₂ solution-treated scaffolds, the tensile strain was not significantly decreased, however, become significantly stiffer after the sterilization. No cytotoxic effects were observed after the 5 % v/v H₂O₂ solution sterilization as confirmed by cell viability assessed by Alamar Blue assay. The results suggest that low concentration of 5 % v/v hydrogen peroxide solution can be used as an alternative method for sterilizing biodegradable 3D porous scaffold with micro/nano-architecture without structural deformation. This study provides the understanding of the sterilization effects on biomechanical profile and cell proliferation of 3D POSS-EDSS-PU scaffolds.

Keywords: biodegradable, hydrogen peroxide solution, POSS-EDSS-PU, sterilization

Procedia PDF Downloads 159

Authors: Daniel Todorov, Anton Hinkov, Petya Angelova, Kalina Shishkova, Venelin Tsvetkov, Stoyan Shishkov

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Human herpes viruses (HHV) are ubiquitous pathogens with a pandemic spread across the globe. HHV type 1 is the main causative agent of cold sores and fever blisters around the mouth and on the face, whereas HHV type 2 is generally responsible for genital herpes outbreaks. The treatment of both viruses is more or less successful with antivirals from the nucleoside analogues group. Their wide application increasingly leads to the emergence of resistant mutants In the past, medicinal plants have been used to treat a number of infectious and non-infectious diseases. Their diversity and ability to produce the vast variety of secondary metabolites according to the characteristics of the environment give them the potential to help us in our warfare with viral infections. The variable chemical characteristics and complex composition is an advantage in the treatment of herpes since the emergence of resistant mutants is significantly complicated. The screening process is difficult due to the lack of standardization. That is why it is especially important to follow the mechanism of antiviral action of plants. On the one hand, it may be expected to interact with its compounds, resulting in enhanced antiviral effects, and the most appropriate environmental conditions can be chosen to maximize the amount of active secondary metabolites. During our study, we followed the activity of various plant extracts on the viral replication cycle as well as their effect on the extracellular virion. We obtained our results following the logical sequence of the experimental settings - determining the cytotoxicity of the extracts, evaluating the overall effect on viral replication and extracellular virion.During our research, we have screened a variety of plant extracts for their antiviral activity against both virus replication and the virion itself. We investigated the effect of the extracts on the individual stages of the viral replication cycle - viral adsorption, penetration and the effect on replication depending on the time of addition. If there are positive results in the later experiments, we had studied the activity over viral adsorption, penetration and the effect of replication according to the time of addition. Our results indicate that some of the extracts from the Lamium album have several targets. The first stages of the viral life cycle are most affected. Several of our active antiviral agents have shown an effect on extracellular virion and adsorption and penetration processes. Our research over the last decade has shown several curative antiviral plants - some of which are from the Lamiacea family. The rich set of active ingredients of the plants in this family makes them a good source of antiviral preparation.

Keywords: human herpes virus, antiviral activity, Lamium album, Nepeta nuda

Procedia PDF Downloads 154

Authors: Priyanka Bhowmik, Subrata Majumdar, Debprasad Chattopadhyay

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Background: Cervical cancer is the third major cause of cancer in women and the second most frequent cause of cancer related deaths causing 300,000 deaths annually worldwide. Evasion of immune response by Human Papilloma Virus (HPV), the key contributing factor behind cancer and pre-cancerous lesions of the uterine cervix, makes immunotherapy a necessity to treat this disease. Objective: A Heat killed fraction of Mycobacterium indicus pranii (MIP), a non-pathogenic Mycobacterium has been shown to exhibit cytotoxic effects on different cancer cells, including human cervical carcinoma cell line HeLa. However, the underlying mechanisms remain unknown. The aim of this study is to decipher the mechanism of MIP induced HeLa cell death. Methods: The cytotoxicity of Mycobacterium indicus pranii against HeLa cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and Propidium iodide (PI) staining. The assessment of reactive oxygen species (ROS) generation and cell cycle analysis were measured by flow cytometry. The expression of apoptosis associated genes was analyzed by real time PCR. Result: MIP could inhibit the proliferation of HeLa cell in a time and dose dependent manner but caused minor damage to normal cells. The induction of apoptosis was confirmed by the cell surface presentation of phosphatidyl serine, DNA fragmentation, and mitochondrial damage. MIP caused very early (as early as 30 minutes) transcriptional activation of p53, followed by a higher activation (32 fold) at 24 hours suggesting prime importance of p53 in MIP-induced apoptosis in HeLa cell. The up regulation of p53 dependent pro-apoptotic genes Bax, Bak, PUMA, and Noxa followed a lag phase that was required for the transcriptional p53 program. MIP also caused the transcriptional up regulation of Toll like receptor 2 and 4 after 30 minutes of MIP treatment suggesting recognition of MIP by toll like receptors. Moreover, MIP caused the inhibition of expression of HPV anti apoptotic gene E6, which is known to interfere with p53/PUMA/Bax apoptotic cascade. This inhibition might have played a role in transcriptional up regulation of PUMA and subsequently apoptosis. ROS was generated transiently which was concomitant with the highest transcription activation of p53 suggesting a plausible feedback loop network of p53 and ROS in the apoptosis of HeLa cells. Scavenger of ROS, such as N-acetyl-L-cysteine, decreased apoptosis suggesting ROS is an important effector of MIP induced apoptosis. Conclusion: Taken together, MIP possesses full potential to be a novel therapeutic agent in the clinical treatment of cervical cancer.

Keywords: cancer, mycobacterium, immunity, immunotherapy.

Procedia PDF Downloads 249

Authors: Ahlam Ali, Katrina Lappin, Jaine Blayney, Ken Mills

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Leukaemia is the most frequently (30%) occurring type of paediatric cancer. Of these, approximately 80% are acute lymphoblastic leukaemia (ALL) with acute myeloid leukaemia (AML) cases making up the remaining 20% alongside other leukaemias. Unfortunately, children with AML do not have promising prognosis with only 60% surviving 5 years or longer. It has been highlighted recently the need for age-specific therapies for AML patients, with paediatric AML cases having a different mutational landscape compared with AML diagnosed in adult patients. Drug Repurposing is a recognized strategy in drug discovery and development where an already approved drug is used for diseases other than originally indicated. We aim to identify novel combination therapies with the promise of providing alternative more effective and less toxic induction therapy options. Our in-silico analysis highlighted ‘cell death and survival’ as an aberrant, potentially targetable pathway in paediatric AML patients. On this basis, 83 apoptotic inducing compounds were screened. A preliminary single agent screen was also performed to eliminate potentially toxic chemicals, then drugs were constructed into a pooled library with 10 drugs per well over 160 wells, with 45 possible pairs and 120 triples in each well. Seven cell lines were used during this study to represent the clonality of AML in paediatric patients (Kasumi-1, CMK, CMS, MV11-14, PL21, THP1, MOLM-13). Cytotoxicity was assessed up to 72 hours using CellTox™ Green reagent. Fluorescence readings were normalized to a DMSO control. Z-Score was assigned to each well based on the mean and standard deviation of all the data. Combinations with a Z-Score <2 were eliminated and the remaining wells were taken forward for further analysis. A well was considered ‘successful’ if each drug individually demonstrated a Z-Score <2, while the combination exhibited a Z-Score >2. Each of the ten compounds in one well (155) had minimal or no effect as single agents on cell viability however, a combination of two or more of the compounds resulted in a substantial increase in cell death, therefore the ten compounds were de-convoluted to identify a possible synergistic pair/triple combinations. The screen identified two possible ‘novel’ drug pairing, with BCL2 inhibitor ABT-737, combined with either a CDK inhibitor Purvalanol A, or AKT/ PI3K inhibitor LY294002. (ABT-737- 100 nM+ Purvalanol A- 1 µM) (ABT-737- 100 nM+ LY294002- 2 µM). Three possible triple combinations were identified (LY2409881+Akti-1/2+Purvalanol A, SU9516+Akti-1/2+Purvalanol A, and ABT-737+LY2409881+Purvalanol A), which will be taken forward for examining their efficacy at varying concentrations and dosing schedules, across multiple paediatric AML cell lines for optimisation of maximum synergy. We believe that our combination screening approach has potential for future use with a larger cohort of drugs including FDA approved compounds and patient material.

Keywords: AML, drug repurposing, ABT-737, apoptosis

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Authors: Belgheis Ebrahimi, Jun Lu

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In recent years, there has been a growing recognition of the potential of marine-based functional foods and combination therapies in promoting a healthy lifestyle and exploring their effectiveness in preventing or treating diseases. The combination of marine bioactive compounds or extracts offers synergistic or enhancement effects through various mechanisms, including multi-target actions, improved bioavailability, enhanced bioactivity, and mitigation of potential adverse effects. Both the green-lipped mussel (GLM) and fucoidan derived from brown seaweed are rich in bioactivities. These two, mussel and fucoidan, have not been previously formulated together. This study aims to combine GLM oil from Perna canaliculus with low molecular weight fucoidan (LMWF) extracted from Undaria pinnatifida to investigate the unique mixture’s anti-inflammatory and antioxidant properties. The cytotoxicity of individual compounds and combinations was assessed using the MTT assay in (THP-1 and RAW264.7) cell lines. The anti-inflammatory activity of mussel-fucoidan was evaluated by treating LPS-stimulated human monocyte and macrophage (THP1-1) cells. Subsequently, the inflammatory cytokines released into the supernatant of these cell lines were quantified via ELISA. Antioxidant activity was determined by using the free radical scavenging assay (DPPH). DPPH assay demonstrated that the radical scavenging activity of the combinations, particularly at concentrations exceeding 1 mg/ml, showed a significantly higher percentage of inhibition when compared to the individual component. This suggests an enhancement effect when the two compounds are combined, leading to increased antioxidant activity. In terms of immunomodulatory activity, the individual compounds exhibited distinct behaviors. GLM oil displayed a higher ability to suppress the cytokine TNF- compared to LMWF. Interestingly, the LMWF fraction, when used individually, did not demonstrate TNF- suppression. However, when combined with GLM, the TNF- suppression (anti-inflammatory) activity of the combination was better than GLM or LWMF alone. This observation underscores the potential for enhancement interactions between the two components in terms of anti-inflammatory properties. This study revealed that each individual compound, LMWF, and GLM, possesses unique and notable bioactivity. The combination of these two individual compounds results in an enhancement effect, where the bioactivity of each is enhanced, creating a superior combination. This suggests that the combination of LMWF and GLM has the potential to offer a more potent and multifaceted therapeutic effect, particularly in the context of antioxidant and anti-inflammatory activities. These findings hold promise for the development of novel therapeutic interventions or supplements that harness the enhancement effects.

Keywords: combination, enhancement effect, perna canaliculus, undaria pinnatifida

Procedia PDF Downloads 81

Authors: Kai-Cheng Yang, Yen-Ling Chen, Er-Chieh Cho, Kuen-Chan Lee

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Titanium dioxide nanomaterials offer superior protection for human skin against the full spectrum of ultraviolet light. However, some literature reviews indicated that it might be associated with adverse effects such as cytotoxicity or reactive oxygen species (ROS) due to their nanoscale. The surface of fullerene is covered with π electrons constituting aromatic structures, which can effectively scavenge large amount of radicals. Unfortunately, fullerenes are poor solubility in water, severe aggregation, and toxicity in biological applications when dispersed in solvent have imposed the limitations to the use of fullerenes. Carboxyfullerene acts as the scavenger of radicals for several years. Some reports indicate that carboxyfullerene not only decrease the concentration of free radicals in ambience but also prevent cells from reducing the number or apoptosis under UV irradiation. The aim of this study is to decorate fullerene –C70-carboxylic acid (C70-COOH) on the surface of titanium dioxide nanoparticles (P25) for the purpose of scavenging ROS during the irradiation. The modified material is prepared through the esterification of C70-COOH with P25 (P25/C70-COOH). The binding edge and structure are studied by using Transmission electron microscope (TEM) and Fourier transform infrared (FTIR). The diameter of P25 is about 30 nm and C70-COOH is found to be conjugated on the edge of P25 in aggregation morphology with the size of ca. 100 nm. In the next step, the FTIR was used to confirm the binding structure between P25 and C70-COOH. There are two new peaks are shown at 1427 and 1720 cm-1 for P25/C70-COOH, resulting from the C–C stretch and C=O stretch formed during esterification with dilute sulfuric acid. The IR results further confirm the chemically bonded interaction between C70-COOH and P25. In order to provide the evidence of scavenging radical ability of P25/C70-COOH, we chose pyridoxine (Vit.B6) and terephthalic acid (TA) to react with singlet oxygen and hydroxyl radicals. We utilized these chemicals to observe the radicals scavenging statement via detecting the intensity of ultraviolet adsorption or fluorescence emission. The UV spectra are measured by using different concentration of C70-COOH modified P25 with 1mM pyridoxine under UV irradiation for various duration times. The results revealed that the concentration of pyridoxine was increased when cooperating with P25/C70-COOH after three hours as compared with control (only P25). It indicates fewer radicals could be reacted with pyridoxine because of the absorption via P25/C70-COOH. The fluorescence spectra are observed by measuring P25/C70-COOH with 1mM terephthalic acid under UV irradiation for various duration times. The fluorescence intensity of TAOH was decreased in ten minutes when cooperating with P25/C70-COOH. Here, it was found that the fluorescence intensity was increased after thirty minutes, which could be attributed to the saturation of C70-COOH in the absorption of radicals. However, the results showed that the modified P25/C70-COOH could reduce the radicals in the environment. Therefore, we expect that P25/C70-COOH is a potential materials in using for antioxidant.

Keywords: titanium dioxide, fullerene, radical scavenging activity, antioxidant

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Authors: Nima Samie, Sekaran Muniandy, Zahurin Mohamed, M. S. Kanthimathi

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Although number of indole containing compounds have been reported to have anticancer properties in vitro but only a few of them show potential as anticancer compounds in vivo. The current study was to evaluate the mechanism of cytotoxicity of selected indolic compound in vivo and in vitro. In this context, we determined the potency of the compound in the induction of apoptosis, cell cycle arrest, and cytoskeleton rearrangement. HT-29, WiDr, CCD-18Co, human monocyte/macrophage CRL-9855, and B lymphocyte CCL-156 cell lines were used to determine the IC50 of the compound using the MTT assay. Analysis of apoptosis was carried out using immunofluorescence, acridine orange/ propidium iodide double staining, Annexin-V-FITC assay, evaluation of the translocation of NF-kB, oxygen radical antioxidant capacity, quenching of reactive oxygen species content, measurement of LDH release, caspase-3/-7, -8 and -9 assays and western blotting. The cell cycle arrest was examined using flowcytometry and gene expression was assessed using qPCR array. Results displayed a potent suppressive effect on HT-29 and WiDr after 24 h of treatment with IC50 value of 2.52±0.34 µg/ml and 2.13±0.65 µg/ml respectively. This cytotoxic effect on normal, monocyte/macrophage and B-cells was insignificant. Dipping in the mitochondrial membrane potential and increased release of cytochrome c from the mitochondria indicated induction of the intrinsic apoptosis pathway by the compound. Activation of this pathway was further evidenced by significant activation of caspase-9 and 3/7. The compound was also shown to activate the extrinsic pathways of apoptosis via activation of caspase-8 which is linked to the suppression of NF-kB translocation to the nucleus. Cell cycle arrest in the G1 phase and up-regulation of glutathione reductase, based on excessive ROS production were also observed. These findings were further investigated for inhibitory efficiency of the compound on colonic aberrant crypt foci in male rats. Rats were divided in to 5 groups: vehicle, cancer control, positive control groups and the groups treated with 25 and 50 mg/kg of compounds for 10 weeks. Administration of compound suppressed total colonic ACF formation up to 73.4%. The results also showed that treatment with the compound significantly reduced the level of malondialdehyde while increasing superoxide dismutase and catalase activities. Furthermore, the down-regulation of PCNA and Bcl2 and the up-regulation of Bax was confirmed by immunohistochemical staining. The outcome of this study suggest sthat the indolic compound is a potent anti-cancer agent against colon cancer and can be further evaluated by animal trial.

Keywords: indolic compound, chemoprevention, crypt, azoxymethane, colon cancer

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Authors: Juan Bernal-Martinez, Zoe Quinones-Jurado, Miguel Waldo-Mendoza, Elias Perez

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Introduction and Objective: Ag-TiO2 nanoparticles (NP) have been characterized as effective antibacterial compounds against E. aureous, E. coli, Salmonella and others. Because these nanoparticles have been used in plastic-food containers, there is a concern about the toxicity of Ag-TiO2 NP for higher organisms from protozoan, invertebrates, and mammals. The objective of this study is to evaluate the cytotoxic effect of Ag-TiO2 NP on the survival and swimming behavior of the unicellular organism Paramecium tetraurelia. Material and Methods: Preparation of metallic silver on TiO2 surface was based on chemical reduction route of AgNO3. Aqueous suspension of TiO2 nanoparticles was preparing by adding 5 g of TiO2 to 250 ml of deionized water and followed by sonication for 10 min. The required amount of AgNO3 solutions was added to TiO2 suspension, maintaining heating and stirring. Silver concentration was 0.5, 1.5, 5.0, 25, 35 and 45 % w/w versus TiO2. Paramecium tetraurelia (Carolina Biological, Cat. # 131560) was used as a biological preparation. It was cultured in artificial culture media made as follows: Stigmasterol 5 mg/ml of ethanol, Caseaminoacids 0.3 gr/lt.; KCl 4mM; CaCl2 1mM; MgCl2 100uM and MOPS 1mM, pH 7.3. This media was inoculated with Enterobacter-sp. Paramecium was concentrated after 24 hours of incubation by centrifugation. The pellet of cells was resuspended in 4.1.1 solution prepared as follows (in mM): KCl, 4 mM; CaCl2, 1mM and Trizma, 1mM; pH 7.3. Transmission electron microscopy (TEM) studies were performed to evaluate the appropriate dispersion and topographic distribution AgNPs deposited on TiO2. The experimental solutions were prepared as follows: 50 mg of Polyvinyhlpirolidone were added to 5 ml of 4.1.1. solution. Then, 50 mg of powder 25-Ag-TiO2 was added, mixing for 10 min and sonicated for 60 min. Survival of Paramecium and possible toxic effects after 25-Ag-TiO2 treatment was observed through an inverted microscope. The Paramecium swimming behavior and possible dead cells were recorded for periods of approximately 20-50 seconds by using a digital USB camera adapted to the microscope. Results and Discussion: TEM micrographs demonstrated the topographic distribution of AgNPs deposited on TiO2. 25Ag-TiO2 NP was efficiently dissolved and dispersed in 4.1.1 solution at concentrations from 0.1, 1 and 10 mg/ml. When Paramecium were treated with 25Ag-TiO2 NP at 100 ug/ml, it was observed that cells started swimming backwards. This backward swimming behavior is the typical avoiding reaction of the ciliate in response to a noxious stimulus. After 10 min of incubation, it was observed that Paramecium stopped swimming backwards and exploited. We can argue that this toxic effect of 25Ag-TiO2 NP is probably due to the calcium influx and calcium accumulation during the long-lasting swimming backwards. Conclusions: Here we have demonstrated that 25Ag-TiO2 NP has a specific toxic effect on an organism higher than bacteria such as the protozoan Paremecium. Probably these toxic phenomena could be expected to be observed in a higher organism such as invertebrates and mammals.

Keywords: Ag-TiO2, calcium permeability, cytotoxicity, paramecium

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Authors: Z. Karahaliloğlu, E. Yalçın, M. Demirbilek, E.B. Denkbaş

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Critical-sized bone defects caused by trauma, bone diseases, prosthetic implant revision or tumor excision cannot be repaired by physiological regenerative processes. Current orthopedic applications for critical-sized bone defects are to use autologous bone grafts, bone allografts, or synthetic graft materials. However, these strategies are unable to solve completely the problem, and motivate the development of novel effective biological scaffolds for tissue engineering applications and regenerative medicine applications. In particular, scaffolds combined with a variety of bio-agents as fundamental tools emerge to provide the regeneration of damaged bone tissues due to their ability to promote cell growth and function. In this study, a magnetic silk fibroin (SF) hydrogel scaffold was prepared by electrogelation process of the concentrated Bombxy mori silk fibroin (8 %wt) aqueous solution. For enhancement of osteoblast-like cells (SaOS-2) growth and adhesion, basal fibroblast growth factor (bFGF) were conjugated physically to the HSA-coated magnetic nanoparticles (Fe3O4) and magnetic SF e-gel scaffolds were prepared by incorporation of Fe3O4, HSA (human serum albumin)=Fe3O4 and HSA=Fe3O4-bFGF nanoparticles. HSA=Fe3O4, HSA=Fe3O4-bFGF loaded and bare SF e-gels scaffolds were characterized using scanning electron microscopy (SEM.) For cell studies, human osteoblast-like cell line (SaOS-2) was used and an MTT assay was used to assess the cytotoxicity of magnetic silk fibroin e-gel scaffolds and cell density on these surfaces. For the evaluation osteogenic activation, ALP (alkaline phosphatase), the amount of mineralized calcium, total protein and collagen were studied. Fe3O4 nanoparticles were successfully synthesized and bFGF was conjugated to HSA=Fe3O4 nanoparticles with %97.5 of binding yield which has a particle size of 71.52±2.3 nm. Electron microscopy images of the prepared HSA and bFGF incorporated SF e-gel scaffolds showed a 3D porous morphology. In terms of water uptake results, bFGF conjugated HSA=Fe3O4 nanoparticles has the best water absorbability behavior among all groups. In the in-vitro cell culture studies realized using SaOS-2 cell line, the coating of Fe3O4 nanoparticles surface with a protein enhance the cell viability and HSA coating and bFGF conjugation, the both have an inductive effect in the cell proliferation. One of the markers of bone formation and osteoblast differentiation, according to the ALP activity and total protein results, HSA=Fe3O4-bFGF loaded SF e-gels had significantly enhanced ALP activity. Osteoblast cultured HSA=Fe3O4-bFGF loaded SF e-gels deposited more calcium compared with SF e-gel. The proposed magnetic scaffolds seem to be promising for bone tissue regeneration and used in future work for various applications.

Keywords: basic fibroblast growth factor (bFGF), e-gel, iron oxide nanoparticles, silk fibroin

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Authors: Katarzyna Bialik-Wąs, Klaudia Pluta, Dagmara Malina, Małgorzata Miastkowska

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In recent years, biocompatible hydrogels have been used more and more in medical applications, especially as modern dressings and drug delivery systems. The main goal of this research was the characteristics of bio-hybrid hydrogel materials incorporated with the nanocarrier-drug system, which enable the release in a gradual and prolonged manner, up to 7 days. Therefore, the use of such a combination will provide protection against mechanical damage and adequate hydration. The proposed bio-hybrid hydrogels are characterized by: transparency, biocompatibility, good mechanical strength, and the dual release system, which allows for gradual delivery of the active substance, even up to 7 days. Bio-hybrid hydrogels based on sodium alginate (SA), poly(vinyl alcohol) (PVA), glycerine, and Aloe vera solution (AV) were obtained through the chemical crosslinking method using poly(ethylene glycol) diacrylate as a crosslinking agent. Additionally, a nanocarrier-drug system was incorporated into SA/PVA/AV hydrogel matrix. Here, studies were focused on the release profiles of active substances from bio-hybrid hydrogels using the USP4 method (DZF II Flow-Through System, Erweka GmbH, Langen, Germany). The equipment incorporated seven in-line flow-through diffusion cells. The membrane was placed over support with an orifice of 1,5 cm in diameter (diffusional area, 1.766 cm²). All the cells were placed in a cell warmer connected with the Erweka heater DH 2000i and the Erweka piston pump HKP 720. The piston pump transports the receptor fluid via seven channels to the flow-through cells and automatically adapts the setting of the flow rate. All volumes were measured by gravimetric methods by filling the chambers with Milli-Q water and assuming a density of 1 g/ml. All the determinations were made in triplicate for each cell. The release study of the model active substance was carried out using a regenerated cellulose membrane Spectra/Por®Dialysis Membrane MWCO 6-8,000 Carl Roth® Company. These tests were conducted in buffer solutions – PBS at pH 7.4. A flow rate of receptor fluid of about 4 ml /1 min was selected. The experiments were carried out for 7 days at a temperature of 37°C. The released concentration of the model drug in the receptor solution was analyzed using UV-Vis spectroscopy (Perkin Elmer Company). Additionally, the following properties of the modified materials were studied: physicochemical, structural (FT-IR analysis), morphological (SEM analysis). Finally, the cytotoxicity tests using in vitro method were conducted. The obtained results exhibited that the dual release system allows for the gradual and prolonged delivery of the active substances, even up to 7 days.

Keywords: wound dressings, SA/PVA hydrogels, nanocarrier-drug system, USP4 method

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Authors: Ahmed Tawfik

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Oxidation of 1,4 dioxane produces metabolites by-products involving glycolaldehyde and acids that have geno- and cytotoxicity impact on microbial degradation. Thereby, the incorporation of algae with bacteria in the treatment system would eliminate and overcome the accumulation of metabolites that are utilized as a carbon source for the build-up of biomass. Therefore, the aim of the present study is to assess the potential of algae/bacteria-based membrane bioreactor (AB-MBR) for biodegradation of 1,4 dioxane-rich wastewater at a high imposed loading rate. Three identical reactors, i.e., AB-MBR1, AB-MBR2, and AB-MBR3, were operated in parallel at 1,4 dioxane loading rates of 641.7, 320.9, and 160.4 mg/L. d., and HRTs of 6.0, 12 and 24 h. respectively. The AB-MBR1 achieved 1,4 dioxane removal rate of 263.7 mg/L.d., where the residual value in the treated effluent amounted to 94.4±22.9 mg/L. Reducing the 1,4 dioxane loading rate (LR) to 320.9 mg/L.d in the AB-MBR2 maximized the removal rate efficiency of 265.9 mg/L.d., with a removal efficiency of 82.8±3.2%. The minimum value of 1,4 dioxane of 17.3±1.8 mg/L in the treated effluent of AB-MBR3 was obtained at an HRT of 24.0 h and loading rate of 160.4 mg/L.d. The mechanism of 1,4 dioxane degradation in AB-MBR was a combination of volatilization (8.03±0.6%), UV oxidation (14.1±0.9%), microbial biodegradation (49.1±3.9%) and absorption/uptake and assimilation by algae (28.8±2.%). Further, the Thioclava, Afipia, and Mycobacterium genera oxidized and produced the required enzymes for hydrolysis and cleavage of the dioxane ring into 2-hydroxy-1,4 dioxane. Moreover, the fungi, i.e., Basidiomycota and Cryptomycota, played a big role in the degradation of the 1,4 dioxane into 2-hydroxy-1,4 dioxane. Xanthobacter and Mesorhizobium were involved in the metabolism process by secreting alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and glycolate oxidase. Bacteria and fungi produced dehydrogenase (DH) for the transformation of 2-hydroxy-1,4 dioxane into 2-hydroxy-ethoxyacetaldehyde. The latter is converted into Ethylene glycol by Aldehyde hydrogenase (ALDH). Ethylene glycol is oxidized into acids using Alcohol hydrogenase (ADH). The Diatomea, Chlorophyta, and Streptophyta utilize the metabolites for biomass assimilation and produce the required oxygen for further oxidation of the dioxane and its metabolites by-products of bacteria and fungi. The major portion of metabolites (ethylene glycol, glycolic acid, and oxalic acid were removed due to uptake and absorption by algae (43±4.3%), followed by adsorption (18.4±0.9%). The volatilization and UV oxidation contribution for the degradation of metabolites were 8.7±0.7% and 12.3±0.8%, respectively. The capabilities of genera Defluviimonas, Thioclava, Luteolibacter, and Afipia. The genera of Defluviimonas, Thioclava, Luteolibacter, and Mycobacterium were grown under a high 1,4 dioxane LR of 641.7 mg/L.d. The Chlorophyta (4.1-43.6%), Streptophyta (2.5-21.7%), and Diatomea (0.8-1.4%) phyla were dominant for degradation of 1,4 dioxane. The results of this study strongly demonstrated that the bioremediation and bioaugmentation process can safely remove 1,4 dioxane from industrial wastewater while minimizing environmental concerns and reducing economic costs.

Keywords: wastewater, membrane bioreactor, bacterial community, algal community

Procedia PDF Downloads 43

Authors: Faraj M. Elkhatri, Hana Ellafi

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In recent years, there has been a growing recognition of the potential of marine-based functional foods and combination therapies in promoting a healthy lifestyle and exploring their effectiveness in preventing or treating diseases. The combination of marine bioactive compounds or extracts offers synergistic or enhancement effects through various mechanisms, including multi-target actions, improved bioavailability, enhanced bioactivity, and mitigation of potential adverse effects. Both the green-lipped mussel (GLM) and fucoidan derived from brown seaweed are rich in bioactivities. These two, mussel and fucoidan, have not been previously formulated together. This study aims to combine GLM oil from Perna canaliculus with low molecular weight fucoidan (LMWF) extracted from Undaria pinnatifida to investigate the unique mixture’s anti-inflammatory and antioxidant properties. The cytotoxicity of individual compounds and combinations was assessed using the MTT assay in (THP-1 and RAW264.7) cell lines. The anti-inflammatory activity of mussel-fucoidan was evaluated by treating LPS-stimulated human monocyte and macrophage (THP1-1) cells. Subsequently, the inflammatory cytokines released into the supernatant of these cell lines were quantified via ELISA. Antioxidant activity was determined by using the free radical scavenging assay (DPPH). DPPH assay demonstrated that the radical scavenging activity of the combinations, particularly at concentrations exceeding 1 mg/ml, showed a significantly higher percentage of inhibition when compared to the individual component. This suggests an enhancement effect when the two compounds are combined, leading to increased antioxidant activity. In terms of immunomodulatory activity, the individual compounds exhibited distinct behaviors. GLM oil displayed a higher ability to suppress the cytokine TNF- compared to LMWF. Interestingly, the LMWF fraction, when used individually, did not demonstrate TNF- suppression. However, when combined with GLM, the TNF- suppression (anti-inflammatory) activity of the combination was better than GLM or LWMF alone. This observation underscores the potential for enhancement interactions between the two components in terms of anti-inflammatory properties. This study revealed that each individual compound, LMWF, and GLM, possesses unique and notable bioactivity. The combination of these two individual compounds results in an enhancement effect, where the bioactivity of each is enhanced, creating a superior combination. This suggests that the combination of LMWF and GLM has the potential to offer a more potent and multifaceted therapeutic effect, particularly in the context of antioxidant and anti-inflammatory activities. These findings hold promise for the development of novel therapeutic interventions or supplements that harness the enhancement effects.

Keywords: formation damage, porosity loses, pore throat, quartz cement

Procedia PDF Downloads 56

Authors: Kirti Hira, A. Sajeli Begum, S. Mahibalan, Poorna Chandra Rao

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Introduction: Cancer is one of the leading causes of death worldwide. Although existing therapy effectively kills cancer cells, they do affect normal growing cells leading to many undesirable side effects. Hence there is need to develop effective as well as safe drug molecules to combat cancer, which is possible through phyto-research. The currently available plant-derived blockbuster drugs are the example for this. In view of this, an investigation was done to identify cytotoxic lead molecules from Hedyotis umbellata (Family Rubiaceae), a widely distributed weed in India. Materials and Methods: The methanolic extract of the whole plant of H. umbellata (MHU), prepared through Soxhlet extraction method was further fractionated with diethyl ether and n-butanol, successively. MHU, ether fraction (EMHU) and butanol fraction (BMHU) were lyophilized and were tested for the cytotoxic effect using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay against non-small cell lung cancer (NSCLC) A549 cell lines. The potentially active EMHU was subjected to chromatographic purification using normal-phase silica columns, in order to isolate the responsible bioactive compounds. The isolated pure compounds were tested for their cytotoxic effect by MTT assay against A549 cells. Compound-3, which was found to be most active, was characterized using IR, 1H- and 13C-NMR and MS analysis. The study was further extended to decipher the mechanism of action of cytotoxicity of compound-3 against A549 cells through various in vitro cellular models. Cell cycle analysis was done using flow cytometry following PI (Propidium Iodide) staining. Protein analysis was done using Western blot technique. Results: Among MHU, EMHU, and BMHU, the non-polar fraction EMHU demonstrated a significant dose-dependent cytotoxic effect with IC50 of 67.7μg/ml. Chromatography of EMHU yielded seven compounds. MTT assay of isolated compounds explored compound-3 as potentially active one, which inhibited the growth of A549 cells with IC50value of 14.2μM. Further, compound-3 was identified as cedrelopsin, a coumarin derivative having molecular weight of 260. Results of in vitro mechanistic studies explained that cedrelopsin induced cell cycle arrest at G2/M phase and down-regulated the expression of G2/M regulatory proteins such as cyclin B1, cdc2, and cdc25C, dose dependently. This is the first report that explores the cytotoxic mechanism of cedrelopsin. Conclusion: Thus a potential small lead molecule, cedrelopsin isolated from H. umbellata, showing antiproliferative effect mediated by G2/M arrest in A549 cells was discovered. The effect of cedrelopsin against other cancer cell lines followed by in vivo studies can be performed in future to develop a new drug candidate.

Keywords: A549, cedrelopsin, G2/M phase, Hedyotis umbellata

Procedia PDF Downloads 175

Authors: Aniruddha Mitra, Abbas Rashidi, Shane Lewis, Jefferson Doehling, Alexis Pawlak, Jacob Schwartz, Imaobong Ekpo, Atin Adhikari

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Working or living close to demolition sites can increase risks of dust-related health problems. Demolition of concrete buildings may produce crystalline silica dust, which can be associated with a broad range of respiratory diseases including silicosis and lung cancers. Previous studies demonstrated significant associations between demolition dust exposure and increase in the incidence of mesothelioma or asbestos cancer. Dust is a generic term used for minute solid particles of typically <500 µm in diameter. Dust particles in demolition sites vary in a wide range of sizes. Larger particles tend to settle down from the air. On the other hand, the smaller and lighter solid particles remain dispersed in the air for a long period and pose sustained exposure risks. Submicron ultrafine particles and nanoparticles are respirable deeper into our alveoli beyond our body’s natural respiratory cleaning mechanisms such as cilia and mucous membranes and are likely to be retained in the lower airways. To our knowledge, how various demolition tasks release nanoparticles are largely unknown and previous studies mostly focused on course dust, PM2.5, and PM10. General belief is that the dust generated during demolition tasks are mostly large particles formed through crushing, grinding, or sawing of various concrete and wooden structures. Therefore, little consideration has been given to the generated submicron ultrafine and nanoparticles and their exposure levels. These data are, however, critically important because recent laboratory studies have demonstrated cytotoxicity of nanoparticles on lung epithelial cells. The above-described knowledge gaps were addressed in this study by a novel newly developed nanoparticle monitor, which was used for nanoparticle monitoring at two adjacent indoor and outdoor building demolition sites in southern Georgia. Nanoparticle levels were measured (n = 10) by TSI NanoScan SMPS Model 3910 at four different distances (5, 10, 15, and 30 m) from the work location as well as in control sites. Temperature and relative humidity levels were recorded. Indoor demolition works included acetylene torch, masonry drilling, ceiling panel removal, and other miscellaneous tasks. Whereas, outdoor demolition works included acetylene torch and skid-steer loader use to remove a HVAC system. Concentration ranges of nanoparticles of 13 particle sizes at the indoor demolition site were: 11.5 nm: 63 – 1054/cm³; 15.4 nm: 170 – 1690/cm³; 20.5 nm: 321 – 730/cm³; 27.4 nm: 740 – 3255/cm³; 36.5 nm: 1,220 – 17,828/cm³; 48.7 nm: 1,993 – 40,465/cm³; 64.9 nm: 2,848 – 58,910/cm³; 86.6 nm: 3,722 – 62,040/cm³; 115.5 nm: 3,732 – 46,786/cm³; 154 nm: 3,022 – 21,506/cm³; 205.4 nm: 12 – 15,482/cm³; 273.8 nm: Keywords: demolition dust, industrial hygiene, aerosol, occupational exposure

Procedia PDF Downloads 423

Authors: Lina Paola Orozco Marin, Yuliet Montoya Osorio, John Bustamante Osorno

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Ischemic events can culminate in acute myocardial infarction by irreversible cardiac lesions that cannot be restored due to the limited regenerative capacity of the heart. Cell therapy seeks to replace these injured or necrotic cells by transplanting healthy and functional cells. The therapeutic alternatives proposed by tissue engineering and cardiovascular regenerative medicine are the use of biomaterials to mimic the native extracellular medium, which is full of proteins, proteoglycans, and glycoproteins. The selected biomaterials must provide structural support to the encapsulated cells to avoid their migration and death in the host tissue. In this context, the present research work focused on developing a natural thermosensitive hydrogel, its physical and chemical characterization, and the determination of its biocompatibility in vitro. The hydrogel was developed by mixing hydrolyzed bovine and porcine collagen at 2% w/v, chitosan at 2.5% w/v, and beta-glycerolphosphate at 8.5% w/w and 10.5% w/w in magnetic stirring at 4°C. Once obtained, the thermosensitivity and gelation time were determined, incubating the samples at 37°C and evaluating them through the inverted tube method. The morphological characterization of the hydrogels was carried out through scanning electron microscopy. Chemical characterization was carried out employing infrared spectroscopy. The biocompatibility was determined using the MTT cytotoxicity test according to the ISO 10993-5 standard for the hydrogel’s precursors using the fetal human ventricular cardiomyocytes cell line RL-14. The RL-14 cells were also seeded on the top of the hydrogels, and the supernatants were subculture at different periods to their observation under a bright field microscope. Four types of thermosensitive hydrogels were obtained, which differ in their composition and concentration, called A1 (chitosan/bovine collagen/beta-glycerolphosphate 8.5%w/w), A2 (chitosan/porcine collagen/beta-glycerolphosphate 8.5%), B1 (chitosan/bovine collagen/beta-glycerolphosphate 10.5%) and B2 (chitosan/porcine collagen/beta-glycerolphosphate 10.5%). A1 and A2 had a gelation time of 40 minutes, and B1 and B2 had a gelation time of 30 minutes at 37°C. Electron micrographs revealed a three-dimensional internal structure with interconnected pores for the four types of hydrogels. This facilitates the exchange of nutrients, oxygen, and the exit of metabolites, allowing to preserve a microenvironment suitable for cell proliferation. In the infrared spectra, it was possible to observe the interaction that occurs between the amides of polymeric compounds with the phosphate groups of beta-glycerolphosphate. Finally, the biocompatibility tests indicated that cells in contact with the hydrogel or with each of its precursors are not affected in their proliferation capacity for a period of 16 days. These results show the potential of the hydrogel to increase the cell survival rate in the cardiac cell therapies under investigation. Moreover, the results lay the foundations for its characterization and biological evaluation in both in vitro and in vivo models.

Keywords: cardiac cell therapy, cardiac ischemia, natural polymers, thermosensitive hydrogel

Procedia PDF Downloads 191

Authors: Sweta Agrawal, Sachin Chaugule, Gargi Rane, Shashank More, Madhavi Indap

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Angiogenesis and metastasis are two of the most important hallmarks of cancer. Hence, most of the cancer therapies nowadays are multi-targeted so as to reduce resistance and have better efficacy. As synthetic molecules arise with a burden of their toxicities and side-effects, more and more research is being focussed on exploiting the vast natural resources of drugs, in the form of plants and animals. Although, the idea of using marine organisms as a source of pharmaceuticals is not new, the pace at which marine drugs are being discovered, has definitely up surged! In the present study, we have assessed the anti-angiogenic and in vitro anti-metastatic activity of aqueous fraction from the extract of marine gastropod Euchelus asper. The soft body of Euchelus Asper was extracted with methanol and named EAME. Partition chromatography of EAME gave three fractions EAME I, II and III. Biochemical analysis revealed the presence of proteins in EAME III. Preliminary analysis had revealed the anti-angiogenic activity was exhibited by EAME III out of the three fractions. Hereafter, EAME III (concentration 25µg/ml-400µg/ml) was tested on chick chorioallantoic membrane (CAM) model for the detailed analysis of its potential anti-angiogenic effect. In vitro testing of the fraction (concentration 0.25µg/ml - 1µg/ml), involved cytotoxicity by SRB assay, cell cycle analysis by flow cytometry and anti-proliferative effect by scratch wound healing assay on A549 lung carcinoma cells. Apart from this, a portion of treated CAM as well as conditioned medium from treated A549 were subjected to gelatin zymography for assessment of matrix metalloproteinases MMP-2 and MMP-9 levels. Our results revealed that EAME III exhibited significant anti-angiogenic activity on CAM which was also supported by histological observations. During histological studies of CAM, it was found that EAME III caused reduction in angiogenesis by altering the extracellular matrix of the CAM membrane. In vitro analysis disclosed that EAME III exhibited moderate cytotoxic effect on A549 cells and its effect was not dose-dependent. The results of flow cytometry confirmed that EAME III caused cell cycle arrest in A549 cell line as almost all of the treated cells were found in G1 phase. Further, the migration and proliferation of A549 was significantly reduced by EAME III as observed from the scratch wound assay. Moreover, Gelatin zymography analysis revealed that EAME III caused suppression of MMP-2 in CAM membrane and reduced MMP-9 and MMP-2 expression in A549 cells. This verified that the anti-angiogenic and anti-metastatic effects of EAME III were correlated with the suppression of MMP-2 and -9. To conclude, EAME III shows dual anti-tumour action by reducing angiogenesis and exerting anti-metastatic effect on lung cancer cells, thus it has the potential to be used as an anti-cancer agent against lung carcinoma.

Keywords: angiogenesis, anti-cancer, marine drugs, matrix metalloproteinases

Procedia PDF Downloads 231

Authors: Elena Filova, Ondrej Kaplan, Marie Markova, Helena Dragounova, Roman Matejka, Eduard Brynda, Lucie Bacakova

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Decellularized vessels have been evaluated as small-diameter vascular prostheses. Reseeding autologous cells onto decellularized tissue prior implantation should prolong prostheses function and make them living tissues. Suitable cell types for reseeding are both endothelial cells and bone marrow-derived stem cells, with a capacity for differentiation into smooth muscle cells upon mechanical loading. Endothelial cells assure antithrombogenicity of the vessels and MSCs produce growth factors and, after their differentiation into smooth muscle cells, they are contractile and produce extracellular matrix proteins as well. Fibrin is a natural scaffold, which allows direct cell adhesion based on integrin receptors. It can be prepared autologous. Fibrin can be modified with bound growth factors, such as basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). These modifications in turn make the scaffold more attractive for cells ingrowth into the biological scaffold. The aim of the study was to prepare thin surface-attached fibrin assemblies with bound FGF-2 and VEGF, and to evaluate growth and differentiation of bone marrow-derived mesenchymal stem cells on the fibrin (Fb) assemblies. Following thin surface-attached fibrin assemblies were prepared: Fb, Fb+VEGF, Fb+FGF2, Fb+heparin, Fb+heparin+VEGF, Fb+heparin+FGF2, Fb+heparin+FGF2+VEGF. Cell culture poly-styrene and glass coverslips were used as controls. Human MSCs (passage 3) were seeded at the density of 8800 cells/1.5 mL alpha-MEM medium with 2.5% FS and 200 U/mL aprotinin per well of a 24-well cell culture. The cells have been cultured on the samples for 6 days. Cell densities on day 1, 3, and 6 were analyzed after staining with LIVE/DEAD cytotoxicity/viability assay kit. The differentiation of MSCs is being analyzed using qPCR. On day 1, the highest density of MSCs was observed on Fb+VEGF and Fb+FGF2. On days 3 and 6, there were similar densities on all samples. On day 1, cell morphology was polygonal and spread on all sample. On day 3 and 6, MSCs growing on Fb assemblies with FGF2 became apparently elongated. The evaluation of expression of genes for von Willebrand factor and CD31 (endothelial cells), for alpha-actin (smooth muscle cells), and for alkaline phosphatase (osteoblasts) is in progress. We prepared fibrin assemblies with bound VEGF and FGF-2 that supported attachment and growth of mesenchymal stem cells. The layers are promising for improving the ingrowth of MSCs into the biological scaffold. Supported by the Technology Agency of the Czech Republic TA04011345, and Ministry of Health NT11270-4/2010, and BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University” project (CZ.1.05/1.1.00/02.0109), funded by the European Regional Development Fund for their financial supports.

Keywords: fibrin assemblies, FGF-2, mesenchymal stem cells, VEGF

Procedia PDF Downloads 325

Authors: Gabriela Wyszogrodzka, Przemyslaw Dorozynski, Barbara Gil, Maciej Strzempek, Bartosz Marszalek, Piotr Kulinowski, Wladyslaw Piotr Weglarz, Elzbieta Menaszek

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MOFs (Metal-Organic Frameworks) belong to a new group of porous materials with a hybrid organic-inorganic construction. Their structure is a network consisting of metal cations or clusters (acting as metallic centers, nodes) and the organic linkers between nodes. The interest in MOFs is primarily associated with the use of their well-developed surface and large porous. Possibility to build MOFs of biocompatible components let to use them as potential drug carriers. Furthermore, forming MOFs structure from cations possessing paramagnetic properties (e.g. iron cations) allows to use them as MRI (Magnetic Resonance Imaging) contrast agents. The concept of formation of particles that combine the ability to transfer active substance with imaging properties has been called theranostic (from words combination therapy and diagnostics). By building MOF structure from iron cations it is possible to use them as theranostic agents and monitoring the distribution of the active substance after administration in real time. In the study iron-MOF: Fe-MIL-101-NH2 was chosen, consisting of iron cluster in nodes of the structure and amino-terephthalic acid as a linker. The aim of the study was to investigate the possibility of applying Fe-MIL-101-NH2 as inhalable theranostic particulate system for the first-line anti-tuberculosis antibiotic – isoniazid. The drug content incorporated into Fe-MIL-101-NH2 was evaluated by dissolution study using spectrophotometric method. Results showed isoniazid encapsulation efficiency – ca. 12.5% wt. Possibility of Fe-MIL-101-NH2 application as the MRI contrast agent was demonstrated by magnetic resonance tomography. FeMIL-101-NH2 effectively shortening T1 and T2 relaxation times (increasing R1 and R2 relaxation rates) linearly with the concentrations of suspended material. Images obtained using multi-echo magnetic resonance imaging sequence revealed possibility to use FeMIL-101-NH2 as positive and negative contrasts depending on applied repetition time. MOFs micronization via ultrasound was evaluated by XRD, nitrogen adsorption, FTIR, SEM imaging and did not influence their crystal shape and size. Ultrasonication let to break the aggregates and achieve very homogeneously looking SEM images. MOFs cytotoxicity was evaluated in in vitro test with a highly sensitive resazurin based reagent PrestoBlue™ on L929 fibroblast cell line. After 24h no inhibition of cell proliferation was observed. All results proved potential possibility of application of ironMOFs as an isoniazid carrier and as MRI contrast agent in inhalatory treatment of tuberculosis. Acknowledgments: Authors gratefully acknowledge the National Science Center Poland for providing financial support, grant no 2014/15/B/ST5/04498.

Keywords: imaging agents, metal-organic frameworks, theranostics, tuberculosis

Procedia PDF Downloads 251

Authors: Atin Adhikari, Aniruddha Mitra, Abbas Rashidi, Imaobong Ekpo, Jefferson Doehling, Alexis Pawlak, Shane Lewis, Jacob Schwartz

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Building constructions in the US involve numerous wooden structures. Woods are routinely used in walls, framing floors, framing stairs, and making of landings in building constructions. Cross-laminated timbers are currently being used as construction materials for tall buildings. Numerous workers are involved in these timber based constructions, and wood dust is one of the most common occupational exposures for them. Wood dust is a complex substance composed of cellulose, polyoses and other substances. According to US OSHA, exposure to wood dust is associated with a variety of adverse health effects among workers, including dermatitis, allergic respiratory effects, mucosal and nonallergic respiratory effects, and cancers. The amount and size of particles released as wood dust differ according to the operations performed on woods. For example, shattering of wood during sanding operations produces finer particles than does chipping in sawing and milling industries. To our knowledge, how shattering, cutting and sanding of woods and wood slabs during new building construction release fine particles and nanoparticles are largely unknown. General belief is that the dust generated during timber cutting and sanding tasks are mostly large particles. Consequently, little attention has been given to the generated submicron ultrafine and nanoparticles and their exposure levels. These data are, however, critically important because recent laboratory studies have demonstrated cytotoxicity of nanoparticles on lung epithelial cells. The above-described knowledge gaps were addressed in this study by a novel newly developed nanoparticle monitor and conventional particle counters. This study was conducted in a large new building construction site in southern Georgia primarily during the framing of wooden side walls, inner partition walls, and landings. Exposure levels of nanoparticles (n = 10) were measured by a newly developed nanoparticle counter (TSI NanoScan SMPS Model 3910) at four different distances (5, 10, 15, and 30 m) from the work location. Other airborne particles (number of particles/m3) including PM2.5 and PM10 were monitored using a 6-channel (0.3, 0.5, 1.0, 2.5, 5.0 and 10 µm) particle counter at 15 m, 30 m, and 75 m distances at both upwind and downwind directions. Mass concentration of PM2.5 and PM10 (µg/m³) were measured by using a DustTrak Aerosol Monitor. Temperature and relative humidity levels were recorded. Wind velocity was measured by a hot wire anemometer. Concentration ranges of nanoparticles of 13 particle sizes were: 11.5 nm: 221 – 816/cm³; 15.4 nm: 696 – 1735/cm³; 20.5 nm: 879 – 1957/cm³; 27.4 nm: 1164 – 2903/cm³; 36.5 nm: 1138 – 2640/cm³; 48.7 nm: 938 – 1650/cm³; 64.9 nm: 759 – 1284/cm³; 86.6 nm: 705 – 1019/cm³; 115.5 nm: 494 – 1031/cm³; 154 nm: 417 – 806/cm³; 205.4 nm: 240 – 471/cm³; 273.8 nm: 45 – 92/cm³; and 365.2 nm: Keywords: wood dust, industrial hygiene, aerosol, occupational exposure

Procedia PDF Downloads 189

Authors: Marcel Verwey, Anna M. Joubert, Elsie M. Nolte, Wolfgang Dohle, Barry V. L. Potter, Anne E. Theron

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A tetrahydroisoquinolinone (THIQ) core can be used to mimic the A,B-ring of colchicine site-binding microtubule disruptors such as 2-methoxyestradiol in the design of anti-cancer agents. Steroidomimeric microtubule disruptors were synthesized by introducing C'2 and C'3 of the steroidal A-ring to C'6 and C'7 of the THIQ core and by introducing a decorated hydrogen bond acceptor motif projecting from the steroidal D-ring to N'2. For this in vitro study, four non-steroidal THIQ-based analogues were investigated and comparative studies were done between the non-sulphamoylated compound STX 3450 and the sulphamoylated compounds STX 2895, STX 3329 and STX 3451. The objective of this study was to investigate the modes of cell death induced by these four THIQ-based analogues in A549 lung carcinoma epithelial cells and metastatic breast adenocarcinoma MDA-MB-231 cells. Cytotoxicity studies to determine the half maximal growth inhibitory concentrations were done using spectrophotometric quantification via crystal violet staining and lactate dehydrogenase (LDH) assays. Microtubule integrity and morphologic changes of exposed cells were investigated using polarization-optical transmitted light differential interference contrast microscopy, transmission electron microscopy and confocal microscopy. Flow cytometric quantification was used to determine apoptosis induction and the effect that THIQ-based analogues have on cell cycle progression. Signal transduction pathways were elucidated by quantification of the mitochondrial membrane integrity, cytochrome c release and caspase 3, -6 and -8 activation. Induction of autophagic cell death by the THIQ-based analogues was investigated by morphological assessment of fluorescent monodansylcadaverine (MDC) staining of acidic vacuoles and by quantifying aggresome formation via flow cytometry. Results revealed that these non-steroidal microtubule disrupting analogues inhibited 50% of cell growth at nanomolar concentrations. Immunofluorescence microscopy indicated microtubule depolarization and the resultant mitotic arrest was further confirmed through cell cycle analysis. Apoptosis induction via the intrinsic pathway was observed due to depolarization of the mitochondrial membrane, induction of cytochrome c release as well as, caspase 3 activation. Potential involvement of programmed cell death type II was observed due to the presence of acidic vacuoles and aggresome formation. Necrotic cell death did not contribute significantly, indicated by stable LDH levels. This in vitro study revealed the induction of the intrinsic apoptotic pathway as well as possible involvement of autophagy after exposure to these THIQ-based analogues in both MDA-MB-231- and A549 cells. Further investigation of this series of anticancer drugs still needs to be conducted to elucidate the temporal, mechanistic and functional crosstalk mechanisms between the two observed programmed cell deaths pathways.

Keywords: apoptosis, autophagy, cancer, microtubule disruptor

Procedia PDF Downloads 253

Authors: D. Malouch, M. Berchel, C. Dreanno, S. Stachowski-Haberkorn, P-A. Jaffres

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Biofouling is a complex natural phenomenon that involves biological, physical and chemical properties related to the environment, the submerged surface and the living organisms involved. Bio-colonization of artificial structures can cause various economic and environmental impacts. The increase in costs associated with the over-consumption of fuel from biocolonized vessels has been widely studied. Measurement drifts from submerged sensors, as well as obstructions in heat exchangers, and deterioration of offshore structures are major difficulties that industries are dealing with. Therefore, surfaces that inhibit biocolonization are required in different areas (water treatment, marine paints, etc.) and many efforts have been devoted to produce efficient and eco-compatible antifouling agents. The different steps of surface fouling are widely described in literature. Studying the biofilm and its stages provides a better understanding of how to elaborate more efficient antifouling strategies. Several approaches are currently applied, such as the use of biocide anti-fouling paint (mainly with copper derivatives) and super-hydrophobic coatings. While these two processes are proving to be the most effective, they are not entirely satisfactory, especially in a context of a changing legislation. Nowadays, the challenge is to prevent biofouling with non-biocide compounds, offering a cost effective solution, but with no toxic effects on marine organisms. Since the micro-fouling phase plays an important role in the regulation of the following steps of biofilm formation, it is desired to reduce or delate biofouling of a given surface by inhibiting the micro-fouling at its early stages. In our recent works, we reported that some amphiphilic compounds exhibited bacteriostatic or bactericidal properties at a concentration that did not affect mammalian eukaryotic cells. These remarkable properties invited us to assess this type of bio-inspired phospholipids to prevent the colonization of surfaces by marine bacteria. Of note, other studies reported that amphiphilic compounds interacted with bacteria leading to a reduction of their development. An amphiphilic compound is a molecule consisting of a hydrophobic domain and a polar head (ionic or non-ionic). These compounds appear to have interesting antifouling properties: some ionic compounds have shown antimicrobial activity, and zwitterions can reduce nonspecific adsorption of proteins. Herein, we investigate the potential of amphiphilic compounds as inhibitors of bacterial growth and marine biofilm formation. The aim of this study is to compare the efficacy of four synthetic phospholipids that features a cationic charge or a zwitterionic polar-head group to prevent microfouling with marine bacteria. Toxicity of these compounds was also studied in order to identify the most promising compounds that inhibit biofilm development and show low cytotoxicity on two links representative of coastal marine food webs: phytoplankton and oyster larvae.

Keywords: amphiphilic phospholipids, biofilm, marine fouling, non-toxique assays

Procedia PDF Downloads 134

Authors: M. Markova, E. Filova, O. Kaplan, R. Matejka, L. Bacakova

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The porcine pericardium is used as a material for cardiac and aortic valves substitutes. Current biological aortic heart valve prosthesis have a limited lifetime period because they undergo degeneration. In order to make them more biocompatible and prolong their lifetime it is necessary to reseed the decellularized prostheses with endothelial cells and with valve interstitial cells. The endothelialization of the prosthesis-surface may be supported by suitable chemical surface modification of the prosthesis. The aim of this study is to prepare bioactive fibrin layers which would both support endothelialization of porcine pericardium and enhance differentiation and maturation of the endothelial cells seeded. As a material for surface adjustments we used layers of fibrin with/without heparin and some of them with adsorbed or chemically bound FGF2, VEGF or their combination. Fibrin assemblies were prepared in 24-well cell culture plate and were seeded with HSVEC (Human Saphenous Vein Endothelial Cells) at a density of 20,000 cells per well in EGM-2 medium with 0.5% FS and without heparin, without FGF2 and without VEGF; medium was supplemented with aprotinin (200 U/mL). As a control, surface polystyrene (PS) was used. Fibrin was also used as homogeneous impregnation of the decellularized porcine pericardium throughout the scaffolds. Morphology, density, and viability of the seeded endothelial cells were observed from micrographs after staining the samples by LIVE/DEAD cytotoxicity/viability assay kit on the days 1, 3, and 7. Endothelial cells were immunocytochemically stained for proteins involved in cell adhesion, i.e. alphaV integrin, vinculin, and VE-cadherin, markers of endothelial cells differentiation and maturation, i.e. von Willebrand factor and CD31, and for extracellular matrix proteins typically produced by endothelial cells, i.e. type IV collagen and laminin. The staining intensities were subsequently quantified using a software. HSVEC cells grew on each of the prepared surfaces better than on control surface. They reached confluency. The highest cell densities were obtained on the surface of fibrin with heparin and both grow factors used together. Intensity of alphaV integrins staining was highest on samples with remained fibrin layer, i.e. on layers with lower cell densities, i.e. on fibrin without heparin. Vinculin staining was apparent, but was rather diffuse, on fibrin with both FGF2 and VEGF and on control PS. Endothelial cells on all samples were positively stained for von Willebrand factor and CD31. VE-cadherin receptors clusters were best developed on fibrin with heparin and growth factors. Significantly stronger staining of type IV collagen was observed on fibrin with heparin and both growth factors. Endothelial cells on all samples produced laminin-1. Decellularized pericardium was homogeneously filled with fibrin structures. These fibrin-modified pericardium samples will be further seeded with cells and cultured in a bioreactor. Fibrin layers with/without heparin and with adsorbed or chemically bound FGF2, VEGF or their combination are good surfaces for endothelialization of cardiovascular prostheses or porcine pericardium based heart valves. Supported by the Ministry of Health, grants No15-29153A and 15-32497A, and the Grant Agency of the Czech Republic, project No. P108/12/G108.

Keywords: aortic valves prosthesis, FGF2, heparin, HSVEC cells, VEGF

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Authors: Maria Zulema Morquecho Vega, Fabiola CarolinaMiranda Castro, Rafael Verdugo Miranda, Ignacio Yocupicio Villegas, Ana lidia Barron Raygoza, Martin enrique MArquez Cordova, Jose Alberto Duarte Moller

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Background: Anemopsis californica, also called (tame grass) belongs to the Saururaceae family small, green plant. The blade is long and wide. Gives a white flower. The plant population is only found in humid, swampy habitats, it grows where there is water, along the banks of streams and water holes. In the winter, it dries up. The leaves, rhizomes, or roots of this plant have been used to treat a range of diseases. Some of its healing properties are used to treat wounds, cold and flu symptoms, spasmodic cough, infection, pain and inflammation, burns, swollen feet, as well as lung ailments, asthma, circulatory problems (varicose veins), rheumatoid arthritis, purifies blood, helps in urinary and digestive tract diseases, sores and healing, for headache, sore throat, diarrhea, kidney pain. The tea made from the leaves and roots is used to treat uterine cancer, womb cancer, relieves menstrual pain and stops excessive bleeding after childbirth. It is also used as a gynecological treatment for infections, hemorrhoids, candidiasis and vaginitis. Objective: To study the cytotoxicity of gels prepared with silver nanoparticles in AC extract combined with chitosan, collagen and hyaluronic acid as an alternative therapy for skin conditions. Methods: The Ag NPs were synthesized according to the following method. A 0.3 mg/mL solution is prepared in 10 ml of deionized water, adjust to pH 12 with NaOH, stirring is maintained constant magnetic and a temperature of 80 °C. Subsequently, 100 ul of a 0.1 M AgNO3 solution and kept stirring constantly for 15 min. Once the reaction is complete, measurements are performed by UV-Vis. A gel was prepared in a 5% solution of acetic acid with the respective nanoparticles and AC extract of silver in the extract of AC. Chitosan is added until the process begins to occur gel. At that time, collagen will be added in a ratio of 3 to 5 drops, and later, hyaluronic acid in 2% of the total compound formed. Finally, after resting for 24 hours, the cytotoxic effect of the gels was studied. in the presence of highly positive bacteria Staphylococcus aureus and highly negative for Escherichia coli. Cultures will be incubated for 24 hours in the presence of the compound and compared with the reference. Results: Silver nanoparticles obtained had a spherical shape and sizes among 20 and 30 nm. UV-Vis spectra confirm the presence of silver nanoparticles showing a surface plasmon around 420 nm. Finally, the test in presence of bacteria yield a good antibacterial property of this nanocompound and tests in people were successful. Conclusion: Gel prepared by biogenic synthesis shown beneficious effects in severe acne, acne vulgaris and wound healing with diabetic patients.

Keywords: anemopsis californica, nanomedicina, biotechnology, biomedicine

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Authors: Suman Chaudhary, Rupinder K. Kanwar, Jagat R. Kanwar

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Azadirachta indica, also known as Neem belonging to the mahogany family is actively gaining interest in the era of modern day medicine due to its extensive applications in homeopathic medicine such as Ayurveda and Unani. More than 140 phytochemicals have been extracted from neem leaves, seed, bark and flowers for agro-medicinal applications. Among the various components, neem leaf protein (NLP) is currently the most investigated active ingredient, due to its immunomodulatory activities against tumor growth. However, these therapeutic ingredients of neem are susceptible to degradation and cannot withstand the drastic pH changes under physiological environment, and therefore, there is an urgent need of an alternative strategy such as a nano-delivery system to exploit its medicinal benefits. This study hypothesizes that guar gum (GG) derived biodegradable nano-carrier based encapsulation of NLP will improve its stability, specificity and sensitivity, thus facilitating targeted anti-cancer therapeutics. GG is a galactomannan derived from the endosperm of the guar beans seeds. Synthesis of guar nanocapsules (NCs) was performed using nanoprecipitation technique where the GG was encapsulated with NLP. Preliminary experiments conducted to characterize the NCs confirmed spherical morphology with a narrow size distribution of 30-40 nm. Differential scanning colorimetric analysis (DSC) validated the stability of these NCs even at a temperature range of 50-60°C which was well within the physiological and storage conditions. Thermogravimetric (TGA) analysis indicated high decomposition temperature of these NCs ranging upto 350°C. Additionally, Fourier Transform Infrared spectroscopy (FTIR) and the SDS-PAGE data acquired confirmed the successful encapsulation of NLP in the NCs. The anti-cancerous therapeutic property of this NC was tested on colon cancer cells (caco-2) as they are one of the most prevalent form of cancer. These NCs (both NLP loaded and void) were also tested on human intestinal epithelial cells (FHs 74) cells to evaluate their effect on normal cells. Cytotoxicity evaluation of the NCs in the cell lines confirmed that the IC50 for NLP in FHs 74 cells was ~2 fold higher than in caco-2 cells, indicating that this nanoformulation system possessed biocompatible anti-cancerous properties Immunoconfocal microscopy analysis confirmed the time dependent internalization of the NCs within 6h. Recent findings performed using Annexin V and PI staining indicated a significant increase (p ≤ 0.001) in the early and late apoptotic cell population when treated with the NCs signifying the role of NLP in inducing apoptosis in caco-2 cells. This was further validated using Western blot, Polymerase chain reaction (PCR) and Fluorescence activated cell sorter (FACS) aided protein expressional analysis which presented a downregulation of survivin, an anti-apoptotic cell marker and upregulation of Bax/Bcl-2 ratio (pro-apoptotic indicator). Further, both the NLP NC and unencapsulated NLP treatment destabilized the mitochondrial membrane potential subsequently facilitating the release of the pro-apoptotic caspase cascade initiator, cytochrome-c. Future studies will be focused towards granting specificity to these NCs towards cancer cells, along with a comprehensive analysis of the anti-cancer potential of this naturally occurring compound in different cancer and in vivo animal models, will validate the clinical application of this unprecedented protein therapeutic.

Keywords: anti-tumor, guar gum, nanocapsules, neem leaf protein

Procedia PDF Downloads 177

Authors: E. Locatelli, I. Monaco, R. C. Martin, Y. Li, R. Pini, M. Chiariello, M. Comes Franchini

Abstract:

Despite the many advantages of application of nanomaterials in the field of nanomedicine, increasing concerns have been expressed on their potential adverse effects on human health. There is urgency for novel green strategies toward novel materials with enhanced biocompatibility using safe reagents. Photothermal ablation therapy, which exploits localized heat increase of a few degrees to kill cancer cells, has appeared recently as a non-invasive and highly efficient therapy against various cancer types; anyway new agents able to generate hyperthermia when irradiated are needed and must have precise biocompatibility in order to avoid damage to healthy tissues and prevent toxicity. Recently, there has been increasing interest in magnesium as a biomaterial: it is the fourth most abundant cation in the human body, and it is essential for human metabolism. However magnesium nanoparticles (Mg NPs) have had limited diffusion due to the high reduction potential of magnesium cations, which makes NPs synthesis challenging. Herein, we report the synthesis of Mg NPs and their surface functionalization for the obtainment of a stable and biocompatible nanomaterial suitable for photothermal ablation therapy against cancer. We synthesized the Mg crystals by reducing MgCl2 with metallic lithium and exploiting naphthalene as an electron carrier: the lithium–naphthalene complex acts as the real reducing agent. Firstly, the nanocrystal particles were coated with the ligand 12-ethoxy ester dodecanehydroxamic acid, and then entrapped into water-dispersible polymeric micelles (PMs) made of the FDA-approved PLGA-b-PEG-COOH copolymer using the oil-in-water emulsion technique. Lately, we developed a more straightforward methodology by introducing chitosan, a highly biocompatible natural product, at the beginning of the process, simultaneously using lithium–naphthalene complex, thus having a one-pot procedure for the formation and surface modification of MgNPs. The obtained MgNPs were purified and fully characterized, showing diameters in the range of 50-300 nm. Notably, when coated with chitosan the particles remained stable as dry powder for more than 10 months. We proved the possibility of generating a temperature rise of a few to several degrees once MgNPs were illuminated using a 810 nm diode laser operating in continuous wave mode: the temperature rise resulted significant (0-15 °C) and concentration dependent. We then investigated potential cytotoxicity of the MgNPs: we used HN13 epithelial cells, derived from a head and neck squamous cell carcinoma and the hepa1-6 cell line, derived from hepatocellular carcinoma and very low toxicity was observed for both nanosystems. Finally, in vivo photothermal therapy was performed on xenograft hepa1-6 tumor bearing mice: the animals were treated with MgNPs coated with chitosan and showed no sign of suffering after the injection. After 12 hours the tumor was exposed to near-infrared laser light. The results clearly showed an extensive damage to tumor tissue after only 2 minutes of laser irradiation at 3Wcm-1, while no damage was reported when the tumor was treated with the laser and saline alone in control group. Despite the lower photothermal efficiency of Mg with respect to Au NPs, we consider MgNPs a promising, safe and green candidate for future clinical translations.

Keywords: chitosan, magnesium nanoparticles, nanomedicine, photothermal therapy

Procedia PDF Downloads 270

Authors: Kazuhiro Tateda, Elisa Gonzalez, Shuhei Ito, Kirstin Heinrich, Kevin Sweetland, Pingping Zhang, Catia Ferreira, Michael Pride, Jennifer Moisi, Sharon Gray, Bennett Lee, Fred Angulo

Abstract:

Clostridium difficile (C. difficile) is the most common cause of antibiotic-associated diarrhea and infectious diarrhea in healthcare settings. Japan has an aging population; the elderly are at increased risk of hospitalization, antibiotic use, and C. difficile infection (CDI). Little is known about the population-based incidence and disease burden of CDI in Japan although limited hospital-based studies have reported a lower incidence than the United States. To understand CDI disease burden in Japan, CLOUD (Clostridium difficile Infection Burden of Disease in Adults in Japan) was developed. CLOUD will derive population-based incidence estimates of the number of CDI cases per 100,000 population per year in Ota-ku (population 723,341), one of the districts in Tokyo, Japan. CLOUD will include approximately 14 of the 28 Ota-ku hospitals including Toho University Hospital, which is a 1,000 bed tertiary care teaching hospital. During the 12-month patient enrollment period, which is scheduled to begin in November 2018, Ota-ku residents > 50 years of age who are hospitalized at a participating hospital with diarrhea ( > 3 unformed stools (Bristol Stool Chart 5-7) in 24 hours) will be actively ascertained, consented, and enrolled by study surveillance staff. A stool specimen will be collected from enrolled patients and tested at a local reference laboratory (LSI Medience, Tokyo) using QUIK CHEK COMPLETE® (Abbott Laboratories). which simultaneously tests specimens for the presence of glutamate dehydrogenase (GDH) and C. difficile toxins A and B. A frozen stool specimen will also be sent to the Pfizer Laboratory (Pearl River, United States) for analysis using a two-step diagnostic testing algorithm that is based on detection of C. difficile strains/spores harboring toxin B gene by PCR followed by detection of free toxins (A and B) using a proprietary cell cytotoxicity neutralization assay (CCNA) developed by Pfizer. Positive specimens will be anaerobically cultured, and C. difficile isolates will be characterized by ribotyping and whole genomic sequencing. CDI patients enrolled in CLOUD will be contacted weekly for 90 days following diarrhea onset to describe clinical outcomes including recurrence, reinfection, and mortality, and patient reported economic, clinical and humanistic outcomes (e.g., health-related quality of life, worsening of comorbidities, and patient and caregiver work absenteeism). Studies will also be undertaken to fully characterize the catchment area to enable population-based estimates. The 12-month active ascertainment of CDI cases among hospitalized Ota-ku residents with diarrhea in CLOUD, and the characterization of the Ota-ku catchment area, including estimation of the proportion of all hospitalizations of Ota-ku residents that occur in the CLOUD-participating hospitals, will yield CDI population-based incidence estimates, which can be stratified by age groups, risk groups, and source (hospital-acquired or community-acquired). These incidence estimates will be extrapolated, following age standardization using national census data, to yield CDI disease burden estimates for Japan. CLOUD also serves as a model for studies in other countries that can use the CLOUD protocol to estimate CDI disease burden.

Keywords: Clostridium difficile, disease burden, epidemiology, study protocol

Procedia PDF Downloads 261

Authors: D. Malouch, M. Berchel, C. Dreanno, S. Stachowski-Haberkorn, P-A. Jaffres

Abstract:

Biofilm is a predominant lifestyle chosen by bacteria. Whether it is developed on an immerged surface or a mobile biofilm known as flocs, the bacteria within this form of life show properties different from its planktonic ones. Within the biofilm, the self-formed matrix of Extracellular Polymeric Substances (EPS) offers hydration, resources capture, enhanced resistance to antimicrobial agents, and allows cell-communication. Biofouling is a complex natural phenomenon that involves biological, physical and chemical properties related to the environment, the submerged surface and the living organisms involved. Bio-colonization of artificial structures can cause various economic and environmental impacts. The increase in costs associated with the over-consumption of fuel from biocolonized vessels has been widely studied. Measurement drifts from submerged sensors, as well as obstructions in heat exchangers, and deterioration of offshore structures are major difficulties that industries are dealing with. Therefore, surfaces that inhibit biocolonization are required in different areas (water treatment, marine paints, etc.) and many efforts have been devoted to produce efficient and eco-compatible antifouling agents. The different steps of surface fouling are widely described in literature. Studying the biofilm and its stages provides a better understanding of how to elaborate more efficient antifouling strategies. Several approaches are currently applied, such as the use of biocide anti-fouling paint6 (mainly with copper derivatives) and super-hydrophobic coatings. While these two processes are proving to be the most effective, they are not entirely satisfactory, especially in a context of a changing legislation. Nowadays, the challenge is to prevent biofouling with non-biocide compounds, offering a cost effective solution, but with no toxic effects on marine organisms. Since the micro-fouling phase plays an important role in the regulation of the following steps of biofilm formation7, it is desired to reduce or delate biofouling of a given surface by inhibiting the micro fouling at its early stages. In our recent works, we reported that some amphiphilic compounds exhibited bacteriostatic or bactericidal properties at a concentration that did not affect eukaryotic cells. These remarkable properties invited us to assess this type of bio-inspired phospholipids9 to prevent the colonization of surfaces by marine bacteria. Of note, other studies reported that amphiphilic compounds interacted with bacteria leading to a reduction of their development. An amphiphilic compound is a molecule consisting of a hydrophobic domain and a polar head (ionic or non-ionic). These compounds appear to have interesting antifouling properties: some ionic compounds have shown antimicrobial activity, and zwitterions can reduce nonspecific adsorption of proteins. Herein, we investigate the potential of amphiphilic compounds as inhibitors of bacterial growth and marine biofilm formation. The aim of this study is to compare the efficacy of four synthetic phospholipids that features a cationic charge (BSV36, KLN47) or a zwitterionic polar-head group (SL386, MB2871) to prevent microfouling with marine bacteria. We also study the toxicity of these compounds in order to identify the most promising compound that must feature high anti-adhesive properties and a low cytotoxicity on two links representative of coastal marine food webs: phytoplankton and oyster larvae.

Keywords: amphiphilic phospholipids, bacterial biofilm, marine microfouling, non-toxic antifouling

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Authors: Nkechi V. Enwuru, Bullum Nkeki, Elizabeth A. Adekoya, Olumide A. Adebesin, Rebecca F. Peters, Victoria A. Aikhomu, Mendie E. U.

Abstract:

Background: Probiotics are live microbial feed supplement beneficial for host. Probiotics and their postbiotic products have been used to prevent or treat various health conditions. However, the products cell viability is often low due to harsh conditions subjected during processing, handling, storage, and gastrointestinal transit. These strongly influence probiotics’ benefits; thus, viability is essential for probiotics to produce health benefits for the host. Microencapsulation is a promising technique with considerable effects on probiotic survival. The study is aimed to formulate a microencapsulated probiotic and evaluate its viability, antimicrobial efficacy, and cytotoxic activity of its postbiotic on the MCF-7 breast cancer cell line. Method: Human and animal raw milk were sampled for lactic acid bacteria. The isolated bacteria were identified using conventional and VITEK 2 systems. The identified lactic acid bacterium was encapsulated using spray-dried and extrusion methods. The free, encapsulated, and chitosan-coated encapsulated probiotics were tested for viability in simulated-gastric intestinal (SGI) fluid and different storage conditions at refrigerated (4oC) and room (25oC) temperatures. The disintegration time and weight uniformity of the spray-dried hard gelatin capsules were tested. The antimicrobial property of free and encapsulated probiotics was tested against enteric pathogenic isolates from antiretroviral therapy (ART) treated HIV-positive patients. The postbiotic of the free cells was extracted, and its cytotoxic effect on the MCF-7 breast cancer cell line was tested through an MTT assay. Result: The Lactobacillus plantarum was isolated from animal raw milk. Zero-size hard gelatin L. plantarum capsules with granules within a size range of 0.71–1.00 mm diameter was formulated. The disintegration time ranges from 2.14±0.045 to 2.91±0.293 minutes, while the average weight is 502.1mg. Simulated gastric solution significantly affected viability of both free and microcapsules. However, the encapsulated cells were more protected and viable due to impermeability in the microcapsules. Furthermore, the viability of free cells stored at 4oC and 25oC were less than 4 log CFU/g and 6 log CFU/g respectively after 12 weeks. However, the microcapsules stored at 4oC achieved the highest viability among the free and microcapsules stored at 25oC and the free cells stored at 4oC. Encapsulated cells were released in the simulated gastric fluid, viable and effective against the enteric pathogens tested. However, chitosan-coated calcium alginate encapsulated probiotics significantly inhibited Shigella flexneri, Candida albicans, and Escherichia coli. The Postbiotic Metabolites (PM) of L. plantarum produced a cytotoxic effect on the MCF-7 breast cancer cell line. The postbiotic showed significant cytotoxic activity similar to 5FU, a standard antineoplastic agent. The inhibition concentration of 50% growth (IC50) of postbiotic metabolite K3 is low and consistent with the IC50 of the positive control (Cisplatin). Conclusions: Lactobacillus plantarum postbiotic exhibited a cytotoxic effect on the MCF-7 breast cancer cell line and could be used as combined adjuvant therapy in breast cancer management. The microencapsulation technique protects the probiotics, improving their viability and delivery to the gastrointestinal tract. Chitosan enhances antibacterial efficacy; thus, chitosan-coated microencapsulated L. plantarum probiotics could be more effective and used as a combined therapy in HIV management of opportunistic enteric infection.

Keywords: probiotics, encapsulation, gastrointestinal conditions, antimicrobial effect, postbiotic, cytotoxicity effect

Procedia PDF Downloads 123