Search results for: in vitro model

Authors: K. S. Hemant Yadav, H. G. Shivakumar

Abstract:

The present study investigation was to prepare and evaluate the mucoadhesive multi-particulate system for nasal drug delivery of anti-histaminic drug. Ebastine was chosen as the model drug. Drug loaded nanoparticles of Ebastine were prepared by ionic gelation method using chitosan as polymer using the drug-polymer weight ratios 1:1, 1:2, 1:3. Sodium tripolyphosphate (STPP) was used as the cross-linking agent in the range of 0.5 and 0.7% w/v. FTIR and DSC studies indicated that no chemical interaction occurred between the drug and polymers. Particle size ranged from 169 to 500 nm. The drug loading and entrapment efficiency was found to increase with increase in chitosan concentration and decreased with increase in poloxamer 407 concentration. The results of in vitro mucoadhesion carried out showed that all the prepared formulation had good mucoadhesive property and mucoadhesion increases with increase in the concentration of chitosan. The in vitro release pattern of all the formulations was observed to be in a biphasic manner characterized by slight burst effect followed by a slow release. By the end of 8 hrs, formulation F6 showed a release of only 86.9% which explains its sustained behaviour. The ex-vivo permeation of the pure drug ebastine was rapid than the optimized formulation(F6) indicating the capability of the chitosan polymer to control drug permeation rate through the sheep nasal mucosa. The results indicated that the mucoadhesive nanoparticulate system can be used for the nasal delivery of antihistaminic drugs in an effective manner.

Keywords: nasal, nanoparticles, ebastine, anti-histaminic drug, mucoadhesive multi-particulate system

Procedia PDF Downloads 392

Authors: Lacramioara Ochiuz, Aurelia Vasile, Iulian Stoleriu, Cristina Ghiciuc, Maria Ignat

Abstract:

Topical administration of chemotherapeutic agents (eg. carmustine, bexarotene, mechlorethamine etc.) in local treatment of cutaneous T-cell lymphoma (CTCL) is accompanied by multiple side effects, such as contact hypersensitivity, pruritus, skin atrophy or even secondary malignancies. A known method of reducing the side effects of anticancer agent is the development of modified drug release systems using drug incapsulation in biocompatible nanoporous inorganic matrices, such as mesoporous MCM-41 silica. Mesoporous MCM-41 silica is characterized by large specific surface, high pore volume, uniform porosity, and stable dispersion in aqueous medium, excellent biocompatibility, in vivo biodegradability and capacity to be functionalized with different organic groups. Therefore, MCM-41 is an attractive candidate for a wide range of biomedical applications, such as controlled drug release, bone regeneration, protein immobilization, enzymes, etc. The main advantage of this material lies in its ability to host a large amount of the active substance in uniform pore system with adjustable size in a mesoscopic range. Silanol groups allow surface controlled functionalization leading to control of drug loading and release. This study shows (I) the amino-grafting optimization of mesoporous MCM-41 silica matrix by means of co-condensation during synthesis and post-synthesis using APTES (3-aminopropyltriethoxysilane); (ii) loading the therapeutic agent (carmustine) obtaining a modified drug release systems; (iii) determining the profile of in vitro carmustine release from these systems; (iv) assessment of carmustine release kinetics by fitting on four mathematical models. Obtained powders have been described in terms of structure, texture, morphology thermogravimetric analysis. The concentration of the therapeutic agent in the dissolution medium has been determined by HPLC method. In vitro dissolution tests have been done using cell Enhancer in a 12 hours interval. Analysis of carmustine release kinetics from mesoporous systems was made by fitting to zero-order model, first-order model Higuchi model and Korsmeyer-Peppas model, respectively. Results showed that both types of highly ordered mesoporous silica (amino grafted by co-condensation process or post-synthesis) are thermally stable in aqueous medium. In what regards the degree of loading and efficiency of loading with the therapeutic agent, there has been noticed an increase of around 10% in case of co-condensation method application. This result shows that direct co-condensation leads to even distribution of amino groups on the pore walls while in case of post-synthesis grafting many amino groups are concentrated near the pore opening and/or on external surface. In vitro dissolution tests showed an extended carmustine release (more than 86% m/m) both from systems based on silica functionalized directly by co-condensation and after synthesis. Assessment of carmustine release kinetics revealed a release through diffusion from all studied systems as a result of fitting to Higuchi model. The results of this study proved that amino-functionalized mesoporous silica may be used as a matrix for optimizing the anti-cancer topical therapy by loading carmustine and developing prolonged-release systems.

Keywords: carmustine, silica, controlled, release

Procedia PDF Downloads 222

Authors: Brajesh Tiwari, Yuvraj Dangi, Abhishek Jain, Ashok Jain

Abstract:

The purpose of the investigation was to evaluate the potential of sphingosomes as nanoscale drug delivery units for site-specific delivery of anti-cancer agents. Doxorubicin Hydrochloride (DOX) was selected as a model anti-cancer agent. Sphingosomes were prepared and loaded with DOX and optimized for size and drug loading. The formulations were characterized by Malvern zeta-seizer and Transmission Electron Microscopy (TEM) studies. Sphingosomal formulations were further evaluated for in-vitro drug release study under various pH profiles. The in-vitro drug release study showed an initial rapid release of the drug followed by a slow controlled release. In vivo studies of optimized formulations and free drug were performed on albino rats for comparison of drug plasma concentration. The in- vivo study revealed that the prepared system enabled DOX to have had enhanced circulation time, longer half-life and lower elimination rate kinetics as compared to free drug. Further, it can be interpreted that the formulation would selectively enter highly porous mass of tumor cells and at the same time spare normal tissues. To summarize, the use of sphingosomes as carriers of anti-cancer drugs may prove to be a fascinating approach that would selectively localize in the tumor mass, increasing the therapeutic margin of safety while reducing the side effects associated with anti-cancer agents.

Keywords: sphingosomes, anti-cancer, doxorubicin, formulation

Procedia PDF Downloads 267

Authors: Akwu N. A., Naidoo Y., Salau V. F., Olofinsan K. A.

Abstract:

Grewia lasiocarpa E. Mey. ex Harv. (Malvaceae) is a Southern African medicinal plant indigenously used with other plants for birthing problems. The anti-diabetic properties of the hexane, chloroform, and methanol extracts of Grewia lasiocarpa stem bark were assessed using in vitro α-glucosidase enzyme inhibition assay. The predictive in silico drug-likeness and toxicity properties of the phytocompounds were conducted using the pKCSM, ADMElab, and SwissADME computer-aided online tools. The highest α-glucosidase percentage inhibition was observed in the hexane extract (86.76%, IC50= 0.24 mg/mL), followed by chloroform (63.08%, IC50= 4.87 mg/mL) and methanol (53.22%, IC50= 9.41 mg/mL); while acarbose, the standard anti-diabetic drug was (84.54%, IC50= 1.96 mg/mL). The α-glucosidase assay revealed that the hexane extract exhibited the strongest carbohydrate inhibiting capacity and is a better inhibitor than the standard reference drug-acarbose. The computational studies also affirm the results observed in the in vitroα-glucosidaseassay. Thus, the extracts of G. lasiocarpa may be considered a potential plant-sourced compound for treating type 2 diabetes mellitus. This is the first study on the anti-diabetic properties of Grewia lasiocarpa hexane, chloroform, and methanol extracts using in vitro and in silico models.

Keywords: grewia lasiocarpa, α-glucosidase inhibition, anti-diabetes, ADMET

Procedia PDF Downloads 69

Authors: Parnaz Boodagh, Danila Vella, Antonio Damore, Laura Modica De Mohac, Sang-Ho Ye, Garret Coyan, Gaetano Burriesci, William Wagner, Federica Cosentino

Abstract:

The use of cardiac patches is among the main therapeutic solution for cardiovascular diseases, a leading mortality cause in the world with an increasing trend, responsible of 19 millions deaths in 2020. Several classes of biomaterials serve that purpose, both of synthetic origin and biological derivation, and many bioengineered treatment alternatives were proposed to satisfy two main requirements, providing structural support and promoting tissue remodeling. The objective of this paper is to compare the mechanical properties and the characterization of four cardiac patches: the Adeka, PhotoFix, CorPatch, and CardioCel patches. In vitro and in vivo tests included: biaxial, uniaxial, ball burst, suture retention for mechanical characterization; 2D surface topography, 3D volume and microstructure, and histology assessments for structure characterization; in vitro test to evaluate platelet deposition, calcium deposition, and macrophage polarization; rat right ventricular outflow tract (RVOT) models at 8- and 16-week time points to characterize the patch-host interaction. Lastly, the four patches were used to produce four stented aortic valve prosthesis, subjected to hydrodynamic assessment as well as durability testing to verify compliance with the standard ISO.

Keywords: cardiac patch, cardiovascular disease, cardiac repair, blood contact biomaterial

Procedia PDF Downloads 106

Authors: Abobkar Saad, Qasmia Abdalla, Fatma Emhemed

Abstract:

Eighty-four of Calligonum comosum were cultured on Murashige and Skoog medium on every combination supplemented with different concentrations of IAA, BA, Zeatin, and GA3. When 84 seeds were inoculated on MS free hormones, different types of cells contain dense cytoplasm were observed ater 23 days and long thick wall cells arranged in layers. In case of using MS +BA(0.5mg/L), different types and shapes of parenchyma cells contain dense cytoplasm were detected after four weeks. In the case of using MS + BA(1mg/L) + GA3 (3mg/L), thick wall parenchyma cells contain dense cytoplasm after 19 days, but many layers of parenchyma cells contain dense cytoplasm after 28 days. When MS +kin(0.5mg/L) a thick cells wall as Sclereids were observed after 29 days. No any response were observed on Zeatin (0.5, 1 mg/L).

Keywords: anatomy, Calligonum comosum, in vitro, aeeds

Procedia PDF Downloads 381

Authors: Mengjie Qu, Yi Wang, Jiawei Ding, Siyu Chen, Yanan Di

Abstract:

Marine ecosystem is facing intensified multiple stresses caused by environmental contaminants from human activities. Xenobiotics, such as benzo(α)pyrene (BaP) have been discharged into marine environment and cause hazardous impacts on both marine organisms and human beings. As a filter-feeder, marine mussels, Mytilus spp., has been extensively used to monitor the marine environment. However, their genomic alterations induced by such xenobiotics are still kept unknown. In the present study, gills, as the first defense barrier in mussels, were selected to evaluate the genetic instability alterations induced by the exposure to BaP both in vivo and in vitro. Both random amplified polymorphic DNA (RAPD) assay and comet assay were applied as the rapid tools to assess the environmental stresses due to their low money- and time-consumption. All mussels were identified to be the single species of Mytilus coruscus before used in BaP exposure at the concentration of 56 μg/l for 1 & 3 days (in vivo exposure) or 1 & 3 hours (in vitro). Both RAPD and comet assay results were showed significantly increased genomic instability with time-specific altering pattern. After the recovery period in 'in vivo' exposure, the genomic status was as same as control condition. However, the relative higher genomic instabilities were still observed in gill cells after the recovery from in vitro exposure condition. Different repair mechanisms or signaling pathway might be involved in the isolated gill cells in the comparison with intact tissues. The study provides the robust and rapid techniques to exam the genomic stability in marine organisms in response to marine environmental changes and provide basic information for further mechanism research in stress responses in marine organisms.

Keywords: genotoxic impacts, in vivo/vitro exposure, marine mussels, RAPD and comet assay

Procedia PDF Downloads 247

Authors: Vanessa Funk, Steven Blum, Stephanie Cole, Jorge Maciel, Matthew Lux

Abstract:

Paper-based synthetic gene circuits offer a new paradigm for programmable, fieldable biodetection. We demonstrate that by freeze-drying gene circuits with in vitro expression machinery, we can use complimentary RNA sequences to trigger colorimetric changes upon rehydration. We have successfully utilized both green fluorescent protein and luciferase-based reporters for easy visualization purposes in solution. Through several efforts, we are aiming to use this new platform technology to address a variety of needs in portable detection by demonstrating several more expression and reporter systems for detection functions on paper. In addition to RNA-based biodetection, we are exploring the use of various mechanisms that cells use to respond to environmental conditions to move towards all-hazards detection. Examples include explosives, heavy metals for water quality, and toxic chemicals.

Keywords: cell-free lysates, detection, gene circuits, in vitro

Procedia PDF Downloads 361

Authors: Uftah Ali M. Shushni, Viola Stuppec, Ulrike Lindequist

Abstract:

Because of the evolving resistance of microorganisms to existing antibiotics, there is an increasing need for new antibiotics not only in human but also in veterinary medicine. As part of our ongoing work on the secondary metabolites produced by marine fungi, the organic extract of the culture filtrate of an Ascomycetous fungus, which was found on driftwood collected from the coast of the Greifswalder Bodden, Baltic Sea, Germany displayed antimicrobial activity against some fish and human pathogenic bacteria. Bioactivity-guided column chromatographic separation led to the isolation of 6-Deoxybostrycoidin. The structure was determined from the interpretation of spectroscopic data (UV, MS, and NMR). 6-Deoxybostrycoidin exhibited in vitro activity against Bacillus subtilis, Staphylococcus aureus and Flexibacter maritimus with minimal inhibitory concentrations of 25, 12.5 and 12.5 μg/ml respectively.

Keywords: marine fungi, fish pathogenic bacteria, microorganism, medicine

Procedia PDF Downloads 496

Authors: Zishan Ahmad, Anwar Shahzad

Abstract:

The present studies describe a protocol for high frequency in vitro propagation through nodal segments and shoot tips in D. arayalpathra, a critically endangered medicinal liana of the Western Ghats, India. Nodal segments were more responsive than shoot tips in terms of shoot multiplication. Murashige and Skoog’s (MS) basal medium supplemented with 2.5 µM 6-benzyladenine (BA) was optimum for shoot induction through both the explants. Among different combinations of plant growth regulator (PGRs) and growth additive screened, MS medium supplemented with BA (2.5 µM) + indole-3-acetic acid (IAA) (0.25 µM) + adenine sulphate (ADS) (10.0 µM) induced a maximum of 9.0 shoots per nodal segment and 3.9 shoots per shoot tip with mean shoot length of 8.5 and 3.9 cm respectively. Half-strength MS medium supplemented with Naphthaleneacetic acid (NAA) (2.5 µM) was the best for in vitro root induction. After successful acclimatization in SoilriteTM, 92 % plantlets were survived in field conditions. Acclimatized plantlets were studied for chlorophyll and carotenoid content, net photosynthetic rate (PN) and related attributes such as stomatal conductance (Gs) and transpiration rate during subsequent days of acclimatization. The rise and fall of different biochemical enzymes (SOD, CAT, APX and GR) were also studies during successful days of acclimatization. Moreover, the effect of acclimatization on the synthesis of 2-hydroxy-4-methoxy benzaldehyde (2H4MB) was also studied in relation to the biomass production. Maximum fresh weight (2.8 gm/plant), dry weight (0.35 gm/plant) of roots and 2H4MB content (8.5 µg/ ml of root extract) were recorded after 8 weeks of acclimatization. The screening of in vitro raised plantlet root was also carried out by using GC-MS analysis which witnessed more than 25 compounds. The regenerated plantlets were also screened for homogeneity by using RAPD and ISSR. The proposed protocol surely can be used for the conservation and commercial production of the plant.

Keywords: 6-benzyladenine, PGRs, RAPD, 2H4MB

Procedia PDF Downloads 159

Authors: Wiam El Kheir, Anais Dumais, Maude Beaudoin, Bernard Marcos, Nick Virgilio, Benoit Paquette, Nathalie Faucheux, Marc-Antoine Lauzon

Abstract:

The high infiltrative pattern of glioblastoma multiforme cells (GBM) is the main cause responsible for the actual standard treatments failure. The tumor high heterogeneity, the interstitial fluid flow (IFF) and chemokines guides GBM cells migration in the brain parenchyma resulting in tumor recurrence. Drug delivery systems emerged as an alternative approach to develop effective treatments for the disease. Some recent studies have proposed to harness the effect CXC-lchemokine-ligand-12 to direct and control the cancer cell migration through delivery system. However, the dynamics of the brain environment on the delivery system remains poorly understood. Nanoparticles (NPs) and hydrogels are known as good carriers for the encapsulation of different agents and control their release. We studied the release of CXCL12 (free or loaded into NPs) from an alginate-based hydrogel under static and indirect perfusion (IP) conditions. Under static conditions, the main phenomena driving CXCL12 release from the hydrogel was diffusion with the presence of strong interactions between the positively charged CXCL12 and the negatively charge alginate. CXCL12 release profiles were independent from the initial mass loadings. Afterwards, we demonstrated that the release could tuned by loading CXCL12 into Alginate/Chitosan-Nanoparticles (Alg/Chit-NPs) and embedded them into alginate-hydrogel. The initial burst release was substantially attenuated and the overall cumulative release percentages of 21%, 16% and 7% were observed for initial mass loadings of 0.07, 0.13 and 0.26 µg, respectively, suggesting stronger electrostatic interactions. Results were mathematically modeled based on Fick’s second law of diffusion framework developed previously to estimate the effective diffusion coefficient (Deff) and the mass transfer coefficient. Embedding the CXCL12 into NPs decreased the Deff an order of magnitude, which was coherent with experimental data. Thereafter, we developed an in-vitro 3D model that takes into consideration the convective contribution of the brain IFF to study CXCL12 release in an in-vitro microenvironment that mimics as faithfully as possible the human brain. From is unique design, the model also allowed us to understand the effect of IP on CXCL12 release in respect to time and space. Four flow rates (0.5, 3, 6.5 and 10 µL/min) which may increase CXCL12 release in-vivo depending on the tumor location were assessed. Under IP, cumulative percentages varying between 4.5-7.3%, 23-58.5%, 77.8-92.5% and 89.2-95.9% were released for the three initial mass loadings of 0.08, 0.16 and 0.33 µg, respectively. As the flow rate increase, IP culture conditions resulted in a higher release of CXCL12 compared to static conditions as the convection contribution became the main driving mass transport phenomena. Further, depending on the flow rate, IP had a direct impact on CXCL12 distribution within the simulated brain tissue, which illustrates the importance of developing such 3D in-vitro models to assess the efficiency of a delivery system targeting the brain. In future work, using this very model, we aim to understand the impact of the different phenomenon occurring on GBM cell behaviors in response to the resulting chemokine gradient subjected to various flow while allowing them to express their invasive characteristics in an in-vitro microenvironment that mimics the in-vivo brain parenchyma.

Keywords: 3D culture system, chemokines gradient, glioblastoma multiforme, kinetic release, mathematical modeling

Procedia PDF Downloads 47

Authors: Rajesh A. Rane, Shital S. Naphade, Rajshekhar Karpoormath

Abstract:

Thiozolidin-4-one, a mimic of thiazolobenzimidazole (TBZ) has drawn many attentions due to its potent and selective inhibition against the HIV-1 and low toxicity by binding to the allosteric site of the reverse transcriptase (RT) as a non-nucleoside RT inhibitor (NNRTI). Similarly, marine bromopyrrole alkaloids are well known for their diverse array of anti-infective properties. Hence, we have reported synthesis and in vitro HIV-1 RT inhibitory activity of a series of 4-thiazolidinone-bromopyrrole alkaloid hybrids tethered with amide linker. The results of in vitro HIV-1 RT kit assay showed that some of the compounds, such as 4c, 4d, and 4i could effectively inhibit RT activity. Among them, compounds 4c having 4-chlorophenyl substituted 4-thiazolidione ring was the best one with the IC50 value of 0.26 µM. The sturdy emerges with key structure-activity relationship that pyrrole-NH-free core benefited inhibition against HIV-1 RT inhibition. This study identified conjugate 4c with potent activity and selectivity as promising compound for further drug development to HIV.

Keywords: antiviral drugs, bromopyrrole alkaloids, HIV-1 RT inhibition, 4-thiazolidinone

Procedia PDF Downloads 422

Authors: Alessandra N. Santos, Max P. Ferreira, Alexandra R. P. Silva, Agda A. R. de Oliveira, Marivalda M. Pereira

Abstract:

The literature describes experiments, in which ceria nanoparticles in the bioactive glass significantly improve differentiation of stem cells into osteoblasts and increase production of collagen. It is not known whether this effect observed due to the presence of nanoceria can be also observed in the presence of cerium in the bioactive glass network. The effect of cerium into bioactive glasses using the sol–gel route is the focus of this work, with the goal to develop a material for tissue engineering with the potential to enhance osteogenesis. A bioactive glass composition based on 70% SiO2–30% CaO is produced with the addition of cerium. The analyses XRD, FTIR, SEM/EDS, BET/BJH, in vitro bioactivity test and the Cell viability assay were performed. The results show that cerium remains in the bioactive glass structure. The obtained material present in vitro bioactivity and promote the cell viability.

Keywords: bioactive glass, bioactivity, cerium salt, material characterization, sol-gel method

Procedia PDF Downloads 192

Authors: P. S. Rajinikanth, Shobana Mariappan, Jestin Chellian

Abstract:

The objective of the study was to prepare and characterize a water-in-oil nano emulsion of 5-Fluorouracil (5FU) to enhance the skin penetration. The present study describes a nano emulsion of 5FU using Capyrol PGMC, Transcutol HP and PEG 400 as oil, surfactant and co-surfactant, respectively. The optimized formulations were further evaluated for heating cooling cycle, centrifugation studies, freeze thaw cycling, particle size distribution and zeta potential in order to confirm the stability of the optimized nano emulsions. The in-vitro characterization results showed that the droplets of prepared formulation were ~100 nm with ± 15 zeta potential. In vitro skin permeation studies was conducted in albino mice skin. Significant increase in permeability parameters was also observed in nano emulsion formulations (P<0.05). The steady-state flux (Jss), enhancement ration and permeability coefficient (Kp) for optimized nano emulsion formulation (FU2, FU1, 1:1 S mix were found to be 24.21 ±2.45 μg/cm2/h, 3.28±0.87 & 19.52±1.87 cm/h, respectively), which were significant compared with conventional gel. The in vitro and in vivo skin deposition studies in rat indicated that the amount of drug deposited from the nano emulsion (292.45 µg/cm2) in skin was significant (P<0.05) an increased as compared to a conventional 5FU gel (121.42 µg/cm2). The skin irritation study using rat skin showed that the mean irritation index of the nano emulsion reduced significantly (P<0.05) as compared with conventional gel contain 1% 5FU. The results from this study suggest that a water-in-oil nano emulsion could be safely used to promote skin penetration of 5FU following topical application.

Keywords: nano emulsion, controlled release, 5 fluorouracil, skin penetration, skin irritation

Procedia PDF Downloads 464

Authors: Rudra Vaghela, P. K. Kulkarni

Abstract:

The aim of the present research was to develop oral Amphotericin B (AmB) nanocrystals (Nc) grafted with suitable ligand in order to enhance drug transport across the intestinal epithelial barrier and subsequently, active uptake by macrophages. AmB Nc were prepared by liquid anti-solvent precipitation technique (LAS). Poloxamer 188 was used to stabilize the prepared AmB Nc and grafted with mannose for actively targeting M cells in Peyer’s patches. To prevent shedding of the stabilizer and ligand, N,N’-Dicyclohexylcarbodiimide (DCC) was used as a cross-linker. The prepared AmB Nc were characterized for particle size, PDI, zeta potential, X-ray diffraction (XRD) and surface morphology using scanning electron microscope (SEM) and evaluated for drug content, in vitro drug release and cell uptake studies using caco-2 cells. The particle size of stabilized AmB Nc grafted with WGA was in the range of 287-417 nm with negative zeta potential between -18 to -25 mV. XRD studies revealed crystalline nature of AmB Nc. SEM studies revealed that ungrafted AmB Nc were irregular in shape with rough surface whereas, grafted AmB Nc were found to be rod-shaped with smooth surface. In vitro drug release of AmB Nc was found to be 86% at the end of one hour. Cellular studies revealed higher invasion and uptake of AmB Nc towards caco-2 cell membrane when compared to ungrafted AmB Nc. Our findings emphasize scope on developing oral delivery system for passively targeting M cells in Peyer’s patches.

Keywords: leishmaniasis, amphotericin b nanocrystals, macrophage targeting, LAS technique

Procedia PDF Downloads 268

Authors: Chin-Tsu Ma, Yi-Jhen Wu, Hsia Ying Cheng, Han Hsiang Huang, Shyh Ming Kuo

Abstract:

The use of specific molecules including nutrients and pharmacological agents has been tried in modulation of stem cells differentiation (MSCs) to insulin producing cells. The aim of this study is to investigate the ability of nano collagen molecules (nutrient or scaffold) to enhance the MSCs differentiation into insulin-producing cells in combination with nicotinamide and exendin-4 (pharmacological agents) in vitro and in vivo. The results demonstrated that the cells exhibit morphologically islet-like clusters after treatment with nano collagen molecules, nicotinamide and exendin-4. MSCs extra treated with nano collagen molecules showed significant increases in Nkx6.1 and insulin mRNA expression at 14-d and 21-d culture compared with those merely treated with nicotinamide and exendin-4. Early 7-day elevation in PDX-1 mRNA expression was observed. Furthermore, the MSCs exposed to nano collagen molecules produced the highest secretion of insulin (p ＜ 0.05). Type-2 diabetes induced by high-fat diet and low dose of streptozotocin in rat model was built in this study. This rat exhibited higher food intake, water intake, lower glucose tolerance, lower-insulin tolerance, and higher HbA1C (significant increases, p ＜ 0.01) as compared with the normal rat that demonstrated the model of type-2 diabetes was successfully built. Biopsy examinations also showed that obvious destruction of islet. After injection of differentiated MSCs into the destructed pancreas of diabetes rat, more regenerated islet were observed at the rats that treated with nano collagen molecules and exhibited much lower HbA1C as compared with the normal rat and diabetes rat after 4 weeks (significant deceases, p ＜ 0.001). These results indicate that the culturing MSCs with nano collagen molecules, nicotinamide, and exendin-4 are beneficial for MSCs differentiation into islet-like cells. These nano collagen molecules may lead to alternations or up-regulation of gene expression and influence the differentiated outcomes induced by nicotinamide and exendin-4.

Keywords: nano collagen molecules, nicotinamide, MSCs, diabetes

Procedia PDF Downloads 378

Authors: Michael Graz, Natasha Hurril, Andrew Shearer

Abstract:

Research on feed supplementation with natural compounds is currently being intensively pursued with a view to improving energy utilisation in ruminants and mitigating the production of methane by these animals. Towards this end, a novel combination of extracts from garlic and bitter orange was therefore selected for trials on the basis of their previously published in vitro anti-methanogenic potential. Three separate in vitro experiments were conducted to determine energy utilisation and greenhouse gas production. These included use of rumen fluid from fistulated cows and sheep in batch culture, the Hohenheim gas test, and the Rusitec technique. Experimental and control arms were utilised, with 5g extracts per kilogram of total dietary dry matter (0.05g/kg active compounds) being used to supplement or not supplement the in vitro systems. Respiratory measurements were conducted on experimental day 1 for the batch culture and Hohenheim gas test and on day 14-21 for the Rusitec Technique (in a 21-day trial). Measurements included methane (CH4) production, total volatile fatty acid (VFA) concentration, molar proportions of acetate, propionate and butyrate and degradation of organic matter (Rusitec). CH4 production was reduced by 82% (±16%), 68% (±11%) and 37% (±4%) in the batch culture, Hohenheim gas test and Rusitec, respectively. Total VFA production was reduced by 13% (±2%) and 2% (±0.1%) in the batch culture and Hohenheim gas test whilst it was increased by 8% (±2%) in the Rusitec. Total VFA production was reduced in all tests between 2 and 10%, whilst acetate production was reduced between 10% and 29%. Propionate production which is an indicator of weight gain was increased in all cases between 16% and 30%. Butyrate production which is considered an indicator of potential milk yield was increased by between 6 and 11%. Degradation of organic matter in the Rusitec experiments was improved by 10% (±0.1%). In conclusion, the study demonstrated the potential of the combination of garlic and citrus extracts to improve digestion, enhance body energy retention and limit CH4 formation in relation to feed intake.

Keywords: citrus, garlic, methane, ruminants

Procedia PDF Downloads 302

Authors: Eva Tvrdá, Barbora Babečková, Michal Ďuračka, Róbert Kirchner, Július Árvay

Abstract:

This study was designed to assess the in vitro effects of Origanum vulgare L. (oregano) extract on the testicular steroidogenesis. We focused on identifying major biomolecules present in the oregano extract, as well as to investigate its in vitro impact on the secretion of cholesterol, testosterone, dehydroepiandrosterone and androstenedione by murine testicular fragments. The extract was subjected to high performance liquid chromatography (HPLC) which identified cyranosid, daidzein, thymol, rosmarinic and trans-caffeic acid among the predominant biochemical components of oregano. For the in vitro experiments, testicular fragments from 20 sexually mature Institute of Cancer Research (ICR) mice were incubated in the absence (control group) or presence of the oregano extract at selected concentrations (10, 100 and 1000 μg/mL) for 24 h. Cholesterol levels were quantified using photometry and the hormones were assessed by ELISA (Enzyme-Linked Immunosorbent Assay). Our data revealed that the release of cholesterol and androstenedione (but not dehydroepiandrosterone and testosterone) by the testicular fragments was significantly impacted by the oregano extract in a dose-dependent fashion. Supplementation of the extract resulted in a significant decline of cholesterol (P < 0.05 in case of 100 μg/mL; P < 0.01 with respect 100 μg/mL extract), as well as androstenedione (P < 0.01 with respect to 100 and 1000 μg/mL extract). Our results suggest that the biomolecules present in Origanum vulgare L. could exhibit a dose-dependent impact on the secretion of male steroids, playing a role in the regulation of testicular steroidogenesis.

Keywords: mice, Origanum vulgare L., steroidogenesis, testes

Procedia PDF Downloads 123

Authors: Emma R. Arakelova, Stepan G. Grigoryan, Flora G. Arsenyan, Nelli S. Babayan, Ruzanna M. Grigoryan, Natalia K. Sarkisyan

Abstract:

Novel nanosize zinc oxide composites of doxorubicin obtained by deposition of 180 nm thick zinc oxide film on the drug surface using DC-magnetron sputtering of a zinc target in the form of gels (PEO+Dox+ZnO and Starch+NaCMC+Dox+ZnO) were studied for drug delivery applications. The cancer specificity was revealed both in in vitro and in vivo models. The cytotoxicity of the test compounds was analyzed against human cancer (HeLa) and normal (MRC5) cell lines using MTT colorimetric cell viability assay. IC50 values were determined and compared to reveal the cancer specificity of the test samples. The mechanistic study of the most active compound was investigated using Flow cytometry analyzing of the DNA content after PI (propidium iodide) staining. Data were analyzed with Tree Star FlowJo software using cell cycle analysis Dean-Jett-Fox module. The in vivo anticancer activity estimation experiments were carried out on mice with inoculated ascitic Ehrlich’s carcinoma at intraperitoneal introduction of doxorubicin and its zinc oxide compositions. It was shown that the nanosize zinc oxide film deposition on the drug surface leads to the selective anticancer activity of composites at the cellular level with the range of selectivity index (SI) from 4 (Starch+NaCMC+Dox+ZnO) to 200 (PEO(gel)+Dox+ZnO) which is higher than that of free Dox (SI = 56). The significant increase in vivo antitumor activity (by a factor of 2-2.5) and decrease of general toxicity of zinc oxide compositions of doxorubicin in the form of the above mentioned gels compared to free doxorubicin were shown on the model of inoculated Ehrlich's ascitic carcinoma. Mechanistic studies of anticancer activity revealed the cytostatic effect based on the high level of DNA biosynthesis inhibition at considerable low concentrations of zinc oxide compositions of doxorubicin. The results of studies in vitro and in vivo behavior of PEO+Dox+ZnO and Starch+NaCMC+Dox+ZnO composites confirm the high potential of the nanosize zinc oxide composites as a vector delivery system for future application in cancer chemotherapy.

Keywords: anticancer activity, cancer specificity, doxorubicin, zinc oxide

Procedia PDF Downloads 374

Authors: Z. Hormozi Moghaddam, M. Mokhtari-Dizaji, M. Movahedin, M. E. Ravari

Abstract:

Mechanical index (MI) is used for quantifying acoustic cavitation and the relationship between acoustic pressure and the frequency. In this study, modeling of the MI was applied to provide treatment protocol and to understand the effective physical processes on reproducibility of stem cells. The acoustic pressure and MI equations are modeled and solved to estimate optimal MI for 28, 40, 150 kHz and 1 MHz frequencies. Radial and axial acoustic pressure distribution was extracted. To validate the results of the modeling, the acoustic pressure in the water and near field depth was measured by a piston hydrophone. Results of modeling and experiments show that the model is consistent well to experimental results with 0.91 and 0.90 correlation of coefficient (p<0.05) for 1 MHz and 40 kHz. Low intensity ultrasound with 0.40 MI is more effective on the proliferation rate of the spermatogonial stem cells during the seven days of culture, in contrast, high MI has a harmful effect on the spermatogonial stem cells. This model provides proper treatment planning in vitro and in vivo by estimating the cavitation phenomenon.

Keywords: ultrasound, mechanical index, modeling, stem cell

Procedia PDF Downloads 296

Authors: Nishanth Venugopal Menon, Chuah Yon Jin, Samantha Phey, Wu Yingnan, Zhang Ying, Vincent Chan, Kang Yuejun

Abstract:

Cell Migration is a vital phenomenon that the cells undergo in various physiological processes like wound healing, disease progression, embryogenesis, etc. Cell migration depends primarily on the chemical and physical cues available in the cellular environment. The chemical cue involves the chemokines secreted and gradients generated in the environment while physical cues indicate the impact of matrix properties like nanotopography and stiffness on the cells. Mesenchymal Stem Cells (MSCs) have been shown to have a role wound healing in vivo and its migration to the site of the wound has been shown to have a therapeutic effect. In the field of stem cell based tissue regeneration of bones and cartilage, one approach has been to introduce scaffold laden with MSCs into the site of injury to enable tissue regeneration. In this work, we have studied the combinatorial impact of the substrate physical properties on MSC migration. A microfluidic in vitro model was created to perform the migration studies. The microfluidic model used is a three compartment device consisting of two cell seeding compartments and one migration compartment. Four different PDMS substrates with varying substrate roughness, stiffness and hydrophobicity were created. Its surface roughness and stiffness was measured using Atomic Force Microscopy (AFM) while its hydrphobicity was measured from the water contact angle using an optical tensiometer. These PDMS substrates are sealed to the microfluidic chip following which the MSCs are seeded and the cell migration is studied over the period of a week. Cell migration was quantified using fluorescence imaging of the cytoskeleton (F-actin) to find out the area covered by the cells inside the migration compartment. The impact of adhesion proteins on cell migration was also quantified using a real-time polymerase chain reaction (qRT PCR). These results suggested that the optimal substrate for cell migration would be one with an intermediate level of roughness, stiffness and hydrophobicity. A higher or lower value of these properties affected cell migration negatively. These observations have helped us in understanding that different substrate properties need to be considered in tandem, especially while designing scaffolds for tissue regeneration as cell migration is normally impacted by the combinatorial impact of the matrix. These observations may lead us to scaffold optimization in future tissue regeneration applications.

Keywords: cell migration, microfluidics, in vitro model, stem cell migration, scaffold, substrate properties

Procedia PDF Downloads 524

Authors: Ilzira A. Minigalieva, Tatiana V. Bushueva, Eleonore Frohlich, Vladimir Panov, Ekaterina Shishkina, Boris A. Katsnelson

Abstract:

Background: The overwhelming majority of the experimental studies in the field of metal nanotoxicology have been performed on cultures of established cell lines, with very few researchers focusing on animal experiments, while a juxtaposition of conclusions inferred from these two types of research is blatantly lacking. The least studied aspect of this problem relates to characterizing and predicting the combined toxicity of metallic nanoparticles. Methods: Comparative and combined toxic effects of purposefully prepared spherical NiO and Mn₃O₄ nanoparticles (mean diameters 16.7 ± 8.2 nm and 18.4 ± 5.4 nm respectively) were estimated on cultures of human cell lines: MRC-5 fibroblasts, THP-1 monocytes, SY-SY5Y neuroblastoma cells, as well as on the latter two lines differentiated to macrophages and neurons, respectively. The combined cytotoxicity was mathematically modeled using the response surface methodology. Results: The comparative assessment of the studied NPs unspecific toxicity previously obtained in vivo was satisfactorily reproduced by the present in vitro tests. However, with respect to manganese-specific brain damage which had been demonstrated by us in animal experiment with the same NPs, the testing on neuronall cell culture showed only a certain enhancing effect of Mn₃O₄-NPs on the toxic action of NiO-NPs, while the role of the latter prevailed. Conclusion: From the point of view of the preventive toxicology, the experimental modeling of metallic NPs combined toxicity on cell cultures can give non-reliable predictions of the in vivo action’s effects.

Keywords: manganese oxide, nickel oxide, nanoparticles, in vitro toxicity

Procedia PDF Downloads 261

Authors: Mala Nath, Nagamani Kompelli, Partha Roy, Snehasish Das

Abstract:

Two new metal-based anticancer chemotherapeutic agents, [(Ph2Sn)2(HGuO)2(phen)Cl2] 1 and [(Ph3Sn)(HGuO)(phen)]- Cl.CH3OH.H2O 2, were designed, prepared and characterized by analytical and spectral (IR, ESI-Mass, 1H, 13C and 119Sn NMR) techniques. The proposed geometry of Sn(IV) in 1 and 2 is distorted octahedral and distorted trigonal-bipyramidal, respectively. Both 1 and 2 exhibit potential cytotoxicity in vitro against MCF-7, HepG-2 and DU-145 cell lines. The intrinsic binding constant (Kb) values of 1 (2.33 × 105 M-1) and 2 (2.46 × 105 M-1) evaluated from UV-Visible absorption studies suggest non-classical electrostatic mode of interaction via phosphate backbone of DNA double helix. The Stern-Volmer quenching constant (Ksv) of 1 (9.74 × 105 M-1) and 2 (2.9 × 106 M-1) determined by fluorescence studies suggests the groove binding and intercalation mode for 1 and 2, respectively. Effective cleavage of pBR322 DNA is induced by 1. Their interaction with DNA of cancer cells may account for potency.

Keywords: anticancer agents, DNA binding studies, NMR spectroscopy, organotin

Procedia PDF Downloads 220

Authors: Cheila Pereira, Sara C. Cunha, Miguel A. Faria, José O. Fernandes

Abstract:

The increasing world population brings several concerns, one of which is food security and sustainability. To meet this challenge, aquaculture, the farming of aquatic animals and plants, including fish, mollusks, bivalves, and algae, has experienced sustained growth and development in recent years. Recent advances in this industry have focused on reducing its economic and environmental costs, for example, the substitution of protein sources in fish feed. Plant-based proteins are now a common approach, and while it is a greener alternative to animal-based proteins, there are some disadvantages, such as their putative content and intoxicants such as mycotoxins. These are naturally occurring plant contaminants, and their exposure in fish can cause health problems, stunted growth or even death, resulting in economic losses for the producers and health concerns for the consumers. Different works have demonstrated the presence of both AFB1 (aflatoxin B1) and ENNB1 (enniatin B1) in fish feed and their capacity to be absorbed and bioaccumulate in the fish organism after digestion, further reaching humans through fish ingestion. The aim of this work was to evaluate the bioaccessibility of both mycotoxins in samples of Sparus aurata muscle using a static digestion model based on the INFOGEST protocol. The samples were subjected to different cooking procedures – raw, grilled and fried – and different seasonings – none, thyme and ginger – in order to evaluate their potential reduction effect on mycotoxins bioaccessibility, followed by the evaluation of the intestinal transport of both compounds with an in vitro cell model composed of Caco-2/HT-29 co-culture monolayers, simulating the human intestinal epithelium. The bioaccessible fractions obtained in the digestion studies were used in the transport studies for a more realistic approach to bioavailability evaluation. Results demonstrated the effect of the use of different cooking procedures and seasoning on the toxin's bioavailability. Sparus aurata was chosen in this study for its large production in aquaculture and high consumption in Europe. Also, with the continued evolution of fish farming practices and more common usage of novel feed ingredients based on plants, there is a growing concern about less studied contaminants in aquaculture and their consequences for human health. In pair with greener advances in this industry, there is a convergence towards alternative research methods, such as in vitro applications. In the case of bioavailability studies, both in vitro digestion protocols and intestinal transport assessment are excellent alternatives to in vivo studies. These methods provide fast, reliable and comparable results without ethical restraints.

Keywords: AFB1, aquaculture, bioaccessibility, ENNB1, intestinal transport.

Procedia PDF Downloads 19

Authors: Raj K. Gupta, X. Gary Tan, Andrzej Przekwas

Abstract:

Bomb blasts from Improvised Explosive Devices (IEDs) account for vast majority of terrorist attacks worldwide. Injuries caused by IEDs result from a combination of the primary blast wave, penetrating fragments, and human body accelerations and impacts. This paper presents a multiscale computational model of coupled blast physics, whole human body biodynamics and injury biomechanics of sensitive organs. The disparity of the involved space- and time-scales is used to conduct sequential modeling of an IED explosion event, CFD simulation of blast loads on the human body and FEM modeling of body biodynamics and injury biomechanics. The paper presents simulation results for blast-induced brain injury coupling macro-scale brain biomechanics and micro-scale response of sensitive neuro-axonal structures. Validation results on animal models and physical surrogates are discussed. Results of our model can be used to 'replicate' filed blast loadings in laboratory controlled experiments using animal models and in vitro neuro-cultures.

Keywords: blast waves, improvised explosive devices, injury biomechanics, mathematical models, traumatic brain injury

Procedia PDF Downloads 212

Authors: Krishna Poudel, Tahar Katuwal, Sujan Karki

Abstract:

A rapid propagation and acclimatization method of large cardamom was optimized in this study. Sprouted rhizome buds were collected. The excised rhizome bud explants were cultured on semi solid culture media. The explants were cultured on Murashige and Skoog’s (MS) medium supplemented with different concentration and combinations of BAP (6-Benzyl-amino-purine) and IBA (Indole-3-butyric acid) for shoot and root induction. Explants cultured on MS basal medium supplemented with 1.0 mg/l BAP + 0.5 gm/l IBA showed the highest rate of shoot multiplication. In vitro shoots were rooted on to the half-strength MS basal media supplemented with 0.5 mg/l IBA. Rooted shoots were transplanted in the screen house for hardening process. These hardened plants were subsequently shifted into the netted nursery for further multiplication process.

Keywords: concentration, explants, hardening, rhizome

Procedia PDF Downloads 203

Authors: Eun Jeong Yu, Inn Soo Kang, Tae Ki Yoon, Mi Kyoung Koong

Abstract:

Advanced female age is associated with higher cycle cancelation rates, lower clinical pregnancy rate, increased miscarriage and aneuploidy rates in IVF (In Vitro Fertilization) cycles. This retrospective cohort study was conducted at a Cha Fertility Center, Seoul Station. All fresh non-donor IVF cycles performed in women aged 40 years and above from January 2016 to December 2016 were reviewed. Donor/recipient treatment, PGD/PGS (Preimplantation Genetic Diagnosis/ Preimplantation Genetic Screening) were excluded from analysis. Of the 1,166 cycles from 753 women who completed ovulation induction, 1,047 were appropriate for the evaluation according to inclusion and exclusion criterion. IVF cycles were categorized according to age and grouped into the following 1-year age groups: 40, 41, 42, 43, 44, 45 and > 46. The mean age of patients was 42.4 ± 1.8 years. The median AMH (Anti-Mullerian Hormone) level was 1.2 ± 1.5 ng/mL. The mean number of retrieved oocytes was 4.9 ± 4.3. The clinical pregnancy rate and live birth rate in women > 40 years significantly decreased with each year of advancing age (p < 0.001). The clinical pregnancy rate decreased from 21% at the age of 40 years to 0% at ages above 45 years. Live birth rate decreased from 12.3% to 0%, respectively. There were no clinical pregnancy outcomes among 95 patients aged above 45 years of age. The overall miscarriage rate was 40.7% (range, 36.7%-70%). The transfer of at least one good quality embryo was associated with about 4-9% increased chance of a clinical pregnancy rate. Therefore, IVF in old age women less than 46 had a reasonable chance for successful pregnancy outcomes especially when good quality embryo is transferred.

Keywords: advanced maternal age, in vitro fertilization, pregnancy rate, live birth rate

Procedia PDF Downloads 110

Authors: A. Reum Son, Jin Seon Kwon, Seung Hun Park, Hai Bang Lee, Moon Suk Kim

Abstract:

These days, stem cell therapy is focused on for promising source of treatment in clinical human disease. As a supporter of stem cells, in situ-forming hydrogels with growth factors and cells appear to be a promising approach in tissue engineering. To examine osteogenic differentiation of hTMSCs which is one of mesenchymal stem cells in vivo in an injectable hydrogel, we use a methoxy polyethylene glycol-polycaprolactone blockcopolymer (MPEG-PCL) solution with osteogenic factors. We synthesized MPEG-PCL hydrogel and measured viscosity to check sol-gel transition. In order to demonstrate osteogenic ability of hTMSCs, we conducted in vitro osteogenesis experiment. Then, to confirm the cell cytotoxicity, we performed WST-1 with hTMSCs and MPEG-PCL. As the result of in vitro experiment, we implanted cell and hydrogel mixture into animal model and checked degree of osteogenesis with histological analysis and amount of expression genes. Through these experimental data, MPEG-PCL hydrogel has sol-gel transition in temperature change and is biocompatible with stem cells. In histological analysis and gene expression, hTMSCs are very good source of osteogenesis with hydrogel and will use it to tissue engineering as important treatment method. hTMSCs could be a good adult stem cell source for usability of isolation and high proliferation. When hTMSCs are used as cell therapy method with in situ-formed hydrogel, they may provide various benefits like a noninvasive alternative for bone tissue engineering applications.

Keywords: injectable hydrogel, stem cell, osteogenic differentiation, tissue engineering

Procedia PDF Downloads 412

Authors: Deepika Saini, Sandeep Jain, Ajay Kumar

Abstract:

The quinoline scaffold is one of the most widely studied in the discovery of derivatives with various heterocyclic moieties due to its potential antimalarial activities. In the present study, a chalcone series of quinoline derivatives clubbed with pyrazole were synthesized to evaluate their antimalarial property by in vitro schizont maturation inhibition assay against both chloroquine sensitive, 3D7 and chloroquine resistant, RKL9 strain of Plasmodium falciparum. Further, top five compounds were studied for in vivo preclinical study for antimalarial potential against P. berghei in Swiss albino mice. To understand the mechanism of synthesized analogues, they were screened computationally by molecular docking techniques. Compounds were docked into the active site of a protein receptor, Plasmodium falciparum Cysteine Protease Falcipain-2. The compounds were successfully synthesized, and structural confirmation was performed by FTIR, 1H-NMR, mass spectrometry and elemental analysis. In vitro study suggested that the compounds 5b, 5g, 5l, 5s and 5u possessed best antimalarial activity and further tested for in vivo screening. Compound 5u (CH₃ on both rings) with EC₅₀ 0.313 & 0.801 µg/ml against CQ-S & CQ-R strains of P. falciparum respectively and 78.01% suppression of parasitemia. The molecular docking studies of the compounds helped in understanding the mechanism of action against falcipain-2. The present study reveals the binding signatures of the synthesized ligands within the active site of the protein, and it explains the results from in vitro study in their EC₅₀ values and percentage parasitemia.

Keywords: antimalarial activity, chalcone, docking, quinoline

Procedia PDF Downloads 377

Authors: P. S. Percin, O. Inanli, S. Karakaya

Abstract:

Bitter melon (Momordica charantia) is a medicinal vegetable, which is used traditionally to remedy diabetes. Bitter melon contains several classes of primary and secondary metabolites. In traditional Turkish medicine bitter melon is used for wound healing and treatment of peptic ulcers. Nowadays, bitter melon is used for the treatment of diabetes and ulcerative colitis in many countries. The main constituents of bitter melon, which are responsible for the anti-diabetic effects, are triterpene, protein, steroid, alkaloid and phenolic compounds. In this study total phenolics, total carotenoids and β-carotene contents of mature and immature bitter melons were determined. In addition, in vitro α-amylase and α-glucosidase activities of mature and immature bitter melons were studied. Total phenolic contents of immature and mature bitter melon were 74 and 123 mg CE/g bitter melon respectively. Although total phenolics of mature bitter melon was higher than that of immature bitter melon, this difference was not found statistically significant (p > 0.05). Carotenoids, a diverse group of more than 600 naturally occurring red, orange and yellow pigments, play important roles in many physiological processes both in plants and humans. The total carotenoid content of mature bitter melon was 4.36 fold higher than the total carotenoid content of immature bitter melon. The compounds that have hypoglycaemic effect of bitter melon are steroidal saponins known as charantin, insulin-like peptides and alkaloids. α-Amylase is one of the main enzymes in human that is responsible for the breakdown of starch to more simple sugars. Therefore, the inhibitors of this enzyme can delay the carbohydrate digestion and reduce the rate of glucose absorption. The immature bitter melon extract showed α-amylase and α-glucosidase inhibitory activities in vitro. α-Amylase inhibitory activity was higher than that of α-glucosidase inhibitory activity when IC50 values were compared. In conclusion, the present results provide evidence that aqueous extract of bitter melon may have an inhibitory effect on carbohydrate breakdown enzymes.

Keywords: bitter melon, in vitro antidiabetic activity, total carotenoids, total phenols

Procedia PDF Downloads 212