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Authors: Mubashar Iqbal, Iqra Anjum, Muhammad Dildar Gogi, Muhammad Jalal Arif

Abstract:

The present study was carried to screen out the effective entomopathogenic fungi (EPF) inoculation method in maize and to evaluate pathogenicity and oviposition-choice in C. partellus. Three entomopathogenic fungi (EPF) formulations Pacer® (Metarhizium anisopliae), Racer® (Beauveria bassiana) and Meailkil® (Verticillium lecanii) were evaluated at three concentrations (5000, 10000 and 20000 ppm) for their endophytism in maize and pathogenicity in C. partellus. The stock solution of the highest concentration (20,000 ppm) was prepared and next lower from stock solution. In the first experiment, three EPF was inoculated in maize plant by four methods, i.e., leaf-inoculation (LI), whorl-inoculation (WI), shoot-inoculation (SI) and root-inoculation (RI). Leaf-discs and stem-cutting were sampled in all four inoculation methods and placed on fungus growth media in Petri dishes. In the second experiment, pathogenicity, pupal formation, adult emergence, sex ratio, oviposition-choice, and growth index of C. partellus were calculated. The leaves and stem of the inoculated plants were given to the counted number of larvae of C. Partellus. The mortality of larvae was recorded on daily basis till the pupation. The result shows that maximum percent mortality (86.67%) was recorded at high concentration (20000ppm) of Beauveria bassiana by leaf inoculation method. For oviposition choice bioassay, the newly emerged adults were fed on diet (water, honey and yeast in 9:1:1) for 48 hours. One pair of C. Partellus were aspirated from the rearing cages and were detained in large test tube plugged with diet soaked cotton. A set of four plants for each treatment were prepared and randomized inside the large oviposition chamber. The test tubes were opened and fitted in the hole made in the wall of oviposition chamber in front of each treatment. The oviposition chamber was placed in a completely dark laboratory to eliminate the effect of light on moth’s behavior. The plants were removed from the oviposition chamber after the death of adults. The number of eggs deposited on the plant was counted. The results of 2nd experiment revealed that in all EPF and inoculation methods, the fecundity, egg fertility and growth index of C. partellus decreased with the increase in concentration being significantly higher at low concentration (5000ppm) and lower at higher concentration (20000ppm). Application of B. bassiana demonstrated that minimum fecundity (126.83), egg fertility (119.52) and growth index (15%) in C. partellus followed by M. anisopliae with fecundity (135.93), egg fertility (132.29) and growth index (17.50%) while V. lecanii show higher values of fecundity (137.37), egg fertility (1135.42) and growth index (20%). Overall leaf inoculation method showed least fecundity (123.89) with egg fertility (115.36) and growth index (14%) followed by whorl, shoot inoculation method and root inoculation method show higher values of fecundity, egg fertility and growth index.

Keywords: Beauveria bassiana, Chilo partellus, entomopathoganic, Metarhizium anisopliae, Verticillium lecanii

Procedia PDF Downloads 105

Authors: Hilal Turkoglu Sasmazel, Ozan Ozkan, Seda Surucu

Abstract:

Tissue engineering is the field of treating defects caused by injuries, trauma or acute/chronic diseases by using artificial scaffolds that mimic the extracellular matrix (ECM), the natural biological support for the tissues and cells within the body. The main aspects of a successful artificial scaffold are (i) large surface area in order to provide multiple anchorage points for cells to attach, (ii) suitable porosity in order to achieve 3 dimensional growth of the cells within the scaffold as well as proper transport of nutrition, biosignals and waste and (iii) physical, chemical and biological compatibility of the material in order to obtain viability throughout the healing process. By hybrid scaffolds where two or more different materials were combined with advanced fabrication techniques into complex structures, it is possible to combine the advantages of individual materials into one single structure while eliminating the disadvantages of each. Adding this to the complex structure provided by advanced fabrication techniques enables obtaining the desired aspects of a successful artificial tissue scaffold. In this study, fibroblast compatibility of poly(ε-caprolactone) (PCL)/chitosan core-shell electrospun hybrid scaffolds with proper mechanical, chemical and physical properties successfully developed in our previous study was investigated. Standard 7-day cell culture was carried out with L929 fibroblast cell line. The viability of the cells cultured with the scaffolds was monitored with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay for every 48 h starting with 24 h after the initial seeding. In this assay, blank commercial tissue culture polystyrene (TCPS) Petri dishes, single electrospun PCL and single electrospun chitosan mats were used as control in order to compare and contrast the performance of the hybrid scaffolds. The adhesion, proliferation, spread and growth of the cells on/within the scaffolds were observed visually on the 3rd and the 7th days of the culture period with confocal laser scanning microscopy (CSLM) and scanning electron microscopy (SEM). The viability assay showed that the hybrid scaffolds caused no toxicity for fibroblast cells and provided a steady increase in cell viability, effectively doubling the cell density for every 48 h for the course of 7 days, as compared to TCPS, single electrospun PCL or chitosan mats. The cell viability on the hybrid scaffold was ~2 fold better compared to TCPS because of its 3D ECM-like structure compared to 2D flat surface of commercially cell compatible TCPS, and the performance was ~2 fold and ~10 fold better compared to single PCL and single chitosan mats, respectively, even though both fabricated similarly with electrospinning as non-woven fibrous structures, because single PCL and chitosan mats were either too hydrophobic or too hydrophilic to maintain cell attachment points. The viability results were verified with visual images obtained with CSLM and SEM, in which cells found to achieve characteristic spindle-like fibroblast shape and spread on the surface as well within the pores successfully at high densities.

Keywords: chitosan, core-shell, fibroblast, electrospinning, PCL

Procedia PDF Downloads 138

Authors: Elvin P. Chizenga, Heidi Abrahamse

Abstract:

Cancer cell resistance to therapy is the main cause of treatment failures and the poor prognosis of cancer convalescence. The progression of cervical cancer to other parts of the genitourinary system and the reported recurrence rates are overwhelming. Current treatments, including surgery, chemo and radiation have been inefficient in eradicating the tumor cells. These treatments are also associated with poor prognosis and reduced quality of life, including fertility loss. This has inspired the need for the development of new treatment modalities to eradicate cervical cancer successfully. Photodynamic Therapy (PDT) is a modern treatment modality that induces cell death by photochemical interactions of light and a photosensitizer, which in the presence of molecular oxygen, yields a set of chemical reactions that generate Reactive Oxygen Species (ROS) and other free radical species causing cell damage. Enhancing PDT using modified drug delivery can increase the concentration of the photosensitizer in the tumor cells, and this has the potential to maximize its therapeutic efficacy. In cervical cancer, all infected cells constitutively express genes of the E6 and E7 HPV viral oncoproteins, resulting in high concentrations of E6 and E7 in the cytoplasm. This provides an opportunity for active targeting of cervical cancer cells using immune-mediated drug delivery to maximize therapeutic efficacy. The use of nanoparticles in PDT has also proven effective in enhancing therapeutic efficacy. Gold nanoparticles (AuNps) in particular, are explored for their use in biomedicine due to their biocompatibility, low toxicity, and enhancement of drug uptake by tumor cells. In this present study, a biomolecule comprising of AuNPs, anti-E6 monoclonal antibodies, and Aluminium Phthalocyanine photosensitizer was synthesized for use in targeted PDT of cervical cancer. The AuNp-Anti-E6-Sulfonated Aluminium Phthalocyanine mix (AlPcSmix) photosensitizing biomolecule was synthesized by coupling AuNps and anti-E6 monoclonal antibodies to the AlPcSmix via Polyethylene Glycol (PEG) chemical links. The final product was characterized using Transmission Electron Microscope (TEM), Zeta Potential, Uv-Vis Spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), and X-ray diffraction (XRD), to confirm its chemical structure and functionality. To observe its therapeutic role in treating cervical cancer, cervical cancer cells, HeLa cells were seeded in 3.4 cm² diameter culture dishes at a concentration of 5x10⁵ cells/ml, in vitro. The cells were treated with varying concentrations of the photosensitizing biomolecule and irradiated using a 673.2 nm wavelength of laser light. Post irradiation cellular responses were performed to observe changes in morphology, viability, proliferation, cytotoxicity, and cell death pathways induced. Dose-Dependent response of the cells to treatment was demonstrated as significant morphologic changes, increased cytotoxicity, and decreased cell viability and proliferation This study presented a synthetic biomolecule for targeted PDT of cervical cancer. The study suggested that PDT using this AuNp- Anti-E6- AlPcSmix photosensitizing biomolecule is a very effective treatment method for the eradication of cervical cancer cells, in vitro. Further studies in vivo need to be conducted to support the use of this biomolecule in treating cervical cancer in clinical settings.

Keywords: anti-E6 monoclonal antibody, cervical cancer, gold nanoparticles, photodynamic therapy

Procedia PDF Downloads 99

Authors: B.L. España-Sánchez, E. Luna-Hernández, R.A. Mauricio-Sánchez, M.E. Cruz-Soto, F. Padilla-Vaca, R. Muñoz, L. Granados-López, L.R. Ovalle-Flores, J.L. Menchaca-Arredondo, G. Luna-Bárcenas

Abstract:

Treatment of burn injured has been considered an important clinical problem due to the fluid control and the presence of microorganisms during the healing process. Conventional treatment includes antiseptic techniques, topical medication and surgical removal of damaged skin, to avoid bacterial growth. In order to accelerate this process, different alternatives for tissue regeneration have been explored, including artificial skin, polymers, hydrogels and hybrid materials. Some requirements consider a nonreactive organic polymer with high biocompatibility and skin adherence, avoiding bacterial infections. Chitin-derivative biopolymer such as chitosan (CS) has been used in skin regeneration following third-degree burns. The biological interest of CS is associated with the improvement of tissue cell stimulation, biocompatibility and antibacterial properties. In particular, antimicrobial properties of CS can be significantly increased when is blended with nanostructured materials. Silver-based nanocomposites have gained attention in medicine due to their high antibacterial properties against pathogens, related to their high surface area/volume ratio at nanomolar concentrations. Silver nanocomposites can be blended or synthesized with chitin-derivative biopolymers in order to obtain a biodegradable/antimicrobial hybrid with improved physic-mechanical properties. In this study, nanocomposites based on chitosan/silver nanoparticles (CS/nAg) were synthesized by the in situ chemical reduction method, improving their antibacterial properties against pathogenic bacteria and enhancing the healing process in thermal burn injuries produced in an animal model. CS/nAg was prepared in solution by the chemical reduction method, using AgNO₃ as precursor. CS was dissolved in acetic acid and mixed with different molar concentrations of AgNO₃: 0.01, 0.025, 0.05 and 0.1 M. Solutions were stirred at 95°C during 20 hours, in order to promote the nAg formation. CS/nAg solutions were placed in Petri dishes and dried, to obtain films. Structural analyses confirm the synthesis of silver nanoparticles (nAg) by means of UV-Vis and TEM, with an average size of 7.5 nm and spherical morphology. FTIR analyses showed the complex formation by the interaction of hydroxyl and amine groups with metallic nanoparticles, and surface chemical analysis (XPS) shows low concentration of Ag⁰/Ag⁺ species. Topography surface analyses by means of AFM shown that hydrated CS form a mesh with an average diameter of 10 µm. Antibacterial activity against S. aureus and P. aeruginosa was improved in all evaluated conditions, such as nAg loading and interaction time. CS/nAg nanocomposites films did not show Ag⁰/Ag⁺ release in saline buffer and rat serum after exposition during 7 days. Healing process was significantly enhanced by the presence of CS/nAg nanocomposites, inducing the production of myofibloblasts, collagen remodelation, blood vessels neoformation and epidermis regeneration after 7 days of injury treatment, by means of histological and immunohistochemistry assays. The present work suggests that hydrated CS/nAg nanocomposites can be formed a mesh, improving the bacterial penetration and the contact with embedded nAg, producing complete growth inhibition after 1.5 hours. Furthermore, CS/nAg nanocomposites improve the cell tissue regeneration in thermal burn injuries induced in rats. Synthesis of antibacterial, non-toxic, and biocompatible nanocomposites can be an important issue in tissue engineering and health care applications.

Keywords: antibacterial, chitosan, healing process, nanocomposites, silver

Procedia PDF Downloads 260