Elvis K. Tiburu

Crystallization of Linde Type A Nanomaterials at Two Temperatures Exhibit Differential Inhibition of HeLa Cervical Cancer Cells In Vitro

This work reports evidence of the synthesis of zeolite A at two different temperatures (60 °C and 105 °C) from kaolin. XRD spectral analysis revealed percent crystallinity of 74 ± 2 and 71 ± 3 in LTA60 and LTA105 respectively. The average crystallize size of LTA60 and LTA105 was also estimated using Scherrer’s equation to be 57 nm. FTIR analysis showed signature peaks characteristics of zeolite A in the two materials, except the appearance of absorbance peak at 2380 cm− 1 in the LTA60 spectrum which was not observed in LTA105. Nitrogen porosity measurements of LTA60 and LTA105 gave the following parameters: average pore width of 2.45 and 1.01 nm respectively for LTA60 and LTA105, surface areas of 19.18 and 8.00 m2/g and pore volume of 0.012 and 0.002 cm3/g in that order. The influence of the materials on HeLa cancer cell growth was also investigated and the results showed differential inhibitory effects with LTA60 revealing pronounced inhibitory effects compared to LTA105 after 72 hours of cell incubation. This studies highlighted the importance of crystallization conditions that could influence the physicochemical parameters including structure, porosity, size, and morphology of zeolite materials on cell activity. The work also provided unique opportunities for utilizing natural deposits of kaolin globally to fabricate biomimetic materials for various biological applications.

Investigating the Influence of Temperature on the Kaolinite-Base Synthesis of Zeolite and Urease Immobilization for the Potential Fabrication of Electrochemical Urea Biosensors

Temperature-dependent zeolite synthesis has revealed a unique surface morphology, surface area and pore size which influence the immobilization of urease on gold electrode supports for biosensor fabrication. XRD characterization has identified zeolite X (Na) at all crystallization temperatures tested. However, N2 adsorption and desorption results showed a pore size and pore volume of zeolite X (Na) 60 °C, zeolite X (Na) 70 °C and zeolite X (Na) 90 °C to range from 1.92 nm to 2.45 nm and 0.012 cm3/g to 0.061 cm3/g, respectively, with no significant differences. The specific surface area of zeolite X (Na) at 60, 70 and 90 °C was 64 m2/g, 67 m2/g and 113 m2/g, respectively. The pore size, specific surface area and pore volumes of zeolite X (Na) 80 °C and zeolite X (Na) 100 °C were dramatically increased to 4.21 nm, 295 m2/g, 0.762 cm3/g and 4.92 nm, 389 m2/g, 0.837 cm3/g, in that order. The analytical performance of adsorbed urease on zeolite X (Na) surface was also investigated using cyclic voltammetry measurements, and the results showed distinct cathodic and anodic peaks by zeolite X (Na) 80 °C and zeolite X (Na) 100 °C. These zeolites’ molar conductance was measured as a function of urea concentration and gave an average polynomial regression fit of 0.948. The findings in this study suggest that certain physicochemical properties, such as crystallization temperature and pH, are critical parameters for improving the morphological properties of zeolites synthesized from natural sources for various biomedical applications.

Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques

This work was intended to develop self-assembly lipids for incorporating G-protein coupled receptors (GPCRs) in order to improve the success rate for nuclear magnetic resonance spectroscopy (NMR) structural elucidation. We hereby report the expression and purification of uniformly 15N-labeled human cannabinoid receptor-2 domain in insect cell media. The domain was refolded by screening several membrane mimetic environments. Different q ratios of isotropic bicelles were screened for solubilizing transmembrane helix 6, 7 and 8 (TMH67H8). As the concentration of dimyristoylphosphocholine (DMPC) was increased such that the q ratio was between 0.16 and 0.42, there was less crowding in the cross peaks with increasing q ratio. In bicelles of q = 0.42, the maximum number of cross peaks were obtained and the cross peaks were uniformly dispersed. The receptor domain in bicelles beyond q = 0.42 resulted in peak crowding. These studies demonstrate that GPCRs folding especially in bicelles is protein-specific and requires the right mix of the longer chain and shorter chain lipids to provide the right environment for proper folding. These findings will allow further development of novel membrane mimetics to provide greater diversity of lipid mixtures than those currently being employed for GPCR stability and folding, which are critical for both X-ray and NMR studies of GPCRs.

Formation of Chitosan Nanoparticles Using Deacetylated Chitin Isolated from Freshwater Algae and Locally Synthesized Zeolite A and their Influence on Cancer Cell Growth

This work reports the isolation and characterization of chitin from green algae using XRD, 13C CP/MAS NMR, FTIR and Microscopy. The XRD diffraction pattern confirmed orthorhombic structure of the crystalline polysaccharide, whereas the FTIR spectra revealed strong absorption bands at 896.9 cm-1 and 852 cm-1 typical of C–H axial and C–H equatorial vibrations within the anomeric center of the glucopyranosicyclic moiety. Another strong absorption band was observed at 1039.9 cm-1 and was assigned to C–O–C, C–O stretching bands. The purity and structure of the deacetylated chitin was confirmed using 13C NMR, showing overlapping peaks around 65 ppm assigned to both the sugar carbon at C2, as well as a methylene carbon at C6. An intense peak at 74 ppm is assigned to C3 and C5 with corresponding resonances at 81 and 104 ppm assigned to C4 and C1 respectively. Zeolite/Chitosan nanocomposites were synthesized by ionic cross-linking of chitosan with sodium tripolyphosphate. Chitosan nanoparticles and LTA of different concentrations were incubated with HeLa cancer cells to investigate their cytotoxicity effects. The exposure of the cells to chitosan nanoparticles resulted in a decreased in cell growth and this was concentration-dependent. Our results revealed the utility of locally available materials to produce new biodegradable nanoparticles to investigate their biological nanotoxicity.

Electrochemical Evaluation of Ion Substituted-Hydroxyapatite on HeLa Cells Plasma Membrane Potential

This study reports the electrochemical activities of a novel ion substituted-Hydroxyapatite material in contact with HeLa cells. The work was performed to evaluate the inhibitory effects of various concentrations of the material on the ion transfer mechanisms in HeLa cells. The materials (n=2: HAp1 and HAp3) were prepared at different stirring times from Achatina achatina snail shells and phosphate-containing solution. The structure of the materials and the trace elements concentration were evaluated using x-ray diffractometry and infrared spectrometry as well as atomic absorption spectroscopy. Electrochemical studies conducted on the cells, after 30 min of exposure to the materials, demonstrated differential responses as elucidated by cyclic voltammetry. The voltammograms revealed HAp1 to be non-redox whereas HAp3 was redox active. Minimal concentrations of HAp1 showed high anodic peak current when compared to the HeLa cells alone, indicating a hyperpolarization of the cells. The peak current gradually reduced as the concentration of HAp1 was increased, and then a sudden rise suggesting inhibition of the cell action potential. HAp3 showed a wavy pattern of the anodic peak current when the material concentration was varied. Peak currents of 0.92 and 0.57 nA were recorded for HAp1 and HAp3, respectively at the highest concentration of 5 μL. The results suggest that different inhibitory mechanisms are at play on the voltage-gated ion channels of the cells, indicating the possibility of using the materials to achieve different cancer proliferation inhibition.

Investigating the Conformation of S100β Protein Under Physiological Parameters Using Computational Modeling: A Clue for Rational Drug Design

Background: Physiochemical factors such as temperature, pH and cofactors are well known parameters that confer conformational changes in a protein structure. With S100β protein being a metal binding brain-specific receptor for both extracellular and intracellular functions, a change in conformation due to the above-mentioned factors, can compromise their cellular functions and therefore result in several pathological conditions such as Alzheimer’s disease, Ischemic stroke, as well as Myocardial Infarction. Objective: The studies conducted sought to elucidate the effect of these physiological factors on the conformational dynamics of S100β protein using computational modeling approaches. Method: Temperature-dependent and protein-cofactor complexes molecular dynamics simulations were conducted by varying the temperature from 100 to 400K using GROMACS 5.0.3. Additionally, the conformational dynamics of the protein was studied by varying the pH at 5.0, 7.4 and 9.0 using Ambertools17. This was done by preparing the protein molecule, solvating and minimizing its energy level as well as heating it to the required temperature, equilibrating and simulating under desired conditions (NVT and NPT ensembles). Results: The results show that the protein misfolds as a function of increasing temperature with alpha helical content at 100K and 400K being 57.8% and 43.3%, respectively. However, the binding sites of the protein was not appreciably affected by temperature variations. The protein displayed high conformational instability in acidic medium (pH ~5.0). The binding sites of Ca2+, Mg2+ and Zn2+ were identified and each exhibited different groupings of the secondary structural elements (binding motifs). The secondary structure analysis revealed different conformational changes with the characteristic appearance of two beta hairpins in the presence of Zn2+and Mg2+. Conclusion: High temperatures, different cofactors and acidic pH confer conformational changes to the S100β structure and these results may inform the design of novel drugs against the protein.

Chitosan Composites Synthesized Using Acetic Acid and Tetraethylorthosilicate Respond Differently to Methylene Blue Adsorption

The sol-gel and cross-linking processes have been used by researchers to synthesize silica-based nanostructures and optimize their size and morphology by changing either the material or the synthesis conditions. However, the influence of the silica nanostructures on the overall physicochemical and mechanistic properties of organic biopolymers such as chitosan has received limited attention. The present study used a one-step synthetic method to obtain chitosan composites to monitor the uptake and release of a basic cationic dye (methylene blue) at two different pH values. Firstly, the composites were synthesized and characterized by Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD) to ascertain their chemical identity. Adsorption studies were conducted using methylene blue and these studies revealed that Acetic Acid-Chitosan (AA-CHI), Tetraethylorthosilicate-Chitosan (TEOS-CHI), Acetic Acid-Tetraethylorthosilicate-Chitosan (AA-TEOS-CHI), and Acetic Acid-Chitosan-Tetraethylorthosilicate (AA-CHI-TEOS) had comparatively lower percentage adsorbances in acidic media after 40 h, with AA-CHI adsorbing most of the methylene blue dye. In contrast, these materials recorded higher percentage adsorbances of methylene blue in the basic media. The release profiles of these composites were fitted with an exponential model. The R-squared values obtained indicated that the AA-CHI at pH ~ 2.6 and AA-TEOS-CHI at pH ~ 7.2 of methylene blue had steady and consistent release profiles. The release mechanisms were analyzed using Korsmeyer-Peppas and Hixson-Crowell models. It was deduced that the release profiles of the majority of the synthesized chitosan beads were influenced by the conformational or surface area changes of the methylene blue. This was justified by the higher correlation coefficient or Pearson’s R values (R ≥ 0.5) computed from the Hixson-Crowell model. The results from this study showed that two of the novel materials comprising acetic acid-chitosan and a combination of equimolar ratios of acetic acid-TEOS-chitosan could be useful pH-sensitive probes for various biomedical applications, whereas the other materials involving the two-step synthesis could be found useful in environmental remediation of toxic materials.

Preparation and characterization of hydroxyapatite from Achatina achatina snail shells: effect of carbonate substitution and trace elements on defluoridation of water

ABSTRACT A novel hydroxyapatite [HAp: Ca10 (PO4)6(OH)2] material for defluoridation was prepared from Achatina achatina (AA) snail shells using a modified chemical precipitation method. X-ray diffractometry and atomic absorption spectrometry revealed carbonate substitution as a function of stirring conditions. Stirring time was varied to control crystallite size and trace element concentrations. In addition, Infrared spectra, cyclic voltammograms, and ion exchange profiles confirmed the functional groups, the surface mass concentration and the fluoride removal efficiency, respectively. It was observed that the samples prepared after 1 hr optimal stirring times reduced fluoride concentration from to without affecting the overall pH conditions of the water, whereas beyond this time frame, low uptake of the fluoride ions was obtained with increasing pH conditions. It was also observed that crystallite size did not affect the removal capacity of the samples. The results demonstrated herein the possibility of using locally prepared AA shells for water purification and other environmental remediation applications.

Novel Nanocrystal Clay Materials with Potential Bone Cells Growth Enhancement or Inhibition Characteristics In Vitro

The application of clay nanocrystals in healing has gained notoriety in recent years. The objective of this work was to investigate whether two medical clay nanocrystals obtained from different geographical locations could exhibit differential cell growth. X-ray diffraction analyses of both nanocrystal materials revealed orthorhombic chamosite structure with lattice parameters: a =15 Å, b= 8 Å and c=7 Å whereas energy dispersive x-ray results showed the presence of Al, Si, Fe and O in both materials. However the porosity measurements of the two materials revealed different pore structures. Both materials were tested on human fetal osteoblast cells and the results showed differential cell growth in vitro. The results underscore the significance of pore structures in cell response as against the chemical composition or the structure of the material. Future mechanistic evaluation would be conducted to better understand the pathways leading to the increased/decrease osteoblast adhesion and proliferation by these materials and possible modification of the clay materials for biomedical applications.