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Sperm attributes and morphology on Rusa timorensis: light and scanning electron microscopy

Mahre MB, Wahid H, Rosnina Y, Jesse FF, Azlan CA, Khumran AM, et al.

Anim. Reprod. Sci., 2014 Aug;148(3-4):245-50.
PMID: 25015846 DOI: 10.1016/j.anireprosci.2014.06.006

This study provides standard information on the attributes of sperm and describes the surface structure of normal and abnormal spermatozoa of Rusa timorensis. Two fertile stags were used as the source of semen collected during the first breeding season commencing from April 5 to July 2, 2012. Another five stags were used as the source of semen collected during the second breeding season commencing from April 1 to June 27, 2013. Semen samples were collected from the stags using an electro-ejaculator. The ejaculate was processed and samples prepared for light and scanning electron microscopy (SEM) according to standard methods. No significant difference (P>0.05) was found between sperm attributes in comparison between different stags and different months of the fertile seasons. The results of this study have also demonstrated that there are no differences in size, shape and surface structure between spermatozoa of the different stags and different months of the fertile seasons. Sperm attributes (volume, pH, sperm concentration, general motility, progressive motility and viability) were 2.2±0.29 ml, 7.2±0.17, 886.3±39.7×10(6) spermatozoa/ml, 78.7±2.01%, 80.8±1.85% and 83.2±0.85%, respectively. Morphological analysis showed low percentage of abnormal spermatozoa 13.9±2.88%. Scanning electron microscopy revealed spermatozoa which consisted of a flat paddle-shaped head, short neck and a tail, which was subdivided into midpiece, principal piece and endpiece. The average spermatozoon was 66.2±0.69 μm in total length. The flat paddle-shaped head was 7.8±0.28 μm long, 4.2±0.15 μm at its widest width, 2.4±0.18 μm basal width and 0.7±0.0 2μm thick. As for the tail, the midpiece length was 13.2±0.14 μm, 0.6±0.04 μm in diameter; the principal piece was 42.6±0.04μm, and 2.8±0.06 μm for the endpiece. Abnormal spermatozoa such as tapered head, microcephalic head, decapitated spermatozoa and bent tails were observed. Results provide standard information useful for development of strategies for semen cryopreservation and assisted reproductive technology in this species.
Fulltext Modified methods of nanoparticles synthesis in pH-sensitive nano-carriers production for doxorubicin delivery on MCF-7 breast cancer cell line

Hamidu A, Mokrish A, Mansor R, Razak ISA, Danmaigoro A, Jaji AZ, et al.

Int J Nanomedicine, 2019;14:3615-3627.
PMID: 31190815 DOI: 10.2147/IJN.S190830

Purpose: Modified top-down procedure was successfully employed in the synthesis of aragonite nanoparticles (NPs) from cheaply available natural seawater cockle shells. This was with the aim of developing a pH-sensitive nano-carrier for effective delivery of doxorubicin (DOX) on MCF-7 breast cancer cell line. Methods: The shells were cleaned with banana pelts, ground using a mortar and pestle, and stirred vigorously on a rotary pulverizing blending machine in dodecyl dimethyl betane solution. This simple procedure avoids the use of stringent temperatures and unsafe chemicals associated with NP production. The synthesized NPs were loaded with DOX to form DOX-NPs. The free and DOX-loaded NPs were characterized for physicochemical properties using field emission scanning electron microscopy, transmission electron microscopy, zeta potential analysis, Fourier transform infrared spectroscopy, and X-ray diffraction. The release profile, cytotoxicity, and cell uptake were evaluated. Results: NPs had an average diameter of 35.50 nm, 19.3% loading content, 97% encapsulation efficiency, and a surface potential and intensity of 19.1±3.9 mV and 100%, respectively. A slow and sustained pH-specific controlled discharge profile of DOX from DOX-NPs was observed, clearly showing apoptosis/necrosis induced by DOX-NPs through endocytosis. The DOX-NPs had IC50 values 1.829, 0.902, and 1.0377 µg/mL at 24, 48, and 72 hrs, while those of DOX alone were 0.475, 0.2483, and 0.0723 µg/mL, respectively. However, even at higher concentration, no apparent toxicity was observed with the NPs, revealing their compatibility with MCF-7 cells with a viability of 92%. Conclusions: The modified method of NPs synthesis suggests the tremendous potential of the NPs as pH-sensitive nano-carriers in cancer management because of their pH targeting ability toward cancerous cells.
Preparation and characterization of cockle shell aragonite nanocomposite porous 3D scaffolds for bone repair

Mahmood SK, Zakaria MZAB, Razak ISBA, Yusof LM, Jaji AZ, Tijani I, et al.

Biochem Biophys Rep, 2017 Jul;10:237-251.
PMID: 28955752 DOI: 10.1016/j.bbrep.2017.04.008

The demands for applicable tissue-engineered scaffolds that can be used to repair load-bearing segmental bone defects (SBDs) is vital and in increasing demand. In this study, seven different combinations of 3 dimensional (3D) novel nanocomposite porous structured scaffolds were fabricated to rebuild SBDs using an extraordinary blend of cockle shells (CaCo3) nanoparticles (CCN), gelatin, dextran and dextrin to structure an ideal bone scaffold with adequate degradation rate using the Freeze Drying Method (FDM) and labeled as 5211, 5400, 6211, 6300, 7101, 7200 and 8100. The micron sized cockle shells powder obtained (75 µm) was made into nanoparticles using mechano-chemical, top-down method of nanoparticles synthesis with the presence of the surfactant BS-12 (dodecyl dimethyl bataine). The phase purity and crystallographic structures, the chemical functionality and the thermal characterization of the scaffolds' powder were recognized using X-Ray Diffractometer (XRD), Fourier transform infrared (FTIR) spectrophotometer and Differential Scanning Calorimetry (DSC) respectively. Characterizations of the scaffolds were assessed by Scanning Electron Microscopy (SEM), Degradation Manner, Water Absorption Test, Swelling Test, Mechanical Test and Porosity Test. Top-down method produced cockle shell nanoparticles having averagely range 37.8±3-55.2±9 nm in size, which were determined using Transmission Electron Microscope (TEM). A mainly aragonite form of calcium carbonate was identified in both XRD and FTIR for all scaffolds, while the melting (Tm) and transition (Tg) temperatures were identified using DSC with the range of Tm 62.4-75.5 °C and of Tg 230.6-232.5 °C. The newly prepared scaffolds were with the following characteristics: (i) good biocompatibility and biodegradability, (ii) appropriate surface chemistry and (iii) highly porous, with interconnected pore network. Engineering analyses showed that scaffold 5211 possessed 3D interconnected homogenous porous structure with a porosity of about 49%, pore sizes ranging from 8.97 to 337 µm, mechanical strength 20.3 MPa, Young's Modulus 271±63 MPa and enzymatic degradation rate 22.7 within 14 days.
Fulltext Safety assessments of subcutaneous doses of aragonite calcium carbonate nanocrystals in rats

Jaji AZ, Zakaria ZAB, Mahmud R, Loqman MY, Hezmee MNM, Abba Y, et al.

J Nanopart Res, 2017;19(5):175.
PMID: 28553160 DOI: 10.1007/s11051-017-3849-z

Calcium carbonate nanoparticles have shown promising potentials in the delivery of drugs and metabolites. There is however, a paucity of information on the safety of their intentional or accidental over exposures to biological systems and general health safety. To this end, this study aims at documenting information on the safety of subcutaneous doses of biogenic nanocrystals of aragonite polymorph of calcium carbonate derived from cockle shells (ANC) in Sprague-Dawley (SD) rats. ANC was synthesized using the top-down method, characterized using the transmission electron microscopy and field emission scanning electron microscope and its acute and repeated dose 28-day trial toxicities were evaluated in SD rats. The results showed that the homogenous 30 ± 5 nm-sized spherical pure aragonite nanocrystals were not associated with mortality in the rats. Severe clinical signs and gross and histopathological lesions, indicating organ toxicities, were recorded in the acute toxicity (29,500 mg/m2) group and the high dose (5900 mg/m2) group of the repeated dose 28-day trial. However, the medium- (590 mg/m2 body weight) and low (59 mg/m2)-dose groups showed moderate to mild lesions. The relatively mild lesions observed in the low toxicity dosage group marked the safety margin of ANC in SD rats. It was concluded from this study that the toxicity of CaCO3 was dependent on the particulate size (30 ± 5 nm) and concentration and the route of administration used.
Fulltext Synthesis, characterization, and cytocompatibility of potential cockle shell aragonite nanocrystals for osteoporosis therapy and hormonal delivery

Jaji AZ, Bakar MZ, Mahmud R, Loqman MY, Hezmee MN, Isa T, et al.

Nanotechnol Sci Appl, 2017;10:23-33.
PMID: 28176933 DOI: 10.2147/NSA.S113030

Calcium carbonate is a porous inorganic nanomaterial with huge potential in biomedical applications and controlled drug delivery. This study aimed at evaluating the physicochemical properties and in vitro efficacy and safety of cockle shell aragonite calcium carbonate nanocrystals (ANC) as a potential therapeutic and hormonal delivery vehicle for osteoporosis management. Free and human recombinant parathyroid hormone 1-34 (PTH 1-34)-loaded cockle shell aragonite calcium carbonate nanocrystals (PTH-ANC) were synthesized and evaluated using standard procedures. Transmission electron microscopy and field emission scanning electron microscopy results demonstrated highly homogenized spherical-shaped aragonite nanocrystals of 30±5 nm diameter. PTH-ANC had a zeta potential of -27.6±8.9 mV. The encapsulation efficiency of the formulation was found to be directly proportional to the concentrations of the drug fed. The X-ray diffraction patterns revealed strong crystallizations with no positional change of peaks before and after PTH-ANC synthesis. Fourier transform infrared spectroscopy demonstrated no detectable interactions between micron-sized aragonite and surfactant at molecular level. PTH-ANC formulation was stabilized at pH 7.5, enabling sustained slow release of PTH 1-34 for 168 h (1 week). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytocompatibility assay in Human Foetal Osteoblast Cell Line hFOB 1.19 showed that ANC can safely support osteoblast proliferation up to 48 h whereas PTH-ANC can safely support the proliferation at 72 h and beyond due to the sustained slow release of PTH 1-34. It was concluded that due to its biogenic nature, ANC is a cytocompatible antiosteoporotic agent. It doubles as a nanocarrier for the enhancement of efficacy and safety of the bone anabolic PTH 1-34. ANC is expected to reduce the cost, dosage, and dose frequency associated with the use of PTH 1-34 management of primary and secondary forms of osteoporosis.
Formulation of a Sustained Release Docetaxel Loaded Cockle Shell-Derived Calcium Carbonate Nanoparticles against Breast Cancer

Hammadi NI, Abba Y, Hezmee MNM, Razak ISA, Jaji AZ, Isa T, et al.

Pharm Res, 2017 06;34(6):1193-1203.
PMID: 28382563 DOI: 10.1007/s11095-017-2135-1

PURPOSE: Here, we explored the formulation of a calcium carbonate nanoparticle delivery system aimed at enhancing docetaxel (DTX) release in breast cancer.
METHODS: The designed nano- anticancer formulation was characterized thorough X-ray diffraction (XRD), Fourier transformed infrared (FTIR), transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM) and Brunauer-Emmett-Teller (BET) methods. The nano- anticancer formulation (DTX- CaCO3NP) was evaluated for drug delivery properties thorough in vitro release study in human body simulated solution at pH 7.4 and intracellular lysosomal pH 4.8.
RESULTS: Characterization revealed the successful synthesis of DTX- CaCO3NP, which had a sustained release at pH 7.4. TEM showed uniformly distributed pleomorphic shaped pure aragonite particles. The highest entrapment efficiency (96%) and loading content (11.5%) were obtained at docetaxel to nanoparticles ratio of 1:4. The XRD patterns revealed strong crystallizations in all the nanoparticles formulation, while FTIR showed chemical interactions between the drug and nanoparticles with negligible positional shift in the peaks before and after DTX loading. BET analysis showed similar isotherms before and after DTX loading. The designed DTX- CaCO3NP had lower (p 0.05) effects at 48 h and 72 h. However, the DTX- CaCO3NP released less than 80% of bond DTX at 48 and 72 h but showed comparable effects with free DTX.
CONCLUSIONS: The results showed that the developed DTX- CaCO3NP released DTX slower at pH 7.4 and had comparable cytotoxicity with free DTX at 48 and 72 h in MCF-7 cells.
Fulltext Antibacterial Activity of Ciprofloxacin-Encapsulated Cockle Shells Calcium Carbonate (Aragonite) Nanoparticles and Its Biocompatability in Macrophage J774A.1

Isa T, Zakaria ZA, Rukayadi Y, Mohd Hezmee MN, Jaji AZ, Imam MU, et al.

Int J Mol Sci, 2016;17(5).
PMID: 27213349 DOI: 10.3390/ijms17050713

The use of nanoparticle delivery systems to enhance intracellular penetration of antibiotics and their retention time is becoming popular. The challenge, however, is that the interaction of nanoparticles with biological systems at the cellular level must be established prior to biomedical applications. Ciprofloxacin-cockle shells-derived calcium carbonate (aragonite) nanoparticles (C-CSCCAN) were developed and characterized. Antibacterial activity was determined using a modified disc diffusion protocol on Salmonella Typhimurium (S. Typhimurium). Biocompatibilittes with macrophage were evaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Bromo-2'-deoxyuridine (BrdU) assays. Transcriptional regulation of interleukin 1 beta (IL-1β) was determined using reverse transcriptase-polymerase chain reaction (RT-PCR). C-CSCCAN were spherical in shape, with particle sizes ranging from 11.93 to 22.12 nm. Encapsulation efficiency (EE) and loading content (LC) were 99.5% and 5.9%, respectively, with negative ζ potential. X-ray diffraction patterns revealed strong crystallizations and purity in the formulations. The mean diameter of inhibition zone was 18.6 ± 0.5 mm, which was better than ciprofloxacin alone (11.7 ± 0.9 mm). Study of biocompatability established the cytocompatability of the delivery system without upregulation of IL-1β. The results indicated that ciprofloxacin-nanoparticles enhanced the antibacterial efficacy of the antibiotic, and could act as a suitable delivery system against intracellular infections.
Fulltext LC-MS/MS Proteomic Study of MCF-7 Cell Treated with Dox and Dox-Loaded Calcium Carbonate Nanoparticles Revealed Changes in Proteins Related to Glycolysis, Actin Signalling, and Energy Metabolism

Ahmed H, Ajat M, Mahmood RI, Mansor R, Razak ISA, Al-Obaidi JR, et al.

Biology (Basel), 2021 Sep 13;10(9).
PMID: 34571787 DOI: 10.3390/biology10090909

One of the most prevalent death causes among women worldwide is breast cancer. This study aimed to characterise and differentiate the proteomics profiles of breast cancer cell lines treated with Doxorubicin (DOX) and Doxorubicin-CaCO3-nanoparticles (DOX-Ar-CC-NPs). This study determines the therapeutic potential of doxorubicin-loaded aragonite CaCO3 nanoparticles using a Liquid Chromatography/Mass Spectrometry analysis. In total, 334 proteins were expressed in DOX-Ar-CC-NPs treated cells, while DOX treatment expressed only 54 proteins. Out of the 334 proteins expressed in DOX-CC-NPs treated cells, only 36 proteins showed changes in abundance, while in DOX treated cells, only 7 out of 54 proteins were differentially expressed. Most of the 30 identified proteins that are differentially expressed in DOX-CC-NPs treated cells are key enzymes that have an important role in the metabolism of carbohydrates as well as energy, including: pyruvate kinase, ATP synthase, enolase, glyceraldehyde-3-phosphate dehydrogenase, mitochondrial ADP/ATP carrier, and trypsin. Other identified proteins are structural proteins which included; Keratin, α- and β-tubulin, actin, and actinin. Additionally, one of the heat shock proteins was identified, which is Hsp90; other proteins include Annexins and Human epididymis protein 4. While the proteins identified in DOX-treated cells were tubulin alpha-1B chain and a beta chain, actin cytoplasmic 1, annexin A2, IF rod domain-containing protein, and 78 kDa glucose-regulated protein. Bioinformatics analysis revealed the predicted canonical pathways linking the signalling of the actin cytoskeleton, ILK, VEGF, BAG2, integrin and paxillin, as well as glycolysis. This research indicates that proteomic analysis is an effective technique for proteins expression associated with chemotherapy drugs on cancer tumours; this method provides the opportunity to identify treatment targets for MCF-7 cancer cells, and a liquid chromatography-mass spectrometry (LC-MS/MS) system allowed the detection of a larger number of proteins than 2-DE gel analysis, as well as proteins with maximum pIs and high molecular weight.