Abstracts of “Institute for Research and Medical Consultations (IRMC) - Summer Research Program - 2023” presented at Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, on August 28, 2023

IRMC-SRP-2023-01: Mining of biosynthetic gene clusters in the bacterial genome with antifungal potential against multi-drug resistant Candida auris

Hussain Anwar Alkatheri^1,2, Noor B. Almandil³, Rahaf Alquwaie⁴, Razan Aldahhan¹, Norah F Alhur¹, Reem AlJindan⁵, Sayed AbdulAzeez¹, J. Francis Borgio^1*

¹ Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ³Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Master Program of Biotechnology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁵ Department of Microbiology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: fbalexander@iau.edu.sa

Introduction: Candida auris is a multidrug-resistant (MDR) fungal pathogen. It was initially discovered in 2009 in Japan from an external ear. C. auris poses a significant global health challenge due to its resistance to major antifungal agents, including azoles, echinocandins, and polyenes. As a result, treatment options against C. auris are limited, emphasizing the necessity for novel therapeutic drugs. One valuable approach for discovering new antimicrobial drugs involves exploring natural compounds produced by bacteria, known as secondary metabolites. These secondary metabolites are synthesized through the activity of a set of genes called biosynthetic gene clusters (BGC).

Objectives: This study aims to perform complete sequencing of the entire bacterial genome of IRMC361 for taxonomical identification of the bacterial strain using a genomic approach and to analyze BGCs within the bacterial genome using bioinformatics tools. Furthermore, the study aims to stimulate the production of bacterial secondary metabolites through the implementation of co-culturing techniques.

Methods: In this study, the DNA of bacterial strain IRMC361 was isolated using the Gentra Puregene Yeast/Bact. Kit. We utilized 16S rRNA and whole-genome sequencing (WGS), in conjunction with advanced computational genomic tools, to determine the bacterial species and identify biosynthetic gene clusters in the bacterial genome. WGS was conducted using Oxford Nanopore technology, and the quality of the reads was evaluated and simultaneously converted into fastQ files. Subsequently, these files were assembled to detect biosynthetic gene clusters and predict metabolic products. Mutations were detected, and 3D models of wild and mutant proteins were constructed. Co-culturing techniques were employed to stimulate the production of secondary metabolites in bacteria. Furthermore, a biofilm inhibition assay was performed to assess the effectiveness of the crude extract from the methanol fraction against C. auris biofilm.

Results and Discussion: DNA extraction was successfully performed, and the presence of bacterial DNA was confirmed through gel electrophoresis of 16S rRNA PCR amplification. Analysis of WGS revealed the presence of four contigs and a bacterial genome length of 3,810,792 bp. The specific bacterial strain identified in this study was Bacillus amyloliquefaciens IRMC361. A comprehensive genomic analysis was conducted, which uncovered the existence of six BGCs coded for antimicrobial potential. Notably, when the bacteria were co-cultured with C. auris, the formation of an inhibition zone was observed, indicating potential anti-C auris activity. The biofilm inhibition assay of the crude compound exhibited significant results in suppressing the growth of C. auris biofilm.

Conclusion: The bacterial isolate IRMC361 was categorized as Bacillus amyloliquefaciens through the application of whole genome phylogeny. Using WGS data mining, biosynthetic gene clusters were successfully detected in the bacterial genome. These clusters generate compounds with potential antifungal properties against multidrug-resistant C. auris. These compounds, coded in the clusters, could serve as a foundation for developing new antifungal drugs and will be deployed in the future for pre-clinical and clinical trials.

Ethical approval reference number: IRB-2022–13-462

IRMC-SRP-2023-02: Characterization of the morphological features of fetal nucleated red blood cells in maternal blood in pregnancy

Shikhah Abdullah Alomran^1,2, Norah F Alhur¹, Nourah H Al Qahtani³, Fahd M AlShehri³, Zahra Alsafwani³, Esra Ahdal³, Noor B. Almandil⁴, Sayed AbdulAzeez¹, J. Francis Borgio^1*

¹Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ³Department of Obstetrics and Gynecology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: fbalexander@iau.edu.sa

Background: Fetal nucleated red blood cells (fNRBCs) are cells of fetal origin and are present in maternal peripheral blood during pregnancy. fNRBCs have distinctive morphological features as they have a diameter of 9 to 13 μm, an intact and dense nucleus and a low nucleus-to-cytoplasm ratio. Researchers proposed the use of fNRBCs as a non-invasive prenatal testing (NIPT) alternative to invasive procedures such as amniocentesis or chorionic villus sampling. The full genome of the fetus is encapsulated within fNRBCs, enabling the detection of genetic variations. However, the primary challenge lies in the scarcity of the fNRBCs, and limited number of biomarkers that differ from the maternal cells.

Objective: The study aims to use live cell imaging to identify fNRBC’s unique characteristics in the maternal blood during pregnancy.

Methods: The samples from pregnant mothers at full term were collected in EDTA vacutainers from the obstetrics and gynecology clinics at KFUH. Informed consent was obtained from all participants. The double-density gradient centrifugation method was employed to isolate the fNRBCs. Visualization of the collected fNRBCs was done with light microscope live-cell imaging. Collected cells were subjected to direct-cell gender multiplex PCR using three Y-chromosome biomarker genes (SRY, DAZ, and DYS14) and ACTB as housekeeping genes and in-house HbF staining to confirm the fetal origin of the cells.

Results and Discussion: At a magnification of 40, the fNRBCs’ distinctive nucleus is easily visible. The mean diameter of the fNRBCs was measured to be 12.04 micrometers using live cell imaging. Additionally, the fNRBCs had a peripheral nucleus with a low cytoplasm-to-nucleus ratio. The presence of at least two Y-chromosome biomarkers and ACTB confirmed the male gender of the fetus and was confirmed with a clinical ultrasound examination. Moreover, HbF staining confirmed the fetal origin of the visualized fNRBCs. Moreover, the diameter of fNRBCs (12.041 ± 1.257 µm) is larger than the diameter of normal red blood cells (RBCs) (7.48575 ± 0.428 µm).

Conclusion: fNRBCs were distinguished from WBCs based on the size of the cells, the position of the nucleus, and the cytoplasm-to-nucleus ratio. More morphological studies are mandatory to enable the utilization of fNRBCs advantage potential in NIPT.

Ethical approval reference number: IRB-2017-13-137

IRMC-SRP-2023-03: Drug repurposing through structure-based drug design for discovering novel anti-Candida treatments

Adel S. Almutairi^1,2, Sarah Almofty³, Rahaf Alquwaie⁴, Norah F Alhur¹, Reem AlJindan⁵, Noor B. Almandil⁶, J. Francis Borgio¹, Sayed AbdulAzeez^1*

¹Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ³Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Master Program of Biotechnology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁵Department of Microbiology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁶Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: asayed@iau.edu.sa

Background: Drug repurposing is one of the most promising research fields due to the rising global interest surrounding the topic of resource management. Candida auris is a multidrug-resistant fungus first identified in Japan in 2009. Unlike other Candida species, C. auris has the potential to easily spread in a hospital setting, and its drug-resistant nature makes it difficult to diagnose. With over 90% of all identified strains being resistant to multiple drugs, it also lacks a gold standard for treatment.

Objectives: This research aims to discover FDA (Food and Drug Administration)-approved drugs that carry anti-Candida auris potential to be repurposed as novel treatments using state-of-the art in silico analytical tools supported by advanced bioinformatic programs and laboratory experiments.

Methods: For this study, homology modeling, which is a structure-based drug design methodology, was utilized. Advanced computational tools were used to conduct in silico docking analysis using a virtual screening technique for prioritizing FDA-approved drug ligands retrieved from various drug data banks against a designated potential druggable Candida auris protein target, A0A2H1A279, which possesses high antigenic potential. The results were then processed based on the highest affinity (n = 10), and an exclusionary criterion was applied to omit all drugs with established anti-candidal properties prior to our experimentation. Subsequently, the results were validated experimentally through rigorous susceptibility testing.

Results and Discussion: FDA-approved drugs (n = 1614) were successfully screened via the “SHAHEEN” supercomputer using a virtual screening technique on the druggable protein target A0A2H1A279. Among the processed 1614 drug ligands, the docking results were arranged based on the highest affinity. Out of the resultantly screened and processed drugs, we report two (ADD2 and ADD3) drugs for the first time with anti-Candida auris potential. The selected approach of designing treatments through drug repurposing showed promise in reporting anti-Candida properties in selected market-available drugs.

Conclusion: Both ADD2 and ADD3 showcased previously undocumented anti-Candida potential during experimentation. Due to the nature of the drugs experimented on being FDA-approved with no perceived hazardous effects, we can accelerate moving into animal testing. If animal testing displays favourable outcomes, human clinical trials can be conducted accordingly.

Ethical approval reference number: IRB-2022–13-462

IRMC-SRP-2023-04: Excavating new antifungal compound coded biosynthetic gene cluster derived from Macrococcus sp. strain against multi-drug resistant Candida auris

Dalal Mohammed-Akram Motabagani^1,2, Noor B. Almandil³, Eman AlHasani⁴, Rahaf Alquwaie⁴, Norah F Alhur¹, Reem AlJindan⁵, J. Francis Borgio¹, Sayed AbdulAzeez^1*

¹Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Medicine, King Faisal University, Al Ahsa, Saudi Arabia, ³Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Master Program of Biotechnology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁵Department of Microbiology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: asayed@iau.edu.sa

Background: In 2009, Candida auris was first isolated from the external ear canal. It is considered one of the most mucocutaneously invasive strains that leads to high mortality due to its resistance to various typical anti-fungal medications. With the emergence of C. auris resistance against repeatedly used antifungal drugs, there is an undeniable dearth. Most of the clinically used antibiotics are manufactured from the secondary metabolites of bacteria. With the advancement of nanopore whole genome sequencing, it became promising to identify novel strains of bacteria against C. auris.

Objective: The main objective of the study is to describe and characterize the genome of a particular bacteria that has anti-Candida auris capabilities through whole genome sequencing and extensive bioinformatics analysis.

Methods: A bacterial strain (ESH58) from fish liver was used for the study. The DNA was extracted, and 16S rRNA PCR was performed. Then, a co-culture inhibition zone test was performed on the SDA plate for 48 hours to test the ESH58’s activity against C. auris. Finally, Oxford Nanopore whole genome sequencing technology and the quality of the reads were converted to FastaQ. The data was analyzed via bioinformatics tools to map and assemble the genome and mine the biosynthetic gene clusters and protein products. In addition, three-dimensional modeling tools were used to structure the protein product of ESH58.

Results and Discussion: The 16sR RNA gene amplification identified our novel organism as bacteria. ESH58 successfully inhibited the growth of C. auris with an inhibition zone of 20 mm. In addition, ESH-58 was successfully sequenced using Oxford Nanopore sequencing and got a median read quality of 11.2 and 2,069,622 bp. The bioinformatics tools classified ESH58 as a novel strain of Macrococcus caseolytics. In addition, the analysis revealed one BGC coded for surfactin. ESH58 underwent the pathogenicity test, and it was classified as a non-human pathogen. The three-dimensional protein structure of BAH16750.1 revealed multiple mutations, including A99M, compared with the wild type. Finally, the protein product of M. caseolytics ESH58 was compared to the product of the closest strain that was chosen based on the phylogenetic tree, M. caseolyticus JCSC5402, and several mutations were detected.

Conclusion: Oxford Nanopore whole genome sequencing classified ESH58 as M. caseolyticus. It inhibited the growth of C. auris with a 20 mm inhibition zone. Bioinformatics analysis labeled ESH58 as a non-human pathogen and revealed BGC-coded surfactin. ESH58 indicated a promising future for utilizing its secondary metabolites to produce new antifungal medications against C. auris.

Ethical approval reference number: IRB-2022–13-462

IRMC-SRP-2023-05: The repurposing approach discovers a novel way to utilize an FDA-approved drug as an antifungal agent against Candida auris infection

Showq H. Alshammery^1,2, Sarah Almofty³, Rahaf Alquwaie⁴, Norah F Alhur¹, Reem AlJindan⁵, Noor B. Almandil⁶, J. Francis Borgio⁴, Sayed AbdulAzeez^4*

¹ Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ² College of Science, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia.

³ Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴ Master Program of Biotechnology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁵ Department of Microbiology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁶ Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: asayed@iau.edu.sa

Background: The emergence of multidrug-resistant Candida auris represents a significant global health concern. The first global case was reported in Japan in 2009, while Saudi Arabia recorded its first case of C. auris infection in 2018. C. auris mainly colonizes the skin but can also spread to internal organs via the bloodstream, which is associated with high mortality. Furthermore, the ability of C. auris to form biofilms is a virulence factor associated with drug resistance, making it a challenging pathogen to treat. Addressing this public health issue requires a stronger effort, as traditional drug discovery methods have proven insufficient due to lengthy processes. However, repurposing existing drugs represents a promising tool for treating C. auris.

Objective: This study aims to identify the suitable ligand for C. auris surface proteins by utilizing a range of bioinformatics tools to prioritize drugs for lab confirmation tests to suggest the approved drug for treating C. auris infection.

Methods: The KRE9 surface protein on the C. auris amino acid sequence was retrieved from the UniProt database in FASTA format. The MODELSWISS-MODEL server was used to create a homology model, visualized using PyMOL. We screened 600 FDA-proven drugs by using Visual software on the KAUST supercomputing-HPC, and we analyzed protein-ligand interactions and efficiently predicted the most appropriate ligand. Selected (KRD9) FDA-approved drugs’ binding affinities were validated using Discovery Studio software. We conducted an ELISA test measuring the optical density OD to study the impact of KRD9 on biofilm formation and how our drugs can effectively address it.

Results and Discussion: The SWISS-MODEL server has successfully predicted the 3D structure of the KRE9 surface protein, with 94.81% of its amino acid residues located within the favored region and a MolProbity score of 1.24. Our molecular docking analysis revealed that the KRD9 ligand has a high binding affinity of -9.7 to the target protein, KRE9. In an ELISA test, KRD9 exhibited the ability to prevent biofilm formation, as evidenced by the low OD in wells treated with KRD9, where the absorption reading value is 0.208, and a notable increase in OD in the control well. Statistical analysis showed a direct correlation between drug concentration and inhibition ratio, with the IC₅₀ recorded at 1.8850 mg.

Conclusion: Based on our findings, KRD9 could potentially be used as an antifungal agent against Candida auris, with a binding affinity to KRE9 of up to -9.7. Understanding the three-dimensional structures of proteins and their interactions with different molecules could lead to more effective disease treatments.

Ethical approval reference number: IRB-2022–13-462

IRMC-SRP-2023-06: Genomic analysis of Enterococcus faecalis for identifying the leading cause of multi-drug resistance

Lujeen H Alghourab^1,2, Noor B. Almandil³, Reem AlJindan⁴, Rahaf Alquwaie⁵, Razan Aldahhan¹, Norah F Alhur¹, Sayed AbdulAzeez¹, J. Francis Borgio^1*

¹Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ³Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Department of Microbiology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁵ Master Program of Biotechnology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: fbalexander@iau.edu.sa

Background: Enterococcus faecalis is a non-sporulating, facultatively anaerobic, and gram-positive bacteria. It is a ubiquitous inhabitant of the human intestinal flora. E. faecalis has gained worldwide attention in the last century due to its ability to horizontally transfer antimicrobial-resistant genes across gram-positive bacteria. Moreover, E. faecalis has shown the capability to sustain the evolution of antimicrobial-resistant genes by developing nucleic acid mutations.

Objective: To sequence the whole genome of E. faecalis and characterize its genome organization, identify the genes associated with antimicrobial resistance, and recognize the phenotypic mutations in E. faecalis IRMC827A.

Methods: E. faecalis was isolated from a fecal sample of a patient who was clinically diagnosed with infective gastroenteritis and had a recent history of diarrhea upon exposure to different types of antibiotics, including ciprofloxacin, gentamicin, flagyl, meropenem, vancomycin, and tazocin. The DNA was extracted using the Puregene Yeast/Bact kit and confirmed by a 16S rRNA sequence. The whole genome of E. faecalis IRMC827A was sequenced using Oxford nanopore technology and assembled and annotated using Prokka methods and the RAST^© tool kit, respectively. The antimicrobial-resistant genes and virulence factors were identified using CARD, Victor, VDFD, and ResFinder databases. Whereas the phenotypic mutation and mobile genetic elements (MGE) were predicted by executing ResFinderFG 2.0 and PathogenFinder 1.1, respectively. Lastly, linezolid resistance was detected using LRE-finder 1.0.

Results and Discussion: Genome mapping revealed aligned reads of antimicrobial-resistant genes, transporters, drug targets, and virulence factors with E. faecalis species. The observed virulence factors of E. faecalis IRMC827A include adherence, biofilm formation, sortase-assembled pili, manganese uptake, anti-phagocytosis, and the spreading factor of multidrug resistance. The IRMC827A isolate had ten genes associated with antimicrobial resistance (ANT(6)-I, tet(M), Tet(L), dfrG, dfrE, lsa(A), efrA, efrB, CatA8, EF-Tu) according to k-mer-based detection methods. The isolate had two antimicrobial-resistant genes (gyrA and parC) with phenotypic mutations and two other mutations (G2505A and G2576T) in 23S rRNA associated with linezolid resistance. Additionally, the prediction of MGEs revealed the presence of two MGEs: the integrative conjugative element (Tn6009) and the insertion sequence (ISS1N).

Conclusion: The escalating prevalence of hospital-acquired infections in Saudi Arabia could be explained by the identified resistome, mobilome, and virulome in E. faecalis IRMC827A. Despite the serious implications of multidrug-resistant microorganisms, no previous research has investigated E. faecalis in terms of genomic organization in Saudi Arabia. This study emphasizes the importance of refining policies on monitoring multidrug resistance infections in hospitals, as it is critical to limit the dissemination of antimicrobial-resistant genes across the Kingdom of Saudi Arabia.

Ethical approval reference number: IRB-2022-01-398

IRMC-SRP-2023-07: RBC depletion mediated novel isolation and characterization of fetal nucleated red blood cells

Norah Fahad Alamri^1,2, Norah F Alhur¹, Nourah H Al Qahtani³, Fahd M AlShehri³, Zahra Alsafwani³, Esra Ahdal³, Noor B. Almandil⁴, Sayed AbdulAzeez¹, J. Francis Borgio^1*

¹Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ³Department of Obstetrics and Gynecology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: fbalexander@iau.edu.sa

Background: Congenital diseases cause an estimated 240,000 infant deaths each year worldwide. Genetic defects are one of the main causes of congenital abnormalities that can be detected during pregnancy. Fetal nucleated red blood cells (fNRBCs) provide access to the fetal whole genome that can be used in NIPT, which helps detect genetic defects in early pregnancy. Several techniques have been recently developed for the isolation of fNRBCs from maternal circulation; however, isolation of fNRBCs from maternal blood has not been clinically standardized due to limitations such as a shortage in number and low purity of the isolated fNRBCs, which will severely restrict the fetal cell-based NIPT.

Objectives: This study aims to develop a novel protocol to isolate fNRBCs from maternal and cord blood based on an RBC depletion reagent.

Methods: EasySep RBC depletion reagent was used to isolate red blood cells from whole blood. In summary, 500 µL of PBS medium was added to 500 µL of cord blood sample, and then 100 µL of RBC depletion reagent was added and mixed with the blood sample. After that, the sample mix was incubated for 10 minutes in a magnetic rack. Cell suspension was poured into a new tube, and isolated RBCs were retained in the old tube. This process was repeated twice. The isolated RBC were washed using plasma-like medium, and the fetal origin of the cells was confirmed using live cell imaging, gender multiplex PCR, and HbF staining.

Results and Discussion: fNRBCs were partially isolated using the RBC depletion protocol after three rounds of separation. Results showed that fNRBCs were retained in a higher amount in the final cell suspension tube along with WBCs using cord blood. CD235a was used to isolate fNRBCs from WBCs, and gender multiplex PCR was conducted to confirm male fetal origin. In addition, live cell imaging using a light microscope demonstrated the mean size of fNRBCs (12.345 µM) and shape in maternal and cord blood samples. Moreover, HbF stain was utilized to visualize fNRBCs, confirming their presence.

Conclusion: The novel RBC depletion protocol (RDP1) helped to partially isolate fNRBCs from cord and maternal blood samples. However, CD235a and RDP1 exhibit further isolation of fNRBCs from white blood cells in the final cell suspension. When this protocol is fully developed, it can be used for the separation of fNRBCs from early-pregnancy maternal blood samples for prenatal diagnosis.

Ethical approval reference number: IRB-2017-13-137

IRMC-SRP-2023-08: Identifying gene clusters coding bioactive compounds in IRMC143B against Candida auris

Wojod Alothman^1,2, Sarah Almofty³, Rahaf Alquwaie⁴, Norah F Alhur¹, Reem AlJindan⁵, Noor B. Almandil⁶, Razan Aldahhan¹, J. Francis Borgio¹, Sayed AbdulAzeez^1*

¹Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ² College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ³ Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Master Program of Biotechnology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁵ Department of Microbiology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁶Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: asayed@iau.edu.sa

Background: Candida auris is an emerging multidrug-resistant fungal pathogen that belongs to the Saccharomycetaceae family. Saudi Arabia has experienced the highest mortality rate caused by multidrug-resistant microbes, including the C. auris species. One of its concerning aspects is its ability to survive on various surfaces, particularly, the healthcare environment, leading to its persistence and transmission. What makes C. auris challenging to control and treat is its resistance to fluconazole, a commonly used antifungal, and other azoles.

Objectives: The aim of this study is to identify a biosynthetic gene cluster of novel native bacteria against Candida auris using whole genome sequencing and comprehensive computational analysis.

Methods: A Bacillus amyloliquefaciens strain was isolated from a home garden basil tree. The total DNA was extracted using the Gentra Purgene Yeast/Bact. Kit. The extracted DNA was analyzed using Nanodrop 2000 DNA to assess its purity, quality, and quantity. Subsequently, the DNA isolate underwent PCR amplification, and to confirm the identity of the isolate, 16S rRNA was sequenced. The whole genome sequencing of the isolate IRMC143B was done using a Nanopore device, followed by bioinformatic tool analysis.

Result and Discussion: The comprehensive genome analysis revealed that the assembled genome of IRMC143B had 1 contig with a total length of 3,981,673 bp. The Bacillus amyloliquefaciens genome has 5,722 protein-coding sequences (CDS). Additionally, IRMC143 was analyzed with antiSMASH using the FASTA file of sequence, resulting in the identification of 13 cluster regions, each with a unique variety of genes. Five clusters encode the biosynthesis of non-ribosomal peptide (NRP), two clusters encode the synthesis of polyketide synthase (PKS), and one cluster encodes the synthesis of saccharide. 2 clusters code for the biosynthesis of hybrid NRP/PKS compounds. Utilizing the NCBI blast tool for gene mutation identification within each cluster, it was shown that the surfactin region coding for the srfaa gene exhibited both insertion and point mutation. These mutations could potentially confer antifungal activity. Prediction of a bacteria’s pathogenicity towards human hosts using a pathogen finder tool showed no relation. Furthermore, no plasmid in IRMC143B was found using the plasmid finder tool.

Conclusion: The metabolic gene cluster found through whole genome sequencing data mining in the IRMC143B genome suggests that the predicted compounds present in the bacteria could potentially be used for producing an anti-Candida auris compound and as a novel anti-Candida auris drug. Further studies can be conducted to assess the toxicity using MTT assays for the selected compounds.

Ethical approval reference number: IRB-2022–13-462

IRMC-SRP-2023-09: Identification and characterization of biosynthetic gene clusters in bacterial stain IRMC272 to discover antifungal compounds against Candida auris

Mohammad H. Albakhit^1,2, Sarah Almofty³, Rahaf Alquwaie⁴, Norah F Alhur¹, Reem AlJindan⁵, Noor B. Almandil⁶, J. Francis Borgio¹, Sayed AbdulAzeez^1*

¹Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ² College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ³Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Master Program of Biotechnology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁵Department of Microbiology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁶ Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: asayed@iau.edu.sa

Background: Candida auris is a multidrug-resistant fungus that poses a global health risk. Its resistance to antifungal drugs and misidentification in standard laboratory methods complicate treatment and control. The spread of C. auris has reached Saudi Arabia and is linked to international travel and healthcare-associated transmission. To address this challenge, targeted discovery of bioactive compounds through analysis of biosynthetic gene clusters (BGCs) is promising. Nanopore sequencing enables rapid analysis of complex microbial communities, unveiling insights into BGC-encoded biosynthetic machinery.

Objectives: The research aims to discover novel anti-C. auris compound gene clusters through nanopore sequencing and explore co-culture techniques for inducing novel secondary metabolite production in the IRMC272 bacterial strain.

Methods: DNA extraction of IRMC272 bacterial strain was performed, followed by amplification of the 16S rRNA gene. Nanopore whole-genome sequencing was conducted to analyze the genetic profile. Further bioinformatics analysis was done to mine and identify the biosynthetic gene clusters (BGCs) and detect any mutations. Co-cultivation of IRMC272 and C. auris was carried out to study their interactions and antifungal activity. Disc diffusion assays were performed to evaluate the inhibition zones in co-culture samples. Finally, a 96-well biofilm inhibition assay was conducted by inoculating preformed C. auris biofilms with the IRMC272 bacterial strain crude extract from methanol fractionation and quantifying the biofilm inhibition using crystal violet staining and absorbance measurements.

Results and Discussion: DNA was extracted from IRMC272, and the presence of bacterial DNA was confirmed through gel electrophoresis of 16S rRNA PCR amplicon. Nanopore sequencing and bioinformatics analysis of the data identified two contigs, revealing a total genome length of 3,787,328 bp. The strain was classified as Bacillus amyloliquefaciens. The analysis focused on six BGCs responsible for secondary metabolite biosynthesis. Mutations were detected in two genes within one BGC. 3D modeling of the mutated proteins enhanced understanding of their structure. Co-culturing experiments showed inhibition of C. auris, indicating potential anti-C. auris activity. A biofilm inhibition assay using a crude extract from the strain also exhibited promising results, suggesting its ability to inhibit biofilm formation.

Conclusion: By combining the strengths of nanopore sequencing technology and the analysis of BGCs, we accelerated the discovery of novel anti-C. auris compounds. This research has the potential to contribute to the development of effective therapies for the treatment of C. auris infections and address the rising challenge of antifungal resistance. The clusters identified could be the root cause of the anti-C. auris activity, most notably surfactin.

Ethical approval reference number: IRB-2022–13-462

IRMC-SRP-2023-10: A comparative morphological analysis of fetal nucleated red blood cells from cord blood using live cell imaging and scanning electron microscopy

Amani A. AlHejji^1,2, Norah F Alhur¹, Nourah H Al Qahtani³, Fahd M AlShehri³, Zahra Alsafwani³, Esra Ahdal³, Sarah Almofty⁴, Noor B. Almandil⁵, Sayed AbdulAzeez¹, J. Francis Borgio^1*

¹Department of Genetic Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ² College of Medicine, King Faisal University, Al Ahsa, Saudi Arabia, ³Department of Obstetrics and Gynecology, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁵Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: fbalexander@iau.edu.sa

Background: Over the past decade, there has been a significant advancement in prenatal screening for fetal aneuploidies, such as trisomy 21, 18, and 13, through the utilization of non-invasive prenatal testing (NIPT). This groundbreaking technology has revolutionized the field by not only enhancing the detection rate for fetal chromosomal abnormalities but also reducing the necessity for invasive tests. Fetal-nucleated red blood cells (fNRBCs) offer a unique opportunity as biomarkers for non-invasive prenatal diagnosis (NIPD) due to their ability to carry the complete genetic information of the fetus. Understanding the morphology of fNRBCs holds great importance in various medical conditions. Thus, their identification and characterization can provide valuable insights for prenatal diagnosis and monitoring.

Objectives: The aim of this study is to identify the morphological characteristics of fNRBC through the utilization of live-cell imaging. This will be further compared with scanning electron microscope analysis and gender multiplex PCR for molecular confirmation.

Methods: Umbilical cord blood samples were obtained at the Department of Obstetrics and Gynecology, King Fahd University Hospital in Khobar, Saudi Arabia. Informed consent was obtained from the mothers of the participants prior to sample collection. To isolate fNRBCs from the umbilical cord blood, double-density Percoll gradients with varying osmolalities were used. The resulting sample was then divided for imaging and direct multiplex gender PCR to confirm the fetal origin.

Results and Discussion: fNRBC demonstrated a substantial increase in diameter when observed using live cell imaging, in contrast to the analysis performed using scanning electron microscopy, and a 26% increase when compared to regular red blood cells (RBCs). The size of fNRBCs observed through live cell imaging was significantly larger than that of RBCs and fNRBCs observed using scanning electron microscopy. Additionally, the PCR of fetal DNA successfully amplified Y chromosome markers, providing confirmation of the male origin of the fetus.

Conclusion: fNRBC showed a larger diameter under live cell imaging compared to scanning electron microscope analysis. The male origin of the fetal DNA in the male samples was confirmed using gender multiplex PCR. By using the identified morphologies, fNRBCs can be reliably separated from maternal blood samples taken during early pregnancy. This facilitates the early prenatal diagnosis of genetic disorders.

Ethical approval reference number: IRB-2017-13-137

IRMC-SRP-2023-11: Efficacy of Lawsonia inermis silver nanoparticles in the detection of melamine in milk and its anti-microbial potential

Omar Sami Alzamil^1,2, Mohammad Azam Ansari¹*

¹Department of Epidemic Disease Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, 31441, Dammam, Saudi Arabia, ²College of Medicine, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia. *Email: maansari@iau.edu.sa

Background: The adulteration of milk and its derivatives with the toxic compound, melamine is a well-known issue that has resulted in many crises in the past, most notably the 2008 Chinese milk scandal. The use of silver nanoparticles (AgNPs) has been an emerging method for visually detecting melamine in milk. In addition, AgNPs have been reported to have significant anti-microbial effects against bacteria and fungi. Many methods exist to synthesize AgNPs, the green method is commonly used due to having many advantages, most notably its cost effectiveness. In this study, we produced AgNPs using the green method through the extract of Lawsonia inermis.

Objectives: Develop a technique to visually detect the presence of melamine in milk using L. inermis AgNPs and to assess it’s the anti-microbial activity.

Methods: AgNPs were synthesized through green method using leaf extract of L. inermis. Characterization of AgNPs was done through Scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive x-ray (EDX) and fourier-transform infrared (FTIR) spectroscopy. For the visual detection of melamine, we used eppendorf tubes containing 200-500 µl mixed solution of AgNPs and raw milk. 300 µl of melamine was serially diluted in each tube except the control. Subsequently, 200-500 µl of L. inermis extract was added in each tube. Lack of color change in the tubes is an indicator of the interruption of AgNPs synthesis due to the presence of melamine. Furthermore, anti-microbial activity against MRSA, Pseudomonas aeruginosa, E. coli ATCC25922 and Candida albicans was assessed through well diffusion method.

Results and Discussion: Eppendorf tubes that had a melamine concentration of more than 10 ppm had interrupted color change, which indicates interference of melamine in the synthesis of AgNPs. As for the tubes that had a melamine concentration of less than 10ppm and the control tube, color change occurred indicating synthesis of AgNPs. For well diffusion method, L. inermis AgNPs concentrations ranging from 25-150 µl were used for all bacteria and C. albicans. Zone of inhibition for bacterial strains were ranged 6 to 23.55 mm. For C. albicans, zone of inhibition ranged from 11 – 17 mm was observed at same dose.

Conclusion: AgNPs synthesized from L. inermis provide a cost-effective and fast method to visually detect melamine in milk. In addition, L. inermis AgNPs were shown to have effective anti-microbial activity against emerging drug-resistant microorganisms.

IRMC-SRP-2023-12: Exploring the Bioactive properties of green synthesized silver nanoparticles using leaf extract of Tribulus cistoides

Faisal Mohammed Alanazi^1,2, B. Rabindran Jermy³, Mohammad Azam Ansari¹*

¹Department of Epidemic Disease Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, 31441, Dammam, Saudi Arabia, ²College of Medicine Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia, ³Department of NanoMedicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam 31441, Saudi Arabia. *Email: maansari@iau.edu.sa

Background: Bacterial resistance is accelerating in a such way that is considered a substantial global threat. Out of 71 antibiotic resistance surveillance programs, three of the most commonly reported species were Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Additionally, those organisms were attributed to a total of 965,980 deaths globally with the highest load in low-income countries. While pharmaceutical drugs seem to be a heavy impact on developing countries’ economies, there are several ways to address this issue, including green synthesis of nanoparticles using herbs which is cost-effective.

Objectives: Characterization of silver nanoparticles synthesized from Tribulus cistoides leaf extract using green nanotechnology and assessing its biological activity against resistant/susceptible bacteria and fungi, and biofilm production amongst biofilm producers.

Methods: Synthesized AgNPs were characterized by using X-ray diffraction analysis (XRD), and ultraviolet-visible spectroscopy. Then the synthesized NPs were utilized to evaluate their potential bioactivity against methicillin-resistant S. aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa (MDR-PA), E. coli ATCC 25922, and Candida albicans using the microtiter broth dilution method to determine the minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). Antibiofilm capacity was assessed by employing the crystal violet tube method at their MIC and MBC values. Furthermore, inhibition of biofilm formation was also examined by confocal laser microscopy (CLSM) and scanning electron microscopy (SEM).

Results and Discussion: AgNPs exhibit excellent antimicrobial activity against all the tested pathogens. MRSA, MDR-PA, E. coli, and C. albicans have a MIC ranging between 31 µg/ml, 31 µg/ml, 15 µg/ml, and 156 µg/ml, respectively, while the MBC values were 250 µg/ml, 62 µg/ml, 15 µg/ml, and 625 µg/ml, respectively. There was a significant reduction in biofilm production for all the bacteria. Furthermore, disruption of morphology with loss in integrity was shown in SEM for all the bacteria. Interestingly, CLSM analysis shows the AgNPs significant inhibition of biofilm formation in all the tested strains.

Conclusion: Green synthesis of silver nanoparticles exploiting Tribulus leaves demonstrated significant bioactivity against the most well-known drug-resistant pathogens with the least MIC and MBC for the susceptible strain of E. coli which reflects the highest efficacy against this organism. Further investigations should be considered for the validation of usage in mice and rats and subsequently in humans.

IRMC-SRP-2023-13: Antimicrobial potential of herbal extract in the prevention of drug resistant gram positive and gram-negative bacteria

Yousef Ahmed Alanazi^1,2, Mohammad Azam Ansari¹*

¹Department of Epidemic Disease Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, 31441, Dammam, Saudi Arabia, ²College of Medicine, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia. *Email: maansari@iau.edu.sa

Background: As long as there is no drug without side effects, many researchers are concerned about the use of herbs as natural drugs with lesser side effects. In this study, we focus on the use of two different herbal extracts against drug-resistant bacteria particularly multidrug-resistant Pseudomonas aeruginosa (MDR-PA), and methicillin-resistant Staphylococcus aureus (MRSA).

Objectives: The study aims to find alternative treatments and promote the evolution of conventional medicine applications. Also, to increase the use of drugs of choice and provide safer natural options against resistant microorganisms and provide better prescribed drugs.

Methods: Microbroth dilution and well diffusion methods were used to assess the antibacterial and antifungal potential of herbal extract. Briefly, two-fold serial dilution of herbal extract with various concentrations was made in 96 well plates. After overnight incubation, inhibition of bacterial growth was examined by determining minimum inhibition concentration (MIC). Subsequently, to assess the minimum bactericidal concentration (MBC), 10 µl of culture from each well were further streaked on nutrient agar (NA) plates and after 24 h of incubation MBC values were calculated. Additionally, the effect of the herbal extract on the biofilm-forming ability of bacteria was also assessed by the crystal violate tube method. Moreover, scanning electron microscopy (SEM) and transmission electron (TEM) techniques were used to find out the effect of herbal extract on the morphology of tested pathogens.

Results and Discussion: The findings of microbroth dilution show that the MIC and MBC value of extract was 10 and 20 mg/ml, respectively against MDR-PA. The well diffusion methods show a zone of inhibition of 10 – 20 mm against the MDR-PA and MRSA. Additionally, it was found that plants extract significantly inhibit biofilm formation of MDR-PA and MRSA. Furthermore, SEM analysis shows that MRSA bacterial cells treated with plant extract exhibits that cell surface was irregular and appeared non intact, indicating damage of cell membrane and wall that may cause bacterial cell death.

Conclusion: The current finding revealed that herbal extracts were effectively inhibit the growth of MRSA and MDR-PA and their biofilm development. However, regarding Escherichia coli and Candida albicans the herbal extracts did not show any effective activity.

IRMC-SRP-2023-14: Probiotic Bimetallic Nanoparticle for Prevention of Drug Resistant Bacterial Infection in Human

Faten Ahmed Al Dossary^1,2, Mohammad Azam Ansari¹*

¹Department of Epidemic Disease Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, 31441, Dammam, Saudi Arabia, ²College of Medicine Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia. *Email: maansari@iau.edu.sa

Background: The benefits of utilizing the biological technique for preparing the NPs are non-toxic, biocompatible, and environmental friendliness. Bacteria can produce intra- and extracellular nanoparticles. Probiotic bacteria are recognized to enhance human health.

Objective: The aim of this study was to synthesized bimetallic NPs by probiotic and to investigate their potential antibacterial and antibiofilm activities against various bacterial strains using different microbiological methods.

Methods: Green synthesized bimetallic (Ag-Fe) nanoparticles were characterized by TEM, SEM, EDX and FTIR. The microbially produced NPs was used to study the antibacterial and antibiofilm activity against multidrug-resistant Pseudomonas aeruginosa (MDR-PA), Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, the effects of synthesized NPs on the morphological structure of bacterial cells were investigated by SEM.

Results and Discussion: TEM showed NPs were spherical and were 10-50 nm in size. The SEM analysis showed that NPs significantly damaged the cell membrane and wall of the tested bacteria. The antibacterial and antibiofilm potential of synthesized NPs was investigated, and it was found that NPs were more effective against E. coli and MRSA. The antibiofilm result shows that NPs significantly inhibit biofilm formation of bacterial strains by 95-98%.

Conclusion: As a result of this finding, it has been shown that NPs have great antibacterial potential and antibiofilm effect, suggesting they can be used for preventing the development of biofilms in drug resistant bacteria.

IRMC-SRP-2023-15: Effects of Essential Oils on Candida albicans growth and their Virulence Attributes

Khadija Mohamed Elmamy^1,2, Mohammad Azam Ansari¹*

¹Department of Epidemic Disease Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, 31441, Dammam, Saudi Arabia, ²College of Medicine Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia. *Email: maansari@iau.edu.sa

Background: Candida albicans is an opportunistic pathogenic yeast responsible for the majority of fungal infections in humans. It can lead to several diseases, ranging from superficial to life-threatening invasive illnesses. Its pathogenesis is associated with biofilm formation and morphological transformation from yeast to hyphae. Therefore, they considered the key virulence factors associated with increased resistance of C. albicans to traditional antifungal drugs. Essential oils [EOs], as an alternative antimicrobial therapy, are the focus of current studies due to their antifungal properties.

Objective: This study aims to investigate the effects of two essential oils [EO] on C. albicans growth, as well as their antibiofilm and anti-hyphal properties.

Methods: Anticandidal activity of EO was assessed by determining their minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) using CLSI M27-A4 method. Moreover, the antibiofilm activities were assessed using the crystal violet-tube method. The anti-virulent activities of EO were also studied at MIC and ×2 MIC values using yeast extract peptone dextrose (YPD) and Spider media.

Results and Discussion: Our findings showed that the MIC values of clove and eucalyptus oils were 0.5 and 14 mg/ml, respectively, while their MFC values were 1 and 28 mg/ml, respectively. Furthermore, it has been found that EO significantly inhibited C. albicans growth, biofilm formation, and yeast-to-hyphal transition at MIC and 2x MIC values.

Conclusion: Our findings revealed that essential oils were effective in suppressing the various virulence factors of C. albicans.

IRMC-SRP-2023-16: Utilizing Zinc Based Metal Organic Framework as Targeted Drug Delivery System for Cancer Treatment

Rana Faisal Alkhudhayri^1,2, Muhammad Nawaz³, Firdos A. Khan⁴, and Sultan Akhtar^1*

¹Department of Biophysics Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ²Department of Biomedical Engineering, College of Engineering, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ³Department of Nanomedicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ⁴Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia. *Email: suakhtar@iau.edu.sa

Background: Nanomaterial-based drug delivery systems are widely investigated due to their promising potential. In this research, zinc-based metal-organic framework (MOF) was utilized as a drug delivery system for breast cancer treatment. The flexible nature of MOF and its ability to tune its shape, morphology, and porosity makes them a good candidate for a drug delivery system.

Objectives: Because of the shortcomings of current traditional cancer treatment methods, this study aims to construct a drug delivery system that lessens those side effects.

Methods: A Zinc based Metal-organic framework (Zn-MOF) was synthesized using the solvo-thermal method using zinc nitrate and terephthalic acid. The prepared materials were then characterized by XRD, EDX, SEM, TEM, FTIR, UV Spectroscopy, and Zeta-sizer. The Zn-MOF was then loaded with 5-Flurouracil drug and further characterized by the aforementioned methods to verify that the loading is successful. Afterwards, breast cancer cells (MCF-7) were cultured and then treated with loaded Zn-MOF at different concentrations (0.02, 0.04, 0.08, 0.12 mg/mL) for 48 hours. Healthy cells (HFF) were used as the control sample in order to compare the results with treated cells. The anticancer results were collected using a confocal light microscope. Finally, MTT Assay was performed, and cell viability (%) was calculated.

Results and Discussion: XRD, EDX, and FTIR results showed the successful synthesis of Zn based MOF nanomaterial. SEM and TEM analysis showed that the prepared nanomaterial exhibited an irregular shaped morphology. After the loading of 5-fluorouracil drug, the morphology of the material was changed, and the presence of the loaded organic material was apparent. The EDX results further confirmed the successful loading of the drug onto the Zn-MOF. MTT assay results showed that there was an obvious trend in cell viability. The cell viability was found to be inversely correlated with the concentration of the loaded Zn-MOF. Finally, further tests are recommended to be conducted with different agents to determine conclusive results.

Conclusion: MOFs are well-known nanomaterials used for many applications such as medical and environmental. The aim of the study is to investigate the efficacy of zinc-based MOFs as drug delivery carriers for breast cancer (MCF-7) treatment. Our results showed the successful loading of 5-Fluororacil into Zn-MOF. The MTT assay of treated MCF-7 cells showed a clear trend where the cell viability is dose-dependent. The lesser toxicity and the ability of Zn-MOF to encapsulate the drug is reason to study MOF systems. Further study is suggested to investigate the mechanism in which the Zn-MOFs work as a targeted drug delivery system.

IRMC-SRP-2023-17: Anticancer activity of gum Arabic and cinnamon coated gold nanoparticles against colorectal cancer cells

Fatimah Zuhair Alabdrabalnabi^1,2, Muhammad Nawaz³, Firdos A. Khan⁴, and Sultan Akhtar^1*

¹Department of Biophysics Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ²College of Public Health, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ³Department of Nanomedicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ⁴Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia. *Email: suakhtar@iau.edu.sa

Background: Colorectal cancer is considered one of the most common, death-leading cancers worldwide, regardless of all the efforts and advancements in diagnosis and treatment. Gold nanoparticles (AuNPs) are one of the most effective anticarcinogenic nanoparticles commonly synthesized by using green methods by utilizing multiple extracts such as gum Arabic and cinnamon.

Objectives: This study aims to investigate the effect of green synthesized gold nanoparticles against colorectal cancer cells (HCT-116) using different characterization techniques.

Methods: Gum Arabic and cinnamon coated gold nanoparticles were prepared from gum-Arabic and cinnamon powder. The nanoparticles were characterized by using scanning electron microscopy/energy dispersive X-rays spectroscopy (SEM/EDX) and transmission electron microscopy (TEM). The colorectal cancer cells (HCT-116) were cultured in 96 well plates and treated with various concentrations (60, 40, 20, 10 and 5 µM corresponding to 9.8, 6.55, 3.28, 1.64, and 0.82 µL, respectively) of gum Arabic encapsulated gold nanoparticles (AuNPs-GA) and cinnamon encapsulated gold nanoparticles (AuNPs-CNM), then incubated for 24 hours. The cell morphology of the treated and control cells was determined by using the light microscopy method. The cell cytotoxicity of the AuNPs-GA and AuNPs-CNM was assessed through an MTT assay using the multi-mode reader. The IC50 was calculated from the cell viability data. Furthermore, independent sample t test was applied for statistical analysis by IBM SPSS.

Results and Discussion: The structure and the morphology of the prepared AuNPs-GA and AuNPs-CNM were confirmed by SEM, EDX, and TEM. TEM results revealed that the synthesized nanoparticles exhibited different shapes and morphology such as spherical, diamond and hexagonal etc. TEM characterization has shown that the size of AuNPs-GA was estimated to ~12 nm, and ~17 nm for AuNPs-CNM. SEM-EDX analysis confirmed the successful preparation of AuNPs (using gum Arabic and cinnamon) where the gold peak was observed with a considerable intensity for both the specimens along with C and O, and P, confirming the presence of organic compounds (gum Arabic and cinnamon).

MTT assay and light microscopy results showed that there was a noticeable reduction in the cell viability after treating the cells with multiple compounds for 24 hrs. The synthesized AuNPs-GA and AuNPs-CNM products produced an anticancer efficacy in the HCT - 116 cells with IC50 of 19.20 and 64.24 µM/mL, respectively after a treatment of 24h. The AuNPs-GA specimen showed its potency by exhibiting a lower inhibitory concentration than AuNPs-CNM. AuNPs-GA has showed a statistically significant difference (P – value <0.05) as compared to AuNPs-CNM across different concentrations.

Conclusion: The MTT results showed that the synthesized nanoparticles induced cell viability reduction in colorectal cancer cell lines (HCT-116).

IRMC-SRP-2023-18: Engineering of Quercetin Loaded Silica Spheres as Targeted Drug Delivery System for Breast Cancer Treatment

Kawther Altalaq^1,2, Muhammad Nawaz³, Firdos A. Khan⁴, and Sultan Akhtar^1*

¹Department of Biophysics Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ²Department of Biomedical Engineering, College of Engineering, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ³Department of Nanomedicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ⁴Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia. *Email: suakhtar@iau.edu.sa

Background: Targeted drug delivery systems have become a focal point in the field of nanomedicine. Unlike conventional chemotherapies and immunotherapies, targeted drug delivery systems have shown great potential in specifically targeting the cancer cells while minimizing the side effects on healthy cells. Among the emerging drug carriers, silica particles (SiO2) have gained significant attention due to their unique properties, including low toxicity, high surface area, and large loading capacity. These characteristics make silica particles highly suitable for delivering a wide range of pharmaceutical agents.

Objectives: In this study, we focused on evaluating the potential of silica spheres (SiO₂) as targeted drug delivery platforms for delivering the anticancer agent Quercetin, with its applications in breast cancer (MCF-7) treatment.

Methods: Silica (SiO₂) spheres were synthesized using the chemical sol-gel route. Tetra-orthosilicate, Cetyltrimethylammonium bromide (CTAB) and ammonia were used as the starting materials. The final treatment of the SiO₂ particles included drying at 60 ^oC for 24 h and calcination in furnace at 550 ^oC for 6 h. Quercetin, an anticancer drug, was subsequently dissolved in ethanol and added to the SiO₂ particles. After filtration, the sample was analyzed using different characterization techniques, namely SEM, TEM and XRD. Breast cancer cells (MCF-7) were cultured and treated with loaded silica spheres using the 96-well plate with the concentrations (5, 10, 20, 40, and 60 µl). Finally, MTT assay was conducted to analyze cell viability of the treated MCF-7 cancer cells.

Results and Discussion: The morphology of the synthesized silica (SiO₂) spheres was characterized using SEM and TEM. The analysis revealed that the silica spheres exhibited an amorphous spherical structure with a smooth surface and a diameter range of 200-500 nm. Moreover, XRD results displayed a broad peak at an angle (2θ) ~ 22^o, indicating the absence of any crystalline and confirming the amorphous nature of both the loaded and unloaded silica spheres. The identity of the synthesized spheres was further confirmed using (FTIR) analysis. After the treatment of breast cancer cells (MCF-7) with different concentrations of Quercetin loaded silica spheres, MTT assay was performed. The cytotoxicity results showed a significant reduction in cell viability and displayed a dose-dependent behavior.

Conclusion: This study investigated the potential of SiO₂ as targeted drug platforms. Quercetin loaded SiO₂ were successfully prepared and tested against MCF-7 cells. The analysis showed that the synthesized SiO₂ had a spherical and amorphous structure with a diameter range of 200-500 nm. The anticancer results revealed that Quercetin loaded silica particles resulted in a concentration-dependent decrease in cell viability. The results suggested that the silica spheres have great potential as drug delivery carriers. Further research is recommended to study the effect of silica in vivo.

IRMC-SRP-2023-19: Study the Antibacterial Capacity of Green Synthesized Silver Nanoparticles

Zainab N. Aldandan^1,2, Mohammad Azam Ansari³ and Sultan Akhtar^1*

¹Department of Biophysics Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ²Department of Physics, College of Science, King Abdulaziz University, Jeddah, Saudi Arabia, ³Department of Epidemic Disease Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, 31441 Dammam, Saudi Arabia, *Email: suakhtar@iau.edu.sa

Background: The spread of bacteria is considered a global risk. Bacteria is a major cause of many diseases and crises. Recently, it has caused concern in the medicine field due to its ability to resist antibiotic drugs. Nanoparticles have made life easier in all aspects such as developed equipment, technology, and antibiotics. In this research, we used silver nanoparticles (AgNPs) that can damage many types of bacteria due to their unique physical properties.

Objective: This study works about alternative solutions to make the antibiotic more effective by using nanoparticles with size less than 100 nm. The main aim of this study was to prepare the nanoparticles using affordable and non-hazardous materials i.e., gum plant, Boswellia serrata instead of chemicals.

Methods: Silver nanoparticles (AgNPs) were prepared by using aqueous extract of B. serrata. 10 ml extract was added in 40 ml of 1mM silver nitrate (AgNO₃) solution. The prepared NPs were then dried and characterized by SEM, TEM and EDX. The antibacterial activity of NPs was investigated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using standard microbroth dilution method.

Results and Discussion: In this study, the SEM and TEM results showed that the cell membrane of bacteria becomes damaged after treatment with AgNPs indicates that AgNPs prepared by green method might be used as an alternative antibiotic. The EDX spectrum showed the percentage of elements in the prepared material, the results were 60 % of Ag in B. serrata. The MIC value for E. coli and S. aureus was ≈ 0.18 & 0.75 mg/mL, respectively. While, MBC was ≈ 0.37 and 6 mg/mL, respectively.

Conclusion: The AgNPs were synthesized by using B. serrata as a stabilizer agent and were successfully tested against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. The prepared NPs were spherical in shape with an average size of 12 nm. MIC and MBC results confirmed the inhibitory and bactericidal potential of synthesized AgNPs as antibacterial agent.

IRMC-SRP-2023-20

Evaluation of Antibacterial Activity of Zinc Oxide Nanoparticles Prepared by Pulsed Laser Ablation in Liquid

Ahmed Behisi^1,2, Mohammad Azam Ansari³, Muidh Alheshibri⁴, Khaled A. Elsayed⁵ and Sultan Akhtar^1*

¹Department of Biophysics Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ²College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia, ³Department of Epidemic Disease Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, 31441 Dammam, Saudi Arabia, ⁴Department of Basic Sciences, Deanship of Preparatory Year and Supporting Studies, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia, ⁵Department of Basic Engineering Sciences, College of Engineering, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia. *Email: suakhtar@iau.edu.sa

Background: Every year 700,000 deaths are caused by multi-drug resistant (MDR) bacteria, this number is expected to increase to 10 million by 2050. In order to combat this issue many drugs and novel materials have been developed. Nanoparticles (NPs) are particles that range in size from 1-100 nm that have unique physical and chemical properties. Previously, Zinc Oxide (ZnO)NPs have been previously used to combat this issue yielding good results. Although it has been successful for initial studies it suffers from a major setback, being its tendency to aggregate significantly decreasing their antibacterial activity.

Objectives: The aim of this study was to increase the antibacterial efficacy of ZnO NPs via modifying its physical nature by pulsed laser ablation in liquid (PLAL).

Methods: The NPs were prepared by dissolving 100 mg of ZnO NPs powder in 10 ml of deionized water. The prepared mixture was then irradiated with a focused laser beam at 355 nm and 120 mJ for 0 min, 30 second, 60 second and 90 second while the solution was stirred by a magnetic stirrer in order to ablate all the powder. Then SEM and TEM were used in order to characterize the material. Antibacterial activity was determined by broth dilution and well diffusion methods. Furthermore, the effects of NPs on morphology of bacterial cells were investigated by SEM and TEM examination.

Results and Discussion: TEM and SEM of ZnO NPs suggested that longer the exposure times of laser leads to spherical and smaller nanoparticles along with nanosheets. The broth dilution study showed that the ZnO NPs inhibits the growth of E. coli. SEM and TEM results showed that ZnO NPs damaged the bacterial cell membrane and wall that lead the death of the cells. On the contrary, the well diffusion study showed that there was not any substantial bactericidal activity as no obvious zone of inhibition was observed. This result is contrary to the literature as ZnO NPs have previously shown bactericidal activity. Further experiments are required in order to understand the treatment mechanism of ZnO interaction with E. coli bacteria.

Conclusion: ZnO NPs were successfully modified by PLAL technique. SEM and TEM analysis shows that NPs severely damaged the bacterial cell membrane and wall that causes cell death.

IRMC-SRP-2023-21: Title Synthesis of novel benzodioxane analogues for potential inhibition of Alzheimer’s disease

Saud Adel alshnabir^1,2, Muhammad Taha¹*, and Muhammad Nawaz ³

¹Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam 31441, Saudi Arabia, ²College of Medicine, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia, ³Department of Nanomedicine Research Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: mtaha@iau.edu.sa

Background: Alzheimer’s disease is an age-related neurodegenerative disease and is the main cause of dementia. Owing to the fact of it being an age-related disease, cases are rising and expected to rise even further in the future. Thus, developing new improved drugs to combat it is crucial. Benzodioxane is a chemical template that is employed by researchers for decades, to design compounds with various biological activity. Due to, its structural properties that allows for vast variety of compounds to be derived from it. Many drugs which are in current use or development, have used benzodioxane as a scaffold in their chemical structure of which are antihypertensive, serotonin receptors agonists and antagonists, antibacterial agents, cancer chemotherapeutic, (nAChRs) agonists and antagonists and many more.

Objective: The objective of this study is to evaluate the potency of sixteen thiourea analogs synthesized based on benzodioxane for their inhibition potency against acetylcholinesterase (AchE) and butyrylcholinesterase (BchE).

Methods: The synthesis begins with reacting (benzo [d] ^[1,3] dioxal) 0.5 mmol and 0.5mmol isothiocyanate in the presence of 10mL CHCl₃. The reaction mixture kept on stirring for 4 hours. After completion of the reaction crude product was transferred into a baker and kept in a fume hood for solvent evaporation. The crude product was recrystallized in ethanol to get pure product. All synthesized compounds were characterized using different spectroscopic methods IR, ¹HNMR, ¹³CNMR and melting point. All synthesized compounds were also tested for inhibition potential of acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) with reported protocol.

Results and Discussion: All the synthesized analogs (1-16) were screened for their acetylcholinesterase and butyrylcholinesterase inhibition activities. Most of the analogs of the series exhibited good inhibition potential when compared to the standard drug donepezil having an IC₅₀ value of 0.016 ± 0.01 for acetylcholinesterase and 0.30 ± 0.010 for butyrylcholinesterase. In addition, five compounds showed better activity than standard for acetylcholinesterase and butyrylcholinesterase. Furthermore, the fluorinated compounds have shown potent activity for both enzymes.

In the contrary, brominated compounds showed moderate activity, due to, the weak interaction with enzyme. Additionally, methoxy compounds showed good activity, this may be due to the polarization of methoxy substituted ring. Moreover, chlorinated compounds showed good activity. Lastly, CN, methyl and nitro substituted compounds showed good to moderate activity. Whereas unsubstituted compounds showed least activity it means substitution enhance activity.

Conclusion: 1-16 Synthesized compounds out of which 5 compounds shown high potency in comparison with stander drug (donepezil). Additionally, all compounds were fully characterized.

IRMC-SRP-2023-22: Synthesis of benzimidazole thiourea to explore new diabetics II inhibitors

Dina Alrastal^1,2, Muhammad Taha¹*, and Muhammad Nawaz ³

¹Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam 31441, Saudi Arabia, ²College of Clinical Pharmacy, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia, ³Department of Nanomedicine Research Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: mtaha@iau.edu.sa

Background: α-Amylase and α-Glucosidase enzymes are involved in the process of carbohydrates digestion process, and it’s one of the therapeutic targets for drugs that lower the post-prandial hyperglycemia (PPHG) in the management of type-2 diabetes mellitus. However, the currently available medications suffer from G.I. side effects that limit their usage. Benzimidazole moiety has excellent stability, biological activity, and bioavailability properties, and it’s a moiety of choice for many different therapeutic indications.

Objective: Synthesize a newer anti-diabetic benzimidazole thiourea analog agent with fewer side effects and better efficacy.

Methods: Sixteen (1-16) benzimidazole thiourea analogs were synthesized. The reaction begins with taking benzimidazole hydrazide 0.5 mmol with various aryl isothiocyanates 0.5mmol and the chloroform solvent 10 mL, all of which added in a round bottom flask and then get subjected to stirring for at least four hrs. and then transferred into a beaker and dried in the fume hood. After that, the solutes dissolve in methanol and are put on a heater till the solution becomes transparent. Fast filtration was used to get rid of any residue. It’s then allowed to dry slowly in the open air. All the synthesized compounds are characterized using different spectroscopic techniques, bioassay testing was carried out.

Results and Discussion: All sixteen (1-16) compounds were tested for α-Amylase and α-Glucosidase inhibition, of which ten (10) compounds showed better activity than the standard drug. Three compounds showed potent activity for both enzymes; 1, 2 and 3; IC₅₀ in the range of 2.10 ± 0.10 to 3.20 ± 0.10 against α-amylase, and IC₅₀ = 2.40 ± 0.10 to 4.30 ± 0.10 against α- glucosidase. Unsubstituted compound 11 showed the least activity (IC₅₀ = 15.40 ± 0.50 for α-amylase) and (IC₅₀ = 16.20 ± 0.60 for α- glucosidase). Furthermore, five compounds showed good activity; 7, 8, 9, 12 and 13; IC₅₀ in the range of IC₅₀ = 4.80 ± 0.20 to 9.10 ± 0.20 for α-amylase, and IC₅₀ = 5.20 ± 0.20 to 9.40 ± 0.20 for α- glucosidase). However, two compounds showed good to moderate activity; 4 and 16; IC₅₀ in the range of IC₅₀ = 9.40 ± 0.30 to 9.50 ± 0.30 for α-amylase), and IC₅₀ = 9.70 ± 0.30 for α- glucosidase).

Conclusion: This study has identified ten compounds that have better efficacy than the standard drug, among which the three analogue shows a powerful inhibitory activity of both enzymes; this may be due to the high electronegativity of the fluorine atom, that made molecules polarized and made appropriate for interaction with the enzymes. Also, any substitution on benzene ring increased activity, as proven by the IC₅₀ value of compound 11. Further investigation of SAR will be done with the help of a molecular docking study.

IRMC-SRP-2023-23: An Effort to Develop a Novel Functionalized Thiazole-Based Derivatives Drug as Dual α-Amylase and α-Glucosidase Enzyme Inhibitors for Type-II Diabetes Management

Faisal Alharamlah^1,2, Muhammad Taha¹*, and Muhammad Nawaz ³

¹Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam 31441, Saudi Arabia, ²College of Dentistry Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabi, ³Department of Nanomedicine Research Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: mtaha@iau.edu.sa

Background: diabetes II is the most occurring disease which involves the blood sugar to rise and cause diabetes with many complications including weight loss, kidney failure, heart disease etc. The current drugs available for managing diabetes II have side effects and cause problems. We design the new thiazole compounds to inhibits the enzymes that involves in digestion of sugar (α-Amylase and α-Glucosidase) and cause in high blood sugar.

Objective: Synthesize a new thiazole-base compounds for managing diabetes II with minimized side effects and improved applicability

Methods: Fifteen (1-15) thiazole-based compounds were prepared. The 0.5 mmol 2-(2-bromophenyl) thiazole-5-carbohydrazide treated with various aryl isothiocyanates 0.5mmol in the chloroform 10 mL, stirred the reaction mixture for 3-4 hours. After completion reaction the solvent was evaporated to afford crude products 1-15. The crude products were recrystallized in methanol to afford 2-(2-bromophenyl) thiazole-5-carbohydrazide-based (1-15). All synthesized compounds were fully characterized by using different methods. All synthesized compounds also evaluated for α-Amylase and α-Glucosidase inhibition as reported.

Results and Discussion: All fifteen (1-15) compounds were tested for α-amylase and α-glucosidase inhibition. Nine compounds showed better activity than standard drug. Fluorinated compounds showed potent activity for both enzymes, this may be due to high electronegativity of fluorine atom which made whole molecules polar and made suitable for interaction with enzyme. Cholorinated compounds also showed good activity for both enzymes due to high electronegativity. Brominated compounds showed good to moderate activity. Methoxy compounds also showed good activity due to high electronegativity of oxygen attached with methoxy for both enzymes. Nitro and methyl showed good activity but less than standard drug. Unsubstituted showed least activity it means any substitution on benzene ring increased activity.

Conclusion: In this study we synthesized 1-15 compounds and evaluated for α-amylase and α-glucosidase inhibition and we found that out of 15 compounds 9 showed potent activity. It’s also revealed that the compound has fluorine or other electronegative atom showed good inhibition.

IRMC-SRP-2023-24: Discovery and synthesis of novel drugs as potent urease inhibitors

Leena Alhajri^1,2, Dr. Muhammad Taha¹*, Dr. Muhammad Nawaz³

¹Department of Clinical Pharmacy, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Clinical Pharmacy Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia, ³Department of Nanomedicine Research Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: mtaha@iau.edu.sa

Background: Urease is a metal-containing enzyme that catalyzes the breakdown of urea into ammonia and carbon dioxide. It is present in a wide variety of organisms, including plants, fungi, archaea, and algae. Urease-producing bacteria have a significant negative impact on human health. As reported previously, the notorious cancerous bacterium Helicobacter pylori (H. pylori) relies on this enzyme as a colonizing factor, leading to diseases such as gastric ulcers, gastritis, and, in advanced cases, stomach cancer. Recently, Antibiotic-resistant bacteria have become prevalent worldwide. Therefore, there is an urgent need for novel urease inhibitors capable of controlling infection by H. pylori and other pathogenic bacteria.

Objective: This study aims to discover and synthesize novel drugs as Aceto-hydrazide analogues (1-16) and test them for urease inhibition activity.

Methods: During the experiment, we synthesized the new derivatives of 1-(2-(2,4-dichlorophenoxy) acetyl)-4-phenylthiosemicarbazide and evaluated them using in vitro biological assays. First, the 2-(2,4-dichlorophenoxy) acetohydrazide 0.5mmol was treated with various isothiocyanate 0.5mmol in the presence of chloroform as solvent 10 mL. After completion of reaction, the solvent was evaporated to get crude 1-(2-(2,4-dichlorophenoxy) acetyl)-4-arylthiosemicarbazide. The crude 1-(2-(2,4-dichlorophenoxy) acetyl)-4-arylthiosemicarbazide analogues were recrystallized in ethanol to obtain pure 1-(2-(2,4-dichlorophenoxy) acetyl)-4-arylthiosemicarbazide analogues. All synthesized analogues 1-16 were fully characterized by different method of spectroscopy. All analogues also tested for urease inhibition potential.

Results and Discussion: All the newly synthesized compounds (1-16) were tested in vitro for their urease inhibitory profiles. It is remarkable that all the newly developed scaffolds illustrated excellent to moderate inhibition profiles on evaluation against urease with IC₅₀ values in the range of 5.40 ± 0.30 to 24.10 ± 1.10 µM as compared to standard thiourea as a reference inhibitor (IC₅₀ = 21.30 ± 0.80 µM). The inhibition depends on the numbers, types, and positions of substituents. We also noticed that without any substituted compound showed least activity.

Conclusion: There are ten (10) compounds with better and more promising anti-urease activity as compared to the standard thiourea. The fluorinated compounds had the highest anti-urease activity, and this may be due to the high electronegativity of the fluorine atom, which made whole molecules polar and suitable for interaction with the enzyme, so this can be considered for further studies and investigation to develop novel anti-urease drugs.

IRMC-SRP-2023-25: Synthesis of new 2-(4-methyIthiazol-2-yl) acetohydrazide compounds as potent Alzheimer’s disease inhibitors

Leen Alshaikh^1,2, Muhammad Taha¹*, Muhammad Nawaz³

¹Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²School of Mechanical Engineering, Purdue University, West Lafayette, Indiana, United States, ³Department of Nanomedicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: mtaha@iau.edu.sa

Background: Alzheimer’s disease (AD) is a neurogenerative disorder that gradually affects the mechanism of cognitive impairment, orientation, and memory. This disease is triggered by several factors such as the alteration of different genes and lifestyle of the individual. Patients with AD have a deficiency of Acetylcholine (ACh) in the cholinergic neurons, hence, both enzymes Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) are targeted to increase the concentration of ACh and prolong its activity in the synapse as a treatment.

Objective: This study investigates the synthesis of 1-16 different derivatives of a thiazole-based compound and their inhibition activity in AChE and BuChE compared to the standard drug donepezil as a potential treatment of Alzheimer’s.

Methods: The 2-(4-methylthiazol-2-yl) acetohdrazide compound was treated with 16 different isothiocyanate derivatives in order to obtain the product LA-I-(1-16). For the synthesis, both the parent compound and the reagent are mixed with 10-mL of chloroform and placed in the stirring plate for approximately 3-4 hours. Afterwards, the reaction mixture was transferred into a beaker. Then, recrystallization process is preformed to obtain crystals. Finally, characterization takes place by the use of NMR spectroscopy, mass spectroscopy, FT-IR spectroscopy, and melting point. All synthesized compounds were tested for both enzymes (AChE and BuChE) inhibition.

Results and Discussion: All the synthesized analogs (1-16) were screened for their potential acetylcholinesterase and butyrylcholinesterase inhibition activities. Most of the analogs of the series exhibited good inhibition potential when compared to the standard drug donepezil having an IC₅₀ value of 0.016 ± 0.01 µM for acetylcholinesterase and 0.30 ± 0.010 µM for butyrylcholinesterase. The most potent analog among the series was analog 3 (4-fluoro) on the phenyl ring, and it showed 0.15 ± 0.01 µM for AChE IC₅₀ and 0.20 ± 0.01 µM of BuChE IC₅₀.

Conclusion: All synthesized compounds showed activity, however 4-flouro isothiocyanate is the most potent analog. Furthermore, the nature of the element fluorine is distinguished to be the most electronegative atom in the periodic table, meaning, it has a capacity to is polarize the whole molecules. Therefore, fluorine was demonstrated the most potent due to its nature that inhibits

IRMC-SRP-2023-26: Preparation of magnetic ZnAgFeO₄ through sonication technique, characterization for antifungal and anti-cancer therapy

Shoug Saleh Alhbabi^1,2, B. Rabindran Jermy¹*, Dana Almohazey³, Thamer Alshammari ⁴, Munirah A. Almessiere⁵

¹Department of Nano-medicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²Deanship Preparatory Year, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ³Department of Stem Cell Research Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Department of Genetics Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam, 34221, Saudi Arabia, ⁵Department of Biophysics, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia. *Email: rjermy@iau.edu.sa

Background: Cancer patients are people who suffer from a decrease in immunity, so they are more susceptible to infection with fungi like Candida albicans. The Candida albicans is a yeast infection effect on oral cavity, digestive system, skin and genitals. The real danger of candida albicans is that they are highly drug resistant. Nanoparticles (1 – 100 nm ) widely used for delivery drug and antifungal. Some studies have proven the effectiveness of minerals such as zinc and iron to treat this type of fungus, and at the same time some research has used this type of minerals as Complementary treatment for cancer.

Objective: Preparation of novel magnetic nanoparticles based on zinc and silver spinel ferrite for multifunctional antifungal and cancer therapy.

Methods: Zinc nitrate, silver nitrate and ferric nitrate were dissolved in (DW) at room temperature with constant stirring. Thereafter, the pH of solution was converted by sodium hydroxide. After that, the solution was sonicated for 30 minutes. Then, the solution was filtered by filtration paper and dried using the oven at 100 °C at 5h. Thus, we took from the final product of the process for preparing magnetic/silica Nano composite. In this step, magnetic particles was dissolved them in DW by sonication . Then CTAB was dissolved in solution using stirrer with steady state. Subsequently, silica was added to the solution gradually and we left it on stirrer overnight. The solution was separated by centrifuge and dried by oven at 100 °C for 5 h. Then, the sample was calcined in furnace to remove CTAB. Finally, we used well diffusion test and biofilm inhibition essay to test out nanoparticles with different concentrations of silver (x=0.0 , x=0.02 , x=0.04 , x=0.06 , x=0.08).

Results and Discussion: After synthesis of NPs, XRD of ZnAgFeO₄ shows pure phase of spinel ferrite nanoparticles without impurities. SEM-EDX analysis provides information about sample composition, size, shape, and morphology. In the present study, elemental analysis using SEM-EDX confirm the presence of various elements: Zn, Ag, Fe and O. SEM analysis shows the presence of magnetic nanoparticles in crystalline agglomerated form, while formation of silica nanocomposite can be visibly seen with the surface textural modification on the external surface of magnetic nanoparticles. Nanoparticles had BET surface area: 96 m²/g, single point adsorption total pore volume of pores: 0.2 cm³/g and average pore diameter: 8.3 nm. The well diffusion test shows inhibition zone around discs about 1cm. Thus, the biofilm inhibition essay shows percentage of biofilm inhibition by ninety-six percent average.

Conclusion: In this study, we succeeded in preparing ZnAgFeO₄ (X = 0.00 – 0.08). XRD, BET and SEM-EDX verified the silica nanocomposite formation with ferrite spinel. The efficacy of nanoparticles on fungi was verified by well diffusion test and biofilm inhibition essay. Moreover, the study is ongoing to verify the efficacy of nanoparticles on a cancer cell line.

IRMC-SRP-2023-27: Synthesis of Si/NiCuZnSeFeO4 Magnetic Spinel Ferrite Nanoparticles: Characterization for Antifungal Therapeutics and Cancer Therapy

Rakan Aljohani^1,2, B. Rabindran Jermy¹*, Dana Almohazey³, Thamer AlShammari⁴, Munirah A. Almessiere⁵

¹Department of Nano-medicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Nursing, King Abdulaziz University, Jeddah, Saudi Arabia, ³Department of Stem Cell Research Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ⁴Department of Genetics Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam, 34221, Saudi Arabia, ⁵Department of Biophysics, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia. *Email: rjermy@iau.edu.sa

Background: Cancer is a disease in which the cells continuously divide abnormally. According to the World Health Organization (WHO), the total number of new cancer cases in Saudi Arabia is reported as 27,885 in 2020. The common treatment for cancer is chemotherapy, but it has numerous side effects. These side effects include the destruction of healthy cells, including white blood cells that play a crucial role in protecting the body against infectious agents like various types of bacteria and fungi. Opportunistic fungi can take advantage of the weakened immune system in cancer patients undergoing chemotherapy, as the treatment targets both cancerous and healthy cells. In recent years, Nanotherapeutics has shown promising potential. It focuses on using nanoparticles for therapeutic purposes and has numerous applications in various areas, including cancer and fungal treatments. Mesoporous silica magnetic nanoparticles have demonstrated significant selectivity in destroying cancer cells without affecting healthy cells.

Objective: This study aims to create a magnetic spinel ferrite with antifungal properties and form silica nanocomposite with the precise objective of selectively targeting cancer cells to reduce the potential for opportunistic fungal infections.

Methods: In the initial phase of the fungi study, NiCuZnSeFeO4 magnetic NPs (X = 0.02, 0,04, 0,08, 0,1) were synthesized using nitrates of cobalt (Co), copper (Cu), zinc (Zn), selenium (Se), iron (Fe), using the hydrothermal technique. Then nanocomposite formation with silica was done using a hydrothermal technique using TEOS and CTAB. This process facilitated the creation of pores and a substantial surface area, enhancing their potential for drug delivery applications. For the cancer study, three distinct nanoparticles were synthesized: NiCuZnSeFeO4, Si/NiCuZnSeFeO4, and Si/NiCuZnSeFeO4/Cp/PEG. The synthesis and characterization of these nanoparticles were conducted using techniques such as Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Analysis (EDX), Brunauer-Emmett-Teller (BET), and X-ray diffraction analysis (XRD). To assess their effectiveness, the antifungal activity of nanoparticles with varying ratios was evaluated against Candida albicans using a biofilm inhibition assay. In the context of cancer research, two cell lines, HFF-1 and HCT-116, were treated. These cell lines were treated to various concentrations (0.025, 0.050, 0.10, 0.50) mg\mL of the nano-formulation to investigate its impact.

Results and Discussion: After synthesis, the formation of silica mesoporous nanoparticles was confirmed through X-ray diffraction analysis (XRD), which indicated the purity of the sample. Energy-dispersive X-ray spectroscopy (EDX) revealed the chemical composition and concentration of the sample, which includes nitrates of cobalt (Co), copper (Cu), zinc (Zn), selenium (Se), and iron (Fe). The scanning electron microscopy (SEM) study shows that the nanoparticles are coated with silica. The Fungal study indicates high biofilm inhibitory activity across all ratios, reaching a plateau phase at the lowest concentration. At the lowest concentration (X=0.01), it exhibited 95.18% biofilm inhibition, showing promise for future investigations. The HCT116 study demonstrates that loading cisplatin into Si/NiCuZnSeFeO4 significantly reduces cell viability. At the highest concentration used (0.1 mg/mL), only 14.8% of cell viability remained. Conversely, the HFF study reveals that loading cisplatin into Si/NiCuZnSeFeO4 at the lowest concentration (0.025 mg/mL) maintained 94.6% cell viability. However, at the highest concentration (0.1 mg/mL), cell viability decreases to 6.47%.

Conclusion: A magnetic spinel ferrite nanoparticle (NiCuZnSeFeO4) nano-formulation was synthesized using the hydrothermal technique for drug delivery targeting colon cancer and opportunistic fungal infections. The nanocarriers for cancer cells were loaded with cisplatin and PEG. Following analysis, the nanoparticles exhibited promising results for further study.

IRMC-SRP-2023-28: Development of Metformin/insulin-based drug deliverable nanoformulation and characterization for potential treatment of type 2 diabetes

Roaa Abdulaziz Almehman^1,2, B Rabindran Jermy¹*, Dana Almohazey³

¹Department of Nano-medicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Medicine, Fakeeh College for Medical Sciences, Jeddah, Saudi Arabia, ³Department of Stem Cell Research Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: rjermy@iau.edu.sa

Background: Diabetes mellitus involves metabolic disorders with high blood sugar levels. Saudi Arabia faces a significant diabetes challenge, ranking seventh globally and second in the Middle East. Metformin hydrochloride is a primary type 2 diabetes treatment, but its limited absorption, requiring high dosages due to its poor absorption, leads to uncomfortable gastrointestinal side effects. In this research, we are investigating an advanced drug delivery based on Mesocellular foam (MSU-F). Nano therapy offers new hope for diabetic management in the form of “Artificial Pancreas”. MSNs possess large porous pores capable of efficiently holding and releasing small and large protein medications, offering potential solutions to improve treatment outcomes.

Objectives: This study seeks a better diabetes treatment using Mescellular foam type silica nanoparticles (MCF). Goals: Develop a sensor-based drug delivery system mimicking “Artificial Pancreas”. Study the controlled drug release pattern, reduce the dose dependent toxicity, and improved drug effectiveness for enhanced diabetes management.

Methods: In this study, we formulated a nanostructured drug delivery system using metformin, insulin, Au/foam silica, normal saline solution (NSS), and phosphate buffer saline (PBS) with a pH of 7.4. The synthesis process involved combining calculated amount of metformin with 10 ml of NSS, followed by loading onto foam type nanocarrier. Subsequently, the samples were stirred at room temperature, subjected to centrifugation, washed, and then dried. The samples were collected at regular intervals and replaced with fresh PBS solution. UV-visible spectrophotometry was used to examine the extent of protein or drug absorption and release. Characterization was performed using XRD, TGA, FTIR, and DS-UV techniques. For drug release analysis, a dialysis membrane technique followed. To assess drug toxicity, we employed human foreskin fibroblast (HFF) cells. Metformin/insulin loaded MCF silica was tested at four different concentrations in a 96-well plate. Stock solutions were prepared by dissolving 5 mg of the drug powder in 1 ml of solvent. Control wells included both no treatment controls and MTT controls.

Results and Discussion: Nano formulation based on Au/MSU-F was prepared using wet-impregnation technique. The cubic phase of Au was confirmed using XRD. The surface area analysis of Au/MSU-F indicated the presence of large surface area of 411 m2/g with pore size distribution of 8.29 nm. Sensor doped Au/MSU-F showed a passive insulin release of about 10.4% in 72 h.

The metformin profile showed a lower sustained release of about 1.8% in 72 h. The toxicity effect of nano formulation was assessed using human foreskin fibroblast (HFF) cells. It was visible that as the concentration of the nano formulation increases then that would very likely decrease the cell viability. At lower nano formulation dose (0.5µL), we observe a 100% cell viability, while increasing the dose (10µL), still 60% of the cell viability remained.

Conclusion: Developed an effective metformin/insulin drug delivery system based on mesocellular foam. The sensor doped nano formulation showed passive insulin and metformin drug release. The study shows that the nano formulation can further tuned to potentially treat type 2 diabetes.

IRMC-SRP-2023-29: Developing Multifunctional In-Situ ZnAgFe2O4/Silica Nanocomposite for Target Specific Cancer Therapy

Farooq Hassan Dandal^1,2, B. Rabindran Jermy¹*, and Dana Almohazey³

¹Department of Nano-medicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²Department of Biomedical Engineering, College of Engineering, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia^{, 3}Department of Stem Cell Research Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. *Email: rjermy@iau.edu.sa

Background: Colon cancer, or colorectal cancer, originates in the colon or rectum cells, posing a major oncology and public health concern. In Saudi Arabia, it constitutes 14.4% of cancer cases. Chemotherapy is a common cancer treatment, using potent drugs to eliminate fast-dividing cancer cells. Yet, it brings side effects such as toxicity, organ failure, and weakened immunity. Targeted cancer therapy, an alternative, employs nanocarriers to precisely attack cancer cells, reducing harm to healthy ones. Magnetic/Silica nanocomposite offer a multifunctional advantage guiding the nanocarrier towards cancer cells, minimize toxic to normal cells and reduce dose dependent side effects.

Objectives: This study aims to develop a novel spinel ferrite magnetic nanoparticles composed of ZnAgxFe(2-x)O4 (x=0.04) coated with silica for cisplatin loading, targeting colon cancer effectively. The prepared nanocarrier characteristics will be analyzed using various techniques (BET, XRD, SEM-EDX) to ascertain the spinel ferrite formation with Ag doping and effective silica coating and cisplatin functionalization. Additionally, drug release and anti-cancer activity were studied in vitro.

Methods: The magnetic ZnAgFe2O4 nanoparticles were synthesized through sonication using nitrate form of Zn, Ag and Fe precursor, followed by the addition of tetraethyl orthosilicate (silica source) and cetyltrimethylammonium ammonium bromide (CTAB) to form a nanocomposite with magnetic nanoparticles. The formation of mesopores was exploited for loading cisplatin, while the introduction of PEG aimed to enhance biocompatibility and pH sensitive drug release. The functionalized nano-formulation underwent comprehensive characterization using techniques such as SEM, EDX, XRD, surface analysis, and UV-visible spectroscopy. To evaluate drug release, dialysis membrane experiments mimicking tissue behavior was conducted using tumor acidic pH 6.6 at different intervals, coupled with UV analysis. Cancer cell studies employing the MTT assay showed that the synthesized nano-formulation induced color change in treated cells due to the addition of MTT, enabling quantification of viability through absorbance measurements.

Results and Discussion: XRD spectrum showed the presence of spinel ferrite characteristic peaks, which reveals that the nanoparticle has no impurity phase. SEM images of the magnetic nanoparticle revealed the intricate surface morphology and structure characteristics at high magnifications, with pure elemental compositions (Zn, Ag, Fe and O) and the arrangement of nanoscale feature. Also, EDX spectrum confirm that the nanoparticle is mainly composed of silver, zinc, iron, and oxygen with no other impurities. Surface analysis results showed the presence of mesoporous silica formation as nanocomposite with large BET surface area of 212 m2/g, total pore volume of 0.37 cm3/g with average pore diameter of 7.0 nm. Drug release study showed the sustained cisplatin release with percentage cumulative release of about 19% in 24 hours. MTT study revealed low cancerous cell viability of about 17% when treated with the developed cisplatin loaded magnetic/silica nanocomposite.

Conclusion: This study showed the development of a versatile nanocomposite for precise cancer therapy. Using a sonication technique, a novel ZnAgFe2O4 spinel ferrite nanoparticle was synthesized, and further modified with silica for magnetic/silica nanocomposite formation. Cisplatin and PEG were integrated in-situ, enhancing targeted drug delivery. This formulation displayed about 19% sustained cisplatin release in 24 h and exhibited low cancerous cell viability at higher concentrations. In conclusion, our developed nanocomposite holds promise as an effective and targeted approach for cancer therapy.

IRMC-SRP-2023-30: In-situ preparation of magnetic Spinel Ferrites ZnAgFeO4/Silica nanocomposite for Targeted Delivery of Tamoxifen Breast Cancer Therapy

Sajidah Jaffar Alalwan1^1,2, B. Rabindran Jermy¹*, and Dana Almohazey³

¹Department of Nanomedicine Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, ²College of Medicine, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia, ³Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, 31441, Dammam, Saudi Arabia. *Email: rjermy@iau.edu.sa

Background: Breast cancer (BC) is a significant global issue, with a 53% prevalence in Saudi Arabia. Tamoxifen (TAM) is a widely used selective estrogen modulator (SERM) in hormonal BC. However, TAM has conventional dose side effects, carcinogenic effects, and risk of TAM-mediated tumors. Moreover, magnetic and mesoporous nanoparticles have emerged as a potential solution to deliver TAM to tumors site, limiting its dose-dependent toxicity.

Objective: This study aims to investigate the potential of a multifunctional in-situ preparation of magnetic spinel ferrites/silica nanocomposite for targeted delivery of TAM against human BC cells (MCF7).

Methods: Our study was done by synthesizing ZnAgxFe(2-x)O4 (x=0.02) magnetic nanoparticles via sonication. The obtained nanoparticles were in-situ coated with mesoporous silica, followed by TAM drug functionalization along with PLGA addition to improve the biocompatibility. A drug release study was performed using the dialysis membrane technique at tumor acidic pH condition. Various characterization techniques such as SEM, XRD, EDX, BET, and UV-visible spectroscopy were used to characterize the nano formulation’s morphology, phase, texture and chemical state. Finally, anti-cancer activity was tested using in-vitro BC cells (MCF-7) and normal cells (HFF) via MTT assay.

Results and Discussion: The synthesis of ZnAgFeO4 spinel ferrites with no impurities was validated by XRD, and the existence of various elements, including Zn, Fe, Ag, and O was confirmed by SEM-EDX. SEM morphological analysis revealed aggregated microsphere particles of various sizes. The surface area measurement of nanoparticles indicated an average pore diameter of 5.25 nm, a total pore volume of 0.20 cm3/g, and a BET surface area of 151 m2/g. The results of the drug release study at a pH of 6.6 showed a passive drug release profile of about 9.5% cumulative TAM release in 24 h, indicating the need to perform the study at a longer duration. The MTT results of the nano formulation showed that MCF-7 cells viability of 46.9% at the highest dose of 0.5mg/mL. Similarly, at the same dose HFF cells had a viability of 46.8%. This indicates the cytotoxic effect of the nano formulation against MCF-7 cells. In case of TAM and PLGA alone, MCF-7 cells showed high viability with 62.8% and 82.8%, respectively. When treated with TAM and PLGA alone HFF cells showed 70.6% and 83.65% cell viability subsequently. The cytotoxic effect of nano formulation is higher than TAM indicating the effectiveness of targeted drug delivery.

Conclusion: We have successfully prepared TAM loaded ZnAgxFe(2-x)O4 (x=0.02)/silica nanocomposite. The silica nanocomposite has been characterized using different physio-chemical techniques. The nanocarrier has the cytotoxic effect against MCF-7 cells in dose dependent manner.