Capstone Project
Title: Synthesis, characterization and biological study of water-soluble Silver Pyrazolates
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Introduction integrating Global Experiences
The emergence in antimicrobial resistance towards current available antibiotics has endangered the ability to prevent and treat a wide variety of infections, prompting the research efforts towards finding alternatives. Antibiotic resistance occurs when bacteria or fungi gain the ability to defeat drugs aimed to kill them. In other words, the infection causing agent could persist even upon antibiotic intervention. This is a global public health threat that may impact the lives of many individuals in developing and developed nations. As per the CDC, in the United States alone, at least 2.8 million people were infected with antibiotic resistant bacteria or fungi, and 35,000 fatalities resulted from it. In developing nations around the world, if you add factors like the lack of surveillance of resistance development, water quality, food scarcity, and poor-quality control of available antibiotics, the impacts of antibiotic resistance are devastating (Chokshi et al., 2019). In fact, antibiotic resistant diseases like tuberculosis (TB) has already impacted developing countries (Chokshi et al., 2019). It was reported in 2013, that there were around 480,000 cases of multidrug-resistant TB in countries like India and South Africa (Chokshi et al., 2019). Key factors like socioeconomic and political factors, may also contribute to the rise of antibacterial resistance which demonstrates many factors play a role in the development of this public health crisis. Experts predict that antimicrobial-resistant infections would continue to rise, ultimately leading to nearly 10 million deaths per year by 2050 (Chokshi et al., 2019). This is the reason our research efforts would not only impact the scientific community, but global public health as a whole.
Figure 1: Predicting the deaths attributed to antibiotic resistance by 2050.
Figure 2: As per the CDC, this is a simplified way to demonstrate how antibiotic resistance occurs.
In our research, we focused on the antibacterial effect of silver ions (Ag+), which its use to treat wounds, stomach pain, and even sterilize water dates back to the ancient Greek era (Barras et al., 2018). It is also believed that the king of Persia, when going to war, would take containers made of silver to disinfect the water. With that said, the first modern description of Silver’s antibacterial properties was given by Raulin in 1869, in which he observed that Aspergillus niger (fungus) would not grow in a silver flask (Clement & Jarrett, 1994). This scientist described silver as oligodynamic because with just small concentrations, it had a powerful way to eradicate microbes and fungi. Therefore, in our research lab we decided that silver was a compound that needed further investigation and would be a great start in trying to solve antibiotic resistance.
As it has been shown, silver’s antibacterial properties have long been known and its toxicity to human cells is considerably lower than bacteria itself (Clement & Jarrett, 1994). Prophylactic treatment of burn wounds has been highly documented to prevent Pseudomonas aeruginosa (gram negative bacterium). It should be noted that Silver binds to many cellular components, specifically membrane components instead of nucleic acids. This strong binding could be one of the important features that gives silver this oligodynamic property, yet the exact mechanism of action is still unknown. Nonetheless, in our experimental design, we aimed to further study silver complexed with certain ligands and determine it antibacterial efficacy.
Figure 4: Silver plated with bacterium showing growth inhibition of bacterium.
Figure 3: Silver flask utilized by the ancient greek era to disinfect water.
Figure 5: Silver (1) has multiple mechanisms to eradicate bacteriums and fungi.
Research Project Reflection
During my first year in college in my honors introduction course, I was introduced to the annual undergraduate research conference at FIU. I attended the conference with hopes to learn and figure out what research lab I wanted to join. At the end of the conference I decided I wanted to join a biochemical research lab that had some application to medicine. After months emailing and trying to join research labs of interest, I was given the opportunity to join Dr. Raptis research lab in the department of chemistry and biochemistry. I was then assigned a project on Silver pyrazolato compounds to fight antibiotic resistance with a graduate student. In the lab, everything began with synthesizing the compounds, which was the fundamental part of our study. Throughout my time in the lab, I started to witness how my knowledge learned in my courses like general chemistry and organic chemistry could be applied at the research level. I learned how to use mathematical computations, utilize concepts of solubility in the lab, and even utilize spectroscopic techniques. In addition, I learned to utilize X-ray diffractometer to characterize molecular structures in conjunction with Infrared spectroscopy to study and identify chemical functional groups along with their locations. After several months, my team and I had been trying to synthesize, characterize, and conduct antibacterial studies on Silver Pyrazolates with minor success. Yet, after each trial, I sat with my mentor to reflect and adapt our procedures. Research is a slow process and within it there is lots of failure built in. I learned that to find success in research, you must adapt procedures and persist, eventually hard work will pay off with good results. It was trial 359 that led us to our first successful synthesis, which initiated us to go in the right direction. My team and I felt very accomplished in what we had just done. This research fueled my innate desire to learn as much as I could and take advantage of every situation to learn from my own mistakes. This growth mindset will allow me to continue persevering and finding success through the rigors of a medical education in my future.
After months of data analysis and repeating trials routinely, my team and I were ecstatic to share our results with the scientific community. I led our team at the annual undergraduate research conference (CURFIU 2019), guiding inquiring professionals through our project. I discussed the effectiveness of our compounds against bacteria commonly found in burn wound infections. We were approached with difficult questions that revealed more work still had to be done. However, our project was admired by many, specifically by a professor who had been a victim of a burn infection. I quickly realized the impact our research can have universally and were humbled to have received a certificate of recognition. As a researcher, I still have much room for improvement in analyzing work and integrating concepts learned in the classroom to the research side. Nevertheless, through my undergraduate research experience, I have learned to be patient, collect and analyze spectroscopic data, collaborate with a research team, and report data in writing. The next chapter of my life would take place in Herbert Wertheim College of medicine, where I aim to also get involved in research and continue to build on the skills I have gained as an undergraduate.
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Research Project Summary
Silver (1) [Ag(I)] has multiple mechanisms of eradicating microbes, making it a great alternative to currently used antibiotics. In this research, we aim to prepare, characterize and assess the antimicrobial efficacy of a family of water soluble silver pyrazolato complexes as effective antimicrobial agents. Four new water soluble silver pyrazolato complexes, namely [Ag2(-4-Cl-pz)2(PTA)4] (1), [Ag2(4-Cl-pz)2(PTA)2] (2), [Ag2(4-CH3-pz)2(PTA)2] (3) and [Ag2(3-CH3-pz)2(PTA)2] (4) (where, PTA = 1, 3, 5-triaza-7-phosphaadamantane) were synthesized and isolated as white crystalline solids. The two step synthesis involves the formation of polymeric [Ag(pz*)]n species, followed by the addition of PTA in varied molar ratio. PTA has been chosen for two reasons: i) to impart aqueous solubility of the complexes, which is crucial for their physiological acceptability and ii) the lipophilic nature of the adamantyl moiety was also expected to facilitate the cellular internalization of these complexes. Certain pyrazole derivatives are part of many NSAIDs (Non-steroidal Anti inflammatory drugs), while PTA is also found to be biocompatible and constitute a crucial part in few anticancer drugs that are currently in Phase II clinical trials. Molecular structures of all complexes reported herein were authenticated by single crystal X-ray crystallography. All the complexes have also been characterized by 1H and 31P NMR spectroscopy. A qualitative antibacterial assay with a soft skin and tissue infection (SSTI) model (in Agar plate) indicated superior growth inhibition for the colony of a notorious nosocomial Gram-negative bacterial strain, namely, Pseudomonas aeruginosa (ubiquitous within burn wound infections). The efficacy of growth inhibition of our trials has been found to be approximately four times superior compared to AgNO3, a known antibacterial used for burn wound infections in current hospital settings.
Research Poster presented at CURFIU 2019
Figure 6: Dr. Raptis Research team. We thank Florida International University for the infrastructure facilities. Special thanks to Dr. Suparna Dhar and Ms. Jenny Stenger-Smith for collaborating with us for the antibacterial studies.
Works Cited:
Barras, F., Aussel, L., & Ezraty, B. (2018). Silver and Antibiotic, New Facts to an Old Story. Antibiotics (Basel, Switzerland), 7(3), 79. https://doi.org/10.3390/antibiotics7030079
Clement, J. L., & Jarrett, P. S. (1994). Antibacterial silver. Metal-based drugs, 1(5-6), 467–482. https://doi.org/10.1155/MBD.1994.467
Chokshi, A., Sifri, Z., Cennimo, D., & Horng, H. (2019). Global Contributors to Antibiotic Resistance. Journal of global infectious diseases, 11(1), 36–42. https://doi.org/10.4103/jgid.jgid_110_18