Periodic Reporting for period 1 - HyperABCD (Hyperaccumulator-grass-derived Antibacterial Boron-functionalized Carbon Dots: Synthesis and Cytotoxicological Evaluation)
Periodo di rendicontazione: 2023-07-01 al 2025-01-31
The excessive use of conventional antibiotic drugs cause selective pressure on microbial pathogenes, leading to the evolution of resistant strains. To help prevent expected outbreaks of drug-resistant bacteria, alternative strategies, thus, should be developed. Among the alternatives, metal-based antimicrobial agents, including but not limited to silver nanoparticles, showed a certain level of practical success both traditionally and in modern times. Nevertheless, they do not escape from microbial resistance and also suffer from considerable environmental and human toxicity problems. At this point, carbon-based nanomaterials attracted growing attention in the last decade with their performance and potentially low risk of resistance development. The initial works on fullerenes (zero-dimensional carbon "nanoballs") and carbon nanotubes, followed by carbon nanosheets (known as "graphenes"), laid the foundation. And one of the latest additions to the family of carbon nanomaterials, carbon dots (CDs), increased the expectations because of their small size, good solubility, and relative affordability. Still, CDs are at their infancy in terms of both synthesis and fundamental understanding as a materials group. Above all, their activity also seem relatively limited. Accordingly, there is urgent need to better understand the synthesis as well as biological evaluation of CDs. Going one step further, it is of particular interest to explore chemically doped (hetereatom-doped) CDs as they would exhibit higher potency. Past research focused on nitrogen doping as it is identified as an antimicrobial activity booster. While being relatively underexplored, the roles of nitrogen, oxygen, sulfur, and phosphorus functionalities have also been studied several times. Nevertheless, despite its growing utility in drug design, there is only a handful of studies on boron-doped CDs, exclusively using synthetic origin precursors.
Motivated by the background portrayed above and worsening sustainability concerns, HyperABCD program explored the synthesis and biological evaluation of boron-doped CDs. To enhance the potential environmental benefit, we opted for a renewable precursor (e.g. hyperaccumulator plant biomass) rather than a synthetic small molecules. Considering that hyperaccumulator plants can absorb and store "toxic" elements in their structure, we consider them an untapped potential for nanocarbon synthesis. Due to its wide availability accross Europe and boron-accumulating character, we selected Puccinella distans (also known as European alkaligrass) as the precursor. There has been no prior research on the utilization of a hyperaccumulator species in carbon dot synthesis. Therefore, our main objective was to successfully employ P. distans in synthesizing boron-doped CDs and evaluating the biological activity of those using model bacterial pathogens and mammalian cell cultures. By doing so, we aimed to pave the way for utilizing boron-doped hyperaccumulator plants in remediating contaminated soils in boron mining sites in the long term.
Motivated by the background portrayed above and worsening sustainability concerns, HyperABCD program explored the synthesis and biological evaluation of boron-doped CDs. To enhance the potential environmental benefit, we opted for a renewable precursor (e.g. hyperaccumulator plant biomass) rather than a synthetic small molecules. Considering that hyperaccumulator plants can absorb and store "toxic" elements in their structure, we consider them an untapped potential for nanocarbon synthesis. Due to its wide availability accross Europe and boron-accumulating character, we selected Puccinella distans (also known as European alkaligrass) as the precursor. There has been no prior research on the utilization of a hyperaccumulator species in carbon dot synthesis. Therefore, our main objective was to successfully employ P. distans in synthesizing boron-doped CDs and evaluating the biological activity of those using model bacterial pathogens and mammalian cell cultures. By doing so, we aimed to pave the way for utilizing boron-doped hyperaccumulator plants in remediating contaminated soils in boron mining sites in the long term.
Due to challenges associated with sourcing alkaligrass grown in boron mining sites, we have worked with Puccinella distans samples obtained from Limagne, France (boron-deficient soil compared to boron mining sites). We have carefully dissected several bunches of plants into their main anatomical structures (briefly, the flower, leaf, and root systems). After discarding the flower systems, roots (soil-contaminated), most exposed outer blades, we grinded the rest of the plant into a powder. Using this powder, we studied a wide range of hydrothermal treatment conditions, varying the temperature and precursor/dopant concentrations (dopant/digester: boric acid and/or sulfuric acid). We purified all samples and conducted comprehensive materials characterizations employing several thermal, spectroscopic, as well as microscopic methods. We achieved brightly fluorescent sub-20-nm particles, which are considered considered CDs. Using a model bacterial pathogen, Escherichia coli, we have observed that boron-doped CDs exhibit promising antibacterial activity. Nevertheless, given the product yield was always too low for further cell-material interaction tests (mammalian cell toxicity assays), we put intense efforts to produce desired CDs at a substantial amount, which turned out to be possible via microwave treatment under certain conditions. In these successful studies with higher yields, we have employed pre-carbonized biomass and co-digester acids, yielding smaller CDs (compared to hydrothermal route). As a result, for the first time, we achieved hyperaccumulator-plant-derived CDs, which are promising as antimicrobial agents.
To the best of our knowledge, hyperaccumulator plants have not been employed for CD synthesis before. We have successfully produced sub-20-nm fluorescent particles with or without boron doping, and boron-doped CDs showed promising antibacterial activity in the preliminary studies. Our research showed that the most common method, hydrothermal carbonization, unexpectedly underperforms in obtaining the desired CDs at a practically reasonable yield. However, we have developed an alternative way, taking advantage of low-heat pre-carbonization and microwave digestion. The developed method is likely to be scalable, which will be further explored collaborating with the industrial project partner of HyperABCD. To ensure the impact of next steps, it is desirable to work with hyperaccumulators grown on boron-rich soil, which will be subjected to the final approval of the government-owned mining company in Türkiye, the largest boron producer of the world.