Feasibility
During the initial project planning phase, we conducted a systematic feasibility assessment of the experiments, with particular attention to time constraints and resource availability. Undergraduate students face significant limitations in accessing laboratory instruments, equipment, and materials. Certain precision instruments must be borrowed or leased, while key reagents for nanozyme assembly require specialized procurement channels. Furthermore, this research integrates multiple disciplines, such as nano-synthetic technology and microbiology, necessitating substantial time investment by students to acquire foundational knowledge and master experimental techniques. Given their relatively limited hands-on experience, additional time must be allocated for iterative experimentation to refine and optimize procedural protocols.
In light of these considerations, this project will focus on the design of antibiotic nanozymes to investigate their potential in reversing common drug resistance mechanisms in most bacterial species. The primary objective is to develop a functionally effective nanozyme system by streamlining experimental procedures and minimizing technical complexity, thereby improving the overall feasibility of the approach. Previous studies have demonstrated that constructing antibiotic nanozyme composites through molecular self-assembly under laboratory conditions is highly feasible. This method allows for the directional immobilization of antibiotic molecules on the nanozyme surface, resulting in synergistic antibacterial complexes. Moreover, the axial coordination strategy employing heme as the catalytic center has been successfully implemented in multiple nanozyme systems, exhibiting peroxidase-like catalytic efficiency comparable to, and in some cases exceeding, that of natural enzymes.
Prior to initiating formal experimentation, it is imperative to finalize the integration strategy between antibiotics and nanozymes and to complete the design of the hemin-based catalytic system. Hemin-based nanozymes are particularly promising due to their intrinsic biocompatibility and multifunctional enzyme-mimicking activities, including peroxidase (POD), oxidase (OXD), and catalase (CAT) functionalities, which are critically involved in regulating cellular redox homeostasis. The design finalization process requires a comprehensive review of relevant literature in the fields of nanomaterials, synthetic assembly methods, microbiology, and biochemistry, along with in-depth consultations with the faculty advisor. This rigorous approach is essential to ensure both the originality of the proposed design and the structural optimization of the nanozyme, although it inherently demands a significant investment of time.
All preparatory work is scheduled to be completed within a two-week period prior to the main experimental phase. This includes three key aspects:
1. Procurement and Logistics:
Key reagents, including gentamicin, fluconazole, and hemin, have been confirmed to be commercially available and of a purity grade that meets experimental specifications.
2. Instrumental Access:
Essential equipment for characterization, such as advanced spectrometers, electron microscopes, and confocal microscopes, will be accessed via the shared instrumentation platforms at our university and affiliated research institutions.
3. Methodology Finalization:
Detailed experimental protocols for each step, from nanozyme synthesis to the assessment of microbial resistance reversal, will be finalized.
A team of six researchers will undertake the experimental work, with the following assigned responsibilities: two members will focus on the assembly and synthesis of antibiotic nanozymes; two will conduct microbiological experiments; and the remaining two will investigate the mechanisms underlying drug resistance reversal. All personnel will collaborate closely under the guidance of the faculty advisors, participating collectively in experimental design, preparation, and follow-up studies. Chemical and microbiological experiments will be conducted in parallel to ensure efficient progress.
The first 30 days will be dedicated to the synthesis of antibiotic nanozymes and the assessment of their bactericidal and bacteriostatic activities. The following 30 days will concentrate on investigating the mechanisms through which these nanozymes counteract drug resistance, with particular emphasis on biofilm degradation and modulation of redox states, aiming to elucidate their specific mode of action and perform biosafety evaluations. Biofilms are a major contributor to persistent infections and antimicrobial resistance, serving as a significant barrier to effective therapeutic intervention; thus, their disruption is essential for enhancing antimicrobial efficacy.
This phase will be carried out by two researchers over a period of two to three weeks, under the supervision of the faculty advisors and informed by a systematic review of relevant literature.
1.1 Assembly Process Optimization
Preliminary trials will determine optimal synthesis conditions and component ratios. Each synthetic cycle requires approximately three days, with three to four cycles (approx. 12 days total) required to obtain structurally sound antibiotic nanozymes in sufficient quantity.
1.2 Structural and Chemical Characterization
The synthesized nanozymes will be comprehensively characterized by techniques including transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS). Acquiring high-quality TEM images necessitates careful sample preparation, encompassing quality control, concentration adjustment, and negative staining. This typically requires 5–6 independent trials, with the entire process taking approximately five days.
This stage focuses on microbial cultivation and assessment of the antibacterial activity of antibiotic nanozymes. Multiple replicates will be performed to ensure statistical reliability.
2.1 Microbial Cultivation
Strains will be reactivated and cultured to the logarithmic phase, typically in three-day cycles (longer for fungi), prior to experimentation.
2.2 Efficacy Assessment
Antibacterial assays will be conducted over 3–5 days per cycle. Five independent cycles will be performed, totaling approximately 20 days.
This phase integrates multiple experimental approaches, including enzymatic activity tests, biofilm disruption studies, and CCK-8 biosafety assays. A key focus is the bacterial resistance mechanism involving intracellular hydrogen sulfide-mediated redox regulation and ROS mitigation. Glutathione (GSH) depletion assays will also be performed, given its critical role in biofilm formation, alongside systematic investigation of the nanozymes’ bactericidal mechanisms.
3.1 Model Establishment and Experimental Design
Relevant experimental models will be established in 3-4 days, followed by 2 days for detailed experimental planning.
3.2 Replication and Mechanistic Analysis
Experiments will be repeated in five rounds, each lasting 3-4 days. Data will be analyzed to elucidate resistance reversal mechanisms, with scheduling optimized to ensure reliability and accuracy.
3.3 Biosafety Verification
Relevant cell cultures will be prepared (approx. 5 days per cycle) in advance. Safety assays (2-4 days per cycle) will be conducted in triplicate, totaling about 10 days.