Multidrug-resistant bacterial infections are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics. Metal nanoparticles can induce cell death, yet the toxicity effect is typically nonspecific. Here, we show that photoexcited quantum dots (QDs) can kill a wide range of multidrug-resistant bacterial clinical isolates, including methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Salmonella typhimurium. The killing effect is independent of material and controlled by the redox potentials of the photogenerated charge carriers, which selectively alter the cellular redox state. We also show that the QDs can be tailored to kill 92% of bacterial cells in a monoculture, and in a co-culture of E. coli and HEK 293T cells, while leaving the mammalian cells intact, or to increase bacterial proliferation. Photoexcited QDs could be used in the study of the effect of redox states on living systems, and lead to clinical phototherapy for the treatment of infections.

Engineered nanoparticle for controlled superoxide flux potentiates antibiotics in MDR clinical isolates.

An important goal of synthetic biology involves the extension and standardization of novel biological elements for applications in medicine and biotechnology. Transcriptional interference, occurring in sets of convergent promoters, offers a promising mechanism for building elements for the design of tunable gene regulation. Here, we investigate the transcriptional interference mechanisms of antisense roadblock and RNA polymerase traffic in a set of convergent promoters as novel modules for synthetic biology. We show examples of elements, including antisense roadblock, relative promoter strengths, interpromoter distance, and sequence content that can be tuned to give rise to repressive as well as cooperative behaviors, therefore resulting in distinct gene expression patterns. Our approach will be useful toward engineering new biological devices and will bring new insights to naturally occurring cis-antisense systems. Therefore, we are reporting a new biological tool that can be used for synthetic biology.

The devastating effects of the coronavirus disease 2019 (COVID-19) pandemic have made clear a global necessity for antiviral strategies. Most fatalities associated with infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) result at least partially from uncontrolled host immune response. Here, we use an antisense compound targeting a previously identified microRNA (miRNA) linked to severe cases of COVID-19. The compound binds specifically to the miRNA in question, miR-2392, which is produced by human cells in several disease states. The safety and biodistribution of this compound were tested in a mouse model via intranasal, intraperitoneal, and intravenous administration. The compound did not cause any toxic responses in mice based on measured parameters, including body weight, serum biomarkers for inflammation, and organ histopathology. No immunogenicity from the compound was observed with any administration route. Intranasal administration resulted in excellent and rapid biodistribution to the lungs, the main site of infection for SARS-CoV-2. Pharmacokinetic and biodistribution studies reveal delivery to different organs, including lungs, liver, kidneys, and spleen. The compound was largely cleared through the kidneys and excreted via the urine, with no accumulation observed in first-pass organs. The compound is concluded to be a safe potential antiviral treatment for COVID-19.

Reactive oxygen species (ROS) represent a broad range of chemical species including superoxide, hydroxyl, singlet oxygen, and hydrogen peroxide. Each species behaves differently in the cellular environment. Some can play specific roles as intracellular signaling molecules, while others act primarily as indiscriminate oxidants. Several recent reports have promoted the use of exogenous ROS as therapeutic agents with applications from cancer therapies to novel antimicrobials. However, therapeutics, specifically antibiotics, should either kill or inhibit the growth of harmful cells (bacteria here) without harming the host cells, and hence selectivity of action is of vital importance. Here, we show that among different ROS, only superoxide was found to be bactericidal, killing a range of multidrug-resistant (MDR) pathogens without affecting the viability or growth of mammalian cells. Superoxide has a high thermodynamic capacity to be a strong oxidant. However, its lack of reactivity with cellular components at a physiological pH, except for the inactivation of biosynthetic enzymes containing labile iron−sulfur clusters, is key to its selectivity. The role of iron in bacterial pathogenesis also makes superoxide a strong candidate for antimicrobial therapy. Additionally, using a series of selective scavengers, we show that the superoxide radical is therapeutically effective and selective compared to other ROS like hydroxyl radicals, confirming previous results that used Escherichia coli gene knockouts to show that superoxide selectively deactivates some enzymes rather than causing indiscriminate damage of cellular components. In our in vitro studies, intracellular superoxide generation using light-activated quantum dots yielded highly selective and effective antimicrobial action. We screened 45 clinical MDR bacterial isolates and observed inhibition/therapeutic action in all strains, highlighting the applicability of such nanoparticle superoxide therapy. These results can pave the way for rational design of nanoscale therapies as precision medicine.

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The rapid emergence of superbugs, or multi-drug resistant (MDR) organisms, has prompted a search for novel antibiotics, beyond traditional small-molecule therapies. Nanotherapeutics are being investigated as alternatives, and recently superoxide-generating quantum dots (QDs) have been shown as important candidates for selective light-activated therapy, while also potentiating existing antibiotics against MDR superbugs. Their therapeutic action is selective, can be tailored by simply changing their quantum-confined conduction-valence band (CB-VB) positions and alignment with different redox half-reactions—and hence their ability to generate specific radical species in biological media. Here, we show the design of superoxide-generating QDs using optimal QD material and size well-matched to superoxide redox potential, charged ligands to modulate their uptake in cells and selective redox interventions, and core/shell structures to improve their stability for therapeutic action. We show that cadmium telluride (CdTe) QDs with conduction band (CB) position at −0.5 V with respect to Normal Hydrogen Electron (NHE) and visible 2.4 eV bandgap generate a large flux of selective superoxide radicals, thereby demonstrating the effective light-activated therapy. Although the positively charged QDs demonstrate large cellular uptake, they bind indiscriminately to cell surfaces and cause non-selective cell death, while negatively charged and zwitterionic QD ligands reduce the uptake and allow selective therapeutic action via interaction with redox species. The stability of designed QDs in biologically-relevant media increases with the formation of core-shell QD structures, but an appropriate design of core-shell structures is needed to minimize any reduction in charge injection efficiency to adsorbed oxygen molecules (to form superoxide) and maintain similar quantitative generation of tailored redox species, as measured using electron paramagnetic resonance (EPR) spectroscopy and electrochemical impedance spectroscopy (EIS). Using these findings, we demonstrate the rational design of QDs as selective therapeutic to kill more than 99% of a priority class I pathogen, thus providing an effective therapy against MDR superbugs.

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase Klebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae, and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST platform offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells.