Functionalized nanoclusters and their use in the treatment of bacterial infections - Patents.com

Abstract

Compositions, methods, and kits are provided for treating bacterial infections with nanoclusters comprising metallic cores conjugated to nucleotides. Refractory infections are often difficult to treat due to the presence of viable cells, a subpopulation of bacterial cells that are highly resistant to conventional antibiotics. The viable cells are dormant, which makes them less susceptible to many antibiotics designed to kill proliferating cells. Administration of nanoclusters comprising nucleotides has been found to be highly effective in eradicating viable cells and in treating infections for a wide range of bacterial species, including gram-positive and gram-negative bacteria. Such treatments have been effective not only in eradicating planktonic bacteria, but also bacteria in biofilms.

Description

[Background technology]

[0001] Antibiotics are the mainstay of modern clinical medicine. However, bacteria develop resistance to both natural and synthetic antibiotics within a few years of their first clinical use (Walsh (2003) Nature Reviews Microbiology 1:65-70). Current mechanisms of antibiotic resistance include reduced uptake through changes in outer membrane permeability, excretion of antibiotics through activation of efflux pump proteins, enzymatic modification of antibiotics, modification of antibiotic targets, and bacterial physiology such as biofilms (van Hoek et al. (2011) Front Microbiol 2:203).

[0002] In the United States and Europe alone, over 50,000 people die each year from resistant infections (The Review on Antimicrobial Resistance. Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations (2014), amr-review.org / Publications.html). Length of stay in hospitals is increased by antibiotic-resistant infections, and these same infections are often acquired in hospitals. The economic impact of antibiotic-resistant infections is estimated to be between US$5 billion and US$24 billion per year in the United States alone (Hall (2004) Nature Reviews Microbiology 2:430-435). However, pharmaceutical companies' drug pipelines have not kept pace with the evolution of antibiotic resistance. In 2004, only 1.5% of all drugs under development by the world's 15 largest pharmaceutical companies were antibiotics (Smith and Coast, "The economic burden of antimicrobial resistance: why it is more serious than current studies suggest" (2012), researchgate.net / publication / 291413454). The new reality we must face is that pharmaceutical companies are not currently lining up to discover new antibiotics. A strategy to protect our existing antibiotics is through the use of antibiotic adjuvants, compounds that enhance the activity of current drugs and minimize or even directly block resistance (Lu et al. (2009) Proc. Natl. Acad. Sci. USA 106(12):4629-4634, Gonzalez-Bello (2017) Bioorg. Med. Chem. Lett. 27(18):4221-4228). Another strategy is the use of antivirulence agents.These drugs can circumvent antibiotic resistance by disarming pathogens of virulence factors that drive human disease, but remain bacterial growth pathways (Dickey et al. (2017) Nat. Rev. Drug Discov. 16(7):457-471).

[0003] Bacterial cells attached to a surface can aggregate with each other to form a biofilm. Bacteria growing biofilms can show increased tolerance to antimicrobial agents that are very difficult or substantially reduced to remove. Biofilm bacteria have two dormant phenotypes: a viable but non-culturable (VBNC) state and a viable state. The dormant phenotypes (VBNC and viable) allow bacteria to survive conditions that are lethal to the rest of their genetically identical lineage. Once in a biofilm, they can evade the immune system. Thus, one of the main roles of biofilms is to provide a protective habitat for viable and VBNC by shielding them from the immune system (Lewis (2010) Microbe (Washington, DC) 5 (10): 429-437). Another property of biofilms is their ability to be more resistant to antimicrobial agents than planktonic cells (Spoering et al. (2001) J. Bacteriol. 183(23):6746-6751). Thus, there is an ongoing unmet need for improved approaches to treat antibiotic-resistant infections. Summary of the Invention

[0004] Compositions, methods, and kits are provided for treating bacterial infections with nanoclusters comprising metallic cores conjugated to nucleotides. Refractory infections are often difficult to treat due to the presence of viable cells, a subpopulation of bacterial cells that are highly resistant to conventional antibiotics. The viable cells are dormant, which makes them less susceptible to many antibiotics designed to kill proliferating cells. Administration of nanoclusters comprising nucleotides has been found to be highly effective in eradicating viable cells and in treating infections for a wide range of bacterial species, including gram-positive and gram-negative bacteria. Such treatments have been effective not only in eradicating planktonic bacteria, but also bacteria in biofilms.

[0005] In one aspect, a nanocluster is provided that comprises a metallic core conjugated to a nucleotide. In some embodiments, the metallic core comprises a noble metal. In some embodiments, the nanocluster comprises a gold metallic core. In some embodiments, the nanocluster is biocompatible with human cells.

[0006] In certain embodiments, the nucleotide is adenosine triphosphate (ATP) or its phosphorothioate analog, deoxyribonucleotide analog, 7-deazapurine nucleotide analog, or phosphomethylphosphonate adenylate ester. Exemplary phosphorothioate analogs include, but are not limited to, ATPαS, ATPβS, or ATPγS. Exemplary deoxyribonucleotide analogs include, but are not limited to, deoxyadenosine triphosphate (dATP). Exemplary 7-deazapurine nucleotide analogs include, but are not limited to, 7-deaza-adenosine-5′-triphosphate (7-deaza-ATP). Exemplary phosphomethylphosphonate adenylate analogs include, but are not limited to, β,γ-methylene adenosine 5′-triphosphate (AMP-PCP). Antimicrobial activity can be enhanced by high temperature synthesis (e.g., at about 100° C.).

[0007] In certain embodiments, the nanoclusters have a median diameter distribution ranging from about 1 nm to about 10 nm, including any diameter within this range, such as 0.5 nm, 0.75 nm, 1 nm, 1.25 nm, 1.5 nm, 1.75 nm, 2 nm, 2.25 nm, 2.5 nm, 2.75 nm, 3 nm, 3.25 nm, 3.5 nm, 3.75 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8 nm, 8.5 nm, 9 nm, 9.5 nm, or 10 nm. In some embodiments, the nanoclusters have a diameter of less than 4 nm. In some embodiments, the nanoclusters have a diameter of about 1 nm to about 2 nm.

[0008] In certain embodiments, the nanocluster is linked to an internalization sequence, a protein transduction domain, or a cell penetrating peptide.

[0009] In another aspect, there is provided a composition comprising the nanoclusters described herein for use in a method of treating an infectious disease. In certain embodiments, the composition further comprises a pharma- ceutically acceptable excipient or carrier. In some embodiments, the infectious disease is a bacterial infection, such as, but not limited to, a Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, or Escherichia coli infection.

[0010] In certain embodiments, the composition further comprises an antibiotic. Exemplary antibiotics include, but are not limited to, fluoroquinolones, aminoglycosides, penicillins, tetacyclines, cephalosporins, macrolides, sulfonamides, carbapenems, ansamycins, carbacephems, carbapenems, lincosamides, monobactams, and oxazolidinones. For example, the antibiotic may include fluoroquinolones, such as ofloxacin, moxifloxacin, ciprofloxacin, gemifloxacin, levofloxacin, or finafloxacin, or derivatives thereof.

[0011] In another aspect, methods are provided for treating an infection in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising a nanocluster as described herein, hi some embodiments, the method further comprises administering a therapeutically effective amount of at least one antibiotic in combination with the composition comprising the nanocluster.

[0012] Exemplary antibiotics include, but are not limited to, fluoroquinolones, aminoglycosides, penicillins, tetacyclines, cephalosporins, macrolides, sulfonamides, carbapenems, ansamycins, carbacephems, carbapenems, lincosamides, monobactams, and oxazolidinones. For example, the antibiotic may include a fluoroquinolone, such as ofloxacin or a derivative thereof.

[0013] In certain embodiments, the subject has a chronic infection. In some embodiments, the subject has an infection, including, but not limited to, an ear infection, a skin infection, a lung infection, chronic suppurative otitis media (CSOM), an infection associated with cystic fibrosis, tuberculosis, or an infection in a wound. In some embodiments, the infection is associated with the formation of a bacterial biofilm in the subject. In certain embodiments, the infection comprises a pathogenic bacterium that is resistant to one or more antibiotics. In some embodiments, the subject has previously been treated for an infection with one or more antibiotics that have not been successful in clearing the infection. In another embodiment, the infection is an infection (e.g., Pseudomonas) in a subject with cystic fibrosis.

[0014] In certain embodiments, the treatment eradicates all or most of the biofilm and planktonic bacteria. In some embodiments, the treatment eradicates all or most of the viable cells, which may be, for example, in a biofilm or internalized by a macrophage. In some embodiments, the viable cells eradicated by the treatments described herein are multi-drug resistant viable cells. The treatment may eradicate viable cells, including either gram-negative or gram-positive bacteria, including, but not limited to, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, or Escherichia coli viable cells.

[0015] In certain embodiments, multiple cycles of treatment are administered to a subject. For example, the nanoclusters described herein can be administered alone or in combination with antibiotics, either intermittently or according to a daily dosing regimen.

[0016] The composition comprising the nanoclusters may be administered by any suitable mode of administration. For example, the composition may be administered intravenously, subcutaneously, by inhalation, or topically. Alternatively, the composition may be administered locally to the site of the infected tissue. For example, in an ear infection, the composition comprising the nanoclusters may be administered locally into the ear canal.

[0017] In another embodiment, a method of eradicating bacteria in a biofilm is provided, comprising contacting the biofilm with an effective amount of a composition comprising the nanoclusters described herein. In some embodiments, the method further comprises contacting the biofilm with an effective amount of at least one antibiotic. The methods described herein can be used to eradicate bacteria in biofilms, for example, on medical devices, personal hygiene products, toiletries, cosmetics, disinfectants, cleaning solutions, or in water treatment or distribution systems.

[0018] In another embodiment, a method of eradicating dormant bacteria, including viable cells, is provided, comprising contacting the dormant bacteria with an effective amount of a composition comprising a nanocluster as described herein. In some embodiments, the method further comprises contacting the dormant bacteria with an effective amount of at least one antibiotic. The dormant bacteria may be present, for example, in a biofilm, in a liquid culture, or on an inanimate surface.

[0019] In another embodiment, a method of inhibiting a virulence factor of a bacterium is provided comprising contacting the bacterium with an effective amount of a composition comprising a nanocluster as described herein. In some embodiments, the bacterium is selected from the group consisting of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. In some embodiments, the virulence factor is Pseudomonas aeruginosa pyocyanin (PYO).

[0020] In another aspect, a kit is provided comprising a nanocluster as described herein and instructions for treating a bacterial infection, hi some embodiments, the kit further comprises an antibiotic.

[0021] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings, in which: It is emphasized that, according to common practice, the various features of the drawings are not drawn to scale. Conversely, dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures: [Brief description of the drawings]

[0022] [Figure 1A] Characterization of adenosine triphosphate-coated gold nanoclusters (AuNC@ATP). Schematic diagram of AuNC@ATP, photograph of solution of synthesized AuNC@ATP were analyzed using UV-Vis spectroscopy to demonstrate the absence of plasmon resonance band at 520 nm. [Figure 1B]Transmission electron microscope (TEM) image of AuNC@ATP. Magnified view of the photo and scale bar (5 nm). [Figure 1C] The particle distribution of AuNC@ATP was measured by TEM. [Figure 2A] Exposure to AuNC@ATP results in the activation of stress that disrupts the outer membrane (OM) and cytoplasmic membrane permeability (CM). First, stationary-phase cultures of Gram-negative bacteria were exposed to AuNC@ATP and colistin (positive control). Then, the permeability of the OM and IM was assessed by measuring the fluorescence of (Figure 2A) 8-anilino-1-naphthalenesulfonic acid (ANS) and (Figure 2B) propidium iodide (PI), respectively. ANS is a compound that changes fluorescence depending on the polarity of its surrounding environment. In the presence of intact Gram-negative bacterial cells in an aqueous environment, ANS fluoresces weakly. If the OM is disrupted, ANS can still penetrate the non-polar phospholipid bilayer, resulting in a measurable increase in fluorescence. PI is a membrane-impermeable DNA dye that can only label bacteria with compromised CM. If the CM is disrupted, PI can still penetrate the CM and bind to DNA, resulting in a measurable increase in fluorescence. [Figure 2B] The permeability of the OM and IM was assessed by measuring propidium iodide (PI) fluorescence. [Figure 3A] AuNC@ATP kills growth-arrested Gram-negative bacteria without causing bacterial cell lysis. Stationary-phase cultures of Gram-negative bacteria resuspended in phosphate-buffered saline (PBS) and exposed to either AuNC@ATP or ofloxacin for 4 h. After treatment, the drug was removed and the number of surviving bacteria was assessed by measuring colony forming units per milliliter (CFU / mL). [Figure 3B]AuNC@ATP-mediated nonlytic cell death of stationary-phase cultures of Gram-negative bacteria. After treatment of stationary-phase cultures of Gram-negative bacteria with PBS, AuNC@ATP (16.8 μM), ofloxacin (8.3 μM), and colistin (1.3 mM), the presence of protein in the collected supernatants of each treatment was evaluated by using the Pierce BCA Protein Assay kit. [Figure 4A] Accumulation of unfolded outer membrane proteins (OMPs) causes the lethality of AuNC@ATP. Inhibition of growth of P. aeruginosa (PA14) and its genetic mutant with ClpXP protease gene deletion (ΔClpXP) incubated with AuNC@ATP at different concentrations. Growth of P. aeruginosa in lysogen broth (LB) was assessed by measuring the optical density at 600 nm (OD600nm) (N=3). [Figure 4B] Inhibition of growth of P. aeruginosa (PA14) and its genetic mutant carrying gene deletion of ClpXP protease (ΔClpXP) incubated with AuNC@ATP at different concentrations. [Figure 4C] Schematic diagram showing that AuNC@ATP exert their antibacterial activity mainly by inducing multiple disruptive stresses that lead to the accumulation of toxic unfolded OMPs in the periplasmic space. [Figure 5A] Viable cells are more sensitive to AuNC@ATP than metabolically active bacterial cells. Schematic showing the isolation of viable cells from a stationary-phase culture of P. aeruginosa (PA14) using ofloxacin. [Figure 5B] ATP levels were measured in isolated viable cells and in exponentially growing PA14. [Figure 5C]Surviving cells and exponentially growing PA14 were resuspended in phosphate-buffered saline (PBS) containing AuNC@ATP (without carbon source). After AuNC@ATP treatment, the number of surviving bacteria was assessed by measuring colony forming units per milliliter (CFU / mL). Dose-response curves were generated by plating cells in AuNC@ATP treatment at different concentrations (N=3). [Figure 6A] P. aeruginosa cannot produce pyocyanin in the presence of sublethal doses of AuNC@ATP. Pyocyanin production by P. aeruginosa (PA14) in lysogeny broth (LB) containing AuNC@ATP (N=3). The inset shows the chemical structure of pyocyanin. After centrifugation, pyocyanin was collected and the optical density at 520 nm (OD520nm) was measured. The pyocyanin concentration was determined by multiplying the OD520 value by 17.072, and the results were expressed in μg / mL. [Figure 6B] A photo of extracted pyocyanin, whose color has been converted to red by HCl. [Figure 7A] Bacteria do not develop resistance to AuNC@ATP, preventing sublethal antibiotic treatment from inducing resistance. Schematic showing serial passaging experiment. The fold change in minimum inhibitory concentration (MIC) was measured as the ratio between MIC at n passages / initial MIC. [Figure 7B] Resistance development of susceptible PAO1 during serial passaging at sub-MIC doses of Ofloxacin, Tobramacy, and AuNC@ATP after 21 passages (1 passage per 24 h). [Figure 7C] Resistance development of susceptible PAO1 during serial passaging at sub-MIC doses of ciprofloxacin in the absence or presence of AuNC@ATP (0.56 μM). [Figure 8A]AuNC@ATP prevents cross-resistance induction by sublethal doses of fluoroquinolones. Schematic diagram showing the disk diffusion assay used to determine the antimicrobial susceptibility profile of PAO1 isolates after 21 passages in medium containing subinhibitory concentrations of ciprofloxacin without AuNC@ATP (PAO1Cip21) and with AuNC@ATP (PAO1Cip21-AuNC@ATP). [Figure 8B] Cross-resistance of PAO1Cip21 and PAO1Cip21-AuNC@ATP against different anti-P. aeruginosa antibiotics. Labels on the vertical axis indicate the antibiotics tested for cross-tolerance, and labels on the horizontal axis indicate the fold change in the inhibition zone compared to the susceptible P. aeruginosa (PAO1 ancestor). [Figure 9A] Multiple dose administration of AuNC@ATP is not toxic to mice. Effect of AuNC@ATP on hematology (FIG. 9B) and clinical chemistry parameters (FIG. 9C) at day 14 after treatment with 38.19 mg / kg dose administered intraperitoneally (IP) three times a day for 14 days. The parameters evaluated are listed in the figures. Ten mice (5 females and 5 males) were used. Phosphate-buffered saline (PBS) was used as a vehicle control. [Figure 9B] Effect of AuNC@ATP on hematology on day 14 after treatment with a dose of 38.19 mg / kg administered intraperitoneally (IP) three times a day for 14 days. [Figure 9C] Effect of AuNC@ATP on clinical chemistry parameters on day 14 after treatment with a dose of 38.19 mg / kg administered intraperitoneally (IP) three times a day for 14 days. [Figure 10A] Quantification of the amount of ATP per AuNC@ATP. Schematic showing how the bioluminescent ATP assay works. [Figure 10B] Linear correlation between luminescence and ATP concentration. [Figure 10C] Linear correlation between luminescence and AuNC@ATP concentration. [Figure 10D] Linear correlation between ATP concentration and AuNC@ATP concentration. [Figure 11A]AuNC@ATP-mediated cell death occurs without releasing periplasmic proteins and cytoplasmic components into the supernatant. Schematic diagram showing how the bioluminescent ATP assay works. [Figure 11B] Quantification of protein concentration in the supernatant after treatment with cells treated with AuNC@ATP, colistin, and AuNC@ATP exposed to colistin. The data demonstrate the absence of cell lysis after AuNC@ATP treatment. [Figure 12A] Killing by AuNC@ATP is independent of reactive oxygen species (ROS). Schematic showing how intracellular ROS was determined using the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). [Figure 12B] Comparison of ROS production after treatment of P. aeruginosa with ofloxacin and AuNC@ATP. Knowing that killing by bactericidal antibiotics is not dependent on ROS, ofloxacin was used as a comparative control. The 1.2 fold change in ROS production upon treatment with both ofloxacin and AuNC@ATP demonstrates that AuNC@ATP-mediated cell death is not associated with ROS production. [Figure 13] ATP is not an anti-survival compound. Representative Petri dishes showing regrowth of viable cells after treatment of ofloxacin-induced viable cells (108 CFU / ml) with ATP (10 mM) and AuNC@ATP (4.2 μM), respectively. Images represent Petri dishes from three independent experiments (n=3) for each condition. We note that the ATP ligand alone did not show bactericidal activity, confirming that the antibacterial effect did not come from the surface ligands. Eradication of viable cells by AuNC@ATP is not determined by surface ligand density, but by the totality of AuNC@ATP as a whole entity and compound. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Compositions comprising nanoclusters comprising a metallic core conjugated to a nucleotide and methods of their use in the treatment of bacterial infections are provided.

[0024] Before describing the present compositions comprising nanoclusters comprising metallic cores conjugated to nucleotides and methods of using them in the treatment of bacterial infections, it is to be understood that the invention is not limited to the particular methods or compositions described, as such may, of course, vary. Also, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0025] Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is to be understood that each intervening value between the upper and lower limits of that range is also specifically disclosed to the tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, it should be understood that the present disclosure supersedes any disclosure of the incorporated publication.

[0027] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0028] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "bacterial cells" includes a plurality of such bacterial cells, a reference to a "nanocluster" includes a reference to one or more nanoclusters and equivalents thereof known to those skilled in the art, and so forth.

[0029] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0030] The term "nanocluster" refers to organic, inorganic, or hybrid nanoclusters having a size of 10 nm or less in length. Nanoclusters can have dimensions of 4 nm or less, including 3 nm or less, or 2 nm or less, or 1 nm or less. In some examples, nanoclusters have dimensions of 2 nm or less. In certain embodiments, nanoclusters have diameters ranging from about 1 nm to about 10 nm, including any diameter within this range, such as 0.5 nm, 0.75 nm, 1 nm, 1.25 nm, 1.5 nm, 1.75 nm, 2 nm, 2.25 nm, 2.5 nm, 2.75 nm, 3 nm, 3.25 nm, 3.5 nm, 3.75 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8 nm, 8.5 nm, 9 nm, 9.5 nm, or 10 nm. In some embodiments, nanoclusters have diameters of about 1 nm to about 2 nm.

[0031] "Diameter" when used in reference to a geometric structure (e.g., a nanocluster, a nanocluster, etc.) refers to a length that describes the overall size of the structure. The length may generally approximate the diameter of a circle or sphere that encircles the structure.

[0032] The term "survivor cell" refers to a cell that has entered a physiological state of non-growth (i.e., dormancy) or very slow growth that renders it less susceptible or resistant to antimicrobial agents. Such cells may "survive" after planktonic bacterial cells have been eradicated by the immune system or conventional treatment with antimicrobial agents. Survivor cells are commonly found in biofilms.

[0033] As used herein, the term "antibacterial agent" is interchangeable with the term "antibiotic" and refers to any agent capable of having a bactericidal or bacteriostatic effect on growth. Antibiotics include, but are not limited to, β-lactam antibiotics, aminoglycosides, aminocyclitols, quinolones, tetracyclines, macrolides, lincosamides, glycopeptides, lipopeptides, polypeptide antibiotics, sulfonamides, trimethoprim, chloramphenicol, isoniazid, nitroimidazoles, rifampicin, nitrofurans, methenamine, and mupirocin.

[0034] The term "antimicrobial effect" means the killing of bacteria or the inhibition or cessation of their growth and / or reproduction.

[0035] As used herein, the term "efflux pump" refers to a protein assembly that transports or exports substrate molecules from the cytoplasm or periplasm of a cell in an energy-dependent or energy-independent manner. As used herein, the term "efflux pump activity" refers to the mechanism responsible for exporting substrate molecules (including antibacterial agents) out of the cell. As used herein, the term "efflux pump inhibitor" refers to a compound that interferes with the ability of an efflux pump to transport or export substrates (including antibacterial agents).

[0036] As used herein, the term "treatment" refers to (1) the prevention of infection or reinfection (prophylaxis), (2) the eradication of an existing infection, or (3) the reduction or elimination of symptoms of the infectious disease of interest (therapy).

[0037] By "therapeutically effective dose or amount" of nanoclusters is intended an amount that, when administered alone or in combination with an antibiotic, as described herein, results in a positive therapeutic response, such as improved recovery from an infection, including any infection caused by gram-positive or gram-negative bacteria. In addition, a therapeutically effective dose or amount may eradicate viable cells as well as other bacterial cells, including planktonic bacteria and bacteria in biofilms, increase ROS accumulation in macrophages, stimulate TNF-α secretion from activated macrophages, restore autophagy, and / or deplete glutathione, catalase, and hydroperoxide reductase. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs used, the mode of administration, etc. The appropriate "effective" amount in any individual case can be determined by one of skill in the art using routine experimentation, based on the information provided herein.

[0038] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in a composition of the present invention and that causes no significant adverse toxicological effects to a patient.

[0039] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salt, salt prepared with inorganic acid, such as chloride, sulfate, phosphate, diphosphate, bromide and nitrate, or salt prepared from any of the above corresponding inorganic acid forms, such as hydrochloride, or salt prepared with organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, paratoluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate.Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium and ammonium (including substituted ammonium).

[0040] "Substantially purified" generally refers to the isolation of a component, such as a substance (a compound, nanocluster, nucleic acid, polynucleotide, RNA, DNA, protein, or polypeptide), that constitutes a large proportion of the material present in a sample. Typically, the substantially purified component in a sample constitutes 50%, preferably 80%-85%, and more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, gel filtration, and sedimentation according to density.

[0041] "Isolated" refers to an entity of interest that is in an environment different from that in which it may naturally occur. "Isolated" is meant to include an entity within a sample in which the entity of interest has been substantially enriched and / or in which the entity of interest has been partially or substantially purified. "Isolated," when referring to a polypeptide, means that the indicated molecule is separate and distinct from the whole organism in which it is found in nature, or exists in the substantial absence of other biological macromolecules of the same type. With reference to a polynucleotide, the term "isolated" refers to a nucleic acid molecule that lacks all or a portion of sequences that are normally associated with it in nature, or a sequence that is naturally occurring but has heterologous sequences associated with it, or a molecule that is dissociated from the chromosome.

[0042] The terms "recipient", "individual", "subject", "host" and "patient" are used interchangeably herein and refer to any vertebrate subject, particularly humans, for whom diagnosis, treatment or therapy is desired. By "vertebrate subject" is meant any member of the subphylum Chordata, including, but not limited to, humans and other primates, including non-human primates such as chimpanzees and other ape and monkey species; domestic animals such as cows, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs; birds, including chickens, turkeys and other poultry birds, domestic birds such as ducks, geese, wild birds and game birds. The terms do not denote a particular age. Thus, both adult and newborn individuals are intended to apply.

[0043] "Biocompatible" generally refers to a material and any metabolic or decomposition products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects to the subject.

[0044] "Homology" refers to the percent identity between two polynucleotides or two polypeptide molecules. Two nucleic acid or two polypeptide sequences are "substantially homologous" to each other if the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences that exhibit complete identity to a specified sequence.

[0045] In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid match of two polynucleotide or polypeptide sequences, respectively. Percent identity can be determined by directly comparing the sequence information between the two molecules by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. For peptide analysis, readily available computer programs can be used to assist in the analysis, such as ALIGN, Dayhoff, MO, in Atlas of Protein Sequence and Structure MO Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482 489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity, such as the BESTFIT, FASTA, and GAP programs, which also rely on the Smith and Waterman algorithm, are available in the Wisconsin Sequence Analysis Package, version 8 (available from Genetics Computer Group, Madison, WI).These programs are easily used with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package mentioned above.For example, the percent identity of a particular nucleotide sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default scoring table and a gap penalty of 6 nucleotide positions.

[0046] Another method for establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this set of packages, the Smith-Waterman algorithm can be used with default parameters (e.g., gap opening penalty 12, gap extension penalty 1, and gap 6) used in the scoring table. From the data generated, the "match" value reflects "sequence identity". Other programs suitable for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs are readily available.

[0047] Alternatively, homology can be determined by polynucleotide hybridization under conditions that form stable double strands between homologous regions, followed by digestion with single-strand specific nucleases and sizing of digested fragments. Substantially homologous DNA sequences can be identified, for example, in Southern hybridization experiments under stringent conditions defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, for example, Sambrook et al. (supra); DNA cloning (supra); Nucleic Acid Hybridization (supra).

[0048] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin that is not associated by its origin or manipulation with all or a portion of a polynucleotide with which it is naturally associated. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. Generally, a gene of interest is cloned and then expressed in a transformed organism, as further described below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0049] The term "derived from" is used herein to identify the original source of a molecule, but is not meant to limit the manner in which it is made (which may be, for example, by chemical synthesis or by recombinant means).

[0050] A polynucleotide "derived from" a specified sequence refers to a polynucleotide sequence that contains a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides, that corresponds to, i.e., is identical to or complementary to, a region of the specified nucleotide sequence. A derived polynucleotide need not necessarily be physically derived from the nucleotide sequence of interest, but may be generated in any manner, including but not limited to chemical synthesis, replication, reverse transcription, or transcription, based on information provided by the sequence of bases in the region from which the polynucleotide is derived. Thus, it may represent either the sense or antisense orientation of the original polynucleotide.

[0051] The term "hydrophilic polymer" refers to a material that has the property of dissolving in, absorbing, or mixing easily with water and contains repeating units constituting a molecular weight of at least 200 to 8,000 or more. Hydrophilic polymers include, but are not limited to, polyethylene glycol (PEG) as well as other materials that may be used to solubilize nanoclusters. Materials for this purpose include polyethylene glycol (PEG), polyoxyethylene, polymethylene glycol, polytrimethylene glycol, polyvinylpyrrolidone, polylysine (D or L) and derivatives, and polyoxyethylene-polyoxypropylene block polymers and copolymers. Hydrophilic polymers may be linear or multi-branched and may include multi-arm block copolymers. Hydrophilic polymers, when attached in sufficient numbers to the nanoclusters, render the nanoclusters soluble.

[0052] Nucleotide-functionalized nanoclusters for the treatment of bacterial infections Compositions comprising functionalized nanoclusters and methods of using them in the treatment of bacterial infections are provided. In particular, functionalized nanoclusters comprising nucleotides are useful for treating chronic infections associated with the production of bacterial biofilms that are not responsive to conventional antibiotic treatment. Without being bound by theory, bacteria in biofilms tend to be more resistant to treatment with antibiotics, in part because the biofilm extracellular matrix and outer layer of cells protect the bacterial cells inside. In addition, many bacterial cells in biofilms adopt a dormant phenotype and become metabolically inactive, which makes them less susceptible to antibiotics that need to be metabolized to be effective (e.g., penicillin requires cell wall remodeling in active bacterial cells to cause cell death). Dormant cells in biofilms that have entered a physiological state of non-growth or very slow growth, and thus become resistant to antimicrobial agents, are referred to herein as "survivor cells" due to their ability to survive after other active bacterial cells have been eradicated by the immune system and antimicrobial agents. Viable cells are often associated with chronic infections due to the difficulty of eradicating them using conventional antibiotic treatments. The methods described herein are particularly useful for treating chronic infections that render viable cells in biofilms more susceptible to antibiotic treatment.

[0053] In certain embodiments, the nanoclusters are conjugated to adenosine triphosphate (ATP) or a nucleotide such as, but not limited to, its phosphorothioate analogs, deoxyribonucleotide analogs, 7-deazapurine nucleotide analogs, or phosphomethylphosphonate adenylate. Exemplary phosphorothioate analogs include, but are not limited to, ATPαS, ATPβS, or ATPγS. Exemplary deoxyribonucleotide analogs include, but are not limited to, deoxyadenosine triphosphate (dATP). Exemplary 7-deazapurine nucleotide analogs include, but are not limited to, 7-deaza-adenosine-5′-triphosphate (7-deaza-ATP). Exemplary phosphomethylphosphonate adenylate analogs include, but are not limited to, β,γ-methylene adenosine 5′-triphosphate (AMP-PCP).

[0054] The nanoclusters are typically spherical in shape, although nanoclusters having other shapes may also be used. For example, the nanoclusters may have shapes such as, but not limited to, spheres, spheres (e.g., flattened or elongated spheres), ellipsoids, rods, cones, cubes, cuboids (e.g., hexahedrons), pyramids, icosahedrons, truncated icosahedrons, or irregular shapes. In certain instances, a combination of nanoclusters of different shapes may be included in the composition. In some embodiments, the nanoclusters may be substantially spherical in shape and thus have dimensions measured as the diameter of a sphere. In certain embodiments, the nanoclusters have a median diameter distribution ranging from about 1 nm to about 10 nm, including any diameter within this range, such as 0.5 nm, 0.75 nm, 1 nm, 1.25 nm, 1.5 nm, 1.75 nm, 2 nm, 2.25 nm, 2.5 nm, 2.75 nm, 3 nm, 3.25 nm, 3.5 nm, 3.75 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8 nm, 8.5 nm, 9 nm, 9.5 nm, or 10 nm. In some examples, the substantially spherical nanoclusters have an average diameter of 2 nm or less. In some embodiments, the substantially spherical nanoclusters have an average diameter of about 1 nm to about 2 nm.

[0055] The nanoclusters may include, for example, metals, ceramics, carbon-based nanomaterials, silicon or silica, boron, polymers, lipids, or proteins. In certain embodiments, the nanoclusters include a metallic core conjugated to a nucleotide. The metallic core may include two or more types of metal atoms, such as a single type of metal atom, or two or more different types of metal atoms. In some embodiments, the nanoclusters include metals, including but not limited to, one or more of gold, silver, platinum, titanium, palladium, rhodium, ruthenium, tin, nickel, copper, aluminum, or oxides, carbides, nitrides, or alloys thereof. In other embodiments, the nanoclusters are composed of oxides of silicon, aluminum, transition metals (e.g., titanium, zirconium, etc.), aluminosilicates, boron nitride, or combinations thereof. Exemplary materials that may be used in the nanoclusters include, but are not limited to, silicon dioxide (e.g., silica), titanium dioxide, silicon-aluminum-oxide, aluminum oxide, and iron oxide. In some examples, the nanoclusters are composed of other inorganic materials, such as, but not limited to, diatomaceous earth, calcium hydroxyapatite, etc. The nanoclusters may also be composed of hydrophobic polymers, such as, but not limited to, polylactides, polylactic acid, polyolefins (e.g., polyethylene, poly(isobutene), poly(isoprene), poly(4-methyl-1-pentene), polypropylene, ethylene-propylene copolymers, and ethylene propylene-hexadiene copolymers), ethylene-vinyl acetate copolymers, and styrene polymers (e.g., poly(styrene), poly(2-methylstyrene), styrene-acrylonitrile copolymers, and styrene-2,2,3,3,-tetrafluoro-propyl methacrylate copolymers).The nanoclusters may also be composed of natural polymers such as proteins, including but not limited to albumin, silk, keratin, collagen, elastin, corn zein, and soy protein-based nanoclusters; or polysaccharide-based polymers, including but not limited to chitosan, hyaluronic acid, alginate, glucan, dextran, and cyclodextrin-based nanoclusters. Carbon-based nanoclusters may include, but are not limited to, carbon nanotubes, graphite, graphene, fullerenes, and nanodiamonds. Combinations of the above materials may also be included in the nanoclusters. In certain embodiments, the nanoclusters are biocompatible with human cells.

[0056] In certain embodiments, the nanoclusters are linked to internalization sequences, protein transduction domains, or cell penetrating peptides to facilitate entry into cells. Cell-penetrating peptides that can be used include, but are not limited to, human immunodeficiency virus (HIV)-Tat, penetratin, transportan, octaarginine, nonaarginine, antennapedia, TP10, buforin II, MAP (model amphipathic peptide), K-FGF, Ku70, melittin, pVEC, Pep-1, SynB1, Pep-7, CADY, GALA, pHLIP, KALA, R7W, and HN-1, which can readily transport nanoclusters across the plasma membrane (see, e.g., Lai et al. (2023) Bioconjug. Chem. 34(1):228-237; Peng et al. (2014) Biomaterials 35(21):5605-5618; Jones et al. (2012) J. Control Release 161(2):582-591; Fonseca et al. (2012) J. Control Release 161(2):582-591; (see, for example, Schwarze et al. (2009) Adv. Drug Deliv. Rev. 61(11):953-64; Schwarze et al. (1999) Science. 285(5433):1569-72; Derossi et al. (1996) J. Biol. Chem. 271(30):18188-18193; Fuchs et al. (2004) Biochemistry 43(9):2438-2444; and Yuan et al. (2002) Cancer Res. 62(15):4186-4190).

[0057] Conjugation Surface functionalization of nanoclusters can be performed by any method known in the art. Functionalization of nanoclusters includes conjugation of nucleotides (e.g., ATP, dATP, ATPαS, ATPβS, ATPγS, 7-deaza-ATP, or AMP-PCP) to molecules on the outer surface of the nanocluster. Surface coatings can be applied to the nanoclusters to introduce functional groups and promote attachment of drugs. For example, gold nanoclusters with surface coatings containing thiol, carboxyl, amine, aldehyde, hydroxyl, or azide groups, PEG, dextran, streptavidin, or maleimide, and compounds that promote bioconjugation are commercially available from several companies (e.g., SigmaAldrich (St. Louis, MO), and Cytodiagnostics (Burlington, Ontario, Canada), Creative Diagnostics (Shirley, NY), and Nanocs (New York, NY)). Drugs can be conjugated to the nanoclusters directly or indirectly through linkers. Exemplary linkers include, but are not limited to, thio C6 linkers (thiohexyl), PEG polymers, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and hydrazide compounds. For a discussion of bioconjugation techniques, see, for example, Chemistry of Bioconjugates: Synthesis, Characterization, and Biomedical Applications (R. Narain ed., Wiley, 2014), GT Hermanson Bioconjugate Techniques (Academic Press, 3 rd edition, 2013) and Bioconjugation Protocols: Strategies and Methods(Methods in Molecular Biology, SSMark ed., Humana Press, 2 ndedition,2011), Avvakumova et al.(2014)Trends Biotechnol.32(1):11-20., Couto et al.(2017)Crit Rev Biotechnol.37(2):238-250, Sivaram et al.(2018)Adv.Healthc Mater.7(1), van Vught et al.(2014)Comput Struct Biotechnol J.9:e201402001, Massa et al. (2016) Expert Opin Drug Deliv 13:1-15, Yeh et al. (2015) PLoS One 10(7):e0129681, Freise et al. (2015) Mol Immunol.67(2 Pt A):142-152.

[0058] Nucleotides or other agents can be conjugated to nanoclusters using a variety of conjugation methods and chemistries. A variety of zero-length, homobifunctional, and heterobifunctional cross-linking reagents can be used. Zero-length cross-linking reagents involve the direct conjugation of two endogenous chemical groups without the introduction of exogenous materials. Agents that catalyze the formation of disulfide bonds belong to this category. Another example is an agent that induces the condensation of carboxyl and primary amino groups to form amide bonds, such as carbodiimides, ethyl chloroformate, Woodward's reagent K (2-ethyl-5-phenylisoxazolium-3'-sulfonate), and carbonyldiimidazole. Homo- and heterobifunctional reagents generally contain two identical or two non-identical sites, respectively, that can be reactive with amino, sulfhydryl, guanidino, indole, or non-specific groups.

[0059] Suitable amino-reactive groups include, but are not limited to, N-hydroxysuccinimide (NHS) esters, imidoesters, isocyanates, acyl halides, aryl azides, p-nitrophenyl esters, aldehydes, and sulfonyl chlorides. Suitable sulfhydryl-reactive groups include, but are not limited to, maleimides, alkyl halides, pyridyl disulfides, and thiophthalimides. In other embodiments, carbodiimides, which are soluble in both water and organic solvents, are used as carboxyl-reactive reagents. These compounds react with free carboxyl groups to form pseudoureas, which can then be coupled to available amines to provide amide bonds.

[0060] In some embodiments, nucleotides or other agents are conjugated to nanoclusters using homobifunctional crosslinkers. In some embodiments, the homobifunctional crosslinkers are reactive with primary amines. Homobifunctional crosslinkers reactive with primary amines include NHS esters, imidoesters, isothiocyanates, isocyanates, acyl halides, aryl azides, p-nitrophenyl esters, aldehydes, and sulfonyl chlorides. Non-limiting examples of homobifunctional NHS esters include disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis-2-(succinimidooxycarbonyloxy)ethyl sulfone (BSOCOES), bis-2-(sulfosuccinimidooxycarbonyloxy)ethyl sulfone (sulfo-BSOCOES), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), dithiobis(succinimidyl propionate (DSP), and dithiobis( Examples of homobifunctional imidoesters include sulfosuccinimidyl propionate (sulfo-DSP). Non-limiting examples of homobifunctional imidoesters include dimethyl malonimidate (DMM), dimethyl succinimidate (DMSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-oxydipropionimidate (DODP), dimethyl-3,3'-(methylenedioxy)dipropionimidate (DMDP), dimethyl-,3'-(dimethylenedioxy)dipropionimidate (DDDP), dimethyl-3,3'-(tetramethylenedioxy)dipropionimidate (DTDP), and dimethyl-3,3'-dithiobispropionimidate (DTBP).

[0061] Non-limiting examples of homobifunctional isothiocyanates include p-phenylene diisothiocyanate (DITC) and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). Non-limiting examples of homobifunctional isocyanates include xylene-diisocyanate, toluene-2,4-diisocyanate, toluene-2-isocyanate-4-isothiocyanate, 3-methoxydiphenylmethane-4,4'-diisocyanate, 2,2'-dicarboxy-4,4'-azophenyl diisocyanate, and hexamethylene diisocyanate. Non-limiting examples of homobifunctional aryl halides include 1,5-difluoro-2,4-dinitrobenzene (DFDNB) and 4,4'-difluoro-3,3'-dinitrophenyl-sulfone. Non-limiting examples of homobifunctional aliphatic aldehyde reagents include glyoxal, malondialdehyde, and glutaraldehyde. Non-limiting examples of homobifunctional acylating reagents include nitrophenyl esters of dicarboxylic acids. Non-limiting examples of homobifunctional aromatic sulfonyl chlorides include phenol-2,4-disulfonyl chloride and alpha-naphthol-2,4-disulfonyl chloride. Non-limiting examples of additional amino-reactive homobifunctional reagents include erythritol biscarbonate, which reacts with amines to give biscarbamates.

[0062] In some embodiments, the homobifunctional crosslinker is reactive with free sulfhydryl groups. Homobifunctional crosslinkers reactive with free sulfhydryl groups include, for example, maleimides, pyridyl disulfides, and alkyl halides. Non-limiting examples of homobifunctional maleimides include bismaleimidohexane (BMH), N,N'-(1,3-phenylene)bismaleimide, N,N'-(1,2-phenylene)bismaleimide, azophenyldimaleimide, and bis(N-maleimidomethyl)ether. Non-limiting examples of homobifunctional pyridyl disulfides include 1,4-di-3'-(2'-pyridyldithio)propionamidobutane (DPDPB). Non-limiting examples of homobifunctional alkyl halides include 2,2'-dicarboxy-4,4'-diiodoacetamidoazobenzene, α,α'-diiodo-p-xylenesulfonic acid, α,α'-dibromo-p-xylenesulfonic acid, N,N'-bis(b-bromoethyl)benzylamine, N,N'-di(bromoacetiphenylhydrazine), and 1,2-di(bromoacetiamino-3-phenylpropane).

[0063] In some embodiments, nucleotides or other agents are conjugated to the nanoclusters using heterobifunctional reagents. Suitable heterobifunctional reagents include amino-reactive reagents that contain a pyridyl disulfide moiety, amino-reactive reagents that contain a maleimide moiety, amino-reactive reagents that contain an alkyl halide moiety, and amino-reactive reagents that contain an alkyl dihalide moiety.

[0064] Non-limiting examples of heterobifunctional reagents containing a pyridyl disulfide moiety and an amino-reactive NHS ester include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-3-(2-pyridyldithio)propionamidohexanoate (LC-SPDP), sulfosuccinimidyl 6-3-(2-pyridyldithio)propionamidohexanoate (sulfo-LCSPDP), 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), and sulfosuccinimidyl 6-α-methyl-α-(2-pyridyldithio)toluamide hexanoate (sulfo-LC-SMPT).

[0065] Non-limiting examples of heterobifunctional reagents containing a maleimide moiety and an amino-reactive NHS ester include succinimidyl maleimidyl acetate (AMAS), succinimidyl 3-maleimidyl propionate (BMPS), N-gamma-maleimidobutyryloxy succinimide ester (GMBS), N-gamma-maleimidobutyryloxy sulfosuccinimide ester (sulfo-GMBS), succinimidyl 6-maleimidyl hexanoate (EMCS), succinimidyl 3-maleimidyl benzoate (SMB), m-maleimidobenzoyl-N -hydroxysuccinimidyl ester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

[0066] Non-limiting examples of heterobifunctional reagents containing an alkyl halide moiety and an amino-reactive NHS ester include N-succinimidyl-(4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate (sulfo-SIAB), succinimidyl-6-(iodoacetyl)aminohexanoate (SIAX), succinimidyl-6-(6-(((iodoacetyl)-amino)hexanoylamino)hexanoate (SIAXX), succinimidyl-6-(((4-(iodoacetyl)-amino)methyl)-cyclohexane-1-carbonyl)aminohexanoate (SIACX), and succinimidyl-4-((iodoacetyl)-amino)methylcyclohexane-1-carboxylate (SIAC).

[0067] A non-limiting example of a heterobifunctional reagent containing an amino-reactive NHS ester and an alkyl dihalide moiety is N-hydroxysuccinimidyl 2,3-dibromopropionate (SDBP). A non-limiting example of a heterobifunctional reagent containing an alkyl halide and an amino-reactive p-nitrophenyl ester moiety is p-nitrophenyl iodoacetate (NPIA).

[0068] In another example, a 3-thio C6 linker can be used to functionalize nucleotides or other agents with thiol groups to facilitate attachment to nanoclusters. For example, a 3-thio C6 linker can be used to add a thiol group to a nucleotide. The free thiol can be used as a reactive functional group to attach a maleimide compound or for conjugation through a disulfide bond.

[0069] An alternative bioconjugation method uses click chemistry, which includes cycloaddition reactions such as the copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), the Diels-Alder reaction, nucleophilic substitution reactions (especially for small strained rings such as epoxy and aziridine compounds), reactions involving the formation of urea compounds, and reactions involving carbon-carbon double bonds such as alkynes in thiol-yne reactions. See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31; Evans, 2007, Aust J Chem 60:384-95; Millward et al. (2013) Integr Biol(Camb) 5(1):87-95); Lallana et al. (2012) Pharm Res 29(1):1-34; Gregoritza et al. (2015) Eur J Pharm Biopharm. 97(Pt B):438-453; Musumeci et al. (2015) Curr Med Chem. 22(17):2022-2050; McKay et al. (2014) Chem Biol 21(9):1075-1101; Ulrich et al. See, e.g., Wangler et al. (2014) Chemistry 20(1):34-41, Pasini (2013) Molecules 18(8):9512-9530, and Wangler et al. (2010) Curr Med Chem. 17(11):1092-1116.

[0070] Pharmaceutical Compositions The functionalized nanoclusters conjugated to nucleotides (e.g., ATP, dATP, ATPαS, ATPβS, ATPγS, 7-deaza-ATP, or AMP-PCP) described herein can be optionally formulated into pharmaceutical compositions containing one or more pharma- ceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids, and surfactants. Carbohydrates such as sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and / or sugar polymers can be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, etc. Excipients may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0071] The compositions may also include antimicrobial agents to prevent or inhibit microbial growth. Non-limiting examples of antimicrobial agents include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0072] Similarly, antioxidants may also be present in the composition.Antioxidants are used to prevent oxidation and thereby prevent deterioration of the nanoclusters or other components of the preparation.Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0073] Surfactants may be present as excipients.Exemplary surfactants include polysorbates such as "Tween 20" and "Tween 80" and Pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (but preferably not in liposomal form), fatty acids, and fatty esters; steroids such as cholesterol; chelating agents such as EDTA; and zinc and other such suitable cations.

[0074] As excipient, acid or base may be present in the composition.Non-limiting examples of acids that can be used include the acid selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.Examples of suitable bases include, but are not limited to, the base selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

[0075] The amount of nanoclusters in a composition (e.g., when contained in a drug delivery system) will vary depending on several factors, but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial). A therapeutically effective dose can be determined empirically by repeated administration of increasing amounts of the composition to determine which amount results in a clinically desired endpoint.

[0076] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and the specific needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing various amounts of the excipient (ranging from low to high amounts), examining stability and other parameters, and then determining the range in which optimal performance is achieved without significant side effects. Generally, however, the excipient is present in the composition in an amount of about 1% to about 99% by weight of the excipient, preferably about 5% to about 98% by weight, more preferably about 15 to about 95% by weight, and most preferably the concentration is less than 30% by weight. These aforementioned pharmaceutical excipients, along with other excipients, are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician's Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, AH, Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, DC, 2000.

[0077] The compositions include all kinds of formulations, especially formulations suitable for injection, such as powders or lyophilized products that can be reconstituted with a solvent before use, as well as ready-to-inject solutions or suspensions, dry insoluble compositions for combining with a vehicle before use, and emulsions and liquid concentrates for dilution before administration.Examples of suitable diluents for reconstituting solid compositions before injection include bacteriostatic water for injection, 5% dextrose in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.For liquid pharmaceutical compositions, solutions and suspensions are envisioned.Further preferred compositions include compositions for oral, ocular, or topical delivery.

[0078] The pharmaceutical agents herein may also be contained in syringes, implantation devices, etc., depending on the intended mode of delivery and use. Preferably, the compositions comprising the nanoclusters are in unit dosage form, by which is meant an amount of the composition appropriate for a single dose in premeasured or prepackaged form.

[0079] The compositions herein may optionally include one or more additional agents, such as antibiotics, adjuvants, immunostimulants, vaccines, and / or other medicines used to treat a subject for an infectious disease. The combination preparations may include a combination of nanoclusters and an antibiotic, including broad-spectrum, bactericidal, or bacteriostatic antibiotics, such as penicillins, including penicillin G, penicillin V, procaine penicillin, benzathine penicillin, veetid (Pen-Vee-K), piperacillin, pipracil, Pfizer Pen, temocillin, negaban, ticarcillin, and Ticar; amoxicillin / clavulanic acid, augmentin, ampicillin / sulbactam, unasyn, piperacillin / tazobactam, penicillin combinations such as zosyn, ticarcillin / clavulanic acid, and timentin; tetracyclines such as chlortetracycline, doxycycline, demeclocycline, eravacycline, lymecycline, meclocycline, methacycline, minocycline, omadacycline, oxytetracycline, rolitetracycline, seracycline, tetracycline, and tigecycline; cefacetrile (cefacetrile ephacetrile), cefadroxil (cefadroxyl; durisef), cefalexin (cephalexin; keflex), cefaloglycin (cephaloglycin), cefalonium (cephalonium), cephaloridine (cefaloridine) idine) (cephalorazine), cefalotin (cephalothin; kefrin), cefapirin (cephapirin; cephadryl), cefatrizine, cefazaflour, cephazedone, cefazolin (cephazolin; ansef, kefzole), cefradine (cephradine;Verocef), cefroxadine, ceftezole, cefaclor (ceclor, distachlor, kefrol, raniclor), cefonicid (monocid), cefprozil (cefproxil; cefzil), cefuroxime (zef, ginnat, dinacef, ceftin, biofuroksym, xorimax), cefuzonam, loracarbef (lorabido), cefbuperazone, cefmetazole (zefazone), cefminox, Cefotetan (Cefottan), cefoxitin (Mefoxin), cefotiam (Pansporin), cefcapene, cefdaloxime, cefdinir (Cefdin, Dinir, Omnicef, Kefnir), cefditoren, cefetamet, cefixime (Fix, Zyfi, Suprax), cefmenoxime, cefodizime, cefotaxime (Claforan), cefovecin (Convenia), cefpimizole, cefpodoxime (Vantin, Pesef, Simplicef) ), cefteram, ceftamers (enshort), ceftibuten (cedax), ceftiofur (naxel, exenel), ceftiolene, ceftizoxime (cefizox), ceftriaxone (rocephin), cefoperazone (cefobid), ceftazidime (meezat, fortum, fortaz), latamoxef (moxalactam), cefclidin, cefepime (maxipime), cefluprenam, cefo Cephalosporins such as Ceris, Cefozopran, Cefpirome (Cefrom), Cefquinome, Flomoxef, Ceftobiprole, Ceftaroline, Ceftolozane, Cephaloram, Cefaparol, Cefcanel, Cephedrolol, Cefenpidon, Cefetrizole, Cefibitril, Cefmatilen, Cefmepidium, Cefoxazole, Cefrotil, Cefsumide, Ceftioxide, Cefuracetime, and Nitrocefin;Flumequine (Flubactin), oxolinic acid (Uroxine), losoxacin (Eladasil), cinoxacin (Cinovac), nalidixic acid (NegGam, Wintmyron), piromidic acid (Panacid), pipemidic acid (Dolcol), ciprofloxacin (Zoxan, Ciprobay, Cipro, Ciproxin), fleroxacin (Megalon, Rokinol), lomefloxacin (Maxaxquin), nadifloxacin (Aquachim, Nadoxine, Nade Ixa), norfloxacin (Rexol, Noroxin, Kinabic, Janascin), ofloxacin (Floxin, Oxaldine, Tarivid), pefloxacin (Peflacin), rufloxacin (Uroflox), enoxacin (Enroxil, Penetrex), balofloxacin (Baloxin), grepafloxacin (Laxal), levofloxacin (Cravit, Levaquin), pazufloxacin (Pasil, Pazucross), sparfloxacin ( Zagam), temafloxacin (Omnifloxacin), tosufloxacin (Ozex, Tosacin), clinafloxacin, gatifloxacin (Zigat, Tekin, Zymar Ophthalmic), moxifloxacin (Avelox, Vigamox), sitafloxacin (Gracevit), prulifloxacin (Kisunon), besifloxacin (Besivance), delafloxacin (Baxdera), gemifloxacin (Factive), and trovafloxacin quinolones / fluoroquinolones such as (Troban), ozenoxacin, danofloxacin (Advosin, Advocid), difloxacin (Dicural, Betequinone), enrofloxacin (Baytril), ivafloxacin (Ibafrin), marbofloxacin (Marbocil, Zenequin), orbifloxacin (Orbax, Victas), and sarafloxacin (Floxazol, Saraflox, Sarafin);Macrophages such as azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin / midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin / tylocine, roxithromycin, telithromycin, cethromycin, solithromycin, tacrolimus, pimecrolimus, sirolimus, amphotericin B, nystatin, and cluentaren. sulfonamides such as sulfacetamide, sulfadiazine, sulfadimidine, sulfafurazole (sulfisoxazole), sulfisomidine (sulfaisodimidine), sulfamethoxazole, sulfamoxole, sulfanitran, sulfadimethoxine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfadoxine, sulfamethpyrazine, and terephthyl; kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin B, C, Aminoglycosides such as neomycin E (paromomycin), streptomycin, plazomycin, amiquin, garamycin, cantrex, neo-flazine, netromycin, nebcin, fumatin, spectinomycin (Bs), and trobicin; carbapenems such as imipenem, meropenem, ertapenem, doripenem, panipenem / betamipron, biapenem, tebipenem, razupenem (PZ-601), lenapenem, tomopenem, and thienamycin (thiempenem); geldanamycin, herbimycin, rifaximin, and xifamycin. ansamycins such as oxazolamide; carbacephems such as loracarbef and lorabide; carbapenems such as ertapenem, invanz, doripenem, dorivax, imipenem / cilastatin, primaxin, meropenem, and melem; glycopeptides such as teicoplanin, thalgoside, vancomycin, vancocin, telavancin, bibativ, dalbavancin, dalvans, oritavancin, and orbactive; lincosamides such as clindamycin, cleocin, lincomycin, and lincocin; lipopeptides such as daptomycin and cubicin;Macrolides such as Azithromycin, Zithromax, Sumamed, Xytron, Clarithromycin, Biaxin, Dirithromycin, Dynavac, Erythromycin, Erythosin, Erythroped, Roxithromycin, Troleandomycin, Tao, Telithromycin, Ketek, Spiramycin, and Lobamycin; Monobactams such as Aztreonam and Azactam; Nitrofurans such as Furazolidone, Floxon, Nitrofurantoin, Macrodantin, and Macrobid; Linezolid, Zyvox, Vrsa ( vrsa), oxazolidinones such as posizolid, radezolid, and torezolid; polypeptides such as bacitracin, colistin, coli-mycin-S, and polymyxin B; clofazimine, lamprene, dapsone, abrosulfone, capreomycin, capastat, cycloserine, ceromycin, ethambutol, miambutol, ethionamide, trecatol, isoniazid, INH, pyrazinamide, aldinamid, rifampicin, rifadin, rimactane, rifabutin, mycobutin, rifampic ... Drugs against mycobacteria such as fapentine, priftin, and streptomycin; and arsphenamine, salvarsan, chloramphenicol, chloromycetin, fosfomycin, monulol, monulil, fusidic acid, fucidin, metronidazole, flagyl, mupirocin, bactroban, platensimycin, quinupristin / dalfopristin, synercid, thiamphenicol, tigecycline, tigacyl, tinidazole, tindamax facidin, trimethoprim, proloprim, and other antibiotics such as Trimpex; adjuvants including aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate; oil-in-water emulsion formulations; saponin adjuvants; complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); cytokines such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12), interferons, macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor (TNF);and one or more other agents for treating infectious diseases, including, but not limited to, vaccines against bacteria and infectious diseases, including bacterial antigenic proteins or attenuated or killed bacteria, and any vaccines, optionally including adjuvants to enhance the immune response against bacteria, such as vaccines against tuberculosis, diphtheria, tetanus, whooping cough, Haemophilus influenzae type B, cholera, typhoid, Streptococcus pneumoniae, etc.;

[0080] Alternatively, such agents may be contained in a composition separate from the composition comprising the nanoclusters and may be co-administered simultaneously with, before, or after the composition comprising the nanoclusters.

[0081] Administration For the treatment of a bacterial infection, at least one therapeutically effective cycle of treatment with a composition comprising a functionalized nanocluster conjugated to a nucleotide (e.g., ATP, dATP, ATPαS, ATPβS, ATPγS, 7-deaza-ATP, or AMP-PCP) described herein is administered to the subject. Bacterial infections that may be treated by the methods described herein include Acinetobacter (e.g., Acinetobacter baumannii), Actinobacillus, Bordetella, Brucella, Campylobacter, Cyanobacteria, Enterobacter (e.g., Enterobacter cloacae), Erwinia, Escherichia coli, Franciscella, Helicobacter (Helicobacter pylori), Hemophilus (e.g., Hemophilus influenzae), Klebsiella (e.g., Klebsiella pneumoniae), Legionella (e.g., Legionella pneumophila), Moraxella (e.g., Moraxella catarrhalis), Neisseria (e.g., Neisseria gonorrhoeae, Neisseria meningitidis), Pasteurella, Proteus (e.g., Proteus mirabilis), Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella (e.g., Salmonella enteritidis, Salmonella typhi), Serratia (e.g., Serratia marcescens), Shigella, Treponema, Vibrio (e.g., Vibrio cholerae), and Yersinia (e.g., Yersinia pestis), as well as gram-negative bacteria, including, but not limited to, Actinobacteria, such as Actinomyces (e.g., Actinomyces israelii), Arthrobacter, Bifidobacterium, Corynebacterium (e.g., Corynebacteriumdiphtheriae), Frankia, Micrococcus, Micromonospora, Mycobacterium (e.g., Mycobacterium tuberculosis, Mycobacterium leprae), Nocardia, Propionibacterium, and Streptomyces; Firmicutes, e.g., Bacilli; the order Bacillales, including Bacillus, Listeria (e.g., Listeria monocytogenes), and Staphylococcus (e.g., Staphylococcus aureus, Staphylococcus epidermidis), Bacilli (e.g., Bacilli anthracis, Bacilli cereus); the order Lactobacillales, including Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus (e.g., Streptococcus pneumoniae, Streptococcus mutans, Streptococcus sanguinis, Streptococcus pyogenes), Clostridia (e.g., Clostridioides difficile, Clostridium perfringens, Clostridium botulinum, Clostridium tetani, Clostridium sordellii), including Acetobacterium, Clostridium, Eubacterium, Heliobacterium, Heliospirillum, Megasphaera, Pectinatus, Selenomonas, Zymophilus, and Sporomusa, Mollicutes, including Mycoplasma (e.g., Mycoplasma pneumoniae), Spiroplasma, Ureaplasma, and Erysipelothrix.

[0082] By "therapeutically effective dose or amount" of nanoclusters conjugated to nucleotides (e.g., ATP, dATP, ATPαS, ATPβS, ATPγS, 7-deaza-ATP, or AMP-PCP), as described herein, is intended an amount that, when administered alone or in combination with an antibiotic, results in a positive therapeutic response, such as improved recovery from an infection, including any infection caused by gram-positive or gram-negative bacteria. In addition, a therapeutically effective dose or amount may eradicate viable cells as well as other bacterial cells (including planktonic bacteria and bacteria in biofilms). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular type of nanocluster and its functionalization, other antibacterial agents or drugs used in combination, the mode of administration, etc. An appropriate "effective" amount in any individual case may be determined by one of skill in the art using routine experimentation based on the information provided herein.

[0083] In certain embodiments, multiple therapeutically effective doses of a composition comprising the nanoclusters and / or one or more other therapeutic agents, such as antibiotics, adjuvants, immunostimulants, vaccines, and / or other drugs to treat infections, or other pharmaceuticals, are administered. Compositions comprising the nanoclusters are typically (but not necessarily) administered orally, via injection (subcutaneously, intravenously, or intramuscularly), by infusion, topically, or locally. Additional modes of administration, such as intra-arterial, intravascular, pulmonary, intralesional, intraparenchymal, rectal, transdermal, transmucosal, intrathecal, intraocular, intraperitoneal, etc., are also contemplated.

[0084] Preparations according to the invention are also suitable for localized treatment. For example, compositions containing the nanoclusters can be administered directly to the site of infected tissue. By choosing a particular preparation and an appropriate method of administration, the nanoclusters can be targeted to sites of chronic infections and sites of bacterial biofilms where viable cells typically reside and require eradication.

[0085] The pharmaceutical agent may be in the form of a liquid solution or suspension immediately prior to administration, but may also take another form such as a syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, etc. Pharmaceutical compositions containing nanoclusters and / or other agents may be administered using the same or different routes of administration according to any pharma- ceutically acceptable method known in the art.

[0086] In another embodiment, pharmaceutical compositions containing nanoclusters and / or other agents are administered prophylactically, for example, to prevent infectious diseases. Such prophylactic use is of particular value to subjects who are immunocompromised, patients being treated with immunosuppressants, or patients with a genetic predisposition or disease that makes them prone to developing infectious diseases (e.g., acquired immune deficiency syndrome (AIDS), cancer, diabetes, or cystic fibrosis), or subjects in environments where they are likely to be exposed to infectious bacteria.

[0087] In another embodiment, the pharmaceutical compositions containing the nanoclusters and / or antibiotics and / or other agents are in sustained release formulations or formulations that are administered using sustained release devices. Such devices are well known in the art and include, for example, transdermal patches and miniature implantable pumps that can provide drug delivery in a continuous, steady-state manner over time at various doses to achieve a sustained release effect in non-sustained release pharmaceutical compositions.

[0088] Those skilled in the art will appreciate the conditions that the nanoclusters can effectively treat. The actual dose administered will vary depending on the age, weight, and general condition of the subject, as well as the severity of the condition being treated, the judgment of the health care professional, and the conjugate being administered. Therapeutically effective amounts can be determined by those skilled in the art and will be adjusted according to the particular requirements of each particular case.

[0089] In certain embodiments, multiple therapeutically effective doses of the composition comprising the nanoclusters are administered according to a daily dosing regimen or intermittently. For example, the therapeutically effective dose may be administered 1 day per week, 2 days per week, 3 days per week, 4 days per week, or 5 days per week, etc. By "intermittent" administration, it is intended that the therapeutically effective dose may be administered, for example, every other day, every 2 days, every 3 days, every week, every other week, etc. For example, in some embodiments, the composition comprising the nanoclusters will be administered once per week, twice per week, or three times per week for an extended period of time, such as 1, 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, etc. By "twice per week" or "twice per week" it is intended that two therapeutically effective doses of the agent are administered to the subject within a 7-day period, starting on day 1 of the first week of administration, with a minimum of 72 hours between doses and a maximum of 96 hours between doses. By "three times a week" or "three times per week" is intended that three therapeutically effective doses are administered to a subject within a seven day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For the purposes of the present invention, this type of administration is referred to as "intermittent" therapy. According to the methods of the present invention, a subject may receive intermittent therapy (i.e., administration of a therapeutically effective dose once a week, twice a week, or three times a week) for one or more weekly cycles until a desired therapeutic response is achieved. The agent may be administered by any acceptable route of administration as described herein below. The amount administered will depend on the potency of the nanoclusters and the type of their functionalization, the magnitude of the effect desired, and the route of administration.

[0090] The nanoclusters (again, preferably provided as part of a pharmaceutical product) may be used alone or in combination with antibiotics, including broad-spectrum, bactericidal, or bacteriostatic antibiotics such as penicillins, including penicillin G, penicillin V, procaine penicillin, benzathine penicillin, veetid (Pen-Vee-K), piperacillin, pipracil, Pfizer Pen, temocillin, negaban, ticarcillin, and Ticar; amoxicillin / clavulanic acid, augmentin, ampicillin / sulbactam, unasyn, piperacillin / tazobactam, zocillin ... penicillin combinations such as cyclosporine, ticarcillin / clavulanate, and timentin; tetracyclines such as chlortetracycline, doxycycline, demeclocycline, eravacycline, lymecycline, meclocycline, methacycline, minocycline, omadacycline, oxytetracycline, rolitetracycline, seracycline, tetracycline, and tigecycline; cefacetrile (cephacetrile), cefadroxil xil (cefadroxyl; durisef), cefalexin (cephalexin; keflex), cefaloglycin (cephaloglycin), cefalonium (cephalonium), cefaloridine (cephalorazine), cefalotin (cephalothin; ke furin), cefapirin (cephapirin; cefadryl), cefatrizine, cefazaflour, cefazedone, cefazolin (cephazolin; ansef, kefzole), cefradine (cephradine; velocef), cefroxadine, ceftezole, cefaclor (ceclor, distachlor, kefrol, raniclor), cefonicid (monocid), cefprozil (cefproxil;Cefdil), cefuroxime (Zef, Zinnat, Dinacef, Ceftin, Biofloccim, Zolimax), cefuzonam, loracarbef (Lorabid), cefbuperazone, cefmetazole (Zefazon), cefminox, cefotetan (Cefottan), cefoxitin (Mefoxin), cefotiam (Pansporin), cefcapene, cefdaloxime, cefdinir (Cefdin, Zinir, Omnicef, Kefnir), cefditoren, cefetamet, cefixime (Fix, Zyfi, Suprax), cefmenoxime, cefodizime, cefotaxime (Claforan), cefovecin (Convenia), cefpimizole, cefpodoxime (Vantin, Pesef, Simplicef), cefteram, ceftamers (Enshorth), ceftibuten (Sedax) ), ceftiofur (Naxxel, Exenel), ceftiolene, ceftizoxime (Cefizox), ceftriaxone (Rocephin), cefoperazone (Cefobide), ceftazidime (Meezat, Fortam, Fortaz), latamoxef (Moxalactam), cefclidin, cefepime (Maxipime), cefluprenum, cefoselis, cefozopran, cefpirome (Cefpirome) cephalosporins such as flom, cefquinome, flomoxef, ceftobiprole, ceftaroline, ceftolozane, cephaloram, cefaparol, cefcanel, cephedrolol, cefenpidon, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide, cerfacetim, and nitrocefin;Flumequine (Flubactin), oxolinic acid (Uroxine), losoxacin (Eladasil), cinoxacin (Cinovac), nalidixic acid (Neggam, Wintmyron), piromidic acid (Panacid), pipemidic acid (Dolcol), ciprofloxacin (Zoxan, Ciprobay, Cipro, Ciproxin), fleroxacin (Megalon, Rokinol), lomefloxacin (Maxaxquin), nadifloxacin (Aquachim, Nadoxine, Nadi Kusa), norfloxacin (Requinol, Noroxin, Kinabic, Janasin), ofloxacin (Floxin, Oxaldine, Tarivid), pefloxacin (Peflacin), rufloxacin (Uroflox), enoxacin (Enroxil, Penetrex), balofloxacin (Baloxin), grepafloxacin (Laxal), levofloxacin (Cravit, Levaquin), pazufloxacin (Pasil, Pazucross), Sparf loxacin (Zagam), temafloxacin (Omnifloxacin), tosufloxacin (Ozex, Tosacin), clinafloxacin, gatifloxacin (Zigat, Tekin, Zymar Ophthalmic), moxifloxacin (Avelox, Vigamox), sitafloxacin (Gracevit), prulifloxacin (Kisnon), besifloxacin (Besivance), delafloxacin (Baxdela), gemifloxacin (Factive) , and quinolones / fluoroquinolones such as trovafloxacin (Troban), ozenoxacin, danofloxacin (Advosin, Advocid), difloxacin (Dicular, Betequinone), enrofloxacin (Baytril), ivafloxacin (Ibafrin), marbofloxacin (Marbocil, Zenequin), orbifloxacin (Orbax, Victas), and sarafloxacin (Floxazol, Saraflox, Sarafin);Macrophages such as azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin / midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin / tylocine, roxithromycin, telithromycin, cethromycin, solithromycin, tacrolimus, pimecrolimus, sirolimus, amphotericin B, nystatin, and cluentaren. sulfonamides such as sulfacetamide, sulfadiazine, sulfadimidine, sulfafurazole (sulfisoxazole), sulfisomidine (sulfaisodimidine), sulfamethoxazole, sulfamoxole, sulfanitran, sulfadimethoxine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfadoxine, sulfamethpyrazine, and terephthyl; kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin B, C, Aminoglycosides such as neomycin E (paromomycin), streptomycin, plazomycin, amiquin, garamycin, cantrex, neo-flazine, netromycin, nebcin, fumatin, spectinomycin (Bs), and trobicin; carbapenems such as imipenem, meropenem, ertapenem, doripenem, panipenem / betamipron, biapenem, tebipenem, razupenem (PZ-601), lenapenem, tomopenem, and thienamycin (thiempenem); geldanamycin, herbimycin, rifaximin, and xifamycin. ansamycins such as oxazolamide; carbacephems such as loracarbef and lorabide; carbapenems such as ertapenem, invanz, doripenem, dorivax, imipenem / cilastatin, primaxin, meropenem, and melem; glycopeptides such as teicoplanin, thalgoside, vancomycin, vancocin, telavancin, bibativ, dalbavancin, dalvans, oritavancin, and orbactive; lincosamides such as clindamycin, cleocin, lincomycin, and lincocin; lipopeptides such as daptomycin and cubicin;Macrolides such as Azithromycin, Zithromax, Sumamed, Xytron, Clarithromycin, Biaxin, Dirithromycin, Dynavac, Erythromycin, Erythosin, Erythroped, Roxithromycin, Troleandomycin, Tao, Telithromycin, Ketek, Spiramycin, and Lobamycin; Monobactams such as Aztreonam and Azactam; Nitrofurans such as Furazolidone, Floxon, Nitrofurantoin, Macrodantin, and Macrobid; Linezolid, Zyvox, Vrsa, Pozizolid, Radezolid, and torezolid; polypeptides such as bacitracin, colistin, coli-mycin-S, and polymyxin B; mycobacteria agents such as clofazimine, lamprene, dapsone, abrosulfone, capreomycin, capastat, cycloserine, ceromycin, ethambutol, miambutol, ethionamide, trecatol, isoniazid, INH, pyrazinamide, aldinamid, rifampicin, rifadin, rimactane, rifabutin, mycobutin, rifapentine, priftin, and streptomycin. drugs; and other antibiotics such as arsphenamine, salvarsan, chloramphenicol, chloromycetin, fosfomycin, monulol, monulil, fusidic acid, fucidin, metronidazole, flagyl, mupirocin, bactroban, platensimycin, quinupristin / dalfopristin, sinercid, thiamphenicol, tigecycline, tigacyl, tinidazole, tindamax facidin, trimethoprim, proloprim, and trimpex; aluminum hydroxide, aluminum phosphate, aluminum sulfate, and other antibiotics; adjuvants including aluminum salts (alum); oil-in-water emulsion formulations; saponin adjuvants; complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); cytokines such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12), interferons, macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor (TNF); detoxified variants of bacterial ADP-ribosylating toxins such as cholera toxin (CT), pertussis toxin (PT), or E. coli heat-labile toxin (LT);It may be administered in combination with one or more other therapeutic agents, such as other agents for treating infectious diseases, including, but not limited to, vaccines, such as vaccines against tuberculosis, diphtheria, tetanus, whooping cough, Haemophilus influenzae type B, cholera, typhoid, and Streptococcus pneumoniae, as well as other vaccines containing bacterial antigen proteins or attenuated or killed bacteria to boost the immune response against bacteria, or other medicines used to treat a particular condition or disease according to a variety of administration schedules depending on the clinician's judgment, the patient's needs, and the like. Particular administration schedules will be known by those of skill in the art or can be determined empirically using routine methods. Exemplary administration schedules include, but are not limited to, 5 times a day, 4 times a day, 3 times a day, 2 times a day, once a day, 3 times a week, 2 times a week, once a week, twice a month, once a month, once a month, and any combination thereof. Preferred compositions are those requiring administration no more than once a day.

[0091] The nanoclusters may be administered before, at the same time, or after the other agent. When provided simultaneously with the other agent, the nanoclusters may be provided in the same or different compositions. Thus, the nanoclusters and one or more other agents may be presented to an individual by simultaneous therapy. By "combination therapy" is intended administration to a subject such that the therapeutic effect of the combination of substances is triggered in the subject undergoing therapy. For example, simultaneous therapy may be achieved by administering a dose of a pharmaceutical composition comprising the nanoclusters and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating an infection, according to a particular administration regimen, which in combination comprise a therapeutically effective dose. Similarly, the nanoclusters and one or more other therapeutic agents may be administered in at least one therapeutic dose. The administration of the separate pharmaceutical compositions may be performed at the same time or at different times (i.e., on the same or different days, sequentially in any order), so long as the therapeutic effect of the combination of these substances is triggered in the subject undergoing therapy.

[0092] kit The kit may include one or more containers of a composition described herein that includes functionalized nanoclusters conjugated to a nucleotide (e.g., ATP, dATP, ATPαS, ATPβS, ATPγS, 7-deaza-ATP, or AMP-PCP), or reagents for preparing such compositions, and optionally, one or more antibiotics for treating bacterial infections. The compositions may be in liquid form or may be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials, including glass or plastic. The containers may have a sterile access port (e.g., the containers may be intravenous solution bags or vials with a stopper pierceable by a hypodermic needle). The kits may further include a container that includes a pharma- ceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, or dextrose solution. The kits may also contain other materials useful to the end user, including other pharma- ceutically acceptable formulation solutions, such as buffers, diluents, filters, needles, and syringes or other delivery devices. The kits may also provide a delivery device pre-filled with the functionalized nanoclusters.

[0093] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods (i.e., instructions for treating bacterial infections with the nanoclusters described herein). These instructions may be present in a variety of forms within the subject kits, and one or more instructions may be present in the kit. One form in which these instructions may be present is as information printed on a suitable medium or substrate, such as a piece of paper (or pieces) on which the information is printed, on the kit packaging, on a package insert, or the like. Yet another form in which these instructions may be present is as a computer readable medium having the information recorded thereon, such as a diskette, compact disc (CD), DVD, Blu-ray, flash drive, and the like. Yet another form in which these instructions may be present is as a website address that may be used via the internet to access the information at a remote location.

[0094] Examples of Non-Limiting Aspects of the Disclosure Aspects including embodiments of the subject matter described above may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the present disclosure, numbered 1-48, are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or succeeding individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below. 1. A nanocluster comprising a metallic core conjugated to a nucleotide, the metallic core having a diameter of less than 10 nm. 2. The nanocluster of aspect 1, wherein the nucleotide is adenosine triphosphate (ATP) or a phosphorothioate analogue thereof, a deoxyribonucleotide analogue, a 7-deazapurine nucleotide analogue, or a phosphomethylphosphonate adenylate ester. 3. The nanocluster of embodiment 2, wherein the phosphorothioate analog is ATPαS, ATPβS, or ATPγS. 4. The nanocluster of embodiment 2, wherein the deoxyribonucleotide analog is deoxyadenosine triphosphate (dATP).

[0095] 5. The nanocluster of embodiment 2, wherein the 7-deazapurine nucleotide analog is 7-deazaadenosine-5'-triphosphate (7-deaza-ATP). 6. The nanocluster of embodiment 2, wherein the phosphomethylphosphonate adenylate is β,γ-methylene adenosine 5′-triphosphate (AMP-PCP). 7. The nanocluster of any one of aspects 1-6, wherein the diameter of the nanocluster is in the range of about 1 nm to about 2 nm, as measured using a transmission electron microscope. 8. The nanocluster of any one of aspects 1-7, wherein the metallic core comprises a noble metal. 9. The nanocluster according to embodiment 8, wherein the metallic core is a gold metallic core. 10. The nanocluster of any one of aspects 1-9, wherein the nanocluster is biocompatible with human cells. 11. The nanocluster according to any one of aspects 1-10, wherein the nanocluster is linked to an internalization sequence, a protein transduction domain, or a cell penetrating peptide.

[0096] 12. A composition comprising a nanocluster according to any one of aspects 1-11 for use in a method of treating an infectious disease. 13. The composition according to aspect 12, further comprising a pharma- ceutically acceptable excipient or carrier. 14. The composition of aspect 12 or 13, further comprising an antibiotic. 15. The composition according to any one of aspects 12 to 14, wherein the infection is a bacterial infection. 16. The composition of aspect 15, wherein the bacterial infection is a Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, or Escherichia coli infection.

[0097] 17. A method for treating an infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of a composition according to any one of aspects 12-16. 18. The method of embodiment 17, wherein the infection is a bacterial infection. 19. The method of embodiment 18, wherein the bacterial infection is a Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, or Escherichia coli infection. 20. The method of any one of aspects 17-19, further comprising administering a therapeutically effective amount of at least one antibiotic in combination with the composition. 21. The method according to any one of aspects 17 to 20, wherein the infection is a chronic infection.

[0098] 22. The method of any one of aspects 17-21, wherein the infection is an ear infection, a skin infection, a lung infection, a catheter-associated urinary tract infection, or a gastrointestinal infection. 23. The method of any one of aspects 17 to 22, wherein the infectious disease is associated with the formation of a bacterial biofilm in the subject. 24. The method of embodiment 23, wherein the biofilm is in a chronic wound in the subject. 25. The method of any one of aspects 17-24, wherein the subject has chronic suppurative otitis media (CSOM), cystic fibrosis, or tuberculosis. 26. The method of any one of aspects 17 to 25, wherein the infection comprises a pathogenic bacterium that is resistant to one or more antibiotics.

[0099] 27. The method of embodiment 26, wherein the subject has previously been treated for an infection with one or more antibiotics that have not been successful in clearing the infection. 28. The method of any one of aspects 17 to 27, wherein said treating eradicates all or a majority of biofilm and planktonic bacteria. 29. The method of any one of aspects 17-28, wherein said treating eradicates all or a majority of viable cells. 30. The method of embodiment 29, wherein the surviving cells are in a biofilm or internalized by a macrophage. 31. The method of aspect 29 or 30, wherein the survivor cells are multidrug-resistant survivor cells.

[0100] 32. The method of any one of aspects 29 to 31, wherein the surviving cells comprise gram-negative or gram-positive bacteria. 33. The method of embodiment 32, wherein the surviving cells comprise Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, or Escherichia coli. 34. The method of any one of aspects 17-33, wherein multiple cycles of treatment are administered to the subject. 35. The method of any one of aspects 17-34, wherein the composition is administered intravenously, subcutaneously, by inhalation, or topically. 36. The method of any one of aspects 17-34, wherein the composition is administered locally to the site of the infected tissue. 37. The method of embodiment 36, wherein the infection is an ear infection and the composition is administered locally into the ear canal. 38. The method of any one of aspects 17 to 37, wherein the infection is a Pseudomonas infection in a subject with cystic fibrosis. 39. A kit comprising the nanocluster of any one of aspects 1-11 and instructions for treating a bacterial infection.

[0101] 40. A method of eradicating bacteria in a biofilm, comprising contacting the biofilm with an effective amount of the nanocluster of any one of embodiments 1-11. 41. The method of embodiment 40, further comprising contacting the biofilm with an effective amount of at least one antibiotic. 42. The method of aspect 40 or 41, wherein the biofilm is on a medical device, a personal hygiene product, a toiletry, a cosmetic product, a disinfectant, a cleaning solution, or in a water treatment or distribution system. 43. A method of eradicating dormant bacteria, including surviving cells, comprising contacting the dormant bacteria with an effective amount of the nanocluster of any one of embodiments 1-11. 44. The method of embodiment 43, further comprising contacting the dormant bacteria with an effective amount of at least one antibiotic.

[0102] 45. The method of aspect 43 or 44, wherein the dormant bacteria are in a biofilm, in a liquid culture, or on an inanimate surface. 46. ​​A method of inhibiting a virulence factor of a bacterium, comprising contacting the bacterium with an effective amount of a nanocluster of any one of embodiments 1-11. 47. The method of claim 46, wherein the bacteria is selected from the group consisting of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. 48. The method of embodiment 47, wherein the virulence factor is Pseudomonas aeruginosa pyocyanin (PYO).

[0103] It will be apparent to those skilled in the art that various changes and modifications can be made to the present invention without departing from the spirit or scope of the present invention.

[0104] experiment The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0105] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0106] The present invention has been described with respect to specific embodiments found or provided by the inventors to include preferred modes for carrying out the invention. Those skilled in the art will understand in light of this disclosure that numerous modifications and changes can be made in the specific embodiments exemplified without departing from the intended scope of the invention. For example, codon redundancy allows changes in the underlying DNA sequence to be made without affecting the protein sequence. Furthermore, biological functional equivalence considerations allow changes to be made in the protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.

[0107] Example 1 Eradication of bacterial viable cells by exploiting their low metabolic activity using adenosine triphosphate-coated gold nanoclusters Introduction The rise of antibiotic resistance and the decline of new antibiotic discovery are causing a global health crisis [1-3]. In addition, it is known that bacteria can survive lethal doses of antimicrobial agents by reducing their metabolism, resulting in depletion of intracellular adenosine triphosphate (ATP) levels, not through genetic mutations that confer resistance [4]. In the process, bacteria enter a low metabolic state called surviving cells, which exhibit multidrug tolerance [5, 6]. Surviving cells do not grow in the presence of antimicrobial agents but can be resuscitated after treatment [7, 8]. Thus, recurrent and chronic infections are commonly associated with surviving cells [8-12].

[0108] Here, accumulating evidence has demonstrated that viable cells drive antibiotic resistance rates [13, 14]. In addition, it is now known that bacteria that encounter antibiotics first become tolerant and then through this tolerance genetic resistance emerges [15, 16]. Due to this link between antibiotic tolerance and resistance and the rise of antibiotic resistance, there is a pressing need to develop therapies to eradicate viable cells. However, killing viable cells is an unmet clinical need, due to the fact that Food and Drug Administration (FDA) approved antibiotics are not effective against viable cells and there are no potential prospects in the global preclinical bacterial pipeline

[17] . Ongoing research on the application of ultrasmall gold nanoclusters (AuNCs have a total diameter of <4 nm) exhibiting antibacterial activity for the treatment and diagnosis of bacterial infections has demonstrated their benefits as a potential alternative solution to conventional antibiotics in the fight against multidrug-resistant Gram-negative bacteria [18–24].

[0109] Most antibacterial AuNCs were discovered in experiments that tested the ability of compounds to inhibit bacterial growth [25–28]. However, they are often ineffective in treating non-growing bacteria with low metabolic activity. For example, previous attempts to kill bacterial cells with low metabolic activity using antibacterial AuNCs have failed, with a 97% reduction in antibacterial efficacy compared to that achieved in bacteria with active metabolism

[28] . This finding demonstrates that existing antibacterial AuNCs are insufficient to address viable cells with depleted intracellular ATP. Another important translational hurdle is the production of intracellular reactive oxygen species (ROS), the main mechanism of action leading to bacterial death induced by existing AuNC strategies, which risks turning metabolically active cells into viable cells [29, 30].

[0110] Supporting this idea, numerous independent studies have shown that compounds that induce ROS production in bacteria promote the formation of persister cells. For example, paraquat, a potent inducer of ROS and a widely used herbicide, encourages metabolically active bacterial cells to become persister cells

[31] . Another example is salicylic acid-induced ROS, which also causes increased persister cell formation

[32] . Sublethal antibiotic treatment has also been shown to result in multidrug resistance through ROS-induced mutagenesis

[33] . These results raise doubts about the appropriateness of developing antibacterial AuNCs, relying on ROS production as the primary mechanism of action to combat antibiotic resistance [23, 28, 34, 35]. Therefore, eradicating persister cells, one root cause of antibiotic resistance, is essential for antibacterial AuNCs to remain relevant in the clinical battlefield of combating antibiotic resistance. In addition, it is necessary to design a new class of AuNCs with a new antibacterial mechanism of action that is not dependent on ROS.

[0111] Current anti-survival cell strategies are based on the paradigm of “awakening” surviving cells from a hypometabolic state before attempting eradication with conventional antibiotics

[36] . Thus, we created a non-conventional antibacterial chemotherapy strategy based on AuNCs as an adjuvant to eradicate surviving cells by conventional antibiotics

[37] . However, surviving cells formed by multidrug-resistant bacteria can easily survive this therapeutic approach, since the AuNC / antibiotic combination cannot eradicate surviving cells once the bacteria become resistant to the partner antibiotic. Here, we show that the low metabolic activity of surviving cells can be exploited to target them over and above their metabolically active counterparts, resulting in complete eradication.

[0112] Results and Discussion Characterization of adenosine triphosphate coated gold nanoclusters (AuNC@ATP). Characterization data of AuNC@ATP are displayed in Figure 1. Transmission electron microscopy (TEM) confirms that AuNC@ATP are highly uniform, with an average core size of about 2.45 ± 0.43 nm. In addition, UV-visible spectra show the absence of a significant surface plasmon peak at about 500 nm, consistent with the small size of the particles

[38] . AuNC@ATP are negatively charged, with a zeta potential in phosphate buffer of -30 ± 2 mV, respectively. The molecular weight of AuNCs (g / mol) is calculated as follows: (i) weight of one AuNC (g) = volume (nm 3 )*Gold density (19.32g / cm 3 )*10 -21 cm 3 / nm 3 =1.487*10 -19 (ii) Molecular weight (g / mol) = weight of AuNC (g) * 6.022 × 10 23 Mol -1 =8.95*10 4 g / mol. The ATP molecular weight is 507 g / mol, which means that AuNCs (with a diameter of 2.45 nm) are 176 times heavier than ATP. Therefore, 99.4% of the weight of AuNCs@ATP is attributed to AuNCs. Furthermore, the amount of ATP in AuNCs@ATP (1 μg / ml or 11.2 nM) was estimated to be 1079 nM based on an ATP bioluminescence assay kit (FIG. 10). Based on this calculation, we estimate that one AuNCs@ATP contains 96 ATP molecules.

[0113] AuNC@ATP disrupts membrane integrity by increasing permeability without causing bacterial cell lysis. The outer membrane (OM) of Gram-negative bacteria is asymmetric, with phospholipids (PL) in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet

[39] . PL biosynthesis is completed in the cytoplasm (CM), also known as the inner membrane (IM), making translocation from the IM to the OM (anterograde transport) essential for filling the OM

[40] . PL translocation from the OM to the IM (retrograde transport) is thought to maintain the asymmetric LPS / PL structure of the OM

[40] . In the stationary phase, cells are no longer able to replace PL lost from the IM through stimulating new synthesis or retrograde transport. Therefore, a balance between anterograde and retrograde transport is essential for Gram-negative bacteria in the viable cell state to ensure the OM permeability barrier and cytoplasmic membrane integrity required for cell survival [41, 42]. It has previously been observed that anterograde transport is ATP-independent but can be abolished by ATP hydrolysis

[43] .

[0114] Moreover, in contrast to anterograde transport, ATP drives retrograde PL transport

[43] . Stationary-phase AuNC@ATP-mediated cell death has been shown to occur through the disruption of lipid homeostasis and activate a novel cell death pathway

[44] . We first investigated whether AuNC@ATP induces changes in OM permeability using 8-anilino-1-naphthalenesulfonic acid (ANS), a fluorescent lipophilic dye that exhibits enhanced fluorescence in hydrophobic environments [45–49]. We chose colistin (polymyxin E), which disrupts the OM, as a positive control [50, 51]. Stationary-phase cultures of multidrug-resistant Gram-negative bacteria, including Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Klebsiella pneumoniae (K. pneumoniae), were treated with either AuNC@ATP or colistin. We found that the interaction between Gram-negative bacteria and AuNC@ATP carried negative charges on the surface, as evidenced by the increase in ANS fluorescence, leading to impaired OM permeability (Fig. 2A). In P. aeruginosa, the impact of AuNC@ATP on OM permeability was comparable to that induced by colistin. Meanwhile, in E. coli and K. pneumoniae, AuNC@ATP induced more damage than colistin, which is positively charged (Fig. 2A). We next examine whether AuNC@ATP induces cytoplasmic membrane (CM) disruption. Propidium iodide (PI) is a membrane-impermeant stain that labels only bacteria with compromised IM

[52] . We found that after AuNC@ATP treatment, PI could penetrate the CM, suggesting damage to the CM (Fig. 2B). In P. aeruginosa and K. pneumoniae, the impact of AuNC@ATP on OM permeability was comparable to that induced by colistin. Meanwhile, in E. coli, AuNC@ATP induced more damage than colistin ( Figure 2 A).

[0115] To demonstrate that AuNC@ATP is active against bacterial cells in the growth-arrested (surviving cell) phase, we compared the bactericidal activity of AuNC@ATP and ofloxacin against stationary-phase Gram-negative bacteria. We found that AuNC@ATP sterilized stationary-phase cultures of P. aeruginosa, E. coli, and K. pneumoniae (Figure 3A). Conversely, ofloxacin failed to eradicate stationary-phase cultures (Figure 3A), broadly supporting other research findings in this field linking bacterial metabolic state with antibiotic lethality [4, 53]. Furthermore, these results suggest that exposure to AuNC@ATP induces Gram-negative cell envelope stress that activates cell death.

[0116] Considering that one of the most striking features of cell death induced by the disruption of PL homeostasis is that cell death does not occur by cell lysis

[44] , we therefore analyzed whether AuNC@ATP caused cell lysis using the presence of proteins in the supernatant of Gram-negative bacteria treated with AuNC@ATP as a proxy for bacterial cell membrane lysis. Colistin and ofloxacin cause bacterial cell lysis by forming membrane pores and peptidoglycan composition [54–56]. However, in contrast to these conventional antibiotics, no cell lysis was observed after exposure of Gram-negative bacteria to AuNC@ATP, as seen by the absence of proteins in the supernatant (Figure 3B). To further prove that the entire protein pool was still within the bacteria after AuNC@ATP treatment, we lysed bacterial cells treated with AuNC@ATP with colistin. As expected, protein levels were comparable to those obtained from the supernatant of cells treated with colistin (Figure 11). Furthermore, AuNC@ATP did not mediate lytic cell death, in contrast to the mechanism of action of existing antimicrobial AuNCs, which results in lysis. [28, 57] This effort demonstrated that AuNC@ATP functions through a mechanism distinct from known classes of bactericidal antibiotics, including cationic antimicrobial polypeptides and existing antimicrobial nanoparticles.

[0117] To further support that the mechanism of action of AuNC@ATP is different from existing antibacterial AuNCs, in which ROS production is essential to achieve bacterial killing [28, 35], we use the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) to confirm that the internalization of AuNC@ATP in bacterial cells does not increase intracellular ROS. As expected, no change in DCFH-DA fluorescence intensity was observed in stationary-phase cultures of P. aeruginosa (Pa14) treated with AuNC@ATP compared to ofloxacin and untreated PA14 cells (Figure 12). Cumulatively, the data in this section support that after translocation through the OM, AuNC@ATP leads to fusion of the inner leaflet of the OM with the outer leaflet of the cytoplasmic membrane, which leads to bacterial death without cell lysis.

[0118] Accumulation of unfolded outer membrane proteins (OMPs) causes lethality of AuNCs@ATP. We next evaluated the ability of AuNCs@ATP to induce a stress response that causes the lethal accumulation of unfolded outer membrane proteins (OMPs). Bacteria express sigma factor (σ) in the cytoplasm, a stress gene. E )-dependent transcription. The β-barrel structure of OMPs is not found in the cytoplasmic membrane, since it represents an open pore that allows the diffusion of water, ions, and hydrophilic molecules up to 600 Da

[58] . E To activate the response, unfolded OMPs in the periplasm typically E It triggers a proteolytic cascade involving ClpXP, an ATP-dependent cytoplasmic protease (RseA) that binds to and destroys inhibitory transmembrane proteins [58, 59]. E When the system is activated, the gene is transformed into σ E It is transcribed from a σ-dependent promoter, leading to upregulation of the OMP folding pathway, preventing the accumulation of highly toxic unfolded OMPs

[58] . However, in cells lacking ClpXP, σ EWhen the response is triggered, it fails to activate the upregulation of the OMP folding pathway, leading to cell death due to the accumulation of unfolded OMPs in the periplasm. Therefore, to test whether exposure to AuNC@ATP accumulates unfolded OMPs in the periplasm and causes envelope damage, we compared the antibacterial activity of AuNC@ATP against P. aeruginosa(PA14) and PA14(ΔClpXP). We found that PA14(ΔClpXP) was 8-fold more sensitive to AuNC@ATP than PA14(ClpXP). The minimum inhibitory concentrations (MICs) of PA14(ClpXP) and PA14(ΔClpXP) were 2.23 μM and 0.28 μM, respectively (Figures 4A and 4B). Cumulatively, this supports that the internalization of AuNC@ATP in bacterial cells induces the lethal accumulation of unfolded OMPs. In line with the fact that AuNC@ATP-mediated cell death occurs without releasing periplasmic proteins and cytoplasmic components into the supernatant, the hypersensitivity of PA14(ΔClpXP) to AuNC@ATP indicates that cell death occurs through the lethal accumulation of unfolded OMPs in the periplasmic space (Figure 4C and Figure 11). We conclude that AuNC@ATP has antibacterial activity that can be attributed to the stress-response accumulation of unfolded OMPs in the periplasm, which disrupts the bacterial membrane by altering lipid homeostasis and asymmetry.

[0119] AuNC@ATP-mediated cell death is through the lethal accumulation of unfolded OMPs, which is involved in σ E The molecular mechanisms by which this response is impaired remain unclear. EThe regulon includes periplasmic chaperones that maintain OMPs in an unfolded state in the periplasm, members of the β-barrel assembly machine (BAM) complex responsible for inserting OMPs into the OM, and periplasmic proteases that degrade misfolded OMPs. If both processes are defective, unfolded OMPs may accumulate in the periplasm. Further investigation of the open questions regarding the molecular mechanism of action will bring interesting insights regarding the potential interactions between AuNC@ATP and the BAM complex or periplasmic proteases. A remarkable feature of the periplasmic space of Gram-negative bacteria is that it contains more than 300 proteins and is devoid of ATP, thus providing a unique protein folding and stabilization environment [60, 61]. ATP has been found to keep proteins in their soluble form and prevent their aggregation

[62] . Furthermore, proteome-wide profiling analysis suggested that ATP regulates the solubility of a remarkably large set of proteins

[63] . Computational simulations have demonstrated that ATP can unfold single chains of hydrophobic macromolecules

[64] . Thus, these findings partially explain why AuNC@ATP induces the unfolding of OMPs in the periplasm.

[0120] Surviving cells are more sensitive to AuNC@ATP than metabolically active bacterial cells. Recent studies have shown a reduction in ATP levels in bacterial surviving cells [10, 65, 66]. The reduction in ATP levels correlates with reduced proteolysis of functional proteins by different ATP-dependent proteases

[67] . Because AuNC@ATP induces lethal accumulation of unfolded OMPs, the lethality of AuNC@ATP should increase as bacteria transition from a metabolically active state to a metabolically suppressed state (i.e., low ATP levels) due to reduced proteolysis of unfolded OMPs. Furthermore, surviving cells will be unable to synthesize new PL to replace the PL lost from the OM when stimulating retrograde transport. We reasoned that these features could be exploited to eradicate surviving cells with low metabolic activity. To test this hypothesis, we compared the bactericidal activity of AuNC@ATP against exponential and surviving cells. Passaging through stationary phase is usually associated with the formation of viable cells, which represent approximately 1% of the total bacterial population [9, 12]. Therefore, 48-h-old stationary phase was treated with ofloxacin for 24 h to eradicate non-viable cells (Figure 5A). Ofloxacin was used at 415 μM, which corresponds to 30-fold the MIC of the PAO1 wild-type strain. The surviving viable cells were concentrated and resuspended in phosphate-buffered saline (PBS) to prevent viable cells from waking up from a metabolically inhibited state. We confirmed that viable cells had reduced ATP levels compared to exponentially growing bacterial cells (Figure 5B). We found that AuNC@ATP (2.2 μM) resulted in a 7-log reduction in bacterial numbers (CFU / ml), compared to less than a log reduction when bacteria were in exponential growth phase (Figure 5C). In addition, we found that the viable cell inoculum (10 8 CFU / ml) were sterilized with 4.45 μM AuNC@ATP. However, when bacteria reached the exponential growth phase (10 8 ~10 5 CFU / ml), only a 3 log reduction was observed.

[0121] No eradication was observed in viable cells after exposure to up to 10 mM ATP, proving that the entire entity of AuNC@ATP was required to eradicate viable cells (Figure 13). Furthermore, in contrast to previous studies reporting a 97% reduction in the antibacterial efficacy of AuNCs when bacteria exhibit low metabolic activity, our findings demonstrate that AuNC@ATP-mediated cell death increases as bacteria transition from a metabolically active to a metabolically inhibited state (i.e., low ATP levels). This supports the conclusion that AuNC@ATP is a new class of antibacterial nanoclusters with a unique and novel mechanism of action. This AuNC@ATP-mediated viable cell death contrasts with the bactericidal activity of conventional antibiotics, whose lethality decreases as bacteria transition from a metabolically active to a metabolically inhibited state [4]. In contrast to current anti-viable cell strategies, this effort is based on the paradigm of “waking” viable cells from a low metabolic state prior to eradication attempts with conventional antibiotics, demonstrating that the low metabolic activity of viable cells can be exploited for eradication over and above their metabolically active counterparts. AuNC@ATP is presented as a benchmark nanocluster to prove the feasibility of this concept.

[0122] P. aeruginosa cannot produce pyocyanin in the presence of sublethal doses of AuNC@ATP. During infection, P. aeruginosa often comes into contact with other pathogens

[68] . Pyocyanin (PYO), a small molecule produced by P. aeruginosa, has been shown to increase the viable cell population of other pathogens in contact with P. aeruginosa. The Gram-negative coccobacillus Acinetobacter baumannii (A. baumannii) is currently at the forefront of the WHO list of pathogens in critical need of new therapeutic development. A. baumannii formed 0.07 and 0.02% viable cells in the presence of amikacin and carbenicillin

[69] . However, in the presence of PYO, this increased 4- and 3-fold

[69] . Considering that PYO promotes vivisection formation from neighboring bacteria in coinfection with P. aeruginosa, our next goal was to elucidate whether AuNC@ATP can act as an inhibitor of PYO production. The chloroform-HCl method was used to evaluate PYO production. We consequently found that PYO production by P. aeruginosa (PA14) was 1.15 ± 0.20 μg / mL and 0.23 ± 0.19 μg / mL in the presence of 0.42 and 0.56 μM AuNC@ATP, respectively, compared to 5.92 ± 0.25 μg / mL in the absence of AuNC@ATP (Figure 6). We concluded that AuNC@ATP is a multifunctional platform. Thus, apart from being used to eradicate or prevent bacterial growth, AuNC@ATP can function as an antivirulence agent that can attenuate PYO production of P. aeruginosa. Several infections associated with the cytotoxic effect of PYO have been reported

[70] . For example, PYO increases interleukin-8 expression by human airway epithelial cells

[71] and mediates tissue injury leading to necrosis during pulmonary infection

[72] .Recently, the FDA approved five antivirulence drugs, including two immunoglobulins (BabyBIG and BAT for Clostridium botulinum) and three monoclonal antibodies (raxibacumab and obiltoxaximab for Bacillus anthracis, and bezlotoxumab for C. difficile)

[73] . In line with this, our data indicate that AuNC@ATP, although an antibacterial nanocluster, can also be used as an antivirulence drug for P. aeruginosa. In future investigations, it may be possible to use AuNC@ATP for antivirulence therapy. The rationale is that once the virulence traits are removed, the bacteria are rendered benign and more easily eliminated by the host immune system

[74] .

[0123] Bacteria do not develop resistance to AuNC@ATP, which can prevent sublethal antibiotic treatment from inducing multidrug resistance. An ideal antibiotic would exhibit a low potential for resistance development alongside the ability to eradicate surviving cells. To address the resistance rate more quantitatively, we serially passaged two biologically independent cultures of P. aeruginosa from (PAO1) in sublethal concentrations of AuNC@ATP, as well as two control antibiotics: tobramycin (targeting protein synthesis) and ofloxacin (targeting DNA gyrase). During 21 passages, we successfully isolated mutants resistant to all control antibiotics, indicating that no AuNC@ATP-resistant mutants emerged (Figure 7A). For tobramycin and ofloxacin, resistance gradually increased throughout the experiment, with MICs increasing by 30-fold and more than 1500-fold, respectively. On the other hand, for AuNC@ATP, the resistance level remained constant, indicating that P. aeruginosa did not acquire partial resistance to AuNC@ATP. Notably, PAO1 resistance gradually increased after 20 passages in culture medium containing subinhibitory concentrations of silver nanoparticles

[75] . This further supports the conclusion that AuNC@ATP is a new class of antibacterial nanoclusters with a distinct mechanism of action.

[0124] It has been suggested that surviving cells are the main cause of the emergence of genetic antibacterial resistance

[14] . Considering that AuNC@ATP targets surviving cells over their metabolically active counterparts, it may serve as a valuable research tool to kill multidrug-resistant subpopulations within homogeneous cultures of bacteria genetically susceptible to antibiotics to demonstrate that resistance emerges from multidrug-resistant subpopulations of bacterial cells. To test this idea, we repeated our serial passage study with two biologically independent cultures of PAO1. Throughout 21 passages, PAO1 rapidly acquired resistance to ciprofloxacin, accompanied by a >250-fold increase in MIC (Figure 7B). However, PAO1 resistance to ciprofloxacin was dramatically reduced in the presence of AuNC@ATP (1.12 μM), which has been demonstrated earlier in this study to selectively kill surviving cells (i.e., a >2-log reduction in CFU / ml over metabolically active counterparts) (Figure 7B). This result supports our conclusion that AuNC@ATP could be a new research tool to demonstrate the link between viable cells and antibiotic resistance development. In addition, this study supports evidence from previous observations that viable cells promote antibiotic resistance rates [14, 66].

[0125] AuNC@ATP prevents cross-resistance, which triggers the emergence of superbugs upon exposure to sublethal doses of antibiotics. Superbugs are bacterial strains that are resistant to several types of antibiotics. Cross-resistance refers to the situation where one antibiotic confers resistance to other drugs within an antibiotic class or to unrelated drugs with different mechanisms of action

[76] . Cross-resistance to β-lactam antibiotics is observed in bacterial populations evolving during exposure of P. aeruginosa to sublethal concentrations of ciprofloxacin

[77] . We demonstrate that strategies addressing surviving cells are a promising approach to combat the emergence of multidrug-resistant superbugs. Thanks to AuNC@ATP, we can now prove that the presence of surviving cells in homogenous cultures of bacteria genetically sensitive to ciprofloxacin is the main cause of cross-resistance triggering the emergence of superbugs, and that their metabolically active counterparts do not play a major role. We investigated the effect of ciprofloxacin without subinhibitory concentrations of AuNCs@ATP (i.e., PAO1 Cip21 ) and ciprofloxacin containing AuNC@ATP (i.e., PAO1 Cip21-AuNC@ATP This was demonstrated by examining the antibiotic resistance profile of the PAO1 isolate after 21 passages in medium containing

[0126] We found that PAO1 was significantly more potent than PAO1, as evidenced by a reduced inhibition zone diameter test for antibacterial activity compared to the ancestral PAO1. Cip21 We found that the P. aeruginosa superbug strain (PAO1) was multidrug-resistant to carbapenems, atypical β-lactam antibiotic classes (meropenem and imipenem), aminoglycosides (tobramycin and amikacin), polymyxins (colistin and polymyxin-B), aztreonam, and cephalosporins (cefepime and ceftazidime) (Figure 8). Cip21 ) can be assumed to emerge during exposure to sublethal concentrations of ciprofloxacin. In contrast, PAO1 Cip21-AuNC@ATPshow similar inhibition zone diameters as observed in the ancestral PA01 (Figure 8). It can therefore be suggested that persister cells are the main cause of the emergence of P. aeruginosa superbug strains during exposure to sublethal concentrations of ciprofloxacin. Furthermore, this study confirms that persister cells are associated with enhanced antibiotic resistance development from fluoroquinolones

[78] . Cumulatively, the data in this section lay the foundation for developing novel nanoantibiotic adjuvants such as AuNC@ATP that would thwart the development of superbugs with the advantage of extending the life span of current antibiotics.

[0127] Nonclinical safety and toxicity of AuNC@ATP after multiple doses of intravenous and intraperitoneal injection. The quantitative parameters of toxicity of AuNC@ATP in mice are presented in Table 1. The median lethal dose (LD ) of a single injection of AuNC@ATP administered intravenously (IV) and intraperitoneally (IP) in mice was 0.01 mg / kg / day. 50 ) were 205.12 and 346 mg / kg, respectively. For comparison, the LD of a single injection of tobramycin sulfate administered to mice by IV and IP was 50were 77 and 262 mg / kg, respectively

[79] . Furthermore, the highest tolerated dose of AuNC@ATP that could be administered intravenously to mice without causing any signs of toxicity (i.e., IV-MTD) was 38.19 mg / kg. The IP-MTD was estimated to be between 95 mg / kg (no deaths) and 195 mg / kg (1 / 10 of mice died) for intraperitoneal injection. No clinical laboratory signs of toxicity were found after 14 days of IP injection of AuNC@ATP at a dose equivalent to the IV-MTD 3 times / day. As shown in Figure 9, we observe that AuNC@ATP did not affect the hematology and clinical chemistry parameters measured. Since AuNCs are cleared from the body through the liver and kidneys [80, 81], one particular interest focuses on liver and kidney toxicity. Changes in alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin (TBIL) levels typically indicate liver damage. Changes in creatinine (CR) and blood urea nitrogen (BUN) levels are associated with kidney damage. No abnormal liver and kidney function-related parameters were observed compared to the control group (PBS) (Figure 9). Cumulatively, the data in this section demonstrate the lack of toxicity of AuNC@ATP.

[0128] conclusion In summary, we engineer adenosine triphosphate-coated gold nanoclusters (AuNC@ATP) as a benchmark nanocluster that outperforms exponentially growing bacterial cells and eradicates viable cells, proving that the low metabolic activity of viable cells can be exploited for eradication and modulation of P. aeruginosa virulence. We also demonstrate that AuNC@ATP can serve as a valuable research tool to prove the link between viable cells and the development of antibiotic resistance. Finally, repeated dose toxicity studies in mice demonstrate its potential safety. Cumulatively, these findings show the promise of AuNC@ATP to eradicate viable cell-driven infectious diseases. EXAMPLES

[0129] Synthesis of AuNC@ATP Freshly prepared aqueous solutions of HAuCl4 (20 mM, 5 mL) and ATP (50 g, 50 mL) were mixed in water (790 mL). After that, aqueous NaOH solution (1 M, 60 mL) was added to the mixture. The mixture was then boiled for 5 min, and the AuNC@ATP solution was cooled to room temperature. After synthesis, AuNC@ATP was collected by centrifugation (4000 g, 60 min) of AuNC@ATP in a Pall Macrosep Advance centrifugation device (membrane, MWCO = 3000). Finally, AuNC@ATP was washed three times with deionized water by centrifugation. The resulting AuNC@ATP was freeze-dried and completely dried before further use.

[0130] AuNC@ATP characterization The absorption spectra of AuNC@ATP were recorded in the visible region of the electromagnetic spectrum (400–800 nm) using an absorption spectrophotometer (spectramMax M2, Molecular Devices, Downington, PA). Furthermore, transmission electron microscopy (TEM) images of AuNC@ATP were acquired to analyze the morphology and measure the core size. In addition, the zeta potential of AuNC@ATP was measured using a Malvern Zetasizer Nano ZS at nanoComposix, San Diago, CA 92111.

[0131] Bacterial strains, growth media, and conditions We used multidrug-resistant bacteria from the CDC & FDA Antibiotic Resistance Isolate Bank. In addition, P. aeruginosa strain PA01 was obtained from ATCC, and PA14 and PA14(ΔClpXP) were obtained from Newman's lab at the California Institute of Technology. In all experiments, bacterial cells were grown in 10 mL of lysogeny broth at 37°C and aerated at 200 rpm in 50 mL plastic polypropylene tubes. Exponential phase cultures were prepared as follows: stationary overnight cultures were diluted 1:1000 in LB and the optical density at 600 nm (OD 600The mixture was incubated at 37° C. with aeration at 200 rpm until the optical density at 600 nm (OD )=0.3 was reached. 600 ) were read every 15 min in a microplate reader (spectramMax M2, Molecular Devices, Downington, PA) to generate growth curves.

[0132] Tolerance Development Serial passage MICs were performed on 96-well microtiter panels. First, an aliquot of the well was taken at the highest concentration that allowed growth and diluted (1 / 100) in fresh medium from the inoculated microtiter panel. After overnight incubation at 37°C, this suspension was diluted to 0.5 McFarland standard turbidity and used to inoculate a new MIC panel, yielding 1.5 × 10 6 A final concentration of CFU / ml was obtained. The panel was incubated according to CLSI guidelines, the MIC was recorded, and the next inoculum was prepared from the well containing the highest drug concentration that allowed the same growth as above. 21 repeated passages were performed.

[0133] Bacterial susceptibility using disk diffusion assay The PA01 strain (the ancestor), ciprofloxacin without AuNC@ATP at subinhibitory concentrations (i.e., PAO1) and ciprofloxacin at subinhibitory concentrations (i.e., 100–2000 μg / mL) were analyzed for susceptibility to a panel of anti-Pseudomonas aeruginosa drugs using the disk diffusion method according to the Clinical Laboratory Standard Institute (CLSI) guidelines. Cip21 ) and ciprofloxacin containing AuNC@ATP (i.e., PAO1 Cip21-AuNC@ATPPAO1 isolates after 21 passages in medium containing 100% glycerol were tested. Briefly, glycerol stocks of each bacteria were streaked onto LB plates and grown overnight at 37°C. Inocula were prepared by diluting several individual colonies in PBS to 0.5 McFarland standard turbidity. The inocula were then spread with a sterile cotton swab onto Mueller Hinton agar plates supplemented with 5% sheep blood. Disks containing 5 μg of antipseudomonal drug were dispensed onto the surface of the plates. After 24 h of incubation at 37°C, the zones of inhibition were measured using digital calipers.

[0134] Survival cell generation Viable cells of P. aeruginosa (PA14) were isolated by treating 250 ml of stationary culture with ofloxacin (final concentration of 415 μM). After 24 h of treatment, samples were washed with PBS and then viable cells were concentrated in 10 ml of PBS. The number of viable cells was estimated by serial dilutions to determine colony forming units per milliliter (CFU / ml).

[0135] Assessment of intracellular ATP levels and total protein ATP levels were measured in exponentially surviving and ofloxacin-induced surviving cells using the BacTiter Glo kit (Promega) according to the manufacturer's instructions. Protein levels were determined using the bicinchoninic acid (BCA) assay (Thermo Scientific, Pierce) according to the manufacturer's instructions.

[0136] Bactericidal activity against surviving cells All bactericidal tests were performed on PBS without a carbon source to prevent surviving cells from awakening from low metabolic activity. Ofloxacin-induced surviving cells were challenged with antimicrobial agents at concentrations listed in the text. Viable bacteria were washed three times with PBS, after which the pellet was resuspended in 100 μL of PBS and then spread on LB plates. Plates were incubated at 37°C for 72 h before growth was assessed.

[0137] In vivo cytotoxicity Animal treatment and sample collection. The Stanford University Administrative Committee approved all animal work in the Laboratory Animal Care. BALB / c mice, aged 10-12 weeks, were purchased from Jackson Laboratories (Sacramento, CA) and housed in the Stanford University Animal Resources Facility according to standard guidelines with food and water provided ad libitum in rooms maintained on a 12-h dark / light cycle. Mice (male = 5 per group and female = 5 per group) BALB / c mice were divided into two groups, including control (PBS) and AuNC@ATP. Treatments were administered intravenously (IV) or intraperitoneally (IP) at doses listed in the text. AuNC@ATP was administered by IP three times a day for 14 consecutive days for subacute toxicity studies. Mice were then sacrificed 14 days after the last injection. Blood was collected for further investigation of serum chemistry and hematology. Blood samples were subjected to toxicity analysis. Inferior vena cava blood collection was performed at the time of sacrifice. Blood (150 μl) was placed in K2 EDTA tubes for hematological analysis and the remaining blood sample was placed in 1.5 mL Eppendorf tubes for serum extraction. For liver and kidney function tests, serum was separated by centrifuging the blood and removing the cellular fraction.

[0138] Generation of intracellular reactive oxygen species (ROS) The intracellular ROS concentration was tested by applying DCFH-DA (2',7'-dichlorofluorescein diacetate) dye, which can be cleaved to DCFH by intracellular nonspecific esterases. DCFH is further oxidized to fluorescent DCF (2',7'-dichlorofluorescein) in the presence of ROS. DCFH-DA (10 μM) in DMSO was added to the treated bacterial solution and further incubated at 37 °C and 200 rpm for 15 min. Then, the bacterial cells were centrifuged at 8000 g for 5 min, washed three times with PBS, and resuspended to the original volume (1 mL) in ultrapure water. The concentration of generated DCF was measured using a microplate reader with excitation / emission wavelengths of 488 / 525 nm. In this experiment, the fluorescence intensity of DCF directly reflects the amount of ROS generated. The amount of ROS was then normalized to the total cell number, which was expressed as the optical density at 600 nm (OD 600 Finally, the relative ROS production levels were calculated by normalizing the ROS levels from the treatment groups with the production levels in the PBS-treated group.

[0139] Outer membrane permeability assay After treatment with antimicrobial agents, stationary-phase cultures of Gram-negative bacteria were diluted with PBS and analyzed for optical density at 600 nm (OD 600 A suspension with a pH of 0.5 was formed and 50 μl of 3 mg / ml 8-anilino-1-naphthalenesulfonic acid (ANS) was added. After equilibration at 37 °C for 30 min, the cells were washed with PBS by centrifugation (8000 g, 5 min) and resuspended in fresh PBS solution (1 ml). The fluorescence intensity from 450 to 650 nm was then measured with excitation at 380 nm.

[0140] Cytoplasmic membrane permeability assay After treatment with antimicrobial agents, stationary-phase cultures of Gram-negative bacteria were diluted with PBS and analyzed for optical density at 600 nm (OD 600A suspension with pH = 0.5 was formed and 5 μl of 1 mg / ml propidium iodide (PI dissolved in sterile deionized H2O) was added. After equilibration at 37 °C for 30 min, the cells were washed with PBS by centrifugation (8000 g, 5 min) and resuspended in fresh PBS solution (1 ml). The fluorescence intensity from 550 to 800 nm was then measured with excitation at 530 nm.

[0141] Pyocyanin quantification assay Pyocyanin concentrations were determined as described by Essar et al.

[82] . First, the optical density at 600 nm (OD 600 Aliquots of exponentially growing cultures of PA14 at 0.5 were taken and diluted (1 / 100) in 50 mL of lysogeny broth (LB) in the absence and presence of sublethal doses of AuNC@ATP, incubated at 37 °C, and aerated at 200 rpm to maximize pyocyanin production. After 24 h, bacterial cells were removed from the cultures by centrifugation (8000 g, 5 min). Pyocyanin was then extracted from the supernatant by adding chloroform to a total of 50% of the total volume. The mixture was vortexed vigorously for 30 s and the sample was allowed to settle for 10 min to allow the aqueous phase to separate. The bluish chloroform fraction was then carefully transferred to a new tube. 0.1 N HCl was then added to 20% of the total volume and vortexed vigorously for 30 s. After this step, the chloroform fraction turned from blue to clear, and the small aqueous fraction of the added acid turned from clear to pink. Again, the samples were allowed to settle for 10 min to allow the aqueous phase to separate. Finally, the aqueous fraction was removed and the OD 520 The absorbance at OD 520 The concentration, expressed as micrograms of pyocyanin produced per milliliter of culture supernatant, was determined by multiplying OD by 17.072. The concentration values ​​were then normalized to the cell density of each sample (OD 600 ).

[0142] statistical analysis Data are expressed as mean ± sd. Statistical analyses (details in figure legends) were calculated using GraphPad Prism Ver. 9 (GraphPad, San Diego, CA). A p value of <0.05 was considered statistically significant. [Table 1] [Technical field]

[0143] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Contract AI154097 awarded by the National Institutes of Health. The Government has certain rights in this invention.

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Claims

1. A nanocluster comprising a metallic core conjugated to a nucleotide, said metallic core having a diameter of less than 10 nm.

2. 2. The nanocluster of claim 1, wherein the nucleotide is adenosine triphosphate (ATP) or its phosphorothioate analog, a deoxyribonucleotide analog, a 7-deazapurine nucleotide analog, or a phosphomethylphosphonate adenylate ester.

3. 3. The nanocluster of claim 2, wherein the phosphorothioate analog is ATPαS, ATPβS, or ATPγS; the deoxyribonucleotide analog is deoxyadenosine triphosphate (dATP); the deoxyribonucleotide analog is deoxyadenosine triphosphate (dATP); or the phosphomethylphosphonate adenylate ester is β,γ-methylene adenosine 5′-triphosphate (AMP-PCP).

4. 3. The nanocluster of claim 1 or 2, wherein the diameter of the nanocluster ranges from about 1 nm to about 2 nm as measured using transmission electron microscopy.

5. 3. The nanocluster of claim 1 or 2, wherein the metallic core comprises a noble metal.

6. 6. The nanocluster of claim 5, wherein the metallic core is a gold metallic core.

7. 3. The nanocluster of claim 1 or 2, wherein the nanocluster is linked to an internalization sequence, a protein transduction domain, or a cell-penetrating peptide.

8. 10. A composition comprising the nanocluster of claim 1 for use in a method of treating an infectious disease.

9. 9. The composition of claim 8, further comprising a pharmaceutically acceptable excipient or carrier and / or an antibiotic.

10. 10. The composition of claim 8 or 9, wherein the infection is a bacterial infection, optionally wherein the bacterial infection is a Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, or Escherichia coli infection.

11. 10. A method for eradicating bacteria in a biofilm or dormant bacteria containing surviving cells, comprising contacting said biofilm or said dormant bacteria with an effective amount of the nanocluster of claim 1 or 2.

12. 12. The method of claim 11, further comprising contacting the biofilm or the dormant bacteria with an effective amount of at least one antibiotic.

13. 12. The method of claim 11, wherein the biofilm or the dormant bacteria are on a medical device, personal hygiene product, toiletry, cosmetic product, disinfectant, cleaning solution, or in a water treatment or distribution system.

14. The method described in claim 11, wherein the biofilm or the dormant bacteria are in a liquid culture or on an inanimate surface.

15. 10. A method of inhibiting bacterial virulence factors, comprising contacting said bacteria with an effective amount of the nanocluster of claim 1 or 2.

16. 16. The method of claim 15, wherein the bacterium is selected from the group consisting of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli.

17. 17. The method of claim 16, wherein the virulence factor is Pseudomonas aeruginosa pyocyanin (PYO).

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