Nitric oxide-releasing liposomes as therapy for respiratory infections

Abstract

Described herein are diazeniumdiolate nitric oxide (NO) donors encapsulated in liposomes. Also described are methods of making the liposomes and methods of treating respiratory infections, such as nontuberculosis Mycobacterium (NTM) with the NO-releasing liposomes.

Description

Attorney Docket No.035052 / 626621 NITRIC OXIDE-RELEASING LIPOSOMES AS THERAPY FOR RESPIRATORY INFECTIONS BACKGROUND

[0001] Non-tuberculosis mycobacteria (NTM) are ubiquitous, opportunistic pathogens that cause severe respiratory infection in the elderly and immunocompromised populations. Unlike other mycobacterial infections (e.g., tuberculosis, leprosy), the instance of NTM infections have risen in developed nations over the last two decades. The most common pathogenic strains of NTM include members of the Mycobacterium avium complex (MAC) and the Mycobacterium abscessus (M. abscessus) groups.

[0002] The current standard of care for NTM pulmonary infection is amikacin, an aminoglycoside that is administered intravenously or via inhalation independently or in combination with other antibiotics. Amikacin treatment is administered while the individual is hospitalized and can last up to 24 months, with high rates of renal and ototoxicity reported. Furthermore, culture conversion (i.e., three consecutive negative cultures at least four weeks apart) is low (between 13 and 40%) even when combined with other antibiotic classes.

[0003] The limitations of traditional antibiotics are due to the intrinsic properties of NTM. Because of their hydrophobic cellular envelopes, NTM survive within immune cells following phagocytosis, prompting the immune system to form a nodulous mass of cells called a granuloma, which most antibiotics cannot cross. As a result, even if the extracellular infection of NTM has been controlled, intracellular NTM can still persist and multiply, necessitating innovation in pulmonary therapeutic delivery. There remains a need for new therapeutics that can successfully treat intracellular NTM infections. BRIEF SUMMARY

[0004] Described are nitric oxide (NO)-releasing liposomes comprising a lipid bilayer comprising an inner leaflet and an outer leaflet; and a NO donor moiety in an aqueous core encapsulated by the lipid bilayer, wherein the NO donor moiety comprises one or more diazeniumdiolate groups. Compositions comprising a plurality of the liposomes in an aqueous solution are also provided.

[0005] In some embodiments, the NO donor moiety is a C-diazeniumdiolate. In some embodiments, the C-diazeniumdiolate comprises two or more diazeniumdiolate groups. In someAttorney Docket No.035052 / 626621 embodiments, C-diazeniumdiolate comprises three diazeniumdiolate groups. In some embodiments, the NO donor moiety is methyl tris diazeniumdiolate (MD3). In some embodiments, the NO donor moiety is a N-diazeniumdiolate. In some embodiments, the N-diazeniumdiolate is 1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate (SPER / NO), N-[bis(3-aminopropyl)amino]diazen-1-ium-1,2-diolate (DPTA / NO), or N-[bis(2- aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA / NO). In some embodiments, the one or more diazeniumdiolate groups comprise one or more pharmaceutically acceptable cations independently selected from the group consisting of sodium, potassium, lithium, calcium, magnesium, and ammonium.

[0006] In some embodiments, the lipid bilayer comprises one or more phospholipids. In some embodiments, at least one phospholipid is a diacyl-phosphatidylcholine. In some embodiments, the lipid bilayer comprises dipalmitoylphosphatidylcholine (DPPC). In some embodiments, at least one phospholipid is a diacyl-phosphatidylethanolamine. the lipid bilayer comprises dipalmitoylphosphatidylethanolamine (DPPE). In some embodiments, one or more phospholipid is a PEGylated phospholipid. In some embodiments, the lipid bilayer comprises a polyethylene glycol-functionalized dimyristoyl-phosphorylethanolamine (DMPE-PEG). In some embodiments, the lipid bilayer further comprises cholesterol. In some embodiments, the lipid bilayer comprises a molar ratio of about 1:1 to about 5:1 of phospholipid to cholesterol. In some embodiments, the lipid bilayer comprises a molar ratio of about 2:1 of DPPC to cholesterol.

[0007] In some embodiments, the aqueous core encapsulated by the lipid bilayer further comprises a buffer. In some embodiments, wherein the buffer comprises one or more of sodium phosphate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (TRIS). In some embodiments, the concentration of the buffer is about 25 mM. In some embodiments, the aqueous core encapsulated by the lipid bilayer has a pH of about 7 to about 9. In some embodiments, the aqueous core encapsulated by the lipid bilayer has a pH of about 7.

[0008] In some embodiments, the liposomes are unilamellar liposomes. In some embodiments, the diameter of the liposomes is less than 300 nm. In some embodiments, the diameter of the liposome is about 150 nm to about 250 nm. In some embodiments, at least 95% of the NO donor moiety encapsulated by the lipid bilayer retain the diazeniumdiolate groups. In some embodiments, theAttorney Docket No.035052 / 626621 liposome releases at least 5 µmol / mg, at least 6 µmol / mg, at least 7 µmol / mg, at least 8 µmol / mg, at least 9 µmol / mg, or at least 10 µmol / mg of NO over 24 hours following administration.

[0009] In some embodiments, the concentration of the NO donor moiety is greater than 1 mg / mL, greater than 2 mg / mL, greater than 3 mg / mL, greater than 4 mg / mL, greater than 5 mg / mL or greater than 6 mg / mL. In some embodiments, the plurality of liposomes has a dispersity of less than 0.3. In some embodiments, the plurality of liposomes has a zeta potential of less than -5 mV. In some embodiments, the composition further comprises amikacin, or a pharmaceutically acceptable salt thereof. In some embodiments, the composition comprises amikacin encapsulated in the aqueous core of a liposome.

[0010] Provided are methods for treating respiratory infections in patients in need thereof comprising administering to the patients an effective amount of the NO-releasing liposomes. In some embodiments, the respiratory infection is caused by an intracellular pathogen. In some embodiments, the intracellular pathogen is an intracellular bacterium. In some embodiments, the intracellular bacterium is a Mycobacterium. In some embodiments, the Mycobacterium is a nontuberculosis Mycobacterium (NTM). In some embodiments, the NTM is selected from the group consisting of Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium haemophilum, Mycobacterium^gordonae, Mycobacterium simiae, Mycobacterium marinum, Mycobacterium malmoense, Mycobacterium ulcerans, Mycobacterium bolletii, Mycobacterium massiliense, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium porcinum, Mycobacterium smegmatis, Mycobacterium vaccae, and Mycobacterium mucogenicum. In some embodiments, the Mycobacterium has a smooth morphology. In some embodiments, the Mycobacterium has a rough morphology.

[0011] In some embodiments, the method comprises aerosolizing the composition comprising the liposomes and administering to the lungs of the patient. In some embodiments, the method further comprises administering to the patient an effective amount of amikacin.

[0012] Described are methods of making a plurality of the NO-releasing liposomes by encapsulating the NO donor moiety in the aqueous core by the lipid bilayer as the liposome is formed in an aqueous solution. In some embodiments, the method comprises forming a thin film of one or more lipids and adding an aqueous solution comprising the NO donor moiety. In some embodiments, the aqueous solution comprises a buffer. In some embodiments, the aqueousAttorney Docket No.035052 / 626621 solution comprising the liposomes is extruded through a filter. In some embodiments, an encapsulation efficiency of the NO donor moiety is greater than 20%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, or greater than 50%. BRIEF DESCRIPTION OF FIGURES

[0013] Figure 1 shows a schematic of an NO donor moiety encapsulated in a liposome.

[0014] Figure 2 shows the chemical structures and pKa values of aqueous buffers for liposome formulations.

[0015] Figure 3A-B shows the minimum bactericidal concentration (MBC) of methyl tris diazeniumdiolate (MD3) against a representative smooth and rough strain of M. abscessus evaluated at 4 h (left) and 24 h (right) via microdilution. Data are expressed as the mean ± standard deviation of 3-5 biological replicates. Statistical analysis was performed using a two-way ANOVA followed by Dunnett’s multiple comparisons test (GraphPad Prism software). Differences are considered significant at the 95% level of confidence.

[0016] Figure 3C-D shows minimum bactericidal concentration of NO-releasing treatments against a representative smooth and rough strain of M. abscessus at (A) 4 and (B) 24 h evaluated via microdilution. Treatments include non-liposomal MD3 (orange), Lip-MD3-9-TRIS (purple), Lip-MD3-9-PB (purple, striped) Lip-MD3-8-HEPES (blue), Lip-MD3-8-PB (blue, striped), and Lip-MD3-7-PB (green, striped). Data are expressed as the mean ± standard deviation of n ≥ 3 replicates. Statistical significance was determined by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

[0017] Figure 4A-C shows the (A) average size distribution, (B) dispersity, and (C) charge of MD3-loaded liposomes as a function of buffer and pH. Data are expressed as the mean ± standard deviation of n ≥ 3 formulations.

[0018] Figure 5A-C shows (A) representative cumulative release profile of MD3 (orange, solid), Lip-MD3-9-TRIS (purple, solid), Lip-MD3-9-PB (purple, striped) Lip-MD3-8-HEPES (blue, solid), Lip-MD3-8-PB (blue, striped), and Lip-MD3-7-PB (green, striped). (B) Acid liberated totals and (C) % NO released in 24 h comparing Lip-MD3-7-PB and MD3. Statistical significanceAttorney Docket No.035052 / 626621 was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (A) and Student’s unpaired t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

[0019] Figure 6A-D shows bactericidal activity of MD3 (orange), Lip-MD3-7-PB (green), empty liposomes (yellow), amikacin (red), and Lip-AMK-7-PB (blue) on smooth (A, C) and rough (B, D) strains of M. abscessus at 4 h (A, B) and 24 h (C, D) in THP-1m cells. Dotted lines represent a 3-log (99.9%) decrease in CFU relative to the growth control. Results are shown as the mean ± standard deviation of n ≥ 3 biological replicates.

[0020] Figure 7 shows quantitative uptake of liposomal pyranine into THP-1m cells. Dotted line represents the fluorescent intensity of non-liposomal pyranine at each time point. Error bars represent standard deviation of n ≥ 3 biological replicates. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

[0021] Figure 8A-F shows representative confocal microscopy images of THP-1m cells treated with of 0.1 mg mL-1MD3 (A, B, C) and liposomal MD3 (D, E, F) for 2 h. Intracellular liposomal cargo and NO represented by rhodamine B (red; A, D), DAF-FM (green; B, E), and overlaid (C, F) fluorescence, respectively. Scale bar is 50 µm.

[0022] Figure 9 shows time course study of fluctuations in intracellular NO fluorescence (green) after 30 minutes of treatment with MD3 (top) and liposomal MD3 (bottom) on THP-1 cells. Rhodamine channel omitted for clarity. Scale bar is 20 µm.

[0023] Figure 10A-B shows confocal microscopy image (A) and Z-stack with orthogonal slices (B) of THP-1 cells treated with of 0.1 mg mL-1liposomal MD3 for 2 h. Intracellular liposomal cargo and nitric oxide represented by Rhodamine B (red) and DAF-FM (green) fluorescence, respectively. Orthogonal slices depict a 50 x 50 x 19.5 µm cross-section of the square field. DETAILED DESCRIPTION

[0024] Nitric oxide-releasing liposomes show potential as an improved treatment for NTM pulmonary disease. Liposomal encapsulation facilitates delivery to infected immune cells, allowing for improved treatment of intracellular mycobacteria. Additionally, nitric oxide’s multiple antimicrobial mechanisms of action can overcome the resistance of M. abscessus, allowing for improved rates of culture conversion with fewer serious side effects compared to amikacin treatment.Attorney Docket No.035052 / 626621

[0025] However, there are unique challenges to forming a high-concentration NO-releasing liposome, tuning it to release NO, controlling the effects of NO aerosolization on the liposome architecture and releasing an efficacious amount of NO. One challenge is encapsulating a high- concentration of NO donor along with mitigation of premature NO release. Also, the liposomal architecture must remain robust in order to preclude fusing and bursting. Described herein are methods for preparation and resultant liposomal compositions that are shown to possess the desired characteristics.

[0026] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents and other references mentioned herein are incorporated by reference into their entirety. Definitions

[0027] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular aspects only and isAttorney Docket No.035052 / 626621 not intended to be limiting of the invention. In case of a conflict in terminology, the present specification is controlling.

[0028] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0029] The term “or” refers to any one member of a particular list.

[0030] Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ± 0.5%, 1%, 5%, or 10% from a specified value.

[0031] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0032] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients.

[0033] As used herein, the term “liposome” refers to a microscopic, closed vesicle having an internal phase or aqueous core enclosed by lipid bilayer. Liposomes are self-assembling, substantially spherical vesicles comprising a lipid bilayer that encircles a core, which can be aqueous, wherein the lipid bilayer comprises amphipathic lipids having hydrophilic headgroups and hydrophobic tails, in which the hydrophilic headgroups of the amphipathic lipid molecules are oriented toward the core or surrounding solution, while the hydrophobic tails orient toward the interior of the bilayer. The lipid bilayer structure thereby comprises two opposing monolayers that are referred to as the “inner leaflet” and the “outer leaflet,” wherein the hydrophobic tails are shielded from contact with the surrounding medium. The “inner leaflet” is the monolayer wherein the hydrophilic head groups are oriented toward the core of the liposome. The “outer leaflet” is the monolayer comprising amphipathic lipids, wherein the hydrophilic head groups are oriented towards the outer surface of the liposome.

[0034] As used herein, the terms “internal phase” and “aqueous core” refer to an aqueous region enclosed in the lipid bilayer of the liposome.

[0035] As used herein, the term encapsulated is used as it is known in this art for liposomes. The lipid bilayer encapsulates the inner core but can also be penetrable or contain holes.Attorney Docket No.035052 / 626621

[0036] As used herein, the term “unilamellar liposome” means a spherical liposome bounded by a single bilayer enclosing the aqueous core. An example of a moiety encapsulated in a unilamellar liposome is shown in Figure 1. In contrast, a “multilamellar liposome” includes multiple lipid bilayers which may involve concentric lipid spheres separated by layers of water.

[0037] As used herein, the term “phospholipid” refers to a phosphorous-containing lipid that is amphipathic. Non-limiting examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoyl phosphatidic acid, and dilinoleoylphosphatidylcholine. A diacyl-phospholipid such as a diacyl-phosphatidylcholine or diacyl-phosphatidylethanolamine comprise two esterified fatty acids.

[0038] As used herein, the term “PEGylated phospholipid” refers to a phospholipid which is covalently attached to polyethyleneglycol which comprises repeating units of -OCH2CH2-.

[0039] As used herein, the term “NO donor moiety” refers to a compound containing one or more diazeniumdiolate groups, which release nitric oxide (NO).

[0040] As used herein, the term “diazeniumdiolate group” refers a functional group with the following chemical structure:. A diazeniumdiolate group is anionic and may include a cation as the counterion. The cation may be selected from the group consisting of sodium, potassium, lithium, calcium, magnesium, and ammonium. Upon protonation, a diazeniumdiolate group releases two molecules of nitric oxide (NO).

[0041] As used herein, the term “C-diazeniumdiolate” refers to a molecule containing a diazeniumdiolate group covalently bound to a carbon atom. A C-diazeniumdiolate may include one or more diazeniumdiolate groups covalently bound to an alkyl group, such as but not limited to methyl. Methyl tris diazeniumdiolate (MD3) is a C-diazeniumdiolate.

[0042] As used herein, the term “N-diazeniumdiolate” or “NONOate” refers to a molecule containing a diazeniumdiolate group covalently bound to an amine. N-diazeniumdiolate include but are not limited to 1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]diazen- 1-ium-1,2-diolate (SPER / NO), N-[bis(3-aminopropyl)amino]diazen-1-ium-1,2-diolate (DPTA / NO), or N-[bis(2-aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA / NO).Attorney Docket No.035052 / 626621

[0043] As used herein, the term “dispersity” refers to a measure of the heterogeneity of sizes of a plurality of liposomes in a composition. A low dispersity indicates uniformity in the size of the plurality of liposomes in the composition.

[0044] As used herein, the term “encapsulation efficiency” refers to the difference between the concentration of the NO donor moiety in a liposomal composition and the initial concentration of the aqueous solution of the NO donor moiety used to form the liposomal, expressed as a percentage. The encapsulation efficiency describes the amount of the NO donor moiety that is internalized into the aqueous core and / or lipid bilayer after all stages of liposome formation and separation have been completed. Overview

[0045] Liposomes are a class of drug delivery vehicles composed of phospholipid bilayers that form spherical vesicles around an aqueous core. These phospholipid bilayers facilitate delivery of hydrophobic and hydrophilic drugs intracellularly via uptake or membrane fusion, while also mitigating drug toxicity (Gonzalez Gomez, A.; Hosseinidoust, Z. ACS Infectious Diseases 2020, 6, 896–908). Recently, liposomal amikacin formulations have been developed to treat Mycobacterium avium complex (MAC) pulmonary infections, however their utility against M. abscessus infection in limited (Rose, S. J., et al., PLoS One 2014, 9, 1–7; Zhang, J., et al., Frontiers in Microbiology 2018, 9, 1–12; Meers, P., et al., Journal of Antimicrobial Chemotherapy 2008, 61, 859–868). Mycobacterium abscessus is more refractory than MAC infections due to the fast- growing nature of the bacteria (i.e., 6-10 h doubling time as compared to a 18-24 h double time for M. avium) and its simplistic genome, making it more likely to develop adaptive and acquired resistance to antibiotics (Strnad, L.; Winthrop, K. L. Seminars in Respiratory and Critical Care Medicine 2018, 39, 362–376). Given the unique challenges of non-tuberculosis mycobacteria pulmonary disease (NTM-PD), the current standard of care is variable and often administered with a goal of symptom reduction versus culture conversion. In this care, a patient is given an antibiotic cocktail consisting of at least three antibiotic classes (e.g., fluoroquinolones, aminoglycosides, macrolides), with treatment lasting from 12 months to several years (Kurz, S. G., et al., Ann Am Thorac Soc 2020, 17, 1033–1039). Patients are often hospitalized during initial dosing as these antibiotics have narrow therapeutic windows and require frequent dosage adjustment or stoppage (Benwill, J. L.; Wallace, R. J., Curr Opin Infect Dis 2014, 27, 506–510). Despite the long durationAttorney Docket No.035052 / 626621 and intensity of treatment, the average rate of culture conversion (i.e., three consecutive negative sputum cultures four weeks apart) is less than 50% (Daniel-Wayman, S., et al., Am J Respir Crit Care Med 2019, 199, 947–951; Luthra, S.; Rominski, A.; Sander, P., Front Microbiol 2018, 9, 1– 13). Among NTM-PD, M. abscessus has the lowest culture conversion rates, as its combination of fast growth and simplistic genome result in intrinsic and acquired resistance to antibiotics. Moreover, the inability of most antibiotic classes to pass through the mammalian cellular membrane makes latent, intracellular NTM-PD another contributing factor to low cure rates (Lambert, P. A., Journal of Applied Microbiology Symposium Supplement 2002, 92, 46–54). Thus, a therapy that is both targeted intracellularly and has the potential to overcome antibiotic resistance towards M. abscessus is needed.

[0046] is an endogenous free radical that is involved in a number ofphysiological processes, including pathogenic killing. Nitric oxide exerts antimicrobial effects by inducing nitrosative and oxidative stresses on bacteria via reactive byproducts of NO, resulting in protein modification, lipid peroxidation, and DNA cleavage. Unlike antibiotics, which target one bacterial mechanism, NO has several antimicrobial mechanisms of action, and is therefore unlikely to elicit resistance (Privette, B. J., et al., Nitric Oxide 2014, 23, 1–7; Schairer, D.O., et al., Virulence 2012, 3, 271–279). Gaseous exogenous NO has been attempted as a treatment for NTM infection, but is limited in efficacy due to its short half-life and toxicity concerns. Nitric oxide donors, which provide sustained release of solution-phase NO, are promising methods of administering long- lasting NO to patients while mitigating toxicity. It has been demonstrated that N-diazeniumdiolates (NONOates) are capable of being encapsulated in liposomes, and NO-releasing liposomes have been explored for anticancer and vasodilatory purposes (Suchyta, D. J.; Schoenfisch, M. H. ACS Biomaterials Science and Engineering 2017, 3, 2136–2143; Suchyta, D. J.; Schoenfisch, M. H. Molecular Pharmaceutics 2015, 12, 3569–3574; Rashid, J., et al., Molecular Pharmaceutics 2018, 15, 1755–1765; Nahar, K., et al., Pharmaceutical Research 2016, 33, 1696–1710). However, the amount of NO donor needed to elicit anticancer and vasodilatory effects (i.e. less than 1 mg NO donor / mL) is less than those needed for antibacterial purposes. NO-releasing liposomes have not yet been used for antimicrobial applications and high liposomal concentrations of NO donor are likely needed to effectively treat for NTM pulmonary diseases. Additionally, NO donors are prone to spontaneous NO release at physiological pH and temperature, thus methods of preparingAttorney Docket No.035052 / 626621 liposomal formulations which maintain the stability of the NO donors during liposome formation and storage are needed.

[0047] Provided herein are compositions of liposomal formulations of NO donors with high liposomal concentrations. Also provided are methods for the preparation of liposomal formulations with high encapsulation efficiency and stability of NO donors and methods of treating respiratory diseases such as NTM with said liposomal formulations. Physiochemical properties of liposomal MD3 were determined, exploring the effects of internal pH and buffer capacity on NO release and planktonic antimicrobial activity. In vitro studies were undertaken to probe the performance of liposomal MD3 compared to common treatments on intracellular infection. Lastly, cellular uptake studies were carried out to understand the mechanism and quantity of small molecule internalization over the course of treatment. Compositions

[0048] Provided are formulations of nitric oxide (NO)-releasing liposomes for the treatment of respiratory infections. Compositions comprising NO-releasing liposomes in an aqueous solution are described. The liposomes comprise a lipid bilayer, which comprises an inner leaflet and an outer leaflet. The liposomes comprise an NO donor moiety in an aqueous core encapsulated by the lipid bilayer, as shown in Figure 1. The NO donor moiety is a compound which comprises one or more diazeniumdiolate groups.

[0049] In some embodiments, the NO donor moiety is a C-diazeniumdiolate. In some embodiments, the chemical structure of the C-diazeniumdiolate is , wherein R is an optionally substituted alkyl group. In some embodiments, R is methyl, ethyl or propyl. In some embodiments, the C-diazeniumdiolate comprises two or more diazeniumdiolate groups. In some embodiments, the C-diazeniumdiolate comprises three diazeniumdiolate groups. In some embodiments, the NO donor moiety is methyl tris diazeniumdiolate (MD3). The chemical structure of MD3 isAttorney Docket No.035052 / 626621 has a net -3 charge and may further comprise one or more pharmaceutically independently selected from the group consisting of sodium, potassium,magnesium, and ammonium. In some embodiments, one or more pharmaceutically acceptable cations of MD3 are sodium. In some embodiments, MD3 comprises three sodium cations and the NO donor moiety has the chemical structure .

[0050] In some embodiments, the NO donor moiety is a N-diazeniumdiolate. In some embodiments, the chemical structure of the N-diazeniumdiolate is , wherein R1and R2are each independently C1-C10optionally substituted alkyl. In some embodiments R1and R2are each independently methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl. In some embodiments, R1and R2are each independently C1-C10 alkyl substituted with one or more amine groups. In some embodiments, the N-diazeniumdiolate is 1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate (SPER / NO) with the chemical structure of . In some embodiments, the N-diazeniumdiolate is N-1,2-diolate (DPTA / NO) with the chemical structure of (2-Attorney Docket No.035052 / 626621 aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA / NO) with the chemical structure of . can be formed with NO donor moieties in the form of asalt. Any of the NO donor moieties described herein may include one or more pharmaceutically acceptable cations independently selected from the group consisting of sodium, potassium, lithium, calcium, magnesium, and ammonium as the counterion to the anionic diazeniumdiolate.

[0052] In some embodiments, the liposomes or lipid bilayers can contain co-lipids that are negatively charged or neutral. As used herein, a “co-lipid” refers to a non-cationic lipid, which includes neutral (uncharged) or anionic lipids. The term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH. The term “anionic lipid” encompasses any of a number of lipid species that carry a net negative charge at physiological pH. Co-lipids can include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols, phospholipid-related materials, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidic acid, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), palmitoyloleyolphosphatidylglycerol (POPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylchol-ine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoyl phosphatidic acid (DOPA), stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl- aryl sulfate polyethyloxylated fatty acid amides, lysophosphatidylcholine, and dioctadecyldimethyl ammonium bromide and the like. Co-lipids also include polyethylene glycol- based polymers such as PEG 2000, PEG 5000 and polyethylene glycol conjugated toAttorney Docket No.035052 / 626621 phospholipids or to ceramides, as described in U.S. Pat. No. 5,820,873, herein incorporated by reference in its entirety.

[0053] In some embodiments, the liposome or lipid bilayer comprises cationic lipids. As used herein, the term “cationic lipid” encompasses any of a number of lipid species that carry a net positive charge at physiological pH, which can be determined using any method known to one of skill in the art. Such lipids include, but are not limited to, the cationic lipids of formula (I) disclosed in International Application No. PCT / US2009 / 042476, entitled “Methods and Compositions Comprising Novel Cationic Lipids,” which was filed on May 1, 2009, and is herein incorporated by reference in its entirety. These include, but are not limited to, N-methyl-N-(2- (arginoylamino)ethyl)-N,N-Di octadecyl aminium chloride or di stearoyl arginyl ammonium chloride] (DSAA), N,N-di-myristoyl-N-methyl-N-2[N′—(N6-guanidino-L-lysinyl)]aminoethyl ammonium chloride (DMGLA), N,N-dimyristoyl-N-methyl-N-2[N2-guanidino-L- lysinyl]aminoethyl ammonium chloride, N,N-dimyristoyl-N-methyl-N-2[N′—(N2,N6-di- guanidino-L-lysinyl)]aminoethyl ammonium chloride, and N,N-di-stearoyl-N-methyl-N-2[N′— (N6-guanidino-L-lysinyl)]aminoethyl ammonium chloride (DSGLA). Other non-limiting examples of cationic lipids that can be present in the liposome or lipid bilayer of the presently disclosed delivery system complexes include N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); N- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTMA”) or other N—(N,N-1- dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; N,N-distearyl-N,N- dimethylammonium bromide (“DDAB”); 3-(N—(N′,N′-dimethylaminoethane)- carbamoyl)cholesterol (“DC-Chol”) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (“DMRIE”); 1,3-dioleoyl-3-trimethylammonium-propane, N- (1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1 ammonium trifluoro-acetate (DOSPA); GAP-DLRIE; DMDHP; 3-β[4N—(1N,8N-diguanidino spermidine)- carbamoyl]cholesterol (BGSC); 3-β[N,N-diguanidinoethyl-aminoethane)-carbamoyl]cholesterol (BGTC); N,N1,N2,N3Tetra-methyltetrapalmitylspermine (cellfectin); N-t-butyl-N′-tetradecyl-3- tetradecyl-aminopropion-amidine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); 1,3-dioleoyloxy-2-(6-carboxyspermyl)-propyl amide (DOSPER); 4-(2,3-bis- palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM) N,N,N′,N′-tetramethyl-N,N′-bis(2- hydroxyethyl)-2,3 dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50); 1,2 dioleoyl-3-(4′-Attorney Docket No.035052 / 626621 trimethylammonio) butanol-sn-glycerol (DOBT) or cholesteryl (4′ trimethylammonia) butanoate (ChOTB) where the trimethylammonium group is connected via a butanol spacer arm to either the double chain (for DOTB) or cholesteryl group (for ChOTB); DL-1,2-dioleoyl-3- dimethylaminopropyl-β-hydroxyethylammonium (DORI) or DL-1,2-O-dioleoyl-3- dimethylaminopropyl-β-hydroxyethylammonium (DORIE) or analogs thereof as disclosed in International Application Publication No. WO 93 / 03709, which is herein incorporated by reference in its entirety; 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl phosphatidylethanolamylspermine (DPPES) or the cationic lipids disclosed in U.S. Pat. No.5,283,185, which is herein incorporated by reference in its entirety; cholesteryl-3β- carboxyl-amido-ethylenetrimethylammonium iodide; 1-dimethylamino-3-trimethylammonio-DL- 2-propyl-cholesteryl carboxylate iodide; cholesteryl-3-β-carboxyamidoethyleneamine; cholesteryl-3-β-oxysuccinamido-ethylenetrimethylammonium iodide; 1-dimethylamino-3- trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxysuccinate iodide; 2-(2-trimethylammonio)- ethylmethylamino ethyl-cholesteryl-3-β-oxysuccinate iodide; and 3-β-N-(polyethyleneimine)- carbamoylcholesterol.

[0054] The lipid bilayer of the liposomes may comprise one or more phospholipids. In some embodiments, one of more of the phospholipids are diacyl phospholipids, comprising two esterified fatty acid chains. In some embodiments, one or more diacyl-phospholipid is a diacyl- phosphatidylcholine or a diacyl-phosphatidylethanolamine. In some embodiments, the lipid bilayer comprises dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the lipid bilayer comprises dipalmitoylphosphatidylethanolamine (DPPE).

[0055] In some embodiments, one or more phospholipid is a PEGylated phospholipid. The size of PEG chain may vary. In some embodiments, the PEGylated phospholipid is a polyethylene glycol- functionalized dimyristoyl-phosphorylethanolamine (DMPE-PEG). In some embodiments, the PEGylated phospholipid is a polyethylene glycol-functionalized dimyristoyl- phosphorylethanolamine, with a molecular weight of about 2000 (DMPE-PEG2000).

[0056] In some embodiments, the lipid bilayer comprises two or more phospholipids.

[0057] In some embodiments, the lipid bilayer comprises cholesterol. In some embodiments, the lipid bilayer comprises a molar ratio of about 1:1 to about 5:1 of phospholipid to cholesterol. In some embodiments, the lipid bilayer comprises a molar ratio of about 1:1 to about 3:1 ofAttorney Docket No.035052 / 626621 phospholipid to cholesterol. In some embodiments, the lipid bilayer comprises a molar ratio of about 2:1 of phospholipid to cholesterol. In some embodiments, the lipid bilayer comprises a molar ratio of about 2:1 of DPPC to cholesterol.

[0058] In some embodiments, the aqueous core encapsulated by the lipid bilayer and / or the aqueous solution comprising the liposomes (e.g., exterior to the liposomes) further comprises a buffer. The addition of the buffer may help to stabilize the diazeniumdiolate during formation of the liposome. In some embodiments, the buffer comprises one or more of sodium phosphate, N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (TRIS). In some embodiments, the buffer comprises phosphate. In some embodiments, the buffer is sodium phosphate. In some embodiments, the concentration of the buffer is about 10 mM to about 150 mM. In some embodiments, the concentration of the buffer is about 25 mM. In some embodiments, the aqueous core encapsulated by the lipid bilayer has a pH of about 7 to about 10. In some embodiments, the aqueous core encapsulated by the lipid bilayer has a pH of about 7 to about 9. In some embodiments, the aqueous core encapsulated by the lipid bilayer has a pH of about 8, about 7.9, about 7.8, about 7.7, about 7.6, about 7.5, about 7.4, about 7.3, about 7.2, or about 7.1. In some embodiments, the aqueous core encapsulated by the lipid bilayer has a pH of about 7.

[0059] In some embodiments, a composition of a plurality of the liposomes further comprises a cryoprotectant. The addition of a cryoprotectant may improve the stability of liposomal compositions during freeze-drying and / or storage at low temperatures by preventing ice crystal formation when water freezes and expands. The cryoprotectant may be located in the aqueous core of the liposomes and / or the aqueous solution exterior of the liposomes. In some embodiments, the cryoprotectant is selected from the group consisting of sucrose, trehalose, dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the liposomal composition comprises about 10 mM to about 1 M of the cryoprotectant. In some embodiments, the liposomal composition comprises about 160 mM of sucrose.

[0060] In some embodiments, the liposomes are unilamellar.

[0061] In some embodiments, an average diameter of the liposomes is less than 300 nm, less than 250 nm, or less than 200 nm. In some embodiments, an average diameter of the liposome is aboutAttorney Docket No.035052 / 626621 150 nm to about 250 nm. In some embodiments, an average diameter of the liposome is about 200 nm.

[0062] For the treatment of respiratory infections, it is desirable to formulate liposomes with a high concentration of NO donor moiety. The concentration needed to effectively treat respiratory infections such as NTM depends on the type of NO donor moiety. It has been found that non- liposomal MD3 can kill NTM at concentrations of 0.25-1.0 mg / mL (McDonald R.A., et al., Antimicrob Agents Chemother. 2024, 68(2):e0132723), whereas SPER / NO, DPTA / NO, and DETA / NO have been shown to kill NTM at concentrations of about 1-4 mg / mL.

[0063] In some embodiments, a concentration of the NO donor moiety in a composition of a plurality of the liposomes is greater than 1 mg / mL, greater than 2 mg / mL, greater than 3 mg / mL, greater than 4 mg / mL, greater than 5 mg / mL or greater than 6 mg / mL. In some embodiments, a concentration of the NO donor moiety in a composition of a plurality of the liposomes is greater than 0.01 M, greater than 0.015 M, greater than 0.02 M, greater than 0.025 M, or greater than 0.03 M.

[0064] As diazeniumdiolates are prone to spontaneous NO release at physiological pH and temperature, it is useful for NO-releasing liposomes to retain the diazeniumdiolate groups on the NO donor moieties during preparation and storage of the liposomes. In some embodiments, at least 95% of the NO donor moiety encapsulated by the lipid bilayer retain the diazeniumdiolate groups.

[0065] In some embodiments, the liposomes release at least 5 µmol / mg, at least 6 µmol / mg, at least 7 µmol / mg, at least 8 µmol / mg at least 9 µmol / mg, or at least 10 µmol / mg of NO over 24 hours following administration.

[0066] In some embodiments, a composition of a plurality of the liposomes has a dispersity of less than 0.3. In some embodiments, a composition of a plurality of the liposomes has a dispersity of less than 0.2, less than 0.1, less than 0.09, less than 0.08, or less than 0.07.

[0067] In some embodiments, a composition of a plurality of the liposomes has a zeta potential of less than -5 mV.

[0068] In some embodiments, the compositions described herein are NO-releasing liposomes comprising a lipid bilayer comprising an inner leaflet and an outer leaflet, and MD3 in an aqueous core encapsulated by the lipid bilayer.

[0069] Administration of the NO-releasing liposomes disclosed herein with amikacin may provide synergistic effects for the treatment of respiratory infections such as NTM and may help toAttorney Docket No.035052 / 626621 overcome resistance to amikacin, especially for M. abscessus infections. In some embodiments, a pharmaceutical composition of NO-releasing liposomes further comprises amikacin. In some embodiments, the amikacin is liposomal amikacin, wherein amikacin is in an aqueous core encapsulated by a lipid bilayer. In some embodiments, amikacin is encapsulated with the NO donor moiety in the aqueous core of liposomes. In some embodiments, a composition comprises a first liposome, wherein the first liposome is an NO-releasing liposome, and a second liposome, wherein the second liposome comprises amikacin in an aqueous core encapsulated by a lipid bilayer. Methods of Preparing Liposomal Formulations

[0070] Described are methods for preparing NO-releasing liposomes with high concentrations of a NO donor moiety for the treatment of respiratory infections. The use of buffers such as sodium phosphate to control the pH of the aqueous solution may help to form liposomes with a majority of diazeniumdiolate groups retained on the NO donor moiety. In some embodiments, the liposomal formulations disclosed herein are prepared by passive encapsulation. Passive encapsulation may allow for the formation of liposomes with a greater concentration of the NO donor moiety as compared to active encapsulation methods. During passive encapsulation, the NO donor moiety is encapsulated in the lipid bilayer as the liposome is formed. In contrast, active encapsulation involves the encapsulation of the NO donor moiety after the formation of the liposome. Ion or pH gradients may be used for active encapsulation.

[0071] In some embodiments, the NO donor moiety is encapsulated in the lipid bilayer as the liposome is formed (e.g., passive encapsulation) in an aqueous solution. In some embodiments, the method involves forming a thin film of the components of the lipid bilayer (e.g., one or more phospholipids such as DPPC and optionally cholesterol). The formation of the thin film of the one or more lipids may involve dissolving the components of the lipid bilayer in an organic solvent such as chloroform and evaporating the solvent. The solvent may be evaporated under reduced pressure (e.g., vacuum) and optionally at an elevated temperature such as about 50 °C. Then, the thin film comprising the one or more lipids may be hydrated by adding an aqueous solution comprising the NO donor moiety. In some embodiments, the composition is heated above 25 °C after the addition of the aqueous solution comprising the NO donor moiety to form the liposomes.Attorney Docket No.035052 / 626621

[0072] In some embodiments, the composition is heated at about 50 °C for about 1 hour after the addition of the aqueous solution comprising the NO donor moiety to the thin film of one or more lipids.

[0073] In some embodiments, the aqueous solution comprising the NO donor moiety further comprises a buffer. In some embodiments, the aqueous solution comprising the NO donor moiety comprises one or more of sodium phosphate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (TRIS). In some embodiments, the aqueous solution comprising the NO donor moiety comprises sodium phosphate. In some embodiments, the aqueous solution comprising the NO donor moiety comprises the buffer at a concentration of about 25 mM. In some embodiments, the aqueous solution comprising the NO donor moiety comprises sodium phosphate is at a concentration of about 25 mM. In some embodiments, the aqueous solution comprising the NO donor moiety has a pH of about 7 to about 9. In some embodiments, the aqueous solution comprising the NO donor moiety has a pH of about 7. In some embodiments, the method further comprises sonicating the aqueous solution comprising the liposomes. In some embodiments, the method further comprises extruding the aqueous solution comprising the liposomes through a filter. Extruding the solution through a filter may provide small liposomes of a homogeneous size (e.g., a diameter of about 150 nm to about 200 nm) that are primarily unilamellar liposomes. In some embodiments, the filter is polycarbonate filter. In some embodiments, the filter has a pore size of about 200 nm. The methods described herein may be used to prepare NO-releasing liposomes comprising a lipid bilayer comprising an inner leaflet and an outer leaflet, and MD3 in an aqueous core encapsulated by the lipid bilayer. Methods of Treating Respiratory Diseases

[0074] Provided are methods of treating respiratory infections with NO-releasing liposomes. An effective amount of the NO-releasing liposomes described herein may be administered to a patient in need thereof. Liposomal formulations may be particularly useful for the treatment of intracellular infections, as the liposomes may facilitate the delivery of the NO donor to infected immune cells, such as macrophages.

[0075] In some embodiments, the respiratory infection is caused by an intracellular pathogen. In some embodiments, the respiratory infection is caused by an intracellular bacterium. In some embodiments, the respiratory infection is caused by a Mycobacterium. In some embodiments, the respiratory infection is caused by Mycobacterium is a nontuberculosis Mycobacterium (NTM). InAttorney Docket No.035052 / 626621 some embodiments, the NTM is selected from the group consisting of Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacteriumkansasii, Mycobacterium xenopi, Mycobacterium haemophilum, Mycobacterium^ gordonae,Mycobacterium simiae, Mycobacterium marinum, Mycobacterium malmoense, Mycobacterium ulcerans, Mycobacterium bolletii, Mycobacterium massiliense, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium porcinum, Mycobacterium smegmatis, Mycobacterium vaccae, and Mycobacterium mucogenicum. In some embodiments, the respiratory infection is caused by a Mycobacterium avium complex (MAC). In some embodiments, the Mycobacterium has a smooth morphology. In some embodiments, the Mycobacterium has a rough morphology.

[0076] In some embodiments, a composition comprising the NO-releasing liposomes is aerosolized and administered to the lung of the patient. In some embodiments, a nebulizer is used for administration of the NO-releasing liposomes. In some embodiments, the nebulizer is a vibrating mesh nebulizer. For pulmonary administration of the composition, the aerosolized particles should be less than 5 µm to facilitate delivery to the alveolar space of the lung.

[0077] In some embodiments, the methods of treating respiratory infections comprise administering NO-releasing liposomes comprising a lipid bilayer comprising an inner leaflet and an outer leaflet, and MD3 in an aqueous core encapsulated by the lipid bilayer. EXAMPLES Example 1: Formation and Characterization of N-diazeniumdiolate Liposomes

[0078] Nitric oxide donors spermine / NO (SPER / NO), dipropylenetriamine / NO (DPTA / NO) and diethylenetriamine / NO (DETA / NO) (Figure 1) were selected as they exhibit a range of NO release kinetics and have demonstrated bactericidal activity of planktonic NTM species. Liposomes were synthesized using the thin film hydration method. Compared to the reverse-phase evaporation method (Suchyta, D. J.; Schoenfisch, M. H. Controlled Release of Nitric Oxide from Liposomes. ACS Biomaterials Science and Engineering 2017, 3, 2136–2143; Suchyta, D. J.; Schoenfisch, M. H. Molecular Pharmaceutics 2015, 12, 3569–3574), thin film hydration makes use of polycarbonate extrusion to exhibit control over liposome size (Pattni, B. S., et al., Chemical Reviews 2015, 115, 10938–10966). While preliminary formulations demonstrated NOAttorney Docket No.035052 / 626621 encapsulation was possible, the system suffered from low encapsulation (~25%) and fast lipid degradation via hydrolysis due to high (>12) pH. To improve this system, aqueous solutions ranging from pH 7-12 were examined for lipid hydrolysis using thin layer chromatography, and no hydrolysis was observed up to pH 9. Thus, active and passive encapsulation protocols ranging from pH 7-9 were tested. Briefly, a 2:1 molar ratio of dipalmitoylphosphatidylcholine (DPPC) to cholesterol was dissolved in a round-bottom flask in chloroform and evaporated under vacuum at 50°C to develop a thin lipid film. The films were subsequently hydrated for 60 minutes at 50°C with sodium chloride solution (active encapsulation) or buffered solution containing dissolved SPER / NO (passive encapsulation). The NO donor SPER / NO was selected for initial study over the others as it has the fastest NO release and is therefore most likely to be affected by elevated temperatures required for liposome formation. Both sets of liposomes were sonicated, extruded through 200 nm filters, and separated from unencapsulated material using a Sephedex column. For active loading, an extra hydration and purification step was used to load and remove unencapsulated NONOate from the liposomal system using an ammonium sulfate gradient. The resulting formulations had consistent size and homogeneity, with 169 ± 6 nm diameters and 0.07 ± 0.02 polydispersity indices across all empty and NO donor-loaded liposomes. All formulations were characterized immediately after formation.

[0079] Liposomal NO donor concentration was determined using ultraviolet–visible (UV-Vis) spectroscopy. Briefly, a stock solution was made from each of the NO donors in 100 mM NaOH and diluted to form standards. Liposome samples were prepared by dissolving liposomes in methanol and sonicating briefly to degrade the liposomal bilayer. The sonicated solution was then centrifuged, and the supernatant containing the NO donor was collected and further diluted in 100 mM NaOH. Absorbance measurements at 260 nm were taken for standards and samples and a calibration curve was made to determine sample concentration (Table 1).

[0080] Table 1. Liposome concentration and encapsulation of SPER / NO using various formation techniques. Standard deviations represent n ≥ 3 separate formations. Formation Liposomal Encapsulation Method SPER / NO Efficiency (mg / mL) (%) Active 7 + 8.5 1.04 ± 0.20 21.1 ± 4.0 Active 8.5 + 9 1.50 ± 0.03 32.4 ± 1.5 Passive 8 1.78 ± 0.16 46.5 ± 4.8 Passive 9 2.18 ± 0.18 52.1 ± 7.3Attorney Docket No.035052 / 626621

[0081] Out of the four formation methods tested, passive encapsulation at pH 9 resulted in the highest concentration (2.18 mg / mL) and was significantly higher than the two active encapsulation methods (1.04 and 1.50 mg / mL). The active encapsulation methods are likely not as effective for NO donors compared to other weak bases as the pH gradient must remain basic. Unlike weak base systems, NO donors may not undergo charge change as they enter the liposome as premature NO release is mitigated through the use of an alkaline solution. Given these data, passive encapsulation at pH 9 was selected while scaling up the initial NO donor concentration (16 mg / mL) for all three NO donors (Table 2). The resulting formulations had similar liposomal concentrations (5.04-5.40 mg / mL), indicating that the NO donors behave similarly during passive encapsulation. The resulting liposomal concentrations of NO donor are consistent with the concentrations needed to impart antimicrobial effects on a number of bacterial species.

[0082] Table 2. Liposome concentration and encapsulation of NONOates using passive encapsulation at pH 9. Standard deviations represent n ≥ 4 separate formations. NONOate Liposomal Encapsulation Encapsulated NONOate Efficiency (mg / mL) (%) SPER / NO 5.17 ± 0.28 44.1 ± 1.2 DPTA / NO 5.14 ± 0.68 40.3 ± 5.1 DETA / NO 5.40 ± 0.34 50.1 ± 3.4

[0083] One of the challenges of quantifying liposomal encapsulation with NO donors is the ability of NONOates to spontaneously release NO at elevated temperatures, which the system is exposed to during formation. To control for this property, samples of NO donor were dissolved in solutions akin to each formation technique and exposed to the same temperature conditions as the liposomal formulation, which are then subsequently prepared for UV-Vis as described above. Encapsulation efficiency (EE) was determined by taking the difference between the liposomal concentration and the concentration of the temperature-exposed NO donor. Following a similar trend to the liposomal concentration, passive encapsulation at pH 9 had significantly higher EE (52.1%) relative to the active methods (21.1 and 32.4%). Interestingly, as the liposomes were scaled up, the EE still remained above 40%, indicating higher concentrations of NO donor could potentially be encapsulated within the liposome.Attorney Docket No.035052 / 626621

[0084] Nitric oxide release kinetics were evaluated in real-time using a Sievers 280i chemiluminescence Nitric Oxide Analyzer (NOA) at 37^C in pH 7.0 phosphate buffered saline (PBS) to mimic physiological conditions in an NTM-infected lung. Both the liposomal and unencapsulated solutions of NO donor were injected into the NOA at volumes corresponding to 1 mg of NO donor and were measured at 37^C in pH 7.0 phosphate buffered saline (PBS) to mimic physiological conditions in an NTM-infected lung (T). Encapsulating the NO donors into the liposomes increased the time of release for each of the NO donors (Table 3).

[0085] Table 3. Nitric oxide-release profiles for free and liposomal NONOates in pH 7.0 PBS at 37^C.a[NO]t andbNOmax represents the total amount of NO released and the maximum instantaneous flux of NO per mg of NONOate, respectively.cHalf-life (t1 / 2) represents the time it takes for half of the [NO]t to be released anddtotal duration (td) describes the length of time until NO release is below the limit of quantitation (10 ppb). [NO]tNOmaxTdt1 / 2(µmol mg-1)a(PPB mg-1)b(hours)c(mins)dSPER / NO 5.35 ± 0.24 16900 ± 346 10.7 ± 0.2 31 ± 2 Lip-SPER / NO 5.07 ± 0.31 13933± 3000 21.9 ± 1.6 32 ± 4 DPTA / NO 6.00 ± 0.32 9833 ± 874 18.9 ± 0.2 83 ± 4 Lip-DPTA / NO 9.45 ± 0.29 15130 ± 381 42.3 ± 4.9 46 ± 17 DETA / NO 9.17 ± 0.84 963 ± 158 85.8 ± 3.9 871 ± 124 Lip-DETA / NO 14.6 1700 93.6 875

[0086] Without wishing to be bound by theory, it is believed that the proton-induced liberation of NO from N-diazeniumdiolates is slowed by the high internal pH of the aqueous core, and the internal solution is additionally shielded from the protonated media by the phospholipid bilayer of the liposomes. Given that NO totals are integrated from time and flux data, the increased duration of release also correlates to higher NO totals, which can be seen in DPTA / NO and DETA / NO. The comparable NO totals seen in SPER / NO may be due to the high initial burst release, which would encompass a higher percentage of NO totals relative to the other donors. Interestingly, the half-life for liposomal DPTA / NO decreased, which may also be explained by the significantly higher initial burst that is present from the liposomes compared to the unencapsulated DPTA / NO. The overallAttorney Docket No.035052 / 626621 increase in NO-totals and release duration across the liposomal NO donors is desired for antimicrobial applications as it would allow more time for the liposomal system to be integrated into the intracellular space while still releasing bactericidal levels of NO. Example 2: Antimicrobial Activity of N-diazeniumdiolate Liposomes

[0087] While killing planktonic bacteria is a general indicator of therapeutic potential, the ability of mycobacteria to proliferate inside macrophages remains a challenge for the current antibiotic treatments of NTM. Additionally, as infection progresses, the mycobacteria can irreversibly shift from a smooth to rough morphology, with each morphology having their own pathogenesis and characteristics. To demonstrate that NO-releasing liposomes are capable of eradicating intracellular mycobacteria, macrophage experiments were conducted for liposomal SPER / NO, DPTA / NO, and DETA / NO, unencapsulated SPER / NO, DPTA / NO, and DETA / NO, unencapsulated amikacin, and empty liposome (Lip-Tris) controls. Monolayers of THP-1 monocytes were differentiated into macrophages (THP-1m) using phorbol myristate acetate. Smooth and rough M. abscessus were prepared at 5×104CFU mL-1and added to the cells to establish intracellular infection. The macrophages were treated for 24 h with 0.04-20 μmol of NO or amikacin treatment in media. The macrophages were then lysed and the CFU was counted to determine MBC (Table 4).

[0088] Table 4. Intracellular MBC of empty liposomes, amikacin, as well as unencapsulated and liposomal NO donors. n=1 for amikacin and empty liposomes; n=2 for all unencapsulated and liposomal NO donors. Treatment Smooth MBC Rough MBC (µmol) (µmol) Lip-Tris -- -- Amikacin 0.36 0.72 SPER / NO 10.0 5.00 Lip-SPER / NO 10.0 2.50 DPTA / NO 10.0 5.00 Lip-DPTA / NO 10.0 2.50 DETA / NO 20.0 5.00 Lip-DETA / NO 20.0 2.50

[0089] The empty liposomes exhibited no killing, indicating that the lipid bilayer and the Tris buffer present in each liposomal formulation does not elicit any antimicrobial properties againstAttorney Docket No.035052 / 626621 intracellular M. abscessus. Amikacin exhibited killing at 0.72 µmol; however, this is 8x more concentrated than the recommended clinical dose, and no killing is observed at clinically relevant concentrations, highlighting the difficulty of intracellular treatment with amikacin. Treatment with NO-releasing material resulted in a threshold of 10 µmol of NO needed to kill smooth M. abscessus for SPER / NO and DPTA / NO while DETA / NO required 20 µmol. Interestingly, all of the NO- releasing material were more effective at killing rough M. abscessus, with liposomal formulations all exhibiting killing as low as 2.5 µmol of NO. Rough M. abscessus develops due to a mutation that affects the growth of glycopeptidolipids (GPL) on the cell wall; the absence of GPL may make the cell wall allow increased NO diffusion. Without wishing to be bound by theory, the improved liposomal killing may be due to liposomal delivery allowing all of the NO-releasing material to be delivered intracellularly, rather than relying on diffusion through the cellular membrane after the NO has been liberated. Example 3: Formation and Characterization of MD3 Liposomes

[0090] Materials: Phorbol myristate acetate (PMA), N-2-hydroxyethylpiperazine-N’-2- ethanesulfonic acid (HEPES) buffer salt, rhodamine B, and cholesterol was purchased from Millipore Sigma (Allentown, PA). Agar, bacterial broth, and supplements were purchased from VWR (Middlebrook; Radnor, PA). 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and lipid extruder apparatus was purchased from Avanti Polar Lipids (Alabaster, AL). Methyl tris diazeniumdiolate (MD3) (83.1 mg / mL in water) was gifted from Vast Therapeutics (Durham, NC). Sephadex spin columns were purchased from Cytiva Life Sciences (Marlborough, MA). Diacetate 4-amino-5-methylamino-2’,7’-difluoroscein (DAF-FM-DA), sodium phosphate, tris base, and sodium hydroxide salts were purchased from Fisher Scientific (Waltham, MA). Nitric oxide, carbon dioxide, diatomic oxygen, and diatomic nitrogen tanks were purchased from Airgas (Durham, NC). Amikacin (AMK) and Tween-80 were purchased from Research Products International (Prospect, IL). All reagents were used as purchased without further modification. Formation of liposomes: Liposomes were formed using the thin film hydration method as described in Gonzalez Gomez, A.; Hosseinidoust, Z. ACS Infect Dis 2020, 6, 896–908. Briefly, a 2:1 molar ratio of dipalmitoylphosphatidylcholine (DPPC) to cholesterol (60 µmol total lipid) was dissolved in a round-bottom flask in chloroform and evaporated under vacuum at 50°C to develop a thin lipid film. The films were allowed to further dry under vacuum overnight. The films wereAttorney Docket No.035052 / 626621 hydrated for 30 minutes at 50°C with 3 mL of 25 mM phosphate buffer (phosphate, HEPES, or tris) at various pH values (7, 8, and 9) containing 16 mg mL-1of dissolved MD3 and 160 mM sucrose. To homogenize the formulations, the liposomes were sonicated for 1 min at room temperature while the formulation was still warm and extruded 11 times each at 50°C through 400 nm and 200 nm polycarbonate filters. Unencapsulated MD3 was removed using a Sephadex G-25 M spin column at 6000xg for 5 min at room temperature, following manufacturer instructions. Where necessary, liposomes were concentrated using ultracentrifugation at 15,000xg using an Amicon filter with a 30 kDa cutoff (Sigma). For intracellular infection and uptake studies of amikacin or pyranine-containing liposomes, the liposomes were formed following a similar method. For confocal studies, MD3 was co-encapsulated with rhodamine B (0.41 mg). Physicochemical characterization: Dynamic light scattering (DLS; Malvern Zetasizer Nano; UK) was used to analyze mean hydrodynamic diameter, dispersity, as well as zeta-potential of the formulations. Liposome samples were prepared for DLS by diluting 1:200 by volume in Milli-Q water (1 mL total volume) and subsequently measured on a Malvern Zetasizer Nano (UK) in disposable plastic (VWR) or folded capillary (Fisher Scientific) cuvettes for size and zeta potential, respectively.

[0091] Entrapment of MD3 was quantified by UV-Visible spectroscopy, modifying an established protocol for a different small-molecule diazeniumdiolate described in Nahar, K, et al., Pharm Res 2016, 33, 1696–1710. Briefly, a 50 µL aliquot of liposomes was added to methanol (1 mL total volume) to disrupt the lipid bilayer. The lipid-methanol solution was then sonicated briefly and spun down at 15,000xg for 3 minutes. Following centrifugation, the supernatant was diluted and measured at 260 nm using a SpectraMax M2 (Molecular Devices; San Jose, CA). Likewise, amikacin was quantified at 340 nm following derivatization. Encapsulation efficiency (EE) was determined by taking the difference between the liposomal concentration (Druglip) and the initial concentration (Druginit) of the aqueous MD3 solution (Equation 1).

[0092] ^^^^ ^%^ ൌ^^^௨^^^౦^ൈ100% (1)same temperature conditions as liposomes during formation was similarly quantified in order to confirm retention of the diazeniumdiolate groups. Retention was determined by taking the difference between the concentration of the temperature control (Drugtemp) and the initial concentration (Druginit) of the aqueous MD3 solution (Equation 2).Attorney Docket No.035052 / 626621

[0094] ^^^^^^^^^^^^^^^^^^ ^%^ ൌ^^^௨^౪^^౦^^^^௨^^^^౪^ൈ 100% (2)in real-time using a Chemiluminescent NO analyzer. Prior to analysis, the instrument was calibrated with air passed through a zero-gas filter (0 ppm of NO) followed by a NO-gas standard balanced with N2 (Airgas; 25.87 ppm). Samples of NO-releasing material were normalized to 1 mg of MD3 from their concentration and volume and released in 10 mM phosphate buffered saline (PBS) at pH 7.0 and 37°C to mimic the physiology of an infected lung. Diatomic nitrogen was used as a carrier gas for liberated NO to reach the reaction cell. Measurements were initially terminated after 24 h of release for all NO-releasing species. Where stated, acid liberation studies consisted of adding 1 mL addition of acidified ethanol (6.1 M H2SO4 in EtOH) to samples first releasing NO for 24 h under neutral conditions. The acidified samples were allowed to release for an additional 24 h before termination.

[0096] Planktonic antimicrobial assay: After preparation of bacteria cultures, NO-releasing materials (MD3, Lip-MD3-9, Lip-MD3-8, Lip-MD3-7) were prepared at 4 mg mL-1in 7H9 + OADC media pH corrected to pH 7 and subjected to 2-fold dilutions across a 96-well plate. Smooth and rough cultures were added to produce a bacterial load of 5 × 104CFU well-1. Each plate included untreated culture and cultureless wells to serve as growth and sterility controls, respectively. The plates were incubated at 37°C for 4 and 24 h, after which the contents of each well were plated on 7H10 + OADC agar and incubated at 37°C until visible colony formation in the growth control occurred (approximately 48-72 h). Additionally, pH measurements were taken at 0 and 24 h using 5 µL aliquots blotted onto pH paper. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of NO-releasing material required for a 3-log reduction in bacterial viability measured in CFU mL-1. At least three biological replicates were completed for each experiment.

[0097] Intracellular antimicrobial assay: To determine the potential drug delivery benefits of liposomal MD3 on intracellular mycobacteria, macrophage experiments were performed following an established protocol with slight modification (Rose, S. J.; Neville, M. E.; Gupta, R.; Bermudez, L. E., PLoS One 2014, 9, 1–7; Mcdonald, R. A.; et al., Antimicrob Agents Chemother 2024, 68, 1– 21). Briefly, human blood monocytes (THP-1) were seeded at 5 × 105cells well-1 into a 24-well plate and differentiated to type 0 macrophages (THP-1m) over 3 days using 25 ng mL-1of PMAAttorney Docket No.035052 / 626621 in RPMI medium. The THP-1m cells were then allowed to incubate in PMA-free media for 1-2 days. The cells were subsequently inoculated with media containing either smooth (ATCC 19977) or rough (103) M. abscessus at a multiplicity of infection (e.g., the ratio of cells to bacteria) of 0.1 overnight. Extracellular bacteria were removed by washing with sterile PBS, and the cells were treated with media containing 63-1000 µg mL-1of either MD3, Lip-MD3, AMK, Lip-AMK, or empty liposomes and incubated for 24 h. The cells were then lysed using tween-80 and the bacteria were subsequently diluted and plated onto 7H10 + OADC agar plates for counting.

[0098] Pyranine cellular uptake: Uptake of a membrane impermeable fluorophore with and without liposomal encapsulation in THP-1m cells was quantified using fluorescence spectroscopy following a previous protocol with slight modification (Grayton, Q. E.; Phan, T. T.; Kussatz, C. C.; Schoenfisch, M. H., ACS Appl Bio Mater 2024, 7, 3796–3809). Initial experiments identified the pH of the pyranine solutions and the optimal excitation and emission wavelengths. Monocytes were seeded at 1 × 105cells well-1in a 96-well plate and exposed to 25 ng mL-1of PMA for 3 days to differentiate them into macrophages. After allowing the cells to rest in PMA-free media for 1-2 days, the cells were exposed to 0.1 mg mL-1of either pyranine or liposomal pyranine in phenol- red free RPMI media and allowed to incubate. After 0.5, 2, 4, and 24 h, the cells were washed 3x with sterile PBS to remove any extracellular material before incubation with PBS containing 0.1% tween-80 for 15 min to lyse the cells. Cells only exposed to cell media were cultured, incubated, washed, and lysed at the given time points on a separate plate to determine the autofluorescence of the cell lysate. The cell lysate fluorescence was read at 512 nm with a 400 nm excitation wavelength using a microplate reader (Molecular Devices Spectra Max M2). The relative fluorescence intensity was determined using Equation 3. Where Flip, Fpyra, and Fblankrefer to the fluorescence of the liposomal pyranine, non-liposomal pyranine, and the autofluorescence of the cells, respectively, at each time point.

[0099] ^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^ ൌி^^౦ିி^^^^ౡி౦౯౨^ିி^^^^ౡ(3)

[0100] The experiment was run with 10 technical replicates and 3 biological replicates for each time point.

[0101] Confocal microscopy: Confocal microscopy was used to further visualize the uptake of both MD3 and liposomes into THP-1m cells. Cells were seeded at 1 × 106cells well-1on a Nunc Lab-Tek II chamber slide (Fisher Scientific). The THP-1 cells were differentiated and rested as described in the pyranine experiments. After resting, the cell media was aspirated and replacedAttorney Docket No.035052 / 626621 with DAF-FM-DA in phenol red-free RMPI media supplemented with 1 vol% GlutaMAX (henceforth called imaging media). The cells were incubated for 60 min before aspirating and replacing with fresh imaging media. The cells were incubated for an additional 30 min to allow the esterases in the cell to cleave the diacetate bonds, preventing dye efflux. The media was once again aspirated and replaced with imaging media containing 0.1 mg mL-1of MD3 or fluorescent liposomal MD3. Untreated wells containing only the DAF-FM probe and cells lacking the probe were used as a control and an autofluorescent baseline, respectively. Treated and untreated wells were incubated for 2 h before the cell media was removed, and the cells were washed with cell media to remove any extracellular material before replacing with fresh imaging media. Images were collected on an Andor Dragonfly spinning disk confocal microscope (Oxford Instruments; Carteret, New Jersey, U.S.) with a HC PL APO 63X / 1.4 oil objective at 37°C, choosing distinct locations throughout the well and acquiring a stitched 3x3 grid of images at each location. Subsequent Z-stack images were also collected. The excitation wavelengths for DAF-FM and rhodamine B were set to 488 and 561 nm, respectively. Emission was detected at 521 nm (DAF- FM) and 594 nm (rhodamine B) using an iXon Life 888 EMCCD with a 40 µm pinhole diameter. Time-course studies were completed as described above but with a 30 min incubation with unstitched, z-stabilized images collected every 15 min. Images were displayed and processed in Fiji (ImageJ) with consistent fluorescent settings used in each experiment to compare treatments. The Z-stack was processed in Imaris Viewer. All images have a pixel size of 0.199 µm pixel-1.

[0102] Statistical analysis: All results were presented as mean ± standard deviation of at least three independent experiments or biological replicates. Student’s t-test was used to determine statistical significance across two means. One-way and two-way analysis of variance (ANOVA) were used with Dunnett’s post-hoc test to compare multiple groups. All statistical and post-hoc tests were performed in GraphPad Prism 10. Statistical significance was considered at the following p-values: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0103] Liposomes can be formed in a number of ways (Gonzalez Gomez, A., ACS Infect Dis 2020, 6, 896–908). Thus, the optimal method for preparing liposomes for a given application must be considered. It has been previously reported on NO-releasing liposomal systems that were created using the reverse-phase evaporation (RPE) method (Suchyta, D. J.; Schoenfisch, M. H., ACS Biomater Sci Eng 2017, 3, 2136–2143; Suchyta, D. J.; Schoenfisch, M. H., Mol Pharm 2015, 12,Attorney Docket No.035052 / 626621 3569–3574). While RPE makes unilamellar vesicles without extrusion, the lack of control over size and the encapsulation efficiency being capped at 50% makes this method impractical for consistent systems requiring larger (antimicrobial) concentrations of NO. Herein, thin film hydration was pursued with the intent to prepare NO-releasing liposomes with a narrow size distribution and high NO payloads (Gonzalez Gomez, A.; Hosseinidoust, Z., ACS Infect Dis 2020, 6, 896–908; Bhowmick, S.; Mazumdar, T.; Sinha, R.; Ali, N., Journal of Controlled Release 2010, 141, 199–207). Additionally, active encapsulation and passive encapsulation methods can be explored. While active encapsulation might result in greater encapsulation efficiencies (up to 100%), this method frequently requires acidic pH gradients, which would result in NO donor degradation (to NO) (Zucker, D.; Marcus, D.; Barenholz, Y.; Goldblum, A., Journal of Controlled Release 2009, 139, 73–80). Passive encapsulation was thus utilized to avoid premature NO donor breakdown.

[0104] Liposomes were formed using the thin film hydration method, as it offers high degree of control over liposome size. A 2:1 molar ratio of DPPC to cholesterol was selected initially for lipid film composition, as it consists of the most prevalent pulmonary liposome components in a balanced ratio (Blanchard, J. D.; Elias, V.; Cipolla, D.; Gonda, I.; Bermudez, L. E., Antimicrob Agents Chemother 2018, 62, 1–14; Rose, S. J.; Neville, M. E.; Gupta, R.; Bermudez, L. E., PLoS One 2014, 9, 1–7). The liposomes were then hydrated with MD3 dissolved in a variety of 25 mM aqueous buffers. The buffer concentration was selected to be 25 mM so the final liposomal aqueous medium (including MD3) would be isotonic with physiological medium. As diazeniumdiolates liberate NO at physiological pH, the buffers were selected to be, neutral to alkaline (Davies, K. M.; Wink, D. A.; Saavedra, J. E.; Keefer, L. K., J Am Chem Soc 2001, 123, 5473–5481). Phosphate buffer (PB), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and tris base (TRIS) were selected for the buffer systems as they are frequently used in alkaline liposome formation and offer buffering capacities corresponding to pH 7, 8, and 9, respectively (Figure 1). Greater pH buffers were not evaluated as high alkalinity has been reported to significantly break down phospholipids to carboxylate salts (Brockerhoff, H., J Lipid Res 1963, 4, 96–99). Additionally, systems of liposomes were made with phosphate buffer (PB) at pH 8 and 9 in order to study the effects of pH outside of its effective buffering capacity. Formulation Lip-MD3-9-TRIS was formed in TRIS buffer at a pH of 9, formulation Lip-MD3-8-HEPES was formed in HEPES buffer at a pH of 8, formulation Lip-MD3-7-PB was formed in phosphate buffer at a pH of 7, formulationAttorney Docket No.035052 / 626621 Lip-MD3-8-PB was formed in phosphate buffer at a pH of 8, and formulation Lip-MD3-9-PB was formed in phosphate buffer at a pH of 9.

[0105] Once the liposomes were formed, they were first characterized by their size, dispersity, and charge using DLS as shown in Table 5a. All of the liposomal formulations had acceptable dispersity values of less than 0.3 and similar diameters of about 200 nm (Figure 4A). This result shows polycarbonate extrusion offers good control over liposome size (Ren, H., et al., ACS Appl Mater Interfaces 2019, 11, 20304–20315). It is generally reported that pulmonary liposomes should be larger than 50 nm but smaller than 500-1000 nm to avoid premature lung clearance and sustain drug release, respectively (Ma, S.; Cong, Z.; Wei, J.; Chen, W.; Ge, D.; Yang, F.; Liao, Y., Journal of Controlled Release 2022, 350, 132–145; Andra, V. V. S. N. L.; Pammi, S. V. N.; Bhatraju, L. V. K. P.; Ruddaraju, L. K., Bionanoscience 2022, 12, 274–291). Thus, liposomes roughly 200 nm in size were targeted for preliminary formulations. An inversely proportional trend was observed for both dispersity and charge (Figure 4B, 4C) with increasing pH (from 7 to 9) leading to less dispersity and greater negative charge. The decrease in dispersity is expected, as aggregation is avoided due to charge repulsion (Nsairat, H.; Khater, D.; Sayed, U.; Odeh, F.; Al Bawab, A.; Alshaer, W., Heliyon 2022, 8, e09394). Nevertheless, aggregation was seen as a small second peak around 8000 nm in the Lip-MD3-7-PB and Lip-MD3-8-HEPES size intensity plots (Figure 4A). However, all liposomal formulations are considered stable and homogeneous, as their dispersity values are below 0.3 (Danaei, M.; et al., Pharmaceutics 2018, 10, 1–17). The dispersity decreased as pH increased regardless of buffer identity, which corresponded to a slight increase of negative zeta (ζ) potential at higher pH values. While DPPC is a neutral lipid, the slight negative charge across all formulations is likely due to the charge of the cholesterol, which trends more negative as alkalinity increases.

[0106] Table 5a. Size and distribution of MD3-loaded liposomes as a function of buffer.Attorney Docket No. 035052 / 626621 Additional experiments expressed all zeta (ζ) potentials as the mean ± standard deviation of n ≥ 3 formulations (Table 5b).

[0107] Table 5b. Size and distribution of MD3-loaded liposomes as a function of buffer.

[0108] Liposomes loaded with small molecule NO-releasing donors face a unique challenge compared to traditional small-molecule therapeutics. Given that diazeniumdiolate donors release NO in a proton-initiated mechanism which is affected by temperature, it is advantageous for the liposomal formulation be performed at elevated temperatures with strategies, such as elevated pH, to minimize premature NO release during the formation process. Thus, in addition to MD3 loading and encapsulation efficiency, retention of NO donor (MD3) was determined, as shown in Table 6a.Attorney Docket No. 035052 / 626621

[0109] Table 6a. Loading and retention of MD3 into liposomes as a function of buffer.

[0110] Regardless of the pH or identity of the solvent buffer, MD3 retains a large portion (e.g., greater than 95%) of its NO-releasing groups after the formation process. Additionally, all three pHs tested were capable of loading MD3 at concentrations greater than 3.0 mg / mL, corresponding to 25-45% encapsulation efficiency across the formulations. Lip-MD3-8-HEPES had lower encapsulation compared to other formulations. Additional experiments were performed on the Lip- MD3-8-HEPES formulation (Table 6b).

[0111] Table 6b. Loading and retention of MD3 into liposomes as a function of buffer.a>5.0 mg mL-1, corresponding to a 33-45% encapsulation efficiency across the formulations. No significant difference in liposomal MD3 concentration was observed between the formulations, even at pH 7, which is interesting considering that NO-release kinetics should increase as pH isAttorney Docket No.035052 / 626621 lowered. Such chemistry would typically lessen the amount of NO donor in the liposomes (Suchyta, D. J.; Schoenfisch, M. H., Mol Pharm 2015, 12, 3569–3574; Davies, K. M.; Wink, D. A.; Saavedra, J. E.; Keefer, L. K., J Am Chem Soc 2001, 123, 5473–5481). However, MD3 had a NO-release duration of ~27 h at pH 7 and 37°C in bulk solution (Mcdonald, R. A., et al., Antimicrob Agents Chemother 2024, 68, 1–21). Considering that the liposomal concentration is measured immediately after formation and use for characterization and in biological assays, the pH of the buffer does not confer a negative effect on the liposomal MD3 concentration during the formation process at pH values as low as 7. Example 4: NO release from MD3 liposomes

[0113] NO release from liposomes: Nitric oxide storage and release kinetics were evaluated in real-time using a Sievers 280i chemiluminescent nitric oxide analyzer (Zysense, Boulder, CO). Prior to analysis, the instrument was calibrated with air passed through a zero-gas filter (0 ppm of NO) as well as a NO-gas standard balanced with N2 (Airgas; 25.87 ppm). Samples of NO-releasing material were normalized to 1 mg of MD3 and released in 10 mM phosphate buffered saline (PBS) at pH 7.0 and 37°C to mimic the physiology of an infected lung. Diatonic nitrogen was used as a carrier gas for liberated NO to reach the reaction cell. Measurements were terminated after 24 h of release.

[0114] After the liposomes were characterized, the NO-release properties of the liposomal system were compared to non-liposomal MD3 over a 24 h window using a real-time, chemiluminescent nitric oxide analyzer (Table 7a). All NO-releasing materials released between 5.7 and 7.0 µmol of NO mg-1in a 24 h window, with the exception of the Lip-MD3-8-HEPES, which was significantly higher (10.33 µmol mg-1). These data, combined with the outlying low liposomal concentration, could point to quenching of absorbance by the HEPES molecules during quantification. Since all NO release measurements are normalized by mass, underestimation of concentration would produce larger NO totals.

[0115] Table 7a. NO release of liposomal and non-liposomal MD3 over a 24 h period.Attorney Docket No. 035052 / 626621

[0116] Additional experiments were performed on the Lip-MD3-8-HEPES formulation. All NO- releasing formulations released between 5.7 and 7.5 µmol NO mg-1over 24 h with similar maximum instantaneous fluxes (Table 7b).

[0117] Table 7b. Native NO release properties of liposomal and non-liposomal MD3.a

[0118] The mechanism of NO release is hypothesized to be driven by water entering the liposomes, protonating the diazeniumdiolate group, and releasing NO which subsequently passes through the bilayer, given that the larger and charged MD3 is unlikely to penetrate the liposomal bilayer to the same extent (Davies, K. M.; Wink, D. A.; Saavedra, J. E.; Keefer, L. K., J Am Chem Soc 2001, 123, 5473–5481; Monteiro, N.; Martins, A.; Reis, R. L.; Neves, N. M. Journal of the Royal Society Interface. Royal Society of London 2014). The only formulation to have aAttorney Docket No.035052 / 626621 significant change in NO release over 24 h compared to non-liposomal MD3 was Lip-MD3-7-PB (Figure 5A).

[0119] Excluding the HEPES formulation, NO totals and half-life decreased as the pH of the formulation decreased, with both Lip-MD3-9-TRIS and Lip-MD3-9-PB exhibiting the most similar behavior to non-liposomal MD3. Liposomal formulations often slow down the release of drug, so it may follow that Lip-MD3-7-PB may be lower than non-liposomal MD3 at 24 h because it is slowing down the release of MD3 (and consequently, NO) from the formulation or because the liposome contains less NO capacity due to premature NO donor breakdown in the pH 7 buffer. To determine which condition is more prominent, acidified ethanol was added to both Lip-MD3- 7-PB and MD3 at 24 h to rupture the liposomes and liberate NO donor (MD3) with liberated NO measured for an additional 24 h. Data collected over the total 48 h revealed that both systems released similar NO payloads (Figure 5B) with the pH 7 liposome MD3 system releasing NO more slowly than MD3 alone (Figure 5C). Liposomal formulations are known to sustain drug release by limiting the permeation of the drug into bulk solution via the liposomal bilayer, which corroborates the acid liberation data (Akbarzadeh, A.; Rezaei-sadabady, R.; Davaran, S.; Joo, S. W.; Zarghami, N. Nanoscale Res Lett 2013, 8, 1–9). Previously reported NO-releasing liposomal formulations were prepared using high intraliposomal pH with the goal of prolonging NO release, as it was believed that the NONOate would be stabilized under the intraliposomal alkaline conditions (Suchyta, D. J.; Schoenfisch, M. H., Mol Pharm 2015, 12, 3569–3574; Nahar, K.; Rashid, J.; Absar, S.; Al-Saikhan, F. I.; Ahsan, F., Pharm Res 2016, 33, 1696–1710; Tahara, Y., et al., Medchemcomm 2017, 8, 415–421). Lip-MD3-7-PB released the smallest amount of NO in 24 h, indicating that intraliposomal pH is not the only factor in dictating NO-release kinetics. Of note, the free NO donor in prior formulations was not encapsulated in liposomes of neutral pH for comparison. As such, the sustained NO-release cannot be attributed solely to intraliposomal pH. Rather, the protection of the NO donor from the bulk solution via the liposomal bilayer must also be considered as the permeability of the liposomal bilayer may change (increase) outside of neutral conditions (Pasarin, D.; Ghizdareanu, et al., Polymers. MDPI February 1, 2023). For NO-releasing liposomes containing MD3, the stability of the bilayer dictates NO release in the first 24 h more than the intraliposomal pH. The total NO released from the liposomes over 24 h corresponds to antimicrobial thresholds in other bacterial species, indicating its potential for treating mycobacteria.Attorney Docket No.035052 / 626621 Example 5: Antimicrobial activity of MD3 liposomes

[0120] Bacteria and cell culture: The strains of M. abscessus chosen for these studies include ATCC 19977, a commercial strain with a smooth morphology, and 103, a clinical isolate that exhibits a rough morphology, provided by Diane Ordway. Cultures were prepared for in vitro assays by streaking on 7H10 agar plates supplemented with 10% Middlebrook oleic albumin dextrose catalase (OADC) and incubating at 37°C until colonies formed (2-3 days). Isolated colonies were sub-cultured in 7H9 + OACD broth at 37°C until turbid and then diluted to 5 × 106colony-forming units per mL (CFU mL-1) using 7H9 + OACD broth or RMPI 1640 media supplemented with 20% glycerol for planktonic and intracellular assays, respectively. To ensure consistency in bacterial growth kinetics for intracellular assays, bacteria was stored at -80° at least one day prior to infection of macrophages and thawed immediately before use.

[0121] Human monocyte cell line THP-1 was cultured in Roswell Park Memorial Institute (RPMI) 1640 culture medium (Corning; VWR), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Omega Scientific; Fisher Scientific), as well as 1% β-mercaptoethanol (Gibco; Fisher Scientific), GlutaMAX (Gibco; Fisher Scientific), non-essential amino acids (Gibco; Fisher Scientific), and HEPES buffer (Corning; VWR). Cells were maintained at 37°C with 5% carbon dioxide and split when population exceeded 9 × 105cells mL-1using 10% of the original media to encourage growth.

[0122] Planktonic antimicrobial assay: After preparation of the cultures as described above, the NO-releasing material (MD3, Lip-MD3-9, Lip-MD3-8, Lip-MD3-7) was prepared at 4 mg mL-1and pH 7 in 7H9 + OADC media and subjected to 2-fold dilutions across a 96-well microtiter plate. Smooth (ATCC 19977) and rough (103) cultures were added to produce a bacterial load of 5 × 105CFU well-1. Each plate included untreated culture and cultureless wells to serve as growth and sterility controls, respectively. The plates were incubated at 37°C for 4 and 24 h, after which the contents of each well were plated on 7H10 + OADC agar and incubated at 37°C until visible colony formation in the growth control occurred (approximately 48-72 h). Additionally, pH measurements were taken at 0 and 24 h using 5 uL aliquots blotted onto pH paper. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of NO-releasing material required for a 3-log reduction in bacterial viability measured in colony-forming units per mL (CFU mL-1). Biological replicates were done at least in triplicate for each experiment.Attorney Docket No.035052 / 626621

[0123] To elucidate the antimicrobial activity of Lip-MD3 solutions against mycobacteria, planktonic cultures of M. abscessus were treated and plated via microdilution (Figure 3A-B). Data from additional experiments performed on the Lip-MD3-8-HEPES formulation are shown in Figure 3C-D. The planktonic bactericidal assays were run at 4 h and 24 h of treatment to simulate typical residence time of a pulmonary formulation in the lung as well as standard treatment time of other in vitro assays, respectively. The 24 h treatment resulted in a 1-2-fold decrease in MBC values across all treatments relative to the 4 h treatments. This is likely due to the slow growth of M. abscessus (doubling time of ~6-10 h), requiring longer time periods for NO to be most effective. Additionally, the rough phenotype had lower average MBC values (0.125-0.875 mg MD3 mL-1) consistently across treatments than that of the smooth phenotype (0.250-1.125 mg MD3 mL-1). The rough phenotype is often less susceptible to traditional antibiotics due to the fact that the phenotype is mutated, which usually correlates to higher rates of acquired antimicrobial resistance. However, this mutation in the rough morphology downregulates production of glycopeptidolipid (GPL), which results in a more porous bacterial cell wall, which could increase drug permeation and consequently increase its NO susceptibility.

[0124] The efficacy of the NO-releasing treatments is mainly dependent of the final pH (i.e. pH at 24 h) of the formulation. The bacterial medium has a pH of 6.6, which corresponds to the initial pH of all of the systems. Any increase in pH can be attributed to the biproducts of NO release and the buffering properties of the NO-releasing formulations. Non-liposomal MD3, which is added to the bacterial medium without any formulation buffer, raises the pH from 6.6 to 7.2 over 24 h. The liposomal systems introduce buffer compositions corresponding to pKa values of 7.21, 7.65, and 8.30 for phosphate, HEPES, and tris buffers, respectively (Figure 2). The final pH across all five liposomal systems corresponded roughly to these pKa values. As shown in Table 8, the HEPES and TRIS formulations have final pH values of 7.8 and 8.8, which correspond roughly to the liposomal formulation pH. Conversely, phosphate buffer formulations maintain a final pH (encompassing treatment, formulation buffer, media, and cultures) of 7.6 and 7.8, despite the liposomal formulation pH of the solutions being 8 or 9, respectively.

[0125] Table 8. Change in pH for NO-releasing treatments during planktonic assays.Attorney Docket No. 035052 / 626621

[0126] The initial pH of the system (e.g. treatment, cultures, and media) mimics physiological conditions, and the liposomal formulations do not rise past the effective pH range of their respective buffers over 24 h. As NO is released from MD3, it also produces several low molecular weight metabolites, including formate, which is basic (Mcdonald, R. A., et al., Antimicrob Agents Chemother 2024). Given that diazeniumdiolates release NO via a proton-initiated mechanism, the higher pH slows down the release of MD3 over time. Consequently, the Lip-MD3-9-TRIS formulation had a significant decrease in bactericidal efficacy compared to non-liposomal MD3 due to slower, more sustained of NO release. Minimizing the overall increase in pH by using lower pH buffers in liposomal formulations is expected to enhance the bactericidal activity.

[0127] Aside from Lip-MD3-9-Tris, all other liposomal formulations killed M. abscessus with the same efficacy as non-liposomal MD3. This comparable activity is likely because bacteria in a planktonic assay are free-floating in the medium without a drug-delivery barrier like when intracellular. Nevertheless, mycobacterial cell walls are hydrophobic (Johansen, M. D.; Herrmann, J. L.; Kremer, L., Nat Rev Microbiol 2020, 18, 392–407). In this regard, the lipophilicity imparted by liposomal encapsulation of MD3 could lead to marginal improvements in antimycobacterial efficacy. Thus, it follows that the combination of minimizing the overall increase in pH and improved lipophilicity correlates to higher bactericidal efficiencies. Notably, the Lip-MD3-7-PB system showed a significant decrease in 24 h MBC in the rough phenotype. While the pH of non- liposomal MD3 was the lowest at 24 h, the increase in lipophilicity by encapsulating MD3 into a liposomal system may confer drug delivery benefits into the hydrophobic cell wall ofAttorney Docket No.035052 / 626621 mycobacteria. Based on the marked improvement in antimycobacterial efficacy in the planktonic experiments, subsequent intracellular assays were carried out with Lip-MD3-7-PB. Example 6: Intracellular antimicrobial assay of MD3 liposomes

[0128] While planktonic assays are a good general indicator of antimicrobial behavior, the intracellular infection of mycobacteria makes it particularly hard to cure. While MD3 has been shown to kill intracellular mycobacteria (Mcdonald, R. A., et al., Antimicrob Agents Chemother 2024), it is unlikely that the highly charged molecule is capable of permeating the cell membrane. Rather, NO is likely diffusing into the cells after release from MD3. Liposomal encapsulation would make MD3 more lipophilic and the ability to mimic the endogenous cell membrane should incorporate higher amounts of the small molecule itself into the cell, which should increase intracellular efficacy further.

[0129] To determine the potential drug delivery benefits of Liposomal MD3 on intracellular mycobacteria, macrophage experiments are performed following an established protocol with slight modification (Mcdonald, R. A., et al., Antimicrob Agents Chemother 2024; Rose, S. J., et al., PLoS One 2014, 9, 1–7). Briefly, human blood monocytes (THP-1) are seeded at 5 × 105cells mL-1into a 24-well plate and differentiated to type 0 macrophages (THP-1m) over 3 days using 25 ng mL-1of phorbol myristate acetate (PMA) in RPMI medium. The THP-1m cells are then allowed to incubate in PMA-free media for 1-2 days. The cells are subsequently inoculated with media containing either smooth (ATCC 19977) or rough (103) M. abscessus at a multiplicity of infection of 0.1 overnight. Extracellular bacteria are removed, and the cells are treated with media containing either MD3, Lip-MD3, AMK, Lip-AMK, or empty liposomes and incubated 24 h. The cells are then lysed using Tween-80 and the bacteria are subsequently diluted and plated onto 7H10 + OADC agar plates for counting. Example 6A: Intracellular infection study

[0130] The performance of Lip-MD3-7-PB in planktonic culture combined with its favorable physiochemical properties prompted further research in an intracellular infection model. Chronic NTM can reside intracellularly, even if the extracellular infection is eradicated, so an intracellular model better represents the challenge of the disease (Johansen, M. D.; Herrmann, J. L.; Kremer, L., Nat Rev Microbiol 2020, 18, 392–407). Liposomal and non-liposomal treatments of MD3 wereAttorney Docket No.035052 / 626621 tested against smooth and rough M. abscessus that were internalized overnight in THP-1m cells. Liposomes only containing buffer (i.e., empty liposomes), liposomal and free (non-liposomal) amikacin were used to determine MD3’s performance against a formulation control and traditional NTM antibiotic, respectively. As in the planktonic study, treatments were evaluated at 4 and 24 h.

[0131] The empty liposomes did not confer any antimicrobial activity at a volume corresponding to the maximum concentration of the other formulations (Figure 6). This data, combined with an earlier report on NO-liberated MD3 not killing NTM, implicates NO as the sole driver of antimicrobial activity (Mcdonald, R. A., et al., Antimicrob Agents Chemother 2024, 68, 1–21). Non-liposomal MD3 was able to achieve a three-log reduction in bacterial load compared to controls at 0.125-0.500 mg mL-1, depending on strain and time allotted. Again, increased incubation time with the treatment (Figure 6C, 6D) resulted in enhanced antimicrobial activity (lower MBC values), but without differences between smooth and rough phenotypes (Figure 6B, 6D). Regardless, the behavior of the liposomal MD3 was either similar or better than free (non- liposomal) MD3 at all time points for both strains tested.

[0132] When tested with a formulation comparable to Lip-MD3-7-PB, liposomal amikacin was the only bactericidal species compared to its non-liposomal counterpart (Rose, S. J.; Neville, M. E.; Gupta, R.; Bermudez, L. E., PLoS One 2014, 9, 1–7). These data infer that the liposomal formulation proposed for treating M. abscessus intracellularly works as intended for traditional antibiotics. However, it is possible that this assay alone is not sufficient for demonstrating the efficacy of the liposomal system since MD3 is capable of releasing membrane permeable NO and the diffusion distance of NO is ~100 µm, 5x bigger than the diameter of macrophages (Möller, M. N.; Denicola, A., Free Radic Biol Med 2018, 128, 137–143). Example 7: In vitro granuloma assay of MD3 liposomes

[0133] An in vitro granuloma assay provides a more complex form of infection to better mimic latent mycobacterial infection. In vitro Granuloma-like cellular aggregates are cultured in THP- 1m cells. THP-1 cells are seeded at 5 × 105cells mL-1into a 24-well plate and differentiated to type 0 macrophages (THP-1m) over 3 days using 25 ng mL-1of phorbol myristate acetate (PMA) in RPMI medium. The THP-1m cells are then allowed to incubate in PMA-free media for 1-2 days. The cells are subsequently inoculated with media containing either smooth (ATCC 19977) or rough (103) M. abscessus at a multiplicity of infection of 0.1 and the cells are left to incubateAttorney Docket No.035052 / 626621 at 37°C for 4-5 days without removal of the bacteria. Formation of the aggregates are confirmed via light microscopy images taken daily. Once the aggregates are established, they are treated with 100 µL of treated media containing MD3, Lip-MD3, AMK, Lip-AMK, or empty liposomes and incubated 24 h. The cells were then lysed using Tween-80 and the bacteria were subsequently diluted and plated onto 7H10 + OADC agar plates for counting. Example 8: Quantitative cellular uptake

[0134] A fluorophore was encapsulated into the liposomes to quantify the amount of cell uptake in a formulation comparable to Lip-MD3-7-PB formulation, since NO is capable of permeating into the cell membrane independent of liposomal encapsulation. Pyranine was selected as a model fluorophore for encapsulation as it, like MD3, is triply anionic, largely hydrophilic, and membrane impermeable (Nandi, R.; Amdursky, N., Acc Chem Res 2022, 55, 2728–2739). The chemical structure of . Pyranine has a net neutral charge and may further comprise onecations independently selected from the group consisting of sodium, potassium, lithium, calcium, magnesium, and ammonium. In some embodiments, one or more pharmaceutically acceptable cations of pyranine are sodium. In some embodiments, pyranine comprises three sodium cations. In this respect, it should not readily enter cells in free form. The fluorescence intensity of intracellular liposomal pyranine was compared to intracellular non-liposomal pyranine at 0.5, 2, 4, and 24 h and calculated as the relative fluorescence intensity. As shown in Figure 7, the liposomal fluorescence intensity initially started lower than the non-liposomal baseline but maintained ~2-fold increase in intensity at 2 and 4 h. At 24 h, this relative intensity increased significantly (~3.5x greater), indicating that liposomal uptake was cumulative over a 24 h period, consistent with other uptake studies (Quagliariello, V., et al., Oncol Rep 2019, 41, 1476–1486). This experiment also demonstrated that the proposed liposomal formulation can successfully deliver membrane impermeable molecules to the insides of cells, which should correlate to increased intracellular MD3 concentrations. Example 9: Confocal microscopy studiesAttorney Docket No.035052 / 626621

[0135] The fluorescent microscopy experiments confirmed that the proposed liposomal formulation facilitated intracellular uptake of membrane impermeable cargo. Increased intracellular concentration of MD3 from liposomal delivery would also increase intracellular NO, as NO could be released directly within the cell instead of passing through the cell membrane. In order to test this hypothesis, confocal microscopy was performed with THP-1m cells pretreated with 5 µM of DAF-FM, a fluorescent probe that fluoresces green in the presence of NO. The cells were then treated with either 0.1 mg mL-1MD3 or liposomal MD3 co-encapsulated with rhodamine B to track the liposomes (red fluorescence) and incubated for 2 h. After incubation, the treatment media was aspirated and the cells washed to remove extracellular material before imaging. As shown in the confocal images in Figure 8A-F, the red fluorescence from Lip-MD3-7- PB shows intracellular localization of the liposomes into the cells. The green fluorescence from the DAF-FM revealed a higher population of cells containing NO compared to treatment with free MD3.

[0136] Based on its size and neutrality, NO would be expected to permeate through the cell membrane on its own. It is thus surprising to see the free MD3-treated cell population showing minimal green fluorescence. The cells may be metabolizing or effluxing NO over time, which would result in a decrease in fluorescence if the extracellular source of NO is removed and no intracellular NO donor is present (as we expect for non-liposomal MD3 treatment). This hypothesis was corroborated by a time-course study in which the extracellular material was removed and subsequently imaged for 45 min across both treatments. While the cells treated with MD3 showed fluorescence that dissipated markedly over time, those treated with Lip-MD3-7-PB maintained constant fluorescence throughout the 45 min duration (Figure 9). The implications of having not only NO but also MD3 within the cells highlights the potential benefits of the drug delivery system. Chronic mycobacterial lung infections are characterized by granuloma formation on the millimeter scale, which exceeds the typical diffusion distance of NO (Wells, G., et al., Am J Respir Crit Care Med 2021, 204, 583). The ability of the NO-releasing prodrug to permeate into the interior of the cell cluster would extend the effective range of NO in killing granuloma- affiliated bacteria.

[0137] Finally, it is likely that simple membrane fusion or endocytosis are the main mechanisms of uptake since these liposomes are not targeting a particular receptor on the macrophages (Akbarzadeh, A.; Rezaei-sadabady, R.; Davaran, S.; Joo, S. W.; Zarghami, N. Nanoscale Res LettAttorney Docket No.035052 / 626621 2013, 8, 1–9). The location of the liposomal cargo after delivery is thus important. Looking at an isolated, enlarged image of a Lip-MD3-7-PB-treated cell (Figure 10A), the DAF-FM fluorescence appears consistent (i.e., of similar intensity) throughout the cell. Likewise, the liposomal cargo appearing speckled, is distributed non-specifically across the cell surface. Given that two- dimensional imaging does not allow for discernment for penetration of drug cargo, a Z-stack with orthogonal slicing was taken for a liposome-treated cell population (Figure 10B). Three- dimensional imaging revealed that the red liposomal cargo was not sequestered at the cell membrane, but rather distributed throughout the cytosol. Given that intracellular bacteria will likely be localized to phagosomes, the distribution of liposomal material throughout the cytosol would be adequate proximity to confer antimicrobial activity (Awuh, J. A.; Flo, T. H., Cellular and Molecular Life Sciences 2017, 74, 1625–1648).

[0138] The formation of NO-releasing liposomes is described using a simple thin film hydration technique to passively encapsulate MD3. This manner of liposome formation facilitates robust and stable loading of MD3. The liposomes in these buffers displayed favorable size, drug loading, retention, and drug release parameters. Planktonic bacterial studies revealed that lower pKa buffers correlated with improved antimicrobial activity. Liposomal encapsulation of MD3 produced similar or marginally better antimicrobial efficacy compared to free (non-liposomal) MD3 in an in vitro intracellular infection model. Probing cellular uptake revealed that liposomes increased the uptake of impermeable species, correlating to greater intracellular NO levels in the cells compared to that achievable with free MD3. The favorable physicochemical and therapeutic potential of NO- releasing liposomes suggest their utility for the treatment of NTM-PD. Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which the inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

Attorney Docket No.035052 / 626621 CLAIMS What is claimed is:

1. A nitric oxide (NO)-releasing liposome comprising, a lipid bilayer comprising an inner leaflet and an outer leaflet; and a NO donor moiety in an aqueous core encapsulated by the lipid bilayer, wherein the NO donor moiety comprises one or more diazeniumdiolate groups.

2. The liposome of claim 1, wherein the NO donor moiety is a C-diazeniumdiolate.

3. The liposome of claim 2, wherein the C-diazeniumdiolate comprises two or more diazeniumdiolate groups.

4. The liposome of claim 3, wherein the C-diazeniumdiolate comprises three diazeniumdiolate groups.

5. The liposome of claim 4, wherein the NO donor moiety is methyl tris diazeniumdiolate (MD3).

6. The liposome of claim 1, wherein the NO donor moiety is a N-diazeniumdiolate.

7. The liposome of claim 6, wherein the N-diazeniumdiolate is 1-[N-[3-aminopropyl]-N-[4- (3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate (SPER / NO), N-[bis(3- aminopropyl)amino]diazen-1-ium-1,2-diolate (DPTA / NO), or N-[bis(2- aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA / NO).

8. The liposome of any one of claims 1-7, wherein the one or more diazeniumdiolate groups comprise one or more pharmaceutically acceptable cations independently selected from the group consisting of sodium, potassium, lithium, calcium, magnesium, and ammonium.

9. The liposome of any one of claims 1-8, wherein the lipid bilayer comprises one or more phospholipids.Attorney Docket No.035052 / 626621 10. The liposome of claim 9, wherein at least one phospholipid is a diacyl- phosphatidylcholine.

11. The liposome of claim 10, wherein the lipid bilayer comprises dipalmitoylphosphatidylcholine (DPPC).

12. The liposome of claim 9, wherein at least one phospholipid is a diacyl- phosphatidylethanolamine.

13. The liposome of claim 11, wherein the lipid bilayer comprises dipalmitoylphosphatidylethanolamine (DPPE).

14. The liposome of claim 9, wherein one or more phospholipid is a PEGylated phospholipid.

15. The liposome of claim 14, wherein the lipid bilayer comprises a polyethylene glycol- functionalized dimyristoyl-phosphorylethanolamine (DMPE-PEG).

16. The liposome of any one of claims 1-15, wherein the lipid bilayer further comprises cholesterol.

17. The liposome of claim 16, wherein the lipid bilayer comprises a molar ratio of about 1:1 to about 5:1 of phospholipid to cholesterol.

18. The liposome of claim 17, the lipid bilayer comprises a molar ratio of about 2:1 of DPPC to cholesterol.

19. The liposome of any one of claims 1-18, wherein the aqueous core encapsulated by the lipid bilayer further comprises a buffer.

20. The liposome of claim 19, wherein the buffer comprises one or more of sodium phosphate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (TRIS).

21. The liposome of claim 19 or 20, wherein the concentration of the buffer is about 25 mM.Attorney Docket No.035052 / 626621 22. The liposome of any one of claims 1-21, wherein the aqueous core encapsulated by the lipid bilayer has a pH of about 7 to about 9.

23. The liposome of claim 22, wherein the aqueous core encapsulated by the lipid bilayer has a pH of about 7.

24. The liposome of any one of claims 1-23, wherein the liposome is a unilamellar liposome.

25. The liposome of any one of claims 1-24, wherein the diameter of the liposome is less than 300 nm.

26. The liposome of claim 25, wherein the diameter of the liposome is about 150 nm to about 250 nm.

27. The liposome of any one of claims 1-26, wherein at least 95% of the NO donor moiety encapsulated by the lipid bilayer retain the diazeniumdiolate groups.

28. The liposome of any one of claims 1-27, wherein the liposome releases at least 5 µmol / mg, at least 6 µmol / mg, at least 7 µmol / mg, at least 8 µmol / mg, at least 9 µmol / mg, or at least 10 µmol / mg of NO over 24 hours following administration.

29. A composition comprising a plurality of liposomes of any one of claims 1-28 in an aqueous solution.

30. The composition of claim 29, wherein a concentration of the NO donor moiety is greater than 1 mg / mL, greater than 2 mg / mL, greater than 3 mg / mL, greater than 4 mg / mL, greater than 5 mg / mL, or greater than 6 mg / mL.

31. The composition of claim 29 or 30, wherein the plurality of liposomes has a dispersity of less than 0.

3.

32. The composition of any one of claims 29-31, wherein the plurality of liposomes has a zeta potential of less than -5 mV.Attorney Docket No.035052 / 626621 33. The composition of any one of claims 30-31, wherein the composition further comprises amikacin, or a pharmaceutically acceptable salt thereof, optionally wherein the amikacin is encapsulated in the aqueous core of a liposome.

34. A method for treating a respiratory infection in a patient in need thereof, the method comprising administering to the patient an effective amount of the liposome of any one of claims 1-29 or the composition of any one of claims 30-33.

35. The method of claim 34, wherein the respiratory infection is caused by an intracellular pathogen.

36. The method of claim 35, wherein the intracellular pathogen is an intracellular bacterium.

37. The method of claim 36, wherein the intracellular bacterium is a Mycobacterium.

38. The method of claim 37, wherein the Mycobacterium is a nontuberculosis Mycobacterium (NTM).

39. The method of claim 38, wherein the NTM is selected from the group consisting of Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium haemophilum, Mycobacterium^gordonae, Mycobacterium simiae, Mycobacterium marinum, Mycobacterium malmoense, Mycobacterium ulcerans, Mycobacterium bolletii, Mycobacterium massiliense, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium porcinum, Mycobacterium smegmatis, Mycobacterium vaccae, and Mycobacterium mucogenicum.

40. The method of claim 39, wherein the NTM is Mycobacterium abscessus.

41. The method of any one of claims 37-40, wherein the Mycobacterium has a smooth morphology.

42. The method of any one of claims 37-40, wherein the Mycobacterium has a rough morphology.Attorney Docket No.035052 / 626621 43. The method of any one of claims 34-42, wherein the composition is aerosolized and administered to the lungs of the patient.

44. The method of any one of claims 34-43, wherein the method further comprises the administering to the patient an effective amount of amikacin.

45. A method of making a plurality of the liposomes of any one of claims 1-29 or the composition of any one of claims 30-33, wherein the NO donor moiety is encapsulated in the aqueous core by the lipid bilayer as the liposome is formed in an aqueous solution.

46. The method of claim 45, wherein the method comprises forming a thin film of one or more lipids and adding an aqueous solution comprising the NO donor moiety.

47. The method of claim 46, wherein the aqueous solution comprises a buffer.

48. The method of any one of claims 45-47, wherein the aqueous solution comprising the liposomes is extruded through a filter.

49. The method of any one of claims 45-48, wherein an encapsulation efficiency of the NO donor moiety is greater than 20%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, or greater than 50%.

50. The liposome of any one of claims 1-28, wherein the aqueous core encapsulated by the lipid bilayer contains a fluorophore.

51. The liposome of claim 50, wherein the fluorophore is pyranine.

52. The composition of any one of claims 29-33, wherein the plurality of liposomes has a fluorescence intensity about 2-3.5x greater than a non-liposomal fluorophore control.

53. The liposome of any one of claims 1-28, wherein the aqueous core further comprises rhodamine B.

54. The liposome of claim 53, wherein the NO donor moiety is MD3.Attorney Docket No.035052 / 626621 55. The composition of any one of claims 53-54, wherein the plurality of liposomes shows intracellular localization greater than the non-liposomal NO donor moiety as measured by confocal microscopy.

Images