Nanoparticle-mediated DNA repair through cellular delivery of NAD+ precursors

Abstract

Various implementations described herein relate to methods and compositions for delivery of small molecules to the brain. According to some implementations, small molecules are encapsulated in a nanoparticle. Small molecules include NAD+ precursors. In some implementations, the nanoparticle includes poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG). Various implementations described herein can be used for research, diagnostic, or therapeutic uses.

Description

NANOPARTICLE-MEDIATED DNA REPAIR THROUGH CELLULAR DELIVERY OF NAD+ PRECURSORSCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the priority of U.S. Provisional Patent Application No. 63 / 538,779, filed on September 15, 2023, and which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] This application relates to nanoparticle-mediated DNA repair through cellular delivery of NAD+ and NAD+ precursors. The small molecule-loaded nanoparticles can be used in the treatment of neurological disorders.BACKGROUND

[0003] The brain has a large metabolic demand, accounting for over 20% of total oxygen metabolism, and requires the continuous delivery of glucose, oxygen, and other nutrients. Oxygen and glucose delivered to the cell cytoplasm and to mitochondria are processed through glycolysis and the tricarboxylic acid (TCA) cycle to produce energy in the form of adenosine triphosphate (ATP). In humans, ATP levels and the rate of energy production are positively associated with brain nicotinamide adenine dinucleotide (NAD+) levels and the NAD+ / NADH ratio. NAD+ is the cell’s hydrogen carrier for redox enzymes and is required for over 500 enzymatic reactions, playing a role in cellular bioenergetics for metabolism and ATP production. NAD+ has several precursors, including nicotinamide (NAM), nicotinic acid (NA, also referred to as niacin), nicotinamide mononucleotide (NMN), and nicotinamide riboside (NR), or it can be synthesized de novo from tryptophan via the kynurenine pathway. The NAD+ / NADH ratio is closely related to the cell metabolic state that determines cytosolic and mitochondrial metabolic homeostasis. NAD+ is essential in glycolysis and the TCA cycle. NAD+ accepts hydride equivalents, which form NADH during ATP production. NAD+ levels can be affected by various factors including age, diet, and health status, and NAD+ dependent enzymes are involved in synaptic plasticity and neuronal stress resistance. Previous studies indicate that many neurological diseases lead to a decrease in total NAD+ levels, NAD+ / NADH redox ratio, or both in the brain.

[0004] Acute injuries to the brain such as perinatal asphyxia, stroke, and traumatic brain injury lead to ischemia, excitotoxicity, and inflammation that result in DNA damage. DNA damage triggers cell death through activation of the poly(ADP-ribose) polymerase-1 (PARP-1 ) cell death pathway, which consumes NAD+. An increase in PARP-1 activity decreases cytosolic NAD+ pools, which decreases ATP production, impairs mitochondrial function, and inactivates cellular metabolism leading to cell death. Increasing NAD+ levels immediately after acute brain injury could restore cellular NAD+ levels to aid in DNA repair and reduce cell death.

[0005] In most cells, it is more common for NAD+ to be recycled via salvage pathways, such as from NAM or NA in the diet, rather than synthesized de novo from tryptophan. In the brain, NAD+ is more likely to be made by NAM- derived NMN available locally to brain cells, rather than from NR or NMN given via intravenous injection. NAM candiffuse across the cell membrane and could be a potent NAD+ precursor to direct delivery of NAD+ to brain cells. However, mammalian cells cannot import NAD+, the cell-specific targeting of NAD+ and NAM is limited, and the conversion rate of precursors like NAM to NAD+ in the brain is not fully understood. Therefore, cellular delivery strategies are needed to capture the potential of an NAD+-restorative therapeutic approach.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0007] FIG. 1 illustrates an example environment for treatment of NAD+-related conditions.

[0008] FIG. 2 illustrates that free NAM and NAD are not effective at improving cell viability.

[0009] FIG. 3 illustrates that in healthy BV-2 cells, blank PLGA-PEG particles and PLGA-PEG nanoparticle- encapsulated NAM (NAM / PLGA-PEG nanoparticles) have minimal effect on cell viability.

[0010] FIG. 4 ill ustrates the effect of NAM / PLGA-PEG nanoparticles on cell viability in oxygen glucose deprivation (OGD)-exposed BV-2 cells.

[0011] FIG. 5 illustrates NAM / PLGA-PEG nanoparticles long term stability and thermal stability.

[0012] FIG. 6A illustrates results of a lactase dehydrogenase (LDH) assay showing % cell cytotoxicity with and without NAM-loaded PLGA-PEG nanoparticles under healthy conditions (n=6). FIG. 6B illustrates regional % cell death of OGD slices treated with and without NAM-loaded PLGA-PEG nanoparticles (n=3-4).

[0013] FIGs. 7A-7C illustrate confocal images of NAM-loaded PLGA-PEG nanoparticles conjugated with CF555 colocalizing with microglia (costained with iba and dapi) in the (7A) cortex and (7B, 7C) striatum.

[0014] FIG. 8 illustrates a Z stack image of NAM loaded PLGA-PEG nanoparticles colocalizing with microglia (costained with iba and dapi).DETAILED DESCRIPTION

[0015] Implementations of the present disclosure relate to diseases and conditions where nicotinamide adenine dinucleotide (NAD+) levels need to be increased to promote DNA repair. Various implementations of the present disclosure provide methods and compositions for targeted delivery of NAD+ and NAD+ precursors that would address the underlying mechanism of NAD+ depletion in response to organ injury (e.g., brain injury) and other energy-depletion conditions. For instance, the disclosed delivery strategies could be applied in other energydepletion conditions, such as in COVID-19 treatment. Implementations of the present disclosure could have applications in aging and neurodegeneration, where NAD+ levels steadily decline, resulting in altered metabolism and increased disease susceptibility. Various implementations of the present disclosure provide methods and compositions for targeted delivery of inhibitors associated with increased availability of NAD+, such as PARP-1inhibitors. Various implementations of the disclosed delivery strategies can be extended to target other organs and diseases.

[0016] Various implementations of the present disclosure replenish cellular energy to enable DNA repair by using a nano-based delivery strategy. The disclosed nanoparticle-mediated delivery of NAD+ precursors promotes cellular function and increases cytosolic NAD+ levels following acute injury, resulting in an improved NAD+ / NAD hydrogen (NADH) redox ratio and DNA repair. Various implementations described herein relate to methods and compositions for delivery of small molecules to the central nervous system. In various implementations described herein, small molecules are delivered to the cytosol of brain cells. In various implementations, the disclosed nanoparticles are loaded with NAD+ precursors to replenish NAD+ levels in acutely-injured, energy-depleted brain ceils with a primary target outcome to see reduction in DNA damage and cell death.

[0017] In some implementations of the present disclosure, small molecules are encapsulated in a nanoparticle. Implementations of the present disclosure describe nanoparticles including poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG). The present disclosure describes the biocompatibility of the disclosed NAD+-replenishing nanoparticles as well as the long-term and thermal stability of the disclosed nanoparticles. Implementations described herein can be used for research, diagnostic, or therapeutic uses.

[0018] Various implementations of the present disclosure utilize polymers that are approved by the United States Food and Drug Administration (FDA) are safe and biodegradable. The use of FDA-approved polymers may facilitate rapid clinical translation of the disclosed nanoparticles. Furthermore, various implementations of the present disclosure include nanoparticles that specifically target particular cell types without additional modification. Avoiding additional modification can reduce manufacturing complexity and potentially expedite regulatory approval for clinical use.

[0019] Embodiments of the present disclosure will now be described with reference to the accompanying figures.

[0020] FIG. 1 illustrates an example environment 100 for treatment of NAD+-related conditions. In this example, a subject 102 suffers from a pathological condition associated with NAD+. The subject 102, in various cases, is a neonate, an infant, a child, or an adult. In some examples, the pathological condition includes a neurological condition. In various cases, the neurological conditions include an acute brain injury. Examples of an acute brain injury include asphyxia, a stroke, circulatory arrest, a traumatic brain injury, or the like. In various cases, the pathological condition includes another acute organ injury. For instance, the acute organ injury may include acute liver injury, acute liver failure, acute spleen injury, acute kidney injury, acute lung injury, a myocardial infarction, acute coronary syndrome, acute heart failure, or the like. In some examples, the pathological condition includes a neurodegenerative disease, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), motor neuron disease, multiple system atrophy, progressive supranuclear palsy, ataxia, dementia, Creutzfeldt-Jakob disease, and the like. In some examples, the pathological condition includes an aging-related condition, such as osteoporosis, osteoarthritis, cardiovascular disease, diabetes,vision problems or loss, hearing problems or loss, sarcopenia, neurodegenerative disease, or other conditions associated with aging.

[0021] In various implementations, the pathological condition is an energy-depleted condition. Energy-depletion condition,” as used herein, refers to a condition associated with a lack of energy (e.g., fatigue, exhaustion, etc.). Energy-depletion conditions can include conditions associated with mitochondrial dysfunction. Mitochondrial dysfunction, in some cases, results in insufficient cellular energy production, which can present as a lack of energy of an individual. Various conditions are associated with mitochondrial dysfunction, including metabolic disorders, cardiovascular diseases, cancer, neurodegenerative diseases, and others.

[0022] Currently, there is no effective way to achieve targeted cell energy regeneration in the brain. Treatment of acute brain injuries and other energy-depletion conditions is generally focused on symptom management (e.g., swelling reduction, pain management, etc.). NAD+ is a coenzyme that has an integral role in cellular metabolism. NAD+ is generally depleted in response to brain injury and other organ injuries, as well as in systemic conditions, such as sepsis, inflammatory diseases, and aging-related conditions. NAD+ can be synthesized from NAD+ precursors, some of which are bioavailable through dietary intake. NAD+ precursors include nicotinic acid (NA), nicotinamide (NAM), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), and tryptophan. Inhibitors associated with increased NAD+ availability include poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors and CD38 inhibitors. “NAD+ boosters,” as used herein, refers to NAD+, NAD+ precursors, and inhibitors associated with increased NAD+ availability.

[0023] Targeted delivery of NAD+ boosters has not been successfully demonstrated, limiting the development of NAD+-based treatments. In particular, targeted delivery into the brain could have significant impacts on the treatment of acute brain injuries. In addition, NAD+ boosters delivered via intravenous injection are often not effectively used by cells. Accordingly, there is a lack of effective techniques for targeted delivery of NAD+ boosters for treatment of organ injuries and other energy-depletion conditions.

[0024] These issues, in various implementations, are addressed by encapsulating NAD+ and NAD+ boosters in a synthetic polymeric nanoparticle. “Nanoparticles,” “nanocarriers,” and their equivalents, as used herein, refer to particles with dimensions (e.g., diameter, height, length, width, etc.) less than or equal to 1000 nanometers (nm). Nanoparticles may have various shapes, including spheres, tubes, dendritic shapes, amorphous shapes, or any other shape. Nanoparticles may be hollow, solid, porous, or have a core-shell structure. A core-shell structure refers to a particle with a core component that is encased in a shell component. The core component may include different materials and / or have different properties (e.g., density, molecular weight, porosity, zeta-potential, etc.) than the shell component. Polymeric nanoparticles refer to nanoparticles that include one or more polymers. In various examples, the polymers are biocompatible. Examples of biocompatible polymers include poly (lactide-co-glycolide) (PLGA), polyethylene glycol) (PEG), polylactic acid (PLA), and polyglycolic acid (PGA).

[0025] Nanomaterials, including liposomes and polymeric nanoparticles, have shown great promise for being used as drug delivery systems. Yet, for liposomes and biodegradable polymers, it is difficult to obtain uniformly sized small nanoparticles or tailor the shape and composition of nanocarriers, which can limit their application in targeted intracellular delivery. Furthermore, improved therapeutic efficiency and target specificity can be dependent on surface chemistry modification of nanocarriers, such as the incorporation of targeting ligands that specifically bind particular cell types. Accurately controlling the density, location, and stereochemistry of conjugated ligands of nanocarriers remains a challenge, resulting in low affinity ligand binding with targeted cells.

[0026] These issues are addressed, in some examples, by using small molecule-loaded nanoparticles with uniform size distribution, low polydispersity, and a near-neutral surface charge (e.g., zeta potential). According to various implementations, the small molecule-loaded nanoparticles efficiently target particular cell types, enabling use of polymeric nanoparticles without additional modification (e.g., the incorporation of additional targeting ligands). In various cases, avoiding additional modification can reduce manufacturing complexity and / or potentially expedite regulatory approval for clinical use. The disclosed nanoparticles, according to various implementations, are associated with low cytotoxicity and immunogenic response. Various implementations of the present disclosure describe nanoparticles that include PLGA and PEG. PLGA and PEG are both approved by the United States Food and Drug Administration (FDA) are safe and biodegradable. The use of FDA-approved polymers may facilitate rapid clinical translation of the disclosed nanoparticles.

[0027] In various implementations of the present disclosure, an NAD+ booster 104 is encapsulated in a polymeric nanoparticle. The NAD+ booster 104, in various examples, includes NAD+ or an NAD+ precursor. NAD+ precursors include NAM, NMN, NA, NR, or the like. In some examples, the NAD+ booster 104 includes an inhibitor associated with increased NAD+ availability, such as a PARP-1 inhibitor, a CD38 inhibitor, or the like.

[0028] The nanoparticles 106, in various examples, are polymeric nanoparticles. According to some implementations, the nanoparticles 106 include PLGA and / or PEG. In some cases, a molecular weight of the PLGA is in a range of 15 kilodaltons (kDa) to 45 kDa. A ratio of lactic acid to glycolic acid, in various examples, in the PLGA is in a range of 3:1 to 3:9. For instance, the ratio of lactic acid to glycolic acid in the PLGA may be 1 :1. In some cases, a molecular weight of the PEG is between 3 kDa and 7 kDa. For example, the molecular weight of the PEG may be 5 kDa. In various examples, a zeta-potential of the nanoparticles 106 is near-neutral. For instance, the zetapotential of the nanoparticles 106 may be in a range of -15 millivolts (mV) to +10 mV. In some examples, the zetapotential of the nanoparticles 106 is in a range of -10 mV to 10 mV. In some cases, the zeta potential of the nanoparticles 106 is in a range of -8 to 0 mV.

[0029] According to various implementations, a size, a size distribution, and a zeta potential of the nanoparticles 106 enables effective intracellular delivery. For instance, at least one dimension of the nanoparticles 106 is in a range of 20 nanometers (nm) to 100 nm. In some examples, the dimension(s) of the nanoparticles 106 is in a range of 40 nm to 80 nm. In various instances, a shape of the nanoparticles 106 is spherical. Some implementations of thenanoparticles 106 have a low polydispersity index, wherein the polydispersity index indicates a distribution of size in a sample, such that a lower polydispersity index indicates a more uniform distribution. The control of the size distribution facilitates uniform intracellular delivery and in vivo activity. In some examples, the polydispersity index of the nanoparticles 106 is in a range of 0.01 to 0.25. In various cases, a standard deviation of a size distribution of the nanoparticles 106 is in a range of 0 nm to 15 nm.

[0030] In some examples, a surfactant is disposed on the outer surface of the synthetic nanoparticles 106. The surfactant includes, for instance, cholic acid, poloxamer 407, polysorbate 20, polysorbate 80, or the like. In various examples, the surfactant affects the cellular targeting of the nanoparticles 106. In some examples, the surfactant affects diffusion (e.g., diffusion kinetics) of the nanoparticles 106 through tissue to reach target cells. Accordingly, the surfactant can, in some cases, affect cellular targeting (e.g., the cells and cell types that the nanoparticles 106 come in contact with) and the degree of uptake of the nanoparticles 106 into cells. For instance, the nanoparticles 106 that include cholic acid may be delivered to a first cell type, while the nanoparticles 106 that include polysorbate 20 may be delivered to a second cell type and not to the first cell type.

[0031] In some examples, the nanoparticles 106 are functionalized to improve stability, activity, loading efficiency, loading capacity, or the like. In some examples, the functionalization alters the mechanism of internalization of the nanoparticles 106 into cells. In various cases, the nanoparticles 106 are functionalized with at least one of a carboxyl group, a methyl group, a methoxy group, or a hydroxyl group. For instance, an end chain of PEG of the nanoparticles 106 may be conjugated to or include a functional group. The functional group, in various cases, has a neutral or negative charge.

[0032] In various instances, the nanoparticles 106 are delivered into one or more cells in the subject 102. In some examples, the nanoparticles 106 are delivered to one or more cell types. “Cell type,” as used herein, refers to cells with a similar phenotype and / or genotype, or cells associated with a particular organ. For instance, cell types include brain cells, liver cells, spleen cells, kidney cells, or the like. In some cases, cell types include epithelial cells, osteocytes, adipocytes, chondrocytes, muscle cells, red blood cells, white blood cells, or the like. In some cases, cell types include glial cells, astrocytes, oligodendrocytes, Schwann cells, microglial cells, ependymal cells, neuroendocrine cells, neurons, or the like. In particular examples, the nanoparticles 106 are delivered into a particular cell type. For instance, in various implementations of the present disclosure, the nanoparticles 106 are delivered to astrocytes.

[0033] In various cases, the nanoparticles 106 are administered to the subject 102 for treatment of the pathological condition. In some implementations, a dosage schedule of the nanoparticles 106 is determined based on the pathological condition. In various cases, a first dose of the nanoparticles 106 is administered to the subject 102 within 6 hours of an acute organ injury, such as an acute brain injury. In various cases, additional doses of the nanoparticles 106 are administered to the subject 102 after 6 hours from the acute organ injury. For example, the additional doses of the nanoparticles 106 may be determined based on a response of the subject 102 to thetreatment. For instance, cellular NAD+ levels and / or NAD+ / NADH ratios of the subject 102 may enable determination of how well the subject 102 is responding to the treatment, and whether the subject 102 may benefit from more frequent doses, additional doses, higher doses, or the like.

[0034] in some examples, an additional treatment may be administered to the subject 102. The additional treatment may include, for instance, an antioxidant, a DNA repair enhancer, an anti-inflammatory agent, a co-factor, a redox substrate, a neurotrophic factor, or another therapy for the treatment of the pathological condition. In various cases, the nanoparticles 106 are administered concurrently with the additional treatment. In various cases, the nanoparticles 106 are administered before and / or after the administration of the additional treatment.

[0035] Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0036] An "effective amount" is the amount of a formulation used to result in a desired physiological change in the subject. For example, an effective amount can provide a change in a metric associated with a pathological disease associated with NAD+ (e.g., a severity or frequency of symptoms or a change in a marker associated with the pathological disease). Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically significant effect in an animal model or in vitro assay relevant to the assessment of a pathological disease’s development or progression.

[0037] A "prophylactic treatment" includes a treatment administered to a subject (e.g. , the subject 102) who does not display signs or symptoms of a pathological disease associated with NAD+ or displays only early signs or symptoms of a pathological disease associated with NAD+ such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the pathological disease further. Thus, a prophylactic treatment functions as a preventative treatment against a pathological disease associated with NAD+. In particular embodiments, prophylactic treatments reduce or delay physical symptoms associated with a pathological disease associated with NAD+.

[0038] A "therapeutic treatment" includes a treatment administered to a subject (e.g., the subject 102) who displays symptoms or signs of a pathological disease associated with NAD+ and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the pathological disease associated with NAD+. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the pathological disease associated with NAD+ and / or reduce control or eliminate side effects of the pathological disease associated with NAD+.

[0039] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0040] In particular embodiments, therapeutically effective amounts induce a reduced progression of a pathological disease associated with NAD+. In particular embodiments, the reduced progression includes reduced or stabilized symptoms, reduced or stabilized levels of one or more markers associated with the pathological disease, or a changein another metric known in the art. For instance, the reduced progression may include a reduction in systemic and / or localized inflammation.

[0041] In particular embodiments, therapeutically effective amounts induce a physiological effect in the subject 102. For instance, the physiological effect comprises an increased NAD+ level or NAD+ / NADH ratio in a cell within the subject compared to a benchmark NAD+ level or NAD+ / NADH ratio. In various cases, the increased NAD+ level or NAD+ / NADH ratio is at least 1%, 2%, 5%, 10%, 15%, or 20% greater than the benchmark NAD+ level or NAD+ / NADH ratio. The benchmark level, in various cases, includes a NAD+ level or a NAD+ / NADH ratio of the subject at a prior time, a NAD+ level or a NAD+ / NADH ratio of an untreated cell from the subject, or a NAD+ level or a NAD+ / NADH ratio of a cell from the subject treated with free NAD+, free NAM, or another free NAD+ precursor. In some implementations, the benchmark level includes an NAD+ level or a NAD+ / NADH ratio of one or more control subjects who have not been treated with free NAD+, free NAM, another free NAD+ precursor, or a NAD+ booster encapsulated in the polymeric nanoparticle.

[0042] In some examples, the physiological effect includes decreased DNA damage, which can lead to susceptibility to pathological conditions (e.g., neurodegenerative conditions, organ injury and / or dysfunction) and increased severity of symptoms related to the pathological condition (e.g., neurological symptoms). In some examples, the physiological effect is associated with at least one of maintaining or improving mitochondrial function, preventing oxidative and reductive stress, or maintaining capacity for synthetic processes, for instance, via the pentose-phosphate pathway. In some examples, the physiological effect includes preventing or reducing symptoms associated with systemic inflammation, oxidative stress, aging, or metabolic disease.

[0043] For administration, therapeutically effective amounts (also referred to herein as “doses” or “dosages”) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of condition, stage of condition, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0044] Useful doses can range from 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pig / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0045] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., hourly, every 2 hours, every 4 hours, every 6 hours, every 12 hours, daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months,every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol.

[0046] Various compositions described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, subcutaneous, and / or sublingual administration. Formulations are generally be administered by injection.

[0047] In various implementations of the present disclosure, a composition including the NAD+ booster 104 and the nanoparticles 106 can be administered by, for example, injection, infusion, perfusion, or lavage. Routes of administration can include bolus intravenous, intradermal, intraarterial, intraparenteral, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, and / or subcutaneous administration.

[0048] In some implementations, the nanoparticles 106 are administered with a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety and purity standards as required by United States FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0049] Exemplary generally used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and / or lubricants.

[0050] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers and / or trimethylamine salts.

[0051] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

[0052] Exemplary isotonic agents include poiyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol or mannitol.

[0053] Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur- containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers or polysaccharides.

[0054] In particular implementations, treatments described herein (e.g., the nanoparticles 106) can be formulated into a carrier in a therapeutically-effective amount. As described herein, exemplary carriers include saline, bufferedsaline, physiological saline, water, Hanks' solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), and combinations thereof.

[0055] Compositions including the nanoparticles 106, the NAD+ booster 104, and / or other components described herein may be administered in a formulation that includes one or more carriers, stabilizers, anesthetics, preservatives, or any combinations thereof.

[0056] In particular implementations, carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In particular implementations, a carrier for infusion includes buffered saline with 5% HSA or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0057] Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0058] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent cell adherence to container walls. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran.

[0059] Where beneficial, formulations can include a local anesthetic such as lidocaine to ease pain at a site of injection.

[0060] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.EXPERIMENTAL EXAMPLEMethods

[0061] Nanoparticle synthesis: PLGA-PEG was commercially purchased via Akina, Inc. (West Lafayette, IN).Nicotinamide (NAM) was purchased via Millipore Sigma (Burlington, MA). NAM-loaded nanoparticles were prepared by the water 1 / organic / water 2 (w1 / o / w2) double emulsion method. The organic phase was prepared by dissolving 25 mg of Poly(lactic-co-glycolic acid) (PLGA; MW = 45K, lactic acid (LA): glycolic acid (GA) = 50:50) copolymerized withPEG (MW = 5K) (Akina) polymers dissolved in 1 mL of dichloromethane (DCM, Fisher Scientific, Waltham, MA). Five mg of NAM was dissolved in 200 L of 1 wt% of cholic / deoxycholic acid sodium salt (bile salts, Sigma) in deionized water (DI) or 1 weight percentage (wt%) of CHA in 1x phosphate buffered saline (PBS, Corning, Corning, NY) to create w1. The w1 and organic phases were then added together, and the incompatible w1 / o phases were emulsified by 20 kHz probe sonication at 30% amplitude with 1 second (s) on: 1s off pulses for 30s or 40s on. After adding 4 mL 3% CHA in DI or PBS as w2, the second sonication was applied with 1s on: 1s off pulses at either 30% for 30s or 20% for 30s. The mixture was transferred to 25 mL of 1% CHA in DI, PBS, or 1 % CHA in PBS (sink) and stirred at 500 rotations per minute (rpm) for 3 hours to allow organic solvent evaporation. Then, the nanoparticles were collected with a 1 hour 100k relative centrifugal unit (RCF) centrifugation step, followed by 5 mL DI resuspension, and 25 minute 100k RCF centrifugation. The final nanoparticles were resuspended in a microcentrifuge tube using 1 mL of DI and stored at 4°C until future usage.

[0062] Assessment of free NAM and NAD on cell viability. BV-2 cells (immortalized microglia cell line) were exposed to 10 minutes of oxygen glucose deprivation (OGD) to induce acute injury. Immediately after OGD, NAM or Nicotinamide adenine dinucleotide (NAD+) treatment was applied to cells - 3 doses were tested: 0.011 pig / mL, 1.1 pig / mL, and 2.7 pig / mL. The assay used (AlamarBlue, Thermo Fisher Scientific, Waltham, MA) is a colorimetric cell metabolism assay - live cells should internalize the Alamarblue reagent.

[0063] Assessment of NAM loading and release: Encapsulation efficiency (EE, %), loading capacity (LC, %), and the release kinetics of NAM was assessed using liquid chromatography-mass spectrometry (LC-MS) measurement of released NAM. Drug release profiles were calculated by performing dialysis over 7 days, taking samples at 1 hour, 4 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days, and analyzing the supernatant using LC-MS for NAD+ and NAD hydrogen (NADH).

[0064] In vitro toxicity and immunogenic response: The cytotoxicity of nanoparticles was evaluated on BV-2 microglia cells and primary astrocytes in vitro using the standard 3-(4, 5-dimethylthi azo-2-y l)-2, 5-di- phenyltetrazoliumromide (MTT) assay. BV-2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Primary astrocytes were isolated from neonatal rats, and cell type was confirmed via GFAP+ staining. The cells will be incubated in 96-well plates with a density of 1.0 xio5per mL for 24 hours. Then, the medium was discarded and treated with Dulbecco's Modified Eagle Medium (DMEM): F-12 Media containing the NAM / PLGA-PEG with various concentrations for 24 hours. After that, cells were incubated with 20 pL of MTT (5 mg / mL in PBS) for 4 hours. Then, 150 pL DMSO was added in each well and the optical density was recorded by a microplate reader at the wavelength of 490 nm. Cells exposed to NAM / PLGA-PEG treatment were assessed for genotoxicity and cytokine response. Cells exposed to blank PLGA-PEG (without NAM), free NAD+, free NAM, and no treatment served as controls. At 4-8 hours after NAM / PLGA-PEG treatment, cells were collected to evaluate DNA damage by comet assay, following standard manufacturing guidelines. Proteome profiler rat cytokine array kit (R&DSystems, Minneapolis, MN) was used according to manufacture guidelines to measure cytokine release profiles to confirm that a cytokine storm is not observed in microglia or astrocyte cultures as one metric of immune activation.

[0065] Assessment of cell viability of NAM-loaded nanoparticles in healthy cells. BV-2 cells were seeding at 20k cells, and proliferated for 2 days. PLGA-PEG nanoparticles without NAM or PLGA-PEG particles containing NAM (NAM / PLGA-PEG) were added to the BV-2 cells at 3 doses; healthy control (HC) cells not exposed to NAM or PLGA- PEG were controls. 24 hours after particle exposure, metabolic activity was measured via AlamarBlue assay, a colorimetric assay that quantifies cell viability through cell metabolism.

[0066] Assessment of cell viability of NAM-loaded nanoparticles in OGD-exposed cells. BV-2 cells were seeding at 20,000 cells, and proliferated for 2 days. 10 minutes of OGD were applied to BV-2 cells. Immediately after OGD, PLGA-PEG or NAM / PLGA-PEG were added to the BV-2 cells at 3 doses; non-treated OGD exposed cells (NT) were controls. NAM / PLGA-PEG with two slightly different formulations were tested: Particles made in 1% CHA in DI water, Particles made in 1 % CHA in PBS.

[0067] Assessment of long-term and thermal stability of NAM-loaded nanoparticles. The particle formulations were characterized by dynamic light scattering (DLS) analysis for size distribution, polydispersity index (PDI), and net surface charge, and by scanning electron microscopy (SEM) for particle morphology, distribution, and surface characteristics. Formulations were lyophilized and lyophilized powders were stored at -20°C and 4°C, dissolved in water, and characterized by high-performance liquid chromatography (HPLC) for stability. For the nanoparticle longterm stability, five aliquots were thawed on day 7, 14, 21 , 28, and 35, respectively and DLS measurements were performed on size and PDI. The other aliquot was also thawed on every measurement day and refrozen after each measurement to assess the freeze-thaw cycle.

[0068] OWH slice preparation and OGD injury model: 300 pm-thick coronal OWH slices were prepared using a vibratome from male and female postnatal 10 (P10) rat brains. Briefly, rats were euthanized at P10 with an overdose of pentobarbital, and the brain was removed under sterile conditions immediately and placed on ice in dissecting media. Slices were separated in dissecting media (0.64% w / v glucose, 100% Hank’s Balanced Salt Solution (HBSS), 1 % penicillin) and transferred onto 35-mm 0.4-pm-pore-sized membrane inserts (Millipore) and placed within a 1.5 ml 6-well plate (CytoOne, Ocala, FL) containing 1 mL 37°C pre-warmed slice culture media (SOM; 48% MEM (minimum essential media), 5% horse serum, 45% HBSS, 1 % GlutaMAX, and 1 % penicillin). Slices were cultured for 4 days in vitro (DIV) at 37°C in 5% CO2. Fresh SCM was replenished for all slices at 24 hours and 72 hours after plating. OGD models in vivo hypoxia-ischemia, and it has been shown that OGD-exposed OWH slices have neuronal cell death, inflammation and oxidative stress, and excitotoxicity. At 4 DIV, slices were transferred into deoxygenated buffered saline solution (BSS; 150 mM NaCI, 2.8 mM KOI, 1 mM CaCh, and 10 mM 2-(4-(2-hydroxyethyl)-1 -piperazinyl)- ethanesulfonic acid (HEPES)) and then placed in a hypoxic chamber, which was flushed with 37°C N2 for 10 minutes, in a 37°C incubator for 0.5 hours. After 0.5 hours OGD, slices were placed into their previous culture conditions.

[0069] NAM dosing study: NAM / PLGA-PEG treatments were applied to OGD-exposed slices immediately after OGD-exposure. Treatment groups were determined and investigated to determine an effective NAM dose that increases total NAD+ and NAD+ / NADH redox ratio, and decreases DNA damage. Cell viability was measured using lactate dehydrogenase (LDH) assay (Caymen Chemical, Ann Arbor, Ml) for global cell viability assessment and propidium iodide (PI) staining for regional cell death. Percent cell death was calculated by counting the number of PI+ cells compared to total cell nuclei stained with DAPI within each region of interest. Cell-specific death was quantified by counting PI+ and cell antibody (Ab+) colocalization with specific cell types.

[0070] Total NAD+ was measured using an Amplite Colorimetric Total NAD+ and NADH Assay Kit (AAT Bioquest, Pleasanton, CA). The NAD+ / NADH redox ratio was measured using the Amplite Colorimetric NAD+ / NADH Ratio Assay Kit (AAT Bioquest). To prepare samples for these assays, after exposure to the NAM / PLGA-PEG treatment, slices were washed with PBS and mechanically homogenized then lysed using lysis buffer. The samples will be centrifuged to remove cellular debri and the collected supernatant will be analyzed using the assay kits according to the manufacturer’s instructions.

[0071] To assess DNA damage, a separate set of brain slices will be snap frozen using liquid nitrogen after exposure to NAM / PLGA-PEG treatment and stored at -80°C until used in the comet assay. Tissue was homogenized in ice cold Merchant’s medium in a cylindrical sieve, to create a cell suspension. An aliquot of the cell suspension was immediately mixed with agarose and places on a microscope slide. When all the samples were placed on the slides, the slides were immersed into 4°C cold lysing solution (2.5M NaCI, 10mM Tris, 100mM Na?- ethylenediaminetetraacetic acid (EDTA), 1% sodium sarcosinate, 10% DMSO, 1 % Triton X-100, pH 10) and kept overnight at 4°C. Slides were rinsed in cold electrophoresis buffer, alkaline treated in ice-cold electrophoresis buffer directly in electrophoresis chamber (0.3M NaOH, 1mM Na2EDTA, pH 13.2) placed on ice and subjected to electrophoresis (pH >13) with 4°C cold circulating buffer. Slides were then rinsed and neutralized (0.4M Tris, pH 7.5). The slides were fixed (95% ethanol), stained with SYBR® Gold or SYBR® Green and comets will be scored. DNA damage was quantified as %DNA in tail (%TDNA) and tail length (TL), where mean values for all cells counted per sample. Electrophoresis efficiency was evaluated by including healthy control and OGD-exposed samples, as negative and positive controls.

[0072] NAM / PLGA-PEG localization in OGD slices: To investigate the mechanism of effect in OGD-exposed slices, Cy3-I abeled NAM / PLGA-PEG were topically applied to OGD-exposed slices immediately after OGD. At 4 hours and 24 hours after NAM / PLGA-PEG application, slices were fixed with 10% formalin for 1 hour and prepared for immunolabeling. Slices were co-stained with primary antibody solutions overnight with anti-l ba-1 (microglia, 1 :250), anti-0lig2 (oligodendrocytes, 1 :300), anti-GFAP (astrocytes, 1 :300), and anti-MAP2 (neurons, 1 :300) followed by secondary antibody staining. Fluorescent confocal images of lba-1 +, Olig2+, and MAP2+ cells were processed using Python-based analysis package combined with VAMPIRE, based on established protocols. 20X tile scans of the entire hemisphere were collected to calculate cell density, and 40X z-stack images, 30-50 pm thick with 3-5 locationsper region of interest in 5 slices per sample were collected for cell morphology and high resolution colocalization analysis.Results

[0073] BV-2 cells (immortalized microglia cell line) were used to evaluate cell viability after treatment with free NAM and NAD+. BV-2 cells were exposed to 10 minutes of oxygen glucose deprivation (OGD) to induce acute injury. There is no significant improvement in cell viability after treatment with free NAM and NAD+ (FIG. 2).

[0074] FIG. 2 illustrates that free NAM and NAD are not effective at improving cell viability. BV-2 cells (immortalized microglia cell line) were exposed to 10 minutes of OGD to induce acute injury. Immediately after OGD, NAM or NAD treatment was applied to cells - 3 doses were tested. There is no significant improvement in cell viability (y-axis is reported as the % change from a non-treated (NT) OGD control); an increase in cell viability would be expected if the drug was beneficial - lack of response with free drug motivated incorporation of NAM or NAD into a nanoparticle delivery system. The assay used (AlamarBlue) is a colorimetric cell metabolism assay - live cells internalize the Alamarblue reagent.

[0075] NAM-loaded nanoparticles were formulated and characterized. For acute brain injury as an application area: nanoparticle formulation physicochemical criteria for effective brain targeting must be: <100nm in diameter (number mean), near-neutral surface charge (Z-potential), low (<0.25) polydispersity (PDI), stable in storage, and high NAM loading. The characteristics of the disclosed NAM-loaded nanoparticles are shown in Table 1.Table 1 : PLGA-PEG nanoparticle encapsulation of NAM.

[0076] Cell viability was evaluated in healthy BV-2 cells and OGD-exposed BV-2 cells. In healthy BV-2 cells, PLGA-PEG nanoparticles without NAM (PLGA-PEG) and PLGA-PEG nanoparticles containing NAM (NAM / PLGA- PEG) did not significantly impact cell viability. No cytotoxicity was observed at these doses (FIG. 3). The % change from the HC is reported on the y-axis; an increase shows an increase in cell viability. In OGD-exposed BV-2 cells, 24 hours after treatment, NAM / PLGA-PEG particles made in 1 % CHA in PBS showed a dose dependent increase in cell viability compared to no treatment (NT) controls (FIG. 4). 20ug / mL and 50ug / mL proved to be the most effective doses in this experimental example.

[0077] FIG. 3 illustrates that in healthy BV-2 cells, blank PLGA-PEG particles and NAM-PLGA-PEG particles have minimal effect on cell viability. BV-2 cells were seeded at 20,000 cells and proliferated for 2 days. PLGA-PEG orNAM / PLGA-PEG were added to the BV-2 cells at 3 doses; healthy control (HC) cells not exposed to NAM or PLGA- PEG were controls. 24 hours after particle exposure, metabolic activity was measured via AlamarBlue assay, a colorimetric assay that quantifies cell viability through cell metabolism. The % change from the HC is reported on the y-axis; an increase shows an increase in cell viability. PLGA-PEG and NAM / PLGA-PEG did not significantly impact cell viability of healthy BV-2 cells. No cytotoxicity was observed at these doses.

[0078] FIG. 4 illustrates the effect of PLGA-PEG nanoparticle-encapsulated NAM on cell viability in OGD-exposed BV-2 cells. BV-2 cells were seeded at 20k cells and proliferated for 2 days. 10 minutes of OGD were applied to BV-2 cells. Immediately after OGD, PLGA-PEG or NAM / PLGA-PEG were added to the BV-2 cells at 3 doses; non-treated OGD exposed cells (NT) were controls. NAM / PLGA-PEG with two slightly different formulations were tested: nanoparticles made in 1 % cholic acid (CHA) in DI water and nanoparticles made in 1 % CHA in PBS. 24 hours after treatment, NAM / PLGA-PEG particles made in 1 % CHA in PBS showed a dose dependent increase in cell viability in OGD-exposed cells compared to NT controls; 20ug / mL and 50ug / mL proved to be the most effective doses.

[0079] Particle size and polydispersity index were measured to evaluate long-term and thermal stability (FIG. 5). In this experimental example, NAM / PLGA-PEG in 1% CHA in DI water or in 1 % CHA in PBS showed improved stability compared to NAM / PLGA-PEG in PBS alone.

[0080] FIG. 5. NAM / PLGA-PEG long term stability and thermal stability. NAM / PLGA-PEG particles made in PBS leads to higher instability compared to NAM / PLGA-PEG particles made in 1% CHA in DI water or 1 % CHA in PBS.

[0081] FIG. 6A illustrates results of a lactase dehydrogenase (LDH) assay showing % cell cytotoxicity with and without NAM-loaded PLGA-PEG nanoparticles under healthy conditions (n=6). Total LDH release was quantified by adding 1 % triton x for 2 h. The aborbance was measured at 490 nm using a plate reader.

[0082] FIG. 6B illustrates regional % cell death of OGD slices treated with and without NAM-loaded PLGA-PEG nanoparticles (n=3-4). Dead cells were stained with propidium iodide and nuclei were stained with dapi. The quantification was done by calculating the ratio of PI positive and dapi countings.

[0083] FIGs. 7A-7C illustrate confocal images of NAM-loaded PLGA-PEG nanoparticles conjugated with CF555 colocalizing with microglia and nuclei costained with iba and dapi, respectively, in the (7 A) cortex and (7B, 7C) striatum. Images were taken at 160X magnification

[0084] FIG. 8 illustrates a Z stack image of NAM loaded PLGA-PEG nanoparticles colocalizing with microglia (costained with iba and dapi) at 80X magnification.EXAMPLE CLAUSES1 . A synthetic nanoparticle including nicotinamide adenine dinucleotide (NAD+) or a NAD+ precursor encapsulated within a carrier including poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG).2. The synthetic nanoparticle of clause 1 , wherein the NAD+ precursor includes nicotinamide (NAM).3. The synthetic nanoparticle of clause 1 or 2, wherein the carrier includes a spherical shape.4. The synthetic nanoparticle of any of clauses 1-3, wherein a diameter of the carrier includes a range of about 20 nanometers (nm) to about 100 nm.5. The synthetic nanoparticle of any of clauses 1-4, wherein a diameter of the carrier includes a range of about 40 nm to about 80 nm.6. The synthetic nanoparticle of any of clauses 1-5, wherein a zeta potential of the synthetic nanoparticle includes a range of about -15 millivolts (mV) to about +10 mV.7. The synthetic nanoparticle of any of clauses 1-6, wherein a polydispersity index of the synthetic nanoparticle includes a range of about 0.01 to about 0.25.8. The synthetic nanoparticle of any of clauses 1-7, wherein a molecular weight of the PLGA is in a range of about 15 kilodaltons (kDa) to about 45 kDa.9. The synthetic nanoparticle of any of clauses 1-8, wherein a ratio of lactic acid to glycolic acid in the PLGA is in a range of about 3:1 to about 3:9.10. The synthetic nanoparticle of any of clauses 1 -9, wherein a molecular weight of the PEG is about 5 kDa.11 The synthetic nanoparticle of any of clauses 1-10, wherein an end chain of the PEG includes a functional group with a neutral or negative charge.12. The synthetic nanoparticle of clause 11 , wherein the functional group includes a carboxyl group, a methyl group, a methoxy group, or a hydroxyl group.13. The synthetic nanoparticle of any of clauses 1-12, further including a surfactant disposed on an outer surface of the carrier including the PLGA and the PEG.14 The synthetic nanoparticle of clause 13, wherein the surfactant includes at least one of cholic acid, poloxamer 407, polysorbate 20, or polysorbate 80.15. A composition including the synthetic nanoparticle of any of clauses 1 -14 and a pharmaceutically acceptable carrier.16. A method of delivering a small molecule to a central nervous system of a subject including administering the synthetic nanoparticle of any of clauses 1-14 or the composition of clause 15 to the subject.17 The method of clause 16, wherein the administering includes intravenous, intranasal, intraperitoneal, intramuscular, or oral administration.18. The method of clause 16 or 17, wherein the administering includes intravenous administration.19. The method of any of clauses 16-18, wherein a physiological effect occurs following administration to the subject.20. The method of clause 19, wherein the physiological effect includes an increased NAD+ level or NAD+ / NAD+ hydrogen (NADH) ratio in a cell of the subject compared to a benchmark NAD+ or NAD+ / NADH ratio.21 . The method of clause 20, wherein the increased NAD+ level or the NAD+ / NADH ratio is at least 5% greater than the benchmark NAD+ level or the benchmark NAD+ / NADH ratio.22. The method of clause 20 or 21 , wherein the benchmark NAD+ or NADH level includes: a NAD+ level of the subject at a prior time; a NAD+ level of an untreated cell of the subject; or a NAD+ level of a cell of the subject treated with free NAM or a synthetic nanoparticle without NAM , and wherein the benchmark NAD+ / NADH ratio includes: a NAD+ / NADH ratio of the subject at a prior time; a NAD+ / NADH ratio of an untreated cell from the subject; or a NAD+ / NADH ratio of a cell from the subject treated with free NAD+ or free NAM.23. A method of treating a condition in a subject in need thereof, the method including administering, to the subject in need thereof, a therapeutically effective amount of: a synthetic nanoparticle including NAD+ or a NAD+ precursor encapsulated within a carrier including PLGA and PEG; or a composition including the synthetic nanoparticle and a pharmaceutically acceptable carrier, thereby treating the subject.24. The method of clause 23, wherein the condition is a neurological condition.25. The method of clause 24, wherein the neurological condition is an acute brain injury.26. The method of clause 25, wherein the acute brain injury includes asphyxia, a stroke, circulatory arrest, or a traumatic brain injury.27 The method of clause 25 or 26, wherein the administering includes administering a first dose within about 6 hours of the acute brain injury.28. The method of clause 27, wherein the administering includes administering a second dose after 6 hours after the acute brain injury.29. The method of any of clauses 23-28, wherein the condition includes an acute organ injury.30. The method of clause 29, wherein the acute organ injury is an acute liver injury, an acute liver failure, an acute spleen injury, an acute kidney injury, an acute lung injury, a myocardial infarction, acute coronary syndrome, or acute heart failure.31 . The method of any of clauses 23-30, wherein the administering includes intravenous, intranasal, intraperitoneal, intramuscular, or oral administration.32. The method of any of clauses 23-31 , wherein the administering includes intravenous administration.33. The method of any of clauses 23-32, wherein the subject is a neonate.34 The method of any of clauses 22-33, wherein the subject is an infant or a child.35. The method of any of clauses 23-34, wherein the subject is an adult.36. The method of any of clauses 23-36, wherein the synthetic nanoparticle includes a spherical shape.37. The method of any of clauses 23-36, wherein a diameter of the synthetic nanoparticle includes a range of about 20 nm to about 100 nm.38. The method of any of clauses 23-37, wherein a diameter of the synthetic nanoparticle includes a range of about 40 nm to about 80 nm.39. The method of any of clauses 23-38, wherein a zeta potential of the synthetic nanoparticle includes a range of about -15 mV to about +10 mV.40. The method of any of clauses 23-39, wherein a polydispersity index of the synthetic nanoparticle includes a range of about 0.01 to about 0.25.41 . The method of any of clauses 23-40, wherein a molecular weight of the PLGA is in a range of about 15 kDa to about 45 kDa.42. The method of any of clauses 23-41 , wherein a ratio of lactic acid to glycolic acid in the PLGA is in a range of about 3: 1 to about 3:9.43. The method of any of clauses 23-42, wherein a molecular weight of the PEG is about 5 kDa.44 The method of any of clauses 23-43, wherein an end chain of the PEG includes a functional group with a neutral or negative charge.45. The method of clause 44, wherein the functional group includes a carboxyl group, a methyl group, a methoxy group, or a hydroxyl group.46. The method of any of clauses 23-45, wherein the synthetic nanoparticle further includes a surfactant disposed on an outer surface of the carrier including the PLGA and the PEG.47 The method of clause 46, wherein the surfactant includes at least one of cholic acid, poloxamer 407, polysorbate 20, or polysorbate 80.48. The method of any of clauses 23-47, wherein the NAD+ precursor includes NAM.49. The method of any of clauses 23-48, further including administering a therapeutically effective amount of at least one of an antioxidant, a DNA repair enhancer, an anti-inflammatory agent, a co-factor, a redox substrate, or a neurotrophic factor to the subject.

[0085] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be used for realizing implementations of the disclosure in diverse forms thereof.

[0086] As will be understood by one of ordinary skill in the art, each implementation disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, or component. Thus, the terms “include” or "including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of" limits the scope of the implementation to the specified elements, steps, ingredients or components and to those that do not materially affect the implementation. As used herein, the term “based on” is equivalent to “based at least partly on,” unless otherwise specified.

[0087] Unless otherwise indicated, all numbers expressing quantities, properties, conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.

[0088] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0089] The terms “a,” “an,” “the” and similar referents used in the context of describing implementations (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate implementations of the disclosure and does not pose a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of implementations of the disclosure.

[0090] Groupings of alternative elements or implementations disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims

[0091] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described inthe following publications. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes- Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021).

[0092] Certain implementations are described herein, including the best mode known to the inventors for carrying out implementations of the disclosure. Of course, variations on these described implementations will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for implementations to be practiced otherwise than specifically described herein. Accordingly, the scope of this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by implementations of the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS1 . A synthetic nanoparticle comprising nicotinamide adenine dinucleotide (NAD+) or a NAD+ precursor encapsulated within a carrier comprising poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG).

2. The synthetic nanoparticle of claim 1, wherein the NAD+ precursor comprises nicotinamide (NAM).

3. The synthetic nanoparticle of claim 1 , wherein the carrier comprises a spherical shape.

4. The synthetic nanoparticle of claim 1, wherein a diameter of the carrier comprises a range of about 20 nanometers (nm) to about 100 nm.

5. The synthetic nanoparticle of claim 1, wherein a diameter of the carrier comprises a range of about 40 nm to about 80 nm.

6. The synthetic nanoparticle of claim 1, wherein a zeta potential of the synthetic nanoparticle comprises a range of about -15 millivolts (mV) to about +10 mV.

7. The synthetic nanoparticle of claim 1, wherein a polydispersity index of the synthetic nanoparticle comprises a range of about 0.01 to about 0.25.

8. The synthetic nanoparticle of claim 1, wherein a molecular weight of the PLGA is in a range of about 15 kilodaltons (kDa) to about 45 kDa.

9. The synthetic nanoparticle of claim 1, wherein a ratio of lactic acid to glycolic acid in the PLGA is in a range of about 3: 1 to about 3:9.10 The synthetic nanoparticle of claim 1, wherein a molecular weight of the PEG is about 5 kDa11. The synthetic nanoparticle of claim 1, wherein an end chain of the PEG comprises a functional group with a neutral or negative charge.

12. The synthetic nanoparticle of claim 11, wherein the functional group comprises a carboxyl group, a methyl group, a methoxy group, or a hydroxyl group.

13. The synthetic nanoparticle of claim 1, further comprising a surfactant disposed on an outer surface of the carrier comprising the PLGA and the PEG14. The synthetic nanoparticle of claim 13, wherein the surfactant comprises at least one of cholic acid, poloxamer 407, polysorbate 20, or polysorbate 80.

15. A composition comprising the synthetic nanoparticle of claim 1 and a pharmaceutically acceptable carrier.

16. A method of delivering a small molecule to a central nervous system of a subject comprising administering the synthetic nanoparticle of claim 1 or the composition of claim 15 to the subject.17 The method of claim 16, wherein the administering comprises intravenous, intranasal, intraperitoneal, intramuscular, or oral administration.

18. The method of claim 16, wherein the administering comprises intravenous administration.

19. The method of claim 16, wherein a physiological effect occurs following administration to the subject.

20. The method of claim 19, wherein the physiological effect comprises an increased NAD+ level or NAD+ / NAD+ hydrogen (NADH) ratio in a cell of the subject compared to a benchmark NAD+ or NAD+ / NADH ratio.21 . The method of claim 20, wherein the increased NAD+ level or the NAD+ / NADH ratio is at least 5% greater than the benchmark NAD+ level or the benchmark NAD+ / NADH ratio.

22. The method of claim 20, wherein the benchmark NAD+ or NADH level comprises: a NAD+ level of the subject at a prior time; a NAD+ level of an untreated cell of the subject; or a NAD+ level of a cell of the subject treated with free NAM or a synthetic nanoparticle without NAM , and wherein the benchmark NAD+ / NADH ratio comprises: a NAD+ / NADH ratio of the subject at a prior time; a NAD+ / NADH ratio of an untreated cell from the subject; or a NAD+ / NADH ratio of a cell from the subject treated with free NAD+ or free NAM.

23. A method of treating a condition in a subject in need thereof, the method comprising administering, to the subject in need thereof, a therapeutically effective amount of: a synthetic nanoparticle comprising NAD+ or a NAD+ precursor encapsulated within a carrier comprising PLGA and PEG; or a composition comprising the synthetic nanoparticle and a pharmaceutically acceptable carrier, thereby treating the subject.

24. The method of claim 23, wherein the condition is a neurological condition.25 The method of claim 24, wherein the neurological condition is an acute brain injury.

26. The method of claim 25, wherein the acute brain injury comprises asphyxia, a stroke, circulatory arrest, or a traumatic brain injury.

27. The method of claim 25, wherein the administering comprises administering a first dose within about 6 hours of the acute brain injury.

28. The method of claim 27, wherein the administering comprises administering a second dose after 6 hours after the acute brain injury.

29. The method of claim 23, wherein the condition comprises an acute organ injury.

30. The method of claim 29, wherein the acute organ injury is an acute liver injury, an acute liver failure, an acute spleen injury, an acute kidney injury, an acute lung injury, a myocardial infarction, acute coronary syndrome, or acute heart failure.31 . The method of claim 23, wherein the administering comprises intravenous, intranasal, intraperitoneal, intramuscular, or oral administration.

32. The method of claim 23, wherein the administering comprises intravenous administration.

33. The method of claim 23, wherein the subject is a neonate.

34. The method of claim 23, wherein the subject is an infant or a child.35 The method of claim 23, wherein the subject is an adult.

36. The method of claim 23, wherein the synthetic nanoparticle comprises a spherical shape.

37. The method of claim 23, wherein a diameter of the synthetic nanoparticle comprises a range of about 20 nm to about 100 nm.

38. The method of claim 23, wherein a diameter of the synthetic nanoparticle comprises a range of about 40 nm to about 80 nm.39 The method of claim 23, wherein a zeta potential of the synthetic nanoparticle comprises a range of about - 15 mV to about +10 mV.

40. The method of claim 23, wherein a polydispersity index of the synthetic nanoparticle comprises a range of about 0.01 to about 0.25.

41. The method of claim 23, wherein a molecular weight of the PLGA is in a range of about 15 kDa to about 45 kDa.42 The method of claim 23, wherein a ratio of lactic acid to glycolic acid in the PLGA is in a range of about 3:1 to about 3:9.

43. The method of claim 23, wherein a molecular weight of the PEG is about 5 kDa.

44. The method of claim 23, wherein an end chain of the PEG comprises a functional group with a neutral or negative charge.

45. The method of claim 44, wherein the functional group comprises a carboxyl group, a methyl group, a methoxy group, or a hydroxyl group.

46. The method of claim 23, wherein the synthetic nanoparticle further comprises a surfactant disposed on an outer surface of the carrier comprising the PLGA and the PEG.

47. The method of claim 46, wherein the surfactant comprises at least one of cholic acid, poloxamer 407, polysorbate 20, or polysorbate 80.

48. The method of claim 23, wherein the NAD+ precursor comprises NAM.49 The method of claim 23, further comprising administering a therapeutically effective amount of at least one of an antioxidant, a DNA repair enhancer, an anti-inflammatory agent, a co-factor, a redox substrate, or a neurotrophic factor to the subject.

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