Enzyme encapsulated nanoparticles

Abstract

Provided herein are compositions, systems, kits, and methods for treating a patient with a disease or condition by administering nanoparticles comprising a biocompatible polymer, wherein the nanoparticles fully, or almost fully, encapsulate: i) tissue-type plasminogen activator (tPA), ii) at least one antioxidant enzyme selected from: superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase, and optionally iii) plasmin protein. In certain embodiments, the disease or condition is selected from: thromboembolism, ischemia-reperfusion injury, stroke, spinal cord injury (SCI), Alzheimer's disease, Muscular Dystrophy, Hypercoagulability, Vaso-occlusive conditions, a fibrotic condition, internal tissue scarring, peritoneal scarring, wound associated scarring, surgical incision associated scarring, and / or dermal scarring.

Description

[0001] ENZYME ENCAPSULATED NANOPARTICLES

[0002] The present application claims priority to U.S. Provisional application serial number 63 / 663,302, filed June 24, 2024, and which is herein incorporated by reference in its entirety.

[0003] FIELD

[0004] Provided herein are compositions, systems, kits, and methods for treating a patient with a disease or condition by administering nanoparticles comprising a biocompatible polymer, wherein the nanoparticles fully, or almost fully, encapsulate: i) tissue-type plasminogen activator (tPA), ii) at least one antioxidant enzyme selected from: superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase, and optionally iii) plasmin protein. In certain embodiments, the disease or condition is selected from: thromboembolism, ischemia-reperfusion injury, stroke, spinal cord injury (SCI), Alzheimer's disease, Muscular Dystrophy, Hypercoagulability, Vaso-occlusive conditions, a fibrotic condition, internal tissue scarring, peritoneal scarring, wound associated scarring, surgical incision associated scarring, inflammation-associated scaring, or that caused by oxidative stress, mitochondrial dysfunction, and organ damage, and / or dermal scarring. In certain embodiments, the fibrotic tissue is formed in body organs, including the liver, lungs, eyes, kidneys, skin, colon, brain, and bone joints.

[0005] BACKGROUND

[0006] Traumatic Spinal Cord Injury (SCI) is a devastating condition, often resulting in permanent disability, disrupting every aspect of the victim’s life and the family. In addition to obvious paralysis, SCI is associated with a risk of developing secondary complications that can be debilitating and even life-threatening (e.g., deep vein thrombosis, urinary tract infections, muscle spasms, osteoporosis, pressure ulcers, chronic pain, and respiratory complications). An estimated 20-30% of people with SCI show clinically significant signs of depression, which in turn has a negative impact on recovery and overall health. The risk of cognitive decline after SCI is 13 times higher than in healthy individuals [1], Several long-term studies have documented the tendency for individuals with SCI to age more rapidly than the general population, due to a decline in overall body physiology, which is similar to the natural aging process [2], Hence, people with SCI are 2 to 5 times more likely to die prematurely than the normal population. Traumatic SCI can be temporally divided into acute (<48 hours), subacute (48 hours to 14 days), intermediate (14 days to 6 months), and chronic (>6 months) phases [3]. With each progressing phase, there is a significant transformation in the spinal cord. The pathophysiology of SCI involves the initial physical impact, which damages the spinal cord tissue. This damage triggers a rapidly progressing cascade of degenerative cellular and molecular events known as “secondary injury.” The secondary injury progression expands the initial lesion site and, with time, affects the entire spinal cord, exacerbating disability. This progressive degeneration of the spinal cord also diminishes the prospect of achieving neurological and functional recovery [3].

[0007] Two critical post-injury events at the acute stage of SCI are: a) excess reactive oxygen species (ROS, highly toxic molecules) formation at the lesion site, which triggers the secondary injury progression, and b) hemorrhage at the lesion site due to ruptured / damaged blood vessels caused by trauma. The clot formed within the spinal cord is highly inflammatory and interferes with axonal growth, leaving cavities within the spinal cord [4] .

[0008] Excess reactive oxygen species (ROS) formation at the lesion site is an important component of the secondary injury cascade [5]. The ROS-mediated damage to cell-membrane lipids, proteins, and DNA triggers a cascade of degenerative events, causing inflammation and overexpression of apoptotic factors, resulting in further spinal tissue death [6,7]. In addition to the loss of neuronal connectivity, SCI triggers the systemic release of ROS and inflammatory cytokines, damaging vital organs such as the liver, lungs, heart, kidneys, and brain, thereby affecting overall body physiology and function. Therefore, SCI is commonly referred to as a multi-organ disease, which is referred to as a secondary complication of SCI.

[0009] In this regard, the nanoparticle formulation encapsulating antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) (nano-SOD / CAT) has been developed and investigated in a rat model of SCI (US Patent 11,439,690; EU Patent 2953640, both of which are herein incorporated by refernece). In certain embodiments, the SOD is human SOD. In other embodiments, the Catalase is a human catalase. A single-dose intravenous administration of nano-SOD / CAT significantly mitigated the impact of secondary injury [8]. The efficacy of nano-SOD / CAT is attributed to its localization at the lesion site following intravenous injection, forming a depot [9], thus sustaining the effect of the encapsulated antioxidant enzymes [8]. The treatment also reduced the lesion volume and demonstrated improved functional recovery. However, this composition of nano-SOD / CAT does not target the thrombus formed within the spinal cord. At a chronic stage of SCI, overexpression of inhibitory factors and glial scar tissue formation at the lesion site impede axonal sprouting and connectivity. Neurons also lose their ability to regenerate, thus significantly diminishing the prospects of regaining neurological and functional recovery. These events prevent the establishment of new neuronal circuitry that has been lost due to injury. Despite significant efforts, no approved regenerative therapy for chronic SCI exists. Rehabilitation remains the primary strategy with limited impact on the quality of life. Epidural spinal cord electrical stimulation or intrathecal injection (e.g., baclofen) can control pain and muscle spasms, respectively.

[0010] SUMMARY

[0011] Provided herein are compositions, systems, kits, and methods for treating a patient with a disease or condition by administering nanoparticles comprising a biocompatible polymer, wherein the nanoparticles fully, or almost fully, encapsulate: i) tissue-type plasminogen activator (tPA), ii) at least one antioxidant enzyme selected from: superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase, and optionally iii) plasmin protein. In certain embodiments, the disease or condition is selected from: thromboembolism, Ischemia-reperfusion injury, stroke, spinal cord injury (SCI), Alzheimer's disease, Muscular Dystrophy, Hypercoagulability, Vaso-occlusive conditions, a fibrotic condition, internal tissue scarring, peritoneal scarring, bone joint tissue scarring, wound associated scarring, surgical incision associated scarring, and / or dermal scarring.

[0012] In some embodiments, provided herein are methods of treating a subject with a condition or disease comprising: administering to the subject a composition comprising nanoparticles, wherein the nanoparticles comprise a biocompatible polymer, and wherein the nanoparticles: a) fully, or almost fully, encapsulate a plurality of tissue-type plasminogen activator (tPA) proteins, and / or biologically active functional mutants or fragments thereof, or bioengineered and b) fully, or almost fully, encapsulate a plurality of at least one antioxidant enzyme selected from the group consisting of: superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase; and / or biologically active functional mutants or fragments thereof, and wherein the condition or disease is selected from: a spinal cord injury (SCI), Alzheimer's disease, Muscular dystrophy, Hypercoagulability, a fibrotic condition, bone joint tissue scaring, internal organ tissue scarring, and / or dermal scarring. In certain embodiments, the subject has a spinal cord injury, and wherein the spinal cord injury is selected from: i) acute phase, which is less than 48 hours, ii) sub-acute phase, which is 48 hours to 14 days, iii) intermediate phase, which is more than 14 days to 6 months, and iv) chronic phase, which is more than 6 months. In additional embodiments, the subject has a spinal cord injury having a scar, and the composition is injected into the scar or near the scar. In further embodiments, the composition is injected into the core of the scar, and / or areas rostral and caudal to the scar.

[0013] In some embodiments, administration is performed at least once, three times (e.g., over one week), or at least six times over two weeks (e.g., approximately evenly spaced in time), or as needed. In other embodiments, the subject has the internal organ tissue scarring selected from: vascular scarring, pulmonary scarring, bone-joint tissue scarring, cardiac scarring, neurological scarring, ocular tissue scarring, and spinal cord scarring; and optionally wherein the composition is administered directly at or near the internal scarring. In additional embodiments, the subject has the fibrotic condition, and wherein the fibrotic condition is selected from: Idiopathic pulmonary fibrosis (IPF), Liver cirrhosis, Cardiac fibrosis, Kidney fibrosis, Systemic sclerosis (SSc), Sclerodermatous graft vs. host disease, Nephrogenic systemic fibrosis, Radiation-induced fibrosis, Injury -induced fibrosis, Surgical dissection of tissue / vessel induced fibrosis, Peritoneal Cavity, Implantable device-induced fibrosis, ageing -related, bone-joint tissue scaring, ocular, and Non-alcoholic steatohepatitis (NASH). Impactable electrodes cause scar tissue around the tissueelectrode interface (e.g., brain and spinal cord electrical stimulators); hence, over time, these begin to develop resistance and malfunction, shortening the device's lifespan.

[0014] In some embodiments, the composition further comprises a sugar (e.g., sucrose or mannose). In additional embodiments, the biocompatible polymer comprises polyesters such as poly (D,L-lactide co-glycolide (PLGA)), Poly lactide (PLL). In certain embodiments, the nanoparticles further comprise polyvinyl alcohol (PVA). In certain embodiments, the nanoparticles comprise pores, and / or a pore-forming agent. In certain embodiments, the nanoparticles comprise an inert protein, such as albumin.

[0015] In particular embodiments, at least one antioxidant enzyme comprises at least two of the antioxidant enzymes. In further embodiments, the at least two antioxidant enzymes comprise superoxide dismutase (SOD) and catalase (CAT), and / or biologically active fragments thereof. In certain embodiments, the nanoparticles further fully, or almost fully, encapsulate an imaging agent. In additional embodiments, the nanoparticlcs have an average transmission electronic microscopic (TEM) diameter of about 90-110 nm (e.g., about 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 nm). In certain embodiments, hydrodynamic diameters of the nanoparticlcs measured in water range from 225 to 325 nm (e.g., about 200, 250, 275, 300 nm). It measures the diameter of nanoparticles and associated water. In further embodiments, the nanoparticles have a zeta potential of about -20 mV to +10 mV (e.g., about -5 to +5 mV).

[0016] In some embodiments, the tissue plasminogen activator (tPA) and the at least one antioxidant enzyme are all human enzymes or biologically active functional mutants or fragments thereof. In additional embodiments, the nanoparticles almost fully encapsulates the tPA proteins, and / or biologically active functional mutants or fragments thereof, engineered such that at least some of the plurality of tPA proteins, and / or biologically active functional mutants or fragments thereof, are available at the surface of the nanoparticles in order to serve as targeting ligands.

[0017] In certain embodiments, the subject has Alzheimer's Disease, Muscular Dystrophy or Hypercoagulability. In further embodiments, the subject is a human. In other embodiments, the administering provides at least 5 pg / Kg of tPA to the subject (e.g., at least 5, 10, or 15 pg / Kg). In further embodiments, the administering provides at least 5 mg / Kg of the nanoparticles to the patient.

[0018] In further embodiments, the administration is performed intravenously, intramuscularly, subcutaneously, intra-arterially, intra-organically, via the lymphatic system, inhalation, or nasally. In other embodiments, the subject has dermal scarring, and the composition is administered topically to the dermal scarring. In further embodiments, the subject has the SCI, and wherein administering the composition prevents or reduces secondary complications of the SCI that would have otherwise been expected without administering the composition. In other embodiments, the secondary complications of SCI are selected from: deep vein thrombosis, urinary tract infections, muscle spasms, osteoporosis, pressure ulcers, chronic pain, respiratory complications, and neurological complications such as cognitive decline.

[0019] In some embodiments, provided herein are compositions comprising: nanoparticles comprising a biocompatible polymer, wherein the nanoparticles: a) fully, or almost fully, encapsulate a plurality of tissue-type plasminogen activator (tPA) proteins, and / or biologically active functional mutants, engineered (tenecteplase, TNKase) or fragments thereof, b) fully, or almost fully, encapsulate a plurality of plasmin proteins and / or biologically active functional mutants or fragments thereof, and c) fully, or almost fully, encapsulate a plurality of at least one antioxidant enzyme selected from the group consisting of: superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase; and / or biologically active functional mutants or fragments thereof.

[0020] In other embodiments, the compositions further comprise a sugar (e.g., sucrose or mannose). In further embodiments, the biocompatible polymer comprises polyesters or derivatives, such as poly (D, L-lactide co-glycolide (PLGA) or poly lactide (PLL). In additional embodiments, the nanoparticles further comprise polyvinyl alcohol (PVA). In other embodiments, the nanoparticles comprise pores and / or a pore-forming agent. In other embodiments, the nanoparticles comprise an inert protein, such as albumin, used as a stabilizing agent for active therapeutic proteins (tPA or antioxidant enzymes).

[0021] In certain embodiments, the at least one antioxidant enzyme comprises at least two of the antioxidant enzymes. In further embodiments, the at least two antioxidant enzymes comprise superoxide dismutase (SOD) and catalase (CAT), and / or biologically active fragments thereof. In additional embodiments, the nanoparticles further fully, or almost fully, encapsulate an imaging agent. In some embodiments, the combination of antioxidant enzymes could vary from about 0:9 to 9:1, or between about 4:6 and 6:4, or about 1:1 based on their catalytic activities.

[0022] In further embodiments, the nanoparticles have an average diameter of about 90-110 (e.g., about 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 nm) as measured using transmission electron microscopy. In additional embodiments, the nanoparticles have a zeta potential of about -20 mV to +10 mV (e.g., -5 mV to 10 mV).

[0023] In some embodiments, the tPA, the plasmin, and the at least one antioxidant enzyme are all human enzymes or biological functional mutants or fragments thereof. In other embodiments, the nanoparticles almost fully encapsulates the tPA proteins, a natural or recombinant form of it (e.g., A alteplase from Genentech, a lyophilized composition of tPA mixed with L-arginine, phosphoric acid, and polysorbate, as wells as salts and buffers), Tenecteplase (TNKase) is a bioengineered alteplase variant, a recombinant DNA-derived version of naturally occurring tPA Genentech), and / or biologically active functional mutants or fragments thereof, such that at least some of the plurality of tPA proteins, and / or biologically active functional mutants or fragments thereof, are available at the surface of the nanoparticles in order to serve as targeting ligands.

[0024] In some embodiments, at least one antioxidant enzyme is combined with at least one other therapeutic enzyme, where enzyme replacement therapy is needed. In particular embodiments, the composition is in a lyophilized form and is optionally sterilized. In other embodiments, the composition further comprises: a dry powder, a dry power ready for reconstitution in saline or a suitable physiological buffer, a spray, a gel, an ointment, or a cream. In additional embodiments, the composition is in the form of a liquid. In some embodiments, the composition further comprises a buffer or saline.

[0025] In particular embodiments, provided herein are methods of treating a patient with a disease or condition comprising: administering the composition described above or herein to a subject with a thromboembolism, reperfusion injury, stroke, spinal cord injury (SCI), Alzheimer's disease, Muscular Dystrophy, Hypercoagulability, Osteoarthritis, fibrotic condition, internal tissue scarring, peritoneal, wound associated, surgical incision associated, spinal stenosis, epidural fibrosis, electrode-tissue contact induced scarring, bone-joint, and / or dermal scarring.

[0026] In certain embodiments, the subject has a spinal cord injury, and wherein the spinal cord injury is selected from: i) acute phase, which is less than 48 hours, ii) sub-acute phase, which is 48 hours to 14 days, iii) intermediate phase, which is more than 14 days to 6 months, and iv) chronic phase, which is more than 6 months. In further embodiments, the subject has a spinal cord injury having a scar, and the composition is injected into the scar. In other embodiments, the composition is injected into the core of the scar, into an area rostral to the scar, and / or into an area caudal to the scar.

[0027] In some embodiments, the administration is performed at least once, at least 3 times over one week or at least six times over two weeks (e.g., generally evenly spaced apart in time) or as needed. In particular embodiments, the subject has the internal scarring, and the internal scarring is selected from: vascular scarring, pulmonary scarring, cardiac scarring, neurological scarring, and spinal cord scarring, and optionally wherein the composition is administered directly to the internal scarring.

[0028] In other embodiments, the subject has the fibrotic condition, and wherein the fibrotic condition is selected from: Idiopathic pulmonary fibrosis (IPF), Liver cirrhosis, Cardiac fibrosis, Kidney fibrosis, Systemic sclerosis (SSc), Sclerodermatous graft vs. host disease, Nephrogenic systemic fibrosis, Radiation-induced fibrosis, device-induced, surgical dissections, incisions associated, and Non-alcoholic steatohepatitis (NASH). In other embodiments, the thromboembolism comprises a platelet-rich clot, freshly formed or an aged clot. In certain embodiments, the thromboembolism comprises a pulmonary embolism, deep vein embolism, a cardiac embolism, a neurologic embolism, or a vascular embolism.

[0029] In particular embodiments, the subject is a human. In other embodiments, the administering provides at least five pg / Kg of tPA to the subject, and / or at least 0.1 pg / Kg amount of the plasmin to the subject. In further embodiments, the administering provides at least 5 mg / kg of the nanoparticles to the subject. In additional embodiments, the administering is performed intravenously, intramuscularly / lymphatically, locally, pulmonary, nasal, or intraglandularly. In some embodiments, the subject has dermal scarring, and the composition is administered topically to the dermal scarring. In some embodiments, the subject has a wound, and the composition is used to promote wound healing. The composition facilitates cell migration and tissue remodeling in wound healing.

[0030] In certain embodiments, the subject has the SCI, and administering the composition prevents or reduces secondary complications of the SCI that would have otherwise been expected without administering the composition. In other embodiments, the secondary complications of SCI are selected from: deep vein thrombosis, urinary tract infections, muscle spasms, osteoporosis, pressure ulcers, chronic pain, cognitive decline, and respiratory complications.

[0031] In some embodiments, vaso-occlusive conditions are created due to the aggregation of blood cells, such as red blood cells (RBCs), in patients with Sickle Cell Disease (SCD). Oxidative stress damages RBCs, affecting their membrane structure and function, which leads to decreased defonnability, increased hemol sis, and anemia, as well as organ damage. SCD is characterized by a hypercoagulable state, meaning there is an increased tendency for blood to clot.

[0032] In some embodiments, the compositions here are used for enzyme replacement therapy (ERT). Reactive oxygen species (ROS) and the body's weak antioxidant defenses can significantly impact enzyme activity. This impact can be both direct, through ROS modifying enzymes and their biological effects, and indirect, by altering the cellular environment and affecting enzyme production or stability. Oxidative stress can impact the activity of enzymes involved in cell signaling pathways, potentially leading to alterations in cell growth, differentiation, and apoptosis. Oxidative stress can impair the activity of various metabolic enzymes involved in the metabolism of carbohydrates, lipids, and proteins. Diseases such as mucopolysaccharidosis I (MPS I) syndrome, Hunter syndrome, Fabry disease, cholesteryl ester disease, Wolman disease, alpha-mannosidosis, Pompe disease, molybdenum cofactor deficiency, adenosine deaminase severe combined immune deficiency, and acid sphingomyelinase deficiency are linked to enzyme deficiency. Oxidative stress can negatively impact the effectiveness of enzyme replacement therapy. For example. Pompe disease, a lysosomal storage disorder, is associated with increased oxidative stress, which can affect the effectiveness of ERT. Elevated oxidative stress levels, observed in both Pompe disease mouse models and patient cells, correlate negatively with the ability of the recombinant enzyme (rhGAA) to correct the GAA deficiency. Similarly, Fabry disease is linked to oxidative stress, which plays a significant role in its pathophysiology, particularly in cardiovascular and renal damage. Accumulation of the lipid globotriaosylceramide (Gb3), a hallmark of Fabry disease, contributes to oxidative stress and inflammation, leading to organ damage.

[0033] In some embodiments, provided herein are systems and kits comprising: a) a composition as described above and herein, and b) a composition storage or delivery device or device to be coated (with the composition) selected from: i) a syringe vial, ii) a syringe, iii) an intracranial delivery device, iv) an intracardiac delivery device, v) a stents / scaffold / mesh, vii) an electroporation device, viii) an ultrasonic device, x) an infusion pump, xi) a catheter or pump, xii) a needle / microneedle or microneedle patch, xiii) a porous catheter, xiv) a balloon, xv) an image-guided injection device, xvii) Inhalational devices, xviii) self-injectable devices, and xix) electrodes. In other embodiments, the delivery device is selected from a catheter, a pump, a needle, a self-injector, or an image-guided local and tissue injection device. In some embodiments, the composition is in the storage device, or delivery device, or on the device as a coating.

[0034] DESCRIPTION OF THE FIGURES

[0035] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0036] Figure 1 shows the results of the clot lysis experiment in vitro with tPA and T-nano- SOD / CAT. Example 1A shows the lysis of the clot, which is 3 hours old, and Example IB shows the lysis of the clot, which is 24 hours old. T-nano-SOD / CAT causes clot lysis of fresh and old clots, whereas tPA is ineffective on old / aged clots. Moreover, T-nano-SOD / CAT treatment is seen to disintegrate and dissolve the clot rapidly, whether it is fresh or old. Figure 2A shows images of the spinal cords with reduced clot accumulation in T-nano- SOD / CAT-trcatcd animals compared to nano-SOD / CAT-trcatcd animals. In this experiment, a single-dose treatment was given 6 hours after inducing the spinal cord injury at T10 using an impactor (severe injury). After cardiac reperfusion with saline to remove blood from the circulation, the harvested spinal cords were analyzed ~22 hours post-treatment. Following imaging, spinal cords were incubated with excess tPA to dissolve the clot, and the supernatants were analyzed by spectrophotometry to quantify lysed clots. The reading was lower for the spinal cord of the animals treated with T-nano-SOD / CAT than with nano-SOD / CAT treated (1.6 vs. 2.34), thus confirming the visual observation.

[0037] Figure 2B shows the effect of the treatment with T-nano SOD / CAT compared to nano- SOD / CAT (without tPA) on functional recovery in the SCI model. In this experiment, a singledose intravenous treatment was given 6 hours after inducing the spinal cord injury at T10 using an impactor (severe injury). The animals were evaluated for post-injury weight recovery, locomotor functions (Basso-Beattie-Bresnahan, BBB; Grid-walk for foot fall and distance traveled), and for sensory perception (using hot plate, which measures latency to respond to the heat and Randall-Selitto test, which measures response to the paw pressure). Figure 2Ba shows post-injury % weight loss and gain following treatment with T-nano-SOD / CAT, nano- SOD / CAT, and untreated control (Saline). Figure 2 Bb shows locomotive recovery based on the Basso, Beattie, and Bresnahan (BBB) scale of 0 to 21, 21 being the normal (uninjured animals). Figure 2 Be shows the BBB scale from 24 to 32 weeks for T-nano-SOD / CAT and nano- SOD / CAT-treated animals. Figure 2 Bd shows a grid walk where foot falls are counted, whereas Figure 2 Be shows the distance travelled. Figure 2 Bf shows the response of the animals to heat sensation using a hotplate, whereas Figure 2 Bg shows the pressure response to the pow-pressure using the Randall-Selitto device. This device is used to determine the pain threshold.

[0038] Figure 2C shows the effect of the treatment with T-nano SOD / CAT compared to saline injection (control) in chronic SCI model. The treatment was administered 6 weeks post-SCI, by which time the seal' tissue had formed at the lesion site, a) T-nano SOD / CAT formulation with incorporated near-infrared dye as a marker was used. The injection of T-nano SOD / CAT was tested in two doses, 10 pl and 30 pl at a concentration of pg / pl T-nano SOD / CAT. One injection was directly injected into the scar tissue at the lesion site, and two additional injections were made adjacent to the scar tissue (rostral and caudal side) directly in the spinal tissue. Figure 2C- a) shows images of the spinal cord harvested at 1 day, 30 pl post-injection with a total of 3 injections. The spinal cords were imaged using a Maestro Optical imaging system for the presence of T-nano-SOD / CAT. Figure 2C-b shows quantification of the signal measured using Maestro with the dose of T-nano-SOD / CAT and time post-treatment. Figures 2C-c to 2C-i show the effect of T-nano-SOD / CAT treatment compared to saline control. In this experiment, 10 pl injection was administered into the spinal tissue as above, directly into the lesion site (scar tissue), and 10 pl on each side adjacent to the lesion site as above. The animals were evaluated for locomotor functions using the BBB scale, Grid-walk (foot fall and distance travelled), and for sensory perception (using Randall-Selitto test and hot plate) (Figure 2C-c to h). The bladder weights were taken at the end of the study and normalized to body weight (Figure 2C-i). Figure 2C-c shows the change in the BBB scores with time post-treatment, whereas Figure 2C-d shows the differences in the BBB scores between T-nano-SOD / CAT-treated and saline control. Figures 2C-e and 2C-f show footfall and distance travelled, respectively, using the grid walk test. Figures 2C-g and 2C-h show the response to the pressure using the Randall-Selitto test and heat sensation using the hot plate, respectively. Figure 2C-i shows bladder weight normalized to the body weight taken at the end of the study time point.

[0039] Figure 3A shows the effect of treatment with T-nano-SOD / CAT on the prevention of thrombin-induced coagulation of fibrinogen. In this experiment, T-nano SOD / CAT was added prior to thrombin-indicated fibrinogen coagulation. Increasing concentrations of hypochlorous acid used as an oxidant show an increase in the coagulation of fibrin, whereas that effect was mitigated when T-nano SOD / CAT was added due to its antioxidant as well as thrombolytic effect. Figure 3B shows the effect of T-nano SOD / CAT on thrombolysis. T-nano SOD / CAT (25 pg) was added one hour after coagulation of thrombin-induced fibrinogen, and reading were taken at different time points. This group contained hypochlorous acid (50 pM). Control is saline. Background signals were subtracted.

[0040] Figure 4A shows an example of rabbit recovery from stroke after treatment with T-nano- SOD / CAT compared to tPA. In this experiment, a single dose treatment of T-nano SOD / CAT was administered 3 hours after stroke induction. The endpoint of the study was one week posttreatment. The animal began to drink and eat by day 1 post-treatment and showed complete neurological recovery by day 3 post-treatment. This is based on the neurological scale, where 0 = full recovery and 10 = death. The animal also started to regain its weight after 6 days as shown in the graph that shows the weight change before and after surgery and treatment. The brain sections at the end of the study show no infarction and very small edema. Figure 4B shows the treatment effect with tPA. Similar to T-nano-SOD / CAT treatment, tPA was administered 3 hours post-stroke induction. The animal died 30 minutes post-tPA treatment, the brain section showed 10.7% edema and 49.2% infarct volume. A section with an arrow shows one section where the infarction is seen (white area), and this is seen in all the sections of the stroke side of the brain. Figure 4C is the compiled neurological scores with time post-treatment for T-nano-SOD / CAT (n=4) and tPA (n=3) treated animals. The T-nano-SOD / CAT-treated animals show complete recovery, except one animal that developed pulmonary effusion after the initial recovery. This effect of pulmonary effusion may not be related to the treatment or stroke, because the animal’s brain did not show infarction. The tPA-treated animals showed neurological scores ranging from 10 to 5, and none of the animals reached the endpoint of 7 days. A similar effect was seen in saline-treated control animals, where the neurological scores were 5-6, and the mortality was seen within 2 days after the stroke induction.

[0041] Figure 5: A) Human tissue-type plasminogen activator amino acid sequence (SEQ ID NO:1). B) Human Plasmin amino acid sequence (SEQ ID NO:2). C) Human superoxide dismutase amino acid sequence (SEQ ID NO: 3). D) Human catalase amino acid sequence (SEQ ID NO:4). E) Native human Plasmin protein (SEQ ID NO:5)

[0042] DETAILED DESCRIPTION

[0043] Provided herein are compositions, systems, kits, and methods for treating a patient with a disease or condition by administering nanoparticles comprising a biocompatible polymer, wherein the nanoparticles fully, or almost fully, encapsulate: i) tissue-type plasminogen activator (tPA), ii) at least one antioxidant enzyme selected from: superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase, and optionally iii) plasmin protein. In certain embodiments, the disease or condition is selected from: thromboembolism, reperfusion injury, stroke, spinal cord injury (SCI), Alzheimer's disease, Muscular Dystrophy, Hypercoagulability, Vaso-occlusive conditions, a fibrotic condition, internal tissue scarring, peritoneal scarring, spinal stenosis, epidural fibrosis, wound associated scarring, surgical incision associated scarring, electrode-tissue contact associated scarring, bone-joint scarring, and / or dermal scarring.

[0044] In certain embodiments, provided herein are compositions of nanoparticles encapsulating antioxidant enzyme(s) and a thrombolytic agent(s) (e.g., T-nano SOD / CAT, which is nanoparticles mostly or totally encapsulating tPA, SOD, and CAT, or TP-nano SOC / CAT which further partially or totally encapsulates plasmin proteins) for treating vascular, neurological, neurodegenerative, and neuro-vascular conditions. Vascular conditions include, for example, thromboembolism, whereas neurological conditions include, for example, post-injury neurodegeneration, scar tissue build-up, and neurodegenerative diseases. Other pathological conditions that may be treated by the nanoparticles herein include both vascular and neurodegenerative diseases, which require neurogenesis, vasculogenesis, and thrombolysis. For example, stroke, spinal cord injury, and ischemia / reperfusion injury, where both vascular embolism leads to neuronal tissue death may be treated with the nanoparticles herein (e.g., T- nano SOD / CAT or TP-nano SOD / CAT). The co-encapsulated thrombolytic agent promotes neuronal sprouting and neurogenesis, vasculogenesis, lesion healing, and synaptic connectivity. In contrast, antioxidant enzymes protect the tissue from reactive oxygen species and prevent dysfunctioning enzymatic reactions in cells / tissue. In other circumstances, the encapsulated thrombolytic agent causes clot lysis, induces neuronal sprouting, and / or overcomes the scar tissue barrier to promote neurogenesis and vasculogenesis in tissue regeneration.

[0045] Antioxidant enzymes enhance the thrombolysis efficacy of the encapsulated thrombolytic agent by mitigating oxidative stress to prevent, halt, and / or reverse translational modification of clotting factors (e.g., plasminogen) that make clot lysis resistant to thrombolysis. The composition of nanoparticles with antioxidant enzymes and thrombolytic agent(s) plays complementary roles in protecting tissue from oxidative stress, promoting neuronal sprouting, and disrupting the scar tissue barrier.

[0046] Plasmin's primary role is in fibrinolysis, the process of breaking down blood clots. It's a serine protease that degrades fibrin, the protein that forms the meshwork of a clot. Plasmin also plays a role in resolving inflammation by transforming microphages from an Ml phenotype, which are inflammatory, to an M2 phenotype, which are regenerative.

[0047] Fibrotic (Scar) tissue is formed after surgical dissection of organ tissue, skin incision, wound healing, or underlined pathological conditions. The nanoparticles herein (e.g., T-nano SOD / CAT and TP-nano SOD / CAT) could be used in such conditions to prevent scar tissue formation or promote normal tissue healing. The thrombolytic agent used, for example, is a tissue serine protease, as an example, a tissue plasminogen activator (tPA) and / or plasmin, an autologous serum protease that is a key component of the fibrinolysis cascade, whereas antioxidant enzymes used as an example are superoxide dismutase (SOD) and catalase (CAT).

[0048] In pathological conditions such as following neuronal injuries (e.g., Stroke, SCI), scar tissue formation at the lesion site is a barrier to restoring neuronal connectivity. These events significantly diminish the prospects of regaining neurological and functional recovery. In such cases, disrupting the scar tissue barrier and promoting neuronal sprouting and growth arc crucial for restoring the lost neuronal circuitry following injury and tissue regeneration. For the regeneration of healthy tissue, vasculogenesis is also vital in providing the necessary nutrients and growth factors to the regenerating tissue. tPA can promote the formation of new axonal varicosities during regenerative processes after axon degeneration, as well as the dissociation of scar tissue. Chondroitin sulfate proteoglycans, a set of extracellular matrix proteins, a component of the scar- tissue with inhibitory action on axon regrowth, are a significant impediment in chronic SCI. tPA activates the chondroitin sulfate proteoglycan-degrading protease, thus promoting axonal growth. The nanoparticle composition, T-nano-SOD / CAT, encapsulates tPA and a combination of antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT). Plasmin is involved in the degradation of extracellular matrix components, thereby contributing to tissue remodeling processes such as wound healing and angiogenesis. While the present disclosure is not limited to any particular mechanism, and understanding the mechanism is not necessary to practice this disclosure, the dual-action T-nano-SOD / CAT or TP-nano- SOD / CAT (with plasmin), which can disrupt the scar tissue and promote neuronal sprouting, would establish the lost neuronal circuitry due to the injury or scar tissue formation to regain functional recovery.

[0049] In yet another condition, thrombolytic agents promote the dissociation and clearance of amyloid-beta depositions in the brain in Alzheimer’s disease (AD), as well as fibrin clearance to prevent disease initiation or progression, such as in Alzheimer’s disease (AD) and multiple sclerosis (MS), with encapsulated antioxidant enzymes facilitating the process. The polymer used for encapsulation is biodegradable, for example, PLGA (poly-lactide co-glycolide), PLL (poly lactide), or modifications thereof.

[0050] In certain embodiments, the nanoparticle compositions herein are injectable via the intravenous route, directly into the targeted and / or near the target area, SQ, intrathecally, intracerebrally, intraspinal, intra-arterially, transdermal, topically, systemically, locally, lymphatic, nasal route, or via inhalation, etc. The injection can be via infusion or implantable pumps, microneedles, syringe, bolus or slow or localized infusion, or a combination of both, delivery using coating onto devices (e.g., balloons, stents, stent retrievers, catheters, inhalation devices, via nasal route, electrodes, electrical leads, etc.), coated onto implantable devices, delivered in a gel formulation, to prevent coagulation and scar tissue build-up or disrupt the formed scar tissue to promote regeneration.

[0051] In certain embodiments, provided herein are nanoparticle encapsulating human tissue plasminogen activator (tPA) (and optionally plasmin) and at least one antioxidant enzyme selected from the group consisting of: superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, and catalase (CAT). In certain embodiments, the nanoparticles comprise plasmin and at least one antioxidant enzyme. In particular embodiments, the nanoparticles herein encapsulate human tPA, human SOD, and human CAT. In some embodiments, this nanoparticle composition protects against the risk of secondary complications associated with spinal cord injury (e.g., deep vein thrombosis, urinary tract infections, muscle spasms, osteoporosis, pressure ulcers, chronic pain, organ damage, and respiratory complications).

[0052] In certain embodiments, provided herein are nanoparticle encapsulating human tissue plasminogen activator (tPA) and plasmin (TP-nano-SOD / CAT) to regain the balance between the plasminogen activator, tPA / plasmin, and the plasminogen activator inhibitor 1 (PAI-1). The increased expression of PAI- 1 leads to fibrotic diseases. The plasminogen activator, such as the tPA / plasmin system, plays a role in extracellular matrix degradation, thereby preventing fibrous tissue formation.

[0053] In certain embodiments, the dual action of the nanoparticles herein (e.g., T-nano- SOD / CAT) is a protective therapy from ROS that could act on the injured spinal cord as well as help prevent deep vein thrombosis (DVT) that leads to pulmonary embolism. In addition, because of the encapsulated antioxidant enzymes, the nanoparticles herein could protect the spinal cord and other embolized tissue / organs from ROS-mediated ischemia-reperfusion injury. In some embodiments, the nanoparticles herein overcome the above issue due to their sustained release, thereby reducing the risk of bleeding complications by avoiding peak high levels of the thrombolytic agent in the circulation. In addition, the encapsulated antioxidant could provide a protective effect on vascular endothelium from ROS-mediated vascular damage. The nanoparticles herein, in certain embodiments, target the embolized tissue to be more effective, and have a small size (-100 nm in diameter as measured by transmission electron microscopy) to permeate and / or bind to the clot to cause clot lysis and / or dissociate the clot by acting on the fibrin network. Furthermore, in some embodiments, at least some tPA is present at the nanoparticle interface and acts as a targeting ligand to the thrombus (e.g., antioxidant enzymes and tPA act synergistically to dissociate the fibrin-rich, old clot). Antioxidant enzymes present in the composition prevent the oxidation of the factors involved in the clot-lysis process (e.g., oxidation of plasminogen, other enzymes, and co-factors that arc required for the clot lysis effect of tPA or plasmin).

[0054] By encapsulating antioxidant enzymes and tPA, which generally have very short biological half-lives (<10 min), are stabilized from rapid clearance. Another reason for the short half-life of tPA is that it binds to the inhibitor of tPA, plasminogen activator inhibitor- 1 (PAI-1), present in the blood, thereby rendering it therapeutically inactive. Thus, the nanoparticles herein work on the thrombus deposit in the spinal cord as well as prevent DVT and pulmonary embolism, in certain embodiments. In particular embodiments, the nanoparticles herein are used to treat vascular embolism in various conditions, such as catheter-related thrombosis

[0013] , cardiovascular disorders, heart stroke, pregnancy, lower limb embolism due to underlying pathological conditions, or disease-related embolism, including sickle cell disease, vascular damage due to inflammation, etc.

[0014] . In certain embodiments, the nanoparticles described herein are effective in protecting the injured spinal cord from secondary injury progression, addressing the issue of thromboembolism, and facilitating regeneration, which ultimately results in rapid and improved functional recovery. The addition of plasmin to the nanoparticles herein may act directly on the clot to cause fibrinolysis.

[0055] Apart from its thrombolytic effect, tPA has an antiapoptotic effect on neurons and oligodendrocyte progenitors through a so-called cytokine — or growth factor-like effect. This effect of tPA is independent of its proteolytic effect, which causes clot lysis. Additionally, tPA is a key regulator of cellular migration and extension of cellular processes during development by promoting the degradation of extracellular matrix (ECM) and cell adhesions. Similarly, tPA can promote the formation of new axonal varicosities during regenerative processes after axon degeneration. Chondroitin sulfate proteoglycans, a set of ECM proteins with inhibitory action on axon regrowth, is a major impediment in chronic SCI. Accordingly, tPA activates the chondroitin sulfate proteoglycan-degrading protease, thus promoting axonal growth and functional recovery. With the dual effect of the nanoparticles herein, tPA, causing degradation of the ECM, could create a more permissive environment as well as promote axonal sprouting and growth, which could promote the formation of new neuronal circuitry and connectivity.

[0056] Glial scar formation (gliosis) is a reactive cellular process involving astrogliosis that occurs after injury to the central nervous system (CNS). In the CNS, glial scars form a major physical and chemical barrier to neuronal regeneration, creating a dense isolation and inhibitory environment. This results in the limitation of optimal neural function and permanent deficits in the human body. Conditions such as chronic ncuroinflammation, brain stroke (SC), traumatic brain injury (TBI), brain tumor, and epileptogenesis can result in scar tissue formation. Fibrosis (Scar tissue) is formed after injury in most organs as a common and complex response that profoundly affects the regeneration of damaged tissue. In disease conditions, fibrosis can occur in various organs, including the lungs, liver, and kidneys. The nanoparticles herein can be used to promote post-stroke recovery as well as other indications where scar tissue is inhibitory to neuronal growth and the regenerative process. The process of scar tissue formation involves an excessive production of reactive oxygen species, inflammation, and / or excess production of plasminogen activator inhibitor 1 (PAI-1).

[0057] It is problematic to employ tPA in a solution form as it has the following issues: a) tPA has a very short half-life (~ 5 min); hence its delivery and retention to the scar tissue to promote neuronal sprouting as well as permeate scar tissue is challenging, b) at high doses tPA has the propensity to cause bleeding, and it is neurotoxic, c) inhibitors of tPA in blood and tissue can bind immediately and impair its effect (e.g. PAI-1), and d) oxidative stress in the injured tissue does not provide conducive environment to promote neurogenesis or regeneration. In certain embodiments, the nanoparticles herein (e.g., T-nano SOD / CAT or TP-nano SOD / CAT) can address the above issues. For example, in chronic SCI conditions, the nanoparticles herein can be directly injected into the scar tissue, near scar tissue, intrathecally, or a combination of all the above methods of administration. In other conditions where scar tissue formation occurs, such as following radiation, sun UV rays, or ageing-associated oxidative stress, the nanoparticles described herein can be used locally, applied topically, or injected subcutaneously (SQ) in a gel or cream formulation. The topical application can be used, for example, as a prophylactic to protect against skin damage or the development of skin cancer. In certain embodiments, during the acute phase (e.g., of SCI), the nanoparticles herein can be administered intravenously to dissolve the clot, where the encapsulated agents are released slowly, thus minimizing the risk of toxicity and sustaining the treatment effect.

[0058] Plasma fibrinogen is an important coagulation factor and is susceptible to post- translational modification by oxidants

[0020] . These modifications affect the structure of fibrinogen and the interactions between various factors involved in the coagulation process. Oxidative stress conditions affect fibrin clot formation and its structure. For example, patients with ischemic stroke show higher levels of oxidative stress markers than controls. The clot from such patients shows prolonged clot lysis time. The electron microscopic pictures of the fibrin network show thicker fibers from the blood collected from ischemic stroke patients

[0021] . The overall results show that oxidative stress-mediated changes in fibrin clot formation, structure, and dissolution affect the effectiveness of thrombolytic therapy

[0021] , In summary, oxidative stress influences the structure of a fibrin clot and its resistance to enzymatic degradation, such as with tPA or plasmin.

[0059] Fibrin deposition is a significant factor in the development and progression of rheumatoid arthritis and other forms of arthritis. Fibrin, a protein involved in blood clotting, is found in the synovial fluid and tissue of arthritic joints, often along with inflammatory cells and other proteins. Fibrin deposition contributes to the inflammation and tissue damage characteristic of arthritis. Therefore, reducing fibrin deposition could lead to less inflammation, chondro- synovial adhesion, and cartilage damage. This is one condition where vascular permeability is impaired; the increased vascular permeability is linked to endothelium integrity, similar to the blood-brain barrier, blood-spinal cord banner, or blood-synovial barrier, allowing blood proteins such as fibrin to infiltrate the tissue and cause inflammation and tissue damage. The compositions described here in, such as T-nano-SOD / CAT or TP-nano-SOD / CAT, address these conditions, as has been demonstrated in stroke and spinal cord injury.

[0060] Due to poor blood and oxygen supply, reactive oxygen species (ROS) production increases after several hours of stroke onset, creating an oxidative stress condition. In addition, tPA treatment increases oxidative stress

[0022] , plays a role in reperfusion injury, and increases the permeability of the blood-brain barrier (BBB) that causes hemorrhage. Diabetic subjects have increased oxidative stress and are at high risk of developing stroke and other embolic conditions

[0023] , Due to oxidative stress, diabetic patients display an increased thrombogenicity that results in a different thrombus composition with respect to nondiabetic patients

[0024] ,

[0061] Erythrocytes produce high quantities of intracellular reactive oxygen species (ROS) through NADPH oxidase activation and hemoglobin autoxidation. This oxidative milieu can alter erythrocyte membrane structure, leading to impaired erythrocyte function. It can also promote erythrocyte lysis, bind to endothelial cells, activate platelets and coagulation factors, expose phosphatidylserine, and release microvesicles. Oxidized erythrocytes contribute to thrombosis and also impair the clot lysis effect of tPA

[0025] .

[0062] Plasminogen activator inhibitor 1 (PAI-1) functions as the primary blocker of plasminogen activator in plasma. Increased levels of PAI- 1 could lead to excessive blocking of tissue-plasminogen activator, which could decrease clot breakdown and eventually cause an unwanted blood clot. Intravenous (i.v.) delivery of a recombinant form of tPA remained until recently the only treatment approved for acute ischemic stroke. In 2025, the FDA approved tenecteplase (TNKase, Genentech), a t-PA analog, for the treatment of acute ischemic stroke (AIS) in adults. The advantage of TNKase is that it can be administered as a single bolus injection, unlike tPA, which is administered as a bolus followed by slow infusion. However, both agents have limitations, including a high risk of hemorrhage. Due to the presence of endogenous inhibitors in circulation and rapid hepatic clearance, traditional tPA (alteplase) exerts a short half-life of 4-6 min

[0026] . This requires a large intravenous dose followed by continuous infusion to achieve therapeutic efficacy, causing risks of inducing hemorrhagic complications. Additionally, fibrin-rich and old clots, which are platelet-rich, produce PAI-1, making tPA resistant to lysis. With the nanoparticles herein (e.g., with mostly or full encapsulated tPA), the above issue can be mitigated as it is not free to bind to the PAI-1, and may localize with the clot, retaining tPA to cause clot lysis.

[0063] Acute ischemic stroke is a serious, life-threatening disease that affects almost 600 million people each year throughout the world, with a mortality of more than 10%, while two-thirds of survivors remain disabled. However, the available treatments for ischemic stroke are still limited to thrombolysis and / or mechanical thrombectomy, and there is an urgent need to develop a new therapeutic target. Recently, intravascular oxidative stress, derived from endothelial cells, platelets, and leukocytes, has been found to be tightly associated with stroke-related thrombosis. It not only promotes primary thrombus formation by damaging endothelial cells and platelets but also affects thrombus maturation and stability by modifying fibrin components. Thus, oxidative stress is expected to be a novel target for the prevention and treatment of ischemic stroke

[0028] .

[0064] Hypercoagulability is a state of increased risk for thrombosis. Hypercoagulability is also designated “thrombophilia,” and there are inherited and acquired hypercoagulable states. Hypercoagulability can occur as a result of the normal physiologic responses to surgical stress and trauma, or it can be due to genetic factors. It has been repeatedly shown that surgery and injury produce a hypercoagulable state. Hypercoagulability and venous thromboembolism in bum / trauma patients are common occurrences. A hypercoagulable state is also associated with sickle cell disease due to the hardening of RBCs, as hemoglobin within the RBC is polymerized. Following certain major operative procedures, large amounts of tissue factor may be released from damaged tissues into venous blood. Mechanical and chemical injury to the collecting veins exposes subendothelial procoagulant proteins, initiating a marked local hypercoagulable process

[0065]

[0029] . Hypercoagulability and advanced vascular sclerotic changes may contribute to the increased incidence of thrombosis in the elderly. Vascular surgeons have to recognize the potential for limb- and life-threatening arterial occlusion events in patients with SARS-CoV-2

[0066]

[0030] . There are, thus, multiple clinical conditions where the nanoparticles herein may be employed.

[0067] Alzheimer’s disease (AD) is the leading cause of dementia among the elderly population. AD is characterized by the accumulation of beta-amyloid ( A|3 ) peptides, which aggregate over time to form AP plaques in the brain. Reducing soluble A|3 levels and, consequently, amyloid plaques constitutes an attractive therapeutic avenue to at least stabilize AD pathogenesis and potentially reverse it. The brain possesses several mechanisms involved in controlling cerebral A0 levels, including the tissue-plasminogen activator (t-PA) / plasmin system and microglia. However, these mechanisms are impaired and ineffective in AD, one of which could be oxidative stress conditions in AD, which impair the lysis capacity of tPA to dissociate A plaques and / or their clearance by microglia / macrophages, as both these processes are influenced by oxidative stress conditions in the brains of AD patients. Serine protease converts plasminogen into plasmin, an enzyme involved in fibrin degradation, which can also dissociate / promote degradation of Ap microaggregates / plaques in the brain. T-nano SOD / CAT, due to the lysis ability of tPA and antioxidant enzymes alleviating oxidative stress, can facilitate the lysis of aggregate deposits of A and clarence from the brain.

[0068] The neurotrophin brain-derived neurotrophic factor (BDNF) is synthesized as a precursor (pro-BDNF), which is cleaved extracellularly by plasmin to release the mature form. The conversion from pro-BDNF to BDNF is an important process that regulates neuronal activity and memory processes. tPA can convert plasminogen into plasmin. However, this proteolytic cascade is counteracted by the expression of the inhibitors PAI-1 and neuroserpin. Inhibitors of tPA are overexpressed in the AD brain, thus mitigating the effect of tPA. In such cases, direct delivery of plasmin in the nanoparticles herein can overcome the issue associated with the inhibitors of tPA. Plasmin is capable of cleaving, degrading, and reducing both non-aggregated monomeric and aggregated fibrillar forms. Additionally, Ap oligomers stimulate fibrin production that complexes with amyloid plaques. This fibrin- Ap complex is implicated in neuroinflammation and neurodegeneration associated with AD pathology. These conditions also increase blood-brain barrier (BBB) permeability and inhibit the repair mechanisms. Multiple sclerosis (MS) is a long-lasting (chronic) disease of the central nervous system. It is thought to be an autoimmune disorder, a condition in which the body attacks itself by mistake. Under healthy conditions, plasma proteins like fibrinogen do not enter the brain parenchyma because of the BBB. Disruption of the BBB is observed in a variety of neurological conditions in humans, including MS and Alzheimer's disease (AD). Such pathologies that are associated with BBB breakdown are indicated by persistent fibrin deposition inside the CNS. Therefore, fibrin has emerged as a potential target for therapeutic strategies in MS and AD pathologies. Histopathology of human brain tissue reveals focal fibrin deposition in MS plaques, which is considered a major contributor to neuroinflammation. Neurological disorders and head trauma can result in the disruption of the neurovasculature and the entry of fibrin and other blood components into the brain, which may contribute to further neurological dysfunction. Therefore, inhibitors of fibrin formation can be protective in MS. In this regard, the nanoparticles described herein (e.g., T-nano-SOD / CAT or TP-nano-SOD / CAT) can provide a therapeutic and protective approach to these diseases.

[0069] The nanoparticles herein, in various embodiments, provide certain advantages. First, such nanoparticles allow the release of tPA, antioxidant enzymes, and plasmin in active form. Second, as shown in the examples, there is enhanced thrombolytic efficacy of T-nano SOD / CAT than tPA (e.g., at >100 fold lower dose of tPA to induce clot lysis in vitro experiment) (Fig. 1A) and is also effective in lysis of old clot where tPA is ineffective (Fig. IB). Third, mitigating the oxidative stress condition to promote neuronal sprouting and to make the scar tissue permissive to neuronal growth. Fourth, in certain embodiments, some of the encapsulated tPA is present at the interface of nanoparticles and acts as a targeting ligand for the clot. Fifth, in particular embodiments, the nanoparticles are small enough to pass through the fibrin network (e.g., fresh clot) or disrupt the fibrin network (e.g., old clot or fibrin-rich clots) to cause better lysis than tPA (Figure 1). Platelet-rich clots are resistant to tPA because they produce PAI-1. Sixth, the nanoparticles herein may be used in preventing and / or treating non-target embolism, embolism caused by tissue injury, increased systemic inflammation and oxidative stress caused by injuries such as SCI or stroke (Figure 2) or other embolic conditions where the risk is reduced. Seventh, where thrombolysis is impaired due to oxidative stress or oxidation, causes hypercoagulability (Figure 3A) or impairs lysis factors (e.g., plasminogen) (Figure 3B). Eighth, the nanoparticles herein may be used in conditions where there is a propensity to form scar tissue (fibrosis), such as after surgical dissection of organs, surgical procedures, spinal cord injury (Figure 2C) skin incision, etc., and diseases associated with hypercoagulability, e.g., hypercoagulability vasculitis, excessive fibrin deposition, wound ischemia, vascular thrombosis (Figure 4). Eighth, in some embodiments, the nanoparticles herein can be employed in neurodegenerative and other inflammatory diseases (e.g., rheumatoid arthritis) implicated due to fibrin deposits. These conditions may be linked to impaired endothelial barrier permeability, allowing blood proteins, such as fibrin, to deposit.

[0070] Several diseases are linked to oxidative stress, resulting from the excessive production of reactive oxygen species (ROS) or impaired body antioxidant defense mechanisms. These include, but are not limited to, neurodegenerative diseases, cardiovascular diseases, cancer, and diabetes. Furthermore, oxidative stress plays a role in conditions like atherosclerosis, chronic obstructive pulmonary disease, and even some aspects of autoimmune diseases.

[0071] Oxidative stress can impair the activity of various enzymes involved in the metabolism of carbohydrates, lipids, and proteins. Oxidative stress can have both direct and indirect impacts, acting through reactive oxygen species (ROS) that modify enzymes, and indirectly by altering the cellular environment and affecting enzyme production or stability. In enzyme replacement therapy, the delivery of antioxidant enzymes in said composition containing antioxidant enzymes can retain or regain the impaired enzymatic activity by mitigating the oxidative stress.

[0072] In certain embodiments, the nanoparticles herein are provided in a formulation that is ready for immediate reconstitution in normal saline and intravenous administration as a bolus followed by slow infusion, similar to how tPA is administered (e.g., a 10% bolus and the remaining amount over 1 hour via slow infusion). In other embodiments, the nanoparticle formulations include at least one cryoprotectant (e.g., sugar's such as glucose, trehalose, fructose, and sucrose) that can easily redisperse the nanoparticles, as well as help in its stability. In general, nanoparticles have a propensity to aggregate during lyophilization (drying step) which is generally the last step in the production process. These sugars are also commonly used to protect proteins and antibodies from aggregation during lyophilization. Sugars may be employed, for example, at a concentration range of 0 to 20%. The tPA nanoparticles herein can be tested with and without sugars added, pre- and post-lyophilization, for mean particle size, size distribution, polydispersity index (a measure of aggregation), and zeta potential (a measure of surface charge).

[0073] The enzymes in the nanoparticles herein may be natural versions (wild type), or engineered, or may be truncations or mutated versions (e.g., deletions, additions, substitutions) thereof that have the same or similar biological activity as the wild type enzymes. In certain embodiments, the wild-type version of five of the enzymes herein arc shown in Figure 5 as follows: A) human tissue-type plasminogen activator amino acid sequence (SEQ ID NO:1); B) human Plasmin amino acid sequence (SEQ ID NO:2); C) human superoxide dismutase amino acid sequence (SEQ ID NO: 3); D) human catalase amino acid sequence (SEQ ID NO:4), and E) Native human Plasmin protein (SEQ ID NO: 5). Mutants and truncations of the sequences in SEQ ID NOs:l-5 are contemplated (e.g., that have the same or generally same biological activity as the wild-type enzymes). In certain embodiments, the enzymes herein have one, two, more conservative or non-conservative amino acid changes (e.g., mutations or truncations of SEQ ID NOs:l-5). Changes to the amino acid sequences the enzymes herein may be generated by changing the nucleic acid sequence encoding the amino acid sequence. A nucleic acid sequence encoding a mutant or truncation of a corresponding wild-type enzyme may be prepared by methods known in the art using the guidance of the present specification for particular sequences. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared nucleic acid encoding the particular enzyme. For example, Tenecteplase (TNKase - Genentech), a tPA analog recently approved by the FDA, has a longer half-life: 20-24 minutes, compared to alteplase's 4-5 minutes. This allows for a single IV bolus administration, while alteplase requires a bolus followed by a 1-hour infusion. Tenecteplase is a modified form of alteplase (recombinant tissue plasminogen activator), with three amino acid substitutions. These modifications, at positions 103, 117, and 296-299, result in a longer half-life, greater fibrin specificity, and increased resistance to plasminogen activator inhibitor 1 (PAI-1) compared to alteplase. Conservative modifications in the amino acid sequences of the enzymes herein (e.g., SEQ ID NOs:l-5) may also be made. Naturally occurring residues are divided into classes based on common side-chain properties:

[0074] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0075] (2) neutral hydrophilic: cys, ser, thr;

[0076] (3) acidic: asp, glu;

[0077] (4) basic: asn, gin, his, lys, arg;

[0078] (5) residues that influence chain orientation: gly, pro; and

[0079] (6) aromatic: trp, tyr, phe. Conservative substitutions will entail exchanging a member of one of these classes for another member of the same class in a particular enzyme amino acid sequence (c.g., exchanging 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acids).

[0080] The above mutations and truncations to the enzymes can be made and tested for biological activity to see if a candidate mutant or truncation has suitable enzyme activity that is similar to the wild-type enzymes. For example, the following screening assays can be employed.

[0081] For tissue-type plasminogen activator (e.g., human tPA) one can screen a candidate mutant or truncated version of this enzyme. The tPA assay protocol (abeam) measures the ability of Tissue type Plasminogen Activator to activate the plasminogen to plasmin in coupled or indirect assays that contain Tissue type Plasminogen Activator, plasminogen, and a plasminspecific synthetic substrate. The amount of plasmin produced is quantitated using a highly specific plasmin substrate releasing a yellow para-nitroaniline (pNA) chromophore. The change in absorbance of the pNA in the reaction solution at 405 nm is directly proportional to the Tissue type Plasminogen Activator enzymatic activity.

[0082] For plasmin (e.g., human plasmin) one can screen a candidate mutant or truncated version of this enzyme using Plasmin Activity Assay Kit (Fluorometric) (abeam). Plasmin Activity Assay Kit (Fluorometric) (ab204728). The assay is based on the ability of Plasmin to proteolytically cleave a synthetic plasmin substrate and release a fluorophore, AMC, which can be easily quantified by fluorescence microplate readers at Ex / Em = 360 / 450 nm. This assay kit is simple and rapid and can detect plasmin activity as low as 10 ng in a variety of samples. For superoxide dismutase (e.g., human SOD) one can screen a candidate mutant or truncated version of this enzyme in a colorimetric activity kit that contains standard for SOD (Cat# EIASODC, Invitrogen, Waltham, MA). The assay is carried out as per the protocol described in the kit. The kit is designed to quantitatively measure SOD activity in a variety of samples. The assay measures all types of SOD activity, including Cu / Zn, Mn, and FeSOD types. The substrate is added followed by Xanthine Oxidase Reagent and incubated at room temperature for 20 minutes. The xanthine oxidase generates superoxide in the presence of oxygen, which converts a colorless substrate in the detection reagent into a yellow colored product. The colored product is read at 450 nm. Increasing levels of SOD in the samples causes a decrease in superoxide concentration and a reduction in yellow product.

[0083] For catalase (e.g., human catalases) one can screen a candidate mutant or truncated version of this enzyme using an amplex™ red catalase assay kit that contains standard for catalase (Cat#A22180, Invitrogen, Waltham, MA). The assay is carried out as per the protocol described in the kit. The Catalase Activity kit is designed to quantitatively measure catalase activity in a variety of samples. Samples arc diluted in the provided Assay Buffer and added to the wells of a half-area clear plate. Hydrogen peroxide is added to each well and the plate incubated at room temperature for 30 minutes. The supplied Colorimetric Detection Reagent is added, followed by diluted horseradish peroxidase, and incubated at room temperature for 15 minutes. The HRP reacts with the substrate in the presence of hydrogen peroxide to convert the colorless substrate into a pink-colored product. The colored product is read at 560 nm. Increasing levels of catalase in the samples cause a decrease in hydrogen peroxide concentration and a reduction in pink product.

[0084] Any of these candidate mutant or truncated enzymes that perform well can be further screened (e.g., in vivo) in the Examples below as part of the nanoparticles.

[0085] EXAMPLES

[0086] EXAMPLE 1

[0087] Formulation and characterization of T-nano SOD / CAT and TP-nano SOD / CAT

[0088] This Example describes various nanoparticle formulations that contain encapsulated tPA (tissue-type plasminogen activator (tPA), SOD (superoxide dismutase), and CAT (and catalase) (can be called "T-nano SOD / CAT") and encapsulated tPA (tissue-type plasminogen activator (tPA), SOD (superoxide dismutase), and CAT (and catalase) and plasmin (can be called "TP- nano SOD / CAT", but percentages and such may vary). All three components, antioxidant enzymes, SOD + CAT, and thrombolytic agent, tPA in T-nano SOD / CAT formulation are encapsulated in a single nanoparticle formulation, and in case of “TP-nano SOD / CAT” all four components, antioxidant enzymes, SOD + CAT, thrombolytic agent, tPA thrombolytic agents, tPA and plasmin are encapsulated in a single nanoparticle formulation (Table 1-A). They are released in the active form under simulated physiological conditions. Four formulations are prepared with varying amounts of tPA (0 to 1.1 mg) per batch, as shown in Table 1-B. TP-nano SOD / CAT formulation contains all the components of Formulation #3 T-nano-SOD / CAT plus 1 mg plasmin as described below. All the formulations contain human serum albumin as a stability agent.

[0089] In a typical 1-gram batch preparation, 810 mg PLGA and 90 mg dimethyl tartaric acid (DMT), used as a plasticizer, are dissolved in 10 ml ethyl acetate (Polymer Phase). Dimethyl tartaric acid could be substituted with diethyl tartaric acid. Separately, 1.65 mg recombinant human CAT and 5 mg recombinant human SOD, and 240 mg human serum albumin as a stabilizing agent to incorporated active proteins (SOD, CAT and tPA) from interfacial inactivation and facilitate the release of the encapsulated therapeutic proteins, and 200 mg tPA formulation (Activase®, Genentech) are dissolved in 2 ml water. Typically, SOD and CAT are combined based on their activities in a 1:1 ratio. Activase®, 200 mg formulation contains 4.43 mg tPA, and the remaining components are additives. This aqueous phase is slowly added to the polymer phase and overtaxed for 4-5 min to form a water-in-oil (w / o) emulsion. This emulsion was further sonicated over an ice bath with a probe sonicator for 2 minutes, each with a total of 4 cycles. This emulsion is further emulsified into 2% w / v 120 ml polyvinyl alcohol solution to form water-in-oil-water (w / o / w / ) emulsion, first by sonication followed by high-speed homogenization for 15 min at 15,000 psi pressure. The organic solvent, ethyl acetate, present in the emulsion was evaporated via stirring for ~6 hours under a vacuum. T-nano-SOD / CAT was formed and lyophilized to remove residual ethyl acetate. The formulation was resuspended and passed through a Tangential Flow Filtration (TFF) system to remove excess PVA and unencapsulated tPA and antioxidant enzymes. Glucose was added as a cryoprotectant prior to lyophilization for 2-3 days. Table 1-A describes the composition for a batch size that is one- fourth of the batch size described above. For TP-nano SOD / CAT formulation, human plasmin or recombinant human plasmin

[0090] (ABcam (ab90928) Native human Plasmin protein (Active), Native human Plasmin protein (Active) is a Human Full Length protein with >=95% purity, Biological Activity, 15 units / mg protein) 1 mg of plasmin was dissolved along with tPA, SOD, and CAT (Formulation #4 in Table 1-B). The mean diameter of TP-nano SOD / CAT nanoparticles was 223 nm and zeta potential of -6.36 mV. The release study in buffer showed 20.4% release within 0-3 days and 33.8% release from 3 to 7 days, with a cumulative release of 54.3% within 7 days. The assay measures the active form of plasmin (ABcam ab273301, Plasmin Activity Assay Kit (Colorimetric)) (Table 1-B).

[0091] EXAMPLE 2

[0092] Clot Lysis in Vitro

[0093] The clot formed in vitro was incubated at 37 °C either with T-nano-SOD / CAT or tPA.

[0094] The clot size was 10 mm. The blood was collected in 3.2% sodium citrate prior to loading into the tubing. A Thrombin 25 Unit was used to form a clot in the tubing. The clot was allowed to form for 3 hours. In the second experiment, 4 a 24-hour-old clot was used. The dose of T-nano- SOD / CAT was 2.5 mg / ml, which is equivalent to 0.007 mg tPA or 4060 Units of tPA. tPA as a control was 1 mg / ml, equivalent to 580,000 Units of tPA. Clot lysis was significantly better with T-nano-SOD / CAT in both fresh and old clots than with tPA. The clot incubated with T-nano- SOD / CAT almost dissolved at 30 min in T-nano-SOD / CAT, whereas it remained unchanged until 60 min when incubated with tPA. It has been reported that tPA is ineffective in old / aged clots. The results are shown in Figure 1A clot lysis of a 3-hour-old clot. Figure IB shows a clot lysis of a 24-hour-old clot. The tPA dose in T-nano-SOD / CAT is ~ 142-fold lower than the tPA dose. Based on the amount of tPA released from T-nano-nano-SOD / CAT during the experimental period (estimated based on 3-day release profiles), the above difference is -1450- fold.

[0095] Example 3 Experiment in spinal cord injury model

[0096] A single-dose intravenous treatment (of T-nano-SOC / CAT formulation 3 in Table 1A) was given 6 hours after inducing the spinal cord injury in rats at T10 using Infinite Horizon (IH) impactor (Mode IH-0400, Precision Systems and Instrumentation, LLC, VA) with an impact force set-up at 250 KDyne and a velocity of 10 mm / s with a dwelling time of 15 sec. These conditions induce a “severe” contusion SCI. Blood clotting time was determined before euthanizing animals at 18 hours post-treatment. Blood was collected, and plasma was used to analyze tPA levels. Animals were perfused prior to harvesting spinal cords and imaging. The image of the spinal cords in Figure 2A shows reduced clot accumulation in T-nano-SOD / CAT- treated animals compared to nano-SOD / CAT-treated animals. Following imaging, spinal cords were incubated with excess tPA to dissolve the clot, and the supernatants were analyzed by spectrophotometry to quantify lysed clots. The spinal cord reading of the animals treated with T- nano-SOD / CAT was lower than that of the nano-SOD / CAT-treated animals (1.6 vs. 2.34), thus confirming the visual observation.

[0097] The blood clotting time measured by giving a small incision to the tail and time it takes to stop bleed was longer, 30-45 sec (n=2) for T-nano SOD / CAT vs. 15 sec for nano-SOD / CAT, indicating the tPA effect in T-nano-SOD / CAT treated animals. The serum tPA level for T-nano- SOD / CAT-treated animals was 5,171 IU, whereas for nano-SOD / CAT-treated animals, it was 755 IU (endogenous tPA). The long-term treatment effect of T-nano SOD / CAT on functional recovery was determined following a singlc-dosc intravenous treatment of T-nano-SOD / CAT (dosc=30 mg / kg) administered 6 hours post-injury. The results were compared with nano-SOD / CAT (without tPA encapsulated) to determine the effect of the encapsulated tPA along with SOD and CAT. In a few analyses, sham control (underwent all surgical procedures but without inducing SCI) and untreated control (saline injection) were included.

[0098] To evaluate the efficacy of the treatment in SCI on functional recovery, animals are tested for locomotor recovery and sensory perception (Figure 2B).

[0099] For locomotor recovery, the following tests were performed. a) Basso-Beattie-Bresnahan (BBB) locomotor score is a behavioral test used to assess motor function. The BBB scale ranges from 0 to 21 and reflects the degree of functional recovery after SCI. Scores from 0 to 7 indicate no or minimal hindlimb movement, 8 to 13 indicate intermittent stepping with no or little coordination, and scores from 14 to 21 indicate increasing coordination and functional recovery. b) Grid Walk: Footfalls and Distance Travelled: Animals were examined in a custom-made chamber consisting of 32 inches in length and 18 inches in breadth with 3 cm square spaced metal rungs over which animals traversed to move on the surface (Cleveland Clinic, Machine shop). Animals were allowed to move on metal rungs for five minutes. Footfalls were considered when the hind limb failed to grasp a bar and fell between the bars. Ethovision XT (Noldus, Leesburg, VA) video tracking system determined the distance traveled in 5 minutes. The data were computed as the number of footfalls / inch of distance traveled.

[0100] For sensory perception, the following tests were performed. a) Thermal Sensitivity Evaluation (Hot Plate): Sensory stimulation of hind paws was assessed using a preheated (50 °C) plate (IITC Inc. Life Science, CA). The animal's latency to respond to the heat, such as lifting or licking a hind paw, jumping, and / or vocalizing, was recorded with a 20-second cut-off for monitoring the response. The data were calculated in seconds as hind paw latency to the reaction. b) Randall-Selitto Test: Analgesy-meter was used to assess neuropathic pain response. It exerts an increasing force at a constant rate and is continuously monitored by a pointer moving along a linear scale. Each animal was acclimatized to the device and immobilized in a soft cotton cloth to place the hind paw under the cone-shaped pusher with a rounded tip. Next, a force (ranging from 0 to 250 g) was applied to the animal's hind paw, which was placed on a small plinth under the cone-shaped pusher with a rounded tip. The pedal switch was used to start the test and released when the animal responded to the force, such as paw withdrawal, struggling to withdraw the paw from the tip, or vocal response. A digital meter recorded the data in gram force (gf). The test was performed on each animal with a 30-minute gap, and an average of three readings was taken.

[0101] The overall results show a slight improvement with T-nano-SOD / CAT treatment over nano-SOD / CAT. The T-nano-SOD / CAT-treated animals show better weight gain (Figure 2B-a), the BBB score improvement (Figure 2B-b), particularly at a later stage (Figure 2B-c), reduced number of foot falls (Figure 2B-d), and increased distance travelled on grid walk (Figure 2B-e) over nano-SOD / CAT-treated animals. All these improvements in locomotor recovery were significantly better compared to the untreated control. In terms of sensory perception evaluations, T-nano-SOD / CAT-treated animals were less sensitive to heat stimulus (Figure 2B-f) and showed greater threshold to pain perception (Figure 2B-g) than nano-SOD / CAT-treated animals. Pain perception depends on the time elapsed after the spinal cord injury. During the early stages of the spinal cord injury, there is a high propensity to pain perception. Increasing the threshold to pain perception demonstrates the treatment effect of T-nano-SOD / CAT over nano-SOD / CAT- treated animals. Considering that the formulations were tested in a severe SCI model, the late differentiation in certain parameters (e.g., BBB score, Figure 2B-c) between T-nano-SOD / CAT and nano-SOD / CAT treated animals is expected, as it takes a long time to rebuild the lost neuronal circuitry and learning. The BBB scores, ranging from 14 to 21, indicate increasing coordination and functional recovery. The T-nano-SOD / CAT-treated animals achieved a mean score of 16.3 at 32 weeks on the scale of 0 to 21, with a further trend of improvement showing significant coordination. The nano-SOD / CAT-treated animals showed a mean BBB score of 14.8 at 32 weeks, which is 1.5 points lower than that achieved with nano-SOD / CAT (Figure 2B-b). In summary, tPA encapsulated in the formulation had played a role by affecting the clot deposit in the spinal cord (Figure 2A), thus achieving better recovery. Further, the systemic increase in tPA levels in T-nano-SOD / CAT could reduce the risk of deep-vein thrombosis after SCI. Example 4

[0102] Six Weeks Post Spinal Cord Injury Treatment

[0103] Six weeks post-spinal cord injury, at which the scar tissue is formed at the lesion site and represents a chronic condition, three doses of T-nano-SOD / CAT were injected into the spinal cord. Post laminotomy, one dose of T-nano-SOD / CAT was administered directly into the scar tissue, and the second and third doses were administered adjacent to the scar tissue towards the cranial and caudal sides. For injection, the T-nano-SOD / CAT formulation was prepared in saline at 1 mg / ml concentration. In one experiment, 10 pl or 30 pl (1 mg / pl) dose of T-nano- SOD / CAT was injected directly at the lesion site, one dose rostral to the lesion site, and another dose caudal to the lesion site, a total of 3 doses. The dose of T-nano-SOD / CAT was injected slowly at each site using a Hamilton syringe. The imaging was used to determine localization and retention of T-nano-SOD / CAT within the spinal cord. For this study, near-infrared dye- loaded T-nano-SOD / CAT was used. The dye acts as a marker for T-nano-SOD / CAT, and the signal can be quantified using the Maestro Optical Imaging System.

[0104] Figure 2C-a demonstrates the localization of the dye-loaded T-nano-SOD / CAT within and around the scar tissue. Figure 2C-b shows a dose-dependent increase in signal around the injection sites and the signal retention with time. The control spinal cord, which received saline, did not show any significant signal (Figure 2C-a and b). There is a reduction in the signal with time at the injection site, which could be due to further diffusion of the injected dose of the formulation within the spinal cord.

[0105] In a second experiment, a 10 pL (1 mg / pL) dose of T-nano-SOD / CAT was administered as above, with a total of three doses (directly into the lesion site, caudal, and rostral to the lesion site). The control group received saline. There was no noticeable bleeding from the injection site in animals that received T-nano-SOD / CAT. Post-injection, animals recovered very well. Animals were monitored for locomotive and sensory functions over time.

[0106] The initial drop in the BBB scores in the T-nano-SOD / CAT over saline group was followed by an improvement in the BBB scores in the treated group over saline (Figure 2C-c and d). The potential reason for the initial drop in the BBB scores over saline could be due to disruption in the scar tissue (lesion cyst), which is fdled with inflammatory cytokines and cells. However, the later gain in the BBB scores in the treated group is potentially mediated by overcoming the barrier and triggering the neuronal connectivity. Overall, the treated animals showed a reduced number of footfalls (Figure 2C-c) and increased distance travelled (Figure 2- C-f). Similarly, the treated animals were less sensitive to pain stimuli (Figure 2C-g) and heat stimuli (Figure 3C-h). The bladder weight is also reduced in the treated animal, which is an indication of recovery (Figure 2C-i). The analysis of the spinal cords from the selected T-nano- SOD / CAT-treated and saline control animals shows a higher % of proliferating Ki67 +ve (4.8% vs. 6.3%) and NeuN +ve cells (17.9% vs. 23.3%) for the treated vs. saline control. NeuN is a marker for newly formed mature neurons. This immunohistochemical analysis was carried out for the spinal section from the lesion site and the immediate proximity to the lesion site. These sites also correspond to the injection sites for the treatment, or saline was administered. The overall analysis suggests the initiation of healing of the lesion cavity.

[0107] EXAMPLE 5

[0108] Fibrinogen is a coagulation factor and is susceptible to modification by oxidants such as ROS (reactive oxygen species). These modifications in fibrinogen can impact the fibrin structure (a component of the clot) and hence the efficacy of the thrombolytic agent, such as tPA, to cause clot lysis. In ischemic conditions, such as those found in spinal cord injury, stroke, or other thromboembolic conditions, there is an increase in ROS levels, which can oxidize plasminogen. Additionally, under these conditions, there is an increase in systemic levels of reactive oxygen species (ROS). T-nano SOD / CAT, due to its dual action, antioxidant and thrombolytic, can prevent oxidation of fibrinogen, thus improving the lysis of clot than with tPA alone.

[0109] Thus, oxidative stress-mediated changes in fibrin clot formation, structure, and dissolution may affect the effectiveness of thrombolytic therapy. The results of the experiments will likely demonstrate that the antioxidant enzymes present in T-nano-SOD / CAT address the above issue, thereby enhancing the thrombolytic efficacy of the encapsulated tPA.

[0110] Materials

[0111] Fibrinogen from human plasma (Millipore Sigma, Cat# F3879, 50-70% protein with >80% of protein is clottable), thrombin (from bovine plasma, Cat# T4648-1KU, Sigma Aldrich), Sodium hypochlorite solution (Millipore Sigma, Cat# 13440, 6-14% active chlorine basis).

[0112] Lyophilized human fibrinogen was dissolved in phosphate buffer saline (PBS, pH 7.4) at 10 mg / ml. In 96-well plates. Fibrinogen solution (100 pL) was added to each well. To this, 25 pL volume of hypochlorous acid was added so that the final concentration of it ranges from 0 to 150 pM. Depending upon the experimental protocol, 25 pL of T-nano SOD / CAT suspension prepared in PBS (containing 25 or 50 pg NP) was added. To promote fibrin polymerization, 100 pL of 2X coating buffer (140 mM NaCl, 2 mM CaCh, 44 mM HEPES buffer with pH 7.4) containing 0.16 NIH U / mL thrombin was added. The final volume of each well was adjusted to 250 pL. The plates were incubated for 1 h at 37° C. To determine the thrombolysis effect of T- nano SOD / CAT, the formulation was added one hour after polymerization of fibrin. The absorbance reading was taken at 350 nm wavelength using a Cytation 5 (BioTek) plate reader.

[0113] Figure 3A shows the effect of treatment with T-nano SOD / CAT on prevention of thrombin-induced coagulation of fibrinogen. In this experiment, T-nano SOD / CAT was added prior to thrombin-indicated fibrinogen coagulation. Increasing concentrations of hypochlorous acid used as an oxidant show an increase in the coagulation of fibrin, whereas that effect was mitigated when T-nano SOD / CAT was added due to its antioxidant as well as thrombolytic effect. These results demonstrate that T-nano SOD / CAT can inhibit the hypercoagulability effect of thrombin under oxidative stress conditions. Figure 3B shows the effect of T-nano SOD / CAT on thrombolysis. T-nano SOD / CAT (25 pg) was added one hour after coagulation of thrombin-induced fibrinogen and reading were taken at different time points. This group contained hypochlorous acid (50 pM). Control is saline. Background signals were subtracted. The results demonstrate the efficacy of T-nano SOD / CAT in inducing clot lysis in the presence of oxidative stress conditions.

[0114] EXAMPLE 6

[0115] A thromboembolic rabbit stroke was created by introducing a thrombin-induced clot (~ 5 mm long) in the middle cerebral artery. Stroke induction was confirmed using fluoroscopic imaging, which showed blockage in blood flow. Treatment was administered intravenously 3 hours after stroke induction. The tPA dose was 2 mg / kg, administered as a 20% bolus, followed by the remaining dose over 30 minutes using an infusion pump. Similarly, a T-nano-SOD / CAT dose of 15 mg / Kg in saline was administered. This dose of T-nano SOD / CAT contains 0.042 mg tPA. Based on the amount of tPA present in T-nano SOD / CAT, this is about a 48-fold lower dose than the tPA dose used in tPA in solution treated rabbits. Post-recovery, animals were monitored for vital signs and neurological scores. Score =0 is full recovery, whereas score =10 is when the animal died. The harvested brains, either when the animal died or at the endpoint of the study (7 or 8 days), were analyzed for infarct volume using TTC staining, and the edema was calculated. Figure 4A is an example of the rabbit treated with T-nano-SOD / CAT. In this experiment, a single dose treatment of T-nano SOD / CAT was administered 3 hours after stroke induction. The endpoint of the study was one week post-treatment. Animals were evaluated for neurological scores (0= full recovery, =10 died). The animal treated with T-nano-SOD / CAT began to drink and eat by day 1 post-treatment, and complete neurological recovery by day 3 post-treatment. The brain sections at the end of the study show no infarction and very low edema. Figure 4B shows the treatment effect with tPA. It was administered 3 hours post-stroke induction. The animal died 30 minutes post-tPA treatment, the brain section showed 10.7% edema and 49.2% infarct volume. Since the animal died right after the treatment, the neurological score is 10. Figure 4C is the compiled neurological scores with time posttreatment. T-nano-SOD / CAT (n=4) and tPA (n=3). The T-nano-SOD / CAT-treated animals show complete recovery, except one animal that developed pulmonary effusion after the initial recovery, whereas the tPA-treated animals showed neurological scores between 10 and 5, and none of the animals reached the endpoint of 7 days. In tPA-treated animals, the cause of mortality was also seen to be due to hemorrhage, but no such incidence was noted in T-nano- SOD / CAT rabbits. A similar effect was seen in saline-treated control animals, where the neurological scores were 5-6, with large infarct size, and the mortality was seen within 1 day after the stroke induction.

[0116] The rabbit stroke model is considered important for the clinical translation of thrombolytic agents, as tPA was approved based on data from this model. Additionally, correlative analyses indicate a therapeutic window ratio of 2.4 to 3 between the rabbit stroke model data and acute ischemic stroke patients. The therapeutic window in the rabbit stroke model for tPA is 1-1.5 hours, and in humans, it is 3 to 4.5 hours. In this study, T-nano- SOD / CAT treatment was administered at 3 hours post-stroke induction. Based on the therapeutic window ratio of 2.4 to 3, the window for treatment in humans for T-nano-SOD / CAT treatment could be 7.2 to 9 hours post-stroke. Apart from a short window for treatment (<3 hours, off-label <4.5 hours), which limits the number of patients that benefit from tPA treatment (<5%), a significant fraction of patients who receive tPA treatment achieve no or incomplete recanalization, 34% experienced early re-occlusion, and they are at high risk of developing brain edema and hemorrhagic transformation (Transl. Stroke Res. (2010) 1:96-107). In that regard, T-nano-SOD / CAT treatment could be safe and effective even with a delayed treatment, considering its thrombolysis ability even on an old and tPA-rcsistant clot (Figure IB). REFERENCES:

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[0148] All publications and patents mentioned in the specification and / or listed below are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope described herein.

Claims

CLAIMSWc Claim:

1. A method of treating a subject with a condition or disease comprising: administering to said subject a composition comprising nanoparticles, wherein said nanoparticles comprise a biocompatible polymer, and wherein said nanoparticles: a) fully, or almost fully, encapsulate a plurality of tissue-type plasminogen activator (tPA) proteins, and / or biologically active functional mutants or fragments thereof, and b) fully, or almost fully, encapsulate a plurality of at least one antioxidant enzyme selected from the group consisting of: superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase; and / or biologically active functional mutants or fragments thereof, and wherein said condition or disease is selected from: a spinal cord injury (SCI), Alzheimer's disease, Muscular dystrophy, Hypercoagulability, Vaso-occlusive conditions, Ischemic conditions, a fibrotic condition, internal organ tissue scarring, and / or dermal scarring.

2. The method of claim 1, wherein said subject has a spinal cord injury, and wherein said spinal cord injury is selected from: i) acute phase, which is less than 48 hours, ii) sub-acute phase, which is 48 hours to 14 days, iii) intermediate phase, which is more than 14 days to 6 months, and iv) chronic phase, which is more than 6 months.

3. The method of claim 1, wherein said subject has a spinal cord injury having a scar, and said composition is injected into said scar.

4. The method of claim 3, wherein said composition is injected into the core of said scar, into an area rostral to said scar, and / or an area caudal and cranial to said scar.

5. The method of any of claims 1-4, wherein said administration is performed at least once, or multiple times over several weeks.

6. The method of any of claims 1-5, wherein said subject has said internal organ tissue scarring, and said internal organ tissue scarring is selected from: vascular scarring, pulmonaryscarring, cardiac scarring, neurological scarring, and spinal cord scarring; and optionally wherein said composition is administered directly at or near said internal scarring.

7. The method of claim 1, wherein said subject has said fibrotic condition, and wherein said fibrotic condition is selected from; Idiopathic pulmonary fibrosis (IPF), Liver cirrhosis, Cardiac fibrosis, Kidney fibrosis, Systemic sclerosis (SSc), Sclerodermatous graft vs. host disease, Nephrogenic systemic fibrosis, Radiation-induced fibrosis, Injury-induced fibrosis, Surgical dissection of tissue / vessel induced fibrosis, bone-joints tissue fibrosis, in osteoarthritis, Peritoneal Cavity, Implantable device-induced fibrosis, Non-alcoholic steatohepatitis (NASH), and due to ageing.

8. The method of any of claims 1-7, wherein said composition further comprises a sugar and / or human albumin.

9. The method of any of claims 1-8, wherein said biocompatible polymer comprises polyesters such as poly (D,L-lactide co-glycolide (PLGA)), Polylactide (PLL).

10. The method of any of claims 1-9, wherein said nanoparticles further comprise polyvinyl alcohol (PVA).

11. The method of any of claims 1-10, wherein said nanoparticles comprise pores, and / or a pore-forming agent.

12. The method of any of claims 1-11, wherein said at least one antioxidant enzyme comprises at least two of said antioxidant enzymes.

13. The method of claim 12, wherein said at least two antioxidant enzymes comprise superoxide dismutase (SOD) and catalase (CAT), and / or biologically active fragments thereof.

14. The method of any of claims 1-13, wherein said nanoparticles further fully, or almost fully, encapsulate an imaging agent.

15. The method of any of claims 1-1 , wherein said nanoparticles have an average transmission electronic microscopic (TEM) diameter of about 90-110 nm, and / or, wherein said nanoparticles have a zeta potential of about -20 mV to +10 mV.

16. The method of claim 15, wherein said nanoparticles are administered in an amount such that said subject receives about 0.09 mg / Kg, or 0.036 - 0.18 mg / Kg, of said tPA.

17. The method of any of claims 1-16, wherein said tissue plasminogen activator (tPA) and said at least one antioxidant enzyme are all human enzymes or biologically active functional mutants or fragments thereof.

18. The method of any of claims 1-17, wherein said nanoparticles almost fully encapsulates said tPA proteins, and / or biologically active functional mutants or fragments thereof, such that at least some of said plurality of tPA proteins, and / or biologically functional mutants or fragments thereof, are available at the surface of said nanoparticles in order to serve as targeting ligands.

19. The method of claim 1, wherein said subject has Alzheimer's Disease, Muscular Dystrophy.

20. The method of claim 1, wherein said subject has Hypercoagulability.

21. The method of any of claims 1-20, wherein the subject is a human.

22. The method of any of claims 1-21, wherein said administering provides at least 5 pg / Kg of tPA to said subject.

23. The method of any of claims 1-22, wherein said administering provides at least 5 mg / Kg of said nanoparticles to said patient.

24. The method of claim of claims 1-23, wherein said administering is performed intravenously or intra-muscle or subcutaneously or intra-organ.

25. The method of claim 1 , wherein said subject has said dermal scarring, and said composition is administered topically to said dermal scarring.

26. The method of claim 1, wherein said subject has said SCI, and wherein said administering said composition prevents or reduces secondary complications of said SCI that would have otherwise been expected without said administering.

27. The method of claim 26, wherein said secondary complications of SCI are selected from; deep vein thrombosis, urinary tract infections, muscle spasms, osteoporosis, pressure ulcers, chronic pain, and respiratory complications.

28. A composition comprising: nanoparticles comprising a bio-compatible polymer, wherein said nanoparticles; a) fully, or almost fully, encapsulate a plurality of tissue-type plasminogen activator (tPA) proteins, and / or biologically active functional mutants or fragments thereof, b) fully, or almost fully, encapsulate a plurality of plasmin proteins and / or biologically active functional mutants or fragments thereof, and c) fully, or almost fully, encapsulate a plurality of at least one antioxidant enzyme selected from the group consisting of: superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase; and / or biologically active functional mutants or fragments thereof.

29. The composition of claim 28, further comprising a sugar, and / or human albumin.

30. The composition of any of claims 28-29, wherein said bio-compatible polymer comprises polyesters or derivatives, such as poly (D,L-lactide co-glycolide (PLGA) or poly lactide (PLL).

31. The composition of any of claims 28-30, wherein said nanoparticles further comprise polyvinyl alcohol (PVA).

32. The composition of any of claims 28-31, wherein said nanoparticles comprise pores and / or a pore- forming agent.

33. The composition of any of claims 28-32, wherein said at least one antioxidant enzyme comprises at least two of said antioxidant enzymes.

34. The composition of any of claims 28-33, wherein said at least two antioxidant enzymes comprise superoxide dismutase (SOD) and catalase (CAT), and / or biologically active fragments thereof.

35. The composition of any of claims 28-34, wherein said nanoparticles further fully, or almost fully, encapsulate an imaging agent.

36. The composition of any of claims 28-35, wherein said nanoparticles have an average diameter of about 90-110 nm as measured using transmission electron microscopy.

37. The composition of any of claims 28-36, wherein said nanoparticles have a zeta potential of about -20 mV to +10 mV.

38. The composition of any of claims 28-37, wherein said tPA, said plasmin, and said at least one antioxidant enzyme are all human enzymes or biological functional mutants or fragments thereof.

39. The composition of any of claims 28-38, wherein said nanoparticles almost fully encapsulates said tPA proteins, and / or biologically active functional mutants or fragments thereof, such that at least some of said plurality of tPA proteins, and / or biologically active functional mutants or fragments thereof, are available at the surface of said nanoparticles in order to serve as targeting ligands.

40. The composition of claim 28, wherein said composition is in a lyophilized form.

41. The composition of claim 40, wherein said composition further comprises: a dry powder, a gel, an ointment, or a cream.

42. The composition of claim 28, wherein said composition is in the form of a liquid.

43. The composition of claim 42, wherein said composition further comprises a buffer or saline.

44. A method of treating a patient with a disease or condition comprising: administering said composition of any of Claims 28-43 to a subject with a thromboembolism, reperfusion injury, stroke, spinal cord injury (SCI), Alzheimer's disease, Muscular Dystrophy, Hypercoagulability, fibrotic condition, internal tissue scarring, peritoneal, wound associated, surgical incision associated, bone-tissue joints, and / or dermal scarring.

45. The method of claim 44, wherein said subject has a spinal cord injury, and wherein said spinal cord injury is selected from: i) acute phase, which is less than 48 hours, ii) sub-acute phase, which is 48 hours to 14 days, iii) intermediate phase, which is more than 14 days to 6 months, and iv) chronic phase, which is more than 6 months.

46. The method of claim 44, wherein said subject has a spinal cord injury having a scar, and said composition is injected into said scar.

47. The method of claim 46, wherein said composition is injected into the core of said scar, into an area rostral to said scar, and / or an area caudal to said scar.

48. The method of any of claims 44-47, wherein said administration is performed at least 3 times over one week.

49. The method of any of claims 44-48, wherein said subject has said internal scarring, and said internal scarring is selected from: vascular scarring, pulmonary scarring, cardiac scarring, neurological scarring, and spinal cord scarring, and optionally wherein said composition is administered directly to said internal scarring.

50. The method of claim 44, wherein said subject has said fibrotic condition, and wherein said fibrotic condition is selected from: Idiopathic pulmonary fibrosis (IPF), Liver cirrhosis, Cardiac fibrosis, Kidney fibrosis, Systemic sclerosis (SSc), Sclerodermatous graft vs. hostdisease, Nephrogenic systemic fibrosis, Radiation-induced fibrosis, device-induced, surgical dissections, incisions associated, and Non-alcoholic steatohepatitis (NASH).

51. The method of claim 44, wherein said thromboembolism comprises a platelet-rich clot.

52. The method of claim 44, wherein said thromboembolism comprises a pulmonary embolism, a cardiac embolism, a neurologic embolism, or a vascular embolism.

53. The method of any of claims 44-52, wherein the subject is a human.

54. The method of any of claims 44-53, wherein said administering provides at least 5 pg / Kg of tPA to said subject, and / or at least 0.1 pg / Kg amount of said plasmin to said subject.

55. The method of any of claims 44-54, wherein said administering provides at least 5 mg / kg of said nanoparticles to said subject.

56. The method of any of claims 44-55, wherein said administering is performed intravenously or intra-muscle or intra-organ.

57. The method of claim 44, wherein said subject has said dermal scarring, and said composition is administered topically to said dermal scarring.

58. The method of claim 44, wherein said subject has said SCI, and wherein said administering said composition prevents or reduces secondary complications of said SCI that would have otherwise been expected without said administering.

59. The method of claim 58, wherein said secondary complications of SCI are selected from; deep vein thrombosis, urinary tract infections, muscle spasms, osteoporosis, pressure ulcers, chronic pain, and respiratory complications.

60. A system or kit comprising: a) a composition of any of Claims 28-43, and b) a composition storage or delivery device or device to be coated with the composition: i) a syringe vial, ii) a syringe, iii) an intracranial delivery device, iv) an intracardiac delivery device, v) an intracardiac delivery device, vi) a stents / scaffold / mesh, viii) an electroporation device, x) an ultrasonic device, xi) an infusion pump, xii) a catheter or pump, implantable pump xiii) a needle / microneedle or microneedle patch, xiv) a porous catheter, a porous polymer film xv) a balloon, vascular access device xvi) an image-guided injection device, xvi) coated electrodes, xvii) inhalation device, xviii) a nasal spray, and xx) a self-injector.

64. The kit or system of claim 60, wherein said delivery device is selected from a catheter, pump, a needle, or image-guided injection device.

65. A method of treating a subject in need of enzyme replacement therapy comprising: administering to said subject a composition comprising nanoparticles, wherein said nanoparticles comprise a bio-compatible polymer, and wherein said nanoparticles comprise at least one of the following: a) fully, or almost fully, encapsulate a plurality of tissue-type plasminogen activator (tPA) proteins, and / or biologically active functional mutants or fragments thereof,b) fully, or almost fully, encapsulate a plurality of plasmin proteins and / or biologically active functional mutants or fragments thereof, and c) fully, or almost fully, encapsulate a plurality of at least one antioxidant enzyme selected from the group consisting of: superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase; and / or biologically active functional mutants or fragments thereof.

66. The method of claim 65, wherein said subject has Pompe disease.

67. The method of claim 65, wherein said subject is a human.

68. The method of claim 65, wherein said nanoparticles are administered in an amount such that said subject receives about 0.09 mg / Kg, or 0.036 - 0.18 mg / Kg, of said tPA.

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