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WO2019113184A1 - Nanoparticules destinées à pénétrer dans un nerf et leurs utilisations - Google Patents

Nanoparticules destinées à pénétrer dans un nerf et leurs utilisations Download PDF

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Publication number
WO2019113184A1
WO2019113184A1 PCT/US2018/064032 US2018064032W WO2019113184A1 WO 2019113184 A1 WO2019113184 A1 WO 2019113184A1 US 2018064032 W US2018064032 W US 2018064032W WO 2019113184 A1 WO2019113184 A1 WO 2019113184A1
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Prior art keywords
nerve
composition
blocker
nanoparticle
diameter
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Inventor
Daniel S. Kohane
Qian Liu
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Boston Childrens Hospital
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Boston Childrens Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/501—Inorganic compounds

Definitions

  • nerve blockers The effectiveness of nerve blockers is limited by poor penetration through various tissue barriers to their site of action.
  • the high concentrations of nerve blockers required to overcome those barriers and achieve useful degrees and durations of nerve block can entail considerable systemic toxicity.
  • the nanoparticle(s) described herein can penetrate into the nerve, resulting in highly efficacious nerve blockage with low toxicity.
  • the nanoparticle is a silica nanoparticle (e.g., a hollow silica nanoparticle).
  • the penetration of the nanoparticle into the nerve is size-dependent.
  • the nanoparticle may be less than 70 nm in diameter (e.g., as measured by
  • the nanoparticle is 10-40 nm in diameter (e.g., as measured by TEM).
  • Methods of inducing local anesthesia using a nerve blocker(s) encapsulated in the nanoparticle(s) are also provided.
  • Methods of promoting neuroregeneration using a nerve growth factor encapsulated in the nanoparticles are also provided.
  • Some aspects of the present disclosure provide compositions comprising a nerve blocker encapsulated in a silica nanoparticle (SN).
  • the nerve blocker is a botulinum toxin.
  • the nerve blocker is a site 1 sodium channel blocker (S1SCB).
  • Non-limiting examples of SlSCBs include neosaxitoxin, saxitoxin, decarbamoyl STX, tetrodotoxin, and gonyautoxin.
  • the S1SCB is tetrodotoxin (TTX).
  • the SN has a diameter of less than 70 nm as measured by transmission electron microscopy (TEM). In some embodiments, the SN has a diameter of 10- 40 nm as measured by TEM. In some embodiments, the SN has a diameter of 10-30 nm as measured by TEM. In some embodiments, the SN has a diameter of 28 nm as measured by TEM.
  • TEM transmission electron microscopy
  • the SN has a diameter of less than 80 nm as measured by dynamic light scattering (DLS). In some embodiments, the SN has a diameter of 36.7 nm as measured by DLS.
  • DLS dynamic light scattering
  • the SN has a negatively charged surface.
  • the SN is porous. In some embodiments, pores on SN wall have a diameter of less than 10 nm. In some embodiments, the pores on SN wall have a diameter of less than 2 nm.
  • the silica nanoparticle is a hollow silica nanoparticle (HSN).
  • HSN hollow silica nanoparticle
  • the HSN has a hollow core with a diameter of 2-60 nm. In some embodiments, the HSN has a hollow core with a diameter of 10-20 nm.
  • the S1SCB is loaded in the hollow core.
  • compositions comprising a nerve growth factor encapsulated in a silica nanoparticle (SN).
  • SN silica nanoparticle
  • compositions comprising a drug for the nervous system encapsulated in a nanoparticle having a diameter of less than 70 nm, wherein the drug is selected from the group consisting of: nerve blocker(s), nerve growth factor(s), steroid(s), anti inflammatory drug(s), anti-infective(s), and agents that modulate neurotransmission, neuron apoptosis and excito toxicity.
  • the drug is selected from the group consisting of: nerve blocker(s), nerve growth factor(s), steroid(s), anti inflammatory drug(s), anti-infective(s), and agents that modulate neurotransmission, neuron apoptosis and excito toxicity.
  • the encapsulated drug enters the nerve.
  • the nanoparticle enhances the entry of the drug into the nerve, compared to free drug.
  • the nanoparticle is selected from the group consisting of silica nanoparticle(s), nanoparticle(s) coated with silica, gold nanoparticle(s), iron based
  • compositions described herein further comprise a pharmaceutically acceptable carrier.
  • aspects of the present disclosure provide method(s) of inducing local anesthesia, the method comprising administering to a subject in need thereof an effective amount of the composition comprising a nerve blocker encapsulated in a silica nanoparticle.
  • the composition is administered locally at a nerve of the subject.
  • the nerve is a peripheral nerve.
  • the peripheral nerve is a sciatic nerve.
  • the SN and the encapsulated nerve blocker enters the nerve.
  • the SN enhances the entry of the nerve blocker into the nerve, compared to free nerve blocker.
  • the nerve blocker is released into the nerve from the SN.
  • the nerve blocker blocks a neuronal signal.
  • the SN enhances the rate of nerve blockade, compared to free nerve blocker.
  • a lower dose of nerve blocker is needed for the same rate of nerve blockade, compared to free nerve blocker.
  • the SN prolongs the nerve blockade by the nerve blocker, compared to free nerve blocker.
  • the nerve blocker encapsulated in the SN is not toxic to the nerve.
  • the method further comprises administering to the subject an effective amount of a second nerve blocker.
  • the second nerve blocker is an amino-amide and/or amino-ester local anesthetic.
  • the second nerve blocker is lidocaine, tetracaine, capsaicin, and analogs thereof.
  • the method further comprises administering to the subject an effective amount of an adjuvant.
  • the adjuvant is a glucocorticoid receptor agonist, an adrenergic agonist, or a vasoconstrictor.
  • the glucocorticoid receptor agonists is dexamethasone.
  • the vasoconstrictor is epinephrine or dexmedetomidine.
  • the subject is a mammal.
  • the mammal is a human.
  • the mammal is a rodent.
  • the rodent is a mouse or a rat.
  • aspects of the present disclosure provide methods comprising contacting a neuron with an effective amount of the composition comprising a nerve blocker encapsulated in a silica nanoparticle.
  • an axonal surface of the neuron contacts the SN in the composition.
  • the encapsulated nerve blocker is released and contacts the neuron.
  • the nerve blocker blocks a neuronal signal.
  • the SN prolongs the neuronal signal blockade by the nerve blocker.
  • the nerve blocker encapsulated in the SN is not toxic to the neuron.
  • the neuron is in vitro. In some embodiments, the neuron is ex vivo. In some embodiments, the neuron is in vivo.
  • the neuron is from a mammal.
  • the mammal is a human.
  • the mammal is a rodent.
  • the rodent is a mouse or a rat.
  • the method comprising administering to a subject in need thereof an effective amount of the composition comprising a nerve growth factor encapsulated in the nanoparticle described herein.
  • the encapsulated nerve growth factor enters the nerve.
  • the nanoparticle enhances the entry of the nerve growth factor into the nerve, compared to free nerve growth factor.
  • FIGs. 1A to ID Characterizations of HSN 30 .
  • FIGs. 1A, 1B show TEM images of HSN 30 at low (FIG. 1 A) and high magnification (FIG. 1B).
  • FIG. 1C shows a representative (of > 10) histogram of the size distribution of HSN 30 measured by TEM.
  • FIG. 1D shows a representative (of > 10) size intensity- weighted distribution of HSN 30 as determined by dynamic light scattering measurement.
  • FIGs. 4A to 4F Representative fluorescence images of sciatic nerves and surrounding tissues 4 hours after injection of FITC- HSN30 (FIGs. 4A and 4B) and free FITC (FIG. 4C). Non-injected leg (FIG. 4D).
  • FIGs. 5A to 5E Characterization and performance of FITC-SN10 and FITC-SN70.
  • FIG. 5A shows a TEM image of FITC-SN10 and
  • FIG. 5B shows a TEM image of FITC-SN70.
  • FIGs. 5C to 5E show representative fluorescence images of sciatic nerves and surrounding tissues 4 hours after injection of FITC-SN10 (FIG. 5C, FIG. 5D) and FITC-SN70 (FIG. 5E).
  • the dotted line indicated the nerve.
  • FIG. 6 Histology of rat tissues injected with free TTX and TTX- HSN30.
  • the top two rows 6 show representative H&E stained sections of muscles at the site of injection 4 and 14 days after injection.
  • the left scale bar is 200 pm, the right is 50 pm.
  • the bottom row shows representative toluidine blue-stained sections of sciatic nerves from animals without (bottom left) and with (bottom center and bottom right) injection of TTX- HSN30.
  • the bottom center was harvested 4 days after injection and bottom right, 14 days after injection.
  • FIG. 7 N 2 adsorption (red)-desorption (black) isotherm of HSN30. Volume is the volume of N 2 absorbed; P, pressure of absorbent; P0, saturation pressure.
  • FIG. 8 N 2 adsorption(red)-desorption(black) isotherm of TTX- HSN30. Volume is the volume of N 2 absorbed; P, pressure of absorbent; P0, saturation pressure.
  • FIG. 9 Pore size distribution curve of HSN30 calculated from the adsorption isotherm by the Non-Local Density Functional Theory (NLDFT) method. 18 The peak between 10 nm and 20 nm reflect the size of the hollow within the HSN30. Pores ⁇ 10 nm peak were from the microporous silica wall, and pores > 20 nm peaks reflect space between aggregated HSN30. 7
  • NLDFT Non-Local Density Functional Theory
  • FIG. 10. TEM image of TTX- HSN 30 .
  • FIG. 11. Cytotoxicity of 60 mg/mL TTX- HSN30 containing 6 mg TTX, suspended in cell culture media.
  • FIGs. 12A to 12C Synthesis and characterization of FITC- HSN30.
  • FIG. 12A shows the synthesis of FITC-APTES.
  • FIG. 12B shows TEM of FITC- HSN30.
  • FIG. 12C shows absorption (grey) and emission (black) spectra of FITC- HSN30.
  • the inset are photographs of brightfield (left) and fluorescence images of suspensions of FITC- HSN30.
  • FIGs. 13A to 13B FITC- HSN30 at the sciatic nerve.
  • FIG. 13A shows a representative photograph of tissues 4 hours after injection of FITC- HSN30. The black arrow indicates the faint light yellow FITC- HSN30.
  • FIG. 14 Measurement of distribution of fluorescence across the nerve. See
  • FIG. 15 Representative photograph of the sciatic nerve and surrounding tissue 4 days after injection of TTX- HSN30.
  • FIG. 16 Schematics showing the hollow silica nanoparticle loaded with tetrodotoxin penetrates the nerve.
  • compositions comprising a drug for the nervous system encapsulated in a nanoparticle having a diameter of less than 70 nm.
  • the nanoparticles described herein can penetrate into the macroscopic bundle of nerves and contact axons/neurons directly. Drugs encapsulated in the nanoparticles can thus be delivered across the tissue barriers and released to their site of action (e.g., axon/neurons).
  • Non-limiting, exemplary drugs for the nervous system include nerve blockers, nerve growth factors, steroids (e.g., glucocorticoid receptor agonists), anti-inflammatory drugs, anti-infectives, and agents that modulate neurotransmission, neuron apoptosis and excitotoxicity.
  • the drug for the nervous system is a nucleic acid (e.g., DNA, mRNA, siRNA, miRNA, or shRNA) or a polypeptide.
  • the drug(s) for the nervous system may be loaded in a smaller nanoparticle before being encapsulated in the nanoparticles described herein.
  • the drug for the nervous system is a drug for a peripheral nervous system.
  • exemplary drugs for the peripheral nerve system include: muscarinic agonists, muscarinic antagonists (e.g., atropine, oxybutinin (ditropan), tolerodine (detrol), scopolamine, ipratropium, dicyclomine (bentyl)), ganglionic stimulants, ganglionic blockers, neuromuscular blockers, cholinesterase inhibitors (e.g., neostigmine, and
  • adrenergic agonists e.g., epinephrine, norepinephrine, isoprotenerenol, dopamine, dobutamine, terbutaline, phenylephrine, and ephedrine
  • adrenergic antagonists e.g., alpha l-antagogonists such as prazosin, doxazosin, terazosin, tamsulosin, and
  • phentolamine beta antagonists such as propranolol, nadolol, pindilol, metoprolol, atenolol, bisoprolol, labetalol, carvedilol, metoprolol, and carvedilol, and indirect adrenergic antagonists such as guanethidine.
  • beta antagonists such as propranolol, nadolol, pindilol, metoprolol, atenolol, bisoprolol, labetalol, carvedilol, metoprolol, and carvedilol
  • indirect adrenergic antagonists such as guanethidine.
  • the drug for the nervous system is a drug for a central nervous system.
  • exemplary drugs for the central nerve system include: analgesics (e.g., analgesic combinations, antimigraine agents, cox-2 inhibitors, miscellaneous analgesics, narcotic analgesic combinations, narcotic analgesics, nonsteroidal anti-inflammatory agents, salicylates), anorexiants, anticonvulsants (e.g., AMPA receptor antagonists, barbiturate anticonvulsants, benzodiazepine anticonvulsants, carbamate anticonvulsants, carbonic anhydrase inhibitor anticonvulsants, dibenzazepine anticonvulsants, fatty acid derivative anticonvulsants, gamma-aminobutyric acid analogs, gamma-aminobutyric acid reuptake inhibitors, hydantoin anticonvulsants, miscellaneous anticonvulsants, neuro
  • Nanoparticle refers to a small object that behaves as a whole unit with respect to its transport and properties. Nanoparticles are typically between 1 to 100 nm in size. Nanoparticles are widely used for drug delivery. Types of nanoparticles that may be used in accordance with the present disclosure include, without limitation: lipid-based nanoparticles (e.g., micelles or liposomes), silica nanoparticles (e.g., mesoporous silica nanoparticles, hollow silica nanoparticles), metal-based nanoparticles (e.g., gold nanoparticles, iron-based nanoparticles), rare-earth-based upconversion nanoparticles, quantum dots, nano diamond, and polymer-based nanoparticles (e.g., nanoparticles based on poly(d,l-lactide), poly(lactic acid) PLA, poly(d,l-glycolide) PLG, poly(lactide-co-glycoli
  • A“silica nanoparticle,” as used herein, refers to a silica (silicon dioxide)-based nanosphere structure.
  • the silica used for producing nanoparticles suitable for drug delivery is porous, e.g., microporous (pore size of less than 2 nm) or mesoporous (pore size of 2 nm to 50 nm).
  • the most common types of mesoporous silica nanoparticles are MCM-41 and SBA-15.
  • the silica nanoparticle has a hollow core in the center. Such a silica nanoparticle is referred to as is a“hollow silica nanoparticle (HSN).”
  • silica nanoparticles can be synthesized by reacting tetraethyl orthosilicate with a template made of micellar rods.
  • silica nanoparticles can be synthesized using a sol-gel method (e.g., as described in Nandiyanto et al., Microporous and Mesoporous Materials. 120 (3): 447-453, 2009, incorporated herein by reference) or a spray drying method (e.g., as described in Nandiyanto et al., Chemistry Letters. 37 (10): 1040-1041, 2008, incorporated herein by reference).
  • tetraethyl orthosilicate is also used with an additional polymer monomer as a template.
  • a sol-gel method is the commonly used Stober process (as described in Stober et al., Journal of Colloid and Interface Science. 26 (1): 62-69, 1968, incorporated herein by reference), which is used to produce silica particles of controllable and uniform size.
  • Stober process a molecular precursor (typically tetraethylortho silicate) is first reacted with water in an alcoholic solution, the resulting molecules then joining together to build larger structures.
  • Another process was reported later, which allowed controlled formation of silica particles with small holes (as described in Boissiere et al., Chemical Communications (20): 2047-2048, 1999; and Boissiere et al., Chemistry of Materials. 12 (10): 2902-2913, 2000, incorporated herein by reference).
  • the process is undertaken at low pH in the presence of a surface-active molecule.
  • the hydrolysis step is completed with the formation of a
  • the ability of a nanoparticle to penetrate into the nerve depends to its size.
  • a nanoparticle e.g., a silica nanoparticle.
  • Such methods include, without limitation, transmission electron microscopy (TEM) and dynamic light scattering (DLS).
  • Transmission electron microscopy is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image.
  • the specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid.
  • An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen.
  • the image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a charge-coupled device.
  • Transmission electron microscopes are capable of imaging at a significantly higher resolution than light microscopes, enabling it to capture fine detail— even as small as a single column of atoms. Transmission electron microscopy is a major analytical method in the physical, chemical and biological sciences. TEMs find application in cancer research, virology, and materials science as well as pollution, nanotechnology and semiconductor research.
  • DLS Dynamic light scattering
  • the size of a nanoparticle may be indicated by its diameter.“Diameter” of a nanoparticle is the length of a straight line passing from side to side through the center of spherical particle. Different methods of measuring may lead to slightly different diameters for the nanoparticle. For example, the diameter obtained using DLS is larger than the diameter obtained by TEM.
  • a nanoparticle e.g., silica nanoparticle
  • a nanoparticle that can penetrate into the nerve has a diameter of less than 70 nm (e.g., as measured by TEM).
  • the nanoparticle (e.g., silica nanoparticle) may have a diameter of less than 70 nm, less than 65 nm, less than 60 nm, less than 55 nm, less than 50 nm, less than 45 nm, less than 40 nm, less than 35 nm, less than 30 nm, less than 25 nm, less than 20 nm, less than 15 nm, or less than 10 nm (e.g., as measured by TEM).
  • the nanoparticle (e.g., silica nanoparticle) has a diameter of 69 nm, 68 nm, 67 nm, 66 nm, 65 nm, 64 nm, 63 nm, 62 nm, 61 nm, 60 nm, 59 nm, 58 nm, 57 nm, 56 nm, 55 nm, 54 nm, 53 nm, 52 nm, 51 nm, 50 nm, 49 nm,
  • the nanoparticle has a diameter of 10- 70 nm (e.g., as measured by TEM).
  • the nanoparticle e.g., silica nanoparticle
  • the nanoparticle e.g., silica nanoparticle
  • the nanoparticle has a diameter of 10-40 nm (e.g., as measured by TEM). In some embodiments, the nanoparticle (e.g., silica nanoparticle) has a diameter of 10- 30 nm (e.g., as measured by TEM). In some embodiments, the nanoparticle (e.g., silica nanoparticle) has a diameter of 28.2+0.9 nm (e.g., as measured by TEM). In some
  • the nanoparticle e.g., silica nanoparticle
  • the nanoparticle has a diameter of 30 nm (e.g., as measured by TEM).
  • the nanoparticle e.g., silica nanoparticle
  • the nanoparticle has a diameter of 28 nm (e.g., as measured by TEM).
  • the nanoparticle (e.g., silica nanoparticle) has a diameter of less than 80 nm as measured by DLS.
  • the nanoparticle e.g., silica nanoparticle
  • the nanoparticle (e.g., silica nanoparticle) has a diameter of 79 nm, 78 nm, 77 nm, 76 nm, 75 nm, 74 nm, 73 nm, 72 nm, 71 nm, 70nm, 69 nm, 68 nm, 67 nm, 66 nm, 65 nm, 64 nm, 63 nm, 62 nm, 61 nm, 60 nm, 59 nm, 58 nm, 57 nm, 56 nm, 55 nm, 54 nm, 53 nm, 52 nm, 51 nm, 50 nm, 49 nm, 48 nm, 47 nm, 46 nm, 45 nm, 44 nm, 43 nm, 42 nm, 41 nm, 40 nm, 39 nm, 38 nm, 37 nm, 37
  • the nanoparticle (e.g., silica nanoparticle) has a diameter of 20- 80 nm, as measured by DLS.
  • the nanoparticle e.g., silica nanoparticle
  • the nanoparticle e.g., silica nanoparticle
  • the nanoparticle has a diameter of 20-50 nm, as measured by DLS. In some embodiments, the nanoparticle (e.g., silica nanoparticle) has a diameter of 20-40 nm, as measured by DLS. In some embodiments, the nanoparticle (e.g., silica nanoparticle) has a diameter of 36.7 nm, as measured by DLS.
  • the nanoparticle is a silica nanoparticle that is porous.
  • the pores on the wall of the silica nanoparticle have a diameter of 10 nm or less than 10 nm.
  • the pores on the wall of the silica nanoparticle may have a diameter of less than 10 nm, less than 9 nm, less than 8 nm, less than 7 nm, less than 6 nm, less than 5 nm, less than 4 nm, less than 3 nm, less than 2 nm, or less than 1 nm.
  • the pores on the wall of the silica nanoparticle have a diameter of 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, or less. In some embodiments, the pores on the wall of the silica nanoparticle have a diameter of less than 2 nm. In some embodiments, the pores on the wall of the silica nanoparticle have a diameter of 2-10 nm.
  • the pores on the wall of the silica nanoparticle may have a diameter of 2-10 nm, 2-9 nm, 2-8 nm, 2-7 nm, 2-6 nm, 2-5 nm, 2-4 nm, 2-3 nm, 3-10 nm, 3-9 nm, 3-8 nm, 3-7 nm, 3-6 nm, 3-5 nm, 3-4 nm, 4-10 nm, 4-9 nm, 4-8 nm, 4-7 nm, 4-6 nm, 4-5 nm, 5-10 nm, 5-9 nm, 5-8 nm, 5-7 nm, 5-6 nm, 6-10 nm, 6-9 nm, 6-8 nm, 6-7 nm, 7-10 nm, 7-9 nm, 7-8 nm, 8-10 nm, 8-9 nm, or 9-10 nm.
  • the silica nanoparticle is a hollow silica nanoparticle.
  • the hollow core has a diameter of 2-60 nm.
  • the hollow core may have a diameter of 2-60 nm, 2-55 m, 2-50 nm, 2-45 nm, 2-40 nm, 2-35 nm, 2-30 nm, 2-25 nm, 2-20 nm, 2-15 nm, 2-10 nm, 2-5 nm, 5-60 nm, 5-55 m, 5-50 nm, 5-45 nm, 5-40 nm, 5-35 nm, 5-30 nm, 5-25 nm, 5-20 nm, 5-15 nm, 5-10 nm, 10-60 nm, 10-55 m, 10-50 nm, 10-45 nm, 10- 40 nm, 10-35 nm, 10-30 nm, 10-25 nm, 10-20 nm, 10-15 nm, 15-60 nm, 15
  • the hollow core has a diameter of 10-20 nm.
  • the hollow core may have a diameter of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm.
  • the hollow core has a diameter of 10 nm.
  • the nanoparticles described herein can be loaded with drugs (e.g., nerve blocker(s) or nerve growth factor(s)).
  • drugs e.g., nerve blocker(s) or nerve growth factor(s)
  • One skilled in the art is familiar with methods of“loading” or “encapsulating” a drug in a nanoparticle, depending on the particular nanoparticle being used.
  • the plurality of pores throughout the silica body and the hollow core are suitable for receiving drugs (e.g., as demonstrated in FIG. 16.
  • Drugs e.g., a nerve blocker or a nerve growth factor
  • the loading efficiency can be calculated based on the total amount of the drug added to the nanoparticle and the amount of the free drug after loading, by using this equation:
  • loading efficiency (total amount of drug - free drug after loading)/total amount of drug.
  • the loading efficiency is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more. In some embodiments, the loading efficiency is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more.
  • Various drugs may be encapsulated in the silica nanoparticles for delivery into the nerve, e.g., a nerve blocker or a growth factors.
  • A“nerve blocker,” as used herein, refers to an agent that interrupts and decreases signals traveling along a nerve (e.g., for the purpose of pain relief).
  • the nerve blocker of the present disclosure is a local anesthetic nerve blocker, which blocks a nerve signal short-term (e.g., hours or days), after being administered (e.g., injected) onto or near a nerve.
  • the nerve blocker of the present disclosure may be a local anesthetic (e.g., an amino-amide and amino-ester local anesthetic).
  • Non-limiting examples of local anesthetics include: (i) the aminoacylanilide group, such as lidocaine, prilocaine, bupivacaine, mepivacaine and related local anesthetic compounds having various substituents on the ring system or amine nitrogen; (ii) the aminoalkyl benzoate group, such as procaine, chloroprocaine, propoxycaine, hexylcaine, tetracaine, cyclomethylcaine, benoxinate, butacaine, proparacaine, and related local anesthetic compounds; (iii) cocaine and related local anesthetic compounds; (iv) the amino carbamate group, such as diperodon and related local anesthetic compounds; (v) the N-phenylamidine group, such as phenacaine and related local anesthetic compounds; (vi) the N-aminoalkyl amide group, such as dibucaine and related local anesthetic compounds; (vii) the aminoketone
  • the nerve blocker of the present disclosure is a protein.
  • Known proteins that may be used as nerve blockers include neurotoxins produced by an organism (e.g., a bacterium or a shellfish), and analogs thereof.
  • the protein nerve blocker is a botulinum toxin.
  • A“botulinum toxin” refers to a family of bacterial toxins produced by Clostridium Botulinum. There are seven well-established serotypes of botulinum toxins (serotypes A-G). Local injections of minute amounts of botulinum toxins can attenuate neuronal activity in targeted regions.
  • Botulinum toxins have been used to treat a growing list of medical conditions, including muscle spasms, chronic pain, overactive bladder problems, as well as for cosmetic
  • botulinum toxin encompasses any functional fragments or variants of botulinum toxins.
  • the nerve blocker of the present disclosure is a site 1 sodium channel blocker (S1SCB).
  • An“ion channel” is a pore-forming membrane protein expressed on the surface of a cell (e.g., a neuron). Ion channels on the surface of a cell (e.g., a neuron) have various biological functions including: establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of ions across the cell membrane, controlling the flow of ions across secretory and epithelial cells, and regulating cell volume. Activated transmembrane ion channels allow ions into or out of cells.
  • Ion channels e.g., a sodium ion channel
  • the trigger that opens the channel for such ions i.e. either a voltage-change ("Voltage-gated”, “voltage-sensitive”, or “voltage- dependent” ion channel) or a binding of a substance (a ligand) to the channel (ligand-gated ion channels).
  • A“sodium channel” refers to an integral membrane protein that form ion channels that conduct sodium ions (Na+) through a cell's plasma membrane (e.g., as described in Bertil et al., Ion Channels of Excitable Membranes (3rd ed.). Sunderland, Mass: Sinauer. pp. 73-7, incorporated herein by reference).
  • sodium channels are responsible for the rising phase of action potentials. These channels go through 3 different states called as resting, active and inactive states. Even though the resting and inactive states wouldn't allow the ions to flow through the channels the difference exists with respect to their structural conformation.
  • Non-limiting examples of sodium channels include, without limitation: NaVl.l (Genebank ID: AB093548), NaVl.2 (Genebank ID: AB208888), NaVl.3 (Genebank ID: AF035685), NaVl.4 (Genebank ID:
  • NaVl.5 Genebank ID: AJ310893
  • NaVl.6 Genebank ID: AB027567
  • NaVl.7 Genebank ID: X82835
  • NaVl.8 Genebank ID: AF117907
  • NaVl.9 Genebank ID:
  • sodium channels serve as specific targets for many nerve blockers (e.g., neurotoxins).
  • nerve blockers e.g., neurotoxins
  • Different neurotoxins occupy different receptor sites on the sodium channel.
  • Five neurotoxin receptor sites have been defined on the vertebrate sodium channel as follows: receptor site 1 binds the sodium channel blockers tetrodotoxin and saxitoxin; site 2 binds lipid-soluble sodium channel activators such as veratridine; site 3 binds a-scorpion and sea anemone toxins, which slow sodium channel inactivation; site 4 binds &scorpion toxins, which affect sodium channel activation; and site 5 binds the polyether ladder brevetoxins and ciguatoxin (e.g., as described in Catterall et ah, Annu.
  • the nerve blockers described herein are site“site 1 sodium channel blockers (SlSCBs)” because they bind to receptor site 1 of a sodium channel.
  • SlSCBs include: neosaxitoxin, saxitoxin, decarbamoyl STX, tetrodotoxin, and gonyautoxin.
  • the nerve blocker is tetrodotoxin (TTX). Any functional variants of fragments of a S1SCB may be used as the nerve blocker of the present disclosure.
  • the nerve blocker may be a combination of any of the agents described herein and known to one skilled in the art.
  • Saxitoxin is a potent neurotoxin and the best-known paralytic shellfish toxin (PST).
  • the term saxitoxin refers to the entire suite of more than 50 structurally related neurotoxins (known collectively as "saxitoxins") produced by algae and cyanobacteria which includes saxitoxin itself (STX), neosaxitoxin (NSTX), gonyautoxins (GTX) and decarbamoyl saxitoxin (dcSTX).
  • Saxitoxin is a neurotoxin that acts as a selective sodium channel blocker. As one of the most potent known natural toxins, it acts on the voltage-gated sodium channels of neurons, preventing normal cellular function and leading to paralysis.
  • Neurosaxitoxin is included, as other saxitoxin-analogs, in a broad group of natural neurotoxic alkaloids, commonly known as the paralytic shellfish toxins (PSTs).
  • PSTs paralytic shellfish toxins
  • STX saxitoxin
  • STX tricyclic perhydropurine alkaloid, which can be substituted at various positions, leading to more than 30 naturally occurring STX analogues.
  • NSTX and other PSTs, are produced by several species of marine dinoflagellates (eukaryotes) and freshwater
  • cyanobacteria blue-green algae (prokaryotes).
  • Tetrodotoxin is a potent neurotoxin. Its name derives from Tetraodontiformes, an order that includes pufferfish, porcupinefish, ocean sunfish, and triggerfish; several of these species carry the toxin. Tetrodotoxin is a sodium channel blocker. It inhibits the firing of action potentials in neurons by binding to the voltage-gated sodium channels in nerve cell membranes and blocking the passage of sodium ions (responsible for the rising phase of an action potential) into the neuron. This prevents the nervous system from carrying messages and thus muscles from flexing in response to nervous stimulation.
  • Gonyautoxin gonyautoxins are a few similar toxic molecules that are naturally produced by algae. They are part of the group of saxitoxins, a large group of neurotoxins along with a molecule that is also referred to as saxitoxin (STX), neosaxitoxin (NSTX) and decarbamoylsaxitoxin (dcSTX). Currently eight molecules are assigned to the group of gonyautoxins, known as gonyautoxin 1 (GTX-l) to gonyautoxin 8 (GTX-8).
  • gonyautoxins Ingestion of gonyautoxins through consumption of mollusks contaminated by toxic algae can cause a human illness called paralytic shellfish poisoning (PSP).
  • PPS paralytic shellfish poisoning
  • the gonyautoxins have their structure based on the 2,6-diamino-4-methyl-pyrollo[l,2- c]-purin-lO-ol skeleton (also known as the Saxitoxin-gonyau toxin skeleton).
  • the different molecules only differ from each other by their substituents, some of them only by a mere stereoisomerism such as GTX-2 and GTX-3.
  • Gonyautoxin can bind with high affinity at the site 1 of the a-subunit of the voltage dependent sodium channels in the postsynaptic
  • A“nerve growth factor,” as used herein, refers to a nerve growth factor (NGF) and is a neurotrophic factor and neuropeptide primarily involved in the regulation of growth, maintenance, proliferation, and survival of certain target neurons. NGF is critical for the survival and maintenance of sympathetic and sensory neurons, as they undergo apoptosis in its absence. Nerve growth factor prevents or reduces neuronal degeneration and promotes peripheral nerve regeneration in rats.
  • NGF nerve growth factor
  • A“neuron” or a“nerve cell” is an electrically excitable cell that receives, processes, and transmits information through electrical and chemical signals. These signals between neurons are termed herein as“neuronal signals” or“nerve signals,” and occur via specialized connections called synapses. Neurons can connect to each other to form neural networks.
  • Neurons are the primary components of the central nervous system, which includes the brain and spinal cord, and of the peripheral nervous system, which comprises the autonomic nervous system and the somatic nervous system.
  • Sensory neurons respond to one particular type of stimuli such as touch, sound, or light and all other stimuli affecting the cells of the sensory organs, and converts it into an electrical signal via transduction, which is then sent to the spinal cord or brain.
  • Motor neurons receive signals from the brain and spinal cord to cause everything from muscle contractions and affect glandular outputs.
  • Intemeurons connect neurons to other neurons within the same region of the brain or spinal cord in neural networks.
  • a typical neuron consists of a cell body, dendrites, and an axon.
  • An axon also called a nerve fiber when myelinated
  • axon hillock is a special cellular extension that arises from the cell body at a site called the axon hillock and travels for a distance, as far as 1 meter in humans or even more in other species.
  • Most neurons receive signals via the dendrites and send out signals down the axon.
  • A“nerve” is an enclosed, cable-like bundle of axons in the peripheral nervous system.
  • a nerve provides a common pathway for the electrochemical nerve impulses that are transmitted along each of the axons to peripheral organs.
  • the central nervous system has an analogous structure, known as“tracts.”
  • a nerve contains neurons and non-neuronal Schwann cells that coat the axons in myelin.
  • each axon is surrounded by a layer of connective tissue called the endoneurium.
  • the axons are bundled together into groups called fascicles, and each fascicle is wrapped in a layer of connective tissue called the perineurium.
  • the entire nerve is wrapped in a layer of connective tissue called the epineurium.
  • the individual nerve fibers are surrounded by a low-protein liquid called endoneurial fluid.
  • the composition described herein further comprises a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the patient (e.g., physiologically compatible, sterile, physiologic pH, etc.).
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethylene glyco
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy.
  • unit dose when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • the formulation of the pharmaceutical composition may dependent upon the route of administration.
  • the composition described herein are suitable for administration via injection (e.g., injection into tissues near a nerve) or topical administration.
  • injectable preparations suitable for injection include, for example, sterile injectable aqueous or oleaginous suspensions and may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 propanediol or 1,3 butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the pharmaceutical composition can be formulated into ointments, salves, gels, or creams, as is generally known in the art.
  • Topical administration can utilize transdermal delivery systems well known in the art.
  • An example is a dermal patch.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the anti-inflammatory agent, increasing convenience to the subject and the physician.
  • release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones,
  • polyesteramides polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.
  • Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which the anti-inflammatory agent is contained in a form within a matrix such as those described in U.S. Patent Nos.
  • Long-term sustained release means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • the pharmaceutical compositions used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).
  • preservatives can be used to prevent the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid.
  • composition ordinarily will be stored in lyophilized form or as an aqueous solution if it is highly stable to thermal and oxidative denaturation.
  • the pH of the preparations typically will be about from 6 to 8, although higher or lower pH values can also be appropriate in certain instances.
  • aspects of the present disclosure provide methods of inducing local anesthesia, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a nerve blocker encapsulated in a nanoparticle described herein.
  • Local anesthesia uses medicine to block sensations of pain from a specific area of the body. Local anesthetics (e.g., nerve blockers described herein)are usually given by injection into the body area that needs to be anesthetized. They are not typically not injected into the
  • Local anesthesia can also be applied directly to the skin or mucous membranes as a liquid or gel.
  • Methods of promoting neuronal regeneration comprise administering to a subject in need thereof an effective amount of a composition comprising a nerve growth factor encapsulated in a nanoparticle described herein.
  • the composition is administered locally at a nerve.
  • administering a nerve means administered in close proximity to the nerve, e.g., injected into the tissue surrounding the nerve such that the composition, the nanoparticle with the encapsulated nerve blocker contact the nerve.
  • the nanoparticle loaded with the nerve blocker penetrate into the nerve.
  • “Penetrate into the nerve” means the nanoparticle crosses the tissue barrier (epineurium), enters the macroscopic bundle of nerves, and contact
  • the nanoparticle crosses the tissue barrier via transcytosis.
  • Transcytosis is a type of transcellular transport in which various
  • macromolecules are transported across the interior of a cell. Macromolecules are captured in vesicles (e.g., a nanoparticle) on one side of the cell, drawn across the cell, and ejected on the other side.
  • vesicles e.g., a nanoparticle
  • the composition described herein may be administered to any nerve.
  • the composition is administered to a peripheral nerve (e.g., a sciatic nerve).
  • a “peripheral nerve” is a nerve outside the brain and spinal cord.
  • the sciatic nerve is also called ischiadic nerve, ischiatic nerve, "butt nerve”) is a large nerve in humans and other animals. It begins in the lower back and runs through the buttock and down the lower limb. It is the longest and widest single nerve in the human body, going from the top of the leg to the foot on the posterior aspect.
  • the sciatic nerve provides the connection to the nervous system for nearly the whole of the skin of the leg, the muscles of the back of the thigh, and those of the leg and foot.
  • the nanoparticle with the encapsulated nerve blocker contacts the neuron. In some embodiments, the nanoparticle with the encapsulated nerve blocker contacts the axonal surface of the neuron. In some embodiments, the nerve blocker is released from the nanoparticle and contacts the neuron or the axonal surface of the neuron. In some embodiments, the nerve blocker enters the neuron (e.g., in the cases of small molecule nerve blockers). In some embodiments, the nerve block does not enter the neuron and exerts its effects on the surface of the neuron (e.g., S lSCBs).
  • the penetration of the nerve block into the nerve is enhanced (e.g., by at least 20%) when encapsulated in a nanoparticle described herein, compared to a free nerve blocker.
  • the penetration of the nerve block into the nerve may be enhanced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least lOO-fold, at least lOOO-fold, or more, when encapsulated in a nanoparticle described herein, compared to a free nerve blocker.
  • the penetration of the nerve block into the nerve is enhanced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5- fold, lO-fold, lOO-fold, lOOO-fold, or more, when encapsulated in a nanoparticle described herein, compared to a free nerve blocker.
  • the nanoparticles release the nerve blockers inside the nerve, which contacts the neurons and blocks a neuronal signal.
  • the nerve blocker is released overtime (e.g., hours or days), thereby prolonging the effect of the nerve block (e.g., by at least 20%), compared to a free nerve blocker.
  • the effect of the nerve blocker is prolonged by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least lO-fold, at least lOO-fold, at least lOOO-fold, or more, when encapsulated in a nanoparticle described herein, compared to a free nerve blocker.
  • the effect of the nerve blocker is prolonged by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, lO-fold, lOO-fold, lOOO-fold, or more, when encapsulated in a nanoparticle described herein, compared to a free nerve blocker.
  • the nanoparticle with the encapsulated nerve blocker enhances the rate of nerve blockade (e.g., by at least 20%), compared to a free nerve block. “Enhance the rate of nerve blockade” means when the nanoparticle with the encapsulated nerve blocker is administered, it takes a shorter time to achieve nerve blockade (e.g., at least 20% less time), compared to when a free nerve blocker is administered.
  • it may take at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% less time to achieve nerve blockade when the nanoparticle with the encapsulated nerve blocker is administered, compared to when a free nerve blocker is administered. In some embodiments, it takes 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% less time to achieve nerve blockade when the nanoparticle with the encapsulated nerve blocker is administered, compared to when a free nerve blocker is administered.
  • a lower dose e.g., at least 20% lower
  • a lower dose e.g., at least 20% lower
  • nerve blocker is needed to achieve the same level of nerve blockade when the nanoparticle with the
  • the encapsulated nerve blocker is administered, compared to when a free nerve blocker is administered.
  • the dose of nerve blocker needed to achieve the same level of nerve blockade is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% lower when loaded into a nanoparticle, compared to a free nerve blocker.
  • the dose of nerve blocker needed to achieve the same level of nerve blockade is 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% lower when loaded into a nanoparticle, compared to a free nerve blocker.
  • same dose of a nerve blocker encapsulated in a nanoparticle results in enhanced level of nerve blockade (e.g., by at least 20%), compared to a free nerve blocker.
  • same dose of a nerve blocker encapsulated in a nanoparticle results in a level of nerve blockade that is at least enhanced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least lOO-fold, at least 1000-fold higher, compared to a free nerve blocker.
  • same dose of a nerve blocker encapsulated in a nanoparticle results in a level of nerve blockade that is at least enhanced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, lO-fold, lOO-fold, lOOO-fold higher, compared to a free nerve blocker.
  • Blocks a neuronal signal or“nerve blockade” refers to the blocking or attenuating (e.g., by at least 20%) of a signal that is been transmitted along a nerve in the presence of a nerve blocker (e.g., free or encapsulated in a nanoparticle), compared to without a nerve blocker.
  • a nerve blocker e.g., free or encapsulated in a nanoparticle
  • a signal that is been transmitted along a nerve is reduced by at least 20%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, in the presence of a nerve blocker (e.g., free or encapsulated in a nanoparticle), compared to without a nerve blocker.
  • a signal that is been transmitted along a nerve is reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, in the presence of a nerve blocker (e.g., free or encapsulated in a nanoparticle), compared to without a nerve blocker.
  • the nanoparticles with the encapsulated nerve blocker is not toxic to the nerve. “Not toxic” means it does not cause any pathological condition to the nerve, e.g., inflammation, and/or does not interfere with the structure or function of the never other than nerve blockade.
  • the method of inducing local anesthesia further comprises administering to the subject an effective amount of a second nerve blocker.
  • the second nerve blocker may be any of the nerve blockers described herein.
  • the second nerve blocker is an amino-amide and amino-ester local anesthetic.
  • the second nerve blocker is lidocaine, tetracaine, capsaicin, and analogs thereof.
  • the method described herein further comprises administering to the subject an effective amount of an adjuvant.
  • An“adjuvant,” for the purposes of the present disclosure refers to an agent that enhances the nerve blockade activity of the nerve blockers (e.g., free or encapsulated in a nanoparticle).
  • the nerve blockade activity of the nerve blocker may be increased by the adjuvant by at least at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least lOO-fold, at least 1000-fold, or more, compared to without the adjuvant.
  • the nerve blockade activity of the nerve blocker is increased by the adjuvant by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5- fold, lO-fold, lOO-fold, lOOO-fold, or more, compared to without the adjuvant.
  • Non-limiting examples of adjuvants include glucocorticoid receptor agonists (e.g., dexamethasone), adrenergic agonists, and vasoconstrictors (e.g., epinephrine or dexmedetomidine).
  • glucocorticoid receptor agonists e.g., dexamethasone
  • adrenergic agonists e.g., adrenergic agonists
  • vasoconstrictors e.g., epinephrine or dexmedetomidine
  • composition described herein can also be used to block a nerve signal in vitro (e.g., in cultured neurons) or ex vivo (e.g., in neurons isolated from a subject).
  • a nerve signal in vitro (e.g., in cultured neurons) or ex vivo (e.g., in neurons isolated from a subject).
  • cultured neurons or neurons isolated from a subject may be contacted with the composition for blockade of neuronal signals.
  • the nanoparticles described herein have similar nerve blockade activities in vitro or ex vivo, compared to its in vivo activity (e.g., when administered to a subject).
  • aspects of the present disclosure provide methods of promoting neuronal regeneration or reducing neurodegeneration, the methods comprising administering to a subject in need thereof an effective amount of a composition comprising a nerve growth factor encapsulated in a nanoparticle described herein.
  • Neurodegeneration is the progressive loss of structure or function of neurons, including death of neurons.
  • Many neurodegenerative diseases - including amyotrophic lateral sclerosis, Parkinson's, Alzheimer's, and Huntington's - occur as a result of neurodegenerative processes.
  • the treatment of these neurodegenerative diseases are also within the scope of the present disclosure.
  • Neuroregeneration refers to the regrowth or repair of nervous tissues, cells or cell products.
  • Such mechanisms may include generation of new neurons, glia, axons, myelin, or synapses.
  • Neuroregeneration differs between the peripheral nervous system (PNS) and the central nervous system (CNS) by the functional mechanisms and especially the extent and speed.
  • PNS peripheral nervous system
  • CNS central nervous system
  • the distal segment undergoes Wallerian degeneration, losing its myelin sheath.
  • the proximal segment can either die by apoptosis or undergo the chromatolytic reaction, which is an attempt at repair.
  • synaptic stripping occurs as glial foot processes invade the dead synapse.
  • the nanoparticle with the encapsulated nerve growth factor enhances neuroregeneration (e.g., by at least 20%), compared to a free nerve growth factor.
  • “Enhance neuroregeneration” means when the nanoparticle with the encapsulated nerve blocker is administered, the number of new neurons is increased (e.g., at least 20% less time), compared to when a free nerve growth factor is administered.
  • the number of new neurons may increase by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least lOO-fold, at least 1000-fold, or more, compared to when a free nerve growth factor is administered.
  • the number of new neurons increases by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, lO-fold, lOO-fold, lOOO-fold, or more, compared to when a free nerve growth factor is administered.
  • the nanoparticle with the encapsulated nerve growth factor enhances the rate of neurorenegeration (e.g., by at least 20%), compared to a free nerve growth factor. “Enhance the rate neuroregeneration” means when the nanoparticle with the
  • the rate of new neurons regrowth is increased (e.g., at least 20% less time), compared to when a free nerve growth factor is administered.
  • the rate of new neurons regrowth may increase by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least lO-fold, at least lOO-fold, at least lOOO-fold, or more, compared to when a free nerve growth factor is administered.
  • the rate of new neurons regrowth increases by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5- fold, lO-fold, lOO-fold, lOOO-fold, or more, compared to when a free nerve growth factor is administered.
  • A“subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • a human i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • the non-human animal is a mammal (e.g., rodent (e.g., mouse or rat), primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)).
  • the non-human animal is a fish, reptile, or amphibian.
  • the non-human animal may be a male or female at any stage of development.
  • the non-human animal may be a transgenic animal or genetically engineered animal.
  • the subject is a companion animal (a pet).
  • a companion animal refers to pets and other domestic animals.
  • Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.
  • the subject is a research animal.
  • Non-limiting examples of research animals include: rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.
  • a“subject in need thereof’ refers to a subject that needs local anesthesia.
  • Subjects that need local anesthesia include, without limitation: subjects in need of pain management, subjects undergoing surgery, subjects with nerve trauma or injury, and subjects with neuropathic pain.
  • Subjects that need neuroregeneration include, without limitation: subjects that sustained injuries to the nervous system, subjects having
  • neurodegenerative diseases e.g., amyotrophic lateral sclerosis, Parkinson's, Alzheimer's, and Huntington's.
  • an“effective amount” of a composition described herein refers to an amount sufficient to elicit the desired biological response (e.g., local anesthesia).
  • An effective amount of a composition described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the drug (e.g., nerve blocker), the condition being treated, the mode of administration, and the age and health of the subject.
  • an effective amount is a therapeutically effective amount.
  • an effective amount is a prophylactic treatment.
  • an effective amount is the amount of a compound described herein in a single dose.
  • an effective amount is the combined amounts of a compound described herein in multiple doses.
  • an effective amount of a composition is referred herein, it means the amount is prophylactically and/or therapeutically effective, depending on the subject and/or the disease to be treated.
  • Determining the effective amount or dosage is within the abilities of one skilled in the art.
  • the terms“administer,”“administering,” or“administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a composition described herein, or a composition thereof, in or on a subject.
  • the composition of the described herein may be administered via local injection or topical application to the site that needs local anesthesia.
  • Tetrodotoxin is a very potent local anesthetic that acts by blocking site 1 on the voltage-gated sodium channel, on the axonal surface.
  • Tetrodotoxin - and other site 1 sodium channel blockers such as the saxitoxins - have minimal local toxicity as well as decreased cardiac and central nervous system toxicity. 1
  • the effectiveness of SlSCBs is limited by relatively poor penetration through various tissue barriers to their site of action; this difficulty is probably due to their hydrophilicity and charge.
  • the high concentrations of SlSCBs required to overcome those barriers and achieve useful degrees and durations of nerve block can entail considerable systemic toxicity.
  • TTX was encapsulated in 28 nm hollow silica nanoparticles (TTX-HSN) and then injected at the sciatic nerve in rats. Hollow silica nanoparticles were used to deliver TTX (TTX-HSN) as a model S 1SCB due to its commercial availability.
  • HSN was selected to deliver TTX because their hollow structure could facilitate loading with TTX. Loading could also be assisted by the negative charge of silica (its isoelectric point is about 2 6 ) while TTX is cationic. Sustained release of TTX would prolong the effect, while reducing systemic toxicity.
  • TTX-HSN achieved an increased frequency of successful blocks, prolonged the duration of block, and decreased toxicity compared to free TTX.
  • imaging of frozen sections of nerve demonstrated that HSN could penetrate into nerve, and that the penetrating ability of silica nanoparticles was highly size-dependent.
  • HSN formulation HSN were prepared as reported (see Methods and Materials below). 7 Transmission electron microscopy (TEM; FIGs. 1A and 1B) of HSN confirmed that the HSNs were hollow spheres with uniform particle size (however, it is possible given the size of the particles and their pores [see below] that these are highly porous particles.) A size distribution of 28.2+0.9 nm (FIG. 1C) was determined by measuring the diameters of 100 individual HSN from the high-magnification TEM images. Dynamic light scattering (DFS) measurements showed a diameter of 36.7 nm (FIG. 1D), which agrees well with the result in FIG. 1C.
  • DFS Dynamic light scattering
  • HSN possessed a negatively charged surface with a zeta potential of -9.8 mV, suggesting its possibility for loading cation TTX.
  • silica nanoparticles are described with a subscript denoting their approximate diameter in nanometers, and hollow particles have the prefix“H” (e.g. HSN30).
  • the nitrogen adsorption isotherm curve of HSN 30 FIG. 7 showed a type IV adsorption- desorption isotherm 8 , which is typical of porous materials.
  • the pore volume was calculated to be 1.43 cm 3 /g (by the software of the V-sorb 2800 surface area and porosimetry analyzer) and the Brunauer-Emmett-Teller (BET) surface area was 462 m 2 /g (BET is the most common theory for determining the surface area of powders and porous materials. 9 ). These values suggested that HSN 30 were highly porous and could be suitable as carrier for drug delivery. 7 After loading with TTX, the pore volume and surface area decreased to 1.11 cm 3 /g and 355 m 2 /g (FIG.
  • the HSN 30 had a calculated pore size distribution centered at 13.4 nm (FIG. 9). 10 There was no significant aggregation of HSN 30 after loading with TTX (FIG. 10 ).
  • TTX was loaded by mixing HSN 30 and TTX in aqueous solution. The mixture was stirred at room temperature for 48 hours. The obtained TTX- HSN 30 solution was diluted and used for the subsequent studies without removing the free TTX.
  • TTX- HSN 30 solution was washed with water three times, and the supernatant after centrifugation (12,000 rmp, 20 minutes) was collected and the free TTX was measured by EFISA.
  • the loaded TTX was calculated to be the total amount of TTX added to the HSN 30 minus the free TTX.
  • the loading efficiency of TTX in HSN 30 was 49.0+2.0% TTX.
  • TTX- HSN30 The potential of these TTX- HSN30 to provide sustained nerve blockade was assessed by performing release kinetic studies at 37°C in PBS (FIG. 2).
  • the TTX- HSN30 significantly increased the duration of TTX release from the system (a dialysis device with a 20,000 MW cut-off), compared with free TTX (e.g., -90% for free TTX vs -50% for TTX- HSN 30 at 6 hours, p ⁇ 0.005).
  • TTX- HSN30 C2C12 cells (myotube cell line used to assess myotoxicity) were exposed to TTX- HSN30 for up to 4 days (FIG. 11). TTX- HSN30 did not reduce cell survival at any duration of exposure tested. Similar studies were performed in PC 12 cells (a pheochromocytoma cell line frequently used in neurotoxicity studies). TTX- HSN30 also did not cause any loss in cell viability for up to 4 days. These results suggested that TTX- HSN30 could be safe for the following animal experiments.
  • Rats (4 in each group) were injected at the left sciatic nerve with 0.3 mL of water containing 0 pg to 6 pg of TTX, either free or in TTX- HSN30 (0-60 mg/mL of HSN30). They then underwent neurobehavioral testing to determine the duration of functional deficits in both hindpaws. The duration of deficits on the injected side reflected duration of nerve block. Deficits on the uninjected side (right; contralateral) reflected systemic TTX distribution.
  • Free TTX showed a concentration-dependent increase in the median duration of nerve block (FIG. 3 A) and frequency of successful nerve blockade (FIG. 3B; see Methods and Materials for the definition of successful nerve block).
  • Nerve block from 4 pg of free TTX had a median duration of 79.5 minutes with 80% successful blocks (FIG. 3B) and 30% of animals had contralateral deficits (FIG. 3C). 6 pg of free TTX caused contralateral deficits in all animals and was uniformly fatal.
  • Nerve block duration was significantly prolonged with the TTX- HSN30 formulations (FIG. 3A).
  • Sensory nerve blockade with 4 pg TTX in TTX- HSN30 lasted 274 minutes (p ⁇ 0.005 compared to free TTX); with 6 pg TTX it lasted 362 minutes and no animals died or had contralateral deficits.
  • 5a chemical permeation enhancers, 4 or drugs that enhance the effect of SlSCBs 11
  • TTX- HSN30 resulted in a much higher rate of successful nerve blockade than did free TTX: 100% blockade was observed even at a very low dose of TTX (e.g. 1 pg, FIG. 3B). This increase in the success rate is not the norm for encapsulated TTX, 5a but was similar to the effect of chemical permeation enhancers on TTX nerve block. 4 Encapsulation in TTX- HSN 30 decreased the incidence of systemic toxicity (FIG. 3C and 3D). There was no evidence of systemic toxicity (contralateral sensory deficits or mortality) at any dose ⁇ 6 pg TTX in TTX- HSN 30 . This enhanced safety is attributable to sustained release function from the HSN 30 .
  • HSN 30 was synthesized to which fluorescein isothiocyanate (a fluorescent dye with an excitation wavelength of 488 nm and emission wavelength of 519 nm was covalently conjugated (see Materials and Methods and FIG. 12A) so that the dye would be associated with the particles and not able to diffuse independently; the particles are denoted FITC- HSN 30 .
  • the diameter of FITC- HSN 30 was -28 nm (FIG. 12B), similar to that of HSN 30 .
  • the absorbance and fluorescence emission spectra of FITC- HSN 30 had peaks at 495 nm and 520 nm, respectively (FIG. 12C).
  • Fluorescent imaging was used to track the location of FITC- HSN 30 in tissue. 300 pF (30 mg/mF) of FITC- HSN 30 in water was injected at the sciatic nerve. Four hours later, animals were euthanized and the sciatic nerve was exposed. FITC- HSN 30 were identified as a faintly light yellow material around the nerve (FIG. 13A). The nerve and surrounding tissue were then harvested. Under irradiation of a 365 nm UV lamp, green fluorescence was observed from sciatic nerve and adjacent muscles in the injected leg (FIG. 13B) but not in the un-treated leg. Frozen sections of the tissues were produced, and fluorescent images taken.
  • FITC-labeled silica nanoparticles that were 9.8+0.5 nm (FITC-SN10; FIG. 5A) and 70.0+6.5 nm (FITC-SN70; FIG. 5B) in diameter were prepared as described. 12 They were injected at the sciatic nerve, and were harvested at 4 hours and processed as were the FITC-HSN30. FITC-SN10 dispersed throughout the nerve (FIGs. 5C to 5D), while FITC-SN70 were all located outside the nerve (FIG. 5E). Quantitative analysis also confirmed this difference in distribution (FIG. 4E).
  • TTX- HSN 30 were not observed on gross dissection (FIG. 15). Microscopic examination revealed very mild myotoxicity and inflammation 4 and 21 days after injection in animals injected with free TTX and TTX- HSN 30 samples (FIG. 6). The myotoxicity and inflammation were quantified using a scoring system (Table l). 13 There was no statistically significant difference between the scores for TTX- HSN 30 and free TTX.
  • peripheral nerve blockade the various particulate and other drug delivery systems that have been used to prolong the duration of local anesthetic effect 4b are generally thought of as being essentially depot systems that release local anesthetics in the immediate vicinity of the nerve.
  • the rationale for using nanomaterials for nerve block is not particularly strong, 14 since in general larger particles will encapsulate more drug, have slower release, and will be less likely to degrade or leave the site of administration. 15 It has been demonstrated herein that 28 nm HSN 30 containing TTX can penetrate into nerve. This penetration is believed to contribute to the increase in the number of successful nerve blocks as well as the
  • DDMS dimethyldimethoxysilane
  • FITC fluorescein isothiocyanate
  • APTES 3- Aminopropyl)triethoxysilane
  • HC1 was purchased from Sigma-Aldrich (St. Louis, MO). Deionized water was used throughout the experiments.
  • MWCO molecular-weight cutoff
  • Dynamic light scattering and zeta potential The size of HSN30 and zeta potential were measured with a Delsa Nano C particle analyzer (Beckman Coulter, CA, USA).
  • nanoparticle solution (3.0 mL) was put into a disposable cuvette (Eppendorf, Hauppauge, NY) at 25 °C, the particle diameter was measured 70 times by the particle analyzer for each sample. Three different samples were tested. The hydrodynamic diameter was calculated by averaging the diameters from those three repeated measurements.
  • For zeta potential 850 mL HSN 30 PBS solution was put into flow cell and tested at 25 °C. The sample was tested 7 times. The result was obtained by averaging the 7 times value.
  • the fluorescent spectra and UV-Vis spectra were recorded on an Agilent Cary Eclipse fluorescence spectrophotometer and an Agilent 8453 UV-Vis G1103A spectrophotometer (Agilent, CA, USA).
  • Nitrogen sorption isotherms were measured with a V-Sorb 2800P BET surface area and pore volume analyzer (Gold APP Instruments Corporation China, Beijing, China). Before measurement, the sample was degassed at 120 °C in vacuum for 2 hours. The specific surface area was calculated using the Brumauer-Emmet-Teller (BET) method 12a . The pore size distribution was derived from the adsorption branches of the isotherms based on the Barrett- Joyner-Halenda (BJH) model. 9
  • TTX-HSN 30 0.3 g of HSN 30 and 0.1 mg of TTX were added to 1 mL aqueous solution. The mixture was stirred at room temperature for 48 hours. The obtained TTX-HSN 30 solution was diluted and used in subsequent studies directly.
  • TTX-HSN30 were washed with water three times, and the supernatant after centrifugation (12,000 rmp, 20 minutes) was collected and the TTX content was quantified by ELISA (Reagen LLC, Moorestown, NJ).
  • TTX-HSN30 100 pL of TTX-HSN30 (10 pg/mL TTX) were placed into a Slide- A-Lyzer MINI dialysis device (Thermo Scientific, Columbia, MD) with a 20,000 MW cut-off. The samples were dialyzed with 1.2 ml PBS and incubated at 37°C on a platform shaker (New Brunswick Innova 40, Eppendorf, Hamburg, Germany; 150 rpm). At predetermined intervals, the dialysis solution was refreshed with PBS. The concentration of released TTX was quantified by ELISA (Reagen LLC, Moorestown, NJ).
  • C2C12 mouse myoblasts American Type Culture Collection (ATCC) Manassas, VA
  • PC 12 rat adrenal gland pheochromocytoma cells ATCC, Manassas, VA
  • C2C12 cells were cultured in DMEM with 20% FBS and 1% Penicillin Streptomycin (Invitrogen, Waltham, MA). Cells were seeded onto a 24-well plate at 50,000 cells/ml and incubated for 10-14 days in DMEM with 2% horse serum and 1% Penicillin Streptomycin to differentiate into myotubules.
  • PC12 cells were grown in DMEM with 12.5% horse serum, 2.5% FBS and 1% Penicillin Streptomycin.
  • TTX-HSN30 Cells were seeded onto a 24 well-plate, and 50ng/ml nerve growth factor was added 24 hours after seeding (Invitrogen, Waltham, MA). The cells was exposed in the cell culture medium with TTX-HSN30. Cell viability was evaluated by the MTS (Invitrogen, Waltham, MA) assay at 24, 48, 72, and 96 hours after exposure to TTX-HSN30.
  • Rats Male male Sprague-Dawley rats (350-400 g) were purchased from Charles River Laboratories (Wilmington, MA) and housed in groups of two per cage on a 7 a.m. to 7 p.m. light/dark cycle. The rats were used according to protocols approved by the Animal Care and Use Committee at Boston Children’s Hospital and the Guide for the Care and Use of
  • the rats were euthanized with carbon dioxide, and the sciatic nerve and adjacent tissue were harvested for histology at 4 days and 14 days after injection. Muscle samples were scored for inflammation (0-4 points) and myotoxicity (0-6 points). 13 The inflammation score was a subjective assessment of severity (0: no inflammation, 1: peripheral inflammation, 2: deep inflammation, 3: muscular hemifascicular inflammation, 4: muscular holofascicular inflammation).
  • the myotoxicity score reflected two characteristic features of local anesthetic myotoxicity: nuclear internalization and regeneration. Nuclear internalization is characterized by myocytes normal in size and chromicity, but with nuclei located away from their usual location at the periphery of the cell. Regeneration is characterized by shrunken myocytes with basophilic cytoplasm. Scoring was as follows: 0. normal; 1. perifascicular internalization; 2. deep internalization (>5 cell layers), 3. perifascicular regeneration, 4. deep regeneration,
  • the sciatic nerve samples were processed and fixed in Karnovsky’s KII Solution (2.5 % glutaraldehyde, 2.0 % paraformaldehyde, 0.025 % calcium chloride in 0.1 M cacodylate buffer, pH 7.4). Samples were treated with osmium tetroxide for post-fixation, and were subsequently stained with uranyl acetate, dehydrated in graded ethanol solutions, and infiltrated with propylene oxide/ Epon mixtures. Tissue sections of 0.5 pm were stained with toluidine blue, followed by high- resolution light microscopy.
  • test solution free FITC, FITC-HSN30, FITC-SN10, or FITC-SN70, with equal absorption intensities to ensure that the same doses of FITC were injected
  • test solution 0.3 mL of test solution (free FITC, FITC-HSN30, FITC-SN10, or FITC-SN70, with equal absorption intensities to ensure that the same doses of FITC were injected) was injected at the sciatic nerve of the left leg. Un-injected right legs were used as fluorescence-free control.
  • sciatic nerves were harvested and embedded into OCT compound (VWR, Radnor, PA) and frozen sections prepared. The slides were fixed with 4% Paraformaldehyde (Sigma-Aldrich, St. Fouis, MO) for 20 minutes, washed three times using PBS buffer.
  • the mean fluorescent intensity in Fl ...F10 was measured, and normalized to the intensity at the surface of nerve. Fluorescence intensity across the diameter of the nerve (along the solid line in FIG. 14) was plotted in FIG. 4E, showing the penetration of FITC-HSN30 into the nerve. In FIG. 4E, all nerve diameters were normalized to 1 to allow plotting of nerves with differing diameters. Mean fluorescence throughout the nerve (FIG. 4F) was calculated by ImageJ.
  • nanoporous materials from adsorption and desorption isotherms. Studies in Surface Science and Catalysis 2000, 129, 597-606.
  • Articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include“or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context.
  • the disclosure of a group that includes“or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

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Abstract

L'invention concerne des compositions comprenant des médicaments (par ex. des bloqueurs des nerfs ou des facteurs de croissance nerveuse) encapsulés dans des nanoparticules (par ex., des nanoparticules ayant un diamètre inférieur à 70 nm). Les nanoparticules décrites dans la description peuvent pénétrer dans le nerf et administrer les médicaments dans le nerf. L'invention concerne également des procédés pour bloquer un signal nerveux et/ou induire une anesthésie locale, et des procédés de promotion de la neurorégénération.
PCT/US2018/064032 2017-12-06 2018-12-05 Nanoparticules destinées à pénétrer dans un nerf et leurs utilisations Ceased WO2019113184A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070122466A1 (en) * 2001-08-13 2007-05-31 University Of Pittsburgh Sphingomyelin liposomes for the treatment of hyperactive bladder disorders
WO2008070538A2 (fr) * 2006-12-01 2008-06-12 Anterios, Inc. Nanoparticules à entités amphiphiles
US20150079159A1 (en) * 2012-04-23 2015-03-19 The Children's Medical Center Corporation Formulations and Methods for Delaying Onset of Chronic Neuropathic Pain
US20150147378A1 (en) * 2008-05-30 2015-05-28 Rutgers, The State University Of New Jersy Copolymer-xerogel nanocomposites useful for drug delivery
US20170172923A1 (en) * 2014-07-22 2017-06-22 Cheolhee WON Composition for delivering bioactive material or protein, and use thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070122466A1 (en) * 2001-08-13 2007-05-31 University Of Pittsburgh Sphingomyelin liposomes for the treatment of hyperactive bladder disorders
WO2008070538A2 (fr) * 2006-12-01 2008-06-12 Anterios, Inc. Nanoparticules à entités amphiphiles
US20150147378A1 (en) * 2008-05-30 2015-05-28 Rutgers, The State University Of New Jersy Copolymer-xerogel nanocomposites useful for drug delivery
US20150079159A1 (en) * 2012-04-23 2015-03-19 The Children's Medical Center Corporation Formulations and Methods for Delaying Onset of Chronic Neuropathic Pain
US20170172923A1 (en) * 2014-07-22 2017-06-22 Cheolhee WON Composition for delivering bioactive material or protein, and use thereof

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