NANOPARTICUES FOR NERVE PENETRATION AND USES THEREOF

REUATED APPUI CATION

This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/595566, filed December 6, 2017, and entitled“NANOPARTICLES FOR NERVE PENETRATION AND USES THEREOF,” the entire contents of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This disclosure was made with government support under grant number GM 073626 awarded by the National Institutes of Health. The government has certain rights in this invention.

BACKGROUND

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.

SUMMARY

Provided herein are compositions and method for delivering a nerve blocker(s) or nerve growth factor(s) into a nerve using a nanoparticle(s). The nanoparticle(s) described herein can penetrate into the nerve, resulting in highly efficacious nerve blockage with low toxicity. In some embodiments, the nanoparticle is a silica nanoparticle (e.g., a hollow silica nanoparticle). In some embodiments, the penetration of the nanoparticle into the nerve is size-dependent. For example, the nanoparticle may be less than 70 nm in diameter (e.g., as measured by

transmission electron microscopy (TEM)). In some embodiments, 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). In some embodiments, the nerve blocker is a botulinum toxin. In some embodiments, the nerve blocker is a site 1 sodium channel blocker (S1SCB). Non-limiting examples of SlSCBs include neosaxitoxin, saxitoxin, decarbamoyl STX, tetrodotoxin, and gonyautoxin. In some embodiments, the S1SCB is tetrodotoxin (TTX).

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the SN has a negatively charged surface.

In some embodiments, 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.

In some embodiments, the silica nanoparticle is a hollow silica nanoparticle (HSN). In some embodiments, 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.

In some embodiments, the S1SCB is loaded in the hollow core.

Other aspects of the present disclosure provide compositions comprising a nerve growth factor encapsulated in a silica nanoparticle (SN).

Also provided herein are 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.

In some embodiments, the encapsulated drug enters the nerve. In some embodiments, the nanoparticle enhances the entry of the drug into the nerve, compared to free drug.

In some embodiments, the nanoparticle is selected from the group consisting of silica nanoparticle(s), nanoparticle(s) coated with silica, gold nanoparticle(s), iron based

nanoparticle(s), rare-earth based upconversion nanoparticle(s), quantum dot(s), nano diamond, and polymer nanoparticle(s). In some embodiments, the compositions described herein further comprise a pharmaceutically acceptable carrier.

Other 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.

In some embodiments, the composition is administered locally at a nerve of the subject. In some embodiments, the nerve is a peripheral nerve. In some embodiments, the peripheral nerve is a sciatic nerve.

In some embodiments, the SN and the encapsulated nerve blocker enters the nerve. In some embodiments, the SN enhances the entry of the nerve blocker into the nerve, compared to free nerve blocker. In some embodiments, the nerve blocker is released into the nerve from the SN. In some embodiments, the nerve blocker blocks a neuronal signal. In some embodiments, the SN enhances the rate of nerve blockade, compared to free nerve blocker. In some embodiments, a lower dose of nerve blocker is needed for the same rate of nerve blockade, compared to free nerve blocker. In some embodiments, the SN prolongs the nerve blockade by the nerve blocker, compared to free nerve blocker. In some embodiments, the nerve blocker encapsulated in the SN is not toxic to the nerve.

In some embodiments, the method further comprises administering to the subject an effective amount of a second nerve blocker. In some embodiments, the second nerve blocker is an amino-amide and/or amino-ester local anesthetic. In some embodiments, the second nerve blocker is lidocaine, tetracaine, capsaicin, and analogs thereof.

In some embodiments, the method further comprises administering to the subject an effective amount of an adjuvant. In some embodiments, the adjuvant is a glucocorticoid receptor agonist, an adrenergic agonist, or a vasoconstrictor. In some embodiments, the glucocorticoid receptor agonists is dexamethasone. In some embodiments, the vasoconstrictor is epinephrine or dexmedetomidine.

In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a rodent. In some embodiments, the rodent is a mouse or a rat.

Other 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. In some embodiments, an axonal surface of the neuron contacts the SN in the composition. In some embodiments, the encapsulated nerve blocker is released and contacts the neuron. In some embodiments, the nerve blocker blocks a neuronal signal. In some embodiments, the SN prolongs the neuronal signal blockade by the nerve blocker. In some embodiments, the nerve blocker encapsulated in the SN is not toxic to the neuron.

In some embodiments, the neuron is in vitro. In some embodiments, the neuron is ex vivo. In some embodiments, the neuron is in vivo.

In some embodiments, the neuron is from a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a rodent. In some embodiments, the rodent is a mouse or a rat.

Yet other aspects of the present disclosure provide methods of promoting

neuroregeneration, 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.

In some embodiments, the encapsulated nerve growth factor enters the nerve. In some embodiments, the nanoparticle enhances the entry of the nerve growth factor into the nerve, compared to free nerve growth factor.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various FIGs. is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

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.

FIG. 2. Cumulative release of TTX from TTX- HSN ₃₀ in vitro. Data are means with SDs (n=4).

FIGs. 3A to 3D. Sciatic nerve blockade with free TTX and TTX- HSN30. Effect of TTX dose on the median duration of sensory nerve blocks (FIG. 3A), the frequency of successful blocks (FIG. 3B), the frequency of nerve block in the un-injected (contralateral) extremity (FIG. 3C), and of death (FIG. 3D). Data in FIG. 3A are medians with interquartile ranges. n=4 per experimental group. 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). The dotted line indicated the nerve. The scale bars are 200 pm. n=4 per experimental group. FIG. 4E shows the relationship between normalized mean fluorescence intensity (a metric of fluorescence) and normalized distance from the surface of the nerve in animals injected with FITC- SN10, FITC- HSN30, FITC- SN70 or free fluorescein, n=4. Tissues were harvested 4 hours after injection. The method of determining the distance between the fluorescence area and the surface of the nerves is shown in Supporting

Information. The fluorescence intensity at the nerve surface (periphery) was set to 1 and all values normalized to that. The diameter of nerve was set to 1 and all distances from the nerve surface normalized to that. FIG. 4F shows the mean fluorescence +SD in nerve after treatment with FITC- HSN30 or free FITC, *p<0.005; n = 4.

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. The scale bars are 200 pm. n=4 per experimental group.

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. The scale bars are 100 pm. All animals were injected with 4 pg TTX, free or in TTX-HSN30 formulation. n=4 per experimental group.

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

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. 13B shows representative bright field and fluorescence (irradiated with a 365 nm UV lamp) photographs of sciatic nerve and surrounding muscles harvested 4 hours after injection of FITC- HSN30. Untreated tissues are provided for comparison. n=4 per experimental group.

FIG. 14. Measurement of distribution of fluorescence across the nerve. See

accompanying text in Methods.“Fl ...” indicated fluorescence area,“D” indicated the distance between an fluorescence area to the initial point.

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.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Some aspects of the present disclosure relate to 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. In some embodiments, the drug for the nervous system is a nucleic acid (e.g., DNA, mRNA, siRNA, miRNA, or shRNA) or a polypeptide. In some embodiments, the drug(s) for the nervous system may be loaded in a smaller nanoparticle before being encapsulated in the nanoparticles described herein.

In some embodiments, the drug for the nervous system is a drug for a peripheral nervous system. Non-limiting, 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

physostigmine), 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.

In some embodiments, the drug for the nervous system is a drug for a central nervous system. Non-limiting, 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, neuronal potassium channel openers, oxazolidinedione anticonvulsants, pyrrolidine anticonvulsants, succinimide anticonvulsants, triazine anticonvulsants), antiemetic/antivertigo agents (e.g., 5HT3 receptor antagonists, anticholinergic antiemetics, miscellaneous antiemetics, NK1 receptor antagonists, phenothiazine antiemetics), antiparkinson agents (e.g., anticholinergic antiparkinson agents, dopaminergic antiparkinsonism agents), anxiolytics, sedatives, and hypnotics (e.g., barbiturates, benzodiazepines, miscellaneous anxiolytics sedatives and hypnotics), cholinergic agonists, cholinesterase inhibitors, CNS stimulants, drugs used in alcohol dependence, general anesthetics, miscellaneous central nervous system agents, and muscle relaxants (e.g., neuromuscular blocking agents, skeletal muscle relaxant combinations, skeletal muscle relaxants, VMAT2 inhibitors).

A“nanoparticle,” as used herein, 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-glycolide) PLGA, or poly(cyanoacrylate)PCA). In some embodiments, the nanoparticle used in the present disclosure is a silica nanoparticle. In some embodiments, the nanoparticle used in the present disclosure is a hollow silica nanoparticle.

A“silica nanoparticle,” as used herein, refers to a silica (silicon dioxide)-based nanosphere structure. In some embodiments, 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. In some embodiments, the silica nanoparticle has a hollow core in the center. Such a silica nanoparticle is referred to as is a“hollow silica nanoparticle (HSN).”

Methods of producing silica nanoparticles are described in the Examples herein and are known in the art. For example, in some embodiments, silica nanoparticles can be synthesized by reacting tetraethyl orthosilicate with a template made of micellar rods. In some

embodiments, 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). In these methods, tetraethyl orthosilicate is also used with an additional polymer monomer as a template.

One example of 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. In the 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

microemulsion before adding sodium fluoride to start the condensation process. The non-ionic surfactant is burned away to produce empty pores, increasing the surface area and altering the surface characteristics of the resulting particles, allowing for much greater control over the physical properties of the material. Recent publication describe further improved methods of preparing silica nanoparticles, e.g., Hou et ah, J. Am. Chem. Soc. 128, 6447-7453, 2006; and Yu et al., J. Colloid Interf. Sci. 376, 67-75, 2012, incorporated herein by reference.

In some embodiments, the ability of a nanoparticle to penetrate into the nerve depends to its size. One skilled in the art is familiar with methods for determining the size of a nanoparticle (e.g., a silica nanoparticle). Such methods include, without limitation, transmission electron microscopy (TEM) and dynamic light scattering (DLS).

“Transmission electron microscopy (TEM)” 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.

“Dynamic light scattering (DLS)” is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution. In the scope of DLS, temporal fluctuations are usually analyzed by means of the intensity or photon auto-correlation function (also known as photon correlation spectroscopy or quasi elastic light scattering). In the time domain analysis, the autocorrelation function (ACF) usually decays starting from zero delay time, and faster dynamics due to smaller particles lead to faster decorrelation of scattered intensity trace. DLS measurements can be equally well performed in the spectral domain. DLS can also be used to probe the behavior of complex fluids such as concentrated polymer solutions.

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. In some embodiments, a nanoparticle (e.g., silica nanoparticle) that can penetrate into the nerve has a diameter of less than 70 nm (e.g., as measured by TEM). For example, 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). In some embodiments, 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,

48 nm, 47 nm, 46 nm, 45 nm, 44 nm, 43 nm, 42 nm, 41 nm, 40 nm, 39 nm, 38 nm, 37 nm, 36 nm, 35 nm, 34 nm, 33 nm, 32 nm, 31 nm, 30 nm, 29 nm, 28 nm, 27 nm, 26 nm, 25 nm, 24 nm,

23 nm, 22 nm, 21 nm, 20 nm, 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, or less (e.g., as measured by TEM).

In some embodiments, the nanoparticle (e.g., silica nanoparticle) has a diameter of 10- 70 nm (e.g., as measured by TEM). For example, the nanoparticle (e.g., silica nanoparticle) may have a diameter of 10-70 nm, 10-65 nm, 10-60 nm, 10-55 nm, 10-50 nm, 10-45 nm, 10- 40 nm, 10-35 nm, 10-30 nm, 10-25 nm, 10-20 nm, or 10-15 nm, 15-70 nm, 15-65 nm, 15-60 nm, 15-55 nm, 15-50 nm, 15-45 nm, 15-40 nm, 15-35 nm, 15-30 nm, 15-25 nm, 15-20 nm, 20-70 nm, 20-65 nm, 20-60 nm, 20-55 nm, 20-50 nm, 20-45 nm, 20-40 nm, 20-35 nm, 20-30 nm, 20-25 nm, 25-70 nm, 25-65 nm, 25-60 nm, 25-55 nm, 25-50 nm, 25-45 nm, 25-40 nm, 25-35 nm, 25-30 nm, 30-70 nm, 30-65 nm, 30-60 nm, 30-55 nm, 30-50 nm, 30-45 nm, 30-40 nm, 30-35 nm, 35-70 nm, 35-65 nm, 35-60 nm, 35-55 nm, 35-50 nm, 35-45 nm, 35-40 nm, 40-70 nm, 40-65 nm, 40-60 nm, 40-55 nm, 40-50 nm, 40-45 nm, 45-70 nm, 45-65 nm, 45-60 nm, 45-55 nm, 45-50 nm, 50-70 nm, 50-65 nm, 50-60 nm, 50-55 nm, 55-70 nm, 55-65 nm, 55- 60 nm, 60-70 nm, 60-65 nm, or 65-70 nm (e.g., as measured by TEM). In some embodiments, the nanoparticle (e.g., silica 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

embodiments, the nanoparticle (e.g., silica nanoparticle) has a diameter of 30 nm (e.g., as measured by TEM). In some embodiments, the nanoparticle (e.g., silica nanoparticle) has a diameter of 28 nm (e.g., as measured by TEM).

In some embodiments, the nanoparticle (e.g., silica nanoparticle) has a diameter of less than 80 nm as measured by DLS. For example, the nanoparticle (e.g., silica nanoparticle) may have a diameter of less than 80 nm, less than 75 nm, 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 as measured by DLS. In some embodiments, 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, 36 nm, 35 nm, 34 nm, 33 nm, 32 nm, 31 nm, 30 nm, 29 nm, 28 nm, 27 nm, 26 nm, 25 nm, 24 nm, 23 nm, 22 nm, 21 nm, 20 nm, 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, or less as measured by TEM DLS.

In some embodiments, the nanoparticle (e.g., silica nanoparticle) has a diameter of 20- 80 nm, as measured by DLS. Lor example, the nanoparticle (e.g., silica nanoparticle) may have a diameter of 20-80 nm, 20-75 nm, 20-70 nm, 20-65 nm, 20-60 nm, 20-55 nm, 20-50 nm, 20-45 nm, 20-40 nm, 20-35 nm, 20-30 nm, 20-25 nm, 25-80 nm, 25-75 nm, 25-70 nm, 25-65 nm, 25-60 nm, 25-55 nm, 25-50 nm, 25-45 nm, 25-40 nm, 25-35 nm, 25-30 nm, 30-80 nm, 30-75 nm, 30-70 nm, 30-65 nm, 30-60 nm, 30-55 nm, 30-50 nm, 30-45 nm, 30-40 nm, 30-35 nm, 35-80 nm, 35-75 nm, 35-70 nm, 35-65 nm, 35-60 nm, 35-55 nm, 35-50 nm, 35-45 nm, 35-40 nm, 40-80 nm, 40-75 nm, 40-70 nm, 40-65 nm, 40-60 nm, 40-55 nm, 40-50 nm, 40-45 nm, 45-80 nm, 45-75 nm, 45-70 nm, 45-65 nm, 45-60 nm, 45-55 nm, 45-50 nm, 50-80 nm, 50- 75 nm, 50-70 nm, 50-65 nm, 50-60 nm, 50-55 nm, 55-80 nm, 55-75 nm, 55-70 nm, 55-65 nm, 55-60 nm, 60-80 nm, 60-75 nm, 60-70 nm, or 60-65 nm, 65-80 nm, 65-75 nm, 65-70 nm, 70- 80 nm, 70-75 nm, or 75-80 nm, as measured by DLS. In some embodiments, the nanoparticle (e.g., silica 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.

In some embodiments, the nanoparticle is a silica nanoparticle that is porous. In some embodiments, the pores on the wall of the silica nanoparticle have a diameter of 10 nm or less than 10 nm. Lor example, 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. In some embodiments, 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. For example, 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.

In some embodiments, the silica nanoparticle is a hollow silica nanoparticle. In some embodiments, the hollow core has a diameter of 2-60 nm. For example, 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, 15-55 m, 15-50 nm, 15-45 nm, 15-40 nm, 15-35 nm, 15-30 nm, 15-25 nm, 15-20 nm, 20-60 nm, 20-55 m, 20-50 nm, 20-45 nm, 20-40 nm, 20-35 nm, 20-30 nm, 20-25 nm, 25-60 nm, 25-55 m, 25-50 nm, 25- 45 nm, 25-40 nm, 25-35 nm, 25-30 nm, 30-60 nm, 30-55 m, 30-50 nm, 30-45 nm, 30-40 nm, 30-35 nm, 35-60 nm, 35-55 m, 35-50 nm, 35-45 nm, 35-40 nm, 40-60 nm, 40-55 m, 40-50 nm, 40-45 nm, 45-60 nm, 45-55 m, 45-50 nm, 50-60 nm, 50-55 nm, or 55-60 nm. In some embodiments, the hollow core has a diameter of 10-20 nm. For example, the hollow core may have a diameter of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm. In some embodiments, 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)). 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. In a non-limiting example, when a porous and hollow silica nanoparticle is 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) can be loaded into a nanoparticle efficiently (e.g., with a loading efficiency of at least 30%). 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.

In some embodiments, 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). In some embodiments, 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, in some embodiments, 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 group, such as falicain, dyclonine and related local anesthetic compounds; and (viii) the aminoether group, such as pramoxine, dimethisoquine, and related local anesthetic compounds.

In some embodiments, 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.

In some embodiments, 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

applications. The term“botulinum toxin,” as used herein, encompasses any functional fragments or variants of botulinum toxins.

In some embodiments, 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) are classified according to 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). In excitable cells such as neurons, myocytes, and certain types of glia, 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:

U24693), NaVl.5 (Genebank ID: AJ310893), NaVl.6 (Genebank ID: AB027567), NaVl.7 (Genebank ID: X82835), NaVl.8 (Genebank ID: AF117907), NaVl.9 (Genebank ID:

AF 126739), and NaX (Genebank ID: M91556), all of which are voltage-gated sodium channels.

As an important element in the transduction of neuronal signals and nerve excitability, sodium channels serve as specific targets for many 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. Reu. Biochem. 66,953-985,1986; Couraud et ah, Handbook of Natural Toxins (Tu, A. T., ed) Vol. 2, p. 659-678,1984; and Baden et ah, FASEB J. 3,1807-1817,

1989, incorporated herein by reference).

The nerve blockers described herein, in some embodiments, are site“site 1 sodium channel blockers (SlSCBs)” because they bind to receptor site 1 of a sodium channel. Non limiting examples of SlSCBs include: neosaxitoxin, saxitoxin, decarbamoyl STX, tetrodotoxin, and gonyautoxin. In some embodiments, 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. In some embodiments, 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.

“Neosaxitoxin” is included, as other saxitoxin-analogs, in a broad group of natural neurotoxic alkaloids, commonly known as the paralytic shellfish toxins (PSTs). The parent compound of PSTs, saxitoxin (STX), is a tricyclic perhydropurine alkaloid, which can be substituted at various positions, leading to more than 30 naturally occurring STX analogues.

All of them are related imidazoline guanidinium derivatives. 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 (GTX) 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).

Ingestion of gonyautoxins through consumption of mollusks contaminated by toxic algae can cause a human illness called paralytic shellfish poisoning (PSP). As part of the group of satoxins, 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

membrane. These channels are responsible for initiating the action potentials, after the synapse. The binding of PSP toxins prevents the generation and propagation of these potentials and hence blocks the synaptic function.

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.

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.

There are many types of specialized neurons. 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) 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. Within a nerve, 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. Finally, the entire nerve is wrapped in a layer of connective tissue called the epineurium. Within the endoneurium, the individual nerve fibers are surrounded by a low-protein liquid called endoneurial fluid.

In some embodiments, the composition described herein further comprises a pharmaceutically acceptable carrier. The phrase“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. The phrase “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.). The term“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. Some examples of 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 ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.

The pharmaceutical 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. The term "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. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, 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.

For topical administration, 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. Many types of 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. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,832,253, and 3,854,480. In addition, pump- based hardware delivery systems can be used, some of which are adapted for implantation.

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions (e.g., chronic pain). Long-term release, are used herein, 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.

In some embodiments, 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). Alternatively, 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. The cyclic Psap peptide and/or the

pharmaceutical 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.

Other drug delivery systems for local anesthetics have been described, e.g., in de Paula et ah, Recent Pat Drug Deliv Formul. 2010 Jan;4(l):23-34, incorporated herein by reference.

Other 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

bloodstream. Local anesthesia can also be applied directly to the skin or mucous membranes as a liquid or gel.

Methods of promoting neuronal regeneration are also provided. Such methods 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.

In some embodiments, the composition is administered locally at a nerve. “Administer at 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. In some embodiments, 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

axons/neurons directly. In some embodiments, 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.

The composition described herein may be administered to any nerve. In some embodiments, 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.

In some embodiments, 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).

In some embodiments, 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. For example, 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. In some embodiments, 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. In some embodiments, 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. For example, 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. In some embodiments, 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.

In some embodiments, 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. For example, 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.

In some embodiments, a lower dose (e.g., at least 20% lower) of nerve blocker is needed to achieve the same level of nerve blockade when the nanoparticle with the

encapsulated nerve blocker is administered, compared to when a free nerve blocker is administered. For example, 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. In some embodiments, 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.

In some embodiments, 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. For example, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, the second nerve blocker is an amino-amide and amino-ester local anesthetic. In some embodiments, the second nerve blocker is lidocaine, tetracaine, capsaicin, and analogs thereof. In some embodiments, 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). For example, 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. In some embodiments, 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).

The 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). For example, 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).

Other 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. When an axon is damaged, 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. In the CNS, synaptic stripping occurs as glial foot processes invade the dead synapse.

In some embodiments, 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. For example, when a nerve growth factor encapsulated in a nanoparticle described herein 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. For example, when a nerve growth factor encapsulated in a nanoparticle described herein 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.

In some embodiments, 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

encapsulated nerve blocker is administered, 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. For example, when a nerve growth factor encapsulated in a nanoparticle described herein 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. For example, when a nerve growth factor encapsulated in a nanoparticle described herein 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. In some embodiments, 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)). In some embodiments, 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.

In some embodiments, the subject is a companion animal (a pet). “A companion animal,” as used herein, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is a prophylactic treatment. In some embodiments, an effective amount is the amount of a compound described herein in a single dose. In some embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses. When 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. Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the Examples below. The Examples are intended to illustrate some of the benefits of the present disclosure and to describe particular embodiments, but are not intended to exemplify the full scope of the disclosure and, accordingly, do not limit the scope of the disclosure.

EXAMPLES

Tetrodotoxin (TTX) 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 (SlSCBs), 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. ² Efforts to overcome the barriers have included disrupting them by osmotic shock ³ or permeabilizing them with chemical permeation enhancers. ⁴ Another approach has been to use sustained release systems, extending the period during which the nerve is exposed to SlSCBs, and maintaining a high local concentration. ⁵ To address these issues, it was hypothesized that nanoencapsulation could enhance S1SCB penetration into nerve and provide extended duration of nerve block. 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. In animals injected with fluorescently labeled HSN, 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. These results demonstrated that HSN could deliver TTX into nerve, enhancing efficacy while improving safety.

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. HSN possessed a negatively charged surface with a zeta potential of -9.8 mV, suggesting its possibility for loading cation TTX. Below, all 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. 8), respectively, demonstrating that the TTX was adsorbed inside the pores of the HSNs. 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 ).

Loading efficiency and TTX release profile of TTX- HSN 30

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.

To measure the loading efficiency of TTX, the obtained 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.

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).

Cytotoxicity

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.

Nerve blockade with TTX and TTX- HSN ₃₀

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. In this animal model it is not possible, due to limiting toxicity, to achieve such long nerve blocks with TTX in the absence of sustained release, ^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 .

Silica nanoparticles distribution

The resemblance of some characteristics of nerve block from TTX- HSN ₃₀ to that from TTX with chemical permeation enhancers led to investigating the possibility that the HSN ₃₀ were enhancing TTX flux into nerve. To evaluate that possibility, 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. In the animals injected with FITC- HSN ₃₀ , FITC fluorescence was observed in the nerve (FIGs. 4A and 4B), whereas no FITC fluorescence was observed in the nerve in animals injected with the same dose of free FITC (FIG. 4C) or in untreated legs (FIG. 4D). Quantitative analysis showed the fluorescence signal penetrated deep into the nerve in animals injected with FITC- HSN ₃₀ (FIG. 4E and FIG. 14), while it remained at the nerve perimeter in animals injected with free FITC. The total fluorescence intensity inside the nerve perimeter was much higher in animals injected with FITC- HSN ₃₀ than in those injected with free FITC (FIG. 4F, p<0.005). These results demonstrated that HSN ₃₀ can cross the perineurial barrier and penetrate into the nerve.

To evaluate the influence of particle size on penetration into nerve, 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). At a normalized distance of 0.1 (one tenth of the diameter of the nerve), the fluorescent intensity in the FITC- SN10 and FITC-HSN30 groups were 72+4.3% and 21+4.0% of the fluorescent intensity at the surface of the nerve, respectively, while that of the FITC-SN70 was only 4+0.5% (both p<0.005, compared to FITC-SN10 and FITC-HSN30, respectively), similar to that obtained with free FITC (p=0.48). These result demonstrated that the nerve penetrating ability of silica nanoparticles was highly size-dependent.

Tissue reaction

Animals injected with TTX and TTX- HSN ₃₀ were euthanized 4 and 14 days after injection (n=4 at each time point), and the sciatic nerve and surrounding tissues were harvested, sectioned for histology and stained with hematoxylin-eosin (H&E). These time points are useful in that they can capture acute and chronic inflammatory responses to injected materials.

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.

Since H&E-stained are relatively insensitive for identifying nerve injury, toluidine blue-stained Epon-embedded sections of the sciatic nerve were obtained in animals injected with TTX- HSN30. Nerves from those animals were normal in appearance (FIG. 6).

Table 1. Myotoxicity and inflammation from free TTX and TTX- HSN30. The range of scores is 0-4 for inflammation and 0-6 for myotoxicity. P values were determined by Mann-Whitney U test Conclusion

In 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. In that view, 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

prolongation of nerve block. The penetrating ability of silica nanoparticles was size-dependent. The sustained release properties of HSNs ₃ o also contributed to the extension of nerve block and enhanced safety by slowing release. This ability to penetrate peripheral nerve is believed to be useful in delivering a range of therapeutics, including combinations of drugs that enhance the activity of local anesthetics ⁴¹³ .

Materials and Methods

All the starting materials were obtained from commercial suppliers and used as received. Pluronic F108, F127, NaAc.3H20, cetyltrimethylammonium chloride solution 25 wt. % in H20 (CTAC), l,3,5-Trimethylbenzene, tetraethyl orthosilicate (TEOS),

dimethyldimethoxysilane (DMDMS), fluorescein isothiocyanate (FITC), (3- Aminopropyl)triethoxysilane (APTES) and HC1 were purchased from Sigma-Aldrich (St. Louis, MO). Deionized water was used throughout the experiments.

Synthesis of hollow silica nanospheres (HSN ₃₀ )

In a typical synthesis, ⁷ 1.0 g of pluronic F108, 1.0 g of l,3,5-trimethylbenzene, and 30 mL 2.0 M HC1 were mixed and stirred for 6 hours at room temperature to form a homogeneous emulsion. Then 1.157 mL of TEOS was added to the homogeneous emulsion under vigorous stirring. After 6 hours, 0.537 mL of DMDMS was added and the reaction was continued for another 48 hours. Then the milky mixture was dialyzed with a semipermeable membrane (molecular-weight cutoff, MWCO = 3,500) in 5 L water for 72 hours, and the water was refreshed every 24 hours. The dialysate was evaporated at 80 °C, and the obtained white powder was calcined at 350 °C for 5 hours to get the final HSN ₃₀ .

Synthesis of FITC-HSN 30, FITC-SN 10, and FITC-SN70 The FITC-conjugated hollow silica nanoparticles (FITC-HSN30) were synthesized by a similar strategy to that for HSN30 except that a mixture of DMDMS and FITC-APTES (total volume is 0.537 mL, volume ratio is 1:1) was used instead of DMDMS. Then the sample was dialyzed with a semipermeable membrane (molecular-weight cutoff, MWCO = 3,500) in 5 L water for 72 hours, and the water was refreshed every 24 hours. The FITC-HSN30 sample was not calcined. FITC-APTES was synthesized by mixing FITC (10 mg) and APTES (1 mL) for 4 hours, and the mixture was used for the preparation of FITC-HSN30 directly.

The FITC-SN10 was synthesized as described. ¹²²¹ 0.6 g F127 was dissolved in 10 mL HC1 (0.85 M) with stirring at room temperature for 2 hours. Then 1.0 g TEOS was added to the homogeneous solution. The mixture was stirred at room temperature for 3 hours, then 0.05 g DEDMS and 0.05 g FITC-APTES were added. Stirring was continued at room temperature for 20 hours. Then the mixture was dialyzed with a semipermeable membrane (molecular- weight cutoff, MWCO = 3,500) in 5 L water for 72 hours, and the water was refreshed every 24 hours. The FITC-SN10 sample was used for animal study without calcining.

In a typical synthesis for FITC-SN ₇ o, ^12b 53.4 g of water, 6.24 g of CTAC (25 wt.% solution), and 0.3 g of NaAc.3H ₂ 0 were mixed and stirred in a silicon oil bath at 80°C for 2 hours. Then 4.35 mL of TEOS was added into the above mixture dropwise under steadily stirring at 400 RPM. The solution was stirred for another 20 hours before adding 200 pL DEDMS and 200 pL FITC-APTES. The solution was stirred for 24 hours then cooled to room temperature and centrifuged at 18,000 RPM for 10 minutes to isolate the products from the suspension. After washing with water 3 times, the sample was dispersed into methanol and shaken at room temperature over night to remove CTAC. The final product was dispersed in water and used in animal studies.

Characterizations of silica nanoparticles.

Transmission electron microscopy: A 10 pL aliquot of the nanoparticle solution was deposited on a copper grid coated by a carbon film. After 2 minutes, excess solution was blotted with filter paper. The sample was dried at room temperature and then imaged on a Tecnai G2 Spirit BioTWIN transmission electron microscope, operating at 100 kV.

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). For dynamic light scattering, 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

The loading ofTTX in HSN30 (TTX-HSN30)

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.

To measure the loading of TTX in HSN30, the obtained 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). The loading efficiency of TTX in HSN30 was calculated as: Loading efficiency = (total TTX-free TTX)/total TTX.

Investigation of the release of TTX

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).

Cell Viability

Cell culture of C2C12 mouse myoblasts (American Type Culture Collection (ATCC) Manassas, VA) and PC 12 rat adrenal gland pheochromocytoma cells (ATCC, Manassas, VA) was performed as reported . ¹⁶ In brief, 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. 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.

Animal care

Adult 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

Laboratory Animal of the US National Research Council.

Sciatic Nerve Blockade and N eurobehavior al Testing

Under isoflurane-oxygen anesthesia, 0.3 mL of test solution was injected at the sciatic nerve of the left leg as described. ¹⁷ Thermal nociception was assessed by a modified hotplate test. The hindpaws were exposed sequentially to a 56 °C hot plate (Stoelting Co., Wood Dale, IL). The time until the animal withdrew its hindpaw (termed latency) was measure with a stopwatch. If the rat kept its paw on the hot plate longer than 12 seconds, it was removed to avoid injury. If a latency longer than 7 seconds occurred, the animal was considered to have an effective/successful nerve block. (Seven seconds is the midpoint between ~2 seconds [baseline thermal latency] and 12 seconds [maximal latency]). The duration of nerve blockade was calculated as the time until thermal latency returned to 7 seconds. The right (uninjected, contralateral) leg was also tested as control for systemic drug distribution, i.e. systemic toxicity would be reflected in an increase in the latency in the right hindpaw. Each hindpaw was tested three times at each time point and the average was recorded.

Tissue Harvesting and Histology

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,

5. hemifascicular regeneration, 6. holofascicular regeneration.

To evaluate the neurotoxicity of the TTX-HSN30 formulations, 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.

Silica nanoparticles distribution

Under isoflurane-oxygen anesthesia, 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. After 4 hours, 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. Afterwards, one drop of ProFong Gold Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA) was applied onto each slide. A coverslip was placed, and the slides imaged by Zeiss FSM 710 multiphoton confocal microscopy.

In order to measure the depth that the injected particles penetrated into the nerve, quantitative analysis was performed on the confocal fluorescence images across the diameter of the nerve. Since silica nanoparticles were injected at the periphery of the nerve (in the loose connective tissue between muscle and nerve), the fluorescence intensity was highest there, at the surface of the nerve. That location, indicated by a dot at the surface of the nerve in FIG. 14, served as the starting point to measure the diameter (solid line across the figure) of the nerve. The nerve was divided into 10 fluorescence areas (labeled Fl to F 10) by yellow lines equally separated by distances Dl ...D10 along the diameter of the nerve. (Note the distances D could be different from nerve to nerve if the nerves differed in diameter.) 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.

Statistical Analysis

The majority of data were reported as medians with 25th and 75th percentiles (n=4 for all experiments), and were compared using the Mann- Whitney U test. This method was selected because the data were ordinal in nature (inflammation scores, myotoxicity scores), or because the data were not normally distributed (e.g. neurobehavioral data with many zero- duration blocks. TTX release and microscopy fluorescent imaging data were reported as means with standard deviations. Statistical comparisons were made with t-tests. Statistical significance was defined as a p< 0.05. Statistical analyses were performed with Origin 8.0 software (Origin Lab Corp. Northampton, MA)

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All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

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. It is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps.

It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well.

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.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

Where websites are provided, URL addresses are provided as non-browser-executable codes, with periods of the respective web address in parentheses. The actual web addresses do not contain the parentheses. In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.