Ultrasound-triggered anesthetic compositions and methods of use

Abstract

Pharmaceutical compositions comprising hydrogen bonded organic framework (HOF) molecules and an anesthetic (e.g., bupivacaine) are provided herein. Drug release from the HOF can be induced using ultrasound such as focused ultrasound (FUS) in an anesthesia or analgesia procedure, such as treatment of pain after anesthesia or to inhibit a peripheral nerve.

Description

DESCRIPTIONULTRASOUND-TRIGGERED ANESTHETIC COMPOSITIONS AND METHODS OF USEBACKGROUND

[0001] This application claims the benefit of United States Provisional Patent Application Nos. 63 / 755,407, filed February 7, 2025, and 63 / 860,298, filed August 8, 2025, the entirety of each of which is incorporated herein by reference.

[0002] The invention was made with government support under Grant No. R35 GM147408 awarded by the National Institutes of Health and Grant No. DMR2340964 awarded by the National Science Foundation. The government has certain rights in the invention.1. Field

[0003] The present invention relates generally to the field of molecular biology and medicine. More particularly, it concerns compositions that can be used to treat pain via release of an anesthetic by application of ultrasound.2. Description of Related Art

[0004] Traumatic nerve injuries cause severe pain and functional impairments. Furthermore, chronic pain is a significant clinical challenge, often requiring long-term opioid use, which carries risks of addiction and adverse side effects. Chronic pain causes significant problems clinically, including the risk of addiction to opioids. Clearly, there is a need for new methods for treating pain.

[0005] Focused ultrasound (FUS) techniques have been used in various clinical therapeutic contests including drug activation, but significant limitations for these approaches exist. FUS can provide effective tissue penetration depth, millimetric spatial resolution, and a good safety profile (Huo et al., 2021; Wang et al., 2018; Bar-Zion et al., 2021). Recent advancements have introduced acoustically-triggered microbubbles, nanoemulsions and liposomes as possible candidates for controlled medication release in mechanotherapy and local anaesthesia (Rwei etal., 2017; Airan et al., 2017; Chen and Hwang, 2013a; Kiessling et al., 2014). However, the ultrasound-triggered drug release mechanism employed by these systems primarily relies on the occurrence of undetermined breaking events on nanoparticle- 1 - 4909-0910-3750, v. 3membranes, consequently limiting their capacity to attain precise and programmable drug control (Rwei et al., 2017; Cravotto et al., 2013; Zhuang et al., 2023). Additionally, these systems also often suffer from low drug loading capacity. Herrmann et al. tested ultrasound for selectively targeting the scission of labile covalent or non-covalent bonds to activate specific drugs (Huo et al., 2021; Shi et al., 2020). Such mechanochemical activations involve subjecting polymer frameworks to ultrasound-induced mechanical stress and strain, which in turn act on some specific chemical bonds to induce targeted scission and rearrangement spontaneously (Cravotto et al., 2013; Shi et al., 2020; Huo et al., 2022; Sha et al., 2022; Ghanem et al., 2021; Akbulatov et al., 2017). Ultrasound mechanochemical activation can exhibited selectivity and molecular programmability (Cravotto et al., 2013; Ghanem et al., 2021; Akbulatov et al., 2017). However, the robust covalent bonding and excessive non-covalent bonds present in each chain of polymer frameworks often lead to high cohesive energy. Consequently, these frameworks typically require a high ultrasound power density to facilitate drug activation, resulting in a prolonged responsive latency on the order of hours. This extended latency is not conducive to the precise modulation of cellular events (Huo et al., 2021; Shi et al., 2020; Shi et al., 2020). The topologically complex nature of polymer frameworks poses challenges in establishing a comprehensive theory to visualize the intrinsic relationships between scission efficiency, framework molecular structure, and ultrasound power (Li et al., 2015; Chen et al., 2021).- 2 - 4909-0910-3750, v. 3SUMMARY

[0006] The present disclosure provides compositions and methods for delivery of an anesthetic comprised in utilize hydrogen-bonded frameworks (HOF) compositions, such as nanoparticles, that can be released from the HOF based on stimulation with FUS. The anesthetic may be released from the HOF nanoparticles due to stimulation with ultrasound such as FUS. For examples, the FUS may be applied during an anesthesia procedure such as for example a nerve block procedure, application to an injured peripheral nerve, during epidural anesthesia, after an injury, or after or during a surgery. In some aspects, the anesthetic may be a local anesthetic such as bupivacaine. The HOF may serve as mechanoresponsive vehicles to achieve ultrasound programmable drug loading and activation release of the anesthetic in a desired region of the body or nerve bundles, such as near peripheral nerves. As shown in the figures, HOF provided herein (e.g., HOF-TATB, HOF-BTB) exhibited beneficial loading, stability, release, and inhibition of neural activity both in vitro and in vivo (e.g., see FIGS. 1-5). These approaches were effective in inhibiting pain in vivo using a sciatic nerve injury model in rats (e.g., FIGS 5-6). The compositions provided herein can be used in a variety of methods of anesthesia, such as local anesthesia, and for treatment of pain and / or analgesia.

[0007] HOF nanoparticles capable of encapsulating local anesthetics such as lidocaine and bupivacaine are provided herein. These nanoparticles may be held together by multivalent hydrogen bonding and π-π stacking interactions, which may improve or ensure structural integrity under physiological conditions. Upon exposure to focused ultrasound (FUS), the framework may dissociate, releasing the encapsulated anesthetic at the target site in a controlled manner. The compositions, methods and systems described herein may thus provide spatial and temporal control over anesthetic release, offering a safer and non-invasive alternative for managing pain including chronic pain.

[0008] An aspect of the present disclosure relates to a method of delivering a drug to a mammalian subject comprising: (i) administering to the subject a hydrogen bonded organic framework (HOF) comprising an anesthetic, preferably a local anesthetic; and (ii) applying oscillating ultrasonic waves or focused ultrasound (FUS) to a portion of the mammalian subject, thereby releasing the anesthetic from the HOF. In some aspects, the anesthetic is a local anesthetic, such as, for example: bupivacaine, lidocaine, mepivacaine, ropivacaine, chloroprocaine, tetracaine, benzocaine, articaine, prilocaine, etidocaine, procaine, cinchocaine,- 3 - 4909-0910-3750, v. 3levobupivacaine, proparacaine, oxybuprocaine, amylocaine, prilocaine, hexylresorcinol, tapentadol, valenfaxine, levacetylmethadol, disopyramide, or a compound from Table 4.

[0009] The HOF may further comprise one or more additional therapeutic agent that is not an anesthetic such as, e.g., a NSAID such as ibuprofen. The local anesthetic may be selected from bupivicaine, mepivicaine, and chloroprocaine. In some aspects, the local anesthetic is bupivacaine. The HOF may comprise or consist of a compound of the formula:wherein: A is a C6-16 aromatic ring or a C3-15 heteroaromatic ring; and each of B, B', and B" are independently a Ce-i6 aromatic ring or a C3-15 heteroaromatic ring, wherein each may further comprise -S(O)2Ra, -NH2, or -C(O)Rawherein Rais hydroxy or amino. A may comprise 1, 2, 3 or 4 rings, preferably 1 ring. A may be an aromatic(c<<^i3) ring or a heteroaromatic(c=3-i2) ring. In some preferred embodiments, A is an aromatic(ce) or heteroaromatic(c3-5) ring. In some embodiments, A is:- 4 - 4909-0910-3750, v. 3In some embodiments, B, B', and B" are each independently substituted aryl(c<i2) or substituted heteroaryl c<i2). In some embodiments, B, B', and B" are each independently an aryl(c=6) or heteroaryl(C=3-5) ring. In some embodiments, B, B', and B” are eachindependently:In some embodiments, B, B', and B" are each independently:The HOF may have intrinsic cavities with sizes between about 1 A and about 500 A. In some preferred embodiments, B, B', and B" have the same structure.In some embodiments, B, B', and B" are each:-5 - 4909-0910-3750, v. 3COOHThe HOF may comprise a compound of the formula:wherein: X₁, X₂ and X₃ are each independently CH or N; R₁, R₂ and R₃ are each independently –S(O)₂Raor -C(O)Ra, wherein: Rais hydroxy or amino; R₄–R₁₅ are each independently –H, –F, –Cl, –Br, –I or –CH₃; or a pharmaceutically acceptable salt thereof. In some preferred embodiments, Xi, X2and X3 are each independently CH or N. In some preferred embodiments Ri, R2 and R3 are each independently -C(O)Ra, wherein Rais hydroxy or amino. In some embodiments, Ri, R2 and R3 are -C(O)NH2. In some embodiments, Ri, R2 and R3 are -COOH. In some embodiments, R4-R15 are -H. The compound may be further defined as:R2- 6 - 4909-0910-3750, v. 3In some embodiments, the compound is further defined as:The compound may be further defined as:R2In some embodiments, Xi is CH, X2 is CH, and / or X3 is CH. In some embodiments, Xi is N, X2is N, and / or X3 is N. In some embodiments, Ri, R2 and R3 are -COOH. The compound may be further defined as:- 7 - 4909-0910-3750, v. 3The compound may be further defined as:In some preferred embodiments, the drug or active pharmaceutical ingredient is encapsulated in the HOF. The HOF may encapsulate from about 1-40 wt% of the drug or active pharmaceutical ingredient relative to the weight of the HOF. In some preferred embodiments, the HOF is further defined as a nanoparticle, and wherein the drug or therapeutic is comprised within the nanoparticle. The nanoparticle may be about 40-800 nm in diameter or size (e.g., about 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 500, 600, 700, or 800 nm in diameter, or any range derivable therein). The mammalian subject may be a human. The hydrogen bonded organic framework (HOF) comprising the anesthetic may be injected into the mammalian subject near a peripheral nerve. The peripheral nerve may be the sciatic nerve, median nerve (e.g., wherein the subject has carpal tunnel syndrome), ulnar nerve (e.g., wherein the subject has cubital tunnel syndrome), radial nerve (e.g., wherein the subject has Saturday night palsy), peroneal nerve (e.g., wherein the subject has foot drop), femoral nerve, tibial nerve, or the lateral femoral cutaneous nerve (e.g., wherein the subject has meralgia paresthetica). The peripheral nerve may be the sciatic nerve. The method may comprise inducing local anesthesia. The method may comprise or be further defined as a nerve block procedure. The method may comprise inducing local anesthesia or analgesia. The hydrogen bonded organic framework (HOF) comprising the anesthetic may be injected into the- 8 - 4909-0910-3750, v. 3mammalian subject to treat post-surgery pain or pain associated with an injury or peripheral neuropathy. The injury is may be a peripheral nerve injury. The pain may be chronic pain.

[0010] Another aspect of the present disclosure relates to a pharmaceutical composition comprising: (i) a compound of the formula:wherein: X₁, X₂ and X₃ are each independently CH or N; R₁, R₂ and R₃ are each independently –S(O)₂Raor - C(O)Ra, wherein: Rais hydroxy or amino; R₄–R₁₅ are each independently –H, –F, –Cl, –Br, –I or –CH₃; or a pharmaceutically acceptable salt thereof; (ii) a local anesthetic, and (iii) an excipient. The local anesthetic may be bupivacaine, lidocaine, mepivacaine, ropivacaine, chloroprocaine, tetracaine, benzocaine, articaine, prilocaine, etidocaine, procaine, cinchocaine, levobupivacaine, proparacaine, oxybuprocaine, amylocaine, prilocaine, hexylresorcinol, tapentadol, valenfaxine, levacetylmethadol, disopyramide, or a compound from Table 4. The local anesthetic may be selected from the group comprising bupivicaine, mepivicaine, and chloroprocaine. In some aspects, the local anesthetic is bupivacaine. In some embodiments, Xi, X2 and X3 are each independently CH or N. In some embodiments, Ri, R2 and R3 are each independently -C(O)Ra, wherein Rais hydroxy or amino. In some embodiments, Ri, R2 and R3 are -C(O)NH2. In some embodiments, Ri, R2 and R3 are -COOH. In some embodiments, R4-R15 are -H. The compound may be further defined as:R2The compound may be further defined as:- 9 - 4909-0910-3750, v. 3CK. OHThe compound may be further defined as:The compound may be further defined as:(V).In some embodiments, Xi is CH, X2 is CH, and / or X3 is CH. In some embodiments, Xi is N, X2 is N, and / or X3 is N. In some embodiments, Ri, R2 and R3 are -C(O)NH2. In some embodiments, Ri, R2 and R3 are -COOH. The compound may be further defined as:- 10 - 4909-0910-3750, v. 3The compound may be further defined as:The local anesthetic may be encapsulated in the HOF. The HOF may encapsulate from about 1-40 wt% of the drug or active pharmaceutical ingredient relative to the weight of the HOF. In some embodiments, the HOF is further defined as a nanoparticle, and wherein the drug or therapeutic is comprised within the nanoparticle. The nanoparticle may be about 200-900 nm in size.

[0011] Yet another aspect of the present disclosure relates to a method of treating pain (e.g., chronic pain) or inducing local anesthesia in a subject or patient in need thereof comprising administering to the patient a pharmaceutically effective amount of a composition described above or herein. The subject or patient is preferably a mammal. The subject or patient may be a human. The pain may result from a peripheral nerve (e.g., damage to or pinching or a peripheral nerve). The peripheral nerve may be a sciatic nerve, median nerve, ulnar nerve, radial nerve, peroneal nerve, femoral nerve, tibial nerve, or the lateral femoral cutaneous nerve.

[0012] Another aspect of the present disclosure relates to a pharmaceutical composition comprising: (i) a hydrogen-bonded organic framework (HOF) compound preferably comprises one or more ^-conjugated polycarboxylic acids, preferably wherein the HOF compound is4909-0910-3750, v. 3HOF-TATB, HOF-BTB, HOF-101, or HOF-102 as described herein; and (ii) a local anesthetic, preferably wherein the local anesthetic is bupivacaine, lidocaine, procaine, tetracaine, ropivacaine, mepivacaine, articaine, chloroprocaine, or etidocaine; even more preferably wherein the local anesthetic is bupivacaine or lidocaine; optionally wherein the pharmaceutical comprises a pharmaceutically acceptable excipient. The local anethetic may be, hexylresorcinol, tapentadol, valenfaxine, levacetylmethadol, disopyramide, or a compound from Table 4.

[0013] A further aspect of the present disclosure relates to a method of treating pain in a mammalian subject comprising administering a pharmaceutical composition as described herein to the mammalian subject and preferably applying ultrasound to a region of the mammalian subject, wherein the administration is preferably via injection, such as injection near a peripheral nerve such as a sciatic nerve, or topical administration, wherein the ultrasound may comprise or consist of focused ultrasound (FUS) or sequential ultrasound pulses, where the FUS is preferably applied topically to the mammalian subject, wherein the mammalian subject may be a human, and wherein the method may optionally comprise a method of treating pain (e.g., such as post-operative pain, chronic pain, acute pain, hyperalgesia, or neuropathic pain), a neurological disease e.g., epilepsy, or neuroinflammation), or other disease.

[0014] Yet another aspect of the present disclosure relates to a method of producing a pharmaceutical composition comprising a hydrogen-bonded organic framework (HOF) compound and a therapeutic agent, comprising (i) using a machine learning algorithm to select the therapeutic agent for use with the HOF compound, wherein the machine learning algorithm comprises one of more parameters for the therapeutic agent (e.g., the volume of molecule, the radius of gyration, molecular weight, the number of rotatable bonds, the ratio of sp3 hybridization carbons, total polar surface area, logP, and / or the net charge at pH 7) and the HOF compound (e.g., number of hydrogen bonding sites, crystal port volume, crystal port size, crystal BET surface area, and / or logP); wherein the HOF compound preferably comprises one or more ^-conjugated polycarboxylic acids, preferably wherein the HOF compound is HOF-TATB, HOF-BTB, HOF-101, or HOF-102 as described herein; and wherein the therapeutic agent is preferably a small molecule (e.g., a FDA approved small molecule drug); wherein the pharmaceutical composition may optionally be the pharmaceutical composition of claim 1; and (ii) combining the HOF compound and the therapeutic agent to produce the pharmaceutical composition; wherein the therapeutic agent may be a local anesthetic, preferably wherein the- 12 - 4909-0910-3750, v. 3local anesthetic is bupivacaine, lidocaine, procaine, tetracaine, ropivacaine, mepivacaine, articaine, chloroprocaine, or etidocaine; even more preferably wherein the local anesthetic is bupivacaine or lidocaine. The local anesthetic may be hexylresorcinol, tapentadol. valenfaxine, levacetylmethadol, disop yramide, or a compound from Table 4.

[0015] As used herein the specification, “a’’ or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0016] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0017] Throughout this application, the tern “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0018] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0019] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] The terms “subject,” “host,” “patient,” and “individual” are used interchangeably herein to refer to any mammalian subject for whom therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.- 13 - 4909-0910-3750, v. 3

[0021] The term “effective amount” is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this application, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0022] An effective response of a patient or a patient’s “responsiveness” to treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder. Such benefit may include decrease in pain perception, regional anesthesia, or analgesia.

[0023] “Treatment” and “treating" refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.

[0024] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.- 14 - 4909-0910-3750, v. 3

[0026] FIGS.1 A-C: provide illustrations of the preparation of ultrasound mechanically responsive hydrogen-bonded organic frameworks (HOFs). (A) Four representative organic monomers and the self-assembled porous HOF structures: HOF-TATB, HOF-BTB, HOF-101 and HOF- 102. (B) Schematic illustration of the ultrasound mechanical stress triggered dissociation of HOFs. (C) shows the drug loading capacity of different HOF materials for lidocaine and bupivacaine.

[0027] FIGS. 2A-C: provide a depiction of size control and release testing of HOF-TATB nanoparticles. (A) Adjust the size of HOF-TATB nanoparticles by H3TATB concentrations. (B) dye / drug release from TATB HOF nanoparticles as a function of size after a single 10s pulse at 1.5 MHz and 3 MPa (C) The amount of dye / drug released per ultrasound stimulation.

[0028] FIGS. 3A-C: provide a depiction of versatile drugs loading of HOF-TATB nanoparticles and stability testing. (A) Versatile hydrophobic drugs were loaded in HOF nanoparticles by double-solvent method. (B) Long-term stability of hydrophobic drug (lidocaine)-loaded HOF nanoparticles in 10% FBS, measured as the remaining drug loading percentage over 40 d. (C) Short-term stability of hydrophobic drug (lidocaine)-loaded HOF nanoparticles in 50% FBS, measured as the remaining drug loading percentage over 50h.

[0029] FIGS. 4A-D: provide a depiction of ultrasound controlled lidocaine release from HOF-TATB nanoparticles and their in vitro inhibition of neural activity. (A) Ultrasound triggered drug release from the HOF-TATB nanoparticles. (B) Fluorescence images of the primary cortical neurons expressing hSyn:: GCaMP6s-WPRE-SV40. Scale bar: 20 pm. (C) Heat maps of normalized GCaMP6s fluorescence imaging from 50 cultured neurons expressing hM3D (Gq) under ultrasound inhibition of lidocaine (lido) release from TATB nanoparticles (1.5 MHz, 1.40 MPa, pulse 10s). (D) Normalized in vitro neuron spiking latency statistical analysis under FUS stimulation.

[0030] FIGS. 5A-E: depict ultrasound-controlled lidocaine release in HOF HOF-TATB nanoparticles for in vivo inhibition of pain. (A) Ultrasound-triggered local drug release in the sciatic nerve. (B) Illustration of the dorsal von Frey test in Dr. Bittner’s lab. (C) Longer ultrasound exposure (8 min > 4 min > 1 min) enhances drug release from HOF nanoparticles, increasing and prolonging VF thresholds, demonstrating precise ultrasound control of anesthesia. (D) Von Frey data analysis from four rats injected with TATB ©lidocaine HC1,- 15 - 4909-0910-3750, v. 3followed by an 8-minute ultrasound treatment over one week. (E) Same analysis for a 4-minute ultrasound treatment.

[0031] FIGS. 6A-F: depicts ultrasound controlled Bupivacine release in HOF HOF-TATB nanoparticles for in vivo inhibition of pain and the effects drug release on animal behaviors. (A) Von Frey mechanical data from 5 rats injected with TATB@bupivacaine, showing sustained hyposensitivity. (B) Statistical analysis of Von Frey thresholds in the same rats, confirming significant differences post-FUS. (C) Von Frey data from 3 control rats (TATB@RhB), showing no hyposensitivity. (D) Statistical analysis of Von Frey data from the same control rats, showing no significant changes. (E) Toe chewing assessments in rats treated with TATB@bupivacaine and TATB ©lidocaine, indicating reduced pain. (F) Sciatic functional index (SFI) assessments showing improved functional recovery in treated rats.

[0032] FIGS. 7A-E: Ultrasound-triggered delivery of local anesthetics using HOF nanoparticles and evaluation of drug loading efficiency, (a) Schematic illustration of focused ultrasound (FUS)-mediated delivery of local anesthetics (EAs) loaded in HOF nanoparticles to the sciatic nerve in mice. Upon ultrasound activation, FA molecules are released from the HOF carriers and interact with sodium channels to block neural signal transmission, (b) Schematic of drug loading mechanism into HOFs using a double-solvent method (DSM). Hydrophilic drugs diffuse into HOFs via aqueous solution, while hydrophobic drugs are loaded using organic solvent (CH₂Cl₂) to enable high loading efficiency. The chemical structures of lidocaine HC1 and bupivacaine HC1 are shown, (c) Chemical structures of four organic linkers (H₃TATB, H₃BTB, H₄TBAPy, and H₄PTTNA) used to synthesize HOF-TATB, HOF-BTB, HOF-101, and HOF-102, respectively, along with corresponding crystal structures, (d) Eoading efficiency of hydrophilic lidocaine HC1 and bupivacaine HC1 into different HOFs using a passive diffusion method in aqueous solution, (e) Eoading efficiency of the neutral forms of lidocaine and bupivacaine (free bases) into HOFs using the dual -solvent method (DSM), highlighting markedly higher loading efficiencies compared to the aqueous method. All data are presented as mean ± s.d., n = 3.

[0033] FIGS. 8A-F: Stability and ultrasound-responsive drug release of HOF-TATB nanoparticles loaded with various therapeutics via the dual-solvent method, (a) Long-term storage stability of HOF-TATB nanoparticles loaded with bupivacaine free base via the DSM, showing minimal drug loss over a 30-day period at 25 °C. (b) Serum stability of HOF-TATB @Lid evaluated in 50% fetal bovine serum (FBS) at 25 °C, revealing sustained- 16 - 4909-0910-3750, v. 3drug encapsulation over 48 hours with limited passive leakage, (c) Powder X-ray diffraction (PXRD) patterns of HOF-TATB@Bup before and after incubation in 50% FBS for 48 hours. The retention of diffraction peaks confirms the structural integrity of the framework under biological conditions, (d) Ultrasound-triggered release of bupivacaine from HOF-TATB nanoparticles in vitro under varying ultrasound intensities (1.5 MHz), demonstrating a dose-dependent increase in drug release with increasing acoustic power (30%, 50%, 80%). (e) Pulsatile release profile of HOF-TATB@Bup under repeated focused ultrasound (FUS) exposures (1.5 MHz, 50% duty cycle, 1 min pulses at 5-minute intervals), showing stepwise increases in cumulative release with each stimulus, indicating the feasibility of on-demand release, (f) Drug loading efficiencies of six representative small-molecule therapeutics into HOF-TATB using the DSM protocol, including local anesthetics (lidocaine, bupivacaine), antiinflammatory agents (aspirin, ibuprofen), a calcium channel blocker (isradipine), and an antidiabetic drug (glibenclamide). Lipophilic compounds exhibited markedly higher loading efficiency, highlighting the potential of HOF-TATB for tailored encapsulation of hydrophobic drugs. All data are presented as mean ± s.d., n = 3 independent replicates.

[0034] FIGS 9A-D: Ultrasound-triggered release of lidocaine from HOF-TATB nanoparticles and resulting inhibition of neuronal activity in vitro, (a) Schematic illustration of the mechanism by which ultrasound (FUS) stimulation induces the release of lidocaine (LA) from HOF-TATB nanoparticles, enabling its interaction with voltage-gated sodium channels and leading to channel blockade, (b) Representative fluorescence image of primary cortical neurons expressing the calcium indicator GCaMP6s (hSyn:: GCaMP6s-WPRE-SV40), used to monitor real-time neuronal activity. Scale bar, 20 pm. (c) Heat maps of normalized GCaMP6s fluorescence intensity from 50 neurons in different experimental conditions (n = 50 neurons examined over three independent experiments for each group), including (1) TATB@Lido / FUS / (+) / (+), (2) TATB@Lido / FUS / (+) / (-), (3) TATB@Lido / FUS / (-) / (+) and (4) TATB@Lido / FUS / (-) / (-). OFF = ultrasound off; ON = ultrasound on (1.5 MHz, 1.08 MPa, 10 s pulse), (d) Quantification of ΔF / F₀ responses across neuron populations under control (TATB ©Lidocaine, no FUS) and FUS-treated conditions. FUS-triggered release of lidocaine significantly inhibited neuronal activity (****p < 0.0001, two-tailed unpaired t-test). All data are presented as mean ± s.d., n = 50 neurons.

[0035] FIGS. 10A-I: Ultrasound-triggered analgesia in vivo using HOF-TATB nanoparticles loaded with local anesthetics, (a) Schematic illustration of ultrasound-- 17 - 4909-0910-3750, v. 3triggered analgesic delivery. HOF-based nanoparticles loaded with lidocaine HC1 (TATB ©Lidocaine HC1) were locally injected near the sciatic nerve of rats. Upon external FUS stimulation, the nanoparticles release lidocaine on demand to block nerve conduction, and mechanical sensitivity was evaluated using von Frey filament testing, (b) Acute analgesic response following FUS treatment (1.5 MHz, 1.4 MPa) in rats injected with HOF-TATB© lidocaine HC1. Increasing FUS durations (1, 4, and 8 minutes) induced dose-dependent elevations in von Frey thresholds (n = 4 rats). (c) Mass spectrometry and histological validation of ultrasound-triggered lidocaine release.(1) Representative mass spectrum showing detection of lidocaine at m / z = 235.1801 ± 15 ppm. (2) H& E-stained sciatic nerve sections and corresponding mass spectrometry imaging (MSI) maps (3) were collected from three treatment groups: Sample 1 (no lidocaine-loaded nanoparticles, no FUS), Sample 2 (lidocaine-loaded nanoparticles without FUS), and Sample 3 (lidocaine-loaded nanoparticles with FUS). Lidocaine accumulation is only observed in Sample 3 following ultrasound stimulation. MSI signals are shown at m / z = 235.1801 with a ±15 ppm window, and intensity is normalized to total ion current. Scale bar: 3 mm. (d) Day-by-day comparison of nociceptive thresholds before and after 8-min FUS in rats treated with HOF-TATB ©lidocaine HC1. A significant increase in threshold was observed across all 7 days (n = 4, P < 0.05 to ***P < 0.0001, paired t-test). (e) The same formulation with a reduced FUS duration of 4 min also led to moderate but significant increases in mechanical threshold over several days (n = 4, P < 0.05 to P < 0.01), indicating a tunable dose-response relationship based on ultrasound exposure, (f) Sustained and repeatable analgesic responses in rats treated with HOF-TATB ©bupivacaine and stimulated with FUS pulses once every 24 hours. Elevated von Frey thresholds were observed after each stimulus (n = 5 rats), (g) Statistical comparison of thresholds before and after FUS across 5 consecutive days for the HOF-TATB ©bupivacaine group, confirming consistent and reversible analgesia (*P < 0.05 to **P < 0.01). (h) Rats injected with unloaded HOF-TATB nanoparticles and stimulated with FUS served as nanoparticle-only controls, showing no elevation in mechanical thresholds (n = 3 rats), (i) Corresponding statistical analysis for the HOF-TATB control group revealed no significant nociceptive changes (ns, not significant). All data represent mean ± s.d. from biological replicates as indicated.- 18 - 4909-0910-3750, v. 3

[0036] FIGS. 11A-C: Ultrasound-responsive HOF-TATB nanocarriers mitigate neuropathic pain and enhance motor recovery in a sciatic nerve transection model, (a) Schematic of the rat sciatic nerve transection and repair procedure. The sciatic nerve was surgically transected (1), fully separated (2), and subsequently reconnected via microsurgical neurorrhaphy (3), with or without local nanoparticle administration. Scale bar, 1 mm. (b) Longitudinal self-mutilation scores (toe -chewing behavior) in rats receiving HOF-TATB loaded with bupivacaine (blue, n = 9) or lidocaine (red, n = 7), compared to untreated controls (black, n= 18). Both treatment groups showed markedly reduced self-injury behavior over 6 weeks post-operation (PO), indicating effective attenuation of neuropathic pain, (c) Sciatic Functional Index (SFI) scores over 6 weeks PO reveal improved locomotor recovery in rats treated with HOF-TATB ©bupivacaine (blue) relative to both negative controls and the lidocaine-treated group. HOF-TATB ©lidocaine HC1 (red) showed limited improvement in functional outcomes, suggesting shorter analgesic duration or lower motor-support efficacy. All data represent mean ± s.d.; statistical analyses were performed using two-way ANOVA with Tukey’s multiple comparisons test unless otherwise noted.

[0037] FIGS. 12A-F: Machine-learning-guided identification of high-loading drug-HOF pairs, (a) Overview of the computational workflow. Drug molecules and HOF scaffolds are encoded into multilevel descriptors, including first-level HOF structural metrics (BET surface area and pore volume) and second-level drug physicochemical features (logP, TPS A, HBD / HBA, molecular weight, molecular volume). RD Kit-based featurization generates unified descriptor vectors describing host-guest interaction-relevant chemical and structural features. A random forest regression model maps these descriptors to drag loading predictions across four representative HOFs (HOF-TATB, HOF-BTB, HOF-101 and HOF-102), (b) Chemical-feature landscape of the training set. Distribution of key drag descriptors — molecular volume, logP, and TPSA — illustrates the broad and chemically diverse training space, ensuring that the model generalizes beyond narrow structural classes and captures a wide range of host-guest interaction modes, (c) Structural diversity of HOF descriptors. Representative HOF features, exemplified by distributions of HOF logP and BET surface area, highlight the intrinsic heterogeneity across the HOF family. Variations in pore geometry (short / long pore sizes) and pore volume collectively define the structural environment governing guest accommodation at the molecular scale, (d) Hydrophilic drugs are incorporated into HOF-TATB through simple aqueous diffusion, where solvated molecules migrate into the hydrophilic pore network driven by concentration gradients. In contrast, hydrophobic drags- 19 - 4909-0910-3750, v. 3require a double-solvent method (DSM), in which a small amount of organic solvent solvates the drug and enables efficient partitioning into the HOF’s hydrophobic pore environment. Together, these complementary loading pathways accommodate a wide polarity range and enable uniformly high loading efficiencies across diverse chemical structures, (e) Experimentally measured loading capacities across four HOF materials using both DSM and aqueous diffusion methods. Loading values were obtained for 15 structurally diverse molecules spanning hydrophilic, moderately lipophilic, and highly lipophilic chemical classes. Distinct loading distributions across HOF-TATB, HOF-BTB, HOF-101, and HOF-102 are consistent with the physicochemical compatibility between each drug class and the corresponding HOF scaffold, n = 3 independent experiments for each sample; data are presented as mean ± s.d. (f) Descriptor-loading relationship heatmap. A normalized heatmap integrates drug descriptors (MolVol, Rg, Mw, RotB, FCsp3, TPSA, logP, charge) and HOF descriptors (HOF ligand logP, number of hydrogen-bonding sites, pore volume, short / long pore sizes, BET surface area) with experimentally measured loading efficiencies for each drug-HOF pair. All descriptor values were independently normalized to a 0-1 scale, as indicated by the color bar. Rows are ordered by increasing loading, revealing chemical and structural patterns associated with high-capacity encapsulation. In particular, HOFs with larger pore volumes, higher BET surface areas, and greater hydrophobicity (higher HOF logP) tend to support enhanced loading across diverse drug chemistries.

[0038] FIGS. 13A-H: Machine learning-guided screening of high-loading anesthetic drugs for HOFs. (a) Schematic representation of small-molecule drugs used in model training and screening. Key molecular descriptors include molecular weight, logP, TPSA, rotatable bonds, and net charge, (b) Crystal structures of four representative HOF materials (HOF-TATB, HOF-101, HOF-BTB, and HOF-102) used for drug loading studies. Structural descriptors such as pore size, surface area (BET), and number of hydrogen bonding sites were extracted for model input, (c) Experimentally measured drug loading capacities for 15 molecules across four HOFs using two loading strategies: double solvent method (DSM) and diffusion method. Each point represents a drug-HOF combination, (d) Benchmarking performance of four machine learning models — Random Forest (RF), Support Vector Machine (SVM), AdaBoost, and Neural Network (NN) — using RMSE and correlation coefficients (Pearson, Spearman, Kendall) under five-fold cross-validation, (e) Scatter plot comparing predicted vs. experimentally measured loading capacities using the best-performing model (Random Forest). The model achieves a mean RMSE of 8.213 ± 1.975 and a Kendall’s r of- 20 - 4909-0910-3750, v. 30.756 ± 0.156. (f) Feature importance analysis of the Random Forest model. On the HOF side, pore size, lipophilicity (logP), and hydrogen bonding were key predictors. On the drug side, lipophilicity (logP), total polar surface area (TPSA), and molecular volume were most influential, (g) Workflow of virtual screening pipeline. Approximately 3,000 FDA-approved drugs were filtered to -250 analgesics / anesthetics. The Random Forest model was used to predict loading capacity, followed by experimental validation of the top 10 candidates. Bupivacaine and Lidocaine HC1 were ultimately selected based on model score, interaction energy, and translational potential, (h) Conceptual illustration of drug-loaded HOF nanoparticles administered for local pain therapy. High drug loading enables long-term, on-demand release in vivo, offering a non-opioid alternative for chronic pain management.

[0039] FIG. 14A-L: Integrated machine-learning pipeline for identifying high-loading anesthetic candidates for HOF-TATB nanoparticles, (a) Principal component analysis (PCA) of drug physicochemical descriptors showing partially separable clusters across four loading ranges (0-20%, 20-40%, 40-60%, 60-80%). Higher-loading molecules occupy a more confined region, indicating that loading efficiency correlates with specific descriptor combinations.(b) Kernel density distributions along PCI further illustrate systematic shifts in feature space across loading groups, supporting that high loading is associated with distinct physicochemical patterns, (c) Benchmarking of four machine-learning models. Random Forest (RF) achieved the lowest prediction error (RMSE) among all tested models, outperforming Support Vector Machine (SVM), AdaBoost, and Neural Network (NN), (d) Correlation metrics (Pearson, Spearman, Kendall) consistently identified RF as the most reliable predictor of loading, (e) Predicted versus experimentally measured loading capacities using the RF model, showing strong agreement (mean RMSE = 8.213 ± 1.975; Kendall’s i = 0.756 ± 0.156). (f) Permutationbased feature importance analysis. On the HOF side, pore size, hydrophobicity (ligand logP), and hydrogen-bonding density were the strongest contributors. On the drug side, logP, TPSA, and molecular volume were dominant determinants, (g) Virtual screening workflow. From -3,000 FDA-approved small molecules, indication-based filtering yielded -250 analgesic or anesthetic candidates. ML-based screening narrowed this to a small panel of high-priority molecules, followed by GFN2-xTB host-guest interaction energy evaluation and biosafety assessment. Bupivacaine (DSM loading) and lidocaine HC1 (diffusion loading) emerged as top candidates.(h) ML-guided ranking of candidate molecules based on predicted loading efficiencies.- 21 - 4909-0910-3750, v. 3(i) Predicted loading distribution of -250 candidates for the dual-solvent method (DSM). (j) Predicted loading distribution of the same candidate set for diffusion loading. (k) Experimental validation showing strong agreement between predicted and measured loading for the top-ranked candidates. (1) GFN2-xTB interaction energy validation of drug-HOF binding, supporting the ML-identified top-performing candidates for each loading mode.

[0040] FIGS. 15A-J: Ultrasound-triggered therapeutic release of HOF-TATB nanoparticles and lidocaine-induced inhibition of neuronal activity in vitro, (a) Hydrodynamic size distribution measured by DLS of HOF-TATB nanoparticles and loaded with bupivacaine and lidocaine HC1. n = 3 independent experiments for each sample, (b) TEM images of HOF-TATB nanoparticles before and after loading with bupivacaine and lidocaine HC1, showing that the particle morphology and crystalline structure were retained after drug loading. Scale bars, 200 nm. n = 3 per group, (c) Powder X-ray Diffraction (PXRD) patterns of HOF-TATB nanoparticles before and after loading with bupivacaine or lidocaine HC1, showing that the characteristic diffraction peaks were preserved, indicating retention of the crystalline structure after drug loading, n - 3 independent experiments for each sample, (d) Long-term storage stability of HOF-TATB @ bupivacaine prepared via the dual-solvent method (DSM), showing minimal decline in drug loading over 30 days in 10% fetal bovine serum (FBS) at 25 °C. (e) Ultrasound-triggered release of bupivacaine from HOF-TATB nanoparticles in vitro under varying ultrasound intensities at 1.5 MHz, demonstrating a dose-dependent increase in drug release with increasing acoustic power (0.79MPa, 1.40MPa, 2.45MPa). (f) Pulsatile release profile of HOF-TATB @Bup under repeated FUS exposures (1.5 MHz, 1.40MPa, 1 min pulses at 5-minute intervals), showing stepwise increases in cumulative release with each stimulus, indicating the feasibility of on-demand release. All data are presented as mean ± s.d., n = 3 independent replicates, (g) Schematic illustration of the mechanism by which FUS stimulation induces the release of the local anesthetic lidocaine (LA) from HOF-TATB nanoparticles, enabling its interaction with voltage-gated sodium channels and leading to channel blockade, (h) Representative fluorescence images of primary cortical neurons expressing GCaMP6s (hSyn:: GCaMP6s-WPRE-SV40) before and after FUS. Prior to sonication (top), neurons displayed robust calcium activity as indicated by strong GCaMP6s fluorescence. Following FUS (bottom), ultrasound-triggered release of lidocaine HC1 from HOF-TATB nanoparticles markedly reduced neuronal activity, resulting in diminished calcium signals. Scale bar, 50 pm. (i) Heat maps of normalized GCaMP6s fluorescence intensity from individual neurons under different experimental conditions (n =50 neurons per group, three- 22 - 4909-0910-3750, v. 3independent experiments), including (1) TATB@Lido HCl / FUS / (+) / (+), (2) TATB@Lido HCl / FUS / (+) / (-), (3) TATB@Lido HCl / FUS / (-) / (+) and (4) TATB@Lido HCl / FUS / (-) / (-). FUS (-) = ultrasound off; FUS (+) = ultrasound on (1.5 MHz, 1.40 MPa, 10 s per pulse), (j) Quantification of ΔF / F₀ responses across neuron populations under control (TATB@ Lidocaine, no FUS) and FUS-treated conditions.< 0.0001, two-tailed unpaired t-test). All data are presented as mean ± s.d.

[0041] FIGS. 16A-L: Ultrasound-triggered analgesia in vivo using HOF-TATB nanoparticles loaded with local anesthetics, (a) Schematic overview of ultrasound-triggered analgesic delivery. HOF-TATB nanoparticles loaded with lidocaine HC1 were perineurally injected near the sciatic nerve. External focused ultrasound (FUS) triggered drug release to achieve temporally precise, on-demand nerve block, (b) MALDI-MSI maps and corresponding H& E-stained longitudinal sciatic nerve sections from three experimental conditions: (1) no nanoparticles, no FUS; (2) nanoparticles without FUS; (3) nanoparticles with FUS. A strong and spatially confined lidocaine signal (m / z = 235.1801 ± 15 ppm, TIC-normalized) was detected only in the FUS-activated group (1.5 MHz, 1.40 MPa, 0.2 s on / 0.8 s off, 8 min). Representative images from n = 3 rats per group. Scale bar: 3 mm. (c) Schematic of von Frey (VF) testing for quantifying mechanical nociceptive thresholds, (d) Time-course of VF thresholds following single FUS stimulation at different intensities. Minimal changes were observed at 0.79 MPa (n = 4), whereas 1.40 MPa (n = 6) and 2.45 MPa (n = 4) produced rapid threshold elevations at 0.5 h followed by gradual decline. Mean ± s.e.m. (e) Grouped VF thresholds before and after FUS. Significant increases were observed at 1.40 and 2.45 MPa (p < 0.05). Two-way ANOVA with Tukey’s test, (f) Time-course of VF thresholds following different FUS durations (1, 4, 8 min; n = 6, 5, 8). Only 8-min stimulation produced a significant elevation at 0.5 h. Mean ± s.e.m. (g) Comparison of VF thresholds before and after FUS at different durations. Significant elevation was observed only in the 8-min group (p < 0.05-0.0001). (h) Day-by-day nociceptive thresholds before and after daily 8-min FUS in rats treated with HOF-TATB @lidocaine HCl (n = 4). Significant increases were observed on all 7 days (paired t-test). (i) Statistical comparison of thresholds before and after FUS across 5 consecutive days in rats treated with HOF-TATB @bupivacaine (n = 5), confirming consistent and reversible analgesia (*p < 0.05-**p < 0.01). (j) VF time-course of the HOF-TATB @bupivacaine group. Each FUS activation (blue shaded area) reliably increased mechanical thresholds, followed by gradual decline until the next stimulus, (k 1) Control animals injected with Rhodamine-B-loaded HOF-TATB nanoparticles showed no change in- 23 - 4909-0910-3750, v. 3thresholds with or without FUS (n = 3). No significant differences were detected. Statistical notation: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All values represent mean ± s.d. unless otherwise stated.

[0042] FIGS. 17A-G: Ultrasound-responsive HOF-TATB nanocarriers mitigate neuropathic pain and enhance motor recovery in a sciatic nerve transection model. Ultrasound-responsive HOF-TATB nanocarriers mitigate neuropathic pain and enhance motor recovery in a sciatic nerve transection model, (a) Overview of the sciatic nerve transection-repair model and treatment timeline. After complete transection and microsurgical neurorrhaphy, rats received perineural injections of analgesic-loaded HOF-TATB nanoparticles (lidocaine HC1 or bupivacaine) beginning at postoperative week 1. Focused ultrasound (FUS) was applied daily to trigger local drug release, and nanoparticle administration was repeated weekly from week 2 to week 5. (b-c) Behavioral assessments included toe-chewing (TC) scoring as an index of spontaneous neuropathic pain and the sciatic functional index (SFI) as a quantitative measure of locomotor recovery, (d) TC scores in rats treated with HOF-TATB @bupivacaine (blue, n = 9) or HOF-TATB @lidocaine HCl (red, n = 7) compared with negative controls (black, n = 18). Both drug-loaded groups exhibited markedly reduced self-injury behavior over six weeks, indicating sustained mitigation of neuropathic pain, (e) Representative photographs of hind paws from control and treated animals. Severe digit mutilation in the control group contrasts with minimal or absent damage in the HOF-TATB ©lidocaine HC1 group, (f) SFI scores over six weeks showing progressive locomotor recovery in the HOF-TATB ©bupivacaine group, exceeding both negative controls and the lidocaine-treated group. The lidocaine group showed modest functional improvement, (g) Axon density quantification at week 6 post-treatment. The HOF-TATB @lidocaine HCl group exhibited significantly higher axon density than controls (*p < 0.05), whereas bupivacaine showed a similar upward trend without reaching significance. All values represent mean ± s.d. Statistical analysis: Two-way ANOVA with Tukey’s multiple comparison test unless otherwise indicated.

[0043] FIG. 18: Following the machine learning-based screening, the top 5 drug candidates for HOF-TATB were selected for further evaluation using two different encapsulation methods: double-solvent and diffusion methods. For each method, the ranking was based on the predicted loading efficiency, and the interaction energies (Einteraction) between the drug molecules and HOF-TATB channels were calculated using GFN2-xTB with implicit- 24 - 4909-0910-3750, v. 3water solvation. The top 5 drugs identified via double-solvent method included hexylresorcinol, tapentadol, bupivacaine, ropivacaine, venlafaxine, whereas the diffusion method yielded amylocaine, lidocaine, levacetylmethadol, disopyramide, hexylresorcinol.

[0044] FIG. 19: Dynamic light scattering (DLS) measurements showed that HOF-TATB nanoparticles maintained a relatively consistent size distribution when dispersed in DI water over a period of 10 days. The initial average hydrodynamic diameter (day 0) exhibited only a slight shift after 7 and 10 days of storage, with no significant aggregation or formation of large particles observed. These results indicate that HOF-TATB possesses good colloidal stability in aqueous environments over extended periods.

[0045] FIG. 20: ’H NMR spectrum of HOF-TATB loaded with bupivacaine in DMSO-ds (400 MHz), showing characteristic peaks of both the HOF-TATB framework and bupivacaine (5 = 7.05 and 2.13 ppm), confirming successful drug loading.

[0046] FIG. 21: ’H NMR spectrum of HOF-TATB loaded with lidocaine- HC1 in DMSO-ds (400 MHz). Characteristic proton signals from lidocaine- HC1 are observed at 5 = 7.05 and 2.13 ppm, together with peaks from the HOF-TATB framework, confirming successful drug encapsulation.

[0047] FIG. 22: Ultrasound-guided injection of HOF-TATB nanoparticles.Representative ultrasound image showing the position of the needle (top arrow) and the sciatic nerve (side arrow) during perineural injection of HOF-TATB nanoparticles. Real-time ultrasound guidance was used to ensure accurate needle placement adjacent to the sciatic nerve for localized delivery of the nanoparticle formulation.

[0048] FIG. 23: Schematic illustration of ultrasound-triggered local anaesthetic (LA) release from HOF nanoparticles for sciatic nerve blockade. HOF nanoparticles loaded with local anaesthetics (LA) were locally administered adjacent to the sciatic nerve. In the absence of focused ultrasound (FUS), LA molecules remain encapsulated within the HOF framework, and sodium channels in the neuronal membrane remain open, allowing Na+influx and normal nerve conduction. Upon FUS stimulation, the HOF structure releases the encapsulated LA, which binds to sodium channel sites, blocking Na+influx and thereby inhibiting action potential propagation, resulting in reversible sciatic nerve blockade.- 25 - 4909-0910-3750, v. 3

[0049] FIG. 24: Mass spectrometry identification of lidocaine Mass spectrometry imaging (MSI) analysis revealed a strong signal at m / z = 235.1801, which corresponds to the [M+H]+of lidocaine. This peak, together with its characteristic fragment ions, is consistent with the reference mass spectrum, confirming the presence of lidocaine in the sample.

[0050] FIG. 25: Representative hematoxylin and eosin (H& E) staining images of sciatic nerve sections from rats treated with TATB ©Lidocaine HC1 without focused ultrasound (FUS) activation (a) and with FUS activation (b). For the FUS group, treatment was applied at 1.5 MHz and 1.40 MPa for 8 min. Sciatic nerves were harvested 7 days after treatment. In both conditions, the nerve fibers exhibited normal morphology with intact myelin structure and no signs of degeneration, inflammatory infiltration, or tissue damage, indicating that the application of TATB ©Lidocaine HC1, with or without LUS, did not induce noticeable histopathological changes in sciatic nerve tissue (scale bar: 100 pm).

[0051] FIG. 26: Representative immunofluorescence images of rat sciatic nerve sections 7 days after treatment, stained with Hoechst (blue, nuclei) and Ibal (red, macrophages / microglia). Top: TATB ©Lidocaine HC1 without FUS activation; Bottom: with FUS activation (1.5 MHz, 1.40 MPa, 8 min). Both groups show only sparse Ibal -positive cells, indicating no significant immune cell infiltration or inflammatory response (scale bar: 50 pm).

[0052] FIG. 27: Representative immunofluorescence images of rat sciatic nerve sections 7 days after treatment, stained with Hoechst (blue, nuclei) and cleaved Caspase-3 (red, apoptotic marker). Top: TATB ©Lidocaine HC1 without FUS activation; Bottom: with FUS activation (1.5 MHz, 1.40 MPa, 8 min). Both groups exhibited minimal Caspase-3-positive staining, indicating no significant apoptosis in sciatic nerve tissue (scale bar: 50 pm).DESCRIPTION OF ILLUSTRATIVE EMBODIMENTSI. HOFs

[0053] Hydrogen-bonded organic frameworks (HOFs) are a class of porous molecular materials that rely on the assembly of organic building blocks by means of hydrogen-bonding interactions to form two-dimensional (2D) and three-dimensional (3D) crystalline networks (Li et al., 2020. The reversible nature of the hydrogen-bond formation endows HOFs with the attributes of solution processability and simple regeneration. High-quality single crystals of HOFs can be grown for unambiguous superstructure determination by single-crystal X-ray- 26 - 4909-0910-3750, v. 3diffraction, which can be used to elucidate superstructure-property relationships. During the past decade, considerable progress has been achieved in realizing stable HOFs with permanent porosities by focusing on the design of molecular building blocks in order to introduce rigidity, auxiliary [π···π] interactions, and interpenetration of their frameworks to sustain the extended networks. HOFs have been used in a wide variety of applications, including catalysis, energy, biomedical products, and the storage and separation of chemicals.

[0054] Porous frameworks, including metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), have garnered significant attention as drug delivery platforms due to their remarkable drug loading capacity and well-defined structures (Wu et al., 2017; Bhunia et al., 2020). Among them, hydrogen-bonded organic frameworks (HOFs) have recently emerged as a particularly promising class of porous materials with both high structural homogeneity and programmability, self-assembled from organic molecular building units (OMBUs) via hydrogen-bonding and Jt-Tt stacking interactions (Lin et al., 2019; Yan et al., 2021; Lin and Chen; 2022). Unlike strong metal-ligand coordination and covalent bonding interactions in MOFs and COFs, the relatively weak non-covalent interactions make HOFs excellent candidates for ultrasensitive mechanochemical activation under FUS stimulation. In addition, the abundant diversities of building units made HOFs easily feasible for fine-tuning of their compositions and functionalities to cater custom-design applications. For instance, the density of hydrogen bondings and the presence of aromatic fused rings in the backbone of OMBU have a substantial impact on the properties of HOFs, particularly their stability under aqueous conditions (Li et al., 2020). If the HOFs can be precisely tailored and selectively activated through non-invasive ultrasound, this technique would serve as a significant advancement in the field of drug delivery. By providing exceptional versatility and excellent temporal resolution, it would expand the scope of this technology to numerous disease applications, thereby advancing the field as a whole.

[0055] In some aspects, HOFs can be synthesized by methods including the use of basic hydrogen bonding synthesizer motifs (Chen et al., 2022). Building blocks of HOFs can include including diaminotriazine (DAT), carboxylic acid (-COOH), sulfonic acid (–SO₃H), pyrazolre, imidazole, pyridine, and urea (Jones et al., 2011; Pulido et al., 2017; Zhao et al., 2021). It should be noted that these organic groups can be assembled into many polymorphs due to the flexibility of hydrogen bonds. HOFs are a kind of solvated crystals that crystallize slowly in a solvent. However, the crystallization of HOFs is easily affected by precursor concentration,- 27 - 4909-0910-3750, v. 3reaction time, temperature, and other factors. Therefore, most of the HOFs are polymorphs. In theory, kinetic isomers can be produced by crystallization at higher concentrations or at shorter reaction times. However, slowing down the crystallization rate or increasing the heat energy can be a straightforward solution to obtain a thermodynamically stable phase. Various methods are available for synthesizing HOFs are possible, such as liquid / vapor diffusion, evaporation / cooling, electrophoretic deposition, and others. Select methods that can be used to synthesize HOFs are described in U. S. patent 11,479,482, Yang et al. (2020), and Wang et al. (2020).

[0056] The present disclosure includes the methods to prepare HOF as described above that may be coupled with the production of anesthetic loaded HOF. After the preparation of the HOFs, the HOFs are dissolved in a solution and admixed with a solution of the drug or active pharmaceutical ingredient. After these solutions were then admixed together, the solutions are subjected to an application of external energy such as agitation, vibration, sonication, or centrifugation. These solutions may then be subjected to one or more of these external energy applications. After the formation of the drug loaded HOFs, excess drug may be removed through washing or dialysis. After removing excess drug, the drug loaded HOFs may be converted into a dry powder through evaporation or freeze drying such as lyophilization. The dry powder form of the drug loaded HOFs may then be resuspended into solution before use.

[0057] The strength of bonding of HOFs can be modified such that a desired level of ultrasound stimulation can disrupt the HOF hydrogen bonding and release a drug or therapeutic agent from inside the HOF. HOFs frameworks assembled from the organic units may include both the pure organic and metal-containing organic moieties through hydrogen bonding interactions, and the frameworks can be further strengthened or weakened by including or removing other weak interactions such as it... it interactions, van der Waals interactions, C-H...it interactions etc. The HOF may comprise a compound of the formula:- 28 - 4909-0910-3750, v. 3wherein: A is a Ce-i6 aromatic ring or a C3-15 heteroaromatic ring; and each of B, B', and B" are independently a Ce-i6 aromatic ring or a C3-15 heteroaromatic ring comprising -S(O)2Ra, -NH2, or - C(O)Rawherein Rais hydroxy or amino.

[0058] In some aspects the HOF comprises a compound having the formula:wherein: X₁, X₂ and X₃ are each independently CH or N; R₁, R₂ and R₃ are each independently –S(O)₂Raor - C(O)Ra, wherein: Rais hydroxy or amino; R₄–R₁₅ are each independently –H, –F, –Cl, –Br, –I or –CH₃; or a pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound has the structure:IL Ultrasound

[0059] Ultrasound may be applied to a mammalian subject to release an anesthetic, preferably a local anesthetic (e.g., bupivacaine), from a HOF as described herein at an area of interest within the subject. It is anticipated that ultrasound or focused ultrasound can be applied to a variety of tissues in the mammalian subject including the brain, a solid tumor, breast, uterine, heart, liver, kidney, other organs, and subcutaneous tissue.

[0060] The terms “ultrasound signal” and “ultrasound” are used interchangeably to refer to sound waves with frequencies that are above what a human can hear, e.g., above 20- 29 - 4909-0910-3750, v. 3kHz. Sound waves are physical vibrations that propagate through a medium, e.g., through air, water, bone, and muscle. In some cases the ultrasound has a frequency ranging from 20 kHz to 100 MHz, such as from 100 kHz to 15 MHz, or from 500 kHz to 5 MHz. The ultrasound signal can be a focused ultrasound signal (FUS) or an unfocused ultrasound signal. In focused ultrasound, the ultrasound emitting device is typically configured such that the highest intensity ultrasound signal is located not only at a particular angle relative to the device, but also at a particular distance away from the ultrasound emitter part of the ultrasound emitting device. For instance, the highest intensity ultrasound signal can be located between 10 mm and 150 mm away from the end of the ultrasound emitter, e.g., 10 mm to 150 mm below the skin, such as between 30 mm and 75 mm. A medical professional can change the settings on the ultrasound emitting device, thereby changing the desired depth of highest ultrasound intensity. This can allow for lower intensity ultrasound applied to a more superficial location, such as the prefrontal cortex of the brain, while also providing for high intensity ultrasound at an interior location, such as Broca’ s area of the brain, which is located underneath the prefrontal cortex. In contrast, in unfocused ultrasound the highest intensity is at or near the surface of the skin or tissue, with ultrasound intensity decreasing with increasing distance from the emitter. One of skill can modify the ultrasound or FUS signal to stimulate particular tissues within a subject such as a mammalian subject. In some embodiments, the ultrasound or FUS is applied to the brain, spinal cord, peripheral nerves, or other tissue in the subject.

[0061] The ultrasound can be applied to a HOF comprising a drug in proximity to the tissue of the subject. As used herein, “in proximity to” means that at least some of the drug can be released due to ultrasound perturbation or stimulation of the HOF. For example, the tissue can be a group of neuron cells in a dopaminergic neuronal pathway of the brain, and the HOF comprising a drug can be located inside a blood vessel adjacent to the dopaminergic neuronal pathway (e.g., the striatum, the subthalamic nucleus, the globus pallidus interna.

[0062] The term “tissue” refers to any region of the subject’s body. For example, the tissue can be a brain tissue, e.g., the auditory complex, the prefrontal cortex, or the hippocampus, ventral tegmental area, thalamus, spinal cord, a muscle tissue, a region of bone, and a liver tissue. In cases wherein the tissue is a brain tissue, the term “brain tissue” includes neurons, glia cells, extracellular fluid, and other components of the brain. In some cases the tissue is a cancerous tumor of any part of the body.- 30 - 4909-0910-3750, v. 3

[0063] The amount of time between administering the nanoparticles or liposomes to the subject and the application of light can be any suitable length. In some cases, such a time is 180 minutes or less, 120 minutes or less, 60 minutes or less, 30 minutes or less, 10 minutes or less, of 5 minutes or less.

[0064] An ultrasound can be applied for the desired amount of time, for example from ranges from about 1 second to about 30 minutes, such as from 1 second to 10 minutes, or from 1 second to 1 minute. The nanoparticles or liposomes may disperse in the blood of body of the subject after administration, so it may be desirable to administer ultrasound to a region for a period of time sufficient to cause the nanoparticle or liposomes to generate light. In some preferred embodiments, the ultrasound is not applied to a tissue for so long that significant toxicity to local tissues occurs from ROS generation by the sonosensitizer.

[0065] The emission of light from the photoexcited mechanoluminescent particle can be triggered by any suitable mechanical stimulus, e.g., by ultrasound. In some cases, the ultrasound is focused ultrasound. In some cases, the ultrasound signal has a frequency ranging from 150 kHz to 15 MHz, such as from 300 kHz to 5 MHz, or from 600 kHz to 2 MHz. The ultrasound signal can be repeated, i.e. wherein the signal is emitted for a time and then not emitted for a time, at any suitable repetition. In some cases, the ultrasound signal is repeated at a rate ranging from 0.05 repetitions per second to 20 repetitions per second, such as from 0.2 repetitions per second to 5 repetitions per second, or from 0.5 repetitions per second to 2 repetitions per second.

[0066] In some cases, the ultrasound signal has a spatial peak pulsed average intensity (ISPPA) at a target neuron ranging from 1 W / cm2to 100 W / cm2, such as from 2 W / cm2to 50 W / cm2, or from 5 W / cm2to 15 W / cm2. As used herein, W / cm2refers to the units watts per centimeter square.

[0067] In some cases, the time interval between application of the ultrasound signal and the emission of light from the mechanoluminescent particle is 9 ms or less, such as 7 ms or less, 5 ms or less, or 3 ms or less. The subject can be, for example, a human, a primate, a rat, a mouse, a horse, a dog, or a cat.- 31 - 4909-0910-3750, v. 3III. Pharmaceutical Compositions

[0068] In some embodiments, a HOF and anesthetic, preferably a local anesthetic, as described herein are included in a pharmaceutical composition. Pharmaceutical compositions of the present invention comprise an effective amount of one or more compounds of the present disclosure, e.g., a mechanoluminescent nanoparticle or mechanoluminescent liposome, or additional agent dissolved or dispersed in a pharmaceutically acceptable earner. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains a mechanoluminescent nanoparticle or mechanoluminescent liposome as described herein or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21stEd., Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should typically meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0069] As used herein, "pharmaceutically acceptable earner" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington - 23rd Edition, October 2020, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0070] The mechanoluminescent nanoparticle or mechanoluminescent liposome may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally,- 32 - 4909-0910-3750, v. 3inhalation (e.g., aerosol inhalation), via injection, infusion, continuous infusion, or localized perfusion bathing target cells directly.

[0071] In some embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0072] In other non-limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above.Parenteral Compositions and Formulations

[0073] In further embodiments, a composition of the present invention may be administered via a parenteral route. As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U. S. Pat. Nos.- 33 - 4909-0910-3750, v. 36,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).

[0074] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U. S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0075] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, " Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the- 34 - 4909-0910-3750, v. 3condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0076] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent. In some preferred embodiments, the HOF can be stored with or without a drug or therapeutic agent, e.g., in a solution aqueous 5% glucose (w / v). In some embodiments, the HOF comprising a drug or therapeutic agent is dissolved in an aqueous 5% glucose (w / v) prior to administration by injection.A. Liposomes

[0077] In some embodiments, the HOF and drug are comprised in a liposome or liposomal formulation. Attributing to its natural constituents, liposomes are effectively metabolized in the body. Liposomes were the first nanodrugs in FDA clinical trials and have been extensively applied in nanomedicines since the first liposomal formulation was approved by FDA in mid-1990s.1 19 211More recently, the liposome based mRNA vaccine developed by BioNTech / Pfizer and Moderna was clinically applied against COVID-19.1221

[0078] The liposomes may comprise a variety of lipid components. In some preferred embodiments, the liposomes contain one or more an aqueous internal chambers. The liposomes are preferably biodegradable. The liposomes may comprise phospholipid, 1, 2-distearoryl-sn-glycero- 3 -phosphatidyl choline (DSPC), sphingomyelin, phosphatidylcholine (e.g., egg phosphatidylcholine or soy phosphatidylcholine), monosialoganglioside, cholesterol, polyethylene glycol (PEG), PEG-succinyl cysteine (PEG-SC), poly(lactic-co-glycolic acid)- 35 - 4909-0910-3750, v. 3(PLGA), dioleoylphosphatidylethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), choline phosphate. In some embodiments, the liposomes may comprise polyethylene glycol (PEG) or be stealth liposomes. In some embodiments, the following liposomes can be used for drug delivery, bioimaging, light induced catalyst, etc.

[0079] The liposomes or nanoparticles may be a variety of sizes. For example, in some embodiments, the liposomes are about 10-1000 nm, 20-750 nm, 50-500 nm, 25-250 nm, 50-300 nm, 25, 50, 75, 100, 125, 150, 175, 200, 250, 275, 300, 350, 400, 500, 600, 750 nm, or any ranger derivable therein. In some preferred embodiments, the liposomes or nanoparticles are 100 -200 nm.

[0080] The organic nanoparticles or liposomes can be administered to a subject via a variety of administration routes. For example, the nanoparticles or liposomes can be administered parenterally, intravenously, intracerebrally, subcutaneously, or intranasally.

[0081] A variety of dosages of nanoparticles or liposomes may be administered to a mammalian subject. For example, the nanoparticles or liposomes may be administered to the subject in an amount of about 5- 10 mg / kg, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 mg / kg, or any range derivable therein.V. Definitions

[0082] The term "hydrogen bond" means a bond that results from an attractive interaction between a hydrogen atom and an electronegative atom, wherein the hydrogen atom itself is covalently attached to another electronegative atom, for example, but not limited to, oxygen (0). nitrogen (N), chlorine (Cl), fluorine (F), etc.

[0083] The term "hydrogen-bonded organic framework" or " HOF" is defined as a material assembled from organic linker molecules through intermolecular hydrogen bonding. The HOF may contain least two moieties capable of promoting hydrogen bonds with at least one other organic linker. The organic linker may comprise heteroatoms, for example, oxygen, nitrogen, silicon etc.- 36 - 4909-0910-3750, v. 3

[0084] The terms “applying” and “administering” are used interchangeably to refer to causing a subject to receive a treatment. For example, applying an ultrasound signal means generating an ultrasound signal and directing it into a region of the subject's body. As another example, administering a particle to the subject means moving the particle inside the body of the subject.

[0049] The terms active agent, active pharmaceutical ingredient, pharmacologically active agent, and drug are used interchangeably herein to refer to a chemical material or compound which, when administered to an organism (human or animal) induces a desired pharmacologic and / or physiologic effect by local and / or systemic action.

[0085] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and nonhuman primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; and the like. " Mammal" means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans. Non-human animal models, e.g., mammals, e.g. non-human primates, murines, lagomorpha, etc. may be used for experimental investigations.

[0086] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect, such as reduction of the effects of a neurological disease. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having; (b) inhibiting progression of a disease; and (c) reducing a symptom of the disease or causing regression of the disease (e.g., reduction in neurological effects or functioning of a neurological disease).

[0087] A “therapeutically effective amount”, a "therapeutically effective dose" or “therapeutic dose” is an amount sufficient to effect desired clinical results (e.g., achieve therapeutic efficacy, achieve a desired therapeutic response). A therapeutically effective dose can be administered in one or more administrations. For puiposes of this disclosure, a therapeutically effective dose of a compositions is an amount that is sufficient, when- 37 - 4909-0910-3750, v. 3administered to the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of a disease state (e.g., neurodegenerative disease, etc.) present in the subject.

[0088] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations·

[0089] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for unit dosage forms depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0090] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. " A pharmaceutically acceptable excipient, diluent, carrier and adjuvant" as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0091] As used herein, a "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.

[0092] The terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially. In one embodiment, the agents are present in the cell or in the subject’s body at- 38 - 4909-0910-3750, v. 3the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.

[0093] The terms “cell,” and “cells,” and “cell population,” used interchangeably, intend one or more mammalian cells. The term includes progeny of a cell or cell population. Those skilled in the art will recognize that “cells” include progeny of a single cell, and there are variations between the progeny and its original parent cell due to natural, accidental, or deliberate mutation or change.

[0094] The term “cancer cell” as used herein refers to a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth or development in an immunocompromised non-human animal model, or any appropriate indicator of cellular transformation. “Cancer cell” may be used interchangeably herein with “tumor cell” or “cancerous cell” and encompasses cancer cells of a solid tumor and a liquid tumor. “Cancer” may be used interchangeably herein with “tumor”.

[0095] When used in the context of a chemical group: “hydrogen” means -H; “hydroxy” means -OH; “oxo” means =0; “carbonyl” means -C(=O)-; “carboxy” means -C(=O)OH (also written as -COOH or -CO2H); “halo" means independently -F, -Cl, -Br or -I; “amino” means -NH2; “hydroxyamino” means -NHOH; “nitro” means -NO2; imino means =NH; “cyano” means -CN; “isocyanyl” means -N=C=O; “azido” means -N3; in a monovalent context “phosphate” means -OP(O)(OH)2 or a deprotonated form thereof; in a divalent context “phosphate” means -OP(O)(OH)O- or a deprotonated form thereof; “mercapto” means -SH; and “thio” means =S; “sulfonyl” means -S(O)2~; and “sulfinyl” means -S(O)-.- 39 - 4909-0910-3750, v. 3

[0096] In the context of chemical formulas, the symbolmeans a single bond, “=” means a double bond, and “=” means triple bond. The symbol “ - ” represents an optional bond, which if present is either single or double. The symbol “ ==” represents a single bondor a double bond. Thus, the formulacovers, for example, ooooand. And it is understood that no one such ring atom forms part of more than one double bond. Furthermore, it is noted that the covalent bond symbol when connecting one or two stereogenic atoms, does not indicate any preferred stereochemistry. Instead, it covers all stereoisomers as well as mixtures thereof. The symbol “' A ”,when drawn perpendicularly across a bond (e.g., CH3for methyl) indicates a point of attachment of the group. It is noted that the point of attachment is typically only identified in this manner for larger groups in order to assist the reader in unambiguously identifying a point of attachment. The symbol” means a single bond where the group attached to the thick end of the wedge is “out of the page.” The symbol “"" HI” means a single bond where the group attached to the thick end of the wedge is “into the page”. The symbol “* AA ” means a single bond where the geometry around a double bond (e.g., either E or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.

[0097] When a variable is depicted as a “floating group” on a ring system, for example, the group “R” in the formula:then the variable may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed. When a variable is depicted as a “floating group” on a fused ring system, as for example the group “R” in the formula:- 40 - 4909-0910-3750, v. 3then the variable may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals -CH-), so long as a stable structure is formed. In the example depicted, R may reside on either the 5-membered or the 6-membered ring of the fused ring system. In the formula above, the subscript letter “y” immediately following the R enclosed in parentheses, represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.

[0098] For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: “Cn” defines the exact number (n) of carbon atoms in the group / class. “C<n” defines the maximum number (n) of carbon atoms that can be in the group / class, with the minimum number as small as possible for the group / class in question. For example, it is understood that the minimum number of carbon atoms in the groups “alkyl(c<8)”, “cycloalkanediyl(c<s)”, “heteroaryl(c<8)”, and “acyl(c<8)” is one, the minimum number of carbon atoms in the groups “alkenyl(c<8 ”, “alkynyl(c<8)”, and “heterocycloalkyl(c<s " is two, the minimum number of carbon atoms in the group “cycloalky l(c<si” is three, and the minimum number of carbon atoms in the groups “aryl(c<8)” and “arenediyl(c<8)” is six. “Cn-n'” defines both the minimum (n) and maximum number (n') of carbon atoms in the group. Thus, “alkyl(C2-io)” designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms “C5 olefin”, “C5-olefin”, “olefines / ’, and “olefines” are all synonymous. When any of the chemical groups or compound classes defined herein is modified by the term “substituted”, any carbon atom in the moiety replacing the hydrogen atom is not counted. Thus methoxyhexyl, which has a total of seven carbon atoms, is an example of a substituted- 41 - 4909-0910-3750, v. 3alkyl(ci-6). Unless specified otherwise, any chemical group or compound class listed in a claim set without a carbon atom limit has a carbon atom limit of less than or equal to twelve.

[0099] The term “saturated” when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine / enamine tautomerism are not precluded. When the term “saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.

[0100] The term “aliphatic” signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic compound or group. In aliphatic compounds / groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds / groups can be saturated, that is joined by single carboncarbon bonds (alkanes / alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes / alkenyl) or with one or more carbon-carbon triple bonds (alkynes / alkynyl).

[0101] The term “aromatic” signifies that the compound or chemical group so modified has a planar unsaturated ring of atoms with 4n +2 electrons in a fully conjugated cyclic n system.

[0102] The term “alkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups -CH3 (Me), -CH2CH3 (Et), -CH2CH2CH3 (z / -Pr or propyl), -CH(CH₃)₂ (i-Pr, iPr or isopropyl), -CH2CH2CH2CH3 (zi-Bu), -CH(CH₃)CH₂CH₃ (sec-butyl), -CH2CH(CH3)2 (isobutyl), -C(CH₃)₃ (tert-butyl, t-butyl, t-Bu or tBu), and -CH₂C(CH₃)₃ (neopentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. The term “alkylidene” refers to the divalent group =CRR' in which R and R' are independently- 42 - 4909-0910-3750, v. 3hydrogen, alkyl, aryl, or heteroaryl. Non-limiting examples of alkylidene groups include: =CH2, =CH(CH₂CH₃), and =C(CH3)2. An “alkane” refers to the class of compounds having the formula H-R, wherein R is alkyl as this term is defined above.

[0103] The term “cycloalkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forming part of one or more nonaromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to a carbon atom of the non-aromatic ring structure. The term “cycloalkanediyl” refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double ortriple bonds, and no atoms other than carbon and hydrogen. The groupjs a non_ limiting example of cycloalkanediyl group. A “cycloalkane” refers to the class of compounds having the formula H-R, wherein R is cycloalkyl as this term is defined above.

[0104] The term “alkenyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2(allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term “alkenediyl” refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched acyclic structure, at least one nonaromatic carboncarbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms “alkene” and “olefin” are synonymous and refer to the class of compounds having the formula H-R, wherein R is alkenyl as this term is defined above. Similarly, the terms “terminal alkene” and “a-olefin” are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.- 43 - 4909-0910-3750, v. 3

[0105] The term “alkynyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH₃, and -CH₂C≡CCH₃ are non-limiting examples of alkynyl groups. An “alkyne” refers to the class of compounds having the formula H-R, wherein R is alkynyl. When any of these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H. -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0106] The term “aryl” refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structures, each with six ring atoms that are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e.g., 4-phenylphenyl). The term “arenediyl” refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structures, each with six ring atoms that are all carbon, and wherein the divalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. Non-limiting examples of arenediyl groups include:- 44 - 4909-0910-3750, v. 3An “arene” refers to the class of compounds having the formula H-R, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes.

[0107] The term “aralkyl” refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.

[0108] The term “heteroaryl” refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused. The term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms. Nonlimiting examples of heteroaryl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl.

[0109] The term “heteroarenediyl” refers to a divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, said atoms forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused; however, the term heteroarenediyl does not preclude the presence of one or more alkyl or aryl groups (carbon- 45 - 4909-0910-3750, v. 3number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroarenediyl groups include:The term ‘W-heteroaryl” refers to a heteroaryl group with a nitrogen atom as the point of attachment. A “heteroarene” refers to the class of compounds having the formula H-R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.

[0110] The term “heteroaralkyl” refers to the monovalent group -alkanediyl-heteroaryl, in which the terms alkanediyl and heteroaryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: pyridinylmethyl and 2-quinolinyl-ethyl.

[0111] The term “heterocycloalkyl” refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the non-aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings are fused. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl.

[0112] The term “heterocycloalkanediyl” refers to a divalent cyclic group, with two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the two points of attachment, said atoms forming part of one or more ring structure(s) wherein at least one of the ring atoms of the non-aromatic ring stmcture(s) is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings are fused. As used herein, the term- 46 - 4909-0910-3750, v. 3heterocycloalkanediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include:The term ‘W-heterocycloalkyl” refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. A-pyrrolidinyl is an example of such a group.

[0113] The term “acyl” refers to the group -C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, or aryl as those terms are defined above. The groups, -CHO, -C(O)CH3 (acetyl, Ac), -C(O)CH₂CH₃, -C(O)CH(CH3)2, -C(O)CH(CH₃)₂, -C(O)C6H5, and -C(O)C₆H₄CH₃ are non-limiting examples of acyl groups. A “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group -C(O)R has been replaced with a sulfur atom, -C(S)R. The term “aldehyde” corresponds to an alkyl group, as defined above, attached to a -CHO group.

[0114] The term “alkoxy” refers to the group -OR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), or -OC(CH3)3 (tert-butoxy). The terms “cycloalkoxy”, “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”, “heterocycloalkoxy”, and “acyloxy”, when used without the “substituted” modifier, refers to groups, defined as -OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term “alkylthio” and “acylthio” refers to the group -SR, in which R is an alkyl and acyl, respectively. The term “alcohol” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. The term “ether” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group.

[0115] The term “alkylamino” refers to the group -NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -NHCH3 and -NHCH2CH3. The term “dialkylamino” refers to the group -NRR', in which R and R' can be the same or different alkyl groups. Non-limiting examples of dialkylamino groups include: -N(CH3)2and- 47 - 4909-0910-3750, v. 3-N(CH3)(CH2CH3). The terms “cycloalkylamino”, “alkenylamino”, “alkynyl amino”, “arylamino”, “aralkylamino”, “heteroarylamino”, “heterocycloalkylamino”, and “alkoxyamino” when used without the “substituted” modifier, refers to groups, defined as -NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkoxy, respectively. A non-limiting example of an arylamino group is -NHCeHs. The terms “dicycloalkylamino”, “dialkenylamino”, “dialkynylamino”, “diarylamino”, “di aralkylamino”, “diheteroarylamino”, “diheterocycloalkylamino”, and “dialkoxyamino”, refers to groups, defined as -NRR', in which R and R' are both cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkoxy, respectively. Similarly, the term alkyl(cycloalkyl)amino refers to a group defined as -NRR', in which R is alkyl and R' is cycloalkyl. The term “amido” (acylamino), when used without the “substituted” modifier, refers to the group -NHR, in which R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(O)CH3.

[0116] When a chemical group is used with the “substituted” modifier, one or more hydrogen atom has been replaced, independently at each instance, by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. For example, the following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2C1, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2C1. The term “haloalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. -F, -Cl, -Br, or -I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, -CH2C1 is a nonlimiting example of a haloalkyl. The term “fluoroalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3are nonlimiting examples of fluoroalkyl groups. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-l-yl. The groups, -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3(methylcarboxyl), -CO2CH2CH3, -C(O)NH2(carbamoyl), and -CON(CH3)2, are non-limiting examples of substituted acyl groups. The groups -NHC(O)OCH3and -NHC(O)NHCH3are non-limiting examples of substituted amido groups.- 48 - 4909-0910-3750, v. 3IV. Examples

[0117] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Ultrasound Programmable Drug Release

[0118] An ultrasound-responsive drug delivery system using HOF nanoparticles was developed to achieve precise, on-demand pain relief and functional recovery in nerve injury. By leveraging a double-solvent method, both hydrophilic and lipophilic anesthetic drugs (lidocaine hydrochloride, bupivacaine) were encapsulated with high efficiency (20%-52%), enabling targeted and controlled drug release upon FUS stimulation.

[0119] HOF nanoparticles were synthesized using a double-solvent method, enabling the co-loading of lipophilic and hydrophilic drugs, including lidocaine hydrochloride and bupivacaine. These nanoparticles achieved a high drug-loading efficiency (20%-52%) and were characterized using dynamic light scattering analysis to confirm size distribution and stability. FUS stimulation was applied to trigger localized, controlled drug release, with high-performance liquid chromatography (HPLC) used to quantify release kinetics. In vitro studies employed AAV-transfected neuronal models expressing GCaMP6s, where calcium imaging was performed to assess neuronal activity suppression following ultrasound-triggered drug release.

[0120] FIG. 1 provides illustrations of the preparation of ultrasound mechanically responsive hydrogen-bonded organic frameworks (HOFs). Four representative organic monomers and the self-assembled porous HOF structures tested are HOF-TATB, HOF-BTB, HOF-101 and HOF-102 (FIG. 1A). Ultrasound mechanical stress may trigger dissociation of HOFs, as depicted in FIG. IB. Different HOF materials show differing drug loading capacities for lidocaine and bupivacaine (FIG. 1 C).- 49 - 4909-0910-3750, v. 3

[0121] Size and release control was tested for HOF-TATB nanoparticles. The size of HOF-TATB nanoparticles was controlled by H3TATB concentrations (FIG. 2A). The dye / drug release from TATB HOF nanoparticles correlated to the size after a single 10s pulse at 1.5 MHz and 3 MPa (FIG. 2B). Finally, the amount of dye / drug released per ultrasound stimulation was monitored (FIG. 2C).

[0122] The versatility and stability of drug loading of HOF-TATB nanoparticles was tested. Hydrophobic drugs, including lidocaine, bupivacaine, isradipine, ibuprofen, and glibenclamide were loaded in HOF nanoparticles by a double-solvent method, and percent loading was measured (FIG. 3A). The long-term stability of lidocaine-loaded HOF nanoparticles in 10% FBS was measured as the remaining drug loading percentage over 40 d (FIG. 3B). Short-term stability of lidocaine-loaded HOF nanoparticles in 50% FBS, was also measured as the remaining drug loading percentage over 50h (FIG. 3C).

[0123] Ultrasound controlled lidocaine release from HOF-TATB nanoparticles, and their in vitro inhibition of neural activity, was tested. FIG. 4A is a schematic of ultrasound triggered drug release from the HOF-TATB nanoparticles. Fluorescence images were taken of the primary cortical neurons expressing hSyn:: GCaMP6s-WPRE-SV40 (FIG. 4B). FIG. 4C depicts heat maps of normalized GCaMP6s fluorescence imaging from 50 cultured neurons expressing hM3D (Gq) under ultrasound inhibition of lidocaine (lido) release from TATB nanoparticles (1.5 MHz, 1.40 MPa, pulse 10s). FIG. 4D depicts normalized in vitro neuron spiking latency statistical analysis under FUS stimulation.Example 2 - Applications for Pain Management

[0124] In a rat sciatic nerve injury model, this strategy demonstrated prolonged pain relief, with VF tests confirming sustained anesthetic effects up to one-week post-treatment. Moreover, ultrasound-triggered drug release allowed precise control over anesthesia duration, while behavioral assessments (TC rates, SFI scores, VF tests) highlighted significant improvements in pain management and motor function recovery.

[0125] For in vivo evaluation, a rat sciatic nerve injury model was used to assess the therapeutic efficacy of HOF-TATB@lidocaine hydrochloride and HOF-TATB@bupivacaine nanoparticles. These formulations demonstrated strong anesthetic effects, with von Frey (VF) tests showing sustained pain relief even one-week post-treatment. Additionally, VF tests- 50 - 4909-0910-3750, v. 3revealed that ultrasound-triggered drug release enabled precise, time-dependent anesthesia, with prolonged ultrasound exposure enhancing the anesthetic duration. Behavioral assessments, including TC rates, SFI scores, and VF tests, were conducted to evaluate the impact of ultrasound-controlled pain management and motor function recovery.

[0126] Ultrasound controlled lidocaine release in HOF HOF-TATB nanoparticles for in vivo inhibition of pain was tested. A schematic of ultrasound-triggered local drug release in the sciatic nerve is depicted in FIG. 5A. The dorsal von Frey test was performed on mice (FIG.5B). Longer ultrasound exposure (8 min > 4 min > 1 min) enhances drug release from HOF nanoparticles, increasing and prolonging VF thresholds, demonstrating precise ultrasound control of anesthesia (FIG. 5C). Von Frey data analysis from four rats injected with TATB ©lidocaine HC1, followed by an 8-minute ultrasound treatment over one week demonstrates efficacy of the treatment (FIG. 5D). The same analysis was conducted for a 4-minute ultrasound treatment (FIG. 5E).

[0127] Ultrasound controlled Bupivacine release in HOF HOF-TATB nanoparticles for in vivo inhibition of pain and the effects of drug release on animal behaviors was also tested. Von Frey mechanical data from 5 rats injected with TATB@bupivacaine, show sustained hyposensitivity (FIG. 6A). Statistical analysis of Von Frey thresholds in the same rats confirmed significant differences post-FUS (FIG. 6B). Von Frey data from 3 control rats (TATB@RhB) show no hyposensitivity (FIG. 6C). Statistical analysis of Von Frey data from the same control rats show no significant changes (FIG. 6D). Toe chewing assessments in rats treated with TATB@bupivacaine and TATB@ lidocaine indicate reduced pain (FIG. 6E). Sciatic functional index (SFI) assessments show improved functional recovery in treated rats (FIG. 6F).

[0128] This work presents a novel, non-invasive approach for trauma -induced nerve damage and chronic pain management, offering a safer, tunable alternative to conventional treatments. Future studies will focus on clinical translation, pharmacokinetic optimization, and broader neurological applications.- 51 - 4909-0910-3750, v. 3Example 3 - Ultrasound -triggered hydrogen-bonded organic frameworks for long-term, on-demand, and precisely controlled anesthetic release reduce pain responses to traumatic injury of rat sciatic nerves

[0129] Spatiotemporally precise and long-acting drug delivery remains a critical unmet challenge in the treatment of chronic pain and neurological disorders (Guzman et al. 2018; Choudhury et al. 2024; Ghosh et al. 2021). However, current approaches, such as systemic administration, suffer from poor spatial precision, off-target effects, and the need for frequent dosing, which limits their clinical applicability (El Badry Mohamed et al. 2025; Khan et al.2021). While implantable drug delivery systems can improve localization, they require invasive procedures, posing risks of tissue damage, inflammation, and long-term complications (Naqvi et al. 2020; Rwei et al. 2017; Li et al. 2023a; Zhang et al. 2020; Li et al. 2023b). Here, an ultrasound-triggered hydrogen-bonded organic framework (HOF)-based drug delivery system is presented that enables long-term drag stability and precise, on-demand anesthetic release. Unlike conventional liposomal or hydrogel-based carriers prone to burst release, the described HOF nanoparticles provide long-term controlled and sustained drag retention, with ultrasound activation ensuring localized release only when needed. Using a double-solvent synthesis approach, efficient loading of hydrophilic and lipophilic anesthetic agents (lidocaine hydrochloride and bupivacaine) was achieved. In vitro studies demonstrated ultrasound-triggered release kinetics and neuronal suppression in adeno-associated virus (AAV)-transfected neurons, confirming precise anesthetic control. In vivo, a rat sciatic nerve injury model showed prolonged pain relief and improved motor recovery, validated by von Frey tests, toe chewing analysis, and Sciatic Functional Index (SFI) measurements. This programmable and non-invasive HOF-based system offers a transformative solution for long-term pain management, with potential extensions to other neurological indications, including epilepsy and neuroinflammation.

[0130] Advanced drug delivery strategies should ensure stable and sustained release but also enable targeted neuronal modulation to achieve controlled inhibition or activation as needed (Jain et al 2009; Özdemir et al. 2017). Incorporating neuromodulatory capabilities into drug delivery systems, such as ultrasound-triggered release for precise neural inhibition, can further enhance therapeutic efficacy (Delaney et al. 2022; Nayak et al. 2021). The advancement of stable, on-demand drug release systems holds significant potential in revolutionizing neuromodulation and long-term therapeutic strategies (Delaney et al. 2022). By maintaining- 52 - 4909-0910-3750, v. 3stable drug levels and enabling precise, externally controlled dosing, such platforms improve disease management across conditions like chronic pain and epilepsy. Importantly, long-term, responsive drug delivery is essential for adaptable neuromodulation and enhanced patient autonomy (Delaney et al. 2022; Ali et al. 2023; Achar et al. 2021; Singh et al. 2019; Dai et al.2025).

[0131] While optogenetics remains the gold standard for spatiotemporal neural control in preclinical models, its reliance on light with poor tissue penetration and the need for invasive fiber implantation hinder its clinical utility (Deisseroth et al. 2015; Zhu et al. 2016; Vollmer et al. 2022; Fougere et al. 2021; Etter et al. 2019; Hou et al. 2023; Creed et al. 2015). Similarly, chemogenetic strategies require viral vectors and systemic agonists, limiting their precision and translational potential. These limitations underscore the need for a programmable, minimally invasive delivery system capable of repeatable, localized neuromodulation with clinical translatability.In parallel, chronic pain — particularly neuropathic pain — remains a widespread and intractable condition that necessitates sustained, site-specific analgesia to minimize the need for frequent dosing and avoid systemic side effects. A range of advanced delivery strategies has been investigated to meet this clinical demand. However, traditional drug carriers such as liposomes, polymeric nanoparticles, and micelles typically suffer from low drug-loading efficiencies, requiring high doses or frequent re-administration to maintain therapeutic effects (Huo et al. 2021a; Wang et al. 2022; Kim et al. 2019; Ghanem et al. 2021; Sha et al. 2022). Among clinically approved options, EXPAREL — a liposomal bupivacaine formulation — provides extended release for up to 72 hours, yet its reliance on passive diffusion results in non-programmable dosing, burst release, and inability to re-activate once administered (Vyas et al. 2016). Moreover, EXPAREL exhibits limited spatial precision and often requires high initial loading to achieve effective analgesia, limiting its utility for dynamic or repeatable pain control in chronic settings.

[0132] Polymeric hydrogels and prodrug-based depots have shown improved drug retention and mechanical tunability, especially in postoperative pain models (Zhang et al. 2020; Choi et al. 2024). However, these systems often require in situ gelation or surgical implantation, and lack responsiveness to external stimuli, precluding their use for dynamic, on-demand modulation. Similarly, polymeric prodrugs rely on intrinsic degradation kinetics, which are difficult to tailor to patient-specific or activity-dependent pain. Exosome-based- 53 - 4909-0910-3750, v. 3delivery platforms offer a high degree of biocompatibility and the potential for endogenous targeting (Cai et al. 2024). Nonetheless, they are hampered by poor manufacturing scalability, batch-to-batch variability, and uncontrolled drug release profiles, limiting their translational utility (Cai et al. 2024).

[0133] Ultrasound-triggered liposomes and micelles represent a non-invasive strategy with potential for external activation (Rwei et al. 2017; Li et al. 2023). However, these nanocarriers generally suffer from limited structural robustness, resulting in premature degradation under physiological conditions. Their low drug loading capacity further restricts long-term therapeutic efficacy, often requiring repeated administration. Other remotely activated platforms, such as light- or magnetically triggered drug earners, enable spatiotemporal control in principle but are inherently constrained by limited tissue penetration (light: <1 mm, magnetics: low spatial resolution), and require specialized hardware, which complicates clinical deployment (Zhang et al. 2020; Zhang et al. 2023). Despite significant progress, no existing platform simultaneously fulfills the four key criteria for clinical-grade chronic pain control: long-term drug retention, high drug-loading capacity, non-invasive administration, and precise, re-triggerable release on demand. A delivery system capable of integrating these attributes remains a critical unmet need in pain medicine.

[0134] Metal-organic frameworks (MOFs) and covalent organic frameworks (COFs) have attracted interest as high-capacity drug carriers due to their tunable porosity and modularity (Zheng et al. 2021; Liang et al. 2021; Ge et al. 2024). However, their drug release profiles are often constrained by strong coordination bonds (in MOFs) or covalent linkages (in COFs), which limit release tunability and require harsh external stimuli or prolonged degradation for drug liberation (Albolkany et al. 2021; Wang et al. 2024). Additionally, the incorporation of metal ions in MOFs raises concerns regarding biocompatibility, long-term toxicity, and potential ions leaching in vivo.

[0135] HOFs provide a unique metal-free, highly porous, and biocompatible alternative for drug delivery (Etter et al. 2019; Hou et al. 2023; Creed et al. 2015; Galaj et al.2018). Unlike coordination- or covalent-bonded frameworks (e.g., MOFs, COFs), the architecture of HOFs relies on dynamic and reversible non-covalent interactions, including n-7i stacking and multivalent hydrogen bonding, which collectively endow them with exceptional drug-loading capacities (-32.1 wt%) and responsive mechanical adaptability (Huo et al. 2021a; Wang et al. 2022; Kim et al. 2019; Ghanem et al. 2021; Sha et al. 2022). Importantly, these- 54 - 4909-0910-3750, v. 3weak but structured interactions enable HOF nanoparticles to undergo ultrasound-triggered disassembly, achieving rapid and efficient drug release within seconds under clinically relevant parameters, in stark contrast to disulfide-based polymeric systems or platinum prodrugs, which typically demand high-energy (>15 W / cm2) ultrasound or prolonged exposure (>30 minutes) for activation (Albolkany et al. 2021; Wang et al. 2024). Beyond release responsiveness, HOFs offer molecular programmability. By tuning their building blocks and hydrogen-bonding patterns, release kinetics can be precisely modulated, ranging from single -pulse burst delivery to multi-phase sustained release (Huo et al. 2021a, 2021b, and 2022).

[0136] This release programmability is further enhanced by the described adaptable drug-loading strategies. Using a diffusion-based approach for hydrophilic anesthetics (e.g., lidocaine HC1) and a dual-solvent technique for lipophilic agents (e.g., bupivacaine), the HOF nanocarriers flexibly accommodate diverse drug chemistries while preserving structural integrity and ultrasound sensitivity. By integrating high drug payloads, tunable release profiles, and rapid, externally triggered activation under clinically relevant ultrasound parameters, this HOF-based delivery system offers a unified platform for both short- and long-acting anesthetic delivery. Collectively, the system described herein addresses a longstanding gap in pain management, bridging the need for non-invasive, long-term, and spatiotemporally precise drug release. It opens new therapeutic avenues not only for peripheral nerve injuries, but also for broader neuromodulation and neurological disorder interventions.Synthesis and Characterization of HOFs

[0137] To construct a programmable drug delivery system with both high drugloading efficiency and ultrasound responsiveness, a library of hydrogen-bonded organic frameworks (HOFs) was synthesized using various carboxyl-functionalized aromatic linkers. Four representative frameworks, HOF-TATB, HOF-BTB, HOF-101, and HOF-102 were assembled via self-assembly of 7r-conjugated polycarboxylic acids (Fig. 7c), resulting in crystalline architectures with diverse hydrogen-bonding patterns and pore topologies. These HOFs are held together through dynamic non-covalent interactions, including directional hydrogen bonding and π–π stacking, which not only enable structural tunability but also impart sensitivity to external mechanical stimuli such as focused ultrasound.

[0138] Two complementary drug-loading strategies were employed tailored to the physicochemical properties of the anesthetics (Fig. 7b). For hydrophilic drugs such as lidocaine hydrochloride and bupivacaine hydrochloride, a passive diffusion method was used, immersing- 55 - 4909-0910-3750, v. 3the HOF nanoparticles in aqueous drag solutions. In contrast, lipophilic drag molecules (free-base lidocaine and bupivacaine) were loaded using a double-solvent method (DSM), in which dichloromethane (CHaChj-dissol ved drug was added dropwise to a water suspension of HOFs to enable interfacial diffusion and pore penetration. These methods allow selective and efficient encapsulation of drags with distinct solubility profiles. As shown in Fig. 7d, HOF-TATB and HOF-BTB showed moderate loading capacities (-10 wt%) for both lidocaine HC1 and bupivacaine HC1. In contrast, HOF-101 and HOF-102 achieved the highest drag payloads (-25-30 wt%) via passive diffusion. This enhanced loading performance is likely due to their extended 7i-conjugated systems and larger pore volumes, which promote improved dragframework interactions compared to HOF-TATB and HOF-BTB. For lipophilic bupivacaine, DSM loading yielded even higher retention (-32-35 wt%) (Fig. 7e), significantly outperforming the HC1 salt form bupivacaine HC1. Moreover, the drag remained stably retained even after sequential solvent washes (DCM and acetone) and overnight soaking, highlighting the strong host-guest interactions between the hydrophobic drag and the HOF pore environment.

[0139] Together, these results establish that HOFs provide a highly modular and chemically adaptable platform capable of encapsulating both hydrophilic and hydrophobic anesthetics with high payload, structural stability, and tunable release kinetics, laying the foundation for responsive and long-term therapeutic applications.Ultrasound-Triggered Drug Release and Stability Evaluation

[0140] Next, the stability and ultrasound-responsiveness of bupivacaine or lidocaine -loaded HOFs was investigated to evaluate their potential for sustained and controllable anesthetic delivery. Drug retention under physiological conditions was first assessed by incubating HOF-TATB loaded with lidocaine in 10% and 50% fetal bovine serum (FBS) at 25 °C. The 10% FBS condition mimics a physiologically relevant environment commonly used in cell-based assays, whereas 50% FBS serves as a stringent, protein-rich medium that closely simulates in vivo serum exposure. HOF-TATB loaded with lidocaine retained over most of its payload after 35 days in 10% FBS (Fig. 8a), indicating excellent longterm stability. In 50% FBS, a more challenging environment, drug leakage remained minimal over a 48-hour period (Fig. 8b), underscoring the framework’s strong retention capabilities. Powder X-ray diffraction (PXRD) analysis further demonstrated that the crystalline structure- 56 - 4909-0910-3750, v. 3of HOF-TATB remained largely intact post-serum incubation, with only modest reduction in diffraction peak intensity (Fig. 8c), confirming structural resilience under physiological stress.

[0141] The ultrasound-triggered release profile of bupivacaine-loaded HOFs was then tested under varying acoustic power. The ultrasound-triggered release experiments demonstrated that the percentage of drug released increased with the ultrasound peak pressure used (Fig. 8c), confirming the system’s capacity for on-demand, repeatable, and programmable dosing. Upon exposure to focused ultrasound, bupivacaine release increased with acoustic intensity, achieving 22.3 ± 1.7% release within 150 s under 80% amplitude (Fig. 8c). In contrast, only 16.4 ± 0.8% release was observed under 50% amplitude, highlighting the tunability of drug release with ultrasound strength. Moreover, bupivacaine release from HOF-TATB could be repeatedly triggered by sequential ultrasound pulses (20 s per pulse), demonstrating stepwise and cumulative drug release. Four successive activations led to incremental release levels of 5.5 ±0.5%, 7.4 ±0.5%, 9.2 ±0.4%, and 10.4 ±0.6%, respectively (Fig. 8e), highlighting the programmable and re -triggerable nature of this delivery system. In contrast to conventional passive release formulations, this ultrasound-triggered strategy enables dynamic dosing control in response to clinical needs. To evaluate the drug compatibility and generalizability of the platform, the loading efficiency of multiple therapeutic compounds with distinct hydrophobicity were tested, including lidocaine and bupivacaine (local anesthetics), isradipine (a calcium channel blocker for hypertension and neuroprotection), ibuprofen (a non-steroidal anti-inflammatory drug), and glibenclamide (a sulfonylurea for type 2 diabetes and neuroinflammation) (Fig. 8f).

[0142] As shown in Fig. 8f, most tested drugs demonstrated high encapsulation efficiencies, with ibuprofen and isradipine exceeding 30% wt, underscoring the broad chemical compatibility of the HOF-based carriers. Notably, among the tested frameworks, HOF-101 and HOF-102 consistently outperformed others in drag loading for both lidocaine and bupivacaine, reaching up to -30% under optimized conditions (Fig. 7e). This performance is attributed to their extended ^-conjugated systems and enlarged pore volumes, which facilitate favorable host-guest interactions across structurally diverse small molecules. Together, these findings validate the HOF platform as a serum-stable, ultrasound-responsive, and structurally tunable delivery vehicle capable of non-invasive, long-term, and spatiotemporally controlled anesthetic release — representing a clinically translatable strategy for chronic pain and potentially other neurological indications.- 57 - 4909-0910-3750, v. 3

[0143] To validate the neuromodulatory functionality of the HOF-based nanocarriers, calcium imaging in cultured neurons was next performed. Such in vitro testing provides a critical proof-of-concept for evaluating the platform's ability to modulate neuronal excitability in a spatially and temporally controlled manner, which is essential for future neurotherapeutic translation. Primary neurons were transduced with AAV carrying GCaMP6s, enabling real-time optical recording of intracellular Ca2+fluctuations as a proxy for neuronal firing activity (Fig. 9a).

[0144] Neurons were first incubated with HOF-TATB@ lidocaine nanoparticles, during which robust and spontaneous calcium oscillations persisted, indicating that the encapsulated drug remained inactive under basal conditions (Fig. 9b). Upon exposure to a focused ultrasound pulse (1.5 MHz, 1.40 MPa, 10 s), a rapid and widespread suppression of calcium transients was observed across the majority of neurons (Fig. 9c-d). This outcome confirms effective ultrasound-triggered release of lidocaine from the HOF matrix, resulting in acute silencing of neuronal activity. Notably, such inhibition was achieved without direct chemical stimulation or fiber implantation, underscoring the noninvasive nature of this approach. These findings establish a foundational demonstration of ultrasound-triggered neuronal modulation that is spatially precise, temporally controllable, and potentially reversible upon anesthetic clearance — offering a promising strategy for remote, noninvasive regulation of neural excitability. This result not only validates the successful construction and functional delivery of the anesthetic payload but also highlights the therapeutic potential of the platform in programmable neuromodulation scenarios, such as peripheral pain relief or CNS circuit silencing. Subsequent in vivo studies were performed to further explore these translational implications.In Vivo Ultrasound-Triggered Analgesia and Programmable Pain Modulation

[0145] Lidocaine hydrochloride is widely used for local anesthesia and nerve block due to its rapid onset of action. However, its short half-life (~90 minutes), hydrophilic nature, and rapid systemic clearance severely limit its utility for sustained or programmable analgesia. Conventional injection leads to transient pain relief, necessitating frequent re-administration and increasing the risk of systemic toxicity. Although liposomal encapsulation and polymeric prodrug approaches have extended anesthetic residence times, these systems lack external controllability and re-triggerability, making them unsuitable for dynamic pain management.- 58 - 4909-0910-3750, v. 3To evaluate the translational potential of the HOF-based delivery platform under stringent conditions, lidocaine HC1 was selected as a model drug. Using the diffusion-loading method, lidocaine HC1 was efficiently encapsulated in HOF-TATB nanoparticles (loading ~9.8 wt%), aided by multivalent hydrogen bonding and π–π stacking interactions with the framework scaffold. This enabled stable retention in physiological conditions and enabled ultrasound-responsive, on-demand drug release in vivo.

[0146] Sprague-Dawley rats were employed to establish a sciatic nerve block model, with ultrasound imaging-guided perineural injection of HOF-TATB ©lidocaine HC1 (Fig. 10a). Following administration, von Frey (VF) filament tests were conducted to quantify nociceptive thresholds (Fig. 10b). Upon focused ultrasound stimulation (1.5 MHz, 1.40 MPa), a significant and programmable increase in pain threshold was observed, with the analgesic duration and intensity strongly correlated with ultrasound exposure time. As shown in Fig. 10c, a single 8-minute ultrasound stimulus elicited a marked increase in nociceptive threshold that persisted for over 4 hours, whereas shorter exposures (1 or 4 minutes) produced only transient elevations lasting less than 1 hour. This dose-dependent effect underscores the programmable nature of the system, in which the duration of ultrasound can be finely tuned to modulate the intensity and longevity of analgesia. Such temporal scalability enables personalized treatment strategies for episodic or chronic pain management with minimal invasiveness.

[0147] To assess the durability and reusability of analgesia from the HOF-TATB ©lidocaine platform, daily FUS stimulation was administered over a 7-day period. An 8-minute ultrasound regimen elicited a consistent and significant elevation of nociceptive thresholds after each stimulation (Fig. lOd), indicating robust and repeatable analgesic activation. Notably, even a shorter 4-minute stimulation produced transient analgesic responses, particularly on the first day (Fig. lOe), but failed to sustain significance in subsequent sessions. These findings underscore the importance of sufficient acoustic dosing to mobilize lidocaine from the HOF matrix and further demonstrate the platform’s dose-tunable and re-triggerable drug release profile, enabling customizable therapeutic windows for chronic pain management.

[0148] To assess the spatial localization and retention of anesthetic agents, mass spectrometry imaging (MSI) on excised sciatic nerve tissue post-FUS was performed. This label-free, high-resolution imaging modality provides chemical mapping of lidocaine distribution, serving as orthogonal validation of drug localization and minimizing concerns of- 59 - 4909-0910-3750, v. 3systemic leakage. While MSI data are currently being processed, this analysis will further confirm the spatiotemporal precision and local targeting capability of the nanocarrier system. Collectively, these findings establish HOF-TATB ©lidocaine HC1 nanoparticles as an effective and non-invasive platform for programmable, on-demand pain management. The use of lidocaine HC1 — despite its inherently rapid clearance — demonstrates the capacity of the HOF system to prolong local drug presence and control release kinetics. In subsequent studies, this platform was extended to encapsulate lipophilic anesthetics such as bupivacaine, which can further enhance duration and depth of nerve block via deeper pore penetration and sustained retention.

[0149] Having established the feasibility of ultrasound-triggered release of hydrophilic anesthetics using lidocaine HC1, it was sought to evaluate the long-term stability and therapeutic durability of the platform using a lipophilic anesthetic — bupivacaine. Due to its higher hydrophobicity, bupivacaine is expected to exhibit stronger retention within the hydrophobic HOF-TATB pore channels, thereby enabling sustained in vivo localization and prolonged release upon ultrasound activation. To assess this, Sprague-Dawley rats received ultrasound imaging-guided perineural injections of HOF-TATB ©bupivacaine nanoparticles near the sciatic nerve. Using previously optimized FUS parameters (1.5 MHz, 1.40 MPa, 0.2 s on I 0.8 s off pulse cycles, total 8 min), the nanoparticle depot was stimulated once daily for five consecutive days. To evaluate the long-term in vivo performance of the system, repeated von Frey tests were performed following daily ultrasound activation. Animals injected with HOF-TATB ©bupivacaine exhibited a robust and repeatable elevation in nociceptive threshold following each FUS pulse, sustained across the entire five-day testing period (Fig. lOf-g). These results confirm the capability of the system for durable drug retention and reliable, on-demand analgesia over extended timescales. In contrast, control animals receiving unloaded HOF-TATB nanoparticles showed no significant change in mechanical sensitivity despite identical FUS stimulation (Fig. lOh-i), ruling out material-related or sonomechanical effects. Importantly, the consistent efficacy across multiple days underscores the long-term structural stability of the HOF nanocarrier in vivo and its potential for chronic pain applications requiring repeated intervention.- 60 - 4909-0910-3750, v. 3Modeling Neuropathic Pain and Motor Dysfunction after Sciatic Nerve Injury

[0150] Chronic peripheral nerve injury, such as trauma to the sciatic nerve, remains a significant clinical challenge (Dahlhamer et al. 2018; Reif et al. 2018). Each year, approximately 559,000 cases of peripheral nerve injury (PNI) are reported in the United States alone, contributing to a global market exceeding $1.68 billion in surgical and pharmacologic interventions (Gaskin et al. 2012; Smith et al. 2019; Da Silva et al. 2014). However, traditional strategies, including neurorrhaphy, suffer from low regenerative success, while systemic antipain medications pose risks of addiction, side effects, and low spatial specificity (Rehni et al.2013; Corder et al. 2018; Kiyatkin et al. 2019). Patients often develop neuropathic pain and sensorimotor dysfunction, manifesting as self-mutilation (toe chewing) and loss of locomotor control, emphasizing the need for localized, on-demand drug delivery solutions (Sporel-Ozakat et al. 1991; Jubran and Widenfalk 2003; Al-Adawi et al. 2002; Ji et al. 2023; Wang et al. 2016).

[0151] To evaluate the long-term therapeutic efficacy and clinical relevance of the described ultrasound-responsive HOF nanoplatform, a rat model of sciatic nerve transection and repair was employed — an established paradigm that recapitulates key hallmarks of chronic neuropathic pain, including spontaneous pain behavior and motor dysfunction (Brushart et al. 2011). Following surgical transection and microsuture-based repair of the sciatic nerve, HOF-TATB nanoparticles encapsulating either bupivacaine or lidocaine were perineurally injected at the injury site (Fig. Ila), and animals subsequently received daily focused ultrasound stimulation (FUS, 1.5 MHz, 8 min) to trigger on-demand anesthetic release. Neuropathic pain progression was quantified by assessing self-mutilation (toe-chewing, TC), a hallmark behavioral correlate of spontaneous dysesthesia in rodent models. Compared to untreated controls, both HOF-TATB@bupivacaine and HOF-TATB@lidocaine significantly suppressed TC severity, with the lidocaine group exhibiting near-complete behavioral rescue over the 6-week observation window (Fig. 1 lb). This sustained suppression of maladaptive pain behavior underscores the platform’s ability to durably modulate aberrant neural activity in a noninvasive, programmable manner. Importantly, these results not only highlight the analgesic efficacy of the HOF delivery system under chronic pathological conditions but also reveal its potential as a clinically translatable strategy for long-term neuromodulation following peripheral nerve trauma or repair.

[0152] Motor recovery was assessed in parallel using the Sciatic Functional Index (SFI), a well-established metric of hindlimb coordination that reflects neuromuscular integrity- 61 - 4909-0910-3750, v. 3following peripheral nerve injury. Animals treated with HOF-TATB@bupivacaine demonstrated marked and sustained improvements in SFI scores over time, indicative of functional reinnervation and recovery of locomotor control (Fig. 11c). Moderate gains were also observed in the HOF-TATB@lidocaine-treated group, whereas untreated controls showed negligible recovery. The concurrent restoration of both sensory (toe-chewing) (Ji et al. 2023) and motor (SFI) (Brushart et al. 2011) functions strongly suggests that ultrasound-programmed anesthetic delivery not only relieves neuropathic pain but also facilitates neurofunctional rehabilitation in the chronic phase of injury. This may result from the minimization of aberrant afferent activity and inflammation at the nerve repair site, thereby enabling more favorable conditions for axonal regeneration and functional reintegration. Taken together, these findings position the HOF-based delivery system described herein as a unique neuromodulatory platform — capable of non-invasively reshaping pathological nerve activity, restoring behavioral output, and offering a programmable alternative to current chronic pain and neurorehabilitation therapies.Comparison with current anesthetic delivery systems and clinical implications

[0153] Current anesthetic delivery platforms — including liposomes (e.g., EXPAREL), hydrogels, micelles, and polymeric depots — each address specific challenges in sustained pain management, yet none simultaneously achieve long-term retention, high loading efficiency, non-invasive reactivation, and precise spatiotemporal control. Most rely on passive release mechanisms, lack modular programmability, or require surgical implantation, ultimately limiting their adaptability for chronic or activity-dependent pain scenarios.

[0154] By contrast, the ultrasound-triggered HOF nanoplatform uniquely integrates all four critical attributes into a single system: (1) exceptional drug-loading capacities (-32.1 wt%) via tailored hydrogen-bonding and π–π stacking, (2) robust retention in serum-rich environments for over one month, (3) re-triggerable release under clinically safe acoustic conditions, and (4) modular adaptability for both short- and long-acting anesthetics. This integrative capability enables both acute and chronic pain modulation with a single injectable carrier, offering dynamic responsiveness to changing pain states without invasive readministration. Moreover, the ability to fine-tune drug kinetics and release timing through framework engineering and external stimulation elevates this strategy from a drug depot to a programmable neuromodulation interface.- 62 - 4909-0910-3750, v. 3

[0155] Together, these findings position the described HOF-based nanoplatform as a clinically relevant and mechanistically distinct alternative to current anesthetic systems, with potential applications extending from perioperative care to long-term management of neuropathic and inflammatory pain.Conclusions

[0156] The data provided herein and above establishes a versatile, ultrasound-triggered HOF nanoplatform for on-demand and long-term delivery of anesthetic agents. By engineering the framework’s hydrogen-bonding network and π–π interactions, high drugloading efficiencies, exceptional aqueous stability, and fast, repeatable drug release under clinically relevant ultrasound parameters were achieved. Through dual-solvent loading, both hydrophilic (lidocaine HC1) and lipophilic (bupivacaine) anesthetics were efficiently encapsulated and released with spatial and temporal precision. In vivo studies confirmed effective peripheral nerve blockade and pain suppression, both in unoperated and sciatic nerve injury models, sustained over multiple weeks. Compared to clinically used passive -release systems such as EXPAREL and experimental ultrasound-responsive liposomes, the HOF-based platform uniquely offers programmable activation, high payload retention, and tunable pharmacokinetics — without the need for implantable depots or repeated dosing.

[0157] This programmable system bridges a critical gap in neuromodulation and pain management by enabling non-invasive, externally controlled therapy with minimal systemic exposure. Beyond anesthetic delivery, the modularity of the HOF framework suggests broad applicability for treating neurological, inflammatory, and metabolic conditions that demand precise spatiotemporal drug control, including epilepsy, where localized, on-demand suppression of aberrant neuronal firing could offer significant therapeutic advantage. As such, this work lays the foundation for next-generation, ultrasound-guided theranostic platforms in both clinical and translational settings.Example 4 - A machine-learning-guided hydrogen-bonded organic framework for longterm, ultrasound-triggered pain therapy

[0158] Effective treatment of chronic pain remains hindered by the lack of dmg delivery systems that simultaneously achieve long-term stability, high spatial precision, and non-invasiveness (Guzman et al. 2018; Choudhury et al. 2024; Ghosh et al. 2021). Here, a programmable, ultrasound-responsive drug delivery platform based on hydrogen-bonded organic framework (HOF) nanoparticles was utilized, enabling on-demand anesthetic release- 63 - 4909-0910-3750, v. 3with long-term and durable analgesic efficacy. A machine learning (ML)-guided screening pipeline was developed to evaluate over 250 FDA-approved drugs, spanning both hydrophilic and lipophilic agents, and identified bupivacaine (lipophilic) and lidocaine hydrochloride (hydrophilic) as optimal candidates. Both agents were efficiently encapsulated into HOF nanoparticles via diffusion and double-solvent methods. Ultrasound-triggered drug release in vitro transiently suppressed calcium signaling in adeno-associated virus (AAV)-transfected neurons. In vivo, ultrasound-activated release of bupivacaine or lidocaine HC1 at the sciatic nerve site significantly elevated mechanical nociceptive thresholds for up to seven days, reduced autotomy (self-mutilaion) behavior, and improved motor function in a rat model of chronic pain. Notably, ML-selected candidates exhibited both high encapsulation efficiency and superior therapeutic performance, directly linking computational prediction to biological efficacy. This work establishes a clinically translatable, non-invasive, and externally programmable strategy for spatiotemporally precise pain management, with broader potential for neurological and neuromodulatory applications.

[0159] Effective management of pain remains hindered in part due to the lack of drug delivery systems that can simultaneously provide high spatiotemporal precision, long-term efficacy, and non-invasiveness (Guzman et al. 2018; Choudhury et al. 2024). In particularly, chronic pain affects over 20% of adults worldwide and 6.9% of adults have experienced high-impact chronic pain, leading to major healthcare costs and decreased productivity (Dahlhamer 2018; Reif et al 2018). Among chronic pain conditions, peripheral nerve injury (PNI) affects hundreds of thousands annually (Gaskin and Richard 2012; Smith et al. 2019; Da Silva 2014). Surgical repair often fails to restore full function, and systemic analgesics carry risks of addiction, adverse effects, and poor spatial specificity (Rehni et al. 2013; Corder et al. 2018; Kiyatkin et al. 2019). Patients frequently develop neuropathic pain and motor deficits, including self-mutilation and impaired locomotion (Sporel-Ozakat et al. 1991; Jubran et al.2003; Al-Adawi et al. 2002; Ji et al 2023; Wang et al. 2016), underscoring the need for localized, on-demand drug delivery.

[0160] While conventional analgesics — including non-steroidal anti-inflammatory drugs and opioid-based medications — provide rapid symptom relief, their long-term use raises serious concerns regarding addiction, tolerance, and systemic side effects (Zeng et al. 2025; Flavin et al. 2025; Rosner et al. 2023). Opioids, in particular, have driven a major public health crisis due to misuse and dependence. These safety concerns have motivated interest in localized- 64 - 4909-0910-3750, v. 3or non-pharmacological strategies such as electrical stimulation, cooling, and optogenetic neuromodulation; however, their clinical applicability remains restricted by invasiveness, hardware complexity, or short-lived efficacy. Together, these limitations highlight the need for next-generation therapeutic platforms that offer non-invasive, spatiotemporally precise, and durable modulation of peripheral nerve activity (Jayathilake et al. 2025).

[0161] To address these unmet needs, next-generation drug delivery platforms must not only ensure long-term drug stability and sustained release but also provide spatiotemporal control over therapeutic action (Jain et al. 2009; Özdemir et al. 2017). In particular, the integration of neuromodulatory capabilities — such as externally triggered, localized drug release — enables precise inhibition or activation of neural circuits, offering significant advantages for treating pain and other neurological disorders (Delaney et al. 2022; Nayak et al.2021). Among various modalities, ultrasound provides a promising non-invasive trigger for deep-tissue drug release, allowing on-demand modulation with minimal systemic exposure. By combining prolonged retention, responsive release, and precise targeting, such systems can significantly improve patient autonomy and therapeutic outcomes in chronic disease management (Delaney et al. 2022; Ali and Baboota 2023; Achar et al. 2021; Singh et al 2019; Dai et al. 2025).

[0162] Currently, most ultrasound-sensitive platforms rely on gas-filled microbubbles or thermosensitive lipids, which exhibit poor stability and are difficult to precisely control in vivo. Recently, hydrogen-bonded organic frameworks (HOFs) have emerged as promising metal-free, biocompatible, and highly porous materials for drug delivery (Galaj et al. 2018; Etter et al. 2019; Hou et al. 2023; Creed et al. 2015), featuring dynamic π–π stacking and hydrogen bonding interactions that enable high drug-loading capacity (—30 wt%) and mechanical responsiveness (Huo et al. 2021; Wang et al. 2022; Kim et al. 2019; Ghanem et al. 2021; Sha et al. 2022). However, three critical challenges remain for advancing HOFs toward clinically meaningful pain therapeutics. First, the encapsulation of hydrophobic drugs in HOFs has not been demonstrated for potent anesthetics or neuromodulators. Many clinically important pain therapeutics are highly lipophilic — facilitating membrane penetration but also causing poor aqueous solubility, rapid systemic clearance, and off-target effects (Creed et al. 2015). Achieving high-capacity loading together with long-term retention and spatiotemporally precise release would greatly expand the therapeutic scope of HOFs. Second, no systematic, predictive framework exists to identify drugs most compatible with a given- 65 - 4909-0910-3750, v. 3HOFs structure for specific biomedical applications. Current studies rely on trial-and-error screening, which is time-consuming, resource-intensive, and prone to overlooking high-potential candidates. Third, ultrasound-activated drug release for pain management has not been validated in clinically relevant injury models. Most studies are conducted in healthy animals, which fail to replicate the inflammatory, neuropathic, and motor-deficit environment following nerve injury (Rwei et al. 2017; Mannhold et al. 2008).

[0163] A variety of non-invasive or minimally invasive analgesic formulations have been reported — including microneedles, liposomes, hydrogels, and light- / pH-responsive systems — but none simultaneously provide high loading, durable stability, deep-tissue triggerability, and programmable on-demand release (Table 1). Notably, recent acoustically activatable liposomes can achieve ultrasound-triggered uncaging of local anesthetics, but their low drug-loading capacity and short-lived analgesia underscore the continued need for a more durable and programmable delivery platform (Purohit et al. 2025). Most current platforms rely on passive diffusion or require shallow-penetration stimuli, limiting clinical utility (Li et al.2023; Xue et al. 2024; Li et al. 2024). Therefore, a robust, externally controllable, long-lasting analgesic system remains unmet. Existing clinical pain-management options — including liposomal bupivacaine (EXPAREL), peripheral nerve catheters, intrathecal pumps, and systemic analgesics — also exhibit limitations in duration, invasiveness, or controllability (Table 2).- 66 - 4909-0910-3750, v. 3Table 1. Summary of reported non-invasive / minimally invasive pain management.Materials Drug loading content Application Stimulus Threshold Stimulus- Drug release Programmabl Reference (wt%) site energy for triggered kinetics (typical eactivation drug response time / modulation?release extent)sensitivityHOF Lido (-9.8% wt%); Bupi: Sciatic nerve FUS (1.5 >0.79 MPa High Repeatable 7 d; Yes This study (-30% wt%) MHz) (opt. 1.40 duration tunable byMPa) US intensity / timeliposomes Ropivacaine (wt% N / A, Peripheral FUS (250 -0.9 MPa High Rapid release Yes Purohit et al., ac nerve (Sciatic kHz) within 1 min Nat. tive loading ) Nanotechnol.nerve); Brain sonication 2025 regions)Microneedle Ropivacaine microcrystals Hind paw pH (6.8- pH 6.8 Low Sustain No Zhang et al., s Adv. Funct.(hydrophobic) (wt% N / A) 7.0) triggers shell (negligible ed Mater. 2023 rupture at pH 7.4; release;active at in vivopH 6.8) analgesia -72 h- 67 - 4909-0910-3750, v 3Hydrogel Ropivacaine, up to ~41 Sciatic nerve Passive N / A Moderate Sustained release; No Li et al., Adv. wt% diffusion (high in vivo analgesia Sci. 2024 loading, -48 hslowdiffusion)Liposome TTX (wt% N / A) Sciatic nerve Ultrasound Effective Dependent In vivo: initial Yes Rwei et al.,(1 MHz) release on block -8 h; Nat. Biomed.above -1 intensity, repeatable up to 3- Eng. 2017 W / cm2, duty cycle, 4 times within 2typically 3 insonation daysW / cm2, 10 duration.mininsonationMacromolec N / A(T tetracaine covalently Hind paw Blue light 200-300 Highly In vivo: anesthesia Yes. Zhang et al.,Nat.ular prodrug linked prodrug) (400 nm) mW- cm2, sensitive; -20-80 min Repeatable up Commun.irradiation depending to ~5x 2020 2-5 min on energy.liposomes TTX Sciatic nerve None N / A Low; rele In vivo anesthesia No Li et al., Nano (-0.15 (depot) ase slowed up to 70h Lett. 2023 wt%)- 68 - 4909-0910-3750, v 3Aromatized TTX (-0.13 wt%) Sciatic nerve None N / A N / A TTX@Lipo-Ph No Li et al., Nat. liposomes (passive enabled nerve Commun.sustained block up to 70 h. 2023 release)Hydrogel Bupivacaine (wt% N / A) Sciatic nerve None N / A Passive In vivo block 39.9 No Choi et al., sustained h Biomaterials release 2024 Liposomes TTX (<0.1 wt%) Sciatic nerve Passive N / A High in vivo block up to No Huang et al.,Adv. Mater. diffusion (strongly 6.6 d2025 reducedreleas)DNA TTX (N / A wt%) Sciatic nerve Passive N / A High In vivo: block 7.1 h No Wang et al., aptamer / diffusion (sequenceNat.TTX specific Commun. (tetrodotoxi binding) 2023 n) complexesPolymeric TTX (1.2 wt%) Sciatic nerve Passive N / A High In vivo block -9.7 No Xue et al., prodrug diffusion (sequenceh Angew.specific Chem. hit. binding) Ed. 2024- 69 - 4909-0910-3750, v 3Peptides TTX (N / A wt%) Sciatic nerve Passive N / A Passive In vivo block up to No Ji et al., Nat.diffusion sustained ~18 h Biomed. Eng.release 2021 Exosome Bupivacaine (N / A wt%) Sciatic nerve Passive N / A Passive In vivo block up to No Cai et al., diffusion sustained ~12h Adv. Funct.release Mater. 2024PLGA Lidocaine (15.5 wt%) Sciatic nerve Ultrasound ~6 min High In vivo only tested Yes, repeated Xu et al.,(1 MHz) (intensity (repeatabl 2 US triggers (1 h, triggers Front.not reported) e in vivo) 3 h) with transient deplete drug Bioeng.analgesia; repeated early Biotechnol. stimulation may 2022 deplete drug early

[0164] Table 2 Comparison of commonly used clinical pain-management modalities.Treatment ClinicalInjection Frequency Duration of Effect Release Control Invasiveness Comments Modality BurdenEXPAREL Low (local FDA-approved, limited Single local injection ~72 hours (3 days) Passive, burst release Low(liposomal) injection) duration- 70 - 4909-0910-3750, v 3Epidural or HighContinuous infusion or Days to weeks (withperipheral Partial (via pump) Moderate-high (catheter Effective but invasive daily pump)nerve catheter care)Intrathecal High (surgicalContinuous infusion Long-term Programmable Very high Suitable for chronic pain drug pump implant)Systemic oralModerate- analgesics Daily (often multiple Side effects, nonHours None (systemic) None high (side(NSAIDs, times / day) localizedeffects)opioids)Used herein~3-4 injections over 4 >1 week (per Low Low- Precise, tunable, long- (HOF@Drug + High (FUS-triggered)weeks injection) (subcutaneous) moderate acting FUS)-71 - 4909-0910-3750, v 3

[0165] To address key limitations in current pain management platforms, a HOF-based nanocarrier system was developed featuring high drug loading capacity, structural stability, and precise ultrasound-triggered release. Specifically, we: (i) demonstrate, for the first time, the encapsulation of a hydrophobic anesthetic within HOF-TATB; (ii) establish a machine learning (ML)-guided screening pipeline to identify drugs with optimal loading and release characteristics; and (iii) validate ultrasound-activated, on-demand anesthetic release in a rodent model of neuropathic pain. The solid-state, modular design of HOF-TATB enables tunable drug encapsulation while preventing premature leakage and maintaining ultrasound responsiveness. The ML-driven approach evaluates drug physicochemical properties to predict compatibility, while versatile loading strategies accommodate a broad range of drug chemistries. By uniting Al-guided drug selection, high payloads, and spatiotemporally precise release, this platform offers a non-invasive, controllable solution for long-term pain relief and holds broader potential for neuromodulation and neurological disorder therapies (Fig. 12a).Methods

[0166] HOF-TATB nanoparticles preparation. HOF-TATB nanoparticles were synthesized following a previously reported method with slight modifications. Briefly, 30 mg of H₃TATB was dissolved in 3 mL of dimethylformamide (DMF) under constant stirring at 1,000 rpm. Subsequently, 12 mL of distilled water was added dropwise, and the mixture was stirred for an additional 10 minutes. The resulting precipitate was collected by centrifugation at 12,000 rpm (13,523 × g) for 5 minutes using an Eppendorf Centrifuge 5420. The collected precipitate was washed three times with acetone and distilled water to remove residual impurities. The final product, with an approximate yield of 30%, was dispersed in water at the desired concentration. The concentration of HOF-TATB in the dispersion was determined using a UV-Vis calibration curve of the H₃TATB solution.

[0167] HOF-BTB nanoparticles preparation. HOF-BTB nanoparticles were synthesized using a modified procedure. Specifically, 30 mg of H₃BTB was dissolved in 2 mL of dimethylformamide (DMF) under continuous stirring. Subsequently, 12 mL of distilled water was added dropwise, and the mixture was stirred for an additional 5 minutes. The resulting precipitate was collected by centrifugation at 12,000 rpm (13,523 × g) for 5 minutes using an Eppendorf Centrifuge 5420. The collected nanoparticles were washed three times with methanol and distilled water to remove any residual impurities. The final yield of HOF-BTB nanoparticles was approximately 25%.- 72 - 4909-0910-3750, v. 3

[0168] HOF-101 nanoparticles preparation. HOF-101 nanoparticles were synthesized following a modified procedure. Specifically, 30 mg of H4TBAPy was dissolved in 3 mL of dimethylformamide (DMF) under constant stirring at 1,000 rpm. Subsequently, 12 mL of distilled water was added dropwise to the solution, and the mixture was stirred for an additional 5 minutes. The resulting precipitate was collected by centrifugation at 12,000 rpm (13,523 × g) for 5 minutes using an Eppendorf Centrifuge 5420. The collected nanocrystals were washed three times with acetone, ethanol, and distilled water to remove any residual impurities. The final product, with a yield of approximately 95%, was resuspended in distilled water at the desired concentration for future use.

[0169] HOF-102 nanoparticles preparation. HOF-102 nanoparticles were synthesized using a modified procedure. Specifically, 10 mg of H₄PTTNA monomer was dissolved in 2 mL of dimethylformamide (DMF) under constant stirring. Subsequently, 8 mL of methanol was added, and the mixture was stirred for an additional 5 minutes. The resulting precipitate was collected by centrifugation at 12,000 rpm (13,523 × g) for 5 minutes using an Eppendorf Centrifuge 5420. The collected nanocrystals were washed three times with methanol and distilled water to remove any residual impurities. The final product exhibited a yield of approximately 85%.

[0170] Preparation of hydrophilic lidocaine HO or bupivacaine-HCl-loaded HOF nanoparticles. Lidocaine HC1 or bupivacaine HCl-loaded HOF nanoparticles were prepared using a solution immersion method. Briefly, 3 mg of lidocaine HC1 or bupivacaine HC1 was dissolved in 2 mL of a 5 mg / mL HOF-TATB or other HOF nanocrystal suspension. The mixture was incubated under gentle vibration at 30°C overnight to facilitate drug loading. Following incubation, the nanocrystals were collected by centrifugation at 12,000 rpm (13,523 × g) for 5 minutes using an Eppendorf Centrifuge 5420. The resulting pellets were washed three times with distilled water to remove any unloaded drug molecules. A 1 mL suspension of the drug-loaded nanocrystals was subjected to freeze-drying to obtain a dry powder. Subsequently, 1 mg of the dried powder was dissolved in 0.2 mL of a DMSO and methanol mixture to completely release the loaded drug and TATB ligands. The drug-loading content was determined using high-performance liquid chromatography (HPLC).

[0171] Preparation of lipophilic lidocaine or bupivacaine-loaded HOF nanoparticles. Lipophilic lidocaine or bupivacaine-loaded HOF nanoparticles were prepared using a dual-solvent method. Specifically, 4 mg of lidocaine or bupivacaine was dissolved in- 73 - 4909-0910-3750, v. 3100 juL of dichloromethane (CH₂Cl₂). This solution was then added dropwise to 2 mL of a 10 mg / mL HOF-TATB or other HOF nanoparticle suspension under continuous stirring at 700 rpm for 4 hours to facilitate drug loading. Following the loading process, the nanoparticles were collected by centrifugation at 12,000 rpm (13,523 × g) for 5 minutes using an Eppendorf Centrifuge 5420. The resulting pellets were washed three times with acetone and distilled water to remove any unloaded drug molecules. A 1 mL suspension of the drug-loaded nanoparticles was subsequently subjected to freeze-drying to obtain a dry powder. For drug content determination, 1 mg of the dried powder was dissolved in 0.2 mL of a DMSO and methanol mixture, ensuring complete release of the loaded drug and the amount of the TATB ligands. The drug-loading content was quantified using HPLC.

[0172] Computational Methods. The interaction energy between a drug molecule and a HOF channel was computed the following way with GFN2-xTB2. The HOF channel was extracted from the crystal structure and relaxed by restrained minimization. The drug was then placed in the middle of the channel and minimized at the presence of the channel while the channel is completely fixed during the process, which provided the total energy of the drug in the complex. The total energy of the drug minimized without the complex was also obtained. The difference is calculated as the interaction energy. All calculations were done with implicit solvation of water.

[0173] In vitro ultrasound-controlled drug uncaging. The in vitro release of drugs from HOF nanoparticles under ultrasound stimulation was evaluated using a FUS system. Freshly prepared drug-loaded HOF nanoparticles were suspended in distilled water (pH adjusted to 2) at a concentration of 1 mg / mL and transferred into glass vials. These vials were placed directly on the ultrasound transducer, and the suspension was subjected to FUS irradiation at a frequency of 1.5 MHz under the specified parameters and for predetermined durations. At designated time intervals, 100 pL of the suspension was carefully extracted from the vial and centrifuged at 8,000 rpm (6,010 × g) for 5 minutes using an Eppendorf Centrifuge 5420. The released drug was detected in the supernatant, and its concentration was determined using HPLC. The percentage of drug release was calculated based on the initial drug loading content.

[0174] Long-term drug stability evaluation in HOF nanoparticles. The long-term stability of drug-loaded HOF nanoparticles was evaluated under physiological conditions. Drug-loaded HOF nanoparticles were prepared at a concentration of 3 mg / mL in fetal bovine- 74 - 4909-0910-3750, v. 3serum (FBS) solutions of either 10% or 50% and stored in glass vials at room temperature. At predetermined time intervals, 100 pL of the nanoparticle suspension was withdrawn and centrifuged at 8,000 rpm (6,010 × g) for 5 minutes using an Eppendorf Centrifuge 5420. The collected nanoparticles were washed three times with acetone and distilled water (DIW) to remove any loosely bound drug. A 1 mL suspension of the washed nanoparticles was then subjected to freeze-drying, resulting in a dry powder. For drag content determination, 1 mg of the dried powder was dissolved in 0.2 mL of a dimethyl sulfoxide (DMSO) and methanol mixture, ensuring complete release of the loaded drag and dissociation of the TATB ligands. The drag-loading content was quantified using HPLC.

[0175] In vitro calcium imaging. Calcium imaging was performed using primary cortical neurons transduced with pAAV(adeno-associated virus)-hSyn-GCaMP6s-WPRE-SV40. Following a 6-day transduction period, the neuron cultures were placed on the stage of a Leica DMi8 fluorescence microscope equipped with a 20x air objective. The water balloon membrane of the FUS transducer was brought into direct contact with the neuron culture medium atop the plate. Fresh TATB@Lido HC1 nanoparticles were added to the medium at a final concentration of 5 pg / mL Lido. FUS stimulation (1.08 MPa, 1.40 MHz, 10 s duration) was applied to trigger the release of Lido HC1, leading to neuronal inhibition. Fluorescence imaging videos were captured using the Leica DMi8 microscope under the green fluorescence channel (Ex: 450-490 nm) with a 5 ms exposure time. For data analysis, the transient increase in green fluorescence (AF / F) was determined by manually segmenting 50 neurons and extracting the fluorescence time-series data. The imaging videos were converted to grayscale using ImageJ software, and the fluorescence data were processed using a custom MATLAB algorithm. This algorithm performed detrending and normalization of the fluorescence timeseries data through second-order polynomial curve fitting and baseline maximum fluorescence value extraction, effectively compensating for photobleaching effects.

[0176] Animal experiments. All animal studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th ed., National Research Council, 2011) and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas at Austin (AUP-2022-00278, approved 02 / 07 / 2023). Male outbred Sprague-Dawley (SD) rats aged 3-6 months were housed 2-3 / cage under a 12-hour light / dark cycle. Food and water were provided ad libitum. Surgical and behavioral procedures were conducted during the day. The rats were randomly assigned to each- 75 - 4909-0910-3750, v. 3group. Under brief isoflurane-oxygen anesthesia, operated (see below in Surgical Procedures) or unoperated (for in vivo nanoparticle evaluation) rats were injected with 500pL of HOF-TATB with or without lidocaine HCL or bupivacaine using a 23G needle. The injection was performed next to the sciatic nerve with the needle introduced posteromedial to the greater trochanter. Ten minutes after the injection, focused ultrasound activation was performed at the injection site.

[0177] Ultrasound-triggered sciatic nerve blockade in unoperated Sprague Dawley rats. Following local injection of nanoparticles (either unloaded, HOF-TATB@Lidocaine HCl, or HOF-TATB@ Bupivacaine) at the sciatic nerve, focused ultrasound (FUS; 1.5 MHz, 1.40 MPa) was applied with a pulsed sequence of 0.2 s on / 0.8 s off for the designated activation period. 30 minutes after ultrasound stimulation, von Frey (VF) testing was performed under anesthesia to assess nerve blockade. Mechanical nociceptive thresholds were determined by measuring paw withdrawal responses to VF filaments applied to the dorsal region of the hindpaw between digits 4 and 5.

[0178] Sensory and motor behavioral testing. Dorsal sensory von Frey (VF) testing was performed as previously described (Jayathilake et al. 2025; Mannhold et al. 2008). Briefly, prior to the administration of drugs (unoperated group) or surgery (peripheral nerve injury group), rats were handled and trained daily for at least 4 sessions. VF testing was performed at predetermined time points for unoperated rats or weekly time points for operated rats. The second tester applied the VF filaments when the rat was calm. The first tester gently wrapped and restrained rats in a towel and elevated them to an upright position to expose the dorsal surface of their hindpaws. The second tester applied VF filaments (Stoelting Co., Wood Dale, IL, USA) between digits 4 and 5 when rats were calm. The beginning filament was presented verticallyperpendicular to the targeted dorsal region until bent for two 1-2 seconds and was repeated on the alternate paw. A positive or negative withdrawal response was recorded for each trial on each side hindpaws. A positive and negative withdrawal responses were respectively followed by a lower and higher force filament on the subsequent trial. Five trials were tested on each side hind paw. Rats were allowed to rest at least 30 seconds between trials of both hindpaws. The final VF thresholds depended on the last response obtained and were converted to gram force for analysis. Any response was voided and the trial was repeated if the filament slipped off of the dorsal surface, the rat moved or picked up its hind paw, or if the rat- 76 - 4909-0910-3750, v. 3struggled in the towel during application of the filament. VF filaments were routinely tested on a scale measuring grams and replaced to accurately represent intended gram forces.

[0179] Sciatic functional index (SFI) testing was performed as previously described. All rats were trained daily for three days prior to surgery. SFI testing was conducted weekly until their post-operative experimental endpoints, and all assessments were performed by testers blinded to group allocation. For footprint collection, the operated hindpaw was inked blue and the contralateral hindpaw red. Rats were then allowed to walk along a slightly inclined 100-mm-wide wooden runway lined with white paper and permitted to return to their home cage. For each testing day, two successful trial runs were required. A successful trial was defined as a run containing at least three consecutive steps on each hindpaw without stopping or hesitation. An average score was calculated for that day using the following formula: 73*((NPL-EPL) / EPL+(ETS-NTS) / NTS-i-(EIT-NIT) / NIT, where NPL stands for normal footprint length, EPL for experimental footprint length, NTS for normal toe spread, ETS for experimental toe spread, NIT for normal intermediary toe spread, and EIT for experimental intermediary toe spread.

[0180] Surgical procedures. Surgical procedures were performed as previously described (Jayathilake et al. 2025; Mannhold et al. 2008). Rats were anesthetized with isoflurane (4% induction, 2% maintenance (RXISO-250; Animal Health International, Roanoke, TX, USA)) / oxygen mixture at 1.5 L / min (Handlebar Anesthesia, Pflugerville, TX, USA). The lateral aspect of the left hindlimb was shaved and sterilized, and a 1.5-2 cm incision was made through the skin and the biceps femoris to expose the sciatic nerve. The sciatic nerve was sharply transected at the mid-thigh level with microscissors. Sciatic nerve stumps were irrigated with 0.5% methylene blue followed by a hypotonic (250 mOsm), calcium-free diluted Normosol-R (ICU Medical, San Clemente, CA, USA) saline solution. Sciatic nerve stumps were trimmed, closely apposed, and secured with at least four 10-0 microsutures (neurorrhaphy) through the epineurium sheaths.

[0181] Following neurorrhaphy, lesion sites were flushed several times with Ca2+-containing normal saline. The surgical site was closed with 5-0 sutures through the muscle, and the skin was closed with wound clips. Rats were allowed to recover on heat pads before being returned to standard housing. Rats received 5 mg / kg subcutaneous injections of carprofen during surgery and for three days PO.- 77 - 4909-0910-3750, v. 3

[0182] Self-mutilation scoring. Self-mutilation often first presented as nibbling of the most distal parts of the toe nails with continued chewing in the proximal direction of the hind paw digits. The severity and the time of onset of self-mutilation to the operated hind limb were monitored and recorded daily until the PO endpoint was reached by either euthanasia criteria set by the IACUC or by reaching an experimental endpoint. Self-mutilation severity ranged from nail bed bleeding to bone exposure and received different scores. Animals received a score of 1 for each digit with significant tissue damage that required additional PO antiinflammatory carprofen administration, as determined by veterinarian staff. An additional score of 5 was given when any digit had bone exposure. For example, when 3 digits had tissue damage and one digit had bone exposure, the rat received a score of 8; two digits had tissue damage and 2 digits had bone exposure, the rat received a score of 12. Self-mutilation rates are presented as percentages and scores are presented as mean ± SEM.

[0183] Morphological analyses for the sciatic nerve and the soleus muscle. After rats were deeply induced with 4% isoflurane / oxygen euthanized by an intracardiac KC1 injection, sciatic nerves and soleus muscles were harvested for following analyses.

[0184] Nerve morphometric analysis: sciatic nerves harvested from operated limbs were placed in 0.1 M sodium cacodylate buffer and then fixed by 2% paraformaldehyde / 3% glutaraldehyde fixative overnight. The next day, tissues were washed with buffer, trimmed and postfixed in 1% osmium tetroxide / 1% potassium ferrocyanide in 0.1 M sodium cacodylate buffer for 3 to 5 hours, washed in water, stained in 1% aqueous uranyl acetate for 1 to 2 hours, and then washed and held in water57. Nerves were dehydrated using graded alcohols, exchanged to absolute acetone, placed in increasing concentrations of Hard Plus Resin 812 (Electron Microscopy Sciences, Hatfield, PA), and then embedded in fresh resin and polymerized at 60°C. Glass knife thick sections (0.5 mm) were stained in toluidine blue and imaged using confocal microscopy. Axon numbers per square micrometer (mm2) were counted and averaged from at least 3 different regions of interest containing at least 150 axons per sample.

[0185] Soleus muscle isolation: Soleus muscles from both unoperated and operated limbs were weighed immediately after isolation. The muscle weight ratio was calculated by dividing the weight of the operated side by that of the unoperated side.- 78 - 4909-0910-3750, v. 3

[0186] Ultrasound- triggered pain management and motor functional recovery after sciatic nerve injury (SNI). Self-mutilation (autotomy) of the hind limbs in SD rats following SNI is a well-established model for studying neuropathic pain (Sporel-Ozakat et al.1991: Al-Adawi et al. 2002; Wang et al. 2016). In this model, rats underwent unilateral sciatic nerve transection followed by end-to-end microsuture repair under an operating microscope. Post-surgery, animals were monitored daily for signs of distress and self-mutilation scores were recorded until 6 weeks post-operation (PO). To evaluate the therapeutic effects of HOF-TATB@ lidocaine and HOF-TATB@bupivacaine, nanoparticles were perineurally injected at the repair site, and focused ultrasound stimulation (1.5 MHz, 1.40 MPa, 8 min) was applied daily to trigger on-demand anesthetic release. Motor functional recovery was assessed weekly for 6 weeks PO using the SFI.

[0187] Mass Spectrometry Imaging. Frozen nerve specimens were longitudinally sectioned at 12 pm thickness using a Thermo NX50 CryoStar cryostat and collected onto ColorFrost Plus™ slides. Serial sections were collected for H& E staining. Fiducial points were placed at the corners of the slides for MSI prior to collection of an optical image using an Epson Perfection V600 flatbed document scanner at 4800 dpi. The sections were coated with 40 mg / mL 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% trifluoracetic acid using an HTX M5 Robotic Reagent Sprayer over 10 passes with the following parameters: a flow rate of 100 pL / min, a track speed of 1200 mm / min, a track spacing of 2 mm, a nozzle temperature of 75°C, a nozzle height of 40 mm, a heated tray temperature of 50°C, a nitrogen pressure of 10 psi, and a CC track pattern. Mass spectrometry image data were acquired at 30 pm resolution using FlexImaging 7.0 on a Bruker timsTOF fleX QTOF mass spectrometer in positive ion mode with the following parameters: 300 shots per pixel, an m / z range of 50-1000, a Funnel 1 RF of 150.0 Vpp, a Funnel 2 RF of 200.0 Vpp, a Multipole RF of 200.0 Vpp, a Collision Energy of 5.0 eV, a Collision RF of 700.0 Vpp, a Transfer Time of 80.0 ps, and a Pre Pulse Storage of 8.0 ps. MSI data was loaded into SciLS Lab 2025b and normalized to Total Ion Current for visualization of the lidocaine distribution in the nerve sections.

[0188] Statistical analyses. Excel was used to calculate means and standard deviations. Graphpad Prism (version 9, GraphPad Software, Boston, MA, USA, www.graphpad.com) was used to perform linear regressions and t-test comparisons.- 79 - 4909-0910-3750, v. 3Materials and Instrumentation

[0189] Chemicals. Unless otherwise specified, all reagents and solvents were obtained from commercial suppliers and used without further purification. The following chemicals were used in this work: concentrated hydrochloric acid (HC1), acetone, dimethylformamide (DMF), dichloromethane (DCM), methanol, ethanol, and dimethyl sulfoxide (DMSO). 1,3,5-Tris(4-carboxyphenyl)benzene (H3BTB), 4,4',4"-(l,3,5-triazine-2,4,6-triyl)tribenzoic acid (H3TATB), 1,3,6,8-tetrakis(benzoic acid)pyrene (H₄TBAPy), and 1,3,6,8-tetra(6-carboxynaphthalen-2-yl)pyrene (H₄PTTNA) were purchased from Chemscene.

[0190] Instruments. Transmission electron microscopy (TEM) images of the HOFs were acquired using a JEOL NEOARM low-kV, aberration-corrected TEM / STEM (30-200 kV) at the Texas Materials Institute, The University of Texas at Austin, in TEM mode at an accelerating voltage of 200 kV. The powder X-ray diffraction (PXRD) patterns were collected using a Rigaku Miniflex 600 diffractometer equipped with a Cu Ka radiation source (X = 1.54184 A) operating at 40 kV and 15 mA. Data were recorded over a 20 range of 2.5-30°. An Agilent MR400 spectrometer (1H, 400 MHz) was used for the collection of1H NMR spectra. UV-Vis spectra were recorded on an Eppendorf BioSpectrometer® Basic. The drug release percentage from HOFs was measured using HPLC (Agilent 6120 Single Quadrupole LC / MS).

[0191] Bioreagents. AAV-hSyn-GCaMP6s-WPRE-SV40 (Addgene viral prep #100843-AAV9; RRID: Addgene_100843) was obtained from the Douglas Kim & GENIE Project (n2t.net / addgene: 100843) (Chen et al. 2013b). Lidocaine, lidocaine hydrochloride, bupivacaine, bupivacaine hydrochloride, aspirin, benzocaine, carbamazepine, ilomastat (GM6001), ibuprofen, isradipine, tetracaine, deschloroclozapine (DCZ), (S)-3,5-dihydroxyphenylglycine (DHPG), dopamine, L-dopa, methylene blue, methylprednisolone, rhodamine B, and scopolamine were purchased from Sigma-Aldrich and used without further purification.

[0192] Determination of Drug Loading Content. The drug loading content was determined either by HPLC or by ¹H NMR, depending on the analytical suitability of each drug. For HPLC analysis, DMSO was used to dissolve the samples prior to injection, unless otherwise specified. For1H NMR analysis, samples were dissolved in DMSO-de, and the drug content was calculated based on the integration of characteristic peaks.- 80 - 4909-0910-3750, v. 3

[0193] Preparation of different (lipophilic drug) drug-loaded HOF-TATB and evaluation of ultrasound-triggered drug release through high-performance liquid chromatography (HPLC). To prepare drug-loaded TATB-HOF, 2 mg of drug was dissolved in 2 mL of HOF-TATB suspension (5 mg / mL). The mixtures were incubated at 37 °C for 10 h and centrifuged at 12,000 rpm (13,523 × g) for 5 min. The resulting pellets were washed three times with distilled water to remove unloaded cargoes and then re-suspended. To evaluate the drug-loading content, 1 mL of the suspension was collected and freeze-dried. The resulting powder (~1 mg) was dissolved in 0.2 mL of DMSO to completely release the cargo, followed by dilution with 0.4 mL of methanol. The drug-loading content was measured using HPLC (Agilent 6120 Single Quadrupole LC / MS).

[0194] For evaluating the ultrasound-triggered drug release, freshly prepared drug-loaded HOF nanocrystal suspensions (10 mg / mL) were transferred into glass vials and placed on an ultrasound transducer. The samples were exposed to focused ultrasound (FUS) at 0.79 MPa, 1.40 MPa, 2.45 MPa and 1.5 MHz for the designated duration under the specified parameters. At predetermined time points, 100 μL aliquots were withdrawn and centrifuged at 8,000 rpm (6,010 × g) for 5 min. The supernatants containing the released drug were collected, and the release percentage was determined based on HPLC calibration curves.

[0195] Preparation of HOF-BTB loaded with various drugs (lipophilic drug) and analysis of ultrasound-responsive release via HPLC. HOF-BTB suspensions (5 mg / mL, 2 mL) were combined with 2 mg of the desired drug and gently agitated to ensure uniform mixing. The mixtures were maintained at 37 °C for 10 h, after which they were centrifuged at 12,000 rpm (13,523 × g) for 5 min. The resulting pellets were washed three consecutive times with distilled water to remove excess, non-incorporated drug and then dispersed again in water. For drug -loading measurements, a 1 mL portion of the suspension was collected, freeze-dried, and the obtained powder (~1 mg) was first dissolved in 0.1 mL DMSO to fully release the encapsulated molecules, followed by dilution with 0.5 mL methanol. The final solutions were analyzed using HPLC (Agilent 6120 Single Quadrupole LC / MS) to determine the drug content.

[0196] Preparation of HOF-101 loaded with various drugs (lipophilic drug) and analysis of ultrasound-responsive release via HPLC. HOF-101 suspensions (5 mg / mL, 2 mL) were mixed with 2 mg of the selected drug under gentle agitation to achieve homogeneous dispersion. The mixtures were incubated at 37 °C for 10 h and then centrifuged at 12,000 rpm (13,523 × g) for 5 min. The pellets obtained were washed three times with distilled water to- 81 - 4909-0910-3750, v. 3remove unbound drug and subsequently re-suspended. For drug-loading quantification, 1 mL of the suspension was freeze-dried, and the resulting powder (~1 mg) was dissolved directly in 0.5 mL DMSO to completely release the encapsulated drug. The drug content was determined using HPLC (Agilent 6120 Single Quadrupole LC / MS).

[0197] Preparation of HOF-102 loaded with various drugs (lipophilic drug) and analysis of ultrasound-responsive release via HPLC. A suspension of HOF-102 (5 mg / mL, 2 mL) was combined with 2 mg of the target drug and stirred gently to ensure even dispersion. The mixture was kept at 37 °C for 10 h, followed by centrifugation at 12,000 rpm (13,523 × g) for 5 min. The precipitates were rinsed three times with distilled water to eliminate residual, unbound drug and then re-dispersed in water. For determining the drug loading, 1 mL of the suspension was taken, freeze-dried, and the obtained solid (~1 mg) was dissolved in 0.5 mL DMSO to fully release the encapsulated molecules. The drug concentration was quantified by HPLC (Agilent 6120 Single Quadrupole LC / MS).

[0198] Primary neuron culture. C57BL / 6 mice (8 weeks old, 20-26 g; Jackson Laboratory) were used in this study. Primary cortical neurons were isolated from embryos at embryonic day 15.5. Briefly, 24-well cell culture plates were coated with poly-L-ornithine (0.2 mg / mL) and incubated at 37 °C for 2 h. The plates were then rinsed three times with PBS and pre- warmed in a cell incubator for 15 min before use. Dissociated neuronal cells were seeded onto the coated plates and maintained in Neurobasal medium supplemented with B27, glutamine, penicillin, and streptomycin. Cultures were incubated at 37 °C in a humidified atmosphere containing 7% CO₂. After 2 days in vitro, a glial inhibitor, 5-fluoro-2'-deoxyuridine (0.1 mM), was added to the culture medium. On day 4, 1 μL of pAAV-hSyn-GCaMP6s-WPRE-SV40 was transfected into the neurons. Calcium imaging experiments were performed after an additional 6 days of incubation.

[0199] Ultrasound-triggered sciatic nerve blockade in unoperated Sprague Dawley rats. After confirming the spatial localization and retention of anesthetic agents in the sciatic nerve of Sprague Dawley (SD) rats as described above, sciatic nerve blockade was evaluated via local administration of anesthetic formulations. Nanoparticles without drug (negative control) or loaded with either HOF-TATB ©Lidocaine HC1 or HOF-TATB@Bupivacaine were injected locally at the sciatic nerve. This was followed by focused ultrasound (FUS; 1.5 MHz, 1.40 MPa) treatment and von Frey (VF) testing. Blockade of mechanical nociceptive responses was assessed by measuring paw withdrawal thresholds in- 82 - 4909-0910-3750, v. 3response to VF filament stimulation applied to the dorsal surface of the hindpaw between digits 4 and 5.

[0200] Ibal and Caspase-3 Staining. Following the in vivo ultrasound activation procedures, rats were deeply anesthetized with 4% isoflurane in oxygen and euthanized via intracardiac KCl injection. Sciatic nerves were harvested, fixed in 4% paraformaldehyde overnight at 4 °C, and sectioned using a cryostat. Longitudinal sciatic nerve sections (10 pm thickness) were washed with 0.3% TBS and blocked with 5% bovine serum albumin in TBS for 30 min at room temperature. The blocking buffer was then replaced with one of the following primary antibody solutions (prepared in TBS): rabbit anti-Ibal (013-27691, Wako Chemicals, 1:500), rabbit anti-cleaved Caspase-3 (9661, Cell Signaling Technology, 1:500). After overnight incubation at 4 °C, sections were washed three times with TBS and incubated for 2 h at room temperature in the dark with a secondary antibody solution containing donkey anti-rabbit Alexa Fluor 594 (A32754, Invitrogen, 1:500) and Hoechst 33342 (17535, ATT Bioquest, 1:5000). Finally, sections were washed three times with TBS, mounted on glass slides with mounting medium, and coverslipped. Fluorescence images were acquired using a Nikon AXR-NSPARC confocal fluorescence microscope.

[0201] H& E staining. Following the in vivo ultrasound activation procedures, rats were deeply anesthetized with 4% isoflurane in oxygen and euthanized via intracardiac KCl injection at 14 days post-treatment. Sciatic nerves were harvested, fixed in 4% paraformaldehyde overnight at 4 °C, and sectioned into 10 pm slices using a cryostat. Hematoxylin and eosin (H& E) staining was performed on the sciatic nerve sections according to previously described protocols. The stained sections were mounted on glass slides with mounting medium and coverslipped. Images were acquired using a Nikon Upr-FLM compound light microscope (Nikon Instruments).

[0202] Computational Methods. Structural descriptors for HOFs and drugs were selected based on the preliminary understanding that non-specific interactions, such as hydrophilicity, play a dominant role in drug loading into HOF crystals. For drugs, the descriptors included molecular volume, radius of gyration, molecular weight, number of rotatable bonds, ratio of sp3-hybridized carbons, total polar surface area, logP, and net charge at pH 7. For HOFs, the descriptors included the number of hydrogen-bonding sites, crystal pore volume, crystal pore size, BET surface area, and logP. All non-crystal features were calculated using RD Kit (doi.org / 10.5281 / zenodo).- 83 - 4909-0910-3750, v. 3

[0203] The interaction energy between a drug molecule and a HOF channel was calculated using the GFN2-xTB method (Bannwarth et al. 2019). A HOF channel was extracted from the crystal structure and relaxed via restrained minimization. The drug molecule was positioned at the center of the channel and minimized in the presence of the fixed channel to obtain the total energy of the complex. Separately, the drug molecule was minimized without the HOF channel to obtain its total energy in isolation. The interaction energy was determined as the difference between these two values. All calculations were performed with implicit solvation in water.ResultsMachine Learning-Guided Identification of High-Loading Anesthetic Candidates

[0204] Achieving high drug loading in porous nanomaterials remains fundamentally empirical, as host-guest compatibility depends on a complex interplay of hydrophobicity, polarity, steric fit, and pore accessibility. To transform this empirical process into a predictive one, an interpretable machine -learning framework was developed that maps multilevel molecular descriptors to drug-loading outcomes in HOF materials.

[0205] Complementary descriptors were first curated that capture the dominant forces driving HOF-guest interactions, including drug lipophilicity (logP), polarity (TPSA), steric size, conformational flexibility, and framework hydrophobicity and pore geometry (FIG.12A). These descriptors collectively represent the chemical and structural determinants governing non-specific adsorption within HOF channels.

[0206] Using experimentally measured loadings across 15 structurally diverse molecules and four HOF scaffolds, a Random Forest regressor was trained to predict drug loading from concatenated host-guest descriptor vectors. This multilevel representation enabled the model to learn cooperative effects between drug chemistry and framework architecture, rather than treating the two independently. Descriptor distributions revealed broad chemical diversity among drugs and clear structural heterogeneity among HOFs (FIGS. 12B-C), ensuring that the model was trained across a sufficiently wide design space. To accommodate polarity differences, hydrophilic drugs were loaded through diffusion, whereas hydrophobic drugs required a double-solvent method (DSM; FIG. 12D), yielding 45 experimentally validated drug-HOF combinations (Table 3).- 84 - 4909-0910-3750, v. 3Table 3. The Training Dataset for machine learning models.loadin g_mol Encapsulation Number Name HOF(%) Method1 Aspirin 12.1 HOF-TATB DSM2 Benzocaine 26.7 HOF-TATB DSM3 Benzocaine 68.5 HOF- 101 DSM4 Benzocaine 72.1 HOF- 102 DSM5 Carbamazepine 28.6 HOF-TATB DSM6 Carbamazepine 71.3 HOF-101 DSM7 Carbamazepine 76.2 HOF- 102 DSM8 GM6001 31 HOF-TATB DSM9 Ibuprofen 38.3 HOF-TATB DSM10 Ibuprofen 30.1 HOF-BTB DSM11 Ibuprofen 64.5 HOF-101 DSM12 Ibuprofen 73.2 HOF- 102 DSM13 Isradipine 30.8 HOF-TATB DSM14 Isradipine 16.7 HOF-BTB DSM15 Isradipine 53.2 HOF-101 DSM16 Isradipine 66.2 HOF- 102 DSM17 Tetracaine 32.2 HOF-TATB DSM18 Tetracaine 26.2 HOF-BTB DSM- 85 - 4909-0910-3750, v. 319 Tetracaine 65.9 HOF-101 DSM20 Tetracaine 76.5 HOF- 102 DSM21 DCZ 12.8 HOF-TATB Diffusion22 DHPG 6.1 HOF-TATB Diffusion23 Dopamine 15 HOF-TATB Diffusion24 Dopamine 18 HOF-101 Diffusion25 Dopamine 43 HOF- 102 Diffusion26 L-dopa 9.2 HOF-TATB Diffusion27 L-dopa 32.4 HOF-101 Diffusion28 L-dopa 41 HOF- 102 Diffusion29 MethylenebBlue 8.6 HOF-TATB Diffusion30 MethylenebBlue 11.5 HOF-BTB Diffusion Methylprednisolon31 e 3.1 HOF-TATB Diffusion Methylprednisolon32 e 1.5 HOF-BTB Diffusion Methylprednisolon33 e 41.4 HOF- 101 Diffusion Methylprednisolon34 e 60.3 HOF- 102 Diffusion35 RhodamineB 14.2 HOF-TATB Diffusion36 RhodamineB 14.6 HOF-BTB Diffusion- 86 - 4909-0910-3750, v. 337 RhodamineB 34.3 HOF-101 Diffusion38 RhodamineB 44.3 HOF- 102 Diffusion39 Scopolamine 5.8 HOF-TATB Diffusion40 Scopolamine 24.8 HOF- 101 Diffusion41 Scopolamine 36.6 HOF- 102 Diffusion42 Tetracaine 16.7 HOF-TATB Diffusion43 Tetracaine 20.2 HOF-BTB Diffusion44 Tetracaine 42.9 HOF-101 Diffusion45 Tetracaine 45.2 HOF- 102 Diffusion

[0207] Analysis of descriptor-loading relationships uncovered distinct structureproperty trends, with larger pore volumes, higher BET surface areas, and more hydrophobic frameworks consistently supporting higher loading (FIGS. 12E-F). These emergent patterns align with established host-guest principles and validate that the model captures mechanistically meaningful features rather than statistical correlations alone.

[0208] To assess whether molecular descriptors encode meaningful loading behavior, the distribution of the training compounds was mapped using principal component analysis (PCA). Drugs with low, medium, and high loading formed partially separated clusters (FIG.14A), indicating that loading capacity reflects systematic variation in physicochemical properties rather than experimental noise. Density profiles along PC1 further revealed clear, non-overlapping trends across loading groups (FIG. 14B), continuing that descriptor space contains predictive chemical structure.

[0209] Benchmarking multiple machine -learning models, including Support Vector Machines (CiteSeerX), Random Forests (Ho 2002), AdaBoost (Freund and Schapire 1997), and neural networks (Zell 1994), identified the Random Forest regressor as the most accurate predictor of loading (FIGS. 14C-D). Predicted and measured values exhibited strong agreement- 87 - 4909-0910-3750, v. 3(FIG. 14E), demonstrating that cooperative host-guest interactions — driven by steric accessibility, hydrophobic matching, and surface polarity — can be reliably learned from multilevel descriptors. Feature -importance analysis (Breiman 2001) revealed that pore accessibility and framework hydrophobicity dominated HOF contributions, while drug sterics, polarity, and lipophilicity were the primary molecular drivers (FIG. 14F), consistent with established host-guest principles.

[0210] Next the optimized model was applied to a library of -3,000 FDA-approved small molecules (Knox et al. 2024) (FIG. 14G). An indication-based filter yielded -250 clinically relevant analgesics or anesthetics (Table 4), which were independently ranked for diffusion-based and double-solvent loading modes (FIG. 14H). Two distinct high-performing groups emerged: (1) hydrophobic drugs with high DSM-predicted loading, and (2) hydrophilic drugs with strong diffusion-predicted loading (FIGS. 14I-J).-88 - 4909-0910-3750, v. 3Table 4. Complete list of 246 compounds in the Focused Library used for machine-leaming- based screening.CompoundNumber Compound name Number Number Compound name name21 Benzhydrocod 411 Aceclofenacone Chlorobutanol 2 Acemetacin 22 Benzonatate 42 Chloroprocaine 3 Acetaminophen 23 Benzydamine 43 Chlorphenesin 4 Acetylsalicylic acid 24 Berotralstat 44 Chlorzoxazone 25 45 Choline 5 Alclofenac magnesium Bromfenac trisalicylate 6 Alfentanil 26 Bronopol 46 Choline salicylate 7 Almotriptan 27 Bupivacaine 47 Cilostazol 28 Buprenorphin 488Alverine e Cinchocaine 9 Alvimopan 29 Butalbital 49 Cisatracurium 10 Amitiptyline 30 Butamben 50 Clidinium 31 51 Clobetasol 11Amitriptyline Butorphanol propionate 32 Butylscopola 5212Amphetamine mine Clomipramine 13 Amylocaine 33 Caffeine 53 Clonidine 14 Anileridine 34 Cannabidiol 54 Cocaine 15 Antrafenine 35 Capsaicin 55 Codeine- 89 - 4909-0910-3750, v. 316 Articaine 36 Carisoprodol 56 Cyclobenzaprine 17 Atracurium besylate 37 Carprofen 57 Decamethonium 18 Baclofen 38 Celecoxib 58 Desflurane 39 Chloral 5919Bazedoxifene hydrate Desipramine 20 Bendazac 40 Chlorcyclizine 60 Dexketoprofen CompoundNumber Compound name Number Number Compound name name61 Dextropropoxyphene 81 Enflurane 101 Ganaxolone 62 Dezocine 82 Ephedrine 102 Glucosamine 63 Diamorphine 83 Epinephrine 103 Glycol salicylate 64 Diclofenac 84 Ethanol 104 Glycopyrronium 65 Dienogest 85 Etidocaine 105 Guaiacol 66 Diflunisal 86 Etodolac 106 Halothane 67 Difluprednate 87 Etomidate 107 Hexafluronium 68 Dihydrocodeine 88 Etoricoxib 108 Hexylcaine 69 Dimethyl sulfoxide 89 Eugenol 109 Hexylresorcinol 70 Disopyramide 90 Fenoprofen 110 Hyaluronic acid 71 Domperidone 91 Fentanyl 111 Hydrocodone 72 Doxacurium 92 Flavoxate 112 Hydromorphone 73 Doxepin 93 Flecainide 113 Hydroxyurea 74 Dronabinol 94 Flumazenil 114 Hyoscyamine 75 Droperidol 95 Flurbiprofen 115 Imipramine- 90 - 4909-0910-3750, v. 376 Duloxetine 96 Fospropofol 116 Indecainide 77 Dyclonine 97 Gabapentin 117 Indomethacin 78 98 Gallamine118Edaravone triethiodide Isoflurane 79 99 gamma- Hydroxybutyri 119Elagolix c acid Isoprenaline 80 100 Gamolenic120Eluxadoline acid Ketamine CompoundNumber Compound name Number Number Compound name name121 Ketoprofen 141 Meloxicam 161 Morphine 122 142 Menthyl 162Ketorolac salicylate Nabilone 123 Lamotrigine 143 Meperidine 163 Nalbuphine 124 144 Mephentermin 164Levacetylmethadol e Naloxegol 125 Le vobupi vacaine 145 Mepivacaine 165 Naloxone 126 146 166 Nandrolone Levomenthol Metamizole decanoate 127 Levorphanol 147 Metaraminol 167 Naproxen 128 Lidocaine 148 Metaxalone 168 Nepafenac 129 Linzagolix 149 Methadone 169 Nicoboxil 130 150 Methocarbam 170Lofexidine ol Nimesulide 131 Lorazepam 151 Methohexital 171 Nitroglycerin- 91 - 4909-0910-3750, v. 3132 152 Methoxyflura 172Lomoxicam ne Norethisterone 133 Loteprednol 153 Methyl 173etabonate nicotinate Nortriptyline 134 154 Methyl 174Loxoprofen salicylate Oliceridine 135 155 Methyltestoste 175Lubiprostone rone Orphenadrine 136 156 Metocloprami 176Meclizine de Oxaprozin 137 157 Metocurine 177Meclofenamic acid iodide Oxetacaine 138 Medifoxamine 158 Midazolam 178 Oxybuprocaine 139 Medroxyprogesterone 159 179acetate Milnacipran Oxycodone 140 Mefenamic acid 160 Mivacurium 180 Oxymetazoline CompoundNumber Compound name Number Number Compound name name181 Oxymorphone 201 Pramocaine 221 Sufentanil 182 Ozanimod 202 Prasterone 222 Sugammadex 183 Pancuronium 203 Pregabalin 223 Sulindac 184 Parecoxib 204 Prilocaine 224 Tannic acid 185 Pentaerythritol 225205tetranitrate Procaine Tapentadol 186 Procaine 226206 benzylpenicilli Technetium Tc- Pentazocine n 99m disofenin- 92 - 4909-0910-3750, v. 3187 Pentosan polysulfate 207 Promethazine 227 Tenoxicam 188 Pentoxifylline 208 Proparacaine 228 Thiamylal 189 Pentoxyverine 209 Propoxycaine 229 Thiethylperazine 190 Phenazopyridine 210 Pyrithione 230 Thiopental 191 Phenol 211 Remifentanil 231 Tiaprofenic acid 192 Phentolamine 212 Remimazolam 232 Tirofiban 193 Phenyl salicylate 213 Rocuronium 233 Tizanidine 194 Phenylephrine 214 Rofecoxib 234 Tolfenamic acid 195 Phenyltoloxamine 215 Ropivacaine 235 Tramadol 196 Pinaverium 216 Salicylamide 236 Triamcinolone 197 Samarium 237217 (153Sm)Pipecuronium lexidronam Trichloroethylene 198 Pipoti azine 218 Sevoflurane 238 Triflusal 199 Piritramide 219 Sildenafil 239 Trimebutine 200 Succinylcholi 240220Piroxicam ne Trimethaphan CompoundNumber Compound name Number Number Compound name name241 Trolamine salicylate 243 Vecuronium 245 Zavegepant 242 Tryptophan 244 Venlafaxine 246 Zucapsaicin- 93 - 4909-0910-3750, v. 3

[0211] To refine these predictions, top-scoring molecules from each group (FIG. 18) were further evaluated using GFN2-xTB interaction energies (FIG. 14L) and clinical safety considerations. Bupivacaine (DSM category) and lidocaine hydrochloride (diffusion category) emerged as the most promising candidates based on the convergence of ML ranking, hostguest interaction strength, and established medical safety profiles. Experimental measurements confirmed the model’s predictive accuracy, showing close agreement between predicted and actual loading capacities (FIG. 14K). Together, these results demonstrate that the integrated ML-simulation pipeline enables reliable, mechanism-informed prioritization of both hydrophobic and hydrophilic anesthetics for HOF-based delivery, providing a scalable and conceptually generalizable framework for therapeutic design.Synthesis and Characterization of HOF@ Lidocaine HO & Bupivacaine

[0212] To experimentally validate the machine learning-guided selection of high-loading anesthetic candidates, HOF-TATB was synthesized and encapsulated the top two predicted drugs — lidocaine hydrochloride (hydrophilic) and bupivacaine (lipophilic) — into its porous framework. Among several candidate materials, HOF-TATB, assembled from 4, 4', 4''-s-triazine-2,4,6-triyl-tribenzoic acid (H3TATB), was selected for its structural robustness, colloidal stability and strong responsiveness to ultrasound stimulation (Freund and Schapire 1997) (FIG. 13B). This framework has previously been validated as an ultrasound-responsive drug-release platform, further supporting its suitability for testing the ML-identified candidates. The framework is stabilized through directional hydrogen bonding and π–π stacking interactions, which facilitate efficient drug encapsulation and enable mechanical energy-triggered release.

[0213] To accommodate differences in hydrophilicity, lidocaine HC1 was loaded via aqueous diffusion, whereas bupivacaine required a dual-solvent method to achieve high loading, the measured loading efficiencies closely matched ML predictions (FIG. 14K). DLS profiles show modest size increases after incorporation (FIG. 15A). Transmission electron microscopy (TEM) revealed well-defined, crystalline nanoparticles for both formulations, with diameters ranging from -500 to 700 nm (FIG. 15B), confirming the preservation of structural integrity post-loading. Powder X-ray diffraction (PXRD) further verified that the crystallinity of HOF-TATB was retained after drug encapsulation (FIG. 15C), indicating that guest incorporation does not compromise framework order. The stability and ultrasoundresponsiveness of these drug-loaded HOFs was investigated to evaluate their potential for sustained and controllable anesthetic delivery. Drug retention under physiological conditions- 94 - 4909-0910-3750, v. 3was first assessed by incubating HOF-TATB loaded with bupivacaine in 10% fetal bovine serum (FBS) at 25 °C. HOF-TATB loaded with bupivacaine showed little change in drug loading over 30 days in 10% FBS at 25 °C (FIG. 15D), indicating excellent stability in a protein-rich environment. Collectively, these results demonstrate that ML-prioritized anesthetic candidates can be efficiently and stably encapsulated within structurally robust HOF systems, establishing a foundation for subsequent ultrasound-responsive release and in vivo therapeutic testing.In vitro Ultrasound-Triggered Drug Release and Stability Evaluation

[0214] The ultrasound-triggered release profile of bupivacaine -loaded HOFs was then tested under varying acoustic power. The ultrasound-triggered release experiments demonstrated that the percentage of drug released increased with the ultrasound peak pressure applied (FIG. 15E), confirming the system’s capacity for on -demand, repeatable, and programmable dosing. Upon exposure to focused ultrasound, bupivacaine release increased with acoustic intensity, achieving 22.3 ± 1.7% release within 150 s under 2.45 MPa (FIG. 15E). In contrast, only 16.4 ± 0.8% release was observed under 1.40 MPa, highlighting the tunability of drug release with ultrasound strength. Moreover, bupivacaine release from HOF-TATB could be repeatedly triggered by sequential ultrasound pulses (20 s per pulse), demonstrating stepwise and cumulative drug release. Four successive activations led to incremental release levels of 5.5 ± 0.5%, 7.4 ±0.5%, 9.2 ±0.4%, and 10.4 ±0.6%, respectively (FIG. 15F), highlighting the programmable and re-triggerable nature of this delivery system. In contrast to conventional passive release formulations, this ultrasound-triggered strategy enables dynamic dosing control in response to clinical needs.In vitro Ultrasound-Triggered Drug Release and Neuron Evaluation

[0215] To validate the neuromodulatory functionality of the HOF-based nanocarriers, calcium imaging was performed in cultured neurons. Such in vitro testing provides a critical proof-of-concept for evaluating the platform’s ability to modulate neuronal excitability in a spatially and temporally controlled manner, which is essential for future neurotherapeutic translation. Primary neurons were transduced with AAV encoding GCaMP6s, a genetically encoded calcium indicator, enabling real-time optical recording of intracellular Ca2+fluctuations as a proxy for neuronal activity (FIG. 15G).

[0216] Neurons were first incubated with HOF-TATB @ Lido HC1 nanoparticles, during which robust and spontaneous calcium oscillations persisted, indicating that the- 95 - 4909-0910-3750, v. 3encapsulated drug remained inactive under basal conditions (FIG. 15H, panel (1)). Upon exposure to a focused ultrasound pulse (1.5 MHz, 1.40 MPa, 10 s), a rapid and widespread suppression of calcium transients was observed across the majority of neurons at the experimental groups TATB@Lido HCl / FUS / ( +) / (+) (FIG. 15H, panel (2)) but not in the other 3control groups (FIGS. 15I-J). This outcome confirms effective ultrasound-triggered release of lidocaine from the HOF matrix, resulting in acute silencing of neuronal activity.In Vivo Ultrasound-Triggered Analgesia and Programmable Pain Modulation

[0217] Building on these in vitro findings, the in vivo analgesic performance of HOF-TATB@ lidocaine HC1 was next evaluated. To this end, Sprague-Dawley rats were employed to establish a sciatic nerve block model, with ultrasound imaging-guided perineural injection of HOF-TATB@ lidocaine HC1 (FIG. 16A, FIG. 22).

[0218] To confirm that drag release occurred only upon ultrasound activation — rather than through passive diffusion or systemic leakage - matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI-MSI) was performed on longitudinal sciatic nerve sections (FIG. 16B). As shown in FIG. 16B and FIG. 24, a strong and spatially confined lidocaine signal was detected exclusively in the FUS-activated HOF-TATB ©lidocaine HC1 group, whereas all non-activated groups showed negligible signal. These results validate that ultrasound precisely triggers localized drug release at the sciatic nerve. It was next examined whether this spatially localized release translated into functional analgesia. Mechanical nociceptive sensitivity was quantified using the von Frey assay (FIG. 16C). Following baseline threshold acquisition, it was evaluated how FUS intensity modulates lidocaine release in vivo (1.5 MHz, 0.2 s on / 0.8 s off, total 8 min). VF time-course profiles (FIG. 16D) showed a clear intensity -dependent effect: even 0.79 MPa elicited a rapid increase in withdrawal thresholds within 0.5 h, whereas 1.40 MPa and 2.45 MPa generated substantially stronger and longer-lasting analgesia, persisting for ~5 h. Group-wise comparisons (FIG. 16E) further confirmed this trend, with all intensities producing significant threshold elevations at 0.5 h and higher pressures yielding greater peak responses and more sustained effects at 2 h and 5 h (p < 0.05). Together, these data demonstrate that the magnitude and duration of analgesia can be precisely tuned by adjusting FUS intensity. Notably, 1.40 MPa is well below the U. S. FDA limit for clinical diagnostic ultrasound intensity (ISPTA), highlighting its translational safety margin. Therefore 1.40 MPa was selected to examine the effect of FUS duration, revealing a clear timedependent enhancement in nociceptive threshold from 1 min to 8 min stimulation (FIG. 16F-- 96 - 4909-0910-3750, v. 3G). These findings demonstrate that both FUS intensity and duration can be tuned to achieve graded, on-demand anesthesia, offering a level of temporal precision currently unattainable with conventional local anesthetic injections. Clinically, such a noninvasive, remotely controllable system could enable personalized pain management — delivering brief, targeted analgesia for short procedures or sustained blockade for extended interventions — while minimizing systemic drug exposure and associated side effects.

[0219] Importantly, histological evaluation revealed preserved nerve morphology, underscoring the safety of this localized activation. Hematoxylin and eosin (H& E) staining of sciatic nerve sections 7 days post -treatment showed intact myelin structure and normal fiber morphology in both FUS-treated and untreated groups, with no evidence of degeneration, inflammatory infiltration, or tissue damage (FIG. 25). Immunofluorescence analysis further confirmed minimal Ibal -positive macrophage / microglia infiltration (FIG. 26) and negligible cleaved Caspase-3 staining (FIG.27), indicating no significant immune or apoptotic responses. These results collectively support that TATB ©Lidocaine HC1, with or without FUS activation, does not induce noticeable histopathological changes in sciatic nerve tissue.

[0220] To assess the reproducibility and stability of the ultrasound-triggered anesthetic effect, daily FUS stimulation (1.40 MPa, 8 min) was administered for 7 consecutive days following perineural injection of HOF-TATB ©lidocaine HC1. VF test scores in Sprague Dawley rats consistently revealed a robust and statistically significant elevation in nociceptive threshold after each FUS session compared to baseline, with no apparent decline in efficacy across the treatment period (FIG. 4H). This indicates that the nanoparticle platform maintains its responsiveness to repeated ultrasound activation over multiple days, without evident tachyphylaxis or functional degradation. Such sustained, repeatable on-demand analgesia could be particularly advantageous in clinical scenarios requiring periodic pain relief — such as post-operative recovery or chronic pain flare-ups — while avoiding the need for continuous systemic drug administration.

[0221] Having established the feasibility of ultrasound-triggered release of hydrophilic anesthetics using lidocaine HC1, the long-term stability and therapeutic durability of the second machine learning-guided drug platform was next evaluated with a lipophilic anesthetic — bupivacaine. Due to its higher hydrophobicity and greater loading capacity (-30.0 wt%), it was hypothesized that bupivacaine would exhibit stronger retention within the hydrophobic HOF-TATB pore channels than lidocaine HC1, thereby enabling sustained in vivo localization and- 97 - 4909-0910-3750, v. 3prolonged release upon ultrasound activation. To test this, Sprague-Dawley rats received ultrasound imaging-guided perineural injections of HOF-TATB@bupivacaine nanoparticles near the sciatic nerve. Using previously optimized FUS parameters (1.5 MHz, 1.40 MPa, total 8 min), the nanoparticle depot was stimulated once daily for five consecutive days and performed repeated von Frey tests following each activation. Animals injected with HOF-TATB@bupivacaine exhibited a robust and repeatable elevation in nociceptive threshold in VF tests after each FUS pulse, sustained across the entire five-day testing period (FIG. 16I-J). Compared with lidocaine HC1, bupivacaine produced similarly strong immediate analgesia and suggested more consistent multi-day repeatability, potentially attributable to its higher hydrophobicity, greater loading capacity, and stronger retention within the HOF-TATB framework. These results confirm the capability of the system for durable drug retention and reliable, on-demand analgesia over extended timescales. In contrast, control animals receiving unloaded HOF-TATB nanoparticles showed no significant change in mechanical sensitivity despite identical FUS stimulation (FIG. 16K-L), ruling out material-related or sonomechanical effects. Importantly, the consistent efficacy across multiple days underscores the long-term structural stability of the HOF nanocarrier in vivo and its potential for chronic pain applications requiring repeated intervention.Modeling Neuropathic Pain and Motor Dysfunction after Sciatic Nerve Injury

[0222] To further assess the long-term therapeutic efficacy and clinical relevance of the ultrasound-responsive HOF nanoplatform, an often-used rat model of sciatic nerve transection and repair was employed - a well-established protocol that involves key hallmarks of chronic neuropathic pain, including spontaneous pain behavior and motor dysfunction (Brushart 2011). Following surgical transection and microsuture-based repair of the sciatic nerve, HOF-TATB nanoparticles encapsulating either bupivacaine or lidocaine HC1 were perineurally injected at the injury site (FIG. 17A) once weekly, from week 2 to week 5. FUS (1.5 MHz, 8 min) was applied daily throughout this period to trigger on-demand anesthetic release. Motor function recovery was assessed using the Sciatic Functional Index (SFI)(Brushart 2011), a widely adopted gait analysis metric that quantitatively primarily evaluates locomotor deficits following sciatic nerve injury (FIG. 17B). SFI values enable precise tracking of functional restoration over time. In parallel, toe -chewing (TC), self-mutilation behavior — a hallmark of spontaneous neuropathic pain in Sprague-Dawley rat — was evaluated as a robust indicator of dysesthesia severity (Ji et al. 2023) (FIG. 17C). Compared to untreated controls, both HOF-TATB@bupivacaine and HOF-TATB@lidocaine- 98 - 4909-0910-3750, v. 3HC1 significantly suppressed TC severity, with the lidocaine group exhibiting near-complete behavioral rescue over the 6-week observation window (FIG. 17D-E).

[0223] As for motor recovery, as expected, animals receiving neurorrhaphy alone (negative controls) showed minimal improvement in SFI scores over the 6-week period, consistent with previous reports that spontaneous functional recovery is rare in this model. Animals treated with HOF-TATB@bupivacaine demonstrated marked and sustained improvements in SFI scores over time, indicative of functional reinnervation and recovery of locomotor control (FIG. 17F). Moderate gains were also observed in the HOF-TATB@ lidocaine HC1 treated group, whereas untreated controls showed negligible recovery. The concurrent restoration of both TC and motor primarily (SFI) functions strongly suggests that ultrasound-programmed anesthetic delivery not only relieves neuropathic pain but also facilitates neurofunctional rehabilitation in the chronic phase of injury (Ji et al. 2023 and Brushart 2011). This result might be due to the minimization of aberrant afferent activity and inflammation at the nerve repair site, thereby enabling more favorable conditions for axonal regeneration and functional reintegration. Taken together, these findings position the HOF-based delivery system described herein as a unique neuromodulatory platform — capable of non-invasively reshaping pathological nerve activity, restoring behavioral output, and offering a programmable alternative to current chronic pain and neurorehabilitation therapies. Moreover, HOF-TATB@ Lidocaine HC1 produced the highest axon density, significantly greater than negative controls (p < 0.05). HOF-TATB@Bupivacaine showed a similar upward trend but was not significantly different from control groups (FIG. 17G).

[0224] This sustained suppression of maladaptive pain TC behavior according to von Frey tests and motor sensory recovery behavior as assessed by the SFI underscores the HOF-TATB platform’s ability to durably modulate aberrant neural activity in a noninvasive, programmable manner. Importantly, these results not only highlight the analgesic efficacy of the HOF drug delivery system under chronic pathological conditions but also reveal its potential as a clinically translatable strategy for long-term neuromodulation following peripheral nerve trauma or repair.

[0225] The in vivo behavioral results — derived from machine learning-guided drug selection — provide critical evidence for the translational potential of the HOF-TATB-based ultrasound-responsive drug delivery system. Both lidocaine HC1 and bupivacaine were prioritized by the predictive model for their high loading capacity and favorable release- 99 - 4909-0910-3750, v. 3profiles, and the in vivo testing confirmed their strong analgesic efficacy. Specifically, von Frey testing demonstrated robust, repeatable, and on-demand analgesia, confirming that ultrasound activation can reliably suppress neuropathic pain hypersensitivity in a targeted and temporally precise manner. TC behavior was sustainably suppressed over the multi-week observation period, indicating that the platform effectively mitigates aberrant sensory signaling beyond transient pain relief. Significant improvements in the SFI scores revealed concurrent restoration of voluntary motor function, suggesting that alleviating neuropathic pain not only improves quality of life but also facilitates neurofunctional rehabilitation by reducing maladaptive afferent input, dampening local inflammation, and creating a permissive environment for axonal regeneration. These combined nocioceptive, sensory and motor outcomes — achieved without systemic drug exposure — directly address key clinical requirements for post-operative and chronic pain management. Such outcomes are rarely achieved in current pain control strategies. Importantly, these behavioral results validate the predictive accuracy of the described machine learning-guided drug selection, as both model-prioritized candidates demonstrated the high loading, favorable release, and in vivo efficacy predicted by the algorithm. This Al-assisted screening framework could be further extended to accelerate drugcarrier pairing for a wide range of therapeutic agents.

[0226] Although both lidocaine HC1 and bupivacaine showed strong behavioral efficacy, further studies in larger preclinical models can be performed to confirm long-term stability, biodistribution, and optimal dosing. Regarding the size of the machine-learning training dataset: although the model demonstrated strong predictive performance, the current training set spans a limited number of drug-HOF combinations, which may constrain the chemical diversity the algorithm can fully learn. Expanding the dataset with additional drug chemotypes and HOF architectures can be performed to improve model generalizability and predictive robustness. PEGylation and other rational HOF modifications can be included to enhance drug loading, extend in vivo persistence, and / or allow for personalized, programmable neuromodulation for diverse peripheral nerve injuries and chronic pain conditions.

[0227] In comparison to the current anesthetic delivery platforms — including liposomes (e.g., EXPAREL), hydrogels, micelles, and polymeric depots — most address specific challenges in sustained pain management but none of the presently available therapies simultaneously achieve long-term drug retention, high loading efficiency, non-invasive reactivation, and precise spatiotemporal control. Clinical options such as liposomal anesthetics,- 100 - 4909-0910-3750, v. 3peripheral nerve catheters, intrathecal pumps, and systemic analgesics also exhibit well-recognized limitations in duration, invasiveness, and controllability (Table 2). Most rely on passive release mechanisms, lack modular programmability, or require surgical implantation, limiting adaptability for chronic or activity -dependent pain. By contrast, the ultrasound-triggered HOF nanoparticle platform described herein integrates all four features into a single injectable system: exceptional drug loading (~30 wt%) via tailored hydrogen-bonding and π–π stacking, robust in vitro retention in serum-rich environments (10% FBS) for over a month, sustained in vivo retention for at least one week, re-triggerable release under clinically safe ultrasound, and modular adaptability for both short- and long-acting anesthetics. This capability enables dynamic responsiveness to changing pain states without invasive readministration and elevates the system from a static drug depot to a programmable neuromodulation interface.

[0228] In summary, the programmable system described herein bridges a critical gap in neuromodulation and pain management by enabling non-invasive, externally controlled therapy with minimal systemic exposure. Beyond anesthetic delivery, it is anticipated that the modularity of the HOF framework can be utilized in and is broadly applicability for other neurological, inflammatory, and metabolic disorders that can benefit from precise spatiotemporal drug control, including epilepsy, post-stroke spasticity, and / or localized inflammatory pain. Given that the ultrasound parameters used in the above studies are within clinically approved safety limits and compatible with existing imaging platforms, these approaches could be rapidly adapted for translational and clinical deployment. As such, this data supports the use of these FUS-activated, HOF nanoparticle drug delivery platforms as next-generation, ultrasound-guided theranostic platforms in both clinical and translational settings.* * *

[0229] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents- 101 - 4909-0910-3750, v. 3described herein while the same or similar results would be achieved. 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Claims

WHAT IS CLAIMED IS:

1. A method of treating pain in a mammalian subject comprising:(i) administering to the subject a hydrogen bonded organic framework (HOF) comprising an anesthetic, preferably a local anesthetic; and(ii) applying oscillating ultrasonic waves or focused ultrasound to a portion of the mammalian subject, thereby releasing the anesthetic from the HOF.

2. The method of claim 1, wherein the anesthetic is a local anesthetic, preferably bupivacaine, lidocaine, mepivacaine, ropivacaine, chloroprocaine, tetracaine, benzocaine, articaine, prilocaine, etidocaine, procaine, cinchocaine, levobupivacaine, proparacaine, oxybuprocaine, amylocaine, or prilocaine; more preferably bupivicaine, mepivicaine, chloroprocaine, hexylresorcinol, tapentadol, valenfaxine, levacetylmethadol, or disopyramide.

3. The method of any one of claims 1-2, wherein the HOF comprises a compound of the formula:wherein:A is a Ce-16 aromatic ring or a C3-15 heteroaromatic ring; andeach of B, B', and B" are independently a Ce-i6 aromatic ring or a C3-15 heteroaromatic ring, wherein each may further comprise -S(O)2Ra, - NH2, or — C(O)Ra wherein Rais hydroxy or amino;preferably wherein A comprises 1, 2, 3 or 4 rings, preferably 1 ring, preferably an aromatic(c<6-i3)ring or a heteroaromatic(c=3-i2) ring.- 110 - 4909-0910-3750, v.

34. The method of claim 3, wherein A is:preferably wherein B, B', and B" are each independently substituted aryl(c i2> or substituted heteroaryl(c<i2). preferably wherein B, B', and B" are each independently an aryl(c=6) or heteroaryl(c=3-5) ring;preferably wherein B, B', and B" are each independently- I ll - 4909-0910-3750, v. 3preferably wherein the HOF has intrinsic cavities with sizes between 1 A and 500 A; preferably wherein B, B', and B" have the same structure, such as preferably wherein B, B', and B" are eachCOOH.The method according to any one of claims 3-4, wherein the HOF comprises a compound of the formula:(II),wherein:Xi, X2 and X3 are each independently CH or N;- 112 - 4909-0910-3750, v. 3Ri, R2 and R3 are each independently -S(O)2Raor -C(O)Ra, wherein: Rais hydroxy or amino;R4-R15 are each independently -H, -F, -Cl, -Br, -I or -CH3;or a pharmaceutically acceptable salt thereof.preferably wherein Xi, X2 and X3 are each independently CH or N;preferably wherein Ri, R2 and R3 are each independently -C(O)Ra, wherein Rais hydroxy or amino; such as wherein Ri, R2 and R3 are -C(O)NH2 or wherein Ri, R2 and R3 are -COOH;preferably wherein R4-R15 are -H.

6. The method according to any one of claim 5, wherein the compound is further defined as:preferably- 113 - 4909-0910-3750, v. 3preferably wherein Xi is CH, X2 is CH, and / or X3 is CH; preferably wherein Xi is N, X2 is N, and / or X3 is N; preferably wherein Ri, R2 and R3 are -COOH;preferably wherein the compound is further defined as:- 114 - 4909-0910-3750, v.

37. The method of any one of claims 1-6, wherein the drug or active pharmaceutical ingredient is encapsulated in the HOF;preferably wherein the HOF encapsulates from about 1-40 wt% of the drug or active pharmaceutical ingredient relative to the weight of the HOF;preferably wherein the HOF is further defined as a nanoparticle, and wherein the drug or therapeutic is comprised within the nanoparticle;such as wherein the nanoparticle is about 40-800 nm in size;preferably wherein the mammalian subject is a human.

8. The method of any one of claims 1 -7, wherein the hydrogen bonded organic framework (HOF) comprising the anesthetic is injected into the mammalian subject near a peripheral nerve;preferably wherein the peripheral nerve is the sciatic nerve, median nerve (e.g., carpal tunnel syndrome), ulnar nerve (e.g., cubital tunnel syndrome), radial nerve (e.g., Saturday night palsy), peroneal nerve (e.g., foot drop), femoral nerve, tibial nerve, or the lateral femoral cutaneous nerve (e.g., meralgia paresthetica);preferably wherein the method comprises inducing local anesthesia or analgesia, and / or wherein the method is further defined as a nerve block procedure; such as, wherein the hydrogen bonded organic framework (HOF) comprising the anesthetic is injected into the mammalian subject to treat post-surgery pain (e.g., chronic pain) or pain associated with an injury (e.g., peripheral nerve injury) or peripheral neuropathy.

9. A pharmaceutical composition comprising:(i) a compound of the formula:(II),wherein:- 115 - 4909-0910-3750, v. 3Xi, X2 and X3 are each independently CH or N;Ri, R2 and R3 are each independently -S(O)2Raor -C(O)Ra, wherein:Rais hydroxy or amino;R₄–R₁₅ are each independently –H, –F, –Cl, –Br, –I or –CH₃;or a pharmaceutically acceptable salt thereof;(ii) a local anesthetic, and(iii) an excipient;preferably wherein the local anesthetic is bupivacaine, lidocaine, mepivacaine, ropivacaine, chloroprocaine, tetracaine, benzocaine, articaine, prilocaine, etidocaine, procaine, cinchocaine, levobupivacaine, proparacaine, oxybuprocaine, amylocaine, prilocaine, hexylresorcinol, tapentadol, valenfaxine, levacetylmethadol, or disopyramide.preferably wherein Xi, X2 and X3 are each independently CH or N;preferably wherein wherein Ri, R2 and R3 are each independently -C(O)Ra, wherein Rais hydroxy or amino;preferably wherein Ri, R2 and R3 are -C(O)NH2 or -COOH;preferably wherein R4-R15 are -H;preferably wherein the compound is further defined as:R- 116 - 4909-0910-3750, v. 3CK J3Hpreferably wherein the compound is further defined as:preferably wherein Xi is CH, X2 is CH, and / or X3 is CH; preferably wherein Xi is N, X2 is N, and / or X3 is N; preferably wherein Ri, R2 and R3 are -C(O)NH2 or -COOH;- 117 - 4909-0910-3750, v. 3preferably wherein the compound is further defined as:preferably wherein the local anesthetic may be encapsulated in the HOF, such as wherein the HOF encapsulates from about 1-40 wt% of the drug or active pharmaceutical ingredient relative to the weight of the HOF;preferably wherein wherein the HOF is further defined as a nanoparticle, wherein the drag or therapeutic is comprised within the nanoparticle;preferably wherein the nanoparticle is about 200-900 nm in size.

10. A method of treating treating pain or inducing local anesthesia in a patient in need thereof comprising administering to the patient a pharmaceutically effective amount of a composition of claim 9;preferably wherein the patient is a mammal, preferably a human;preferably wherein the pain results from a peripheral nerve (e.g., a sciatic nerve, median nerve, ulnar nerve, radial nerve, peroneal nerve, femoral nerve, tibial nerve, or the lateral femoral cutaneous nerve).- 118 - 4909-0910-3750, v.

311. A pharmaceutical composition comprising:(i) a hydrogen-bonded organic framework (HOF) compound preferably comprises one or more n-conjugated polycarboxylic acids, preferably wherein the HOF compound is HOF- TATB, HOF-BTB, HOF-101, or HOF-102 as described herein; and(ii) a local anesthetic, preferably wherein the local anesthetic is bupivacaine, lidocaine, procaine, tetracaine, ropivacaine, mepivacaine, articaine, chloroprocaine, etidocaine, hexylresorcinol, tapentadol, valenfaxine, lev acetylmethadol, or disopyramide; even more preferably wherein the local anesthetic is bupivacaine or lidocaine; optionally wherein the pharmaceutical comprises a pharmaceutically acceptable excipient.

12. A method of treating pain in a mammalian subject comprising administering the pharmaceutical composition of claim 11 to the mammalian subject and preferably applying ultrasound to a region of the mammalian subject;wherein the administration is preferably via injection, such as injection near a peripheral nerve such as a sciatic nerve, or topical administration;wherein the ultrasound may comprise or consist of focused ultrasound (FUS) or sequential ultrasound pulses, where the FUS is preferably applied topically to the mammalian subject;wherein the mammalian subject may be a human; andwherein the method may optionally comprise a method of treating pain (e.g., such as post-operative pain, chronic pain, acute pain, hyperalgesia, or neuropathic pain), a neurological disease (e.g., epilepsy, or neuroinflammation), or other disease.

13. A method of producing a pharmaceutical composition comprising a hydrogen-bonded organic framework (HOF) compound and a therapeutic agent, comprising:(i) using a machine learning algorithm to select the therapeutic agent for use with the HOF compound, wherein the machine learning algorithm comprises one of more parameters for the therapeutic agent (e.g., the volume of molecule, the radius of gyration, molecular weight, the number of rotatable bonds, the ratio of sp3 hybridization carbons, total polar surface area, logP, and / or the net charge at pH 7) and the HOF compound (e.g., number of hydrogen bonding sites, crystal port volume, crystal port size, crystal BET surface area, and / or logP);- 119 - 4909-0910-3750, v. 3wherein the HOF compound preferably comprises one or more ^-conjugated polycarboxylic acids, preferably wherein the HOF compound is HOF-TATB, HOF- BTB, HOF-101, or HOF-102 as described herein; andwherein the therapeutic agent is preferably a small molecule (e.g., a FDA approved small molecule drug);wherein the pharmaceutical composition may optionally be the pharmaceutical composition of claim 11; and(ii) combining the HOF compound and the therapeutic agent to produce the pharmaceutical composition;wherein the therapeutic agent may be a local anesthetic, preferably wherein the local anesthetic is bupivacaine, lidocaine, procaine, tetracaine, ropivacaine, mepivacaine, articaine, chloroprocaine, etidocaine, hexylresorcinol, tapentadol, valenfaxine, levacetylmethadol, or disopyramide; even more preferably wherein the local anesthetic is bupivacaine or lidocaine.- 120 - 4909-0910-3750, v. 3

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