Hydrogen bonded organic frameworks for drug delivery with ultrasound

Abstract

Pharmaceutical compositions comprising hydrogen bonded organic framework (HOF) molecules and a drug are provided herein. Drug release from the HOF can be induced using ultrasound such as focused ultrasound (FUS). Methods of ultrasonic drug delivery using a HOF are provided herein and can be used to treat a variety of diseases.

Description

PATENT UTFB.P1344WO HYDROGEN BONDED ORGANIC FRAMEWORKS FOR DRUG DELIVERY WITH ULTRASOUND BACKGROUND OF THE INVENTION

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 519,490, filed August 14, 2023, the entirety 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. The government has certain rights in the invention. 1. Field of the Invention

[0003] The present invention relates generally to the field of molecular biology and medicine. More particularly, it concerns HOF nanoparticles that can be used for ultrasound programmable drug activation. 2. Description of Related Art

[0004] It has long been a goal of modern medicine to non-invasively and precisely deliver a medicine to specific target tissues or cells involved in the disease. Such targeted delivery of a medication may improve a concentration and duration that yields an intended physiological response while avoiding one or more excess or inadequate effects (Mirvakili et al., 2021; Boulatov, 2021; Rao et al., 2019). However, the limitations of existing therapeutic modalities, including low temporal resolution, noticeable off-target effects or invasiveness, have hindered efficacy in achieving desirable and specific cellular events control (Mirvakili et al., 2021; Boulatov, 2021). Technologies like light-responsive optogenetics can enable spatiotemporal control of medication, but they have limited applicability in deep tissue due to the inefficient delivery of photons within organisms (Marshel et al., 2019; Chen et al., 2021; Lee et al., 2021). Non-invasive techniques, such as magnetic technology, have been tested for achieving activation in deep tissue, but they suffer from the temporal resolution and the increased engineering complexity involved in thedesign of magnetic systems (Rao et al., 2019; Lee et al., 2021; Sebesta et al., 2022; Romero et al., 2022).

[0005] While recent advancements in these non-invasive controls over drug activation hold great promise for clinical therapeutic applications, challenges remain in terms of limited programmable control of drug activation, modality complexity, and restricted drug bioavailability in deep tissue, particularly in large animals such as non-human primates and humans (Rao et al., 2019; Rwei et al., 2017; Huo et al., 2021).

[0006] 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 et al., 2017; Airan et al., 2017; Chen and Hwang, 2013; 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 membranes, 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 - 2 - 4860-4443-2856, v.1et 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). While significant progress has been made in acoustically- triggered system, it is still difficult to simultaneously achieve high programmability, high responsive sensitivity, and high drug loading capacity, which are required for many practical biomedical applications. Clearly, there is a need for new and improved methods of drug delivery and activation. - 3 - 4860-4443-2856, v.1SUMMARY OF THE INVENTION

[0007] The present disclosure provides compositions and methods for drug delivery that utilize hydrogen-bonded frameworks (HOF), such as nanoparticles, that comprise a drug or therapeutic agent, which can be released from the HOF based on stimulation with FUS. In some preferred embodiments, the HOF nanoparticles comprise a drug or therapeutic agent that can be released due to stimulation with ultrasound such as FUS. The HOF may serve as mechanoresponsive vehicles to achieve ultrasound programmable drug activation or release in a desired region of the body or in discrete or specific tissues. As shown in the figures, HOF provided herein (e.g., HOF-TATB, HOF-BTB) exhibited beneficial porosity, aqueous stability, topology, biocompatibility, and can be used with ultrasound for delivery of a drug or therapeutic agent in vivo. Crystal structures were determined and targeted 10%, 20%, 30%, 40%, 50% and 60% dissociation of HOFs can be achieved using ultrasound to trigger drug release (e.g., of DCZ, dopamine, or procaine) in the brain, central nervous system, or other tissues. In some aspects, the HOF can thus be used in combination with FUS to deliver the drug or therapeutic agent to a variety of tissues in a mammalian subject including, e.g., the brain, breast, uterus, liver, kidney, heart and other organs, and subcutaneous tissue.

[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 a drug or active pharmaceutical ingredient; and (ii) applying oscillating ultrasonic waves to a portion of the mammalian subject, thereby releasing the drug or active pharmaceutical ingredient from the HOF. The HOF may comprise or consist of a compound of the formula:wherein: A is a C6-16 aromatic ring or a 15 ring; and each of B, B′, and B′′ are independently a C6-16 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 - 4 - 4860-4443-2856, v.1or 4 rings, preferably 1 ring. A may be an aromatic(C≤6−13)ring or a heteroaromatic(C=3−12)ring. In some preferred embodiments, A is an aromatic(C6)or heteroaromatic(C3−5)ring. In some embodiments, A is: ,heteroaryl(C≤12). 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 each independently: , , 4860-or In som . Thewith sizes between about 1 Å and about 500 Å. In some preferred embodiments, B, B′, and B′′ have the same structure. In some embodiments, B, B′, and B′′ are each: .The may a compound of the formula: , 4860-4443-2856, v.1wherein: X1, X2and X3are each independently CH or N; R1, R2and R3are each independently −S(O)2Ra or −C(O)Ra, wherein: Ra is 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, X1, X2and X3are each independently CH or N. In some preferred embodiments R1, R2 and R3 are each independently −C(O)Ra, wherein Ra is hydroxy or amino. In some embodiments, R1, R2 and R3 are -C(O)NH2. In some embodiments, R1, R2 and R3 are -COOH. In some embodiments, R4-R15are −H. The compound may be further defined as: . In some embodiments, the. The compound may be. 4860-4443-2856, v.1The compound may be further defined as: . In some embodiments, X1isembodiments, X1is N, X2is N, and / or X3 is N. In some embodiments, R1, R2 and R3 are -COOH. The compound may be further defined as: . The compound may be.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 - 8 - 4860-4443-2856, v.1defined as a nanoparticle, and wherein the drug or therapeutic is comprised within the nanoparticle. The nanoparticle may be about 40-800 nm in size. The mammalian subject may be a human. The method may further comprise treating a disease in the mammalian subject. The disease may be a cancer, a neurological disorder, a neurodegenerative disease, a cardiac disease, a lung disease, a heart disease, a kidney disease, inflammation, pain relief, or an inflammatory disease. The cancer may be, e.g., a brain cancer (e.g., a glioma or glioblastoma), a breast cancer, a skin cancer, a uterine cancer, a liver cancer, a lung cancer, or a pancreatic cancer. The neurodegenerative disease may be Parkinson’s disease, or Alzheimer's disease. The neurological disorder may be pain (e.g., neuropathic pain), epilepsy, depression, or addiction. The drug or active pharmaceutical agent may be a chemotherapeutic, a neuroactive drug, or an anti-inflammatory drug， an analgesic, or an anesthetic.

[0009] Another aspect of the present disclosure relates to a pharmaceutical composition comprising: (i) a compound of the formula: , wherein: X1, X2 and X3 areand R3 are each independently −S(O)2Ra or −C(O)Ra, wherein: Ra is hydroxy or amino; R4-R15 are each independently −H, −F, −Cl, −Br, −I or −CH3; or a pharmaceutically acceptable salt thereof; (ii) a drug or an active pharmaceutical agent, and (iii) an excipient. In some embodiments, X1, X2and X3are each independently CH or N. In some embodiments, R1, R2 and R3 are each independently −C(O)Ra, wherein Rais hydroxy or amino. In some embodiments, R1, R2and R3are –C(O)NH2. In some embodiments, R1, R2and R3are -COOH. In some embodiments, R4-R15are −H. The compound may be further defined as: - 9 - 4860-4443-2856, v.1. The compound may be further The compound may beThe compound may be further4860-4443-2856, v.1In some embodiments, X1is CH, X2is CH, and / or X3is CH. In some embodiments, X1is N, X2is N, and / or X3 is N. In some embodiments, R1, R2 and R3 are –C(O)NH2. In some embodiments, R1, R2 and R3 are −COOH. The compound may be further defined as: . The compound may be. In some embodiments, theanesthetic, chemotherapeutic, or an anti-inflammatory drug, an analgesic, a painkiller, or an enzyme. 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 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.

[0010] Yet another aspect of the present disclosure relates to a method of treating or preventing a disease or disorder 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 disease - 11 - 4860-4443-2856, v.1or disorder may be a cancer, a neurological disorder, a neurodegenerative disease (e.g., Parkinson’s disease or Alzheimer's disease), a lung disease, a heart disease, a kidney disease, inflammation, or pain. The neurological disorder may be pain (e.g., neuropathic pain), epilepsy, depression, addiction, stroke, or spinal cord injury. The cancer may be a brain cancer (e.g., glioma or glioblastoma), a breast cancer, a skin cancer, a uterine cancer, liver cancer, lung cancer, or pancreatic cancer. In some embodiments, the neurological disorder is pain, epilepsy, depression, or addiction.

[0011] 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.

[0012] 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.

[0013] Throughout this application, the term “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.

[0014] 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.

[0015] 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 - 12 - 4860-4443-2856, v.1“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.

[0016] 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. - 13 - 4860-4443-2856, v.1BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] FIGS. 1A-B: Ultrasound mechanically responsive hydrogen-bonded organic frameworks (HOFs) preparation. show a schematic illustration of the ultrasound mechanical stress triggered dissociation of HOFs, where the HOFs were stable in solution but disassociated when triggered by the ultrasound power (Eus) exceeding HOFs scission threshold (Eus> Ethreshold) FIG. 1A, Schematic illustration of the ultrasound mechanical stress triggered dissociation of HOFs, where the HOFs were stable in solution but disassociated when triggered by the ultrasound power (Eus) exceeding HOFs scission threshold (Eus > Ethreshold). (FIG. 1B) Four representative organic monomers and the self-assembled porous HOF structures: HOF-TATB, HOF-BTB, HOF- 101 and HOF-102. The organic molecular building units (OMBUs) of HOFs self-assemble through hydrogen-bonding and π-π stacking interactions, resulting in the formation of 3D porous frameworks.

[0019] FIGS. 2A-I: Ultrasound mechanically triggers the time-resolved dissociation curve of different HOFs (mean ± SEM, n=3 independent samples) at various peak pressure, including (FIG. 2A) HOF-TATB, (FIG. 2B) HOF-BTB, (FIG. 2C) HOF-101 and (FIG. 2D) HOF-102. (FIG. 2E) Cohesive energy of different HOFs, including their hydrogen-bonding interactions and π-π stacking interactions in each unit. HOFs were constructed from organic building units through intermolecular non-covalent hydrogen-bonding interactions and π-π stacking interactions (Pi-Pi), where the bonding energy of one unit was denoted as the cohesive energy of HOFs. Moreover, the HOFs displayed varying ultrasound thresholds for disassociation, which were associated with the characteristics of the organic building units. (FIG. 2F) The prediction heatmap of ultrasound-controlled dissociation of HOFs. By referring to the provided heatmap, one can determine the optimal cohesive energy needed to attain a dissociation percentage at a specific ultrasound power. This will guide the structural design of HOFs at the molecular level to achieve the cohesive energy for subsequent programmable control of HOFs dissociation at - 14 - 4860-4443-2856, v.1certain ultrasound peak pressure. (FIG. 2G) The free dye release from HOF particles without ultrasound stimulation. (FIG. 2H) Schematic diagram of ultrasound triggered drug release from the HOF particles, with drug release occurring when Eus > Ethreshold. (FIG.2I) Ultrasound triggered dye release from the HOF particles after 90 s stimulus. The HOF particles exhibited distinct ultrasound thresholds for drug activation, with the order of sensitivity being HOF-TATB@RB (0.51 MPa) < HOF-BTB@RB (1.55 MPa) < HOF-101@RB (3.94 MPa) < HOF-102@RB (8.04 MPa).

[0020] FIGS. 3A-H show ultrasound-controlled cargo release from HOF-TATB nanoparticles and their in vitro modulation of neural activity. TEM images and hydrodynamic size distribution measured by dynamic light scattering of the HOF-TATB nanoparticles (i) before loading of clozapine-N-oxide (CNO) and (ii) after loading of CNO, and (iii) after irradiating by ultrasound (1.5 MHz, 1.55 MPa, 60 s). Scale bar: 200 nm (FIG. 3A). Ultrasound triggered dye release (mean ± SEM, n=3 independent samples) from HOF-TATB nanoparticles (1.5 MHz, 1.55 MPa) over time (FIG. 3B). Ultrasound triggered dye release (mean ± SEM, n=3 independent samples) from HOF-TATB after 60 s irradiation (1.5 MHz) at different peak pressure (FIG. 3C). Ultrasound-triggered dye release (mean ± SEM, n=3 independent samples) at different frameworks disassociation percentage of the HOF-TATB nanoparticles (FIG.3D). Fluorescence images of the primary cortical neurons expressing hSyn::hM3D (Gq)-mCherry and hSyn::GCaMP6s-WPRE- SV40. Scale bar: 40 μm (FIG. 3E). Heat maps of normalized GCaMP6s fluorescence intensity from 100 neurons in different experimental conditions. The experiments were independently repeated at least three times. F0is the mean fluorescence intensity of baseline, hM3D(Gq)+means neurons with hM3D(Gq) expression, hM3D(Gq)- means neurons without hM3D(Gq) expression, FUS+means the neurons are treated with the focused ultrasound and FUS- means the neurons without focused ultrasound treatment, TATB@CNO+means neurons with CNO loaded HOF- TATB nanoparticles, and TATB@CNO- means neurons HOF-TATB nanoparticles only without CNO. OFF means ultrasound is off. ON means ultrasound is on (1.5 MHz, 1.08 MPa, pulse 10 s) (FIG.3F). Statistical analysis of calcium signal changes in 100 primary neurons under the different conditions. Mean ± SEM, n ≥ 3 independent tests. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (3G). Normalized in vitro neuron spiking latency under sono-chemogenetics stimulation. The hM3D(Gq) and GCaMP6s expressing neurons (100 neurons) were conducted to analyze the - 15 - 4860-4443-2856, v.1latency between the neuron firing and ultrasound stimulation. The results determined that the latency of sono-chemogenetics stimulation is around 1.6 s (FIG. 3H). Mean ± SEM; 3 times independent tests (n ≥ 3).

[0021] FIGS. 4A-O show in vivo sono-chemogenetic deep brain stimulation in mouse. Experimental scheme of the in vivo fiber photometry in the VTA area (FIG. 4A). Confocal fluorescence images of the co-expression of hM3D(Gq) and GCaMP6s in mouse VTA area. Scale bar: 20 μm (FIG. 4B). Normalized GCaMP6s fluorescence intensity change (ΔF / F0) in mice VTA under the different experimental conditions. Remarkable fluorescence increase was observed only when the FUS was applied to the mice with hM3D(Gq) expressed in VTA neurons. The pink area represents the FUS irradiation (1.5 MHz, 1.40 MPa, pulse 10s). Solid line, mean; shade area, SEM; at least 3 mice in each group (n ≥ 3). One-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 4C). Statistical analysis of calcium signal changes in mice VTA region under different conditions. Mean ± SEM, n ≥ 3 independent mouse. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG.4D). Normalized in vivo neuron spiking latency in mice VTA under sono-chemogenetics stimulation, where the sono-chemogenetics exhibited temporal resolution with around 3.5 s latency (FIG. 4E). C-fos expression confocal fluorescence images in the VTA after the mice are treated with different conditions. Remarkable increase of c-fos expression was observed only when the mice with hM3D(Gq) expression in VTA neurons were treated with FUS (1.5 MHz, 1.40 MPa, pulse 20 s, focus length 5 mm). Scale bar: 20 μm (FIG. 4F). Quantification of the c-fos expression percentage among hM3D(Gq)+ neurons. Mean ± SEM, n ≥ 3 independent samples. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 4G). Scheme of conditioned place preference tests with sono-chemogenetics (FIG. 4H). Traces of mouse freely exploring apparatus (i) before and (ii) after sono-chemogenetic stimulation. Red one is the conditioned chamber (FIG. 4I). Statistical analysis of time spent in the FUS stimulation chamber. Mean ± SEM, n, number of the mice in each group. Paired t tests and two-tailed comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 4J). Preference score of mice with different conditions. Mean ± SEM, n, number of the mice in each group. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 4K). Scheme of forced swimming test (FST) with sono- - 16 - 4860-4443-2856, v.1chemogenetics (FIG. 4L). Presentative traces of mice in FST under the different conditions (FIG. 4M). Time-resolved mouse immobility score curve (FIG.4N) and statistical analysis of immobility time of mice in the FST tests (FIG.4O). Only the mice with hM3D(Gq) expression in VTA neurons and TATB@CNO nanoparticles only exhibited reduced immobility score after FUS stimulation. Mean ± SEM, n, number of the mice in each group. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001).

[0022] FIGS. 5A-J show in vivo sono-chemogenetic deep brain stimulation in rats. Experimental scheme of the in vivo fiber photometry in rat VTA area (FIG. 5A). Confocal fluorescence images of the co-expression of hM3D(Gq) and GCaMP6s in rat VTA area. Scale bar: 20 μm (FIG. 5B). Normalized GCaMP6s fluorescence intensity change (ΔF / F0) in rat VTA under the different experiment conditions. Remarkable fluorescence increase was observed only when the FUS was applied to treat the mice with hM3D(Gq) expressed in VTA neurons. The pink area represents the FUS irradiation ((1.5 MHz, 2.45 MPa, pulse 20 s, focus length 9 mm). Solid line, mean; shade area, SEM; at least 3 rats in each group (n ≥ 3). One-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 5C). Statistical analysis of calcium signal changes in rat VTA region under the different conditions. Mean ± SEM, n ≥ 3 independent rats. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 5D). Normalized in vivo neuron spiking latency in rat VTA under sono-chemogenetics stimulation, where the sono-chemogenetic stimulation exhibited around 8.8 s latency at depth of 9 mm (5E). C-fos expression in confocal fluorescence images in the VTA after the rat is treated with different conditions. Scale bar: 20 μm (FIG. 5F). Quantification of the c-fos expression percentage among the hM3D(Gq)+ neurons. Mean ± SEM, n ≥ 3 independent samples. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 5G). Rat conditioned place preference tests (FIG.5H), scheme of conditioned place preference tests with sono-chemogenetics. Traces of rat freely exploring apparatus (ii) before and (iii) after sono-chemogenetic stimulation. Red one is the conditioned chamber. (i) Statistical analysis of time spent in the FUS stimulation chamber. Mean ± SEM, n, number of the rat in each group. Paired t tests and two-tailed comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 5I). Preference score of rats at different conditions. Mean ± SEM, - 17 - 4860-4443-2856, v.1n, number of the rats in each group. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 5J).

[0023] FIG. 6 shows the1H NMR spectrum of HOF-TATB build unit (H3TATB).

[0024] FIG. 7 shows the1H NMR spectrum of HOF-BTB build unit (H3BTB).

[0025] FIG. 8 shows the1H NMR spectrum of HOF-101 build unit (H4TBAPy).

[0026] FIG. 9 shows the1H NMR spectrum of HOF-102 build unit (H4PTTNA).

[0027] FIGS. 10A-D show the x-ray diffraction tests of nanosized HOFs, HOF-TATB nanoparticles (FIG. 10A), HOF-BTB nanoparticles (FIG. 10B), HOF-101 nanoparticles (FIG. 10C), HOF-102 nanoparticles (FIG. 10D).

[0028] FIG.11 shows the simulated PXRD pattern (black) of crystal structure of nanosized HOF-TATB obtained from electron diffraction tests, and the experimental PXRD patterns of HOF- TATB samples before and after ultrasound stimulation.

[0029] FIGS. 12A-B show single-component sorption isotherms of nitrogen at 77 K of nanosized HOF-TATB. BET Surface Area: 166.5 m2g-1, Langmuir Surface Area: 292.11 m2g-1(11A). The pore size distribution of nanosized HOF-TATB, pore volume: 0.21 cm3g-1(11B).

[0030] FIGS. 13A-H show that FUS triggered the dissociation of HOFs. The HOFs nanoparticles were irradiated with different ultrasound power, and the dissociation of HOFs was determined through UV-Vis tests. The characteristic spectrum of different HOFs (FIG. 13A), HOF-TATB (FIG.13B), HOF-BTB (FIG.13C), HOF-101 (FIG.13D), HOF-102. The dissociation curves of HOFs at different ultrasound peak pressure (FIG. 13E), HOF-TATB (FIG. 13F), HOF- BTB (FIG. 13G), HOF-101 (FIG. 13H), HOF-102.

[0031] FIGS. 14A-D show the thermal dissociation tests of HOFs, HOF-TATB (FIG. 14A), HOF-BTB (FIG. 14B), HOF-101 (FIG. 14C), HOF-102 (FIG. 14D). The HOFs nanoparticles were incubated at different temperatures for 5 min. After that, the HOFs solution was extracted out and centrifuged, the supernatant was conducted to do UV-Vis tests for HOFs dissociation determination. The thermal dissociation occurred around 60ºC. Only around a 2% - 18 - 4860-4443-2856, v.1increase was observed at HOF-TATB and HOF-BTB, and no thermal dissociation was observed in HOF-101 and HOF-102 the temperature at 100ºC.

[0032] FIG.15 shows that a linear model fits the relationship between the Ecohesive of HOFs and the Ln[k] at fixed Eus. With 1.55, 1.72, 3.94, 6.49, 8.03 MPa peak pressure, Ln[k] of HOF- TATB, HOF-BTB, HOF-101, and HOF-102 correlates to their cohesion energy linearly, respectively.

[0033] FIG. 16 shows that when Ln[k] is held constant, a linear correlation is observed between the ultrasound peak pressure and the cohesive energy of HOFs. To achieve a targeted 10%, 20%, 30%, 40%, 50% and 60% dissociation of HOFs at a fixed ultrasound peak pressure, it is possible to calculate the corresponding Ecohesiveof HOFs using the established linear relationship.

[0034] FIGS. 17A-C show TEM images and hydrodynamic size distribution measured by dynamic light scattering of HOF-BTB nanoparticles (FIG. 17A), HOF-101 nanoparticles (FIG. 17B), and HOF-102 nanoparticles (FIG. 17C).

[0035] FIG. 18 shows the biostability evaluation of HOF-TATB nanoparticles. The hydrodynamic size distribution was measured by dynamic light scattering with or without 10% FBS.

[0036] FIGS. 19A-D show the ultrasound triggered drug release (mean ± SEM, n=3 independent samples) from HOF-TATB. The fluorescence dye Rhodamine B (RB) was loaded into the HOF-TATB nanoparticles (TATB@RB) at first. After that, the TATB@RB nanoparticles were irradiated by the ultrasound with different power densities, including 0.51 MPa (FIG. 19A), 0.89 MPa (FIG. 19B), and 1.08 MPa (FIG. 19C). The quantification of drug release percentage without ultrasound and with ultrasound for 90 s. Mean ± SEM, n ≥ 3 independent samples. One- way ANOVA and Dunnett’s multiple comparison tests (P(ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 19D).

[0037] FIGS. 20A-C show the in vitro biosafety evaluation of HOF-TATB nanoparticles. The cell viability tests of HOF-TATB nanoparticles in human embryonic kidney 293 (HEK-293) cells. Mean ± SEM; at least 3 times independent tests (n ≥ 3). The hemolysis tests of HOF-TATB - 19 - 4860-4443-2856, v.1nanoparticles (FIG.20A), photograph (FIG.20B) and hemolysis statistical analysis, mean ± SEM; at least 3 times independent tests (n ≥ 3) (FIG. 20C).

[0038] FIG.21 shows normalized GCaMP6s fluorescence intensity increase (ΔF / F0) from 100 of hM3D(Gq) and GCaMP6s expressing neurons in response to free CNO containing neuron culture medium (20 µM). Solid line, mean; shade area, SEM; at least 3 times independent tests (n ≥ 3).

[0039] FIG. 22 shows normalized GCaMP6s fluorescence intensity from 100 neurons in different experimental conditions. The experiments were independently repeated at least three times. F0was the mean fluorescence intensity of baseline, hM3D(Gq)+means neurons with hM3D(Gq) expression, hM3D(Gq)- means neurons without hM3D(Gq) expression, FUS+means the neurons that are irradiated by the focused ultrasound and FUS- means the neurons without focused ultrasound irradiation, TATB@CNO+means HOF-TATB nanoparticles loaded with CNO is added to the neurons, TATB@CNO- means only HOF-TATB nanoparticles are added to treat the neurons. OFF, ultrasound is off. ON, ultrasound is on (1.5 MHz, 1.08 MPa, pulse 10 s).

[0040] FIG. 23 shows ultrasound power deposition in the tissue. To determine the ultrasound power transfer efficiency in the tissue, porcine skin with different depths was first placed on the FUS transducer (1.5 MHz, 2.40 MPa primary ultrasound power), and the ultrasound power was detected by the hydrophone. The results determined that the 1.5 MHz ultrasound could achieve around 20 mm tissue penetration, and the ultrasound power transfer efficiency was still at 37% even at 10 mm tissue depth.

[0041] FIG. 24 shows the in vivo ultrasound power transfer in the mouse head with FUS focus length of 5 mm. The ultrasound power heatmap in the mouse head shows that around 0.9 MPa was delivered to the mouse VTA when 1.40 MPa primary ultrasound power was used.

[0042] FIG. 25 shows the normalized GCaMP6s fluorescence intensity change (ΔF / F0) in mice VTA under the repeated sono-chemogenetic stimulation after 5 days injection. Multiple increases in GCaMP6s signal were recorded in mouse VTA under the repeated irradiation of sono- chemogenetic stimulation. FUS (1.5 MHz, 1.40 MPa, pulse 10 s), TATB@CNO nanoparticles (2 μg / mL). The blue area represents the FUS irradiation. - 20 - 4860-4443-2856, v.1

[0043] FIG. 26A-D shows the in vivo stability evaluation of HOF-TATB nanoparticles. Rhodamine B (RB) dye-loaded HOF-TATB nanoparticles (TATB@RB) were injected into the mouse VTA.50 µm thick brain slices were prepared 1 day (FIG.26A), 3 days (FIG.26B), 2 weeks (FIG. 26C) and 3 weeks (FIG. 26D) following injection. a-d, fluorescence images of TATB@RB injection sites. Scale bar: 50 µm. Blue signal is DAP and, red signal is TATB@RB.

[0044] FIG. 27 shows the in vivo ultrasound power transfer in rat heads with FUS focus length of 10 mm. The ultrasound power heatmap in the rat head shows that around 1.30 MPa was delivered to the rat VTA when 2.45 MPa primary ultrasound power was used.

[0045] FIG. 28 shows an in vivo biosafety evaluation by H&E staining after sono- chemogenetics, scale bar: 100 μm.

[0046] FIGS. 29A-B show an in vivo biocompatibility evaluation of the sono- chemogenetics via determining microglia (Iba1) activation. Fluorescence images of Iba1 of the brain slices in mouse VTA under the different conditions 7 days after sono-optogenetic stimulation. Scale bar :50 µm. Blue signal is DAPI and red signal is Iba1 (FIG. 29A). Statical analysis of the Iba1 intensity. Mean ± SEM, n >3, mice in each group. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 29B).

[0047] FIGS. 30A-B show an in vivo biocompatibility evaluation of the sono- chemogenetics via determining neuron apoptosis (Caspase-3). Fluorescence images of Caspase-3 of the brain slices in mouse VTA under the different conditions 7 days after sono-optogenetic stimulation. Scale bar: 50 µm. Blue signal is DAPI and red signal is Caspase-3 (FIG.30A). Statical analysis of the Caspase-3 intensity. Mean ± SEM, n >3, mice in each group. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001) (FIG. 30B).

[0048] FIGS. 31A-B show an in vivo biocompatibility evaluation of the sono- chemogenetics via determining astrocytes (GFAP) activation. Fluorescence images of GFAP of the brain slices in mouse VTA under the different conditions 7 days after sono-optogenetic stimulation. Scale bar: 50 µm. Blue signal is DAPI and red signal is GFAP. Statical analysis of the - 21 - 4860-4443-2856, v.1GFAP intensity. Mean ± SEM, n >3, mice in each group. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), * 0.01≤ P < 0.05, ** 0.001≤ P < 0.01, **** P< 0.0001).

[0049] 32: H-bond motifs in HOF-TATB (Guest solvent molecule is denoted as G).

[0050] FIG. 33A-C: Topology analysis of HOF-TATB. (FIG. 33A) Structures of two different hydrogen bonding motifs and their simplified forms. (FIG. 33B) 3D structure of the interpenetrated network in HOF-TATB and its simplified 3,4-connected topology viewed from the c-axis (FIG. 33C) Perspective view of a simplified single net.

[0051] FIG.34A-B: Calculated pore surface and view of cubic cage and 1D pore channel of HOF-TATB. (FIG. 34A) view along a-axis; (FIG. 34B)view along b-axis. (Connolly surface with pore radius of 1.2Å)

[0052] FIG. 35A-C: Crystal structure of HOF-TATB. (FIG. 35A) view along a-axis; (FIG. 35B) view along b-axis; (FIG. 35C) view along c-axis.

[0053] FIG. 36: Aqueous stability test of HOF-TATB. The HOF-TATB was stored in a solution and collected at fixed time points for PXRD tests to evaluate crystal stability.

[0054] FIGS. 37A-B: The porosity characterization of HOF-TATB particles. Single- component sorption isotherms of nitrogen at 77 K of nanosized HOF-TATB. BET Surface Area: 166.5 m2g-1.

[0055] FIG. 38: The porosity characterization of HOF-BTB particles. Single-component sorption isotherms of nitrogen at 77 K of nanosized HOF-BTB. BET Surface Area: xx m2g-1.

[0056] FIGS. 39A-B: The porosity characterization of HOF-101 particles. Single- component sorption isotherms of nitrogen at 77 K of nanosized HOF-101. BET Surface Area: 390.9 m2g-1.

[0057] FIGS. 40A-B: The porosity characterization of HOF-102 particles. Single- component sorption isotherms of nitrogen at 77 K of nanosized HOF-102. BET Surface Area: 1294.3 m2g-1. - 22 - 4860-4443-2856, v.1

[0058] FIGS.41A-K: FUS triggered dissociation of TATB-HOFs. The TATB-HOFs were irradiated with different ultrasound powers. The dissociation of HOFs was located in the supernatant after centrifuge, and determined through UV-Vis tests. The UV-Vis characteristic spectrum of dissociated TATB-HOFs at different peak pressure, (FIG. 41A) 0 MPa, (FIG. 41B) 0.26 MPa, (FIG. 41C) 0.51 MPa, (FIG. 41D) 0.89 MPa, (FIG. 41E) 1.08 MPa, (FIG. 41F) 1.39 MPa, (FIG.41G) 1.55 MPa, (FIG.41H) 1.72 MPa, (FIG.41I) 3.94 MPa, (FIG.41J) 6.49 MPa and (FIG. 41K) 8.03 MPa.

[0059] FIGS. 42A-K: FUS triggered dissociation of BTB-HOFs. The BTB-HOFs were irradiated with different ultrasound powers. The dissociation of HOFs was located in the supernatant after centrifuge, and determined through UV-Vis tests. The UV-Vis characteristic spectrum of dissociated BTB-HOFs at different peak pressure, (FIG.42A) 0 MPa, (FIG.42B) 0.26 MPa, (FIG. 42C) 0.51 MPa, (FIG. 42D) 0.89 MPa, (FIG. 42E) 1.08 MPa, (FIG. 42F) 1.39 MPa, (FIG.42G) 1.55 MPa, (FIG.42H) 1.72 MPa, (FIG.42I) 3.94 MPa, (FIG.42J) 6.49 MPa and (FIG. 42K) 8.03 MPa.

[0060] FIGS. 43A-F: FUS triggered dissociation of HOF-101. The HOF-101 were irradiated with different ultrasound powers. The dissociation of HOFs was located in the supernatant after centrifuge, and determined through UV-Vis tests. The UV-Vis characteristic spectrum of dissociated HOF-101 at different peak pressure, (FIG. 43A) 0 MPa, (FIG. 43B) 1.55 MPa, (FIG.43C) 1.72 MPa, (FIG.43D) 3.94 MPa, (FIG.43E) 6.49 MPa and (FIG.43F) 8.03 MPa.

[0061] FIGS. 44A-F: FUS triggered dissociation of HOF-102. The HOF-102 were irradiated with different ultrasound powers. The dissociation of HOFs was located in the supernatant after centrifuge, and determined through UV-Vis tests. The UV-Vis characteristic spectrum of dissociated HOF-102 at different peak pressure, (FIG. 44A) 0 MPa, (FIG. 44B) 1.55 MPa, (FIG.44C) 1.72 MPa, (FIG.44D) 3.94 MPa, (FIG.44E) 6.49 MPa and (FIG.44F) 8.03 MPa.

[0062] FIGS. 45A-D: The thermal dissociation tests of HOFs. (FIG. 45A) HOF-TATB, (FIG.45B) HOF-BTB, (FIG.45C) HOF-101, (FIG.45D) HOF-102. The HOFs nanoparticles were incubated at different temperatures for 5 min. After that, the HOFs solution was extracted out and centrifuged, the supernatant was conducted to do UV-Vis tests for HOFs dissociation determination. The thermal dissociation occurred around 60 °C. Only around a 2% increase was - 23 - 4860-4443-2856, v.1observed at HOF-TATB and HOF-BTB, and no thermal dissociation was observed in HOF-101 and HOF-102 the temperature at 100 °C.

[0063] FIG.46: A linear model fits the relationship between the ultrasound peak pressure and the Ln[k] of HOFs when the peak pressure is below 1.55 MPa.

[0064] FIG. 47: A linear model fits the relationship between the ultrasound peak pressure and the Ln[k] of HOFs when the peak pressure is up 1.55 MPa.

[0065] FIG. 48: A linear model fits the relationship between the Ecohesiveof HOFs and the Ln[k] at fixed Eus. With 1.72, 3.94, 6.49, and 8.04 MPa peak pressure, Ln[k] of HOF-TATB, HOF- BTB, HOF-101, and HOF-102 correlate to their cohesion energy linearly, respectively.

[0066] FIG. 49: When Ln[k] is held constant, a linear correlation is observed between the ultrasound peak pressure and the cohesive energy of HOFs. To achieve a targeted 10%, 20%, 30%, 40%, 50% and 60% dissociation of HOFs at a fixed ultrasound peak pressure, it is possible to calculate the corresponding Ecohesive of HOFs using the established linear relationship.

[0067] FIG. 50: Theoretical ultrasound power threshold for triggered dissociation of HOFs. The thresholds for HOFs’ dissociation (HOF-TATB, HOF-BTB, HOF-101, and HOF-102) are 0.07 MPa, 0.82 MPa, 4.55 MPa, and 7.33 MPa. The shadow represents the absence of any dissociation, and the line indicates the theoretical dissociation phase change boundary.

[0068] FIGS. 51A-C: Ultrasound triggered drugs release, including (FIG. 51A) deschloroclozapine (DCZ), (FIG. 51B) dopamine and (FIG. 51C) procaine from HOF-TATB at 1.5 MHz, 1.55 MPa (mean ± SEM, n=3 independent samples).

[0069] FIG. 52: Ultrasound triggered CNO release from HOF-TATB at 1.5 MHz, 1.55 MPa (mean ± SEM, n=3 independent samples).

[0070] FIGS.53A-C: Ultrasound-induced blood-brain barrier opening evaluation through Evans blue staining. (FIG. 53A) Brain extracted from mice injected with microbubbles and exposed to 20 s ultrasound stimulus at peak pressures of 1.0 MPa (left) and 0.75 MPa (right); (FIG. 53B) Brain from mice without microbubble injection exposed to 20 s ultrasound stimulus at peak pressures of 1.0 MPa (left) and 1.0 MPa (right). (FIG.53C) Brain from mice without microbubble - 24 - 4860-4443-2856, v.1injection exposed to 20 s ultrasound stimulus at peak pressures of 1.5 MPa (left) and 1.5 MPa (right). The red circles indicate the ultrasound-treated lesions.

[0071] FIG. 54: The evaluation of ultrasound-induced thermal effects at the focus. Real- time temperature detection was conducted at the mice ventral tegmental area (VTA) during focused ultrasound (FUS) stimulation (1.5 MHz, 1.55 MPa, duration 20 s). No significant temperature changes were observed during the initial 10 s of ultrasound exposure, with only a slight increase of approximately 1.25 ℃ detected after the 20 s stimulus.

[0072] FIG. 55: The in vivo ultrasound power transfer in rat heads with FUS focus length of 10 mm. The ultrasound power heatmap in the rat head shows that around 1.18 MPa was delivered to the rat VTA when 2.45 MPa primary ultrasound power was used. - 25 - 4860-4443-2856, v.1DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. HOFs

[0074] 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 diffraction, 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.

[0075] 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 π-π 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 - 26 - 4860-4443-2856, v.1technique 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.

[0076] In some embodiments, 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, reaction 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).

[0077] The present application includes the methods to prepare HOF as described above that may be coupled with the production of drug 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.

[0078] 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 - 27 - 4860-4443-2856, v.1agent 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 π…π interactions, van der Waals interactions, C-H…π interactions etc. The HOF may comprise a compound of the formula: wherein: A is a C6-16aromatic ring orring; and each of B, B′, and B′′ are independently a C6-16 aromatic ring or a C3-15 heteroaromatic ring comprising −S(O)2Ra, –NH2, or −C(O)Rawherein Rais hydroxy or amino.

[0079] In some embodiments the HOF comprises a compound having the formula: , wherein: X1, X2 and X3 areand R3 are each independently −S(O)2Ra or −C(O)Ra, wherein: Ra is 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: - 28 - 4860-4443-2856, v.1. . asou

[0080] Ultrasound may be applied to a mammalian subject to release a drug 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.

[0081] 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 kHz. 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 - 29 - 4860-4443-2856, v.1from 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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 - 30 - 4860-4443-2856, v.1is 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.

[0087] 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.

[0088] 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. III. Diseases

[0089] Focused ultrasound with a pharmaceutical composition comprising a HOF and a drug as described herein can be used to treat a variety of diseases in a mammalian subject. In some preferred embodiments, the disease is a brain disease, a cancer, essential tremor, uterine fibroids, a fibroadenomas of the breast, melanoma, solid tumor, Alzheimer's disease, Parkinson's disease, stroke, epilepsy, depression, pain relief, or local anesthesia.

[0090] In some embodiments, the disease is a breast cancer or a brain cancer. The brain cancer may be a glioma, glioblastoma (also called glioblastoma multiforme), ependymoma, astrocytoma, protoplasmic astrocytoma, fibrillary astrocytoma, astroblastoma, oligodendroglioma, oligodendroblastoma, primitive neuroectodermal cancer, cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, diffuse Pontine glioma (DIPG). The brain cancer may be malignant, metastatic, or non-metastatic.

[0091] The cancer can be a cancer of the bladder, blood, bone, bone marrow, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, - 31 - 4860-4443-2856, v.1skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; - 32 - 4860-4443-2856, v.1lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. IV. Pharmaceutical Compositions

[0092] In some embodiments, a HOF and drug 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 carrier. 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. - 33 - 4860-4443-2856, v.1

[0093] As used herein, "pharmaceutically acceptable carrier" 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.

[0094] 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, inhalation (e.g., aerosol inhalation), via injection, infusion, continuous infusion, or localized perfusion bathing target cells directly.

[0095] 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.

[0096] 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 - 34 - 4860-4443-2856, v.1weight, 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. A. Parenteral Compositions and Formulations

[0097] 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. 6,7537,514, 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).

[0098] 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 - 35 - 4860-4443-2856, v.1the 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.

[0099] 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 condition 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 Biologics standards.

[0100] 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 - 36 - 4860-4443-2856, v.1embodiments, the HOF comprising a drug or therapeutic agent is dissolved in an aqueous 5% glucose (w / v) prior to administration by injection. A. Liposomes

[0101] 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.[19–21]More recently, the liposome based mRNA vaccine developed by BioNTech / Pfizer and Moderna was clinically applied against COVID-19.

[0022]

[0102] 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) (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.

[0103] 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.

[0104] 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. - 37 - 4860-4443-2856, v.1

[0105] 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. B. Drugs and Therapeutic Agents

[0106] A variety of drugs and therapeutic agents can be included with or encapsulated in a HOF as described herein. The drug may be a small molecule, a biological or protein therapeutic, or enzyme. In some embodiments, the drug is an anesthetic, a volatile anesthetic, an anticancer drug or chemotherapeutic, an anti-inflammatory drug (e.g., NSAID etc.), an analgesic drug or a painkiller. The drug or therapeutic agent and HOF may be comprised in a pharmaceutical formulation that includes an excipient.

[0107] In some embodiments, the drug may be a drug for the treatment of Parkinson’s disease (PD) such as, e.g., selegiline, rasagiline, safinamide, or 4-Dihydroxyphenylalanine（L- DOPA). V. Definitions

[0108] 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.

[0109] 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.

[0110] 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 - 38 - 4860-4443-2856, v.1example, 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.

[0111] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human 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.

[0112] 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).

[0113] 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 purposes of this disclosure, a therapeutically effective dose of a compositions is an amount that is sufficient, when administered 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. - 39 - 4860-4443-2856, v.1

[0114] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations·

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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 the 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 - 40 - 4860-4443-2856, v.1therapeutic 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.

[0119] 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.

[0120] 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”.

[0121] When used in the context of a chemical group: “hydrogen” means −H; “hydroxy” means −OH; “oxo” means =O; “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)−. - 41 - 4860-4443-2856, v.1

[0122] In the context of chemical formulas, the symbol “−” means 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 bond or a double bond. Thus, the formula covers, for example, , , , and . And it is understood such ring atomFurthermore, 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 “ ”, when drawn perpendicularly across a bond (e.g., for methyl) indicates a pointof 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 ofThe symbol “ ” means a single bond where the group attached to the thick end of the wedge is “into the page”. The symbol “ ” 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.

[0123] 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: - 42 - 4860-4443-2856, v.1(R)y, then the variable may replace anyany 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.

[0124] 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≤8)”, “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≤8)” is two, the minimum number of carbon atoms in the group “cycloalkyl(C≤8)” 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(C2-10)” 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”, “olefin(C5)”, and “olefinC5” 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 - 43 - 4860-4443-2856, v.1of a substituted alkyl(C1-6).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.

[0125] 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.

[0126] 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 carbon-carbon 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).

[0127] 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 π system.

[0128] 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 (n-Pr or propyl), −CH(CH3)2(i-Pr,iPr or isopropyl), −CH2CH2CH2CH3(n-Bu), −CH(CH3)CH2CH3(sec-butyl), −CH2CH(CH3)2 (isobutyl), −C(CH3)3 (tert-butyl, t-butyl, t-Bu ortBu), and −CH2C(CH3)3 (neo- pentyl) 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 - 44 - 4860-4443-2856, v.1−CH2CH2CH2− are non-limiting examples of alkanediyl groups. The term “alkylidene” refers to the divalent group =CRR′ in which R and R′ are independently hydrogen, 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.

[0129] 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 non-aromatic 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 or triple bonds, and no atoms other than carbon and hydrogen. The is a non-limiting example of cycloalkanediyl group. A “cycloalkane” refers tohaving the formula H−R, wherein R is cycloalkyl as this term is defined above.

[0130] 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 carbon-carbon 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 - 45 - 4860-4443-2856, v.1alkene” and “α-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.

[0131] 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≡CCH3are 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.

[0132] 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: - 46 - 4860-4443-2856, v.1nd An “arene” l as that term is defined above. Benzene and toluene are non-limiting examples of arenes.

[0133] 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.

[0134] 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. Non-limiting 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.

[0135] 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 - 47 - 4860-4443-2856, v.1preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroarenediyl groups include: N N N . The term “N-heteroaryl” atom as the point ofattachment. 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.

[0136] 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.

[0137] 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.

[0138] 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 structure(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 heterocycloalkanediyl - 48 - 4860-4443-2856, v.1does 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 “N- atom as the pointof attachment. N-pyrrolidinyl is an example of such a group.

[0139] 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(CH2)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.

[0140] 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.

[0141] 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 - 49 - 4860-4443-2856, v.1groups. Non-limiting examples of dialkylamino groups include: −N(CH3)2and −N(CH3)(CH2CH3). The terms “cycloalkylamino”, “alkenylamino”, “alkynylamino”, “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 −NHC6H5. The terms “dicycloalkylamino”, “dialkenylamino”, “dialkynylamino”, “diarylamino”, “diaralkylamino”, “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.

[0142] 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, −CH2Cl, −CF3, −CH2CN, −CH2C(O)OH, −CH2C(O)OCH3, −CH2C(O)NH2, −CH2C(O)CH3, −CH2OCH3, −CH2OC(O)CH3, −CH2NH2, −CH2N(CH3)2, and −CH2CH2Cl. 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, −CH2Cl is a non-limiting 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 −CH2CF3 are non-limiting examples of fluoroalkyl groups. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-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. - 50 - 4860-4443-2856, v.1IV. Examples

[0143] 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 – HOF NANOPARTICLES - PREPARATION AND TESTING

[0144] As a demonstration of the ultrasound programmable drug activation technology (UPDATE), the CNO was loaded into optimal HOF nanoparticles in order to achieve genetically- targeted neural modulation through chemogenetics with designer receptors exclusively activated by designer drugs (DREADDs) (Boesmans et al., 2018; Gomez et al., 2017; Nagai et al., 2020). By precisely adjusting the responsiveness of CNO-loaded HOF nanoparticles to FUS in the mice and rat VTA, spatiotemporal control of neural activity was effectively attained with a short latency of mere seconds. The findings demonstrate that UPDATE can achieve high temporal resolution and allow for targeted control of drug activation in specific types of cells while retaining the benefits of minimal invasiveness. In particular, UPDATE will be very promising for non-invasive control of neural activity in larger animals and human applications.

[0145] Four different HOF nanoparticles HOF-TATB, HOF-BTB, HOF-101, and HOF- 102 were successfully generated, with varying density of hydrogen bonding and aromatic rings in respective building units (FIG. 1). The nuclear magnetic resonance (NMR) spectrum of corresponding ligands are shown in FIGS.6-9. Further powder X-ray diffraction has demonstrated the high crystallinity and phase purity of nanoscale HOF particles (FIG. 10). Specifically, HOF- 101 and HOF-102 crystalize in square lattice topology, where each molecule interacts with four neighboring molecules via carboxlylic acid dimers through eight OーH···O hydrogen bonds, extending into a two-dimensional (2D) layer (Wang et al., 2019; Yin et al., 2018). Moreover, these 2D square layers further stack with adjacent layers through π-π stacking interactions (FIG.10 and Table 2) (Wang et al., 2019; Yin et al., 2018). Into the tetracarboxylic building units, - 51 - 4860-4443-2856, v.1hexagonal honeycomb motif structures are observed in HOF-BTB and HOF-TATB. H3BTB molecules prefer to form hexagonal honeycomb layers and each individual hexagonal sheet assembling in stacks several molecular thick and aligning in parallel stacks to each other.40 Notably, the self-assembly structure of HOF-TATB exhibits variations depending on the choice of solvents during the preparation process (Li et al., 2020). In comparison to previous research, we observed a new crystalline phase in the obtained HOF-TATB nanoparticles (Li et al., 2020). The crystal structure of HOF-TATB nanoparticles was determined by electron diffraction crystallography studies, which revealed that each H3TATB unit was held together by π-π stacking and a unique combination of hydrogen bonding motifs into a doubly interpenetrated three- dimensional porous structure (FIG. 11). The experimental 77 K nitrogen adsorption tests revealed a high Brunauer−Emmett−Teller (BET) surface area of 166.5 m2g-1and a pore volume of 0.21 cm3g-1for HOF-TATB (FIG.12A-B). We targeted four different organic molecular building units (OMBUs) with varying density of hydrogen bonding and aromatic rings in respective building units for the synthesis of four porous HOF particles HOF-TATB, HOF-BTB, HOF-101, and HOF- 102 (FIG.1B). The nuclear magnetic resonance (NMR) spectrum of corresponding OMBUs were obtained and confirmed the structures. We employed a precipitation method to produce HOFs particles by fine-tuning solvent polarity. Both dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses confirmed the successful fabrication of HOF particles. The results revealed that the size of these HOFs particles ranged from 250 to 600 nm, with a polydispersity index (PDI) of approximately 0.2 to 0.3. Due to the planar nature, the OMBUs spontaneously assembled into two-dimensional layers via classic carboxylic acid H-bonded R22(8) dimers, where the tetracarboxylic OMBUs form a square lattice topology layer in HOF- 101 and HOF-102. More specifically, the strong π-π stacking of pyrene moieties in HOF-101 and HOF-102 drives the 2D square layers to further stack with adjacent layers into 3D crystals with rhombus 1D channel (FIG.1B) (Wang et al., 2019; Yin et al., 2018). The layers of self-assembled tricarboxylic acids in HOF-BTB are first stacked to align the aromatic motifs and further interwoven to maximize the overall molecular packing. (Zentner et al., 2015) The crystal structure of HOF-BTB, HOF-101 and HOF-102 were previously reported and the phase purity was confirmed by the powder X-ray diffraction (PXRD) (FIGS. 10A-D). (Wang et al., 2019; Yin et al., 2018; Zentner et al., 2015) We note that the crystal structure of downsized HOF-TATB particles was not yet reported, which herein was determined by micro-crystal electron diffraction - 52 - 4860-4443-2856, v.1(microED; FIGS. 32-35). In HOF-TATB, H3TATB OMBUs self-assemble into a doubly- interpenetrated three-dimensional hydrogen-bonded (3,4)-nets through two types of intermolecular hydrogen bonding motifs and inclined π-π stacking (FIG.32). Other than the typical carboxylic acid dimer (O···H–O 2.675-2.686 Å) formed by one of the three carboxylic acid, the other two carboxylic acid groups participate in the formation of a unique hydrogen-bonded D44(15) tetramer motifs (O·· ·H–O 2.575-2.762 Å) with a dangling weak hydrogen bond with guest methanol and the adjacent tetramer in the neighboring interpenetrated net (O···H–O 3.051- 3.767 Å) (FIG. 32, 35). The tetramer motifs from two interpenetrated nets are associated by the inversion center rendering an inclined stacked geometry of H3TATB molecules with a distance of 3.32 Å. Upon removal of the guest solvents, HOF-TATB exhibits a one-dimensional pore channel along the crystallographic b-axis with a size of 12.2 ^ 23.9 Å2 and a 50.3% solvent-accessible void of the crystal volume, indicating excellent potential for drug-loading properties. The bulk purity and prolonged hydrolytic stability (up to one week) of as-synthesized HOF-TATB particles were confirmed by PXRD (FIG. 36). The 77 K N2 or 195 K CO2 adsorption isotherms of four HOFs demonstrated their porous structures (FIGS. 37-40, and Table 1). Table 1. The HOFs dissociation constant k at different ultrasound power densities. HOFs Ultrasound peak pressure (MPa)

[0146] The ultrasound-triggered scission of HOF particles was next verified in an aqueous medium by monitoring the framework dissociation percentage over time (FIG. 2A-D, - 53 - 4860-4443-2856, v.1FIGS.16A-K, and FIGS.44A-F). Following ultrasound stimulation, the OMBUs cleaved from the HOFs were dissolved and found in the supernatant after centrifugation. Their concentration was then measured using UV-Vis spectroscopy to calculate the framework dissociation percentage (FIGS.16A-K and FIGS.44A-F). The results indicated that different HOFs exposed to ultrasound with varying peak pressures will reach characteristic dissociation equilibrium, as illustrated in FIGS. 2A-D and FIGS. 16A-K and FIGS. 44A-F. For instance, irradiation of the HOF particles with ultrasound (1.5 MHz) at a power of 8.04 MPa led to approximately 91.8%, 45.3%, 11.6%, and 4.7% dissociation after equilibrium in HOF-TATB, HOF-BTB, HOF-101, and HOF-102, respectively. This suggests that the ultrasound-triggered HOF dissociation may be a thermodynamic process. However, no obvious dissociation was observed even when these HOFs were heated at 100 °C for 5 minutes, demonstrating their high thermal stability (FIGS. 14A-D, FIGS.45A-D). These results determined that the ultrasound stress provides the main driving force to shear the intramolecular noncovalent bonds and dissociate the frameworks instead of ultrasound thermal effect. In fact, HOFs demonstrate a remarkable capability for ultrasound power programmable behavior through mechanochemical activation. The percentage of dissociation equilibrium increases in response to higher ultrasound peak pressure, and different HOFs with specific building units exhibit distinct ultrasound responsive thresholds. Activation of HOFs occurs exclusively when the ultrasound power reaches a specific peak pressure, with the ultrasound power activation thresholds being contingent upon the type of OMBU used (FIGS. 2A-D). In addition, the dissociation percentage increases in response to higher ultrasound peak pressure for all 4 different HOFs, with difference in increasing rate depending on the specific type of HOF, as shown in FIGS.2A-D.

[0147] The structure-property relationships of the HOFs were further investigated using density functional theory (DFT) calculations, by calculating the cohesive energy (Ecohesive) of, from the highest to the lowest, HOF-TATB, HOF-BTB, HOF-101, and HOF-102 in solution, indicating increasing relative stability of the HOFs (FIGS. 1-2A-I) This observation is consistent with the experimental findings where the dissociation equilibrium constant (k) decreases sequentially in these four HOFs (see Table 2 below). Gibbs Free Energy provides a means of defining equilibrium or the tendency of a reaction. In the context of the ultrasound-triggered HOF dissociation process, the acoustic field can be considered to alter the reaction free energy change. Consequently, the change in ln(k) should exhibit a linear correlation with ultrasound power. The experimental data - 54 - 4860-4443-2856, v.1revealed an approximate linear correlation between the ultrasound peak pressure and ln(k), as illustrated in FIGS.46-47, supporting the notion that the ultrasound-triggered dissociation of HOF is indeed likely a thermodynamic process. Our reaction model indicated a linear relationship between ln(k) and Ecohesiveof the HOFs (FIG. 15), and thus we can extrapolate the required minimum Ecohesive of HOF at any desired ln(k). Additionally, we found a linear relationship between the required minimum Ecohesive and the ultrasound peak pressure (EUS) of the HOFs (FIG. 16 and Table 3) at any ln(k). From these linear relationships between ln(k) and Ecohesive, and between EUS and Ecohesive, we developed a three-variable model to represent how these variables are correlated. The heat map in FIG. 1, predicts the minimum Ecohesive of HOFs for achieving a given dissociation percentage at a specific ultrasound power, thus providing a guideline for designing HOFs for on-demand drug activation by ultrasound. Since the sensitivity of HOFs to ultrasound is governed by the overall strength of weak interactions encoded in the HOF structure, primarily contributed by hydrogen-bonds and π-π interactions among OMBUs, the relationship between the OMBU structures and ultrasound sensitivity is important for elucidating the acoustic scission principles of HOFs at the molecular level. The DFT results (FIG. 1) show that the hydrogen-bonding energy (EHB) between two OMBUs is similar for all four OMBUs, and that the hydrogen-bond strength is largely independent of the molecular structure of the OMBU. The contribution of hydrogen-bonding to a HOF’s stability arises from the number of H-bonds in each OMBU. For π-π interactions, the energy (Eπ-π) varies with the structure of the OMBU. Higher Eπ-π was found in HOF-101 and HOF-102 due to the increased number of aromatic fused rings in the OMBU (FIG. 1). Since mechanochemical bond scission is more likely to occur in weak bonds to trigger the dissociation of frameworks, it is reasonable to suggest that the OMBUs with fewer H-bonds and aromatic fused rings in the backbone should be sought after to make HOFs with a higher ultrasound dissociation constant. - 55 - 4860-4443-2856, v.1Table 2. The HOFs dissociation constant k at different ultrasound power densities. HOFs Ultrasound peak pressure (MPa)Table 3. The parameters for the heatmap prediction model. Dissociation Dissociation Ln k EUS = aECohesive energy R- 56 - 4860-4443-2856, v.19% 0.098901 -2.3136349 -0.5698 -2.297 0.9767- 57 - 4860-4443-2856, v.1-- 58 - 4860-4443-2856, v.141% 0.694915 -0.3639654 -0.5948 0.8063 0.9014- 59 - 4860-4443-2856, v.160% 1.500000 0.40546511 -0.6047 2.031 0.8602- 60 - 4860-4443-2856, v.179% 3.761905 1.32492541 -0.6164 3.494 0.8076

[0148] HOF-TATB nanoparticles display the highest ultrasound sensitivity and could offer the most desirable temporal resolution to gate drug activation compared with other HOF nanoparticles (FIGS. 3A-H and FIGS. 17A-C and Table 3). Their porous structure enables high drug loading content (14.6 ± 2.5 wt%), and their negative surface potential provides excellent biostability, even with 10% fetal bovine serum (FIG. 18). Using fluorescence dye as a payload, gradual release occurs with increased ultrasound duration, whereas no significant release occurs without ultrasound (FIGS. 3A-H and FIGS. 19A-D). The percentage of released drug also increases with the ultrasound peak pressure, with an activation threshold of around 0.51 MPa (FIGS.3A-H and 19A-D). We also determined that the payload release percentage is in a positively linear relationship with the frameworks dissociation, where the acoustic scission leads to the temporal release of payloads (FIG. 3A-H). Furthermore, we determined the biosafety of the HOF- TATB nanoparticles through the hemolysis and cell viability tests, which implied that our HOF- TATB nanoparticles generally are biocompatible and biosafe as drug delivery platforms (FIG. 20A-C).

[0149] Next, we applied our HOF-TATB nanoparticles for loading of CNO (TATB@CNO) for ultrasound-triggering of CNO release to activate DREADDS in spatiotemporal neuromodulation. We first evaluate our system for activation of mouse primary neurons. The neurons were transduced with endogenous “designer receptors” hM3D(Gq) with red fluorescent reporter mCherry (AAV9-hSyn-hM3D(Gq)-mCherry) and green fluorescent calcium indicator (AAV9-hSyn-GCaMP6s-WPRE-SV40), as shown in FIG. 3A-H. We initially validated the activation of hM3D(Gq) expressing neurons with the free designer drug CNO using fluorescence imaging using GCaMP6s calcium indicators (FIG. 21). Then, by subjecting CNO-encapsulating nanoparticles (TATB@CNO) to ultrasound irradiation, over 90% of hM3D(Gq)+ neurons were found to fire with a latency of 1.6 s and continuous activation was observed for ~60 s but only sporadic neuronal activation was observed in the absence of ultrasound stimulus or in hM3D(Gq)- neurons (FIGS. 3A-H and FIG. 22). The burst release of CNO from HOF-TATB nanoparticles - 61 - 4860-4443-2856, v.1under ultrasound stimulus enables the rapid triggering of hM3D(Gq), inducing long-term neuron membrane depolarization. This sono-chemogenetic approach provides a novel method for achieving fast and continuous neuronal activation with minimal invasiveness (Deisseroth, 2015).

[0150] Precisely timed activation of genetically targeted neurons is important for researchers to understand the links between brain activity and behavior (Deisseroth, 2015; Adamantidis et al., 2015). We subsequently assessed real-time sono-chemogenetic neural excitation through fiber photometry in the ventral tegmental area (VTA) of mice, a region known for its role in regulating reward learning and depression (Tye et al., 2013; Witten et al., 2011; Airan et al., 2009). Ultrasound energy propagates through tissue as a traveling pressure wave, with penetration depth increasing with frequency but at the cost of decreased resolution (Meng et al., 2021). In our experiments, a 1.5 MHz transducer achieved a maximum penetration depth of 20 mm, with 40% delivery efficiency at a tissue depth of 10 mm (FIG. 23). The ultrasound energy heatmap in the mouse brain showed that a primary power of 1.4 MPa produced an acoustic pressure of around 0.9 MPa at the VTA of mice (FIG. 24), which is sufficient to activate CNO release (FIGS. 3A-H and FIGS. 19A-D). Compared to the visible and near-infrared light, ultrasound energy transfer efficiency is orders of magnitude higher, making sono-chemogenetics a promising approach for achieving deep brain stimulation compared to optogenetics and NIR-based optogenetics (Wu et al., 2022; Finlayson et al., 2022; Chen et al., 2018; Comprehensive Biometidcal Physics, 2014). We performed unilateral transduction of neurons in VTA using the AAV9-hSyn-hM3D(Gq)-mCherry and AAV9-hSyn-GCaMP6s-WPRE-SV40 virus, followed by the injection of TATB@CNO in the same region 4 weeks later (FIG. 4A-O). We used fiber photometry to record Ca2+influx-induced GCaMP6s signal increase in mice to assess neuronal excitation in response to ultrasound-gated release of CNO after 2 days of injection. The GCaMP6s signal increased significantly upon ultrasound irradiation of the VTA region where neurons were transduced with hM3D(Gq), but remained unchanged without hM3D(Gq) expression or without ultrasound stimulus (FIG.4A-O). This sono-chemogenetics approach demonstrated high temporal resolution, with a 3.5 s latency for neural activations upon ultrasound stimulus (FIG.4A-O), faster than other recently reported minimally invasive methods such as magnetic modulation and photothermal modulation (Rao et al., 2019; Wu et al., 2022). We observed over 120 s of continuous excitation of neural activity with each only 10 s sono-chemogenetic stimulation, and multiple increases in GCaMP6s fluorescent signal were observed upon repeated exposure to - 62 - 4860-4443-2856, v.1ultrasound even after 5 days injection due to the long-term biostability of TATB@CNO in the brain (FIGS. 25 and 26A-D). Immunofluorescence analysis of the expression of c-fos, an immediate early gene and a marker for neural excitation (Wang et al., 2023) further revealed that neurons excitation in the VTA was only triggered by the FUS stimulus in mice with both expression of hM3D(Gq) and injected TATB@CNO nanoparticles (FIG. 4A-O).

[0151] Based on the notable neural excitation observed in the VTA, we subsequently assessed the ability of sono-chemogenetic stimulation in modulating the reward learning behavior of mice (FIG. 4A-O). After injecting TATB@CNO nanoparticles in the VTA region with hM3D(Gq) expression on Day 0, mice were allowed to freely explore the apparatus without any stimulus on Day 2 (Pre-tests). On Day 3, mice received sono-chemogenetic stimulus (1.5 MHz, pulse 20s, 1.40 MPa) in the designated conditioned chambers with two sessions. On Day 4, mice again freely explored the apparatus without any stimulus (Post-test). Trail tracing revealed that mice expressing hM3D(Gq) preferentially explored the conditioned side of the apparatus after receiving sono-chemogenetic stimulation (FIG. 4A-O), resulting in an approximately 2-fold increase in time spent in the conditioned chamber (FIG. 4A-O). Notably, in the absence of one of hM3D(Gq) expression, TATB@CNO nanoparticles, or ultrasound stimulus, the preference did not vary significantly within the experimental trail (FIG. 4A-O).

[0152] Based on the previous evidence of the antidepressant effects of neural activity in the VTA (Tye et al., 2013), we aimed to investigate whether sono-chemogenetic stimulation could impact mouse behavior in the forced swim test (FST). To achieve this, VTA neurons were transduced with hM3D(Gq) and mice were allowed to recover from TATB@CNO injection surgery (Day 0) for two days before undergoing a 5-minute pre-FST test (Day 2). On Day 3, mice received ultrasound stimulus (1.5 MHz, pulse 20s, 1.40 MPa) before the 5-minute FST assay (FIG. 4A-O). We tracked the mice's swimming patterns during the FST period using motion cameras. The results showed that hM3D(Gq)+mice exhibited increased activity, swimming longer distances during the FST period (FIG. 4A-O). Furthermore, the dynamic motion analysis showed that hM3D(Gq)+mice exhibited higher mobility throughout the FST period, with a slight increase in immobility preference only after swimming for 2 minutes. On the other hand, in the absence of one of hM3D(Gq) expressions, TATB@CNO nanoparticles, or ultrasound stimulus, the - 63 - 4860-4443-2856, v.1immobility time significantly increased in mice (FIG. 4A-O). These findings suggest that sono- chemogenetic stimulation can effectively modulate mouse behavior in the FST.

[0153] Utilizing the high energy transmission efficiency of ultrasound in tissues and the high ultrasound sensitivity of HOF-TATB nanoparticles, we conducted an investigation into the efficacy of sono-chemogenetic modulation in rats. Our analysis of the rat head ultrasound heatmap revealed that a peak ultrasound pressure of 1.30 MPa was achieved to gate the CNO release, by applying a primary ultrasound power of 2.45 MPa (FIG. 27). Prior to TATB@CNO nanoparticle injection and optical fiber implantation, hM3D(Gq) and GCaMP6s were transduced into the rat VTA neurons (FIG.5A-J). After a recovery period of two days, we recorded the green fluorescent signal of GCaMP6s under ultrasound stimulation (1.5 MHz, pulse 20 s, 2.45 MPa) to evaluate the change in neuron activity. We observed a significant increase in GCaMP6s signal in hM3D(Gq)+rats with stimulation, but not in the absence of hM3D(Gq), TATB@CNO nanoparticles, or ultrasound stimulus (FIG. 5A-J). Notably, the sono-chemogenetic approach achieved neuron activation with a latency of 8.8 s from the application of ultrasound and continuous activation for over 60 s (FIG. 5A-J). In addition, we evaluated neuron activation in post-hoc brain slices via c- fos immunostaining. Remarkable c-fos signals were observed in neurons expressing hM3D(Gq) in VTA, whereas few c-fos signals were observed in the absence of hM3D(Gq), TATB@CNO nanoparticles, or ultrasound stimulus (FIG. 5A-J). These results confirm that our sono- chemogenetic approach can achieve remote brain stimulation even in deep brain regions of rats.

[0154] Lastly, we further assessed the potential of sono-chemogenetics in shaping behaviors in rats using a 4-day conditioned place preference assay. Rats transduced with hM3D(Gq) in the VTA region received TATB@CNO nanoparticles on Day 0 and were allowed to freely explore the chambers after a two-day recovery period. On Day 3 and Day 4, the rats were subjected to ultrasound stimulus (1.5 MHz, pulse 20 s, 2.45 MPa) in the designated conditioned chambers, with two sessions each day. On Day 5, post-tests were conducted, where the rats were allowed to freely explore the chamber without any stimulus (FIG. 5A-J). The rats treated with sono-chemogenetics exhibited a preference to stay in the conditioned chamber compared to other groups in post-tests (FIG. 5A-J). Our results demonstrate a statistically significant difference in the preference score between the hM3D+ / FUS+ / TATB@CNO+group and the other control groups without hM3D(Gq), TATB@CNO nanoparticles, or ultrasound stimulus (FIG. 5A-J), indicating - 64 - 4860-4443-2856, v.1that sono-chemogenetics can effectively modulate reward learning behaviors in rats through non- invasive sono-optogenetic deep brain stimulation.14 days after stimulation, we also evaluated the in vivo biocompatibility of the sono-chemogenetic approach by immunostaining brain sections. These results demonstrate that the sono-chemogenetics approach did not cause any significant cell toxicity through H&E staining (FIG. 28). Additionally, we did not observe any activation of glial cells or astrocytes, nor did we detect any neuron apoptosis (FIGS. 29A-B and FIGS. 31A-B). Furthermore, we did not observe any evident blood-brain barrier opening and uncontrollable thermal effects at the targeted area under the pressure at the focus spot we used for sono- chemogenetics (FIGS. 53-54).

[0155] Single crystal structures of HOF-TATB was determined by Electron Diffraction (ED):

[0156] Sample preparation: Grains of HOF-TATB in methanol were placed on a lacey carbon copper grid (300 mesh) grid. The methanol was evaporated at RT and the grid was clipped. The TEM grid was flash frozen in liquid nitrogen. The frozen grid was placed into the autoloader cartridge of the Glacios microscope (Thermo Fisher Scientific) under liquid nitrogen. MicroED data were collected in a Glacios TEM (operating voltage of 200 keV, wavelength of 0.025 Å), equipped with a Ceta-D CMOS camera (Thermo Fisher Scientific).

[0157] MicroED data collection: Crystals were identified in low magnification search mode and selected for diffraction screening so that they were well separated from other crystals and grid bars. Diffraction of these crystals was tested by acquiring a still diffraction frame by switching the microscope into diffraction mode using the 50 µm C2 aperture and a pre-set camera length of 0.67. Once a suitable crystal was detected, the microscope was moved back into imaging mode and the maximum tilt range for data collection of the crystal was explored. MicroED data collection was controlled through the program EPU-D (Thermo Fisher Scientific). Data was collected by continuously rotating the crystal in the beam as the camera acquired diffraction frames (1 ° / sec) over a tilt range of 50 to 100 º. Diffraction data were recorded in rolling shutter mode (0.5 s integration per frame, binned by 2).

[0158] Data Processing and structure determination: Data sets were indexed, integrated, and scaled using the software XDS (Pawley, 1981; Sheldrick 2015). Diffraction data - 65 - 4860-4443-2856, v.1from multiple crystals were merged to obtain an almost complete diffraction data set. The structure of HOF-TATB was determined by ShelxT (Sheldrick et al., 2015) using direct methods. The dataset was solved in space group P2 / c. All non-hydrogen atom locations were identified in the asymmetric unit, and atom types were assigned through chemical knowledge. The structure was refined using ShelxL (Hübschle et al., 2011) and the Shelxle (Hübschle et al., 2011) and Olex2 (Dolomanov et al., 2009) software for viewing and manipulating the structure during refinement. All nonhydrogen atoms were refined anisotropically by full-matrix least-squares. All hydrogen atoms were placed using a riding model. Refinement statistics can be found in Table 4. The structure was deposited under 2338302. Table 4. Crystal data and structure refinement for HOF-TATB CCDC number 2338302 E i i l f l C4H4N O1≤- 66 - 4860-4443-2856, v.1Reflections collected 40592 Independent reflections 6430 [Rint = 0.1937]EXAMPLE 2 – HOF NANOPARTICLES - PREPARATION AND TESTING

[0159] HOF-TATB nanoparticles. 30 mg H3TATB was dissolved in 3 mL dimethylformamide (DMF), and then 12 mL distilled water was added with a stirring speed of 1000 rpm. After stirring for 10 min, the nanoparticles were collected via centrifugation at 12000 rpm (13523 x g) for 5 min (Centrifuge 5420, Eppendorf), and then washed with acetone and distilled water 3 times, respectively. The final products with around 30% yield were dispersed in water at the desired concentration for future use, where the concentration was determined through the UV-Vis calibration curve of H3TATB solution.

[0160] HOF-BTB nanoparticles. 30 mg H3BTB was dissolved in 2 mL DMF, and then 12 mL distilled water was added with stirring. After 5 min stirring, the products were collected through centrifuge 5 min at 12000 rpm (13523 x g), and washed with methanol and water 3 times, respectively. The yield was around 25%.

[0161] HOF-101 nanoparticles. Briefly, 30 mg H4TBAPy was dissolved in 3 mL DMF, and then dropwise added into 12 mL distilled water with stirring at 1000 rpm. After 5 min stirring, the nanoparticles were separated via centrifugation at 12000 rpm (13523 x g) for 5 min, and then washed with acetone, ethanol, and distilled water 3 times, respectively. The final products (yield: 95%) were resuspended with distilled water at the desired concentration for future use.

[0162] Nano-HOF-102. 10 mg H4PTTNA monomer was dissolved in 2 mL DMF, and then 8 mL methanol was added with stirring. After stirring for 5 min, the pallets were collected - 67 - 4860-4443-2856, v.1through centrifuge 5 min at 12000 rpm (13523 x g), and washed with methanol and water 3 times, respectively, with a yield 85%.

[0163] Focused ultrasound (FUS) controlled scission of HOFs. Briefly, 2 mL fresh HOFs solution was loaded in glass vials, and placed on the surface of FUS transducer (Del Piezo Inc, DL-47, and Image Guided Therapy System). Ultrasound peak pressure was determined via hydrophone (Onda Corporation, HGL-0200) connected to a pre-amplifier (Onda Corporation, AG- 2010) with a gain of 20 dB. The HOFs solution was irradiated under the FUS stimulation with different parameters. After that, 100 μL solution was extracted at a fixed time, and centrifuged at 8000 rpm for 5 min (Centrifuge 5430, Eppendorf), where the dissociated monomers or oligomers were separated and mainly located in the supernatant. The supernatant was extracted to do the UV- Vis tests (Eppendorf BioSpectrometer® Basic spectrophotometer) to determine the HOF dissociation percentage according to the following formula: ^^^^^^^^^^ ^^ ^^^^^^^^^^^ ^^^^ ^^^^^^^^^^^^ ^^^^^^^^^^ = × 100% ^^^^^^^^^^ ^^ ^^^^^^^ ^^^ ^^^^^^^^

[0164] Theoretical calculation of the cohesive energy of HOFs and prediction model building. Density functional theory (DFT) calculations done with the Vienna Ab-Initio Package (Kresse and Hafner, 1993; Kresse and Furthmuller, 1996; Kresse and Joubert, 1999; Furthmuller et al., 1996). Core electrons are described within the projected augmented wave framework; valence electrons are described with a plane wave basis set up to an energy cutoff of 400 eV (Blöchl et al., 1994). The generalized gradient approximation in the form of the Perdew, Burke and Ernzehof functional is used to model electronic exchange and correlation. Van der Waals interactions are calculated using the DFT-D2 method (Grimme et al., 2006). Solvation energies are calculated using the VASPsol implicit solvation model (Fishman et al., 2013; Sundararaman and Schwarz, 2017; Zan et al., 2021).

[0165] Crystal structures of HOF-TATB, HOF-BTB, HOF-101 and HOF-102 were obtained for cohesive energy calculation. For each HOF, a monomer is isolated from the crystal structure and optimized to find its energy, Emono. The crystal structures are used to calculate the energy of the HOF, EHOF, except for BTB, where a fraction of the experimental crystal structure is used as an approximation. - 68 - 4860-4443-2856, v.1

[0166] The dissociation of the HOF is modeled as reaction (1) where a constructional unit breaks from the bulk HOF state and becomes a dissociated dissolved monomer. Constructional unit (HOF)→ dissociated monomer (aq) (1)

[0167] Given the experimentally measured dissociation percentage, x, at equilibrium, the dissociation equilibrium constant can be calculated using equation (2) below. ^ =^^^^ (2)

[0168] The cohesion energy Ecohesionof each HOF in an aqueous environment is calculated using equation (3) Ecohesion = EHOF - n. Edm (3)

[0169] Where EHOFis the energy for a unit cell of the HOF, n is the number of constructional units in the unit cell, and Edmis energy of a dissociated monomer. For the crystal structures used in this study, n are 8, 2, 2, and 2 for HOF-TATB, HOF-BTB, HOF-101 and HOF- 102, respectively.

[0170] To characterize the hydrogen-bonding energy EHB and π-π interaction energy Eπ- π between two constructional units, a dimer bonded through hydrogen-bonding or π-π interaction is isolated from the crystal structure. The energy of the relaxed dimer is Edimer-HB and Edimer- π-π for the hydrogen-bonded dimer and the π-π bonded dimer, respectively. They are used to calculate EHB and Eπ-π using equations (4) and (5). EHB = Edimer-HB - 2 Edm (4) Eπ-π= Edimer-π-π- 2 Edm(5)

[0171] It is worth noting that EHBis the energy per hydrogen-bonded dimer. One HOF- TATB building monomer can form 3 hydrogen bonds while one HOF-101 building monomer can form 4 hydrogen bonds. Therefore, the total amount of hydrogen bonding energy a monomer can have is 1.5 EHB for HOF-TATB and 2 EHB for HOF-101. Structures of the dissociated monomers, hydrogen-bonded dimers, and π-π bonded dimers are shown in Table 5 below. - 69 - 4860-4443-2856, v.1Table 5. Structures of relaxed models for HOF-TATB, HOF-BTB, HOF-101 and HOF-102.4860-4443-2856, v.1

[0172] Preparation of drug-loaded HOF nanoparticles. 2 mg of Rhodamine B (RB) or CNO was dissolved in 2 mL of a 5 mg / mL HOF-TATB nanoparticle solution. The mixtures were vibrated at 40 ℃ for 2 hours and then centrifuged at 12,000 rpm (13523 x g) for 5 minutes. The pellets were washed three times with distilled water to remove unloaded cargoes and then suspended in PBS. A 1 mL solution of nanoparticles was extracted for freeze-drying. Subsequently, the resulting powder (1 mg) was dissolved in 1 mL of water (pH 13) to completely release the cargo. The drug loading content was then measured using UV-Vis spectroscopy.

[0173] In vitro ultrasound controlled drug uncaging. A solution of fresh drug-loaded HOF nanoparticles at a concentration of 1 mg / mL was loaded into glass vials and positioned on the ultrasound transducer. The solution was irradiated with FUS at a frequency of 1.5 MHz for a specific duration and under desired parameters. At predetermined time intervals, 100 μL of the solution was extracted and centrifuged at 8,000 rpm (6010 x g) for 5 minutes. The released drugs were found in the supernatant, and the percentage of drug release was calculated using UV-Vis spectroscopy.

[0174] In vitro calcium imaging. Calcium imaging was performed using primary cortical neurons that were transduced with AAV-9-hSyn-hM3D(Gq)-mCherry and pAAV-hSyn- GCaMP6s-WPRE-SV40. After 6 days of transduction, the neurons were fixed on a fluorescence microscope. The water balloon member of the FUS transducer was brought into contact with the neuron culture medium on top of the plate. Fresh TATB@CNO nanoparticles with a final concentration of 5 μg / mL CNO were added to the medium. Subsequently, FUS stimulation (1.08 MPa, 1.5 MHz, 10 s duration) was applied to trigger the release of CNO for neuron activation. Imaging videos were captured using a Leica DMi8 fluorescence microscope equipped with a 20X air objective, with a 5 ms exposure time, and green channel (Ex: 450-490 nm). The transient increase in green fluorescence (ΔF / F) was calculated by extracting the fluorescence time-series data of 100 neurons through manual segmentation and conversion of the video into grayscale using ImageJ software. The raw data was then processed using a custom MATLAB algorithm that detrends and normalizes the fluorescent time-series data through second-order polynomial curve fits and baseline maximum fluorescent value extraction, compensating for photobleaching effects.

[0175] Stereotaxic injection of virus. All procedures designed according to the National Institute of Health Guide for the Care and Use of Laboratory Animals, approved by the Institutional Animal Care and Use Committee at the University of Texas at Austin (AUP-2021-00086, AUP- 2021-00162), and were supported via the Animal Resources Center at the University of Texas at Austin. All the surgery tools were sterilized before the experiments. The hair was shaved before the injection, and the skin on the head was sterilized three times with 80% ethanol and iodophor. All the virus injections were conducted via the micro-injection system (World Precision Instruments, UMP3 Microinjection Syringe Pump) with 300 nL / min. After the injection, the needles remain inside the brain for at least 5 min to ensure the efficient diffusion of the virus, and then slowly withdraw in 5 min. The skin was closed with sutures after the injections. After the surgery, the animals were placed on the 37 ℃ heating pad and returned to the cage until fully recovered.

[0176] Mouse model. C57BL / 6 mice (20-26 g; 12-16 weeks old; Jackson laboratory) were used in our research. Mice were anesthetized with 2.5% isoflurane using anesthesia machine (Vaporizer Sales & Service Inc) and the head was fixed in stereotaxic frame (Kopf Stereotaxic Instruments). Each animal received a subcutaneous injection of meloxicam (5 mg / kg) and Ethiqa (3.25 mg / kg), and ophthalmic ointment was used to cover the eyes before surgery. For the photometry tests, 1200 nL AAV-9-hSyn-hM3D(Gq)-mCherry (100 µL at titer ≥ 1×10¹³ vg / mL)) and pAAV-hSyn-GCaMP6s-WPRE-SV40 (100 µL at titer ≥ 1×10¹³mixture were unilaterally injected into the VTA, with the coordinatesbregma: anteroposterior (AP) -3.08 mm, mediolateral (ML) + 0.40 mm, and dorsoventral (DV) -5.0 mm.3,47 Of note, for the behavior tests, 1000 nL AAV-9-hSyn-hM3D(Gq)-mCherry solutions were unilaterally injected into the VTA. After 4 weeks, these mice were used for experiments.

[0177] Rat model. 3-4 months old Long-Evans Rats (Charles River) were used in our experiments. Rats were anesthetized with 5% isoflurane, and received a subcutaneous injection of meloxicam (2 mg / kg) and Ethiqa (0.65 mg / kg) before surgery, respectively. For the photometry tests, 1200 nL AAV-9-hSyn-hM3D(Gq)-mCherry and pAAV-hSyn-GCaMP6s-WPRE-SV40 mixture were unilaterally injected into the VTA, with the coordinates relative to bregma: anteroposterior (AP) -5.0 mm, mediolateral (ML) + 1.0 mm, and dorsoventral (DV) -8.6 mm.44 - 73 - 4860-4443-2856, v.1For the behavior tests, 1000 nL AAV-9-hSyn-hM3D(Gq)-mCherry was unilaterally injected into the VTA. After 4 weeks, these rats could be used for experiments.

[0178] Mouse Photometry tests. The hM3D(Gq) and GCaMP6s transduced mice were used for the photometry tests.2 μL of TATB@CNO (2 mg / mL) nanoparticles were unilaterally injected into the VTA with the same coordinates to the viral injection. Then, the optical fibers were implanted at the virus transduced VTA area (-3.08 mm AP, + 0.5 mm ML, -5.0 mm DV). After recovery for 2 days, the mouse head was head-fixed and the FUS (1.5 MHz, 1.40 MPa, 10 s duration) was used to irradiate the VTA area. FUS focus length was set to be 5 mm through control of the FUS water bubble. The signal was recorded through the R810 Dual Color Multi-Channel Fiber Photometry System (RWD life science) (Zan et al., 2021)

[0179] Mouse FST evaluation. hM3D (Gq) transduced mice (20-24 weeks old) were used. A cylindrical tank (20 cm diameter x 30 cm height) was used for mice FST. The water level was around 15 cm, and the water temperature was around 23-25 ºC (Can et al., 2012). The cylindrical tank was surrounded by a white background when we were ready to do the tests. On day 0, 2 μL of TATB@CNO (2 mg / mL) nanoparticles were unilaterally injected into the VTA (-3.08 mm AP, + 0.5 mm ML, -5.0 mm DV). After 2 days of recovery, the mouse was subjected to a 5 min pre- test without FUS stimulation on day 2. On day 3, FUS (1.5 MHz, 1.40 MPa, 20 s duration) was used to irradiate the VTA area through control of the FUS focus length (-3.08 mm AP, + 0.5 mm ML, -5.0 mm DV). After that, the mouse was placed in the FST container, and 5 min FST videos were recorded to analyze the immobility. Trajectory data was obtained from pre-built software integrated into the recorded FST videos using (R820 Tricolor Multi-Channel Fiber Photometry System, RWD). A custom-made algorithm in MATLAB was developed for FST data analysis. The difference in position between subsequent frames of the video was used to determine the displacement of the mice, which was then divided by the FPS of the video to determine the velocity. A threshold value of the velocity of 0.1 pixels / second was determined to be the optimal value for determining the state of mobility and immobility for mice in FST across groups. For every 5 second window, the time accrued between frames where the velocity is less than the threshold is accumulated and was used as proportion to the 5 second window to determine the immobility percentage over time. The total immobility time was determined similarly by accumulating the total time where the velocity between frames were below the threshold. - 74 - 4860-4443-2856, v.1

[0180] Mouse Conditioned place preference tests: After the injection AAV (AAV9-hSyn- hM3D (Gq)-mCherry) for 4 weeks. 2 μL of TATB@CNO (2 mg / mL) nanoparticles were unilaterally injected into the VTA (-3.08 mm AP, + 0.5 mm ML, -5.0 mm DV) at day 0. Briefly, the two side boxes (30 cm X 60 cm X 30 cm) were made of plexiglas, and conjugated with one medium chamber (15 cm X 30 cm X 30 cm). One side box was covered with white and red striped papers, and another was covered with yellow and blue striped papers (Airan et al., 2009; Cunningham et al., 2006). On day 2, the mice were placed in the medium chamber to allow them freely to explore the apparatus for 15 min (pre-test). On day 3, mice were conditioned with 2 sessions. At first, the mice were irradiated with FUS (1.5 MHz, 1.40 MPa, 20 s duration), and then restricted in designated conditioned chambers for 30 min. In the second session around 3 h later, mice received the same treatment and were then restricted in designated conditioned chambers for 30 min. On day 4, the mice were placed and restricted in the medium chamber for 5 min. After that, the mice were allowed to freely move. The videos were recorded through a trail tracking camera. The data was analyzed through R810 Dual Color Multichannel Fiber Photometry with a behavior tracking system (RWD life science). For place preference tests, mice were chosen for tests only if the baseline preference for either side chamber is between 10%-70%, or for the medium chamber is <40% (Airan et al., 2009; Olson et al., 2006)

[0181] Rat Photometry tests: hM3D(Gq) and GCaMP6s transduced rats were used. 2 μL of TATB@CNO (10 mg / mL) nanoparticles were unilaterally injected into the VTA with similar coordinates (-5.0 mm AP, + 1.0 mm ML, -8.6 mm DV). Then, the optical fibers were implanted into a similar area. After recovery for 2 days, the rat head was fixed, and FUS (1.5 MHz, 2.45 MPa, 20s duration) was used to irradiate the VTA area. The FUS focus length was set to 9 mm through control of the FUS water bubble. The signal was recorded through the R810 Dual Color Multichannel Fiber Photometry System (RWD life science).

[0182] Rat Conditioned place preference tests: After 4 weeks of expression of hM3D(Gq) in VTA neurons in rats, 2 μL of TATB@CNO (10 mg / mL) nanoparticles were unilaterally injected into the VTA (-5.0 mm AP, + 1.0 mm ML, -8.6 mm DV) at day 0. Similar to mice conditioned place preference tests, the two side boxes (30 cm X 60 cm X 30 cm) were made of plexiglass, but conjugated with one medium chamber (25 cm X 30 cm X 30 cm). On day 2, the rats were placed in the medium chamber, and stable for 5 min. After that, we opened the door to allow them freely - 75 - 4860-4443-2856, v.1to explore the apparatus for 15 min (pre-test). On day 3, rats were conditioned with 2 sessions. At first, the rats were irradiated with FUS (1.5 MHz, 2.45 MPa, 20s duration), and then restricted in designated conditioned chambers for 30 min. In the second session around 3 h later, rats received the same treatment and were then restricted to designated conditioned chambers for 30 min. On day 4, repeat the 2 pairing sessions similar to day 3. After two days of training, we started the tests on day 5, the rats were placed and restricted in the medium chamber for 5 min. After that, the rats were allowed to freely move for 15 min. The data analysis methods were similar to the mice conditioned place preference tests.

[0183] c-fos staining in mice / rats brain sections. Specifically, mice / rats expressing hM3D (Gq) in VTA neurons and wild-type mice / rats were first subjected to sono-chemogenetics treatment. After 60 minutes, the mice were anesthetized with ketamine (16 mg / kg) administered intraperitoneally. Following induction of deep anesthesia, perfusion was performed using PBS, followed by 4% paraformaldehyde. The brains were then extracted and stored in 4% paraformaldehyde at 4℃ overnight, and sliced using a vibrating blade microtome (Leica VT1200). Brain slices with a depth of 60 μm were washed with 0.3% Triton-X PBS (TBS) solution and subsequently blocked with 5% bovine serum albumin TBS solution for 30 minutes at room temperature. In the case of mouse brain sections, following the blocking step, the samples were incubated with rabbit anti-c-Fos antibody (ab222699, Abcam, 1:500) / mouse anti-tyrosine hydroxylase antibody (MA1-24654, Fisher Scientific, 1:1000) / 0.3% Triton-X in PBS. The samples were then incubated at 4°C overnight and washed three times with TBS solution. Next, a mixture of TBS and secondary antibodies, including goat anti-rabbit Alexa Fluor 405 (ab175652, Abcam, 1:500), goat anti-mouse Alexa Fluor 488 (ab150113, Abcam, 1:1000), and Hoechst 33342 (17535, ATT Bioquest, 1:5000), was added and the slices were incubated for 2 hours at room temperature in a dark room. The slices were then washed three times with TBS, mounted on slides using mounting media (9990402, Fisher Scientific), and covered with a coverslip. Confocal images were obtained using a Zeiss 710 laser scanning microscope. For rat brain sections, the procedure was identical except for the use of rabbit anti-c-Fos antibody (ab289723, Abcam, 1:500) instead of ab222699. Detailed information on the antibodies used in this study can be found in Table 6 below. - 76 - 4860-4443-2856, v.1Table 6. Antibodies used in this work Primary antibodies Secondary antibodies- 77 - 4860-4443-2856, v.1

[0184] Preparation of different drug-loaded TATB-HOF and evaluation of ultrasound-triggered drug release through high-performance liquid chromatography (HPLC):. To prepare drug-loaded TATB-HOF, 2 mg of deschloroclozapine, dopamine, or procaine was dissolved in 2 mL of 5 mg / mL HOF-TATB solution. The mixtures were then incubated at 40 ℃ for 2 hours and centrifuged at 12,000 rpm (13523 x g) for 5 minutes. The resulting pellets were washed three times with distilled water to remove unloaded cargoes and then suspended. To evaluate the drug-loading content, 1 mL of the mixture solution was extracted and freeze-dried. The resulting powder (1 mg) was dissolved in 1 mL of water (pH 13) to completely release the cargo, and the drug loading content was measured using HPLC.

[0185] For the evaluation of ultrasound-triggered drug release performance, the solution of fresh drug-loaded HOF nanoparticles at a concentration of 10 mg / mL was loaded into glass vials and positioned on the ultrasound transducer. The solution was irradiated with focused ultrasound (FUS) at a pressure of 1.5 MPa and a frequency of 1.5 MHz for a specific duration and under desired parameters. At predetermined time intervals, 100 μL of the solution was extracted and centrifuged at 8,000 rpm (6010 x g) for 5 minutes. The released drugs were found in the supernatant, and the percentage of drug release was calculated using HPLC calibration curves.

[0186] Ultrasound power heatmap determination in mouse and rat brain. 57BL / 6 mice (20-26 g; 12-16 weeks old; Jackson Laboratory) and Long-Evans rats (200-400 g; 12-16 weeks old; Charles River) were used in this study. The animals were sacrificed, and their heads were collected. The heads were then perfused with a solution of 4% paraformaldehyde (PFA) over 24 hours to preserve the tissue. After perfusion, the heads were sliced into different thicknesses. To assess the ultrasound transmission efficiency in the head, a transducer (Image Guided Therapy System) was positioned in front of the head sections, and ultrasound gel (Aquasonic 100 Ultrasound Gel) was applied to ensure good contact between the transducer and the head sections. A hydrophone (HGL-0200, Onda Corporation) connected with a data acquisition oscilloscope (2204A-D2, Pico Technology) was placed behind the sections to accurately record the signals. The voltage measurements obtained from the hydrophone were then converted to absolute pressure values using the calibration specifications provided by the hydrophone vendor. - 78 - 4860-4443-2856, v.1

[0187] Ultrasound induced blood-brain barrier (BBB) opening evaluation. The evaluation of ultrasound-induced BBB opening under sono-chemogenetics was conducted using C57BL / 6 mice (20-26 g; 12-16 weeks old; Jackson Laboratory). The mice were anesthetized with 2.5% isoflurane, and their heads were immobilized. Before surgery, each animal received subcutaneous injections of meloxicam (5 mg / kg) and Ethiqa (3.25 mg / kg), with ophthalmic ointment applied to cover their eyes. Subsequently, the hair on the scalp was shaved to prepare for the ultrasound-induced BBB opening evaluation. Ultrasonic gel was applied to ensure efficient coupling of ultrasound energy between the transducer and the skull. For the positive control group, mice were injected with 0.1 mL of microbubbles at a concentration of 1 x 108bubbles / mL, prepared according to established methods. Following microbubble injection, focused ultrasound (FUS) exposures were conducted on targeted brain areas, specifically the right ventral tegmental area (VTA) (AP: -3.08 mm, ML: +0.40 mm, DV: -5.0 mm) with a pressure of 0.75 MPa, and the left VTA (AP: -3.08 mm, ML: -0.40 mm, DV: -5.0 mm) with a pressure of 1.0 MPa. The exposure duration at the targeted sites was set to 20 s. For the experimental groups, procedures were similar to those of the positive control groups, except for microbubble injection. Peak pressures were adjusted to 1.0 MPa and 1.5 MPa. Following ultrasound stimulation, 40 μL of Evans blue (4 mg / mL in saline) was injected via the tail vein. One hour post-injection, mice were deeply anesthetized with intraperitoneal administration of ketamine (16 mg / kg). Subsequently, perfusion was conducted using PBS, followed by 4% paraformaldehyde. The brains were then extracted for imaging.

[0188] The evaluation of ultrasound-induced thermal effects. Type-K thermocouple wire with (TP-01-NIST, Reed Instrument) was inserted into the ventral tegmental area for mice anteroposterior (AP) -3.08 mm, mediolateral (ML) + 0.40 mm, and dorsoventral (DV) -5.0 mm. Thermocouple wire was connected to a cold-junction compensated analog-to-digital converter (ADC) with 12-bit resolution and interfaced with an Arduino Uno to sample and store the data at 4Hz. The ultrasound transducer (1.5MHz, Image Guided Therapy System) was placed concentrically with the thermocouple wire on the mouse’s exposed scalp with ultrasound gel (Aquasonic 100, Parker) for acoustic coupling. The ultrasound stimulation (1.55 MPa) was applied to treat the ventral tegmental area (VTA), and real-time thermal effects were monitored using a thermocouple. - 79 - 4860-4443-2856, v.1EXAMPLE 3 – MATERIALS AND METHODS

[0189] Chemicals: Unless otherwise mentioned, all reagents and solvents were purchased from commercial sources and used as received without further purification. Details: (4- (methoxycarbonyl)phenyl)boronic acid, 1,3,6,8-tetrabromopyrene, palladium tetrakis(triphenylphosphine), potassium carbonate, 1,4-dioxane, sodium hydroxide（NaOH), potassium hydroxide (KOH), tetrahydrofuran (THF), concentrated hydrochloric acid (HCl), acetone, dimethylformamide (DMF), methanol, ethanol, palladium (II) acetate, methyl 6-bromo- 2-naphthoate, sodium acetate (NaOAc), Palladium(II) chloride (PdCl2), dichloromethane (CH2Cl2), magnesium sulfate (MgSO4), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane), dimethyl sulfoxide (DMSO), silica gel, potassium phosphate (K3PO4), hexane, ethyl acetate, aluminum chloride (AlCl3), 1,3,5-triphenylbenzene, acetyl chloride, bromine, 1-propanol, 1- butanol, sodium bicarbonate, Sodium thiosulfate, 4-Methylbenzonitrile, tri-fluoro- methanesulfonic acid, aqueous ammonia, acetic acid, sulfuric acid (98%), chromium (VI)-oxide, acetic anhydride.

[0190] Instruments: Gas adsorption / desorption isotherms were measured by the volumetric method using a Micromeritics ASAP2020 surface area and pore analyzer. The powder X-ray diffraction patterns were collected by a Panalytical X’Pert powder diffractometer equipped with a Cu-sealed tube (λ = 1.54184 Å) at 40 kV and 40 mA over the 2θ range of 5−40°. Bruker 400 MHz Avance III HD Nano was used for collections of 1H NMR spectra (1H NMR, 400 MHz).

[0191] Bioreagents: pAAV-hSyn-hM3D(Gq)-mCherry was a gift from Bryan Roth (Addgene viral prep # 50474-AAV9; n2t.net / addgene:50474;RRID:Addgene_50474). (AAV- hSyn-GCaMP6s-WPRE-SV40 was a gift from Douglas Kim & GENIE Project (Addgene viral prep # 100843-AAV9; n2t.net / addgene:100843; RRID:Addgene_100843)1 Clozapine N-oxide (CNO) dihydrochloride (water soluble) was ordered from Hello Bio, Rhodamine B was ordered from Sigma-Aldrich, - 80 - 4860-4443-2856, v.1

[0192] Synthesis of the HOF-BTB building units: 1,3,5-Tris(4-carboxyphenyl)benzene (H3BTB): 1,3,5-Tribromobenzene (1.00 g, 3.1 mmol), 4-ethoxycarbonylphenylboronic acid (2.22 g, 11.4 mmol) and K3PO4 (5.06 g, 23.8 mmol) were mixed in 1,4-dioxane (80 mL) and the mixture was degassed under argon for 10 min. [Pd(PPh3)4] (0.03 g, 0.031 mmol) was added to the reaction mixture with stirring and the mixture was heated to 85ºC for 4 days under argon. The solution was evaporated to dryness and the residue was extracted with chloroform. The final product H3L was obtained by hydrolyzing the crude product with 2M aqueous NaOH, followed by acidification with concentrated HCl (1.18 g, 85 %).1H-NMR (400 MHz, DMSO-d6): δ = 13.03 (s, 3H), 8.08 (d, 9H), 8,02 (d, 6H).

[0193] Synthesis of HOF-TATB building unit 4,4’,4’’-(1,3,5-Triazine-2,4,6-triyl)- tribenzoic acid (H3TATB): 1, 4-Methylbenzonitrile (10.0 g, 86.0 mmol) was slowly added to tri-fluoro-methanesulfonic acid (20.9 mL, 236 mmol). After stirring at room temperature for 17 h, the red brown reaction mixture was neutralized with aqueous ammonia solution (2 M) with ice. The white precipitate was filtered and washed with a large amount of deionized water and acetone. The crude product was then recrystallized from toluene and dried under vacuum conditions. After that, 2,4,6-tris(4-methylphenyl)-1,3,5-triazine (8.1 g, 23.0 mmol, yield: 80 %) in the form of white crystals was obtained. Then, 2,4,6-tris(4-methylphenyl)-1,3,5-triazine (7.92 g, 22.5 mmol) was poured into a 500 mL flask, acetic acid (217 mL, 3.79 mol) and sulfuric acid (12.5 mL, 234 mmol) were added, the mixture solution was stirred, then chromium (VI)-oxide (20.5 g, 205 mmol) and acetic anhydride (13.7 mL, 145 mmol) were added carefully in small amounts (1.5 g and 1 mL, respectively). The dark-brown slurry was stirred at room temperature for 72 h, then poured into cold water (900 mL), well mixed, and then separated by centrifugation. The precipitates were collected, washed with water and dissolved in aqueous sodium hydroxide solution (2 M, 500 mL). Unreacted starting material was removed by filtration, then the solution was acidified with hydrochloric acid (2 M) and washed with water to yield a slightly yellow crude product. After recrystallization from N, N-dimethylformamide and H2O, 7.37 g (16.7 mmol, 74 %) of as a white powder product was obtained.1H-NMR (400 MHz, DMSO-d6): δ = 13.24 (s, 3H), 8.63 (d, 6H), 8,06 (d, 6H).

[0194] Synthesis of HOF-101 building unit 1,3,6,8-Tetrakis(benzoic acid)pyrene (H4TBAPy) Ligand: A mixture of (4-(methoxycarbonyl)phenyl)boronic acid (5 g, 32.9 mmol), - 81 - 4860-4443-2856, v.11,3,6,8-tetrabromopyrene (2.85 g, 5.5 mmol), palladium tetrakis(triphenylphosphine) (0.1 g 0.09 mmol), and potassium carbonate (6 g, 44 mmol) in dry dioxane (100 mL) was stirred under the protection of N2 for 72 h at 85 °C. The reaction mixture was poured into a solution of ice water and concentrated hydrochloric acid (v / v = 3:1). The organic phase was extracted with chloroform, and the combined extraction was dried over magnesium sulfate. After filtration, the solvent was removed under vacuum to give 3.44 g (4.6 mmol) 1,3,6,8-tetrakis(4- (methoxycarbonyl)phenyl)pyrene (yield: 84%). Then, 1g (17.8 mmol, 12 equiv) KOH was added to a suspension of 1 g (1.465 mmol) of 1,3,6,8-tetrakis(4-(methoxycarbonyl)phenyl)pyrene in 100 mL of THF / dioxane / H2O (v / v = 5:2:2), and the mixture was stirred under reflux at 85 °C for 12 hours. The solvent was removed under vacuum, and then 100 mL H2O was added to the residue. The mixture (yellow clear solution) was stirred at room temperature for 2 h. The pH value was adjusted to 2 using concentrated HCl. The resultant yellow solids were collected by filtration and washed with water several times. After drying under vacuum, 0.88 g (1.29 mmol) product was obtained with a yield of 97%. 1H-NMR (DMSO-d6): δ= 13.10 (s, 4H), 8.20 (s, 4H), 8.15 (d, 8H), 8.08 (s, 2H), 7.85 (d, 8H).

[0195] Synthesis of HOF-102 building unit 1,3,6,8-Tetra(6-carboxynaphthalen-2- yl)pyrene(H4PTTNA): Dioxane (250 mL) was placed in a 500 mL three-necked round-bottom flask and purged with argon for 1.5 h. With argon purging and the use of a mechanical stirrer, tetrabromopyrene (5.0 g, 9.7 mmol), methyl 6-(pinacolboryl)-2-naphthoate (13.3 g, 42.5 mmol), potassium phosphate tribasic (16.5 g, 77.7 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.55 g, 0.48 mmol) were added. The reaction was heated to 90°C for 72 h. The reaction mixture was allowed to cool to room temperature, and then 250 mL of water was added. The yellow solid was filtered using a glass Büchner funnel (medium frit) and washed with water (2×500 mL), followed by 500 mL of acetone. The filter flask was then emptied, and the solid was collected by passing hot chloroform (6×500 mL) through the frit and collecting the filtrate. Purification by flash column chromatography (silica gel) afforded the L1-OMe product as a light yellow solid (5.28 g, 58%). Then, L1-OMe (5g, 5.3 mmol) was added to a 1000 mL single-necked flask, and dioxane (250 mL) was then added while stirring. A solution of potassium hydroxide (3.0 g, 53 mmol; 250 mL of water) was added, and the reaction mixture was heated to reflux while being vigorously stirred for 18 h (at this point, a clear solution was observed). The reaction was allowed to cool to room temperature. The organic solvent was removed using rotary evaporation, and 500 mL of - 82 - 4860-4443-2856, v.1water was added to dissolve the solid obtained. Concentrated HCl was added to the solution dropwise with stirring until the solution reached pH 1. After being stirred for an additional hour, the yellow precipitate was collected via centrifugation and washed with water (3×50 mL). The final solid product was recrystallized from DMF, filtered, and dried (3.6 g, 68%).1H-NMR (DMSO-d6): δ= 13.14 (s, 4H), 8.71 (s, 4H), 8.49 (s,4H), 8.32 (d, 4H), 8.29 (s, 6H), 8.11 (d, 4H), 8.08 (d, 4H), 7.94 (d, 4H).

[0196] Thermolysis of HOFs. 1 mg / mL fresh HOFs solution was loaded into a centrifuge tube, and heated with a heater (Fisherbrand™ Isotemp™ Digital Dry Baths / Block Heaters) for 5 min at the designed temperature. After that, the solution was centrifuged at 8000 rpm for 5 min, and the supernatant was extracted to do the UV-Vis tests to determine the HOF dissociation percentage.

[0197] Cell viability tests. Human embryonic kidney 293 (HEK293T) cells were seeded into 96 well plates coated with 10 μg / mL Poly-L-Ornithine solution and cultured in DMEM with 10% fetal bovine serum at 37 °C under 5% CO2. Then, complete medium containing TATB nanoparticles at various concentrations were added into the plate when the confluency was around 90%. After incubation for 24 h, Cell-Titer blue reagent (10 μL, Promega Corporation) was added into each well. Microplate reader (BioTek Synergy H1, 560ex / 590em nm) was used to measure the cell viability after 4 h incubation according to the following formula: ^^^^^^^^^^^^ ^^^^^^^^" ^^ ^^%^^^ ^^^ !^^^^^^^" #%$ = × 100% ^^^^^^^^^^^^ ^^^^^^^^" ^^ ^^^^^^^

[0198] Hemolysis tests of TATB nanoparticles. According to the previous method, 0.3-0.5 mL fresh blood was extracted from the mouse heart, and then washed 3 times with 5 mL PBS after centrifuge at 8000 rpm for 5 min. The red blood cells (RBCs) were suspended with 2 mL PBS. To evaluate the biocompatibility of nanoparticles, 1 mL TATB nanoparticles at different concentrations were mixed with 30 µL RBCs. The mixture was incubated at 37 ºC for 2 h, and then centrifuged at 12000 rpm for 5 min. The supernatant was extracted out for UV-Vis tests at absorption 541 nm. Notably, 30 μL RBCs were added to 1 mL distilled water as positive control or 1 mL PBS as negative control. The hemolysis percentage was calculated through the following formula: - 83 - 4860-4443-2856, v.1&' − '^^ ^^^^^^^^^^ ^^ ^^%^^^ ^^%^^"^^^ ^^^^^^^^^^ #%$ = × 100% &' − '^^ ^^^^^^^^^^ ^^ ^^^^^^!^ ^^^^^^^

[0199] Primary neuron culture. C57BL / 6 mice (8 weeks old, weighing 20-26 g; Jackson Laboratory) were utilized for our research. Primary cortical neurons were prepared from mice at embryonic day 15.5. Initially, 24-well cell culture plates were coated with poly-l-ornithine (0.2 mg / mL) and incubated at 37 ºC for 2 hours. The plates were then washed three times with PBS and pre-warmed in a cell incubator for 15 minutes prior to use. Dissociated neural cells were plated on the coated plates and cultured in neurobasal medium supplemented with B27, glutamine, penicillin, and streptomycin. The neurons were incubated at 37°C under 7% CO2. After 2 days of incubation, a glial inhibitor, 5-fluoro-2'-deoxyuridine (0.1 mM), was added to the culture medium. Following 4 days of incubation, 1 μL of AAV-9-hSyn-hM3D(Gq)-mCherry and 1 μL of pAAV- hSyn-GCaMP6s-WPRE-SV40 were transfected into the neurons. Calcium imaging experiments were conducted after an additional 6 days of incubation.

[0200] Ultrasound power heatmap determination in mouse and rat brain. 57BL / 6 mice (20-26 g; 12-16 weeks old; Jackson Laboratory) and Long-Evans rats (200-400 g; 12-16 weeks old; Charles River) were used in this study. The animals were sacrificed, and their heads were collected. The heads were then perfused with a solution of 4% paraformaldehyde (PFA) over a period of 24 hours to preserve the tissue. After perfusion, the heads were sliced into different thicknesses. To assess the ultrasound transmission efficiency in the head, a transducer (Image Guided Therapy System) was positioned in front of the head sections, and ultrasound gel (Aquasonic 100 Ultrasound Gel) was applied to ensure good contact between the transducer and the head sections. A hydrophone (HGL-0200, Onda Corporation) connected with a data acquisition oscilloscope (2204A-D2, Pico Technology) was placed behind the sections to accurately record the signals. The voltage measurements obtained from the hydrophone were then converted to absolute pressure values using the calibration specifications provided by the hydrophone vendor.

[0201] Iba1, GFAP and Caspase-3 Staining: Following the in vivo sono-optogenetics procedures, mice were subjected to anesthesia via intraperitoneal injection of ketamine (16 mg / kg) after 14 days. Perfusion was then performed using PBS and 4% paraformaldehyde. The brain was extracted and stored in 4% paraformaldehyde overnight at 4ºC, and sliced using a vibrating blade microtome. Brain slices with a depth of 60 µm were washed with TBS solution and blocked with - 84 - 4860-4443-2856, v.15% bovine serum albumin TBS solution for 30 minutes at room temperature. Subsequently, the blocking buffer was replaced by rabbit anti-Iba1 antibody (013-27691, Wako Chem, 1:500) / TBS or rabbit anti-Cleaved Caspase-3 antibody (9661, Cell Signaling Tec., 1:500) / TBS or rabbit anti- GFAP antibody (13-0300, Invitrogen, 1:500) / TBA After incubation overnight at 4 ºC, the slices were washed three times with TBS solution. Fresh TBS solution containing the secondary antibody Donkey anti-rabbit Alexa Fluor 594 (A32754, Invitrogen, 1:500) and Hoechst 33342 (17535, ATT Bioquest, 1:5000) was added and incubated for 2 hours at room temperature in a dark room. Finally, the slices were washed three times with TBS, mounted on slides with mounting media, and covered with a coverslip. Fluorescence images were obtained using a Leica DMi8 fluorescence microscope.

[0202] H&E staining. Following the in vivo sono-optogenetics procedures, the mice were anesthetized with i.p. injection of ketamine after 14 days, and perfused with PBS and 4% paraformaldehyde. The brains were extracted, fixed in 4% paraformaldehyde overnight at 4 ºC, and sliced into 10 µm sections using a vibrating blade microtome. H&E staining was performed on the brain sections according to previously described protocols. The stained sections were then mounted onto slides with mounting media and covered with a coverslip. Images were captured using a Nikon Eclipse Ni Compound Light Microscope. Detailed information on the antibodies used in this study can be found in Table 6. * * *

[0203] 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 described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. - 85 - 4860-4443-2856, v.1REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Adamantidis, A. et al. Nat. Neurosci. 18, 1202–1212 (2015). Airan, R. D. et al. Nano Lett. 17, 652–659 (2017). Airan, R. D., Thompson, K. R., Fenno, L. E., Bernstein, H. & Deisseroth, K. Nature 458, 1025– 1029 (2009). Akbulatov, S. et al. Science 357, 299–303 (2017). Bansal, A., Shikha, S. & Zhang, Y. Nat Biomed Eng (2022) doi:10.1038 / s41551-021-00829-3. Bar-Zion, A. et al. Nat. Nanotechnol. 16, 1403–1412 (2021). Bhunia, S., Deo, K. A. & Gaharwar, A. K. 2D Adv. Funct. Mater. 30, 2002046 (2020). Blöchl, P. E. Projector augmented-wave method. Phys. Rev. B Condens. Matter 50, 17953– 17979 (1994). Boesmans, W., Hao, M. M. & Vanden Berghe, P. Nat. Rev. Gastroenterol. Hepatol. 15, 21–38 (2018). Boulatov, R. Nature chemistry vol. 13112–114 (2021). Can, A. et al. J. Vis. Exp. e3638 (2012). Chen et al., Mater. Adv. 3, 3680 (2022). Chen, H. & Hwang, J. H. J Ther Ultrasound 1, 10 (2013). Chen, R. et al. Nat. Biotechnol. 39, 161–164 (2021). Chen, S. et al. Science 359, 679–684 (2018). Chen, Y., Mellot, G., van Luijk, D., Creton, C. & Sijbesma, R. P. Chem. Soc. Rev. 50, 4100– 4140 (2021). Comprehensive Biomedical Physics. (Newnes, 2014). Cravotto, G., Gaudino, E. C. & Cintas, P. Chem. Soc. Rev. 42, 7521–7534 (2013). Cunningham, C. L., Gremel, C. M. & Groblewski, P. A. Nat. Protoc. 1, 1662–1670 (2006). D. A. Tyrrell, T. D. Heath, C. M. Colley, B. E. Ryman, Biochim. Biophys. Acta 457, 259 (1976). D. Bobo, K. J. Robinson, J. Islam, K. J. Thurecht, S. R. Corrie, Pharm. Res. 33, 2373 (2016). Deisseroth, K. Nat. Neurosci. 18, 1213–1225 (2015). - 86 - 4860-4443-2856, v.1Dolomanov et al., “OLEX2: a complete structure solution, refinement and analysis program”. J. Appl. Crystallogr, 42, 339-341 (2009). Duque, M. et al. Nat. Commun. 13, 600 (2022). Finlayson, L. et al. Photochem. Photobiol. 98, 974–981 (2022). Fishman, M., Zhuang, H. L., Mathew, K., Dirschka, W. & Hennig, R. G. Phys. Rev. B Condens. Matter 87, 245402 (2013). Furthmüller, J., Hafner, J. & Kresse, G. Phys. Rev. B Condens. Matter 53, 7334–7351 (1996). Ghanem, M. A. et al. Nat. Rev. Mater. 6, 84–98 (2021). Gomez, J. L. et al. Science 357, 503–507 (2017). Grimme, S. J. Comput. Chem. 27, 1787–1799 (2006). Hübschle, C. B. et al., “ShelXle: a Qt graphical user interface for SHELXL”, J. Appl. Crystallogr. 44, 1281-1284 (2011). Huo, S. et al. Adv. Sci. 9, e2104696 (2022). Huo, S. et al. Nat. Chem. 13, 131–139 (2021). Jones et al., Nature, 474, 367–371 (2011). Kiessling, F. et al. Adv. Drug Deliv. Rev. 72, 15–27 (2014). Kim, C. K., Adhikari, A. & Deisseroth, K. Nat. Rev. Neurosci. 18, 222–235 (2017). Kresse, G. & Furthmüller, J. Comput. Mater. Sci. 6, 15–50 (1996). Kresse, G. & Furthmüller, J. Phys. Rev. B Condens. Matter 54, 11169–11186 (1996). Kresse, G. & Hafner, J. Phys. Rev. B Condens. Matter 47, 558–561 (1993). Kresse, G. & Joubert, D. Phys. Rev. B Condens. Matter 59, 1758–1775 (1999). L. Schoenmaker, D. Witzigmann, J. A. Kulkarni, R. Verbeke, G. Kersten, W. Jiskoot, D. J. A. Crommelin, Int. J. Pharm. 601, 120586 (2021). Lee, J.-U. et al. Nat. Mater. 20, 1029–1036 (2021). Li et al., ACC Mater Res, 1, 77-87 (2020). Li, J., Nagamani, C. & Moore, J. S. Acc. Chem. Res. 48, 2181–2190 (2015). Li, Y.-L. et al. J. Am. Chem. Soc. 142, 7218–7224 (2020). Lin, R.-B. & Chen, B. Chem 8, 2114–2135 (2022). Lin, R.-B. et al. Chem. Soc. Rev. 48, 1362–1389 (2019). Marshel, J. H. et al. Science 365, 5202 (2019). Matsubara, T. et al. Nat. Commun. 13, 1950 (2022). - 87 - 4860-4443-2856, v.1Meng, Y., Hynynen, K. & Lipsman, N. Nat. Rev. Neurol. 17, 7–22 (2021). Mirvakili, S. M. & Langer, R. Nature Electronics 4, 464–477 (2021). Nagai, Y. et al. Nat. Neurosci. 23, 1157–1167 (2020). Olson, V. G. et al. Science 311, 1017–1020 (2006). Pawley, G.S. “Unit-cell refinement from powder diffraction scans”, J. Appl. Crystallogr.14, 357- 361 (1981). Pulido et al., Nature, 543, 657–664 (2017). Rao, S. et al. Nat. Nanotechnol. 14, 967–973 (2019). Romero, G., Park, J., Koehler, F., Pralle, A. & Anikeeva, P. Nature Reviews Methods Primers 2, 1–21 (2022). Rwei, A. Y. et al. Nat Biomed Eng 1, 644–653 (2017). Sebesta, C. et al. Nat. Mater. (2022) doi:10.1038 / s41563-022-01281-7. Sha, Y. et al. Angew. Chem. Int. Ed Engl. 61, e202203169 (2022). Sheldrick, G. M. “Crystal structure refinement with SHELXL.” Acta Crystallogr. B71, 3-8 (2015). Sheldrick, G. M. “SHELXT – integrated space-group and crystal-structure determination”, Acta Crystallogr A Found Adv. 71, 3-8 (2015). Shi, Z., Song, Q., Göstl, R. & Herrmann, A. Chem. Sci. 12, 1668–1674 (2020). Shi, Z., Wu, J., Song, Q., Göstl, R. & Herrmann, A. J. Am. Chem. Soc. 142, 14725–14732 (2020). Sundararaman, R. & Schwarz, K. J. Chem. Phys. 146, 084111 (2017). Szablowski, J. O., Lee-Gosselin, A., Lue, B., Malounda, D. & Shapiro, M. G. Nat Biomed Eng 2, 475–484 (2018). Allen, Theresa M., Christian B. Hansen, and Daniel E. Lopes de Menezes. "Pharmacokinetics of long-circulating liposomes." Advanced Drug Delivery Reviews 16.2-3 (1995): 267-284. Tye, K. M. et al. Nature 493, 537–541 (2013). Wang B, Lin RB, Zhang Z, Xiang S, Chen B. Hydrogen-Bonded Organic Frameworks as a Tunable Platform for Functional Materials. J Am Chem Soc.,142(34):14399-14416, 2020. Wang, B. et al. Chem. Commun. 56, 66–69 (2019). Wang, B. et al., “A novel mesoporous hydrogen-bonded organic framework with high porosity and stability. Chem. Commun. 56, 66-69 (2019). Wang, J. B., Aryal, M., Zhong, Q., Vyas, D. B. & Airan, R. D. Neuron 100, 728–738.e7 (2018). - 88 - 4860-4443-2856, v.1Wang, W. et al. J. Am. Chem. Soc. 145, 1097–1107 (2023). Witten, I. B. et al. Neuron 72, 721–733 (2011). Wu, M.-X. & Yang, Y.-W. Adv. Mater. 29, (2017). Wu, X. et al. Nat Biomed Eng 6, 754–770 (2022). Xian, Q. et al. Proc. Natl. Acad. Sci. U. S. A. 120, e2220575120 (2023). Yang et al. “Porous hydrogen-bonded organic frameworks (HOFs): From design to potential applications” Chemical Engineering Journal 399(1):125873, 2020. Yang, Y. et al. Nat. Chem. 13, 933–939 (2021). Yin, Q. et al. Angew. Chem. Weinheim Bergstr. Ger. 130, 7817–7822 (2018). Yin, Q., et al., “An ultra-robust and crystalline redeemable hydrogen-bonded organic framework for synergistic chemo-photodynamic therapy. Angew. Chem. Weinheim Bergstr. Ger. 130, 7817-7822 (2018). Zan, G.-Y. et al. Cell Rep. 37, 109913 (2021). Zentner, C. A. et al. Chem. Commun. 51, 11642–11645 (2015). Zentner, C.A. et al., “High surface area and Z’ in a thermally stable 8-fold polycatnated hydrogen- bonded framework.” Chem. Commun. 51, 11642-11645 (2015). Zhao et al., Nat. Commun., 12, 817 (2021). Zhuang, F., Xiang, H., Huang, B. & Chen, Y. Adv. Mater. e2303158 (2023). - 89 - 4860-4443-2856, v.1

Claims

WHAT IS CLAIMED IS:

1. A method of delivering a drug to a mammalian subject comprising: (i) administering to the subject a hydrogen bonded organic framework (HOF) comprising a drug or active pharmaceutical ingredient; and (ii) applying oscillating ultrasonic waves to a portion of the mammalian subject, thereby releasing the drug or active pharmaceutical ingredient from the HOF.

2. The method of claim 1, wherein the HOF comprises a compound of the formula: wherein:A is a C6-16aromatic ring or a C3-15heteroaromatic ring; and each of B, B′, and B′′ are independently a C6-16aromatic ring or a C3-15heteroaromatic ring, wherein each may further comprise −S(O)2Ra, –NH2, or −C(O)Rawherein Rais hydroxy or amino.

3. The method of claim 2, wherein A comprises 1, 2, 3 or 4 rings, preferably 1 ring.

4. The method of claim 3, wherein A is an aromatic(C≤6−13) ring.

5. The method of claim 3, wherein A is a heteroaromatic(C=3−12) ring.

6. The method of either claim 2 or claim 3, wherein A is an aromatic(C6) or heteroaromatic(C3−5) ring. - 90 - 4860-4443-2856, v.

17. The method of claim 6, wherein A is: ,.

9. Theeach independently substituted aryl(C≤12)or substituted heteroaryl(C≤12).

10. The method of claim 9, wherein B, B′, and B′′ are each independently an aryl(C=6) or heteroaryl(C=3−5) ring.

11. The method of claim 10, wherein B, B′, and B′′ are each independently , , 4860-or 12. . 13.one of claims 1-12, wherein the HOF has intrinsic cavities with sizes between 1 Å and 500 Å.

14. The method of any one of claims 1-13, wherein B, B′, and B′′ have the same structure.

15. The method of claim 14, wherein B, B′, and B′′ are each . 16.according to any one of claims 1-15, wherein the HOF comprises a compound of the formula: - 92 - 4860-4443-2856, v.1I), wherein: X1, X2 and X3 are each independently CH or N; R1, R2 and R3 are each independently −S(O)2Ra or −C(O)Ra, wherein: Ra is hydroxy or amino; R4-R15 are each independently −H, −F, −Cl, −Br, −I or −CH3; or a pharmaceutically acceptable salt thereof.

17. The method of claim 16, wherein X1, X2and X3are each independently CH or N.

18. The method of either claim 16 or claim 17, wherein R1, R2and R3are each independently −C(O)Ra, wherein Ra is hydroxy or amino.

19. The method of claim 18, wherein R1, R2and R3are -C(O)NH2.

20. The method of claim 18, wherein R1, R2and R3are -COOH.

21. The method of claim 20, wherein R4-R15 are −H.

22. The method according to any one of claims 16-21, wherein the compound is further defined as: . 4860-4443-2856, v.

123. The method according to claim 16-22, wherein the compound is further defined as: .

24. The methodcompound is further defined as: .

25. The method accordingthe compound is further defined as: .

26. The method of any27. The method according to any one of claims 16-23 and 26, wherein X2 is CH.

28. The method according to any one of claims 16-23, 26, and 27, wherein X3 is CH.

29. The method according to any one of claims 16-23, wherein X1is N. - 94 - 4860-4443-2856, v.

130. The method according to any one of claims 16-23 and 29, wherein X2is N.

31. The method according to any one of claims 16-23, 29, and 30, wherein X3 is N.

32. The method of any one of claims 16-31, wherein R1, R2 and R3 are -COOH.

33. The method according to any one of claims 16-32, wherein the compound is further defined as: .

34. The methodthe compound is further defined as: .

35. The method of any one drug or active pharmaceutical ingredient is encapsulated in the HOF.

36. The method of claim 35, wherein the HOF encapsulates from about 1-40 wt% of the drug or active pharmaceutical ingredient relative to the weight of the HOF.

37. The method of any one of claims 1-36, wherein the HOF is further defined as a nanoparticle, and wherein the drug or therapeutic is comprised within the nanoparticle.

38. The method of claim 37, wherein the nanoparticle is about 40-800 nm in size. - 95 - 4860-4443-2856, v.

139. The method of any one of claims 1-38, wherein the mammalian subject is a human.

40. The method of any one of claims 1-39, wherein the method comprises treating a disease in the mammalian subject.

41. The method of claim 40, wherein the disease is a cancer, a neurological disorder, a neurodegenerative disease, a cardiac disease, a lung disease, a heart disease, a kidney disease, inflammation, pain relief, or an inflammatory disease.

42. The method of claim 41, wherein the cancer is a brain cancer, a breast cancer, a skin cancer, a uterine cancer, a liver cancer, a lung cancer, or a pancreatic cancer.

43. The method of claim 42, wherein the brain cancer is a glioma or glioblastoma.

44. The method of claim 42, wherein the neurodegenerative disease is Parkinson’s disease, or Alzheimer's disease.

45. The method of claim 42, wherein the neurological disorder is pain, epilepsy, depression, or addiction.

46. The composition of any one of claims 1-44, wherein the drug or active pharmaceutical agent is a chemotherapeutic, a neuroactive drug, or an anti-inflammatory drug， or an analgesic, or an anesthetic.

47. A pharmaceutical composition comprising: (i) a compound of the formula: ,wherein: X1, X2and X3are each independently CH or N; - 96 - 4860-4443-2856, v.1R1, R2and R3are 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 drug or an active pharmaceutical agent, and (iii) an excipient.

48. The pharmaceutical composition of claim 47, wherein X1, X2and X3are each independently CH or N.

49. The pharmaceutical composition of either claim 47 or claim 48, wherein R1, R2 and R3 are each independently −C(O)Ra, wherein Rais hydroxy or amino.

50. The pharmaceutical composition of claim 49, wherein R1, R2and R3are –C(O)NH2.

51. The pharmaceutical composition of claim 49, wherein R1, R2 and R3 are -COOH.

52. The pharmaceutical composition of claim 51, wherein R4-R15 are −H.

53. The pharmaceutical composition according to any one of claims 47-52, wherein the compound is further defined as: .

54. The pharmaceuticalof claims 47-53, wherein the compound is further defined as: - 97 - 4860-4443-2856, v.1I).

55. The pharmaceutica f claims 47-54, wherein the compound is further defined as: .

56. The pharmaceuticalof claims 47-54, wherein the compound is further defined as: .

57. The pharmaceuticalclaims 47-54, wherein X1is CH.

58. The pharmaceutical composition according to any one of claims 47-54 and 57, wherein X2is CH.

59. The pharmaceutical composition according to any one of claims 47-54, 57, and 58, wherein X3is CH. - 98 - 4860-4443-2856, v.

160. The pharmaceutical composition of any one of claims 47-54 and 57-59, wherein X1is N.

61. The pharmaceutical composition according to any one of claims 47-54 and 57-60, wherein X2 is N.

62. The pharmaceutical composition according to any one of claims 47-54 and 57-61, wherein X3 is N.

63. The pharmaceutical composition of any one of claims 47-53 and 55-62, wherein R1, R2 and R3are –C(O)NH2.

64. The pharmaceutical composition of any one of claims 47-53 and 55-62, wherein R1, R2 and R3 are −COOH.

65. The pharmaceutical composition according to any one of claims 47-64, wherein the compound is further defined as: .

66. The pharmaceuticalof claims 47-64, wherein the compound is further defined as: . 4860-4443-2856, v.

167. The pharmaceutical composition of any one of claims 47-66, wherein the drug or active pharmaceutical agent is anesthetic, chemotherapeutic, or an anti-inflammatory drug, an analgesic, a painkiller, or an enzyme.

68. The pharmaceutical composition of any one of claims 47-67, wherein the drug or active pharmaceutical ingredient is encapsulated in the HOF.

69. The pharmaceutical composition of claim 68, wherein the HOF encapsulates from about 1-40 wt% of the drug or active pharmaceutical ingredient relative to the weight of the HOF.

70. The method of any one of claims 47-69, wherein the HOF is further defined as a nanoparticle, and wherein the drug or therapeutic is comprised within the nanoparticle.

71. The method of claim 70, wherein the nanoparticle is about 200-900 nm in size.

72. A method of treating or preventing a disease or disorder in a patient in need thereof comprising administering to the patient a pharmaceutically effective amount of a composition according to any one of claims 47-71.

73. The method of claim 72, wherein the patient is a mammal.

74. The method of claim 73, wherein the patient is a human.

75. The method according to any one of claims 72-74, wherein the disease or disorder is a cancer, a neurological disorder, a neurodegenerative disease, a lung disease, a heart disease, a kidney disease, inflammation, or pain.

76. The method of claims 75, wherein the neurological disorder is pain, epilepsy, depression, addiction, stroke, or spinal cord injury.

77. The method of claim 75, wherein the cancer is a brain cancer, a breast cancer, a skin cancer, a uterine cancer, liver cancer, lung cancer, or pancreatic cancer.

78. The method of claim 77, wherein the brain cancer is a glioma or glioblastoma.

79. The method of claim 75, wherein the neurodegenerative disease is Parkinson’s disease or Alzheimer's disease.

80. The method of claim 75, wherein the neurological disorder is pain, epilepsy, depression, or addiction. - 100 - 4860-4443-2856, v.1

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