Hydrogen-bonded frameworks nanoparticles for SONO-optogenetic treatment of brain diseases

Abstract

Hydrogen-bonded framework nanoparticles comprising a chemiluminescent compound (e.g., L-012) are provided and can be used, e.g., for sono-optogenetic treatment of neurodegenerative diseases such as Parkinson's disease. Related in vivo and therapeutic methods are also provided.

Description

DESCRIPTION HYDROGEN-BONDED FRAMEWORKS NANOPARTICLES FOR SONO- OPTOGENETIC TREATMENT OF BRAIN DISEASES BACKGROUND This application claims the benefit of United States Provisional Patent Application No. 63 / 606,481, filed December 5, 2023, the entirety of which is incorporated herein by reference.

[0001] 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

[0002] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, it concerns hydrogen-bonded organic frameworks containing a chemiluminescent compound. 2. Description of Related Art

[0003] Cell-type-specific activation of parvalbumin-expressing neurons in the external globus pallidus (PV-GPe) through optogenetics has been tested for movement dysfunction recovery in mice with Parkinson's disease, in an attempt to treat this neurodegenerative disorder. However, the applicability of these approaches have been significantly hindered by adverse effects and risks stemming from the implantation of optical fibers into the brain.

[0004] Parkinson's disease (PD) represents an essential challenge to the global healthcare community and leads to various motor dysfunctions and cognitive impairments in the later stage of PD.1,2 Deep brain stimulation (DBS) in either the subthalamic nucleus (STN) or the globus pallidus interna (GPi) in PD patients, achieved through high-frequency electrical stimulation with implanted microelectrodes, has emerged as an effective clinical intervention for alleviating motor symptoms.3,4 However, a significant limitation of the existing DBS therapies is that they treat the disease's symptoms but do not correct the underlying circuit dysfunction responsible for the symptoms, consequently leading to off-target activation.5 Furthermore, it is an invasive procedure that can cause partial damage to brain tissue and induce indiscriminate activation of surrounding tissues. Finally, DBS is costly, necessitates a4905-0304-6915, v.1specialized surgical setup, and demands highly skilled surgeons, making it significantly less accessible for rural areas and underrepresented populations with limited income. Therefore, there is a strong need to develop a Parkinson's treatment solution that offers long-term therapeutic efficiency with minimal invasiveness.

[0005] Parkinson's disease (PD) represents an essential challenge to the global healthcare community and leads to various motor dysfunctions and cognitive impairments in the later stage of PD. Deep brain stimulation (DBS) in either the subthalamic nucleus (STN) or the globus pallidus interna (GPi) in PD patients, achieved through high-frequency electrical stimulation with implanted microelectrodes, has emerged as an effective clinical intervention for alleviating motor symptoms. However, a significant limitation of the existing DBS therapies is that they treat the disease's symptoms but do not correct the underlying circuit dysfunction responsible for the symptoms, consequently leading to off-target activation. Furthermore, it is an invasive procedure that can cause partial damage to brain tissue and induce indiscriminate activation of surrounding tissues. Finally, DBS is costly, necessitates a specialized surgical setup, and demands highly skilled surgeons, making it significantly less accessible for rural areas and underrepresented populations with limited income. Therefore, there is a strong need to develop a Parkinson's treatment solution that offers long-term therapeutic efficiency with minimal invasiveness.

[0006] Focused ultrasound (FUS)-triggered mechanoluminescent nanoparticles has been tested for non-invasive light delivery in deep brain regions, leveraging ultrasound's tissue penetration and established clinical safety. Recent developments have introduced piezoelectric inorganic nanoparticles capable of generating light emission upon FUS stimulation, successfully applied for the non-invasive mouse motor cortex modulation, named sono-optogenetics. These nanoparticles ingeniously harness photoenergy stored within lattice defects, releasing it under ultrasonic mechanical stress. Yet, their application has been encumbered by the need for prerequisite photocharging, particularly in settings necessitating prolonged and repeated stimulation after a single injection. More recently, the emergence of photocatalytic organic nanoparticles, driven by ultrasound-induced cascade reactions between sonosensitizers and chemiluminescent reagents in liposomes, has enabled the achievement of active glow emission without charging. This innovation holds promise for sustainable and repeatable sono-optogenetics, bolstered by improved biocompatibility and system simplicity. Nonetheless, the restricted drug loading capacity within these nanoparticles has curtailed the - 2 - 4905-0304-6915, v.1efficiency of cascade reactions, resulting in diminished luminescence intensity and duration, particularly within deeper tissues under ultrasound stimulation. The development of sono- mechanoluminescent nanoparticles with higher photon yields and longer duration remains a primary challenge in advancing non-invasive sono-optogenetics.

[0007] Hydrogen-bonded organic frameworks (HOFs), assembled through intermolecular multivalent hydrogen bonds and π-π stacking interactions of organic building units, have recently emerged as a promising class of porous materials with high structural homogeneity and programmability. They exhibit high drug loading capacity, biocompatibility, and functional programmability achieved by adjusting building units for various bioapplications. Possible application of these compounds in some treatment of neurodegenerative diseases remains largely unexplored. There is a clear need for new therapies to treat neurodegenerative diseases. - 3 - 4905-0304-6915, v.1SUMMARY

[0009] The present disclosure provides, in some aspects, non-invasive sono- optogenetic approaches utilizing focused ultrasound (FUS)-triggered mechanoluminescent nanoparticles that can enable remote photon delivery deep within the brain for targeted neural modulation. These approaches overcome limitations in the prior art because they can allow for the stimulation of circuitry involved in neurological disorders, including neurodegenerative diseases (e.g., modulation of PV-GPe neurons with optogenetics for movement dysfunction recovery and treatment of Parkinson's disease) without implantation of optical fibers into the brain of a mammalian subject that can damage the brain and risk infection of the central nervous system. These mechanoluminescent nanoparticles preferably include sonosensitized hydrogen-bonded frameworks (HOF) and a chemiluminescent compound (e.g., preferably L012) that can serve as a nanoscale light source through ultrasound-induced cascade reactions. This system can advantageously provide increased brightness and longer-lasting light emission as compared to previous liposomal systems, thus facilitating repeatable deep brain stimulation. These sono-optogenetic approaches demonstrated effective modulation in vivo for the treatment of neurodegenerative diseases; these approaches can be used to modulate the mouse motor cortex for limb motion control and, notably, was employed for the first time to activate PV-GPe neurons, rescuing movement dysfunction in dopamine-depleted Parkinson's disease rats. These approaches can be used for precise and minimally invasive treatments for neurodegenerative disorders, and can be extended to larger non-primate models including human clinical applications. The HOF nanoparticles provided herein can also be used as photon delivery systems, e.g., for non-invasive bioimaging and photocatalysis.

[0010] As shown in the below examples, sonosensitized HOF incorporating chemiluminescent L012 are provided and can be used as a nanoscale light source to enable efficient photon delivery deep within the brain with high temporal resolution (e.g., FIGS. 1a- h, FIGS. 2a-l, FIGS. 3a-g). This can allow for precise control of neurons in neural circuit modulation. Data is provided showing targeted activation of PV-GPe neurons through these nanotransducers under focused ultrasound (FUS) stimulation (e.g., FIGS. 4a-g), ultimately ameliorating motor dysfunction in a Parkinson's disease rat model (e.g., FIGS.5a-g). Although the HOF can preferably include L012, it is anticipated that a variety of chemiluminescent compounds can be used. These findings establish the feasibility of non-invasive neural modulation through sono-optogenetics and underscore their potential as therapeutic techniques - 4 - 4905-0304-6915, v.1for the treatment of neurological diseases. The examples support the use of these non-invasive ultrasound-induced photon delivery systems for a variety of purposes, which may include bioimaging, photocatalysis, and biosensors. Crystal structures (FIGS. S1a-d), stability (FIGS. S2a-c, FIGS. 3a-b) and other properties including generation of hydroxyl radicals and1O2from HOF ultrasound stimulation (e.g., FIGS. S4a-b, FIGS. S5a-b), light emission from L012 containing HOF (FIGS. S6-S9) support the use of these HOF for clinical use and treatment of neurological or neurodegenerative diseases. HOF containing L012 demonstrated remarkably good biosafety (FIGS. S10a-b) and in vivo biocompatibility based on evaluation of evaluation of microglia (Iba1) and astrocytes (GFAP) activation and neuronal apoptosis (Caspase-3) (e.g., FIGS. S13a-e). The sono-optogenetics can be used in the treatment of various neurological diseases, and the biocompatible results further support clinical use. In some aspects, the HOF can be used to deliver a drug using ultrasound to treat a neurodegenerative disease. Without wishing to be bound by any theory, the inventors hypothesized that a HOF constructed from sonosensitizer building units could serve as porous carriers to load a high percentage of chemiluminescent reagents, acting as ultrasound-induced cascade reaction containers to achieve higher photon yields for deep brain sono-optogenetics due to increased local substrate concentration. The integration of sono-optogenetics with the targeted modulation of PV-GPe neural circuits in a Parkinson's disease rat model support the idea that these approaches can be used in clinical therapeutic interventions in the realm of neurodegenerative disorders.

[0011] An aspect of the present disclosure relates to a method of treating a brain disease in a mammalian subject comprising: (i) administering to the subject a hydrogen bonded organic framework (HOF) comprising: a chemiluminescent compound and / or a drug; and (ii) applying oscillating ultrasonic waves to the head or central nervous system of the mammalian subject, thereby releasing the chemiluminescent compound or drug from the HOF; wherein if the HOF comprises the chemiluminescent compound, then the brain of the mammalian subject comprises a neuron expressing a photosensitive protein, and wherein the emitted light modulates the photosensitive protein. The HOF may comprise a compound of the formula:- 5 - 4905-0304-6915, v.1wherein: A is a C6-16aromatic ring or a C3-15heteroaromatic ring; 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. A may comprise 1, 2, 3 or 4 rings, preferably 4 rings. A may be an aromatic(C=6−16) ring, preferably an aromatic(C=16) ring. In some embodiments, A has the structure: .In some B, B′, and B′′ are each independently substituted aryl(C≤12) or substituted 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 , , or- 6 - 4905-0304-6915, v.1B′′ may have the same structure. In some embod . Thea compound of the formula:wherein: R1, R2and R3are each independently −S(O)2Raor −C(O)Ra, wherein: Rais hydroxy or amino; R4-R20 are each independently −H, −F, −Cl, −Br, −I or −CH3; or a pharmaceutically acceptable salt thereof. R1, R2and R3may each independently be −C(O)Ra, wherein Rais hydroxy or amino. R1, R2 and R3 may be -C(O)NH2. R1, R2 and R3 may preferably be - COOH. R4-R20may preferably be −H. The HOF may preferably comprise a compound of the formula: - 7 - 4905-0304-6915, v.1or a pharmaceutically acceptable salt thereof. y emit light in response to free radicals or a reactive oxygen species (ROS). The reactive oxygen species (ROS) may be singlet oxygen (1O2) or hydroxyl radical (•OH). In some preferred embodiments, the chemiluminescent compound is L012, a dioxetane, luminol, isoluminol, an imidazopyrazinone, a lophine, or an acridinium. In some preferred embodiments, the chemiluminescent compound is L012. The chemiluminescent compound or drug may be encapsulated in the HOF. The HOF may encapsulates from about 1-30 wt% of the drug or active pharmaceutical ingredient relative to the weight of the HOF. In some preferred embodiments, the HOF is further defined as a nanoparticle, and wherein the drug or therapeutic is comprised within the nanoparticle. The nanoparticle may be about 50-900 nm in size. The mammalian subject may be a human. The brain disease may be Parkinson’s disease (PD), Alzheimer's disease, ataxia, Huntington's disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, essential tremor, epilepsy, stroke, or traumatic brain injury. In some embodiments, the brain disease is Parkinson’s disease. The oscillating ultrasonic waves may preferably comprise focused ultrasound (FUS). The focused ultrasound (FUS) may comprise oscillating ultrasonic waves of 0.5-5 Mhz and / or a pulse of 5-30 seconds. The focused ultrasound (FUS) may comprises oscillating ultrasonic waves of 0.5 – 20 MHz and / or a pulse of 10-1000 ms. The focused ultrasound (FUS) may have a focus length of about 5-120 mm. The HOF may be comprised in or preferably forms nanoparticles. The nanoparticles may be about 100-1000 nm across or in diameter. The nanoparticles may be about 200-800 nm across or in diameter. The HOF may further comprises a sonosensitizer (e.g., IR-780, DCPH-P-Na(I), hematoporphyrin, zinc protoporphyrin, methylene blue, TiO2, Chlorin e6, indocyanine Green (ICG), hematoporphyrin derivative, or photofrin. The HOF may further comprise a fluorescent dye or imaging agent. The drug may be a neuroactive drug, an anti-inflammatory drug, a NSAID drug, an analgesic, an anesthetic, a chemotherapeutic, or an anticancer drug. The neuroactive drug may be a drug for the treatment of Parkinson’s disease (PD) (e.g., selegiline, rasagiline, - 8 - 4905-0304-6915, v.1safinamide, 4-Dihydroxyphenylalanine（L-DOPA), apomorphine, rotigotine, amantadine, or entacapone).

[0012] Another aspect of the present disclosure relates to a method of contacting a tissue of a subject with light, comprising: applying an ultrasound signal to a nanoparticle in proximity to the tissue of the subject, wherein the nanoparticle comprises a hydrogen bonded organic framework (HOF) as described above or herein, comprising: a chemiluminescent compound and / or a drug as described above or herein; and wherein the ultrasound signal causes the photosensitizer to induce the chemiluminescent to emit light that contacts the tissue. The subject may preferably be a mammalian subject, even more preferably a human.

[0013] Yet another aspect of the present disclosure relates to a composition comprising a hydrogen bonded organic framework (HOF) described above or herein, comprising: a chemiluminescent compound and / or a drug described above for use in the treatment of a brain disease. The brain disease may be Parkinson’s disease (PD), Alzheimer's disease, ataxia, Huntington's disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, essential tremor, epilepsy, stroke, or traumatic brain injury; preferably wherein the brain disease is Parkinson’s disease.

[0014] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a hydrogen bonded organic framework (HOF) comprising: a chemiluminescent compound and / or a drug; wherein the HOF comprises a compound of the formula:- 9 - 4905-0304-6915, v.1wherein: R1, R2and R3are each independently −S(O)2Raor −C(O)Ra, wherein: Rais hydroxy or amino; R4-R20 are each independently −H, −F, −Cl, −Br, −I or −CH3; or a pharmaceutically acceptable salt thereof. R1, R2and R3may each independently be −C(O)Ra, wherein Rais hydroxy or amino. R1, R2 and R3 may be -C(O)NH2. R1, R2 and R3 may preferably be - COOH. R4-R20may preferably be −H. The HOF may preferably comprise a compound of the formula: or achemiluminescent compound may emit light in response to free radicals or a reactive oxygen species (ROS). The reactive oxygen species (ROS) may be singlet oxygen (1O2) or hydroxyl radical (•OH). The chemiluminescent compound may be L012, a dioxetane, luminol, isoluminol, an imidazopyrazinone, a lophine, or an acridinium. The chemiluminescent compound may preferably be L012. The HOF may be comprised in or may preferably form nanoparticles. The nanoparticles may be about 100- 1000 nm across or in diameter. The nanoparticles may be about 200-800 nm across or in diameter. The HOF may further comprises a sonosensitizer (e.g., IR-780, DCPH-P-Na(I), hematoporphyrin, zinc protoporphyrin, methylene blue, TiO2, Chlorin e6, indocyanine Green (ICG), hematoporphyrin derivative, or photofrin). The HOF may further comprise a fluorescent dye or imaging agent. The drug may be a neuroactive drug, an anti-inflammatory drug, a NSAID drug, an analgesic, an anesthetic, a chemotherapeutic, or an anticancer drug. The neuroactive drug may be a drug for the treatment of Parkinson’s disease (PD) (e.g., selegiline, rasagiline, safinamide, 4-dihydroxyphenylalanine（ L-DOPA), apomorphine, rotigotine, amantadine, or entacapone). The composition may be formulated for parenteral administration or injection.

[0015] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. - 10 - 4905-0304-6915, v.1

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

[0017] Throughout this application, the 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.

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

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

[0020] 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. - 11 - 4905-0304-6915, v.1BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] FIGS. 1a-h. Ultrasound-triggered generation of reactive oxygen species (ROS) in HOF nanoparticles for light emission. (a) Schematic illustration of the sonosensitized HOF nanoparticles preparation. (b) The morphology of HOF nanoparticles tested via TEM, scale bar: 200 nm. (c) Hydrodynamic size distribution of HOF nanoparticles at different conditions. (d) Schematic illustration of the ultrasound-triggered cascade reaction in HOF nanoparticles. (e) The evaluation of O generation by HOF nanoparticles under ultrasound irradiation (1.5 MHz, 1.55 MPa) over time through monitoring the UV-Vis spectra of the DPBF probe. (f) The quantitative analysis of O generation through monitoring DPBF decomposition at conditions with or without ultrasound irradiation (n > 3 per group). (g) The determination of O generation in L012-loaded HOF nanoparticles and (h) the quantitative analysis of O generation.

[0023] FIGS. 2a-l. Ultrasound-triggered light emission and in vitro neuron activation. (a) Mechanoluminescence spectra of HOF@L012 nanoparticles. The emission spectrum of the nanotransducers is mainly overlaid with the ChR2 opsin absorption spectrum (blue dot curve). (b) Blue light is emitted from HOF@L012 nanotransducers even at a frequency of 5 Hz under the ultrasound stimulus (1.5 MHz, 1.55 MPa, pulse 50 ms on, 150 ms off). (c) The latency between light emission and ultrasound stimulus at different frequencies. (d) The light emission half-time determination of HOF@L012 nanotransducers under the ultrasound stimulus (1.5 MHz, 1.55 MPa). (e) Ultrasound peak pressure dependent light emission in HOF@L012 nanotransducers. (f) Normalized ultrasound energy deposition in the tissue and (g) normalized light emission at different tissue depths (1.5 MHz, 1.55 MPa). (h) The scheme of ChR2 opsin activation under the irradiation of mechanoluminescence. (i) Fluorescent images of mouse primary neurons expressing hSyn::ChR2-EYFP and hSyn::JRGECO1a, scale bar: 50 μm. (j) The determination of ChR2 expressing primary neurons activation through monitoring Ca2+indicator (JRGECO1a) fluorescence signal changes in different experimental conditions, (i) FUS -, HOF@L012 -; (ii) FUS +, HOF@L012 - 12 - 4905-0304-6915, v.1-; (iii) FUS -, HOF@L012 +; (iv) FUS +, HOF@L012 +, FUS stimulation (1.5 MHz, 1.55 MPa, pulse 100 ms on 900 ms off). (k) Statistical analysis of JRGECO1a signal changes at different conditions (n=3 per group, two-way ANOVA, and multiple comparisons test). (l) Spike probability of ChR2 expressing primary neurons under the mechanoluminescene irradiation (n=3 per group, two-way ANOVA and multiple comparisons test). All plots show mean ± SEM unless otherwise mentioned. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

[0024] FIGS. 3a-g. Non-invasive activation of motor cortex neurons for behavior modulation in ChR2 transgenic mice. (a) Scheme of non-invasive motor cortex stimulation via sono-optogenetics. (b) In vivo light generation from HOF@L012 nanotransducers in mice motor cortex under the ultrasound stimulation (1.55 MPa, pulse 100 ms on 900 ms off). (c) Time-resolved limb’s motion tracking when the mice were treated under the different experimental conditions,(i) ChR2 -, FUS +, HOF@L012 +;(ii) ChR2 +, FUS -, HOF@L012 +; (iii) ChR2 +, FUS +, HOF@L012 - and (iv) ChR2 +, FUS +, HOF@L012 +; (d) Statistical analysis of limbs’ motions in different groups of subjects (n=5 per group, two-way ANOVA and multiple comparisons test). (e) The activation probability of sono-optogenetics in mice motor cortex relative behavior modulation. (f) Confocal imaging of the expression of immediate early gene marker c-Fos in the mice motor cortex, scale bar: 30 μm. (g) Statistical analysis of c-Fos signals at different conditions at the M2 motor cortex region (n=4 per group, two-way ANOVA, and multiple comparisons test). All plots show mean ± SEM unless otherwise mentioned. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

[0025] FIGS. 4a-g. Sono-optogenetc activation of parvalbumin-expressing globus pallidus neurons in rat. (a) Experimental scheme of the in vivo fiber photometry of the parvalbumin-expressing globus pallidus externa neurons (PV-GPe) activity. (b) In vivo light generation from HOF@L012 nanotransducers in rat GPe region under the ultrasound stimulation (2.45 MPa, pulse 100 ms on 900 ms off).(c) Confocal fluorescence images of the co-expression of ChR2 and jRGECO1a in rat GPe area. Scale bar: 16 μm. (d) Normalized jRGECO1a fluorescence intensity change (ΔF / F0) in rat GPe under the different experimental conditions. Remarkable fluorescence increase was observed only when the FUS was applied to the rat with ChR2 expressed in PV-GPe neurons. The purple patterned area represents the FUS irradiation (1.5 MHz, 1.40 MPa, pulse 10s). Solid line, mean; shade area, SEM; 3 rats in each group (n = 3). One-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), - 13 - 4905-0304-6915, v.1**** P < 0.0001). (e) Statistical analysis of calcium signal changes in rat GPe region under different conditions. Mean ± SEM, n = 3 independent mouse. Two-way ANOVA and Tukey’s multiple comparison test (P ≥ 0.05 (ns), *** 0.0001≤ P < 0.001). (f) c-fos expression confocal fluorescence images in the GPe after the rats are treated with different conditions. Remarkable increase of c-fos expression was observed only when the rats with ChR2 expression in PV-GPe neurons were treated with FUS (1.5 MHz, 1.40 MPa, pulse 20 s, focus length 5 mm). Scale bar: 20 μm. (g) Quantification of the c-fos expression cells density (i), c-fos expression percentage among PV+ neurons (ii) and c-fos expression percentage among ChR2+ neurons (iii). 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, *** 0.0001≤ P < 0.001, **** P< 0.0001).

[0026] FIGS.5a-i. Non-invasive sono-optogenetic selective stimulation of PV-GPe neurons for long-lasting motor recovery in hemiparkinsonian rats. (a) Experimental scheme of hemiparkinsonian model creation and PV-GPe sono-optogenetic stimulation’ efficacy assessment using cylinder test. (b) Sono-optogenetic stimulation (red line) demonstrated a significant effect, comparable with optogenetics (green dashed line), in terms of motor recovery of the lesioned side and an increase in contralateral touches percentage. Mean ± SEM, n = 5. Two-way ANOVA and Tukey’s multiple comparison test (*** 0.0001 ≤ P < 0.001, **** P < 0.0001). (c) Individual contralateral touches percentage changes for each rat after FUS stimulation. Mean ± SEM, n = 5. Two-way ANOVA and Tukey’s multiple comparison test (*** 0.0001 ≤ P < 0.001, **** P < 0.0001). (d) Experimental scheme of hemiparkinsonian model creation and PV-GPe sono-optogenetic stimulation’ efficacy assessment using apomorphine-induced rotation test. (e) Sono-optogenetic stimulation (red line) showed a significant effect, comparable with optogenetics (green dashed line), in terms of attenuation of rotation rate (angular speed). Mean ± SEM, n = 5. Two-way ANOVA and Tukey’s multiple comparison test (*** 0.0001 ≤ P < 0.001). (f) Individual angular speed changes for each rat after FUS stimulation. Mean ± SEM, n = 5. Two-way ANOVA and Tukey’s multiple comparison test (*** 0.0001 ≤ P < 0.001). (g) (i-iii) Individual rat data with a comparison of rotation rate before (i) and after (ii) FUS stimulation through movement tracking and angular speed. (iv) Comparison of angular displacement during rotation test in rats before and after FUS (n = 5). The rats’ movement tracking, Angular Speed, Angular Velocity, and Angular Displacement were calculated using DeepLabCut (a markerless position tracking algorithm based on transfer learning with neural networks for 2D and 3D videos). For (b) and (e), the positive control groups were represented by hemi-parkinsonian rats with PV- - 14 - 4905-0304-6915, v.1GPe optogenetic stimulation, while the negative control groups - hemi-parkinsonian rats with GPe FUS stimulation without mechanoluminescent nanoparticles injection. . (g) Individual angular speed changes for each rat after FUS stimulation. Mean ± SEM, n = 5. Two-way ANOVA and Tukey’s multiple comparison test (*** 0.0001 ≤ P < 0.001). (h) Sono-optogenetic stimulation (red line) showed a significant effect, comparable with optogenetics (green dashed line), in terms of attenuation of rotation rate (angular speed). Mean ± SEM, n = 5. Two-way ANOVA and Tukey’s multiple comparison test (*** 0.0001 ≤ P < 0.001). articles and the ensuing sono-optogenetic stimulation on brain tissue integrity.

[0027] FIGS. S1a-d. The crystal structure determinations of HOF nanoparticles. (FIG. S1a) Powder X-Ray Diffraction (PXRD) tests of HOF nanoparticles, where the as- synthesized HOF nanoparticles were phase-pure and highly crystalline, which fitted very well with the calculated one. (FIG. S1b)N sorption isotherm (adsorption and desorption) at 77 K for HOF nanoparticles, where the pore volume of HOF nanoparticles is 34.7 cmmol . (FIG. S1c) Pore size distribution of HOF nanoparticles, and the dominant pore size is around 1.63 nm and 1.84 nm. (FIG. S1d)The BET surface areas for as-synthesized HOF nanoparticles (497.5 mg) were obtained from the N adsorption isotherm at 77 K.

[0028] FIGS. S2a-c. The stability evaluation of HOF nanoparticles. (FIG. S2a) The dynamic light scattering tests of HOF nanoparticles after 1 day or 7 days storage, (FIG. S2b) or stimulated with / without ultrasound for 60 s (1.55 MPa, 1.5 MHz). (FIG. S2c) The TEM images of HOF nanoparticles after 60 s ultrasound stimulation (1.55 MPa, 1.5 MHz), scale bar: 500 nm.

[0029] FIGS. S3a-b. The stability evaluation of dye-loaded HOF nanoparticles. (FIG. S3a) The dye release from HOF nanoparticles under ultrasound stimulation. (FIG. S3b) The long-term stability evaluation of dye-loaded HOF nanoparticles, where only around 8% free due was released after incubating 36 h at 37 ℃.

[0030] FIGS. S4a-b. The evaluation of the hydroxyl radicals (∙OH) generation through HOF nanoparticles under ultrasound irradiation. (FIG. S4a) The evaluation of hydroxyl radicals (∙OH) generation by HOF nanoparticles under ultrasound irradiation (1.5 MHz, 1.55 MPa) over time through monitoring the UV-Vis spectra of the SA probe. (FIG. S4b) The quantitative analysis of ∙OH generation through monitoring SA decomposition at conditions with or without ultrasound irradiation (n > 3 per group). - 15 - 4905-0304-6915, v.1

[0031] FIGS. S5a-b. Ultrasound peak pressure dependent1O2 generation by HOF nanoparticles under ultrasound irradiation. (FIG. S5a) The UV-Vis spectra of the DPBF probe after the HOF nanoparticles were irradiated by ultrasound for 60 s at different peak pressure. (FIG. S5b) The quantitative analysis of1O2 generation through monitoring DPBF decomposition at conditions with or without ultrasound irradiation (n > 3 per group).

[0032] FIGS. S6a-d. Ultrasound triggered light emission from HOF@L012 nanoparticles at different frequencies (1.55 MPa,1.5 MHz, pulse 50 ms on). (FIG. S6a) 1 Hz, (FIG. S6b) 2 Hz, (FIG. S6c) 4 Hz and (FIG. S6d) 10 Hz.

[0033] FIGS. S7a-f. Ultrasound triggered light emission from HOF@L012 nanoparticles at different pulses (1.55 MPa, 1.5 MHz). (FIG. S7a) 50 ms on 950 ms off, (FIG. S7b) 100 ms on 900 ms off, (FIG. S7c) 200 ms on 800 ms off, (FIG. S7d) 500 ms on 500 ms off, (FIG. S7e) 1000 ms on 1000 ms off, (FIG. S7f) static analysis of light intensity at different pulse.

[0034] FIGS. S8a-d. Ultrasound triggered light emission from HOF@L012 nanoparticles at different peak pressure. (FIG. S8a) 0.89 MPa. (FIG. S8b) 1.08 MPa. (FIG. S8c) 1.40 MPa. (FIG. S8d) 1.55 MPa.

[0035] FIGS. S9a-e. Ultrasound triggered light emission from HOF@L012 nanoparticles at different pork skin depths. (FIG. S9a) 0 mm. (FIG. S9b) 3mm. (FIG. S9c) 5 mm and (FIG. S9d) 10 mm and (FIG. S9e) 15 mm.

[0036] FIGS. S10a-b. The in vitro biosafety tests of HOF@L012 nanoparticles. (FIG. S10a) The cell viability tests of HOF@L012 nanoparticles in HEK-293T cells with / without ultrasound stimulation. (FIG. S10b) The hemolysis tests of HOF@L012 nanoparticles.

[0037] FIG. S11. The latency between sono-optogenetic stimulation and limb motion.

[0038] FIG. S12. Tyrosine hydroxylase (TH) immunofluorescence staining in hemiparkinsonian rat’s basal ganglia. Unilateral 6-OHDA lesion resulted in dopamine neurons degeneration and death in the right substantia nigra pars compacta (SNpc) and surrounding regions with a significant decrease of TH+ dopamine neurons. VTA (ventral tegmental area), SNpr (substantia nigra pars reticulata). - 16 - 4905-0304-6915, v.1

[0039] FIGS. S13A-F. In vivo biocompatibility evaluation of the sono-chemogenetics via determining microglia (Iba1) and astrocytes (GFAP) activation and neuronal apoptosis (Caspase-3). Fluorescence images of Iba1 (FIG. S13A), GFAP (FIG. S13C) and Caspase-3 (FIG. S13E) of the brain slices in rat GPe without stimulation and 7 days after sono-optogenetic stimulation. Scale bar: 100 μm. Blue signal is DAPI and the red signal is Iba1 (FIG. S13A), GFAP (FIG. S13C) and Caspase-3 (E). (FIG. S13B, FIG. S13D, FIG. S13F) Statical analysis of the Iba1 (FIG. S13B), GFAP (FIG. S13D) and Caspase-3 (FIG. S13F) intensity. Mean ± SEM, n = 3 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).

[0040] FIG. S14: The in vivo ultrasound power transfer in rat heads with FUS focus length of 5 mm. The ultrasound power heatmap in the rat head shows that around 1.50 MPa was delivered to the rat GPe when 2.45 MPa primary ultrasound power was used. - 17 - 4905-0304-6915, v.1DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0041] Porous HOFs can be used in function-tunable and programmable drug delivery systems thanks to their building unit designability and well-defined structures. Sonosensitized HOF nanoparticles, loaded with a chemiluminescent agent (e.g., preferably L012) are provided herein and can be used as ultrasound-triggered nano light sources in a tissue in a subject (e.g., in the brain of a mammalian subject). As shown in the below examples, these sono-HOF nanoparticles not only exhibited exceptional L012 loading capacity due to their high porosity but also acted as ROS generators, resulting in longer-lasting and brighter light emissions. The integration of sono-mechanoluminescent nanoparticles with cell-type-specific optogenetics, termed sono-optogenetics, enabled precise and minimally invasive neural modulation deep within the brain and can be used in neurological disease treatments. Spatiotemporal modulation of the motor cortex in mice was achieved through sono-optogenetics using these sono-HOF nanoparticles, allowing control of limb movements. Furthermore, the sono-HOF nanoparticles were used with sono-optogenetics to specifically activate PV-GPe neurons in dopamine-depleted Parkinson's disease rats, resulting in a comparable alleviation of motor symptoms when compared to optogenetic stimulation. Thus, these in vivo experiments in an animal model of Parkinson’s disease support the use of these particles for treating brain diseases and neurodegenerative diseases including Parkinson’s disease. The sono-HOF nanoparticles provided herein can also be used in bioimaging, photocatalysis, biosensors, and related applications.16,21,27HOF nanoparticles comprising a chemiluminescent compound provided herein can be used in combination with sono-optogenetics to treat a variety of brain or neurological diseases, and may allow for targeted modulation of neuronal activity. As shown in the below examples, biocompatibility of the sono-HOF nanoparticles was notable and further supports clinical use. I. HOFs

[0042] 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 - 18 - 4905-0304-6915, v.1past 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 extendednetworks. HOFs have been used in a wide variety of applications, including catalysis, energy, biomedical products, and the storage and separation of chemicals.

[0043] 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 carter custom-design applications. For instance, the density of hydrogen bondings and the presence of aromatic fused rings in the backbone of OMBU have a substantial impact on the properties of HOFs, particularly their stability under aqueous conditions (Li et al., 2020). If the HOFs can be precisely tailored and selectively activated through non-invasive ultrasound, this technique would serve as a significant advancement in the field of drug delivery. By providing exceptional versatility and excellent temporal resolution, it would expand the scope of this technology to numerous disease applications, thereby advancing the field as a whole.

[0044] 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 - 19 - 4905-0304-6915, v.1HOFs 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).

[0045] 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.

[0046] The strength of bonding of HOFs can be modified such that a desired level of ultrasound stimulation can disrupt the HOF hydrogen bonding and release a drug or therapeutic agent from inside the HOF. HOFs frameworks assembled from the organic units may include both the pure organic and metal-containing organic moieties through hydrogen bonding interactions, and the frameworks can be further strengthened or weakened by including or removing other weak interactions such as π…π interactions, van der Waals interactions, C- H…π interactions etc. The HOF may comprise a compound of the formula:- 20 - 4905-0304-6915, v.1wherein: A is a C6-16aromatic ring or a C3-15heteroaromatic ring; 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.

[0047] In some embodiments the HOF comprises a compound having the formula:(II), wherein: R1, R2,R3,and R4are each independently −S(O)2Raor −C(O)Ra, wherein: Rais hydroxy or amino; R4-R20 are each independently −H, −F, −Cl, −Br, −I or −CH3; or a pharmaceutically acceptable salt thereof. R4-R20may preferably be −H. In some preferred embodiments, the compound has the structure:. - 21 - 4905-0304-6915, v.1II. Chemiluminescent Compounds

[0048] Chemiluminescent compounds (also referred to as chemiluminescence compounds) as used herein refers to compounds that can generate light in response to free radicals (e.g., ROS) that can be produced by a sonosensitizer. In some preferred embodiments, a nanoparticle (e.g., a lipid nanoparticle) or a liposome of the present disclosure comprises a chemiluminescent compound that produces light in response to a singlet oxygen (1O2) or hydroxyl radical (•OH).

[0049] It is anticipated that a variety of chemiluminescent compounds can be used. For example, chemiluminescent compounds that may be used include L012, dioxetane derivatives, luminol, isoluminol, imidazopyrazinone derivatives, lophine derivatives and acridinium derivatives.

[0050] In some embodiments, the chemiluminescent compound is L012 (also called L- 012). L012 (8-amino-5-chloro-7-phenyl-pyrido[3,4-d]pyridazine-1,4(2H,3H)dione) is a luminol-based molecule that has been reported to produce much stronger chemiluminescence than other probes (lucigenin, luminol, and MCLA) (e.g., Nishinaka et al. “A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells.” Biochem Biophys Res Commun. 1993;193(2):554-559.). L-012 has been used as a CL probe for measuring superoxide (O2·−) and other reactive oxygen species (ROS) derived particularly from NADPH oxidases (Nox) (Zielonka et al. “On the use of L-012, a luminol-based chemiluminescent probe, for detecting superoxide and identifying inhibitors of NADPH oxidase: a reevaluation.” Free Radic Biol Med.65:1310-1314, 2013). L012 has the structure: .

[0051] The chemiluminescent compound may emit a range of wavelengths of light in response to stimulation by a free radical or ROS. For example, the chemiluminescent compound may emit a blue wavelength of about 430-550 nm. The wavelength of the light emitted may range from 250 nm to 650 nm, such as from 300 nm to 550 nm or from 350 nm to 450 nm. In some cases, the emitted photon has a wavelength ranging from 350 nm to 2000 - 22 - 4905-0304-6915, v.1nm, such as from 400 nm to 1700 nm, from 400 nm to 1300 nm, from 400 nm to 1000 nm, from 400 nm 800 nm, from 400 nm to 650 nm, or from 450 nm to 500 nm. In some cases, 80% or more of the emitted photons have such wavelengths, such as 90% or more or 95% or more. As used herein, the term “Stokes shift” refers to the difference between the absorption and emission wavelengths, and the Stokes shift can range in some embodiments from 20 nm to 100 nm, such as from 60 nm to 80 nm. In some embodiments, 80% or more of the wavelength of light emitted by the nanoparticles or liposomes is from about 400 – 700 nm, 400, 450, 500, 550, 600, 650, 700 nm, or any range derivable therein.

[0052] The light emitted by the chemiluminescent compound can be utilized in a variety of methods. Contacting a tissue inside a subject with light can be used in applications including optogenetics, photodynamic therapy, selective gene editing, and fluorescent imaging. In optogenetics, a neuron that includes a photosensitive protein can be contacted with light, thereby modulating the neuron. Thus, optogenetics allows for the study of neural circuits by specifically activating or deactivating particular neurons. III. Ultrasound

[0053] Ultrasound may be applied to a mammalian subject to release a chemiluminescent compound (e.g., preferably L012) and / or drug from a HOF as described herein within the brain or central nervous system of a mammalian subject. It is anticipated that ultrasound or focused ultrasound can be applied to a variety of regions in the brain of the mammalian subject including the striatum (e.g., caudate nucleus, putamen), thalamus, Globus pallidus, subthalamic nucleus, basal ganglia, cerebral cortex, or substantia nigra (e.g., substantia nigra pars reticulata (SNr), substantia nigra pars compacta (SNc)).

[0054] 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 - 23 - 4905-0304-6915, v.1mm away from the end of the ultrasound emitter, e.g., 10 mm to 150 mm below the skin, such as between 30 mm and 75 mm. A medical professional can change the settings on the ultrasound emitting device, thereby changing the desired depth of highest ultrasound intensity. This can allow for lower intensity ultrasound applied to a more superficial location, such as the prefrontal cortex of the brain, while also providing for high intensity ultrasound at an interior location, such as Broca’ s area of the brain, which is located underneath the prefrontal cortex. In contrast, in unfocused ultrasound the highest intensity is at or near the surface of the skin or tissue, with ultrasound intensity decreasing with increasing distance from the emitter. One of skill can modify the ultrasound or FUS signal to stimulate particular tissues within a subject such as a mammalian subject. In some embodiments, the ultrasound or FUS is applied to the brain, spinal cord, peripheral nerves, or other tissue in the subject.

[0055] 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.

[0056] 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.

[0057] 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, or 5 minutes or less.

[0058] 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, 10-240 seconds, or any range derivable therein. The nanoparticles or liposomes may disperse in the blood of body of the subject after administration, so it may be - 24 - 4905-0304-6915, v.1desirable 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 physical damage or toxicity to local tissues occurs from ROS generation by the chemiluminescent compound or sonosensitizer.

[0059] The emission of light from the photoexcited mechanoluminescent particle can be triggered by any suitable mechanical stimulus, e.g., by ultrasound. In some cases, the ultrasound is focused ultrasound. In some cases, the ultrasound signal has a frequency ranging from 150 kHz to 15 MHz, such as from 300 kHz to 5 MHz, or from 600 kHz to 2 MHz. The ultrasound signal can be repeated, i.e. wherein the signal is emitted for a time and then not emitted for a time, at any suitable repetition. In some cases, the ultrasound signal is repeated at a rate ranging from 0.05 repetitions per second to 20 repetitions per second, such as from 0.2 repetitions per second to 5 repetitions per second, or from 0.5 repetitions per second to 2 repetitions per second.

[0060] 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.

[0061] 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. IV. Pharmaceutical Compositions

[0062] 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 an pharmaceutical composition that contains a mechanoluminescent nanoparticle or mechanoluminescent liposome as - 25 - 4905-0304-6915, v.1described 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.

[0063] 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.

[0064] 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.

[0065] In some embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an 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 is 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 - 26 - 4905-0304-6915, v.1art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0066] In other non-limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. A. Parenteral Compositions and Formulations

[0067] 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.

[0068] 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). The form is preferably sterile and should be fluid to the extent that easy injectability exists. Conditions of manufacture and storage is preferably 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 - 27 - 4905-0304-6915, v.1thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0069] 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.

[0070] 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 - 28 - 4905-0304-6915, v.1solution aqueous 5% glucose (w / v). In some embodiments, the HOF comprising a drug or therapeutic agent is dissolved in an aqueous 5% glucose (w / v) prior to administration by injection. A. Liposomes and Nanoparticles

[0071] If desired, the HOF and chemiluminescent compound and / or drug are comprised in a liposome or liposomal formulation. In some embodiments, the HOF and chemiluminescent compound and / or drug are not comprised in liposomes or any lipid formulation, but the HOF forms nanoparticles that preferably comprise or contain the chemiluminescent compound and / or drug. Depending on the route of administration and the desired pharmacokinetics, both of these approaches

[0072] Attributing to its natural constituents, liposomes are preferably 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 (Bobo et al., 2016; Allen et al., 1995). More recently, the liposome based mRNA vaccine developed by BioNTech / Pfizer and Moderna was clinically applied against COVID-19 (Schoenmaker et al., 2021). In some instances the HOF and chemiluminescent compound and / or drug are preferably not included in liposomes, e.g., in the interest of simplicity or to increase generation of light from the chemiluminescent compound comprised in the HOF in response to ultrasound stimulation.

[0073] The liposomes may comprise a variety of lipid components. The liposomes can 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. - 29 - 4905-0304-6915, v.1

[0074] 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.

[0075] 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.

[0076] 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

[0077] A variety of drugs and therapeutic agents (e.g., to treat a neurodegenerative disease such as Parkinson’s disease) 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.

[0078] In some aspects, the drug is preferably a drug for the treatment of Parkinson’s disease (PD) such as, e.g., selegiline, rasagiline, safinamide, 4-Dihydroxyphenylalanine（L- DOPA), or apomorphine, rotigotine, amantadine, and / or entacapone. V. Neurodegenerative Diseases

[0079] Focused ultrasound with a pharmaceutical composition comprising a HOF and chemiluminescent compound (e.g., preferably L012) and / or a drug as described herein can be used with FUS or sono-optogenetic approaches, to treat a variety of neurodegenerative diseases. In some preferred embodiments, the neurodegenerative disease is Parkinson’s disease (PD). - 30 - 4905-0304-6915, v.1

[0080] The neurodegenerative disease may be Parkinson’s Disease (PD). The HOF containing a chemiluminescent compound (preferably L012) and / or drug can be administered to the patient alone or in combination with an additional therapy to treat PD such as, for example deep brain stimulation, administration of DA neurons to the subject, or an additional therapeutic to treat PD (e.g., an anti-inflammatory agent, a diuretic, levodopa, carbidopa- levodopa, a dopamine agonist, a monoamine oxidase (MAO) B inhibitor, a catechol O- methyltransferase (COMT) inhibitor, an anticholinergic, or amantadine). The HOF-based FUS or sono-optogenetic therapy as described herein can be administered before, after, or concomitantly with the additional therapy.

[0081] A variety of clinical parameters can be used to diagnose PD. For example, a subject having PD may have at least one motor symptom associated with PD such as, e.g., tremors, rigidity, bradykinesia, postural instability, walking or gait difficulties, dystonia, and / or vocal symptoms disturbances. A subject having PD may have at least one additional non-motor symptom associated with PD. Non-limiting examples of non-motor symptoms associated with PD include, but are not limited to, excessive salivation, insomnia, constipation, forgetfulness, Depression and Anxiety, pain, fatigue, hyposmia and urinary urgency.

[0082] Other neurodegenerative diseases that can be treated with a HOF-based FUS or sono-optogenetic therapy as described herein include Alzheimer's disease, ataxia, Huntington's disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, essential tremor, epilepsy, stroke, or traumatic brain injury. VI. Optogenetic methods

[0083] Optogenetics is a method that employs light to modulate tissue, such as neurons, that have been genetically modified to express light-sensitive proteins. In some cases, the neurons are neurons in the brain. Thus, by stimulating these brain neurons with light, brain processes and brain regions can be studied. In some cases, the photosensitive proteins are ion channels located in a cell membrane.

[0084] Brain studies with optogenetics traditionally employed, for example, fiber optics implanted into the brain to provide the excitatory light. However, such implantation causes various complications associated with the surgery and use, along with disadvantages such as infection risk and damage to brain tissue resulting from the surgery and implantation. - 31 - 4905-0304-6915, v.1

[0085] Any suitable photosensitive protein can be employed, such as channelrhodopsin-2 (ChR2), VChRl, iC++, ChRmine, or halorhodopsin (HPHR), e.g., a halorhodopsin from Natronomonas (NpHR). In some cases, the method also includes genetically modifying the neuron to express the photosensitive protein, e.g. with CRISPR gene editing.

[0086] The method can result, based on configuration, in the hyperpolarization or depolarization of the target neuron or neurons. Hyperpolarization includes partial hyperpolarization and complete hyperpolarization, whereas depolarization includes partial depolarization and complete depolarization. VII. Fluorescent Imaging

[0087] Compositions and methods provided herein can allow for light emission deep within the body. The methods can be used in a deep-tissue photodynamic therapy. Such methods, which utilize a nanoparticle or liposome as disclosed herein, are distinct from functional ultrasound imaging that purely relies on ultrasound to directly image a part of the subject.

[0088] For example, the nanoparticle or liposome can produce light due to application of ultrasound in an internal region of the subject, such as the intestines of the patient. The emission of light from the particles (e.g., fluorescent light) can be measured in order to image the region. In some cases, the device measuring the fluorescent light is outside the patient, and in other cases it is inside the patient. For example, a fluorescent light measuring device can be inserted into the gastrointestinal tract, e.g. the intestines, of the patient, and can thereby measure the objects or features between the ultrasound stimulated mechanoluminescent particles and the detection device. In such cases, the mechanoluminescent particles can be administered to the subject in any suitable area. For example, if the intestines are to be imaged, the mechanoluminescent particles can be administered in the leg, and optionally photoexcited in the leg, where after they travel through veins to an intestine region, at which they are contacted with ultrasound signal. VIII. Gene Editing

[0089] In some aspects, light generation via the nanoparticles and liposomes provided herein can be utilized in a gene editing method (e.g., deep-tissue gene editing mediated by ultrasound). These methods, which employ the nanoparticles or liposomes provided herein, - 32 - 4905-0304-6915, v.1are distinct from nonspecific gene editing that purely relies on systemically delivered CRISPR- Cas9 for editing the genome without spatiotemporal precision in the body.

[0090] As such, in some cases the tissue comprises a group of compounds that causes genetic modification to the tissue after absorbing the emitted light. In such cases, the method can be referred to as a method of selectively genetically modifying tissue by selectively applying an ultrasound signal to the tissue, i.e. surrounding tissues that do not receive the ultrasound signal or that do not comprise photoexcited liposomes or photoexcited nanoparticles are not genetically modified because they do not receive the light emitted by the nanoparticles or liposomes. The method can also be referred to as light-inducible gene editing. The gene editing can be referred to as spatially-selective gene editing since the gene editing will only occur in locations where the emitted light can penetrate.

[0091] For example, the photoexcited mechanoluminescent particle can be contacted with ultrasound in an internal region of the subject, such as the liver or the brain of the patient. In turn, the emission of light from the particles can be used to control the function of photoswitchable Cas9 in order to activate the CRISPR-Cas9 system for localized gene editing. In this case, the mechanoluminescent particles can be administered to the subject via intravenous injection. For example, if the genome in the liver is to be edited, the mechanoluminescent particles can be administered in the leg, and optionally photoexcited in the leg, where after they travel through veins to the liver, at which they are contacted with ultrasound signal to produce localized light emission and gene editing. Examples of such a photoswitchable genome editing methods are described by Moroz-Omori et al (ACS Central Science, 2020, 6, 5, 695, doi:10.1021 / acscentrasci.9b01093) and Zhou et al (ACS Chemical Biology, 2018, 13, 2, 443, doi:10.1021 / acschembio.7b00603), which are incorporated herein by reference. IX. Definitions

[0092] 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.

[0093] The term "hydrogen-bonded organic framework" or "HOF" is defined as a material assembled from organic linker molecules through intermolecular hydrogen bonding. - 33 - 4905-0304-6915, v.1The 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.

[0094] The terms “applying” and “administering” are used interchangeably to refer to causing a subject to receive a treatment. For example, applying an ultrasound signal means generating an ultrasound signal and directing it into a region of the subject’s body. As another example, administering a particle to the subject means moving the particle inside the body of the subject.

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

[0095] 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.

[0096] 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).

[0097] A “therapeutically effective amount”, a "therapeutically effective dose" or “therapeutic dose” is an amount sufficient to effect desired clinical results (e.g., achieve - 34 - 4905-0304-6915, v.1therapeutic 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.

[0098] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitativedeterminations·

[0099] 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.

[0100] 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.

[0101] 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. - 35 - 4905-0304-6915, v.1

[0102] 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 therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.

[0103] 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.

[0104] 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)2or 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)−.

[0105] In the context of chemical formulas, the symbol “−” means a single bond, “=”means a double bond, and “≡” means triple bond. The symbol “ ” represents an optionalbond, which if present is either single or double. The symbol “ ” represents a single bondor a double bond. Thus, the covers, for- 36 - 4905-0304-6915, v.1and . And it is understood that no one such ring atom forms part of more than one double it is noted that the covalent bond symbol “−”, when connecting one or twoatoms, does not indicate any preferred stereochemistry. Instead, it covers allstereoisomers as well as mixtures thereof. The symbol “ ”, when drawn perpendicularlyacross a bond (e.g., for methyl) indicates a point of attachment of the group. It is notedthat the point of is typically only identified in this manner for larger groups in orderto assist thein unambiguously identifying a point of attachment. The symbol “ ”means a single bond where the group attached to the thick end of the wedge is “out of theThe symbol “ ” means a single bond where the group attached to the thick end of the wedgeis “into the page”. The symbol “ ” means a single bond where the geometry around adouble 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.

[0106] 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 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: (R)y,then the variable may replace any to any of the ring atoms of either of the fused rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring - 37 - 4905-0304-6915, v.1atom (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.

[0107] 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 of 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.

[0108] 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 - 38 - 4905-0304-6915, v.1“saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.

[0109] 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).

[0110] 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.

[0111] 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 −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.

[0112] 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 - 39 - 4905-0304-6915, v.1the 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 group is a non- limiting example of cycloalkanediyl group. A “cycloalkane” refers compoundshaving the formula H−R, wherein R is cycloalkyl as this term is

[0113] 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 alkene” 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.

[0114] The term “alkynyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups −C≡CH, −C≡CCH3, and −CH2C≡CCH3 are non-limiting examples of alkynyl groups. An “alkyne” refers to the class of compounds having the formula H−R, wherein R is alkynyl. When any of these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by −OH, −F, −Cl, −Br, −I, −NH2, −NO2, −CO2H, −CO2CH3, −CN, −SH, −OCH3, −OCH2CH3, −C(O)CH3, −NHCH3, −NHCH2CH3, −N(CH3)2, −C(O)NH2, −C(O)NHCH3, −C(O)N(CH3)2, −OC(O)CH3, −NHC(O)CH3, −S(O)2OH, or −S(O)2NH2. - 40 - 4905-0304-6915, v.1

[0115] 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: Anas that term is defined above. Benzene and toluene are non-limiting examples of arenes.

[0116] 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.

[0117] 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 - 41 - 4905-0304-6915, v.1sulfur, 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.

[0118] The term “heteroarenediyl” refers to a divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, said atoms forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused; however, the term heteroarenediyl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroarenediyl groups include: N N . The term “N-heteroaryl”atom as the point of attachment. A “heteroarene” refers to the class of compounds having the formula H−R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.

[0119] 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.

[0120] 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, - 42 - 4905-0304-6915, v.1wherein 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.

[0121] 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 does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include: . The term “N-a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group.

[0122] 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. - 43 - 4905-0304-6915, v.1

[0123] 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.

[0124] The term “alkylamino” refers to the group −NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: −NHCH3and −NHCH2CH3. The term “dialkylamino” refers to the group −NRR′, in which R and R′ can be the same or different alkyl groups. Non-limiting examples of dialkylamino groups include: −N(CH3)2and −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.

[0125] 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, - 44 - 4905-0304-6915, v.1−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 −CH2CF3are 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)OCH3 and−NHC(O)NHCH3 are non-limiting examples of substituted amido groups. IV. Examples

[0126] 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 – Development of sonosensitized HOF nanoparticles

[0127] The sonosensitized HOF (Sono-HOF) was constructed using 1,3,6,8-tetrakis(p- benzoic acid) pyrene (H4TBAPy) units, a planar molecule with a large aromatic fused ring and four carboxylate acid groups (Fig.1a). Nano-sized Sono-HOF particles were prepared through a solvent-exchange approach. Powder X-ray diffraction confirmed the high crystallinity and phase purity of these Sono-HOF nanoparticles (Supplementary Fig.1a). Each H4TBAPy unit formed interactions with four neighboring units through eight O-H⋯O hydrogen bonds, resulting in the formation of a two-dimensional layer. These layers then interacted with adjacent layers through π-π stacking interactions, self-assembling into three-dimensional - 45 - 4905-0304-6915, v.1frameworks. Experimental nitrogen adsorption tests at 77 K revealed that the Sono-HOF nanoparticles possessed a high Brunauer−Emmett−Teller (BET) surface area of 497.5 m g and a pore volume of 34.7 cm mol (Supplementary Fig.1), endowing them with a high drug loading capacity. Transmission electron microscopy (TEM) showed regular multi-layered frameworks in these nanoparticles (Fig. 1b). Dynamic light scattering tests confirmed a well- distributed particle size with an average of 533.7 ± 14.9 nm (Fig.1c and Supplementary Table 1). Of note, the extensive π-conjugated system favored strong π-π stacking interactions, while multivalent hydrogen bonds reinforced intermolecular interactions, ensuring the stability of the framework in solution. Remarkably, no significant changes in size or morphology were observed after 7 days of incubation, even under ultrasound stimulation (Supplementary Fig. 2). Additionally, the negative surface potential of these nanoparticles ensures their stability even in the presence of 10 % fetal bovine serum, as demonstrated in Fig. 1c and Supplementary Table 1. These sono-HOF nanoparticles exhibited excellent drug loading capacity, approximately ~25 wt%, attributed to their high porosity, with no noticeable size changes observed after drug loading (Supplementary Table 1). To assess drug stability within the HOF nanoparticles, dye release tests were conducted. The results revealed that no dye was released, even under ultrasound stimulation, indicating long-term drug stability and enhancing their suitability for clinical applications (Supplementary Fig.3). Supplementary Table 1. Dynamic light scattering tests and drug loading content tests of various sono-HOF nanoparticles. entries Nanoparticles Size PDI Zeta potential DLC (d nm) (mv) (wt%) .6 4- 46 - 4905-0304-6915, v.1Example 2 – Sono-HOF nanoparticles act as cascade reaction containers and ROS sources for light emission

[0128] Pyrene served as an efficient photosensitizer to generate reactive oxygen species (ROS). It was hypothesized that the periodic integration of H4TBAPy units into the frameworks would function as a sonosensitizer to generate ROS under ultrasound stimulation (Fig. 1d). It was also hypothesized that the periodic integration of H4TBAPy units into the frameworks would serve as a sonosensitizer, producing ROS under ultrasound stimulation. ROS production was assessed and quantified using colorimetric probes, specifically 1,3- diphenylisobenzofuran (DPBF) and salicylic acid (SA), to detect singlet oxygen (O) and hydroxyl radical (•OH), respectively. During ultrasound irradiation, the characteristic UV-Vis absorption peak of DPBF at 420 nm rapidly decreased, while no changes were observed in the characteristic UV-Vis absorption peak of SA at 297 nm. These results indicated the generation of O but not •OH by the Sono-HOF nanoparticles (Fig.1e,f, and Supplementary Fig. 4). Furthermore, the yields of O increased with higher ultrasound peak pressure and displayed ultrasound power-dependent behavior (Supplementary Fig. 5). It was anticipated that L012 would efficiently consume the generated O to produce light, preventing any potential leakage since excessive O could damage cell membranes and lead to cell death. Consequently, ROS generation was also evaluated in L012-loaded Sono-HOF nanoparticles (HOF@L012). The results confirmed no O leakage during ultrasound stimulation in the HOF@L012 solution (Fig. 1g, h). L012, a potent ROS scavenger, effectively quenched ROS for light emission, safeguarding normal tissue against ultrasound-induced damage.

[0129] Ultrasound-induced cascade reaction dominated the triggered light emission from HOF@L012 nanoparticles. The fluorescence spectrum from HOF@L012 showed the emission wavelength at around 470 nm, which mainly overlapped with the channelrhodopsin- 2 (ChR2) for optogenetic stimulation (Fig. 2a). The real-time light emission from the HOF@L012 nanoparticles was also evaluated via photons recording system (Supplementary Video 1). Compared to luminol and dioxetane derivatives, chemiluminescent L012 demonstrated a remarkable increase in ROS sensitivity and reaction rate constant, with a responsive threshold for activation that was thousands of times lower, which ensured the temporal light was generated once under the ultrasound stimulus. In addition, opsins activation in optogenetics usually requires temporal light control and tunable pulse frequency in neural modulation for different disease treatments, such as 40 Hz flickering light-induced memory rescuement in Alzheimer’s disease. Time-resolved mechanoluminescence determined that this - 47 - 4905-0304-6915, v.1system exhibited excellent controllability and temporal resolution, from pulse frequency 1 to 10 Hz, with less than 4 ms latency (Fig.1b, c and Supplementary Figs.6-7), and showed great potential in optogenetic applications. The long-term activation of HOF@L012 nanoparticles was also assessed. As depicted in Fig. 2d, the decay half-time of light intensity was approximately 120 seconds. Remarkably, approximately 3000 repeated emissions (pulse 100 ms on 900 ms off) for practical application in optogenetics was achieved. It's worth noting that the light intensity decreased with each irradiation cycle due to the continuous and irreversible consumption of L012, marking an approximately three-fold increase compared to previous liposome systems. On the other hand, the light intensity is positive with ultrasound peak pressure due to the energy-dependent ROS generation (Fig. 2e and Supplementary Fig. 8). Ultrasound energy typically travels through tissue as a propagating pressure wave, attenuating exponentially with tissue depth. As depicted in Fig. 2f, approximately 20 mm of tissue penetration was achieved using a 1.5 MHz ultrasound wave, with nearly 40% of the energy effectively delivered to tissues at a depth of 10 mm, ensuring efficient activation for light emission (Fig. 2g and Supplementary Fig. 9). This level of tissue penetration is orders of magnitude greater than what can be achieved with both visible and NIR light sources.

[0130] The light emission from HOF@L012 nanoparticles was predominantly governed by ultrasound-induced cascade reactions. The fluorescence spectrum of HOF@L012 exhibited an emission wavelength of around 470 nm, which closely aligned with the activation range of channelrhodopsin-2 (ChR2) used in optogenetic stimulation (Fig.2a). Real-time light emission from HOF@L012 nanoparticles was recorded using a photon recording system (Supplementary Video 1). When compared to luminol and dioxetane derivatives, chemiluminescent L012 displayed a remarkable increase in sensitivity to reactive oxygen species (ROS) and reaction rate constants, with a responsive threshold for activation thousands of times lower, ensuring that temporal light emission occurred precisely under ultrasound stimulation. Furthermore, optogenetics often necessitates precise control over the timing and tunable pulse frequencies for neural modulation in various disease treatments, such as the use of 40 Hz flickering light to rescue memory in Alzheimer's disease. Time-resolved mechanoluminescence analysis revealed that this system exhibited exceptional controllability and temporal resolution, supporting pulse frequencies ranging from 1 to 10 Hz with a latency of less than 4 ms (Fig. 1b, c, and Supplementary Figs. 6-7), making it highly suitable for optogenetic applications. The long-term activation of HOF@L012 nanoparticles was also assessed. As shown in Fig. 2d, the decay half-time of light intensity was approximately 120 - 48 - 4905-0304-6915, v.1seconds, and approximately 3000 repeated emissions (pulse 100 ms on 900 ms off) were achieved, making it practical for optogenetic applications. It's important to note that light intensity decreased with each irradiation cycle due to the continuous and irreversible consumption of L012, marking an approximately three-fold increase compared to previous liposome systems. Additionally, light intensity was positively correlated with ultrasound peak pressure due to the energy-dependent ROS generation (Fig. 2e and Supplementary Fig. 8). Ultrasound energy typically propagates through tissue as a traveling pressure wave, attenuating exponentially with tissue depth. As illustrated in Fig. 2f, approximately 20 mm of tissue penetration was achieved using a 1.5 MHz ultrasound wave, with nearly 40% of the energy effectively delivered to tissues at a depth of 10 mm, ensuring efficient activation for light emission (Fig. 2g and Supplementary Fig. 9). This level of tissue penetration far surpasses what can be achieved with both visible and NIR light sources. Example 3 – Sono-optogenetic activation of ChR2-expressing neurons for non-invasive motor cortex modulation in mice

[0131] In vitro opsin activation experiments were also conducted using primary neurons under Sono-optogenetics (Fig. 2h). Initially, neurons were transduced with AAV9- hSyn::ChR2-EYFP and AAV9-hSyn::NES-JRGECO1a.WPRE.SV40 to express ChR2 opsins and calcium indicators (Fig. 2i). As shown in Figs. 2j-l, efficient spiking was observed in ChR2-expressing neurons, as evidenced by the tracking of calcium fluorescence signals under sono-mechanoluminescence irradiation, resulting in an approximately 85% spike probability. In contrast, only sporadic activation was observed in the absence of ultrasound stimulus or in ChR2-negative neurons.

[0132] The spatiotemporal activation of genetically targeted neurons is of utmost importance for researchers aiming to uncover the intricate links between brain activity and behavior. Consequently, the assessment of sono-chemogenetic activation in the mouse secondary motor cortex (M2) was conducted to modulate limb motion in mice (Fig. 3a). Initially, the light emission in the motor cortex area under ultrasound stimulation following tail vein injection was measured. Synchronous blue light with a higher relative power intensity of 1.44 mW.mm was generated under FUS stimulation compared to the previous liposome system (1.01 mW.mm ) due to the integrated design within the Sono-HOF cascade reaction containers (Fig. 3b). This level of light emission was sufficient to activate ChR2 opsins for neural circuit modulation. Given that the motor cortex governs higher-order control of body - 49 - 4905-0304-6915, v.1movement, real-time mouse limb motions were monitored using cameras during Sono- optogenetics in Thy1-ChR2-YFP transgenic mice. As depicted in Fig. 3c-e and Supplementary Video 2, DeepLabCut analysis of videos revealed that controlled limb motion was achieved following sono-optogenetic stimulation, with a high 60% activation probability. Conversely, only sporadic motions were observed in the absence of FUS stimulation or liposome injection or in wild-type mice. Neuron activation signals in post-hoc tissue samples was also assessed by examining the expression of the immediate early gene marker c-Fos. As shown in Figs. 3f,g, remarkable c-Fos signals were observed in the motor cortex area under sono-optogenetics, while few signals were found in other control groups. These results confirm the efficacy of the sono-optogenetic system in controlling neuron activation in a non-invasive manner, thus enabling remote and spatiotemporal brain modulation for potential clinical applications. Example 4 – Sono-optogenetic activation of PV-GPe neurons restores movement in hemiparkinsonian rats

[0133] A recent study has identified PV-GPe neurons as crucial components of the basal ganglia circuit, capable of inducing long-lasting attenuation of pathological activity in the SNr. Targeted interventions within the Globus Pallidus externa (GPe), rather than broad global interventions, have proven effective in sustaining lasting improvements in behavior, such as reducing immobility and bradykinesia, and in restoring normal physiological basal ganglia output in the 6-OHDA mouse model. Consequently, targeted activation of PV-GPe neurons through non-invasive sono-optogenetics holds great promise for ameliorating movement dysfunction in Parkinson's disease. The photon delivery efficiency in the rat GPe area was first evaluated. The system described herein achieved efficient photon delivery to the deep brain, owing to the excellent tissue penetration capabilities of FUS. As shown in Fig.4a, the light with a relative light intensity of around 1.22 mW.mm was emitted following the ultrasound stimulation in GPe after two days of intracranial injection of HOF@L012 nanoparticles. This level of light emission should be sufficient to activate ChR2 opsins, thereby promoting neuron spiking. Subsequently, neuron excitation responses to sono-optogenetic stimulation were recorded within the GPe region using fiber photometry. The photon delivery efficiency of our system was evaluated in the rat GPe brain area with our FUS system of focus length of 5 mm (Supplementary Fig. 14). PV-GPe neurons were transduced with ChR2 opsins, along with a fluorescence reporter (pAAV-Ef1a-DIO hChR2(E123T / T159C)-EYFP) - 50 - 4905-0304-6915, v.1and a red fluorescent calcium reporter (pAAV.Syn.NES-JRGECO1a.WPRE.SV40), as illustrated in Fig.4b. The JRGECO1a signal exhibited a substantial increase upon ultrasound stimulation of the GPe region with ChR2 expression, while no changes were observed in the absence of ultrasound stimulation or ChR2 expression (Fig. 4c,d). Furthermore, neuron activation in post-hoc tissue samples were assessed by examining the expression of the immediate early gene marker c-Fos. As depicted in Fig.4e,f, the results revealed significant c- fos signals within ChR2-expressing PV-GPe neurons, whereas sporadic c-fos signals were detected in the absence of ChR2 opsins, ultrasound stimulus, or nanoparticles.

[0134] Given the efficient activation of specific PV neurons in GPe, it was next evaluated whether the sono-optogenetics could rescue the movement dysfunction in PD rats. Transgenic rats expressing Cre recombinase in PV-expressing neurons were used to build the hemiparkinsonian PD rat model. AAV9 viral vectors carrying ChR2 genes (pAAV-Ef1a- DIO hChR2(E123T / T159C)-EYFP) were intracranially injected into the GPe to target the expression of ChR2 in PV neurons. Subsequently, the neurotoxin 6-hydroxydopamine (6- OHDA) was administered unilaterally into the medial forebrain bundle (MFB) to induce a hemiparkinsonian model (Fig. 5a) further verified through TH staining (Supplementary Fig. 12). After an incubation period of 4 weeks, HOF@L012 nanoparticles were injected into the same region. Sono-optogenetic stimulation of PV-GPe neurons in the hemiparkinsonian rat model was conducted to validate the alleviation of movement dysfunction through the cylinder test (Fig. 5a-c) and the apomorphine-induced rotation test (Fig. 5d-g) after two days of recovery from the surgery. Similar to the effects of optogenetic stimulation in PV-GPe neurons, the sono-optogenetics also yielded notable improvements in movement dysfunction and increased paw usage, as evidenced by a sustained increase in contralateral touch percentages, which endured for over 15 minutes following PV-GPe FUS stimulation (Fig.5b-c). Subsequent to the cylinder test, PV-GPe sono-optogenetic stimulation elicited a significant reduction in the apomorphine-induced rotation rate (angular speed) (Fig. 5e-f). Moreover, the DeepLabCut movement track analysis visualized a conspicuous reduction in rotations following PV-GPe FUS stimulation (Fig. 5g). Both behavioral assessments demonstrated consistent outcomes with the effects of selective optogenetic stimulation (Fig. 5b,e). Quantification of angular speed indicated a sustained reduction in rotations over 15 minutes following PV-GPe FUS stimulation (Fig. 5h, i). These behavioral assessments consistently demonstrated the effectiveness of sono-optogenetics in rescuing motor dysfunction in dopamine-depleted PD rats. - 51 - 4905-0304-6915, v.1

[0135] In the PV-GPe optogenetics and sono-optogenetics experiments described herein, motor treatment effects that persisted beyond stimulation were achieved, extending up to 15 minutes (20 minutes in some cases) in duration. However, it is worth noting that the longevity of this effect was lower compared to a previously demonstrated study conducted in a dopamine-depleted (DD) mouse model. Several factors may contribute to this discrepancy, including differences in the Parkinson's disease models employed, the extent of dopamine depletion, and the specific behavioral tests conducted. In the previous study, authors reported a prolonged and persistent effect during the open field test, but only in the mouse model with bilateral advanced dopamine loss. They were unable to demonstrate a persistent component of behavioral rescue in mice with partial dopamine depletion (mice with >20% striatal tyrosine hydroxylase remaining on either side). Furthermore, in unilaterally dopamine-depleted mice, PV-ChR2 stimulation did not induce enduring alterations in behavior. In the instant results, using a hemi-parkinsonian rat model with unilateral partial dopamine depletion, a stable effect that endured beyond the cessation of PV-GPe neuronal activation was observed. Notably, the sono-optogenetic effect closely paralleled that of optogenetics in terms of motor rescue and longevity. This suggests that sono-optogenetics could achieve a stable behavioral motor effect comparable to invasive optogenetic techniques. Finally, a series of histological biosafety studies were also conducted, encompassing Iba-1 (a marker for microglia activation), GFAP (a marker for astrocytic cells), and cleaved Caspase-3 (an apoptosis marker) staining (Supplementary Fig. 13) to evaluate the biosafety of this sono-optogenetic approach. The results collectively affirmed the safety of the administered nanoparticles and the ensuing sono- optogenetic stimulation on brain tissue integrity, exhibiting great potential for future clinical application in non-invasive deep brain stimulation. Example 6 – Methods

[0136] The following methods were used in the experiments of Examples 1-5. HOF nanoparticles preparation

[0137] The HOF ligand, 1,3,6,8-Tetrakis(benzoic acid)pyrene (H4TBAPy), was generously provided by Dr. Banglin Chen's laboratory. In a summarized procedure, 30 mg of H4TBAPy was dissolved in 3 mL of DMF at 60 ℃, followed by the gradual addition of 12 mL of distilled water under stirring (1000 rpm / min). After 15 minutes, the solution was harvested and subjected to centrifugation at 12000 rpm (13523 x g) for 5 minutes. The resultant yellow precipitate was collected, resuspended in acetone, and subjected to centrifugation at 12000 rpm - 52 - 4905-0304-6915, v.1(13523 x g) for 5 minutes. This washing step was repeated three times, followed by subsequent washes with ethanol and distilled water, each performed three times. Finally, the collected precipitate was resuspended in 3 mL of distilled water and stored at room temperature in a dark environment. This material was utilized for dynamic light scattering, UV-Vis spectrum, and TEM tests. Drug-loaded HOF nanoparticles preparation

[0138] L012-loaded and fluorescent dye (Rhodamine B, RB)-loaded HOF nanoparticles were synthesized. In a brief procedure, 3 mg of L012 or RB powder was introduced into 2 mL of HOF nanoparticles solution (5 mg / mL). The solution was gently agitated to facilitate the dissolution of L012 or RB. Subsequently, the solution was subjected to incubation in a water bath at 60 ℃ for 6 hours. Following this incubation, the solution was retrieved and subjected to centrifugation at 12000 rpm (13523 x g) for 5 minutes. The resulting pellets were subjected to at least four washes with distilled water to eliminate any unloaded drugs. Subsequently, the collected pellets were resuspended in distilled water for subsequent tests. The generation of O under the FUS stimulation in HOF nanoparticles.

[0139] One mL of either blank HOF nanoparticles or L012-loaded HOF nanoparticles (HOF@L012) at a concentration of 0.1 mg / mL was mixed with 30 μL of a 1 mg / mL 1,3- Diphenylisobenzofuran (DPBF) methanol solution in the dark and placed in a glass vial. The mixture was then irradiated with or without focused ultrasound (FUS, 1.5 MHz, Image Guided Therapy) at 1.5 MHz. At a fixed time interval, 20 μL of the solution was extracted for UV-Vis spectrum tests. The characteristic absorption of DPBF around 420 nm was monitored and used to quantify the generation of singlet oxygen (O) due to the selective consumption of DPBF byO. Ultrasound peak pressure was measured using a hydrophone (Onda Corporation, HGL- 0200). At least three independent tests were conducted in the experiments. The generation of •OH under the FUS stimulation in HOF nanoparticles.

[0140] Similar to the O detection, 1 mL of either blank HOF nanoparticles or HOF@L012 nanoparticle solution was mixed with 50 μL of a 1 mg / mL salicylic acid (SA) methanol solution in the dark. After being stimulated for a fixed time under ultrasound, 20 μL of the solution was extracted for UV-Vis spectrum tests. The generation of •OH was evaluated by tracking the consumption of SA using UV-Vis spectrum tests, where the characteristic absorption of SA was around 297 nm. - 53 - 4905-0304-6915, v.1The sono-mechanoluminescence spectrum evaluation of HOF@L012.

[0141] 2 mL HOF@L012 (2 mg / mL) were loaded into a cuvette and placed in the Fluorolog3 Fluorometer fluorescence spectrometer. The FUS transducer was positioned in contact with the cuvette. Subsequently, the solution was irradiated at a peak pressure of 1.55 MPa with a 10 s pulse. The emission was recorded using the fluorometer. The ChR2 absorption spectra were referenced from previous research and extracted using GetDataGraphDigitizer software. Blue light emission from the HOF@L012 nanoparticles.

[0142] To assess real-time sono-mechanoluminescence, light emissions were monitored using a video camera (CS165MU1 / M - Zelux® 1.6 MP Monochrome CMOS Camera, Thorlabs) in a dark environment. One milliliter of HOF@L012 (2 mg / mL) was loaded into a glass vial, which was then positioned atop an ultrasound transducer (1.5 MHz) with ultrasound gel filling. The video camera was placed in front of the glass vial to record the emissions. The solution was exposed to ultrasound stimulation with various pulse settings, including 1 Hz (50 ms pulse on, 950 ms off), 2 Hz (50 ms pulse on, 450 ms off), 4 Hz (50 ms pulse on, 200 ms off), 5 Hz (50 ms pulse on, 150 ms off), and 10 Hz (50 ms pulse on, 50 ms off), all at a peak pressure of 1.55 MPa. Alternatively, different peak pressures were tested with a 1-second pulse on and a 1-second pulse off or different pulse frequencies at a constant 1.55 MPa peak pressure. All parameters remained constant during video capture. To investigate the time delay between FUS stimulation and light emission, the LED indicator light for the FUS pulse and the mechanoluminescence light were simultaneously recorded. The time difference between these two emissions represented the latency. Data analysis was performed using ImageJ software. Ultrasound power deposition in the tissue.

[0143] Ultrasound energy propagates through tissues in the form of waves. To assess the efficiency of ultrasound energy transmission within the tissue, pork skin was used as a surrogate for normal tissue. Pork skin samples of varying depths were positioned on the ultrasound transducer, with an ultrasound hydrophone subsequently placed behind the pork skin and filled with ultrasound gel. The primary ultrasound power was controlled by adjusting the output power, and the peak pressure behind the pork skin was detected in real-time via the hydrophone. The ultrasound transmission efficiency within the tissue was calculated by dividing the measured peak pressure by the initial peak pressure. - 54 - 4905-0304-6915, v.1In vitro neuron activation via sono-mechanoluminescence.

[0144] Primary cortical neurons were isolated from mice in the experiments. In brief, pregnant C57BL / 6 mice were euthanized when the pups reached 15.5 days of gestation. Subsequently, the pups were carefully removed from the abdominal cavity and placed in a dissection medium. After removing the meninges, the cortex was collected, subjected to a 12- minute trypsin incubation at 37°C, washed three times with the dissection medium, and homogenized by pipetting. The resulting solution was filtered using a cell strainer. The cells were then diluted in a neuron culture medium consisting of a Neurobasal medium supplemented with B27, glutamine, and penicillin-streptomycin. Following cell counting, approximately 2 x 10 cells were seeded into each well of poly-l-ornithine-coated 24-well plates (Day In Vitro 0, DIV 0). The cells were placed in an incubator with 7.5% CO2, and a glial inhibitor was added on DIV 3. On DIV 4, 0.5 μL of pAAV-hSyn-hChR2(H134R)-EYFP (Addgene viral prep # 26973-AAV9; http: / / n2t.net / addgene:26973; RRID:Addgene_26973) and 0.5 μL of pAAV.Syn.NES-JRGECO1a.WPRE.SV40 (Addgene viral prep # 100854- AAV9; http: / / n2t.net / addgene:100854; RRID:Addgene_100854) were added to each well. After an additional 5 days of incubation, ChR2 opsins (green) and the JRGECO1a calcium indicator (red) were successfully expressed in the neurons, enabling in vitro calcium imaging. To activate the neurons, glass vials filled with 2 mL of HOF@L012 nanoparticles (5 mg / mL) were positioned over the cells. Ultrasound stimulation (1.5 MHz, 1.55 MPa, pulse 100 ms on 900 ms off) was applied to generate blue light for neuron activation. The red JRGECO1a fluorescence signal was recorded using a Leica DMi8 fluorescence microscope equipped with a 20X air objective and a 30 ms exposure time in the red channel (Ex: 548-573 nm). The transient increase in red fluorescence (ΔF / F) was calculated by extracting fluorescence time- series data from neurons through manual segmentation using ImageJ software. The raw data were further processed using a custom MATLAB algorithm that detrended and normalized the fluorescent time-series data through second-order polynomial curve fits and baseline maximum fluorescent value extraction, compensating for photobleaching effects. In vitro biosafety evaluation, including cell viability tests and hemolysis tests. In vitro cell viability.

[0145] Human Embryonic Kidney 293 (HEK-293T) cells were employed in the experiments. The 96-well plates were pre-incubated with a 10 μg / mL Poly-L-Ornithine solution before use. HEK-293T cells were seeded into the 96-well plates at a cell density of 10,000 cells per well in a complete DMEM medium. Subsequently, the cells were incubated - 55 - 4905-0304-6915, v.1overnight at 37°C in a 5% CO environment. After this incubation period, the culture medium was removed, and a fresh, complete medium containing HOF@L012 nanoparticles at various concentrations was added. The cells were then subjected to ultrasound treatment (1.5 MHz, 1.55 MPa, pulse 100 ms on, 900 ms off) for 20 s. Following this treatment, the cells were incubated for an additional 24 hours. After that, 10 μL of Cell-Titer Blue reagent (Promega Corporation) was added to each well, and the cells were incubated for another 4 h. Fluorescence intensity was measured using a Microplate reader (BioTek Synergy H1, 560ex / 590em nm), and cell viability was calculated according to the following formula: Cell viability (%) =Fluorescence intensity of sampleFluorescence intensity of control 100% Hemolysis tests of HOF@L012 nanoparticles.

[0146] 0.5 mL of fresh blood was collected from the mice's heart. The red blood cells (RBCs) were isolated by centrifugation at 8000 rpm for 5 minutes and subsequently washed with cold PBS at least three times. They were then suspended in 2 mL of PBS. Next, 1 mL of HOF@L012 nanoparticles at various concentrations was mixed with 20 μL of RBCs and incubated at 37°C for 2 hours. The resulting solution was then centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected for UV-Vis spectrum testing. The characteristic absorption peak at 541 nm was used to calculate the percentage of hemolysis. In addition, for control purposes, 20 μL of RBCs were added to 1 mL of distilled water to create the positive control group or 1 mL of PBS to create the negative control group. The hemolysis percentage was determined using the following formula: Hemolysis percentage (%) =UV-Vis absorbance at 541 nm of sampleUV-Vis absorbance at 541 nm of positive control 100% In vivo motor cortex modulation via sono-optogenetics.

[0147] The study was conducted using Thy1-ChR2-YFP transgenic mice (20-26 g; 4 weeks old; Jackson Laboratory). All procedures were designed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. They were approved by the Institutional Animal Care and Use Committee at the University of Texas at Austin (AUP- 2021-00086) and supported by the Animal Resources Center at the University of Texas at Austin. The mice were initially anesthetized with 2.5% isoflurane using an anesthesia machine from Vaporizer Sales & Service Inc. Their heads were securely fixed in a stereotaxic frame, and they were placed on a heating pad set to 37 ℃ to maintain body temperature. Ophthalmic ointment was applied to cover their eyes before surgery. Subsequently, their fur was shaved, - 56 - 4905-0304-6915, v.1and an FUS (Focused Ultrasound) transducer, filled with ultrasound gel, was positioned over the motor cortex. The coordinates of the transducer relative to bregma were as follows: anteroposterior (AP) 0.0 mm, mediolateral (ML) + 0.50 mm, and dorsoventral (DV) -0.5 mm. Next, 0.2 mL of HOF@L012 nanoparticles (10 mg / mL) in saline was injected through the tail vein. The concentration of isoflurane was then reduced to 0.5% to ensure that the mice were under light anesthesia before stimulation. To confirm the mice's anesthesia status, the mice's limbs were gently manipulated while checking for any movement. If there was a body movement response indicating light anesthesia, ultrasound pulses (1.5 MHz, 1.55 MPa, pulse 100 ms on, 900 ms off) were administered to activate the nanoparticles around the motor cortex, modulating limb motion. The mice's limb movements were recorded using a video camera. Subsequently, the limb motion data were analyzed using DeepLabCut, following the previously established method. Stereotaxic injection of the virus into the external globus pallidus (GPe) of rats.

[0148] All procedures were conducted in strict accordance with the guidelines outlined in the National Institute of Health Guide for the Care and Use of Laboratory Animals. The research protocols received approval from the Institutional Animal Care and Use Committee at the University of Texas at Austin (Approval ID: AUP-2021-00162) and were carried out with the support of the Animal Resources Center at the University of Texas at Austin. All surgical instruments were thoroughly sterilized prior to each procedure. Before injections, the fur on the surgical area was carefully shaved, and the skin on the head underwent a meticulous sterilization process involving three rounds of cleaning with 80% ethanol and iodophor. 1500 nL of AAV9 virus (pAAV-Ef1a-DIO hChR2(E123T / T159C)-EYFP, Addgene 35509) was injected into the GPe of Parvalbumin (PV)-Cre transgenic rats for expression of EF1a-driven, Cre-dependent, humanized channelrhodopsin E123T / T159C mutant fused to EYFP for further optogenetic activation. Virus injections were precisely administered using a micro-injection system (World Precision Instruments, UMP3 Microinjection Syringe Pump) at a rate of 300 nL / min. Following each injection, the needles were left in place inside the brain for a minimum of 5 minutes to facilitate efficient virus diffusion. They were then slowly withdrawn over a 5- minute period. Sutures were used to close the incision in the skin after the injections were completed. Following the surgical procedures, the animals were placed on a 37 ℃ heating pad and monitored in their cages until they had fully recovered. Transgenic Rat Breeding and Genotyping - 57 - 4905-0304-6915, v.1

[0149] At first, male Parvalbumin (PV)-Cre transgenic rats (the gift from the Loren Frank lab, UCSF) were bred with wild-type Long Evans female rats (Charles River Laboratories). Tissue samples from rat pups were collected using an ear-punching device and genotyped to identify PV-Cre transgenic animals (Transnetyx genotyping service), which were further used in experiments. Rat model for photometry tests.

[0150] PV-Cre rats (3-4 months old, Charles River) were used in the 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. 1500 nL pAAV-Ef1a-DIO hChR2(E123T / T159C)-EYFP and pAAV.Syn.NES-JRGECO1a.WPRE.SV40 mixture (1:1, v / v) was unilaterally injected into the external globus pallidus (GPe), with the coordinates relative to bregma: anteroposterior (AP) -0.9 mm, mediolateral (ML) +3.00 mm, and dorsoventral (DV) -5.30 mm. Parkinson's Disease Rat Model Creation

[0151] To induce Parkinson's disease in PV-Cre rats, a unilateral hemiparkinsonian rat model was created through stereotaxic injection of 6-hydroxydopamine (6-OHDA, Hello Bio, HB1889) into the medial forebrain bundle (MFB) region. Under isoflurane anesthesia, a unilateral incision was made, exposing the skull. A hole was drilled unilaterally, and 2 μl of 4 μg / μl 6-OHDA in 0.9% saline was injected into the MFB (-4.0 mm AP, +1.2 mm ML, -8.1 mm DV) using a microliter syringe with a 33G needle. The infusion rate was controlled by a digital micro-syringe pump at 0.25 μl / min. The micro-syringe was left in place for an additional 5-7 minutes to ensure proper diffusion of the solution. The hemiparkinsonian symptoms were observed in 2-3 weeks after the 6-OHDA injection, and the hemiparkinsonian model was confirmed through behavioral tests (cylinder test and apomorphine-induced rotation test) and tyrosine hydroxylase (TH) immunofluorescence staining in the rat's basal ganglia. Photometry tests to record the neuron activation under sono-optogenetics.

[0152] After a 4-week period following virus injection, the rats were utilized for photometry tests. Similar to the virus injection procedures, 2 μL of HOF@L012 nanoparticles at a concentration of 100 mg / mL were unilaterally injected into the GPe using coordinates of (-0.9 mm AP, +3.0 mm ML, -5.3 mm DV). Subsequently, optical fibers (200 μm core, R-FOC- BF200c-39NA, sourced from RWD Life Science) were implanted with corresponding coordinates in the GPe region. Following a recovery period of 2 days, the rat was restrained, - 58 - 4905-0304-6915, v.1and its head was immobilized. A FUS transducer (1.5 MHz, 2.45 MPa) was positioned above the head and filled with ultrasound gel. A pulse sequence (100 ms on, 100 ms off, with a duration of 10 seconds) was administered to irradiate the GPe area according to predefined FUS parameters. The resulting signal was recorded and analyzed using the R810 Dual Color Multichannel Fiber Photometry System from RWD Life Science. Behavior tests in PD rats under Sono-optogenetics

[0153] Cylinder and Apomorphine-induced rotation tests were performed in four rat groups: naive (n=5), hemiparkinsonian with PV-GP optogenetic stimulation (n=5), hemiparkinsonian with PV-GP sono-optogenetic stimulation (n=5), hemiparkinsonian without stimulation. Cylinder test during optogenetic and sono-optogenetic stimulations

[0154] A cylinder test is traditionally used to measure asymmetric forelimb use in hemi-parkinsonian rats, with the extent of asymmetry indicating the severity of the unilateral lesion induced by the 6-OHDA injection. Each rat was placed in a cylindrical environment (inner diameter: 20 cm, height: 30 cm) and allowed to behave spontaneously while a video camera was positioned directly above the cylinder. Rats were recorded for five minutes with no prior habituation to the cylinder, and the number of wall touches with each paw was analyzed from the resulting videos. The data were then analyzed to calculate contralateral touch percentages for each rat, represented as the number of contralateral touches over the sum of contralateral and ipsilateral touches, multiplied by 100.

[0155] Next, PV-GPe optogenetic stimulation during the cylinder test was studied. The LED Driver (Thorlabs, LEDD1B, and M405F1) was connected to the implanted fiber optic cannula using a patch cable. For optogenetic stimulation, various parameters were tested for motor recovery efficacy: frequencies of 5-20 Hz, pulse widths of 20-40 ms, power levels of 5- 7 mW, and stimulation periods of 30-60 seconds with a 30-180 second pause. The most effective parameters for the cylinder test were found to be 7 mW, 20 Hz, with either a 15 ms on / 35 ms off pattern or a 20 ms on / 30 ms off pattern, with a 30-second stimulation period and a 30-60 second interpulse interval, resulting in a total stimulation period of 7 minutes.

[0156] PV-GPe sono-optogenetic stimulation was next explored. Similar to the virus injection procedures, 2 μL of HOF@L012 nanoparticles at a concentration of 100 mg / mL was unilaterally injected into the GPe using coordinates of (-0.9 mm AP, +3.0 mm ML, -5.3 mm - 59 - 4905-0304-6915, v.1DV). Then, on the 2nd and 3rd days after nanoparticle injection, FUS stimulation (sono- optogenetics) of PV-GPe neurons (5-10 Hz frequency, 60-100 ms pulse width, 30-90 sec stimulation period) was performed. To achieve effective FUS stimulation while the rat was awake, the rat was securely placed in a medical-grade plastic bag with holes for the nose and the top of the head. This approach minimized stress for the rat and ensured proper positioning of the FUS transducer at the target stimulation site. After a 30-90 second FUS stimulation period, the rat was placed back into the cylinder to record wall touches. The contralateral touches percentages were calculated for each group as described above. Apomorphine-induced rotation test during optogenetic and sono-optogenetic stimulations

[0157] The apomorphine rotation test is commonly used to assess the extent of motor impairment induced by the lesion. Apomorphine, as a dopamine receptor agonist, acts post- synaptically and, due to the hyperstimulation of supersensitive dopamine receptors in the denervated striatum, induces rotation in the opposite contralateral direction. To induce rotation, 0.1 mg / kg of apomorphine dissolved in a sodium chloride solution was injected subcutaneously. After the injection, the rat was placed inside a glass cylinder (inner diameter: 25 cm, height: 50 cm). The rats typically began to rotate within about 5-10 minutes, reaching their maximum rotation rate in approximately 15 minutes. The effects of apomorphine lasted for 60-65 minutes (confirmed in the control hemiparkinsonian group). Once the maximum rotation rate was achieved (maintained for a constant 5-minute period), the baseline rate was recorded for 5 minutes. Afterward, optogenetic or sono-optogenetic stimulation was performed as described above. Optogenetic stimulation was conducted for 7 minutes, and the post- stimulation period was recorded for 15 minutes. For sono-optogenetics, video recordings were made for 20 minutes after stimulation. Angular speed (revolutions per minute) was calculated for each group. Additionally, video recordings were processed using DeepLabCut software to track rotations within different groups. C-fos staining in the mice / rat brain section after Sono-optogenetics.

[0158] The animals initially underwent sono-optogenetic treatment. After 60 minutes, the animals were anesthetized intraperitoneally using ketamine (16 mg / kg) and subsequently perfused with cold PBS, followed by 4% paraformaldehyde. The brains were carefully extracted and immersed in 4% paraformaldehyde at 4 ℃, where they were left to soak overnight. The brains were then sectioned into 60 μm thick slices using a vibrating blade microtome (Leica VT1200). These brain slices were rinsed with a 0.3% Triton-X PBS (TBS) - 60 - 4905-0304-6915, v.1solution and subsequently subjected to a 30-minute blocking step with a 5% bovine serum albumin TBS solution at room temperature. In the case of mouse brain sections, after the 30- minute blocking, the slices were incubated with a rabbit anti-c-Fos antibody (ab222699, Abcam, 1:500) in TBS. The samples were left to incubate at 4°C overnight and underwent three subsequent washes with TBS solution. Following this, a mixture of TBS and secondary antibodies (goat anti-rabbit Alexa Fluor 594, ab175652, Abcam, 1:500) was added, and the slices were incubated for 2 hours at room temperature in a dark environment. The slices were then washed three times with TBS, mounted on slides using mounting media (9990402, Fisher Scientific), and covered with coverslips. Confocal images were acquired using a Zeiss 710 laser scanning microscope.

[0159] For rat brain sections, the procedure was essentially the same, except for the choice of antibodies. After the initial 30-minute blocking step at room temperature using a 5% bovine serum albumin TBS solution, the samples were incubated with a rabbit anti-c-Fos antibody (ab289723, Abcam, 1:500) or a mouse anti-Parvalbumin antibody (P3088-100UL, Sigma-Aldrich, 1:1000) in TBS. Subsequently, a mixture of TBS and secondary antibodies (goat anti-rabbit Alexa Fluor 647, A32733, Fisher Scientific, 1:500) or (goat anti-mouse Alexa Fluor 594, A21125, Invitrogen, 1:1000) was applied, and the slices were again incubated for 2 hours at room temperature in a dark environment. After three washes with TBS, the slices were mounted on slides using mounting media (9990402, Fisher Scientific) and covered with coverslips for confocal imaging. Biosafety evaluation via immunostaining.

[0160] The rats initially underwent sono-optogenetic treatment. After 14 days, the rats were anesthetized intraperitoneally using ketamine (16 mg / kg) and subsequently perfused with cold PBS, followed by 4% paraformaldehyde. The brains were carefully extracted and immersed in 4% paraformaldehyde at 4 ℃, where they were left to soak overnight. The brains were then sectioned into 60 μm thick slices using a vibrating blade microtome (Leica VT1200). These brain slices were rinsed with a 0.3% Triton-X PBS (TBS) solution and subsequently subjected to a 30-minute blocking step with a 5% bovine serum albumin TBS solution at room temperature. After that, the samples were incubated with a rabbit anti-Iba1 antibody (013- 27691, FUJIFILM Wako Chemical, 1:1000) or rabbit anti-GFAP antibody (13-0300, Invitrogen, 1:1000) or rabbit anti-Cleaved Caspase-3 (9661, Cell signaling Technology, 1:1000) in TBS. The samples were left to incubate at 4°C overnight and underwent three - 61 - 4905-0304-6915, v.1subsequent washes with TBS solution. Following this, a mixture of TBS and secondary antibodies (goat anti-rabbit Alexa Fluor 594, ab175652, Abcam, 1:500) was added, and the slices were incubated for 2 hours at room temperature in a dark environment. The slices were then washed three times with TBS, mounted on slides using mounting media (9990402, Fisher Scientific), and covered with coverslips. Confocal images were acquired using a Zeiss 710 laser scanning microscope. * * *

[0161] 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. - 62 - 4905-0304-6915, 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. Ascherio, A. & Schwarzschild, M. A. The epidemiology of Parkinson’s disease: risk factors and prevention. Lancet Neurol.15, 1257–1272 (2016). Bloem, B. R., Okun, M. S. & Klein, C. Parkinson’s disease. Lancet 397, 2284–2303 (2021). Okun, M. S. Deep-Brain Stimulation for Parkinson’s Disease. N. Engl. J. Med.367, 1529– 1538 (2012). Volkmann, J. Deep brain stimulation for the treatment of Parkinson’s disease. J. Clin. Neurophysiol.21, 6–17 (2004). Gittis, A. H. & Yttri, E. A. Translating Insights From Optogenetics To Therapies For Parkinson’s Disease. 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Claims

WHAT IS CLAIMED IS:

1. A method of treating a brain disease in a mammalian subject comprising: (i) administering to the subject a hydrogen bonded organic framework (HOF) comprising: a chemiluminescent compound and / or a drug; and (ii) applying oscillating ultrasonic waves to the head or central nervous system of the mammalian subject, thereby releasing the chemiluminescent compound or drug from the HOF; wherein if the HOF comprises the chemiluminescent compound, then the brain of the mammalian subject comprises a neuron expressing a photosensitive protein, and wherein the emitted light modulates the photosensitive protein.

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

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

4. The method of claim 3, wherein A is an aromatic(C=6−16) ring.

5. The method of claim 4, wherein A is an aromatic(C=16)ring.

6. The method of claim 4, wherein A has the structure: - 66 - 4905-0304-6915, v.

1.

7. The e o of any one of claims 2-6, wherein B, B′, and B′′ are each independently substituted aryl(C≤12) or substituted heteroaryl(C≤12).

8. The method of claim 7, wherein B, B′, and B′′ are each independently an aryl(C=6)or heteroaryl(C=3−5) ring.

9. The method of claim 8, wherein B, B′, and B′′ are each independently , , or10. - 67 - 4905-0304-6915, v.

1.

11. 1-10, wherein B, B′, and B′′ have the same structure.

12. The method of claim 11, wherein B, B′, and B′′ are each . 13.according to any one of claims 1-12, wherein the HOF comprises a compound of the formula:wherein: R1, R2 and R3 are each independently −S(O)2Ra or −C(O)Ra, wherein: Ra is hydroxy or amino; R4-R20are each independently −H, −F, −Cl, −Br, −I or −CH3; or a pharmaceutically acceptable salt thereof. - 68 - 4905-0304-6915, v.

114. The method of claim 13, wherein R1, R2and R3are each independently −C(O)Ra, wherein Ra is hydroxy or amino.

15. The method of claim 14, wherein R1, R2 and R3 are -C(O)NH2.

16. The method of claim 14, wherein R1, R2 and R3 are -COOH.

17. The method of claim 16, wherein R4-R20are −H.

18. The method of claim 1, wherein the HOF comprises a compound of the formula:

19. The method of any one of claims 1-18, wherein the chemiluminescent compound emits light in response to free radicals or a reactive oxygen species (ROS).

20. The method of claim 19, wherein the reactive oxygen species (ROS) is singlet oxygen (1O2) or hydroxyl radical (•OH).

21. The method of any one of claims 19-20, wherein the chemiluminescent compound is L012, a dioxetane, luminol, isoluminol, an imidazopyrazinone, a lophine, or an acridinium.

22. The method of claim 7, wherein the chemiluminescent compound is L012.

23. The method of any one of claims 1-22, wherein the chemiluminescent compound or drug is encapsulated in the HOF.

24. The method of claim 23, wherein the HOF encapsulates from about 1-30 wt% of the drug or active pharmaceutical ingredient relative to the weight of the HOF.

25. The method of any one of claims 1-24, wherein the HOF is further defined as a nanoparticle, and wherein the drug or therapeutic is comprised within the nanoparticle. - 69 - 4905-0304-6915, v.

126. The method of claim 25, wherein the nanoparticle is about 50-900 nm in size.

27. The method of any one of claims 1-26, wherein the mammalian subject is a human.

28. The method of any one of claims 1-27, wherein the brain disease is Parkinson’s disease (PD), Alzheimer's disease, ataxia, Huntington's disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, essential tremor, epilepsy, stroke, or traumatic brain injury.

29. The method of claim 28, wherein the brain disease is Parkinson’s disease.

30. The method of any one of claims 1-29, wherein the oscillating ultrasonic waves comprise focused ultrasound (FUS).

31. The method of claim 30, wherein the focused ultrasound (FUS) comprises oscillating ultrasonic waves of 0.5-5 Mhz and / or a pulse of 5-30 seconds.

32. The method of claim 31, wherein the focused ultrasound (FUS) comprises oscillating ultrasonic waves of 0.5 – 20 MHz and / or a pulse of 10-1000 ms.

33. The method of claim 32, wherein the focused ultrasound (FUS) has a focus length of about 5-120 mm.

34. The method of any one of claims 1-33, wherein the HOF is comprised in or forms nanoparticles.

35. The method of claim 34, wherein the nanoparticles are about 100-1000 nm across or in diameter.

36. The method of claim 35, wherein the nanoparticles are about 200-800 nm across or in diameter.

37. The method of any one of claims 1-36, wherein the HOF further comprises a sonosensitizer.

38. The method of claim 37, wherein the sonosensitizer is IR-780, DCPH-P-Na(I), hematoporphyrin, zinc protoporphyrin, methylene blue, TiO2, Chlorin e6, indocyanine Green (ICG), hematoporphyrin derivative, or photofrin.

39. The method of any one of claims 1-38, wherein the HOF further comprises a fluorescent dye or imaging agent. - 70 - 4905-0304-6915, v.

140. The method of any one of claims 1-39, wherein the drug is a neuroactive drug, an anti-inflammatory drug, a NSAID drug, an analgesic, an anesthetic, a chemotherapeutic, or an anticancer drug.

41. The method of claim 40, wherein the neuroactive drug is a drug for the treatment of Parkinson’s disease (PD).

42. The method of claim 41, wherein the drug is selegiline, rasagiline, safinamide, 4- Dihydroxyphenylalanine（L-DOPA), apomorphine, rotigotine, amantadine, or entacapone.

43. A method of contacting a tissue of a subject with light, comprising: applying an ultrasound signal to a nanoparticle in proximity to the tissue of the subject, wherein the nanoparticle comprises the hydrogen bonded organic framework (HOF) of any one of claims 1-18 comprising: a chemiluminescent compound and / or a drug of any one of claims 19-42; and wherein the ultrasound signal causes the photosensitizer to induce the chemiluminescent to emit light that contacts the tissue.

44. The method of claim 43, wherein the subject is a mammalian subject, preferably a human.

45. A composition the hydrogen bonded organic framework (HOF) of any one of claims 1-18 comprising: a chemiluminescent compound and / or a drug of any one of claims 19-42 for use in the treatment of a brain disease.

46. The composition of claim 45, wherein the brain disease is Parkinson’s disease (PD), Alzheimer's disease, ataxia, Huntington's disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, essential tremor, epilepsy, stroke, or traumatic brain injury; preferably wherein the brain disease is Parkinson’s disease.

47. A pharmaceutical composition comprising a hydrogen bonded organic framework (HOF) comprising: a chemiluminescent compound and / or a drug; wherein the HOF comprises a compound of the formula: - 71 - 4905-0304-6915, v.1wherein: R1, R2and R3are each independently −S(O)2Raor −C(O)Ra, wherein: Rais hydroxy or amino; R4-R20are each independently −H, −F, −Cl, −Br, −I or −CH3; or a pharmaceutically acceptable salt thereof.

48. The pharmaceutical composition of either claim 47, wherein R1, R2and R3are each independently −C(O)Ra, wherein Ra is hydroxy or amino.

49. The pharmaceutical composition of claim 48, wherein R1, R2 and R3 are -C(O)NH2.

50. The pharmaceutical composition of claim 48, wherein R1, R2and R3are -COOH.

51. The pharmaceutical composition of claim 50, wherein R4-R20are −H.

52. The pharmaceutical composition of claim 47, wherein the HOF comprises a compound of the formula:- 72 - 4905-0304-6915, v.1or a pharmaceutically acceptable salt thereof.

53. The pharmaceutical composition of any one of claims 47-52, wherein the chemiluminescent compound emits light in response to free radicals or a reactive oxygen species (ROS).

54. The pharmaceutical composition of claim 53, wherein the reactive oxygen species (ROS) is singlet oxygen (1O2) or hydroxyl radical (•OH).

55. The pharmaceutical composition of any one of claims 53-54, wherein the chemiluminescent compound is L012, a dioxetane, luminol, isoluminol, an imidazopyrazinone, a lophine, or an acridinium.

56. The pharmaceutical composition of claim 55, wherein the chemiluminescent compound is L012.

57. The pharmaceutical composition of any one of claims 47-56, wherein the HOF is comprised in or forms nanoparticles.

58. The pharmaceutical composition of claim 57, wherein the nanoparticles are about 100-1000 nm across or in diameter.

59. The pharmaceutical composition of claim 58, wherein the nanoparticles are about 200-800 nm across or in diameter.

60. The pharmaceutical composition of any one of claims 47-56, wherein the HOF further comprises a sonosensitizer.

61. The pharmaceutical composition of claim 60, wherein the sonosensitizer is IR-780, DCPH-P-Na(I), hematoporphyrin, zinc protoporphyrin, methylene blue, TiO2, Chlorin e6, indocyanine Green (ICG), hematoporphyrin derivative, or photofrin.

62. The pharmaceutical composition of any one of claims 47-61, wherein the HOF further comprises a fluorescent dye or imaging agent.

63. The pharmaceutical composition of any one of claims 47-60, wherein the drug is a neuroactive drug, an anti-inflammatory drug, a NSAID drug, an analgesic, an anesthetic, a chemotherapeutic, or an anticancer drug.

64. The pharmaceutical composition of claim 63, wherein the neuroactive drug is a drug for the treatment of Parkinson’s disease (PD). - 73 - 4905-0304-6915, v.

165. The pharmaceutical composition of claim 64, wherein the drug is selegiline, rasagiline, safinamide, 4-dihydroxyphenylalanine（L-DOPA), apomorphine, rotigotine, amantadine, or entacapone.

66. The pharmaceutical composition of any one of claims 47-65, wherein the composition is formulated for parenteral administration or injection. - 74 - 4905-0304-6915, v.1

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