Differential acoustic impedance particles for ultrasonic drug uncaging

Abstract

Compositions and methods are provided for ultrasound-triggered drug release that enables high drug loading potential for varied drugs of interest, has minimal drug release without ultrasound, can be activated following intravenous administration, and utilizes an ultrasound-activation mechanism that is mechanically-based, without requiring heating above body temperature or cavitation, and is activated with a low duty cycle and shorter ultrasound protocol than is used for heat or sonodynamic activation. These parameters are accomplished by tuning the acoustic impedance and osmolarity of the liquid core of an acoustically activatable particle, e.g. liposomes or emulsions, to maximize ultrasound responsiveness while maintaining overall stability of the structure in the absence of ultrasound.

Description

DIFFERENTIAL ACOUSTIC IMPEDANCE PARTICLES FOR ULTRASONIC DRUG UNCAGING Cross-Reference to Related Applications

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No.63 / 539,504 filed September 20, 2023, the disclosure of which application is herein incorporated by reference. Government Support Research

[0002] This invention was made with Government support under contracts NS114438 and NS115637 awarded by the National Institutes of Health. The Government has certain rights in the invention. Background

[0003] Externally-triggered stimulus-responsive drug delivery systems promise targeted, on- demand drug delivery to regions of interest throughout the body. Ultrasound-gated drug delivery systems are of particular interest as they can leverage clinical therapeutic focused ultrasound systems, which can target sonication to millimeter-sized regions of interest throughout the body and are in active clinical use for a variety of applications worldwide. With ultrasonic drug uncaging using such systems, following intravenous infusion of ultrasound- sensitive drug-loaded nanocarriers, focused ultrasound is applied to a region of interest in the brain or body with a protocol sufficient to induce drug uncaging from the nanocarriers while they are circulating in the blood volume or while they are resident in the parenchyma of the region of interest. The freed drug is then able to enter the otherwise unperturbed tissue parenchyma, as it would normally do when given in an unencapsulated form.

[0004] Several drug-loaded nanocarriers have been described for enabling ultrasonic drug uncaging including perfluorocarbon-based systems that incorporate drug into the shell of gas- phase microbubbles or liquid-phase nanodroplets. For microbubble carriers, the micron-scale gas phase increases the risk of inertial cavitation with ultrasound application, which while may be effective for inducing drug release may also induce sonoporation or other forms of injury to the surrounding tissue, raising safety and practical clinical implementation. For liquid perfluorocarbon nanodroplets, vaporization to a gas phase is many times the proposed drug uncaging mechanism, which also then confers a risk of inertial cavitation. For both liquid and gas-based perfluorocarbon systems, since most drugs show negligible solubility in perfluorocarbons, the drug is loaded in the particle shell limiting the drug loading potential of the system and likely leading to nonspecific uncaging of the agent upon exposure to body temperature plasma via diffusion of the drug across the few nanometers thick particle shell.

[0005] A non-perfluorocarbon-based ultrasound-triggered delivery system is offered by heat- sensitive liposomes which have a differential melting behavior of some lipid shell components upon heating. In this system, ultrasound or radiofrequency irradiation is applied to heat the medium temperature, inducing drug uncaging from the liposome core. While effective, heating the tissue to the transition temperature of these liposomes may induce a local heat shock of the background tissue, which is not always desired. Further, maintaining a higher tissue temperature may present challenges in highly vascularized tissue or in organs that move with cardiorespiratory motion, and requires high intensity ultrasound or radiofrequency application, with high actuating device performance requirements.

[0006] Alternatively, a different liposomal system incorporates a porphyrin or related molecule, producing reactive oxygen species upon sonodynamic ultrasound application, inducing encapsulated therapeutic leakage. While this does not necessarily require or risk inertial cavitation or heating, this system requires continuous ultrasound application for several minutes, providing similar technical constraints as with heat-activated liposomes. Another recently-described alternative liposomal system for ultrasound-triggered uncaging uses supercritical carbon dioxide encapsulated into a liposome, with bubble generation with ultrasound application, which also raises risks of inertial cavitation and system instability, as with microbubbles.

[0007] Alternative externally-triggered stimulus-responsive drug delivery systems that are activated at physiologically safe levels of ultrasound radiation, using generally recognized as safe components, are of great interest for clinical purposes. The present disclosure addresses this need. SUMMARY

[0008] Compositions and methods are provided for ultrasound-triggered drug release that enables high drug loading potential for varied drugs of interest, has minimal drug release without ultrasound, can be activated following intravenous administration, and utilizes an ultrasound-activation mechanism that is mechanically-based, without requiring heating above body temperature or cavitation, and is activated with a low duty cycle and shorter ultrasound protocol than is used for heat or sonodynamic activation. These parameters are accomplished by tuning the acoustic impedance and osmolarity of the liquid core of an acoustomechanically activatable particle, e.g. liposomes or emulsions, to maximize ultrasound responsiveness while maintaining overall stability of the structure in the absence of ultrasound. In some embodiments the compositions utilize generally recognized as safe (GRAS) materials. In some embodiments effective ultrasound uncaging is performed with ultrasound intensities within FDA guidelines for safe diagnostic ultrasound application.

[0009] The acoustically activatable particles (AAP) are vesicles comprising a polymer or lipid coated emulsion (single layer shell) or polymersome / liposome (double layer shell) where the core material has an acoustic impedance that differs from the acoustic impedance of the physiologic media that would surround the formulation droplets after parenteral administration. Physiologic media of interest include, for example, plasma, cerebrospinal fluid, synovial fluid, etc. In some embodiments the AAP is a liposome, referred to herein as an AAL. A drug substance of interest is loaded into the core and / or shell of the particle. Upon ultrasound irradiation, the leakiness of the formulation droplets to the drug substance is increased, allowing elution of the drug substance into the medium. The core material may be aqueous or oily, and is typically free of perfluorocarbons. The drug substance of interest may be a small molecule, or larger molecules such as peptides, proteins, or nucleic acids.

[0010] AAP of the disclosure are useful for imaging, oxygen delivery for ischemia, micro- embolization, thermal ablation, blood-brain barrier opening, and as drug delivery vehicles, such as for delivery of chemotherapeutics, psychotropic compounds, and / or anesthetics. AAP allow encapsulation of various small molecules. The AAP meet the demands of clinical manufacturing methods, as they are practically feasible, scalable, compatible with current good manufacturing practices (cGMP), and produce particles that are sufficiently stable for a variety of uses and for storage.

[0011] In some embodiments, the core material of an AAP is an aqueous solution comprising a GRAS solute, which solute causes the solution to have acoustic impedance that differs from the acoustic impedance of the surrounding physiologic fluid, e.g. plasma, CSF, etc. Solutes of interest include, without limitation, sugars, e.g. monosaccharide, disaccharides, polysaccharides, etc.; polymers, dendrimers, and other viscous agents that shift the viscosity and acoustic impedance of the medium to be different from that of plasma. Monosaccharides of interest include, for example, glucose, galactose, fructose, hexose, pentose, ribose, mannose, xylose, deoxyribose, glyceraldehyde, arabinose, tagatose, altrose, etc. Disaccharides of interest include, for example, sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, kojibiose, nigerose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, trehalulose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, mannobiose, melibiose, allolactose, melibiulose, lactulose, rutinose, rutinulose, xylobiose, etc. Polysaccharides of interest include, for example, starch, glycogen, cellulose, heparin, and peptidoglycan, chitin, hyaluronic acid, amylopectin, amylose, etc. Polymers of interest include, for example, poly-lactate, poly-glycolate, poly-lacto-co-glycolic acid (PLGA), poly-ethyleneglycol (PEG), polyvinyl alocohol (PVA), etc.

[0012] Acoustic parameters that yield ultrasound induced release have been shown to be effective at 250 kHz sonication, with a dose-response with duty cycle and the appliedultrasound intensity / pressure. Embodiments may be tuned to allow activation with from about 50 to about 1000 kHz ultrasound, 1-50% duty cycle, and mechanical indices of 0.2-5.0.

[0013] In some embodiments, the core material of an AAP is an aqueous saccharide solution, where the acoustic impedance differs from the acoustic impedance of plasma by about 0.02 MPa*s / m or more. In certain embodiments the core material is an aqueous sucrose solution, where sucrose is present at a concentration of from about 2% to about 10%, e.g. at least about 2%, at least about 3%, at least about 4%, and not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%. In some embodiments, a solution of from about 4% to 6%, e.g. about 5% sucrose is used. The level of free (non-encapsulated) drug in a targeted tissue after exposure to ultrasound (US) may be from about 1.25X the level in the absence of US, from about 1.5X, from about 1.75X, from about 2X, from about 2.5X, or more. In some embodiments the increase is from about 1.75X to about 2.5X the level in the absence of US.

[0014] In some embodiments, the level of ultrasound required for uncaging the drug of interest from an AAP is from about 50 to about 1000 kHz ultrasound, from about 100 to about 500 kHz, e.g. from about 200 kHz, from about 250 kHz, up to about 500 kHz, up to about 400 kHz, up to about 300 kHz, and may be from about 200 to 300 kHz, for a period of time of from about 1 to about 5 minutes, e.g. for about 1, 2, 3, 4, 5, minutes, with a duty cycle between 1-50%, and a pressure that yields a mechanical index between 0.2-5.0.

[0015] In some embodiments a formulation is provided, comprising an AAP of the disclosure, wherein the particle core comprises a drug substance of interest in a solution where the acoustic impedance differs from the acoustic impedance of plasma; and a pharmaceutically acceptable carrier. In some embodiments the formulation is provided in a unit dose, where the dose comprises an effective amount of the AAP for targeted drug delivery.

[0016] In some embodiments, a method is provided for ultrasound-triggered drug release to a tissue of interest, the method comprising administering an individual in need thereof an effective dose of a drug of interest encapsulated in AAP, and subjecting the targeted tissue to a dose of ultrasound effective to uncage the drug. In some embodiments the targeted tissue is the brain. In some embodiments, including targeting to the brain, administration of the AAP is parenteral. In some embodiments the targeted tissue is solid organs of the periphery; the spinal cord; peripheral nervous system; etc.

[0017] In some embodiments a method is provided for ultrasound-triggered drug release to the brain of an individual, the method comprising parenteral administration of an effective dose of a drug of interest encapsulated in an AAP to an individual in need thereof, and subjecting the targeted brain tissue to a dose of ultrasound effective to uncage the drug. The dose of ultrasound is preferably at a level that is sufficiently low as to avoid parenchymal damage. For example, the frequency of ultrasound may be from about 200 to 300 kHz, for a period of timeof from about 1 to about 5 minutes. The AAP may comprise liposomes, where the core comprises a sucrose solution of from about 2.5% to about 7.5%. The drug of interest may be a neurally active therapeutic agent, e.g. propofol, ketamine, nicardipine, verapamil, dexmedetomidine, modafinil, doxorubicin, temozolomide, rivastigmine, galantamine, donepezil, bupivacaine, ropivacaine, lidocaine, clonidine, xylazine, dexmedetomidine, cocaine, amphetamine, methamphetamine, ibogaine, psilocin, psilocybin, methyl-d- methamphetamine, lysergic acid, cisplatin, tacrolimus, p-aminophenyl-α-D-mannoside, levodopa, anti-NRI, anti-ICAM1, anti-HSP72, anti-actin, anti-VEGF; GDNF, NGF, H102 peptide, TF peptide, α-Mangostin, matrix metalloproteinase-9, superoxide dismutase, VEGF, transferrin, tissue plasminogen activator, or an oligonucleotide or polynucleotide. In some embodiments, the therapeutic agent is a drug with logP >1. In some embodiments, the therapeutic agent is a drug with logP >0. In some embodiments, the therapeutic agent is an anesthetic. In some embodiments, the therapeutic and / or contrast agent is a vasodilator. In some embodiments, the composition comprises an imaging agent, contrast agent, and / or dye, optionally in combination with the therapeutic agent.

[0018] In some aspects, provided herein is a method of treating or ameliorating a neurological disease or disorder selected from Alzheimer's Disease, epilepsy, tremors, seizures, CNS cancers and tumors (gliomas, glioblastoma multiforme (GBM), medulloblastoma, astrocytoma, diffuse intrinsic pontine glioma (DIPG)), pain, and psychiatric diseases (e.g., PTSD, anxiety disorder, depression, bipolar disease, suicidality, substance use disorder, schizophrenia, chronic pain), wherein an AAP composition comprising a neurologically active therapeutic agent, as described herein, is administered intravenously or into the cerebrospinal fluid (CSF) of a subject and an ultrasound pulse is subsequently delivered to the brain or brain vasculature of the subject with an intensity sufficient to yield particle activation, at a time when the AAP are present in the brain or brain vasculature. In some embodiments, the AAP is administered intravenously or intraparenchymally in the central nervous system, a peripheral limb, or a solid organ after which ultrasound is applied to the region of interest.

[0019] In some embodiments, the composition or method described herein is used in combination with one or more methods of imaging (e.g. fMRI or PET), measuring electrophysiology (e.g. EEG), and / or behavioral assessment of brain function, following focal drug release. In some embodiments, the methods disclosed herein for targeted drug release can be combined with methods of imaging (e.g. fMRI), methods of measuring electrophysiology (e.g. EEG), or methods of behavioral assessment of brain function, following focal drug release.

[0020] These and other objects, advantages, and features of the disclosure will become apparent to those persons skilled in the art upon reading the details of the compositions and methods as more fully described below. BRIEFDESCRIPTION OF THEFIGURES

[0021] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0022] FIGS.1A-1I. Acoustically activatable liposomes (AALs) for on-demand drug uncaging. a-b Hypothesized scheme for AAL-mediated ultrasonic drug uncaging in which drug-loaded ultrasound-sensitive nanocarriers release their cargo upon ultrasound activation while they are circulating in the blood pool. Sucrose incorporated into the internal medium of liposomes alters the acoustic characteristics of the internal buffer, increasing interaction with applied ultrasound, which increases drug permeability across the lipid membrane. c In vitro ketamine uncaging from liposomes with 0% (KL), 5% (S5-KL), or 10% (S10-KL) added sucrose in the liposome internal medium. Sonicated (250 kHz center frequency, 1.7 MPa estimated in situ peak negative pressure at room temperature, 25% duty cycle, PRF 5 Hz, 60 s) samples with more sucrose internally uncaged more drug. d Spontaneous ketamine release from KL, S5- KL, S10-KL with 1 hour incubation at 37 ºC in plasma and buffer shows different stability characteristics, with 10% additional sucrose showing reduced plasma stability at 37 ºC. e-f In vitro ketamine uncaging from liposomes with internal buffers composed with equiosmolar additional sodium chloride (NaCl5-KL), glucose (Glu5-KL), or sucrose (S5-KL) yields differing amounts of drug uncaging with the same ultrasound protocol, in correlation to their differences in internal acoustic impedances, confirming that differential acoustic characteristics contribute most to AAL ultrasound responsiveness rather than other physicochemical characteristics; r2=0.97, p = 0.002. g Ultrasonic ketamine uncaging from S5-KL with varying peak negative pressure / mechanical index (MI; 250 kHz, 25% duty cycle, PRF 5 Hz, 60 s) at room temperature (RT) or 37 ºC. h Stability of S5-KL for both drug retention while exposed to plasma and for ultrasonic uncaging across 90 days in 4 ºC storage. No change in size / PDI by DLS was observed in these experiments; a representative batch showed size / PDI of 165.8 nm / 0.061 at day 1 post production and 167.7 nm / 0.074 at day 90 post production. i Representative transmission electron microscopy images show spherical shape of the KL and S5-KL liposomes with primarily unilamellar vesicles and occasional, <5% multilamellar vesicles, and that the size distribution of the particles decreased with sonication. No gas elements or voids within the AALs were noted with or without sonication. S5-KL was selectedfor future experiments and referred to as ‘SonoKet.’ Data presented as mean ± SD of three or more independent experiments. Comparisons between two groups was performed by two- tailed, two-sample Student's t-test, and that among multiple groups by one way analysis of variance (ANOVA); ns = non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001.

[0023] FIGS. 2A-2F. In vivo drug delivery performance of SonoKet (S5-KL) mediated ketamine uncaging. Top: While under anesthesia, rats received an i.v. infusion of 1.5 mg / kg SonoKet (n=3) or dose-matched ketamine HCl (n=3) for 5 min, with sonication started at t=2.5 min and continued for 2.5 min. Separate rats also received SonoKet with no FUS applied to either region (n=5). Bottom: Simulated (Fig.5) ultrasound beamplots overlaid onto the mPFC or RsC sonication target (schematics adapted using Paxinos atlas sections) with SPME sampling targets indicated by asterisks (white asterisk = sonication on, black asterisk = no sonication); sites are 7 mm apart. Immediately after treatment, ketamine and its metabolites were repeatedly sampled via new SPME pins every 5 minutes up to 25 minutes post-infusion start. b Amount of ketamine extracted at each brain target from 0-5 min post-sonication. Significantly higher ketamine was extracted from both the mPFC and the RsC when sonication was applied with SonoKet versus Ketamine-HCl or versus Sonoket without sonication. mPFC: KetHCl vs SonoKet (p=0.0028, Hedge’s g = 3.66); KetHCl+FUS vs SonoKet+FUS (p=0.0041, Hedge’s g = 3.24); SonoKet vs SonoKet+FUS (p=7e-6, Hedge’s g = 7.31). RsC: KetHCl vs SonoKet (p=0.0016, Hedge’s g = 45.46); KetHCl+FUS vs SonoKet+FUS (p=0.0018, Hedge’s g = 2.69); SonoKet vs SonoKet+FUS (p=2.99e-5, Hedge’s g = 5.37). Tukey’s post-hoc HSD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. c Ketamine and norketamine levels at each brain sampling site were quantified for rats receiving Ketamine HCl. There was no significant difference in the pharmacodistribution or pharmacokinetics of either compound when sonication was applied to either the mPFC or RsC. Hydroxynorketamine brain levels were below the limit of detection. d Ketamine and norketamine brain levels in rats who received SonoKet. Sonication applied to the mPFC (top left; 5-min: p=0.0043, 10-min: p=0.0057) or to the RsC (middle left; 5-min: p=0.010, 10-min: p=0.022) resulted in higher ketamine levels compared to the non-sonicated regions. Pharmacokinetics cleared at the same rate in both regions at 15 minutes and beyond. Hydroxynorketamine brain levels were below the limit of detection. Paired t-tests, *p < 0.05, **p < 0.01. e Blood clearance pharmacokinetics of ketamine, norketamine, and hydroxynorketamine metabolites extracted every 5 min post- treatment; pharmacokinetic modeling results in Table 3. Data presented as mean ± SD. f Histological and immunohistochemical analyses of brain tissue 24 h after sonication with SonoKet show no indication of parenchymal damage in comparison to healthy saline- administered controls at both the mPFC (top) and RsC (bottom). Left: Representative magnified images (20x) and quantification of GFAP immunostaining shows no indication of gliosis or change in astrocytic activation (GFAP positive cells, green). Middle: Representativemagnified images (20x) and quantification of IBA-1 immunostaining indicate no change in microglial activation (IBA-1 positive cells, purple). Right: Representative magnified images (10x) and quantification of Fluoro-Jade C staining shows no change in neuronal degeneration (FJ-C positive cells, green). Not pictured: Gross examination of hematoxylin and eosin (H&E) stained slices demonstrated no tissue damage. Two-tailed unpaired t-test; no significance, n = 3 rats / group. Data are presented as mean ± SD.

[0024] FIGS.3A-3D. Unique electrophysiologic signatures of regional ketamine uncaging. a Left: Recording electrode schematic for each site. Right: Recording completed in fully awake rats undergoing acute restraint stress. Following a 5-min baseline, an infusion was administered i.v. at t=5-10 min and FUS (250 kHz; 1.1 MPa est. in situ peak neg. pressure; 50 ms / 5 Hz PRF / 2.5 min) was applied at t=2.5-5 min. Recording continued for another 35 minutes after treatment. b Spectrograms depicting electrophysiological response to saline with ultrasound, ketamine HCl (0.75 mg / kg; repeated from dose-response evaluation Fig. 8), SonoKet (0.75 mg / kg) without ultrasound, and SonoKet with ultrasound applied to and with recording from the mPFC (top row; n=4-5) or RsC (bottom row; n=5-6). Power spectral density percent change from the baseline (0-5 mins) were calculated. c Average percent change in power spectral density from baseline versus frequency for each of the three indicated time periods for each recording site and treatment: time of sonication (7.5-10 min), time immediately after treatment (10-25 min), and in a delayed period (25-45 min). d Power spectral density percent change from baseline area under the curve for each spectral band: theta (4-8 Hz), alpha (8-15 Hz), beta (15-25 Hz), and gamma (25-55 Hz). Percent change was calculated for each of the three indicated time periods. Data presented as box plots. Tukey’s post hoc HSD, *p<0.05, **p < 0.01.

[0025] FIGS. 4A-4G. Regional ketamine uncaging selectively activates affective behavioral efficacy. a Rats undergoing awake restraint stress were administered an i.v. infusion of SonoKet (0.75 mg / kg), dose-matched ketamine HCl, or saline for 5 min, with sonication applied to either the mPFC or RsC at t=2.5 min for 2.5 min. Rats were then released and placed into an open-field test box and filmed from above in a well-lit room. b Tracks for all rats overlaid for each group, separated by treatment type and sonicated region. All treatment groups received ultrasound at either the mPFC (top) or RsC (bottom).c Total distance traveled within each treatment group averaged across subjects in 1-min time bins. d Cumulative distance traveled averaged over treatment group per region. Data presented as box plots. Tukey’s post hoc HSD test showed no significance; n = 6 rats / group. d Average speed over the first 5 min of recording for each treatment group averaged across subjects. e Average speed for each treatment group per region for the first 5 min of recording. Data presented as box plots. Tukey’s post hoc HSD test showed no significance; n = 6 rats / group. f Percentage of time spent in the center versus edge of the field for each treatment averaged acrosssubjects in 1-min time bins. g Cumulative percentage of time spent in the center versus edge of the field averaged over treatment group per region. mPFC: saline vs SonoKet (p=0.037, Hedges g = 1.24); KetHCl vs SonoKet (p=0.028, Hedges g = 1.35). Data presented as box plots. Tukey’s post hoc HSD Test; *p < 0.05; n = 6 rats / group.

[0026] FIGS.5A-5B. Simulations of focused ultrasound that was applied in the reported rat experiments with skull attenuation estimated using micro-CT and the same transducer frequency, aperture, and f#. a. Visualization of beamform simulations overlaid on the rat skull. b. Simulated pressure fields along the center of the field with and without skull across intact rat skull with the top of the skull and the bottom of the skull indicated.

[0027] FIGS. 6A-6D. In vivo ultrasonic dose response relationship of AAL-mediated drug uncaging a. Experimental schematic (top) and timeline (bottom). Sonication was applied to a frontal cortical target with SPME sampling completed at the center of the intracranial ultrasound field (white asterisk) or the non-sonicated contralateral (Contra) target (black asterisk). b, c. Dose-response of ketamine delivery following AAL uncaging with ultrasound duty cycle (b) or AAL dose (c). d. Increased blood concentration with liposome-encapsulated ketamine versus unencapsulated ketamine. e. Increased drug delivery in vivo with sucrose incorporated into the liposomes vehicles (S5-KL) versus no sucrose incorporation (S0-KL). f. Simulated pressure fields with and without skull across intact rat skull for this transducer and sonication target with the top of the skull and the bottom of the skull indicated as in Fig.5.

[0028] FIGS.7A-7C. In vivo pharmacokinetics and biodistribution of SonoKet (S5-KL) versus dose-matched free ketamine following intravenous bolus administration. a. Experimental timeline (top) and blood concentration of ketamine and its major metabolites following bolus i.v. administration of 1 mg / kg of unencapsulated ketamine (Ketamine HCl) or SonoKet (S5- KL). b, c. Biodistribution analysis of solid organ uptake of ketamine, norketamine, and hydroxyketamine following 1.5 mg / kg i.v. bolus administration of unencapsulated ketamine (Ketamine HCl) or SonoKet (S5-KL).

[0029] FIGS.8A-8C. Rat brain electrophysiological dose-response analyses of Ketamine HCl i.v. infusion. a. Non-normalized time-frequency spectrograms of unencapsulated ketamine dose response recording from mPFC (top row) and RsC (bottom row) following 0.5, 0.75, 1, and 1.5 mg / kg i.v. infusion. b. Spectrograms of power percent change from the baseline (0-5 minutes of the recording). c. Percent power change from baseline (0-5 min) was plotted versus frequency (Hz). Percent change was averaged within time bins during sonication (7.5-10 min), immediately after treatment (10-25 min), and after clearance time point (25-45 min).

[0030] FIG. 9. Ketamine HCl power percent change from baseline. Quantification of area under the curve for Fig.8 data, as in Fig.3d calculated for change within delta (1-4 Hz), theta (4-8 Hz), alpha (8-15 Hz), beta (15-25 Hz), and gamma (25-55 Hz) bands. Percent change was averaged within three time bins: time of sonication (7.5-10 min), time immediately aftertreatment (10-25 min), and from 25 min after treatment until the end of the recording (25-45 min).

[0031] FIGS.10A-10B. Non-normalized power spectrograms and delta band quantificaitons for rat electrophysiology experiments in Fig 3. a. Non-normalized time-frequency spectrograms of ketamine dose response recording from mPFC (top row) and RsC (bottom row) following SonoKet 0.75 mg / kg infusion with and without sonication, dose-matched ketamine HCl (KetHCl) or saline (Sal). b. Area under the curve (AUC) was calculated for percent change within the delta band (1-4 Hz) in mPFC (left column) and RsC (right column). AUC was averaged within three time bins: time of sonication (7.5-10 min), time immediately after treatment (10-25 min), and 25 minutes after treatment until the end of the recording (25- 45 min).

[0032] FIG. 11. Comparisons of percent change of power spectral density between mPFC and RsC in Fig 3. Area under the curve (AUC) for percent change within each band comparing recordings from mPFC (gray) and RsC (black). AUC was averaged within three time bins: time of sonication (7.5-10 min), time immediately after treatment (10-25 min), and 25 minutes after treatment until the end of the recording (25-45 min).

[0033] FIGS. 12A-12B. No change in animal speed during open field testing (Fig. 4) with treatment following acute restraint stress. a. Group average speed of animals in initial 1 or 2 minutes of the open field test. b. Time-resolved speed across each group for 0-1 min and 0-2 min.

[0034] FIG. 13. No change in distribution of animal speed during open field testing (Fig.4) with treatment following acute restraint stress. Histogram of speed distribution for each group over initial (a) 1, (b) 2, or (c) 5 min of the open field test.

[0035] Fig. 14A-E. Altering the acoustic impedance of the internal medium of liposomes enhances their ultrasound sensitivity for on-demand drug uncaging. (A) Hypothesized scheme of acoustomechanically activatable liposomes (AAL) and proposed mechanism for ultrasonic drug uncaging. Sucrose incorporated into the internal medium of liposomes increases both the acoustic impedance and the osmolarity of the internal buffer. Ultrasound mechanically affects the intravenously injected AAL, due to the differential acoustic impedance (ΔZ) of the liposome internal medium, increasing drug permeability across the lipid membrane. Then, the difference in osmotic pressure (ΔΠ) across the lipid membrane accelerates the rate of water entry into the liposome and drug uncaging into the blood, after which the drug diffuses into the tissue that received sufficient ultrasound intensity. (B) In vitro ketamine uncaging from liposomes with 0% (KL), 5% (S5-KL), or 10% (S10-KL) added sucrose in the liposome internal medium. Sonicated (250 kHz center frequency, 1.7 MPa peak negative pressure at room temperature, 25% duty cycle, PRF 5 Hz, 60 s) samples with more sucrose internally uncaged more drug. (C, D) In vitro ketamine uncaging from liposomes with internal buffers composedof ammonium sulfate with equiosmolar additional sodium chloride (NaCl5-KL), glucose (Glu5- KL), or sucrose (S5-KL) yields differing amounts of drug uncaging with the same ultrasound protocol, in correlation to their differences in internal acoustic impedances; r2=0.97, p = 0.002. (E) Spontaneous ketamine uncaging from KL, S5-KL, S10-KL with 1 hour incubation at 37 ºC in plasma and buffer shows different stability characteristics, with 10% additional sucrose showing reduced plasma stability at 37 ºC. Data presented as mean ± SD of three or more independent experiments. Comparisons between two groups was performed by two-tailed Student's t-test, and that among multiple groups by one way analysis of variance (ANOVA); ns = non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001.

[0036] Fig. 15A-H. Investigating the mechanism of ultrasonic drug uncaging from AALs. Ultrasonic ketamine uncaging from S5-KL with varying peak negative pressure / mechanical index (MI; 25% duty cycle, PRF 5 Hz, 60 s) at room temperature (RT) or 37 ºC with (A) 250 kHz or (D) 650 kHz center frequency. Increased ketamine uncaging observed at 250 kHz across different pressures / MIs and temperatures compared to 650 kHz. (B, C, E, F) Magnitude of the received echo spectrum following ultrasound applied to gas-filled microbubbles (black) or S5-KL (red), with the corresponding spectrum from saline sonication subtracted, following sonication with 37 ºC medium temperature with a center frequency of 250 kHz (B, C) or 650 kHz (E, F), 1.8 MI, and 25% duty cycle. Spectra and area under the curve (AUC) quantification (with corresponding spectra / AUC from saline sonication subtracted) indicate wideband acoustic scatter from microbubbles, indicative of inertial cavitation, in all conditions. With S5- KL, differences in harmonic (H) and ultraharmonic (UH) but not subharmonic (SH) bands nor wideband scatter (a pattern indicating particle oscillation) were observed with 250 kHz sonication at 37 ºC, but with no response at these bands with 650 kHz sonication nor with 250 kHz sonication at RT (Fig. S3). Statistical significance testing completed by comparing the non-subtracted AUCs for each of microbubble and of S5-KL sonication with the corresponding AUCs from saline sonication. All microbubble comparisons were statistically significantly different. (G) Representative Cryo-EM images show spherical shape of the S5-KL liposomes with primarily unilamellar vesicles and occasional, <5% multilamellar vesicles, and that the size distribution of the particles decreased with sonication. (H) Stability of S5-KL for both drug retention while exposed to plasma and for ultrasonic uncaging across 90 days in 4 ºC storage. No change in size / PDI by DLS was observed in these experiments; a representative batch showed size / PDI of 165.8 nm / 0.061 at day 1 post production and 167.7 nm / 0.074 at day 90 post production. Data presented as mean ± SD of three or more independent experiments. Comparisons between two groups were performed by two-tailed Student's t-tests; ns = non- significant, *p < 0.05, **p < 0.01 and ***p < 0.001.

[0037] Fig.16A-B. AALs are a platform for ultrasonic uncaging of a variety of drugs. (A) To confirm the generalizability of the concept, several agents – ketamine, ropivacaine,bupivacaine and lidocaine – were loaded into the liposome with or without 5% additional sucrose internally. Each drug loaded formulation shows higher in vitro ultrasonic drug uncaging with sucrose incorporated versus the no sucrose control formulations. Data presented as mean ± SD of three independent experiments. Comparisons between two groups were performed by two-tailed Student's t-test; ns = non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001. (B) Cryo-EM images of each formulation confirm their spherical morphology, without a specific morphologic change with sucrose incorporation, with primarily unilamellar vesicles and occasional, <5% multilamellar vesicles. Ketamine data partly repeated from Figs.14 and 15.

[0038] Fig. 17A-B. In vivo AAL pharmacokinetics and biodistribution. (A) Ketamine quantification in blood samples collected at different timepoints after 1 mg / kg body weight intravenous bolus injection of free, unencapsulated ketamine-HCl (FK) or S5-KL (n=4 adult rats each) shows differental blood clearance with S5-KL compared to FK for ketamine itself and its major metabolites norketamine and hydroxynorketamine (Quantification in Table 11). (B) Ketamine quantification in different organs after 60 min following 1.5 mg / kg body weight intravenous bolus injection of FK or S5-KL shows higher solid organ drug accumulation with ketamine-HCl (FK) compared to the ketamine-loaded AAL (S5-KL; n=4 adult rats each). Data presented as mean ± SD. Comparisons between multiple groups performed by one way analysis of variance (ANOVA), ns = non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001.

[0039] Fig.18A-J. In vivo uncaging of ketamine-loaded AALs. (A) Schematic and timeline for the in vivo pharmacodistribution study. Ultrasound (250 kHz, 0.9 MPa est. peak in situ neg. pressure at the sampling site center (white asterisk), 5-25% duty cycle, 5 Hz PRF; or sham) was applied to a frontal cortical / hippocampal brain region unilaterally following a bolus or during the latter 2.5 min of 5 min intravenous infusion of 0.625-1.5 mg / kg body weight of either free unencapsulated ketamine-HCl (FK), or ketamine liposomes with 0% (KL), 5% (S5-KL), or 10% (S10-KL) added sucrose in the internal buffer. Sonication beam map constructed based on hydrophone beam mapping of the transducer with intensity scaled according to the applied intensity derated for rat skull insertion loss. After sonication, solid phase microextraction (SPME) was used to sample both the sonicated brain (white asterisk) and a contralateral control region (Contra; black asterisk) separated 5 mm apart. A venous blood sample was taken at the end of the SPME sampling period. (B) Ketamine extracted from the sonicated (red) or contralateral control region (Contra; grey) region when varying the ultrasound protocol duty cycle and keeping the AAL dose constant showed increasing ketamine delivery to the brain with increasing ultrasound duty cycle. (C) Ketamine extracted from the sonicated (red) or contralateral control region (Contra; grey) region, or from sham treated animals when varying the total AAL dose and keeping the ultrasound protocol constant showed increasing ketamine delivery to the brain with increasing AAL dose. (D) Ketamine quantification in bloodshowed higher levels of drug with the liposomal formulations (KL, S5-KL, & S10-KL) compared to the free drug (FK) infusion. (E) Ketamine quantification following free drug infusion shows no differential ketamine delivery with or without ultrasound application. With each liposomal formulation infusion, ultrasound application significantly increased the drug concentration in the sonicated brain region compared to the contralateral or sham controls, with increased uncaging seen with sucrose incorporation. (F) Schematic representation of the rat brain depicting the region of ultrasound sonication and histological sampling for safety analysis. Histological and immunohistochemical analyses of brain tissue 72 hrs after sonication with AALs (n=3 / group) show no indication of parenchymal damage in comparison to healthy controls. Presented transverse image is from section level 2 in the schematic. All levels were assessed in the analysis. (G) Representative magnified images (4x, large, and 20x, inset, zoom; derived from section 1 in the schematic) of hematoxylin and eosin staining shows no alteration in tissue and cellular morphology. (H) Representative magnified images (10x, large, and 20x, inset, zoom; derived from section 1 in the schematic) of Fluoro-Jade C staining for neurodegeneration shows no change in neuronal degeneration (FJ-C positive cells, green). (I) Representative magnified images (10x, large, and 40x, inset, zoom; derived from section 1 in the schematic) of IBA-1 immunostaining indicate no change in microglial activation (IBA-1 positive cells, purple). (J) Representative magnified images (10x, large, and 40x, inset, zoom; derived from section 1 in the schematic) of GFAP immunostaining shows no indication of gliosis or change in astrocytic activation (GFAP positive cells, green). Data presented as mean ± SD. Comparisons between two groups was performed by two-tailed Student's t-test, and that among multiple groups by one way analysis of variance (ANOVA); Group n: 3-8. ns = non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001.

[0040] Fig.19A-K. Noninvasive nerve block with in vivo AAL-mediated ultrasonic ropivacaine uncaging. (A) Adult rats (n=8 / group) received a 5 min intravenous infusion of saline or a ropivacaine-loaded AAL (S5-RL, 5 mg / kg), with focused ultrasound (FUS; 250 kHz, 25% duty cycle, 5 Hz PRF, 0.9 MPa est. in situ peak neg. pressure) applied to the sciatic nerve of the left limb for 2.5 min during the latter half of a 5 min infusion. Frequency of paw withdrawal to a mechanical stimulus (von Frey test) was assayed before and after infusion and sonication. (B) In saline and FUS treated animals, no significant difference in mechanical sensitivity was seen across time or between the limb ipsilateral (ipsi) to sonication vs. the limb contralateral (contra) to sonication. (C) In S5-RL and FUS treated animals, significant differences in mechanical sensitivity were seen in the limb ipsilateral to sonication at time points after sonication and infusion vs. the baseline (red asterisks), as well as at certain time points between the limb ipsilateral to sonication vs. contralateral to sonication (black asterisks). (D- F) No significant differences were seen of key electrocardiographic parameters with a 5 min intravenous infusion of 5 mg / kg ropivacaine-HCl or S5-RL (n=3 / group). (G-K) No concerningparenchymal damage noted by inspection of H&E stained tissue harvested from n=3 rats / group receiving S5-RL AAL infusion and sham or ultrasound applied unilaterally to their sciatic nerves. Comparisons between multiple groups performed by two way analysis of variance (ANOVA); ns = non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001.

[0041] FIG.20. Schematic (top) and pictures (bottom) of the in vitro drug uncaging experiment and apparatus. Note, to prevent water intrusion into the 250 kHz transducer housing, this transducer is wrapped in a condom with degassed ultrasound gel (blue material visible in the pictures) coupling the transducer to the condom. The whole wrapped transducer is submerged in degassed water to couple to the sample tube. The 650 kHz transducer housing is water tight and is directly submerged in the degassed water.

[0042] Fig. 21. Schematic (left) and picture of the apparatus used for measuring acoustic scatter during in vitro AAL ultrasonic drug uncaging. Note, to prevent water intrusion into the 250 kHz transducer housing, this transducer is wrapped in a condom with degassed ultrasound gel coupling the transducer to the condom. The whole wrapped transducer is submerged in degassed water to couple to the sample tube. The 650 kHz transducer housing is water tight and is directly submerged in the degassed water.

[0043] Fig.22A-B. (A) Acoustic scatter spectra observed during 250 kHz, 1.8 MI, 25% duty cycle sonication at room temperature of microbubbbles (dark grey) or S5-KL (red), with the corresponding spectrum from saline sonication subtracted. (B) Quantification of area under the curve (AUC) for each indicated spectral band, with the AUC for each band from saline sonication subtracted. For statistical comparisons, the non-subtracted AUC for each condition and spectrum was compared to the set of saline control AUCs for each band. SH: subharmonic, H: harmonic, UH: ultraharmonic. ns: not significant. The wideband signal differences between microbubble vs. saline sonication were statistically significantly different for all conditions. No statistically significant band differences were observed for S5-KL sonication under these conditions.

[0044] Fig 23. Norketamine & hydroxynorketamine amounts extracted from each organ of the biodistribution experiment reported in Fig.17.

[0045] Fig.24A-B. Quantification of brain histological markers to assess for safety following ultrasonic ketamine uncaging (Fig.18). (a) Mean fluorescence intensity of total FluoroJade C positive cells (marker of neuronal degeneration). In comparison to healthy control brains, there was no difference in levels of FluoroJade C positive cells in either Ultrasound (US), liposome, or liposome+US groups. (b) Mean fluorescence intensity of total IBA-1 positive cells (marker of microglial activation). In comparison to healthy control brains, there was no difference in levels of IBA-1 positive cells in either US, liposome, or liposome+US groups. (c) Mean fluorescence intensity of total GFAP positive cells (marker of astrocytic activation and gliosis). In comparison to healthy control brains, there was no difference in levels of GFAP positivecells in either US, liposome, or liposome+US groups. Data presented as mean ± S.D., analyzed by one-way ANOVA with post-hoc Dunnett's multiple comparisons test; n=3 for each group. DETAILEDDESCRIPTION

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, some potential and preferred methods and materials are now described. All patents, patent applications and non- patent publications mentioned herein are incorporated herein by reference in their entirety to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0047] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range and any other stated or intervening value in that stated range, is encompassed and specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0048] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nanoparticle" includes a plurality of such nanoparticles, and reference to "the therapeutic agent" includes reference to one or more therapeutic agents and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0049] Furthermore, it is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separatelyor in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub- combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0050] Certain aspects, including embodiments, of the present subject matter may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the following detailed description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of these aspects may be used or combined with any of the preceding or following aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0051] The patents, patent applications and publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and are incorporated by reference herein in their entirety. Nothing disclosed herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0052] 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 compounds / therapeutic agents of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.

[0053] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient. Such a carrier medium is essentially chemically inert and nontoxic.

[0054] As used herein, the phrase "pharmaceutically acceptable" means approved by a regulatory agency of the Federal government or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly for use in humans.

[0055] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such carriers can be sterile liquids, such as saline solutions in water, or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously or intothe cerebrospinal fluid (CSF). Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The carrier, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.

[0056] Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such pharmaceutical compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration.

[0057] The compositions disclosed herein may be administered through any mode of administration. In some aspects, the compositions is administered intracranially or into the cerebrospinal fluid (CSF). In some aspects, the compositions are used for parenteral administration. These compositions may be administered, for example, intraperitoneally, intravenously, or intrathecally. In some aspects, the compositions are injected intravenously. In some embodiments, the compositions are injected into the lymphatic system. In some embodiments, the compositions may be administered enterally or parenterally. Compositions may be administered subcutaneously, intravenously, intramuscularly, intranasally, by inhalation, orally, sublingually, by buccal administration, topically, transdermally, or transmucosally. Compositions may be administered by injection. In some embodiments, compositions are administered by subcutaneous injection, orally, intranasally, by inhalation, into the lymphatic system, or intravenously. In certain embodiments, the compositions disclosed herein are administered by subcutaneous injection.

[0058] The terms "individual," "subject," "host," and "patient," to which administration is contemplated, are used interchangeably herein; these terms typically refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets, but can also include commercially relevant birds such as chickens, ducks, geese, quail, and / or turkeys. A mammalian subject may be human or other primate (e.g., cynomolgus monkey, rhesus monkey), or commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs. The subject can be a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g.,young adult, middle-aged adult or senior adult). In some embodiments, the subject may be murine, rodent, lagomorph, feline, canine, porcine, ovine, bovine, equine, or primate. In some embodiments, the subject is a human. In some embodiments, the subject is female. In some embodiments, the subject is male. In some embodiments, the subject is an infant, child, adolescent or adult.

[0059] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. 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, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0060] A "therapeutically effective amount" or "efficacious amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0061] The term “uncaging” refers to the process of inducing oscillations and / or expansion of the core of the nanoparticles, which allows the encapsulated therapeutic agent to be released from the nanoparticles.

[0062] The terms "polypeptide," "peptide," and "protein", used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N- terminal methionine residues; immunologically tagged proteins; and the like.

[0063] The terms “nucleic acid” and "polynucleotide" are used interchangeably herein, and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of nucleic acids and polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides, vectors, probes, primers, single-, double-, or multi-stranded DNA or RNA, genomic DNA, DNA-RNA hybrids, chemically or biochemically modified, non-natural, or derivatized nucleotide bases, oligonucleotides containing modified or non-natural nucleotide bases (e.g., locked-nucleic acids (LNA) oligonucleotides), and interfering RNAs.

[0064] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi(dot)nlm(dot)nih(dot)gov / BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol.48: 443-453 (1970).

[0065] The terms “double stranded RNA,” “dsRNA,” “partial-length dsRNA,” “full-length dsRNA,” “synthetic dsRNA,” “in vitro produced dsRNA,” “in vivo produced dsRNA,” “bacterially produced dsRNA,” “isolated dsRNA,” and “purified dsRNA” as used herein refer to nucleic acid molecules capable of being processed to produce a smaller nucleic acid, e.g., a short interfering RNA (siRNA), capable of inhibiting or down regulating gene expression, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner. Design of a dsRNA or a construct comprising a dsRNA targeted to a gene of interest is routine in the art, see e.g., Timmons et al. (2001) Gene, 263:103-112; Newmark et al. (2003) Proc Natl Acad Sci USA, 100 Supp 1:11861-5; Reddien et al. (2005) Developmental Cell, 8:635-649; Chuang & Meyerowitz (2000) Proc Natl Acad Sci USA, 97:4985-90; Piccin et al. (2001) Nucleic Acid Res, 29:E55-5; Kondo et al. (2006) Genes Genet Syst, 81:129-34; and Lu et al. (2009) FEBS J, 276:3110-23; the disclosures of which are incorporated herein by reference.

[0066] The terms “short interfering RNA”, “siRNA”, and “short interfering nucleic acid” are used interchangeably may refer to short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and other short oligonucleotides useful in mediating an RNAi response. In some instances siRNA may be encoded from DNA comprising a siRNA sequence in vitro or in vivo as described herein.When a particular siRNA is described herein, it will be clear to the ordinary skilled artisan as to where and when a different but equivalently effective interfering nucleic acid may be substituted, e.g., the substation of a short interfering oligonucleotide for a described shRNA and the like.

[0067] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of a therapeutic (i.e., first therapeutic agent) and the compounds disclosed herein. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0068] "Concomitant administration" of a known therapeutic agent with a pharmaceutical composition of the present invention means administration of the therapeutic agent and agent of the disclosure at such time that both the known therapeutic agent and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention. Therapeutic agents contemplated for concomitant administration according to the methods of the present invention include any other agent for use in treatment.

[0069] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.

[0070] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0071] "Pharmaceutically acceptable excipient "means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0072] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0073] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0074] The phrase “determining the treatment efficacy” and variants thereof can include any methods for determining that a treatment is providing a benefit to a subject. The term “treatment efficacy” and variants thereof are generally indicated by alleviation of one or more signs or symptoms associated with the disease and can be readily determined by one skilled in the art. “Treatment efficacy” may also refer to the prevention or amelioration of signs and symptoms of toxicities typically associated with standard or non-standard treatments of a disease. Determination of treatment efficacy is usually indication and disease specific and can include any methods known or available in the art for determining that a treatment is providing a beneficial effect to a patient. For example, evidence of treatment efficacy can include but is not limited to remission of the disease or indication. Further, treatment efficacy can also include general improvements in the overall health of the subject, such as but not limited to enhancement of patient life quality, increase in predicted subject survival rate, decrease in depression or decrease in rate of recurrence of the indication (increase in remission time). (See, e.g., Physicians' Desk Reference (2010).)

[0075] As used herein, “polydispersity index” (PDI) is defined as the ratio of weight average molecular mass (MW) to the number average molecular mass (Mn), and represented by the equation: PDI = MW / Mn. The ratio of the two can be used to describe how far away the encountered distribution is from a uniform distribution. Thus, PDI is used to describe the degree of homogeneity or non-uniformity of a distribution (herein, a population of nanoparticles). For a perfectly uniform (“monodisperse”) sample consisting of exactly one and only one molecular weight both the Mw and the Mn would be the same value. For synthetic molecules / particles, however, the two numbers are not the same, and MW is usually greater than Mn, and therefore the PDI is greater than one. The larger the polydispersity index, the broader the molecular weight range. In calculating PDI of nanoparticles in a distribution of nanoparticles made by the methods described herein, for example, MW describes their average molecular weight by mass and Mn describes their average molecular weight by number. Mn may be determined by employing methods which depend upon the number ofmolecules present in the polymer sample. For example, colligative property such as osmotic pressure is used. Weight average molecular mass (MW) may be measured using methods such as light scattering and ultracentrifugation, sedimentation, etc. which depend upon the mass of individual molecules.

[0076] In certain instances, following generation of the AAP, they may be size selected, isolated and / or purified according to any convenient method known for isolation and / or purification of vesicles. Thus, the isolated and purified AAP may be delivered to a subject unprocessed or they may be size selected, isolated and / or purified by any convenient method described herein or known in the art. The methods of manufacturing the AAP of the present disclosure may involve purification steps, such as membrane extrusion to select for a size under 1 micron, and / or to select for nanoparticles under 500 nm, and / or to select for nanoparticles under 250 nm. Purification may additionally or alternatively be performed using sequential centrifugation and resuspension in fresh aqueous solution.

[0077] The blood-brain barrier (BBB) is a system of vascular structures, enzymes, receptors and transporters designed to prevent access of potentially toxic molecules into the CNS, and to enable passage of nutrients, such as glucose, into brain tissues / structures. The continuous capillaries forming the BBB are sealed and have no fenestrations (openings), forming special tight junctions that restrict paracellular transport. Molecules are restricted from passing between the adjacent cells in capillaries of the CNS by these tight junctions, and pinocytosis is also limited across these capillaries; thus, the main mechanism by which molecules / drugs / imaging agents can pass through the capillaries of the CNS into the brain is passive transcellular diffusion. The molecules transported by passive transcellular diffusion are limited to low molecular weight lipophilic molecules, and this permeability of the BBB is proportional to the lipophilicity of the low molecular weight molecules. However, above a certain molecular weight, the permeability of lipophilic molecules across the BBB is substantially reduced.

[0078] Compared with the vasculature of many other organs, the normal BBB severely restricts the passage of most drugs from plasma to the extracellular space, with more than an 8-log difference in the entry rate of small, lipid-soluble molecules compared with large proteins. A few macromolecules are able to enter the brain tissue from the blood by a receptor-mediated process; for example, brain cells require a constant supply of iron to maintain their function and the brain may substitute its iron through transcytosis of iron-loaded transferrin (Tf) across the brain microvasculature. Other biologically active proteins, such as insulin and immunoglobulin G, are actively transcytosed through BBB endothelial cells. The presence of receptors involved in the transcytosis of ligands from the blood to the brain offers opportunities for developing new approaches to the delivery of therapeutic compounds across the BBB (Jain, K., (2012) Nanomedicine. 7(8):1225-1233). Several potentially effective therapeuticagents for neurological disorders are available but their use is limited because of insufficient delivery across the BBB (Jain, K., (2012) Nanomedicine.7(8):1225-1233). Compositions

[0079] Liposomes. Liposome size can vary from about 0.025 μm to about 2.5 μm and may have one (single layer) or bilayer membranes. On the basis of their size and number of bilayers, liposomes can be classified as multilamellar vesicles (MLV) and unilamellar vesicles, and can be further classified by size. Methods of preparation include passive and / or active loading techniques. Passive loading techniques can include mechanical dispersion, e.g. sonication, extrusion, freeze-thaw, lipid film hydration, micro-emulsification, membrane extrusion, dried reconstitution, etc.; solvent dispersion method, and detergent removal.

[0080] Drug loading can be attained either passively (i.e., the drug is encapsulated during liposome formation) or actively (i.e., after liposome formation). Hydrophobic drugs can be directly combined into liposomes during vesicle formation, where the amount of uptake and retention is governed by drug-lipid interactions. Passive encapsulation of water-soluble drugs depends on the ability of liposomes to trap aqueous buffer containing a dissolved drug during vesicle formation. Trapping effectiveness is limited by the trapped volume delimited in the liposomes and drug solubility. Water-soluble drugs that have protonizable amine functions can be actively entrapped by employing pH gradients, which can result in trapping effectiveness approaching 100%. Alternatively, drug with an acid group may be actively loaded using a calcium acetate or similar gradient.

[0081] In some embodiments, drug loading utilizes a transmembrane gradient for active loading. Liposome are generated, and the external medium is replaced to establish a transmembrane ammonium gradient is an iso-osmolar environment. A tangential flow filtration (TFF) system may be used in multiple rounds. The drugs of interest are then added into the treated liposomes, and a similar TFF procedure used to remove the uncapsulated drug from the formulation.

[0082] Liposome typically comprise phospholipids, which consist of a glycerol unit that is bonded to a phosphate group (PO42−) and to two fatty acid molecules. Natural phospholipids useful in liposomes include those with phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA) polar head groups. Natural phospholipids can comprise a variety of fatty acids, e.g. palmitic acid; margaric acid; oleic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, etc.

[0083] The major saturated synthetic phospholipids are based on either stearic and / or palmitic fatty acid, e.g. HSPC, DPPE, DPPA, DPPG, DPPC, DSPE, DSPA, DSPG, DSPC, DOPE, DOTAP, DOPC, POPC, DMPC, etc.

[0084] Cholesterol is the major steroid usually used in liposome preparation, usually in a ratio less than about 30% of the total lipids to improve liposome rigidity and stability, although β- Sitosterol is also used.

[0085] Surfactants can be utilized in liposomes formulations to modify the encapsulation and release properties of liposomes through surface tension reduction between different immiscible phases. Commonly utilized surfactants in liposomes formulations include sodium cholate, Span 60, Span 80, Tween 60, Tween 80, etc.

[0086] Oleic acid and N-[1(2,3-dioleoyloxy) propyl]-N,N,N-trimethylam-monium chloride (DOTAP) are usually used to prepare anionic and cationic liposomes, respectively. Charged liposomes showed higher liposomal stability during the storage, as charged particles repel each other and reduce aggregation abilities. Cationic liposomes are suitable for delivering various negatively charged macromolecules such as DNA, RNA, and oligonucleotides.

[0087] Second-generation liposomes may comprise surface synthetic polymers, glycoproteins, polysaccharides, or specific receptors ligands, e.g. hyaluronic acid, polyvinyl alcohol (PVA), polyethylene glycol (PEG)), etc.

[0088] As an alternative to liposomes, similar vesicular particles may be obtained with amphiphilic diblock-copolymers. Exemplary block copolymers include: polyethylene glycol- poly(lactic-co-glycolic acid) (PEG2k-PLGA2k), MW: PEG=2kDa and PLGA=2kDa; polyethylene glycol-poly(lactic-co-glycolic acid) (PEG2k-PLGA5k), MW: PEG=2kDa and PLGA=5kDa; polyethylene glycol-poly(ε-caprolactone) (PEG2k-PCL2k), MW: PEG=2kDa and PCL=2kDa; polyethylene glycol- poly(ε-caprolactone) (PEG2k-PCL5k), MW: PEG=2kDa and PCL=5kDa; polyethylene glycol-poly(L-lactic acid) (PEG2k-PLLA2k), MW: PEG=2kDa and PLLA=2kDa; polyethylene glycol-poly(L-lactic acid) (PEG2k-PLLA5k), MW: PEG=2kDa and PLLA=5kDa.

[0089] The acoustic impedance and osmolarity of the liquid core of an AAP is tuned to maximize ultrasound responsiveness while maintaining overall stability of the structure in the absence of ultrasound. The core material has an acoustic impedance that differs from the acoustic impedance of the physiologic media that would surround the formulation droplets after parenteral administration, e.g. plasma, CSF, etc. In some embodiments, the core material of an AAP is an aqueous solution comprising a GRAS solute, which solute causes the solution to have acoustic impedance that differs from the acoustic impedance of plasma. Solutes of interest include, without limitation, sugars, e.g. monosaccharide, disaccharides, polysaccharides, etc.; polymers, dendrimers, and other viscous agents that shift the viscosity and acoustic impedance of the medium to be different from that of plasma. Monosaccharides of interest include, for example, glucose, galactose, fructose, hexose, pentose, ribose, mannose, xylose, deoxyribose, glyceraldehyde, arabinose, tagatose, altrose, etc.Disaccharides of interest include, for example, sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, kojibiose, nigerose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, trehalulose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, mannobiose, melibiose, allolactose, melibiulose, lactulose, rutinose, rutinulose, xylobiose, etc. Polysaccharides of interest include, for example, starch, glycogen, cellulose, heparin, and peptidoglycan, chitin, hyaluronic acid, amylopectin, amylose, etc. In some embodiments the solute is sucrose.

[0090] In some embodiments, the core material of an AAP is an aqueous saccharide solution, where the acoustic impedance differs from the acoustic impedance of plasma by about 0.02 MPa*s / m or more, for example from about 0.02 MPa*s / m to about 2 MPa*s / m, from about 0.02 to about 1 MPa*s / m, from about 0.02 to about 0.5 MPa*s / m, from about 0.02 to about 0.2 MPa*s / m, and may be at least about 0.02 MPa*s / m, at least about 0.04 MPa*s / m, at least 0.06 MPa*s / m, at least 0.08 MPa*s / m up to about 0.2 MPa*s / m, up to about 0.1 MPa*s / m.

[0091] In certain embodiments the core material is an aqueous sucrose solution, where sucrose is present at a concentration of from about 2% to about 10%, e.g. at least about 2%, at least about 3%, at least about 4%, and not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%. In some embodiments, a solution of from about 4% to 6%, e.g. about 5% sucrose is used. The level of free (non-encapsulated) drug in a targeted tissue after exposure to ultrasound (US) may be from about 1.25X the level in the absence of US, from about 1.5X, from about 1.75X, from about 2X, from about 2.5X, or more. In some embodiments the increase is from about 1.75X to about 2.5X the level in the absence of US.

[0092] As shown in Table 2, the addition of salts, such as NaCl or ammonium sulfate, will change the osmolarity of the solution, but will not change the impedance, and does change the release profile during ultrasound. In contrast, sucrose at 5% or 10% changed the impedance by 0.04 MPa*s / m and 0.08 MPa*s / m, respectively, and did alter the release profile during ultrasound.

[0093] A drug substance of interest is loaded into the core and / or shell of the particle. In some embodiments, the agent encapsulated within the AAP of the present disclosure is a therapeutic agent, for example a neurally active therapeutic agent, e.g. propofol, ketamine, nicardipine, verapamil, dexmedetomidine, modafinil, doxorubicin, temozolomide, rivastigmine, galantamine, donepezil, bupivacaine, lidocaine, cisplatin, tacrolimus, p-aminophenyl-α-D- mannoside, levodopa, etc. The drug of interest may be a protein, e.g. antibodies such as anti- NRI, anti-ICAM1, anti-HSP72, anti-actin, anti-VEGF, etc.; growth factors such as GDNF, NGF, etc.; H102 peptide, TF peptide, α-Mangostin, matrix metalloproteinase-9, superoxide dismutase, VEGF, transferrin, tissue plasminogen activator, etc.; or a an oligonucleotide orpolynucleotide. In some embodiments, the therapeutic agent is a drug with logP >1. In some embodiments, the therapeutic agent is a drug with logP >0. In some embodiments, the therapeutic agent is an anesthetic. In some embodiments, the therapeutic and / or contrast agent is a vasodilator. In some embodiments, the composition comprises an imaging agent, contrast agent, and / or dye, optionally in combination with the therapeutic agent.

[0094] A fluorophore may be present in the core of an AAP, optionally combined with a therapeutic agent. Fluorophores absorb light at a characteristic wavelength and then re-emit the light most typically at a characteristic different wavelength. Fluorophores are well known to those of skill in the art and include, but are not limited to rhodamine and rhodamine derivatives, fluorescein and fluorescein derivatives, coumarins and chelators with the lanthanide ion series. A fluorophore is distinguished from a chromophore which absorbs, but does not characteristically re-emit light. “Fluorophore" refers to a molecule that, when excited with light having a selected wavelength, emits light of a different wavelength, which may emit light immediately or with a delay after excitation. Fluorophores, include, without limitation, fluorescein dyes, e.g., 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4', 5',7',1,4-hexachlorofluorescein (HEX), and 2',7'- dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE); cyanine dyes, e.g. Cy3, CY5, Cy5.5, etc.; dansyl derivatives; 6-carboxytetramethylrhodamine (TAMRA), BODIPY fluorophores, tetrapropano-6-carboxyrhodamine (ROX), ALEXA dyes, Oregon Green, and the like. Combinations of fluorophores also find use, e.g. where transfer or release of a fluorophore leads to a color change.

[0095] In some embodiments the agent encapsulated within the AAP is a contrast or imaging agent for imaging of the brain or brain vasculature. In some embodiments, the contrast or imaging agent is a dye. In some embodiments, the contrast or imaging agent is a fluorophore. In some embodiments, the contrast or imaging agent is selected from gadolinium-containing compounds, iodine-containing compounds, and superparamagnetic iron oxide.

[0096] The compositions disclosed herein may comprise contrast agents to enhance contrast in MRI or fMRI, as well as may be used for analyte detection. The early and widely implemented MRI contrast agents are small-molecule chelates that incorporate paramagnetic ions that alter T1, such as gadolinium (Gd3+) or manganese (Mn2+or Mn3+). In some embodiments, the contrast agent may comprise gadolinium (Gd). Non-limiting examples of Gd-comprising contrast agents are gadoterate, adodiamide, gadobenate, gadopentetate, gadoteridol, gadoversetamide, gadoxetate, gadobutrol, gadoterate, gadodiamide, gadobenate, gadopentetate, gadoteridol, gadofosveset, gadoversetamide, gadoxetate, and gadobutrol. In some embodiments, the contrast agent comprises 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In other embodiments, the contrast agent is DOTA-Gd. The contrast agent may be GdNP-DO3A (gadolinium 1-methlyene-(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triAcetate). In some embodiments, the contrast agent is pH sensitive. For example, 1,4,7,10 tetraazacyclododecane-1,4,7,10- tetraacetic acid (DOTA) may be used for pH sensing. This molecule contains a p-nitrophenol on a twelve-member ring. Under basic conditions, only one water molecule is involved in the coordination, while under acidic conditions, two water molecules will coordinate to Gd. The contrast agent may be an iron oxide, iron platinum, or manganese contrast agent. The contrast agent may be protein contrast agent. The contrast agent should be capable of providing appropriate response to whatever MRI resolution is desired and whatever MRI intensity is used. Additional contrast agents may be found in U.S. Pat. No.6,321,105, and U.S. Patent Publication US 2015 / 0202330, each of which is incorporated in their entirety.

[0097] Imaging agents can include fluorescent molecules, radioisotopes, nucleotide chromophores, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and combinations thereof. In some embodiments, the composition further comprises a fluorescent dye. The fluorescent dye may be a derivative of rhodamine, erythrosine or fluorescein. The fluorescent dye may be a xanthene derivative dye, an azo dye, a biological stain, or a carotenoid. The xanthene derivative dye may be a fluorene dye, a fluorone dye, or a rhodole dye. The fluorene dye may be a pyronine dye or a rhodamine dye. The pyronine dye may be chosen from pyronine Y and pyronine B. The rhodamine dye may be rhodamine B, rhodamine G and rhodamine WT. The fluorone dye may be fluorescein or fluorescein derivatives. The fluorescein derivative may be phloxine B, rose bengal, or merbromine. The fluorescein derivative may be eosin Y, eosin B, or erythrosine B. The azo dye may be methyl violet, neutral red, para red, amaranth, carmoisine, allura red AC, tartrazine, orange G, ponceau 4R, methyl red, or murexide-ammonium purpurate. Exemplary fluorescent dyes include, but not limited to Methylene Blue, rhodamine B, Rose Bengal, 3-hydroxy-2, 4,5, 7-tetraiodo-6-fluorone, 5, 7- diiodo-3-butoxy-6-fluorone, erythrosin B, Eosin B, ethyl erythrosin, Acridine Orange, 6′-acetyl- 4, 5, 6, 7-tetrachloro-2′,4′, 5′, 6′, 7′-tetraiodofluorescein (RBAX), fluorone, calcein, carboxyfluorescein, eosin, erythrosine, fluorescein, fluorescein amidite, fluorescein isothiocyanate, indian yellow, merbromin, basic red 1, basic red 8, solvent red 45, rhodamine 6G, rhodamine B, rhodamine 123, sulforhodamine 101, sulforhodamine B, and Texas Red (sulforhodamine 101 acid chloride).In some embodiments, the compositions and methods disclosed herein may include lipid or protein emulsifiers that improve the stability, drug loading, and drug release efficacy of the system.

[0098] According to the present invention, compositions can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, aerosol, or intramuscular means. The most typical route of administration is intravenous although other routes can be equally effective.

[0099] For parenteral administration, compositions of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies and / or polypeptides can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient. An exemplary composition comprises polypeptide at 1 mg / mL, formulated in aqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mM L-arginine, 0.01% polysorbate 20, adjusted to pH 7.4 with HCl or NaOH.

[0100] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[0101] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

[0102] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.

[0103] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Preferably, a therapeutically effective dose will provide therapeutic benefit without causing substantial toxicity.

[0104] Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of thepopulation). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1).

[0105] Also within the scope of the invention are kits comprising the compositions of the invention and instructions for use. The kit can further contain a least one additional reagent. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit. Methods of Use

[0106] In some embodiments, a method is provided for ultrasound-triggered drug release to a tissue of interest, the method comprising administering an individual in need thereof, an effective dose of a drug of interest encapsulated in AAP, and subjected the targeted tissue to a dose of ultrasound effective to uncage the drug. In some embodiments the targeted tissue is the brain. In some embodiments, including targeting to the brain, administration is parenteral.

[0107] In some embodiments a method is provided for ultrasound-triggered drug release to the brain of an individual, the method comprising parenteral administration of an effective dose of a drug of interest encapsulated in AAP to an individual in need thereof, and subjecting the targeted brain tissue to a dose of ultrasound effective to uncage the drug. The dose of ultrasound is preferably at a level that is sufficiently low as to avoid parenchymal damage. For example, the dose of ultrasound may be from about 200 to 300 kHz, for a period of time of from about 1 to about 45 minutes. The AAP may comprise liposomes, where the core comprises a sucrose solution of from about 2.5% to about 7.5%.

[0108] In other embodiments a method is provided for ultrasound-triggered drug release to a nerve of an individual, the method comprising parenteral administration of an effective dose of a drug of interest encapsulated in AAP to an individual in need thereof, and subjecting the targeted nerve tissue to a dose of ultrasound effective to uncage the drug. Nerves suitable for targeting include, for example, peripheral nerves, spinal cord, optical nerves, nerves in the brain, and the like. The dose of ultrasound is preferably at a level that is sufficiently low as to avoid nerve damage. For example, the dose of ultrasound may be from about 200 to 300 kHz,for a period of time of from about 1 to about 45 minutes. The AAP may comprise liposomes, where the core comprises a sucrose solution of from about 2.5% to about 7.5%.

[0109] The drug of interest may be a neurally active therapeutic agent, e.g. propofol, ketamine, nicardipine, verapamil, dexmedetomidine, modafinil, doxorubicin, temozolomide, rivastigmine, galantamine, donepezil, bupivacaine, lidocaine, cisplatin, tacrolimus, p- aminophenyl-α-D-mannoside, levodopa, etc. The drug of interest may be a protein, e.g. antibodies such as anti-NRI, anti-ICAM1, anti-HSP72, anti-actin, anti-VEGF, etc.; growth factors such as GDNF, NGF, etc.; H102 peptide, TF peptide, α-Mangostin, matrix metalloproteinase-9, superoxide dismutase, VEGF, transferrin, tissue plasminogen activator, etc.; or a an oligonucleotide or polynucleotide. In some embodiments, the therapeutic agent is a drug with logP >1. In some embodiments, the therapeutic agent is a drug with logP >0. In some embodiments, the therapeutic agent is an anesthetic. In some embodiments, the therapeutic and / or contrast agent is a vasodilator. In some embodiments, the composition comprises an imaging agent, contrast agent, and / or dye, optionally in combination with the therapeutic agent.

[0110] In addition to therapeutic delivery, brain imaging and presurgical mapping of functional brain regions to identify a surgical tract between critically important brain regions and a lesion to be resected and a margin around the lesion, the compositions and methods described herein are useful in basic research and clinical applications in psychiatry. Some other applications for the compositions and methods described herein include pre-hoc validation of a brain region to be intervened upon with, for example, deep brain stimulation (DBS), radiosurgery, radiofrequency ablation (RFA), laser ablation, or focused ultrasound (FUS) ablation.

[0111] For example, the compositions and methods described herein can be used for validating the location of the ventral intermediate (VIM) thalamic nucleus prior to ablation for essential tremor or tremor-dominant Parkinson disease (PD), or for validating a focus as a principal seizure generator prior to resection or ablation.

[0112] Another application for the compositions and methods described herein is adjunctive focal pharmacotherapy for psychiatric treatment; for example, modulating processes in the amygdala in real time using anti-adrenergic therapeutic agents during talk or exposure therapy sessions for PTSD or anxiety disorder. Alternatively, ketamine may be infused locally into the ventromedial prefrontal cortex (vmPFC) of an acutely depressed or suicidal patient in order to isolate ketamine’s antidepressant action over its anesthetic, addictive, and psychotogenic actions. Similarly, the compositions and methods described herein can be used in focused delivery of epileptogenic treatments or to focally decrease activity of a pathologic neural circuit.

[0113] Another application for the compositions and methods described herein is to determine which peripheral nerves most contribute to a complex regional pain syndrome throughsequential anesthesia of each nerve, or to ablate certain targets, wherein the composition comprising the nanoparticles described herein can be used for thermal ablation via super- heating at the sonication focus.

[0114] A basic research application for the compositions and methods described herein is for validating / testing a hypothesis of the role of a brain region in the performance of a particular brain function, or a receptor’s action in a specific brain region (e.g. validation of insular subfields as necessary for certain risk calculations in decision making).

[0115] Another application for the compositions and methods described herein is for focal delivery of vasoactive substances to treat alterations of perfusion, e.g. focally delivering calcium channel antagonists like verapamil and / or nicardipine to treat cerebrovascular disorders such as stroke, cerebral vasospasm, or reversible cerebral vasoconstriction syndrome (RCVS).

[0116] Another application for the compositions and methods described herein is for the focal delivery of therapeutic agents to treat a cardiovascular disease or disorder selected from hypertension, arterial spasm or blockage, cerebral vasospasm, and myocardial or other end organ infarction or ischemia.

[0117] These techniques for exquisitely focalized delivery of a therapeutic agent / drug and / or imaging agent to an organ or organ substructure, such as the cardiovascular system, heart, blood vessels, brain or brain vasculature, lungs and / or alveoli, for example, can transform both basic research and clinical science. The AAP described herein have been adapted for encapsulation and focal delivery of drugs and / or imaging agents using focused ultrasound (FUS).

[0118] The presently described compositions and methods are particularly useful in clinical settings for focal drug-delivery, as they are easily adapted to be specific for the encapsulation and delivery of a wide range of brain-active neuromodulating agents and accounting for certain chemical features of the drug. The drug is not released generally into the brain or brain vasculature until after a FUS pulse uncages and releases the drug to a very defined region of the brain. Thus, specific noninvasive neuromodulation can be achieved. EXAMPLES

[0119] The following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present compositions and methods, and are not intended to limit the scope of what the inventors regard as their invention nor are the examples intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Standard abbreviations may be used, (e.g., “bp” refers to base pair(s); “kb” refers to kilobase(s); “ml” refers to milliliter(s); “s” or “sec” refers to second(s); “min” refers to minute(s); “h” or “hr” refers to hour(s); “aa” refers to amino acid(s); “nt” refers to nucleotide(s); “i.v.” or “IV” refers to intravascular(ly); and the like. Example 1 Acoustomechanically activatable liposomes for ultrasonic drug uncaging

[0120] There is increasing interest in repurposing psychedelic drugs like ketamine to treat affective disorders. However, the non-therapeutic effects of these drugs – including their hallucinogenic, sedative, and abusable properties – limit mainstream acceptance and practical utilization. Targeting delivery of the unmodified drug to the brain regions thought to most contribute to the desired functional effects could selectively amplify the therapeutic effects of these drugs. To validate this approach, we developed a clinically translatable ultrasound- sensitive liposomal formulation of ketamine, SonoKet, which selectively uncages ketamine upon application of focused ultrasound. We confirmed in rats that SonoKet uncaging safely enables noninvasive selective delivery of ketamine to regional targets like the medial prefrontal cortex, a mediator of the affective efficacy of ketamine, or the retrosplenial cortex, a mediator of its dissociative effects. Uncaging ketamine in either region elicited unique electrophysiological signatures and differential behavioral responses to acute stress, with a decreased behavioral indicator of stress specifically following medial prefrontal cortical ketamine uncaging. These results confirm the regional specificity of ketamine action, both physiologically and behaviorally, and support that noninvasive regionally targeted drug delivery such as with ultrasonic drug uncaging could indeed selectively activate the therapeutic effects of psychotropic drugs like ketamine over their recreational properties.

[0121] Ketamine is a multimodal drug with a complex pharmacologic profile that yields diverse clinical applications including for anesthesia, analgesia, and antidepressant treatment, but which also carries abuse potential as ketamine is a hallucinogenic recreational drug. Recently, the utility of ketamine for mental health treatment has gained much attention, especially following the FDA approval of one of its isomers for treatment-resistant depression. In addition to depression, ketamine has been proposed as having therapeutic utility in post-traumatic stress disorder (PTSD), acute and chronic pain, and substance use disorders among other indications. However, its hallucinogenic, recreational, and sympathomimetic properties have raised concerns regarding its abuse liability and safety profile.

[0122] These multitude of actions of ketamine and similar drugs has led to efforts to optimize treatment protocols to maximize a given therapeutic action (e.g. for affective disorder treatment) over their less medically desirable actions (e.g. dissociation, hallucinations,hypertension). These efforts generally vary administration parameters like total dose, route and rate of administration, the isomer or metabolite that is administered, or chemical features of the drug itself to attempt to amplify the desired therapeutic efficacy of the drug over its side effects. An alternate strategy is suggested by preclinical and clinical data that show that the different functional effects of ketamine are attributable to its action at spatially distinct brain regions. For instance, the affective actions of ketamine have been attributed to limbic brain regions like the medial prefrontal cortex (mPFC) including cingulate subfields and the lateral habenula, while the dissociative actions of ketamine are attributed to regions like the retrosplenial cortex (RsC). Meanwhile, its cardiovascular and genitourinary effects are attributable to action outside of the nervous system. This suggests that if one were to target ketamine delivery to the brain regions most central to the desired therapeutic effect, one could maximize its therapeutic efficacy over its variety of side effects that may otherwise be counteractive.

[0123] Conventional approaches for region-specific targeting of psychoactive substances typically involve invasive methods that may disrupt adjacent neural circuits and present challenges in translating findings to larger animal models and ultimately to humans. To validate the proposed approach of selectively activating a desired functional effect of a drug of interest through targeted drug delivery to key brain targets, we developed an ultrasound- sensitive biocompatible drug-loaded liposomal vehicle that enables noninvasive targeted drug delivery to millimeter-sized regions of interest following activation by low intensity focused ultrasound (FUS) protocols achievable by current clinically utilized FUS systems. After intravenous (i.v.) infusion of these acoustically-activatable liposomes (AALs), FUS is applied to the bodily region of interest, uncaging the drug from the AALs as they circulate in the blood volume of the tissue, after which the freed drug is able to enter the organ as it otherwise does normally, thereby selectively targeting drug delivery to the sonicated region of interest (Fig. 1a, b). We loaded this drug delivery platform with ketamine to create an ultrasound-sensitive ketamine formulation, SonoKet, to investigate whether targeted delivery of ketamine to specific brain targets may indeed selectively amplify signatures of affective efficacy. Results

[0124] Development and in vitro validation of ketamine-loaded acoustically activatable liposomes. To enable noninvasive targeted drug delivery to brain regions of interest, we designed an ultrasound-gated vehicle, given the availability and performance of clinical transcranial focused ultrasound systems that can target ultrasound fields to essentially any brain region of interest with high spatiotemporal resolution. In developing an ultrasound- sensitive drug delivery vehicle to enable this regional brain targeting for drugs like ketamine, which are lipophilic but water-soluble under physiologic conditions, we first noted it would bedesirable for the vehicle to have an aqueous core to enable stable loading of the drug. Next, to maintain eventual clinical translation potential, we sought to only use excipients already in approved drug products, as listed in the FDA inactive ingredients database (IID). We therefore chose to start with liposomes as they are now commonly used drug delivery vehicles with aqueous cores that may be formulated with excipients in the IID. While liposomes have been suggested as ultrasound-sensitive vehicles in certain prior formulations, loading ketamine into a usual unmodified liposome showed no significant response to ultrasound in these conditions (Fig.1c), suggesting we would need to incorporate a material into the liposome to increase its sensitivity to ultrasound. We noted that ultrasound-material interactions are determined by parameters like speed of sound (c) and density (ρ), which may be encapsulated as the acoustic impedance (Z=ρc) or bulk modulus (K= ρc2), and therefore sought to incorporate materials that yield substantive differences in these parameters compared to the usual salt buffers used to load drugs into liposomes. We chose to incorporate sugars into the internal buffer of the liposome since moderate percentages of sugars have been shown to substantively shift the acoustic impedance of aqueous buffers (Fig. 1a; Tables 1, 2). Additionally, since these sugars are GRAS (Generally Recognized As Safe) excipients for pharmaceutical formulations, adding sugars would maintain high clinical translational potential for the eventual vehicle. Compared with a ketamine-loaded liposome with no added internal excipient other than an ammonium sulfate loading battery, liposomes incorporating varied percentages of sucrose showed significantly increased ultrasonic drug uncaging performance: with 5% sucrose added to the internal buffer yielding ~40% drug uncaging with a 250 kHz, 25% duty cycle, 1 min ultrasound protocol, and 10% added sucrose yielding ~60% drug uncaging with this same protocol, compared to a nonsignificant trend towards ~20% estimated free drug with no added sucrose when the formulation was incubated with plasma (Figs.1c; Tables 1, 2). Notably, with sucrose incorporation there were no significant differences in key physicochemical properties including size, polydispersity, zeta potential, drug loading, or unencapsulated drug percent (Table 1). However, while 10% additional sucrose internally significantly increased the amount of ultrasonic drug uncaging relative to 5% additional sucrose, it did so at the expense of displaying a significant rate of drug release without ultrasound when incubated with plasma at 37 ºC, indicating that there is a relative tradeoff of ultrasound responsiveness and formulation stability with this scheme (Fig.1d).

[0125] We next noted that increasing sucrose percentages would yield shifts in both the acoustic parameters and the osmolarity of the internal buffer. To discern which physical parameters – the acoustic characteristics or osmolarity – contributed more to our results we next prepared liposomes with equiosmolar internal buffers that have different acoustic characteristics (Table 2). Drug-loaded liposomes with internal buffers composed of ammoniumsulfate with equiosmolar additional sodium chloride, glucose, or sucrose were prepared and found to have similar physicochemical characteristics with each other (Table 1). Despite similar internal osmolarities, they showed differing amounts of drug uncaging with the same ultrasound protocol, in correlation to their differences in internal acoustic impedance (Fig.1e, f; Table 1). This indicates that the differential acoustic characteristics of the inner medium of the liposome, rather than another physical parameter, most contribute to the ultrasound- responsiveness of this drug-loaded nanocarrier. To the degree that (hyper)osmolarity of the internal medium affects uncaging performance, it may contribute to a baseline leak or instability (Fig.1d) or to accelerate drug release by driving water influx into the internal buffer following the initial ultrasound-induced increase in membrane permeability. For further experiments, 5% sucrose added internally was chosen as a relative optimum of ultrasound responsiveness with stability of the formulation with body temperature incubation in plasma. We dubbed this ultrasound-sensitive ketamine-loaded formulation ‘SonoKet’.

[0126] With this formulation, in vitro dose-response relationships were noted between drug uncaging and varying ultrasound parameters such as in situ peak negative pressure and mechanical index (MI; Fig. 1g). Intuitively, there was increased drug uncaging seen with a higher medium temperature of 37 ºC versus room temperature (~22 ºC; Fig. 1g). While medium temperature does contribute to drug uncaging with this system, the response at some ultrasound intensities at room temperature indicates that medium heating is not necessary for the uncaging performance of this system. Additionally, at these ultrasound intensities and duty cycles, the level of ultrasound-induced medium heating is at most +0.1-0.2 ºC per the bio-heat equation, arguing against a thermal type response mechanism. Instead, a mechanical cavitation (stable / oscillatory or inertial) type response mechanism is likely.

[0127] Importantly, this formulation shows stability for both drug loading and ultrasonic uncaging across months of time in refrigerated storage (Fig.1h). By electron microscopy, the formulation particles show spherical morphology as expected, but with no internal precipitation of the drug (Fig.1i). With sonication, the size distribution of the particles decreased, cohering with an ultrasound-induced leak of the internal contents (Fig. 1i). Notably, no gas phase bubbles or voids were noted within the formulation with or without sonication.

[0128] In vivo pharmacokinetics and pharmacodistibution of ketamine uncaging using SonoKet. We next validated that SonoKet can be used in vivo to locally deliver ketamine to brain regions targeted with focused ultrasound (FUS). Following intravenous (i.v.) infusion, SonoKet would be activated by FUS while the formulation particles circulated in the blood volume of the tissue of interest, releasing ketamine from the formulation particles, thereby allowing the freed drug to enter the parenchyma of the sonicated brain region (Fig 1b).

[0129] To assess the regional specificity of the pharmacodistribution induced with SonoKet ultrasound activation, we used solid-phase microextraction (SPME) to directly measure theconcentration of ketamine delivered to each brain region. First, male rats were prepared in a stereotaxic frame and kept under anesthesia. The medial prefrontal cortex (mPFC) and retrosplenial cortex (RsC) were targeted given the importance of each region in mediating the affective (mPFC) and dissociative (RsC) actions of ketamine (Fig. 2a, 5). A surgical craniectomy was performed to expose two brain sampling sites at 3.2 mm and -4 mm relative to bregma to sample the mPFC and RsC, respectively. Following an initial in vivo dose- response evaluation in a separate pilot cohort (Fig. 6), subjects received a drug-loaded nanoparticle infusion (1.5 mg / kg, i.v.) or a dose-matched free unencapsulated drug infusion for 5 minutes, with or without ultrasound application to the brain (Fig.2a). In conditions that received ultrasound (250 kHz, 25% duty cycle pulsed, 1.1 MPa estimated peak in situ negative pressure derated for the skull insertion loss, 2.5 min; Fig.5), sonication was applied to either the mPFC or RsC during the last 2.5 min of infusion. Immediately following treatment, SPME pins were inserted to align the portion of the pin with the SPME coating to the sonication target with an additional pin sampling the non-sonicated control region, for 5-min samplings repeated through 20 min following the end of sonication and infusion (Fig. 2a). The mPFC and RsC sonication and sampling sites are separated by 7 mm along the anteroposterior axis.

[0130] As expected, with free unencapsulated ketamine (Ketamine HCl) infusion, no differences in ketamine delivery and clearance were observed between the two sampled regions, with or without ultrasound application (Fig. 2b). In contrast, with SonoKet infusion, significant increases in ketamine delivery were observed selectively in the sonicated brain regions (mPFC: 2.1x, RsC: 2.0x, comparing SonoKet with and without FUS) with no difference of ketamine delivery to the non-sonicated brain region relative to the control sham treated animals, and no difference of the amount of ketamine delivery or clearance between the mPFC and the RsC (Fig.2b, d). With ultrasonic ketamine uncaging using SonoKet, higher ketamine levels were extracted at the site of ultrasound application compared to dose-matched free ketamine (KetHCl) administration at the initial time point (mPFC: 1.2x; RsC: 1.5x) (Fig. 2b). While a non-zero amount of ketamine was delivered to the brain with SonoKet without ultrasound activation, sonicated regions consistently had two-fold or higher amounts of ketamine extracted than non-sonicated regions, providing a suitable therapeutic window for clinical application. In each experiment, a similar clearance profile was noted, with ketamine and its metabolites clearing from the brain and blood by 15-20 min post-treatment (Fig.2c-e; 7; Tables 3-4). The levels of major ketamine metabolites in either the brain or blood were negligible in comparison to the amounts of unmetabolized ketamine delivered in each experiment, with no significant differences of metabolite distribution or clearance between each condition (Fig. 2c-e, 7; Tables 3-4). Given the lack of increase of ketamine delivery to the non-sonicated sampling sites in both the pilot (Fig.6) and the main cohorts (Fig.2) relative to sham treated animals, an in vivo spatial resolution for this drug delivery technique of <5-7mm is estimated for 250 kHz activating ultrasound. Importantly, biodistribution analyses demonstrate lower solid organ uptake of ketamine with SonoKet administration compared to dose-matched unencapsulated ketamine (Fig.7) confirming that this approach should lower systemic side effects when targeting a particular brain regional site concentration of a drug of interest. These results validate that ultrasonic ketamine uncaging with SonoKet can indeed noninvasively and selectively deliver ketamine locally to these two distinct brain targets, while displaying otherwise similar pharmacokinetics compared to acute systemic ketamine administration.

[0131] Safety analysis for this novel targeted drug delivery platform. To assess the safety of this targeted drug delivery platform, we conducted an immunohistochemistry panel for tissue damage. The histological evaluations for morphological tissue damage, neuronal degeneration, microglial / astrocytic activation, and gliosis did not reveal any signs of brain parenchymal injury associated with the localized ultrasonic drug uncaging protocol directed to either the mPFC or the RsC (Fig.2f). Quantification of the immunohistochemical data revealed no significant differences between tissues in the saline control and SonoKet and ultrasound conditions (Fig.2f). These results confirm that the pharmacodistribution and pharmacokinetics measurements were not biased by physiological tissue damage from application of this targeted drug delivery platform nor from the focused ultrasound itself, consistent with prior studies.

[0132] Unique site-specific electrophysiological signatures evoked with localized ketamine delivery. To evaluate the functional efficacy of localized ketamine delivery via ultrasonic ketamine uncaging in the affective context, we measured electrocorticography (ECoG) potentials over the mPFC and RsC targets while the subjects underwent acute restraint stress (Fig. 3a). Notably, different oscillatory band patterns have been associated with different functional effects of ketamine, with gamma band activity from frontal regions being a biomarker of the affective efficacy of ketamine and slower band activity from the retrosplenial cortex correlated to the dissociative actions of ketamine. Awake rats were placed in a noninvasive restraint for the entirety of a 45-minute recording, similar to prior investigations using restraint as an acute behavioral stressor. ECoG measurements were recorded from 5 minutes prior and continued until 35 minutes after i.v. infusion and sonication treatment (Fig.3a). The ECoG potentials were processed to determine the power spectral density across 1-55 Hz before, during, and after treatment following similar analyses of intracranial recordings from humans receiving a ketamine infusion.

[0133] We first characterized the dose-response relationship for each recording site during free unencapsulated Ketamine HCl administration at 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, and 1.5 mg / kg. With increasing free ketamine administered systemically there was an increase principally in the gamma (25-55 Hz) band for both the mPFC and RsC recording sites (Fig.8-5). A power decrease was also observed in slower bands for the RsC recording site, with significance at the 1.5 mg / kg dose (Fig.9). The sustained response of gamma-band power for the duration of the recordings, despite the relatively more rapid clearance of the drug (Fig. 2c,e, 7) was expected given similar prior characterizations.

[0134] Following these dose-response characterizations, we selected 0.75 mg / kg as the dose of SonoKet and Ketamine HCl for subsequent experiments as the blood plasma ketamine levels achieved with 0.75 mg / kg infused over 5 minutes with either formulation (~200 ng / ml; Fig.2, 6d) are similar to the plasma levels achieved with the 0.5 mg / kg over 40-min infusions typically used in humans for subanesthetic ketamine treatments for affective disorders. In the awake restraint acute stress model, rats were administered SonoKet with and without sonication, dose-matched Ketamine HCl, or saline infusion with sonication in a random ordering and with at least one week for washout between successive experiments. Differential ECoG patterns were recorded between the mPFC and the RsC when ketamine was uncaged from SonoKet (Fig.3b-d, 10-11). With this protocol, notably no significant change in ECoG potentials were seen with SonoKet infusion without ultrasound application compared to saline infusion. However, with mPFC ketamine uncaging, increases in gamma band (25-55 Hz) powers were observed to a similar to a trend-wise greater degree and similar overall spectral pattern compared to dose-matched free ketamine infusion for the mPFC recording site (Fig. 3b-d, 8). In contrast, with RsC ketamine uncaging, acute increases in principally slower theta (4-8 Hz), alpha (8-15 Hz), and beta (15-25 Hz) bands were observed with sustained activities in beta and low gamma bands, in an overall distinct spectral pattern to that achieved with both dose-matched free unencapsulated ketamine administration and with mPFC uncaging (Fig. 3b-d, 8-11). While RsC SonoKet uncaging showed apparent increases in delta band activity (Fig.3b-c, 10), cohering with prior characterizations, this comparison was driven by changes in the saline control condition, suggesting a nonspecific effect of this experimental paradigm completed during acute restraint stress rather than directly of ketamine delivery to the RsC. Nonetheless, this pattern of principally higher-frequency band induction in the mPFC and lower frequency band induction in the RsC with ketamine administration coheres with analogous intracranial characterizations seen with ketamine administrations in both humans and rodents, with the frontal gamma band induction being proposed as a biomarker for the affective efficacy of ketamine and the slower band activity in the retrosplenial cortex correlated to its dissociative effects. Additionally, given the relatively broad and rapid propagation of these potentials through the brain and cerebrospinal fluid, these results demonstrate how targeted uncaging may be used to noninvasively determine the localized response of a portion of the brain to a given pharmacological stimulus, given the challenges otherwise of source localization using surface electrical recordings. Overall, this differential functional electrophysiologic response to ketamine uncaging in the mPFC versus RsC, despite similarlevels of ketamine delivery to each brain region (Fig.2), confirms the site-specific physiologic action underlying ketamine’s multiple functional effects.

[0135] Ultrasonic ketamine uncaging in the mPFC selectively affects behavioral responses to stress. After observing unique electrophysiologic signatures of ketamine uncaging between the mPFC and RsC, we next evaluated if these site-specific electrophysiological effects were reflected in behavioral effects indicative of affective efficacy. Behavioral responses of rodents to subanesthetic ketamine are variable and influenced by factors like sex that do not necessarily have parallel human effects. Nonetheless certain rodent behavioral responses to subanesthetic ketamine are considered indicative for reporting relevant neurobiological limbic and affective circuit activity. Namely, in rodents, ketamine is known to have locomotor induction and sensitization effects that are correlated to mesolimbic dopaminergic circuit modulation as well as effects relevant for stress and affective processing that are correlated to limbic circuit activity. To evaluate for both domains of functional behavioral effects of subanesthetic ketamine, we used the open field test (OFT) assay and quantified parameters related to locomotion (e.g. total distance traveled, speed) and stress / affective processing (e.g. percent time in the field center). Rats with a surgically implanted target marker for sonication were placed in a restraint while awake to induce a brief, acute stress for 5 minutes, equating to the length of treatment (Fig.4a). Subjects received either saline, unencapsulated Ketamine HCl, or SonoKet infusion and all rats received sonication directed to either the mPFC or RsC. After receiving the treatment, subjects were placed in an open field chamber and recorded by video for 20 minutes. There were no significant effects in locomotion parameters like total distance traveled or speed (Fig.4b-h, Fig.12-13). In contrast, subjects receiving ultrasonic ketamine uncaging directed to the mPFC spent significantly more time in the center of the field compared to rats that received either dose-matched Ketamine HCl or saline (Fig. 4b, g, h; multiple comparison corrected p-values: saline vs SonoKet p=0.037, Hedges g = 1.24; KetHCl vs SonoKet p=0.028, Hedges g = 1.35). Notably, no significant differences were observed between conditions for rats sonicated at the RsC. The lack of locomotion induction at this dose coheres with previous similar dose studies and indicates no significant mesolimbic dopaminergic system modulation. Meanwhile the result of increased center time – typically interpreted as a lower stress, anxiolytic-like phenotype following in this case the stressor provided by acute restraint – specifically with mPFC ketamine uncaging coheres with the identification of the mPFC as critical to the affective efficacy of ketamine.

[0136] For this study, we developed a novel technology for noninvasive targeted drug delivery by conferring ultrasound sensitivity to the commonly clinically utilized vehicle of liposomes with the modification of incorporating sucrose into the liposome inner core. After validating the in vitro and in vivo performance of this technology to enable noninvasive targeted drug deliveryto regions of interest in the brain, we then used this system to confirm that ketamine indeed induces distinct functional responses depending on which brain region it is targeted to, with regionally-distinct functional effects identified by both electrophysiology and behavior. This technology is safe, noninvasive, and preserves the integrity and architecture of the brain with equivalence of drug targeting performance across brain regions – features that are difficult to achieve with microcannulation or other current means for regionally targeted drug application in vivo.

[0137] Recently there has been great interest in repurposing recreational psychotropic drugs like ketamine for mental health applications, based on preclinical and clinical evidence supporting varied psychiatric indications. However, tempering that excitement are concerns regarding the abuse liability, ethics, and safety of utilizing potent hallucinogenic recreational drugs in a clinical context, with specific such concerns documented in relation to the approval and labeling of an isomer of ketamine for depression treatment. To address these concerns, efforts have been made to limit or separate the recreational properties of these drugs from their therapeutic actions, either through optimizing drug administration protocols or through synthesizing or screening new chemical entities that recapitulate some of the key pharmacologic profiles of these drugs. This study supports and provides the technological means for an alternate strategy to achieve the desired functional separation of the varied actions of ketamine by targeting drug delivery to the brain regions thought to be most critical for the desired therapeutic action. In principle, a similar approach is utilized for a wide variety of neuropsychiatric drugs of interest that have potent therapeutic effects but dose-limiting side effects due to drug action at off-target regions of the brain or body. Notably, as current approved clinical protocols for administration of drugs likes ketamine in psychiatric care require close clinical monitoring of the subject by trained medical personnel, applying ultrasonic drug uncaging instead of usual protocols for administration adds minimal complication to the current clinical workflow – namely adding ultrasound device placement and operation but potentially saving substantively on the need for close patient monitoring if dissociative, hallucinogenic, and sympathomimetic properties are minimized.

[0138] Towards a translational end, we note that the ultrasound-sensitive liposomal formulation of ketamine used in this study is composed of excipients in the FDA inactive ingredients database, within levels used in already approved formulations of other drugs, and is produced using methods compatible with current good manufacturing practices (cGMP). Additionally, the ultrasound protocols used here for successful SonoKet activation in vivo are well within the capacity of current clinically utilized transcranial focused ultrasound systems. In translating these results to larger animals and humans, care will need to be taken in accounting for some of these rodent results to be complicated by the potential for standing wave generation at the 250 kHz ultrasound frequency used in these studies, although thecorrelation between the in vitro and in vivo uncaging performance with this ultrasound protocol (Fig.1, 2, 6) mitigates some of these concerns and our simulations confirm that the effects of standing wave generation in the rat skull in these protocols is minimal (Fig.2a, 5). Efforts will explicitly translate this technology and these results to larger animal models and to humans, and will extend this drug delivery platform to other drugs of neuropsychiatric interest. Methods

[0139] Acoustically activatable liposomes (AALs) synthesis and characterization. Materials. All the chemicals and reagents used in the study were of the highest purity grade with sourcing as follows. Lipoid: Hydrogenated Soy Phosphatidylcholine (HSPC) (Cat no: Lipoid S PC-3) and [N-(methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt] (DSPE-PEG 2000) (Cat no: PE18:0 / 18:0 – PEG 2000). Evonik: Cholesterol (PhytoChol Inject HU). Ketamine-HCl injectable solution (100 mg / mL; Dechra Pharmaceuticals, procured through Stanford University Environmental Health & Safety). Hiemdia: Ammonium Sulfate (Cat no: PCT0003). Fisher Scientific: Absolute Ethanol (200 proof) (Cat no: BP2818-100), Sucrose (Cat no: S5-500), HPLC and LC / MS grade Water, Methanol, Acetonitrile, 2-Propanol, Formic Acid. Sigma Aldrich: HEPES buffer solution (Cat no: 83264-500ML-F) and L-Histidine monohydrochloride monohydrate (Cat no: H8125). Repligen: TFF filters (C02-S05U-05-N (SN: 20020493-03 / 21-057). Lampire Biological Laboratories: Male Canine Plasma (Cat no.7302009). Cytiva: PD-10 column Sephadex G-25 M (Cat no.17085101), Sephacryl S-500 (Cat no.1706130). Milli-Q water was used to prepare all the buffers. Millipore Sigma: Cerilliant® certified standard solutions of ketamine hydrochloride, ketamine-D4 hydrochloride, norketamine hydrochloride, norketamine-D4 hydrochloride, and hydroxynorketamine hydrochloride; Supel™ BioSPME 96-Pin Devices (product number 59680-U).

[0140] Liposome production. An ammonium sulfate remote loading scheme was utilized for each reported liposome, following published protocols. Briefly, small unilamellar vesicles were prepared by dissolving lipid components (HSPC:DSPE-PEG2000:cholesterol 52.8:42.3:4.8 molar ratio) in heated ethanol and then diluting the mixture to 10% ethanol with 250 mM ammonium sulfate alone or with 5-10% (by weight) sucrose, 5% glucose, or 73 mM NaCl depending on the experiment. Then the solution was extruded (Avestin LF-50 with 200 nm pore polycarbonate Whatman filter at 65-70 ºC) ten times to generate unilamellar liposomes. Then, the sample was processed with tangential flow filtration (5x 5-fold dilution / reconcentration) against a buffer of 10 mM HEPES, 145 mM NaCl (pH 7.4) with 0, 5, or 10% sucrose depending on the internal buffer osmolarity, to generate a transmembrane ammonium gradient. For remote loading, the liposomes were diluted 10-fold and then the drug was added as a hydrochloride salt to a 1 mg / ml concentration and the mixture was heated to55 ºC for 1.5 h. Then TFF (4x 5-fold dilution / reconcentration) was repeated against a buffer of 10 mM histidine (pH 7.4) with 10 or 15% sucrose. Finally, the samples were filtered through a sterile 220 nm PVDF syringe filter inside a sterile hood and the sterilized filtrate were dispensed into aliquots, sealed, and stored at 4 ºC for future use.

[0141] Sizing & Zeta potential: The Z-average diameter, polydispersity index (PDI) and zeta potential of liposomes were measured by dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS90 (Malvern, United Kingdom), reading a 1:100 diluted sample of liposomes. All measurements were carried out in triplicates with the auto mode with the following settings: 20 to 30 runs per measurement, temperature = 25 ºC, refractive index of Lipid = 1.5.

[0142] Cryo-electron microscopy (cryo-EM): Cryo-EM was performed on a ThermoFisher Scientific Glacios™ Transmission Electron Microscope (Cryo-TEM) operating at 200 kV.3 μL of purified samples were applied on glow-discharged (40 sec, 15 mA, PELCO easiGlow™, TED PELLA Inc.) holey carbon grids (Quantifoil Cu R1.2 / 1.3, 200 mesh). The grids were blotted using a Vitrobot Mark IV (FEI) with 3 sec blotting time, blot force 2 at 22°C in 100% humidity, and plunge-frozen in liquid ethane. The plunge frozen grids were clipped and loaded into the Glacios EM. The specimen was examined at 36,000x magnification and images were recorded with a pixel size of 1.15 Å using SerialEM software (University of Colorado, Boulder) and a K3 direct electron camera (Gatan). Autofocusing performed with defocus range set to 1.5-3 microns.

[0143] High-Performance Liquid Chromatography (HPLC): HPLC was carried out using an Agilent 1260 Infinity II HPLC System (Agilent Technologies, San Jose, CA) equipped with a multiple wavelength UV detector (G7165A) using Agilent OpenLab CDS software. The chromatographic separation was executed at ambient room temperature using a reverse- phase Agilent 5 HC-C18(2) column (250 mm x 4.6 mm, 5 µm particle size) (Cat no.588905- 902) equipped with an Agilent 5 HC-C18(2) guard column (12.5 mm x 4.6 mm, 5 µm) (Cat no. 520518-904). Water and acetonitrile with 0.1% trifluoroacetic acid were used as the mobile phase. A linear gradient elution was used with detection wavelength of 270 nm for ketamine (Table 8).

[0144] Drug concentration quantification. Samples were diluted 1:15 in HPLC grade Methanol and measured using HPLC according to the USP method for each drug.

[0145] Unencapsulated drug quantification. First, 2.5 mL of 1:4 diluted liposomes were loaded onto a PD10 size exclusion column. Then, buffer was added. The initial 4.5 mL eluted fraction contained the liposomes and the next 9 mL of elute contained the unencapsulated drug. The drug concentration in each fraction was determined by HPLC and the unencapsulated fraction determined as the percent in the 2ndeluted fraction versus the total in the 1stand 2ndfractions.

[0146] In vitro ultrasonic drug uncaging. First 200 μl of liposomes diluted 1:4 in canine plasma were loaded into 0.2 mL thin-walled PCR tubes placed in a custom 3D printed holder held at the focus of a 250 or 650 kHz hydrophone-calibrated focused ultrasound transducer (Fig.5). Degassed water of either 25 or 37 ºC was used to couple the transducer to the sample tube. Then ultrasound (60 s, 25% duty cycle, 5 Hz PRF, varying peak pressure) was applied. For analysis, each applicated was repeated 5x and these fractions were collected together into 1 mL. Liposomes were separated from unencapsulated drug by a homemade Sephacryl S-500 column, with PBS as the elution buffer, collecting the first 5.5 mL of elute as the liposome fraction and the next 8 mL as the uncencapsulated drug fraction. Drug concentration in each elute was quantified by HPLC.

[0147] Speed of sound measurement. A clean 20-gallon fish tank was filled with deionized water and degassed overnight. A 650 kHz 30 mm aperture f1.0 focused ultrasound transducer (Sonic Concepts Inc, Bothell, WA), a 35.6 cm long PVC cylinder with a 1.5-inch diameter, and a capsule hydrophone (Onda Corp., Sunnyvale, CA) were placed in a row underwater. Both devices were linked to an oscilloscope (Keysight Technologies, Santa Rosa, CA) to view the time of flight between the transducer and the hydrophone. The PVC pipe was wrapped in an ultrasound-compatible plastic probe cover and sealed with O-rings to create a separate internal fluid compartment. First, the pipe was loaded with 37 ºC DI water, and the ultrasound pulse arrival time was used as a reference, along with the known speed of sound in DI water at 37 ºC, for subsequent measurements. Each buffer was sequentially loaded after heating, and the difference in pulse arrival time was recorded. A temperature measurement after each run confirmed minimal heat loss. The differences in pulse arrival time due to different speeds within the length of the pipe were translated into speeds of sound of the various buffers. Finally, measurements of density at 37 ºC were performed by weighing 10 mL of each buffer using a balance.

[0148] Animals. All animal experiments were carried out in accordance with the Stanford IACUC and Administrative Panel on Laboratory Animal Care (APLAC). Male Long-Evans rats between 7 – 10 weeks old with body weight 250 – 450 g (Charles River Laboratories, Wilmington, MA; Envigo, Indianapolis, IN) were used in all in vivo studies.

[0149] Animal treatment preparation. Ketamine hydrochloride (Dechra Veterinary Products) was diluted in 0.9% sterile saline to obtain 1 mg / mL solutions. All treatments were administered via i.v. tail vein infusion continuously over 5 minutes with an infusion pump (World Precision Instruments, Sarasota, FL).

[0150] In vivo ultrasound protocol. A custom 250 kHz focused ultrasound transducer (designed and constructed by Ronald Watkins, Stanford University) powered by an amplifier (240L, E&I Ltd, Rochester, NY) was utilized in all in vivo experiments. Calibration of voltages was conducted using a hydrophone (ONDA corp). Skull attenuation was accounted for andcalculated based on weight96to achieve the desired in situ pressure. In all experiments where ultrasound was applied, continuous focused ultrasound was applied to either the mPFC or RsC after 2.5 minutes of drug infusion for 2.5 minutes (250 kHz, 25% duty cycle, 1.1 MPa estimated peak in situ pressure, 50ms pulse width).

[0151] Ultrasound simulations. To simulate the pressure-field distributions during FUS treatment, a male Long Evans rat (weight: 453 g) was imaged on a Quantum GX micro-CT (Waltham, Massachusetts, USA). The obtained micro-CT image was 1024x1024x553 with a cubic voxel size of 0.086 mm. The images were resampled linearly to a cubic voxel size of 0.34 mm. The bone, soft tissue, and water were isolated based on their Hounsfield units (1200 HU for bone / soft tissue and 930 HU for soft tissue / water threshold). Density and sound speed were linearly interpolated in each region using hounsfield2density, a predefined function through the k-Wave MATLAB (Natick, Massachusetts, USA) toolbox97. The region surrounding the animal was defined as water. The transducer was defined as a bowl with a diameter and radius of curvature of 100 mm. At 250 kHz center frequency the points per wavelength was 17.44 in water and Courant–Friedrichs–Lewy stability criterion of 0.1 leading to a time step of 22.8 ns. The simulation was run for 85 µs allowing the initial wave to travel to the length of the simulation grid (125 mm).

[0152] Solid Phase Microextraction (SPME) and Pharmacokinetics. Chemical and materials. The Cerilliant® certified standard solutions of ketamine hydrochloride, ketamine-D4 hydrochloride, norketamine hydrochloride, norketamine-D4 hydrochloride, and hydroxynorketamine hydrochloride, were purchased from Millipore Sigma, USA. The Supel™ BioSPME 96-Pin Devices (product number 59680-U) were also procured from the same source. For the LC / MS grade water, acetonitrile, 2-propanol, methanol, and formic acid, the procurement was made through Fisher Scientific.

[0153] Animal preparation. Rats were fixed into a stereotaxic frame and anesthetized under isoflurane for the entire terminal experiment. They were administered with 2mL of saline subcutaneously and kept on a heating pad at 37ºC. After dorsal scalp exposure, 2-mm burr holes were drilled into the skull for SPME pin insertion (3.2 mm posterior to bregma for mPFC or 4mm anterior to bregma for RsC, both at -1mm lateral to midline). A durotomy was performed with a 32g needle. For rat subjects that received ultrasound, the ultrasound transducer was positioned directly above the desired burr hole via a 3-axes positioning system (ThorLabs, Newton, NJ), and ultrasound gel was used for coupling. Rats received 1.5 mg / kg of Sonoket, ketamine HCl, or saline vehicle.

[0154] SPME sample preparation and sampling. The SPME pins were removed from commercially available BioSPME 96-pin devices, cleaned using a solution of methanol, acetonitrile, and isopropanol (in a 2:1:1 v / v / v ratio). Pins were preconditioned overnight in a methanol: water solution (1:1 v / v) and then stored in water until sampling.

[0155] During the experiment, the pins were loaded into a custom-designed, 3D-printed stereotaxic holder that had been tailored for precise positioning and sampling. Pins were inserted 3 mm ventrally into the brain via the burr holes (SPME absorptive medium centered at ~2.5 mm ventral) and left in contact with the rat brain tissue for 5 minutes immediately following the drug administration. Repeated sampling with new SPME pins occurred every 5 minutes post-treatment for 4 times to measure tissue pharmacokinetics over time (Fig 2b-c). The SPME pins passively sampled the drug and its metabolites through diffusion. Post- sampling, the SPME pins were removed and washed for 5 seconds via static immersion in water to effectively remove any residual blood. Following washing, the SPME pins were desorbed into 50 µL of MeOH: H2O (9:1 v / v) solvent containing 1% formic acid and deuterated internal standards. The desorption process occurred for 30 minutes with agitation at 1500 rpm at room temperature. The desorbed aliquots were quantified by LC-MS / MS analysis employing an instrumental calibration curve.

[0156] Blood pharmacokinetics. Blood samples (~400 μL) were collected for adult rats (n=3 / group) who received 1.5 mg / kg i.v. infused SonoKet without ultrasound and ketamine HCl in the same cohort sampled for SPME (Fig.2e). Samples were collected via tail snipping into Eppendorf tubes loaded with EDTA (Millipore sigma; 50 μL Na2EDTA) at 0, 5-, 10-, 15-, 20-, and 25-min post-treatment. Another 8 adult rats (n=4 / group), following anesthesia, received an intravenous bolus dose of 1 mg / kg of ketamine as either free ketamine-HCl or ketamine liposomes (Fig 7). Then, blood samples (approximately ~400 μL) were collected by tail snipping into Eppendorf tubes pre-loaded with 50 μL Na2EDTA at 1, 2.5, 5, 10, and 15 min.

[0157] Blood sample processing. Blood samples (100 μL) from each time point were aliquoted into three individual Eppendorf tubes, followed by the addition of 5 μL of deuterated internal standards (ketamine-D4 and norketamine-D4 at a concentration of 1 μg / mL) to each tube. Protein precipitation was carried out with 500 μL of cold acetonitrile. To facilitate extraction, the samples were subjected to an hour of extraction at room temperature using a mechanical shaker with agitation at 1500 rpm. Following this extraction, the samples underwent centrifugation at 12000 rpm and 4°C for 10 minutes. The resulting supernatant was transferred to new Eppendorf tubes, and the solvent was evaporated to dryness. Each dried sample was then reconstituted with 100 μL of MeOH: H2O (9:1 v / v) solvent containing 1% formic acid and then centrifuged once more under identical conditions for 10 min. The final supernatant was transferred to an insert containing HPLC vials and quantified by LC-MS / MS using the instrumental calibration curve. The Calibration curve standards ranging from 0.1 to 100 ng were prepared in MeOH: H2O (9:1) solvent containing 1% formic acid and internal standards at a concentration 50 ng / mL.

[0158] Biodistribution. Adult rats (n=4 / group) received either free or liposomal ketamine as an intravenous bolus dose of 1.5 mg / kg. Rats were sacrificed at 1 hour from the time of administration and perfused with 1x phosphate buffer (PBS) via transcardial perfusion to remove blood from the systemic circulation. Afterward, the brain, liver, kidney, spleen, lung, heart, and spinal cord, were collected and stored at -80 °C.

[0159] The solid organ samples were homogenized in equal-weight volume of 1x phosphate buffer (PBS) using a handheld homogenizer.100 μL of tissue homogenate was aliquoted from each organ in triplicate per organ. Similarly, 100 μL of blood sample from each time point was aliquoted into triplicates. Deuterated internal standards (ketamine-D4 and norketamine-D4) were added to each blood and tissue homogenate sample. Protein precipitation was carried out with 500 μL of cold acetonitrile. The samples were subjected to an hour of extraction using a mechanical shaker, followed by centrifugation at 12000 rpm and 4°C for 10 minutes. The resulting supernatant was collected and after evaporation of the organic solvent, the sample was reconstituted with 100 μL of MeOH:H2O (9:1) containing 1% formic acid and quantified by LC-MS / MS analysis using an instrumental calibration curve.

[0160] LC-MS / MS analysis. The LC-MS / MS analysis was carried out using the Agilent 1290 Infinity LC System coupled to the 6490 Triple Quadrupole LC / MS System with iFunnel technology (Agilent Technologies, San Jose, CA, U.S.A.). The LC-MS / MS analysis was executed in the positive mode, utilizing Agilent Jet Stream technology for electrospray ionization (AJS-ESI), operating under the optimized MS-MS conditions (Table 5). The fragmentor voltage of 380 V and a cell accelerator voltage of 5 V was consistently applied for all SRM transitions. Data acquisition and processing were performed using Agilent MassHunter Workstation Data Acquisition (B.08.00) and MassHunter Workstation Quantitative Analysis (B.07.01) software (Agilent Technologies, San Jose, CA, U.S.A.) respectively. The configuration of the MS source and the optimized parameters (Table 6). The chromatographic separation was performed at room temperature using Agilent ZORBAX RRHD Eclipse Plus C18 (5 cm x 2.1mm, 1.8 µm particle size) column (Part Number: 959757- 902) equipped with an Agilent ZORBAX RRHD Eclipse Plus C18 guard column (2.1 mm, 1.8 µm) (Part Number: 821725-901). The aqueous mobile phase (A) consisted of LC / MS grade water with 0.1 % formic acid, while the organic mobile phase (B) consisted of LC / MS grade acetonitrile with 0.1% formic acid. The injection volume was 5 μL. A constant flow rate of 300 µL / min was applied with a chromatographic gradient (Table 7). The column pressure and autosampler temperature were set at a maximum of 800 bar and 5 °C respectively. The analytical response, represented as relative peak area ratios (analyte to internal standard) was converted to amounts extracted by employing an instrumental calibration curve consisting of analytes in the desorption solvent in the range of 0.1-100 ng / mL.

[0161] Awake-Restraint Electrophysiology Recording and Analysis. Surgical setup. Animals were anaesthetized with isoflurane (5% induction, 1.5–2.5% maintenance) and positioned in a stereotaxic frame for implantation of custom electrocorticography (ECoG) electrodes. A midline incision was made over the scalp and five holes were drilled through the skull with stereotaxic guidance. Electrodes for mPFC were positioned at 1 mm anterior, and 2 mm right lateral to bregma (positive electrode); 6 mm posterior and 2 mm right lateral to bregma (ground electrode); and 6 mm posterior and 2 mm left lateral to bregma (reference electrode). Two stabilizing screws were implanted at 2 mm posterior and 3mm bilaterally to bregma for structural integrity. Electrodes for RsC were positioned at 5 mm anterior, and 3 mm right lateral to bregma (positive electrode); 11 mm posterior and 3 mm right lateral to bregma (ground electrode); and 11 mm posterior and 3 mm left lateral to bregma (reference electrode). Two stabilizing screws were implanted at 8 mm posterior and 4mm bilaterally to bregma for structural integrity. Electrodes were then screwed into the skull without breaching the dura, and dental cement applied to the skull to fix the electrodes in place. After recovery from anesthesia, animals were housed in separate cages for the duration of the experiment and allowed for at least 7 days of recovery before recordings.

[0162] ECoG Recording. At the beginning of the recording session, rats were briefly anesthetized with isoflurane and catheterized via tail vein. While under anesthesia, animals were placed in a thin, flexible plastic restraint cone (Amazon.com) and positioned in a custom head-restraining apparatus. Rats receive oxygen via nose cone to prevent hypoxia. Recordings were performed with an 8 Channel Cyton Biosensing Board at a sampling frequency of 1000 Hz. For rat subjects that received ultrasound, the ultrasound transducer was positioned directly above either the mPFC (+3.2mm A / P, -0.5mm M / L from bregma) or the RsC (-4mm A / P, 0mm M / L from bregma) via the 3-axes positioning system and coupled with ultrasound gel. Implanted screws were utilized for precise targeting according to the rat brain atlas. Data acquisition began 25-30 minutes after the animal is in the restraint to allow for complete isoflurane clearance. A baseline acquisition (5 min) was recorded prior to starting the treatment via i.v. infusion. Rats received 0.75 mg / kg of Sonoket, ketamine HCl, or saline vehicle. We acquired data continuously for 35 mins following a 5-minute baseline and a 5- minute treatment infusion protocol, for a total recording of 45 minutes.

[0163] Data Analysis. Electrophysiological data was filtered using MNE-Python with a bandpass filter with a low cutoff of 1 Hz and a high cutoff of 200 Hz. The data was then denoised by decomposing into 5 levels of Daubechies 8 wavelets, zeroing outlier coefficients, then reconstructing the modified data. For full session spectrograms, the first 45 minutes of the recording was defined as a single epoch and time-frequency representation was computed with a multitaper technique and adjusted to baseline (initial 0-5 min of recording) with percent change from baseline using MNE-Python. For band power trace plots, the time-frequencyrepresentation was averaged within frequency band cutoffs (delta: 1-4 Hz, theta: 4-8 Hz, alpha: 8-15 Hz, beta: 15-25 Hz, gamma: 25-55 Hz) and a moving average of the band power was computed with a 2-minute-long convolving window. One recording within the RsC 1 mg / kg ketamine HCl condition was excluded due to excessive artifacts that could not be corrected by denoising. Area under the curve (AUC) was calculated by integrating the percent of power change within each frequency band as indicated above in time-bins indicated as time of sonication (7.5-10 min), time immediately after treatment (10-25 min), and time after clearance (25-45 min) and depicted in arbitrary units.

[0164] Behavioral Open-Field Analysis. Rats were surgically implanted with a targeting screw and given at least 7 days to recover prior to behavioral tests. To acclimate to the experimenter and reduce stress, rats were handled 3 days prior to recording. All behavioral tests were performed in an environmentally controlled room. Open-field locomotor activity was recorded from above in a custom-built white Plexiglas apparatus (90 cm × 90 cm × 40 cm). Animals were placed at the center of the field and randomized for treatment group.

[0165] Rats were placed under isoflurane (2%) briefly to be catheterized via tail vein and loaded into the awake restraint. They were administered oxygen via nose cone within the restraint to prevent hypoxia. After ensuring the rats are fully awake prior to starting treatment, rats received an i.v. infusion of 0.75 mg / kg SonoKet, ketamine HCl, or saline vehicle for 5 minutes. Ultrasound was applied to the mPFC or RsC beginning at 2.5 minutes after the start of infusion and continued till the end of infusion. After treatment, rats were immediately placed at the center of the arena, where they were allowed to freely explore for 20 minutes while locomotor activity was recorded.

[0166] Prior to analysis, videos were first reencoded for format compatibility and clipped to 20 minutes starting at 5 seconds after the initialization of the recording with FFmpeg. ToxTrac was then used to track animal position with tracking settings matching the ToxId algorithm and detection settings adjusted ad hoc. Custom Python scripts were then used to quantify and plot cumulative frame-to-frame distance travelled and time spent in the center of the open field, which was defined as a concentric square field with half the width of the full square field.

[0167] Histological safety analysis. Drug administration and tissue collection. Rats were administered an i.v. infusion of 0.75 mg / kg SonoKet, dose-matched ketamine HCl, or vehicle over 5 minutes (n=3 / group), followed by the same ultrasound application to either the mPFC or RsC. After 24 h post-treatment, the animals were anesthetized with isoflurane (5%) and transcardially perfused with 1x phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) diluted in PBS. Brains were extracted and stored overnight in 4% PFA, cryopreserved in 15% sucrose for the next 48 hours, and lastly, in 30% sucrose for the last 48 hours. They were then subsequently washed in PBS and frozen in embedding medium. Coronal brain sections (40 µm thick) were cut using a CM1800 Cryostat (Leica Microsystems).These sections were stored in a solution of 30% sucrose and 30% ethylene glycol in 0.1 M PB at -20 °C until processed for immunohistochemistry.

[0168] Microscopy and image analysis. Sections were stained with hematoxylin and eosin (H&E), Fluorojade-C, GFAP and IBA-1 to evaluate for parenchymal damage, neuronal degeneration, and astrocytic or microglial activation, respectively. Alexa Fluor® 488 secondary antibody was used after primary anti-GFAP incubation. Cy5® secondary antibody was used after primary anti-IBA1 incubation. Tissue sections were free-float mounted on microscope glass slides (Fisher, Pittsburgh, PA). All histology images were collected with a fluorescence microscope (BZ-X800, Keyence Corp., Itasca, IL). For quantifiable histological markers (Fluoro-Jade C, IBA-1+, and GFAP+ cells), signals above the threshold background were used for manual ROI segmentation to calculate the total mean fluorescent area of cells using BZ-X Advanced Analysis Software (Keyence Corp., Itasca, IL). Mean fluorescent intensity is reported in arbitrary units.

[0169] General statistical analysis. Rats were randomly assigned to treatment conditions. Data where n ≤ 4 were plotted as barplots and n ≥ 5 were plotted as box plots. For comparisons between two groups, a two-tailed, two-sample Student's T-test was conducted. For comparisons between multiple groups, a one-way ANOVA was conducted, followed by a post- hoc Tukey's honestly significant difference (HSD) test for pairwise comparisons. Effect size was calculated using Hedge’s g. All comparisons were two-tailed. Statistical tests, sample sizes N, corrected p values, and effect sizes g are reported for each analysis in the text and figure captions. All statistical analyses were performed using custom scripts in Python. References

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[0268] Rodriguez, A. et al. ToxTrac: A fast and robust software for tracking organisms. Methods Ecol. Evol.9, 460–464 (2018). Table 1 Formulation Average PDI Zeta Drug Free Entrapment pKa LogP Size (nm) Potential Loading Drug EfficiencyNote: S5 = 5% additional sucrose, S10 = 10% additional sucrose in the internal buffer of the liposome. The pKa and logP values were derived from PubChem and DrugBank. Table 2 Buffers Density Speed of Acoustic Bulk Osmolarity Sound Impedance Modulus )Table 2. Measured acoustic parameters and osmolarity of plasma and the varied liposome internal and external media. Table 3 KetamFree Ketamine P-value AAL vs Unitsine AALTmaxm 1 ± 0 1 ± 0 NA C0ng / mL 4708.81 ± 720.06 1074.98 ± 138.67 * 0.010 dAAL or free ketamine bolus intravenous administration. Cmax: maximum concentration; Tmax: time of maximum concentration; C0: concentration extrapolated to t0; t1 / 2: half life; AUC0-t: area under the curve from 0 to last time point; AUC0-∞: area under the curve from 0 to infinity; MRT0- t: mean residence time from 0 to last time point; MRT0-∞: mean residence time from 0 to infinity; VZ: volume of distribution; Cl: clearance; VSS: volume of distribution at steady state; *: p < 0.05; **: p < 0.01; ***: p<0.001 Table 4 Ketamine AAL Free Ketamine P-value AAL vs Free UnitsTmaxm 1 ± 0 1 ± 0 NA C0ng / mL 4708.81 ± 720.06 1074.98 ± 138.67 * 0.010 nextrapolated to t0; t1 / 2: half life; AUC0-t: area under the curve from 0 to last time point; AUC0-∞: area under the curve from 0 to infinity; MRT0-t: mean residence time from 0 to last time point; MRT0-∞: mean residence time from 0 to infinity; VZ: volume of distribution; Cl: clearance; VSS: volume of distribution at steady state; *: p < 0.05; **: p < 0.01; ***: p<0.001. Pharmacokinetic analysis of ketamine and its metabolites following ketamine-loaded AAL or free ketamine 1.5 mg / kg bolus intravenous administration for Fig 7. Table 5 Precursor Dwell Time Collision Energy CompoundsProduct (m / z)Ketamine 238.21 220 60 10 Ketamine-D4 (Internal Standard) 242.21 129 60 26Table 6. Optimized MS parameters Source name AJS-ESI Drying Gas Temperature (°C) 225 °Ca e . o aogap c g a e o - ayss Time (min) 0 1 4.5 4.9 5 6.0Table 8. Chromatographic gradient for HPLC Analysis Time (min) 0.0 0.3 10.30 10.50 10.75 12

[0269] This disclosure provides a system for ultrasonic drug uncaging that 1) enables high drug loading potential for varied drugs of interest, 2) has minimal drug uncaging without ultrasound, 3) can be activated following intravenous administration and not necessarily intraparenchymal injection, 4) is formulated with common pharmaceutical excipients, and 5) has an ultrasound-activation mechanism that is mechanically-based without necessitating medium heating above body temperature, nor risk of inertial cavitation, and could be activated with a lower duty cycle, shorter ultrasound protocol than is used for heat or sonodynamic activation. We have been able to accomplish this by tuning the acoustic impedance and osmolarity of the liquid core of liposomes to maximize ultrasound responsiveness whilemaintaining overall stability of the liposome structure without ultrasound. Further, we have been able to formulate these acoustomechanically-activatable liposomes (AALs) with common pharmaceutical excipients while seeing effective ultrasonic drug uncaging with ultrasound intensities within FDA and society safety guidelines, indicating that this system has high clinical translational potential. RESULTS

[0270] Design and development of acoustomechanically activatable liposomes (AALs). In designing a next-generation ultrasound-activatable drug carrier for ultrasonic drug uncaging, we first considered prior successful formulations that utilize a liquid perfluorocarbon nanodroplet as the ultrasound-responsive element to allow uncaging of small lipophilic drugs. We noted that in some of these implementations, while inertial cavitation or a liquid-to-gas phase transition may be involved with the drug uncaging mechanism, neither inertial cavitation nor gas phase induction was observed during drug uncaging with our prior formulation. We then hypothesized that we could retain ultrasound sensitivity in the system by replacing the core perfluorocarbon with a liquid droplet that has a differential acoustic impedance to mediate an ultrasound interaction. Further, by choosing a replacement liquid that can also be loaded with a drug, we would increase the drug loading potential of the system and likely also the overall stability of drug binding, particularly for water soluble drugs. Additionally, by replacing the perfluorocarbon for more commonly used pharmaceutical excipients, we would increase the clinical translatability of the system. Indeed, several candidate aqueous buffers show significantly varying acoustic impedance measurements in the literature, suggesting that a liposome could be optimized for ultrasound response by incorporating elements to shift the acoustic impedance of its internal buffer. Finally, if the internal buffer has a substantially different osmolarity compared to the surrounding medium, following the membrane leakiness induced by the initial ultrasound application, this osmotic gradient would induce a fluid shift thereby accelerating further drug uncaging with ultrasound (Fig.14A).

[0271] To implement this hypothetical system, we made liposomes containing varied percentages of sugars in its internal loading battery. We chose to start with liposomes as they are now commonly used drug delivery vehicles formulated with excipients in the FDA Inactive Ingredients Database. We further chose to incorporate sugars into the internal buffer of the liposome since moderate percentages of sugars have been shown to substantively shift the acoustic impedance of aqueous buffers. Additionally, since these sugars are GRAS (Generally Recognized As Safe) excipients for pharmaceutical formulations, this system would maintain high clinical translational potential for the eventual vehicle. We tested this system with loading of the anesthetic and antidepressant ketamine, a lipophilic amphipathic weak base that we found could be actively loaded into liposomes with an ammonium sulfate loading battery(Table 9). Compared with a ketamine-loaded liposome with no added internal excipient other than the ammonium sulfate loading battery, liposomes incorporating varied percentages of sucrose showed significantly increased ultrasonic drug uncaging performance: with 5% sucrose added to the internal buffer yielding ~40% drug uncaging with a 250 kHz, 25% duty cycle, 1 min ultrasound protocol, and 10% added sucrose yielding ~60% drug uncaging with this same protocol, compared to a nonsignificant trend towards ~20% drug uncaging with no added sucrose (Fig. 14B). Notably, there were no significant differences in key physicochemical properties including size, polydispersity, zeta potential, drug loading, or unencapsulated drug percent (Table 9).

[0272] We next noted that increasing sucrose percentages would yield increases in both the acoustic impedance and the osmolarity of the internal buffer. To discern which parameter, acoustic impedance or osmolarity, contributed more to our results we next prepared liposomes with equiosmolar internal buffers, but differential acoustic impedances (Table 10). Drug- loaded liposomes with internal buffers composed of ammonium sulfate with equiosmolar additional sodium chloride, glucose, or sucrose were prepared and found to have similar physicochemical characteristics with each other (Table 9). Despite similar internal osmolarities, they showed differing amounts of drug uncaging with the same ultrasound protocol, in correlation to their differences in internal acoustic impedance (Fig.14C, D; Table 10). This indicates that acoustic impedance differences of the inner medium of a liposome likely contribute to the ultrasound-responsiveness of the drug-loaded nanocarrier. Potentially, an internal-external osmolarity gradient then further contributes to increase this drug uncaging following the initial ultrasound-induced membrane leakiness. Notably, while 10% additional sucrose internally significantly increased the amount of ultrasonic drug uncaging relative to 5% additional sucrose, it did so at the expense of displaying a significant rate of drug uncaging without ultrasound when incubated with plasma at 37 ºC, indicating that there is a relative tradeoff of ultrasound responsiveness and formulation stability with this scheme (Fig.14E). For further experiments, 5% sucrose added internally was chosen as a relative optimum of ultrasound responsiveness with stability of the formulation with body temperature incubation in plasma.

[0273] Mechanism of AAL-mediated ultrasonic drug uncaging. To further explore the mechanism of drug uncaging with this system, the ultrasound frequency was varied between either 250 kHz or 650 kHz, usual frequencies for transcranial therapeutic ultrasound application clinically. Significant drug uncaging was seen with 250 kHz sonication, with increasing peak pressure / mechanical index (MI) correlating to increased drug uncaging, similar to prior results with liquid perfluorocarbon based systems (Fig.15A). Intuitively, there was increased drug uncaging seen with a higher medium temperature of 37 ºC versus 25 ºC (Fig.15A). In contrast, with 650 kHz sonication, only marginal drug uncaging was seen withultrasound, with a minimal dose-response relationship with ultrasound peak pressure / MI, and with only minimal variation with medium temperature (Fig.15D). This is in distinct contrast to perfluorocarbon-based systems that show significant drug uncaging across a range of frequencies up to and beyond 1 MHz. This increased responsiveness at 250 kHz compared to 650 kHz argues against both a thermal or radiation force type mechanism for the drug uncaging, since both thermal and radiation force transfer mechanisms tend to increase with increasing acoustic attenuation coefficients, which in turn increase with the acoustic frequency. Arguing against inertial cavitation as an underlying drug uncaging mechanism, the liquid phase of the formulation particles has a low vapor pressure and we observed no evidence of wideband acoustic scatter with sonication of the liposomal formulation, in contrast to sonication of gas-phase microbubbles, which readily generate these signals of inertial cavitation (Fig. 15B, C, E, F). Instead, with sonication conditions sufficient for robust drug uncaging (250 kHz, 0.9 MPa estimated peak in situ pressure, 37 ºC) we observe increases of the harmonic and ultraharmonic bands in the observed acoustic scatter, consistent with an oscillation of the particles in these conditions (15B, C); with no such differential signals seen with sonication with 650 kHz (Fig.15E,F) nor at room temperature, conditions in which no or minimal ultrasonic drug uncaging was observed (Fig. 15A, D). By electron microscopy, the formulation particles show the expected spherical morphology, but with no internal precipitation of the drug that is shown to correlate to stability of drug loading with other liposomal formulations. With sonication, the size distribution of the particles decreased, cohering with an ultrasound-induced leak of the internal contents (Fig.15G). Nonetheless, this formulation shows stability for both drug loading and ultrasonic uncaging across months of time in refrigerated storage (Fig.15H). Together, this frequency response difference and these other characterizations suggest an oscillation-based acoustomechanical type drug uncaging mechanism, mediated by the differential acoustic impedance of the internal core droplet, contributing to increased permeability of the liposome membrane to the loaded drug with ultrasound. Notably, effective ultrasonic uncaging was seen at body temperature with 250 kHz ultrasound and an in situ peak negative pressure of 0.9 MPa, or a mechanical index (MI) of 1.8, which is within FDA and AIUM guidelines for safe ultrasound application (without microbubbles present, as in this application). Accordingly, we chose this ultrasound protocol for subsequent evaluations.

[0274] AALs as a generalized platform for ultrasonic drug uncaging. To confirm that these results were not somehow specific to the particular drug used in these initial experiments, we prepared liposomes with or without 5% additional sucrose internally, and loaded with several other agents: lidocaine, bupivacaine, and ropivacaine. These liposomes show similar size, polydispersity, and zeta potential as the ketamine-loaded liposomes (Table 9). They showed differential drug loading rates that likely reflect the specific chemical characteristics of eachdrug, such as their logP and pKa, as they relate to the ammonium sulfate active loading mechanism (Table 9). Each of these liposomes showed increased ultrasonic drug uncaging with the formulations incorporating added sucrose internally, while formulations without added sucrose showed no significant or minimal drug uncaging with ultrasound (Fig.16).

[0275] In vivo targeted drug delivery achieved by AAL-mediated ultrasonic drug uncaging. With these in vitro characterizations demonstrating in vitro stability and ultrasonic drug uncaging efficacy, we next characterized their performance in vivo. Following a bolus intravenous injection, the blood drug concentration showed differential blood pharmacokinetics with an acoustomechanically activatable liposome (AAL) versus a dose- matched administration of a free unencapsulated drug formulation (Fig. 17A, Table 11). Notably, there were also higher drug metabolite blood levels with the AAL formulation compared to the unencapsulated drug formulation. This behavior was reflected in the biodistribution at 1 hour post bolus administration, in which the solid organs showed increased accumulation of the drug with the free unencapsulated form administration compared to the AAL formulation (Figs. 17B). The combination of the higher blood drug metabolite concentration yet lower unmetabolized drug accumulation in the solid organs with the AAL formulation suggests that some of the blood clearance of the drug with the AAL formulation is likely due to the sequestration and metabolization of the whole particle by end organs like the liver. Further, it is noted that this end organ sequestration and metabolism could contribute to some degree of nonspecific leak of the drug into circulation, and may be a less present mechanism in humans vs. rodents given the lower hepatic blood flow and hepatic mass (normalized by body weight) in humans.

[0276] To assess the differences induced of in vivo pharmacodistribution with ultrasonic drug uncaging with AALs, in adult rats we used solid-phase microextraction (SPME) to measure the amounts of ketamine delivered to the brain either with a free unencapsulated drug infusion or an AAL infusion, with or without ultrasound application to the brain. Ultrasound (or sham administration) was applied to a frontal cortical and hippocampal region unilaterally following a bolus administration or during the latter 2.5 min of a 5 min intravenous infusion of either free, unencapsulated ketamine-HCl or of ketamine-loaded liposomes (Fig.18A). After the stop of infusion and sonication, SPME was used to sample both the sonicated brain and a contralateral control region. A venous blood sample was taken at the end of the SPME sampling period. Varying either ultrasound parameters such as duty cycle or the total dose of the AAL yielded a dose-response relationship between each parameter and the amount of drug delivered to the brain (Fig.18B, C). Comparing the different liposomal formulations, the blood samples showed higher blood levels of ketamine with each liposomal formulation compared to free drug, confirming our blood pharmacokinetics results following bolus administration (Fig. 18D, 17A). Nonetheless, despite the higher blood concentrations ofketamine with liposomal formulation administration, the ketamine brain levels seen either with sham treatment or in the contralateral control region were less than half of that seen following a dose-matched free unencapsulated ketamine infusion (Fig. 18E). While ultrasound application did not measurably alter the pharmacodistribution of ketamine following a free ketamine infusion, ultrasound application yielded significantly higher brain ketamine levels with the liposomal infusions compared to the sham condition or to the contralateral non- sonicated region (Fig. 18E). Additionally, the significantly higher brain concentration in the sonicated region versus the contralateral non-sonicated brain that was separated by 5 mm (Fig.18A, E) confirmed the spatial resolution of this technique to be at least 5 mm with 250 kHz ultrasound. Notably, this up to 5 mm of spatial localization of the uncaging effect is close to the full-width at half-maximum of the 250 kHz ultrasound beam used in this application (FWHM = 4 mm, or 2 mm half-width at half-maximum, at this focal distance per hydrophone based beam characterization), confirming that this uncaging is limited spatially by the applied ultrasound field. Furthermore, significantly higher brain ketamine levels were seen with sonication with liposomes made with additional sucrose internally compared to liposomes without added sucrose, confirming that this relatively simple formulation manipulation increases the ultrasound sensitivity of the system both in vitro and in vivo. Notably, despite higher ultrasonic drug uncaging efficacy in vitro, no significant difference was seen in vivo between liposomes made with 10% added sucrose internally, compared to those made with 5% added sucrose, likely reflecting the relative instability in plasma of the 10% added sucrose formulation seen in vitro (Fig. 14E). Importantly, histological assessment for morphological tissue damage, neuronal degeneration, microglial activation, and astrocytic activation and gliosis yielded no evidence of brain parenchymal injury seen with AAL-mediated brain ultrasonic drug uncaging (Figs.18F-J).

[0277] Functional noninvasive nerve block achieved by AAL-mediated ultrasonic drug uncaging. After confirming that the AAL strategy yielded targeting of the pharmacodistribution of the drug to the sonicated field in the brain (Fig. 18), in looking to confirm the functional efficacy and the generalizability of this strategy we first noted that the functional response of the brain to drugs such as ketamine may be highly variable and turned to targeted drug delivery to a peripheral site. Specifically, we tested whether uncaging an AAL loaded with a local anesthetic could be used to induce anesthesia of a peripheral nerve, as a form of a noninvasive needle-less nerve block. Adult rats were administered a 5 min intravenous infusion of either saline or a ropivacaine-loaded AAL (S5-RL). With 2.5 min of pulsed sonication applied to the sciatic nerve of one limb during the latter half of the infusion, a dense anesthesia to a mechanical (von Frey fiber) stimulus was observed only in the sonicated limb of the ropivacaine-AAL administered animals, with this anesthesia lasting at least 4 hours following infusion and sonication (Fig.19A-C). No such change was seen in either limb of thesaline infused animals or in the contralateral non-sonicated limb of the ropivacaine-loaded AAL treated animals (Fig.19B, C). Notably, in rats receiving this same dose of ropivacaine- loaded AALs, no significant electrocardiographic abnormalities or visible intolerances were noted (Fig.19D-F) and histologic evaluation of the sonicated region confirmed no concerning anatomic damage (Fig.10G-K), confirming the safety of this approach.

[0278] With these results, we have demonstrated a system for ultrasonic drug uncaging that can be loaded with varied drugs with high drug loading, minimal drug uncaging without ultrasound, and effective drug uncaging with pulsed low-intensity ultrasound both in vitro and, importantly, in vivo for multiple drugs of interest. To analyze the underlying mechanism of AAL-mediated ultrasonic drug uncaging we turn to the framework provided in a recent review of ultrasound-mediated drug delivery that identified thermal absorption, radiation force, acoustic streaming, oscillation (stable cavitation), or inertial cavitation as the main plausible mechanisms for ultrasound-material interactions. The frequency response we observe with more effective drug uncaging at the lower ultrasound frequency (Fig. 15) argues against thermal absorption and radiation force type mechanisms as both these processes tend to increase with ultrasound frequency. In addition, the length scales and tortuosity of the capillary vasculature compared to the in situ ultrasound wavelength argue against acoustic streaming underlying the effective in vivo drug uncaging we observe with AALs (Figs.18,19). Finally, the spectra of acoustic signals scattered during AAL-mediated ultrasonic drug uncaging (Figs.15) argue against inertial cavitation as the underlying mechanism and instead are more consistent with an ultrasound-induced oscillation of the liposomes.

[0279] Regarding why this oscillation of AALs occurs with 250 kHz ultrasound specifically and not with a similar pressure or MI of 650 kHz ultrasound, we note that the bulk modulus and therefore the resonant frequency of a liposome-like structure depends on the shear modulus of the shell and the bulk modulus of the internal fluid medium, with the combined modulus being intermediate between the two. The relevant modulus of the lipid bilayer shell is different between room and body temperature, likely accounting for the differential uncaging efficacy and observed acoustic scatter spectra between the room and body temperature experiments (Figs. 15). In adding sucrose to the core of a liposome, we increase its internal acoustic impedance and bulk modulus (Table 10), and therefore the resonant frequency of the whole structure. In this implementation, this resonant frequency appears to be closer to 250 kHz than 650 kHz. While we have focused our attention on implementing this system using common pharmaceutical excipients to maximize clinical translatability, an implication of this oscillation- based acoustomechanical uncaging mechanism is that an even further ultrasound-responsive system could be achieved by replacing sucrose with equiosmolar amounts of a different substance that even further shifts the core bulk modulus, which in turn would shift the optimaldrug uncaging resonant frequency. We contend that this ultrasound induced oscillation of the liposomes increases permeability of the lipid bilayer to both the loaded drug and to water, allowing drug egress into the external medium and water ingress into the hyperosmolar core fluid, thereby inducing and then accelerating drug uncaging (Fig.14A).

[0280] Underlying the clinical translatability of this system is that the ultrasound intensities and protocols sufficient for drug uncaging in vitro and in vivo in these demonstrations are within FDA and AIUM guidelines for safe ultrasound application and are readily achievable using current clinically utilized therapeutic focused ultrasound systems. Additionally, the AAL formulation constituents are commonly used and validated pharmaceutical excipients, with the simple disaccharide sucrose being the main additional ingredient yielding increased ultrasound sensitivity in this current AAL implementation. Furthermore, the manufacturing methods for liposome production used here are compatible with Good Manufacturing Practices (GMP) and the formulation ingredients are each validated excipients in the FDA Inactive Ingredients Database, together indicating a ready path to clinical translation of this system. In addition, by equilibrating this formulation in a storage buffer whose tonicity and pH are matched to that of plasma (Table 10), we are able to avoid an osmotic shock to the liposomes upon infusion in vivo and see that this formulation is stable for months in refrigerated storage (Fig. 15H), a decided practical advantage over prior formulations that necessitated frozen storage or fresh preparation. Finally, these AALs enable targeted drug delivery to within a few millimeters resolution in the rat brain following an intravenous infusion (Fig.18), a notable practical advantage over systems necessitating direct intraparenchymal injection of the drug-loaded formulation.

[0281] While an ammonium sulfate based active loading scheme was demonstrated here, the strategy of incorporating elements to increase the acoustic responsiveness of the liposome is applicable to any liposome with any internal core buffer and loading battery. Of note, there was a nonzero rate of drug leak without ultrasound both in vitro and in vivo. The in vivo ultrasonic uncaging effect size seen with these AALs is sufficient to identify a relevant therapeutic index for each drug tested, and are commensurate with or exceed the demonstrated on- vs. off-target delivery of other benchmark systems that have been so characterized. MATERIALS AND METHODS

[0282] Materials. All the chemicals and reagents used in the study were of the highest purity grade with sourcing as follows. Lipoid: Hydrogenated Soy Phosphatidylcholine (HSPC) (Cat no: Lipoid S PC-3) and [N-(methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3- phosphoethanolamine, sodium salt] (DSPE-PEG 2000) (Cat no: PE18:0 / 18:0 – PEG 2000). Evonik: Cholesterol (PhytoChol Inject HU). Ketamine-HCl injectable solution (100 mg / mL;Dechra Pharmaceuticals, procured through Stanford University Environmental Health & Safety). Astatech: Ropivacaine-HCl (Cat no. 42084) and Bupivacaine-HCl (Cat no. 42055). Hiemdia: Ammonium Sulfate (Cat no: PCT0003). Fisher Scientific: Absolute Ethanol (200 proof) (Cat no: BP2818-100), Sucrose (Cat no: S5-500), HPLC and LC / MS grade Water, Methanol, Acetonitrile, 2-Propanol, Formic Acid. Sigma Aldrich: Lidocaine-HCl (Cat no. L5647), HEPES buffer solution (Cat no: 83264-500ML-F) and L-Histidine monohydrochloride monohydrate (Cat no: H8125). Repligen: TFF filters (C02-S05U-05-N (SN: 20020493-03 / 21- 057). Lampire Biological Laboratories: Male Canine Plasma (Cat no.7302009). Cytiva: PD- 10 column Sephadex G-25 M (Cat no.17085101), Sephacryl S-500 (Cat no.1706130). Milli- Q water was used to prepare all the buffers. Millipore Sigma: Cerilliant® certified standard solutions of ketamine hydrochloride, ketamine-D4 hydrochloride, norketamine hydrochloride, norketamine-D4 hydrochloride, and hydroxynorketamine hydrochloride; Supel™ BioSPME 96-Pin Devices (product number 59680-U).

[0283] Liposome production. An ammonium sulfate remote loading scheme was utilized for each reported liposome, following published protocols. Briefly, small unilamellar vesicles were prepared by dissolving lipid components (HSPC:DSPE-PEG2000:cholesterol 52.8:42.3:4.8 molar ratio) in heated ethanol and then diluting the mixture to 10% ethanol with 250 mM ammonium sulfate alone or with 5-10% (by weight) sucrose, 5% glucose, or 73 mM NaCl depending on the experiment. Then the solution was extruded (Avestin LF-50 with 200 nm pore polycarbonate Whatman filter at 65-70 ºC) ten times to generate unilamellar liposomes. Then, the sample was processed with tangential flow filtration (5x 5-fold dilution / reconcentration) against a buffer of 10 mM HEPES, 145 mM NaCl (pH 7.4) with 0, 5, or 10% sucrose depending on the internal buffer osmolarity, to generate a transmembrane ammonium gradient. For remote loading, the liposomes were diluted 10-fold and then the drug was added as a hydrochloride salt to a 1 mg / ml concentration and the mixture was heated to 55 ºC for 1.5 h. Then TFF (4x 5-fold dilution / reconcentration) was repeated against a buffer of 10 mM histidine (pH 7.4) with 10 or 15% sucrose. Finally, the samples were filtered through a sterile 220 nm PVDF syringe filter inside a sterile hood and the sterilized filtrate were dispensed into aliquots, sealed, and stored at 4 ºC for future use.

[0284] Sizing & Zeta potential: The Z-average diameter, polydispersity index (PDI) and zeta potential of liposomes were measured by dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS90 (Malvern, United Kingdom), reading a 1:100 diluted sample of liposomes. All measurements were carried out in triplicates with the auto mode with the following settings: 20 to 30 runs per measurement, temperature = 25 ºC, refractive index of Lipid = 1.5.

[0285] Cryo-electron microscopy (cryo-EM): Cryo-EM was performed on a ThermoFisher Scientific Glacios™ Transmission Electron Microscope (Cryo-TEM) operating at 200 kV.3 μLof purified samples were applied on glow-discharged (40 sec, 15 mA, PELCO easiGlow™, TED PELLA Inc.) holey carbon grids (Quantifoil Cu R1.2 / 1.3, 200 mesh). The grids were blotted using a Vitrobot Mark IV (FEI) with 3 sec blotting time, blot force 2 at 22°C in 100% humidity, and plunge-frozen in liquid ethane. The plunge frozen grids were clipped and loaded into the Glacios EM. The specimen was examined at 36,000x magnification and images were recorded with a pixel size of 1.15 Å using SerialEM software (University of Colorado, Boulder) and a K3 direct electron camera (Gatan). Autofocusing performed with defocus range set to 1.5-3 microns.

[0286] High-Performance Liquid Chromatography (HPLC): HPLC was carried out using an Agilent 1260 Infinity II HPLC System (Agilent Technologies, San Jose, CA) equipped with a multiple wavelength UV detector (G7165A) using Agilent OpenLab CDS software. The chromatographic separation was executed at ambient room temperature using a reverse- phase Agilent 5 HC-C18(2) column (250 mm x 4.6 mm, 5 µm particle size) (Cat no.588905- 902) equipped with an Agilent 5 HC-C18(2) guard column (12.5 mm x 4.6 mm, 5 µm) (Cat no. 520518-904). Water and acetonitrile with 0.1% trifluoroacetic acid were used as the mobile phase. A linear gradient elution was used with detection wavelength of 270 nm for ketamine, and 263 nm for lidocaine, bupivacaine, and ropivacaine.

[0287] Drug concentration quantification. Samples were diluted 1:15 in HPLC grade Methanol and measured using HPLC according to the USP method for each drug.

[0288] Unencapsulated drug quantification. First, 2.5 mL of 1:4 diluted liposomes were loaded onto a PD10 size exclusion column. Then, buffer was added. The initial 4.5 mL eluted fraction contained the liposomes and the next 9 mL of elute contained the unencapsulated drug. The drug concentration in each fraction was determined by HPLC and the unencapsulated fraction determined as the percent in the 2ndeluted fraction versus the total in the 1stand 2ndfractions.

[0289] In vitro ultrasonic drug uncaging. First 200 μl of liposomes diluted 1:4 in canine plasma were loaded into 0.2 mL thin-walled PCR tubes placed in a custom 3D printed holder held at the focus of a 250 or 650 kHz hydrophone-calibrated focused ultrasound transducer. Degassed water of either 25 or 37 ºC was used to couple the transducer to the sample tube. Then ultrasound (60 s, 25% duty cycle, 5 Hz PRF, varying peak pressure) was applied. For analysis, each applicated was repeated 5x and these fractions were collected together into 1 mL. Liposomes were separated from unencapsulated drug by a homemade Sephacryl S-500 column, with PBS as the elution buffer, collecting the first 5.5 mL of elute as the liposome fraction and the next 8 mL as the uncencapsulated drug fraction. Drug concentration in each elute was quantified by HPLC.

[0290] Speed of sound measurement: A clean 20-gallon fish tank was filled with deionized water and degassed overnight. A 650 kHz 30 mm aperture f1.0 focused ultrasound transducer (Sonic Concepts Inc, Bothell, WA), a 35.6 cm long PVC cylinder with a 1.5-inch diameter, anda capsule hydrophone (Onda Corp., Sunnyvale, CA) were placed in a row underwater. Both devices were linked to an oscilloscope (Keysight Technologies, Santa Rosa, CA) to view the time of flight between the transducer and the hydrophone. The PVC pipe was wrapped in an ultrasound-compatible plastic probe cover and sealed with O-rings to create a separate internal fluid compartment. First, the pipe was loaded with 37 ºC DI water, and the ultrasound pulse arrival time was used as a reference, along with the known speed of sound in DI water at 37 ºC, for subsequent measurements. Each buffer was sequentially loaded after heating, and the difference in pulse arrival time was recorded. A temperature measurement after each run confirmed minimal heat loss. The differences in pulse arrival time due to different speeds within the length of the pipe were translated into speeds of sound of the various buffers. Finally, measurements of density at 37 ºC were performed by weighing 10 mL of each buffer using a balance.

[0291] Acoustic scatter measurements. A similar setup as was used for in vitro ultrasonic drug uncaging was applied with a capsule hydrophone (HGL-0400, Onda Corp., Sunnyvale, CA) placed beside the PCR tube perpendicular to the ultrasound focus Z-axis. The emitted and scattered signals from the samples inside the PCR tube was detected by the hydrophone, recorded, and processed by Fourier transform by the oscilloscope, using the first 2 ms of signal. Custom lipid perfluorobutane microbubbles were used in a 1:50 dilution in saline.

[0292] Animal preparation. All animal experiments were carried out in accordance with the Stanford IACUC and Administrative Panel on Laboratory Animal Care (APLAC). Male Long- Evans rats 7-10 weeks old, body weight 250-400 g (Charles River Laboratories, Wilmington, MA; or Envigo, Indianapolis, IN) were used in all in vivo studies. Rats were anesthetized under 2% isoflurane for the entire experiment and fixed into a stereotaxic frame. They were administered 2 mL of saline subcutaneously and kept on a heating pad at 37 ºC. Ketamine- loaded liposomes, ketamine-HCl, or saline vehicle were administered by tail vein catheter. A 250 kHz focused ultrasound transducer (custom design; 10 mm aperture, f=1.0, single element 250kHz transducer with 50 Ohm termination, Sonic Concepts, Bothell, WA; calibrated with hydrophone HGL-0400, Onda Corp, Sunnyvale, CA) powered by an amplifier (240L, E&I Ltd, Rochester, NY) was utilized in all in vivo ultrasound experiments. Skull insertion loss was accounted for and estimated based on weight to achieve the desired in situ pressure.

[0293] In vivo ultrasonic drug uncaging. Before animal experiments, commercial Supel™ BioSPME 96-Pin Devices (product number 59680-U) were carefully detached from their housing, cleaned with 2:1:1 v / v / v methanol:acetonitrile:isopropanol, then preconditioned overnight in 1:1 v / v methanol:water, then transferred to water until sampling. After anesthesia induction, the rat dorsal scalp was exposed and 2-mm burr holes were drilled bilaterally into the skull for BioSPME pin insertion (5 mm posterior to bregma, + / - 2.5 mm lateral to midline). A durotomy was performed with a 32g needle. The ultrasound transducer was positioneddirectly above the right burr hole via a 3-axes positioning system (ThorLabs, Newton, NJ), and ultrasound gel was used for coupling. Drugs were administered as a bolus or infused over 5 minutes intravenously via a tail vein catheter with an infusion pump (World Precision Instruments, Sarasota, FL). Focused ultrasound was applied after 2.5 minutes of drug infusion for 2.5 minutes (250 kHz, 0.9 MPa estimated peak in situ pressure, 5 Hz PRF, 25% duty cycle). For sampling, the BioSPME pins were loaded into a custom-designed, 3D-printed stereotaxic holder and the BioSPME pins were inserted 3 mm ventrally into the brain via the burr holes (SPME absorptive medium centered at ~2.5 mm ventral to bregma). Following 5 min exposure to the rat brain tissue to allow drug / metabolite diffusion to the device, the SPME pins were retrieved carefully, washed for 5 seconds via static immersion in water, and desorbed into 50 µL of MeOH:H2O (9:1 v / v) containing 1% formic acid and deuterated internal standards. Following 30 min of desorption with agitation at 1500 rpm at room temperature, the desorbed aliquots were quantified by LC-MS / MS analysis employing an instrumental calibration curve.

[0294] Pharmacokinetics & Biodistribution. For the pharmacokinetics experiment, 8 adult rats (n=4 each group), following anesthesia, received an intravenous bolus dose of 1 mg / kg of ketamine as either free ketamine-HCl or ketamine liposomes. Then, blood samples (approximately ~400 μL) were collected by tail snipping into Eppendorf tubes pre-loaded with 50 μL Na2EDTA at 1, 2.5, 5, 10, and 15 min. For the biodistribution experiment, another 8 adult rats (n=4 each group) received either free or liposomal ketamine as an intravenous bolus dose of 1.5 mg / kg. Rats were sacrificed at 1 hour from the time of administration and perfused with 1x phosphate buffer (PBS) via transcardial perfusion to remove blood from the systemic circulation. Afterward, the tissues, including the brain, liver, kidney, spleen, lung, heart, and spinal cord, were collected and stored at -80 °C.

[0295] The solid organ samples were homogenized in equal-weight volume of 1x phosphate buffer (PBS) using a handheld homogenizer.100 μL of tissue homogenate was aliquoted from each organ in triplicate per organ. Similarly, 100 μL of blood sample from each time point was aliquoted into triplicates. Deuterated internal standards (ketamine-D4 and norketamine-D4) were added to each blood and tissue homogenate sample. Protein precipitation was carried out with 500 μL of cold acetonitrile. The samples were subjected to an hour of extraction using a mechanical shaker, followed by centrifugation at 12000 rpm and 4°C for 10 minutes. The resulting supernatant was collected and after evaporation of the organic solvent, the sample was reconstituted with 100 μL of MeOH:H2O (9:1) containing 1% formic acid and quantified by LC-MS / MS analysis using an instrumental calibration curve.

[0296] LC-MS / MS analysis. The LC-MS / MS analysis was conducted using an Agilent 1290 Infinity LC System coupled to a 6490 Triple Quadrupole LC / MS System with iFunnel technology (Agilent Technologies, San Jose, CA). The MS / MS analysis was executed in the positive mode, utilizing Agilent Jet Stream technology for electrospray ionization (AJS-ESI),operating under the conditions of selected reaction monitoring (SRM). The fragmentor voltage of 380 V and a cell accelerator voltage of 5 V were consistently applied for all SRM transitions. Data acquisition and processing were performed using Agilent MassHunter Workstation Data Acquisition and MassHunter Workstation Quantitative Analysis software (Agilent Technologies, San Jose, CA), respectively. The chromatographic separation was using Agilent ZORBAX RRHD Eclipse Plus C18 column (5 cm x 2.1mm, 1.8 µm particle size; cat no. 959757-902) equipped with an Agilent ZORBAX RRHD Eclipse Plus C18 guard column (2.1 mm, 1.8 µm; cat no.821725-901). Water and acetonitrile with 0.1% trifluoroacetic acid were used as the mobile phase. The analytical response, represented as relative peak area ratios (analyte to internal standard), was converted to amounts extracted by employing an instrumental calibration curve consisting of analytes in the desorption solvent in the 0.1-100 ng / mL range.

[0297] Safety analysis: For the ketamine uncaging safety analysis, adult rats were administered either saline vehicle (n=3) or ketamine-loaded liposomes (n=3) via IV infusion. Ultrasound was applied to all animals after 2.5 minutes of drug infusion for 2.5 minutes (250 kHz, 0.9 MPa estimated in situ peak negative pressure, 50 ms pulse length, 5 Hz PRF, 25% duty cycle). The targeted brain site was 5 mm posterior to bregma and +2.5 mm lateral to midline to the right side of the brain. At 72 hours post-infusion, rats were euthanized and fixed via transcardial perfusion with 1x PBS and 4% paraformaldehyde (PFA). Brains were extracted and stored in 4% PFA for 24 hours, cryopreserved in 15% sucrose for 48 hours, and lastly, in 30% sucrose for 48 hours. Brains were frozen in optimal cutting medium compound and stored at -80 °C until serially sectioned with a cryostat at 30 μm in the horizontal plane. Every 12th section (360 μm apart) was stained with hematoxylin and eosin (H&E; Vector Laboratories Inc, Newark, CA), Fluorojade-C (Biosensis, Thebarton, SA, Australia), recombinant anti-GFAP antibody (ab33922, Abcam), and recombinant IBA-1 (ab178846, Abcam) to evaluate for parenchymal damage, neuronal degeneration, and astrocytic or microglial activation, respectively. Alexa Fluor® 488 secondary antibody (Thermo Fisher Scientific, Waltham, MA) was used after primary anti-GFAP incubation. Cy5® secondary antibody (Thermo Fisher Scientific, Waltham, MA)) was used after primary anti-IBA1 incubation. Tissue sections were free-float mounted on microscope glass slides (Fisher, Pittsburgh, PA). All histology images were collected with a fluorescence microscope (BZ- X800, Keyence Corp., Itasca, IL). For quantifiable histological markers (Fluoro-Jade C, IBA- 1+, and GFAP+ cells), signals above thresholded background were used for manual ROI segmentation to calculate the total mean fluorescent area of cells using BZ-X Advanced Analysis Software (Keyence Corp., Itasca, IL). For the ropivacaine uncaging safety analysis (Fig.6), a similar process was complete except for targeting and harvesting of the sciatic nerve and surrounding thigh tissues following ropivacaine-HCl (n=3) or ropivacaine loaded AAL(n=3) administration. Muscle and nerve tissues beneath the targeted site were extracted and stored in 4% PFA for 24 hours. Tissues were then frozen in optimal cutting medium compound and stored at -80 °C until serially sectioned. Tissue sections were flash-thawed onto microscope slides immediately after sectioning with a cryostat at 7 μm. Every 20thsection (140 μm apart) was stained with hematoxylin and eosin (H&E) for gross examination of tissue damage.

[0298] Mechanical sensitivity behavior experiment: Adult Sprague Dawley rats were acclimated to a raised stainless steel mesh table for 30 minutes. Then, baseline paw withdrawal responses were obtained using monofilaments and the von Frey percent response method (ten pokes per filament 1g-26g), as previously described. Based on baseline responses, the 26g monofilament was selected to evaluate the effects of local uncaging ropivacaine. On the procedure day, rats were anesthetized and kept under 2% isoflurane during the experiment. Saline vehicle or ropivacaine-loaded liposomes was administered, followed by focused ultrasound applied using a dorsal approach to target the sciatic nerve. Paw withdrawal response was then evaluated using the 26g monofilament and the von Frey percent response method 30 minutes, 1 hour, and 4 hours post treatment.

[0299] Electrocardiographic analysis: Rats were anesthetized using 2% isoflurane. Three alligator electrodes were placed on the rats in limb lead II position: the negative electrode was placed on the front paws and the positive electrode on the left hind paws. ECG recordings were taken at baseline, immediately, and 5 minutes after injection of either free ropivacaine HCl or ropivacaine-loaded AAL using a Sy-W002 Vet 3 Channel ECG Machine (Sunny Medical Equipment Limited, China). The following changes in ECG pattern were assayed: heartrate (bpm), duration of QRS complex and QT interval (corrected for given heart rate in each animal), and Periodic Repolarization Dynamics (PRD).

[0300] Data analysis. For Table 11, pharmacokinetic parameters were estimated using the NonCompart package and comparisons were made with two-tailed Student’s t-tests in R version 4.1.3. GraphPad Prism 10 (GraphPad Software, La Jolla, CA) was used for the remaining statistical analysis. Comparisons between two groups were performed by two-tailed Student's t-test, and that among multiple groups by one way analysis of variance (ANOVA) with Dunnett’s post hoc multiple comparisons tests. A p-value of 0.05 or less was considered statistically different and the p-values were categorized as *: p < 0.05, **: p < 0.01, and ***: p < 0.001. TABLE 9 Typical physicochemical characteristics of different drug-loaded AALs.Formulation Average PDI Zeta Drug Free Entrapment pKa LogP Size (nm) Potential Loading Drug EfficiencyFIG.10 Measured acoustic parameters and osmolarity of plasma and the varied liposome internal and external media. Buffers Density Speed of Acoustic Bulk Osmolarity Sound Impedance Modulus )Table 11 Pharmacokinetic analysis of ketamine and its metabolites following ketamine-loaded AAL or free ketamine bolus intravenous administration. UnitsKetamine AALFree Ketamine P-value AAL vsC0ng / mL 4708.81 ± 720.06 1074.98 ± 138.67 * 0.010 t1 / 2m 11.22 ± 2.52 8.82 ± 1.07 0.236 tionextrapolated to t0; t1 / 2: half life; AUC0-t: area under the curve from 0 to last time point; AUC0-∞: area under the curve from 0 to infinity; MRT0-t: mean residence time from 0 to last time point; MRT0-∞: mean residence time from 0 to infinity; VZ: volume of distribution; Cl: clearance; VSS: volume of distribution at steady state; *: p < 0.05; **: p < 0.01; ***: p<0.001. Table 12 AAL performance and practicality compared to benchmark ultrasonic drug uncaging systems. Ultrasonic In Vivo Use and Performance Clinical Translatability and PracticalityAcousto- Mechanical Frequency: <5 mm @ 250 None 3 mg / mL 4 ºC storage, mechanical particle 250 kHz kHz ketamine stability >3 mo l oscillation Dut C cle ge; mo No ) ge ility e; >7 e; >1 ge ilitySupercritic Cavitation Frequency: Spatial None API conc.4 ºC storage; al CO2 237-1570 kHz localization in N / A; 5-46% Stability N / A loaded PNP: vivo: N / A loadinExample 3 Acoustomechanically activatable liposomes for ultrasonic drug uncaging

[0301] Ultrasound-activatable drug-loaded nanocarriers have been developed that enable noninvasive and spatiotemporally-precise on-demand drug delivery throughout the body. However, most systems for ultrasonic drug uncaging utilize cavitation or heating as the drug release mechanism and often incorporate relatively exotic excipients into the formulation that together limit the drug-loading potential, the stability, and the clinical translatability and applicability of these systems. Here we describe an alternate strategy for the design of such systems in which the acoustic impedance and osmolarity of the internal liquid phase of a drug- loaded particle is tuned to maximize ultrasound-induced drug release. No gas phase, cavitation, or medium heating is necessary for the drug release mechanism. Instead, a non- cavitation-based mechanical response to ultrasound mediates the drug release. Importantly, this strategy can be implemented with relatively common pharmaceutical excipients, as we demonstrate here by implementing this mechanism with the inclusion of a few percent sucrose into the internal buffer of a liposome. Further, the ultrasound protocols sufficient for in vivo drug uncaging with this system are achievable with current clinical therapeutic ultrasound systems and with intensities that are within FDA and society guidelines for safe ultrasound application. Finally, this current implementation of this mechanism is versatile and effective for the loading and uncaging of any therapeutic that may be loaded into a liposome, as we demonstrate here for four different drugs. These acoustomechanically activatable liposomes formulated with common pharmaceutical excipients provide a system with high clinical translational potential for ultrasonic drug uncaging of myriad drugs of clinical interest.

[0302] This disclosure relates to a system for ultrasound-triggered drug release that 1) enables high drug loading potential for varied drugs of interest, 2) has minimal drug release without ultrasound, 3) can be activated following intravenous administration and not necessarily intraparenchymal injection, and 4) an ultrasound-activation mechanism that is mechanically-based without necessitating medium heating above body temperature, nor cavitation, and could be activated with a lower duty cycle and shorter ultrasound protocol than is used for heat or sonodynamic activation. As we demonstrate here, we have been able to accomplish this by tuning the acoustic impedance and osmolarity of the liquid core ofliposomes to maximize ultrasound responsiveness while maintaining overall stability of the liposome structure without ultrasound. Further, we have been able to formulate these acoustomechanically-activatable liposomes with common pharmaceutical excipients and see effective ultrasonic drug uncaging with ultrasound intensities within FDA guidelines for safe diagnostic ultrasound application, indicating that this system has high clinical translational potential. Results

[0303] In designing a next-generation ultrasound-activatable drug carrier for ultrasonic drug uncaging, we first considered prior successful formulations that utilize a liquid perfluorocarbon nanodroplet as the ultrasound-responsive element. We noted that in some of these implementations, while cavitation or a liquid-to-gas phase transition may be involved with the drug release mechanism, such cavitation or gas phase induction is not necessarily always present during drug release. We then hypothesized that we could retain ultrasound sensitivity in the system by replacing the core perfluorocarbon with a liquid droplet that has an acoustic impedance difference with the surrounding medium, to mediate an ultrasound interaction with the droplet relative to the medium. Further, by choosing a replacement liquid that can also be loaded with a drug, we would increase the drug loading potential of the system and likely also the overall stability of drug binding. Additionally, by replacing the perfluorocarbon for more commonly used pharmaceutical excipients, we would increase the clinical translatability of the system. Indeed, several candidate aqueous buffers show significantly varying acoustic impedance measurements in the literature, suggesting that a liposome with an internal compartment composed of a differential acoustic impedance buffer could be optimized for ultrasound response. Finally, if the internal buffer has a substantially different osmolarity compared to the surrounding medium, this osmotic gradient would induce a fluid shift following the membrane leakiness induced by the initial ultrasound application, thereby accelerating further drug release with ultrasound.

[0304] To implement this system, we made liposomes containing varied percentages of sugars in the internal loading battery. We chose to start with liposomes as they are now relatively commonly used drug delivery vehicles formulated with excipients in the FDA Inactive Ingredients Database. We further chose to incorporate sugars into the internal buffer of the liposome since moderate percentages of sugars can substantively shift the acoustic impedance of aqueous buffers. Additionally, since these sugars are GRAS (Generally Recognized As Safe) excipients for pharmaceutical formulations, this system would maintain high clinical translational potential for the vehicle. We tested this system with loading of the anesthetic and antidepressant ketamine, a lipophilic amphipathic weak base that we found could be actively loaded into liposomes with an ammonium sulfate loading battery (Table 1).

[0305] Compared with a ketamine-loaded liposome with no added internal excipient other than the ammonium sulfate loading battery, liposomes incorporating varied percentages of sucrose showed significantly different amounts of ultrasound-induced drug release: with 5% sucrose added to the internal buffer yielding ~40% drug release with a 250 kHz 25% duty cycle, 1 min ultrasound protocol, and 10% added sucrose yielding ~60% drug release with this same protocol, compared to a nonsignificant trend towards ~20% drug release with no added sucrose. Notably, there were no significant differences in key physicochemical properties including size, polydispersity, zeta potential, drug loading, or unencapsulated drug percent (Table 9).

[0306] We next noted that increasing sucrose percentages would yield increases in both the acoustic impedance and the osmolarity of the internal buffer, and the differences in both parameters with the surrounding medium. To help discern which parameter, acoustic impedance or osmolarity, was more contributory to our results we next prepared liposomes with equiosmolar internal buffers, but differential acoustic impedances. Drug-loaded liposomes with internal buffers composed of ammonium sulfate with equiosmolar additional sodium chloride, glucose, or sucrose were prepared and found to have similar physicochemical characteristics with each other (Table 9). Indeed, despite similar internal osmolarities, they showed differing amounts of drug release with the same ultrasound protocol, in correlation to their differences in internal acoustic impedances. This indicates that acoustic impedance differences across the shell of a liposome likely contribute to the ultrasound-responsiveness of the drug-loaded nanocarrier, potentially in addition to an internal-external osmolarity gradient.

[0307] Notably, while 10% additional sucrose internally did significantly increase the amount of ultrasound-induced drug release relative to 5% additional sucrose, it did so at the expense of displaying a significant rate of drug release without ultrasound when incubated with plasma at 37 ºC, indicating that there is a relative tradeoff of ultrasound responsiveness and formulation stability with this scheme. For further experiments, 5% sucrose added internally was chosen as a relative optimum of ultrasound responsiveness with stability of the formulation with body temperature incubation in plasma.

[0308] To further explore the mechanism of drug release with this system, the ultrasound frequency was varied between either 250 kHz or 650 kHz, usual frequencies for transcranial therapeutic ultrasound application clinically. Significant drug release was seen with 250 kHz sonication, with increasing peak pressure / mechanical index correlating to increased drug release, similar to prior results with liquid perfluorocarbon based systems. Intuitively, there was increased drug release seen with a higher temperature medium of 37ºC versus 25ºC. In contrast, with 650 kHz sonication, only marginal drug release was seen with ultrasound, witha minimal dose-response relationship with ultrasound peak pressure / mechanical index, and with only minimal variation with medium temperature. This is in distinct contrast to perfluorocarbon-based systems that show significant drug release across a range of frequencies up to and beyond 1 MHz.

[0309] This increased responsiveness at 250 kHz compared to 650 kHz argues against both a thermal or radiation force type mechanism for the drug release, since both thermal and radiation force transfer mechanisms tend to increase with increasing acoustic attenuation coefficients, which in turn increases with the acoustic frequency. Arguing against cavitation as an underlying drug release mechanism, the liquid phase of the formulation particles has a low vapor pressure and we observe no evidence of wide-band or ultraharmonic acoustic scatter with sonication of the liposomal formulation with a sonication protocol that is effective for drug release, in contrast to sonication of gas-phase microbubbles, which readily generate these signals of cavitation.

[0310] By electron microscopy, the formulation particles show the expected spherical morphology, but with no internal precipitation of the drug that is shown to correlate to stability of drug loading with other liposomal formulations. However, with sonication, the size distribution of the particles relatively decreased, likely indicating an ultrasound-induced leak of the internal contents. Nonetheless, this formulation shows stability for both drug loading and ultrasound-induced release across months of time in storage. Together, this frequency response difference and the other characterizations suggests a non-cavitation-based acoustomechanical type drug release mechanism, such as an ultrasound-induced rarefaction or oscillation of the internal droplet relative to the surrounding medium, as mediated by the differential acoustic impedance of the internal droplet, contributing to increased permeability of the liposome membrane to the loaded drug with ultrasound. Notably, at body temperature, effective ultrasound-induced release was seen with 250 kHz ultrasound and an in situ peak negative pressure of 0.9 MPa, or a mechanical index of 1.8, which is below the FDA and AIUM guidelines for safe ultrasound application (without microbubbles present, as in this application). Accordingly, we chose this ultrasound protocol for subsequent evaluations.

[0311] To confirm that these results were not specific to the particular drug used in these initial experiments, we prepared liposomes with or without 5% additional sucrose internally, and loaded with several other agents: lidocaine, bupivacaine, and ropivacaine. These liposomes show similar size, polydispersity, and zeta potential as the ketamine-loaded liposomes (Table 9). They showed differential drug loading rates that likely reflect the specific chemical characteristics of each drug, such as their logP and pKa, as they relate to the ammonium sulfate active loading mechanism (Table 9). Each of these other liposomes showed increased ultrasonic drug uncaging with the formulations incorporating added sucrose internally, whileformulations without added sucrose showed no significant or minimal drug uncaging with ultrasound.

[0312] With these in vitro characterizations demonstrating in vitro stability and ultrasonic drug uncaging efficacy, we next characterized their performance in vivo. Following a bolus intravenous injection, the blood drug concentration showed slower blood clearance pharmacokinetics with the acoustomechanically activatable liposome (AAL) versus a dose- matched administration of a free unencapsulated drug formulation. Notably, there were also higher metabolite blood levels with the AAL formulation compared to the unencapsulated drug formulation. This behavior was reflected in the biodistribution at 1 hr post bolus administration, in which the solid organs showed increased accumulation of the drug with free unencapsulated form administration compared to the AAL formulation administration. The combination of the higher blood metabolite concentration of the drug, yet lower unmetabolized drug accumulation in the solid organs with the AAL formulation suggests that some of the blood clearance of the drug with the AAL formulation is likely due to the sequestration and metabolization of the whole particle by end organs like the liver. Further, this end organ sequestration and metabolism could contribute to some degree of nonspecific leak of the drug into circulation.

[0313] To assess the differences induced in the in vivo pharmacodistribution with ultrasonic drug uncaging with AALs, in adult rats, we used solid-phase microextraction (SPME) to measure the amounts of ketamine delivered to the brain either with a free unencapsulated drug infusion or an AAL infusion, with or without ultrasound application to the brain. Ultrasound (or sham administration) was applied to a frontal cortical and hippocampal region unilaterally during the latter 2.5 min of a 5 min intravenous infusion of either free, unencapsulated ketamine-HCl or ketamine-loaded liposomes. After the stop of infusion and sonication, SPME was used to sample both the sonicated brain and a contralateral control region. A venous blood sample was taken at the end of the SPME sampling period. The blood samples showed higher blood levels of ketamine with each liposomal formulation compared to the free drug infusion, confirming our blood pharmacokinetics results following bolus administration (Fig. 5b, 4a). Nonetheless, despite the higher blood concentrations of ketamine with liposomal formulation administration, the ketamine brain levels seen with sham ultrasound administration or in the contralateral control region were less than half of that seen following a free unencapsulated ketamine infusion.

[0314] While ultrasound application did not measurably alter the pharmacodistribution of ketamine following a free ketamine infusion, ultrasound application yielded significantly higher brain ketamine levels with the liposomal infusions compared to the no ultrasound condition or the contralateral non-sonicated brain that was separated by 5 mm. Furthermore, significantly higher brain ketamine levels were seen with sonication with liposomes made with additionalsucrose internally compared to liposomes without added sucrose, confirming that this relatively simple formulation manipulation increases the ultrasound sensitivity of the system both in vitro and in vivo. Notably, despite higher ultrasonic drug uncaging efficacy in vitro, no significant difference was seen in vivo between liposomes made with 10% added sucrose internally, compared to those made with 5% added sucrose, likely reflecting the relative instability in plasma of the 10% added sucrose formulation seen in vitro. Importantly, histological assessment for morphological tissue damage, neuronal degeneration, microglial / astrocytic activation, and gliosis yielded no evidence of brain parenchymal injury seen with AAL-mediated ultrasonic drug uncaging.

[0315] With these results, we have demonstrated a system for ultrasonic drug uncaging that can be loaded with varied drugs with high drug loading, minimal drug release without ultrasound, and effective drug release with pulsed low-intensity ultrasound both in vitro and, importantly, in vivo. The ultrasound-induced drug release mechanism with this system does not necessitate medium heating or cavitation, and shows a frequency response characteristic indicating a mechanical type interaction, with significant ultrasound-induced drug release achievable with pulsed ultrasound with low intensities that are within FDA and AIUM guidelines for safe ultrasound application. Crucially, we have been able to achieve these features with formulation constituents that are commonly used and validated pharmaceutical excipients, with the simple disaccharide sucrose being the main additional ingredient yielding increased ultrasound sensitivity in this current implementation of acoustomechanically activatable liposomes (AALs).

[0316] The addition of sucrose in the core of the formulation increases the acoustic impedance of the inner core droplet of the liposome relative to the surrounding medium, and this acoustic impedance difference permits an ultrasound interaction that increases drug leak across the liposome membrane. The osmotic pressure difference across the lipid membrane may further accelerate drug release following the initial acoustic interaction induced lipid membrane leakiness. While we have focused our attention on implementing this system using common pharmaceutical excipients to maximize clinical translatability, an implication of this acoustomechanical release mechanism is that an even further ultrasound-responsive system could be achieved by replacing sucrose with equiosmolar amounts of a different substance that even further shifts the acoustic impedance away from that of plasma.

[0317] We also maintained clinical translatability of this system. Indeed, the manufacturing methods for liposome production used here are compatible with Good Manufacturing Practices (GMP) and the formulation ingredients are each validated excipients in the FDA Inactive Ingredients Database, together indicating a ready path to clinical translation of this system. Additionally, this formulation is stable for months in refrigerated storage, a decidedpractical advantage over prior formulations that necessitated frozen storage. In addition, the ultrasound intensities sufficient for drug release with this system have a mechanical index within the FDA and AIUM guidelines of safe ultrasound application; indeed no evidence of parenchymal damage was observed in our in vivo experiments. Further, this ultrasound protocol is readily achievable using current clinically utilized therapeutic focused ultrasound systems. Finally, these AALs enable targeted drug delivery to a few millimeters resolution in the rat brain following an intravenous infusion, a notable practical advantage over systems necessitating direct intraparenchymal injection of the drug-loaded formulation.

[0318] While an ammonium sulfate based active loading scheme was demonstrated here, the strategy of incorporating elements to increase the acoustic impedance of the liposome core to increase its ultrasound responsiveness is applicable to any liposome with any internal core buffer and loading battery. Methods

[0319] Materials: All the chemicals and reagents used in the study were of the highest purity grade. Hydrogenated Phosphatidylcholine (HSPC) from Soybean (Cat no: Lipoid S PC-3) and [N-(Carbonul-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3- phosphoethanolamine, sodium salt] (DSPE-PEG 2000) (Cat no: PE18:0 / 18:0 – PEG 2000) were procured from Lipoid. Cholesterol (PhytoChol Inject HU) was purchased from Evonik. Ketamine-HCl injectable solution (100 mg / mL) is a controlled substance and was purchased via Stanford University Environmental Health & Safety. Ropivacaine-HCl (Cat no.42084) and Bupivacaine-HCl (Cat no.42055) were purchased from Astatech. Ammonium Sulphate (Cat no: PCT0003) was obtained from Hiemdia. Absolute Ethanol (200 proof) (Cat no: BP2818- 100), Sucrose (Cat no: S5-500), HPLC grade Water, Methanol & Acetonitrile were purchased from Fisher scientific. Lidocaine-HCl (Cat no. L5647), HEPES buffer solution (Cat no: 83264- 500ML-F) and L-Histidine monohydrochloride monohydrate (Cat no: H8125) were obtained from Sigma Aldrich. Repligen filters (C02-S05U-05-N (SN: 20020493-03 / 21-057) were used for tangential flow filtration (TFF). Male Canine Plasma (Cat no.7302009) was obtained from Lampire biological laboratories. PD-10 column Sephadex G-25 M (Cat no. 17085101), Sephacryl S-500 (Cat no. 1706130) were purchased from Cytiva. Milli-Q water was used to prepare all the buffers. The Cerilliant® certified standard solutions of ketamine hydrochloride, ketamine-D4 hydrochloride, norketamine hydrochloride, norketamine-D4 hydrochloride, and hydroxynorketamine hydrochloride, were purchased from Millipore Sigma, USA. The Supel™ BioSPME 96-Pin Devices (product number 59680-U) were also procured from the same source. For the LC / MS grade water, acetonitrile, 2-propanol, methanol, and formic acid, the procurement was made through Fisher Scientific.

[0320] Production of AAL: All the liposome used in the study were synthesized using a multistep process, divided into four major steps as described here under:

[0321] Buffers: Buffer A: 250 mM ammonium sulfate solution (pH= 7.4) with 0, 5 &10% of sucrose. For liposome prepared with added NaCl and Glucose in internal medium, 5% sucrose (146 mOsmol) were replaced with 73 mM NaCl and 146 mM Glucose respectively. Buffer B: 10 mM HEPES buffer including 145 mM of NaCl (pH= 7.4) with 0, 5 &10 % of sucrose. Buffer C: 10 mM histidine Buffer (pH= 7.4) with x% of sucrose, where x= 10% (when buffer A & B contain 0 or 5% sucrose) and x=15% (when buffer A & B contain 10% sucrose).

[0322] Step 1: Preparation of liposome encapsulating ammonium sulphate and sucrose: HSPC= 387 mg, Cholesterol= 153 mg, DSPE-PEG= 126 mg (Molar ratio 52.8: 42.3: 4.8) were weighed into a 20 ml glass vial and 1.5 ml absolute ethanol was added into it. The resulting mixer was heated up using a water bath (70 ℃) for solubilization of lipids. Then, 13.5 ml of buffer A was mixed with the lipid solution to form multilamellar vesicles (MLVs) filled with sucrose and ammonium sulfate. The vesicle suspension was extruded repeatedly ten times using Avestin LF-50 equipped with 200 nm pore size polycarbonate filter at 70 ℃ to obtain unilamellar liposome with low polydispersity at the size range of 150-170 nm.

[0323] Step 2: Purification and transmembrane Ammonium gradient generation: Once the liposomal encapsulating ammonium sulphate and sucrose were produced, the next step was to remove the ethanol while simultaneously replacing liposomal external medium with buffer B to establish a transmembrane ammonium gradient for the active loading of drugs. Similar % of sucrose that used in internal buffer was added in the external buffer to create an iso-osmolar environment. For purification and buffer exchange, a tangential flow filtration (TFF) system using Masterflex® L / S® Peristaltic Pump (Model no. 07522-20, 0.1-600 RPM 0.1 HP) with multichannel pump head (model no. 7535-04), MasterFlex 06503-14 tubes and modified polyethersulfone (mPES) column from Repligen (C02-S05U-05-N (SN: 20020493-03 / 21-057) was used. For 1 round of TFF process, 10 mL liposome was diluted 5 times using buffer B to make it 50 mL and concentrated back the retentate to original volume of 10 ml (which generates 40 mL filtrate) using TFF. With 40 mL / min flow rate, a total of 8 rounds of TFF process mentioned above were applied for complete buffer exchange (total 390,625-fold dilution).10 ml of buffer exchanged blank liposome with (NH4)2SO4gradient was produced at the end of this step.

[0324] Step 3: Remote loading of drugs: 10 ml blank liposome was diluted 10 times with buffer B to make 100 mL total volume.100 mg drug (final concentration 1 mg / mL) was added into the blank liposome and heated the solution at 55℃ using a water bath for 1.5 h. After the completion of the drug loading process, the solution was cooled down to room temperature and repeatedly washed using TFF to get the final product.

[0325] Step 4: Washing and sterilization: To remove the unencapsulated drug from the liposomes, a similar TFF process as described in the gradient generation section was followed. First, 100 ml drug loaded liposomes were concentrated to 10 ml using TFF (which generates 90 mL filtrate). For 1 round of TFF process, the above 10 mL dug loaded liposome was diluted again 5 times using 40 mL of buffer C to make it 50 mL and concentrated back the retentate to original volume of 10 ml using TFF (which generates 40 mL filtrate). With 40 mL / min flow rate, a total of 4 rounds of TFF process mentioned above were applied to wash out the unencapsulated drug (total 625-fold dilution). Finally, the purified samples were filtered through sterile 220 nm PVDF syringe filter inside the biosafety cabinet. The filtrate final liposomes were sealed and stored at 4 ℃ for future use.

[0326] Physicochemical characterization of AAL: DLS size & Zeta potential: The Z-average diameter, polydispersity index (PDI) and zeta potential of liposomes were measured with a Malvern Zetasizer Nano ZS90 (Malvern, United Kingdom).10 µL of liposomes were dispersed in 990 µL of water for analysis. All measurements were carried out in triplicates with the auto mode of the measurement where the following settings were used; 20 to 30 runs per measurement, temperature: 25 ºC, refractive index of Lipid 1.5.

[0327] Cyro-electron microscopy (cryo-EM): Cyro-EM was performed on a ThermoFisher Scientific Glacios™ Transmission Electron Microscope (Cryo-TEM) operating at 200 kV.3 μL of purified samples were applied on glow-discharged (40 sec, 15 mA, PELCO easiGlow™, TED PELLA Inc.) holey carbon grids (Quantifoil Cu R1.2 / 1.3, 200 mesh). The grids were blotted using a Vitrobot Mark IV (FEI) with 3 sec blotting time, blot force 2 at 22°C in 100% humidity, and plunge-frozen in liquid ethane. The plunge frozen grids were clipped and loaded into the Glacios EM. The specimen was examined at 36,000x magnification and images were recorded with a pixel size of 1.15 Å using SerialEM software (University of Colorado, Boulder) and a K3 direct electron camera (Gatan). Autofocusing performed with defocus range set to 1.5-3 microns.

[0328] Drug Loading measurement: 20 µL of liposome were mixed with 300 µL High pressure liquid chromatography (HPLC) grade Methanol in an HPLC vial to destroy the liposomal structure and extract out the total drug encapsulated in liposome. Drug concentration was measured using HPLC.

[0329] Free Drug assessment: Free drug were separated out from the liposomes using PD10 size exclusion column. For that, 550 µL of liposome was diluted with 2200 µL of same buffer that used to store the liposome (10 mM Histidine buffer pH 7.4, with appropriate amount of sucrose).2.5 ml of diluted liposome were loaded on top of pre-equilibrized PD10 column and let it drain. Thereafter, using the storage buffer, initial 4.5 mL and then 9 mL of column eluent were collected in two different fractions. The total drug present in the 9 mL fraction was calculated using HPLC, which represents the free drug amount in liposome.

[0330] High Performance Liquid Chromatography: The HPLC analysis was carried out using the Agilent 1260 Infinity II HPLC System (Agilent Technologies, San Jose, CA, U.S.A.) equipped with a multiple wavelength UV detector (G7165A). Both data acquisition and processing were performed using Agilent OpenLab CDS software. The chromatographic separation was executed at ambient room temperature using a reverse-phase Agilent 5 HC- C18(2) column (250 mm x 4.6 mm, 5 µm particle size) (Part Number: 588905-902). Water and acetonitrile with 0.1 trifluoroacetic acid were used as the mobile phase. For ketamine analysis, a linear gradient elution was used and the detection wavelength was set at 270nm.

[0331] Acoustic characterization of AAL: In vitro ultrasonic drug uncaging: 250 µL of liposome was diluted with 750 µL of canine plasma and aliquoted in 5 different 200 μl aliquots in Fisherbrand™ 0.2 ml PCR tubes (Fisher Scientific. NJ, USA). The PCR tubes were placed (one at a time) in a custom 3D printed holder and coupled using degassed water to a focused ultrasound transducer at room temperature and sonicated with ultrasound for 60 s total, with varying peak negative pressure, using cycles of 50 ms ultrasound on and 150 ms off, i.e., pulse repetition frequency of 5 Hz. The center frequency of the transducer was 250 kHz or 650 kHz. Following ultrasound application, liposome was collected back to 1 mL and the uncaged drug was separated from liposome using size exclusion column. Sephacryl S-500 homemade column were used to separate plasma bound uncaged drug.1 ml of sonicated and control samples were loaded on top of pre-equilibrized with phosphate buffer saline (pH 7.4) (PBS) column and let it drain. Thereafter, using the PBS, initial 5.5 mL and then 8 mL of column eluent were collected in two different fractions. Drugs were extracted out from the plasma using methanol precipitation method and the total drug present in the 2nd fraction was calculated using HPLC, which represents the uncaged drug amount.

[0332] Speed of sound measurement: A clean 20-gallon fish tank was filled with deionized water and degassed overnight. A 650 kHz focused ultrasound transducer with a 1-inch diameter (Sonic Concepts Inc, Bothell, WA, USA), a 35.6 cm long PVC cylinder with a 1.5 inch diameter, and a capsule hydrophone (Onda Corp, Sunnyvale, CA, USA) were placed in a row underwater. Both devices were linked to an oscilloscope (Keysight Technologies, Santa Rosa, CA, USA) to view the time of flight between transducer and hydrophone. The PVC pipe was wrapped in an ultrasound-compatible plastic probe cover and sealed with O-rings to create a separate internal fluid compartment. First, the pipe was loaded with 37 ºC DI water, and the ultrasound pulse arrival time was used as a reference, along with the known speed of sound in DI water at 37 ºC, for subsequent measurements. Each buffer was sequentially loaded after heating and the difference in pulse arrival time was recorded. A temperature measurement after each run confirmed minimal heat loss. The differences in pulse arrival time due to different speeds within the length of the pipe were translated into speeds of sound of thevarious buffers. Finally, measurements of density at 37 ºC gave the acoustic impedance value for each buffer.

[0333] Cavitation assessment: The same setup used for in vitro ultrasonic drug uncaging was applied with the addition of a capsule hydrophone (Onda Corp, Sunnyvale, CA, USA) which was placed beside the PCR tube perpendicularly to the ultrasound focus Z-axis. The emitted and scattered signals from the samples inside the PCR tube was detected by the hydrophone, recorded, and fast Fourier transformed by the oscilloscope. For every sample, only the first 2 ms of signal was processed.

[0334] Animal model: Animal preparation: All animal experiments were carried out in accordance with the Stanford IACUC and Administrative Panel on Laboratory Animal Care (APLAC). Male Long-Evans rats between 7 – 10 weeks old with body weight 250 – 400 g (Charles River Laboratories, Wilmington, MA, USA; Envigo, Indianapolis, Indiana, USA) were used in all in vivo studies. Rats were fixed into a stereotaxic frame and anesthetized under isoflurane for the entire terminal experiment. They were administered with 2mL of saline subcutaneously and kept on a heating pad at 37C. 1.5 mg / kg ketamine, ketamine-loaded liposomes, or saline vehicle was used for all animal experiments. A 250 kHz focused ultrasound transducer powered by an amplifier (240L, E&I Ltd) was utilized in all in vivo ultrasonic drug uncaging and pharmacokinetics experiments. Skull attenuation was accounted for and calculated based on weight to achieve the desired in situ pressure.

[0335] Solid phase microextraction protocol: SPME pins were carefully detached from commercial BioSPME 96-pin devices, and subsequently cleaned with a mixture of methanol: acetonitrile: isopropanol. The SPME pins were preconditioned overnight in methanol: water (1:1 v / v) solution and then were kept in water until sampling. Immediately before SPME sampling, the SPME pins were loaded into a custom-designed, 3D-printed stereotaxic holder that had been tailored for precise positioning and sampling. The SPME pins were exposed to the rat brain tissue for a duration of 5 minutes immediately after the drug administration. The SPME pins sample the drug and metabolites through diffusion. Post-sampling, the SPME pins were retrieved carefully and washed for 5 seconds via static immersion in water to effectively remove any residual blood. Following washing, the SPME pins were desorbed into 50 µL of MeOH: H2O (9:1 v / v) solvent containing 1% formic acid and deuterated internal standards. This desorption process took place for 30 minutes with agitation at 1500 rpm at room temperature. The desorbed aliquots were quantified by LC-MS / MS analysis employing an instrumental calibration curve.

[0336] In vivo ultrasonic drug uncaging: The rat dorsal scalp was exposed with surgical scissors. Bilateral 2-mm burr holes were drilled into the skull for SPME pin insertion (5 mm posterior to bregma, + / - 2.5 mm lateral to midline). A durotomy was performed with a 32g needle. The ultrasound transducer was positioned directly above the right burr hole via a 3-axes positioning system (ThorLabs, Newton, NJ, USA) and ultrasound gel was used for coupling. Drugs were infused over 5 minutes intravenously via tail vein catheter with an infusion pump (World Precision Instruments). Focused ultrasound was applied after 2.5 minutes of drug infusion for 2.5 minutes (250 kHz, 0.9MPa, 50ms pulse length, 25% duty cycle). A 3D printed stereotaxic holder is used to insert the polymer-coated SPME pins 3 mm ventrally into the brain via the burr holes. Blood samples were collected during time of sacrifice or via cutting of the tail at different time points for the pharmacokinetics experiments.

[0337] Pharmacokinetics & biodistribution. For both experiments, rats were administered 2mL of saline subcutaneously before being anesthetized under 2% isoflurane for the entire terminal experiment. They were kept on a heating pad at 37C to maintain body heat. For the pharmacokinetics (blood clearance) experiment, a total of 8 rats were employed, and evenly distributed into two groups (N=4 each). One group received free unencapsulated ketamine- HCl while the other group received ketamine liposomes. Following anesthesia, the rats were administered a dose of 1 mg / kg of ketamine HCl for the free drug group and ketamine liposomes for the liposomal drug group. Drugs were injected bolus intravenously via tail vein catheter. Blood samples (approximately ~400 μL) were collected by tail snipping into Eppendorf tubes including with EDTA (Millipore sigma; 50 μL Na2EDTA) at 1, 2.5, 5, 10, and 15 min. Animals were sacrificed via decapitation after blood collection.

[0338] For the biodistribution experiment, N=8 rats were used and evenly distributed into two groups so that the free drug and liposomal drug groups each contained N=4 rats. Following anesthesia, the rats were administered a dose of 1.5 mg / kg of ketamine HCl for the free drug group and liposomal ketamine for the liposomal drug group, bolus intravenously via tail vein catheter. Rats were sacrificed at 1 hour from the time of administration and perfused with 1x phosphate buffer (PBS) via transcardial perfusion to remove blood from the systemic circulation. Afterward, the tissues including the brain, liver, kidney, spleen, lung, heart, and spinal cord were collected and stored at -80 °C.

[0339] Sample Preparation: For processing the tissue samples, collected tissue samples were homogenized in equal weight volume of 1x phosphate buffer (PBS) using a disposable mechanical homogenizer. 100 μL of tissue homogenate was aliquoted into three individual Eppendorf tubes from each organ, creating triplicate samples for each organ. Similarly, 100 μL of blood sample from each time point was aliquoted into three individual Eppendorf tubes. Subsequently, 5 μL of deuterated internal standards (ketamine-D4 and norketmine-D4) at a concentration of 1 μg / mL were added to each 100 μL blood and tissue homogenate samples. Protein precipitation was carried out with 500 μL of cold acetonitrile. To facilitate extraction, the samples were subjected to an hour of extraction using a mechanical shaker. Following this extraction, the samples underwent centrifugation at 12000 rpm and 4°C for 10 minutes. The resulting supernatant was transferred to new Eppendorf tubes, and the solvent wasevaporated to dryness. Following this, each sample tube was reconstituted with 100 μL of MeOH: H2O (9:1) solvent containing 1% formic acid and then centrifuged once more under identical conditions for 5 min. The supernatant was transferred to an insert containing HPLC vials and quantified by LC-MS / MS using the instrumental calibration curve. The Calibration curve standards ranging from 0.1 to 100 ng were prepared in MeOH: H2O (9:1) solvent containing 1% formic acid and 50 ng of deuterated internal standards.

[0340] Safety analysis: Rats were administered either saline vehicle or ketamine-loaded liposomes via IV infusion. Ultrasound was applied to all animals after 2.5 minutes of drug infusion for 2.5 minutes (250 kHz, 0.9MPa, 50ms pulse length, 25% duty cycle). The targeting site was 5 mm posterior to bregma and +2.5 mm lateral to midline to the right side of the brain. Animal subjects were then sacrificed at 24 hours via transcardial perfusion of 1x PBS and fixed in 4% paraformaldehyde (PFA). Brains were extracted and kept in 4% PFA for 24 hours, transferred into 15% sucrose for 48 hours, and lastly, into 30% sucrose for 48 hours. Brains were frozen in OCT compound and kept at -80C prior to slicing.

[0341] Statistical analysis: GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. Comparisons between two groups was performed by two-tailed Student's t-test, and that among multiple groups by one way analysis of variance (ANOVA). A p-value of 0.05 or less was considered statistically different and the p-values were categorized as *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

[0342] The preceding merely illustrates the principles used in the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the present disclosure and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the disclosure and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the disclosure as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present disclosure, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present disclosure is embodied by the appended claims.

Claims

What is claimed is:

1. An acoustically activatable particle (AAP) for ultrasound-triggered compound release, comprising: a polymer or lipid coated vesicle with a bilayer or unilayer shell; and a core material having an acoustic impedance that differs from the acoustic impedance of the physiologic media; and comprising a compound of interest for release.

2. The AAP of claim 1, wherein the AAP is a liposome.

3. The AAP of claim 1 or claim 2, wherein the core is an aqueous solution.

4. The AAP of any of claims 1-3, wherein the core is an aqueous solution incorporating a solute that shifts the core acoustic impedance.

5. The AAP of claim 4, wherein the solute is a compound generally recognized as safe (GRAS).

6. The AAP of any of claims 1-5, wherein the core is an aqueous sucrose solution.

7. The AAP of claim 6, wherein sucrose is present at a concentration of from about 2% to about 10%.

8. The AAP of any of claims 1-7, wherein the AAP uncages at an ultrasound intensity of from about 50 kHz to 1 MHz.

9. The AAP of any of claims 1-7, wherein the AAP uncages at an ultrasound frequency of from about 200 kHz to about 500 kHz.

10. The AAP of any of claims 1-9, wherein following exposure to ultrasound, the level of non-encapsulated drug in a targeted tissue is at least 1.25X the level in the absence of ultrasound.

11. The AAP of any of claim 1-11, wherein the compound of interest is a therapeutic agent and / or an imaging agent.

12. The AAP of any of claims 1-11, wherein the compound of interest is a neurally active drug, protein, or polynucleotide.

13. The AAP of claim 12, wherein the compound of interest is selected from propofol, ketamine, nicardipine, verapamil, dexmedetomidine, modafinil, doxorubicin, temozolomide, rivastigmine, galantamine, donepezil, bupivacaine, ropivacaine, lidocaine, clonidine, xylazine, dexmedetomidine, cocaine, amphetamine, methamphetamine, ibogaine, psilocin, psilocybin, methyl-d-methamphetamine, lysergic acid, cisplatin, tacrolimus, p-aminophenyl-α-D- mannoside, levodopa, anti-NRI, anti-ICAM1, anti-HSP72, anti-actin, anti-VEGF; GDNF, NGF, H102 peptide, TF peptide, α-Mangostin, matrix metalloproteinase-9, superoxide dismutase, VEGF, transferrin, tissue plasminogen activator, or an oligonucleotide or polynucleotide.

14. A pharmaceutical formulation comprising an AAP of any of claims 1-13, and a pharmaceutically acceptable buffer.

15. A method of delivering a compound to a tissue of interest, the method comprising: administering an effective dose of a pharmaceutical formulation of claim 14; and administering a dose of ultrasound to the tissue of interest, sufficient to uncage the compound from the AAP.

16. The method of claim 15, wherein the tissue of interest is neural tissue.

17. The method of claim 15, wherein the neural tissue is one or both of the brain and spinal cord.

18. The method of claim 16, wherein the neural tissue is a peripheral nerve.

19. The method of any of claims 15-18, wherein the administration is performed parenterally.

20. The method of any of claims 15-19, wherein the administration is combined with imaging, measuring electrophysiology (e.g. EEG), and / or behavioral assessment of brain function.

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