LOCALIZED DELIVERY OF DIAGNOSTIC OR THERAPEUTIC AGENTS USING FOCUSED ULTRASOUND
20240009436 · 2024-01-11
Assignee
Inventors
- Mehmet Fatih YANIK (Fällanden, CH)
- Mehmet OZDAS (Zürich, CH)
- Aagam SHAH (Washington, DC, US)
- Paul JOHNSON (oLTEN, CH)
- Shashank SIRSI (Frisco, TX, US)
Cpc classification
A61M37/0092
HUMAN NECESSITIES
A61K9/0009
HUMAN NECESSITIES
International classification
A61M37/00
HUMAN NECESSITIES
A61K41/00
HUMAN NECESSITIES
Abstract
In a method for enhancing localized delivery of a diagnostic or therapeutic agent using focused ultrasound (FUS), ultrasound-controllable drug carriers are administered into a blood vessel. Each drug carrier comprises an ultrasound-sensitive microbubble loaded with the diagnostic or therapeutic agent. The drug carriers are aggregated inside the vessel both along a radial direction and a longitudinal direction by application of an aggregation FUS sequence. Subsequently the diagnostic or therapeutic agent is released from the drug carriers by application of an uncaging FUS sequence. The aggregation and uncaging sequences are applied using FUS below a threshold power level such that harmful cavitation is avoided, as evidenced by the absence of broadband emissions and preferably non-integer harmonics from an emission spectrum of the drug carriers. Thereby damage to the vasculature by the FUS sequence is avoided. The method can in particular be employed to deliver drugs to the brain without opening the blood-brain barrier.
Claims
1. A system for enhancing localized delivery of a diagnostic or therapeutic agent to a human or animal body using focused ultrasound, the system comprising: a source of ultrasound-controllable drug carriers, each drug carrier comprising an ultrasound-sensitive microbubble, the microbubble comprising a microbubble shell and a gas encapsulated by the microbubble shell, the microbubble being loaded with a diagnostic or therapeutic agent; an administration device for administering the ultrasound-controllable drug carriers into a blood vessel of the human or animal body; a focused ultrasound source for creating FUS radiation in a target region of the human or animal body; and a controller configured to operate the focused ultrasound source to apply an aggregation FUS sequence to the drug carriers in the target region, the aggregation FUS sequence generating radiation forces that cause the drug carriers to aggregate inside the blood vessel.
2. The system of claim 1, wherein the controller is configured to operate the focused ultrasound source to apply the aggregation FUS sequence at FUS power levels below a threshold power level such that broadband emissions are essentially absent from an emission spectrum of the drug carriers in the target region.
3. The system of claim 1, wherein the controller is further configured to operate the focused ultrasound source to apply an uncaging FUS sequence to the target region, the uncaging FUS sequence being applied subsequent to the aggregation FUS sequence, the uncaging FUS sequence causing the diagnostic or therapeutic agent to be released from drug carriers in the target region.
4. The system of claim 3, wherein the controller is configured to operate the focused ultrasound source to apply both the aggregation FUS sequence and the uncaging FUS sequence at FUS power levels below a threshold power level such that broadband emissions are essentially absent from an emission spectrum of the drug carriers in the target region.
5. A system for enhancing localized delivery of a diagnostic or therapeutic agent to a human or animal body using focused ultrasound, the system comprising: a source of ultrasound-controllable drug carriers, each drug carrier comprising an ultrasound-sensitive microbubble, the microbubble comprising a microbubble shell and a gas encapsulated by the microbubble shell, the microbubble being loaded with a diagnostic or therapeutic agent; an administration device for administering the ultrasound-controllable drug carriers into a blood vessel of the human or animal body; a focused ultrasound source for creating FUS radiation in a target region of the human or animal body; and a controller configured to operate the focused ultrasound source to apply an uncaging FUS sequence to the drug carriers in the target region, the uncaging FUS sequence causing the diagnostic or therapeutic agent to be released from the drug carriers, wherein the controller is configured to operate the focused ultrasound source to apply the uncaging FUS sequence at FUS power levels below a threshold power level such that broadband emissions are essentially absent from an emission spectrum of the drug carriers in the target region.
6. The system of claim 5, wherein the administration device is configured for administering the ultrasound-controllable drug carriers into a blood vessel of the brain of the human or animal body, wherein the target region is a region of said brain, and wherein the controller is configured to operate the focused ultrasound source to apply the uncaging FUS sequence at FUS power levels below a threshold power level such that the blood-brain barrier remains intact.
7. A method for enhancing localized delivery of a diagnostic or therapeutic agent to a human or animal body using focused ultrasound, the method comprising: a) administering ultrasound-controllable drug carriers into a blood vessel of the human or animal body, each drug carrier comprising an ultrasound-sensitive microbubble, the microbubble comprising a microbubble shell and a gas encapsulated by the microbubble shell, the microbubble being loaded with a diagnostic or therapeutic agent; and b) aggregating the drug carriers in a target region inside the blood vessel by exposing the drug carriers to radiation forces, the radiation forces being generated by applying an aggregation FUS sequence to the target region.
8. The method of claim 7, wherein the aggregation FUS sequence is applied at FUS power levels below a threshold power level such that broadband emissions are essentially absent from an emission spectrum of the drug carriers in the target region.
9. The method of claim 7, further comprising: c) releasing the diagnostic or therapeutic agent from the drug carriers after aggregation by application of an uncaging FUS sequence to the target region.
10. The method of claim 9, wherein both the aggregation FUS sequence and the uncaging FUS sequence are applied at FUS power levels below a threshold power level such that broadband emissions are essentially absent from an emission spectrum of the drug carriers in the target region.
11. A method of enhancing localized delivery of a diagnostic or therapeutic agent to a human or animal body using focused ultrasound, the method comprising: a) administering ultrasound-controllable drug carriers into a blood vessel of the human or animal body, each drug carrier comprising an ultrasound-sensitive microbubble, the microbubble comprising a microbubble shell and a gas encapsulated by the microbubble shell, the microbubble being loaded with a diagnostic or therapeutic agent; and b) releasing the diagnostic or therapeutic agent from the drug carriers by application of an uncaging FUS sequence to a target region, wherein the uncaging FUS sequence is applied at FUS power levels below a threshold power level such that broadband emissions are essentially absent from an emission spectrum of the drug carriers in the target region.
12. The method of claim 11, wherein the ultrasound-controllable drug carriers are administered into a blood vessel of the brain of the human or animal body, wherein the target region is a region of said brain, wherein the uncaging FUS sequence is applied at FUS power levels below a threshold power level such that the blood-brain barrier remains intact.
13. The method of claim 7, comprising: recording ultrasound emissions from the drug carriers in the target region; obtaining a frequency spectrum of the recorded ultrasound emissions; and setting FUS power levels such that broadband emissions are essentially absent from the obtained frequency spectrum.
14. Use of an ultrasound-controllable drug carrier for enhancing localized delivery of a diagnostic or therapeutic agent to a human or animal body, the ultrasound-controllable drug carrier comprising an ultrasound-sensitive microbubble, the microbubble comprising a microbubble shell and a gas encapsulated by the microbubble shell, the microbubble being loaded with a diagnostic or therapeutic agent, the enhancement of localized delivery of the diagnostic or therapeutic agent being carried out by a method according to claim 7.
15. An ultrasound-controllable drug carrier for use in a method for enhancing localized delivery of a diagnostic or therapeutic agent to a human or animal body according to claim 7, the ultrasound-controllable drug carrier comprising an ultrasound-sensitive microbubble, the microbubble comprising a microbubble shell and a gas encapsulated by the microbubble shell, the microbubble being loaded with a diagnostic or therapeutic agent.
16. The system of claim 1, wherein each drug carrier comprises a plurality of microparticles or nanoparticles attached to the microbubble, the microparticles or nanoparticles comprising the diagnostic or therapeutic agent.
17. The system of claim 1, wherein the controller is configured to operate the focused ultrasound source in such a manner that the aggregation FUS sequence generates radiation forces that cause the drug carriers to aggregate inside the blood vessel both along a radial direction and a longitudinal direction of the blood vessel.
18. The system of claim 1, wherein the controller is configured to operate the focused ultrasound source to apply the aggregation FUS sequence at FUS power levels below a threshold power level such that broadband emissions and non-integer harmonics are essentially absent from an emission spectrum of the drug carriers in the target region.
19. The system of claim 3, wherein the controller is configured to operate the focused ultrasound source to apply both the aggregation FUS sequence and the uncaging FUS sequence at FUS power levels below a threshold power level such that broadband emissions and non-integer harmonics are essentially absent from an emission spectrum of the drug carriers in the target region.
20. The system of claim 4, wherein the administration device is configured for administering the ultrasound-controllable drug carriers into a blood vessel of the brain of the human or animal body, wherein the target region is a region of said brain, wherein the controller is configured to operate the focused ultrasound source to apply both the aggregation FUS sequence and the uncaging FUS sequence at FUS power levels below a threshold power level such that the blood-brain barrier remains intact.
21. The system of claim 20, wherein the diagnostic or therapeutic agent is capable of crossing the blood-brain barrier without opening of the blood-brain barrier.
22. The system of claim 5, wherein each drug carrier comprises a plurality of microparticles or nanoparticles attached to the microbubble, the microparticles or nanoparticles comprising the diagnostic or therapeutic agent.
23. The system of claim 5, wherein the controller is configured to operate the focused ultrasound source to apply the uncaging FUS sequence at FUS power levels below a threshold power level such that broadband emissions and non-integer harmonics are essentially absent from an emission spectrum of the drug carriers in the target region.
24. The system of claim 6, wherein the diagnostic or therapeutic agent is capable of crossing the blood-brain barrier without opening of the blood-brain barrier.
25. The method of claim 7, wherein each drug carrier comprises a plurality of microparticles or nanoparticles attached to the microbubble, the microparticles or nanoparticles comprising the diagnostic or therapeutic agent.
26. The method of claim 7, wherein the drug carriers are aggregated in the target region both along a radial direction and a longitudinal direction of the blood vessel.
27. The method of claim 7, wherein the aggregation FUS sequence is applied at FUS power levels below a threshold power level such that broadband emissions and non-integer harmonics are essentially absent from an emission spectrum of the drug carriers in the target region.
28. The method of claim 9, wherein both the aggregation FUS sequence and the uncaging FUS sequence are applied at FUS power levels below a threshold power level such that broadband emissions and non-integer harmonics are essentially absent from an emission spectrum of the drug carriers in the target region.
29. The method of claim 10, wherein the ultrasound-controllable drug carriers are administered into a blood vessel of the brain of the human or animal body, wherein the target region is a region of said brain, wherein both the aggregation FUS sequence and the uncaging FUS sequence are applied at FUS power levels below a threshold power level such that the blood-brain barrier remains intact.
30. The method of claim 29, wherein the diagnostic or therapeutic agent is capable of crossing the blood-brain barrier without opening of the blood-brain barrier.
31. The method of claim 11, wherein each drug carrier comprises a plurality of microparticles or nanoparticles attached to the microbubble, the microparticles or nanoparticles comprising the diagnostic or therapeutic agent.
32. The method of claim 11, wherein the uncaging FUS sequence is applied at FUS power levels below a threshold power level such that broadband emissions and non-integer harmonics are essentially absent from an emission spectrum of the drug carriers in the target region.
33. The method of claim 12, wherein the diagnostic or therapeutic agent is capable of crossing the blood-brain barrier without opening of the blood-brain barrier.
34. The method of claim 11, comprising: recording ultrasound emissions from the drug carriers in the target region; obtaining a frequency spectrum of the recorded ultrasound emissions; and setting FUS power levels such that broadband emissions are essentially absent from the obtained frequency spectrum.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
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DESCRIPTION OF PREFERRED EMBODIMENTS
Definitions
[0090] In the present disclosure, references in the singular may also include the plural. Specifically, the word a or an may refer to one, or one or more, unless the context indicates otherwise.
[0091] The expression in particular is to be understood as being non-limiting, referring to an example.
[0092] The term mammal includes both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, rodents, bovines, equines and porcines.
[0093] The expression administering into a vessel is to be understood as encompassing both direct administration into the vessel of interest and indirect administration, i.e., administration into another vessel that is in fluid communication with the vessel of interest.
[0094] Focused ultrasound (FUS) is ultrasound radiation that is focused onto a small region in space, which is called the target region. Transducers for generating focused ultrasound are well known in the art. Focused ultrasound radiation causes acoustic pressure oscillations in the target region. The amplitude of the pressure oscillations can be characterized by a parameter called peak negative pressure, i.e., the maximum amplitude of the negative pressure in the ultrasound field relative to ambient pressure. The dependence of peak negative pressure in a medium on the electric power or voltage amplitude supplied to the transducer can be readily determined by calibration measurements that are well known in the art.
[0095] In the present disclosure, the term cavitation is to be understood as broadly relating to the expansion and contraction or collapse behavior of gas-filled bubbles in an ultrasonic field. There are two different types of cavitation: inertial cavitation and stable (non-inertial) cavitation. Inertial cavitation is when a bubble rapidly expands and violently collapses in a liquid medium. Inertial cavitation generally occurs at relatively high acoustic pressures. During an inertial collapse, the speed of the gas-liquid interface may become supersonic in the gas and/or in the liquid. These supersonic motions may produce shock waves and jets in the surrounding fluid, which will propagate outward and can be damaging to surrounding biological tissue, like the blood-tissue barrier of a blood vessel. Stable cavitation is when a bubble expands and contracts in an oscillatory manner without collapsing. Stable cavitation generally occurs at lower acoustic pressures. Inertial cavitation causes both non-integer harmonics and broadband emissions in the ultrasound emission spectrum from the bubbles. Stable cavitation does not cause broadband emissions, but may cause non-integer harmonics above a certain level of acoustic pressure.
[0096] In the present disclosure, the term radiation force is to be understood as relating to a force that acts onto an ultrasound-sensitive (i.e., acoustically active) object like a microbubble as a result of ultrasound radiation. Primary radiation forces are forces that result from pressure gradients in the incident ultrasound field. They typically act in the direction of the incident ultrasound waves, i.e., away from the ultrasound transducer that generates the incident ultrasound field. Secondary radiation forces are forces that result from pressure gradients in the ultrasound field that is scattered by ultrasound-sensitive objects. They can cause attraction or repulsion between the ultrasound-sensitive objects.
[0097] In the present disclosure, the term aggregation refers to a process in which the concentration of ultrasound-controllable drug carriers in a target region is increased. In some embodiments, this increase may lead to the formation of aggregates, i.e., clusters of drug carriers, in which adjacent drug carriers are so close that they essentially touch one another. In other embodiments, aggregation encompasses an increase of concentration without the formation of aggregates.
[0098] In the present disclosure, the term uncaging refers to a process in which an agent is released from a carrier that encapsulates the agent. In particular, the term uncaging refers to a process in which an agent is released from a liposome. Before being uncaged, the agent can be located in the interior of the liposome that is delimited by the lipid shell (e.g., if the agent is hydrophilic), or it can be located within the lipid bilayer that forms the lipid shell (e.g., if the agent is hydrophobic). In both cases, the agent is considered to be encapsulated by the liposome.
[0099] A diagnostic or therapeutic agent is a compound that can be used for diagnostic purposes or for therapeutic purposes or for both. In the present disclosure, a diagnostic or therapeutic agent is also called a drug for short. The agent can be, for instance, a small molecule, a nucleic acid, an oligosaccharide, a polysaccharide, an oligopeptide, a polypeptide, a protein, a virus, or a combination of these. The term small molecule refers to organic compounds having a molecular weight of no more than 900 daltons.
[0100] Concept of Focal Aggregation and Uncaging of Ultrasound-Controlled Drug Carriers
[0101]
[0102] Ultrasound-Controlled Drug Carriers (UC-Carriers)
[0103]
[0104] In an embodiment, DSPC and DSPE-PEG2k formed the lipid shells of the microbubbles (monolayer) and liposomes (bilayer). The microbubbles (1.5 m mean diameter) were stabilized with a perfluorobutane (PFB, C.sub.4F.sub.10) gas core and had DSPE-PEG5k-Mal on the surface for conjugation with liposomes (116 nm mean diameter; conjugated UC-carriers have 1.7 m mean diameter), which contained DSPE-PEG5k-SH on the surface, with a PBS core. Small molecules were actively loaded into the liposomes prior to conjugation. For the experiments that will be subsequently described, either sodium fluorescein dye (model drug for in vitro experiments) or muscimol (for in vivo experiments) were loaded into the core of the liposomes.
[0105] While this embodiment has proven advantageous, the presently proposed method is expected to yield good results also for microbubbles and liposomes of different composition and with different sizes. In particular, it is expected that the presently proposed method yields good results for conjugated UC-carriers having a size ranging from about 1 to 5 m, with liposomes having a size in the range from about 50 to 200 nm. Other lipid systems than DSPC/DSPE may be employed, and the PEG moieties do not need to be PEG2k and PEG5k, respectively. Instead of being encapsulated in a liposome, the drug may be loaded to a microbubble also in another manner.
[0106] AU-FUS Sequence
[0107]
[0108] In Vitro Characterization of AU-FUS Sequence
[0109] For in vitro characterization of the AU-FUS sequence, dye-loaded UC-carriers flowed through microdialysis tubing (13 kDa cut-off, single pass), embedded in low-melt agarose. The system was confocally aligned to an inverted water immersion 60 objective lens and a 2.5 MHz FUS transducer. The entire setup was inside a custom-made water tank, filled with deionized, degassed water.
[0110] Fluorescein-loaded UC-carriers flowed continuously through the dialysis tubing at speeds (10 L/min, corresponding to 5 mm/s of flow speed) that mimicked the highest blood flow rates in brain capillaries. The carriers were then exposed to either commonly used BBB-opening FUS (standard-FUS sequence) or the novel AU-FUS sequences.
[0111] The effect of the novel AU-FUS sequences is illustrated in
[0112] Dye release into the agarose was measured both within the focus and outside the focus of the FUS transducer. Standard-FUS ultrasound sequences as well as multi-component sequences were used, with the parameters provided in Table 1 below.
TABLE-US-00001 TABLE 1 FUS parameters P.sub.A t.sub.A P.sub.U t.sub.U t.sub.OFF PRF Condition (MPa) (ms) (MPa) (ms) (ms) (Hz) 25000 Standard.sub.1-FUS 1.25 1 25000 Standard.sub.2-FUS 2.5 1 25000 Standard.sub.3-FUS 0.75* 1 25000 Standard.sub.4-FUS 1.5* 1 25000 AU.sub.1-FUS 0.3 500 0.5 90 300 30 10000 (in vitro) AU.sub.2-FUS 0.38 500 0.63 90 300 50 10000 (in vitro) AU.sub.3-FUS 0.25 1000 0.63 90 300 100 1000 (in vitro) AU-FUS 0.075* 1000 0.188* 90 300 100 1000 (in vivo) P.sub.A, P.sub.Upeak negative pressure in megapascal (MPa) for aggregation and uncaging sequences, respectively (*accounting for ~70% skull attenuation; see Methods), t.sub.Apulse duration (in milliseconds) of aggregation sequence, t.sub.Uduration (in milliseconds) of uncaging sequence, t.sub.OFFduration (in milliseconds) of delay between the end of the uncaging sequence and the start of the following aggregation sequence, PRFpulse repetition frequency in Hz, NOCnumber of cycles.
[0113] The performance of the AU-FUS sequence was compared to the performance of standard, single-component FUS sequences. Results are illustrated in
[0114] The AU-FUS sequences not only delivered more molecules than the standard-FUS sequences, but also required several-fold lower ultrasound pressures than both the standard-FUS sequences and the reported fragmentation pressures of Ferrara and colleagues, thus having a much reduced damage potential for the vasculature or BBB when used in vivo. Even doubling the pressures of standard-FUS sequences did not result in greater deposition, suggesting the acoustic radiation forces generated by the first component of the novel AU-FUS sequences played a role in efficient delivery of the molecules.
[0115] Optimization for In Vivo Experiments
[0116] When tested in vivo (with FUS-parameters adjusted to account for the attenuation of ultrasound waves by the skull), even the best in vitro optimized AU-FUS parameters [AU.sub.1-FUS (in vitro) and AU.sub.2-FUS (in vitro)] still caused weak BBB opening. Since neither in vitro deposition nor artificial BBB models sufficiently mimic in vivo BBB, capillaries, drug, and blood-plasma interactions, the AU-FUS pulse sequences were further optimized by iterations between in vivo and in vitro experiments. It is remarked that the in vitro studies were performed using commercially available microdialysis tubing, which is larger than the diameter of brain capillaries. Due to the large size of ultrasound transducers and the optical objective lenses, it is currently infeasible to image microparticles in brain capillaries during FUS. Many parameters were systematically varied, including the number of cycles, pulse-repetition frequencies, amplitudes for each component of AU-FUS, pulse-to-pulse delays (FUS OFF period), UC-carrier concentrations, and UC-carrier lipid chemistries and compositions.
[0117] Based on these optimizations, an in vivo AU-FUS sequence [AU-FUS (in vivo); see Table 1] was found that was able to deliver small molecules with extremely high efficiency while also completely avoiding BBB damage. The effects of the in vivo optimized AU-FUS sequence on UC-carriers using the in vitro setup were studied. It was observed that neither aggregation nor uncaging sequences alone were effective in releasing small molecules and that both components are essential for low-pressure drug delivery when ultrasound power levels as low as in the in vivo AU-FUS sequence are used.
[0118] This is illustrated in
[0119] Receptor-Specific Focal Modulation of a Cortical Network In Vivo
[0120] The AU-FUS sequence [AU-FUS (in vivo); see Table 1] was tested in vivo by manipulating a specific cortical network without opening the BBB. Rat vibrissa motor cortex (vM1) receives whisker sensory information (80%) through projections from vibrissa sensory cortex (vS1, Barrel cortex). When rodent whiskers are mechanically deflected, evoked activity propagates from brainstem via thalamus to vS1, and then to vM1. During simultaneous recordings the peaks of whisker-evoked potentials (wEPs) were observed first in vS1 and 3.0 ms later in vM1. It was tested whether inhibiting vS1 by the novel technique would suppress wEPs in vM1 in anesthetized rats. The FUS transducer was positioned above the intact skull and focused on vS1. Using a multielectrode array, the wEPs and the whisker-evoked multi-unit activity in vM1 were monitored online. In parallel, muscimol-loaded UC-carriers were continuously (i.v.) injected. The AU-FUS sequence was applied repeatedly on vS1 to aggregate the UC-carriers and to uncage muscimol. Muscimol is an agonist of ionotropic GABA.sub.A receptors, the major receptor responsible for fast inhibitory transmission in the brain, which readily crosses the BBB.
[0121] Setup for In Vivo Experiments
[0122]
[0123] The FUS transducer 136 was operated by a function generator 132 via an RF amplifier 134. The function generator 132 was configured by a computer 120. The output from the function generator 132 was monitored by an oscilloscope 122.
[0124] Signals from the recording probes 140 were amplified by a pre-amplifier 150 and digitized using an analog-to-digital converter 152. Digitized signals were recorded by a computer 154, which may or may not be identical with computer 120.
[0125] Whiskers were mechanically stimulated by a whisker/LED stimulator 160. The whisker/LED stimulator 160 also drove an LED 162 for visually stimulating an eye of the animal. Contralateral whiskers or eye were mechanically deflected or visually stimulated, respectively, at 0.3 Hz.
[0126] Muscimol-loaded UC-carriers were injected intravenously through the tail vein using an infusion pump 110. vM1 and V1 recordings were performed on separate cohorts.
[0127] Results of In Vivo Experiments
[0128] The experimental results showed that FUS-mediated delivery of muscimol inhibits vS1 and reduces the whisker evoked multi-unit activity and wEPs in its projection target vM1. This is illustrated in
[0129] A complete set of control experiments was performed in order to confirm the specificity of the novel approach and to verify that only muscimol-loaded UC-carriers with AU-FUS (in vivo) results in local inhibition. The results of these control experiments are illustrated in
[0130] To rule out that the observed modulation of vM1 could be due to spreading of muscimol from vS1, signals were recorded from a cortical area close to vS1 but without significant connectivity with vS1: Muscimol was delivered to vS1 using AU-FUS, and the visually evoked potentials (VEPs) were measured from primary visual cortex (V1). No statistically significant changes in VEPs recorded from V1 were observed. This is illustrated in
[0131] The experimental results were also consistent with the initial estimates that small molecules cannot be spreading large distances to induce physiological responses: Spreading could happen through two different means. First, uncaged muscimol could perfuse through the capillaries to distant regions. Assuming the diffusion coefficient of a small molecule even in water as D=1.510.sup.5 cm.sup.2 s.sup.1, and a capillary radius of r=5 m, small molecules would take T=16 ms (T=r.sup.2/D) on average to reach to the capillary walls from anywhere within the capillary. Assuming an average blood flow speed in capillaries of v=1.5 mm/sec, free muscimol is expected to flow only 25 m (=T*v) beyond its release site before it reaches the blood brain barrier. Even if free muscimol does not enter the brain tissue immediately and remains in the circulation, it cannot reach far away tissues before entry to veins because the maximal length of capillaries in the rat brain is only about 250 m and drug uptake to the brain is mainly confined to the capillaries. Importantly, muscimol's concentration will be negligible after entering the vein and redistributing in systemic circulation. Second, muscimol could diffuse within the interstitial space. The diffusion coefficient of muscimol in rat barrel cortex is D=8.710.sup.6 cm.sup.2 s.sup.1. Given that the maximum inhibition in vM1 occurs within 20 mins, muscimol can diffuse in tissue only up to 1 mm. This diffusion distance has been confirmed by measuring the spread of fluorescent muscimol in the rat brain tissue. In deeper subcortical structures, the observed rostrocaudal spread of muscimol has been shown to be 1.7 mm on timescales comparable to muscimol action in the present work. Since vM1 is 7 mm away from vS1 in rats, muscimol diffusion in the tissue from release site to recording site cannot be the cause of observed neuronal inhibition. Additionally, radial diffusion of muscimol also rapidly dilutes muscimol (d.sup.3) with distance (d) from the delivery locus, thus making its concentration too low to induce physiological responses. These estimates support the experimental finding above. Thus, drug delivery is highly local and the observed neuromodulations cannot be due to the spreading of uncaged drug.
[0132] 1300-Fold Focal Aggregation of Drug by AU-FUS
[0133] The total amount of muscimol injected in a single dose of AU-FUS treatment was 200 ng, as estimated using Liquid Chromatography-High Resolution Tandem Mass Spectroscopy (LC-HR-MS/MS). The results from LC-HR-MS/MS are shown in
[0134] The effect of systemically administered muscimol on the wEPs in vM1 (without AU-FUS or UC-carriers) was tested. A comparable reduction of wEPs was observable only after systemically administering at least 1300 times the measured payload of the UC-carriers (at least 260 g systemic muscimol). This is illustrated in
[0135] This is consistent with the amount of systemic muscimol required for brain inactivation which is 1.6 mg/kg.
[0136] Preservation of Blood-Brain Barrier Integrity and Normothermia
[0137] BBB integrity was assessed to determine the safety of the proposed sequence, as BBB-opening can be accompanied by inflammation and cell death. BBB opening was evaluated by measuring the extravasation of Evans Blue dye (EB) through IVIS spectrum epifluorescence imaging, Gadolinium (Gd)-enhanced T1-weighted Magnetic Resonance (MR) imaging, and extravasation of Immunoglobulin G (IgG) by immunohistochemical staining, none of which easily crosses the intact BBB. Tracer (EB, Gd, and IgG) extravasation was measured in regions of interest (ROIs) ipsilateral and contralateral to FUS in vS1 to determine BBB opening.
[0138] Results are illustrated in
[0139] There was no statistically significant difference in EB or IgG extravasation or Gd contrast enhancement when comparing ROIs contralateral and ipsilateral to FUS treatment site for the animals undergoing AU-FUS. In contrast, marked EB, Gd, and IgG labelling demonstrated profound BBB opening with standard-FUS parameters (standard.sub.2-FUS in Table 1).
[0140] Since it is known that FUS can cause rapid temperature increases in the brain, which can have adverse effects on the BBB, the temperature within the AU-FUS focal volume was monitored. An average temperature increase of only 0.12 C. during the sonication was measured, which is within the normal range of temperature fluctuations in the awake behaving rats and is significantly below the threshold for alterations in BBB permeability, cell damage, or changes in cell activity.
[0141] Passive Cavitation Detector Responses Show No Spectral Signatures of BBB Opening
[0142] Passive cavitation responses of UC-carriers were measured under standard-FUS sequences at different pressures, and under the presently proposed AU-FUS sequence. The results are shown in
[0143] Part A of
[0144] Part B of
[0145] Part A of
[0146] Part B of
[0147]
[0148]
[0149] Upon injection of UC-carriers, the standard.sub.4-FUS sequence (1.5 MPa) caused strong broadband emissions, indicating inertial cavitation, along with ultra-harmonic emissions (see part A of
[0150] Sonicating with the standard 3-FUS sequence (0.75 MPa) caused ultra-harmonic emissions, which are signatures of stable cavitation, with negligible broadband emissions (see part B of
[0151] In contrast, sonication of UC-carriers with the presently proposed AU-FUS sequence caused neither ultra-harmonics nor broadband emissions (see
DISCUSSION
[0152] Technological advancements that enable safe and robust manipulation of specific neural circuits involved in disease pathologies can address both efficacy and molecular specificity challenges of existing treatments. In this study, a novel technique is demonstrated that allows efficacious and non-invasive modulation of specific brain circuits by spatially targeted delivery of receptor-specific small molecules. Several fundamental challenges were overcome to make this possible: 1) Devising a means to trap and concentrate sufficiently high numbers of UC-carriers in circulation, 2) Uncaging ample amounts of drug with minimal ultrasound energies to induce significant physiological responses, 3) Avoiding opening/damaging BBB and tissue heating, 4) Producing stable microbubble-liposome complexes suitable for use with multi-component FUS which can carry sufficient payloads with diverse physical properties, and 5) Identifying FUS sequences that do not cause nonspecific neuronal responses (i.e. without molecular specificity) due to the application of FUS alone (i.e. in the absence of UC-carriers).
[0153] The AU-FUS sequences concentrate the drug carriers not only along the radial axis of the capillaries (primary radiation forces), but also along the longitudinal axis of the capillaries (secondary radiation forces). While the applicant does not wish to be bound by theory, the aggregation component of the AU-FUS sequence likely utilizes secondary radiation forces, known as Bjerknes forces, to drive the UC-carriers into close proximity to each other at low-pressure amplitudes. The dynamics of aggregated UC-carriers in a cluster are complex as they continue to volumetrically oscillate, coalesce, break up, and re-form repeatedly under continuous low-intensity ultrasound. While this physical response of UC-carriers could lead to enhanced drug delivery (as liposomes may be de-stabilized in this process), the exact mechanisms responsible for facilitating drug release from conjugated liposomes are difficult to elucidate due to the complex nature of UC-carrier aggregation, which is highly dependent on UC-carrier size distributions, concentrations, ultrasound frequencies and pressures. Additionally, the application of higher intensity fragmentation pulses in the second part of the AU-FUS sequence, as the microbubble shells destabilize, may promote better drug release from liposomes in UC-carrier clusters compared to individual UC-carriers, since gas from the microbubbles would leak out at high velocities, causing shear effects on the liposomal bilayer, thereby releasing its contents. It is also important to note that the present UC-carriers are expected to respond differently to FUS compared to microbubbles alone as liposome-attached bubbles have different physical properties than microbubbles alone.
[0154] The vS1-vM1 circuit is a good model for in vivo validation of FUS-mediated delivery of drugs. First, the functional and anatomical connectivity between vS1-vM1 is well described. In this manner spatial specificity of FUS application and receptor specificity of drug loaded UC-carriers was demonstrated. Second, multiple studies demonstrate that inhibiting vS1 results in attenuation of evoked responses in vM1 as well. Finally, this circuit allowed to rule out the effects of uncaged muscimol spreading as a cause of the observed vM1 inhibition using a neighboring cortical area (V1) as control.
[0155] A major challenge in the chronic use of ultrasound-mediated drug release in the brain has been that previous approaches either required or caused unavoidable BBB opening. Recently, it has been shown that BBB opening can induce sterile inflammation under certain conditions, has strong effects on cell activity and behavior, and is implicated in neurodegenerative diseases. It is therefore highly desirable to achieve FUS-mediated drug release without compromising the BBB, especially for use in chronic treatments of many brain disorders. Therefore, passive cavitation monitoring of UC-carriers was performed to ensure the absence of inertial cavitation signals which otherwise indicate BBB opening or cell death. The intactness of BBB integrity was demonstrated by multiple measures: Evans Blue dye, Gadolinium extravasation through IVIS spectrum and MR imaging, respectively, as well as ImmunoglobulinG (IgG) immunohistochemical staining. To rule out even highly transient BBB opening, tracers (Evans Blue dye and Gadolinium) were also injected prior to sonication. No extravasation of the tracers was observed.
[0156] Recent studies by Airan and colleagues (see Background section) suggested that FUS-sensitive perfluoropentane (PFP) nanoemulsions (instead of the microbubbles+liposomes used here) might be used to deliver lipophilic-only agents to the brain without opening the BBB. However, unlike the gas-filled microbubbles used in the present study, nanoemulsions cannot be spatially concentrated due to their significantly lower responsiveness to acoustic radiation forces, making further improvements in their efficacy and specificity more challenging. Since the presently proposed UC-carriers require 1/1300 times or less drug than that required for systemic delivery to yield significant response, even if the payload is to be completely released systemically, it causes no detectable effect.
[0157] While the mechanistic understanding of psychiatric and neurological disorders has advanced in the preceding decades, translation of the knowledge or testing of hypotheses towards viable treatments has been almost non-existent. This has been in part due to the vast complexity and heterogeneity of the brain and an inability to selectively target regions and circuits of interest. Circuit-selective neuromodulation in humans may offer a strategic path towards effective treatment of many CNS disorders. Moreover, targeted delivery may offer a new avenue for small molecules that have failed due to toxicity or lack of efficacy, by allowing drugs to be delivered only to a desired region and possibly at higher concentrations than achievable through systemic delivery. Approved drugs should also benefit from focal delivery as this can likely eliminate, many if not all, side effects. The ability to enhance drugs' concentrations by 1300 fold over systemic levels demonstrates the power of our technology, and represents a fundamental leap forward in non-invasive targeted drug delivery.
[0158] Existing clinical ultrasound systems for targeting the brain are expensive and bulky, which would make chronic treatments challenging. However, they can be significantly scaled down with the use of integrated MEMS (Micro-Electro-Mechanical Systems) ultrasound transducers, which are capable of generating sufficient ultrasound power and can also be chronically implanted under the scalp or beneath/within the skull. Use of such technologies beneath the skull can also allow the use of higher frequency ultrasound waves (e.g. 2.5 MHz) with higher spatial resolution in clinical applications. The other option would be to use drugs with long lasting effects such as the NMDAR antagonist ketamine (recently approved as a rapid-onset antidepressant for patients with treatment resistant depression) which is efficacious for weeks following acute administration. Targeted delivery of ketamine could reduce its side-effects (such as psychotomimetic and perceptual disturbances, in addition to heart rate and blood pressure complications), while significantly enhancing its therapeutic index. While further refinement of our technology (e.g. reducing the sonication time required by further ultrasound sequences and/or drug loading and employing closed-loop, real-time passive cavitation detection for acoustic monitoring) can advance it to the clinic, the present approach presents a viable path forward, since individual chemical and ultrasound components of the presently proposed technology are already FDA approved. Regardless, the technology presented here enables the most efficacious, non-invasive, receptor-specific, millimeter-precision manipulation of brain circuits.
[0159] Methods
[0160] Ultrasound-Controlled Drug Carriers (UC-Carriers)
[0161] Ultrasound controlled-drug carriers were created as follows. The UC-carriers contained a backbone of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti Polar Lipids) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-polyethylene glycol (PEG)2000 (Corden Pharma) in 90:5 molar ratio. The remaining 5% was DSPE-PEG5000-Thiol (SH) for liposomes and DSPE-PEG5000-Maleimide (MAL) (both from Nanocs) for the bubbles. The lipids were dissolved in chloroform and mixed in appropriate volumes to achieve a total concentration of 2 mg/mL for the bubbles and 10 mg/mL for liposomes. Chloroform was then evaporated under nitrogen and kept overnight under vacuum. The resultant lipid films were stored at 20 C. till further use.
[0162] For the bubbles, the lipid films were rehydrated with 1 PBS containing 10% propylene glycol and 10% glycerol. The solution was then heated at 70 C. for at least 30 mins, and bath sonicated for at least 20 mins or until the solution was clear. The headspace in the vial was filled with perfluorobutane (PFB; SynQuest Laboratories), and microbubbles were formed through a probe tip sonicator (70% power; Branson SLPe with 3 mm tip). The microbubble solution was then size isolated by centrifugation at 300g for 3 mins, 3 times. After each centrifugation the wash solution was discarded and the remaining bubbles were resuspended in PBS:EDTA (1 mM EDTA; pH 6.5).
[0163] The liposome lipid films were rehydrated with 1 PBS, heated at 70 C. for at least 30 mins, and bath sonicated for 3 hrs. Drug (muscimol from Hellobio, sodium fluorescein from Sigma-Aldrich) was added at a drug/lipid ratio of 0.3 and 15 freeze-thaw cycles were performed in liquid nitrogen and 37 C. water bath, for 2.5 mins each (all steps performed in the dark for fluorescein). The liposomes yielded a mean size of 116 nm.
[0164] The resultant bubbles and liposomes were mixed and allowed to conjugate overnight at 4 C. Next day the solution was washed 2 times by centrifugation at 300g for 3 mins. The concentration and size distribution were analyzed in triplicate using Multisizer 3 (Beckman Coulter). The mean size of the UC-carriers was 1.7 m.
[0165] Animal Preparation
[0166] Female Long Evans Rats (200-300 g, Charles Rivers Laboratories, Research Models and Service, Germany and Janvier Labs, Rodent Research Models and Associated Services, France) were used. The animals were housed in groups in standard IVC cages (Allentown), and had ad libitum access to food and water, and were on an inverted light cycle (12 hour dark/12 hrs light). All procedures were approved by the Veterinary Office, Canton Zrich, Switzerland.
[0167] Surgery Animals were anesthetized in an induction chamber with 4-5% oxygenated isoflurane for 3-4 mins. They were then moved to a preparation area where the tail vein was catheterized with a winged 27 G catheter (Terumo), and the head was shaved. 2 mg/kg Meloxicam (Metacam) and 7 mL/kg warmed Lactated Ringers solution (Fresenius Kabi, AG) were subcutaneously injected. The rat was moved to a stereotaxic frame (David Kopf), and an incision was performed on scalp to expose skull surface. A layer of eye cream was put on the eyes. A craniotomy was performed with a micro-drill above vM1 (Coordinates AP: 0-2.5 mm and ML: 0-2 mm, with respect to bregma), and dura was carefully opened. During the craniotomy, the skull was frequently flushed with Ringers solution (B. Braun) to prevent heating. After dura removal, a piece of gel foam (Pfizer) was put on brain and Ringers solution was regularly applied to keep the brain moisturized until electrode insertion.
[0168] Whisker Stimulation
[0169] Whiskers were cut to around 15 mm length. The 8-12 largest whiskers were inserted into a glass capillary tube which was attached to a piezo actuator (T223-H4CL-503X, Piezo Systems). The piezo actuator was shielded with a custom-made copper cover. The whiskers were deflected with 120 Hz cosine pulses (292 mm/s velocity, displaced 2.34 mm in 8 ms) which were generated in LabView (National Instruments) and converted into an analog signal (DAC NI, USB-6211) which then drove the piezo actuator. Stimulus presentation was synchronized with the electrophysiological recordings with a TTL signal at stimulus onset. Whiskers were continuously stimulated at a repetition rate of 0.3 Hz.
[0170] FUS Setup and Transducer Calibration
[0171] A custom-made transducer (Sonic Concepts) with 2.5 MHz center frequency, 40 mm diameter, 30 mm working distance/20.65 mm focal depth, and 0.50.52.5 mm theoretical focus was used, along with a custom impedance matching network for the transducer (Sonic Concepts). Calibration was done in a degassed and deionized water filled chamber with a mm needle hydrophone (Acoustic Precision). The ultrasound pulses were generated with a function generator (Agilent 33210A, Keysight technologies) and PicoScope (3205B) oscilloscope and controlled by a custom MATLAB script. The signal was amplified 50 dB through a power amplifier (E&I 325LA).
[0172] Electrophysiology and FUS Drug Delivery for vS1-vM1 Measurements
[0173] All electrophysiological data were recorded with a RHD2000 system (Intan Technologies) with 30 kS/s sampling rate. All stereotaxic coordinates were determined with respect to bregma. A 32 channel Neuronexus probe (A2x16-10 mm-100-500-177-A32, 15 or 50 m thick) attached to a motorized 3D arm (StereoDrive-960HD, Neurostar), fixed outside of the stereotaxic frame, was inserted into the vM1 (Coordinates AP: 1-2 mm, ML: 0.5-1 mm, depending on the vasculature, DV: 1.5-2 mm from pia) at 50 to the coronal plane. The probe was initially inserted 250-500 m below the cortical surface. The FUS transducer was integrated with an acoustic collimator which was filled with degassed and deionized water and contained a polystyrene film (McMaster-Carr) at the end. The collimator's shape was designed according to the FUS transducer's geometry so that it would not interfere with the FUS beam. This assembly was stereotaxically positioned such that the FUS focal volume was targeted to vS1 (Coordinates AP: 2.3 mm, ML: 6 mm, DV: 3.3 mm from skull surface) at a 30 angle with respect to the sagittal plane such that the focal volume of FUS beam targeted cortical layers of vS1. A sufficient amount of warmed (to body temperature) sterile ultrasound gel (Parker Laboratories) was put on the skull over vS1 for acoustic coupling. After positioning the FUS transducer, the recording probe was further inserted below cortical surface to reach a final DV position of 1.5-2 mm (tip). Following this, there was a period of about 1-2 hrs during which the wEP amplitudes stabilized. Baseline wEP responses were acquired for 10 mins, followed by the intravenous injection of small molecule- or vehicle-loaded UC-carriers (2-2.510.sup.9 total UC-carriers per animal) and/or muscimol (250 ng,
[0174] Electrophysiology and FUS Drug Delivery for vS1-V1 Measurements
[0175] The protocol used for vS1-V1 was the same as vS1-vM1 paradigm with the following exceptions: FUS sonication coordinates were changed to AP: 2.3 mm, ML: 6.5 mm, DV: 3.3 mm from skull surface and FUS angle was changed from 30 to 34 to create space for electrode insertion to V1. A more lateral region of vS1 was sonicated to achieve better coupling with skull with the new angling of the FUS transducer. Electrode insertion coordinates were changed for V1 recording to AP: 5.5-6.0 mm, ML: 3.2-3.5 mm, DV: 1.6-2.0 mm to ensure that recording is done from cortical layers of V1. The angle of the probe insertion was kept at 50 as in vM1 recording. Instead of wEPs, VEPs were recorded by visual stimulation. The amount of muscimol-loaded UC-carriers per animal was changed from 2-2.510.sup.9 to 2.5-3.010.sup.9 and sonication period was changed from 20-25 to 30-35 mins to show that even excessive muscimol delivery does not diffuse off-target brain circuits.
[0176] Visual Stimulation
[0177] A thin layer of eye cream was put on the contralateral eye. A 5 mm green LED was inserted in custom made black rubber cone. The cone was positioned on the contralateral eye such that the LED illuminates on the eye 3-4 mm away from the cornea. This configuration allowed sufficient intensity light stimulation to the eye, as the cone covered the eye and blocked any light from outside. The LED was triggered with a 10 ms TTL pulse which was generated in LabView (National Instruments) and buffered through a NI-USB-6211 (National Instruments) board. Stimulus presentation was synchronized with the electrophysiological recordings with the same TTL signal used for stimulation. The eye was continuously stimulated at a repetition rate of 0.3 Hz. The ipsilateral eye was covered with a black rubber after putting sufficient amount of eye cream. The whole recording session was done while all the lights in the room were turned off.
[0178] Simultaneous vS1 and vM1 wEP Recordings
[0179] Animals were prepared for craniotomy as indicated above and stereotaxic coordinates were determined with respect to bregma. An incision was made on the scalp and craniotomies were performed on vS1 (Coordinates AP: 2.3 mm, ML: 6 mm, DV: 1.1 mm from pia) then on vM1 (Coordinates AP: 1.5 mm, ML: 1 mm, DV: 0.8 mm from pia). vS1 was covered with gel foam, and continuously supplied with Ringers solution to keep the brain fresh until the vM1 craniotomy was completed. 8-12 whiskers were inserted into a capillary tube attached to a piezo stimulator. The first probe was inserted to vS1 with a 30 angle to the sagittal plane, 5 mins later the second electrode was inserted to vM1 parallel to the sagittal plane. Neuronexus, A4x8-5 mm-100-200-177 probes were used for both recordings in the experiment. Once both probes were inserted, whiskers were continuously stimulated at 1 Hz, wEPs were allowed 1-2 hrs to stabilize and responses were subsequently recorded for 8 mins.
[0180] In Vitro FUS Characterization
[0181] UC-carriers (510.sup.8 MB/mL) flowed through a porous (13 kDa pore-size, 200 m ID) microdialysis tube (132294, Spectra/Por) which was surrounded by agarose gel (0.6% in DI water+0.9% NaCl, 16500500, UltraPure Invitrogen) in a custom-built channel. The custom-built channel that held the agarose gel had five marked sites, each of which was sequentially brought in the confocal alignment of a water immersion objective (CFI APO NIR 60X W, Nikon) and the FUS transducer in a water tank containing degassed and deionized water. The first four of the five sites in direction of flow were sonicated with the test FUS pulse sequences and the last site served as a control site. For either standard-FUS or AU-FUS characterizations, each site was sonicated for the same amount of time (5 mins). The tubing was then retracted from the agarose gel and each of the five agarose-gel sites were cut out and melted in heated (80 C.) deionized water, and fluorescence was measured with a plate reader (the control site was subtracted from all readings) (Gen5 Microplate Reader, BioTek).
[0182] 3D Scanning of Skull Effects on FUS
[0183] Rat skulls were extracted, and tissue was removed from the surface before degassing in a chamber for 30 mins to remove any trapped air inside the skull cap. The skulls were then positioned inside a deionized and degassed water chamber and controlled manually by mounting them on a 3D stage (PT3/M, Thorlabs). The water chamber was at the base of a stereotaxic system (Neurostar). A metal pointer was mounted on the motorized arm of the stereotaxic system to find bregma. The FUS transducer subsequently replaced the metal pointer on the stereotaxic arm such that the focal point matches precisely with tip of metal pointer. The FUS transducer was then positioned such that the focus of the ultrasound was aligned at 4 mm rostral to bregma and 7 mm ventral from skull surface. A needle hydrophone (9 m thick gold electrode PVDF film; 0.2 mm tip size, Precision Acoustics) was mounted on a motorized stage (PT3/M-Z8), and then moved to find focal point of the transducer. The hydrophone scanned an area (4 mm6 mm5 mm) in 3D with skull and (4 mm6 mm4 mm) without skull. The motors were moved with 100 m step sizes to scan the AP and DV axes, and ML axis moved continuously to decrease time required for scanning, while data was collected with 14 m resolution. ML values were then sampled every 100 m. An optical displacement sensor (SICK, OD Mini) was used to find the position of ML motor, and an analog-to-digital-converter (ADC) (NI 6009) was used to gather sensor data to the computer. Hydrophone pressure readings were collected with a PicoScope (3205B), and the entire setup was controlled with a custom MATLAB script.
[0184] Calculation of Skull Transmission Factor
[0185] To measure the transmission factor of the skull, the peak negative pressure was measured with the hydrophone at the focal point of the transducer (P1), following which the skull was moved in between. Because the skull acts as a lens, the focal area changes slightly (depending on skull region and thickness). Hence, the hydrophone was repositioned to find the new focal area and peak negative pressure was measured again (P2). The transmission factor is calculated as P2/P1100. For the brain region 4 mm rostral and 7 mm ventral to bregma, the transmission factor was 0.43 (57% loss), however for skull region above vS1 we expect 70% loss due to greater skull thickness. This data is in agreement with previous findings.
[0186] Electrophysiology Data Analysis
[0187] All electrophysiological data analysis was done in Python, version 3.6, using custom scripts. For evoked potential (wEP.fwdarw.vM1 and VEP.fwdarw.V1) analysis, the raw data was low pass filtered (3rd order Butterworth filter) at 300 Hz. The wEPs and VEPs were extracted based on the time stamp of the whisker/visual stimulus. The waveforms were then corrected for amplitude offset by taking the mean of a 25 ms time-window preceding the stimulus onset. The peak negative value for the wEP and VEP was considered to be the amplitude of the wEP and VEP response and used for analysis and data visualization. The four recording sites (i.e. electrodes) with the highest response amplitudes were then automatically selected for each experiment. Extracellular spike detection and sorting was done with Klustakwik, an open source software. PSTHs were then extracted through a custom code in Python. For wEP and VEP analysis, moving averages were calculated for a window step size of 180 whisker deflections/visual stimuli (moving step size is 1 deflection/visual stimulus). Responses were normalized for each electrode to the average response of the min window preceding FUS. For statistical analysis, in order to keep the number of data points for baseline (10 mins, 152 peaks) and post treatment (30 mins, 457 peaks) same, 152 peak values from post treatment were randomly selected. Data was visualized using Prism 7.0 and 8.0.
[0188] Measurement of Brain Temperature at the Sonication Site
[0189] A temperature probe (0.4 mm diameter, IT-21, Harvard Apparatus) was inserted through a 21G metal needle such that the tip of the probe stayed in the open cavity of the needle at the tip. This diameter (0.4 mm) was selected because it is smaller than the ultrasound wavelength (0.62 mm). A small craniotomy was performed on the skull in the following coordinates of AP: 2.3 mm, ML: 2-2.5 mm and the probe was inserted at a 52 angle. The probe was connected to a portable thermocouple thermometer with 0.1 C. resolution (Harvard Apparatus). The FUS traducer was positioned with a collimator and coupling gel on vS1 at 34, and the temperature was allowed to stabilize for 1-2 hrs. The output of the thermocouple was digitized through an Analog-to-Digital Converter (ADC) (NI-6009, National Instruments). The corresponding output voltage from the ADC was converted to temperature and further analyzed using a custom MATLAB (Mathworks) script based on the probe's calibration sheet from the manufacturer. The data was collected at 10 Hz, a moving average was applied (window size of 10 s and step size of 0.1 s), and then further downsampled to 1 Hz for analysis. The data was visualized in Prism 8 (GraphPad).
[0190] IVIS Spectrum Imaging
[0191] At the end of the experiment, animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) prior to transcardial perfusion. Blood was cleared with phosphate-buffered saline (PBS) solution and animals were perfused with 4% paraformaldehyde solution (PFA; in PBS at pH 7). Brains were removed and placed in 4% PFA for at least 72-h before sectioning with a compresstome. Sections were cut at 100 m thickness into a PBS bath and mounted onto microscope slides in MilliQ water. Sections were dried in the dark and subsequently imaged using the IVIS Spectrum (Living Image). The following parameters were used: Epi-Illumination, FOV: 6.6, FSTOP: 2, Binning: (M) 8, Exposure time: 1 s, Excitation: 465 nm, Emission: 680 nm, and scales are presented as radiant efficiency. Slides were imaged from bregma 2.280.7 mm (AP) and ROI analysis was employed using dimensions slightly larger than the theoretical FUS focal volume (3.5 mm DV1.5 mm ML oval angled at 30) at locations 6 mm (to top of ROI) from midline. Radiant efficiency values ((photons/sec/cm2/sr)/(W/cm2)) for ROIs in vS1 for regions ipsilateral and contralateral to FUS were measured and values were normalized to the mean value of the contralateral side.
[0192] MRI Imaging
[0193] Contrast enhanced T1-weighted imaging was employed to visualize BBB disruption. Two groups of animals were compared (AU-FUS vs standard-FUS, n=3 each). After sonication animals were transferred into the MR scanner (Bruker 7T PharmaScan). Pre-Contrast T1-weighted images were acquired (TE/TR: 4.5 ms/146 ms; NEX=3; FOV: 35 mm35 mm; matrix=256256; slice thickness: 0.5 mm; Flip Angle=82). These images were repeated (post-contrast) after injecting a bolus of the MRI contrast-agent Gd-DTPA (Omniscan) at 0.3 mL/kg. Additionally, TurboRARE anatomical images were acquired as a reference (TE/TR: 24 ms/4095 ms; NEX=10; echo spacing factor=8; rare factor=8; slice thickness=0.45 mm; matrix: 180120; FOV 20 mm12 mm). The animals were kept at a constant anesthesia level of 2.5% and sacrificed at the end of the experiment. A signal enhancement analysis was done. A region of interest (ROI) (1 mm1 mm) was drawn around 3 adjacent post-contrast T1-weighted slices, based on the FUS reference stereotactic coordinates, while excluding ventricles from the ROI. The difference in pre- and post-sonication T1-weighted images was calculated for ipsilateral and contralateral to FUS sites. The values for each group were pooled across animals, subsequently plotted and compared. In the case of Gd-DTPA injection before sonication, there was no pre-scan image, and the total signal intensity was measured for ROIs ipsilateral and contralateral to FUS, and values were normalized to the mean value for the contralateral side.
[0194] IgG Staining
[0195] Rats were treated with either AU-FUS or standard-FUS and remained under 2% isoflurane anesthesia for 3 hrs while maintaining body temperature with a thermometric heating blanket with rectal probe. Rats were injected with ketamine (100 mg/kg) and xylazine (10 mg/kg) prior to transcardial perfusion. Following perfusion, the brains were post-fixed for 18 hrs before preparing sections with a compresstome (50 m thickness). Sections were blocked with 0.3% H.sub.2O.sub.2 in PBS for 10 mins at room temperature (RT), rinsed with PBS containing 0.25% triton X-100 (PBST), and blocked with PBST containing 10% normal goat serum. The tissue was then incubated with Biotinylated anti-rat IgG (Vector labs BA-9400) diluted 1:1500 in PBST overnight at 4 C. Sections were rinsed 3 times with PBST, incubated in PBST containing horseradish peroxidase avidin D (diluted 1:8000; Vector labs A-2004) for 1 hr at RT, and washed 3 times with PBST before adding a solution containing 0.05% diaminobenzidine, 0.01% H.sub.2O.sub.2, and 0.3% imidazole in PBS for 10 mins at RT. The sections were immediately rinsed 3 times and mounted on glass slides before applying Fluoroshield with DAPI and sealing cover slips.
[0196] IgG Image Acquisition and Analysis
[0197] All the slides were imaged using Nikon Eclipse TI microscope with Ander Neo sCMOS camera (DC-152Q-COO-FI) and NIS Elements software (v14.13.04 64 bit). Brightfield simages were acquired using 4 objective (Plan Fluor 4/0.13) and LED 0.8% intensity with 10 ms exposure and 11 binning. Single image tiles were acquired with 25% overlap, and automatic stitch blending, image registration, and shading correction was performed. All analysis was performed using FIJI. Three sets of 3 slices were analyzed, corresponding to the focal center (3 sections; 2.3 mm relative to bregma) and the anterior and posterior ends of the focal volume (3 sections each; 700 M posterior or anterior to the focal center). Oval shaped ROIs (3.5 mm1.5 mm, 30 angle) 6 mm medial to midline at the surface of the cortex were added and average pixel intensity was measured. As increased staining decreases mean intensity, the normalized intensities are presented as the ROI contralateral to FUS treatment over the ROI ipsilateral to treatment.
[0198] PCD Experiments and Analysis
[0199] Rats were anesthetized with isoflurane, the head was shaved, and 2 mg/kg meloxicam and 2 mL of Lactated ringers were injected subcutaneously. Animals were then fixed on a stereotaxic setup (Kopf) and a midline incision was made to expose the skull for brain coordinates. A metal pointer was attached to a motorized arm of stereotaxic system to find bregma. Sterilized ultrasound gel was then applied on the skull for coupling. A water box with ultrasound transparent polystyrene film at the bottom was positioned on the skull and filled with degassed water. The ultrasound transducer was then attached to the motorized stereotaxic arm with a custom-made metal pointer such that its focal point precisely targets bregma. The transducer was then moved to the target brain regions with the motorized arm using Neurostar software.
[0200] A single element FUS transducer (H-147, Sonic Concepts, USA) at 2.5 MHz frequency with focal volume of 0.510.513.28 mm was used. The transducer was calibrated with a 0.2 mm hydrophone (Acoustic Precision, UK) such that it generates pressures at the focal point identical to the custom-made transducer used for the drug delivery experiments. A broadband (10 kHz-15 MHz) passive cavitation detector (PCD, Y-107, Sonic Concepts, USA) was confocally aligned with the focal area of the transducer through a 20 mm center hole of the transducer. The data was amplified with a 20 dB RF amplifier (Ramsey Electronics), and digitized with the PicoScope (5242D, Pico Technology, UK) at 15 bits resolution with 125MS/s rate.
[0201] The data was recorded with the PicoScope's graphical user interface (PicoScope 6.14.10), and converted to MATLAB (.mat) format. For the standard.sub.3,4-FUS sequences, the first 2 ms from the PCD was used in the analysis. For the AU-FUS sequence, the data was analyzed from two different segments separately since the AU-FUS sequence consists of two distinct sequences; AU-FUS Aggregation and AU-FUS Uncaging sequences. Because UC-carriers are expected to aggregate most strongly towards the end of the sequence, the last 2 ms of the AU-FUS Aggregation sequence was used for analysis. For the AU-FUS Uncaging sequence, since the first pulse sequence impinging on the aggregated bubbles is expected to give the strongest response, this pulse sequence (0.4 ms) is used for analysis. All the data was subjected to a Hamming window and plotted in the frequency domain after Fast Fourier Transform (FFT) in MATLAB 2015b.
[0202] 50 kHz before and after the ultra-harmonics (3.75 and 6.25 MHz) was considered for area under-the-curve (AUC) calculations. For standards-FUS (1.5 MPa), the ultra-harmonics were detrended with a median filter to eliminate signal elevation due to the broadband emission background. For broadband emission calculations, 250 kHz before and after the integer and ultra-harmonics were excluded and the total range between 2.5 MHz to 10 MHz was covered.
[0203] Extraction of Muscimol from Loaded UC-Carriers for LC-HR-MS/MS Quantification
[0204] Muscimol-loaded UC-carriers were prepared as stated above. After overnight conjugation of the microbubbles and liposomes, the solution was washed and centrifuged twice at 300 g for 3 mins to remove any unencapsulated muscimol in PBS:EDTA (1 mM EDTA; pH 6.5). The final microbubble cake was resuspended in PBS:EDTA (1 mM EDTA; pH 6.5) and the concentration and size distribution were analyzed in triplicates using Multisizer 3 (Beckman Coulter). The total volume was also noted. 0.5 mL of absolute ethanol was added to the UC-carriers to dissolve the lipids and extract the muscimol. Following this, 2.5 mL running buffer (RB; 95% acetonitrile, 5% water, 0.1% formic acid) used in LC-HR-MS/MS detection was added. The solution was then bath sonicated at 70 C. After this, 1.5 mL of the solution was centrifuged at 13,300 rpm for 10 mins, and the resultant supernatant was aliquoted in triplicate (400 L) and frozen at 80 C. until LC-HR-MS/MS detection. The dilutions were noted and factored in while calculating the total amount of muscimol.
[0205] Quantification of Muscimol with LC-HR-MS/MS
[0206] The stock solutions of muscimol (HelloBio) and internal standard (methanamine hydrochloride, Sigma-Aldrich, ISTD) were prepared in a mixture of acetonitrile/H.sub.2O 1:1 (v/v) (ULC-MS grade, Biosolve BV) at a concentration of 200 and 100 g/mL, respectively. The stock solutions were stored at 4 C. until use. The solutions used for the quantification calibration curve were prepared as a dilution series at the concentrations of 1000, 500, 250, 50, 10, 2, and 0.5 ng/mL in acetonitrile/H.sub.2O 1:1 (v/v) supplemented with ISTD at a final concentration of 200 ng/mL. The muscimol-loaded UC-carrier samples were diluted with acetonitrile/H.sub.2O 1:1 (v/v) by a factor of 1:50. A volume of 5 L was injected for quantification.
[0207] Liquid chromatography was performed on an UltiMate 3000 UHPLC (Thermo Fisher, Waltham, MA, USA) build from a binary RS pump, an XRS open autosampler, a temperature-controllable RS column department and a diode array detector, all from the series Dionex UltiMate 3000. Compound separation was achieved at 25 C. on an ACQUITY UPLC Amide Column (100 , 1.7 m, 2.1100 mm; Waters, Milford, MA, USA). Eluent A consisted of H.sub.2O and eluent B was acetonitrile, both acidified with 0.1% formic acid (VWR International bvba). The following conditions were applied for elution at a constant flow rate of 0.3 mL: (i) linear decrease starting from 90% to 50% B during 3.5 min; (ii) switch to 10% B from 3.5 to 3.7 min (iii) holding 10% B until 7.0 min (iv) change until 7.2 min to the starting conditions of 90% B; (v) equilibration for 2.8 min until the next measurement run.
[0208] Mass spectrometry was conducted on a QExactive quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated ESI source operating under following conditions: needle voltage of 3.5 kV, sheath, auxiliary and sweep gas (N.sub.2) flow rates of 30, 15 and 0 (arbitrary units), respectively. The capillary and the auxiliary gas heater temperature amounted 280 C. and 250 C., respectively. The data independent MS/MS mode (DIA) in positive ionization mode was selected including an inclusion list of the precursor ion corresponding to protonated molecules of muscimol (m/z 115.05020, 62 CE [collision energy], tR=2.25-5.00 min) and ISTD (m/z 113.07094, 20 CE, tR=0-2.25 min). A precursor ion isolation window of 3.0 m/z and a resolution of 70000 at full width at half maximum (FWMH) were selected together with a maximum IT of 400 ms and an AGC target of 210.sup.5. The ion chromatograms corresponding to the signals of the fragment ions of muscimol and the ISTD were extracted at m/z 98.02-98.03 and 96.04-96.05, respectively. Xcalibur 4.1 and QuanBrowser 4.1 (Thermo Fisher Scientific) software were employed for data acquisition, and for peak-area integration and quantitation, respectively.
[0209] The recovery was estimated by first adding the standard solution containing 4.86 ng/mL muscimol in a 50 diluted sample containing 1.527 ng/mL. A spiked concentration of 6.32 ng/mL was obtained (98.6% recovery). In the second standard addition, 19.44 ng/mL were added to another sample containing 1.589 ng/mL. A spiked concentration of 22.212 ng/mL was obtained in this case (106.1% recovery).
[0210] Quantification was performed with the addition of the internal standard and the calibration curves were constructed by least-squares linear regression analysis. Thereby, peak area ratios of the signals from the analyte and the internal standard were plotted against the concentration of the analyte. The set of calibrators at 0.5, 2, 10, 50, 250, 500, and 1000 ng/mL concentration were measured to determine the dynamic range and the linearity of the quantification method. The quadratic fitting and the weighting function of 1/X2 were selected and correlation coefficient values R2>0.999 was obtained. A deviation below 5% was obtained by comparing the weighted and the measured concentrations.
[0211] Statistical Analysis
[0212] Non-parametric statistical tests (pairwise Mann-Whitney rank sum test, or Wilcoxon matched-pairs signed rank test) were performed for electrophysiological data and imaging data. All statistical analysis was performed using GraphPad Prism version 7 and 8 for Mac, GraphPad Software, San Diego, California USA.
[0213] Modifications
[0214] While the invention has been described in conjunction with specific embodiments, it is to be understood that the invention is not restricted to these embodiments, and that many modifications are possible without leaving the scope of the present invention. In particular, it is possible to apply the uncaging FUS sequence without prior application of the aggregation FUS sequence. It is also possible to deliberately open the BBB before application of the AU-FUS sequence in order to enhance delivery of drugs that are not able to cross the intact BBB. Other drug carriers than those described may be used, in particular, drug carriers comprising other types of microbubbles with or without liposomes attached to them.