Acoustically activatable liposomes as a translational nanotechnology f…

Materials

All chemicals and reagents were of the highest purity grade. Lipoid: hydrogenated soy phosphatidylcholine (HSPC) (catalogue number Lipoid S PC-3) and [N-(methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt] (DSPE-PEG 2000) (catalogue number PE18:0/18:0, PEG 2000). Evonik: cholesterol (PhytoChol Inject HU). KetHCl injectable solution (100 mg ml⁻¹; Dechra Pharmaceuticals, procured through Stanford University Environmental Health & Safety). Hiemdia: ammonium sulfate (catalogue number PCT0003). Fisher Scientific: absolute ethanol (200 proof) (catalogue number BP2818-100), sucrose (catalogue number S5-500), HPLC and LC/MS grade water, methanol, acetonitrile, 2-propanol and formic acid. Sigma-Aldrich: HEPES buffer solution (catalogue number 83264-500ML-F) and l-histidine monohydrochloride monohydrate (catalogue number H8125). Repligen: TFF filters (C02-S05U-05-N (SN 20020493-03/21-057). Lampire Biological Laboratories: male canine plasma (catalogue number 7302009). Cytiva: PD-10 column Sephadex G-25 M (catalogue number 17085101) and Sephacryl S-500 (catalogue number 1706130). Milli-Q water was used to prepare all 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).

AAL synthesis and characterization

Liposome production

Initially, large multilamellar vesicles were prepared by dissolving lipid components (HSPC/DSPE-PEG 2000/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. The solution was extruded (Avestin LF-50 with 200 nm pore polycarbonate Whatman filter at 65–70 °C) ten times to generate unilamellar liposomes. Samples were processed with tangential flow filtration (5 × 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 loading, the drugs were added to 10-fold diluted liposome at final 1 mg ml⁻¹ concentration and heated to 55 °C for 1.5 h. To remove the unencapsulated drug, repeated TFF (4 × 5-fold dilution/reconcentration) was performed against a buffer of 10 mM histidine (pH 7.4) with 10% or 15% sucrose. Finally, the samples were sterilized using a 220 nm PVDF filter and stored at 4 °C.

Physiochemical characterization

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). Cryo-electron microscopy (cryo-EM) was used for structural analysis. Drug loading efficiency was measured by destructing the liposome using methanol followed by HPLC to quantify the total drug presented in the sample and reported as DL in mg ml⁻¹ concentration. For the free drug measurement, initially unencapsulated drug was separated from liposome using PD10 column followed by HPLC to quantify the percent free drug using the following formula: FD(%) = (drug in free fraction/sum of drug in free and liposome fractions) × 100 (method details in Supplementary Information).

In vitro ultrasonic drug uncaging

PCR tube containing liposomes (1:4 diluted in canine plasma) was placed in a custom 3D-printed holder held at the focus of a 250 kHz or 650 kHz hydrophone-calibrated FUS transducer and degassed water of either 25 °C or 37 °C was used for coupling. For the flow uncaging, the focal zone of a 250 kHz FUS transducer was aligned with tubing through which liposomes, diluted 30-fold with canine plasma, were flowed at a rate of 130 μl min⁻¹. An ultrasound (60 s, 25% duty cycle, 5 Hz pulse repetition frequency (PRF), varying peak pressure) was applied and a total volume of 1 ml was collected after sonication for each condition. Liposomes after uncaging were separated from unencapsulated free 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 unencapsulated drug fraction. Drug concentration in each elute was quantified by HPLC. The % drug uncaging was calculated using the following formula:

In vitro acoustic emissions recordings during sonication of an AAL and its internal buffer

To characterize acoustic emissions during ultrasonic uncaging, the magnitude of the received echo spectrum was measured using a flow chamber set-up. In brief, the focal zone of a 250 kHz FUS transducer was aligned with a tubing segment through which liposomes flowed at a constant rate of 130 μl min⁻¹. Ultrasound was delivered with a 0.1% duty cycle and a 1 Hz pulse repetition frequency, with peak pressures varying across experimental conditions. A hydrophone (ONDA) was positioned 2.5 cm from the tubing at a 90° angle relative to the transducer axis to capture acoustic backscatter during sonication. The echo signals were collected by PicoScope and subjected to fast Fourier transform analysis. The resulting spectra were plotted to assess the frequency-dependent magnitude of the acoustic emissions.

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 FUS transducer (Sonic Concepts), a 35.6 cm long PVC cylinder with a 1.5 inch diameter, and a hydrophone were placed in a row underwater. Both devices were linked to an oscilloscope (Keysight Technologies) 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 °C deionized water, and the ultrasound pulse arrival time was used as a reference, along with the known speed of sound in deionized water at 37 °C, 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 owing 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 °C were performed by weighing 10 ml of each buffer using a balance.

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 (Charles River Laboratories; Envigo) and male Sprague–Dawley rats (Charles River Laboratories) were used in all in vivo studies with ketamine and ropivacaine, respectively. All rats were between 7 and 10 weeks old with body weight 250–450 g. Isoflurane was used to anaesthetized animals for surgical and terminal procedures and briefly for awake animal experimental set-up.

Animal treatment preparation

Ketamine hydrochloride (Dechra Veterinary Products) was diluted in 0.9% sterile saline to obtain 1 mg ml⁻¹ solutions. All treatments were administered via intravenous tail vein infusion continuously over 5 min with an infusion pump (World Precision Instruments).

In vivo ultrasound protocol and blood–brain barrier (BBB) opening verification

A custom 250 kHz FUS transducer (designed and constructed by R. Watkins, Stanford University) powered by an amplifier (240 L, E&I) was utilized in all in vivo experiments. Calibration of voltages was conducted using a hydrophone (ONDA). Skull attenuation was accounted for and calculated based on weight⁴⁴ to achieve the desired in situ pressure. In all experiments where ultrasound was applied, continuous FUS was applied to either the mPFC or RsC after 2.5 min of drug infusion for 2.5 min (250 kHz, 25% duty cycle, 1.1 MPa estimated peak in situ pressure, 50 ms pulse width). For BBB opening verification, 4 ml kg⁻¹ of 2% Evans Blue dye was administered via the tail vein immediately after FUS treatment and ketamine-loaded liposome infusion. The rat was then anaesthetized and perfused with PBS before the brain was extracted. As a positive control for BBB opening, Definity microbubble infusion was used instead of ketamine-loaded liposomes, with ultrasound applied at 0.5 MPa with 1% duty cycle and 1 Hz PRF for 3 min.

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. The obtained micro-CT image was 1,024 × 1,024 × 553 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 (1,200 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 toolbox⁴⁵. 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 centre 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).

In vivo measurements of temperature change with ultrasound

To assess temperature changes during in vivo ultrasound-mediated drug uncaging, thermal measurements were taken adjacent to the skull at both the sonicated and contralateral brain sites. After dorsal scalp exposure, 2 mm burr holes were drilled into the skull to allow for insertion of a thermocouple probe. Animals underwent the in vivo ultrasound protocol listed above. Thermocouple probes were inserted 3 mm vertically into the brain parenchyma through the burr holes to measure temperature at three time points: immediately before ultrasound exposure, immediately after and 1 min post-exposure.

SPME

Rats were fixed into a stereotaxic frame, administered 2 ml of saline subcutaneously and kept on a heating pad at 37 °C. After dorsal scalp exposure, 2 mm burr holes were drilled into the skull for SPME pin insertion (relative to bregma, −5 mm A/P and ±2.5 mm M/L for Fig. 4; +3.2 mm A/P for mPFC, or −4 mm for RsC, both at +1 mm M/L to the right). A durotomy was performed with a 32 g needle. Burr holes were used only in the SPME and thermal probe experiments to permit passage of the probes for direct assessment of the brain immediately after treatment. For rat subjects that received ultrasound, the ultrasound transducer was positioned directly above the desired burr hole via a three-axis positioning system (ThorLabs), with a coupling cone and ultrasound gel for coupling. Rats received 1.5 mg kg⁻¹ of SonoKet, KetHCl or saline vehicle. Pins were loaded into a custom-designed, 3D-printed stereotaxic holder for precise positioning and sampling, inserted 3 mm ventrally into the brain via the burr holes (SPME absorptive medium centred at ~2.5 mm ventral) and left in contact with the rat brain tissue for 5 min (refs. ^46,47,48,49). Post-sampling, SPME pins were washed to remove residual blood, desorbed into 50 µl of MeOH/H₂O (9:1 v/v) solvent containing 1% formic acid and quantified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS; method details in Supplementary Information).

Blood pharmacokinetics

Blood samples from rats (n = 3 per group) that received 1.5 mg kg⁻¹ intravenous bolus or infused SonoKet or dose-matched KetHCl were collected via tail snipping into EDTA at different time points, and 3–4× cold acetonitrile was mixed to precipitate the plasma protein, which was then removed using a centrifuge. The collected supernatant was dried, reconstituted with 100 μl of MeOH/H₂O (9:1 v/v) solvent containing 1% formic acid and quantified by LC-MS/MS.

Biodistribution

Adult rats (n = 4 per group) received either free or liposomal ketamine as an intravenous bolus dose of 1.5 mg kg⁻¹. Rats were killed at 1 h from the time of administration and perfused with 1× PBS via transcardial perfusion to remove blood from the systemic circulation. The collected brain, liver, kidney, spleen, lung, heart and spinal cord were homogenized in equal-weight volume of 1× PBS. Similar to blood processing, 3–4× cold acetonitrile was mixed to precipitate the protein, which was then removed using a centrifuge. The collected supernatant was dried, reconstituted with 100 μl of MeOH/H₂O (9:1 v/v) solvent containing 1% formic acid and quantified by LC-MS/MS.

Awake-restraint electrophysiology recording and analysis

Surgical set-up

Animals were anaesthetized and positioned stereotaxically for implantation of custom ECoG electrodes. A midline incision was made over the scalp and five holes were drilled through the skull with stereotaxic guidance⁴³. All electrodes were positioned relative to bregma. mPFC electrodes were positioned at +1 mm A/P and +2 mm M/L to the right (positive electrode), and −6 mm A/P and ±2 mm M/L (ground and reference electrodes, respectively). To provide enough ultrasound clearance without sonicating the electrodes in experiments where the RsC was sonicated while recording from the mPFC, the ground and reference electrodes were both moved to −11 mm A/P and respectively ±2 mm M/L. Two stabilizing screws were implanted at −2 mm A/P and ±3 mm M/L for structural integrity. RsC electrodes for all recordings from the RsC were positioned at −5 mm A/P and +3 mm M/L to the right (positive electrode), and −11 mm A/P and ±3 mm M/L (ground and reference electrodes, respectively). Two stabilizing screws were implanted at −8 mm A/P and ±4 mm M/L for structural integrity. Electrodes were screwed into the skull without breaching the dura and dental cement applied to fix the electrodes in place. Animals were housed in separate cages afterwards and allowed for at least 7 days of recovery before recordings.

ECoG recording

At the beginning of each recording session, rats were briefly anaesthetized to be catheterized via the tail vein, placed in a thin, flexible plastic restraint cone (Amazon.com), and positioned in a custom head-restraining apparatus⁵⁰. Rats received oxygen via the nose cone to prevent hypoxia. Recordings were performed with an 8-Channel Cyton Biosensing Board^51,52 at a sampling frequency of 1,000 Hz. For subjects that received ultrasound, the transducer was positioned directly above either the mPFC (+3.2 mm A/P, −0.5 mm M/L) or the RsC (−4 mm A/P, 0 mm M/L) via the 3-axis positioning system and coupled with ultrasound gel. Implanted screws were utilized for precise targeting according to the rat brain atlas⁴³. Data acquisition began 25–30 min after the animal is in the restraint to allow for complete isoflurane clearance. A baseline acquisition (5 min) was recorded before starting the treatment. Rats received 0.75 mg kg⁻¹ of SonoKet, KetHCl or saline vehicle. Data were acquired continuously for 35 min following a 5 min baseline and a 5 min treatment infusion protocol, for a total recording of 45 min.

Data analysis

Electrophysiological data were filtered using MNE-Python with a band-pass filter with a low cut-off of 1 Hz and a high cut-off of 200 Hz (ref. ⁵³). The data were then denoised by decomposing into 5 levels of Daubechies 8 wavelets, zeroing outlier coefficients and then reconstructing the modified data⁵⁴. The first 45 min 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–frequency representation was averaged within frequency band cut-offs (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-min-long convolving window²⁷. One recording within the RsC 1 mg kg⁻¹ KetHCl condition was excluded owing to excessive artefacts that could not be corrected by denoising. The 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.

Behavioural open-field analysis

Rats were surgically implanted with a targeting screw and given at least 7 days to recover before behavioural tests. To acclimate to the experimenter and reduce stress, rats were handled 3 days before recording. All behavioural 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 centre of the field and randomized for the treatment group.

Rats were placed under isoflurane briefly to be catheterized via the tail vein and loaded into the awake restraint⁵⁰. They were administered oxygen via the nose cone within the restraint to prevent hypoxia. After ensuring that the rats are fully awake before starting treatment, rats received an intravenous infusion of 0.75 mg kg⁻¹ SonoKet, KetHCl or saline vehicle for 5 min, followed by the in vivo ultrasound protocol. After treatment, rats were immediately placed at the centre of the arena, where they were allowed to freely explore for 20 min while locomotor activity was recorded.

Before analysis, videos were first reencoded for format compatibility and clipped to 20 min starting at 5 s 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^55,56. Custom Python scripts were then used to quantify and plot cumulative frame-to-frame distance travelled and time spent in the centre of the open field, which was defined as a concentric square field with half the width of the full square field.

Ropivacaine in vivo experiments

Mechanical sensitivity

Rats were acclimated to a raised stainless steel mesh table for 30 min. Baseline paw withdrawal responses were obtained using monofilaments and the von Frey percent response method (10 pokes per filament 1–26 g)^57,58. On the basis of baseline responses, the 26 g monofilament was selected to evaluate the effects of local uncaging ropivacaine. On the procedure day, rats were anaesthetized during the experiment. Saline vehicle or ropivacaine-loaded liposomes were administered, followed by application of ultrasound using a dorsal approach to target the sciatic nerve. Paw withdrawal response was then evaluated using the 26 g monofilament and the von Frey percent response method 30 min, 1 h and 4 h post-treatment.

Electrocardiographic analysis

Under anaesthesia, 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 paw. ECG recordings were taken at baseline, immediately and 5 min after injection of either free ropivacaine-HCl or ropivacaine-loaded AAL using a Sy-W002 Vet 3 Channel ECG Machine (Sunny Medical Equipment Limited). The following changes in the ECG pattern were assayed: heart rate (bpm), duration of QRS complex and QT interval (corrected for given heart rate in each animal), and periodic repolarization dynamics (PRD).

Histological safety analysis

Drug administration and tissue collection

Rats were administered an intravenous infusion of 0.75 mg kg⁻¹ SonoKet, dose-matched KetHCl or vehicle over 5 min (n = 3 per group), followed by ultrasound application (−5 mm A/P and +2.5 mm M/L to the right relative to bregma). After 72 h post-treatment, the animals were anaesthetized and transcardially perfused with 1× PBS followed by 4% paraformaldehyde (PFA) diluted in PBS. Brains were extracted and stored in 4% PFA for 24 h, 15% sucrose for the next 48 h and 30% sucrose for the last 48 h. They were washed in PBS and frozen in embedding medium. Coronal brain sections (30 µm thick) were cut using a CM1800 cryostat (Leica Microsystems) and were stored in 30% sucrose and 30% ethylene glycol in 0.1 M PB at −20 °C until processed for immunohistochemistry.

Microscopy and image analysis

Every 12th section (360 μm apart) was stained with haematoxylin and eosin (H&E; Vector Laboratories), Fluoro-Jade C (Biosensis), 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) was used after primary anti-GFAP incubation. Cy5 secondary antibody (Thermo Fisher Scientific) was used after primary anti-IBA1 incubation. Tissue sections were free-float mounted on microscope glass slides (Fisher). All histology images were collected with a fluorescence microscope (BZ-X800, Keyence). For quantifiable histological markers (Fluoro-Jade C, IBA-1⁺ and GFAP⁺ cells), signals above thresholded background were used for manual region of interest segmentation to calculate the total mean fluorescent area of cells using BZ-X Advanced Analysis Software (Keyence). For the ropivacaine uncaging safety analysis, a similar process was complete except for targeting and collecting 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 h. Tissues were frozen in optimal cutting medium compound and stored at –80 °C until serially sectioned with a cryostat at 7 μm. Every 20th section (140 μm apart) was stained with H&E for gross examination of tissue damage.

General statistical analysis

Rats were randomly assigned to treatment conditions. Data where n ≤ 4 were plotted as bar plots 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, two-sided, analysis of variance (ANOVA) was conducted, followed by a post hoc Tukey’s honestly significant difference (HSD) test for pairwise comparisons. Effect size was calculated using Hedges 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. Pharmacokinetic parameters were estimated using the NonCompart package⁵⁹ and comparisons were made with two-tailed Student’s t-tests in R version 4.1.3. The remaining statistical analyses were performed using GraphPad Prism 10 (GraphPad Software) and custom scripts in Python.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.