Amphiphilic anchors and methods for imaging, analysis, and cargo delivery

Abstract

Described herein are compositions comprising "amphiphilic click reactive anchors" (ACRAs) that efficiently incorporate into natural and synthetic membranous structures on cells, vesicles, liposomes, lipid nanoparticles, and other materials, and methods of use thereof, e.g., for biological and chemical analysis, drug delivery, and imaging.

Description

[0001] AMPHIPHILIC ANCHORS AND METHODS FOR IMAGING, ANALYSIS, AND CARGO DELIVERY

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 710,327, filed on October 22, 2024. The entire contents of the foregoing are hereby incorporated by reference.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with Government support under Grant Nos.

[0006] CA259675 and CA 232103 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0007] TECHNICAL FIELD

[0008] Described herein are compositions comprising “amphiphilic click reactive anchors” (ACRAs) that efficiently incorporate into natural and synthetic membranous structures on cells, vesicles, liposomes, lipid nanoparticles, and other materials, and methods of use thereof, e.g., for biological and chemical analysis, drug delivery, and imaging.

[0009] BACKGROUND

[0010] Nano-sized particles with different compositions, including polymers, lipids, and inorganic materials, have been demonstrated to be a promising mode of delivery for therapeutics and diagnostic agents. Further, delivery of cargo including therapeutic nucleic acids such as mRNA has proven possible thanks to encapsulation into, for example, synthetic lipid nanoparticles (LNPs), which decrease mRNA immunogenicity and protect mRNA from degradation. However, the lack of sitespecificity of LNP delivery in the body, when they are given systemically, remains a major bottleneck in their translation as delivery mode for therapeutic mRNA. A challenging aspect that applies to nano-sized, cargo delivery vectors including LNPs is that when administered intravenously, these particles can passively accumulate in tumors thanks to enhanced permeability and retention (EPR) effect, which is characterized by leaky tumor vasculature and reduced lymphatic drainage. However, only 5% of the administered particles accumulate in the tumor and there is a large heterogeneity in the EPR effect between tumors as well as within each tumor.

[0011] Therefore, an active delivery approach augmenting drug delivery into tumors and other cells and tissues that may exhibit even less prominent EPR effect is critically needed.

[0012] SUMMARY

[0013] Provided herein are amphiphilic click reactive anchors (ACRAs) comprising: (a) a fusogenic lipid that inserts into lipid membranes; (b) a hydrophilic polyethylene (PEG) polymer to improve solubility and provide uniform membrane labeling, and (c) a reactive bioorthogonal click-chemistry moiety, wherein components (a), (b), and (c) are covalently bonded.

[0014] In some embodiments, the fusogenic lipid of component (a) is DOPE (dioleoylphosphatidylethanolamine).

[0015] In some embodiments, the hydrophilic PEG polymer of component (b) is linear PEG with an average molecular weight between about 1000 and 5000 kd, preferably about 2000 kd.

[0016] In some embodiments, the reactive bioorthogonal click-chemistry moiety of component (c) is transcyclooctene TCO or methyltetrazine mTz.

[0017] Also provided herein are compositions comprising one or more membranebound carriers with an ACRA as described herein incorporated into an external membrane of the carrier, preferably wherein the reactive bioorthogonal clickchemistry moiety is on the surface of the carrier.

[0018] In some embodiments, the membrane-bound carrier is selected from the group consisting of liposomes (optionally a lipid nanoparticle (LNP)), extracellular vesicles (EVs) or exosomes, and cells.

[0019] In some embodiments, the liposomes are microbubbles or nanobubbles comprising a membrane comprising l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC); cholesterol; l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly ethylene glycol)-2000] (DSPE-PEG2K), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly ethylene glycol)-2000]-TCO (DOPE-PEG2k-TCO), wherein the membrane surrounds a center comprising gas or gas-precursor.

[0020] In some embodiments, the gas or gas-precursor is selected from perfluorohexane, perfluoropropane (CsFs), perflubutane (C4F10), or octafluoropropane disposed inside the liposomes. Additionally, provided herein are methods for creating an ultrasound-targeted delivery vehicle. The methods comprise providing a composition as described herein, comprising amphiphilic click reactive anchors (ACRAs) comprising: (a) a fusogenic lipid that inserts into lipid membranes; (b) a hydrophilic polyethylene (PEG) polymer to improve solubility and provide uniform membrane labeling, and (c) a reactive bioorthogonal click-chemistry moiety, wherein components (a), (b), and (c) are covalently bonded, comprising a first half of a click pair in component (c); contacting the composition with a cargo comprising a second half of the click pair that reacts with the first half in the first composition, whereby the reaction of the first and second halves of the click pair creates an ultrasound-targeted delivery vehicle.

[0021] In some embodiments, the cargo is a diagnostic or therapeutic agent.

[0022] In some embodiments, the therapeutic agent comprises nucleic acids (optionally in an expression vector or naked); small molecules; large molecules (optionally antibodies); or premade protein complexes, e.g. CRISPR-Cas based gene editors such as nucleases, base editors, or prime editors.

[0023] In some embodiments, the diagnostic agent comprises dyes, fluorescent probes, or radionuclides.

[0024] In some embodiments, the cargo is encompassed within a carrier, optionally is inside a LNP or EV, or is conjugated to the second half of the click pair, either directly or via a linker.

[0025] Also provided herein are ultrasound-targeted delivery vehicles made by a method described herein.

[0026] Additionally, provided herein are methods for promoting delivery of a cargo to a target cell or tissue. The methods comprise administering an ultrasound-targeted delivery vehicle as described herein to the target cell or tissue, and administering focused ultrasound to the target cell or tissue to disrupt the micro / nanobubbles, such that the lipid of component (a) of the ACRA can be incorporated into the membranes of cells in the target tissue, thereby promoting delivery of the cargo to the target cell or tissue.

[0027] Further, provided herein are methods for promoting delivery of a cargo to a target cell or tissue. The methods comprise administering a composition as described herein, comprising a first half of a click pair in component (c); administering focused ultrasound to the target cell or tissue; administering a composition comprising a cargo comprising a second half of a click pair that reacts with the first half in the first composition, whereby the reaction of the first and second halves of the click pair promote delivery of the cargo to the target cell or tissue.

[0028] In some embodiments, the cargo is a diagnostic or therapeutic agent. In some embodiments, the therapeutic agent comprises nucleic acids (optionally in an expression vector or naked); small molecules; large molecules (optionally antibodies); or premade protein complexes, e.g. CRISPR-Cas based gene editors such as nucleases, base editors, or prime editors. In some embodiments, the diagnostic agent comprises dyes, fluorescent probes, or radionuclides.

[0029] In some embodiments, the cargo is encompassed within a carrier, optionally is inside a LNP or EV, or is conjugated to the second half of the click pair, either directly or via a linker.

[0030] In some embodiments, the target cell or tissue is in a musculoskeletal, tumor, nervous system, or vascular tissue, or any other target tissue in vivo.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0032] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0033] DESCRIPTION OF DRAWINGS FIGs. 1A-D. A) Amphiphilic reactive anchors allow for a “tuning” of cell membranes by decorating cell membranes with click chemistry handles following burst of ultrasound-responsive nanobubbles containing ACRA. These would then allow for “striking” delivery as result of biorthogonal capture of lipid nanoparticles displaying the counter click incorporated with counter ACRA. ACRAs are synthesized starting from DOPE lipid conjugated on the amine group with click reagents trans-cyclo-octene or methyl-tetrazine. B) To assess ACRA binding kinetics, we used a "two-step ” approach with click-reaction in cell culture conditions, whereby DOPE-PEGik-TCO was added to cells followed by the addition of cognate bioorthogonally-reactive fluorophore (mTz-AF488). This experiment verifies that (a) ACRA can display a reactive TCO click handle on the surface of live cells in culture, (b) the TCO handle can efficiently react with a partner mTz-construct on the surface of cells. C) We optimized the composition of bubbles to enhance both binding and delivery efficiency to the target cells. We specifically adjusted the molar ratio of DSPE-PEG and DOPE-PEG-TCO (trans-cyclooctene) while fixing the composition of other components, DPSC (67.4 mol%) and cholesterol (27.6 mol%). The molar ratio composition was 67.4% DSPC, 27.6% cholesterol, 3% DSPE-PEG, and 2% TCO-PEG-DOPE. D) The results show that nanobubbles can be burst to label cell membranes with ACRA glues, as showed by clicking on site with click reagent conjugated to dye. Note that successful labelling of cell membrane only occurs in condition that underwent both US and nanobubble treatment.

[0034] FIGs. 2A-E. A) Experimental setup to test LNPs delivery in cells mediated by nanobubbles and focused ultrasound in YUMM1.7 cell line. B) Display of the 4 reagents used for LNPs formulation, including ionizable lipid, b-sitosterol, PEG, and helper lipid, with the respective molar ratio. C) Characterization of co-localization of dye encapsulated in the LNPs and dye clicked on LNPs post-formulation, indicating successful encapsulation of cargo as well as click handle modification of the surface.

[0035] D) Characterization of size by NTA (left), click handle displayed on the surface of LNPs (middle) and lack of micelles due to DOPE-mTz addition to LNPs (black line is only DOPE-AF488, indicating no particle presence in fractions of SEC 6-10, where nanoparticles are collected). E) Experiment results with Cy5 LNPs delivered to cells after + / - US, nanobubbles and Liposome treatment. The results shows that all components are necessary to enhance delivery of LNPs to cells, namely nanobubbles with DOPE-PEG2k-TCO in formulation, ultrasound, and LNPs displaying DOPE-mTz.

[0036] FIGs. 3A-B. A) In this experiment, cells express a red fluorescent protein derivative DsRed (“RFP”); upon Cre recombination, cells begin expressing GFP. The pretreatment of cells with DOPE-PEG-mTz leads to a statistically significant higher number GFP expressing cells compared to cells treated only with LNPs. The bar plot on the right panel represents number of GFP positive cells and ** a p value of 0.0033 from unpaired t-test. B) In this experiment, Cre reporter cells shift from BFP to iRFP upon Cre recombination. The pretreatment of cells with burst bubbles containing DOPE-PEG2K-TCO leads to a higher number of Cre-activated cells, as shown in the fluorescence imaging panel (iRFP cells) and in the flow cytometry panels showing the percentage of single iRFP positive cells over the entire cell population.

[0037] FIGs. 4A-B. A) Six to 12 weeks old nu / nu mice (MGH Cox-7) under anesthesia were treated on their left hindlimb with ultrasound (“FUS”, Chattanooga, 5W, 50% duty cycle, 1.2W / cm2, 1MHz) with 30s on and 10s pulse for 3 mins. B) Test of delivery of Cy5-loaded LNPs displaying mTz (left cartoon), following a) pretreatment with nanobubbles with ACRA and US, then i.v. injection of LNPs, or b) prereacting nanobubbles and LNPs prior to i.v. injection. Quantification of Cy5 dye in skin (right panel) showing higher delivery in skin tissue that was treated with US. See Figure 24for schematic on “one-step” and “two-step” delivery methods using ACRA.

[0038] FIGs. 5A-E. Clickable ACRA lipid structures. A-E) Multiple lipid-PEG conjugates that contain TCO or mTz click reactive groups have been synthesized and tested. Several are shown here, such as DOPE-PEG-TCO, with varying molecular lengths of PEG linker.JH NMR spectra offer chemical characterization of the newly synthesized lipid-conjugates. Clickable amphiphile design and synthesis: ACRAs contain three main components (Figure 1): (i) a lipid that inserts into lipid membranes on cells and drug delivery vehicles, DOPE (dioleoylphosphatidylethanolamine); (ii) a hydrophilic PEG polymer to improve solubility, uniform membrane labeling, and glue reactivity, and (iii) a reactive bioorthogonal click-chemistry handle (transcyclooctene TCO, or methyltetrazine mTz). Together, these three chemical components formed DOPE-PEG-TCO and DOPE-PEG-mTz as a matched pair of ACRA components that react with each other. These “click”-modified DOPE-PEG amphiphilic lipids are applied to target cells or used for conjugations of vectors, to obtain two click handles (mTz and TCO) on the outer membranes that, following ultrafast inverse-demand Diels-Alder cycloaddition reaction, would bring the membranes in such proximity as to cause efficient cytosolic delivery or ultrasound-triggered release.

[0039] FIGs. 6A-C.1H NMR spectra of synthesized ACRAs.

[0040] FIG. 7. DOPE outperforms DSPE as an ACRA lipid component. To perform this test, 7,500 M0C2 cells were plated in 96 well plates and O / N incubated. Cell media was replaced, after which 20 pM of DOPE-PEGIK-TCO or DSPE-PEGIK-TCO was administered to cells in a complete cell culture medium and incubated for 60 min. Cells were then washed 2x with PBS, then 20 pM mTz-AF488 was added to cells in a complete cell culture medium and incubated for 10 min. Cells were washed 2x with PBS and imaged by fluorescence microscopy. With this experiment, we have been able to verify that DOPE is surprisingly superior to DSPE in labeling live cell membranes.

[0041] FIG. 8. Characterization of cell membrane binding kinetics of ACRA (DOPE-PEGIK-TCO) on live cell cultures. To assess ACRA binding kinetics, we used two approaches; a “ two-step ” approach with click-reaction in cell culture conditions, whereby DOPE-PEGik-TCO was added to cells followed by the addition of cognate bioorthogonally-reactive fluorophore (mTz-AF488); and "pre-react” conditions with DOPE-PEGik-TCO pre-reacted with mTz-AF488 prior to addition to the cells. Representative images (left) and corresponding quantification (right) are shown. This experiment verifies that (a) ACRA can display a reactive TCO click handle on the surface of live cells in culture, (b) the TCO handle can efficiently react with a partner mTz-construct on the surface of cells, and (c) both 2-step and “pre¬ react” l-step conditions are effective in delivering ACRA to cells. The rapid kinetics are nonobvious and motivated us to continue development.

[0042] Procedure: 7500 M0C2 cells were plated in wells of a 96-well microtiter plate. Following 24 h, cell medium was replaced with 20 pM of DOPE-PEGik-TCO or DOPE-PEGik-AF488 (the latter being the reaction product of DOPE-PEGik-TCO with mTz-AF488). Cells were incubated for 5, 15, 30 minutes and 1 hour, after which culture media was discarded, cells washed 2x PBS. For the two-steps condition, 20 pM mTz-AF488 was added to the cells for 15 minutes, followed by 2x PBS-wash. Following these washing steps, both conditions were treated with culture media containing Hoechst 33342 nuclear counterstain. Cells were then imaged using a 20x magnification objective lens by fluorescence microscopy. Results: The intensity of the fluorophore on live cells had a time-dependent increase and was detectable within 5 minutes of treatment. Error bars from intensity quantification of single cells in 3 fields of view per condition, per time of ACRA incubation.

[0043] FIGs. 9A-9B. ACRA cell labeling efficiency depends on PEG chain length and branching structure, with DOPE-PEGik-TCO showing best results. We studied whether a different length of the PEG between DOPE and click reagent would affect cell labelling. In addition, we tested whether a branched PEG, which would display 3 more reactive groups on the DOPE lipid, would improve the intensity of labeling of cells. (A) DOPE-TCO (circles), DOPE-PEGik-TCO (triangles), and DOPE-PEG2k-TCO (squares) were pre-reacted with mTz-AF488 for 10 minutes and treated to HEK293T cells at various concentrations for 30 minutes. Cells were washed and analyzed for AF488 fluorescence by flow cytometry. (B) DOPE-PEGik-TCO and branched tri -functional DOPE-(PEGik-TCO)3 were incubated with mTz-AF488, treated to cells as in A, and analyzed by flow cytometry. A and B are separate, and Y-axis scales are not directly comparable.

[0044] Findings from these experiments are nonobvious. One might have hypothesized that the branched PEG would be more effective due to potential multivalent / avidity effects. It is nonobvious why PEG2k outperforms PEGik and DOPE-TCO structures lacking large PEG linkers.

[0045] Procedure: HEK293T cells were kept in suspension in PBS. The various ACRA compounds were pre-reacted with mTz-AF488 for 10 minutes. Then, 20, 10, 5, 2.5, 1.2 pM of each pre-reacted DOPE and AF488 were added to cells and incubated for 30 minutes. Wells were pelleted at 300g for 5 min, the supernatant was discarded, and cells were resuspended in PBS. After repeating the washing 2 more times, cells were analyzed in an Accuri flow cytometer. As shown in the left plot, DOPE with 2K PEG outperformed the other DOPE lipids, indicating it as the best PEG length for our cell labeling applications. As indicated in the right panel, the comparison between linear IkPEG was better than a branched 3xlkPEG. This study informed us that the DOPE-2K-TCO is the best option to include in the nanobubbles for our cell pre-targeting applications mediated by ultrasound.

[0046] FIG. 10. ACRA (DOPE-PEGik-TCO) labels YUMM1.7 cancer cells. This experiment confirms that ACRA is able to label live cell cultures using a distinct model from M0C2 or HEK293T cells. 7.500 cells were plated in 96 well plates and incubated O / N. Cell medium was replaced, then 20, 10, 5, 2.5, 1.2 pM DOPE-PEG2K-TCO were administered to cells in complete cell culture media. Cells were incubated for 30 min, after which cells were 2x washed with PBS and 20 pM of mTz-AF488 was added in all conditions, as well as in a control with only mTz-AF488. As indicated in the fluorescence microscopy images in the left panel of the figure, DOPE- PEG2K-TCO efficiently labelled YUMM1.7 cell membranes and captured mTz-AF88, as compared to images with cells only exposed to mTz-AF488. The right panel shows concentration-dependent increase in single-cell AF488 intensity as measured by flow cytometry of treated cells as in the images at left.

[0047] FIG. 11. Liposome and bubble preparation. Liposomes and bubbles were produced using the thin-film hydration method. Briefly, a predetermined ratio of each lipid component was dissolved in chloroform and gently mixed. The mixture was placed under a vacuum to remove the solvent, which led to the formation of a thin lipid film. The film was dissolved in phosphate-buffered saline (PBS) buffer via vigorous vortexing and sonication, achieving a 10 mg / mL lipid concentration. To obtain downsized and homogeneous liposomes, we passed the mixture through a series of polycarbonate Nuclepore track-etch membranes. The membrane pore size was sequentially decreased from 400 to 100 nm (400, 200, 100 nm) or 400 to 50 nm (400, 200, 100, 50 nm).

[0048] Before preparing the bubbles, 1 mL of liposome solution (10 mg / ml lipid concentration) and 10 pL of perfluorohexane were chilled in an ice bath. The 10 pL of perfluorohexane was carefully added to the liposome solution. To encapsulate perfluorohexane into the liposomes, we sonicated the mixture for 30 seconds (3 seconds on / 1 second off) in the ice bath using a probe-type sonicator. The tube containing the mixture was inverted a few times, and the sonication step was repeated one more time.

[0049] This experiment characterizes bubbles for ultrasound-enhanced drug delivery.

[0050] FIG. 12. Confirming conditions to burst bubbles using ultrasound. We optimized the ultrasound (US) parameters for bubble bursting. Bubbles containing 1% v / v perfluorohexane were exposed to ultrasound under various conditions. The power was varied from 0 to 20 W, and the duty cycle was adjusted between 20% and 50%, with a total exposure duration of 5 minutes. Bubbles generated strong US contrast, and their bursting by focused US (FUS) resulted in detectable contrast changes in US imaging. In parallel, cytotoxicity tests were performed at each FUS condition to measure the potential adverse effect of US on cell viability. About 104HEK293T cells were dispersed in the culture medium and exposed to ultrasound under various conditions for 5 minutes. The cells were then seeded in a 96-well plate, incubated for 24 hours, and cell viability was quantitatively analyzed using the CCK-8 assay. From these experiments, the optimal FUS condition was set as 5W (50%).

[0051] This experiment characterizes ultrasound-responsiveness of bubbles.

[0052] FIGs. 13A-B. Assessing bubble popping by ultrasound. We incorporated a 1% molar ratio of FITC-labeled DOPE-PEG into the liposome or bubbles to visualize DOPE-containing bubbles. The protocols for preparing liposomes and bubbles are the same as before. The hydrodynamic diameter was measured by dynamic light scattering (DLS) on samples containing liposomes or bubbles in PBS (0.1 mg / ml lipid concentration).

[0053] After confirming the size distribution, we obtained bright field and fluorescence microscope images (A) of FITC-labeled bubbles under ultrasound exposure. 2 mL of FITC-labeled bubbles (0.1 mg / ml of total lipids concentration) were exposed to FUS by a wand-type ultrasound device with 5 W of power, a 50% duty cycle, and a frequency of 1 MHz. Thirty seconds after FUS exposure, bubbles were observed to enlarge. After one minute, most bubbles disappeared, indicating their burst. (B) quantification of the liposomes and bubbles.

[0054] This experiment characterizes ultrasound-responsiveness of bubbles and disruption of DOPE-PEG-FITC from bubble structures during ultrasound.

[0055] FIG. 14. Optimization of bubble composition. We optimized the composition of bubbles to enhance both binding and delivery efficiency to the target cells. We specifically adjusted the molar ratio of DSPE-PEG and DOPE-PEG-TCO (trans-cyclooctene) while fixing the composition of other components, DPSC (67.4 mol%) and cholesterol (27.6 mol%). Since the 3-arm TCO moiety is able to increase the likelihood of capturing the methyl tetrazine (mTz)-conjugated therapeutic vehicle, we compared the 1-arm and 3-arm TCO in two different molar ratios. All bubbles were prepared using the same extrusion method as described before.

[0056] We first measured the hydrodynamic size of each bubble at a lipid concentration of 0.1 mg / ml in PBS. All bubbles exhibited similar hydrodynamic size (-140 nm) regardless of different DSPE-PEG and DOPE-PEG-TCO ratios and the number of TCO arms. Next, we reacted bubbles (2 mg / ml lipid concentration) with mTz-Cy5 fluorescent dyes (40 nmol). After 1 hour of reaction, we removed excess mTz-Cy5 using a PD-10 desalting column and dissociated bubbles by adding dimethyl sulfoxide to measure Cy5 fluorescence intensity. From this measurement, we could estimate the amount of clicked dyes per lipid concentration.

[0057] This experiment characterizes ACRA-functionalized bubbles and optimization.

[0058] FIG. 15. Formulation of lipid nanoparticles (LNP) loaded with mRNA. We formulated lipid nanoparticles by vortex and sonication methods. We pre-mixed ionizable lipid (SM-102, 35 mol%), helper lipid (DOPC, 16 mol%), pegylated lipid C-14 (PEG2000-DMG, lmol%) and non-lipid structural component (P-sitosterol, 48mol%) in 95% ethanol. 1.5ug / ml Cy5 labelled mRNA was added to 20mM citrate buffer at pH=4. The two solutions were then mixed and vortexed for 30s, after which sonicated for 5 min in a water bath sonicator. This was repeated 3 times, after which the nanoparticles were concentrated in 30-kD amicon filter at 2000g for 20 min. To assess encapsulation of mRNA, we quantified Cy5 fluorescence in the filtered (containing LNPs) and flow-through (containing the materials that did not complex in nanoparticles including non-encapsulated mRNA). As shown in the plot on the right, most mRNA was successfully encapsulated in LNPs (95%).

[0059] This figure supports our development of ACRA-functionalized LNP.

[0060] FIG. 16. Ultrasound enhances ACRA-mediated Cy5 delivery to treated tissue in mice at different ACRA bubble doses. Methods follow the “2-step” method in Figure 4C in the main text, but comparing different doses of bubbles, and using mTz-Cy5 rather than mTz-LNP LS, LM, RS, RM: Left skin, left muscle, right skin, right muscle.

[0061] FIGs. 17A-B. Bubbles containing single-arm DOPE-PEG-TCO ACRA are more effective than bubbles containing DOPE-(PEG-TCO)3 branched ACRA. Methods follow those of Figure 4C in the main text, but using mTz-Cy5 fluorophore as the bubble-conjugating agent rather than mTz-labeled LNP. In both 1-step (A) and 2-step (B) administrations, the single-armed DOPE-PEG-TCO (“1RM-DOPE”) shows more uptake in ultrasound-treated tissue; in contrast, the 3-armed DOPE-(PEG-TCO)3 (“3RM-DOPE”) shows no increase in uptake in ultrasound treated versus non-treated skin tissue. LS, LM, RS, RM: Left skin, left muscle, right skin, right muscle.

[0062] FIG. 18. Bubbles enhance ultrasound-mediated probe delivery. Methods follow those of Figure 4C with the two-step procedure, but without bubble administration to mice and using mTz-Cy5 rather than LNP. Ultrasound-mediated increase in mTz-Cy5 dye accumulation was diminished in the absence of bubbles. LS, LM, RS, RM: Left skin, left muscle, right skin, right muscle.

[0063] FIGs. 19A-B. Methods to label EVs with click chemicals. A, B) Single EV-click event assessment: Analysis was done using ExoView allowing us to profile our EV population based on tetraspanin EV markers and fluorescence, with the ATTO488 positive population representing clicked EVs. ExoView was done in accordance with the manufacturer's protocol. ATTO488 fluorescence between unclicked and clicked (17 uM and 690 uM) shows an increase in ATTO488 fluorescence. These experiments demonstrate and characterize maleimide-TCO as a vesicle functionalization strategy applicable to ACRA bubbles.

[0064] FIG. 20. Experimental methods to functionalize EVs with click chemistry moiety by DOPE-PEG-TCO. As shown in the graph panel labeled 1, there was a higher ratio of AF488 / Nanoluc, which was decreased by further separation with Exodisc and quantified in the graph panel labeled 2. This experiment showed that EVs can be conjugated efficiently by ACRA (DOPE-PEG-TCO) to obtain EVs with a click chemistry handle. In addition, this method does not compromise the biological activity of EVs, as the nanoluciferase was not compromised across the samples.

[0065] This study demonstrates that ACRA can be utilized for labelling extracellular vesicles with click moieties. A number of ligands can be clicked on the EVs, such as mTz-conjugated antibodies, small molecules, etc.

[0066] FIGs. 21A-E. Experimental methods for conjugation of EVs with ACRA (DOPE-PEG-TCO) followed by click with ATTO647 and AF488 fluorescent dyes. A-E) Different plots demonstrate loading of DOPE-PEG-TCO into multiple EV solutions. DOPE-PEG-TCO loaded EVs were conjugated with mTz-AF488 dye. The SEC profile with AF488 fluorescence showed that excess amounts of DOPE-PEG-TCO result in more micelle formation that can be eliminated with SEC but not jeopardize EV labeling. These experiments demonstrate and characterize ACRA-enabled vesicle functionalization.

[0067] FIG. 22. ACRA improves intracellular protein cargo delivery with engineered extracellular vesicles. We tested the applicability of ACRA for bio-orthogonal capture of drug delivery vehicles other than LNP. As shown in the fluorescence microscopy at bottom, DOPE-PEG-TCO treatment (“With TCO-DOPE”) increases the number of iRFP+ cells compared to the negative control cells (“without TCO-DOPE”). “On” denotes iRFP and “Off’ denotes BFP. Similarly, it can be seen by the column “mCherry” that there was a considerable difference in the number of cells that had mCherry in the “With TCO-DOPE” as compared to “Without TCO-DOPE”. mCherry is loaded in EVs to monitor their uptake in cells and for cargo loading by mCherry-nanobody conjugates. Thus, ACRA improves intracellular delivery of an EV protein cargo.

[0068] FIG. 23. Bio-orthogonal capture of Cy5-labelled EVs in vivo aided by ultrasound and nanobubbles containing DOPE-PEG-TCO. We studied the applicability of DOPE-PEG-TCO to pre-label cells in vivo with a click moiety by incorporating DOPE-PEG-TCO in nanobubbles, which would aid the local delivery of extracellular vesicles (EVs). As shown on the right panel, the left skin resulted in higher intensity of Cy5 than in the negative control (-US) muscle and skin, indicating that the DOPE-PEG-TCO in nanobubbles enhanced the delivery of Cy5 EVs in the US -treated tissue.

[0069] FIG. 24. One-step and two-step delivery strategies using ACRA. Top: In the 2-step strategy, an ACRA glue component “A” is incorporated within and / or on the surface of gas-filled bubbles. Focused ultrasound pops the bubbles and disrupts the liposome structure, freeing the ACRA and allowing it to incorporate into targeted tissues and cell membranes. A second glue component “B” reacts with the ultrasound-released component “A”, which promotes its localized retention and cargo delivery.

[0070] Bottom: In the l-step strategy, the glue components “A” and “B” form pre-reacted bubble-anchored cargo. Focused ultrasound pops the bubbles at target tissue sites and allows the anchored cargo to perform its intended function. Experimental data showing the function of both one-step and two-step procedures are provided, including in Fig 4C.

[0071] FIG. 25. Ultrasound enhances LNP delivery and mRNA expression in mouse tissue. Representative bioluminescence imaging (left) and quantification (right) indicating enhanced luciferase mRNA expression with focused ultrasound (FUS) and nanobubble-TCO, but not with ACRA-free nanobubbles. Data are means + / - std. dev (n:2-4); *P<0.05 (Two way ANOVA and Tukey’s Multicomparison test).

[0072] FIG. 26. Quantification of relative luminescence index (RLI) in each flank normalized to liver after i.v. injections with 5 pg FLuc mRNA loaded-mTz-LNP (mTz-LNP-FLuc) with (right hand circle in each pair) and without (left hand circle in each pair) focused ultrasound (+ / - FUS). Luminescence obtained by In Vivo Imaging System 3 hours post-injection of Flue mRNAloaded-mTz-LNP. *P < 0.05 (paired t-test) and n=4.

[0073] FIG. 27. Expression of FLuc mRNAin flanks and liver of mice after i.v. injections with mTz-LNP loaded with Flue mRNA.

[0074] FIGs. 28A-C. Ultrasound enhances mRNA-LNP expression in the mouse heart. A) Schematic. B) Rep. confocal axial heart cross-sections imaged 1 week posttreatment. C) Quantification of tdTomato+ cells per cm2of heart tissue. Data are mean ± std. dev (n = 2-4).

[0075] FIG. 29. Microbubble-TCO combined with ultrasound enhances the cellular response to mTz-LNP-Cre. Left: Experimental schematic. Right:

[0076] Quantification of iRFP-positive cells following treatment with microbubble-TCO and mTz-LNP-Cre at the indicated dose. One mg / ml of Microbubble-TCO was burst by ultrasound for 5 minutes in cell medium, then 50 pL of this medium was added with 50 pl to Cre reporter cells and incubated for 30 minutes. Cells were then washed with PBS and different doses of mTz-Cre-LNP were administered to cells and incubated for 30 min, after which cells were washed and incubated for 3 days until imaging by fluorescence microscopy. Cells were imaged and the ratio of iRFP to BFP was quantified and reported as positive cell ratio. (Mean ± SD; n=3-4; *P<0.05, **P<0.01, One way Anova and Tukey’s multicomparison test).

[0077] FIGs. 30A-E. B-mode and contrast-enhanced ultrasound (CEUS) imaging of circulating microbubble-TCO (“MB-TCO”) (A) before, (B) at injection, (C) during high power microbubble destruction, and (D) after destruction. Bottom right quantification shows time series data with mean contrast signal intensity (contrast mean power) changing with microbubble-TCO administration and doppler-mode destruction.

[0078] FIGs. 31A-B. Size distribution and stability of microbubbles in physiological conditions. A) Size distribution of microbubbles dispersed in PBS and hydrodynamic diameter at 25 and 37 C in PBS and PBS containing 5% serum. Mean ± SD, n=3. B) Microscopic illustration of microbubbles between 0 and 3 hours and at 25°C in PBS, or 37°C in PBS and PBS containing 5% serum. DETAILED DESCRIPTION

[0079] Drug delivery facilitated by ultrasound (US) and contrast nanobubbles is a promising active delivery approach, whereby vessel walls and / or cell membranes can be opened as a result of cavitation due to US and nanobubble interaction, allowing for increased particle permeability across natural barriers. In addition, the application of US at the target site has been shown to be a promising mode for site-specific delivery of drug vectors. However, higher US energy is required to deliver larger carriers across barriers and, given the short half-life of contrast nanobubbles, higher doses might be needed thus increasing risk of systemic toxicity. A delivery platform that also increases retention by rapid capture of drug carriers in the tumor site could be beneficial for clinical outcomes.

[0080] Described herein is the development of “Amphiphilic Click Reactive Anchors” (ACRAs). ACRAs efficiently incorporate into natural and synthetic membranous structures on cells, vesicles, liposomes, lipid nanoparticles, and other materials. ACRAs present functional chemical groups, namely bio-orthogonally reactive click chemical moieties, on the membranes for bioconjugation application. They support applications for biological and chemical analysis, drug delivery, and imaging. ACRAs can be used to make a site where US is applied more “sticky” and reactive towards LNPs, EVs, or other reagents carrying various cargo, e.g., different therapeutics including mRNA. ACRA are synthesized from fusogenic lipids conjugated via PEG with a click moiety, e.g., mTz or TCO, e.g., for biorthogonal capture of LNPs. We demonstrate that ACRAs incorporate into cell membranes and display a click handle in the extracellular space. ACRAs can be incorporated in contrast nanobubbles for US-assisted “tuning” of the cell membrane followed by “striking” delivery of click chemistry-modified LNPs or other agents. ACRAs also support the US-enhanced delivery of pre-formed nanobubble-anchored cargo complexes in tissue. We demonstrate the broad applicability of ACRAs for sitespecific delivery mediated by US, using model small molecule cargos (fluorescent dye), LNPs, and engineered extracellular vesicles (EVs) functionalized to display click moieties. As shown herein, these can be preferentially delivered to specific tissues in live mice where US is applied. Amphiphilic Click Reactive Anchors (ACRAs)

[0081] Described herein are ACRAs that can act as molecular glue with multiple applications, including promoting accumulation of a desired cargo in a target cell or tissue. ACRAs contain three main components (see, e.g., Figure 1 A, Figures 5A-E and Figures 6A-C): (a) a fusogenic lipid that inserts into lipid membranes on cells and drug delivery vehicles, preferably DOPE (dioleoylphosphatidylethanolamine); (b) a hydrophilic PEG polymer to improve solubility and provide uniform membrane labeling, and (c) a reactive bioorthogonal click-chemistry handle (transcyclooctene TCO, or methyltetrazine mTz). Full characterizations of the exemplary ACRAs synthesized herein, including DOPE-PEG2k-TCO and DOPE-PEG2k-mTz, are shown in Figures 5A-E and Figures 6A-C.

[0082] Fusogenic Lipids

[0083] While the ACRA exemplified herein preferably use DOPE as a lipid, other lipids can also be used including l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC); 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC); and l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC).

[0084] Polyethylene glycol (PEG) polymers

[0085] As noted above, the ACRA also include a hydrophilic PEG polymer, to improve solubility and provide uniform membrane labeling. PEG lipids for use with the instant ACRA can have the general structure: — (CFECFEOjn — or — (CH2CH2O)nCH2CH2 — . Poly(ethylene glycol) methyl ether (mPEG) can also be used. Preferably, the polyethylene glycol molecule can weigh from about 1,000 kilodaltons (kd) to about 5,000 kd on average, and preferably is about 2,000 kd average weight. Branched and linear PEG can be used, but preferably the PEG is linear. As used herein, “about” means plus or minus 10%.

[0086] Preferably the PEG comprises an amine group to react with a click moiety. DOPE-PEG-NH2conjugates are commercially available, e.g., DOPE-PEG(2000)amine, from Avanti Research (Cat. No. 880234) or BroadPharm (Cat. No. BP-26171). Click Moiety

[0087] Bioorthogonal “click” chemistries are widely used in chemical biology for myriad applications such as activity-based protein profiling, crosslinking of proteins, monitoring cell proliferation, generation of novel enzyme inhibitors, monitoring the synthesis of newly formed proteins, protein target identification, and studying glycan processing. The third component of the ACRA described herein is a reactive bioorthogonal click-chemistry handle (also referred to herein as a click moiety), preferably trans-cyclooctene (TCO) or methyltetrazine (mTz), although dibenzocyclooctyne (DBCO) and bicyclo[6.1.0]nonyne (BCN) can also be used. Bioorthogonal click moieties include those groups that can undergo “click” reactions between azides and alkynes, traceless or non-traceless Staudinger reactions between azides and phosphines, and native chemical ligation reactions between thioesters and thiols. Additionally, the click moiety can be any of an azide, a cyclooctyne, a nitrone, a norbomene, an oxanorbornadiene, a phosphine, a dialkyl phosphine, a trialkyl phosphine, a phosphinothiol, a phosphinophenol, a cyclooctene, a nitrile oxide, a thioester, a tetrazine, an isonitrile, a tetrazole, a quadricyclane, and derivatives thereof. Selectively reactive moieties of members of a set of corresponding haplomers are selected such that they will react with each other to produce an active effector agent. Numerous click pairs can be used with the methods and compositions disclosed herein. Examples include, but are not limited to, azide-alkyne pairs, azide-activated alkyne pairs (e.g., cyclooctynes), azide-phosphine Staudinger / non-traceless Staudinger / traceless Staudinger chemistry pairs, traceless phosphinophenol Staudinger ligation, traceless phosphinomethanethiol Staudinger ligation, and native chemical ligation pairs, such as those described in, for example, PCT Publication WO 14 / 197547.

[0088] Compositions

[0089] The ACRAs described herein can be incorporated into compositions for a number of applications. The ACRA can, for example, be incorporated into the membranes of carriers such as liposomes, extracellular vesicles or exosomes, or cells, e.g., by mixing the ACRAs with the carrier, and allowing the lipid portion of the ACRA to fuse with and thus be incorporated into the membrane of the carrier, with the click moiety on the outside of the carrier. In some compositions, the ACRA are incorporated into the surface membranes of liposomes, which are preferably microbubbles (typically 1-3 pm in diameter, optionally 0.1-10 pm or 1-10 or 1-5 pm) or nanobubbles (<1 pm, typically 90-110 nm in diameter, optionally 50-150 or 50-200 nm in diameter), e.g., for use with focused ultrasound (FUS), which can have gas or a poorly water-soluble gas / gas-precursor core stabilized by shell-forming agents at the surface. The gas / gas-precursor core can include, for example, a perfluorocarbon such as perfluorohexane, perfluoropropane (CsFs), perflubutane (C4F10, also referred to as perfluorobutane), or octafluoropropane; this core is also referred to as an ultrasound contrast gas or gasprecursor, and the gas / gas-precursor filled micro / nanobubbles are also referred to as ultrasound contrast agents. A variety of microbubble shell and gas / gas-precursor compositions are available, many of which are clinically approved. Shell components can include polymers (Macrogol 4000 polyethylene glycol), proteins (e.g., human albumin), and lipids such as hydrogenated egg phosphatidylserine, distearoylphosphatidylcholine, dipalmitoylphosphatidyl-glycerol sodium, palmitic acid, which are listed components of SONAZOID and SONOVUE marketed products. Gas components can include air, nitrogen, sulfur hexafluoride, and perfluorocarbons. See e.g., Zhang et al., Eur J Radiol. 2023 Oct: 167: 111060; Aryal et al., Ultrasound Med Biol. 2019 Jul;45(7): 1733-1742; Hughes et al., Ultrasound Med Biol. 2023 Jul;49(7): 1479-1490. In some embodiments, the microbubble composition is a perflutren lipid microsphere suspension (e.g., DEFINITY). In some embodiments, the microbubble composition comprises nitrogen and perfluorobutane, encapsulated in particles comprising polyethylene glycol (or derivatives thereof), phospholipid, and fatty acid substituents, e.g., VEVO microbubbles (MicroMarker, VisualSonics).

[0090] Alternatively, the ACRA can be incorporated into the surface membranes of, or used to create conjugates comprising nanobubbles with, carriers comprising therapeutic or diagnostic cargo, such as liposomes, lipid nanoparticles (LNP), extracellular vesicles (EVs) or exosomes, or cells that comprise cargo such as therapeutic nucleic acids (e.g., in expression vectors or naked); small molecules; large molecules (optionally antibodies);premade protein complexes, e.g. CRISPR-Cas based gene editors such as nucleases, base editors, prime editors, and so on; and diagnostic agents such as dyes, fluorescent probes, or radionuclides. In some embodiments, the cargo is encompassed by the carrier (e.g., is inside a LNP or EV). Methods of Use

[0091] The ACRAs described herein can be used for a number of applications. An exemplary use is for the delivery of therapeutic cargo in vivo / in vitro by enhancing fusion of carriers such as cells (e.g., donor cells), EVs, nanoparticles (e.g., LNPs), or micro / nanobubbles carrying therapeutic or diagnostic cargo as described herein with recipient cells. In these methods, first ACRAs with a first half of a click pair are administered (e.g., systemically administered), e.g., fused to FUS microbubbles or nanobubbles, and then FUS is administered to the target tissue, disrupting the micro / nanobubbles such that the ACRA can be incorporated into the membranes of cells in the target tissue. Second components (which can comprise ACRA or not) with the second half of the click pair incorporated onto the surface, e.g., into the membranes, of the carriers are then administered (e.g., systemically administered), and when they reach the target cells or tissue the click components react, bringing the carriers into proximity with the cells or target tissue and promoting fusion of the carrier into the target cells or tissue. This is also referred to herein as a two-step method. Alternatively, the first ACRAs with a first half of a click pair fused to a microbubble or nanobubble are mixed with the second components comprising the carrier and the second half of a click pair (optionally a second ACRA) to create conjugates that have a carrier at one end and a microbubble or nanobubble at the other end. These can then be administered to a subject and targeted to a tissue using FUS. This is also referred to herein as a one-step method.

[0092] Figure 24 exemplifies the two-step method and one-step method. In the 2-step strategy shown at the top of Figure 24, a first ACRA glue component “A” (comprising the first half of a click pair) is incorporated on the surface of gas-filled micro / nanobubbles. Focused ultrasound pops the bubbles and disrupts the liposome structure, freeing the ACRA and allowing it to incorporate into targeted tissues and cell membranes. A second glue component “B” (comprising the second half of the click pair, and which can be an ACRA but need not be) reacts with the ultrasound-released component “A”, which promotes its localized retention and cargo delivery. In the l-step strategy, shown in the bottom panel of Figure 24, the glue components “A” and “B” form pre-reacted bubble-anchored cargo. Focused ultrasound pops the bubbles at target tissue sites and allows the anchored cargo to perform its intended function. Experimental data showing the function of both one-step and two-step procedures are provided, including in Fig 4C.

[0093] The ACRAs can also be used for the targeting of donor cells, EVs, nanoparticles (e.g., LNPs), or micro / nanobubbles to desired cell types by clicking antibodies, targeting peptides, etc., onto the surface of the delivery vehicles, using a method similar to that described above.

[0094] The ACRAs are also useful for imaging by clicking dyes or fluorescent probes or radionuclides (e.g. via chelator) on cells, EVs, nanoparticles (e.g., LNPs), or micro / nanobubbles, and for analytical methods by enabling facile labeling, bioconjugation, purification / separation, identification, and other analytical steps, on either synthetic or natural structures with hydrophobic membranes, including cells, EVs, liposomes, and others.

[0095] As one example, LNPs comprising the ACRAs described herein, including the “click”-modified DOPE-PEG amphiphilic lipids, can be applied to target cells or used for conjugations of vectors, to obtain click handles (e.g., mTz and / or TCO) on the outer membranes of the cells that, following ultrafast inverse-demand Diels-Alder cycloaddition reaction, would bring the LNP and cell membranes into sufficient proximity as to cause efficient cytosolic delivery or ultrasound-triggered release.

[0096] The methods can be used to deliver therapeutic or diagnostic agents to any tissue in the body, including musculoskeletal, tumor, nervous system, and vascular tissues, e.g., to treat or diagnose musculoskeletal, oncologic, neurologic, and vascular diseases.

[0097] Thus, the present methods can include systemic (e.g., intravascular) administration of microbubbles or nanobubbles comprising an ACRA. Following systemic (e.g., IV) administration of the ACRA-linked micro / nanobubbles, focused ultrasound is directed to the desired tissue. In some embodiments, the ultrasound carrier frequency is from 200 kHz to 1.5 MHz, e.g., 200 kHz to 1.0 MHz, 200 kHz to 500 kHz, 500 kHz to 1.5 MHz, 500 kHz to 1.0 MHz, or 1.0 MHz to 1.5 MHz. In some embodiments, the burst duration of the ultrasound frequency is 10 ms or about 10 ms. In some embodiments, the number of bursts is 30 to 180, e.g., 30 to 150, 30 to 100, 30 to 50, 50 to 180, 50 to 150, 50 to 100, 100 to 180, 100 to 150, or 150 to 180. In some embodiments, the burst repetition frequency is from 0.25 to 100 Hz, e.g., 0.25 to 1.5 Hz, 0.25 to 1 Hz, 0.25 to 0.5 Hz, 0.5 to 2 Hz, 0.5 to 1.5 Hz, 0.5 to 1.0 Hz, 1 to 2 Hz, 1 to 1.5 Hz, or 1.5 to 2 Hz. In some embodiments, the FUS Pressure is from 0.25 to 0.5 MPa. In some embodiments, the acoustic power is from 0.1W to 50W.

[0098] EXAMPLES

[0099] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0100] MATERIALS AND METHODS

[0101] The following materials and methods were used in the Examples set forth below.

[0102] General Information

[0103] Unless stated otherwise, all materials were used as received from commercial sources. N,N-diisopropylethylamine, anhydrous dichloromethane (DCM), sodium hydroxide, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and HBTU were purchased from Sigma Aldrich (St. Louis, MO, USA). l,2-Dioleoyl-sn-glycero-3-PE (DOPE), DOPE-PEGik-amine-methyltetrazine-NHS ester, DOPE-PEG2k-amine, TCO-NHS, and acid-PEG5-amide-tri(3-methoxypropanamide-PEG4-azide) methane were purchased from BroadPharm (San Diego, CA, USA). CDCh was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). 'H, and13C NMR spectra were recorded on a Bruker AC-400 MHz spectrometer.

[0104] Cell lines: HEK293T cells were purchased from the American Type Culture Collection (ATCC) and cultured in complete cell culture medium containing Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin (PS). Cells were passaged when 80 / 90% confluent. HT1080 cells were from ATCC, and HT1080 lox Cre stable transductants were generated using pLV-CMV-LoxP-DsRed-LoxP-eGFP (Addgene plasmid # 65726; n2t.net / addgene:65726; RRTD:Addgene_65726). Cells were cultured in DMEM with 10% FBS and 1% P / S and passaged when 80 / 90 % confluent. Melanoma cell line (YUMM1.7) was purchased from ATCC and cultured in DMEM / F12 supplemented with 10 % FBS and 1% P / S. YUMM1.7 were passaged 2 times per week or when at 80% confluence. M0C2 mouse oral squamous cell carcinoma was obtained from Kerafast and cultured in cell medium DMEM supplemented with 10 % FBS and 1% P / S. MOC2 cells were passaged 2 times per week or when at 80% confluence.

[0105] Mice: Animal research was performed with approval from the Institutional Animal Care and Use Committee (IACUC) at Mass General Hospital (MGH).

[0106] Experiments were performed using male C57BL / 6(JAX) or nude(Cox7, MGH) mice that were 6-12 weeks old. Mice were fed with autoclaved food and water and maintained in ventilated cages in a light-dark cycle, temperature (18-23 °C) and humidity (40-60%) controlled vivarium at MGH. All mice procedures were performed with anesthesia with isoflurane (Isoflurane: 3-4% for induction and 1-3% for maintenance).

[0107] Microscope : In vitro images were taken by fluorescent microscopy [lOx Plan lens (Olympus), DAPI filter(lex= 380 / 30, lem= 450 / 50), FITC filter (lex= 470 / 40, kem = 525 / 50), TRITC filter (lex= 530 / 40, lem= 605 / 70), and Cy5 filter(AeX= 630 / 40, kem = 700 / 75) Revolve, Echo],

[0108] Synthesis of DOPE-PEGlk-mTz (1).

[0109] DOPE-PEGik-amine (35 mg, 19.1 pmol) and methyltetrazine-NHS ester (15 mg, 28.1 pmol) were dissolved in 1 mL anhydrous DCM, and DIPEA (30 pL) was added before the mixture was shaken at room temperature for 18 hours. IM NaOH (1 mL) was then added, and the mixture was shaken for a further 1 h before the solution was washed sequentially with water and brine (10 mL each). The organic layer was removed and evaporated to provide the product as a pink solid (34 mg, 73% yield).

[0110] Synthesis of DOPE-PEG2k-TCO (2)

[0111] DOPE-PEG2k-amine (50 mg, 27.3 pmol) and TCO-NHS ester (10 mg, 37.4 pmol) were dissolved in 1 mL anhydrous DCM, and DIPEA (30 pL) was added before the mixture was shaken at room temperature for 18 hours. IM NaOH (1 mL) was then added, and the mixture was shaken for a further 1 h before the solution was washed sequentially with water and brine (10 mL each). The organic layer was removed and evaporated to provide the product as a colorless solid (52 mg, 95% yield).

[0112] Synthesis of branched DOPE-(PEG-TCO) 3 (3)

[0113] DOPE (10 mg, 13.4 pmol) and acid-PEG5-amide-tri(3-methoxypropanamide-PEG4-azide) methane (22.2 mg, 17.1 pmol) were dissolved in anhydrous DCM and HBTU (6.5 mg, 17.1 pmol) and DIPEA (5 pL, 28.7 pmol) were added before the mixture was shaken at room temperature for 18 hours. TCEP hydrochloride (10 mg, 34.9 pmol) was added to the solution, which was then stirred for 2 hours at room temperature, followed by washing sequentially with water and brine. The organic phase was isolated and TCO-NHS (21.5 mg, 80.4 pmol) and DIPEA (10 pL, 57.4 pmol) were added, and the mixture was then incubated at room temperature for 20 hours. IM NaOH (to achieve 1 : 1 DCM:water) was then added and the mixture was shaken for a further 1 h before the solution was washed sequentially with IM HC1, water and brine. The organic layer was removed and evaporated to provide the product as a colorless semisolid (53% yield).

[0114] Labelling of cell membrane by ACRA glues

[0115] M0C2 cells were plated in 96 well plates, after which 20 pM of the lipids were added to cells and these were incubated for Ih. Then, the medium was removed, cells were washed twice with PBS, and mTz-AF488, used instead of vectors, was added to the cells for assessing click reaction in cell culture. After 10 min, cells were washed and fresh medium was added to the cells. As control, cells were exposed only to 20 pM of mTz-AF488. To assess ACRA binding kinetics, we used a “two-step” approach with click-reaction in cell culture conditions, whereby DOPE-PEGik-TCO was added to cells followed by the addition of cognate bioorthogonally-reactive fluorophore (mTz-AF488). 7500 M0C2 cells were plated in wells of a 96-well microtiter plate. Following 24 h, cell medium was replaced with 20 pM of DOPE-PEGik-TCO. Cells were incubated for 5, 15, 30 minutes and 1 hour, after which culture medium was discarded and cells were washed 2x PBS. For the two-steps condition, 20 pM mTz-AF488 was added to the cells for 15 minutes, followed by 2x PBS-wash. Following these washing steps, both conditions were treated with culture media containing Hoechst 33342 nuclear counterstain (1 :500 dilution added for 10 minutes to cells in culture). Cells were then imaged using a 20x magnification objective lens by fluorescence microscopy. We studied whether a different length of the PEG between DOPE and click moiety would affect cell labelling. In addition, we tested whether a branched PEG, which would display 3 more reactive groups on the DOPE lipid, would improve the intensity of labelling of cells. The various ACRA compounds were pre-reacted with mTz-AF488 for 10 minutes. Then, HEK293T cells were incubated with 20, 10, 5, 2.5, 1.2 pM of each pre-reacted DOPE and AF488 in PBS for 30 minutes. Wells were pelleted at 300g for 5 min, the supernatant was discarded, and cells were resuspended in PBS. After repeating the washing 2 more times, cells were analyzed in Accuri flow cytometer. We tested ACRA glues also on mouse melanoma cell line YUMM1.7. Cells (7500) were plated in 96 well plates and incubated overnight. Cell medium was replaced, then 20, 10, 5, 2.5, 1.2 pM DOPE-PEG2K-TCO were administered to cells in complete cell culture media. Cells were incubated for 30 min, after which cells were 2x washed with PBS and 20 pM of mTz-AF488 was added in all conditions, as well as in a control with only mTz-AF488.

[0116] Synthesis of nanobubbles incorporating ACRA glues

[0117] Liposomes and bubbles were prepared using lipid thin film and hydration methods. Briefly, DSPC, cholesterol, DSPE-PEG2k, and DOPE-PEG2k-TCO were dissolved in chloroform and combined at a specific molar ratio of 67.4:27.6:3:2 mol%. The solvents were then removed by evaporation under vacuum at 60°C, allowing for the formation of a uniform lipid film. The lipid film was hydrated in PBS (pH 7.4) at a lipid concentration of 10 mg / ml with vigorous vortexing for 10 minutes, followed by sonication using a probe-type sonicator for 2 minutes. To obtain downsized and homogeneous liposomes, the solution was extruded for 21 cycles through a series of polycarbonate Nuclepore track-etch membranes with pore sizes sequentially decreased from 400 to 50 nm (400, 200, 100, 50 nm). To prepare the bubbles, 10 pL of perfluorohexane was carefully added to 1 mL of the liposome solution (10 mg / ml lipid concentration). To encapsulate perfluorohexane into the liposomes, the mixture was sonicated for 30 seconds (3 seconds on / 1 second off) in an ice bath using a probe-type sonicator. The tube containing the mixture was gently inverted a few times, and the sonication step was repeated one more time. The hydrodynamic diameter was measured by dynamic light scattering (DLS) on samples containing liposomes or bubbles in PBS (0.1 mg / ml lipid concentration).

[0118] Confirming conditions to burst nanobubbles using ultrasound Nanobubbles containing 1% v / v perfluorohexane were exposed to ultrasound under various conditions. The power was varied from 0 to 20 W, and the duty cycle was adjusted between 20% and 50%, with a total exposure duration of 5 minutes. Bubbles generated strong US contrast, and their bursting by focused US (FUS) resulted in detectable contrast changes in US imaging in wet-type high intensity-focused ultrasound system (VIFU-2000, Alpinion medical system, Korea). In parallel, cytotoxicity tests were performed at each US condition to measure the potential adverse effect of US on cell viability. About 104HEK293T cells were dispersed in the culture medium and exposed to US under various conditions for 5 minutes. The cells were then seeded in a 96-well plate, incubated for 24 hours, and cell viability was quantitatively analyzed using the CCK-8 assay. From these experiments, the optimal FUS condition was set as 5W (50%).

[0119] Assessing bubble popping by ultrasound

[0120] We incorporated a 1% molar ratio of FITC-labeled DOPE-PEG into the liposome or bubbles to visualize DOPE-containing bubbles. The protocols for preparing liposomes and bubbles are the same as described above. We obtained bright field and fluorescence microscope images of FITC-labeled bubbles under ultrasound exposure. 2 mL of FITC-labeled bubbles (0.1 mg / ml of total lipids concentration) were exposed to ultrasound by a wand-type ultrasound device with 5 W of power, a 50% duty cycle, and a frequency of 1 MHz. Thirty seconds after US exposure, bubbles were observed to enlarge. After one minute, most bubbles disappeared, indicating their successful burst.

[0121] Optimization of bubble composition to incorporate ACRA glues

[0122] We optimized the composition of bubbles to enhance both binding and delivery efficiency to the target cells. We specifically adjusted the molar ratio of DSPE-PEG and DOPE-PEG2K-TCO (trans-cyclooctene) while fixing the composition of other components, DPSC (67.4 mol%) and cholesterol (27.6 mol%). We tested whether a 3 -arm TCO moiety was able to increase the efficiency in capturing the methyl tetrazine (mTz)-conjugated therapeutic vehicle. We therefore compared the 1-arm and 3-arm TCO in two different molar ratios. All bubbles were prepared using the same extrusion method as described above. We first measured the hydrodynamic size of each bubble at a lipid concentration of 0.1 mg / ml in PBS. All bubbles exhibited similar hydrodynamic size (-140 nm) regardless of different DSPE-PEG and DOPE-PEG2K-TCO ratios and the number of TCO arms. Next, we reacted bubbles (2 mg / ml lipid concentration) with mTz-Cy5 fluorescent dyes (40 nmol). After 1 hour of reaction, we removed excess mTz-Cy5 using a PD-10 desalting column and dissociated bubbles by adding dimethyl sulfoxide to measure Cy5 fluorescence intensity. From this measurement, we could estimate the amount of clicked dyes per lipid concentration. Labelling of cell membrane by ACRA glues incorporated in US-responsive nanobubbles

[0123] We pre-reacted D0PE-PEG2k-TC0 with mTz-AF647 prior to mixing all lipids for liposome and nanobubbles formulation as described above. After formulation of nanobubbles or liposomes, these were split into two samples; one to apply directly onto the cells, and one to undergo treatment with ultrasound by a wand-type ultrasound device with 5 W of power, a 50% duty cycle, and a frequency of 1 MHz. After burst of the latter, the media containing US-treated liposomes or nanobubbles were added to the cells. This setup compares AF647 uptake in cells treated with liposomes or nanobubbles, with and without US.

[0124] Synthesis of lipid nanoparticles incorporating ACRA glues and mRNA We formulated lipid nanoparticles by vortex and sonication methods. We premixed ionizable lipid (SM-102, 35 mol%), helper lipid (DOPC, 16 mol%), pegylated lipid C-14 (PEG2000-DMG, lmol%) and non-lipid structural component (P-sitosterol, 48mol%) in 95% ethanol. 1.5ug / ml Cy5 labelled mRNA was added to 20mM citrate buffer at pH 4. The two solutions were mixed and vortexed for 30s, and then sonicated for 5 min in a water bath sonicator. This was repeated 3 times, after which the nanoparticles were concentrated in 3 OK amicon filter at 2000g for 20 min. To assess encapsulation of mRNA, we quantified Cy5 fluorescence in the LNPs retained in the filter and flow-through (containing the materials that did not complex in nanoparticles including non-encapsulated mRNA). We also synthesized LNPs containing Cy5 dye for uptake kinetics studies. Briefly, the same formulation as described above, only without mRNA, was mixed with lipid C16 conjugated to Cy5 at 5 molar ratio and the LNPs were vortexed and sonicated as described above. To decorate LNPs surface with click chemistry moieties, 40 uM of the ACRA glue DOPE-PEGik-mTz was added to the concentrated LNPs and incubated for 2 hours, after which the unlabelled free DOPE-PEGik-mTz was removed by centrifugation at 2.000g for 20 min in 30K amicon filter. Size of LNPs, were determined by Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA). Number concentration of mRNA-LNPs was determined by NTA.

[0125] Use of ACRA glues for enhanced mRNA delivery with LNPs

[0126] Cre-reporter cells were plated in 96 well plates overnight. After 24 hours, 15 pM DOPE-PEG2k-TCO was added to adherent cells and incubated for 30 minutes. Cells were washed two times with PBS, after which fresh media was added and an equal amount (of mTz-labeled LNP loaded with Cre mRNA was added to the cells in all conditions. Cells were incubated for 72 hours after which BFP and iRFP were imaged by fluorescence microscopy and iRFP-positive cells quantified. In order to test the delivery platform in vivo, the same in vitro test as described above with HT1080Lox was performed on mouse melanoma YUMM1.7 cell line transduced with BFP-iRFP Cre reporter system by lentivirus. This cell line expresses a blue fluorescent protein, and when floxed by recombinant Cre it expresses iRFP protein. Same concentrations of DOPE-PEG2K-TCO and LNPs were used as with HT1080Lox cells.

[0127] In-vitro testing of bubble popping and ACRA-enhanced LNP delivery We tested whether ACRA-labeled bubbles (bubbles containing DOPE-PEG2k-TCO) increase uptake of mTz-modified therapeutic agents. We used mouse melanoma cell line (YUMM1.7) as a model cell line and Cy5-conjugated LNPs as a model therapeutic vehicle and produced as described above. Cells were seeded at a density of ~105cells / well in a 12-well plate. Liposomes (non-US responsive) and bubbles were prepared as described before. First, ultrasound was applied to the liposomes or bubbles containing media at a concentration of 5* 109parti cles / mL. Then, 1 mL of each conditioned media was incubated with YUMM1.7 cells for 30 minutes. After removing the culture media and washing the cells twice with prewarmed PBS, the cells were further incubated with Cy5 / mTz-labeled LNPs for 30 minutes. The LNP-containing media was then removed, and the cells were washed twice with PBS. Finally, the cells were fixed with 4% paraformaldehyde solution for 20 minutes and stained with DAPI 0.5 ug / ml, 20 min, dark condition, RT, to visualize the nuclei. We investigated qualitative analysis of mTz-LNPs uptake by using confocal laser scanning microscopy. The mean fluorescence intensity of Cy5 per cell was quantified using ImageJ.

[0128] LNPs were loaded with Cre mRNA and labelled with DOPE-PEGik-mTz as described above. Nanobubbles incorporating TCO were pre-reacted with Cre mRNA-LNPs displaying mTz. Then the combined particles were either US-treated or non-US treated as described above. After that, particles were added to YUMM1.7 Cre reporter cells and incubated for 15 minutes, after which medium was discarded, cells 2x washed with PBS and incubated for 72h until quantification of iRFP positive cells in imaging by fluorescence microscopy.

[0129] In-vivo testing of bubble popping and ACRA-enhanced LNP delivery.

[0130] Six to 12 weeks old nu / nu mice (MGH Cox-7) under anesthesia (Isoflurane: 3-4% for induction and 1-3% for maintenance) were treated on their left hindlimb with US using a “Chattanooga” portable apparatus and applying the US-probe on limbs for 3 min with 5W, 50% duty cycle, 1.2W / cm2, 1MHz, with 30s on and 10s pulse. For the 1-step method, lOOpl of 4mg / ml DOPE-PEG -2k-TCO was pre-reacted with lOOpl of ~30pM mTz-LNP-Cy5 under room temperature for 30mins; mice were intravenously injected with 200pl of the mixture. Then, the ultrasound treatment was applied to the same spot for 5 minutes. For the two-step method, mice were first intravenously injected lOOpl of 4mg / ml DOPE-PEG2K-TCO bubble, then followed by 5 mins ultrasound treatment to the same spot with the same condition as before, then intravenously injected with lOOpg of mTz-LNP-Cy5. 3 hrs after the treatment, mice were perfused with 100ml of PBS with a peristaltic pump by cardiac puncture to remove the remaining materials in circulation, and the muscle and skin were harvested for immediate imaging using a Sapphire Biomolecular Imager (Azure Biosystems). For the in vivo experiment, the bubble preparation followed the same extrusion methods as previously described. For the l-step in vivo experiments, we prepared Cy5-labeled liposomes. We first reacted mTz-Cy5 with DOPE-PEG2k-TCO for 30 minutes. For the 1-arm DOPE-PEGIK-TCO, the molar ratio was 1.2:1 (mTz-Cy5 :DOPE-PEG2k-TCO); for the 3-arm DOPE-(PEG-TCO)3, the ratio was 3.6: 1. Unreacted mTz-Cy5 was removed using a centrifugal filter (1 kDa, cut-off). After purifying the pre-reacted mTz-Cy5 with DOPE-PEG2K-TCO lipid, this product was mixed with other lipid components, and the mixture was used to produce liposomes. For the 2-step in vivo experiments, we used unlabeled DOPE-PEG2K-TCO and prepared liposomes. Once the liposomes were ready, we followed the same protocol as before to prepare bubbles. Specifically, 10 pL of perfluorohexane was added to 1 mL of the liposome solution (10 mg / mL lipid concentration), and the mixture was subjected to probe-type sonication for 1 minute in an ice bath. The final bubbles were diluted 2.5 times (4 mg / ml) and 25 times (0.4 mg / ml) for intravenous injection. Labelling of extracellular vesicles with click reagents

[0131] Maleimide PEG-4 trans-cyclooctene (Mal-TCO) was conjugated to engineered EVs (mCherry expression) as a click handle that will react with mTz-labeled recipient cells. Mal-TCO was diluted in DMSO to a stock concentration of 5 mM. Once added to EVs, incubation of 2 hours on the hula mixer in the dark. 40k Zeba desalting column filtration to allow for the removal of excess Mal-TCO. Determining Mal-TCO concentration: Labeling efficiency curve of increasing concentrations of Mal-TCO (0.0017 uM - 690 uM). After the removal of excess Mal-TCO, pM of mTz-ATTO488 was added to the TCO-conjugated EVs and the reaction proceeded for 15 minutes at 4C on the hula mixer in the dark. 40k Zeba desalting column filtration followed to allow for the removal of excess mTz-ATTO488. Following removal of excess dye, samples were loaded into a 96-well clear bottom plate and the ATTO488 (Click fluorescence) and mCherry (EV fluorescence) was measured using a Synergy Plate reader. For quantification, clicked events on EVs were calculated (ATTO488 / mCherry fluorescence).

[0132] We assessed whether ACRA could be used to functionalize EVs with click chemistry moieties. We used the supernatant media of engineered cells (ENoMi red), which release EVs displaying nanoluc and a flag tag on the surface. The nanoluc is useful for quantification and the flag tag allows separation of engineered EVs (EnomiEVs) from the wild type EVs, given the heterogeneity in the EVs secreted. EnomiEVs were added to three different concentrations of DOPE-PEGIK-TCO. Then, the unlabeled DOPE-PEGIK -TCO was separated by amicon filter. The produced EnomiEVs-TCO were added to IpM of mTz-AF488 for 30 minutes, after which the un-clicked dye was separated from the rest by amicon filter and following Exodisc. To verify whether conjugation of EVs with DOPE-PEGIK -TCO occurred, we quantified nanoluciferase and fluorescence by microplate reader, and normalized the intensity of the fluorophore to the nanoluc signal. In this way, we would take the varying number of EVs across the samples into account when assessing the DOPE-PEGIK -TCO concentration dependent increase in the fluorophore intensity. Different concentrations of DOPE-PEGIK -TCO was added to EV preparations, demonstrating the affinity for DOPE-PEGIK -TCO to EVs. Association of DOPE-PEGIK -TCO with EVs was validated with size-exclusion chromatography (SEC) and mTz-ATTO647 dye exposure. ACRA for enhanced delivery of extracellular vesicles in vitro We used engineered extracellular vesicles (EVs), loaded with Cre recombinase and mCherry proteins, and engineered to display a flag-tag on the outer surface to aid isolation (upper figure). To perform this study, we used a reporter cell HEK293T transduced with a Cre reporter cassette (HEK-BFP) to shift from BFP to iRFP when floxed by Cre. We prelabelled HEK-BFP with 15 pM DOPE-PEGIK -TCO in complete cell culture medium for 30 min, after which cells were washed and incubated with EVs for 72h, after which cells were imaged to assess fluorescence.

[0133] ACRA for enhanced delivery of extracellular vesicles in vivo Engineered EVs were labelled with 200 pM NHS-Cy5 to display the fluorescent probe on the EV surface lysine. In addition, EVs were labelled with mal-mTz to display the click handle on the EV surface cysteine. After EVs labelling, these were clicked with nanobubbles (top-left figure) that incorporate DOPE-PEGIK -TCO. C57B16 mice were injected intravenously with nanobubbles-EVs, after which ultrasound was applied on left muscle. Following 1 hour post-treatment, mouse tissue was harvested and imaged to assess biodistribution of Cy5.

[0134] Synthesis of Lipid-PEGn-mTz

[0135] Lipid-PEGn-amine (1 eq) and mTz-NHS ester (1.5 eq) were dissolved in anhydrous DCM and DIPEA (2 eq) was added before the mixture was shaken at room temperature for 18 hours. IM NaOH (to achieve 1 : 1 DCM: water) was then added and the mixture was shaken for a further 1 h before the solution was washed sequentially with IM HC1, water and brine. The organic layer was removed and evaporated to provide the product (70-85% yield).

[0136] Synthesis of Lipid-PEGn-TCO

[0137] Lipid-PEGn-amine (1 eq) and TCO-NHS ester (1.5 eq) were dissolved in anhydrous DCM and DIPEA (2 eq) was added before the mixture was shaken at room temperature for 18 hours. IM NaOH (to achieve 1 : 1 DCM: water) was then added and the mixture was shaken for a further 1 h before the solution was washed sequentially with IM HC1, water and brine. The organic layer was removed and evaporated to provide the product (80-95% yield).

[0138] Synthesis of branched DOPE-PEG n-TCO.

[0139] DOPE (1 eq) and branched NHS-PEGn-azide (1.5 eq) were dissolved in anhydrous DCM and DIPEA (2 eq) was added before the mixture was shaken at room temperature for 18 hours. TCEP (2 eq) was added to the solution, which was then stirred for 2 hours at room temperature, followed by washing sequentially with water and brine. TCO-NHS (6 eq) and DIPEA (4 eq) were added to the organic phase, which was then incubated at room temperature for 20 hours. IM NaOH (to achieve 1 : 1 DCM:water) was then added and the mixture was shaken for a further 1 h before the solution was washed sequentially with IM HC1, water and brine. The organic layer was removed and evaporated to provide the product (40-60% yield).

[0140] Synthesis of branched DOPE-PEG n-DBCO-TCO.

[0141] DOPE (1 eq) and branched NHS-PEGn-azide (1.5 eq) were dissolved in anhydrous DCM and DIPEA (2 eq) was added before the mixture was shaken at room temperature for 18 hours. DBCO-PEG-TCO (6 eq) was added and the solution was shaken for a further 2 hours at room temperature. The mixture was washed sequentially with IM HC1, water and brine, then the organic layer was removed and evaporated to provide the product (64% yield).

[0142] In vivo testing of nanobubble popping and ACRA-enhanced LNP delivery.

[0143] Six to 12 weeks old nu / nu mice (MGH Cox-7) under anesthesia (Isoflurane: 3-4% for induction and 1-3% for maintenance) were treated on their right hindlimb with US using the wand portable transducer (Chattanooga, TN, USA) and applied on limbs for 3 min with 5W, 50% duty cycle, 1MHz, with 30s on and 10s off pulse. For the in vivo experiment, the nanobubble was prepared with 10 pL perfluorohexane added to 1 mL of the liposome solution (10 mg / mL lipid concentration), and the mixture was subjected to probe-type sonication for 1 min in an ice bath. The final nanobubbles were diluted 5 times (4 mg / ml) for intravenous injection. Mice were first intravenously injected with 100 pl of 4 mg / ml nanobubble-TCO, then followed by 3 min ultrasound treatment to the same spot with the same condition as before, then intravenously injected with LNP displaying mTz (150 pg total lipid mass) and loaded with firefly luciferase (FLuc) mRNA (5 pg total, TriLink Biotechnologies). Following 4 hr, mice were imaged in an in vivo imaging system (Perkin Elmer IVIS system). The expression of mRNA in the flanks was normalized to the flank without FUS and the data reported as percentage increase resulting from nanobubbles-TCO and FUS on right hindlimbs. Modification and characterization of commercial microbubbles with ACRA glues.

[0144] We modified VEVO Microbubbles with 5.1 mM ACRA-TCO (DOPE-PEG2k-TCO). The vial was subsequently mixed with saline and agitated vigorously, following the manufacturer’s recommendations (VisualSonics). Microbubble stability was characterized by DLS, and their bursting capacity was verified. The microbubbles displayed a hydrodynamic size of 2100 ± 26 nm and were readily visualized under a microscope. We assessed stability of the modified microbubbles over 0-3 hours when suspended in PBS or PBS containing 5% serum, at either 25 or 37 °C. At time 0, all conditions showed a similar hydrodynamic diameter range (1,500-3,000 nm). By 0.5 hours, a size reduction was observed only at 37 °C, and by 1 and 3 hours, all microbubbles exhibited a diameter of approximately 1,100 nm.

[0145] In vitro testing of bubble popping and ACRA-enhanced LNP delivery.

[0146] To evaluate whether ACRA-labeled bubbles enhance functional mRNA delivery, we used YUMM1.7 Cre-reporter cells that switch from BFP to iRFP upon Cre-mediated recombination. YUMM1.7 cells transduced into Cre reporter with lentivirus transduction and selected by puromycin antibiotic. Reporter cells

[0147] (~1 x 10A5 / well, 12-well plate) were treated with mTz-LNP loaded with Cre mRNA at varying doses to generate dose-response curves. Two experimental conditions were compared: LNP only (-US) and LNP + ACRA-labeled nanobubbles or microbubbles with ultrasound (+US). Bubbles were prepared as described previously and applied at 5xlOA9 particles / mL during ultrasound exposure. Following 30 min of incubation with media containing burst bubbles, cells were washed with pre-warmed PBS and maintained in fresh growth medium. Cre activity was quantified as the proportion of cells undergoing BFP-to-iRFP conversion using fluorescence microscopy and ImageJ analysis.

[0148] Microbubble-TCO preparation.

[0149] 650 pL of 0.9% NaCl saline was added to 50 pL of a 10 mM DOPE-PEG2k-TCO solution in ethanol. Using a 25G 5 / 8" syringe, we injected the resulting 700 pL mixture into a fresh microbubble vial (MicroMarker, VisualSonics) and shook it well to ensure thorough mixing. The microbubble solution was diluted by mixing 1:4 contrast to saline. Following reconstitution, mixing, and dilutions as above, 100 pL of the microbubble-Cy5 contrast was intravenously injected. Ultrasound-Mediated Microbubble bursting.

[0150] We performed myocardial contrast echocardiography (MCE) using an MX250S ultra-high-frequency linear-array transducer (central frequency, 21 MHz; axial resolution, 75 pm; acoustic power: 10% B-mode) coupled to a VEVO 3100 Imaging System (both FUJIFILM VisualSonics, Toronto, ON, Canada). We administered 50 pL of diluted microbubble-TCO via tail vein catheter while performing MCE on mice under 1.5% isoflurane anesthesia on a heated stage.

[0151] Microbubble-TCO inflow perfusion into heart tissue, and its peak accumulation, were monitored over a 30-second period. For localized microbubble-TCO destruction and bursting, we increased the acoustic power to 100% within the same imaging frame for 60-90 seconds by switching to Color Doppler Mode on the instrument. We then reverted to imaging contrast mode and recorded an additional 15 seconds of contrast imaging. Plotting a curve of ultrasound contrast intensity over time revealed minimal residual microbubble contrast in the left ventricular cavity and myocardium following the high power-doppler mode application. The Nonlinear Contrast mode in this study provided a transmit frequency of 18 MHz, a mechanical index of 0.7, and a 10% acoustic power. Amplitude modulation was used to isolate non-linear signals from microbubble contrast agents, and imaging was conducted at a frame rate of 25 fps with a depth setting of 14 mm using an MX250S transducer. Color Doppler mode was conducted to destruct the microbubbles using the same probe, with a transmit frequency of 16 MHz, a mechanical index of 1.2, a 100% acoustic power, a frame rate of 18 fps, and a depth of 14 mm.

[0152] Cardiac LNP delivery.

[0153] Ai9 Cre-reporter mice (B

[0154]

[0155] 6.Cg-Gt(ROSA)26Sortm9(CAG~tdTo^ 10-14 weeks old, female, homozygous) were used to evaluate cardiac delivery. Anesthetized mice underwent ultrasound imaging as described above to confirm transducer placement over the heart. 100 pL of microbubble-TCO was administered via tail vein catheter and the transducer was set to color doppler mode to trigger microbubble bursting. Immediately after ultrasound, mTz-LNP-Cre (5 pg RNA) was administered by the catheter. For this experiment, LNP from lonis Pharmaceuticals were used, composed of ALC-0315 (50% mol), DSPC (10%mol), Cholesterol (38.5% mol), and DMG-PEG2000 (1 ,5%mol) with an N / P ratio of 7. A solution of 100 pL LNP in PBS (0.05 mg / mL RNA) were incubated with 0.8 pL of a 40 pM DOPE-PEGik-mTz solution in PBS for 1 hr. LNP were then centrifuged in 30kDA MWCO filters and the size characterized.

[0156] To explore the optimal application to the heart, we compared the following groups: microbubble-TCO with US, followed by mTz-LNP-Cre; mTz-LNP-Cre with ultrasound; mTz-LNP-Cre only; and vehicle control. Mice were maintained under standard housing conditions for 1 week, after which they were intravenously injected with 20 pL lectin-AF647 conjugate (Thermo Fisher) and 1 hour later terminally dissected for analysis of Cre recombination, following PBS perfusion via cardiac puncture under isoflurane anesthesia. Livers and axially -bisected hearts were imaged by confocal microscopy after overnight fixation in PF A. Tissues were stained with DAPI and then imaged with confocal microscope. Quantification of Cre recombination on a single-cell level was performed by counting the number of tdTomato-positive cells per cm2of visible heart tissue using ImageJ with two independent researchers.

[0157] Example 1. Synthesis of ACRA glues

[0158] ACRAs contain three main components (Figure 1 A, Figures 5A-E and Figures 6A-C): (a) a lipid that inserts into lipid membranes on cells and drug delivery vehicles, DOPE (dioleoylphosphatidylethanolamine); (b) a hydrophilic PEG polymer to improve solubility and provide uniform membrane labeling, and (c) a reactive bioorthogonal click-chemistry handle (transcyclooctene TCO, or methyltetrazine mTz). The full characterizations of the synthesized ACRAs are shown in Figures 5A-E and Figures 6A-C.

[0159] Example 2. Use of ACRA to label cell membranes with a click moiety available for biorthogonal reactions

[0160] We first tested whether DSPE-PEGlk-TCO or DOPE-PEGlk-TCO label cell membranes. This test showed that only DOPE-PEGlk-TCO successfully anchored cell membranes as demonstrated by the green-fluorescent dye following click with mTz-AF488 (Figure 7). To assess the binding capacity of ACRA glues on cells in vitro by reacting the click displayed on the cell membrane with the counter click conjugated to a dye and evaluated the binding kinetics (Figure IB). It is possible to verify that, compared to the dye alone, ACRA penetrate the cell membrane already after 5 minutes incubation of cell with ACRA. From the imaging up to 1 hour, it is possible to verify that ACRA stays anchored to the cell membrane, with increased intensity of the dye. Further, we demonstrated from the kinetics studies that fast anchoring of ACRAs occur with either “one-step” or “two-steps” approach (Figure 8).

[0161] In order to identify the optimal ACRA for labeling cells, we compared singlearm PEGylation with PEGik or PEG2k to branched PEGylation with 3 arms ( DOPE-(PEG-TCO)3 ) (Figure 9). From the flow cytometry analysis to quantify the amount of dye clicked on cells, we could observe that the single-arm PEG2K outperformed the smaller PEGylations (Figure 9).

[0162] We tested whether the ACRA glues anchor other cell types and administered DOPE-PEG2k-TCO to a melanoma cell line (YUMM1.7) as a model for assessing the delivery mode developed in this study. As shown in Figure 10, the imaging and the flow cytometry analysis demonstrated labelling in the concentration range between 5 and 20 uM (Figure 10). We used these concentrations in this range in the in vitro tests performed in Examples 3 and 4.

[0163] We tested whether the ACRA glue could be incorporated in ultrasound-responsive nanobubbles for labelling of cell membranes by US (Figures 11-13).

[0164] ACRA was therefore incorporated in liposomes comprising DSPC (1,2-distearoyl-sn-glycero-3 -phosphocholine), cholesterol, DSPE-PEG2K (,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000), and DOPE-PEG-TCO (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]-TCO), resulting in particles of 98 nm, with a shift to 124 nm after loading with gas precursor (Figure 1C). We also verified incorporation of the ACRA as branched DOPE-(PEG-TCO)3 in nanobubbles (Figure 14). We tested the difference in cell membrane labelling by ACRA between liposome (no gas) and nanobubbles (with gas), with and without US (Figure ID). As demonstrated by fluorescence imaging, cell membranes were labelled by ACRA only in the conditions that included US and nanobubbles containing ACRA (Figure ID).

[0165] Example 3. Use of ACRA glues for enhanced delivery of LNPs

[0166] We tested whether ACRA glues enhance the delivery of LNPs surface-modified with a click handle and loaded with a reporter dye (Figure 2). For this test (Figure 2A), we used nanobubbles incorporating ACRA as DOPE-PEG2K-TCO. After bursting these nanobubbles in cell culture media, this was added to cells and incubated for Ih for “tuning” cells. Then, cells were washed and treated with LNPs. The LNPs were synthesized with 4 main components (Figure 2B) and were modified with click handles by ACRA as DOPE-PEGik-mTz after the formulation was made. As shown in Figure 2C, the colocalization of Cy5 inside LNPs and the AF488 clicked on their surface indicates successful encapsulation of dye and click handle on the surface. The LNPs had a size of ca. lOOnm (Figure 2D), and we assessed that the modification with sufficient ACRA does not lead to formation of lipid micelles with hydrophilic head sticking out (Figure 2D). Further, we demonstrated that the LNPs formulation retained >95% of the mRNA added to formulation (Figure 15). We then performed the experiment as described in Figure 2A and demonstrated that only in the condition that underwent treatment with both US and nanobubbles had increased uptake of Cy5-LNPs (Figure 2E).

[0167] Example 4. Use of ACRA glues for US-assisted, enhanced delivery of LNPs containing mRNA to cancer cells

[0168] We first assessed whether ACRA could enhance delivery of mRNA-loaded LNPs, using Cre mRNA and human HT1080 Cre-reporter cells as a model system (Figure 3 A). As shown in Figure 3 A, there is a significant increase in cells that are GFP positive because of ACRA treatment prior to administration of Cre mRNA LNPs. Then, we tested whether incorporating ACRA glues in US-responsive nanobubbles allow for enhanced mRNA delivery in cells, using mouse melanoma cell line with Cre reporter system (YUMM1.7-BFP). As shown in Figure 3B, there is an increase in the uptake of Cre mRNA-LNPs following treatment with nanobubbles and ultrasound based on increased amount of iRFP positive cells, as a result of flox by Cre, demonstrated by fluorescence imaging and flow cytometry.

[0169] Example 5. ACRA for US-assisted delivery of LNPs in mouse tissue

[0170] We assessed the delivery of LNPs in mouse tissue mediated by US and nanobubbles including ACRA glues (Figure 4A). We used the same mouse for testing the effects of US for the delivery of dye, by comparing the skin and muscle that was treated with US with the skin and muscle without US. We used Cy5-loaded LNPs to assess US-mediated delivery of LNPs. For this test, we used either “1-step” or “two -steps” approach. With “l-step”, we pre-reacted nanobubbles with LNPs. With the “2-steps”, we first i.v. injected nanobubbles, then applied US, then i.v. injected Cy5-LNPs. As shown in Figure 4B, a higher amount of Cy5-LNPs was detected in the tissue of the mouse that was treated with US, in both “1-step” as well as in the “2-steps” approach. In this test we compared nanobubble doses (0.4 and 4 mg / ml injected in mouse. We observed that both doses were able to label tissue with ACRA in vivo (Figure 16). In addition, when we compared the efficiency of delivery by ACRA with different PEGylations, we demonstrated that the single arm-DOPE-PEGik-TCO worked better in vivo than branched-DOPE-(PEG-TCO)3 in labelling tissue in vivo (Figures 17-18).

[0171] Example 6. ACRA applicability to other delivery vectors

[0172] We tested whether ACRA could be used for US-assisted delivery of extracellular vesicles (EVs). For this, we first demonstrated that we could modify the surface of EVs with click handles by using a maleimide-mTz / TCO conjugate that covalently binds to surface cysteine proteins (Figure 19). We also demonstrated that ACRA could be used to label EVs with a click handle by mixing it with EVs (Figure 20) supported by further characterization to show that we could successfully label EVs with ACRA and purify EVs samples from unwanted micelles by use of size exclusion chromatography (Figure 21).

[0173] After labelling EVs with click reagents, we demonstrated enhanced EVs cargo delivery in cells pretreated with ACRA as compared to cells that were not pre-treated, as shown in Figure 22. Motivated by the in vitro results, we tested whether nanobubbles with ACRA could be used for US-assisted delivery of EVs in vivo. The results demonstrate higher uptake of Cy 5 -labelled EVs in the flank of mice that were treated with the US as compared to control (Figure 23).

[0174] Example 7. Ultrasound-pretargeting with ACRA enhanced the expression of mRNA cargo delivered by lipid nanoparticles (LNP) in mice

[0175] This example investigated whether ultrasound-pretargeting with ACRA could enhance the expression of mRNA cargo delivered by lipid nanoparticles (LNP) in mice.

[0176] We used an ultrasound-responsive nanobubble formulation as described above combined with LNP delivering mRNA encoding Firefly Luciferase, which we detected by bioluminescence imaging (Figs. 25-27). Ultrasound was applied to the flank and resulted in enhanced Luciferase expression compared to untreated contralateral flank. The nanobubbles and general LNP delivery scheme were described previously, but this example used the luciferase reporter to monitor localized in vivo enhancement of mRNA expression.

[0177] As a second application, we used an ultrasound-responsive microbubble formulation combined with LNP delivering mRNA encoding Cre recombinase, which we detected using a genetic reporter mouse model that expresses tdTomato fluorescent protein upon Cre recombination (Fig. 28-30). Ultrasound was applied to the heart, and resulted in enhanced Cre activity compared to mice that did not receive the ACRA pre-targeting (Fig. 31).

[0178] OTHER EMBODIMENTS

[0179] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An amphiphilic click reactive anchor (ACRA) comprising:(a) a fusogenic lipid;(b) a hydrophilic polyethylene (PEG) polymer, and(c) a reactive bioorthogonal click-chemistry moiety, wherein components (a), (b), and (c) are covalently bonded.

2. The ACRA of claim 1, wherein the fusogenic lipid of component (a) is DOPE (dioleoylphosphatidylethanolamine).

3. The ACRA of claim 1 or 2, wherein the hydrophilic PEG polymer of component (b) is linear PEG with an average molecular weight between about 1000 and 5000 kd, preferably about 2000 kd.

4. The ACRA of any of claims 1 to 3, wherein the reactive bioorthogonal clickchemistry moiety of component (c) is transcyclooctene TCO or methyltetrazine mTz.

5. A composition comprising one or more membrane-bound carriers with the ACRA of any of claims 1-4 incorporated into an external membrane of the carrier, preferably wherein the reactive bioorthogonal click-chemistry moiety is on the surface of the carrier.

6. The composition of claim 5, wherein the membrane-bound carrier is selected from the group consisting of liposomes (optionally a lipid nanoparticle (LNP)), extracellular vesicles (EVs) or exosomes, and cells.

7. The composition of claim 6, wherein the liposomes are microbubbles or nanobubbles comprising a membrane comprising l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC); cholesterol; l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(poly ethylene glycol)-2000] (DSPE-PEG2K), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)- 2000]-TCO (DOPE-PEG2k-TCO), wherein the membrane surrounds a center comprising gas or gas-precursor.

8. The composition of claim 7, wherein the gas or gas-precursor is selected from perfluorohexane, perfluoropropane (CsFs), perflubutane (C4F10), or octafluoropropane disposed inside the liposomes.

9. A method for creating an ultrasound-targeted delivery vehicle, the method comprising:providing the composition of claim 7, comprising a first half of a click pair in component (c);contacting the composition with a cargo comprising a second half of the click pair that reacts with the first half in the first composition, whereby the reaction of the first and second halves of the click pair creates an ultrasound-targeted delivery vehicle.

10. The method of claim 9, wherein the cargo is a diagnostic or therapeutic agent.

11. The method of claim 10, wherein the therapeutic agent comprises nucleic acids (optionally in an expression vector or naked); small molecules; large molecules (optionally antibodies); or premade protein complexes, e.g. CRISPR-Cas based gene editors such as nucleases, base editors, or prime editors.

12. The method of claim 10, wherein the diagnostic agent comprises dyes, fluorescent probes, or radionuclides.

13. The method of any of claims 9 to 12, wherein the cargo is encompassed within a carrier, optionally is inside a LNP or EV, or is conjugated to the second half of the click pair, either directly or via a linker.

14. An ultrasound-targeted delivery vehicle made by the method of any of claims 9 to 13.

15. A method for promoting delivery of a cargo to a target cell or tissue, the method comprising:administering the ultrasound-targeted delivery vehicle of claim 14 to the target cell or tissue, andadministering focused ultrasound to the target cell or tissue to disrupt the micro / nanobubbles, such that the lipid of component (a) of the ACRA can beincorporated into the membranes of cells in the target tissue, thereby promoting delivery of the cargo to the target cell or tissue.

16. A method for promoting delivery of a cargo to a target cell or tissue, the method comprising:administering the composition of any of claims 5 to 8, comprising a first half of a click pair in component (c);administering focused ultrasound to the target cell or tissue;administering a composition comprising a cargo comprising a second half of a click pair that reacts with the first half in the first composition, whereby the reaction of the first and second halves of the click pair promote delivery of the cargo to the target cell or tissue.

17. The method of claim 15 or 16, wherein the cargo is a diagnostic or therapeutic agent.

18. The method of claim 17, wherein the therapeutic agent comprises nucleic acids (optionally in an expression vector or naked); small molecules; large molecules (optionally antibodies); or premade protein complexes, optionally CRISPR-Cas based gene editors such as nucleases, base editors, or prime editors.

19. The method of claim 18, wherein the diagnostic agent comprises dyes, fluorescent probes, or radionuclides.

20. The method of any of claims 15 to 19, wherein the cargo is encompassed within a carrier, optionally is inside a LNP or EV, or is conjugated to the second half of the click pair, either directly or via a linker.

21. The method of any of claims 15 to 19, wherein the target cell or tissue is in a musculoskeletal, tumor, nervous system, or vascular tissue.

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