WO2019200354A1 - Therapeutic nanodroplet double emulsions and methods of use thereof - Google Patents

Abstract

A double emulsion includes a phospholipid, a non-aqueous shell, a non-ionic surfactant, water, a therapeutic agent in the water, and a continuous aqueous phase surrounding the phospholipid. The phospholipid encloses the non-aqueous shell. The non-aqueous shell encloses the non-ionic surfactant. The non-ionic surfactant encloses the water and the therapeutic agent. The double emulsion has an average diameter of at most 50 μm. A targeting agent may be conjugated to the phospholipid.

Description

THERAPEUTIC NANODROPLET DOUBLE EMULSIONS AND

METHODS OF USE THEREOF

BACKGROUND

[01] Standard clinical treatments for cancer patients include surgery, radiation, and chemotherapy. Administration of chemotherapeutic drugs has been used for cancer treatment since the 1940s but targeted anti-cancer therapies have only recently been developed [10, 11]. Currently, conventional chemotherapy drugs are typically delivered systemically and cause serious side effects in other organs, including reduced immune activity and damage to organs such as the heart and kidneys [12]. Therefore, the maximum dose that can be administered is limited. To address this problem, targeted delivery strategies are in development to increase the efficacy of chemotherapy while reducing systemic toxicity. One such method of targeted delivery utilizes targeting agents that bind to nucleolin.

[02] Nucleolin [8] is an abundant, non-ribosomal protein of the nucleolus, the site of ribosomal gene transcription and packaging of pre-ribosomal RNA. This 710 amino acid phosphoprotein has a multi-domain structure consisting of a histone-like N-terminus, a central domain containing four RNA recognition motifs and a glycine/arginine-rich C-terminus, and has an apparent molecular weight of 110 kD. While nucleolin is found in every nucleated cell, the expression of nucleolin on the cell surface has been correlated with the presence and aggressiveness of neoplastic cells [3].

[03] The correlation of the presence of cell surface nucleolin with neoplastic cells has been used for methods of determining the neoplastic state of cells by detecting the presence of nucleolin on the plasma membranes [3]. This observation has also provided new cancer treatment strategies based on administering compounds that specifically target nucleolin [4].

[04] Nucleic acid aptamers are short synthetic oligonucleotides that fold into

unique three-dimensional structures that can be recognized by specific target proteins. Thus, their targeting mechanism is similar to monoclonal antibodies, but they may have substantial advantages over these, including more rapid clearance in vivo, better tumor penetration, non-immunogenicity, and easier synthesis and

storage.

[05] Guanosine-rich oligonucleotides (GROs) designed for triple helix formation are known for binding to nucleolin. This ability to bind nucleolin has been suggested to cause their unexpected ability to effect antiproliferation of cultured prostate carcinoma cells [6]. The antiproliferative effects are not consistent with a triplex- mediated or an antisense mechanism, and it is apparent that GROs inhibit proliferation by an alternative mode of action. It has been surmised that GROs, which display the propensity to form higher order structures containing G-quartets, work by an aptamer mechanism that entails binding to nucleolin due to a shape- specific recognition of the GRO structure; the binding to cell surface nucleolin then induces apoptosis. The antiproliferative effects of GROs have been demonstrated in cell lines derived from prostate (DU145), breast (MDA-MB-231 , MCF-7), or cervical (HeLa) carcinomas and correlates with the ability of GROs to bind cell surface nucleolin [6].

[06] AS1411 , a GRO nucleolin-binding DNA aptamer that has antiproliferative activity against cancer cells with little effect on non-malignant cells, was previously developed. AS1411 uptake appears to occur by macropinocytosis in cancer cells, but by a nonmacropinocytic pathway in nonmalignant cells, resulting in the selective killing of cancer cells, without affecting the viability of nonmalignant cells [9]. AS141 1 was the first anticancer aptamer tested in humans and results from clinical trials of AS141 1 (including Phase II studies in patients with renal cell carcinoma or acute myeloid leukemia) indicate promising clinical activity with no evidence of serious side effects. Despite a few dramatic and durable clinical responses, the overall rate of response to AS1411 was low, possibly due to the low potency of AS1411.

[07] Ultrasound-responsive nanodroplet emulsions have been used for targeted delivery of molecular therapeutics [47]. In one study, an aptamer (sgc8c) was used to target nanodroplets loaded with doxorubicin to CCRF-CEM cells. High-intensity focused ultrasound (HIFU) was introduced to trigger targeted acoustic droplet vaporization, to cause the doxorubicin to chemically treat the cells and cause mechanical damage to the cells. SUMMARY

[08] In a first aspect, the invention is a targeted double emulsion comprising a first phospholipid, a second phospholipid, a targeting agent conjugated to the first phospholipid, a non-aqueous shell, a non-ionic surfactant, water, a therapeutic agent in the water, and a continuous aqueous phase surrounding the first phospholipid and the second phospholipid. The first phospholipid and the second phospholipid enclose the non-aqueous shell. The non-aqueous shell encloses the non-ionic surfactant. The non-ionic surfactant encloses the water and the therapeutic agent. The double emulsion has an average diameter of at most 5 pm.

[09] In a second aspect, the invention is a double emulsion comprising a

phospholipid, a non-aqueous shell, a non-ionic surfactant, water, a therapeutic agent in the water, and a continuous aqueous phase surrounding the phospholipid. The phospholipid encloses the non-aqueous shell. The non-aqueous shell encloses the non-ionic surfactant. The non-ionic surfactant encloses the water and the

therapeutic agent. The double emulsion has an average diameter of at most 50 pm.

[10] DEFINITIONS

[1 ] The term“CRO" means a control aptamer.

[12] An "anti-nucleolin agent" includes any molecule or compound that interacts with nucleolin. Such agents include, for example, anti-nucleolin antibodies, peptides, pseudopeptides, aptamers such GROs and nucleolin targeting proteins.

[13] Tumors and cancers include solid, dysprol iterative tissue changes and diffuse tumors. Examples of tumors and cancers include melanoma, lymphoma,

plasmocytoma, sarcoma, glioma, thymoma, leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, esophageal cancer, brain cancer, lung cancer, ovary cancer, endometrial cancer, bladder cancer, kidney cancer, cervical cancer, hepatoma, and other neoplasms. For more examples of tumors and cancers, see, for example Stedman [1].

[14] "Treating a tumor” or "treating a cancer" means to significantly inhibit growth and/or metastasis of the tumor or cancer, and/or killing cancer cells. Growth inhibition can be indicated by reduced tumor volume or reduced occurrences of

metastasis. Tumor growth can be determined, for example, by examining the tumor volume via routine procedures (such as obtaining two-dimensional measurements with a dial caliper). Metastasis can be determined by inspecting for tumor cells in secondary sites or examining the metastatic potential of biopsied tumor cells in vitro.

[15] A“chemotherapeutic agent” is a chemical compound that can be used

effectively to treat cancer in humans.

[16] A "pharmaceutically acceptable carrier" includes any and all solvents,

dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents which are compatible with pharmaceutical administration. Preferred examples of such carriers or diluents include water, saline, Ringer’s solutions and dextrose solution. Supplementary active compounds can also be incorporated into the compositions.

[17] "Medicament,"“therapeutic composition,” and“pharmaceutical composition” are used interchangeably to indicate a compound, matter, mixture or preparation that exerts a therapeutic effect in a subject, which is preferably sterile and ready for use, for example in a unit dosage form.

[18] “Therapeutically active compound” or“therapeutic agent" means an active agent used to treat a disease or condition, or exert an effect on cells of a patient, such as a chemotherapeutic agent or a cytotoxic agent.

[19] “Nanodroplets”,“nanoemulsions” or“double emulsions” are composed of a biocompatible phospholipid shell encapsulating an inert, non-toxic perfluorocarbon that further encapsulates a non-ionic surfactant surrounding an aqueous core containing a therapeutic agent.

[20] Particle size means average particle diameter as determined by a particle size analyzer using light scattering, for example, a NanoBrook 90Plus Particle Size Analyzer. [21] The amounts and ratios of compositions described herein are all by weight, unless otherwise stated.

BRIEF DESCRIPTION OF THE DRAWINGS

[22] The invention can be better understood with reference to the following

drawings and description.

[23] FIG. 1 illustrates a non-targeted double emulsion.

[24] FIG. 2 illustrates a cartoon representation of a targeted double emulsion.

[25] FIG. 3 illustrates the experimental set up for treatment of MDA-MB-231 cells using ultrasound to vaporize double emulsions.

[26] FIG. 4 is a scanning electron microscope image showing TNBC cells prior to treatment.

[27] FIG. 5 is a scanning electron microscope image showing TNBC cells after treatment with ultrasound-responsive double emulsions.

[28] FIG. 6 is a graph showing the relative fluorescence of low dose, medium dose and high dose double emulsions normalized to the low dose treatment.

[29] FIG. 7 is an image showing double emulsions one minute after being placed in culture.

[30] FIG. 8 is a graph showing the effect of ultrasound on fluorescein uptake

normalized to no ultrasound treatment.

[31] FIG. 9 illustrates the structural backbone of FluorN561 and FluorN562.

[32] FIG. 10 is a graph showing the release profile for Poloxamer 188 and N562 at different surfactant concentrations after being stored at 4°C.

[33] FIG. 11 is a graph showing the release profile for Poloxamer 188 and N562 at different surfactant concentrations after being stored at 21 °C. [34] FIG. 12 is a graph showing the release profile for Poloxamer 188 and N562 at different surfactant concentrations after being stored at 37°C.

[35] FIG. 13 is a graph showing the relative fluorescence for Poloxamer188 and

FluorN562 after being stored at 4°C, 21 °C and 37°C, normalized to the last measured point at 72 hours.

[36] FIG. 14 illustrates a schematic of a microfluidic device for creating

monodispersed double emulsions.

[37] FIG. 15 is a photograph showing the monodispersity of the double emulsions.

[38] FIG. 16 is a photograph showing the generation of double emulsions in the orifice of a microfluidic device at steady state.

[39] FIG. 17 is a photograph showing a microfluidic device set up.

[40] FIG. 18 is a schematic illustrating an ultrasonic flow system.

[41] FIG. 19 is a schematic illustrating intracellular delivery of a therapeutic agent induced by double emulsion rupture.

[42] FIG. 20 is a graph illustrating intracellular delivery of fluorescein at varying flow rates.

[43] FIG. 21 is a graph illustrating the results of an MTT assay.

[44] FIG. 22 is a schematic illustrating the in vivo therapeutic use of targeted

double emulsions.

DETAILED DESCRIPTION

[45] The invention described in International Publication Number WO 2018/026958 published 8 February 2018 makes use of perfluorocarbon-based micelles that are conjugated to targeting agents, causing an antiproliferative effect on cancers and tumors. The micelles contain a therapeutic agent. The targeting agent targets the micelles to cancer cells or tumors, by binding to nucleolin. Once the micelles enter the tumor area, ultrasound may be used to induce a phase change of the perfluorocarbon, from liquid to gas, causing the micelles to rupture and release the therapeutically active compound. Optionally, the use of a light-absorbing dye, in conjunction with a light delivery method (such as a laser), may be used to induce phase change of the perfluorocarbon and cause the rupture of the micelles. The micelles may enter the cells via endocytic pathways where their components are metabolized and the therapeutic agent is released. Furthermore, the micelles may be used in ultrasound imaging, where the formation of a gas phase within the micelles enhances the contrast of the ultrasound image.

[46] The present application further includes a non-ionic surfactant and an

aqueous phase within the micelles, forming a double emulsion. Such a double emulsion is larger than the perfluorocarbon-based micelles, having a particle size of up to 100 pm (microns), although smaller sizes of 1 to 5 pm are preferred for in vivo use. The larger size offers the opportunity to incorporate a larger amount of a therapeutic agent, such as a cytotoxic agent or anti-cancer agent. The double emulsions may be used with or without a targeting agent.

[47] Targeted, ultrasound-responsive double emulsions may be used to deliver hydrophilic molecular compounds to a cell of interest. Historically, double emulsions have been used for over a hundred years in food and cosmetics industries, but limited research has been conducted for biomedical use. Emulsions consist of two immiscible liquids, in which one liquid is dispersed within the other causing a droplet to form. A major limitation of double emulsions is that double emulsions tend to coalesce without an effective surfactant. However, recent developments in synthesis of new biocompatible surfactants has helped improve the stability of double emulsions allowing further research to be conducted. However, lack of an effective controlled release methodology hindered translation to clinical use. To address this limitation, targeted, ultrasound-responsive formulated double emulsions have been developed to enable spatial and temporal control over release of encapsulated compounds.

[48] Ultrasound has been widely used for decades in medical and engineering applications, including non-destructive testing of materials. More recently, ultrasound applications in therapeutics have been investigated. Studies have demonstrated ultrasound-enhanced therapeutic delivery to cells in vitro and in vivo. This delivery technique generally facilitates transmembrane transport of drug by causing oscillatory behavior of double emulsions in response to ultrasound. Ultrasound- based therapies have several distinct advantages, which includes being non- invasive, portable, and targeted. Ultrasound can induce oscillation, rupture, and collapse of the double emulsion nanodroplets, which releases the payload from the aqueous core of the emulsion. The perfluorocarbon shell undergoes a phase change during vaporization causing a change from liquid to gas. It is theorized, due to anisotropic pressures, that inertial cavitation will occur, which can cause transient pores in nearby cell membranes and enable transport of therapeutic compounds directly into the cytoplasm of cells.

[49] FIG. 1 illustrates a non-targeted double emulsion 100. The double emulsion includes an aqueous core 110 surrounded by a non-aqueous shell 120. A

therapeutic agent 130 is contained within the aqueous core. A phospholipid layer 150 surrounds the non-aqueous shell. A non-ionic surfactant 160 surrounds the aqueous core. The double emulsion is present in an aqueous phase 140, forming a water-in-oil-in-water or W/O/W emulsion.

[50] The aqueous core includes the therapeutic agent dissolved in an aqueous solution. Preferably, the water is deionized water. The therapeutic agent may be any water-soluble compound. The therapeutic agent may also be a compound that has been modified to be water soluble.

[51] The therapeutic agent may be a chemotherapeutic agent used for the

treatment of cancer. Examples of commonly used chemotherapeutic agents include vinorelbine (Navelbine®), mitomycin, camptothecin, cyclophosphamide (Cytoxin®), methotrexate, tamoxifen citrate, 5-fluorouracil, irinotecan, doxorubicin, flutamide, paclitaxel (Taxol®), docetaxel, vinblastine, imatinib mesylate (Gleevec®), anthracycline, letrozole, arsenic trioxide (Trisenox®), anastrozole, triptorelin pamoate, ozogamicin, irinotecan hydrochloride (Camptosar®), leuprolide acetate implant (Viadur), bexarotene (Targretin®), exemestane (Aromasin®), topotecan hydrochloride (Hycamtin®), gemcitabine HCL (Gemzar®), daunorubicin

hydrochloride (Daunorubicin HCL®), toremifene citrate (Fareston), carboplatin (Paraplatin®), cisplatin (Platinol® and Platinol-AQ®), oxaliplatin and any other

platinum-containing oncology drug, trastuzumab (Herceptin®), lapatinib (Tykerb®), gefitinib (Iressa®), cetuximab (Erbitux®), panitumumab (Vectibix®), temsirolimus (Torisel®), everolimus (Afinitor®), vandetanib (ZactimaTM), vemurafenib

(ZelborafTM), crizotinib (Xalkori®), vorinostat (Zolinza®), and bevacizumab

(Avastin®). Preferred chemotherapeutic drugs include doxorubicin, cisplatin and carboplatin. A particularly preferred chemotherapeutic drug is doxorubicin.

[52] It is well known that lipophilic and lipophobic properties of chemotherapeutic agents may be adjusted using well known chemical techniques, such as

esterification. Multiple chemotherapeutic agents may be administered together, such as 2 or 3 chemotherapeutic agents, either by producing double emulsions with multiple chemotherapeutic agents, or by mixing batches of double emulsions, each containing a different chemotherapeutic agent.

[53] The therapeutic agent may be a cytotoxic agent, such as pore-forming toxins

(PFT), SN-38, radionuclides or magnetic spin-vortex discs, which are magnetized only when a magnetic field is applied to avoid self-aggregation that can block blood vessels, begin to spin when a magnetic field is applied, causing membrane disruption of target cells.

[54] The therapeutic agent may be a nucleic acid, including DNA and RNA.

Examples of suitable nucleic acids include DNA plasmids, messenger RNAs

(mRNAs), small interfering RNAs (siRNAs), micro RNAs (miRNA) mimics or inhibitors and long non-coding RNAs (IncRNAs). RNA is particularly well-suited for delivery in a double emulsion since the double emulsion will encapsulate the RNA and prevent its degradation in vivo.

[55] The therapeutic agent may be a nanoparticle for drug or gene delivery.

Examples of suitable nanoparticles include liposomes, metallic nanoparticles such as gold, silver and iron oxide, and polymeric vesicles such as polyethylenimine (PEI) and poly(lactic-co-glycolic) acid (PLGA).

[56] The non-ionic surfactant may be any biocompatible surfactant. Examples of suitable non-ionic surfactants include poloxamers such as poloxamer 188 and fluorosurfactants such as FluorN561 (Cytonix), FluorN562 (Cytonix) and

perfluoropolyethers with PEG or Tris. Preferably, the non-ionic surfactant is poloxamer 188 or FluorN562. The non-ionic surfactant may be present in an amount of 0.1 - 10% (w/v), including 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1 %, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0% and 9.5%.

[57] The non-aqueous shell may be any biocompatible, inert substance.

Preferably, the non-aqueous shell is a perfluorocarbon. Examples of suitable perfluorocarbons include perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, and perfluorononane. Preferably the perfluorocarbon is

perfluoropentane or perfluorohexane. Ultrasound or light can be used to initiate boiling of the perfluorocarbon in the double emulsion and release the therapeutically active compound. While some perfluorocarbons, such as perfluoropentane, have a boiling point lower than human body temperature, the perfluoropentane remains in the liquid phase after being introduced into a human subject, because the pressure inside the double emulsion raises the boiling point of the perfluorocarbon; The perfluorocarbon acts like a superheated fluid, transforming into the gas phase upon application of the ultrasound in a sudden and complete phase change. For perfluorocarbons with higher boiling points, such as perfluorooctane, ultrasound, such as high-intensity focused ultrasound (HIFU) may not be sufficient to increase the temperature of the double emulsions to induce a phase change. In these instances, a light-absorbing dye, such as a cyanine dye or FITC, may be included as part of the double emulsion, and light may be used to induce a phase change from liquid to gas in the double emulsion, for example infrared light, visible light or UV light.

[58] The phospholipid layer includes one or more phospholipids, each having a hydrophilic phosphate head and lipophilic tail. The phosphate heads of the phosphate layer face the outside of the double emulsion (the outer water phase), while the lipophilic tails face the inside of the double emulsion (the non-aqueous shell). Additional phospholipids, as well as other compounds such as cholesterol, may be present and form the phospholipid layer. Examples of suitable phospholipids include 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), PEGylated

phospholipids (such as 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-PEG2000)), phospholipids having a linking agent (such as 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-maleimide)), and

phospholipids having a dye (such as 1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG2000-FITC)).

[59] The double emulsion may optionally include a dye conjugated to the

phospholipid layer. The dye may be any light-absorbing dye that disrupts the double emulsion after exposure to a suitable light source, such as a laser.

[60] The double emulsion may have a size of 800 nm - 100 pm, including 850 nm,

900 nm, 950 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm and 95 pm. The double emulsion preferably has a size of at most 5 pm when used in in vivo to permit the double emulsion to travel through the capillaries. The double emulsion preferably has a size of at most 50 pm when used in vitro.

[61] To make the double emulsions generally uniform in size, the double

emulsions may optionally be extruded through a membrane having a specific pore size, such as a membrane having 50 pm or 5 pm diameter openings. The double emulsions may be extruded through the membrane multiple times, such as 1-20 times, more preferably 5-15 times, to produce double emulsions having a more uniform size and a narrower size distribution.

[62] FIG. 2 illustrates a cartoon representation of a targeted double emulsion 200.

The double emulsion includes an aqueous core 210 surrounded by a non-aqueous shell 220. As in FIG. 1, a phospholipid layer (not shown) surrounds the non-aqueous shell and a non-ionic surfactant (not shown) surrounds the aqueous core. A therapeutic agent 230 is contained within the aqueous core. A targeting agent 240 is conjugated to the outer surface of the phospholipid layer using a linker 250. The double emulsion is present in an aqueous phase 260, forming a water-in-oil-in-water or W/O/W emulsion.

[63] The therapeutic target of the targeting agent may be any receptor that is

unique to a specific cell type. For example, the target cell may be a T-cell for immunotherapies, especially cancer or HIV treatments, a B-cell for immunotherapies, immune cells for immunotherapies, cancer cells and endothelial cells for

cardiovascular therapies. Examples of suitable T-cell receptors include CD3, CD4, CD8, CD28 and TNF receptors. Examples of suitable B-cell receptors include CD19 receptors. Examples of suitable immune cell receptors include CTLA-4 and PD-1 receptors. Examples of suitable cancer cell receptors include EGF and folate receptors and nucleolin. Examples of suitable endothelial cell receptors include CD54 (ICAM-1), CD106 (VCAM-1 ), integrins, selectins, and VEGF receptors.

[64] The targeting agent may be any compound that specifically binds to target receptors on a therapeutic target. For example, the targeting agent may be an aptamer, a peptide/protein (including viral proteins) or an antibody. Examples of suitable aptamers include guanosine-rich oligonucleotides, such as AS1411.

Examples of suitable peptide/proteins include Arg-Gly-Asp (RGD) peptides.

Examples of suitable antibodies include anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD28 antibodies, anti-TNF antibodies, anti-CD19

antibodies, anti-CTLA-4 antibodies, anti-PD1 antibodies, anti-EGF antibodies, folate, anti-CD3 antibodies, anti-CD54 antibodies, anti-CD106 antibodies and anti-VEGF antibodies.

[65] When targeting cancer cells or tumors, the targeting agent is preferably an anti-nucleolin agent. Anti-nucleolin agents may include antibodies, proteins, GROs, aptamers, or other compounds that bind to nucleolin. Targeting agents include aptamers, such as GROs. Examples of aptamers include guanosine-rich

oligonucleotides (GROs). Examples of suitable oligonucleotides and assays are also given in Miller et al. [7]. Characteristics of GROs include:

(1) having at least 1 GGT motif,

(2) preferably having 4-100 nucleotides, although GROs having many more nucleotides are possible,

(3) optionally having chemical modifications to improve stability.

[66] Especially useful GROs form G-quartet structures, as indicated by a

reversible thermal denaturation/renaturation profile at 295 nm. Preferred GROs also compete with a telomere oligonucleotide for binding to a target cellular protein in an electrophoretic mobility shift assay [6]. In some cases, incorporating the GRO nucleotides into larger nucleic acid sequences may be advantageous; for example, to facilitate binding of a GRO nucleic acid to a substrate without denaturing the nucleolin-binding site. Examples of oligonucleotides are shown in Table 1 ; preferred oligonucleotides include SEQ IDs NOs: 1-7; 9-16; 19-30 and 31 from Table 1. Most preferably, the targeting agent is AS1411. AS1411 advantages over other aptamers include increased internalization into the cancer or tumor cells and near-universal targeting of various tumor types.

[67] Table 1: Non-antisense GROs that bind nucleolin and non-binding

controls¹·²·³.

[68] ¹lndicates a good plasma membrane nucleolin-binding GRO. indicates a nucleolin control

(non-plasma membrane nucleolin binding). ³GRO sequence without ¹ or ² designations have some anti-proliferative activity.

[69] Any antibody that binds nucleolin may also be used. In certain instances, monoclonal antibodies are preferred as they bind single, specific and defined epitopes. In other instances, however, polyclonal antibodies capable of interacting with more than one epitope on nucleolin may be used. Many anti-nucleolin antibodies are commercially available, and are otherwise easily made. Table 2 lists a few commercially available anti-nucleolin antibodies.

[70] Table 2: commercially available anti-nucleolin antibodies

[71] Human antibodies, such as those described in U.S. Patent No. 9,260,517 [44] may also be used.

[72] Nucleolin targeting proteins are proteins, other than antibodies, that

specifically and selectively bind nucleolin. Examples include ribosomal protein S3, tumor-homing F3 peptides and myosin H9 (a non-muscle myosin that binds cell surface nucleolin of endothelial cells in angiogenic vessels during tumorigenesis).

[73] The linker may be any linker capable of coupling the targeting agent to the phospholipid layer. Preferably, the phospholipid layer includes a first phospholipid, coupled to the linker, and a second phospholipid, not coupled to the linker. Suitable linkages include maleimide-thiol linkages (thioether linkage), biotin-streptavidin bonds, amide linkage (for example reacting NHS ester with primary amine), a hydrazone linkage, and click-chemistry. Preferably, the linker is a maleimide-thiol linkage or biotin-streptavidin bond. A standard protocol involves using biotinylated linkers in the shell to enable covalent binding to avidins, which are then covalently bound to biotinylated antibodies. Typically, each reaction occurs by incubating the biotinylated component and the avidin component together for 60 min at 4 °C. After incubation the unbound components are removed via a centrifugation wash. Additional details of the linking process may be found in references [76], [77], [78] and [79].

[74] FIG. 22 is a schematic illustrating the therapeutic in vivo use of targeted

double emulsions. The targeted double emulsion travels through the capillaries to the target cells. The targeting agent binds the targeted double emulsion to the target cells. The application of ultrasound results in rupture of the double emulsion, allowing the therapeutic agent (also known as the payload) to pass through the endothelial layer and enter the cell membrane.

[75] Ultrasound may be administered using any suitable ultrasound generation and application device. The ultrasound is preferably applied at a frequency, intensity and duration that is sufficient to vaporize the non-aqueous shell and release the therapeutic agent without damaging the surrounding tissue. For example, the ultrasound may be high intensity focused ultrasound (HIFU) administered for less than 10 ps.

[76] Appropriate ultrasound parameters may be determined by simple

experimentation. For example, ultrasound may be applied to a cell culture

containing a double emulsion until the double emulsion releases the therapeutic agent. The cells are observed before and after ultrasound application to determine the effect of the ultrasound. One or more ultrasound parameters may be varied and the ultrasound reapplied. The cells are then observed to determine the effect of the modified ultrasound parameters on the release of the therapeutic agent. This process may be repeated until a desired outcome is achieved. Determining appropriate ultrasound parameters is a simple process for a skilled artisan since ultrasound is a well-studied mechanical technique that produces predictable results.

[77] The double emulsions may be used as a medicament. A pharmaceutical composition is formulated to be compatible with its intended route of administration, including intravenous, intradermal, subcutaneous, oral, inhalation, transdermal, transmucosal, and rectal administration. Solutions and suspensions used for parenteral, intradermal or subcutaneous application can include a sterile diluent, such as water for injection, saline solution, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

[78] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL® (BASF; Parsippany,

NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid so as to be administered using a syringe. Such

compositions should be stable during manufacture and storage and are preferably preserved against contamination from microorganisms such as bacteria and fungi. The carrier can be a dispersion medium containing, for example, water, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and other compatible, suitable mixtures. Various antibacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain microorganism contamination. Isotonic agents such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride can be included in the composition.

Compositions that can delay absorption include agents such as aluminum

monostearate and gelatin.

[79] Sterile injectable solutions can be prepared by incorporating the active

agents, and other therapeutic components, in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization. Methods of preparation of sterile solids for the preparation of sterile injectable solutions include vacuum drying and freeze-drying to yield a solid.

[80] An appropriate dosage level of the therapeutic agent will generally be about

0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. The compounds may be administered on a regimen of 1 to 4 times per day. When administered with a second form of treatment, such as radiotherapy (RT) for treating cancer, the double emulsions are preferably administered once per day prior to RT. Administration by continuous infusion is also possible. All amounts and concentrations of double emulsion are based on the amount or concentration of the therapeutic agent.

[81] Pharmaceutical preparations may be pre-packaged in ready-to-administer form, in amounts that correspond with a single dosage, appropriate for a single administration referred to as unit dosage form. Unit dosage forms can be enclosed in ampoules, disposable syringes or vials made of glass or plastic.

[82] However, the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the patient undergoing therapy.

[83] The medicaments of the present invention may be administered in

combination with other treatments such as chemotherapy, radiation therapy (RT), hyperthermia, gene therapy and photodynamic therapy.

[84] The medicament may be administered to any patient in need of treatment who could be treated with a water-soluble therapeutic agent. Preferred conditions for treatment include cancer, cardiovascular diseases such as heart failure, acute myocardial infarction, fibrosis and atherosclerosis, and other diseases that are localized in organs such as the liver, lungs, kidneys, bladder, stomach and intestines. After administration, the patient may be examined to determine if the administration has been effective to treat the condition. Further administration to the patient may be desirable to further treat the condition. The patient may be any species in need of treatment, such as a human, monkey, dog, cat, rabbit, cow, horse, camel, alpaca, pig, goat, guinea pig, mouse, rat or sheep. Preferably, the patient is a human.

[85] Non-targeted double emulsions may be used for in vitro treatment of tissue cultures or cell cultures. A preferred in vitro treatment is nucleic acid transfection. Non-targeted double emulsions containing DNA or RNA may be introduced to a cell culture. The application of ultrasound ruptures the double emulsion, which permeates the cell membrane and allows the DNA or RNA to enter the cell or cell nucleus. Similarly, non-targeted doubled emulsions may be used to introduce therapeutic agents into cells ex vivo and the treated cells may be returned to the patient.

[86] The double emulsions may be prepared using microfluidic systems. In

microfluidic systems, multiple pumps continuously deliver a solution of the aqueous core containing the therapeutic agent, a solution of the non-aqueous shell and the phospholipid layer, and a second aqueous solution through a microfluidic device to form water-in-oil-in-water double emulsions. The pump rates may be varied to achieve a desired double emulsion profile. Preferably, the double emulsions are monodisperse. Alternatively, double emulsions may be prepared using standard chemical synthetic processes, such as mixing the components followed by agitation (see Example 1 below).

[87] EXAMPLES

[88] Example 1 - Targeted Drug Delivery to Cancer Cells in Static Culture

Conditions

[89] Our objective was to evaluate whether chelating a tumor-targeting aptamer onto the double emulsion improved uptake of the payload in the cells compared to untargeted particles. Secondly, we wanted to see if ultrasound improved the delivery of the payload compared to control samples which did not receive application of ultrasound. We selected an aptamer that has the ability to target nucleolin- expressing cancer cells, such as the human triple-negative breast cancer (TNBC) cell line MDA-MB-231 , which we selected for our experimental testing. [90] Generation of Polydisperse Targeted Double Emulsions

[91] We placed 375 pl_ of deprotected AS141 1 maleimide solution in a

microcentrifuge tube. By deprotecting AS141 1 , it will allow the thiol group to react with the maleimide group on polar head of the phospholipid surfactant. The thiol group on AS1411 was deprotected with 10mM of (tris)(2-carboxyethyl)phosphine) (TCEP) for 1 hour and immediately added to lipid solutions for overnight incubation at 4°C to allow the reaction to occur. For untargeted control samples we placed 300 pl_ of 2% maleimide solution and 75 mI_ of PBS in a microcentrifuge. Both solutions were used as the surfactant. AS1411 has been shown to target nucleolin receptors while the maleimide solution without AS141 1 has no functional tumor-targeting moieties. We agitated the solutions using a micropipette to ensure thorough mixing. We then added 375 mI_ of Poloxamer 188 into both solutions and stored them at 4 °C. We placed 400 mI_ of 100 mg/mL fluorescein sodium salt in deionized water and 800 mI_ of perfluorohexane (PFH) in a 15 mL centrifuge tube. Using a 20 kHz sonicator, we set the amplitude to 30% for 30 seconds and sonicated the samples while on an ice bath. We removed the surfactants from 4 °C storage and placed the entire contents of each solution in separate 2 mL glass vials. We pipetted the fluorescein sodium salt/PFH solution up and down multiple times to ensure that the solution was well mixed. We then pipetted 250 pL into each surfactant solution. A septum cap was crimped onto each glass vial, and the solution was amalgamated for 45 seconds. We removed each vial from the amalgamator and left it to sit for 20 minutes to allow the double emulsions to settle at the bottom of the vial.

[92] Treatment of TNBC Cells at Physiological Conditions

[93] Seven different treatments were used on MDA-MB-231 cells. The objective of this study was to observe if a dose dependent effect occurred, to determine the effect of a targeting aptamer on uptake of molecular compound, and to determine the effect of ultrasound on release and delivery of the molecular compound from double emulsions. Three different doses were used with AS1411 -conjugated double emulsions and untargeted double emulsions: low dose (25 pL), medium dose (50 pL), and high dose (100 pL). Each dose was added at a 1 :20 ratio with PBS before treatment to ensure double emulsions were well distributed amongst cells. Treatments were added to a petri dish containing MDA-MB-231 cells. Following

treatment with compound, the petri dishes were placed back in the CO2 incubator for 4 hours at 37 °C. Following 4 hours in the incubator, groups were treated with ultrasound or no ultrasound based on experimental group specification.

[94] In groups that were treated with ultrasound, the petri dish was scanned across the ultrasound beam to ensure triggered release of molecular compound occurred. FIG. 3 illustrates the experimental set up for treatment of MDA-MB-231 cells using ultrasound to vaporize double emulsions. Ultrasound pulses were delivered with an ultrasound imaging system (2.5 MHz, ATL P4-1 probe, Verasonics Vantage 64LE system, 3 MPa peak negative pressure). Following ultrasound treatment, cells were washed with PBS. Cells were trypsinized to remove cells that were adhered to the bottom of the petri dish. Media was used to neutralize the trypsin, and the cells were placed in a vial. Cells were spun down at 1000 rpm for 5 minutes using a centrifuge. Supernatant was removed and cells were resuspended in 1 ml_ before assessing fluorescein uptake using flow cytometry. All data was normalized to AS141 1 conjugated low dose treatment. Statistical analysis was performed in Minitab using a two-way ANOVA. For samples imaged with scanning electron microscopy (SEM), cells were immediately fixed (< 1 min) in 2.5% glutaraldehyde after ultrasound treatment. FIG. 4 is a scanning electron microscope image showing TNBC cells prior to treatment. FIG. 5 is a scanning electron microscope image showing TNBC cells after treatment with ultrasound-responsive double emulsions. The arrow in FIG. 5 indicates a hole in the cell membrane.

[95] Treatment of TNBC with Ultrasound

[96] The objective of this study was to determine the effect of ultrasound on

release and delivery of the molecular compound from double emulsions. Two groups were used for the treatment of MDA-MB-231 cells: double emulsions (70mI_) vaporized with ultrasound and double emulsions (70pL) with no treatment. Once MDA-MB-231 cells were treated with double emulsions, the petri dishes were stored at 21 °C for 5 minutes. This allowed all the double emulsions to sink to the bottom of the petri dish, and it was stored at 21 °C to reduce the amount of passive release of the payload. In the group that was treated with ultrasound, the petri dish was scanned across the ultrasound beam to ensure triggered release of molecular

compound occurred, shown in FIG. 3. Ultrasound pulses were delivered with an ultrasound imaging system (2.5 MHz, ATL P4-1 probe, Verasonics Vantage 64LE system, 3 MPa peak negative pressure). Following ultrasound treatment, cells were washed with PBS. Cells were trypsinized to remove cells that were adhered to the bottom of the petri dish. Media was used to neutralize the trypsin, and the cells were placed in a vial. Cells were spun down at 1000 rpm for 5 minutes using a centrifuge. Supernatant was removed and cells were resuspended in 250 pl_ before assessing fluorescein uptake using flow cytometry. All data was normalized to double

emulsions where ultrasound was not applied. Statistical analysis was performed in Minitab using student T-test.

[97] Results and Discussion

[98] Tumor-targeted double emulsions versus non-targeted double emulsions

showed no statistically significant difference (p=0.628). Previous studies have shown that AS1411 has the ability to target and induce uptake through micropinocytosis in cancer cells. Multiple factors could explain the reasoning for the lack of a statistical difference. The most plausible explanation is that double emulsions sink in cell culture due to their higher density compared to the surrounding media, which allows the emulsion droplets to contact the cells without being dependent on ligand-receptor binding. For future studies, a dynamic flow model could be used to prevent emulsions from accumulating at the bottom of the container before treating with ultrasound.

[99] Ultrasound treatment also did not show any statistically significant increase compared to untreated control groups (p=.054). Additional testing is necessary to determine if ultrasound has an effect on uptake. In fact, in this study it appears that ultrasound may actually have a detrimental effect on uptake. This may be an artifact of the experimental setup in which high passive uptake of molecular compounds occurs. In this case, ultrasound treatment may be inducing leakage of the molecular compounds from the cells. The purpose of ultrasound treatment was to cause a phase change in perfluorocarbon allowing the release of the compound from the double emulsion droplets. It was theorized that double emulsions proximal to a cell will induce a collapse of the perfluorocarbon phase which can potentially facilitate rapid transport of payload directly to the cell by causing transient pores in the cell’s membrane. This led us to further investigate the induced interaction between the double emulsion and cell membrane that may be caused by ultrasound treatment. Our finding, shown in FIG. 5, indicates the presence of transient pores in the cell membrane following treatment of ultrasound. This confirms that ultrasound could have a potential use as a therapeutic delivery method from double emulsions. In this circumstance, however, double emulsions had the ability to interact with the cancer cells for approximately four hours. This is ample time for endocytosis-mediated uptake by the cell. Thus, a high concentration of payload is already inside the cell. When vaporization occurs, the payload can actually leave the cell by the newly formed transient pores.

[100] High dose treatment was shown to be statistically different compared to low dose (p<.01) and medium dose (p<.01). No statistical difference was found between low dose and medium dose (p=0.074), although there was a trend toward

significance and a larger sample size may have revealed a statistical difference.

FIG. 6 is a graph showing the relative fluorescence of the low dose, medium dose and high dose double emulsion treatments normalized to the AS1411 low dose treatment.

[101] The results of this study indicated that the double emulsions were not stable after 4 hours of incubation at 37 °C and spontaneously released their payload prior to ultrasound treatment. In addition, molecular compound (fluorescein) was taken up by cells even without AS1411 targeting, indicating non-specific delivery (likely by endocytosis) in the static culture system. The SEM image of FIG. 5 showing pores in the membrane is a positive finding since this indicates that molecular compounds could enter the cytosol of the cancer cell more easily. Nevertheless, due to limitations of the static cell culture system and double emulsion stability we did not observe any significant differences in uptake of the molecular compound by cells between experimental groups.

[102] Following the results, surfactants were investigated to determine the ability for it to stabilize the particle to reduce the passive release of the payload, which is discussed in further detail in the next section. A study was performed to further

analyze the effect that ultrasound had on uptake of the payload. Due to the density of the double emulsion, we observed that double emulsions sank to the bottom of the container within the first minute. FIG. 7 is an image showing the double emulsions one minute after being placed in culture. A group was treated with ultrasound after 5 minutes and was compared to a group that was not treated with ultrasound to determine if ultrasound had an impact on uptake of molecular compound. It was determined that ultrasound treatment induced more uptake of fluorescein (p<0.05). FIG. 8 is a graph showing the effect of ultrasound on fluorescein uptake normalized to no ultrasound treatment.

[103] Example 2 - Characterization of Surfactant Effects on Double Emulsion

Stability

[104] Different surfactant types, concentrations, and temperatures were tested to see the effect on the release profile of the double emulsions and increase the storage shelf-life of double emulsions. For double emulsions to be translated to the clinic, they must be able to be synthesized and stored for a duration of time prior to treatment without releasing the payload. With a more stable surfactant, a slower release profile will take place. Also, the effect of temperature on double emulsion stability was investigated to determine leakage of the internal payload over time at different temperatures.

[105] Synthesis of Double Emulsions

[106] We evaluated three different surfactants for experimental testing: Poloxamer

188 (Sigma-Aldrich), FluorN561 (Cytonix), and FluorN562 (Cytonix). Poloxamer 188 is a nonionic copolymer composed of a hydrophobic chain flanked by two hydrophilic chains. Poloxamer 188 has been commonly used as a surfactant for research due to its biocompatibility. However, for double emulsion use, it was unclear if it would provide enough stability for drug delivery applications. Thus, other surfactants were investigated. FluorN561 and FluorN562 are non-ionic, ethylene glycol-based fluorosurfactants and have the same structural backbone. FIG. 9 illustrates the structural backbone of FluorN561 and FluorN562, with A and ^* indicating the locations where functional groups may be attached. Fluor561 , commonly denoted as N561 , contains one perfluro group and three polyethylene glycol groups, and

FluorN562, commonly denoted as N562, contains two perfluoro groups and two polyethylene glycol groups. Both of these surfactants appeared advantageous since both are expected to be biocompatible and had longer chains compared to

Poloxamer 188. The longer chain lengths may reduce coalescence of the drug delivery vehicles.

[107] We diluted each solution to three different concentrations for testing.

Poloxamer 188 concentrations were tested at 1%, 2.5%, and 5% (w/v). N561 and N562 concentrations were tested at 0.2%, 0.5%, and 1% (w/v). Stock solutions were prepared at 10% (w/v) for Poloxamer 188 and 2% (w/v) for FluorN561 and

FluorN562. For the lowest concentration of each solution, 200 pL was added to 1.8 ml_ of PBS. For the middle concentration, 500 pl_ was added to 1.5 ml_ of PBS. For the highest concentration, 1 ml_ was added to 1 ml_ of PBS. In a 2 ml_ glass vial, 200 pL of fluorescein deionized water solution at a concentration of 200 mg/ml_ was added to 1.5 ml_ of diluted surfactant. A septum cap was crimped onto the glass vial and the solution was amalgamated for 45 seconds. Following amalgamation the solution was left undisturbed on the bench for 20 minutes at room temperature. This allowed the double emulsions to settle on the bottom of the solution.

[108] Washing Procedure for Double Emulsions

[109] Double emulsion washing steps were included to remove unencapsulated compounds from the solution. Prior to removing the supernatant that contained the free compounds, additional solutions were made for washing the emulsions.

Resuspension in PBS is a common methodology for most methods; however, it was discovered that in a PBS solution without surfactants, the double emulsion would release its compound very quickly. To prevent that from occurring, we prepared 5 ml_ solutions of 1%, 2.5%, and 5% Poloxamer 188, 5 ml_ solutions of 0.2%, 0.5%, and 1% FluorN561 , and 5 ml_ solutions of 0.2%, 0.5%, and 1% FluorN562 using PBS.

We removed 1 ml_ aliquots of each surfactant concentration and pipetted it into a separate vial. We then removed double emulsions of each solution and gently pipetted them into corresponding vials that contained 1 ml_ of surfactant. We allowed 5 minutes for the double emulsions to settle, then we remove the supernatant from the solution. We carefully washed an additional three times and removed the supernatant, allowing double emulsions to settle for 3 minutes each time.

[110] Release Profile of Double Emulsion

[111] For each surfactant type and concentration, three samples were prepared.

Each sample was covered with foil to prevent photobleaching of the fluorescence in the solution. For N562 and Poloxamer 188 samples, the three concentrations were stored at three different temperatures to observe the effect that temperature has on the release profile for each emulsion type. N561 was not sampled since stable formation of double emulsions did not occur with this surfactant. The conditions were 4 °C, 21 °C, and 37 °C. 20 mI_ aliquots were taken from the supernatant of each sample and placed in a 96 well plate. We then added 180 mI_ of deionized water to dilute each sample in the 96 well plate. Fluorescence was measured with a spectrofluorometer using an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The measurements were acquired at 1 hr, 2 hr, 4 hr, 24 hr,

72 hr, and 72hr post treatment. All data was normalized to the last data point taken.

[112] FIG. 10 is a graph showing the release profile for Poloxamer 188 and N562 at different surfactant concentrations after being stored at 4°C. FIG. 1 1 is a graph showing the release profile for Poloxamer 188 and N562 at different surfactant concentrations after being stored at 21 °C. FIG. 12 is a graph showing the release profile for Poloxamer 188 and N562 at different surfactant concentrations after being stored at 37°C. The data shown in FIG. 10, FIG. 1 1 and FIG. 12 was normalized to 72 hour post-treatment with ultrasound.

[113] Results and Discussion

[114] Initial testing showed that temperature may have a significant effect on the passive release profile of the emulsion droplets but was not statistically significant. For Poloxamer 188 and N562 emulsions at 37 °C (FIG. 12), almost all of the fluorescein was released from the double emulsions within 1 hour. This suggests that an additional surfactant is necessary to have a more sustained release profile at that temperature. For Poloxamer 188 and FluorN562 emulsions at 4 °C (FIG. 10) and 21 °C (FIG. 11), a slower release profile occurred. This suggests that either 4 °C or 21 °C may be used for emulsion storage before treatment. FluorN561 is not a viable option as a surfactant for double emulsions due to the inability for stable double emulsions to be formed.

[115] Additional testing was performed to further examine the effect that surfactant and temperature has on release profile of double emulsions. FIG. 13 is a graph showing the relative fluorescence for Poloxamer188 and FluorN562 after being stored at 4°C, 21 °C and 37°C, normalized to the last measured point at 72 hours. FluorN562 performed better than Poloxamer188 at each temperature (p<0.05).

FluorN562 showed a slower release profile. In order for ultrasound-responsive, targeted double emulsions to be used clinically, a slow release profile is needed so that the emulsion remains intact until it is vaporized by ultrasound at the target site. This will allow a potentially high amount of payload to be delivered to the tumor. FluorN562 at 21 °C exhibited the best release profile (p<0.05).

[116] Example 3 - Microfluidic Synthesis of Monodisperse Double Emulsions

[117] Microfluidic devices were developed to synthesize monodispersed double emulsions that are under 5 microns. Monodisperse double emulsions have many advantages compared with polydisperse double emulsions. With monodisperse double emulsions, the size of each droplet in the emulsions is known and can be reproduced consistently, thus it is possible to perform quality control to determine if the emulsions could be used clinically based on the size, but with polydisperse double emulsions there can be a large variance in size and the distribution can vary between batches.

[118] Design

[119] Multiple components were considered in the design of the microfluidic system, which included flow type and channel size. Flow characteristics have an important impact on output of double emulsions. If flow is turbulent, steady production of double emulsions will not occur since coalescence of droplets is more likely to occur. Channel size is an important factor in double emulsion production. If channels are too small, clogging is likely to occur. We modified a special design recently described in the literature, which employs a double-layer format to enable formation of very small double emulsions (less than 10 microns in diameter) through an orifice. FIG. 14 illustrates a schematic of the microfluidic device for creating monodispersed double emulsions.

[120] Fabrication of Microfluidic Devices

[121] The SU8 master was fabricated on a silicon wafer at the UofL Micro/Nano

Technology Center using standard photolithography techniques. PDMS-based microfluidic devices were fabricated using previously established methods. Briefly,

60 g of silicone base and 6 g of curing agent was added to a cup and was mixed thoroughly using a stir stick. The cup containing the mixture was degassed by placing it in the desiccator until air bubbles were no longer visible. All of the PDMS was poured over the SU8 master and placed back into the desiccator to remove any remaining air bubbles. Following the removal of all bubbles, the device was baked for 2 hours at 60 °C in a lab oven. The PDMS was carefully peeled off the SU8 master before removing PDMS devices. Each individual device was cut from the PDMS block using a razor blade. The microfluidic inlets and outlets were punched in the PDMS using a 2 mm biopsy puncher. Once devices were prepared, they were treated with oxygen plasma (100 V for 60 s, 0.5 atm of O2) and immediately bonded onto glass microscope slides.

[122] System Preparation

[123] The microfluidic device was placed on the stage of an inverted microscope coupled with a digital camera that can take frames less than 100 ps apart. Syringes were mounted on a syringe pump for continuous infusion into the microfluidic device. We found that syringes of 10 mL or larger were optimal. Syringe were primed before attaching approximately 45 cm lengths of flexible tygon PVC tubing (1/16” ID, 1/8” OD) to the syringes. We connected the loose ends into the appropriate input ports in the device. We also inserted a 15 cm length of the tygon PVC tubing into the output port. We primed the device by running the syringe pump at high rates of speed (4 mL/min) until fluid in the tubing reaches the inlet channels of the device.

[124] Generation of Monodisperse Double Emulsions [125] We focused the microscope on a region of the device where the 10 pm wide channel converged with the 50 pm channel and contained the 50 pm by 50 pm orifice. We set the syringe pumps to 500 pl/hr for the inner phase and 500 pL/hr for the middle phase. We allowed continuous flow for 5 minutes to reach steady state. We ran the syringe pump at 2000 pL/hr for the continuous (outer) phase, also allowing 5 minutes to reach steady state. We maintained flow rates of the inner and middle phase and incrementally increased the flow at 200 pL/hr each minute for the continuous phase until 5,000 pL/hr was reached. We allowed steady state to be reached for the continuous double emulsion generation and then acquired videos and images as double emulsions were generated in the device.

[126] Emulsion imaging

[127] We pipetted 50 pL of solution from the bottom of the collecting vial, then

placed the solution in 1 ml_ of phosphate-buffered saline (PBS). We ensured that the solution was well-mixed, then pipetted 5 pL of PBS solution mixture onto a

microscope slide for imaging under a fluorescent microscope. We adjusted the optical filter to the appropriate setting to ensure detection of the double emulsion encapsulated payload. We placed a scale bar on the image to determine droplet size. ImageJ was used to analyze the diameter of the double emulsion.

[128] Results and Discussion

[129] PDMS microfluidic devices are capable of generating water/oil/water (w/o/w) double emulsions using coaxial flow. In order to have laminar flow, it is essential that the channels are aligned properly. When the channels were properly aligned and no clogging of the channels occurred, double emulsions were able to be generated as small as 1 pm and ranged from 1 pm to 2 pm in size. FIG. 15 is a photograph showing the monodispersity of the double emulsions (scale bar = 100 pm).

[130] We found that the current design state of the microfluidic device has many limitations. The system is very sensitive to changes in pressure and takes a significant amount of time to reach steady state. FIG. 16 is a photograph showing the generation of double emulsions in the orifice at steady state. An introduction of an air bubble to the system will cause the device to stop producing double emulsions for a period of time due to the variability in pressure. Another limitation is the amount of time that it takes for the microfluidic device to create enough double emulsions for treatment.

[131] Changes in design can potentially address these issues. A pressure regulator can be added to maintain steady pressure, allowing a constant production of double emulsions. Designing a new microfluidic system with multiple outputs can increase the droplet production rate significantly. Both of these design changes could address the current limitations compared to other synthesis methodologies.

[132] Example 4 - Enhanced Intracellular Delivery of Molecular Compounds Using

Non-Targeted Double Emulsions

[133] Various techniques have been developed to enhance intracellular delivery such as electroporation and phage delivery. These techniques, however, lack spatial control and uniform delivery. To address these limitations, an ultrasound-driven microfluidic device has been developed. The ultrasound-driven microfluidic device uses acoustic forces to actively transport molecular compounds intracellular through a process called sonoporation. Sonoporation induces transient membrane pores which facilitates intracellular transport of molecular compounds. To improve the efficiency of molecular delivery in the device, exogenous ultrasound-responsive double emulsions have been developed that can release their payload as they align with cells inside the device.

[134] Experimental Procedure

[135] Double emulsions were synthesized by amalgamation to form droplets with fluorescein loaded inner phase (1 mg/ml_), perfluorohexane middle phase for ultrasound activation, and a non-reactive polymeric fluorosurfactant solution. FIG.

17 is a photograph showing the microfluidic device set up. 100 pL of double emulsions were added to 1 mL of media containing 100,000 human breast carcinoma cells (MDA-MB-231 ). Multiple flow rates (30 ml/hr, 60 ml_/hr, and 120 mL) and ultrasonic conditions (no ultrasound and ultrasound) were utilized to determine optimal parameters for maximum molecular delivery to cells. Ultrasound pulses were delivered with a piezoelectric ultrasound device (5 MHz, continuous mode, 5 V output). FIG. 18 is a schematic illustrating the ultrasonic flow system.

FIG. 19 is a schematic illustrating intracellular delivery induced by double emulsion rupture. Fluorescein uptake was assessed using flow cytometry. FIG. 20 is a graph illustrating intracellular delivery of fluorescein. Cell viability was assessed using standard MTT assay protocol. FIG. 21 is a graph illustrating the MTT assay. For statistical analysis of cell viability and MTT assay, two-way ANOVA was utilized.

[136] Results and Discussion

[137] Ultrasound generally enhanced intracellular delivery and there was a trend toward significance (p=0.057). Statistical significance for fluorescein uptake by cells was not observed between different flow rates. Higher sample sizes are needed to reach statistical significance. Additionally, flow rates will have an important impact on cellular delivery due to pressure generation and exposure time to ultrasound. However, the optimal flow rate has yet to be determined. Inconsistent results were seen across treatment groups, especially the ultrasound groups. It was observed that cells and double emulsions tend to separate from solution due to density differences. MTT Assay showed cell proliferation/viability decreased across all groups compared to the no treatment group (p<0.05). No statistical difference was observed for different ultrasound parameters nor for different flow rates. This indicates, with the current concentration of double emulsions, that ultrasound has minimal effect on cell viability.

[138] The ultrasound-driven microfluidic device has the ability for efficient and

consistent delivery to each cell. However, the device is currently limited by inability to maintain a homogenous solution before treatment. Further modifications need to be made to the system to ensure consistent treatment for each cell.

[139] Example 5 - In vivo administration of targeted double emulsion to a cancer patient

[140] A patient presents with triple negative breast cancer. The location of the cancer is identified using standard diagnostic and imaging techniques. A targeted double emulsion containing doxorubicin as the therapeutic agent and AS1411 as the targeting agent is administered to the patient by infusion. AS1411 binds to cell- surface nucleolin on triple negative breast cancer cells. Ultrasound is applied to the region of the patient’s breast where the cancer is located. The ultrasound disrupts the non-aqueous shell, releasing the doxorubicin. The doxorubicin enters the triple negative breast cancer cells. The triple negative breast cancer cells are killed and the patient recovers.

[141] Example 6 - In vivo administration of targeted double emulsion to a cardiac patient

[142] A patient presents with cardiovascular disease. A targeted double emulsion containing RNA as the therapeutic agent and an anti-CD54 antibody as the targeting agent is administered to the patient by infusion. The anti-CD54 antibody binds to damaged endothelial cells in the heart. Ultrasound is applied to the patient’s heart. The ultrasound disrupts the non-aqueous shell, releasing the RNA. The RNA enters the damaged endothelial cells. The endothelial cells produce proteins encoded by the RNA that repair the heart and the patient recovers.

[143] Example 7 - In vitro transfection

[144] A non-targeted double emulsion containing DNA as the therapeutic agent is added to a cell culture. Ultrasound is applied to the cell culture. The ultrasound disrupts the non-aqueous shell, releasing the DNA. The non-aqueous shell disruption permeates the cell membrane and allows the DNA to enter the permeated cells. The cells are transfected by the DNA.

[145] REFERENCES

[146] [1] Stedman, T.L. 2000. Stedman's medical dictionary. Lippincott Williams

& Wilkins, Philadelphia, xxxvi, [127], 2098.

[147] [2] Miller, D., P. Bates, and J. Trent. 2000. Antiproliferative activity of G- rich oligonucleotides and method of using same to bind to nucleolin, Int’l Pub. No. WO 00/61597.

[148] [3] Bates P J, Miller D M, Trent J O, Xu X,“A New Method for the

Diagnosis and Prognosis of Malignant Diseases” International Application, Int’l Pub. No. WO 03/086174 A2 (23 Oct. 2003).

[149] [4] Bates P J, Miller D M, Trent J O, Xu X,“Method for the Diagnosis and

Prognosis of Malignant Diseases” U.S. Patent App. Pub., Pub. No. US

2005/0053607 A1 (10 Mar. 2005).

[150] [5] Derenzini M, Sirri V, Trere D, Ochs R L, "The Quantity of Nucleolar

Proteins Nucleolin and Protein B23 is Related to Cell Doubling Time in Human Cancer Cells" Lab. Invest. 73:497-502 (1995).

[151] [6] Bates P J, Kahlon J B, Thomas S D, Trent J O, Miller D M,

"Antiproliferative Activity of G-rich Oligonucleotides Correlates with Protein Binding" J. Biol. Chem. 274:26369-77 (1999).

[152] [7] Miller D M, Bates P J, Trent J O, Xu X,“Method for the Diagnosis and

Prognosis of Malignant Diseases” U.S. Patent App. Pub., Pub. No. US

2003/0194754 A1 (16 Oct. 2003).

[153] [8] Bandman O, Yue H, Corley N C, Shah P,“Human Nucleolin-like

Protein” U.S. Pat. No. 5,932,475 (3 Aug. 1999).

[154] [9] Reyes-Reyes E M, Teng Y, Bates P J,“A New Paradigm for Aptamer

Therapeutic AS141 1 Action: Uptake by Macropinocytosis and Its Stimulation by a Nucleolin-Dependent Mechanism” Cancer Res 70(21 ): 8617-29 (2010). [155] [10] E. Perez-Herrero, A. Fernandez-Medarde, Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy, Eur J Pharm Biopharm, 93 (2015) 52-79.

[156] [1 1] C. Sawyers, Targeted cancer therapy, Nature, 432 (2004) 294-297.

[157] [12] O. Tacar, P. Sriamornsak, C.R. Dass, Doxorubicin: an update on

anticancer molecular action, toxicity and novel drug delivery systems, J Pharm Pharmacol, 65 (2013) 157-170.

[158] [13] D.A. Fernandes, D.D. Fernandes, Y. Li, Y. Wang, Z. Zhang, D.

Rousseau, C.C. Gradinaru, M.C. Kolios, Synthesis of Stable Multifunctional

Perfluorocarbon Nanoemulsions for Cancer Therapy and Imaging, Langmuir, (2016).

[159] [14] J.A. Kopechek, E. Park, C.S. Mei, N.J. McDannold, T.M. Porter,

Accumulation of phase-shift nanoemulsions to enhance MR-guided ultrasound- mediated tumor ablation in vivo, J Healthc Eng, 4 (2013) 109-126.

[160] [15] N. Rapoport, Phase-shift, stimuli-responsive perfluorocarbon

nanodroplets for drug delivery to cancer, Wiley Interdiscip Rev Nanomed

Nanobiotechnol, 4 (2012) 492-510.

[161] [16] R. Gupta, J. Shea, C. Scafe, A. Shurlygina, N. Rapoport, Polymeric

micelles and nanoemulsions as drug carriers: Therapeutic efficacy, toxicity, and drug resistance, J Control Release, 212 (2015) 70-77.

[162] [17] N. Rapoport, R. Gupta, Y.S. Kim, B.E. O'Neill, Polymeric micelles and nanoemulsions as tumor-targeted drug carriers: Insight through intravital imaging, J Control Release, 206 (2015) 153-160.

[163] [18] J.P. Marshalek, P.S. Sheeran, P. Ingram, P.A. Dayton, R.S. Witte, T.O.

Matsunaga, Intracellular delivery and ultrasonic activation of folate receptor-targeted phase-change contrast agents in breast cancer cells in vitro, J Control Release, 243 (2016) 69-77. [164] [19] C.H. Wang, S.T. Kang, Y.H. Lee, Y.L. Luo, Y.F. Huang, C.K. Yeh,

Aptamer-conjugated and drug-loaded acoustic droplets for ultrasound theranosis, Biomaterials, 33 (2012) 1939-1947.

[165] [20] J.A. Kopechek, E.J. Park, Y.Z. Zhang, N.l. Vykhodtseva, N.J. McDannold,

T.M. Porter, Cavitation-enhanced MR-guided focused ultrasound ablation of rabbit tumors in vivo using phase shift nanoemulsions, Phys Med Biol, 59 (2014) 3465- 3481.

[166] [21] O.D. Kripfgans, J.B. Fowlkes, D.L. Miller, O.P. Eldevik, P.L. Carson,

Acoustic droplet vaporization for therapeutic and diagnostic applications, Ultrasound Med Biol, 26 (2000) 1177-1189.

[167] [22] E. Strohm, M. Rui, I. Gorelikov, N. Matsuura, M. Kolios, Vaporization of perfluorocarbon droplets using optical irradiation, Biomed Opt Express, 2 (2011) 1432-1442.

[168] [23] K. Wilson, K. Homan, S. Emelianov, Biomedical photoacoustics beyond thermal expansion using triggered nanodroplet vaporization for contrast-enhanced imaging, Nat Commun, 3 (2012) 618.

[169] [24] P.J. Bates, D.A. Laber, D.M. Miller, S.D. Thomas, J.O. Trent, Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer, Exp Mol Pathol, 86 (2009) 151-164.

[170] [25] P.J. Bates, E.M. Reyes-Reyes, M.T. Malik, E.M. Murphy, M.G. O'Toole,

J.O. Trent, G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms, Biochim Biophys Acta, (2016).

[171] [26] E.M. Reyes-Reyes, Y. Teng, P.J. Bates, A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism, Cancer Res, 70 (2010) 8617-8629.

[172] [27] J.E. Rosenberg, R.M. Bambury, E.M. Van Allen, H.A. Drabkin, P.N. Lara,

Jr., A.L. Harzstark, N. Wagle, R.A. Figlin, G.W. Smith, L.A. Garraway, T. Choueiri, F. Erlandsson, D.A. Laber, A phase II trial of AS1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal cell carcinoma, Invest New Drugs, 32 (2014) 178-187.

[173] [28] A. Latorre, C. Posch, Y. Garcimartin, A. Celli, M. Sanlorenzo, I. Vujic, J.

Ma, M. Zekhtser, K. Rappersberger, S. Ortiz-Urda, A. Somoza, DNA and aptamer stabilized gold nanoparticles for targeted delivery of anticancer therapeutics,

Nanoscale, 6 (2014) 7436-7442.

[174] [29] M.T. Malik, M.G. O'Toole, L.K. Casson, S.D. Thomas, G.T. Bardi, E.M.

Reyes-Reyes, C.K. Ng, K.A. Kang, P.J. Bates, AS1411-conjugated gold

nanospheres and their potential for breast cancer therapy, Oncotarget, 6 (2015) 22270-22281.

[175] [30] Y.S. Shiao, H.H. Chiu, P.H. Wu, Y.F. Huang, Aptamer-functionalized gold nanoparticles as photoresponsive nanoplatform for co-drug delivery, ACS Appl Mater Interfaces, 6 (2014) 21832-21841.

[176] [31] M. Khader, P.M. Eckl, Thymoquinone: an emerging natural drug with a wide range of medical applications, Iran J Basic Med Sci, 17 (2014) 950-957.

[177] [32] C.C. Woo, A.P. Kumar, G. Sethi, K.H. Tan, Thymoquinone: potential cure for inflammatory disorders and cancer, Biochem Pharmacol, 83 (2012) 443-451.

[178] [33] L. Li, J. Hou, X. Liu, Y. Guo, Y. Wu, L. Zhang, Z. Yang, Nucleolin- targeting liposomes guided by aptamer AS1411 for the delivery of siRNA for the treatment of malignant melanomas, Biomaterials, 35 (2014) 3840-3850.

[179] [34] Z.X. Liao, E.Y. Chuang, C.C. Lin, Y.C. Ho, K.J. Lin, P.Y. Cheng, K.J.

Chen, H.J. Wei, H.W. Sung, An AS1411 aptamer-conjugated liposomal system containing a bubble-generating agent for tumor-specific chemotherapy that overcomes multidrug resistance, J Control Release, 208 (2015) 42-51.

[180] [35] X. Li, Y. Yu, Q. Ji, L. Qiu, Targeted delivery of anticancer drugs by

aptamer AS1411 mediated Pluronic F127/cyclodextrin-linked polymer composite micelles, Nanomedicine, 11 (2015) 175-184. [181] [36] J. Zhang, R. Chen, F. Chen, M. Chen, Y. Wang, Nucleolin targeting

AS1411 aptamer modified pH-sensitive micelles: A dual-functional strategy for paclitaxel delivery, J Control Release, 213 (2015) e137-138.

[182] [37] A. Aravind, P. Jeyamohan, R. Nair, S. Veeranarayanan, Y. Nagaoka, Y.

Yoshida, T. Maekawa, D.S. Kumar, AS1411 aptamer tagged PLGA-lecithin-PEG nanoparticles for tumor cell targeting and drug delivery, Biotechnol Bioeng, 109 (2012) 2920-2931.

[183] [38] J. Wu, C. Song, C. Jiang, X. Shen, Q. Qiao, Y. Hu, Nucleolin targeting

AS1411 modified protein nanoparticle for antitumor drugs delivery, Mol Pharm, 10 (2013) 3555-3563.

[184] [39] W.J. Duncanson, L.R. Arriaga, W.L. Ung, J.A. Kopechek, T.M. Porter,

D.A. Weitz, Microfluidic fabrication of perfluorohexane-shelled double emulsions for controlled loading and acoustic-triggered release of hydrophilic agents, Langmuir, 30 (2014) 13765-13770.

[185] [40] M.L. Fabiilli, K.J. Haworth, I.E. Sebastian, O.D. Kripfgans, P.L. Carson,

J.B. Fowlkes, Delivery of chlorambucil using an acoustically-triggered

perfluoropentane emulsion, Ultrasound Med Biol, 36 (2010) 1364-1375.

[186] [41] Kopechek J A, Park E, Mei C S, McDannold N J and Porter T M 2013

Accumulation of phase-shift nanoemulsions to enhance MR-guided ultrasound- mediated tumor ablation in vivo J. Healthc. Eng. 4 109-26.

[187] [42] Kopechek J A, Park E, Zhang Y, Vykhodtseva N I, McDannold N J and

Porter T M, 2014 Cavitation-enhanced MR-guided focused ultrasound ablation of rabbit tumors in vivo using phase shift nanoemulsions Phys. Med. Biol. 59 (2014) 3465-3481.

[188] [43] Kopechek, J. A., Zhang, P., Burgess, M. T., Porter, T. M. Synthesis of

Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation. J. Vis. Exp. (67), e4308, DOI: 10.3791/4308 (2012). [189] [44] U.S. Patent No. 9,260,517.

[190] [45] Bates, et al.,“Discovery and Development of the G-rich Oligonucleotide

AS1411 as a Novel Treatment for Cancer”, Experimental and Molecular Pathology, Vol. 86, pp. 151-164 (2009).

[191] [46] Malik, et al.,“AS1411-conjugated gold nanospheres and their potential for breast cancer therapy”, Oncotarget, Vol. 6, pp. 22270-22281 (2015).

[192] [47] Wang, et al.,“Aptamer-conjugated and drug-loaded acoustic droplets for ultrasound theranosis”, Biomaterials, Vol. 33, pp. 1939-1947 (2012).

[193] [48] Tannock, I., Cell kinetics and chemotherapy: a critical review. Cancer

Treat Rep, 1978. 62(8): p. 1117-33.

[194] [49] Iwamoto, T., Clinical application of drug delivery systems in cancer

chemotherapy: review of the efficacy and side effects of approved drugs. Biol Pharm Bull, 2013. 36(5): p. 715-8.

[195] [50] Tiwari, G., et al., Drug delivery systems: An updated review. Int J Pharm

Investig, 2012. 2(1): p. 2-11.

[196] [51] Lakhin, A.V., V.Z. Tarantul, and L.V. Gening, Aptamers: problems,

solutions and prospects. Acta Naturae, 2013. 5(4): p. 34-43.

[197] [52] Maeda, H., T. Sawa, and T. Konno, Mechanism of tumor-targeted

delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS. J Control Release, 2001. 74(1- 3): p. 47-61.

[198] [53] Maeda, H., H. Nakamura, and J. Fang, The EPR effect for

macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv Drug Deliv Rev, 2013. 65(1): p. 71-9. [199] [54] Maeda, H., Vascular permeability in cancer and infection as related to macromolecular drug delivery, with emphasis on the EPR effect for tumor-selective drug targeting. Proc Jpn Acad Ser B Phys Biol Sci, 2012. 88(3): p. 53-71.

[200] [55] Pitt, W.G., G.A. Husseini, and B.J. Staples, Ultrasonic drug delivery--a general review. Expert Opin Drug Deliv, 2004. 1(1): p. 37-56.

[201] [56] Schoellhammer, C.M., et al., Ultrasound-mediated gastrointestinal drug delivery. Sci Transl Med, 2015. 7(310): p. 31 Oral 68.

[202] [57] Bekeredjian, R., et al., Ultrasound targeted microbubble destruction

increases capillary permeability in hepatomas. Ultrasound Med Biol, 2007. 33(10): p. 1592-8.

[203] [58] Garti, N., Progress in Stabilization and Transport Phenomena of Double

Emulsions in Food Applications. LWT - Food Sci Tech, 1997. 30(3): p. 222-235.

[204] [59] Chavez-Paez, M., et al., Coalescence in double emulsions. Langmuir,

2012. 28(14): p. 5934-9.

[205] [60] Holtze, C., et al., Biocompatible surfactants for water-in-fluorocarbon

emulsions. Lab Chip, 2008. 8(10): p. 1632-9.

[206] [61] Datta, S.S., et al., 25th anniversary article: double emulsion templated solid microcapsules: mechanics and controlled release. Adv Mater, 2014. 26(14): p. 2205-18.

[207] [62] Chiu, Y.L., et al., Synthesis of fluorosurfactants for emulsion-based

biological applications. ACS Nano, 2014. 8(4): p. 3913-20.

[208] [63] Fabiilli, M.L., et al., Delivery of water-soluble drugs using acoustically triggered perfluorocarbon double emulsions. Pharm Res, 2010. 27(12): p. 2753-65.

[209] [64] Fabiilli, M.L., et al., Assessment of the biodistribution of an [(18) F]FDG- loaded perfluorocarbon double emulsion using dynamic micro-PET in rats. Contrast Media Mol Imaging, 2013. 8(4): p. 366-74. [210] [65] Shpak, O., et al., Ultrafast dynamics of the acoustic vaporization of

phase-change microdroplets. J Acoust Soc Am, 2013. 134(2): p. 1610-21.

[211] [66] Park, J., et al., The kinetics of blood brain barrier permeability and

targeted doxorubicin delivery into brain induced by focused ultrasound. J Control Release, 2012. 162(1): p. 134-42.

[212] [67] Fan, Z., et al., Spatiotemporally controlled single cell sonoporation. Proc

Natl Acad Sci U S A, 2012. 109(41 ): p. 16486-91.

[213] [68] Kooiman, K., et al., Sonoporation of endothelial cells by vibrating targeted microbubbles. J Control Release, 2011. 154(1 ): p. 35-41.

[214] [69] Brujan, E.-A., Jets from pulsed-ultrasound-induced cavitation bubbles near a rigid boundary. J. Phys. D: Appl. Phys, 2017. 50(21 ): p. 215302.

[215] [70] Soundararajan, S., et al., Plasma membrane nucleolin is a receptor for the anticancer aptamer AS141 1 in MV4-11 leukemia cells. Mol Pharmacol, 2009. 76(5): p. 984-91.

[216] [71] Chen, Z. and X. Xu, Roles of nucleolin. Focus on cancer and anti-cancer therapy. Saudi Med J, 2016. 37(12): p. 1312-1318.

[217] [72] Wise, J.F., et al., Nucleolin inhibits Fas ligand binding and suppresses

Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex. Blood, 2013. 121 (23): p. 4729-39.

[218] [73] Farin, K„ et al., Oncogenic synergism between ErbB1 , nucleolin, and mutant Ras. Cancer Res, 201 1. 71 (6): p. 2140-51.

[219] [74] Cole, R.H., T.M. Tran, and A.R. Abate, Double Emulsion Generation

Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device. J Vis Exp, 2015(106): p. e53516.

[220] [75] Centner, C.S.,“Tumor-targeted double emulsions for ultrasound-triggered delivery of molecular therapeutics", Electronic Theses and Dissertations, Paper 3002 (2018). [221] [76] Kilbanov, A.L.,“Preparation of targeted microbubbles: ultrasound contrast agents for molecular imaging”, Medical & Biological Engineering & Computing, Vol. 47, Issue 8, pp. 875-882 (2009).

[222] [77] Sakahara, H. et al.,“Avidin-biotin system for delivery of diagnostic

agents”, Advanced Drug Delivery Reviews, Vol. 37, Issues 1-3, pp. 89-101 (1999).

[223] [78] Korpanty, G. et al.,“Targeting vascular endothelium with avidin

microbubbles”, Ultrasound in Medicine and Biology, Vol. 31 , Issue 9, pp. 1279-1283 (2005).

[224] [79] Loughrey, H.C. et al.,“Preparation of streptavidin-liposomes for use in ligand-specific targeting applications”, Liposome Technology Volume III, Chapter 11 , pp. 163-178 (1993).

[225] [80] Riley, R.S. et al.,“Delivery technologies for cancer immunotherapy",

Nature Reviews Drug Discovery, Vol. 18, pp. 175-196 (2019).

[226] [81] Yewale, C. et al.,“Epidermal growth factor receptor targeting in cancer: a review of trends and strategies”, Biomaterials, Vol. 34, Issue 34, pp. 8690-8707 (2013).

[227] [82] Cheung, A. et al.,“Targeting folate receptor alpha for cancer treatment”,

Oncotarget, Vol. 7, No. 32, pp. 52553-52574 (2016).

[228] [83] Khodabandehlou, K. et al.,“Targeting cell adhesion molecules with

nanoparticles using in vivo and flow-based in vitro models of atherosclerosis", Experimental Biology and Medicine, Vol. 242, No. 8, pp. 799-812 (2017).

Claims

WHAT IS CLAIMED IS:

1. A targeted double emulsion, comprising:

a first phospholipid,

a second phospholipid,

a targeting agent, conjugated to the first phospholipid,

a non-aqueous shell,

a non-ionic surfactant,

water,

a therapeutic agent, in the water, and

a continuous aqueous phase, surrounding the first phospholipid and the second phospholipid,

wherein the first phospholipid and the second phospholipid enclose the non- aqueous shell,

the non-aqueous shell encloses the non-ionic surfactant,

the non-ionic surfactant encloses the water and the therapeutic agent, and the double emulsion has an average diameter of at most 5 pm.

2. A double emulsion, comprising:

a phospholipid,

a non-aqueous shell,

a non-ionic surfactant,

water,

a therapeutic agent, in the water, and

a continuous aqueous phase, surrounding the phospholipid,

wherein the phospholipid encloses the non-aqueous shell,

the non-aqueous shell encloses the non-ionic surfactant,

the non-ionic surfactant encloses the water and the therapeutic agent, and the double emulsion has an average diameter of at most 50 pm.

3. The double emulsion of any of the preceding claims, wherein the therapeutic agent is dissolved in the water.

4. The double emulsion of any of the preceding claims, wherein the non- aqueous shell comprises a perfluorocarbon.

5. The double emulsion of any of the preceding claims, wherein the peril urorocarbon comprises perfluoropentane or perfluorohexane.

6. The double emulsion of any of the preceding claims, further comprising a dye, conjugated to the phospholipid.

7. The double emulsion of any of the preceding claims, wherein the targeting agent is selected from the group consisting of aptamers, peptides/proteins, viral proteins, and antibodies.

8. The double emulsion of any of the preceding claims, wherein the targeting agent comprises AS1411.

9. The double emulsion of any of the preceding claims, wherein the therapeutic agent is selected from the group consisting of chemotherapeutic agents, cytotoxic agents, nucleic acids, and nanoparticles for drug or gene delivery.

10. The double emulsion of any of the preceding claims, wherein the therapeutic agent comprises a first chemotherapeutic agent and a second

chemotherapeutic agent.

1 1. The double emulsion of any of the preceding claims, wherein the therapeutic agent comprises doxorubicin, cisplatin, or carboplatin.

12. The double emulsion of any of the preceding claims, wherein the targeting agent is conjugated to the phospholipid via a thiol-maleimide linkage, biotin-streptavidin bonds, an amide linkage, a hydrazone linkage, or click chemistry.

13. A pharmaceutical composition for use in treating cancer, comprising the double emulsion of any of the preceding claims, and a pharmaceutically acceptable carrier, wherein the therapeutic agent comprises a chemotherapeutic agent.

14. A method of treating cancer, comprising:

administering an effective amount of the pharmaceutical composition of any of the preceding claims to a patient in need thereof;

followed by administering ultrasound to the patient.

15. A method of imaging cancer or a tumor, comprising:

(1) administering the double emulsion of any of the preceding claims to a patient; and

(2) imaging the cancer or tumor with ultrasound.

16. A pharmaceutical composition for use in treating cardiovascular disease, comprising the double emulsion of any of the preceding claims, and a pharmaceutically acceptable carrier,

wherein the therapeutic agent comprises a nucleic acid.

17. A method of treating cardiovascular disease, comprising:

administering an effective amount of the pharmaceutical composition of any of the preceding claims to a patient in need thereof;

followed by administering ultrasound to the patient.

18. The method of any of the preceding claims, wherein the patient is selected from the group consisting of humans, monkeys, dogs, cats, rabbits, cows, horses, camels, alpaca, pigs, goats, guinea pigs, mice, rats, and sheep.

19. The method of the preceding claims, wherein the patient is a human.

20. A method of transfecting a tissue culture or cell culture, comprising: administering the double emulsion of any of the preceding claims to the tissue culture or cell culture;

followed by administering ultrasound to the tissue culture or cell culture.