WO2018053601A1

WO2018053601A1 - Method for preparing a lipid bubble

Info

Publication number: WO2018053601A1
Application number: PCT/AU2017/051042
Authority: WO
Inventors: Harendra PAREKH; Sachin Thakur
Original assignee: The University Of Queensland
Priority date: 2016-09-23
Filing date: 2017-09-25
Publication date: 2018-03-29

Abstract

The present invention relates to a method for preparing a lipid bubble encapsulating a gas including the steps of forming a lipid bubble; vortexing the lipid bubble in a liquid environment, under an atmosphere of the gas and at an elevated temperature, to form a gas-containing lipid bubble; and subjecting the gas-containing lipid bubble to snap cooling. These lipid bubbles are stable, have a well-defined size and narrow size distribution for reproducibility and predictability in drug delivery, diagnostics, and theranostics applications.

Description

TITLE

Method for Preparing a Lipid Bubble

FIELD OF THE INVENTION

[0001 ] The present invention relates to the field of drug delivery, diagnostics, and theranostics. More particularly, the invention relates to a method for preparing a lipid bubble encapsulating a gas and the uses thereof.

BACKGROUND TO THE INVENTION [0002] Microbubbles are defined as bubbles with a diameter between 1 μιη and 1 mm, and nanobubbles are defined as bubbles with a diameter between 1 nm and 1 μιη. It has been found that bubbles which are suspended in liquids can be used as contrast agents in medical diagnostics (sonography), as drug delivery vehicles, or as a combination of these two approaches (theranostics). [0003] Sonography utilizes sound pressure waves with a frequency of around 20 kHz to several gigahertz to see inside the body. These sound waves are pulsed non-invasively into a patient's tissue and the reflected sound (the "echo") from parts of the tissue or organs are recorded and displayed. The resultant image depicts the difference in the reflected sound. The body consists of around 80% water and it has been found that the echogenicity of water is vastly different to the echogenicity of a gas. Substances that are more efficient at reflecting sound waves are said to have a higher echogenicity, and contrasting echogenicity leads to accurate sonographic images. A method of achieving a sonographic image with higher contrast is contrast-enhanced ultrasound which involves injecting a subject with a high echogenicity substance prior to imaging.

[0004] A bubble has a high echogenicity because the gas component of the bubble compresses and oscillates in an ultrasonic frequency and results in greater echo. As such, the echogenicity of a bubble is dependent on the amount of gas that is encapsulated by the bubble.

[0005] The bubbles presently utilized in the art are unstable, unable to encapsulate or accommodate therapeutic agents in their core, have a short lifetime, and have a broad size distribution, i.e. they are typically heterogeneous in nature. The distribution of the size of these bubbles is important to ensure that the echogenicity of the contrast agent remains constant and predictable. The instability of these prior art bubbles means that the bubbles need to be injected shortly after preparation. [0006] These bubbles can also be utilized to deliver active agents to specific regions of the body by utilizing bioactive materials coated or tethered to the surface of the bubble. When these bubbles reach the region to be treated they can be collapsed or burst to release the active agent. The size distribution of these bubbles is important to ensure that the extent of the active agent delivery is reproducible. This technique allows for targeted drug delivery, and in turn requires a lower amount of active agent for effective treatment. This is particularly advantageous for delivering drugs with hazardous side effects.

[0007] Therefore, it is advantageous to have bubbles that are more stable to ensure that they have sufficient time to reach the desired region (for drug delivery, diagnostics and theranostic purposes), are stable so that they can be transported/stored and do not need to be injected shortly after preparation, and have a well-defined size (μιη and nm) and narrow size distribution for reproducibility and predictability in drug delivery, diagnostics, and theranostics applications.

SUMMARY OF THE INVENTION

[0008] In a first aspect, the invention resides in a method for preparing a lipid bubble encapsulating a gas including the steps of:

a) forming a lipid bubble; b) vortexing the lipid bubble in a liquid environment, under an atmosphere of the gas and at an elevated temperature to form a gas- containing lipid bubble; and

c) subjecting the gas-containing lipid bubble to snap cooling,

to thereby prepare a lipid bubble encapsulating a gas.

[0009] In a second aspect, the invention resides in a method of targeted delivery of a lipid bubble encapsulating a gas to a target area of a tissue in a subject, including the steps of:

a) injecting the lipid bubble encapsulating the gas into the tissue; and

b) subjecting the lipid bubble encapsulating the gas to an ultrasound stream,

wherein the ultrasound stream is directed generally toward the target area, to thereby deliver the lipid bubble encapsulating the gas to the target area of the tissue in the subject, and wherein the lipid bubble encapsulating the gas is produced by the method of the first aspect.

[0010] In a third aspect, the invention resides in a method of delivery of the contents of a lipid bubble encapsulating a gas to a tissue in a subject, including the steps of:

a) injecting the lipid bubble encapsulating the gas into the subject systemically; and

b) subjecting the tissue to ultrasound,

wherein the lipid bubble encapsulating the gas is disrupted when it is subjected to the ultrasound, thereby releasing its contents to the tissue, and wherein the lipid bubble encapsulating the gas is produced by the method of the first aspect.

[001 1 ] In a fourth aspect, the invention resides in a method of delivery of an active agent within a topical composition comprising a lipid bubble encapsulating a gas to a tissue of a subject, including the steps of:

a) applying the topical composition to the subject; and

b) subjecting the topical composition to an ultrasound stream, to thereby deliver the active agent into the tissue of the subject, and wherein the lipid bubble encapsulating the gas is produced by the method of the first aspect.

[0012] The various features and embodiments of the present invention referred to in the individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections as appropriate.

[0013] Further features and advantages of the present invention will become apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] To assist in understanding the invention and to enable a person skilled in the art to put the invention into practical effect, preferred embodiments of the invention will be described by way of example only with reference to the accompanying drawings, in which:

FIG 1 shows a depiction of a porcine eye injected with rhodamine-tagged lipid bubbles and not subjected to an ultrasound stream;

FIG 2 shows a graphical representation of the amount of rhodamine in each quadrant of the porcine eye depicted in FIG 1 .

FIG 3 shows a depiction of a porcine eye injected with rhodamine-tagged lipid bubbles that has been subjected to an ultrasound stream directed from above the cornea toward the posterior of the eye;

FIG 4 shows a graphical representation of the amount of rhodamine in each quadrant of the porcine eye depicted in FIG 3;

FIG 5 shows a depiction of a porcine eye injected with rhodamine-tagged lipid bubbles that has been subjected to an ultrasound stream directed toward the side of the eye;

FIG 6 shows a graphical representation of the amount of rhodamine in each quadrant of the porcine eye depicted in FIG 5. FIG 7 shows a series of images of an eye injected with lipid bubbles (D-F) and non-echogenic emulsion (A-C), subsequent ultrasound application (B and E), and after ultrasound application (C and F);

FIG 8 shows an image of the movement of the lipid bubbles upon application of ultrasound;

FIG 9 shows a graphical representation of amount of migration following ultrasound application;

FIG 10 shows a series of images of migration after the application of multiple ultrasound pulses, and the results of these pulses;

FIG 1 1 shows the echogenic profiles of sonicated bubbles (left) and vortexed bubbles (right);

FIG 12 shows a graphical representation of the temperature decline over time for the lipid bubble encapsulating a gas being snap cooled;

FIG 13 shows a graphical representation of the loading efficiency vs the duration of loading;

FIG 14 shows the echogenicity of the ciprofloxacin lipid bubble over 4 weeks;

FIG 15 shows graphical representations of the ciprofloxacin concentration, lipid bubble size, and PDI of the ciprofloxacin lipid bubble encapsulating the gas;

FIG 16 shows a microscopy image of a topical composition comprising 1 % v/v of the lipid bubble encapsulating a gas;

FIG 17 shows the echogenic profile of topical compositions comprising (A) 0%, (B) 1 %, (C) 3%, (D) 5% v/v lipid bubbles; with 0.1 % w/v carbomer;

FIG 18 shows the echogenic profile of topical compositions comprising (A) 0% lipid bubbles and 0.1 % w/v carbomer; (B) 0.1 %, (C) 0.2% (D) 0.3%, (E) 1 % and (F) 1 .5% w/v carbomer, with 1 % v/v lipid bubbles; FIG 19 shows the effect of exposing 0.1 % w/v carbomer without lipid bubbles (A-C) and with 3% v/v lipid bubbles (D-F) to continuous ultrasound.

FIG 20 shows an ultrasound image of the topical composition comprising the lipid bubble encapsulating a gas before disruptive ultrasound was applied;

FIG 21 shows an ultrasound image of the topical composition comprising the lipid bubble encapsulating a gas after disruptive ultrasound was applied;

FIG 22 shows a graphical representation of rhodamine B penetration into 1 .3% w/v agar phantoms after exposing a composition comprising only carbomer (1 % w/v) compared to a topical composition comprising carbomer (1 %, w/v) and lipid bubbles (1 % v/v);

FIG 23 shows echogenic profiles of a topical composition on (A) the day of preparation; (B) 1 day, (C) 2 days, (D) 7 days, (E) 15 days, (F) 21 days and (G) 28 days after preparation;

FIG 24 shows a graphical representation of the distribution of rhodamine B co-formulated with FF formulation following pars plana ultrasound administration; and

FIG 25 shows a graphical representation of rhodamine tagged FF formulation following convection, pars plana and corneal ultrasound administration.

DETAILED DESCRIPTION OF THE INVENTION

[0015] In this specification, adjectives such as anterior, posterior, at least, and the like may be used solely to distinguish one element or action from another element or action without necessarily requiring or implying any actual such relationship or order. Words such as "comprises", "comprising", "includes", or "including" are intended to define a non-exclusive inclusion, such that a method that comprises a list of elements does not include only those elements but may include other elements not expressly listed, including elements that are inherent to such a method.

[0016] As used in this specification the indefinite articles "a" and "an" may refer to one entity or a plurality of entities (e.g. components) and are not to be read or understood as being limited to a single entity.

[0017] The term "subject" as used herein, includes both human and veterinary subjects. For example, targeted delivery of a lipid bubble encapsulating a gas to a target area in a subject can include targeted delivery to a human subject or a veterinary subject. Preferably, the subject is a human. However, the targeted delivery may also be applicable to mammals such as domestic and companion animals, performance animals such as horses, livestock, and laboratory animals.

[0018] The term "bubble" as used herein, can be interchanged with "nanobubble" and/or "microbubble". [0019] The term 'topical composition' refers to a composition that is applied to body surfaces such as the skin or mucous membranes. Mucous membrane includes, but is not limited to, eyes, ears, inside of the nose, inside of the mouth, tongue, lips and cervix.

[0020] The term "about" as used herein in relation to the amount of a component, means that the amount is nominally the number following the term "about" but the actual amount may vary from this precise number to an unimportant degree.

[0021 ] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as would be commonly understood by those of ordinary skill in the art to which this invention belongs.

[0022] In a first aspect, the invention resides in a method for preparing a lipid bubble encapsulating a gas including the steps of:

c) subjecting the gas-containing lipid bubble to snap cooling,

to thereby prepare a lipid bubble encapsulating a gas.

[0023] It will be appreciated that the formation of lipid bubbles is, generally, known in the art and the following merely describes one possible method for the preparation thereof. Therefore, the first step in the first aspect of forming the lipid bubble may be achieved by a number of means which would be known to the person skilled in the art.

[0024] The lipid bubble is prepared by dissolving a lipid in a volatile liquid and transferring the resultant solution to a suitable vessel. Typical lipids have a hydrophobic tail and a hydrophilic head, and are generally fatty acids or a derivative of a fatty acid that are generally insoluble in water. Suitable lipids include amphiphiles, fats, fatty acids, sterols, vitamins, waxes, glycerides, and phospholipids. More particularly, the lipid is phosphocholine. Preferably, the lipid is selected from the group consisting of 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-PEG(2k)-OMe), 1 ,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), and 1 ,2-disteroyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-PEG(5k)- OMe). It will be understood by a person skilled in the art that the lipid bubble can comprise of more than one lipid, and also that the list provided is not an exhaustive list, but merely demonstrates the types of molecules that could be utilized. The lipid can also include charged lipids.

[0025] In one embodiment, the lipid comprises a mixture of DPPC and DSPE-PEG(2k)-OMe. The ratio of DPPC to DSPE-PEG(2k)-OMe is suitably between about 88:12 and about 99:1 , more suitably between about 90:10 and about 95:5, preferably between about 93:7 and about 94:6, and most preferably about 94:6. [0026] In another embodiment, the lipid comprises a mixture of DPPC and DSPE-PEG(5k)-OMe. The ratio of DPPC to DSPE-PEG(5k)-OMe is suitably between about 88:12 and about 99:1 , more suitably between about 90:10 and about 95:5, preferably between about 93:7 and about 94:6, and most preferably about 94:6.

[0027] The volatile liquid is selected from the group consisting of halogenated hydrocarbons, hydrocarbons, and organic liquids. Preferably, the volatile liquid is selected from chloroform, ethanol, isopropanol, methanol, hexane, ether, acetone, or dichloromethane. A person skilled in the art will appreciate that a mixture of two of more volatile liquids can be used.

[0028] The volatile liquid is then removed from the lipid. It will be understood by a person skilled in the art that the volatile liquid can be removed from the lipid by a number of different methods, such as evaporation, or passing a stream of gas over the volatile liquid, or subjecting the volatile liquid to reduced pressure, or a combination thereof. A suitable liquid is then added to the lipid to place the lipid in a liquid environment.

[0029] The liquid forming the liquid environment is suitably a buffer, a co- solvent or a mixture thereof. Preferably, the liquid is glycerol, propylene glycol, or a buffer. The buffer is capable of maintaining the pH in a range of from about 7 to about 8, and more preferably about 7.4. It will be appreciated by a person skilled in the art that the buffer can be any buffer that does not interact with the lipid. The person skilled in the art will also understand that the buffering range is dependent on the lipid because the formation of the lipid bubble can be pH dependent. The buffer is selected from the group consisting of phosphate buffered solutions, HEPES, acetate and TRIS buffers. Preferably, the buffer is phosphate buffered saline.

[0030] Additional components can be added to the lipid to improve the stability, structure and incorporation of drugs and other compounds into or of the resulting lipid bubble. These additional components can include glycerol, charged nanoparticles, propylene glycol, sugars, and acids. One non-limiting example of a compound that assists with structure and stability is cholesterol. A person skilled in the art will understand that more than one additional component can be added to improve the properties of the resultant lipid bubble, and that the list provided is not an exhaustive list, but merely a list that exemplifies some examples of the additional components that can be utilized. Further to this, additives to improve cryopreservation can be added. Suitable additives to improve cryopreservation include sugars such as mannitol, sucrose, and trehalose.

[0031 ] Another component that could be incorporated into the resulting lipid bubble is, but is not limited to, polyethylene glycol (PEG). Various PEGs, including but not limited to hyperbranched PEGs and methacrylate PEGs, can be used for targeting nanoparticles to disease related targets using bi-specific antibodies. The use of hyperbranched PEGs leads to the number of targeting sites being significantly increased. It is postulated that the hyperbranched PEG moieties on the surface of the lipid bubbles can be used to target and bind with a target site. WO2016/123675 discusses such hyperbranched PEGs, the disclosure of which is incorporated by reference herein in its entirety. Therefore, in one embodiment, the lipid bubble comprises a PEG. In embodiments, the PEG may be a hyperbranched PEG and/or a methacrylate PEG.

[0032] The liquid, such as for example a buffer, and lipid are then mixed at suitably at least 1 ^QC above the phase transition temperature of the lipid, more suitably about 1 ^QC to about 20 ^QC above the phase transition temperature of the lipid, preferably about 5 ^QC to about 15 ^QC above the phase transition temperature of the lipid, more preferably about 7 ^QC to about 12 ^QC above the phase transition temperature of the lipid, and most preferably about 10 ^QC above the phase transition temperature of the lipid. The advantages of this temperature are discussed hereinafter.

[0033] The liquid and lipid are then agitated to form the lipid bubbles. The method of agitation can include shaking, sonication, or stirring. This lipid bubble encapsulates atmospheric air, unless bubble formation was carried out under a different gaseous environment, and has a wide size distribution. The encapsulated air will need to be displaced with another gas and the size distribution standardized to prepare the lipid bubble for therapeutic/diagnostic/theranostic use. It will be appreciated by a person skilled in the art that the gaseous environment can be the gas to be encapsulated and in such an embodiment this gas will not need to be replaced in step b). [0034] Therefore, in one embodiment, the step a) of forming a lipid bubble includes a step of subjecting the lipid in a liquid environment to agitation.

[0035] The lipid bubbles in the liquid environment are then transferred to an air tight vessel with a suitable amount of headspace. The air in the headspace is then removed, by means of a vacuum pump, although a person skilled in the art will understand that the air in the headspace can be removed in a number of different ways, such as displacement. The gas to be encapsulated is introduced into the headspace under positive pressure, so that the lipid bubbles in the liquid environment are under an atmosphere of the gas.

[0036] A person skilled in the art will understand that any gas may potentially be utilized as the gas to be encapsulated. It is known in the art that heavy gases have a lower solubility and their use can alleviate the problems associated with the gas dissolving in the bloodstream. It is postulated that larger gas molecules, such as perfluoropropane (PFP) and hydrophobic gases, will be more stably encapsulated within the lipid bubble. It is believed that the larger molecules are less likely to diffuse, and that hydrophobic gases have a higher affinity for lipids. In contrast, smaller molecules are expected to diffuse more easily from the lipid bubble. Therefore, it is preferable that the gas to be encapsulated is a heavy gas that is not toxic. It has been found that suitable gases include halogenated hydrocarbons, sulphur hexafluoride, air, noble gases, carbon dioxide, and nitrogen. Preferably, the gas is selected from the group consisting of perfluoropropane, oxygen, nitrogen, hydrogen, nitric oxide, carbon monoxide, hydrogen sulfide, and carbon dioxide. Most preferably, the gas is selected from the group consisting of halogenated hydrocarbons. A person skilled in the art will appreciate that the list provided is not an exhaustive list, but merely demonstrates the types of gases that can be encapsulated. The person skilled in the art will also realize that more than one gas can be utilized simultaneously. [0037] It is postulated that chelating agents may further assist in maintaining the gas within the bubbles. In this regard, in one embodiment, the lipid bubble further comprises a chelating agent.

[0038] The vessel, and hence the lipid bubbles in the liquid environment under the gas atmosphere, are then subjected to vortex-heating. Vortex-heating is the act of simultaneously subjecting the lipid bubbles in the liquid environment to a rapid swirling motion and an elevated temperature. The inventors have found that vortex-heating significantly improves the amount of gas encapsulated by the lipid bubble, and is discussed hereinafter. [0039] The elevated temperature is selected based on the lipid(s) utilized in forming the lipid bubble. It is preferred that the elevated temperature is a temperature above the phase transition temperature of the lipid or lipids forming the lipid bubble. The phase transition temperature is the temperature at which the lipid changes from one state to another. For example, if the temperature is above the phase transition temperature of the lipid, then the lipid undergoes a phase change from an ordered gel phase to a disordered liquid crystalline phase.

[0040] The elevated temperature is suitably at least 1 ^QC above the phase transition temperature of the lipid, more suitably from about 1 ^QC to about 30 ^QC above the phase transition temperature of the lipid, preferably from about 5 ^QC to about 20 ^QC above the phase transition temperature of the lipid, more preferably from about 7 ^QC to about 15 ^QC above the phase transition temperature of the lipid, and most preferably about 10 ^QC above the phase transition temperature of the lipid. For instance, the glass transition temperature of 1 ,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC) is about 41 ^QC and so the temperature at which vortex-heating is carried out may be about 51 ^QC. In another example, the glass transition temperature of 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-A/-[amino(polyethylene glycol)-2000] (DPSE-PEG(2K)- OMe) is about 55 ^QC and so the temperature at which vortex-heating is carried out may be about 65 ^QC.

[0041 ] By maintaining the temperature of the liquid environment above the phase transition temperature of the lipid, the lipid bubble is more malleable and permeable because it is in the disordered liquid crystalline phase. This more malleable and permeable lipid bubble allows for efficient interaction with the gas, and leads to minimal resistance to optimal gas entrapment and exchange. The permeable shell allows gas to enter and exit the bubble, and this allows for efficient displacement of gas. Further to this, there is a greater volume of gas encapsulated by the lipid bubble compared to the originally encapsulated gas (air in the earlier discussion), and there will be a net outward movement of air and a net inward movement of the gas to be encapsulated which leads to more encapsulated gas in the lipid bubble.

[0042] A typical prior art approach to forming a lipid bubble encapsulating a gas involves subjecting the lipid bubble to sonication, whereby sound waves are used to agitate the lipid bubbles in a sample. Sonication is able to rupture and reform lipid based bubbles. While sonication can achieve small bubble sizes, the technique is limited by the amount of gas that can be encapsulated by the lipid bubble. Sonication does not provide a means for improved gas encapsulation and leads to lipid bubbles with a lower volume of encapsulated gas. Sonication only agitates the surface of the liquid environment in which the lipid bubble is contained, and there is no significant increase in the surface area of the liquid environment which is placed in contact with the gaseous atmosphere. As a result there is a minimal increase in the contact between the liquid environment, and the lipid bubbles therein, with the gas atmosphere. As a result there is less interaction between the lipid bubbles in the liquid environment and the gas atmosphere, and this leads to less efficient gas exchange. As previously mentioned, sonication can rupture and compromise a lipid bubble. The compromised lipid bubble will result in encapsulated gas escaping into the atmosphere. These bubbles have a reduced volume of encapsulated gas and a lower echogenicity. The rupturing of the lipid bubble also leads to problems associated with stability. Over time, due to the compromised lipid bubble, the encapsulated gas will escape, making them less efficient as contrast-enhancing agents. The compromised lipid bubble will also result in more gas being lost during the gas tight membrane extrusion step discussed hereinafter. [0043] The formation of a vortex, as described herein, is distinctly different to sonication as it applies centrifugal forces to the liquid environment to thereby generate a vortex in which the gas and lipid bubbles interact more efficiently and to a far greater extent. [0044] The vortex formed by the swirling motion of the liquid environment increases the surface area of the liquid environment, and hence the number of lipid bubbles, in contact with the gaseous atmosphere. The centrifugal forces move the liquid environment upward and outward. This upward and outward force results in a vortex or a whirlpool, whereby the lipid bubbles in the liquid environment are constantly being turned over and exposed to the gas to ensure efficient mixing and exposure. This leads to a more efficient displacement of the air within the lipid bubble with the gas to be encapsulated. An advantage to this approach compared to traditional methods is that the present approach is more efficient and a greater amount of gas is encapsulated. It will be appreciated by a person skilled in the art that any approach that forms a vortex within the liquid environment can be utilized. Alternatively, a person skilled in the art will also realize that this advantage arises from the increased surface area of the liquid environment in contact with the gas to be encapsulated. As such, any method of increasing the surface area of the liquid environment will be suitable. This increased surface area can be achieved by any other manner such as shaking, and swirling. Another advantage of the method of the present disclosure is that substantially all of the lipid bubbles formed during vortex-heating can be used in the following gas-tight membrane extrusion step, discussed hereinafter. This allows the method of producing a lipid bubble encapsulating a gas to be more efficient and commercially viable.

[0045] In one embodiment, the vortex is formed by subjecting the lipid bubble in a liquid environment to a suitable rotational speed of about 500 RPM to 3000 RPM, more suitably about 1000 RPM to about 2000 RPM, and most preferably about 1500 RPM. It will be understood by a person skilled in the art that the rotation speed will depend on the viscosity of the liquid environment. Multiple vessels can be subjected to a vortex-heating at the same time by utilizing a device that can subject the vessels to elevated temperature and a rapid swirling motion. One method of achieving this is to use a heated block that can accommodate multiple vessels. The heated block, and the vessels, is subjected to a swirling motion to form a vortex at elevated temperature.

[0046] It is important to note that the degree of echogenicity arises from the amount of gas encapsulated by the lipid bubble. The greater the amount of gas encapsulated results in a higher echogenicity, and will result in a better contrasting agent. However, one disadvantage with the prior art lipid bubbles is that when the lipid bubbles are subjected to gas tight membrane extrusion they lose some of the encapsulated gas, resulting in a loss of echogenicity. [0047] It is, therefore, important to maximise the amount of encapsulated gas in the lipid bubbles prior to the process of gas tight membrane extrusion. The maximized amount of encapsulated gas will alleviate the problems associated with the gas lost during gas tight membrane extrusion and size standardization.

[0048] The gas-containing lipid bubble is then subjected to snap cooling. The term "snap cooling" refers to rapidly cooling the gas-containing lipid bubble to a desired temperature. However, it will be appreciated by a person skilled in the art that the temperature that the gas-containing lipid bubbles is cooled to will depend on the lipid used. The gas-containing lipid bubble is preferably cooled to a temperature below the phase transition temperature of the lipid or lipids forming the lipid bubble but above the freezing temperature of the liquid environment. If the liquid environment is frozen then this can compromise the integrity of the lipid bubble. The cooling of the gas-containing lipid bubble will lower the temperature to below the phase transition temperature of the lipid or the lipids forming the lipid bubble, and the lipid bubble will undergo a phase transition from a disordered liquid crystalline phase to an ordered gel phase which effectively "seals" around the encapsulated gas. The rapid cooling results in a rapid contraction of the gas-containing lipid bubble which leads to improved gas entrapment and retainment. The gas-containing lipid bubble is cooled to a temperature suitably from about 1 ^QC to about 15 ^QC, and more suitably from about 2 ^QC to about 8 ^QC.

[0049] The time taken for the gas encapsulated lipid bubble to be cooled to the desired temperature is suitably within about 7.5 minutes, more suitably within about 6 minutes, and most preferably within about 5 minutes.

[0050] The rate of cooling the gas-containing lipid bubble is suitably about 15 ^QC to about 45 ^QC/minute for the first minute of cooling, preferably about 20 ^QC/minute to about 40 ^QC/minute for the first minute of cooling, and most preferably 25 ^QC/minute to about 30 ^QC/minute for the first minute of cooling; followed by suitably about 5 ^QC to about 25 ^QC/minute for the second minute of cooling , and preferably about 5 ^QC/minute to about 15 ^QC/minute for the second minute of cooling; and about 5 °C/minute of cooling for the remaining time until the desired storage temp of 2-8 °C is achieved. The inventors of the present invention have found that the cooling rate of the gas-containing lipid bubble is non-linear and the rate of cooling decreases as the temperature nears the desired temperature. It will be appreciated by a person skilled in the art that the cooling rate can be adjusted by adjusting the external temperature surrounding the vessel containing the gas-containing lipid bubble.

[0051 ] It will be understood by a person skilled in the art that the time taken to cool the gas-containing lipid bubble and the rate of cooling the gas-containing lipid bubble will also depend on the volume of the liquid environment, as a greater amount of lipid bubbles will take longer to cool to the desired temperature.

[0052] In one embodiment, the invention further includes a step, after step b), of subjecting the lipid bubble encapsulating the gas to gas tight membrane extrusion at an elevated temperature.

[0053] In an alternative embodiment, the step of subjecting the lipid bubble encapsulating the gas to gas tight membrane extrusion can occur after step c). It will be understood that the lipid bubble encapsulating the gas can undergo multiple gas tight membrane extrusion steps to improve size distribution and maximize gas entrapment. Further to this, the lipid bubble encapsulating a gas will be reheated to the elevated temperature and maintained at this temperature during gas tight membrane extrusion to ensure efficient gas entrapment.

[0054] Most of the presently utilized methods of standardizing the bubble size distribution utilize centrifugation, where the centrifuged solution is usually separated into three layers; the uppermost layer of the centrifuged solution has the smallest diameter lipid bubbles, the lowermost layer has the largest diameter lipid bubbles and the middle layer has the midrange sized lipid bubbles. Generally, the midrange bubbles are isolated and the other layers are discarded, which leads to wastage and an inefficient process. This problem is not observed in the method of the present disclosure.

[0055] However, centrifugation is unsuitable for smaller sized lipid bubbles. It has been found that smaller sized lipid bubbles, e.g., nanobubbles, simply disintegrate or dissolve, and larger sized lipid bubbles tend to stabilize over time to a bubble diameter of about 1 -2 μιη.

[0056] The gas tight membrane extrusion step of the present invention is completed at an elevated temperature. As previously mentioned, the elevated temperature is selected based on the lipid(s) utilized in forming the lipid bubble. The elevated temperature is a temperature above the phase transition temperature of the lipids forming the lipid bubble, and the advantages of completing gas tight membrane extrusion is discussed hereinabove. Gas tight membrane extrusion involves passing the lipid bubbles with encapsulated gas through a membrane. The membrane has pores which the lipid bubbles must pass through. As the lipid bubbles pass through the membrane they reduce in diameter to fit through the pores, and therefore their size is limited. The pores in the membrane have a diameter that is suitably from about 30 nm to about 200 nm, alternatively about 200 nm to about 600 nm, alternatively about 600 nm to about 1 μιη, alternatively about 1 μιη to about 400 μιη, or alternatively about 400 μιη to about 1000 μιη. Suitable pore diameters can be selected from the group consisting of about 30 nm, about 50 nm, about 100 nm, about 200 nm, about 400 nm, about 800 nm, about 1 μιη, about 2 μιη, about 4 μιη, about 8 μιη, about 10 μιη, about 20 μιη, about 50 μιη, about 100 μιη, about 200 μιη, about 400 μιη, and about 800 μιη. It will be understood by a person skilled in the art that the pore size will depend on the desired size distribution of the lipid bubble. The extrusion process results in lipid bubbles with a narrow distribution of lipid bubble size. The lipid bubbles can be passed through the membrane multiple times to improve the distribution of lipid bubble size.

[0057] Alternatively, another method of standardizing the bubble size distribution is to filter the lipid bubbles to isolate the desired bubble size. For instance, the lipid bubbles can be filtered through differently sized membranes to remove larger bubbles. Preferably, the filtration should be performed under gas tight conditions.

[0058] The lipid bubbles encapsulating the gas can be stored at a temperature below the phase transition temperature to ensure that the encapsulated gas does not escape. It has been found that the stored lipid bubbles are stable, and are able to maintain their size distribution and echogenicity.

[0059] It has also been found that the extrusion process was able to reduce the average size and improve the polydispersity index (PDI) of the lipid bubbles encapsulating a gas. It was also noted that the extruded lipid bubbles were able to retain their narrow size distribution over time and are homogeneous. These lipid bubbles were compared to crude lipid bubbles which are those lipid bubbles that were formed using the present method, but not subjected to gas tight membrane extrusion. In comparison, the crude lipid bubbles were unable to retain their size distribution characteristics and had a poorer echogenicity profile. It should be understood that the present method results in lipid bubbles with encapsulated gas that are more stable and have a narrower PDI. It has been found that gas tight extrusion enhances echogenicity. The inventors have further found that the extrusion process improves the overall arrangement of lipids that form the lipid bubble, and the more ordered arrangement of lipids in the membrane leads to better retention of gas.

[0060] The extruded lipid bubbles exhibited visibly distinct properties compared to the crude formulation. The crude formulation possessed a small heterogeneous foam layer on top of the solution, whereas a larger more uniform and paste-like foam was observed with the extruded lipid bubbles. The small heterogeneous foam layer of the crude formulation was difficult to re-suspend for prolonged intervals, whereas the extruded lipid bubbles were easily re- suspended and resulted in a homogeneous milky white mixture. Although the re- suspended extruded lipid bubbles reverted back to a paste-like foam layer after 10-15 minutes, it was readily re-suspended with agitation.

[0061 ] A comparison of the crude formulation and the extruded lipid bubbles showed that the extrusion process increased the total number of bubbles per given volume approximately 10-fold. The increase in the number of bubbles was attributed to nanosized bubbles because there was no apparent/detectable change in micron-sized bubble numbers when extruded at 400 and 1000 nm.

[0062] The extruded lipid bubbles were then tested to determine the effect of the foam layer. In this regard, the foam layer was removed to give a foam -free formulation (FF), and this was compared to the formulation with the foam layer still present (the foam-mixed formulation, FM).

[0063] Upon a vigorous shake, the FM formulation comprises a proportion of micron-sized and nano-sized bubbles. In contrast, the FF formulation comprised only low-nanosized bubbles and was devoid of microbubbles. As such, this appeared to indicate that a significant amount of micron-sized lipid bubbles were present in the foam layer, and the liquid layer comprised mostly nano-sized lipid bubbles. It was found that shaking increased the echogenicity of the formulation. Additionally, a 10-fold greater dose of the FF formulation was required to obtain comparable echogenicity to the FM formulation dose.

[0064] The elimination of the foam layer did not completely eradicate formulation echogenicity, and confirmed that the gas was adequately retained in the lipid bubbles that resided in the body (solution) of the formulation, and separate from the foam. Both the FF and FM formulations demonstrated similar decay in echogenic signal over 30 minutes, and indicated that gas escape from the formulation was not dependent on bubble size.

[0065] Storage conditions for both the FM and FF formulations were optimized to confer long term stability. Firstly, different storage gases were tested. Formulations stored with PFP in the headspace of the vials experienced slow decay in echogenicity. In contrast, formulations stored under completely gastight conditions with no headspace experienced no significant drop in echogenic signal over the same timeframe. Storage under an N₂ gas atmosphere was deemed least suitable as this saw the greatest decrease in echogenic signal. In one embodiment, the lipid bubbles encapsulating a gas are stored under an atmosphere of nitrogen or PFP. In another embodiment, the lipid bubbles encapsulating a gas are stored in conditions where there is essentially no headspace.

[0066] Secondly, the FM and FF formulations were tested to see their effect on stability. In this regard, the echogenic stability of the FF formulation was lower when compared to the FM formulation. As such, the foam layer was advantageous to the echogenic stability of the lipid bubbles. In one embodiment, the formulation of lipid bubbles encapsulating a gas comprises a foam layer.

[0067] Further to this, the extruded lipid bubbles encapsulating the gas demonstrated superior retention of echogenicity over the crude formulation in all instances. Crude FM formulation echogenicity dropped below 90% of the original echogenic reading within 2 months, whereas the FM formulations extruded through 1000 nm and 400 nm membranes retained >90% of their echogenicity for up to 6 months.

[0068] It has been postulated that viscosity enhancers can be added to the liquid environment of the lipid bubble to improve retention of echogenicity. Non- limiting examples of viscosity enhancers include glycerol and propylene glycol, either alone or in combination. Additionally, enhancement of the buffer viscosity can also improve retention of echogenicity. In this regard, it has been found that some lipid bubble formulations containing about 2% to about 20% v/v load of viscosity enhancer resulted in retaining greater than 90% of their echogenicity for up to a month. These formulations were monitored over 6 months and were able to retain their echogenicity during this timeframe. In this regard, the liquid environment of the lipid bubble encapsulating a gas or the mixture containing the lipid bubble encapsulating the gas can further comprise the viscosity enhancer.

[0069] The gas encapsulated by the lipid bubble is not only useful in diagnostics but can serve as a therapeutic or theranostic agent to certain ailments. It is possible for the encapsulated gas to be delivered to a specific region to treat diseases. Tumours are generally less well oxygenated due to their fast rate of growth, and this is known as tumour hypoxia. This hypoxia leads to resistance to radiotherapy and chemotherapy. It is known in the art that treatment of these hypoxic tumours with oxygen prior to radiotherapy and chemotherapy can improve a subject's response. It will be clear that the present lipid bubbles can be used to transport oxygen gas to a specific region to improve a subject's response to chemotherapy and radiotherapy. Therefore, a person skilled in the art will understand that the gas itself can be a therapeutic agent where the gas itself is delivered to the target area. [0070] In another embodiment, the lipid bubbles with encapsulated gas further comprise an active agent. The active agent is introduced during step a). By introducing active agents to/into the lipid bubbles, the active agent is prevented from being absorbed directly into the bloodstream. The active agents can be tethered to, or encapsulated by, the lipid bubble. This allows the active agent to be transported to a target area where the lipid bubble can release the active agent. The lipid bubble can be burst, or disrupted, using ultrasound waves of a certain frequency to release the active agent near the target area. Suitable active agents include chemoactive drugs, or genes. The lipid bubbles can also encapsulate agents for theranostic purposes, these agents include PET agents, MRI agents, and ultrasound contrast agents. An example of the PET contrast agent is Gallium and its salts, and an example of the MRI agent is Gadolinium and its salts. However, it will be appreciated by a person skilled in the art that these lists are not exhaustive lists but merely lists of suitable active agents and theranostic agents that can be encapsulated in the lipid bubble. [0071 ] Additional compounds can be used in step a) to remotely load charged drug molecules into the liposome. Suitable additional compounds include citric acid and ammonium sulfate. In this regard, once the liposome containing ammonium sulfate or citric acid are formed, dialysis is performed with a buffer at a temperature of about 2^QC to about 8^QC to remove any ammonium sulfate in the extraliposomal environment. The removed ammonium sulfate is replaced with the buffer. Dialysis is then performed at an elevated temperature, in the presence of the additional compound(s) in buffer (e.g., isosmotic phosphate buffered saline). During dialysis, the additional compound(s) migrates into the liposome and encounters the ammonium sulfate which renders the additional compound(s) insoluble and precipitates inside the liposome. As a result, the additional compound(s) can no longer freely migrate across the liposome and is effectively trapped in the liposome. Dialysis is performed again to remove any residual unencapsulated additional compound from the extraliposomal environment.

[0072] In another embodiment, the lipid bubble encapsulating the gas can further comprise a bioactive agent. The bioactive agent is also introduced during step a). The bioactive agent can form part of the lipid, and hence lipid bubble, and bind to a desired target area located on the tissue of a subject. Alternatively, the bioactive agent may coat the lipid bubble. The lipid bubble encapsulated gas will therefore remain at the target area until the encapsulating shell breaks down, is burst or disrupted to release its contents. Suitable bioactive agents include ligands, bioactive functional groups, or targeted antibodies. Preferable bioactive agents include macromolecules, and small molecule bioactive agents. Preferably, the bioactive agent is selected from the group consisting of genes, peptides, proteins, antibodies, anti-infective agents, anti-cancer agents or chemotherapeutic agents. It will be appreciated that the macromolecules may include compounds such as siRNA, plasmid DNA, small molecule agonists and antagonists, and/or targeted genome/gene editing constructs (e.g., CRISPR- Cas9, TALENS, Zinc-finger nucleases, etc.). These macromolecules can be delivered using a lipid bubble encapsulating a gas to a target area. As such, it will be appreciated by the person skilled in the art that the lipid bubbles encapsulating the gas can be used for gene and therapeutic peptide delivery applications.

[0073] Further to this, the force of the destruction of the bubble can also force the active agent into the pores of nearby tissue and cells to provide better delivery of the active agent. This improves the penetration of the active agent to the target area/tissue and increases the efficacy of the active agent. The force of this destruction is proportional to the amount of gas encapsulated, and so the greater the amount of gas encapsulated the more efficient the delivery and deposition of active agent into nearby cells and tissue.

[0074] It will be appreciated by a person skilled in the art that lipid bubbles encapsulating a gas with a smaller diameter are more desirable from a therapeutic perspective because they can permeate deeper into tissue through the enhanced permeation and retention (EPR) effect. This is particularly useful when the volume to be injected is limited, for example, drug delivery to the eye is typically limited to a maximum injection volume of 100 μΙ_. By improving the delivery and retention this problem is alleviated. [0075] In a second aspect the invention resides in a method of targeted delivery of a lipid bubble encapsulating a gas to a target area of a tissue in a subject, including the steps of:

a) injecting the lipid bubble encapsulating the gas into the tissue; and

b) subjecting the lipid bubble encapsulating the gas to an ultrasound stream,

[0076] It has been found that a lipid bubble encapsulating a gas can be delivered to a target area of tissue by subjecting the lipid bubble to an ultrasound stream. The inventors have found that the lipid bubble encapsulating the gas can undergo acoustic streaming in a fluid driven by the absorption of high amplitude acoustic oscillations. Preferably, the high amplitude acoustic oscillation is ultrasound.

[0077] Acoustic streaming is made possible by the gas encapsulated in the lipid bubble, and the size distribution of the lipid bubble is important to ensure that this acoustic streaming is reproducible and predictable. [0078] A person skilled in the art will understand that an advantage of the use of the lipid bubbles encapsulating a gas, produced by the method of the first aspect, lies in the increased amount of gas encapsulated by the lipid bubble.

[0079] In order to utilize acoustic streaming, the ultrasound stream should be directed generally toward the target area, wherein the lipid bubble is located in between the ultrasound probe and the target area. [0080] The known methods of delivering lipid bubbles with an active agent to a target area often utilize natural diffusion of the active agent to the surrounding area. In viscous liquids or regions of the subject, the diffusion of active agents is particularly ineffective and a high loading of the active agent is required so that a suitable amount is delivered to the target area. This is expensive, inefficient and can have adverse effects on the subject. By directing an ultrasound stream to the present lipid bubbles encapsulating the gas and the target area, it is possible to move the lipid bubbles efficiently and quickly to the target area. The use of the ultrasound stream allows for an active agent to be delivered closer to the target area and therefore the delivery of the active agent is greater. [0081 ] It will be appreciated that the active agent can be encapsulated by, tethered to, or injected with, the lipid bubbles.

[0082] In one embodiment, the lipid bubbles encapsulating the gas can be injected as a mixture with an active agent and then subjected to an ultrasound stream to deliver the active agent towards the desired target area whereby delivery of the active agent is driven by physical movement of the associated lipid bubbles. It will be appreciated that the lipid bubbles encapsulating a gas can be combined with an injectable. A non-limiting example of the injectable is saline solution or water. In this regard, the lipid bubbles encapsulating a gas may be diluted further in a similar biocompatible solvent just prior to administration, if required for dose titration purposes. However, it will be appreciated that certain applications can only have a limited volume of injectable substances. In this regard, it will be appreciated that the lipid bubble may be administered 'as prepared' without any dilution or an injectable.

[0083] In another embodiment, the target area of the tissue is located in the eye. For example, administering an active agent to the posterior of the eye is extremely difficult due to its location, neurosensory/cellular barriers and vitreous rheology. A typical method of administering an active agent to the posterior of the eye requires the injection of the active agent into the anterior of the vitreous humour and allowing it to diffuse through the vitreous humour. A control experiment was completed using a porcine eye, where the porcine eye was injected with rhodamine tagged lipid bubbles encapsulating a gas, allowed to diffuse for 60 seconds and snap frozen before the distribution of the rhodamine was determined (Figure 1 ). The porcine eye was monitored to determine the relative amounts of rhodamine in the eye (Figure 2). The porcine eye was separated into four quadrants, the anterior vitreous near injection site (AN), the anterior vitreous far from injection site (AF), the posterior vitreous near injection site (PN), and the posterior vitreous far from injection site (PF). In another experiment, the same rhodamine tagged lipid bubbles encapsulating the gas was injected into a porcine eye in the same area and subjected to an ultrasound stream directed from above the cornea towards the posterior of the eye (Figure 3) for 60 seconds. The eye was then snap frozen before the distribution of the rhodamine was determined (Figure 4).

[0084] In another experiment, the same rhodamine tagged lipid bubbles encapsulating the gas was injected into a porcine eye in the same area and subjected to an ultrasound stream directed towards the side of the eye (Figure 5) for 60 seconds. The eye was then snap frozen before the distribution of rhodamine was determined (Figure 6).

[0085] It is clear from these experiments that acoustic streaming of lipid bubbles encapsulating a gas can be used to deliver an active agent towards a target area. The results clearly show that there is a greater amount of rhodamine in the area in which the ultrasonic stream is directed compared with that when simple diffusion is relied upon. For instance, when the ultrasound stream is directed from above the cornea there is an increase in rhodamine located in the PN quadrant compared to the control experiment. In the experiment where the ultrasound stream was directed towards the side of the eye an increase was observed in the AF quadrant compared to the control experiment. Preferably, the target area is in the eye. More preferably, the target area is the posterior of the eye. [0086] The lipid bubbles encapsulating the gas can be injected at a number of injection depths. For instance, the lipid bubbles can be injected at any depth into the eye. Non-limiting injection depths include 4 mm and 8 mm. The length of the ultrasound application can be about 60 seconds. However, it will be appreciated by the person skilled in the art that different lengths of ultrasound application can be applied to achieve acoustic streaming. The dosage of lipid bubbles encapsulating a gas for intravitreal dose is suitably less than 100 μΙ_, more suitable from between about 10 μΙ_ and about 80 μΙ_, preferable from between about 20 μΙ_ and about 70 μΙ_, and most preferably between about 20 μΙ_ and about 50 μΙ_. It will be appreciated by the person skilled in the art that the dosage of the lipid bubble is not limited to the above listed values, and that the above listed values and ranges merely exemplify various dosage amounts. Additionally, it will be appreciated that greater doses of lipid bubbles encapsulating a gas may be used for other applications. [0087] The FM formulation was visible to the naked eye when injected into the bovine vitreous and could be readily visualized through the cornea (Figure 7D). Pars plana ultrasound applied to the eye was able to move the FM bolus in the direction of wave propagation (Figure 7E). Following removal of the ultrasound stimulus, the FM formulation was observed to have some degree of recoil and moved back towards the starting location of the bolus (Figure 7F). In contrast, a non-echogenic white emulsion was injected into a separate eye as a control to assess whether this formulation could be mobilized using identical ultrasonic parameters (Figure 7A). In the control, no migration was observed during (Figure 7B), or after removal of (Figure 7C), ultrasound application. Figure 7 shows dots that reference the location of the bolus in each stage. It should be clear that the FM formulation moved upon application of ultrasound whereas the non-echogenic emulsion did not.

[0088] Recoil was identified as a function of ultrasonic power used, placement of the probe on the eye as well as site of injection. It appeared that injection of the lipid bubbles bolus into the central vitreous (away from the lens or retinal walls) would minimize the impact of recoil and improve ultrasound responsiveness. The vitreous of a 'live' eye has a natural flow which migrates/moves slowly from the central anterior region to the posterior region of the eye and around the fringes back towards the front. In contrast, a freshly excised eye has a stagnant vitreous that is more resistant to flow. It is postulated that this was largely responsible for the observed 'recoil' effect in the excised eye, and this may be mitigated in a live functioning eye.

[0089] Fluorescence evaluations were attempted to enable the concurrent visualization of FF formulation and liposomes. All particles were successfully tracked using optical fluorescence (Figure 8). Figure 8 shows an image indicating the location of the bolus of the lipid bubbles before ultrasound ("-US"), and the migration of the bolus after the application of ultrasound ("+US"). It should be clear that migration of the bolus was observed when ultrasound is applied to the FF formulation. In contrast, ultrasound was unable to impact on the intravitreal distribution of dye or non-echogenic liposomes. Ultrasound significantly promoted movement of both FF (p<0.001 ) and FM formulations (p<0.0001 ) towards the target region.

[0090] Overall, fluorescence studies indicated that ultrasound administration caused a 15.0 ± 5.1 % and 17.8 ± 1 .8% net increase in migrated distance of dye (from the lens to the posterior pole) when it was incorporated into the FF and FM formulations, respectively. The results of these migration studies are shown in Figure 9 in a graphical representation showing the distance migrated by the bolus towards the posterior pole of the eye following the application of ultrasound. It should clear that the use of the FF and FM formulations resulted in significant increase in migration distance.

[0091 ] In one embodiment, the step of subjecting the lipid bubble encapsulating the gas to an ultrasound stream comprises subjecting the lipid bubble encapsulating the gas to multiple ultrasound pulses. It is postulated that multiple pulses of ultrasound separated by ultrasound-free intervals to allow for tissue recovery, or through use of a dispersed ultrasound array that accommodates for axial and lateral blur, may allow for a gentler administration protocol.

[0092] To this end, three 60-second ultrasound pulses were separated by 2 minutes of no ultrasound application to the lipid bubbles. No significant/very little increase in migration distance was observed following the first ultrasound pulse. However, when the anterior and posterior hemispheres of the eye were considered separately, it was observed that multiple pulses increased bulk transfer of the formulation to the posterior hemisphere of the vitreous humour. Region of interest analysis indicated that following bolus administration, <10% of the administered dose was present in the posterior half of the vitreous humor in all cases. While a single pulse of ultrasound resulted in 28.6 ± 8.5% of the dose transferring to the posterior region, this could be further increased after multiple pulses with 45.1 ±1 1 .4% and 47.8 ± 15.2% of the dose having reached the posterior vitreous after 2 and 3 pulses, respectively (Figure 10). Figure 10 also shows a graphical representation of distance migration of the present lipid bubbles compared to convection. It should be apparent that the presence of the lipid bubbles significantly increased the distance migrated.

[0093] In a third aspect, the invention resides in a method of delivery of the contents of a lipid bubble encapsulating a gas to a tissue in a subject, including the steps of:

a) injecting the lipid bubble encapsulating the gas to the subject systemically;

b) subjecting the tissue to ultrasound,

[0094] It is known in the art that lipid bubbles can be used for the delivery of active agents. As mentioned previously, the lipid bubbles encapsulating a gas can encapsulate active agents to prevent them from dissolving in the bloodstream. The active agent is as substantially described in the first aspect of the invention.

[0095] The ultrasound can be directed in a standard manner to the tissue of interest and so the lipid bubbles circulate through the bloodstream until they enter the region being subjected to ultrasound and are disrupted to release the active agent. The bursting of a lipid bubble can be achieved by subjecting the lipid bubble to an ultrasound stream of a particular frequency and intensity/power. Therefore the lipid bubble can be burst at a specific area to deliver the active agent to said area. As mentioned previously, the encapsulated gas itself can be a therapeutic agent and so the lipid bubble can be disrupted to deliver the gas to the tissue.

[0096] Furthermore, the lipid bubbles can also have bioactive agents tethered to, or coated on, the lipid bubble which can bind to the target area. The bioactive agents are as substantially described in the first aspect of the invention. This directs the lipid bubble to or keeps it near the target area at which point it can be disrupted by ultrasound. It will be appreciated by a person skilled in the art that bioactive agents can be used in conjunction with ultrasound to provide enhanced targeting. It will be apparent to the person skilled in the art that the advantages discussed hereinbefore apply equally to the third aspect of the invention. In one embodiment, the lipid bubbles can be co-formulated with an active agent. The co-formulation comprises the lipid bubbles mixed with an active agent. The co-formulation may include non-covalent surface coatings to the outer lipid shell (e.g., through ionic interactions and/or hydrogen bonding), and/or covalent surface tethering (e.g., through chemical ligation to the lipids/PEG composition of the lipid bubble). In another embodiment, the lipid bubbles may be tagged with the active agent. A lipid bubble that is tagged with the active agent refers to the active agent being within the lipid bubble. A lipid bubble tagged with the active agent may be generally described as incorporation of an active agent within the lipid shell of the lipid bubble. This incorporation may be a 'non-covalent incorporation'.

[0097] As previously mentioned, the lipid bubbles encapsulating a gas can further include compounds such as siRNA, plasmid DNA, small molecule agonists and antagonists, and/or targeted genome/gene editing constructs (e.g., CRISPR-Cas9, TALENS, Zinc-finger nucleases, etc.). These compounds can be delivered using the lipid bubble encapsulating a gas to a target area. As such, it will be appreciated by the person skilled in the art that the lipid bubbles encapsulating the gas can be used for gene and therapeutic peptide delivery applications.

[0098] In a fourth aspect, the invention resides in a method of delivery of an active agent within a topical composition comprising a lipid bubble encapsulating a gas to a tissue of a subject, including the steps of

c) applying the topical composition to the subject; and

d) subjecting the topical composition to an ultrasound stream,

to thereby deliver the active agent into the tissue of the subject, and wherein the lipid bubble encapsulating the gas is produced by the method of the first aspect. [0099] In one embodiment, the topical composition is applied to an area of the subject selected from the eyes, ears, inside of the nose, inside of the mouth, tongue, lips and cervix. In one embodiment, the topical composition is applied to the skin. In another embodiment, the topical composition is applied to the eye. In another embodiment, the topical composition is applied to a site of infection. [00100] Foot infections are a common and disabling complication of diabetes and can cause high rates of morbidity and mortality. Diabetes is also a significant contributor to the cost burden of healthcare. Typically, extended courses of systemic antibiotics are required to treat diabetic foot infections. Infections may also develop antibiotic resistance, which adds to the cost burden of healthcare. [00101 ] A diabetic foot may compromise vascularisation and so many patients may be receiving inefficient tissue penetration at the infection site and thus sub- optimal therapy. It would be advantageous to refine the treatment methods to improve the efficacy of antibiotics. Such refined treatments could also be used in enhancing the efficacy of other topical compositions. [00102] It is envisaged that a lipid bubble encapsulating a gas can be used to promote active agent delivery. It is postulated that transdermal delivery of drugs could be enhanced by the use of the present lipid bubbles. As shown in Liao et al. (Ultrasound in Med. & Biol., (2016) Vol. 42, No. 8, pp. 1976-1985), the combination of ultrasound and microbubbles can be utilized to enhance transdermal permeation of an active agent. Furthermore, as shown in Thakur et al. (Thakur SS, Ward MS, Popat A, Flemming NB, Parat M-O, Barnett NL, et al. (2017) Stably engineered nanobubbles and ultrasound - An effective platform for enhanced macromolecular delivery to representative cells of the retina. PLoS ONE 12(5): e0178305. https://doi.Org/10.1371 /journal.pone.0178305), nanobubbles and ultrasound can be used in enhancing macromolecular permeation through three distinct cell types/bodies comprising the retina. In this regard, a topical composition comprising a lipid bubble encapsulating a gas and one or more active agents can be topically applied to a subject and cavitated using disruptive ultrasound. As previously mentioned, the force of the destruction of the lipid bubble can force the active agent into the wound or skin and therefore improve penetration and delivery of the active agent. Alternatively, it is envisaged that the topical composition can be subjected to an ultrasound stream to simply move the active agent across barriers (e.g., biofilms), and towards the desired tissue.

[00103] The lipid bubble encapsulating a gas is substantially as described in any of the previous aspects of the invention. Topical compositions are typically viscous so that the composition does not drip off the subject, as such the topical composition can further comprise viscosity enhancers and the additional components mentioned hereinabove. In an embodiment, the topical composition further comprises a rheology modifier. Non-limiting examples of the rheology modifier include carbopol and carbomer. In an embodiment, the carbomer is carbomer 941 . In one embodiment, the topical composition further comprises one or more active agents.

[00104] A viscosity enhancer trialed with the topical composition was carbomer 941 . Various concentrations of carbomer 941 were trialed to identify the optimal gel properties. It was found that 0.1 % w/v of carbomer 941 formed an easily pourable composition. An increase of carbomer 941 to 0.5% w/v resulted in a minimal gel-like substance. The topical compositions comprising carbomer 941 in a concentration of 1 .0% w/v and 1 .5% w/v formed a viscous gel, holding its structure whilst maintaining its fluidity. In one embodiment, the amount of viscosity enhancer in the topical composition is suitably between about 0.1 % and about 10%, more suitably between about 0.1 % and about 5%, preferably between about 0.1 % and about 2%, and most preferably selected from the group consisting of 0.1 %, 0.5%, 1 % and 1 .5% or therebetween. It will be appreciated that the amount of viscosity enhancer in the topical composition may vary depending on the varying components in the topical composition, and the type of viscosity enhancer utilized. [00105] Furthermore, the topical composition comprising carbomer 941 in an amount of about 1 .0% exhibited shear-thinning behavior which is ideal for the purpose of a topical wound gel because it will flow easily when sheared and remain viscous (at the site of application) when the shear stress is removed.

[00106] Gentamicin could then be added to the topical composition. The pH of the topical composition was maintained, or subsequently adjusted, to a pH of 5.5 to ensure that the gentamicin was successfully incorporated into the topical composition. Furthermore, this pH maintained the viscosity at a suitable level. Furthermore, this pH also closely mimics the slightly acidic nature of the skin.

[00107] It will be appreciated that the above steps can be completed in any order. For instance, the lipid bubbles could be incorporated into a composition comprising carbomer 941 and gentamicin.

[00108] The active agent can be incorporated into the lipid bubble encapsulating the gas as either a co-formulation, or part of the lipid bubble, or encapsulated by the lipid bubble. In the case where the topical composition is a co-formulation the lipid bubbles encapsulating the gas can be mixed with a suitably viscous compatible medium comprising the active agent.

[00109] The topical composition may comprise the lipid bubble encapsulating the gas in an amount suitably between about 0.01 % and about 20%, more suitably between about 0.1 % and about 10%, preferably between about 0.1 % and about 5%, more preferably between about 1 .5% and about 5%, and most preferably selected from the group consisting of about 0.5%, about 1 %, about 3% and about 5% or therebetween by volume of the topical composition. Additionally, a microscopy image of the topical composition comprising 1 % v/v of the lipid bubble encapsulating a gas was taken to confirm the presence of the lipid bubbles encapsulating a gas (Figure 16). It should be clear that the topical composition comprises the lipid bubbles encapsulating the gas. [001 10] Various lipid bubble concentrations (0.5, 1 , 3 and 5% v/v) were incorporated into a 0.1 % w/v carbomer composition. Imaging was performed on these compositions and all compositions comprising the lipid bubbles were found to be echogenic (Figure 17, (A) 0%, (B) 1 %, (C) 3%, (D) 5% v/v lipid bubbles with 0.1 % w/v carbomer). Some of the images have larger regions of white noise due to the presence of air bubbles being incorporated during the mixing process. These air bubbles could be eliminated by refrigerating the gel overnight.

[001 1 1 ] A number of topical compositions comprising 1 % v/v lipid bubbles and various amounts of carbomer were prepared to determine the effect of viscosity on the echogenicity of the topical composition. These topical compositions contained (A) 0% w/v carbomer, (B) 0.1 % w/v carbomer, (C) 0.2% w/v carbomer 941 , (D) 0.3% w/v carbomer, (E) 1 %w/v carbomer, and (F) 1 .5% w/v carbomer. Imaging was performed on these compositions and topical compositions (B)-(F) were shown to be echogenic (Figure 18), It should be clear that the viscosity is an important aspect of the topical application.

[001 12] Different concentrations of lipid bubbles (1 %, 3% and 5% v/v) were incorporated into the 0.1 % w/v carbomer composition and exposed to various ultrasound settings. The range of frequencies trialed was either 1 MHz or 3 MHz at the highest intensity obtainable from the device (i.e. 2.5 W/cm²). A number of ultrasound parameters were tested, however the optimal conditions were found to be one minute of continuous ultrasound exposure at a frequency of 1 MHz and a power intensity of 2.5 W/cm² (Figure 19). Figure 19 shows the effect of exposing a topical compositions without lipid bubbles (A-C) and topical compositions with lipid bubbles (D-F) to continuous ultrasound for 2 minutes at 1 MHz, 2.5W/cm². The images before ultrasound are presented in (A) and (D), the effects of ultrasound are shown in (B) and (E), and after agitating the topical composition (C) and (F). Furthermore, it appears that the penetration depth of ultra is shown to be greater at 1 MHz compared to 3MHz. Figure 1 9 appears to show a path of white noise that is cleared by the ultrasound waves from the body of the composition, and a large reduction in echogenicity.

[001 13] To further assess the effect of disruptive ultrasound on the lipid bubbles encapsulating a gas, a topical composition comprising 1 % v/v of the lipid bubble encapsulating a gas was prepared. The topical composition was imaged to see echogenicity of the topical composition before being subjected to disruptive ultrasound (Figure 20). The topical composition was then subjected to disruptive ultrasound to burst the lipid bubbles encapsulating a gas and the resulting composition was imaged to see the echogenicity of the resultant composition (Figure 21 ). A comparison of Figures 20 and 21 clearly show that there is a large reduction in the echogenicity and thus the lipid bubbles encapsulating a gas. It is clearly shown that there is a noticeable void through the centre of the sample, and this provides a visual image of the applied path of disruptive ultrasound waves.

[001 14] Additionally, visible lipid bubbles could be observed in the topical composition. As such, the number of bubbles in a sample of the topical composition comprising the lipid bubbles encapsulating a gas could be counted (within a certain area) before and after disruptive ultrasound. In this regard, the amount of the topical composition (both disrupted and undisrupted) that was observed was 10 μΙ_. Additionally, there was no noticeable difference in morphology or size of the lipid bubbles encapsulating a gas. The control sample (topical composition not subjected to disruptive ultrasound) was found to have 1 14 bubbles in the 10 μΙ_ aliquot, whereas the topical composition subjected to disruptive ultrasound was found to have 12±15 bubbles (range 3-30 bubbles, n=3) in the 10 μΙ_ aliquot. As such, there was about an 89% reduction in bubble count following disruptive ultrasound.

[001 15] An agar-based barrier model was explored to provide an indication of penetration of a topical composition comprising lipid bubbles. In this regard, topical compositions comprising 1 % v/v carbomer, 1 % lipid bubbles and Rhodamine B was prepared. The rhodamine was infused into the topical composition. The abovementioned topical composition was compared to a composition comprising 1 % v/v carbomer and Rhodamine B without lipid bubbles as a control test. It was found that the presence of the lipid bubbles impacts on the penetration of Rhodamine B dye into the agar barrier phantoms (Figure 22). As such, it is postulated that the lipid bubbles can be used to target biofilms. A clinical advantage of the present lipid bubbles may be the treatment of diabetic leg ulcers or other infected chronic wounds, where friction and skin abrasion with a cream may not be a desirable cutaneous delivery method for an antibiotic formulation. [001 16] The stability of the topical compositions comprising lipid bubbles was assessed over a period of one month. The topical composition was still echogenic after one month from preparation (Figure 23). Figure 23 shows echogenicity of the topical composition on (A) the day of preparation of the topical composition; and (B) 1 day, (C) 2 days, (D) 7 days, (E) 15 days, (F) 21 days and (G) 28 days after preparation. These results indicate that the topical composition show echogenicity over time.

EXAMPLES

Sample preparation of the lipid bubbles

[001 17] A mixture of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1 ,2- distearoyl-sn-glycero-phosphoethanolamine-A/-[amino(polyethylene glycol)-2000] in a 94:6 ratio was prepared at a lipid concentration of 4 img/mL in chloroform. The chloroform was evaporated to obtain a thin lipid film. Nitrogen gas was blown over the film to ensure complete removal of solvent. The lipid film was rehydrated in phosphate buffered saline for 15 minutes at 51 °C. The resultant suspension was then sonicated or extruded to form the lipid bubbles. In the case of sonication, the suspension was transferred to a thin walled test tube and subjected to sonication (43 ± 2 kHz, sweep bandwidth with 20Hz pulses) at 51 °C for 10 minutes (Soniclean™ Ultrasonic Cleaner 160HD, 220/240V, 50/60 Hz, 170W and pulse swept power: 70W). In the case of extrusion, the formulation was serially extruded through polycarbonate membranes (5 x 800 nm, 5 x 400 nm, 5 x 200nm and 5 x 100nm) under N₂ gas using LIPEX™ extruder (Northern Lipids Inc., Burnaby, BC).

Sample preparation of a gas-containing lipid bubble

[001 18] 2.5 imL of the suspension was transferred to a gas tight headspace vial and crimp sealed. Following air evacuation of the head space, 15 imL PFP gas at 10 psi was added to the crimp vial. The vial was vortexed using a Benchmark Scientific MultiTherm Shaker at 1500 RPM and 51 °C for 15 minutes to prepare gas-containing liquid bubbles.

Sample preparation of lipid bubble encapsulating a gas

[001 19] The crude lipid bubbles separated into a liquid phase and a foam phase, and following rigorous mixing 1 .2 mL of formulation (corresponding to 0.9 mL of liquid and 0.3 mL of froth) was drawn up using a 1 mL gastight syringe. Particle size was standardized using gas tight extrusion (Avanti^® Mini-Extruder, Avanti^® Polar Lipids, Alabaster, AL, USA), at a temperature of 51 ^QC, by passing the formulation 31 times through 400 nm polycarbonate membranes prior to snap cooling and storage at 2-8 °C.

Sample snap cooling

[00120] Directly following heated extrusion the formulations were transferred into: a) head space vials (9mL) containing -2.5 mL of the lipid bubbles encapsulating a gas, the vials were sealed and air evacuated, with the head space replaced with perfluoropropane gas (PFP) or b) shell vials (1 mL) containing 1 mL of the lipid bubbles encapsulating a gas with a blanket of PFP blown over prior to capping.

[00121 ] The vials were slotted into a pre-cooled (2 ^QC - 8 ^QC) custom-made cooling block where the dimensions of each cylindrical orifice (two sizes) accommodated either the 9 mL and 1 mL vials. The cooling block ensures a snug/tight fit to each vial, ensuring maximum and rapid head transfer into the pre-cooled block, and this leads to efficient snap-cooling of the lipid bubbles encapsulating a gas. This also prevents any likelihood of freezing which would compromise the lipid bubble integrity.

[00122] The rate of snap cooling was measured by monitoring the temperature of the 2.5 mL of lipid bubbles in head space vials that sat within the custom-made cooling block. The final formulation storage temperature of less than 8^QC was achieved within 386.7±1 1 .1 seconds (n=3). Shown in Figure 12 is the declining temperature of the lipid bubbles encapsulating a gas in relation to time in the pre-cooled block. Stability Results

[00123] In order to compare stability results, lipid bubbles encapsulating the gas with a mean lipid bubble diameter of about 200 nm and 400 nm were stored under the same conditions as a sample of crude lipid bubble encapsulating the gas. As mentioned above, the 'Extruded lipid bubbles V were the lipid bubbles that were subjected to the heat extrusion through 400 nm membranes. The 'Extruded lipid bubbles 2' were the crude lipid bubbles that were passed through small 450 nm syringe filters at room temperature. The 'crude lipid bubbles' were those which were not passed through a gastight membrane. The polydispersity index and Z-average particle size were tested after 4 weeks and 8 weeks after storage. (Table 1 )

Extruded lipid Extruded lipid Crude lipid bubbles bubbles 1 bubbles 2

Z-ave PDI Z-ave PDI Z-ave PDI

(nm) (nm) (nm)

Day 1 212.3 ± 0.293 ± 379.2 ± 0.253 ± 606.4 ± 0.496 ±

15.9 0.030 14.1 0.010 132.4 0.096

Day 28 220.8 ± 0.284 ± 353.8 ± 0.232 ± 522.3 ± 0.470 ±

12.3 0.030 7.1 0.019 48.2 0.050

Day 56 218.4 ± 0.332 ± 391 .0 ± 0.258 ± 439.8 ± 0.388 ±

12.0 0.049 67.1 0.034 55.0 0.069

Table 1 - Stability Results [00124] It is clear from the results that the lipid bubbles that were subjected to gas tight membrane extrusion were able to retain their polydispersity and average particle size. It is also clear that there was little to no stabilization to form 1 -2 μιη diameter bubbles upon storage. It can be seen that the Z-average particle size of the lipid bubbles encapsulating a gas maintain their narrow size distribution compared to the crude lipid bubbles encapsulating a gas.

Echogenic profile [00125] The echogenic profile of the sonicated lipid bubbles encapsulating a gas were then compared to the echogenic profile of the lipid bubbles encapsulating the PFP prepared using the method of the present disclosure. The sonicated lipid bubbles were prepared according to the method of Suzuki et al. (Suzuki et al., J. Control. Release (2007) 1 17(1 ) pg. 130-136). The lipid bubbles with higher echogenicity are the white spots in Figure 1 1 , and it can be seen that that the echogenicity of the heat-vortexed lipid bubbles of the present invention is far greater than the echogenicity of the sonicated lipid bubbles encapsulating a gas.

Sample incorporation of Ciprofloxacin into the lipid bubble

[00126] Ciprofloxacin Hydrochloride, a known antibiotic belonging to the fluoroquinolone class, exerts its bactericidal effect by inhibiting enzymes required for DNA replication, transcription, repair, and recombination (i.e. topoisomerase II (DNA gyrase) and topoisomerase IV). A sample preparation of ciprofloxacin in a lipid bubble was prepared using the following method.

(Ciprofloxacin Hydrochloride Monohydrate)

[00127] DSPC and DSPE-PEG (molar ratio of 94:6; 1 1 mM) were dissolved in ethanol and transferred to a rotary evaporator to remove the organic solvent causing formation of a lipid film. The lipid film was then hydrated using ammonium sulfate solution (135 mM) at 65 ^QC to make a lipid solution containing large and polydisperse lipid bubbles.

[00128] The crude lipid bubbles were then extruded 10 times through a 800 nm PC membrane, and then 5 times through a 200 nm PC membrane to form nanosized («150 nm) lipid bubbles with a low polydispersity index (PDI ~ 0.07). The external ammonium sulfate buffer was then replaced with isosmotic saline (0.9% w/v) via dialysis (48 hr; 2-8 ^QC), which was performed such that no residual ammonium sulfate remained in the external buffer solution.

[00129] Following dialysis, ciprofloxacin HCI monohydrate (2 grams in powder form) was added to 2 mL of the formulation and remote loading was performed for 10 minutes at 65 °C (10 ^QC above the lipid transition temperature of DSPC). The formulation was snap-cooled in ice, immediately after remote loading, and cooled to 2-8 ^QC until supercharging (adding the gas) was performed.

[00130] Different loading times were examined to determine the best drug loading times. Figure 13 depicts loading efficiencies achieved with different loading times.

[00131 ] To introduce a contrast agent (perfluoropropane - C3F8) to the formulation, 1900 μΙ_ of the prepared formulation was transferred to a 9 mL crimp sealed vial, where the air was evacuated from the headspace by applying a vacuum for 3 minutes through a needle. Next, 25 mL of perfluoropropane (at 10 psi) was then injected into the vial headspace using a gastight syringe. The vial was then vortexed for 1 5 minutes (at 1500 RPM and 65 ^QC), after which the resulting solution («1 mL) was subjected to 31 times extrusion at 65 ^QC through a 400 nm PC filter membrane.

[00132] After extrusion, the sample was transferred into 1 mL shell vials (with no headspace), sealed and snap-cooled in ice water followed by storage in a custom made pre-cooled block (2-8 ^QC), and stored. Stability testing was conducted over 28 days.

[00133] To confirm echogenicity, 50 μΐ of supercharged, extruded and cooled formulation was added to 50 mL of isosmotic saline and imaging was performed using an Ellex Eye Cubed ultrasound unit. Figure 14 shows that the echogenic formulations create contrast, observed as white noise even after a prolonged period of time. This was an encouraging result because despite a 1000-fold dilution, appreciable echogenicity was still observed.

[00134] The ciprofloxacin suspension was monitored over 4 weeks and the results are shown in Figure 15. It should be clear that the ciprofloxacin concentration, lipid bubble size, and polydispersity of the resultant lipid bubbles did not change substantially during this time.

Sample incorporation of Doxorubicin into the lipid bubble

[00135] DSPC and DSPE-PEG (molar ratio of 94:6; 21 .5 mM) were dissolved in ethanol and transferred to a rotary evaporator to remove the organic solvent resulting in the formation of a lipid film. The lipid film was then hydrated using an ammonium sulfate solution (135 mM) at 65 ^QC to make a lipid solution containing large and polydisperse lipid bubbles.

[00136] The crude lipid bubbles were sonicated using a probe sonicator at an amplitude of 80 to form nanosized lipid bubbles (<155 nm) with a low polydispersity index (PDI ~ 0.3). The external ammonium sulfate buffer was replaced with isosmotic phosphate buffered saline (150 mM, pH adjusted to 6.5 using 0.1 N HCI) via dialysis (48 hr; 2-8 ^QC), which was performed such that no residual ammonium sulfate remained in the external buffer solution. Following dialysis, doxorubicin HCI (4 grams in powder form) was added to 2 imL of the formulation and remote loading performed for 130 minutes at 65 °C (10 ^QC above the lipid transition temperature of DSPC). The formulation was snap-cooled in ice water, immediately after remote loading, and kept at 2-8 ^QC until supercharging was performed.

[00137] To introduce the contrast agent (perfluoropropane - C₃F₈) to the formulation, 1900 μΙ_ of liposomal doxorubicin prepared earlier was transferred to a 9 imL crimp sealed vial, where the air was evacuated from the headspace by applying a vacuum for 3 minutes. 25 imL of perfluoropropane (at 10 psi) was then injected into the vial headspace using a gastight syringe. The vial was immediately vortexed for 15 minutes (at 1500 RPM and 65 ^QC). [00138] After extrusion, the sample was transferred into 1 imL shell vials (with no headspace), sealed and snap-cooled in ice water followed by storage in a custom made pre-cooled block (2-8 ^QC), and stored.

[00139] Multiple samples were analyzed to determine the concentration of doxorubicin, and the drug loading efficiency was also determined (Table 2). Concentration Final Concentration

j Dox-NBs Calculated ^g/mL) without ^g/mL) with % Drug Loading

AUC

I (2mg/mL) Cone. final correction theoretical correction Efficiency factor factor (0.95)

i Sample 1 | 2117830 1881.565 2051.257837 1948.694945 97% I j Sample 2 ί 2204943 1957.119 2157.113436 2049.257764 102% j i Sample 2 j 2278867 2021.233 2181.93971 2072.842724 102%x = doxorubicin; NB = nanobubbles; AUC = Area under curve.

Table 2: Doxorubicin concentrations and loading efficiency of Doxorubicin and the lipid bubble encapsulating a gas

Sample preparation of rhodamine tagged DPPC lipid bubble and the co- formulation of rhodamine and DPPC lipid bubble

[00140] Hydrophilic (co-formulation) and lipophilic (rhodamine tagged) variants of rhodamine with the lipid bubbles described hereinabove were prepared using the following method.

[00141 ] The lipid bubbles were prepared as described hereinabove. In the lipophilic variant, lipophilic 5-carbonyl-tetramethylrhodamine N-succinimidyl ester (5-TAMRA) was incorporated with the lipid mixture at 2 mol% and the remaining steps were followed. In the hydrophilic variant, hydrophilic rhodamine B dye was mixed with the lipid bubbles encapsulating a gas at a concentration of 2 mg/mL prior to injection. Optical fluorescence evaluations

[00142] Optical fluorescence evaluations were employed to visualize intravitreal propagation of rhodamine containing FM formulation, FF formulation and liposomes when compared to unbound rhodamine. In this regard, particle migration in the presence and absence of ultrasound was observed. Migration distance was calculated by mapping co-ordinates of the leading edge of the particle bolus in relation to fixed sections of the eye before and after ultrasound administration. The distance between co-ordinates was converted into a length in millimeters by using an object of known dimensions in the image for reference. Region of interest evaluations were also carried out by analyzing the relative degree of dye fluorescence in anterior and posterior regions of the vitreous humour before and after ultrasound exposure.

Sample topical composition comprising a lipid bubble encapsulating a gas

[00143] Carbomer 941 was dispersed in deionised water and mixed using a magnetic stirrer until a uniform gel was formed. The pH of the gel was increased to 5.5 by adding approximately 0.4 g of sodium hydroxide (NaOH) for every gram of carbomer 941 and the resulting gel was mixed using a magnetic stirrer. The gel was left to hydrate overnight in the fridge (2 - 8 °C) to ensure the carbomer was well hydrated and to eliminate any air bubbles introduced from the mixing process.

[00144] If the desired topical composition comprised gentamicin then gentamicin was added to the abovementioned topical composition, and the resultant mixture was mixed using a magnetic stirrer. If the desired topical composition comprised rhodamine B then rhodamine B was added to the abovementioned topical composition, and the resultant mixture was mixed using a magnetic stirrer.

[00145] The desired amount of lipid bubbles were added to the abovementioned composition, and stirred at 400 RPM for 5 minutes.

Bolus migration evaluated using a snap freezing ex vivo porcine eye [00146] Ultrasound was unable to impact the movement of intravitreally injected rhodamine dye in the porcine model in the absence of lipid bubbles, this observation being apparent in both fresh and aged (liquefied vitreous) eyes. Subsequently, the co-formulation approach was evaluated whereby a mixture of unbound rhodamine dye and FF formulation was injected and followed by administration of ultrasound. Ultrasound clearly impacted the migration of injected lipid bubbles in fresh eyes. In the co-formulation approach, ultrasound administration resulted in the dye and FF lipid bubbles moving (Figure 24). In this instance, pars plana administration of the ultrasound stimulus resulted in significant migration of the rhodamine dye from the AN region to the AF region (p<0.05). [00147] With ultrasound clearly able to impact FF formulation migration in fresh vitreous humour, the effect of ultrasound problem orientation was further examined. The lipid bubbles were tagged with 5-TAMRA to allow their distribution to be quantified. In the case of pars plana administration, the FF formulation were primarily moved into the AF region of the vitreous, with 56.5 ± 8.9% of the injected dose being transferred to this region (p<0.0001 ). In contrast, corneal administration resulted in the FF formulation more readily moving into the PN region, in this case 36.3 ± 4.2% of injected dose had transferred to this region (p<0.0001 , Figure 25). Neither of the investigative approaches elicited significant movement of the formulation to the PF region, which contained <10% of the administered formulation dose in all instances.

[00148] The above description of various embodiments of the present invention is provided for purposes of description to one of ordinary skill in the related art. It is not intended to be exhaustive or to limit the invention to a single disclosed embodiment. As mentioned above, numerous alternatives and variations to the present invention will be apparent to those skilled in the art of the above teaching. Accordingly, while some alternative embodiments have been discussed specifically, other embodiments will be apparent or relatively easily developed by those of ordinary skill in the art. Accordingly, this invention is intended to embrace all alternatives, modifications and variations of the present invention that have been discussed herein, and other embodiments that fall within the spirit and scope of the above described invention.

Claims

1 . A method for preparing a lipid bubble encapsulating a gas including the steps of:

a) forming a lipid bubble;

b) vortexing the lipid bubble in a liquid environment, under an atmosphere of the gas and at an elevated temperature to form a gas- containing lipid bubble; and

c) subjecting the gas-containing lipid bubble to snap cooling,

to thereby prepare a lipid bubble encapsulating a gas.

2. The method of claim 1 , wherein the liquid environment is selected from the group consisting of a buffer, glycerol, propylene glycol.

3. The method of claim 1 or 2, wherein the liquid environment comprises more than one liquid.

4. The method of any one of the preceding claims, wherein the gas is selected from the group consisting of air, a perfluorocarbon gas, and oxygen.

5. The method of any one of the preceding claims, wherein the gas comprises more than one gas.

6. The method of any one of the preceding claims, wherein the elevated temperature is from about 5 ^QC to about 15 ^QC above the phase transition temperature of the lipid forming the lipid bubble.

7. The method of any one of the preceding claims, wherein the snap cooling reduces the temperature of the gas-containing lipid bubble to about 2 ^QC to about 8 ^QC.

8. The method of any one of the preceding claims, wherein the snap cooling reduces the temperature of the gas encapsulated lipid bubble at a rate of about 25 ^QC/minute to 30 ^QC/minute for the first minute.

9. The method of claim 7, wherein the about 2 ^QC to about 8 ^QC temperature is reached within 7.5 minutes.

10. The method of any one of the preceding claims, wherein the vortex is generated at a speed of about 1000 RPM to about 2000 RPM.

1 1 . The method of any one of the preceding claims, further including a step, after step b), of subjecting the lipid bubble encapsulating the gas to gas tight membrane extrusion.

12. The method of claim 1 1 , wherein the membrane comprises pores with an average diameter selected from the group consisting of about 30 nm, about 50 nm, about 100 nm, about 200 nm, about 400 nm, about 800 nm, about 1 μιη, about 2 μιη, about 4 μιη, about 8 μιη, about 10 μιη, about 20μιη, about 50 μιη, about 100 μιη, about 200 μιη, about 400 μιη, and about 800 μιη.

13. The method of claim 12, wherein the lipid bubble encapsulating a gas is passed through the membrane more than once.

14. The method of any one of the preceding claims wherein the lipid bubble further comprises an additional component.

15. A lipid bubble encapsulating a gas when produced by the method of any one of the preceding claims.

16. A method of targeted delivery of a lipid bubble encapsulating a gas to a target area of a tissue in a subject, including the steps of: a) injecting the lipid bubble encapsulating the gas into the tissue; and

b) subjecting the lipid bubble encapsulating the gas to an ultrasound stream,

wherein the ultrasound stream is directed generally toward the target area, to thereby deliver the lipid bubble encapsulating the gas to the target area of the tissue in the subject, and wherein the lipid bubble encapsulating the gas is produced by the method of any one of claim 1 to claim 16.

17. The method of claim 16, wherein the target area of the tissue in the subject is located in the eye.

18. A method of delivery of the contents of a lipid bubble encapsulating a gas to a tissue in a subject, including the steps of:

b) subjecting the tissue to ultrasound,

wherein the lipid bubble encapsulating the gas is disrupted when it is subjected to the ultrasound thereby releasing its contents to the tissue, and wherein the lipid bubble encapsulating the gas is produced by the method of any one of claim 1 to claim 16

19. The method of claim 18, wherein the lipid bubble further encapsulates an active agent.

20. The method of claim 18 or claim 19, wherein the lipid bubble further comprises a bioactive agent on an exterior surface thereof.

21 . A method of delivery of an active agent within a topical composition comprising a lipid bubble encapsulating a gas to a tissue of a subject, including the steps of a) applying the topical composition to the subject; and b) subjecting the topical composition to an ultrasound stream,

to thereby deliver the active agent into the tissue of the subject, and wherein the lipid bubble encapsulating the gas is produced by any one of claim 1 to claim 16