INTRODUCTION:

Microbubbles are miniature gas bubbles of less than 10 micron diameter (equal to size of RBC). The microbubbles which mostly contain oxygen or air can remain suspended in water for extended period. Gradually, the gas within microbubbles dissolved into water and bubbles disappear due to their compressibility, they undergo volumetric oscillation and scatter. Much more energies than rigid spheres of same size would do. Coated microbubbles have advantage of being stable in body for a significant period of time; as shells serves to protect the gases of microbubbles from diffusion into blood stream.¹ The shell may be composed of surfactants, lipids, proteins, polymers, or combination of these materials. The interior gas is poor solvent for drug so that drug is loaded within or onto the shell.²Figure 1 shows biomedical structure of microbubble.

Received on 01.02.2013 Accepted on 20.03.2013

Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 2, Pg 56-65

Fig. 1 : Biomedical structure of microbubble¹

Fig. 2: Release of Agents From Regular Bubbles And Microbubbles¹

Microbubbles are sensitive to destruction by ultrasound. In process of destruction; the gas to liquid interface may be achieved linear speed of about 700 m/s. In such condition, closely located liposomes may be ruptured; and their contain release and/or deposited in surrounding cells and tissue.³Figure 2 shows release of agents from bubbles and microbubbles.

On application of low frequency ultrasound, these microbubbles start oscillating and undergo a process of cavitation resulting in bursting or break up of the bubble, drug molecules if incorporated within the bubble are released by this process and these are useful in drug delivery.⁴ Two factors which are taken into account for drug delivery are:

1. Incorporation of drug into the microbubbles

2. Drug release from microbubbles: Drug molecules can be incorporated in a variety of ways within the microbubble as follows

a. Drug molecules can be incorporated within the bubble

b. Drug molecules can also be incorporated within the bubble membrane or shell material of the microbubble.

c. Drugs can also be attached to the shell of the microbubble (for eg. by non covalent bonds)

d. These can also be attached to the microbubble surface via a ligand (for eg. avidin-biotin complex).

e. Also if the microbubble is made up of multiple layers it can also be incorporated within the various layers of this microbubble.^5-7

Composition of microbubbles:

Microbubbles basically composed of three phases

A. Gas Phase

B. Shell Materials

C. Outermost Liquid or Aqueous Phase

D. Other Components

A.Gas Phase:

The gas phase can be a single gas or a combination of gases can be used. Combination gases are used to cause differentials in partial pressure and to generate gas osmotic pressures which stabilize the bubbles. When a combination of gases is used two types of gases are involved one is the Primary Modifier Gas also known as first gas. Air is preferably used as primary modifier gas, sometimes nitrogen is also used as first gas. The other gas is Gas Osmotic Agent also known as second gas; it is preferably a gas that is less permeable through the bubble surface than the modifier gas. It is also preferable that the gas osmotic agent is less soluble in blood and serum.^8,9

B.Shell Material:

The shell material encapsulates the gas phase. It plays a major role in the mechanical properties of microbubble as well as diffusion of the gas out of the microbubble.¹⁰

Various types of shell material that can be used are protein shell, surfactant shell, lipid shell, polymer shell, polyelectrolyte multilayer shell.

Fig. 3: Components of Microbubbles

Fig.4: Various Layers In Microbubble Composition Protein Shells

Albumin shelled microbubbles were a pioneering formulation used in contrast ultrasound imaging. They paved the way for several subsequent formulations that could pass the lung capillaries and provide contrast in the left ventricle of the heart. The first albumin microbubble formulation was prepared by GE healthcare USA which is approved by US Food and Drug administration.¹¹

Albumin-coated microbubbles are formed by sonication of a heated solution of 5% (w/v) human serum albumin in the presence of air. During sonication, microbubbles of air are formed which become encapsulated within a 15-nm thick shell of aggregated albumin. Heating is necessary to denature the albumin prior to sonication and facilitate encapsulation. Biochemical analysis suggested that the shell is a monomolecular layer of native and denatured albumin in multiple orientations.¹² The albumin shell is held together through disulfide bonds between cystein residues formed during cavitation.¹³Covalent cross-linking may explain the relative rigidity of albumin shells observed during ultrasonic insonification.¹⁴

Several proteins other than albumin have been used to coat microbubbles. This is not surprising given the amphipathic nature of many proteins, which makes them highly surface active. Disulfide bridging occurs between thiol groups found in cystein amino acid residues, which are present on most proteins.¹⁵

Surfactant Shells:

Microbubbles stabilized by mixtures of the synthetic surfactants SPAN-40 and TWEEN-40 were formulated by Wheatley et al.^16,17The SPAN/TWEEN solution was sonicated in the presence of air to form stable microbubbles. Using a Langmuir trough, they were able to establish the correct ratio of SPAN to TWEEN (roughly 1:1) to use for maximum film stability. Interestingly, they showed that surfactant derived from sonicated microbubbles was more stable (i.e., was capable of reaching higher collapse pressures on the Langmuir trough) than that used in the precursor solution, indicating that the sonication process modified the surfactant to form a more stable film.¹⁷

Dressaire et al.¹⁸ reported on stable microbubbles formed from sucrose stearate (mono- and di-ester) formed by a blending process at 70 °C in 75 wt% glucose syrup. These microbubbles were stable in suspension for over a year and showed remarkable polygonal domains on their surface. These microbubbles were not stable upon dilution, and therefore have limited biomedical utility.

Lipid Shells:

Lipid-coated microbubbles are one of the most interesting and useful formulations used for biomedical imaging and drug delivery. The lipid shell is inspired by nature, as stable microbubbles found ubiquitously in the oceans and fresh waters of Earth are known to be stabilized by acyl lipids and glycoproteins.¹⁹ The lipid shell of a microbubble is also bio inspired, as it mimics the remarkable stability and compliance of lung surfactant

as it mimics the remarkable stability and compliance of lung surfactant.²⁰

Lipid shells have several advantages. Phospholipids spontaneously self-assemble into a highly oriented monolayer at the air-water interface, such that their hydrophobic acyl chains face the gas and their hydrophilic head groups face the water. Thus, the lipid monolayer will form spontaneously around a newly entrained gas bubble, just as for surfactants and proteins. Saturated di-acyl phospholipids are capable of laterally compressing within the monolayer plane to reach a very low surface tension when below their main phase transition temperature, which is the temperature at which the membrane transforms from a crystalline or ‘gel-like’ state to a liquid-crystalline or ‘fluid-like’ state. This is important, because surface tension at the curved interface induces a Laplace overpressure which, in turn, forces the gas core to dissolve.²¹ Thus; the low surface tension reached by the lipid monolayer stabilizes the microbubble.²²

The lipid molecules are held together by ‘weak’ physical forces, without chain entanglement, which makes the shell compression during ultrasound insonification. So, lipid-coated microbubbles have exhibited favorable ultrasound characteristics.^{14, 23-26}

Polymer Shell:

The term “polymer microbubble” is stabilized by a thick shell comprising cross-linked or entangled polymeric species. The bulk nature of the polymer shell makes it more resistant to area compression and expansion than its lipid and albumin counterparts, which reduces the echo-genicity and drug delivery activity. For example, polymer microbubbles have been observed to fracture during insonification, thereby releasing their gas core via extrusion through the shell defect.²⁵ The resulting gas bubble was unstable and rapidly dissolved according to the classical Epstein and Plesset equation.²¹ The shell, on the other hand, remained intact and often propelled away from the gas core; this effect may be useful for drug delivery.

In 1990, Wheatley et al.²⁷ reported on a new polymer shelled microbubble, in which the shell was formed by the ionotropic gelation of alginate. The microbubbles were formed by concentric jets of air and alginate solution that were sprayed into a reservoir. The alginate adsorbed to the gas/liquid interface. Microbubble diameters ranged between 30 and 40 μm and were therefore too large for intravenous administration.

In 1997, Bjerknes et al.²⁸ described a method for making microbubbles encapsulated by a proprietary double-ester polymer with ethylidene units using an emulsification, solvent evaporation method. The polymer microbubbles had a broad size distribution ranging from 1– 20 μm diameter. The polymer shell was typically 150–200 nm thick.

In 2005, Cavalieri et al.²⁹ described a method to prepare microbubbles coated with poly (vinyl alcohol) (PVA). PVA microbubbles were prepared by chemical cross-linking at the air/water interface during high-speed stirring (8000 RPM) of an acidic solution of telechelic PVA. The mean diameter was approximately 6 ± 1 μm. The shell thickness could be decreased from 0.9 to 0.7 μm by decreasing the operating temperature from room conditions to 4 °C. PVA microbubbles had a shelf life of several months and are capable of carrying hydrophobic drugs, charged polymers (e.g., DNA) and targeting ligands.

Polyelectrolyte Multilayer Shells:

A new class of polymer-surfactant shell hybrids was recently introduced that involves polyelectrolyte multilayer (PEM) shells on preformed microbubbles. The preformed microbubbles are coated with a charged surfactant or protein layer, which serves as a substrate for PEM deposition. The layer-by-layer assembly technique is used to sequentially adsorb oppositely charged polyions to the microbubble shell.

C. Aqueous or Liquid Phase:

The external, continuous liquid phase in which the bubble resides typically includes a surfactant or foaming agent. Surfactants suitable for use include any compound or composition that aids in the formation and maintenance of the bubble membrane by forming a layer at the interphase. The foaming agent or surfactant may comprise a single component or any combination of compounds, such as in the case of co surfactants.³⁰

Eg: Block copolymers of polyoxypropylene, polyoxyethylene, sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acid esters and their salts, dodecyl poly (ethyleneoxy) ethanol, etc.

• Nonionic Surfactants: Polyoxyehylene polyoxypropylene copolymers Eg. Pluronic F-68, polyoxyethylene stearates, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerolpolyethylene glycol oxystearates, glycerol polyethylene glycol ricinoleate etc.

• Anionic Surfactants: Fatty acids having 12 -24 carbon atoms Eg. Sodium Oleate.

D. The Other Components:

The various other components that may be incorporated in the formulation include osmotic agents, stabilizers, chelators, buffers, viscosity modulators, air solubility modifiers, salts and sugars can be added to fine tune the microbubble suspensions for maximum shelf life and contrast enhancement effectiveness. Such considerations as sterility, isotonicity and biocompatibility may govern the use of such conventional additives to injectable compositions.³¹

METHODS TO PREPARE MICROBUBBLES:^32-35

The various methods that can be used for the preparation of these microbubbles include:

1) Cross Linking Polymerization

2) Emulsion Solvent Evaporation

3) Atomization and Reconstitution

4) Sonication

1) Cross Linking Polymerisation:

In this a polymeric solution is vigorously stirred, which results in the formation of a fine foam of the polymer which acts as a colloidal stabilizer as well as a bubble coating agent. The polymer is then cross linked, after cross linking microbubbles float on the surface of the mixture. Floating microbubbles are separated and extensively dialyzed against Milli Q water.

2) Emulsion Solvent Evaporation:

In this method two solutions are prepared; one is an aqueous solution containing an appropriate surfactant material which may be amphilic biopolymer such as gelatin, collagen, albumin or globulins. This becomes the outer continuous phase of the emulsion system. The second is made from the dissolution of a wall forming polymer in a mixture of two water immiscible organic liquids. One of the organic liquids is a relatively volatile solvent for the polymer and the other is relatively non-volatile non-solvent for the polymer. The polymer solution is added to the aqueous solution with agitation to form an emulsion. The emulsification step is carried out until the inner phase droplets are in the desired size spectrum. It is the droplet size that will determine the size of the microbubble. As solvents volatilizes, polymer conc. in the droplet increases to a point where it precipitates in the presence of the less volatile non-solvent. This process forms a film of polymer at the surface of the emulsion droplet. As the process continues, an outer shell wall is formed which encapsulates an inner core of non-solvent liquid. Once complete, the resulting microcapsules can then be retrieved, washed and formulated in a buffer system. Subsequent drying, preferably by freeze-drying, removes both non-solvent organic liquid core and water to yield air filled hollow microbubbles.

In 2011, Steven J. Siegel described new method to prepare microbubbles by emulsion solvent evaporation. Oil-in-water emulsification processes are examples of single emulsion processes. Polymer in the appropriate amount is first dissolved in a water immiscible, volatile organic solvent (e.g., dichloromethane (DCM)) in order to prepare a single phase solution. The drug of particle size around 20–30 _m is added to the solution to produce a dispersion in the solution. This polymer dissolved drug dispersed solution is then emulsified in large volume of water in presence of emulsifier (polyvinyl alcohol (PVA) etc.) in appropriate temperature with stirring. The organic solvent is then allowed to evaporate or extracted to harden the oil droplets under applicable conditions. In former case, the emulsion is maintained at reduced or atmospheric pressure with controlling the stir rate as solvent evaporates. In the latter case, the emulsion is transferred to a large quantity of water (with or without surfactant) or other quench medium to diffuse out the solvent associated with the oil droplets. The resultant solid microspheres are then washed and dried under appropriate conditions to give a final injectable microsphere formulation.³⁶

3) Atomization and Reconstitution:

A spray dried surfactant solution is formulated by atomizing a surfactant solution into a heated gas this result in formation of porous spheres of the surfactant solution with the primary modifier gas enclosed in it. These porous spheres are then packaged into a vial; the headspace of the vial is then filled with the second gas or gas osmotic agent. The vial is then sealed, at the time of use it is reconstituted with a sterile saline solution. Upon reconstitution the primary modifier gas diffuses out and the secondary gas diffuses in, resulting in size reduction. The microbubbles so formed remain suspended in the saline solution and are then administered to the patient.

In 2006 Farook U et al. described novel method, based on co-axial electro hydrodynamic jetting, for the preparation of microbubble suspensions containing bubbles <10 _m in size and having a narrow size distribution. No selective filtration is necessary and the suspensions are produced directly by the process. To demonstrate the method, glycerol was used as the liquid medium, flowing in the outer needle of the co-axial twin needle arrangement and undergoing electro hydrodynamic atomization in the stable cone-jet mode while air flowed through the inner needle at the same time. At zero applied voltage a hollow stream of liquid flowed from the outer needle. When the applied voltage was increased, eventually the hollow stream became a stable cone-jet and emitted a micro thread of bubbles, which were collected in a container of glycerol to obtain microbubble suspensions.³⁷

4) Sonication:

Sonication is preferred for formation of microbubbles, i.e. through an ultrasound transmitting septum or by penetrating a septum with ultrasound probe including ultrasonically vibrating hypodermic needle. sonication can be accomplished in a number of ways for example: a vial containing a surfactant solution and gas in headspace of the vial can be sonicated through a thin membrane. Sonication can be done by contacting or even depressing the membrane with an ultrasonic probe or with a focused ultrasound “beam”. Once sonication is accomplished, the microbubble solution can be withdrawn from the vial and delivered to patient. Sonication can also be done within a syringe with a low power ultrasonically vibrated aspirating assembly on the syringe.

PROPERTIES OF MICROBUBBLES:

The ideal properties of microbubbles of divided into 2 classes.

1. Functional Properties^8,25,37-39

The functional properties are those which can render them useful for performing their various functions; Such as:-

a) Injectability: Since microbubbles are to be injected in the body to exert their effects they must be injectable.

b) Ultrasound scattering efficiency: As microbubbles become larger they become more ecogenic.

c) Biocompatibility: Microbubbles interact with vital organ of body at cellular levels they should be bio-compatibles.

d) Rheology: Microbubble possess rheologic property similar to red blood cells, Bubble perfusion data reflect red blood cell flow.

2. Structural Properties:

This refers to the structure or physical properties of microbubbles as follows:³⁹

a) It should have an average external diameter between the ranges of 1 to 10 micrometer narrow size distribution so as to avoid complication when injected into the body.

b) Density and compression difference between themselves and the surrounding body tissues to create an acoustic impendance an to scatter ultrasound at a much higher intensity than the body tissue so as to be used as contrast agent.

c) The microbubbles acoustic back scatter signal is dependent on the compressibity of the gas, the size of microbubbles the thickness, viscocity and density of the bubble shell. The properties f surrounding medium and the frequency and power of the applied ultrasound

d) At even higher acoustic pressure the microbubbles undergoes forced expansion and compression, which result in its destruction by either outward diffusion of the gas during the compression Phase or diffusion via large shell defects, or by complete fragmentation of the microbubble shell and gas core.

e) Sufficient surface chemical properties to be modified for the attachment of various ligands to target them to specific tissues or organs.

f) Uniformity of the shell thickness. The shell material also affects microbubble mechanical elasticity. The more elastic material, the more acoustic energy it can withstand before bursting.

WORKING OF MICROBUBBLES:

Microbubbles work by resonating in an ultrasound beam, rapidly contracting and expanding in response to the pressure changes of the sound wave. By a fortunate coincidence, they vibrate particularly strongly at the high frequencies used for diagnostic ultrasound imaging. This makes them several thousand times more reflective than normal body tissues. In this way they enhance both grey scale images and flow mediated Doppler signals. As well as being useful in itself, the resonance that microbubbles produce has several special properties that can be exploited to improve diagnoses. Just as with a musical instrument, multiple harmonic signals or overtones are produced. Ultrasound scanners can be tuned to "listen" to these harmonics, producing strong preferential imaging of the microbubbles in an image. The selective excitation produced can also destroy microbubbles relatively easily, an effect that can be useful both in imaging and in emerging therapeutic applications.⁴⁰

Mechanisms for microbubble destruction by ultrasound,¹

a) Gradual diffusion of gas at low acoustic power

b) Formation of a shell defect with diffusion of gas

c) Immediate expulsion of the microbubble shell at high acoustic power

d) Dispersion of the microbubble into several smaller bubbles.

DRUG–LOADED MICROBUBBLES: ULTRASOUND – TRIGGERED DELIVERY SYSTEMS:

The microbubbles themselves have been proposed as carrier vehicles for drugs and genes. Microbubbles can have drug molecules incorporated within the thick polymer shell or inside the gas core (or multiple cores) of thick-shelled bubbles. Drug could be attached to the external surface of the thin lipid monolayer bubbles by covalent or non-covalent bonds, or incorporated in liposomes or nanoparticles that are then associated with the bubble surface.⁴¹

Microbubbles as gene delivery vehicles:

Lipid microbubbles are currently the most commonly used form of contrast agents in ultrasonography. The positively charged groups of some synthetic lipids make electrostatic interactions possible between the lipids and plasmid DNA, which possesses an overall negative charge. To load DNA on the charged microbubble shell, a plasmid is simply mixed with the lipid microbubbles immediately prior to use. DNA coupled to the surface of the microbubbles remains intact even after insonation.^42-44 The adherence of DNA to the microbubbles enhances the transfection efficiency compared to co-administration of bubbles and DNA. A higher local concentration is obtained by releasing the gene from the bubbles by focused ultrasound in the immediate proximity to permeabilized vessels and cells. The loading capacity of the microbubbles is restricted to their surface area, and therefore, high amounts of microbubbles have to be injected to provide the desired effect. Plasmid DNA was incorporated inside the gas core of biodegradable polymer microbubbles.⁴⁵ This approach will not only protect the plasmid from host nucleases, but it will also attain high plasmid load per bubble.

Microbubble as Hydrophilic and Hydrophobic drug Carrier:

Polymeric microbubbles possess a thick, hard shell, which permits a much higher loading capacity. They are usually manufactured by a double water-in-oil-emulsion technique resulting in a microsphere, the precursor of the microbubble, with an internal water-phase and an external organic phase containing the dissolved polymer (e.g. poly (lactide-co-glycolide) or poly (lactide-co-ethylene glycol)). Polymers of this kind are fully biocompatible, biodegradable, and approved by the FDA for parenteral applications. The organic solvent is removed by evaporation or extraction, while the internal water-phase is eliminated during lyophilization or spray-drying. Ideally, in such a bubble a single hollow core is present, surrounded by a polymeric shell. Most often, though, the thin section samples analyzed with scanning or transmission electron microscopy reveal an internally porous structure with multiple voids. Volatile compounds such as camphor or ammonium bicarbonate can be added to the preparation to help increase the porosity of the particles.⁴⁶ These bubbles can be stored for long periods (perhaps for many years) in sealed vials in dry lyophilized state; they are gradually degraded through hydrolysis when resuspended in water. The advantage of polymeric microbubbles is that both hydrophobic and hydrophilic drugs can be incorporated. Depending on its solubility, the drug is added in either the organic or aqueous phase prior to emulsification. Proteins (enzymes, hormones) are encapsulated with high efficiency, and they are still intact after release, with limited loss of activity.^47-54 The rate of drug release from the polymer microbubbles may also depend on the lipophilicity and water solubility of the drug. After destruction of the microbubble by ultrasound, the weak interactions between a hydrophilic drug and the polymer fragments will be broken easily, and the drug will be liberated rapidly.

Multi-particle assemblies for ultrasound-mediated drug Delivery:

If one wants to broaden the spectrum of drugs associated with bubbles to include proteins, enzymes, antibodies and hydrophilic substances, wants to improve loading capacity, or if the drug can't survive the harsh conditions of microbubble preparation, one could use multi-particle assemblies. Liposomes or nanoparticles that entrap the drug can be prepared separately and then coupled to the surface of the microbubbles.^55-58 When the microbubble is destroyed by ultrasound in the target tissue, the energy released in the form of high shear flow, microjets, and micro streaming, will cause rupture of the membrane of microbubble-associated liposomes and subsequently release the encapsulated drug.^57-59

CHARACTERIZATION OF MICROBUBBLES:

Once prepared these microbubbles are characterized as per the following parameters,³³

a) Microbubble Diameter and Size Distribution: The average diameter as well as size distribution of these microbubbles can be determined by Laser light Scattering, Scanning Electron Microscopy, Transmission Electron Microscopy.

b) Shell Thickness: Shell thickness is determined by coating the shell with a fluorescent dye like Red Nile, this is then determined by Fluorescent Microscopy against a dark background.

c) Microbubble Concentration: The microbubble concentration is determined by counting the no. of microbubbles per ml by using the Coulter Counter Machine.

d) Air Content by densitometry: The content of air encapsulated within the microbubbles in the suspension samples is measured by oscillation U-tube densitometry.

e) Ultrasound Reflectance Measurement: Experimental set up consists of transducer, microbubble contained in a vessel consisting of metallic reflector and cellophane membrane, this vessel is in turn kept in another vessel containing water. The signals which are reflected are evaluated for the ultrasound reflecting capacity of these microbubbles.

THERAPEUTIC APPLICATION:

Bio-effects of ultrasound in the presence of microbubbles:

high acoustic pressures can lead to rapid (microsecond) disintegration of microbubbles. This phenomenon is widely used in diagnostic ultrasonography, but it can also induce bio-effects in certain conditions. The extent of the undesired bio-effects can be accentuated by a combination of several factors, such as high concentration of contrast agent, the delivery method (intra-arterial vs. intravenous), the ultrasound system transducer and settings (low ultrasound frequency and high pressure), low attenuation by the tissues located between the insonation focus and the transducer, the ultrasound imaging mode (intermittent), and the type of targeted tissue.^59-66

Thrombolysis via ultrasound-assisted microbubble destruction:

Intravenously administered microbubbles were able to enhance the thrombolytic effects of ultrasound, either with or without the presence of these fibrinolytic agents, often resulting in complete vessel recanalization, progressed from animal and in vitro experimentation to clinical trials^67-69. This phenomenon is explained by the ability of microbubbles to lower the threshold energy needed for cavitation⁷⁰. Cavitation creates high-speed micro streams (or microjets) of sufficient magnitude to promote clot lysis. The induced changes in the fibrin mesh accelerate the transport and penetration of fibrinolytic agents into the clot, provide more efficient use of thrombolytic enzymes, and achieve faster clot dissolution without the release of large amounts of the potentially hazardous clot fragments into the medium. The importance of this technique goes beyond simply lowering the dose of expensive thrombolytic agents; it helps reduce the existing risk of haemorrhaging, while accelerating clot dissolution and limiting ischemic damage to the tissues, and thus improving patient outcome.

Enhancement of vascular permeability by insonated microbubbles

The endothelial lining in the brain, the blood-brain barrier, which is normally impenetrable even for the majority of low molecular weight drugs, can be reversibly “softened” by ultrasound- mediated microbubble destruction (using a transcranial ultrasound transducer), and thus the entry of drugs into the central nervous system can be improved. Compared to other strategies that have known limitations, this technique shows only minimal vascular effects tiny regions of extravasated red blood cells⁷¹ and is safer because of its non-invasive character.^72-74

Enhancement of cell membrane permeability by insonated microbubbles

The ability of microbubbles to act as cavitation nuclei when destroyed can increase cell membrane permeability, if cells are located in close proximity to microbubbles.⁷⁵ This phenomenon called ‘sonoporation’. First, the microjets cause shear stress on the cell membrane and create transient, non-lethal holes in the plasma membrane, through which a drug or gene is able to diffuse.^76,77

Second, the generation of intracellular reactive oxygen species, following the application of ultrasound, might contribute to permeabilization of the cell membrane^78-80 without affecting the cell viability. The local, transient temperature increase due to the absorption and dissipation of ultrasound energy may also influence phospholipid bilayer fluidity and thus cell permeability.⁸¹ Other scenarios are the involvement of active transport mechanisms, such as endocytosis and phagocytosis in the uptake of microbubbles, and the fusion of lipid-based microbubbles with the phospholipid cell membrane.

Rationale for Using Microbubbles for Gene and Drug Delivery

The inability to deliver nucleic acids to target cells via systemic delivery is the biggest rate limiting barrier in gene therapy applications. The use of ultrasound with microbubbles, however, is beginning to largely overcome this limitation, enabling target specific nucleic acid delivery following systemic delivery. Targeted gene delivery by co-injection of plasmid DNA with microbubbles can be effective for producing detectable levels of gene expression, but it often requires large amounts of DNA in order to produce quantifiable results.⁸² This is due to the fact that poly (nucleic acids) are prone to nuclease mediated degradation and rapid clearance by the reticulo endothelial system (RES) when introduced into the blood stream⁸³. Therefore, carriers are required to facilitate delivery. Several methods have been developed to utilize microbubbles as carriers for nucleic acids in order to increase their circulation time in the bloodstream, protect them from degradation, and improve specificity of targeted delivery.

Targeted drug delivery can be accomplished by the incorporation of small molecules into the microbubble shell. The drug can be released upon destruction of the microbubble through ultrasound-mediated cavitation. Ultrasound focusing allows exquisite tissue selectivity. The same rationale for conjugating nucleic acids to microbubbles for gene delivery also applies to molecules for drug delivery applications. Unlike nucleic acids for gene delivery applications, drugs are rarely electrostatically bound to the microbubble surface. Rather, they are incorporated within or just beneath the microbubble shell. Alternatively, they can be loaded into a carrier which can then be linked to the microbubble surface.

Protein Microbubbles for Gene and Drug delivery:

As described above, protein microbubbles are formed by a relatively simple method of disulfide cross linking of the proteins during sonication. The relative simplicity of the formulation procedure lends this class of microbubbles to be an attractive tool for both drug and gene delivery applications. The 15-nm thick protein shell can accommodate the loading of nucleic acids or other macromolecules without significantly disrupting the acoustic properties of the microbubble. These macromolecules may be fully or partially incorporated within the shell during covalent cross linking of proteins during the formulation stage. Alternatively, the charged protein surface is amenable to adsorption of nucleic acids without significantly altering the acoustic response.

Polyelectrolyte Multilayer microbubbles (PEM) may have important applications in gene delivery by improving the overall loading capacity of nucleic acids onto the microbubble surface, thus presumably increasing the payload that is delivered following ultrasonic destruction. Research has demonstrated that PEM assembly on microbubbles may have a potential advantage of increasing the stability and payload capacity. Both effects are expected to improve transfection efficiency in vivo.²

Lipid Microbubbles for Gene and Drug Delivery:

Lipid microbubbles are more frequently used in drug and gene delivery applications than protein microbubbles. Fragmentation of the lipid monolayer following microbubble destruction is critical in targeted delivery applications where the drug being carried needs to be easily released when an ultrasound trigger is applied. Lipid microbubbles are more acoustically responsive and may serve as more desirable vehicles for ultrasound-triggered drug release.

One of the earliest strategies to incorporate drugs into lipid-stabilized microbubbles was the development of acoustically active lipospheres (AALs). AALs are similar to lipid microbubbles, but they contain a thick oil layer separating the lipid shell from the gas core. Hydrophobic molecules can be loaded within the oil layer to create drug loaded AALs capable of releasing their contents upon microbubble disruption. AALs were acoustically active, but they were not as stable as microbubbles formulated without the entrapped layer of oil.

Cationic lipids were introduced into the microbubble during formulation, which enabled them to electrostatically bind the negatively charged phosphate backbone of plasmid DNA. The size of the microbubbles was approximately 3 μm in diameter, and the microbubbles were able to carry 0.001 pg of pDNA/μm². Cationic microbubbles were loaded with plasmid DNA.²

Polymer Microbubbles for Drug Delivery:

Thick shelled gas-filled polymer microspheres have repeatedly been shown to be viable contrast agents for in vivo imaging and molecular targeting. However, the utilization of the agents as drug and gene delivery carriers is rarely found in published literature. One may infer that the thick cross linked polymer shell may be more resistant to fragmentation and thus less effective for depositing genes/drugs, making lipid or protein microbubbles more attractive carriers. A recent publication by Mahier-Humbert et al.,⁸⁴ however, does showed evidence that hard-shell polymer microbubbles are promising candidates for ultrasound-mediated gene delivery. Higher levels of transfection by sonoporation were achieved when comparing microbubbles with thick polymer shells (made using triglyceride or polystyrene) to lipid-based microbubbles, although higher acoustic pressures were required for the polymer shells. The authors suggest that the nature of fragmentation and jetting may be more violent for polymer microbubbles when higher acoustic pressure are applied, which may make them more efficient at propelling DNA into cells. Another advantage of polymer microbubble is that they are more favorable for incorporating a wide variety of hydrophobic or hydrophilic macromolecules within the thick cross linked polymer matrix.

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