INTRODUCTION:

Ultrasound-mediated microbubbles destruction has been planed as an innovative method of immediately delivering of drugs and genes to different tissues until a particular area of interest is reached and after this the ultrasound is used to rupture microbubbles, to cause site specific delivery of bioactive materials.¹Microbubbles are also small sphere shaped bubbles comprising of gas and they remain separated from each other that is they do not agglomerate, and also have their size range in micrometers usually 1-100µm.²There has been a lot of investigation on microbubbles in recent years. Miccrobubbles are minute gas bubbles of less than 50 microns diameter in water. The microbubbles mostly containing oxygen or air can remain suspended in the water for longer period. Gradually, the gas within microbubbles dissolves into the water, thus the bubbles disappear.

In the medical field, microbubbles are used as diagnostic aids to scan the various organs of body and recently are being proposed to be used as drug or gene carriers for treatment in cancer therapy.^{3, 4}Biomedically microbubbles are defined as small spherical gas bubbles prepared of phospholipids or biodegradable polymers, which are approximately the size of RBCs and are as diagnostic aids, as drug and gene carriers in combination with ultrasound.⁵This review focuses on the components of micro bubbles; it’s method of preparation, mechanism by which ultrasound contrast agents facilitate the delivery of bioactive substance and application in cancer therapy.

Components of micro bubbles:⁶

Micro bubbles mainly comprise of three phases:

1) Innermost Gas phase⁷

2) Shell material enclosing the gas phase⁸

3) Outermost liquid or aqueous phase⁹

In addition to this formulation may also compose of other components.

Fig.1: Components of Microbubbles

1) Gas phase:

In this gas phase a single gas or combination of gases can be used. Combination gases are used to cause differentials in partial pressure and to produce gas osmotic pressures which stabilize the bubbles. When a combination of gases is used, these are of two types first is primary modifier gas also known as first gas.¹⁰Air is preferably used as primary modifier gas; Nitrogen is also used as first gas. The vapor pressure of first gas is (760-X) mm of Hg, where X is the vapor pressure of the second gas. Another gas is Gas osmotic agent also known as second gas. It is less permeable through bubble surface than first gas. Examples of second gas are perflurocarbons or sulfur hexafluoride.

2) Shell material:¹¹

This shell material encapsulates the gas phase. It plays an important role in the mechanical properties and diffusion of gas out of microbuuble. Also, the shell acts as region for encapsulation of drug molecules and ligands can be attached to the shell membrane for achieving targeting of these microbubbles to different organs or tissues. It account for the elasticity or compressibility of microbubbles. More acoustic energy it can resist before breaking up, which increases the residence time of these microbubbles in body. More hydrophilic the shell material, more easily it is used by the body this in the body. Eg. The different types of shell materials that are used are proteins like albumin, carbohydrate like galactose, phospholipids like phosphotidylcholine, phosphotidylethanolamine etc.

3) Outermost liquid or aqueous phase:

The external, continuous aqueous phase in which the bubble resides typically include a surfactant or foaming agent. The surfactants that are used include any compound or composition that helps in the creation and preservation of the bubble membrane by forming a film at interphase. The foaming agent or surfactant may consist of a single component or any combination of compounds, as in case of co surfactants. The decrease in the surface tension acting on the bubble increases the persistence time of bubble in the body. Eg: Block copolymers of polyoxypropylene, sugar esters, fatty alcohols etc.

Non ionic surfactants like polyoxyehylene, polyoxypropylene copolymers. Eg: pluronic f-68, polyoxyethylene sterats, etc.

Anionic surfactants: fatty acids having 12-24 carbon atoms. Eg –sodium oleate.

4) Other components:

The various other components that may be incorporated in the formulation contain osmotic agents, chelators, stabilizers, viscosity modulators, buffers, air solubility modifiers, salts and sugar.

Methods to prepare microbuble:

There are various methods that can be used for preparation of these microbubbles are as follows:

1. Cross linking polymerization^5,6

2. Emulsion solvent evaporation^12,13

3. Atomization and reconstitution¹⁴

4. Sonication¹⁵

1) Cross linking polymerization:

In this a fine foam of polymer is formed where a polymeric solution is stirred vigorously, acting as a colloidal stabilizer as well as a bubble coating agent. The polymer is then cross linked, after this microbubbles float on the surface of mixture. Then, these floating microbubbles are separated and dialyzed against Milli Q water.

Eg: 2% aqueous solution of telechelic PVA is vigorously stirred at room temperature for 3 hrs at a pH of 2.5 by an ultra turrax T-25 at 8000 rpm equipped with a Teflon coated tip, which results in formulation of fine foam of PVA. This fine foam of PVA is then cross linked at room temperature and at 5 0 c by adding Hcl as a catalyst. The cross linking reaction is stopped by neutralization of the mixture and then this formed microbubbles are separated.

2) Emulsion solvent evaporation:

In this method two solutions are prepared, one is aqueous solution containing and appropriate surfactant material may be amphillic. For eg. In the emulsion system, Gelatin, collagen, albumin or globulin, which acts as outer continuous phase? Then the second solution is prepared by dissolving a wall forming polymer in a mixture of two water immiscible organic liquid, from which one of the liquid is a volatile solvent and other is nonvolatile nonsolvent for the polymer. The emulsification step is formed by adding the polymer solution to the aqueous solution with agitation to form an emulsion. This emulsification step is carried out till the inner phase droplets are in the preferred size spectrum; the droplet size will determine the size of microbubble.

The polymer concentration in droplet precipitates in the presence of less volatile non solvent increases to a point, as solvent volatizes. This process forms a film of polymers at surface of the emulsion droplet. An outer shell wall is formed as the process continues, which encapsulates an inner core of nonsolvent liquid. As the process is finished, the formed microcapsules can thereafter be removed, washed and formulated within buffer system. Successive drying, remove the water and nonsolvent organic liquid core to give up air filled hollow microbubbles.

3) Atomization and reconstitution:

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

4) Sonication:

This method is preferred for formation of microbubbles by introducing a septum with an ultrasound probe including a hypodermic needle which vibrates ultrasonically or through an ultrasound transmitting spectrum. A number of ways are used for accomplishing sonication, for eg: a vial containing a surfactant solution and the gas in headspace of vial which can be sonicated through a thin film. As sonication is completed, the microbubble solution is withdrawn from vial and delivered to the patient.

How microbubble works: ^16-23

Microbubble works by resonating in an ultrasound beam, swiftly expanding and contracting in response to the pressure changes of the ultrasound waves. Unfortunately, microbubbles vibrate strongly at high frequencies used for diagnostic ultrasound imaging; this makes the microbubbles more reflective than normal body tissues. By this way microbubbles develop both flow mediated Doppler signals and grey scale images. The resonance that microbubbles create has several properties that can be exploited for improving diagnosis as being useful in itself. Just as a musical instrument can produce multiple harmonic signal or overtones, ultrasound scanners can be tuned to “listen” to these harmonics, which produce strong preferential imaging of microbubbles in an image. The selective excitation created can also destroy microbubbles relatively easily and an effect is produced that can be of use both in imaging and in rising therapeutic application in cancer therapy.

Application of Microbubble in Cancer therapy:

Diagnostic Aids:

Microbubbles are elastic and compressible, these undergo compression and rarefaction. This creating an acoustic impedance difference between biological tissues and fluids as these are efficient reflectors of ultrasound, hence used as contrast agents and diagnostic aids for cancer and also used to scan the tumors arising in the body.

Increase the efficiency of Cancer treatment:

The laser-induced bubble formation around nanoparticles may play an essential role in selective laser nanophotothermolysis of cancer cells targeted with nanoparticles. We proposed theoretically, a new dynamic mode for selective cancer treatment which involves the overlapping of bubbles inside the cell volume. These bubbles- overlapping mode (BOM) can considerably increase the efficiency of cancer treatment by laser-heated nanoparticles as a result of the large damage range. On the basis of nanoparticle optics below the diffraction limit and the kinetic model of bubble dynamics, we found the condition and criteria (inter particle distance and particle size and concentration) for BOM initiation in cancer cells by laser radiation .Using MDA-MB-231 breast cancer cells, we showed that the optimal size range of the gold nanoparticles for effective laser initiation of BOM is 30-40nm and the lower concentration limit is n2.44×1011cm–3 (i.e. the absolute number of particles homogeneously distributed inside a tumour cell is n 430). It was established that the formation of nano-clusters on the cell surface with sizes larger than the sizes of individual nanoparticles, may further increases the efficiency of the laser treatment of cancer.²⁴

Prostate cancer detection:

The diagnosis of prostate cancer is now-a-days limited by the low sensitivity and specificity of Systemic conventional grey-scale ultrasonography. We assessed contrast enhanced colour Doppler ultrasonography by means of a microbubble ultrasound contrast agent to detect tumour vascularity and improve the diagnosis of prostate cancer. The use of a microbubble ultrasound contrast agent for transrectal colour Doppler targeted biopsy significantly enhanced the detection of prostate cancer compared with systematic biopsy following conventional grey-scale ultrasonography (p<0.001 ). Contrast- agent enhanced colour Doppler imaging may allow for limited targeted biopsy, which reduce costs and morbidity. The accuracy of screening for prostate cancer can be improved by new ultrasound techniques involving Color-enhanced Doppler imagings with microbubble contrast. This is so, that physicians are better able to decide the presence and accurate location of a mass within the prostate. The differences in velocity can be sensed by the Doppler imaging and it also transmits these differences through different color pixels to produce a picture on a screen. Microbubbles are minute bubbles of gas and without creating any harm they can permeate through small blood vessels. Further the microbubbles by increasing the intensity of back scatter signal enhance the imaging. Microbubbles tend to concentrate in the cancer, as blood vessels and blood flow both are more prevalent in cancerous tissues than regular tissues; which is exposed on the produced picture. This allows the physicians to more accurately locate for biopsies. As we compared with conventional ultrasonography the detection rate of prostate cancer was 27% with Doppler guided biopsies and the overall core biopsy detection rate was 13% for Doppler guided biopsies when compared with only 4.9% for conventional ultrasonography. Both these results indicate that the Doppler-guided biopsies with microbubble contrast may allow physicians to more accurately decide the optimal location for a biopsy. Microbubble contrast-enhanced studies demonstrate a clear relationship between contrast enhancement in the prostate and in analysis of clinically significant cancer. ^25-28

Leukemia Treatment:

Acute myeloid leukemia (AML) is a rapid progressive cancer which is characterized by neoplastic of myeloid cells. The current diagnostics of AML with therapeutically and prognostically implication includes cellular morphology, and in particular gene mutation analysis, immunological markers, cytogenetic, and response after chemotherapy. Normally, the high doses of cytotoxic drugs and in preferred cases hematopoietic stem cell transplantation can be included in treatments. A lot of patients over 60 years involve toxicity problems with these high intensive therapy regimens and only 20-30 % of the patients reach to a long time survival. The need for new, targeted and effective treatment is therefore great. Imaging modalities plays an important role in evaluating disease expansion, progression in hematological malignancies and also has a very essential role in pre-clinical trials using animal models (Fig.2.). To overcome the limitations of a single imaging modality the use of different modalities has resulted in protocols this multimodality provides a thorough view of anatomical, physiological and/ or molecular processes in vivo and also allows quantitative measurements and visualization of processes in specific targeted tissue or organ. This makes it a very essential tool to detect early cancer and decide direct treatment of patient with cancer. By using specific and sensitive imaging modalities, treatment can be given previous to the malignant cell load becomes too large for existing therapeutics. As the treatment can be so exhausting, the administration of direct drug delivery may permit the patient to accept disease controlling treatment with soothing intentions. The use of stronger drugs gives the opportunity by the direct drug delivery than otherwise it would be too toxic for the patient.²⁹

Fig.2: Gas-filled microbubbles enclosed with a bioactive substance pass undamagingly and unexcitingly through blood vessels until they are exposed to ultrasound. After this the bubbles rupture, causing the release of the bioactive substance and the opening of holes in the cells that line the vessel.¹

Blood vessel growth in tumors:

Imaging being able to rapidly detect and diagnosed blood vessel growth in cancerous tumors, and even expect how fast the tumors might metastasize. The millions of minute microbubbles injected into the blood stream of the animal models and then joined with contrast-enhanced ultrasound. It is an economical and commonly-used method using ultra sound waves to “examine” inside the body. They found that blood flow has a high velocity slower velocity inside a tumor than on the periphery of the tumors. The velocity of blood flow is a essential marker for detection tumor, treating malignant brain tumors can be difficult. The protective blood-brain barrier blocks many drugs from acting on brain cells. Now, a new method for delivering drugs directly to the affected area may increase effectiveness of chemotherapy in brain tumors and reduces its toxic effect on healthy cells. Researchers at the University of California are developing a novel technique that uses ultrasound and drug-laden “microbubbles” to deliver concentrated chemotherapy drugs to the inner lining of blood vessels. Doctors already use ultrasound to identify tumors and guide biopsy procedures. Ultrasound pulse sequences can also guide micro-packaged medications to specific parts of the body.^{24, 30, 31.}

Breast cancer:

Conventional ultrasound provides high resolution anatomic detail, and breast ultrasound plays an important role in breast cancer diagnosis.³² The responsibility of ultrasound, microbubble contrast ultrasound and colour Doppler ultrasound in detection of breast carcinomas is not fully established. The differential diagnosis of breast diseases has a recommended role for colour Doppler ultrasound. Studies have demonstrated an improved sensitivity in vascularity with microbubble contrast, but with differ in the specificity of differentiating malignant and benign lesions.^{33, 34}All these studies were performed by using common machine settings. The analysis of breast masses with microbubble contrast may become a useful tool as the newer harmonic and phase inversion techniques are developed. The microbubble contrast study helps in the estimation of radiofrequency ablation in breast tumors and has a similar use in radiofrequency ablation of liver tumours.³⁵ The treatment of any cancer is based on the identification of diseases participation of the sentinel node, which is the first node to drain a tumor into the lymphatic system. ³⁶ This predicts that the ragional lymph nodes should be detached. Microbubble contrast may play an important role in this development; sentinel nodesin swine models with melanoma confirmed sentinel node improvement in 28 of 31 sentinel lymph nodes, and some of in peritumor injection of microbubble contrast. This is established that the signal voids within the lymph nodes representative of intranodal metastasis with 95% sensitivity by the authors , using low MI grey scale pulse-inversion imaging .³⁷This method of detection of sentinel node is the better alternative technique without the adverse effects of these established technique. The blue invasive technique has a relatively more rate of allergic reaction and it has 20% of a technical failure rate, whereas technetium 99m scintigraphy has a reported 12% of failure rate.³⁸The non-sentinel nodes can be detected by both these techniques, without leading extensive nodal dissection. The stimulated acoustic emission ultrasound imaging method has successfully developed by another group, using a specific microbubble that targets lymph nodes.³⁹For evaluation of these techniques with application, particularly within the axilla of breast cancer patients which is an important potential clinical use required further studies.⁴⁰The paper by Bazan-Peregrino et al.⁴¹ describe a detailed in vitro study of microbubble-assisted delivery of viral particles to breast cancer cells embedded in a novel flow phantom. Use of the phantom allowed the authors to illustrate the effects of ultrasound intensity and a co-injection of commercial microbubbles on virus particle delivery to cells in the hydrogel matrix far from the vessel walls. The use of stable cavitations increased the viral infection rate by a factor of 10, while inertial cavitation increased infection by a factor of 60.⁴²

CONCLUSION:

Microbubble ultrasound contrast agents offer a wide range of potential benefits for both diagnostic and therapeutic applications. Microbubbles are an important therapeutic tool for cancer treatments. As a result, they have become the subject of a broad and rapidly developing field of research. The application of microbubble with ultrasound which gives a synergistic effect for drug/DNA delivery is currently in its infancy. The use of ultrasound microbubbles is a great step forward and has created various challenging therapeutics options, in cancer. Microbubbles have rapidly evolved from a diagnostic adjuvant to a possible therapeutic agent. At present, however, their behavior is by no means fully understood, and consequently their effectiveness has yet to be maximized. Moreover, while no definite evidence of harmful effects has been obtained, there remain some concerns as to their safty. The aim of this review is to explain the application of microbubble drug delivery system in cancer therapy. In the coming years, this promising technique needs further development to make it available for clinical applications.

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Received on 14.08.2013 Modified on 10.09.2013

Research J. Pharm. and Tech. 6(11): November 2013; Page 1279-1284