**2.1 Protein as stabilizing agent in formation of microbubbles**

Albumin-shelled microbubbles were a pioneering formulation used in contrast ultrasound imaging. For perfusion in capillary and microvessels, albumin-shelled microbubbles are very effective. The size of albumin-shelled microbubbles ranges from 1 to 15 μm in diameter in 7 × 108 microbubbles/mL which is stable for 2 years. To formulate albumin-coated microbubbles by sonication method, 5% w/v human serum albumin with air is required and encapsulated within 15 nm thick shell of aggregated albumin. For better encapsulation process, the denaturation of albumin by heating is essential [9, 10]. The albumin shell is held together through disulfide bonds between cysteine residues formed during cavitation [11]. Covalent cross-linking may explain the relative rigidity of albumin shells observed during ultrasonic insonification [12]. Apart from albumin, several proteins are used to coat microbubbles.

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*Using Microbubbles as Targeted Drug Delivery to Improve AIDS*

The proteins which are amphipathic in nature are highly surface-active. In most of the proteins, the disulfide bridge between two thiol groups is present. Cavalieri and co-workers prepared microbubbles by using lysozyme which retain their enzymatic activity for several months and found to be stable [13]. Korpanty et al. [14] developed microbubble by incorporating avidin into albumin shell. **Figure 2A** illustrates target-

*Microbubble shell morphologies. (A) A lysozyme protein microbubble imaged with SEM (Calaveri et al. (13)). The microbubble diameter is roughly 1 μm. (B) A diC20:0 phospholipid microbubble imaged with fluorescence microscopy taken from Borden et al. Scale bar denotes 20 μm. (C) A PLA-PFO polymer* 

SPAN-40 and TWEEN-40 are used as stabilizing agent in the preparation of microbubbles [15, 16]. For the formation of stable microbubbles, the SPAN/ TWEEN solution was sonicated in the presence of air. For maximum film stability, a Langmuir trough was used in the ratio of SPAN to TWEEN (roughly 1:1). By using sonicated microbubbles, modified surfactant was formed which was more stable film due to higher collapse pressure on the Langmuir trough [16]. Dressaire et al. recently reported stable microbubbles formed from a blending process at 70°C in

75 wt% glucose syrup, sucrose stearate (mono- and di-ester) formed [17].

For biomedical imaging and drug delivery, lipid-coated microbubbles are one of the most interesting and useful formulations. The lipid shell is inspired by nature, as stable microbubbles found ubiquitously in the oceans and freshwaters of earth

During ultrasound and sonication technique, the lipid molecules which are held together by weak physical forces form the microbubble shell having property of expansion and compression without chain entanglement. Lipid-coated microbubbles therefore reduce the damping effect on resonance and reseal around the gas core during fragmentation process [12]. Thus, the lipid-coated microbubble itself is a versatile platform technology. An example of lipid microbubble is shown in **Figure 2B**, which depicts heterogeneity and phase separation of phosphatidylcholine and lipopolymers that are typically used to stabilize lipid microbubble [19].

The term, "polymer microbubble" typically refers to a special class of microbubbles that are stabilized by a thick shell comprising cross-linked or entangled polymeric species. Polymer shells are more resistant to expansion and compression;

**2.3 Lipid as stabilizing agent in the formation of microbubbles**

are known to be stabilized by acyl lipids and glycoproteins [18].

**2.4 Polymer as a stabilizing agent in the formation of microbubbles**

ing vascular endothelium in biotin-mediated coupling of antibodies.

**2.2 Surfactant as stabilizing agent in formation of microbubbles**

*DOI: http://dx.doi.org/10.5772/intechopen.87157*

**Figure 2.**

*microbubble imaged with SEM.*

#### **Figure 2.**

*Pharmaceutical Formulation Design - Recent Practices*

It mostly contains oxygen or air and remains suspended in the water for an

enhanced drug delivery [55]. Microbubbles are usually injected intravenously which is a safe process as compared to the use of conventional method like magnetic resonance imaging and radiography. Microbubble is used in the medical field as diagnostic aids to scan the various organs of the body, and recently they are being proposed to be used as drug or gene carriers and also for treatment in cancer therapy. It is also used to improve the fermentation of soil, to increase the hydroponic plant growth, to increase the aquaculture productivity, and to improve the

**2. Compositions and physicochemical properties of microbubbles**

Albumin-shelled microbubbles were a pioneering formulation used in contrast ultrasound imaging. For perfusion in capillary and microvessels, albumin-shelled microbubbles are very effective. The size of albumin-shelled microbubbles ranges

To formulate albumin-coated microbubbles by sonication method, 5% w/v human serum albumin with air is required and encapsulated within 15 nm thick shell of aggregated albumin. For better encapsulation process, the denaturation of albumin by heating is essential [9, 10]. The albumin shell is held together through disulfide bonds between cysteine residues formed during cavitation [11]. Covalent cross-linking may explain the relative rigidity of albumin shells observed during ultrasonic insonification [12]. Apart from albumin, several proteins are used to coat microbubbles.

microbubbles/mL which is stable for 2 years.

**2.1 Protein as stabilizing agent in formation of microbubbles**

quality of water, in sewage treatment.

from 1 to 15 μm in diameter in 7 × 108

extended period. The gas present in the microbubbles dissolves into the water, and the bubble disappears. Incorporation of drug in microbubble includes (1) binding of drug to microbubble shell and (2) attachment of drug at specific site of ligand. In ultrasound-mediated microbubbles, application of high intensity ultra sound can rupture capillary blood vessels resulting in deposit of protein and genetic material into the tissue, ultrasonic rupture of microvessels with diameter 7 μm. Ultrasound forms pores in the membrane of shell. Ultrasound microbubble causes transient hole in the cell surface resulting in rapid translocation of plasmid DNA from the outside to cytoplasm. Low-intensity ultrasound microbubble (0.6 W/cm<sup>2</sup>

*Illustration describe various shell compositions of microbubbles. The diameter between 0.5 and 10 μm is applied for biomedical use so that it can pass through the capillary of the lung. Microbubbles compose of total particle volume which act as single chamber so that the shell of the microbubble separate encapsulated gas and the surrounding aqueous medium by using various shell materials like lipid with thickness~3 nm thick, protein having 15–20 nm thick and polymer of 100–200 nm thick. Hydrophobic and Vander Waals interactions binds the lipid molecule together and by covalent disulphide bonding the protein molecules get cross-linked so that the* 

) caused

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**Figure 1.**

*formation of bulk like material.*

*Microbubble shell morphologies. (A) A lysozyme protein microbubble imaged with SEM (Calaveri et al. (13)). The microbubble diameter is roughly 1 μm. (B) A diC20:0 phospholipid microbubble imaged with fluorescence microscopy taken from Borden et al. Scale bar denotes 20 μm. (C) A PLA-PFO polymer microbubble imaged with SEM.*

The proteins which are amphipathic in nature are highly surface-active. In most of the proteins, the disulfide bridge between two thiol groups is present. Cavalieri and co-workers prepared microbubbles by using lysozyme which retain their enzymatic activity for several months and found to be stable [13]. Korpanty et al. [14] developed microbubble by incorporating avidin into albumin shell. **Figure 2A** illustrates targeting vascular endothelium in biotin-mediated coupling of antibodies.
