BIOSURFACTANT AND THEIR BIOSURFACTANT AND THEIR

APPLICATION APPLICATION

IN MICROBUBBLE PREPARATION IN MICROBUBBLE PREPARATION

BY ,MISS ANKITA.K.GURAOM.Sc BIOTECHNOLOGY II

CONTENTS1)Biosurfactant-an intro

2)Types of biosurfactants

3)Action of biosurfactants

4)What are microbubbles?

5)Properties of microbubbles

6)Methods of generating microbubbles

7)Types of microbubbles

8)Why do we prefer biosurfactant for microbubbles?

9)Applications of biosurfactant stabilized microbubbles in biomedical field

10)Industrial applications.

Biosurfactants- An intro

●Biosurfactants are surface-active substances synthesised by living cells.●Chemical composition of biosurfactant includes glycolipids, lipopeptides, protein complexes and fatty acids.●Fatty acid esters of sugars and fatty acid esters or amides of amino acids. ●Several microorganisms are known to synthesise surface-active agents; most of them are bacteria and yeasts.

Types of biosurfactants

Class of organic compound Biosurfactant type Micro organism

1.LIPOPEPTIDES Surfactin A & B Bacillus subtilis

Inturin A Bacillus subtilis

Pumilacidin Bacillus pumilus

2.GLYCOLIPIDS Rhamnolipids Pseudomanas sp.

Sophorolipids Sacchromyces sp.

Trehalolipids Mycobacterium sp.

3.POLYMERIC SURFACTANT

Bioemulsan Acinobacter sp.

4.SECONDARY METABOLITE

Viscosinamide Pseudomonas flouroscens

Action of biosurfactant

● Biosurfactant tend to interact with the phase boundry between two phases in heterogenous system.

● They diffuse in water and adsorb at interphase between air and water.

● Like soaps they form micelle.

● This conditioning film changes the substratum properties like wettability and surface tension.

● In natural conditions, the organic molecule from aqeous phase tend to immobilize at solid surface and form the conditioning film.

What are microbubbles?● Microbubbles are bubbles smaller than one

millimetre in diameter, but larger than one micrometre.They are actually the colloidal bubbles.

● Microbubbles used for biomedical purposes are typically between 0.5 and 10 μm diameter (the upper limit for passage through the lung capillaries).

● The gas core is a single chamber and comprises a large majority of the total particle volume. The shell acts as a barrier between the encapsulated gas and the surrounding aqueous medium.

● Different shell materials may be used, including lipid (~3 nm thick), protein (15–20 nm thick) and polymer (100–200 nm thick).

● The lipid molecules are held together through physical force fields, such as hydrophobic and van der Waals interactions.

● Wheareas protein shell held by disulfide bond.

Properties of microbubbles

The following properties make microbubble a promising concept for biomedical purposes -

● Unlike ordinary bubbles they don't burst at water surface rather they shrink as they rises upward.

● Interior gas pressure higher than ordinary bubble.

● Surface electricity produced due to the accumulation of ions(mainly H+ & OH-) on surface.

● Free radical generation on collapsing of microbubble.

Methods to generate microbubbles

1)Hand-agitation method: Used in clinical studies results in rather large and unstable microbubbles. These microbubbles with unstable variable diameter cannot pass through the capillaries.

2)Ultrasonic cavitation: or sonication, a technique generally reserved for in vitro tissue disruption has recently been used to create relatively small and stable microbubbles in physiologic solutions.

Contd..

3)Microchannel emulsification: is a novel technique for producing monodispersed emulsions in which droplets are formed by spontaneous transformation caused by interfacial tension. An MC structure consists of a narrow channel and a slitlike terrace.

Types of microbubbles

Based on the chemical nature of shell there are 4 types of microbubbles.

1) PROTEIN SHELL

2) SURFACTANT SHELL

3) LIPID SHELL

4) POLYMER SHELL

CONTD..

1)Protein shell

● Albumin shell were the first to be produced.

● The first albumin microbubble formulation to be a was Albunex (GE Healthcare) with a size range from 1 to 4.5 μm diameter.

● Albumin-coated microbubbles are formed by sonication of a heated solution of 5% (w/v) human serum albumin in the presence of air.

2)Surfactant shell

● Microbubbles stabilized by mixtures of the synthetic surfactants SPAN-40 and TWEEN-40.

● The SPAN/TWEEN solution was sonicated in the presence of air to form stable microbubbles.

● The correct ratio of SPAN to TWEEN (roughly 1:1) to use for maximum film stability.

● They have limited biomedical application.

3)Lipid shell

● Bio inspired since microbubbles stabilized by acyl lipids & glycoprotein already found in marine ecosystem.

● Imitates the stability & compliance of lung surfactants.

● Among biosurfactants rhamnolipids have been widely used as lipid alternative.

● Commercially available formulations includes Definity (Lantheus Medical Imaging) and Sonovue® (Bracco Diagnostics).

4)Polymer shell

● Stabilized by a thick shell comprising cross-linked or entangled polymeric species.

● The bulky in nature so more resistant to area compression and expansion, which reduces the echogenicity and drug delivery activity.

● Diameter- 30 to 40 μm and were therefore too large for intravenous administration.

● Overall they are not suitable for biomedical applications.

Why do we prefer biosurfactant stabilized microbubbles?

● Environmental freindly and cost effective.

● In biomedical application it is safe to use non toxic biosurfactant instead of synthetic surfactant.

● Chemical diversity of biosurfactants provides a wide range of choices to meet specific applications.

● They are biodegradable, so safe to use for drug delivery and gene delivery.

Applications of biosurfactant stabilized microbubbles.

1) CEUS

2) Targeted CEUS

3) Drug delivery

4) Gene delivery

1)CEUS● A medical contrast agent is a substance used to

enhance the contrast of structures or fluids within the body in medical imaging.

● Microbubbles composing tiny amounts of nitrogen or perfluorocarbons strengthened and supported by a protein or lipid.

● Small enough to pass through the capillaries and increase contrast, so used in echocardiography.

● The drop in density on the interface between the gas in the bubble and the surrounding liquid strongly scatters and reflects the ultrasound back to the probe.

Contd..● This process of backscattering gives the liquid

with these bubbles a high signal, which can be seen in the resulting image.

2)Targeted CEUS● An ultrasound molecular imaging scan goes as

follows:

1) The microsphere may be targeted to specific tissue by incorporating protein ligands on the surface.

2) After dwell time, the target tissue is scanned and the contrast signal in the region of interest is determined.

3) A pulse is then applied to fragment and dissolve all microbubbles within the field of view.

Contd..

4) Free microbubbles are allowed to flow back into the field and again video intensity is determined.

5)The signal from adherent microbubbles is delineated from that of freely circulating microbubbles .

6)The GPIIb/IIIa receptors play a key role in the formation of vascular clots, lipid-coated perfluorocarbon gas containing microbubble with bioconjugated ligands inserted into the membrane to dissolve the vascular clots.

3)Drug delivery

● Cavitation of bubbles in an ultrasound field can increase the permeability of an endothelial vasculature, allowing small molecules to enter into tissue from the blood stream, a technique known as sonoporation.

● Once the shells are destroyed, the contents of the microbubbles spill into the surrounding area and the drugs reach the target directly instead of going through the whole bloodstream.

● This localized release technique prevents the drugs from influencing other systems in the body.

Contd.. ● This system finds vast application in

chemotherapy

4)Gene delivery

● Protein microbubbles used for this purpose because:

1)The nucleic acids may be incorporated in the shell during covalent crosslinking of proteins during the formulation stage.

2)The charged protein surface is amenable to adsorption of nucleic acids without significantly altering the acoustic response.

● Gene delivery mediated by albumin microbubble first attempted by Shohet et al(2000).

Contd..● The following strategy was used to deliver

adenoviral vector to the cardiac region of the mice. This is based on adsorption of the compounds to the microbubble shell.

1)Albumin microbubbles were formed by sonicating a solution of containing 1% human serum albumin and 5% fructose with perflouropropane gas.

2)The microbubbles were then mixed with

adenoviral vector( encoding a ß galactosidase reporter gene) for 2 hours.

3) Buoyant microbubbles rose to the top of this solution and were collected while unattached adenovirus in the subphase was discarded.

Contd..

4) The adenovirus-loaded microbubbles were injected systemically into rats, and the cardiac region was exposed to ultrasound.

5)The results of the study showed that ß-galactose expression was seen only in the myocardium following ultrasound-mediated destruction of microbubbles.

● Yet another strategy includes coating the microbubble but it is inefficient due to the negative charges on both the protein shell and the nucleic acid backbone.

Industrial applications of biosurfactant stabilized microbubble

● In sludge treatment by using the micro bubbles to capture and float organic matters, thus decreasing the time required for the treatment.

● In Japan micro bubbles of concentrated oxygen containing about 2% ozone used to inactivate norovirus in shellfish and oysters for preservation.

● Due to high volume surface ratio, cleaning effect of micro bubbles is used in cleaning the inside of vegetables such as cabbage and radish sprout, as well as maintenance of freshness with vegetables.

Contd..

● The micro bubbles can penetrate deeply into skin for a good scrub without the need for any shampoo or soap.

● Suwa company(Singapore) has developed a small handy micro bubble generator which can be used at home.

Conclusion

● Biosurfactant along with it's wide range of utility in microbial world it too finds scope in medical and industrial field.

● Especially biosurfactant stabilized microbubbles has revolutionized the medical imaging sector and delivery systems.

● Future prospective of these microbubbles includes various domestic applications.

References

1)Biosurfactants for Microbubble Preparation and Application Int J Mol Sci. 2011; 12(1): 462–475. Published online 2011 January 17.

2)Biosurfactants for Microbubble Preparation and Application Int J Mol Sci. 2011; 12(1): 462–475.Published online 2011 January 17.

3)Microbubble generation-By W.B. Zimmermann, http://www.eyrie.shef.ac.uk.

4)http://www.wikipedia.com

5)Ultrasound activated microbubble drug delivery-http://www.wellsphere.com

6) http://suwaprecision.com