A beginner level presentation on surface active agents produced by microbes that can substitute synthetic ones for microbubble based applications.

1. BIOSURFACTANT AND THEIR
APPLICATION
IN MICROBUBBLE PREPARATION
BY ,
MISS ANKITA.K.GURAO
M.Sc BIOTECHNOLOGY II

2. CONTENTS
1)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.

3. 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.
●

4. 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
Rhamnolipids
Pseudomanas sp.
Sophorolipids
Sacchromyces sp.
Trehalolipids
Mycobacterium sp.
3.POLYMERIC
SURFACTANT
Bioemulsan
Acinobacter sp.
4.SECONDARY
METABOLITE
Viscosinamide
Pseudomonas flouroscens
2.GLYCOLIPIDS

6. 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.
●
In natural conditions, the organic molecule from
aqeous phase tend to immobilize at solid surface
and form the conditioning film.
This conditioning film changes the substratum
properties like wettability and surface tension.
●

8. 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).

9. ●
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.

10. 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.

11. 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.

12. 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.

13. 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..

14. 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.

15. 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.

16. 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).

17. 4)Polymer shell
●
Stabilized by a thick shell comprising crosslinked 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.

19. 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.

20. Applications of biosurfactant
stabilized microbubbles.
1) CEUS
2) Targeted CEUS
3) Drug delivery
4) Gene delivery

21. 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.

22. Contd..
● This process of backscattering gives the liquid
with these bubbles a high signal, which can be
seen in the resulting image.

23. 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.

24. 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.

26. 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.

27. Contd
.. ● This system finds vast application in
chemotherapy

28. 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).

29. 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.

30. 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 ßgalactose 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.

31. 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.

32. 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.

33. 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.

34. 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 deliveryhttp://www.wellsphere.com
6) http://suwaprecision.com