Microbubble preparation and characterisation for the biomedical applications

Ketan Pancholi, School of Engineering, Robert Gordon University, Schoolhill, Aberdeen ,AB10 1FR

T-junction

• T-junction1,2 three dimensional aligned and easy to assemble.• For each oil of given viscosity , the effect of varying the gas pressure (Pg)

from 0.64 KPa to 17 KPa was determined for different liquid flow rates (Ql) from 1.66 x 10-10 to 8.33 x 10-8 m3/s.

20m

Focussing Region

1 K.Pancholi, M.Edirisinghe (2007) Microbubbling of viscous media, American Physical Society, March Meeting , Denver, USA2 *International patent. PCT filed on 28/03/08 (PCT/GB2008/001050)

Experimental Set-upHigh pressure withstanding T-junction enclosed in polymer block with guiding holes to self align tubes on its own axis. Total set up is shown including high speed camera, stainless steel syringe able to withstand 50MPa pressure.

•First air thread enters liquid column downstream to establish two phase flow*•Air column reduces effective area for liquid to flow•This increases pressure at air liquid interface as liquid is supplied at constant flow rate by pump to maintain flow rate.•A sharp increase in liquid pressure normal to interface starts decreasing air column width

*K.Pancholi, E.Stride, M.Edirisinghe (2008) Dynamics of bubble in viscous liquid, Langmuir, 24 (8), 4388 -4393

100µm

Optical micrograph of monodisperse bubbles produced in Lipid-water suspension using the T-junction*.

Ultrasonic transmission response to bubbles produced using different techniques. A) Blue and grey plot shows water and suspension response. B) Red dotted line shows T-junction bubbles response*.

*K.Pancholi, U.Farook, R.Moaleji, E.Stride,M.Edirisinghe (2008),Novel Method for preparing phospholipids coated microbubbles, European Biophysics Journal, Volume 37, Number 4.

Use and application of Microbubbles*

• When ultrasound pulse is transmitted through microbubble suspension backscatter from the microbubbles only contain one frequency

• Backscatter depends on microbubble radius, gas core and microbubble coatings

• Particles surrounding increases stiffness of the bubble in response to ultrasound

• Results in response containing multiple frequency

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*E. Stride, K. Pancholi, M. J. Edirisinghe and S. Samarasinghe (2008) Increasing the nonlinear character of microbubble oscillations at low acoustic pressures, Journal of Royal Society Interface, 5(24):807-11

Ultrasound response of nano-particle coated microbubbles

• Experimental measurements of backscattered ultrasound power showing the amplification of nonlinear behaviour. The black curve shows the frequency spectrum for the backscattered power from a bubble suspension without nano-particle and the grey curve shows

a spectra for a bubble suspension coated with gold nano-particles.

• Experimental measurements of backscattered ultrasound power showing the amplification of nonlinear behaviour. The black curve shows the frequency spectrum for the backscattered power from a bubble suspension without nano-particle and the grey curve shows

a spectra for a bubble suspension coated with gold nano-particles.

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Physics of fluid

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• Slender body theory applied to drop• Drop placed in hyperbolic flow•Tangential flow boundary condition•Normal stress balance •Plotted radius of droplet against length of droplet in direction of flow• Both liquid viscosity (µl) and the ratio of liquid to gas flow rate (Ql/Qg) influences the size of the bubble •with higher flow rates and higher viscosities smaller bubbles form but only up to a limiting value of Ql/Qg and/or µl.

• Slender body theory applied to drop• Drop placed in hyperbolic flow•Tangential flow boundary condition•Normal stress balance •Plotted radius of droplet against length of droplet in direction of flow• Both liquid viscosity (µl) and the ratio of liquid to gas flow rate (Ql/Qg) influences the size of the bubble •with higher flow rates and higher viscosities smaller bubbles form but only up to a limiting value of Ql/Qg and/or µl.

*K.Pancholi, E.Stride, M.Edirisinghe (2008) Dynamics of bubble in viscous liquid, Langmuir, 24 (8), 4388 -4393

Two liquid supply capillaries. The angle between liquid supply and gas supply could vary from 0-90 degrees

The angle between liquid supply and gas supply is shown 45 degrees.

The angle between liquid supply and gas supply is shown 0 degrees.

The angle between liquid supply and gas supply is shown 0 degrees and no third capillary. In all other cases third capillary can be avoided as well.

*UK patent application No. 02776963. PCT filed on 28/03/08

Possible designs of the T-junctionPossible designs of the T-junction

(PCT/GB2008/001050)

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Harvard PHD-4400 syringe pump

Collection vial

T-junction

Manometer

Pressurized Air Tank

Potential electrical difference applied

Dimple

Hybrid method*•Without electrospraying bubble size of 30 mm with ± 0.1μm standard deviation.

•After electrospraying bubble size obtained was 5.1μm with ± 2μm standard deviation.

• Stability of bubbles found to be 2 hours without any change in size.

•Bubbles produced with this method guarantees that all bubbles are less than 7 mm which is better than currentlyavailable FDA approved microbubbles.

20µm

Optical micrograph of microbubbles collected directly from the outlet of the T-junction a) without an applied electric field and b) with electric field.

*K.Pancholi, E.Stride, M.Edirisinghe (2008),Journal of targeted drug delivery, 16(6):494-501.

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Insonation of chitosan suspension and subsequent electrospraying of foam in sodium hydroxide solution.•Increase in chitosan concentration in solution controls the release of drug.

•Variation in acetic acid in chitosan solution can reduce surface tension without decreasing viscosity.

•Electrospray of highly viscous liquid increases the particle size but lowering surface tension results in smaller particles.

Chitosan particle size distribution for different surface tension but nearly same viscosity.

Ketan Pancholi, Eleanor Stride and Mohan Edirisinghe (2008), Novel preparation of porous chitosan particles for drug delivery, Journal of Materials in Medicine (submitted)

a cb

d

SEM images of chitosan particles made using electrohydrodynamic atomisation of chitosan foam. Foam was prepared by insonation of chitosan suspension. (a) Particle is of 500 nm range (b) Bulk particles (c-d) one particle out of bulk (e-f) probable porous membrane or void created by exposure of foam bubbles on surface.

e f

Image correlation analysis

1. Ketan Pancholi, Eleanor Stride and Mohan Edirisinghe (2008) Spatio-temporal image correlation spectroscopy for ultrasound assisted drug diffusion measurement.

4m

•Useful for measuring kinetics of particles at micrometer scale.• Enables counting number of nano-particles present on image.• Velocity, diffusion coefficient and direction of molecules can be measured. •The method can be extended to other types of characterising techniques.• Possible applications measurement of drug diffusion, interaction of signalling molecules with receptors and intracellular transport of any vector.

Re-distribution of receptors after exposure to receptor specific antibodies and confocal image of microbubbles

Image correlation spectroscopy to measure diffusion

1. Ketan Pancholi, Eleanor Stride and Mohan Edirisinghe (2008) Spatio-temporal image correlation spectroscopy for ultrasound assisted drug diffusion measurement.

•Experimental procedure•Real time image obtained using high speed camera•Fluorescence intensity of images were auto-correlated spatially and temporally1

•Simulation showing the temporal fluorescent intensity fluctuations due to diffusion of fluorescent dye from particle in water. •Darkest blue is lowest intensity and darkest red represents highest intensity

Estimation of bubbles diffusion• Microbubbles containing

fluorescent dye immobilised in water like gel

• Fluorescence intensity of images were auto-correlated spatially and temporally*

• Nonlinear data fitting to function to find out diffusion constant

*Ketan Pancholi, Eleanor Stride and Mohan Edirisinghe (2008) Spatio-temporal image correlation spectroscopy for ultrasound assisted drug diffusion measurement,