The outline and latest status of fine bubble measurement techniques - - PowerPoint PPT Presentation

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The outline and latest status of fine bubble measurement techniques and UK bubble applications including advances in drug delivery using bubbles

Dr Stephen Ward-Smith Malvern Instruments Ltd

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Agenda

• An introduction to fine / nanobubbles
• Standardising fine bubbles
• A brief overview of how we measure fine bubbles
• Applications of fine bubbles

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Definitions

• An ultrafine bubble or nanobubble is a bubble (or bubble distribution) that is

predominantly <1 micron (most is less than 100nm).

• That is 1/1000 of a mm
• A microbubble is 1 – 100 microns in size

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Standardising fine bubbles

• ISO committee TC281(4 years old)
• 2nd Chairman
• 3 work groups – definitions, characterisation methods, applications
• Why standardise?

– Speak the same language – Do things the same way – Evaluate performance of “same” technologies

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Techniques used for characterising fine and ultrafine bubbles

• Particle tracking analysis (aka Nanoparticle tracking

analysis, NTA, PTA)

• Resonance Mass Measurement
• Dynamic Light Scattering (aka Photon Correlation

Spectroscopy)

• Laser Diffraction
• Zeta potential
• Others (electrozone sensing, ultrasonics, static multiple

light scattering, image analysis)

• All are sizing techniques, bar Zeta Potential which is a

measure of particle charge

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Brief Summary

Technique Size Range Laser Diffraction <100nm to >2mm Dynamic Light Scattering <1nm to >1 micron NTA <30nm to >1 micron RMM (bubbles) <100nm to > 2micron Electrozone sensing <100nm to >3 mm

• Particle size ranges will depend on the

sample and the sensor used.

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NTA

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NanoSight provides data on particle size distribution, concentration and aggregation, with much higher resolution than has been previously

• possible. The addition of a fluorescence options dramatically extends

the capabilities of the instruments, allowing truly multi-parametric characterisation of nanoparticles. Endorsed by an exponential growth in scientists citing its use in scientific papers, and applicable in a wide range of fluids, including complex biological systems, NanoSight instruments provide scientists with detailed data and knowledge of nanoparticle systems that was previously unavailable.

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Introduction to NanoSight

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Particles are Visualised Directly, in Real Time

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Bubbles in flow

› Particles are too small to be imaged by the microscope › The Particles seen as light points moving under Brownian motion › This is visualisation of scatter (not a resolved image) › Speed of particles varies directly with particle size

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Principle of Measurement

• Nanoparticles move under Brownian

motion due to the random movement

• f water molecules surrounding them.
• Small particle move faster than larger

particles.

• Diffusion Coefficient can be calculated

by tracking the movement of each particle and then through application

• f the Stokes-Einstein equation

particle size can be calculated.

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Nanoparticle Tracking Analysis (NTA) is the gathering of unique information and comes from assessment of individual particles, rather than averaging over a bulk sample.

analysis tracking capture

Nanoparticle Tracking Analysis

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The NanoSight NTA (nano-particle tracking) analysis suite allows for captured video footage to be simultaneously tracked and analysed…

Nanoparticles being tracked and analysed by NanoSight NTA

Particle Sizing in action - Software Analysis

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Minimum size limit is related to:

› Material type › Wavelength and power of illumination source › Sensitivity of the camera

Size Concentration

Minimum concentration is related to:

› Poor statistics (Requiring longer analysis time)

10 – 50 nm (50nm bubbles)

1000 – 2000 nm

Maximum Size limit is related to:

› Limited Brownian motion › Viscosity of solvent

Appr

• x 106 / mL

Maximum concentration is related to:

› Inability to resolve neighboring particles › Tracks too short before crossing occurs

Approx 109 / mL NTA Detection Limits

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Importance of NTA Statistics:

• In order to standardize measurements with NTA, it is likely that

some measure of minimum particle counts will need to be established with community.

• More particles analyzed results in better repeatability.
• In addition more narrow size distributions result in better
• reproducibility. Height : base ratio??
• 5 x 60s Runs
• Mode size 54.7 ±1.1nm
• 3.91x10^8 part/ml ± 1.5x10^7
• 6768 particles per analysis
• 5 x 180s Runs
• Mode size 84.9 ±4.8nm
• 3.35x10^8 part/ml ± 8.2x10^6
• 6187 particles per analysis

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First batch of results with IDEC generator

• Since December 2016 have had a kind loan of an IDEC generator
• Freezing removed all sub micron material from the samples (so freeze / thaw

appears to be a good “proof” of the existence of bubbles

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Time experiment

• Concentration increases over time. Getting into area where dilution needed for

NTA

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Importance of NTA Statistics:

• Sample flowing because
• f syringe pump.
• Particle size and

concentration still measured accurately despite flow.

• Flowing the liquid means

that more particles are measured - better sampling.

• More particle analyzed =

better reproducibility.

• The broader the size

distribution the more particles should be analyzed

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UFB sample 3 months on – still 3 x10^8 left

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Resonant Mass Measurement for Bubble Measurements

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An Introduction to Resonant Mass Measurement

Microchannel Resonator

• 8x8µm or 2x2µm Microfluidic channel embedded inside

resonator in the form of a cantilever design

• A particle passing through the resonator changes the

total mass of the resonator and shifts the resonant frequency

• Excursion in resonant frequency gives an accurate and

precise measure of particle’s buoyant mass, which can be converted in to dry mass and size using the fluid and particle density

resonator bypass channel

MEMS Sensor

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Measuring Particle Mass in Fluid

• 150
• 100
• 50

50 100 150

• 400
• 300
• 200
• 100

Frequency Shift (mHz) Time (msec)

200

1. 3. 2. 1. 2. 3.

Relates to particle mass

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Archimedes Instrument

PC Signal Processing Frequency Measurement and Feedback Sensor Optics Pneumatics Fluidics

sample waste pressure source

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Archimedes for Bubble Measurements

• Why use RMM with bubble samples:

– RMM is the only technique which offers large scale characterisation of bubbles – This is particularly useful when bubbles are coated and therefor Archimedes can differentiate between droplets of the coating material and bubbles

• How does the Archimedes system cater for bubbles:

– Ability to customise the pneumatic pressures used for loading to ensure the sample is not damaged during pneumatics operations. Typical pressures used for bubbles are around 20.7kPa (3 PSI). – Our accessible vials allow sample to be stirred to avoid creaming during the measurement – Sample can be frequently reloaded throughout the measurement

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Current Limitations for bubble measurements with RMM

• The very smallest bubble that can be seen at present using RMM are around 120nm.
• To improve this we are investigating:

– Improving measurement noise – Improving system sensitivity – Surface interactions which may be preventing the bubbles moving through the resonator – Pressures used to load the sample

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Bubble UK

• Applications include

– Ultrasound contrast agents – Cleaning of contaminants – Washing of meat and vegetables – Agrochemical sprays (bubbles in drops control drift) – And…..

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Bubble World

• Hydroponics (10 - 20% more growth)
• Fish farming (growth, fish delivery)
• Cleaning (floors, machine oil off metal parts)
• Seed Germination (earlier, more )
• Nanobubble washing machine
• Spa baths / showers

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Barley seed germination

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Extracorporeal and Injectable Medical Devices to Enhance Oncological Drug Delivery

Constantin Coussios & Eleanor Stride

Institute of Biomedical Engineering, University of Oxford

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And when the press get hold of it!!

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A Universal Problem Across Drug Classes 15nm 100- 300nm

TREND TOWARDS BIOLOGICS REPRESENTS GROWING CLINICAL NEED

CHEMO- THERAPEUTICS ANTIBODIES / IMMUNO-ONCOLOGY ONCOLYTIC VIRUSES / IMMUNO-ONCOLOGY <1nm

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Tumour Physiology as a Barrier to Drug Delivery

• Size selectivity of the tumour

endothelium (100-600 nm)

• Elevated intratumoral pressure

(~ 20 mm Hg)

• Highly irregular tumour

vasculature

• Increased distance between

nearest blood vessel and farthest cell (~ 200 um in tumours vs 90 um in tissue)

Irregular Vasculature

high vascular density low vascular density (hypoxic region) high vascular density

100 µm

vasculature cancer cells therapeutic agents

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Key Challenges in Oncological Drug Delivery

• Off-target toxicity
• Poor accumulation of drug in tumour for a given systemic dose

(typically 0.5-3% of injected dose)

• Poor penetration of drug in tumour
• The larger the therapeutic, the greater the delivery challenges…

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Cavitation of microbubbles can drive drugs out of the blood vessels and into the perivascular space of the tumours

Microbubble Endothelial cells within healthy tissue Endothelial cells within cancerous tissue

Micropumping from within the vasculature

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Submicron cavitation nuclei propel both drug and cavitation nuclei out of the ‘leaky’ vasculature and deep into the tumour

Sub micron cavitation nuclei

Micropumping from within the vasculature and the tumour

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Mean hydrodynamic diameter ~ 400 nm Mean cavity diameter ~ 200 nm Cavitation threshold at 0.5 MHz ~ 0.5 MPa

Particle size distribution Cavity size distribution Cavitation threshold

10 20 30 40 50 10 100 1,000 Proportion (%) Particle diameter (nm) 10 20 30 40 50 10 100 1,000 Proportion (%) Particle diameter (nm) 20 40 60 80 100 1 2 3 Probability of cavitation (%) Acoustic pressure (MPa) Kwan, J. J., Myers, R., Coviello, C. M., Graham, S. M., Shah, A. R., Stride, E., Carlisle, R. C. & Coussios, C. C. (2015). Ultrasound-propelled nanocups for drug delivery. Small, 39 (11) 5305-5314.

Sub-micron cavitation nuclei (polymeric cups)

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Virus alone caused regression in just 1 of 4 mice but cavitation-enhanced delivery of a virus caused regression in 4 of 4 mice

Myers, R., Kwan,J., Coviello, C., Carlisle, R. & Coussios, C. C. et al., (2016) Polymeric cups for cavitation mediated delivery of oncolytic vaccinia virus. Molecular Therapy

Cavitation-Mediated Delivery Enhances Viral Efficacy

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Enhanced IgG Antibody Delivery in CT- 26/Balbc (IV)

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CONTROL TREATMENT

Tumour Cells (BLUE) Blood Vessels (RED) Antibody (GREEN)

Kwan, J. J., Myers, R., Coviello, C. M., Graham, S. M., Shah, A. R., Stride, E., Carlisle, R. C. & Coussios, C. C. (2015). Ultrasound-propelled nanocups for drug delivery. Small, 39 (11) 5305-5314.

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Enhanced Anti-PD-L1 Therapy in CT-26/Balbc (IV)

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Anti-PD-L1 + Cups Anti-PD-L1 + Cups + US

= 100μg anti-PD-L1 dose administered with or without SonoTran

1st anti-PD-L1 dose administered 10 days post-implantation (once tumours were established with volumes ~ 50mm3).Mice culled if tumour >1000mm3 or if there was ulceration of the tumour.

200 400 600 800 1000 10 20 200 400 600 800 1000 10 20

• Day 28: 1 / 5 mice surviving in the control group
• Day 28: 4 / 5 mice surviving in the SonoTran treatment group

Days Post-Implantation Days Post-Implantation Tumour Size (mm3) Tumour Size (mm3)

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• Ultrasound responsive

core (gas or volatile liquid)

• Surfactant or

polymer shell to stabilise the bubble and improve pharmacokinetics

• Drugs can be incorporated into the

shell and released by exposure to focused ultrasound at the target location

• Targeting

species

Courtesy of Dr RJ Eckersley

Drug Encapsulation

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Ultrasound + bubbles Ultrasound only No exposure Bubbles only

Stride et al. Ultrasound in Medicine & Biology 35:861-868 (2009)

Localised Delivery

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Cavitation Agents

Size Coating Gas

• Design parameters
• Stability
• Acoustic

response

• Clearance time
• Extravasation
• Payload capacity
• Stability
• Acoustic

response

• Clearance time
• Targeting
• Drug

incorporation

• Stability
• Acoustic

response

• Therapeutic action
• Cell interaction

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Role of the core gas

• Reduced levels of oxygen in tissue (hypoxia) play a major

role in the development, progression and treatment of many diseases.

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• SDT involves the use of a drug that is only activated upon

exposure to ultrasound.

• It thus has excellent potential for delivering targeted therapy with

minimal side effects.

• However, drug activation also requires the presence of oxygen.

+ =

• 2

Reactive

• xygen

species

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4 3 SDT with SF6 microbubbles SDT with O2 microbubbles no ultrasound with ultrasound with ultrasound no ultrasound Standard microbubbles Oxygen microbubbles with ultrasound no ultrasound

McEwan et al. J. Cont. Rel. 203:51-56 (2015)

Pilot Study

• Human primary pancreatic adenocarcinoma (BxPC3)

xenograft tumours in male Balb/c SCID mice (n = 8/group).

• Rose Bengal with Perfluorocarbon or oxygen microbubbles

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Summary

• The thermal and mechanical effects
• f ultrasound can be exploited to

enhance the localisation, delivery and efficacy of therapeutic agents, including:

– small molecules – viruses – antibodies – oligonucleotides

• Acoustically responsive micro and

nano-scale particles can be used to further enhance these effects by promoting cavitation.

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Summary

• These particles can be engineered

to encapsulate therapeutic material as well as targeted to specific sites.

• They can also be designed to

modify the tissue environment in

• rder to promote therapy.
• The approach is non-invasive and

facilitates real-time monitoring of delivery and release.

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Acknowledgements

• Bubble team at Malvern Instruments
• Dr Eleanor Stride at her team at Oxford University
• IDEC – Dr Fujita and his team for loan of generator
• Japanese fine and ultrafine bubble community at the FBIA

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Thank you for your attention

• Any Questions?

Steve Ward–Smith - stephen.ward-smith@malvern.com

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