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

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 Malvern PANalytical

Agenda  An introduction to fine / nanobubbles  Standardising fine bubbles  A brief overview of how we measure fine bubbles  Applications of fine bubbles Malvern PANalytical

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 Malvern PANalytical

Standardising fine bubbles  ISO committee TC281(4 years old)  2 nd Chairman  3 work groups – definitions, characterisation methods, applications  Why standardise? – Speak the same language – Do things the same way – Evaluate performance of “same” technologies Malvern PANalytical

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 Malvern PANalytical

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. Malvern PANalytical

NTA Malvern PANalytical

Introduction to NanoSight 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. Malvern PANalytical 2

Particles are Visualised Directly, in Real Time › 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) Bubbles in flow › Speed of particles varies directly with particle size Malvern PANalytical 2

Principle of Measurement  Nanoparticles move under Brownian motion due to the random movement of 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 of the Stokes-Einstein equation particle size can be calculated. Malvern PANalytical 3

Nanoparticle Tracking Analysis Nanoparticle Tracking Analysis (NTA) is the gathering of unique information and comes from assessment of individual particles, rather than averaging over a bulk sample . capture tracking analysis Malvern PANalytical 4

Particle Sizing in action - Software Analysis 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 Malvern PANalytical 5

NTA Detection Limits Size Concentration Minimum size limit is related to: Minimum concentration is related to: › Material type › Wavelength and power of illumination › Poor statistics (Requiring longer analysis source time) › Sensitivity of the camera ox 10 6 / mL Appr 10 – 50 nm (50nm bubbles) Maximum concentration is related Maximum Size limit is related to: to: › Limited Brownian motion › Inability to resolve neighboring particles › Viscosity of solvent › Tracks too short before crossing occurs Approx 10 9 / mL 1000 – 2000 nm Malvern PANalytical 6

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 5 x 180s Runs Mode size 54.7 ± 1.1nm • Mode size 84.9 ± 4.8nm • 3.91x10^8 part/ml ± 1.5x10^7 • 3.35x10^8 part/ml ± 8.2x10^6 • • 6768 particles per analysis • 6187 particles per analysis Malvern PANalytical

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 Malvern PANalytical

Time experiment  Concentration increases over time. Getting into area where dilution needed for NTA Malvern PANalytical

Importance of NTA Statistics: • Sample flowing because of 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 Malvern PANalytical

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

Resonant Mass Measurement for Bubble Measurements Malvern PANalytical

An Introduction to Resonant Mass Measurement MEMS Microchannel Resonator Sensor • 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 bypass channel resonator Malvern PANalytical

Measuring Particle Mass in Fluid 0 Frequency Shift (mHz) 1. 3. -100 -200 1. Relates -300 to 2. particle mass 3. -400 2. -150 -100 -50 0 50 100 150 200 Time (msec) Malvern PANalytical

Archimedes Instrument Signal Processing Optics Pneumatics Fluidics Frequency Measurement PC and Feedback pressure source Sensor sample waste Malvern PANalytical

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 Malvern PANalytical

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 Malvern PANalytical

Bubble UK  Applications include – Ultrasound contrast agents – Cleaning of contaminants – Washing of meat and vegetables – Agrochemical sprays (bubbles in drops control drift) – And….. Malvern PANalytical

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 Malvern PANalytical

Barley seed germination Malvern PANalytical

Extracorporeal and Injectable Medical Devices to Enhance Oncological Drug Delivery Constantin Coussios & Eleanor Stride Institute of Biomedical Engineering, University of Oxford Malvern PANalytical

And when the press get hold of it!! Malvern PANalytical

A Universal Problem Across Drug Classes TREND TOWARDS BIOLOGICS REPRESENTS CHEMO- ANTIBODIES / ONCOLYTIC VIRUSES / GROWING IMMUNO-ONCOLOGY THERAPEUTICS IMMUNO-ONCOLOGY CLINICAL 100- <1nm 15nm NEED 300nm Malvern PANalytical

Tumour Physiology as a Barrier to Drug Delivery Irregular Vasculature high vascular  Size selectivity of the tumour density endothelium (100-600 nm) low vascular density  Elevated intratumoral pressure (hypoxic region) high vascular (~ 20 mm Hg) density  Highly irregular tumour vasculature vasculature 100 µm  Increased distance between nearest blood vessel and farthest cell (~ 200 um in tumours vs 90 um in tissue) cancer cells therapeutic agents Malvern PANalytical