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Non-Invasive Therapy & Drug Delivery

Therapeutic Ultrasound & Drug Delivery

BUBBL: Non-Invasive Therapy & Drug Delivery

Externally applied energy forms such as ultrasound, shock-waves, magnetic or radiation fields can be deployed under real-time image guidance for non-invasive surgery or to achieve targeted release and enhanced delivery of therapeutic agents from stimulus-responsive drug carriers.

A key focus of the research in the biomedical ultrasonics, biotherapy and biopharmaceuticals laboratory (BUBBL) is the identification, optimization and modeling of key mechanisms that can simultaneously enhance non-invasive therapy and enable real-time monitoring and control of the therapeutic process deep within the body.

The BUBBL research facility is unique by virtue of its capability to manufacture a wide array of stimulus-responsive micro- and nano-carriers, ranging from liposomes to multi-layered vesicles for multi-modality and theranostic use, as well as having the capacity  to design, build and test the customized therapeutic devices, which provide the stimuli to impact upon these carriers.

Key application areas include the treatment of cancer and solid tumours, delivery through the blood-brain barrier and stroke therapy, and spinal therapies, whilst therapeutic agents being delivered range from conventional chemotherapeutics to siRNA and oncolytic viruses.

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BUBBL Facilities

Diagnostic and Therapeutic Ultrasound Laboratory Equipment

  • 2 large beamplotting tanks for fully automated 3-D acoustic characterization measurements.
  • 11 High Intensity Focussed Ultrasound (HIFU) transducers (0.25-3.3 MHz)
  • 9 Power Amplifiers (50-1000W) for HIFU delivery
  • 1 PVDF membrane, 8 PVDF needle, 2 PZT, and 1 fiber-optic hydrophones for validated charcterization of acoustic fields.
  • 3 different platforms for diagnostic ultrasound and therapy monitoring

 

Clinical Therapeutic Ultrasound Equipment

BUBBL is conducting a number of clinical trials in collaboration with the clinical HIFU unit in the Cancer Centre of the Oxford University Hospitals Trust, which is located on the same site as the BUBBL laboratory. The following clinical devices are in routine clinical use: 

Biopharmaceuticals and Encapsulation Equipment

  • FTS Lyostar I freeze-drying system
  • EHD (Electrohydrodynamic) spraying
  • Microfluidic devices for ultrasound contrast agent manufacturing
  • Virtis Advantage Plus freeze-drying system
  • Sorvall ST16R centrifuge
  • Buchi B205 Rotavapor
  • Furnace with temperature control (ThermoScientific)


Analytical Equipment

  • Varian Cary 50 UV/VIS spectrophotometer
  • Varian Prostar dual pump HPLC system with UV detector
  • Asymmetric Flow Field Flow Fractionation system (Postnova Analytics)
  • 880 nm laser for Raman scattering
  • Malvern Zetasizer Nano ZS ZEN3600
  • Malvern Mastersizer S laser light diffraction system
  • Mettler-Toledo DL39 Karl-Fischer-Titrator with Stombuli Oven
  • ATC Pycnomatic helium pycnometer

Triggered Drug Release

Conventional methods of drug administration such as tablets or intravenous injection typically distribute a drug throughout the body. This may be undesirable, however, in the case of drugs which show poor uptake in certain types of tissue and/or produce unwanted side effects.

The aim of the research being carried out in BUBBL is to develop systems which enable drugs to be encapsulated, targeted to a specific region and released “on demand” in response to an external stimulus, for example exposure to ultrasound. Such systems provide not only a means of controlling the drug concentration and reducing the risk of harmful side-effects, but also control over treatment location and timing.

Vehicle Design & Fabrication

One of the key areas of research is the design and fabrication of stimuli responsive vehicles for drug delivery at the nanoscale and microscale. These include: engineered microbubbles, thermally and/or acoustically sensitive liposomes, polymeric capsules, sonosensitive nanoparticles and stabilized nanoemulsions.

A variety of manufacturing techniques are available in the laboratory for fabricating these vehicles, for example:

  • Microfluidics
  • Electrospraying
  • Spray drying
  • Freeze drying
  • Ultrasonic emulsification
  • Templating methods
  • Extrusion
  • Thin film hydration

Targeting Strategies

Magnetic MicrobubblesIn addition to encapsulating the drug, drug carriers can also be engineered to enable them to be concentrated in a target region. Strategies include:

  • Controlling the particle size distribution to exploit biological uptake mechanisms such as the enhanced permeability and retention (EPR) effect exhibited by certain types of tumour.
  • Incorporating magnetic nanoparticles to enable vehicles to be manipulated in vivo using and externally applied magnetic field.
  • Attaching targeting ligands to the vehicle surface to facilitate binding to target cells.
  • Incorporating acoustically active components and/or conjugating with microbubbles to exploit acoustic radiation force.

See also enhanced drug delivery.

Release Mechanisms

Release of the drug may be induced by exposing the vehicles to various external stimuli. The majority of the research currently being conducted in BUBBL is focused on the use of ultrasound to trigger release through:

  • Generation of heat to activate thermally sensitive carriers.
  • Generation of cavitation to physically disrupt carriers.
  • Mechanical oscillation of acoustically active vehicles.

See also enhanced drug delivery.

Other methods are also being investigated including:

  • Photosensitivity through the inclusion of gold nanoparticles
  • Sensitivity to certain chemical species/tissue environments
  • Charge sensitivity

The presence of stimuli responsive components may also be exploited to generate a signal detectable outside the body that can be utilized for monitoring of drug delivery.

For further details please see our publications pages.

Enhanced Drug Delivery

We have developed technologies to ensure liposomal and virus-based therapeutics can achieve good circulation following injection into the bloodstream, decreasing uptake into non-target tissue and allowing a level of accumulation in tumour deposits. However, at present, release of the tumor-cell-killing cargo and penetration of that cargo deep into the tumour is still sub-optimal.

In response, we have developed systems which utilise focused ultrasound as a stimulus to trigger release of therapeutic payload and propel it deep into tumours. The ultrasound induced phenomena that drive this release and movement are the shock waves microstreaming created by inertial cavitation, an event resulting from the rapid expansionand violent collapse of a gas bubble in response to the rarefactional and compressional pressures exerted by an ultrasound wave.

Inertial cavitation can be detected and mapped in real-time and in 3D using a technique developed in Oxford, known as Passive Acoustic Mapping (PAM). The location of the stimulus driving drug delivery can thus be correlated to the location of the delivery. See also drug delivery monitoring.

adenovirus in green remains close to blood vessels in red   adenovirus is detected hundreds of microns from the blood vessels

Ultrasound mediated penetration of adenovirus vector deep into tumours. In the absence of inertial cavitation (left image) adenovirus in green remains close to blood vessels in red. In the presence of inertial cavitation (right image) adenovirus is detected hundreds of microns from the blood vessels.

For further details please see our publications pages.

Therapy Monitoring

In many ultrasound-based therapies, cavitation (the collapse of bubbles) can play a major role in treatment:

  • inertial cavitation greatly enhances heating during HIFU
  • cavitation acts in various ways to aid localised drug delivery
  • inertial cavitation is a key factor in tissue fractionation (or histotripsy) and also important in shock wave lithotripsy

Mapping the spatial and temporal extent of cavitation is therefore an important concern when monitoring ultrasound treatments. This is usually attempted using B-mode ultrasound imaging, where the presence of bubbles creates increased backscatter (hyperechoes) from the probing ultrasound pulses.  Alternatively, we have developed array systems and algorithms to conduct passive acoustic mapping (PAM) that alternatively can provide accurate spatio-temporal maps of the bubble activity in real-time during the therapy.  Since cavitation can act to promote positive therapeutic effects, this activity should spatially correspond to the treated region, and this has been confirmed through both in-vitro and in-vivo experiments.

PAM System Development:
Systems have been developed for real-time fully 3D PAM processing.  These systems consist of custom arrays made of PVDF coupled to low-noise preamplifiers and rapid data collection system.  The arrays have been primarily designed to not inhibit the existing CE mark of the clinical equipment.  Developed software allows for PAM in the three primary planes at real-time clinical frame rates.

Therapy Monitoring 1

Figure: Custom sparse 2D array systems for performing 3D PAM for both HIFU (left) and drug delivery (right) applications where the sensor array system design is made to not remove existing CE marking from the existing ultrasound equipment.  The 32 channel PAM array for HIFU has been used in an in-vivo clinical trial and the 64 channel PAM array for drug delivery has been used in in-vitro experiments.

PAM Algorithm Development:
Both sparse linear weighting and optimal beamforming algorithms have been incorporated into improving PAM.  Several issues exist to reduce the resolution and image quality of PAM images including low channel count systems and interference from scatterers and multi-bubble interaction.  Improvements in the algorithms can aid in improving the resulting images to correlate the cavitation activity to the location of the bioeffect allowing for improved monitoring.

Therapy monitoring 2

Figure: Cumulative PAM maps of in-vivo experimental drug delivery for conventional (left) and optimal (right) processing collected over 20 HIFU pulses. The white ellipse shows the bounds of the 3dB HIFU focus, which corresponds best to the cavitation activity using the optimal processing (right).

For further details please see our Publications pages.

Ablative Therapies

High-amplitude ultrasound waves, generated outside the body, can be focused deep within tissue onto a region about the size of a grain of rice. In that region, conversion of the mechanical energy carried by the ultrasound wave into heat can lead to cell death by thermal necrosis, whilst leaving tissue outside the HIFU focal region unaffected. The potential of this technique to destroy deep-seated tumours non-invasively is currently being explored in the Clinical HIFU Unit at the Churchill Hospital in Oxford.

Contrast MR of a large tumour in the left kidney (left) and 12 days following HIFU treatment (right)

Contrast MR of a large tumour in the left kidney (left) and 12 days following HIFU treatment (right). Photo courtesy of the Clinical HIFU Unit, Churchill Hospital, Oxford.

The research being carried out in the IBME is aimed at further improving the speed, resolution, targeting and real-time monitoring of HIFU treatments, as applied to cancer therapy and to a range of novel HIFU applications. The laboratory is involved in research at all levels, ranging from the basic science of understanding cell death by ultasound-induced heating, to the clinical trials (in collaboration with the clinical HIFU unit) of novel methods for improved treatment delivery and treatment monitoring. The thermal dose that results in cell death, changes in the material properties of tissue in response to heating and ablation, and method.

One area of research is related to acoustic cavitation, a phenomenon that occurs when high amplitude acoustic waves propagating through tissue spontaneously nucleate and excite small, micrometre-sized bubbles. The presence of such bubbles at the HIFU focus has been shown to greatly enhance the local rate of heating. This excess heating can be exploited to reduce treatment times or, through the use of cavitation promoting agents, increase the specificity of HIFU therapy. In addition, the acoustic emissions associated with cavitation can be used to provide additional monitoring during a treatment. Work performed in excised tissue has shown that spatial localisation of cavitation events through a technique called passive acoustic mapping (PAM) provides a better means of discriminating whether or not a lesion has been formed. This is being utilized in several of the projects ongoing in BUBBL with the aim of enabling faster, safer procedures to be performed.

Ex-vivo Inertial cavitation mapping

Ex-vivo Inertial cavitation mapping during 1.1 MHz HIFU using a commercial ultrasound probe (L10-5, Zonare Medical Systems). A lesion was formed whose location was well correlated with passive acoustic maps reconstructed from both the broadband and harmonic parts of the spectrum. These are considered to be representative of two regimes of cavitation, inertial and stable. In contrast, conventional assessment through detection of B-mode hyper-echo did not detect the lesion.

Lesions of different shapes and sizes created by a HIFU transducer in a polyacrylamide-BSA tissue-mimicking material

Lesions of different shapes and sizes created by a HIFU transducer in a polyacrylamide-BSA tissue-mimicking material.

A further area of interest pertains to the development of our understanding of heat-induced cell death. In 1984, Sapareto and Dewey proposed the thermal dose cumulative equivalent minutes (CEM) metric as a means of quantifying cell damage induced by mild heating. Even though the model provides a good descriptor at the temperatures and rates of heating encountered in mild hyperthermia (5-10 C above body temperature), its applicability in the context of the very high rates of heating and high temperatures achieved during ultrasound-induced ablation has yet to be demonstrated. Through the development of a broad range of cell-embedding tissue-mimicking materials, research carried out in BUBBL aims at acquiring a basic understanding of the limitations of the CEM model and at the development of adequate means of quantifying heat-induced damage under conditions relevant to HIFU therapy.

A third area of interest involves monitoring tissue ablation by tracking changes in material properties of the tissue. The current state of the art is MR guided HIFU but this involves delivering HIFU within an MR system, which is both expensive and technically challenging.  Properties that are known to change when tissue is heated or ablated include its sound speed, acoustic attenuation, stiffness, optical and electromagnetic scattering, absorption and dielectric constants. We are developing various ultrasound or dual-wave modalities for imaging the changes in these properties such that ablation can be monitored in real time.

Tissue Fractionation

In addition to enhancing thermal ablation of tissue, the intense activity of cavitation bubbles induced by focused ultrasound can also produce mechanical effects. These can be exploited for the minimally invasive removal of diseased tissue.

One of the projects ongoing in the IBME seeks to apply this to the treatment of spinal disease. Four out of five adults will suffer from low back pain during their lifetime and around 5% of sufferers become chronically disabled. This imposes a high economic and social burden on society because the disorder affects people of working age as well as the elderly, with the total cost being estimated to be over 1 % of the UK's GDP. Low back pain is strongly associated with degeneration of the intervertebral discs, the soft tissues that connect the spinal vertebrae and allow the spine to articulate. Current surgical treatments for low back pain are highly invasive and have relatively low long term success rates. The present work aims to develop a novel, minimally invasive therapy for disc replacement without the need for surgical incision. If successful, it has the potential to revolutionise clinical practice for the treatment of back pain, thus improving quality of life and reducing the economic impact of this major disease.

In this project we will employ high intensity focused ultrasound to fractionate degenerated tissue in a tightly controlled region so that it can be easily removed by minimally invasive needle. A new class of self assembling peptide gels will then be injected through the same needle into the cavity to restore the disc's mechanical function. These developments will involve a combined programme of computational and experimental modelling of the acoustics and mechanics of the disc and the surrounding vertebral structures in order to optimise the performance of the procedure across the likely variance in disc properties seen in a typical patient population.

Shock Wave Therapies

Acoustic shock waves use high amplitude acoustic pulses (normally in the range of 10 to 100 MPa) with durations on the order of 1 microsecond.  They can induce therapeutic effects in tissue by mechanical means: either through direct stress/strain or by acoustic cavitation.  The main use of shock waves in medicine has been lithotripsy in which shock waves are used to fragment kidney stones so that they can be passed naturally.  However, shock waves have also been considered for other applications such as treatment of soft-tissue pain (e.g. tendonitis and heel spurs), promoting repair or growth of bone, neo-vascularisation and wound healing. The efficacy of these other applications is not always clear and the mechanisms are poorly understood.

Areas of research carried out in BUBBL include:

  • Understanding shock wave interaction with elastic objects, in particular kidney stones and bone. This image is a snapshot showing the stress field created in a natural kidney stone.
    Shock Wave Interaction
  • Interaction of shock waves with soft tissue and how shock waves may damage tissue. This image is a B-mode ultrasound image of a pig kidney immediately after being subject to a shock wave - the circled region is cavitation created by the shock wave in the collecting system.
    Shock Wave Damage
  • Tracking of kidney stones during lithotripsy.
    Tracey
  • How shock waves may promote repair of bone fracture.
 This image shows a fractured rat femur that has been subject to shock waves.  The circled region shows the initial formation of new bone.
    Shock waves may promote repair of bone fracture