James J. Kwan

Ultrasound‐Propelled Nanocups for Drug Delivery

Ultrasound-induced bubble activity (cavitation) has been recently shown to actively transport and improve the distribution of therapeutic agents in tumors. However, existing cavitation-promoting agents are micron-sized and cannot sustain cavitation activity over prolonged time periods because they are rapidly destroyed upon ultrasound exposure. A novel ultrasound-responsive single-cavity polymeric nanoparticle (nanocup) capable of trapping and stabilizing gas against dissolution in the bloodstream is reported. Upon ultrasound exposure at frequencies and intensities achievable with existing diagnostic and therapeutic systems, nanocups initiate and sustain readily detectable cavitation activity for at least four times longer than existing microbubble constructs in an in vivo tumor model. As a proof-of-concept of their ability to enhance the delivery of unmodified therapeutics, intravenously injected nanocups are also found to improve the distribution of a freely circulating IgG mouse antibody when the tumor is exposed to ultrasound. Quantification of the delivery distance and concentration of both the nanocups and coadministered model therapeutic in an in vitro flow phantom shows that the ultrasound-propelled nanocups travel further than the model therapeutic, which is itself delivered to hundreds of microns from the vessel wall. Thus nanocups offer considerable potential for enhanced drug delivery and treatment monitoring in oncological and other biomedical applications.

Polymeric Cups for Cavitation-mediated Delivery of Oncolytic Vaccinia Virus

Oncolytic viruses (OV) could become the most powerful and selective cancer therapies. However, the limited transport of OV into and throughout tumors following intravenous injection means their clinical administration is often restricted to direct intratumoral dosing. Application of physical stimuli, such as focused ultrasound, offers a means of achieving enhanced mass transport. In particular, shockwaves and microstreaming resulting from the instigation of an ultrasound-induced event known as inertial cavitation can propel OV hundreds of microns. We have recently developed a polymeric cup formulation which, when delivered intravenously, provides the nuclei for instigation of sustained inertial cavitation events within tumors. Here we report that exposure of tumors to focused ultrasound after intravenous coinjection of cups and oncolytic vaccinia virus , leads to substantial and significant increases in activity. When cavitation was instigated within SKOV-3 or HepG2 xenografts, reporter gene expression from vaccinia virus was enhanced 1,000-fold (P < 0.0001) or 10,000-fold (P < 0.001), respectively. Similar increases in the number of vaccinia virus genomes recovered from tumors were also observed. In survival studies, the application of cup mediated cavitation to a vaccinia virus expressing a prodrug converting enzyme provided significant (P < 0.05) retardation of tumor growth. This technology could improve the clinical utility of all biological therapeutics including OV.

Ultrasound-mediated drug release from nanoscale liposomes using nanoscale cavitation nuclei

We have previously presented a liposomal formulation of mean size 140 nm, manufactured using DSPE, cholesterol, DSPC and DSPE-PEG at ratios of 65:25:3:7, which exclusively releases encapsulated doxorubicin in the presence of inertial cavitation nucleated by microbubbles (SonoVue®, Bracco) at peak rarefactional pressures in excess of 1.2 MPa at 0.5 MHz (Graham et al., J. Controlled Release, 2014). However, the benefits of cavitation-sensitive liposomes small enough to pass through the leaky tumor vasculature can only be fully realized if they can be triggered by cavitation nuclei which are also small enough to extravasate into the tumor mass. In the present work, we demonstrate that liposomal release comparable to that mediated by SonoVue® microbubbles can be achieved using gas-stabilizing polymeric nanocups of mean diameter 400 nm, at peak rarefactional pressure amplitudes in excess of 1.5 MPa at 0.5 MHz or 4 MPa at 1.6 MHz. Mechanistically, we hypothesize that release occurs once a threshold peak shear rate is exceeded in the fluid surrounding the collapsing microbubble, thus exceeding the critical shear stress on the liposomal surface. This is confirmed experimentally by demonstrating a correlation between release and the maximum power of broadband acoustic emissions received by a passive cavitation detector.

Evaluation of sub-micron, ultrasound-responsive particles as a drug delivery strategy

Substantial portions of tumors are largely inaccessible to drugs due to their irregular vasculature and high intratumoral pressure. The enhanced permeability and retention effect causes drug carriers within the size range of 100–800 nm to passively accumulate within tumors; however, they remain localized close to the vasculature. Failure to penetrate into and throughout the tumor ultimately limits treatment efficacy. Ultrasound-induced cavitation events have been cited as a method of stimulating greater drug penetration. At present, this targeting strategy is limited by the difference in size between the nano-scale drug carriers used and the cavitation nuclei available, i.e., the micron-scale contrast agent SonoVue. In vivo this results in spatial separation of the two agents, limiting the capacity for one to impact upon the other. Our group has successfully formulated two different monodisperse suspensions of nanoparticles that are of a size that will permit better co-localization of cavitation nuclei an...

Inertial cavitation at the nanoscale

Our group has recently developed novel nano-sized drug carriers that spatially target a tumour and release their payload in the presence of ultrasound-induced inertial cavitation. To maximize drug release and distribution within the tumour, co-localisation of the drug carrier and cavitation nuclei is necessary. We have recently demonstrated that rough-patterned silica nanoparticles can reduce inertial cavitation thresholds to clinically relevant levels, and will extravasate in tumours alongside the liposomes by virtue of their size. We now report on the underlying mechanisms that these nanoparticles, which are orders of magnitude smaller than the acoustic wavelength, can instigate inertial cavitation. The rough surface of the nanoparticle is modelled as a plane with a crevasse that traps a nanobubble. Using this model, we predict the motion of a gas bubble as it emerges from the cavity in response to the compressional and rarefactional ultrasonic pressures. We show that cavitation occurs when the nanobubb...

In situ hydrogel formation for biomedical applications using acoustic cavitation from high intensity focused ultrasound

There is a growing interest in polymer mechanochemistry for their industrial applications. For example, stress-induced crosslinking gel formation from polymer networks is a rapidly growing field of study. Recent work utilizes a variety of different polymer structures and crosslinking mechanisms. However, these polymers are typically soluble in only organic solvents and require the use of a sonicating probe or bath at frequencies below 100 kHz. These requirements limit their use in biomedical applications that require in situ gel formation within a patient (e.g., blocking of varicose veins, internal wound healing, etc.). Here we report on the development of a water soluble block copolymer that forms a hydrogel in the presence of acoustic cavitation from high intensity focused ultrasound. These block copolymers are comprised of hydrophilic polyethylene glycol methyl methacrylate units and hydrophobic tridentate crosslinkers. The tridentate crosslinker forms bonds with free metal ions in solution only in the presence of acoustic cavitation induced mechanical stress. We show that the block copolymer is capable of forming a hydrogel in under 90 seconds and will also block a liquid channel formed in an agarose cylinder.There is a growing interest in polymer mechanochemistry for their industrial applications. For example, stress-induced crosslinking gel formation from polymer networks is a rapidly growing field of study. Recent work utilizes a variety of different polymer structures and crosslinking mechanisms. However, these polymers are typically soluble in only organic solvents and require the use of a sonicating probe or bath at frequencies below 100 kHz. These requirements limit their use in biomedical applications that require in situ gel formation within a patient (e.g., blocking of varicose veins, internal wound healing, etc.). Here we report on the development of a water soluble block copolymer that forms a hydrogel in the presence of acoustic cavitation from high intensity focused ultrasound. These block copolymers are comprised of hydrophilic polyethylene glycol methyl methacrylate units and hydrophobic tridentate crosslinkers. The tridentate crosslinker forms bonds with free metal ions in solution only in the...

The induction of inertial cavitation from polymeric nanocups—From theory to observation

The inability for therapeutics to distribute throughout the entirety of the tumor is a major challenge in cancer therapy. Acoustic cavitation from microbubbles promotes drug distribution and improves therapeutic efficacy. Yet microbubbles are too large to navigate the microvasculature of the tumor, and are destroyed by the ultrasound wave. Thus, there is a need for submicron cavitation nucleation agents. Recently, we have developed a submicron polymeric nanocup capable of trapping a nanobubble within the surface crevice. Using a modified Rayleigh-Plesset model that accounts for the size and shape of the crevice on the surface of the nanocup, we predicted that the cavity trapped bubble expands and contracts yet remains in the cavity. With a sufficient peak negative pressure amplitude, the model indicated that the surface bubble extends beyond and detaches from the crevice before inertial collapse. To verify our predictions, we exposed nanocups to high intensity focused ultrasound at different driving frequencies and observed bubble nucleation from nanocups using the Brandaris ultra high-speed camera. Acoustic emissions were recorded using a passive cavitation detector. These direct observations of the induction of inertial cavitation from nanocups verified the predictions made by the modified Rayleigh-Plesset crevice model.The inability for therapeutics to distribute throughout the entirety of the tumor is a major challenge in cancer therapy. Acoustic cavitation from microbubbles promotes drug distribution and improves therapeutic efficacy. Yet microbubbles are too large to navigate the microvasculature of the tumor, and are destroyed by the ultrasound wave. Thus, there is a need for submicron cavitation nucleation agents. Recently, we have developed a submicron polymeric nanocup capable of trapping a nanobubble within the surface crevice. Using a modified Rayleigh-Plesset model that accounts for the size and shape of the crevice on the surface of the nanocup, we predicted that the cavity trapped bubble expands and contracts yet remains in the cavity. With a sufficient peak negative pressure amplitude, the model indicated that the surface bubble extends beyond and detaches from the crevice before inertial collapse. To verify our predictions, we exposed nanocups to high intensity focused ultrasound at different driving frequ...

The effects of high intensity focused ultrasound on biofilms formed by Pseudomonas aeruginosa

Bacterial infections are increasingly difficult to treat due to their growing resistance to antibiotics. Most of these bacterial infections form a biofilm that limits the effectiveness of the antibiotic. Biofilms are microbial cells that are protected by a self-generated matrix of extracellular polymeric substances. In addition to their intrinsic antibiotic resistance, these biofilms are able to respond to the stresses from the antibiotic by inducing drug resistance mechanisms. Currently, the strategy to combat drug resistance is to develop novel drugs, however, the rate of drug development is being surpassed by the rate of drug resistance. There is therefore a need for alternative means in enhancing the efficacy of current drug therapeutics. We propose to use of high intensity focused ultrasound (HIFU) to disrupt the biofilm and promote drug penetration. However, the effects of HIFU on these bacterial communities remain unknown. Here we report on microstructural changes within biofilms formed by Pseudomo...

Triggered Drug Release and Enhanced Drug Transport from Ultrasound-Responsive Nanoparticles

Conventional systemic drug therapy across all drug classes does not adequately provide safe and efficacious treatment for a broad range of fatal diseases. To address this challenge, there have been major advances in stimulus-responsive technologies for active drug delivery. Ultrasound-mediated drug therapy in particular has garnered much attention because it is highly accessible, cost effective, drug agnostic, and noninvasive. Broadly speaking, ultrasound is capable of providing thermal and mechanical effects. As a result, ultrasound-responsive nanoparticles have been developed to react to specific ultrasound stimuli. In this chapter, we discuss the current challenges that face drug delivery to cancer, cardiovascular, and neurological disorders. We then explore the different means by which ultrasound enables drug release, drug transport, and sonoporation of cell membranes from ultrasound-responsive nanoparticles.

Cavitation enhanced drug delivery in-vivo using combined B-mode guidance and real-time passive acoustic mapping: Challenges and results

Inertial cavitation nucleated by nano-scale sonosensitive particles (SSPs) at modest peak negative pressures (~1 MPa at 500 kHz) and monitored by passive acoustic mapping (PAM) has been recently shown to improve the dose and distribution of anti-cancer agents during ultrasound (US) enhanced delivery (Myers 2016, Kwan 2015). As applications of therapy monitoring using PAM have advanced rapidly including its use in clinical trials, means of validating the performance of PAM in-vivo remains a major focus of efforts. For drug delivery, PAM should not only quickly and reliably detect and localize desired and undesired cavitation, but it should provide some predictor of successful delivery. In-vivo experiments using PAM in subcutaneous tumor implanted murine models across a range of cancer cell lines (HEPG2, SKOV, EMT6, CT-26) demonstrate the detection of inertial cavitation by SSPs in the target regions when sonicated by US, but no cavitation with US alone. Additionally when SSPs are co-administered with an on...