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Gene delivery will cure all. That was the hope of many since the discovery and therapeutic implementation of genetic research. Although some of that hope has been realised, the delivery of nucleotides to cells in the body remains a major barrier in wider application. Ultrasound-mediated delivery of drugs has been investigated in various forms for decades and continues to be an area of engineering and science that is much explored. The body of work presented in this thesis strides to bring the two areas of research endeavour together. Ultrasound-mediated delivery of drugs and antibody particles, normally in conjunction with cavitation nucleation agents, has been shown to be effective both in vitro and in vivo. In this work, we focus on nucleotide delivery, in particular mRNA, which has yet to be fully explored, especially in the context of achieving delivery of non-encapsulated, 'free' mRNA. Data presented here explores the relationship between cavitation and the transfer of, and protein expression from, free mRNA. The protein expression exhibited in various cell lines in vitro was further tested in vivo, using either sub-micron or micron-sized cavitation nuclei and the effects on transfection efficiency and expression time were examined. A luciferase reporter gene assay was used to characterise events in vitro across different cell lines including cancer and non-cancerous types. The impact and varying acoustic exposure parameters and cavitation on expression were assessed, and differences in expression time and transfection rate were observed. These data were used to determine appropriate acoustic parameters for in vivo delivery. Cavitation-mediated delivery was examined in vivo in two types of cancer tumours, and mRNA expression was quantified using in vivo fluorescence imaging and using excised homogenised tumour tissue. The results presented here lay the foundations for cavitation-mediated delivery and transfection of free mRNA both in vitro and in vivo by identifying appropriate acoustic parameters and cavitation nucleation agents, which result in enhanced delivery and by identifying the appropriate time windows for measurements to quantify expression.

DNA-based vaccines represent a powerful technology that could be well-suited to the quick development and rapid, large-scale production needed for effective response to rapidly emerging or mutating pathogens. However, DNA vaccines have not yet been widely accepted or utilised as a vaccine technology due to the lack of immunogenicity observed. Intramuscular injection is the most commonly used vaccination dosing route but the skin represents an immune-rich environment that has been shown to enable significant dose-sparing and broader immunogenicity advantages. However, traditional needle and syringe methods are not adept at accessing this potentially powerful vaccination site. Cavitation has been shown to be able to permeabilise the stratum corneum and hence, provides a method for accessing the epidermis in a reliable and pain-free way. In this thesis, cavitation-mediated delivery of DNA vaccines into the dermal compartment is explored as a strategy for improved vaccination. This thesis begins by exploring the formulation and ultrasound parameters of needle-free, cavitation- mediated delivery of DNA into the skin. Data shows that relatively low quantities of DNA are delivered into the skin compared to direct injection but that cavitation can be a mechanism to drive increased delivery into the skin. It is also demonstrated that, once cavitation is achieved, exposure of skin to higher ultrasound pressure amplitudes provides no additional benefit. It is also proposed that cavitation-mediated delivery targets a different cell population compared to intradermal injection, based on the observation that the balance of anti-transgene antibody isotypes is altered. Specifically, for cavitation mediated delivery an IgG1-dominant IgG profile is produced compared to a balanced IgG1/IgG2a response observed upon injection. An initial exploration of improved DNA vectors for vaccination, showcases the scope for improvement. Hence, subsequent chapters are devoted to three avenues of optimisation. The potential of protein cavitation nuclei (p-CaN) is demonstrated, both as cavitation nuclei and as an immune-stimulatory particle. It is shown p-CaN can match delivery of DNA compared to polymeric cavitation nuclei produced to GMP standard, as well as induce an immune response against the protein used to formulate the p-CaN. A number of p-CaN iterations are validated in this work, including p-CaN produced by the scalable technology of hydrodynamic shearing. The second optimisation explored was an improved ultrasound set-up (V2). Crucially, the ultrasound transducer was inverted to enable easier handling and the scruff of the mouse is treated to prevent accidental ultrasound exposure of internal structures. Optimisation of delivery parameters using this improved set-up are determined and validated through reporter gene expression, anti-transgene antibody assays, as well as qPCR for DNA delivery quantification. The final optimisation explored is in terms of DNA vectors used. In particular, perforin-encoding DNA was identified as a powerful adjuvant for cavitation-mediated delivery and shown to improve antibody titres and avidity compared to a non-adjuvanted DNA vaccine. A self-amplifying vector was also explored with the transgene expression profile defined and the immunogenic potential characterised. This showed that the self-amplifying vector has a marked increase in protein production after 5 days and can produce more reproducible antibody titres, higher avidity and a more IgG2a-skewed immune response compared to a non-amplifying vector. In the final chapter, the potential of the self-amplifying vector and the perforin-encoding DNA vector are tested in a challenge study, which shows that the self-amplifying vector improves survival, and reduces symptomatic disease in a mouse model.

Vaccinia Virus (VV) is an attractive vector for oncolytic virotherapy given its potential for tumour-selective tropism, efficient cell-to-cell spread, rapid replication in cancer cells, large transgene coding capacity and stimulation of anti-tumour immunity. It has a well-defined safety profile and is being assessed in late�stage clinical trials. Systemic delivery of VV has shown limited clinical success in patients due to a knockdown in infectivity, as a result of rapid immune-mediated neutralisation, and poor penetration into tumours. These factors have often restricted its route of delivery to intratumoural (IT) injection. For VV to become applicable across a wider range of patients/cancer indications, amenability to systemic delivery is required. This may be achieved using strategies that modulate VV by genetic or chemical means and/or that alter the physiology of target tumours. Chemical stealthing has not previously been applied to enhance the pharmacokinetics (PK) of enveloped viruses such as VV. Ultrasound (US)-mediated cavitation has been previously shown to effectively enhance the delivery of therapeutics, including VV, to solid tumours. In this thesis, US-mediated cavitation was applied to DLD and CAPAN-2 xenografts after IT administration of VV with the aim of producing more homogenous intratumoural infection. The limited success observed in the IT context provided motivation to instead enhance the stability of VV for improved IV administration. Flow virometry demonstrated that VV could be effectively modified using an amphiphilic polymer (to create PCVV). This PCVV showed reduced natural tropism, a 20% decrease in anti-VV antibody binding and could be used as a platform for the attachment of an antibody to permit retargeting of infection to the tumour associated antigen mucin-1 (MUC1). PK analysis of VV, PCVV and aMUC1-PCVV showed circulation kinetics for PCVV and aMUC1-PCVV were enhanced at two different doses (approximately 5-fold higher circulating dose at 5 minutes vs VV). The hypothesis that the application of US-mediated cavitation during this 5 minute window could be used to enhance extravasation of PCVV and aMUC1-PCVV into and throughout tumours, was explored. When samples were administered IV and US-mediated cavitation was applied to CAPAN-2 xenografts, increased infection (as assessed by transgene expression) was observed. The impact of cavitation on aMUC1-PCVV transgene expression was highest (a mean increase of 530-fold compared to non US exposed tumours). Tumour growth studies showed that combining aMUC1-PCVV, SSPs and US enabled substantial and significant tumour regression (-73%). The work presented in this thesis brings to light the challenges of achieving effective and uniform delivery to tumours after both IT and IV administration of VV and contributes to the necessary progress VV needs to make if it is going to achieve its full clinical potential.

Vaccines are arguably the most important medical technology developed to date, and have provided dramatic reductions in disease morbidity and mortality over the last 200 years. Although vaccines have been undeniably successful, improvements in vector production, delivery and ease of use would be of great benefit. Historically, vaccine development has been based on the "three Is" paradigm of Louis Pasteur (isolate, inactivate, inject). However, an improved understanding of immunology, pathology and microbiology is now helping vaccine development adopt a more 'rational design' approach. This thesis builds on previous work in the development of a plug-and-play universal vaccination platform. Previously, this platform has been reported to evoke a rapid, high titre, isotype switched, epitope-specific antibody response against an epitope of the circumsporozoite protein from the malaria parasite. Here we describe repurposing the platform to vaccinate against ErbB-2 overexpressing tumours and H3N2 influenza viruses. Cancer is among the leading causes of morbidity and mortality today. ErbB-2 is a popular target for cancer treatment as it is localised to the cell surface and has raised expression in 15 - 30% of all breast cancers, as well as in some ovarian, renal, colon and lung cancers. Influenza is a common pathogen that annually infects 5 - 10% of adults and 20 - 30% of children worldwide. Despite influenza (flu) vaccines being available since the 1940s' they have had restricted success due to flu proteins being subject to a high rate of antigenic drift and shift. However, many groups have reported conserved regions of the viral proteins with low immunogenicity. If such low immunogenicity can be overcome then this would pave the way for a flu vaccine with long lasting efficacy. This thesis begins by first giving an overview of novel vaccination technologies before describing the formulation methods of the vaccine platform developed herein. The encapsulation efficiency of liposomal-vaccine particles after all purification steps was found to be approximately 0.1, 0.3, and 0.5% for the three formulation methods used. This low percentage is due to dilution factors in the purification steps and inherent poor passive loading efficiency of liposomes. The surface conjugation efficiency of the target peptide was demonstrated to increase from 0.4% in the first two formulation methods to 92.8% in the third method. The immunogenicity of this vaccination platform, after subcutaneous injection, is then demonstrated by the elicitation of a rapid (within 1 week) raised (10.5-fold, p<0.001) humoral immune response specific for the ErbB-2 peptide. This process is shown to be driven by non-cognate T helper cells specific for an ovalbumin peptide, OVA323-339. Lastly, to demonstrate the plug-and-play adaptability of the platform the formulation is repurposed to show raising of a class-switched, high-avidity, antibody response to a H3N2 influenza virus epitope, with titres reaching 98.7-fold higher than control groups (p<0.001) just one week after initial vaccination.

The monocarboxylate transporter 1 (MCT1) has received special attention as a potential therapeutic target in cancer thanks to its role in lactate shuttling. In breast cancer (BC), high MCT1 expression has been correlated with basal-like phenotype and a ‘triple-negative’ status, an aggressive subgroup of tumours with no identified molecular targets. In this thesis, MCT1 was targeted with a brominated pyruvate derivative, 3-bromobyruvate (3BP). 3BP is an alkylating agent known to inhibit glycolysis. Here, it was shown that 3BP is selectively toxic to cells expressing MCT1. Metabolomics, used to investigate metabolic effects, is a high-throughput, unbiased approach that requires no assumptions on possible targets. Inhibition of glyceraldehyde phosphate dehydrogenase (GADPH), an established 3BP target, was confirmed. Accumulation of pentose phosphate pathway (PPP) intermediates, inhibition of nucleotide synthesis, ATP and thiol-based antioxidant depletion were found to be profound in MCT1- expressing cells. The reactivity of 3BP towards thiols necessitates the use of a delivery system. 3BP was encapsulated in cavitation-sensitive liposomes and co-administered with lipid microbubbles (MBs) as ultrasound contrast agents (UCAs). Ultrasound locally triggers the release of the therapeutic, which was confirmed using an in vitro apparatus developed in house. With radiotherapy being one of the main modalities used in BC treatment, the next question was whether a radio-labelled therapeutic agent could be delivered via MCT1. A radio-iodinated lactate derivative (‘123I-HPLA’) was synthesised and tested for selectivity towards MCT1- expressing cells with positive outcomes. Finally, Poly(lactic-co-glycolic acid) (PLGA) MBs were investigated for the development of a novel delivery system. Clinically relevant ultrasound pressures resulted in persistent cavitation, fragmentation and accelerated monomer release from PLGA MBs. The findings of this study support the use of small halogenated carboxylic acids to selectively target MCT1 and addresses the delivery challenges associated with such compounds.

Tumour and tumour microenvironment exploitation of immunologic checkpoints like PDL1 can enable immune evasion. Recent advances in oncology, have brought forward antibody drugs, such as anti-PD-L1, capable of blocking and/or mediating the eradication of cells that express PD-L1. Despite promise across a wide range of indications, clinically such agents meaningfully extend survival only in a fraction of patients treated. Immature, heterogenous blood vessels, elevated interstitial pressures, and stromal barriers severely limit the accumulation and extravasation of drugs within the tumour microenvironment. We hypothesize that the physical size of antibody therapeutics, such as antiPD-L1, compounded by delivery barriers could be a factor limiting their efficacy. Recently, submicron cavitation nucleation agents, sonosensitive particles (SSPs), activated by focused ultrasound have been shown to improve the delivery of large, anti-cancer therapeutics via microstreaming-mediated transport. Our aim was to pre-clinically investigate whether systemically-dosed anti-PD-L1 followed by SSPs and the application of focused ultrasound could improve the intratumoural delivery and therapeutic efficacy of anti-PD-L1. Efficacy studies revealed that pairing anti-PD-L1 with ultrasound-mediated cavitation can significantly extend median survival time. Cavitation was also found to enhance the intratumoural accumulation of anti-PD-L1 and three other antibodies. Further, the binding intensity and distribution of anti-PD-L1 improved in cavitation-treated tumours. Immunological analysis suggests that anti-PD-L1 paired with cavitation enables the innate and adaptive immune system to leverage the modest cavitation-induced inflammation and improved anti-PDL1 delivery to facilitate tumour attack. Taken in a broader context, this pre-clinical work suggests that ultrasound-mediated cavitation warrants further investigation as viable tool to improve drug delivery clinically.

Injections are a commonly used and widely accepted method of drug administration to prevent and treat diseases. Although using a needle and syringe provides a rapid, lowcost, and direct way of administering almost any kind of therapeutic agent into the body, it is a painful and invasive method leading to issues of patient compliance and needle-phobia. The delivery of drugs through the skin (transdermal delivery) is an attractive alternative approach because it is non-invasive and has low associated risks. However, the penetration of molecules into/through the skin is severely restricted by the outermost layer of the skin (stratum corneum). Initial studies of ultrasound-mediated, cavitation-enhanced transdermal drug delivery have shown great promise by utilising a gel formulation containing polymeric cavitation nuclei to help deliver a model immunogen (Ovalbumin) into the skin. However, enhanced adoption of this technology to permit delivery of several classes of therapeutic agents requires further validation and optimisation and is currently stymied by the lack of real-time monitoring when used across a broad range of therapeutic classes and environmental conditions. Such monitoring is especially important in situations where the dose of the delivered therapeutic agent needs to be precisely defined. Therefore, the focus of this thesis is to achieve transdermal delivery using cavitation-enhanced transport and develop methodologies to monitor or at least confirm successful delivery in real-time.

Drug development for cancer is heading towards increased use of highly-targeted and complex biological therapeutics. As the complexity and costs of drug development increase, the importance of ensuring good tumoural delivery and distribution of these drugs is paramount. The tumour microenvironment, which possesses many characteristics distinguishing it from healthy tissue, presents both challenges to and opportunities for drug delivery. Countless novel nanoscale formulations or reformulations of existing drugs have been created to harness the potential of the 'enhanced permeability and retention' of tumours. The scale of this effect has, however, come under recent scrutiny, and a clear role for alternative delivery mechanisms has emerged. Ultrasound-mediated cavitation has been evaluated as a non-invasive external stimulus to improve delivery of drugs to solid tumours. Cavitation nuclei can be used to seed cavitation at relatively low pressures to achieve cavitation levels capable of enabling enhanced drug transport, without safety concerns, suggesting a path for clinical translation. In this thesis, the effects of ultrasound-mediated cavitation on the delivery of a range of therapeutic drug classes to solid tumours was investigated: a cancer-targeting antibody (cetuximab), an actively targeted nanoparticle (cetuximab-GNP), and an oncolytic virus (ColoAd). Two classes of cavitation nuclei were employed: a microbubble ultrasound contrast agent (SV) and sonosensitive gas-entrapping nanoparticles (SSPs). Ultrasound-mediated cavitation was shown to enhance the delivery of all therapeutics tested here, though the scale of the effect varied. Cavitation activity achieved with SV or SSPs in vivo showed different characteristics, with SSPs demonstrating more sustained cavitation. Delivery of a long-circulating antibody was significantly more enhanced with SSPs than with SV, but the two cavitation nuclei provided a comparable benefit to the shorter-circulating targeted nanoparticle and oncolytic virus. Furthermore, site-specific density-modification of ColoAd was investigated, in an attempt to further enhance its cavitation-mediated transport, though the conclusion of this work was that the proposed modification strategy was not viable. The work presented in this thesis highlights the challenge of achieving effective tumoural delivery of systemically administered biological therapeutics, and provides compelling evidence for the further investigation of ultrasound-mediated cavitation as an effective tool for enhancing the delivery of biological therapeutics into tumours.

Gene therapy is the process of introducing genes to augment or minimise the expression of absent or defective genes, respectively. Non-viral gene delivery systems for the treatment of genetic diseases, have evolved into highly appreciable nucleic acid-based therapies due to considerably less risk of host immunogenicity and induction of inflammatory responses. However, they are challenged by limited delivery. Thus, more efficient strategies are continually being sought. Lithotripter shock waves (LSW) are powerful acoustic waves that are an attractive choice of delivery system, as they offer a non-invasive, targeted and safe approach. Furthermore, the delivery of messenger ribonucleic acid (mRNA) possesses several advantages over commonly delivered plasmid deoxyribonucleic acids (pDNA), because it does not require opening of the nuclear envelope, thereby reducing the level of cell injury necessary for transfection. This work presents the first investigation on the efficacy of LSW mediated mRNA delivery, based on optimised SW parameters that balance the desired enhanced permeability of cell membranes against undesired cytotoxicity, and maintain the structural and biological stability of the RNA. A transfectability measure that defines the ability of SWs to permeabilise a cell whilst keeping it alive was established for dissimilar cell types, as a function of the acoustic pressure and number of SWs. Statistically significant RNA uptake was recorded in a tissue mimicking system, and using RNA analogues at various concentrations, the SW induced bio-distribution was characterised. In addition to LSW induced gene augmentation using mRNA, it was shown that LSWs could be used to effect gene inhibition through the delivery of siRNA. Kinetic experiments were carried out to measure mRNA uptake during shock wave exposure and indicated that rate of delivery was highest at the start of the SW dose and decreased during treatment. The results also suggested that the enhancement of cell permeability was significantly transient, and that mRNA was highly susceptible to degradation in its naked state. Furthermore, mRNA-based gene expression was shown to be predictive but quantal. The in vitro tissue model was improved from a gel-based system, to one that incorporated multi-cellular spheroids which capture aspects of 3-D tumours. Static overpressure was applied during SW exposure in order to suppress cavitation effects and isolate effects that could be attributed to shear due to cell-to-cell coupling. The results showed that mild overpressure improved RNA uptake the most, but that at higher overpressure, the level of increase in RNA uptake relative to controls, was dependent on the type of RNA nucleotide being delivered. This suggested that a complex interaction between LSW cavitation and direct stress dominates delivery. A final report was on the significant improvement of gene delivery when mRNA was encapsulated within a lipid nanoparticle vector, and SW exposure was assisted by cavitation agents. Also, by exposure to another acoustic stimulus - focused ultrasound (US), direct comparisons were made between SWs and US on the efficiency of delivery and tissue penetration. In conclusion, this thesis has shown that by choosing parameters appropriately, shock waves can be a promising strategy for the delivery of genes to cells.