no / The duocarmycins belong to a class of agent that has fascinated scientists for over four decades. Their exquisite potency, unique mechanism of action, and efficacy in multidrug-resistant tumour models makes them attractive to medicinal chemists and drug hunters. However, despite great advances in fine-tuning biological activity through structure-activity relationship studies (SARS), no duocarmycin-based therapeutic has reached clinical approval. In this review, we provide an overview of the most promising strategies currently used and include both tumour-targeted prodrug approaches and antibody-directed technologies.

Yes / Boron dipyrromethene derivates (BODIPYs) are promising photosensitisers (PSs) for cancer treatment using photodynamic therapy (PDT). This study investigates the functionalisation of graphene oxide (GO) with a BODIPY derivate for glutathione (GSH) depletion and PDT. The functionalisation of GO with a 3,5-dichloro-8-(4-boronophenyl) BODIPY via a diol derivatisation with the phenyl boronic acid moiety at the meso position of the BODIPY core, allowed to preserve the intrinsic properties of GO. We demonstrated that both chlorine atoms were substituted by GSH in the presence of glutathione transferase (GST), inducing a relevant bathochromic shift in the absorption/emission features and thus generating the active PS. Ex vitro assessment using cell lysates containing cytoplasmatic GST revealed the intracellular catalytic mechanism for the nucleophilic substitution of the GO-BODIPY adduct with GSH. Confocal microscopy studies showed important differences in the cellular uptake of free BODIPY and GO-BODIPY and revealed the coexistence of GO-BODIPY, GO-BODIPY-GS, and GO-BODIPY-GS2 species inside vesicles and in the cytoplasm of the cells after 24 h of incubation. In vitro biocompatibility and safety of GO and GO-BODIPY were evaluated in 2D and 3D models of prostate adenocarcinoma cells (PC-3), where no toxicity was observed up to 100 µg ml−1 of GO/GO-BODIPY in all treated groups 24 h post-treatment (cell viability > 90%). Only a slight decrease to 80% at 100 µg ml−1 was observed after 48 h of incubation. We demonstrated the efficacy of a GO adduct containing an α-chlorine-substituted BODIPY for the simultaneous depletion of intracellular GSH and the photogeneration of reactive oxygen species using a halogen white light source (5.4 mW cm−2) with a maximum in the range of 500–800 nm, which significantly reduced cell viability (<50%) after irradiation. Our study provides a new vision on how to apply BODIPY derivates and potentiate the toxicity of PDT in prostate and other types of cancer.

One of the major challenges in cancer treatment is delivering high enough doses of active substance specifically to cancer cells without accumulation in healthy organs. Pretargeting has emerged as a potential solution, where the delivery of a cancer recognizing (primary) agent and a cancer killing (secondary) agent are separated. Pretargeted cancer therapy utilizing PNA probes has proved to be a promising approach to selectively deliver toxic payloads to cancer cells while minimizing accumulation in healthy organs. The aim of this project was to develop a new set of secondary PNA probes specifically designed for PNA pretargeted delivery of cytotoxic drugs. A HER2-specific Affibody molecule, ZHER2:2891-SR-H6, was recombinantly produced in E. coli before being conjugated to a primary PNA hybridization probe, HP9, through sortase A-mediated ligation, to produce the primary agent, ZHER2:2891-SR-HP9. Circular dichroism (CD) spectroscopy confirmed the stability of the constructs with high melting temperatures of 71.2 and 73.7 °C. Surface plasmon resonance (SPR) analysis demonstrated high binding affinity to HER2, slightly affected by PNA conjugation. Three new secondary PNA hybridization probes were designed, differing mainly in prevalence and position of a hydrophilic PEG molecule. The probes were produced by solid phase peptide synthesis and conjugated to the cytotoxic drug DM1 through maleimide-cysteine coupling. Analytical RP-HPLC evaluation revealed a slightly higher apparent hydrophobicity for the probe with PEG in the main chain. All three secondary probes displayed high affinity to the primary probe with KD values between 498–505 pM. In vitro cytotoxicity studies on HER2-overexpressing cells demonstrated comparable potent cytotoxic activity for pre-incubated primary and secondary probes with IC50 values of 10–14 nM. These results indicate the successful development of three PNA-drug conjugates for pretargeted delivery of cytotoxic drugs.

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The lack of efficacy of existing chemotherapeutic treatments of solid tumors is partially attributed to the limited diffusion distance of therapeutics and the low selectivity of anti-cancer drugs with respect to cancerous tissue, which also leads to high levels of systemic toxicity in patients. Thus, chemotherapy can be enhanced through improving anti-cancer drug carrier selectivity and transport properties. Several strains of gram positive (e.g. Clostridium and Bifidobacterium) and gram-negative (e.g. Salmonella Typhimurium and Escherichia coli) bacteria have been shown to possess the innate ability to preferentially colonize tumor tissues. The overall goal of this dissertation is to characterize the transport and sensing of Bacteria-Enabled Drug Delivery Systems (BEADS) in select relevant environments and to investigate the associated underlying principles. BEADS consist of an engineered abiotic load (i.e. drug-laden micro or nano-particles) and a living component (i.e. bacteria) for sensing and actuation purposes. Findings of this dissertation work are culminated in experimental demonstration of deeper penetration of the NanoBEADS within tumor tissue when compared to passively diffusing chemotherapeutic nanoparticles. Lastly, the transport mechanisms that Salmonella Typhimurium VNP20009 utilize to preferentially colonize solid tumors are also examined with the ultimate goal of engineering intelligent and more efficacious drug delivery vehicles for cancer therapy. / Ph. D.

This dissertation describes the development of the fiberoptic microneedle device (FMD), a microneedle technology platform for fluid and light delivery, from general engineering characterization to specific applications in treating bladder and brain cancers. The central concept of the FMD is physical modification of silica fiberoptics and capillary tubes into sharp microneedles capable of penetrating a tissue's surface, enabling light and fluid delivery into the interstitial spaces. Initial studies sought to characterize the mechanical penetration and optical delivery of multimode fiberoptics and capillary tubes modified through a custom, CO2 laser melt-drawing technique. Additional work with multimode fibers investigated using an elastomeric lateral support medium to ensure robust penetration of small diameter fibers. These early experiments laid an engineering foundation for understanding the FMD technology. Subsequent studies focused on developing the FMD to treat specific diseases. The first such investigation sought to leverage the high aspect ratio nature of FMDs made from long capillary tubes as a therapy delivery device deployable through the instrument channel of a urological cystoscope. The therapeutic strategy was to infuse single-walled carbon nanohorns (SWNHs), a carbon-based nanoparticle allowing surface modification and drug encapsulation, into the infiltrating front of later stage bladder tumors. The SWNHs primarily serve as exogenous chromophores, enabling a fluid-based control of photothermal heat generation created when the SWNHs interacted with laser energy from an interstitial FMD or a light-emitting fiber in the bladder's interior. The study described here primarily sought to characterize the dispersal of the infused SWNHs and the photothermal response of the particles when heated with a 1064 nm laser. The FMD was also developed as a platform capable of conducting convection-enhanced delivery (CED), a therapeutic approach to treat invasive tumors of the central nervous system such as malignant glioma (MG). Intracranial CED involves the placement of small catheters local to the tumor site and slow infusion of a chemotherapeutic over long timeframes (12-72 hours). A primary challenge of this treatment approach is infused chemotherapeutics not dispersing sufficiently to reach the infiltrating cells in the tumor's margins. The hypothetical improvement provided by the FMD technology is using sub-lethal photothermal heating to sufficiently increase the diffusive and convective transport of an infusate to reach infiltrative cells in the tumor's periphery. Initial experiments sought to demonstrate and characterize a heat-mediated increase of volumetric dispersal in Agarose tissue phantoms and ex vivo tissue. Subsequent studies with in vivo rodent models determined the best laser parameters to achieve the desired levels of diffuse, sub-lethal heat generation and then demonstrated the hypothesis of increasing the rate of volumetric dispersal though concurrent local hyperthermia. This research was the first demonstration of photothermal augmentation of an interstitially infused fluid's dispersal rate, which may have uses outside of the CED approach to brain cancer exhibited here. Taken in sum, this manuscript describes the potency and versatility of the FMD technology platform through its development in various biomedical applications. / Ph. D.

The limited transport of conventional chemotherapy within the tumor microenvironment (TME) is due to irregular vascularization, increased tumor interstitial pressure, and a dense extracellular matrix (ECM). The lack of selectivity of anticancer drugs often leads to systemic toxicity and damage to healthy tissues. Bacteria-based cancer therapy (BBCT) is a promising alternative, as tumor-targeting bacteria have been shown to preferentially colonize primary and metastatic tumors and induce anti-tumor effects. In this dissertation, we focus on several aspects of bacteria-nanoparticle conjugates, wherein BBCT is synergistically combined with nanomedicine to augment the efficacy of both treatment modalities. We explore biofabrication of our bacteria-nanoparticle conjugates called NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems) and their interaction with the TME. Specifically, (1) we investigate the effects of two bacteria-NP conjugation chemistry and assembly process parameters of mixing method, volume, and duration, on NP attachment density and repeatability. We evaluate the influence of linkage chemistry and NP size on NP attachment density, viability, growth rate, and motility of NanoBEADS. (2) We investigate the effect of dense stroma and ECM production on the intratumoral penetration of bacteria with a mathematical model of bacterial intratumoral transport and growth. (3) We develop a microfluidic device with multicellular tumor spheroids to study the transport of tumor-targeting bacteria and support real-time imaging and long-term experiments. (4) We develop a new type of bacteria-based bio-hybrid drug delivery system using engineered cell surface display for enhancing the attachment of nanoparticles. / Doctor of Philosophy / Chemotherapy faces challenges in effectively reaching tumors due to factors like irregular blood vessel distribution, increased tumor pressure, and the presence of dense structures such as the extracellular matrix (ECM). This often results in collateral damage to healthy tissues. Bacteria-based cancer therapy (BBCT) offers a promising alternative, utilizing tumor-targeting bacteria to selectively attack tumors. This dissertation focuses on optimizing NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems), which are chemotherapy encapsulating nanoparticle-bacteria assemblies to overcome these challenges and characterizing its behavior in tumors. Firstly, we investigated the optimization of bacteria-nanoparticle attachment, exploring various linkage chemistries and assembly processes to enhance attachment density, viability, and motility. Secondly, we examine how dense stroma and ECM affect bacterial penetration providing insights into intratumoral transport dynamics. Thirdly, we develop a microfluidic device integrated with multicellular tumor spheroids to enable real-time imaging and long-term experimentation on bacteria and drug transport. Lastly, we explore the potential of engineered cell surface display to enhance nanoparticle attachment in NanoBEADS, paving the way for self-propelled and highly targeted drug delivery systems. This dissertation strives to contribute to the transformation of current approaches to cancer treatment by refining drug delivery precision and efficacy while minimizing systemic toxicity.

In vitro protein selection has had major impacts in the field of protein engineering. Traditional screens assay individual proteins for specific function. Selection, however, analyzes a pool of mutants and yields the best variants. Phage display, a successful selection technique, also provides a reliable link between variant phenotype and genotype. It can also be coupled with high throughput sequencing to map protein mutations; potentially highlighting vital mutations in variants. We propose to apply this technique to cancer therapy. RAF, a serine/threonine kinase, is critical for cell regulation in mammals. RAF can be activated by oncogenic RAS, found in over 30% of cancers, to drive cancer proliferation. Rigosertib, a benzyl styryl sulfone in phase III clinical trials for myelodysplastic syndrome (MDS), is an inhibitor of the RAS binding domain (RBD) in RAF. Phage display can be used to select RAF mutants for RAS binding affinity, in the presence of Rigosertib. High-throughput sequencing of these variants can provide a means of anticipating, and mapping resistance to this anti-cancer drug.