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Polymer-anticancer drug conjugates display passive tumour targeting by the enhanced permeability and retention effect. Several conjugates containing traditional chemotherapy (doxorubicin (Dox), paclitaxel and platinates) have entered clinical trials. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-Dox (PK1) has shown activity in anthracycline-resistant breast cancer patients and was 5 times less toxic than Dox. The use of polymer conjugates to deliver endocrine agents is largely unexplored, therefore, the aim of this study was to design a novel HPMA copolymer conjugate carrying a combination of endocrine therapy (the aromatase inhibitor aminoglutethimide (AGM)) and chemotherapy (Dox) on the same polymer backbone. As patients with advanced metastatic breast cancer have a relatively poor prognosis (< 20 % survival at 5 years), it was hoped that this conjugate would elicit increased antitumour activity. A library of conjugates was synthesised to contain Dox, AGM, or both drugs attached to the same polymer chain. First, the conjugates were prepared by aminolysis of polymeric intermediates (Mw 20,000-25,000 g/mol Mw/Mn = 1.3- 1.5) where the C-terminus of the peptide side-chains were esterified with p-nitrophenol. The lower reactivity of the aromatic amine of AGM, however, favoured use of 1,3-dicyclohexylcarbodiimide (DCC)-mediated coupling, leading to improved yield of conjugation. NMR confirmed product identity and nuclear Overhauser effect (NOE) measurements verified the covalent binding of AGM. Total and free Dox were measured by HPLC and AGM content using an indirect method. Overall, the drug content of the conjugates was in the range 2.8 - 8.6 % w/w (free drug content < 1.4 % w/w total). Cytotoxicity of the conjugates was determined using the human oestrogen-dependent breast cancer cell line (MCF-7) and the aromatase-transfected derivative MCF-7ca combined with the MTT assay (72 h). Although HPMA copolymer-Dox was much less active than free Dox, and neither the HPMA copolymer-AGM nor mixtures of drug conjugates bearing AGM or Dox caused an elevation in cytotoxicity in either cell line, when AGM and DOX were covalently linked to the same polymeric backbone, cytotoxicity was significantly enhanced (more evident for MCF-7ca cells). To better understand the precise mechanism of action, the ability to inhibit aromatase was evaluated in 3 different in vitro systems. Free AGM and HPMA copolymer-AGM inhibited aromatase, but drug release was probably essential for the activity of the HPMA copolymer-AGM. To establish whether the rate or mechanism of uptake of HPMA copolymer-Dox ( AGM) were responsible for the different cytotoxicity, the uptake of the conjugates was evaluated by FACS. Internalisation and marked membrane binding were seen for both conjugates suggesting that the different cytotoxicity could not be attributed to differences in the uptake. Finally, immunocytochemistry studies showed that the HPMA copolymer-AGM-Dox reduced the level of the anti-apoptotic protein Bcl-2 in MCF-7, while the HPMA copolymer-Dox had no effect on this cellular marker. To conclude, the HPMA copolymer-AGM-Dox conjugate is the first polymer therapeutic to combine chemotherapy and endocrine therapy. The fact that AGM and Dox can act synergistically to produce markedly enhanced cytotoxicity in vitro compared to the HPMA copolymer-Dox conjugate, that has already shown activity in breast cancer patients clinically, underlines the potential importance of this polymer- drug combination.

Introduction: Variable rate intravenous insulin infusions (VRIIIs) are very effective in managing hyperglycaemia in hospital inpatients when used correctly. However, if used incorrectly, they can cause harm or even death. Unlike traditional safety approaches that focus on ensuring adherence to predefined standards and identifying errors to prevent their recurrence, Resilient Health Care (RHC) shifts the focus to understanding variability in work, including both successes and failures. RHC achieves this by comparing Work as Imagined (WAI) (what people expect themselves and others to do) with Work as Done (WAD) (what people actually do) with the aim of realigning WAI with WAD in order to reflect reality. Aim: To develop a model of VRIII use in adult inpatients informed by the RHC framework. Methods: This mixed methods study was conducted in three phases at a Vascular Surgery Unit in a tertiary teaching hospital in England, UK. Phase I: Exploring WAI. Analysis of all documents related to VRIII use, and focus groups with users of VRIII. Documents and transcripts were analysed using inductive/deductive analysis. Phase II: Exploring WAD. Video Reflexive Ethnography methodology and quantitative data were used. The qualitative data were analysed using thematic analysis and the quantitative data were used to judge the outcomes of the observed tasks. Phase III: Model development. Two separate hierarchical task analyses (HTAs) were produced, one for each phase, to identify similarities and differences and to inform the development of the model of VRIII use. Results: WAI was perceived as a complex process that involved iterative tasks of producing hospital-specific guidelines and a multi-pronged approach to ensuring implementation of and adherence to guidelines. WAD was accomplished by using standardised practices (adhering to VRIII guidelines) and context-dependent adaptations (assigning blood glucose monitoring to other colleagues during busy shifts). Reflexive meetings revealed that a lack of knowledge in the selection of intravenous fluids and monitoring of blood glucose tasks was the main challenge faced while using VRIII. Suggestions from healthcare practitioners designed to overcome this challenge focused mainly on the need for face-to-face, VRIII-focused training that is tailored to their need. The comparison between WAI and WAD HTAs highlighted that most of the tasks in both HTAs were mostly aligned. The comparison also assisted in developing a model for the use of VRIII. The model showed that standardisation (WAI) and flexibility (context dependent adaptations) must complement each other to ensure the delivery of patient care while using VRIII. The model also highlighted two key points to be considered when monitoring and continuously reviewing everyday clinical work and its resultant outcomes: 1) Understanding the permanence status of adaptations is a crucial step in differentiating between work that has long-term and short-term success. 2) Using monitoring tools such as checklists, clinical audits and VRE, corresponding to the type of task being performed is fundamental in reviewing everyday work and designing and implementing effective interventions that help in improving patient care while using VRIIIs. Conclusion: This research explored safety and complexity in the use of VRIIIs by integrating innovative methodologies inspired by the RHC construct of comparing WAI with WAD and by the VRE principles of exnovation, care, reflexivity and collaboration. The methods employed and findings of this thesis could help researchers as well as healthcare practitioners to better understand the use of VRIIIs in situ and move beyond describing concepts to providing practical recommendations, based on practical data obtained from a Vascular Surgery Unit, for improving safety and patient care delivery while using VRIII.

Polymer-drug conjugates (PDCs) are drug delivery systems in which drug molecules are covalently linked to hydrophilic polymers. These systems have initially been developed to improve the anticancer efficacy and safety of chemotherapeutic agents based on the enhanced permeability and retention (EPR) effect. In these applications, the PDCs require the release of the conjugated drugs within the tumour tissues. However, there are few cases in which the release of the drug is not the aim. For example, Movantik®, the only available PDC on the market, was developed to prevent the unfavourable penetration of naloxol across the bloodbrain barrier (BBB) while retaining its effect in the intestines. This system was designed as a non-prodrug employing PEG (as a polymer) and an ether bond as a linker. The current project focuses on the biological evaluation of utilising the PEGylation strategy to develop non-prodrug PDCs of haloperidol providing compartmentalisation of haloperidol at the intended site of action, which could allow potential non-CNS applications of the conjugated haloperidol. In Chapter 1, a general introduction to the biological barriers, PDCs and their applications are discussed. The rationale behind this project is also provided. Chapter 2 identifies the various non-cancer applications of PDCs and key features influencing the design of such systems for specific diseases are recognised. Chapter 3 represents a systematic analysis of clinical studies of nanomedicines (including PDCs) used to treat solid tumours of different origins based on the EPR effect. From all studied cancers, ovarian, brain, stomach, breast, colon and colorectal, and pancreatic cancers showed the highest levels(up to >8-fold) of accumulation of nanomedicines compared to other tumours. Moreover, tumour size was another factor that impacted the accumulation of nanomedicines, with high levels of accumulation observed in large tumours (~5-fold) compared to medium or very large tumours. Other parameters such as perfusion levels, the presence of angiogenesis and inflammation in tumour tissues were identified as factors that might influence the magnitude of the EPR effect and, as a consequence, the accumulation of nanomedicines within tumour tissues. The chapter proposes two strategies to select patients who could potentially benefit from the increased accumulation of nanomedicines based on the EPR effect, which might enhance the clinical outcomes of using nanomedicines as personalised anticancer agents. In Chapter 4, the feasibility of utilising the PEGylation strategy to prevent haloperidol diffusing through the BBB is demonstrated using different in silico, in vitro and in vivo approaches. The synthesis of a PEG-haloperidol conjugate was carried out (using PEG 6000 Da) by applying a protocol slightly modified based on our previously reported protocol. The in vitro binding assay indicated that the PEG-haloperidol conjugate had a retained activity through D2 receptors, however, this was lower than that of the free drug (~18-fold at 10 nM). Molecular docking (MD) studies indicated that the conjugates exhibited a retained binding affinity for the D2 receptors, and the binding pattern of the conjugate in the binding pocket explained the loss of the biological activity of the conjugates compared to the free haloperidol. In vivo studies on rats revealed that rats treated with PEG-haloperidol were not cataleptic in contrast to the free haloperidol treated rats, which indicated the prevented crossing of PEG-haloperidol into the CNS. Chapter 5 describes potential applications of PEG-haloperidol conjugates in the field of cancer (acting via s receptors) assessed using in vitro and in silico approaches. PEGs of two MWs (2000 and 6000 Da) were synthesised. PEG (2000 Da) enhanced the haloperidol's loading in the conjugate (~25% w/w) by ~3-fold compared with the loading of haloperidol in PEG (6000 Da) conjugate. The cytotoxicity of the conjugates was evaluated using breast cancer cell lines (MCF-7 and MDA-MB 231). The application of the conjugates as potential antiproliferative agents was limited as their IC50 values were > 100 µM (compared to ~50 µM for the free haloperidol) for both cell lines. The conjugates were also tested for potential antimigratory activity in vitro on vascular endothelial cells (HUVECs). The conjugates significantly inhibited the VEGF-stimulated migration of HUVECs (> 65% inhibition) although at a lower level compared to the free haloperidol (91% of inhibition). MD studies were performed and explained the loss of the biological activity of the conjugates compared to free haloperidol. Chapter 6 indicates a preliminary evaluation of potential cardiovascular applications of PEG-haloperidol by studying its effects on human platelets' aggregation induced by CRP-XL or ADP. The results indicated that free and conjugated haloperidol (at all tested concentrations) did not significantly abrogate the platelets aggregation stimulated by the CRP-XL (mediated by GPVI receptors). Moreover, haloperidol and PEG-haloperidol inhibited, however, not significantly, ADP-induced aggregation of human platelets at concentrations ³12.5 µM haloperidol equivalent, probably through P2Y1 receptors. However, further studies by employing other agonists and/or increasing the incubation time, and using different methodologies are required to identify the final conclusion. Chapter 7 represents the feasibility of using PEG-based PDC (designed as a non-prodrug system) to decrease or avoid the transfer of conjugated drugs through the human placenta. PEG, as a polymeric carrier, did not significantly affect the apoptosis or proliferation rates within placental explants when incubated up to 48 h, as indicated via immunohistochemistry staining. No signs of necrosis were observed when the explants were challenged with PEG as the released levels of lactate dehydrogenase from the explants did not significantly change. Treatment with PEG did not alter the normal function of the placental tissues where the secreted levels of hCG hormone from the explants were not significantly influenced by the polymer. Moreover, the cellular uptake studies of the PEG-Cy5.5 (dye) and PEG-haloperidol conjugates, used as model drugs, using fluorescent microscopy and RP-HPLC, respectively, showed complete absence of PEG-Cy5.5 from the placental tissues and limited uptake of PEG-haloperidol by the tissues compared to the free Cy5.5 and haloperidol, respectively. This indicated the potential efficiency of PEGylation strategy to design non-prodrug systems to treat illnesses during pregnancy without inducing negative effects on the developing fetus. In Chapter 8, the key findings of this PhD project are summarised, critical evaluation of work-related aspects and potential future work is suggested. Specifically, taken together, the data presented in this thesis demonstrated the feasibility of using PEGylated macromolecules (designed as non-prodrug systems) to reduce or prevent the transfer of conjugated drugs across biological barriers while retaining their activity. This strategy would form a platform to design drug delivery systems for applications where specific compartmentalisation of the effects of drugs is required. Future work will look to investigate this further by exploring PEGylated systems of therapeutic agents of different classes for their potential clinical applications.

Poly(amidoamine) (PAMAM) dendrimers are nanosized, highly defined, hyperbranched polymeric vehicles designed for targeted delivery of drugs or bioactive molecules. Whilst heavily researched, their mechanism of interaction with biological systems is barely understood. Here, medium-generation PAMAMs (-COOH-terminus (G4.5); -NH2-terminus (G5)) were characterized by UV-Vis and fluorescence, small-angle X-ray scattering and computational simulations. Biophysical (surface pressure measurements, neutron reflectometry) and microbiological techniques were used to assess modulating factors (i.e. solvent pH, residual methanol) of dendrimer-membrane interactions and related toxicity. PAMAM surface groups are charged at pH 7, and in phosphate buffer solution the dendrimers assumed a compact, near-globular shape (radius ≈2.5 - 3 nm). PAMAM G5 penetrated anionic phosphatidylglycerol (DPPG, model bacterial lipid) monolayers rapidly but showed lesser membrane-activity on supported bilayers. Electrostatic effects could be potential drivers but also hinder PAMAM G4.5 penetration into DPPG monolayers. Across techniques, it was shown that solvent pH influenced dendrimer core (tertiary amines) and surface charge, with higher impact on structural characteristics of zwitterionic PAMAM G4.5 than of G5. At pH 4, the charge effect led to higher penetration levels into DPPG monolayers and bilayers for both dendrimers (G5 > G4.5) compared to pH 7. Methanol as co-solvent affected PAMAM radii and spectroscopic properties across the pH range tested. Additionally, it increased DPPG monolayer penetration compared to the methanol-free buffer environment, which could be explained by both, the impact on PAMAMs and on lipid layer organization. Anti-bacterial efficacy was studied on gram-negative and gram-positive bacteria. Amine-terminated PAMAMs led to growth-inhibition of most strains, but specifically gram-positive Staphylococcus spp. which are rich in anionic membrane lipids (i.e. PG lipids). Whilst overall less inhibitory than G5, PAMAM G4.5 was bactericidal against Staphylococcus saprophyticus. Taken together, our findings highlight the significance of the PAMAM characteristics and the solvent-PAMAM-lipid interplay and explain, at least partially, potential drivers of PAMAM membrane-toxicity.