The effect of surface functionalisation on cancer cells internalisation and selective cytotoxicity of zirconium metal organic frameworks

Abánades Lázaro, Isabel (2018) The effect of surface functionalisation on cancer cells internalisation and selective cytotoxicity of zirconium metal organic frameworks. PhD thesis, University of Glasgow.

Abstract

A considerable amount of effort has been directed to develop porous materials as drug delivery systems (DDSs) – one of the most promising emerging applications in healthcare, as most anticancer therapeutics have toxic dose dependence due to a lack of tumour selectivity – as their hierarchical porosity can be used to store and release challenging drugs.

Among them, Metal-Organic Frameworks (MOFs) – emerging hybrid, highly porous crystalline structures – offer several advantages compared to other available DDS, as they combine desirable features from both organic (biocompatibility, e.g. porous polymers) and inorganic (high loadings, e.g. mesoporous silica) porous materials. MOFs are highly amenable to functionalisation, meaning fine control over their physical properties can be achieved, and thus they have experienced tremendous development during the past decade in many applications. Despite surface engineering being advantageous for diverse fields – in biomedicine, it can both improve stability and dispersion, and provide the possibility of targeted carriers, decreasing the immune system recognition – surface functionalization of MOFs is underdeveloped. The multiple synthetic steps – synthesis, drug loading and surface modification – and the lack of orthogonality between them hinder their industrial manufacturing as DDSs.

This thesis focuses on the development of surface functionalisation protocols of Zirconium MOFs, particularly UiO-66, a Zr-terephthalate MOF, the study of their cell internalisation fate and routes and the correlation with their therapeutic activity.
During Chapter 1, an introduction to the use of DDSs in anticancer therapy, followed by examples of the most relevant MOFs from a coordination chemistry point of view, is given, in which zirconium MOFs and their synthesis are highlighted. Particular focus is given to the coordination modulation process, in which monodentate modulators are introduced to the MOFs synthesis to compete with the multidentate linkers during nucleation, enhancing properties such as porosity through the induction of defects. Then, the most relevant examples of surface functionalization of Zr MOFs for drug delivery are discussed with respect to the effects on properties such as colloidal dispersion in aqueous solvents, physiological stability, and drug release kinetics.

In Chapter 2 different functionalised modulators (i.e p-functionalised benzoic acids, folic acid or biotin) are introduced to UiO-66 synthesis to obtain surface-functionalised UiO-66 with the appropriate size for drug delivery by one-pot synthesis. Full characterisation of the materials shows them to be remarkably porous due to the defects formed when modulators attach to available zirconium positions in the pores and on the surfaces of the MOFs. Furthermore, the use of a carboxylate-containing anticancer metabolic target (dichloroacetic acid, DCA) as a modulator of UiO-66 synthesis is explored, and co-modulated samples, in which both DCA and functionalised modulators are introduced to UiO-66 synthesis, are synthesised and fully characterised, resulting in drug-containing (ca. 20% w/w) surface-functionalised MOFs by one pot syntheses. Importantly, DCA modulation induces a high number of defects, and consequently highly charged nanoparticles which are colloidally stable in aqueous solvents. Particle size control in the DCA modulated synthesis of the UiO family of isoreticular MOFs – including UiO-66 and its bromo, amino and nitro derivatives, and extended structures Zr-Naphthalenedicarboxylate (NDC) and Zr-Biphenyldicarboxylate (BPDC) – is achieved, obtaining ca. 100 nm particles of UiO-66 derivatives and microcrystals of Zr-NDC and Zr-BPDC when ZrCl4 is the metal precursor, and mesoporous < 20 nm UiO-66 derivatives and ca. 200 nm Zr-NDC and Zr-BPDC when ZrOCl2 is used as the metal precursor. The high porosity of the DCA modulated samples, due to DCA attachment to the inner and outer surface at defect sites, allows the loading of a second drug, the well-known anticancer drug 5-fluorouracil (5-FU), into the pores of the isoreticular MOFs to create dual DDSs.

Different postsynthetic modes of surface coating, based in both coordination and covalent chemistry, are studied during Chapter 3. The functionalities of the p-functionalised benzoic acid modulators, introduced to UiO-66 structure during Chapter 2, are used to covalently attach short-chain alkanes and long-chain polymers to UiO-66 surface through copper-catalysed azide-alkyne cycloaddition. Exhaustive characterisation confirms that the attachment occurs through covalent chemistry and not through surface adhesion or electrostatic forces. Folic acid and biotin, which are introduced to UiO-66 surface as synthetic modulators during Chapter 2, are also introduced to UiO-66 surface postsynthetically. Colloidal dispersion and stability towards phosphates are investigated and compared to bare MOFs, in order to gain insights into the effect of both surface chemistry and mode of attachment on physical properties.

A comprehensive overview of in vitro studies of cellular internalisation of zirconium MOFs is given in Chapter 4, focussing on the relevance of the endocytosis internalisation routes, which are strictly correlated with therapeutic efficacy. The postsynthetic surface functionalisation protocols investigated in Chapter 3 are applied to analogous calcein-loaded UiO-66 samples. Calcein is a fluorescent molecule not able to efficiently cross the cell membrane by itself, and hence serves as an in ideal probe of MOFs cellular internalisation. Its release from bare and poly(ethylene glycol) coated UiO-66 into phosphate buffered saline at pH 7.4 and 5.5, in order to simulate extracellular and intracellular conditions, is found to be pH responsive (more pronounced at 5.5) for all MOFs, but an ideal decrease in calcein release at pH 7.4 occurs only for PEGylated MOFs. Internalisation of calcein-loaded MOFs by HeLa cervical cancer cells is studied by fluorescence assisted cell sorting, highlighting the effects of surface chemistry on endocytosis efficiencies and internalisation mechanisms.

A discussion of in vitro studies into anticancer drug delivery from Zr MOFs is provided in Chapter 5, alongside a summary of the therapeutic effects of DCA and approaches to enhance its anticancer efficacy. Experimental assessment of the in vitro anticancer performance towards MCF-7 breast cancer cells of the DCA-containing MOFs of the UiO family of different sizes (ca. 100 nm and <20 nm), synthesised by coordination modulation during Chapter 2, is given. The effect of dual-drug containing MOFs (DCA and 5-FU) is also examined, to investigate the possible synergic effect of the drug combination. Then, the cytoxicity of bare and surface functionalised, DCA-loaded and empty UiO-66 MOFs is studied at first upon incubation with HeLa cells, for which the cellular routes of internalisation were elucidated in Chapter 4. The most promising MOFs are then tested for selective anticancer activity against a series of cancerous and healthy cells lines, and their macrophage uptake and ROS production is also analysed, to determine the effect of surface functionalization. The selective anticancer cytotoxicity of folate-coated MOFs is attributed to a combination of cancer cell targeting and optimal cell internalisation routes.

To summarise, the one-pot synthesis of drug-loaded, surface functionalised UiO-66 has been successfully performed, resulting in porous, crystalline MOFs with the appropriate size for drug delivery. The use of a carboxylate-containing anticancer metabolic target as a modulator has been explored for the UiO family of isoreticular MOFs, resulting in well-dispersed nanoMOFs with enhanced anticancer activity, into which a second drug can be loaded, enabling the creation of dual DDSs. A series of postsynthetic surface modifications are performed, enabling the study of the MOF’s properties (colloidal dispersion, physiological stability and biocompatibility) with respect to their surface chemistry and coating mode, but more importantly providing valuable insights into correlations between surface chemistry, routes of cellular internalisation and therapeutic effect.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Metal organic frameworks, controlled release, drug delivery systems, anticancer activity, endocytosis routes, dichloroacetic acid.
• Subjects: Q Science > QD Chemistry
• Colleges/Schools: College of Science and Engineering > School of Chemistry
• Supervisor's Name: Forgan, Dr. Ross S.
• Date of Award: 2018
• Depositing User: Dr Isabel Abánades Lázaro
• Unique ID: glathesis:2018-9099
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 16 May 2018 14:02
• Last Modified: 18 Jun 2018 09:04
• URI: https://theses.gla.ac.uk/id/eprint/9099

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