Chapter Summary

Telechelic, Reversible Addition-Fragmentation Chain Transfer (RAFT)-derived macromonomers with a pyridyl disulfide end-group were converted into high molecular weight, disulfide-linked polymers using a polycondensation, step-growth procedure. The applicability of this method to polycondense a library of macromonomers with different functionalities including (meth)acrylates and acrylamides was investigated. Side-chain sterics were found to be important as non- linear poly(ethylene glycol) analogues proved incompatible with this synthetic methodology, as did methacrylates due to their pendant methyl group. This method was used to incorporate disulfide bonds into poly(N-isopropylacrylamide), pNIPAM,

precursors to give dual-responsive (thermo- and redox) materials. These polymers were shown to selectively degrade in the presence of intracellular concentrations of glutathione, but be stable at low, extracellular concentrations. Due to the molecular weight-dependent cloud point of pNIPAM, the lower critical solution temperature behaviour could be switched off by a glutathione gradient without a temperature change; an isothermal transition.

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2.2 Introduction

Responsive or ‘smart’ materials are capable of undergoing a physical response upon the application of an external stimulus. These materials are finding application in a diverse range of fields including switching surfaces and adhesives, artificial muscles, sensors,1 and biomedical fields such as drug delivery,2 gene delivery3 and tissue engineering.4 Synthetic polymers exhibiting a lower critical solution temperature (LCST) have been extensively investigated as smart, thermo-responsive materials. Upon increasing the solution temperature above the cloud point (the measureable property of an LCST) an aqueous polymer solution becomes insoluble and aggregates/precipitates. This property can be exploited for either drug release5 or hyperthermia-triggered cellular uptake due to increased lipophilicity above the

LCST.5-7 Examples of polymers displaying this behaviour include

poly[(oligoethyleneglycol)methyl ether methacrylate] (pOEGMA),8, 9 poly(N-

isopropylacrylamide) (pNIPAM)10 and poly(N-vinylpiperidone).11 While

temperature changes are useful, some applications may require a change in polymer solubility (i.e. to switch between “active” and “inactive” states) without applying a

thermal gradient. “Isothermal” transitions have previously been demonstrated by

Alexander and co-workers based on salt concentration gradients,12, 13 Steinhauer et

al. following aminolysis of RAFT-derived polymer chains14 and by Rimmer and co-

workers due to bacterial binding.15 Other examples include the use of light,16, 17 protein binding18 and the application of dissolved gases.19 We have demonstrated that selective cleavage of a single polymer end-group results in a shift in pNIPAM cloud point allowing an isothermal transition based on bioreduction.20

54 Polymer degradation is a critical consideration for in vivo applications such as drug

or gene delivery. This is typically achieved by the incorporation of one or more cleavable linkers into either the polymer backbone, side-chain or as a cross-linker.21,

22

Common degradation triggers include hydrolysis, thermolysis or enzymatic action.23-26 A major challenge associated with the development of biodegradable polymers is the introduction of functional groups onto the polymer backbone. For example ring opening polymerisation of N-carboxyanhydrides or cyclic esters, which

give degradable poly(amides) or poly(esters) respectively, are incompatible with most functional groups.27-29 Conversely, controlled radical polymerisation processes enable a vast range of functional groups to be incorporated into a polymer structure, but give rise to an all-carbon backbone which cannot degrade.30 To address the above paradox, we have previously reported a synthetic route towards degradable, main-chain disulfide bond-containing pNIPAM. This was achieved by polymerising NIPAM using a RAFT chain transfer agent containing a pyridyl disulfide moiety at the α-terminus. Following aminolysis of the ω-terminal dithioester, a polycondensation-type, step-growth polymerisation with release of pyridine thione occurred (Figure 2.1). The degradability of this material was demonstrated by the addition of a reducing agent which produced polymer chains with a higher LCST than the disulfide-linked counterpart. Hence, a novel method to ‘switch off’ LCST behaviour using a secondary chemical stimulus was illustrated.31 A similar procedure combining aminolysis and thiol-disulfide exchange has also been recently applied to alkyl disulfides.32

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Figure 2.1 Preparation of disulfide-linked polymers using a polycondensation-type methodology: (i) Ethanolamine (1 eq.); triethylamine (2 eq.); THF; N2; 25 °C; 24h.31

The redox-sensitive nature of disulfide-containing polymers is of particular interest for triggered cellular delivery applications. The main reducing agent (anti-oxidant) inside cells is glutathione (GSH) where it is present at mM concentrations. Conversely, the extracellular GSH concentration is only µM hence this differential provides a unique and selective trigger to promote intracellular degradation, whilst ensuring stability in the circulation.33, 34 The use of disulfide-containing polymers has seen a particular focus on gene delivery therapies to date35-40 though there are also reports utilising redox-sensitive materials for targeted, drug delivery applications.41-47

This chapter explores the scope of our synthetic methodology as a means of preparing controlled radical polymerisation-derived, highly functionalised disulfide- linked polymers. The biodegradability of these materials using biorelevant glutathione concentrations is investigated to ensure selectivity for intracellular conditions. Finally, thermally responsive, degradable polymers are tested for their

56 isothermal response to glutathione gradients to enable their LCST behaviour to be ‘switched off’ once inside a cell (Figure 2.2).

Figure 2.2 Study concept: Disulfide-linked, biodegradable pNIPAM will be prepared and the in vivo GSH concentration gradient used to trigger selective

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2.3 Results and Discussion

The aim of this work was to exemplify our previous report on the synthesis of disulfide-linked pNIPAM, obtained by a polycondensation-type reaction of pyridyl disulfide-terminated, RAFT-derived polymers, Scheme 2.1.31 Previously we used a dithioester chain transfer agent (Scheme 2.2, compound 1) which, although an excellent mediator for the polymerisation of a wide range of monomers, is less synthetically accessible than trithiocarbonate RAFT agents. We therefore prepared the trithiocarbonate propanoic acid 2-{[(dodecylthio)thioxomethyl]thio}-2-methyl- 2-(2-pyridinyldithio)ethyl ester (PADE, 4) using a method modified from that described by Skey and O’Reilly.48 First, 2-(dodecylthiocarbonothioylthio)-2- methylpropanoic acid, 2, was prepared by the reaction of dodecane thiol with carbon disulfide and 2-bromo methyl propionic acid in the presence of potassium phosphate. This compound was then coupled to pyridyl disulfide-containing alcohol 3 in the presence of N,N-diisopropylcarbodiimide and 4-dimethylaminopyridine to give

PADE (Scheme 2.2, compound 4), which was isolated using column chromatography on silica to give a yellow oil in good yield (78 %).

Structure and purity was confirmed by 1H NMR, 13C NMR spectroscopies and high resolution mass spectrometry. As shown in Figure 2.3, analysis by 1H NMR spectroscopy revealed 4 peaks between 7.10 and 8.47 ppm and two triplets at 4.36 and 3.03 ppm corresponding to the pyridyl protons and two CH2 groups on the

starting alcohol. Signature peaks from the carboxylic acid fragment include a triplet and doublet at 0.88 and 3.27 ppm corresponding to the CH3 and terminal CH2 group

58 carbon at 221.5 ppm and the CH3 carbon at 25.3 ppm from the carboxylic acid

fragment together with the pyridyl carbons between 119.8 and 159.9 ppm.

Scheme 2.1 Synthetic strategy for the preparation of a disulfide-linked polymer from