To test the versatility of the chemistry, the conjugation of three further polymers was attempted. First, poly(DMA) and poly(4-AM) were synthesised as described in Section 5.2.ii containing a terminal azide group. Both of these polymers are permanently hydrophilic (i.e. they exhibit no temperature responsive behaviour) and there is some evidence that they may be useful for the synthesis of biocompatible coatings.29 Other
hydrophilic polymers, most notably poly(ethylene oxide) (commonly referred to as PEO or PEG) have been used extensively in the literature to protect DNA from degradationin vivo, and also to impart so-called stealth properties to polymer nanoparticles intended for use as drug delivery agents.30-35 It was therefore thought that the efficient conjugation of these
164 Figure 5.17 shows PAGE analyses of the conjugation reactions between s0–alkyne and azide-functionalised poly(DMA) and poly(4-AM). The reactions proceeded efficiently, with yields of around 50 % estimated by densitometry. These results proved that the CuAAC reaction is generally applicable to the synthesis of hydrophilic DNA–polymer conjugates.
Figure 5.17 15 % native PAGE analyses of the poly(DMA) (P18, left) and poly(4-AM) (P19, right) DNA–polymer conjugates. Yields were around 50 % in both cases, as assessed by densitometry.
Next, the synthesis of DNA–poly(styrene) conjugates was attempted using the poly(styrene)–alkyne synthesised in Section 5.2.i (P12). Poly(styrene) has practically no solubility in water, so the synthesis of these conjugates has always proved to be very difficult, with yields typically below 5 %, even when solid phase synthesis techniques are employed.36 Since the CuAAC system used in this work employs 5 % water as a DNA
solubiliser, there was some concern that the polymer would become insoluble. However, it was found that the poly(styrene) used could tolerate a small amount of water, and was fully solubilised in the 95 % DMF solution used as the reaction solvent.
Despite the incompatibility of the polymer and DNA species, the conjugate was still observed in approximately 74 % yield by 2 % agarose gel electrophoresis (see Figure 5.18). Agarose was used instead of polyacrylamide for these analyses because its wider pores allow the migration of even relatively large aggregates.
Figure 5.18 2 % agarose gel analysis showing the successful synthesis of a poly(styrene)– DNA conjugate using CuAAC. The yield by densitometry (left) was estimated to be 74 %.
It was hypothesised that, upon transfer of the conjugate into the aqueous loading buffer used for electrophoresis, the poly(styrene) segments would aggregate to form large, micellar structures and it is these that were observed in the gel and not the free conjugate itself. To test this hypothesis, the solution was analysed by dynamic light scattering (DLS). The results are shown in Figure 5.19 and revealed the presence of a significant population of particles around 50 nm in diameter.
Figure 5.19 DLS analysis by number of a solution of the s0–poly(styrene) conjugate in water (correlation function inset). The main population had a hydrodynamic diameter of 48 nm and a dispersity of 0.24.
166 These particles were imaged by TEM, dry on a graphene oxide support, without staining.37
Well-defined nanoparticles with diameters around 20 nm were observed (Figure 5.20 and Figure 5.21), confirming the amphiphilic nature of the DNA–poly(styrene) conjugate.
Figure 5.20 TEM micrograph of the nanoparticles formed when the s0–poly(styrene) conjugate was transferred from DMF (a good solvent for both blocks) to water (a poor solvent for poly(styrene)). The sample was dried directly onto the graphene oxide-coated TEM grid without staining. Scale bar: 50 nm.
Figure 5.21 TEM micrograph (left) and particle size analysis (right) of the s0–poly(styrene) nanoparticles. Particle analysis of 372 nanoparticles.
The CuAAC reaction was thus shown to be an excellent chemistry for the conjugation of DNA to a wide range of functional polymers, including hydrophilic, temperature- responsive and hydrophobic.
5.3 Conclusions
The copper catalysed azide–alkyne cycloaddition (CuAAC) reaction was shown to be an excellent technique for the synthesis of DNA–polymer conjugates. An alkyne dithioester CTA was used to successfully polymerise styrene and NIPAM with good control over molecular weights and dispersity. NMR spectroscopy confirmed the presence of the alkyne functional group at the polymer chain termini. An azide-functionalised CTA was synthesised by esterification of the common trithiocarbonate CTA DDMAT, and was used to polymerise DMA, NIPAM and styrene with control. Whilst the CTA itself was observed to be stable under RAFT polymerisation conditions in the absence of monomer, FTIR studies of the polymers revealed only low incorporation of the azide group. A different route was therefore adopted, whereby azide-terminated polymers were synthesised by post- polymerisation modification of a PFP activated ester located at the polymer chain terminus. Using this method, azide-functionalised polymers of NIPAM, DMA, 4-AM and styrene were all synthesised. NMR spectroscopy revealed efficient substitution of the PFP ester, and FTIR confirmed an increase in incorporation of the azide group compared to polymers synthesised using the azide-functionalised CTA.
A large number of catalyst and solvent combinations were then tested for the DNA– polymer conjugation reaction. Only one – copper iodide triethylphosphite – was found to be effective in organic solvents. The reaction conditions were optimised and used to conjugate polymers of NIPAM, DMA, 4-AM and styrene to azide- and alkyne- functionalised DNA in up to 90 % yield. Coupling of poly(styrene) to azide DNA led to the formation of self-assembled hybrid DNA–polymer micelles, which were observed by agarose gel electrophoresis, DLS and TEM.
168