Polymer/DNA Bio-conjugates via Grafting-from and Grafting-to

Author: Simon Bou

Bou, Simon, 2017 Polymer/DNA Bio-conjugates via Grafting-from and Grafting-to, Flinders University, School of Chemical and Physical Sciences

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Polymer bio-conjugates can be prepared by the grafting-from, grafting-through and the grafting-to approach. Grafting-from involves the preparation of a bio-macro initiator, where a functional group attached to the bio-molecule is used to initiate the polymerisation of a monomer. The grafting-through approach, involves the preparation of bio-monomer that is polymerisable; and the grafting to approach is where the polymer is prepared separately with specific functionality that can be used to attach bio-molecules. This thesis investigated grafting-from and grafting-to as a method of making polymer bio-conjugates that have potential use in bio-medical applications.

Polymer bio-conjugates were initially prepared by the grafting-from approach. First N-hydroxyl succinimide/N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide (NHS/EDC) coupling chemistry transformed the end groups of a trithiocarbonate reversible addition fragmentation chain transfer (RAFT) agent. Solution proton nuclear magnetic resonance (1H NMR) spectroscopy and electrospray ionisation (ESI) mass spectrometry (MS) confirmed the synthesis of the RAFT ester agent. Amide coupling between an amine functionalised oligonucleotide and the synthesised activated ester RAFT agent yielded a bio-macro initiator agent. RAFT polymerisation of 2-hydroxyethyl acrylate (HEA) in solution, using the bio-macro agent under a range of reaction conditions was performed to grow p(HEA) from the oligonucleotide. The purification of the polymer bio-conjugates was found to be straight-forward, however, growth from the bio-macro agent was found to be slow and limited.

Polymer bio-conjugates were also prepared by the grafting-to approach. Five poly(N-isopropyl acrylamide)-co-hydroxymethlyacrylamide (pNIPAAm-co-HMAAm) copolymers were prepared by RAFT polymerisation. It was confirmed by 1H NMR spectroscopy and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy that the feed ratio of the monomer in the copolymerisation was retained in the final polymer composition. The polydispersity (Đ) of between 1.3-1.6 for the pNIPAAm-co-HMAAm copolymers indicated that good control over molecular weight was achieved by RAFT polymerisation. The temperature response of the pNIPAAm-co-HMAAm copolymers was determined by measuring their lower critical solution temperatures (LCSTs). The results clearly showed that the LCSTs of the pNIPAAm-co-HMAAm copolymers could be controlled by the changing the ratio of HMAAm monomer in the pNIPAAm-co-HMAAm copolymer. It was proposed that the pNIPAAm-co-HMAAm copolymers form a domain at their LCST that is favourable for SYBR Green (SG) intercalation. The interaction between SG and pNIPAAm-co-HMAAm copolymers was measured by the monitoring the change in SG intensity when they were mixed together in solution. The SG fluorescence intensity for all synthesised pNIPAAm-co-HMAAm copolymers increased with polymer concentration. The initial increase in SG fluorescence intensity for the five pNIPAAm-co-HMAAm copolymers occurred at the calculated LCSTs of the copolymers. The critical micelle concentration (CMC) of the pNIPAAm-co-HMAAm copolymers was determined by extrapolation from a graph of SG intensity versus polymer concentration. The CMC concentration of the pNIPAAm-co-HMAAm copolymers was found to increase as the HMAAm composition increased. Dynamic light scattering (DLS) measured the diameters of the pNIPAAm-co-HMAAm copolymer aggregates at different concentrations. The results indicated that aggregation was dependent on temperature, concentration and HMAAm content. The pNIPAAm-co-HMAAm copolymer aggregates sizes were also measured by atomic force microscopy (AFM). The results suggested that the copolymers with the increased HMAAm content wet the surface more readily. This was thought to be due to a greater amount of hydrophilic character as the HMAAm content is increased. It was proposed that the interaction between the polar head groups of the HMAAm copolymer will wet the surface of the mica more readily leading to greater spreading of the particle.

The hydroxyl functionality of the pNIPAAm-co-HMAAm copolymers were transformed by Steglich esterification to introduce a strained alkyne (SA) functionality dibenzylcyclooctyne-acid (DBCO). For the first time, the LCSTs of SA functionalised pNIPAAm-co-HMAAm copolymers were measured using a newly developed light scattering method. This was performed using a thermal cycler that can measure light scattering in real-time. This light scattering method can also measure the LCSTs at very low volumes and in high-throughput. Making this a very useful method of measuring polymer bio-conjugates, since they are often synthesised in limited quantities. In a grafting-to approach, DNA end functionalised with azides were coupled via click chemistry to pNIPAAm-co-HMAAm copolymers. The melt temperature of DNA was found to be impacted by its conjugation to pNIPAAm-co-HMAAm copolymers, which has potential implications for drug delivery applications.

NHS functionalised polyethylene glycol (PEG) copolymers were prepared by free radical polymerisation (FRP). The NHS-PEG copolymers were transformed into alkene functionalised PEG copolymers. 1H NMR spectroscopy and ATR-FTIR spectroscopies confirmed the transformation. Cross-linked hydrogel disks were prepared by thiol-ene photo polymerisation of the alkene functionalised PEG copolymers. Increasing the cross-link density decreased the swelling profile of the hydrogel disk in water. At low cross-link density, a sustained release of a model drug was observed. When the cross-link density was increased the release, profile changed to burst release. The PEG copolymers thus display tuneable cross-link properties which could make them useful in drug delivery applications.

Keywords: Polymer bio-conjugates, drug delivery, RAFT, Temperature responsive polymers, Hydrogels

Subject: Chemistry thesis

Thesis type: Doctor of Philosophy
Completed: 2017
School: School of Chemical and Physical Sciences
Supervisor: Professor Claire Lenehan