Reversible addition-fragmentation chain transfer emulsion polymerisation for preparation of biologically compatible nanoparticles

Reversible deactivation radical polymerisation represents a versatile route to prepare well-defined polymeric materials with complex architecture, controlled molecular weight and tuneable end-groups. RDRP techniques have now been translated to heterogeneous polymerisations (emulsion, dispersion, suspension etc.) allowing large scale preparation of nanoparticles with tuneable cores and shells in an aqueous environment. Such systems show great promise in biomedical applications due to their long circulation time, passive tumour accumulation, and core-shell architecture capable of drug loading and controlled release. The overall aim of this thesis is to assess RAFT emulsion polymerisation as a route to prepare nanoparticles for potential biomedical applications, and to study their physical properties, cytotoxicity, cellular uptake and in vivo distribution. Firstly, the synthesis of nanoparticles from amphiphilic block copolymers via RAFT emulsion polymerisation is explored, revealing optimum conditions. Preliminary in vitro and in vivo cytotoxicity and biodistribution studies indicated high biocompatibility with significant liver accumulation post-injection. Following this, a systematic study identifying the effect of nanoparticle rigidity on cellular uptake is explored using a library of hard, intermediate or soft cores tuned with their glass transition temperature. Intracellular fluorescence studies display an increasing amount of uptake with decreasing nanoparticle rigidity, with mechanistic studies suggesting this could be due to a preference of the harder nanoparticles to be internalised via clathrin and caveolae-mediated endocytosis. In the next chapter, alkyne functional RAFT agents are prepared to impart functionality at the nanoparticle surface. It is found that by replacing the initial carboxylate with other functionality significantly reduces colloidal stability. Finally, polysulfonated macro-RAFT agents are used to synthesise heparin-mimicking nanoparticles, via RAFT emulsion polymerisation, capable of stabilising growth factors. The nanoparticles outperform linear analogues and heparin itself, suggesting that the high local concentration at the particle surface significantly improves bioactivity. Overall, this thesis describes how aspects such as particle size, core and shell composition, and corona functionality can be modified individually for specific biological applications

Synthesis of mannosylated and PEGylated nanoparticles via RAFT emulsion polymerisation, and investigation of particle-lectin aggregation using turbidimetric and DLS techniques

Recent developments in controlled radical polymerisation presents an attractive way of producing biocompatible polymeric nanoparticles for a wide range of applications. With this motivation, well defined P (ManAm) and P(PEGA) coated nanoparticles in a range of different sizes have been synthesised via RAFT emulsion polymerisation. The particles were used to precisely investigate the effect of particle size on lectin binding with Concanavalin A, and validate the use of online DLS measurements for lectin-glycoparticle aggregation studies. Larger particles were found to have an enhanced aggregation by both UV–Vis turbidimetric and DLS aggregation studies. The DLS technique was shown to be robust up to an aggregate diameter of c.500 nm for aggregation tests, and was not affected by any dilution or light scattering effects that typically hinder the common use of turbidimetry in particle aggregation studies

Controlled radical polymerization in dispersed systems for biological applications

Polymeric nanoparticles show great promise in a range of biomedical applications, improving pharmacokinetic properties, dose requirements and immune response in drug delivery and bioimaging. Common synthesis techniques such as self-assembly, while prevalent, are unscalable and require the use of organic solvents, or extensive purification. In contrast, recent developments in dispersed state reversible deactivation radical polymerization allow the preparation of well-defined nanomaterials in fully aqueous environments often achieving full monomer conversion, and thus direct use in biological environments without purification in high quantities. These techniques have allowed the preparation of a variety of nanoparticle architectures (nanogel, latex, micelle, nanoworms, vesicles), using ATRP, RAFT and NMP, which in many cases perform significantly better than free radical alternatives. This review focuses on the biological relevance of RDRP in dispersed systems, covering miniemulsion, dispersion, suspension and emulsion polymerizations

A study on the preparation of alkyne functional nanoparticles via RAFT emulsion polymerisation

The multivalent presentation of functional groups on nanoparticle surfaces has long been exploited to attach biologically active moieties. The conventional chemistries typically used (amide, ester, disulfide) however, are non-selective and inefficient. The Huisgen azide alkyne [1,4] cycloaddition (CuAAC) ‘click’ reaction has paved the way for atom economic, and orthogonal conjugation chemistries, and is now widely used in nanoparticle science. In this work, alkyne functionalised nanoparticles were prepared, without lengthy post-nanoparticle synthesis modification procedures, exploiting RAFT emulsion polymerisations stabilised by functional macro-RAFT agents. Our results indicated that ester derived RAFT agents and addition of pendant charged groups are vital to retain colloidal stability and narrow molecular weight distributions. Finally the nanoparticles and model polymers were functionalised with an azido functional polymer and fluorescent dye, showing the surfaces were easily accessible for rapid and efficient post-polymerisation functionalisation

Self-assembly and dis-assembly of stimuli responsive tadpole-like single chain nanoparticles using a switchable hydrophilic/hydrophobic boronic acid cross-linker

Living systems are driven by molecular machines that are composed of folded polypeptide chains, which are assembled together to form multimeric complexes. Although replicating this type of system is a longstanding goal in polymer science, the complexity the structures impose is synthetically very challenging, and generating synthetic polymers to mimic the process of these assemblies appears to be a more appealing approach. To this end, we report a linear polymer programmable for stepwise folding and assembly to higher order structures. To achieve this, a diblock copolymer composed of 4-acryloylmorpholine and glycerol acrylate was synthesised with high precision via reversible addition fragmentation chain transfer polymerisation (Đ < 1.22). Both intramolecular folding and intermolecular assembly were driven by a pH responsive cross-linker, benzene-1,4-diboronic acid. The resulting intramolecular folded single chain nanoparticles were well defined (Đ < 1.16) and successfully assembled into a multimeric structure (Dh = 245 nm) at neutral pH with no chain entanglement. The assembled multimer was observed with a spherical morphology as confirmed by TEM and AFM. These structures were capable of unfolding and disassembling either at low pH or in the presence of sugar. This work offers a new perspective for the generation of adaptive smart materials

Sulfonated copolymers as heparin-mimicking stabilizer of fibroblast growth factor : size, architecture, and monomer distribution effects

Fibroblast growth factors (FGF) are involved in a wide range of biological processes such as cell proliferation and differentiation. In living organisms, the binding of FGF to its receptors are mediated through electrostatic interactions between FGF and naturally occurring heparin. Despite its prevalent use in medicine, heparin carries notable limitations, namely; its extraction from natural sources (expensive, low yield and extensive purification), viral contamination, and batch-to-batch heterogeneity. In this work a range of synthetic homopolymers and copolymers of sodium 2-acrylamido-2-methylpropane sulfonate (AMPS®) were evaluated as potential FGF stabilisers. This was studied by measuring the proliferation of BaF3-FR1c cells, as a model assay, and the results will be compared with the natural stabilisation and activation of FGF by heparin. This study explores the structure-activity relationship of these polysulfonated polymers with a focus on the effect of molecular weight, co-monomer type, charge dispersion and polymer architecture on protein stabilisation

Pyridyl disulfide reaction chemistry : an efficient strategy toward redox-responsive cyclic peptide–polymer conjugates

Cyclic peptide–polymer conjugates are capable of self-assembling into supramolecular polymeric nanotubes driven by the strong multiple hydrogen bonding interactions between the cyclic peptides. In this study, we have engineered responsive nanotubes by introducing a cleavable bond that responds to a reductant utilizing pyridyl disulfide reaction chemistry. Reactions between a cysteine containing cyclic peptide (CP-SH) and pyridyl disulfide containing polymers were initially studied, leading to the quantitative formation of cyclic peptide–polymer conjugates. An asymmetric cyclic peptide–polymer conjugate (PEG-CP-S-S-pPEGA) was then synthesized via orthogonal pyridyl disulfide reaction chemistry and NHS coupling chemistry. The disulfide linker formed by the pyridyl disulfide reaction chemistry was then selectively reduced to thiols in the presence of a reductant, enabling the transition of the conjugates from nonassembling unimers to self-assembled supramolecular polymeric nanotubes. It is anticipated that the pyridyl disulfide reaction chemistry will not only enrich the methodology toward the synthesis of cyclic peptide–polymer conjugates, but also lead to the construction of a new family of redox-responsive cyclic peptide–polymer conjugates and supramolecular polymeric nanotubes with tailored structures and functionalities

Hybrid Poly(<i>β</i>‐amino ester) Triblock Copolymers Utilizing a RAFT Polymerization Grafting‐From Methodology

The biocompatibility, biodegradability, and responsiveness of poly(β‐amino esters) (PBAEs) has led to their widespread use as biomaterials for drug and gene delivery. Nonetheless, the step‐growth polymerization mechanism that yields PBAEs limits the scope for their structural optimization toward specific applications because of limited monomer choice and end‐group modifications. Moreover, to date the post‐synthetic functionalization of PBAEs has relied on grafting‐to approaches, challenged by the need for efficient polymer–polymer coupling and potentially difficult post‐conjugation purification. Here a novel grafting‐from approach to grow reversible addition–fragmentation chain transfer (RAFT) polymers from a PBAE scaffold is described. This is achieved through PBAE conversion into a macromolecular chain transfer agent through a multistep capping procedure, followed by RAFT polymerization with a range of monomers to produce PBAE–RAFT hybrid triblock copolymers. Following successful synthesis, the potential biological applications of these ABA triblock copolymers are illustrated through assembly into polymeric micelles and encapsulation of a model hydrophobic drug, followed by successful nanoparticle (NP) uptake in breast cancer cells. The findings demonstrate this novel synthetic methodology can expand the scope of PBAEs as biomaterials

Functionalisation and stabilisation of polymeric arsenical nanoparticles prepared by sequential reductive and radical cross-linking

The chemical reactivity of arsenic is diverse and distinctive depending upon its interchangeable oxidation states. Alkyl and aryl arsines (As(I)) exist as oligomers, composed of labile and redox responsive As–As bonds which have been exploited to form reactive and responsive materials. Here, the lability and reactivity of As(I)-functional polymeric nanoparticles, derived from thermoresponsive polymers P(PEGA20-b-[NIPAm80-n-co-AsAmn]) (P1, n = 4; P2, n = 11; P3, n = 15; P4, n = 18), is elaborated by in situ reaction with functional acetylenes, resulting in the formation of vinylene–arsine cross-linked polymeric arsenical nanoparticles (NPV–As). Spherical particles with sizes <35 nm have been prepared, which are advantageous for potential drug-delivery (e.g. tumour accumulation) applications. Functional acetylenes enable the introduction of reactive amine, acid and alcohol functional groups into the particles, while the use of propargyl-O-rhodamine ester results in the formation of fluorescent nanoparticles. The vinylene–arsine cross-linking confers increased stability of the polymeric arsenical nanoparticles in model biological redox conditions (GSH, H2O2, 5 mM) compared to those reported previously, with nanoparticle structures retained over 7 days. The parent polymeric arsenicals and the resulting nanoparticles were all shown to exhibit limited cytotoxicity in vitro and cell uptake was confirmed by incubating fluorescent-labelled nanoparticles with PC3 cells. Furthermore, fluorescent confocal microscopy using the PC3 cell-line, confirmed that the nanoparticles were internalised by the cells with evidence of mitochondrial co-localisation, which supports a mitochondria-targeting of arsenic hypothesized based on work involving organoarsenical chemotherapeutics. Thus, this work demonstrates a novel strategy for the preparation of polymeric arsenical nanoparticles, with broad functional group tolerance, and expands our emerging understanding of the in vitro behaviour of this family of nanomaterials

Therapeutic potential of miRNAs in Clostridioides difficile infection

Treating Clostridioides difficile infection with miRNAs alone or combined with live biotherapeutic products may augment therapeutic efficacy and help counteract drug resistance in the future