Developing Cationic Nanoparticles for Gene Delivery

PhDGene delivery can potentially treat acquired and genetic diseases such as cystic fibrosis, haemophilia and cancer. Non-viral gene delivery vectors are attractive candidates over viral vectors such as recombinant viruses, due to their lower cytotoxicity and immunogenicity, despite significantly lower transfection efficiencies. To improve efficiency of non-viral vectors, the investigation of the various parameters influencing DNA transfection is essential. The present study developed a versatile gene delivery system with tailored physicochemical and biological properties. The system used polymer brushes synthesised via atomic transfer radical polymerisation (ATRP), grafted from silica nanoparticles, whose charge density, grafting density, chemistry, length of brush, the size and shape can be altered. The primary focus of the study was poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), known for its positive charge and DNA condensation. The ability of PDMAEMA to interact with DNA was characterised using dynamic light scattering, electrophoretic light scattering methods, surface plasmon resonance and in situ ellipsometry whilst its interaction with cells was studied via cell viability assays. The brush behaviour in response to pH and ionic strength was also studied. The charge density was altered by copolymerising with poly[oligo(ethylene glycol) methyl ether methacrylate](POEGMA) and the effect of such modification on DNA interaction was studied. PDMAEMA-grafted nanoparticles gave the highest transfection efficiency compared to other synthesised polymer brushes, but still displaying almost 2-fold lower transfection efficiency than the commercially available reagent jetPEI®. Different brush chemistries were also investigated. Poly(glycidyl methacrylate) (PGMA) decorated with oligoamines: allylamine, diethylenetriamine and pentaethylene hexamine, and PDMAEMA quaternized with alkyl halides: methyl iodide, allyl iodide and ethyl iodoacetate did not show any significant transfection, despite their performance reported in the literature. The robust system developed is a promising platform for further investigation of parameters influencing cellular uptake and gene expression, and important milestone to develop non-viral gene delivery systems.This work was supported by the Royal Society and the Engineering and Physical Sciences Research Council [Grant EP/J501360/1]

Uptake and release of cargo molecules from responsive surfaces

The development of smart surfaces is of interest for a wide range of applications such as drug-delivery, anti-fouling and lubrication. These layers are stimuli-responsive enabling the controlled uptake and release of a cargo molecule. The aim of this work is to develop methods for monitoring such processes in thermoresponsive systems. To do so, it is required to produce responsive surfaces and design methods to probe their responsiveness as well as the binding of cargos. I present synthetic strategies to graft poly(N-isopropylacrylamide) (pNIPAM), a pNIPAM-based diblock copolymer and polyvaline from silica substrates as well as a procedure for generating mixed surfactant layers of didodecyldimethylammonium bromide (DDAB), and deuterated sodium dodecylsulfate (d-SDS) with defined composition. The main technique for investigating these films is Total Internal Reflection (TIR) Raman spectroscopy. This chemically specific and surface selective technique allows the monitoring of phase transitions inside the layers. It also provides insight on the accumulation of cargo molecules or changes in the layers' composition. A novel Raman imaging technique provides insight into the uniformity of the layers and cargo distribution within them. First, I studied the potential of the cationic surfactant DDAB to transport the anionic surfactant d-SDS onto a silica surface. I show that the adsorption was possible only when the mixture is on the cationic side implying the electrostatic interactions between the vesicles and the surface govern the adsorption. Once the coadsorption had taken place, I triggered the gel-liquid phase transition of DDAB in order to induce a phase separation on the surface. No preferential desorption of d-SDS was observed. Second, I looked at grafted pNIPAM at the silica-water interface and present data on the phase transition showing a change in the hydration of the polymer. I then introduce three cargo molecules (benzamide, d-malonamide and potassium thiocyanate) which have the potential to bind onto one state of pNIPAM via hydrophobic interactions, hydrogen bonding or specific ion interactions. TIR Raman spectroscopy was shown to be able to detect small quantities of the molecules but ultimately showed no selective binding. Third, the focus shifts to grafted polyvaline layers since they are less hydrophobic than pNIPAM. I determine the secondary structure of the grafted chains and present the impact of temperature. All adsorption measurements with the cargo molecules introduced in the previous chapter showed no selective binding. Fourth, I report on a diblock copolymer with an inner cationic block poly((2-dimethylamino) ethyl methacrylate) (pDMAEMA) and an outer neutral thermoresponsive block, pNIPAM. The copolymer was thermoresponsive. Cargo adsorption measurements showed accumulations of each molecule inside the film. The electrostatics driven adsorption of KSCN was stronger at higher temperatures

Functional Hydrophilic Polymers for Solution Assembly and Non-Viral Gene Therapy

This thesis examines functional hydrophilic polymers designed in linear and comb architectures and that carry functional moieties in the context of solution assembly and non-viral gene therapy. Specifically, polymers containing cations, zwitterions, and reactive groups are investigated as non-viral gene therapy reagents and at oil-water interfaces on droplets. Cations facilitate complexation of nucleic acids and interaction with cellular and nuclear membranes, while zwitterions impart stimuli-responsive solution properties and biocompatibility. Reactive groups, including alkenes, alkynes, and benzylic methylenes, permit post-polymerization modification leading to tunable polymer properties in solution and at interfaces. This work expands the knowledge base related to solution, interfacial, and DNA complexation properties imparted by the inclusion and arrangement of functional groups within hydrophilic polymers. Chapters 2-4 discuss polymers having a comb architecture designed for non-viral gene therapy. These polymers contain a polycyclooctene backbone and oligopeptide and zwitterionic pendent groups. Chapter 2 describes comb polymers with Simian Virus 40 nuclear localization sequence (SV40 NLS) pendent groups that interact with receptors on the nuclear membrane to provide enhanced nuclear uptake of complexed DNA. Cell culture experiments revealed a marked effect of oligopeptide orientation on the resulting gene expression levels provided by the NLS-containing comb polymers. Moreover, these polymers outperformed commercial transfection reagents, both in gene expression levels and cell viability. Chapter 3 describes the introduction of zwitterions into these comb polymers and investigates the impact of zwitterions on polymer-DNA complex (or ‘polyplex’) properties. Dispersing small amounts of zwitterion into comb polymers with SV40 NLS and oligolysine pendent groups improved gene expression levels. Chapter 4 describes the synthesis of comb polymers having oligoarginine sequences as pendent groups and properties of the corresponding polyplexes. Compared to oligolysine sequences, oligoarginine facilitated stronger polymer-DNA binding and allowed inclusion of higher loadings of biocompatible zwitterions without compromising the ability of the polymer to complex DNA. Chapters 5 and 6 investigate zwitterionic polymers at the oil-water interface of emulsion droplets. Experiments in Chapter 5 demonstrate the ability of zwitterionic sulfobetaine methacrylate polymers to stabilize oil-in-water droplets in pure water at room temperature, followed by coalescence upon increasing salt concentration or temperature. Integrating alkene and alkyne groups directly into the zwitterionic moiety allowed inclusion of high loadings of functional groups without interrupting the salt-triggered droplet coalescence exhibited by sulfobetaine homopolymers. Functional group placement was found to greatly impact interfacial properties. Chapter 6 describes the preparation of adhesive droplets from novel sulfur-based zwitterionic polymers. Droplet adhesion was modulated by the presence of salt and nucleophiles, and the resultant stability and adhesiveness of the droplets. The interdroplet interactions were found to dictate the mechanical properties of the emulsion and its stability upon application of centrifugal force

Renewable Bio-Based Polymers and Degradable Functional Polymers

In this dissertation, polymers derived from renewable bio-based resources and degradable functional polymers with stimuli-responsive properties by various polymerization techniques were investigated. The properties of these polymeric materials were characterized and discussed. In Chapter 1, the overall background and recent development of renewable bio-based polymers as well as degradable stimuli-responsive polymers was introduced. Major research objectives of my doctoral work were described. The first section of the dissertation, on the preparation of renewable bio-based polymers was provided from Chapter 2 to Chapter 4. In Chapter 2, the preparation of novel polymers derived from renewable gum rosin by atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) polymerization was described. Chapter 3 described the preparation of different rosin containing polycaprolactone (PCL) by a combination of ring-opening polymerization (ROP) and click chemistry. The rosin containing PCL showed excellent hydrophobicity, elevated glass transition temperature, low water uptake and full degradability. Also the polymers exhibited good biocompatibility and low cytotoxicity, suitable for potential biomedical applications. In Chapter 4, sustainable graft copolymers derived from renewable cellulose, rosin and fatty acid as novel thermoplastic elastomers were accomplished by ATRP and mechanical properties of the polymers were characterized by tensile stress-strain and creep compliance testing. The second part of the dissertation is the preparation and characterization of degradable salt-responsive polymers. In Chapter 5, degradable cationic random copolymers containing a PCL skeleton and quaternary ammonium side groups were synthesized by a combination of ring-opening polymerization and copper-catalyzed click reaction. These random copolymers exhibited ion strength-dependent solubility in water. In salt-free water or water with low ionic strength, random copolymers were completely soluble while in high salt concentration solution, the solubility of random copolymers decreased. Also these cationic random copolymers showed good degradability in dilute acid solution. Chapter 6 described the preparation of high molecular weight cationic salt-responsive bottle-brush polymers by ring-opening polymerization, ring-opening metathesis polymerization, and click reaction. These cationic bottle brush polymers exhibited not only good salt responsive properties but also better mechanical properties due to the high molecular weight of the polymers. Both the random copolymers and bottle brush polymers with salt responsive properties showed potential applications in personal hygiene products. Finally, a summary is given in Chapter 7. In addition, some suggestions about future research directions on the renewable polymer materials and degradable stimuli-responsive polymers are provided

Recent Advances in Hybrid Biomimetic Polymer-Based Films: from Assembly to Applications

Biological membranes, in addition to being a cell boundary, can host a variety of proteins that are involved in different biological functions, including selective nutrient transport, signal transduction, inter- and intra-cellular communication, and cell-cell recognition. Due to their extreme complexity, there has been an increasing interest in developing model membrane systems of controlled properties based on combinations of polymers and different biomacromolecules, i.e., polymer-based hybrid films. In this review, we have highlighted recent advances in the development and applications of hybrid biomimetic planar systems based on different polymeric species. We have focused in particular on hybrid films based on (i) polyelectrolytes, (ii) polymer brushes, as well as (iii) tethers and cushions formed from synthetic polymers, and (iv) block copolymers and their combinations with biomacromolecules, such as lipids, proteins, enzymes, biopolymers, and chosen nanoparticles. In this respect, multiple approaches to the synthesis, characterization, and processing of such hybrid films have been presented. The review has further exemplified their bioengineering, biomedical, and environmental applications, in dependence on the composition and properties of the respective hybrids. We believed that this comprehensive review would be of interest to both the specialists in the field of biomimicry as well as persons entering the field

SYNTHESIS AND CHARACTERIZATION OF THERMALLY RESPONSIVE POLYMER LAYERS

Future devices such as biomedical and microfluidic devices, to a large extent, will depend on the interactions between the device surfaces and the contacting liquid. Further, biological liquids containing proteins call for controllable interactions between devices and such proteins, however the bulk material must retain the inherent mechanical properties from which the device was fabricated from. It is well known that surface modification is a suitable technique to tune the surface properties without sacrificing the bulk properties of the substrate. In the present study, surface properties were modified through temperature responsive polymer layers. After the modification, the surfaces gained switchability toward protein interaction as well as surface wettability properties. Poly(N-isopropylacrylamide) (PNIPAM), a well studied thermo-responsive polymer was utilized in the subsequent work. Firstly, thermally responsive brushes made from well defined block copolymers incorporating NIPAM and the surface reactive monomer, glycidyl methacrylate (GMA) were fabricated in a single step process. Reaction of the PGMA block with surface hydroxyl groups anchors the polymers to the surface yet allows PNIPAM to assemble at the interface at high enough concentration to exhibit thermally responsive properties in aqueous solutions. Surface properties of the resulting brushes prepared the 1-step process are compared to characteristics of PNIPAM brushes synthesized by already established methods. The thickness, swelling, and protein adsorption of the PNIPAM films were studied by ellipsometry. Chemical composition of the layer was studied by angle-resolved x-ray photoelectron spectroscopy. Film morphologies and forces of adhesion to fibrinogen were examined using atomic force microscopy (AFM) tapping mode and colloidal probe technique. Block copolymer (BCP) and conventional brush films were abraded and subsequently examined for changes in thermally responsive behavior. The results show that deposition of PNIPAM-b-PGMA provides an effective route to create thermally responsive brushes via a 1-step process, with properties equaling and surpassing that of traditional brushes obtained in multiple steps. Further, the 1-step deposition of reactive BCPs can be extended to fabricate mixed block copolymer films. Well defined BCP containing ethylene glycol and GMA were deposited from a joint solution with PNIPAM-b-PGMA. Mixed brush films were also fabricated via a 2-step process for comparison of the resulting properties. PNIPAM BCP layers were utilized as the grafted primary layer with which end-functionalized PEG was grafted in a second step. Protein adhesion and adsorption of the resulting mixed brush films were studied by AFM colloidal probe technique and ellipsometry. In the next part of the work reported, monolayers of PNIPAM containing nanogels were anchored to the surface of silicon wafers, glass slides, polyvinylidene fluoride (PVDF) fibers, and tungsten wires using a \u27grafting to\u27 approach. The particles of were synthesized with different diameters by free radical precipitation polymerization and reversible addition chain transfer polymerization (RAFT) techniques. The behavior of the synthesized grafted layers with the behavior of PNIPAM brushes (densely end-grafted) is compared. Indeed, the grafted monolayer swells and collapses reversibly at temperatures below and above the transition temperature of PNIPAM. AFM in aqueous environment was utilized to study the actuation behavior of the nanogel films. Wettability studies of the grafted layers were performed using various contact angle measurement methods to determine the contact angle changes on different substrates. New methods for the development of thermally responsive polymer films are described. The methods enable the grafting of films with tunable film thickness, temperature response, and well defined biological interaction. The complete grafting of the responsive polymer films require no organic rinsing after grafting step

Advances in Thermoresponsive Polymers

Thermoresponsive polymers, materials able to undergo sharp and often reversible phase separations in response to temperature stimuli, are introducing new paradigms in different fields, including medicine, advanced separations and oil and gas. In "Advances in Thermoresponsive Polymers", a clear picture of the frontiers reached in the understanding of the mechanistic behavior associated with temperature-induced phase separation, the influence of the polymer structure in regulating the macroscopic behavior of these materials and the latest applications for which thermoresponsive polymers show great potential is provided