Microgels are a class of colloids that are mechanically soft, and while in many cases can behave similarly to their hard-sphere counterparts, their interaction potentials are quite different. The softness of the interaction between microgels makes them capable of deformation and compression into more concentrated assemblies. This concentrated regime is interesting because little, if any, experimental work has been done to see how the bulk properties of soft-sphere assemblies deviate from those of hard-spheres at the point where their interaction potentials begin to diverge. In this thesis the effects on assembly phase behavior and dynamics of both particle compression and softness of the interaction potential are addressed. Poly(N-isopropylacrylamide) (pNIPAm) microgels are an excellent model system in which to study these effects. The thermoresponsivity of the polymer provides the experimentalist with a dial to tune the volume fraction of an assembly, while maintaining a constant particle number density in the system. Optical microscopy, particle tracking analysis and rheology have been used to investigate the effects of packing and particle structure on equilibrium phase behavior and localized perturbations to the phase of the assembly of this soft-sphere system. It has been elucidated from these experiments and others involving deswelling of large microgel particles in the presence of high concentrations of smaller microgels, that the soft, repulsive interaction between microgels is caused by a longer-range repulsion than was previously believed. The particles are acting on each other from a distance through the osmotic pressure of the assembly, which causes each particle to deswell without coming into direct contact with a neighboring particle.

abstract: Current treatment methods for cerebral aneurysms are providing life-saving measures for patients suffering from these blood vessel wall protrusions; however, the drawbacks present unfortunate circumstances in the invasive procedure or with efficient occlusion of the aneurysms. With the advancement of medical devices, liquid-to-solid gelling materials that could be delivered endovascularly have gained interest. The development of these systems stems from the need to circumvent surgical methods and the requirement for improved occlusion of aneurysms to prevent recanalization and potential complications. The work presented herein reports on a liquid-to-solid gelling material, which undergoes gelation via dual mechanisms. Using a temperature-responsive polymer, poly(N-isopropylacrylamide) (poly(NIPAAm), the gelling system can transition from a solution at low temperatures to a gel at body temperature (physical gelation). Additionally, by conjugating reactive functional groups onto the polymers, covalent cross-links can be formed via chemical reaction between the two moieties (chemical gelation). The advantage of this gelling system comprises of its water-based properties as well as the ability of the physical and chemical gelation to occur within physiological conditions. By developing the polymer gelling system in a ground-up approach via synthesis, its added benefit is the capability of modifying the properties of the system as needed for particular applications, in this case for embolization of cerebral aneurysms. The studies provided in this doctoral work highlight the synthesis, characterization and testing of these polymer gelling systems for occlusion of aneurysms. Conducted experiments include thermal, mechanical, structural and chemical characterization, as well as analysis of swelling, degradation, kinetics, cytotoxicity, in vitro glass models and in vivo swine study. Data on thermoresponsive poly(NIPAAm) indicated that the phase transition it undertakes comes as a result of the polymer chains associating as temperature is increased. Poly(NIPAAm) was functionalized with thiols and vinyls to provide for added chemical cross-linking. By combining both modes of gelation, physical and chemical, a gel with reduced creep flow and increased strength was developed. Being waterborne, the gels demonstrated excellent biocompatibility and were easily delivered via catheters and injected within aneurysms, without undergoing degradation. The dual gelling polymer systems demonstrated potential in use as embolic agents for cerebral aneurysm embolization. / Dissertation/Thesis / Ph.D. Bioengineering 2012

abstract: Aqueous solutions of temperature-responsive copolymers based on N-isopropylacrylamide (NIPAAm) hold promise for medical applications because they can be delivered as liquids and quickly form gels in the body without organic solvents or chemical reaction. However, their gelation is often followed by phase-separation and shrinking. Gel shrinking and water loss is a major limitation to using NIPAAm-based gels for nearly any biomedical application. In this work, a graft copolymer design was used to synthesize polymers which combine the convenient injectability of poly(NIPAAm) with gel water content controlled by hydrophilic side-chain grafts based on Jeffamine® M-1000 acrylamide (JAAm). The first segment of this work describes the synthesis and characterization of poly(NIPAAm-co-JAAm) copolymers which demonstrates controlled swelling that is nearly independent of LCST. The graft copolymer design was then used to produce a degradable antimicrobial-eluting gel for prevention of prosthetic joint infection. The resorbable graft copolymer gels were shown to have three unique characteristics which demonstrate their suitability for this application. First, antimicrobial release is sustained and complete within 1 week. Second, the gels behave like viscoelastic fluids, enabling complete surface coverage of an implant without disrupting fixation or movement. Finally, the gels degrade rapidly within 1-6 weeks, which may enable their use in interfaces where bone healing takes place. Graft copolymer hydrogels were also developed which undergo Michael addition in situ with poly(ethylene glycol) diacrylate to form elastic gels for endovascular embolization of saccular aneurysms. Inclusion of JAAm grafts led to weaker physical crosslinking and faster, more complete chemical crosslinking. JAAm grafts prolonged the delivery window of the system from 30 seconds to 220 seconds, provided improved gel swelling, and resulted in stronger, more elastic gels within 30 minutes after delivery. / Dissertation/Thesis / Ph.D. Bioengineering 2012

Orientador: Watson Loh / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-05T11:31:09Z (GMT). No. of bitstreams: 1 Teixeira_LucianaAkissuedeCamargo_M.pdf: 592549 bytes, checksum: bcab0d8b03c6105e79e7174dd9b9c308 (MD5) Previous issue date: 2004 / Mestrado / Físico-Química / Mestre em Química

So-called, “Dynamic biomaterials” comprised of stimuli-responsive hydrogels are useful in a wide variety of biomedical applications including tissue engineering, drug delivery, and biomedical implants. More than 150,000 peer-reviewed articles (as of 2016) have been published on these materials, and more specifically, over 100,000 of these are on the most widely studied, poly(N-isopropylacrylamide). This thermoresponsive polymer in a crosslinked hydrogel network undergoes a large volume phase transition (𝑉/𝑉0 ~ 10 − 100) within a small temperature range (𝑇 ~ 1 − 3𝐾) making it particularly useful for tissue engineering applications because of the ability to control the topographical configuration of cells into tissue modules which can be applied in multiple layers to form three dimensional constructs. Nevertheless, applications with poly(N-isopropylacrylamide) hydrogels are hindered by two key obstacles: 1. there is presently no quantitative prediction of mechanical properties over the volume phase transition and 2. the mechanisms of cell attachment and detachment remain controversial and unclear. Current polymer-solution theory, first postulated by Paul Flory and Maurice Huggins in 1942, successfully predicts hydrogel swelling for non-stimuli-responsive polymers based on an empirically derived interaction parameter. However, for stimuli-responsive polymer hydrogels, this theoretical framework fails to quantitatively predict swelling and mechanical properties of the polymer. Currently, only qualitative agreement with experiment has been shown. Cell-cell and cell-matrix interactions are mediated through proteins collectively known as cell adhesion molecules. For cell-matrix interactions, these are generally the transmembrane protein, integrin, and the serum protein, fibronectin. It is widely accepted that nearly all molecular mechanisms of cell-matrix interactions are dependent on recognition of the peptide sequence Arg-Gly-Asp. However, much less is known about mechanical mechanisms involved in cell-cell and cell-matrix interactions. Obstacles to the advancement of these applications are 1) unclear mechanisms of cell release and 2) extended exposure of cells to hypothermic conditions. The author, in collaboration with others, has published work demonstrating reduced cell exposure to hypothermic conditions during tissue module release and elucidated a mechanism of tissue module release: mechanical strain. The central hypothesis of work in this proposal is that tissue module release occurs due to a mechanical strain-rate coinciding with critical force needed overcome the dynamic bond strength of cell adhesion molecules. Advances in this area could improve biomaterial design and accelerate the field of regenerative medicine by reducing or eliminating the need for allograft transplants. This dissertation project, then, seeks to address these two obstacles through biophysical characterization methods and analysis including: atomic force microscopy, scanning electron microscopy, laser-scanning confocal micrscopy, phase-contrast microscopy, and mass-balance analysis. It is hypothesized that, (1) mechanical properties of PNIPAAm hydrogels are quantitatively predicted based on crosslinker ratio in the water-rich phase, (2) release of cells from micropatterned PNIPAAm hydrogels occurs when the lateral strain in the surface exceeds ϵ > 0.25, and (3) the molecular mechanism of rapid cell release from micro-patterned PNIPAAm hydrogels is mediated by the transmembrance protein integrin and its extracellular matrix receptor, fibronectin. Results from these studies could be useful for improving the design of biomaterials based on PNIPAAm hydrogels for applications in tissue engineering.

Stimuli-responsive polymers and self-assembling peptides represent two classes of materials with interesting properties and great potential to be used as biomaterials. The conjugation of polymer with peptide offers a way to combine the controlled chemical, mechanical, and thermal properties of polymer with the functionality of designed bioactive group. Pure hybrid materials with the characteristics of individual components or systems containing hybrid materials became attractive for applications in drug delivery and tissue engineering. This work focused on systems where the thermo-responsive properties of a polymer were combined with the gelling properties of two different ionic-complementary peptides via conjugation. The prototypical thermo-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) was chosen due to its lower critical solution temperature (LCST) ~32°C being close to body temperature. Ionic-complementary oligo-peptides, containing the alternating hydrophobic/hydrophilic and charged/uncharged amino acids, phenylalanine (F), glutamic acid (E) and lysine (K), were selected as they are known to form β-sheet rich fibrillar networks at low concentrations. Two peptide sequences with different charge distribution were chosen: FEFEFKFK and FEFKFEFK which form self-supporting gels at ~17 and 10 mg ml-1 respectively. Polymer-peptide conjugates were used to confer self-assembling and thermo-responsive behaviour to the system.Thermo-responsive PNIPAAm-rich hydrogels were obtained by targeting different degrees of functionalisation of PNIPAAm with the self-assembling peptides. Two series of such systems were prepared by using either a thiol-modified FEFEFKFK or a thiol-modified FEFKFEFK peptide as the chain-transfer agent in the free radical polymerisation of NIPAAm. The resulting polymer/conjugate mixtures were studied by proton nuclear magnetic resonance (1H NMR). The polymer/conjugate ratios were calculated and showed that the conjugate fraction in the mixtures increased with increasing concentration of peptide used for the polymerisation. Static light scattering (SLS) and viscometry showed the aggregation of the polymer/conjugate mixtures presumably due to the presence of peptide. The values from gel permeation chromatography (GPC), which were mostly attributed to the unconjugated polymers, were higher than those obtained from 1H NMR and centrifugation for the conjugates. The polymer/conjugate mixtures formed self-supporting gels where the critical gelation concentration decreased with increasing conjugate content. Oscillatory rheology experiments confirmed gels had formed and revealed that their elastic modulus, G' varied from ~ 10 to 400 Pa depending on the sample. TEM and AFM studies proved the formation of β-sheet fibres of ~ 4.5 ± 1.5 nm in diameter. The PNIPAAm-rich hydrogels were also characterised by micro DSC to reveal their thermo-responsiveness and phase separation and showed the LCST at ~ 30°C. The results of the study showed that varying the peptide sequence did not have an effect on thermal, mechanical or morphological properties of the hydrogels. By exploiting the self-assembly of the ionic-complementary peptides, it was possible to create PNIPAAm-rich, thermo-responsive hydrogels with controllable properties.Further in the study pure PNIPAAm-FEFEFKFK conjugate was incorporated into the FEFEFKFK peptide matrix to create peptide-rich thermo-responsive composite gels. Two series of the composite gels were prepared by varying separately the peptide matrix and polymer-peptide conjugate concentration. Micro DSC measurements revealed an endothermic peak at ~ 30ºC characteristic of the LCST of PNIPAAm. Oscillatory rheology studies showed that the composite gels became stronger with increasing conjugate concentration (G' ~ 20 - 200 Pa). Network morphology was studied by SANS. Using contrast variation and contrast matching techniques it was possible to distinguish between the peptide fibres and the PNIPAAm chains. Below and above the LCST the scattering curves showed a q-1 behaviour which is typical of rod-like objects. TEM and AFM also proved the formation of fibres of ~4.0 ± 0.8 nm and ~4.5 ± 1 nm respectively. AFM studies showed that the fibres of the composite gels were decorated with polymer chains. The thermo-responsiveness and the gelation properties of these conjugate-based scaffolds have potential for use as drug delivery vehicles or tissue engineering scaffolds.

Conventionally, adherent cells are cultured in vitro using flat 2D cell culture trays. However the 2D cell culture method is tedious, unreliable and does not replicate the complexity of the 3D dynamic environment of native tissue. Nowadays 3D scaffolds can be used to culture cells. However a number of challenges still exist, including the need for destructive enzymes to release confluent cells. Poly(Nisopropylacrylamide) (PNIPAAm), a temperature responsive polymer, has revolutionised the cell culture fraternity by providing a non-invasive means of harvesting adherent cells, whereby confluent cells can be spontaneously released by simply cooling the cell culture medium and without requiring enzymes. While PNIPAAm monolayer cell culturing is a promising tool for engineering cell sheets, the current technology is largely limited to the use of flat 2D substrates, which lacks structural and organisational cues for cells. The aim of this project was to develop a 3D PNIPAAm scaffold which could be used efficiently for non-invasive 3D culture of adherent cells. This project was divided into three phases: Phase 1 (preliminary phase) involved development and characterisation of cross-linked PNIPAAm hydrogels; Phase 2 involved development and characterisation of PNIPAAm grafted 3D non-woven scaffolds, while Phase 3 focused on showing proof of concept for non-invasive temperature-induced cell culture from the 3D PNIPAAm grafted scaffolds. In Phase 1, PNIPAAm was cross-linked with N,N’-methylene-bis-acrylamide (MBA) using solution free-radical polymerisation to form P(PNIPAAm-co-MBA) hydrogels. A broad cross-link density (i.e. 1.1 - 9.1 Mol% MBA) was investigated, and the effect of using mixed solvents as the co-polymerisation medium. The P(PNIPAAm-co-MBA) gels proved unsuitable as a robust cell culture matrix, due to poor porosity, slow swelling/deswelling and poor mechanical properties. Subsequently, in Phase 2, polypropylene (PP), polyethylene terephthalate (PET), and nylon fibers were processed into highly porous non-woven fabric (NWF) scaffolds using a needle-punching technology. The NWF scaffolds were grafted with PNIPAAm using oxyfluorination-assisted graft polymerisation (OAGP). The OAGP method involved a 2 step process whereby the NWF was first fluorinated (direct fluorination or oxyfluorination) to introduce new functional groups on the fibre surface. The functionalised NWF scaffolds were then graft-polymerised with NIPAAm in an aqueous medium using ammonium persulphate as the initiator. Following oxyfluorination, new functional groups were detected on the surface of the NWF scaffolds, which included C-OH; C=O; CH2-CHF, and CHF-CHF. PP and nylon were both easily modified by oxyfluorination, while PET displayed very little changes to its surface groups. Improved wetting and swelling in water was observed for the oxyfluorinated polymers compared to pure NWF scaffolds. PP NWF showed the highest graft yield followed by nylon and then PET. PNIPAAm graft yield on the PP NWF was ~24 ±6 μg/cm2 on grafted pre-oxyfluorinated NWF when APS was used; which was found to be significantly higher compared to when pre-oxyfluorinated NWF was used without initiator (9 ±6 μg/cm2, p= 1.7x10-7); or when grafting was on pure PP with APS (2 ±0.3 μg/cm2, p = 8.4x10-12). This corresponded to an average PNIPAAm layer thickness of ~220 ±54 nm; 92 ± 60 nm; and 19 ± 3 nm respectively. Scanning electron microscopy (SEM) revealed a rough surface morphology and confinement of the PNIPAAm graft layer to the surface of the fibers when oxyfluorinated NWF scaffolds were used, however when pure NWF scaffolds were used during grafting, homopolymerisation was observed as a loosely bound layer on the NWF surface. The OAGP method did not affect the crystalline phase of bulk PP as was determined by X-ray diffraction (XRD), however, twin-melting thermal peaks were detected from DSC for the oxyfluorinated PP and PP-g-PNIPAAm NWF which possibly indicated crystal defects. Contact angle studies and microcalorimetric DSC showed that the PP-g-PNIPAAm NWF scaffolds exhibited thermoresponsive behaviour. Using the 2,2-Diphenyl-1-1-picrylhydrazyl (DPPH) radical method and electron-spin resonance (ESR), peroxides, as well as trapped long-lived peroxy radicals were identified on the surface of the oxyfluorinated PP NWF, which are believed to be instrumental in initiating graft polymerisation from the NWF. A free radical mechanism which is diffusion controlled was proposed for the OAGP method with initiation via peroxy radicals (RO•), as well as SO4•- and OH• radicals, whereby the latter result from decomposition of APS. In Phase 3 of this study, proof-of-concept is demonstrated for use of the PNIPAAm grafted NWF scaffolds in non-invasive culture of hepatocytes. Studies demonstrated that hepatocyte cells attached onto the 3D PNIPAAm scaffolds and remained viable in culture over long periods. The cells were released spontaneously and non-destructively as 3D multi-cellular constructs by simply cooling the cell culture medium from 37°C to 20°C, without requiring destructive enzymes. The PP-g- PNIPAAm NWF scaffolds performed the best in 3D cell culture. Additionally the CSIR is developing a thermo responsive 3D (T3D) cell culturing device, whereby the 3D thermo responsive NWF scaffolds are used in the bioreactor for cell culture. Temperature-induced cell release was also verified from the 3D Thermo responsive scaffolds in the bioreactor. This technology could lead to significant advances in improving the reliability of the in vitro cell culture model. Please cite as follows: Chetty, AS 2012, Thermoresponsive 3D scaffolds for non-invasive cell culture, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-06112013-151344/ > D13/4/713/ag / Thesis (PhD)--University of Pretoria, 2012. / Chemical Engineering / unrestricted

PNIPAM (poly(N-Isopropylacrylamide)) is a well-known thermoresponsive polymer. Dissolved in water, it shows a structural change at 32 oC, above which the polymer folds together, and a phase separation occurs. The temperature where the polymer changes structure is known as the LCST (Lower Critical Solution Temperature), and can be modified by adding certain salts to the solution [1]. The mechanism by which the ionic components of the salts affect the LCST is not yet completely understood. The purpose of this master thesis is to study this mechanism. In order to investigate the mechanism, a combination of diffusion NMR and electrophoretic NMR was used, giving the effective charge per molecule which is directly proportional to the grade of association of ions to the polymer. The salts tested were: NaCl, NaClO4, NaSO4, NaI, NaSCN and CaCl2 from which the ClO4-, SCN-, and I- ions, as well as Cl- ions from CaCl2, were found to bind to PNIPAM.

Yes / Highly branched poly(N-isopropylacrylamide) (HB-PNIPAM), functionalized with vancomycin at the chain ends, acted as a bacterial adhesive and was incorporated into polyurethane foams to form semi-interpenetrating networks. The poly(N-isopropylacrylamide) was labelled with a solvatochromic dye, Nile red. It was found that the thermal response of the polymer was dependent on architecture and temperature dependent color changes were observed within the foam. The foams had open pore structures and the presence of the HB-PNIPAM substantially reduced the shrinkage of the foam as the temperature was increased upto 20 °C. The foams were selectively adhesive for Staphylococcus aureus (Gram-positive bacteria) compared to Pseudomonas aeruginosa (Gram-negative bacteria) and the presence of S. aureus was indicated by increased fluorescence intensity (590 to 800 nm).

<p>Efficient delivery of therapeutic cell and pharmaceutical suspensions to the posterior segment of the eye remains an elusive goal. Delivery is made difficult by blood ocular barriers that separate the eye from systemic circulation, the compartmentalized structure of the eye that limits diffusion across the globe, and effective clearance mechanisms that result in short drug residence times. The work presented in this thesis focuses on the design, synthesis, evolution and refinement of novel biomaterial scaffolds ultimately intended to facilitate the minimally invasive delivery of therapeutic payloads into the posterior segment of the eye. The first generation materials presented in this work (Chapter 2) consist of linear chains of temperature-sensitive amine-terminated poly(N-isopropylacrylamide) (PNIPAAm) grafted onto the backbone of type I collagen. Second generation materials (Chapter 3) saw the inclusion of the lubricious polysaccharide, hyaluronic acid (HA), and replacement of the bulky collagen backbone, which was observed to impede scaffold gelation, with small cell adhesive RGD peptide sequences. The introduction of degradability was the emphasis of third generation copolymers (Chapter 4) and was achieved through copolymerization with dimethyl-γ-butyrolactone acrylate (DBA). The DBA lactone side group was found to undergo a hydrolysis dependent ring opening, which raises copolymer LCST above physiologic temperature, triggering the gelled scaffold to solubilize and be excreted from the body via renal filtration without the liberation of any degradation by-products. Degradation was found to occur slowly, which is favourable for long-term release scaffolds intended to decrease the frequency of injections required to maintain therapeutically relevant concentrations within the vitreous. Finally, the design of a fourth generation material is discussed (Chapter 5), in which optical transparency is achieved through copolymerization of third generation materials with polyethylene glycol (PEG) monomers of varying molecular weight. Synthesis, design and characterization of the various copolymers is described herein.</p> / Doctor of Philosophy (PhD)