reading

Chapter 2: Light-Triggered Azobenzenes: From Molecular Architecture to Functional Materials

2.4 Photodeformable Azobenzene-Based Materials: Artificial Muscle-like Actuation

Chapter 3: Functionalization with Interpenetrating Smart Polymer Networks by Gamma Irradiation for Loading and Delivery of Drugs

Chapter 5: Stimuli-Responsive Polymers as Adjuvants and Carriers for Antigen Delivery

5.3 Factors Affecting Adjuvant Potential of Stimuli-Responsive Polymeric Adjuvant

Chapter 6: Cyclodextrins as Advanced Materials for Pharmaceutical Applications

9.3 Results Related to Use of Smart Nanostructured Materials to Control Cancers Cells

Chapter 10: Quantum Cutter and Sensitizer-Based Advanced Materials for their Application in Displays, Fluorescent Lamps and Solar Cells

11.3 Electrical Conductive Properties of Nanofibers of Conducting Polymer Nanocomposites

Chapter 13: Commodity Thermoplastics with Bespoken Properties using Metallocene Catalyst Systems

Chapter 14: Elastic Constants, Structural Parameters and Elastic Perspectives of Thorium Mono-Chalcogenides in Temperature Sensitive Region

The Advance Materials Series is intended to provide recent advancements of the fascinating field of advanced materials science and technology, particularly in the area of structure, synthesis and processing, characterization, advanced-state properties, and applications. The volumes will cover theoretical and experimental approaches of molecular device materials, biomimetic materials, hybrid-type composite materials, functionalized polymers, super-amolecular systems, information- and energy-transfer materials, biobased and biodegradable or environmental friendly materials. Each volume will be devoted to one broad subject and the multidisciplinary aspects will be drawn out in full.

Series Editor: Dr. Ashutosh Tiwari
Biosensors and Bioelectronics Centre
Linköping University
SE-581 83 Linköping
Sweden
E-mail: ashutosh.tiwari@liu.se

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Published simultaneously in Canada.

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The development of tuned materials by environmental requirements is the recent arena of materials research. It is a newly emerging, supra-disciplinary field with great commercial potential. Stimuli-responsive materials answer by a considerable change in their properties to small changes in their environment. They are becoming increasingly more prevalent as scientists learn about the chemistry and triggers that induce conformational changes in material structures and devise ways to take advantage of and control them. New responsive materials are being chemically formulated that sense specific environmental changes and adjust in a predictable manner, making them useful tools.

Stimuli-responsive materials are in widespread demand among researchers because they can be customized via chemistry to trigger induced conformational changes in structures or be taken advantage of in the form of structural or molecular regime via minute external environmental changes. Their effectors are both i) physical, i.e., temperature, electric or magnetic fields, mechanical stress; and ii) chemical, i.e., pH, ionic factors, chemical agents, biological agents. Thermoresponsive polymers represent an important class of “smart” materials as they are capable of responding dramatically to small temperature changes. The chapter on “Smart Thermoresponsive Biomaterials” describes a range of thermoresponsive polymers and the criteria that influence their thermoresponsive character for surface modifications and applications, in particular for cell culture and chromatography. In the chapter “Light-Triggered Azobenzenes: From Molecular Architecture to Functional Materials,” the principle of light-triggered materials is covered, for example, azobenzene-based materials, their photochromic switching and oscillation ability, and potential biological and artificial muscle-like actuation applications. The chapter entitled “Functionalization with Interpenetrating Smart Polymer Networks by Gamma Irradiation for Loading and Delivery of Drugs,” discusses the γ-irradiation assisted graft copolymerization containing interpenetrating polymer networks and other architectures, mainly focusing on the performance of materials modified with stimuli-responsive components capable of high loading therapeutic substances and their control release properties. The recently investigated applications of smart or intelligent polymeric materials for tissue engineering, regenerative medicine, implants, stents, and medical devices are overviewed in “Biomedical Devices Based on Smart Polymers.” The chapter “Stimuli Responsive Polymers as Adjuvants and Carriers for Antigen Delivery,” illustrates the promising advantages of responsive materials in immunology as carriers for an antigen and adjuvant for enhancing immunogenicity of an antigen. “Cyclodextrins and Advanced Materials for Pharmaceutical Applications” highlights the combination of cyclodextrins and pharmaceutical excipients or carriers such as nanoparticles, liposomes, etc., and fosters the progress of the advanced dosage forms with the improved physicochemical and biopharmaceutical properties.

“Recent Advances in Smart Wearable Systems,” presents an overview of the smart nanoengineering that yields state-of-the-art wearable systems and sensor technologies, and underlying challenges are overviewed. The high surface functionalities available in such materials provide an opportunity to modify their outer surfaces and achieve multivalent effects. The chapter on “Functionalization of Smart Nanomaterials” describes the surface nanoengineering aimed at coupling advanced features for a range of optoelectronic applications. A thrust towards the development of novel nanoparticles has paved the way for sucessful cancer diagnosis and treatment. The chapter “Role of Smart Nanostructured Materials in Cancers,” summarizes different types of nanoparticles currently available for cancer therapy. Smart nanomaterials including visible quantum cutting and near-infrared quantum cutting phosphors such as fluoride phosphors, oxide phosphors, phosphate phosphors and silicate phosphors, and their potential application for PDPs and Hg-free fluorescent lamps, are the focus of “Quantum Cutter and Sensitizer-Based Advanced Materials for Their Application in Displays, Fluorescent Lamps and Solar Cells.” The chapter on “Nanofibers of Conducting Polymer Nanocomposites” focuses on the preparative strategies of nanofibers of conducting polymers and nanocomposites and their electrical conductive properties and applications.

The biocompatible smart polymeric architect has significantly in creased attention in biodevice and system managements. “Stimuli-Responsive Redox Biopolymers” investigates Arabic-co-polyaniline as pH-responsive redox copolymers and their properties for biosensor applications. The development of the metallocene catalysts, from their discovery to their present state-of-the-art, is portrayed in “Commodity Thermoplastics with Bespoke Properties Using Metallocene Catalyst Systems,” with an emphasis on weighing up discrete catalysts for stereo-specific polymerization and technologically important processes.

The study of elastic properties provides information about the magnitude of the forces and nature of bonding between the atoms. The impact of solids on the world of science and technology has been enormous, covering such diverse applications as solar energy, image processing, energy storage, computer and telecommunication technology, thermoelectric energy conversion, and new materials for numerous applications. The chapter “Elastic Constants, Structural Parameters and Elastic Perspectives of Thorium Monochalcogenides in Temperature Sensitive Region” predicts the anharmonic elastic properties of thorium chalcogenides having NaCl-type structure under high temperature using Born-Mayer repulsive potentials and the long- and short-range interaction approach.

This book is written for a large readership including university students and researchers from diverse backgrounds such as chemistry, materials science, physics, pharmacy, medical science, and biomedical engineering. It can be used not only as a textbook for both undergraduate and graduate students, but also as a review and reference book for researchers in the materials science, bioengineering, medical, pharmaceutical, biotechnology, and nanotechnology fields. We hope the chapters of this book will provide valuable insight in the important area of responsive materials and cutting-edge technologies.

PART 1

STIMULI-RESPONSIVE POLYMERIC MATERIALS

Chapter 1

Smart Thermoresponsive Biomaterials

Biological Physics Group, School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom

Abstract

Thermoresponsive materials represent an important class of advanced materials that have evolved over the past few decades. These materials are also designated as “smart” materials as they are capable of responding dramatically to small temperature changes. In this chapter we will present a select range of polymers that exhibit thermoresponsive behavior, with a particular focus on polyacrylamide-based polymers. We also review the criteria that influence their thermoresponsive character. Inherently, many materials are not thermoresponsive, thus approaches for surface modification of such materials resulting in unique thinly-coated thermoresponsive surface layers or films are also shown. Finally, select biological applications of thermoresponsive biomaterials are presented, in particular for cell culture and chromatography applications.

Keywords: Temperature responsive, functional polymers, nanofilms, cell culture, chromatography

1.1 Introduction

Synthetic polymers that can respond to external stimuli in a controlled manner are increasingly of interest to science and industry. Such polymers have been designed to mimic natural biopolymers, such as proteins, polysaccharides and nucleic acids in living organisms within which responses to stimuli are common processes. Such “smart” or “intelligent” stimuli-responsive polymers are capable of undergoing relatively large and abrupt changes in response to small external environmental changes. The exemplar stimuli are often classified as either physical (temperature, electric or magnetic fields, and mechanical stress) or chemical effectors (pH, ionic factors, chemical agents, biological agents), resulting in changes of the interactions between polymer chains or between chains and solvents at the molecular level (Figure 1.1). Such changes in the physiochemical properties of the polymers can subsequently affect their interactions with other systems, for example, adherent cells. These stimuli-responsive polymer systems are attractive to bio-related applications such as cell expansion, tissue engineering, controlled drug delivery, non-viral gene transfection, enzymatic activity control, biotechnology and chromatography for bio molecular separation and purification [1, 2].

Figure 1.1 A schematic representation of stimuli-responsive polymer change for (a) free polymer in aqueous bulk environment, and (b) surface immobilized polymer. The temperature-dependent soluble (hydrated below the LCST) to insoluble (dehydrated above the LCST) change of polymer in aqueous media is shown.

Significant scientific research towards the understanding and development of dynamically responsive materials has resulted in a number of excellent reviews by other authors on the general topic of thermoresponsive polymer materials and related areas. The references in this chapter are hence primarily provided as starting points for further reading [3–7]. In this chapter we will describe the development of a select range of temperature-responsive polymers that exhibit thermoresponsive behavior. In particular we will review the use of polyacrylamide-based polymers and also the criteria that influence their temperature-responsive character. Inherently, many materials are not thermoresponsive, thus approaches for surface modification of such materials need to be taken to produce unique thinly-coated thermoresponsive surface layers or films. Finally, we will present cell culture and chromatographic purification as select biological applications of thermoresponsive biomaterials.

1.2 Temperature-Responsive Polymers

1.2.1 Thermoresponsive Polymers Based on LCST

The change of temperature is a relatively easy and widely used stimulus for causing responsive behavior of polymers. A common phenomenon is the change in solubility when the temperature is shifted across the critical solution temperature at which the phase of a polymer solution or composite changes discontinuously. In general, solutions that appear as monophasic (isotropic state) below a specific temperature and turn biphasic above it, exhibit a lower critical solution temperature (LCST). LCST is hence the critical temperature beyond which immiscibility or insolubility occurs. Acquisition and control of LCST within the physiological temperature range is essential for applications such as cell culture and drug delivery. LCST is dependent on factors such as the ratios of monomers, their hydrophobic and hydrophilic nature, polydispersity, branching and the degree of polymerization [5]. Thus the LCST of polymers in water can be altered by incorporating hydrophilic or hydrophobic moieties. For example, the copolymerization of N-isopropylacrylamide (NIPAAm) with hydrophilic monomers results in the increase of the LCST [7, 8]. In contrast, the LCST decreases when copolymerized with hydrophobic monomers, but this process may also affect the temperature sensitivity of NIPAAm-based copolymers. The copolymerization of ionizable groups such as acrylic acid (AAc) or N, N’-dimethylacrylamide (DMAAm) with NIPAAm can result in the discontinuous alternation or even disappearance of LCST at the pKa of the ionizable group [9].

For polymers such as poly(N-isopropylacrylamide) (PNIPAAm), an important characteristic is its intermolecular interaction with water molecules. Depending on its physical states, e.g., macromolecular solution, micellar aggregation or gel, changes in temperature across LCST have a huge impact on hydrogen bonding and hydrophobic interactions resulting in big differences in their amphiphilic properties. The extent of hydrophobic interaction can be manipulated by tuning the balance of monomer ratios and common examples are often seen from different diblock poly(ethylene oxide)–poly(propylene oxide) (PEOm-co-PPOn) and their triblock copolymers (PEOm-co-PPOn-co-PEOm), where changes in the ratio of m to n can lead to very different physiochemical properties including thermoresponsive behavior.

Homopolymers: A number of other polymers can also display thermoresponsive behavior across their LCST with examples given in Figure 1.2. With some adjustments, the range of their thermoresponsive switches can be made useful for cell thermoresponsive detachment upon confluence. For example, poly(vinyl methyl ether) (PVME) has the LCST of around 36°C and is usually synthesized via solution polymerization (Figure 1.2ii) [10]. Another exemplar thermoresponsive polymer is poly(N-vinyl caprolactam) which can be easily prepared by free radical polymerization of N-vinyl caprolactam in solution, and has LCST of 32–34°C (Figure 2iii) [11].

Figure 1.2 Chemical structures of polymers showing LCST; (i) poly(N-isopropylacrylamide) (PNIPAAm); (ii) poly(vinyl methyl ether) (PVME); (iii) poly(N-vinyl caprolactam); (iv) poly(N-(L)-(1-hydroxymethyl) propylmethacrylamide (P(L-HMPMAAm)); (v) poly(N, N’-diethylacrylamide) (PDEAAm); (vi) poly(2-carboxyisopropylacrylamide) (PCIPAAm); (vii) poly(N-acryloyl-N’-alkylpiperazine).

Poly (N-substituted acrylamide) polymers are by far the most popular and well-researched thermoresponsive polymers. PNIPAAm is the most well known of the thermo responsive polymers having a sharp phase transition in water (LCST) within the physiological range of about 32°C (Figure 1.2i). These polymers are also prevalent because of the fact that poly(N-substituted acrylamide) polymers are easy to prepare by radical polymerization [12, 13].

Other poly (N-substituted acrylamide) polymers shown in Figure 1.2 include poly(N, N’-diethylacrylamide) (PDEAAm) with LCST in the range of 25–35°C [14], poly(2-carboxyisopropylacrylamide) (PCIPAAm) composed of a isopropylacrylamide group and carboxyl group, thus having the advantage of temperature response and additional functionality in its pendant groups [15]. Interestingly, the polymer poly(N-(L)-(1-hydroxymethyl) propylmethacrylamide) (P(L-HMPMAAm)) (Figure 1.2iv) has optical activity associated with it and shows a different thermosensitive phase transition from that of optically inactive P(DL-HMPMAAm) [16]. The polymer poly(N-acryloyl-N’-propylpiperazine) with the LCST of 37°C is both temperature and pH responsive [17]. However, the Poly(N-acryloyl-N’-propylpiperazine) homopolymers based on methylpiperazine and ethylpiperazine were found not to exhibit LCST due to their weak hydrophobicity [18].

Copolymers: The co-polymerization of different N-substituted acrylamide monomers can provide further copolymer functionality and LCST tuning potential arising from the hydrophilic-hydrophobic balance of monomer units. The copolymer PNIPAAm-co-PCIPAAm has similar sensitivity and LCST to the homopolymer PNIPAAm [15, 19]. It has structural similarity to PNIPAAm-co-poly(acrylic acid), but the two have very different temperature-responsive behavior. Triblock copolymers such as PEOm-co-PPOn-co-PEOm also exhibit temperature-responsive micellization and gelation arising from their amphiphilic balance [20, 21]. The replacement of the PPO block with other hydrophobic groups such as the poly(1,2-butylene oxide) (PBO) results in a shift of the thermoresponsive LST behavior [22]. Likewise, its substitution by poly(L-lactic acid) (PLLA) and (DL-lactic acid-co-glycolic acid) (PLGA) can also result in the shift of the thermoresponsive performance with the added benefit of biodegradable ester group incorporation [23, 24].

1.2.2 Biopolymers and Artificial Polypeptides

Temperature-responsive behavior is common in some biopolymers such as gelatin, agarose and gellan benzyl ester [25–27]. These polypeptides can form helix conformations, leading to physical crosslinking. Gelatin is obtained from collagen by breaking its triple-helix structure into single-stranded molecules. It is a thermally reversible hydrogel and its film stability at 37°C is poor. Thus, it is not ideal for direct use as thermoresponsive cell culture substrate under the normal cell culture conditions. Stable hydrogels of gelatin have, however, been obtained by chemical crosslinking or conjugation with chitosan by tyrosinases [28]. A substrate based on a triblock copolymer, poly (N-isopropylacrylamide)-co-poly[(R)-3-hydroxybutyrate]-co-poly (N-isopropylacrylamide) (PNIPAAm-co-PHB-co-PNIPAAm), co-coated with gelatin, has been developed for thermoresponsive cell culture. It was found to be superior to the PNIPAAm homopolymer coating in terms of film stability, surface coating and cell growth [29]. Surface deposition of collagen in low density on PNIPAAm was also found to enhance cell adhesion but did not affect cell detachment compared to uncoated PNIPAAm [30].

Biomimetic polypeptides such as elastin-like polypeptides (ELPs), composed of Val-Pro-Gly-Xaa- Gly amino acid repeat units (where Xaa is a guest residue, not proline), have shown thermally reversible phase transition behavior. ELPs are water-soluble below their transition temperature. But above the transition temperature they precipitate, driven by hydrophobic aggregation. For example, a block co-polypeptide composed of ELPs segment and silk-like segment has been reported to undergo sol-gel transition [31, 32].

1.2.3 Temperature Sensitivity of Polymers

For the versatility of applications, temperature-responsive polymers require high sensitivity or fast response over a narrow temperature. The incorporation of phase-separated structures can result in rapid swelling/deswelling within hydrogels, resulting in the change of physical form associated with a large shift in surface area and amphiphilicity [33]. The inclusion of hydrophilic moieties can also increase the deswelling rate of PNIPAAm hydrogel network. For example, the random copolymerization of NIPAAm with acrylic acid (AAc) or methacrylic acid (MAAc) provides the hydrogels with faster deswelling kinetics than PNIPAAm hydrogel by itself [34]. However, for AAc-content above 1.3 wt%, the deswelling rate decreased when more AAc segments were added. An increase in the AAc content divided the long linear NIPAAm segments into short ones, causing the decrease of the driving force for hydrophobic aggregation and the subsequent disappearance of the LCST. In contrast, hydrophilic PEO grafts similarly introduced onto the PNIPAAm backbone were found not to interfere with long PNIPAAm sequences.

The copolymerization of PNIPAAm with poly(ethylene glycol) (PEG) onto porous culture membranes was carried out by electron beam irradiation to provide better detachment of the cells. In this case, the NIPAAm monomers and PEG macromonomers (PEG methacrylate, MW = 4000) were dissolved in propanol containing 0.05% distilled water at a total concentration of 60 wt/wt%. This monomer-containing solution mixture was spread uniformly over the surface of a porous membrane (Cell Culture Insert) and irradiated using an electron beam resulting in the covalently bound polymer. In cell sheet detachment experiments, only 19 min were required to detach the cell sheets from PNIPAAm co-grafted with 0.5wt% of PEG, compared to approximately 35 min incubation at 20°C to completely detach the cell sheets from PNIPAAm coated on the same porous culture membranes. When the porous membranes were used, water molecules could access PNIPAAm molecules grafted on the surfaces from both underneath and peripheral to the attached cell sheets, resulting in more rapid hydration of grafted PNIPAAm molecules and faster detachment of cell sheet than nonporous tissue culture polystyrene (TCPS) dishes [35].

Alternatively, rapid deswelling (faster acceleration of the polymer shrinking rate) was shown by PNIPAAm hydrogels having a comb-type molecular architecture rather than a linear-type structure [36]. However, in the case of surface-immobilized PNIPAAm films, the free mobile linear PNIPAAm showed a more rapid phase transition than PNIPAAm randomly crosslinked onto the surface, due to their different chain mobilities [37].

1.3 Development of Thermoresponsive Surfaces

Many applications utilize thermoresponsive polymers in solution or in the bulk state, however, temperature-responsive surface or interface has important biomedical applications such as temperature-modulated membranes, chromatography and cell culture dishes [38–40]. PNIPAAm polymers have been extensively investigated for the development of various temperature-responsive surfaces because of their specific advantages in biomedical applications. An important advantage includes a reversible temperature-dependent phase transition (LCST) in aqueous solution within the physiological range. Thus copolymers based on NIPAAm have been investigated and further developed for chromatography, tissue engineering and cell culture applications through controlled surface modification, but not all surfaces are directly amenable to modification.

1.3.1 Surface Modifications Using Energetic Oxidation

Surface properties of materials with no functional oxygen or nitrogen groups are often required to be altered prior to monomer or polymer grafting. The modification of surfaces is also required to facilitate cell attachment and growth. Surfaces for cell culture or tissue culture applications, other than polystyrene, include thermoplastic polymers such as polyethylene terephthalate (PET) which are also easy to mold and manufacture. However, they are hydrophobic in nature, so will exhibit a very different surface topology and chemical nature from the extracellular matrix (ECM). Other surfaces that require some form of surface modification to further facilitate cell attachment and growth include polycarbonate and glass. An important route for surface modification of materials includes the use of high-energy irradiation.

A number of methods that use high-energy irradiation are available for modifying the surface of polymers. The resulting surface oxidation can make it more hydrophilic by introduction of hydroxyl and carboxyl functional groups. There are a variety of treatments that can be used to do this, such as UV, corona discharge, gamma irradiation, plasma treatments and electron beam irradiation. X-rays and electron beams are more penetrating than heat, light and microwave. Electrons, X-rays and gamma rays ionize the material they strike by stripping electrons from the atoms of the exposed material. This ionized environment is very damaging to bacteria or viruses and can also change the chemical composition and surface structure of a material. Generally, these techniques can all effectively bombard the surface with ions, electrons or photons, resulting in a subtle difference between the chemical groups formed on the surface. The bombarding radiation breaks some of the bonds in the polymer chains as well as the gaseous material surrounding them, to produce readily available free radicals on the culture surface, and these have the ability to quench nearby molecules. For a particular material, each technique can result in different density of surface oxidized groups, such as hydroxyl or carboxyl groups. For the polystyrene surface, research has shown that plasma treatment produces a wide variety of functional groups such as alkylperoxide, aldehyde, and carboxylic, and also up to 15% of hydroxyl groups, as shown in Figure 1.3. The percentage of hydroxyl groups formed by UV was 12%, and 8% by corona, but gamma radiation was found to produce very little of the hydroxyl groups. In summary, by using an energetic source for irradiation the surface of polymers like polystyrene can be oxidized. The resulting interactions with polystyrene lead to carbon-carbon scissions and generation of a variety of oxygen-containing functionalities at the polymer surface.

Figure 1.3 Schematic drawing showing that the surface of polystyrene is hydrophobic but the oxidized surface has both hydroxyl and carboxyl functional groups making it more hydrophilic and susceptible to polymer attachment.

Modification of polymer surfaces can be performed cleanly and rapidly by plasma treatment, also leading to the formation of various active species on the surface of polymers such as polyethylene (PE), polypropylene (PP), and polytetrafluoroethylene (PTFE) [41]. However, this method also results in polymerization, functionalization, etching, roughening and crosslinking. Plasma can be described as a partially ionized gas consisting of free radicals, ions, photons and electrons. It can be created by gases such as oxygen and argon excited by an external energy such as heat or electric discharge. The effectiveness of plasma treatment depends on the chemical monomers or gases used to generate the reactive species. The plasma treatment of polyethylene terephthalate (PET) using air as the gas results in a large increase in hydrophilicity [42]. The treatment of poly(hydroxymethylsiloxane) surfaces by either O₂ plasma or 6 keV Ar+ beams also resulted in different adhesion, proliferation and spreading of normal human dermal fibroblast cells. Low cell adhesion and scarce viability was found from O₂ plasma treated surfaces, but complete cell confluence, optimal spreading and proliferation were observed in the case of 6 keV Ar+ beams. The observed differences in cell responses were attributed to the relative surface free energy as a result of the two different plasma beams applied [43].

The high energy modification treatments such as gamma radiation and lasers for surface modification can lead to the modification of polymers as well as providing improvement to surface biocompatibility [44]. To achieve a specific gamma radiation effect it is necessary to apply a specific dose to the material. The radiation dose is a measure of the radiation energy deposited in unit mass of the material, measured in Gray (Gy) (1 Gy means 1 joule of radiation energy deposited in each kilogram of material). For example, to sterilize medical devices low doses of the order of 25 kGy are required, but the control of pathogens can be achieved with doses of 1.5 to 3.0 kGy, to allow preservation of the material and to render it biologically inert. The crosslinking of plastics and polymers requires much higher doses of up to 200 kGy, and certain polymers are known to undergo chain scission whilst others predominantly crosslink. The susceptibility of different polymeric materials to radiation cross-linking depends mainly on their chemical structure and some can be crosslinked at low doses, while others containing non-reactive groups, such as styrene monomers, require higher doses that may lead to polymer degradation. The gamma-ray irradiation on polystyrene in the presence of polyfunctional monomers such as pro-pyleneglycol bis-allylcarbonate(PGBAC) and dimethyleneglycol bis-allylcarbonate (DEGBAC) can reduce the dose required for cross-linking of polystyrene polymer as well as improving the mechanical and physical properties of the irradiated material. The effect of gamma radiation on the surface chemical properties of polystyrene was studied by ESCA and FT-IR. Gamma radiation was found to produce surface -C=O- and C-O-containing functional groups and to also cause oxidation in depths of greater than 10 nm [45].

In another investigation the surface of polyethylene and polystyrene was made hydrophilic by a two-step method. The polymer surface was first hydroxylated by treatment with an aqueous sodium persulfate solution or by gamma irradiation in water, after which grafting was initiated by the thermal decomposition of hydroxyl groups formed at the surface. Surface modification of polyethylene but not of polystyrene was found as a result of gamma irradiation in the presence of a concentrated aqueous acrylamide solution, thus indicating the difficulty in modifying polystyrene by this route [46].

1.3.2 Surface Grafting of Polymers

The modification of substrate polymeric surfaces by surface- reactive polymers represents a major approach for surface functionalization and can be attempted by several methods. Such procedures can increase the substrate polymer stability and robustness and avoid the mixing of these polymers with cells. To have functional polymers chemically bound to the substrate polymer there must be a sufficiently activated substrate surface with polar groups for possible bonding and crosslinking. Alternatively, such surface functional groups can be generated in situ through input of energy to break and form chemical bonds so that surface functional groups are created and bound with functional groups from polymers or monomers. This route has been followed by Watanabe et al. [47]. These researchers formed a cell culture support polymer coating by spreading a layer of PNIPAAm polymer and NIPAAm monomer via a solution onto polystyrene, drying and then using electron beam treatment. By coating with a mixture of homopolymer and monomer via solution rinsing followed by irradiation, they managed to avoid surface crystallization with better film uniformity. Similar processes have also been attempted by UV treatment, though the reported studies have so far always incorporated initiators to improve the generation of functional groups from polymer substrates such as polystyrene.

Hence, a polymer substrate with very little or no surface active groups can be activated for grafting either by (a) high energy irradiation (UV, plasma, electron beam, gamma) to induce the formation of surface active groups (free radicals, hydroxyl and carboxyl groups), (b) by use of surface initiator/photoinitiators such as benzophnone, phenylglyoxylates, hydroxyl alkylphenones or acylphosphine oxides, or (c) crosslinking agents including either monomeric or polymeric materials having at least two latent but reactive activatable groups that are capable of forming covalent bonds with other materials when subjected to a source of thermal or radiation energy. Other multifunctional photocrosslinkers based on trichloromethyl triazine such as 2,4,6-tris(trichloromethyl)-1,3,5 triazine and 2-(methyl)-4,6-bis(trichloromethyl)-1,3,5-triazine have also been reported. These crosslinking agents have been used in combination with thermal stimuli responsive polymers to prepare responsive nono-fibers for surface modification [48].

1.3.3 Graft Polymerization

Once the surface has been activated the grafting of the functional polymer can be attempted either by “grafting to” or “grafting from” the substrate. The modification of polymer surfaces by graft polymerization can be used to produce specific surface properties. Graft polymerization is mainly based on free radical reactions and this technique can also be referred to as a “grafting from” method, but in this route high energy is often used to generate immobilized initiators followed by polymerization. The monomer may react with the free radical sites on the polymer substrate surface leading to the selective graft attachment. The initial grafting step involves the generation of reactive surface function groups by chemical or energetic irradiation. Low molecular weight polymers or selected monomers can then be attached to the reactive surface groups. In the “grafting from” approach an initiator is immobilized on a surface, after which active species are generated on the surface (radicals initiated) to initiate the subsequent polymerization of monomers from the surface. Compared with the “grafting to” method this method incorporates relatively more polymer onto a surface and is more versatile.

The “grafting to” approach has been widely used for the modification of surfaces with stimuli-responsive polymers and has the advantage of controlling the molecular weights of the grafted polymer chains by adjusting the polymerization conditions. It consists of two procedures, (i) preparing the polymer substrate with functional groups, and (ii) reaction of the polymer with the surface. The reaction with the substrate surface requires appropriate functional groups to be present on the polymer (amine or carboxyl) and the presence of initiators such as N, N’-azobis (isobutyronitrile) (AIBN) or crosslinkers such as imidoester, N-Hydroxysuccinimide and carbodiimide. The exact requirement for crosslinker or initiator is dependent on what functional groups are present on the polymer and surface.

Surface-initiated living radical polymerization (atom transfer radical polymerization [ATRP]) is a controlled polymerization technique that enables the preparation of surfaces with dense polymer brushes from surface-immobilized ATRP initiators. The dense polymer brush layers formed exhibit specific properties different from the dilute brush layers prepared by conventional “grafting from” or “grafting to” approaches.

1.4 Surface Characterization

Once a thermoresponsive polymer has been grafted onto the surface it is important to determine whether the surface immobilization of the polymer, for example, PNIPAAm, has been successful. Qualitative and quantitative analyses of grafted PNIPAAm on inorganic substrates such as silicon, glass, and quartz can be performed relatively easily by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), atomic force microscopy (AFM), ellipsometry, surface plasmon resonance, X-ray reflectometry, or neutron reflectometry (NR).

In contrast, thinly-coated polymers like PNIPAAm grafted on polymeric substrates require sensitive and selective detection because of similar signals from grafted PNIPAAm and polymeric substrates, such as polystyrene. In such circumstances XPS is one of the best means of characterizing surface-oriented PNIPAAm on polymeric substrates qualitatively. For example, the angle-dependent intensity of PINPAAm signal and the absence of π-π^* shake-up peaks derived from polystyrene were used in XPS for analysis of PNIPAAm surface characterization [49]. Attenuated total reflection Fourier transform infrared spectroscopy (ATR/FT-IR) was used to perform quantitative characterization of the amount of grafted PNIPAAm on the surface of thermoresponsive tissue culture polystyrene (TCPS). The absorption arising from mono-substituted aromatic rings observed at 1600 cm⁻¹ and the amide carbonyl derived from PNIPAAm in the region of 1650 cm⁻¹ were monitored [50]. The ratio of peak intensities at (I₁₆₅₀)/(I₁₆₀₀) was used to determine the amount of grafted PNIPAAm on the surface. AFM has also been used to observe dense nanoparticle-like domains of PNIPAAm on the surface of TCPS.

1.5 Cell Culture and Tissue Engineering Applications

A number of factors can influence the attachment of cells to a particular surface. The cells can initially stick to the surface of a material through nonspecific physicochemical interactions such as hydrophobic interactions, van der Waals forces and coulombic forces. This is followed by specific binding to site (such as RGD) on adhesive extracellular matrix (ECM) proteins. This latter binding is via integrin receptor proteins which also play an important role in cell signaling through protein kinases and require ATP metabolism. As a result of such integrin binding, significant effects on cell physiology can occur. Conventionally cell culture is carried out on a TCPS dish; cells are subsequently harvested by enzymatic proteolysis of the ECM using protease such as trypsin and by chelating Ca²⁺ ions to disrupt cell-cell junctions with, for example, EDTA. This kind of enzymatic treatment can result in integrin and basal ECM disruption as well as the possibility of changing the cells biological properties. This concern is particularly relevant to stem cells because their differentiation is strongly dependent on ECM properties.

By introducing temperature-responsive polymers such as PNIPAAm-grafted polymers onto cell culture dish surfaces, the use of digestive enzymes and chelating agents are avoided. Cultured cells can be harvested by lowering the culture temperature below LCST. This principle relies on the sharp transformation of surface properties from cell favored attachment and growth at 37°C to disfavored surface conditions causing fast detachment at ambient temperature around 20°C. Confluent cells on a PNIPAAm-grafted surface can be recovered as contiguous intact cell monolayer or sheet for tissue engineering or as individual cells as in normal cell culture applications [51, 52]. This thermoresponsive process as applied in cell culture is shown schematically in Figure 1.4.

Figure 1.4 (a) A schematic representation of temperature-responsive cell attachment and detachment; (b) actual 3T3 cells are shown attached at 37°C and being detached 20°C as large sheets.

Surface-based applications heavily hinge on how temperature-responsive surfaces are fabricated. For cell culturing the monomer composition, graft configuration, graft density, chain crosslinking within surface film, grafting to substrate surface, chain length, film thickness and uniformity are among important factors that could affect surface performance relating to cell adhesion and detachment through changes of film hydration and dehydration. A number of groups have attempted to develop thermoresponsive cell culture dishes with PNIPAAm grafted on the surface of polystyrene. Noteworthy studies have been undertaken by Okano et al.; these authors have developed electron beam irradiation to treat NIPAAm monomers uniformly spread onto the surface of the tissue culture polystyrene dishes (TCPS) to achieve polymerization and covalent surface immobilization [52–54]. In their typical work a NIPAAm solution of 40 wt% dissolved in isopropyl alcohol was added to each TCPS dish (culture area: 8cm²). This was then irradiated with a 25 Mrad (0.25 MGy) electron beam (210 kV, 19 mA, under 4×10⁻⁴ Pa) using an Area Beam Electron Processing System (Nisshin-High Voltage Co. Ltd.) to form a homopolymer (PNIPAAm) coating. The PNIPAAm-grafted dishes were then rinsed with distilled water to remove remaining NIPAAm monomers and free copolymers and dried in nitrogen gas. The PNIPAAm-grafted dishes were gas sterilized by ethylene oxide before using in-cell culture experiments. This method has been used for large-scale production of thermo-responsive TCPS dishes and marketed globally by Nunc^TM [55]. However, electron irradiation requires expensive equipment to undertake coating in a well-designated lab and the actual coating must be done under vacuum, bringing up the cost of the products. Okano et al. have shown that using this method to graft TCPS with NIPAAm, a dry thickness of 15 nm performed well with respect to cell adhesion and detachment. However, cell adhesion dramatically decreased on surfaces grafted with PNIPAAm layers thicker than 30 nm. As for polymer PNIPAAm grafting density, they found that grafting density of greater than 1.4 mgcm⁻² resulted in the cells not adhering to the surface, even though the contact angles had not changed to a significantly hydrophilic range at 37°C [53]. However, surface wettability changes were observed for all grafting densities between 20 and 37°C, yet temperature-dependent cell adhesion and detachment were only observed on PNIPAAm-grafted surfaces that were 15–20 nm thick. When the grafted layers were thicker than 30 nm, and the grafting densities were greater than 1.4 μg/cm², no cell adhesion occurred. Interestingly, Takezawa et al. reported the lack of adhesion and proliferation of fibroblasts on TCPS coated with only PNIPAAm, whereas cells adhered and proliferated on TCPS coated with a mixture of PNIPAAm and collagen [56]. Their results imply that fibroblasts cannot adhere and proliferate on coatings composed only of PNIPAAm, but require the interactions of collagen for initiating cell attachment.

Plasma polymerization has also been used to prepare PNIPAAm-grafted surfaces. Plasma glow discharge of the NIPAAm monomer vapor was used to deposit PNIPAAm onto silicon and glass surfaces as well as on TCPS [57, 58]. Retention of the polymer structure was confirmed by spectroscopic data from Fourier transform infrared spectroscopy (FTIR) and electron spectroscopy for chemical analysis. This one-step method can coat on any substrate. However, it is not suitable for large-scale production due to the difficulties of continuous treatment and size.

Photopolymerization and photografting of PNIPAAm were performed on polystyrene petri dishes using UV irradiation in the presence of benzophenone as initiator. The NIPAAm monomer of up to 40 wt% in 2-propanol with 0.1 per cent benzophenone as a photosensitizer was poured into petri dishes and irradiated by UV light (365 nm) for 30 min. XPS analyses revealed the existence of grafted PNIPAAm [59]. The use of initiators requires further surface cleaning to assure removal of residuary chemicals particularly in cell culture applications, and thus ideally there would be advantages to avoiding the use of initiators.