XXXI cycle (2016 – 2018)
PhD thesis
Study of structural, colloidal and dynamic
properties of stimuli-responsive microgels
and their application as thin films
Supervisor:
Candidate:
“Smart” microgels are attracting more and more interest in the last years, thanks to their great versatility and therefore possibility to be used in many technological fields. The key aspect of these systems is that they can change their behaviour from hard to soft spheres simply by acting on external stimuli such as temperature, pH or solvents. In the present work, the attention is mainly focused on thermo- and pH-responsive microgels made of poly(N-isopropylacrylamide) (PNIPAM) and poly(acrylic acid) (PAAc). A preliminary study aiming at the preparation of stimuli responsive peptide-based microgels was also carried out.
PNIPAM microgels were prepared by precipitation polymerization at different particle size, evaluating the effect of the concentration of surfactant (SDS) on the final dispersion by dynamic light scattering (DLS). Then the physical behaviour was investigated through different techniques, such as differential scanning calorimetry (DSC) and elastic incoherent neutron scattering (EINS). Analysing dispersions in deuterated water throughout a wide concentration range (10 – 95% wt), the typical LCST curve of PNIPAM was found out and the dynamic of the volume phase transition (VPT) was observed. Moreover, exploiting the high concentrated sample, at low temperature an unexpected dynamic transition was discovered, that can be associated to the one of the proteins. Solvents also affect the hydrophilic – hydrophobic transition, thus various mixtures of hydrogenated or deuterated solvents were used to disperse PNIPAM microgels. The variation in the swelling behaviour was observed through DLS and DSC analyses, finding out a dependence of the VPT temperature with the hydrophobicity of the used solvent.
Adding acrylic acid to the preformed PNIPAM microgels, PNIPAM-PAAc interpenetrated polymer networks (IPN) microgels were prepared. Particular attention was focused on the synthesis conditions and how they affected the structural and chemical composition of the final particles. IPNs were synthetized
scattering techniques and electrophoretic measurements. The insertion of the PAAc network affects the compactness of the particles, allowing to tune their nature from the one of the hard-sphere to the one of swollen coils. A PAAc threshold concentration has also been found, beyond which the behaviour of the particles begins to quite differ from that of the PNIPAM.
The suitability of PNIPAM and IPN microgels for tissue engineering was evaluated by preparing thin films for cell culturing tests. Film were prepared by spin coating ene-functionalized microgels dispersions and anchoring them on glass supports by thiol-ene reaction. Films made of P(NIPAM-co-AAc) copolymer were prepared also. The thickness was well controlled by the spin coating conditions and the different topographies of the microgels films during the preparation procedure were observed by atomic force microscopy (AFM). Films were also prepared through drop casting for comparison. How the cell growth was affected by the different types of substrate was finally evaluated by culturing tests. The obtained results could lead in the future, after further investigations, to use PNIPAM and PAAc-based films as a novel substrate for the growth and differentiation of cardiac tissues, fundamental especially in myocardial tissue reconstruction.
Innovative systems able to replace the classic PNIPAM microgels have been taken into consideration also. Elastin-like peptides (ELPs) have theoretically the same thermo-responsive behaviour and higher biocompatibility. Peptides with Z(XPAVG)n sequence, where Z is propargylglycine and X is either 60% or 80% valine with the remain isoleucine, were synthetized by microwaved-assisted solid phase peptide synthesis (SPPS) at three different molecular weights. The chemical composition of the samples was investigated in dried condition, but it was not possible to study the thermal behaviour in solution since they showed a limited solubility in most of solvents. Thus these preliminary results need to be further investigated in future works.
1. Introduction ... 1
1.1. Microgels of PNIPAM and of PNIPAM-PAAc IPN... 1
1.1.1. Overview and definitions ... 1
1.1.2. Properties of microgels ... 4
1.1.3. Stimuli-responsive microgels ... 7
1.1.4. PNIPAM microgels ... 11
1.1.4.1. Peculiarity ... 11
1.1.4.2. PNIPAM phase behaviour ... 16
1.1.4.3. Synthesis ... 21
1.1.5. Interpenetrated Polymer Network (IPN) of PNIPAM and PAAc ... 25
1.1.5.1. Phase behaviour of IPN PNIPAM-PAAc ... 28
1.1.5.2. Synthesis ... 30
1.1.6. Applications of stimuli-responsive materials ... 32
1.2. Elastin-like peptides ... 39
1.3. Aim of the thesis ... 43
2. Results and Discussion on PNIPAM and IPN microgels ... 47
2.1. PNIPAM microgels ... 48
2.1.1. Microgel synthesis and characterization ... 48
2.1.2. Effect of concentration and solvent on PNIPAM behaviour ... 57
2.1.2.1. Effect of concentration ... 58
2.1.2.2. Effect of the solvent ... 71
2.2. IPN microgels ... 78
2.2.1. Investigation of the colloidal properties of IPNs ... 86
2.2.2. PAAc/PNIPAM ratio effect on temperature behaviour ... 93
2.3.1. Functionalization of microgels and substrate ... 103
2.3.1.1. Ene-functionalization of microgels ... 103
2.3.1.2. Modification of the substrate ... 110
2.3.2. Films preparation by spin coating... 112
2.3.2.1. Topography and thickness control ... 112
2.3.2.2. Attachment of the films onto the surface ... 117
2.3.3. Cell culturing ... 121
2.3.4. Films preparation by drop casting ... 123
3. Conclusions on PNIPAM and IPN microgels ... 126
4. Experimental ... 131
4.1. Materials ... 131
4.2. Synthesis ... 132
4.2.1. Synthesis of PNIPAM microgels ... 132
4.2.2. Synthesis of PNIPAM macrogel... 133
4.2.3. Synthesis of IPN microgels ... 133
4.2.4. Synthesis of P(NIPAM-co-AAc) microgel ... 134
4.2.5. Synthesis of IPN-ENE microgel ... 135
4.2.6. Synthesis of P(NIPAM-co-AAc)-ENE microgel ... 135
4.3. Instruments and methods ... 135
5. Appendix: Elastin-like peptides ... 155
5.1. Results and Discussion ... 155
5.2. Conclusions on Elastin-like peptides ... 164
5.3. Experimental... 164
5.3.1. Materials ... 164
5.3.2. Peptides synthesis ... 165
1. Introduction
In this chapter, the systems studied in this thesis are introduced and it is divided in two sections to analyse the two main topics: microgels of PNIPAM and of PNIPAM-PAAc IPN and elastin-like peptides.
The first section reports some definitions and the characteristic of microgel system. In particular, the nature of microgels and their features will be explained, such as the ability of some of them to respond to external stimuli. Since this work is focused on PNIPAM-based microgel, its particular physical behaviour will be illustrated. Then the peculiarity of the IPN microgel made of PNIPAM and PAAc will be showed, with an emphasis on some interesting applications of these systems.
In the second section, a different system, elastin-like peptides, is introduced. It is related to the previous one due to the thermo-responsive ability, that is still to be fully understood for these materials.
1.1. Microgels of PNIPAM and of PNIPAM-PAAc IPN
1.1.1. Overview and definitions
According to the IUPAC definition, a gel is a nonfluid polymer or a colloidal network that is expanded throughout its whole volume by a fluid.1 In fact, gels are regarded as three-dimensional networks of cross-linked macromolecules, typically surrounded by a large number of solvent molecules. This network consists of several elements, including bridging strands (a polymer chain that connects one cross-link to another), crosslinks (or junctions), dangling ends, and
loops.2 The latter two elements can be regarded as defects in the network, the former two are indispensable for maintaining a network. Three or more strands must be connected to one cross-link to form a network structure.3,4
This structure can be made by a covalent polymer network or by a polymer network that is formed through the physical aggregation of polymer chains. In the first case, the polymer chains are cross-linked through covalent bonds, which are irreversible if no chemical degradation occurs. These gels behave as elastic solids, with the conservation of the permanent shape under self-weight (no flowing phenomenon) and no physical modification. In the case of physically cross-linked gels, the aggregation is caused by hydrogen bonds, crystallization, helix formation, complexation, etc., that results in regions of local order acting as the network junction points.5 The junction points can also be provided by glassy domains in the network, for examples in the case of block copolymers, or through lamellar structures including mesophases (e.g., soap gels, phospholipids, and clays). For these gels, the interactions between the polymer chains at the cross-linking points are reversible.
Polymer gels typically contain a large amount of solvent6 and virtually any fluid can be used as an extender, such as organic solvent or air (xerogel and aerogel, respectively). However, the widest range of gels consists of hydrogels, where the network is swollen with water, even up to 90% by weight.
The ability of hydrogels to absorb water arises from hydrophilic functional groups attached to the polymeric backbone, while obviously their resistance to dissolution arises from cross-links between the network chains. Moreover, to achieve high degrees of swelling, it is common to use polymers that are water-soluble when in non-cross-linked form.7
The capacity to contain a large amount of solvent gives hydrogels a characteristic softness originated from the liquid nature. On the other hand, they can maintain shape like solid materials, unless excessive stress is applied. Consequently, the combination of softness and shape retention ability provides unique properties, particularly mechanical properties.8–10 Moreover, their high-water content makes them biocompatible and allows the diffusion of high-water
able to absorb, store and release large amount of water, as well as aqueous solutions containing active substances, various uses of hydrogels have been explored in hygienic products11, drug delivery systems11,12, food additives13, biomedical applications14,15, tissue engineering and regenerative medicine16, separation of biomolecules or cells17 and biosensor18.
In this work, a particular type of gels, which are definite as microgels (the term was first introduced by Baker19), have been studied. Microgels are colloidally stable, solvent swollen microparticles with size ranging from 1 nanometre to 1 micrometre.
The most common monomers used to prepare microgels are based on derivates of methacrylate, acrylate, acrylamide and styrene compounds.20 Other monomers frequently used are N-vinyl caprolactam, derivatives of 2-oxazoline, ethylene glycol and ethylene imine.21,22 Same examples are shown in Figure 1.1.
Figure 1.1. Structure of the monomers frequently used to prepare microgel particles: (a) methylmethacrylate, (b) methacrylic acid, (c) acrylic acid, (d) styrene, (e)
N-isopropylacrylamide, (f) N,N’-methylene bisacrylamide, (g) ethyleneglycol dimethacrylate and (h) divinylbenzene.
The peculiarity of these materials is that they link the concept of gel with the concept of colloidal system. In fact, a colloidal system is described as a dispersion of small insoluble particles in a continuous phase and, in microgels,
the dispersed phase is the three-dimensional network that is swollen with the solvent and that doesn’t dissolve in the environment.
Microgels cannot be described only as classical colloidal systems, because, like bulk hydrogels, they are mechanically soft, and due to this softness, they exhibit properties that differ significantly from hard spheres.23
They can be better definite as soft colloids, because they exhibit intermediate properties between soluble polymers, macrogel (as in hydrogel with larger size than microgels) and insoluble colloidal particles. On one hand the typical feature of the microgel particles are strictly related to the balance between polymer-polymer and polymer-polymer-solvent interactions, as observed for polymer-polymers soluble in water. On the other hand, they exhibit a cross-linking density, a degree and a characteristic time of swelling that are typical of aqueous macrogels. Finally, like colloids based on hydrophobic polymers, colloidal microgels can be prepared to obtain a monodisperse size distribution. Therefore, both their colloidal and polymer-like nature must be taken into account to describe their behaviour.24
1.1.2. Properties of microgels
Consisting in a colloidal dispersion, an important parameter to describe microgels is the volume fraction occupied by the microgel particles (φd). This
value depends on the volume fraction of polymer in the dispersion (φp) and the
volume fraction of polymer within each particle (φ2),20 according equation (1.1):
𝜙𝑑 =𝜙𝑝 𝜙2 (1.1) where: 𝜙𝑝 = 𝑚𝑝 𝜌𝑝 ⁄ 𝑚𝑝 𝜌𝑝 ⁄ +𝑚𝑠 𝜌𝑠 ⁄ (1.2)
with mp and ms mass of polymer and solvent respectively and ρp and ρs densities
The volume fraction can be also expressed as a function of the number density of the particles n and their volume Vp (equation (1.3)), where the radius changes
according to the degree of swelling.25
𝜙 = 𝑛𝑉𝑝 = 𝑛4𝜋𝑅 3
3 (1.3)
In the case of microgels made of stimuli-responsive networks (see chapter 1.1.3), another main characteristic property of microgel is the swelling behaviour and the extent of such swelling. This is usually determined from the change in the hydrodynamic diameter of the particles when they shift from a swollen to a collapsed state in response to a change in the environment.20 The swelling change is reported with respect to a reference diameter (d0), which is
often chosen as that of the collapsed state. The extent of particle swelling is expressed as the swelling ratio α, which is simply:
𝛼 = 𝑑
𝑑0 (1.4)
where d is the measured hydrodynamic diameter at a given degree of swelling. The swelling ratio and φ2 are related by:
𝛼 = 1
𝜙2 (1.5)
Microgels are considered as ideal model systems for studying interparticle interactions, exploiting their swelling/de-swelling capability. The value for α is therefore of considerable importance for understanding the behaviour of microgel dispersions.
Flory–Rehner's theories of swelling of cross-linked polymer networks were developed for macroscopic gels.6 However, at the single-particle level, they remain good models for predicting the α value for microgels and provide a reasonable description of the elastic properties of microgel particles.26–28 The extent of swelling of microgels can be affected by various parameters. One of these is the internal structure of microgel particles, for example considering
the cross-linking degree: a particle with a higher degree of cross-linking tends to swell less, due to the more rigid network, than a particle with a lower degree of crosslinking. Another important structural information is the distribution of the cross-links inside each particle. Although the theories of microgel swelling assume uniform swelling due to a uniform cross-link density, the situation is highly unlikely. Often the cross-link density decreases from the centre of the particles toward the periphery. In the case of copolymerization of difunctional and tetrafunctional monomers, for instance, this means that the larger proportion of the cross-linking tetrafunctional monomer is incorporated during the initial growth of the particles. This is not surprising if the solubility of the polymer chain decreases with increasing molecular weight and addition of cross-linking monomer facilitates a substantial increase in its length.20 Following the confirmation of the heterogeneous nature of most microgel particles, an empirical modification of the Flory-Rehner theory has been proposed by Hino and Prausnitz29, applicable both for macro- and microgels. Applying the modification to the relation that links the number of segments between junction points to the monomer concentration at preparation, it allows to correlate the particle diameter to temperature and to calculate the phase diagram by using a first-order perturbation theory for the fluid phase and an extended cell model for the solid phase.
The degree of swelling also influences the optical properties of microgel dispersions. The refractive index of an individual microgel increases as the particle de-swells; its refractive index is essentially equal to that of a solvent when highly swollen, whereas it approaches that of a dehydrated polymer network when de-swollen.30
Another interesting feature of microgel particles is their rapid swelling/de-swelling kinetics in comparison to macrogels. It has been shown that the driving force and the equilibrium extent of the swelling behaviour is the same for microgels and macrogels, while the dynamics result highly sensitive to the gel size.31 Indeed, microgel particles retain the unique physical properties of bulk hydrogels, while their swelling/shrinking kinetics is much faster with respect to macrogels: in response to temperature changes, microgels achieve the swollen
because shrinking of the exterior layer prevents water transport from the interior.
The deformable nature of microgel particles instead has important implications for their rheological properties. It was reported that the rheological behaviour of microgel particles is equivalent to that of hard particles with a thin, soft shell.20 Dilute microgel dispersions exhibit Newtonian flow, whereas concentrated dispersions are highly shear thinning.32 When compared at the same number concentration to hard-sphere particles, swollen microgel particles greatly increase the dispersion viscosity, due to the larger effective hydrodynamic diameter of the swollen particles.33 At low crosslinking densities, microgel exhibit no longer yielding due to cage breaking, as typically found in colloidal particle systems, but instead, they show an alternative continuous and less pronounced yielding process at higher strain and shear rate, interpreted as disentanglement of long dangling chains at the particle surface. Moreover, when particles size decreases under 100 nm, this transition from colloid-like to polymer-like yielding shifts at even higher crosslinker content.34 It is for these reasons that microgel particles have potential application in the surface coatings industry as filler materials.
Finally, due to their colloidal nature, lately microgels are largely studied for understanding atomic and molecular behaviour and to address questions in the field of statistical mechanics and condensed matter physics. This has been possible because colloidal particles are slower and larger than their atomic and molecular counterparts, making them amenable to imaging via video microscopy and light-scattering techniques, with a resolution up to a single-particle.35
1.1.3. Stimuli-responsive microgels
Among soft colloids, system with stimuli-responsive properties are interesting materials for a variety of different application, due to their smart response to changes in the external environment.
Aqueous dispersions of responsive microgel allow to modulate the interaction between the network and the solvent through easily accessible parameters usually not relevant in ordinary colloids.24 In particular, responsive microgels can reversibly change their volume (swelling/shrinking behaviour), undergoing a Volume Phase Transition (VPT) (Figure 1.2), in response to slight changes in the properties of the medium, such as temperature, pH, salt concentration, light, electric or magnetic field. A slight change in the environmental conditions may in turn generate great modifications of their macroscopic properties. The reversibility of the volume transition is an additional advantage for the applications of these hydrogels as responsive materials.
Figure 1.2. Schematic picture of the volume phase transition of microgel particles from a swollen hydrated phase to a shrunken dehydrated one by crossing the VPT.
The sharp volume phase transition of microgels is the key point during the responsive process. There are three major mechanisms that are responsible for the stimuli-induced volume change: changes in osmotic pressure or charge density (e.g., pH-responsive microgels); changes in solvent affinity of the polymer chains (e.g., thermo-responsive microgels); changes in the cross-linking density of polymer chains.36
The volume phase transition affects the solvent mediated interparticle forces and leads to a novel phase-behaviour drastically different from those of conventional hard-spheres-like colloidal systems. Indeed, due to their softness, microgels can be highly packed to effective volume fraction far above those of hard colloids, with interesting consequences on their structural and dynamical behaviour. Moreover, the swelling behaviour has been shown to be the driving mechanism for tuning the effective packing fraction, thus enabling an experimental control parameter to explore the phase behaviour.
In fact, for hard-sphere colloids, which interact only as a result of excluded volume, phase behaviour is controlled by the volume fraction, φ; an increase in φ drives the system towards its glassy state, analogously to a decrease in temperature, T, in molecular systems. At volume fractions below 0.49, hard-sphere suspensions are in a fluid phase, in which particle positions are uncorrelated over long distances. Between φ = 0.49 and 0.55, an ordered crystal phase coexists with a less dense fluid phase. Above φ = 0.55, the thermodynamically stable phase is the crystal. Interestingly, the densest closed-packed arrangement of spheres, corresponding to φ = 0.74, is rarely seen. Instead, with increasing particle volume fraction, an increase in viscosity is observed, indicating the increased interactions between particles and a concomitant decrease in the particle diffusion coefficient. This condition eventually results in the formation of a glassy, jammed state, in which the particles are forced into close contact, and the system is kinetically arrested and held out of equilibrium.24
Instead, in stimuli-responsive microgel suspensions, the polymer-like nature of the particles enables external control of the particle size and concomitant tuning of the suspension volume fraction. Therefore, they are good candidates as ideal model systems for providing new insight into the glass formation in molecular systems, as they provide an elegant way to tune φ without having to change the particle number concentration.25
Some of most known thermo-responsive microgels are based on poly(N-isopropylacrylamide) (PNIPAM), due to its biocompatibility and volume phase transition around 32 °C.21,30 The main characteristic and behaviour of this polymer will be thoroughly shown in the following chapter (1.1.4).
Another interesting thermo-responsive polymer is poly(N-vinyl caprolactam) (PNVC), since it possesses very interesting properties for medical and biotechnological applications, e.g. solubility in water and organic solvents, biocompatibility, high absorption ability and a transition temperature within the settings of these applications (33 °C).37
A class of thermo-responsive polymer consist of poly(2-oxazoline)s ((POx)s). The living cationic ring-opening polymerization of 2-oxazolines provides easy and
direct access to a wide variety of well-defined polymers. Furthermore, the properties of poly(2-oxazoline)s can be tuned simply by varying the side chain of the 2-oxazoline monomer. The earlier applications of poly(2-oxazoline)s were as stabilizers/surfactants and compatibilizers and recently the use of poly(2-oxazoline)s in biomedical applications has evolved as a result of their biocompatibility as well as their stealth behaviour.38 In particular, the most known (POx)s is poly(2-ethyl-2-oxazoline) (PEtOx).21,38
The stimuli responsiveness of the polyurethanes (PU) is often exploited to prepare Shape Memory Polymers (SMP). Indeed, PU may have reversible amorphous or crystalline phase. PU has a unique structural characteristic because of the presence of inherent incompatibility due to microphase inhomogeneity in its chain molecules. It possesses a wide-ranging temperature for shape recovery, high recoverable strain (up to 400%), inherent soft-hard segments, high control on the softening and retraction temperatures with good biophysical properties. Further, the properties including switching transition temperature like glass transition temperature (Tg) or melting temperature (Tm) can be tuned much easily by proper choice and using suitable composition of the components during polymerization process.39,40
Triblock copolymers of poly(ethylene poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO, the poloxamers/Pluronics) are widely used reverse thermal gelation polymers. The block copolymers with different numbers of hydrophilic ethylene oxide and hydrophobic propylene oxide units are characterized by different hydrophilic-lipophilic balance (HLB). Due to their amphiphilic character these copolymers display surfactant properties including ability to interact with hydrophobic surfaces and biological membranes. In aqueous solutions at concentrations above critical micelle concentration (CMC) these copolymers self-assemble into micelles.41 The possibility to create a wide variety of polymers with different composition leads the transition to range from 32 °C for L62 with a low PEO content to above 100 °C for F127, possessing a high PEO content.42
against which every new polymer is being tested, even though a large number of investigated polymers perform better in terms of cytotoxicity and transfection efficiency.43 Poly(acrylic acid) (PAAc) is another polymer that has a relevant role between the pH-responsive gels and microgels, mostly regarding the application in the fields of drug delivery and soft robotics.44,45 A more extensive explanation of the behaviour and applications of PAAc will be shown in the chapters 1.1.5 and 1.1.6.
Moreover, the combination of various stimuli-responsive polymers makes it possible to explore different stimuli-responsive macro and microgels. In this study, the attention is focused on two kinds of polymers: a thermo-responsive polymer, poly(N-isopropylacrylamide) (PNIPAM), and a pH-responsive polymer, poly(acrylic acid) (PAAc).
In particular, the systems studied are a homopolymeric microgel of PNIPAM, described in more details in chapter 1.1.4, and interpenetrated polymer networks (IPNs) of PNIPAM and PAAc (chapter 1.1.5).
1.1.4. PNIPAM microgels
As one of the most widely studied smart material, thermo-responsive polymers such as poly(N-isopropylacrylamide) have attracted an increasing interest and they have been more and more studied in the past several years, because of the easy controllability of temperature and more importantly their promising applications in various fields.21 Below, the main characteristics and behaviour of microgels made of this polymer will be explained.
1.1.4.1. Peculiarity
Normally, thermo-responsive microgels are prepared from polymers that exhibit a critical solution temperature in water, around which the interactions between polymer chains and water change between hydrophilic state and hydrophobic state dramatically within a small temperature range. Resulting from the expansion or contraction of polymer chains in water, thermo-responsive microgels indicate volume phase transition (VPT) around the lower
critical solution temperature (LCST) or upper critical solution temperature (UCST) depending on the polymer. General phase diagrams describing these behaviours are shown in Figure 1.3.
Figure 1.3. Temperature vs. polymer volume fraction (φ). Schematic illustration of phase diagrams for polymer solution (a) lower critical solution temperature (LCST) behaviour and (b)
upper critical solution temperature (UCST) behaviour.
For microgels made of polymers with LCST, such as poly(N-isopropylacrylamide) (PNIPAM), when the temperature lowers below LCST, the structure swells due to the diffusion of water into the polymer network, because water molecules form hydrogen-bonds with the polar groups on the polymer chains. However, the efficiency of the hydrogen-bonding decreases with the rise in temperature, and when the temperature is above LCST, the hydrophobic property of the polymer chains starts dominating, causing the network to shrink. So, the VPT is driven by the coil-to-globule transition of the polymer chains.
In particular, PNIPAM particles display a volume phase transition temperature (VPTT) at about 32°C.30 The VPTT of the microgels is directly associated with the LCST of the polymer comprising the microgel. It is to be stressed that the two terms are often used interchangeably, given that they coincide for pure PNIPAM microgels. However, the VPTT is the temperature associated with the thermally induced collapse of the microgel, whereas the LCST is related to the thermally induced de-solvation of the polymer chains; the two values are related but not necessarily identical.24
The temperature at which the actual transition appears can be tuned by introducing different additives such as salts, non-electrolytes, hydrotropes and
surfactants or by synthesis, for instance by copolymerization with other monomers or by controlling the degree of crosslining.46
For example, PNIPAM forms mixed hydrophobic aggregates with surfactants, especially in case of anionic ones, such as sodium dodecyl sulfate (SDS). The electrostatic repulsion between the charged polymer-bound aggregates contrasts the polymer coil collapse at high temperatures, thus leading to a remarkable increase of the LCST, whilst no changes are observed for cationic or non-ionic surfactants. In addition PNIPAM microgels can be prepared by employing cationic free radical initiators, thus obtaining microgels with positive charge on their backbone30 or by adding several inorganic salts such as NaI, NaBr, NaCl, NaF, Na2SO4 and Na3PO4 for investigating their effect on the cloud point (defined as the temperature where the mixture starts to phase separate, thus becoming cloudy).46 The electrolytes are known to increase or decrease the LCST of the polymer solutions, thus affecting the phase behaviour of uncharged polymers due to the destruction of the hydration structure surrounding the dissolved polymer chains.
The addition of additives has effect on the swelling degree of microgels particles also. Indeed, de-swelling may be induced by adding free (i.e. non-adsorbing) polymer to the continuous phase, provided that the free polymer is excluded from the microgel particle interior.47
Particle de-swelling may also be induced by addition of a co-solvent that competes for the water molecules that hydrate the polymer chains.48 The solvent may affect the transition of the system also. PNIPAM linear polymers and microgels display interesting phase behaviour in aqueous mixtures with other solvents, called "co-non-solvency", a phenomenon whereby a polymer has poor solubility in a mixture of solvents that are individually good solvents for that polymer.46 Indeed, it was reported that PNIPAM microgel particles and macrogels de-swell when alkanols are added to the water dispersion, even though the alkanols are good solvents for the polymer.49
The competition between the solvents can result in a poorer solubility of the polymer in the mixture than in the individual solvents; this depends on the relative affinity between the solvents themselves and between the solvents and
the polymer. For instance, in alcohol/water mixtures, the transition temperature of PNIPAM shifts to lower temperatures and the microgels de-swell as the alcohol concentration increases, as shown in Figure 1.4. This behaviour was observed in methanol/water, ethanol/water, and propanol/water mixtures.50
Figure 1.4. Size change of PNIPAM microgels as a function of temperature, for different methanol concentrations. By increasing the alcohol concentration of the water/methanol
mixtures, the solvent becomes progressively poorer and the particle size decreases.49
Interestingly, if the volume fraction of the alcohol is increased above about 0.4, a re-entrant swelling is observed, as shown in Figure 1.5 for PNIPAM microgels in isopropanol/water mixtures.51 Indeed, at low isopropanol volume fraction, the particle size decreases with the increase of isopropanol. Then, after reaching a minimum value, particle size increases with any further increase in the alcohol volume fraction. In addition, increasing the temperature the particle size decreases, as expected for any thermosensitive microgel with a LCST.
Figure 1.5. Microgel particle size versus alcohol volume fraction, for different temperatures. At each temperature the swelling degree of the particles changes, as indicated by the size change for zero alcohol content. The experimental system is based on PNIPAM microgels dispersed in propanol/water mixtures. The dashed line corresponds to the particle size measured in pure
water at 50 °C.51
There are more sophisticated theories to explain these co-non-solvency effects. For example, the lattice fluid hydrogen bound theory (LFHB)52 accounts for
specific hydrogen bonding between the solvents and the solvent and polymer; this theory has been successfully applied to the swelling of PNIPAM macrogels,53
and should in principle also be applicable to other microgel systems.
It is interesting to discuss also the deuterium isotope effects on the swelling kinetics and volume phase transition of polymer microgels. In particular, a slower swelling kinetics of microgels in D2O than in H2O have been observed and ascribed to the high viscosity of the medium.54 Therefore, the LCST of PNIPAM microgels dispersed in D2O solutions are expected to be slightly shifted forward with respect to the H2O solutions.
In the end, it can be declared that the response of polymer microgels in water-miscible solvents is believed to be important for understanding the role played by hydrophobicity and hydrogen bonding in the polymer-solvent interactions in the dynamics of polymer solutions.
Among synthesis parameters the spatial distribution of the cross-linking density was demonstrated to have almost no effect on the volume phase transition temperature, even if it markedly affects the swelling capacity of PNIPAM
microgels at low temperatures.55 Indeed it has been shown that the swelling capability decreases with increasing the N,N’-methylenebisacrylamide (BIS) fraction in PNIPAM networks, due to the topological constraints introduced within the PNIPAM microgel particles.56 Therefore the elastic response of the system can be deeply controlled by changing the cross-linker concentration, leading to microgel particles characterized by a different degree of softness. Moreover, copolymerization with charged monomers, such as acrylic acid (AAc), methacrylic acid (MAAc) or similar affects both the swelling and the VPTT of the microgels, due to local electrostatic repulsion.56 The effective charge density can be controlled by the amount of comonomer, the pH or the ionic strength of the medium. In particular, it has been shown that the reduction of the swelling capability of PNIPAM microgel deeply depends on the amount of AAc comonomer.56,57 As regards the VPTT, it depends on the macromolecular architecture also, being the response of the P(NIPAM-co-AAc) microgels strictly related to the mutual interference between the two monomers, as it will better explained in chapter 1.1.5. For example, for randomly copolymerized P(NIPAM-r-AAc) microgels, the volume phase transition temperature is observed to increase with AAc fraction, whilst by interpenetrating the hydrophilic PAAc into the PNIPAM microgels network a little influence on the globule-to-coil transition of the PNIPAM chains has been reported.56,58
1.1.4.2. PNIPAM phase behaviour
PNIPAM phase behaviour and its liquid-to-solid transition has been intensively investigated by using different techniques, such as DLS, UV-visible transmission spectroscopy, rheometry, calorimetry and so on.57,59–61 These works have shown that since the particle size decreases with increasing temperature, the volume fraction can be changed by varying the temperature of a colloidal dispersion, thus exhibiting a phase transition that leads to a novel phase behaviour.
Indeed, at low polymer concentration, PNIPAM dispersion looks like a clear liquid, since the absence of spatial constraints allows particle to freely diffuse,
slightly higher concentrations the phase behaviour of such colloidal suspension resembles that for hard spheres below the VPTT, where the microgel spheres are fully swollen and the van der Waals attractive interactions are attenuated by the presence of the solvent inside and outside each particle.25,62 By further increasing concentration, a cage between neighbouring particles gradually forms and limits their motion, even if in this regime of concentration, the particles are still independent, since interpenetration or compression doesn’t take place. The system is in a glass state where the microgel particles exhibit repulsion due to their charge or to steric stabilization. At this concentration above the VPT instead, van der Waals attractions become dominant, originating a fluid-fluid arrested phase separation, which is maintained also when the concentration increases.60–62
For the system below the VPT, at even higher concentration, the interpenetration of the outer and less-linked regions of the PNIPAM microgels occurs as well as soft particles compression, leading to a deviation from the spherical shape and to a favourite direction of interaction between particles. At this point the cage structure is destroyed and the system finally percolates, yielding the gel transition, as shown in Figure 1.6.59
Figure 1.6. Concentration and temperature dependent phase diagram of PNIPAM microgel suspensions.59
As mentioned above, for suspension of hard spheres, the liquid-to-solid transition occurs at packing fraction of 0.64, but for soft microgels no universal behaviour has been observed.24 Following same rheological studies34,63,64 it has been reported that the particle size and the degree of crosslinking seems to be
an important parameter governing the physics of the liquid-to-solid transition in dispersions of soft colloidal particles.
Indeed, at low crosslinking density, microgels are soft and can be compressed and deformed to adopt non-spherical, polyhedral shapes upon packing to high concentrations, leading to a liquid-to-solid transition and effective particle volume fractions that may far exceed the hard-sphere value.25 Recent study denotes that the interpenetration of the particles at high packing fraction plays an important role, especially for microgels with low crosslinker content,65 indicating microgels in suspension to have less of a hard-sphere nature, but instead, more that of interpenetrating non-crosslinked polymer coils at similarly high concentration66. Furthermore, it has been discovered that this phenomenon is particularly accentuated for ultra-soft small particles (with radius of only about 50 nm),34 which undergo a liquid-to-solid transition at unusually high effective particle volume fractions much larger than 10, comparable to the concentration range where solutions of linear polymers exhibit a liquid-to-solid transition due to chain entanglements. Therefore, it appears impossible to describe these very small PNIPAM microgels as effective colloidal particles any longer. In Figure 1.7 it is illustrated how large microgels (top, left), even at low crosslinking density, behave similar to colloidal hard-sphere dispersions (top, right), and instead small microgels (bottom, left) exhibit the rheological behaviour of semi-dilute solutions of linear polymer chains (bottom, right).
This unusual behaviour is attributed to dangling polymer chain ends in the corona of these small microgels that can interpenetrate considerably upon increasing particle packing, an effect that becomes more important the smaller the particles are.
The phase behaviour of PNIPAM in water has been studied by calorimetric techniques also67,68 using the heat capacity signal to investigate the behaviour of polymer chains, especially during the demixing process (phase separation), in the entire composition range. These studies analyse a different scale compared to the previous ones, since in the phase diagram reported in Figure 1.6 the behaviour of microparticles was observed, while in this case the focus is at the level of polymer chains, that is at nanoscale.
Regarding the phase separation, the system PNIPAM/water is classified as a type II demixing, that is when the LCST is nearly independent with the molar mass of the polymer.69 Indeed, a broad LCST miscibility gap was obtained experimentally with a minimum at molar fraction of PNIPAM between 0.4 and 0.5, independent of chain length (Figure 1.8 (c)). It was also proved that there isn’t any difference in the behaviour between linear polymer chains and crosslinked network67, indicating that the phase separation occurs in the same way, independently of the kind of structure of the polymer.
Figure 1.8. (a): State diagram of PNIPAM Mw=74000 g/mol, demixing curve (O), Tg-composition curve (●), melting (Tm: X) and crystallization (Tcr: □) temperature of water; (b): State diagram of PNIPAM Mw=74000 g/mol, demixing curve: threshold in cpapp 0.01 J g-1 K-1
(O), 0.1 J g-1 K-1 (●) and cloud point curve (X); (c): demixing curves for PNIPAM Mw=18000 g/mol (Δ), PNIPAM 74000/water (O) and PNIPAM 186800/water (*). Demixing temperatures are calculated from threshold in cpapp (0.01 or 0.1 J g-1 K-1) upon heating; Tg from cpapp upon
cooling; Tm and Tcr from the total heat flow, upon heating and cooling, respectively.68
Moreover, a typical glass transition appears for the PNIPAM/water mixture when the system crosses the transition temperature Tg, but this value changes depending the polymer concentration in solution. Indeed, the higher the polymer concentration, the smaller the rates of diffusion (and thus the rate of
1.8 (a)). Vice versa, Tg lowers with increasing water content, due to the plasticizing effect of water on PNIPAM.68
Regarding the kinetic of the phase separation, after increasing and then decreasing the polymer concentration in the solution, remixing appears to be slower than demixing. This effect is magnified by the partial vitrification of the PNIPAM-rich phase, which is usually not taken into account for polymer/water mixtures. However, a higher degree of partial vitrification increases the remixing time from less than 1 h up to 1 day or even more, indicating the importance of this phenomenon with respect to applications. At higher polymer concentration the rate of phase separation is slowed down as well.68
Despite these findings, the phase diagram is far from being completely clear and in particular the understanding of the phase behaviour at high temperature and concentrations is still lacking, despite the evidences of formation of an attractive glass.59 Indeed, until now the exact nature of the interaction potential to describe the behaviour of microgel particles and therefore the role of repulsive interactions and the origin of attractive ones, such as the real nature (gel or attractive glass) of the state at high concentration, are still ambiguous.62
1.1.4.3. Synthesis
The synthesis of temperature–responsive microgels of poly(N-isopropylacrylamide) was first reported in 198670 and, since then, there have been hundreds of publications describing the preparation, characterization and applications of these systems.47,49,67,71
Monodisperse PNIPAM-based microgel particles are usually prepared by precipitation polymerization of N-isopropylacrylamide (NIPAM). The major requirement for the preparation of the microgel particles is that the reaction is conducted above the LCST of PNIPAM, usually around 70°C, due to the higher reaction rate at high temperatures (Figure 1.9).72,73 Under these conditions the water-soluble monomers form water insoluble polymers, which in turn aggregate until they form a colloidally stable particle. The intact particle nature of the PNIPAM microgels is ensured by chemical cross-linking achieved by the
copolymerization of N,N’-methylenebisacrylamide (BIS), although NIPAM is in principle able to form microgels via self-cross-linking74.
The polymer chain growth in aqueous NIPAM/BIS microgel synthesis takes place via radical polymerization. The reaction starts with an initiator and then chain propagation occurs through the addition of a NIPAM or BIS monomer to the terminal radical site of an existing polymer chain, that could be a NIPAM or a BIS radical. The rate of reaction between these two monomers determines the distribution of crosslinking within the particle. The earliest results72 related to the internal structure of PNIPAM microgels, which investigated the kinetics of particle formation, reported that during the particle formation the cross-linker monomer was incorporated into the microgel structure faster than the NIPAM monomer (Figure 1.9). This result implies that the different polymerization rates of the monomers lead to the formation of inhomogeneously cross-linked microgel particles.
Figure 1.9. N,N’-methylenebisacrylamide (BA in this figure) and N-isopropylacrylamide (NIPAM) conversion as function of reaction time for reactions conducted at 50°C and 70°C.72
However, latter studies revealed that the structural inhomogeneity depends on the degree of cross-linking: the most highly cross-linked microgels retain a particle character and exhibit a Gaussian segment density distribution in their swollen state. Instead, with decreasing cross-link density the microgel particles can be better described as core/shell structures formed by a highly cross-linked core and a shell of dangling polymer chains.75
To fully understand the reaction involved during the formation of the cross-linked network, recently the polymerization rate constants of the major chain propagation reactions were calculated quantum-mechanically.76 The results confirm that BIS is preferentially consumed at the beginning of the microgel synthesis process, as the value of reaction rate constant of BIS is slightly higher than the one of NIPAM. Summarizing, difference in the reaction rate constants influences the linker density within microgels and results in a higher cross-linking density in the core of microgels and a less cross-linked microgel shell. In principle, initiation and termination could also have an impact on the inhomogeneous cross-linker distribution. However, this seems not likely: the reaction conditions are similar to those of the termination reactions and the vast majority of termination reactions will be radical combination reactions. In general, the reaction rate constant of these reactions is quite large. In other words, whenever two radicals “meet”, they will recombine. As a consequence, the termination reactions are limited mostly by diffusion. Diffusion on the other hand depends mainly on the size of the molecules. The sizes of the NIPAM and BIS are very similar. Therefore, the size of the radical chains will depend on their length but not on their composition. Hence, the diffusion will be of the same order for all radicals. Thus, the effective rate constants for termination will be nearly the same for all radical pairs in the two considered systems. That is why, termination reactions do not favour any of the monomers. Consequently, an effect that affects all monomers equally cannot be the origin of an inhomogeneous cross-linker distribution.76
Another key aspect in microgel synthesis is the control over particle size. The standard method to achieve this size control is to use surfactants, such as sodium dodecyl sulfate (SDS), in the synthesis, which also enhances colloidal stabilization during the sensitive nucleation stage.49 In general, the average particle diameter decreases with SDS concentration, up to 0.58 g/L.
Figure 1.10. Average diameter of microgel particles at 25°C as a function of SDS concentration used in polymerization.49
During the nucleation process, the small initially formed particles are colloidally unstable, therefore, they tend to nucleate to form the coagulative association of precursor particles. Since the SDS concentration during the reaction is usually below the critical micelle concentration (2.36 g/L), the micellar nucleation is not possible, but since PNIPAM is less hydrophilic above the LCST, it would be favourable for SDS to adsorb onto the surface of phase separated PNIPAM particles. In summary, PNIPAM microgel latex particles form by homogeneous nucleation and the role of SDS in particle nucleation is to increase the colloidal stability of precursor particles and thus lower the diameter of primary particles. Moreover, particle volumes are linear with conversion, so this implies that for most of the polymerization the number concentration is constant. In other words, the particle nucleation stops at low conversions. Further support for a rapid nucleation process is the fact that the particle size distributions, as determined by dynamic light scattering, is usually narrow.72
This behaviour doesn’t longer occur when the amount of SDS exceed the critical micelle concentration. More recent works demonstrated that could be an advantage to use a higher concentration of SDS, such as 5.3 mM and 6.7 mM, as it gives even smaller nanogels of size 50 nm and 35 nm at room temperature, respectively.77,78 Furthermore, high SDS concentration shifts the polymerization system closer to the good-solvent conditions and the microgel structure is significantly affected in the process. Indeed, as indicated by SANS studies,78 the
appear to be more homogeneously structured than the samples prepared in low surfactant concentration.
1.1.5. Interpenetrated Polymer Network (IPN) of PNIPAM and PAAc
As mentioned above, the internal structure of microgel particles is a fundamental parameter that influence the swelling behaviour and the mechanical properties of these systems. Over the years, increasingly complex structures have been developed in order to obtain well-controlled properties over a large range. Indeed, very different kind of architecture were well studied in recent years beside the conventional homogeneous network, such as network with a tetrahedral geometry, interpenetrated polymer network (IPN), semi-interpenetrated polymer network, double network, nano-composite hydrogel, microgels-filled network, microgel reinforced network and double crosslinked network.79
The introduction of specific morphologies across different length scales has resulted in micro and macrogels with mechanical properties that space from brittle materials to synthetic rubbers-like materials.80
Increasing the complexity of the microgel structures also leads to being able to incorporate more elements inside a single particle. For example, it is possible to have different polymers with different responses to external stimuli within the same system.
Precisely, in this work, the architecture taken in consideration is the interpenetrating polymer network (IPN), as shown in Figure 1.11, in which in each particles two networks are present, one of PNIPAM and one of poly(acrylic acid) (PAAc).
Figure 1.11. Schematic representation of an interpenetrating polymer network (IPN).81
In general, an interpenetrating polymer networks is a system composed of two or more cross-linked networks that are interlaced at the molecular scale but not covalently bonded to each other. In this way they can’t be separated unless chemical bonds are broken.82
Adding acrylic acid (AAc) to PNIPAM microgel under polymerization condition leads to the formation of a second homopolymeric network that provides a pH-sensitivity to the system and that leads to a more complex phase behaviour. In this way a thermo- and pH-sensitive system made of two interpenetrated networks is obtained.
The response to the external environment of this kind of architecture is very different from that of a system with an equal chemical composition but synthetized by copolymerization of NIPAM and AAc. Indeed, by random copolymerization of two monomers, a single network of both monomer is obtained exhibiting properties dependent on the two monomers ratio.56,83 Instead, the IPN particles exhibit the same independent response as the two components to different external stimuli, since their interpenetration leads to a largely reduction of the mutual interference between the repeating units, making the temperature dependence of the VPT unchanged with respect to the case of pure PNIPAM microgel, as is shown in Figure 1.13 (left).84–86
Regarding the pH-responsive behaviour of the pH-responsive polymer, it is due to the ionizable pendant moieties that can accept and donate protons in
the degree of ionization in a polymer bearing weakly ionizable groups is dramatically altered at pH values around the pKa. This rapid change in net charge causes an alteration of the hydrodynamic volume of the polymer chains. The transition from collapsed state to expanded state is explained by the osmotic pressure exerted by mobile counterions neutralizing the network charges.87
In weak polyacids such as poly(acrylic acid), the carboxylic pendant groups accept protons at low pH, while releasing them at high pH. When ionizable groups are protonated and electrostatic repulsion forces disappear within the polymer network, hydrophobic properties dominate, causing aggregation of the polymer chains from the aqueous environment. Another way to explain the collapse mechanism is based on the hydrogen bonding between the hydrogen in the protonated carboxylic group and an electrondonating atom (e.g. oxygen or nitrogen) in other functional groups, when the ionizable pendants groups are uncharged. By this way it is possible to control the precipitation/ solubilization of molecular chains, deswelling/ swelling of hydrogels, or hydrophobic/hydrophilic characteristics of surfaces. Poly(acrylic acid), that has a pKa around 4.5, shows a relatively continuous phase transition from hydrophilic to hydrophobic state87, as shown in Figure 1.12.
Figure 1.12. Titration curves and pH vs. dissociation degree (αa) or dissociation degree in
presence of metal ion (αm) plots for PAA and PAA–Ni systems (solid square, PAA; open square,
1.1.5.1. Phase behaviour of IPN PNIPAM-PAAc
As mentioned above, the pH-sensitivity of the acrylic acid, due its different solubility at acidic and neutral pH, introduces in the IPN system an additional pH and ionic strength tunability, which gives rise to a complex phase behaviour, since they affect the pair interaction between colloidal particles. Indeed, at acidic pH the PAAc chains are not effectively solvated by water and the formation of H-bonds between the protonated carboxylic groups of PAAc and the amide groups of PNIPAM is favoured.89 At neutral pH instead, the acrylic acid is almost deprotonated, soluble in water, thus forming H-bonds with water molecules. Both compounds are therefore solvated by water, that mediates their interaction, and the lower number of interchain H-bonds between PAAc and PNIPAM, makes the two networks completely independent one to each other. So, the temperature and pH-tunability of the microgel swelling properties allows the system to exist as a liquid, a colloidal crystal or a disordered state depending on temperature, pH and concentration.24,57
For highly diluted IPN microgel suspensions the interaction between particles can be neglected and the particle size variation with temperature is the same of LCST around 32-34°C as pure PNIPAM microgels (Figure 1.13, left).56,58,90 However, the swelling capability of the IPN microgel is reduced with respect to the pure PNIPAM, due to presence within IPN microgel particles of the PAAc network that partially hinders the shrinking of PNIPAM skeleton.
Figure 1.13. On the left, temperature-induced volume phase transition for PNIPAM and IPN microgels. They both exhibit the same volume phase transition temperature. On the right,
pH-induced volume phase transition for PNIPAM and IPN microgels.90
Moreover, the additional pH sensitivity due to the PAAc leads to different response of the IPN microgels at different pH values (Figure 1.13, right). For instance, the diameter of the particles at pH < 5 is lower than at neutral pH because of the strong hydrophobicity of the IPN at acidic pH, as at this pH the acrylic acid is undissociated and tends to form hydrogen bonds with the PNIPAM chains rather than with water molecules, thus extruding water from its interior. This results in a decrease of the particle size. At pH > 5, the carboxylic groups of the PAAc chains become deprotonated, leading to a strong charge repulsion force between the chains in the network that limits the hydrogen-bonding capability of the microgel. Indeed, in the range of pH between 5 and 10 an almost constant size of the IPN particles is found.
By visual inspection, turbidity and viscosity measurements it has been shown57,90 that at low polymer concentrations the IPN dispersions undergo a transition from a translucent and easily flowing state, at temperature below the LCST, where the IPN microgels particles are fully swollen, to a shrunken state as temperature increases above the VPT. Nevertheless, also at low concentrations the formation of small aggregates without flocculation was observed, thus suggesting the presence of an attractive interparticle potential.
By increasing the microgel concentration57 the system evolves from a diffusive liquid to a subdiffusive one, and by further increasing concentration a transition to a face-centered cubic (FCC) crystal was observed also at temperature far
above the intrinsic LCST, in contrast to pure PNIPAM crystals. Indeed, at room temperature a colloidal crystal formed, which was indicated by an iridescent pattern due to Bragg diffraction. The results are due to a delicate balance between soft repulsive interactions and short-range weak attractions. In particular, the former interactions presumably arise from the solvation repulsion between solvated PNIPAM-PAAc coronas around the particles, from the compression and/or interpenetration of PNIPAM-PAAc coronas, and from the deformation of microgels upon close contact. Short-range attractive interactions instead have three sources: van der Waals attractions, hydrogen bonding between protonated carboxylic groups of PAAc at the surfaces of neighboring microgels and hydrophobic interactions between isopropyl groups of PNIPAM and/or the main chain of PNIPAM-PAAC coronas. Therefore, the result is that at high concentration, interactions are dominated by attractive short-range interactions, such as hydrophobic interactions and H-bonding.57,62 Under this concentration regime, as the pH is increased above 5, the crystal region of the phase diagram collapses and the dispersion undergoes a transition from an ergodic fluid to a glassy state. At further high particle concentrations the system exists in a glassy state with a drastic increase of the viscosity above the VPT.62
Notice that, the swelling behaviour of IPN microgels is highly influenced by the effective charge density, which can be experimentally controlled by the content of AAc monomers. However, the spatial distribution of the overall charge in IPN microgels is still matter of investigation. Even if many aspects of the IPN microgel behaviour have been clarified, a systematic investigation of its phase behaviour as a function of temperature, pH and concentration are still poorly understood and the IPN microgel intra-particle response to changes in the external parameters has not been deeply investigated, in particular at high temperatures and/or concentrations.
1.1.5.2. Synthesis
cross-linked at the same time using orthogonal chemistry.91 Another way to produce IPN systems is to use a sequential polymerization, where the second polymer network is prepared in the presence of the first one, which results in the formation of interpolymer complexes due to physical interaction, like hydrogen bonding.90,92,93 This last procedure, developed first by Xia and Hu90,92, is the one used to prepare IPN particles of PNIPAM and PAAc in this work: the second monomer, namely the acrylic acid, was polymerized in the presence of a preformed colloidal PNIPAM network.
During the reaction, the interaction through hydrogen bonds between the acrylic acid and the PNIPAM chains is supposed to be strong enough within each individual PNIPAM nanoparticle to keep the second monomer inside the particle and not in the aqueous environment outside them. The complexation is expected to be promoted by the high density of amide (CONH-) groups of PNIPAM into the particles. Under these circumstances, the polymerization of AAc would occur primarily within each single nanoparticle and, with further proceeding of the reaction, every single particle act as a skeleton for the polymerization of acrylic acid. However, with the growth of the second network it is possible that the acrylic acid will continue to polymerize even outside the particle, thus leading to the fast growing of the particle size. The nanoparticles therefore undergo a structural transition from a homogeneous network to IPN microgels, which will be characterized by a highly dense core of interpenetrated PNIPAM and PAAc networks, surrounded by a low-density shell mainly populated by PAAc chains. In the case that the hydrogen bonds aren’t strong enough to keep the acrylic acid inside the PNIPAM preformed network, it is possible that poly(acrylic acid) grows even outside the particles resulting in isolated PAAc chains.
However, even if different paper already reported the synthesis of IPN PNIPAM-PAAc microgels,85,86,90,92 a real understanding of the synthesis and a way to control the final composition and the structure of the particles is still lacking.
1.1.6. Applications of stimuli-responsive materials
Responsive polymer materials can adapt to surrounding environment, regulate transport of ions and molecules, change wettability and adhesion of different species on external stimuli, or convert chemical and biochemical signals into optical, electrical, thermal and mechanical signals, and vice versa. Thus, these materials are playing an increasingly important part in a diverse range of applications, such as drug delivery, diagnostics, tissue engineering and ‘smart’ optical systems, as well as biosensors, microelectromechanical systems, coatings and textiles.94
These materials can be constructed with various types of architecture depending on the type of application they are intended for, ranging from two-dimensional (films) to three-two-dimensional (particles) structures. However, all these systems exploit the swelling/deswelling of the polymer chains as a response of external stimuli.
For example, microgel dispersions, as 3D stimuli-responsive materials, are widely used for controlled drug delivery and release systems.95,96 Indeed, they have attracted great interest because of the broad opportunities for in vivo applications. Such nanosized particles in the swollen state could store a large quantity of various drugs and release them inside cells after the particle has been internalized and has collapsed in the shrunken state. A smart drug-delivery polymeric system undergoes a complex chain of responses to survive in vivo, deliver the cargo, release the drug into the target cells and match the desired kinetics of the release.94 Drug release can be activated on demand by local changes in temperature or pH or by remote physical stimuli, so microgels gain particular importance in anti-cancer and anti-inflammatory drug delivery, as cancer and inflammation are associated with heat generation, acidic pH and change in ionic content.97,98 It is prudent to stress that the optimal size for an efficient delivery to cells and internalization within them is considered to be in the range 25–100 nm.94
As it will be seen in this work, microgel particles could be used to create also a two-dimensional system, like a stimuli-responsive surface. These films have a
wide range of applications, such as in coatings99, biointerface and bioseparation100, in micro- and nano actuators101 and as sensors102.
One of the most interesting application is in tissue engineering. Tissue Engineering (TE) is an interdisciplinary new field, involving different subjects, aiming to design, build and develop in vitro living tissue models towards in vivo tissue substitutes (regenerative medicine). Tissue engineering involves the use of a tissue scaffold on which cells are seeded for the formation of new viable tissue for medical purpose. So, to develop an engineered tissue, it is crucial to study and to design scaffolds, as they must have some specific properties (physical, chemical and mechanical) that can emulate the extracellular matrix (ECM) and, among these, the elasticity of the materials is one of the most important, as it has a profound effect on cell spreading, morphology and function.
Cells adhere not only to solid substrates that range in stiffness but also they are sensitive to substrates that vary in topography and thickness. In literature, there are different attempts and techniques to design scaffolds with different topography, depending mostly on the type of cells.103 Among these materials, polymers provide a versatile platform for this purpose, mimicking various cues of extracellular matrix such as chemical composition, rigidity, and topography. Many are biocompatible and easily processable, allowing to create patterned surfaces that can promote cell growth and guide tissue regeneration in the same way of the ECM.
As mentioned earlier, the elastic modulus of a material plays an important role in regulating cellular behaviour as seen from directed differentiation of stem cells into various cell types in accordance to differential rigidity.104 In Figure 1.14, the elastic modulus of tissues in human body, as well as of various synthetic biomaterials used in cell and tissue engineering, are summarized. Human tissues have their own rigidity based on specific cell types and structural organization, ranging from few kPa to few tens of GPa, so also the scaffold used to construct similar microenvironment in vitro must have equivalent range in stiffness.105
Figure 1.14. Mechanical properties of natural tissues and synthetic polymers. (a) Range of the elastic modulus of various tissues in human body.(b) The same of various biocompatible
polymers used for in vitro studies with respect to patterning resolution and mechanical properties.105
In particular, PNIPAM stiffness ranges from about 10 to 100 kPa and can mimic the elasticity of a lot of natural tissues, such as brain, heart, kidney, vein and arteria. Moreover, PNIPAM stiffness can be easily tuned by changing the temperature: for example PNIPAM elastic modulus is 9.8 kPa at 25 °C and around 170 kPa at 40°C, where it is dehydrated.106
The thermo-responsivity of PNIPAM has attracted a huge interest in biomedical fields, as the LCST of this polymer is around 32°C that is in the physiological range of body temperature. Furthermore, in addition to the tunable stiffness, PNIPAM-based films above the LCST show a relatively hydrophobic surface while below the LCST, they are hydrophilic.107
