Layer-by-layer assembled microgels can combine conflicting properties: Switchable stiffness and wettability without affecting permeability

Responsive interfacial architectures of practical interest commonly require the combination of conflicting properties in terms of their demand upon material structure. Switchable stiffness, wettability and permeability, key features for tissue engineering applications, are in fact known to be exclusively interdependent. Here we present a nanoarchitectonic approach that decouples these divergent properties by the use thermo-responsive microgels as building blocks for the construction of three-dimensional arrays of interconnected pores. Layer-by-layer assembled poly(N-isopropylacrylamide-co-methacrylic acid) (PNIPAM-co-PMAA) microgel films were found to exhibit an increase in hydrophobicity, stiffness and adhesion properties upon switching the temperature from below to above the lower critical solution temperature, while the permeability of redox probes through the film remained unchanged. Our findings indicate that the switch in hydrophilicity and nanomechanical properties undergone by the microgels does not compromise the porosity of the film, thus allowing the free diffusion of redox probes through the polymer-free volume of the submicrometer pores. This novel approach for decoupling conflicting properties provides a strategic route for creating tailorable scaffolds with unforeseen functionalities.


Introduction
The possibility of creating interfacial architectures displaying conflicting and seemingly mutually exclusive properties to achieve multiple functionalities is one of the grand challenges for materials science in the 21st century. [1][2][3] One broad challenge is to combine switchable stiffness and wettability with high permeation properties from aqueous solutions. 4,5 The strong interest in combining this set of properties arises from their potential applications in biomedical research and, in particular, in tissue engineering as well as cell adhesion control and manipulation. 6,7 As is well known, the design and fabrication of tailorable scaffolds has been a key to the success of tissue engineering. [8][9][10] This notion is even more relevant if we consider that the integration of thermoresponsive units in the scaffold and/or platform facilitates remote control over cell-surface interactions by switching the environmental temperature. This concept was pioneered by Okano an co-workers 11 employing poly(Nisopropylacrylamide) (PNIPAM) as thermoresponsive units and led to the development of the so-called temperature-responsive cell culture surfaces. PNIPAM undergoes a solubility transition at the lower critical solution temperature (LCST) of 32 ° C. The PNIPAM surface has the extraordinary property that at 32 °C, the surface becomes hydrophobic allowing for cell attachment and proliferation. Then, upon decreasing temperature below the LCST, the surface becomes hydrophilic creating a hydration layer such that the cell sheet with deposited extracellular matrix detaches spontaneously. Since then, diverse groups exploited the temperature-dependent interactions between proteins and PNIPAM-coated surfaces to create substrates that reversibly capture proteins or cells. However, it is now widely accepted that these types of substrates should exhibit not only switchable wettability and stiffness to control cell adhesion, but they should also display high permeability in aqueous solution in order to maximize nutrient delivery and metabolite removal. This poses conflicting property requirements in multiple physico-chemical domains, i.e.: hydrophobicity and stiffness versus aqueous permeability.
In the case of PNIPAM-based polymer films, it has been demonstrated that the thermoactuation of the polymer chains strongly compromise the permeation properties. As already observed in other branches of materials science, 12 stiffness and permeability are competing materials properties. In 2005, Jaber and Schlenoff 13 studied the permeation properties of thermally responsive polyelectrolyte multilayers constituted of charged PNIPAM copolymers. They observed that the transport of an electrochemically active probe molecule, potassium ferricyanide, through the thin film was substantially suppressed as temperature increased above the LCST. The same group studied the correlation between permeation properties and stiffness in polyelectrolyte multilayers corroborating the conflicting nature of these two properties. They observed that an increase in modulus was unambiguously correlated with a decrease in the permeability to iodide ion. 14 The same correlation was also observed in PNIPAM brushes 15 and hydrogels 16 ; a thermo-induced increase in stiffness or hydrophobicity was associated with a concomitant decrease in permeability. In principle, the permeability and mechanical properties of water-swollen systems such as polyelectrolyte multilayers, polymer brushes or hydrogels are expected to be strongly correlated because softer, more hydrated materials also tend to be more permeable. These observations suggest that permeability properties in polymer thin films cannot be decoupled from their nanomechanical or even wetting properties. This has strong implications for consistent application of switchable, thermoresponsive polymer films in cell and tissue engineering. As recently noted by Volodkin and coworkers, 17 there is a strong need to design polymer films possessing reservoir and permeation capacity, and cell adhesive properties. Within this framework, the real challenge is to design polymer films with switchable nanomechanical, wetting and adhesion properties but keeping the reservoir and permeation properties of the films unaffected.
Most materials design problems of practical interest involve solutions with property/response sets that conflict in terms of their demand upon material structure at various scales. In recent years, computational modeling has demonstrated that microstructural composites represent a promising strategy to design and fabricate multifunctional materials combining conflicting properties. According to these computational studies, a multiphase microstructural configuration might facilitate the combination of properties from different building blocks without disrupting their own functions. 18 With this in mind, we drew upon a "nanoarchitectonic approach" [19][20][21][22] to develop switchable polymer films displaying conflicting properties, such as stiffness and permeability. The goal of our research was to integrate materials and length scales in a rational manner in order to decouple divergent functional requirements, thus far considered to be strongly correlated and exclusively interdependent functional attributes. To this end, we have exploited the submicrometer structure of microgels and the versatility of layer-by-layer assembly to build-up nanoarchitectured, opal-like thermoresponsive films that act as matrices containing arrays of three-dimensional interconnected pores. Layer-by-layer assembled poly(N-isopropylacrylamide-co-methacrylic acid) (PNIPAM-co-PMAA) microgel films exhibited an increase in hydrophobicity, stiffness and adhesion properties upon switching the temperature from below to above the LCST, but the permeability of redox probes through the film remained unaffected.
Our results indicate that due the nanoarchitectured nature of the film, the switch in hydrophilicity and nanomechanical properties undergone by the microgels does not compromise the porosity of the film, thus permitting the free diffusion of redox probes through the polymer-free volume of the submicrometer pores. To the best of our knowledge, this is the first attempt to rationally combine conflicting properties in thin polymer films using a layer-by-layer assembly approach. We envision that our approach for decoupling divergent functional properties using hierarchically organized films would provide a facile route from creating interfacial architectures with unforeseen functional combinations.

Results and Discussion
The use of microgels as building blocks to construct polymeric films via layer-by-layer assembly was first reported by Lyon and co-workers. 23 Since then, extensive research was conducted on the construction of responsive microgel films. [24][25][26][27][28][29] One of the most appealing aspects of using thermoresponsive PNIPAM-co-PMAA microgels as polyanions in layer-by-layer assembled microgel films relies on the fact that we can create interfacial architectures with hierarchical microscale-tonanoscale structures that can actuate and change their properties upon varying the temperature. In the case of PNIPAM-co-PMAA microgels, they exhibit a well-defined LCST at 32 o C where the system undergoes a volume phase transition to a collapsed state with a concomitant release of solvent (water) from the interior of the microgel. The layer-by-layer construction of the hierarchically organized microgel films was accomplished following the protocol described by Decher and co-workers 30,31 using PNIPAM-co-PMAA microgels and poly(diallyldimethylammonium chloride) (PDADMAC) as polyanions and polycations, respectively.
The build-up of PNIPAM-co-PMAA microgel/PDADMAC multilayer films ( fig. 1) was monitored by microgravimetric measurements using a quartz crystal microbalance (QCM). Assembly of films of anionic PNIPAM-co-PMAA microgel and cationic PDADMAC was done by 15 min alternate adsorption from their respective solutions at pH 6.5 (at room temperature). QCM monitoring of dry films showed a monotonic increase in frequency upon increasing the number of adsorption steps. The frequency shift is proportional to the mass of each adsorbed layer and indicates an increase of film mass on the QCM sensor with increasing number of deposition cycles (see Figure S1).  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57 58 59 Figure 1. Schematic of the LbL deposition process using PDADMAC and PNIPAM-co-PMAA microgels as polycations and polyanions, respectively. PNIPAM-co-PMAA microgel samples were characterized by TEM imaging (scale bar: 100 nm), and films were characterized by in-situ AFM imaging (height image 2 x 2 µm 2 and phase image 1 x 1 µm 2 , color represents a total range of 100 nm in the height image). Note that the interfacial architecture depicted in the figure corresponds to a simplified representation of the film structure, in which anionic microgels are surrounded by polycation chains. However, partial diffusion of the polyelectrolytes into the microgels is also a plausible configuration that should be taken into account.
Once assembled, films were characterized by in-situ atomic force microscopy. Figure 1 shows an AFM image of a (PNIPAM-co-PMAA/PDADMAC) 9 /PNIPAM-co-PMAA multilayered film. It can be observed that the layer-by-layer assembly process led to a film exhibiting close-packed microgel on the outermost layer.
Previous studies reported by von Klitzing and coworkers suggest that, in principle, microgels containing charged monomers cannot lead to the formation of close-packed films due to strong interparticle microgel-microgel repulsion. 29,32 However, the same authors conclude that there are two electrostatic contributions that control the formation of the microgel monolayer: (i) electrostatic repulsion between charged microgels, and (ii) attractive electrostatic interactions between the substrate and the microgels. According to their results, when pH conditions range between 6 and 10, there is a trade-off between attractive and repulsive interactions. From the results, we can infer that the assembly of the microgel on the PDACMAC layer rather than microgel-microgel repulsion governs the formation of each layer. This could be ascribed to the presence of salt in the assembly solution that ultimately screens the repulsive electrostatic interactions between charged microgels.
Once confirmed the structure of the film at room temperature (below LCST) we proceeded to study the morphological changes taking place in the sample upon increasing temperature above the LCST. We should note that, thus far, in-situ AFM imaging of morphological transformations as well as changes in adhesion and nanomechanical properties of LbL-assembled microgel multilayers in response to temperature have not been reported in the literature.  We should bear in mind that precise topographic measurement of the outermost microgel layer is not trivial due to the softness of the imaged sample. 33 However, we were able to reliably image the surface by using the high-frequency force tapping mode (Bruker's Quantitative Nanomechanics, QNM), for which the AFM tip briefly interacts with the sample surface thus minimizing the possible deformation of the microgels during the imaging process. 34 Figure 2 depicts the morphology of the (PNIPAM-co-PMAA/PDADMAC) 9 /PNIPAM-co-PMAA multilayered film as obtained from in-situ AFM characterization performed at 25 and 40 o C. It is observed that no significant morphological changes take place in the sample upon increasing temperature above LCST. Indeed, by evaluating AFM height profiles of individual microgel particles at the outermost layer of the film ( Figure 2C), we obtained mean FWHM diameters of (390 ± 50) nm and (370 ± 40) nm for 25 and 40 °C respectively. The absence of major morphological changes across the LCST also implies that the film does not undergo significant changes in the dimensions of the submicron interparticle cavities (as observed in the AFM images). Or, in other words, the temperature increase does not significantly affect the volume of the macroporous space generated by the three-dimensional interconnected cavities. Dynamic light scattering measurements of these microgels dispersed in solution confirmed that the hydrodynamic diameter varies from 1400 to 200 nm in the same conditions in which they were LbL-assembled, i.e.: microgels in solution deswell by a factor of 1/7 in diameter. 35 Differences observed in deswelling between microgels dispersed in solution and integrated in LbL assemblies are likely attributable to the interaction with the underlying polyelectrolyte layer. For instance, Höfl et al. 28 observed that poly(Nisopropylacrylamide)-co-vinylacetic acid microgel particles adsorbed onto a solid substrate display a similar volume phase transition as their dispered counterparts. However, surprisingly, their swelling  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 capacity was reduced by one order of magnitude compared to value of microgels in solution. Similarly, Schmidt et al. 36 reported that negatively charged PNIPAM microgels adsorbed on positively charged polyethyleneimine layers exhibit a reduced swelling/deswelling capacity.
From these results, we infer that the microgel integration and confinement into the multilayer assembly -a scenario in which these building blocks are surrounded by polyelectrolyte layers -exerts a marked influence on the shape and dimensional actuation of the microgels, which in turn defines not only the texture but also the hierarchical micro/nanotopography of the interfacial architecture.
Then, we proceeded to study the influence of the thermal conditions on the nanomechanical and adhesion properties of the LbL-assembled microgel multilayers. Probing of the surface mechanical properties with nanometer-scale resolution was accomplished via atomic force microscopy characterization. Despite some technical issues associated with this AFM probing (e.g.: nanoscale contact area or high local pressure), a number of successful applications of this experimental approach have been demonstrated to date. [37][38][39][40][41][42] Figure 3A depicts typical AFM force-distance curves (FDC) performed on the microgel multilayer under different temperature conditions, i.e.: below and above LCST. We defined an upper force threshold of 2 nN, which led to deformations on the order of 100 nm. High agreement on subsequent measurements on the same spot indicated that the surface was not plastically deformed during nanomechanical characterization. Visual inspection of the FDC showed a clear difference on the mechanical response of the film for temperatures below and above the LCST. Conversion of the FDCs into load penetration curves further confirmed that a more compliant surface is associated with the 25 °C state, as indentation is almost twice as deep under a normal load of 1nN ( Figure 3B).  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59 Elastic response of AFM force-distance data was analyzed by the Sneddon's contact mechanics model, assuming a parabolic tip and a plane surface contact. Knowledge of the force applied to the indenter as a function of the penetration during the loading experiment allows for the calculation of Young's modulus E using a numerical form of the Sneddon's equation for AFM data, which has been successfully applied in previous reports 39,43 (further technical details are described in the SI). An analysis of these data indicated a 6-fold increase of the modulus when increasing the temperature from 25 to 40 °C (75 ± 41 kPa and 450 ± 210 kPa, respectively). The variation in modulus observed in microgel multilayer is comparable to a previous study on thermoreponsive microgel monolayers. 36 This variation on nanomechanical properties is fully reversible (see Figs. S2 and S3 in the SI file). Furthermore, analyzing force curves from a linear profile along the section of a microgel within the opal-like structure, we observed that the elastic modulus increases at positions closer to the apex (see Figure 4 A-D). A similar observation was previously reported by Schmidt et al. in a set of experiments performed on microgel monolayers 36 . These results could be explained considering that in many cases the polymerization of PNIPAM microgels results in the formation of a highly crosslinked core and a shell with lower density. In our case, we have corroborated the existence of this structural feature in our PNIPAM-co-PMAA microgel samples by TEM imaging (see Fig. 1).  Langmuir   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 section (blue and red lines in A and B respectively). (E-H) 2 x 2 µm 2 adhesion force maps of the imaged regions, and adhesion force cross-sections along the red and blue lines.
In a similar vein, we explored the adhesion response of the microgel multilayer by AFM nanomechanical mapping. QNM adhesion force maps at 25 and 40 o C showed a significant adhesion increase above the LCST, particularly on microgels surfaces, which was evidenced by the adhesion images contrast as well as in their cross-sections (Figure 4 E-H).
In order to avoid possible artifacts from the fast rate nanomechanical mapping scans, we proceed to quantify the adhesion from single FDC taken at random locations of the multilayer surface. We evaluated the work required to pull of the AFM probe from the surface as a measure of the adhesion energy. Work of adhesion can be read from the AFM retraction curves as the area enclosed by the force curve and the x-axis (Figure 5 A). We observed a gradual increase in the mean work of adhesion from (0.53 ± 0.15) × 10 -16 J to (1.33 ± 0.25) × 10 -16 J when temperature was increased from 25 °C to 40 o C respectively ( Figure 5B). This behavior, observed in previous studies on PNIPAM microgel monolayers, 44,45 is in close resemblance to the adhesion behavior of thermoresponsive polymer brushes [46][47][48] and can be ascribed to the fact that above the LCST the solvent is depleted from the macromolecular environment and the polymer chains/segments collapse. This process leads to large adhesion forces due to van der Waals interactions between the polymer layer and the AFM tip. On the other hand, below the LCST the microgel is swollen and the microgel-tip interaction is mainly governed by repulsive osmotic forces which in turn lead to a decrease in adhesion forces.
In addition, and as expected, the switching in the adhesion properties of the (PNIPAM-co-PMAA/PDADMAC) 9 /PNIPAM-co-PMAA assembly upon increasing temperature from 25 to 40 o C was accompanied by an increase in the contact angle of the microgel assembly from 71 to 95 o ( Figure  5 C-D), i.e.: an increase in hydrophobicity. Note that this wettability switch is fully reversible and multilayer assemblies exhibit an increase in hydrophilicity upon decreasing the working temperature below LCST (see Fig. S4 in the SI file). So far, these results are similar to those previously reported in the literature using PNIPAM-based films. Next, we studied the transport properties of the multilayered microgel films under different temperature conditions. These studies were carried out using redox probes, Fe(CN) 6 4-/3-, that diffuse across the microgel multilayer grown on a conductive gold substrate. Electrochemical experiments were performed in 0.1 M KCl as supporting electrolyte. We should note that this saline concentration did not affect the structural integrity of the self-assembled film, as corroborated by AFM characterization (see Fig. S5 in the SI file). Figure 6 describes the voltammograms of (PNIPAM-co-PMAA/PDADMAC) 9 /PNIPAM-co-PMAA multilayers in contact with electrolyte solutions containing Fe(CN) 6 4-/3-. We observed that -in stark contrast with the vast literature on PNIPAM films 14-16 -a temperature increase above the LCST does not promote changes in the permeation of the electroactive Fe(CN) 6 4-/3ions across the nanoarchitectured film. The electrochemical characterization conclusively reveals that the magnitude of the voltammetric signal, which reflects the transport of Fe(CN) 6 4-/3ions towards the underlying conductive support, is not affected by the switching from below to above LCST. Interestingly, the only electrochemical feature that changes upon increasing temperature is the redox potential of Fe(CN) 6 4-/3ions which displays a marked cathodic shift upon increasing temperature above LCST. This can be explained by considering previous work by Anson and co-workers. 49 These authors demonstrated that the interaction of Fe(CN) 6 4-/3with polyelectrolytes bearing quaternary amines promote a strong cathodic shift in the redox potential of the electroactive ions. As a consequence, we hypothesize that upon increasing temperature above the LCST, the collapse of the microgel domains within the multilayer leads to the exposure of PDACMAC layers to the incoming Fe(CN) 6 4-/3ions, this being reflected by the shift in the redox potential.   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 Taken all together, the set of experiments can be interpreted considering that the opal-like microgel multilayer act as thermoresponsive matrix containing arrays of three-dimensional interconnected pores. Upon switching the temperature from below to above the LCST, films exhibit an increase in hydrophobicity, stiffness and adhesion properties. However, due the hierarchically organized, nanoarchitectured nature of the film, the switch in hydrophilicity and nanomechanical properties undergone by the self-assembled microgels does not compromise the porosity of the film, thus facilitating the free diffusion of redox probes through the polymer-free volume of the submicrometer pores generated in the inter-microgel cavities.
To the best of our knowledge, this is the first experimental report on layer-by-layer assembled films combining conflicting properties, such as stiffness and permeability. Our experimental results demonstrate that by drawing upon a "nanoarchitectonic approach" it is possible to decouple strongly correlated properties in order to create platforms that can switch stiffness, adhesion and wettability without affecting their high permeability properties.

Conclusions
We have demonstrated that by integrating organized microgel domains in polyelectrolyte multilayers we were able to decouple divergent, but strongly correlated functional properties in thin film architectures. In particular, we have shown that the layer-by-layer construction of nanoarchitectured, opal-like thermoresponsive films offer an interesting interfacial design in which the archetypical switching in hydrophobicity, stiffness and adhesion properties displayed by PNIPAM-based films can be implemented without affecting or disrupting the high permeability of the nanostructured thermoresponsive layer. We interpreted the results and concluded that the unique properties ascribed to these films arise from the hierarchical structure of the microgel multilayer that facilitates the coexistence of conflicting events and mechanisms operating at macroscopic, nanoscopic and/or molecular scales. In other words, the nanoarchitectured nature of the film displaying spatiallyaddressed 3D domains is the key to manipulate functions that are predominant at different structural dimensions, and consequently to harmonize conflicting properties. We consider that this conceptual framework based on "interfacial nanoarchitectonics" opens a new dimension: the possibility to create phase separated regions (functional domains) and concomitantly controlling their collective properties in order to decouple conflicting functions operating at different length scales.
This work offers a general concept that can be extended to other conflicting functions, thus opening new pathways to the fabrication of ultrathin multilayers displaying advanced concerted functions for potential use in a range of applications. The latter is particularly relevant if we consider that the use of microgels in LbL assembly retains the inherent simplicity of traditional LbL assembly, yet extends the range of nano-and mesoscale structural control that can be imparted to the interfacial architecture.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60