Three-Dimensional Microstructured Azobenzene-Containing Gelatin

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3D Microstructured Azobenzene-Containing Gelatin as Photoactuable Cell Confining System Fabrizio Andrea Pennacchio, Chiara Fedele, Selene De Martino, Silvia Cavalli, Raffaele Vecchione, and Paolo A Netti ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13176 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 25, 2017

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ACS Applied Materials & Interfaces

3D Microstructured Azobenzene-Containing Gelatin as Photoactuable Cell Confining System Fabrizio A. Pennacchio,a,b,‡ Chiara Fedele,a,b,‡,† Selene De Martino,a,b Silvia Cavalli,a* Raffaele Vecchione,a,b* Paolo A. Nettia,b* a Center for Advanced Biomaterials for Healthcare, IIT@CRIB, Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci, 53 80125Napoli, Italy b Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industriale, DICMAPI, Università degli Studi di Napoli Federico II, Piazzale Tecchio, 80 80125 Napoli, Italy. KEYWORDS. Azobenzene, Photoactuation, Two-Photon Lithography, Hydrogel, Cell Confinement

ABSTRACT. In materials science there is a considerable interest in the fabrication of highly engineered biomaterials that can interact with cells and control their shape. In particular, from the literature the role played by physical cell confinement in cellular structural organization and thus in the regulation of its functions has been well established. In this context, the addition of a dynamic feature to physically confining platforms aiming at reproducing in vitro the well-known dynamic interaction between cells and their microenvironment would be highly desirable. To this aim, we have developed an advanced gelatin based hydrogel able to be finely micro-patterned by two-photon polymerization (2PP) and stimulated in a controlled way by light irradiation thanks

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to the presence of an azobenzene crosslinker. Light-triggered expansion of gelatin microstructures induced an in-plane nuclear deformation of physically confined NIH-3T3 cells. The microfabricated photoactuable gelatin shown in this work paves the way to new “dynamic” caging culture systems that can find application, for example, as “engineered stem-cell niches”.

Introduction Engineered biomaterials able to confine cells represent a relevant tool to control cell functions and guide their behavior in a precise way. In particular, the scientific literature reports examples of cell caging platforms where the physical confinement is used, for instance, to regulate the embryonic stem cell spherogenenesis or to get a deeper comprehension over the mechanisms involved in cellular mechanotransduction pathways.1-2 However, up to now, the use of biomaterials whose properties cannot be changed in time has been mostly explored providing just “static” interactions between cells and the material itself, thus limiting the possibility to reproduce in vitro the well-known dynamic interaction between cells and their microenvironment.3 Therefore, it would be highly desirable to develop engineered “smart” materials able to generate in vitro spatio-temporal complex cell signaling events and regulate “on demand” cell-material dynamic interplay.4-5 In the literature there are examples of stimuli-responsive materials able to change their properties in response to several external stimuli, such as pH, temperature,6 electrical and magnetic fields,7-8 enzymes9 and light10-11 and offer the possibility to introduce a temporal control over cell signaling. In particular, among all these stimuli, light represents the ideal source for biological investigations, since it can be precisely localized over a substrate, it is finely spatio-temporally tunable, and remotely addressable, without altering the environmental cell culture conditions.12-14 For these reasons, there is accumulating evidence that in the near future light-responsive materials, in particular


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those containing azobenezenes, will drive forward the research in the biological field.15 For example, azopolymer thin film photopatterning has been recently used for controlling in realtime cell behavior,16 while azobenzene-containing poly(ethylene-glycol)-based hydrogels, whose elastic modulus was affected by azobenzene photoinduced isomerization, were found biocompatible and promising for valvular interstitial cell differentiation into myofibroblasts.17 Recently, photoresponsive liquid crystalline elastomers have been elegantly exploited as dynamic cell culture supports.18-20 In this scenario, to the best of our knowledge, no examples of light-responsive materials used for the dynamic stimulation of physically confined cells have been reported so far. Motivated by all these reasons we have modified and fabricated gelatin in order to produce a novel light-responsive photoactuable hydrogel platform, designed to confine and deliver a local mechanical stimulation at a single cell level. Here, cell positioning was obtained microstructuring the material as an array of interconnected microchannels and combining this topographical guidance with the material adhesive properties. Gelatin is an attractive candidate as starting material for preparing hydrogels for cell culture as it presents important biochemical cues of the extracellular matrix (ECM), enhancing cell-material interaction. However, the use of native gelatin in physiological condition is limited by a reversible sol-gel transition at 37 °C, a drawback that we overpassed by modifying the material with acrylic groups, which allowed photopolymerization by means of a 2PP process.21-22 Additionally, adding an azobenzene crosslinker molecule in the mixture, besides increasing microscale-patterning resolution, allowed the custom made gelatin photoresist to perform as a photodeformable gelatin-based microstructured platform. Indeed, once irradiated with an adequate laser source, these structures underwent a deformation able to stimulate living cells. To demonstrate the feasibility of our


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approach, we used immortalized fibroblasts (NIH-3T3 cells) and we assessed aspects of biocompatibility of the substrate, including cell viability/cytotoxicity, cell adhesion and morphological compatibility. Photostimulation by means of the multiphoton laser of a confocal microscope allowed a real-time observation of cell response to the biomechanical cue in a cellfriendly environment. Results and Discussion Gelatin fabrication and photostimulation Gelatin is a natural hydrogel extensively used in biological applications and obtained from the partial hydrolysis and denaturation of collagen. As such, it is inherently biocompatible, biodegradable and promotes cell adhesion through collagen motifs.23 Conversely, the fabrication of well-defined gelatin microstructures that resemble the extracellular microenvironment structural features results challenging and, as a consequence, the application of gelatin in the production of advanced cell instructive platforms are limited.24-25 In this work, the combination of the cell-adhesive characteristics of gelatin with lightresponsiveness in precisely fabricated structures is proposed. More in details, through the design of an acrylamide-modified gelatin containing azobenzene-based crosslinkers, a smart photoactuable platform aimed at stimulating cells on demand was fabricated by means of a 3D lithography system. When illuminated with a specific wavelength, the azobenzene molecule undergoes an isomerization from the more stable trans isomer to the less stable cis isomer, the former having a near planar while the latter a bent conformation. From a materials science perspective, this isomerization leads to interesting photomechanical effects when azobenzenes are incorporated into polymer networks, such as crosslinked hydrogel matrices.26 The light-induced isomerization


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can change different material properties such as the mesh size and/or its swelling behavior and, in specific conditions, it can trigger also notable shape deformations.10 Our photoresist mixture was formulated including Irgacure 369 as photoinitiator (PI), acrylamide-modified gelatin, and, finally, an azobenzene-based bisacrylamide crosslinker (Scheme S1 in the Supporting Information). The presence of the azobenzene moiety gave the possibility to deform the polymerized and microstructured material with the use of light. The synthesized molecule was characterized in solution by means of UV-vis spectroscopy, showing a reversible isomerization behavior under specific illumination conditions (See Figure S3 in the Supporting Information). The photodeformation process of gelatin microstructures was studied employing the multiphoton laser of a confocal microscope. Gelatin parallelepipeds (30 x 30 x 10 µm3) were fabricated with the Nanoscribe 2PP system on a glass substrate and used as simple test structure to study the photomechanical response of the microstructured material. The structures were designed in order to be on a dimensional scale interesting for the localization and manipulation of single cells. Additionally, gelatin blocks were used as simple structures for the investigation of the photomechanical effect in order to work as modular elements for cell stimulation. The employment of the microscope laser was not only a finely tunable stimulus for the photoactuation, but also represented a powerful tool to observe, record and evaluate in real-time the material microdeformations with spatio-temporal accuracy, even in the presence of living cells. Furthermore, with the use of the multiphoton laser, we were able to precisely localize the UV absorption in order to set the experimental conditions for the biological studies. In fact, since UV light is known to be harmful for cells, this method minimized cytotoxic effects, while maximizing the light penetration depth. Exposing the structures to a 700 nm laser (voxel


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wavelength 350 nm) at 13 ± 0.5 mW output power and at a fixed z position for 10 minutes, an inplane expansion of the structures of about 10% was registered, as shown in Figure 1. All the stimulation experiments were conducted under water immersion of the samples. Using a laser power higher than 15 ± 0.5 mW the material was instead damaged.

Figure 1. Deformation of the gelatin structures upon MP illumination at 700 nm for 10 minutes. A) Before illumination and B) after illumination. As shown in figure, aided by the grid, the illuminated structure increased its x and y dimensions (10 %). Scale bars are 10 µm. As a negative control the same structure was exposed to a laser tuned at 780 nm (voxel wavelength 390 nm), a wavelength at which the azobenzene molecule presents an adsorption valley (see Figure S3 in the Supporting Information) and does not isomerize. In these conditions (780 nm and 13.5 ± 0.5 mW) no shape change was observed, confirming that the deformation was related to the azobenzene isomerization (Figure S4 in the Supporting Information). In order to characterize possible photostimulation-related variations in the mechanical properties of the material, we measured the Young modulus of the microstructures before and immediately after the photoactuation through an AFM force spectroscopy analysis. From the collected data, a decrease of the Young modulus from 6.5 to 3.89 kPa was observed (Figure S5 in the Supporting Information). All the works present in the literature reporting hydrogel azobenzene-based photoactuation describe a variation of the material mechanical properties.


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However, each study observed different material behaviors upon photostimulation probably due to differences in molecular architecture of each material, as, for example, shrinking accompanied by an increase in the storage modulus in an azobenzene-containing poly(amide acid)27 or a decrease in elastic modulus in branched PEG-azobenzene.17 In hydrogel matrices, it is worth noticing that material stiffness plays a fundamental role in mediating cell behavior and function.28 Local variations in hydrogel stiffness may cause, for example, a preferential cell migration on hydrogel substrates. Therefore, the capability of our smart gelatin-based material to change its mechanical properties upon light exposure could be further exploited for the development of stiffness-tunable platforms, usable to precisely control cell fate and study cell behavior in response to stiffness-related dynamic mechanical stimuli.11, 29 Besides conferring photoactuability to the gelatin, the azobenzene moiety was opportunely designed to act also as a crosslinker participating to the material structuration and compatible with the working specifications of our 3D lithography system.30 Presenting in fact an adsorption valley at 390 nm, the two-photon wavelength at which the Nanoscribe works, this molecule does not interfere with the adsorbed radiation and the consequent excitation of the photoinitiator, a primary condition for the material 2PP fabrication process.30 As it is evident from the images (Figure 2), the azo-crosslinker improved the CAD reproducibility and the structural stability of the fabricated microstructures, two critical issues in the photopatterning of gelatin by means of DLW-2PP technique. This result considerably improved the material potentialities in the reproduction of ECM microfeatures and gives then the possibility to evaluate their effect over the cellular behavior down to the single cell level.25


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Figure 2. Bright field images of micrometric structures obtained with the same writing parameters A) with azo-crosslinker in the photoresist mixture and B) without it. Scale bars are 10 µm. Biological investigation For our biological investigations, an established cell line such as immortalized NIH-3T3 fibroblasts has been used, since it is a well-recognized approach to gain general information on cellular behavior.16,

18, 31

Combining the ability to microstructure and photoactuate our gelatin

with its cell adhesive properties, we have built up a remotely controllable platform for the mechanical stimulation of cells. In our strategy, cells resulted physically confined between adjacent gelatin blocks, whose photoexpansion compressed them at the nuclear site (Figure 3A). Here, thanks to the adhesive motives of gelatin we did not need to selectively functionalize the microstructured platform to obtain cell confinement as done in a previous work.32


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Figure 3. A) Graphical representation of the gelatin platform. B) CAD structures of the entire platform. C) Z-stack maximum intensity projection of a fixed cell cultured on gelatin structures (nuclei were stained in blue with HOECHST, while the cytoskeleton is colored in red with rhodamin-phalloidin). Scale bar is 10 µm. D) Lower magnification image of cells confined between gelatin microchannels. Scale bar is 30 µm. The distance between structures was chosen following preliminary results (data not shown) showing that the 10 µm channel width was the best distance for cell confinement. Moreover, the well-known phenomenon of contact guidance was exploited, for which nano- or micropatterns together with cell adhesive motives influence cell orientation and morphology.33-37 More in details, a network of interconnected 10 µm wide channels was produced with the Nanoscribe system, fabricating an array of acrylate gelatin parallelepipeds (30 x 30 x 10 µm3) directly attached to a glass substrate. Here, taking advantage of the unique 3D fabrication ability of a 2 photon polymerization equipment such as the Nanoscribe system, we included a linear topography (3 µm pitch) on the lateral surface of each gelatin parallelepiped (Figure 3A-B). Introducing such a pattern on the channel walls, an enhancement of the cell confinement was


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observed (Figure S6 in the Supporting Information). This result is in line with literature data that have highlighted the capability of micrometric linear topographies to align fibroblasts.38 Confined cells showed a mature cytoskeletal organization (Figure 3 C-D), an essential condition for stress transmission to the nucleus, activating molecular pathways that regulate important cell functions such as gene expression, migration, proliferation, and differentiation.39-41 Material biocompatibility was tested with a live/dead assay that showed no cytotoxic effects after 24 h (see Figure S7 in the Supporting Information). Moreover, from other preliminary results we observed that the material did not compromise cellular viability even after longer periods, i.e. up to 25 days of cell culture (data not shown), thus showing the potential use of this platform also for investigations requiring longer culture timing. Once confined, living cells were mechanically and selectively stimulated by photoactuating their surrounding structures. Laser scanning was precisely restricted using the region-of-interest (ROI) editor of the microscope software, thus selecting the structures to be stimulated and avoiding cell exposure to UV radiation. To observe the cell body and nucleus during the stimulation, cells were stained with, respectively, the vital Cell Tracker Deep Red and HOECHST. The photoinduced expansion of the structures provoked the deformation of the nuclear area (Figure 4), a factor that could deeply influence cellular behavior and function. After stimulation, living cells were followed every hour, up to 4 hours, to evaluate possible cellnoxious effects eventually related to the stimulation process. Indeed, from the collected data (Figure S8, Supporting Information), we did not observe cytotoxic effects related to the UV light exposure in the two-photon excitation event.


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Figure 4. MP images of NIH-3T3 living cells. A and C) before light irradiation. B and D) after 10 minutes of MP stimulation. Cells are stained with the vital CellTracker Deep Red for the cell body, while HOECHST is used for nucleus staining. Scale bars are 10 µm. Comparing cell nuclei before and after stimulation (Figure 5A), a decrease of the nuclear area was observed and calculated as described in the Experimental Section. From our investigation, a range of deformations between 3 and 20% was registered.


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Figure 5. A) Cell nuclei before, immediately after light stimulation and an overlay of the two images. Cell nuclei are stained with HOECHST. B) Correlation between the nucleus polarization and its area variation (12 cells). The variability of these results was related to the nuclear polarization before the stimulation. The variation in the nuclear area was quantified by comparing the fluorescent signal of the nucleus before and after the stimulation and the initial polarization degree of the nucleus was calculated as the ratio between its two main axes (Imax/Imin). By fitting experimental points with a power law function a correlation between nuclear area contraction and Imax/Imin was evidenced, as shown in Figure 5B. Our results match with the data presented in the literature, which reported, in fact, that a higher polarization of the nucleus is associated to a lower cellular compliance.42 Potentially, the capability to finely modulate the nucleus deformation controlling its polarization degree can give the possibility to study with high accuracy cell response in a broad range of conditions. To this end, next developments will include the obtainment of a higher control on the nucleus polarization, therefore increasing the reproducibility of the results. Chemical, mechanical and topographical properties of the cell microenvironment are able to determine cell shape and, thus, its fate.43 Consequently, the ability to locally trigger a cellular mechanotrandsuctive pathway through nuclear deformation is fundamental to understand the role of molecular forces in the modulation of cell functions. In the call for new experimental platforms that allow the smart manipulation of force-responsive structures in cell-friendly conditions, engineered hydrogels, such as our gelatin photoresist, emerge as an extremely advantageous tool for the realistic evaluation of cellular behavior. In this context, the use of azobenzene-based polymers as smart cell-instructive platform designed to dynamically interact with cells has allowed in the last years to expand scientific knowledge about mechanisms behind


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the regulation of cell functions.16-18,

44

However, following this strategy, despite notable steps

done towards the comprehension of the cellular dynamic behavior, up to now the majority of the conducted studies have been based on the employment of 2D interfaces, which, as well-known, do not faithfully replicate the physiological environment. To overcome this limitation, in our proposed approach, we combined the advantages of the azobenzene-based materials with those related to the use of cell-instructive hydrogel microstructures. More in details, by means of a 3D lithography system we have given a first example of a microstructured photoactuable hydrogel platform that could represent a valuable tool and a starting point for an evaluation of cell reactions to dynamic modulation of cell shape in a more realistic environment. This approach could be appealing, for instance, for closely mimicking the stem cell niche, which orchestrates stem cell phenotype, proliferation, and differentiation through the modulation of key elements of ECM such as composition and architecture.45-46 This strategy would aim in perspective at designing sophisticated 3D engineered stem cell niche, whose properties could be tuned through an external light trigger on demand, regulating interplay between cells and their associated ECM and analyze this crosstalk effect on stem cell self-renewal, differentiation, quiescence and apoptosis.47 For this reason, we are currently investigating the behavior of human mesenchymal stem cell (hMSC) upon in situ photostimulation of the proposed hydrogel-based platform. Therefore, we foresee the use of such photoactuating systems as a novel paradigm in stem cell research. Conclusion In this work, we have exploited the photoactuation of azobenzene-containing hydrogel microstructures opportunely designed to alter on demand the shape of NIH-3T3 fibroblasts, a cell line commonly used as model for mechanotransduction studies. To this end, we have added an


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azobenzene molecule as crosslinker to a photopolymerizable gelatin that was structured by means of 2PP direct laser writing technique. Here, gelatin was the material of choice thanks to its strong and well-known capability to promote cell adhesion. Our gelatin-based photoresist was found highly efficient for 2PP lithography. The fabrication results highlighted the role played by the azobenzene crosslinker that has substantially improved the CAD reproducibility and the structural stability of the material on the micrometric scale, two main issues in gelatin fabrication. Moreover, the results shown in this work demonstrated the possibility to photoactuate for the first time gelatin to be used as “dynamic” microstructured hydrogel for cell culture. Thus, combining the light-sensitivity of the material with the possibility to microfabricate it, we have built up an array of interconnected microchannels for the confinement and mechanical stimulation of living cells upon material expansion as consequence of the illumination. Choosing the multiphoton laser of a confocal microscope as light source, we were able to selectively deform the single cell with high spatio-temporal accuracy in a biocompatible environment. Our results evidenced that the photostimulation was efficiently transferred from the structures to the cell nuclei, provoking a decrease of the cell nuclear area. Moreover, from preliminary AFM nanoindentation analysis, the material showed a variation in stiffness in response to the light stimulus. Taken altogether, these findings highlighted the applicability of our proposed platform as versatile, smart hydrogel micro-scaffold for studying and guiding the cell mechanotransduction process in real-time. Overall, our results encourage the microfabrication of more complex 3D light-responsive hydrogel-based structures for the development of “dynamic” engineered platforms, going from static to dynamic intelligent materials for cell culture applications. Experimental Section


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Gelatin photoresist preparation: Acrylamide-modified gelatin B (20 % w/v) (for its synthesis and characterization see the Supporting Information, Figure S1)22 was dissolved in a citrate buffer (pH 3.1) at 40 °C overnight by gently stirring. When the solution became clear, 4 wt% of azo-crosslinker (for its synthesis and characterization see the Supporting Information, Scheme S1 and Figure S2)48-49 and 3 wt% of Irgacure 369 were added. Direct laser writing two-photon polymerization (DLW-2PP) process: DLW-2PP was performed on a Nanoscribe Photonic Professional GT system (Nanoscribe GmbH). This system uses a 780 nm Ti-Sapphire laser emitting ≈100 fs pulses at 80 MHz with a maximum power of 150 mW and is equipped with a 63x, 1.4 NA oil immersion objective. The substrate was placed in a holder that fits into a piezoelectric x/y/z stage. A galvo scanner determines the laser trajectories. Gelatin photoresist was first heated at 40 °C and then poured on a circular glass coverslip (30 mm diameter, 0.17 mm thickness) previously washed with 2-propanol and dried with nitrogen. To minimize solvent evaporation from the photoresist, gelatin was dropped in a closable homemade poly(dimethylsiloxane) (PDMS) reservoir carefully placed on the glass surface. Gelatin was fabricated at room temperature (solid state) with an output power of 24 mW and writing-speed of 7500 µm/s. To minimize the optical aberrations related to the already polymerized gelatin, structures were written in a “top-down” sequence (the first layer was the farthest from the substrate). Thanks to the solid state of the resist during the fabrication, this writing sequence could be used without recurring to supporting structures. After exposure, gelatin was developed in water at 45 °C for 20 minutes. After development, the sample was stored in water at room temperature to prevent solvent evaporation. Cell culture and staining: NIH-3T3 fibroblasts (ATCC CRL-1658) were cultured in high glucose DMEM and incubated at 37°C in a humidified atmosphere at 5% CO2. Prior to cell


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seeding, substrates were sterilized in a solution of Penicillin-Streptomycin in PBS (Phosphate Buffered Saline) (1:2, v:v) for 4 hours. On each sample 7000 cells/cm2 were seeded and left adhering overnight in the incubator. Live cell staining was performed for nuclei by using HOECHST (150 µl cell culture medium of a solution 1:10000 v:v in PBS) and for cell body with a treatment in a vital CellTracker Deep Red solution (1:1000 in cell culture medium without FBS) for 30 minutes in the incubator. Fixed cell staining was performed as follows: cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 3 min. Actin filaments were stained with rhodamin-phalloidin. Samples were incubated for 30 min at room temperature in the phalloidin solution (dilution 1:200), and, finally, cells were incubated for 15 min at 37 °C in HOECHST solution (dilution 1:1000) to stain cell nuclei. Photostimulation: A multiphoton (MP) confocal microscope (TCS SP5 MP, Leica Microsystems, Germany) was used to photoactuate the hydrogel 3D structures. The azobenzene isomerization and the consequent structure deformation were activated by using a MP laser, tuned at 700 nm wavelength (two-photon absorption wavelength at 350 nm, 13 ± 0.5 mW output power). The structures were immersed in water-based media and photostimulated by controlling the laser beam focusing and position. The exposed area was determined through the region-ofinterest (ROI) editor of the microscope software. The deformation of the structures was live monitored and recorded via software, by time-lapse imaging. Quantification of cell deformation: Cell nucleus polarization and area were evaluated from stained

cells

analyzed

with

the

ImageJ

MomentMacroJ

version

1.3

script

(hopkinsmedicine.org/fae/mmacro. htm). Based on a fluorescence threshold, this macro evaluates the nuclear area and its principal moments of inertia (i.e. maximum and minimum).


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The anisotropy index was calculated as the ratio of the principal moments (Imax/Imin), where the higher Imax/Imin, the higher the nucleus polarization. ASSOCIATED CONTENT Supporting Information. Supporting Information material: 1H NMR spectrum of modified gelatin B, Synthesis of Azo-crosslinker, 1H NMR spectrum of Azo-crosslinker, UV/Vis absorption spectra of Azo-crosslinker, Negative control of photostimulation, AFM analysis of mechanical properties, Topographical cell confinement, Live/Dead assay, Cell viability after gelatin photostimulation. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *[email protected], [email protected]; [email protected]; Present Addresses † Laboratory of Chemistry and Bioengineering, Tampere University of Technology, P. O. Box 541, Tampere FI 33101, Finland Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources Financial support was provided by IIT (Istituto Italiano di Tecnologia).


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ACKNOWLEDGMENT Dr. Valeria Panzetta is acknowledged for her important guidance during AFM nanoindentation measurements and Lucia Rossano for her appreciated help in some biological experiments reported in Supporting Information. We thank Dr. Fabio Formiggini for his precious support during photostimulation experiments and Dr. Luca Raiola for his valuable contribution in recording NMR data. Pietro Melone is instead acknowledged for the fruitful discussion regarding chemical synthesis and Valentina La Tilla for her irreplaceable aid in the production of the ToC figure. REFERENCES (1)

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Three-Dimensional Microstructured Azobenzene-Containing Gelatin

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