Communication pubs.acs.org/cm
Tethered Pyro-Electrohydrodynamic Spinning for Patterning WellOrdered Structures at Micro- and Nanoscale Sara Coppola,†,‡ Veronica Vespini,† Giuseppe Nasti,‡ Oriella Gennari,† Simonetta Grilli,† Maurizio Ventre,‡ Maria Iannone,§ Paolo A. Netti,‡,§ and Pietro Ferraro*,† †
CNR − Istituto Nazionale di Ottica, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy Department of Chemical Materials and Production Engineering, University of Naples Federico II, Piazzale Tecchio 80, 80138 Naples, Italy § Center for Advanced Biomaterials Fore Health Care @CRIB, Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, 80125 Napoli, Italy ‡
S Supporting Information *
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opening the way to the multijetting spinneret modality for multiplexing and speeding up the fabrication process. Printing of micro- and nanofibers directly from a polymer drop with unprecedented order, direct writing of sharp/straight edges, and easy multijets electrospinning are demonstrated and reported. Experimental fabrication of patterns embedded with active molecules ensures that functionalized patterns preserve their functionalities after the TPES process. Results regarding the use of smart patterns for keeping alive and viable cultured cells are discussed. This study opens the way to innovative optogenesys analysis, guiding light for generating or transporting optical/electronic signals from and to cells.17−21 The setup proposed in this work, unlike the conventional ES, is electrode-free and nozzle-free, Figure 1a,b. The method allows polymer nanofibers to be printed directly from a polymer drop overpassing the viscosity border of nozzle clogging in conventional inkjet systems.22 The drop reservoir is placed directly on a flat substrate (base) while an electric field,
lthough electrospinning (ES) allows the production of unsurpassed nanoscale polymer fibers, the major drawbacks are the nozzle-clogging and single-jet spinneret, respectively. This is a real limitation in terms of usable polymers and for patterning active organics. Nowadays the micro-engineering of smart materials could represent a new route for many fields of technology ranging from the production of electronic and photonic devices1−3 to regenerative medicine and tissue engineering.4−7 An enormous technological interest is related to the possibility of patterning fibers directly in well-ordered patterns avoiding the deposition of nonwoven submicrometer mats often occurring in ES.8,9 In the past decade several attempts have been made using fieldinduced10−13 and near-field ES,14,15 but only very recently, with the introduction of mechano ES,16 has the production of wellordered fiber patterns been achieved. Nevertheless, some drawbacks related to the complexity of the setup, the operating temperature, and the selection of usable materials for problems related to nozzle clogging still persist. Moreover, high temperature can cause deterioration of the optical and electronic properties of active organic materials eventually embedded in the functionalized fibers. On the other side, interfering effects due to closeness of multiple electrified nozzles ban working with multiple spinnerets. Here we introduce a revolutionary nozzle-free approach, the tethered pyro-electrodynamic spinning (TPES) operating in wireless modality, i.e., without electric circuit, electrodes, and voltage supply. This novel approach definitively simplifies the ES apparatus extending the nanofiber spinning also to active organic polymers preserving at the same time all the properties of conventional systems. Fiber drawing from the liquid polymer is driven through the pyroelectric charge generated into a ferroelectric crystal (i.e., LiNbO3) able to induce the electrohydrodynamics (EHD) pressure required for polymer manipulation without wires. The approach is highly flexible, simple, compact, and cost-effective when compared with classical ES, and last but not least, it allows working safely, avoiding the use of high-voltage equipment at kVolts scale. For the first time, in situ observation of fiber drawing is provided allowing real-time adjustments and full control of the process. Moreover the TPES adds to the capabilities of conventional ES the chance of printing polymer fibers even in the case of multiple drops © 2014 American Chemical Society
Figure 1. (a) Set-up of conventional ES apparatus with nozzle and high voltage power supply. (b) Wireless ES system made of three principle components: thermal source, LN crystal, and polymer drop. (d) Finite element simulation of sharp-cone electric field lines (arrows) compared with (c) conventional ES field. Received: April 9, 2014 Revised: May 12, 2014 Published: May 14, 2014 3357
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it is comparable with the nanofibers produced by conventional ES. In particular, here we show experiments of direct writing of the poly-co-glycolic acid (PLGA) ink using the pyroelectrodynamic approach.29,30 DMC (dimethyl carbonate) was used as solvent while the fluorochrome Nile Red was introduced for modeling active compounds. The substrate is conveniently chosen for various applications, and no special requirements are needed. Because of the intrinsic stability of the system we can produce fiber patterns with uniform diameter and regular geometry in real time, as demonstrated in the Supporting Information Movies (nos. 2 and 3) and shown in Figure 3.
induced by the pyroelectric effect activated onto a Lithium Niobate (LN) crystal,23 exerts an attractive force on the polymer drop deforming it into a Taylor’s cone thus generating liquid jet emission. The process of polymer deformation starts as a consequence of the temperature gradient in more of a safety condition than in case of external high-voltage supply. The fibers stretched from the elongation of the Taylor cone are deposited directly onto the target substrate (collector) facing the base at a distance d and mounted onto computer-controlled x,y axes translation stage. The experiments are carried out at room temperature and in air atmosphere. It is important to note that the pyro-electric field, 2.7 × 107 V m−1 < Epyro < 5.5 × 107 V m−1, exerts its attractive force over the drop volume in total and not only over the tip of a small capillary, like in case of conventional ES, having intrinsically an optimum arrangement of the electric field lines.24 The process works in a sort of upside-down (in respect to the vertical axis) experimental configuration when compared to traditional ES. As a consequence, the sharp-cone distribution of the electric field lines (Figure 1d) provides a suitable electric field 3D distribution with higher selectivity for the attraction force on a single fiber, thus improving the stability and resolution in the deposition process. In fact, the pyro-electric field lines converge (i.e., are focused) just at one point, enhancing the electric attraction on the polymer drop, whereas in the conventional ES system they clearly diverge from the electrode to the collector, Figure 1c. Under the action of the Epyro the elongated tip of the Taylor’s cone is focused and put in direct contact with the collector, so that the jet is finely controlled in space defining a stable condition of work25−27(Supporting Information and Supporting Information Movie no. 1). This contact has two effects. First, the adhesion allows the fiber emerging from the Taylor’s cone to be fixed, avoiding the bending instabilities and tethering continuous liquid flow to the collector. Once the polymer jet is tethered to the substrate of interest, it could be used to realize well-ordered patterns completely avoiding the whipping perturbations driven by the lateral electric force and the aerodynamic interactions. By controlling d it is possible to keep the jet diameter uniform, thus avoiding the stretching and thinning effect associated with the electrified jet in conventional ES28 (Supporting Information). Moreover, TPES, when working under stable conditions, presents a good spatial resolution. In fact the deflection of the jet typically caused by the transiently charged nanofibers is reduced with respect to the instability region of conventional ES. Using the intrinsic viscoelasticity of the polymer and the versatility of the process, we can print regular but long wave forms of instability, such as whipping instabilities or printing of beads-on-a-string.29 In Figure 2 we display SEM images, evidencing the printing of knotted fibers with a good alignment. The minimum diameter obtainable is 300 nm (Figure 2b), and
Figure 3. (a) Large view of polymer pattern in comparison with 1 euro cent. (b) Steady cone-jet. (c) Printing sharp and precise 90° corners. (d) Writing parallel and uniform lines with diameters up to 50 μm and (e) side view of the real time fiber drawing. (f) Optical fluorescence microscope image of an ordered square grid; the inset represents the corresponding bright field image.
The emission visible in the fluorescence images reported in Figure 3d is uniform along the fiber, evidencing the absence of morphological defects and the high control of the fiber shape and positioning achieved through TPES. It is important to note that a continuous fiber of over 1 cm length could be printed starting form a drop reservoir of 500 μL without interrupting the process in a stable condition (Figure 3a). No syringes are required for the formation of the spinning cone, making the multi-jetting applications easier,30 even in case of simultaneous processing of different materials, reducing the time needed for the routine maintenance and cleaning of conventional apparatus and controlling the nonaxisymmetric fiber instabilities. In the main time, the aerodynamic interactions and the bending torque produced from jet dipole charge interaction with the external electric field and the repulsion of surface charges with the multi-jetted fibers are finely controlled, making possible simultaneous polymer printing starting from multiple polymer drops, Figure 4. In order to show the potentialities in processing multifunctional biomaterials, we conducted some experiments regarding the growth, the contact guidance, and cell polarization of cells
Figure 2. (a) SEM image of well-aligned beads-on-a-string polymer fibers. (b) SEM image of individual nanofiber with a diameter of ∼300 nm.
Figure 4. (a) Multiple jets for simultaneous printing of polymer lines and bright field image of the pattern obtained through TPES (b). 3358
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over the PLGA fibers. We analyzed the morphological features of human mesenchymal stem cells (hMSCs). In order to limit cell adhesion discouraging nonspecific attachment on the supporting substrate, the polymer was deposited on a PTFE coated glass slide. hMSCs were cultivated for 24 h and then fixed and stained for the visualization of cytoskeletal stress fibers and nuclei. Cell bodies were predominantly located within the interfiber gap, and actin stress fibers were strongly coaligned with the pattern direction, Figure 5b−d.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank Ing. L. Ambrosio. This work was supported by FIRB RBFR10FKZH, EFOR-CABIR CNR, and by the MAAT project.
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REFERENCES
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Figure 5. (a) 3D rendering of TRITC−Phalloidin z-stack of cells coaligned with the parallel uniform PLGA lines. (b) Cell body coaligned with the pattern direction. (c) Side fluorescence view of cells clung to the fiber. (d) Optical fluorescence image of nuclei patterning interfiber gap.
Interestingly, the 3D reconstruction of the z-stack revealed that the basal surface of the cells was not in contact with the substrate, but cells clung to PLGA fibers (Figure 5c) approximately halfway through the fiber thickness, Supporing Information Movie no. 4. Accordingly, the vast majority of nuclei were located between the fibers, displaying a prolate elliptical shape, Figure 5d. Curiously, hMSC acquired an elongated shape coaligned with the direction of the polymer deposition. This distribution would be the object of further study. In fact, although the biomolecular mechanisms by which this phenomenon occurs is still unclear, it is tempting to speculate that cell adhesion confinement controls the magnitude and direction of the cell’s contractile forces, which eventually causes the cell body to lay in complex microarchitectures in which forces equilibrate, stretching the nucleus. In summary, we introduce a novel approach in ES allowing the fabrication of well-ordered microscale patterns with all the facilities and properties of conventional systems. The advantages in terms of compactness and safety because of electrode-less properties make it a promising technology for the direct printing of high resolution polymer structures avoiding all the problems of single-jet spinneret and nozzle clogging. We demonstrate the fabrication of active ordered patterns in the case of highly viscous materials and their functionality after the TPES process in terms of allowing cell growing. New intriguing perspectives for patterning active organic materials for optogenesis studies and for constituting integrated arrays of sensors in the human body can be foreseen.
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ASSOCIATED CONTENT
S Supporting Information *
Additional details, figures, and movies. This material is available free of charge via the Internet at http://pubs.acs.org. 3359
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(30) Coppola, S.; Vespini, V.; Grilli, S.; Ferraro, P. Lab Chip 2011, 11, 3294−3298.
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