Design of a low cost spinneret assembly for coaxial electrospinning Anant Raheja, T. S. Chandra, and T. S. Natarajan Citation: Applied Physics Letters 106, 254101 (2015); doi: 10.1063/1.4922948 View online: http://dx.doi.org/10.1063/1.4922948 View Table of Contents: http://scitation.aip.org/content/aip/journal/apl/106/25?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Study of polyvinyl alcohol nanofibrous membrane by electrospinning as a magnetic nanoparticle delivery approach J. Appl. Phys. 115, 17B908 (2014); 10.1063/1.4867600 Large effect of titanium precursor on surface reactivity and mechanical strength of electrospun nanofibers coated with TiO2 by atomic layer deposition J. Vac. Sci. Technol. A 31, 061506 (2013); 10.1116/1.4817718 Modeling of solvent evaporation from polymer jets in electrospinning Appl. Phys. Lett. 98, 223108 (2011); 10.1063/1.3585148 Collection method for extra aligned nanofibers deposited by electrospinning Rev. Sci. 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Most laboratories purchase the spinneret from commercial manufacturers at a significant expense, or design it indigenously to save costs but compromise on manufacturing precision. Therefore, the present work suggests the use of a relatively lower priced McIntyre cannula needle, conventionally used for ophthalmic surgeries, as a coaxial spinneret for electrospinning. The McIntyre cannula needle was modified to synthesize hollow fibers of nylon 6, which acted as sheath with hydrogen peroxide as core during electrospinning. In addition, encapsulation of bioactives, viz., red blood cells, bacterial cells, and lysozyme (enzyme protein) was attempted, using their aqueous suspensions as core, with polycaprolactone solution as sheath. Resulting fibers had an integral core-shell structure with the bioactives encapsulated in the core. This indicated that the modified McIntyre cannula functions suitably as a spinneret for coaxial electrospinning. Thus, apart from being a clinical device, the modified McIntyre cannula needle C 2015 provides an economic alternative to conventional coaxial spinneret assemblies. V AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4922948] Coaxial electrospinning is a technique to obtain coreshell nanofibers by electrostatic drawing of two or more independent polymer solutions in a coaxial stream. This requires a spinneret with concentrically arranged needle orifices to enable simultaneous drawing of core and shell fluids. Many researchers prefer to build their own in-house spinneret due to high cost of commercial spinnerets or their incompatibility with existing equipment. Although it is cheaper to fabricate a coaxial spinneret in-house, it lacks the mechanical robustness of commercial designs. In house designs take-up valuable person-hours for development and still end-up having defects in fabrication. This leads to faults such as leakage of polymer solutions during electrospinning, breakage at vulnerable joints, and clogging of tubes. This causes undesirable defects in the final fiber morphology such as polymorphic micro-particles, heterogeneous core morphology and fiber diameters. Therefore, the present study proposes a low cost scheme of modifying standard medical grade cannula needles, to fabricate a coaxial spinneret. A McIntyre coaxial cannula is used for simultaneous irrigation and aspiration in ophthalmic surgeries.1 It is available with most surgical product suppliers at a price of 20–40 USD, which is approximately five times lower than that of commercial coaxial spinneret assemblies (prices as advertised by international manufacturers like Ramehart, Spraybase, Linari, etc.). The McIntyre cannula comprises the above-mentioned concentric arrangement of an outer 18 G needle and an inner 23 G needle (variants available). Since, medical cannulation procedures require an uninhibited flow of core and shell fluids, the outer (shell) and inner (core) needles are held in position by means of a T-shaped plastic joint a) Electronic mail: tsn@iitm.ac.in 0003-6951/2015/106(25)/254101/4/$30.00 as shown in Figure 1. The T-joint acts as an adaptor for the two needles wherein, the core needle penetrates through the centre of the plastic block forming a leak-proof assembly. This in turn lodges into the shell needle by Luer lock mechanism. The T-joint maintains an annular gap of 0.2 mm between outer wall of the core needle and inner wall of the shell needle. Both the outer and inner needles are sealed at the tip (Figure 1) such that the inner aspirating needle is extended relative to the irrigating tip. The seal provides orifices for irrigation and aspiration on the side, at the bevel end of respective needles. However, this is not suitable for coaxial electrospinning, as the seal would obstruct the flow of core and sheath fluids if pumped through this arrangement. The only modification needed to convert the cannula into a coaxial spinneret was, grinding its bevel, to allow both inner and outer liquids to flow out in a coaxial stream. Hence, the sealed bevel was ground gently without distorting FIG. 1. Illustration depicts modification of a coaxial cannula needle (left) to a coaxial spinneret (right) with a concentric arrangement of needle orifices. 106, 254101-1 C 2015 AIP Publishing LLC V This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 131.111.164.128 On: Sat, 15 Aug 2015 06:04:35 254101-2 Raheja, Chandra, and Natarajan the annular spacing between needles. Further, both the outer and inner needle tips were ground until above the aspiration orifice, so that the open ends of the needles were in the same plane, perpendicular to their axis. After grinding, the needles were held in place by the T-joint. An annular gap of 0.2 mm for passage of sheath fluid and the inner core orifice of 0.32 mm allowed a higher core to sheath flow volume ratio of 1.6:1, thus promoting the encapsulation efficiency. However, this arrangement also necessitated maintaining higher sheath flow rate relative to the core flow to obtain a stable Taylor cone during electrospinning. In addition to flow rate, the inner needle tube was made to obtrude compared to the outer needle tube. Since, frequent breaks in the stream occurred during the coaxial flow when the inner needle was kept at the same level as the outer needle or protruding outside. Various reasons could be attributed to this behaviour including transverse electrostatic interactions between the inner and outer fluids due to charge imbalance in two solutions, instability of fluid stream due to difference in viscosity of outer and inner solutions, etc. A recent study corroborates this phenomenon, based on numerical simulations on behaviour of a coaxial stream from varying exit pipe lengths of a coaxial spinneret.2 Hollow tubes of nylon 6 were synthesised using the proposed spinneret to validate its capability for use in coaxial electrospinning. Hollow nano-tubes are desirable in areas of microfluidics, optical wave-guides, and molecular separation. Many reports demonstrate template synthesis of ceramic hollow nanotubes using inorganic/polymer composites.3,4 However, only a few researchers have been able to develop and characterize electrospun polymer tubes with micron size diameters.5 This could be because of inherent flexibility of the polymer tubes at nanoscale, which tend to collapse internally, losing their hollowness and forming monolithic ribbon like flat fibers, during post-spinning. Therefore, the present study used 30% hydrogen peroxide as the core fluid and 19 wt. % nylon 6 in formic acid as the sheath. This combination prevented collapse of the hollow tubes. Moreover, after formation of fibers, hydrogen peroxide innocuously degraded into water and oxygen leaving behind intact nanotubes as shown in Figure 2. A microtome cross section of the fibers was used for the scanning electron microscope (SEM) image analysis, which revealed fibers with a mean outer diameter of 382 nm. Fibers with diameters as low as 108 nm were also observed. However, it was difficult to capture sharp micrographs of multiple fiber cross sections due to charging under the electron beam. Further, to confirm the hollowness of the fibers, a TEM characterization was done, which is shown in the image. However, the bright lines seen at the edge of the fibers were Fresnel fringes (pointed in Figure 2(b) by black arrows) and did not correspond to hollowness of the fiber. These fringes commonly occur in carbon holey films or carbon nanotubes due to suboptimal focussing.6 In this case, the fibers displayed such fringes due to charging and mechanical movement under the electron beam. Therefore, a line scan of a 180 nm fiber in the TEM image was done with respect to its gray value using GATAN digital micrograph software (Figure 2(c)), as per an earlier report.7 This revealed that the fiber was thicker at the edges compared to its central axis, which is an indication of Appl. Phys. Lett. 106, 254101 (2015) FIG. 2. (a) HR-SEM image of hollow nylon 6 nanotube with a diameter of 108 nm, (b) HR-TEM image of nylon 6 hollow electrospun fibers showing Fresnel fringes (black arrow), and (c) corresponding line scan with respect to gray value of the 180 nm fiber indicates a hollow central portion in the fiber. a hollow core. Thus, after correlating the SEM image with the TEM image analysis, it was concluded that hollow nylon 6 nanotubes can be synthesized using a modified McIntyre cannula as a coaxial spinneret. Coaxial electrospinning can be used to form core-shell polymer fibers, wherein both the core and shell polymers perform independent functions. This is a valuable method for integrating distinct functional capabilities into a single nanofiber by combining two independent polymers or nonpolymeric entities through coaxial electrospinning.8 In the present case, an aqueous mixture of acridine orange (1 mg/ ml) in 75 mg/ml of poly ethylene glycol (PEG) was pumped through the core of the coaxial spinneret assembly. Here, the sheath was a non-fluorescent solution of 20 wt. % polycaprolactone (PCL) dissolved in dichloroethane. On observing the electrospun fibers through optical and fluorescence microscopy, the fibers showed a red fluorescent core of PEG with acridine orange and a transparent sheath of PCL (Figure 3). In addition, most fibers had a continuous core and some showed septum formation in the core. These images provide significant evidence that the cannula spinneret can be used for synthesis of core-shell fibers. FIG. 3. Images of PCL-PEG/acridine orange coaxial electrospun fibers under a (a) bright-field and (b) fluorescence microscope showing the red stained PEG core. This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 131.111.164.128 On: Sat, 15 Aug 2015 06:04:35 254101-3 Raheja, Chandra, and Natarajan Appl. Phys. Lett. 106, 254101 (2015) TABLE I. Dimensions and broad functions of bioactive agents encapsulated in core-shell electrospun fibers using the modified McIntyre cannula needle. Core units Red blood cells Dimensionsa Broad function 6–8 lm Oxygen delivering cells in vertebrates Bacterium found as normal flora of soil Industrial enzyme with antibacterial activity B. subtilis 1 lm 1.5 lm Lysozyme 2.6 nm 4.5 nm a The dimensions are close approximations. FIG. 4. Optical microscope image of RBCs encapsulated in electrospun PCL fibers. Here, the black arrow indicates intact RBCs seen as bulges in the fiber, while the red arrow points to fibers with lysed RBCs. The yellow arrow indicates a typical electrosprayed bead that may or may not contain RBCs. Another application of core-shell electrospun fibers in biology is for the purpose of encapsulation. Core-shell electrospun fibers are being intensely studied for developing drug delivery systems and immobilization matrices.9 Therefore, it was pertinent to evaluate potential of the modified cannula to encapsulate bio-functional molecules and live cells inside core-shell electrospun fibers. The representative units, chosen for coaxial electrospinning as core, differed broadly in size and biological functionality as shown in Table I. Red blood cells (RBCs) carry out the essential function of oxygen delivery in vertebrates. There is a constant need for efficient RBC storage systems due to an ever-increasing demand for blood in clinical transfusions.10 RBCs measure about 6–8 lm in diameter and are significantly larger than bacterial cells and protein molecules (Table I). Healthy RBCs were isolated from human blood premixed with an anti-coagulant. These were then suspended under physiological buffer conditions for encapsulation. The suspension was fed through the core of coaxial spinneret, while a 30 wt. % PCL in dichloroethane was used as the sheath solution for electrospinning. The resulting electrospun fibers were observed through an optical microscope (Figure 4). The irregular bulges in some fibers indicate that a significant number of RBCs retained their native morphology. However, a majority of cells inside the fibers lysed, displaying only the red haemoglobin pigment. This lysis occurred due to dissolution of the cell membrane, a lipid bilayer, which is highly susceptible to organic solvents present in the coaxial stream. In addition, the flow of a coaxial jet stream exposes the cells to shear forces, which in combination to the stretching process would cause significant cell lysis. Except for the lack of cell integrity, it was otherwise possible to encapsulate this relatively large cell type in electrospun fibers. Unlike RBCs, which only have a cell membrane, bacteria possess an additional cell wall made of peptidoglycan, which maintains the cell’s turgidity and integrity even under high osmotic pressure. Therefore, bacteria are better equipped to undergo the process of electrospinning without losing significant viability. Hence, a culture of Bacillus subtilis was used for encapsulation through coaxial electrospinning by means of the modified McIntyre cannula. B. subtilis is a rod shaped, gram positive, endospore forming bacterium. It is a good model for studying viability of encapsulated microorganisms, owing to its ability to resist harsh environmental conditions. Log phase culture of B. subtilis in Luria Bertani (LB) broth with an optical density of 0.4 was used as core solution, while a 30 wt. % solution of PCL in dichloroethane was used as sheath fluid for coaxial electrospinning. Electrospinning was carried out under aseptic atmosphere at a flow rate of 0.4 ml/h for both the sheath and core. An electric field of 1 kV/cm was applied to obtain a stable coaxial stream. The viability of B. subtilis was confirmed by inoculating a piece of the electrospun mat in sterile growth medium and observing it under optical and scanning electron microscopes. Exposure to organic solvents causes inactivation of enzymes by removal of water molecules.11 In addition, it may lead to dissolution of cell membranes causing cell death as seen above for RBCs.12 In the present study, B. subtilis (Figure 5(a)) showed sufficient growth from the fiber matrices even after exposure to organic solvents during electrospinning. This is because, though its vegetative forms succumbed to the dehydrating effects of organic solvents, the spores survived and propagated in culture media. In addition, the cells over grew the surrounding sheath and degraded it, causing segmentation of fibers (Figure 5(b)). This also FIG. 5. (a) HR-SEM image of nonencapsulated B. subtilis. (b) HR-SEM image, and (c) optical microscope image of monochrome stained B. subtilis encapsulated in PCL by coaxial electrospinning and grown in LB broth for 24 h. This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 131.111.164.128 On: Sat, 15 Aug 2015 06:04:35 254101-4 Raheja, Chandra, and Natarajan FIG. 6. (a) TEM image of a coaxial fiber with a core of PVA-lysozyme enclosed in a PCL sheath, (b) SDS-PAGE gel showing identical electrophoretic mobility of lysozyme before (N-native) and after (T-test) encapsulation, and (c) UV absorption spectra indicating the presence of lysozyme in PCL-PVA core-shell fibers. accounts for numerous spores seen under optical microscope (Figure 5(c)). Core-shell fibers have shown great potential for developing delivery systems for protein and drug molecules.13 Therefore, a representative enzyme protein, namely, lysozyme, was encapsulated in electrospun PCL fibers by means of the modified McIntyre cannula, to validate its use as a coaxial spinneret. A 9 wt. % solution of PCL in chloroform with 20% dimethylformamide was used as sheath solution and 5 wt. % poly vinyl alcohol (PVA) in milliQ filtered water as the core. Lysozyme concentration of 20 mg/ml was incorporated in the core solution. A flow rate of 1.0 ml/h for the sheath and 0.3 ml/h for the core was used, to have a visually stable Taylor cone without any dripping. An electric field of 1 kV/cm was applied between the spinneret and a grounded aluminium foil for collection of fibers. As seen in Figure 6(a), the mixture of PVA-lysozyme was entrapped in the core (57.9 nm) with PCL as sheath (21.2 nm). The structure of a protein is defined by its amino acid sequence and the resulting folded conformation. A change in protein structure could occur if it degrades into multiple chains, due to free radical attack, enzymatic cleavage, thermal degradation, etc. Therefore, the encapsulated lysozyme was tested for degradation through SDS-PAGE (sodium dodecyl sulphate assisted polyacrylamide gel electrophoresis). It was seen that both the native and encapsulated lysozyme had the same electrophoretic mobility (Figure 6(b)). This suggests that lysozyme retained its primary structure even after electrospinning. In addition, the activity of lysozyme was quantified by measuring its ability to lyse Micrococcus luteus cells.14 It was observed that an average of 95% of the lysozyme activity was retained even after encapsulation. Further, an UV spectrophotometry of the lysozyme encapsulated PCL-PVA nanofiber mat recorded an Appl. Phys. Lett. 106, 254101 (2015) absorption peak at 272 nm, which is close to the absorption of native lysozyme at 282 nm (Figure 6(c)). The blue shift of 10 nm was attributed to the stabilizing interaction between lysozyme and PVA. The energetically favourable interaction exposed the otherwise buried aromatic amino acid residues resulting in absorption at a lower wavelength. As demonstrated above through multiple iterations and trials, the modified McIntyre cannula offers an economical and competent spinneret design for coaxial electrospinning. Moreover, just like any other spinneret assembly whether commercial or indigenous, it is capable of synthesising both hollow and core-shell nanofibers. Further, as demonstrated above, the modified cannula can be used for encapsulation of biomolecules and live cells. Therefore, the study concludes the modified cannula as validated and equivalent to a standard coaxial spinneret for the purpose of electrospinning. 1 D. J. McIntyre, “Instrument for aspirating and irrigating during ophthalmic surgery,” U.S. patent 4,014,333 (1975). B. Lee, S. Jeon, H. Park, G. Lee, H. Yang, and W. 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