Design of a low cost spinneret assembly for coaxial electrospinning

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
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APPLIED PHYSICS LETTERS 106, 254101 (2015)
Design of a low cost spinneret assembly for coaxial electrospinning
Anant Raheja,1 T. S. Chandra,1 and T. S. Natarajan2,a)
1
Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036,
India
2
Department of Physics, Indian Institute of Technology Madras, Chennai 600036, India
(Received 12 February 2015; accepted 15 June 2015; published online 24 June 2015)
Coaxial electrospinning makes use of a concentric arrangement of spinneret orifices for synthesis
of core-shell polymer nanofibers. 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
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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.
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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.
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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. Yu, “New electrospinning nozzle to reduce jet instability and its application to manufacture of
multi-layered nanofibers,” Sci. Rep. 4, 6758 (2014).
3
D. Li and Y. Xia, “Direct fabrication of composite and ceramic hollow
nanofibers by electrospinning,” Nano Lett. 4(5), 933–938 (2004).
4
A. Natarajan, S. C. Mahavadi, T. S. Natarajan, J. H. Masliyah, and Z. Xu,
“Preparation of solid and hollow asphaltene fibers by single step electrospinning,” J. Eng. Fibers Fabr. 6(2), 1–6 (2011).
5
K. L. Ou, C. S. Chen, L. H. Lin, J. C. Lu, Y. C. Shu, W. C. Tseng, J. C.
Yang, S. Y. Lee, and C. C. Chen, “Membranes of epitaxial-like packed,
super aligned electrospun micron hollow poly(l-lactic acid) (PLLA)
fibers,” Eur. Polym. J. 47(5), 882–892 (2011).
6
I. M. Watt, The Principles and Practice of Electron Microscopy
(Cambridge University Press, 1997).
7
Y. Qiao, F. Polzer, H. Kirmse, E. Steeg, S. Kirstein, and J. P. Rabe, “In
situ synthesis of semiconductor nanocrystals at the surface of tubular Jaggregates,” J. Mater. Chem. C 2(43), 9141–9148 (2014).
8
B. Sun, B. Duan, and X. Yuan, “Preparation of core/shell PVP/PLA ultrafine fibers by coaxial electrospinning,” J. Appl. Polym. Sci. 102(1), 39–45
(2006).
9
H. Jiang, Y. Hu, Y. Li, P. Zhao, K. Zhu, and W. Chen, “A facile technique
to prepare biodegradable coaxial electrospun nanofibers for controlled
release of bioactive agents,” J. Controlled Release 108(2–3), 237–43
(2005).
10
G. Pilwat, P. Washausen, J. Klein, and U. Zimmermann, “Immobilization
of human red blood cells,” Z. Naturforsch., C: J. Biosci. 35(3–4), 352–356
(1980).
11
L. A. Gorman and J. S. Dordick, “Organic solvents strip water off
enzymes,” Biotechnol. Bioeng. 39(4), 392–397 (1992).
12
J. Sikkema, J. A. de Bont, and B. Poolman, “Mechanisms of membrane
toxicity of hydrocarbons,” Microbiol. Rev. 59(2), 201–222 (1995).
13
C. Wang, K. W. Yan, Y. D. Lin, and P. C. H. Hsieh, “Biodegradable core/
shell fibers by coaxial electrospinning: Processing, fiber characterization,
and its application in sustained drug release,” Macromolecules 43(15),
6389–6397 (2010).
14
Y. C. Lee and D. Yang, “Determination of lysozyme activities in a microplate format,” Anal. Biochem. 310(2), 223–224 (2002).
2
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