EFFECT OF ATMOSPHERIC PRESSURE NON-EQUILIBRIUM PLASMA
TREATMENT ON POLY-L-LACTIC ACID ELECTROSPINNABILITY
V. Colombo1,2, D. Fabiani2,3, M.L. Focarete4, E. Ghedini1,2, M. Gherardi1,
C. Gualandi2, R. Laurita1, P. Sanibondi1, M. Zaccaria3
Alma Mater Studiorum – Università di Bologna
1
Department of industrial engineering (DIN)
Advanced Mechanics and Materials - Interdepartmental Center for Industrial Research (CIRIMAM),
Via Saragozza 8-10, 40123 Bologna, Italy
3
Electric, Electronic and Information Engineering Department, viale Risorgimento 2, 40136 Bologna, Italy
4
Department of Chemistry “G. Ciamician”, via Selmi 2, 40126 Bologna, Italy
2
Abstract: Three different plasma sources (Argon jet, direct liquid phase discharge reactor and
a gas phase discharge reactor with liquid electrode) have been used to treat Poly-L-lactic
(PLLA) acid solution in order to verify the effect on its electrospinnability. Polymeric
solutions can be electrospun to fabricate fibrous mats, but it is not guaranteed total solvent
evaporation: plasma treatment of polymeric solution not containing high boiling point and
polar solvent allows the production of good quality fibers. Electrospinning of a solvent-free
nanofibrous PLLA scaffolds has been carried out through the exposure of polymeric solution
to a plasma jet. This result leads to a very important improvement for this kind of electrospun
mats in tissue engineering and bio-medical field.
Keywords: Biomaterials, solvent-free scaffold, tissue engineering, atmospheric pressure
not-thermal plasma, plasma in liquids.
1. Introduction
Electrospinning is a suitable method to produce
continuous polymeric fibers, with diameters ranging from
several nanometers to a few microns. This process is
based on spinning a polymeric solution electrostatically
charged by a DC bias between a needle, through which
the solution flows, and a grounded plate[1].
The presence of bead-like defects along the fibers,
which can weaken mechanical properties of the
electrospun mat, or the absence of fiber formation are
usually due to a not optimized polymeric solution
preparation, mainly in terms of solution viscosity and/or
conductivity, the latter aspect related to the use of solvents
with a low dielectric constant [2]. One of the approaches
typically implemented to prepare solutions with a good
electrospinnability is the dissolution of the polymer in a
mixture of a specific solvent and non-solvent for the
polymer. Polymer solubilisation is carried out by its
specific solvent, while the non-solvent component for the
polymer is usually a high dielectric constant substance
that is added to increase the charge density of the entire
solution.
Electrospun scaffolds are widely used in tissue
engineering applications. A strict requirement in the
biomedical field is the biocompatibility of the electrospun
mat, that must be free of toxic solvent traces [3]. Usually
solvents added to increase the dielectric constant have a
high boiling point and for this reason it is difficult to
ensure their complete removal through evaporation during
the electrospinning process, thus the final scaffold might
show traces of organic solvents.
Atmospheric pressure not-thermal plasmas are a
cocktail of charged particles, reactive species, UV
radiation, thermal flux and electric field. A not-thermal
plasma treatment of the electrospinning polymeric
solutions can avoid the formation of beads in the mat [4]:
therefore can be used to avoid or reduce the use of the
aforementioned high boiling point organic solvents.
Poly-L-lactic acid (PLLA) is a biocompatible and
biodegradable polymer, which is soluble in organic
solvents,
such
as
dichloromethane
(DCM).
Dimethylformamide (DMF) is usually added to DCM in
appropriate amount in order to increase the dielectric
constant of the solution [5]. However, the boiling
temperature of DMF is around 153°C (much higher than
that of DCM which is about 40°C), and it is therefore
difficult to completely avoid traces of residual DMF in
the scaffold after the electrospinning process. The aim of
this work is to investigate the effect of different plasma
sources on a PLLA solution in DCM before the
electrospinning process (pre-electrospinning solution).
Different plasma treatments have been implemented and
the effect of such treatments has been investigated.
Fiber morphology (fiber diameter distribution, presence
of defect such as beads along fiber axis, etc.) of mats
produced from plasma treated solutions are compared
with those of mats fabricated from an untreated PLLA
solution, in order to verify the improvement of
electrospinnability induced by plasma.
2. Materials and methods
Poly(L-lactic acid) (PLLA) (Lacea H.100-E) (Mw = 8.4
104 g/mol, PDI = 1.7) was supplied by Mitsui Fine
Chemicals. Dichloromethane (DCM) was purchased from
Sigma-Aldrich and employed without any further
purification. PLLA was dissolved at three different
concentrations, 8%, 10% and 13% w/v, in pure DCM.
The electrospinning (ES) apparatus, made in house, was
composed of a high voltage DC power supply (Spellman,
SL 50 P 10/CE/230), a syringe pump (KDScientific 200
series), a glass syringe, a stainless steel blunt-ended
needle (inner diameter: 0.84mm) connected with the
power supply electrode and a grounded aluminium
plate-type collector (7 × 7 cm2). The polymer solution
was dispensed through a Teflon tube to the needle that
was vertically placed on the collecting plate. PLLA
solutions were electrospun by using the following
conditions: voltage = 15 kV, needle-to-collector distance
= 15 cm, flow rate = 0.015 ml/min.
Three different plasma sources operated at atmospheric
pressure, in order to generate plasma inside or above the
liquid surface [6]: (i) a novel multi-gas plasma jet
developed by the authors, (ii) a direct liquid phase
discharge reactor (LFDR) and (iii) a gas phase discharge
reactor with liquid electrode (GPDR). A tungsten needle
(1 mm diameter) has been used as a high voltage
electrode for (ii) and (iii), submerged and above the liquid
surface respectively. Two separate gas inlets are employed
to control the production of reactive species generated by
the plasma jet; the primary gas is usually Ar, while O2, N2
or Air can be used as secondary gas. In this work Ar is
used as primary gas, while no secondary gas is used, the
typical aspect of the plasma jet working in the
aforementioned conditions is shown in Figure 1.
The plasma sources have been alimented by: (i) a
microsecond unipolar pulse generator, in-house developed
by the authors, producing high voltage pulses with a rise
time of about 100 µs, a pulse repetition frequency (PRF)
of 50-3500 Hz and a peak voltage (PV) of 15-25 kV and
(ii) a pulse generator (FID GmbH – FPG 20-1NMK)
producing high voltage pulses with a rise time of few
kV/ns, a PV of 7-20 kV into a 100-200 Ω load impedance
and a maximum PRF of 1000 Hz.
The conductivity of polymeric solutions was measured
using a conductivity meter (Eutech Instruments, COND
6+).
Scanning Electron Microscope (SEM) observations
were carried out using a Philips 515 SEM by applying an
accelerating voltage of 15 kV on samples sputter coated
with gold. The distribution of fiber diameters was
determined through the measurement of about 250 fibers
by means of an acquisition and image analysis software
(EDAX Genesis) and the results were given as the
average diameter±standard deviation.
Figure 1. Picture of the plasma jet operated in Ar at flow rate 2
slpm, driven by the nanopulsed power generator, PRF 1000 Hz
and PV 17 kV.
3. Results
In order to evaluate the effect of plasma treatments on
fiber formation a mat electrospun from a PLLA solution
not exposed to the plasma sources has been used as
control. It is possible to observe in the SEM images of
Figure 2, that at PLLA concentration of 8, 10 and 13%
w/v, the removal of DMF from the solution didn’t allow
the complete fiber formation.
Figure 2. Mats electrospun from an untreated PLLA in 100%
DCM solution: 8% w/v (A), 10% w/v (B), 13% w/v (C).
Fiber morphology of mats electrospun from a solution
treated by means of LFDR and GPDR is reported in
Figure 3.
Figure 3. Mats electrospun from a treated solution (treatment
time=300 seconds): LFDR operating at PV=23 kV, PRF=0,4
kHz on a 10% w/v solution (A), LFDR operating at PV=14,4
kV, PRF=1,3 kHz on a 13% w/v solution (B), GPDR operating
at PV=6 kV, PRF=1,4 kHz on a 10% w/v solution (C), GPDR
operating at PV=16 kV, PRF=1,3 kHz on a 13% w/v solution
(D). Plasma sources driven by the microsecond unipolar pulse
generator.
Figure 3A shows that low PRF value avoided complete
fiber formation, whereas in the other cases good quality
fibers were obtained, although with non-uniform diameter
distribution. When LFDR and GPDR have been used as
plasma sources a spark regime in some operating
conditions was observed, inducing the polymer
degradation (change in polymer solution color has been
observed, probably due to polymer burning): for this
reason those solutions were not electrospun.
The best results in terms of fiber quality and
preservation of polymer integrity were achieved with the
exposure of the PLLA solutions to the plasma jet: further
experiments were carried out using this plasma source
driven by the nanopulsed power supply and operating at
the following conditions: 2 slpm argon flow rate, PV = 27
kV, PRF = 1000 Hz.
SEM image of fibers obtained from a solution treated
with the plasma jet is reported in Figure 4A. Good quality
fibers without defects have been produced, demonstrating
a significant improvement in electrospinnability. However,
this effect is temporary: the improvement of the
electrospinnability of the PLLA solution lasts until 210
minutes, as shown in Figure 4C.
shown in Table 1. The treatment carried out with the
plasma jet can enhance the polymeric solution
conductivity; however this property changed up to one
hour. Conductivity increase caused by the treatment is not
the only property of the solution modified by the plasma:
a viscosity increase has been qualitatively observed after
the treatments.
The effect of plasma exposure on fiber diameter
distribution was investigated and reported in Table 2: this
analysis was carried out only on mats properly formed
with defect-free fibers. Mats electrospun starting from 6
ml of polymeric solutions (10% w/v) showed fibers
having a diameter in the range of micrometers, due to the
increase of the polymer concentration in the solution.
Indeed, solution weight decreased after plasma exposure,
due to the evaporation of the solvent. Mat electrospun
from 12 ml of polymeric solution and treated for 120
seconds showed nanometric fibers: in this case the effect
of solvent evaporation was less influent. Fiber diameter
measurement of mats electrospun with a delay between
the plasma exposure and the spinning process are reported
in Table 3. It is possible to observe that fiber diameter was
not affected by the increase of the delay.
Exposure
time [s]
0
10
30
120
Conductivity
[μS]
0
0,27
0,22
0,25
Table 1. Polymeric solution conductivity after different
treatment times of a dual gas plasma jet exposure
Exposure
time [s]
10
30
120
Average fiber diameter [μm]
Treated volume:
Treated volume:
6 ml
12 ml
1,88 ± 0,90
*
1,35 ± 0,48
*
1,71 ± 0,40
0,39 ± 0,14
Table 2. Average fiber diameter distribution after different
treatment times of a dual gas plasma jet exposure (treatment
time = 120 seconds). * in these cases measurement was not
carried out due to the large amount of beads along the fibers
Figure 4. SEM images of fibers obtained from 12 ml of plasma
treated polymeric solution (treatment time = 120 seconds): mats
have been electrospun after 0 minutes (A), 180 minutes (B) and
210 minutes (C) delay between plasma treatment and the
electrospinning process. Plasma source driven by the
nanopulsed power supply.
Polymeric solution conductivity was measured in order
to evaluate its effect on the treatment. Results for 12 ml of
polymer solution (10% w/v) after plasma jet exposure are
Average fiber diameter [μm]
ES delay:
ES delay:
ES delay:
0 minutes
30 minutes
180 minutes
0,39 ± 0,14
0,52 ± 0,16
0,53 ± 0,18
Table 3. Average fiber diameter distribution of mats electrospun
after a dual gas plasma jet exposure (treatment time = 120
seconds) at three different delays between treatment and
spinning process.
Figure 5 shows SEM images of mats obtained from
polymeric solutions at different concentrations exposed to
plasma using the aforementioned parameters. Fibre
morphology improvement is observed for all investigated
polymer concentrations (compare Figure 2 with Figure 5).
Mat obtained form from the 8% w/v polymeric solution
showed bead-on-string morphology, however the mat has
been formed and collected. The electrospun fibers
obtained from the 10% w/v and 13% w/v polymeric
solutions did not show any bead and defect. Solutions
prepared at different polymer concentration (8, 10 and
13% w/v) were also electrospun after several delays
between plasma exposure and the spinning process. This
study was carried out in order to highlight the evolution of
the plasma exposure effect on the improvement of the
electrospinnability. Indeed, spinning procedure requires a
characteristic time to the realization of a membrane
having the desired thickness according to its specific
application field.
plasma effects started to decay, because some beads were
formed, as previously shown in Figure 4.
The solution at 13% w/v was exposed to the plasma and
mats were electrospun without beads along the fiber axis
until 26 hours (Figure 7).
Figure 7. SEM images of 13% w/v PLLA electrospun
membranes after dual gas plasma jet exposure (treatment time =
120 seconds) of 12 ml. Mats have been electrospun after 180
minutes (A) and 26 hours (B) delay between plasma treatment
and the electrospinning process.
It is noteworthy that fibers with a diameter in the range
of a few hundreds of nanometers could be electrospun
from a treated solution at 10% w/v, while micrometric
fibers could be electrospun from a 13% w/v treated
solution (1,54±0,6 µm).
5. SEM images of electrospun membranes after dual gas plasma
jet exposure (treatment time = 120 seconds) of 12 ml of PLLA at
8% w/v (A), 10% (B), 13% (C).
The PLLA solution at 8% w/v plasma exposed and
electrospun after different delay times generates a large
amount of beads (Figure 6).
4. Conclusion
The removal of a high boiling point solvent, such as
DMF, in the electrospinning process, is an important issue
in order to increase the bio-compatibility of electrospun
PLLA mats for bio-medical applications. The possibility
of electrospinnig mats without beads is a strict
requirement to preserve mat mechanical properties. In this
work we highlighted that exposing a PLLA in 100%
DCM solution to a plasma jet, driven by a nanopulsed
power supply working at 2 slpm Ar flow rate, PV=27 kV,
PRF=1000 Hz, treatment time = 120 seconds, it is
possible to produce nanofibrous solvent-free scaffolds
without defects, that represent high added value materials.
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Figure 6. SEM images of 8% w/v PLLA electrospun membranes
after dual gas plasma jet exposure (treatment time = 120
seconds) of 12 ml. Mats have been electrospun after 30 minutes
(A) and 90 minutes (B) delay between plasma treatment and the
electrospinning process.
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plasma and good quality fibers without defects were
produced within 180 minutes, while after 210 minutes
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