Applied Surface Science 457 (2018) 247–255

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Applied Surface Science

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Full Length Article

Preparation of photodynamic P(MMA-co-MAA) composite nanofibers doped T

with MMT: A facile method for increasing antimicrobial efficiency
⁎ ⁎
Qingqing Wanga, Wangbingfei Chena, Quan Zhanga, Reza A. Ghiladib, , Qufu Weia,
a
Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, Wuxi 214122, China
b
Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA

A R T I C LE I N FO A B S T R A C T

Keywords: We report the preparation of photodynamic materials from electrospun nanofibers of P(MMA-co-MAA) co-
Montmorillonite polymer that were doped with montmorillonite (MMT), and further functionalized with the cationic photo-
Photodynamic sensitizer methylene blue (MB). The resultant composite nanofibers were characterized by spectroscopic (in-
Nanofiber frared, UV − vis, fluorescence) and physical (SEM/TEM, gravimetric) methods. Addition of MMT (0–6 wt%) in
Electrospinning
the electrospinning process increased the adsorption of the methylene blue photosensitizer from 21.5 mg
Photosensitizer
(67.1 μmol)/g P(MMA-co-MAA) parent copolymer to 30.0 mg (93.8 μmol)/g P(MMA-co-MAA)/MMT-6 compo-
Antibacterial
site nanofibers. The MB-decorated P(MMA-co-MAA)/MMT-6 composite nanofibers showed a 99.997% (4.8 log
units) and 97% (1.8 log units) reduction in CFU/mL against Staphylococcus aureus (ATCC-6538) and Escherichia
coli strain 8099, respectively, after visible light illumination (LED lamp, 30 min, 35 ± 5 mW/cm2), demon-
strating that the increased photosensitizer loading attributable to MMT doping led to a 1–2 log unit increase in
photodynamic inactivation efficacy over the non-doped MB-decorated P(MMA-co-MAA) parent copolymer
(99.9% and 84% reduction for S. aureus and E. coli, respectively). The results demonstrate that MMT and similar
additives may be a universal method for increasing the adsorption capacity of cationic photosensitizers in
electrospun nanofibers, thereby generating materials with improved photodynamic inactivation efficacy without
significantly changing the fiber morphology, cost, or ease of production.

1. Introduction materials: namely, the non-specific cellular damage caused by singlet

From biodefense to general population security against existing and
emerging infections, as well as for preventing nosocomial infections in
hospitals, the development of materials that possess antimicrobial or
self-disinfecting properties has been garnering increasing interest [1,2].
While efforts in the design and development of these materials have
incorporated a diverse array of biocidal agents ranging from natural
products to heavy metals [3–12], photodynamic materials have
emerged as a promising platform for anti-infective materials [13].
Based upon antibacterial photodynamic inactivation (aPDI) [14,15],
these materials employ photosensitizers (PS) that, when illuminated by
visible light in the presence of molecular oxygen, generate powerful
cytotoxic reactive oxygen species (ROS), leading to the inactivation of a
broad range of microorganisms, including bacteria [14,16–19], fungi
[20–23], viruses [24–27], and parasites [28,29]. As the primary bio-
cidal agent, singlet oxygen (1O2) [30,31] not only confers this broad
spectrum anti-infective mode of action, it endows the photodynamic
materials with a significant advantage over other antimicrobial
oxygen (and other ROS) makes aPDI equally effective against drug-re-
sistant strains as drug-susceptible ones, and without giving rise to new
resistance from repeated photosensitization [14,20,32]. Moreover, the
photosensitizers, visible light, and material scaffolds themselves are
non-toxic, and are therefore not expected to have an adverse effect on
human health, while the short half-life [33], limited diffusion distance
in solution (< 200 nm) [34,35], and decay back to harmless, breathable
oxygen together make singlet oxygen an environmentally benign bio-
cidal agent.
Driven by the promise of photodynamic materials, a number of re-
cent studies have reported the immobilization of different classes of
photosensitizers (e.g., porphyrins, phthalocyanines, phenothiazines
[36,37]) onto both natural (e.g., cellulose, chitosan, cotton fabrics
[38–44]) and artificial (e.g. silicone, polyurethane, polystyrene, poly-
ethylene, polycaprolactone, and polyacrylonitrile [25,45–50]) scaf-
folds. While photosensitizer immobilization via covalent attachment is
considered more robust than non-covalent methods [19,38,41–43], the
scalability needed for commercial applications often precludes covalent

⁎
Corresponding authors.
E-mail addresses: qqwang@jiangnan.edu.cn (Q. Wang), cwbfsophie@outlook.com (W. Chen), 877749003@qq.com (Q. Zhang), Reza_Ghiladi@NCSU.edu (R.A. Ghiladi),
qfwei@jiangnan.edu.cn (Q. Wei).

https://doi.org/10.1016/j.apsusc.2018.06.041
Received 30 March 2018; Received in revised form 24 May 2018; Accepted 7 June 2018
Available online 07 June 2018
0169-4332/ © 2018 Elsevier B.V. All rights reserved.
Q. Wang et al. Applied Surface Science 457 (2018) 247–255

conjugation strategies where modification of both the photosensitizer increased photosensitizer adsorption compared to the MMT-free parent
and scaffold may be tedious and/or synthetically challenging. As such, co-polymer. Methylene blue (MB) was selected as a model cationic
non-covalent PS-immobilization methods have been studied, including photosensitizer owing to its low cost, lack of toxicity when used in
electrostatic attraction, physical blending, interior encapsulation, and therapeutic doses (< 2 mg/kg) [16,57], and it has already been in-
surface coating [25,47,48,50–54]. Generally speaking, these studies corporated into medical grade polymers used to fabricate catheters
have provided a number of critical insights into the design principles [58]. Notably, it will be shown that the increased MB photosensitizer
behind photodynamic materials, including the need for a high material loading afforded by the MMT additive leads to a 1–2 log unit increase in
surface area [48,54], and the use of cationic charges (either on the photodynamic inactivation efficacy against model strains of Gram-po-
material scaffold or the photosensitizer itself [36]) for improved anti- sitive Staphylococcus aureus and Gram-negative Escherichia coli bacteria.
bacterial efficacy. Although photophysical properties (e.g., singlet The results here will demonstrate that by including additives such as
oxygen quantum yield, robustness towards photobleaching) are an MMT in their production, it may be possible to universally improve the
important consideration, the aforementioned studies on photodynamic antimicrobial properties of a range of photodynamic materials without
materials have also collectively shown, not surprisingly, that photo- negatively impacting their means of production, cost or physical
sensitizer loading directly correlates with antimicrobial efficacy. Thus, properties.
an inherent challenge in the development of photodynamic materials is
to increase photosensitizer loading, while at the same time minimizing
2. Materials and methods
the costs associated with the production of the material.
Importantly – and relevant to the present study – to the best of our
2.1. Materials
knowledge no studies have explored the use of additives as the means to
increase photosensitizer loading in the design and production of pho-
Organically-modified MMT [modified with bis(2-hydroxyethyl)me-
todynamic materials. To address this knowledge gap, here we introduce
thyltallow ammonium salts, commercial name DK2] was supplied by
a simple, economical, and scalable approach employing montmor-
Zhejiang FengHong Clay Co. (China), and was produced from the ion
illonite as an additive in electrospinning to facilely structure nanofibers
exchange of Na+-MMT with a mixture of bis(2-hydroxyethyl)methyl-
with both internal and external sites for PS functionalization (Fig. 1).
tallow ammonium salts. Methyl methacrylate (MMA, AR) was pur-
We surmised that inexpensive montmorillonite (MMT) clay, consisting
chased from Sinopharm Chemical Reagent Co., Ltd. Prior to use, the
of a lamellar stack of aluminosilicate sheets, was a promising additive
MMA was washed by 10 wt% NaOH/water solution for three times and
for increasing photosensitizer adsorption due to (i) its layered structure,
distilled under nitrogen atmosphere to remove inhibitors and trace
within whose interlayers exist exchangeable cations [55] that can be
water. Methacrylic acid (MAA), potassium persulfate (K2S2O8), sodium
replaced with cationic photosensitizers, and (ii) MMT is designated by
dodecylbenzenesulfonate (SDBS), chloroform, N,N-dimethylformamide
the US FDA as a Generally Recognized as Safe (GRAS) food additive up
(DMF), potassium chloride, Tween 80, disodium hydrogen phosphate
to 2% w/w [56], and is thus unlikely to have any toxicity in the topical
(Na2HPO4), and monopotassium phosphate (KH2PO4) were purchased
applications envisioned here. To that end, poly(methyl methacrylate-
as reagent grade chemicals (when available) from Sinopharm Chemical
co-methacrylic acid) polymer [henceforth abbreviated as P(MMA-co-
Reagent Co., Ltd., and were used as received. Tryptone, soy peptone,
MAA)], was synthesized by emulsion polymerization, blended with
1,5-dihydroxynaphthalene, fluorescein isothiocyanate (FITC) were
MMT, and then electrospun into P(MMA-co-MAA)/MMT composite
purchased from Shanghai Vita Chemical Reagent Co. Ltd.
nanofibers possessing a high specific surface area. We will show that the
Staphylococcus aureus (ATCC-6538) and Escherichia coli strain 8099
MMT additive enables internally (within the MMT interlayers) and
were obtained from Shanghai Xiejiu Biotech. Co., Ltd. A NOVA II power
externally (surface functionalized) bound PSs to both exist within a
meter (Orphir Optronics, Israel) was used to determine the fluence rate
single nanofiber, thus endowing the composite nanofibers with
of the light intensity.

Fig. 1. Top: Preparation of composite nanofibers via electrospinning and photosensitizer (PS) adsorption. Shown are the photographic images of the P(MMA-co-
MAA)/MMT-6 and MB-decorated P(MMA-co-MAA)/MMT-6 nanofibrous membranes. Bottom: a schematic representation of the MB-decorated P(MMA-co-MAA)/
MMT-6 composite nanofiber showing electrostatic surface functionalization and MMT interlayer adsorption of the cationic MB (depicted as the blue oval). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

248
Q. Wang et al.
2.2. Polymerization of P(MMA-co-MAA) Co-polymers
P(MMA-co-MAA) co-polymers were synthesized by an emulsion co-
polymerization approach with a mass ratio of MMA to MAA of 4:1, as
follows: a pre-emulsified monomer solution was prepared upon addi-
tion of 40 g MMA, 10 g MAA and 1.145 g SDBS to 15 mL deionized
water, followed by vigorous stirring, while the initiator solution was
separately prepared upon addition of 0.458 g K2S2O8 to 10 mL deio-
nized water. Under N2 atmosphere, to a 500 mL three-neck flask con-
taining 200 mL deionized water at 80 °C was added 1/3 of the pre-
emulsified monomer solution and 1/2 of the initiator solution, and
stirred until a slight blue color was observed, after which the remaining
monomer and initiator solutions were then added to the flask. The re-
action mixture was kept under N2 atmosphere and reacted for 4 h, after
which a 1 wt% HCl aqueous solution was added until demulsification
was observed (∼15 mL). Centrifugation (400 rpm) was employed to
wash the as-synthesized polymer solids with deionized water three
times, the polymer cake was obtained by filtration, and then dried in a
vacuum oven at 35 °C to constant weight. The as-synthesized P(MMA-
co-MAA) co-polymer was found to contain 6.64 wt% carboxylic acid
groups as determined by Boehm titration [59,60].
Gel permeation chromatography (GPC) analysis was carried out
with an Agilent Model 1100 HPLC equipped with a UV/vis detector.
The analysis was conducted at 25 °C using THF as the eluent at a flow
rate of 1.7 mL/min. A total of 20 μL of a 5 mg/mL solution of the ana-
lyte in THF was injected onto two Styragel columns (Styragel HR 2 THF
and Styragel HR 4 THF, Waters) connected in series for analysis.
Standard monodisperse polystyrenes were used for calibration.
2.3. Electrospinning and characterization of P(MMA-co-MAA)/MMT
composite nanofibers
2.3.1. Electrospinning of P(MMA-co-MAA)/MMT composite nanofibers
Variable weight ratios, 100:0, 98:2, 96:4 and 94:6, of P(MMA-co-
MAA) and MMT, respectively, were initially mixed as dry solids, and
then dissolved in DMF/chloroform (7:3 w/w) blend solution to a final
concentration of 5 wt%. After vigorous stirring for 2 h, upon which a
homogeneous solution was obtained, the solution was placed in a syr-
inge equipped with a blunt-end stainless steel needle. The applied
voltage was set as 15 kV with a working distance of 15 cm from the
stainless steel needle tip to the collector site (circular rotating drum
covered with aluminum foil), and the flow rate was kept at 0.5 mL h−1.
The as-electrospun nanofibrous membranes were collected, and de-
noted as pristine P(MMA-co-MAA) (100:0), P(MMA-co-MAA)/MMT-2
(98:2), P(MMA-co-MAA)/MMT-4 (96:4) and P(MMA-co-MAA)/MMT-6
(94:6).
2.3.2. Characterization of P(MMA-co-MAA)/MMT composite nanofibers
Scanning electron microscopy (SEM) of the electrospun P(MMA-co-
MAA) and P(MMA-co-MAA)/MMT composite nanofibers was performed
on a Quanta 200 (Holland FEI Company). The samples were coated
with a thin layer of gold nanoparticles by sputtering before the SEM
imaging, where the magnification size used was ×104. Transmission
electron microscopy (TEM) was conducted on P(MMA-co-MAA)/MMT-
6 composite nanofibers using a JEOL 2010 (Hitachi Co., LTD). In lieu of
aluminum foil, a copper wire mesh was used to collect the nanofibers
during electrospinning for 2–3 s. Fourier transform infrared (FTIR)
spectra were recorded on a Nicolet Nexus (Thermo Electron
Corporation) within the range of 4000–400 cm−1 using a KBr pellet (32
scans, 4 cm−1 resolution). Thermogravimetric analysis (TGA) was
performed with a TA-Q500 (TA Instruments Company) from room
temperature to 800 °C, and at a heating rate of 10 °C/min under N2.
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Applied Surface Science 457 (2018) 247–255
2.4. Preparation and characterization of MB-decorated P(MMA-co-MAA)/
MMT composite nanofibers
2.4.1. Preparation of MB-decorated P(MMA-co-MAA)/MMT composite
nanofibers
To a conical flask containing 40 mL methylene blue aqueous solu-
tion (100 mg/L) was added 100 mg P(MMA-co-MAA)/MMT composite
nanofibrous membranes. The conical flask was then placed in a shaking
bath at 30 °C (80 rpm). After 6 days, the membranes were placed in
100 mL phosphate-buffered saline solution (pH 6.5) to achieve deso-
rption equilibrium (∼3 days). Periodic sampling of the conical flask
solution enabled determination of the adsorption capacity of methylene
blue per g composite membrane by UV–visible spectroscopy using the
known molar absorptivity coefficient of methylene blue
(ε664 = 95,000 M−1 cm−1) [61].
2.4.2. Characterization of MB-decorated P(MMA-co-MAA)/MMT
composite nanofibers
Confocal laser scanning microscopy was performed on a TCS SP8
instrument (Leica Microsystems GmbH, Germany): for visualization of
the MB-decorated composite nanofibers, an excitation wavelength of
514 nm and an emission wavelength of 694 nm were used, and fluor-
escein isothiocyanate (FITC, λex = 488 nm, λem = 518 nm) labeling
was employed to visualize S. aureus. Samples were prepared by placing
a very thin electrospun nanofibrous membrane on a glass slide, adding
one drop of water to smooth the surface, and then a cover glass was
added and whose four sides were sealed with nail polish. The electronic
absorption spectra of the MB-decorated P(MMA-co-MAA)/MMT com-
posite nanofibrous membrane and a MB aqueous solution (10 mg/L)
were recorded using a UV–vis spectrophotometer (UV-9600, Beijing
Beifen-Ruili Analytical Instrument (Group) Co., Ltd).
2.5. Water absorption behavior
Per literature protocol [62], the dynamic wetting behavior of the
samples was tested by a DCAT 21 combined dynamic contact angle
measuring device and tensiometer (DataPhysics Instruments GmbH).
Samples measuring 1 x 5 cm were prepared and then immersed into an
aqueous MB solution (100 mg/L, 313 μM). The weight of the sample
was recorded during the process, and the mass of water absorbed was
plotted as a function of time.
2.6. Photooxidation studies
Both 1,5-dihydroxynaphthalene (1,5-DHN) and potassium iodide
were chosen as model substrates [49,63] to investigate the photo-
oxidation properties of MB-decorated P(MMA-co-MAA)/MMT-6 com-
posite nanofibers, as follows: to a 50 mL beaker was added a 10 mL
solution of either 300 μM 1,5-DHN in acetonitrile or 0.1 M aqueous KI.
A square sample of MB-decorated P(MMA-co-MAA)/MMT composite
nanofibrous membrane (50 mg) was then horizontally suspended in a
beaker using cotton yarn at each of the four corners, enabling a mag-
netic stir bar to be placed beneath the membrane (80 rpm). The sample
was illuminated with visible light produced by an LED lamp at a fixed
distance of 8 cm (35 ± 5 mW/cm2), and the temperature was main-
tained at 25 °C. The UV–vis spectra were recorded at regular intervals
(UV9600, Beifen-Ruili Analytical Instruments Co., Ltd., Beijing, China).
2.7. Photodynamic inactivation assays
Photoinactivation studies employing the model bacteria
Staphylococcus aureus (ATCC 6538) and Escherichia coli strain 8099 were
performed in triplicate as previously described [19,40,64]. Briefly,
three samples of the nanofibrous membranes [P(MMA-co-MAA)/MMT-
0, P(MMA-co-MAA)/MMT-2, P(MMA-co-MAA)/MMT-4, P(MMA-co-
MAA)/MMT-6), with and without MB decoration] were individually
Q. Wang et al.
placed into adjacent wells of two identically prepared 24-well flat-
bottom plates. Samples were cut to precisely fit the well bottom
(∼1.8 cm in diameter, ∼0.1 mm thickness). A 100 μL aliquot of PBS
containing 1–5 × 108 CFU/mL of bacteria was added to each of the
three wells per plate. Once inoculated, one 24-well plate was illumi-
nated for 30 min with an LED lamp (400–700 nm, 35 ± 5 mW/cm2
fluence rate), while the other 24-well plate was kept without illumi-
nation for an identical time period as a dark control. Following illu-
mination, 0.9 mL of sterile PBS was added to each well in both the il-
luminated and the dark control plates, and the plates were vigorously
vortexed for 5 min to re-suspend the bacteria. Then each well was 1:10
serially diluted (100 μL in 0.9 mL aliquots of PBS) five times, and 10 μL
from the undiluted and each diluted well were separately plated in
columns on gridded six column square plates (TSB-agar plates for S.
aureus; LB-agar plates for E. coli), followed by overnight dark incubation
at 37 °C. The survival rate was determined by the ratio of CFU/mL of
the illuminated plate versus that of the corresponding dark control (set
to 100%). The minimum detection limit was 100 CFU/well (based on
the plated 10 μL aliquot from the 1 mL undiluted well), and the detec-
tion limit range of bacterial survival was 0.0001%. Statistical sig-
nificance (two tailed P value) was assessed via an unpaired Student’s t-
test.
3. Results and discussion
3.1. Preparation and characterization of MB-decorated P(MMA-co-MAA)/
MMT composite nanofibrous membranes
The preparation of P(MMA-co-MAA)/MMT composite nanofibers
was accomplished by thoroughly mixing P(MMA-co-MAA) and MMT at
variable composition ratios prior to electrospinning. Application of a
high voltage and collection on an aluminum foil target yielded the P
(MMA-co-MAA)/MMT composite nanofiber materials as shown in
Fig. 1. Gel permeation chromatography (data not shown) determined
the molecular weight of the parent P(MMA-co-MAA) co-polymer to be
in the range of 415,000–462000 g mol−1, with a Mw/Mn of 2.26. Fol-
lowing electrospinning, MB decoration was performed via a simple
adsorption process, during which MB was electrostatically attracted to
the negatively charged carboxylate (CO2−) groups on the nanofiber
surface, or adsorbed within the nanofiber-embedded MMT interlayers
via a cation exchange process. Such adsorption of MB has been pre-
viously shown to be feasible for pristine MMT [65,66], however here
we show that MB can be adsorbed by MMT within nanofibers them-
selves. The amount of methylene blue adsorbed per g composite
membrane was determined to be 21.5 mg (67.1 μmol), 23.5 mg
(73.5 μmol), 24.1 mg (75.3 μmol), and 30.0 mg (93.8 μmol) for P(MMA-
co-MAA), P(MMA-co-MAA)/MMT-2, P(MMA-co-MAA)/MMT-4, P
(MMA-co-MAA)/MMT-6, respectively (Fig. S1), confirming that the
MMT doping increased the photosensitizer loading of the resultant
composite nanofibers by 40% above that achievable solely by surface
functionalization of the parent P(MMA-co-MAA) co-polymer. In line
with the adsorption of the photosensitizer, the MB-decorated composite
nanofibers exhibited a strong blue color as depicted by its UV/visible
absorption spectrum (Fig. S2), with an absorption maximum of 683 nm,
a bathochromic shift of 19 nm compared to the solution spectrum of
MB. When compared to their solution spectra, such bathochromic shifts
observed for photosensitizers immobilized on solid supports have been
previously [39,40] attributed to the differences in the local environ-
ment (e.g., polarity, solvation) of the photosensitizer, and we suggest a
similar phenomenon here.
3.1.1. Scanning electron microscopy (SEM)
Characterization by scanning electron microscopy (SEM) of the P
(MMA-co-MAA) co-polymer and P(MMA-co-MAA)/MMT composite
nanofibers revealed the effect of MMT on the morphology of the na-
nofibers (Fig. 2). The pure P(MMA-co-MAA) nanofibers exhibited a
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Applied Surface Science 457 (2018) 247–255
Fig. 2. SEM images of P(MMA-co-MAA), P(MMA-co-MAA)/MMT-2, P(MMA-co-
MAA)/MMT-4, and P(MMA-co-MAA)/MMT-6 composite nanofibers.
smooth surface and a relatively uniform diameter distribution, with an
average fiber diameter of 564 nm (Fig. 2a). Upon addition of MMT
yielding the P(MMA-co-MAA)/MMT-2 composite (Fig. 2b), an increase
in the average fiber diameter to 786 nm was noted, as was a minor
change in the fiber morphology: the MMT intercalated nanofibers had
bulges and protrusions, with an uneven surface and irregular diameter
along the axial direction. Similarly, P(MMA-co-MAA)/MMT-4 (Fig. 2c)
and P(MMA-co-MAA)/MMT-6 (Fig. 2d) also showed a comparable
morphology to that of P(MMA-co-MAA)/MMT-2, with an increased
fiber diameter and non-uniform structure.
Transmission electron microscopy (Fig. S3) images further con-
firmed the SEM results: the P(MMA-co-MAA) co-polymer exhibited a
smooth, uniform fiber morphology, whereas the P(MMA-co-MAA)/
MMT-6 composite nanofibers showed a non-uniform distribution with
features (bulges, protrusions) attributable to MMT intercalation.
3.1.2. Confocal laser scanning microscopy (CLSM)
The structure of the electrospun P(MMA-co-MAA)/MMT composite
nanofibers was visualized via confocal laser scanning microscopy
(Fig. 3). As expected, P(MMA-co-MAA) showed no significant fluores-
cence emission spectrum (Fig. 3a), however the CLSM image of P(MMA-
co-MAA)/MMT composite nanofibers did exhibit some slight, dis-
continuous fluorescence (Fig. 3b), which was attributed to the em-
bedded montmorillonite: the pristine MMT nanoparticles themselves
exhibited a similarly weak fluorescence (Fig. S4) [66], likely due to the
fact that organically-modified MMT was employed in the present study.
Importantly, those fluorescence signals originated exclusively from the
nanofibers in P(MMA-co-MAA)/MMT-6, indicating that the MMT na-
noparticles were uniformly well-incorporated within the composite
nanofibers rather than being scattered in-between the fibers. As ex-
pected, adsorption of the methylene blue photosensitizer yielding MB-
decorated P(MMA-co-MAA) (Fig. 3c) and MB-decorated P(MMA-co-
MAA)/MMT-6 (Fig. 3d) showed strong fluorescence signals for both
that were coincident with the nanofibers, however it was not possible to
distinguish between MB that was immobilized on the surface of the
nanofibers vs. within the MMT interlayers.
Confocal laser scanning microscopy was also employed to visualize
the MB-decorated P(MMA-co-MAA)/MMT-6 composite nanofibers in
the presence of the Gram-positive bacterium Staphylococcus aureus
(ATCC-6538) (Fig. S5). Visualization of fluorescein-labeled (FITC,
λex = 488 nm, λem = 518 nm) S. aureus showed the bacteria to be dis-
persed throughout the material (Fig. S5(a)). When excited at 514 nm
and monitored at 694 nm, the MB photosensitizer within the composite
nanofibers was also visualized (Fig. S5(b)). Importantly, as shown in the
Q. Wang et al. Applied Surface Science 457 (2018) 247–255

Fig. 3. CLSM images (λex = 514 nm, λem = 694 nm) of (a) P(MMA-co-MAA), (b) P(MMA-co-MAA)/MMT-6, (c) P(MMA-co-MAA)/MB and (d) P(MMA-co-MAA)/
MMT-6/MB composite nanofibers.

fluorescence signal overlay of panels A and B (Fig. S5(c)), no fluores-

cence intensity ascribed to methylene blue overlapped the bacteria
outside of the nanofibers, demonstrating that no leaching of the pho-
tosensitizer from the composite nanofibers to the bacteria was occur-
ring, and establishing the composite nanofibers as a photodynamic
material rather than as a carrier of a solution-based photosensitizer.
Finally, scanning electron microscopy was able to successfully visualize
S. aureus as being spherical in shape and forming grape-like clusters
while on the MB-decorated P(MMA-co-MAA)/MMT-6 composite nano-
fibers (Fig. S5(d)).

3.1.3. Fourier-transform infrared (FTIR) spectroscopy

Fourier-transform infrared spectroscopy provided further evidence
for the presence of MMT within the P(MMA-co-MAA)/MMT composite
nanofibers (Fig. 4). In pristine MMT, the absorption peak observed at
3633 cm−1 could be attributed to the stretching vibration of AleAleOH
groups in the octahedral layer. The sharp peak at 1037 cm−1 was at-
tributed to SieO stretching vibration. OeH bending vibrations in
dioctahedral 2:1 layer silicates located at 916 cm−1, 854 cm−1, and Fig. 4. FT-IR spectra (KBr pellet, 400–4000 cm−1) of P(MMA-co-MAA) (blue), P
(MMA-co-MAA)/MMT-6 (red), and MMT (black). (For interpretation of the
803 cm−1 could be assigned to AleAleOH, AleMgeOH and Mge-
references to colour in this figure legend, the reader is referred to the web
FeeOH, respectively. In addition, tetrahedral bending modes were also
version of this article.)
observed at 520 cm−1 for SieOeAl and at 468 cm−1 for SieOeMg
[67]. The P(MMA-co-MAA) and P(MMA-co-MAA)/MMT-6 composite
nanofibers both exhibited a broad absorption band at 3600–3100 cm−1 mode of eCH3 and eCH2e groups. The peaks located at 1725 cm−1 are
due to the eOH group on the MAA moiety. The absorption peaks at ascribed to C]O stretching vibration, indicating the existence of eC]
2998 cm−1 and 2948 cm−1 can be attributed to the stretch vibration Oe groups in MMA and MAA [68]. Compared with pure P(MMA-co-

251
Q. Wang et al. Applied Surface Science 457 (2018) 247–255

increase to possibly representing the exchange of the quaternary am-

monium salts within the MMT interlayers with the cationic methylene
blue photosensitizer. After the samples were retracted from the MB
solution (650–680 s), the overall mass increases were found to follow
the trend P(MMA-co-MAA)/MMT-0 < MMT-2 < MMT-4 < MMT-6,
indicating that the concentration of MMT embedded within the
polymer nanofibrous matrix directly correlated with an increased water
absorption capacity.

3.2. Photo-oxidation of model substrates

The mechanism of photodynamic inactivation by methylene blue

Fig. 5. Thermal gravimetric analysis of P(MMA-co-MAA) (black, P(MMA-co- has been previously shown to proceed via generation of singlet oxygen
MAA)/MMT-6 (red), and MMT (blue). (For interpretation of the references to in studies employing both Gram-negative E. coli and Gram-positive S.
colour in this figure legend, the reader is referred to the web version of this aureus [69–71], the bacteria under investigation here (see below). To
article.) confirm the ability of the MB-decorated P(MMA-co-MAA)/MMT com-
posite nanofibers to generate singlet oxygen, photooxidation studies of
MAA) nanofibers, P(MMA-co-MAA)/MMT-6 composite nanofibers the model substrates 1,5-dihydroxynaphthalene (1,5-DHN) and KI were
showed two new absorption peaks at 520 cm−1 and 468 cm−1, in- investigated. Specifically, the oxidation of 1,5-dihydroxynaphthalene
dicating the presence of MMT within the P(MMA-co-MAA) matrix. (1,5-DHN) to 5-hydroxy-1,4-naphthoquinone (5-HNQ) as catalyzed by
singlet oxygen [63,72] that was photo-generated upon illumination of
the MB-decorated P(MMA-co-MAA)/MMT-6 composite nanofibrous
3.1.4. Thermal gravimetric analysis
membranes is shown in Fig. 6. The progress of the photooxidation re-
Thermal gravimetric analysis was performed on the parent P(MMA-
action was clearly observed in the UV–visible spectra (Fig. 7), where the
co-MAA) copolymer and the P(MMA-co-MAA)/MMT-6 composite na-
absorption maximum of 1,5-DHN at 330 nm decreased with the ap-
nofibers to gain an understanding of the thermal stability of these
pearance of a new peak at 420 nm, corresponding to the formation of 5-
materials (Fig. 5). Initially, a minor weight loss (∼5%) was observed
HNQ (ε415 = 3600 M−1 cm−1 [73]). As expected, no 5-HNQ product
for both materials up to 240 °C. From ∼250 to 347 °C, a slightly greater
was observed in the absence of illumination (data not shown). Simi-
thermal degradation of the P(MMA-co-MAA)/MMT-6 composite nano-
larly, the oxidation of I- to I3- as catalyzed by singlet oxygen [74] that
fibers (red trace) was noted compared to the P(MMA-co-MAA) copo-
was generated upon illumination of the MB-decorated P(MMA-co-
lymer (black trace), which was ascribed to the decomposition of the
MAA)/MMT-6 composite nanofibrous membrane is shown in Fig. S6.
surfactant bis(2-hydroxyethyl)methyltallow ammonium salts contained
The appearance of two new spectral features at 298 and 350 nm, at-
within the MMT interlayer of the composite nanofiber, and matches
tributable to I3- formation [75], were observed that increased in an il-
well with the thermal degradation of MMT itself (blue trace). The onset
lumination time-dependent manner. Taken together, the results from
decomposition temperature (Tonset) for P(MMA-co-MAA)/MMT-6 com-
the photooxidation studies of 1,5-DHN and KI strongly suggested that
posite nanofibers was 347 °C, a negligible difference to that of neat P
MB-decorated P(MMA-co-MAA)/MMT-6 composite nanofibrous mem-
(MMA-co-MAA) (349 °C). In addition, the P(MMA-co-MAA)/MMT-6
branes were able to generate singlet oxygen upon visible light illumi-
achieved pyrolysis equilibrium within a relatively sharp period of
nation (see Fig. 7).
343–424 °C, which was at a slightly lower temperature than that of the
P(MMA-co-MAA) copolymer (352–443 °C). Finally, virtually identical
thermal degradation patterns to that of P(MMA-co-MAA)/MMT-6 were 3.3. Evaluation of antibacterial efficacy
noted for P(MMA-co-MAA)/MMT-2 and P(MMA-co-MAA)/MMT-4
composite nanofibers (data not shown). Taken together, the thermal In vitro aPDI studies employing the MB-decorated P(MMA-co-MAA)/
gravimetric analysis data suggest that the addition of MMT does not MMT composite nanofibrous membranes were performed against
significantly alter the thermal stability of the parent co-polymer. Gram-positive S. aureus (ATCC 29213) (Fig. 8A) and Gram-negative E.
coli 8099 (Fig. 8B) using LED lamp illumination (30 min, 35 ± 5 mW/
cm2). As expected, the photosensitizer-free P(MMA-co-MAA)/MMT
3.1.5. Dynamic wetting behavior
composite nanofibers showed no significant antibacterial activity
The dynamic wetting behavior of the P(MMA-co-MAA) co-polymer
(< 0.5 log units), neither in the dark (Fig. 8, black) nor when illumi-
and P(MMA-co-MAA)/MMT composite nanofibers is represented by the
nated (grey). Demonstrating the necessity for the photosensitizer, the
water absorption curves in Fig. 5. A detailed analysis of such absorption
photodynamic inactivation of S. aureus using the MB-decorated P
behavior has been well illustrated in our previous work [62]. Here, the
samples were subjected to wetting by an aqueous methylene blue so-
lution. Initially, a sudden negative weight change was observed for all
samples when breaking the solution/air interface, which was attributed
to the hydrophobicity of the samples where the surface tension of the
water/air interface resists interruption, a factor that contributes to this
negative force input. For P(MMA-co-MAA), a mass increase occurred
after ∼40 s, while it took ∼55 s for the P(MMA-co-MAA)/MMT com-
posite nanofibers to show a similar mass increase, suggesting MMT
addition yields a more hydrophobic surface. The samples were then
held static within the MB solution for 10 min, after which absorption
equilibrium was achieved within 100 s. From 100 to 650 s, a further
increase in mass was observed, the magnitude of which scaled with the
wt% MMT in the samples: i.e., the P(MMA-co-MAA)/MMT-6 composite
nanofibrous membrane showed the greatest mass increase while P Fig. 6. Water absorption behavior of P(MMA-co-MAA), P(MMA-co-MAA)/
(MMA-co-MAA) was relatively stable after 450 s. We ascribe this mass MMT-2, P(MMA-co-MAA)/MMT-4, and P(MMA-co-MAA)/MMT-6.

252
Q. Wang et al. Applied Surface Science 457 (2018) 247–255

2.5 1.2 (MMA-co-MAA)/MMT composite nanofibrous membranes was found to

317 nm
0.9

Abs420
331 nm
0.6
be moderately effective, achieving 99.9% reduction (3 log units,
2.0 0.3 P = 0.004) in CFU/mL after 30 min of visible light illumination for the
0 MMT-0 material (parent copolymer), but increased to a more im-
Absorbance

0 30 60 90 120
1.5 Time (min) pressive 99.997% (4.8 log units, P = 0.0005) pathogen inactivation for
420 nm the MMT-6 containing composite nanofibers (Fig. 8a, light blue). An
1.0 0 min improvement in photodynamic efficacy was noted for the materials that
20 min
40 min contained MMT, suggesting that the MB within the MMT interlayers
0.5 60 min played an additional photocytotoxic role beyond that attributable to the
80 min
100 min
surface-immobilized photosensitizer. Against E. coli, however, a re-
0.0 120 min duced efficacy was noted, achieving only an 84% reduction (0.9 log
275 300 325 350 375 400 425 450 475 500 units, P = 0.001) in CFU/mL after 30 min illumination for the MMT-0
Wavelength (nm) material, which increased to 97% (1.8 log units, P = 0.0015) pathogen
Fig. 7. UV–visible spectroscopic monitoring of the oxidation of 1,5-dihydrox-
inactivation for the MMT-6 containing composite nanofibers (Fig. 8b,
ynaphthalene (1,5-DHN) to 5-hydroxy-1,4-naphthoquinone (5-HNQ) as cata- light blue). The differential efficacy noted between Gram-positive S.
lyzed by MB-decorated P(MMA-co-MAA)/MMT-6 composite nanofibers, prior to aureus and Gram-negative E. coli was not unexpected, as Gram-negative
(red; t = 0 min) and post (purple; t = 120 min) illumination (LED lamp, bacteria possess an additional outer membrane of highly impermeable
35 ± 5 mW/cm2); each interval represents a 20 min timepoint. Inset: single lipopolysaccharides that makes them more resistant than Gram-positive
wavelength absorbance (420 nm) of 5-HNQ as a function of illumination time. species to photodynamic inactivation [76]. Overall, however, our re-
(For interpretation of the references to colour in this figure legend, the reader is sults demonstrate that the increase in MB photosensitizer loading from
referred to the web version of this article.) the addition of MMT in the electrospinning process increased the bio-
cidal activity of the resultant composite nanofibers with a 1–2 log units
A 100 improvement in photodynamic inactivation efficacy.
Remarkably, the dark controls also exhibited inactivation against S.
10 aureus that was most pronounced for the MMT-6 containing composite
nanofibers with 30 min dark incubation (24%, ∼0.4 log units,
Survival %

1 P = 0.0013; Fig. 8a, dark blue). We attributed this dark inactivation to

the incidental photodynamic production of singlet oxygen arising from
the minimal ambient room light present during the assay. Such in-
0.1
activation under dark control conditions has been noted previously for
several photodynamic materials, including Por(+)-paper (a cellulose-
0.01
porphyrin conjugate [40]), PAN-Por(+) (a porphyrin-embedded poly-
acrylonitrile non-woven textile [54]), RC-TETA-PPIX-Zn (a regenerated
0.001
MMT-0 MMT-2 MMT-4 MMT-6 cellulose nanofiber-porphyrin conjugate [77]), and MB-polyester fab-
MMT-0 MMT-2 MMT-4 MMT-6
rics (unpublished results). In each of these aforementioned materials, a
P(MMA-co-MAA)/MMT (dark)
high photosensitizer loading results in sensitivity to the minimal light
P(MMA-co-MAA)/MMT (+ light)
conditions employed during the aPDI assay (i.e., serial dilution, plating
MB-decorated P(MMA-co-MAA)/MMT (dark)
procedures), and we suggest a similar phenomenon is occurring here
MB-decorated P(MMA-co-MAA)/MMT (+ light)
with the MB-decorated P(MMA-co-MAA)/MMT composite nanofibers.
B 100 The dark controls for the assays employing E. coli, however, did not
exhibit any dark inactivation (Fig. 8b, dark blue), in line with the above
10 discussion that Gram-negative bacteria are more robust towards pho-
Survival %

todynamic inactivation than their Gram-positive counterparts.

1

0.1 4. Conclusion

0.01 We have successfully synthesized a series of P(MMA-co-MAA)/MMT

0.001
MMT-0
1 MMT-2
2 MMT-4
3 MMT-6
4
Fig. 8. Photodynamic inactivation studies employing MB-decorated P(MMA-co-
MAA)/MMT nanofibrous membranes with 0–6 wt% MMT against (A) S. aureus
(ATCC 29213) and (B) E. coli 8099. Displayed is the % survival (vs. material-
free dark control) for photosensitizer-free controls of P(MMA-co-MAA)/MMT in
the dark (black) and illuminated (grey), and for MB-decorated P(MMA-co-
MAA)/MMT in the dark (dark blue) and illuminated (light blue). Illumination
conditions were as follows: LED lamp, 30 min, 35 ± 5 mW/cm2. The detection
limit was 0.0001%, and in the cases where error bars cannot be visualized, the
error bars themselves were smaller than the marker employed in the plot. (For
interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
composite nanofibers that contained both internal and surface-func-
tionalizable sites for cationic photosensitizer immobilization. Our re-
sults demonstrated that such materials are capable of achieving a
∼99.997% photodynamic inactivation of Gram-positive Staphylococcus
aureus in under 30 min of visible light illumination from a standard LED
bulb, albeit a more modest level of inactivation was observed against
Gram-negative E. coli. While other photodynamic materials have shown
better efficacy owing to the use of more costly or synthetically chal-
lenging photosensitizers and/or grafting methods, the results reported
here nevertheless demonstrate the utility of this approach: when com-
pared to the parent P(MMA-co-MAA) copolymer, we found that the
increase in photosensitizer adsorption attributed to the MMT doping led
to a 1–2 log unit increase in photodynamic inactivation efficacy. Given
this improvement that comes with a minimal cost and impact on pro-
duction methods, consideration should be given to revisiting previously
studied photodynamic materials to now include additives such as MMT,
or in the production of next-generation materials for improved photo-
dynamic inactivation efficacy.

253
Q. Wang et al.
Acknowledgements
We thank the financial support from the 111 Project (B17021),
Recruitment Program of Foreign Experts B (JSB2017016), Natural
Science for Youth Foundation (51603090), Natural Science Foundation
of Jiangsu Province (BK20150155) and Fundamental Research Funds
for the Central Universities (JUSRP115A04).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.apsusc.2018.06.041.
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