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org/journal/acsodf Article

Antifouling Silicone Hydrogel Contact Lenses with a Bioinspired

2‑Methacryloyloxyethyl Phosphorylcholine Polymer Surface
Kazuhiko Ishihara,* Kyoko Fukazawa, Vinay Sharma, Shuang Liang, Amanda Shows,
Daniel Chuck Dunbar, Yang Zheng, Junhao Ge, Steve Zhang, Ye Hong, Xinfeng Shi,
and James Yuliang Wu*
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Metrics & More Article Recommendations sı Supporting Information

ABSTRACT: Inspired by the cell membrane surface as well as the ocular tissue,
a novel and clinically applicable antifouling silicone hydrogel contact lens material
was developed. The unique chemical and biological features on the surface on a
silicone hydrogel base substrate were achieved by a cross-linked polymer layer
composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), which was
considered important for optimal on-eye performance. The effects of the polymer
layer on adsorption of biomolecules, such as lipid and proteins, and adhesion of
cells and bacteria were evaluated and compared with several conventional silicone
hydrogel contact lens materials. The MPC polymer layer provided significant
resistance to lipid deposition as visually demonstrated by the three-dimensional confocal images of whole contact lenses. Also,
fibroblast cell adhesion was decreased to a 1% level compared with that on the conventional silicone hydrogel contact lenses. The
movement of the cells on the surface of the MPC polymer-modified lens material was greater compared with other silicone hydrogel
contact lenses indicating that lubrication of the contact lenses on ocular tissue might be improved. The superior hydrophilic nature
of the MPC polymer layer provides improved surface properties compared to the underlying silicone hydrogel base substrate.

■ INTRODUCTION
Contact lenses are medical devices widely used for correcting
which is physically adsorbed to the surface of the lens,
increasing the hydrophilicity and lubrication of the lens.11,12
vision. The most advanced polymer materials have been However, if contact lenses are worn for a prolonged period,
then the improved wettability and lubricity from the water-
utilized to create contact lenses with good optical character-
soluble polymers is reduced as the substance is washed away.
istics, comfort of wearing, and safety for various patients
Therefore, it is important that a polymer, developed for surface
regardless of their ages.1−3 In the past 20 years, many high
modification of silicone hydrogel lens materials, creates a stable
oxygen permeable silicone hydrogel materials have been
surface that may resist lipid deposition, protein adsorption, and
developed for supplying increased oxygen levels to the ocular
bacterial adhesion.
tissue so that they can be worn for an extended period of
A series of polymers containing 2-methacryloyloxyethyl
time.4,5 Generally, silicone hydrogel materials contain poly-
phosphorylcholine (MPC) unit have attracted great attention
dimethylsiloxane (PDMS) in addition to hydrophilic mono-
due to the bioinspired chemical structure of the MPC unit also
mers. The PDMS phase and the hydrophilic polymer phase
found in phospholipids on the cell membrane surface.13,14 Due
form a micro-phase-separated structure. Oxygen gas diffuses
to the zwitterionic nature of the phosphorylcholine head group
through the PDMS phase and is supplied to the ocular
in the MPC polymer, water molecules surrounding the MPC
tissue.6,7 However, PDMS can pose some problems in the
unit take a unique hydration structure.13,15 Therefore, it has
ocular environment due to its hydrophobic nature.8−10 The
been observed that water lubrication properties on the
first problem is the adsorption of biomolecules. Lipids and
substrates are improved on MPC polymer surfaces, which
proteins in tears are adsorbed to the PDMS phase, where a could contribute to a more comfortable contact lens.16−20 In
stable adsorption layer is formed. This can not only affect the addition, the MPC unit has an overall neutral charge, allowing
optical properties, surface wettability of the contact lens, and
oxygen permeability as an imperfection on the contact lens
surface but also act as attachment points for bacterial adhesion. Received: December 30, 2020
Furthermore, the hydrophobic PDMS phase can reduce the Accepted: February 3, 2021
lubricating properties of the contact lens. In order to solve Published: February 26, 2021
these problems, many silicone hydrogel contact lenses have
been treated by adding surfactants or a water-soluble polymer,
such as poly(N-vinylpyrrolidone), to the packaging solution,

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the polymer to be excellent at suppressing protein adsorption,
cell adhesion, and reduce blood coagulation and immune
responses.13,21,22 Therefore, the MPC polymers have been
applied as a surface treatment for medical devices including
conventional soft contact lenses.16 These characteristics of the
MPC polymer might be used to improve the lubricity and
antifouling properties of silicone hydrogel lenses. We have
reported the stable chemical reaction of the MPC polymer at
the surface of the silicone hydrogel material and character-
ization of the surface in the aqueous medium.23 When the
surface is covered with the MPC polymer on the base silicone
hydrogel material, a water-rich and higher lubricious surface is
formed. In this study, we evaluated the surface biological
reactions of several silicone hydrogel contact lenses including
the MPC polymer-modified contact lens material.
■ RESULTS AND DISCUSSION
AFM Imaging of Contact Lens Substrates. In Table 1,
the base monomer chemicals and hydration degree of silicone
Table 1. Chemical Components of Contact Lens Materials
Used in this Studya
contact lens materials, United hydration
States adopted name base monomers degree (%)
MPC polymer-modified lens mPDMS, G-PDMS, NVP, 55
material and its base substrate TEGDMA, MMA,
EGMEMA
senofilcon C mPDMS, SiGMA, DMA, 41
HEMA, TEGDMA,
b
PVP
samfilcon A TRIS, Ma2D37, M1- 46
(EDS)n-TMS, NVP,
DMA, HEMA
comfilcon A M3U, FMM, VMA, 48
HBMA, IBM, NVP,
TAIC
a
MPC, 2-methacryloyloxyethyl phosphorylcholine; mPDMS, poly-
dimethylsiloxane monomethacrylate; G-PDMS, glycerol-function-
alized polydimethylsiloxane dimethacrylate; SiGMA, [methyl bis-
(trimethylsiloxy)silyl] propyl glycerol methacrylate; NVP, N-vinyl
pyrrolidone; TEGDMA, tetraethylene glycol dimethacrylate; MMA,
methyl methacrylate; EGMEMA, 2-methoxyethyl methacrylate;
DMA, N,N-dimethylacrylamide; HEMA, 2-hydroxyethyl methacry-
late; PVP, poly(N-vinylpyrrolidone); TRIS, trimethylsiloxy silane;
Ma2D37, silicone bis(meth)acrylamide monomer; M1-(EDS)n-TMS,
mono ethylenically unsaturated polymerizable group containing
polycarbosiloxane monomer; M3U, α-ω-bis(methacryloyloxyethyl
iminocarboxy ethyloxypropyl)-poly(dimethylsiloxane)-poly-
(trifluoropropylmethylsiloxane)-poly(ω-methoxy-poly-
(ethyleneglycol)propylmethylsiloxane); FMM, 2-ethyl E2-E(2-meth-
ylprop-2-enoyl)oxyethylcarbamate; VMA, N-vinyl-N-methylaceta-
mide; HBMA, 4-hydroxybutyl methacrylate; IBM, isobornyl
methacrylate; TAIC, 1,3,5-tripop-2-enyl-1,3,5-triazine-2,4,6-
(1H,3H,5H)-trione. bWetting reagent.
hydrogel contact lenses used in this study are summarized.24,25
We developed a new contact lens material using the
bioinspired MPC polymer.23 The surface modification of the
base material with the MPC polymer was carried out by
chemical reaction at the surface. By conducting the XPS
analysis under high vacuum conditions, XPS signals of specific
atoms, that is, carbon at 285 eV, oxygen at 532 eV, nitrogen at
399 eV, and silicone at 102 eV, were observed on the base
substrate (data was not shown). After surface modification
with the MPC polymer, new XPS signals were observed at 133
and 403 eV.23 They were attributed to the phosphorus atom in
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the phosphate group and the nitrogen atom in the
trimethylammonium group, respectively, in the phosphor-
ylcholine group.26,27 These results indicated that the MPC
polymer layer was formed on the surface.12,13
AFM is one of the most promising surface characterization
tools that is capable of revealing the nanoscopic morphological
features with high resolution. This imaging technique has
already been employed extensively in the field of contact lens
research to study different surfaces and contact lens
materials.28−35 In this study, AFM surface imaging was
conducted on different contact lenses including the new lens
material and its non-surface modified base substrate, senofilcon
C, samfilcon A, and comfilcon A. The surface of the contact
lens was kept in a fully hydrated state during experiments. The
AFM topographical side view images of a 5.0 μm × 5.0 μm
scan area from each of these contact lenses are displayed in
Figure 1 where all the images were on the same Z scale, i.e., ±
150 nm. As shown in the figure, the surface of this new lens
was considerably different than the other contact lenses. The
surfaces of the non-surface modified base substrate and the
other three contact lenses, i.e., senofilcon C, samfilcon A, and
comfilcon A, looked relatively flat with an interspersed domain
feature, which is typical for silicone hydrogel contact lens
materials. Conversely, the new lens exhibited distinctive
surface features and topographies resulting from densely
packed MPC polymer units attached to the contact lens
surface. Additional AFM images of all the contact lenses with
the top view angle are provided in Figure S1 as supporting
information. This MPC polymer layer has been well
characterized in our previous report.23
A similar polymer surface gel layer with a near 100% water
content on the surface was also described by Dunn et al.36
Such highly hydrated gel surfaces are as smooth as the surface
of silicone hydrogel substrates or contact lenses, which
typically have 20−80% water contents.1
It is well accepted and reported in the literature that the
grafting polymer layer including the polymer brush structure,
extending normal to the underlying substrate can provide
novel and/or enhanced surface properties such as wettability,
lubricity, reduced biological responses, i.e., antifouling proper-
ties, and so on.37−44 The MPC polymers on the lens surface
can provide super-hydrophilic and antifouling properties. Thus,
we investigated if the MPC polymer layer would provide
unique antifouling surface properties to this new contact lens
in the following sections.
Lipid Adsorption on Contact Lenses. Each contact lens
was incubated in fluorescently labeled ATS for 3 days followed
by a cleaning in OPTI-FREE PUREMOIST MPDS. Whole
lenses were submerged in PBS within a cylinder-shaped lens
holder so that the spherical shape of the lens was maintained.
Figure 2 displays the top views of the three-dimensional (3D)
confocal imaging of this new lens and its base substrate,
senofilcon C, samfilcon A, and comfilcon A. The blue color
came from the fluorescence of the lens material and
background at 405 nm laser excitation. The red color
corresponded to the fluorescently labeled non-polar lipid,
Rh-TAG, excited by a 561 nm laser. To fully demonstrate the
lipid deposition on contact lens, a side view and a 45° tilted
angle view are included in the Supporting Information (Figures
S2 and S3). As shown in the figures, this new lens had no
observable Rh-TAG deposition. As a comparison, senofilcon C
had some Rh-TAG depositions near the contact lens center
while the new lens non-surface modified base substrate and
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Figure 1. AFM topographical images (side view, 5 μm × 5 μm) of the contact lenses used in this study.

chromatography to measure cholesterol sorption on conven-

Figure 2. 3D confocal images (top view, 14.5 mm × 14.5 mm) of the
whole lenses after being soaked in ATS and cleaned by OPTI-FREE
PUREMOIST MPDS.
comfilcon A had a few Rh-TAG deposition near the contact
lens center as well as on the lens edge. Samfilcon A had some
large, bright Rh-TAG signals, mostly on the lens edge (Figure
2). These results demonstrate that the new lens had no
detectable Rh-TAG lipid deposition compared to the other
four contact lens materials. Several studies of lipid sorption on
contact lenses have been reported. Zhao et al. used thin layer
tional silicone hydrogel contact lenses including senofilcon A
after 30 days of wear and reported both lens type, which
averaged from 0.1 to 8.2 μg/lens.45 Also, Pitt et al. reported
that in the case of senofilcon A, 5.3 μg/lens of cholesterol and
4.4 μg/lens of phosphatidylcholines are absorbed, which was
determined by a radio-labeled method.46 Regarding the lipid
adsorption characteristics of the contact lenses used in this
study, a preliminary study was conducted in which lipids
adsorbed were extracted from the contact lenses after being
worn for 30 days on human eye and evaluated using liquid
chromatography equipped with mass spectroscopy. As a result,
the amount of absorbed non-polar lipid was approximately 40
nmol/lens in senofilcon C and decreased in the order of
samfilcon A (20 nmol/lens) and comfilcon A (6 nmol/lens).
Furthermore, the contact lens material modified with the MPC
polymer showed the lowest non-polar lipid adsorption amount,
approximately 3 nmol/lens.
Protein Adsorption Behavior. It is considered that the
adsorption of protein to the surface of the contact lens affects
the characteristics of the contact lens and is one of the factors
that can deteriorate comfort during long-time wear.47−49
Furthermore, it is also known that the adsorption of a specific
protein induces an infection or an immune response due to

Figure 3. Protein adsorption on various silicone hydrogel contact lenses. (A) Amount of protein adsorbed on the surface from artificial tear protein
solution and (B) amount of lysozyme adsorbed from single lysozyme solution (n = 5, mean ± SD). * represents p < 0.01 versus base substrate
without the MPC polymer.

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bacterial adhesion.50,51 The state of protein adsorption to the
contact lens was observed using mixed protein solutions.
Figure 3A shows the amount of proteins adsorbed on
various contact lenses from the artificial tear protein solution.
The adsorption amount ranged from 0.2 to 1.2 μg/cm2,
depending on the type of contact lens material. Compared to
senofilcon C, this new lens showed significantly more protein
adsorption. It is generally known that the amount of protein
adsorbed depends on the hydrophobicity of the surface and the
charge density. On the other hand, in the case of hydrogels,
attention must be paid to the diffusion into the materials. The
water content of contact lens materials is low, senofilcon C
having a water content of 41%, while this new lens is 55%.
Lysozyme has a relatively small molecular weight, which makes
it easy to diffuse into the contact lens, and as a result, the
amount of adsorbed protein increases. Thus, the amount of
lysozyme adsorbed on the contact lens from a single protein
solution was measured. Even with a relatively hydrophobic
base material, there is no significant difference between the
amount of protein adsorbed from artificial tear protein solution
and the amount of adsorbed lysozyme alone, as shown in
Figure 3B. On the other hand, senofilcon C and comfilcon A
reduced the amount of protein adsorbed from artificial tear
protein solution. These results suggest that protein adsorption
from artificial tear protein solution relies on different modes of
protein adsorption to the surface and diffusion into the
interior. That is, in senofilcon C and comfilcon A, albumin and
immunoglobulin, with relatively high molecular weights, were
adsorbed on the surface from artificial tear protein solution,
and a stable adsorption layer was formed so that the diffusion
of lysozyme into the substrate was suppressed. For this new
lens, the protein adsorption might occur only on the
hydrophobic surface of the contact lens, the amount of protein
adsorption was almost the same from the artificial tear protein
solution or the lysozyme alone solution. Although the
adsorption itself was suppressed by the MPC polymer layer,
it was hypothesized that the lysozyme was trapped inside the
hydrophobic substrate material because the diffusion of
lysozyme into the inside could not be prevented. Considering
the results of the protein permeability of the MPC polymer
hydrogel membrane reported previously, the permeability
depends on the molecular weight, and the permeability of a
protein of about 104 Da is relatively high.52 On the other hand,
albumin and immunoglobulin (< 6 × 104 Da) hardly permeate.
In addition, the protein adsorption amount is reduced without
depending on the molecular weight, which supports the above
hypothesis.
We evaluated protein adsorption of a specific protein on the
substrate from the protein mixture by a gold-labeled
immunoassay, which is a good methodology for visualization
of proteins on the surface.20 Also, the immunoassay provides
us the information from the most outer surface of contact lens
because the molecular size of an antibody is large compared
with the network structure of the hydrogel contact lens.52
Figure 4 shows the SEM image of adsorption of lysozyme on
the contact lens surface from the artificial tear protein solution
detected by immunoassay.21,53,54 The number of the white
particles corresponds to the adsorbed lysozyme molecules.
This MPC polymer-modified contact lens appears to reduce
the amount of adsorbed lysozyme at the surface. In Figure 4,
only lysozyme among the proteins adsorbed on the surface of
contact lenses is selectively visualized by colloidal gold labeling
immunization.53,54 If protein adsorption occurs in multiple
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Figure 4. SEM image of adsorbed lysozyme on various contact lens
substrates. White particles correspond to adsorbed lysozyme detected
by gold-labeled immunoassay. The bar indicates 100 μm.
layers, or if the protein is absorbed inside the contact lens, then
it is not observed in Figure 4. In fact, the density of colloidal
gold particles is not high until it completely covers the surface.
This does not completely correspond to the result in Figure 3.
That is, the results shown in Figure 3 quantified whole
amounts of the proteins in the adsorption layer.
Cell Adhesion Inhibition on the Contact Lens
Surface. Cell adhesion on various contact lens materials was
evaluated. Due to the effect of autofluorescence on the contact
lens material, the cells could not be fluorescently labeled and
observed with a fluorescence microscope. Therefore, observa-
tion was performed with an optical phase-contrast microscope.
Figure 5A shows optical microscopic images of contact lens
surfaces after 24 h of culturing. Adhered cells were observed on
the base substrate, samfilcon A, and comfilcon A. On the other
hand, a small number of cells adhered on senofilcon C and
almost no cells observed on the new lens itself. By quantitative
counting, the number of adherent cells on the base substrate
was 1.2 ± 0.3 × 104 cells/cm2, senofilcon C was 0.2 ± 0.3 ×
104 cells/cm2, samfilcon A was 1.7 ± 0.3 × 104 cells/cm2,
comfilcon A was 1.8 ± 0.4 × 104 cells/cm2, and MPC polymer-
modified lens was 0.1 ± 0.1 × 102 cells/cm2. No significant
growth of the adhered cells was observed on any of the contact
lens materials during 24 h of culturing. After the cells were
seeded on the surface, the cells weakly adhered to the surface
and then migrated on the surface.55,56 Initial adhesion of cells
to the contact lens surface was significantly different depending
on the contact lens material. The migration of adhered cells on
this new contact lens surface tended to be greater compared to
other contact lenses. Figure 5B demonstrates a series of
pictures of adhered cells on this new lens base substrate
surface. After the cells were seeded and attached to the surface,
cells could migrate along the surface slowly, and adhered cells
were observed in the examination area even after 120 min.
This phenomenon was also observed on the surface of
samfilcon A and comfilcon A. For this new lens, as shown in
Figure 5C, movement of adhered cells was quick, and no cells
were observed in the observation area after 90 min. The
surface of the MPC polymer has been reported to inhibit cell
adhesion.16,37,55,56 Thus, on the surface of the new lens, the
MPC polymer layer at the surface had improved resistance to
cell adhesion due to the slipping property.16 This property
might improve compatibility of this new contact lens material
with ocular tissue due to improved cell adhesion resistance and
reduced friction at the interface.
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Figure 5. Phase contrast microscopic images of cells adhered on various silicone hydrogel contact lenses. (A) 24 h cell-culturing period for various
silicone hydrogel contact lenses. (B, C) Time-lapse images of adherent cells cultured for 2 h on the base substrate and MPC polymer-modified lens
material, respectively.

Bacterial Adhesion Resistance. Bacterial adhesion to
contact lenses must be minimized to maintain optimal ocular
health. In particular, when a contact lens is used for multiple
days, the bacterial growth over time could become a problem.
There are not many reports that have been made on the effect
of bacterial types on bacterial adhesion to hydrogel materials.
Absolom et al. analyzed the adhesion of five types of bacteria to
polymers with different degrees of hydrophilicity using a
thermodynamic model.57,58 As a result, it is concluded that
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bacterial adhesion is determined by the difference in bacterial
surface properties and surface tension of the substrate and
suspension medium, that is, the surface free energy. However,
it can be said that the bacteria adhesion occurs in a very
complex manner.
MPC polymers have been reported to reduce bacterial
adhesion, including the common nosocomial pathogens such
as Staphylococcus aureus, S. epidermidis, Candida albicans, and
Pseudomonas aeruginosa.55,56,59−62 It has been reported that
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Figure 6. Number of bacteria adhered on various silicone hydrogel contact lenses. (A) Adhesion of E. coli and (B) adhesion of P. aeruginosa. (n = 6,
mean ± SD) * represents p < 0.01 versus base substrate without the MPC polymer.
intraocular lenses coated with the MPC polymer suppress the
adhesion of S. aureus and S. epidermidis. Also, Selan et al.
compared typical materials for contact lenses, silicone
hydrogel, poly(2-hydroxyethyl methacrylate (HEMA)), and
MPC polymer-coating materials in terms of their susceptibility
to biofilm formation by S. epidermidis and P. aeruginosa and
concluded that the MPC polymer can inhibit biofilm formation
significantly.62
Figure 6 shows the number of Escherichia coli and P.
aeruginosa adhered to various contact lens surfaces. The
poly(ethylene terephthalate) (PET) substrate served as a
control sample. A little number of E. coli was adhered on the
MPC polymer-modified lens material and senofilcon C as well
as the new lens base substrate compared to the PET substrate,
and no significant differences were observed among these test
samples. On the other hand, it was found that the adhesion of
P. aeruginosa was lower on the MPC polymer-modified lens
material than the other contact lenses. Although there are still
many unclear points about the influence of the physicochem-
ical properties of the material on bacterial adhesion,57,58 the
MPC polymer layer was considered to play a role in
suppressing P. aeruginosa adhesion. As previously reported,
the hydrophilic nature and water-compatible characteristics of
the MPC polymer coating contribute to the reduction of
bacterial adhesion on the new lens surface.15,27,63
■ CONCLUSIONS
The properties of silicone hydrogel contact lenses were studied
with a focus on the biological response at the surface. A cell
membrane-like structure was formed by adding an MPC
polymer layer on the new lens contact lens surface. The effects
of reduced lipid and protein adsorption were observed on this
surface. In addition, clear effects were also exhibited for the
inhibition of cell and bacterial adhesion. These results suggest
that effective methodologies of preparing a new surface for a
silicone hydrogel contact lens that can lead to significant
improvements on antifouling properties, which were consid-
ered important for cleanness and safety benefits.
■
EXPERIMENTAL SECTION
Preparation of the MPC Polymer-Modified Lens
Material. The base substrate was prepared by radical
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polymerization of a monomer mixture under general
manufacture conditions using 2,2′-azobisisobutyronitrile as
an initiator.23 Mixture of monomers, polydimethylsiloxane
monomethacrylate, glycerol-functionalized polydimethylsilox-
ane dimethacrylate, N-vinylpyrrolidone, tetra(ethylene glycol)
dimethacrylate, methyl methacrylate, and ethylene glycol
methyl ether methacrylate was prepared, and the initiator
was added. Then, the mixture was heated and cured by
polymerization in the mold. The surface modification of the
base substrate with the MPC polymer was carried out by the
following two-step procedure.23,64 At first, the cured base
substrate was put into a solution of commercial-grade
poly(methacrylic acid) (weight-averaged molecular weight is
up to 100 kDa) to produce an interpenetrating anchoring layer
on the base substrate surface. Second, the treated substrate was
immersed into an aqueous solution containing 0.2 wt %
polyaminoamide-epichlorohydrin (PAE) (Ashland, Wilming-
ton, DE, USA) and 0.2 wt % poly(MPC-co-2-aminoethyl
methacrylate) (PMA) (NOF, Co., Tokyo, Japan) having the
MPC units and the amino groups. The PAE and PMA can
react with carboxylic groups in the poly(methacrylic acid), and
stable amide-bond cross-linking was formed. The specific
atoms at the surface of the MPC polymer-modified lens
material were confirmed by X-ray photoelectron spectroscopy
(XPS).23
Monomer species and hydration degree of conventional
silicone hydrogel contact lenses examined in this study and the
MPC polymer-modified lens material are summarized in Table
1.
XPS Measurement. The surface elemental composition of
the samples was analyzed using XPS, AXIS-His 165 Kratos/
Shimadzu Co., Kyoto, Japan. The takeoff angle of the
photoelectrons was set to be 90°. The binding energies of
the elements were corrected with reference to the carbon atom
(C1s) in alkyl groups at 285.0 eV, and the peak areas of the
corresponding elements were calculated to evaluate the
elemental composition of the samples.
Atomic Force Microscopy (AFM) Imaging. Surface
morphology of contact lens substrates, in their respective
lens packaging solution, was examined using a Dimension
FastScan Bio Icon atomic force microscope and PFQNM-
HIRS-F-A probe (AFM, Bruker Nano, Santa Barbara,
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California, USA). The AFM images for the samples were
captured at a 0.5 Hz scan rate in the “PeakForce QNM in
Fluid” operating mode in an anti-vibration enclosure and at
room temperature.
In Vitro Lipid Adsorption Experiment. Lipid Adsorption
on Contact Lenses in Artificial Tear Solution. An artificial tear
solution (ATS) based on the literature65−69 was used for the in
vitro lipid adsorption experiment. A fluorescently labeled
triglyceride, 1,2-dioleoyl-3-[16-N-(lissamine rhodamine B
sulfonyl) amino]palmitoyl-sn-glycerol (Rh-TAG, Avanti Polar
Lipids, Inc., AL, USA), was added to the ATS as a model non-
polar lipid for confocal imaging of the in vitro lipid uptake and
distribution on contact lenses. Each contact lens was incubated
in an amber glass vial with 5.0 mL of ATS on an incubating
plate shaker set to 35 °C and 150 rpm for 3 days. The contact
lens was then cleaned by submerging lenses in 5.0 mL of
OPTI-FREE PUREMOIST multi-purpose disinfecting solution
(MPDS) at ambient temperature overnight. After overnight
cleaning, each contact lens was rinsed in a fresh aliquot of 4.0
mL of OPTI-FREE PUREMOIST MPDS and followed by 4.0
mL of PBS to remove any loosely bound lipids.
Lipid Imaging by Confocal Microscopy. The confocal
imaging was performed on a Zeiss LSM880 laser scanning
microscope (Carl Zeiss Microscopy, LLC., NY, USA). Whole
lens images were acquired by a Fluar 2.5x/0.12 M27 objective
(Carl Zeiss Microscopy, LLC., NY, USA) with z-stack and tile
scan modes. The contact lens was imaged while submerged in
a phosphate buffered saline solution (PBS, pH 7.4) in a lens
holder using a 561 nm diode pumped solid state laser and a
405 nm diode laser. The imaging conditions were kept
consistent for all contact lenses.
Protein Adsorption Experiments. Measurement of the
Amount of Lysozyme Adsorbed on the Contact Lens
Substrate. A previously reported micro bicinchoninic acid
(BCA) protein assay method (Thermo Scientific Inc.,
Waltham, MA, USA) was used to determine the concentration
of the adsorbed proteins.70 Lysozyme (human, Sigma-Aldrich,
St. Louis, MO, USA) was dissolved in PBS at a concentration
of 1.20 mg/mL. The solution was used for the lysozyme
adsorption test. The contact lens substrates were inserted into
a 24-well tissue culture plate and were pre-wetted with double-
distilled water overnight at 37 °C. After the removal of water,
1.0 mL of lysozyme solution was added into each well, and the
plate was incubated at 37 °C for different time points. The
contact lenses were then rinsed with PBS several times to
remove the lysozyme solution and weakly adsorbed proteins.
The adsorbed lysozyme was detached using 1.0 wt % sodium
dodecyl sulfate (SDS) solution and a 5 min sonication. The
lysozyme was measured using a 150 μL aliquot of SDS solution
gently mixed with BCA reagents in a 96-well tissue culture
plate. The reaction was incubated at 37 °C for 2 h, and the
absorbance was determined at a wavelength of 562 nm with a
microplate reader (Wallac 1420; Perkin Elmer Co., Ltd., MA,
USA). The amount of proteins adsorbed on the lens materials
was calculated using a calibration curve with a standard
solution of bovine serum albumin (BSA, Sigma-Aldrich). Each
measurement was performed six times for each lens material.
The same procedure was carried out to determine the total
amount of proteins adsorbed on the contact lens from artificial
tear protein solution, which contained three kinds of major
proteins in tear, including lysozyme (1.20 mg/mL), BSA (3.88
mg/mL), and immunoglobulin (IgG) from bovine serum
(Sigma-Aldrich) (1.66 mg/mL).
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Visualization of the Proteins Adsorbed at the Tear-
Contacting Surface. Densities of lysozyme, albumin, and IgG
adsorbed on the tear-contacting surface of the lens materials
were examined using the gold colloid-labeled immunoassay
method.21,53,54 Contact lenses were placed in a 24-well tissue
culture plate. To equilibrate the substrate surfaces, each lens
was soaked overnight in PBS. The PBS was removed, and the
artificial tear protein solution was added into each well
incubated at 37 °C for 24 h.71 The contact lenses were
sufficiently rinsed with PBS and treated overnight with 1.0 wt
% BSA solution at 4 °C. The lenses were rinsed again with PBS
and incubated in PBS solution containing a primary antibody
(1.0 wt % BSA solution containing IgG from rabbit and anti-
human lysozyme) for 1 h at 37 °C. The lenses were rinsed with
PBS and incubated overnight with 2.5 wt % of glutaraldehyde
in PBS at 4 °C. After that, the contact lenses were immersed in
50 mM glycine solution. The lenses were rinsed with PBS and
subjected to the secondary antibody (anti-rabbit IgG and goat
IgG labeled with 10 nm gold colloid) treatment for 1 h at 37
°C. The size of the gold colloid retained after rinsing increased
to 200−300 nm as detected using a silver enhancer kit (SE-
100, Sigma-Aldrich). The surface of the contact lens was
observed using scanning electron microscopy (SEM, SM-200,
TOPCON, Tokyo, Japan) after being freeze dried and
sputtered with gold. The amount of gold particle-labeled
proteins on the surface was calculated based on SEM images.
Cell Adhesion Experiment. Preparation of Cell
Suspensions. Mouse fibroblast L929 cells were used as
model cells in this study.26 The L929 cells were purchased
from RIKEN BRC through the National Bio-Resource Project
of the MEXT, Japan. The cells were cultured routinely in
Dulbecco’s modified Eagle medium (DMEM) containing 10%
fetal bovine serum (FBS) and 1.0% penicillin at 37 °C in a
5.0% CO2 atmosphere. The cell density was counted after
trypsinization.
Cell Morphology Observation on the Boundary of the
Contact Lens and Dish Surface. A sterilized contact lens was
cut into strip to keep them horizontal and placed on the
bottom of a glass bottom cell culture dish (35 mm in diameter,
IWAKI, ASAHI GLASS Co., Ltd., Japan) and then fixed using
a metal ring (external diameter 13 mm, inner diameter 6 mm).
L929 cells, with 4 × 105 cells/dish, were added into each
sample and cultured in a stage top incubator for microscopy
(Tokai Hit., Co, Ltd.) with 3.0 mL of DMEM containing 10%
FBS and 1.0% penicillin at 37 °C in a 5.0% CO2 atmosphere.
After 24 h, the adhesion states of the cells on the surfaces of
the various contact lenses were examined using a phase
contrast microscope (IX71, Olympus Co., Ltd., Tokyo, Japan).
The number of cells adhered on the surface was counted from
the corresponded microscopic image. Four microscopic images
of each contact lens were used to determine the number of
adherent cells.
The initial attachment of the cells was observed for over 3 h.
After the cell suspension was put on the contact lens surface
using the same procedure described above, the cell movement
was observed with a time-lapse video for 3 h. Time-lapse
videos were recorded using a phase contrast mode of the
confocal laser scanning microscope (FV1000-D IX81,
Olympus Co., Ltd., Tokyo, Japan). Picture frames were
taken every 30 min.
Bacterial Adhesion Experiment. The antibacterial
activity of various contact lens substrate samples was evaluated
using a previously reported procedure.27,63 Two Gram-negative
https://dx.doi.org/10.1021/acsomega.0c06327
ACS Omega 2021, 6, 7058−7067
ACS Omega http://pubs.acs.org/journal/acsodf
bacteria were used as bacteria models, Escherichia coli (E. coli)
and Pseudomonas aeruginosa (P. aeruginosa). To control the
number of bacteria, they were first incubated in a Luria−
Bertani (LB) medium at 37 °C for 12−16 h. The optical
density of the LB medium at 600 nm (OD600) was measured,
and the number of bacteria was calculated as follows: 1.0
OD600 = 8.0 × 108 bacteria/mL. After the suspension was
centrifuged several times, bacteria were collected and washed
with PBS. A bacteria suspension in PBS was prepared, and the
number of bacteria was adjusted to the range of 3.0−10.0 ×
106 bacteria/mL. The contact lens samples were soaked in PBS
at 37 °C overnight to reach equilibrium, the surface liquid was
blown off, and the lenses were then added to a 24-well cell
culture plate. Then, 50 μL of bacteria suspension was spread
onto each contact lens sample, and PBS was added to all the
wells of the cell culture plate to prevent the suspension from
drying. The culture plate was covered with a lid and was
maintained at 37 °C for 1 h in an incubator under normal
atmospheric conditions. Subsequently, 950 μL of PBS was
added to each well, and the culture plate was gently shaken for
3 min manually. The suspension was removed from the
microplate and added to a fresh 24-well cell culture plate.
Statistical Analysis. The results were evaluated by
Student’s t test. The significance of differences was evaluated
by p < 0.01 against the control. All the statistical analyses were
performed using Microsoft Excel 2016 (Microsoft Corp.,
Redmond, WA, USA).
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.0c06327.
Representative AFM topographical images (top view) of
the contact lenses used in this study, 3D confocal images
(side view) of the whole lenses after being soaked in
ATS and cleaned by OPTI-FREE PUREMOIST MPDS,
and 3D confocal images (45° tilted angle view) of the
whole lenses after being soaked in ATS and cleaned by
OPTI-FREE PUREMOIST MPDS (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Kazuhiko Ishihara − Department of Materials Engineering,
School of Engineering, The University of Tokyo, Bunkyo-ku,
Tokyo 113-8656, Japan; orcid.org/0000-0002-4196-
5488; Email: ishihara@mpc.t.u-tokyo.ac.jp
James Yuliang Wu − Alcon Vision LLC, Fort Worth, Texas
76134, United States; Email: james.wu@Alcon.com
Authors
Kyoko Fukazawa − Department of Materials Engineering,
School of Engineering, The University of Tokyo, Bunkyo-ku,
Tokyo 113-8656, Japan; orcid.org/0000-0001-6332-
9522
Vinay Sharma − Alcon Vision LLC, Fort Worth, Texas 76134,
United States
Shuang Liang − Alcon Vision LLC, Fort Worth, Texas 76134,
United States
Amanda Shows − Alcon Vision LLC, Fort Worth, Texas
76134, United States
Daniel Chuck Dunbar − Alcon Vision LLC, Fort Worth,
Texas 76134, United States
7065
Article
Yang Zheng − Alcon Vision LLC, Duluth, Georgia 30097,
United States
Junhao Ge − Alcon Vision LLC, Duluth, Georgia 30097,
United States
Steve Zhang − Alcon Vision LLC, Duluth, Georgia 30097,
United States
Ye Hong − Alcon Vision LLC, Duluth, Georgia 30097, United
States
Xinfeng Shi − Alcon Vision LLC, Fort Worth, Texas 76134,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.0c06327
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare the following competing financial
interest(s): V.S., S.L., A.S, D.C.D., Y.Z., J.G., S.Z., Y.H., X.S.,
and J.Y.W. are employees of Alcon Vision, LLC, Fort Worth,
TX, and Duluth, GA, USA. The authors declare no competing
financial interest.
■
ACKNOWLEDGMENTS
The authors would like to thank Ms. Keiko Oai, Mr. Katsuya
Mitera, Dr. Ren Zhang, and Mr. Bohan Cheng, The University
of Tokyo, for their significant technical support on the
evaluations of bacteria adhesion, protein adsorption, and cell
adhesion, respectively. We also would like to thank Dr. George
Yao, Dr. Rebecca Rice, Dr. Stephen “Paul” Shannon, and Dr.
John Pruitt, Alcon Vision LLC, for the in-depth and productive
discussions.
■
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