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Broad-spectrum Antimicrobial/Antifouling Soft Material Coatings using Poly(ethylenimine) as a Tailorable Scaffold Wei Cheng, Chuan Yang, Xin Ding, Amanda C Engler, James L Hedrick, and Yi Yan Yang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00359 • Publication Date (Web): 03 Jun 2015 Downloaded from http://pubs.acs.org on June 17, 2015
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Broad-spectrum Antimicrobial/Antifouling Soft Material Coatings using
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Poly(ethylenimine) as a Tailorable Scaffold
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Wei Cheng,†,§ Chuan Yang,†,§ Xin Ding,† Amanda C. Engler‡ , James L. Hedrick,*,‡ and Yi
4
Yan Yang*,†
5
†
6
Singapore
7
‡
IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120, USA
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§
These authors contributed equally to this work.
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669,
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ABSTRACT: Microbial colonization and biofilm formation is the leading cause of contact
2
lens-related keratitis. Treatment of the condition remains a challenge because of the need for
3
prolonged therapeutic course and high doses of antimicrobial agents especially for biofilm
4
eradication. The development of strategies to prepare non-fouling contact lens surfaces is a
5
more practical way to ensure users’ safety and relieve the excessive public healthcare burden.
6
In this study, we report a series of polymers that were modified to introduce functionality
7
designed to facilitate coating adhesion, antimicrobial and antifouling properties. Cyclic
8
carbonate monomers having different functional groups including adhesive catechol,
9
antifouling poly(ethylene glycol) (PEG), and hydrophobic urea/ethyl were conjugated onto
10
branched polyethylenimine (bPEI, 25 kDa) at various degrees in a facile and well-controlled
11
manner using a simple one step, atom economical approach. Immersion of contact lenses into
12
an aqueous solution of the catechol-functionalized polymers at room temperature resulted in
13
robust and stable coating on the lens surfaces, which survived the harsh condition of
14
autoclaving and remained on the surface for a typical device application life time (7 days).
15
The deposition of the polymer was unambiguously confirmed by static contact angle
16
measurement and X-ray photoelectron spectroscopy (XPS). Polymer coating did not change
17
light transmission significantly. Combinatorial optimization demonstrated that lenses coated
18
with bPEI functionalized with catechol, PEG (5 kDa) and urea groups at 1:12:3:23 molar
19
ratio for 18 h provided the highest antifouling effect against four types of keratitis-causing
20
pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and
21
Fusarium solani, after 7 days of incubation. The polymer coating also inhibited protein
22
adsorption onto the contact lens surfaces after exposure to bovine serum albumin solution for
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up to 24 h owning to the flexible and large PEG constituent. Notably, all the polymer
24
coatings used in this study were biocompatible, achieving ≥90% cell viability following
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direct contact with human corneal epithelial cells for 24 h. Hence, these polymer coatings are
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envisaged to be promising for the prevention of contact lens-related keratitis.
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KEYWORDS. Functional cyclic carbonates; PEI modification; Contact lens coating; Antifouling; Broad spectrum
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INTRODUCTION
7
Severe corneal infection (keratitis) from contact lens wear has led to corneal scarring,
8
impaired vision, light sensitivity, tearing, blindness, and cornea damage.1 Microbial adhesion
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and biofilm formation on the contact lens surface is believed to be one of the triggering
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factors of the subsequent corneal infiltration events.2 Keratitis can be caused by various
11
microorganisms, including bacteria such as Staphylococcus aureus (S. aureus) and
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Pseudomonas aeruginosa (P. aeruginosa), and fungi such as Candida albicans (C. albicans)
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and Fusarium solani (F. solani). Due to the sight-threatening nature of the complication and
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the escalating number of incidences since the introduction of soft lenses in the 1970s, it is
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imperative to develop effective means for prevention of contact lens-related keratitis.
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Some common strategies have been used to prevent biofilm formation on contact lens
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surface. For example, disinfecting multipurpose solutions have been used to reduce microbial
18
colonization, but alexidine containing contact lens cleaning solutions ReNu with
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MoistureLoc were found to be implicated in certain cases of fungal keratitis.3 Also, leachable
20
antimicrobial agents such as silver and antibiotics have been incorporated into contact lens.
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Despite being highly efficacious, their antimicrobial activity faded over time upon antibiotic
22
leaking. In addition, contact lens coating material with inherent antimicrobial and antifouling
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functions, such as the cationic peptide melimine, successfully reduced the severity of
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bacterial infection during lens wear in rabbit and pig models. However, the tedious
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conjugation procedure and high peptide synthesis cost hindered its large-scale application.4, 5
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Another well-known clinical complication related to contact lens use is the formation of
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protein deposits derived from the tear film or artificial tear solutions, leading to reduced
5
vision, discomfort, and increased chance of inflammation and microbial infection.6, 7 Proteins
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adhere to contact lens surfaces via non-specific interactions, which usually involve
7
hydrophobic and electrostatic interactions.8 Biocompatible surface modification to reduce the
8
non-specific adsorption of proteins is critical. It is well known that poly(ethylene glycol)
9
(PEG) has antifouling property, which has been exploited to prevent protein adhesion on
10
various biomedical device surfaces, eliminating protein fouling-associated adverse effects.9
11
The protein repelling ability of PEG is attributed to its large excluded volume,
12
configurational entropy, surface coverage by grafted chains, and the thickness of grafted
13
layers.10
14
The aim of this study was to synthesize and evaluate coating materials that have antifouling
15
activity against both microbes and proteins, and adhesion property for attachment to contact
16
lenses. Specifically, branched polyethylenimine (bPEI) was used as a scaffold for subsequent
17
modification owing to its inherent antimicrobial activity11 and the abundance of surface
18
primary amine groups (~ 58) due to its branched chain topology. Both the primary and
19
secondary amines (~116) have been readily modified by Michael addition12 and ring-opening
20
chemistries.13, 14 Therefore using bPEI as a general scaffold, rationally designed materials can
21
readily be made to achieve wet adhesion, antimicrobial/biofilm formation, antifouling and
22
low toxicity. The bPEI was modified in a facile and efficient way using cyclic carbonate
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monomers functionalized with catechol, PEG, and hydrophobic urea or ethyl groups through
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ring opening of the cyclic carbonate by the bPEI primary amines to various degrees. As
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hydrophobic modification of bPEI was reported to enhance antimicrobial activity,15, 16 the
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hydrophobic urea or ethyl group was introduced in anticipation that the polymer’s membrane-
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disrupting antimicrobial property can be strengthened. The adhesive property of the cationic
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polyamines were imparted through the conjugation of multiple catechol-containing cyclic
5
carbonates as catechol groups were reported to facilitate adhesion to most substrates.17
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Moreover, PEG is envisaged to provide antifouling properties against proteins. The collective
7
functional group modification on the bPEI scaffold is believed to mitigate biofilm formation
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and toxicity.
9
These modified bPEI polymers were coated on the contact lens surface via a single step
10
immersion procedure. After autoclaving, a series of studies were carried out to investigate
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coating stability and light transmittance, antifouling activity against clinically relevant
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microbes and bovine serum albumin protein, as well as the toxicity against human corneal
13
epithelial cells. Through carefully modulating the identity and number of functional groups
14
conjugated on the PEI backbone and the lens coating time, protein adhesion and fouling of a
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broad range of keratitis-causing pathogenic microbes on coated contact lenses were
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effectively mitigated. Furthermore, negligible cytotoxicity to human corneal epithelial cells
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was achieved. These polymer coatings can be potentially used to prevent contact lens-related
18
keratitis.
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EXPERIMENTAL SECTION
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Materials. Reagents were purchased from Sigma-Aldrich and used as received unless
21
otherwise noted. Poly(ethylene glycol) mono methyl ether (PEG, Mn=5,000, PDI: 1.04) was
22
purchased from Polymer Source Inc., Canada. The PEG and urea-functionalized cyclic
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carbonates (MTC-PEG and MTC-urea) used in this study were prepared according to our
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previous protocol.18-20 Contact lenses (Air Optix, Singapore) were purchased in their original 5 ACS Paragon Plus Environment
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packaging, and had a diameter of 14.2 mm and a curvature of 8.6 mm. All the
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microorganisms used in this study were purchased from ATCC (American Type Culture
3
Collection) and cultured according to the instructions provided.
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Synthesis of MTC-Catechol (Scheme S1). Synthesis of 3,4-bis(benzyloxy)benzoic acid.
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3,4-Dihydroxy benzoic acid (5 g, 32.44 mmol), benzyl bromide (15.41 mL, 129.76 mmol),
6
and potassium carbonate (17.93 g, 129.73 mmol) in 100 mL acetonitrile were refluxed for 18
7
h. The reaction solution was cooled to room temperature and the salts were filtered out. The
8
solution was then concentrated down to 20 mL and diluted with 100 mL of hexanes. A solid
9
was formed, which was collected by filtration. The solid was then recrystallized out of
10
THF/hexanes to yield a pure product. Yield: 7.88 g (57%). 1H NMR (400 MHz, CDCl3,
11
22 °C): δ 7.68 (m, 2H, H of benzoic acid), 7.38 (m, 10H, -OCH2PhH), 6.95 (d, 1H, H of
12
benzoic acid), 5.23 (d, 4H, -OCH2PhH).
13
Synthesis of 2-hydroxyethyl 3,4-bis(benzyloxy)benzoate. 3,4-Bis(benzyloxy)benzoic acid (4
14
g, 9.43 mmol) was suspended in ethylene glycol (50 mL) and the solution was heated to 80
15
ºC. A catalytic amount of KOH (0.106 g, 1.86 mmol) was added and the solution was left to
16
react until all solids were dissolved (8-10 h). The solution was precipitated into cold water
17
(500 mL) and left to stir for several hours. A white solid was formed. The solid was filtered
18
out and rinsed with water. The solid was then freeze dried to remove any remaining water.
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Yield: 2.81 g, (79%). 1H NMR (400 MHz, CDCl3, 22 °C): δ 7.68 (d, 2H, H of benzoate), 7.39
20
(m, 10H, -OCH2PhH), 6.96 (d, 1H, H of benzoate), 5.25 (d, 4H, -OCH2PhH), 4.44 (t, 2H, -
21
COOCH2CH2OH), 3.95 (t, 2H, -COOCH2CH2OH).
22
Synthesis of pentafluorophenyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC- OC6F5).
23
A 100 mL round bottom flask was charged with 2,2-bis(hydroxymethyl)propionic acid (bis-
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MPA) (3.00 g, 22 mmol), bis-(pentafluorophenyl)carbonate (PFC) (21.70 g, 55 mmol), CsF 6 ACS Paragon Plus Environment
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(0.7 g, 4.6 mmol), and 70 mL of anhydrous THF. Initially the reaction was heterogeneous, but
2
after one hour a clear homogeneous solution was formed that was allowed to stir for 20 h.
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The solvent was removed in vacuo. The residue was re-dissolved in methylene chloride, and
4
after 10 min, a byproduct was precipitated, and filtered out. The filtrate was extracted with
5
sodium bicarbonate and water, and dried with MgSO4. The solvent was evaporated in vacuo
6
and the product was recrystallized from ethyl acetate/hexane mixture to give MTC-OC6F5 as
7
a white crystalline powder. Yield: 5.50 g (75%). 1H NMR (400 MHz, CDCl3, 22 °C): δ 4.85
8
(d, 2H, -CH2OCOO-), 4.36 (d, 2H, -CH2OCOO-), 1.55 (s, 3H, -CH3).
9
Synthesis
of
2-(3,4-bis(benzyloxy)benzoyloxy)ethyl
5-methyl-2-oxo-1,3-dioxane-5-
10
carboxylate (MTC-catechol). 2-Hydroxyethyl 3,4-bis(benzyloxy)benzoate (2.81 g, 7.42
11
mmol), MTC-OC6F5 (2.54 g, 7.79 mmol), and proton sponge (1.59, 7.42 mmol) were
12
dissolved in THF (10 mL). The reaction solution was stirred overnight at room temperature.
13
Once the reaction was complete as indicated by 1H NMR (~18 h), the solution was added to a
14
mixture of diethylether (100 mL) and hexanes (10 mL), and left for several hours at -40 ºC.
15
White crystals were recovered and washed with diethylether and hexanes. Yield: 2.59 g,
16
(67%). 1H NMR (400 MHz, CDCl3, 22 °C): δ 7.64 (d, 2H, H of benzoate), 7.43 (m, 10H, -
17
OCH2PhH), 6.97 (d, 1H, H of benzoate), 5.24 (d, 4H, -OCH2PhH), 4.68 (d, 2H, -CH2OCOO-
18
), 4.54 (s, 4H, -COOCH2CH2O-), 4.19 (d, 2H, -CH2OCOO-), 1.32 (s, 3H, -CH3).
19
Synthesis of catechol-, PEG- and urea-functionalized bPEI (Scheme 1). bPEI
20
(Mn=10,000, PDI: 2.5, 0.2 g, 0.02 mmol) was dissolved in 2 mL of dry DCM, followed by
21
addition of a solution of MTC-catechol (83.2 mg, 0.16 mmol), MTC-PEG (Mn = 5,142; 0.206
22
g, 0.04 mmol) and MTC-urea (103.1 mg, 0.32 mmol) in 2 mL of dry DCM. The reaction
23
solution was stirred for one hour before being concentrated to dryness. Then, the mixture of
24
the functional bPEI, MeOH (7.5 mL), THF (7.5 mL), and Pd-C (10% w/w, 0.2 g) was swirled
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under H2 (7 atm) overnight. After evacuation of H2, the mixture was filtered using syringe
2
and the filtrate was concentrated to dryness. MeOH (10 mL) and 1 M HCl (10 mL) were
3
added sequentially and the reaction solution was stirred for 2-3 h. After acidification, the
4
solution was purified by centrifugal filtration (MWCO = 3,000) and washed twice with de-
5
ionized (DI) water. Finally, the concentrated solution in the tube was freeze-dried, giving rise
6
to the deprotected and acidified catechol-, PEG- and urea-functionalized bPEI H. Similarly,
7
functional bPEI derivatives with various compositions were synthesized and their respective
8
compositions, NMR data and yield are listed in Table 1.
MTC-Fg
Fg: Catechol-bz protected Urea or Ethyl PEG (750 Da or 5 kDa)
PEI - 10kDa Antifouling Hydrophobic or
Adhesive
9 10 11
Scheme 1. Synthesis procedures and chemical structure of catechol-containing functional bPEI derivatives.
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Table 1. Compositions, NMR data and yield of the functional bPEI derivatives. 1 Polymer Compositions H NMR data (400MHz, D2O, 22 °C) δ 7.20 (s, br, 36H, PhH), 3.85-4.45 (m, br, 72H, H12 catechol groups a, H-f and H-g), 2.50-3.65 (m, br, 954H, H-b and A H of bPEI), 1.08 (m, 36H, H-c). δ 7.17 (d, br, 33H, PhH), 3.95-4.50 (m, br, 76H, H11 catechol and 5 a, H-f and H-g), 3.55 (s, 341H, H of PEG), 2.60PEG (Mn 750 Da) B 3.52 (m, br, 962H, H-b and H of bPEI), 1.08 (m, groups 48H, H-c). δ 7.15 (m, 27H, PhH), 3.90-4.60 (m, br, 56H, H-a, 9 catechol and 1 H-f and H-g), 3.58 (s, 455H, H of PEG), 2.50-3.53 PEG (Mn 5 kDa) C (m, br, 950H, H-b and H of bPEI), 1.01 (m, 30H, groups H-c). δ 7.17 (s, br, 88H, PhH), 4.05 (s, br, 110H, H-a, H11 catechol and 11 e, H-f and H-g), 2.30-3.70 (m, br, 996H, H-b, H-d D urea groups and H of bPEI), 0.96 (m, 66H, H-c). δ 6.85-7.20 (m, br, 72H, PhH), 4.06 (m, br, 92H, 9 catechol, 1 PEG H-a, H-e, H-f and H-g), 3.57 (s, 1364H, H of (Mn 5 kDa) and 9 E PEG), 2.45-3.53 (m, br, 986H, H-b, H-d and H of urea groups bPEI), 1.03 (s, br, 57H, H-c). δ 6.90-7.40 (m, br, 27H, PhH), 4.05 (m, br, 96H, 9 catechol, 1 PEG H-a, H-f, H-g and -COOCH2CH3 of MTC-ethyl), (Mn 5 kDa) and 10 3.56 (s, 1364H, H of PEG), 2.48-3.54 (m, br, F ethyl groups 970H, H-b and H of bPEI), 1.09 (s, br, 87H, H-c and -COOCH2CH3 of MTC-ethyl). δ 6.80-7.15 (m, br, 85H, PhH), 4.06 (m, br, 110H, 10 catechol, 3 PEG H-a, H-e, H-f and H-g), 3.56 (s, 1364H, H of (Mn 5 kDa) and 11 G PEG), 2.50-3.54 (m, br, 1000H, H-b, H-d and H of urea groups bPEI), 1.06 (s, br, 114H, H-c). δ 6.80-7.20 (m, br, 151H, PhH), 4.08 (m, br, 170H, 12 catechol, 3 PEG H-a, H-e, H-f and H-g), 3.58 (s, 1364H, H of (Mn 5 kDa) and 23 H PEG), 2.45-3.50 (m, br, 1052H, H-b, H-d and H of urea groups bPEI), 1.06 (s, br, 114H, H-c).
Yield 92%
82%
89%
84%
87%
90%
88%
84%
2
3
1
H NMR spectroscopy. 1H NMR spectra of polymers were recorded on a Bruker Advance
4
400 NMR spectrometer at 400 MHz at room temperature to analyze polymer compositions.
5
The 1H NMR measurements were carried out with an acquisition time of 3.2 s, a pulse
6
repetition time of 2.0 s, a 30° pulse width, 5208-Hz spectral width, and 32 K data points.
7
Chemical shifts were referred to the solvent peaks (δ = 4.70 ppm for D2O).
8
Polymer Coating on Contact Lens Surfaces. Contact lenses were punched into small
9
discs with diameter of 3 mm by Integra™ Miltex™ Standard Biopsy Punches (Fisher 9 ACS Paragon Plus Environment
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Scientific, Singapore). After thoroughly cleaning with sterile phosphate-buffered saline (PBS)
2
and drying with nitrogen flow, the contact lens samples were soaked in a Tris-buffered saline
3
(10 mM, pH 8.0) containing various polymers at 1 mM (acidified unmodified bPEI: 10
4
mg/mL, PEG-catechol: 8.5 mg/mL, A: 13.9 mg/mL, B: 17.8 mg/mL, C: 19.2 mg/mL, D: 16.9
5
mg/mL, E: 22.5 mg/mL, F: 21.4 mg/mL, G: 29.6 mg/mL, and H: 34.3 mg/mL) for 4, 8, 18,
6
or 24 h with gentle shaking at room temperature. The uncoated and coated contact lens
7
samples were then rinsed three times with PBS, soaked in PBS, and autoclaved for 20 min at
8
121°C (Hirayama, Japan). Immediately after autoclaving, these coated or uncoated contact
9
lenses were rinsed with PBS and put in 96 well plates for subsequent experiments. All the
10
polymer solutions were colorless, and the coated contact lenses were transparent and showed
11
no significant difference in appearance as compared to the uncoated contact lens.
12
Light Transmittance Measurement. The various polymers were coated onto contact lens
13
surfaces for 18 h, followed by autoclaving. Each coating was done in triplicates. The pristine
14
control lens and polymer-coated lenses were rinsed three times in PBS and immersed in 100
15
µL of PBS in 96-well plates. The lenses all sat flat on the bottom of the wells so that they did
16
not float around during the measurement. The measurement was performed with the contact
17
lenses in a fully hydrated state whilst immersed in PBS. The absorbance of the samples was
18
recorded using a microplate reader (TECAN, Sweden) at a wavelength of 600 nm, which falls
19
within the visible light range. After subtracting the background absorbance of pure PBS, the
20
transmittance of the lenses was calculated using the following formula:
21
%T = 100(10-A) where %T is the percentage of transmittance, and A is the absorbance.
22
Static Contact Angle Measurements. An OCA30 contact angle measurement device
23
(Future Digital Scientific Corp., U.S.A.) was used to measure the static contact angle on
24
uncoated and polymer-coated contact lens surfaces, 1 day and 7 days after coating to evaluate 10 ACS Paragon Plus Environment
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if coating was successful and stable. DI water (1 µL droplet) was used for the measurements.
2
All samples were measured in triplicates. The static contact angle data were presented as
3
Mean ± SD.
4
Polymer Coating Assessment Using X-ray Photoelectron Spectroscopy (XPS).
To
5
further verify the effective coating of polymers and the coating stability, XPS spectra of
6
uncoated and polymer-coated contact lens surfaces were analyzed. Uncoated or coated
7
contact lenses were submerged in autoclaved water, and incubated at room temperature under
8
gentle shaking. At 1 day and 7 days after coating, the surface chemistry of uncoated and
9
coated contact lenses was analyzed by XPS (Kratos Axis His, Kratos Analytical, Shimadzu,
10
Japan) with Al Kα source (hν = 1486.71 eV). The angle between the sample surface and
11
detector was kept at 90°. The survey spectrum ranging from 1100 to 0 eV was acquired with
12
pass energy of 80 eV.
13
Treatment of Contact Lens Surface with Microbes. Fungi Candida albicans (C. albicans,
14
ATCC10231) and Fusarium solani (F. solani, ATCC36031) cells were cultured in Yeast
15
Mold Broth (YMB, BD Singapore) and Sabouraud Dextrose Broth (SDB, Sigma-Aldrich,
16
Singapore) at room temperature, respectively. Bacteria Staphylococcus aureus (S. aureus,
17
ATCC6538) and Pseudomonas aeruginosa (P. aeruginosa, ATCC9027) cells were cultured
18
in BBL™ Mueller Hinton Broth (MHB, BD Singapore) at 37°C. All the microorganisms
19
were grown overnight to reach mid-logarithmic growth phase. For C. albicans, S. aureus and
20
P. aeruginosa, the concentrations of the microbes were adjusted by obtaining optical density
21
(O.D.) reading of 0.07 at the wavelength of 600 nm on a microplate reader (TECAN,
22
Switzerland), which corresponds to the concentration of Mc Farland 1 solution (3 x 108
23
CFU/mL), before the microbe solutions were further diluted by 105 times to achieve an initial
24
loading of 3 x 103 CFU/mL. For F. solani, due to its characteristic clumpy culture, its 11 ACS Paragon Plus Environment
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solution was first filtered using 100 µm Falcon® Cell Strainer (Fisher Scientific, Singapore).
2
The F. solani cell concentration in the flow-through was determined by counting using a
3
hemocytometer, and diluted to a final concentration of 3 x 103 CFU/mL. The microbial
4
solution (100 µL) was added to each well of 96-well plates containing the autoclaved
5
uncoated or polymer-coated contact lens, and incubated at room temperature for the fungi and
6
at 37°C for the bacteria, under constant shaking. After 24 h, the lenses were rinsed three
7
times with sterile PBS, and 100 µL of 3 x 103 CFU/mL fresh microbial suspension was added
8
to the lenses. This was repeated every day for 7 days to allow biofilm formation. Otherwise,
9
C. albicans suspension was diluted to 3 x 108 CFU/mL and incubated with the lenses for 4 h
10
before fresh medium was added to replace the microbial solution, and the incubation went on
11
for a further 48 h. After 7 days or 48 h of incubation, the contact lenses were rinsed three
12
times with PBS, before being used for further analysis.
13
Antifouling Activity Analysis of the Uncoated and Coated Contact Lenses by XTT
14
Reduction Assay. A (2-methoxy-4-nitro-5-sulfo-phenol)-2H-tetrazolium-5-carboxanilide
15
(XTT) reduction assay was used to quantify the live microbes on contact lens surfaces by
16
measuring the mitochondrial enzyme activity in live cells. In this assay, mitochondrial
17
dehydrogenases of the viable microbial cells reduced XTT to an orange colored formazan
18
derivative, and change in optical density (O.D.) reading was recorded to analyze the viability
19
of cells on the surfaces. The uncoated or polymer-coated contact lens surface after incubation
20
with various microbes, and washed three times in PBS, was submerged in a mixture solution
21
of 100 µL PBS, 10 µL XTT (1mg/mL), and 2 µL menadione (0.4 mM). After incubation at
22
37°C for 4 h, the absorbance at a wavelength of 490 nm of the samples was measured using a
23
microplate reader (TECAN, Sweden) with 600 nm as the reference wavelength.
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LIVE/DEAD Baclight Bacterial Viability Assay of Contact Lens Surfaces. To visualize
2
the viable microbes on uncoated and polymer-coated contact lens surfaces, a LIVE/DEAD
3
Baclight bacterial viability kit (L-7012, Invitrogen) with 2 staining agents was used.
4
Propidium iodide, a red nucleic acid staining agent, was used to label dead bacterial cells by
5
penetrating damaged cell membrane. Meanwhile, SYTO® 9, a green fluorescent nucleic acid
6
staining agent, was used to label both live and dead microbes by penetrating cells with either
7
intact or damaged membrane. After incubation with various microbes, the supernatant was
8
removed and the lenses were rinsed 3 times with PBS, and soaked in a dye solution (5.01 mM
9
of SYTO® 9 and 30 µM of propidium iodide in PBS) at room temperature in the dark for 15
10
min. The stained microbes on the contact lenses were then observed using oil immersed 10x
11
or 63x objective lens of a Zeiss LSM 5 DUO laser scanning confocal microscope (Germany).
12
Protein Adsorption. The extent of adsorption of bovine serum albumin (BSA, as a model
13
protein) on uncoated and polymer-coated contact lens surfaces was determined by micro
14
BCA™ protein assay. Briefly, uncoated and polymer-coated contact lens pieces (3 mm
15
diameter) were immersed in 1 mL of PBS containing 50 mg/mL of BSA for 12 or 24 h at
16
37°C. Each sample was then rinsed three times with PBS, and subsequently placed in a tube
17
with 100 µL of PBS containing 1 wt% of sodium dodecyl sulfate (SDS). The adsorbed BSA
18
on the sample surfaces was removed by sonication of the samples in the SDS solution for 30
19
min. The bicinchoninic acid (BCA) method was applied to quantify the amount of BSA in the
20
SDS solution using Micro BCA™ protein assay reagent kit (Pierce, U.S.A.). The amount of
21
BSA was calculated by measuring the absorbance at 562 nm. A standard curve was generated
22
by plotting the average blank-corrected 562 nm reading for each BSA standard (included in
23
the kit) vs. its concentration in µg/mL, based on which the protein concentration of each
24
unknown sample was calculated.
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1
Cell Culture and Biocompatibility of Polymer-coated Contact Lenses. HCE-2 [50.B1]
2
(ATCC® CRL11135™), which is a human corneal epithelial cell line transformed by
3
epithelial adenovirus 12-SV40 hybrid, was cultured at 37°C in a 5% CO2 humid incubator in
4
keratinocyte serum-free medium (GIBCO®, Life Technologies, Singapore) supplemented
5
with 0.05 mg/mL bovine pituitary extract, 5 ng/mL epidermal growth factor (GIBCO®, Life
6
Technologies, Singapore), 500 ng/mL hydrocortisone (Sigma-Aldrich, Singapore) and 5
7
µg/mL insulin (Life Technologies, Singapore). Fresh medium was added every other day and
8
the cells were grown to 90% confluency in tissue culture flasks pre-coated with a mixture of
9
0.01 mg/mL fibronectin (Biopolis Shared Facilities, BSF, A*STAR, Singapore), 30 µg/mL
10
bovine collagen type I (Life Technologies, Singapore) and 10 µg/mL bovine serum albumin
11
(Sigma-Aldrich, Singapore) before sub-culturing.
12
To assess the cytotoxicity of the uncoated and various coated contact lenses on HCE-2 cells,
13
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cellular viability
14
assay was performed to examine the percentage of metabolically active cells in the
15
uncoated/coated contact lenses-placed wells relative to control wells with untreated cells.
16
Briefly, HCE-2 cells were seeded onto a 96-well tissue culture plate pre-coated in the same
17
way as the tissue culture flasks, at 10,000 cells in 100 µL medium per well. The cells were
18
left to adhere for 18-24 h at 37°C in a humid CO2 incubator, which resulted in the formation
19
of a monolayer of HCE-2. Then, small pieces of autoclaved uncoated and polymer-coated
20
contact lenses were directly placed on top of the monolayer, and incubated for 24 h. After
21
that, lenses were carefully removed by tweezers, and placed under the light microscope to
22
check the presence of any adherent cells on their surfaces. The cells in each well were
23
incubated with 100 µL of growth medium and 20 µL of MTT solution (5 mg/mL in PBS) for
24
4 h at 37°C. Formazan crystals formed in each well were solubilized using 150 µL of DMSO
25
upon removal of growth media. A 100 µL aliquot from each well was then transferred to a 14 ACS Paragon Plus Environment
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1
new 96-well plate for determination of absorbance using a microplate spectrophotometer
2
(TECAN, Sweden) at wavelengths of 550 nm and 690 nm. Relative cell viability was
3
expressed as [(A550-A690)sample / (A550-A690)control] x 100%. Data were expressed as mean ±
4
standard deviations of six replicates.
5
RESULTS AND DISCUSSION
6
Synthesis and Characterization of Functional Cyclic Carbonates and bPEI Polymers.
7
As shown in Scheme 1, branched PEI was modified to various degrees using cyclic
8
carbonates functionalized with catechol (MTC-catechol), PEG (MTC-PEG), as well as urea
9
(MTC-urea) or ethyl (MTC-ethyl) through ring opening of the cyclic carbonate by the
10
primary amine groups of bPEI. These bPEI derivatives were then subjected to hydrogenolysis
11
to remove the protecting benzyl groups from the catechol, followed by acidification with 1 M
12
HCl solution to improve polymer aqueous solubility and stability. MTC-catechol was
13
designed and synthesized by a three-step reaction as shown in Scheme S1. Briefly, both
14
phenolic hydroxyl groups of protocatechoic acid were protected by benzyl groups, followed
15
by esterification of its aliphatic carboxylic acid group with a large excess of ethylene glycol
16
(also used as solvent in the reaction). Then, the resulted 2-hydroxyethyl 3,4-
17
bis(benzyloxy)benzoate was reacted with a reactive monomer, MTC-OC6F5 to give MTC-
18
catechol in high yield.
19
The compositions of these bPEI derivatives (Table 1) were estimated from 1H NMR
20
spectroscopy by quantitative comparisons between integral intensities of the peak of phenyl
21
protons of MTC-catechol and MTC-urea, those of the ethylene groups in MTC-PEG, as well
22
as those of ethylene protons in bPEI. For example, Figure 1 shows the proton NMR spectra of
23
polymer H in D2O. The molar ratio of MTC-catechol to bPEI was determined to be 12 by
24
comparison of the integral intensities of the peaks of phenyl groups in MTC-catechol ranging 15 ACS Paragon Plus Environment
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Page 16 of 34
1
within 6.80-7.20 ppm with those of ethylene protons in bPEI ranging within 2.45-3.50 ppm.
2
Similarly, 3 units of MTC-PEG and 23 units of MTC-urea were conjugated to bPEI by
3
comparison of the integral intensities of the peak of ethylene groups in MTC-PEG at 3.58
4
ppm and those of the peak of methylene protons at 4.08 ppm with those of ethylene protons
5
in bPEI ranging within 2.45-3.50 ppm, respectively. It was also clearly observed that the peak
6
of the protons of benzyl protecting groups of MTC-catechol disappeared after hydrogenolysis.
7
8 9
Figure 1. (a) 1H NMR spectra of protected catechol-containing functional bPEI conjugate in MeOD and (b) deprotected catechol-containing bPEI conjugate H in D2O.
10 11
Characterization of Polymer Coatings. Visible Light Transmission and Surface
12
Wettability of Uncoated and Polymer-Coated Contact Lenses. The transmittance of both the
13
pristine and polymer-coated contact lenses at 600 nm were above 95%, demonstrating that
14
the various polymer coatings did not affect the transparence of the surface-modified contact
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lenses or visible light transmission (Table S1). The polymer-coated contact lenses were
2
further evaluated for static water contact angle in comparison with pristine contact lens. The
3
pristine soft contact lens was made of 67% lotrafilcon B and 33% water and therefore
4
hydrophilic, with a static contact angle of 44.4°±0.8°. Upon coating with various polymers
5
and in agreement with previous findings,21 the surfaces became relatively more hydrophobic
6
with significantly increased static contact angle (53.9°±2.6°, 55.2°±1.1°, 66.7°±1.1°,
7
61.7°±0.6°, 62.6°±5.3°, 65.6°±3.4°, 65.9°±0.4°, 67.5°±3.1°, 68.1°±0.7°, and 74.9°±4.9° for
8
acidified unmodified bPEI (AbPEI), PEG-catechol, and A, B, C, D, E, F, G and H-coated
9
contact lens surface, respectively), indicating successful coating of the polymers (Table 2 and
10
Figure 2). The fact that higher static contact angle was observed for coated contact lens
11
surfaces was anticipated due to the deposition of polymers that were relatively more
12
hydrophobic than the contact lens surface. The attachment of the AbPEI was by non-specific
13
adsorption, which might be leached out as a function of time.22 Indeed, after being
14
continuously immersed in DI water for 7 days, the contact angle of the lens treated with
15
AbPEI without catechol group dropped significantly to the base level of the pristine control
16
(45.6°±1.6° for AbPEI-treated lens vs. 43.1°±2.6° for the pristine control), while the static
17
contact angles for all catechol-containing polymer-coated contact lens surfaces did not
18
change (Figure 2). This finding indicates the importance of catechol groups in anchoring
19
coating materials onto the surface of the lens substrate. Taken together, the results showed
20
successful and stable coating of the polymers on contact lens surfaces over 7 days following
21
coating and autoclaving.
22 23 24 25 26 27
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Table 2. Static contact angle of the polymer-coated contact lens surfaces at 1 day and 7 days after coating and autoclaving. Static Contact Angle Polymer 1 day after autoclaving
7 days after autoclaving
Pristine Control
44.4° ± 0.8°
43.1° ± 2.6°
AbPEI
53.9° ± 2.6°
45.6° ± 1.6°
PEG-Catechol
55.2° ± 1.1°
53.5° ± 0.8°
A
66.7° ± 1.1°
67.0° ± 1.3°
B
61.7° ± 0.6°
62.0° ± 0.3°
C
62.6° ± 5.3°
61.7° ± 3.2°
D
65.6° ± 3.4°
67.1° ± 1.3°
E
65.9° ± 0.4°
62.6° ± 3.4°
F
67.5° ± 3.1°
66.7° ± 2.5°
G
68.1° ± 0.7°
70.7° ± 2.0°
H
74.9° ± 4.5°
73.0° ± 0.5°
3
4
5 6
Figure 2. Static contact angle of uncoated and polymer-coated contact lens surfaces. Static contact angle was measured using DI water. (n=3, **p
