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Reducing Friction in the Eye: A Comparative Study of Lubrication by Surface-Anchored Synthetic and Natural Ocular Mucin Analogues Olof Sterner,† Chrysanthi Karageorgaki,† Massimiliano Zürcher,† Stefan Zürcher,†,‡ Charles W. Scales,§ Zohra Fadli,§ Nicholas D. Spencer,‡ and Samuele G. P. Tosatti*,† †
SuSoS AG, Lagerstrasse 14, CH-8006 Dübendorf, Switzerland Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich, Vladimir-Prelog-Weg 5, CH-8093 Zurich, Switzerland § Johnson & Johnson Vision Care Inc., Jacksonville, Florida 32256, United States ‡
S Supporting Information *
ABSTRACT: Biomaterials used in the ocular environment should exhibit specific tribological behavior to avoid discomfort and stress-induced epithelial damage during blinking. In this study, two macromolecules that are commonly employed as ocular biomaterials, namely, poly(vinylpyrrolidone) (PVP) and hyaluronan (HA), are compared with two known model glycoproteins, namely bovine submaxillary mucin (BSM) and α1-acid glycoprotein (AGP), with regard to their nonfouling efficiency, wettability, and tribological properties when freely present in the lubricant, enabling spontaneous adsorption, and when chemisorbed under low contact pressures. Chemisorbed coatings were prepared by means of photochemically triggered nitrene insertion reactions. BSM and AGP provided boundary lubrication when spontaneously adsorbed in a hydrophobic contact with a coefficient of friction (CoF) of ∼0.03−0.04. PVP and HA were found to be excellent boundary lubricants when chemisorbed (CoF ≤ 0.01). Notably, high-molecular-weight PVP generated thick adlayers, typically around 14 nm, and was able to reduce the CoF below 0.005 when slid against a BSM-coated poly(dimethylsiloxane) pin in a tearlike fluid. KEYWORDS: poly(vinylpyrrolidone), hyaluronan, glycoprotein, friction, tears, ocular lubricants, perfluorophenyl azide
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INTRODUCTION Soft biological interfaces, including the respiratory and gastrointestinal tracts, the oral cavity, and the eye,1 are predominantly lubricated by mucus. The tribological performance of such systems is often dependent on complex interactions between several components, both surfaceanchored and dispersed. A recurring element is the proteoglycana “bottle-brush” structure consisting of a protein backbone densely decorated with glycosaminoglycans (GAGs). The lubricating properties of proteoglycans are believed to be related to their general structure, which contains dense and highly hydrated polysaccharide chains that are covalently attached to a protein backbone. These structures provide elastic, electrostatic, and osmotic resistance to compression and form an interface that can easily be sheared.2,3 Mucins are a diverse family of proteoglycans, omnipresent in soft lubricated interfaces, existing both as membrane-bound and as gel-forming species.4 For example, in the eye, both the eyelid and the cornea are lined with a mucus membrane, that is, the conjunctiva, so that during a blink, the shear plane is shifted away from the two epithelia, minimizing stress.5 An important component of this system is the tear film: a complex lubricant © XXXX American Chemical Society
consisting of glycoproteins, proteins, and lipids. Tears spontaneously form a stratified liquid structure, where the aqueous phase is covered with a thin film of lipids, which contribute to the reduced evaporation rate,6 rheological modulation in association with proteins,7 and to the overall stability of the tear film.8 In addition, lipids have been shown to be boundary lubricants.9 A compromised tear film, especially with regard to tear-film break-up, is correlated with dry-eye symptoms.8 In this regard, an ophthalmic biomaterial (e.g., a contact lens) should preferably have good tribological properties against the conjunctiva and, in particular, favorable compatibility with the amphiphilic tear film.10 The aim of this study was to evaluate the importance of molecular weight, morphology (surface-anchored vs dissolved), contact geometry (self-mated vs against mucin), and lubricant composition (simple buffer vs complex tearlike fluid (TLF)) on the lubricating performance of two common ocular biomaterials: hyaluronan (HA) and poly(vinylpyrrolidone) (PVP). Received: December 21, 2016 Accepted: May 15, 2017
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DOI: 10.1021/acsami.6b16425 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
acetate (ACS grade), toluene (99.9%), 2-propanol (99.8%), and potassium carbonate (K2CO3, 99%). N-Hydroxysuccinimide-PFPA (PFPA-NHS) and TLF prepared without MgCl2 and CaCl2 were provided by SuSoS AG (Dübendorf, Switzerland). TLF is a complex buffer consisting of proteins, mucin, and lipids and prepared from phosphate-buffered saline (PBS).31 4-(2-Hydroxyethyl)piperazine-1ethane sulfonic acid (HEPES, 99%) was acquired from BDH Biochemical (Switzerland). PVP (MW = 10, 55, 360, and 1300 kDa), HA sodium salt from Streptococcus equi (MW = 30−50, 150− 300, 1000−1250 kDa), PAAm hydrochloride (MW = 15 kDa), polyethyleneimine (PEI) (branched MW = 25 kDa), diiodomethane (99%), Dulbecco’s PBS without MgCl2 and CaCl2 (PBS), lyophilized lysozyme from chicken egg white (≥90%), and AGP from bovine plasma (99%) were purchased from Sigma Aldrich (Switzerland). Lyophilized BSM was purchased from Merck Millipore (Germany). BSM was used without further purification, which could lead to additional species, mainly albumin, also being present.32,33 The molecular weight of the commercially acquired PVP and HA were determined by means of size-exclusion chromatography combined with multiangle light scattering (SEC-MALS, PSS Polymer Standards Service GmbH, Mainz, Germany) (see Table 1).
Such information may aid in the development or improvement of biomaterials situated between soft lubricated interfaces, with a focus on the ocular environment. Two model glycoproteins having lubricious properties, bovine submaxillary mucin (BSM) and α-1 acid glycoprotein (AGP),11 were also included in the study for comparative purposes. The efficiency of HA as a boundary lubricant has been disputed in the literature, and it appears to have a stronger wear-reducing role than a friction-reducing one under conditions similar to those in articular joints.12−15 However, Singh et al. demonstrated that surface-anchored HA was efficient in reducing the coefficient of friction (CoF) to below 0.1 when covalently linked to cartilage tissue.16 HA is also a constituent of the tear fluid, with a potential role in wound healing.17 In addition, corneal epithelial cells have been reported to express the CD44 receptor, which specifically binds HA.18,19 The potential role of HA as a tear-film lubricant has been clinically demonstrated with regard to mediating dryeye symptoms, when used in artificial tear drops.20 PVP is an extensively used polymer in the fields of pharmaceuticals and biomaterials, being applied, for example, as a binder for pharmaceuticals, a wetting agent for contact lenses,21 and as a complexation agent (e.g., with iodine).22,23 PVP has also been shown to be biocompatible when present as a hydrogel.24 One explanation for the wide applicability of PVP is its amphiphilic nature, arising from the side chain, which has both a hydrophilic protein-like lactam bond and a lipophilic methylene component. This leads to good solubility in water and organic solvents, as well as compatibility with both hydrophilic and hydrophobic compounds. Surface-grafted chains of PVP have been shown to be nonadhesive to proteins, cells, marine bacteria, and algae spores.25−27 In addition, hydrogel coatings containing PVP have lubricious properties.28 To comprehensively compare the lubricating mechanisms and efficiency of the four compounds (i.e., PVP, HA, BSM, and AGP) under the same conditions, three tribological contacts were considered. In a first instance, the CoF between two hydrophilic or two hydrophobic surfaces was evaluated with the compounds added as solutes in the aqueous solution. In the second instance, the intrinsic lubricating properties were further evaluated by surface tethering the compounds to both the surface and the countersurface and sliding them against each other. To ensure that the binding strength and coating architecture were similar, and thus comparable, chemisorbed monolayer films were prepared by means of nitrene insertion, as triggered by the photochemical decomposition of surface-anchored perfluorophenyl azides (PFPA). Singlet nitrenes insert rapidly into C−H/N−H bonds, forming covalent adducts with compounds in close proximity.29 Poly(allylamine) (PAAm) or poly(ethyleneimine) (PEI) grafted with PFPA acted as a cationic self-assembled adhesion promoter on negatively charged substrates, as previously described.25,30 Polymers and proteins were covalently linked to the PAAm- or PEI-PFPA adhesion promoter layer from the dry state, after deep UV exposure. Finally, both the adlayers were tested in an eye-mimicking model to evaluate the performance under conditions similar to those existing between the cornea and eyelid, in terms of the lubricant, countersurface, and contact pressure.
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Table 1. Molecular Weight of the Polymers (MW), Polydispersity Index (PDI), Molecular Weight of the Monomeric Units (MWmon), and the Degree of Polymerization (DP) of the Polymer Samples polymer
MW (g/mol)b
PDIb
MWmon (g/mol)
DPa
PVP 10 kDa PVP 55 kDa PVP 360 kDa PVP 1300 kDa HA 30 kDa HA 150 kDa HA 1000 kDa
16 100 66 000 478 000 657 000 40 400 240 000 1 170 000
1.55 2.03 2.16 2.42 1.79 1.36 1.65
111 111 111 111 379 379 379
90 495 3243 11 712 106 634 3080
a
DP of HA is based on the disaccharide subunit. bAs determined with SEC-MALS. Synthesis of PAAm-g-PFPA and PEI-g-PFPA. The synthesis of the PAAm-graft-PFPA (PAAm-g-PFPA) adhesion promoter has been previously described.25,34 Briefly, to prepare 10 mL of the stock solution at 3 mg/mL, 18.9 mg of PAAm·HCl was weighed together with 47.5 mg of K2CO3 in a bottle with a stir bar and dissolved in 3.8 mL of ultrapure water (UPW; Milli-Q, Merck Millipore, Germany) by heating. In a separate vial, 16.8 mg of PFPA-NHS was dissolved in 6.2 mL of ethanol by brief sonication. The ethanol solution was added dropwise to the aqueous solution under vigorous stirring and left stirring overnight. A modified synthesis route for PAAm-g-PFPA has also been presented.35 At 100% yield, a stoichiometric grafting ratio of 4 was targeted. However, from a previous study by Serrano et al., the effective grafting ratio is expected to be slightly higher.25 Further, Serrano et al. calculated a PFPA surface density of approximately 1.65 PFPA groups per nm2 for a 1.8 nm thick film of PAAm-g-PFPA synthesized according the protocol above. To prepare 5 mL of 10 mg/mL PEI-graf t-PFPA (PEI-g-PFPA) stock solution, 0.27 mL of the PEI stock solution (100 mg/mL in ethanol) was added to a clean bottle with a stir bar. In a separate bottle, 34.9 mg of PFPA-NHS was dissolved in 4.7 mL of ethanol under brief sonication. The PFPA-NHS solution was added to the PEI solution under vigorous stirring and left stirring overnight. A grafting ratio of one PFPA per six ethyleneimine monomers was targeted, leading to a surface density of PFPA groups of 2.3 nm2, assuming a similar thickness as for PAAm-g-PFPA. Preparation of Chemisorbed Coatings on Silicon Wafers. Silicon wafers (10 mm × 8 mm; POWATEC, Switzerland) were cut and sonicated for 10 min, twice in toluene and twice in 2-propanol, and then dried with filtered (0.45 μm) N2 gas. Before surface functionalization, the wafers were exposed to oxygen plasma (
