Synthetic mucin lubricant

Abstract

A lubricant comprising a mucin with a structure of poly(Gal-Thr)n and a liquid solvent, wherein n is an integer from 18-26.

Description

SYNTHETIC MUCIN LUBRICANTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and is a non-provisional of, U. S. Patent Application 63 / 761,058 (filed February 20, 2025) the entirety of which is incorporated herein by reference.STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant numbers DMR-2212139, DMR 2212162, TI-2528187 and TI-2304237 awarded by the National Science Foundation and FA9550-23-1-0230 awarded by the Air Force Office of Scientific Research. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Mucus is a ubiquitous hydrogel produced across the animal kingdom, with every animal producing at least three different mucuses. Mucus has many functions, but in most cases an important function is lubrication. In humans, mucus provides lubrication between the eyelid and the eye, for food bolus transport, and for sexual function among many other functions. Many of the advantageous tribological properties of mucus are directly attributed to mucins, an important protein found in all mucus The effectiveness of mucins in solution in lubricating soft contacts has motivated its use in numerous applications, including catheters and medical tubes, contact lenses, artificial eye drops, and joint lubrication. Mucin solutions have also been reported to lubricate hard-hard contacts, and applications in such contacts have been proposed, such as metal-based artificial heart valves and ceramic-based artificial joints.

[0004] Fundamental understanding of the nanoscale mechanisms by which mucins lubricate at nanomaterial interfaces has the potential to assist in the development of diagnostics and therapies for the aforementioned medical conditions. It also can assist inthe rational development of purified forms of natural mucins and rationally designed synthetic mucins for such purposes, and for additional engineering and commercial applications.

[0005] In mammals, mucins are composed of heavily glycosylated domains that give the molecule a bottlebrush morphology, along with cysteine-rich domains and von Willebrand terminal assemblies. The glycosylated regions have steric crowding between neighboring sugar groups, leading to a rel tively stiff and linear structure on the scale of nm to tens of nm The sugar groups in natural mucin often have ionizable sialic acid and sulfate terminations, which gives mucin a net negative charge near neutral pH. This electrostatic repulsion, along with the affinity of the sugar groups for water, reduces aggregation of the mucin. Cross-linking of the mucins by disulfide or ionic bridges can generate a semisolid hydrogel structure, anchored to the surface by transmembrane mucins such as MUC1.

[0006] A difficulty in achieving a better understanding of mucus lubrication can be attributed to the complexity of mucus, which, in addition to containing multiple different mucin proteins, can also contain hundreds of other proteins produced by the body which in some cases may participate in lubrication, along with other constituents such as microbes and solids originating from outside the body.

[0007] Synthetic mucins offer an attractive alternative path for understanding the lubricity of mucins generally, including the degree to which stress-induced modifications lead to tribofilm formation. They also are promising for fulfilling application needs because their structures are well-defined, they can be produced at scale, they can be tailored to specific applications, and have minimal batch-to-batch variability. Such work has been limited to molecules with non-natural backbones and brushes so far, although some synthetic mucins have been constructed from fragments of natural mucin and achieved impressive lubrication results. An improved synthetic mucin is therefore desirable.

[0008] The discussion above is merely provided for general background information and is not intended to be used as an aid in determining the scope of the claimed subject matter.SUMMARY

[0009] A lubricant comprising a mucin with a structure of poly(Gal-Thr)nand a liquid solvent, wherein n is an integer from 18-26. The technical problem to be solved is the formation of a lubricant using a scalable process that provides low batch-to-batch variability while still providing acceptable level of lubrication An advantage that may be realized in the practice of some disclosed embodiments is the formation of a tribofilm that results in persistent lubrication, even after the solution has been washed away.

[0010] In a first embodiment, a method of treating a subject for an eye condition is provided. The method comprising steps of: adding a pharmaceutically acceptable lubricant to a subject’s eye, the lubricant comprising a mucin with a structure of poly(Gal-Thr)nand a liquid solvent, wherein Gal is galactose, Thr is threonine and n is an integer from 18-26 and wherein the lubricant is cysteine-free.

[0011] In a second embodiment, a method of lubricating a subject’s body is provided. The method comprising steps of: coating at least a portion of a subject’s skin with a pharmaceutically acceptable lubricant comprising a mucin with a structure of poly(Gal-Thr)nand a liquid solvent, wherein Gal is galactose, Thr is threonine and n is an integer from 18-26 and wherein the lubricant is cysteine-free.

[0012] In a third embodiment, a method of lubricating a subject’s body is provided. The method comprising steps of: coating at least a portion of a condom, a tampon or a pessary with a pharmaceutically acceptable lubricant comprising a mucin with a structure of poly(Gal-Thr)nand a liquid solvent, wherein Gal is galactose, Thr is threonine and n is an integer from 18-26 and wherein the lubricant is cysteine-free.

[0013] In a fourth embodiment, a lubricant is provided. The lubricant comprising: a mucin with a structure of poly(Gal-Thr)nand a liquid solvent, wherein Gal is galactose, Thr is threonine and n is an integer from 18-26, wherein the lubricant is cysteine-free.

[0014] This brief description of the invention is intended only to provide a brief overview of subject matter disclosed herein according to one or more illustrative embodiments and does not serve as a guide to interpreting the claims or to define or limit the scope of the invention, which is defined only by the appended claims. This brief description is provided to introduce an illustrative selection of concepts in a simplified form that are further described below in the detailed description. This brief description is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. The claimed subject matter is not limited to implementations that solve any or all disadvantages noted in the background.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] So that the manner in which the features of the invention can be understood, a detailed description of the invention may be had by reference to certain embodiments, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the drawings illustrate only certain embodiments of this invention and are therefore not to be considered limiting of its scope, for the scope of the invention encompasses other equally effective embodiments. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the features of certain embodiments of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views. Thus, for further understanding of the invention, reference can be made to the following detailed description, read in connection with the drawings in which:

[0016] FIG. 1 A is a graph of friction vs sliding speed for DI water control and different concentrations of poly(Gal-Thr)22 aqueous solution for a macroscale PDMS ball on three SiO₂ plates with a 1 N applied load using triborheometry.

[0017] FIG. 1B depicts an order of testing performed to generate the data reported in FIG. 1C, inset shows a structural model of the poly(Gal-Thr)22 monomer unit,

[0018] FIG. 1 C is a graph of friction vs sliding speed first in 50 mg / mL solution, and then directly afterward in DI water. Low friction persists even in mucin-free solution, indicating the presence of a durable mucin-derived tribofilm on the surfaces. Error envelopes are the standard deviation across at least three sweeps of the sliding speed range.

[0019] FIG. 2 depicts an order of testing performed in the AFM SiCh ball on PDMS testing of aqueous poly(Gal-Thr)₂₂ solutions.

[0020] FIG. 3A is a graph of friction vs applied load for a succession of sliding environments including 1 mg / mL poly(Gal-Thr)22 solution for a SiO₂ colloid on a PDMS substrate (the order of the measurements is as listed in the legend). The load is ramped from high (positive, compressive) to low (negative tensile) in the measurements. The legend is listed in chronological order.

[0021] FIG. 3B shows topography and FIG. 3C shows phase channels of a tapping mode image captured during inverse imaging of the colloidal probe immediately after completion of sliding in the 1 mg / mL poly(Gal-Thr)22 solution. There is no discernible topographic contrast on the colloid surface; weak variations in the phase signal are observed.

[0022] FIG. 4A is a graph of friction vs applied load for a succession of sliding environments for a SiO₂ colloid sliding on PDMS flat. The legend is listed in chronological order.

[0023] FIG. 4B shows topography and FIG. 4C shows phase channels of a tapping mode image captured during inverse imaging of the colloid probe immediately after completion of sliding in the 10 mg / mL poly(Gal-Thr)22 solution.

[0024] FIG. 5A is a graph showing friction vs applied load for a succession of sliding environments for a microscale SiO₂ colloid- PDMS flat contact The legend is listed in chronological order.

[0025] FIG. 5B shows topography and FIG. 5C shows phase channels of a tapping mode image captured by scanning across an inverted AFM probe immediately after completion of sliding in the aqueous 50 mg / mL poly(Gal-Thr)22 solution The topography channel has been flattened with a quadratic polynomial to improve the contrast of the tribofilm region.

[0026] FIG. 6A and FIG. 6B are graphs of friction vs applied load for a succession of sliding environments for a microscale SiO₂ colloid-AhCh flat contact where the relevant poly(Gal-Thr)22 concentration is 1 mg / mL (FIG. 6 A) and 50 mg / mL (FIG. 6B). The legends are listed in chronological order.

[0027] FIG. 6C shows topography of a tapping mode image captured by scanning across an inverted AFM probe immediately after completion of sliding in the aqueous 50 mg / mL poly(Gal-Thr)22 solution The green circle represents the maximum nominal Hertzian contact area of the initial SiO₂-Al₂O₃ contact.

[0028] FIG. 7 depicts an FTIR spectrum of wear track (“contact’ ’) and noncontact region of PDMS ball after triborheometry testing in 50 mg / mL poly(Gal-Thr)22. Error envelope is the standard deviation of four spectra captured on separate regions of the ball. Inset shows synthetic peak fitting of the OH stretch region of the noncontact region of PDMS ball, which can separate contributions from bound and mobile water molecules.

[0029] FIG. 8A is a reproduction of selected data from FIG. 3 A for the 1 mg / mL poly(Gal-Thr)22 sliding experiment for PDMS on SiO₂, with the addition of JKR fits theto the poly(Gal-Thr)22 data. Solid line fits have unconstrained work of adhesion, W, and interfacial shear strength. Dashed lines, which are only fit to the region with applied load <500 nN, have unconstrained W, but r is constrained to match that found for the DI, prepoly(Gal-Thr)22 friction vs load test.

[0030] FIG. 8B is a reproduction of selected data from FIG. 4A for the 10 mg / mL poly(Gal-Thr)22 sliding experiment, with the addition of a JKR fit to the final poly(Gal- Thr)22 sliding test for applied loads less than 750 nN.

[0031] FIG. 9 is a schematic illustration of poly(Gal-Thr)₂₂ concentrating along contact boundary of SiO₂−PDMS contact without penetrating contact interior.

[0032] FIG. 10A, FIG. 10B is a schematic illustration of side (FIG. 10A) and top (FIG. 10B) views of the experimental geometry of the ball-on-3-plates triborheometry setup.DETAILED DESCRIPTION OF THE INVENTION

[0033] This disclosure provides a mucin with an amino acid backbone of polythreonine, which is one of the amino acids that is the site of O-glycosylation in natural mucins. Each of these threonines (Thr) is modified at the O-position with the monosaccharide galactose (Gal), which is the most common terminal carbohydrate residue on many mucin glycans. The resulting synthetic mucin, poly(Gal-Thr)22, is shorter than natural mucin, with a monodisperse Mwof 6 kDa vs 0.5−20 MDa for natural mucins, but is entirely made of natural biological structures resulting in inherent biocompatibility (see FIG. IB, inset for structural formula). Poly(Gal-Thr)22 exhibits the typical shear thinning rheological response of natural mucin solutions.. Poly(Gal-Thr)22 is also able to achieve an order of magnitude reduction in friction, demonstrating nanoscale lubrication as an additional important facet of its interfacial behavior.

[0034] This disclosure provides a lubricant that comprises a mucin and a liquid solvent. In one embodiment, the mucin is a poly(Gal-Thr)nmucin, wherein n is aninteger (e.g.. from 18-26, from 9-25, from 20-24, from 21-23, or 22). The mucin may be present in a liquid solvent (e.g. water, oil, etc.) at a concentration of, for example, from 5-80 mg per mL, from 40-60 mg per mL or from 5-15 mg per mL The mucin may be cysteine-free. The disclosed lubricants may be used in a variety of methods such as for the treatment of an eye condition (e.g. dry eye disease), as a coating for an implant, as a personal lubricant (e.g as a sexual lubricant, a sexual lubricant for a condom such as a male condom or a female condom, a lubricant for a tampon, a pessary or other similar device ) as a joint / articular lubricant.

[0035] In one embodiment, the lubricant consists essentially of the mucin and water.. Additional materials may be present provided they do not significantly decrease the lubricity of the lubricant Examples of additional materials include buffers (e.g. stabilizing at a pH of about 7), stabilizers, etc. A stabilizer helps maintain stability of the composition. Examples include lecithin, sodium stearoyl lactylate, polysorbates (Tween 20, Tween 80, polysorbate 60), lecithin, sodium citrate, tris buffer, sodium metabisulfite, ascorbic acid, ethylenediaminetetraacetic acid (EDTA), citric acid, butylated hydroxytoluene (BHT), tocopherols. A buffer is a compound that maintains the pH of the composition. Examples include phosphate buffer (e.g. sodium phosphate), citrate buffer (e.g. citric acid), carbonate buffer (e.g. sodium bicarbonate), acetate buffer, tris buffer, HEPES buffer. In one embodiment, the lubricant contains sufficient salt to render it isotonic for ophthalmic uses (e.g. 0.9 wt% saline).

[0036] In another embodiment, a mucin is provided that consists of polymeric galactose-threonine. The mucin is cystine-free and has a molecular weight of, for example, IkDa to lOkDa: 3kDa to 8kDa; 4kDa to 8kDa; 5kDa to 7kDa and 6kDa±0.5kDa, The mucin may be present with a liquid solvent to provide a lubricant.

[0037] This disclosure demonstrates aqueous solutions of a novel synthetic mucin that mimics the structure of biological mucin domains but with a low molecular weight and no specific surface anchoring groups lubricates PDMS-SiO₂ (PDMS, polydimethylsiloxane) contacts in the boundary contact regime. For a macroscalePDMS-SiO₂ contact in the boundary lubrication regime, friction coefficients for 50 mg / mL poly(Gal-Thr)₂₂ solutions were more than an order of magnitude below those found for DI water. This shows that poly(Gal-Thr)22 can be a viable alternative for applications which utilize natural mucin lubricants, thus avoiding complications with natural mucins.

[0038] By performing microscale friction testing using colloidal probe AFM which directly probes nanoscale contact mechanics, an unanticipated, quantitative agreement is shown with the degree of lubrication found at macroscale. Further analysis using contact mechanics shows that reduction of interfacial adhesion is a potent mechanism for friction reduction at the microscale that is operative at low poly(Gal-Thr)22 solution concentrations. More importantly, at higher poly(Gal-Thr)22 concentrations, effective lubrication is provided by the formation of a functional nanoscale interfacial structure via stress-induced modification of the mucin, with its structure indicating a nucleation and growth mechanism. The tribofilm reduces the average interfacial shear strength at 10 mg / mL by 56% and at 50 mg / mL, the apparent friction coefficient is reduced by 97%. FTIR-ATR analysis indicates that the tribofilm is compositionally identical to, yet denser than, passively adsorbed poly(Gal-Thr)22. A denser structure, produced by the tribological stresses, implies H-bonding within the tribofilm, which in turn, increases its durability in the tribological contact.

[0039] Additionally, in microscale hard-hard SiO₂-Al₂O₃ contacts, while I mg / mL poly(Gal-Thr)22 did not show a lubrication advantage relative to DI water, 50 mg / mL poly (Gal -Thr)22 provided a friction reduction exceeding 95% at the highest loads probed due to the formation of thick, filamentous tribofilm that functions as a stress-induced nanomaterial separating the counterfaces. This performance makes poly(Gal-Thr)₂₂ a promising material for tribological applications in hard-hard contacts.

[0040] The performance of poly(Gal-Thr)22 is remarkable given its very low molecular weight relative to natural mucins, that it only mimics a single domain structure, and that it has no specific surface anchoring groups like cysteine domains andvon Willebrand terminal domains. While such features may further enhance lubrication, this study shows that they are not necessary to achieve good lubrication. The poly(Gal-Thr)22 tribofilm ensures the sliding surface is densely decorated with glycan OH groups. ~OH groups promote lubrication in numerous contexts via bound hydration shells which separate the surfaces, mitigating adhesion and providing a low shear stress sliding interface. This insight, along with the other findings presented here, could prove useful in understanding how synthetic mucin structures can be tailored to provide further improved lubrication. In some studies bovine submaxillary mucin (BSM) can achieve another order of magnitude reduction in friction at a lower mucin solution mass concentration than observed here for poly(Gal-Thr)22. Adhesion of the poly(Gal-Thr)22 to the surfaces appears to be an important ingredient for ensuring low friction with mucin, given that the passively adsorbed poly(Gal-Thr)22 can be easily swept from the counterface prior to tribofilm nucleation. Adding hydrophobic domains to the poly(Gal-Thr)22 would enhance adhesion to hydrophobic counterfaces and create additional attraction between hydrophobic sites in adjacent tribofilm molecules which will enhance cross-linking, improving both the durability and stability of the resulting tribofilms.

[0041] Macroscale Response

[0042] To investigate the macroscale lubricating performance of poly(Gal-Thr)22 under aqueous conditions, p was measured using triborheometry (a rheometer operated in a tribology mode) with a rotating PDMS ball on three stationary SiO₂ plates, across a range of sliding speeds in deionized water and in aqueous solutions of poly(Gal-Thr)22 at varying concentrations. FIG. 1A presents results from this testing. Focusing on the measurement in deionized (DI) water, a Stribeck curve response is observed: at low speeds, the boundary p is very high because of the large real contact area between the PDMS and SiO2. At increasing speeds, p is lower as more water is entrained into the contact, providing hydrodynamic load support, which brings a subset of microscale and nanoscale asperities out of contact and reduces the real area of contact. This is referred to as the mixed regime.

[0043] The poly(Gal-Thr)22 solutions, at concentrations which span the physiological range in the human body (1-5 wt %), show progressively decreasing p in both the boundary and mixed regimes as the poly(Gal-Thr)22 concentration increases. There is also a large reduction in p between 10 and 50 mg / mL. At 50 mg / mL, in the boundary regime below 20 pm / s, p < 0.1, which is similar to that found in many natural mucin studies for PDMS contacts, although some studies with BSM have achieved p < 0.01. Sliding in 50 mg / mL solution reduces friction compared to sliding in DI water by a factor of 84~93% for speeds in the boundary regime below 1 mm / s.

[0044] In some tribological systems, such as diamond-like carbon or fully formulated motor oil, low friction in the boundary regime is achieved because of stress-induced formation of a molecularly interconnected thin film which forms on one or both of the counterfaces. Such films are referred to as tribofilms, and are robust enough to survive the normal and shear stresses of the tribological contact and subsequently provide lubrication. In some cases the tribofilm may experience ’ear, but the tribofilm is replenished by fresh precursor molecules from the surrounding solution that react under the applied contact stresses to contribute to tribofilm growth. In other systems such as fatty acid-containing oils, a passively adsorbed monolayer from additive molecules in the oil is robust enough on its own to provide lubrication to the contact, again assisted by readsorption that compensates for removal that occurs during sliding. No studies have determined whether mucins form such a stress-modified tribofilm during tribological sliding.

[0045] To examine the mechanism of boundary lubrication of the disclosed synthetic mucin, a test was performed with an aqueous 50 mg / mL solution, and then the poly(Gal-Thr)22-exposed PDMS ball and Si O2 plates were tested in pure DI water. The testing order is illustrated in FIG. IB. The p in 50 mg / mL solution of poly (Gal -Thr)22 in FIG. 1C is 50% higher than was found in FIG. 1 A, which may be attributed to a suspected lower roughness of the PDMS ball used in this test as a result of having been subjected to a longer run-in period in DI water. This explains the 80% higher boundary p in DI waterand may also explain why experimental scatter was smaller in this testing Despite this difference, consistent with the data in FIG. 1 A sliding in 50 mg / mL poly(Gal-Thr)22 reduces p by 91-93% below 1 mm / s. When sliding these mucin-exposed surfaces in DI water, p is indistinguishable from sliding in 50 mg / mL poly (Gal -Thr)22, except at the highest speeds where it is higher by only 25%, still far lower than the initial p when sliding fresh surfaces in DI water. This indicates that exposing the ball and plates to poly(Gal-Thr)22 under sliding conditions provides enduring lubrication. The enduring lubrication indicates the presence of a tribofilm with submicrometer to nanoscale structural extent on one or both surfaces. The persistence of this low friction tribofilm for the 4.3 m of continuous sliding during the test indicates that the tribofilm has a sufficient volume and durability to retain its low friction functionality despite some degree of wear that may occur during the test. A poly(Gal-Thr)22 concentration threshold for growth of a full tribofilm may explain the large decrease in friction observed between 10 and 50 mg / mL in FIG. 1 A.

[0046] The value of p in the boundary regime is 80% higher for the tests in DI water FIG. 1C compared to those in FIG. 1 A and is 50% higher for the tests in 50 mg / mL solution. This is attributable to the expected lower roughness of the PDMS ball used in the tests in FIG. 1C since it was subjected to an extended run-in period in DI water for these tests. This would lead to higher true contact area with the plates and thus higher friction. Lower roughness may also explain why experimental scatter was smaller in this test.

[0047] Microscale Response

[0048] To further examine the tribological response of the poly(Gal-Thr)22 solutions, AFM experiments were performed using SiO₂ colloid probes. The goals were (1) to show that the macroscale response trends can be replicated at smaller length scales; (2) to observe the proposed tribofilm growth dynamics in a welldefmed contact with known contact pressures and nanoscale contact geometry; and (3) to take advantage of well- defined contact to pursue a mechanistic analysis of the friction response.1

[0049] FIG. 2 shows the sequence of testing for microscale friction measurements using AFM, which is essentially the same as the macroscale testing shown in FIG. 1C, again for a PDMS / SiO₂ contact: friction is initially measured in DI water alone to establish a friction baseline for the particular probe used in the experiment, measurements were then performed in poly(Gal-Thr)₂₂ solution to evaluate the lubricity in poly(Gal-Thr)₂₂ including extended sliding of at least 1400 cycles to ensure adequate time and stress history for the possible nucleation and growth of tribofilm, and finally the poly(Gal-Thr)₂₂-exposed SiO₂ ball is again slid in pure DI water to evaluate the lubricity and durability of any tribofilm formed during the sliding in poly ( Gal -Thr)22 solution. The measurements are conducted at three concentrations: 1, 10, and 50 mg / mL. The speed range used, 0.01-100 pm / s, ensures the contact is firmly in the boundary contact regime. For these measurements, the primary focus was on the load dependence of friction since such AFM measurements can access the tensileloaded (negative loads) regime, providing information about interfacial adhesion in the contact, and can permit analysis using adhesive contact mechanics models.

[0050] While the spherical body is now a SiCh colloid instead of a PDMS sphere, in both cases the PDMS experiences migrating contact (any given region of PDMS within the sliding track intermittently experiences contact with the SiO₂ followed by periods of no contact, where it is exposed to the solution), and in both cases the SiO experiences continuous contact (the SiO₂ regions are always in contact with PDMS).

[0051] 1 mg / mL poly(Gal-Thr)₂₂: FIG. 3A presents the friction vs load response for a colloidal AFM probe in DI water, 1 mg / mL solution, and then again in pure DI water. For the initial measurement of friction vs load in DI water, a large negative (i.e., tensile) load of −1100 nN is reached before the SiO₂ colloid pulls away from PDMS substrate. Only data prior to pulloff is included for clarity of presentation. This large tensile force indicates that adhesion is strong There is also a sublinear dependence of friction on the load. This sublinear friction-load response is characteristic of a single asperity contact, which can often be modeled analytically for simple geometries such as a sphere on flat,even in the presence of adhesive forces. To see whether this response has single asperity scaling, a fit using a generalized friction fitting algorithm, which can accommodate the range of expected single asperity adhesive contact mechanics models, was performed. The analysis demonstrated that the Johnson-Kendall-Roberts (JKR) model fits the data best. The JKR model is indeed expected to apply for PDMS-SiCh contacts, as it applies for compliant materials with relatively strong, short-range adhesion and larger probe radii. The work of adhesion, W, for PDMS−SiO₂ underwater calculated from this fit using the measured 11.4 μm radius of the colloid is 21 mJ / m². It is within the range of values expected for nonspecific, i.e., van der Waals, interactions between these surfaces.

[0052] Sliding in 1 mg / mL solution produces an immediate and substantial reduction in both friction and adhesion compared to DI water alone. In the first sliding test measuring in 1 mg / mL solution, friction is immediately reduced with respect to the measurement in DI water (FIG. 3 A, white squares). For example, at zero applied load in FIG. 3B, friction in DI water alone is 1730 nN, but for the initial sliding test in the poly(Gal-Thr)22 solution friction is 800 nN for zero applied load, a decrease by more than a factor of 2. Adhesion is also significantly reduced, as evident in the maximum tensile (negative) load that is reached prior to the point of separation (the pull -off force). In DI water, this maximum tensile load is -1110 nN; for the initial sliding test in poly(Gal-Thr)22 it is -115 nN, a nearly 10-fold decrease. After measuring the speed dependence of friction at an applied load of 50-100 nN, a long period of sliding was performed at 50~100 nN applied load for a total sliding distance of 74 cm, over a time of 40 min. This step allows for any slow evolution of the contact geometry and chemistry to take place. Friction was then measured as a function of load again to check for any changes. Friction is further reduced (FIG. 3B, black squares), indicating progressive evolution of the interface that further improved lubrication. Upon swapping back to pure DI water with a fresh PDMS substrate, friction is immediately restored to its prior level in pure DI water. This suggests that any tribofilm formed in 1 mg / mL poly(Gal-Thr)22 solution is not durable in a mucin-free liquid environment.

[0053] To directly check for the presence of a tribofilm or other changes to the contact zone of the AFM colloid, a tapping mode AFM image of the contact zone on the colloid directly after the 1 mg / mL poly(Gal-Thr)22 sliding tests was obtained via the inverse imaging method, FIG. 3C. No contrast that would suggest an adsorbed film or probe modification is observed. This image was taken in air after drying of the contact zone, so there could be a reduction in any adsorbed film thickness from a loss of the poly(Gal-Thr)22 hydration shell. The associated phase image (FIG. 3C), which can be sensitive to local mechanical and compositional properties, does show some contrast, which is not present in images of the colloid performed after the initial sliding in DI water, before any exposure to mucin. This suggests that, after sliding in 1 mg / mL solution, some adsorbed mucin may be present but it is undetectable in the topography image and thus likely very thin.

[0054] 10 mg / mL poly(Gal-Thr)22: Sliding in a higher concentration of 10 mg / mL, poly(Gal-Thr)22 leads to a more pronounced and persistent reduction of friction than the 1 mg / mL case. FIG. 4A presents results from the same test protocols as in FIG. 3A, but with 10 mg / mL solution. Initial sliding in DI water again exhibits sublinear growth of friction vs load, which is well fit with the JKR model, yielding W = 32.5 mJ / m², somewhat larger than the value of 21 mJ / m2found for sliding in the 1 mg / mL (tested with a different probe) and indistinguishable from the literature value for glass in contact with PDMS in DI water referenced earlier (32.5 mJ / m²) (FIG. 3 A, white circles). In the very- first sliding test in the 10 mg / mL solution, friction is reduced by a factor of 20 at zero applied load (vs a factor of 2 in the 1 mg / mL testing) (FIG. 3 A, white squares). After prolonged low-load (80 nN) sliding to allow for the possibility of a tribofilm to form, loaddependent friction is lowered further (FIG. 3A, black squares). After returning to the pure DI water, unlike in the 1 mg / mL case, friction is not restored to its prior high levels (FIG. 3 A, black circles). Friction and adhesion remain substantially reduced relative to that found for the initial sliding test in DI water.

[0055] To understand the friction evolution across the experiment, tapping mode topography (FIG. 4B) and phase (FIG. 4C ) images of the colloid via inverse imaging were acquired immediately after sliding in the 10 mg / mL solution. The topography shows that two isolated islands of tribofilm, from 60-110 nm thick, placing them within the regime of functional nanostructured tribofilms. These islands coexist with numerous smaller, filamentous structures which are predominantly about 2 nm tall; their presence is more discernible in the phase channel (FIG. 4C. The inverse imaging method cannot capture the entire contact zone at the highest loads that were calculated because of the 2.12 pm pitch between the spikes of the TGT1 grating, so comparison with the noncontact regions cannot be performed within the same image (for reference, for PDMS-Si and the 12.1 pm diameter of this probe, at zero applied load for the high adhesion prepoly(Gal-Thr)22 DI water sliding condition, the contact diameter calculated from the JKR model is 2.2 pm, for the highest load while sliding in poly(Gal-Thr)22 once adhesion is greatly reduced, the JKR contact diameter is 1.6 pm). The coexistence of very thin and much thicker regions without any intermediate thickness islands is consistent with a stress-activated nanoscale nucleation process. The much thicker islands must contribute significant lubrication to the interface, given the significant friction reduction that comes from extended sliding. Imaging of the probe surface after sliding in DI water post-poly(Gal-Thr)22 didnot reveal any structure or contrast on the colloid surface to explain the evident continuing lubrication of the contact presented in FIG. 4A.

[0056] 50 mg / mL poly(Gal-Thr)22: At 50 mg / mL, poly(Gal-Thr)22 provides the strongest and most durable microscale lubrication of all concentrations tested, FIG. 5 A presents the same type of tests as those in FIG. 3A and FIG. 4A, but in 50 mg / mL solution. The initial friction response in DI water, while similar at high loads to what is seen with the probes measured at lower poly(Gal-Thr)22 concentration, does not show large adhesion or a JKR-like response at low loads. This is attributed to sparse contamination of the fresh probe that would be expected to disrupt adhesion; these contaminants could be detected in the inverse imaging. Because even brief ambient exposure prior to immersion leads to adsorption of adventitious contaminants on bothhydrophobic and hydrophilic surfaces, the present measurements reflect a practically relevant condition, with results indicating that contact and sliding displace preexisting adsorbates. Regardless, sliding in 50 mg / mL solution, as with the tests at lower concentrations, shows an immediate improvement in lubrication of the contact (FIG. 5 A, white squares). After extended sliding in 50 mg / mL solution, lubrication improves further (FIG. 4A, black squares). For example, at 1600 nN applied load (near the upper limit of applied loads tested), the friction force is reduced by 97% relative to that in DI water prior to poly(Gal-Thr)22 exposure, and the slope of the friction vs load data (the effective p) is reduced by 97%. Upon sliding in DI water after sliding in the 50 mg / mL solution, the friction rises, but residual lubrication remains with respect to DI water (FIG.5A, black circles).

[0057] To evaluate interfacial structural changes responsible for the increased lubrication, topographical characterization of the colloid surface via inverse imaging is again performed, but this time using the sharp tip of an inverted tapping mode AFM probe to capture the entire contact region along with a portion of the out-of-contact region (FIG. 5B, FIG. 5C). The topography image has been flattened by subtracting a quadratic surface matching the noncontact region of the image to effectively remove the overall curvature of the spherical probe and thus provide better contrast for identifying the resulting topography changes. A circular tribofilm that spans the entire nominal contact area at the highest applied load is observed. The thickness of tribofilm exceeds 50 nm at the center. The phase image shows clear contrast for the tribofilm, attributable to substantially different composition and mechanical properties than the underlying stiff silica substrate. Additionally, there is a contrast change near the center of the tribofilm which is attributed to evolution of the tribofilm structure during the extended low-load sliding, where only a subset of the tribofilm would be in contact. The presence of the mucinderived tribofilm following sliding indicates its ability to favorably adsorb to the counterfaces and displace preexisting surface contamination under tribological loading. After sliding again in DI water, as in the 10 mg / mL case, this tribofilm is almost entirely removed. Some lateral phase contrast is present, which may be a sign of poly(Gal-Thr)22that remains in the contact region and continues to provide modest lubrication in pure DI water sliding conditions.

[0058] Comparison with a Hard-Hard Contact Interface

[0059] The observation of tribofilm formation on the silica colloidal probes suggests that poly(Gal-Thr)₂₂ may lubricate hard-hard interfaces as well. This is desirable for applications as mentioned earlier, but such experiments also allow us to probe the lubrication and tribofilm formation behavior up to higher contact pressures. Accordingly, experiments were also performed using a hard -hard Al₂O₃−SiO₂ interface. AI2O3 substrates were utilized since SiCh-SiOz sliding experiments led to gross adhesive transfer of SiOz to the colloid surface. Experiments were performed with 1 and 50 mg / mL poly(Gal-Thr)₂₂ concentration solutions (FIG. 6A, FIG. 6B, FIG. 6C). For 1 mg / mL concentration, an initially high adhesion of 660 nN was found when sliding in pure DI water, but the adhesion was quickly extinguished upon sliding, leading to the stable friction-load response in FIG. 6A (white circles).

[0060] This sudden reduction in adhesion was a common phenomenon in both the hard-hard sliding experiments and in the hard-soft experiments (e.g., in the 50 mg / mL poly(Gal-Thr)₂₂ experiment shown in FIG. 5A, FIG. 5B, FIG. 5C) and is attributed to trace contaminants present in the DI water entering the contact and strongly adhering to the colloid after experiencing high contact stresses. Single-asperity friction-load scaling was not observed, likely the result of the high stiffness of the counterfaces which precludes conformal contact and leads to a multiasperity interface. The higher modulus of AI2O3 relative to PDMS ensures that the nominal Hertzian contact area is orders of magnitude smaller at similar applied normal loads than in the PDMS SiOz contact, which can largely explain why the magnitude of the friction forces for all loads and sliding environments are more than an order of magnitude smaller than that observed for PDMS-SiOz contacts using a SiOz colloidal sphere of the same diameter. The high stiffness also leads to a much smaller nominal contact radius than in the PDMS-SiOz experiments, with a maximum nominal Hertz radius at the highest applied load (Esi02~70 GPa, vSiO„ = 0.17, PAI2O3— GPa,vAi2o3— 0.28, negligible roughness assumed) of merely 41 nm, corresponding to a highly confined nanoscale contact, with an average pressure (85 MPa) far exceeding that in the soft-hard system.

[0061] Upon sliding in the 1 mg / mL poly(Gal-Thr)22 solution, friction was not reduced, with a very similar friction-load response to that observed in DI water alone (FIG. 6 A, black triangles). The friction response was likewise unchanged when sliding in DI water after poly (Gal -Thr)22 exposure (FIG. 6A, black circles).

[0062] The response for the sliding experiment in the 50 mg / mL concentration poly(Gal-Thr)22 is shown in FIG. 6B. The initial friction-load response in DI water is very similar to the 1 mg / mL experiment (FIG. 6A, white circles). However, in this case, upon sliding, friction is suppressed to near the limits of detectability, with friction reduction exceeding 95% at the higher applied loads (FIG. 6B, black triangles). Upon returning to sliding in pure DI water, friction remains suppressed. The friction suppression can be confidently attributed to the formation of tribofilm separating the Al₂O₃ and SiO₂ counterfaces, as shown in the topographic inverse image of the Al₂O₃ colloid in FIG. 6C. The lateral extent of the tribofilm, unlike in the hard-soft experiments, greatly exceeds the nominal Hertzian contact diameter: the 2.12 pm pitch between the TGTl spikes used to capture the topographic image does not allow for imaging the entire tribofilm, but its diameter exceeds the contact diameter at the maximum load estimated from the Hertz model (82 nm) by more than a factor of 20. While the thickness of the tribofilm cannot be precisely determined, it exceeds 250 nm based on topographic relief in the image. The tribofilm possesses a striking filamentous structure that bears at least a superficial similarity to the filamentous structure often observed to form spontaneously by gel-forming mucins. In this case, the structure is formed as a result of cyclic shear stress.

[0063] The formation of a mucin-derived tribofilm at a hard-hard sliding contact is consistent with recent literature for a natural system. Mucin-derived tribofilm enhanced the wear resistance of tooth enamel in fluid s with and without porcine gastric mucin(PGM), presenting evidence of stress induced tribofilm formation. Additionally, the tribofilm regions were found to be better protected from demineralization than areas coated with a passively adsorbed pellicle. Protection from demineralization is a major function of salivary pellicles on tooth enamel. This indicates that the methodology, and the behavior of the synthetic mucin under stress, both have potential to advance the understanding of mucin’s behavior in natural systems.

[0064] FTIR-ATR Spectroscopy of Tribofilm

[0065] To interrogate the structure and chemistry of the lubricating tribofilm formed during friction testing, Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy was performed on one of the SiO₂ wafers and the PDMS ball utilized in the macroscale trib orheom etry of the 50 mg / mL poly(Gal-Thr)₂₂ solution (FIG. 1 A, FIG. IB, FIG. 1C). After rinsing the samples with DI water to remove weakly bound poly(Gal-Thr)22 and allowing the ball to air-dry, FTIR-ATR spectra were captured in both the contact and noncontact regions of the samples, and the background PDMS signal was subtracted from the spectra using a spectrum obtained from a clean PDMS ball, scaled to match the intensity of the 2960 cm⁻¹ PDMS peak in the sample spectra. The resulting spectra for both regions of the PDMS ball are shown in FIG. 7. All spectral features are reproduced between the contact and noncontact regions of the sample with two exceptions, discussed further below. Otherwise, peak positions match within 5 cm⁻¹ and line shapes are indistinguishable between the two regions. From 3000 to 3600 cm-1, there is a broad peak attributed to -OH stretching from adsorbed water and galactose, ~CH stretching peaks are present at 2915 and 2850 cm-1, and amide peaks from amide groups in the threonine backbone are present between 1500 and 1700 cm⁻¹.

[0066] However, the more intense poly(Gal-Thr)22 peaks in the noncontact region, along with stronger attenuation of the PDMS substrate spectrum (subtracted from spectra in FIG. 7) imply that the tribofilm layer is thinner than the passively adsorbed poly(Gal-Thr)22 layer in the noncontact region. This agrees with the observation that in the microscale AFM contacts, any passively adsorbed poly(Gal-Thr)22 is initially swept awayand that tribofilm formation occurs progressively with accumulated stress and sliding history in the contact region. In both the contact and noncontact regions, the amide I peak is observed at 1628 ± 3 cm−1. The amide I peak is frequently used to evaluate secondary structure in biomolecules, and this position is consistent with a p sheet or aggregated strand conformation. Since the synthetic mucins are likely shorter than the persistence length of the bottlebrush region of mucin, which has been estimated to exceed 30 nm, the polv(Gal-Thr)22 cannot fold to form a P sheet. The amide I position thus implies that there is significant intermolecular H-bonding in both the tribofilm and in the passively adsorbed poly(Gal-Thr) 2 layer.

[0067] A number of studies examining water adsorption on surfaces, in nanocapillaries, and into hygroscopic polymers indicate that the broad ~OH stretching peak can be fit with multiple peaks corresponding to the strengths of H-bond interactions with the material. An FTIR study on water responsive silk films for example suggested that five components capture the diversity of H-bond configurations, with three components corresponding to water molecules bound to the solid at 3000, 3210, and 3290 cm−1, and two components corresponding to loosely bound mobile water at 3410 and 3550 cm. The FTIR-ATR results are in reasonable agreement with this: fitting the spectra with five components yielded peaks at 3070, 3200, 3280, 3400, and 3516 cm−1. The component at 3280 cm1is proportionally much larger than seen in prior water adsorption studies, so this component is assigned to OH stretching in the poly(Gal-Thr)22 molecule, in addition to bound water.

[0068] The key result from this fitting is that there is significantly less mobile water present in the tribofilm region relative to the noncontact regions This can be quantified by the ratio of the combined mobile water peak areas at 3400 and 3516 cm−1to the poly(Gal-Thr)22 and bound water peak areas at 3280 cm1. For the tribofilm region, this ratio is 0.20, whereas for the noncontact region it is 0.43. This indicates that the passively adsorbed poly (Gal -Thr)22 film can capture twice as much mobile water as the tribofilm in 50% RH ambient conditions.

[0069] DISCUSSION

[0070] The Agreement between Macro- and Microscopic Friction Measurements Suggests Conformal Contact Occurs across These Scales: The microscale and macroscale SiO2−PDMS tribological results are consistent with each other. The average Hertzian contact pressure in the macroscale experiments, 161 kPa, falls within the range of the AFM SiO2−PDMS experiments which reached 216 kPa at the maximum applied load of 1850 nN (and was equal to 161 kPa at 1200 nN). Comparing the friction force in poly (Gal -Thr)22 solution normalized by the corresponding value in pure DI water at the same applied load between microscale and macroscale measurements, reasonable quantitative agreement is achieved (Table 1). Such agreement is unexpected as there are numerous differences between macroscale and microscale frictional contacts. Most importantly, as a result of the typical multiasperity nature of macroscale contacts even with small amounts of roughness, the macroscale adhesion force is usually negligible.Table 1. Comparison of Friction Reduction by Poly(Gal-Thr)22 during Macroscale and Microscale for Load Corresponding to 161 kPa Average Hertz Contact Pressure Fractional Friction Reduction vs DI water (%) in SiO2−PDMS contactspoly(Gal-Thr)22 Concentration Macroscale Microscale1 mg / mL 39 41 10 mg / mL 68 80 50 mg / mL 90 98

[0071] However, macroscale ’DMS-glass contacts are known to retain adhesion, as the low elastic modulus of PDMS leads to conformal contact even in the presence of some counterface roughness. The instrumentation did not allow for direct adhesion measurements but a single-asperity friction-load scaling was found indicating that conformal contact was achieved. This potentially explains the agreement between macro- and microscale testing.

[0072] At Low Concentrations, Friction Reduction in poly (Gal -Thr)22 Solutions Is Mostly Driven by Reduced Adhesion: The microscale SiO2−PDMS AFM friction measurements allow us to rationalize some of the mechanisms by which poly (Gal -Thr)22 provides lubricity. Since JKR friction”! oad scaling was shown to be appropriate in FIG.3A, FIG. 3B, FIG. 3C, FIG. 4A, FIG. 4B, FIG. 4C the poly(Gal-Thr)22 sliding curves were fit with the JKR area (A) relationship directly, which, helpfully, involves W and the interfacial shear strength T: where the only free parameters are T and W. The known parameters are the colloid radius R and reduced elastic modulus E*, calculated for PDMS−SiO2 under the assumption any tribofilm will have a small effect on the contact mechanics. This equation can be w'ell fit to the initial and final poly(Gal-Thr)22 sliding data for the 1 mg / ml. solution (white squares and black squares in FIG. 8A). The fits show- a large reduction in adhesion relative to sliding in DI water, as expected. However, both fits correspond to larger T values than were found when sliding in DI water: 143 and 100 kPa for the initial poly(Gal-Thr)22 and final poly(Gal-Thr)22 cases, respectively, vs 74 kPa for DI water. There is no reason to expect such an increase in T. These fits are believed to be based on invalid assumptions. If T is constrained to not exceed that found in the case of sliding in DI water (dotted line and dash-dot-dot line in FIG. 8A), reasonable fits were only achieved for loads below approximately 500 nN (FIG. 8A), as the data depart substantially above the fitted response at higher loads. This suggests that JKR friction−load scaling is only followed for the poly(Gal-Thr)22 friction vs load measurements below' a threshold load.

[0073] JKR fitting was performed for the final 10 mg / ml., sliding test. Similar to the 1 mg / mL data, JKR fitting is only successful below' a threshold applied load, in this case of approximately 750 nN. The departure of the friction vs load data from the JKR fit above this load is clearly observable. The fit is only reasonable if T is reduced to 52 kPa, much lower than the 117 kPa value obtained with the same tip sliding in DI water. This shows that lubrication is achieved both via a large reduction in adhesion (which reduces the contact area) as well as a reduction in r.

[0074] The different r obtained with different probes while sliding in DI water in FIG. 8 A, FIG. 8B, 74 and 117 kPa, is attributable to imprecision in the lateral calibration technique, the wedge method, where uncertainty has been estimated at 10−20%. The wedge method uncertainty may be modestly higher as a repurposed optical grating with a sufficiently large pitch (32 pm) was used to accommodate the colloidal probe underwater, rather than the microfabricated test grating (with a pitch of 10 pm), which is commonly used with sharp probes in air.

[0075] At Low Concentrations, High Loads Drive Increased Friction, Indicating that Adsorbed poly(Gal-Thr)22 Is Reversibly Displaced from the Sliding Contact: For the 1 mg / mL experiment, poly(Gal-Thr)22 dramatically reduces adhesion to negligible levels without any measurable decrease of T, yet both parameters are usually coupled.Similarly, for the 10 mg / mL experiment, while T has decreased by approximately a factor of 2, adhesion again has been nearly eliminated. Without wishing to be bound to any particular theory, adsorbed poly(Gal-Thr)22 just inside and outside the edge of the contact zone is believed to locally perturbs surface and interfacial energies. In the JKR model, the contact area is controlled by an equilibrium between adhesion energy and elastic energy, leading to a compressively loaded central contact region surrounded by a tensile- loaded annulus. The work of adhesion is specified by the Dupre equation: W = y12~ Yi “ Y2, where Yi is the free energy of the PDMS-water interface, Y2 is the free energy of the SiO2~water interface, and y12is the free energy of PDMS-SiCL interface. In the absence of any adsorbed poly(Gal-Thr)22, strong adhesion is observed between clean PDMS and SiCh in pure water. While two hydrophilic interfaces in water experience net repulsion and two hydrophobic interfaces experience net attraction, hydrophobic interactions are generically stronger and longer ranged than hydrophilic repulsion. As a result, when a hydrophobic surface (PDMS) is brought into contact with a hydrophilic surface (SiO2) in water, the hydrophobic interaction can dominate, leading to net adhesion between the interfaces. The surface of PDMS exposed to bulk mucin solution attracts a significant mass of adsorbed mucin molecules, illustrated schematically in FIG.9. Additionally, sliding may serve to increase the mass of poly(Gal-Thr)22 near thecontact boundary' through pileup. Individual poly(Gal-Thr)22 molecules that manage to penetrate the tensile-loaded region of PD S S1O2 interface will sterically separate the PDMS and SiO2locally. Since the hydrophobic interaction is short-ranged with a decay length of less than 2 nm, such interactions are attenuated locally; this will reduce γ12and thus W.

[0076] Recent work also shows that the introduction of polar species to an otherwise hydrophobic surface can weaken the strength and range of hydrophobic adhesion. The reduction in W near the contact boundary necessitates a smaller equilibrium elastic energy, leading to a smaller equilibrium contact radius and thus a reduced total contact area. The latter is inferred from the observation of reduced friction when sliding in low concentration mucin solutions. Because the contact pressure distribution varies across the contact radius, poly(Gal-Thr)22 molecules that penetrate the outer rim of the contact might not reach the more highly loaded interior and instead may be redirected around the contact periphery. Hence, the average r will remain relatively invariant as W decreases. In this way, adsorbed hydrophilic poly(Gal-Thr)22molecules near the contact boundary can serve to suppress W without penetrating the interior of the contact where they would affect T for the PDMS Si O2 contact.

[0077] Examining FIG. 8A, in 1 mg / mL concentration solution, the initial and final poly (Gal -Thr)22 trends (white squares and black squares, respectively), grow more rapidly than the prediction of the JKR model above a threshold load (dotted line and dash-dot-dot line), provided that one makes the reasonable assumption that T is constrained to not exceed the value found when sliding in pure DI water. This is consistent with poly(Gal-Thr)22molecules being excluded from the contact at high contact pressures. In other words, at low concentrations, passively adsorbed mucin on the surfaces reduces adhesion and friction but is then expelled from regions of the contact where contact pressure becomes too large. The effect is reversible: upon reducing the load, friction follows the same trajectory as the increasing load segment.

[0078] With the proposed friction reduction mechanism for low concentration poly ( Gal -Thr)22 solutions in hand, it is worth revisiting the surprising agreement between the magnitude of the friction reduction for the macroscale and the microscale cases at the 1 mg / mL concentration. Recall that there was no evidence of tribofilm mediation of friction observed at the microscale, hence only passively adsorbed mucin is believed to be involved. In the macroscale case, if the interfacial shear strength remains invariant because of the lack of a tribofilm, only the adhesion reduction mechanism is possible to reduce friction An increase in the roughness of the counterfaces could also cause a reduction in the friction, but one would expect poly (Gal -Thr)22 to accumulate in any low spots of the counterfaces and increase the real area of contact, thereby increasing friction, not reducing it. There is also the possibility that poly(Gal-Thr)22 molecules could act as pinning sites between the incommensurate counterfaces, but again, that would cause the friction to increase, not decrease. In the limit of adhesion reduction to zero, an upper bound was estimated for the macroscale contact area change that occurs if adhesion is eliminated. Using 32.5 mJ / m2for W calculated for the microscale contact, the 6.35 mm radius of the PDMS ball, and the applied load of 1 N, JKR contact mechanics estimated that elimination of adhesion reduces the contact area by only 6%. This is not enough to explain the 39% reduction in friction for 1 mg / mL poly(Gal-Thr)22 relative to pure DI water sliding. Therefore, additional lubrication mechanisms must be operative to achieve the observed friction reduction at the macroscale.

[0079] Enhanced H-Bonding Is Likely Responsible for Formation and Persistence of Tribofilm Seen at High Concentrations: At 10 and 50 mg / mL, friction is modified by the formation of tribofilm within the contact area on the colloidal probe. The morphology of the tribofilm immediately after sliding in poly(Gal-Thr)22. suggests that tribofilm formation is a nucleation and growth process. The morphology of the colloid’s contact region in the 10 mg / mL poly(Gal-Thr)22 experiment, FIG. 4B, is composed of a small number of rather large islands of tribofilm and numerous small islands with <1% the volume of any large island. The lack of islands in the intermedi ate size range isconsistent with classical nucleation theory where there is some energy barrier to cross before a small island can rapidly become a large one.

[0080] Mammalian mucins are expected to maintain a net negative charge near neutral pH from partial ionization of sialic acid terminations. Poly(Gal-Thr)22 does not contain sialic acid groups, but zeta potential measurements of the poly(Gal-Thr)22 show that they are also negatively charged, although the reason is unclear. This charge, along with hydration shells around the synthetic polymers expected as a result of the poly(Gal-Thr)22 hydrophilicity, are anticipated to maintain mutual repulsion between poly(Gal-Thr)22molecules, which aids in water solubility and prevents poly(Gal-Thr)22 aggregation. Additionally, the poly(Gal-Thr)22 solutions lacked any of the features proposed to stabilize mucin association in the context of mucus gels, such as ions, other proteins, hydrophobic domains within the mucin, or cysteine groups. Nevertheless, a relatively stable aggregate of poly (Gal -Thr)22 tribofilm forms to lubricate the contact. Since the functional groups of poly(Gal-Thr)22 are heavily hydroxylated and therefore compatible with direct poly(Gal-Thr)22-poly(Gal-Thr)22 intermolecular H-bonding, one could attribute the formation of a stable or metastable tribofilm to direct H-bonding between the poly(Gal-Thr)22. While evidence shows that in an aqueous environment hydrophilic repulsion between surfaces should persist all the way to direct contact, H-bonding alone can stabilize hydrogel structures. Given the low strength of isolated H-bonds, the stability of molecular complexes must depend on H-bond cooperativity.Excluding water, as the hydrophobic PDMS−SiO2interaction does in the current context, can help stabilize cooperative intermolecular H-bonds against dissolution via competition with water. Such H-bonds have been well documented between glycans. In a rheological study of poly(Gal-Thr)22, while the bulk fluid was found to have a low viscosity close to that of water, the poly (Gal -Thr)22 also segregated to the air- water interface to form a surface gel. This is an analogous interfacial phenomenon where dehydration of poly(Gal-Thr)22 may strengthen H-bonds leading to persistent structure formation.

[0081] The FTIR-ATR results strongly support the idea that Hbonds do indeed stabilize the tribofilm structure against dissolution and wear. The FTIR-ATR spectra in FIG. 7 show that all peaks, peak positions, and peak lineshapes, other than those for free water, are indistinguishable between the tribofilm and the layer of passively adsorbed poly(Gal-Thr)22. This implies that poly(Gal-Thr)22 molecules in the tribofilm are essentially intact and have not been damaged by the tribological stress. Such molecular damage and the generation of reactive free radicals would otherwise be a plausible mechanism for cross-linking and bonding to the substrate, as has been seen in other tribofilm-forming systems under similar contact pressure regimes as these PDMS / SiCh experiments. The presence of a layer of passively adsorbed poly(Gal-Thr)22 that is thicker than tribofilm on the PDMS, implied by its higher absorption intensity, shows that the poly(Gal-Thr)22 molecules have no difficulty adsorbing to the SiO2and, especially, the PDMS. Furthermore, it indicates that the mucin molecules can associate with each other to form multiple layers of poly(Gal-Thr)22. The only appreciable difference between the contact and noncontact FTIR-ATR spectra is in the OH stretching region, where the analysis indicates that the passively adsorbed poly(Gal-Thr)22 film retains or captures from ambient much more water than the tribofilm region. Since the chemical composition is indistinguishable between these two films, there must be some structural difference between them.

[0082] Free volume in polymers has been identified as a strong predictor of water sorption capacity in the absence of chemical differences. The higher prevalence of mobile water in the passively adsorbed poly(Gal-Thr)22 thus indicates that the tribofilm possesses less free volume and a denser structure, whose formation is attributable to the applied shear and compressive stresses during contact. Such close packing of the poly(Gal-Thr)22 molecules will encourage the formation of stronger and more numerous H-bonds, which may contribute to the subsequent durability of the tribofilm that is stabilized by cooperative H-bonds.

[0083] EXPERIMENTAL METHODS

[0084] Poly(Gal-Thr)22 was synthesized using the synthetic protocol described in International Patent Publication WO2023 / 014875 (filed August 4, 2022) and corresponding U. S. national stage application 18 / 681,351 (filed February 5, 2024), the contents of which are hereby incorporated by reference. Also see Lerna et al.; Scalable Preparation of Synthetic Mucins via Nucleophilic Ring-Opening Polymerization of Glycosylated N-Carboxyanhydrides. Macromolecules, Vol. 55, Issue 11 (May 19, 2022), DOI 10.1021 / acs. macromol.1C02477, the content of which is hereby incorporated by reference. After synthesis, the poly(Gal-Thr) 2 was lyophilized and stored as powder below 0°C until used. Prior to experiments, the powder was mixed with deionized water to achieve the specified concentration and ultrasonicated to fully dissolve the poly(Gal-Thr)22. This choice to use DI water for friction testing as opposed to a buffering solution was made based on prior work showing that buffering solutions can have significant effects on measured friction due to surface interactions.

[0085] Macroscale tribometry measurements were performed in a triborheometer (DHR3, TA Instruments, New Castle, DE) using the ball-on-3-plates geometry, as shown in FIG 10A, FIG. 10B. The ball rotates on a central axis while remaining in contact with three stationary silica glass (SiO2) plates. Thus, a circular strip around the PDMS experiences contact as the ball rotates, while each of the three SiO2plates experience sliding contact in a constant circular contact patch. This is the same instrument used to characterize the rheological response of this poly (Gal -Thr (22 molecule in recent work, but using cone-plate, Couette (concentric cylinder), and double-walled ring geometries, as used commonly for rheological studies. Here, the ball was composed ofpoly dimethylsiloxane (PDMS) produced from Sylgard 184 (Dow Corning, Midland, MI) mixed at a 10:1 (wt:wt %) base:curing agent ratio, vacuum degassed, and cured at RT in a custom spherical mold. The plates were composed of diced silicon wafer with a 300 nm thick thermal oxide layer (University Wafer, Boca Raton, FL) The diced wafer pieces were sonicated in acetone then isopropyl alcohol then blown dry with N2gas flow prior to experiments. An applied vertical load of 1 N was utilized in the experiments, leading to a nominal maximum Hertzian pressure of the 3 contact spots on the 45° verticallyangled plates of 242 kPa, creating a contact spot approximately 1.8 mm in diameter, based on a Hertz contact mechanics calculation using PDMS Young’s modulus and Poisson’s ratio of 2.5 MPa and 0.48, respectively. This calculation assumes negligible roughness. This macroscale contact pressure was the lowest that could be achieved with good repeatability and matches a number of prior studies using PDMS-SiCh tribocouples to study mucin lubrication. At least 500 pL of poly(Gal-Thr)22 solution was used for each measurement to ensure the contacts were fully submerged. Six sequential velocity sweeps were performed with alternating increasing and decreasing speeds. The presented data are composed of an average of the three decreasing speed sweeps. Repeatable hysteresis was observed between increasing and decreasing speeds in the low-speed regime, attributed to static friction effects on startup from the lowest speeds. The poly(Gal-Thr)22 solution itself and the three silica plates were replaced after every test. The PDMS ball was reused after wiping clean with methanol and DI water because the ball-to-ball variability could obscure trends when swapping to a fresh ball after each test. The cleaning regimen was deemed adequate based on replicable Stribeck curves which could be produced when sliding in DI water alone after cleaning.

[0086] Microscale AFM friction testing was performed in an MFP-3D AFM (Asylum Research, Santa Barbara, CA). Commercial AFM probes (Multi75DLC, Budget Sensors, Sofia, Bulgaria) were used. For each one used, a very smooth (Rq= 0.8 nm across 1 × 1 μm2) ~25 pm diameter silica colloid (SiO2-R-25.0, Microparticles GMBH, Berlin, Germany) was attached using a micromanipulator and epoxy ( JB Weld, Sulphur Springs, TX).

[0087] The cantilever normal spring constant was determined using the Sader method18prior to colloid attachment, and corrected for the colloid offset and cantilever tilt, yielding knorm= 2.6 ± 0.2 N / m for the probes in the study. The normal calibration constant β to convert from volts to nanonewtons was determined by measuring the inverse optical level sensitivity (InVOLS) from a force-distance curve when submerged in DI water on a stiff Si wafer after colloid attachment, and the Sader calibration constant(kSader) after correction for both the 11° cantilever tilt and the colloid offset from the end of the cantilever:, j.3 / L \ ksader InVOLSfr^-ie-. P = o:

[0088] Lateral forces were calibrated using the extended wedge method which accounts for the large radius of the colloid. This calibration was performed under DI rvater to ensure an accurate lateral InVOLS. Since the large colloid is incompatible with typical gratings used for the wedge method, an optical grating (G31.6E63QEAC, Optometries, Ayer, MA) was utilized for this purpose after etching off the aluminum coating using ferric chloride (MG Chemicals, Burlington, Canada) which improved the lateral homogeneity of the lateral force signal.

[0089] AT'M experiments were performed inside a humidity cell maintained at saturation pressure of H2O to allow for working inside a droplet of poly(Gal-Thr)22 solution without a significant evaporation rate which would shift the poly(Gal-Thr)22 concentration during the experiment. PDMS flat substrates were prepared from Sylgard 184 at 10: 1 (wt.wt %) base. curing agent ratio, vacuum degassed for 15 min, cast to a thickness of approximately 2 mm in polystyrene Petri dishes, cured at 65 °C for 4 h, stored under clean dry N2 flow from the boiloff of a liquid N2 dewar, and used without solvent extraction, supported on a steel puck (measured roughness was less than 5 nm across 90 * 90 pm2). Sapphire flat substrates were singlecrystal sapphire wafer (C-plane, prime. University Wafer, measured roughness was less than 0.6 nm across 5 x 5 pm2) sonicated under acetone and isopropyl alcohol then ozone cleaned to ensure a hydroxylated surface prior to each experiment. PDMS solvent extraction to remove uncross-linked oligomers which are known to be present in Sylgard 184 was not performed. Inverse imaging of the colloidal ATM probes was performed using tapping mode in air on a “sample” composed of either an array of silicon spikes (TGT1 grating, NT-MDT, Tempe, AZ) or on an inverted tapping mode AFM probe (Tap300- G, Budget Sensors, Sofia, Bulgaria). The measurement principle is equivalent for both samples.However, imaging an inverted tapping mode AFM probe allowed for imaging a larger area of the colloid apex, which was needed to see the full extent of the tribofilm at 50 mg / mL. Speed-dependence and load-dependence of friction measurements were performed using custom measurement codes; load was always varied by starting at a maximum load and then progressively decreasing it until the colloid separated from the substrate to capture the adhesive regime of contact. Microscale contact pressures were chosen to be similar to those used in the macroscale testing, i.e., the load-dependence tests were performed with a range that crosses the contact pressure of the macroscale testing.

[0090] As the AFM is a linear sliding experiment, the PDMS substrate experiences a linear strip of contact as the tip slides, while the SiO2tip experience sliding contact in a contact patch at the end of the tip. For each friction vs load measurement obtained, the tip was moved to a new location on the PDMS substrate. This was to avoid wear of the PDMS, but it also meant that, unlike for the triborheometer, each PDM S location was unlikely to build up a tribofilm due to a limited amount of sliding (as opposed to the colloid apex, which was always in contact and could form a tribofilm).

[0091] Experiments were run as follows: 1. Slide in pure DI water to establish load¬ dependent and speed-dependent friction baselines for the particular probe; 2. Slide in poly(Gal-Thr)22 solution; 3. Slide in DI water again to establish whether any persistent changes have occurred in the colloid contact zone. Sliding in poly(Gal-Thr)22 included an extended period of constant low load (<100 nN) to allow for any possible slow evolution of the contact zone such as nucleation and growth of a lubricating tribofilm, which has been shown to occur in other mucin-lubricated systems.

[0092] FTIR-ATR spectra were collected on a Nicolet iS5 FTIR spectrometer using a diamond ATR attachment under ambient conditions over a range of 4000 cm1to 1300 cm-1at a resolution of 4 cm-1. The measurements were captured at 22 °C with a room humidity of 42 ± 5% RH The Hertz contact diameter of 2 mm in the triborheometry experiments along with the visibility of the wear track by eye allowed for positioning ofthe contact zone (and the noncontact zone) entirely on the 1.8 mm diameter diamond ATR crystal.

[0093] As used herein, the term ‘‘approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0094] As used herein, the term “patient” or “subject” refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human.

[0095] The term “pharmaceutically acceptable” as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. For example, a composition can include a pharmaceutically acceptable ester or amide of a compound herein. In certain implementations, a composition includes a pharmaceutically acceptable salt of a compound herein. Non-limiting examples of pharmaceutically acceptable salts include carboxylate salts, amino acid addition salts and zwitterionic forms thereof, which are known to those skilled in the art as suitable for use with humans and animals. In cases where a compound is sufficiently basic or acidic to form a stable nontoxic acid or base salt, a composition includes a pharmaceutically acceptable salt of the compound. Non-limiting examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, non-limiting examples of which include tosylate, methanesulfonate.acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts are obtained using standard procedures known in the art. For example, pharmaceutically acceptable salts may be obtained by reacting a sufficiently basic compound with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium, magnesium) salts of carboxylic acids and other anionic groups in molecules within a pharmaceutical composition also are contemplated.

[0096] As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the symptom(s) of the disease, disorder, and / or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0097] As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0098] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do notdiffer from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Claims

What is claimed is:

1. A method of treating a subject for an eye condition, the method comprising steps ofadding a pharmaceutically acceptable lubricant to a subject’s eye, the lubricant comprising a mucin with a structure of poly(Gal-Thr)nand a liquid solvent, wherein Gal is galactose, Thr is threonine and n is an integer from 18-26 and wherein the lubricant is cysteine-free,2. The method as recited in claim 1, wherein n is an integer from 20-24.

3. The method as recited in claim 1, wherein the lubricant consists essentially of the mucin and the liquid solvent, wherein the liquid solvent is water.

4. The method as recited in claim 1, wherein the lubricant consists essentially of the mucin, the liquid solvent and a buffer, wherein the liquid solvent is water.

5. The method as recited in claim 4, wherein the buffer is selected from the group consisting of a phosphate buffer, a citrate buffer, a carbonate buffer, an acetate buffer, a tris(hydroxymethyl)aminomethane (tris) buffer, a N-2- hydroxyethylpiperazine-N-2-ethane sulfonic acid (IIEPES) buffer.

6. The method as recited in claim 1, wherein the lubricant forms a tribofilm.

7. A method of lubricating a subject’s body, the method comprising steps of:coating at least a portion of a subject’s skin with a pharmaceutically acceptable lubricant comprising a mucin with a structure of poly(Gal-Thr)nand a liquid solvent, wherein Gal is galactose, Thr is threonine and n is an integer from 18- 26 and wherein the lubricant is cysteine-free.

8. The method as recited in claim 7, wherein the lubricant consists essentially of the mucin and the liquid solvent, wherein the liquid solvent is water.

9. A method of lubricating a subject’s body, the method comprising steps of: coating at least a portion of a condom, a tampon or a pessary with a pharmaceutically acceptable lubricant comprising a mucin with a structure of poly(Gal-Thr)nand a liquid solvent, wherein Gal is galactose, Thr is threonine and n is an integer from 18-26 and wherein the lubricant is cysteine-free.

10. The method as recited in claim 9, wherein the lubricant consists essentially of the mucin and the liquid solvent, wherein the liquid solvent is water.

11. The method as recited in claim 9, wherein the lubricant forms a tribofilm.

12. A lubri cant compri sing:a mucin with a structure of poly (Gal -Thr)nand a liquid solvent, wherein Gal is galactose, Thr is threonine and n is an integer from 18-26, wherein the lubricant is cysteine-free.

13. The lubricant as recited in claim 12, wherein n is from 19-25.

14. The lubricant as recited in claim 12, wherein n is from 20-24.

15. The lubricant as recited in claim 12, wherein n is from 21-23.

16. The lubricant as recited in claim 12, wherein n is from 22,17. The lubricant as recited in claim 12, wherein the mucin is present in the water at a concentration of from 1-100 mg per ml.

18. The lubricant as recited in claim 12, wherein the mucin is present in the water at a concentration of from 5-80 mg per mL.

19. The lubricant as recited in claim 12, wherein the mucin is present in the water at a concentration of from 40-60 mg per mL,20. The lubricant as recited in claim 12, wherein the mucin is present in the water at a concentration of from 5-15 mg per mL.

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