Tear Film Constituents and Medicines for Eyes Investigated as Langmuir Films

The tear film has an important role on the quality of the vision process and is of importance in the fields of ophthalmology and optometry. The lipid layer, the outermost part of the tear film, can be investigated with the Langmuir technique, as well as the interaction of components of the aqueous part of the tear film with those of the lipid layer. Dysfunctions in the stability and composition of the tear film are the causes of eye diseases, as the dry eye syndrome. For the treatment of this disease, the artificial tears are used, and one type of them are the lipid tears, which can be investigated with the Langmuir technique. This technique can also be used to investigate the influence on the lipid layer of other artificial tear components or medicines instilled in the eye. This review presents investigations where the Langmuir technique has been used in relation with the tear film. In particular, the surface properties of natural tears and of four commercial lipid-containing artificial tears have been reported and discussed in connection with their composition.


Introduction
The tear film [1] has a layered structure with three mean parts or layers. The inner layer adjacent to the corneal epithelium, the middle layer or aqueous layer and the outer layer or lipid layer [2,3]. Even this lipid layer is a multilayer of several nanometers formed by several types of lipids, it can be investigated using the Langmuir technique, which can also be used to investigate the interactions of the lipid components with the components of the aqueous layer. These questions are considered in section 2 of this review.
The tear film presents dysfunctions or diseases and some of them, as the dry eye syndrome or ocular irritations, can be treated with artificial tears [4,5]. One type of them are the lipid artificial tears which tray to repair or improve the lipid layer present in the outermost part of the tear film. These artificial tears are able to be studied using the Langmuir technique. These questions are considered in section 3 of this review.
Finally, some conclusions will be presented and a wide list of references for the reader to get more inside the subject.

Structure and Composition
One model of the tear film considers three mean layers or phases [6], the mucin layer, in the inner part, the aqueous layer, and the lipid layer, in the outer part. Other models have been proposed [7,8] where the separation between the aqueous and the mucin layer is not well defined. One of the mean function of the lipid layer is to preserve the aqueous layer from a fast evaporation.
The lipid layer is formed by a great variety of lipids [2,3,[9][10][11]. The mucin layer is formed meanly by mucoglycoproteins (mucins) [3,12]. The aqueous layer contains around 98% of water, and the rest are proteins (as lysozyme and IgG forming part of the immunologic system, lactoferrin and albumin) [3,13], dispersed mucins, electrolytes (as sodium, potassium, calcium and magnesium salts), glucose, vitamins (as vitamins A and C), and cells.
The aqueous layer is that with the most volume and thickness, around 90% of the total thickness of the tear film, established around 6-9 μm but with significant variations. The thickness of the mucin layer is around 0.8-1 μm and the thickness of the lipid layer is around 0.1 μm [14].
Different techniques are used to evaluate clinically the tear film [16][17][18][19][20][21], being the most usual the Schirmer test, the height of the lacrimal meniscus, the break-up time (BUT) and the no-invasive break-up time (NIBUT), fluorescein staining or rose Bengal stain. More recently, the osmolarity test has been also used, especially related with the dry eye syndrome [18,[22][23][24].

The Lipid Layer
The lipid layer is a complex film and in its composition a lot of lipids have been found [25,26], some of them polar (fatty acids, fatty amides, phospholipids) [27] and most of them non-polar (triglycerides, diesters, diacylglicerols, cholesterol and wax esters, hydrocarbons) [28][29][30]. Several compositions have been reported by several authors. Nichols et al. [31] studied the composition on fatty acids and fatty amides, which only represents around 2% of the total lipid content of the lipid layer. These authors found myristic, palmitic, stearic, and oleic acids, as well as myristamide, palmitamide, stearamide, oleamide, and erucamide, being oleamide [32] one of the most abundant which has been related with the dry eye syndrome. Butovich et al. [30] found that the content of oleamide is less than 0.1%, and also found that phospholipids and ceramides are in a low content, being the content of PC less than 0.01%. Borchman et al. [33] found that the composition of lipids in the tear film is different of that in the meibum. For instance, cholesterol and phospholipids represent around 15% in the tear film but only 0-7% in the meibum; meanwhile, cholesterol and wax esters represent around 45% in both. For a detailed lipid composition see references reported above, even composition usually changes from an author to another.
Grazing incidence X-ray diffraction and X-ray reflectivity were applied to meibum films at the air−water interface to identify its molecular organization [34]. The lipid layer interacts with lipocalins of the aqueous layer rendering stability to the tear film by reducing the surface tension [35]. Using several techniques, Rosenfeld et al. [36] proposed a refined structure for the tear-film lipid layer.
The lipid layer can be optically observed using interferometry, and some commercial instruments have been developed based in this principle [19,37,38]. The usual slit-lamp instrument can also be used to observe the lipid layer [39,40]. The interferential patterns have been studied and related to thickness and type of lipid films [21,[41][42][43][44][45]. For images of these interferential patterns, see technical information from Tearscope-Keeler Ltd., and also Guillon and Godfrey [37]. A stroboscopic video color microscope has also been developed using interferometric principles and used for film imaging studies [46,47] and for measuring the tear film lipid layer thickness [48]. A contemporary perspective on the structure-function relationship of the lipid layer of the tear film has been recently reviewed [49].

The Langmuir Film Technique Applied to Natural Tears
It is well known that when amphiphilic molecules, as fatty acids and phospholipids, are placed on water or aqueous solution, they spread forming a film, usually monomolecular, known as Langmuir film or monolayer. In the Langmuir technique, a trough is used to contain this film, provided of mobile barriers that permits to compress or decompress the film obtaining the surface pressure-area isotherms. The surface pressure (Eq. 1) is measured with an electrobalance using a Wilhelmy plate [50,51]. From the isotherms, the compressibility modulus or its inverse, the elastic modulus (Eq. 2), can be calculated obtaining information on the physical states and elasticity of the film [52,53].
Along the years, the Langmuir technique has been applied to a variety of compounds and systems, with several applications [50,51,54,55]. In connection with Langmuir films, the Brewster angle microscopy (BAM) technique has been developed [56]. Recent applications of this technique have been reviewed [57]. Even the lipid layer of the tear is not a monolayer, its lipid nature permits to be studied with the Langmuir technique. Thus, the film formation on the water/ air interface has been applied to study the behavior of Meibomian lipids secreted by the lipid glands, placed in the eyelids [58][59][60][61][62][63][64][65] or its interaction with lachrymal proteins [66][67][68][69][70] (see section 2.4). Tragoulias et al. [71] also applied this technique to study the surface activity of human tears and several of its components.
Kulovesi et al. [62] have studied the impact of lipid composition on the structural and dynamical properties of the tear lipid film using Langmuir films, X-ray diffraction, and coarse-grained molecular dynamics simulations. Georgiev et al. [58] applied surface relaxations as a tool to distinguish the dynamic interfacial properties of films formed by normal and diseased meibomian lipids. The interaction of Meibomian lipids with polar lipids in Langmuir monolayers has been treated by Georgiev et al. [72] and by Rantamäki and Holopainen [73]. It has been pointed that the presence of anionic phospholipids (PS, PI, cardiolipin) in small amounts is necessary [11]. Bland et al. [74] investigated the role of specific tear film lipids connected to the dry eye syndrome. The molecular organization of the tear lipid layer has been simulated by Kulovesi et al. [75] using several techniques, including the Langmuir monolayer technique. Petrov et al. [64] also studied the organization of the tear lipid layer, with bovine meibomian tear film and simulated tear film, using surface pressure-area isotherms and fluorescence microscopy joined to grazing incidence x-ray diffraction (GIXD). [76,77], realized a combined clinical and Langmuir film study of natural tears. This study shows the applicability of the Langmuir technique to collected natural tears, which include the lipid components. Parameters obtained from clinical tests, as Schirmer test and tear film break-up-time (BUT), can be correlated with isotherm characteristics. Additionally, this study highlighted on the importance of not to use eye cosmetics prior to collect natural tears. Images obtained using a Brewster angle microscope showed the presence of excess of lipid components when eye cosmetics are used, Fig. 1, in comparison with BAM images obtained with natural tears without cosmetic contamination, Fig. 2.
The effect of cosmetics has been pointed out by Guillon and Godfrey [37], which can break up the lipid layer and provoke dry eye, and stick to the contact lens surface producing a non-wetting effect. Figure 3 presents surface pressure-area isotherms of collected natural tears spread on water subphase, showing in one case a good isotherm and in another case a weaker isotherm. Values of inverse of the compressibility modulus (inset in Fig. 3) indicate fluidity of the spread tear films, with slight differences in between both tear films. Figure 4 shows BAM images of collected natural tears spread on water surface, and in correlation with different tear patterns. As the thickness of the lipid tear film increases, the reflected light increases and the BAM image becomes brighter.

Interactions of Aqueous Tear Components with the Lipid Layer
It is important, in order to understand the structure and alterations of the lipid layer, to study the interactions of aqueous components of the tear with lipids. The composition of the aqueous layer of the natural tears has been indicated previously in section 2.1. It has been observed that lachrymal proteins have surface activity and that contribute to the decrease of the surface tension of the tear film and to its stability. In the formation of the lipid layer, the presence of ions must be also considered [11] since its presence can stabilize the formation of the monolayer of polar lipids. Bartkowiak et al. [78] studied the interaction of mucin with phospholipids, using the Langmuir technique. The authors found that mucin molecules interact with PC headgroups forming a more stable film. Millar et al. [79] investigated the interaction of ocular mucin with Meibomian lipids. Mudgil and Millar [68] studied the interaction of lipocalin with a bovine Meibomian lipid film, while Millar et al. [67] studied the adsorption of human tear lipocalin to human Meibomian lipid films. Mudgil et al. [69] studied the interaction of lysozyme with phospholipid and Meibomian lipid films, and Nishimura et al. [80] studied the effect of lysozyme on DPPC and cholesteryl myristate films. Miano et al. [81] studied the interaction of lactoferrin with DPPC as a model of the preocular tear film outer interface. The role of lactoferrin in the tear film has been reviewed by Flanagan and Willcox [82]. Miano et al. [66] studied the insertion of tear proteins (lactoferrin, human serum albumin, lysozyme, sIgA and lipocalin) in a Meibomian lipid film from bovine eyelids. Millar and Schuett [26] reviewed the effect of proteins on the tear film lipid layer, and Svitova and Lin [70] reviewed the interactions of human tear-lipid films with model-tear proteins in vitro. Torrent-Burgués and Raju [83] studied the interaction of lysozyme with several lipids, all of them present in the lipid layer of the tear film. They observed strong influence of lysozyme on the surface pressure-area isotherms and on the elastic modulus of the Langmuir films. The influence was greater on fatty acids, as oleic acid or stearic acid, than on phospholipids, as DPPC or POPC, or cholesterol. The interaction of lipocalins of the aqueous layer with the components of the lipid layer has been studied by Gasymov et al. [35]. Interpretation of these effects are based on electrostatic and hydrophobic interactions. For that, the presence and distribution of charges in the biomolecules are essential, as well as the values of isoelectric points (IEP) and the pH of the medium. Guckeisen et al. [84] found that the IEP of proteins at the air/liquid interface is close to that in the bulk solution.
Other studies that investigate the interactions of proteins with lipid films are also of interest in respect to understand the interaction of human tear proteins with the tear lipid layer [85][86][87][88][89][90][91][92][93][94][95][96][97]. Toimil et al. [98] studied with the Langmuir technique the interaction of human serum albumin with phospholipid monolayers. Calvez et al. [99,100] analyzed the contribution of phospholipid monolayers to the binding of proteins, and proposed a new approach to determine factors that governs protein binding onto lipid monolayers. Stefaniu et al. [101] reviewed the applicability of Langmuir monolayers as models to study interactions of lipids with proteins and drugs, between others.

The Dry Eye Syndrome
The dry eye syndrome [102] is a common disease which affects a lot of people and is getting more frequent due to external factors as pollution, screens of devices, air conditioning, or the use of contact lenses. Actually, the dry eye syndrome is classified as a multifactorial disease of the tear film and it can provoke red eye, itchiness, sensation of tired eye, and discomfort or alter the visual quality or the tear stability [5,103], and even can damage the ocular surface.
The dry eye syndrome can be classified as secretion lack or as evaporative. The first one (the former) is due to gland dysfunction [104]; meanwhile, the second one (the later) is due to an excess of evaporation of the aqueous component due to poor quality in the lipid layer [105,106], but also to a poor flicker, poor wetting of the ocular surface or distortion of the tear film caused by contact lenses [107,108] or environment factors [109,110]. Contributions of evaporation and other mechanisms to tear film thinning and break-up were revised by King-Smith et al. [111]. The evaluation of the dry eye is performed with the methods or techniques commented previously in section 2.1 and also reported in other works [112,113].
One of the usual treatments for the dry eye is the use of artificial tears, which are commented in the next section. Other solution is the use of polymer inserts [114] or the use of medicaments. Nichols et al. [115] reviewed the use of four approved dry eye medications in the USA and Europe, the vehicle used to deliver the medication and new treatment options for dry eye disease with novel vehicles.
Benelli [119] studied Systane® lubricant eye drops, Systane Ultra®, and Systane Balance®, which provide symptomatic relief to patients with dry eye. Scaffidi and Korb [135] used two tears, Refresh Dry Eye (Allergan, Inc., Irvine, CA, USA) and Soothe, and concluded that the application of a lipid emulsion eye-drop will increase the lipid layer thickness (LLT). Korb et al. [133] used Soothe and Systane and also concluded that the application of lubricant eye drops increase the LLT. Peters and Millar [134] used three artificial tear fluids: buffered saline alone, one with proteins and mucins, and one containing proteins, mucins and lipids. They concluded that the tear break-up time (BUT) was improved by the presence of phospholipids, in especial by phosphatidylinositol (PI). Steven et al. [136] found that BUT and other clinical signs of Meibomian gland disease improved with the application of semifluorinated alkane eye drops. Rohit et al. [138] studied the effect of lipid supplements on tear lipid biochemistry and their influence on lens wearers, and results suggest a potential role for lysophospholipids and OAHFAs in modulating symptoms during contact lens wear.
Several lipid artificial tears are present in the market and commercialized by several companies. In the composition of some of these lipid tears occurs, as a principal item, a phospholipid component that is soy lecithin, which is composed mainly by phospholipids as phosphocholine or also named phosphatidylcholine (PC). Soy lecithin is obtained from soy grains by mechanical extraction or chemically using hexane. It has a great content of PC, specially refined lecithin, but also other lipids are present as phosphatidylserine (PS) and phosphatidylinositol (PI). The fatty acid chains present in these PC are mainly palmitic or stearic, in C1, and oleic or linoleic, in C2. For example, POPC is palmitoyloleoylphosphatidylcholine (M=760.1), or DLPC that is dilinoleoylphosphatidylcholine (M=782.1) (see Fig. 5), but an average soy polar extract PC is that reported with M=775.0 and phospholipid composition wt/wt%: PC 45.7, PE 22.1, PI 18.4, PA 6.9, unknown 6.9 (Avanti Polar Lipids, https:// avant ilipi ds. com/). Refined lecithin is used mostly for pharmaceutical applications and research.
In commercial lipid artificial tears, other components are also present and have been detailed for some of them in the next section. Glycosaminoglycans have also been used in artificial tears, as hyaluronic acid or chondroitin sulfate. Ceridório et al. [139] have studied how chondroitin sulfate interacts with headgroups in phospholipid monolayers, and Zander et al. [140] studied the influence of hyaluronan on DPPC.

Lipid Artificial Tears and the Langmuir Film Technique
The behavior of several lipid artificial tears has been studied in a few works using the Langmuir technique due to the presence of phospholipid components. Dinslage et al. [132] prepared lipid tears containing phospholipids and realized a clinical and Langmuir film study. The Langmuir technique has been applied to study the behavior of commercial lipid artificial tears [141] presented in dispensers for spray applications, Innoxa, Opticalm, Optrex and Zero, which composition is shown in Table 1. From the surface pressure-area isotherms, the inverse of the compressibility modulus or elastic modulus, C s -1 , is obtained from eq. 2, which give information on the compressibility and fluidity of the film. Figure 6 shows similar behavior of the lipid tears Optrex and Zero but a different behavior for the Opticalm one. Figure 1 in reference Torrent-Burgués [141] shows that the tear Innoxa presents a behavior more similar to Optrex. Analyzing the composition of these tears, which is similar even a bit different for Opticalm, the different behavior of Opticalm seems more related to the raw material of soy lecithin as it is manufactured by different companies.
Values of elastic modulus indicate fluidity of the films, corresponding to the liquid expanded state (LE) [53,141]. These values, which are lower compared with others reported in literature for PCs ([141] and references cited therein), indicate the presence of several unsaturations in the PCs of soy lecithin, especially for Opticalm. Figure 7 shows the behavior of the tear Zero at two temperatures (23 and 32°C) and in two subphases (water and NaCl 0.9%). It can be seen a notable influence of the saline   Fig. 6A and isotherm a in Fig. 7A, which corresponds to different samples. Another result from the isotherms, where it has been assumed for molecular discussion that the main phospholipid component of soy lecithin is POPC, is that the low value of area per molecule attained at the collapse indicates that the liposomes remains partially forming a bilayer on the subphase, it means they do not spread totally as a monolayer. This fact influence on the isotherm position, which will depend on the amount of lipid spread. For more information, see reference Torrent-Burgués [141].
The presence of the lipid artificial tear film can be observed by BAM (Fig. 8), but due to its fluidity and probably to a refractive index similar to that of the subphase, it cannot be distinguished clearly and only big aggregates appears bright.
The behavior of different Optrex tears, see composition in Table 2, has also been investigated by the author's group. These investigations show no significant differences in the Langmuir film behavior of these three Optrex tears  . 7 A Surface pressure-area isotherms for the zero lipid-containing artificial tear at two temperatures and in two subphases: (a, blue) water 23°C, (b, violet) water 32°C, (c, green) NaCl 0.9% 23°C, and (d, red) NaCl 0.9% 32°C. B Inverse of the compressibility modulus for isotherms of Fig. 7(A) (see Fig. 9), which seems reasonable due to the similar composition they present. There is a subphase influence when comparing water, 0.9% saline solution and phosphate buffer solution (PBS). The influence of temperature was also studied: a temperature increase expands the Langmuir film and fluidizes it. More interesting are the effect due to the presence of proteins in the subphase at pH=7.4, with a marked difference in between bovine serum albumin (BSA) and lysozyme (Fig. 10). BSA produces a shift of the Optrex tear isotherm to lower areas and close to that of BSA alone; meanwhile, lysozyme produces a shift to higher areas and with a marked increase in the surface pressure. An explanation to this different behavior is related to the different isoelectric point (IEP) of both proteins, IEP≈4.8 for BSA and IEP≈11 for lysozyme, that leads to a negative charge for BSA and a positive charge for lysozyme at pH=7.4.
Weisenberger et al. [48] performed a clinical study using Systane Complete®, which contains lipids, and Systane Ultra® eye drops. This study found that Systane Complete increases the lipid layer thickness and benefit subjects with dry eye symptoms. In the author's laboratory a physicochemical study allowed to obtain some properties of Systane Complete: surface tension of 43.9 mN/m and pH of 7.23 at 23°C. The presence of the lipid components was observed with BAM ( Fig. 11). Interactions of some eyedrop formulations with human meibum lipids have been reported by Eftimov et al. [142] using the Langmuir technique and BAM.

The Influence of Instilled Medicines on the Tear Film
The tear film can be altered by the instillation of medicines and artificial tears [115,137,[143][144][145], especially for the action of some components, but also for the action of medicines dispensed orally or by other methods that can affect the gland secretion of the natural tear components. When a drop of an artificial tear or a pharmacological product, with a volume around 20-40 L, is instilled in the eye, with a tear volume of around 10 L, obviously, the tear film is perturbed, not only by the enormous quantity of instilled liquid but also because the structure of the tear is momentarily altered, which can provoke alterations in the vision quality. But at each blind new tear is supplied by the eye glands and the tear structure, composition and functionality is restored or improved with the afforded active ingredients. The application of an lipid artificial tear as a spray, which is done with the closed eyes, do not perturb the tear film but the quantity of artificial tear that reach the tear film is reduced. Usually, the sprayed volume in each application is around 100 L, and as the usual concentration of PC is 10 mg/mL, the amount of PC is around 1 mg, which is enormous if all of it will reach the tear film. But the eyelids acts as a barrier and thus the amount that reach the tear film and incorporates in the lipid layer is small. Cell membranes can be affected by a drug, and this effect can be studied by the Langmuir technique and by insertion experiments of drugs into lipid films. The effect of drugs is a wide field and is out of the target of this work, but some references and comments will be done here. A list of common drugs used in ophthalmology can be found [146][147][148]. Ophthalmic drugs are used for three main purposes: diagnostic, therapeutic, or prophylactic. Previous sections have treated the case of artificial tears. Sometimes, inserts are used to deliver drugs to the eye [114,149], but viscous drops, gels, and ointments are also available. Preservatives in eye-drops, as benzalconium chloride, polidronium, cetrimide, biguanides, chlorhexidine, or polyhexanide [150], or surfactant agents in contact lens solutions can also affect the cell membranes of the corneal or conjunctival epithelia. Mishra et al. [137] reviewed the use of liposome preparations for ophthalmic drug delivery. These formulations are mainly composed of phosphatidylcholine (PC) and other constituents such as cholesterol and lipid-conjugated hydrophilic polymers.
Sanfilippo et al. [151] presented a study in vitro on bacterial infections of the ocular surface commonly treated empirically with broad spectrum antibiotics. For glaucoma treatment or glaucoma prevention, some prescribed medicaments are instilled on the eye. Ocular surgery, as cataract surgery, which usually use eye drops before and after surgery, can Fig. 9 Surface pressure-area isotherms for three Optrex tears (see composition in Table 2) in NaCl 0.9% subphase at 23°C: Optrex red (a, red), Optrex yellow (b, yellow), Optrex green (c, green)  also provoke changes in the tear film [152,153]. Fangueiro et al. [154] studied epigallocatechin gallate lipid nanoparticles for ocular instillation for the future treatment of several diseases, such as dry eye, age-related macular degeneration, glaucoma, diabetic retinopathy and macular edema. The use of antimicrobial peptides in ocular surface has been reviewed by Mohammed et al. [155]. Studies published about the interaction of some drugs and antimicrobials with mimetic biomembranes using the Langmuir technique can be useful in ophthalmology. Nano-formulations with antimicrobial activity has been developed recently ( [156][157][158], ; [159]; and references cited therein) and its effect on biomimetic membranes has been studied using the Langmuir technique. Caseli et al. [160] investigated, using the Langmuir technique, the influence of the components of a dye solution on model membranes.
The presence of surfactants and preservatives in the eye drops can alter the surface tension of the tear film and/or modify the epithelial cells and its environment [144]. Surfactants can alter meanly the lipid layer, preservatives can alter meanly the epithelium and the mucin layer. Souza et al. [161] studied the biocide action of poly(hexamethylene biguanide), a common component in contact lens solution, using Langmuir monolayers of dipalmitoylphosphatidylglycerol. Sandez-Macho et al. [162] studied the interaction of poloxamine block copolymers, usual components in contact lens solutions, with lipid membranes. Di Pascuale et al. [143] observed the changes in the tear lipid layer using interferometry after instillation of emulsion eye drops. Other works have also used the Langmuir technique to study interactions of drugs with lipid and phospholipid films, which could be useful for the reader [163][164][165][166][167][168][169][170][171][172][173][174][175][176]. Zhang et al. [177] studied, using the Langmuir technique, the interaction between Lycium barbarum polysaccharide (LBP) and model lipids (PC, PE, PS, and PI) of the human retinal pigment epithelial cells (RPEs), located at the outermost layered structure of the retina. Oxidative stress damages retinal cells. The LBP are a type of exogenous biological macromolecule that can be used to treat the disease and play an important role in the thickness of the retina and the tightness of retinal cell connections. For that, LBP has been used to treat oxidative damage of RPE cells.

Conclusions
The Langmuir technique can be used to investigate the following: (a) the lipid layer of the tear film and its interaction with components of the aqueous part of the tear film; (b) lipid tears, containing lipid components, used for the treatment of dry eye syndrome; and (c) the influence of other artificial tear components or medicines instilled in the eye on the lipid layer.
Parameters obtained from clinical tests, as Schirmer test and tear film break-up-time (BUT), can be correlated with isotherm characteristics. BAM images show on the importance of not to use eye cosmetics prior to collect natural tears.
Lachrymal proteins have surface activity and contribute to the decrease of the surface tension of the tear film and to its stability. The effects of proteins on lipid layers are very important, and can be interpreted based on electrostatic and hydrophobic interactions. The presence of BSA and lysozyme proteins in the subphase, at pH=7.4, shows a marked difference in between them. BSA produces a shift of the Optrex artificial tear isotherm to lower areas and close to that of BSA alone; meanwhile, lysozyme produces a shift to higher areas and with a marked increase in the surface pressure. An explanation to this different behavior is related to the different IEP of both proteins, that leads to a negative charge for BSA and a positive charge for lysozyme at pH=7.4.
The surface behavior of several studied lipid-containing artificial tears shows similarities in between some of them but important differences in respect to others. As the reported composition of these tears is similar, the different behavior seems more related to the raw material of soy lecithin, as different companies manufacture it.
Acknowledgements The author thanks PhDs E. Guaus and J. Hoyo, and MD C. Serés, for collaborating along several years.
Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.

Declarations
Ethical Approval Not applicable.

Competing Interests
The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.