When examining the specific solutions in which the lenses were incubated, it was found that PC deposition on balafilcon A and senofilcon A followed the order SLTS > ATS > PrTS > LTS after 3 and 14 days of incubation. However, etafilcon A deposited the most PC when incubated in SLTS > LTS > PrTS > ATS after 3 days and SLTS > ATS > LTS > PrTS after 14 days. Etafilcon A PC deposition was lower and more variable between the time points and solutions. The orders listed above represent the ranking for the overall deposition amounts. However, when the statistical differences between the individual solutions are examined using a Tukey's post hoc analysis (Table 9), it is found that not all solution comparisons are statistically different.
This study was designed to examine lipid binding to contact lenses in the presence or absence of other macromolecules. Specifically, the contact lens competitive binding profiles for cholesterol and phosphatidylcholine were examined by varying the components and complexity of the incubation solutions used through use of a radiochemical carbon-14 in vitro model. The simpler incubation solutions used in this study (SLTS, LTS, and PrTS) were used to represent the complexity and composition of previously used simple incubation solutions dominating in vitro experiments in the literature over the past 20 years,1,6,40 – 42 whereas the composition and complexity of the more complex ATS was more similar to that of human tear fluid. Many other researchers are now beginning to incorporate a more complex artificial tear fluid into their in vitro models for material, deposition, and solution testing.
The results from this study show quite clearly that experiments performed with simple, moderately complex, or complex incubation solutions will exhibit different deposition results. Cholesterol and phosphatidylcholine behaved differently in their deposition profiles between the four different incubation solutions and between silicone hydrogel and conventional hydrogel contact lens materials. For the two silicone hydrogel contact lens materials, cholesterol deposition was highest when the lenses were incubated in the PrTS followed by the ATS and then SLTS. The lowest deposition was found with the LTS incubation. This implies that the cholesterol is outcompeted for binding sites when it is in the presence of other lipids, but when protein is present in the solution and most likely depositing on the material, protein deposition increases cholesterol deposition, as is seen with the deposition profile in the PrTS and ATS. This is likely occurring because protein deposition and denaturation is making the lens surface more hydrophobic during binding, thus providing additional binding sites for cholesterol. Protein denaturation is thought to be more prevalent on silicone hydrogels than on conventional hydrogel materials, where a much higher percentage of the protein remains in its native state.5,43,44 This trend of increasing non-polar lipid deposition was also seen by Bontempo and Rapp12 – 14 in their solution composition studies with conventional hydrogel lenses.
When examining the cholesterol deposition on etafilcon A, a different trend was seen. In this case, when incubating in the SLTS solution for 14 days, the greatest amount of lipid was deposited followed closely by the PrTS. Incubation in the LTS deposited significantly less cholesterol, and the ATS incubation deposited only minute amounts of cholesterol. However, the only statistical differences in cholesterol deposition on etafilcon A were found between the SLTS solution and the other three. Therefore, these results imply that cholesterol is easily outcompeted for the hydrophobic binding sites by other non-polar lipids on this material and that protein deposition does more to encourage deposition than other lipids.
When examining the phosphatidylcholine deposition, a different trend of deposition was seen. The highest amount of PC was deposited on all materials using the SLTS solution followed by ATS, PrTS, and the LTS depositing the least amount of PC. This order of deposition was statistically significant for both silicone hydrogel materials, but this was not the case for etafilcon A. For the two silicone hydrogel lens materials, phosphatidylcholine was deposited in the highest masses when it did not have to compete with any other tear film constituents, proteins or lipids alike. PC is in fairly low concentration in the tear film and does not have a strong attraction toward these hydrophobic materials and thus is easily outcompeted by more prevalent, hydrophobic, and attractive lipids and proteins. This is evident when incubating in the LTS, as the other lipids available in the solution restrict the deposition of PC. When PC is surrounded with proteins, as is the case when incubating in the PrTS and ATS, PC deposition decreases when compared with SLTS deposition levels but increases significantly when compared with the LTS. This shows that protein deposition alters the lens surface chemistry so that it is less hydrophilic and thus creates more sites for PC to bind.
Etafilcon A results show a similar trend, with higher deposition of PC occurring when it is the only lipid present; however, no significant differences were seen in deposition masses for the other three solutions, p > 0.05. Therefore, these results show that phosphatidylcholine has little affinity for etafilcon A, especially with other tear film components present.
When the overall deposition relating to lens material is analyzed, it is seen that balafilcon A usually accumulates the most lipid for both cholesterol and PC; however, senofilcon A deposited the most cholesterol when incubating in the PrTS. Balafilcon A's propensity to deposit higher masses of lipid has also been found by other researchers and has been attributed to its polymer composition, polymeric structure, and its plasma oxidation process.44 – 46 As seen in Table 3, one of the monomeric constituents of balafilcon A is N-vinyl pyrrolidone (NVP),47 – 50 a monomer known to be lipophilic and a cause of increased lipid deposition for FDA group II conventional hydrogel lens materials12,13,51,52 which incorporate it. In addition to NVP, the incorporation of silicone, a very hydrophobic molecule, also increases the lens' lipophilic nature. To mask the hydrophobic matrix of balafilcon A, the lens is subjected to a plasma oxidation process which converts the surface into silicate.53 However, this does not create a continuous silicate surface but creates silicate “glassy” islands across the entire surface of the lens (Fig. 4A).53 Therefore, there are portions of the polymer surface that have not been converted into silicate and are therefore still relatively hydrophobic. Finally, the balafilcon A material also contains a vast number of pores (Fig. 4B).48,54 – 57 These pores are classified as macropores and are much larger in size than what is common in other silicone hydrogels as network pores.48,55,56 These macropores are thought to be continuous from the anterior to the posterior surface of the lens material and therefore are another area for lipid to deposit.48,55,56 For a lipid such as cholesterol, which is non-polar, balafilcon A still provides many available hydrophobic sites and thus results in higher levels of cholesterol deposition. When analyzing phosphatidylcholine deposition, balafilcon A is also an ideal deposition matrix. Phosphatidylcholine is an amphiphilic molecule that contains both a hydrophilic “water loving” head group (in this case choline) and a hydrophobic “water hating” tail group which contains two fatty acid chains, one saturated and one unsaturated. This dual nature of PC allows it to then deposit not only on the hydrophobic portions of the surface but also on the more hydrophilic silicate islands.
Senofilcon A is a second-generation silicone hydrogel lens and unlike balafilcon A does not have a silicate surface coating. However, it does have an internal wetting agent incorporated into its polymeric structure which is designed to aid with surface wettability. This wetting agent is a high-molecular-weight molecule of polyvinylpyrrolidone.58 Polyvinylpyrrolidone is a polymer of the monomer NVP which is known to be lipophilic in nature (as discussed with balafilcon A), and thus senofilcon A may also deposit increased amounts of cholesterol and phosphatidylcholine when compared with a conventional hydrogel such as etafilcon A.
Etafilcon A is a FDA group IV conventional hydrogel material composed of poly-2-hydroxyethyl methacrylate (HEMA) and methacrylic acid (MA). Conventional hydrogel materials based on HEMA have a long history of proving to be relatively low lipid depositors, especially when compared with silicone hydrogels. However, ionic materials such etafilcon A tend to deposit large amounts of proteins, specifically lysozyme. This is because the MA in etafilcon A gives the lens material a net negative charge and therefore attracts positively charged lysozyme through electrostatic interactions.3,59,60 These electrostatic interactions cause heavy deposits of lysozyme onto these materials, thus allowing little lipid to deposit in comparison.
Some of the other newly published in vitro lipid articles have quantified higher masses of phospholipids and cholesterol depositing on these materials. For example, in vitro work from Pucker et al.6 and Carney et al.1 both published higher lipid deposition masses than the work presented in this article. However, the experimental procedures in these other in vitro studies had at least one of these variations: altered concentrations of lipids in the ATS, different incubation durations and volumes, the use of different incubation solution compositions, and replenishment of the ATS throughout incubation.1,6,9 Therefore, these alterations in method may help to explain the differences in lipid deposition found.
When the cholesterol and phosphatidylcholine deposition results found in this in vitro experiment are compared with recent ex vivo data, it is seen that the results tend to fall within the same range of deposition. However, direct comparisons should not be made as the wear time of the ex vivo lenses are much longer than the incubation time of this experiment, ex vivo lenses are cleaned nightly, and it would be incorrect to extrapolate the data.46,61 In addition, many of the published ex vivo and in vitro studies on lipid deposition have quantified different lipid species and examined different contact lens materials than those in this study.
The use of a more complex ATS for in vitro contact lens studies has been a major topic of discussion for many years, specifically at contact lens conferences over the past year or so. Many researchers believe a more complex ATS better represents the composition of the natural tear film and will better predict and model lipid deposition during an in vitro experiment. However, the cost of creating a more complex tear fluid can be high and therefore the question becomes “Is the increase in ATS complexity worth the cost and will it impact the in vitro deposition of the tear film components?”
The experiment designed and described in this article examined the effect that the ATS composition had on the resulting deposition of two different model lipids. The four different tear solution compositions that were examined in this study were meant to represent the main solution compositions and complexities often utilized in previous literature (simple, moderate, and complex) and have allowed us to examine the impact that various individual components have on deposition.
The results from this experiment show that the composition of the incubation solution, the lipids under examination, length of incubation, and the lenses used will all have an influence on the overall deposition profile. The interactions between the components of the tear film and the contact lens surface will dictate the deposition that occurs. If in vitro models are really meant to mimic in vivo conditions then it is imperative that more complex models are utilized and that every attempt is made to make the in vitro conditions as similar as possible to human contact lens wear. It is only by completing these types of experiments that we can improve an in vitro model's usefulness and systematically explore the relationships that are occurring during human contact lens wear and then test and incorporate them into the in vitro models.
This in vitro study demonstrates that cholesterol and phosphatidylcholine deposition is cumulative over time and that silicone hydrogel materials deposit more lipid than a conventional hydrogel FDA group IV material. It also clearly demonstrates that deposition of cholesterol and phosphatidylcholine is influenced by the composition of the incubation medium. Specifically, cholesterol exhibited significant increases in deposition with protein-rich incubation solutions; however, significant competition with other lipids decreased deposition in the lipid-rich LTS and ATS solutions. Phosphatidylcholine deposited extremely well when it was the only component in the incubation solution, only moderately well with a protein-rich ATS and ATS solutions, and very poorly when it competes with other lipids in the LTS.
These results prove that in vitro models must use more physiologically relevant incubation solutions that mimic the natural tear film if in vitro data is to be extrapolated to the in vivo situation.
The authors thank Dr. Zoya Leonenko for the use of AFM and Liz Drolle for assistance with imaging. Lyndon Jones has received funding over the past 3 years from the following companies which are either directly involved in products used in this manuscript or involved in the manufacture of competing products: Alcon, AMO, B&L, CIBA Vision, CooperVision, and Johnson & Johnson.
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