Therapeutic Hydrogel Lenses and the Antibacterial and Antibiotic Drugs Release

The aim of this research was to evaluate the effects of different lens types on the availability and efficacy of anti-inflammatory and antibiotic drugs. Three lens types were examined: (1) nonionic hydrogel lenses; (2) ionic hydrogel lenses; and (3) silicone hydrogel lenses. The lenses were incubated with (a) dexamethasone; (b) betamethasone; (c) bromophenacyl bromide; and (d) chloramphenicol. Drug availability was quantified by gradient HPLC, and chloramphenicol antibacterial activity was quantified by testing the inhibition of Salmonella typhimurium growth on agar. The lens allowing the most abundant passage of betamethasone was the ionic hydrogel lens, followed by the silicone hydrogel lens and nonionic hydrogel lens. The lens allowing the most abundant passage of dexamethasone was the ionic hydrogel lens, but only at 0.5 h and 1 h. Regarding chloramphenicol, the ionic hydrogel lens and silicone hydrogel lens allowed more abundant passage than the nonionic hydrogel lens. These results highlight the relevance of adapting lenses to anti-inflammatory therapy, thus allowing a personalized medical approach.


Introduction
Therapeutic lenses provide analgesic and protective effects by shielding epithelial defects and acting as a physical bandage for the corneal surface. The coverage of the corneal surface reduces the symptoms caused by the exposure of the corneal nerve endings. In the last decade, membrane hydrogel used has further expanded due to both the improvement of lens materials and the expansion of therapeutic indications. Furthermore, hydrophilicity could increase the bioavailability of pharmacological preparations in solution.
These lenses, which can incorporate and absorb water-soluble molecules, are soaked with eye drops to increase the contact time between the pharmacological preparation and the ocular surface [1]. Multiple investigations of various commercially available lenses and different drugs have allowed for conclusions to be made into the factors that influence drug uptake and release in vitro such as nature of the material backbone, the overall lens water content and surface charge, [2].
Daily-wear lenses are manufactured with silicone hydrogel. Other hydrogel materials offer lens manufacturers a very positive and consistent patient response compared to silicone hydrogel lens materials [3]. A series of poly (2-hydroxyethyl methacrylate) (pHEMA) hydrogels containing crosslinked β-cyclodextrin-hyaluronan (β-CD-crHA) with tear-protein adsorption resistance and sustained drug delivery have been developed as contact lens materials for eye diseases. Copolymers of HEMA and methacrylic acid are the most used materials for disposable soft lenses and typically have a water content in the 55-60% range (in the vial or blister). During wear, these lenses lose approximately 10% of their water content and accumulate a substantial amount of protein because positively charged tear proteins and lysozyme precipitate onto the negatively charged lens [4]. Poly-HEMA is a hydrophilic hydrogel with high water content and has been widely used in biomedical applications, including contact lenses and ophthalmic drug delivery systems, due to its safety and excellent biocompatibility [5]. Ionic lenses can absorb a thicker layer of protein than nonionic lenses, with the former adsorbing primarily lysozyme [6]. Hydrogel lenses with higher water content adsorb larger amounts of protein [7]. Hydrogel materials were the most common lens materials for decades, until the recent successful development of truly complex soft lenses (i.e., the silicone hydrogel polymer complex), which account for up to 65% of today's modern contact lenses. These hydrogels show adequate light transparency, oxygen permeability, and swelling for use as contact lenses [8]. Oxygen transmission through the silicone hydrogel complex is six times higher than through lenses made with "simple hydrogel materials." However, instillation of the silicone molecular component carries with it an inherent hydrophobicity, meaning that a silicone hydrogel polymer contact lens does not intimately interact with the precorneal tear film. Indeed, this complex polymer repels the tear film, much as the surface of a newly waxed car repels water. This hydrophobic characteristic is directly responsible for the high (up to 50%) frequency of dry eye symptoms in silicone hydrogel polymer lens wearers [9]. Silicone hydrogel contact lenses were found to be effective and well-tolerated bandage lenses [10]. However, the penetration of a drug can be greatly improved by prolonging its contact time with the eye. Hydrophilic contact lenses can be used as an adjuvant to drug delivery. Drugs penetrate hydrophilic contact lenses at a rate that depends on the pore size between the cross-linkages of the three-dimensional structure of the lens, the concentration of the drug in the solution, and the molecular size of the drug. [11]. In rabbits' eyes, an increased intraocular gentamicin level was found in eyes fitted with hydrophilic contact lenses [12]. Frequently, alterations in corneal sensitivity result from refractive surgery, corneal surgery and cataract surgery. The lenses were tested without drugs and with drugs and are nontoxic to the anterior surface of the eye [13,14].
The latest studies show that contact lenses can achieve an extended-release period of a few weeks without any significant impact on critical lens properties. Furthermore, in vivo animal studies have confirmed the safety, efficacy and increased drug bioavailability of therapeutic lenses compared to eye drops [15]. Therapeutic lenses can be useful in maintaining the trophism of the eye surface and can be designed to convey not only drugs but also natural therapeutic substances [16]. Furthermore, the advantages of these lenses include analgesic action in bullous keratopathy, which is notoriously painful and benefits from the use of anti-inflammatory drugs; the maintenance of corneal hydration, in the case of lagophthalmos; and, finally, a physical bandaging function in the event of wounds or refractive surgery, loss of the filter draft after trabeculectomy, or corneal sutures that need protection from eyelashes in trichiasis. However, further clinical studies should be conducted on this basis to determine the possible effects of their tolerability on the anterior surface of the eye. Additionally, the release duration varies depending on the drug and the properties of the lens; in general, however, the release durations are usually only a few minutes to a few hours for all ophthalmic drugs in the size range of 300 to 500 Dalton's, and most of the drug is released in an initial burst [17].
Corticosteroids and antibiotics administered systemically and/or on the anterior surface of the eye, have long been used to treat and prevent ocular diseases, especially after ophthalmic surgery. Dexamethasone, an anti-inflammatory and immunosuppressive drug, is another example of a hydrophobic glucocorticoid and has a very low loading efficiency by soaking method [18]. However, the interaction between different lens types and ocular drugs has not been well examined under controlled experimental conditions with reference to hydrogel lenses. Release of dexamethasone sodium phosphate, a corticosteroid used in the management of ocular inflammation, has been observed from commercial contact lenses in vitro. Similar to the drug release behavior with antibiotic agents, release is generally uncontrolled and occurs in a rapid burst release in vitro for periods of less than one hour [19]. A greater amount of dexamethasone sodium phosphate is released from hydrogel versus silicone hydrogel materials Since one of the main benefits of using contact lenses is the potential for significant improvement in corneal bioavailability, it is important to measure or model drug transport into the eyes by drug-eluting contact lenses.
The goal of the present study is to evaluate the interaction of pHEMA hydrogel lenses with nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and antibiotics commonly used in ocular therapy. The release of the drugs by the lenses is evaluated at different times after drug application.

Pharmacological Eye Drops
The commercially available anti-inflammatory eye drops tested with the lenses were 0.1% dexamethasone, 0.2% betamethasone and 0.9 mg/mL bromophenacyl bromide. Antibiotic eye drops tested with the lenses included 0.5% chloramphenicol. These eyedrops were tested to evaluate their interaction with the three types of therapeutic lenses over different periods of time (0.5, 1, 2, and 24 h).
The characteristics of the contact lenses and pharmaceutical agents are reported in Table 1. Table 1. Characteristics of the contact lenses and pharmaceutical agents.

HPLC Gradient Analysis
The dexamethasone, betamethasone, bromophenacyl bromide and chloramphenicol contained in the pharmacological eye drops were quantified in the liquid phase upstream and downstream, and their interaction with the lenses was evaluated with highperformance liquid chromatography (HPLC) gradient analysis. The separation was carried out using an HPLC system consisting of a vacuum degasser, an autosampler, a capillary pump and a thermostatically controlled column compartment (Agilent series 1200, Agilent Technologies, Palo Alto, CA, USA). Briefly, five microliters of sample were injected onto a 1.0 × 150 mm, 3.5-µm particle size Symmetry300 TM -C18 column (Waters). Mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was 20 µL/min, and the elution was performed in the following sequence: isocratic 90% A for 10 min, a linear gradient over the course of 50 min to 100% B, maintenance at 100% B for 15 min and, finally, a linear gradient to 90% A over the course of 5 min. The re-equilibration time in 90% A was 20 min. The eluent flow was directly sent to the electrospray ionization (ESI) ion source of a 6210 time-of-flight mass spectrometer (Agilent Technology) to characterize the HPLC peaks. The following operation parameters were applied: capillary voltage, 4300 V; nebulizer pressure, 20 psi; drying gas, 5 L/min; gas temperature, 300 • C; fragmented voltage, 250 V; skimmer voltage, 80 V; octopole RF, 250 V. The full-scan data, recorded using Agilent's Mass Hunter software, were processed with Mass Hunter Qualitative Analysis (Agilent Technology).
We tested the 3 types of lenses by incubating the pharmacological compounds together for different times (0.5, 1, 2 and 24 h). We stratified them over the lens, which acted as a filter, with 100 microliters of eyedrops applied to the upstream side and 50 microliters of physiological solution applied to the downstream side. These volumes guarantee that solution did not dry, did not fix to vial walls and avoided exceeding dilutions hampering drug detection. The areas of the peaks of the compounds, normalized by volume, were measured.

Bacterial Proliferation Assay
The influence of lens interaction on the antibacterial effect of chloramphenicol was tested with a bacterial challenge using Salmonella typhimurium seeded at 2 × 10 9 bacteria/mL on agar-coated plates.
An antibiotic disk was inserted in the middle of the plate, and the zone of inhibition on the plate was measured after 24 h. Both the chloramphenicol eyedrops and the liquid suspending the therapeutic lenses for storage were tested.

Statistical Analysis
Statistical significance of differences recorded in various experimental conditions were evaluated by non-parametric Man -Whitney U and Kruskal-Wallis tests. A threshold p value of <0.05 was considered as statistically significant. Results are presented as mean ± standard error of the mean (SEM). Analyses were performed using Statview software (Abacus Concept Inc., Berkeley, CA, USA).

HPLC Gradient Analysis
HPLC gradient analysis successfully detected the drugs in the aqueous media well, as demonstrated by the examples of drug detection reported in Figure 1. For each tested drug, the specific elution times through the chromatographic column ( Figure 1, X axis) were as follows: dexamethasone, 36 min; chloramphenicol, 29 min; betamethasone, 38 min; bromophenacyl bromide, 46 min. For each tested drug, the specific absorbance intensities (Figure 1, X axis) were as follows: dexamethasone, 239 nm; chloramphenicol and betamethasone, 241 nm; bromophenacyl bromide, 280 nm.

Time Course and Drug Interaction with the Lenses
The time courses of the interactions between the tested drugs and the lenses are reported in Figure 2. Evaluations were performed at time points of 0.5, 1, 2 and 24 h. These time point were selected because longer times did not reflect eyedrop persistence due to the ocular clearance. The amounts of drugs eluted through the lenses at each time course were determined by gradient HPLC as applied to the fluid eluted through the lenses contained in the lower parts of the wells.  At 0.5, 2, and 24 h, the lens allowing the most abundant passage of betamethasone was the G72 HW lens, by the Balafilcon A and C75 lenses. Only at 1 h was the Balafilcon A lens slightly more effective than the G72 HW lens in allowing drug passage. These differences were significant for the G72 HW lens compared to the other lenses at 2 and 24 h.
The lens allowing the most abundant passage of bromophenacyl bromide at 0.5 h was the C75 lens (p = 0.05), while at 1 h, the G72HW lens ranked highest. No significant differences were detected at other time points or between other lenses.
The lens allowing the most abundant passage of dexamethasone was the G72HW lens, but only at 0.5 h and 1 h.
Regarding chloramphenicol, the G72HW and Balafilcon A lenses allowed more abundant passage than the C75 lens at all time points tested, except for 2 h, when no significant difference between the Balafilcon A and C75 lenses was observed.
The drug that passed most readily through all lenses was chloramphenicol. Conversely, for anti-inflammatory drugs, differences were observed only at the latest (2 and 24 h for betamethasone) or earliest (0.5 and 1 h for dexamethasone and bromophenacyl bromide) time points.
The average quantities of drugs that passed through the lenses at all tested time points are reported in Figure 3. Significant differences were observed for the following: (a) the G72HW and Balafilcon A lenses compared to the C75 lens for betamethasone and chloramphenicol; (b) the G72HW lens compared to the Balafilcon A lens for all tested drugs. For the ionic lens, the regression line equation displays a positive angular coefficient with a high R2 value, thus indicating a continuous drug release from the lens increasing from 30 min to 24 h. For the non-ionic lens, the regression line equation displays a negative angular coefficient with a high R2 value, thus indicating a decrease in drugs release from the lens from 30 min 2 to 24 h.

Antibacterial Capacity of Chloramphenicol and Passage through the Lenses
The antibacterial capacity of chloramphenicol was comparatively tested before and after passage through the three lenses examined. The obtained results are reported in Figure 4. The diameter of the zone of inhibition around the chloramphenicol depot area (central white spot) was not different among the six experimental conditions tested. This finding indicates that passage through the lenses does not decrease the antibacterial capacity of chloramphenicol independent of the type of lens tested.

Discussion
The obtained results indicate that passage through the lens affects the quantity of anti-inflammatory drugs, whether steroidal or nonsteroidal. Conversely, lens passage does not affect the antibacterial efficacy of the antibiotic chloramphenicol.
These findings bear clinical relevance because therapeutic contact lenses are an option to treat ocular diseases due to their ability to release drugs for extended periods of time. Therefore, critical lens properties such as water content and ion and oxygen permeability for comfortable day and night wear contact lenses are important topics of research to enable their therapeutic use. We evaluated the interaction between different types of drugs and hydrogel therapeutic lenses. The lenses evaluated in this study are of two different types: ionic and nonionic. The Contaflex 75% nonionic hydrogel lens with 75% water is a type of lens in which the water content is ideally suited to bandaging and other therapeutic lens applications. Hydration of these materials is best performed in buffered saline with a pH of 6.8-7.3. G72HW ionic hydrogel lenses, including G-4X, G-5X and Ultra O 2 , have 72% water content and a refractive index of 1.384, and they meet the criteria for high-performance daily-wear lenses. We analyzed the interaction of four eye drop formulas, including three different anti-inflammatory drugs and one antibiotic, with therapeutic lenses under different conditions and at different times. Silicone hydrogel materials for daily-wear lenses should be supported by sound clinical data]. We measured the volume-normalized areas of the peaks of nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and antibiotics commonly used in ocular therapy. In our experiment, we verified for all the analysis times considered (after 30 min, 60 min, 2 h and 24 h) that then G72HW ionic hydrogel lens released a different quantity of eye drops than the Contaflex 75% nonionic hydrogel lens or the balafilcon A silicone hydrogel lens. Hydrogels have proven to be an extremely versatile class of materials with many potential applications in ophthalmology. Indeed, they are widely applied as soft contact lenses and foldable intraocular lenses [20].
The results of the present study highlight the relevance of adapting lenses to the ongoing therapy administered to each patient to allow a personalized medical approach. Furthermore, our findings emphasize the importance of using hydrogel material to minimize the modification of drug efficacy by the contact lens.
Our results provide evidence that the ionic lens (G72HW) is more effective than other lenses in releasing steroidal and nonsteroidal anti-inflammatory drugs and the antibiotic tested. This result is amenable to the electric charge of the ionic hydrogel. The presence of similar electrical polarities on both lenses and drugs causes reciprocal repulsion decreasing the amount of drugs entrapped inside the hydrogel and increasing the amount of drug released. This situation is well detectable for chloramphenicol but less evident for antiinflammatory drugs. Indeed, the hydrogel ionic lens provides a better release than other lenses for bethamethsone while differences from other lenses, although present, are less evident for bromophenac and dexamethasone. It is likely that the differences observed in the interactions between different anti-inflammatory drugs and the hydrogel ionic lens are due to differences in the electric charges of the anti-inflammatory drugs tested.
HPLC gradient analyses demonstrate differences in the amount of chloramphenicol released from different lenses. However, the dimension of the bacterial inhibition-growth haloes was similar for all three lens types. This finding indicates that the amount of chloramphenicol released from all lenses was anyway enough to cause bacterial growth inhibition anyway. This situation occurs for a pure S. thipymurium strain devoid of any resistance. It is likely that in the clinical situation having a higher amount of antibiotic released for the lens could decrease the risk of antibiotic-resistance occurrence.
Our study has several limitations. Nevertheless, this appears to be the first report to highlight the importance of selecting contact lens parameters to optimize drug therapy to the ocular surface; their hydration can be helpful in minimizing infections, and the properties of the materials (oxygen transmissibility) are important for the development of corneal neovascularity [21]. Corneal and conjunctival alterations can also be found in contact lens wearers. In these cases, the reduced supply of oxygen to the ocular surface interferes with corneal metabolism by inducing corneal hypoesthesia and increasing conjunctival sensitivity. Chronic use of contact lenses can also induce mechanical stimulation of nociceptors and the activation of sub-acute inflammation with the release of inflammatory mediators [22].
The future appears promising for drug-eluting contact lenses, but several challenges remain to be overcome regarding processing and storage issues, lack of use in the elderly population, regulatory issues, and high costs of clinical studies and cost-benefit analyses.
The use of therapeutic contact lenses in the USA involves a very large market (45 million contact lens wearers) that is counteracted by refractive surgery, but this type of lens has been and will be more precisely used because patient compliance is often high, with patients often facing exacerbation of their disease if they do not accept what is prescribed by the doctor. A contact lens with a burst release is superior to eye drops in terms of improved bioavailability, but this benefit is possibly not adequate to justify the increased cost from the use of contact lenses, particularly if more than one lens may be needed each day. However, advances in contact lens design now allow users to wear lenses continuously for a few weeks [23], which provides an ideal platform for extended release of ophthalmic drugs.
Furthermore, numerous other in vitro and in vivo studies have been conducted, including large in vivo studies with significant numbers of humans or animals, using commercial contact lenses soaked in various molecules to release into the eyes, including dexamethasone phosphate, chloromycetin, gentamicin or carbenicillin, kanamycin, tobramycin, ciprofloxacin, floxacin [24] and lomefloxacin [24]. These studies clearly demonstrated an increase in bioavailability with the use of drug-eluting contact lenses compared to eye drops. The increased bioavailability leads to increased drug concentrations in the cornea and aqueous humor, which, in turn, can extend the therapeutic effect compared to that of eye drops [25]. Indeed, these recent advances have led to the development of several approaches for extended delivery of a variety of ophthalmic drugs including the glaucoma drug timolol, the dry eye drug cyclosporine, the anti-inflammatory dexamethasone, the anesthetic lidocaine, antibiotics, antivirals, and antifungals. It is now feasible to design continuous-wear silicone hydrogel contact lenses that release drugs for long durations, thus affording the possibility of continuous drug delivery without lens replacement for 2-4 weeks [26].
However, this evidence-based review has clearly demonstrated that comfort cannot be considered in isolation but is contact lens-specific and depends on the surface and bulk properties of the material, the lens design characteristics, and the modality of use of the contact lens; comfort may also be significantly influenced by the replacement frequency and interaction with any lens care system that may be used.

Conclusions
Our study has definitively shown that the use of therapeutic contact lenses is a viable way to optimize anti-inflammatory therapy by optimizing its administration. Further studies will serve to clarify whether the timing of the release of various drugs on human corneas can produce sensitization and side effects.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.