Imprinted Contact Lenses for Ocular Administration of Antiviral Drugs

polymers Article Imprinted Contact Lenses for Ocular Administration of Antiviral Drugs Angela Varela-Garcia, JoséLuis Gomez-Amoza , Angel Concheiro and Carmen Alvarez-Lorenzo * Departamento de Farmacolog í a, Farmacia y Tecnolog í a Farmac é utica, I+D Farma Group, Facultad de Farmacia and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain; angela.varela.gar[email protected] (A.V.-G.); [email protected] (J.L.G.-A.); [email protected] (A.C.) *Correspondence: carmen.alvarez.lor[email protected]; Tel.: +34-881815239 Received: 7 August 2020; Accepted: 2 September 2020; Published: 4 September 2020   Abstract: A variety of ocular diseases are caused by viruses, and most treatments rely on the use of systemic formulations and eye drops. The efficient ocular barriers that oppose antiviral drug penetration have prompted the development of improved topical delivery platforms. The aim was to design hydrogel contact lenses endowed with an affinity for acyclovir (ACV) and its prodrug valacyclovir (VACV), first-choice drugs against herpes simplex virus (HSV) ocular keratitis, and that can sustain the release of therapeutic doses during daily wearing. Functional monomers suitable for interaction with these drugs were screened using computational modeling. Imprinted and non-imprinted hydrogels were prepared with various contents in the functional monomer methacrylic acid (MAA) and characterized in terms of swelling, transmittance, mechanical properties, and ocular compatibility (hen’s egg test on chorioallantoic membrane (HET-CAM) assay). The values were in the range typical of soft contact lenses. Compared to ACV, the capability to load VACV was remarkably higher due to stronger electrostatic interactions with MAA. The advantages of the imprinting technology were evidenced for VACV. Stability of VACV loading solution/hydrogels under steam heat sterilization and subsequent drug release was investigated. Permeability studies through bovine and porcine cornea and sclera of the drug released from the hydrogels revealed that VACV accumulates in the cornea and can easily cross the sclera, which may facilitate the treatment of both anterior and posterior eye segments diseases. Keywords: drug-eluting contact lens; molecularly imprinted hydrogel; antiviral drug; sustained release; cornea penetration; sclera penetration 1. Introduction Infection by herpes simplex virus (HSV) starts when the virus comes into contact with damaged skin or mucous membranes; the incubation period extends to 4 days [ 1 ]. There are two subtypes of herpes simplex virus, HSV-1 and HSV-2. The main difference is that HSV-1 appears mainly in the orolabial area, while HSV-2 affects the genital area, although, in developed countries, cases of genital conditions due to HSV-1 and orolabial due to HSV-2 are on the increase [ 2 ]. It is estimated that 90% of the world’s population is infected with HSV [ 2 ]. Periodically, the virus can reactivate and travel to the skin or mucous membranes, causing a recurrent symptomatic or asymptomatic infection. Many factors can trigger this reactivation, for example, stress, exposure to heat or cold, menstruation, fever, or immunosuppression [ 3 ]. The clinical manifestations depend on whether the infection is primary or recurrent, the immune status of the host, and the entry portal [4]. Polymers 2020,12, 2026; doi:10.3390/polym12092026 www.mdpi.com/journal/polymers Polymers 2020,12, 2026 2 of 19 At the ocular level, recurrent HSV represents a serious epidemiological cause of infectious and inflammatory disease [ 5 , 6 ]. Herpes disease affects more than 10 million people, and of these, approximately 2 million suffer vision problems in the affected eye. Epithelial keratitis accounts for 50–80% of ocular herpes. Worldwide, about 1 million new or recurrent cases of epithelial keratitis occur annually [ 7 ], and it is the most common cause of irreversible blindness in developed countries [ 8 ]. Ocular herpes is related to primary orofacial herpes (HSV-1); about 56%–58% of patients with ocular herpes have a history of oral herpes [8,9]. Current therapy for the treatment of HSV ocular keratitis includes topical, oral, and intravenous antiviral agents [ 3 , 10 ]. Viral keratitis can become a chronic and recurrent disease, affecting patients’ quality of life due to the limited efficacy of available treatments [ 10 , 11 ]. Most of the approved antivirals are acyclic nucleosides and nucleotide analogs, which interrupt virus replication [12]. Acyclovir (9-(2-hydroxyethoxymethyl) guanine) (ACV) is a purine nucleoside analog that remains the treatment of choice for HSV-1 infections to date [ 10 , 11 ]. It is a selective antiviral agent as it specifically targets virus-infected cells and selectively inhibits the viral DNA polymerase [ 4 , 13 ]. Nevertheless, the inhibition of virus replication may not affect the latency, so the infection may have not been solved [ 3 , 10 , 12 ]. ACV has a good safety profile and is well-tolerated by patients, but its oral bioavailability is low (10%–20%) and its plasma half-life is short, which involves frequent administrations [ 3 – 5 ]. Moreover, there are studies that demonstrate the growing resistance to this drug mainly in immunosuppressed subjects, developed through a mutation of the viral gene thymidine kinase, essential for the phosphorylation of ACV. An additional limitation of oral administration of ACV is renal toxicity in elderly patients, who are unable to excrete the drug properly [ 8 , 10 , 12 ]. An alternative is the topical application of ACV, but its effectiveness depends on its ability to cross the epithelium [ 3 ]. In comparative studies with other non-selective antiviral agents, ACV ointment has been shown to be more effective and less toxic [ 13 ]. The problem with topical forms is their low retention time on the eye surface [ 10 ]. In some cases, corticosteroids are used as adjuvant therapy to antivirals [ 11 ], but many side effects can occur in long-term therapy, including cataract, suppression of the immune response, and possible secondary glaucoma [10,14]. Valacyclovir (VACV) is a l-valine ester of ACV (Figure 1) and acts as a prodrug with improved bioavailability [ 13 , 15 , 16 ], but the oral administration of VACV still does not provide effective concentrations in the eye [ 17 ]. For example, oral administration of VACV (500 mg/day) does not suppress HSV-1 DNA shedding in tears [ 16 , 18 ]. Reports on topical formulations of VACV are still scarce and, so far, they focused on cationic Eudragit microspheres for mucoadhesion to the cornea surface [ 15 ] and solid lipid nanoparticles to penetrate into the eye tissues [ 19 ]. Recent studies have confirmed that VACV binds to the oligopeptide transporter of the corneal epithelium, and that its transcorneal permeability is three times higher than that of ACV. It is transformed to ACV by enzymatic hydrolysis in the eye [ 15 , 20 ]. VACV also shows a higher affinity than ACV for the amino acid transporter ATB 0,+ present in ocular tissues [21]. Polymers 2020, 12, x FOR PEER REVIEW 2 of 19 50–80% of ocular herpes. Worldwide, about 1 million new or recurrent cases of epithelial keratitis occur annually [7], and it is the most common cause of irreversible blindness in developed countries [8]. Ocular herpes is related to primary orofacial herpes (HSV-1); about 56%–58% of patients with ocular herpes have a history of oral herpes [8,9]. Current therapy for the treatment of HSV ocular keratitis includes topical, oral, and intravenous antiviral agents [3,10]. Viral keratitis can become a chronic and recurrent disease, affecting patients' quality of life due to the limited efficacy of available treatments [10,11]. Most of the approved antivirals are acyclic nucleosides and nucleotide analogs, which interrupt virus replication [12]. Acyclovir (9-(2-hydroxyethoxymethyl) guanine) (ACV) is a purine nucleoside analog that remains the treatment of choice for HSV-1 infections to date [10,11]. It is a selective antiviral agent as it specifically targets virus-infected cells and selectively inhibits the viral DNA polymerase [4,13]. Nevertheless, the inhibition of virus replication may not affect the latency, so the infection may have not been solved [3,10,12]. ACV has a good safety profile and is well-tolerated by patients, but its oral bioavailability is low (10%–20%) and its plasma half-life is short, which involves frequent administrations [3–5]. Moreover, there are studies that demonstrate the growing resistance to this drug mainly in immunosuppressed subjects, developed through a mutation of the viral gene thymidine kinase, essential for the phosphorylation of ACV. An additional limitation of oral administration of ACV is renal toxicity in elderly patients, who are unable to excrete the drug properly [8,10,12]. An alternative is the topical application of ACV, but its effectiveness depends on its ability to cross the epithelium [3]. In comparative studies with other non-selective antiviral agents, ACV ointment has been shown to be more effective and less toxic [13]. The problem with topical forms is their low retention time on the eye surface [10]. In some cases, corticosteroids are used as adjuvant therapy to antivirals [11], but many side effects can occur in long-term therapy, including cataract, suppression of the immune response, and possible secondary glaucoma [10,14]. Valacyclovir (VACV) is a L-valine ester of ACV (Figure 1) and acts as a prodrug with improved bioavailability [13,15,16], but the oral administration of VACV still does not provide effective concentrations in the eye [17]. For example, oral administration of VACV (500 mg/day) does not suppress HSV-1 DNA shedding in tears [16,18]. Reports on topical formulations of VACV are still scarce and, so far, they focused on cationic Eudragit microspheres for mucoadhesion to the cornea surface [15] and solid lipid nanoparticles to penetrate into the eye tissues [19]. Recent studies have confirmed that VACV binds to the oligopeptide transporter of the corneal epithelium, and that its transcorneal permeability is three times higher than that of ACV. It is transformed to ACV by enzymatic hydrolysis in the eye [15,20]. VACV also shows a higher affinity than ACV for the amino acid transporter ATB0,+ present in ocular tissues [21]. Figure 1. Structure of acyclovir (ACV) and valacyclovir (VACV) and workflow of the experiments. The aim of this work was to design hydrogels suitable for soft contact lenses (SCLs) with an affinity for ACV and VACV and that can sustainedly release these drugs on the ocular surface during daily wearing (Figure 1). Among the proposed procedures to endow the SCLs with an affinity for specific molecules, the creation of artificial receptors using the molecular imprinting Acyclovir Valacyclovir Structural monomer Functional monomer Figure 1. Structure of acyclovir (ACV) and valacyclovir (VACV) and workflow of the experiments. Polymers 2020,12, 2026 3 of 19 The aim of this work was to design hydrogels suitable for soft contact lenses (SCLs) with an affinity for ACV and VACV and that can sustainedly release these drugs on the ocular surface during daily wearing (Figure 1). Among the proposed procedures to endow the SCLs with an affinity for specific molecules, the creation of artificial receptors using the molecular imprinting technique stands out [ 22 – 24 ]. This technique requires incorporating the drug into the monomers mixture so that the monomers can rearrange according to their affinity. This rearrangement becomes permanent during polymerization. The removal of the template molecules generates cavities with the most appropriate size and chemical groups to host the drug of interest again [ 25 ]. The molecular imprinting approach has been successfully applied to develop SCLs loaded with antiglaucoma [ 22 , 26 ], antiallergic [ 27 , 28 ], and antimicrobial [ 29 , 30 ] drugs, among others, and therapeutic agents that may increase ocular comfort [ 31 ] and even address the management of diabetic eyes [32,33], but not with antiviral drugs yet. To carry out the work, functional monomers suitable for interaction with the antiviral drugs were first screened using computational modeling, a technique that has been shown to be efficient to save time and materials in the development of imprinted materials [ 34 ]. Methacrylic acid (MAA) showed a higher affinity for the drugs than the structural monomer 2-hydroxyethyl methacrylate (HEMA) and other functional monomers. MAA may interact with the side chain of VACV through not only hydrogen bonding with the ring (as in the case of ACV) but also electrostatic interactions with the amino group of the valine chain. Hydrogels were prepared with various contents in the functional monomer in the presence (imprinted) and absence (non-imprinted) of each drug. The hydrogels were characterized in terms of swelling, light transmission, mechanical properties, eye compatibility (hen’s egg test on chorioallantoic membrane (HET-CAM) assay), and capability to load and release the antiviral drugs. The stability of VACV loading solution/hydrogels under steam heat sterilization and subsequent drug release was investigated. Finally, the permeability through bovine and porcine cornea and sclera of the drug released from the hydrogels was evaluated (Figure 1). 2. Materials and Methods 2.1. Materials Acyclovir (ACV; MW 225.21 g/mol; solubility in water 1.02 mg/mL [ 35 ]) was purchased from Farmalabor (Canosa di Puglia, Italy); valacyclovir hydrochloride (VACV; MW 360.80 g/mol; solubility in water 174 mg/mL [ 36 ]) was from Acros Organics (Geel, Belgium); 2,2 0 -azo-bis(isobutyronitrile) (AIBN), dichlorodimethylsilane, ethylene glycol dimethacrylate (EGDMA), and methacrylic acid (MAA) were from Sigma-Aldrich (Steinheim, Germany); ethanol absolute and NaOH were from VWR (Leuven, Belgium); 2-hydroxyethyl methacrylate (HEMA) was from Merck (Darmstadt, Germany); acetic acid and NaCl were from Scharlau (Sentmenat, Spain); and methanol was from Fisher (Loughborough, UK). Ultrapure water (resistivity >18 M Ω· cm) was obtained by reverse osmosis (MilliQ ® , Millipore, Madrid, Spain). Simulated lacrimal fluid (SLF) was prepared with the following composition: 6.78 g/L NaCl, 2.18 g/L NaHCO 3 , 1.38 g/L KCl, and 0.084 g/L CaCl 2· 2H 2 O with pH 7.5. Carbonate buffer pH 7.2 was prepared by mixing buffer solution A (6.2 g/L NaCl, 0.355 g/L KCl, 0.1 g/L NaH 2 PO 4· H 2 O, and 2.45 g/L NaHCO3) and buffer solution B (0.115 g/L CaCl2and 0.155 g/L MgCl2·6H2O). 2.2. Computational Modeling A preliminary study was carried out using computer modeling to elucidate interactions between the drugs to be studied (ACV and VACV) and functional monomers used in the synthesis of hydrogels. The tested monomers were acrylamide (AAm), 2-aminoethyl methacrylate hydrochloride (AEMA), N-(3-aminopropyl) methacrylamide hydrochloride (APMA), ethylene glycol phenyl ether methacrylate (EGPEM), butoxyethyl methacrylate (BEM), hydroxyethyl methacrylate (HEMA), and methacrylic acid (MAA). The 3D structure of the functional monomers and ACV and VACV was taken from the PubChem database [ 37 ]. The SDF files were transformed to PDB files using OpenBabel v. 2.4.1 software [ 38 ]. The Autodock Tools v. 4.2.6 software was used to calculate molecular docking. In all Polymers 2020,12, 2026 4 of 19 cases, the grid was generated with default settings around the monomer and the drug, the smallest conformation was used, and the docking was performed using the Lamarckian Genetic Algorithm [ 39 ]. Estimated free energy of binding ( ∆ G binding ) and dissociation constant (Ki) values were obtained. Autodock used a semi-empirical force field to evaluate the binding in two steps. The ligand (e.g., monomer) and receptor (e.g., drug) started in an unbound conformation. First, the intramolecular energetics were estimated for the transition from these unbound states to the conformation in the bound state. Second, the intermolecular energetics of combining the ligand and receptor were estimated. Energies of dispersion/repulsion, hydrogen bonding, electrostatics, and desolvation were evaluated as described in the user guide [ 40 ]. The dissociation constant of the complex (Ki), also known as the inhibition constant, was estimated as follows: Ki =e(∆Gbinding/(RT)) (1) In this equation, Ris the gas constant and Tthe absolute temperature in Kelvin. 2.3. Synthesis of Imprinted and Non-Imprinted Hydrogels Different mixtures of monomers were prepared as shown in Table 1. The components were added to vials and mixed at room temperature and under magnetic agitation (300 rpm) until they were completely dissolved. Finally, the initiator (AIBN) was added, and the solutions were stirred for 15 min more. The solutions were injected, with a needle and syringe, into pre-assembled molds, consisting of two pre-treated glass plates (12 × 14 cm) separated by a 0.45 mm-thick silicone frame. After pre-treatment of the glass plates with dichlorodimethylsilane, the plates were left to dry in a hood for 1 h, thoroughly washed with ethanol, rinsed with water, and dried in an oven at 70 ◦ C for 1 h before being assembled. Polymerization of the monomers inside the molds was carried out for 12 h at 50 ◦ C and then for a further 24 h at 70 ◦C. All hydrogel compositions were prepared in triplicate. Table 1. Composition of the hydrogels (NIP: Non-imprinted hydrogels, MIP: Imprinted hydrogels). Final ethylene glycol dimethacrylate (EGDMA), methacrylic acid (MAA), and 2,20-azo-bis(isobutyronitrile) (AIBN) concentrations were 8, 200 and 10 mM, respectively. Hydrogel HEMA (mL) EGDMA (µL) MAA (mL) ACV (mg) VACV (mg) AIBN (mg) NIP 5 7.55 0 0 0 8.21 NIP200 5 7.55 0.084 0 0 8.21 MIPACV 5 7.55 0 45 0 8.21 MIPA1:5 5 7.55 0.084 45 0 8.21 MIPA1:10 5 7.55 0.084 23 0 8.21 MIPA1:15 5 7.55 0.084 15 0 8.21 MIPVACV 5 7.55 0 0 25 8.21 MIPV1:6 5 7.55 0.084 0 50 8.21 MIPV1:12 5 7.55 0.084 0 25 8.21 MIPV1:32 5 7.55 0.084 0 10 8.21 2.4. Drug Removal After polymerization, each hydrogel sheet was immersed in 500 mL of boiling water for 15 min in order to remove unreacted monomers and template drugs and facilitate the cutting into discs (10 mm in diameter). Further washing was then performed, except for a few discs of each type of hydrogel, which were reserved for the direct drug release test (as explained in Section 2.5). For the washing, the discs were immersed in water, under magnetic agitation (300 rpm) and at room temperature. The medium was replaced every 24 h until no signal was detected in the range of 190–800 nm (UV-Vis spectrophotometer, Agilent 8534, Waldbronn, Germany). When no spectrophotometric signal was detected, the hydrogels were dried in an oven at 70 ◦ C for 24 h and stored protected from Polymers 2020,12, 2026 5 of 19 light and humidity. In parallel, the amount of ACV (MIP ACV , MIP A1:5 , MIP A1:10 , and MIP A1:15 ) and VACV (MIP VACV , MIP V1:6 , MIP V1:12 , and MIP V1:32 ) removed in each washing step was monitored spectrophotometrically at 252 and 253 nm, respectively. 2.5. Direct Drug Release Test from Boiled Hydrogels After the boiling step, three discs of each type of hydrogel were individually placed in vials containing 5 mL of SLF and kept under oscillating agitation (300 rpm) at 35 ◦ C. At preset times (0.5, 1, 2, 6, and 24 h), 3 mL of medium was removed and the absorbance was measured at 252 nm (ACV) and 253 nm (VACV) (UV-Vis spectrophotometer, Agilent 8453, Waldbronn, Germany), returning the samples to the release vial. The experiments were carried out in triplicate. The amounts of drug released were calculated using previously prepared calibration curves and referred to the unit of mass of the dry disc. The calibration curves were prepared by dissolving ACV (30 µ g/mL) in ethanol:water (50:50, v/v) mixture, and VACV (50 µ g/mL) in water. Dilutions of 2, 3, 5, 10, 15, 20, 25, and 30 µ g/mL were made for ACV, and 1.25, 2.5, 5, 10, 15, 20, 25, 30, 40, and 50 µ g/mL for VACV. The calibration curves were validated for absorbances recorded at 252 and 253 nm, respectively (UV-Vis spectrophotometer, Agilent 8453, Waldbronn, Germany). 2.6. Drug Loading and Release ACV loading was tested, in triplicate, on the hydrogels NIP, NIP 200 , MIP ACV , MIP A1:5 , MIP A1:10 , and MIP A1:15 . Each hydrogel disc (approx. 40 mg) previously washed and dried was placed in a tube with 5 or 15 mL of ACV aq. solution (0.3 mg/mL) and kept under oscillating agitation (300 rpm), at room temperature (23–25 ◦ C), for 4 days. The absorbance of the solution was monitored at 252 nm (UV-Vis spectrophotometer, Agilent 8453, Waldbronn, Germany) by taking aliquots of 0.2 mL and diluting to 5 mL with ethanol:water mixture (50:50, v/v) (i.e., 1:25 dilution). The amount of drug loaded was estimated by the difference between the initial and final amount of drug in solution calculated using the previously prepared calibration curve, and referred to the unit of mass of the dry disc. VACV loading was evaluated, in triplicate, on the hydrogels NIP, NIP 200 , MIP VACV , MIP V1:6 , MIP V1:12 , and MIP V1:32 . Each hydrogel disc (approx. 40 mg) previously washed and dried was placed in a tube with 5 mL of VACV (0.3 mg/mL) solution in 0.1 mM NaOH medium (pH 6.6). The loading tubes were kept under the same conditions of agitation, temperature, and time as for the ACV loading. The absorbance of the medium was monitored spectrophotometrically at 253 nm. The drug network/water partition coefficient (K N/W ) was calculated for each hydrogel from the total amount of drug loaded using the following equation Loading (total)=VS+KN/W∗Vp WP ∗C0(2) where V S is the volume of water absorbed by the hydrogel (mL), V p the volume of dry polymer (mL), W p the weight of the dry hydrogel (g), and C 0 the concentration of drug in the loading solution (g/mL). The loaded discs were removed from the tubes and rinsed with water. The surface water was removed with filter paper, and the discs were then immediately placed in release tubes with 15 and 10 mL of SLF (for ACV and VACV, respectively) under oscillating agitation (300 rpm) and at 35 ◦ C. The release kinetics was evaluated for 24 h. Samples of the medium were periodically taken and analyzed following the same protocol as in Section 2.5. Feasibility of the simultaneous loading and sterilization of the hydrogels was also investigated. Dried NIP, NIP 200 , MIP VACV , MIP V1:6 , MIP V1:12 , and MIP V1:32 hydrogels were placed in vials containing 5 mL of VACV (0.3 mg/mL) solution in 0.1 mM NaOH medium (pH 6.6) and kept for 12 h at room temperature. Then, the vials were steam heat sterilized (autoclave, 121 ◦ C, 30 min) and stored at room temperature for 24 h without shaking. VACV solutions without hydrogels were processed as controls. Drug loading and release profiles were recorded as explained above. The sterilization tests were carried out in triplicate for each hydrogel and VACV solution and repeated in two independent runs. Polymers 2020,12, 2026 6 of 19 2.7. Solvent Uptake The uptake of water and SLF was monitored recording the increase in weight of dried discs after being immersed in 4 mL of the corresponding medium at room temperature (23–25 ◦ C). At predetermined times (0.5, 1, 2, 4, 8, and 24 h), each disc was taken from the vial, excess water was removed with blotting paper, and the weight recorded. The discs were immediately returned to the vials. The solvent uptake was calculated as follows: Solvent uptake (%)=Wt−W0 W0 ∗100 (3) where W0and Wtrepresent the weight of the dried and swollen hydrogel, respectively. 2.8. Light Transmission The light transmittance (%) of discs swollen in SLF was measured in a spectrophotometer (Agilent Cary 60 UV-Vis, Waldbronn, Germany) in triplicate from 200 to 800 nm. 2.9. Mechanical Properties The mechanical properties of NIP, NIP 200 , MIP VACV , MIP V1:6 , MIP V1:12 , and MIP V1:32 hydrogels swollen in water were tested in triplicate at room temperature (23–25 ◦ C). Each hydrogel was cut into 16 × 9 mm strips and attached to the upper and lower clamps, with a 7 mm gap, on a TA.XT Plus Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK), equipped with a 5 kg load cell. The crosshead speed applied to record the stress–strain plots was 0.1 mm/s. The Young’s modulus (E) was calculated as the slope of the straight-line part of engineering stress (force per cross-sectional area, N/mm 2 ) versus the engineering strain (change in active length divided by original length, mm/mm) [ 41 , 42 ] as follows: E= F A0 ∆L L0 (4) 2.10. HET-CAM Test The hen’s egg test on chorioallantoic membrane (HET-CAM) was performed by incubating fertilized hen eggs (50–60 g) at 37 ◦ C and 60% RH for 9 days. On the ninth day of incubation, a circular cut was made on the top of the egg of approximately 1 cm diameter with a rotary saw (Dremel 300, Breda, The Netherlands). The shell was removed, and the inner membrane was moistened with 0.9% NaCl for 30 min (time during which the egg remained inside the climatic chamber). The membrane was then removed to expose the chorioallantoic membrane (CAM) [ 33 ]. The test was performed by placing in each CAM a hydrogel disc previously soaked for 4 days in drug loading solution. Aqueous solutions of NaOH 0.1 N and NaCl 0.9% (300 µ L) were used as negative and positive controls, respectively. The blood vessels were observed under white light for 5 min, to detect possible bleeding, vascular lysis, or coagulation. All tests were performed in triplicate and the irritation score calculated as reported previously [33]. 2.11. Cornea and Sclera Permeability Tests Fresh bovine and porcine eyes were collected from a local slaughterhouse and transported according to the Bovine Corneal Opacity/Permeability (BCOP) test protocol [ 33 , 43 ]. During transport, the eyes were kept immersed in PBS with added antibiotics (penicillin 100 IU/mL and streptomycin 100 µ g/mL), in an ice bath. Corneas and scleras were isolated using a scalpel. The tissues were washed with 0.9% NaCl and mounted in vertical diffusion cells (Franz cells). To balance the tissues, the donor and receptor chambers were filled with carbonate buffer pH 7.2 and placed in a bath at 37 ◦ C, with magnetic stirring, for 30 min. After that time, the content of the donor chamber was removed and the corneas and scleras were exposed to VACV-loaded NIP and MIP V1:12 discs (loaded as in Section 2.6). Polymers 2020,12, 2026 7 of 19 The discs were covered with 2 mL of 0.9% NaCl. In parallel, corneas and scleras were exposed to 2 mL of a VACV solution (100 µ g/mL in NaOH 0.1 mM; pH 6.6) as a control, for 6 h. The donor chambers were covered with parafilm to avoid evaporation. Samples (1 mL) of the receptor medium were taken at 0.5, 1, 2, 3, 4, 5, and 6 h, and replaced with carbonate buffer pH 7.2 taking care of preventing bubbles formation in the diffusion cell. The amount of VACV permeated into the receptor chamber was quantified by HPLC (Autosampler Waters 717, Waters Controller 600, Photodiode Detector 996, Milford, MA, USA), equipped with a C18 column (Waters Symmetry C18, 5 µ m, 4.6 × 250 mm) and operated with the Empower2 software. The mobile phase consisted of acetic acid (1:1000): methanol (90:10 v/v) with a flow rate of 1 mL/min. The injection volume was 50 µ L and the column was kept at 30 ◦ C. The calibration was performed with standard solutions of ACV and VACV (6.25–0.19 µ g/mL) in carbonate buffer pH 7.2 [ 44 ], and the absorbance was quantified at 251 nm. Retention times were ~3 min for VACV and 4.8 min for ACV (typical HPLC chromatograms are shown in Figure S1, Supporting Information). The accumulated amounts of drug permeated were calculated from the sum of VACV and ACV peaks [ 20 ]. The steady-state flow (J) and the time delay (t lag ) were obtained from the slope and x-intercept, respectively, of the linear regression of the accumulated amount of drug permeated per area vs. time [ 45 ]. After 6 h of the test, aliquots of the liquid remaining at the donor chamber were taken for further analysis. The coefficients of permeability of the drug through the cornea and sclera were calculated as the ratio of Jto the concentration of drug in the donor chamber [45]. The corneas/scleras were also removed from the diffusion cells after 6 h test, rinsed with 0.9% NaCl, and immersed in 3 mL of an ethanol:water mixture (50:50 v/v) overnight. They were then sonicated for 99 min at 37 ◦ C, centrifuged (1000 rpm, 5 min, 25 ◦ C), filtered, and re-centrifuged (14,000 rpm, 20 min, 25 ◦ C) [ 46 ]. The drug extracted from the corneas/scleras was quantified by HPLC as explained above. 2.12. Statistical Analysis The effects of hydrogel composition on drug loading and permeability through porcine and bovine tissues were analyzed using ANOVA and the multiple range test (Statgraphics Centurion XVII, Stat Point Technologies Inc., Warrenton, VA, USA). 3. Results and Discussion 3.1. Computational Modeling Computational modeling is a versatile tool for the first screening of functional monomers suitable for preparing imprinted hydrogels [ 34 , 47 , 48 ]. Although the conditions during polymerization cannot be precisely resembled, computational modeling has been shown to be useful to identify the monomers with a high affinity for the template drug, which, in turn, may endow the imprinted hydrogels with high rebinding and controlled release performances [ 48 , 49 ]. HEMA, as a main structural monomer of SCLs, and six functional monomers bearing different chemical moieties (amido, amine, phenyl, butyl, and acrylic acid) were screened regarding their interactions with ACV and VACV. Results of computational modeling are summarized in Figure 2. High negative values of ∆ G binding indicated favorable binding interactions between the drug and the monomer. The lower the value of Ki, the lower the likelihood of complex disassembly. Considering both ∆ G binding and Ki, the interaction of ACV and VACV with MAA was predicted to be more favorable than with other monomers (Figure 2). Interestingly, MAA may interact with VACV through hydrogen bonds with the amino groups in the aromatic ring (as in the case of ACV) and also with the primary amine of the valine side chain. The pKa of the amino group of valine has been estimated to be in the 9.1–9.6 range [ 50 ]. Thus, in the aqueous medium used to load the hydrogels and in the lacrimal fluid, this amino group is expected to remain protonated. This opens the possibility of the fact that the MAA mers in the hydrogels may readily interact with VACV through electrostatic interactions. Therefore, MAA was chosen as the functional monomer to prepare the hydrogels. Polymers 2020,12, 2026 8 of 19 Polymers 2020, 12, x FOR PEER REVIEW 8 of 19 Figure 2. Computational modeling results of the interaction of acyclovir (ACV) with the monomers 2-hydroxyethyl methacrylate (HEMA), acrylamide (AAm), 2-aminoethyl methacrylate hydrochloride (AEMA), N-(3-aminopropyl) methacrylamide hydrochloride (APMA), butoxyethyl methacrylate (BEM), ethylene glycol phenyl ether methacrylate (EGPEM), and methacrylic acid (MAA); and the interaction of valacyclovir (VACV) with 2-hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA). 3.2. Synthesis of Hydrogels and Drug Removal Imprinted and non-imprinted hydrogels were synthesized combining HEMA with MAA as the functional monomer. The drug was added at different levels, in ascending mole ratios of ACV:MAA (1:5, 1:10, and 1:15 mol/mol) and VACV:MAA (1:6, 1:12, and 1:32 mol/mol), as explained in Table 1. It should be noted that the total content in MAA was fixed, and only the content in the template drug varied. The ratio VACV:MAA was lower than that of ACV:MAA because of the additional binding points that VACV may establish with MAA according to the computational study. The use of MAA as comonomer should enhance drug–hydrogel interactions through hydrogen bonding of the acrylic acid group with the rings of the drugs (ACV and VACV) and electrostatic interactions with the Figure 2. Computational modeling results of the interaction of acyclovir (ACV) with the monomers 2-hydroxyethyl methacrylate (HEMA), acrylamide (AAm), 2-aminoethyl methacrylate hydrochloride (AEMA), N-(3-aminopropyl) methacrylamide hydrochloride (APMA), butoxyethyl methacrylate (BEM), ethylene glycol phenyl ether methacrylate (EGPEM), and methacrylic acid (MAA); and the interaction of valacyclovir (VACV) with 2-hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA). 3.2. Synthesis of Hydrogels and Drug Removal Imprinted and non-imprinted hydrogels were synthesized combining HEMA with MAA as the functional monomer. The drug was added at different levels, in ascending mole ratios of ACV:MAA (1:5, 1:10, and 1:15 mol/mol) and VACV:MAA (1:6, 1:12, and 1:32 mol/mol), as explained in Table 1. It should be noted that the total content in MAA was fixed, and only the content in the template drug varied. The ratio VACV:MAA was lower than that of ACV:MAA because of the additional binding points that VACV may establish with MAA according to the computational study. The use of MAA as comonomer should enhance drug–hydrogel interactions through hydrogen bonding of the acrylic acid group with the rings of the drugs (ACV and VACV) and electrostatic interactions with the VACV side chain. The highest drug:MAA mole ratio was limited by the poor solubility of the drugs in Polymers 2020,12, 2026 9 of 19 the monomers solution, but also considering that 1:4 to 1:6 mole ratios have been commonly reported as adequate to create imprinted cavities [ 34 , 51 ]. Additionally, non-imprinted hydrogels prepared with the same content in MAA (NIP 200 ) and imprinted hydrogels without MAA (MIP ACV and MIP VACV ) were synthesized under the same conditions in order to elucidate the role of the functional monomer and of the drug template, respectively. Monomers and template drug molecules were easily detected in the washing medium. Monomers mainly absorbed at wavelengths below 220 nm, while ACV and VACV absorbed at 252–253 nm. Both peaks were clearly recorded in the first washing solution after boiling. VACV was easily removed from the VACV-imprinted hydrogels during the washing process in boiling water (Figure 3a). Elution of residual monomers caused minor interferences in the quantification of the amounts of drug extracted. By contrast, the amounts of ACV removed from the hydrogels synthesized using ACV as a template were lower than the amounts added during synthesis (Figure 3a). This could be due to the limited solubility of the ACV in aqueous medium, which may favor hydrophobic association with the hydrogel polymer backbone. Polymers 2020, 12, x FOR PEER REVIEW 9 of 19 VACV side chain. The highest drug:MAA mole ratio was limited by the poor solubility of the drugs in the monomers solution, but also considering that 1:4 to 1:6 mole ratios have been commonly reported as adequate to create imprinted cavities [34,51]. Additionally, non-imprinted hydrogels prepared with the same content in MAA (NIP200) and imprinted hydrogels without MAA (MIPACV and MIPVACV) were synthesized under the same conditions in order to elucidate the role of the functional monomer and of the drug template, respectively. Monomers and template drug molecules were easily detected in the washing medium. Monomers mainly absorbed at wavelengths below 220 nm, while ACV and VACV absorbed at 252– 253 nm. Both peaks were clearly recorded in the first washing solution after boiling. VACV was easily removed from the VACV-imprinted hydrogels during the washing process in boiling water (Figure 3a). Elution of residual monomers caused minor interferences in the quantification of the amounts of drug extracted. By contrast, the amounts of ACV removed from the hydrogels synthesized using ACV as a template were lower than the amounts added during synthesis (Figure 3a). This could be due to the limited solubility of the ACV in aqueous medium, which may favor hydrophobic association with the hydrogel polymer backbone. Figure 3. (a) Amounts of ACV and VACV removed during washing in boiling water and subsequent washings from each of the hydrogels tested (codes as in Table 1), and (b) release profiles of ACV and VACV from the imprinted discs that were previously boiled in water (15 min) and dried to constant weight. The drug released corresponds to that used as a template during synthesis. 3.3. Direct Drug Release Test from Boiled Hydrogels A direct release in SLF was carried out for hydrogels after boiling (without further washing) for the first screening of their ability to release the remaining ACV and VACV template in a sustained way (Figure 3b). The hydrogels polymerized using ACV as a template (MIPACV, MIPA1:5, MIPA1:10, MIPA1:15) released about 0.20 mg of drug per gram of disc. The sum of the amount released plus the amount already removed during boiling was still lower than the total amount added during synthesis (45, 45, 23, and 15 mg of ACV, respectively), and practically no differences were observed between the different types of hydrogel. This finding pointed to ACV template entrapment in the polymer network, favored by the hydrophobicity of the drug. By contrast, the hydrogels polymerized using VACV as the template (MIPVACV, MIPV1:6, MIPV1:12, MIPV1:32) released the small amount of remnant VACV in a sustained way for 24 h. The mass balance of VACV confirmed that the molecules used as the template were mostly removed during the boiling process. A similar release experiment was performed with non-imprinting hydrogels and, as expected, no signal was recorded at the wavelength used for drug quantification. ACV/VACV removed (mg/g) 0 10 20 30 40 50 Boiling Subsequent washings Time (h) 0 4 8 12 16 20 24 ACV/VACV removed (mg/g) 0.0 0.2 0.4 0.6 0.8 1.0 MIPACV MIPA1:5 MIPA1:10 MIPA1:15 MIPVACV MIPV1:6 MIPV1:12 MIPV1:32 MIPACV MIPA1:5 MIPA1:10 MIPA1:15 MIPVACV MIPV1:6 MIPV1:12 MIPV1:32 (a) (b) Figure 3. ( a ) Amounts of ACV and VACV removed during washing in boiling water and subsequent washings from each of the hydrogels tested (codes as in Table 1), and ( b ) release profiles of ACV and VACV from the imprinted discs that were previously boiled in water (15 min) and dried to constant weight. The drug released corresponds to that used as a template during synthesis. 3.3. Direct Drug Release Test from Boiled Hydrogels A direct release in SLF was carried out for hydrogels after boiling (without further washing) for the first screening of their ability to release the remaining ACV and VACV template in a sustained way (Figure 3b). The hydrogels polymerized using ACV as a template (MIP ACV , MIP A1:5 , MIP A1:10 , MIP A1:15 ) released about 0.20 mg of drug per gram of disc. The sum of the amount released plus the amount already removed during boiling was still lower than the total amount added during synthesis (45, 45, 23, and 15 mg of ACV, respectively), and practically no differences were observed between the different types of hydrogel. This finding pointed to ACV template entrapment in the polymer network, favored by the hydrophobicity of the drug. By contrast, the hydrogels polymerized using VACV as the template (MIP VACV , MIP V1:6 , MIP V1:12 , MIP V1:32 ) released the small amount of remnant VACV in a sustained way for 24 h. The mass balance of VACV confirmed that the molecules used as the template were mostly removed during the boiling process. A similar release experiment was performed with non-imprinting hydrogels and, as expected, no signal was recorded at the wavelength used for drug quantification. Polymers 2020,12, 2026 16 of 19 4. Conclusions Acyclovir and valacyclovir behaved differently when incorporated into HEMA-based hydrogels, despite their similar chemical structure and a priori similar binding energy with MAA. The limited solubility of ACV in the monomer mixture together with unspecific hydrophobic interactions may explain why ACV-imprinted hydrogels were not effective in terms of drug loading and release. Moreover, the small amount of ACV loaded was not completely released in SLF, probably because of the hydrophobicity of the drug. By contrast, the use of MAA as functional monomer remarkably increased the affinity of the hydrogels for VACV through electrostatic interactions between the acrylic acid group of the monomer and the drug lateral chain. The use of VACV as a template during polymerization facilitated the arrangement of the polymer network, creating specific cavities that contributed to enhancing the affinity of the drug for the hydrogel in subsequent loading. The degree of swelling, light transmission, and mechanical properties showed common values to daily wear contact lenses. In addition, no potential eye irritation was observed in the HET-CAM assay. VACV-imprinted hydrogels can release the drug in a sustained manner for 10 h, which is a common time of wearing disposable SCLs. Therapeutically relevant amounts accumulated in the cornea. At the sclera level, VACV-loaded hydrogels showed permeability values equivalent to those achieved with the aqueous solution of the drug. VACV permeability through the sclera suggests the possibility of delivery to the posterior segment. Therefore, hydrogels containing MAA and imprinted with VACV are suitable candidates for the preparation of drug-eluting contact lenses. As VACV does not withstand steam heat sterilization, the scale-up may involve the autoclaving of the hydrogels first and then the packaging in VACV solution prepared using sterilizing filtration. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/12/9/2026/s1, Figure S1: Typical HPLC chromatograms of VACV and ACV standard solutions and of a sample of the receptor medium during the permeability tests. Figure S2. HPLC chromatograms of the VACV loading solution before and after steam heat sterilization (autoclave 121 ◦ C, 30 min). Figure S3. Drug (VACV+ACV) release profiles in SLF from non-imprinted and imprinted hydrogels that were loaded by soaking in VACV solution and sterilized by steam heat sterilization. The data are shown as accumulated amounts obtained after conversion from absorbance values recorded at 253 nm. Author Contributions: Conceptualization, A.C. and C.A.-L.; Methodology, A.V.-G. and J.L.G.-A.; Software, J.L.G.-A.; Investigation, A.V.-G. and C.A.-L.; Resources, A.C. and C.A.-L.; Writing—original draft preparation, A.V.-G. and C.A.-L.; Writing—review and editing, A.C., J.L.G.-A. and C.A.-L.; Supervision, A.C. and C.A.-L.; funding acquisition, A.C. and C.A.-L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by MINECO [SAF2017-83118-R] Spain, Agencia Estatal de Investigaci ó n (AEI) Spain, Xunta de Galicia [ED431C 2016/008; AEMAT ED431E 2018/08] Spain, and FEDER. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Whitley, R.J.; Roizman, B. Herpes simplex virus infections. Lancet 2001,357, 1513–1518. 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