Abstract
Naltrexone (NTX) is a potent opioid growth factor receptor (OGFR) antagonist proved to be useful for treatment of ocular surface complications. The aim of this work was to explore the feasibility of designing NTX-imprinted 2-hydroxyethyl methacrylate-based hydrogels for sustained drug release on the ocular surface. Acrylic acid (AAc) and benzyl methacrylate (BzMA) were chosen as functional monomers able to form binding cavities mimicking OGFR binding sites for NTX. Imprinted hydrogels containing functional monomers loaded higher amounts of NTX compared to non-imprinted ones by simple soaking in drug aqueous solution. In addition, possibility of carrying out the loading and sterilization processes in one step was investigated. NTX release was evaluated both under agitated sink conditions and in a microfluidic flow chamber mimicking the hydrodynamic conditions of the eye, namely the small volume of lachrymal fluid and its renovation rate. Sustained release profiles together with adequate swelling degree (46 to 57% w/w), light transparency (over 85%) and oxygen permeability may make these hydrogels suitable candidates to NTX-eluting contact lenses. NTX-loaded and non-loaded discs successfully passed the chorioallantoic membrane test for potential ocular irritation and were cytocompatible with human mesenchymal stem cells. Finally, NTX-imprinted hydrogels tested in the bovine corneal permeability assay provided therapeutically relevant amounts of NTX inside the cornea, reaching drug levels similar to those attained with a concentrated aqueous solution in spite the discs showed sustained release.
Keywords
Naltrexone Contact lenses Imprinted hydrogels Diabetic eye complications Corneal diseases Dry eye
1. Introduction
Cornea, sclera, conjunctiva and tear film are functionally linked as one system, including tear glands, to protect the eye against external adverse events and pathogens [1]. In addition, lachrymal fluid and cornea are the first refracting elements. Thus, cornea integrity and an adequate tear production are essential to preserve a healthy vision [2]. Metabolic changes suffered by persons with diabetes lead to important ocular surface complications such as decreased tear production, diminished corneal sensitivity, and delayed wound healing [3]. Disruption of tear film barrier function and poor tear quality production can also result in irritation, inflammation of the stroma and impaired vision.
Enkephaline is a natural opioid growth factor (OGF) that, when interacts with its receptor (OGFr), negatively regulates cell proliferation and tissue growth. Enkephaline has been found to be at higher levels in persons with diabetes compared to healthy people [4,5]. Elevated concentrations of this peptide, which have also been confirmed in diabetes animal models, are responsible for complications such as ulceration and increased susceptibility to infection due to delayed epithelialization during wound healing. It should be noted that the OGF-OGFr axis plays a key role in the homeostasis of cornea and retina [1], and thus antagonists of OGFr, such as naltrexone (NTX), may revert ocular surface complications by blocking the effects of enkephaline [4,6]. NTX is approved in oral formulations for treatment of addiction to opioids or alcohol, although it undergoes a relevant hepatic first pass effect. Preclinical studies have demonstrated that NTX, either systemically or topically applied, can notably improve corneal wound healing and reverse severe dry eye [6,7]; for example, studies on type I diabetic rats and normal rats with episodic dry eye have shown that one drop of NTX restore tear production in <1 h and the effect persists for 72 h [6]. Importantly, repeated doses of NTX eye drops (50 μM; 4 drops) over a 24 h period did not cause adverse events on healthy human volunteers [2]. However, NTX tends to autoxidation when it is formulated as eye drops [8]. Furthermore, conventional ocular formulations provide low ocular bioavailability because of short precorneal residence time and limited cornea permeability. Thus, to achieve a therapeutic effect, frequent instillations are needed. Alternatively, mucoadhesive in situ gelling formulations [9] and niosomes [10] have been explored.
Contact lenses (CLs) are safe devices widely used to correct refractive errors and are attracting an increasing interest as drug delivery platforms [[11], [12], [13], [14]]. Several approaches have been implemented to endow CLs with ability to host drugs such as timolol, acetazolamide, olopatadine, amphotericin B, ciprofloxacin or epalrestat, among others [[15], [16], [17], [18], [19], [20]]. CLs can act as a reservoir that provides sustained levels of drug in the precorneal area (postlens lachrymal fluid) while minimizes drug loss due to blinking, reflex lachrymation or nasolacrimal drainage [21]. One useful approach to enhance the capability of CLs to load therapeutic amounts of drug is the molecular imprinting [11,22]. This technique relies on the use of the target drug molecules as templates during polymerization in order to induce monomers arrangement as a function of their affinity. After polymerization, the template molecules are removed and specific cavities for the target drug, named imprinted pockets, are revealed. Physical stability of the imprinted cavities is quite limited in the case of loosely crosslinked networks, as occurs for soft CLs. Therefore, a precise selection of functional monomers that can endow the cavities with sufficient affinity for the target molecule even after the swelling of the network is required for the success of the recognition. Bioinspired strategies that rely on mimicking the pharmacological target of the drug in the hydrogel network have been shown useful for the rational selection of functional monomers [17,23,24].
The aim of the present work was to design NTX-imprinted poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels suitable as soft CLs that can load therapeutic amounts of NTX and provide sustained release on the ocular surface. The hydrogels were designed taking into account the information available about the interactions of NTX with the μ-opioid receptor (MOR) [25,26], which are mainly driven by binding to amino acid residues Asp147 (bearing a carboxylic acid group) and Tyr148 (bearing a phenyl group) and polar interactions with Lys233 [27,28]. Taking into account this information, functional monomers were chosen among those bearing carboxylic acid groups (acrylic acid, AAc) and aromatic groups (benzyl methacrylate, BzMA). It can be hypothesized that the addition of NTX before polymerization may drive the adequate spatial arrangement of the monomers for a more efficient formation of ad hoc artificial receptors (imprinted hydrogels). Both imprinted and non-imprinted hydrogels were carefully washed and then loaded with NTX by soaking in aqueous solutions. Feasibility of carrying out loading and sterilization processes in one step was investigated. Since there is not a standardized release test for ocular solid or semisolid formulations, the diversity of setups and conditions reported in literature (some far from mimicking cornea environment) make comparisons and predictions difficult [29]. Thus, to gain an insight into the effect of the tests conditions on the release profiles, NTX release from the hydrogels was recorded using both a conventional test in bulk medium and also a microfluidic device mimicking lachrymal fluid turn over [30] and the information obtained compared to elucidate whether correlations among both methods could be established. Finally, after confirming biocompatibility, most promising formulations were also evaluated regarding corneal accumulation and permeability.
2. Experimental
2.1. Materials
Naltrexone hydrochloride (NTX), ethylene glycol dimethacrylate (EGDMA) and dichlorodimethylsilane were from Sigma-Aldrich (Steinheim, Germany); 2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AAc) were from Merk (Dramstad, Germany); benzyl methacrylate (BzMA) from Polysciences Inc. (Warrington, UK) and 2,2′-azobis(2-methylisopropionitrile) (AIBN) from Across (New Jersey, USA). WST-1 cell proliferation reagent was from Roche (Mannheim, Germany); phosphate buffer saline (PBS), MEM Alpha and fetal bovine serum (FBS) were from Sigma-Aldrich (Saint Louis, USA); penicillin/streptomycin (10,000 U/mL and 10,000 μg/mL), l-glutamine (200 mM) and 0.25% trypsin-EDTA were from Gibco (Paisley, UK). Water was purified using reverse osmosis (resistivity > 18 MΩ·cm, MilliQ, Millipore® Spain). Simulated lachrymal fluid (SLF) was prepared with the following composition: 6.78 g/L NaCl from Scharlau (Barcelona, Spain), 2.18 g/L NaHCO3 from Panreac (Barcelona, Spain), 1.38 g/L KCl, and 0.084 g/L CaCl2·2H2O from Merck (Dramstadt, Germany) with pH 7.8. Carbonate buffer pH 7.2 was prepared mixing buffer solution A (100 mL): 1.24 g NaCl from Scharlau (Barcelona, Spain), 0.071 g KCl from Merck (Dramstadt, Germany), 0.02 g NaH2PO4·H2O from Merk (Dramstadt, Germany) and 0.49 g NaHCO3 from Panreac (Barcelona, Spain); and buffer solution B (100 mL): 0.023 g CaCl2 from Merck (Dramstadt, Germany) and 0.031 g MgCl2·6H2O from Scharlau (Barcelona, Spain).
2.8. HET-CAM test
The Hen's Egg Test on Chorio-Allantoic Membrane (HET-CAM) assay is a validated alternative to animal testing of ocular irritancy [34]. The HET-CAM test was carried out using fertilized hen's eggs (50–60 g; Coren, Spain) incubated at 37 °C and 60% RH during 7 days [34]. A rotator saw (Dremel 300, Breda, The Netherlands) was used to make a circular cut of 1 cm in diameter on the wider extreme (where the air chamber is placed), on the eighth day, to remove the eggshell. The inner membrane was wet with 0.9% NaCl for 30 min and then carefully removed to expose the chorioallantoic membrane (CAM). NTX aqueous solution (0.3 mg/mL, 300 μL) and hydrated drug-loaded discs (imprinted and non-imprinted in duplicate) were placed on the CAM. 0.9% NaCl and 0.1 N NaOH solutions (300 μL) were used in triplicate as negative and positive controls, respectively. The vessels of CAM were observed during 5 min, under white light, for haemorrhage, vascular lysis or coagulation. The irritation score (IS) was calculated as previously reported [20,35].
2.9. Cytocompatibility assay
Human mesenchymal stem cells derived from bone marrow (hMSC, ATCC-PCS-500-012™) were cultured in 175 cm2 culture flask with MEM Alpha supplemented with fetal bovine serum heat inactivated (FBS, 10%), and antibiotics (penicillin/streptomycin 10,000 Units/mL and 10,000 μg/mL respectively, 1%). hMSCs were harvested, at approximately 90% of confluence, with 0.25% trypsin-EDTA. Cells were seeded (10,000 cells/well) in 24 wells plate and kept at 37 °C, 5% CO2 and 95% HR for 12 h allowing cell adhesion.
Pieces of NTX-loaded and non-loaded imprinted hydrogels bearing AAc and BzMA (19.6 mm2) were placed in the 24 wells plate (10,000 cells/well) the plate was kept at 37 °C, 5% CO2 and 95% HR. Solution of NTX (50 μM) in MEM Alpha was used as control. After 72 h, WST-1 cell proliferation assay was carried out following manufacturer instructions. Briefly, the medium was replaced by MEM Alpha (0.5 mL, with no supplements), and WST-1 cell proliferation reagent (50 μL; Roche, Mannheim, Germany) was added. Plates were incubated at 37 °C, 5% CO2 and 95% HR for 1 h and measured spectrophotometrically at 450 nm in a Bio-Rad plate reader 680 (California, USA).
2.10. Bovine corneal permeability test
Fresh bovine eyeballs were collected from the local slaughterhouse and transported following BCOP protocol alternative to in vivo testing [36]. Eyes were carried completely immersed in PBS with antibiotics 1% (100 IU/mL penicillin, 100 μg/mL streptomycin) in an ice bath. Once arrived, corneas were excised with 2–3 mm of surrounding sclera, rinsed with PBS and mounted in vertical diffusion (Franz) cells. Carbonate buffer pH 7.2 (6 mL) was used to fill receptor chamber and a small stir bar was also incorporated. The cornea was placed on the receptor chamber (maintained at 37 °C) and the donor chamber was fixed and filled also with carbonate buffer pH 7.2 (0.785 cm2 area available for permeation). After 1 h equilibration, the buffer in the donor chamber was removed and the corneas were exposed to NTX-loaded discs (C2 and D2 discs with 1 mL of 0.9% NaCl) or to control drug solution (350 μg/mL NTX, 2 mL). All experiments were carried out in triplicate. The donor chamber was covered with parafilm to prevent evaporation. Samples (1 mL) were taken from the receptor chamber at 1, 2, 3, 4, 5 and 6 h, replacing with the same volume of bicarbonate buffer each time and taking care of removing air bubbles from the diffusion cell. NTX in the receptor medium was quantified by means of an HPLC equipment (Waters 717 Autosampler, Waters 600 Controller, 996 Photodiode Array Detector) fitted with a C18 column (Waters Symmetry C18 5 μm; 3.9 × 150 mm) and Empower2 software. Mobile phase consisted of acetonitrile: 10 mM ammonium acetate buffer (40:60 v/v, pH adjusted to 5.6 with acetic acid) at 0.5 mL/min and 30 °C [37]. The injection volume was 50 μL, and naltrexone was quantified at 220 nm (retention time 3.08 min). Calibration was carried out with NTX standard solutions (1–10 μg/mL) in water (filtered through 0.2 μm, 13 mm GMP Minispike filters).
After 6 h of test, the formulations were removed from the donor chambers and the corneas were rinsed at least three times with PBS. Then, the corneas were placed in tubes with 2 mL acetonitrile for 24 h. The amounts of NTX extracted from the cornea were measured by HPLC as explained above.
2.11. Statistical analysis
The effects of sterilization process on NTX loading, and of hydrogels with and without NTX on NTX diffusion coefficients, hMSCs viability and cornea permeability were analyzed using ANOVA and multiple range test (Statgraphics Centurion XVI 1.15, StatPoint Technologies Inc., Warrenton VA).
3. Results and discussion
3.1. Hydrogel synthesis
Naltrexone hydrochloride (NTX) is a white powder soluble in water (up to 100 mg/mL) with a peak of absorbance at 281 nm (Fig. S1, Supporting information). The UV/Vis quantification method was validated regarding linearity, accuracy and precision in water, SLF and 0.9% NaCl medium. NTX is a weak acid that at 32 °C (~ocular surface temperature) has a pKa of 8.20 due to the dissociation of the proton on aliphatic nitrogen and a pKa of 9.63 associated to the dissociation of the phenolic proton [38]. Thus, it is expected that NTX can establish ionic interactions through its positively charged aliphatic nitrogen with the negatively charged AAc. Also, hydrogen bonding may occur between AAc and the hydroxyl and carbonyl groups of NTX. It is known that in the pharmacological receptor (MOR) hydrophobic interactions contribute to stabilize ligands binding [28], and hence BzMA was chosen as an additional functional monomer (Fig. 1).
NTX easily dissolved in the monomers mixture up to 10 mM. Higher concentrations were not tested since that amount of NTX should already provide therapeutic amounts to hydrogel pieces of dimensions in the range typical of CLs. After polymerization, hydrogel sheets were immersed in boiling water (500 mL) and the amount of NTX removed was quantified spectrophotometrically at 281 nm. Boiling is typically used to clean soft CLs after fabrication, and thus both imprinted and non-imprinted hydrogels underwent the same cleaning process. Imprinted hydrogel sheets were polymerized in the presence of 10 mg of NTX (~3.3 mg/g) and nearly the whole amount of template drug was removed during boiling (Fig. 2). Differences in the UV–Vis spectra of washing medium of imprinted and non-imprinted discs were clearly observed despite the large volume of water used for boiling. Interference of residual monomers in the peak absorbance of the drug was minor (Fig. S2, Supporting information). Subsequently, the discs were immersed in SLF pH 7.8 to complete NTX removal before loading assay (Fig. 2). Salts in the SLF medium participated in ionic competition for AAc monomers and thus triggered the release of residual NTX from the polymer network. All hydrogels were prepared in triplicate, and reproducible behavior was observed.
3.2. Hydrogel characterization
Swelling degrees in water and SLF of both imprinted and non-imprinted discs were in the 46–57% range (Fig. S3, Supporting information), which is typical of HEMA hydrogels. Thus, functionalization with small proportions of AAc and BzMA did not alter water uptake. Regarding light transparency, all swollen discs showed transmittance above 85% in the visible range. A decrease in transmittance below 300 nm was recorded for NTX-loaded hydrogels (Fig. 3). This means that NTX loading is not expected to disturb a clear vision but even could protect the eye against UV radiation.
3.3. Naltrexone loading
After the drug removal step (applied to both imprinted and non-imprinted hydrogels), dried discs were immersed in a NTX aq. solution (0.3 mg/mL) to quantify the drug loading ability (Fig. 4a). Non-imprinted hydrogels without functional monomers or with only BzMA (A1 and B1) did not show any affinity for NTX. Differently, non-imprinted hydrogels with AAc could load 8.06 ± 0.27 m/g and 7.28 ± 0.41 mg/g for C1 and D1 (codes as in Table 1) respectively. The FMF values indicated that the functional monomer AAc increases the amount of NTX loaded (CFMF = 41.18) while BzMA has no influence in NTX uptake (BFMF = 0.26). The combination of AAc and BzMA led to a FMF similar to that of hydrogels with AAc (DFMF = 38.14) confirming the minor role of BzMA. Besides, imprinted hydrogels were able to increase the amount of NTX loaded (up to 10.41 ± 0.40 mg/g and 10.83 ± 0.38 mg/g for C2 and D2 respectively), with KN/W of 34.4 for C2 and 35.9 for D2. Calculated imprinting factors (CIF = 1.31 and DIF = 1.46) indicated that hydrogels synthesized with functional monomers in presence of the drug can uptake more amount of drug due to specific cavities formation inside the network. Interestingly D2 hydrogels bearing both functional monomers, although loaded NTX in a similar amount as C2 hydrogels, had a few larger imprinting factor, which means that BzMA when correctly oriented in the imprinted cavity plays a role in the loading, increasing drug affinity for the binding regions.
From a manufacturing point of view, drug loading may involve an additional, time-consuming step in the processing of drug-eluting CLs. To minimize the impact of this step, we then evaluated the feasibility of carrying out both drug loading and final sterilization in one step. First, we verified that NTX aqueous solution can withstand autoclaving by recording the UV–Vis spectrum and the drug concentration before and after sterilization (Fig. S1, Supporting information) and no significant differences were observed. Hydrogels that underwent autoclaving in the NTX solution showed similar loading (Fig. 4b) than those loaded by soaking at room temperature (Fig. 4a) although a slight increase in the amounts loaded was recorded. For comparative purposes the hydrogels autoclaved in the NTX solution were allowed to stabilize at room temperature for 24 h (i.e., under the same conditions as the non-autoclaved hydrogels). After this time, the amount of NTX loaded by autoclaved hydrogels was 8.46 ± 0.72 mg/g for C1, 8.96 ± 1.1 mg/g for D1, 11.03 ± 0.76 mg/g for C2 and 9.25 ± 0.12 mg/g for D2.
3.4. Effect of autoclaving on NTX-loaded discs
Batches of imprinted and non-imprinted discs were loaded at room temperature (as explained above) and then transferred to vials containing fresh NTX storage solution (0.3 mg/mL, 3 mL) and autoclaved (121 °C, 20 min). The purpose of this step was to elucidate whether a subsequent sterilization process may alter the amounts loaded, either by promoting further uptake or by triggering discharge. After autoclaving, samples were stored at room temperature during 2 days. Non-imprinted BzMA/AAc discs (D1) showed a minor decrease in the amount of NTX loaded after autoclaving, but it was reloaded during storage (Fig. S4a, Supporting information). Imprinted discs prepared with BzMA/AAc (D2) were able to uptake a little more amount of NTX (1.71 mg/g; Fig. S4b, Supporting information) in addition to that previously loaded. Overall, the discs retained the initial amount of NTX with minor changes during sterilization and storage.
3.5. Naltrexone release
The lack of standardized methods to evaluate drug release from ocular solid formulations, and particularly CLs, makes comparison among published data difficult. In this regard, two main setups have been identified as the most suitable ones in terms of comparison of the ability of the CLs to provide sustained release and to predict in vivo behavior. These two setups consist in either agitated sink conditions or microfluidic flow devices [11,29,30,39]. For comparison purposes, release from NTX-loaded discs was investigated using both setups. Discs that had been loaded with NTX at room temperature were rinsed with water and the excess of solution on their surface carefully wiped. Infinite sink conditions were achieved using 3 mL of 0.9% NaCl under magnetic stirring (Fig. 5). NTX was sustainably released for 24–48 h; the discs providing almost 100% drug released without showing plateaus of pseudo-equilibria [29]. Discs functionalized with AAc released higher amounts of drug to the medium than those contained in experimental eye drops (10−5 M) [6] already from the first half an hour. Imprinted discs C2 and D2 released 12.11 ± 0.33 and 13.16 ± 0.11 mg/g, respectively, and non-imprinted discs C1 and D1 released 9.90 ± 0.14 and 7.17 ± 0.05 mg/g, at day 8, in good agreement with the total amounts loaded. The well agitated conditions (200 rpm) prevented that a boundary layer was formed between the disc and the surrounding solvent, which could lead to false slow release. Thus, even under agitated sink conditions, which are prone to trigger a rapid discharge of the CLs, the NTX-loaded functionalized discs could cover one-whole day treatment.
3.6. HET-CAM and cytocompatibility tests
Potential ocular irritation effects of NTX-loaded discs and NTX were evaluated on the chorioallantoic membrane (CAM) of fertilized hen eggs. Neither the loaded discs nor NTX aqueous solution (300 μL, 0.3 mg/mL) directly placed on the CAM induced haemorrhage, lysis or coagulation, and behaved as the negative control (0.9% NaCl) (Fig. 7), which is in good agreement with the good ocular tolerance reported for NTX eye drops [2]. Cytocompatibility tests with hMSCs confirmed the high biocompatibility of hydrogels bearing AAc and BzMA after 72 h direct contact (Fig. S6, Supporting information); all hydrogels were as cytocompatible as the D2 group.
3.7. Bovine corneal permeability
Bovine corneal permeability test was carried out by monitoring the amount of NTX that diffused from the CLs (C2 discs loaded with 835.97 ± 33.02 μg, and D2 discs loaded with 866.92 ± 38.21 μg) towards the cornea and the receptor medium mimicking the aqueous humour. As a control, a concentrated solution of NTX (350 μg/mL, 2 mL) in 0.9% NaCl was used; the total amount of drug supplied to the donor compartment (700 μg) being slightly lower to the total amount of drug contained in the CLs.
After 6-h test, NTX concentration in the 0.9% NaCl solution (1 mL) of the donor chamber was 293.82 μg/mL for C2 and 295.93 μg/mL for D2 discs. In the case of the control, the corneas were exposed to a high NTX concentration since the very first minute; after 6 h test the NTX decreased to 306.56 μg/mL. Nevertheless, the amount of NTX in the receptor chamber was quite low and only quantifiable after 3 h (Fig. S7, Supporting information). This means that NTX mainly accumulated into the cornea and only a very small portion of the drug molecules (<1%) reached the receptor compartment that mimicked the aqueous humour.
Amounts accumulated in cornea were 41.23 ± 5.77 μg/cm2 for C2 and 37.66 ± 6.63 μg/cm2 for D2 discs (Fig. 8). These values were not statistically different from those recorded for the NTX solution (32.37 ± 3.91 μg/cm2) in spite that the hydrogel discs sustainedly released the drug.
4. Conclusion
Incorporation of AAc to HEMA network increases affinity for NTX through weak interactions with the aliphatic nitrogen, hydroxyl and carbonyl groups of NTX. The presence of the drug during polymerization facilitates monomers arrangement creating specific cavities that contribute to increase even more NTX affinity. Although OGFr includes hydrophobic interaction with its ligands, BzMA only plays a minor role in the loading of imprinted networks. Swelling degree, oxygen permeability and light transmission of functionalized hydrogels are in the common range for CLs. Besides, no potential ocular irritation is observed on the CAM neither cytotoxicity in hMSCs monolayer. Relevantly from the fabrication point of view, loading and sterilization can take place either simultaneously or in separate steps with minor changes in total amount loaded. Imprinted hydrogels are able to control NTX release for at least 2 days in well-agitated bulk medium and for a more prolonged period in physiological-mimicking dynamic conditions, maintaining therapeutic concentrations in the lachrymal fluid until day 3. Despite the sustained release, the NTX-loaded hydrogels allow relevant amounts of drug accumulate in the cornea in the first 6 h of application. Thus, hydrogels containing AAc as functional monomer may be suitable for preparing NTX-eluting CLs containing therapeutic amounts of drug. The effects of a small change in thickness and curvature of CLs on NTX release rate are expected to be minor but would require further investigation.
