Atropine-eluting silicone contact lenses for myopia control

Preparation of atropine-loaded implants

Atropine implants were prepared by UV-excited radical polymerization using an atropine mixture containing AS, a monomer mixture, and a photo initiator. The photoinitiator solution (1 ml) was prepared with Ir2959 (100 mg) and 50% alcohol (1 ml). The monomer mixture (1 ml) consists of HEMA (900 μl), NVP (50 μl), TRIS (40 μl) and EGDMA (10 μl). Atropine mixture (1 ml) was composed of AS (13 mg), monomer mixture (900 μl) and photoinitiator solution (100 μl). Subsequently, the prepared atropine mixture was vortexed for 5 min to mix well and sonicated at 40 W for 15 min to remove any trapped air bubbles. A total of 0.4 μl (5 μg), 1.0 μl (13 μg), and 2 μl (26 μg) of the atropine mixture were respectively pipetted into homemade polypropylene molds. The mold included a silicone gasket (0.1 mm thick) which was drilled 3 × 2 mm. These molds were then shifted into a UV transilluminator (Lumen Dynamics S2000-XLA, Canada), and the atropine implant was cured for 2 min at a wavelength of 365 nm (500 mW/cm² intensity). The dry implants were kept in airtight glass vials at room temperature, coded as implant-1 (about 50 μl 0.01% AS, containing 5 μg AS), implant-2 (about 50 μl 0.025% AS, containing 13 μg AS), and implant-3 (about 50 μl 0.05% AS, containing 26 μg AS), according to the administrations in clinic.

Preparation of contact lens encapsulated with atropine-loaded implant

Atropine-eluting contact lens was synthesized by implantation technology and free radical polymerization method using a polypropylene lens mold (14 mm outer diameter and 8.5 mm base curve).^28,29 Before this, A liquid monomer mixture (1 ml) was prepared with monomer mixture (900 μl) and photoinitiator solution (100 μl). And then, the atropine implant (implant-1, implant-2, or implant-3) was placed in the concavity of the female mold, keeping it at a distance of at least 2 mm (radial) from the center. The 50 μl volume of the liquid monomer mixture was added to the female mold, and the male mold was inserted into the female carefully. The assembling mold of male and female was exposed to ultraviolet lamp curing for 5 min at 365 nm (500 mW/cm² intensity).

The synthetic atropine-lens was gently removed with a tweezer before being sterilized by a UV lamp (EC-TL01B, Yichenpai, Foshan, China) at 253.7 nm for 2 h and placed in an extraction solution containing 0.02% w/v atropine at 50°C for 30 min to extract away the unreacted monomers. The different inserts (implant-1, implant-2, and implant-3) were embedded in CLs to form drug-lenses with the drug concentration of 5 μg, 13 μg and 26 μg, respectively, coded as CLs-1, CLs-2, and CLs-3. The CLs without any insert were coded as blank contact lenses (BCLs). These CLs were used for measuring the drug release profile.

As for in vivo tests, the CLs were synthesized as above, and sterilized by a UV at 253.7 nm for 2 h in a dry state, followed by immersing in sterile water for 10 min at room temperature. The procedure of fabrication of atropine implant and the atropine-eluting contact lens was schematically drawn in Figure 2(a) and (b).

Swelling and water content

The dry CLs were carefully weighed (stated as W_D), and then put in deionized water for hydration at 37°C. These wet CLs were taken out at different predetermined time points and weighed (stated as W_W) after the free water on the lens surface was blotted by filter paper. The swelling (%) was calculated as follows:

The water content (%) was calculated as follows:

Light transmittance

The whole transmittance (%) of drug-loaded CLs (CLs-1, CLs-2, and CLs-3) and BCLs was scanned from 350 to 800 nm using the UV-vis spectrophotometer. The transmittance was analyzed separately at 600 nm (standard color light wave) for research and comparison. ²⁹ The experiment was conducted three times to attain an average.

In vitro release property

For in vitro release investigation, the CLs were put in 24-well plates with 1 ml PBS in each well, which was shaken at 60 r/min in an incubator at room temperature. The 1 ml aliquots of the release media were regularly removed for drug measurement and replaced with the same volume of the fresh PBS. The amount of atropine was measured at 205 nm using the UV-vis spectrophotometer. Three trials were reported. The cumulative release (μg) and release rate (μg/h) of atropine were plotted, and the drug release rate was determined as followed formula:

Cytotoxicity

RCF were used to evaluate the Cytotoxicity of drugs-lenses by Cell Counting Kit-8 (CCK-8) assay. RCF were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS and grown in an incubator with a humidified atmosphere of 5% CO₂ at 37°C.

The CLs samples (n = 4) were cut into small segments and placed in 2 ml of DMEM for 24 h at 37°C to attain the CLs medium. RCF were seeded in a 96 well plate with a density of 5 × 10³ cells per well for 24 h, then the old medium was swapped out, and 150 μl new media (extract) was administered neat to the cells for 24 h. Then the media was removed, followed by addition of 110 μl fresh medium containing 10 μl of CCK-8 solution to each well and fostered for more 2 h. Subsequently, the absorbance of the cell media was determined at 450 nm using a microplate reader (Dragon Wellscan MK-3, Labsystems, Finland). The cell viability was determined as followed formula:

To further visually analyze drug-lenses' cytocompatibility, the lenses were placed in the 24-well plate with 400 μl of DMEM in each well for hydration. And then, 3 × 10⁴ cells were seeded in each concavity of the CLs. These cells were cultured for 24 h in the incubator with 5% CO₂ at 37°C. The growth state of these cells on CLs was performed and visualized under a light microscope (CKX41SF, Olympus, Japan).

Ocular irritation

New Zealand rabbits were used for the ocular irritation evaluation of the drugs, and drug lenses, which were divided into the AS eye drops groups (0, 0.01, 0.025 and 0.05%, AS/saline, w/w) and the CLs groups (BCLs, CLs-1, CLs-2, and CLs-3). The right eyes of rabbits wore the contact lens containing CLs for eight hours daily, while the lefts were treated with AS eye drops as control. Drugs were given once daily for up to 7 days consecutively. Eight hours after daily administration, the rabbit eyes were examined under a slit lamp (SLM1-2ER, Kang Huaruiming, China) to check for any apparent discomfort, conjunctival hyperemia, swelling, or discharge. Additionally, the pupil diameter (mean of transverse and longitudinal diameters of pupils) was measured with a ruler to further assess the potential mydriasis side effects of the atropine.

After receiving therapy for seven days, the rabbits were executed, and their eyes (eye drops and the lenses groups) were removed and fixed in optimum cutting temperature compound (OCT) for the frozen section. The sections of the eyes were cut using a freeing microtome (Cryostar NX50, Thermo, Shanghai, China) and stained by hematoxylin and eosin (H&E). Corneal and iris sections were evaluated using the light microscopy.

Anti-myopia effect

4-week-old female guinea pigs were applied for the anti-myopia study, housing under standardized conditions with 12 h cycles of light and darkness. Self-made facemasks with white balloons were used to induce monocular form-deprivation myopia (FDM) in guinea pigs. Meanwhile, the pharmaceutical preparations (CLs-1 or 0.01% AS drops) were used to treat the induced eyes. CLs-1 lenses were drilled and cut into the semi-circular tables with a diameter of 4 mm, in which the complete AS implant was included, about 5 μg atropine in each implant. CLs-1 was worn on the myopic eye. Animals were divided randomly into four groups (n = 4): positive control group (normal group), form-deprivation myopia control group (FDM group), form-deprivation + 0.01% AS group (0.01% AS group), and form-deprivation + CLs-1 group (CLs-1 group). The procedure lasted two weeks to complete the induction and therapy process.

The refraction was measured with a streak retinoscope (YZ24, Suzhou Liuliu Technology Co., Ltd, China), and the ocular axial length was obtained with an electronic vernier caliper. Besides, to ensure that the IOP was within normal limits during the experiment, intraocular pressure (IOP) was measured by using finger measurement before and after treatment. Following the experiment, these animals were slain, and the eyeballs were removed for ocular axial measurement and histology (frozen-section). The sections were stained with hematoxylin-eosin (H&E). Corneal and scleral sections were evaluated under optical microscopy.

Swelling and water content

The swelling behavior of CLs in water was measured. As shown in Figure 3(a), the prepared CLs reached an equilibrium hydration state and remained stable within 1 h, with a burst uptake at initial 10 min. It indicates that the CLs can be stored in dry state and worn at any time due to their rapid swelling properties.

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Figure 3.

The characterization of the contact lenses. (a) Curve of swelling equilibrium. (b) The water content after complete hydration. (c) The light transmittance in the visible range (380–780 nm). (d) The light transmittance at 600 nm as a function of lenses with different AS loading. ** indicates significant difference (p < 0.01) with BCLs. *** indicates very significant difference (p < 0.001) with BCLs.

As literature ¹⁴ reported, atropine tends to degrade in an aqueous solution, particularly at high temperatures like ≥ 40°C and long-term storage. For example, it will hydrolyze for 8.1867% after 10-days storage. Contrarily, it degrades more slowly if it was stored in a dry state. Therefore, we set up a novel method to wrap the atropine-loaded implant into contact lens and keep it at dry state for storage. The lens will swell quickly in solution and the drug releases upon usage.

The water content of silicone CLs has a direct relationship with their in situ monomer performance, while the oxygen permeability of the lens is mainly related to the silicon channel. The water content (%) and the physical diagram of the lenses after complete hydration were shown in Figure 3(b). The water content of CLs-1 (34.47 ± 0.78%), CLs-2 (34.35 ± 0.67%), and CLs-3 (34.77 ± 1%) did not fluctuate significantly (p > 0.05), compared with that of BCLs (33.99 ± 0.51%). It indicates that the water content of CLs with or without drug encapsulation is to be similar. This may be due to the fact that the addition of a small amount of drug is not enough to affect the overall water content of the lenses. Additionally, the physical appearance is also almost unaltered after the drug loading (Figure 3(b)). As we know, water content is one of the important parameters of CLs, generally between 30% and 80%. The lens can be divided into low water content (< 50%) and highwater content (> 50%). ³⁰ Realizing that the contact lens with high water content tends to absorb more tears, causing eye discomfort or the disease of dry eye, we chose to prepare silicone hydrogel lens with low water content.^31,32 At the same time, given the inverse relationship between oxygen permeability and water content of silicone hydrogel lenses, many commercial CLs with lower moisture content are being produced, such as Acuvue Oasys with 38% water content, Air Optix with 33% water content, and O₂ Optix with 33% water content. ³³ In addition, a non-ionic monomer (NVP) was added to the prepared lens to resist tear protein precipitation and to keep the eyes comfortable.^34,35 More importantly, lenses with low water content can effectively delay the release of hydrophilic drugs compared with those containing high water content. ³⁶ Thus, we designed the low water content lens, which may reduce the deposition of tear protein to make it more comfortable and can be worn longer time, especially for patients with dry eye symptom. The wettability of the lens surface with modification techniques will be explored to make the lens more comfortable during wearing in our future research.

Light transmittance

In order to ensure clear vision, the CLs’ light transmittance should not alter seriously after drug loading. The transmittance of our silicone CLs was characterized by measuring the transmission spectrum in the range of 350–800 nm. As shown in Figure 3(c), the transmittance (%) of all the CLs is greater than 80% in the visible light range (380–780 nm). At the wavelength of 600 nm, the transmittance (%T) values were measured for comparison. The result revealed the optical transmittance for CLs-1 (%T = 95.44 ± 0.36, p = 0.0055), CLs-2 (%T = 93.51 ± 0.54, p = 0.001) and CLs-3 (%T = 90.37 ± 0.9, p = 0.0005), comparing with that of the BCLs, (Figure 3(d)). That is, the drug loaded in the lens reduces the transmittance to a certain extent. That’s why we design the drug-implant and locate it at the boundary of the lenses, in order to prevent the visual clarity deterioration during the wearing.

In vitro release property

The mechanism of drug release involves the diffusion of drug molecules from the matrix of the inner implant and then release from the cross-linked molecules in the outer layer of the contact lens. AS is soluble in water, and its diffusion properties are highly related to the swelling of the silicone matrix. There have been reported that atropine molecules diffused from the silicon domain to water channels and then was out of the silicone hydrogel material, owing to the silicon domain as the major limiting factor of water-soluble drugs. ²²

In this work, the releasing properties of AS from atropine-lenses with different drug loading (post monomer extraction) were shown in Figure 4. It exhibited an “initial burst-continue-slow” released profile of AS from atropine-lenses. As shown in Figure 4(a) and (c), ∼50–60% AS released within the first hour, and ∼30–40% release continued for the following 12 h. Moreover, the cumulative percentage of the drug released from atropine-lenses at the time points of 0.5, 1, 2, 4, 8, and 12 h was also calculated in detail (Supplementary Table S1). The remaining drugs of drug-lenses slowly released for the next 120 h, except for the drug of CLs-1 which released for the next 72 h (Figure 4(b)). After that, the drug release curve basically reached a steady state. The cumulative release amounts of AS from CLs-1, CLs-3, and CLs-3 were respectively calculated to be 5.04 μg, 14.86 μg, and 25.35 μg, which indicated that the drug loading of the atropine-lenses reached the target value, on the other hand.

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Figure 4.

In vitro drug release from the contact lenses with different drug loading. (a) Cumulative drug release as a function of process time during short term, 12 h. (b) The total cumulative drug release during long term, 240 h. (c) The drug release rate as a function of time, calculated from the amount of drug released per unit time.

With all three CLs, CLs-1∼CLs-3, the drug release rate and release amounts of atropine-lenses increased significantly with the increase of drug loading, gradually approaching the maximum with the release time. All these lenses displayed burst release at the first hour and delayed release after then, during the test interval of 240 h. A burst release of the drugs may from the aqueous channels, followed by a prolonged release from the silicone structure. This release characteristic is similar to previous studies. ²² The burst release is not benefit for the bioavailability of the loaded drug. Thus, we are polishing the fabricating technology and will report in future.

Cytotoxicity

The cytocompatibility of drug-lenses were tested with RCF as the model cell, using CCK-8 assay. The results showed that the cell viability (%) of samples BCLs, CLs-1, CLs-2, and CLs-3 was 99.7 ± 2.89%, 98.03 ± 3.26%, 100.62 ± 3.38%, and 100.9 ± 3.67%, respectively (Figure 5). There is no significant difference between drug-loading and blank control lens (p > 0.05). The morphology and the growth status of RCF cells cultured on these lenses did not display different significantly, which is consistent with the quantitative result of CCK-8 assay (Supplementary Figure S1). These results suggest that our drug-lenses were cytocompatible and safe for ocular treatments. The biocompatibility evaluation was performed with rabbit eyes as the models as follows.

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Figure 5.

Cell viability of the contact lenses with different drug loading and the blank.

Ocular irritation

A study of ocular irritation was conducted to evaluate the potential side effects of atropine-lenses with rabbit eyes as the model, using Draize test. ³⁷ After daily administration for seven consecutive days at all doses from 0.01% to 0.05% AS, the conjunctival hyperemia or corneal edema was not observed in all eyes (Figure 6(a)). However, the pupil diameter of rabbit eyes in the AS and CLs groups increased significantly as the dose increased from 5 to 25 μg, comparing with the control eyes (Figure 6(a) and (b)). And, all the eyes treated with AS drops displayed more enlarged pupils than those with CLs correspondingly. For CLs group, the pupil diameter of CLs-1 treated eyes (7.58 ± 0.24 mm) was not significantly diverse from that of control (6.83 ± 0.31 mm, p > 0.05), while the diameters of CLs-2 (8.08 ± 0.31 mm, p < 0.05) and CLs-3 (8.75 ± 0.2 mm, p < 0.01) were significantly enlarged than that of BCLs. Large pupils will cause glare or/and blurred near vision. Thus, the treatment of CLs-1 was the safest and had the least adverse effects on pupillary response. That’s might be attributed to the sustained drug release of atropine-lenses.

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Figure 6.

Ocular irritation studies. As the references, the normal eyes were treated with saline (0%) and contact lenses without drug (BCLs). (a) Ocular observation. The rabbit’s eyes were treated with CLs and AS drops for seven consecutive days. (b) The pupil diameter after AS and CLs treated. (c) Histological analysis of cornea (HE staining). (d) Histological analysis of iris (HE staining). * indicates statistical difference (p < 0.05) between AS Drops and CLs groups. ** indicates significant difference (p < 0.01) between AS Drops and CLs groups.

The histopathology of eye tissues was examined. The results displayed that no inflammation was observed in the cornea and iris tissues, comparing with the normal eyes (Figure 6(c) and (d)). All eyes exhibited the normal histology structure, indicating that there was almost not irritation after the eyes were treated by AS drops or CLs. Thus, atropine-loaded lenses are safe and suitable for ocular treatment. Particularly, CLs-1 has the least side effects in terms of mydriasis, and would be applied to the study of anti-myopia efficiency.

Anti-myopia efficiency

Axial myopia, caused by excessive axial growth, is the most common in human eye myopia. Abnormal axial growth leads to changes in the scleral structure, including progressive scleral thinning, collagen fiber formation, and fibrous lamellar reconstruction disorder.^38–40 For myopic animal models, although rabbit eyes are suitable for lenses, they are rarely used in anti-myopia studies due to the difficulty of myopia induction, like only −1.00 D of myopia can be induced in 30 days. ⁴¹ Considering that guinea pigs, as one of the most common animal models for myopia, can be induced to have high myopia in a short time, so, in our case, guinea pigs were selected to construct the animal myopia models with FDM induced by the mask method. ⁴² The FDM eyes were treated with 0.01% AS drops or wore CLs-1 lenses that were cut to the proper size to wear and did not shed during wearing. The IOP of all animals’ eyes was within the normal range during treatment period. Compared with the FDM group (negative control), all FDM eyes treated with 0.01% AS drops or wearing CLs-1 lenses became relieved, somewhat, after 14-days treatment, smaller refractive error than that of FDM, though still bigger than that of normal eyes (Figure 7(a)). The diopter of −4.38 ± 0.41 D (p < 0.05) and −3.91 ± 0.36 D (p < 0.01), respectively, compared with −5.47 ± 0.36 D of the FDM negative control. Correspondingly, the ocular axis of 8.525 ± 0.079 mm (p < 0.05) and 8.425 ± 0.071 mm (p < 0.01), respectively, compared with 8.69 ± 0.083 mm of FDM eyes (Figure 7(b)). These findings demonstrate that CLs-1 lenses were superior to 0.01% AS drops in inhibiting the growth of diopter and axial length for myopia animal eyes.

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Figure 7.

In vivo tests against form deprivation myopia. The guinea pig eyes with FDM were treated by 0.01% AS drops or CLs-1 contact lenses for 14 days, with normal eyes as the positive control and FDM as the negative control. (a) The refraction of the eyes. (b) The axial length of the eyes. (c) Histopathology analysis (HE staining). Red arrow indicates the disordered collagen fiber of FDM sclera. “ΔA”, “ΔB”, “ΔC” and “ΔD” indicate sclera tissue widths. * indicates statistical difference (p < 0.05). ** indicates significant difference (p < 0.01). *** indicates very significant difference (p < 0.001).

Histopathological studies for the animal eye tissues were carried out. Figure 7(c) shows that no histological inflammation was observed in all corneas. There was no significant difference in scleral thickness between the all groups, similar value of ΔA, ΔB, ΔC and ΔD in Figure 7(c). However, the disorganized and broken collagen fibers displayed in the sclera tissue of FDM eyes (Figure 7(c), red arrow), while the orderly arranged collagen fibers in that of 0.01% AS and CLs-1 groups, which is close to the normal eyes. This indicates that AS was successfully loaded into the lens and played the same role as AS drops in inhibiting the growth of myopia. Although the therapeutic effects of Cls-1 and 0.01% AS on myopia did not exhibit a significant difference at the scleral fiber level, there were differences in the inhibition of refractive development and ocular axis growth, which indicated that atropine-lens was superior to eye drops in delaying myopic progression. This indicates that the sustained release of atropine-lenses is beneficial to improve the myopia control effect, which is expected to be applied in the prevention and control of myopia in children and adolescents with rapid myopia growth. However, the release properties of atropine in vivo are not known. The in vivo pharmacokinetic study of atropine might more intuitively reflect the continuous release effect of the drug-lens and will explain the reason for the drug-lens about the improved efficacy against myopia. This topic will be study in our future work.