Materials

Erbium chloride (ErCl3, 99.9%), Gadolinium chloride (GdCl3, 99.9%), Ytterbium chloride (YbCl3, 99.9%), Sodium chloride (NaCl), Ammonium fluoride (NH4F), Ethylene glycol, 2-(2-ethoxyethoxy) ethyl acrylate (EA), 2, 2’-Azobis(2-methylpropionitrile) (AIBN) were purchased from Aladdin (Shanghai, China). Polyethylene glycol 4000 (PEG4000) and ethylene glycol phenyl ether methacrylate (EGPEMA) were purchased from Macklin (Shanghai, China). Poly (ethylene glycol) diacrylate (PEGDA) was purchased from Sigma-Aldrich. All chemicals were of analytical grade and used following the manufacturers’ instructions. Cell culture medium DMEM/F12 (1:1), fetal bovine serum (FBS), trypsin, and penicillin–streptomycin were purchased from Gibco. Phosphate-buffered saline (PBS) was purchased from Boster Biotechnology. Cell counter kit-8 (CCK-8) and Calcein/PI Cell Viability/ Cytotoxicity Assay Kit were obtained from Beyotime Biotechnology. Most of the chemical reagents were used without further purification except polymerization monomers, which were purified by the inhibitor remover.

Materials synthesis and preparation

980 nm-excited UCNPs preparation: NaGdF4:Yb/Er UCNPs was synthesized by an improved one-step hydrothermal method [19, 20]. 2.4 mmol NaCl, 0.96 mmol GdCl3, 0.216 mmol YbCl3, 0.024 mmol ErCl3, and 5 mg PEG4000 were dissolved in 20 mL ethylene glycol, followed with 15 mL 6 mmol NH4F in ethylene glycol solution adding and stirred for 30 min. Then, the mixed solution was transferred to stainless-lined autoclave and reacted in an oven at 200 ℃ for 12 h. The product was collected by centrifugation (10,000 rpm, 10 min), washed with ethanol and ultrapure water three times and dried at 60 °C. Thus, the NaGdF4:Yb3+, Er3+ UCNPs were prepared.

PEEU Synthesis: The synthesis of PEEU optical lens materials were similar with the synthesis of PEE optical lens materials except that the UCNPs were added when the reaction monomers mixture were prepared [15]. Briefly, Pretreated EGPEMA and EA were mixed at a mass ratio of 7:3, and then added the crosslinker PEGDA (1 wt%), initiators AIBN (0.5 wt%) and a certain amount of UCNPs. Ultrasonication was carried out for 10 min to ensure a well-dispersion of the mixed solution. Next, the reaction mixture was magnetically stirred for 30 min and bubbled with a stream of nitrogen (N2) for 30 min to remove the oxygen (O2). The degassed reaction mixture was then injected into an optical lens mold and polymerized for 24 h at 65 ℃. Finally, the obtained materials were washed by alcohol and ultra-pure water sequentially. Thus, the PEEU optical lens materials were obtained. For different NIR light conversion efficiency assay, different UCNPs contents, including none (0 wt%), 0.2 wt% or 0.4 wt% UCNPs doped PEEU materials were prepared, and abbreviated as PEE (0 wt%), PEEU (0.2 wt%), PEEU (0.4 wt%).

Characterization process

Malvern Laser Particle Sizer (Malvern Instrument Ltd., Malvern, UK) measurement was used to measure the hydrated particle size and potential size of UCNPs. Fluorescence spectrophotometer (SpectraMax 190, USA) was used to test the fluorescence properties of UCNPs, using a 980 nm laser instead of a xenon lamp as the light source. The water contact angle and wettability were evaluated by contact angle analyzer (Data Physics Instrument GmbH, OCA25). Standard dumbbell-shaped specimens were used to test the mechanical properties, and the tensile test was carried out at the rate of 10 mm/min at 25 ℃ (CJinan Metis Test Technology, MT2503). The optical transmittances of the materials were measured by ultraviolet-visible spectrophotometer (UV-Vis) (Shimadzu, Japan) in the wavelength range of 400–800 nm with air as the reference. The refractive index of the materials was measured by Abbe refractometer (NAR-1 T, ATAGO CO., LTD, JAPAN). The lamp source used in all the experiments was NIR laser 980 nm (Lasever Inc, LSR980NL-3W-FC).

NIR light conversion effect evaluation

As mentioned above, different UCNPs contents doped PEEU optical lens materials, including PEE (0 wt%), PEEU (0.2 wt%), PEEU (0.4 wt%) were synthesized for different NIR light conversion efficiency assay. The different optical lens materials were placed in dark environment, and illuminated by the 980 nm NIR light at different intensities. Photographs were taken and recorded to evaluate the conversion effect of NIR light.

In vitro biocompatibility evaluation

The PEEU optical lens materials were made into intraocular lens disk (Φ = 6 mm) and soaked in 300 μL of PBS (pH = 7.4) for 24 h to obtain the leaching solution. Human lens epithelial cells (HLECs) and human corneal epithelial cells (HCECs) were cultured in DMEM/F12 (1:1) medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37 ℃, 5% CO2 humidified atmosphere. Cells were seeded in the 96 well tissue culture polystyrene plates at density of 8000 cells per well. After incubated for 24 h, 20 μL material leachate solutions were added and incubation for another 24 h. Then the cells were incubated with 10% CCK-8 working solution for 2 h, and the OD value was measured by a microplate reader to calculate cell viability. The fluorescent images of the cells were also taken after live/dead staining.

Scheme 1

Schematic diagram of the PEEU optical lens converts NIR light into visible light

Pupillary light reflex experiment

The animal experiments were carried out according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the experimental animal ethics committee of Wenzhou Medical University. Due to the similarity between the eye structure of rabbit and human, it is widely used in the study of ophthalmology and pathology [21]. Moreover, contrast sensitivity has now become a common indicator for assessing the human visual system’s ability to process spatial frequency information, and some studies have indicated that the rabbit’s contrast sensitivity is significantly lower than that of humans [22]. Therefore, the same light that has a significant stimulating effect on the rabbit’s eye will also have a significant stimulating effect on the human eye. Finally, in order to explore whether rabbits can “see” NIR light by PEEU optical lens, pupillary light reflex (PLR) experiments were carried out. Three New Zealand white rabbits with weight about 2.5–3 kg were used in this study. The PEE and PEEU (0.4 wt%) optical lenses were prepared for use. On the other hand, the rabbits were dark-adapted for 1 h. Then, illuminate the rabbit eye fitted with PEEU or PEE optical lenses with 980 nm NIR light, and the pupil responses were observed by eye images taken as well as the diameter and area of pupil calculation. The light intensity was 1 W/cm2, with a distance of around 15 cm, and the irradiation area was circular, concentrated in the center of the eye.

Subcutaneous implantation experiment

The animal experiments were carried out according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the experimental animal ethics committee of Wenzhou Medical University. Two New Zealand white rabbits with weight about 2.5–3 kg were selected in this study. The PEEU optical lens materials were sterilized by ethylene oxide disinfection. After anesthetizing, the rabbits’ side abdomens were shaved and disinfected with iodophor solution. Three small skin incisions were made and the materials were implanted. After subcutaneous implantation, the incisions were sutured. On the 7th day postoperatively, the rabbits were humanely sacrificed under anesthesia. The adjacent tissue around the implants were isolated, fixed in a paraformaldehyde solution, and stained with hematoxylin − eosin (H&E) for histological observation.

Statistical analysis

Three parallel samples were set in the experiments, and the results were expressed as mean ± standard deviation. One-way analysis of variance was adopted to compare the statistical difference between two groups. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) represent significant differences, and P > 0.05 represent no significance difference (ns).