Introduction

Multifunctional contact lenses can be synthesized by adding different additives to increase their value1. The incorporation of graphene (Gr) has been used to improve water retention in CLs2, which provides superior comfort and extended wearability3. Gr is often investigated as a nanofiller for polymer nanocomposites due to its exceptional mechanical4 and thermal properties5,6 and it can also be used in advanced sensors and electronic components, opening up new avenues for monitoring eye health and providing augmented reality experiences7,8. There are some prior works on CLs with integrated Gr, which predominantly involve attaching Gr grown through Chemical Vapor Deposition (CVD) onto CLs through molding2,9,10. Gr has been explored as an additive in hydrogel CLs as transparent electrodes11,12, sensors13, power generation14, EMI shielding2, drug delivery15,16, therapeutics3, Intra Ocular Pressure sensing (IOP)17, and barrier films2,18,19. Lee et al.2, developed a smart contact lens with Gr and investigated its capabilities for EMI shielding and dehydration protection. Yin et al.12, developed a soft transparent contact lens electrode with Gr for the conformal full-cornea recording of electroretinogram. However, the complexity of the procedure makes it difficult to manufacture in bulk. Also, the decrease in transparency upon increasing Gr layers limits the scope. Although, alternative methods are available for incorporating graphene into CLs, it is necessary to ensure an acceptable level of transparency.

In this work, we are incorporating Gr into commercial CLs through three different techniques. Breath in - Breath out technique (BIBO) previously explored for manufacturing colorblindness correcting gold and silver doped CLs20. The change in hydrogel volume in the protic and aprotic solvent is utilized to incorporate the Gr into CLs. This simple and cost-effective method could synthesize large amounts of functionalized CLs with acceptable transparency throughout the visible spectrum, in a short span. Secondly, we investigated the production of Gr-incorporated CLs through immersion in Gr ink. Even though the procedure is much simpler compared to the BIBO technique, the maximum loading of Gr is limited. Also, we investigated 3D printing graphene structures into commercial CLs using vat photopolymerization. However, due to the instability of Gr in hydrogel resin, the structures showed increased agglomeration and non-uniformity. Further optimization is necessary to stabilize the graphene in the hydrogel, such as functionalization, using suitable surfactants or coupling agents. For any contact lens to be ideal, it is primarily important for it to be antibacterial, biocompatible, and non-cytotoxic. The antibacterial activity prevents possible eye infections if the hands are dirty while wearing and removing the contact lenses. These are evaluated with antibacterial studies against Staphylococcus aureus and Pseudomonas aeruginosa, cell viability tests, and pro-inflammatory cytokine gene expression analysis. Predominantly pro-inflammatory cytokines interleukin-6 and interleukin-1 beta gene expression analysis was performed in this work21,22.

Materials and methods

Materials

Acetone, Perchloric acid (HClO4), Graphite powder, Sodium Deoxycholate, Hydroxyethyl methacrylate (HEMA), and Diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO) were purchased from Sigma Aldrich. The chemicals were used without further purification. 1-Day Acuvue Oasis (Senofilcon A) contact lenses were obtained from Acuvue, Johnson & Johnson. Minimum essential medium (MEM) from Gibco, Human dermal fibroblasts, neonatal (HDFn, C0045C) from Thermo Fisher, fetal bovine serum (FBS), trypan blue (Sigma), 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT), ethanol, Dulbecco’s phosphate-buffered saline (DPBS) from Gibco, HOT FIRE Poi EvaGreen qPCR Mix Plus (Solis Biodyne), high-capacity cDNA reverse transcription kit (Applied Biosystems), micPCR (biomolecular systems), Trizol (Invitrogen) and trypsin-ethylenediaminetetraacetic acid (EDTA). Methicillin Resistant S. Aureus (MRSA) 33,591 and P. aeruginosa 9027 were purchased from ATCC.

Preparation of graphene ink

Graphene ink is produced by expanding graphite flakes through microwave irradiation followed by a liquid phase exfoliation process. Initially, 4 g of graphite powder was intercalated with perchloric acid (HClO4) in the ratio of about 1:4 at 120 °C for 2 h. The intercalated graphite flakes were subjected to 1500 W microwave irradiation for 1 min to produce expanded graphite. The resulting expanded graphite was added to 100 ml of deionized water with 0.4 g of Sodium Deoxycholate (SDC) followed by high speed shear mixing at 3000 rpm for 2 h. The solution is centrifuged to separate exfoliated graphene and unexfoliated graphite flakes. The separated supernatant with the exfoliated graphene at a concentration of 20 mg/ml and density 1.01 g/cm3 is collected and labeled as graphene ink and used further.

Graphene incorporated lens synthesis

The breath in- breath out method was used to incorporate the Gr into the commercial CLs. The Acuvue lenses were soaked in 10 ml of acetone for 2 min, followed by a soak in 10 ml of Gr ink at a 1 mg/ml concentration. This process was repeated to ensure enough loading of the Gr into the contact lens’s gel matrix. As the contact lenses are placed in acetone (aprotic), the hydrogel expands and the pores get enlarged. These lenses were then placed in water based graphene ink (protic solvent) where the hydrogel starts to shrink. The diffusion moves the graphene into the contact lens and the acetone out when the lenses are placed in graphene ink and vice-versa when placed in acetone. The diffusion and the expansion-contraction cycles of the contact lens lead to the graphene being trapped within the contact lenses. After soaking, the lenses were washed with deionized water and sonicated for 10 min to remove any excess colloidal solution on the surface. The samples were stored in a contact lens solution at 25 °C for further analysis. The process is illustrated in Fig. 1a.

The commercial CLs were kept dipped in the Gr ink (20 mg/ml) in a 10 ml solution for 10, 20, 40, and 60 min to obtain Gr dipped CLs (Fig. 1b). In order to obtain 3D printed structures on commercial CLs, Gr-HEMA resin was prepared by adding graphene ink (2% w/w) to the resin containing HEMA as the precursor and TPO (2% w/w) as the photoinitiator. This resin was used in DLP-based vat photopolymerization (Wanhao D8). The commercial contact lens was carefully placed on the build plate followed by printer re-alignment to print the graphene structure onto it (Schematics represented in Fig. 1c). All the contact lenses were thoroughly cleaned and sonicated before further analysis to remove un-attached or loosely held graphene from the contact lenses.

Fig. 1
figure 1

a Schematics of the BIBO technique for Gr incorporated CLs preparation b Schematics of Gr dipped CLs c Schematics of 3D printing on contact lens

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Anti-Bacterial studies

The lens dipped in graphene using the BIBO method (9 cycles) was investigated with the same procedure as Liya et al. For measuring the anti-bacterial activity of the contact lenses, the bacterial strains Staphylococcus aureus and Pseudomonas aeruginosa were cultured at 37 °C for 24 h (1 ml of 1.0 × 106 cfu ml−1 in 10 ml LB broth) in a shaker. To eliminate any influence of the storage solution of commercial contact lenses on the attachment of bacteria, the lenses were stored in deionized water. Clear contact lenses and graphene lenses were used for the experiment to be compared. The contact lenses were rinsed with ethanol and cut into two. Each half was placed onto 12-well plate wells. The bacterial culture solution prepared was added to each well. After shaking for 15 min, the culture with the corresponding lens plate was incubated at 37 °C for 8 h.

Next, the lenses were taken out and washed carefully with PBS to remove any excess bacteria. Further, the lenses (with bacteria attachments) were fixed by immersing in 0.1 mM glutaraldehyde in PBS solution and stored at 4 °C overnight. The lenses removed from the solution were subjected to a set of dehydration steps with 10–100% ethanol solutions. Finally, the samples were mounted onto SEM stubs and were gold coated for image analysis.

MTT assay

The graphene lens prepared using the BIBO method for 9 cycles was further evaluated to determine the level of cytotoxicity, if any. An MTT assay kit from Abcam was employed. 1.8 × 105 ml−1 Neonatal human dermal fibroblasts (HDFn) cells were seeded and grown in MEM media supplemented with 20% FBS for 24 h at 37 °C and 5% CO2 in a 12-well tissue culture plate. Additionally, the cells were incubated with a media-prewashed graphene lens for 24 h. The control sample data was obtained from wells without a lens. A comparison with commercially available clear lenses was also performed. The assay was done in triplicates. The lens was removed and the MTT assay was performed following the kit protocol. Finally, the absorbance measured at 590 nm using a microplate reader (Tecan) was used to calculate the cell viability with the equation below:

$$Cell{\text{ }}viability{\text{ }}\% {\text{ }} = {\text{ }}\left( {Absorbance{\text{ }}of{\text{ }}cells{\text{ }}with{\text{ }}lens/Absorbance{\text{ }}of{\text{ }}cells{\text{ }}without{\text{ }}lens} \right)*100$$
(1)

Trypan blue assay

To determine the cell viability, HDFn cells were seeded onto 12 well plates at a density of 1.8 × 105 cells ml−1. Post 24 h of growth at 37 °C and 5% CO2, the coated lens was washed with serum-free media and incubated with the cells. The lens was removed after 24 h and cells were collected using 0.025% Trypsin (Gibco) to perform the Trypan blue (Sigma) assay. The % of live cells was obtained using Invitrogen countess 3FL. The data retrieved was statistically analyzed using one-way ANOVA. P values lower than 0.05 were considered significant.

Quantitative polymerase chain reaction (qPCR)

To investigate any inflammatory responses in the cells due to the graphene lens prepared, a real time PCR was employed to evaluate the expression levels of pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-1 beta (IL-1β). The pro-inflammatory cytokines, interleukin-1 beta, and interleukin-6 expression analysis were implemented and the sequence is provided in the Table 1. Three graphene lenses and clear lenses each were maintained in HDFn culture plate well (12 well plates) for 24 h and post incubation, the cells were lysed, collected, and pooled. Their RNA was isolated using Trizol. The trizol kit protocol was utilized for the same. The RNA conversion to cDNA was achieved using a High capacity cDNA reverse transcription kit following the manufacturer’s protocol. The primer sequences used are given in the table below. All data obtained was relative to the GAPDH housekeeping gene. The relative expression levels (2 − ΔΔCt) were plotted and statistical analysis was done. P < 0.05 results were considered to be significant21,23.

Table 1 Primer sequence used for gene expression studies.
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Characterization details

The transmission spectrum of both nanoparticles and contact lenses was obtained using a UV-Vis spectrophotometer USB 2500 + and analyzed using OceanView software. Tecnai TEM 200 kV was used to monitor Gr morphology. SEM imaging was conducted using FEI - Helios Nanolab 650 after gold coating the samples before the examination. FTIR analysis was done using Perkin Elmer - Spotlight 200 FTIR microscopy system. Raman Spectroscopy was done using Witec Alpha 300 RAS.

Results and discussion

The prepared graphene ink was diluted to measure the UV-Vis spectrum of the ink. Figure 2a shows the UV-Vis spectrum of Gr ink of 0.04 mg/ml concentration. Throughout the visible range, the transmission was reduced by around 75–85%, indicating uniform dispersion of the Gr ink and broad spectrum absorption. This is in agreement with the observation of graphene dispersed in 100 ppm SDS24.

Figure 2b shows the Raman spectra of Gr; the typical D-band was observed at 1337.21 cm−1, and the G-band was observed at 1576.45 cm−1. The strong D-band may indicate the presence of defects in Gr sheets or the formation of edges as a result of exfoliation from graphitic crystals to graphene sheets. The shear forces in a high-speed mixer are not enough to induce new defects in the graphene sheets as indicated in similar processes before25. Therefore, the D-band indicates defects in graphene sheets inherited from the graphite crystal as well as the new edges formed as a result of exfoliation. Both the G band and D band show a significant shift towards lower wavenumber when compared to a typical peak position of pristine graphite and it shows the presence of graphene. The 2D band at 2683.22 cm−1 was broad, indicating the multilayered graphene. In addition, the ratio between the G band and the 2D band gives information on the number of graphene layers. The I2D/IG is found to be 0.59 confirming the formation of multilayered graphene sheets. The graphene ink showed slightly increased density as approximately 1.01 g/cm3.

In the SEM images shown in Fig. 2c, Gr is observed as transparent at the center and opaque around the edges. The transparency at the center is due to the lack of sufficient material to produce electron matter interaction with Gr, and the edges are more visible due to the edge effect26. Additional SEM images of the graphene showing this effect is provided in the Figure S1. TEM images of Gr shown in Fig. 2d indicated that the Gr layer size ranges from around 150 nm to ~ 5 µms. TEM images also showed that Gr formed neat layers with low agglomeration.

Fig. 2
figure 2

a UV-Vis spectrum of Gr ink. Captured images of graphene ink shown as inset. b Raman spectrum of Gr ink. c SEM image of Gr ink. d TEM image of Gr ink

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Figure 3a shows the transmission spectra of the Gr lens prepared through the BIBO technique. With the increase in cycles, the transmittance % decreases indicating that higher loading could be achieved with an increased number of cycles. The contact lenses showed around 10–20% transmission decrease every 3 cycles. While a single layer graphene absorbs approximately 2.3% light, the 50% transmission decrease in the lens which underwent 9 BIBO cycles could be estimated to be absorption of around 20 to 23 layers of graphene. After nine cycles, CLs were observed to have more Gr agglomeration. A maximum of 40–50% transmission reduction was attained before having observable aggregations within the contact lens. Nine BIBO cycles were the optimum for producing a tinted contact lens without any aggregation of Gr. Figure 3b shows the transmission spectra of the Gr lens prepared through dipping commercial contact lenses within the Gr ink. The maximum reduction in transmission was around 30% achieved by immersing the contact lens in Gr ink for 40 min. This transmission decrease corresponds to approximately 13 layers of graphene. Further increase in immersion time did not decrease the transmission further. This suggests that self-assembly led coating of Gr onto the contact lens might have achieved saturation in 40 min27. The higher concentration of graphene in the liquid and the two-dimensional flexibility of graphene lead to the self-assembly of Gr onto the hydrogel CLs. The transmission spectra indicate a nearly constant transmission loss throughout the visible region for both types of lenses. These techniques could be used to achieve desirable visible light absorption and will be beneficial as broad-spectrum absorber. Figures 3c and d show the captured image of the Gr lenses prepared using the two techniques. Figure 3e and f show the comparison between commercial CLs and graphene incorporated contact lenses. Uniform tinting is observed in the sample. Figure 3g and h show the Gr-HEMA structures printed onto CLs using a DLP-based 3D printer. The instability of graphene within the resin and the targeted exposure of UV in 3D printing have led to non-uniform graphene concentrations within the structures printed on commercial CLs. Further methods to stabilize the graphene within the resin could provide methods to develop graphene containing electrodes or structures on contact lenses.

Fig. 3
figure 3

a UV-Vis Transmission spectra of Gr lens with an increasing number of Breath in - Breath out cycles. b UV-Vis Transmission spectra of Gr lens with increasing dipping time c The CLs changing visible coloration after breath in - breath out cycles d The CLs changing visible coloration after dipping time e Commercial contact lens on an eye model f)Graphene incorporated commercial contact lens on an eye model g 3D printed Gr-HEMA pattern on CLs h 3D printed Gr-HEMA pattern on CLs on an eye model

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The SEM image of contact lenses in Fig. 4a and b showed a uniform distribution of Gr throughout the contact lens surface. Like SEM images of the Gr ink, Gr’s center was more transparent than the edges26. The SEM image of clear lens is shown in Fig. 4c for comparison. The chances of aggregation are higher with repeated cycles of the BIBO. One such aggregation position is shown in Fig. 4g. Solvent interaction with graphene can lead to particle aggregation in graphene ink28.

The FTIR spectra in Fig. 4d show multiple bands at 3420 cm−1 (O–H stretching) and 1721 cm−1 (–C = O stretching). The band at 1467 cm−1 is described by C-H bending. The bands between 2710 cm−1 and 2900 cm−1 can be ascribed to C–H stretching for methylene –CH2 and methyl –CH3 groups. The absorption peak at 1620 cm−1 can be dedicated to ester carbonyl (C = O) groups. The 1261 cm−1, 1625 cm−1, and 1721 cm−1 peaks can be ascribed to the C-N stretch, C = C stretch, and C = O stretch, respectively29. There was no observable change in the C = C stretching at 1625 cm−1 when Gr was added since the Gr concentration was too low to be detected by the equipment.

The contact angle and water retention properties of the lenses were studied (Fig. 4e and f). The contact angle measured showed slight increase in both contact lenses with graphene. However this was comparable to the commercial contact lenses available in the market30,31. The total swelling remained same for the contact lenses however the lenses with graphene showed slight slower hydration rate. These results indicate that the hydration properties stay relatively unchanged after the process. Both contact lenses were exposed to certain duration of UV, then subjected to repeated hydration and drying cycles, and then long term stability was also investigated (Fig. 5). The absorption spectra remained the same indicating the possible use of contact lenses for the desired period.

Fig. 4
figure 4

a SEM image of Gr dipped contact lens b SEM image of Gr lens prepared using breath in – breath out technique. c) SEM image of clear lens d FTIR spectra of clear lens, Gr ink dipped lens and Gr lens prepared using breath in – breath out technique. e Contact angle measurement of contact lenses f Swelling behaviour of the contact lenses g Formation of aggregation in an increased number of breath in – breath out cycles

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Fig. 5
figure 5

Contact lenses prepared using BIBO method undergoing a UV exposure b repeated hydration and re hydration cycles c)Long term storage. Contact lenses prepared using dipping undergoing d UV exposure e)repeated hydration and re hydration cycles f Long term storage. Images captured by camera before and after the tests g BIBO method h Dipping method

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Fig. 6
figure 6

SEM images of surface adhesion of P. aeruginosa bacteria for a Clear lens b Graphene-dipped lens. SEM images of surface adhesion of S. aureus bacteria for c Clear lens d Graphene-dipped lens

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Bacterial culture studies with clear lens and graphene-dipped lens were done and results have been exhibited in Fig. 6. The SEM images suggest lower bacterial attachments onto the graphene lens when compared to a clear lens (pre-washed). The lens displays exceptional anti-bacterial properties relative to the clear lens. In the case of S. aureus, the colony attachments are limited as opposed to clear lens which seems to have significant colony attachments. Previously, there have been works that emphasized the antibacterial and antibiofilm properties of graphene and its derivatives32,33,34.

Fig. 7
figure 7

a Optical image of HDFn cultured without lens exposure post 24 h growth, b Optical image of cells after 24-hour incubation with graphene lens; scale bar: 200 μm, (c) Results from MTT assay (d) Results from Trypan blue assay; data presented as mean percent viability ± s.e. One way ANOVA test results involving multiple comparisons are indicated, *=p < 0.05 (e) qPCR results of IL-6 and (f) qPCR results of IL-β; relative to GAPDH

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The optical microscopy images of cells cultured without lens exposure (Fig. 7a) and those incubated for 24 h with the prepared graphene lens (Fig. 7b) appear comparable, indicating that the presence of the graphene lens does not visibly impair cell attachment or morphology. MTT and Trypan blue assays converge to substantiate the insignificant cytotoxicity of the coated lens. The MTT and Trypan blue assay results are represented in Fig. 7c and d. The assay data obtained from the wells with cells which had clear lens and graphene lens were compared to that of those without any lens. All the statistics were performed with One-way ANOVA using GraphPad Prism software. Dunnett’s Multiple Comparison Test indicated non-significant differences among the clear lens, graphene dipped lens, and the control well without lens. This compares multiple groups against a single control while adjusting for the increased risk of false positives that can occur when making multiple comparisons. The differences were considered statistically significant at p value < 0.05. Over 90% cell viability corresponds to a potentially biocompatible lens. The live cell % was high and comparable to that of the control wells without any lens as well as with the clear lens. This could also validate the stability of the graphene-dipped lens which does not leach into the culture media during the incubation with cells. Moreover, our evaluation of the anti-inflammatory properties of the graphene lens reinforces our claim that the prepared graphene lens does not cause any significant inflammatory responses when compared with commercially available clear lenses (Fig. 7e and f). The IL-6 gene expression levels appear to have significantly reduced in the case of the graphene-dipped lens when compared with the clear lens. There are no significant differences in the expression levels of IL-β between our prepared lens and the clear lens commercially available. Thus suggesting the prepared graphene lens does not induce any pro-inflammatory responses among the cells. The statistics were applied using One-way ANOVA with Dunnett’s multiple comparison test considering p < 0.05 to be statistically significant. The expression levels of pro inflammatory cytokine IL-6 when cells were incubated with graphene lens was observed to be reduced approximately 40% relative to the clear commercially available lens. Whereas, the IL- β levels were comparable to that of clear lens with non-significant changes. Overall, the pieces of evidence collected confirm that the graphene lens prepared using BIBO method is biocompatible and non-toxic. The BIBO method provides a viable approach to prepare graphene incorporated contact lenses with control over the concentration of graphene present through controlled experimental conditions.

Conclusion

Here, we successfully incorporated graphene into commercial contact lenses through three techniques: the breath in - breath out (BIBO) technique, immersion of CLs in graphene ink, and 3D printing of graphene hydrogel composite. Graphene ink prepared using high-speed shear mixing was utilized as the graphene source. This graphene ink was used as the aqueous solution in BIBO cycles and was repeated to obtain the necessary attachment without losing transparency beyond acceptable limits. Around 50% transmission reduction was achieved through incorporation of graphene through BIBO method with around 9 cycles without compromising the optical clarity. The immersion time was changed to control the loading concentration of graphene in the second method involving the self-assembly of graphene onto hydrogels. However, the self-assembly was self-limiting: a 30% transmission reduction was the maximum under the experimental conditions, as further immersion did not increase absorption. This was achieved within 40 min of dipping time. The third method utilized graphene ink dispersed in hydroxyethyl methacrylate to 3D print patterns onto the CLs. The UV-Vis spectroscopy indicated steady absorption throughout the visible region as a tinting additive for visible light, suggesting uses such as broad-spectrum absorbers. This simple and rapid process may offer improved scalability for producing graphene-integrated hydrogel CLs. The prepared graphene lens utilizing BIBO method has exhibited exceptional anti-bacterial properties against P. aeruginosa and S. aureus, thus proving to be significantly unaffected by these bacteria in comparison to commercially available contact lenses. Additionally, there is substantial evidence to prove that the developed lenses do not lead to inflammation and are not cytotoxic, which are ideal properties of a biocompatible contact lens.