Surface-Engineered Contact Lens as an Advanced Theranostic

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Surface engineered contact lens as an advanced theranostic platform for modulation and detection of viral infection Wing Cheung Mak, Kwan Yee Cheung, Jenny Orban, Chyan-Jang Lee, Anthony P. F. Turner, and May Griffith ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08644 • Publication Date (Web): 29 Oct 2015 Downloaded from http://pubs.acs.org on November 4, 2015

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ACS Applied Materials & Interfaces

Surface Engineered Contact Lens as an Advanced Theranostic Platform for Modulation and Detection of Viral Infection Wing Cheung Mak 1,2 *, Kwan Yee Cheung1, Jenny Orban2, Chyan-Jang Lee1, Anthony P.F. Turner2,§, May Griffith1,§ 1

Integrative Regenerative Medicine Centre, Department of Clinical and Experimental Medicine,

Linköping University, SE 58185, Linköping, Sweden 2

Biosensors and Bioelectronics Centre, Department of Physics, Chemistry and Biology,

Linkӧping University, SE 58183, Sweden

Tel.: +46 013286921; Fax: +46 013287568

KEYWORDS: theranostics, contact lens, cornea, anti-viral, diagnostics

ACS Paragon Plus Environment

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ABSTRACT We have demonstrated an entirely new concept of a wearable theranostic device in the form of a contact lens (theranostic lens) with a dual functional hybrid surface to modulate and detect a pathogenic attack with the corneal HSV-1 model. The theranostic lenses were constructed using a facile Layer-by-Layer (LbL) surface engineering technique, keeping the theranostic lenses with good surface wettability and optically transparency, and non-toxic towards human corneal epithelial cells (HCECs). The theranostic lenses were used to capture and concentrate inflammatory cytokines such as interleukin-1α (IL-1α) which is upregulated during HSV-1 reactivation for sensitive, non-invasive diagnostics, as well as incorporating anti-viral coating to serve as a first line of defense to protect patients against disease. Our strategy tackles major problems in tear diagnostics that are mainly associated with sampling of a relatively small volume of fluid and the low concentration of biomarkers. The theranostics lenses show effective anti-HSV-1 activity and good analytical performance for the detection of IL-1α, with a limit of detection of 1.43 pg mL-1 and a wide linear range covering the clinically relevant region. This work offers a new paradigm for “wearable” non-invasive healthcare devices combining “diagnosis" and "protection” against disease, while supporting patient compliance. We believe that this approach holds immense promise as a next-generation point-of-care and de-centralized diagnostic/theranostic platform for a range of biomarkers.

INTRODUCTION A theranostic device is one that has the dual functions of both diagnosis/detection and therapy. Theranostics is a new and rapidly growing area, with most work to date centered around the development of dual-function nanoparticles, such as those serving as drug carriers cum contrast


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agents in medical imaging 1,2,3. Our goal was to develop a wearable theranostic device in form of a contact lens and to test it in a clinically relevant model. We selected a facile surface nanoengineering approach, the so-called Layer-by-Layer (LbL) technique 4, for effecting the theranostic functions in the contact lens. LbL technology is a simple procedure for the construction of biomaterials based on multi-layered thin films with integrated biological functions and activities

5,6,7

. This technique enables the deposition of multiple layers

of biomolecules to create biofunctional thin films under mild aqueous physiological temperature and pH conditions, without limitation on the substrate morphology, allowing the use of, for example, flat surfaces, irregular surfaces or colloid particles. The thickness of each layer or the combined stack of films can be adjusted by altering the adsorption conditions (e.g. ionic strength, temperature and pH conditions) 8. The stability and permeability of the LbL films can be adjusted by simply controlling the number of layers 9. Physical adsorption of bioactives, such as enzymes, antibodies, receptors proteins, poly-nucleic acids and polysaccharides, onto specific layers allows biofunctionalized, multilayer films to be constructed that can then be used for the development of bioanalytical devices or biomedical implants

10,11

. Furthermore, the use of

natural polymers or their synthetic analogs for the thin film deposition can modify the surface attributes of the template materials by improving their biocompatibility and circumventing cytotoxicity 12,13. Recently, LbL multilayer technology was used successfully to load and release antibiotics in a tunable manner 14. Our aim was to demonstrate proof-of-concept for dual functioning LbL surfaces that can detect and modulate a pathogenic attack of corneal Herpes Simplex Virus (HSV) infection. HSV serotype-1 (HSV-1) disease is the main infectious cause of blindness worldwide

15,16

. It is

estimated that over 80% of the global population has been exposed to HSV-1 but only a small


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proportion becomes infected

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. Once infected, the virus can establish latency in the corneal

nerves (trigeminal ganglion) and become reactivated at a later time. The recurrent disease is associated with inflammation and may cause corneal scarring, thinning, neovascularization, and is known as Herpes Simplex Keratitis (HSK)

18,19

. The incidence of corneal HSK in developed

nations per year is estimated to be ~240,000 in 2013, and 1 – 1.5 million per year in developing countries 20. The causes of reactivation is still unknown but a range of identified factors include bright sunlight, some foods, exposure to high temperature, corneal transplantation surgery to treat scarring from HSV-1 and the prophylactic anti-inflammatory steroids prescribed as postoperative medication. In other words, the triggers are varied and viral reactivation leading to disease recurrence is unpredictable. A simple theranostic device that can detect the earliest signs of disease recurrence, e.g. inflammation, and provide an anti-viral effect to dampen viral activity and thereby afford the patient the opportunity to seek help prior to developing a full-blown infection, would be very useful. Tear fluid was used as the diagnostic medium in the current study. Tear fluid is an extracellular fluid secreted from by lachrymal glands (tear glands). Several important biomarkers are found within tear fluid (e.g. glucose, interleukins, lactoferrin, lipocalin and lysozyme

21

and

this has led to their use for diagnosing both local ocular problems such as eye infections, dry eye syndrome and glaucoma, and for systemic issues such as diabetes, hyperglycemia, breast cancer and lysosomal storage diseases

22,23

. In HSK, viral reactivation disease recurrence is

characterized by release of active viral particles and inflammation of the ocular surface. Hence, inflammatory cytokines such as interleukin-1α (IL-1α) which is upregulated during HSV-1 reactivation is a potential biomarker for diagnosis associated with HSV-1 infection

24

. In this

article, we demonstrate the concept of theranostic lens with dual functions for both detection and


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modulation for pathogenic attack of HSV-1 that could provide new paradigm for healthcare combining “diagnosis" and "protection” against cornea diseases with a non-invasive approach.

EXPERIMENTAL Materials Poly(sodium 4-styrenesulfonate) (PSS, M w ≈ 200000 g mol-1), poly(allylamine hydrochloride) (PAH,

M

w

≈

70000 g mol-1), bovine serum albumin (BSA), Tween-20, 3,3,5,5-

tetramethylbenzidine dihydrochloride (TMB), hydrogen peroxide (H2O2) and 4',6-diamidino-2phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Interleukin-1α (IL-1α), goat anti mouse IL-1α immunoglobulin G (anti- IL-1α IgG), biotinylated goat anti mouse IL-1α immunoglobulin G (biotin-anti- IL-1α IgG) and avidin conjugated horseradish peroxidase (avidin-HRP) were purchased from Biolegend. Contact lenses (ACUVUE®, Johnson & Johnson) were purchased from a local pharmacy. Artificial tears was composed of balanced salt solution (sodium chloride 7.14 mg mL-1, potassium chloride 0.38 mg mL-1, calcium chloride 0.15 mg mL-1, magnesium chloride 0.20 mg mL-1 dibasic sodium phosphate 0.42 mg mL-1, sodium bicarbonate 2.10 mg mL-1 and dextrose 0.92 mg mL-1) was doped with 1.7 mg mL-1 lysozyme 25, and adjusted to pH 7.4. Keratinocyte-serum free medium (KSFM) supplemented with L-glutamine, human epidermal growth factor (EGF), and bovine pituitary extract (BPE) and alexa Fluor 488 conjugated goat anti-rabbit antibody were purchased from Life Technologies (California, USA). Anti-HSV-1 antibody was purchased from Thermo Scientific (California, USA). WST-1 assay kit was purchased from Roche (Basel, Switzerland). The HSV-1 Strain F viruses were obtained from Dr. Earl Brown (University of Ottawa, Ontario, Canada).


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Surface functionalization of contact lenses Contact lenses were washed four times by dipping each lens into 1 mL of the PBS (10 mM, pH 7.2). The surfaces of the contact lenses were then functionalized using a Layer-by-Layer (LbL) deposition technique. In brief, contact lenses were dipped into 2 mL anionic PSS polyelectrolyte solution (5 mg mL-1, in 0.5M NaCl) and incubated for 10 minutes to allow adsorption. The excess polyelectrolyte was removed and the contact lenses were washed with 0.5M NaCl for 3 times. Then, cationic PAH polyelectrolyte solution (5 mg mL−1, in 0.5 NaCl) was deposited onto the PSS coated contact lens surfaces using the same procedures and conditions as mentioned for PSS. The contact lenses were coated by alternate deposition of PSS and PAH until the desired number of layers was achieved (typically 4 layers). The LbL-modified contact lenses provided a stable interface for the subsequence addition of theranostics functions. The antibody surface coverage on the [PSS/PAH]-coated theranostic lenses was evaluated. The [PSS/PAH]-coated lenses were incubated with an antibody solution with an initial concentration of 1.25 µg mL-1, and the amount of antibody adsorbed onto the lenses was evaluated by spectrophotometric measurements. The incubation chambers were coated with BSA, which is a common blocking agent, to minimize the non-specific adsorption of antibodies onto the chamber surface in order to improve the accuracy of the calculation.

Preparation of theranostics contact lenses The [PSS/PAH]2 coated contact lenses were functionalized by incubating the lenses in a 200 µL solution mixture containing 0.5 mg mL−1 PSS and 1.25 µg mL-1 anti- IL-1α IgG in 0.1 M phosphate buffer saline (PBS) pH 7.2 for 2 hours; whereas the PSS provides an anti-viral


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function and the anti-IL-1α IgG is the basis for the diagnostic assay. Subsequently, the dualfunction lenses with an outermost surface composed of PSS and anti- IL-1α IgG were washed three times with washing buffer (0.1 M PBS, 0.1% (w/v) BSA and 0.05% Tween-20, at pH 7.2). Finally, the lens surface was blocked by incubation with 1% (w/v) BSA in 10 mM PBS (pH 7.2) for 10 minutes, followed by washing three times with buffer (10 mM PBS, 0.1% BSA, 0.05% Tween-20) and stored in 10 mM PBS (pH 7.2).

Contact lens-based affinity assay for detection of IL-1α An IL-1α calibration curve was obtained by incubating the theranostics contact lenses in 200 µL of IL-1α at concentrations ranging from 0 to 100 pg mL-1 for 1 hour at room temperature. To test the ability of the lens to detect the cytokine, lenses were permeated continuously with artificial tears spiked with IL-1α (0 to 100 pg mL-1) at a flow rate of 1.5 µL min-1 for 6 hours, using an inhouse in-vitro ocular microfluidic model driven by syringe pump to mimic the ocular environment and tear flow. After incubation with cytokine, the lenses were washed twice with washing buffer and subsequently incubated with 200 µL solution mixture of biotin-anti-IL-1α IgG (1.25 µg mL-1). Avidin-HRP (at 1.25 µg mL-1) was applied after which the lenses were washed again with washing buffer. Finally, the lenses were immersed in 200 µL TMB solution for 5 minutes, followed by addition of 200 µL H2O2 (0.1M) and the color intensity was measured at 450 nm using a spectrophotometer.

Cytotoxicity study To determine if the surface engineered contact lenses are cytotoxic, lens samples were cut into identical sized pieces of 6 mm in diameter with a trephine (circular blade). Each sample were


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placed on top of confluent human corneal epithelial cells (HCECs; dish.

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26

) in a 48-well cell culture

The HCECs were supplemented with Keratinocyte Serum-Free Medium (Life

Technologies, Invitrogen, California, USA) containing with L-glutamine, human epidermal growth factor and bovine pituitary extract, and incubated at 37 °C in a humidified incubated with 5% CO2 for a further 24 and 48 hours. Cell viability was then assessed with the WST-1 assay according to the manufacturer’s instructions. The HCECs were incubated with the cell proliferation reagent WST-1 at 37 °C for 1 hour. The absorbance at 450 nm was measured with a microplate reader (Molecular Devices). Triplicate measurements were performed for each contact lens sample.

Anti-HSV-1 activity HSV-1 strain F viruses (a gift from Prof. Earl Brown, University of Ottawa, Canada) were propagated in Vero cells and titered prior to use as we previously described 27. To determine the anti-viral effect of the surface engineered contact lens against HSV-1, different surface-modified lens samples were placed into different wells of a 24 well tissue culture plate, where each well contained a confluent monolayer of cultured HCECs in 150 µL of KSFM medium. To each well, 500 µL of HSV-1 at a multiplicity of infection [MOI]=1 in KSFM was added. Virus and cells were incubated at 37 °C for 1hour, after which the lens samples were removed and the HCECs were washed with phosphate buffered saline (PBS, 0.01M, pH 7.4). Fresh KSFM medium was then added to the HCECs and they were further incubated at 37°C for 24 hours.

An

immunofluorescence assay (IFA) was used to determine the proportion of infected HCECs. This provides an indirect measure of the efficacy of PSS-coated lenses to inactivate the viruses, as inactivated virus is will not actively infect HCECs and replicate. To detect HSV-1 replication


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within HCECs, the cells were fixed with 4% paraformaldehyde for 1 hour at 4°C and then permeabilized with PBS containing 0.5% Triton X-100 for 5 minutes at room temperature. An anti-HSV-1 antibody (Thermo Scientific, California, USA) diluted with PBS (1 : 400) was applied and incubated for 1 hour at room temperature. After washing with PBST (PBS, 0.01 M, pH 7.4, 0.1% Tween 20), the HCECs were incubated in Alexa Fluor 488 conjugated goat antirabbit antibody for visualization. Cell nuclei were visualized by counterstaining with DAPI (4',6diamidino-2-phenylindole). Samples examined under a Zeiss fluorescent microscope (Zeiss Axio Observer Z1). Triplicate measurements were performed for each contact lens sample.

Characterization Optical and fluorescence microscopy images of HCEC were recorded on a Zeiss Vert A1 (Zeiss, Germany) connected to a CCD color digital camera (AxioCam Cm, Zeiss, Germany). Images were captured and analyzed using imaging software (Zen2012, blue edition, Zeiss, Germany). Scanning electron microscopy (SEM) was used to reveal the surface morphology of the surface engineered contact lenses. Contact lens samples were cut into 5 mm2 pieces, mounted with double sized carbon tape onto copper stands and then coated with platinum for visualization. SEM images were taken using a LEO 1550 Gemini microscope (Zeiss, Germany). Energy-dispersive X-ray spectroscopy (EDX) was performed using an INCA X-ray microanalysis system (Oxford Instruments, UK) coupled with the scanning electron microscope (LEO 1550 Gemini, Zeiss, Germany). Optical transparency tests were performed using a custom-built light transmission and backscattering measurement system at room temperature with white light (quartz-halogen lamp source). Contact lens samples were incubated at 35.5 °C for 1 hour and the optical transparency


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of the contact lens samples were immediately measured. The percentage transmission of light through the contact lenses samples were compared with open beam intensity. The relative percent of light back scattered from the collimated beam by the sample was measured with a circular array of 8 photodiodes, 30 degrees off axis. Surface hydrophobicity of the surface modified lenses were characterized using a contact angle instrument (CAM 200 optical angle meter, KSV instrument LTD). The modified lenses were placed under a syringe orifice; a drop of milli-Q water was formed and applied onto the surface of the contact lenses. An image of the water droplet on the applied contact lens surface was captured and the contact angle was calculated by the software. The contact angle measurements using mill-Q water are sensibly independent of the temperature (i.e. measurement at room temperature or cornea physiological temperature of ~33.5 ˚C will be similar) 28.

RESULTS AND DISCUSSION Surface nanoengineering and fabrication of the theranostic lenses The design of the theranostic lens was composed of a dual functional outermost layer consist of hybrid mixture of polystyrene sulfonate (PSS) as the anti-viral agents, as well as target specific anti-IL-1α antibodies, that able to detect inflammation through capture of IL-1α followed by a simple colorimetric affinity assay for detection (Figure 1). The LbL self-assembly process is basically conducted through electrostatic interaction between oppositely charged species. The adsorption process is not limited by the surface morphology and topography. Hence, it provides an ideal facile method for contact lenses modification. A bi-layer coating fashion i.e. [PSS/PAH]n was used to create a stable interface for immobilization of the anti-HSV-1 and bio-recognition molecules. A negatively-charged


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polyelectrolyte, PSS, was used as the first inner-most coating layer due to its relatively hydrophobic backbone, which is more readily deposited onto the contact lens surfaces. Meanwhile, a positively-charged polyelectrolyte, such as PAH, was desirable as the outer-most layer to provide a positively-charged interface for the immobilization of the anti HSV-1 molecules and antibodies, which were negatively charged at physiological pH 7.0. Total protein assays were performed to measure the protein content in the supernatants and to quantify the percentage of antibodies adsorbed onto the contact lens surface. Figure 2A showed that contact lenses without a LbL coating had a relatively low antibody adsorption of ~8%, while increasing the number of LbL coatings increased the antibody adsorption yield. A steady state was observed after coating of 4 polyelectrolyte layers i.e. [PSS/PAH]2 reaching a maximal antibody adsorption of ~33%. We further performed affinity assays with the antibodycoated contact lenses in order to investigate the antibody’s biological function and to optimize the assay performance (Figure 2B). Results revealed that the absorbance signals measured at 450 nm for the affinity assays for detection of IL-1α (100 pg mL-1) increase as the layer number increased, and a maximum signal was obtained after coating of 4 polyelectrolyte layers i.e. [PSS/PAH]2. The increase in signal response is mainly due to an increase in the amount of antibodies immobilized on the contact lens surfaces (Figure 2A). Both the total protein assays and affinity assays imply that 4 polyelectrolyte layers i.e. [PSS/PAH]2 is sufficient to create a stable interface for antibody immobilization and to maximize the performance of the affinity assay. By knowing the surface area of the contact lenses, the antibody surface coverage of the modified contact lenses was calculated to be 63 ng cm-2. The theoretically calculated surface coverage for a closely packed antibody (IgG) monolayer is in the range of 200 - 450 ng cm-2 ,


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depending on the orientation of the IgG molecules

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. By comparing with the theoretical value

and assuming the deposited antibodies form a monolayer, it is estimated that around 14.0% to 31.5% of the total contact lenses surface was covered by the antibodies, depending on the antibodies orientation.

Characterization of the theranostics contact lenses The contact lens surface had to be kept hydrophilic to ensure comfort to the wearer. The contact angle measurements obtained are summarized in Figure 3A. The bare, unmodified contact lens was hydrophilic with a similar contact angle to that previously report for this type of lens 30. The LbL-modified contact lenses and the LbL-modified lenses with antibodies (LbL-Ab) were more hydrophilic than the unmodified lenses (i.e. they had smaller contact angles). This is mainly due to the high density of charged groups within the polyelectrolyte coatings. Nevertheless, the modified contact lenses show improved hydrophilicity and could provide more comfort for wearing. Contact lenses should also be transparent to allow light transmission for good vision. As shown in Figure 3B, there was only small difference in transparency between the bare and LbLmodified lenses. At cornea physiological temperature of 35.5 ˚C, the bare and the LbL-modified contact lenses showed white light transmission of 96.3% and 91.5%, respectively. The LbLmodified contact lens showed a light transmission of 91.5% that is well above the 87% measured in healthy human cornea. This insignificant difference in the optical transparency between the bare and LbL-modified contact lens is most likely due to the self-limiting LbL adsorption process creating a film with an individual single layer thickness of only tens of nanometers

31

.

The influence on the oxygen permeability of the LbL coating was previously reported by


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Leväsalmi et al.

32

It was showed that 10 layers of PAH/PSS coating has no effect on oxygen

permeability, as the oxygen permeability was virtually unchanged compared with the uncoated PMP-COOH polymer substrate. Our theranostic lens is composed of only 4 PSS/PAH layers and an outermost PSS+Ab layer (together 5 layers). Therefore, the influence of the LbL coating on the oxygen permeability of the theranostic lens is likely insignificant. To further investigate the interfacial characteristic of the LbL-modified contact lenses, the surface morphology of the contact lens samples was examined. Bare contact lens had a smooth surface, while the LbL-modified and the LbL-Ab-modified contact lenses had rougher surfaces (Figure 3C), indicative of coating of polyelectrolytes and/or antibodies onto the surfaces. We further analysis the surface topology of the bare, LbL-modified contact lenses with AFM (Figure S1). The AFM images show the LbL-modified contact lenses had rougher surface topologies which are consistence with the SEM data. In parallel to SEM imaging, energy-dispersive X-ray spectroscopy (EDX) was performed to characterize the elemental composition of the bare and modified contact lens surfaces. Upon X-ray irradiation, the back scattered spectrum was collected and analyzed for the elements that were present on the sample (Figure S2). A characteristic sulfur peak was observed in both LbL-modified and LbL-Ab-modified contact lenses, confirming the presence of the polyelectrolyte PSS and/or antibodies on the surfaces, while no sulfur peak was observed in bare contact lens. It is difficult to distinguish the difference between PSS and antibodies as they both contain sulfur. However, the difference in surface morphologies between LbL-modified and LbL-Ab-modified contact lenses shows a larger granulated structure is presence in the LbL-Ab-modified contact lens, which likely resulted from the larger globular structure of the antibodies.


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Cytotoxicity tests The surface-modified contact lenses coated with [PSS/PAH]4-Ab+PSS were tested and compared with the bare contact lenses as a control. We did not observe obvious cell toxicity on the modified surface of contact lens when compared to control after 24 and 48 hours (Figure 4). These results are consistent with another study which demonstrated the low toxicity of PSS 33. In addition, the theranostic lenses are not intended to be worn for more than 24 hours for sample collection. This further minimizes any potential problems with toxicity.

Anti-HSV-1 therapeutic activity The antiviral activity of PSS against a broad range of viruses has been previously reported

34,35

.

For example, it has been demonstrated to be effective against human papillomavirus (HPV) and Herpes simplex viruses (HSV-1 and HSV-2) in in vitro studies

34

. The proposed predominant

antiviral mechanism of multiple sulfated or sulfonated polymers and polysaccharides is the inhibition of virus adsorption onto cell surface receptors

36,37

thereby preventing viral entry.

HSV-1 infection in cornea may cause severe keratitis. To determine the efficacy of immobilized PSS against HSV-1 infection, lenses coated with [PSS/PAH]4-PSS, [PSS/PAH]4-Ab and [PSS/PAH]4-Ab+PSS were tested with HCES infected with HSV-1. The bare contact lenses and pure PSS solution (10 µg mL-1) were used as a negative and positive control, respectively. We found that the lens samples containing PSS as the outermost coating (i.e. the [PSS/PAH]4-PSS, [PSS/PAH]4-Ab+PSS) have obvious anti-HSV-1 activity, indicated by the absence of green fluorescently-labeled HSV-1 viral protein (Figure 5). In contrast, control bare lenses and lenses coated with antibody were not able to inhibit virus infection. These results strongly suggest that HSV-1 infection into the HCECs was effectively blocked since no viral protein expression could


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be found inside the cells. This demonstrated that the PSS coating onto contact lenses could effectively block the entry of HSV-1 into the cells similar to that of PSS alone in solution 36, 37.

Analytical performance of theranostics lenses for IL-1α detection There are two major technical challenges for tear diagnostics associated with the relatively small sample volume of tear fluid and the low concentration of biomarkers compared with the levels in the blood. Despite the availability of highly sensitive enzyme-linked immunosorbent assays (ELISA) that allow detection of minute quantities of IL-1α, the common practice for tear diagnostics still requires extraction of tears and topping up of small volumes of tear sample with addition buffer to provide sufficient sample volume for ELISA. In contrast, the use of contact lenses as a diagnostic tool, would allow for direct intimate contact between the lens and ocular fluid, avoiding the need to extract small volumes of tears from the eyes. The analyte capturing process could therefore take place during daily wear. A daily disposable contact lens is usually worn for 8 to 14 hours, providing sufficient incubation time to capture and concentrate the trace amount of analytes from the tears. To demonstrate this innovative analytical concept and study the performance of the theranostic lenses, an in vitro artificial ocular model composed of a hydrogel-based, mimic human cornea, integrated with a microfluidics system, was developed (Figure 6A). The theranostics lenses were placed on the surface of an artificial hydrogel-based cornea, which was then be placed inside a chamber connected with syringe pump, to mimic tear flow, using artificial tears doped with IL1-α at different known concentrations. The flow rate of the artificial tears was set at 1.2 µL min-1, which is equivalent to the turnover rate of tear production in the human adult 38. Therefore, during the incubation time of 8 hours, an approximate total volume of


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576 µL sample fluids was running through the theranostic lens, allowing the capture and preconcentration of the IL-1α biomarker from a relatively large sample volume. Figure 6B shows the analytical performance of the theranostic lenses for detection of IL-1α from 0.625 to 400 pg mL-1 with a linear correlation coefficient R2 of 0.986. The Limit of Detection, based on three times the standard derivation of the blank signals divided by the calibration sensitivity, was calculated to be 1.43 pg mL-1. According to the literature, the basal IL-1α concentration of healthy subject is 43.1 ± 24 pg mL-1, and increases 5 – 10 folds in patients suffering from chronical viral infection

39

. Therefore, the analytical performance of the theranostic lenses is

within the clinically relevant region. It is important to notice that the theranostic lenses show a relatively wide linear detection range. The wide linear detection range could be explained by the advantage of the relatively large total surface area of the contact lenses of about 3.34 cm2

40

compared with the total surface of a single well in 96 wells plate of about 0.32 cm2. The relatively large surface area of the theranostics lenses extended the saturation limit by capturing a larger amount of IL-1α, and therefore increasing the linear dynamic window. Another important parameter related to the analytical performance is the detection specificity. The specificity of the theranostic lenses was evaluated by comparing the signal responses obtained for IL-1α against two other non-specific biomarkers (IL-6 and IL-8). As can be seen from Figure 6C, a clear signal response was only observed in the presence of IL-1α. In contrast, a significant signal was observed for IL-6 and IL-8 compared to the blank signal. These results demonstrate a good analytical performance and high specificity of the theranostic lenses for detection of IL-1α.


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CONCLUSIONS We demonstrate the first theranostic lens with a dual function hybrid surface composed of both anti-viral and bio-recognition molecules to modulate and detect a pathogenic attack using a corneal HSV-1 model. The theranostic lenses were constructed using a facile LbL surface engineering technique. The theranostic lenses showed good anti-HSV-1 activities as well as good analytical performance for detection of IL-1α, with a Limit of Detection of 1.43 pg mL-1 and a wide linear detection range covering the clinically relevant region. Our concept of using theranostic lenses for capturing and pre-concentrating tear biomarkers, provides a new solution for tear diagnostics that overcomes problems associated with the relatively small sample volume of tear fluid and low concentration of biomarkers, as well as minimizing the effect of reflexive tears from a single extraction. Moreover, the theranostic lenses had good surface wettability and optically transparency, and were non-toxic towards HCECs. This unique combination of features demonstrated the practical utility of the theranostic lens as a new generation of wearable healthcare device.


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FIGURES

Figure 1. Schematic diagram illustrating the surface engineering fabrication processes and the construction of the theranostics lens.


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Figure 2. (A) Antibody adsorption efficiency onto contact lens surface as a function of LbL coating. (B) Signal optimization of the affinity assays as a function of LbL coating.


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Figure 3. The surface engineered contact lens shows similar (A) surface wettability and (B) optical transparency compared to an unmodified contact lens. (C) SEM images showing the surface morphology of the modified contact lenses remains smooth, and the coatings are under 100 nm in thickness.


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Figure 4. Cytotoxicity studies of the LbL modified contact lenses with [PSS/PAH]4-Ab+PSS and the unmodified bare lenses towards HCECs for 24 and 48 hours. Cell control comprises of HCECs alone and positive control comprises of PSS alone were performed. Cell proliferation was determined using the WST-1 assay.


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Figure 5. Efficacy of contact lens coated with (A) [PSS/PAH]4-Ab, (B) [PSS/PAH]4-PSS, or (C) [PSS/PAH]4-Ab+PSS in blocking HSV-1 infection of HCEC as visualized at 24 hours postinfection. An uncoated or bare contact lens (D) served as a negative control, while a 10 µg mL-1 PSS solution (E) served as a positive control for anti-HSV-1 activity. Virus-infected cells are stained green by the anti-HSV-1/2 antibody followed by Alexa Fluor 488 conjugated goat antirabbit secondary antibody (column i) and the cell nuclei are stained with DAPI (column ii).


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Figure 6. (A) An in vitro artificial ocular model consisting of a hydrogel-based human corneal mimic integrated with a microfluidics system. A theranostic lens is placed on the surface of the artificial cornea, and is then later placed inside a chamber connected to a syringe pump to mimic tear flow using artificial tears. (B) Calibration curve for IL-1α used to determine the concentration of the cytokine; inset shows the theranostic lens before and after assay. (C) Specificity studies showing the signal response for detection of IL-1α and non-specific IL-6 and IL-8, respectively. n=3 samples per data point for B and C.


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ASSOCIATED CONTENT Supporting Information. “This material is available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. §These authors contributed equally. ACKNOWLEDGMENT We would like to thanks for the financial supported by Linköping Center for Life Science Technologies (LIST). We would like to thanks Mohammad Mirazul Islam for his help on the transparency measurements.


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Table of Contents Graphic


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Surface-Engineered Contact Lens as an Advanced Theranostic

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