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Peer-reviewed articles

2024

Bose,S., Phan,C.-M., Rizwan,M., Tse,J. W., Yim,E., Jones,L. Fabrication and Characterization of an Enzyme-Triggered, Therapeutic-Releasing Hydrogel Bandage Contact Lens Material Pharmaceutics 2024;16(1):Article 26 [ Show Abstract ]

Purpose: The purpose of this study was to develop an enzyme-triggered, therapeutic-releasing bandage contact lens material using a unique gelatin methacrylate formulation (GelMA+).

Methods: Two GelMA+ formulations, 20% w/v, and 30% w/v concentrations, were prepared through UV polymerization. The physical properties of the material, including porosity, tensile strain, and swelling ratio, were characterized. The enzymatic degradation of the material was assessed in the presence of matrix metalloproteinase-9 (MMP-9) at concentrations ranging from 0 to 300 µg/mL. Cell viability, cell growth, and cytotoxicity on the GelMA+ gels were evaluated using the AlamarBlueTM assay and the LIVE/DEADTM Viability/Cytotoxicity kit staining with immortalized human corneal epithelial cells over 5 days. For drug release analysis, the 30% w/v gels were loaded with 3 µg of bovine lactoferrin (BLF) as a model drug, and its release was examined over 5 days under various MMP-9 concentrations.

Results: The 30% w/v GelMA+ demonstrated higher crosslinking density, increased tensile strength, smaller pore size, and lower swelling ratio (p < 0.05). In contrast, the 20% w/v GelMA+ degraded at a significantly faster rate (p < 0.001), reaching almost complete degradation within 48 h in the presence of 300 µg/mL of MMP-9. No signs of cytotoxic effects were observed in the live/dead staining assay for either concentration after 5 days. However, the 30% w/v GelMA+ exhibited significantly higher cell viability (p < 0.05). The 30% w/v GelMA+ demonstrated sustained release of the BLF over 5 days. The release rate of BLF increased significantly with higher concentrations of MMP-9 (p < 0.001), corresponding to the degradation rate of the gels.

Discussion: The release of BLF from GelMA+ gels was driven by a combination of diffusion and degradation of the material by MMP-9 enzymes. This work demonstrated that a GelMA+-based material that releases a therapeutic agent can be triggered by enzymes found in the tear fluid.

Ganguly,S., Wulff,D., Phan,C-M., Jones,L., Tang,X. Injectable and 3D Extrusion Printable Hydrophilic Silicone-Based Hydrogels for Controlled Ocular Delivery of Ophthalmic Drugs Applied Bio Materials 2024;7(9):6286-6296 [ Show Abstract ]

While silicone elastomers have found widespread use in the biomedical industry, 3D printing them has proven to be difficult due to the material’s slow drying time, low viscosity, and hydrophobicity. Herein, we arrested the hydrophilic silicone (HS) macrochains into a semi-interpenetrating polymer network (semi-IPN) via an in situ photogelation-assisted 3D microextrusion printing technique. The flow behavior of the pregel solutions and the mechanical properties of the printed HS hydrogels were tested, showing a high elastic modulus (approximately 15 kPa), a low tan δ, high elasticity, and delayed network rupturing. The uniaxial compression tests
demonstrated a nearly negligible permanent deformation, suggesting that the printed hybrid hydrogel maintained its elastic properties. Drug loading and diffusion in the microporous hydrogel are shown via the non-Fickian anomalous transport mechanism, leading to highly tunable loading/releasing profiles (approximately 20% cumulative release) depending on the HS concentration. The drug encapsulation exhibits exceptional stability, remaining intact without any degradation even after a storage period of 1 month. As far as we know, this is the first soft biomaterial based on HS that functions as an exceptional controlled drug delivery device.

Phan,C. M., Chan,V. W. Y., Drolle,E., Hui,A., Ngo,W., Bose,S., Shows,A., Liang,S., Sharma,B., Subbaraman,L., Zheng,Y., Shi,X., Wu,J., Jones,L. Evaluating the in vitro wettability and coefficient of friction of a novel and contemporary reusable silicone hydrogel contact lens materials using an in vitro blink model Contact Lens Anterior Eye 2024;47(2):102129 [ Show Abstract ]

Purpose
To evaluate the in vitro wettability and coefficient of friction of a novel amphiphilic polymeric surfactant (APS), poly(oxyethylene)–co-poly(oxybutylene) (PEO-PBO) releasing silicone hydrogel (SiHy) contact lens material (serafilcon A), compared to other reusable SiHy lens materials.

Methods
The release of fluorescently-labelled nitrobenzoxadiazole (NBD)-PEO-PBO was evaluated from serafilcon A over 7 days in a vial. The wettability and coefficient of friction of serafilcon A and three contemporary SiHy contact lens materials (senofilcon A; samfilcon A; comfilcon A) were evaluated using an in vitro blink model over their recommended wearing period; t = 0, 1, 7, 14 days for all lens types and t = 30 days for samfilcon A and comfilcon A (n = 4). Sessile drop contact angles were determined and in vitro non-invasive keratographic break-up time (NIKBUT) measurements were assessed on a blink model via the OCULUS Keratograph 5 M. The coefficient of friction was measured using a nano tribometer.

Results
The relative fluorescence of NBD-PEO-PBO decreased in serafilcon A by approximately 18 % after 7 days. The amount of NBD-PEO-PBO released on day 7 was 50 % less than the amount released on day 1 (6.5±1.0 vs 3.4±0.5 µg/lens). The reduction in PEO-PBO in the lens also coincided with an increase in contact angles for serafilcon A after 7 days (p 0.05). The other contact lens materials had stable contact angles and NIKBUT over their recommended wearing period (p > 0.05), with the exception of samfilcon A, which had an increase in contact angle after 14 days as compared to t = 0 (p < 0.05). Senofilcon A and samfilcon A also showed an increase in coefficient of friction at 14 and 30 days, respectively, compared to their blister pack values (p < 0.05).

Conclusion
The results indicate that serafilcon A gradually depletes its reserve of PEO-PBO over 1 week, but this decrease did not significantly change the lens performance in vitro during this time frame.

Wong,K-Y., Liu,Y., Phan,C-M., Jones,L., Wong,M-S., Liu,J. Selection of DNA aptamers for sensing drugs treating eye disease: atropine and timolol maleate Sensors & Diagnostics 2024;3(10):1679-1688 [ Show Abstract ]

Effective monitoring of ocular drugs is crucial for personalized medicine and improving drug delivery efficacy. However, traditional methods face difficulties in detecting low drug concentrations in small volumes of ocular fluid, such as that found on the ocular surface. In this study, we used capture-SELEX to select aptamers for two commonly used ocular drugs, timolol maleate and atropine. We identified TMJ-1 and AT-1 aptamers with binding affinities of 3.4 μM timolol maleate and 10 μM atropine, respectively. Our label-free TMJ-1 biosensor using thioflavin T staining achieved a limit of detection (LOD) of 0.3 μM for timolol maleate. The AT-1 biosensor showed an LOD of 1 μM for atropine, and exhibited a 10-fold higher sensitivity compared to UV-visible spectroscopy. Future research in this area holds promise in enhancing drug delivery monitoring and improving the treatment of ocular diseases.

2023

Pereira-da-Mota,A. F., Vivero-Lopez, M., Garg,P., Phan,C-M., Concheiro,A., Jones,L., Alvarez-Lorenzo,C. In vitro–in vivo correlation of drug release profiles from medicated contact lenses using an in vitro eye blink model Drug Delivery and Translational Research 2023;13(4):1116-1127 [ Show Abstract ]

There is still a paucity of information on how in vitro release profiles from drug-loaded contact lenses (CLs) recorded in 3D printed eye models correlate with in vivo profiles. This work aims to evaluate the release profiles of two drug-loaded CLs in a 3D in vitro eye blink model and compare the obtained results with the release in a vial and the drug levels in tear fluid previously obtained from an animal in vivo study. In vitro release in the eye model was tested at two different flow rates (5 and 10 µL/min) and a blink speed of 1 blink/10 s. Model CLs were loaded with two different drugs, hydrophilic pravastatin and hydrophobic resveratrol. The release of both drugs was more sustained and lower in the 3D eye model compared to the in vitro release in vials. Interestingly, both drugs presented similar release patterns in the eye model and in vivo, although the total amount of drugs released in the eye model was significantly lower, especially for resveratrol. Strong correlations between percentages of pravastatin released in the eye model and in vivo were found. These findings suggest that the current 3D printed eye blink model could be a useful tool to measure the release of ophthalmic drugs from medicated CLs. Nevertheless, physiological parameters such as the composition of the tear fluid and eyeball surface, tear flow rates, and temperature should be optimized in further studies.

Phan,C. M., Ross,M., Fahmy,M., McEwen,B., Hofmann,I., Chan,V. Clark-Baba,C., Jones,L. Evaluating Viscosity and Tear Breakup Time of Contemporary Commercial Ocular Lubricants on an In Vitro Eye Model Translational Vision Science & Technology 2023;12(6):29 [ Show Abstract ]

Purpose: To evaluate the link between the viscosity of ophthalmic formulation and tear film stability using a novel in vitro eye model.

Methods: The viscosities and noninvasive tear breakup time (NIKBUT) of 13 commercial ocular lubricants were measured to evaluate the correlation between viscosity and NIKBUT. The complex viscosity of each lubricant was measured three times for each angular frequency (ranging from 0.1 to 100 rad/s) using the Discovery HR-2 hybrid rheometer. The NIKBUT measurements were performed eight times for each lubricant using an advanced eye model mounted on the OCULUS Keratograph 5M. A contact lens (CL; ACUVUE OASYS [etafilcon A]) or a collagen shield (CS) was used as the simulated corneal surface. Phosphate-buffered saline was used as a simulated fluid.

Results: The results showed a positive correlation between viscosity and NIKBUT at high shear rates (at 10 rad/s, r = 0.67) but not at low shear. This correlation was even better for viscosities between 0 and 100 mPa*s (r = 0.85). Most of the lubricants tested in this study also had shear-thinning properties. OPTASE INTENSE, I-DROP PUR GEL, I DROP MGD, OASIS TEARS PLUS, and I-DROP PUR had higher viscosity in comparison to other lubricants (P < 0.05). All of the formulations had a higher NIKBUT than the control (2.7 ± 1.2 seconds for CS and 5.4 ± 0.9 seconds for CL) without any lubricant (P < 0.05). I-DROP PUR GEL, OASIS TEARS PLUS, I-DROP MGD, REFRESH OPTIVE ADVANCED, and OPTASE INTENSE had the highest NIKBUT using this eye model.

Conclusions: The results show that the viscosity is correlated with NIKBUT, but further work is necessary to determine the underlying mechanisms.

Ramaswamy,M., Ho,B., Phan,C. M., Qin,N., Ren,C. L., Jones,L. Inexpensive and rapid fabrication of PDMS microfluidic devices for biological testing applications using low cost commercially available 3D printers Journal of Micromechanics and Microengineering 2023;33(10):105016 [ Show Abstract ]

Polydimethylsiloxane (PDMS) elastomers have been extensively used in the development of microfluidic devices, capable of miniaturizing biomolecular and cellular assays to the microliter and nanoliter range, thereby increasing the throughput of experimentation. PDMS has been widely used due to its optical clarity and biocompatibility, among other desirable physical and chemical properties. Despite the widespread use of PDMS in microfluidic devices, the fabrication process typically requires specialized facilities, instruments, and materials only available in a limited number of laboratories. To expand microfluidic research capabilities to a greater scientific population, we developed and characterized a simple and robust method of fabricating relatively inexpensive PDMS microfluidic devices using readily available reagents and commercially available 3D printers. The moulds produced from the 3D printers resolve designed microfluidic channel features accurately with high resolution ( >100 µm). The critical physical and chemical post-processing modifications we outline here are required to generate functional and optically clear microfluidic devices.

Wong,K. Y., Phan,C.M., Chan,Y.T., Chuy-Ying Yuen,A., Zhao,D., Chan,K. Y., Do,C. W., Chuen Lam,T., Han Qiao,J., Wulff,D., Hui,A., Jones,L., Wong,M. S. A review of using Traditional Chinese Medicine in the management of glaucoma and cataract Clinical and Experimental Optometry 2023;107(2):156-170 [ Show Abstract ]

Traditional Chinese Medicine has a long history in ophthalmology in China. Over 250 kinds of Traditional Chinese Medicine have been recorded in ancient books for the management of eye diseases, which may provide an alternative or supplement to current ocular therapies. However, the core holistic philosophy of Traditional Chinese Medicine that makes it attractive can also hinder its understanding from a scientific perspective – in particular, determining true cause and effect. This review focused on how Traditional Chinese Medicine could be applied to two prevalent ocular diseases, glaucoma, and cataract. The literature on preclinical and clinical studies in both English and Chinese on the use of Traditional Chinese Medicine to treat these two diseases was reviewed. The pharmacological effects, safety profile, and drug-herb interaction of selected herbal formulas were also investigated. Finally, key considerations for conducting future Traditional Chinese Medicine studies are discussed.

2022

Kapadia,W., Qin,N., Zhao,P., Phan,C.-M., Haines,L., Jones,L. Ren,C. L. Shear-Thinning and Temperature-Dependent Viscosity Relationships of Contemporary Ocular Lubricants Translational Vision Science & Technology 2022;11(3):Article 1 [ Show Abstract ]

Purpose: To evaluate the shear viscosity of contemporary, commercially available ocular lubricants at various shear rates and temperatures and to derive relevant mathematical
viscosity models that are impactful for prescribing and developing eye drops to treat dry eye disease.

Methods: The shear viscosity of 12 ocular lubricants was measured using a rheometer and a temperature-controlled bath at clinically relevant temperatures at which users may experience exposure to the drops (out of the refrigerator [4.3°C]; room temperature [24.6°C]; ocular surface temperature [34.5°C]). Three replicates for each sample at each temperature were obtained using a standard volume (0.5 mL) of each sample. The viscosity of each ocular lubricant was measured over the full range of shear rates allowed
by the rheometer.

Results: The shear viscosity of the same ocular lubricant varied significantly among the three temperatures. In general, a higher temperature resulted in smaller viscosities than a lower temperature (an average of −48% relative change from 4.3°C to 24.6°C and −21% from 24.6°C to 34.5°C). At a constant temperature, the viscosity of an ocular lubricant over the studied shear rates can be well approximated by a power-law model.

Conclusions: Rheological analysis revealed that the ocular lubricants exhibited shear-thinning behavior at the measured temperatures. Differences in the ocular lubricants’ formulations and measured temperatures resulted in different viscosities.

Translational Relevance: When prescribing eye drops, eye care professionals can select the optimal one for their patients by considering a variety of factors, including its
rheological property at physiologically relevant shear rates and temperatures, which can improve residence time on the ocular surface, while ensuring appropriate comfort and vision. However, care must be taken when using the derived mathematical models in this study because the in vivo shear behavior of the ocular lubricants has not been examined and might show deviations from those reported when placed on the ocular surface.

Pereira-da-Mota,A. F., Phan,C-M., Concheiro,A., Jones,L., Alvarez-Lorenzo,C. Testing drug release from medicated contact lenses: The missing link to predict in vivo performance Journal of Controlled Release 2022;343(March):672-702 [ Show Abstract ]

Contact lenses (CLs) offer a wide variety of advantages as ocular drug-releasing platforms, but the feasibility of medicated CL development is constrained by numerous scientific, technological, and regulatory challenges. One main difficulty is the setting of release rate specifications for each drug, since at present there are no standardized in vitro release models that can appropriately predict the performance of drug-eluting CLs once placed onto the eye. CL-adapted release tests may provide knowledge on how the drug release pattern should perform in vivo to trigger and maintain the therapeutic effects for both anterior and posterior ocular tissues. Moreover, in vitro release tests are valuable tools for quality assessment during production and to investigate the effect of a change in composition or process variables. This review aims to shed light on biorelevant ways of evaluating in vitro drug release from CLs and the feasibility of establishing in vitro-in vivo correlations (IVIVC) to predict in vivo performance. First, general guidelines and Pharmacopeia release tests for topical ophthalmic formulations as well as in vitro release tests implemented for drug-CLs in the last two decades are analyzed. Then, development of an appropriate method to investigate IVIVC is attempted from the few papers simultaneously reporting in vitro release profiles and either in vivo release or therapeutic response. Finally, key points to be considered for in vitro testing drug release from a medicated CL are suggested to pave the way to the clinical arena.

Yee,A., Phan,C-M., Jones,L. Uptake and release of polyhexamethylene biguanide (PHMB) from hydrogel and silicone hydrogel contact lenses using a radiolabel methodology Contact Lens Anterior Eye 2022;45(5):101575 [ Show Abstract ]

Purpose
The purpose of this study was to evaluate the uptake and release of radiolabelled polyhexamethylene biguanide (PHMB) on reusable daily wear contact lenses (CLs) over 7 days.

Methods
Three silicone hydrogel (SH) contact lens materials (lotrafilcon B, balafilcon A, senofilcon A) and two conventional hydrogel (CH) materials (etafilcon A, omafilcon A) were examined. In experiment 1 (1-day study), CLs were soaked in 2 mL of phosphate buffered solution (PBS) containing radiolabelled 14C PHMB (1 µg/mL) for 8 h. The release kinetics of 14C PHMB from the CLs was measured at t = 0.25, 0.5, 1, 2, 4, 8, and 24 h in fresh 2 mL PBS. In experiment 2 (7-day study), the CLs were soaked in the 14C PHMB (1 µg/mL) solution for 8 h followed by a 16-hour release in 2 mL PBS. The lens cycle was repeated daily for 7 days. After both experiments, the residual amount of PHMB remaining within the lenses was extracted to determine the total uptake of PHMB.

Results
In experiment 1, the total uptake of PHMB for etafilcon A was significantly greater than senofilcon A (p = 0.01). There were no significant differences in total uptake of PHMB between other lens materials (p > 0.05). Etafilcon A released more PHMB compared to all other lens types over a 24-hr period (p < 0.001). In experiment 2, all CL materials continued to sorb more PHMB over time (p < 0.001). By day 7, the amount of PHMB sorbed by etafilcon A was significantly greater than senofilcon A (p = 0.02). After day 2, the CH materials released significantly more PHMB than the SH materials (p < 0.01).

Conclusion
The CL materials continued to sorb PHMB with no signs of saturation after 7 days. All lens materials released a consistent amount of PHMB each day. Radioactive labelling provides a sensitive method of assessing the uptake and release of PHMB from CL materials.

2021

Chan,V. W. Y., Phan,C-M., Ngo,W., Jones,L. Lysozyme Deposition on Contact Lenses in an In Vitro Blink-Simulation Eye Model Versus a Static Vial Deposition Model Eye & Contact Lens 2021;47(7):388-393 [ Show Abstract ]

Purpose:
To evaluate active lysozyme deposition on daily disposable (DD) contact lenses (CL) using a novel in vitro blink model.

Methods:
Three conventional hydrogel DD CL materials (etafilcon A, omafilcon A, nelfilcon A) and three silicone hydrogel DD CL materials (delefilcon A, senofilcon A, somofilcon A) were tested. The device blink rate was set to 6 blinks/min with a tear flow rate of 1 μL/min using an artificial tear solution (ATS) containing lysozyme and other typical tear film components. After incubation at 2, 4, or 8 hr, lenses were removed, and lysozyme activity was measured. A separate experiment was conducted with lenses incubated in a static vial containing 480 μL of ATS.

Results:
Etafilcon A deposited significantly higher amounts of active lysozyme (402±102 μg/lens) than other lens materials after 8 hr (P<0.0001). Etafilcon A had a higher amount of active lysozyme using the blink model compared with the static vial (P=0.0435), whereas somofilcon A (P=0.0076) and senofilcon A (P=0.0019) had a higher amount of lysozyme activity in the vial compared with the blink model.

Conclusion:
The blink model can be tuned to provide quantitative data that closely mimics ex vivo studies and can be used to model deposition of lysozyme on CL materials.

Chan,V. W. Y., Phan,C-M., Walther,H., Ngo,W., Jones,L. Effects of Temperature and Blinking on Contact Lens Dehydration of Contemporary Soft Lens Materials Using an In Vitro Blink Model Translational Vision Science & Technology 2021;10(8):11 [ Show Abstract ]

Purpose: The purpose of this studywas to evaluate the effects of temperature and blinking on contact lens (CL) dehydration using an in vitro blink model.

Methods: Three silicone hydrogel (delefilcon A, senofilcon A, and comfilcon A) and two conventional hydrogel (etafilcon A and omafilcon A) CL materials were evaluated at 1
and 16 hours. The water content (WC) of the CLs was measured using a gravimetric method. Lenses were incubated on a blink model, internally heated to achieve a clinically relevant surface temperature of 35°C. An artificial tear solution (ATS) was delivered to the blink model at 4.5 μL/min with a blink rate of 6 blinks/min. A comparison set of lenses were incubated in a vial containing either 2 mL of ATS or phosphate-buffered saline (PBS) at 35°C.

Results: Increasing temperature to 35°C resulted in a decrease in WC for all tested CLs over time (P ≤ 0.0052). For most CLs, there was no significant difference in WC over time between ATS or PBS in the vial (P > 0.05). With the vial system, WC decreased and plateaued over time. However, on the blink model, for most CLs, the WC significantly decreased after 1 hour but returned toward initial WC levels after 16 hours (P > 0.05).

Conclusions: The reduction in WC of CLs on the eye is likely due to both an increase in temperature and dehydration from air exposure and blinking.

Translational Relevance: This study showed that the novel, heated, in vitro blink model could be used to provide clinical insights into CL dehydration on the eye.

Jones,L., Hui,A., Phan,C-M., Read,M. L., Azar,D., Buch,J., Ciolino,J. B., Naroo,S. A., Pall,B., Romond,K., Saknaridurg,P., Schnider,C. M., Terry,L., Willcox,M. CLEAR - Contact lens technologies of the future Contact Lens Anterior Eye 2021;44(2):398-430 [ Show Abstract ]

Contact lenses in the future will likely have functions other than correction of refractive error. Lenses designed to control the development of myopia are already commercially available. Contact lenses as drug delivery devices and powered through advancements in nanotechnology will open up further opportunities for unique uses of contact lenses.

This review examines the use, or potential use, of contact lenses aside from their role to correct refractive error. Contact lenses can be used to detect systemic and ocular surface diseases, treat and manage various ocular conditions and as devices that can correct presbyopia, control the development of myopia or be used for augmented vision. There is also discussion of new developments in contact lens packaging and storage cases.

The use of contact lenses as devices to detect systemic disease has mostly focussed on detecting changes to glucose levels in tears for monitoring diabetic control. Glucose can be detected using changes in colour, fluorescence or generation of electric signals by embedded sensors such as boronic acid, concanavalin A or glucose oxidase. Contact lenses that have gained regulatory approval can measure changes in intraocular pressure to monitor glaucoma by measuring small changes in corneal shape. Challenges include integrating sensors into contact lenses and detecting the signals generated. Various techniques are used to optimise uptake and release of the drugs to the ocular surface to treat diseases such as dry eye, glaucoma, infection and allergy. Contact lenses that either mechanically or electronically change their shape are being investigated for the management of presbyopia. Contact lenses that slow the development of myopia are based upon incorporating concentric rings of plus power, peripheral optical zone(s) with add power or non-monotonic variations in power. Various forms of these lenses have shown a reduction in myopia in clinical trials and are available in various markets.

Phan,C. M., Qiao,H., Yee,A., Jones,L. Deposition of Fluorescently Tagged Lysozyme on Contact Lenses in a Physiological Blink Model Eye & Contact Lens 2021;47(2):127-133 [ Show Abstract ]

PURPOSE: To visualize the deposition of fluorescein isothiocyanate (FITC) lysozyme on daily disposable contact lenses (CLs) using a novel blink model.

METHODS: Three daily disposable conventional hydrogel CLs (etafilcon A, omafilcon A, and nelfilcon A) and three silicone hydrogel CLs (delefilcon A, senofilcon A, and somofilcon A) were evaluated in the study. The CLs were mounted onto a novel blink model and exposed to an artificial tear solution containing FITC lysozyme for 2 and 10 hr. The flow rate and blink speed were set to 1 μL/min and 6 blinks/min, respectively. After the incubation period, a 5-mm-diameter disc was punched out from the center of the lens and mounted on a microscope slide. The slides were imaged using the Zeiss 510 Meta confocal laser scanning microscope, which scanned the lens from the front to the back surface at 5-μm increments.

RESULTS: There was an increase in deposition of FITC lysozyme for all lens types with increasing incubation time (P0.05). The conventional hydrogel CLs deposited higher amounts of FITC lysozyme than the silicone hydrogel CLs (P<0.001), with etafilcon A depositing the highest at all time points (P<0.05). Interestingly, at the 2-hr incubation time, most CLs showed a higher amount of deposition at the front surface than the back surface of the lens. In particular, etafilcon A showed preferred deposition at the front surface at all time points.

CONCLUSION: The results suggest that there is differential deposition at the front surface of the CL, which is exposed to the prelens tear film, compared with the back surface of the CL, which is exposed to the postlens tear film. Therefore, it may be beneficial to design CL materials with differing surface properties for the front and back surfaces of the CL to enhance interactions with the tear film and ocular surface.

Phan,C. M., Shukla,M., Walther,H., Heynen,M., Suh,D., Jones,L. Development of an In Vitro Blink Model for Ophthalmic Drug Delivery Pharmaceutics 2021;13(Article 300):1-10 [ Show Abstract ]

Purpose: The purpose of this study was to develop an advanced in vitro blink model that
can be used to examine the release of a wide variety of components (for example, topical ophthalmic
drugs, comfort-inducing agents) from soft contact lenses. Methods: The model was designed using
computer-aided design software and printed using a stereolithography 3D printer. The eyelid and
eyeball were synthesized from polyvinyl alcohol and silicone material, respectively. Simulated
tear fluid was infused through tubing attached to the eyelid using a syringe pump. With each
blink cycle, the eyelid slides and flexes across the eyeball to create an artificial tear film layer. The
flow-through fluid was collected using a specialized trough. Two contact lenses, etafilcon A and
senofilcon A, were incubated in 2 mL of a water-soluble red dye for 24 h and then placed on the eye
model (n = 3). The release of the dye was measured over 24 h using a tear flow rate of 5 µL/min.
Results: Approximately 25% of the fluid that flowed over the eye model was lost due to evaporation,
nonspecific absorption, and residual dead volume. Senofilcon A absorbed more dye (47.6 ± 2.7 µL)
than etafilcon A (22.3 ± 2.0 µL). For etafilcon A, the release of the dye followed a burst-plateau
profile in the vial but was sustained in the eye model. For senofilcon A, the release of the dye was
sustained in both the vial and the eye model, though more dye was released in the vial (p < 0.05).
Overall, the release of the dye from the contact lenses was higher in the vial compared with the eye
model (p < 0.05). Conclusion: The blink model developed in this study could be used to measure
the release of topical ophthalmic drugs or comfort agents from contact lenses. Simulation of a blink
mechanism, an artificial tear film, and nonspecific absorption in an eye model may provide better
results than a simple, static vial incubation model.

Yee,A., Chan,V., Heynen,M., Phan,C. M., Jones,L. Uptake and release of a multipurpose solution biocide (MAP-D) from hydrogel and silicone hydrogel contact lenses using a radiolabel methodology Eye & Contact Lens 2021;47(5):249-255 [ Show Abstract ]

Purpose:
The purpose of this study was to evaluate the uptake and release of radiolabelled myristamidopropyl dimethylamine (MAP-D) on reusable daily wear contact lenses (CLs) over 7 days.

Methods:
Three silicone hydrogel (SH) CL materials (lotrafilcon B, balafilcon A, senofilcon A) and two conventional hydrogel (CH) materials (etafilcon A, omafilcon A) were tested. A short-term (experiment 1, N=4) and a longer-term (experiment 2, N=3) study was conducted. In experiment 1, the CLs were incubated in 2 mL of phosphate buffered solution (PBS) containing 14C MAP-D (5 μg/mL) for 8 hrs. The release of 14C MAP-D was measured at t=0.25, 0.5, 1, 2, 4, 8, and 24 hr in PBS. In experiment 2, the CLs were incubated in the 14C MAP-D solution for 8 hrs followed by a 16-hr release in PBS. This cycle was repeated daily for 7 days. At the end of both experiments, lenses were extracted to determine the total uptake of MAP-D. The radioactivity was measured using a beta scintillation counter.

Results:
In experiment 1, all three SH lenses sorbed similar amounts of MAP-D (P=0.99), all of which were higher than the two CH materials (P<0.01). However, the CH materials released a greater amount of MAP-D than the SH materials (P<0.01). In experiment 2, the uptake of MAP-D in SH materials increased over 7 days, whereas the amount of MAP-D remained constant in the CH materials (P=0.99). Similar to experiment 1, the CH lenses released more MAP-D than SH lenses after 7 days (P<0.01).

Conclusion:
The SH materials absorbed greater amounts of MAP-D compared to CH materials. However, the CH materials released the greatest amount of MAP-D. Radioactive labelling of MAP-D offers a highly sensitive method of assessing the uptake and release profiles of biocides to CL materials.

2019

Phan,C. M., Subbaraman,L., Jones,L. Uptake and release of polyvinyl alcohol from hydrogel daily disposable contact lenses Optom Vis Sci 2019;96(3):180-186 [ Show Abstract ]

SIGNIFICANCE:
Polyvinyl alcohol is a wetting agent that could reduce the symptoms of dry eye and contact lens discomfort. Currently, only one lens type, nelfilcon A (DAILIES AquaComfort Plus), releases polyvinyl alcohol. The concept of releasing this agent from contact lenses could be applied to other lens materials.

PURPOSE:
The purpose of this study was to measure the release of polyvinyl alcohol from commercially available hydrogel daily disposable contact lenses using refractive index and iodine-borate methods.

METHODS:
Etafilcon A, omafilcon A, and nelfilcon A were soaked in phosphate-buffered saline and 0.2% trifluoroacetic acid/acetonitile for 24 hours to remove residual blister pack components. The lenses were then incubated in a 10-mg/mL solution of polyvinyl alcohol for 24 hours. After the incubation period, the lenses were placed in 2 mL of phosphate-buffered saline. At specified time intervals, t = 0.5, 1, 2, 4, 8, 12, and 24 hours, the samples were evaluated using refractive index and an iodine-borate assay. Polyvinyl alcohol uptake was determined by extracting the lenses with methanol for 24 hours.

RESULTS:
There were no differences in the uptake of polyvinyl alcohol between lens types (P > .05). The release of this wetting agent for all lens types followed a burst-plateau profile after the first 30 minutes (P > .05). Nelfilcon A had a slightly higher release of polyvinyl alcohol (P .05).

CONCLUSIONS:
The results suggest that the contact lenses tested in this study have similar efficiency in delivering polyvinyl alcohol.

Phan,C. M., Walther,H., Qiao,H., Shinde,R., Jones,L. Development of an eye model with a physiological blink mechanism Translational Vision Science & Technology 2019;8(5):Article 1 [ Show Abstract ]

Purpose: To develop an eye model with a physiological blink mechanism.

Methods: All parts of the eye model were designed using computer-aided design software. The eyelid consisted of a unique 3D printed structure containing teeth to physically secure a flexible membrane. Both the eyeball and eyelid membrane were synthesized using polyvinyl alcohol (PVA). Four molecular weights of PVA (89–98, 85–124, 130, and 146–186 kDa) were tested at a range of concentrations between 5% and 30% weight/volume. The wettability and water content of these materials were compared with the bovine cornea and sclera. The model was connected to a microfluidic pump, which delivers artificial tear solution (ATS) to the eyelid. A corneal topographer was used to evaluate the tear break-up and tear film regeneration.

Results: The eyelid flexes and slides across the eyeball during each blink, which ensures direct contact between the two surfaces. When loaded with an ATS, this mechanism evenly spreads the solution over the eyeball to generate an artificial tear film. The artificial tear film in this eye model had a tear break-up time (TBUT) of 5.13 ± 0.09 seconds at 1.4 μL/min flow rate, 6 blinks/min, and <25% humidity.

Conclusions: This model simulates a physiological blink actuation and an artificial tear film layer. Future studies will examine variations in flow rates and ATS composition to simulate clinical values of TBUT.

Translational Relevance: The eye model could be used to study in vitro TBUT, tear deposition, and simple drug delivery.

Phan,C. M., Walther,H., Riederer,D., Lau,C., Lorentz,H., Subbaraman,L., Jones,L. Analysis of polyvinyl alcohol release from commercially available daily disposable contact lenses using an in vitro eye model J Biomed Mater Res B Appl Biomater 2019;107(5):1662-1668 [ Show Abstract ]

The purpose of this work was to determine the release of polyvinyl alcohol (PVA) from etafilcon A, omafilcon A, and nelfilcon A daily disposable hydrogel contact lenses using a novel in vitro model. PVA is an ocular lubricant that can be found in multiple formulations of artificial tears. Nelfilcon A innately contains PVA, so only the release of PVA from this lens was evaluated. Etafilcon A and omafilcon A lenses were incubated in a PBS solution containing PVA. The release of PVA was evaluated using a novel in vitro blink platform with Milli-Q water and PBS under various blink conditions and flow rates. Nelfilcon A lenses significantly released more PVA than other lenses at 0.5 and 1.5 h in both PBS and Milli-Q water (p 0.05). All tested groups and lenses showed a burst release within the first 4.5 h and rapidly plateaued thereafter. The current study demonstrates that releasable PVA (whether through uptake or through being inherently available from the material) is loosely bound on hydrogel lenses, and the majority is released within 4.5 h.

2018

Phan,C. -M, Walther,H., Smith,R. W., Riederer,D., Lau,C., Lorenz,K. O., Subbaraman,L. N., Jones L. Determination of the release of PEG and HPMC from nelfilcon A daily disposable contact lenses using a novel in vitro eye model. J Biomater Sci Polym Ed 2018;29(17):2124-2136 [ Show Abstract ]

The traditional method to measure release of components from CLs is a vial containing a static volume of PBS (phosphate buffered saline). However, this model does not simulate physiologically relevant tear volume and natural tear flow, air exposure, and mechanical rubbing. These factors can significantly impact release kinetics. We have developed an in vitro eye model (OcuFlow) that simulates these parameters. The aim of the study was to measure the release of PEG (polyethylene glycol), and HPMC (hydroxypropyl methylcellulose) from a daily disposable hydrogel contact lens material (nelfilcon A; Dailies AquaComfort PLUS; DACP;) over 24 hrs using the OcuFlow platform. The elution of PEG and HPMC from DACP lenses was analyzed using LCMS (liquid chromatography mass spectrometry). The release of all wetting agents from the lenses followed a burst release pattern, which occurred within the first 1.5 hrs (P < 0.05). The release of PEG was greater than that of HPMC (P < 0.05). The amount of PEG and HPMC released at any given time was less than 1% of the amount in the blister pack solution. Our results suggest that HPMC and PEG are rapidly released from the CL.

Phan,C. -M, Weber,S., Mueller,J., Yee,A., Jones,L. A rapid extraction method to quantify drug uptake in contact lenses Translational Vision Science and Technology 2018;7(2):11 [ Show Abstract ]

Purpose: To develop a simple extraction procedure to quantify the uptake of four topical ocular pharmaceutical drugs into contact lenses (CLs). Methods: Four silicone hydrogel (SH) CLs (balafilcon A, senofilcon A, lotrafilcon B, comfilcon B) and four conventional hydrogel (CH) CLs (nesofilcon A, hilafilcon B, nelfilcon A, etafilcon A) were evaluated. The drugs studied were natamycin, moxifloxacin, timolol maleate, and ketotifen fumarate. For drug incubation, three CLs of each type were placed in 1 mL of 1 mg/mL drug-loading solution for 24 hours. The lenses were then extracted in 2 mL methanol for 2 hours. This process was repeated to obtain a total of three extraction cycles. Detection of natamycin, moxifloxacin, ketotifen fumarate, and timolol maleate were measured by absorbance at 305, 287, 297, and 295 nm, respectively. Results: The majority of the drugs were extracted after the first extraction cycle (P 0.05). Conclusions: This study provides a simple approach to determine drug uptake into CLs. This method can also be modified, such as changing the extraction time, extraction cycles, or extraction solvent to better suit other drugs and CL combinations. Translational Relevance: There is considerable interest in using CLs for ocular drug delivery. Accurately quantifying drug uptake on CLs has been a challenge. Hence, this study provides a simple method to quantify drug uptake in CLs. © 2018 The Authors.

Qiao,H., Phan,C. -M., Walther,H., Subbaraman,L. N., Jones,L. Depth Profile Assessment of the Early Phase Deposition of Lysozyme on Soft Contact Lens Materials Using a Novel In Vitro Eye Model 2018;44(Suppl 2):S11-S18 [ Show Abstract ]

OBJECTIVE: To characterize the location of fluorescently labeled lysozyme on commercial contact lenses (CLs) using an in vitro eye model that simulates tear volume, tear flow, air exposure, and mechanical wear.
METHODS: One commercially available conventional hydrogel CL material (etafilcon A) and three silicone hydrogel CL materials (balafilcon A, lotrafilcon B, and senofilcon A) were evaluated in this study. The CLs were mounted on the in vitro eye model and exposed to artificial tear fluid containing fluorescein isothiocyanate (FITC)-labeled lysozyme for 2 and 10 hrs. After these short incubation periods, circular discs were punched from the CLs at the center and periphery and were prepared for confocal laser scanning microscopy (CLSM). The CLSM captured a series of consecutive images spaced 5 μm apart, and the resulting images were rendered into two dimensional cross-sectional views of the CL. The mean fluorescence at each 5 μm slice was used to generate a histogram depicting the penetration of FITC-lysozyme into CLs.
RESULTS: For both incubation periods, the CLSM images and histogram of etafilcon A showed that FITC-lysozyme is more concentrated at the lens surface, with a moderate amount of deposition in the lens matrix. For balafilcon A, FITC-lysozyme was evenly distributed throughout the lens. For lotrafilcon B, there was a greater amount of FITC-lysozyme deposition on the surfaces of the lens versus the matrix. Senofilcon A had differential FITC-lysozyme distribution profiles depending on the location of the lens. At the lens periphery, FITC-lysozyme primarily deposited on the surface, whereas FITC-lysozyme was uniformly distributed at the center of the lens.
CONCLUSIONS: With the use of a sophisticated in vitro eye model, the study revealed a complex deposition pattern of FITC-labeled lysozyme on various CL materials after short periods of exposure. An understanding of the early deposition pattern of lysozyme on different CL material may elucidate new insights into the processes behind CL discomfort.

Walther,H., Phan,C. -M., Subbaraman,L. N., Jones,L. Differential deposition of fluorescently tagged cholesterol on commercial contact lenses using a novel in vitro eye model Translational Vision Science and Technology 2018;7(2):18 [ Show Abstract ]

Purpose: We evaluate the differences in lipid uptake and penetration in daily disposable (DD) contact lenses (CL) using a conventional “in-vial” method compared to a novel in vitro eye model. Methods: The penetration of fluorescently labelled 22-(N-(7-Nitrobenz-2-Oxa-1,3- Diazol-4-yl)Amino)-23,24-Bisnor-5-Cholen-3beta-Ol (NBD)–cholesterol on three silicone hydrogel (SH) and four conventional hydrogel (CH) DD CLs were investigated. CLs were incubated for 4 and 12 hours in a vial, containing 3.5 mL artificial tear solution (ATS), or were mounted on an in vitro eye-blink platform designed to simulate physiologic tear flow (2 mL/24 hours), tear volume and “simulated” blinking. Subsequently, CLs were analyzed using laser scanning confocal microscopy and ImageJ. Results: Penetration depth and fluorescence intensities of NBD-cholesterol varied between the incubation methods as well as lens materials. Using the traditional vial incubation method, NBD-cholesterol uptake occurred equally on both sides of all lens materials. However, using our eye-blink model, cholesterol penetration was observed primarily on the anterior surface of the CLs. In general, SH lenses showed higher intensities of NBD-cholesterol than CH materials. Conclusions: The traditional “in-vial” incubation method exposes the CLs to an excessively high amount of ATS, which results in an overestimation for cholesterol deposition. Our model, which incorporates important ocular factors, such as intermittent air exposure, small tear volume, and physiological tear flow between blinks, provides a more natural environment for in vitro lens incubation. Translational Relevance: In vitro measurements of CLs are a common approach to predict their interactions and performance on the eye. Traditional methods, however, are rudimentary. Therefore, this study presents a novel in vitro model to evaluate CLs, which consequently will enhance elucidations of the interactions between CLs and the eye.

2017

Hui,A., Bajgrowicz-Cieslak,M., Phan,C. -M, Jones,L. In vitro release of two anti-muscarinic drugs from soft contact lenses Clinical Ophthalmology 2017;11:1657-1665 [ Show Abstract ]

The purpose of this study was to investigate the release of the anti-myopia drugs atropine sulfate and pirenzepine dihydrochloride from commercially available soft contact lenses. Standard ultraviolet (UV) absorbance-concentration curves were generated for atropine and pirenzepine. Ten commercially available contact lenses, including four multifocal lenses, were loaded by soaking in atropine or pirenzepine solutions at two different concentrations (10 mg/mL and 1 mg/mL). The release of the drugs into phosphate-buffered saline was determined over the course of 24hours at 34°C using UV absorbance. Materials with surface charge released the greatest amount of atropine when loaded with either concentration when compared to the other lens types (p<0.05), releasing upward of 1.026±0.035mg/lens and 0.979±0.024mg/lens from etafilcon A and ocufilcon A, respectively. There were no significant differences in the amount of atropine or pirenzepine released from the multifocal and non-multifocal lenses made from the same lens materials. Narafilcon A material demonstrated prolonged release of up to 8 hours when loaded with pirenzepine, although the overall dose delivered from the lens into the solution was among the lowest of the materials investigated. The rest of the lenses reached a plateau within 2 hours of release, suggesting that they were unable to sustain drug release into the solution for long periods of time. Given that no single method of myopia control has yet shown itself to be completely effective in preventing myopia progression, a combination of optical and pharmaceutical devices comprising a drug delivering contact lens presents a novel solution that warrants further investigation. © 2017 Hui et al.

2016

Phan,C. -M, Bajgrowicz-Cieslak,M., Subbaraman,L. N., Jones,L. Release of Moxifloxacin from Contact Lenses Using an In Vitro Eye Model: Impact of Artificial Tear Fluid Composition and Mechanical Rubbing Transl Vis Sci Technol. 2016;5(6):3 [ Show Abstract ]

PURPOSE: The aim of this study was to evaluate and compare the release of moxifloxacin from a variety of daily disposable (DD) contact lenses (CLs) under various conditions using a novel in vitro eye model.
METHODS: Four commercially available DD conventional hydrogel (CH) CLs (nelfilcon A, omafilcon A, etafilcon A, and ocufilcon B) and three silicone hydrogel (SH) CLs (somofilcon A, narafilcon A, and delefilcon A) were evaluated. These lenses were incubated in moxifloxacin for 24 hours. The release of the drug was measured using a novel in vitro model in three experimental conditions: (1) phosphate buffered saline (PBS); (2) artificial tear solution (ATS) containing a variety of proteins and lipids; and (3) ATS with mechanical rubbing produced by the device.
RESULTS: Overall, CH CLs had a higher drug release than SH CLs (P < 0.05) under all conditions. Typically, a higher drug release was observed in PBS than ATS (P < 0.05). For CH, drug release was found to be higher in ATS with rubbing than PBS or ATS (P < 0.05). For most lens types, ATS with rubbing produced higher drug release than ATS alone (P < 0.05). Generally, the release kinetics for all conditions were sustained over the 24-hour testing period, and no burst release was observed (P < 0.05).
CONCLUSIONS: Moxifloxacin release from a CL into ATS is lower when compared to release into PBS. When mechanical rubbing is introduced, the amount of drugs released is increased.
TRANSLATIONAL RELEVANCE: Results suggest that sophisticated in vitro models are necessary to adequately model on-eye drug release from CL materials.

Phan,C. -M, Bajgrowicz,M., Gao,H., Subbaraman,L. N., Jones,L. W. Release of fluconazole from contact lenses using a novel in vitro eye model Optometry and Vision Science 2016;93(4):387-394 [ Show Abstract ]

Purpose. Rapid drug release followed by a plateau phase is a common observation with drug delivery from contact lenses (CLs) when evaluated in a vial. The aim of this study was to compare the release of fluconazole from seven commercially available daily disposable CLs using a conventional vial-based method with a novel in vitro eye model. Methods. An eye model was created using two 3-dimensional printed molds, which were filled with polydimethylsiloxane to obtain an inexpensive model that would mimic the eyeball and eyelid. The model was integrated with a microfluidic syringe pump, and the flow-through was collected in a 12-well microliter plate. Four commercial daily disposable conventional hydrogels (nelfilcon A, omafilcon A, etafilcon A, ocufilcon B) and three silicone hydrogels (somofilcon A, narafilcon A, delefilcon A) were evaluated. These CLs were incubated with fluconazole for 24 h. The drug release was measured in a vial containing 4.8 mL of phosphate-buffered saline and in the polydimethylsiloxane eye model with a 4.8-mL tear flow across 24 h. Results. Overall, conventional hydrogel CLs had a higher uptake and release of fluconazole than silicone hydrogel CLs (p < 0.05). A higher drug release was observed in the vial condition compared with the eye model (p < 0.001). In the vial system, the drugs were rapidly released from the CL within the first 2 h, followed by a plateau phase. In contrast, drug release in the eye model under low tear volume was sustained and did not reach a plateau across 24 h (p < 0.05). Conclusions. Rapid drug release results from using a vial as the release system. Under low tear volume at physiological tear flow, commercial CLs can maintain a sustained drug release profile for up to 24 h. However, eyes with fungal keratitis may have increased tearing, which would significantly accelerate drug release. © 2015 American Academy of Optometry.

Phan,C. -M, Bajgrowicz,M., McCanna,D. J., Subbaraman,L. N., Jones,L. Effects of Antifungal Soaked Silicone Hydrogel Contact Lenses on Candida albicans in an Agar Eye Model Eye and Contact Lens 2016;42(5):313-317 [ Show Abstract ]

Purpose: To evaluate the effects of two commercial silicone hydrogel contact lenses (CLs) soaked with natamycin (NA) or fluconazole (FL) on the growth of Candida albicans in an in vitro eye model. Methods: Three-D printed molds were used as a cast for making eye-shaped models comprising potato dextrose agar. Senofilcon A (SA) and lotrafilcon B (LB) CLs were incubated with either 2 mL of NA or FL at a concentration of 1 mg/mL for 24 hr. To simulate a fungal infection, the eye models were coated with C. albicans. The drug-soaked lenses were placed on top of the eye models. Seven experimental conditions were examined: (1) NA-SA, (2) NA-LB, (3) FL-SA, (4) FL-LB, (5) SA, (6) LB, and (7) control - no lens. At specified time points (t1, 8, 16, 24, 48 hr), the agar eyes from each experimental condition were removed from the incubator and photographed. The yeast cells from the 24 and 48 hr time point were also analyzed using light microscopy. Results: At 24 and 48 hr, there was considerable growth observed for all conditions except for the NA-SA and NA-LB conditions. When observed under the microscope at 24 and 48 hr, the morphology of the yeast cells in the FL-SA and SA condition were similar to that of the control (oval shaped). There was limited hyphae growth observed for LB and significant visible hyphae growth for the NA-LB group. For NA-SA, NA-LB, and FL-LB groups, the cells were significantly smaller compared with the control. Conclusions: For NA-SA and NA-LB, there was limited growth of C. albicans observed on the eye models even after 48 hr. Under the microscope, the cell morphology differ noticeably between each testing condition, and is dependent on drug-lens combinations. © 2015 Contact Lens Association of Ophthalmologists.

Phan,C. -M, Subbaraman,L., Jones,L. W. The use of contact lenses as biosensors Optometry and Vision Science 2016;93(4):419-425 [ Show Abstract ]

The tear film is a complex multilayer film consisting of various proteins, enzymes, and lipids and can express a number of biomarkers in cases of disease. The development of a contact lens sensor presents a noninvasive alternative for the detection and management of various diseases. Recent work has resulted in the commercialization of a device to monitor intraocular pressure for up to 24 h, and there are extensive efforts underway to develop a contact lens sensor capable of continuous glucose tear film monitoring to manage diabetes. This clinical perspective will highlight the major developments within this field and list some of the major challenges that still need to be addressed. © 2015 American Academy of Optometry.

Phan,C. -M, Walther,H., Gao,H., Rossy,J., Subbaraman,L. N., Jones,L. Development of an in Vitro ocular platform to test contact lenses Journal of Visualized Experiments 2016;2016(110):e53907 [ Show Abstract ]

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

2015

Bajgrowicz,M., Phan,C. -M, Subbaraman,L. N., Jones,L. Release of ciprofloxacin and moxifloxacin from daily disposable contact lenses from an in vitro eye model Investigative Ophthalmology and Visual Science 2015;56(4):2234-2242 [ Show Abstract ]

Purpose. To analyze the release of two fluoroquinolones, ciprofloxacin and moxifloxacin, from conventional hydrogel (CH) and silicone hydrogel (SH) daily disposable contact lenses (CLs), comparing release from a fixed-volume vial and a novel in vitro eye model. Methods. Four CH CLs (nelfilcon A, omafilcon A, etafilcon A, ocufilcon B) and three SH CLs (somofilcon A, narafilcon A, delefilcon A) were used. The lenses were incubated in drug solutions for 24 hours. After the incubation period, the lenses were placed in two release conditions: (1) a vial containing 4.8 mL PBS for 24 hours and (2) an in vitro eye model with a flow rate at 4.8 mL over 24 hours. Results. Release in the vial for both drugs was rapid, reaching a plateau between 15 minutes and 2 hours for all lenses. In contrast, under physiological flow conditions, a constant and slow release was observed over 24 hours. The amounts of ciprofloxacin released from the lenses ranged between 49.6 ±0.7 and 62.8 ± 0.3 µg per lens in the vial, and between 35.0 ± 7.0 and 109.0 ± 5.0 µg per lens in the eye model. Moxifloxacin release ranged from 24.0 ± 4.0 to 226.0 ± 2.0 µg per lens for the vial, and between 13.0 ± 2.0 and 151.0 ± 10.0 µg per lens in the eye model. In both systems and for both drugs, HEMA-based CLs released more drugs than other materials. Conclusions. The parameters of the release system, in particular the volume and flow rate, have a significant influence on measured release profiles. Under physiological flow, release profiles are significantly slower and constant when compared with release in a vial. © 2015, The Association for Research in Vision and Ophthalmology, Inc.

2014

Hall,B., Phan,C. -M, Subbaraman,L., Jones,L. W., Forrest,J. Extraction versus in Situ techniques for measuring surface-adsorbed lysozyme Optometry and Vision Science 2014;91(9):1062-1070 [ Show Abstract ]

PURPOSE: To compare two techniques for measuring the activity of lysozyme deposited onto hydrogel contact lens and to image the binding of Micrococcus lysodeikticus to contact lenses. METHODS: Using a previously described protein extraction technique and a recently developed in situ technique, we measured the time-dependent activity of adsorbed lysozyme on six different contact lens materials during the first minute and up to 1 week of interaction with the material surface. Total activity of extracted lysozyme, total in situ activity, and the activity of the outer surface layer of sorbed lysozyme were determined using the two different techniques. Micrococcal cellular interaction with surface-adsorbed lysozyme was imaged using confocal microscopy. RESULTS: The differences between total extracted activities, total in situ activities, and surface activities were both measurable and material specific. In most cases, total extracted activity is greater than total in situ activity, which, in turn, is greater than surface activity. After 1 week, etafilcon A had the highest extracted activity at 137 µg/lens, followed by omafilcon A, balafilcon A, comfilcon A, senofilcon A, and lotrafilcon B at 27.4, 2.85, 2.02, 0.46, and 0.27 µg/lens, respectively. Micrococcal cell adhesion was greatest on contact lenses with high contact angles, such as balafilcon A, omafilcon A, and senofilcon A and lowest on contact lenses with low contact angles, such as etafilcon A, comfilcon A, and lotrafilcon B. Subsequent removal/prevention of adhered micrococcal cells was greatest on balafilcon A, which had the highest surface activity, and lowest on lotrafilcon B, which had the lowest surface activity. CONCLUSIONS: This study has measured and made direct comparisons between two established techniques for measuring the activity of adsorbed lysozyme. The extraction technique determines the activity of underlying layers of lysozyme or lysozyme within the matrix of the material. Conversely, the in situ technique allows conclusions to be drawn about only the biologically relevant lysozyme including the activity of just the outer surface of adsorbed lysozyme. © American Academy of Optometry.

Phan,C. -M, Subbaraman,L. N., Jones,L. In vitro drug release of natamycin from ß-cyclodextrin and 2-hydroxypropyl-ß-cyclodextrin-functionalized contact lens materials Journal of Biomaterials Science, Polymer Edition 2014;25(17):1907-1919 [ Show Abstract ]

Purpose: The antifungal agent natamycin can effectively form inclusion complexes with beta-cyclodextrin (ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (HP-ßCD) to improve the water solubility of natamycin by 16-fold and 152-fold, respectively (Koontz, J. Agric. Food. Chem. 2003). The purpose of this study was to develop contact lens materials functionalized with methacrylated ß-CD (MßCD) and methacrylated HP-ßCD (MHP-ßCD), and to evaluate their ability to deliver natamycin in vitro. Methods: Model conventional hydrogel (CH) materials were synthesized by adding varying amounts of MßCD and MHP-ßCD (0, 0.22, 0.44, 0.65, 0.87, 1.08% of total monomer weight) to a monomer solution containing 2-hydroxyethyl methacrylate (HEMA). Model silicone hydrogel (SH) materials were synthesized by adding similar concentrations of MßCD and MHP-ßCD to N,N-dimethylacrylamide (DMAA)/10% 3-methacryloxypropyltris(trimethylsiloxy)silane (TRIS). The gels were cured with UV light, washed with ethanol and then, hydrated for 24 h (h). The model materials were then incubated with 2 mL of 100 g/mL of natamycin in phosphate buffered saline (PBS) pH 7.4 for 48 h at room temperature. The release of natamycin from these materials in 2 mL of PBS, pH 7.4 at 32 ± 2 °C was monitored using UV-vis spectrophotometry at 304 nm over 24 h. Results: For both CH and SH materials, functionalization with MßCD and MHP-ßCD improved the total amount of drugs released up to a threshold loading concentration, after which further addition of methacrylated CDs decreased the amount of drugs released (p < 0.05). The addition of CDs did not extend the drug release duration; the release of natamycin by all model materials reached a plateau after 12 h (p < 0.05). Overall, DMAA/10% TRIS materials released significantly more drug than HEMA materials (p < 0.05). The addition of MHP-ßCD had a higher improvement in drug release than MßCD for both HEMA and DMAA/10% TRIS gels (p < 0.05). Conclusions: A high loading concentration of methacrylated CDs decreases overall drug delivery efficiency, which likely results from an unfavorable arrangement of the CDs within the polymer network leading to reduced binding of natamycin to the CDs. HEMA and DMAA/10% TRIS materials functionalized with MHP-ßCD are more effective than those functionalized with MßCD to deliver natamycin.

Phan,C. -M, Subbaraman,L., Jones,L. Contact lenses for antifungal ocular drug delivery: A review Expert Opinion on Drug Delivery 2014;11(4):537-546 [ Show Abstract ]

Introduction: Fungal keratitis, a potentially blinding disease, has been difficult to treat due to the limited number of approved antifungal drugs and the taxing dosing regimen. Thus, the development of a contact lens (CL) as an antifungal drug delivery platform has the potential to improve the treatment of fungal keratitis. A CL can serve as a drug reservoir to continuously release drugs to the cornea, while limiting drug loss through tears, blinking, drainage and non-specific absorption. Areas covered: This review will provide a summary of currently available methods for delivering antifungal drugs from commercial and model CLs, including vitamin E coating, impregnated drug films, cyclodextrin-functionalized hydrogels, polyelectrolyte hydrogels and molecular imprinting. This review will also highlight some of the main factors that influence antifungal drug delivery with CLs. Expert opinion: Several novel CL materials have been developed, capable of extended drug release profiles with a wide range of antifungal drugs lasting from 8 h to as long as 21 days. However, there are factors, such as first-order release kinetics, effectiveness of continuous drug release, microbial resistance, ocular toxicity and potential complications from inserting a CL in an infected eye, that still need to be addressed before commercial applications can be realized. © Informa UK, Ltd.

Phan,C. -M, Subbaraman,L., Liu,S., Gu,F., Jones,L. In vitro uptake and release of natamycin Dex -b- PLA nanoparticles from model contact lens materials Journal of Biomaterials Science, Polymer Edition 2014;25(1):18-31 [ Show Abstract ]

Purpose: To evaluate the uptake and release of the antifungal agent natamycin encapsulated within poly(D,L-lactide)-dextran nanoparticles (Dex-b-PLA NPs) from model contact lens (CL) materials. Methods: Six model CL materials (gel 1:poly(hydroxyethyl methacrylate, pHEMA); gel 2:85% pHEMA: 15% [Tris(trimethylsiloxy)silyl]-propyl methacrylate (TRIS); gel 3: 75% pHEMA: 25% TRIS; gel 4: 85% N,N dimethylacrylamide (DMAA): 15% TRIS; gel 5:75% DMAA: 25% TRIS; and gel 6: DMAA) were prepared using a photoinitiation procedure. The gels were incubated in: (1) natamycin dissolved in deionized (DI) water and (2) natamycin encapsulated within Dex-b-PLA NPs in dimethylsulfoxide/DI water. Natamycin release from these materials was monitored using UV-visible spectrophotometry at 304 nm over 7 d. Results: Natamycin uptake by all model CL materials increased between 1 and 7 d (p < 0.001). The uptake of natamycin-NPs was higher than the uptake of the drug alone in DI water (p < 0.05). Drug release was higher in materials containing DMAA than pHEMA (p < 0.05). All gels loaded with natamycin-NPs also released more drug compared to gels soaked with natamycin in DI water (p < 0.001). After 1 h, CL materials loaded with natamycin alone released 28-82% of the total drug release. With the exception of gel 6, this burst released was reduced to 21-54% for CL materials loaded with natamycin-NPs. Conclusions: Model CL materials loaded with natamycin-Dex-b-PLA NPs were able to release natamycin for up to 12 h under infinite sink conditions. DMAA-TRIS materials may be more suitable for drug delivery of natamycin due to the higher drug release observed with these materials. © 2013 Taylor & Francis.

Phan,CM, Hui,A., Subbaraman,L., Jones,L. Insights to Using Contact Lenses for Drug Delivery Clin Exp Pharmacol 2014;3(145):2161-1459 [ Show Abstract ]

There has been considerable interest in the potential application of contact lenses for ocular drug delivery. This short communication provides an overview of the challenges faced by delivering drugs using contact lenses, highlights the solutions to limitations that have already been achieved, and describes the barriers that remain before commercial application can be realized.

2013

Phan,C. -M, Subbaraman,L. N., Jones,L. In vitro uptake and release of natamycin from conventional and silicone hydrogel contact lens materials Eye and Contact Lens 2013;39(2):162-168 [ Show Abstract ]

OBJECTIVES:: To investigate the uptake and release of the antifungal ocular drug, natamycin from commercially available conventional hydrogel (CH) and silicone hydrogel (SH) contact lens (CL) materials and to evaluate the effectiveness of this delivery method. METHODS:: Five commercial SH CLs (balafilcon A, comfilcon A, galyfilcon A, senofilcon A, and lotrafilcon B) and four CH CLs (etafilcon A, omafilcon A, polymacon, vifilcon A) were examined in this study. These lenses were incubated with natamycin solubilized in dimethyl sulfoxide, and the release of the drug from these lenses, in Unisol 4 pH 7.4 at 32±1 C, was determined using UV-visible spectrophotometry at 305 nm over 24 hours. RESULTS:: There was a significant uptake of natamycin between 0 hour and 24 hours (P0.05). There was a significant difference in release between all the SH materials (P0.05). There was a significant difference in release between all the SH materials (P0.05). There was a significant difference in release between all the SH materials (P0.05). Overall, the release of natamycin was higher in CH than SH lenses (P<0.001). CONCLUSIONS:: All CLs released clinically relevant concentrations of natamycin within 30 minutes, but this release reached a plateau after approximately 1 hour. Further CL material development will be necessary to produce a slow and sustained drug releasing device for the delivery of natamycin. © 2013 Lippincott Williams & Wilkins.

Scientific Presentations

2024

Garg P, Shokrollahi P, Phan CM, Jones L. 3D printing of PVA loaded ocular inserts for ocular drug delivery The Association for Research in Vision and Ophthalmology, Seattle, WA, May 9, 2024 [ Show Abstract ][ PDF ]

Purpose: To develop ocular inserts comprised of polyvinyl alcohol (PVA) and gelatin methacrylate (GelMA), using 3D printing technology.

Methods: Inserts were synthesized using a bioink formulation consisting of 10% (w/v) GelMA, 5% (w/v) and 7.5% (w/v) PVA, 0.6% (w/v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and 5% (v/v) yellow dye as a light absorbing agent to improve print resolution. They had a 4mm diameter, 1mm thickness and were fabricated using a commercial masked-stereolithography (mSLA) 3D printer at 95% humidity and 37°C temperature. Morphology of the inserts was investigated by freeze-drying samples and imaging them using a scanning electron microscope (SEM). Release of PVA over 5 hours was studied by incubating at 35°C in PBS in an incubator shaker at 50rpm. The hydrogel samples were freeze dried and their equilibrium swelling was studied in PBS using gravimetric method.

Results: The PVA-loaded ocular inserts were 3D printed within 30 minutes. SEM images showed that 7.5% PVA loaded inserts had more uniform pore size distribution compared to the gels with no PVA. Approximately 37% of PVA was released within the first 2 hrs from the inserts containing PVA, and the release continued up to approximately 4 hrs before reaching a plateau. The release kinetics can be attributed primarily due to passive diffusion. The swelling curves of these hydrogels suggest that they reach equilibrium swelling within 24hr. From the slope of the swelling curve in the first hour, it can be inferred that swelling happens at a slower rate for GelMA/PVA compared to GelMA alone. This slower swelling rate helps to control the release and supports a sustained release of PVA from the combination.

Conclusions: This study showed that a GelMA-PVA based bioink can be used to 3D print ocular inserts that can release PVA for up to 4 hours. Future work will focus on designing 3D scaffolds to increase the release duration of PVA from these gels.

Ho B, Phan CM, Garg P, Shokrollahi P, Jones L. A screening platform for simultaneous evaluation of biodegradation and therapeutic release from an ocular hydrogel and its effect on human corneal epithelial cells The Association for Research in Vision and Ophthalmology, Seattle, WA, May 7, 2024 [ Show Abstract ][ PDF ]

Purpose: To integrate human corneal epithelial cells (HCECs) into a millifluidic screening platform that quantifies biodegradation and release of an entrapped therapeutic from an ocular hydrogel.

Introduction: Biodegradable hydrogels are novel drug delivery methods designed to release entrapped drugs or therapeutics as the gel degrades in situ. The primary challenge in developing biodegradable hydrogels for drug delivery lies in accurately measuring their degradation over time, while simultaneously being able to evaluate the drug release kinetics, which is typically a cumbersome procedure. To properly evaluate the biodegradation of a hydrogel, it is also essential to simulate key factors of the target tissue environment. In the context of the eye, this includes ocular temperature, tear flow, and low tear volume. Recent advances in organ-on-a-chip technologies have made it possible to emulate the human corneal environment. This will allow more accurate measurements of hydrogel degradation rates, subsequent drug or therapeutic release, and ultimately the overall effect on human corneal epithelial cells.

Methods: Gelatin Methacrylate (GelMA) ocular inserts with polyvinyl alcohol (PVA) (10% GelMA 7.5% PVA) were placed inside a custom-designed millifluidic device. Ocular inserts were degraded with up to 200 μg/mL of matrix metallipeptidase 9 (MMP9) for 24 hours at 37oC in PBS. Biodegradation of the ocular insert was quantified using a computational image analysis pipeline. The eluates containing the degradation products were collected to measure PVA released using a spectrophotometric assay, and its toxicity on human corneal epithelial cells (HCECs) was determined by alamarBlueTM assays.

Results: There was significant biodegradation of the GelMA-PVA inserts with increasing concentration of MMP9 in the millifluidic device, which was accurately quantified using a custom computational analysis. Degradation products in the eluate were collected, and there was a ~2-fold increase of PVA released in samples treated with MMP9 compared to the control. The same eluates were non-toxic to HCECs, and interestingly protected HCECs from hyperosmotic conditions mimicking dry eye disease.

Ho B, Phan CM, Jones L, Hui A, Ketelson H. Evaluating Protective Effects of Hyaluronic Acid Containing Ophthalmic Lubricants on an in Vitro Dry Eye Model American Academy of Optometry Meeting, Indianapolis, Nov 6, 2024 [ Show Abstract ]

Purpose: To evaluate the protective effects of hyaluronic acid (HA) with the actives glycerin and polyethylene glycol/propylene glycol in ophthalmic lubricants on an in vitro model for dry eyes.

Methods: Three different topical formulations ((non-HA) containing the active hydroxyethyl cellulose, Optase® Dry Eye Intense (OI) containing the active glycerin and Systane® Hydration (SH)) containing the actives polyethylene glycol/propylene glycol were tested. The OI eye drops contained a single high MW HA polymer whereas the SH formulation contained a dual polymer system of HA and hydroxypropyl guar. The in vitro cell model consisted of immortalized human corneal epithelial cells (HCECs) cultured in DMEM/F12 media with 1% FBS and 1% penicillin/streptomycin. HCECs were seeded into 96-well plates at a density of 15,000 cells/well and incubated for 16 hr at 37°C and 5% CO2. Once confluent, media was aspirated, and HCECs were treated with HA and non-HA-based formulations for 30 min and imaged every min by brightfield microscopy. The formulations were removed, and HCECs were washed with 100 µL of PBS 0-3 times to simulate removal of the formulation from the ocular surface by tear dilution. Cells were then exposed to a dry eye environment by completely removing media for 90 seconds. Morphology changes were observed by time-lapse microscopy, and HCEC viability was determined using alamarBlueTM.

Results: There was a significant reduction in cell viability to 78% (Fig 1A) for cells desiccated in the PBS control condition (p < 0.05, two-tailed T-test). Treatment with the non-HA eyedrop showed no protection of cell viability. HA-containing formulations protected HCECs from desiccation by 98-99% (Fig 1B) compared to respective controls (p < 0.05, two-tailed T-test). This protection was maintained after multiple PBS washes. Despite the SH formulation containing two polymers, the OI treatment showed similar levels of protection against desiccation. However, the OI and SH formulations resulted in different cell morphologies. Under desiccation stress, the morphology of HCECs changed from elongated to a rounded shape (Fig 1C). HCECs treated with OI remained elongated whereas cells in SH were more circular.

Conclusion: This study utilized an in vitro dry eye cell model to evaluate the effects of HA with the actives glycerin and polyethylene glycol/propylene glycol in topical ophthalmic eye drops. Formulations containing HA protected against dry eye conditions, yet those without HA did not. HA also maintained more durable protection when exposed to rinsing mechanisms. Interestingly, protection levels may differ between different formulations of HA with the various actives when based on changes in cell morphology, warranting further investigation to identify HAs with optimal properties for ocular benefits.

2023

Chan V, Drolle E, Phan CM, Hui A, Shi C, Subbaraman L, Wu J, Jones L. Evaluating the activity of lysozyme deposited on contemporary reusable silicone hydrogel contact lenses using an in vitro eye model The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, 2023 [ Show Abstract ][ PDF ]

Purpose: To evaluate lysozyme activity (LA) on five contemporary reusable silicone hydrogel contact lens (CL)materials over their proposed wear period using an advanced in vitro blink model.

Methods: Five CL materials (lotrafilcon B, samfilcon A, comfilcon A, senofilcon A, and serafilcon A) were cycled daily for 16h on an eye model, followed by 8h of soaking in OPTI-FREE PureMoist, to mimic a typical wear cycle. An artificial tear solution containing physiologically representative proteins and lipids was delivered to the model at a rate of 1.2-2.1μl/min. The model includes an artificial eyelid that blinks at a rate of 6blinks/min, which was kept at room temperature and humidity above 50%. Serafilcon A and senofilcon A were tested over 14 days, whereas the other CLs were evaluated for 30 days. At specified time intervals, including after 1, 7, 14 and 30 days, CLs were removed from the model and lysozyme extracted using a solvent containing acetonitrile and trifluoroacetic acid. The LA from the extracts were then evaluated using a micrococcal absorbance assay.

Results: Overall, LA decreased over time, reaching non-detectable levels by day 30 (p<0.05). Serafilcon A (13.9 ± 7.8μg/lens), and samfilcon A (9.6 ± 2.3μg/lens), had the highest LA after 1 day, followed by comfilcon A (4.7 ± 1.8μg/lens), lotrafilcon B (3.3 ± 1.6μg/lens), and senofilcon A (2.2 ± 3.7μg/lens). By day 7, LA for the weekly replacement lens, serafilcon A, decreased to 0.5 ± 0.6μg/lens. By day 14, LA for the biweekly replacement lens, senofilcon A, decreased to 0.6 ± 0.7μg/lens. Lotrafilcon B, samfilcon A, and comfilcon A, all monthly replacement lenses, decreased in activity by day 30 (0.1 ± 0.2μg/lens, 0.5 ± 0.7μg/lens, 0.0 ±0.0μg/lens respectively).

Conclusions: Deposition of biologically active lysozyme has been proposed to be an important factor for biocompatible CL wear. A decline in activity over time as the deposited protein becomes denatured may impact overall CL performance and has been linked to reduced comfort. LA decreases over time and reaches near zero for all lens types by the end of their proposed wearing period, confirming that they should be replaced within their specified replacement intervals. Compared to simple in vitro vial models, using an advanced blink model for CL deposition testing aims to provide more physiologically relevant results prior to clinical testing.

Garg P, Wulff D, Phan CM, Jones L. Evaluation of a biodegradable bioink for the fabrication of ophthalmic devices using 3D printing The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ][ PDF ]

Purpose: To develop a degradable bioink for fabricating ophthalmic devices using 3D printing.

Methods: The bioink formulation consisted of 10% gelatin methacrylate (GelMA), 0.6% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and 5% yellow dye as a light absorbing agent to improve print resolution. The bioink was used to 3D print square sheets (7x7x1 mm) using a commercial masked-stereolithography (mSLA) 3D printer at 95% humidity and 37°C temperature. The degradation of printed sheets was evaluated with different concentrations (0,25,50,100 μg/ml) of matrix metalloproteinase (MMP9) enzyme 37°C. MMP9s are naturally found in the tear film and elevated in various diseased states such as in corneal wounds and dry eye disease. The weights of the sheets were measured at t = 0,4,6,8,12,16,24 hrs. Another set of cubes (1x1x1 cm) was autoclaved and kept sealed in storage at different temperatures (4°C, 25°C, and 37°C) in phosphate buffered saline (PBS) and their weight was measured on day 10. An attempt was made to fabricate a contact lens using this bioink.

Results: Samples that were exposed to MMP9 enzymes showed a time-dependent degradation with increasing enzyme concentration. The samples incubated with 100 and 50 μg/ml of MMP9 were completely degraded by the end of 12 and 16 hrs, respectively. At the end of 24 hrs, the samples incubated at 25 μg/ml enzyme showed 72.8% degradation whereas the control samples did not show any signs of degradation. Interestingly, samples that were autoclaved and kept in storage also did not show any signs of degradation at all temperatures. A 3D-printed CL with overall diameter 14mm and thickness 1mm was printed without any support structures within 1 hour.

Conclusion: This study showed GelMA-based bioink can be used to fabricate biodegradable devices such as contact lenses. The biomaterials degrade in the presence of MMP-9 and future work will work on tuning the degradation kinetics of these materials, as well as incorporating ocular drugs.

Garg P, Wulff D, Phan CM, Jones L. Fabrication of a degradable ocular drug delivery system using 3D printing CBB 2023 Conference: Waterloo for Health, Technology and Society, March, 2023 [ PDF ]

Ho B, Phan CM, Ramasamy M, Hui A, Jones L. PDMS microfluidic devices fabricated from commercial 3D printers support growth of viable HCECs and enable cell biological assays for low-cost high-throughput screening The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ]

Purpose: To integrate human corneal epithelial cells (HCECs) into a PDMS microfluidic chip fabricated from a novel 3D printing method to perform cell biological assays.
Introduction: The advent of microfluidic devices has enabled tight control over the physical and chemical cellular environment in vitro, while allowing for large-scale imaging and biochemical reactions at single-cell resolution. These devices are capable of miniaturizing assays to the microliter and nanoliter range, thereby increasing assay throughput with high sensitivity, a feature that is highly advantageous in high-throughput cell-based screens. Polydimethylsiloxane (PDMS) has been widely used in microfluidics devices due to its optical clarity and non-toxicity to cells, among other desirable features. However, the fabrication of PDMS devices traditionally requires specialized facilities and instruments. Additionally, PDMS itself is highly hydrophobic and does not support mammalian cellular viability and growth.

Methods: PDMS devices were cured in 3D-printed moulds generated using the FormLabs stereolithography (SLA) printer (FormLabs 3B+, FormLabs, Somerville, MA). These devices were sterilized by autoclaving, and coated with 0.01% polydopamine (PDA) and 20μg/mL collagen. HCECs were seeded onto the device, and allowed to grow for 18-36 hours in DMEM/F12 media at 37oC. HCECs were imaged by light microscopy, and viability was assessed by alamarBlue assays.

Results: Here, we present a novel and simple method of generating PDMS microfluidic devices suitable for mammalian cell biology assays using commercial 3D printing. We show that PDMS devices coated with polydopamine (PDA) support the growth of human corneal epithelial cells (HCECs) that are metabolically active (~60-90% viability) and are comparable to HCECs cultured in standard tissue culture plastic consumables. Finally, HCECs cultured in our devices are capable of growth with fluid flow rates of up to 1mm/s.

Conclusion: Our study shows that PDMS devices manufactured through the aid of a novel 3D printing pipeline support the growth of HCECs. We aim to utilize these microfluidic devices as a tool to screen different compounds and formulations while assessing cellular viability and acquiring high resolution microscopic and fluorescence images of HCECs.

Phan C-M, Chan V, Walther H, Pereira da Mota A, Lorenzo CA, Jones L. Developing a High-throughput in vitro Eye Model for Evaluating Ocular Drug Delivery with Contact Lenses XXV Biennial Meeting of the International Society for Eye Research, Feb 21, 2023 [ Show Abstract ]

Purpose: To develop a high-throughput in vitro eye model for evaluating ocular drug delivery with contact lenses (CLs).
Method: The eye model was designed using CAD software and manufactured using a combination of fabrication methods, including moulding, CNC machining, laser cutting, and 3D printing. The model consists of an eyeball, an upper and lower eyelid, and a collection tray to collect flow-through fluid. The portion of the upper eyelid that comes into contact with the eyeball is moulded with a highly wettable and durable polyvinyl alcohol hydrogel. The centre of the eyeball is designed with a 300 µm thick, 15 mm diameter cut-out that allows for a contact lens to be mounted. Simulated tear fluids can be delivered through an inlet located on the upper eyelid using a pump. During each blink cycle, the eyelid slides and flexes across the eyeball to create an artificial tear film layer. The blink distance, speed and rate are actuated using a motor controlled by an Arduino board and software. The release of a red dye from two CLs (etafilcon A and senofilcon A) and the release of two drugs (resveratrol and pravastatin sodium) from drug-loaded CLs were evaluated using the model and compared to the traditional vial testing method. Phosphate buffered saline (PBS) was used as the simulated tear fluid and infused into the model at 5 µL/min, at a blink rate of 1 blink/10 s.
Results: The fluid flows from the inlet, spreads across the eye, accumulates in the lower eyelid and then flows into the collection tray via gravity. During this process, approximately 25% of the fluid originally injected into the model was lost due to evaporation, nonspecific absorption, and residual dead volume. Overall, the release of the dye and drugs from the CLs was higher in a vial compared with the eye model. Interestingly, the drug release profiles from the drug-loaded CLs on the eye model were similar to in vivo results previously collected from a rabbit study, although the total amount of drugs released was significantly less. 9 or 24 CLs can be tested with one syringe or peristaltic pump, respectively.
Conclusion: The current eye model developed from this study could be used to measure the release of ophthalmic drugs or comfort agents from CLs in a high-throughput manner. However, further work is required to fine-tune the parameters of the model, such as the composition of the tear fluid, blink rate, tear flow rates, and temperature, to better simulate in vivo conditions.

Phan CM, Ramasamy M, Ho B, Hui A, Jones L. Fabrication of a microfluidic chip using 3D printing for evaluating ocular cytotoxicity The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ][ PDF ]

Purpose: To develop a PDMS (polydimethyl siloxane) microfluidic chip to evaluate ocular cytotoxicity with ophthalmic formulations and materials.

Methods: The microfluidic chip was designed using CAD software (FreeCAD), and the moulds of the chips were printed using (1) a stereolithography (SLA) and (2) digital light processing (DLP) 3D printer. The printed moulds were washed with isopropyl alcohol (IPA), UV-cured for 1-hour at 60oC, followed by heating in an oven at 120oC for 2 hours to remove any unreacted polymers. The surface of the chips was smoothed with sandpaper with increasing grit, followed by an application of nail polish. The moulds were then cast with PDMS, a gas-permeable and clear polymer commonly used for the fabrication of microfluidic chips. The moulds and chips were imaged using SEM (scanning electron microscopy). The light transmittance of the chips was also measured. The PDMS top half of the chip was adhered to a microscope slide using medical-grade double-sided tape. For a pilot study, the PDMS chips were sterilized via autoclaving, coated with 0.1% polydopamine to improve their surface wettability, and then seeded with immortalized human corneal epithelial cells (HCEC). After 2 days of incubation in a nutrient media broth (no flow), cell adhesion and growth were evaluated using light microscopy.

Results: Both 3D printers were able to print moulds with high resolution, with channel dimensions as low as 50 µm, and with faster print times for the DLP printer. SEM images revealed that moulds that were both sanded and had a nail coating were significantly smoother than the original 3D-printed moulds. The chips cast from the polished moulds were transparent, with >85% transmittance from 450-700 nm, and could be used to image cells through a microscope. The microfluidic chips were able to handle flow rates up to 1 mL/min for 24 hours without any signs of leakage. HCEC cells were able to adhere and grow on the coated PDMS microfluidic chip after 2 days.

Conclusion: This study showed that SLA and DLP printers could be used to fabricate PDMS microfluidic chips as a low-cost rapid prototyping approach. The fabricated chips were clear and could be used to incorporate HCEC cells. Future work will examine the viability of cells under different flow rates and shear stress conditions on these chips.

Ramasamy M, Ho B, Phan CM, Jones L. Fabrication of a microfluidic chip for ophthalmic drug delivery studies using 3D printing The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ]

Purpose: To develop a microfluidic chip for testing the release of ocular drugs from soft contact lenses using 3D printing.

Methods: The microfluidic chips were designed using CAD (computer-aided design) software consisting of a top and bottom portion. The top portion comprised of inlet, outlet, and channels for fluid flow. The lower portion contained a dome-shaped mount to mount a contact lens. The chips were printed using clear resin on a commercial stereolithography (SLA) 3D-printer. The printed chips were washed in isopropyl alcohol (IPA) for 30 minutes, air dried and UV cured for 30 minutes. The top and bottom portions of the chip were fused by applying a thin layer of resin, followed by UV-curing for 10 minutes. In another design iteration, moulds for the chips were 3D printed and casted with polydimethylsiloxane (PDMS). The two halves of the PDMS chips were fused using double-sided adhesive tape. In a preliminary study, two commercial contact lenses, etafilcon A and senofilcon A, were soaked in 2 ml of red food dye for 2 hours. The release of the dye was measured using the PDMS chip with phosphate-buffered saline at a flow rate of 1.5 L/min over 24 hours via absorbance at 520 nm. The dye extraction from both lenses was
performed by incubating the dye-soaked lenses in 1:1 acetonitrile/water solution for 24 hours with gentle shaking.

Results: Both the chip and moulds were printed in less than 5 hours, with a minimum resolution of 50 μm. The resulting resin and PDMS chips can also be sterilized by autoclaving. The top and bottom parts of the chips were completely sealed such that no leakage was detected at a flow rate of up to 100 μL/min for 24 hours. The release kinetics of the dye was linear throughout the 24 h period for both lens types under the current parameters. The total amount of dye released after 24 h was higher for etafilcon A (26.26 mg/lens) than senofilcon A (18.41 mg/lens), which corresponded to approximately 83.1% and 40.01% release, respectively. Both the lens types were still visibly red after 24 hours. The output of the microfluidic chip could be used as an input for subsequent analyses.

Conclusions: This study showed a cost- and time-efficient approach to fabricate a microfluidic chip for evaluating drug release from contact lenses. Future work will examine the release profile of various ocular drugs from contact lenses using different flow conditions.

Wulff D, Phan C-M, Jones L. Development of a 3D-printed hydrogel eye model for evaluating ocular drug delivery The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ][ PDF ]

Purpose: To 3D-print a soft hydrogel eye model using a novel bioink for evaluating ocular drug delivery.

Methods: The eye model was designed using CAD software. It includes several key components made from a hydrogel, including an upper and lower eyelid, a frontal surface to mimic the cornea and sclera, and an internal chamber to mimic the interior of the eye. The components were designed to fit with an existing blink model that was developed previously in our laboratory that allows for automated blinking and tear collection. The eyeball and the lower eyelid were 3D bioprinted using a modified commercial mSLA printer (Photon Mono X, AnyCubic, Shenzhen, China). Various bioinks were tested, consisting of 5-15% gelatin methacryloyl, 1-5% polyvinyl alcohol, 5-30% polyethylene glycol diacrylate, 0.4-0.6% lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and 2-3% of a yellow food-grade dye in phosphate buffer solution. Different formulations were evaluated to create prints that were desirable in terms of print quality, stiffness, and flexibility. Printing was undertaken at 40˚C to ensure the ink remained a liquid and at 90% humidity to protect the parts and ink from desiccation.

Results: Both the eye and the lower eyelid were successfully printed in high resolution using 100 µm layer heights without any support structures within 3 hours. The prints are hydrophilic with a 60-80% water content, are soft and flexible, and are fabricated with biocompatible biomaterials. Both components were able to be incorporated into an in vitro blink model which will allow for improved testing that more closely mimics a human ocular system, in particular, drug absorption through the cornea. A port on the eye allows for the sampling of fluid from the interior eye model for testing the diffusion of drugs to the posterior chamber when released by topical ophthalmic formulations or anterior segment devices such as contact lenses.

Conclusion: This study demonstrated that a modified mSLA 3D printer can be used to fabricate soft, hydrophilic ocular model components using a novel biocompatible bioink.

2022

Chan V, Phan CM, Walther H, Ngo W, Jones L. Effects of temperature and blinking on contact lens dehydration using an in vitro blink model 10th Canadian Optometry School Research Conference, Montréal, Canada, Dec 3, 2022

Chan VWY, Phan CM, Walther H, Jones L. Fabrication of an in vitro eye model for practicing corneal foreign body removal 10th Canadian Optometry School Research Conference, Montréal, Canada, Dec 3, 2022

Jones L, Bose S, Phan CP, Rizwan M, Tse JW, Yim EKF. Fabrication of an enzyme-triggered therapeutic releasing biomaterial for bandage contact lenses American Academy of Optometry, San Diego, 2022 [ Show Abstract ]

Purpose: The use of a soft bandage contact lens in combination with a therapeutic could help improve the treatment of corneal injuries. The purpose of this study was to develop an enzyme-triggered therapeutic release platform using a unique gelatin methacrylate formulation (GelMA+) and bovine-lactoferrin (BLF), a model therapeutic.

Methods: Two formulations of GelMA+, 20% and 30% w/v, were prepared using UV polymerization. The properties of the material, including porosity, tensile strain, and swelling were characterized. The degradation of GelMA+ in the presence of matrix metalloproteinase-9 (MMP-9), typically found upregulated at a wounded sight, from 0 – 300 µg/mL of the enzyme was also evaluated. Cell viability, cell growth, and cytotoxicity on the GelMA+ gels were determined using the AlamarBlueTM assay and the LIVE/DEAD™ Viability/Cytotoxicity Kit staining with immortalized human corneal epithelial cells after 5 days. For a preliminary drug release study, the 30% GelMA+ gels were also loaded with 3 µg of BLF, and the release of the therapeutic was evaluated over 5 days at various MMP-9 concentrations (0, 100, 300 µg/mL) in phosphate-buffered saline (PBS 1X) at 37 °C. The gels were washed for 1 hour at room temperature (22 – 24 °C) before the release phase to remove any loosely bound BLF on the surface. The amount of BLF released was measured using an ELISA kit and UV absorbance at 450 nm, n=4.

Results: The 30% w/v GelMA+ had a higher crosslinking density, tensile strength, smaller pore size, and lower swelling ratio than the 20% w/v GelMA+ (p<0.05). The degradation rate of the 20% w/v gel was much faster (p<0.001), degrading almost completely after 48 hours at 300 µg/mL of MMP9. After 5 days, There was no cytotoxicity detected in the live/dead staining for either concentration, but the 30% w/v GelMA+ showed significantly higher cell viability (p<0.05). In the drug release study, there was no burst release of BLF observed for the 30% w/v gel, and the release of the therapeutic was sustained over 5 days. The rate of release from the gel significantly increased with increasing concentrations of MMP-9 (p<0.001), correlated to the rate of degradation of the gels.

Conclusion: The results showed that degradation of GelMA+ can be tuned by modifying the cross-linking density or exposure to different concentrations of MMP-9. The release of BLF from 30% GelMA+ is driven by a combination of diffusion and degradation of the material by MMP-9 enzymes. Future work will focus on optimizing the materials to deliver other therapeutic agents at physiologically-relevant concentrations of MMP enzymes

Phan C-M, Wulff D, Garg P, Jones L.. Developing a novel in vitro eye model using 3D bioprinting for drug delivery studies The Association for Research in Vision and Ophthalmology, Denver, CO, USA, May 1, 2022 [ Show Abstract ]

Purpose: To develop an in vitro eye model using a novel 3D bioprinting method for testing the release of ophthalmic formulations to the posterior ocular region.

Methods: The eye model was designed using CAD software and includes both an anterior aqueous chamber and a posterior vitreous chamber. The vitreous chamber is surrounded by a blood chamber to mimic vessels that can be used to transport a blood-like substance. Three inlet ports control the flow of fluid into the chambers and the blood channels, and the three outlet ports allow fluids to exit these compartments. The eye model was 3D printed on a commercial mSLA printer (Photon Mono X, AnyCubic), which was retrofitted with a humidity and temperature control module to create a printing environment at 37°C and >80% humidity. The bioink formulation consisted of 10% gelatin methacrylate (GelMa). After printing, the models were incubated at 37°C to remove any uncured GelMa within any hollow compartments. For this study, phosphate-buffered saline was used as an aqueous and vitreous humour mimic. To evaluate the diffusion of a small hydrophilic molecule on the eye model, a contact lens (Air Optix) was soaked with a water-soluble red food dye for 1 hour and then placed on the eye model. The amount of dye in the anterior chamber, posterior chamber, and blood channels was measured using UV spectrophotometry after 24 hours.

Results: The entire model can be printed without any support structures within approximately 3 hours. The 3D printed eye model can also be autoclaved for testing that requires sterility. Because there were no diffusion barriers present in the current model, the red dye was detected in all three chambers after 24 hours. The highest concentration of dye was found in the anterior chamber, followed by the blood chamber and then the posterior chamber.

Conclusions: The prototype developed in this study can be used as a starting point to develop enhanced 3D printed eye models to test drug release kinetics of various devices and formulations. Future work will focus on adding the appropriate diffusion barriers to better simulate drug diffusion through ocular tissues.

Layman Abstract: The aim of the study was to create an advanced eye model using commercial 3D printing methods. Current 3D bioprinters are extremely expensive and regular commercial 3D printers do not have the capabilities to print biological materials. We are developing a method to 3D print sophisticated eye models using inexpensive 3D printers. The models from this research can further be refined for studying drug absorption in the eye. This research will also enable researchers to create their own biological models using 3D printing methods.

Phan C-M, Wulff D, Jones L.. Developing bioinks for commercial mSLA printers and a method for quantifying print quality Canadian Biomaterial Society Conference, Banff, AB, May 25, 2022 [ PDF ]

Phan CM, Chan V, Drolle E, Shi C, Subbaraman L, Wu J, Jones L. Evaluating the in vitro wettability of contemporary reusable soft contact lenses using an in vitro blink model American Academy of Optometry, San Diego, 2022 [ Show Abstract ][ PDF ]

Purpose: To evaluate the in vitro wettability of four contemporary reusable soft contact lens materials (serafilcon A, senofilcon A, senofilcon C, lotrafilcon B, comfilcon A) over a 14-day simulated wearing period using a novel, physiologically relevant in vitro eye model.

Methods: The 14-day wearing period was simulated using an in vitro blink model (OcuBlink). A tear mimic solution containing relevant proteins and lipids was delivered to the eye model at a rate of 1.25 -2.25 µL/min. A tear film was created over the lens via an artificial eyelid that slid across an eyeball surface at 6 blinks/minute, with a lens in-situ, at room temperature and humidity above 50%. For each cycling day, the lenses were incubated on the eye model for 16 hours, followed by a 10 second rub-rinse each side with OPTI-FREE PureMoist Solution, and then incubated overnight for 8 hours in the same solution. Lens wettability was quantified at t = 0 (straight from blister pack), 1, 7, and 14 days via two methods, (1) sessile drop contact angle (CA) and (2) non-invasive keratographic tear-break-up time (NIKBUT), n = 4 for each lens type. Contact angles were measured using the Optical Contact Analyzer (DataPhysics, Germany). NIKBUT measurements were assessed on the OcuBlink via the OCULUS Keratograph 5M (OCULUS, Germany).

Results: At t = 0, comfilcon A (24.0 ± 5.3°) had the lowest contact angle, followed by lotrafilcon B (29.6 ± 6.5°), serafilcon A (67.7 ± 16.0°), senofilcon C (89.2 ± 6.0°), and senofilcon A (92.0 ± 3.1°). All contact lens materials had similar CA out of the blister pack compared to 7 days (p >0.05). There was a slight increase in CA between 7-14 days for serafilcon A and lotrafilcon B, but this was not statistically significant (p>0.05). The NIKBUT for all contact lens materials ranged between 4 – 9 seconds, and there were no significant differences between NIKBUT within the same lens type at any time point (p>0.05). NIKBUT at t = 0 was highest for serafilcon A (8.3 ± 1.7 s) (p<0.05), followed by lotrafilcon B (6.3 ± 1.4 s), comfilcon A (6.1 ± 1.5 s), senofilcon A (5.6 ± 1.1 s), and senofilcon C (5.5 ± 0.8 s).

Conclusion: Lenses with a very low CA did not translate into significantly higher NIKBUT. For some lens materials, notably serafilcon A, there was an increase in CA after 7 days of in vitro testing, which warrants further investigation.

Phan CM, Kapadia W, Qin Nm Zhao P, Ren C, Haines L, Jones L. Development of a microfluidic viscometer for measuring microliter-volume fluid samples American Academy of Optometry, San Diego, 2022 [ Show Abstract ][ PDF ]

Purpose: To develop a microfluidic viscometer capable of measuring the viscosity of microliter-volume fluid samples such as human tear fluid.

Methods: The microfluidic chip was designed using CAD software and laser cut from acrylic sheets into the desired shape and sizes. The various parts of the microfluidic chips were assembled and combined together using medical-grade double-sided tape. The micro-channels were coated with a repellant coating to provide a smooth and hydrophobic surface, which provided a straight flow path for a sample to approach flow equilibrium as it moves through the chip. The chip was attached to a syringe pump to control the flow rate. Additional supporting devices, including a high-speed camera and a pressure transducer, were used for analysis. Based on the difference in capillary pressure at the air-liquid interface, channel dimension, sample length, sample velocity, volumetric flow rate and contact angle, the viscosity of the fluid sample can be derived. For a preliminary experiment, the viscosity of an eye drop (Hydrasense, Bayer Inc., Canada) was measured by the developed microfluidic viscometer as well as a commercial cone/plate rheometer (Model: LVDV-III+, Brookfield Engineering Laboratories Inc, MA, USA) at a shear rate between 27.8 – 308 s-1, n=3.

Results: The minimum sample volume that was needed for measurement was 12 microliters, but this volume can be reduced with further optimization. The results showed that the viscosity values obtained for both the commercial rheometer and the microfluidic viscometer were in strong agreement. At a low shear rate (141 s-1), the viscosity of the eye drop was at 8.03 mPa·s, and at higher shear rates (622 s-1), the viscosity decreased to 6.33 mPa·s. The viscosity of the eye drop decreased with increasing shear rate, which reflects its shear-thinning properties. The maximum shear that was tested was 622 s-1 with no signs of fluid leakage.

Conclusion: The developed microfluidic chip and analysis setup can be used to analyze the viscosity of fluids using very low sample volumes and at very high shear rates. Future work will focus on optimizing the system to further reduce the sample volume requirements to permit testing with human tear samples. This device will provide valuable data in clinical studies investigating dry eye and other ocular surface diseases.

Ramasamy M, Qin N, Phan C-M, Jones L. Developing a microfluidic chip using 3D printing for testing drug release from contact lenses 10th Canadian Optometry School Research Conference, Montréal, Canada, Dec 3, 2022

Shukla M, Phan CM, Jones L. Effect of methacrylic acid on atropine sulfate loading into model contact lens materials 10th Canadian Optometry School Research Conference, Montréal, Canada, Dec 3, 2022

Wulff D, Phan C-M, Jones L. 3D printing using a novel bioink with a commercial mSLA printer to fabricate a model contact lens The Association for Research in Vision and Ophthalmology, Denver, CO, USA, May 2, 2022 [ Show Abstract ]

Purpose: To develop a cost-effective and scalable 3D printing method and novel bioinks to fabricate contact lenses.

Methods: The bioink formulations consisted of GelMA (gelatin methacrylate), LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate), and a yellow food-grade dye. The dye minimizes unwanted light leakage during the photopolymerization process. A commercial mSLA (masked stereolithography) printer, the Photon Mono X (AnyCubic, Shenzhen), was retrofitted with a custom temperature and humidity control kit. The printing process was performed at 40 oC and 90% humidity to ensure that the GelMA remained at a liquid state and to prevent the bioink from drying out, respectively. A set of matrix cubes of varying sizes with holes was used as a standard control. Images of the cubes were taken with a camera, top-down and side-review, analyzed with the ImageJ software and compared with the original CAD designs to derive an overall print quality score. Two print variables, exposure time (5 s to 40 s) and yellow dye concentration (1 – 7%), were analyzed in this study.

Results: The best resolution with the highest print scores were obtained at either 5% yellow dye concentration and 30 seconds exposure time, or 3% yellow dye concentration and 20 seconds exposure time. There was an overall optimal range for both print times (20 - 30 s) and yellow dye concentration (3 - 5%). Values above or below this critical value resulted in lower print quality scores of the standard cubes. A prototype contact lens with a 200 µm thickness was able to be 3D printed using the developed print methods and parameters, with a total print time of approximately 20 minutes. Approximately 28 contact lenses can be printed at the same time using the 3D printer. However, the surface and edges of the 3D printed contact lens were still visually very rough.

Conclusions: The current study demonstrated that a low-cost commercial 3D mSLA printer can be used to fabricate model contact lenses using a hydrogel material. Still, further work is necessary to improve the print quality for fabricating ultra-thin devices such as contact lenses. Future work will use this 3D printing method to fabricate contact lenses for drug delivery.

2021

Bose S, Phan CM, Yim E, Jones L. Fabrication of a MMP-9 triggered biomaterial for corneal wound healing The Association for Research in Vision and Ophthalmology. San Francisco, May, 2021

2020

Chan V, Phan CM, Jones L. Evaluating lysozyme deposition on contemporary daily disposable contact lenses in a novel in vitro blink model The Association for Research in Vision and Ophthalmology, 2020 [ Show Abstract ][ PDF ]

Purpose : To evaluate total lysozyme deposition on daily disposable (DD) contact lenses (CL) using a novel in vitro eye-blink model.

Methods : Three conventional hydrogel (CH) DD CL materials (etafilcon A, omafilcon A, nelfilcon A) and three silicone hydrogel (SH) DD CL materials (delefilcon A, senofilcon A, somofilcon A) were tested. The lenses were removed from their blister packs and placed directly on the eye model. An artificial tear solution (ATS) was flown over the lenses at a flow rate of 1 µl/min. The blink rate was set to 6 blinks per minute and the model was incubated at room temperature (23-26°C) and >45% humidity. After exposure periods of 2, 4 or 8 hours the lenses were removed and the lysozyme was extracted using acetonitrile: 0.2% trifluoroacetic acid (n=3). A separate experiment was conducted with lenses incubated in a vial containing 480 µL of ATS on an orbital shaker at 60 rpm for 8 hours (n=3). The lysozyme activity was measured using a spectrophotometric assay.

Results : Etafilcon A had the highest amount of active lysozyme absorption at all time points (p>0.001). After 8 hours, a cumulative total of 402 ± 102µg/lens of active lysozyme was absorbed on etafilcon A. Delefilcon A had the highest cumulative amount of active lysozyme (26 ± 1µg/lens) for SH materials (p<0.05). Nelfilcon A, senofilcon A and somofilcon A had the lowest amount of lysozyme activity (p0.05). The amount of active lysozyme sorption for certain lens types was different when measured using the eye-blink model as compared to a vial (p<0.05). Etafilcon A had a higher amount of active lysozyme when incubated on the blink model as compared to the vial (p<0.05), whereas somofilcon A and senofilcon A had a higher amount of lysozyme activity in the vial compared to the eye model (p0.05).

Conclusions : The in vitro eye-blink model provides quantitative data that is close to that determined from ex vivo studies and is an excellent model to assess the deposition of lysozyme on DD CL materials.

This is a 2020 ARVO Annual Meeting abstract.

Phan CM, Shukla M, Heynen M, Walther H, Jones L. Development of an In Vitro Blink Model for Measuring Drug and Comfort Agent Elutes from Soft Contact Lens Polymers Academy at Home, 2020 [ Show Abstract ]

Purpose: To develop an advanced in vitro blink model that could be used to examine release of a wide variety of components (topical drugs; comfort agents etc) from soft contact lenses.
Methods: The model was designed using CAD software and 3D printed using an SLA printer. A UV-curable resin polymer was used to fabricate the main components of the eye model to ensure water-sealed parts. The eyelid and eyeball were synthesized from a polyvinyl alcohol and a silicone material respectively. Simulated tear fluid is delivered through tubing attached to the eyelid. With each blink cycle (1 blink/10s), the eyelid slides and flexes across the eyeball to create an artificial tear film. The flow-through fluid is collected in a specialized trough. Two contact lenses, etafilcon A and senofilcon A, were incubated in 2 mL of a water-soluble red dye for 24 hours and then placed on the eye model. The release of the dye was measured at t= 0.5, 1, 2, 4, 8, 12, and 24 hours (n=3) in phosphate buffered saline (flow rate = 5 µL/min). The dye release from the lenses was also evaluated in a vial containing 2 mL of PBS over 24 hours (n=3). The amount of dye in the samples was determined using a UV/Vis spectrophotometer at 520 nm.
Results: All of the contact lenses were visibly red after the incubation with the dye. After the release studies, the etafilcon A lenses became transparent with a slight red tint, whereas the senofilcon A lenses retained a moderate red colour. For the vial study, the release of the dye from the contact lenses was rapid within the first 4 hours, which was then followed by a slower release phase. In contrast, dye release from the lenses on the eye model was significantly slower and was sustained over the 24-hour period. The total amounts of dye released in the eye model were also significantly lower compared to the vial (p<0.05), which could be attributed to the absorption of the dye into the eyelid. From the total amount of tear fluid (7.2 mL) that was infused into the eye model, approximately 5.2 ± 0.6 mL of flow-through was collected. The loss of fluid can be attributed to fluid absorption into the eyelid and contact lenses, as well as evaporation.
Conclusion: The blink model developed in this study could be used to measure the release of topical ophthalmic drugs or comfort agents from contact lenses. The results showed that the release kinetics of a dye from contact lenses on the eye model was significantly different than that of a vial. The model also simulated non-specific absorption of the dye into the eyelid. Future work will focus on developing polymers to simulate the absorption of drugs on the eye.

Yee A, Phan CM, Heynen M, Walther H, Jones L. The uptake and release kinetics of myristamidopropyl dimethylamine (MAP-D) from contact lenses using radioactive labelling he Association for Research in Vision and Ophthalmology, 2020 [ Show Abstract ][ PDF ]

Purpose : Biocides are an important disinfecting component commonly found in contact lens (CL) multipurpose solutions (MPS). During the disinfection step, the uptake of biocides into CL materials can significantly reduce the efficacy of the MPS to disinfect the lens. An increased release of biocides from the CL can lead to clinical complications such as discomfort and corneal staining. However, detection methods using chromatography and spectrometry can be time-consuming and have low sensitivity. The purpose of this study was to evaluate the uptake and release of myristamidopropyl dimethylamine (MAP-D; ALDOX®) using a radiolabel method.

Methods : Radioactive (14C) MAP-D was purchased from Moravek Inc. (CA, USA). Five soft CL materials (lotrafilcon B, balafilcon A, senofilcon A, etafilcon A, omafilcon A) were tested (N=4). The lenses were incubated in PBS (ISO 18369-3) solution containing 2mL of 14C radioactive MAP-D (5µg/mL) for 8 hours, followed by a release period in PBS for 16 hours. The cycle was continued over a 7-day period with a new replenishing solution for each day. The samples were counted for their radioactive signal (CPM) using the LS6500 Beckman Coulter liquid scintillation beta counter (ON, CA). A standard curve was used to convert CPM to µg of MAP-D.

Results : After 7-days, the silicone hydrogel (SH) lenses, lotrafilcon B (36.21±0.42µg), balafilcon A (36.29±1.15µg) and senofilcon A (35.54±1.37µg) had a significantly greater uptake of MAP-D compared to the conventional hydrogel (CH) lenses, etafilcon A (7.66±1.19µg) and omafilcon A (6.54±0.94µg) (p<0.01). However, the percent of MAP-D released was higher for CH lenses, etafilcon A (80%), omafilcon A (93%) compared to SH lenses, lotrafilcon B (25%), balafilcon A (19%) and senofilcon A (19%) over the 7-days (p<0.01).

Conclusions : Radioactive labelling offers a highly sensitive and accurate way of assessing the uptake and release kinetics of MAP-D to CL materials. Greater uptake of MAP-D occurs to SH materials, and the release of MAP-D is relatively minor. While CH materials take up only low amounts of MAP-D, the majority of it is easily released from these materials.

This is a 2020 ARVO Annual Meeting abstract.

2019

Bose S, Phan CM, Rizwan M, Tse J, Yim E, Jones L. Release of FitC-Dextran from a MMP9-triggered material for corneal wound healing ISCLR, Singapore, 2019

Jones L, Yee A, Jabeen A, Subbaraman L, McCanna D, Phan CM. Novel in-vitro method to study bacterial interaction with contact lenses Global Specialty Lens Symposium, Las Vegas, Nevada, 2019 [ PDF ]

Phan C, Walther H, Jones L.. Development of a blink model for testing contact lenses American Academy of Optometry, Orlando, 2019 [ Show Abstract ][ PDF ]

Purpose: To develop an eye model with a physiological blink mechanism to test contact lenses.

Methods: All parts of the eye model were designed using CAD software. The eyeball moulds were CNC machined to ensure a smooth finish for the corneal surface. A chamber to house the eye model was cut using a laser cutter, assembled, and sealed using methylene chloride. The eyelid consisted of a unique 3D printed structure containing teeth to physically secure a flexible membrane. Both the eyeball and eyelid membrane were synthesized using polyvinyl alcohol (PVA). Four molecular weights of PVA (89-98 kDa, 85-124 kDa, 130 kDa, and 146-186 kDa) were tested at a range of concentrations between 5 – 30 % w/v. The wettability and water content of these materials were compared with the bovine cornea and sclera. The rotational motion of the eyelid was controlled by a stepper motor and an Arduino. The model was connected to a microfluidic pump, which delivers artificial tear solution (ATS) to the eyelid. A corneal topographer was used to evaluate the tear break-up and tear film regeneration.

Results: The eyelid flexes and slides across the eyeball during each blink, which ensures direct contact between the two surfaces. When loaded with an ATS, this mechanism evenly spreads the solution over the eyeball to generate an artificial tear film. The speed, degree of actuation, and rate of blinking can be controlled using the open source Arduino software. The artificial tear film layer in this eye model had a tear break-up time (TBUT) of 5.13 ± 0.09 seconds at 1.4 µL/min flow rate, 6 blinks/min, and <25% humidity.

Conclusion: This model simulates a physiological blink actuation and an artificial tear film layer. Future studies will examine variations in flow rates and ATS composition to simulate clinical values of TBUT. The model could be used to study in vitro TBUT, tear deposition, and drug delivery from contact lenses.

Phan CM, Qiao H, Jones L. A simple method to synthesize PVA hydrogels Canadian Biomaterials Society, Quebec, 2019 [ PDF ]

Phan CM, Qiao H, Shinde R, Jones L. Development of an in vitro eye model with polyvinyl alcohol The Association for Research in Vision and Ophthalmology. Vancouver, British Columbia, 2019 [ Show Abstract ][ PDF ]

Purpose: The purpose of this study was to develop a simple method to synthesize an in vitro eye model using polyvinyl alcohol with similar wettability and water content as the cornea, sclera, and lens.  

Methods: Bovine eyes, donated by an abattoir, were used as the ex vivo eye model for comparison. The eyes were dissected fresh, and the wettability and water content for the cornea, sclera, lens, and vitreous humour were measured. Four molecular weights of polyvinyl alcohol, 89-98 kDa, 85-124 kDa, 130 kDa, and 146-186 kDa, were tested. The monomer was added to a mixed solvent of dimethyl sulfoxide and Milli-Q water to achieve a range of concentrations between 5 – 30 % w/v. The mixture was heated at 120oC for 3 hours, and polymerized at -30o C for 3 hours. The gels were then equilibrated with Milli-Q water for 3 days before characterization. A model eyeball from polyvinyl alcohol was also synthesized using a custom mould.

Results: The bovine cornea had the highest wettability, with the lowest advancing contact angle (28.0 ± 7.2°), followed by the lens (64.0 ± 4.6°) and sclera (56.73 ± 6.5°). The contact angles for the polyvinyl alcohol gels ranged between 40.4 ± 2.5° and 61.6 ± 4.7° for various gels. The vitreous humour had the highest water content (98.7 ± 0.2 %), followed by the cornea (80.9 ± 1.2%), sclera (64.8 ± 0.7%), and lens (59.7 ± 7.5%). The water content for the gels decreased with increasing concentrations of the polyvinyl alcohol (p<0.001), and ranged between 66.1 ± 0.1 % to 91.4 ± 0.2 %. The eye models made from polyvinyl alcohol were translucent, smooth, wettable, lubricous, and pliable.

Conclusion: This study presents a simple method to synthesize hydrogels from polyvinyl alcohol, which can be formulated to closely match the wettability and water content of a bovine cornea, sclera, and lens. The method can be easily modified in future studies to incorporate other reagents. The eye model synthesized from this study could be used as a potential model to study in vitro tear-break up or drug absorption.

Phan CM, Walther H, Jones L. Mass spectrometry detection of phosphatidyl choline from delefilcon A Canadian Biomaterials Society, Quebec, 2019 [ PDF ]

Phan CM, Walther H, Jones L. Development of a polymeric eye model for foreign body removal Canadian Biomaterials Society, Quebec, 2019

Phan CM, Walther H, Qiao H, Jones L. Development of a novel in vitro blink model Canadian Biomaterials Society, Quebec, 2019 [ PDF ]

Phan CM, Walther H, Qiao H, Jones L. The development of an eye model that truly blinks ISCLR, Singapore, 2019

Walther H, Chan V, Phan CM, Jones L. Modelling non-invasive tear break-up times of soft lenses using a sophisticated in vitroblink platform Invest Ophthalmol Vis Sci 2019;60, E-abstract 6328 [ Show Abstract ][ PDF ]

Purpose : To evaluate the feasibility of a novel in vitro eye model to determine the pre-lens non-invasive tear breakup times (NIBUT) of two daily disposable (DD) contact lenses (CLs).

Methods : An artificial eyeball synthesized from polyvinyl alcohol (15% w/t, 89-98 kDa) was used on a novel in vitroeye model (OcuBlink), incorporating a blink and constant tear flow. A flat black disc was incorporated into the eyeball for contrast. The OcuBlink’s flexible eyelid design ensures contact with the eyeball, thereby enabling an even spread of the tear solution over the front of the CL. The blink interval was set to 10 seconds, and the flow rate of an artificial tear solution was set to 1.4 µL/min. A silicone hydrogel (SH; delefilcon A), and conventional hydrogel (CH; nelfilcon A) DD were placed over the corneal section and the NIBUT was evaluated after 1 h of blinking. A corneal topographer (Atlas, Zeiss Canada) was used to illuminate the lens surfaces and capture changes of the placido ring images. The humidity during the experiment was between 22-23%.

Results : A stable artificial tear film layer was formed over the artificial eyeball and the CLs. This layer was regenerated after each blink. The NIBUTs for delfilcon A, nelfilcon A, and the artificial eyeball were 7.55 ± 1.59 seconds, 8.16 ± 0.81 seconds, and 5.13 ± 0.09 seconds respectively. There were no significant differences between the two lens types (p>0.05), and between nelfilcon A and the artificial eyeball (p>0.05). However, delfilcon A had a longer NIBUT than the artificial eyeball (p<0.05).

Conclusions : The measured NIBUT values over the artificial eyeball and CLs are similar to those found in vivo. The OcuBlink simulates the blink motion and physiological tear flow rates, and could be used to measure in vitro pre-lens NIBUT values for various CLs.

Walther H, Phan CM, Jones L.. Detection of Phosphatidylcholine from Delefilcon A Contact Lenses using Mass Spectrometry American Academy of Optometry, Orlando, 2019 [ Show Abstract ][ PDF ]

Purpose: The gradual release of phospholipids from contact lenses could potentially stabilise the tear film over the front surface of the lens and increase comfort during wear. Delefilcon A (Dailies Total 1) contact lenses inherently contain phosphatidylcholine (PC), a zwitterionic phospholipid that is a potent wetting agent and lubricant found in the human tear film.Due to its molecular structure, PC is relatively difficult to detect using standard spectrophotometric techniques. However, it has been detected previously at low concentrations using ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS). The aim of this study was to detect the presence of PC from contact lens extracts of delefilcon A lens materials and its blister packaging solution using UHPLC-MS.

Methods: PC was extracted from the contact lens and the blister pack using 3 mL of 2:1 chloroform: methanol and 0.5 mL of monosodium phosphate aqueous buffer. The chloroform layer containing PC was collected, dried down using nitrogen gas, and then resuspended in 100 µL sample buffer. A PC standard, L-α-Phosphatidylcholine (Sigma Aldrich, St. Louis, MO), was prepared at 8 pmol/µL. The samples were analyzed using UHPLC-MS on a Waters and Thermo/Dionex system using a multi-step, reversed-phase gradient consisting of A: 60:40 acetonitrile: water and B: 90:10 isopropanol: acetonitrile, both with 10mM ammonium formate and 0.1% formic acid. The flow was set at 250 uL/min, the column was kept at 45C, sample tray at 4C, and injection volume was 10 uL.

Results: During the electrospray process, L-α-Phosphatidylcholine, also known as 1,2-diacyl-sn-glycero-3-phosphocholine, protonates to form C42H10NO8P+, with a theoretical mass m/z = 758.57. Elution peaks from the UHPLC-MS corresponding to PC were detected in the PC standard at m/z = 758.5-758.6. Other elution peaks for PC were also detected at m/z = 760.58-760.60, 786.60-786.61, and 788.62-788.63. These same peaks, with nearly identical retention times, were detected in both the blister pack solution and contact lens extracts for delefilcon A.

Conclusion: These results indicate that PC is present within the delefilcon A material and also in its blister pack solution. Future studies will determine if PC is released over time from the contact lens using a novel in vitro blink platform.

2018

Yee A, Jabeen A, Subbaraman L, McCanna D, Phan C-M, Jones L. Novel In-Vitro Method to Study Bacterial Interaction with Contact Lenses American Academy of Optometry, San Antonio, USA, 2018 [ Show Abstract ][ PDF ]

Purpose: Previous in-vitro studies have used a “soak” or closed vial method to assess bacterial binding to contact lenses (CL). The purpose of this study was to develop a novel in-vitro drip model to determine if bacterial adhesion to a CL material was possible. The novel in-vitro drip model would more closely resemble an accurate eye model in comparison to current methods undertaken.

Methods: The novel in-vitro drip method consists of a 5.5 mL syringe with saline solution and a flow rate controller dispensing 5 µl of saline solution containing the bacteria. The consistent drip volume is adjustable and mimics the normal human tear volume and flow. The solution flows through a silicone tube and onto a CL. The CL was placed on a sterile glass eyeball in an enclosed container to maintain the environment’s humidity. In the soak method, the CL was placed on top of a sterile glass eyeball and placed in the enclosed container with a 5 mL saline solution of 1.0 x 107 colony forming units (CFU)/mL. For both methods, lenses were incubated in the solution for 16 hours. After removal, the viable cells were diluted in serial dilutions. Aliquots of each dilution were plated on a trypticase soy agar plate and incubated for 24 hours at 37°C. After 24 hours, the CFU per lens were calculated manually under magnification.

Results: Using the in-vitro drip method, adhesion of Staphyloccocus aureus onto senofilcon A was successfully demonstrated. Preliminary analysis showed no significant difference (p = 0.34) between the drip and soak method when compared at high CFU/mL.

Conclusion: The novel drip method is a promising alternative to the conventional soak method, as this model is closer to the contamination that would occur in a human eye. The drip method may be an acceptable method of testing once the method can be further developed and tested in future studies, using a variety of lenses and bacteria.

2017

Gorbet M, Toameh D, Zhang J, Phan C-M, Walther H, Jones L. Development of a dynamic co-culture ocular cell in vitro model for ocular biocompatibility testing Invest Ophthalmol Vis Sci 2017;E-Abstract 4727

2016

Phan C-M, Walther H, Riederer D, Smith R, Subbaraman L, Jones L. Determination of the release of wetting agents from nelfilcon a using a novel in vitro eye model Optom Vis Sci 2016;93: E-abstract 165114 [ PDF ]

Phan C, Bajgrowicz M, Subbaraman L, Jones L. Release of moxifloxacin from daily disposable contact lenses using an in vitro eye model: Impact of artificial tear fluid composition and mechanical rubbing Invest Ophthalmol Vis Sci 2016;57: E-abstract 1474 [ PDF ]

Qiao H, Phan C-M, Walther H, Subbaraman L, Jones L. Localizing lysozyme deposition on contact lenses using a novel in vitro eye model Optom Vis Sci 2016;93: E-abstract 160100

Walther H, Phan C-M, Qiao H, Liu Y, Subbaraman L, Jones L. In vitro eye model to simulate the impact of blinking on contact lens deposition and drug delivery Optom Vis Sci 2016;93: E-abstract 160101

Walther H, Phan C, Subbaraman L, Jones L. Cholesterol Penetration into Daily Disposable Contact Lenses Using a Novel In Vitro Eye-Blink Model Invest Ophthalmol Vis Sci 2016;57: E-abstract 1476 [ PDF ]

2015

Bajgrowicz M, Phan C, McCanna D, Subbaraman L, Jones L. Effects of antifungal soaked silicone hydrogel contact lenses on Candida albicans in an agar eye model ISCLR Budapest, Hungary, 2015

Bajgrowicz M, Phan C, Subbaraman L, Jones L. Release of ciprofloxacin and moxifloxacin from daily disposable contact lenses using an in vitro eye model Invest Ophthalmol Vis Sci 2015;56: E-abstract 6085 [ PDF ]

Phan C, Jones L, Subbaraman L, Bajgrowicz M. Release of fluconazole from daily disposable contact lenses using a novel in vitro eye model Invest Ophthalmol Vis Sci 2015;56: E-abstract 3085

Phan C, Walther H, Gao H, Bajgrowicz M, Subbaraman L, Jones L. Developing a novel in vitro eye-blink platform for drug delivery and deposition research ISCLR Budapest, Hungary, 2015

Walther H, Phan C, Subbaraman L, Jones L. Cholesterol Penetration into Daily Disposable Contact Lenses Using a Novel In Vitro Eye-Blink Model ISCLR Budapest, Hungary, 2015

2014

Phan C, Subbaraman L, Jones L. Delivery of natamycin using cyclodextrin functionalized contact lenses Invest Ophthalmol Vis Sci 2014;55: E-abstract 4643 [ PDF ]

2013

Hall B, Phan C, Subbaraman L, Jones L, Forrest J. Extraction versus in situ techniques for measuring surface adsorbed lysozyme Canadian Optometry Schools Research Conference, Waterloo, Canada, 2013

Hall B, Phan C, Subbaraman L, Jones L. Forrest J. Direct comparison between in situ versus extraction techniques for measuring absorbed proteins: Application to lysozome deposited onto hydrogel contact lenses Invest Ophthalmol Vis Sci 2013;54: E-Abstract 5467

Phan C, Lui S, Gu F, Jones L. In vitro uptake and release of Natamycin dex-b-PLA nanoparticles from silicone hydrogel contact lens materials 20/20 NSERC ophthalmic materials conference, Niagara Falls, Canada, 2013

Phan C, Subbaraman L, Jones L. Delivery of natamycin using cyclodextrin functionalized contact lenses NSERC 20/20 Meeting, 2013

Phan C, Subbaraman L, Jones L, Liu S, Gu F. In vitro uptake and release of natamycin dex-b-pla nanoparticles from silicone hydrogel contact lens materials Invest Ophthalmol Vis Sci 2013;54: E-Abstract 501

Phan C, Subbaraman L, Liu S, Gu F, Jones L. Drug delivery of natamycin from contact lens materials using Dex-b-PLA nanoparticles ISCLR conference, Kyoto, Japan, 2013

Phan C, Subbaraman L, Liu S, Gu F, Jones L. In vitro uptake and release of natamycin Dex-b-PLA nanoparticles from silicone hydrogel contact lens materials Canadian Optometry Schools Research Conference, Waterloo, Canada, 2013

2012

Phan C, Jacob J, Subbaraman L, Jones L. Visualizing the uptake and release of natamycin from commercial contact lenses using confocal microscopy 2020 NSERC ophthalmic materials conference, Burlington, Canada, 2012

Continuing Education Presentations

2023

Phan C-M. Enhanced ocular drug delivery with spherical nucleic acids and screening methods thereof Centre for Eye and Vision Research Conference, Hong Kong, May 18, 2023

Phan C-M. Development of photoresponsive drug delivery systems and in vitro models for testing thereof Centre for Eye and Vision Research Conference, Hong Kong, May 18, 2023

2021

Phan C. Can we simulate the anterior eye to further improve contact lenses of the future? BCLA Virtual Clinical Conference & Exhibition, June 13, 2021

Professional Publications

2023

Phan C-M. Fast Forward to the Future: 3D Printing of Specialty Contact Lenses Contact Lens Spectrum 2023;38, August: 43

2022

Phan C. Smart Contact Lenses for Diagnostic and Therapeutic Applications OPTIK by VuePoint Optik 2022, May-June: 44-47

Phan CM. Fast forward to the future - diagnosing systemic diseases with smart lenses Contact Lens Spectrum 2022;37, February: 11

2021

Phan C. CLEAR report summary: Contact Lens Technologies of the Future https://contactlensupdate.com/2021/06/15/contact-lens-technologies-of-the-future/ 2021;60

2020

Phan C. Contact lens biosensors: Can we sense our tears? https://contactlensupdate.com/2020/02/06/contact-lens-biosensors-can-we-sense-our-tears/ 2020;February.

Phan C. Beyond 20/20 vision: the Mojo smart lens https://contactlensupdate.com/2020/02/06/beyond-20-20-vision-the-mojo-smart-lens/ 2020;February.

2019

Phan C., Walther H., Jones L. Development of a polymeric eye model for foreign body removal Optometric Education 2019;45, 1: 1-6

2012

Phan C. Delivering cyclosporine A from contact lenses: An article review ContactLensUpdate.com 2012