Zwitterionic Polymers

Six novel zwitterionic coatings (F–K) and three control coatings (L–N) were synthesized directly onto 24 well plates at BioInteractions Ltd. (Table 2-5).

Table 2-5: Composition details of zwitterionic coatings

Coating Novel Zwitterionic Polymer Composition

F 90% butyl methacrylate

10% novel zwitterionic monomer G 80% butyl methacrylate

20% novel zwitterionic monomer H 70% butyl methacrylate

30% novel zwitterionic monomer I 58% hydroxypropyl methacrylate

31% hexyl methacrylate

11% novel zwitterionic monomer J 50% hydroxypropyl methacrylate

30% hexyl methacrylate

20% novel zwitterionic monomer K 50% methoxyethyl methacrylate

30% hexyl methacrylate

20% novel zwitterionic monomer L 100% poly (butyl methacrylate)

M 90% butyl methacrylate

10% 2-methacryloyloxyethyl - phosphorylcholine (MPC)

N 70% butyl methacrylate

30% 2-methacryloyloxyethyl - phosphorylcholine (MPC)

2.5.1

Cell Growth Study

Four wells per coating were synthesised, F-K coatings were in one plate and L-N coatings were in an additional plate. N/N1003A LECs were seeded on coated wells at 1x104/cm2, uncoated TCPS wells served as a control. Representative phase contrast micrographs were taken throughout the study starting at day 1 and cells were fed every 2 – 3 days. Cells were fixed at days 1, 4 and 7 with NBF.

Composition of zwitterionic coatings and ratios of zwitterionic monomer: comonomer(s) used.

2.5.2

Cytotoxicity Assay

To test whether the coatings were prohibiting cell attachment via the chemistry and not because the coatings were leaching toxic material into the medium, a cytotoxicity assay was performed, following BS EN ISO 10993-5:2009 using the extraction method. To do this medium was added directly to coatings F-N and incubated at 37° for 48 hours. In addition medium was added to polystyrene coverslips (blank) and TCPS wells (negative control) and incubated at 37° for 48 hours. N/N1003A LECs were seeded at 5x104/cm2 onto 24 well TCPS plates for 24 hours. Medium from the sub confluent LEC monolayer was removed and replaced with medium from the coatings and control wells. 5% DMSO was added to four wells of sub confluent LECs and served as a positive control to kill the LECs. LEC metabolic activity was monitored at days 1 and 3 by a resazurin (Sigma Aldrich, UK) assay. To do this medium was removed and replaced with medium containing 10% resazurin (in the dark). Cells were incubated for 3 hours at 37°. After this time 100µl of the medium containing resazurin was aliquoted from each well into a black plate in triplicate. The plate was read on the FLx800 microplate fluorescence plate reader (Bio-Tek Instruments INC., UK) at 530 excitation/590 emission wavelengths.

2.5.3

Toxicity Assay

An additional toxicity assay was performed to observe if unattached cells from coatings F-N could be removed from the coated wells, seeded into fresh TCPS wells and attach, spread and grow as usual. LECs were seeded onto the coated wells and left for two hours to attach, TCPS served as a control. After two hours medium with floating cells were removed from coated wells and reseeded onto fresh 24 well plates. As some cells attach to coatings J, L and N the original plates were kept and these wells were refilled with fresh medium, meaning any cells that had adhered in the two hours could continue to grow. The metabolic activity was assessed using resazurin again on days 1 and 4.

2.5.4

Cell Staining

Zwitterionic plates were fixed with NBF on days 1, 4 and 7. Cells were stained with DAPI, (2.1.14) and phalloidin (2.1.15). To assess LEC growth three micrographs per well were taken and the cell nuclei was counted using a macro set up in ImageJ. The average cell growth of each coating at each time point was calculated.

2.5.5

Time Lapse Microscopy

Zwitterionic coatings were examined using the IncuCyte time lapse microscopy (Essen BioScience) for a total of 7 days. Micrographs were taken every hour to build up a video of still micrographs. Micrographs were taken in the exact same location each time.

2.5.6

Surface Analysis

Contact angle measurements were taken using the piezo-electric system (2.1.18.1), 9 points per sample were measured, repeated in triplicate. The topography of the coatings was also examined using SEM (2.1.18.2) and WLI (2.1.18.3). For WLI 4 areas per sample were analysed x50 and four at x100 magnification.

2.5.7

Bulk Materials

Coating F was copolymerised with pHEMA and synthesised as a bulk material. Contact lens moulds were used as IOL moulds were not available. The bulk material was cured using UV light. PHEMA contact lenses were used as a control. Contact lenses were placed in 13mm diameter cell crowns to hold the lens in place inside a 24 well plate. N/N1003A LECs were seeded at 1x104/cm2 for seven days. To compare the bulk material to the gold standard, LECs were also seeded onto Acrysof (Alcon) acrylic IOLs. Untreated TCPS wells served as a control. Phase contrast micrographs were taken during the seven days in culture. On day 7 cells were fixed with methanol for 5 minutes and cells were stained with methylene blue (2.1.10) to visualise the cell attachment, morphology and growth.

An additional control of C-Flex (Rayner) pHEMA IOL was separately tested to compare LEC response to bulk material pHEMA provided by BioInteractions Ltd. LECs were seeded on Acrysof IOLs to repeat and confirm cellular response. Uncoated TCPS wells were used as a control. Cells were seeded at 1x104/cm2 for 7 days. Phase contrast micrographs were taken during the 7 days in culture. On day 7 cells were fixed with methanol and cells were stained with methylene blue.