Abstract
Purpose :
Previous work shows that avian lenses treated with blebbistatin, a myosin II inhibitor, leads to a change in actomyosin distribution and an overall loss of stiffness of the lens as a whole. In the present study, blebbistatin was linked to a lens-specific antibody (anti-aquaporin 0; AQP0Ab) to form an antibody-drug (anti-aquaporin 0-blebbistatin; b-AQP0Ab) conjugate. The effects of the b-AQP0Ab on the stiffness of crystalline lenses were investigated.
Methods :
One lens of 7-day-old white leghorn chickens (Gallus gallus domesticus) was treated with either 0.05 mg/ml of AQP0Ab (n=6) or b-AQP0Ab (n=5). The fellow eyes served as controls, and were exposed to vehicle (phosphate buffered saline; PBS). All lenses were treated for 30 minutes at room temperature. After treatment, lenses were placed, anterior side down, onto a pedestal in a chamber containing 10 mL of Tyrode’s solution (TS). Lenses were compressed axially by 0.75 mm and the resultant force exerted back by the lens was registered by a 10N sensor. Force-compression data of individual lenses were adjusted to account for buoyancy of the TS and filtered (running average function; Sigmaplot) to remove high frequency noise. The data were then fit with 3-parameter exponential growth curves (y=y0+aebx) in order to determine the b-coefficient, which is a unitless constant that relates force and compression, and therefore, stiffness of the lens.
Results :
Stiffness values of lenses treated with AQP0Ab were not different than those of their vehicle controls (compare mean ± SD: 3.38±0.97 vs 3.41±1.11, respectively; p=0.957). However, lenses treated with b-AQP0Ab were significantly stiffer than their vehicle counterparts (5.03±0.23 vs 4.07±0.68; p=0.035).
Conclusions :
Lenses treated with b-AQP0Ab showed a significant decrease in stiffness compared to their vehicle counterparts, indicating functionality of the antibody-drug conjugate. As AQP0Ab treatment did not affect the stiffness of the lens, the change in biomechanical integrity is presumably due to the effects of the hybridized blebbistatin. Future directions include optimizing the percentage yield of b-AQP0Ab, and determining the efficacy of the b-AQP0Ab conjugate to unconjugated blebbistatin.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.