Design and evaluation of novel fast forming pilocarpine-loaded ocular hydrogels for sustained pharmacological response

Abstract

Fast forming hydrogels prepared by crosslinking a poly(ethylene glycol) (PEG)-based copolymer containing multiple thiol (SH) groups were evaluated for the controlled ocular delivery of pilocarpine and subsequent pupillary constriction. Physical properties of the hydrogels were characterized using UV-Vis spectrophotometry, transmission electron microscopy (TEM), rheometry, and swelling kinetics. Pilocarpine loading efficiency and release properties were measured in simulated tear fluid. The hydrogel formulations exhibited high drug loading efficiency (approximately 74%). Pilocarpine release was found to be biphasic with release half times of approximately 2 and 94 h, respectively, and 85-100% of the drug was released over 8-days. Pilocarpine-loaded (2% w/v) hydrogels were evaluated in a rabbit model and compared to a similar dose of drug in aqueous solution. The hydrogels were retained in the eye for the entire period of the study with no observed irritation. Pilocarpine-loaded hydrogels sustained pupillary constriction for 24 h after administration as compared to 3 h for the solution, an 8-fold increase in the duration of action. A strong correlation between pilocarpine release and pupillary response was observed. In conclusion, the current studies demonstrate that in situ forming PEG hydrogels possess the viscoelastic, retention, and sustained delivery properties required for an efficient ocular drug delivery system.

Fig. 1

TEM images of 3% (A) and 5% (B) hydrogels prepared using copolymer and crosslinker in 1:1 stoichiometry. The crosslinking networks are clearly visible; the network density increases with increase in copolymer concentration.

Fig. 2

Optical transmissivities of 3, 4, 5 and 6% hydrogels prepared using copolymer and crosslinker in 1:1 or 1:2 stoichiometry. The statistically significant different groups are denoted by * (p<0.05) and ** (p<0.01). The optical transmissivities of the hydrogel decreases with increase in copolymer and crosslinker concentration. All the hydrogels were found to be transparent or close to transparent.

Fig. 3

Influence of strain (A) and frequency (B) on G′ and G″ of hydrogels. The rheological measurements were carried out on 3% and 5% (w/v) hydrogels prepared using copolymer and crosslinker in 1:1 stoichiometry. The strain sweep test establishes the regime of linear viscoelasticity (LVE). The frequency sweep test shows that the hydrogels are more elastic than viscous and that they have the ability to resist structural changes under strain.

Fig. 4

Swelling kinetics of the hydrogel as a function of time. 3, 4 and 6% (w/v) hydrogels were prepared using 1:1 (A) and 1:2 (B) stoichiometries. All measurements were performed in triplicate and plotted as mean±SEM. The degree of hydrogel swelling decreases with increasing copolymer and crosslinker concentrations.

Fig. 5

Fractional release of pilocarpine as a function of time for 3, 4, 5 and 6% (w/v) hydrogels prepared using 1:1 (A) and 1:2 (B) stoichiometries. All measurements were performed in triplicate and plotted as mean±SEM. The release data were fitted using a two-phase exponential association equation in GraphPad Prism 4 software. The goodness of fit (R²) for the different hydrogels varied from 0.76 to 0.93. The initial burst release of pilocarpine appears to correlate well with the swelling phase (~0–3h) as shown in Figure 4. Once swelling terminates, a sustained release phase begins. The higher burst phase appears to also be affected by pore size with the largest pore size hydrogel (3%) having the largest burst effect. The release mechanism is non-fickian or anomalous involving both diffusion and polymer relaxation (0.5<n<1). An increase in copolymer/crosslinker concentration results in a slower pilocarpine release.

Fig. 6

Decrease in pupil diameter vs. time for pilocarpine-loaded hydrogel (4%, w/v, copolymer and crosslinker in 1:2 stoichiometry) and aqueous pilocarpine (2%, w/v) solution in PBS. Mean±SEM of four determinations is reported. The statistically significant differences in pupil diameter changes between both the groups is denoted by * (p<0.05) and ** (p<0.01). The hydrogel shows sustained pharmacological response for a period of 24 h.

Fig. 7

Correlation between in vitro pilocarpine release and pupillary constriction obtained in vivo. A linear correlation is evident with an R² of 0.97. As the amount of pilocarpine available for absorption decreases, a corresponding increase in pupil diameter is observed. Data are reported as mean±SEM. Solid line indicates the best-fit line and dashed line indicates the 95% confidence interval.

Scheme 1

Copolymer synthesis, (i) N, N-dimethylaminopyridine, p-toluene sulfonic acid monohydrate, N, N-diisopropylcarbodiimide/dichloromethane, RT, 24 h; (ii) trifluoroacetic acid, triisopropyl silane/water, RT, 5 h.