(T1230-02-09) Examination of the Diffusion Properties of Hydrogels as Optimized Preclinical In Vitro Models for the Human Vitreous Body

Purpose: The prevalence of age-related eye diseases is increasing, yet many preclinical animal studies are necessary in the development of new therapeutic approaches. Therefore, a biorelevant in vitro model with comparable diffusion properties for therapeutics would be beneficial.
The aim was to compare rheologically optimized hydrogels with different gel formers regarding their diffusion properties for a therapeutically relevant drug. This allows the selection of the most similar vitreous body substitute for the biorelevant in vitro model in the end.

Methods: Fresh porcine vitreous bodies (vitreous) were used as a surrogate for the human vitreous body. Gellan gum-hyaluronic acid¹ (Gellan-HA, gellan gum: Kelcogel^® F, CP Kelco & hyaluronic acid: Caesar & Loretz), hyaluronic acid-agar² (HA-Agar, agar: Kobe I, Carl Roth), and hypromellose³ (HPMC, Methocel E4M®, Caesar & Loretz) were used as gelling agents, and phosphate-buffered saline (PBS) pH 7.47 served as the basis. Diffusion studies were conducted in a USP apparatus 7 (400-DS, Agilent) over 7 days at a temperature of 35 °C and a dipping speed of 30 dips/min. Self-designed gel-baskets (Figure 1) were produced using 3D printing, into which 0.1 ml triamcinolone acetonide suspension (Triam 40 mg/ml Lichtenstein, Zentiva) was injected. A dialysis tubing (25 kDa MWCO, Spectra/Por^® 7, Repligen) was stretched around them and fixed with sealing rings, and an extension rod was screwed in. To maintain sink conditions, the release volume consisted of 10 ml PBS pH 7.47 with an addition of 0.3% sodium dodecyl sulfate, and the entire medium was replaced at each sampling point. The samples were measured by HPLC (pump L-2130, autosampler L-2200, oven L-2300, UV-detector L-2400, VWR Hitachi, column: Eurospher II 100-5 C18A, Knauer) with a mobile phase mixture of water/acetonitrile 72/28 with a flow rate of 1.5 ml/min at 240 nm and 40 °C. For rheological measurements, a 25 mm parallel plate system with a measuring distance of 1 mm (MCR 702e MultiDrive, Anton Paar) was used at 35 °C. Following a predetermined relaxation period after trimming, viscosity tests were conducted at a shear rate of 0.5 s^-1.

Results: In Figure 2, the diffusion rates of various hydrogels are depicted. A more detailed comparison is performed after 8 hours, as it could be shown that the viscosity of vitreous ex vivo is still comparable to fresh material (Figure 3). It is evident that a lower diffusion rate is achieved in PBS. This is most likely caused by the fast sedimentation in the non-gelled media. The diffusion rate is also lower in HPMC. Here, the suspension also sinks to the bottom of the basket over time, and in addition, the gel viscosity is much higher at the end than that of the vitreous (Figure 3). This gel compresses into a small lump at the bottom of the basket, which encloses the suspension. The two Gellan-HA gels behave mostly very similarly, with only the loss factor of the more concentrated gel adjusted to that of the vitreous. The diffusion rate of Gellan-HA is always higher than that of the vitreous, especially in the comparison after 8 hours, as more suspension is retained in the middle of the basket here, whereas a larger part sinks to the bottom in the vitreous. After 7 days, when approximately an equilibrium is reached, the diffusion rates of 7.3 and 7.4 µg/h are similar to that of the vitreous after 8 hours (11.1 µg/h). The diffusion in HA-Agar is consistently similar to the vitreous, and even after 8 hours they are the closest. However, Figure 3 shows that there is almost nothing left of the vitreous after 7 days, and its viscosity is almost at the level of PBS (3 mPa*s). Therefore, its diffusion rate is no longer representative here. Comparing the diffusion rate of HA-Agar towards the end with the vitreous after 8 hours, other gels are closer. When examining the viscosities after 7 days (Figure 3), it is noticeable that the viscosity of the Gellan-HA gels has slightly decreased compared to the fresh ones and porcine vitreous. In contrast, the viscosity of HA-Agar has slightly increased; nevertheless, it is still very similar to that of the vitreous.

Conclusion: We observed that under the conditions here, the diffusion properties of HPMC least resemble those of the vitreous, and additionally, the stability over 7 days is insufficient. HA-Agar most closely resembles the vitreous and has the best stability; however, the diffusion rate in the plateau region is lower than that of the vitreous after 8 hours, which makes it more suitable for shorter experiments. Both Gellan-HA gels exhibit comparable diffusion and stability properties. They consistently show higher diffusion rates, but their plateau value is quite similar to that of the vitreous after 8 hours. Therefore, they appear more suitable for long-term tests, provided sufficient stability.
For a final decision between the gels, further diffusion experiments with varying conditions are necessary.

References: 1. Suri, S. and Banerjee, R. In vitro evaluation of in situ gels as short term vitreous substitutes. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials. 79. 650-664. (2006)
2. Awwad, S.; Lockwood, A.; Brocchini, S. and Khaw, P. T. The PK-Eye: A Novel In Vitro Ocular Flow Model for Use in Preclinical Drug Development. J Pharm Sci. 104. 3330-42. (2015)
3. Fernandez-Vigo, J.; Refojo, M. F. and Verstraeten, T. Evaluation of a viscoelastic solution of hydroxypropyl methylcellulose as a potential vitreous substitute. Retina (Philadelphia, Pa.). 10. 148-152. (1990)

Acknowledgements: The authors thank Theo Keinhörster Großschlachterei mit Fleischhandel e.K. for supply of the fresh porcine eyes and CP Kelco for the gellan gum powder.


Figure 1. Gel-basket with screw-in rod and depicted dimensions in millimeter.


Figure 2. Mean rate of diffusion of triamcinolon acetonide in the porcine vitreous body, phosphate-buffered saline (PBS), gellan gum-hyaluronic acid (Gellan-HA), hypromellose (HPMC) and hyaluronic acid-agar gels (HA-Agar), n = 6 (vitreous body) or 3 (hydrogels and PBS), mean ± sd.


Figure 3. Determination of gel stability during diffusion via viscosity measurements for porcine vitreous body, gellan gum-hyaluronic acid (Gellan-HA), hypromellose (HPMC) and hyaluronic acid-agar gels (HA-Agar) at 35°C and a shear rate of 0.5 s^-1 after 12 seconds, n = 6 (vitreous body) or 3 (hydrogels), mean ± sd.