Development of in situ crosslinked hyaluronan as an adjunct to vitrectomy surgery

Abstract

Ophthalmologists have used hyaluronan (HA) products as adjuncts to ocular surgery since the 1970s. However, HA products are not always functional in surgeries of the posterior eye segment due to their lack of biomechanical strength. In this study, we developed an in situ crosslinked HA (XL-HA) and evaluated its potential as an adjunct to vitrectomy surgery in an in vitro model with a triamcinolone acetonide (TA) layer used as a pseudo residual vitreous cortex (RVC). Within a few minutes at concentrations over 0.9%, XL-HA, generated by the click chemistry of HA-dibenzocyclooctyne and HA-azidoethylamine, formed a hydrogel with the appropriate hardness for tweezers peeling. XL-HA (concentration, 0.76-1.73%) without dispersion successfully entered the TA layer and removed more than 45% of the total TA. Dynamic viscoelasticity helps to explain the rheological behavior of hydrogels, and the assessment results for XL-HA indicated that suitable concentrations were between 0.97% and 1.30%. For example, 1.30% XL-HA hydrogel reached sufficient hardness at 3 min for tweezers peeling, and the TA removal ability exceeded 70%. These results demonstrated that XL-HA was a potential adjunct to successful vitrectomy.

Keywords: In situ crosslinked hyaluronan; Ophthalmic viscoelastic device; Removal; Residual vitreous cortex; Vitrectomy.

Fig. 1

In situ crosslinked hyaluronan hydrogel. Reaction scheme of in situ crosslinked hyaluronan hydrogel (XL-HA)

Fig. 2

Removal of TA, a pseudo-vitreous cortex, with the Beriplast P. Freeze-dried product A (human fibrinogen, 240 mg; human factor XIII, 180 IU; bovine aprotinin, 3000 KIE) and freeze-dried product B (human thrombin, 900 U) were reconstituted in 3 mL of BSS, solution A and B (undiluted), respectively. Besides, BBG was added to solution A to give a final concentration of 270 µg/mL. A TA layer. B The Beriplast P hydrogel was formed by using the multi-layer procedure recommended in the manufacturer’s instructions. Briefly, solution A (175 µL) was placed on the TA layer, and the same volume of solution B was placed on the solution A layer. C After incubating the mixture under ambient conditions for 5 min, the resulting hydrogel was removed with tweezers. D–F Beriplast P prepared by mixing diluted solution A and B was also evaluated. D Just mixing. E After 5 min. F Removing Beriplast P

Fig. 3

Time-course of dynamic viscoelasticity of various viscoelastic materials. A XL-HA: The same volume (140 µL) of 1% solutions of HA-DBCO and HA-AEA prepared as batch 1 were mixed and put on a measuring disc. Dynamic viscoelasticity was measured with a modular compact rheometer: G’, indigo line; G”, maroon line. Effect of concentrations on the dynamic elasticity of XL-HA. (B) Time-course of the G’ of XL-HAs prepared from HA derivatives with the DS of 0.09 (batch 1) at different concentrations. C Time-course of the G’ of XL-HAs prepared from HA derivatives with the DS of 0.30 (batch 6) at different concentrations

Fig. 4

Typical 1H-NMR spectra of HA300, HA-DBCO, HA-AEA, and modified HA-AEA. Modified HA-AEA was prepared by reacting HA-AEA with a sufficient amount of DBCO-amine. HA-AEA, HA-DBCO, and HA300 were evaluated individually. NMR chemical shifts of aromatic protons are observed at around 7–8 ppm and N-acetyl methyl protons are observed at 1.9–2.1 ppm of HA-DBCO or modified HA-AEA

Fig. 5

Removal of pseudo-vitreous cortex with the XL-HA. Batch 1 used in this study. A Picking up XL-HA after standing in BSS for 3 min. B XL-HA was placed on the TA layer (left), and the resulting XL-HA was kept for 5 min and removed with tweezers (right). C Quantitative assay using a 24-well plate

Fig. 6

Effect of HA300, HA-AEA, or HA-DBCO on the growth of ARPE-19 cells. The effect of HA-AEA (batch 1) and HA-DBCO (batch 1) on the proliferation of ARPE-19 cells was tested in comparison with their raw material HA300. Values represent the mean ± standard deviation (n = 3)