Sustained intraocular pressure-lowering effect and biocompatibility of a single subconjunctival administration of hydrogel-encapsulated nano-brinzolamide

Abstract

Brinzolamide is a widely used treatment for glaucoma, but its effectiveness relies on at least twice-daily dosing, which can be challenging for patient adherence. To overcome this limitation, we developed an injectable hydrogel-based delivery system designed to maintain therapeutic drug levels with a single administration. This approach aims to simplify treatment and improve clinical outcomes. Brinzolamide-loaded polyethylene glycol poly (lactic-co-glycolic acid) (PEG-PLGA) nanoparticles were encapsulated within a hydrogel synthesized through the crosslinking of oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMC). In vitro studies were conducted to assess the nanoparticles' characterization, release profile, and biocompatibility. In a steroid-induced high intraocular pressure (IOP) mouse model, the efficacy of a single subconjunctival injection in lowering IOP was evaluated. Additionally, both cellular and animal biocompatibility were assessed. The brinzolamide-loaded hydrogel system (Hydrogel@Brz) contained nanoparticles with an average diameter of 40.76 nm, exhibiting a stable size distribution and a spherical morphology. The hydrogel demonstrated excellent injectability, self-healing properties, and a porous structure conducive to nanoparticle encapsulation. In vitro release studies revealed a sustained drug release of 86% over 14 days. No cytotoxicity was observed in human primary trabecular meshwork cells (HTMCs), human Tenon's capsule fibroblasts (HTFs), or the retinal ganglion cell line R28. In vivo, a single injection led to a prolonged IOP reduction lasting up to 21 days. No signs of drug toxicity were detected in ocular tissue sections, transverse optic nerve sections under transmission electron microscopy, or pathology slides of various organs. The brinzolamide-loaded hydrogel has demonstrated promising potential for sustained drug delivery and effective intraocular pressure reduction while maintaining good biocompatibility. However, further studies in larger animal models and long-term evaluations are needed to confirm its clinical applicability.

Fig. 1

Material characterization. A The drug-loaded nanoparticles have a particle size of 40.76 nm, a PDI of 0.162, and a uniform size distribution; B The particle size and PDI of the nanoparticles are stable within two weeks of storage at 4 °C; C Transmission electron microscopy of the nanoparticles; D Nuclear magnetic resonance spectrum; the three new peaks in the circle represent the successful synthesis of OHA; E 4 wt% OHA and 4 wt% CMC form a stable hydrogel within 3 min at room temperature; F Scanning electron microscopy: The hydrogel has a porous surface structure; G Shear thinning, the viscosity of the hydrogel decreases with the increase of shear rate, and it has good injectability; H Step strain scanning, when the strain is 1%, the storage modulus G′> loss modulus G″, the hydrogel structure; when the strain is 500%, the storage modulus G′< loss modulus G″, the hydrogel structure is destroyed, and when the strain is restored to 1%, the hydrogel structure can be restored. The representative hydrogel has good self-healing properties; I In vitro drug release, the cumulative drug release was 44.63% at two days and 86% at 14 days

Fig. 2

Hydrogel@Brz’s intraocular pressure-lowering effect and preliminary in vivo drug release investigation. A Schematic diagram of the modeling approach; B Changes in intraocular pressure; C Fluorescence and semi-quantitative analysis of frozen sections from eyeballs collected 1 and 2 weeks after subconjunctival injection of hydrogel-loaded Nile red nanoparticles (Week 1 Hydrogel@Nile red = 14.8 ± 1.5, Nile red = 2.4 ± 0.5; Week 2 Hydrogel@Nile red = 6.7 ± 0.4, Nile red = 0.5 ± 0.1). The white arrow points to the ciliary body that produces aqueous humor. Data analyzed by unpaired Student’s t test or Welch’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Cytotoxicity of the drug. A–C CCK8 assay evaluating the relative cell viability of HTFs (Control = 100.0 ± 5.0, Brz = 101.7 ± 3.8, Hydrogel = 99.6 ± 3.6, Hydrogel@Brz = 98.5 ± 7.1), HTMCs (Control = 100.0 ± 2.8, Brz = 100.2 ± 3.1, Hydrogel = 104.3 ± 2.2, Hydrogel@Brz = 105.2 ± 3.34), and R28 cells (Control = 100.0 ± 4.3, Brz = 99.54 ± 2.1, Hydrogel = 102.8 ± 8.4, Hydrogel@Brz = 97.7 ± 6.0); D, E Phalloidin staining showing cytoskeletal structures of HTMCs and HTFs after drug intervention. Data analyzed by unpaired Student’s t test or Welch’s t test; ns = p > 0.05

Fig. 4

Biocompatibility at the animal level. A Schematic diagram of animal toxicology experiments; B Visual performance analysis, where Cyc(Cycles) represents mouse visual ability, with higher values indicating better performance (Control = 188.0 ± 13.0, Hydrogel = 186.0 ± 18.2, Hydrogel@Brz = 188.0 ± 14.8); C Electrophysiological assessment of phNR wave amplitude, which decreases in cases of retinal ganglion cell damage (Control = 4.56 ± 0.80, Hydrogel = 4.46 ± 0.63, Hydrogel@Brz = 4.48 ± 0.60); D, E Immunohistochemical staining and semi-quantitative analysis of the retinal ganglion cell marker Brn3a (Control = 24.0 ± 2.7, Hydrogel = 23.67 ± 3.5, Hydrogel@Brz = 24.33 ± 3.5); F, G Transmission electron microscopy of optic nerve cross-sections and axon counting (Control = 55.3 ± 4.2, Hydrogel = 54.3 ± 6.0, Hydrogel@Brz = 56.3 ± 4.7); H. Histological sections and staining of the eyeball and major systemic organs. Staining includes HE for the eyeball, intestine, spleen, and heart; Masson’s trichrome for the lungs; Sirius Red for the liver; and PAS staining for glycogen in the kidneys. Data analyzed by unpaired Student’s t test or Welch’s t test; ns = p > 0.05