Redox-Responsive Hyaluronic Acid-Based Nanogels for the Topical Delivery of the Visual Chromophore to Retinal Photoreceptors

Abstract

Delivering therapeutics to the posterior segment of the eye is challenging due to various anatomical and physical barriers. While significant improvements have been realized by introducing direct injections to diseased sites, these approaches come with potential side effects that can range from simple inflammation to severe retinal damage. The topical instillation of drugs remains a safer and preferred alternative for patients' compliance. Here, we report the synthesis of penetratin-complexed, redox-responsive hyaluronic acid-based nanogels for the triggered release and delivery of therapeutics to the posterior part of the eye via topical application. The synthesized nanogels were shown to release their load only when exposed to a reducing environment, similar to the cytoplasm. As a model drug, visual chromophore analog, 9-cis-retinal, was loaded into nanogels and efficiently delivered to the mouse retina's photoreceptors when applied topically. Electroretinogram measurements showed a partial recovery of photoreceptor function in all treated eyes versus untreated controls. To the best of our knowledge, this report constitutes the first attempt to use a topically applied triggered-release drug delivery system to target the pigmented layer of the retina, in addition to the first attempt to deliver the visual chromophore topically.

Figure 1

Synthesis steps for the preparation of HA–cys–CH conjugate.

Figure 2

Physical characteristics of nanogel’s (A) size and (B) ζ potential at different stages of synthesis and drug loading steps.

Figure 3

(A) Triggered-release profile of fluorescein from the redox-sensitive HA–cys–CH and HA–cys–CH/P nanogels showing that the presence of 10 mM DTT causes the disintegration of covalent cross-links of nanogels to release their load. (B) Change of nanogel’s size by DLS in response to 10 mM DTT.

Figure 4

Real-time monitoring of cell attachment behavior using ECIS in response to HA–cys–CH and HA–cys–CH/P exposure. The HA-based nanogels were biocompatible with ARPE-19 cells at the exposure concentrations of 1 mg/mL and below.

Figure 5

Confocal microscopy images of ARPE-19 cells exposed to 2 mg/mL carbon dot-conjugated nanogels (A) without and (B) with penetratin at 30 min, 1 h, 2 h, and 4 h. Penetratin coating significantly increased the intracellular concentration of the nanogels.

Figure 6

Acute treatment with nanogels loaded with 9-cis-retinal largely restores the amplitude and sensitivity of chromophore-deficient mouse photoreceptors ex vivo. (A) Representative family of transretinal rod ERG responses from Rpe65^–/– mouse retinas. Test flashes of 505 nm light with intensities of 5.7 × 10⁴, 2.0 × 10⁵, 6.0 × 10⁵, 2.0 × 10⁶, and 5.7 × 10⁶ photons/μm² were delivered at time 0. (B) Representative family of transretinal rod ERG responses from Rpe65^–/– mouse retinas treated with HA–cys–CH nanogels loaded with 9-cis-retinal in the presence of penetratin. Test flashes of 505 nm light with intensities of 14, 33, 114, 392, 1.2 × 10³, 3.9 × 10³, and 1.1 × 10⁴ photons/μm² were delivered at time 0. (C) Representative family of transretinal rod ERG responses from Rpe65^–/– mouse retinas treated with 9-cis-retinal only. Test flashes of 505 nm light with the same intensities as in (B) were delivered at time 0. (D) Averaged rod intensity–response functions (mean ± scanning electron microscopy (SEM)) for untreated Rpe65^–/– isolated retinas (n = 4) and Rpe65^–/– retinas treated either with HA–cys–CH nanogels loaded with 9-cis-retinal in the presence of penetratin (n = 3) or free 9-cis-retinal in the media (n = 7). Error bars for some points are smaller than the symbol size. Hyperbolic Naka-Rushton fits yielded half-saturating intensities (I_1/2) of 4.7 × 10⁵, 175, and 142 photons/μm².

Figure 7

Confocal microscopy images of retinal cross sections after topical application of (A) PBS buffer, (B) fluorescein-only, (C) fluorescein-labeled nanogels without penetratin, and (D) fluorescein-labeled-nanogels with penetratin complexation. The presence of the nanogels can be seen in the marked section. RPE, retinal pigmented epithelium; POS, photoreceptor outer segments; ONL, outer nuclear layer; and INL, inner nuclear layer. Scale bars are 20 μm. The sections are stained only for 4′,6-diamidino-2-phenylindole (DAPI), the nucleus staining (blue). Green particles are the fluorescein-labeled nanogels.

Figure 8

Topical application of nanogels loaded with 9-cis-retinal partially restores ERG a-wave and b-wave responses of chromophore-deficient mice in the presence of the penetratin peptide in vivo. (A, B) Averaged rod (scotopic) intensity–response functions (mean ± SEM) for control untreated Rpe65^–/– mouse left eyes (n = 8) and right eyes of the same animals treated with HA–cys–CH nanogels loaded with 9-cis-retinal in the presence of penetratin (n = 8). (C, D) Lack of response recovery in the absence of penetratin. Averaged rod intensity–response functions (mean ± SEM) for control untreated Rpe65^–/– mouse left eyes (n = 6) and right eyes of the same animals treated with HA–cys–CH nanogels loaded with 9-cis-retinal in the absence of penetratin (n = 6). (E, F) Lack of response recovery after topical application of the visual chromophore alone. Averaged rod intensity–response functions (mean ± SEM) for control untreated Rpe65^–/– mouse left eyes (n = 5) and right eyes of the same animals treated with 9-cis-retinal only (n = 5). Error bars for some points in all panels are smaller than the symbol size. Statistical significance of the data is presented as **P < 0.01.