Chitosan and Hyaluronic Acid Nanoparticles as Vehicles of Epoetin Beta for Subconjunctival Ocular Delivery

Abstract

Neuroprotection in glaucoma using epoetin beta (EPOβ) has yielded promising results. Our team has developed chitosan-hyaluronic acid nanoparticles (CS/HA) designed to carry EPOβ into the ocular globe, improving the drug's mucoadhesion and retention time on the ocular surface to increase its bioavailability. In the present in vivo study, we explored the possibility of delivering EPOβ to the eye through subconjunctival administration of chitosan-hyaluronic acid-EPOβ (CS/HA-EPOβ) nanoparticles. Healthy Wistar Hannover rats (n = 21) were split into 7 groups and underwent complete ophthalmological examinations, including electroretinography and microhematocrit evaluations before and after the subconjunctival administrations. CS/HA-EPOβ nanoparticles were administered to the right eye (OD), and the contralateral eye (OS) served as control. At selected timepoints, animals from each group (n = 3) were euthanized, and both eyes were enucleated for histological evaluation (immunofluorescence and HE). No adverse ocular signs, no changes in the microhematocrits (≈45%), and no deviations in the electroretinographies in both photopic and scotopic exams were observed after the administrations (p < 0.05). Intraocular pressure remained in the physiological range during the assays (11-22 mmHg). EPOβ was detected in the retina by immunofluorescence 12 h after the subconjunctival administration and remained detectable until day 21. We concluded that CS/HA nanoparticles could efficiently deliver EPOβ into the retina, and this alternative was considered biologically safe. This nanoformulation could be a promising tool for treating retinopathies, namely optic nerve degeneration associated with glaucoma.

Keywords: chitosan; epoetin beta; erythropoietin; hyaluronic acid; mucoadhesion; nanoparticles; ocular delivery.

Figure 1

Mean IOP (mmHg) variation during the study after the subconjunctival administration of the nanoparticles. Data represents all groups.

Figure 2

Scotopic luminesce response-mean amplitudes of the a-wave and the b-wave (µV) recorded from the OD and OS in response to increscent light-stimulus intensities (dB). Values correspond to before and after the administration of the nanoparticles (after = before euthanasia). Data represent all groups.

Figure 3

Example of the scotopic luminesce response. The retinal response was recorded from the OD and OS of a rat (group C) before euthanasia in response to crescent light-stimulus intensities (in this example, from –10 dB to +5 dB).

Figure 4

Photopic luminesce response-mean amplitudes of the a-wave and the b-wave (µV) recorded from the OD and OS in response to increscent light-stimulus intensities (dB). Values correspond to before and after the administration of the nanoparticles (after–before euthanasia). Data represent all groups.

Figure 5

Example of a photopic flicker exam. The retinal response was recorded from the OD and OS of a rat (group D) before euthanasia in response to decrescent light-stimulus intensities (from 0 dB to –15 dB), after 10 min of continuous light exposure.

Figure 6

Scotopic Adaptation-mean amplitudes of the a-wave and the b-wave (µV) recorded from the OD and OS in response to light-stimulus after t minutes of dark adaptation (from 0 to 32 min). Values correspond to before and after the administration of the nanoparticles (after–before euthanasia). Data represent all groups.

Figure 7

Example of a Scotopic Adaptation exam. The retinal response was recorded from the OD and the OS of a rat (group E) before euthanasia in response to light-stimulus after 0 to 32 min of dark adaptation. This figure shows timepoints 8, 16, and 32 min.

Figure 8

Immunofluorescence image showing a cross-section of the retina after CS/HA-EPOβ administration: (a) OD from group A; (b) OS from group A (control); (c) OD from group F; (d) OS from group F (control). Green and blue channels were merged, and green tissue auto-fluorescence is visible in (b,d). Red arrows indicate EPOβ and cell nuclei are blue (DAPI) (40$\times$). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 8

Immunofluorescence image showing a cross-section of the retina after CS/HA-EPOβ administration: (a) OD from group A; (b) OS from group A (control); (c) OD from group F; (d) OS from group F (control). Green and blue channels were merged, and green tissue auto-fluorescence is visible in (b,d). Red arrows indicate EPOβ and cell nuclei are blue (DAPI) (40$\times$). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 9

Immunofluorescence image of the HepG2 cells: (a) positive control showing EPO in green and nuclei in blue; (b) negative control showing a very light green (auto-fluorescence) surrounding the nuclei in blue. Green and blue channels were merged (40×).

Figure 10

Photomicrographs of the treated eye cross-sections after CS/HA-EPOβ administration stained with hematoxylin and eosin: (a) ocular globe from group C (magnification 4×); CO, cornea; IR, iris; CB, ciliary body; LE, lens; RE, retina; SC, sclera. (b) cross-section from a rat’s retina from group D (magnification 40×); CH, choroid; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. (c) cross-section from a rat’s retina from group E (magnification 100×).

Figure 11

Sequence of procedures performed in each animal (n = 21). Oph Exam–Ophthalmological examination; Htc–blood collection for microhematocrit; ERG–electroretinography; CS/HA-EPOβ–subconjunctival administration of the nanoformulation.

Figure 12

Pictures the ERG set: (a) active electrodes (red) with silver tips placed on corneas; (b) reference electrodes (blue) placed next to the ears and the ground electrode (black) at the base of the tail; (c) head positioning inside the MiniGanzfeld stimulator.

Figure 13

Photo of the subconjunctival administration of the C/HA-EPOβ in the OD of a rat, under general anesthesia.

Figure 14

Representation of a rat ocular globe painted with tissue dyes: (a) frontal view; (b) caudal view. Photo of a rat ocular globe painted with tissue dyes: (c) lateral view; (d) caudal view, in green we can identify the transected optic nerve.