Microneedle loaded with luteolin-colostrum-derived exosomes: a dropless approach for treatment of glaucoma

Abstract

Glaucoma, a leading cause of irreversible blindness, is marked by elevated intraocular pressure (IOP) and retinal ganglion cell death. Traditional IOP-lowering eye drops often fail to penetrate the ocular barrier, leading to suboptimal outcomes. Microneedles (MN), offer a promising minimally invasive and localized alternative. Our study aimed to formulate a naturally-derived nanodelivery system using Luteolin-loaded colostrum-derived exosomes (LUT-EX) and propolis in MN arrays for better ocular delivery. The isolated exosomes were uniform, averaging 50.83 nm in size, with a zeta potential of -21.89 mV. LUT-EX showed a 48-h sustained release and high safety with an IC50 of 356.3 µg/mL. Integrating LUT-EX and propolis into MN arrays achieved optimal dissolution in over one minute and maintained mechanical strength under 30 N compression. LUT-EX@MN increased LUT permeation through scleral tissues 2.6-fold compared to gel matrix formulations. It also showed a sustained IOP-lowering effect reaching the normal IOP level in the first 3h and sustained over 7 days. The integrated system significantly reversed glaucoma-induced changes in TNF-α, IL-8, MYOC, NRF2, TIMP1, and IL-1β levels, resembling those of the healthy group. It also boosted antioxidant activity, increasing glutathione peroxidase by 1.6-fold compared to glaucomatous rabbits. Thus, our study highlighted that the integration of LUT-EX into microneedle arrays presents a groundbreaking dropless approach for localized glaucoma treatment, offering enhanced therapeutic efficacy. This platform could revolutionize glaucoma management, paving the way for more effective and targeted ocular therapies.

Keywords: Bio-inspired; Extracellular vesicles; Herbal drug; Intraocular pressure; Microneedles; Propolis.

Fig. 1

Characterization of the isolated colostrum milk-derived exosomes including a) Transmission electron microscope (TEM) of unloaded and loaded exosomes, b) Characterization of exosomes surface biomarkers (CD9 and CD81) determined by flowcytometry, c) Characterization of exosomes surface biomarker CD63 determined by flowcytometry, d) Profile of in vitro release of LUT free solution and LUT-loaded exosomes (LUT-EX) in 12mL 25% PEG (v/v) in PBS (pH 7.4) kept at 37 °C and 100 rpm to ensure sink conditions. Results are expressed as % LUT released, and the data presented is the mean of triplicate (mean ± SD)

Fig. 2

Evaluation of CSF: a) Cells’ viability with increasing concentrations of LUT, EX, and LUT-EX after 24 h, b) Images captured by confocal laser microscopy demonstrating the cellular association of C6 free solution and C6-EX 4 h and 24 h post incubation, c) Quantitative representation of the corrected calculated total fluorescence intensity. (n = 6, mean ± SD,) (p ≤ 0.001), d) Representative images for the migration assay: to different treatments’ migration activity (magnification × 20), and e) Calculated percentage closure of scratched area and f) calculated migration index and migration rate of cells after treatment with different formulations after 24h (n = 6, mean ± SD,). One-way ANOVA was utilized for data analysis, followed by Tukey’s post-hoc test to compare groups. Means of similar symbols were statistically insignificant: a > b > c (p ≤ 0.05)

Fig. 3

Characterization of prepared MNs: a) SEM image for morphological examination of unloaded MN, LUT@MN and LUT-EX@MN at magnification × 60 and × 250, scale bar represents 60 μm and 250 μm, b) TEM images demonstrating the preservation of vesicular nature of exosomes after incorporation in MNs, c) Images captured using a stereomicroscope at various times after unloaded MN, LUT@MN, and LUT-EX@MN were inserted into freshly dissected bovine sclera demonstrating their dissolution. Magnification 4.5×, d) Percentage of the length of needle remaining post dissolution at various time intervals for either blank dMN, LUT@MN, or LUT-EX@MN (mean ± SD, n = 3), e) Images showing MN height of unloaded MN, LUT@MN, and LUT-EX@MN before and after compression using the texture analyzer. (mean ± SD, n = 5), f) Graphical representation of the % reduction in length after compression, g) Using Parafilm M^® at a magnification of 4.5x, micrographs of stereomicroscope illustrate the in vitro insertion behavior of MN and display the microneedle array after insertion in three distinct Parafilm layers. and h) A bar graph displaying the percentage of LUT that either penetrated or was deposited in freshly dissected bovine scleral tissue following six hours of exposure to various treatments. (mean ± SD, n = 3)

Fig. 4

a) Images of chorioallantoic membrane test conducted on hen’s eggs (HET-CAM) following different treatments at room temperature for prediction of the potential of ocular discomfort and irritation potential. b) Scoring of ocular irritation in each sample via measuring the red irritated area and c) Percentage reduction in intraocular pressure following topical ocular insertion of a single dosage of different fabricated MN and their corresponding gel matrix over a period of seven days following glaucoma induction in rabbits

Fig. 5

Analysis of glaucoma biomarkers in aqueous humor of different treated modalities. Protein levels of a) TNF-α, b) IL-8, and c) activity of GPx were measured quantitatively by ELISA. Fold change in gene expression levels of d) MYOC, e) IL-1β, f) TIMP, and g) NRF2 measured and normalized to the expression of GAPDH which acts as the housekeeping gene by qRT-PCR using (2^-ΔΔCt). (mean ± SD, n = 3). One-way ANOVA was used for data analysis which was followed by Tukey’s post-hoc test for group comparisons. Means of similar symbols were statistically insignificant a < b < c < d < e and all of the p-values are ≤ 0.05

Fig. 6

Photomicrographs of rabbits’ eyes examining the retina via H&E staining, Scale bar 50µm. A) Negative control consists of the normal healthy retinal layers (1) ganglion cell layer (GCL), (2) inner plexiform layer, (3) inner nuclear layer, (4) outer plexiform layer, (5) outer nuclear layer, and (6) rod and cone lamina; B) positive control confirmed the induction of glaucoma where it showed distinctive damage to the whole identity of retina layers. C) Placebo Gel group showed outer nuclear layer increased thickness (yellow line). D) Placebo MN group showed degenerated layers (red circle) and edema in GCL (black arrows). E) EX@ Gel group showed congestion in GCL layer (black arrow) and abnormal inner nuclear layer (red arrow). F) EX@MN group showed normal layer rods and cones layer (yellow triangle) with edema and congestion of GCL (black arrow). G) LUT@Gel group showed exaggerated inner plexiform layer (yellow line) and edema in GCL layer (black arrow). H) LUT@MN group comprises the normal layers with minimal apoptosis in GCL (black arrow). I) LUT-EX@Gel group showed abnormal layers specifically the GCL without distinctive neurons (yellow line). J) LUT-EX@MN group comprised near normal retina layers