In vitro characterization of bovine colostrum milk-derived exosomes

Milk-derived exosomes have several benefits over exosomes derived from other sources when it comes to treating glaucoma. Milk is a plentiful and easily sustainable source of exosomes. Additionally, collecting large amounts for therapeutic uses is easier and simpler [32]. Moreover, milk-derived exosomes are extremely biocompatible and unlikely to trigger any immunological reaction because they are naturally obtained from a common food source. The development of non-invasive treatment approaches benefits from the stability and integrity of this type of exosomes. Moreover, exosomes derived from milk include bioactive compounds that have anti-inflammatory and neuroprotective qualities, among other potential therapeutic benefits [33]. Thus, milk-derived exosomes are a viable option for additional study and advancement in glaucoma treatment.

In our study, bovine colostrum milk-derived exosomes were successfully isolated using the precipitation method. There are several benefits to utilizing polyethylene glycol (PEG) in the precipitation process to separate colostrum-derived exosomes. PEG-based precipitation reliably produces a large number of exosomes. Additionally, PEG reagents are widely accessible and affordable, making this approach cost-effective as it does not require complex procedures or complicated equipment. It is also worth mentioning that PEG-based precipitation preserves the integrity and biological activity of exosomes, which makes them suitable for a variety of downstream uses [34].

The successful isolation of colostrum-derived exosomes was confirmed, as depicted in Fig. 1, which highlights their morphology, colloidal properties, and surface markers from bovine colostrum milk. TEM imaging verified the uniform spherical shape of these natural nanoparticles (Fig. 1a) both before and after drug loading, with average diameters of 45.18 ± 4.2 nm and 51.74 ± 2.9 nm, respectively. The slight increase in size suggests an insignificant change, indicating that the integrity of exosomes remained intact. The extracted exosomes’ average particle size measured by the Malvern zetasizer was 50.83 nm with a PDI of 0.13. This discrepancy may arise because TEM directly assesses the physical size of air-dried exosomes, while the zetasizer determines their hydrodynamic diameter, which includes the influence of the surrounding liquid layer causing the observed slight increase in particle size [35]. The results of Lei et al. align with our results, who reported a size of 83.2 nm for milk-derived exosomes [36] where both isolated exosomes showed a typical size of exosomes (<100 nm) when compared to other extracellular vesicles [37].

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)

Particle concentration and zeta potential of the isolated colostrum milk-derived exosomes were assessed using the Malvern Zetasizer before. The analysis revealed a particle concentration of 1.54E+10 particles/mL and an average zeta potential of −21.89 mV, indicating that the extracted exosomes exhibit reduced susceptibility to excessive aggregation and precipitation; a desirable characteristic for an effective drug delivery system. These results align with earlier studies that reported a zeta potential of −17 mV for exosomes derived from bovine milk [38]. Additionally, previous studies by Lei et al. reported a yield of 7.24E+9 particles/mL for bovine milk-derived exosomes, which supports and aligns with our findings [36]. Both particle concentration and zeta potential were assessed for LUT-loaded EX to confirm exosomal membrane integrity after drug loading. Particle concentration for loaded EX was 1.52E+ 10 and zeta potential was around −16 mV, confirming that the exosomes kept their structure intact after drug loading.

Bradford protein assay was carried out to measure the content of protein within the isolated exosomes, where protein concentration directly reflected the exosome content [39]. The total protein content was 2.944 mg/mL, confirming the efficient isolation of exosomes using the applied method. This finding aligns with the results documented by González-Sarrías, et al. who reported that the protein content in the developed bovine milk exosomes was 1.49-2.61 mg/mL [40].

The lipid bilayer of exosomes contains specific membrane proteins, such as tetraspanins (e.g., CD81, CD9, and CD63), which are crucial for facilitating the fusion of exosomes with the membranes of recipient cells. Additionally, these proteins serve as molecular markers, aiding in the identification of exosomes across various experimental methods [32]. The milk-derived exosomes tested positive for surface markers CD81, CD9, and CD63, representing 29.77%, 39.99%, and 52.55%, respectively as illustrated in Fig. 1b and 1c. Based on the exosome characterization data, there is clear evidence that the extracellular vesicles we isolated qualify as exosomes in terms of their size, morphology, and surface marker expression, in accordance with the MISEV-2023 guidelines [41].

Determination of entrapment efficiency of LUT

To optimize entrapment efficiency, two different concentrations of LUT solution (3 mg/mL and 5 mg/mL) were tested. The isolated exosomes exhibited entrapment efficiencies of 56.9% for the 3 mg/mL sample and 27.84% for the 5 mg/mL sample, suggesting an inverse relationship between concentration and loading saturation. This inverse relationship has been also observed by Gul et al. where entrapment of methotrexate decreased as its concentration increased upon loading in plasma-derived exosomes due to saturation and precipitation [42]. Similarly, Aqil et al. reported an entrapment efficiency of 53.9% for exosomes isolated from raw bovine milk [23]. Based on these results, the 3 mg/mL LUT concentration was selected for further use.

In vitro release of LUT from LUT-EX

The release profile of LUT from LUT-EX was evaluated in vitro using 25% v/v PEG 400 in PBS (pH 7.4) at 37 °C, showing a biphasic release profile (Fig. 1d). LUT exhibited an initial burst release from LUT-EX within the first 1 h, releasing around 20% of LUT, followed by a sustained release over 48h. In comparison, free drug displayed nearly complete (100%) release within 24 h with an initial burst release of about 50% in the first hour. The burst release behavior of LUT-EX could be attributed to surface-adsorbed drug molecules, while the sustained release is due to the gradual diffusion of LUT encapsulated within the exosomes [43].

In vitro studies of cell cultureIn vitro cytotoxicity studies

CSF cells were used to assess the cytotoxicity of LUT, unloaded EX, and LUT-loaded exosomes on normal corneal tissues. As shown in Fig. 2a, LUT-EX showed higher cell viability compared to free LUT at all concentrations with IC50 values of 10.47, 556, and 356.3 µg/mL for free LUT, unloaded EX and LUT-EX, respectively.

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)

This suggests that loading LUT into exosomes decreases LUT’s cell cytotoxicity. Exosomes are extremely biocompatible and generally safe since they are naturally produced from cells or natural resources and are made up of lipids, proteins, and nucleic acids. Thus, compared to synthesized nanoparticles, they are less prone to cause immunological reactions [44]. The increased cell viability observed with LUT-EX relative to the free drug is in agreement with other studies that showed that drug-loaded exosomes have promising safety profiles in preclinical and clinical studies and are capable of delivering therapeutic payloads such as drugs, siRNA, and proteins with minimal toxicity [45].

Cellular association

The cellular association experiment was assessed to evaluate the effectiveness of the prepared exosomal delivery systems and their capability to reach and be absorbed by target cells in the eye. As mentioned earlier, exosomes are considered potential natural delivery systems for drugs, DNA, and other therapeutic agents. They are promising vehicles for targeted therapy because of their capability to enhance cellular uptake [46]. As shown in Fig. 2b and 2c, Ex-C6 showed significantly higher cellular uptake compared to free C6 at both time points (4 h and 24 h). Cellular uptake was more prominent after 24 h where the uptake of Ex-C6 increased by 3.3-fold compared to a 2.1-fold increase after 4 h. This indicates that EX-C6 is more effective in delivering the compound into cells, resulting in higher fluorescence. This is supported by previous studies showing that exosomes can improve the delivery of therapeutic agents [47]. Exosomes can target particular cells with precision due to their surface proteins and ligands. This targeting capacity helps minimize off-target effects by delivering therapeutic chemicals directly to the diseased cells [48].

Exosomes are known to facilitate the uptake of C6 into cells through clathrin-mediated endocytosis and micropinocytosis, a process where cells engulf the exosomes along with their cargo [49]. Moreover, the exosomal delivery system ensures that C6 is retained within the cells for a longer duration, reflecting the enhancement in the drug’s therapeutic efficacy. The lipid bilayer membrane of the exosomes, which is similar to that of the cell membrane also enables them to fuse with target cells and deliver their cargo directly into the cytoplasm, bypassing endocytic pathways [50].

Cellular proliferation and migration evaluation using scratch assay

In glaucoma research, wound healing assays on corneal fibroblasts are crucial for evaluating the therapeutic potential of drugs such as LUT that target the damage in the trabecular meshwork (TM) or other ocular tissues for glaucoma treatment. These experiments aid in assessing how drugs affect cellular processes that are essential for preserving IOP and aqueous humor outflow, such as migration, proliferation, and extracellular matrix (ECM) remodeling.

In this study, tissue regeneration and cell migration capabilities were assessed via a scratch model assay. The % wound closure, migration index and migration rate were calculated which are crucial for understanding cell motility and repair mechanisms in the TM and RCGs. The migration index helps assess the efficiency of cell movement, indicating how well cells contribute to wound healing and tissue regeneration. On the other hand, the migration rate quantifies the speed of cell migration, providing insights into how quickly damaged tissues may recover [51]. As shown in Fig. 2d-f, LUT-EX showed a significantly higher (p ≤ 0.05) percentage of cell migration after 24 h (75% closure in the scratched area with migration index of 0.8 ± 0.03 and migration rate of 0.3 ± 0.01 mm/day) when compared to the untreated cells, followed by unloaded Ex (65%) and Free LUT (40%). This suggested that LUT-EX significantly enhanced cell migration compared to other treatments. The significant cell migration and proliferation observed with LUT-EX treatment suggest that exosome-mediated delivery of LUT can enhance the cell proliferation and extracellular matrix (ECM) remodeling process. This could be due to the anti-inflammatory and antioxidant properties of LUT, which are more effectively delivered by exosomes. It is worth mentioning that exosomes can promote both the migration and proliferation of fibroblasts and keratinocytes, which are crucial for ECM remodeling [52]. The presence of LUT within exosomes further enhances these processes, leading to faster wound closure.

Fabrication and characterization of MNsFabrication and morphological examination

Microneedles were developed using a polymeric combination of PVP and PVA to ensure mechanical strength and structural stability during storage and flexibility, which is crucial for maintaining structural integrity during skin insertion [53]. Both PVP and PVA are biocompatible and biodegradable, consequently, they are safer for biomedical applications compared to MNs made from non-biodegradable materials like metals. PVP/PVA MNs can be designed to control the release rate of drugs, allowing for sustained and targeted delivery, which is a significant advantage over some other MN types [54]. The combination of these polymers enhances the penetration capabilities of MNs, ensuring the effective delivery of therapeutic agents [53]. In comparison, MNs made from other materials, such as metals or ceramics, may offer higher mechanical strength but lack the biodegradability and biocompatibility of PVP/PVA MNs. Additionally, MNs made from materials like silicon or glass can be more challenging to fabricate and may not provide the same level of controlled drug release [55].

Propolis was incorporated into the polymer blend due to its demonstrated effectiveness in treating ocular diseases, likely due to its antiglaucoma, antioxidant, anti-inflammatory, and neuroprotective properties [56]. The MN arrays (10 × 10 needle arrangement) had a height of 580.72 ± 10.58 μm and contained 100 microneedles. Water evaporation while drying was shown to cause an average 3.3% decrease in height when compared to the actual height of silicone mold (600 μm) [57]. The MN array's appearance and the pyramidal structure of unloaded, LUT-loaded, and LUT-EX-loaded MN are shown in Fig. 3a. It has been reported that pyramidal microneedles were more forceful than conical ones because, at the same base diameter or width, they possess larger cross-sectional area, which makes it easier to introduce into the retina and reduces the risk of bending or breaking during the insertion procedure [5]. The drug content in MN was measured using spectrophotometrically, showing that LUT@MN or LUT-EX@MN array contained approximately 39.7 ± 1.27 μg of LUT. To verify uniform drug distribution, the MN patches were divided into equal halves, and the LUT content in each half was assessed. The analysis confirmed even drug distribution, where each half contained approximately equal amounts with an average of 19.59 ± 1.09 μg of LUT in each half.

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)

To ensure the integrity of LUT-EX following their incorporation into the MN matrix, deionized water was used to dissolve exosome-loaded MNs, where aliquots were analyzed using TEM, which confirmed the preservation of their vesicular structure (Fig. 3b).

Ex vivo dissolution test

To determine the ideal application time for dissolving MNs on scleral tissue, a dissolution study was performed. Figure 3c presents the dissolution times for both blank and exosome-loaded MNs inserted into the sclera. The results indicated that the MNs, whether blank or loaded with exosomes, took over 1 min to fully dissolve (Fig. 3d). This characteristic makes them well-suited for intrascleral drug delivery, as rapid dissolution (less than 1 min) could cause the sharp tips to dissolve before fully penetrating the scleral tissue, resulting in drug deposition on the surface as opposed to inside the sclera thus lowering bioavailability [58].

Mechanical properties

For effective ocular drug delivery, MNs must be strong and sharp in order to effectively penetrate the scleral tissue and endure compression without breaking. Evaluating the mechanical strength of the MN array is thus critical. A predefined force of 30 N was selected as it surpasses the 10 N force necessary for effective implantation into ocular tissue [59]. Figure 3e depicts the percentage of height reduction in MNs when subjected to a 30 N force, demonstrating their ability to withstand sufficient pressure for successful tissue implantation. The MNs displayed adequate mechanical strength, with a height reduction of less than 25% under compression (Fig. 3f). Furthermore, there was no significant difference (p > 0.05) between blank MNs and those loaded with exosomes, implying that the inclusion of drug-loaded exosomes in the MNs did not compromise their durability.

Insertion depth

To evaluate the efficiency of trans-scleral insertion, Parafilm M®; an artificial folded membrane designed to mimic the layers of the sclera was used to test the insertion capability of the prepared MNs [26]. The parafilm was folded to get the three layers with a total thickness of 0.39 mm since the thickness of the parafilm sheet is 0.13 mm, and that of the human scleral tissues is around 0.67 mm [60]. This is to represent the penetration into the mid-scleral region rather than total scleral region without piercing through. The insertion test was conducted using a texture analyzer, previously employed for compression testing in compression mode, to measure the MN’s capability to insert into the layers of Parafilm, as depicted in Fig. 3g. Images in Fig. 3g illustrate complete penetration through three Parafilm layers. According to this, the therapeutic payload can be successfully distributed into the deeper layers of the human sclera once MNs have been inserted and the polymer matrix has biodegraded, thereby avoiding the scleral barrier. Additionally, the MNs penetrate only the mid-layer of the sclera, where they become soft and dissolve, rather than passing through the entire sclera. This is important to prevent getting in contact with or damaging highly sensitive ocular tissues like the retina and the choroid and enable delivery of the drug in a painless and minimally invasive way. Therefore, the insertion depth of the exosome-loaded MNs offers an optimal balance between delivery efficiency and patient comfort, making them ideal for effective transscleral drug administration [29].

Ex vivo permeation study

The impact of MNs on the permeation of LUT across the sclera was evaluated using the Franz-diffusion cells set-up. Fig. 3h illustrates the percentages of LUT that permeated and deposited when delivered either freely or encapsulated in colostrum-derived exosomes, using a gel (MN gel polymer matrix) or MNs. The percentage of LUT permeated following the application of LUT@MN was approximately two times higher than that achieved with the gel formulation. Furthermore, using LUT-EX@MN resulted in 40% of the loaded LUT permeating the sclera within 6 h, compared to just 15% from LUT-EX@gel, representing a ~2.6-fold increase in permeation. This significantly achieved enhancement in the permeation of LUT from MNs in comparison to the gel matrix highlighting their promising ability in improving drug delivery through the sclera.

Ocular tolerance test

The eye is a highly sensitive and delicate organ, vulnerable to foreign substances or chemicals. To ensure safety, the prepared MN matrix was tested using the HET-CAM method; a fast, sensitive, and cost-effective test. As a model positioned between the systems of in vivo and in vitro studies, it circumvents ethical and legal concerns. The CAM of the chick embryo, consisting of veins, arteries, and capillaries, exhibits an inflammatory response to injury similar to that of rabbit conjunctival tissue. As illustrated in Fig. 4a and b, the formulations were evaluated against a negative control (normal saline, considered non-irritant) and a positive control (0.1 M NaOH, considered irritant). The scores were documented following the scoring system outlined by Gupta et al. According to this scoring system, all of the developed formulations exhibited a zero score for confirming their non-irritant properties [61].

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

In vivo evaluation of the efficacy of LUT-EX@MN in the glaucoma-induced rabbit model

The anti-glaucoma potential of LUT-EX@MN was evaluated against various control groups (negative control, positive control, blank MN) and the same groups in the polymeric matrix before casting them into MN (blank gel, EX@MN, EX@gel, LUT@MN, LUT@gel, and LUT-EX@gel). These different formulations were tested in rabbits with glaucomatous right eyes over one week following a single application of the tested formulations. As depicted in Fig. 4c, gel-based formulations did not show a significant decrease in IOP through one week (p > 0.05), likely due to their limited pre-corneal retention time caused by blinking and nasolacrimal drainage [62]. However, LUT-EX@gel significantly lowered IOP within the first three hours (18% ± 3.3%), though this effect was not sustained over seven days as observed from the decrease in percent reduction of IOP (Fig. 4c). In contrast, LUT@gel did not show a comparable reduction in IOP during the same period (10.7% ± 3.5%). This difference may be attributed to the ability of exosomes to efficiently deliver their cargo to ocular tissues. Noticeably, EX@MN lowered IOP over a period of 48 h, highlighting the IOP-reducing properties of milk-derived exosomes (Fig. 4c). This observation is in agreement with previous findings by Seong et al., who reported that exosomes could help manage glaucoma by lowering IOP [63]. Of all the formulations tested, LUT-EX@MN demonstrated the most pronounced reduction in IOP, achieving approximately a 50% decrease within three hours, which was maintained over seven days (Fig. 4c). It is important to note that LUT-EX@gel did not achieve the same effectiveness as LUT-EX@MN, emphasizing the role of MNs in improving LUT-EX permeability. This observation is in line with the ex vivo permeation results. Moreover, this finding supports the capability of dissolving MNs to ensure sustained drug release [55]. The area under the curve (AUC) for IOP reduction across different formulations has been also calculated as shown in Table 3. The use of AUC for IOP reduction is well-established in glaucoma research, as it provides a more comprehensive assessment of treatment efficacy by accounting for both magnitude and duration of pressure control. Unlike single time-point measurements, AUC reflects real-world IOP fluctuations, making it a critical metric in evaluating sustained therapy. AUC is directly correlated with the extent and duration of the therapeutic effect. [64]. As shown in Table 3, AUC, Cmax (highest % reduction in IOP), and Tmax (time taken to achieve Cmax) were calculated. Gels in general exhibited smaller AUC, lower Cmax, and shorter Tmax when compared to their corresponding MN formulations indicating the effect of MN in bypassing the corneal barrier and delivering the drug directly into the deeper ocular tissues. All gels exhibited shorter Tmax compared to MN (3h vs 24 or 48 h in MN) indicating rapid effect. Within gel formulations, there was no significant difference between blank@gel, LUT@gel, and EX@gel with regard to their AUC, Cmax and Tmax. However, LUT-EX@gel showed approximately 1.8-fold increase in AUC reaching a Cmax of 18 ± 4.1% after 3 h. The selected microneedles formulation (LUT-EX@MN) exhibited the highest AUC value among all formulations with 5.3-fold increase in AUC compared to LUT-EX@gel. The increase in Cmax (53.2 ± 4.4%) and delayed Tmax (24h) reflects the potential role of MN or exosome-based systems in sustaining drug release and improving therapeutic efficacy for ocular conditions like glaucoma highlighting the synergistic benefits of trans-scleral delivery and bioactive carrier systems. These findings strongly support the potential of biologically inspired and minimally invasive platforms for effective ocular drug delivery.

Table 3 AUC, Cmax (highest % reduction in IOP) and Tmax (Time taken to achieve Cmax) for IOP reduction over time across different treatment modalities (mean ± SD, n = 6)Assessment of gene expression and protein levels of various biomarkers participating in the management of glaucoma

RGCs exposed to stress caused by IOP elevation undergo apoptosis, leading to progressive atrophy. Oxidative stress and cytokine release further contribute to the deterioration of glaucoma. In glaucomatous eyes, studies have reported increased TNF-α protein levels, elevated TNF-α gene expression in glial cells, and upregulated TNF-α receptor 1 in RGCs [65]. Figure 5a shows that the positive control group receiving no treatment exhibited significantly higher (p ≤ 0.05) TNF-α protein levels (22.8 ± 1.1 pg/mL) in comparison to the negative healthy control group (13.5 ± 2.6 pg/mL). Remarkably, TNF-α levels achieved with LUT-EX@MN treatment (14.1 ± 1.1 pg/mL) were statistically insignificant compared to those of the negative control group. Evidence from research supports this conclusion, suggesting that LUT may be a promising treatment for reducing inflammation and suppressing pro-inflammatory cytokines. Supporting these findings, previous studies done both in vitro and in vivo have shown that LUT modulates multiple signaling pathways associated with inflammation and effectively suppresses key inflammatory mediators, particularly TNF-α [66].

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

Furthermore, several studies examining cytokine concentrations in aqueous humor samples have identified elevated levels of cytokines, such as IL-8 [67]. As illustrated in Fig. 5b, the untreated positive control group showed significantly increased (p ≤ 0.05) IL-8 expression (33.5 ± 5.5 pg/mL) compared to the healthy negative control group (17.8 ± 1.96 pg/mL). Importantly, IL-8 levels following treatment with LUT-EX@MN (17.5 ± 0.9 pg/mL) were close to those of the negative control group. In an attempt to investigate the anti-inflammatory effect of LUT, Hytti et al. examined the effects of LUT on ARPE-19; the human retinal pigment epithelial cell line, assessing its potential to reduce intracellular inflammation. In addition, it has been documented that treatment with LUT led to a decrease in IL-8 levels and suppression of the inflammatory response in stressed ARPE-19 cells [68].

On the other hand, glutathione peroxidase (GPx) is a vital antioxidant enzyme that plays a role in neutralizing reactive oxygen species (ROS). In glaucoma patients, the heightened need for antioxidants like GPx results in their depletion [69]. As depicted in Fig. 5c, the untreated positive control group displayed significantly (p ≤ 0.05) reduced GPx activity (3.2 ± 0.6 U/mL) when compared to the healthy negative control group (5.4 ± 0.1 U/mL). Expression of GPx following treatment with LUT-EX@MN has increased significantly and its levels (5.2 ± 0.5 U/mL) were comparable to those in the negative control group. In line with these findings, Yuan et al. investigated the antioxidant properties of LUT by examining glutathione and glutamate levels in rabbits with glaucoma. The results showed that LUT treatment preserved endogenous aqueous humor glutathione levels, suggesting that LUT is not only effective in reducing IOP but also offers protection against oxidative damage and the advancement of glaucomatous neurodegeneration [15].

The MYOC gene encodes myocilin, a protein predominantly located in the trabecular meshwork (TM) near the cornea of the eye. Overexpression, misfolding, and aggregation of MYOC proteins induce endoplasmic reticulum (ER) stress and initiate the unfolded protein response (UPR) via the ER's homeostatic mechanisms. When the UPR fails to clear misfolded proteins through proteasomal degradation, cells are unable to resolve ER stress, leading to apoptosis and subsequent degradation of TM tissue. This ER stress response and resulting cell toxicity caused by misfolded MYOC are widely recognized as a key mechanism in the pathogenesis of glaucoma [70]. At the gene expression level, Fig. 5d illustrates that the positive control group exhibited a significantly higher fivefold increase in MYOC gene expression when compared to the negative control group. However, treatment with LUT-EX@MN effectively reversed this increase, resulting in the lowest MYOC gene expression, which was a 1.16-fold increase compared to the levels observed in the negative control group. It is noteworthy to mention that, extracellular vesicles and exosomes from diverse sources have been investigated as potential therapeutic options for glaucoma-related damage [71]. Supporting this, Pan et al. demonstrated that exosomes originating from umbilical cord mesenchymal stem cells enhanced RGC survival in a mouse model [