The Effects of Mesenchymal Stem Cell-Derived Exosomes on the Attenuation of Dry Eye Disease in Sjögren Syndrome Animal Model

Abstract

BACKGROUND:

Sjögren’s syndrome (SS) is a chronic autoimmune disease delineated by excessive lymphocyte infiltration to the lacrimal or salivary glands, leading to dry eye and dry mouth. Exosomes secreted from mesenchymal stem cells (MSC) are known to have anti-inflammatory and tissue regeneration abilities. This study endeavored to demonstrate the effect of MSC-derived exosomes on the clinical parameter of dry eyes and associated pathology in SS mouse model.

METHODS:

Exosomes obtained from bone marrow-derived human MSC (Catholic MASTER Cells) were injected into the subconjunctival sac of 17 weeks-old NOD/LtJ female mice once and sacrificed after 7 days, or administered topically as an eyedrop every day for 14 days, then sacrificed. Clinical dry eye parameters, including tear volume and corneal staining scores, density of goblet cells in the conjunctiva, pro-inflammatory cytokine expressions of cornea and conjunctiva, and the lacrimal glands were evaluated. Infiltration of inflammatory foci, and expression of B and T cells in the lacrimal glands were examined.

RESULTS:

Tear volume, corneal stain scores and density of goblet cells in conjunctiva were improved in the exosome-treated groups compared to the control group. Pro-inflammatory cytokine expressions were also reduced in the cornea and conjunctiva of the exosome-treated group. In the lacrimal glands of the exosome-treated mice, inflammatory foci infiltration and B cell marker expressions were significantly decreased.

CONCLUSIONS:

Thus, this study demonstrated the amelioration of dry eyes with the administration of exosomes in SS animal model, suggesting promising therapeutic potential of MSC-derived exosomes in SS dry eyes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13770-025-00755-4.

Introduction

Sjögren’s syndrome (SS) is systemic autoimmune disease that is marked by excessive lymphocyte infiltration into the exocrine glands such as the salivary and lacrimal glands (LG), resulting in reduced secretory function and granular destruction [1]. Dry eye (DE) or dry mouth are the main symptoms that are a result of progressive exocrine gland destruction and have been shown to drastically decrease the quality of life in SS patients [2]. SS predominantly affects middle-aged women, with the etiology of SS being complex in that it involves diverse factors such as genetics, epidemiology, environmental influences, viral infections, and hormonal factors. However, treating SS poses formidable challenges due to the limited efficacy and restricted effectiveness of current therapeutic options[3].

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to self-renew and differentiate into various mesodermal lineages including chondrocytes, osteocytes, and adipocytes [4]. MSCs can promote growth factor secretions, prevent apoptosis, inhibit inflammation, regulate extracellular matrix dynamics, and promote tissue regeneration [5, 6]. Various studies have shown the therapeutic effects of MSCs on various autoimmune diseases, including diabetes and lupus [4, 6–8]. In relation to dry eyes, our previous study demonstrated that bone-marrow human MSCs (hMSCs) alleviated DE in SS murine model [9].

Recently, MSCs-derived exosomes have gained popularity as a therapeutic option for various diseases with findings that show MSCs confer effects in autoimmune disorders by producing paracrine factors such as extracellular vehicles and cytokines [7]. Exosomes are small extracellular vesicles with a diameter ranging from 30 to 150 nm with a lipid bilayer that also includes various bioactive molecules such as genes, lipids, and proteins [10]. The characteristics and functions of exosomes are determined by the type of cell they originate from and the state of the tissue or cell during their formation. As a result, although exosomes share certain biological functions, they contain specific proteins that reflect their cellular origins and the conditions of the cells, leading to their distinct biological properties. An emerging collection of studies point to the potential of MSCs-derived exosomes as treatment options for various diseases in that it possesses anti-inflammatory, neuroprotective, and other immunomodulatory properties similar to MSCs [11, 12]. Human corneal MSC-derived exosomes have been reported to accelerate cornea epithelial wound healing and repair [13–15]. This study aims to investigate, for the first time, the role of hMSC-derived exosomes in the attenuation of dry eyes and lacrimal gland pathology in SS animal model.

Materials and methods

Exosome characterization

Exosomes were obtained from Korean FDA-approved bone-marrow human MSCs (Catholic MASTER Cells) supplied by the Catholic Institute of Cell Therapy (CIC, Seoul, Korea) and approved by the Korean FDA and safety/toxicology tests to be clinically applicable. hMSCs were cultivated as previously mentioned, and the isolation of exosomes was conducted using an aqueous two-phase system (ATPS) with the Exo2D-EV™ isolation kit (Exosomeplus, Seoul, Republic of Korea) [16–18]. Characterization of exosomes was performed by measuring particle size and concentration using Nanoparticle Tracking Analysis (NTA) (ExoCope, Exosomeplus) and using the mean data as well as quantifying the expression of exosome surface markers CD9, CD81, and CD63 via total internal reflection fluorescence (TIRF) microscopy as mentioned previously [16]. Isolation and characterization of exosomes were executed by Exosomeplus (Exosomeplus, Suwon, Korea) and the obtained exosomes were stored at − 80 °C until further use.

Animals

NOD/LtJ female mice was purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained in specific pathogen-free conditions in the animal facility of the Catholic University of Korea. All mice were in a controlled environment with a 12-h light–dark cycle and provided sterilized food and water ad libitum. 17 weeks-old NOD/LtJ female mice were utilized in this study referencing the onset of DE of the SS murine model reported in a previous study [19]. All procedures were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC no. 2023-0202-01).

Exosome administration

Mice were treated with exosomes were administered either by subconjunctival injection or eyedrop. Subconjunctival injection of exosomes containing a concentration of 2.0 × 10⁸ particle # /10μL and the same volume of vehicle without exosomes were injected into the control group and sacrificed after 7 days. Topical administration of exosomes by eyedrop contained a concentration of 1.0 × 10⁸ particle # /5μL and the same volume of vehicle without exosomes were administered daily for 14 days and then sacrificed.

Phenol red thread test

Tear production assessment was conducted with phenol red threads (Zone-Quick; Menicon, Nagoya, Japan) and administered into the lateral canthus of the conjunctival fornix of mice for 1 min. The length of the cotton thread that became wet was measured with a millimeter scale ruler using the i-solution program (IMT Inc, NY, USA).

Corneal surface staining

DE severity was determined by corneal surface staining where one drop of 1% Lissamine Green B (Sigma-Aldrich Corp., St. Louis, MO, USA) was administered into the inferior lateral conjunctival sac for 1 min and washed with PBS. The area of the stained corneal surface was evaluated through a blind test conducted by two people utilizing the National Eye Institute (NEI) grading system which divides the cornea into 5 zones (central, superior, inferior, nasal and temporal) [20]. The severity of punctate staining in each zone was scored on a scale from 0 to 3, and compiled by adding the total score of the 5 zones (the maximum total score of 15).

Periodic acid Schiff (PAS) staining of conjunctival goblet cells

Conjunctival goblet cell histology was assessed in mice by excision of the whole eyeball of mice and fixation in 10% formalin then embedment in paraffin block. The blocks were cut through the superior and inferior conjunctival fornices at a thickness of 4-μm. The sections were dewaxed in xylene for 40 mins and then hydrated in 100%, 95%, 90%, 85% and 70% ethanol and stained with PAS staining kit (Abcam, Cambridge, UK). After staining, the number of goblet cells were counted in four different cuts per 100 μm of the same mice sample. The goblet cell density was calculated by determining the average goblet cell count from each mouse.

Histologic analysis of lacrimal glands

The lacrimal glands of mice were harvested and fixed in 10% formalin, then embedded with paraffin blocks and cut into 4-μm thick sections. The sections were deparaffinized in xylene and hydrated in graded 100%, 95%, 90%, 85% and 70% ethanol, then PBS. After, the LG tissue was stained with hematoxylin and eosin (ab245880, Abcam, Cambridge, UK) and observed under a microscope at ×40 and ×100 magnification. The foci in the lacrimal glands were scored based on the evaluation protocol described previously [21, 22].

For immunofluorescence staining, slides were placed in target retrieval solution (Dako, CA, USA) and microwaved for 15 min. Then, they were washed 3 times for 2 min each in PBS with 0.05% Tween (PBST). The slides were incubated in blocking buffer (10% normal goat serum in PBST) for 1 h, then in B220 (BD Biosciences, CA, USA) and CD3 (Santa Cruz Biotechnology, TX, USA) in PBST overnight at 4 °C. The slides were washed in PBST and incubated with AlexaFluor 488 or 546-conjugated anti-rabbit or mice IgG Ab (Thermo Fisher Scientific, MA, USA). After, they were washed again in PBST and mounted with DAPI (Dako). Slides were viewed using Zeiss LSM 800 confocal microscopy (Carl Zeiss, Overkochen, Germany).

RNA isolation and real-time PCR

For assessment of gene expression, total RNA was extracted from the lacrimal glands and corneas of mice tissues using TRIzol reagent (Gibco-Invitrogen, Grand Island, NY, USA). Complementary DNA (cDNA) were prepared by using reverse transcriptase (Promega, WI, USA) and real-time PCR conducted with SYBR Green I (Takara Bio Inc., Kusatsu, Shiga, Japan). GAPDH was used as a housekeeping gene and for calibration of the average threshold cycle value for the desired target genes. Relative quantitation was calculated by the 2-△△Ct method. The Primer sequences used in this study are listed as shown in Table 1.

Table 1.

Primers and sequences used in real-time PCR

Statistical analysis

The statistical evaluations and significance between the treatment groups were performed using GraphPad Prism 9.1.1 (GraphPad Software, La Jolla, CA, USA) and non-parametric, two-tailed Mann–Whitney t-tests. All quantitative data in this study were representative of as least three independent experiments conducted with six mice in each group and expressed as mean ± SD. p < 0.05 indicated that the analysis results were statistically significant, p < 0.01 as highly significant, and p < 0.001 as extremely highly significant.

Results

Dry eye clinical parameters and goblet cell counts are improved in exosome-treated mice by subconjunctival injection

Exosomes isolated from hMSCs demonstrated an average concentration of 6.0 × 10⁹ particle # /mL and a mean diameter size of 130 ± 2.4 nm, aligning with the established size range of 30–150 nm typically used to define exosomes (Supplement Fig. 1A). Characterization was confirmed by staining and observing the percentage of exosome cell surface markers CD9, CD63, and CD81 by TIRF. CD63-positive exosomes accounted for more than half of the total population of exosomes, with 55.3%. In comparison, 29.9% of exosomes were stained for only CD81, followed by double-stained CD63 and CD81 constituting 6.6% of total exosomes (Supplement Fig. 1B). The effects of the administration of exosomes through subconjunctival injection was evaluated in NOD/LtJ female mice by clinical DE parameters such as tear volume and corneal epithelial defects, as well as the number of goblet cells in the conjunctiva. As shown in Fig. 1A, tear volume levels were significantly higher in the exosome-injected mice compared to the control mice on day 7 (Exosome Injection = 1.63 ± 0.29; Control Injection = 1.33 ± 0.16, p < 0.05). Exosome-injected mice demonstrated a significant reduction of corneal epithelial staining scores on day 7 compared to day 0 (Day 0 = 6.81 ± 2.37; Day 7 = 3.75 ± 0.97; p < 0.01) (Fig. 1B). Corneal epithelial staining scores was significantly lower in exosome-injected mice than control mice on day 7 (Exosome Injection = 3.75 ± 0.97; Control Injection = 7.17 ± 1.21, p < 0.001) (Fig. 1B). Moreover, the number of goblet cells in exosome-injected mice was significantly higher than control mice on day 7 (Exosome Injection = 64.25 ± 12.74; Control Injection = 38.58 ± 10.66, p < 0.01) (Fig. 1C).

Fig. 1.

Examination of clinical parameters and the number of goblet cells in control or exosome-injected mice. A Tear volume of mice treated with subconjunctival injection of control or exosome in NOD/LtJ mice were assessed by phenol red thread on day 0 and day 7. B Representative photomicrographs of corneal epithelial defects by lissamine green staining and corneal stain scores on day 0 and day 7. C Conjunctival goblet cell density analysis by PAS staining results. Scale bar: 100uM. Data are presented from six independent experiments (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001

Dry eye clinical parameters and goblet cell counts are ameliorated in mice administered with exosome by eyedrop

Clinical DE parameters and goblet cell counts were also examined in mice that were administered with exosome or control topically as an eyedrop once a day for 14 days. Tear volume levels of the exosome eyedrop group revealed significant increases on day 14 compared to day 0 (Day 0 = 1.39 ± 0.29; Day 14 = 1.87 ± 0.30, p < 0.01), and was drastically higher than control group on day 14 (Fig. 2A, Exosome Eyedrop = 1.87 ± 0.30; Control Eyedrop = 1.32 ± 0.33, p < 0.01). The NEI score for corneal epithelial defects displayed significant decreases in the exosome eyedrop group on day 14 compared to day 0 (Day 0 = 6.67 ± 1.80; Day 14 = 3.17 ± 1.67, p < 0.01) (Fig. 2B). Corneal epithelial staining scores was significantly lower in the exosome eyedrop group than the control eyedrop group (Exosome eyedrop = 3.17 ± 1.67; Control eyedrop = 8.00 ± 1.58, p < 0.01) (Fig. 2B). The number of goblet cells was also shown to be dramatically recovered in the exosome eyedrop group compared to the control group (Exosome Eyedrop = 67.93 ± 24.10 vs Control Eyedrop = 39.64 ± 12.98, p < 0.01) (Fig. 2C).

Fig. 2.

Analysis of clinical parameters and the number of goblet cells in control or exosome eyedrop-treated mice. A Evaluation of the tear volume of mice conducted by phenol red thread test on day 0 and day 14 for mice administered topically with control or exosomes. B Corneal stain scores and representative photographs of the corneal surface with lissamine green staining on day 0 and day 14. C PAS staining images of the conjunctiva indicating the density and quantity of goblet cells. Scale bar: 100uM. D Tear volume and NEI score comparison between day 0 and day 7 or day 0 and day 14 for mice treated with exosomes by injection or eyedrops, respectively. Data are presented from six independent experiments (n = 6). * p < 0.05, ** p < 0.01, ***p < 0.001

A comparison of the effects of exosome treated by either injection or eyedrop administration in mice was observed by analyzing the differences in clinical phenotypes between day 0 and day 7 or day 14. The results showed that there was a statistically significant increase in comparative tear volume difference for mice administered with exosome eyedrops compared to those treated with exosome injection. Such distinctions were not apparent in comparative NEI score difference for mice treated with either exosome injection or eyedrops (Fig. 2D).

Lacrimal gland lymphocyte infiltration are decreased in exosome-treated mice

In order to investigate the effects of exosome injection or eyedrop in the LG of SS mice, histologic section examinations were observed by confirming the number of foci and the size of the lymphocyte infiltration lesions. The results showed that in the exosome injection group, foci score was significantly decreased compared to the control group (Exosome Injection = 1.17 ± 0.37 vs Control Injection = 2.67 ± 0.47, p < 0.001) (Fig. 3A). Likewise, lacrimal glands of mice in the exosome eyedrop group also demonstrated drastically diminished lymphocyte infiltrates compared to the control group (Exosome Eyedrop = 1.75 ± 0.66, Control eyedrop = 2.88 ± 0.33, p < 0.01) (Fig. 3B). In order to observe the effects of exosome treatment in immune cell infiltration of the lacrimal glands, immunofluorescence staining was performed for B cell marker anti-B220 and T cell marker anti-CD3 (Fig. 3C). As demonstrated in the results, treatment of exosomes by injection or eyedrop showed considerable attenuations of B220⁺ cells compared to control groups, respectively. In contrast, CD3⁺ cells did not demonstrate differences in their expression in the LG.

Fig. 3.

Lacrimal gland histology results of NOD/LtJ female mice treated with control or exosomes by subconjunctival injection or eyedrop administration. A Extraorbital lacrimal gland removed from mice were fixed in 10% formalin and stained with H&E. Representative photographs and arrows indicate leukocytic infiltrations in the lacrimal glands and lacrimal gland foci scores are shown in the graphs below. Scale bar: 500uM. B Representative photographs of the leukocytic infiltrations in the lacrimal glands of mice topically administered with exosomes. Data are presented from six independent experiments (n = 6). C Representative images of lacrimal glands co-stained for B220 and CD3 in control or exosome-administered mice. Effects of hMSCs exosomes in B-cell and T-cell markers shown by photomicrographs stained with B220 and CD3 in control or exosome-treated mice. Scale bar: 100uM. Data are presented from six independent experiments (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001

Expression of proinflammatory cytokines is decreased in the lacrimal glands and in the cornea and conjunctiva of exosome-treated mice

To further explore the anti-inflammatory effects of hMSCs exosomes in a mouse model of SS, real-time PCR was utilized to assess the expression levels of proinflammatory cytokines in both the cornea and conjunctiva as well as the lacrimal glands. As shown in Fig. 4A, gene expression of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, IFN-γ, IL-17A and MMP9) were decreased overall in a statistically significant manner in exosome-injected mice in both the cornea and conjunctiva as well as the lacrimal glands compared to the control group. Additionally, all proinflammatory cytokine gene levels were also shown to be significantly reduced in the cornea and conjunctiva as well as in the lacrimal gland of mice administered with exosome by eyedrop (Fig. 4B).

Fig. 4.

Pro-inflammatory cytokine gene expression in the cornea and conjunctiva and lacrimal glands of mice. A Gene expression levels of inflammatory cytokines in mice treated with control or exosome by subconjunctival injection. B Pro-inflammatory cytokine levels in mice treated with control or exosome eyedrop. Data are presented from six independent experiments (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001

Discussion

The present study demonstrated that administration of human MSCs-derived exosomes by either subconjunctival injection or eyedrop alleviated dry eye clinical parameters and reduction of lacrimal gland inflammatory foci infiltration in SS animal model. Clinical phenotypes, such as tear volume and corneal epithelial defects, were markedly recovered with exosome treatment compared to the control group. Not only that, the number of goblet cells was also recovered in the conjunctiva of mice, as well as attenuations in the infiltration of lymphocytes to the lacrimal glands with exosome injection or eyedrop administration. This implies that human MSCs-derived exosomes hold promise for potentially restoring the clinical signs of DE in SS.

Autoimmune diseases such as SS stem from a multitude of factors and pose significant challenges in terms of finding a treatment, often necessitating prolonged drug therapy to manage disease progression. In SS DE, the immune system is disrupted, resulting in an unstable ocular surface homeostasis and abnormal immune responses [23–25]. To counter these pathologic symptoms, numerous topical therapeutics such as immunosuppressive agents (corticosteroids) or immunomodulatory agents (cyclosporine and tacrolimus) have been considered, but have the potential to cause side effects with long-term use such as intraocular hypertension, cataracts and decreased wound healing [7, 26–29]. A potential therapeutic treatment to countervail SS DE is by utilizing MSCs, which were shown to not only have immunomodulatory and anti-inflammatory properties, but also enhance salivary gland function by inhibiting Th17 and Tfh cells [30]. Our previous study demonstrated that MSCs derived from bone marrow was effective in alleviating the clinical phenotypes of DE and lacrimal gland B cell infiltration in a SS animal model [9].

Adding to the cumulative data regarding MSCs, there has been current studies that have also shown promising potential of exosomes for SS treatment. They are recognized for their roles in cell-to-cell communication, anti-inflammatory actions, and immunomodulation. Of note, the isolated hMSC-derived exosomes demonstrated more than half of exosomes expressed surface marker CD63, followed by CD81. This is in line with past studies that show CD63 to be the most abundant type of tetraspanins in exosomes [31, 32]. Levy [33] marked on the important role of CD81 in the immune response, supporting the function of exosomes on conferring immunomodulatory effects. Li et al. [34] demonstrated that treating SS female NOD mice with labial gland-derived MSC-Exo reduced inflammation in the salivary glands and restored secretory function by suppressing the differentiation of Th17 cells and promoting the induction of Treg cells. In non-SS dry eye Yu et al. [35] administered human adipose tissue stem cells derived extracellular vesicles to DE mice and confirmed its inhibition of NLRP3 inflammasome activity, which protected the tear film and consequently suppressed the progression of DE. Similarly, our current study has shown that administration of human MSCs exosomes either by injection or eyedrop to SS mice significantly decreased clinical phenotypes of DE and lacrimal gland foci infiltration.

Of interest, our results indicated a significant reduction of inflammatory foci and B cell marker B220 expression in the lacrimal glands in exosome-treated mice by subconjunctival injection and eyedrop, while T cell marker CD3 was rarely observed. In vitro co-culture of B cells and exosomes isolated from the plasma of head and neck squamous cell carcinoma patients as well as healthy donors demonstrated that the expression of checkpoint receptors PD-1 and LAG3 increased, and BCR signaling receptor CD19 decreased, resulting in an inhibition of B cell proliferation and survival [36]. It was revealed that PRDM1, a B cell differentiation marker, which is upregulated in patients with primary SS, was decreased with the treatment of labial gland-derived MSC exosomes (LGMSC-Exos). This demonstrated the inhibitory effects of LGMSC-Exos on PRDM1 expression, offering insight into the possibility of exosomes as a therapeutic function for treating SS diseases [37]. These studies support our data demonstrating the inhibition of B cell infiltration in lacrimal gland by hMSC-derived exosomes treatment.

SS patients were shown to have increased levels of Th1, Th2 and Th17-related cytokines such as IFN-γ, TNF-α, IL-6 and IL-17A, with MMP9 surmised to play a pathogenic role in dry eye diseases [38, 39]. This is consistent with studies that have indicated upregulations in MMP9 gene expressions in animal models of autoimmune diseases, as well as reduced tear volume and tissue damage in the lacrimal glands [40, 41]. Our study also identified increased levels of inflammatory cytokines and MMP9 in the control group, which is consistent with those observed in SS patients. Furthermore, we showed that treatment with hMSC-derived exosomes, whether delivered via subconjunctival injection or eyedrops, significantly reduced pro-inflammatory cytokine levels in the lacrimal glands, cornea and conjunctiva.

Exosomes contain a diverse array of bioactive molecules, including proteins, lipids, DNA and various forms of RNA. Through mechanisms such as membrane fusion, endocytosis, and receptor-mediated uptake, they deliver their molecular cargo to recipient cells, thereby playing critical roles in modulating immune responses, promoting tissue repair and regeneration, and serving as vehicles for drug delivery. Furthermore, the analysis of exosomal cargo can reveal valuable information about the cells of origin and holds great potential as a biomarker for diseases diagnosis, prognosis, and therapeutic monitoring [42, 43]. In particular, miRNA and proteins contained within exosomes have attracted significant attention due to their ability to influence gene expression in recipient cells [44–46]. Guifang et al. demonstrated that MSC-derived exosomes carrying miR-223-3p play a protective role in dry eye disease models [47]. Specifically, exosomal miR-223-3p was shown to attenuate ocular surface damage and suppress inflammatory responses by targeting and downregulating Fbxw7, a key regulator involved in inflammation and cell cycle progression. In addition, miRNAs contained in human MSC-derived exosomes effectively alleviated inflammation and restored ocular surface homeostasis by suppressing the IRAK1/TAB2/NF-κB signaling pathway [48]. Exosomes carrying miR-204 modulate the IL-6/IL-6R/STAT3 signaling pathway to reprogram the phenotype of infiltrating macrophages in the corneal tissue of GVHD-associated dry eye disease models, thereby suppressing inflammation and restoring tissue homeostasis [49]. Elucidating the molecular mechanisms of such exosomal cargo is essential for advancing therapeutic strategies based on exosome-mediated delivery. Our study primarily investigated the phenotypic ameliorations induced by exosome treatment. Recognizing the pivotal role of comprehensive analyses of bioactive molecules within exosomal cargo for elucidating their mechanisms of action, future studies will utilize in vitro co-culture systems in conjunction with proteomic profiling and/or RNA sequencing to further investigate the molecular mechanisms involved.

In terms of a clinical perspective, subconjunctival injection is considered to be minimally invasive, facilitating in the swift delivery of drugs to their target by traversing the epithelial cell barrier. However, injections might cause slight discomfort to patients and some would not prefer subconjunctival injections even though it is minimally invasive. Eyedrops, on the other hand, are easy to use but also entails the inconvenience of needing frequent application. This particular study demonstrated that the administration of exosomes via injection or eyedrop routes were shown to be both effective in alleviating clinical DE phenotypes as well as reduction of inflammation of lacrimal gland, cornea and conjunctiva. In our results, both subconjunctival injection and eyedrop administration of exosomes were effective. Nonetheless, the increase in tear volume observed in the exosome eyedrop group from Day 0 to Day 14 was statistically greater than that in the subconjunctival injection group from Day 0 to Day 7. This difference may be attributed to the daily administration of exosomes in the eyedrop group compared to the single-dose injection. It suggests that frequent, non-invasive delivery via topical eyedrops effectively might be more effective to improve dry eye clinical parameters in mice. Interestingly, the injection group received a lower number of exosome particles yet still achieved comparable efficacy, suggesting this method may offer a more cost-effective treatment option. These findings collectively support the therapeutic potential of MSC-derived exosomes in the treatment of SS-associated dry eye. Both subconjunctival injection and eyedrops demonstrated significant efficacy, suggesting that the choice of administration route could be tailored according to patient-specific conditions of preferences.

In this experiment, exosomes were administered via subconjunctival injection followed by mice sacrifice after 7 days, or through daily eyedrop applications over a 14 day period and then mice sacrifice. While our data demonstrated that the therapeutic effect of one-time subconjunctival injection persisted for approximately 1 week in mice, this duration may be extended in humans due to species-specific differences in lifespan and metabolism. Although such short-term observation periods are commonly used in SS-associated dry eye mouse models because of their limited lifespan, they may not sufficiently capture the long-term therapeutic efficacy of exosome-based treatments. Given that exosome therapies are generally associated with low immunogenicity and a favorable safety profile, further investigation is warranted to assess their long-term safety through extended follow-up studies. It may also be beneficial to administer subconjunctival injection in one eye and topical eyedrops in the contralateral eye of the same animal to directly compare the efficacy of different delivery routes under identical systemic conditions.

In conclusion, this study for the first time demonstrated the therapeutic effects of hMSC-derived exosomes on DE in SS mouse model. The current study utilized exosomes secreted from human bone marrow-derived MSCs that were clinically approved to use by the Korean FDA. As it showed in the results that both subconjunctival injection or eyedrop administration of exosomes were effective in relieving dry eyes of SS, it could potentially serve as a foundation for guiding future clinical applications of exosomes in treating all types of dry eyes.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (No. RS-2024-00512879), the Research Fund of Seoul St. Mary’s Hospital, The Catholic University of Korea (ZC23EISI0773) and the Nano Convergence 2020 Plus grant funded by both the Ministry of Trade, Industry & Energy (MOTIE) and Ministry of Science and ICT (MSIT) (No. R202101500).

Declarations

Conflict of interest

So-Hyang Chung has patents issued and pending to The Catholic University of Korea, Industry-Academic Cooperation Foundation, and Jaesung Park has patents issued and pending to POSTECH Research and Business Development Foundation. Other authors declare that they have no known conflict of interest that could have influenced the work reported in this paper.

Ethical approval

All procedures were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC no. 2023-0202-01).