Round window membrane extracellular vesicles facilitate inner ear drug delivery

S Holdsclaw; D Silkstone; F Moazzam; H Jeanneret; N Tehsin; E Norris; E Talic; K Popowski; G Cruse; K Cheng; A Moatti

doi:10.1016/j.jconrel.2025.114153

. Author manuscript; available in PMC: 2026 Jan 28.

Published in final edited form as: J Control Release. 2025 Aug 21;387:114153. doi: 10.1016/j.jconrel.2025.114153

Round window membrane extracellular vesicles facilitate inner ear drug delivery

S Holdsclaw ^a,^b,¹, D Silkstone ^c,^d,¹, F Moazzam ^c,¹, H Jeanneret ^c,^e, N Tehsin ^a, E Norris ^a, E Talic ^e, K Popowski ^e, G Cruse ^e, K Cheng ^e,^f, A Moatti ^c,^g,^*

PMCID: PMC12836284 NIHMSID: NIHMS2125619 PMID: 40848758

Abstract

Hearing loss affects over 430 million people worldwide, yet effective treatments remain limited, in part due to challenges of inner ear drug delivery, a process that is risky, unreliable, and inefficient. Addressing this challenge calls for delivery approaches that ensure therapies reach the inner ear safely and effectively. Intratympanic (IT) injection, which delivers drugs directly into the middle ear, is currently the safest method for preserving cochlear integrity. However, its efficiency is limited by the round window membrane (RWM), a barrier with tight junctions that restricts therapy-related substance passage into the inner ear. We propose that extracellular vesicles (EVs) released by RWM cells could serve as a novel vehicle for drug delivery, as their membrane features and intravesicular components may facilitate IT transport. Importantly, EVs are also expected to elicit minimal immune responses, addressing a key safety concern for inner ear therapies. We isolated and characterized EVs derived from RWM cells, including sequencing and Ingenuity Pathway Analysis to predict drug delivery pathways and immune-related functions. To establish translational relevance, we investigated their uptake in vitro and assessed passage across the RWM ex vivo and in vivo, demonstrating that dexamethasone-loaded EVs effectively crossed the membrane in a porcine model. We further demonstrated functional delivery potential by showing enhanced cytoplasmic retention of brain-derived neurotrophic factor (BDNF) and improved adeno-associated virus (AAV)-mediated transduction in vitro.

Keywords: Inner ear drug delivery, Round window membrane, Extracellular vesicles, Intratympanic

1. Introduction

Globally, more than 430 million individuals are reported to suffer from disabling hearing loss requiring clinical intervention [1]. Moving toward clinical applications to prevent and treat hearing loss requires the establishment of a delivery method that provides a uniform passage, maintains cochlear integrity, and introduces minimal side effects. Gene therapy offers promise for treating deafness, but delivery is one barrier to clinical implementation. To date, several gene therapy clinical trials for the treatment of hearing loss are active, all of which utilize intra-cochlear injections [2,3]. However, perforation and surgical manipulation of the inner ear, such as intracochlear injection, carry a significant risk of deafness and are not suitable for patients with residual hearing. In addition, clinical responses to intracochlear injections are highly variable. For example, differences in surgical techniques during cochleostomy procedures in adult mice have been shown to result in variable hair cell regeneration outcomes [4]. Translation of AAV-involved studies to large animal models has also been controversial, due to technical delivery challenges and stronger immune responses in some primates [5,6]. For instance, while some studies reported that AAV9-PHP-B transduced hair cells when injected through the RWM in non-human primates [7,8], same studies have not consistently reported this finding for all animals and showed strong dose-dependency [8]. Finally, certain intracochlear approaches suggested in small animals (e. g., semicircular canal) [9] are not feasible in humans.

The IT (or transtympanic) injection is considered the safest delivery method, as it preserves cochlear integrity and has been used to administer steroids for sudden sensorineural hearing loss and gentamicin for vertigo associated with Meniere’s disease [10–12]. Although certain substances can traverse the RWM, like cationic ferritin and adenovirus [13], others show low penetrance, including neurotrophins [14] and small molecules such as CHIR99021 and VPA [15], while some do not cross at all, like AAVs [16].

For small molecules, two molecular properties primarily govern the movement of drugs across biological membranes, such as RWM: lipophilicity and surface polar groups [17–19]. Lipophilic, low-polarity compounds may cross the RWM but are rapidly cleared by the inner ear vasculature, preventing therapeutic levels in the cochlear apex and regions critical for speech perception (2–4 kHz). By contrast, macromolecules such as proteins and nanoparticle-based carriers tend to persist longer once inside the cochlea but encounter significant barriers to RWM entry. In addition, synthetic nanoparticles are subject to lysosomal degradation, requiring higher doses that risk toxicity and inflammation, which can further reduce RWM permeability [20–22]. These physicochemical constraints have motivated the development of alternative strategies to improve IT delivery, including chemical or mechanical manipulation of the RWM and the use of synthetic nanoparticle carriers. Such approaches have shown partial success in animal models: for example, RWM manipulation can enhance permeability, but raises concerns about long-term toxicity, perilymph leakage, and infection [23,24]. Similarly, magnetically-guided nanoparticles have demonstrated targeted delivery through RWM, yet clinical translation remains limited due to nanoparticle aggregation, the need for surface modification to ensure biocompatibility, and the technical complexity of generating controlled magnetic fields and maintaining patient orientation [25–28]. Furthermore, there are no preclinical studies with large animal models to demonstrate inner ear drug delivery relevant to human physiological dimensions and immune responses [29]. The high risk and high variability associated with the current solutions, together with translational challenges, underscore the need for a safer and more reliable approach. Given these limitations, biologically derived carriers may offer a more effective and translationally viable alternative.

EVs, which are naturally produced in the endosomal compartment of most eukaryotic cells, possess intrinsic membrane properties and molecular cargos that confer stability, biocompatibility, and reduced immunogenicity [30–32]. These features make EVs an attractive platform for therapeutic delivery to the inner ear compared with synthetic nanoparticles.

Compared with liposomes, a more conventional FDA-approved carrier, EVs demonstrate superior properties both in cellular uptake and drug loading capacity [33–36]. Furthermore, liposomes are limited by instability, off-target effects, premature drug release, oxidation, and the risk of hypersensitivity reactions [37]. In contrast, EVs possess a membrane composition that more closely resembles that of their parent cell in terms of lipid composition, fluidity, and proteins, which contributes to their higher affinity for uptake by homologous cell types [38–44]. Beyond their innate advantages, EVs can also be engineered through surface modifications to enhance targeting specificity and stability [45]. Importantly, EVs have been shown to successfully traverse biological barriers, including the blood-brain and vitreous-retina interfaces, while maintaining the activity of their cargo via transcellular transport and passage through tight junctions [46–48]. These findings suggest that EVs may likewise be capable of crossing the cellular layers of the RWM, providing a promising strategy for inner ear drug delivery [49,50].

EV secretion can be stimulated in cultured cells by stressors such as heat shock or serum deprivation [51–53], enabling the collection of EVs from a specific parent cell population [48,54]. Heat shock, typically achieved by exposing cells to elevated temperatures (42–45 °C), induces protein denaturation and activates heat shock protein (HSP) pathways, leading to a transient increase in EV release [53,55,56]. The resulting vesicles are enriched in HSP70, HSP90, and stress-related RNAs, which have been investigated for immunomodulatory applications [55,57]. By contrast, serum deprivation mimics nutrient starvation and activates autophagy-related pathways, producing EVs enriched in autophagy proteins and regenerative RNAs, although generally at lower yield [51,53,58,59].

EVs can be engineered to carry a wide variety of therapeutic cargo, including mRNA, miRNA, proteins, viruses, and small molecules [60]. Loading inside the EVs can shield cargoes like AAVs from immune recognition in circulation, potentially improving translation to larger animal models with stronger immune responses [61,62]. Once administered, EVs can be internalized by target cells through different mechanisms, such as micropinocytosis, endocytosis, plasma membrane fusion, or direct passage via gap-junction trafficking [49]. Consequently, EVs may either traverse the RWM intact or fuse with RWM cells and release their cargo. Upon uptake/fusion of the exosome, the cargo, such as mRNA, can be translated into protein within recipient cells [50] and secreted into the perilymph of the Scala Tympani, effectively turning the RWM into a local ‘factory’ for therapeutic production.

Previous studies have explored the use of EVs as therapeutic agents in the inner ear [63]. Mesenchymal stem cell (MSC)-derived EVs were shown to attenuate the inflammatory response in microglia and promote spiral ganglion neuron survival/growth, with in-vivo delivery via canalostomy alleviating noise trauma in a mouse model [64]. The same group also demonstrated safe injection of MSC-derived EVs into the cochlea of a human patient through the RWM during a cochlear implantation [65]. Supporting cell-derived EVs (enriched in HSP70) were further shown to protect against ototoxic drug-induced hair cell death, underscoring the protective potential of EVs [66]. Previously, HEK293T-derived EVs associated with AAV1 markedly improved transduction of hair cells in vivo via canalostomy or RWM injection, and delivery of Lhfpl5 using this platform partially restored hearing in a mouse model of hereditary deafness [67]. While these findings highlight both the safety and therapeutic potential of EVs for inner ear applications, they have thus far relied on invasive cochlear or RWM injection and have not addressed the challenge of improving IT drug delivery.

Here, we investigate EVs derived from RWM as a potential platform for IT drug and gene delivery to the inner ear. We compare their uptake and transport properties with MSC-derived EVs and liposomes and examine their ability and potential to deliver both small molecules and macromolecular cargos, including proteins and viral vectors, across the RWM. Using porcine models and relevant cell systems, this study aims to establish the translational potential of RWM-derived EVs for safer and more effective IT delivery.

2. Results

2.1. RWM cells secrete EVs

The RWM consists of two epithelial layers sandwiching a middle fibroblast layer, as shown schematically in Fig. 1A. The RWM was dissected from the porcine inner ear [68]. RWM cells were isolated and sorted using the CD326 (EpCAM) antibody (Supplementary Fig. S1 and Table S1). EVs were then collected from both cell populations of RWM, following i) serum deprivation and ii) heat shock treatments, two commonly used methods to enhance EV release. Although both approaches increase EV yield, they generate vesicles with distinct molecular content (Table S2). As such, we employed both to assess whether these differences influence EV activity. For comparison, EVs were also isolated from porcine MSC following serum deprivation.

EVs were characterized via transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) (Fig. 1B). Both methods confirmed the size of EVs in the expected range, commonly known as exosomes (30–150 nm, up to 200 nm) [62]. Membrane integrity was further verified by TEM following loading with red fluorescent protein (RFP) using electroporation (Fig. 1B), demonstrating that the isolation and loading procedures did not disrupt vesicular structure.

The identity of RWM and MSC EVs was confirmed by flow cytometry detection of exosome marker, CD63, shown in Fig. 1C. ImmunoTEM further verified the presence of canonical exosome markers of CD63, CD81, and CD9 in RWM Fibroblast-derived EVs under heat shock conditions (Fig. 1D). Western blotting analysis (Fig. 1E) also confirmed expression of these markers in RWM-derived EVs generated under serum deprivation and heat shock conditions. To assess post-translational modification, peptide N-Glycosidase (PNGase F) digestion demonstrated N-glycosylation of all three surface proteins.

To evaluate the stability of RWM-derived EVs after long-term storage, we analyzed EV size and particle concentration following two years (at −80 °C, > 10 freeze-thaw cycles), Supplementary Table S3. The EV population was largely preserved, as size distribution and particle concentration remained stable, indicating minimal degradation or loss under these storage conditions. A reduction in average particle size was observed, which may reflect structural changes, such as membrane shrinkage, vesicle fragmentation, or aggregate dissociation, processes known to occur during prolonged storage, even at −80 °C.

2.2. Ingenuity pathway analysis highlights Fibro HSEVs with potential to improve transport and mitigate immune response

To identify the role of EVs released by RWM cells, we performed bulk RNA sequencing of RWM EVs and their parent cells (Supplementary Materials 1–3). Supplementary Figs. S2, S3, and S4 show the detailed canonical pathways.

Specifically, using Ingenuity Pathway Analysis (IPA), we identified pathways related to vesicle-mediated trafficking, as summarized in Fig. 2A and detailed in Supplementary Table S4. Canonical pathway analysis suggests that EVs from RWM epithelial cells (Epi EVs) may upregulate clathrin-mediated endocytosis pathways, while EVs from RWM fibroblasts (Fibro EVs) may enhance intra-Golgi and retrograde Golgi-to-ER trafficking. Notably, EVs from RWM fibroblasts isolated under heat shock (Fibro HSEVs) may further promote gap junction trafficking in addition to the Golgi-associated pathways.

From the intracellular and second messenger signaling pathways, canonical analysis suggests that Epi EVs may downregulate signaling associated with ciliary beating, potentially impacting mucociliary clearance [69].

We also analyzed immune response and cytokine signaling pathways—critical factors for effective drug delivery—as summarized in Fig. 2B and detailed in Supplementary Table S5. Among the groups, canonical pathway analysis suggests that Fibro HSEVs may downregulate inflammatory responses, including cytokine signaling and broader immune system pathways, while potentially upregulating pathways associated with immune tolerance, such as MHC class II antigen presentation. In contrast, Epi EVs may promote activation of cellular immune response and cytokine signaling pathways.

Based on these findings, we primarily focus on the use of the Fibro HSEVs due to their potential upregulation of vesicle-mediated trafficking and immunomodulation (lowering immune response).

2.3. RWM EVs have higher RFP uptake by RWM cells than MSC EVs

To assess uptake efficiency, RWM-derived EVs were loaded with RFP by electroporation and applied to cultured RWM cells (Fig. 3A). Electroporation and ultrasonication are widely used for EV loading; the former is effective for charged, hydrophilic cargos such as proteins (RFP, BDNF) and nucleic acids, whereas the latter is more suitable for small, lipophilic drugs such as dexamethasone [70–72]. Uptake of RFP and RFP-loaded EVs by RWM cells was evaluated at three time points (Fig. 3B), with representative images shown at 24 h (Fig. 3C). RFP-loaded Fibro EVs showed significantly greater uptake than naked RFP at 12 h, and combined Epi + Fibro EVs demonstrated even higher uptake at 24 h. In contrast, MSC-derived EVs did not increase RFP uptake relative to naked RFP at any time point. These findings indicate that fibroblast EVs, and particularly combined Epi + Fibro EVs, exhibit superior uptake by RWM cells, consistent with predictions from RNA sequencing analysis.

2.4. RWM Fibro HSEVs mediate cytoplasmic retention of BDNF

Fibro HSEVs efficiently loaded with BDNF by electroporation (90–100 % efficiency; Supplementary Fig. S5A) were applied to HEK293T cells to assess nuclear localization. After 24 h, BDNF delivered via Fibro HSEVs was predominantly retained in the cytoplasm, with significantly lower nuclear localization compared to naked BDNF. Pearson’s Correlation Coefficient (PCC) analysis confirmed greater co-localization of BDNF with the nuclear marker DAPI in naked BDNF samples, whereas Fibro HSEV-mediated delivery resulted in cytoplasmic retention (Fig. 3D). Representative images at 24 h are shown in Fig. 3E.

2.5. RWM Fibro HSEVs significantly improve dexamethasone fluorescein (DexF) passage across RWM explants

A schematic of the ex-vivo and in-vivo transport assays is shown in Fig. 4A. In the ex-vivo setup, the substances were applied to the surface of porcine RWM explants as previously described [68].

DexF was encapsulated into EVs by ultrasonication, which provided higher loading efficiency than electroporation or passive incubation (Supplementary Fig. S5B). The overall loading efficiency of DexF remained low (~ 5 %) for both RWM-derived EVs and liposomes. Nevertheless, Fibro HSEVs significantly enhanced RWM permeability for DexF compared with free DexF (Fig. 4B). While liposome (Lipo)and MSC-derived EV loading also enhanced the RWM permeability for DexF, the improvement was not significant. Additionally, the concentration of DexF detected after passage was not significantly greater, potentially due to low MSC loading and drug leakage from liposomes.

Passage of Fibro HSEVs across the outer epithelial layer of the RWM explant was confirmed by immunoTEM, which revealed gold-conjugated CD9, CD63, and CD81 immunolabeling within the middle fibroblast layer of RWM (Fig. 4C).

2.6. RWM Fibro HSEVs enhance the passage of dexamethasone sodium phosphate (DSP) ex vivo and in vivo in pigs

Ex-vivo and in-vivo passage of DSP, either free or loaded into Fibro HSEVs, was assessed at 0.5 h and 1 h to approximate clinical waiting times (Fig. 4D). DSP loading was performed as described for DexF.

For in-vivo delivery, DSP formulations were administered via IT injection into the middle ear [73] (Fig. 4A), and perilymph (20 μL) was collected 1 h post-injection from the RWM using capillary tubes. Mass spectrometry analysis detected dexamethasone and DSP in the perilymph. Although the total DSP concentration delivered by EVs was not significantly higher due to low loading efficiency, permeability across the RWM was significantly improved when DSP was encapsulated in Fibro HSEVs, Fig. 4D. Together, these findings demonstrate that Fibro HSEVs enhance DSP transport in both ex-vivo and in-vivo settings.

2.7. RWM Fibro HSEVs improve AAV2 and AAV9 transduction efficiency and eliminate the vector dependency of efficiency

Fibro HSEVs successfully encapsulated AAV2-CMV-GFP with a loading efficiency of ~17 % using either electroporation or ultrasonication (Supplementary Fig. S5C). In HEK293T cells, GFP expression from AAV2-loaded Fibro HSEVs (e8 vg) was significantly higher than from free AAV2 at the same dose and comparable to a 10-fold higher concentration of AAV2 (e9 vg), Fig. 5A–B. Quantification was performed by measuring nuclear GFP signal (% transduction; Supplementary Fig. S6A).

Fig. 5. — Loading in RWM EVs leads to higher transduction in vitro. (A) Confocal imaging, 20× objective, was used to identify the green fluorescent protein (GFP) signal after transduction of HEK cells in culture, 72 h post introduction of naked AAV2-CMV-GFP (e8 vg and e9 vg:10×) and AAV2-CMV-GFP loaded Fibro HSEVs (e8 vg). Blue: DAPI, Green: GFP. (B) Fibro-HSEVs loading enhanced the efficiency of AAV2 transduction comparable to AAV2 with 10× higher concentration (technical replicates n: 16, biological replicates: 4, nested t-test p-value: 0010, p-value<0.0001, and p-value<0.0001, for EP and US, respectively). (C) Confocal imaging, 20× objective, was used to identify the GFP signal after transduction of HEK293T cells in culture after 72 h of introduction of naked AAV9-CMV-GFP (e9 vg:10×) and AAV9-CMV-GFP loaded Fibro HSEVs (e8 vg). Blue: DAPI, Green: GFP. (D) The Fibro HSEVs enhanced the efficiency of AAV9 transduction comparable to the AAV2 10× concentration (technical replicates n: 16, biological replicates: 2, t-test p-value: 0.0062). (E) Confocal imaging, 20× objective, was used to identify the GFP signal after transduction of RWM cells in culture after 72 h of introduction of naked AAV2-CMV-GFP (e9 vg:10×) and AAV2-CMV-GFP loaded Fibro HSEVs (e8 vg). Blue: DAPI, Green: GFP. (F) The Fibro HSEVs AAV2 transduction, comparable to AAV2 10 x higher concentration, was not significantly different (technical replicates n: 16, biological replicates: 2, t-test p-value: 0.1054).

For AAV9-CVM-GFP, which showed lower baseline transduction efficiency in culture, encapsulation into Fibro HSEVs markedly enhanced GFP expression, exceeding even the effect of a 10-fold higher free AAV9 dose (Figs. 5C–D). Notably, Fibro HSEVs encapsulation eliminated the vector dependency of efficiency, as AAV2 and AAV9 achieved comparable transduction when delivered in vesicles (Supplementary Fig. S6B). Although loading efficiency was similar between electroporation and ultrasonication, the encapsulation via electroporation resulted in significantly enhanced early-stage transduction (Supplementary Fig. S6C).

We next examined AAV2-CMV-GFP transduction in primary RWM cell cultures. Similar to the HEK293T results, AAV2-loaded Fibro HSEVs (e8 vg) achieved transduction levels equivalent to a 10-fold higher free AAV2 dose (Figs. 5E–F). Importantly, loading AAVs into Fibbro HSEVs enhanced transduction efficiency in both RWM and HEK293T cells to a similar extent, eliminating any cell type differences (Supplementary Fig. S6D). However, the onset of GFP expression remained slower in RWM cells than in HEK293T cells (Supplementary Fig. S7).

3. Discussion

Given the limitations of synthetic nanovesicles for drug delivery to the inner ear, we investigated RWM cell-derived EVs as drug delivery vehicles across the RWM. We have shown that porcine RWM cells secrete EVs and that these EVs are stable and maintain their integrity after drug loading. Additionally, we have identified the glycosylation of these proteins via PNGase F. Glycosylation plays an important role in EV biology, influencing stability, biodistribution, and uptake. Surface glycans can mediate interactions with lectins, receptors, and components of the extracellular matrix, thereby modulating targeting and internalization pathways [74,75]. In the context of the inner ear, the glycocalyx may facilitate more efficient passage across the RWM and contribute to the superior uptake observed for RWM EVs compared with liposomes. Moreover, glycosylation can shield vesicles from rapid clearance and dampen immune activation, further supporting their translational potential as delivery vesicles [76].

We performed RNA seq and canonical pathway analysis of RWM EVs compared to their parent RWM cells and identified pathways related to vesicle trafficking and immune response. We revealed that Epi EVs may upregulate clathrin-mediated endocytosis pathways, which typically direct internalized molecules toward lysosomal degradation [77,78]. By contrast, Fibro HSEVs are predicted to upregulate the intra-Golgi and retrograde Golgi-to-ER traffic pathways, primally facilitated by coat protein complex I (COPI), which can bypass lysosomal fate [78]. These findings suggest that Fibro EVs may provide more effective drug delivery than Epi EVs. Moreover, Fibro HSEVs, released under heat shock stress, may further enhance gap junction trafficking, offering an additional advantage for direct transport across the RWM.

Among intracellular and second messenger signaling pathways, G-protein–coupled receptors play a role in regulating ciliary beating and mucociliary removal [69], thereby influencing drug removal from the middle ear and round window niche. The Epi EVs appear to downregulate pathways that enhance ciliary beat frequency, which may improve drug delivery efficiency. Combining the Epi and Fibro EVs, in future studies, could yield a higher chance of trafficking to bypass both the mucociliary removal and the lysosomal degradation, respectively.

We have also analyzed the immune-related pathways in EVs, as both up- and down-regulation of the immune response are critical factors for effective drug delivery. For certain therapies, particularly immunotherapies, activation of the immune system is desirable, whereas in other cases, premature immune recognition and clearance of therapeutic agents can reduce efficacy. In addition, inflammatory conditions may alter drug absorption and distribution. Our analysis suggests that Fibro HSEVs may be advantageous for non-immunotherapy applications, as they appear to downregulate inflammatory responses while upregulating pathways that promote immune tolerance. The immunomodulatory properties support their potential use in the inner ear, consistent with prior studies reporting therapeutic benefits of heat shock EVs in treating hearing loss [60]. Conversely, Epi EVs may be more suitable for immunotherapy applications, given their immune-activating profiles.

We demonstrated that MSC EVs are uptaken less efficiently by RWM cells than RWM EVs, with RFP-loaded MSC EVs showing significantly lower uptake, highlighting the tissue-specific role of EVs in targeted delivery. Among RWM EVs, Fibro EVs exhibited higher RFP uptake by RWM cells than Epi EVs. Furthermore, the combined Epi + Fibro EVs achieved greater uptake efficiency than either subtype alone, underscoring the distinct yet complementary role of each EV population in trafficking.

We observed cytoplasmic retention of BDNF when delivered via RWM Fibro HSEVs. BDNF is a promising therapeutic for inner ear disorders due to its neuroprotective and regenerative properties. Although HEK293T cells lack the tropomyosin receptor kinase B (TrkB) to interact with the BDNF, preventing interaction with BDNF, uptake could still be detected. For future signaling studies, transfecting cells with the gene encoding TrkB would allow assessment of downstream activity. Notably, Fibro HSEVs mediated greater cytoplasmic than nuclear localization of BDNF. This may indicate that the EVs promote retention or gradual release within the cytoplasm, limiting their transport to the nucleus. Such retention in cytoplasm may be advantageous for therapeutic applications requiring delayed release and enhanced stability, as in IT delivery.

We revealed that the RWM EVs (Fibro HSEVs) significantly improved the passage of DexF (permeability) across RWM explants compared to liposomes and MSC EVs, underscoring the importance of tissue-specific EVs for targeted drug delivery.

Additionally, we showed that RWM EVs (Fibro HSEVs) significantly enhanced the permeability of RWM for DSP both ex vivo and in vivo in pigs. Our in-vivo measurement was performed in pigs as a translational model for hearing research due to their cochlear similarity to humans. Thus, successful enhancement in drug delivery using EVs in pigs underscores their viability for human use. DSP is clinically administered via IT injection for the treatment of sudden hearing loss and Meniere’s disease at concentrations of 4–24 mg/mL [79]. Despite the low DSP loading efficiency of 5 % (0.2 mg/mL) in EVs, we did not observe any significant changes in the concentration delivered into the inner ear, owing to their higher passage. To achieve a higher concentration of DSP in future applications, either increasing vesicle number or improving loading efficiency will be necessary.

Beyond small molecules or proteins, EVs can be used to deliver other cargo, such as AAV for gene therapy. We found that RWM EVs (Fibro HSEVs) enhanced AAV2 and AAV9 transduction efficiency, irrespective of vector type, in both human (HEK293T) and porcine (RWM) cells in vitro. While AAV2 and AAV9 have been successfully used for inner ear gene therapy in mice and some non-human primates via intracochlear injection, these vectors are unable to cross the RWM. As the initial step, we identified whether encapsulating AAVs inside Fibro HSEVs could facilitate their uptake and transduction efficiency in HEK293T and RWM cells. Remarkably, Fibro HSEV-loaded AAV2 (e8 vg) achieved transduction levels comparable to a 10-fold higher concentration of free AAV2 (e9 vg), and in the case of AAV9, loading into Fibro HSEVs significantly outperformed even the 10-fold free vector concentration (e9 vg). These findings demonstrate that EV-mediated delivery both enhances efficiency and eliminates the dependency of transduction on vector type. Importantly, the ability to reduce the required AAV dose could mitigate host immune responses while maintaining efficacy [67], as EVs inherently provide immune camouflage and Fibro HSEVs specifically downregulate immune response pathways. Thus, EV-mediated AAV delivery not only may circumvent the need for invasive intra-cochlear injection but also holds translational promise by reducing immune barriers across species. Future work will evaluate the ability of Fibro HSEVs-loaded AAVs to cross the RWM ex vivo and in vivo in pigs. To the best of our knowledge, this is the first report demonstrating the use of electroporation and ultrasonication to load AAVs directly into EVs. Previous approaches relied on the passive association of AAVs with EVs secreted from HEK293 cells, a labor-intensive strategy, which yields low quantities and lacks EV specificity [67]. In contrast, our method not only streamlines production but also allows flexibility in selecting EVs from different cell types, thereby enabling greater tissue-specific targeting. Using qPCR, we confirmed loading efficiencies of 15–20 %.

The use of RWM EVs as the vehicle for drug delivery to the inner ear could address several longstanding challenges. 1) These vesicles can efficiently deliver a wide range of molecules, including viral vectors via IT injection, a safe and clinically feasible alternative to intracochlear delivery by upregulating vesicle-mediated transport pathways for both direct and indirect trafficking. 2) Depending on their cellular origin, EVs (such as Fibro HSEVs) can bypass lysosomal degradation pathways, a major limitation of synthetic nanoparticles. 3) They are stable in body fluid such as perilymph and could potentially achieve therapeutic distribution to the apical regions of the cochlea, including frequency ranges critical for speech perception. 4) Origin-specific vesicles, such as Fibro HSEVs, may also downregulate the immune response pathways, thereby minimizing host immunogenicity. 5) They could downregulate and bypass mucociliary removal pathways based on their origin (Epi EVs). Collectively, these findings support RWM EVs as a safe, reliable, and translatable platform for inner ear therapeutics, with the potential to provide sustained and targeted drug delivery.

4. Methods

4.1. RWM cell culture

Cochleae from male and female healthy piglets (1–2 weeks) were utilized. The RWM was identified in each cochlea, and the entire RWM was excised. RWMs were washed in phosphate-buffered saline (PBS) with 1 % antibiotic-antimycotic (AA, Sigma-Aldrich). Cell culture plates were coated with fibronectin (Thermo Fisher) at 2.5 μg of fibronectin per cm². RWM samples were cut into smaller pieces, and 2–3 tissue pieces were placed in each fibronectin-coated well of a 6-well plate. For the growth of fibroblast RWM cells, 1 mL of Fibroblast growth media was added to the well. Fibroblast growth media consisted of Dulbecco’s Modified Eagle Medium (DMEM; Corning Life Sciences) containing 15 % fetal bovine serum (FBS; Cytiva) and 1 % AA.

For the growth of RWM epithelial cells, 1 mL of Epithelial Growth Media was added to the well. Epithelial Growth Media was comprised of EpiLife base media (Thermo Fisher) containing 10 % FBS, 1 % A-A, and 1 % human keratinocyte growth supplement (HKGS; Thermo Fisher). Tissues were incubated at 37 °C and 5 % CO2 for 2 days before an extra 1 mL of media was added to each well. Tissues were incubated for 5 more days to allow cell outgrowth onto the plate. To passage, the cells were gently washed 3 times with PBS to remove tissue pieces. Cells were harvested after a 5-min incubation with 0.05 % Trypsin 0.53 mM EDTA (Corning) and replated into a new fibronectin-coated dish with their respective media. Cells were characterized using immunostaining of each cell line for the fibroblast/MSC marker, vimentin, and the epithelial marker, CD326 (EpCAM).

4.2. RWM single cell isolation

Under sterile conditions, RWM tissues were minced into pieces using a sharp blade. The pieces were transferred into a 5 mL tube containing 4 mL of ice-cold Phosphate Buffered Saline (PBS). The sample pieces were transferred into a new 5 mL tube containing 2 mL of ice-cold digest buffer (PBS, 2 μL of 10 μM ROCK inhibitor Y-27632 2HCL CAT S1049, 10 mg/mL Protease CAT P5380, 2.5 mg/mL DNase 1 CAT 7470). The ratio of digestion buffer to sample was 5:1. The samples were incubated for 10–15 min at 4 °C by rocking or ideally by shaking at 150 rpm in a floor shaker. After enzymatic digestion, an equal amount of ice-cold inactivation buffer (PBS, FBS- 10 %, Antibiotic/Antimycotic1 %) was added. The digested cell suspension was passage through a 70 or 100 μm strainer into 5 mL tubes. The cell isolate was centrifuged at 300 g for 6 min at 4 °C then the supernatant was removed.

4.3. RWM cell sorting

The supernatant was decanted and resuspended in a 100 μL staining cocktail (Anti CD632-FITC CAT 11-579,182). Cells were stained for 20 min at 4 °C. To prepare compensation controls, a single-colour compensation control for each fluorochrome used in the experiment, along with an unstained control sample, was used. 1 μL of control antibody was added. Ex: PE fluorochrome, FITC fluorochrome, etc., to the tube designated for that fluorochrome. No antibody was added to the unstained control. Control samples were incubated for 20 min at 4 °C along with the experimental samples. After staining, cells were resuspended in ~2 mL 1× PBS and spun down at 400 g for 5 min. Pellets were resuspended in 1 mL of Magnetic-activated cell sorting (MACS) buffer (PBS + 2 % FBS/Bovine Serum Albumin (BSA) + 2 mM EDTA). Samples were filtered through a 40–70 μM filter to prevent clumps from clogging the machine. 5 μL diluted propidium iodide (PI) per mL of cells was added immediately before sorting. Cells were sorted using a Beckman MoFlo XDP cell sorter.

Catching buffer for sorting was MACS +1 % Anti-anti. Each catching tube was filled with ~1.5 mL of catching buffer and labeled appropriately. The catching tubes were inverted and vortexed gently before and after cell collection. Cells were collected from the sort in 5 mL round-bottom propylene tubes with caps.

Sorted cells were cultured for an extra three weeks before exosome collection or RNA sequencing.

Fibroblast Growth Media: DMEM, 15 % FBS, 1 % Antibiotic/Antimycotic.

Epithelial Growth Media: EpiLife, 10 % FBS, 1 % Human Keratinocyte Growth Supplement (HKGS), 1 % Antibiotic/Antimycotic.

4.4. MSC cell culture

Bone marrow-derived mesenchymal stem cells (MSCs) were isolated from piglet femurs. Femurs were removed and cleaned of fat and muscle. Both ends of the femur were cut off to reveal the bone marrow. Using a needle and syringe, one end of the femur was punctured, and 10 mL PBS with 1 % A-A was flushed through to collect marrow on the other end. 1× red blood cell lysis buffer (Invitrogen) was added to the marrow for 10 min with constant shaking at room temperature. The marrow was then centrifuged at 400 g for 6 min, and the cell pellet was resuspended in 10 mL of PBS. This was centrifuged again at 400 g for 6 min before the pellet was resuspended in the RWM fibroblast media and plated on non-coated flasks. The media was changed after 6 h and every day after plating for 1 week to remove non-adherent cells. MSCs were then expanded for EV isolation.

4.5. HEK 293T cell culture

HEK293T cells were purchased from the American Type Culture Collection (ATCC; American Type Culture Collection, Manassas, VA, USA) and then expanded as previously described. Briefly, cells were plated in T-75 cell culture treated flasks (VWR) and maintained in Dulbecco’s Modified Eagle Medium (DMEM Thermo Fisher Scientific, Waltham, MA, USA) containing 10 % FBS (Corning), 1 % l-glutamine (Life Technologies), 1 % pen-strep (Life Technologies), 1 % sodium pyruvate (Life Technologies), and 1 % nonessential amino acids (Thermo Fisher Scientific).

Cells were passaged (1:20) every 2–3 days. Media was removed.

from the flask and cells were briefly washed with 5 mL of room temperature 1× PBS (Thermo Fisher Scientific). PBS was removed, then 1 mL of 0.25 % trypsin (Life Technologies) was added, and the solution was swirled gently to coat the entire flask bottom. Trypsin remained until all cells had lifted (no longer than 3 min), then 9 mL of complete HEK media (see above) was added to inactivate trypsin and mixed thoroughly. 9.5 mL of cell solution was discarded, and the remaining 0.5 mL received 9.5 mL of fresh complete HEK media and was returned to the incubator to be cultured at 37 °C and 5 % CO2.

HEK293T cells were allowed to reach 70 %–80 % confluence before.

any transduction experiment. All procedures performed in this study involving human samples were in accordance with the ethical standards of the institutional research committee and with the guidelines set by the Declaration of Helsinki.

4.6. EV collection and characterization

EVs were collected from the RWM cells and the MSCs’ secretome. Cells were allowed to reach 70–80 % confluence in 3 T-175 flasks before secretome isolation. Two methods were used to induce EV release:

Serum deprivation: The FBS-containing media was removed from the flask and replaced with FBS-free media. After 24 h, the cells were washed with serum-free media 3 times for 30 min each before 15 mL of serum-free media was added to the cells. 72 h later, the secretome was collected from the flask.
Heat shock: The cells were stored at 40 °C for 1 h before secretome collection.

The collected secretome was filtered through a 0.22 μm filter to remove cellular debris. The secretome was stored at −20 °C until it was used for EV collection by ultrafiltration. A 100 kDa centrifugal filter (Millipore, SCGP00525) was primed with 5 mL PBS before the PBS was removed and the secretome was added. The filter was spun at 3000 g for 15 min at 4 °C. The flow through was discarded, and this was repeated until all the secretome was filtered. The tube was rotated 180° after each run to prevent aggregation on one side of the filter. 1 mL of PBS + 10 μM Trehalose was used to dislodge EVs from the filter for collection. Isolated EVs were stored at −80 °C. After collection, each EV sample was characterized by nanoparticle tracking analysis (NTA; NanoSight), transmission electron microscopy (TEM), and western blotting for EV markers (CD9, CD63, and CD81). This checked for expected EV size, morphology, and membrane markers. EVs were collected up to three times for each cell line.

4.7. Nanosight

Prior to analysis, samples were diluted to the optimal concentration (e8 particles/mL). We used Malvern NanoSight and a flow rate of approximately 50–100 μL/min.

4.8. Transmission electron microscopyTEM

Fibro-, Epi-, and MSC EVs were fixed with 4 % paraformaldehyde (PFA) and 1 % glutaraldehyde onto 100 mesh copper grids (Electron Microscopy Sciences) before and after loading with RFP. The fixed EVs were stained with Vanadium Negative Stain (ab172780; Abcam) for TEM imaging (JEOL JEM-2000FX).

4.9. Western blotting

EVs suspended in 1× PBS (Thermo Fisher Scientific) + 10 μM Trehalose (Sigma) were thawed at room temperature. Equal amounts of EV suspension (9 μL) and 2× Orange Lysis Buffer (Promega) were mixed to create a 1× protein lysate, then 2 μL of PNGase F (Promega) was added. 15 μL of the 1× samples, alongside 5 μL of Chameleon Duo Pre-stained Protein Ladder (LI-COR), were then loaded into different wells of 4–12 % Bis-Tris polyacrylamide protein gels (NuPage Novex, Thermo Fisher Scientific). Proteins were separated using SDS-PAGE at a constant 180 V for 1 h in 1× MOPS SDS Running Buffer (Invitrogen, MOPS 20× Running Buffer diluted with deionized water). Proteins inside gel slabs were then transferred electrophoretically to 0.45 μm nitrocellulose membranes (Invitrogen) at a constant 0.2 A for 85 min in 1× NuPage Transfer Buffer (Invitrogen, 20× Transfer Buffer, 10 % methanol, diluted with deionized water). Membranes were washed briefly in deionized water twice before being blocked for 1 h at room temperature in Intercept^® (TBS) Blocking Buffer (LI-COR) on a rocker. After which, blots were probed overnight, on a rocker at 4 °C, with a 1:500 dilution of the primary antibody against proteins of interest (CD9, CD63, CD81, Novus) in 5 mL of Intercept^® T20 (TBS) Antibody Diluent (LI-COR). The next day, membranes were washed (3 times 5 min) with TBS-T (Tris-Buffered Saline with TWEEN ^® 20 Detergent), incubated with IRDye^® 800CW Goat anti-Rabbit IgG Secondary Antibody (LICOR) and IRDye^® 680RD Goat anti-Mouse IgG Secondary Antibody (LI-COR) at a 1:2500 dilution in Intercept^® T20 (TBS) Antibody Diluent on rocker for 1 h at room temperature. They were then washed (3 times, 5 min) again in TBS-T before being imaged under the 700 and 800 channels for 10 min using the Odyssey^® Fc imaging system (LI-COR).

4.10. Flow cytometry

We stained the vesicles with FITC-CD63 via immunomagnetic beads (EXOFLOW300A-1), incubating them for 60 min at 4 °C in the dark. After incubation, we washed the vesicles twice in PBS and resuspended them in 1 mL of FACS buffer.

We calibrated the flow cytometer, loaded controls, and set up appropriate gates based on forward scatter (FSC) and side scatter (SSC) to identify populations of interest. The sample was then analyzed, and data were collected for FSC, SSC, and fluorescence channels. We compensated for spectral overlap if necessary and exported the data for analysis. Finally, we analyzed the data by setting gates on populations and quantified positive events, troubleshooting any issues like background signal or low resolution along the way. Flow cytometry was conducted with a CytoFlex Flow Cytometer (Beckman Coulter), and data were analyzed with FCS Express software (De Novo).

4.11. EV fluorescent label-RFP loading

RFP (ab268535; Abcam) was loaded into EVs and Liposome particles (300,202, Avanti) via electroporation, yielding RFP-EVs and RFP-Lipos. One billion nanoparticles from each sample were diluted in Gene Pulser Electroporation Buffer (Bio-Rad, Hercules, CA, USA) at a 1:9 ratio of nanoparticles to buffer. RFP was added to the nanoparticle-buffer solution and transferred to an ice-cold 0.4 cm Gene Pulser/MicroPulser Electroporation Cuvette (Bio-Rad). The electroporation cuvette was inserted into the Gene Pulser Xcell Total System (Bio-Rad) and electroporated. The electroporation buffer was filtered out of the fluorescently labeled nanoparticles by the ultrafiltration method described above.

4.12. EV dexamethasone loading

DexF (D1383, ThermoFisher) or DSP (D4902, Sigma) was added to the 1 billion nanoparticle-buffer solution and transferred to an ice-cold 5 mL tube, and the content was brought up to 5 mL with PBS. The sonication was performed and followed by incubation at 37 °C for 1 h. DexF/DSP was also loaded by electroporation, as described above for RFP loading.

4.13. EV BDNF loading

BDNF (ab206642; Abcam) was loaded into EV particles via electroporation or ultrasonication, yielding EP-BDNF-EVs and US-BDNF EVs, respectively.

Electroporation: One billion RWM-Fibroblast-Derived EVs were diluted in Gene Pulser Electroporation Buffer, and 5 μg of BDNF was added to the nanoparticle-buffer solution and transferred to an ice-cold 0.4 cm Gene Pulser/MicroPulser Electroporation Cuvette. The electroporation cuvette was inserted into the Gene Pulser Xcell Total System and electroporated. The electroporation buffer was filtered out of the EP-BDNF-EVs by the above ultrafiltration method.

Ultrasonication: One billion RWM-Fibroblast-Derived EVs were diluted in 4.2 mL of PBS. 5 μg of BDNF was added to the nanoparticlePBS solution and sonicated at 4 °C. Following ultrasonication, samples were incubated at 37 °C for 30 min. The PBS was filtered out of the US-BDNF-EVs by the above ultrafiltration method. Loaded samples were then stored at −80 °C.

4.14. EVs AAV loading

AAVs (AAV2-CMV-GFP or AAV9-CMV-GFP, UNC Viral Vector Core) were loaded into EV particles via electroporation or ultrasonication, yielding EP-AAV-EVs and US-AAV-EVs, respectively. Electroporation and Ultrasonication were performed as described above.

4.15. In-vitro delivery

To measure the delivery efficiency of RWM EVs, EVs loaded with RFP were co-cultured with RWM epithelial cells and fibroblasts. For the co-culture of epithelial and fibroblast RWM cells, epithelial cells and fibroblasts were plated at a 2:1 ratio into the same well of a chamber slide using a co-culture media of Advanced DMEM/Ham’s F-12 (DMEM/F12, Thermo Fisher) containing 10 % FBS and 1 % A-A. Cells were allowed to grow for 3 days before the media was changed. Cells were cocultured with 200,000 EV particles per well for 0, 12, and 24-h time points. Cells received no EVs (negative control), free-floating RFP, RFP-loaded RWM fibroblast EVs, RFP-loaded RWM epithelial EVs, a mix of RFP-loaded fibroblast and epithelial EVs, or RFP-loaded MSC EVs. At each time point, the media was removed from the wells of the chamber slide, and the cells were washed with PBS before continuing with fixation and staining.

4.16. RFP immunostaining

Chamber slides were fixed using 4 % paraformaldehyde (PFA), permeabilized, and blocked using IHC blocking buffer DAKO protein block (X0909) with 0.4 % Triton for 1 h at RT (room temperature). Cells were then stained with primary antibodies against vimentin (sc-6260; Santa Cruz Biotechnology) and EpCAM (ab71916; Abcam) at a 1:100 dilution. Cells were incubated with primaries overnight at 4 °C. The cells were washed with PBS and incubated with anti-mouse (A21236; Invitrogen) and anti-rabbit (A11008; Invitrogen) secondary antibodies at a 1:2000 dilution for 1.5 h at RT. Two drops of NucBlue (Invitrogen) were added to each well after 30 min of secondary incubation. The cells were washed with PBS and mounted with ProLong Gold (Invitrogen) at RT for 4 h before observation. Cells were imaged using a confocal microscope (Olympus FV3000) to detect nuclei, vimentin, EpCAM, and RFP in the cells. To quantify the amount of RFP delivered to each well, the number of red pixels was counted for each well of the slide using ImageJ and divided by the number of nuclei in that well.

4.17. BDNF uptake

BDNF Uptake: Coverslips (174,950, Thermo Fisher) were dipped in 70 % ethanol for 5 min, placed in well plates, and dried under UV light for 20 min. HEK293T cells were passaged into 24-well plates and cultured for 48 h. At the 48-h mark, BDNF was added. Source BDNF was diluted in sterile PBS, while EP-BDNF-EVs were directly applied. After 72 total hours of passaging, the media was removed, and cells were washed once with PBS and fixed with 4 % PFA in PBS for 20 min. Samples were washed again 3 times with 1 mL PBS.

Immunohistochemistry (IHC): IHC was performed by blocking for 1 h at room temperature with 200 μL of DAKO solution. DAKO solution was composed of 1:1 DAKO (protein block) (Agilent, #X090930-2) to PBS, with 0.4 % Triton X-100 (Sigma, #X100) added. DAKO solution was removed, and 100 μL of a primary antibody solution (1:100 antibody: DAKO solution) and incubated overnight at 4 °C. The primary antibody was rabbit anti-BDNF (Thermo-Fisher, #PA5-85730). The next day, samples were washed 2× quickly with 0.8 mL PBS, and 2 × 10 min with 0.8 mL PBS. Following washes, 100μlL of a secondary antibody solution (1:200 antibody: DAKO solution) was added and incubated for 1.5 h at room temperature. The secondary antibody was donkey anti-rabbit Cy3 (Sigma, #AP182C). 30 min into incubation, 2 drops of NucBlue (DAPI) (Fisher, #R37606) were added per well. After incubation, samples were washed 2 times quickly with 0.8 mL PBS, and 2 times for 10 min with 0.8 mL PBS. Slides were fixed with hydro mount (Fisher, #50-899-90,144) and stored at 4 °C until imaging.

Imaging: Fixed slides were imaged using confocal microscopy (Olympus FV3000). A 60× (oil immersion) objective was used to capture images of each sample for DAPI, brightfield, and Cy3 channels.

4.18. AAV transduction

AAV2-CMV-GFP and AAV9-CMV-GFP were kindly provided by Dr. Chales Askew from UNC Vector Core. Coverslips (48393-026, VWR;174,950, Thermo Fisher) were dipped in 70 % ethanol for 5 min, placed in well plates, and dried under UV light for 20 min. HEK293T or RWM-Epi cells were plated into 6 or 24-well plates. Naked AAV or AAV-EVs were then added for the corresponding vg. 1 X AAV corresponded to 3.3e8 vg. Cells were imaged with a benchtop microscope (Echo RVL-100-G, 10× objective) at 24 and 48-h time points to assess transduction rates. At 72 h, the media was removed, and cells were fixed in 4 % PFA in PBS for 20 min. Slides were washed 3 times in PBS and then mounted with a DAPI stain (Thermo-Fisher, #P36962). Slides were stored at 4 °C until imaged with confocal microscopy (Olympus, FV3000). Mounted samples of in-vitro transduction were imaged using confocal microscopy (Olympus FV3000) at 20× magnification. Map images of samples were obtained and converted to a usable file format using IMARIS software.

4.19. ELISA

Using a commercially available Human BDNF ELISA kit (Abcam, #ab212166), a standard curve was first created using our source BDNF. The standard curve was constructed as a 4-parameter Logistic Regression (4PL) using freely available online software (MyCurveFit.com). Loaded EVs were lysed via a 1:1 addition in RIPA buffer (Fisher, #PI89900) and 3 rounds of 5 min. On: ultrasonication, 5 min, Off: on ice, 5 min. Lysed EVs were tested at a range of dilutions in the same ELISA protocol. Absorbance values were interpolated from the standard curve to yield BDNF concentrations and then adjusted for dilution factors. Loading efficiency was calculated as the concentration of BDNF retained in loaded EVs over the total BDNF added.

4.20. qPCR

SnapGene software was used to estimate our AAV plasmid sequence’s extinction coefficients, which were used in combination with a nanodrop to adjust the initial DNA concentration to 1 ng/μL. Serial dilutions were then performed to create a standard curve of Ct. vs. Log dilution factor following qPCR (qTower 3G). Forward and reverse primers were used for the GFP sequence.

Forward: AGC AGC ACG ACT TCT TCA AGT CC.

Reverse: TGT AGT TGT ACT CCA GCT TGT GCC, Thermo-Fisher. DNA from loaded EVs was extracted using a commercially available.

DNA extraction micro prep kit (D3021, Zymo Research). Viral plasmids were linearized using BamHI (Thermo-Fisher, #FD0054), followed by heat-inactivation of the restriction endonuclease, according to manufacturer recommendations. qPCR was performed with technical duplicates of 3 samples, Ct values were obtained, and the standard curve was interpolated to calculate the DNA concentration of the samples using Microsoft Excel. Loading efficiency was calculated as the concentration of AAV DNA retained in loaded EVs over the total AAV DNA added.

4.21. Quantification

Transduction: From 20× map images obtained from transduction imaging; snapshots were captured for 16 unique locations. Selected areas were segmented using the Ilastik software to create a pixel classification of DAPI masks. 4 images were used per sample to train batch processing. Masked images were input to a custom Matlab code, which 1) created a binary mask of DAPI stains, 2) counted nuclei from connected regions, and 3) removed background GFP that was outside of the nucleus. Lastly, the transduction rate was calculated as the number of DAPI masks with >0 corrected GFP signal over the total number of DAPI masks. Matlab Code can be found attached for AAV2 and AAV9 analysis. Note that different thresholds were applied between AAV serotypes to account for the expected weaker signal of AAV9 GFP expression.

BDNF Uptake: From 60× Images taken of BDNF uptake, IMARIS software was used to capture snapshots of 16 locations per sample. Images were then segmented using the Ilastik software, using pixel classification of DAPI stains. Segmented images were then imported into Matlab, where a custom code was used to 1) create a binary mask of nuclei and cytoplasm regions from segmentation, 2) estimate the number of nuclei based on mask area, and 3) normalize Cy3 intensity per nucleus and cytoplasm regions.

4.22. ImmunoTEM

Antibodies were conjugated to 40 nm gold nanoparticles using a commercially available kit (230-0010, Novus Biologicals). Selected antibodies were CD9 (NB500-327, Novus Biologicals), CD63 (NBP2-42225, Novus Biologicals), CD81 (NB100-65805, Novus Biologicals), and B-Actin (3700, Cell Signaling Technologies). Antibodies were diluted to 0.1 mg/mL using the kit diluent and then conjugated with gold nanoparticles. Removal of non-conjugated antibodies was performed by diluting 1:10 the sample: reaction quencher buffer, centrifuging for 10 min at 9000 g. The supernatant was removed, and samples were diluted in quencher buffer. Conjugation was confirmed with absorbance at 530 nm, with final conjugated antibody concentrations of 5 μg/mL.

An ex-vivo RWM chamber was constructed using previously validated methods. To do so, the skin was removed to visualize the porcine skull, which was then cut using a bone striker. The brain was removed, allowing the inner ear to be visualized. The inner ear was excised by further cutting using a bone striker and a bone cutter. Once removed, the bone surrounding the RWM was drilled away, leaving only the RWM, which was glued inside the bottom of a cut 0.5 mL Eppendorf Tube. The construct was then placed in a 24-well plate transwell with the lower mesh removed and stored overnight (0.15 mL above on RWM, 1.5 mL below) in Day 1 media (DMEM CAT 10014-CV, 1 % Antimycotic CAT A5955, 1 % N2 CAT 17502048). The next day media was changed to Day 2 media (DMEM, 1 % FBS, CAT SH30396.03, 1 % Antimycotic, 1 % N2), and passage testing started. BDNF-EVs were added to the top portion, and following 24 h, the chamber was fixed in 4 % PFA.

TEM Copper grids (01802-F, Ted Pella) were incubated in Aurion blocking buffer (25,595, VWR) for 15 min at room temperature. Washing was performed with 2 times 5-min washes of Aurion Incubation solution (25,561, Electron Microscopy Sciences). Grids were incubated at 4 °C overnight with conjugated primary antibodies. The next day, grids were washed 6 times for 5 min with Aurion incubation solution, followed by 2 times 5 min washes in PBS. Grids were postfixed in 2 % Glutaraldehyde (16,320 Aqueous Glutaraldehyde EM Grade 50 %) in PBS, then washed 3 times for 5 min with ddH2O. For contrast, incubation with 7 % uranyl acetate (22,400 Uranyl Acetate, Reagent, A.C.S.) for 20 min was performed, then washed 3 times for 10 min with ddH2O. Samples were dried overnight prior to imaging.

4.23. Bulk RNA-sequencing

Bulk RNA sequencing was performed externally by GENEWIZ, LLC. (South Plainfield, NJ, USA) using Ultra-low input RNA-seq Library Preparation and Illumina HiSeq Sequencing RNA library according to the manufacturer’s instructions. The cells were sent frozen in 10 % DMSO and media in cryovials for analysis. The EVs were also sent frozen in PBS. QIAamp Circulating Nucleic Acid Kit (50) (Cat. No. / ID: 55114) and ~ 350 M PE reads (~105GB) were used.

4.24. RNA analysis

The resulting FASTQ files were filtered and trimmed using Rfastp (v1.12.0) with a quality cutoff of 20 and forward and reverse primer sequences AGATCGGAAGAGC. Trimmed reads were mapped to the Sus Scrofa reference genome available on ENSEMBL (v11.1.110) using a STAR aligner (v.2.7.3a). Transcript counts were quantified using Salmon (v1.4.0) and annotated according to ENSEMBL (v11.1.110). Differential expression analysis was performed in R using DESeq2 (v1.42.0) with an adjusted p-value cutoff of 0.05.

4.25. IPA analysis

We used QIAGEN Ingenuity Pathway Analysis (IPA) to identify the canonical pathways using the calculated differential expression inputs (p-value, q-value, fold change, and log ratio).

4.26. Animal procedures

We utilized Yorkshire wild-type pigs (male and female) postnatal days P20–30 in this study (7–12 kg). All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at North Carolina State University, following the standards of the National Institute of Health and Committee on Care and Use of Laboratory Animals. We used an intratympanic surgical procedure previously developed to deliver therapeutics into the porcine middle ear [73]. The pigs were euthanized one hour after injection. The perilymph was collected from the RWM via a capillary tube, stored at −80 °C, and sent to Carbon for mass spectrometry analysis to identify dexamethasone concentration.

4.27. Statistical analysis

Statistical analysis was performed using GraphPad Prism analysis software (GraphPad Software, San Diego, CA, USA). Comparisons among the two groups were performed using the t-test, followed by Welch’s correction test. Comparisons among more than two groups were performed using a parametric 1-way ANOVA test followed by Bonferroni’s multiple comparisons test. p ≤ 0.05 was considered statistically significant. The legend is as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns: not significant.

Supplementary Material

Supporting information

NIHMS2125619-supplement-Supporting_information.pdf^{(1.5MB, pdf)}

Acknowledgment

The initial data for this study were generated using the NCSU CMI-Young Scholar Program (YSP) seed grant. Research reported in this publication was supported by the NIDCD of the National Institutes of Health under award numbers K99-R00DC019960 (AM). SH was supported by NCSU OUR (Office of Undergraduate Research) over the summer.

The authors would like to thank Dr. Alon Greenbaum and Dr. Frances Ligler for providing insight, space, and resources to perform part of this study. The authors would like to thank Dr. Phuong-Uyen C. Dinh for her help with the Flow Cytometry. The authors would like to thank Dr. Kathryn Polkoff for her help in mentoring one of the undergraduate students on this project. The authors thank Keith Abbey and Sasank Sivadanam for assistance during early method development and training. We gratefully acknowledge Dr. Charles Askew for providing AAVs.

This work was performed in part at the Analytical Instrumentation Facility (AIF) at North Carolina State University, which is supported by the State of North Carolina and the National Science Foundation (award number ECCS2025064). The AIF is a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), a site in the National Nanotechnology Coordinated Infrastructure (NNCI).

Inclusion and diversity

We worked to ensure sex balance in the selection of non-human subjects. One or more of the authors of this paper self-identifies as a gender minority in their field of research. While citing references scientifically relevant to this work, we also actively worked to promote gender balance in our reference list. We avoided “helicopter science” practices by including the participating local contributors from the region where we conducted the research as authors on the paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jconrel.2025.114153.

Footnotes

CRediT authorship contribution statement

S. Holdsclaw: Writing – review & editing, Visualization, Methodology, Investigation, Formal analysis, Data curation. D. Silkstone: Writing – review & editing, Visualization, Methodology, Investigation, Formal analysis, Data curation. F. Moazzam: Investigation, Data curation. H. Jeanneret: Writing – review & editing, Visualization, Supervision, Methodology, Investigation, Data curation. N. Tehsin: Investigation, Data curation. E. Norris: Investigation, Data curation. E. Talic: Validation, Supervision, Methodology, Investigation. K. Popowski: Writing – review & editing, Supervision, Methodology, Investigation, Conceptualization. G. Cruse: Supervision, Resources. K. Cheng: Supervision, Resources. A. Moatti: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare no competing interests. The authors have assigned rights to a Provisional Application // No. 63/841,625, Round Window Membrane Extracellular Vesicles Improve Drug Delivery to the Inner Ear.

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting information

NIHMS2125619-supplement-Supporting_information.pdf^{(1.5MB, pdf)}

Data Availability Statement

Data will be made available on request.

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PERMALINK

Round window membrane extracellular vesicles facilitate inner ear drug delivery

S Holdsclaw

D Silkstone

F Moazzam

H Jeanneret

N Tehsin

E Norris

E Talic

K Popowski

G Cruse

K Cheng

A Moatti

Abstract

1. Introduction

2. Results

2.1. RWM cells secrete EVs

Fig. 1.

2.2. Ingenuity pathway analysis highlights Fibro HSEVs with potential to improve transport and mitigate immune response

Fig. 2.

2.3. RWM EVs have higher RFP uptake by RWM cells than MSC EVs

Fig. 3.

2.4. RWM Fibro HSEVs mediate cytoplasmic retention of BDNF

2.5. RWM Fibro HSEVs significantly improve dexamethasone fluorescein (DexF) passage across RWM explants

Fig. 4.

2.6. RWM Fibro HSEVs enhance the passage of dexamethasone sodium phosphate (DSP) ex vivo and in vivo in pigs

2.7. RWM Fibro HSEVs improve AAV2 and AAV9 transduction efficiency and eliminate the vector dependency of efficiency

Fig. 5.

3. Discussion

4. Methods

4.1. RWM cell culture

4.2. RWM single cell isolation

4.3. RWM cell sorting

4.4. MSC cell culture

4.5. HEK 293T cell culture

4.6. EV collection and characterization

4.7. Nanosight

4.8. Transmission electron microscopyTEM

4.9. Western blotting

4.10. Flow cytometry

4.11. EV fluorescent label-RFP loading

4.12. EV dexamethasone loading

4.13. EV BDNF loading

4.14. EVs AAV loading

4.15. In-vitro delivery

4.16. RFP immunostaining

4.17. BDNF uptake

4.18. AAV transduction

4.19. ELISA

4.20. qPCR

4.21. Quantification

4.22. ImmunoTEM

4.23. Bulk RNA-sequencing

4.24. RNA analysis

4.25. IPA analysis

4.26. Animal procedures

4.27. Statistical analysis

Supplementary Material

Acknowledgment

Inclusion and diversity

Appendix A. Supplementary data

Footnotes

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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