Assessing the Safety of Liposome Nanocarriers Delivered Intratympanically to the Rat Inner Ear

Background

LPNs are the oldest nanotherapeutic platform in the clinic, successfully used for cancer, infectious disease, inflammation, and pain. Their high payload capacity and proven uptake in the inner ear make them attractive for otologic therapy. Intratympanic administration is a rational, targeted approach that limits systemic exposure and is already employed clinically for Meniere’s disease and sudden sensorineural hearing loss. While prior work demonstrated peptide‑functionalized LPNs reaching the cochlea, the safety profile of LPNs in the inner ear—particularly regarding barrier function, inflammation, and cell viability—remains unknown. This study fills that gap by evaluating the biocompatibility of LPNs in the rat inner ear using Gd‑MRI, ABR, and comprehensive histological analyses.

Hyaluronic acid (HA) is a major extracellular matrix component that binds CD44 and TLR2/4, initiating inflammatory pathways. Accumulation of HA has been linked to increased permeability and inflammation in various tissues, including the cochlea, where silver nanoparticles (AgNPs) have previously induced HA buildup. Therefore, HA, CD44, and TLR2 were examined as critical inflammatory markers in this study.

The objective was to determine whether transtympanic LPNs compromise inner‑ear barrier integrity, induce inflammation, or cause hearing loss. Gd‑MRI assessed barrier function, ABR measured auditory thresholds, and immunohistochemistry quantified HA, CD44, TLR2, and apoptotic DNA fragmentation.

Results

LPNs did not cause functional changes in the rat cochlea

Gd‑MRI demonstrated that LPNs entered the cochlea efficiently, with strong signal in the scala vestibuli and scala tympani within 3 h post‑injection (Fig. 2c‑f). By 6 h, signal distribution became homogeneous across the cochlea, indicating complete equilibration (Fig. 2e‑f). Blood‑perilymph and blood‑endolymph barrier integrity remained intact, as reflected by comparable signal intensities between treated and untreated ears (Table 1). ABR thresholds for click and tone bursts (2–32 kHz) showed no significant shift at 2, 4, or 7 days post‑injection, confirming preserved auditory function (Fig. 3).

LPNs did not induce glycosaminoglycan accumulation in the rat cochlea

Periodic acid–Schiff staining revealed no increase in glycosaminoglycans across cochlear structures (spiral limbus, bony wall, stria vascularis) following LPN or LPN‑Gd treatment compared with deionized water controls (Figs. 5, 6). Quantification confirmed no statistically significant differences (p > 0.05).

Minor impact on hyaluronic acid secretion by LPNs

Immunofluorescence detected HA primarily in spiral ganglion cells, strial basal cells, outer sulcus cells, and capillary endothelial cells (Fig. 7). While HA intensity in spiral ligament fibrocytes decreased slightly after LPN or LPN‑Gd administration, this change did not translate into altered barrier permeability or hearing loss (Fig. 8).

CD44 expression remained unchanged after LPN exposure

CD44‑positive cells were abundant in the stria basal cell layer, spiral ligament, Deiters’ cells, and capillary endothelium across all groups, with no significant differences in staining intensity or cell counts between treated and control ears (Figs. 9, 10).

TLR2 expression was unaffected by LPNs

TLR2 immunoreactivity was high in strial basal cells, spiral ligament fibrocytes, and endothelium but did not differ between LPN, LPN‑Gd, and water‑treated ears (Figs. 11, 12).

No increase in cochlear apoptosis following LPN administration

TUNEL staining revealed sparse apoptotic nuclei distributed throughout the cochlea in all groups, with no elevation in the LPN or LPN‑Gd cohorts compared with controls (Fig. 13). The occasional apoptosis observed in the stapes footplate and oval window niche is considered part of normal cellular turnover.

Discussion

Our data demonstrate that intratympanic LPNs reach the inner ear efficiently, traversing both oval and round window pathways, and do so without compromising the blood‑perilymph or blood‑endolymph barriers. The lack of hearing threshold shifts and absence of inflammatory or apoptotic markers confirm that LPNs are well tolerated in the rat cochlea. The minor reduction in HA secretion from spiral ligament fibrocytes appears clinically irrelevant, likely reflecting a shift toward high‑molecular‑weight, anti‑inflammatory HA without inducing permeability changes. These findings align with prior reports that 95‑nm LPNs are most efficient for inner‑ear delivery and that larger (110–115 nm) LPNs still maintain safety.

Importantly, the study confirms that LPNs do not activate the TLR2/CD44 inflammatory axis, a key pathway implicated in cochlear injury by reactive oxygen species and mitochondrial toxins. Therefore, LPNs represent a safe vehicle for targeted drug delivery to the inner ear, supporting their potential clinical application in sensorineural hearing loss and other otologic conditions.

Conclusions

Transtympanic delivery of liposome nanocarriers is safe for the rat inner ear, preserving barrier integrity, auditory function, and cellular homeostasis. LPNs hold promise as a drug delivery platform for future inner‑ear therapies.

Methods

Materials

Lipids (sphingosine, SOPC, DSPE‑PEG‑2000), fluorescent dyes (DiI, TRITC‑DHPE), Gd‑DOTA (DOTAREM), and silver nanoparticles (AgNPs) were sourced from commercial suppliers. Lipid purity was verified by thin‑layer chromatography; concentrations were determined gravimetrically.

Preparation and Characterization of LPNs

Unilamellar LPNs (~110 nm) encapsulating 500 mM Gd‑DOTA (LPN‑Gd) and blank LPNs (~115 nm) were prepared by a previously described thin‑film hydration method (Refs 6, 3). Size distribution and zeta potential were measured by dynamic light scattering.

Administration of LPNs

Under isoflurane anesthesia, 50 µl of LPN or LPN‑Gd was injected transtympanically into the left middle ear via an operating microscope. The contralateral ear received deionized water as a negative control. Animals were positioned with the injected ear upward for 15 min post‑injection.

Evaluation of Biological Barrier Function Using Gd‑MRI

Gd‑MRI was performed on a 4.7 T Bruker scanner using a RARE protocol. Contrast was delivered intravenously 2 h before imaging. Imaging time points ranged from 5 h to 8 days post‑injection to capture acute and sub‑acute changes.

Auditory Brainstem Response (ABR)

ABR thresholds for click and tone bursts (2–32 kHz) were recorded at baseline, 2, 4, and 7 days post‑injection using a standard electrode montage and sound‑attenuating chamber.

Histological and Immunofluorescence Analyses

After the final ABR, cochleae were harvested, decalcified, and sectioned. Hematoxylin–eosin and periodic acid–Schiff stains assessed inflammation and glycosaminoglycan content. Immunofluorescence for HA, CD44, and TLR2 was performed on cryosections, and confocal imaging quantified signal intensities. Apoptosis was detected by TUNEL labeling.

Statistical Analysis

Data were analyzed with SPSS 20. One‑way ANOVA and Kruskal–Wallis tests compared ABR shifts and staining intensities; post‑hoc LSD tests followed significant findings. p < 0.05 was considered significant.

Abbreviations

ABR

Auditory brainstem response

AgNPs

Silver nanoparticles

dH2O

Deionized water

Gd-DOTA

Gadolinium‑tetra‑azacyclo‑dodecane‑tetra‑acetic acid (DOTAREM)

Gd-MRI

Gadolinium‑enhanced magnetic resonance imaging

LPN

Liposome nanocarrier

LPN-Gd

Gd‑DOTA‑containing LPN

ROI

Region of interest

TLR

Toll‑like receptor