Table 1.

Summary of studies on nanomaterials potential side effects on inner ear cells.

Tested Nanomaterial	Nanomaterial Properties	Type of Study	Type of Cells	Experimental Design	Observations	Refs.
LCNs	Size: 50 nm Polydispersity index: <0.2	In vitro	Cochlear cells isolated from newborn Sprague-Dawley rats	The cells were treated with LNCs at concentrations varying between 0 and 1.5 mg/mL for 24 h.	Survival rates of treated cells, depending on concentration: −1.5 mg/mL—37.96% −0.15 mg/mL—86.41% −0.015 mg/mL—80.06% Surviving cells from all treated groups had fewer LNCs in their cytoplasm.	[82,138]
LCNs	Nanoparticle size: 50 nm Pledget size: 8 mm3 LNC concentration: 20.5 g/L	In vivo	Interdental cells, stria marginal cells, outer hair cells, inner hair cells, semi-circular canal endothelial cells, cochlear nerve of rats	A small piece of gelatin sponge pledget saturated with LNCs was placed on the round window membrane, and it was there for D28 for ABR study and 2 h for neural elements studies.	None of the animals manifested middle ear infections during the study. No inflammation was detected in the inner ear LNC treatment did not induce apoptosis. The inner ear neural elements were preserved.	[138]
Resveratrol-loaded PLGA nanoparticles	Size: 135.5 ± 37.3 nm	In vitro	HEI-OC1 and SVK-1 cell line	The cells were treated with blank nanoparticle, resveratrol, and resveratrol-loaded nanoparticles at concentrations up to 1 mg/mL for 24 h.	No cell line’s viability was affected by blank nanoparticles in concentrations below 0.6 mg/mL At a concentration of 1 mg/mL, blank nanoparticles produced the death of 56% cells from HEI-OC1 cell line Resveratrol and resveratrol-loaded nanoparticles lead to negligible cell death rates.	[82,143]
Polyethylenimine (PEI)-plasmid DNA nanoparticles	Size: ~20–100 nm Shape: almost spherical	In vitro	Cochlear epithelium isolated from C57BL/6J male and female mice	The cochlear explants were treated with linear nanoparticle polyplexes loaded with plasmid DNA at various weight ratios; the cell viability was assessed in a 0–48 h interval after transfection.	The use of a higher linear polyethylenimine-plasmid DNA ratio conducted to a significant time-dependent reduction in hair cell viability Oto-nanotoxicity of the tested material started to manifest immediately after the addition of the poly-plex, especially outer hair cells were noted to be more vulnerable in the acute phase.	[82,139]
SPIONs	Size: 100 and 500 nm	In vitro	EC5V cells derived from the inner ear ampulla of semicircular canals.	The cells were treated with SPIONs at final concentrations, depending on size: 100 nm—3 × 1010, 3 × 109, 3 × 108 NP/mL 500 nm—7 × 107, 7 × 106 NP/mL.	A lower number of surviving cells were reported in the 100 nm treated group than in the 500 nm and control groupsApoptotic cells were more frequently observed in the 100 nm group than in the 500 nm and control groups.	[82,140]
SPIONs	Size: 200 nm	In vivo	Inner ear cells of albino male guinea pigs	In each animal, on one ear, a 0.4 mm scala tympani cochleostomy, 1.5 mm under the round window ridge was performed through a posterior approach and bullostomy and 1 μL of saline serum was injected. In the other ear, a bolus of 1 μL of nanoparticles was performed using the same method.	At day 7, hearing threshold shift showed no difference between saline-treated ears and nanoparticles treated ears.	[140]
AuNPs	Size: 50 nm Shape: spherical	In vitro	HEI-OC1 cell line	The cells were treated with nanoparticles at 0–100 μM for up to 6 days	There were not reported any significant changes in cell viability.	[82,96]
AuNPs	Size: 50 nm Shape: spherical	In vivo	Mouse cochlear cells	Gold nanoparticles were applied in vivo to mouse cochleae	The injected nanoparticles fully diffused throughout the inner ear and were successfully localized within the cells. The presence of nanoparticles had no observable effect on the morphology of the hair cells. Gold nanoparticles do not enhance X-ray attenuation in a significant manner. Hence they are not considered suitable computed tomography imaging contrast agents for the inner ear.	[96]
Methoxy poly (ethylene glycol)-polylactic acid nanoparticles loaded with dexamethasone	Size: 130 nm Shape: spherical	In vivo	Inner ear cells of male guinea pigs	The treatment was administered intraperitoneally at a dose of 10 mg/kg and at a concentration of 10 mg/mL, 1 h before cisplatin injection. Three days after treatment, the animals were euthanized, and their tissues were prepared for the examination.	The auditory brainstem response threshold was not significantly changed, indicating nanoparticles’ nontoxicity. A single injection of nanoparticles was reported to provide significant functional and histological protection of the cochlea from the cisplatin, which was similar to the effect of repeated injection of the free drug for 3 days.	[144]
Unmodified PLGA-nanoparticles, surface modified with poloxamer 407, chitosan, or methoxy poly(ethylene glycol)	Size: 100–200 nm Relatively uniform size distribution	In vitro	HEI-OC1 cell line	The cells were treated with nanoparticles at concentrations varying between 0 and 80 mg/mL for 24 h	IC50 values: (1) unmodified NPs—71.30 ± 4.16 mg/mL (2) P407-modified NPs—60.53 ± 0.55 mg/mL (3) Chitosan-modified NPs—65.39 ± 0.47 mg/mL (4) mPEG-modified NPs—81.70 ± 1.04 mg/mL Uptake efficiencies: (1) unmodified NPs—79.7% (2) P407-modified NPs—91.4% (3) Chitosan-modified NPs—58.3% (4) mPEG-modified NPs—48.1%	[82,142]
Unmodified PLGA-nanoparticles, surface modified with poloxamer 407, chitosan, or methoxypoly (ethylene glycol)	Size: 100–200 nm Relatively uniform size distribution	In vivo	Inner ear cells of albino guinea pigs	The four types of nanoparticles were injected at a concentration of 25 mg/mL into the unilateral tympanic cavity of the guinea pigs. They were examined 24 h after administration	No inflammation was detected in the inner ear Several tissues, such as stria vascularis, spiral ligament, organ of Corti, and spiral ganglion cells, barely underwent morphological alterations after nanoparticles administration The hydrophilic coating of PLGA nanoparticles played an important role in inner ear transport, particularly the P407 modification The surface-modified nanoparticles were considerably localized in the spiral ligament, stria vascularis, organ of Corti, and spiral ganglion cells, while the unmodified particles were distributed marginally	[142]
Chitosan nanoparticles	Average size: 152.7 nm Polydispersity index: 0.135 Shape: spherical	In vitro	HEI-OC1 cell line	The cells were treated with nanoparticles at concentrations varying between 0 and 2.5 mg/mL for 24 h	There was not reported any significant change in cell viability Nanoparticles were internalized in cells with an 89.1% uptake efficiency	[82,130]
Chitosan nanoparticles	Average size: 152.7 nm Polydispersity index: 0.135 Shape: spherical	In vivo	Inner ear cells of guinea pigs	The nanoparticles were injected at a concentration of 2.5 mg/mL into the unilateral tympanic cavity of the guinea pigs. The animals were decapitated 1 h after the treatment and examined after 24 h	The number of surviving hair cells hardly decreased, indicating the safety of the tested nanoparticles. The chitosan nanoparticles were successfully delivered into the vestibule, accumulating significantly more in the vestibular system than in the cochlear tissues The nanoparticle uptake in saccular supporting cells was much higher compared to cochlear hair cells of middle and apical turns	[130]
Lipid nanoparticles-encapsulated brain-derived neurotrophic factor (BDNF) mRNA	Lipid composition: SS-cleavable and pH-activated lipid-like material:dioleolyphosphatidyl ethanolamine (DOPE):cholesterol= 3:3:4	In vivo	Inner ear cells of Hartley guinea pigs	The animals were intramuscularly injected with gentamicin, promptly followed by intravenous injection of ethacrynic acid. On day 1, for early therapy, or day 14, for late therapy, 5 μl of lipid nanoparticles loaded with 0.1 mg/mL BDNF -enhanced green fluorescent protein mRNA was administered	On day 1 after gentamicin exposure, the auditory thresholds of the group administered with nanoparticles significantly improved compared to the sham control group. The auditory thresholds did not differ significantly between the sham control group and animals administered with nanoparticles 14 days after gentamicin exposure. The outer hair cells in the cochlea of the group treated 1 day after gentamicin exposure were significantly decreased compared with those in the control group, while inner hair cells counts had no significant differences in all turns among all groups.	[145]
Lithium niobate NPs	Size range: 200–600 nm Average size: 392.25 nm Polydispersity index: 0.517	In vitro	OC-k3 cell line	(1) Cytotoxicity of nanoparticles was investigated using the MTS assay. The OC-k3 cells were seeded in 96-well plates at the concentration of 7000 cells/well in 100 μL of medium and left to adhere for 24 h at ambient temperature after which were treated with the compound resuspended in a complete medium at three different concentrations: 0.85, 15, and 74 ng/mL. Vitality was analyzed 24, 48, and 72 h after treatment. (2) Similarly, a morphological test was performed by seeding and culturing the cells on a round glass slide.	The tested nanoparticles induced a significant increase in cell viability after 72 h of incubation at the concentrations of 0.0085 and 0.015 μg/mL. The morphological test proved a good state of cellular health. No cell morphology alterations were noticed at any of the tested doses and time points.	[1]