Céline Jaudoin
email: celine.jaudoin@universite-paris-saclay.fr

Florence Agnely
email: florence.agnely@universite-paris-saclay.fr

Yann Nguyen
email: yann.nguyen@inserm.fr

Evelyne Ferrary
email: evelyne.ferrary@inserm.fr

Amélie Bochot
email: amelie.bochot@universite-paris-saclay.fr

Pr Amélie Bochot

Nanocarriers for drug delivery to the inner ear: physicochemical key parameters, biodistribution, safety and efficacy

Keywords: Cochlea, hydrogels, intracochlear administration, intratympanic administration, nanoparticulate systems, round window membrane, targeting Abbreviations Cmax, maximum drug concentration achieved in perilymph, cryoTEM, transmission electron cryomicroscopy, ELISA, enzyme-linked immunosorbent assay, FDA, Food and Drug Administration, FITC, fluorescein isothiocyanate, GFP, green fluorescent protein, GJB2, gap junction protein beta 2, LC-MS, liquid chromatography-mass spectrometry, PdI, polydispersity index, PEG, poly(ethylene glycol), PLA, polylactic acid, PLGA, poly(lactic-co-glycolic acid), RT-PCR, reverse membrane, siRNA, small interfering RNA, SPION, superparamagnetic iron oxide nanoparticles, TEM, transmission electron microscopy, TRITC, tetramethylrhodamine, TTI, transtympanic injection

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

More than 5% of the world's population has disabling hearing loss, and this may double by 2050 (World Health Organization, 2019). Hearing loss can be a consequence of several factors: noise exposure, aging, ototoxicity of drugs, autoimmune response or genetic impairment [START_REF] Smouha | Inner ear disorders[END_REF].

As auditory sensory cells do not regenerate [START_REF] Schilder | Hearing Protection, Restoration, and Regeneration: An Overview of Emerging Therapeutics for Inner Ear and Central Hearing Disorders[END_REF]; hearing is restored by conventional hearing aids for mild and moderate hearing loss or cochlear implants for severe to profound sensorineural hearing loss [START_REF] Roche | On the Horizon: Cochlear Implant Technology[END_REF]. Besides deafness or tinnitus, additional vestibular disorders (balance dysfunctions) may occur. Despite the high incidence of these diseases and their impact on quality-of-life, there is still no dedicated medication on the market and some drugs are used off-label.

The inner ear is a very isolated organ located in the temporal bone and protected by many physiological barriers [START_REF] Nyberg | Delivery of therapeutics to the inner ear: The challenge of the blood-labyrinth barrier[END_REF]. Over the last 30 years, the local administration of drugs has been developed to maximize drug diffusion into the inner ear [START_REF] Plontke | Local drug delivery to the inner ear: Principles, practice, and future challenges[END_REF].

Intratympanic administration by injection of the drug inside the middle ear cavity is a safe and common route of administration used in the clinic [START_REF] Lechner | Intratympanic Steroid Use for Sudden Sensorineural Hearing Loss: Current Otolaryngology Practice[END_REF]. Currently, anti-inflammatory drugs are being evaluated on a wide range of inner ear diseases in clinical trials using intratympanic administration [START_REF] Bento | Residual Hearing Preservation with the Evo® Cochlear Implant Electrode Array: Preliminary Results[END_REF][START_REF] Marshak | Prevention of cisplatin-induced hearing loss by intratympanic dexamethasone: A randomized controlled study[END_REF][START_REF] Patel | Intratympanic corticosteroids in Ménière's disease: A mini-review[END_REF][START_REF] Maria | Hearing Preservation Surgery for Cochlear Implantation-Hearing and Quality of Life After 2 Years[END_REF]. Results are partially mitigated because of the lack of efficient conventional dosage forms for the inner ear. Indeed, solutions and suspensions are rapidly eliminated by the Eustachian tube reducing the drug half-time in the cochlea. Repeated injections are then required, decreasing patient compliance.

Currently, drugs from different therapeutic classes have emerged in clinical trials: antioxidants (Ebselen, Sound Pharmaceuticals), anti-inflammatory (Otividex ® , Otonomy, Inc.), or anti-apoptotic drugs (D-c-Jun kinase inhibitor-1, Auris Medical) [START_REF] Schilder | Hearing Protection, Restoration, and Regeneration: An Overview of Emerging Therapeutics for Inner Ear and Central Hearing Disorders[END_REF]. To give them a chance of success, there is an urgent need for efficient drug delivery systems for the inner ear (El Kechai et al., 2015a;[START_REF] Mäder | Controlled drug release to the inner ear: Concepts, materials, mechanisms, and performance[END_REF].

Nanocarriers are nanoscale drug delivery systems (<1 µm) with tunable surface and physicochemical properties. Due to their numerous advantages, over the past 15 years, they have generated considerable interest for drug delivery to the inner ear [START_REF] Mäder | Controlled drug release to the inner ear: Concepts, materials, mechanisms, and performance[END_REF][START_REF] Mittal | Nanoparticle-based drug delivery in the inner ear: current challenges, limitations and opportunities[END_REF]. Nanocarriers may compensate drug properties such as low solubility, degradation, short halflife and low passage across physiological barriers. They may also offer the possibility to release the drug in a sustained manner and to address it to specific tissues [START_REF] Agrahari | Inner ear targeted drug delivery: what does the future hold?[END_REF]. Several reviews have described the success of nanocarriers to treat or prevent inner ear diseases such as noise-induced hearing loss or drug ototoxicity [START_REF] Kim | Nanomedicine for Inner Ear Diseases: A Review of Recent in Vivo Studies[END_REF][START_REF] Li | Advances in nano-based inner ear delivery systems for the treatment of sensorineural hearing loss[END_REF][START_REF] Mittal | Nanoparticle-based drug delivery in the inner ear: current challenges, limitations and opportunities[END_REF][START_REF] Pyykkö | An overview of nanoparticle based delivery for treatment of inner ear disorders[END_REF].

However, the critical physicochemical characteristics influencing the biodistribution of nanocarriers in the inner ear are not well defined. Drug pharmacokinetics after nanocarrier administration have not been thoroughly compared among nanocarriers and with other formulations such as liquid forms or hydrogels.

This review focuses on nanocarrier characteristics leading to their improved efficacy against inner ear diseases. First, a brief overview of ear anatomy is presented, as well as the advantages of intratympanic administration. Second, the nanocarrier biodistribution, drug pharmacokinetics and key characteristics to deliver drugs from nanocarriers in the inner ear are presented. Then, we give an overview of the present and future advanced strategies to improve nanocarrier entrance into the inner ear. Finally, the safety and therapeutic efficacy of nanocarriers used in different inner ear disorders are discussed.

Anatomy and physiology of hearing and balance

The ear is anatomically divided into three parts: the outer, middle, and inner ear (Fig. 1A). The outer ear is represented by the auricle (visible part of the ear) and the external auditory canal, which is closed by the tympanic membrane. The role of the outer ear is to channel sound waves into the auditory canal to induce vibrations of the tympanic membrane [START_REF] Hayes | Anatomy and physiology of the external, middle and inner ear[END_REF]. This membrane is around 0.6 mm in thickness in humans and consists of three layers: an outer cutaneous layer, a core of connective tissue and an inner layer of mucus [START_REF] Hentzer | Ultrastructure of the human tympanic membrane[END_REF]. Thus, it isolates the air-filled cavity of the middle ear from the environment. The middle ear contains the ossicular chain -the malleus, incus, and stapes -that conducts sound waves from the tympanic membrane to the inner ear. The Eustachian tube ends in the nasopharynx. It maintains equal air pressure on both sides of the tympanic membrane while swallowing or yawning. The inner ear, also named the labyrinth, lies deep within the petrous portion of the temporal bone, the hardest bone in the human body (Fig. 1A).

The inner ear consists of two entangled organs: the vestibular apparatus, which is the organ of balance, and the cochlea, the organ of hearing (Fig. 1A). The vestibular apparatus contains three semicircular canals oriented in three different spatial directions, and two membranous sacs, the utricle and saccule, responding to gravitational forces [START_REF] Mazzoni | The vascular anatomy of the vestibular labyrinth in man[END_REF]. In the utricle and saccule, small calcite crystals called otoliths increase local shearing forces in response to slight displacements of the head, then stimulating the hair cells located underneath. The semi-circular canals hold the ampulla crest located in each canal and which is composed of the cupula (cap gel) and hair cells.

The cochlea is a long tube coiled around the modiolus comprising the cochlear nerve fibers (8 th pair of cranial nerves) [START_REF] Sakamoto | Anatomy of the inner ear[END_REF]. The spiral tube contains three internal compartments: the scala media, which is separated from the scala tympani by the basilar membrane and from scala vestibuli by Reissner's membrane (Fig. 1B). The scala tympani ends on the round window membrane, which separates the inner ear from the middle ear. The scala vestibuli ends on the oval window membrane, on which the stapes rests. Resting on the basilar membrane, the sensory inner hair cells of the organ of Corti respond to the wave stimuli and generate an action potential on nervous fibers, whereas the outer hair cells amplify the signal (Fig. 1B) [START_REF] Corey | Mechanical Transduction Processes in the Hair Cell[END_REF]. The bioelectrical signals are transmitted to the spiral ganglion neurons located in the bony spiral canal, and then conveyed to the brain, where they are interpreted as sounds. High frequency waves stimulate the basal part of the cochlea whereas low frequencies stimulate the apex [START_REF] Sakamoto | Anatomy of the inner ear[END_REF].

Within the inner ear, two separate fluid-filled compartments are present, one inside the other: the scala media, filled with endolymph (~8 µL in guinea pigs or rats), contained within the bony labyrinth filled with perilymph (~70 µL) (Fig. 1B) (El Kechai et al., 2015a). These fluids are totally different in composition: the endolymph, a high K + fluid, bathes the apical ciliated part of the sensory cells, whereas the perilymph, a high Na + fluid, bathes their basolateral synaptic part [START_REF] Wangemann | Ion and Fluid Homeostasis in the Cochlea[END_REF].

Routes of administration for inner ear drug delivery

Several strategies of administration exist to deliver drugs to the inner ear: systemic access (oral or 135 intravenous) and local administration (intracochlear or intratympanic). These routes are described in this section, with the different pharmaceutical forms used. Their advantages and limitations are detailed in Table 1.

Intravenous and oral administrations

In addition to common physiological barriers such as the hepatic passage or gut, drugs administered by intravenous and oral administration must cross the blood-labyrinth barrier to reach the inner ear. This barrier lies between the vasculature and inner ear fluids (perilymph and endolymph) [START_REF] Nyberg | Delivery of therapeutics to the inner ear: The challenge of the blood-labyrinth barrier[END_REF] and dramatically restricts drug access to the inner ear. In the cochlea, the blood-labyrinth barrier is characterized by a continuous capillary endothelium with tight junctions [START_REF] Jahnke | The fine structure of freeze-fractured intercellular junctions in the Guinea pig inner ear[END_REF][START_REF] Juhn | Nature of blood-labyrinth barrier in experimental conditions[END_REF]. The blood-endolymph barrier, localized within the stria vascularis, is even more complex: the tight junctions of the strial endothelium separate the lumen of the capillaries from the strial interstitial fluid, and a second epithelium separates the interstitial fluid from the endolymph compartment [START_REF] Shi | Pathophysiology of the cochlear intrastrial fluid-blood barrier (review)[END_REF]. Furthermore, exchanges between endolymph and perilymph are also restricted by the labyrinthine barrier. The presence of these barriers explains the limitations observed for drugs administered by these routes (Table 1).

Lipophilic and low molecular weight drugs are more susceptible to cross the blood-labyrinth barrier but the percentage of drug passage from the bloodstream is very low (around 0.000005% for methylprednisolone [START_REF] Bird | Intratympanic Versus Intravenous Delivery of Methylprednisolone to Cochlear Perilymph[END_REF]). For some drugs, the blood-labyrinth barrier is even more selective than the blood-brain barrier. However, several conditions can influence the passage of drugs, including inflammation [START_REF] Hirose | Systemic lipopolysaccharide compromises the blood-labyrinth barrier and increases entry of serum fluorescein into the perilymph[END_REF][START_REF] Zhang | Lipopolysaccharide-induced middle ear inflammation disrupts the cochlear intra-strial fluid-blood barrier through down-regulation of tight junction proteins[END_REF], diuretics [START_REF] Liu | Ototoxic destruction by coadministration of kanamycin and ethacrynic acid in rats[END_REF], osmotic agents [START_REF] Le | Mannitol and the blood-labyrinth barrier[END_REF], elevated blood pressure [START_REF] Inamura | Permeability changes of the blood-labyrinth barrier measured in vivo during experimental treatments[END_REF], and noise exposure in guinea pigs [START_REF] Suzuki | Effect of noise exposure on blood-labyrinth barrier in guinea pigs[END_REF] but not in rats [START_REF] Laurell | Intact blood-perilymph barrier in the rat after impulse noise trauma[END_REF].

Intracochlear administration

Intracochlear administration consists of the direct administration of the drug inside the cochlea (Table 1). Thus, there is no physiological barrier for the drug to access the inner ear. A small volume (a few microliters) of drug solution [START_REF] Braun | Protection of inner ear function after cochlear implantation: compound action potential measurements after local application of glucocorticoids in the guinea pig cochlea[END_REF]), suspension (Paasche et al., 2006) or gel [START_REF] De Ceulaer | Long-term evaluation of the effect of intracochlear steroid deposition on electrode impedance in cochlear implant patients[END_REF], is slowly injected with a fine needle inside the cochlea through the round window membrane or by a cochleostomy. In the particular case of cochlear implantation, used to restore the hearing function in the case of severe to profound sensorineural hearing loss, drugs can be included within the coating [START_REF] Richardson | Polypyrrole-coated electrodes for the delivery of charge and neurotrophins to cochlear neurons[END_REF] or the silicone matrix of the electrode array [START_REF] Douchement | Dexamethasone eluting electrodes for cochlear implantation: Effect on residual hearing[END_REF]. Once placed in the scala tympani, the electrode array releases the drug in a sustained manner over several years. Unlike the simple intracochlear injection, this method is not used in humans at present. However, in the case of a drug-loaded electrode array, a long-term release in the perilymph would be obtained, which is not possible with intracochlear injection. Nevertheless, both techniques are extremely invasive and need a surgical approach under general anesthesia (El Kechai et al., 2015a). Because of its limitations (Table 1), intracochlear administration is not the most commonly used method of administration in clinical practice.

Intratympanic administration

The intratympanic route is the administration of a drug in the middle ear, and which must then dlipo This route offers many advantages over intracochlear administration (Table 1). The main technique used in clinical practice is transtympanic injection. The solution, suspension or hydrogel, is injected with a fine needle (~25 G) through the tympanic membrane, and fills the middle ear cavity [START_REF] Liu | Assessment of complications due to intratympanic injections[END_REF]. Then, the patient lies on the other ear for 15 to 30 minutes to maximize contact of the drug formulation with the round window membrane. Another route of intratympanic administration is the deposition of the solution only on the round window niche, using a sponge (Gelfoam ® ) or hydrogel to attain increased residence time in the middle ear. The application of Gelfoam ® is not used by physicians to any great extent because it requires surgery to access the middle ear [START_REF] Enticott | Methylprednisolone applied directly to the round window reduces dizziness after cochlear implantation: A randomized clinical trial[END_REF]. It has also been proposed to insert a wick through the tympanic membrane and place it in the round window region. The wick can be reloaded from ear drops administered to the external auditory canal [START_REF] Silverstein | Silverstein MicroWick[END_REF].

Corticoids are mainly used in clinical practice (off-label) by transtympanic injection to treat sudden hearing loss [START_REF] Lechner | Intratympanic Steroid Use for Sudden Sensorineural Hearing Loss: Current Otolaryngology Practice[END_REF]), Ménière's disease [START_REF] Weckel | Control of vertigo in Ménière's disease by intratympanic dexamethasone[END_REF] or to preserve hearing during cochlear implantation [START_REF] Kuthubutheen | Preoperative steroids for hearing preservation cochlear implantation: A review[END_REF]. New therapeutics such as D-c-Jun kinase inhibitor-1 (AM-111, Auris Medical), peroxisome proliferator-activated receptor-γ agonist (STR001, Strekin AG), progenitor cell activator (FX-322, Frequency Therapeutics), and γ-secretase inhibitor (LY3056480, Audion Therapeutics) are currently in clinical trials for the treatment of sensorineural hearing loss [START_REF] Schilder | Hearing Protection, Restoration, and Regeneration: An Overview of Emerging Therapeutics for Inner Ear and Central Hearing Disorders[END_REF]. Otonomy is developing a formulation containing gacyclidine (OTO-313) for the treatment of subjective tinnitus (phantom sounds only heard by the patient), and Synphora is testing an agonist of prostaglandin receptor, latanoprost (Xalatan ® ) for Ménière's disease (a disease associating vertigo, tinnitus, and low frequency hearing loss, with crisis evolution, linked to endolymphatic hydrops).

To reach the inner ear fluids, the drug must diffuse from the middle ear cavity through the round window and the oval window membranes, but also through areas of the otic capsule where the bone is thin in some animals, such as at the apex of the cochlea [START_REF] Mikulec | Entry of substances into perilymph through the bone of the otic capsule after intratympanic applications in guinea pigs: implications for local drug delivery in humans[END_REF]. Pharmacokinetics studies cannot be performed in humans. Perilymph sampling requires a highly invasive surgery under general anesthesia. The volume and pressure changes induced by the sampling of perilymph through the round window membrane can damage the fragile sensory epithelium of the cochlea, and thus induce non reversible profound hearing loss. However, in animals, it is possible to sample the perilymph to quantify the drug. The cochlea can also be collected, fixed and stained to assess the presence of drug. Knowing the dose administered in the middle ear, the amount of passage of the drug through the main local barriers (round and oval windows) can be quantified. The duration of drug release to the inner ear ranges from a few hours to a few days. of the round window, potential obstruction of the round window with false membranes) -Clearance of liquid formulations through the Eustachian tube -Risk of introducing pathogens in the middle ear -Risk of tympanic membrane perforation too large to heal

Round window membrane

The major barrier between the middle ear and the inner ear was long assumed to be the round window membrane [START_REF] Goycoolea | Clinical aspects of round window membrane permeability under normal and pathological conditions[END_REF]. It is located at the base of the cochlea, with regard to the scala tympani (Fig. 1A). The membrane consists of three layers: the outer epithelium facing the middle ear and comprising a single layer of cuboidal cells with microvilli. These cells are interconnected by tight junctions at the outer surface. The inner epithelium, bathing the perilymph, consists of squamous cells with large extracellular spaces (Goycoolea et al., 1988b;[START_REF] Goycoolea | Clinical aspects of round window membrane permeability under normal and pathological conditions[END_REF]. These two layers are separated by a core of connective tissue made of collagen and elastic fibers that includes fibroblasts, lymphatic and blood vessels. When the middle ear is filled with aqueous content, the round window behaves like a semi-permeable membrane. Its permeability depends on several factors: duration of exposure, drug concentration, molar mass, liposolubility, electrical charge, thickness of the membrane and factors influencing its permeability [START_REF] Goycoolea | Round window membrane. Structure function and permeability: A review[END_REF] and using additives in the formulation composition such as benzyl alcohol [START_REF] Mikulec | Permeability of the round window membrane is influenced by the composition of applied drug solutions and by common surgical procedures[END_REF].

-Electrical charge: cationic ferritin crosses easily through the outer layer of the membrane, carried by pinocytotic vesicles (Goycoolea et al., 1988a), whereas anionic ferritin is not able to pass through in rodents and cats [START_REF] Goycoolea | Clinical aspects of round window membrane permeability under normal and pathological conditions[END_REF][START_REF] Nomura | Otological significance of the round window[END_REF].

-Lipophilic drugs are more likely to diffuse passively through the round window membrane compared to hydrophilic ones [START_REF] Wang | Principles of inner ear sustained release following intratympanic administration[END_REF].

-Drug molar mass: low-molecular weight substances diffuse by paracellular pathways in the first layer of the round window membrane. Large substances follow specific transcellular pathways [START_REF] Goycoolea | Clinical aspects of round window membrane permeability under normal and pathological conditions[END_REF]. If such a specific pathway does not exist, the substance cannot reach the perilymph, as observed with albumin (70 kDa). Specific pathways can include receptor-mediated endocytosis, phagocytosis or channels between cells such as for latex spheres (Goycoolea et al., 1988b).

-The average thickness of the round window is 70 µm in humans [START_REF] Goycoolea | Round window membrane. Structure function and permeability: A review[END_REF] versus 10 to 14 µm in rodents (Goycoolea et al., 1988b). Thus, the barrier is more difficult to cross in humans than in rodents. However, the surface of the membrane is larger in humans (2.3 mm²; [START_REF] Okuno | Anatomy of the round window: A histopathological study with a graphic reconstruction method[END_REF] than in guinea pigs (1.2 mm²; [START_REF] Ghiz | Quantitative anatomy of the round window and cochlear aqueduct in guinea pigs[END_REF].

-In the presence of inflammation, an increase in membrane permeability is observed in the early stages, but then the membrane becomes thicker and permeability decreases [START_REF] Engmér | Immunodefense of the Round Window[END_REF].

-The entry of drugs into the human inner ear after intratympanic application may also be impeded by additional membranes or mucus [START_REF] Engmér | Immunodefense of the Round Window[END_REF]. Obstruction of the round window membrane with false membranes occurs in 33% of cases in humans [START_REF] Alzamil | Extraneous round window membranes and plugs: Possible effect on intratympanic therapy[END_REF].

Oval window membrane

Recently, the oval window has been demonstrated to be a substantial route of access for the inner ear. The amount of gadolinium-DOTA (~560 Da) able to enter the oval window in rats is 90% (Zou et al., 2012a). Amounts of drugs able to pass through the oval window are estimated at 35% against 65%

for the round window membrane in guinea pigs, for both trimethylphenylammonium (~140 Da; [START_REF] Salt | Marker entry into vestibular perilymph via the stapes following applications to the round window niche of guinea pigs[END_REF] and gentamicin (~480 Da; [START_REF] Salt | Perilymph pharmacokinetics of locally-applied gentamicin in the guinea pig[END_REF].

Oval window is located at the beginning of scala vestibuli and is partly obstructed by the stapes, the base of which rests against the window (Fig. 1A). The stapes footplate is attached to the oval window by the annular ligament. This articulation is called the stapediovestibular joint. This joint is like every articulating surface, composed of a hyaline cartilage and a fluidic articular cavity, sealed by epithelial cells with tight junctions [START_REF] Ohashi | Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid[END_REF][START_REF] Ohashi | Three-dimensional regular arrangement of the annular ligament of the rat stapediovestibular joint[END_REF]. The annular ligament has a porous structure, composed of a network of fibrillin, collagen and MAGP-36 (36 kDa microfibril-associated glycoprotein), the pores of which are filled with hyaluronic acid [START_REF] Ohashi | Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid[END_REF]. Drugs with small molecular dimensions can diffuse through the annular ligament, which provides direct access to the perilymph, or even through the different layers of the oval window. The annular ligament thickness is variable in humans (0.26 to 0.64 mm) [START_REF] Mohammadi | Micro-CT analysis of the anatomical characteristics of the stapedial annular ligament[END_REF] whereas it is fivefold thinner in rodents [START_REF] Ohashi | Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid[END_REF]. Despite its possible advantages, the oval window pathway is rarely used.

Distribution and metabolism

The flow of inner ear fluids is very low, between 1.6 nL/min [START_REF] Salt | Perilymph Kinetics of FITC-Dextran Reveals Homeostasis Dominated by the Cochlear Aqueduct and Cerebrospinal Fluid[END_REF] and 30 nL/min [START_REF] Ohyama | Volume flow rate of perilymph in the guinea-pig cochlea[END_REF] in guinea pigs (unknown in humans). Thus, the distribution of drugs, once in the perilymph, is mainly governed by passive diffusion, establishing a gradient from base to apex [START_REF] Salt | Principles of local drug delivery to the inner ear[END_REF]. Drugs present in the scala tympani are distributed quickly into the spiral ligament, scala vestibuli and the vestibule. Therefore, the presence of the active substance in the vestibule does not necessarily indicate a passage through the oval window. The drug can also diffuse through the large pores of the osseous spiral lamina to spread into the modiolus [START_REF] Rask-Andersen | Perilymph/modiolar communication routes in the human cochlea[END_REF]. In humans, the modiolar wall of the scala vestibuli and tympani is porous, composed of a web of connective tissue within the perilymph, forming a perilymphatic route to the modiolar space [START_REF] Salt | Pharmacokinetic principles in the inner ear: Influence of drug properties on intratympanic applications[END_REF]. Diffusion to the endolymph is thought to depend on the charge of the molecule, because cationic markers, such as gadolinium-DOTA (Zou et al., 2012a), are excluded from the endolymph as Reissner's membrane is positively charged (+ 80 mV) [START_REF] Nyberg | Delivery of therapeutics to the inner ear: The challenge of the blood-labyrinth barrier[END_REF].

Metabolism is sometimes crucial for drug efficacy (prodrugs) but can also lead to its rapid degradation. The perilymph contains proteins (2 mg/mL) and enzymes such as lactate dehydrogenase [START_REF] Scheibe | Biochemical differences between perilymph, cerebrospinal fluid and blood plasma in the guinea pig[END_REF], aminotransferases [START_REF] Lysaght | Proteome of human perilymph[END_REF] and phosphodiesterases [START_REF] Swan | Proteomics analysis of perilymph and cerebrospinal fluid in mouse[END_REF]. For instance, dexamethasone phosphate [START_REF] Hargunani | Intratympanic Injection of Dexamethasone: Time Course of Inner Ear Distribution and Conversion to Its Active Form[END_REF] and triamcinolone acetonide [START_REF] Salt | Pharmacokinetic principles in the inner ear: Influence of drug properties on intratympanic applications[END_REF] are metabolized within the ear into their biologically active moieties by phosphodiesterases, but when administered into the middle ear, just a fraction of the total amount of drug is transformed [START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF][START_REF] Salt | Pharmacokinetic principles in the inner ear: Influence of drug properties on intratympanic applications[END_REF].

Elimination

Elimination of drugs starts in the middle ear: the Eustachian tube eliminates liquid forms in less than 30 minutes in humans [START_REF] Plontke | intratympanic versus intravenous delivery of methylprednisolone to cochlear perilymph[END_REF]. In the inner ear, elimination is performed by two main pathways: the vascular system [START_REF] Salt | Principles of local drug delivery to the inner ear[END_REF] and the cochlear aqueduct [START_REF] Salt | Pharmacokinetic principles in the inner ear: Influence of drug properties on intratympanic applications[END_REF]. As the vasculature of the inner ear is not directly in contact with the scalae, but is contained within the bony canals, direct elimination of drugs from the perilymph by blood vessels is unlikely [START_REF] Salt | Principles of local drug delivery to the inner ear[END_REF]. However, it may occur after drug diffusion into the spiral ganglion, the organ of Corti or the lateral wall, for which fluid pathways exist from the perilymph to the blood vessels.

The cochlear aqueduct is a bony channel from the end of the cranium, connected to the scala tympani just next to the round window [START_REF] Gopen | Anatomy of the normal human cochlear aqueduct with functional implications[END_REF][START_REF] Salt | Communication pathways to and from the inner ear and their contributions to drug delivery[END_REF] (Fig. 1A). In rodents, this aqueduct allows fluid efflux (0.5-1 µL/min in guinea pigs) [START_REF] Salt | The effect of cerebrospinal fluid pressure on perilymphatic flow in the opened cochlea[END_REF], thus promoting drug diffusion into the cerebrospinal fluid [START_REF] Salt | Communication pathways to and from the inner ear and their contributions to drug delivery[END_REF]. Drugs can even be found in the brain after intratympanic administration and then in the contralateral ear [START_REF] Chen | Preliminary study on brain-targeted drug delivery via inner ear[END_REF][START_REF] Zhang | Novel multiple agents loaded PLGA nanoparticles for brain delivery via inner ear administration: In vitro and in vivo evaluation[END_REF]. The cochlear aqueduct in humans is longer, and exchanges between the perilymph and cerebrospinal fluid are more restricted, but the duct is often permeable [START_REF] Gopen | Anatomy of the normal human cochlear aqueduct with functional implications[END_REF]. The potential elimination by this pathway in humans remains uncertain.

Characteristics of formulations for transtympanic injection

Formulations injected into the middle ear must be sterile, nonpyrogenic, with an osmolarity around 300 mOsm/L to avoid perilymph leakage, without preservatives and with a physiological pH (7.38-7.42) (Ph. Eur. 10.2, 2020a, 2020b).

Solutions and suspensions are easily injectable into the middle ear, but are also quickly eliminated by the Eustachian tube, thus repeated injections are needed. For this purpose, hydrogels increasingly being developed to prolong the residence time of the drug in the middle ear (El Kechai et al., 2015a;[START_REF] Mäder | Controlled drug release to the inner ear: Concepts, materials, mechanisms, and performance[END_REF]. Shear-thinning hydrogels, based on hyaluronic acid, are attractive because their viscosity decreases under shear during injection and they are rapidly recovered once injected due to their non-thixotropic behavior.

Nanocarriers used in inner ear application

Drug delivery systems designed for intratympanic administration should meet the following specifications to be efficient [START_REF] Mäder | Controlled drug release to the inner ear: Concepts, materials, mechanisms, and performance[END_REF]:

-Protect poorly sensitive drugs;

-Load a sufficient amount of drug to be efficient;

-Avoid rapid clearance by the Eustachian tube;

-Enable close contact with the round window membrane;

-Ensure effective transport of the drug through the round window membrane;

-Achieve a therapeutic dose in the inner ear; -Target specific cells; -Be safe.

Recently, approaches using nanocarriers have been proposed to overcome these hurdles.

Nanocarriers are characterized by a diameter of less than 1 µm. They differ greatly with respect to the materials used (e.g. polymeric, lipid, inorganic), their size, surface charge, shape and biodegradability.

In the pharmaceutical field, nanocarriers provide many advantages in fighting cancer, pain management and antibiotic therapy [START_REF] Gonçalves | A glance over doxorubicin basednanotherapeutics: From proof-of-concept studies to solutions in the market[END_REF][START_REF] Uchegbu | Nanomedicines and nanodiagnostics come of age[END_REF]. Indeed, due to their numerous advantages, they can compensate for disadvantageous drug properties such as low solubility, degradation, and short half-life. They can also sustain the release and provide high surface exchange. Thus, nanocarriers for drug delivery to the inner ear have generated great interest over the last 15 years (El Kechai et al., 2015a;[START_REF] Mäder | Controlled drug release to the inner ear: Concepts, materials, mechanisms, and performance[END_REF][START_REF] Staecker | Developments in delivery of medications for inner ear disease[END_REF]. Polymeric nanoparticles, liposomes, polymersomes, cubosomes, lipid-based nanoparticles, dendrimer-based nanoparticles and superparamagnetic nanoparticles have been evaluated in preclinical studies for inner ear applications to administer small or macromolecules (Fig. 2). Polymeric nanoparticles are mainly based on poly(lactic-co-glycolic acid) (PLGA), a biocompatible and biodegradable polymer approved by the Food and Drug Administration (FDA).

Self-assembled polymeric nanoparticles obtained using drug-conjugated polymers are only tested in preclinical studies. They are based on copolymeric systems of N-vinylpyrrolidone and methacrylic derivatives conjugated to antioxidants (α-tocopherol, α-tocopheryl succinate) or anti-inflammatory drugs (ibuprofen) [START_REF] Palao-Suay | Anticancer and antiangiogenic activity of surfactant-free nanoparticles based on self-assembled polymeric derivatives of vitamin E: Structure-activity relationship[END_REF]. These different types of polymeric nanoparticle allow the encapsulation of hydrophilic or lipophilic drugs in their polymeric matrix for inner ear delivery (see sections 5.1.3 and 7).

Liposomes are biocompatible vesicle-like lipidic nanocarriers, already on the market for other applications [START_REF] Crommelin | The role of liposomes in clinical nanomedicine development. What now? Now what?[END_REF]. Polymersomes have a similar structure, except that the outer shell is composed of self-assembled amphiphilic block copolymers instead of lipids [START_REF] Chidanguro | Polymersomes: Breaking the Glass Ceiling?[END_REF]. In liposomes and polymersomes, hydrophilic drugs are encapsulated in the inner aqueous core and lipophilic drugs in the phospholipid bilayers or polymeric shell.

Cubosomes are nanocarriers formed from a lipid cubic phase and stabilized by a polymer [START_REF] Barriga | Cubosomes: The Next Generation of Smart Lipid Nanoparticles?[END_REF]. The lipid cubic phase is a single lipid bilayer that forms a continuous porous structure containing aqueous medium. Compared to liposomes, the membrane surface area is more effective, allowing high drug loading of both hydrophilic (e.g. nerve growth factor) and lipophilic drugs.

Lipid-based nanocarriers allow the encapsulation of lipophilic drugs such as edaravone (antioxidant) or dexamethasone (anti-inflammatory drug) at higher drug loading [START_REF] Nicolas | Poly(Alkyl Cyanoacrylate) Nanosystems, in: Intracellular Delivery, Fundamental Biomedical Technologies[END_REF]. Solid lipid nanoparticles (solid core), lipid nanocapsules (liquid core) and nanoemulsions (stabilized oil nanodroplets dispersed in aqueous phase) have been described [START_REF] Gao | Solid lipid nanoparticles loaded with edaravone for inner ear protection after noise exposure[END_REF][START_REF] Mohan | Targeted Amelioration of Cisplatin-Induced Ototoxicity in Guinea Pigs[END_REF][START_REF] Yang | Optimized phospholipidbased nanoparticles for inner ear drug delivery and therapy[END_REF].

Dendriplexes are composed of dendrimers, hyperbranched star-shaped macromolecules that exhibit hydrophobic cavities and a cationic surface. This cationic surface can bind to anionic nucleic acids and the hydrophobic regions to hydrophobic drugs [START_REF] Wu | In vivo delivery of Atoh1 gene to rat cochlea using a dendrimer-based nanocarrier[END_REF].

Chitosan-based nanocarriers are stabilized by weak ionic interactions between chitosan and small anionic molecules [START_REF] Vigani | Hyaluronic acid and chitosan-based nanosystems: a new dressing generation for wound care[END_REF]. They can encapsulate hydrophilic drugs in their gel-like matrix.

Inorganic nanoparticles are also used in biodistribution studies, because their material (maghemite, iron oxide or silver) is easily traceable by microtomography [START_REF] Zou | Micro CT visualization of silver nanoparticles in the middle and inner ear of rat and transportation pathway after transtympanic injection[END_REF] or magnetic resonance imaging (Zou et al., 2010b(Zou et al., , 2017b)). Superparamagnetic iron oxide nanoparticles (SPION) are inorganic nanoparticles of small size (5-15 nm) that can be loaded into larger nanoparticles such as PLGA nanoparticles [START_REF] Kopke | Magnetic Nanoparticles: Inner Ear Targeted Molecule Delivery and Middle Ear Implant[END_REF] or chitosan nanocarriers [START_REF] Ramaswamy | Magnetic Nanoparticle Mediated Steroid Delivery Mitigates Cisplatin Induced Hearing Loss[END_REF]. If an additional magnetic field is applied during the administration of the nanoparticles in the inner ear, the SPION are attracted and cross the round window membrane (see section 5.3). All these nanocarriers have been studied to deliver either anti-inflammatory drugs (dexamethasone phosphate), antioxidants (edaravone) or antiapoptotic drugs (D-c-Jun kinase inhibitor-1) to protect the inner ear from noise exposure [START_REF] Gao | Solid lipid nanoparticles loaded with edaravone for inner ear protection after noise exposure[END_REF][START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF][START_REF] Mamelle | Assessment of the efficacy of a local steroid rescue treatment administered 2 days after a moderate noise-induced trauma in guinea pig[END_REF]. Antiinflammatory drugs (dexamethasone, methylprednisolone) and antioxidants (N-acetylcysteine, tocopheryl succinate) have also been tested to protect the inner ear against anticancer drug ototoxicity ( [START_REF] Martín-Saldaña | pH-sensitive polymeric nanoparticles with antioxidant and anti-inflammatory properties against cisplatin-induced hearing loss[END_REF][START_REF] Mohan | Targeted Amelioration of Cisplatin-Induced Ototoxicity in Guinea Pigs[END_REF][START_REF] Sun | A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration[END_REF][START_REF] Wang | A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss[END_REF]. Finally, dexamethasone phosphate is also employed to reduce the trauma induced by cochlear implantation [START_REF] Mamelle | Effect of a liposomal hyaluronic acid gel loaded with dexamethasone in a guinea pig model after manual or motorized cochlear implantation[END_REF].

In the case of gene delivery to treat genetic hearing loss [START_REF] Maeda | In vitro and in vivo suppression of GJB2 expression by RNA interference[END_REF], several nanocarriers have been evaluated: cationic liposomes, hyperbranched polylysine and dendriplexes. Nucleic acids (e.g. siRNA, mRNA), due to their negative charges bind to cationic charges at the surface of liposomes, to the cationic hyperbranched polylysine or to cationic dendrimers [START_REF] Degors | Carriers break barriers in drug delivery: endocytosis and endosomal escape of gene delivery vectors[END_REF].

Nanocarrier biodistribution and pharmacokinetics

The efficacy of nanocarriers after intratympanic administration depends on their ability to accumulate inside the round window membrane or in the inner ear tissues and to release the drug to the perilymph. It raises several issues concerning their biodistribution and drug release in the inner ear.

What is their fate after intratympanic administration? Do they enhance drug bioavailability? What are the key parameters influencing nanocarrier biodistribution and drug release? This section focuses first on nanocarrier biodistribution and drug pharmacokinetics after nanocarrier administration. The key physicochemical characteristics of nanocarriers for inner ear delivery are discussed. Finally, advanced approaches enhancing nanocarrier diffusion through the inner ear barriers are presented.

Passive approaches

Published studies on the biodistribution of nanocarriers and pharmacokinetics are summarized in Table 2. They were performed exclusively in rodents. Different types of nanocarrier were evaluated, based on polymer, lipid or iron with different surfaces (neutral, anionic, cationic or PEGylated). Many of the nanocarriers were PEGylated (Table 2). Nanocarrier size ranged from 10 to 630 nm but most of them had a diameter centered around 140 nm. In vivo studies were carried out over periods ranging from 2 hours to 2 weeks but most often over a short period (3 days).

To assess nanocarrier biodistribution, a fluorescent tracer can be covalently linked to the raw material of the nanocarrier or encapsulated inside it. Then the cochlea can be observed by confocal microscopy to track labeled nanocarriers (Table 2). However, when the tracer is not covalently bonded, it can be released from the nanocarrier depending on its characteristics. When fluorescent dots are observed in tissues, the tracer is probably still inside the nanocarrier whereas if the fluorescence is diffuse, then the probe has probably been released.

To evaluate drug pharmacokinetics, it is essential to have sensitive analytical techniques to quantify the drug in the perilymph. HPLC coupled to a UV or mass spectrometer (LC-MS) is generally used. However, when sampling the perilymph, small volumes (~2 µL) are taken to avoid contamination of the perilymph with cerebrospinal fluid. After sampling, the animal is euthanized, since multiple sampling is very difficult to set up [START_REF] Salt | Pharmacokinetic principles in the inner ear: Influence of drug properties on intratympanic applications[END_REF]. Another approach is to label the cochlea by immunostaining specifically to detect the drug, or alternatively to develop high resolution imaging [START_REF] Zou | Inner ear barriers to nanomedicine-augmented drug delivery and imaging[END_REF].

Biodistribution

When evaluated, nanocarriers are seen inside the round window [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF]Liu et al., 2013b;[START_REF] Tamura | Drug delivery to the cochlea using PLGA nanoparticles[END_REF][START_REF] Yu | Bovine serum albumin nanoparticles as controlled release carrier for local drug delivery to the inner ear[END_REF][START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF]W. Zhang et al., 2011;Y. Zhang et al., 2011a;[START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF] and/or the oval window membranes (Zou et al., 2014b[START_REF] Zou | Micro CT visualization of silver nanoparticles in the middle and inner ear of rat and transportation pathway after transtympanic injection[END_REF](Zou et al., , 2017b) ) (Table 2). Cubosomes, hyperbranched polylysine, PLGA nanoparticles and PEGylated liposomes are distributed in the three layers of the round window membrane suggesting that they might diffuse inside this barrier [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF]Liu et al., 2013b;[START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF]W. Zhang et al., 2011).

Conversely, PEGylated polymersomes are only found in the outer layer of the round window membrane, suggesting that they are not able to cross this barrier (Y. Zhang et al., 2011a;[START_REF] Zhang | Improving the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO4 to quench autofluorescence in the rat inner ear[END_REF]. Nevertheless, they are detected in the inner ear, but only after transtympanic injection (Zhang et al., 2010) and not after round window application (Y. Zhang et al., 2011a). Since the transtympanic administration involved two access routes to the inner ear, the round and the oval window membranes, the entrance of PEGylated polymersomes into the inner ear might be carried out partially by the oval window membrane. Conversely, when PEGylated liposomes are applied to the ossicular chain, they are internalized in chondrocytes and fail to cross the oval window membrane (Zou et al., 2014b).

Thus, the properties of the nanocarriers (size, nature of the surface, rigidity, shape or lipophilicity) may influence their ability to cross the round and/or the oval window membranes (see section 5.2).

Different types of nanocarriers are found in the inner ear after administration: liposomes, polymersomes, lipid nanocapsules, PLGA nanoparticles, cubosomes, inorganic nanoparticles, and hyperbranched polylysine, showing that they are able to cross middle ear barriers (Table 2). However, the proportion of these nanocarriers in the inner ear is not known. Indeed, methods of quantification of the nanocarriers are difficult to set up because their concentration needs to be related for instance to an intensity of fluorescence. The concentration of the nanocarrier raw material is sometimes assessed for inorganic nanoparticles in the perilymph (Zou et al., 2010b[START_REF] Zou | Micro CT visualization of silver nanoparticles in the middle and inner ear of rat and transportation pathway after transtympanic injection[END_REF](Zou et al., , 2017b)), but never related to a concentration of nanocarriers or a percentage of the initial amount of nanocarriers administered. Semiquantification of labeled nanocarriers is done using confocal microscopy [START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF] or spectrofluorometry on the sampled perilymph [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF]. These methods are used for advanced strategies of administration (see section 5.3), but not for standard nanocarrier administrations (Table 2).

Nanocarriers are mostly observed in the cochlea (Table 2); however, the vestibule is not always evaluated. Most of them are found in the basal turn of the cochlea [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF]Liu et al., 2013b;[START_REF] Martín-Saldaña | Polymeric nanoparticles loaded with dexamethasone or α-tocopheryl succinate to prevent cisplatin-induced ototoxicity[END_REF][START_REF] Tamura | Drug delivery to the cochlea using PLGA nanoparticles[END_REF]Zou et al., 2017b) and, when evaluated, in the vestibule [START_REF] Zhang | Improving the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO4 to quench autofluorescence in the rat inner ear[END_REF]Y. Zhang et al., 2011a;Zou et al., 2010bZou et al., , 2014bZou et al., , 2017b)). If the oval window membrane is involved, by direct application on the oval window membrane or by transtympanic injection, nanocarriers are present in the vestibule [START_REF] Ding | Is oval window transport a royal gate for nanoparticle delivery to vestibule in the inner ear?[END_REF]Zou et al., 2014b).

Thus, specific targeting of the vestibule might be possible, for example, to treat Ménière's disease.

When specific tissue biodistribution is assessed, nanocarriers are principally located in the spiral ganglion [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF]W. Zhang et al., 2011;[START_REF] Zhang | Improving the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO4 to quench autofluorescence in the rat inner ear[END_REF][START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF] and around hair cells of the organ of Corti [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF][START_REF] Kim | Development of a drug delivery system for the inner ear using poly(amino acid)-based nanoparticles[END_REF][START_REF] Martín-Saldaña | pH-sensitive polymeric nanoparticles with antioxidant and anti-inflammatory properties against cisplatin-induced hearing loss[END_REF][START_REF] Shi | Pathophysiology of the cochlear intrastrial fluid-blood barrier (review)[END_REF][START_REF] Yang | Optimized phospholipidbased nanoparticles for inner ear drug delivery and therapy[END_REF]W. Zhang et al., 2011;[START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF]. They are also visualized in the spiral ligament and stria vascularis [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF][START_REF] Zhang | Improving the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO4 to quench autofluorescence in the rat inner ear[END_REF]Zou et al., 2012b[START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF].

Liposomes and lipid nanocapsules can also be distributed in nerve fibers or the auditory nerve [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF][START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF], suggesting that they are able to spread in the modiolus to reach other turns of the cochlea. Surprisingly, SPION exhibit a contradictory behavior, by accumulating only in the perilymphatic space of the saccule in the vestibule (Zou et al., 2010b). In this study, SPION are administered in the middle ear without the application of a magnet, as described in section 5.3. They might use an unknown specific pathway mechanism that causes their entrapment in saccule hair cells. Dendriplexes also reach specific tissue such as the hair cells of the organ of Corti [START_REF] Wu | In vivo delivery of Atoh1 gene to rat cochlea using a dendrimer-based nanocarrier[END_REF]. Otherwise, nanocarriers without targeting ligands do not seem to target a specific inner ear tissue (Table 2).

The integrity of nanocarriers once in the inner ear has not been evaluated for some nanocarriers (lipid nanoemulsions, dendriplexes, cubosomes, hyperbranched polylysine, chitosan nanocarriers).

Their integrity has been demonstrate for PEGylated nanocapsules [START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF] and polymersomes [START_REF] Kim | Development of a drug delivery system for the inner ear using poly(amino acid)-based nanoparticles[END_REF] in the organ of Corti, and PEGylated liposomes (Zou et al., 2012b) and PLGA nanoparticles in the perilymph [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF]. However, nanocarriers seem to lose their integrity when found in the spiral ligament (Zou et al., 2012b[START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF] or the supporting cells [START_REF] Kim | Development of a drug delivery system for the inner ear using poly(amino acid)-based nanoparticles[END_REF].

Nanocarriers seem to cross the middle ear barriers quickly, since they are detected in the inner ear for less than 1 hour (Liu et al., 2013b;[START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF][START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF] up to 3 hours (Zou et al., 2014b(Zou et al., , 2017b) ) after administration. For PLGA nanoparticles, the peak concentration in the round window membrane occurs 30 minutes after transtympanic injection.

The persistence of the nanocarriers in the inner ear tissues depends strongly on their nature (Table 2). Lipid nanocapsules remain in the inner ear for 1 week [START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF], unlike to silver nanoparticles which are eliminated in less than 6 hours [START_REF] Zou | Micro CT visualization of silver nanoparticles in the middle and inner ear of rat and transportation pathway after transtympanic injection[END_REF] or to liposomes eliminated/degraded in less than 24 hours (Zou et al., 2012b).

Most of the nanocarriers cross the middle ear barriers quickly to reach the basal turn of the cochlea and the vestibule, without targeting a specific tissue. They do not lose their integrity once in the perilymph [START_REF] Kim | Development of a drug delivery system for the inner ear using poly(amino acid)-based nanoparticles[END_REF][START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF]Zou et al., 2012b[START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF]; however, little is known about the fraction of nanocarriers that effectively reach the inner ear. Further studies are needed to evaluate this essential parameter.

Hypothesis on pathway mechanisms

Nanocarriers seem to enter the inner ear by either the round window [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF][START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF]Zou et al., , 2014b) ) or the oval window (Y. Zhang et al., 2011b;Zou et al., 2010b[START_REF] Zou | Micro CT visualization of silver nanoparticles in the middle and inner ear of rat and transportation pathway after transtympanic injection[END_REF]Zou et al., , 2017b)). The hypothetical mechanisms of entrance through the round window membrane are widely discussed in the literature [START_REF] Mittal | Nanoparticle-based drug delivery in the inner ear: current challenges, limitations and opportunities[END_REF][START_REF] Pritz | Nanomedicine strategies for drug delivery to the ear[END_REF], but there is no detailed description for the mechanisms of entrance through the oval window (Table 2).

Lipid nanocapsules of small size (~50 nm) are thought to cross the first layer of the round window membrane by a paracellular pathway (Fig. 3), because they are found in the inner ear within 30 minutes [START_REF] Zou | Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation[END_REF]. However, tight junctions between cells of the first layer might impede this pathway for larger nanocarriers (~150 nm) such as PLGA nanoparticles, which cross the outer layer of the round window membrane by transcellular pathways (Fig. 3). Multiple mechanisms of cell internalization are involved [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF]. PLGA nanoparticles around 200 nm diameter, are internalized in macropinosomes (0.5-10 µm) using the macropinocytosis pathway (Fig. 3). Smaller PLGA nanoparticles are endocytosed in caveosomes (50-80 nm) by caveolae-mediated endocytosis [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF]. These pathways lead to internalization of the nanoparticles into endosomes and then lysosomes. Once in the lysosomes, nanoparticles are trapped, and this might lead to drug release from nanoparticles or to nanocarrier degradation (Fig. 3). However, exocytosis mediated by the Golgi apparatus and the endoplasmic reticulum is also observed. Early endosomes can be recycled, which leads to the discharge of PLGA nanoparticles in the extracellular matrix (Fig. 3). These transcellular pathways are also described in the inner epithelium of the round window membrane and some fibroblasts [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF].

The mechanisms involved in the transcellular transport of PLGA nanoparticles have not been described for other types of nanocarriers. Clathrin-mediated endocytosis is not involved in the uptake of PLGA nanoparticles in the outer epithelium of the round window membrane [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF], but it can be involved for other types of nanocarriers. Cationic material has been shown to be transported by pinocytotic vesicles [START_REF] Goycoolea | Round window membrane. Structure function and permeability: A review[END_REF], but this has not been clearly demonstrated for cationic dendriplexes [START_REF] Wu | In vivo delivery of Atoh1 gene to rat cochlea using a dendrimer-based nanocarrier[END_REF] and cationic liposomes (Jero et al., 2001b(Jero et al., , 2001a)). As described by [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF], nanocarriers can accumulate within the cells or the connective tissue of the round window membrane without reaching the scala tympani. Liposomes and polymersomes are highly accumulated, respectively, inside the connective tissue and the outer layer of the round window membrane [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF]Zou et al., 2014b). The drug can be released from the nanocarrier accumulated in the round window membrane. Thus, this barrier might act as a drug reservoir, though this has only been is demonstrated for advanced systems combining hydrogels and liposomes [START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF] (see section 5.3).

Concerning access through the oval window, nanocarriers are supposed to cross by the stapediovestibular joint (Fig. 3). Inorganic nanoparticles are clearly visible inside the joint after transtympanic injection [START_REF] Zou | Micro CT visualization of silver nanoparticles in the middle and inner ear of rat and transportation pathway after transtympanic injection[END_REF](Zou et al., , 2017b)). Conversely, liposomes are not observed inside the joint, but are internalized by the chondrocytes (Zou et al., 2014b); however, they might release their content in the inner ear in a sustained manner from the oval window.

Further studies are needed to evaluate which pathways are involved in the transport of nanocarriers across the round and oval window membranes. One must consider that this is a dynamic mechanism, and thus, the time window for observation of the phenomena must be carefully chosen to avoid 530 misleading conclusions.

Pharmacokinetics

One of the goals of using nanocarriers is to increase the drug concentration in the inner ear to 535 enhance drug efficacy. Compared to drug solutions, nanocarriers increase drug concentration and drug persistence in the inner ear in most of the studies [START_REF] Bu | Enhanced bioavailability of nerve growth factor with phytantriol lipid-based crystalline nanoparticles in cochlea[END_REF][START_REF] Cai | Enhanced local bioavailability of single or compound drugs delivery to the inner ear through application of plga nanoparticles via round window administration[END_REF]Liu et al., 2013a;[START_REF] Sun | A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration[END_REF][START_REF] Yang | Optimized phospholipidbased nanoparticles for inner ear drug delivery and therapy[END_REF]. For dexamethasone, therapeutic doses (30-40 ng/mL) [START_REF] Kim | Regulation of ENaC-mediated sodium transport by glucocorticoids in Reissner's membrane epithelium[END_REF] are reached with PEGylated PLGA nanoparticles and the delivery is extended from 12 to 48 hours compared to the solution [START_REF] Sun | A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration[END_REF]. However, therapeutic doses are rarely documented for other drugs (e.g. nerve growth factor, salvianolic acid B, tanshinone IIA and panax notoginsenoside) (Table 2). Since the drug concentration and persistence are increased using nanocarriers, the drug entrance into the inner ear seems to rely on the characteristics of the nanocarrier and not of the drug; however, this is not always demonstrated. PLGA nanoparticles increase the concentration of a hydrophilic drug (panax notoginsenoside) in the perilymph compared to the drug solution, but not for lipophilic drugs (salvianolic acid B and tanshinone IIA) [START_REF] Cai | Enhanced local bioavailability of single or compound drugs delivery to the inner ear through application of plga nanoparticles via round window administration[END_REF].

The biodistribution of nanocarriers and drug pharmacokinetics studies are rarely evaluated simultaneously (Table 2). When studied, the biodistribution of the nanocarrier seems to explain the kinetics of the active molecule. The nanocarrier concentrates the drug in its core, and then diffuses in the inner ear, which increases the drug concentration in the perilymph. For PLGA nanoparticles, the peak concentration of panax notoginsenoside observed around 30 to 60 minutes [START_REF] Cai | Enhanced local bioavailability of single or compound drugs delivery to the inner ear through application of plga nanoparticles via round window administration[END_REF] is consistent with a rapid passage across the round window membrane (~30 minutes) [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF]. For instance, PEGylated liposomes which can cross the round window membrane are more efficient in delivering disulfiram to the cochlea than polymersomes which are accumulated in the outer layer of the round window [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF]. Nevertheless, polymersomes are able to deliver disulfiram to the cochlea from this barrier which may act as a reservoir of polymersomes.

Despite its importance for drug activity, the stability of the drug is seldom documented. For gene delivery, the integrity of nucleic acids is particularly important for activity as this is easily degraded. This can be investigated by adding the green fluorescence protein gene to the vectorized plasmid.

Thus, if a cell emits additional fluorescence at the specific wavelength of the green fluorescence protein, the gene must have been efficiently incorporated into its genome. After complexation with cationic liposomes (Jero et al., 2001a(Jero et al., , 2001b) ) or dendriplexes [START_REF] Wu | In vivo delivery of Atoh1 gene to rat cochlea using a dendrimer-based nanocarrier[END_REF], green fluorescence protein expression is observed in many cochlea tissues for cationic liposomes and in hair cells for dendriplexes (Table 2). Thus, cationic nanocarriers are efficient in delivering non-degraded macromolecules to the inner ear tissues, though their concentration has not been determined in these studies.

Another key point for drug activity is the conversion of the prodrug into its active form. Yet, some studies have not evaluated this point [START_REF] Sun | A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration[END_REF] due to the technical challenges related to the analytical method. However, it is important to evaluate for dexamethasone phosphate, which must be converted into dexamethasone to be active (see section 3.3.3). Polylactic acid nanoparticles [START_REF] Sun | A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration[END_REF] and nanoemulsions [START_REF] Yang | Optimized phospholipidbased nanoparticles for inner ear drug delivery and therapy[END_REF] encapsulated the active form of dexamethasone, but the control group received the prodrug dexamethasone phosphate. [START_REF] Yang | Optimized phospholipidbased nanoparticles for inner ear drug delivery and therapy[END_REF] quantified the active form of dexamethasone in tissues and demonstrated that dexamethasone concentration is equivalent between groups (dexamethasone-loaded nanoemulsions versus dexamethasone phosphate). Thus, this point must be systematically evaluated.

To conclude, nanocarriers seem to improve the drug pharmacokinetics profile in terms of drug persistence and concentration in the inner ear. The nanocarrier can protect the drug [START_REF] Wu | In vivo delivery of Atoh1 gene to rat cochlea using a dendrimer-based nanocarrier[END_REF], diffuse into the inner ear through the middle ear barriers and cause increased drug concentration in the perilymph or inner ear tissues. However, the impact on the therapeutic effect of the drug/nanocarrier degradation in the middle ear barriers is poorly documented. Systematic studies evaluating both the biodistribution of nanocarriers and pharmacokinetics should be carried out to demonstrate clearly whether the increase in drug concentration in the inner ear is the consequence of nanocarrier diffusion and accumulation in the inner ear or to nanocarrier accumulation in the round window membrane and drug release from this membrane.

Key physicochemical characteristics for inner ear delivery using nanocarriers

The key parameters influencing the biodistribution of nanocarriers in the inner ear are difficult to identify because only a few studies have evaluated their influence for the same nanocarrier (Table 2).

The size, the nature of the surface, the rigidity, the shape of the nanocarrier or the lipophilicity of the material used to manufacture the nanocarriers might all play a role in nanocarrier biodistribution. To properly evaluate the effect of physiochemical characteristics on nanocarrier delivery to the inner ear, the parameters should be modified one by one, which is rarely the case.

Influence of size

Most nanocarriers have a diameter centered around 140 nm without any real justification. The size of nanocarriers strongly influences their passage in the inner ear. However, comparisons were only evaluated for liposomes and PLGA nanoparticles [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF]Zou et al., 2012bZou et al., , 2010a)). Indeed, small liposomes (~95 nm hydrodynamic diameter) accumulate more easily in the inner ear compared to larger ones (~240 nm) (Zou et al., 2012b(Zou et al., , 2010a)). Larger liposomes might be too large to cross the round window membrane. Conversely, PLGA nanoparticles demonstrate the opposite behavior [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF]. Only 30 minutes after transtympanic injection, 300 nm-sized nanoparticles are found in the cochlea with higher fluorescence levels than for 150 and 80 nm nanoparticles. However, 1 day after administration, fluorescence levels in the cochlea are similar whatever the size of PLGA nanoparticles [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF]. Those results might be explained by the kinetics of entrance of nanocarriers depending on their size [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF].

Thus, the effect of size might differ according to the type of nanocarrier; however, for a given size, there is an effect of the nanocarrier material. For instance, the round window membrane behaves differently for polymersomes and liposomes despite their similar size (around 90 nm) and vesicular structure [START_REF] Buckiová | Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane[END_REF]. Liposomes reach the inner ear whereas polymersomes are embedded inside the outer layer of the round window. Although other parameters are most likely to be involved, nanocarrier size should be further evaluated because it also strongly influences drug encapsulation, and consequently the dose administered.

Influence of surface composition

The surface properties of nanocarriers can be modified using an anionic or cationic material, or a polymeric hydrophilic coating, such as chitosan, PEG or poloxamer that provides steric protection and a hydrophilic surface to the nanocarrier. Cationic charge seems to induce a higher accumulation of nanocarriers in the inner ear compared to neutral and anionic ones (Table 2). Cationic PLGA nanoparticles are distributed in the inner ear within 30 minutes even though 1 day after administration, fluorescence levels in the cochlea are equivalent between cationic and PEGylated PLGA nanoparticles [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF]. However, for nanoemulsions, the cationic charge of the droplets must be combined with PEGylation to permit the delivery of Nile red to the inner ear [START_REF] Yang | Optimized phospholipidbased nanoparticles for inner ear drug delivery and therapy[END_REF]. The difference between these two studies, aside from the type of nanocarrier used, may be explained by the cationic chitosan layer coating the PLGA nanoparticles.

Chitosan chains provide a supplementary steric protection and a hydrophilic surface to the nanoparticles that might also favor their biodistribution in the inner ear.

Hydrophilic polymeric coating might play an important role. Although PEGylation is often used, its role in the biodistribution is not always evaluated (Table 2). PEGylated PLGA nanoparticles are not more highly accumulated in the inner ear than uncoated or cationic ones [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF], but they deliver coumarin-6 more efficiently into the cochlea [START_REF] Wen | Nanomedicine strategy for optimizing delivery to outer hair cells by surface-modified poly(lactic/glycolic acid) nanoparticles with hydrophilic molecules[END_REF]. PEGylated PLGA nanoparticles may accumulate in the round window membrane and release their content from there, which can be interesting from a safety point of view (see section 6).

The coating of PLGA nanoparticles with poloxamer strongly improves the delivery of coumarin-6 compared to PEGylated, chitosan-coated or uncoated ones [START_REF] Wen | Nanomedicine strategy for optimizing delivery to outer hair cells by surface-modified poly(lactic/glycolic acid) nanoparticles with hydrophilic molecules[END_REF]. These authors suggest that poloxamer adsorption at the surface prevents nanoparticle clearance from the middle ear by the ciliated epithelium. This polymer inhibits hydrophobic interactions, hinders clearance and promotes nanoparticle entrance into the inner ear. In addition, poloxamer chains can interact with cell membranes, decrease the microviscosity of cell membranes and lead to pore formation [START_REF] Demina | Relationship between the structure of amphiphilic copolymers and their ability to disturb lipid bilayers[END_REF]. These authors hypothesize that it permits a higher outer hair cell penetration [START_REF] Wen | Nanomedicine strategy for optimizing delivery to outer hair cells by surface-modified poly(lactic/glycolic acid) nanoparticles with hydrophilic molecules[END_REF] but the integrity of nanoparticles in the outer hair cells has not been proved. Furthermore, one must consider that the uncoated nanoparticles in that study are in fact covered by polyvinyl alcohol (hydrophilic polymer) during manufacturing, as highlighted by [START_REF] Albert | Bare and Sterically Stabilized PLGA Nanoparticles for the Stabilization of Pickering Emulsions[END_REF]. PEG or poloxamer may be more hydrophilic and/or provide more steric protection than polyvinyl alcohol leading to nanocarrier accumulation in the cochlea.

To summarize, coating of the nanocarrier by a hydrophilic polymer (chitosan, PEG or poloxamer) seems to favor the passage of the nanocarrier into the inner ear. Further studies are needed to evaluate the impact of the charge independently of any other parameters (size or polymer coating).

Influence of nanocarrier concentration

Raising the concentration of PLGA nanoparticles from 10 to 90 mg/mL in the administered suspension increases their concentration inside the round window membrane [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF]. The variation of silver nanoparticle concentration from 37 to 370 mM leads to a higher concentration in the perilymph [START_REF] Zou | Micro CT visualization of silver nanoparticles in the middle and inner ear of rat and transportation pathway after transtympanic injection[END_REF]. However, these later nanoparticles induce a local inflammation that enhances the permeability of the round window membrane. Thus, it may not be related to the nanoparticle concentration alone. Nanocarrier concentration does not seem to influence the kinetics of entrance of nanocarriers through the middle ear barriers [START_REF] Zhang | Understanding the translocation mechanism of PLGA nanoparticles across round window membrane into the inner ear: A guideline for inner ear drug delivery based on nanomedicine[END_REF][START_REF] Zou | Micro CT visualization of silver nanoparticles in the middle and inner ear of rat and transportation pathway after transtympanic injection[END_REF]. Further studies are needed to evaluate whether it can prolong their persistence in the inner ear.

Other parameters

Other parameters might have an impact on the biodistribution of nanocarriers in the inner ear, such as the rigidity or the shape of the nanocarrier. However, up to now, this has rarely been evaluated. We can hypothesize that liposomes, due to their aqueous content and flexible lipid bilayers, are more susceptible to deform, unlike rigid PLGA nanoparticles. This might explain the difference in biodistribution observed when changing their size (see section 5.2.1).

In other applications such as cancer, elongated nanoparticles are used to increase the surface of contact with membranes and improve the uptake in cells [START_REF] Cong | Rod-shaped mesoporous silica nanoparticles for nanomedicine: recent progress and perspectives[END_REF]. Cubic cubosomes may migrate through the round window membrane due to their larger contact surface compared to spherical nanocarriers (Liu et al., 2013b). However, the shape parameter has not been thoroughly investigated, though it could be interesting to evaluate.

Advanced approaches

Advanced delivery approaches to the inner ear are defined here as the combination of nanocarriers with an additional strategy. The aim is either to enhance drug release or to limit off-target effects.

Several strategies are used (Table 3): -Incorporation of the nanocarrier into a hydrogel vehicle; -Active targeting allowing specific tissue therapy and which could be useful in the future development of gene therapy; -Increasing the permeability of the round window by chemical (cell-penetrating peptide) or physical (magnetic delivery, ultrasound) triggers. PLGA, poly(lactic-co-glycolic acid); RWM, round window membrane; SPION, superparamagnetic iron oxide nanoparticles; TEM, transmission electron microscopy; TTI, transtympanic injection.

Nanocarrier-loaded hydrogels

Over the past 5 years, hybrid systems combining nanocarriers and hydrogels have been increasingly developed for inner ear drug delivery (Table 3). Unlike sponges, hydrogels fill the whole cavity of the middle ear and their high viscosity reduces the elimination of nanocarriers by the Eustachian tube.

Three polymers are used as a matrix for nanocarrier administration: chitosan-glycerophosphate thermosensitive gel, poloxamer 407 thermosensitive gel and the shear-thinning non-thixotropic hyaluronic acid gel. They are combined with nanocarriers of different sizes (between 146 and 290 nm), different natures (PLGA nanoparticles, chitosan-based or liposomes) or different surfaces (PEGylated, cationic, anionic or neutral) (Table 3).

The incorporation of nanocarriers inside a hydrogel prolongs the drug residence time in the middle ear which increases the drug concentration in the perilymph compared to the drug-loaded hydrogel [START_REF] Ding | Is oval window transport a royal gate for nanoparticle delivery to vestibule in the inner ear?[END_REF][START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF][START_REF] Lajud | A novel chitosan-hydrogel-based nanoparticle delivery system for local inner ear application[END_REF]. When the drug concentration in the perilymph is not enhanced by the hydrogel, the mean residence time of the drug in the inner ear is extended [START_REF] Dai | A novel vehicle for local protein delivery to the inner ear: injectable and biodegradable thermosensitive hydrogel loaded with PLGA nanoparticles[END_REF] (Table 3). Using this strategy, the sustained release of dexamethasone phosphate converted in vivo into dexamethasone [START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF] and interferon-α [START_REF] Dai | A novel vehicle for local protein delivery to the inner ear: injectable and biodegradable thermosensitive hydrogel loaded with PLGA nanoparticles[END_REF] occurs over 1 month and 3 days, respectively.

However, the increase in residence time of the hybrid hydrogel in the middle ear does not always improve inner ear exposure to the drug [START_REF] Kayyali | A Novel Nanoapproach for Targeted Inner Ear Imaging[END_REF]. In this study, the liposomes are quite large (280 nm hydrodynamic diameter) and the attractive interactions between the cationic chitosan polymer and the anionic liposomes might impede their diffusion inside the hydrogel or induce their destabilization [START_REF] Kayyali | A Novel Nanoapproach for Targeted Inner Ear Imaging[END_REF].

If the nanocarriers do not retain their integrity after incorporation in the hydrogel, then they do not provide any protection or sustained release of the drug. Chitosan-glycerophosphate gel exhibits a macroporous microstructure, with well-interconnected pores [START_REF] Dai | A novel vehicle for local protein delivery to the inner ear: injectable and biodegradable thermosensitive hydrogel loaded with PLGA nanoparticles[END_REF][START_REF] Qu | α-Tocopherol liposome loaded chitosan hydrogel to suppress oxidative stress injury in cardiomyocytes[END_REF]. This gel microstructure does not change with anionic liposomes of 240 nm [START_REF] Qu | α-Tocopherol liposome loaded chitosan hydrogel to suppress oxidative stress injury in cardiomyocytes[END_REF] or PLGA nanoparticles of 290 nm [START_REF] Dai | A novel vehicle for local protein delivery to the inner ear: injectable and biodegradable thermosensitive hydrogel loaded with PLGA nanoparticles[END_REF], though the gel has increased roughness. However, PEGylated liposomes of 160 nm lead to a "patchy" microstructure. If anionic liposomes [START_REF] Kayyali | A Novel Nanoapproach for Targeted Inner Ear Imaging[END_REF] or PLGA nanoparticles [START_REF] Dai | A novel vehicle for local protein delivery to the inner ear: injectable and biodegradable thermosensitive hydrogel loaded with PLGA nanoparticles[END_REF] retain their integrity when incorporated into chitosan gels, this has not been demonstrated for PEGylated liposomes [START_REF] Lajud | A novel chitosan-hydrogel-based nanoparticle delivery system for local inner ear application[END_REF].

Nevertheless, PEGylation provides steric protection and hydrophilic surface for the liposomes incorporated in the chitosan hydrogel, preventing liposome destabilization. For hyaluronic acid and PEGylated liposome mixtures, clusters of intact but deformed liposomes are observed (El Kechai et al., 2015b). Poloxamer 407 is organized in a cubic micellar phase that drastically increases the viscosity of the medium [START_REF] Dumortier | A Review of Poloxamer 407 Pharmaceutical and Pharmacological Characteristics[END_REF] whereas weak ionic interactions are involved in the formation of chitosan-based nanocarriers [START_REF] Ding | Is oval window transport a royal gate for nanoparticle delivery to vestibule in the inner ear?[END_REF]. Thus, assessing their integrity within the poloxamer gel is important.

If the liposomes can diffuse within the gel, they might be able to accumulate in the round window membrane and/or to diffuse into the inner ear, which can be an additional benefit of the system. This depends strongly on the electrostatic and steric interactions between the nanocarrier and the polymer network. PEGylated liposomes migrate from the gel towards the round window membrane [START_REF] El Kechai | Mixtures of hyaluronic acid and liposomes for drug delivery: Phase behavior, microstructure and mobility of liposomes[END_REF]. PEGylation provides steric protection to the liposomes and the anionic charges of these vesicles induce repulsive electrostatic interactions with the anionic polymer chains. In addition, the bicontinuous microstructure observed with this system also favors the long-distance migration of liposomes.

Little is known about the integrity of the nanocarriers once they have reached the inner ear. El Kechai et al. ( 2016) quantified the passage in perilymph of liposomes included in a hyaluronic acid gel which filled the middle ear. Despite the high lipidic concentration administered (40-to 80-fold more concentrated than other studies using liposomes), a very low amount of liposomes is detected in the perilymph (0.00003%). However, they are found internalized in the cytoplasm of cells in the round window membrane suggesting that this barrier acts like a reservoir of liposomes [START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF]. It allows the sustained release of dexamethasone phosphate in the inner ear over 1 month. In another study, intact liposomes are found in the organ of Corti [START_REF] Lajud | A novel chitosan-hydrogel-based nanoparticle delivery system for local inner ear application[END_REF]. However, their concentration has not been determined (Table 3). The use of hydrogels combined with nanocarriers seems to be a very promising strategy to sustain drug delivery to the inner ear.

Active targeting to specific inner ear tissues

Active targeting allows drug delivery to specific tissues, reducing off-target effects. The ligand covalently fixed to the surface of the nanocarrier binds to a specific receptor present only on the targeted cells. In the inner ear application, effective targeting is first verified by intracochlear administration. Then, intratympanic administration is performed to evaluate 1) the nanocarrier delivery to the cochlea and 2) the accumulation of the nanocarriers in targeted tissues. Two main tissues are targeted: outer hair cells and spiral ganglion neurons.

-Outer hair cell targeting

The loss of outer hair cells leads to significant hearing loss [START_REF] Dallos | Cochlear amplification, outer hair cells and prestin[END_REF]. The objective of therapeutics is to preserve outer hair cell survival to prevent noise or age-related sensorineural hearing loss. Two peptides have been developed, A665 and A666 peptides, to target the extracellular domain of prestin receptor, exclusively present on outer hair cell membrane [START_REF] Dallos | Cochlear amplification, outer hair cells and prestin[END_REF][START_REF] Surovtseva | Prestin binding peptides as ligands for targeted polymersome mediated drug delivery to outer hair cells in the inner ear[END_REF]. A666-PEGylated PLA (polylactic acid) nanoparticles lead to a specific release to the outer hair cells [START_REF] Wang | A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss[END_REF] but A665-gold nanoparticles do not [START_REF] Kayyali | A Novel Nanoapproach for Targeted Inner Ear Imaging[END_REF]. The absence of specific targeting can be due to a dissociation of the ligand from the nanoparticles inside the round window membrane [START_REF] Kayyali | A Novel Nanoapproach for Targeted Inner Ear Imaging[END_REF]. A modified A665 peptide, called Prestin-Targeting-Peptide-1, is more efficient in targeting the outer hair cells [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF]; 5% of the initially administered targeted PEGylated liposomes are quantified in the perilymph 2 days after administration with the thermosensitive chitosan gel (Table 3). Targeted liposomes reach the apical turn of the cochlea while untargeted ones do not. Incorporation of the nanocarriers in the chitosan hydrogel does not impede their migration into the inner ear. After incorporation of a plasmid inside the liposomes, gene expression is achieved in outer hair cells but only with targeted liposomes [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF].

Thus, the liposomes seem to retain their integrity once in the inner ear. Although it was not discussed by the authors, targeted liposomes are also localized in off-target tissues [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF]. When loaded with c-Jun-N-terminal kinase inhibitor, a significant protection of hearing function is achieved with Prestin-Targeting-Peptide-1-coated liposomes compared to non-targeted ones (see section 7).

Thus, Prestin-Targeting-Peptide-1 seems to be the most efficient ligand to target outer hair cells. As the loss of activity of prestin in outer hair cells can lead to deafness [START_REF] Dallos | Cochlear amplification, outer hair cells and prestin[END_REF], the toxicity of this ligand should be evaluated.

-

Spiral ganglion neuron targeting

Maintaining the survival of the spiral ganglion neurons significantly enhances cochlear implantation outcomes [START_REF] Ramekers | Temporary Neurotrophin Treatment Prevents Deafness-Induced Auditory Nerve Degeneration and Preserves Function[END_REF]. In preclinical studies, neurotrophins (neuronal growth factors) efficiently improve neuronal density in the spiral ganglion and hearing thresholds [START_REF] Kandathil | Effects of brain-derived neurotrophic factor (BDNF) on the cochlear nucleus in cats deafened as neonates[END_REF]. However, these drugs need to be continuously delivered, otherwise the benefit is lost [START_REF] Shepherd | Neurotrophins and electrical stimulation for protection and repair of spiral ganglion neurons following sensorineural hearing loss[END_REF].

A 13-mer Tet1 peptide is used to target the trisialoganglioside clostridial toxin receptor expressed in spiral ganglion neurons [START_REF] Santi | Identification and localization of the GM1 ganglioside in the cochlea using thin-layer chromatography and cholera toxin[END_REF]. This peptide specifically binds to this receptor and allows efficient targeting of brain neurons [START_REF] Park | Neuron-specific delivery of nucleic acids mediated by Tet1-modified poly(ethylenimine)[END_REF]. When conjugated to PEGylated polymersomes [START_REF] Zhang | Targeted delivery of Tet1 peptide functionalized polymersomes to the rat cochlear nerve[END_REF], efficient targeting of the cochlear nerve and spiral ganglion neurons is achieved only by intracochlear administration. Intratympanic administration leads to biodistribution of polymersomes in the cochlea but not restricted to the neurons.

The discovery of new targets in other cell populations, such as the inner hair cells or supporting cells, to induce specific regeneration, could be interesting strategies in the future [START_REF] Zhong | Atoh1 and other related key regulators in the development of auditory sensory epithelium in the mammalian inner ear: function and interplay[END_REF].

Cell-penetrating peptides

Cell-penetrating peptides are composed of 6 to 30 amino acid residues able to increase cellular internalization and penetration in physiological barriers such as the blood-brain barrier [START_REF] Silva | Combination of cell-penetrating peptides with nanoparticles for therapeutic application: A review[END_REF]. In preclinical studies, cell-penetrating peptides promote siRNA [START_REF] Qi | Efficient siRNA transfection to the inner ear through the intact round window by a novel proteidic delivery technology in the chinchilla[END_REF] and protein [START_REF] Kashio | Topical application of the antiapoptotic TAT-FNK protein prevents aminoglycoside-induced ototoxicity[END_REF][START_REF] Takeda | Protein transduction therapy into cochleae via the round window niche in guinea pigs[END_REF] delivery to the inner ear. Thus, combining cell-penetrating peptides with nanocarriers, either linked to their surface [START_REF] Yoon | Intratympanic delivery of oligoarginine-conjugated nanoparticles as a gene (or drug) carrier to the inner ear[END_REF] or mixed with the nanocarrier suspension [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF], should enhance the passage of nanocarriers through the round window membrane. Currently, two peptides have been reported: oligoarginine, a cationic 8-mer peptide, and low molecular weight protamine (Table 3).

When polymersomes are covered with oligoarginine [START_REF] Yoon | Intratympanic delivery of oligoarginine-conjugated nanoparticles as a gene (or drug) carrier to the inner ear[END_REF], the release of Nile red is more visible on confocal microscopy than for uncovered polymersomes in the organ of Corti (Kim et al., 2015). However, no quantification was performed in this study. Regarding low molecular weight protamine, it enhances the entrance of PLGA nanoparticles by the round window [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF].

The concentration of coumarin-6 in the inner ear is slightly increased by the presence of the cellpenetrating peptide on the surface of PLGA nanoparticles (Table 3).

Magnetic delivery of nanocarriers in the inner ear

The magnetic delivery of nanocarriers consists of the injection into the middle ear of SPION (5-15 nm), incorporated or not in a larger nanocarrier, that cross the round window membrane thanks to the application of a magnetic field [START_REF] Kopke | Magnetic Nanoparticles: Inner Ear Targeted Molecule Delivery and Middle Ear Implant[END_REF].

First, the proof-of-concept was established in guinea pigs and fresh human explants [START_REF] Kopke | Magnetic Nanoparticles: Inner Ear Targeted Molecule Delivery and Middle Ear Implant[END_REF]. PLGA nanoparticles containing SPION were efficiently pulled from the middle to the inner ear by a magnet applied on the contralateral ear for 1 hour. A channel of nanoparticles was observed inside the round window membrane and a large amount of nanoparticles was found in the perilymph (Table 3). When dexamethasone acetate was incorporated into 500 nm-large PLGA nanoparticles, 10% of the initial dose applied to the round window membrane was quantified in the cochlea within 1 hour [START_REF] Du | Magnetic targeted delivery of dexamethasone acetate across the round window membrane in guinea pigs[END_REF]. A very high dexamethasone concentration of 18 µg/mL was reported in the perilymph. However, this method was not suitable in humans because the distance between the magnet and the nanoparticle suspension must be less than 2 cm [START_REF] Du | Magnetic targeted delivery of dexamethasone acetate across the round window membrane in guinea pigs[END_REF]. Thus, a new system of four magnets that pushed nanoparticles into the inner ear was set up allowing larger distances of 3 to 5 cm for the application of the magnets on the treated ear [START_REF] Sarwar | Magnetic injection of nanoparticles into rat inner ears at a human head working distance[END_REF]; 300 nm-sized chitosan nanocarriers loaded with SPION and methylprednisolone efficiently protected the inner ear from cisplatin ototoxicity in the long term using this magnetic delivery method [START_REF] Ramaswamy | Magnetic Nanoparticle Mediated Steroid Delivery Mitigates Cisplatin Induced Hearing Loss[END_REF].

In a recent study, another method was used to target the apex of the cochlea [START_REF] Leterme | Superparamagnetic Nanoparticle Delivery to the Cochlea Through Round Window by External Magnetic Field: Feasibility and Toxicity[END_REF].

The magnet was displaced every 10 minutes to promote SPION displacement all along the turns of cochlea. In 2012, Otomagnetics was created to develop the magnetic delivery for the inner ear, eye and skin [START_REF] Otomagnetics ; Paasche | Changes of postoperative impedances in cochlear implant patients: The short-term effects of modified electrode surfaces and intracochlear corticosteroids[END_REF]. Preclinical studies to evaluate polymer-based nanocarriers loaded with SPION are under way for the protection of the inner ear of children undergoing chemotherapy [START_REF] Otomagnetics ; Paasche | Changes of postoperative impedances in cochlear implant patients: The short-term effects of modified electrode surfaces and intracochlear corticosteroids[END_REF].

Nevertheless, this strategy needs to verify safety issues due to the long-term residence of inorganic material within the inner ear. In addition, the clinical application must be adjusted according to the position of the round window membrane, which is highly variable in humans [START_REF] Proctor | Anatomy of the round window niche[END_REF].

Magnetic guidance will require a submillimetric accuracy with coupling to a navigation system to drive the SPION through the magnetic field.

Future strategy: focused ultrasound?

Initially used for diagnosis by echography, ultrasound enhances the permeability of barriers, such as the blood-brain barrier or the skin [START_REF] Tharkar | Nano-Enhanced Drug Delivery and Therapeutic Ultrasound for Cancer Treatment and Beyond[END_REF]. Effective nanocarrier delivery to the inner ear has not been demonstrated yet with focused ultrasound, but it has been evaluated for drug delivery [START_REF] Liao | Ultrasound-induced microbubble cavitation via a transcanal or transcranial approach facilitates inner ear drug delivery[END_REF][START_REF] Shih | Middle-ear dexamethasone delivery via ultrasound microbubbles attenuates noise-induced hearing loss[END_REF][START_REF] Shih | Ultrasound-aided microbubbles facilitate the delivery of drugs to the inner ear via the round window membrane[END_REF]. Microbubbles were mixed into a solution of biotin-FITC and injected into the middle ear of guinea pigs [START_REF] Shih | Ultrasound-aided microbubbles facilitate the delivery of drugs to the inner ear via the round window membrane[END_REF]. The ultrasound transducer was applied on the liquid-filled middle ear cavity. After 1 minute of ultrasound exposure, the biotin-FITC concentration in the inner ear was increased by a factor of 3.5. Similarly, a solution of 0.9 mg of dexamethasone phosphate administered by transtympanic injection led to a 10-fold higher concentration in the perilymph using ultrasound [START_REF] Shih | Middle-ear dexamethasone delivery via ultrasound microbubbles attenuates noise-induced hearing loss[END_REF]. The combined use of ultrasound and nanocarriers might provide sustained release and an increase in the dexamethasone phosphate concentration in the inner ear. Unlike magnetic delivery, the ultrasound technique might require surgery [START_REF] Shih | Middle-ear dexamethasone delivery via ultrasound microbubbles attenuates noise-induced hearing loss[END_REF]. To avoid this, [START_REF] Liao | Ultrasound-induced microbubble cavitation via a transcanal or transcranial approach facilitates inner ear drug delivery[END_REF] are currently developing a "transcranial" approach on which the transducer is applied directly to the skull as well as an approach by application of the transducer on the external auditory membrane. These strategies seem closer to a suitable and easy clinical application [START_REF] Liao | Ultrasound-induced microbubble cavitation via a transcanal or transcranial approach facilitates inner ear drug delivery[END_REF], and thus, are promising, particularly for nanocarriers administration. Nevertheless, the safety of focused ultrasound should be assessed.

Advantages of nanocarriers over drug solutions/hydrogels

The encapsulation of different types of drug (small drugs, siRNA proteins or peptides) within nanocarriers modifies their pharmacokinetic profile by increasing their concentration and/or their residence time in perilymph. This is particularly evidenced for nanocarriers dispersed within hydrogels (Table 3). Nanocarriers also allow the targeting of specific tissues when covered with ligands. For now, the outer hair cells are the only tissue which has been efficiently targeted. Nanocarriers have been proved to migrate into the inner ear, with or without losing their integrity. However, data obtained from longer studies (1 week at least) are needed to quantify the amount of nanocarrier able to cross the barriers to accumulate in the inner ear. Studies evaluating the impact of one unique parameter might allow a better understanding of the key physicochemical parameters governing nanocarrier passage into the inner ear. Clearly, size is key but the surface charge and concentration of the nanocarrier also seem to play an important role.

Safety of nanocarrier administration

Before considering the safety of nanocarriers for inner ear delivery, the composition of the formulation must be adapted to the middle and inner ear compartments. It should have a physiological pH (7.38-7.42) and an osmolality around 300 mOsm/kg (El Kechai et al., 2015a). If the pH is controlled, the osmolality is rarely reported although this parameter is crucial. Nanocarriers are often dispersed in pure water [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF]Liu et al., 2013a;[START_REF] Meyer | Lipidic nanocapsule drug delivery: neuronal protection for cochlear implant optimization[END_REF], which leads to hypotonic suspensions, or directly incorporated in standard buffers such as phosphate-buffered saline [START_REF] Lajud | A novel chitosan-hydrogel-based nanoparticle delivery system for local inner ear application[END_REF][START_REF] Martín-Saldaña | pH-sensitive polymeric nanoparticles with antioxidant and anti-inflammatory properties against cisplatin-induced hearing loss[END_REF][START_REF] Roy | Strategies for drug delivery to the human inner ear by multifunctional nanoparticles[END_REF], which could conversely lead to hypertonic suspensions. Hypotonic or hypertonic suspensions can induce noxious fluid displacements between the perilymph and the suspension via the round window [START_REF] Mikulec | Permeability of the round window membrane is influenced by the composition of applied drug solutions and by common surgical procedures[END_REF]. Hearing is extremely sensitive to changes in fluid volume and ionic composition. Thus, a formulation intended for inner ear delivery should respect the homeostasis of inner ear fluids. Furthermore, according to the monography for parenteral preparations in the European Pharmacopeia (Ph. Eur. 10.2, 2020a), the system must be sterile and endotoxin-free.

The safety of a formulation is the evaluation of its effect without any drugs on healthy animals, and the assessment of the function of the targeted organ. For the inner ear, the main outcome to measure is the hearing function, through auditory brainstem response records. The second outcome is the evaluation of the structure of the organ of Corti by histology or an inflammatory response (macrophages, pro-inflammatory cytokines or other markers) which is very important for the evaluation of nanocarrier toxicity on the inner ear. If some nanocarriers cross the inner ear barriers, they might remain in the cochlea for a long time and induce side effects in the long term. Hearing function must be primarily evaluated, because some defects are not visible from histology such as loss of hair cell bundles or conductivity changes [START_REF] Corey | Mechanical Transduction Processes in the Hair Cell[END_REF].

A summary of studies carried out on the safety evaluation of different nanocarriers (PEGylated, cationic, anionic, neutral) using passive or advanced strategies is presented below (Table 4). The duration of these studies is very variable: from 1 day up to 3 months but centered around 1 week. The toxicity of nanocarriers on the inner ear was evaluated after intratympanic administration, which is well-tolerated in the clinic, and by intracochlear administration to maximize the nanocarrier concentration in the inner ear (Table 4). The latter technique allows the identification of potential side effects. For example, a dose-dependent toxicity and inflammation were observed after intracochlear injection of cationic liposomes [START_REF] Staecker | Gene expression in the mammalian cochlea: A study of multiple vector systems[END_REF] but not after intratympanic administration (Jero et al., 2001b). The toxicity of nanocarriers by intratympanic administration was less pronounced for several reasons: 1) low passage of nanocarriers into the cochlea, 2) potential destabilization of nanocarriers in middle ear barriers, 3) low residence time in the middle ear when they are administered as liquid suspensions. The administration of the nanocarrier is mostly neither repeated nor continuous, although it could be necessary to evaluate the effect of multiple administrations (Table 4).

Furthermore, safety studies must be conducted on a broad concentration range of nanocarriers in terms of material or particle concentration that fit clinical practice, because the nanocarrier concentration is adjusted as a function of the drug loading obtained in efficacy studies.

Safety of the passive administration of nanocarriers

Most of the nanocarriers showed a good safety profile with no impact on hearing function or inner ear structures (Table 4), apart from silver nanoparticles that are ototoxic (Zou et al., 2014a).

PEGylated liposomes exhibit a slight increase in hyaluronic acid expression but all other markers of inflammation are not expressed (Zou et al., 2017a). A high toxicity is observed after administration of 900 cationic liposomes in mice [START_REF] Staecker | Gene expression in the mammalian cochlea: A study of multiple vector systems[END_REF], but the cationic lipid (N-[1-(2, 3dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) used in this study is reported to be quite toxic [START_REF] Campani | Lipid nanovectors to deliver RNA oligonucleotides in cancer[END_REF].

Despite the number of studies on PLGA nanoparticles, so far their safety has been poorly evaluated in inner ear application (Table 4). Nevertheless, the safety of PLGA microparticles is reported after intracochlear administration in guinea pigs [START_REF] Ross | Persistence, distribution, and impact of distinctly segmented microparticles on cochlear health following in vivo infusion[END_REF]. A loss of 10 dB is observed at midrange frequencies (8 and 20 kHz) 1 week after administration. According to hair cell count, there is no toxicity induced on the sensory cells. However, PLGA microparticles degrade over time due to hydrolysis of PLGA chains. This might lead to a potentially toxic acidification of the pH within the cochlea [START_REF] Liu | Less harmful acidic degradation of poly(lactic-coglycolic acid) bone tissue engineering scaffolds through titania nanoparticle addition[END_REF]. Consequently, future studies on PLGA nanoparticles should evaluate these parameters.

Safety of advanced strategies for nanocarrier administration

As these strategies have recently been developed, their safety has not been extensively evaluated.

Cell-penetrating peptides and active targeting did not induce obvious histological changes on inner ear structures but have not been evaluated on hearing function [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF][START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF].

On the other hand, the safety of magnetic delivery has been widely evaluated, even at 3 months [START_REF] Shimoji | Toxicology study for magnetic injection of prednisolone into the rat cochlea[END_REF]. Concerns about the accumulation of iron oxide in the inner ear, with potential noxious oxidative properties, are the major limitation for clinical studies. When injected by the intracochlear route, SPION coated with streptavidin do not induce any hearing loss at 1 week [START_REF] Nguyen | Superparamagnetic nanoparticles as vectors for inner ear treatments: Driving and toxicity evaluation[END_REF]. However, that study might be too short for the coating to be degraded and release its content, which may induce toxicity. Otomagnetics shows that chitosan nanocarriers containing SPION do not induce any hearing loss 3 months after administration by magnetic delivery [START_REF] Shimoji | Toxicology study for magnetic injection of prednisolone into the rat cochlea[END_REF]. Slight inflammation is reported. However, another study, also conducted in rats, reports a mild inflammatory response with the same nanocarriers [START_REF] Lafond | Middle Ear Histopathology Following Magnetic Delivery to the Cochlea of Prednisoloneloaded Iron Oxide Nanoparticles in Rats[END_REF]. Numerous macrophages infiltrate the round window membrane and the inner ear. SPION are identified inside the cytoplasm of the macrophages. Using a hyaluronic acid gel, SPION organized in clusters of 135 nm are administered by magnetic delivery so that there was only the effect of SPION [START_REF] Leterme | Superparamagnetic Nanoparticle Delivery to the Cochlea Through Round Window by External Magnetic Field: Feasibility and Toxicity[END_REF]).

An immediate but transient hearing loss is observed at high frequencies, thus nearby the round window membrane. However, no other adverse effect has been reported. Thus, it is possible that the adverse reaction observed by [START_REF] Lafond | Middle Ear Histopathology Following Magnetic Delivery to the Cochlea of Prednisoloneloaded Iron Oxide Nanoparticles in Rats[END_REF] is due to using chitosan nanocarriers.

Therapeutic efficacy of drug-loaded nanocarriers on inner ear disorders

In this section, we give an overview of the therapeutic efficacy of drug-loaded nanocarriers on inner ear disorders. Several drug-loaded nanocarriers with a size between 87 and 300 nm but most frequently with a mean diameter centered around 150 nm have been tested in preclinical studies. Their characteristics and therapeutic efficacy are summarized in Table 5. 935 Animal models used to evaluate the efficacy of nanocarriers pertain exclusively to the cochlea.

Indeed, models for diseases impacting the vestibule (Ménière's disease) are difficult to set up [START_REF] Kapolowicz | Plasticity in Limbic Regions at Early Time Points in Experimental Models of Tinnitus[END_REF], and consequently, they have not been evaluated with nanocarriers. Hearing function is measured before and after administration of a treatment. The sensitivity of this measurement is of ±5 dB, thus only a hearing improvement of 10 dB is significant.

The most common animal model used is the protection against cisplatin ototoxicity in rodents.

Cisplatin is an anticancer drug widely administered in clinics [START_REF] Duan | Nanoparticle formulations of cisplatin for cancer therapy[END_REF] inducing ototoxicity [START_REF] Paken | A Review of Cisplatin-Associated Ototoxicity[END_REF]. The resulting hearing loss is attributed to oxidative stress and inflammation [START_REF] Gentilin | Cisplatin-Induced Ototoxicity: Updates on Molecular Targets[END_REF]. Other drugs are known to be ototoxic, like aminoglycoside antibiotics (kanamycin, gentamicin) [START_REF] Xie | New developments in aminoglycoside therapy and ototoxicity[END_REF]. Depending on patient inter-variability, these drugs can cause permanent hearing loss in adults and children [START_REF] Lanvers-Kaminsky | Pharmacogenetics of drug-induced ototoxicity caused by aminoglycosides and cisplatin[END_REF]. The rodent model is quite easy to set up, but the level of hearing loss depends on the ototoxic drug dose.

Thus, studies are not always comparable (Table 5).

Another model is noise-induced hearing loss. The rodent is exposed to a high level of noise over a predetermined time. The hearing loss level is not very reproducible among studies or animals due to several factors such as duration of noise exposure, targeted frequencies, and noise intensity. Moreover, rodents can spontaneously recover hearing with time [START_REF] Ma | An efficient strategy for establishing a model of sensorineural deafness in rats[END_REF].

In the case of the cochlear implant model, the trauma resulting from insertion of the electrode array inside the scala tympani is more reproducible [START_REF] Mamelle | Effect of a liposomal hyaluronic acid gel loaded with dexamethasone in a guinea pig model after manual or motorized cochlear implantation[END_REF].

The genetic model of hearing loss is quite rare, but gap junction protein β2 gene mutation has recently been developed. It is the most common cause of recessive prelingual genetic deafness [START_REF] Estivill | Connexin-26 mutations in sporadic and inherited sensorineural deafness[END_REF]. In the mutant murine model, the generation of genetic deafness leads to a shift of 20 dB in hearing thresholds [START_REF] Maeda | In vitro and in vivo suppression of GJB2 expression by RNA interference[END_REF].

In some studies, nanocarriers are administered before the induction of trauma to maximize the amount of drug directly available in the inner ear. PLGA nanoparticles [START_REF] Wang | A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss[END_REF] and PEGylated liposomes [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF] are applied 1 hour and 2 days, respectively before trauma.

However, one must bear in mind that these conditions must reproduce real life clinical practice. This is not the case for noise-induced hearing loss.

Efficacy of the drug-loaded nanocarrier over free drug solution

The duration of studies evaluating the efficacy of drug-loaded nanocarriers in suspension is quite short (3 days to 1 week). Indeed, it is adapted to the duration of exposure to the drug after intratympanic administration of nanocarrier suspension (see section 5.1.3).

The benefit of drug-loaded nanocarriers compared to drug solution has been demonstrated for most of the nanocarriers (Table 5). SiRNA-loaded cationic liposomes [START_REF] Maeda | In vitro and in vivo suppression of GJB2 expression by RNA interference[END_REF], self-assembled nanoparticles [START_REF] Martín-Saldaña | pH-sensitive polymeric nanoparticles with antioxidant and anti-inflammatory properties against cisplatin-induced hearing loss[END_REF], 2017[START_REF] Shi | Pathophysiology of the cochlear intrastrial fluid-blood barrier (review)[END_REF], dexamethasone-loaded PEGylated nanoemulsions [START_REF] Yang | Optimized phospholipidbased nanoparticles for inner ear drug delivery and therapy[END_REF], dexamethasone-loaded PEGylated PLGA nanoparticles [START_REF] Sun | A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration[END_REF][START_REF] Wang | A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss[END_REF] and edaravone-loaded solid lipid nanoparticles [START_REF] Gao | Solid lipid nanoparticles loaded with edaravone for inner ear protection after noise exposure[END_REF] significantly improve hearing (> 10 dB compared to the control group) in different animal models (Table 5).

Nanocarrier efficacy is generally explained by the rise in drug concentration in the inner ear and the drug residence time (Jero et al., 2001b;[START_REF] Maeda | In vitro and in vivo suppression of GJB2 expression by RNA interference[END_REF][START_REF] Sun | A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration[END_REF]. However, these factors are not always related. For example, dexamethasone-loaded nanoemulsions are more efficient in protecting against kanamycin ototoxicity compared to the free drug, despite equivalent inner ear drug delivery [START_REF] Yang | Optimized phospholipidbased nanoparticles for inner ear drug delivery and therapy[END_REF].

Among the factors favoring the efficacy of nanocarriers compared to the free drug, the drug loading and final dose administered are very important. A 50 µg dose of encapsulated dexamethasone is more efficient against cisplatin ototoxicity compared to the same dose of free drug [START_REF] Sun | A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration[END_REF] whereas 0.6 µg of drug (either encapsulated in the nanoparticles or free) is not more efficient in another study with non-targeted PLGA nanoparticles [START_REF] Wang | A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss[END_REF]. For drugs other than corticoids, the dose administered in the control group receiving the free drug is rarely documented [START_REF] Gao | Solid lipid nanoparticles loaded with edaravone for inner ear protection after noise exposure[END_REF][START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF][START_REF] Mohan | Targeted Amelioration of Cisplatin-Induced Ototoxicity in Guinea Pigs[END_REF].

Additional efficacy of advanced strategies

We can highlight a trend of recent studies to test the efficacy of hydrogels containing nanocarriers (Table 5). The duration of these studies is longer, more than 1 week compared to those done with nanocarriers alone (see section 5.3). Two hybrid systems combining a hydrogel (chitosan or hyaluronic acid) with drug-loaded PEGylated liposomes protect the cochlea in different models [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF][START_REF] Mamelle | Effect of a liposomal hyaluronic acid gel loaded with dexamethasone in a guinea pig model after manual or motorized cochlear implantation[END_REF]. However, the benefit of hybrid systems over hydrogels has not been assessed because the hydrogel group is not present in animal studies [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF][START_REF] Mamelle | Effect of a liposomal hyaluronic acid gel loaded with dexamethasone in a guinea pig model after manual or motorized cochlear implantation[END_REF]. This is important especially for drugs like D-c-Jun kinase inhibitor-1 which cross the round window membrane easily [START_REF] Eshraghi | Preclinical and clinical otoprotective applications of cell-penetrating peptide D-JNKI-1 (AM-111)[END_REF]. Indeed, the drug release has been shown to be prolonged for hyaluronic acid gel containing PEGylated liposomes [START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF]. However, most of the reports do not perform drug pharmacokinetic studies [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF][START_REF] Mohan | Targeted Amelioration of Cisplatin-Induced Ototoxicity in Guinea Pigs[END_REF].

The active targeting of outer hair cells is an efficient strategy [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF][START_REF] Wang | A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss[END_REF]. Drug-loaded targeted nanocarriers provide significant protection against noise or cisplatin ototoxicity. Targeted nanocarriers do not seem to modify the drug release in the perilymph, but they specifically accumulate in the outer hair cells [START_REF] Wang | A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss[END_REF]. For the chitosan gel containing targeted liposomes, the small fraction of targeted liposomes reaching the perilymph (5%) is sufficient to protect the outer hair cells from noise [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF].

Another strategy, the magnetic delivery of methylprednisolone-loaded PLGA nanoparticles [START_REF] Ramaswamy | Magnetic Nanoparticle Mediated Steroid Delivery Mitigates Cisplatin Induced Hearing Loss[END_REF], has demonstrated its superiority over drug-loaded nanoparticles alone, with a low dose of drug administered compare to other studies (Table 5). This is coherent with the prolonged drug release and the high drug/nanocarrier concentration achieved in pharmacokinetic studies with similar systems [START_REF] Du | Magnetic targeted delivery of dexamethasone acetate across the round window membrane in guinea pigs[END_REF][START_REF] Kopke | Magnetic Nanoparticles: Inner Ear Targeted Molecule Delivery and Middle Ear Implant[END_REF].

To conclude, all these strategies seem to enhance drug efficacy, by raising the drug concentration in the perilymph and the duration of drug exposure [START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF][START_REF] Ramaswamy | Magnetic Nanoparticle Mediated Steroid Delivery Mitigates Cisplatin Induced Hearing Loss[END_REF].

However, the safety of such systems has not always been evaluated [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF][START_REF] Wang | A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss[END_REF].

Conclusion

Nanocarriers for inner ear therapy have undergone rapid progress over this last decade, particularly in combination with advanced strategies. They sustain the drug delivery to the inner ear after intratympanic administration, whether they cross the round window membrane [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF][START_REF] Ramaswamy | Magnetic Nanoparticle Mediated Steroid Delivery Mitigates Cisplatin Induced Hearing Loss[END_REF] or accumulate in this barrier [START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF]. The size and surface composition of the nanocarriers are key parameters for their passage across this barrier. When evaluated, the improved efficacy of the nanocarrier is related to the sustained release of the drug (Table 5). This review highlights a clear trend towards the development of nanocarriers incorporated in hydrogels [START_REF] Dai | A novel vehicle for local protein delivery to the inner ear: injectable and biodegradable thermosensitive hydrogel loaded with PLGA nanoparticles[END_REF][START_REF] Ding | Is oval window transport a royal gate for nanoparticle delivery to vestibule in the inner ear?[END_REF][START_REF] El Kechai | Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear[END_REF][START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF], coated with a targeting ligand [START_REF] Kayyali | A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss[END_REF][START_REF] Wang | A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss[END_REF], or using a method to increase the permeability of the round window membrane [START_REF] Cai | Engineering PLGA nano-based systems through understanding the influence of nanoparticle properties and cell-penetrating peptides for cochlear drug delivery[END_REF][START_REF] Ramaswamy | Magnetic Nanoparticle Mediated Steroid Delivery Mitigates Cisplatin Induced Hearing Loss[END_REF][START_REF] Yoon | Intratympanic delivery of oligoarginine-conjugated nanoparticles as a gene (or drug) carrier to the inner ear[END_REF]. These advanced strategies have proved to be efficient in long-term studies in different animal models and pave the way for the development of new drug delivery systems for the inner ear.

Nanocarriers appear to be globally safe for the inner ear. However, the toxicity at long term of some promising systems such as PLGA nanoparticles [START_REF] Sun | A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration[END_REF][START_REF] Wen | Nanomedicine strategy for optimizing delivery to outer hair cells by surface-modified poly(lactic/glycolic acid) nanoparticles with hydrophilic molecules[END_REF] needs to be evaluated for side effects due to nanocarrier degradation. In the future, cross-sectional studies evaluating nanocarrier biodistribution, drug delivery, safety and efficacy must be developed.

Sample CRediT author statement

Fig. 1 :

 1 Fig. 1: A) Anatomy of the middle and inner ear, B) Anatomy of the cochlear canals. B: reprinted from Maynard and Downes (2019) with permission from Elsevier.

Fig. 2 :

 2 Fig. 2: Nanocarriers used for inner ear applications. PEG, poly(ethylene glycol); SPION, superparamagnetic iron oxide nanoparticles.

Fig. 3 :

 3 Fig. 3: Nanocarrier pathway mechanisms across middle ear barriers.

  895

Céline JAUDOIN :

 : Conceptualization, Methodology, Investigation, Visualization, Writingoriginal draft. Florence AGNELY: Conceptualization, Supervision, Writing -review & editing, Funding acquisition, Project administration. Yann NGUYEN: Writing -review & editing. Evelyne FERRARY: Writing -review & editing. Amélie BOCHOT: Conceptualization, Supervision, Writing -review & editing, Funding acquisition, Project administration.

Table 1 :

 1 Routes of administration to deliver drugs to the inner ear: benefits and limitations

	Administration	Benefits	Limitations
	Systemic or	-Easy to use in clinic with medical staff	-Blood-labyrinth barrier and temporal bone
	oral	(intravenous) or without medical staff (oral)	localization do not allow high molecular drug
			passage
			-Small lipophilic molecules only
			-Poor drug efficacy
			-Very low drug concentration in inner ear
			-High doses required
			-Huge systemic exposure leading to side effects
	Intracochlear	-Minimized systemic exposure	-Highly invasive
		-Long-term local drug delivery possible (several	-Small volume injected
		weeks to years)	-Requires hospitalization and highly specialized
		-Avoid inner ear barriers, direct access to	medical staff
		cochlea	-Potential toxicity of a high drug concentration in the
		-Drugs can be delivered from electrode array	cochlea
		coating	-Risk of introducing pathogens in the inner ear
		-Adapted for liquid formulations and medical	-Risk of hearing trauma
		devices
		-Useful for safety evaluation and drug efficacy
		in preclinical studies
	Intratympanic	-Minimized systemic exposure	-Requires diffusion through middle ear barriers to
		-Short and middle term local drug delivery	access the cochlea
		(several days to weeks)	-High inter-individual variability (variable thickness
		-Minimally invasive
		-Usually an outpatient procedure
		-Adapted for conventional liquid dosage forms,
		hydrogels, nanocarriers and medical devices
		-Repeated injections possible

Table 2 :

 2 Biodistribution of nanocarriers and pharmacokinetics after intratympanic administration

	Nanocarrier	Nanocarrier	Drug/Tracer	Study design Administration	Nanocarrier biodistribution and	Detection/	Detection/	Hypothesis on	Reference
		characteristics				pharmacokinetics	quantification	quantification	nanocarrier pathway
							method of the	method of the	mechanism
							nanocarrier	drug/tracer
	Liposomes
	Cationic liposome	ns	Plasmid leading to human GFP cell expression	Mouse n = 2/group 3 days	Sponge applied on RWM for 3 days	Gene expression in spiral ganglion, Reissner's membrane, organ of Corti, spiral limbus.		Fluorescence (not quantified)		(Jero et al., 2001b)
	Cationic	ns	Plasmid leading	Mouse	Sponge applied	Gene expression in spiral ganglion, Reissner's		Fluorescence	Cationic charge of
	liposome		to GFP cell expression	n = 1/group 3 days	on RWM for 3 days	membrane, spiral limbus, organ of Corti and vestibular hair cells.		(not quantified)	liposomes may facilitate RWM	(Jero et al., 2001a)
						Higher expression at cochlea base.			passage.
	PEGylated	130 ± 20 nm	Gadolinium-	Rat	TTI	3 h after administration: gadolinium-DOTA detected		Magnetic	Main passage by oval
	liposomes		DOTA	n = 14 for the whole study 2 days		in vestibule and first turn of cochlea. Detection in perilymph but not in endolymph compartment. 6 h: diffusion to second turn of cochlea. No more 21% global passage of gadolinium-DOTA from detection at day 1.		resonance imaging (semi-quantified)	window membrane.	(Zou et al., 2010a)
						middle ear to inner ear.
	PEGylated liposomes	95 ± 10, 130 ± 10 and	Gadolinium-DOTA,	Rat n = 11/group			Fluorescence (not quantified),	Magnetic resonance	Size-dependent passage. Both round
		240 ± 15 nm	TRITC-labeled	2 days			cryoTEM on	imaging	and oval windows
							perilymph	(semi-quantified)	passage. Paracellular pathway.	(Zou et al., 2012b)
	Cationic	105 ± 15 nm	Gadolinium-	Rat	Application on		Fluorescence	Magnetic	RWM application:
	PEGylated liposomes	PdI = 0.04 +14 mV	DOTA or indocarbocyanine	n = 14 for the whole study	RWM or ossicular chain		(not quantified)	resonance imaging	accumulation of cationic PEGylated
			dye	1 day				(semi-quantified)	liposomes inside
									RWM.
									Ossicular chain
									application: cationic
						Ossicular chain application: Gadolinium-DOTA and the round window membranes. Liposomes in the utricle of liposomes, that might diffuse from liposomes in the annular ligament. Slight detection region of the chondrocytes. No detection of localized in the footplate, in nuclei and perinuclear through the ossicular chain to reach both the oval detected in scala tympani at 3 h. Liposomes diffuse			stapes. chondrocytes of the liposomes in the Accumulation of window membrane. PEGylated liposomes do not cross oval	(Zou et al., 2014b)
						the cochlea.

TTI

95 nm-liposomes observed in perilymph without loss of integrity. No more liposome detection at day 2. Gadolinium-DOTA transport in inner ear depends on liposome size: 95 nm > 130 nm > 240 nm Gadolinium-DOTA found mostly in ossicular chain, then in vestibule and scala vestibuli. Liposomes found 6 h after administration in utricle, spiral ligament and spiral ganglion.

For both application sites, gadolinium-DOTA detected in cochlea but not in the vestibule and in endolymph compartment. No more detection of gadolinium-DOTA at day 1.

RWM application: Gadolinium-DOTA detected at 3 h, mainly in the RWM, with a decreasing gradient from basal to apex turns of the cochlea. ns, not specified; cryoTEM, transmission electron cryomicroscopy; GFP, green fluorescent protein; PdI, polydispersity index; PEG, poly(ethylene glycol); RWM, round window membrane; TRITC, tetramethylrhodamine isothiocyanate; TTI, transtympanic injection.

Table 2 :

 2 Biodistribution of nanocarriers and pharmacokinetics after intratympanic administration (continued)

	Nanocarrier	Nanocarrier	Drug/Tracer	Study design Administration	Nanocarrier biodistribution and	Detection/	Detection/	Hypothesis on	Reference
		characteristics				pharmacokinetics	quantification	quantification	nanocarrier pathway
							method of the	method of the	mechanism
							nanocarrier	drug/tracer
	Polymersomes
	PEGylated	82 nm,	Rhodamine-lipid	Mouse	Sponge applied	Both nanocarriers found in the cytoplasm of the	Fluorescence	Disulfiram	Liposomes cross
	liposomes	PdI = 0.05	labeled, disulfiram	n = 30 2 weeks	on RWM for 2 weeks.	spiral ganglion neurons of each turn, hair cells, spiral ligament, stria vascularis, and in all layers of RWM	(not quantified)	toxicity on hearing thresholds	RWM. They reach Rosenthal canal
	Versus		(100 ng)			at day 1.		(quantified)	through the porous
	PEGylated	90 nm	Indocarbocyanine dye			Same distribution for both nanocarriers but liposomes are present in higher number in spiral ganglion (45% versus 35% of neuron cytoplasms at			spiral lamina and spread to other turns of the cochlea.	(Buckiová et al., 2012)
	polymersome		and disulfiram (100 ng)			day 2). High accumulation of polymersomes in RWM outer			cross the RWM. Polymersomes did not
						layer. Liposomes more efficient than polymersomes
						to deliver disulfiram.
	PEGylated	83 ± 17 nm Indocarbocyanine	Rat			Fluorescence
	polymersomes		dye	n = 16 for the			(not quantified)
				whole study 3 to 5 days						(Zhang et al., 2010)
	PEGylated polymersomes	63 ± 10 nm Indocarbocyanine dye	Rat n = 14 for the	Sponge applied on RWM for		Fluorescence (semi-quantified)		Polymersomes failed to cross RWM outer	(Y. Zhang
				whole study	3 days or TTI				layer. Effective	et al.,
				3 days					transport through oval window with TTI.	2011b)
	Poly(amino	27 ± 16 nm	FITC-labeled and	Mouse	Sponge applied		Fluorescence	Fluorescence
	acid)-based polymersomes	-35 mV	Nile red	n = 4/group 1 day	on RWM for 1 day		(not quantified)	(not quantified)		(Kim et al.,
										2015)

TTI

Polymersomes accumulation in the outer layer of RWM. No polymersomes in connective tissue layer.

Higher number of polymersomes in cochlea, 5 days after administration. Distribution in stria vascularis, basilar membrane, spiral ganglion and vestibule. No detection of polymersomes in endolymph.

RWM: polymersomes only found in the outer layer. TTI induces 3-fold more polymersome passage in the inner ear than the sponge vehicle. Vestibule: TTI induces 2.5-fold more polymersome passage than with the sponge vehicle.

Few polymersomes nearby inner hair cells and supporting cells within the organ of Corti. Few polymersomes found in modiolus, without loss of integrity. Nile red released in inner hair cells, but not in supporting cells. Sparse fluorescence in modiolus.

FITC, fluorescein isothiocyanate; LC-MS, liquid chromatography-mass spectrometry; PdI, polydispersity index; PEG, poly(ethylene glycol); RWM, round window membrane; TTI, transtympanic injection.

Table 2 :

 2 Biodistribution of nanocarriers and pharmacokinetics after intratympanic administration (continued)

	Nanocarrier	Nanocarrier	Drug/Tracer	Study design Administration	Nanocarrier biodistribution and	Detection/	Detection/	Hypothesis on	Reference
		characteristics				pharmacokinetics	quantification	quantification	nanocarrier pathway
							method of the	method of the	mechanism
							nanocarrier	drug/tracer
	Lipid-based nanocarriers
	PEGylated lipid	52 ± 5 nm	Indocarbocyanine	Rat	Sponge applied	30 min: nanocapsules found in spiral ganglion and	Fluorescence		Paracellular pathway
	nanocapsules	-55 ± 7 mV	dye or Nile red, rhodamine or	n = 5/point 1 week	on RWM for 30 min	hair cells without loss of integrity. Nanocapsules that lose their integrity found in nerve fibers and lateral	(semi-quantified)		of lipid nanocapsules through RWM.
			FITC-lipid			wall.			Increasing the
			labeled			1 h: nanocapsules found in inner and outer hair cells without loss of integrity. Nanocapsules lose their			residence time of the sponge induces an
						integrity in nerve fibers of organ of Corti and spiral			increase in nanocarrier	(Zou et al.,
						ligament. Day 1: nanocapsules found in the paracellular			accumulation in RWM.	2008)
						pathway of the outer layer of RWM, with a fraction
						that lost its integrity. Day 7: low detection of nanocapsules in spiral
						ganglion but high detection in hair cells and nerf
						fibers of organ of Corti.
	Lipid		Dexamethasone	Mouse			Fluorescence	Dexamethasone	PEGylation promotes
	nanoemulsions:		(4.2 µg)	Number ns			(semi-quantified)	immunostaining	the diffusion of
	-Cationic PEGylated	280 nm 0 mV	or Nile red	3 days				(semi-quantified)	nanocarriers across middle ear mucosa and
	-Cationic -Anionic	225 nm -26 mV +25 mV 211 nm							inner ear barriers.	(Yang et al., 2018)
	-Neutral	190 nm -4 mV
	PLA or PLGA-based nanoparticles
	PLGA nanoparticles	140-180 nm	Rhodamine B	Guinea pig n = 4/group 1 day	Sponge applied on RWM for 1 day	Nanoparticles present in RWM and in round window niche. Nanoparticles mostly in the basal turn of cochlea, in scala tympani and basilar membrane.	Fluorescence (not quantified)			(Tamura et al., 2005)
	PLGA	Labelled	Coumarin-6 or	Guinea pig	RWM	Coumarin-6 released in perilymph up to 6 h by the		UV-quantification	Bioadhesion of PLGA
	nanoparticles	nanoparticles:	salvianolic acid B	n = 168 for the	application	nanoparticles, Cmax = 201 ng/mL versus 18 ng/mL		of coumarin-6,	nanoparticles on the
		135 nm PdI = 0.17	(2 mg), tanshinone IIA	whole study 4 days		for the coumarin-6 solution. Multidrug release: up to 16 h in perilymph, for drug		HPLC-UV for the different drugs	outer surface of the RWM.
		Drug-loaded nanoparticles:	(0.1 mg), panax notoginsenoside (3 mg)			solution and drug-loaded nanoparticles. Cmax in perilymph for drugs-loaded PLGA nanoparticles versus drug solution: salvianolic acid		(quantified)		(Cai et al., 2014)
		154 nm PdI = 0.01				(350 versus 680 µg/mL), tanshinone (10 versus 36 µg/mL),
						panax notoginsenoside (~1000 versus ~100 µg/mL
						for each metabolites)
	ns, not specified; LC-MS, liquid chromatography-mass spectrometry; PdI, polydispersity index; PEG, poly(ethylene glycol); PLA, polylactic acid; PLGA, poly(lactic-co-glycolic acid); RWM, round window membrane; TTI, transtympanic injection.

TTI

24 h after administration: cationic PEGylated nanoemulsions found in inner hair cells (concentration 2-fold higher compared to Nile red solution and cationic, anionic and neutral nanoemulsions). Release of dexamethasone by cationic PEGylated nanoemulsions to the inner ear equivalent to the dexamethasone phosphate solution which is more concentrated (dose ≈ 150 µg).

Table 2 :

 2 Biodistribution of nanocarriers and pharmacokinetics after intratympanic administration (continued)

	Nanocarrier	Nanocarrier	Drug/Tracer	Study design Administration	Nanocarrier biodistribution and	Detection/	Detection/	Hypothesis on	Reference
		characteristics				pharmacokinetics	quantification	quantification	nanocarrier pathway
							method of the	method of the	mechanism
							nanocarrier	drug/tracer
	PLGA		Coumarin-6	Guinea pig	TTI	Coumarin-6 in:		Fluorescence	PLGA nanoparticle
	nanoparticles:			Number ns				(semi-quantified)	surface plays a key
	-Uncoated	158 nm PdI = 0.12		1 day		-Uncoated: very low signal in outer hair cells (1 arbitrary unit)			role. Hydrophilic coating prevents
	-PEGylated -Poloxamer-coating coating -Chitosan-	135 nm PdI = 0.3 PdI = 0.17 170 nm PdI = 0.11 155 nm				-PEGylated: spiral ganglion, outer hair cells 3 turns (1.25 arbitrary units), gradient from base to apex -Poloxamer-coating: spiral ganglion, outer hair cells (2 arbitrary units), stria vascularis, of the			clearance from middle Poloxamer decreases ear by the ciliated epithelia by blocking interactions. hydrophobic	(Wen et al., 2016)
						-Chitosan-coating: spiral ganglion, spiral ligament, very low signal in outer hair cells			micro-viscosity, creating pores in outer
						(1 arbitrary unit)			hair cells.
	PLGA	PdI <2	Indocarbocyanine	Guinea pig	TTI	Effect of size:	Fluorescence		Size-dependent and
	nanoparticles: -Uncoated	155 ± 5 nm, 84 ± 4,	dye	n = 3/point 1 day		nanoparticles. 0.5 h: higher passage of 300 nm-nanoparticles in cochlea compared to 150 nm and 80 nm-sized	(semi-quantified)		hydrophilic-dependent pathway to cochlea.
	-PEGylated	292 ± 15 nm -8 mV 135 ± 5 nm				24 h: same quantification for all. Effect of surface: 0.5 h: chitosan-coated nanoparticles passage in				(Cai et al., 2017)
	-Poloxamer-coating	-12 mV 185 ± 10 nm				cochlea is 2-fold higher than other PLGA-nanoparticles.
	-Chitosan-coating	-16 mV 170 ± 15 nm +18 mV				24 h: passage in cochlea more important for poloxamer > PEGylated ≈ chitosan > uncoated nanoparticles.
	PLGA	160 nm	Coumarin-6	Guinea pig	TTI for 10 to	Nanoparticles present in perilymph 30 min after TTI,	TEM on		Nanoparticles enter in
	nanoparticles	PdI = 0.19 -12 mV		10 to 60 min n = 3/point	60 min	without loss of integrity. Coumarin-6 intensity increases in RWM from 10 to 30 min, and then	perilymph and RWM samples		the inner ear in a concentration-
						drops at 60 min. Raising the concentration of	(not quantified),		dependent manner.
		[PLGA] tested: 10, 30 and				administered nanoparticles increases the amount of nanoparticles inside the RWM.	confocal imaging on RWM		Pathway involved: macropinocytosis,
		90 mg/mL				Nanoparticles are internalized in lysosomes to be	(not quantified),		caveolae-mediated
						degraded but also transported by exocytosis out of the cells. Specific pathway mechanisms involved:	HPLC-fluorescence detector of		endocytosis pathway, exocytosis mediated by endoplasmic	(Zhang et al., 2018)
						macropinocytosis, caveolae-mediated endocytosis pathway, exocytosis mediated by endoplasmic	coumarin-6 in perilymph		reticulum, Golgi apparatus and
						reticulum, Golgi apparatus and recycling endosomes.	(quantified)		recycling endosomes.
						Pathway mechanisms not involved: clathrin-mediated endocytosis, paracellular transport.			Pathway not involved: clathrin-mediated
									endocytosis,
									paracellular transport.

ns, not specified; PdI, polydispersity index; PEG, poly(ethylene glycol); PLGA, poly(lactic-co-glycolic acid); RWM, round window membrane; TEM, transmission electron microscopy; TTI, transtympanic injection.

Table 2 :

 2 Biodistribution of nanocarriers and pharmacokinetics after intratympanic administration (continued)

	Nanocarrier	Nanocarrier	Drug/Tracer	Study design Administration	Nanocarrier biodistribution and	Detection/	Detection/	Hypothesis on	Reference
		characteristics				pharmacokinetics	quantification	quantification	nanocarrier pathway
							method of the	method of the	mechanism
							nanocarrier	drug/tracer
	PEGylated PLA	130 nm	Dexamethasone	Guinea pig	RWM	1 h after RWM application: strong coumarin-6		LC-MS in	Rapid accumulation of
	nanoparticles	PdI = 0.1	(50 µg) or	n = 3-5/point	application	detection in stria vascularis, organ of Corti, spiral		perilymph	coumarin-6 in organ of
		-26 mV	coumarin-6	1 h to 2 days		ganglion in each cochlea turn. Higher dexamethasone concentration (from		(quantified), fluorescence	Corti suggesting easy passage of RWM.	(Sun et al.,
						8 500 ng/mL at 1 h to 300 ng/mL at 48 h) with		(not quantified)		2015)
						nanoparticles compared with dexamethasone phosphate solution (13 000 ng/mL at 1 h to
						850 ng/mL at 6 h).
	Miscellaneous
	Oleic acid and	12 ± 0.5 nm		Rat	Sponge applied	Slight passage through the RWM after sponge	Magnetic
	poloxamer 407-	Neutral charge		n = 23 for the	on RWM for	application. Nanoparticles released in inner ear up to	resonance
	coated iron oxide nanoparticles			whole study 1 week	1 week Without magnet delivery	3 days. Nanoparticles found only in perilymphatic space of the saccule.	imaging (semi-quantified), Prussian blue			(Zou et al., 2010b)
							staining (not quantified)
	Polyvinyl-	117 ± 24 nm		Rat	TTI	At 24 h, nanoparticles found in RWM, oval window	Micro-		Nanocarrier entrance
	pyrrolidone	-20 ± 9 mV		n = 2/point		and scala tympani. No more detection of	tomography		in inner ear by both	(Zou et al.,
	stabilized silver nanoparticles			1 week		nanoparticles in the inner ear at 1 week.	(semi-quantified)		oval and round window membranes.	2015)
	Maghemite	50-60 nm		Rat	TTI	High accumulation of nanoparticles in RWM and	Magnetic		Accumulation and
	nanoparticles	+55 mV		n = 6 for the whole study	Without magnet delivery	oval window up to day 14. High nanoparticle accumulation in the basal turn of	resonance imaging		diffusion of nanoparticles in both
				2 weeks		cochlea and vestibule 3 h post-TTI, starting to	(semi-quantified),		oval and round	(Zou et al.,
						decrease at 6 h. Only a few nanoparticles in scala media. Slight detection of nanoparticles at day 1.	Maghemite staining of RWM		window membranes.	2017b)
							and oval window
							(not quantified)
	Cubic glyceryl monooleate-	211 ± 23 nm PdI = 0.18	FITC-labeled enzyme or	Guinea pig n = 8 for the	TTI	Cubosomes found in RWM and basal turn of scala tympani, 30 min after administration.	Fluorescence (not quantified)	Fluorescence (quantified)	Cubosomes entrance in the inner ear by the
	based	-27 mV	octadecyl	whole study		Labeled enzyme found in perilymph up to 24 h with			RWM.	(Liu et al.,
	cubosomes		rhodamine B	1 day		cubosomes and 12 h in solution. Cmax = 4 µg/mL with cubosomes against 2 µg/mL with the enzyme				2013b)
						solution.
	Bovine serum albumin-loaded cubosomes	215 nm PdI = 0.08 -23 mV	Nerve-growth factor	Guinea pig Number ns 1 day	Sponge applied on RWM for 1 day	At 2 h, Cmax = 13 ng/mL for cubosomes and 2 ng/mL for solution. No more nerve-growth factor in perilymph at 6 h.		ELISA test (quantified)		(Bu et al., 2015)
	Bovine serum albumin -	636 nm +5 mV	Rhodamine B	Guinea pig n = 1 for the	TTI	Nanoparticles adhere to the RWM surface. Rhodamine B seems to be more abundant in the	Scanning electron microscopy of the	Fluorescence (not quantified)	Deposit of nanoparticles on the	(Yu et al.,
	nanoparticles			whole study		inner ear with nanoparticles than with solution	RWM surface.		outer surface of the	2014)
				3 days		3 days after administration.			RWM.

ns, not specified; ELISA, enzyme-linked immunosorbent assay; PdI, polydispersity index; PEG, poly(ethylene glycol); PLA, polylactic acid; RWM, round window membrane; TTI, transtympanic injection.

Table 2 :

 2 Biodistribution of nanocarriers and pharmacokinetics after intratympanic administration (continued)

	Nanocarrier	Nanocarrier	Drug/Tracer	Study design Administration	Nanocarrier biodistribution and	Detection/	Detection/	Hypothesis on	Reference
		characteristics				pharmacokinetics	quantification	quantification	nanocarrier pathway
							method of the	method of the	mechanism
							nanocarrier	drug/tracer
	Dendriplexes	132 ± 20 nm	Plasmid	Rat	Sponge applied	Cyclodextrin modified dendriplexes: 48% and 82%		Fluorescence
	modified or not	PdI = 0.15	containing Atoh1	n = 17 for the	on RWM for	of, respectively inner and outer hair cells showed		(semi-quantified),
	with cyclodextrins	+31 mV	and GFP gene	whole study 1 week	1 week	GFP expression. Atoh1 gene expressed in cochlea sensory epithelium.		western blot and RT-PCR		(Wu et al.,
	versus	300-600 nm				Non-modified dendriplexes: expression < 10% in		(not quantified)		2013)
	Polyethylen-imine /DNA					hair cells. Polyplexes: expression < 1% in hair cells.
	polyplexes
	Hyper-branched polylysine	73 nm PdI = 1.9	FITC-labeled	Rat n = 6 for the whole study 1 day	Sponge applied on RWM for 1 day	Distribution gradient through the 3 layers of RWM. Perinuclear and in vesicle localization in outer layer and connective tissue. Abundant nanocarriers in organ of Corti and spiral ligament, few in spiral ganglion.	Fluorescence (not quantified)		Outer layer of RWM: transport to nucleus via nucleolin binding at cell surface.	(W. Zhang et al., 2011)
	Nanoparticles	128-175 nm	Coumarin-6	Rat	TTI	At 2 h, nanoparticles based on tocopherol or	Fluorescence
	based on drug-conjugated			n = 2/group 2h		tocopheryl succinate observed in inner and outer hair cells. Decreasing gradient from basal to apical turn.	(not quantified)			(Martín-
	polymer					Nanoparticles based on tocopheryl succinate and				Saldaña et
	(tocopherol, tocopheryl					ibuprofen found in organ of Corti. They lose their integrity because coumarin-6 is released.				al., 2018, 2017, 2016)
	succinate,
	ibuprofen)

FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; PdI, polydispersity index; RT-PCR, reverse transcription polymerase chain reaction; RWM, round window membrane; TTI, transtympanic injection.

Table 3 :

 3 Advanced approaches for nanocarrier and drug delivery to the inner ear

	Nanocarrier	Nanocarrier	Drug	Study design Administration	Nanocarrier biodistribution and pharmacokinetics	Detection/	Detection/	Reference
		characteristics	/Tracer				quantification	quantification
							method of the	method of the
							nanocarrier	drug
	Hydrogels containing nanocarriers
	Chitosan gel containing PEGylated	160 nm PdI = 0.16	Carboxy-fluorescein,	Mouse Number ns	RWM application	Liposomes found in perilymph after liposomal gel application, 2-fold more than with liposome suspension.	Fluorescence (semi-	Fluorescence (semi-	(Lajud et al., 2015)
	liposomes		rhodamine-	1 day		Liposomes found in organ of Corti, without loss of their integrity.	quantified)	quantified)
			labeled lipid
	Hyaluronic acid gel	146 ± 50 nm	Dexamethasone	Guinea pig	TTI	Liposome accumulation inside RWM at day 2, in the perinuclear	Fluorescence	LC-MS on
	containing PEGylated	PdI = 0.1	phosphate	n = 42 for the		region of fibroblasts. Low number of intact liposomes in perilymph	(semi-	perilymph
	liposomes	-29 mV	(1.5 mg) or rhodamine-labeled lipid	whole study 1 month		at day 2 (0.00003% of initial lipid concentration administered). Dexamethasone and dexamethasone phosphate quantified in perilymph up to 1 month with the liposomal gel. Conversion of (dexamethasone) of 833 ± 382 ng/mL with liposomal gel against dexamethasone phosphate into dexamethasone: Cmax	quantified)	(quantified)	(El Kechai et al., 2016)
						39 ± 23 ng/mL for gel only at day 15.
						Macroscopic gel persistence in middle ear up to 1 week.
	Chitosan gel	280 nm	Lipiodol-	Mouse	RWM	Slight diffusion of lipiodol-iopamidol in scala tympani observed		Micro-	(Kayyali et
	containing anionic	PdI = 0.23	iopamidol	Number ns	application	when using liposomal gel. Higher diffusion of free-iodine contrast		tomography	al., 2017)
	liposomes	-18 mV	49 mg/mL	1 day		agents in cochlea.		(not quantified)
	Chitosan gel containing PLGA	290 ± 2 nm PdI = 0.08	Interferon α-2b (0.12 µg)	Guinea pig n = 38/point	TTI	Cmax: solution > nanoparticles > hydrogel > nanoparticle-loaded hydrogel, respectively 540 > 470 > 305 > 290 ng/mL		ELISA (quantified)
	nanoparticles			4 days		Release up to 70 h and 36 h, respectively for the nanoparticle-loaded hydrogel and other formulations (solution, nanoparticles, hydrogel). Interferon mean residence time increased with			(Dai et al., 2018)
						nanoparticle-loaded hydrogel (23 h) compared to hydrogel (14 h) or
						nanoparticles alone (10 h).
	Chitosan nanocarriers	153 nm	Nile red	Guinea pig	TTI, RWM or	Suspension administered by TTI:	Fluorescence	HPLC-UV after
	dispersed or not in	PdI = 0.14		n = 18 for the	oval window	3-fold more Nile red in vestibule than in cochlea.	(semi-	tissue extraction
	poloxamer gel	+22 mV		whole study 4 h	application	Accumulation in stria vascularis in the cochlea. Presence of nanoparticles in perinuclear and paracellular pathways in oval window. Nanocarriers observed in the outer layer of RWM. RWM application of nanoparticle-loaded hydrogel:	quantified)	(quantified)	(Ding et al., 2019)
						6 ng Nile red/g cochlear tissue.
						Oval window application of nanocarrier-loaded hydrogel:
						12 ng Nile red/g cochlear tissue. High passage in the vestibule.
	Active targeting of nanocarriers
	PEGylated	105 ± 20 nm	Indocarbocyanine	Rat	TTI	Both targeted and untargeted polymersomes found in RWM, spiral	Fluorescence		(Zhang et
	polymersomes coated	-1 mV	dye	n = 27 for the		ligament, scala vestibuli and tympani and Reissner's membrane. No	(not		al., 2012)
	with Tet1 peptide			whole study 3 days		accumulation in cochlear nerve.	quantified)
	ns, not specified; Cmax, maximum drug concentration achieved in perilymph; ELISA, enzyme-linked immunosorbent assay; LC-MS, liquid chromatography-mass spectrometry; PdI, polydispersity index; PEG,
	poly(ethylene glycol); PLGA, poly(lactic-co-glycolic acid); RWM, round window membrane; TTI, transtympanic injection.
	675

Table 3 :

 3 Advanced approaches for nanocarrier and drug delivery to the inner ear (continued) targeted liposomes contained in the liposomal gel found in perilymph at day 2 (50 nM). Both targeted and untargeted liposomes found in outer hair cells of mid and basal turns of cochlea. Targeted liposomes reached the apical turn unlike untargeted ones. Both TdTomato and carboxyfluorescein are expressed by outer hair cells, but also in other tissues, after application of the gel containing targeted liposomes.

	Nanocarrier	Nanocarrier	Drug	Study design Administration	Nanocarrier biodistribution and pharmacokinetics	Detection/	Detection/	Reference
	characteristics	/Tracer				quantification	quantification
							method of the	method of the
							nanocarrier	drug
	PEGylated PLA	157 ± 16 nm	Coumarin-6 or	Guinea pig	RWM	2 h after application, coumarin-6 found in the perinuclear region of		Fluorescence	(Wang et
	nanoparticles coated	PdI = 0.1	dexamethasone	n = 6 for the	application	outer hair cells and in modiolus, to a greater extent for targeted		(not quantified)	al., 2018)
	with A666 peptide	-30 mV	(0.7 µg)	whole study 2 h to 2 days		nanoparticles than for untargeted ones. Nevertheless, dexamethasone releases are not significantly different between		LC-MS for dexamethasone
						A666-covered and untargeted PEGylated PLA nanoparticles with,		concentration in
						respectively 295 and 444 ng/mL in perilymph 2 days after administration.		perilymph (quantified)
	Chitosan gel	87 ± 5 nm	Plasmid encoding	Mouse	RWM	5% of the Fluorescence	Fluorescence	(Kayyali et
	containing PEGylated liposomes coated with		TdTomato and carboxy-	n = 10/group 2 to 5 days	application		in perilymph sample	(not quantified)	al., 2018)
	prestin-targeting		fluorescein,				(quantified)
	peptide-1		rhodamine or cyanine 5-labeled
			lipid
	Cell-penetrating peptides
	Poly(amino acid)-	103 nm	Nile red or GFP	Mouse	Sponge applied	Gene expression found in modiolus and lateral wall at, respectively		Fluorescence	(Yoon et al.,
	based polymersomes coated with	+22 mV	gene	Number ns 1 to 2 days	on RWM for 1 to 2 days	1 and 2 days after administration. Nanoparticles found in organ of Corti, modiolus, and lateral wall. Loss of nanoparticle integrity for a		and GFP immunostaining	2015)
	oligoarginine peptide					fraction of the nanoparticles administered.		(not quantified)
	PLGA nanoparticles
	-Uncoated
	-Poloxamer

coating Nanoparticles mixed or not with low molecular weight protamine

		PdI <2	Indocarbocyanine	Guinea pig	TTI	Low molecular weight protamine increases nanoparticle passage in	Fluorescence	HPLC-	(Cai et al.,
		155 ± 5 nm -13 mV	dye or coumarin-6	n = 3/point 1 day		cochlear tissue. Both uncoated and poloxamer-coated nanoparticles found in spiral ganglion and stria vascularis, but also in organ of	(quantified)	fluorescence detector	2017)
		185 ± 10 nm				Corti for poloxamer-coated nanoparticles.		(quantified)
		-16 mV				Low molecular weight protamine increases the release of coumarin-6 in perilymph from 4 to 6 h for uncoated and poloxamer-coated
						nanoparticles.
	Magnetic delivery
	PLGA nanoparticles	180 nm		Rat	RWM	Nanoparticles that kept their integrity found in perilymph after	TEM on		(Kopke et
	containing oleic acid-coated SPION	PdI = 0.1		n = 8 Guinea pig	application ± magnet	magnetic field application: 51 ± 13 nanoparticles/µm² of TEM images.	perilymph samples and		al., 2006)
				n = 3		2 nanoparticles/µm² of TEM images without magnetic field.	RWM (semi-
				Fresh human temporal bone		Channel of nanoparticles visible in human RWM and presence of nanocarriers in perilymph.	quantified)
				n = 2
				1 h
	ns, not specified; GFP, green fluorescent protein; LC-MS, liquid chromatography-mass spectrometry; PdI, polydispersity index; PEG, poly(ethylene glycol); PLGA, poly(lactic-co-glycolic acid); PLA, polylactic acid;
	RWM, round window membrane; SPION, superparamagnetic iron oxide nanoparticles; TEM, transmission electron microscopy; TTI, transtympanic injection.

Table 3 :

 3 Advanced approaches for nanocarrier and drug delivery to the inner ear (continued)

	Nanocarrier	Nanocarrier	Drug	Study design Administration	Nanocarrier biodistribution and pharmacokinetics	Detection/	Detection/	Reference
		characteristics	/Tracer				quantification	quantification
							method of the	method of the
							nanocarrier	drug
	PLGA nanoparticles	160-280 nm		Chinchilla	TTI ± magnet	PLGA nanoparticle clusters found with or without exposure to	TEM (not		(Ge et al.,
	containing SPION	-20 mV		n = 3/group 40 min		magnetic field in spiral ligament, perilymph, Reissner's membrane, spiral ligament, stria vascularis, hair cells and RWM. Pinocytosis	quantified)		2007)
						observed in RWM for large clusters.
	PLGA nanoparticles containing SPION	483 ± 158 nm -20 mV	Dexamethasone acetate (0.8 µg)	Guinea pig n = 24 for the	RWM application	2-fold increase in dexamethasone concentration in cochlea (soft tissue, RWM, perilymph) using the magnetic field: 90 ng		HPLC-UV (quantified)	(Du et al., 2013)
				whole study	± magnet	quantified/cochlea, 18 µg/mL in perilymph. 10% of the initial dose
				1 h		delivered in 60 min.
	SPION dispersed in a hyaluronic acid gel	135 ± 5 nm		Rat n = 6 for the	RWM application	High deposition of SPION in the first turn of scala tympani. SPION reached the second and third turns of cochlea. SPION present in	Prussian blue staining (not		(Leterme et al., 2019)
				whole study	+ magnet	organ of Corti, stria vascularis, spiral ganglion and modiolus.	quantified)
				1 month		Nanoparticle-loaded hydrogel still present as a layer at the surface of the round window membrane.

Table 4 :

 4 Safety of nanocarriers administered by intratympanic or intracochlear routes

	Nanocarrier	Nanocarrier	Study	Administration	Outcomes regarding safety	Reference
		characteristics	design
	Passive delivery
	Cationic liposomes	ns 0.01 mM	Mouse n = 1/group 1 week	Sponge applied on RWM for 3 days	No histological damage. Slight increase in hearing thresholds.	(Jero et al., 2001a)
	Cationic	ns	Mouse	Sponge applied	No histological damage to the organ of Corti.
	liposomes		n = 2/group 3 days	on RWM for 3 days or intracochlear		(Jero et al., 2001b)
				injection
	Cationic	ns	Guinea pig	Intracochlear	No inflammation, no lymphocyte or	(Wareing
	liposomes	0.005 mM	Number ns	injection or	macrophage infiltration. Fibrosis and	et al.,
			2 weeks	osmotic pump	immunoreactivity at osmotic pump site.	1999)
	Cationic liposome	ns	Mouse	Intracochlear	Destruction of organ of Corti in a dose-	(Staecker
			n = 3/group	injection	dependent manner. Inflammation at injection	et al.,
			3 days		site.	2001)
	Cationic	105 ± 15 nm	Rat	Application on	No cochlear inflammation and no toxicity up
	PEGylated	PdI = 0.04	n = 14 for	the stapes or on	to 20 days.
	liposomes	+14 mV	the whole	the RWM		(Zou et
		1 mM	study	± continuously		al., 2014b)
			1 or 20 days	with osmotic
				pump
	Cationic	105 ± 15 nm	Rat	TTI	No histological damage. No
	PEGylated	PdI = 0.04	n = 4 for the		glycosaminoglycan expression, no apoptotic	(Zou et
	liposomes	+14 mV	whole study		cells. Slight increase in hyaluronic acid	al., 2017a)
		1 mM	1 week		expression in spiral ligament.
	PEGylated	82 nm,	Mice	Sponge applied	No toxicity of nanocarriers on hearing
	liposomes	PdI = 0.05	n = 30 for	on RWM for	thresholds. No histological damage.	(Buckiová
	versus	1 mM	the whole	2 weeks.		et al.,
	PEGylated	90 nm	study			2012)
	polymersomes	3 mg/mL	2 weeks
	Poly(amino acid)-	27 ± 16 nm	Mouse	Sponge applied	No impact on hearing thresholds. Swollen
	based	-35 mV	n = 4/group	on RWM for	middle ear mucosa in nanocarrier group.	(Kim et
	polymersomes		1 day to	1 day		al., 2015)
			1 week
	PEGylated lipid	52 ± 5 nm	Guinea pig	Intracochlear	No hair cell loss.
	nanocapsules	-55 ± 7 mV	n = 15 for	injection	No hearing thresholds difference between
	versus	1015	the whole		groups but 10 dB global average loss at	(Scheper
	Hyperbranched	particles/mL	study		day 28.	et al.,
	polylysine	10 nm	1 month			2009)
		PdI = 10
		0.04 mM
	PEGylated lipid nanocapsules	52 ± 5 nm 5 ± 1 mV 20.5 mg/mL	Rat Number ns 1 month	Sponge applied on RWM for 3 days	No impact on hearing thresholds at day 7 and day 28. No apoptosis induction. Less innervation of outer hair cells but no impact for inner hair cells.	(Y. Zhang et al., 2011b)
	PLGA		Guinea pig	TTI	No inflammation in the inner ear.
	nanoparticles:	25 mg/mL	Number ns
	-Uncoated	PdI = 0.12 158 nm	1 day
	-PEGylated	135 nm PdI = 0.17				(Wen et al., 2016)
	-Poloxamer-covered	170 nm PdI = 0.11
	-Chitosan-covered	155 nm PdI = 0.3
	ns, not specified; PdI, polydispersity index; PEG, poly(ethylene glycol); PLGA, poly(lactic-co-glycolic acid); RWM, round
	window membrane; TTI, transtympanic injection.

Table 4 :

 4 Safety of nanocarriers administered by intratympanic or intracochlear routes (continued)

	Nanocarrier	Nanocarrier	Study	Administration	Results outcomes regarding safety	Reference
		characteristics	design
	Nanoparticles	128-175 nm	Rat	TTI	No impact on hearing thresholds.
	based on drug-conjugated polymer (tocopherol, tocopheryl succinate,		n = 2/group 3 days			(Martín-Saldaña et al., 2018, 2017, 2016)
	ibuprofen)
	Silver	117 ± 24 nm	Rat	TTI	Barrier permeability changes. Dose-dependent
	nanoparticles	-20 ± 9 mV 0.02-4 mg/mL	n = 14 for the whole study		hearing loss, but reversible 5 h post-administration. At 4 mg/mL, 30 dB loss at all frequencies. Cell death in tissues at low doses.	(Zou et al., 2014a)
			1 week
	Streptavidin-covered SPION	200 nm 3.10 10 -1.5.10 12	Guinea pig n = 28 for	Intracochlear injection	No impact on hearing thresholds.	(Nguyen
		particles/mL	the whole			et al.,
			study			2016)
			1 week
	Advanced delivery
	Hyaluronic acid	146 ± 50 nm	Guinea pig	TTI	No impact on hearing thresholds.
	gel containing	PdI = 0.1	n = 42 for			(El Kechai
	PEGylated	-29 mV	the whole			et al.,
	liposomes	80 mM	study			2016)
			1 month
	A666 peptide-covered gold nanoparticles in hydrogel	52 nm	Mouse Number ns 1 day	TTI	No histological damage induced on hair cells. (Kayyali et al., 2017)
	PLGA		Guinea pig	TTI	No histological change induced on organ of
	nanoparticles		n = 3/group		Corti, spiral ganglion, stria vascularis and
	±		1 day		RWM after both nanoparticle and low
	mixed with protamine: -Uncoated	-13 mV PdI <2 155 ± 5 nm			molecular weight protamine administration. (Cai et al., 2017)
	-Poloxamer-coating	185 ± 10 nm -16 mV
	Chitosan	300 nm	Rat	TTI + magnet	No hearing loss at long-term in single and
	nanocarriers	PdI = 0.67	n = 12/group	Single-dose or	multi-dose study. Repeated administrations	(Shimoji
	containing SPION	25 mg/mL	1-3 months	multi-dose	did not increase hearing loss. Slight	et al.,
				(1/week)	inflammation in cochlea. No iron particles	2019)
					found in other organs.
	Chitosan	300 nm	Rat	TTI + magnet	Mild inflammatory changes. Macrophages
	nanocarriers	25 mg/mL	n = 5/group	Single-dose or	containing intracytoplasmic iron present in the	(Lafond et
	containing SPION		1 month	multi-dose	middle ear. Repeated administrations did not	al., 2018)
				(1/week)	increase inflammation ratio.
	Hyaluronic acid gel containing SPION	135 ± 5 nm 2.5 mg/mL	Rat n = 6 for the whole study 1 month	TTI + magnet	Immediate postoperative shift of hearing thresholds (10-15 dB) at 16 and 32 kHz, reversible at day 7. No impact on hearing thresholds at 2 and 4 kHz.	(Leterme et al., 2019)
	ns, not specified; PdI, polydispersity index; PEG, poly(ethylene glycol); PLGA, poly(lactic-co-glycolic acid); RWM, round
	window membrane; SPION, superparamagnetic iron oxide nanoparticles; TTI, transtympanic injection.

Table 5 :

 5 Therapeutic efficacy of nanocarriers administered by intratympanic route

	Type of nanocarrier Nanocarrier	Drug (dose or	Therapeutic	Study design	Administration	Therapeutic effect	Reference
		characteristics	drug loading %)	application
	Passive delivery
	Cationic liposomes	ns	4 different siRNA	Efficacy to	GJB2-mutant	Sponge applied	Liposomes-siRNA decrease mutant GJB2 expression by 70% in the cochlea and do not	(Maeda et al.,
			(0.25-5 µg)	silence GJB2 mutant gene,	mouse (~20 dB hearing	on RWM	affect endogenous GJB2. 15 dB of hearing improvement at all frequencies.	2005)
				involved in	loss)
				genetic hearing loss	n = 6/group 3 days
	PEGylated PLA	130 nm	Dexamethasone	Efficacy against	Guinea pig	Applied on	Dexamethasone-loaded nanoparticles induce 10 dB of hearing improvement at 4-8 kHz	(Sun et al.,
	nanoparticles	-26 mV	(50 µg, 8%)	cisplatin ototoxicity	n = 6/group 3 days	RWM	compared to non-encapsulated dexamethasone. No effect at high frequencies. Dexamethasone-loaded nanoparticles protect hair cells (65%) at 6 kHz region compared	2015)
							to dexamethasone phosphate solution (40%).
	Methacrylic derivatives of	128 nm -5 mV	Methyl-prednisolone	Efficacy against cisplatin	Rat n = 6/group	TTI	15% methylprednisolone-loaded nanoparticles based on methacrylic derivative of tocopheryl succinate induce 20 dB of hearing improvement at mid-high frequencies.	(Martín-Saldaña et al., 2016)
	tocopheryl succinate		(10 or 15%)	ototoxicity	3 days		Hair cell protection at cochlea base.
	or tocopherol						No efficacy of 10% methylprednisolone-loaded nanoparticles based on methacrylic derivative of tocopheryl succinate or tocopherol.
	Methacrylic	~120-140 nm	Tocopheryl	Efficacy against	Rat	TTI	Dexamethasone-loaded nanoparticles based on methacrylic derivative of tocopherol	(Martín-Saldaña
	derivatives of	-3 to -7 mV	succinate (10%) or	cisplatin	n = 6/group		induce 15 dB of hearing improvement at all frequencies.	et al., 2017)
	tocopheryl succinate or tocopherol		dexamethasone (15%)	ototoxicity	3 days		Tocopheryl succinate-loaded nanoparticles based on methacrylic derivative of tocopheryl succinate induce 10 dB of hearing improvement at 12, 20 and 32 kHz.
							No efficacy of other nanoparticles: dexamethasone-loaded nanoparticles based on
							methacrylic derivative of tocopheryl succinate, tocopheryl succinate loaded nanoparticles based on methacrylic derivative of tocopherol.
	Methacrylic	180-210 nm	Dexamethasone	Efficacy against	Rat	TTI	Dexamethasone-loaded nanoparticles based on methacrylic derivatives of tocopheryl	(Martín-Saldaña
	derivatives of tocopheryl succinate,	-5 to 0 mV	(10%)	cisplatin ototoxicity	n = 3/group 3 days		succinate and ibuprofen induce 10 dB of hearing improvement at all frequencies. No efficacy of dexamethasone-loaded nanoparticles based on methacrylic derivatives of	et al., 2018)
	tocopherol or						tocopherol and ibuprofen.
	ibuprofen
	Solid lipid nanoparticles	94 nm	Edaravone	Chronic noise exposure to	Guinea pig n = 96 for the	TTI	Edaravone-loaded nanoparticles induce 10 dB of hearing improvement compared to untreated and edaravone solution groups. Decrease of free radicals in the inner ear.	(Gao et al., 2015)
				induce hearing	whole study
				loss	1 week
	Cationic PEGylated	143 ± 22 nm Dexamethasone	Efficacy against	Deafened mouse	TTI	Dexamethasone-loaded lipid nanocapsules induced 20 dB of hearing improvement	(Yang et al.,
	lipid nanoemulsions		(93 ± 8%, 4.2 µg)	kanamycin	n = 6/group		compared to dexamethasone suspension group.	2018)
				ototoxicity	1 week
	ns, not specified; GJB2, gap junction protein beta 2; PEG, poly(ethylene glycol); PLGA, poly(lactic-co-glycolic acid); PLA, polylactic acid; RWM, round window membrane; siRNA, small interfering RNA; TTI, transtympanic injection.

Table 5 :

 5 Therapeutic efficacy of nanocarriers administered by intratympanic route (continued)Non-targeted drug-loaded PEGylated liposomes induce 20 dB of hearing improvement compared to unloaded PEGylated liposomes. PEGylated liposomes targeting outer hair cells induce 35 dB of hearing improvement on average at all frequencies. Drug-loaded nanocarriers with magnetic delivery induce 10 dB of hearing improvement compared to drug solution and increased outer hair cell density compared to drug solution (68 versus 50 cells /200 µm). PdI, polydispersity index; PEG, poly(ethylene glycol); PLGA, poly(lactic-co-glycolic acid); RWM, round window membrane; siRNA, small interfering RNA; SPION, superparamagnetic iron oxide nanoparticles; TTI, transtympanic injection.

	Type of nanocarrier Nanocarrier	Drug (dose or	Therapeutic	Study design	Administration	Therapeutic effect	Reference
		characteristics	drug loading %)	application
	Advanced delivery
	Gel containing lipid	ns	N-acetyl-L-	Efficacy against	Guinea pig	TTI	No therapeutic effect on hearing thresholds.	(Mohan et al.,
	nanocapsules		cysteine	cisplatin ototoxicity	n = 45 for the whole study		Lipid nanocapsules not adapted to encapsulate hydrophilic drugs.	2014)
					1-3 weeks
	Hyaluronic acid gel containing PEGylated	146 ± 50 nm PdI = 0.1	Dexamethasone phosphate	Cochlear implantation	Guinea pig n = 5-8/group	TTI	Manual insertion + drug-loaded liposomal gel: 10 dB of hearing improvement at all frequencies.	(Mamelle et al., 2017)
	liposomes	-29 mV	(1.5 mg)	with manual or	1 week		Robotic insertion + unloaded liposomal gel: 10 dB of hearing improvement at all
				robotic insertion			frequencies. Robotic insertion + drug-loaded liposomal gel: no additional effect of dexamethasone
							phosphate.
	Hyaluronic acid gel containing PEGylated	146 ± 50 nm PdI = 0.1	Dexamethasone phosphate	Noise-induced hearing loss	Guinea pig n = 13/group	TTI 2 days post trauma	No hearing recovery at 1 week except in control group. Full recovery at 1 month except at 8 kHz.	(Mamelle et al., 2018)
	liposomes	-29 mV	(1.5 mg)		1 month
	Chitosan-gel	87 ± 5 nm	c-Jun kinase	Permanent	Mouse	Applied on		(Kayyali et al.,
	containing PEGylated liposomes coated with		inhibitor-1	noise-induced hearing loss	n = 10/group 14 days	RWM 2 days before trauma		2018)
	prestin-targeting
	peptide-1
	PEGylated PLGA	158 ± 14 nm	Dexamethasone	Efficacy against	Guinea pig	Applied on	Peptide-covered dexamethasone-loaded nanoparticles induce, respectively 20 and	(Wang et al.,
	nanoparticles coated	-30 mV	(0.6 µg)	cisplatin	n = 10/group	RWM 1h before	10 dB of hearing improvement at low and high frequencies.	2018)
	with A666 peptide			ototoxicity	3 days	cisplatin injection	No efficacy of not-targeted dexamethasone-loaded nanocarriers.
	Chitosan	300 nm	Methyl-	Efficacy against	Mouse	TTI + magnet (Ramaswamy et
	nanocarriers	PdI = 0.67	prednisolone	cisplatin	n = 6/group			al., 2017)
	containing SPION		(0.15 µg)	ototoxicity	1½ months
	ns, not specified; 940

Acknowledgements

47

Céline Jaudoin acknowledges the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation for her PhD grant 2017-110. This work was supported by ANR (The French National Research Agency) (N° ANR-15-CE19-0014-02-04). Yann Nguyen and Evelyne Ferrary acknowledge the "Fondation pour l'Audition" (Hearing Institute starting grant). The authors would like to thank Savina Legrenzi for drawing Figure 1A specifically for this review.

Declaration of interest

The authors declare no conflict of interest.