RESUMEN
BACKGROUND: There exists an unfulfilled requirement for effective cochlear pharmacotherapy. Controlled local drug delivery could lead to effective bioavailability. The round window niche (RWN), a cavity in the middle ear, is connected to the cochlea via a membrane through which drug can diffuse. We are developing individualized drug-eluting RWN implants (RNIs). To test their effectiveness in guinea pigs, a commonly used model in cochlear pharmacology studies, it is first necessary to develop guinea pig RNIs (GP-RNI). METHODS: Since guinea pigs do not have a RWN such as it is present in humans and to reduce the variables in in vivo studies, a one-size-fits-all GP-RNI model was designed using 12 data sets of Dunkin-Hartley guinea pigs. The model was 3D-printed using silicone. The accuracy and precision of printing, distribution of the sample ingredient dexamethasone (DEX), biocompatibility, bio-efficacy, implantability and drug release were tested in vitro. The GP-RNI efficacy was validated in cochlear implant-traumatized guinea pigs in vivo. RESULTS: The 3D-printed GP-RNI was precise, accurate and fitted in all tested guinea pig RWNs. DEX was homogeneously included in the silicone. The GP-RNI containing 1% DEX was biocompatible, bio-effective and showed a two-phase and sustained DEX release in vitro, while it reduced fibrous tissue growth around the cochlear implant in vivo. CONCLUSIONS: We developed a GP-RNI that can be used for precise inner ear drug delivery in guinea pigs, providing a reliable platform for testing the RNI's safety and efficacy, with potential implications for future clinical translation.
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Implantes Cocleares , Dexametasona , Sistemas de Liberación de Medicamentos , Ventana Redonda , Cobayas , Animales , Ventana Redonda/efectos de los fármacos , Ventana Redonda/metabolismo , Dexametasona/administración & dosificación , Dexametasona/farmacocinética , Dexametasona/farmacología , Sistemas de Liberación de Medicamentos/métodos , Liberación de Fármacos , Impresión Tridimensional , Cóclea/efectos de los fármacosRESUMEN
INTRODUCTION: The round window membrane (RWM) presents a significant barrier to the local application of therapeutics to the inner ear. We demonstrate a benchtop preclinical RWM model and evaluate superparamagnetic iron oxide nanoparticles (SPIONs) as vehicles for magnetically assisted drug delivery. METHODS: Guinea pig RWM explants were inset into a 3D-printed dual chamber benchtop device. Custom-synthesized 7-nm iron core nanoparticles were modified with different polyethylene glycol chains to yield two sizes of SPIONs (NP-PEG600 and NP-PEG3000) and applied to the benchtop model with and without a magnetic field. Histologic analysis of the RWM was performed using transmission electron microscopy (TEM) and confocal microscopy. RESULTS: Over a 4-h period, 19.5 ± 1.9% of NP-PEG3000 and 14.6 ± 1.9% of NP-PEG600 were transported across the guinea pig RWM. The overall transport increased by 1.45× to 28.4 ± 5.8% and 21.0 ± 2.0%, respectively, when a magnetic field was applied. Paraformaldehyde fixation of the RWM decreased transport significantly (NP-PEG3000: 7.6 ± 1.5%; NP-PEG600: 7.0 ± 1.6%). Confocal and electron microscopy analysis demonstrated nanoparticle localization throughout all cellular layers and layer-specific transport characteristics within RWM. CONCLUSION: The guinea pig RWM explant benchtop model allows for targeted and practical investigations of transmembrane transport in the development of nanoparticle drug delivery vehicles. The presence of a magnetic field increases SPION delivery by 45%-50% in a nanoparticle size- and cellular layer-dependent manner. LEVEL OF EVIDENCE: NA Laryngoscope, 134:3355-3362, 2024.
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Sistemas de Liberación de Medicamentos , Ventana Redonda , Cobayas , Animales , Ventana Redonda/metabolismo , Oído Interno/metabolismo , Nanopartículas Magnéticas de Óxido de Hierro/química , Microscopía Confocal , Microscopía Electrónica de Transmisión , Nanopartículas de Magnetita , Impresión Tridimensional , Polietilenglicoles/químicaRESUMEN
Countless therapeutic antibodies are currently available for the treatment of a broad range of diseases. Some target molecules of therapeutic antibodies are involved in the pathogenesis of sensorineural hearing loss (SNHL), suggesting that SNHL may be a novel target for monoclonal antibody (mAb) therapy. When considering mAb therapy for SNHL, understanding of the pharmacokinetics of mAbs after local application into the middle ear is crucial. To reveal the fundamental characteristics of mAb pharmacokinetics following local application into the middle ear of guinea pigs, we performed pharmacokinetic analyses of mouse monoclonal antibodies to FLAG-tag (FLAG-mAbs), which have no specific binding sites in the middle and inner ear. FLAG-mAbs were rapidly transferred from the middle ear to the cochlear fluid, indicating high permeability of the round window membrane to mAbs. FLAG-mAbs were eliminated from the cochlear fluid 3 h after application, similar to small molecules. Whole-body autoradiography and quantitative assessments of cerebrospinal fluid and serum demonstrated that the biodistribution of FLAG-mAbs was limited to the middle and inner ear. Altogether, the pharmacokinetics of mAbs are similar to those of small molecules when locally applied into the middle ear, suggesting the necessity of drug delivery systems for appropriate mAb delivery to the cochlear fluid after local application into the middle ear.
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Oído Interno , Pérdida Auditiva Sensorineural , Ratones , Cobayas , Animales , Anticuerpos Monoclonales/metabolismo , Distribución Tisular , Oído Interno/metabolismo , Cóclea/metabolismo , Oído Medio , Ventana Redonda/metabolismo , Pérdida Auditiva Sensorineural/metabolismoRESUMEN
BACKGROUND: We have developed 3D-printed microneedle technology for diagnostic aspiration of perilymph and intracochlear delivery of therapeutic agents. Single microneedle-mediated round window membrane (RWM) perforation does not cause hearing loss, heals within 48-72 h, and yields sufficient perilymph for proteomic analysis. In this study, we investigate the anatomic, physiologic, and proteomic consequences of repeated microneedle-mediated perforations of the same RWM at different timepoints. METHODS: 100-µm-diameter hollow microneedles were fabricated using two-photon polymerization (2PP) lithography. The tympanic bullae of Hartley guinea pigs (n = 8) were opened with adequate exposure of the RWM. Distortion product otoacoustic emissions (DPOAE) and compound action potential (CAP) were recorded to assess hearing. The hollow microneedle was introduced into the bulla and the RWM was perforated; 1 µL of perilymph was aspirated from the cochlea over the course of 45 s. 72 h later, the above procedure was repeated with aspiration of an additional 1 µL of perilymph. 72 h after the second perforation, RWMs were harvested for confocal imaging. Perilymph proteomic analysis was completed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Two perforations and aspirations were performed in 8 guinea pigs. In six, CAP, DPOAE, and proteomic analysis were obtained; in one, only CAP and DPOAE results were obtained; and in one, only proteomics results were obtained. Hearing tests demonstrated mild hearing loss at 1-4 kHz and 28 kHz, most consistent with conductive hearing loss. Confocal microscopy demonstrated complete healing of all perforations with full reconstitution of the RWM. Perilymph proteomic analysis identified 1855 proteins across 14 samples. The inner ear protein cochlin was observed in all samples, indicating successful aspiration of perilymph. Non-adjusted paired t-tests with p < 0.01 revealed significant changes in 13 of 1855 identified proteins (0.7%) between the first and second aspirations. CONCLUSIONS: We demonstrate that repeated microneedle perforation of the RWM is feasible, allows for complete healing of the RWM, and minimally changes the proteomic expression profile. Thus, microneedle-mediated repeated aspirations in a single animal can be used to monitor the response to inner ear treatments over time.
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Pérdida Auditiva , Proteómica , Animales , Cobayas , Cromatografía Liquida , Espectrometría de Masas en Tándem , Ventana Redonda/metabolismo , Cóclea/metabolismo , Perilinfa/metabolismo , Pérdida Auditiva/metabolismoRESUMEN
The application of insulin-like growth factor 1 (IGF-1) to the round window membrane (RWM) is an emerging treatment for inner ear diseases. RWM permeability is the key factor for efficient IGF-1 delivery. Ultrasound microbubbles (USMBs) can increase drug permeation through the RWM. In the present study, the enhancing effect of USMBs on the efficacy of IGF-1 application and the treatment effect of USMB-mediated IGF-1 delivery for noise-induced hearing loss (NIHL) were investigated. Forty-seven guinea pigs were assigned to three groups: the USM group, which received local application of recombinant human IGF-1 (rhIGF-1, 10 µg/µL) following application of USMBs to the RWM; the RWS group, which received IGF-1 application alone; and the saline-treated group. The perilymphatic concentration of rhIGF-1 in the USM group was 1.95- and 1.67- fold of that in the RWS group, 2 and 24 h after treatment, respectively. After 5 h of 118 dB SPL noise exposure, the USM group had the lowest threshold shift in auditory brainstem response, least loss of cochlear outer hair cells, and least reduction in the number of synaptic ribbons on postexposure day 28 among the three groups. The combination of USMB and IGF-1 led to a better therapeutic response to NIHL. Two hours after treatment, the USM group had significantly higher levels of Akt1 and Mapk3 gene expression than the other two groups. The most intense immunostaining for phosphor-AKT and phospho-ERK1/2 was detected in the cochlea in the USM group. These results suggested that USMB can be applied to enhance the efficacy of IGF-1 therapy in the treatment of inner ear diseases.
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Cóclea/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Pérdida Auditiva Provocada por Ruido/tratamiento farmacológico , Factor I del Crecimiento Similar a la Insulina/farmacología , Microburbujas/uso terapéutico , Ventana Redonda/efectos de los fármacos , Ondas Ultrasónicas , Animales , Cóclea/metabolismo , Modelos Animales de Enfermedad , Cobayas , Pérdida Auditiva Provocada por Ruido/metabolismo , Pérdida Auditiva Provocada por Ruido/patología , Ventana Redonda/metabolismoRESUMEN
The cochlea's inaccessibility and complex nature provide significant challenges to delivering drugs and other agents uniformly, safely and efficiently, along the entire cochlear spiral. Large drug concentration gradients are formed along the cochlea when drugs are administered to the middle ear. This undermines the major goal of attaining therapeutic drug concentration windows along the whole cochlea. Here, utilizing a well-known physiological effect of salicylate, we demonstrate a proof of concept in which drug distribution along the entire cochlea is enhanced by applying round window membrane low-frequency micro vibrations with a probe that only partially covers the round window. We provide evidence of enhanced drug influx into the cochlea and cochlear apical drug distribution without breaching cochlear boundaries. It is further suggested that ossicular functionality is not required for the effective drug distribution we report. The novel method presented here of local drug delivery to the cochlea could be implemented when ossicular functionality is absent or impeded and can be incorporated in clinically approved auditory protheses for patients who suffer with conductive, sensorineural or mixed hearing loss.
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Antiinflamatorios/farmacocinética , Cóclea/metabolismo , Ventana Redonda/metabolismo , Salicilatos/farmacocinética , Vibración , Animales , Antiinflamatorios/administración & dosificación , Cobayas , Salicilatos/administración & dosificaciónRESUMEN
N-acetylcysteine is a thiol-containing antioxidant, which has shown otoprotective effects in in vitro as well as in vivo models of cisplatin-induced hearing loss. Systemic administration of antioxidants, however, is associated with the major potential drawback of interference with the tumoricidal effect of cisplatin. This therapeutic limitation can be overcome by local intratympanic injection of the antioxidant N-acetylcysteine, which results in very restricted systemic uptake of the drug, whilst intracochlear drug levels are substantially higher. Furthermore, osmolality and pH properties of formulations for intratympanic injection need to be controlled, as they impact the fraction of drug crossing the barriers of the inner ear and could potentially damage middle and inner ear structures. This study focused on (i) the evaluation of concentration-time profiles of N-acetylcysteine in perilymph, cerebrospinal fluid and plasma after intratympanic administration, (ii) the influence of the dosage form, i.e. a thermoreversible poloxamer 407 hydrogel versus a solution, on N-acetylcysteine pharmacokinetics, and (iii) the development of a pH- and osmolality-adjusted formulation for intratympanic N-acetylcysteine delivery. 49 female albino guinea pigs were randomized into two treatment groups, receiving either a single intratympanic injection of a 4% N-acetylcysteine poloxamer 407 hydrogel or a 4% N-acetylcysteine solution. 8 animals served as untreated controls. N-acetylcysteine levels in perilymph, cerebrospinal fluid and plasma were monitored over a period of 24 h. Samples were taken at 1, 3, 6, 12 and 24 h (poloxamer 407 hydrogel group) and 1, 6 and 24 h (solution group) post injection, and analysed by high performance liquid chromatography-tandem mass spectrometry. Intratympanic application of the 4% N-acetylcysteine poloxamer 407 hydrogel resulted in a 4-fold larger perilymph area under the concentration-time curve (0-24 h) than topical administration of the equally concentrated N-acetylcysteine solution but in similar plasma N-acetylcysteine levels. N-acetylcysteine concentrations in the cerebrospinal fluid were below the level of detection (5 ng/ml) in both treatment groups. N-acetylcysteine-containing formulations applied to the middle ear were isohydric and osmolality was reduced by up to 200 mosmol/kg compared to equally concentrated formulations used in previous studies. In summary, we were able to demonstrate that the intratympanic injection of thermoreversible poloxamer 407 hydrogels increases and sustains N-acetylcysteine delivery to the inner ear. Given the low plasma N-acetylcysteine levels after topical application and the physiological pH and osmolality of the hydrogel, the risk of compromising the antineoplastic effects of cisplatin therapy and of local side effects is minimal.
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Acetilcisteína/administración & dosificación , Antioxidantes/administración & dosificación , Portadores de Fármacos , Poloxámero/química , Ventana Redonda/metabolismo , Acetilcisteína/química , Acetilcisteína/farmacocinética , Animales , Antioxidantes/química , Antioxidantes/farmacocinética , Preparaciones de Acción Retardada , Composición de Medicamentos , Femenino , Cobayas , Hidrogeles , Concentración de Iones de Hidrógeno , Inyecciones , Concentración Osmolar , Perilinfa/metabolismoRESUMEN
Introduction. The objective of this study was to evaluate the feasibility and toxicity of superparamagnetic iron oxide nanoparticles (SPIONs) administered into the cochlea through the round window (RW) by an external magnetic field. Materials and Methods. In 5 Wistar rats, the left RW was punctured. SPIONs suspended in hyaluronic gel (5 mg/mL) were applied in the RW niche and covered by a muscle graft. The nanoparticles were mobilized using a rare earth magnet (0.54 T) held in 4 consecutive positions around the head. The right ear served as control. Hearing function was monitored by auditory brainstem responses (4-32 kHz tone bursts). Results. The auditory thresholds remained unchanged 1 month after the administration. The histological study of the cochleae showed that SPIONs were driven into the scala tympani in the basal turn, the second turn, and the apex. Conclusion. Superparamagnetic nanoparticles can be driven inside the cochlea toward the apex with a preserved hearing up to 1 month in rats.
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Sistemas de Liberación de Medicamentos/métodos , Nanopartículas de Magnetita , Ventana Redonda , Animales , Umbral Auditivo/efectos de los fármacos , Cóclea/efectos de los fármacos , Cóclea/metabolismo , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Estudios de Factibilidad , Campos Magnéticos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/toxicidad , Masculino , Ratas , Ratas Wistar , Ventana Redonda/efectos de los fármacos , Ventana Redonda/metabolismoRESUMEN
Glucocorticoids have direct anti-inflammatory, anti-oxidant and anti-apoptotic effects on cochlear hair cells. Cochlear glucocorticoid therapy has gained particular attention for its ability to enhance the protection of residual hearing following hearing preservation cochlear implantation. Local drug delivery methods achieve high drug concentrations within the inner ear fluids but are reliant upon diffusion across the round window membrane. Diffusion has been shown to demonstrate large individual variability. This study explores the role of "adjuvant agents", which when administered with glucocorticoids, enhance inner ear absorption and distribution. Guinea pig cochleae were administered either dexamethasone alone or in combination with hyaluronic acid, histamine, or combination histamine and hyaluronic acid, targeted at the round window membrane. Control subjects received saline. Perilymph was sampled from the cochlear apex, and basal to apical dexamethasone concentrations recorded with mass spectroscopy. Cochleae were harvested, and immunohistochemistry employed to explore dexamethasone tissue penetration and distribution. Basal to apical gradients were observed along the scala tympani, with higher dexamethasone concentrations observed at the cochlear base. Gradients were more pronounced and uniform when administered on a hyaluronic acid sponge, while histamine increased absolute concentrations reaching the inner ear. Tissue penetration correlated with perilymph concentration. Our results demonstrate that adjuvant agents can be employed to enhance dexamethasone absorption and distribution in the inner ear, thus proposing therapeutic strategies that may enhance steroid facilitated hearing protection.
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Adyuvantes Farmacéuticos/farmacología , Dexametasona/farmacocinética , Glucocorticoides/farmacocinética , Ventana Redonda/efectos de los fármacos , Absorción Fisicoquímica , Animales , Cóclea/anatomía & histología , Cóclea/metabolismo , Difusión , Sistemas de Liberación de Medicamentos , Glicosaminoglicanos/farmacología , Cobayas , Histamina/farmacología , Ácido Hialurónico/farmacología , Perilinfa/metabolismo , Permeabilidad , Ventana Redonda/metabolismo , Distribución TisularRESUMEN
OBJECTIVE: The current investigation evaluated a novel extended release delivery system for treating inner ear diseases. The platform technology consists of a film forming agent (FFA) and microsphere component to localize and extend drug delivery within the ear. STUDY DESIGN: Studies evaluated dissolution kinetics of microspheres with multiple encapsulates, testing of a variety of FFAs, and ability to localize to the round window membrane in mice in vivo. SETTING: Studies were completed at Orbis Biosciences and The University of Kansas Medical Center. SUBJECTS: In conjunction with in vitro characterization, an infrared dye-containing microsphere formulation was evaluated for round window membrane (RWM) localization and general tolerability in C57/BL6 Mus musculus for 35â¯days. METHODS: In vitro characterization was performed using upright diffusion cells on cellulose acetate membranes, with drug content quantified by high performance liquid chromatography. Mus musculus dosing of infrared dye-containing microspheres was performed under anesthesia with a 27 GA needle and 2.0⯵L injection volume RESULTS: In vitro dissolution demonstrates the ability of the FFA with microsphere platform to release steroids, proteins, peptides, and nucleic acids for at least one month, while necroscopy shows the ability of the FFA with dye-loaded microspheres to remain localized to Mus musculus RWM for the same period of time, with favorable tolerability. CONCLUSIONS: Combining FFA and microsphere for localized drug delivery may enable cost-effective, extended release local delivery to the inner ear of new and existing small molecules, proteins, peptides, and nucleic acids.
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Portadores de Fármacos/química , Enfermedades del Laberinto/tratamiento farmacológico , Esteroides/administración & dosificación , Animales , Celulosa/análogos & derivados , Celulosa/química , Preparaciones de Acción Retardada , Liberación de Fármacos , Colorantes Fluorescentes/química , Humanos , Inyección Intratimpánica , Membranas Artificiales , Ratones Endogámicos C57BL , Microesferas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Ventana Redonda/metabolismo , Esteroides/efectos adversosRESUMEN
Irreversible hearing loss induced by aminoglycoside in human through local or systemic administration route negatively impacts quality of life. The aim of this work was to develop and validate an analytical method suitable for the detection and quantification of neomycin in cochlear perilymph of guinea pig after local application. The SupelMIP SPE column was used for the pre-treatment of matrix. Chromatographic separation was conducted by a reversed phase ODS column (100â¯×â¯2.1â¯mm, 3⯵m) at 40⯰C in gradient mode with 0.2 (v/v) HFBA in water and 0.2 (v/v) HFBA in acetonitrile as mobile phase, at a flow rate of 0.30â¯mL/min, with retention time of 3.50 and 3.62â¯min for internal standard tobramycin and analyte neomycin, respectively. The MS was performed with positive ionization mode, with data acquisition in Multi Reaction Monitor (MRM) mode. This method was proved to be specific, accurate (97.1-115% of nominal values) and precise (CV%â¯<â¯15%). Calibration curves for matrix matched standard of neomycin ranged from 1.25 to 200⯵g/mL, with LOD and LLOQ of 0.625 and 1.25⯵g/mL in blank matrix. The matrix effect was corrected to (-0.1) - 1.33 by adding internal standard. The relative SPE recovery values were ≥98.9% in low, medium and high QC samples. Neomycin in matrix proved to be stable under room temperature - and -20⯰C, or under three freeze-thawing cycles, or under processing as well. Finally, the proposed method was successfully applied to a toxicokinetics study of neomycin in perilymph after round window membrane (RWM) administration, which was in accordance with threshold shift of auditory brainstem response (ABR) test related to hearing loss.
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Cromatografía Líquida de Alta Presión/métodos , Neomicina/análisis , Perilinfa/química , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Cobayas , Límite de Detección , Modelos Lineales , Neomicina/aislamiento & purificación , Reproducibilidad de los Resultados , Ventana Redonda/metabolismoRESUMEN
The cochlea, or inner ear, is a space fully enclosed within the temporal bone of the skull, except for two membrane-covered portals connecting it to the middle ear space. One of these portals is the round window, which is covered by the Round Window Membrane (RWM). A longstanding clinical goal is to reliably and precisely deliver therapeutics into the cochlea to treat a plethora of auditory and vestibular disorders. Standard of care for several difficult-to-treat diseases calls for injection of a therapeutic substance through the tympanic membrane into the middle ear space, after which a portion of the substance diffuses across the RWM into the cochlea. The efficacy of this technique is limited by an inconsistent rate of molecular transport across the RWM. A solution to this problem involves the introduction of one or more microscopic perforations through the RWM to enhance the rate and reliability of diffusive transport. This paper reports the use of direct 3D printing via Two-Photon Polymerization (2PP) lithography to fabricate ultra-sharp polymer microneedles specifically designed to perforate the RWM. The microneedle has tip radius of 500 nm and shank radius of 50 µ m, and perforates the guinea pig RWM with a mean force of 1.19 mN. The resulting perforations performed in vitro are lens-shaped with major axis equal to the microneedle shank diameter and minor axis about 25% of the major axis, with mean area 1670 µ m2. The major axis is aligned with the direction of the connective fibers within the RWM. The fibers were separated along their axes without ripping or tearing of the RWM suggesting the main failure mechanism to be fiber-to-fiber decohesion. The small perforation area along with fiber-to-fiber decohesion are promising indicators that the perforations would heal readily following in vivo experiments. These results establish a foundation for the use of Two-Photon Polymerization lithography as a means to fabricate microneedles to perforate the RWM and other similar membranes.
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Sistemas de Liberación de Medicamentos/instrumentación , Agujas , Impresión Tridimensional , Ventana Redonda/metabolismo , Animales , Transporte Biológico , Diseño de Equipo , CobayasRESUMEN
BACKGROUND: The round window membrane (RWM) functions as the primary biological barrier for therapeutic agents in the inner ear via local application. Previous studies on inner ear nano-drug delivery systems mostly focused on their pharmacokinetics and distribution in the inner ear, but seldom on the interaction with the RWM. Clarifying the transport mechanism of nanoparticulate carriers across RWM will shed more light on the optimum design of nano-drug delivery systems intended for meeting demands for their clinical application. METHODS: The poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) encapsulating coumarin-6 were prepared by emulsifying solvent evaporation method. We utilized confocal laser scanning microscope (CLSM) in combination with transmission electron microscope to investigate the transport pathway of PLGA NPs in the RWM. Simultaneously, the concentration and time dependence of NPs across the RWM were also determined. The endocytic mechanism of NPs through this membrane interface was classically analyzed by means of various endocytic inhibitors. The intracellular location of NPs into lysosomes was evaluated using CLSM scanning microscope colocalization analysis. The Golgi-related inhibitors were employed to probe into the function of Golgi and endoplasmic reticulum (ER) in the discharge of NPs out of cells. RESULTS: PLGA NPs were herein transported through the RWM of a sandwich-like structure into the perilymph via the transcellular pathway. NPs were internalized predominantly via macropinocytosis and caveolin-mediated endocytic pathways. After being internalized, the endocytosed cargos were entrapped within the lysosomal compartments and/or the endoplasmic reticulum/Golgi apparatus which mediated the exocytotic release of NPs. CONCLUSION: For the first time, we showed the translocation itinerary of NPs in RWM, providing a guideline for the rational fabrication of inner ear nanoparticulate carriers with better therapeutic effects.
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Sistemas de Liberación de Medicamentos , Ácido Láctico/química , Nanomedicina , Nanopartículas/química , Ácido Poliglicólico/química , Ventana Redonda/metabolismo , Animales , Cumarinas/farmacología , Endocitosis/efectos de los fármacos , Exocitosis/efectos de los fármacos , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Microscopía Confocal , Modelos Biológicos , Nanopartículas/ultraestructura , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Tiazoles/farmacologíaRESUMEN
Dexamethasone (Dex)-loaded PHEA-g-C18-Arg8 (PCA) nanoparticles (PCA/Dex) were developed for the delivery of genes to determine the synergistic effect of Dex on gene expression. The cationic PCA nanoparticles were self-assembled to create cationic micelles containing an octadecylamine (C18) core with Dex and an arginine 8 (Arg8) peptide shell for electrostatic complexation with nucleic acids (connexin 26 [Cx26] siRNA, green fluorescent protein [GFP] DNA or brain-derived neurotrophic factor [BDNF] pDNA). The PCA/Dex nanoparticles conjugated with Arg8, a cell-penetrating peptide that enhances permeability through a round window membrane in the inner ear for gene delivery, exhibited high uptake efficiency in HEI-OC1 cells. This potential carrier co-delivering Dex and the gene into inner ear cells has a diameter of 120-140 nm and a zeta potential of 20-25 mV. Different types of genes were complexed with the Dex-loaded PCA nanoparticle (PCA/Dex/gene) for gene expression to induce additional anti-inflammatory effects. PCA/Dex showed mildly increased expression of GFP and lower mRNA expression of inflammatory cytokines (IL1b, IL12, and INFr) than did Dex-free PCA nanoparticles and Lipofectamine® reagent in HEI-OC1 cells. In addition, after loading Cx26 siRNA onto the surface of PCA/Dex, Cx26 gene expression was downregulated according to real-time polymerase chain reaction for 24 h, compared with that using Lipofectamine reagent. After loading BDNF DNA into PCA/Dex, increased expression of BDNF was observed for 30 h, and its signaling pathway resulted in an increase in phosphorylation of Akt, observed by Western blotting. Thus, Dex within PCA/Dex/gene nanoparticles created an anti-inflammatory effect and enhanced gene expression.
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Péptidos de Penetración Celular/farmacocinética , Dexametasona/farmacocinética , Técnicas de Transferencia de Gen , Nanopartículas/administración & dosificación , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Línea Celular , Péptidos de Penetración Celular/administración & dosificación , Conexina 26 , Conexinas/genética , Oído Interno/citología , Terapia Genética/métodos , Proteínas Fluorescentes Verdes/genética , Lípidos , Ratones , Micelas , Nanopartículas/química , Polihidroxietil Metacrilato/análogos & derivados , Polihidroxietil Metacrilato/química , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ventana Redonda/efectos de los fármacos , Ventana Redonda/metabolismoRESUMEN
Intratympanic gentamicin therapy is widely used clinically to suppress the vestibular symptoms of Meniere's disease. Dosing in humans was empirically established and we still know remarkably little about where gentamicin enters the inner ear, where it reaches in the inner ear and what time course it follows after local applications. In this study, gentamicin was applied to the round window niche as a 20 µL bolus of 40 mg/ml solution. Ten 2 µL samples of perilymph were collected sequentially from the lateral semi-circular canal (LSCC) at times from 1 to 4 h after application. Gentamicin concentration was typically highest in samples originating from the vestibule and was lower in samples originating from scala tympani. To interpret these results, perilymph elimination kinetics for gentamicin was quantified by loading the entire perilymph space by injection at the LSCC with a 500 µg/ml gentamicin solution followed by sequential perilymph sampling from the LSCC after different delay times. This allowed concentration decline in perilymph to be followed with time. Gentamicin was retained well in scala vestibuli and the vestibule but declined rapidly at the base of scala tympani, dominated by interactions of perilymph with CSF, as reported for other substances. Quantitative analysis, taking into account perilymph kinetics for gentamicin, showed that more gentamicin entered at the round window membrane (57%) than at the stapes (35%) but the lower concentrations found in scala tympani were due to greater losses there. The gentamicin levels found in perilymph of the vestibule, which are higher than would be expected from round window entry alone, undoubtedly contribute to the vestibulotoxic effects of the drug. Furthermore, calculations of gentamicin distribution following targeted applications to the RW or stapes are more consistent with cochleotoxicity depending on the gentamicin concentration in scala vestibuli rather than that in scala tympani.
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Gentamicinas/administración & dosificación , Gentamicinas/metabolismo , Gentamicinas/farmacocinética , Perilinfa/metabolismo , Animales , Cobayas , Humanos , Enfermedad de Meniere/tratamiento farmacológico , Modelos Biológicos , Ventana Redonda/metabolismo , Rampa Timpánica/metabolismo , Canales Semicirculares/metabolismo , Vestíbulo del Laberinto/metabolismoRESUMEN
Gene therapy with viral vectors is one of the most promising strategies for sensorineural hearing loss. However, safe and effective administration of the viral vector into cochlear tissue is difficult because of the anatomical isolation of the cochlea. We investigated the efficiency and safety of round window membrane (RWM) application of Sendai virus, one of the most promising non-genotoxic vectors, after pretreatment with hyaluronic acid (HA) on the RWM to promote efficient viral translocation into the cochlea. Sendai virus expressing the green fluorescent protein reporter gene was detected throughout cochlear tissues following application combined with HA pretreatment. Quantitative analysis revealed that maximum expression was reached 3 days after treatment. The efficiency of transgene expression was several 100-fold greater with HA pretreatment than that without. Furthermore, unlike the conventional intracochlear delivery methods, this approach did not cause hearing loss. These findings reveal the potential utility of gene therapy with Sendai virus and HA for treatment of sensorineural hearing loss.
Asunto(s)
Cóclea/metabolismo , Ácido Hialurónico/farmacología , Ventana Redonda/metabolismo , Virus Sendai/genética , Transfección/métodos , Animales , Femenino , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Cobayas , Pérdida Auditiva Sensorineural/terapiaRESUMEN
PURPOSE: Supplementation of exogenous nerve growth factor (NGF) into the cochlea of deafened animals rescues spiral ganglion cells from degeneration. However, a safe and potent delivery of therapeutic proteins, such as NGF, to spiral ganglion cells remains one of the greatest challenges. This study presents the development of self-assembled cubic lipid-based crystalline nanoparticles to enhance inner ear bioavailability of bioactive NGF via a round window membrane route. METHODS: A novel nanocarrier-entrapped NGF was developed based on phytantriol by a liquid precursor dilution, with Pluronic(®) F127 and propylene glycol as the surfactant and solubilizer, respectively. Upon dilution of the liquid lipid precursors, monodispersed submicron-sized particles with a slight negative charge formed spontaneously. RESULTS: Biological activity of entrapped NGF was assessed using pheochromocytoma cells with NGF-loaded reservoirs to induce significant neuronal outgrowth, similar to that seen in free NGF-treated controls. Finally, a 3.28-fold increase in inner ear bioavailability was observed after administration of phytantriol lipid-based crystalline nanoparticles as compared to free drug, contributing to an enhanced drug permeability of the round window membrane. CONCLUSION: Data presented here demonstrate the potential of lipid-based crystalline nanoparticles to improve the outcomes of patients bearing cochlear implants.
Asunto(s)
Cóclea/metabolismo , Alcoholes Grasos/química , Nanopartículas/química , Factor de Crecimiento Nervioso/farmacocinética , Animales , Disponibilidad Biológica , Cóclea/efectos de los fármacos , Cristalización , Femenino , Humanos , Factor de Crecimiento Nervioso/farmacología , Células PC12 , Tamaño de la Partícula , Ratas , Ventana Redonda/efectos de los fármacos , Ventana Redonda/metabolismo , Ventana Redonda/cirugía , Electricidad Estática , Difracción de Rayos XRESUMEN
A drug delivery system to the inner ear using nanoparticles consisting of oligoarginine peptide (Arg8) conjugated to poly(amino acid) (poly(2-hydroxyethyl L-aspartamide; PHEA) was investigated to determine whether the limitations of low drug transport levels across the round window membrane (RWM) and poor transport into inner ear target cells, including hair cells and spiral ganglion, could be overcome. Three types of carrier materials, PHEA-g-C18, PHEA-g-Arg8, and PHEA-g-C18-Arg8, were synthesized to examine the effects of oligoarginine and morphology of the synthesized carriers. Nile red (NR) was used as a fluorescent indicator as well as to model a hydrophobic drug. Compared with PHEA-g-C18-NR nanoparticles, the oligoarginine-conjugated nanoparticles of PHEA-g-C18-Arg8-NR and PHEA-g-Arg8-NR entered into HEI-OC1 cells at significant levels. Furthermore, the strongest fluorescence intensity was observed in nuclei when PHEA-g-C18-Arg8 nanoparticles were used. The high uptake rates of PHEA-g-C18 and PHEA-g-C18-Arg8 nanoparticles were observed in ex vivo experiments using hair cells. After the delivery of PHEA-g-C18-Arg8 nanoparticles with reporter gene transfer, EGFP (enhanced green fluorescent protein) expression was monitored as an indicator of gene delivery. In the inner ear cells, PHEA-g-C18-Arg8 nanoparticles showed comparable or better transfection capabilities than the commercially available Lipofectamine reagent. PHEA-g-C18-Arg8 penetrated in vivo across the RWM of C57/BL6 mice with Nile red staining and GFP expression in various inner ear tissues. In conclusion, PHEA-g-C18-Arg8 nanoparticles were successfully transported into the inner ear through the intratympanic route and are proposed as promising candidates as delivery carriers to address inner ear diseases.
Asunto(s)
Arginina/administración & dosificación , Portadores de Fármacos/química , Oído Interno/efectos de los fármacos , Técnicas de Transferencia de Gen , Nanopartículas/química , Animales , Núcleo Celular/metabolismo , Cóclea/patología , Sistemas de Liberación de Medicamentos , Regulación de la Expresión Génica , Vectores Genéticos/química , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Células Ciliadas Auditivas/metabolismo , Humanos , Lípidos/química , Masculino , Ratones , Ratones Endogámicos C57BL , Oxazinas/química , Polihidroxietil Metacrilato/análogos & derivados , Polihidroxietil Metacrilato/química , Polímeros/química , Ventana Redonda/metabolismo , Solventes/químicaRESUMEN
There is no definitive conclusion concerning the spread of viral vectors to the brain after a cochlear inoculation. In addition, some studies have reported different distribution profiles of viral vectors in the central auditory system after a cochlear inoculation. Thus, rats were grouped into either a mimetic aging group or a young group and transfected with adenoviral vectors (AdVs) by round window membrane injection. The distribution of AdV in central nervous system (CNS) was demonstrated in the two groups with transmission electron microscopy and immunofluorescence. We found that the AdV could disseminate into the CNS and that the neuronal damage and stress-induced GRP78 expression were reduced after transfection with PGC-1α, as compared with the control vectors, especially in the mimetic aging group. We also found that the host immune response was degraded in CNS in the mimetic aging group after transduction through the cochlea, as compared with the young group. These results demonstrate that viral vectors can disseminate into the CNS through the cochlea. Moreover, mimetic aging induced by D-galactose could facilitate the spread of viral vectors into the CNS from the cochlea. These findings may indicate a new potential approach for gene therapy against age-related diseases in the CNS.
Asunto(s)
Adenoviridae/fisiología , Sistema Nervioso Central/virología , Cóclea/virología , Vectores Genéticos/farmacocinética , Ventana Redonda/virología , Factores de Edad , Animales , Sistema Nervioso Central/metabolismo , Cóclea/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Oído Interno/metabolismo , Oído Interno/virología , Femenino , Galactosa/metabolismo , Vectores Genéticos/administración & dosificación , Microscopía Electrónica de Transmisión , Ratas , Ratas Sprague-Dawley , Ventana Redonda/metabolismo , Transfección/métodosRESUMEN
We have previously demonstrated that both age-related and noise-induced hearing loss are reduced in transgenic mice that ubiquitously overexpress X-linked inhibitor of apoptosis protein (XIAP). In view of the therapeutic implications of these findings, we have developed a minimally invasive surgical method to deliver adenoid-associated virus (AAV) across the round window membrane (RWM) of the cochlea, enabling efficient gene transfer to hair cells and sensory neurons in this enclosed structure. This RWM approach was used in the present study to evaluate the effectiveness of AAV-mediated XIAP overexpression in protecting against cisplatin-induced ototoxicity. Two weeks following surgery, AAV-derived XIAP was detected in the majority of inner and outer hair cells, resulting in a threefold elevation of this antiapoptotic protein in the cochlea. The protection of AAV-mediated XIAP overexpression was evaluated in animals treated with cisplatin at a dose of 4 mg kg(-1) per day for 4-7 consecutive days. The XIAP overexpression was found to attenuate cisplatin-induced hearing loss by ~22 dB. This was accompanied by a reduction of the loss of vulnerable hair cells and sensory neurons in the cochlea by 13%.