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Round window membrane and delivery of biologically active agents into the cochlea
Department of Clinical Neoruscience, Karolinska Institutet, Stockholm, Sweden.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Establishing efficient methods for local administration of drugs to the inner ear has great clinical relevance for the management of inner ear disorders. However, the administration route remains a critical issue. The most feasible approach for a non invasive drug delivery to the inner ear is application of medication to the middle ear cavity on the promise that it will diffuse through the thin round window membrane (RWM) separating the inner ear from the middle ear cavity. Gene therapy represents a promising future in otology and offers an exciting therapeutic alternative as it could be used in prevention or management of cochlear disorders. Also for a gene therapy approach, RWM application seems most feasible administration route. Exploring and characterizing the RWM route of administration is thus a fundamentally important area of research for the development of future treatment of inner ear disorders. The objectives of the thesis were to evaluate the efficacy of two drug and gene delivering vehicles to the inner ear, sodium hyaluronate (HYA) and chitosans, which can be applied to the cochlea. Ultimate aim is to establish an efficient drug delivery system and gene transfection for the inner ear. HYA and chitosans loaded with the ototoxic drug neomycin as tracer for drug release have been instilled into the middle ear of the guinea pigs. Effects on RWM and cochlear hair cells were evaluated after a single instillation of HYA (day 7 and 28), chitosans and saline solution (day 7). The hearing organ was analysed for hair cell loss and the thickness and ultrastructural properties of the RWM were analysed by light and transmission electron microscopy. The in vitro transfection efficiency of chitosan was tested by exposing organotypic cultures of the organ of Corti, prepared from postnatal day 2 rats, to chitosan carrying plasmid DNA (pDNA). The in vivo transfection efficiency was tested at one day or seven days after infusing chitosan/pDNA polyplexes with the use of osmotic pumps into the cochlea of adult guinea pigs. Tissue analysis was made by immunohistochemsitry and RT-PCR. HYA and chitosans, especially glycosylated derivative, are safe vehicles that can be used for drug transport into the inner ear through the RWM. Both vehicles successfully released the loaded neomycin, which exerted toxic effects on cochlear hair cells in a degree depending on the concentrations used. The vehicles per se had no noxious effect on the cochlear hair cells but they provoked a comparable effect on the thickness and morphology of the RWM. The thickness of the RWM returned to normal 4 weeks after exposure to HYA. Chitosan as a carrier for inner ear transfection, was associated with inconsistent transfection in vitro and in vivo . The importance of the RWM as a portal for local therapy of inner ear disorders is highlighted in this thesis by focusing on efficiency and effects of the vehicles, applied to the RWM for delivering biologically active agents into the cochlea. The difficulties and variability associated with applying substances to the RWM were explored. The results of this thesis add new knowledge concerning mechanisms of passage of biologically active agents through the RWM and may help us to better understand the role of RWM in the local cochlear therapy and problems of local treatment of inner ear diseases.

Place, publisher, year, edition, pages
Universitetsservice US-AB , 2010. , p. 60
Keywords [en]
Round window membrane, sodium hyaluronate, chitosan, local cochlear drug delivery and gene delivery
National Category
Medical and Health Sciences Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology; Biology
Identifiers
URN: urn:nbn:se:oru:diva-75083ISBN: 978-91-7457-067-0 (print)OAI: oai:DiVA.org:oru-75083DiVA, id: diva2:1336912
Public defence
2010-10-27, 09:10 (English)
Opponent
Supervisors
Available from: 2019-08-06 Created: 2019-07-11 Last updated: 2019-08-07Bibliographically approved
List of papers
1. Middle Ear Application of a Sodium Hyaluronate Gel Loaded with Neomycin in a Guinea Pig Model
Open this publication in new window or tab >>Middle Ear Application of a Sodium Hyaluronate Gel Loaded with Neomycin in a Guinea Pig Model
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2009 (English)In: Ear and Hearing, ISSN 0196-0202, E-ISSN 1538-4667, Vol. 30, no 1, p. 81-89Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: Establishing methods for topical administration of drugs to the inner ear have great clinical relevance and potential even in a relatively short perspective. To evaluate the efficacy of sodium hyaluronate (HYA) as a vehicle for drugs that could be used for treatment of inner ear disorders.

METHODS: The cochlear hair cell loss and round window membrane (RWM) morphology were investigated after topical application of neomycin and HYA into the middle ear. Sixty-five albino guinea pigs were used and divided into groups depending on the type of the treatment. Neomycin was chosen as tracer for drug release and pharmacodynamic effect. HYA loaded with 3 different concentrations of neomycin was injected to the middle ear cavity of guinea pigs. Phalloidin stained surface preparations of the organ of Corti were used to estimate hair cell loss induced by neomycin. The thickness of the midportion of the RWM was measured and compared with that of controls using light and electron microscopy. All animal procedures were pe rformed in accordance with the ethical standards of Karolinska Institutet.

RESULT: Neomycin induced a considerable hair cell loss in guinea pigs receiving a middle ear injection of HYA loaded with the drug, demonstrating that neomycin was released from the gel and delivered to the inner ear. The resulting hair cell loss showed a clear dose-dependence. Only small differences in hair cell loss were noted between animals receiving neomycin solution and animals exposed to neomycin in HYA suggesting that the vehicle neither facilitated nor hindered drug transport between the middle ear cavity and the inner ear. One week after topical application, the thickness of the RWM had increased and was dependent upon the concentration of neomycin administered to the middle ear. At 4 weeks the thickness of the RWM had returned to normal.

CONCLUSION: HYA is a safe vehicle for drugs aimed to pass into the inner ear through the RWM. Neomycin was released from HYA and transported into the inner ear as evidenced by hair cell loss.

Place, publisher, year, edition, pages
Lippincott Williams & Wilkins, 2009
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology; Biology
Identifiers
urn:nbn:se:oru:diva-75077 (URN)10.1097/AUD.0b013e31818ff98e (DOI)000262312000010 ()19125030 (PubMedID)2-s2.0-64849084054 (Scopus ID)
Funder
Swedish Research Council
Note

Funding Agencies:

European Commission

Tysta Skolan

Pettus and Augusta Hedlund Foundation

AFA Insurance

Hawler Medical University-Iraq

Available from: 2019-07-10 Created: 2019-07-10 Last updated: 2019-08-07Bibliographically approved
2. Use of the biodegradable polymer chitosan as a vehicle for applying drugs to the inner ear
Open this publication in new window or tab >>Use of the biodegradable polymer chitosan as a vehicle for applying drugs to the inner ear
2010 (English)In: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 39, no 1-3, p. 110-115Article in journal (Refereed) Published
Abstract [en]

Development of efficient local delivery systems for the auditory organ has an important role in clinical practice for the management of inner ear disorders using pharmacological means. Chitosan, a biodegradable polymer, is a good drug carrier with bioadhesive properties. The aim of this study was to investigate the feasibility of using chitosan to deliver drugs to the inner ear across the round window membrane (RWM). Three structurally different chitosans loaded with a tracer drug, neomycin, were injected into the middle ear cavity of albino guinea pigs (n=35). After 7 days the effect of chitosans and neomycin was compared among the treatment groups. The hearing organ was analysed for hair cell loss and the RWM evaluated in term of thickness. All tested chitosan formulations successfully released the loaded neomycin which then diffused across the RWM, and exerted ototoxic effect on the cochlear hair cells in a degree depending on the concentrations used. Chitosans per se had no noxious effect on the cochlear hair cells. It is concluded that the chitosans, and especially glycosylated derivative, are safe and effective carriers for inner ear therapy.

Place, publisher, year, edition, pages
Elsevier, 2010
Keywords
Round window membrane, Chitosan, Hair cell, Drug delivery
National Category
Pharmaceutical Sciences
Research subject
Oto-Rhino-Laryngology; Biology
Identifiers
urn:nbn:se:oru:diva-75076 (URN)10.1016/j.ejps.2009.11.003 (DOI)000274672300016 ()19931387 (PubMedID)2-s2.0-73749087296 (Scopus ID)
Funder
Forte, Swedish Research Council for Health, Working Life and WelfareSwedish Research Council
Note

Research funders:

European Commission FP6, EUROHEAR (LSHG-CT-20054-512063)

Tysta Skolan Foundation

Petrus and Augusta Hedlund Foundation

Hawler Medical University-Iraq (HMU)

Available from: 2019-07-10 Created: 2019-07-10 Last updated: 2019-08-07Bibliographically approved
3. Assessment of in Vitro and in Vivo Transfection Efficiency of the Biodegradable Polymer Chitosan in the Inner Ear
Open this publication in new window or tab >>Assessment of in Vitro and in Vivo Transfection Efficiency of the Biodegradable Polymer Chitosan in the Inner Ear
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2010 (English)In: The Journal of International Advanced Otology, ISSN 1308-7649, Vol. 6, no 3, p. 307-315Article in journal (Refereed) Published
Abstract [en]

Background: Sensorineural hearing loss is a significant problem worldwide and a condition that is not completely cured by currently available therapy. Gene therapy of the inner ear offers an exciting alternative and it has been suggested that this therapeutic modality could be used in treatment aiming at preventing, reversing or managing cochlear disorders. Because of their desired properties as an alternative to the viral vectors, non-viral vectors have been extensively explored for gene delivery. One example is chitosan, a biodegradable cationic polymer.

Objective: To evaluate the in vitro and in vivo transfection efficiency of chitosan as a non-viral gene carrier for gene delivery to cells of the inner ear.

Materials and Methods: Organotypic cultures of the hearing organ, the organ of Corti, were prepared from postnatal day 2 rats, and exposed to chitosan carrying plasmid DNA (pDNA) encoding for green fluorescent protein (GFP) for 24-48 hours. The in vivo transfection efficiency was tested at two time points, at one day or seven days after infusing chitosan/pDNA polyplexes through osmotic pumps into the cochlea of adult guinea pigs (n=41). The tissue was then processed for anti-GFP immunostaining (in vitro and in vivo) and RT-PCR (in vivo).

Results: The in vitro assessment showed prominent GPF transfection after 24-48 hours, while the in vivo GFP transfection in the inner ear was inconsistent and did not show good correlation with the in vitro transfection. Conclusion: It can be concluded that the using chitosan as a carrier for the in vivo transfection, is associated with varying and in consistent degree of transfection.

Place, publisher, year, edition, pages
European Academy of Otology and Neurotology and the Politzer Society, 2010
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology; Biology
Identifiers
urn:nbn:se:oru:diva-75078 (URN)000285986200002 ()2-s2.0-78650762031 (Scopus ID)
Funder
Swedish Research Council
Note

Funding Agencies:

European Commission

Tysta Skolan Foundation

Petrus and Augusta Hedlund Foundation

Swedish Council for Working Life and Social Research through the FAS Center 

Hawler Medical University-Iraq (HMU)

Available from: 2019-07-10 Created: 2019-07-10 Last updated: 2019-08-07Bibliographically approved
4. Ultrastructural features of the guinea pig round window membrane following round window membrane administration of sodium hyaluronate and chitosan
Open this publication in new window or tab >>Ultrastructural features of the guinea pig round window membrane following round window membrane administration of sodium hyaluronate and chitosan
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(English)Manuscript (preprint) (Other academic)
National Category
Other Medical Sciences not elsewhere specified Otorhinolaryngology
Research subject
Biology; Oto-Rhino-Laryngology
Identifiers
urn:nbn:se:oru:diva-75080 (URN)
Available from: 2019-07-11 Created: 2019-07-11 Last updated: 2019-08-07Bibliographically approved

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Saber, Amanj

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