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Assessment of in Vitro and in Vivo Transfection Efficiency of the Biodegradable Polymer Chitosan in the Inner Ear
Örebro University, School of Medical Sciences. Örebro University Hospital. Center for Hearing and Communication Research and Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; Department of Orolaryngology, Karolinska University Hospital, Stockholm, Sweden.
Center for Hearing and Communication Research and Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; Department of Orolaryngology, Karolinska University Hospital, Stockholm, Sweden.
Center for Hearing and Communication Research and Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; Department of Orolaryngology, Karolinska University Hospital, Stockholm, Sweden.
Center for Hearing and Communication Research and Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; Department of Orolaryngology, Karolinska University Hospital, Stockholm, Sweden.
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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. Vol. 6, no 3, p. 307-315
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology; Biology
Identifiers
URN: urn:nbn:se:oru:diva-75078ISI: 000285986200002Scopus ID: 2-s2.0-78650762031OAI: oai:DiVA.org:oru-75078DiVA, id: diva2:1336878
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
In thesis
1. Round window membrane and delivery of biologically active agents into the cochlea
Open this publication in new window or tab >>Round window membrane and delivery of biologically active agents into the cochlea
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
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:nbn:se:oru:diva-75083 (URN)978-91-7457-067-0 (ISBN)
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

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Saber, Amanj

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