[In vitro and in vivo studies on hydroxyapatite nanoparticles as a novel vector for inner ear gene therapy].
OBJECTIVE
To assess the possibility of hydroxyapatite (HAT) nanoparticles as a gene vector for inner ear gene transfection.


METHODS
The HAT nanoparticles were prepared by the precipitation hydrothermal technique. DNA binding test under series of pH values and HAT concentrations were carried out by means of DNA electrophoresis to show the DNA binding and protective effect of the HAT nanoparticles. The cytotoxicity of HAT was evaluated via methyl thiazolyl tetrazolium assay (MTT) test with cultured Hela cells. The in vitro green fluorescent protein (GFP) reported transfection test and neurotrophin 3 (NT3) Western blot were completed with both Hela cells and primarily cultured mice cochlear neurons, so as to observe HAT mediated in vitro gene transfection efficiency and consequent NT3 gene expression. The complex consisting of HAT nanoparticles and recombinant plasmid with enhanced GFP and NT3 (HAT-pEGFPC2-NT3) was perfused perilymphatically into the cochlea of the guinea pigs with experimental excitotoxic inner ear damage. Inner ear NT3 gene expression was immunohistochemically demonstrated (DAB method) to evaluate the capability of HAT nanoparticles in mediating NT3 gene transfection in living animals and the possibility of future clinical application.


RESULTS
When applied at the lowest concentration of 250 microg/ml and the pH values of 3-7, the 40-50 nm stick-shaped nanoparticles fully loaded the recombinant plasmid pEGFPC2-NT3, and well protected the genes from being destroyed by DNase I. Well biocompatibility between HAT nanoparticles and the cultured Hela cells had been confirmed by methyl thiazolyl tetrazolium assay (MTT) method when the concentration of HAT nanoparticles ranged from 62.5 microg/ml to 500 microg/ml. Immunofluorescence test of the report gene GFP revealed that the transfection efficiency of HAT nanoparticle vector in Hela cells was 15.7% +/- 2.6% (x +/- s). EGFP was immunofluorescently expressed by transfection of HAT-pEGFPC2-NT3 in the primarily cultured cochlear neurons of mice. NT3 immunohistochemical test showed many marked ganglion cells in the cochlea when perilymphatically transfected HAT-pEGFPC2-NT3 complex into the cochlea of guinea pigs with experimental excitotoxic inner ear damage in vivo.


CONCLUSIONS
HAT nanoparticles can mediate NT3 gene transfection both in vitro and in the living animal's cochlea. As a non-viral gene vector without the risk of biological disaster, HAT nanoparticle vector is worthy of further fully researches although its transfection efficiency needs to be improved.
Sun
Hong
H
Department Otorhinolaryngology, Head and Neck Surgery, The Third Xiangya Hospital, Central South University, Changsha 410013, China. shjhaj@vip.163.com
Jiang
Ming
M
Zhu
Shai-Hong
SH
D000818:Animals; D003051:Cochlea / Q000187:drug effects / Q000378:metabolism; D004337:Drug Carriers; D017886:Durapatite / Q000008:administration & dosage*; D007758:Ear, Inner / Q000187:drug effects* / Q000378:metabolism; D015316:Genetic Therapy; D005822:Genetic Vectors; D006168:Guinea Pigs; D006367:HeLa Cells; D006801:Humans; D007759:Labyrinth Diseases / Q000628:therapy*; D051379:Mice; D008815:Mice, Inbred Strains; D053758:Nanoparticles; D010957:Plasmids; D014162:Transfection
D004337:Drug Carriers; D017886:Durapatite