[EN] A PROCESS FOR THE PREPARTION OF NON-VIRAL VECTOR FOR DELIVERY OF NUCLEIC ACIDS BY MUCOSAL ROUTE<br/>[FR] PROCÉDÉ DE PRÉPARATION D'UN VECTEUR NON VIRAL POUR L'ADMINISTRATION D'ACIDES NUCLÉIQUES PAR VOIE MUQUEUSE
申请人:DEPT OF BIOTECHNOLOGY INDIA
公开号:WO2014064710A1
公开(公告)日:2014-05-01
The invention relates to a process for preparation of non-viral vector for delivery of nucleic acids by mucosal route comprising the steps' of (a) synthesis of pDNA loaded calcium phosphate nanoparticles using reverse micro-emulsion method having n- Hexane as oil phase and water as the aqueous phase; (b) addition of excess water to make total volume of water to adjust the molar ratio of water to AOT at 10 before stirriung both the micro-emulsions; (c) taking 1.36M of anhydrous calcium chloride and 0.35 M di-sodium hydrogen phosphate in two separate micro-emulsion system as the precursors; (d) adding 3μg of pDNA of interested into each system followed by continuous stirring for 12 hours; (e) mixing the micro- emulsion with di-sodium hydrogen phosphate to the micro-emulsion with calcium chloride at slow rate with continuous stirring at 4°C; (f) keeping the mixture, as obtained in step (e), undisturbed at low temperature under continuous stirring for 24 hours; (g) removing n- Hexane by using Bucci-evaporator and dissolving the resulting mass of AOT in 10 ml of absolute ethanol (99.9%) by vortexing; (h) centrifuging the solution for half an hour at 800 rpm at 4°C in a cold centrifuge; (i) washing the pelleted nanoparticles with absolute alcohol three times; (j) dispersing the pelleted nanoparticles in double distilled water at 4°C by vortexing to secure clear dispersion; and (k) dialyzing the dispersion, as obtained in step (j), in cold room using 14 kD dialysis membrane bag and followed finally for coating over nanoparticles with the polymers.