The use of the biomaterial chitosan (CS) to form CS/siRNA nanoparticles for gene silencing have been widely investigated. The present study shows that the physico-chemical properties and in-vitro gene silencing of CS/siRNA nanoparticles can be influenced by nanoparticle composition. siRNA loaded CS nanoparticles were obtained starting from (1) CS itself, (2) CS and sodium tripolyphosphate (TPP), (3) CS and hyaluronic acid (HA) (4) CS, HA and TPP. As comparison, polyethyleimmine (PEI) nanoparticles were also produced. CS nanoparticles were prepared by different methods of ionic cross-linking (simple complexation, ionic gelation and complex coacervation) using different CS nanoparticles to siRNA weight ratios (N/P 10, N/P 50, N/P 100). All methods produced positively charged, stable particles with size in the range of 200 nm to 500 nm. Gel retardation assay revealed no migration of siRNA from nanoparticles for higher chitosan to siRNA weight ratio (N/P 50 and 100) thus suggesting a complete binding of siRNA in the presence of an excess of positive charges which can counteract with negatively charged siRNA. Moreover, serum stability tests showed that in contrast with naked siRNA, the siRNA recovered from CS-siRNA and CS-HA-siRNA nanoparticles (N/P 50 and 100) was intact up to 5 days after incubation in 50% serum. In-vitro tests showed that all CS based nanoparticles were non toxic with respect to PEI based particles. Transfection studies revealed poor gene silencing efficiency in adhese neuroblastoma cell line and great gene silencing efficiency in suspension leukemia cell line. The highest gene silencing efficiency in leukemia cell line was achieved using CS/siRNA nanoparticles (N/P 50). This work confirms the application of CS as a non-viral carrier for siRNA particularly for suspension cell lines, which are traditionally difficult to transfect and underlines the importance of nanoparticle composition for the optimisation of gene silencing.

Chitosan nanoparticles for siRNA delivery: influence of nanoparticle composition on gene silencing efficiency

TONELLI, ROBERTO;LUPPI, BARBARA;LOMBARDINI, LORENZA;RAMPELLI, SIMONE;HRELIA, PATRIZIA;PESSION, ANDREA
2010

Abstract

The use of the biomaterial chitosan (CS) to form CS/siRNA nanoparticles for gene silencing have been widely investigated. The present study shows that the physico-chemical properties and in-vitro gene silencing of CS/siRNA nanoparticles can be influenced by nanoparticle composition. siRNA loaded CS nanoparticles were obtained starting from (1) CS itself, (2) CS and sodium tripolyphosphate (TPP), (3) CS and hyaluronic acid (HA) (4) CS, HA and TPP. As comparison, polyethyleimmine (PEI) nanoparticles were also produced. CS nanoparticles were prepared by different methods of ionic cross-linking (simple complexation, ionic gelation and complex coacervation) using different CS nanoparticles to siRNA weight ratios (N/P 10, N/P 50, N/P 100). All methods produced positively charged, stable particles with size in the range of 200 nm to 500 nm. Gel retardation assay revealed no migration of siRNA from nanoparticles for higher chitosan to siRNA weight ratio (N/P 50 and 100) thus suggesting a complete binding of siRNA in the presence of an excess of positive charges which can counteract with negatively charged siRNA. Moreover, serum stability tests showed that in contrast with naked siRNA, the siRNA recovered from CS-siRNA and CS-HA-siRNA nanoparticles (N/P 50 and 100) was intact up to 5 days after incubation in 50% serum. In-vitro tests showed that all CS based nanoparticles were non toxic with respect to PEI based particles. Transfection studies revealed poor gene silencing efficiency in adhese neuroblastoma cell line and great gene silencing efficiency in suspension leukemia cell line. The highest gene silencing efficiency in leukemia cell line was achieved using CS/siRNA nanoparticles (N/P 50). This work confirms the application of CS as a non-viral carrier for siRNA particularly for suspension cell lines, which are traditionally difficult to transfect and underlines the importance of nanoparticle composition for the optimisation of gene silencing.
2010
THIRD BIOTECH WORKSHOP, "Drug delivery systems for biotech products"
22
22
R. Tonelli; B. Luppi; L. Lombardini; S. Rampelli; P. Hrelia; A. Pession
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/88859
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