Chitosan-mediated siRNA delivery in vitro: effect of polymer molecular weight, concentration and salt forms.
The aim of this study was to investigate chitosan/siRNA complexes formulated with various chitosan salts (CS) including chitosan aspartate (CS-Asp), chitosan glutamate (CS-Glu), chitosan acetate (CS-Ac), and chitosan hydrochloride (CS-HCl) for in vitro siRNA delivery into stable and constitutive enhanced green fluorescent protein (EGFP)-expressing HeLa cells. The CS/siRNA complexes were characterized by 2% agarose gel electrophoresis and investigated for their transfection efficiency in stable and constitutive EGFP-expressing HeLa cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The formation of complexes CS/siRNA is mainly dependent on the weight ratio, whereas salt form and molecular weight has less effect. The particle sizes of the complete complexes were in the range of 270-373 nm except the complete complex of CS-Ac, with a slightly positive charge of less than 2 mV. The ability of CS to transfer functionally active siRNA into cell culture is mainly dependent on the weight ratio and molecular weight of CS whereas salt form of CS has less effect. The high gene-silencing efficiency was observed with low MW of CS (20 kDa) and high weight ratio of 32. Over 80% average cell viabilities were observed for CS/siRNA complexes in all weight ratios comparison to untreated cells. This study suggests CS salts have the potential to be used as safe siRNA delivery vectors.
Techaarpornkul
Sunee
S
Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand.
Wongkupasert
Sirirat
S
Opanasopit
Praneet
P
Apirakaramwong
Auayporn
A
Nunthanid
Jurairat
J
Ruktanonchai
Uracha
U
D000818:Animals; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D048271:Chitosan / Q000378:metabolism* / Q000494:pharmacology*; D006224:Cricetinae; D004587:Electrophoresis, Agar Gel; D020868:Gene Silencing; D005822:Genetic Vectors; D005971:Glutamates / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D010316:Particle Size; D011108:Polymers / Q000378:metabolism / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics* / Q000494:pharmacology; D012492:Salts / Q000378:metabolism / Q000494:pharmacology*; D012965:Sodium Chloride / Q000378:metabolism / Q000494:pharmacology; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection
D005971:Glutamates; D011108:Polymers; D034741:RNA, Small Interfering; D012492:Salts; D013778:Tetrazolium Salts; D013844:Thiazoles; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride; D048271:Chitosan; C022616:thiazolyl blue