Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers.
Nonviral gene delivery systems based on conventional high-molecular-weight chitosans are efficient after lung administration in vivo, but have poor physical properties such as aggregated shapes, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery and a slow dissociation and release of plasmid DNA, resulting in a slow onset of action. We therefore developed highly effective nonviral gene delivery systems with improved physical properties from a series of chitosan oligomers, ranging in molecular weight from 1.2 to 10 kDa. First, we established structure-property relationships with regard to polyplex formation and in vivo efficiency after lung administration to mice. In a second step, we isolated chitosan oligomers from a preferred oligomer fraction to obtain fractions, ranging from 10 to 50-mers, of more homogeneous size distributions with polydispersities ranging from 1.01 to 1.09. Polyplexes based on chitosan oligomers dissociated more easily than those of a high-molecular-weight ultrapure chitosan (UPC, approximately a 1000-mer), and released pDNA in the presence of anionic heparin. The more easily dissociated polyplexes mediated a faster onset of action and gave a higher gene expression both in 293 cells in vitro and after lung administration in vivo as compared to the more stable UPC polyplexes. Already 24 h after intratracheal administration, a 120- to 260-fold higher luciferase gene expression was observed compared to UPC in the mouse lung in vivo. The gene expression in the lung was comparable to that of PEI (respective AUCs of 2756+/-710 and 3320+/-871 pg luciferase x days/mg of total lung protein). In conclusion, a major improvement of chitosan-mediated nonviral gene delivery to the lung was obtained by using polyplexes of well-defined chitosan oligomers. Polyplexes of oligomer fractions also had superior physicochemical properties to commonly used high-molecular-weight UPC.
Department of Pharmacy, Uppsala University, Sweden.
M
M
Köping-Höggård
K M
KM
Vårum
M
M
Issa
S
S
Danielsen
B E
BE
Christensen
B T
BT
Stokke
P
P
Artursson
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D048271:Chitosan / Q000737:chemistry; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008168:Lung / Q000378:metabolism; D051379:Mice; D011094:Polyethyleneimine; D011108:Polymers; D013329:Structure-Activity Relationship
D011108:Polymers; D011094:Polyethyleneimine; D048271:Chitosan; D008156:Luciferases