Supplementary Material for
Ternary Polymeric Nanoparticles for Oral siRNA Delivery
Jing Zhang, Chunbai He, Cui Tang*, Chunhua Yin
State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China
*Corresponding author: Tel: +86 21 6564 3556; fax: +86 21 5552 2771.
E-mail address: (C. Tang)
SUPPLEMENTARY METHODS
Gel retardation
Preparation of TTMC/siRNA/HA nanoparticles with different weight ratios of components in nanoparticle formulation: Six hundred microliters of siRNA solution (0.2 mg/mL) was mixed with 1.2 mL or 1.8 mL of HA solution (1 mg/mL) at the siRNA/HA weight ratio of 1:10 or 1:15, respectively. The cationic polymers (6 mg/mL) were added drop-wise into the mixture under magnetic stirring at the polymer/HA weight ratio of 6:1, 7:1, 8:1, 9:1, and 10:1, respectively, followed by constant magnetic stirring for 30 s. The resultant nanoparticles were incubated at 37 °C for 30 min before use.
Agarose gel electrophoresis: The association of siRNA with the nanoparticles was monitored with gel retardation assay on 4 % agarose gel electrophoresis stained with ethidium bromide (0.5 μg/ml). The electrophoresis was performed at 56 V for 1 h.
Preparation and characterization of siRNA loaded nanoparticles via simple complexation
Chitosan, TMC, or TTMC solution (1.5 mL, 2 mg/mL) were added drop-wise into the 1 mL of siRNA solution (0.2 mg/mL) at the polymer/siRNA weight ratio of 15:1, respectively, followed by constant magnetic stirring for 30 s. The resultant nanoparticles were incubated at 37 °C for 30 min before use, and termed as cationic polymer/siRNA nanoparticles.
The particle size and ζ potential of nanoparticles were determined with Zetasizer Nano (Malvern, UK). Stability of nanoparticles against dilution was evaluated in terms of particle size and ζ potential. Chitosan/siRNA, TMC/siRNA, and TTMC/siRNA nanoparticles containing 4 μg of NC siRNA were diluted with DEPC-treated water upon 100 and 250 folds, respectively.
SUPPLEMENTARY FIGURE
Fig. S1 Agarose gel electrophoresis of TTMC/siRNA/HA nanoparticles with different weight ratios of components in nanoparticle formulation.
Fig. S2 Particle size (column) and ζ potential (scatter) of chitosan/siRNA, TMC/siRNA, and TTMC/siRNA nanoparticles after 100 and 250 folds dilution with DEPC-treated water.
Fig. S3 Agarose gel electrophoresis of naked NC siRNA incubated with various intestinal fluids and homogenates at 37 ○C. DF: duodenal fluids; JF: jejunal fluids; IF: ileal fluids; CF: colonic fluids; DTH: duodenal tissue homogenates; JTH: jejunal tissue homogenates; ITH: ileal tissue homogenates; CTH: colonic tissue homogenates; DMH: duodenal mucosa homogenates; JMH: jejunal mucosa homogenates; IMH: ileal mucosa homogenates; CMH: colonic mucosa homogenates.