American Society of Hematology

Figure 1

$Figure 1. Characterization of siILs. (A) Measurement of siIL size. siRNA-bearing immunoliposomes (siILs) were prepared as described in “Preparation of siILs.” siIL mean diameter was approximately 86 nm after conjugation of NLDC-145 mAb to liposome surface. (B) Removal of unencapsulated siRNA. Polyacrylamide gel electrophoresis was used to resolve siRNA-containing liposomes before and after RNase III digestion of unencapsulated siRNA. (C) Fraction elution of siILs. After the encapsulation of Cy3-labeled siRNA and conjugation of liposomes to Alexa647-labeled NLDC-145 mAb (anti-DEC205), siILs were separated from digested siRNA fragments of exteriorized siRNA and unconjugated mAb by Sepharose CL-4B gel filtration chromatography. Eluted fractions were analyzed by spectrofluorometry (excitation/emission 550/570 nm for siRNA and 650/668 nm for mAb) for simultaneous detection of encapsulated siRNA and conjugated mAb. (Left panel) siRNA-bearing siILs containing both siRNA and NLDC-145 mAb, which comigrate through the filtration column (first set of overlapping peaks). (Middle panel) Empty siILs containing no siRNA but conjugated to NLDC-145 mAb. (Right panel) Control stealth liposomes containing siRNA but not conjugated to NLDC-145 mAb. (D) Stability assay of siILs in blood plasma. Cy3-labeled CD40-siILs or naked siRNA was incubated with fresh mouse plasma at 37°C for various time periods. After incubation for 0, 0.5, 3, 12, 24, and 48 hours, siRNA from siILs was extracted with Triton X-100 and detected by polyacrylamide gel electrophoresis. (E) RNase resistance assay of siILs. siILs or naked siRNA (30 pmol) was incubated with 40 ng RNase at 37°C for 6 hours. siRNA was extracted from siILs with Triton X-100 and detected by polyacrylamide gel electrophoresis.$

Characterization of siILs. (A) Measurement of siIL size. siRNA-bearing immunoliposomes (siILs) were prepared as described in “Preparation of siILs.” siIL mean diameter was approximately 86 nm after conjugation of NLDC-145 mAb to liposome surface. (B) Removal of unencapsulated siRNA. Polyacrylamide gel electrophoresis was used to resolve siRNA-containing liposomes before and after RNase III digestion of unencapsulated siRNA. (C) Fraction elution of siILs. After the encapsulation of Cy3-labeled siRNA and conjugation of liposomes to Alexa647-labeled NLDC-145 mAb (anti-DEC205), siILs were separated from digested siRNA fragments of exteriorized siRNA and unconjugated mAb by Sepharose CL-4B gel filtration chromatography. Eluted fractions were analyzed by spectrofluorometry (excitation/emission 550/570 nm for siRNA and 650/668 nm for mAb) for simultaneous detection of encapsulated siRNA and conjugated mAb. (Left panel) siRNA-bearing siILs containing both siRNA and NLDC-145 mAb, which comigrate through the filtration column (first set of overlapping peaks). (Middle panel) Empty siILs containing no siRNA but conjugated to NLDC-145 mAb. (Right panel) Control stealth liposomes containing siRNA but not conjugated to NLDC-145 mAb. (D) Stability assay of siILs in blood plasma. Cy3-labeled CD40-siILs or naked siRNA was incubated with fresh mouse plasma at 37°C for various time periods. After incubation for 0, 0.5, 3, 12, 24, and 48 hours, siRNA from siILs was extracted with Triton X-100 and detected by polyacrylamide gel electrophoresis. (E) RNase resistance assay of siILs. siILs or naked siRNA (30 pmol) was incubated with 40 ng RNase at 37°C for 6 hours. siRNA was extracted from siILs with Triton X-100 and detected by polyacrylamide gel electrophoresis.

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