Figure S2 Intracellular localization of LNPs <Methods> Cells (5 x 104) were seeded on glass bottom dishes (IWAKI, ASAHI GLASS CO., LTD, Japan) in 2 ml of culture medium 1 day before transfection. For transfection, the cells were incubated with a 2 ml aliquot of LNPs that were dually labeled with rhodamine (pDNA) and NBD (lipid envelope) in DMEM containing 10% serum. LNPs were incubated with the cells for 3 h. The nuclei were stained with 5 µg/mL Hoechst33342 (DOJINDO Laboratories, Kumamoto, Japan). Cell surface-bound LNPs were removed by washing 2 times with 1 ml of PBS containing heparin (20 Units/ml). The culture medium was replaced with the buffer, and microscopic observations were then performed. Scale bars indicated 10 µm. <Result> In the case of LNPPalmM, pDNA (red) was dispersed throughout the cytoplasm, and nuclear accumulation was negligable. Also, the pDNA free from the co-localization with lipid envelope (green) was clearly observed. In contrast, pDNA delivered with LNPPalmA was dominantly co-localized with lipid envelope, and furthermore, was extensively accumulated to the nuclear periphery. This support our conclusion that LNPPalmA is associated with the nuclear transport machinery and that the particles remain intact. Figure S3 Effects of retinoic acid on the intracellular localization of LNPs <Methods> Cells (5 x 104) were seeded on glass bottom dishes (IWAKI, ASAHI GLASS CO., LTD, Japan) in 2 ml of culture medium 1 day before transfection. For transfection, the cells were incubated with a 2 ml aliquot of LNPs including 1.6 µg pDNA labeled with rhodamine in DMEM supplied with 10% serum. The LNPs were
Figure S2 Intracellular localization of LNPs <Methods> Cells (5 x 104) were seeded on glass bottom dishes (IWAKI, ASAHI GLASS CO., LTD, Japan) in 2 ml of culture medium 1 day before transfection. For transfection, the cells were incubated with a 2 ml aliquot of LNPs that were dually labeled with rhodamine (pDNA) and NBD (lipid envelope) in DMEM containing 10% serum. LNPs were incubated with the cells for 3 h. The nuclei were stained with 5 µg/mL Hoechst33342 (DOJINDO Laboratories, Kumamoto, Japan). Cell surface-bound LNPs were removed by washing 2 times with 1 ml of PBS containing heparin (20 Units/ml). The culture medium was replaced with the buffer, and microscopic observations were then performed. Scale bars indicated 10 µm. <Result> In the case of LNPPalmM, pDNA (red) was dispersed throughout the cytoplasm, and nuclear accumulation was negligable. Also, the pDNA free from the co-localization with lipid envelope (green) was clearly observed. In contrast, pDNA delivered with LNPPalmA was dominantly co-localized with lipid envelope, and furthermore, was extensively accumulated to the nuclear periphery. This support our conclusion that LNPPalmA is associated with the nuclear transport machinery and that the particles remain intact. Figure S3 Effects of retinoic acid on the intracellular localization of LNPs <Methods> Cells (5 x 104) were seeded on glass bottom dishes (IWAKI, ASAHI GLASS CO., LTD, Japan) in 2 ml of culture medium 1 day before transfection. For transfection, the cells were incubated with a 2 ml aliquot of LNPs including 1.6 µg pDNA labeled with rhodamine in DMEM supplied with 10% serum. The LNPs were
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