Effect of PEG anchor in PEGylation of folate-modified cationic liposomes with PEG-derivatives on systemic siRNA delivery into the Tumor

Abstract

Materials and Methods Accessibility of siRNA in siRNA lipoplexesFA-PEG-modified siRNA lipoplexes and FA-PEG-and PEG-modified siRNA lipoplexes were prepared by mixing at charge ratios (+:-) of 1:1~4:1. Amounts of free siRNA in the siRNA lipoplexs were measured using exclusion assays with SYBR ® Green I Nucleic Acid Gel Stain (Takara Bio Inc., Shiga, Japan) and calculated based on the standard curves of free siRNA as previously reported [1].Cellular uptake of FA -PEG-modified siRNA lipoplexes KB cells were plated into 35-mm culture dishes at a density of 3 × 10 5 cells 24 h prior to each experiment. LP-0.5F5, LP-1F5, LP-2F5, and LP-3F5 lipoplexes with 50 pmol Cy5-siRNA were diluted in 1 mL of folate-deficient RPMI-1640 medium, and they were incubated with KB cells for 3 h. After the incubation, the cells were fixed with 4% formaldehyde. The localization of Cy5-siRNA was visualized using an Eclipse TS100-F microscope (Nikon, Tokyo, Japan).

Recent Advance of Liposome Nanoparticles for Nucleic Acid Therapy

“…In order to solve the above problems, researchers began to try to introduce cell-targeted small molecule compounds to increase the intake of liposomes by target cells. Tang et al introduced folic acid molecules into 1,2-distanoyl- sn -glycero-3-phosphoethanolamine (DSPE) FA-PEG5000-DSPE modified with PEG of different molecular weights (MW: 2.0 kDa, 3.4 kDa, and 5.0 kDa), and studied their delivery effects on siRNA [ 112 ]. The experimental results showed that polo-like kinase 1 (PLK1) siRNA transfection mediated by FA-PEG5000-DSPE could selectively inhibit the growth of human nasopharyngeal carcinoma KB cells.…”

Design of Folate-Containing Liposomal Nucleic Acid Delivery Systems for Antitumor Therapy

“…Nonetheless, PEG spacer with a high molecular weight of 5000 Da inhibited cellular uptake of siRNA lipoplexes compared to a short analog (2000 Da) [ 67 ]. In this case, lower amounts of the FA lipoconjugate are required [ 69 ]. Besides PEG, other hydrophobic (hexamethyl or longer alkyl chains) or hydrophilic (butoxybutoxybutyl) spacers may be used [ 44 , 49 ], providing effective NA delivery in vitro.…”

“…A possible explanation is the greater probability of PEG 5000 chains to interact between each other which lead to “masking” of the ligand [ 72 ]. On the other hand, cationic liposomes containing 0.5 mol% DSPE-PEG 5000 -FA provided more effective siRNA delivery both in vitro and in vivo compared with liposomes containing FA lipoconjugate with shorter PEG spacers [ 69 ]. Moreover, liposomes containing 0.5 mol% Chol-suc-PEG 4000 -FA demonstrated a three-fold higher cellular uptake by HepG2 cells compared with liposomes containing Chol-suc-PEG 2000 -FA with shorter PEG spacer [ 73 ].…”

“…FA-containing cationic liposomes were prepared and optimized for in vivo siRNA delivery [ 69 ]. Liposomes differed both in the length of PEG spacer (2000, 3400 or 5000 Da) and in the molar percentage of FA lipoconjugate.…”

“…Inclusion of 1–2 mol% FA-PEG-DSPE into siRNA lipoplexes might be needed for preventing agglutination during systemic injection. Therefore, 2.5 mol% PEG lipid was added to provide additional lipoplex stabilization in blood circulation, which permitted an increased accumulation of Cy5-labeled siRNA in KB xenografts [ 69 ].…”