Novel PEGylated Liposomes Enhance Immunostimulating Activity of isRNA

Abstract

The performance of cationic liposomes for delivery of therapeutic nucleic acids in vivo can be improved and specifically tailored to certain types of cargo and target cells by incorporation of PEG-containing lipoconjugates in the cationic liposome’s composition. Here, we report on the synthesis of novel PEG-containing lipoconjugates with molecular masses of PEG 800, 1500 and 2000 Da. PEG-containing lipoconjugates were used as one of the components in liposome preparation with the polycationic amphiphile 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetra-azahexacosan tetrahydrochloride (2X3) and the lipid-helper dioleoylphosphatidylethanolamine (DOPE). We demonstrate that increasing the length of the PEG chain reduces the transfection activity of liposomes in vitro, but improves the biodistribution, increases the circulation time in the bloodstream and enhances the interferon-inducing activity of immunostimulating RNA in vivo.

Immunostimulating RNA Delivered by P1500 PEGylated Cationic Liposomes Limits Influenza Infection in C57Bl/6 Mice

“…It was shown [ 13 ] that the antiproliferative activity with respect to tumor cells, as well as activation of cytokine secretion by isRNA, requires the presence of transfection agents. isRNA delivery to the cell, mediated by liposomes, is necessary for the manifestation of the interferon-inducing activity of isRNA, and the effectiveness of both the interferon-inducing and antiproliferative effects depends on the efficiency of isRNA delivery into the cells [ 20 , 21 ]. This indicates that isRNA activates some cytosolic or endosomal sensors, similarly to viral RNA.…”

“…efficiently delivered various nucleic acids, including isRNA, to target cells both in vitro and in vivo [ 22 ]. The addition of PEG (MW 1500) lipoconjugate to the 2X3-DOPE formulation (2 mole%) resulted in the formulation P1500 [ 20 ], providing prolonged circulation of lipoplexes in the bloodstream and increased efficiency of IFN-α induction by isRNA. Hydrodynamic diameters, polydispersity index (PI) and ξ-potentials of liposomes and lipoplexes formed by isRNA and liposomes (N/P = 6/1) are shown in Table 1 .…”

“…Oligoribonucleotides (strand 1: 5′-GUGUCAGGCUUUCAGAUUUUUU-3′; strand 2: 5′-AAAUCUGAAAGCCUGACACUUA-3′) were synthesized, isolated and annealed as described previously [ 20 ].…”

“…injection through the tail vein with isRNA (0.5 µg/g) precomplexed with cationic liposomes at an N/P ratio of 6/1 six hours prior to infection with influenza A/WSN/33 (H1N1; hereafter named IVA). For the delivery of isRNA cationic liposomes 2X3-DOPE [ 17 ] and PEG-containing cationic liposomes P1500 [ 20 ] were used. Complexes of cationic liposomes with isRNA were prepared in a serum-free Opti-MEM medium by mixing equal volumes of isRNA and liposomes, followed by incubation for 20 min at room temperature.…”

“…We have previously developed an efficient liposomal nucleic acid delivery system based on polycationic amphiphile 1,26-bis (cholest-5-en-3-yloxycarbonylamino) -7,11,16,20-tetraazagehexacosane tetrahydrochloride (2X3) and lipid-helper dioleoylphosphatidylethanolamine (DOPE), which efficiently delivers nucleic acids in vitro and in vivo [ 17 , 18 , 19 ]. We synthesized PEG-containing lipoconjugates that differed in the molecular weight of PEG (800, 1500 and 2000 kDa) and showed that increasing the length of PEG in the lipoconjugate decreased the effectiveness of the delivery of nucleic acids by the liposomes containing these lipoconjugates in vitro, but improved biodistribution, duration of circulation in the bloodstream and interferon-inducing effect of isRNA in vivo [ 20 ]. IsRNA exhibited the most pronounced interferonogenic effect in combination with liposomes containing 1500 kDa PEG (P1500); therefore, in the present study, we chose this formulation and 2X3-DOPE core system to study the antiviral effect of isRNA.…”