Nanoparticles evading the reticuloendothelial system: role of the supported bilayer

Abstract

We have previously shown that the PEGylated LPD (liposome-polycation-DNA) nanoparticles were highly efficient in delivering siRNA to the tumor with low liver uptake. Its mechanism of evading the reticuloendothelial system (RES) is reported here. In LPD, nucleic acids were condensed with protamine into a compact core, which was then coated by two cationic lipid bilayers with the inner bilayer stabilized by charge-charge interaction (also called the supported bilayer). Finally, a detergent-like molecule, polyethylene glycol (PEG)-phospholipid is post-inserted into the lipid bilayer to modify the surface of LPD. The dynamic light scattering (DLS) data showed that LPD had improved stability compared to cationic liposomes after incubation with a high concentration of DSPE-PEG(2000), which is known to disrupt the bilayer. LPD prepared with a multivalent cationic lipid, DSGLA, had enhanced stability compared to those containing DOTAP, a monovalent cationic lipid, suggesting that stronger charge-charge interaction in the supported bilayer contributed to a higher stability. Distinct nanoparticle structure was found in the PEGylated LPD by transmission electron microscopy, while the cationic liposomes were transformed into tubular micelles. Size exclusion chromatography data showed that approximately 60% of the total cationic lipids, which were located in the outer bilayer of LPD, were stripped off during the PEGylation; and about 20% of the input DSPE-PEG(2000) was incorporated into the inner bilayer with about 10.6 mol% of DSPE-PEG(2000) presented on the particle surface. This led to complete charge shielding, low liver sinusoidal uptake, and 32.5% injected dose delivered to the NCI-H460 tumor in a xenograft model.

Figure 1

Illustration of the structure and formation of LPD. Cryo-TEM photograph (A), the illustration of the double lipid bilayer structure (B) and proposed mechanism for the formation of the LPD nanoparticles (C). Figure 1A was reproduced from Tan et al. with permission [11].

Figure 2

Stability of the nanoparticle formulations upon different degree of PEGylation. (A) Chemical structures of DOTAP and DSGLA. (B) Size distribution of different PEGylated formulations. Data = mean ± SD, n = 4–6

Figure 3

TEM photographs of liposomes/PEGylated liposomes and LPD/PEGylated LPD. Arrows indicate the “sprouts” of the particles and arrow heads indicate the small particles. Bar = 100 nm.

Figure 4

Size exclusion chromatography of different samples. (A) chromatography of neutral liposomes and pure DSPE-PEG₂₀₀₀ ; (B) chromatography of DOTAP in different formulations; (C) chromatography of DSPE-PEG₂₀₀₀ in different formulations; (D) chromatography of different components in the PEGylated LPD. Data are representative chromatography from 2–3 batches of formulations.

Figure 5

Tissue distribution analysis. (A) Liver sinusoidal uptake of cy3-siRNA in naked LPD (positive control) and PEGylated LPD. Nuclei (blue), F-actin (green), cy3-siRNA (red). Magnification = 1,600 x. Data are representative pictures from 3 mice in each group. (B) Fluorescence signal of FAM-labeled siRNA in different tissues detected by the Xenogen IVIS imaging system. Data are from two representative animals in each group. Data of the NCI-H460 model are reproduced from a previously published article with permission [7].

Figure 6

Proposed model for the formation of PEGylated LPD and the mechanism of favored tumor uptake.