Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor

Abstract

Calcium phosphate (CaP) nanoparticles (NP) with an asymmetric lipid bilayer coating have been designed for targeted delivery of siRNA to the tumor. An anionic lipid, dioleoylphosphatydic acid (DOPA), was employed as the inner leaflet lipid to coat the nano-size CaP cores, which entrap the siRNA, such that the coated cores were soluble in organic solvent. A suitable neutral or cationic lipid was used as the outer leaflet lipid to form an asymmetric lipid bilayer structure verified by the measurement of NP zeta potential. The resulting NP was named LCP-II with a size of about 25 to 30nm in diameter and contained a hollow core as revealed by TEM imaging. PEGylation of NP was done by including a PEG-phospholipid conjugate, with or without a targeting ligand anisamide, in the outer leaflet lipid mixture. The sub-cellular distribution studied in the sigma receptor positive human H460 lung cancer cells indicated that LCP-II could release more cargo to the cytoplasm than our previous lipid/protamine/DNA (LPD) formulation, leading to a significant (~40 fold in vitro and ~4 fold in vivo) improvement in siRNA delivery. Bio-distribution study showed that LCP-II required more PEGylation for MPS evasion than the previous LPD, probably due to increased surface curvature in LCP-II.

Figure 1

The outline for the preparation of LCP-II NP and the structure of DOPA.

Figure 2

(A), TEM image of CaP cores coated with DOPA. (B), Hypothesis of the CaP core growth. (C) and (D),TEM images of LCP-II NPs coated with DOTAP and DSPE-PEG without(C) and with (D) negative staining. Arrows in (D) show lipid bilayer surrounding the CaP core.

Figure 3

(A) Zeta-potential of different liposome and LCP-II formulations. All lipid compositions contained equal amount of cholesterol. (B) Fluorescence quenching of Rhod-PE by trypan blue. N=3.

Figure 4

Subcellular distribution of FAM-labeled dsDNA (model for siRNA) in H460 cells. Cells were incubated with 50 nM dsDNA formulated in LCP-II coated with DOTAP targeted with AA (A), untargeted LCP-II (B), or AA-targeted LPD (C) for 3 h and imaged with confocal microscopy. Short arrows in (A) indicate spread, even distribution of fluorescently labeled dsDNA. Long arrows in (C) indicate punctuate distribution of dsDNA.

Figure 5

In vitro silencing effect of anti-luciferase siRNA formulated in LPD, LCP and LCP-II with DOTAP (dotted line) and DOPC (solid line) as the outer leaflet lipid. Data indicate that LCP-II was about 50-fold more active in delivering siRNA than LPD.

Figure 6

Biodistribution of fluorescence-labeled siRNA delivered by LCP-II coated with DOTAP and modified with different amounts of DSPE-PEG. LCP-II was coated with either 16 or 23 mol% of DSPE-PEG and i.v. injected into nude mice bearing human H460 tumor.

Figure 7

In vivo silencing effect of luciferase siRNA delivered with LCP-II. Mice bearing human H460 tumor stably expressing luciferase were i.v. injected with LCP-II prepared with either DOTAP or DOPC, indicated at the top of the figure, as the outer leaflet lipid. Luc: luciferase siRNA. Con: control siRNA. Numbers in X-axis indicate the injected dose of siRNA in mg/kg. ** indicates p<0.05.