Elsevier

Journal of Controlled Release

Volume 158, Issue 1, 28 February 2012, Pages 108-114
Journal of Controlled Release

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

https://doi.org/10.1016/j.jconrel.2011.10.020Get rights and content

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 30 nm 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.

Introduction

RNA interference (RNAi) therapeutics, such as siRNA, requires a suitable vehicle for in vivo delivery [1]. An ideal vehicle for cancer therapy should meet at least four major criteria. They include evasion of the mononuclear phagocytic system (MPS), extravasation from the blood circulation into the tumor, diffusion through the extracellular matrix to bind with tumor cells, and escape from the endosome to release the cargo siRNA into the cytoplasm [2].Therefore, a well protected nanoparticle (NP) modified with a suitable targeting ligand is considered a typical siRNA delivery vehicle to the tumor if the NP diameter is less than 200 nm [3], [4]. Two major types of lipid-based NPs have been developed for targeted siRNA delivery, such as lipid nanoparticle (LNP)[5], [6], [7], [8] and lipid/polycation/DNA complex (LPD)[9], [10], [11]. Polymers such as transferrin-cyclodextrin polycationare are also effective [12], [13], [14].

To improve the cargo release activity of our previous LPD formulation, we have prepared a pH-sensitive calcium phosphate (CaP) core to replace the protamine/DNA core in the LPD formulation [15]. The Lipid/Calcium/Phosphate type I (LCP-I) formulation was protected by PEG tethered with an anisamide ligand for binding to the sigma receptor over-expressing tumor cells. LCP-I showed a 4-fold improvement of the silencing effect in vitro compared to the previous LPD formulation. There was also a significant target gene silencing activity in a xenograft model with no significant elevation of inflammatory cytokines, i.e., IL-6 and IL-12, in the blood. However, the CaP core in LCP-I is highly hydrophilic and requires an un-scalable column method for purification. In addition, many factors on the surface of the nanoparticle can influence blood residence time and organ-specific accumulation [16].Therefore, it is desirable that a variety of lipid can be used as the outer leaflet lipid. Such flexibility in the choice of the lipid can be important for controlling the pharmacokinetics and tissue distribution properties of the NP.

In the current work, we prepared a novel siRNA delivery vehicle by employing an anionic phospholipid, dioleoylphosphatidic acid (DOPA), as a pre-coating reagent during the formation of the nano-sized CaP core in which siRNA was encapsulated. The lipid coating prevented the CaP core from aggregation during the centrifugal separation step and rendered it soluble in CHCl3. The resulting NP core was very small (25–30 nm) and contained a hollow structure. The DOPA layer on the surface of the CaP core also served as the inner leaflet lipid for the surface lipid bilayer of LCP. Lipids for the outer leaflet could simply be added into the CaP core solution in CHCl3. Since a PEG–lipid conjugate, with or without a tethered ligand, could be included in the outer leaflet lipids, it was not necessary to perform PEGylation of NP by post-insertion [17]. The improved formulation is named Lipid/Calcium/Phosphate type II (LCP-II). Significantly different from the previous LCP-I formulation, LCP-II contains an asymmetric lipid structure verified by the measurement of zeta potential. We report here the preparation, properties, and in vitro and in vivo activity of this formulation.

Section snippets

The preparation of LCP-II NP

The information about the materials is shown in the Supplementary information. Fig. 1 shows a flow diagram for the preparation of siRNA-entrapped LCP-II NPs. The anionic lipid coating CaP cores were prepared by a water-in-oil micro-emulsion method. Briefly, 300 μL of 500 mM CaCl2with 100 μL of 2 mg/mL siRNA was dispersed in 15 mL Cyclohexane/Igepal CO-520 (71/29 V/V) solution to form a very well dispersed water-in-oil reverse micro-emulsion. The phosphate part was prepared by 300 μL of 25 mM Na2HPO4 (pH

The characterizations of LCP-II

In the preparation of LCP-II, an amphiphilic phospholipid DOPA (see Fig. 1) was added into the phosphate part of the reverse micro-emulsion. DOPA is known to strongly interact with cations at the interface [18]. It is expected that the CaP core should be coated with DOPA because excess Ca should be available on the core surface. The C18:1 chains of DOPA were sufficiently hydrophobic such that the coated cores were soluble in a non-poplar solvent, i.e., CHCl3, but not in a polar solvent, i.e.,

Conclusion

CaP core stabilized with DOPA was prepared by water/oil microemulsion and further coated with cationic or neutral lipid to form LCP-II. The vehicle has a hollow spherical structure with a size of about 40 nm and possesses an asymmetric lipid bilayer at the surface. With the targeting ligand anisamide, the new LCP-II showed a 40-fold improved silence activity compared to the previous LPD formulation. The new NP vehicle effectively delivers siRNA to solid tumor in a xenograft model. The

Acknowledgments

This study was supported by NIH grants CA129835, CA149363 and CA151652.We thank Andrew Satterlee for editing the manuscript.

References (30)

  • O. Garbuzenko et al.

    Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer

    Chem. Phys. Lipids

    (2005)
  • J.M. Perkel

    RNAi therapeutics: a two-year update

    Science

    (2009)
  • K.A. Whitehead et al.

    Knocking down barriers: advances in siRNA delivery

    Nat. Rev. Drug Discov.

    (2009)
  • M.E. Davis

    The first targeted delivery of siRNA in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to clinic

    Mol. Pharm.

    (2009)
  • S.C. Semple et al.

    Rational design of cationic lipids for siRNA delivery

    Nat. Biotechnol.

    (2010)
  • Cited by (0)

    1

    On leave from Sichuan University, The People's Republic of China.

    View full text