Scavenger receptors mediate cellular uptake of polyvalent oligonucleotide-functionalized gold nanoparticles

Abstract

Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NP entry in HeLa cells. Biochemical analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NP entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species, as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles.

Figure 1

Cellular internalization of DNA-Au NPs when coated with increasing amounts of BSA. The method for loading a variable number of proteins on DNA-Au NPs is shown in Scheme 1. Cells were grown in media containing 10% FBS with 5 nM (A) and 10 nM (B) of DNA-Au NPs.

Figure 2

(A) Cellular uptake of DNA-Au NPs loaded with varying number of BSA molecules at 5nM (A) and 10nM (B) solution concentration of DNA-Au NPs in serum-free conditions. Serum-free conditions yield significantly higher number of nanoparticles per cell (150% increase) compared with normal 10% serum cultures (shown in Figure 1).

Figure 3

Endocytotic uptake of DNA-Au NPs in HeLa cells is mediated by scavenger receptors. Treatment with agonists of scavenger receptors, fucoidan (A) and polyinosinic acid (poly I, B), prior to nanoparticle addition results in up to 60% inhibition of the cellular uptake of DNA-Au NPs. Pharmacological agents that inhibit other modes of cellular entry, such as bafilomycin A1 and methyl β-cytodextrin, did not inhibit nanoparticle uptake (C, D, Supporting Information, Figure 4).

Figure 4

Polyinosinic acid dependent inhibition of DNA-Au NPs is dependent on the density of DNA on nanoparticles. When the cellular uptake of DNA-Au NPs is lowered with decreasing DNA density, the ability of poly I to inhibit cellular internalization of DNA-Au NPs is lowered as well.

Scheme 1

Stable loading of proteins on DNA functionalized gold nanoparticles. The proteins are covalently attached to tosyl-modified oligonucleotides, which are then hybridized to nanoparticles functionalized with a complementary sequence. The variable loading of proteins is achieved by using different ratios of protein-DNA conjugate to DNA-Au NPs.

Scheme 2

The proposed mechanism of cellular uptake of DNA-Au NPs. In serum containing media, DNA-Au NPs are coated with proteins which reduce nanoparticle interactions with scavenger receptors. Cellular uptake is mediated by the displacement of serum proteins by these receptors. In the absence of serum proteins, these interactions are more easily facilitated, resulting in greater uptake as a result of high DNA density. If the binding to scavenger receptors is inhibited by addition of competitive ligands, then the cellular uptake of nanoparticles is also reduced.