Investigation of enzyme-sensitive lipid nanoparticles for delivery of siRNA to blood-brain barrier and glioma cells

Abstract

Clinical applications of siRNA for treating disorders in the central nervous system require development of systemic stable, safe, and effective delivery vehicles that are able to cross the impermeable blood-brain barrier (BBB). Engineering nanocarriers with low cellular interaction during systemic circulation, but with high uptake in targeted cells, is a great challenge and is further complicated by the BBB. As a first step in obtaining such a delivery system, this study aims at designing a lipid nanoparticle (LNP) able to efficiently encapsulate siRNA by a combination of titratable cationic lipids. The targeted delivery is obtained through the design of a two-stage system where the first step is conjugation of angiopep to the surface of the LNP for targeting the low-density lipoprotein receptor-related protein-1 expressed on the BBB. Second, the positively charged LNPs are masked with a negatively charged PEGylated (poly(ethylene glycol)) cleavable lipopeptide, which contains a recognition sequence for matrix metalloproteinases (MMPs), a class of enzymes often expressed in the tumor microenvironment and inflammatory BBB conditions. Proteolytic cleavage induces PEG release, including the release of four glutamic acid residues, providing a charge switch that triggers a shift of the LNP charge from weakly negative to positive, thus favoring cellular endocytosis and release of siRNA for high silencing efficiency. This work describes the development of this two-stage nanocarrier-system and evaluates the performance in brain endothelial and glioblastoma cells with respect to uptake and gene silencing efficiency. The ability of activation by MMP-triggered dePEGylation and charge shift is demonstrated to substantially increase the uptake and the silencing efficiency of the LNPs.

Keywords: BBB; angiopep; cleavable PEG-lipid; gene therapy; matrix metalloproteinase; nanocarrier.

Figure 1

Schematic presentation showing the gene delivery by dual modified LNPs. Notes: The nanoparticle is modified with angiopep for receptor-mediated uptake in LRP-1 expressing cells (left pathway) and a MMP-cleavable lipopeptide for activation in tumor tissue microenvironment (right pathway). Intra- or extracellular cleavage of the lipopeptide dePEGylates the LNP and reverses the surface charge from negative to positive leading to increased uptake and endosomal escape. Abbreviations: LNPs, lipid nanoparticles; LRP-1, low-density lipoprotein receptor-related protein-1; PEG, poly(ethylene glycol).

Figure 2

Uptake of angiopep-functionalized LNPs. Notes: Representative histograms for bEnd.3 cells treated with A/PE-PEG-LNP (black), PE-PEG-LNP (gray), or buffer (dashed). Insert represents the MFI averaged over three samples. Error bars are SD. Abbreviations: LNPs, lipid nanoparticles; PEG, poly(ethylene glycol); MFI, mean fluorescence intensity; SD, standard deviation.

Figure 3

Proteinase cleaving of LNPs. Notes: (A) The MALDI-TOF mass spectrum shows the size of the PEGylated lipids in PE-PEG-LNP, Chol-PCL-LNP, and DM-PCL-LNP when treated with buffer or thermolysin for cleavage of PCL. (B) ζ-potential of the LNPs when treated with buffer or thermolysin. Abbreviations: LNPs, lipid nanoparticles; PEG, poly(ethylene glycol); PCL, PEGylated cleavable lipopeptide; Chol, cholesterol; DM, dimyristoyl; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.

Figure 4

siRNA delivery with angiopep and Chol-PCL modified LNPs. Notes: (A) Uptake of RhB-labeled LNPs in U87MG cells measured as fluorescence intensity of the cell lysate in arbitrary units (au). (B) Luciferase reporter activity relative to untreated cells after treatment with LNPs containing anti-luciferase siRNA. LNP dose corresponds to 120 nM siRNA. Error bars are SEM of two independent experiments performed in triplicates. *Significant difference from PE-PEG-LNP, **significant difference from Chol-PCL-LNP; determined using independent t-test P<0.05. Abbreviations: PCL, PEGylated cleavable lipopeptide; LNPs, lipid nanoparticles; RhB, rhodamine B; PEG, poly(ethylene glycol); MMP, matrix metalloproteinase; SEM, standard error of mean; Chol, cholesterol.

Figure 5

In vitro uptake of PE-PEG or DM-PCL containing LNPs in bEnd.3 and U87MG cells. Notes: (A) Fraction of administered RhB-labeled lipid vehicle in the cell lysate. (B) Fraction of administered ³³P labeled siRNA in the cell lysate. Error bars are SEM (n=4). Abbreviations: PEG, poly(ethylene glycol); PCL, PEGylated cleavable lipopeptide; LNPs, lipid nanoparticles; RhB, rhodamine B; SEM, standard error of mean; DM, dimyristoyl.

Figure 6

Knockdown of luciferase by LNPs. Notes: (A) bEnd.3 and (B) U87MG cells were incubated for 48 hours with nanoparticles containing 60 (black), 120 (gray), or 240 (white) nM siRNA. The luciferase expression of the cells was normalized to their total protein contents and plotted as percentage of the expression level of nontreated cells. Nonsense siRNA (siGFP) and the commercial transfection agent RNAiMAX served as negative and positive control, respectively. Error bars are SEM (n=4). Abbreviations: PEG, poly(ethylene glycol); PCL, PEGylated cleavable lipopeptide; LNPs, lipid nanoparticles; SEM, standard error of mean; DM, dimyristoyl.

Figure 7

Cytotoxicity of DM-PCL-LNP. Notes: MTS assay is used to analyze the proliferation of bEnd.3 cells treated with DM-PCL-LNP at siRNA concentrations ranging from 10 nM to 2.2 µM (squares). As control, cells treated with RNAiMAX (circles) were included. Error bars are SD. Abbreviations: PCL, PEGylated cleavable lipopeptide; LNP, lipid nanoparticle; SD, standard deviation; DM, dimyristoyl.