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

Bruun J, Larsen TB, Jølck RI, Eliasen R, Holm R, Gjetting T, Andresen TL - Int J Nanomedicine (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Micro- and Nanotechnology, Center for Nanomedicine and Theranostics, Technical University of Denmark, DTU Nanotech, Lyngby, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f5-ijn-10-5995: 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.

Mentions: The uptake of DM-PCL containing LNPs (DM-PCL-LNP and A/DM-PCL-LNP) was tested in the two MMP-2/9 expressing brain cell lines bEnd.3 and U87MG.37,38 Both LNPs had a tenfold higher uptake than PE-PEG-LNP (Figure 5A), which was three times higher than observed for A/Chol-PCL-LNP (Figure 4A). While it was observed that the LNPs with cholesterol-anchored PCL had an additive effect of dual-modification with both angiopep and PCL, this was not the case for DM-PCL-LNPs. Instead the uptake improvement by the cleavable PEG coating was so much higher than the receptor-mediated uptake that it completely overshadowed the effect of angiopep functionalization. This was evident by an equal uptake of the dual functionalized A/DM-PCL-LNP and the nontargeted DM-PCL-LNP (Figure 5A). It was therefore hypothesized that the difference in uptake between the DM- and Chol-anchored PCLs is caused by a difference in orientation and positioning of the lipopeptides in the LNPs due to their individual size and lipophilicity. This difference in orientation presumably provides better accessibility for MMPs to the cleavage site of DM-PCL than Chol-PCL.


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

Bruun J, Larsen TB, Jølck RI, Eliasen R, Holm R, Gjetting T, Andresen TL - Int J Nanomedicine (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4590347&req=5

f5-ijn-10-5995: 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.
Mentions: The uptake of DM-PCL containing LNPs (DM-PCL-LNP and A/DM-PCL-LNP) was tested in the two MMP-2/9 expressing brain cell lines bEnd.3 and U87MG.37,38 Both LNPs had a tenfold higher uptake than PE-PEG-LNP (Figure 5A), which was three times higher than observed for A/Chol-PCL-LNP (Figure 4A). While it was observed that the LNPs with cholesterol-anchored PCL had an additive effect of dual-modification with both angiopep and PCL, this was not the case for DM-PCL-LNPs. Instead the uptake improvement by the cleavable PEG coating was so much higher than the receptor-mediated uptake that it completely overshadowed the effect of angiopep functionalization. This was evident by an equal uptake of the dual functionalized A/DM-PCL-LNP and the nontargeted DM-PCL-LNP (Figure 5A). It was therefore hypothesized that the difference in uptake between the DM- and Chol-anchored PCLs is caused by a difference in orientation and positioning of the lipopeptides in the LNPs due to their individual size and lipophilicity. This difference in orientation presumably provides better accessibility for MMPs to the cleavage site of DM-PCL than Chol-PCL.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Micro- and Nanotechnology, Center for Nanomedicine and Theranostics, Technical University of Denmark, DTU Nanotech, Lyngby, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus