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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

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.
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f4-ijn-10-5995: 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.

Mentions: The combined effect of angiopep modification and cleavable PEG in the formulation of LNPs was initially evaluated with Chol-PCL for uptake and gene knockdown in U87MG cells expressing LRP-1 and MMP-2. Cellular uptake was evaluated by measuring fluorescence intensity of the cell lysate, and these data confirmed the results from the flow cytometry uptake study (Figure 4A). For both angiopep-functionalized LNPs, A/PE-PEG-LNP and A/Chol-PCL-LNP, a significantly higher uptake was obtained compared to their non-targeted counterpart, PE-PEG-LNP and Chol-PCL-LNP, respectively.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4590347&req=5

f4-ijn-10-5995: 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.
Mentions: The combined effect of angiopep modification and cleavable PEG in the formulation of LNPs was initially evaluated with Chol-PCL for uptake and gene knockdown in U87MG cells expressing LRP-1 and MMP-2. Cellular uptake was evaluated by measuring fluorescence intensity of the cell lysate, and these data confirmed the results from the flow cytometry uptake study (Figure 4A). For both angiopep-functionalized LNPs, A/PE-PEG-LNP and A/Chol-PCL-LNP, a significantly higher uptake was obtained compared to their non-targeted counterpart, PE-PEG-LNP and Chol-PCL-LNP, respectively.

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