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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

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.
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f2-ijn-10-5995: 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.

Mentions: PE-PEG-LNP had an insignificant uptake in bEnd.3 cells over 4 hours, and the fluorescence histogram was inseparable from that of the untreated cells (Figure 2). In contrast, angiopep containing A/PE-PEG-LNP had a 2.4-fold higher mean fluorescence intensity than the PE-PEG-LNP without the angiopep, and this clearly shifted the histogram toward higher fluorescence intensity suggesting enhanced cellular uptake of the targeted LNPs.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

f2-ijn-10-5995: 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.
Mentions: PE-PEG-LNP had an insignificant uptake in bEnd.3 cells over 4 hours, and the fluorescence histogram was inseparable from that of the untreated cells (Figure 2). In contrast, angiopep containing A/PE-PEG-LNP had a 2.4-fold higher mean fluorescence intensity than the PE-PEG-LNP without the angiopep, and this clearly shifted the histogram toward higher fluorescence intensity suggesting enhanced cellular uptake of the targeted LNPs.

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