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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: 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).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.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

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.
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f6-ijn-10-5995: 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.

Mentions: The ability to mediate knockdown was examined together with the uptake efficiency, and, as for the uptake, (Figure 5) there was greater knockdown for the DM-PCL containing LNPs in both bEnd.3 cells (Figure 6A) and U87MG cells (Figure 6B). At siRNA concentration of 120 nM, both DM-PCL-LNP and A/DM-PCL-LNP attained a reduction of the luciferase protein expression down to 30% of the expression level of untreated cells (Figure 6), which was a significant improvement from the 90% and 60% previously attained by A/Chol-PCL-LNP and A/PE-PEG-LNP, respectively (Figure 4B). When doubling the concentration (120 nM), it was possible to reach a knockdown of 15% in the bEnd.3 cells that was comparable to the commercial agent RNAiMAX. The increased amount of siRNA did not influence the knockdown in U87MG further as it seemed to have reached a maximum effect at 120 nM (Figure 6B).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

f6-ijn-10-5995: 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.
Mentions: The ability to mediate knockdown was examined together with the uptake efficiency, and, as for the uptake, (Figure 5) there was greater knockdown for the DM-PCL containing LNPs in both bEnd.3 cells (Figure 6A) and U87MG cells (Figure 6B). At siRNA concentration of 120 nM, both DM-PCL-LNP and A/DM-PCL-LNP attained a reduction of the luciferase protein expression down to 30% of the expression level of untreated cells (Figure 6), which was a significant improvement from the 90% and 60% previously attained by A/Chol-PCL-LNP and A/PE-PEG-LNP, respectively (Figure 4B). When doubling the concentration (120 nM), it was possible to reach a knockdown of 15% in the bEnd.3 cells that was comparable to the commercial agent RNAiMAX. The increased amount of siRNA did not influence the knockdown in U87MG further as it seemed to have reached a maximum effect at 120 nM (Figure 6B).

Bottom Line: 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).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.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