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. 2012 Sep 10;162(2):414-21.
doi: 10.1016/j.jconrel.2012.07.005. Epub 2012 Jul 16.

Markedly Enhanced Skeletal Muscle Transfection Achieved by the Ultrasound-Targeted Delivery of Non-Viral Gene Nanocarriers With Microbubbles

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Markedly Enhanced Skeletal Muscle Transfection Achieved by the Ultrasound-Targeted Delivery of Non-Viral Gene Nanocarriers With Microbubbles

Caitlin W Burke et al. J Control Release. .
Free PMC article

Abstract

Our goal was to enhance ultrasound (US)-targeted skeletal muscle transfection through the use of poly(ethyleneglycol) (PEG)/polyethylenimine (PEI) nanocomplex gene carriers and adjustments to US and microbubble (MB) parameters. C57BL/6 mice received an intravenous infusion of MBs and either "naked" luciferase plasmid or luciferase plasmid condensed in PEG/PEI nanocomplexes. Pulsed ultrasound (1 MHz; 0.6 MPa or 0.8 MPa) was applied to the right hindlimb for 12 min. Luciferase activity in both hindlimbs was assessed at 3, 5, 7, and 10 days post-treatment by bioluminescent imaging. When targeted to hindlimb using unsorted MBs and 0.6 MPa US, 7 days after treatment, we observed a >60-fold increase in luciferase activity in PEG/PEI nanocomplex-treated muscles over muscles treated with "naked" plasmid DNA. Luciferase activity was consistently greater after treatment with PEG/PEI nanocomplexes at 0.6 MPa as compared to 0.8 MPa. The combination of small diameter MBs and 0.6 MPa US also resulted in significantly greater gene expression when compared to concentration matched intramuscular injections, a control condition in which considerably more PEG/PEI nanocomplexes were present in tissue. This result suggests that, in addition to facilitating PEG/PEI nanocomplex delivery from the bloodstream to tissue, US enhances transfection via one or more secondary mechanisms, including increased cellular uptake and/or trafficking to the nucleus of PEG/PEI nanocomplexes. We conclude that PEG/PEI nanocomplexes may be used to markedly enhance the amplitude of US-MB-targeted skeletal muscle transfection and that activating "small" MBs with a moderate level (0.6 MPa) of acoustic pressure can further enhance these effects.

Figures

Figure 1
Figure 1
Schematic illustration of the ultrasound pulsing protocol (not drawn to scale with respect to time).
Figure 2
Figure 2
A–C: Image cytometry analysis of PEG/PEI nanocomplex interactions with MBs. A: Scatterplot showing brightfield and fluorescence intensity of particle(s) passing through the image cytometer. B: Set of 10 representative images from the low fluorescence intensity P1 population. MBs are present, but PEG/PEI nanocomplexes are absent. C: Set of 10 representative images from the high fluorescence intensity P2 population. Both MBs and PEG/PEI nanocomplexes are present, but they are only rarely colocalized (arrow). D: Smoothed histograms showing the distribution of MB diameters within single batches of small and unsorted MBs.
Figure 3
Figure 3
PEG/PEI nanocomplexes increase US-MB-targeted transfection. A: Representative IVIS scans from mice in which PEG/PEI nanocomplexes (left) or naked plasmid DNA (right) has been delivered using 0.6 MPa US and unsorted MBs. B: Bar graph of gene expression for animals receiving co-injections of unsorted microbubbles (MBs) and either PEG/PEI or naked plasmid at 3, 5, 7, and 10 days after insonation. *Significantly different than all other groups at same time point (P<0.05).
Figure 4
Figure 4
Influence of peak-negative pressure on gene expression. Line graphs of bioluminescence data for animals receiving co-injections of small (A) and unsorted (B) MBs at 3, 5, 7, and 10 days after insonation at 0.8MPa and 0.6MPa. *Significantly different than 0.8 MPa at same time point (P<0.05).
Figure 5
Figure 5
Transgene expression after IM injection compared to US-MB-targeted delivery. A: IVIS scans showing luciferase gene expression 7 days after treatment. B: Bar graph, with inset, quantifying gene expression as a function of time. *Significantly different than all other groups at same time point (P<0.05). #Significantly different that IM injection (6μg of DNA condensed in PEG/PEI nanocomplexes) at same time point (P<0.05).
Figure 6
Figure 6
H&E stained muscle cross-sections used to evaluate the potential for local toxicity due to PEG/PEI nanocomplex delivery via IM injection (A) or US-MB-targeted delivery (B) at 7 days after treatment. Necrotic and/or regenerating muscle fibers were not observed, and inflammatory cell infiltration was not evident.

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