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. 2011 May 9;7(9):1227-35.
doi: 10.1002/smll.201001934. Epub 2011 Apr 1.

Covalently Linking Poly(lactic-Co-Glycolic Acid) Nanoparticles to Microbubbles Before Intravenous Injection Improves Their Ultrasound-Targeted Delivery to Skeletal Muscle

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Covalently Linking Poly(lactic-Co-Glycolic Acid) Nanoparticles to Microbubbles Before Intravenous Injection Improves Their Ultrasound-Targeted Delivery to Skeletal Muscle

Caitlin W Burke et al. Small. .
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Abstract

Intravenously injected nanoparticles can be delivered to skeletal muscle through capillary pores created by the activation of microbubbles with ultrasound; however, strategies that utilize coinjections of free microbubbles and nanoparticles are limited by nanoparticle dilution in the bloodstream. Here, improvement in the delivery of fluorescently labeled ≈150 nm poly(lactic-co-glycolic acid) nanoparticles to skeletal muscle is attempted by covalently linking them to albumin-shelled microbubbles in a composite agent formulation. Studies are performed using an experimental model of peripheral arterial disease, wherein the right and left femoral arteries of BalbC mice are surgically ligated. Four days after arterial ligation, composite agents, coinjected microbubbles and nanoparticles, or nanoparticles alone are administered intravenously and 1 MHz pulsed ultrasound was applied to the left hindlimb. Nanoparticle delivery was assessed at 0, 1, 4, and 24 h post-treatment by fluorescence-mediated tomography. Within the coinjection group, both microbubbles and ultrasound are found to be required for nanoparticle delivery to skeletal muscle. Within the composite agent group, nanoparticle delivery is found to be enhanced 8- to 18-fold over 'no ultrasound' controls, depending on the time of measurement. A maximum of 7.2% of the initial nanoparticle dose per gram of tissue was delivered at 1 hr in the composite agent group, which was significantly greater than in the coinjection group (3.6%). It is concluded that covalently linking 150 nm-diameter poly(lactic-co-glycolic acid) nanoparticles to microbubbles before intravenous injection does improve their delivery to skeletal muscle.

Figures

Figure 1
Figure 1
Characterization of MNCAs. A: Number weighted NiComp NP size distribution. B: Scanning electron microscopy image of PLAGA NPs. C: Epifluorescence-transmitted light merge image of a single composite delivery agent (MNCA) comprised of 100nm PLAGA polymer NPs adhered to a contrast agent microbubble (MB) using carboiimide chemistry linkers. MB is approximately 2μm in diameter.
Figure 2
Figure 2
Specific and Nonspecific Interactions of NPs with MBs. Left column: Image cytometry scatter plots of microbubble (MB) area vs. fluorescence intensity. Middle Column: Brightfield images of MBs. Right Column: Cy5.5 images of MBs and/or NPs. Top row: Composite agent (MNCA). Middle row: Microbubbles (MBs) incubated with nanoparticles (NPs). Bottom row: Microbubbles (MBs). Arrows denote MB presented in the middle and right columns. Stars denote individual MBs visualized in middle and right columns. The median MB mean pixel intensity was 171.75 for the MNCA group and 0 for MBs incubated with NPs and MBs alone.
Figure 3
Figure 3
Fluorescence-mediated tomography scans showing NP delivery to hindlimb skeletal muscle at various time points after ultrasound application and NP biodistribution 1hr following MNCA infusion. A: Top row: Composite agent (MNCA) injection. Middle row: Co-injection of microbubbles (MBs) and nanoparticles (NPs). Bottom row: NP injection without microbubbles. Ultrasound treated regions are denoted with white asterisks. Enhanced fluorescence intensity is evident with US application for the MNCA and MB+NP groups. B: Bar graph of fluorochrome concentration [% initial dose (ID) per gram of tissue] in “off-target” tissues and organs, as determined by FMT at 1 hr after treatment with MNCAs.
Figure 3
Figure 3
Fluorescence-mediated tomography scans showing NP delivery to hindlimb skeletal muscle at various time points after ultrasound application and NP biodistribution 1hr following MNCA infusion. A: Top row: Composite agent (MNCA) injection. Middle row: Co-injection of microbubbles (MBs) and nanoparticles (NPs). Bottom row: NP injection without microbubbles. Ultrasound treated regions are denoted with white asterisks. Enhanced fluorescence intensity is evident with US application for the MNCA and MB+NP groups. B: Bar graph of fluorochrome concentration [% initial dose (ID) per gram of tissue] in “off-target” tissues and organs, as determined by FMT at 1 hr after treatment with MNCAs.
Figure 4
Figure 4
Microbubbles (MBs) and ultrasound (US) facilitate nanoparticle (NP) delivery to skeletal muscle after intravenous injection. Bar graph of fluorochrome concentration [% initial dose (ID) per gram of tissue] as a function of time. *pairwise comparisons with Holm-Sidak t-tests indicate significantly different than NP+US group at same time point (P<0.05). **Analysis of variance indicates significantly different than MB+NP (P<0.05), but no pairwise differences exist.
Figure 5
Figure 5
Ultrasound application enhances nanoparticle (NP) delivery to skeletal muscle following the intravenous injection of MNCAs. Bar graph of fluorochrome concentration [% initial dose (ID) per gram of tissue] as a function of time. *pairwise comparisons with Holm-Sidak t-tests indicate significantly different than MNCA group at same time point (P<0.05).
Figure 6
Figure 6
Composite agent (MNCA) formulation yields greater ultrasound-mediated nanoparticle delivery to hindlimb skeletal muscle that co-injections of microbubbles (MBs) and nanoparticles (NPs). Bar graph of fluorochrome concentration [% initial dose (ID) per gram of tissue] as a function of time. *pairwise comparisons with Holm-Sidak t-tests indicate significantly different than MB+NP+US group at same time point (P<0.05).
Figure 7
Figure 7
Confocal images of cross-sectioned skeletal muscle at 1hr after ultrasound application. Top row: Composite agent (MNCA) injection. Middle row: Co-injection of microbubbles (MBs) and nanoparticles (NPs). Bottom row: NP injection without microbubbles. Left column: Capillaries labeled with BS-I lectin. Middle column: VT680-conjugated nanoparticles (NPs) that have been delivered to tissue. Right column: Merge images showing delivered NPs (red) with respect to capillaries (green). Filled arrows denote co-localization of NPs and capillary endothelium. Open arrows denote regions where NPs have been delivered beyond the endothelium to the interstitial space between muscle fibers.

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