Poster X8 - Ultrasound-responsive dendrimer-microbubble complexes for precision cancer gene therapy

Introduction: Precision delivery of nucleic acids has emerged as a pivotal therapeutic regimen to combat cancer. In recent years, cancer gene therapy has focused on employing viral or non-viral vectors to transfer exogenous genetic material into cells to induce therapeutic effects [1]. Branched polymeric dendrimer nanoparticles offer high transfection efficiency owing to their high loading capacity, reduced immunogenicity compared to viral vectors, and endosomal escape capability. However, achieving site-specific delivery after systemic administration remains challenging, and presents difficulty in precisely targeting the genes to a specific tissue of interest while minimizing off-target effects. To address this, our objective is to develop a novel, dendrimer-microbubble complex as an ultrasound-activated nanocarrier for spatially-localized cancer gene therapy. Focused ultrasound is an effective stimulus in medicine as it is non-invasive, non-ionizing, offers significant depth penetration within tissues, and is safe for clinical use at low intensities. In our particle design, dendrimers are functionalized with a linker and coupled with plasmid DNA via electrostatic attraction, forming dendriplexes. Dendriplexes are then bound to the surface of functionalized micron-scale ultrasound-responsive lipid microbubbles. Upon application of low-intensity focused ultrasound, microbubbles will collapse, releasing attached nanoparticles for localized delivery. Ultrasound-triggered microbubble cavitation also induces transient pores in nearby cell membranes and can enhance endocytosis, facilitating enhanced nanoparticle uptake [2]. In this study, we aim to characterize the dendrimers and dendrimer-microbubble complexes and assess their ultrasound-responsivity and transfection capability in 2D cell cultures.

Materials and

Methods: Dendrimer nanoparticle functionalization with a linker group was assessed via NMR spectroscopy. Dendrimers were characterized for size via Transmission Electron Microscopy (TEM) and nanoparticle tracking analysis (NTA). These functionalized cationic dendrimers were electrostatically coupled with plasmid DNA (pDNA) coding for green fluorescent protein (GFP) at several N:P (negative to positive charge) ratios to form dendriplexes. To investigate the ability of dendrimers to deliver DNA to in vitro cell cultures, dendriplexes were added to HEK293T cells at 70% cell confluence. After 48 hours of incubation, cells were imaged on a widefield microscope (Leica Thunder Imaging System). Gas-core lipid-shell microbubbles were fabricated by sparging hydrophobic perfluorocarbon gas into a phospholipid mixture under vortex, with subsequent purification from free phospholipid by buoyancy. Microbubble particle size was assessed by image analysis of brightfield images. To create ultrasound-responsive particle complexes, the dendriplexes were loaded on the surface of functionalized gas-core lipid-shell microbubbles (Fig. 1A). After washing to remove unbound dendriplexes, successful conjugation was confirmed using fluorescence microscopy of fluorescently-tagged (AlexaFluor-488) dendriplexes coupled to microbubbles. Microbubble-dendriplex conjugate response to applied focused ultrasound was visualized using a custom ultrasound/fluorescence microscope system with a 2.25 MHz focused ultrasound transducer.

Results, Conclusions, and Discussions: Results and discussion: Unconjugated dendrimers appeared to form clusters with a peak size of 100 nm as visualized by TEM imaging. The presence of a defined linker peak was visualized on the NMR spectra of the functionalized dendrimer, supporting successful linker functionalization. Size measurements via NTA of dendrimers loaded with pDNA at different N:P ratios revealed a 100-200 nm particle size range, which is suitable for endocytosis. After treating HEK293T cells with dendriplexes containing GFP plasmid, cytosolic GFP signal was observed in cells after 48h, supporting successful cellular transfection, with no signal observed in the untreated cells (Fig. 1B). The transfection via dendriplexes showed a general trend of decreasing transfection efficiency with increasing N:P ratio (decreasing the amount of dendrimers while keeping the pDNA constant), supporting the role of dendrimers in facilitating pDNA delivery.
Fabricated ultrasound-responsive functionalized microbubbles had diameters in the 1-5 µm range, suitable for activation at clinical ultrasound frequencies. After dendriplex conjugation, the fluorescently-tagged dendrimers were seen to colocalize with the microbubbles, supporting successful conjugation (Fig. 1C). Microbubbles without dendriplexes displayed no green fluorescent signal. Upon focused ultrasound exposure, dendriplex-conjugated microbubbles collapsed only in the ultrasound focal zone (Fig. 1D). Microbubbles conjugated to fluorescently-labeled dendriplexes were also seen to rupture within the ultrasound focal zone (Fig. 1E), leaving behind a debris cloud, supporting that the microbubble conjugates are responsive to applied ultrasound triggers and supporting their ability to release pDNA-loaded dendrimers.

Conclusion: Our work demonstrates the first example of coupling dendrimers to ultrasound-responsive microbubbles and demonstrates the ability to activate these particles within an ultrasound-defined region for potential use as a remote-triggered gene delivery vehicle. We have shown that the dendriplexes are capable of delivering pDNA to cells and that the microbubble-dendriplex conjugates can be activated within the ultrasound focal zone. Ongoing work will evaluate the efficiency of these ultrasound-responsive gene delivery complexes for spatially-localized gene delivery to cancer cells. Overall, these microbubble-dendrimer complexes offer promise as a new stimuli-responsive gene delivery strategy for precision cancer therapy.

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References:
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2. Meijering BD, Juffermans LJ, van Wamel A, Henning RH, Zuhorn IS, Emmer M, Versteilen AM, Paulus WJ, van Gilst WH, Kooiman K, de Jong N, Musters RJ, Deelman LE, Kamp O. Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation. Circ Res. 2009;104(5):679-87. Epub 20090122. doi: 10.1161/CIRCRESAHA.108.183806. PubMed PMID: 19168443.