U40 - A dendrimer-based ultrasound-responsive gene delivery system for targeted cancer gene therapy

Introduction: : Precision gene therapy has shown considerable promise as a therapeutic strategy to combat cancer. A vector that can selectively and efficiently deliver the gene to target cells with minimal toxicity is critical for successful gene therapy. Viral vectors possess the natural ability to shuttle genetic materials into cells but present concerns with respect to their immunogenicity and lack of spatial specificity. Among non-viral vectors, hyperbranched polymeric dendrimer nanoparticles are efficient nucleic acid delivery vectors due to their reduced immunogenicity, high DNA loading capacity and endosomal escape capability [1-3]. However, dendrimers are typically administered intravenously which can result in off-target effects from delivery to non-tumor cells, presenting a significant challenge in gene therapy. To address the challenge, we have developed a unique dendrimer-microbubble complex as an ultrasound-activated nanocarrier for spatially-localized cancer gene therapy. Focused ultrasound is an effective stimulus for safe and non-invasive particle activation with high tissue penetration depth and tight spatial localization [4]. In our particle design, dendrimers are functionalized with a linker and electrostatically coupled with plasmid DNA (pDNA), forming dendriplexes. Dendriplexes are bound to the surface of functionalized micron-scale microbubbles, forming dendriplex-microbubble conjugates (Fig. 1A) that release the gene-loaded dendrimers and increase their local bioavailability upon ultrasound stimulation. Ultrasound-induced microbubble cavitation also induces transient pores in nearby cell membranes, increasing nanoparticle uptake. This study aims to develop and characterize the dendrimer-microbubble gene delivery complex, assess its response to focused ultrasound, and evaluate the ability to modulate cellular transfection, paving the way for applications in precision gene therapy.

Materials and

Methods: : Unfunctionalized and linker-functionalized dendrimers were characterized via Transmission Electron Microscopy (TEM) and nanoparticle tracking analysis (NTA). The presence of linker groups was verified by NMR spectroscopy and binding of the fluorescent dendrimers to functionalized micron-sized silica beads. The functionalized cationic dendrimers were electrostatically coupled with a green fluorescent protein (GFP)-encoding plasmid DNA (pDNA) at multiple N:P (negative to positive charge) ratios to form dendriplexes and their size and charge were assessed by NTA. The transfection capability of these DNA-dendrimer complexes was assessed in HEK293T cells through delivery of the GFP plasmid. After 48 hours, GFP expression was imaged on a widefield microscope and quantified via image segmentation. Gas-core, lipid-shell microbubbles were fabricated with inclusion of a PEGylated functionalized lipid in their shell and their size was assessed by brightfield image analysis. Functionalized microbubbles were incubated with fluorescent, linker-functionalized dendrimers and washed to remove unbound dendrimers. A custom ultrasound application and fluorescence microscope system was used to expose dendrimer-microbubble conjugates to focused ultrasound (2.25 MHz) and image their response. Transfection efficiency of the dendriplex-microbubble gene delivery system was compared before and after ultrasound exposure in a 2D cell culture setup. A custom enclosed cell culture chamber with an acoustically transparent window was utilized to allow ultrasound transmission for particle activation and monitoring via microscopy.

Results, Conclusions, and Discussions:: Results and discussion: TEM imaging showed that the unfunctionalized and functionalized dendrimers formed particle clusters less than 100 nm. Peak diameters of dendriplexes complexed at multiple N:P ratios measured via NTA were between 120-180 nm, which is suitable for endocytosis. The dendriplexes became more negatively charged with DNA complexation, supporting successful dendriplex formation. Following cell exposure to GFP pDNA-loaded dendrimers, transfection was observed at 48hr, with minimal transfection seen with DNA alone. The transfection via dendriplexes showed a trend of decreasing transfection efficiency with increasing N:P ratio (decreasing the dendrimer amount while keeping the pDNA constant), supporting that dendrimers promote improved pDNA delivery. Gas-core, phospholipid shell microbubbles were fabricated with diameters in the 1-5 µm range, which is suitable for activation at clinical ultrasound frequencies. After incubation with dendrimers, a fluorescent dendrimer signal was observed to colocalize with microbubbles conjugated to linker-functionalized dendrimers, supporting successful conjugation (Fig. 1B). Functionalized microbubbles with unfunctionalized dendrimers did not show dendrimer complexation. Upon low-intensity ultrasound exposure via a custom ultrasound application and fluorescence microscope system, microbubble-dendrimer conjugates collapsed in a tight focal zone, demonstrating their potential for targeted delivery (Fig. 1C). Microbubbles conjugated to fluorescently-labeled dendriplexes were seen to rupture within the ultrasound focal zone and leave behind a debris cloud (Fig. 1D), further supporting that the microbubble conjugates are responsive to the applied ultrasound trigger. In ultrasound-mediated transfection studies, transfection was minimal for dendriplex-microbubble conjugates without ultrasound exposure, and significantly increased post-ultrasound exposure, demonstrating that transfection can be modulated with externally-applied low-intensity ultrasound (Fig. 1E).

Conclusion: Our work demonstrates the development of an innovative acoustically-responsive gene-delivery vehicle based on dendrimer-microbubble conjugates. We have demonstrated the responsiveness of the conjugates to focused ultrasound and shown that the transfection via these particles can be modulated using the ultrasound stimulus. Ongoing work will evaluate the efficiency of these ultrasound-responsive gene delivery complexes for spatially-localized gene delivery to cancer cells and utilize these complexes for delivery of wild-type p53 to triple-negative breast cancer. Overall, ultrasound-responsive dendrimer-microbubbles offer promise as a stimuli-responsive gene delivery strategy for precision cancer therapy.

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