Size-Differentiated Gas-Core Microparticles for Selective Ultrasound-Activated Gene Delivery in Engineered Tissue Constructs

Introduction: : Increasing evidence supports that many cancers have a polyclonal origin,[1] where multiple mutated cell populations contribute to tumor formation. Tissue-engineered in-vitro models utilizing a hydrogel matrix have shown considerable promise to study cancer progression by replicating important aspects of cell-cell and cell-matrix interactions. However, replicating the temporal aspect of the genetic transformations that lead to this polyclonal structure is challenging to achieve in a 3D model system. Recent work from our group has demonstrated the ability to integrate gas-core lipid-shell microbubble particles within tissue-engineered hydrogel constructs for ultrasound-stimulated gene delivery of a single transgene with spatial and temporal control.[2] Noninvasively-applied ultrasound waves stimulate the microbubble particles causing them to rupture and deliver their cargo to nearby cells via sonoporation.[3] In order to model polyclonal cancer progression, there is a need to selectively control activation of different particle populations within a single tissue construct which each carry distinct genetic payloads. Our objective is to develop a technique to isolate and selectively activate different size subpopulations of ultrasound-activatable gene delivery particles to enable the multiplexed sequential delivery of multiple genes within tissue-engineered constructs (Fig. 1A). In this study, differential centrifugation for microbubble size isolation was applied by varying both the relative centrifugal force (RCF) and the times at each applied RCF to acquire three distinct microbubble size populations. Further, we developed techniques to selectively activate these size-isolated subpopulations within cell-seeded hydrogels, paving the way for ultrasound-controlled sequential delivery of multiple genes within tissue-engineered constructs.

Materials and

Methods: : Size-isolated gas core, lipid-shell microparticles were prepared by differential centrifugation (1 minute at 10 RCF, 6 times followed by 5 minutes at 20 RCF, twice) and collection of the buoyant particle fractions after each centrifugation cycle, resulting in three distinct size populations of large, medium and small microbubbles. Each size population of microparticles contained DSTAP (1,2-Stearoyl-3-trimethylammoniumpropane) cationic lipid in the lipid shell, which allowed coupling to plasmid DNA by electrostatic interactions. Microparticles were coupled with GFP or mCherry DNA plasmids and integrated at different particle concentrations into cell-seeded (HEK293T) collagen hydrogels. For multiplexed particle experiments, the ratio between each two particle size populations in the tested combination was varied as well. Microparticle-containing hydrogel samples were subjected to 2.25 MHz focused ultrasound pulses at a range of applied intensities inside the hydrogel. Finally, transfection was assessed at 48 hr post-ultrasound application by confocal fluorescence imaging following DAPI nuclear staining. Transfection efficiency was quantified by image analysis using Volocity software by counting the number of GFP or mCherry-positive cells within the total cell count in an ROI set within the ultrasound focal zone.

Results, Conclusions, and Discussions:: We have successfully isolated three different microparticle size populations with mean sizes of 4.3 +/- 2.5 µm (large), 1.8 +/- 0.3 µm (medium), and 0.8 +/- 0.1 µm (small) using adjustable centrifugal force (Fig. 1B). Dependence of cellular transfection on particle size, concentration, and ultrasound intensity was observed; hydrogels containing medium-sized microparticle populations showed higher levels of transfection at 100% ultrasound intensity at a concentration of 10^9 particles/mL, while large and small microparticles showed less transfection at the same concentration and ultrasound intensity (Fig. 1C, upper row). At lower ultrasound intensity (25%) and a higher concentration of 10^10 particles/mL, small microparticles showed higher levels of transfection compared to large and medium microparticles, supporting selective activation (Fig. 1C, bottom row). Selective gene activation was observed in a preliminary study with hydrogels containing a mixture of small (GFP-coupled) and large (mCherry-coupled) bubble microparticles at a 50-50% ratio within the hydrogel. When the optimal ultrasound conditions established for the small-sized particle population were applied, these particles were observed to preferentially activate and transfect cells twice as much as the large-sized particles (Fig. 1D).
We have developed a method to successfully create gene delivery microparticle subpopulations that have varying degrees of responsivity to 2.25 MHz ultrasound at different ultrasound intensities and particle concentrations, resulting in differential transfection when embedded in cell-seeded hydrogels. Preliminary results support that preferential transfection of a particular gene cargo can be achieved within hydrogels containing multiple gene delivery particle populations by applying ultrasound conditions to selectively activate a single particle type. Ongoing work will utilize this gene delivery platform to control sequential transfection of multiple genes within the same tissue construct and explore differential activation by varying ultrasound frequency. The technique developed in this study paves the way for future sequential and controlled ultrasound-triggered delivery within hydrogels and hydrogel-based in vitro models, which can enable controlling the activation of multiple genetic transformations relevant to polyclonal tumors.

Acknowledgements and/or References (Optional)::

References: [1] Lu Z. Nature. 2024;636:233-240 [2] Lowrey M. Cell Mol Bioeng. 2024;17:401-421. [3] Martinez P. Pharmaceutics. 2022;14:1925