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Abstract IntroductionCoaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection. MethodsPhospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl₂crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression. ResultsCoaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments. ConclusionsOur results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine. 

Abstract Lipid‐coated microbubbles are an important class of gene delivery vehicles activated by ultrasound to locally deliver their DNA payloads to cells. Negatively charged DNA is electrostatically loaded onto the positively charged surface of microbubbles that contain a cationic lipid shell. Characterizing the zeta potential of individual cationic microbubbles to determine a population distribution and how this is affected by DNA complexation is critical to maximize DNA loading and circulation time. Traditional zeta potential analysis provides an ensemble charge measurement for a particle population but cannot measure individual particles to determine a distribution. Here, single‐particle tracking microelectrophoresis technology is applied to measure zeta potentials of individual microbubbles synthesized with different ratios of 1,2‐distearoyl‐3‐trimethylammonium‐propane (DSTAP) cationic lipid as well as loaded with increasing amounts of DNA. Results show that at 0 mol% DSTAP all microbubbles are negatively charged, and at 10 mol% half are positive. All particles are positive at 20 mol% DSTAP but the population shifts to negative values upon incubation with 0.01 pg DNA/microbubble. Analyzing zeta potential on the individual microbubble level is a powerful tool to understand DNA loading across a population of microbubbles and enables microbubble surface charge and nucleic acid loading optimization for delivery applications.