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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.
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Massively parallel microbubble nano-assembly
Abstract Microbubbles are an important tool due to their unique mechanical, acoustic, and dynamical properties. Yet, it remains challenging to generate microbubbles quickly in a parallel, biocompatible, and controlled manner. Here, we present an opto-electrochemical method that combines precise light-based projection with low-energy electrolysis, realizing defined microbubble patterns that in turn trigger assembly processes. The size of the bubbles can be controlled from a few to over hundred micrometers with a spatial accuracy of ~2 μm. The minimum required light intensity is only ~0.1 W/cm2, several orders of magnitude lower compared to other light-enabled methods. We demonstrate the assembly of prescribed patterns of 40-nm nanocrystals, 200 nm extracellular vesicles, polymer nanospheres, and live bacteria. We show how nanosensor-bacterial-cell arrays can be formed for spectroscopic profiling of metabolites and antibiotic response of bacterial assemblies. The combination of a photoconductor with electrochemical techniques enables low-energy, low-temperature bubble generation, advantageous for large-scale, one-shot patterning of diverse particles in a biocompatible manner. The microbubble-platform is highly versatile and promises new opportunities in nanorobotics, nanomanufacturing, high-throughput bioassays, single cell omics, bioseparation, and drug screening and discovery.
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- PAR ID:
- 10617248
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 16
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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