Leveraging 3D Bioprinting and Photon-Counting Computed Tomography to Enable Noninvasive Quantitative Tracking of Multifunctional Tissue Engineered Constructs.

3D bioprinting is revolutionizing the fields of personalized and precision medicine by enabling the manufacturing of bioartificial implants that recapitulate the structural and functional characteristics of native tissues. However, the lack of quantitative and noninvasive techniques to longitudinally track the function of implants has hampered clinical applications of bioprinted scaffolds. In this study, multimaterial 3D bioprinting, engineered nanoparticles (NPs), and spectral photon-counting computed tomography (PCCT) technologies are integrated for the aim of developing a new precision medicine approach to custom-engineer scaffolds with traceability. Multiple CT-visible hydrogel-based bioinks, containing distinct molecular (iodine and gadolinium) and NP (iodine-loaded liposome, gold, methacrylated gold (AuMA), and Gd2 O3 ) contrast agents, are used to bioprint scaffolds with varying geometries at adequate fidelity levels. In vitro release studies, together with printing fidelity, mechanical, and biocompatibility tests identified AuMA and Gd2 O3 NPs as optimal reagents to track bioprinted constructs. Spectral PCCT imaging of scaffolds in vitro and subcutaneous implants in mice enabled noninvasive material discrimination and contrast agent quantification. Together, these results establish a novel theranostic platform with high precision, tunability, throughput, and reproducibility and open new prospects for a broad range of applications in the field of precision and personalized regenerative medicine.

Customized Scaffolds for Direct Assembly of Functionalized DNA Origami.

Functional DNA origami nanoparticles (DNA-NPs) are used as nanocarriers in a variety of biomedical applications including targeted drug delivery and vaccine development. DNA-NPs can be designed into a broad range of nanoarchitectures in one, two, and three dimensions with high structural fidelity. Moreover, the addressability of the DNA-NPs enables the precise organization of functional moieties, which improves targeting, actuation, and stability. DNA-NPs are usually functionalized via chemically modified staple strands, which can be further conjugated with additional polymers and proteins for the intended application. Although this method of functionalization is extremely efficient to control the stoichiometry and organization of functional moieties, fewer than half of the permissible sites are accessible through staple modifications. In addition, DNA-NP functionalization rapidly becomes expensive when a high number of functionalizations such as fluorophores for tracking and chemical modifications for stability that do not require spatially precise organization are used. To facilitate the synthesis of functional DNA-NPs, we propose a simple and robust strategy based on an asymmetric polymerase chain reaction (aPCR) protocol that allows direct synthesis of custom-length scaffolds that can be randomly modified and/or precisely modified via sequence design. We demonstrated the potential of our strategy by producing and characterizing heavily modified scaffold strands with amine groups for dye functionalization, phosphorothioate bonds for stability, and biotin for surface immobilization. We further validated our sequence design approach for precise conjugation of biomolecules by synthetizing scaffolds including binding loops and aptamer sequences that can be used for direct hybridization of nucleic acid tagged biomolecules or binding of protein targets.

Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies.

DNA origami nanocarriers have emerged as a promising tool for many biomedical applications, such as biosensing, targeted drug delivery, and cancer immunotherapy. These highly programmable nanoarchitectures are assembled into any shape or size with nanoscale precision by folding a single-stranded DNA scaffold with short complementary oligonucleotides. The standard scaffold strand used to fold DNA origami nanocarriers is usually the M13mp18 bacteriophage's circular single-stranded DNA genome with limited design flexibility in terms of the sequence and size of the final objects. However, with the recent progress in automated DNA origami design-allowing for increasing structural complexity-and the growing number of applications, the need for scalable methods to produce custom scaffolds has become crucial to overcome the limitations of traditional methods for scaffold production. Improved scaffold synthesis strategies will help to broaden the use of DNA origami for more biomedical applications. To this end, several techniques have been developed in recent years for the scalable synthesis of single stranded DNA scaffolds with custom lengths and sequences. This review focuses on these methods and the progress that has been made to address the challenges confronting custom scaffold production for large-scale DNA origami assembly.