Extracellular pH is important in clinical measurements due to its correlation to cell metabolism and disease progression. In MRI, T 1 /T 2 ratiometric analysis and other methods have been previously applied to quantify pH using conventional pulse sequences. However, for nanoparticle-based approaches, heterogeneity in size and surface functionalization tends toward qualitative rather than quantitative results. To address this limitation, we developed a novel DNA-based MRI contrast agent, pH-DMRCA, which utilizes a highly programmable and reproducible nanostructure. The pH-DMRCA is a dendritic DNA scaffold that is functionalized with a pH-responsive MRI-sensitive construct, Gd(NP-DO3A), at the end of each DNA arm. We first evaluated the r 1 and r 2 response of our pH-DMRCA over a range of pH values (pH = 5−9) to establish a relaxometric model of pH. These MRIbased assessments of pH were validated in a separate set of samples using a pH electrode (n = 18) and resulted in a good linear correlation (R 2 = 0.99, slope = 0.98, intercept = 0). A Bland−Altman analysis of the results also showed reasonable agreement between the calculated pH and measured pH. Moreover, these pH comparisons were consistent across three different pH-DMRCA concentrations, demonstrating concentration-independence of the method. This MRI-based pH quantification methodology was further verified in human blood plasma. Given the versatility of the DNA-based nanostructures, the contrast agent has a potential to be applied to a wide variety of imaging applications where extracellular pH is important including cancer, stroke, cardiovascular disease, and other important diseases.

The development of programmable biomaterials for use in nanofabrication represents a major advance for the future of biomedicine and diagnostics. Recent advances in structural nanotechnology using nucleic acids have resulted in dramatic progress in our understanding of nucleic acid-based nanostructures (NANs) for use in biological applications. As the NANs become more architecturally and functionally diverse to accommodate introduction into living systems, there is a need to understand how critical design features can be controlled to impart desired performance in vivo. In this review, we survey the range of nucleic acid materials utilized as structural building blocks (DNA, RNA, and xenonucleic acids), the diversity of geometries for nanofabrication, and the strategies to functionalize these complexes. We include an assessment of the available and emerging characterization tools used to evaluate the physical, mechanical, physiochemical, and biological properties of NANs in vitro. Finally, the current understanding of the obstacles encountered along the in vivo journey is contextualized to demonstrate how morphological features of NANs influence their biological fates. We envision that this summary will aid researchers in the designing novel NAN morphologies, guide characterization efforts, and design of experiments and spark interdisciplinary collaborations to fuel advancements in programmable platforms for biological applications.

Gadolinium (Gd)-based contrast agents (CA) are widely used to enhance anatomical details in magnetic resonance imaging (MRI). Significant research has expanded the field of CAs into bioresponsive CAs by modulating the signal to image and monitor biochemical processes, such as pH. In this work, we introduce the modular, dynamic actuation mechanism of DNA-based nanostructures as a new way to modulate the MRI signal based on rotational correlation time, τR. We combined a pH-responsive oligonucleotide (i-motif) and a clinical standard CA (Gd-DOTA), to develop a pH-responsive MRI CA. The i-motif folds into a quadruplex in acidic conditions and was incorporated onto gold nanoparticles (iM-GNP) to achieve increased relaxivity, r1, compared to unbound i-motif. In vitro, iM-GNP resulted in a significant increase in r1 over a decreasing pH range (7.5 - 4.5) with a calculated pKa = 5.88 ± 0.01 and a 16.7% change per 0.1 pH unit. In comparison, the control CA with a non-responsive DNA strand (T33-GNP) did not show a significant change in r1 over the same pH range. To demonstrate the potential for performance in tissue, CAs were evaluated in an ex vivo rat brain model. When compared to pre-contrast signal intensity (1/T1), the response to a simulated acidic microenvironment was over 5 times higher than the signal measured in a physiological pH. This approach paves a path for novel programmable, dynamic DNA-based complexes for τR-modulated bioresponsive MRI CAs.

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