Phase-change nanodroplets (PCNDs) continue to generate significant research interest due to their potential to extravasate into tissue, to be targeted for molecular imaging and drug delivery, and to undergo an induced phase-change to "activated" microbubbles (MBs) for ultrasound (US) imaging. To accurately quantify molecular markers, however, one assumes a consistent proportion of PCNDs in a region of interest (ROI) are stably activated and imaged. Herein we present a framework for developing a diagnostic sequence that is optimized for PCND activation uniformity, contrast, and acquisition time. To develop this framework, activation was examined at three scales of increasing complexity: single, adjacent, and full ROI activation(s). First, transmit parameters for a single activation were optimized using PCNDs across concentrations (1.0 × 107-109 PCND/mL) in controlled phantom experiments, considering contrast-to-noise ratio (CNR), area, and offset (e.g., centroid axial distance from transmit focus) of the activation region along with acquisition time. Activation regions were also compared to prospective estimates based on US-beam imaging. Next, overlap and order of adjacent activation regions was optimized by maximizing spacing while preserving uniformity and minimizing signal loss from adjacent transmit interactions. Finally, an optimized raster-scanning scheme was applied to a mock tumor ROI, yielding 1.4-7.0 dB greater contrast enhancement over unoptimized schemes while reducing acquisition time. Further, PCND concentration was found to share a nonlinear relationship with US signal enhancement that differed significantly between linear and harmonic imaging modes. In conclusion, our framework's optimization of contrast, uniformity, acquisition time, and cavitation mitigation for PCND US imaging should help facilitate its eventual clinical translation.

Introduction Clinical adoption of NK cell immunotherapy is underway for medulloblastoma and osteosarcoma, however there is currently little feedback on cell fate after administration. We propose magnetic particle imaging (MPI) for the detection, localization, and quantification of VivoTrax-labeled NK cells. Methods Human-derived NK-92 cells were labeled by co-incubation with VivoTrax for 24 hours then the excess nanoparticles were washed with centrifugation. Cytolytic activity of labeled vs. unlabeled NK-92 cells was assessed after 4 hours of co- incubation with medulloblastoma cells (DAOY) or osteosarcoma cells (LM7 or OS17) using bioluminescent or GFP counts. Labeled NK-92 cells at two different doses (0.5 or 1 x 106) were administered to excised mouse brains (cerebellum), tibias, and lungs then imaged by 3D preclinical MPI (MOMENTUM imager) and localized relative to fiducial markers. NK-92 cells were imaged by clinical-scale MPI under development at Magnetic Insight Inc. Results NK-92 cells were labeled with an average of 3.17 pg Fe/cell with no measured effects on cell viability or cytolytic activity against 3 tumor cell lines. MPI signal was directly quantitative with the number of VivoTrax-labeled NK-92 cells, with preclinical limit of detection of 3.1 x 104 cells on MOMENTUM imager. Labeled NK-92 cells could be accurately localized in mouse brains, tibias, and lungs within < 1 mm of stereotactic injection coordinates with preclinical scanner. Feasibility for detection of a clinically relevant dose of 4 x 107 labeled NK-92 cells was demonstrated on clinical-scale MPI. Conclusion MPI can provide sensitive, quantitative, and accurate spatial information on NK cell delivery, showing its potential to resolve a significant unmet clinical need to track NK cell treatments in patients. ### Competing Interest Statement Olivia C. Sehl, Kelvin Guo, Benjamin Fellows, A. Rahman Mohtasebzadeh, Erica E. Mason, Toby Sanders, Petrina Kim, David Trease, Patrick W. Goodwill, and Joan M Greve report relationship with Magnetic Insight Inc. that includes: employment and stock ownership.

Clinical adoption of NK cell immunotherapy is underway for medulloblastoma and osteosarcoma, however there is currently little feedback on cell fate after administration. We propose magnetic particle imaging (MPI) may have applications for the quantitative detection of NK cells. Human-derived NK-92 cells were labeled by co-incubation with iron oxide nanoparticles (VivoTrax™) for 24 h then excess nanoparticles were washed with centrifugation. Cytolytic activity of labeled versus unlabeled NK-92 cells was assessed after 4 h of co-incubation with medulloblastoma cells (DAOY) or osteosarcoma cells (LM7 or OS17). Labeled NK-92 cells at two different doses (0.5 or 1 × 106) were administered to excised mouse brains (cerebellum), fibulas, and lungs then imaged by 3D preclinical MPI (MOMENTUM™) for detection relative to fiducial markers. NK-92 cells were also imaged by clinical-scale MPI under development at Magnetic Insight Inc. NK-92 cells were labeled with an average of 3.17 pg Fe/cell with no measurable effects on cell viability or cytolytic activity against 3 tumor cell lines. MPI signal was directly quantitative with the number of labeled NK-92 cells, with preclinical limit of detection of 3.1 × 104 cells on MOMENTUM imager. Labeled NK-92 cells could be accurately localized in mouse brains, fibulas, and lungs within < 1 mm of stereotactic injection coordinates with preclinical scanner. Feasibility for detection on a clinical-scale MPI scanner was demonstrated using 4 × 107 labeled NK-92 cells, which is in the range of NK cell doses administered in our previous clinical trial. MPI can provide sensitive, quantitative, and accurate spatial information on NK cells soon after delivery, showing initial promise to address a significant unmet clinical need to track NK cell fate in patients.

Imaging plays a critical role in all stages of cancer care from early detection to diagnosis, prognosis, and therapy monitoring. Recently, photoacoustic imaging (PAI) has started to emerge into the clinical realm due to its high sensitivity and ability to penetrate tissues up to several centimeters deep. Herein, we encapsulated indocyanine green J (ICGJ) aggregate, one of the only FDA-approved organic exogenous contrast agents that absorbs in the near-infrared range, at high loadings up to ∼40% w/w within biodegradable polymersomes (ICGJ-Ps) composed of poly(lactide-co-glycolide-b-polyethylene glycol) (PLGA-b-PEG). The small Ps hydrodynamic diameter of 80 nm is advantageous for in vivo applications, while directional conjugation with epidermal growth factor receptor (EGFR) targeting cetuximab antibodies renders molecular specificity. Even when exposed to serum, the ∼11 nm-thick membrane of the Ps prevents dissociation of the encapsulated ICGJ for at least 48 h with a high ratio of ICGJ to monomeric ICG absorbances (i.e., I895/I780 ratio) of approximately 5.0 that enables generation of a strong NIR photoacoustic (PA) signal. The PA signal of polymersome-labeled breast cancer cells is proportional to the level of cellular EGFR expression, indicating the feasibility of molecular PAI with antibody-conjugated ICGJ-Ps. Furthermore, the labeled cells were successfully detected with PAI in highly turbid tissue-mimicking phantoms up to a depth of 5 mm with the PA signal proportional to the amount of cells. These data show the potential of molecular PAI with ICGJ-Ps for clinical applications such as tumor margin detection, evaluation of lymph nodes for the presence of micrometastasis, and laparoscopic imaging procedures.

An effective strategy for overcoming radiotherapy resistance is to sequester radiosensitizing hafnium oxide or gold nanoparticles (GNPs) within tumors, but desmoplasia, but desmoplasia creates a delivery barrier impeding this approach. Here we report a radiosensitization strategy that overcomes this limitation by applying an atomic-scale agent - ionic gold - for in situ biosynthesis of GNPs within tumors. We showed that the in situ generation of GNPs occurs with higher efficiency in cancerous pancreatic cells than in non-cancerous cells and that these GNPs have high nuclear localization, resulting in the specific radiosensitization of cancer cells. Our studies in a xenograft murine model of pancreatic cancer revealed a uniform distribution of GNPs in the tumor, with insignificant gold content and low toxicity in normal organs. Radiosensitization by biosynthesized GNPs halted tumor growth for more than 30 days and was associated with a significantly higher median survival versus radiation alone (235 vs 102 days, respectively).

Identification of the mouse anatomy from US images.

Perfluorocarbon nanodroplets (PFCs) are a new class of liquid contrast agents that have been explored for ultrasound and photoacoustic imaging applications by the biomedical imaging community. These contrast agents can undergo a reversible or irreversible liquid-to-gas phase transitions upon an external excitation by a light or an ultrasound stimulus. Here, we evaluate the influence of apparently minute changes to the coating of PFCs and of dye-embedding strategies (i.e., for light activation) on the activation efficiency of the phase transition and the corresponding signal generation by PFCs. We discuss implications of these findings for further development of PFC contrast agents.

Legend for Supplementary Figures S1-S2.

Many tissues are composed of layered structures, and a better understanding of the changes in the layered tissue biomechanics can enable advanced guidance and monitoring of therapy. The advent of elastography using longitudinally propagating shear waves (LSWs) has created the prospect of a high-resolution assessment of depth-dependent tissue elasticity. Laser activation of liquid-to-gas phase transition of dye-loaded perfluorocarbon (PFC) nanodroplets (a.k.a., nanobombs) can produce highly localized LSWs. This study aims to leverage the potential of photoactivation of nanobombs to incudce LSWs with very high-frequency content in wave-based optical coherence elastography (OCE) to estimate the elasticity gradient with high resolution. In this work, we used multilayered tissue-mimicking phantoms to demonstrate that highly localized nanobomb (NB)-induced LSWs can discriminate depth-wise tissue elasticity gradients. The results show that the NB-induced LSWs rapidly change speed when transitioning between layers with different mechanical properties, resulting in an elasticity resolution of ∼65 µm. These results show promise for characterizing the elasticity of multilayer tissue with a fine resolution.

MAPS allow differentiation of delivery and molecular interactions.

Lipid-based formulations provide a nanotechnology platform that is widely used in a variety of biomedical applications because it has several advantageous properties including biocompatibility, reduced toxicity, relative ease of surface modifications, and the possibility for efficient loading of drugs, biologics, and nanoparticles. A combination of lipid-based formulations with magnetic nanoparticles such as iron oxide was shown to be highly advantageous in a growing number of applications including magnet-mediated drug delivery and image-guided therapy. Currently, lipid-based formulations are prepared by multistep protocols. Simplification of the current multistep procedures can lead to a number of important technological advantages including significantly decreased processing time, higher reaction yield, better product reproducibility, and improved quality. Here, we introduce a one-pot, single-step synthesis of drug-loaded magnetic multimicelle aggregates (MaMAs), which is based on controlled flow infusion of an iron oxide nanoparticle/lipid mixture into an aqueous drug solution under ultrasonication. Furthermore, we prepared molecular-targeted MaMAs by directional antibody conjugation through an Fc moiety using Cu-free click chemistry. Fluorescence imaging and quantification confirmed that antibody-conjugated MaMAs showed high cell-specific targeting that was enhanced by magnetic delivery.

Abstract Background While microbubble contrast agents (MCAs) are commonly used in ultrasound (US), they are inherently limited to vascular targets due to their size. Alternatively, phase‐changing nanodroplet contrast agents (PNCAs) can be delivered as nanoscale agents (i.e., small enough to extravasate), but when exposed to a US field of sufficient mechanical index (MI), they convert to MCAs, which can be visualized with high contrast using nonlinear US. Purpose To investigate the effect of perfluorocarbon (PFC) core composition and presence of cholesterol in particle coatings on stability and image contrast generated from acoustic activation of PNCAs using high‐frequency US suitable for clinical imaging. Methods PNCAs with varied core compositions (i.e., mixtures of perfluoropentane [C5] and/or perfluorohexane [C6]) and two coating formulations (i.e., with and without cholesterol) were characterized and investigated for thermal/temporal stability and postactivation, nonlinear US contrast in phantom and in vivo environments. Through hydrophone measurements and nonlinear numerical modeling, MI was estimated for pulse sequences used for PNCA activation. Results All PNCA compositions were characterized to have similar diameters (249–267 nm) and polydispersity (0.151–0.185) following fabrication. While PNCAs with majority C5 core composition showed higher levels of spontaneous signal (i.e., not due to US activation) in phantoms than C6‐majority PNCAs, all compositions were stable during imaging experiments. When activating PNCAs with a 12.3‐MHz US pulse (MI = 1.1), C6‐core particles with cholesterol‐free coatings (i.e., CF‐C6‐100 particles) generated a median contrast of 3.1, which was significantly higher (p < 0.001) than other formulations. Further, CF‐C6‐100 particles were activated in a murine model, generating US contrast ≥3.4. Conclusion C6‐core PNCAs can provide high‐contrast US imaging with minimal nonspecific activation in phantom and in vivo environments.