We have developed laser-activated perfluorocarbon nanodroplets containing copper sulfide nanoparticles (CuS NPs) for contrast-enhanced ultrasound and photoacoustic imaging. As potential clinical contrast agents, CuS NPs have favorable properties including biocompatibility, biodegradability, and enhance contrast in photoacoustic images at clinically relevant depths. However, CuS NPs are not efficient optical absorbers when compared to plasmonic nanoparticles and therefore, contrast enhancement with CuS NPs is limited, requiring high concentrations to generate images with sufficient signal-to-noise ratio. We have combined CuS NPs with laser-activated perfluorocarbon nanodroplets (PFCnDs) to achieve enhanced photoacoustic contrast and, more importantly, ultrasound contrast while retaining the favorable clinical characteristics of CuS NPs. The imaging characteristics of synthesized CuS-PFCnD constructs were first tested in tissue-mimicking phantoms and then in in vivo murine models. The results demonstrate that CuS-PFCnDs enhance contrast in photoacoustic (PA) and ultrasound (US) imaging. Upon systemic administration in vivo, CuS-PFCnDs remain stable and their unique vaporization provides sufficient PA/US contrast that can be further exploited for contrast-enhanced background-free imaging. The conducted studies provide a solid foundation for further development of CuS-PFCnDs as PA/US diagnostic and eventually therapeutic agents for clinical applications.

Regenerative therapies using stem cells have great potential for treating neurodegenerative diseases and traumatic injuries in the spinal cord. In spite of significant research efforts, many therapies fail at the clinical phase. As stem cell technologies advance toward clinical use, there is a need for a minimally invasive, safe, affordable, and real-time imaging technique that allows for the accurate and safe monitoring of stem cell delivery in the operating room. In this work, we present a combined ultrasound and photoacoustic imaging tool to provide image-guided needle placement and monitoring of nanoparticle-labeled stem cell delivery into the spinal cord. We successfully tagged stem cells using gold nanospheres and provided image-guided delivery of stem cells into the spinal cord in real-time, detecting as few as 1000 cells. Ultrasound and photoacoustic imaging was used to guide needle placement for direct stem cell injection to minimize the risk of needle shear and accidental injury and to improve therapeutic outcomes with accurate, localized stem cell delivery. Following injections of various volumes of cells, three-dimensional ultrasound and photoacoustic images allowed the visualization of stem cell distribution along the spinal cord, showing the potential to monitor the migration of the cells in the future. The feasibility of quantitative imaging was also shown by correlating the total photoacoustic signal over the imaging volume to the volume of cells injected. Overall, the presented method may allow clinicians to utilize imaged-guided delivery for more accurate and safer stem cell delivery to the spinal cord.

Photoacoustic imaging using exogenous contrast agents has emerged as a hybrid technique that enables the deep imaging of optical properties of tissues with high spatial resolution. The power of this imaging technique can be greatly enhanced by the use of contrast agents that absorb at near-infrared wavelengths and whose optical properties can be modulated in response to the local environment. We have designed contrast agents consisting of gold nanoparticles coated with anisotropic silica nanoshells. The tunable aggregation of these janus particles in cell culture media resulted in a dramatic amplification of photoacoustic signals in the near-infrared region. We also demonstrated imaging using these contrast agents in mammalian cells, including macrophages and breast cancer cells as well as in vivo. The ability to modulate janus particle aggregation in response to a range of stimuli in combination with the high resolution and deep penetration of multiwavelength photoacoustic imaging are attractive for a broad range of applications in diagnostic imaging and theranostics.

A minimally invasive method to detect lymph node metastases using glycol-chitosan-coated gold nanoparticles and ultrasound-guided photoacoustic imaging.

Nanoparticles play an important role in biomedicine. We have developed a method for sizecontrolled synthesis of photomagnetic Prussian blue nanocubes (PBNCs) using superparamagnetic iron oxide nanoparticles (SPIONs) as precursors. The developed PBNCs have magnetic and optical properties desired in many biomedical diagnostic and therapeutic applications. Specifically, the size-tunable photomagnetic PBNCs exhibit high magnetic saturation, strong optical absorption with a peak at approximately 700 nm, and superior photostability. Our studies demonstrate that PBNCs can be used as MRI and photoacoustic imaging contrast agents in vivo. We also showed the utility of PBNCs for labeling and magnetic manipulation of cells. Dual magnetic and optical properties, together with excellent biocompatibility, render PBNCs an attractive contrast agent for both diagnostic and therapeutic applications. The use SPIONs as precursors for PBNCs provides flexibility and allows researchers to design theranostic agents according to required particle size, optical, and magnetic properties.

Lymph node mapping is important in cancer immunotherapy because the morphology of lymph nodes is one of the crucial evaluation criteria of immune responses. We developed new theragnostic glycol-chitosan-coated gold nanoparticles (GC-AuNPs), which highlighted lymph nodes in ultrasound-guided photoacoustic (US/PA) imaging. Moreover, the ovalbumin epitope was conjugated GC-AuNPs (OVA-GC-AuNPs) for delivering tumor antigen to lymph node resident macrophage. In vitro studies proved the vigorous endocytosis activity of J774A.1 macrophage and consequent strong photoacoustic signals from them. The macrophages also presented a tumor antigen when OVA-GC-AuNPs were used for cellular uptake. After the lingual injection of GC-AuNPs into healthy mice, cervical lymph nodes were visible in a US/PA imaging system with high contrast. Three-dimensional analysis of lymph nodes revealed that the accumulation of GC-AuNPs in the lymph node increased as the post-injection time passed. Histological analysis showed GC-AuNPs or OVA-GC-AuNPs located in subcapsular and medullar sinuses where macrophages are abundant. Our new theragnostic GC-AuNPs present a superior performance in US/PA imaging of lymph nodes without targeting moieties or complex surface modification. Simultaneously, GC-AuNPs were able to deliver tumor antigens to cause macrophages to present the OVA epitope at targeted lymph nodes, which would be valuable for cancer immunotherapy.

The past three decades have seen numerous advances in tissue engineering and regenerative medicine (TERM) therapies. However, despite the successes there is still much to be done before TERM therapies become commonplace in clinic. One of the main obstacles is the lack of knowledge regarding complex tissue engineering processes. Imaging strategies, in conjunction with exogenous contrast agents, can aid in this endeavor by assessing in vivo therapeutic progress. The ability to uncover real-time treatment progress will help shed light on the complex tissue engineering processes and lead to development of improved, adaptive treatments. More importantly, the utilized exogenous contrast agents can double as therapeutic agents. Proper use of these Monitoring/Imaging and Regenerative Agents (MIRAs) can help increase TERM therapy successes and allow for clinical translation. While other fields have exploited similar particles for combining diagnostics and therapy, MIRA research is still in its beginning stages with much of the current research being focused on imaging or therapeutic applications, separately. Advancing MIRA research will have numerous impacts on achieving clinical translations of TERM therapies. Therefore, it is our goal to highlight current MIRA progress and suggest future research that can lead to effective TERM treatments.

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