Noninvasive and Highly Multiplexed Five-Color Tumor Imaging of Multicore Near-Infrared Resonant Surface-Enhanced Raman Nanoparticles In Vivo

Abstract

In vivo multiplexed imaging aims for noninvasive monitoring of tumors with multiple channels without excision of the tissue. While most of the preclinical imaging has provided a number of multiplexing channels up to three, Raman imaging with surface-enhanced Raman scattering (SERS) nanoparticles was suggested to offer higher multiplexing capability originating from their narrow spectral width. However, in vivo multiplexed SERS imaging is still in its infancy for multichannel visualization of tumors, which require both sufficient multiplicity and high sensitivity concurrently.Here we create multispectral palettes of gold multicore-nearinfrared (NIR) resonant Raman dyes-silica shell SERS (NIR-SERRS) nanoparticle oligomers and demonstrate noninvasive and five-plex SERS imaging of the nanoparticle accumulation in tumors of living mice. We perform the five-plex ratiometric imaging of tumors by varying the administered ratio of the nanoparticles, which simulates the detection of multiple biomarkers with different expression levels in the tumor environment. Furthermore, since this method does not require the excision of tumor tissues at the imaging condition, we perform noninvasive and longitudinal imaging of the five-color nanoparticles in the tumors, which is not feasible with current ex vivo multiplexed tissue analysis platforms. Our work surpasses the multiplicity limit of previous preclinical tumor imaging methods while keeping enough sensitivity for tumortargeted in vivo imaging and could enable the noninvasive assessment of multiple biological targets within the tumor microenvironment in living subjects.

Expanding the Multiplexing Capabilities of Raman Imaging to Reveal Highly Specific Molecular Expression and Enable Spatial Profiling

“…Due to the LSPR effect of AuNPs, the intensity of Raman signals for adsorbed molecules is enhanced dramatically, allowing them to be detected at extremely low concentrations . Some groups have utilized near-infrared (NIR) resonant Raman dyes to provide additional enhancement when utilized with a NIR laser, , but these resonant SERS NPs exhibit a high-fluorescence background and crowded Raman spectra, which are both properties known to negatively impact the conditioning of a multiplexed system and contribute to degraded performance at higher plexities. Narrow peaks, low background, and unique molecular fingerprints make SERS NPs labeled by small organic molecules one of the best candidates for extracting highplex data from a single biological sample, animal model, or patient .…”

“…After successfully demonstrating 26-plex imaging in vitro , we evaluated the multiplexing capability of our SERS NPs in conjunction with Raman microscopy and spectral unmixing algorithms applied for in vivo imaging. Several reports discuss the potential of utilizing SERS NPs for in vivo clinical applications using either active or passive tumor targeting. ,,− Their ability to sensitively and specifically localize various tumor types could significantly improve patient outcome. Although further testing will be needed for regulatory approval, preliminary toxicity studies have indicated that these gold/silica-based NPs are chemically inert and show no significant toxicity in cell culture or in murine models after intravenous administration. , We wanted to be able to detect and deconvolve all 26 SERS NPs in the presence of the native tissue background in a living subject.…”

“…injection (Figure ). To date, only a plexity of 5 has been previously demonstrated for noninvasive imaging when targets and SERS nanoparticles are colocalized in vivo . , Other in vivo imaging techniques, such as fluorescence, have begun sophisticated work on advanced phasor-space unmixing and denoising algorithms to increase plexity throughput . Difficulty for more fluorescent agents to be spectrally demultiplexed when colocalized in vivo is a significant impediment to its advancement for higher-plex imaging.…”

“…Raman spectral imaging is perfectly suited to perform molecular imaging without suffering from these limitations. − Molecular imaging of biological samples stained with various batches of SERS NP contrast agents, which we call SERS flavors, leverages the chemical specificity of Raman spectroscopy to transmit the state of biomarker expression in the form of multiplexed Raman spectroscopic optical signals. , The enhancement factor of the scattering cross-section achieved by localized surface plasmon resonance (LSPR) of nanoscale gold affords high sensitivity and fast mapping speeds using nondestructive laser intensities . As a result, the Raman spectrum of each SERS flavor can serve as a unique barcode for discovering and localizing a multitude of protein targets simultaneously. − Previously, the largest reported number of SERS NPs multiplexed during Raman imaging was achieved with 10 unique SERS flavors . We are now discovering that far more SERS NPs can be simultaneously present and identified per Raman spectral acquisition.…”

Near-infrared II plasmonic porous cubic nanoshells for in vivo noninvasive SERS visualization of sub-millimeter microtumors

“…This NIR-II SERS probe could also delineate the tumor margin directly in a living mouse model following the intravenous administration. The non-invasive SERS imaging capability of our NIR-II SERS probes in living animals opens up an opportunity to intraoperative guidance of complete tumor resection and longitudinal monitoring of cancer therapeutic response 6 – 8 , 53 . We find that the tumor accumulation of NIR-II SERS probes declines over prolonging time following intravenous administration, so that longitudinal SERS imaging should be performed prior to metabolic elimination of NIR-II SERS probes from the tumors.…”