The use of metallic nanoparticles in applications ranging from drug delivery to consumer electronics has exploded in the last two decades. Although this broad range of use cases has brought about technological revolutions in multiple fields, the effects of widespread production and subsequent human exposure to these nanoparticles have yet to be fully understood. New imaging techniques are a critical part of developing a more complete understanding of chronic exposure and biodistribution. Here we present a novel label free luminescence imaging technique to analyze the biodistribution, content, and biological context of metallic nanoparticles using multiphoton luminescence.
Failure to fully understand the molecular expression and tumor heterogeneity across a patient’s tumor can lead to administration of ineffective therapies that increase patient morbidity and healthcare costs. The -omics era has made it possible to identify several new molecular markers involved in cancer development, survival, invasion and even predicting treatment response. We are developing an entirely new nano-based molecular imaging strategy that has the potential to offer both high content molecular expression and spatial profiling in a single histology image. We have created an expansive library of 26 SERS nanoparticle (NP) batches, each bearing a unique spectral fingerprint with exceptional multiplexing capabilities. Spectral deconvolution was successfully demonstrated with a mixture of all 26 SERS NPs in a single imaging pixel both in vitro and in vivo. This opens up new opportunities to efficiently interrogate the heterogeneous molecular expression found within and across patient tissues offering clinicians a new multiplexed molecular imaging tool with the potential to predict how well a patient is likely to respond to given therapies based on their unique profile.
Profiling the heterogeneous landscape of cell types and biomolecules is rapidly being adopted to address current imperative research questions. Precision medicine seeks advancements in molecular spatial profiling techniques with highly multiplexed imaging capabilities and sub-cellular resolution, which remains an extremely complex task. Surface-enhanced Raman spectroscopy (SERS) imaging offers new promise through the utilization of nanoparticle-based contrast agents that exhibit narrow spectral features and molecular specificity.
Herein, we report the first demonstration of simultaneously multiplexing 26 different nanoparticles in a single imaging pixel with subcellular resolution.
SERS nanoparticles are powerful optical contrast agents for imaging assays. Their highly specific sets of narrow spectral bands make them well suited for multiplexing applications, and their enhanced inelastic scattering cross sections enable rapid, high content imaging. Multiplexed hyperspectral imaging datasets commonly undergo a spectral unmixing postprocessing step using a compensation matrix of reference spectra to produce quantitative image channels. We perform hyperspectral Raman imaging on mixtures with increasing plexity and varying degrees of linear system conditioning and compare against the ground truth to determine the most robust workflow for quantitative biological SERS imaging.
Negative surgical margins can be difficult to confirm intraoperatively. We propose a workflow of immunostaining and surface imaging of fresh excised tissue using highly sensitive and spectrally separable SERS nanoparticles as the targeted contrast agent. The adaptive focus capabilities of an advanced Raman instrument, combined with our rotational accessory tool for exposing each surface of the stained specimen to the objective lens, enables topographic mapping of the entire excised specimen’s surface. Detailed surface renderings color-encoded according to unmixed SERS nanoparticle abundances show a path forward for high-content, interactive surgical margin assessment.
This Conference Presentation, “A resurgence in nanoparticles: accelerating the clinical translation of nano-based imaging contrast agents,” was recorded at SPIE Photonics West held in San Francisco, California, United States
Nanoscale materials are routinely developed, characterized, and evaluated as diagnostic and therapeutic agents for disease treatment in humans. However, the size and composition of many such agents result in poor clearance profiles within biological tissues, thereby posing profound challenges to translational clinical applications. Herein, we present a hyperspectral imaging technique capable of quantifying plasmonic nanoparticle biodistribution with single particle limit of detection and microanatomical detail. Using this method, we find that, although intravenous administration of plasmonic nanoparticles remains largely infeasible from a biodistribution perspective, alternate routes relevant to treatment of oral and gastrointestinal diseases are within translational reach.
Nanoparticles have been explored extensively as potential biomedical imaging and therapeutic agents. One critical aspect of in vivo nanoparticle use is the characterization of biodistribution profiles. Such studies improve our understanding of particle uptake, specificity, and clearance mechanisms. Currently, the most prevalent nanoparticle biodistribution methods provide either aspatial quantification of whole-organ particle accumulation or nanometerresolution images of uptake in single cells. Few existing techniques are well-suited to study particle uptake on the micron to millimeter scales relevant to sub-tissue physiology. Here we demonstrate a new method called Hyperspectral Microscopy with Adaptive Detection (HSM-AD) that uses machine learning classification of hyperspectral dark-field images to study interactions between tissues and administered nanoparticles. This label-free, non-destructive method enables quantitative particle identification in histological sections and detailed observations of sub-organ accumulation patterns consistent with organ-specific clearance mechanisms, particle size, and the molecular specificity of the nanoparticle surface. Unlike studies with electron microscopy, HSM-AD is readily applied for large fields of view. HSM-AD achieves excellent detection sensitivity (99.4%) and specificity (99.7%) and can identify single nanoparticles. To demonstrate HSM-AD’s potential for novel nanoparticle uptake studies, we collected the first data on the sub-organ localization of large gold nanorods (LGNRs) in mice. We also observed differences in particle accumulation and localization patterns in tumors as a function of conjugated molecular targeting moieties. Thus, HSM-AD affords new degrees of detail for the study of nanoparticle uptake at physiological scales. HSM-AD may offer an auxiliary or alternative approach to study the biodistribution profiles of existing and novel nanoparticles.
Topical application and quantification of targeted, surface-enhanced Raman scattering (SERS) nanoparticles offer a new technique that has the potential for early detection of epithelial cancers of hollow organs. Although less toxic than intravenous delivery, the additional washing required to remove unbound nanoparticles cannot necessarily eliminate nonspecific pooling. Therefore, we developed a real-time, ratiometric imaging technique to determine the relative concentrations of at least two spectrally unique nanoparticle types, where one serves as a nontargeted control. This approach improves the specific detection of bound, targeted nanoparticles by adjusting for working distance and for any nonspecific accumulation following washing. We engineered hardware and software to acquire SERS signals and ratios in real time and display them via a graphical user interface. We report quantitative, ratiometric imaging with nanoparticles at pM and sub-pM concentrations and at varying working distances, up to 50 mm. Additionally, we discuss optimization of a Raman endoscope by evaluating the effects of lens material and fiber coating on background noise, and theoretically modeling and simulating collection efficiency at various working distances. This work will enable the development of a clinically translatable, noncontact Raman endoscope capable of rapidly scanning large, topographically complex tissue surfaces for small and otherwise hard to detect lesions.
Adam de la Zerda, Moritz Kircher, Jesse Jokerst, Cristina Zavaleta, Paul Kempen, Erik Mittra, Ken Pitter, Ruimin Huang, Carl Campos, Frezghi Habte, Robert Sinclair, Cameron Brennan, Ingo Mellinghoff, Eric Holland, Sanjiv Gambhir
The difficulty in delineating brain tumor margins is a major obstacle in the path toward better
outcomes for patients with brain tumors. Current imaging methods are often limited by
inadequate sensitivity, specificity and spatial resolution. Here we show that a unique triplemodality
magnetic resonance imaging - photoacoustic imaging - Raman imaging nanoparticle
(termed here MPR nanoparticles), can accurately help delineate the margins of brain tumors in
living mice both preoperatively and intraoperatively. The MPRs were detected by all three
modalities with at least a picomolar sensitivity both in vitro and in living mice. Intravenous
injection of MPRs into glioblastoma-bearing mice led to MPR accumulation and retention by the
tumors, with no MPR accumulation in the surrounding healthy tissue, allowing for a noninvasive
tumor delineation using all three modalities through the intact skull. Raman imaging allowed for
guidance of intraoperative tumor resection, and a histological correlation validated that Raman
imaging was accurately delineating the brain tumor margins. This new triple-modality–
nanoparticle approach has promise for enabling more accurate brain tumor imaging and
resection.
Raman spectra are most commonly analyzed using the ordinary least squares (LS) method. However, LS is sensitive to variability in the spectra of the analyte and background materials. We previously addressed this problem by successfully proposing a novel hybrid least squares and principal components (HLP) algorithm. HLP extended LS by allowing the reference spectra to vary in accordance with the principal components observed in calibration sets. Previously, HLP assumed zero-mean Gaussian measurement noise. In this work, we show that the noise in fact follows a Poisson distribution, and update the mathematical framework of our algorithm accordingly. Since the name ’least squares’ referred to the Gaussian noise model, we also generalize the name of our algorithm to the Hybrid reference Spectrum and Principal component analysis (HSP) algorithm. The performance of the Gaussian and Poisson noise models is compared using both simulated and measured spectra. The simulated spectra were computed by adding various concentrations of Raman-enhanced gold-silica nanoparticles to three different backgrounds (paraffin, glass and quartz). The measured spectra were acquired from a serial dilution of gold-silica nanoparticles placed on an excised pig colon. For the simulated spectra, the Poisson model
consistently outperformed the Gaussian model, on average reducing the mean absolute concentration error as well as its standard deviation by ~15-20%. For the measured data, the Gaussian and Poisson noise models yielded similar concentration estimates. Both HSP algorithms also outperformed the LS algorithm, indicating that the incorporation of the principal components yields a larger improvement in accuracy than the optimization of the noise model. Further comparison between the two HSP algorithms (with Gaussian and Poisson noise models) was precluded by a lack of precise ground truth knowledge of the nanoparticle concentrations on the colon tissue. However, the simulation results already demonstrated that the optimization of noise models can improve the detection accuracy of Raman spectroscopy, and that it may therefore be an important consideration in future high-sensitivity Raman imaging studies.
Photoacoustic imaging of living subjects offers high spatial resolution at increased tissue depths
compared to purely optical imaging techniques. We have recently shown that intravenously injected
single walled carbon nanotubes (SWNTs) can be used as targeted photoacoustic imaging agents in
living mice using RGD peptides to target αvβ3 integrins. We have now developed a new targeted
photoacoustic imaging agent based on SWNTs and Indocyanine Green (SWNT-ICG) with
absorption peak at 780nm. The photoacoustic signal of the new imaging agent is enhanced by ~20
times as compared to plain SWNTs. The particles are synthesized from SWNT-RGD that noncovalently
attach to multiple ICG molecules through pi-pi stacking interactions. Negative control
particles had RAD peptide instead of RGD. We measured the serum stability of the particles and
verified that the RGD/RAD conjugation did not alter the particle's absorbance spectrum. Finally,
through cell uptake studies with U87MG cells we verified that the particles bind selectively to αvβ3
integrin. In conclusion, the extremely high absorption of the
SWNT-ICG particles shows great
promise for high sensitivity photoacoustic imaging of molecular targets in-vivo. This work lays the
foundations for future in-vivo studies that will use the SWNT-ICG particles as imaging agents
administered systemically.
Photoacoustic molecular imaging is an emerging technology offering non-invasive high resolution imaging of the molecular expressions of a disease using a photoacoustic imaging agent. Here we demonstrate for the first time the utility of single walled carbon nanotubes (SWNTs) as targeted imaging agents in living mice bearing tumor xenografts. SWNTs were conjugated with polyethylene-glycol-5000 connected to Arg-Gly-Asp (RGD) peptide to target the αvβ3 integrin that is associated with tumor angiogenesis. In-vitro, we characterized the photoacoustic spectra of the particles, their signal linearity and tested their uptake by αvβ3-expressing cells (U87MG). The photoacoustic signal of SWNTs was found not to be affected by the RGD conjugation to the SWNTs and was also found to be highly linear with concentration (R2 = 0.9997 for 25-400nM). The cell uptake studies showed that RGD-targeted SWNTs gave 75% higher photoacoustic signal than non-targeted SWNTs when incubated with U87MG cells. In-vivo, we measured the minimal detectable concentration of SWNTs in living mice by subcutaneously injecting SWNTs at increasing concentrations. The lowest detectable concentration of SWNTs in living mice was found to be 50nM. Finally, we administered RGDtargeted and non-targeted SWNTs via the tail-vein to U87MG tumor-bearing mice (n=4 for each group) and measured the signal from the tumor before and up to 4 hours post-injection. At 4 hours post-injection, tumors of mice injected with RGD-targeted SWNTs showed 8 times higher photoacoustic signal compared with mice injected with non-targeted SWNTs. These results were verified ex-vivo using a Raman microscope that is sensitive to the SWNTs Raman signal.
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