Spatially offset Raman spectroscopy for biomedical applications.

In recent years, Raman spectroscopy has undergone major advancements in its ability to probe deeply through turbid media such as biological tissues. This progress has been facilitated by the advent of a range of specialist techniques based around spatially offset Raman spectroscopy (SORS) to enable non-invasive probing of living tissue through depths of up to 5 cm. This represents an improvement in depth penetration of up to two orders of magnitude compared to what can be achieved with conventional Raman methods. In combination with the inherently high molecular specificity of Raman spectroscopy, this has therefore opened up entirely new prospects for a range of new analytical applications across multiple fields including medical diagnosis and disease monitoring. This article discusses SORS and related variants of deep Raman spectroscopy such as transmission Raman spectroscopy (TRS), micro-SORS and surface enhanced spatially offset Raman spectroscopy (SESORS), and reviews the progress made in this field during the past 5 years including advances in non-invasive cancer diagnosis, monitoring of neurotransmitters, and assessment of bone disease.

Distorted Phthalocyanines by Click Chemistry: Photoacoustic, Photothermal, and Surface-Enhanced Resonance Raman Studies.

Distortion of nominally planar phthalocyanine macrocycles affects the excited state dynamics in that most of the excited-state energy decays through internal conversion. A click-type annulation reaction on a perfluorophthalocyanine platform appending a seven-membered ring to the ß-positions on one or more of the isoindoles distorts the macrocycle and modulates solubility. The distorted derivative enables photoacoustic imaging, photothermal effects, and strong surface-enhanced resonance Raman signals.

Dual-Modality Surface-Enhanced Resonance Raman Scattering and Multispectral Optoacoustic Tomography Nanoparticle Approach for Brain Tumor Delineation.

Difficulty in visualizing glioma margins intraoperatively remains a major issue in the achievement of gross total tumor resection and, thus, better clinical outcome of glioblastoma (GBM) patients. Here, the potential of a new combined optical + optoacoustic imaging method for intraoperative brain tumor delineation is investigated. A strategy using a newly developed gold nanostar synthesis method, Raman reporter chemistry, and silication method to produce dual-modality contrast agents for combined surface-enhanced resonance Raman scattering (SERRS) and multispectral optoacoustic tomography (MSOT) imaging is devised. Following intravenous injection of the SERRS-MSOT-nanostars in brain tumor bearing mice, sequential MSOT imaging is performed in vivo and followed by Raman imaging. MSOT is able to accurately depict GBMs three-dimensionally with high specificity. The MSOT signal is found to correlate well with the SERRS images. Because SERRS enables uniquely sensitive high-resolution surface detection, it could represent an ideal complementary imaging modality to MSOT, which enables real-time, deep tissue imaging in 3D. This dual-modality SERRS-MSOT-nanostar contrast agent reported here is shown to enable high precision depiction of the extent of infiltrating GBMs by Raman- and MSOT imaging in a clinically relevant murine GBM model and could pave new ways for improved image-guided resection of brain tumors.

Multi-center study finds postoperative residual non-enhancing component of glioblastoma as a new determinant of patient outcome.

INTRODUCTION: The aim of the present study is to assess whether postoperative residual non-enhancing volume (PRNV) is correlated and predictive of overall survival (OS) in glioblastoma (GBM) patients. METHODS: We retrospectively analyzed a total 134 GBM patients obtained from The University of Texas MD Anderson Cancer Center (training cohort, n = 97) and The Cancer Genome Atlas (validation cohort, n = 37). All patients had undergone postoperative magnetic resonance imaging immediately after surgery. We evaluated the survival outcomes with regard to PRNV. The role of possible prognostic factors that may affect survival after resection, including age, sex, preoperative Karnofsky performance status, postoperative nodular enhancement, surgically induced enhancement, and postoperative necrosis, was investigated using univariate and multivariate Cox proportional hazards regression analyses. Additionally, a recursive partitioning analysis (RPA) was used to identify prognostic groups. RESULTS: Our analyses revealed that a high PRNV (HR 1.051; p-corrected = 0.046) and old age (HR 1.031; p-corrected = 0.006) were independent predictors of overall survival. This trend was also observed in the validation cohort (higher PRNV: HR 1.127, p-corrected = 0.002; older age: HR 1.034, p-corrected = 0.022). RPA analysis identified two prognostic risk groups: low-risk group (PRNV < 70.2 cm3; n = 55) and high-risk group (PRNV ≥ 70.2 cm3; n = 42). GBM patients with low PRNV had a significant survival benefit (5.6 months; p = 0.0037). CONCLUSION: Our results demonstrate that high PRNV is associated with poor OS. Such results could be of great importance in a clinical setting, particularly in the postoperative management and monitoring of therapy.

MUC1 Aptamer Targeted SERS Nanoprobes.

Recently, surface-enhanced Raman scattering (SERS) nanoprobes (NPs) have shown promise in the field of cancer imaging due to their unparalleled signal specificity and high sensitivity. Here we report the development of a DNA aptamer targeted SERS NP. Recently, aptamers are being investigated as a viable alternative to more traditional antibody targeting due to their low immunogenicity and low cost of production. We developed a strategy to functionalize SERS NPs with DNA aptamers, which target Mucin1 (MUC1) in human breast cancer (BC). Thorough in vitro characterization studies demonstrated excellent serum stability and specific binding of the targeted NPs to MUC1. In order to test their in vivo targeting capability, we co-injected MUC1-targeted SERS NPs, and as controls non-targeted and blocked MUC1-targeted SERS NPs in BC xenograft mouse models. A two-tumor mouse model with differential expression of MUC1 (MDA-MB-468 and MDA-MB-453) was used to control for active versus passive targeting in the same animals. The results showed that the targeted SERS NPs home to the tumors via active targeting of MUC1, with low levels of passive targeting. We expect this strategy to be an advantageous alternative to antibody-based targeting and useful for targeted imaging of tumor extent, progression, and therapeutic response.

Stable Radiolabeling of Sulfur-Functionalized Silica Nanoparticles with Copper-64.

Nanoparticles labeled with radiometals enable whole-body nuclear imaging and therapy. Though chelating agents are commonly used to radiolabel biomolecules, nanoparticles offer the advantage of attaching a radiometal directly to the nanoparticle itself without the need of such agents. We previously demonstrated that direct radiolabeling of silica nanoparticles with hard, oxophilic ions, such as the positron emitters zirconium-89 and gallium-68, is remarkably efficient. However, softer radiometals, such as the widely employed copper-64, do not stably bind to the silica matrix and quickly dissociate under physiological conditions. Here, we overcome this limitation through the use of silica nanoparticles functionalized with a soft electron-donating thiol group to allow stable attachment of copper-64. This approach significantly improves the stability of copper-64 labeled thiol-functionalized silica nanoparticles relative to native silica nanoparticles, thereby enabling in vivo PET imaging, and may be translated to other softer radiometals with affinity for sulfur. The presented approach expands the application of silica nanoparticles as a platform for facile radiolabeling with both hard and soft radiometal ions.

Lymph Node Micrometastases and In-Transit Metastases from Melanoma: In Vivo Detection with Multispectral Optoacoustic Imaging in a Mouse Model.

Purpose To study whether multispectral optoacoustic tomography (MSOT) can serve as a label-free imaging modality for the detection of lymph node micrometastases and in-transit metastases from melanoma on the basis of the intrinsic contrast of melanin in comparison to fluorine 18 fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT). Materials and Methods The study was approved by the institutional animal care and use committee. Sequential MSOT was performed in a mouse B16F10 melanoma limb lymph node metastasis model (n = 13) to survey the development of macro-, micro- and in-transit metastases (metastases that are in transit from the primary tumor site to the local nodal basin) in vivo. The in vitro limit of detection was assessed in a B16F10 cell phantom. Signal specificity was determined on the basis of a simultaneous lymphadenitis (n = 4) and 4T1 breast cancer lymph metastasis (n = 2) model. MSOT was compared with intravenous FDG PET/CT. The diagnosis was assessed with histologic examination. Differences in the signal ratio (metastatic node to contralateral limb) between the two modalities were determined with the two-tailed paired t test. Results The mean signal ratios acquired with MSOT in micrometastases (2.5 ± 0.3, n = 6) and in-transit metastases (8.3 ± 5.8, n = 4) were higher than those obtained with FDG PET/CT (1.1 ± 0.5 [P < .01] and 1.3 ± 0.6 [P < .05], respectively). MSOT was able to help differentiate even small melanoma lymph node metastases from the other lymphadenopathies (P < .05 for both) in vivo, whereas FDG PET/CT could not (P > .1 for both). In vitro, the limit of detection was at an approximate cell density of five cells per microliter (P < .01). Conclusion MSOT enabled detection of melanoma lymph node micrometastases and in-transit metastases undetectable with FDG PET/CT and helped differentiate melanoma metastasis from other lymphadenopathies. (©) RSNA, 2016 Online supplemental material is available for this article.

Silica nanoparticles as substrates for chelator-free labeling of oxophilic radioisotopes.

Chelator-free nanoparticles for intrinsic radiolabeling are highly desirable for whole-body imaging and therapeutic applications. Several reports have successfully demonstrated the principle of intrinsic radiolabeling. However, the work done to date has suffered from much of the same specificity issues as conventional molecular chelators, insofar as there is no singular nanoparticle substrate that has proven effective in binding a wide library of radiosotopes. Here we present amorphous silica nanoparticles as general substrates for chelator-free radiolabeling and demonstrate their ability to bind six medically relevant isotopes of various oxidation states with high radiochemical yield. We provide strong evidence that the stability of the binding correlates with the hardness of the radioisotope, corroborating the proposed operating principle. Intrinsically labeled silica nanoparticles prepared by this approach demonstrate excellent in vivo stability and efficacy in lymph node imaging.

A "schizophotonic" all-in-one nanoparticle coating for multiplexed SE(R)RS biomedical imaging.

SERS nanoprobes for in vivo biomedical applications require high quantum yield, long circulation times, and maximum colloidal stability. Traditional synthetic routes require high metal-dye affinities and are challenged by unfavorable electrostatic interactions and limited scalability. We report the synthesis of a new near-IR active poly(N-(2-hydroxypropyl) methacrylamide) (pHPMA). The integration of various SERS reporters into a biocompatible polymeric surface coating allows for controlled dye incorporation, high colloidal stability, and optimized in vivo circulation times. This technique allows the synthesis of very small (<20 nm) SERS probes, which is crucial for the design of excretable and thus highly translatable imaging agents. Depending on their size, the "schizophotonic" nanoparticles can emit both SERS and fluorescence. We demonstrate the capability of this all-in-one gold surface coating and SERS reporter for multiplexed lymph-node imaging.

Correction: Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation.

Correction for 'Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation' by Chrysafis Andreou et al., Nanoscale Horiz., 2022, 7, 1540-1552, https://doi.org/10.1039/d2nh00331g.

Near-Infrared Phototheranostic Iron Pyrite Nanocrystals Simultaneously Induce Dual Cell Death Pathways via Enhanced Fenton Reactions in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is considered more aggressive with a poorer prognosis than other breast cancer subtypes. Through systemic bioinformatic analyses, we established the ferroptosis potential index (FPI) based on the expression profile of ferroptosis regulatory genes and found that TNBC has a higher FPI than non-TNBC in human BC cell lines and tumor tissues. To exploit this finding for potential patient stratification, we developed biologically amenable phototheranostic iron pyrite FeS2 nanocrystals (NCs) that efficiently harness near-infrared (NIR) light, as in photovoltaics, for multispectral optoacoustic tomography (MSOT) and photothermal ablation with a high photothermal conversion efficiency (PCE) of 63.1%. Upon NIR irradiation that thermodynamically enhances Fenton reactions, dual death pathways of apoptosis and ferroptosis are simultaneously triggered in TNBC cells, comprehensively limiting primary and metastatic TNBC by regulating p53, FoxO, and HIF-1 signaling pathways and attenuating a series of metabolic processes, including glutathione and amino acids. As a unitary phototheranostic agent with a safe toxicological profile, the nanocrystal represents an effective way to circumvent the lack of therapeutic targets and the propensity of multisite metastatic progression in TNBC in a streamlined workflow of cancer management with an integrated image-guided intervention.

Molecular body imaging: MR imaging, CT, and US. Part II. Applications.

Molecular imaging is expected to have a major impact on the early diagnosis of diseases and disease monitoring in the next decade. Traditionally, nuclear imaging techniques have been the mainstay of molecular imaging in the clinical arena. However, with continued development of molecularly targeted contrast agents for nonnuclear imaging techniques such as magnetic resonance (MR), computed tomography (CT), and ultrasonography (US), the spectrum of clinical molecular imaging applications is expanding. In the second part of this review series, an overview of applications of molecular MR imaging-, CT-, and US-based imaging strategies that show promise for clinical translation is presented, and key challenges that need to be addressed to successfully translate these promising techniques in the future are discussed. © RSNA, 2012.

Molecular body imaging: MR imaging, CT, and US. part I. principles.

Molecular imaging, generally defined as noninvasive imaging of cellular and subcellular events, has gained tremendous depth and breadth as a research and clinical discipline in recent years. The coalescence of major advances in engineering, molecular biology, chemistry, immunology, and genetics has fueled multi- and interdisciplinary innovations with the goal of driving clinical noninvasive imaging strategies that will ultimately allow disease identification, risk stratification, and monitoring of therapy effects with unparalleled sensitivity and specificity. Techniques that allow imaging of molecular and cellular events facilitate and go hand in hand with the development of molecular therapies, offering promise for successfully combining imaging with therapy. While traditionally nuclear medicine imaging techniques, in particular positron emission tomography (PET), PET combined with computed tomography (CT), and single photon emission computed tomography, have been the molecular imaging methods most familiar to clinicians, great advances have recently been made in developing imaging techniques that utilize magnetic resonance (MR), optical, CT, and ultrasonographic (US) imaging. In the first part of this review series, we present an overview of the principles of MR imaging-, CT-, and US-based molecular imaging strategies.

Multiplexed imaging in oncology.

In oncology, technologies for clinical molecular imaging are used to diagnose patients, establish the efficacy of treatments and monitor the recurrence of disease. Multiplexed methods increase the number of disease-specific biomarkers that can be detected simultaneously, such as the overexpression of oncogenic proteins, aberrant metabolite uptake and anomalous blood perfusion. The quantitative localization of each biomarker could considerably increase the specificity and the accuracy of technologies for clinical molecular imaging to facilitate granular diagnoses, patient stratification and earlier assessments of the responses to administered therapeutics. In this Review, we discuss established techniques for multiplexed imaging and the most promising emerging multiplexing technologies applied to the imaging of isolated tissues and cells and to non-invasive whole-body imaging. We also highlight advances in radiology that have been made possible by multiplexed imaging.

Visualizing surface marker expression and intratumoral heterogeneity with SERRS-NPs imaging.

Cell surface marker expression in tumors dictates the selection of therapeutics, therapy response, and survival. However, biopsies are invasive, sample only a small area of the tumor landscape and may miss significant areas of heterogeneous expression. Here, we investigated the potential of antibody-conjugated surface-enhanced resonance Raman scattering nanoparticles (SERRS-NPs) to depict and quantify high and low tumoral surface marker expression, focusing on the surface markers epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in an intracerebral and peripheral setting with an inter- and intratumoral comparison of Raman signal intensities. Methods: ICR-Prkdc mice were injected with glioblastoma, epidermoid carcinoma, or breast tumor cell lines intracerebrally and peripherally. SERRS-NPs were functionalized with cetuximab or trastuzumab and administered via tail vein injection. Raman imaging was performed 18 hours post-injection in excised tumors and in vivo through the skull. Tumors were then fixed and processed for immunohistochemical evaluation. Results: Confirmed by MRI and immunohistochemistry for EGFR and HER2, our results demonstrate that antibody-conjugated SERRS-NPs go beyond the delineation of a tumor and offer clear and distinct Raman spectra that reflect the distribution of the targeted surface marker. The intensity of the SERRS-NP signal accurately discriminated high- versus low-expressing surface markers between tumors, and between different areas within tumors. Conclusion: Biopsies can be highly invasive procedures and provide a limited sample of molecular expression within a tumor. Our nanoparticle-based Raman imaging approach offers the potential to provide non-invasive and more comprehensive molecular imaging and an alternative to the current clinical gold standard of immunohistochemistry.

Design and synthesis of gold nanostars-based SERS nanotags for bioimaging applications.

Surface-enhanced Raman spectroscopy (SERS) nanotags hold a unique place among bioimaging contrast agents due to their fingerprint-like spectra, which provide one of the highest degrees of detection specificity. However, in order to achieve a sufficiently high signal intensity, targeting capabilities, and biocompatibility, all components of nanotags must be rationally designed and tailored to a specific application. Design parameters include fine-tuning the properties of the plasmonic core as well as optimizing the choice of Raman reporter molecule, surface coating, and targeting moieties for the intended application. This review introduces readers to the principles of SERS nanotag design and discusses both established and emerging protocols of their synthesis, with a specific focus on the construction of SERS nanotags in the context of bioimaging and theranostics.

Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation.

Visualizing the presence and distribution of multiple specific molecular markers within a tumor can reveal the composition of its microenvironment, inform diagnosis, stratify patients, and guide treatment. Raman imaging with multiple molecularly-targeted surface enhanced Raman scattering (SERS) nanoprobes could help investigate emerging cancer treatments preclinically or enable personalized treatment assessment. Here, we report a comprehensive strategy for multiplexed imaging using SERS nanoprobes and machine learning (ML) to monitor the early effects of immune checkpoint blockade (ICB) in tumor-bearing mice. We used antibody-functionalized SERS nanoprobes to visualize 7 + 1 immunotherapy-related targets simultaneously. The multiplexed images were spectrally resolved and then spatially segmented into superpixels based on the unmixed signals. The superpixels were used to train ML models, leading to the successful classification of mice into treated and untreated groups, and identifying tumor regions with variable responses to treatment. This method may help predict treatment efficacy in tumors and identify areas of tumor variability and therapy resistance.

Noninvasive Imaging of Cancer Immunotherapy.

Immunotherapy has revolutionized the treatment of several malignancies. Notwithstanding the encouraging results, many patients do not respond to treatments. Evaluation of the efficacy of treatments is challenging and robust methods to predict the response to treatment are not yet available. The outcome of immunotherapy results from changes that treatment evokes in the tumor immune landscape. Therefore, a better understanding of the dynamics of immune cells that infiltrate into the tumor microenvironment may fundamentally help in addressing this challenge and provide tools to assess or even predict the response. Noninvasive imaging approaches, such as PET and SPECT that provide whole-body images are currently seen as the most promising tools that can shed light on the events happening in tumors in response to treatment. Such tools can provide critical information that can be used to make informed clinical decisions. Here, we review recent developments in the field of noninvasive cancer imaging with a focus on immunotherapeutics and nuclear imaging technologies and will discuss how the field can move forward to address the challenges that remain unresolved.

Structurally symmetric near-infrared fluorophore IRDye78-protein complex enables multimodal cancer imaging.

Rationale: Most contemporary cancer therapeutic paradigms involve initial imaging as a treatment roadmap, followed by the active engagement of surgical operations. Current approved intraoperative contrast agents exemplified by indocyanine green (ICG) have a few drawbacks including the inability of pre-surgical localization. Alternative near-infrared (NIR) dyes including IRDye800cw are being explored in advanced clinical trials but often encounter low chemical yields and complex purifications owing to the asymmetric synthesis. A single contrast agent with ease of synthesis that works in multiple cancer types and simultaneously allows presurgical imaging, intraoperative deep-tissue three-dimensional visualization, and high-speed microscopic visualization of tumor margins via spatiotemporally complementary modalities would be beneficial. Methods: Due to the lack of commercial availability and the absence of detailed synthesis and characterization, we proposed a facile and scalable synthesis pathway for the symmetric NIR water-soluble heptamethine sulfoindocyanine IRDye78. The synthesis can be accomplished in four steps from commercially-available building blocks. Its symmetric resonant structure avoided asymmetric synthesis problems while still preserving the benefits of analogous IRDye800cw with commensurable optical properties. Next, we introduced a low-molecular-weight protein alpha-lactalbumin (α-LA) as the carrier that effectively modulates the hepatic clearance of IRDye78 into the preferred renal excretion pathway. We further implemented 89Zr radiolabeling onto the protein scaffold for positron emission tomography (PET). The multimodal imaging capability of the fluorophore-protein complex was validated in breast cancer and glioblastoma. Results: The scalable synthesis resulted in high chemical yields, typically 95% yield in the final step of the chloro dye. Chemical structures of intermediates and the final fluorophore were confirmed. Asymmetric IRDye78 exhibited comparable optical features as symmetric IRDye800cw. Its well-balanced quantum yield affords concurrent dual fluorescence and optoacoustic contrast without self-quenching nor concentration-dependent absorption. The NHS ester functionality modulates efficient covalent coupling to reactive side-chain amines to the protein carrier, along with desferrioxamine (DFO) for stable radiolabeling of 89Zr. The fluorophore-protein complex advantageously shifted the biodistribution and can be effectively cleared through the urinary pathway. The agent accumulates in tumors and enables triple-modal visualization in mouse xenograft models of both breast and brain cancers. Conclusion: This study described in detail a generalized strategic modulation of clearance routes towards the favorable renal clearance, via the introduction of α-LA. IRDye78 as a feasible alternative of IRDye800cw currently in clinical phases was proposed with a facile synthesis and fully characterized for the first time. This fluorophore-protein complex with stable radiolabeling should have great potential for clinical translation where it could enable an elegant workflow from preoperative planning to intraoperative deep tissue and high-resolution image-guided resection.

DNA Nanostructures and DNA-Functionalized Nanoparticles for Cancer Theranostics.

In the last two decades, DNA has attracted significant attention toward the development of materials at the nanoscale for emerging applications due to the unparalleled versatility and programmability of DNA building blocks. DNA-based artificial nanomaterials can be broadly classified into two categories: DNA nanostructures (DNA-NSs) and DNA-functionalized nanoparticles (DNA-NPs). More importantly, their use in nanotheranostics, a field that combines diagnostics with therapy via drug or gene delivery in an all-in-one platform, has been applied extensively in recent years to provide personalized cancer treatments. Conveniently, the ease of attachment of both imaging and therapeutic moieties to DNA-NSs or DNA-NPs enables high biostability, biocompatibility, and drug loading capabilities, and as a consequence, has markedly catalyzed the rapid growth of this field. This review aims to provide an overview of the recent progress of DNA-NSs and DNA-NPs as theranostic agents, the use of DNA-NSs and DNA-NPs as gene and drug delivery platforms, and a perspective on their clinical translation in the realm of oncology.