Intravital optical imaging for immune cell tracking after photoimmunotherapy with plasmonic gold nanostars.

Bladder cancer has been ranked as one of the most commonly occurring cancers in men and women with approximately half of the diagnoses being the late stage and/or metastatic diseases. We have developed a novel cancer treatment by combining gold nanostar-mediated photothermal therapy with checkpoint inhibitor immunotherapy to treat bladder cancer. Experiment results with a murine animal model demonstrated that our developed photoimmunotherapy therapy is more efficacious than any individual studied treatment. In addition, we used intravital optical imaging with a dorsal skinfold window chamber animal model to study immune responses and immune cell accumulation in a distant tumor following our photoimmunotherapy. The mice used have the CX3CR1-GFP receptor on monocytes, natural killer cells, and dendritic cells allowing us to dynamically track their presence by fluorescence imaging. Our proof-of-principle study results showed that the photoimmunotherapy triggered anti-cancer immune responses to generate anti-cancer immune cells which accumulate in metastatic tumors. Our study results illustrate that intravital optical imaging is an efficient and versatile tool to investigate immune responses and mechanisms of photoimmunotherapy in future studies.

Plasmonic Gold Nanostar-Mediated Photothermal Immunotherapy.

Cancer is among the leading cause of death around the world, causing close to 10 million deaths each year. Significant efforts have been devoted to developing novel technologies that can detect and treat cancer early and effectively to reduce cancer recurrences, treatment costs, and mortality. Gold nanoparticles (GNP) have been given particular attention for its use with photo-induced hyperthermia coupled with novel immunotherapy methods to provide a new platform for highly selective and less invasive cancer treatment. Among the various GNP platforms, gold nanostars (GNS) have a unique star-shaped geometric structure that allows superior light absorption and photothermal heating. This photothermal effect have also been found to amplify the anti-tumor immune response and can be exploited with adjuvant treatments using immune checkpoint inhibitors. This combination treatment known as Synergistic Immuno Photo Nanotherapy (SYMPHONY) has been shown to reverse tumor-mediated immunosuppression and has led to effective and long-lasting immunity against not only primary tumors but also cancer metastasis. This overview highlights the development and applications of GNS-mediated therapy developed in our laboratory for cancer treatment. This paper also presents recent results of experimental studies to illustrate the superior performance of GNS for photothermal treatment applications.

Direct SERDS sensing of molecular biomarkers in plants under field conditions.

Molecular biomarkers such as microRNAs (miRNAs) play important roles in regulating various developmental processes in plants. Understanding these pathways will help bioengineer designing organisms for efficient biomass accumulation. Current methods for RNA analysis require sample extraction and multi-step sample analysis, hindering work in field studies. Recent work in the incorporation of nanomaterials for plant bioengineering research is leading the way of an agri-tech revolution. As an example, surface-enhanced Raman scattering (SERS)-based sensors can be used to monitor RNA in vivo. However, the use of SERS in the field has been limited due to issues with observing Raman signal over complex background. To this end, shifted-excitation Raman difference spectroscopy (SERDS) offers an effective solution to extract the SERS signal from high background based on a physical approach. In this manuscript, we report the first application of SERDS on SERS sensors. We investigated this technique on SERS sensor developed for the detection of a microRNA biomarker, miR858. We tested the technique on in vitro samples and validated the technique by detecting the presence of exogenous miR858 in plants directly under ambient light in a growth chamber. The possibility of moving the detection of nucleic acid targets outside the constraints of laboratory setting enables numerous important bioengineering applications. Such applications can revolutionize biofuel production and agri-tech through the use of nanotechnology-based monitoring of plant growth, plant health, and exposure to pollution and pathogens.

SERS in Plain Sight: A Polarization Modulation Method for Signal Extraction.

Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical spectroscopy offering advantages ranging from "vibrational fingerprints" to multiplexed detection. However, the use of this technique in real-world applications has been limited due to difficulties in detecting inherently weak Raman signals often embedded in strong interfering background signals. A variety of plasmonics-active platforms have been developed to increase Raman signals but are not sufficient to extract weak SERS signals from intense interfering background signals. Herein, we describe a practical method, referred to as polarization modulation-SERS (PM-SERS), which utilizes the polarization dependence of anisotropic SERS-active nanostructures to modulate the plasmonic effect to extract SERS signals and remove background. The modulation is obtained by switching the polarization of the excitation source at a specific frequency involving addition of only few optical components such as liquid crystal polarizers to a typical Raman setup. In this work, we characterized the polarization-dependent response of the SERS substrates fabricated using the oblique angle evaporation (OAV) technique and their response under laser excitation using a polarization modulated source. We demonstrated that the PM-SERS method can extract the analyte weak SERS signals from the strong interfering background signal in different situations, involving a fluorescent sample and a strong background light, and we show the possibility of using PM-SERS at a quasi-real time rate (0.5 Hz). We believe that the PM-SERS method will help expand the translation of applications that utilize SERS-substrates to real-world settings.

Non-invasive sensitive brain tumor detection using dual-modality bioimaging nanoprobe.

Despite decades of efforts, non-invasive sensitive detection of small malignant brain tumors still remains challenging. Here we report a dual-modality 124I-labeled gold nanostar (124I-GNS) probe for sensitive brain tumor imaging with positron emission tomography (PET) and subcellular tracking with two-photon photoluminescence (TPL) and electron microscopy (EM). Experiment results showed that the developed nanoprobe has potential to reach sub-millimeter intracranial brain tumor detection using PET scan, which is superior to any currently available non-invasive imaging modality. Microscopic examination using TPL and EM further confirmed that GNS nanoparticles permeated the brain tumor leaky vasculature and accumulated inside brain tumor cells following systemic administration. Selective brain tumor targeting by enhanced permeability and retention effect and ultrasensitive imaging render 124I-GNS nanoprobe promise for future brain tumor-related preclinical and translational applications.

Gold Nanostars Obviate Limitations to Laser Interstitial Thermal Therapy (LITT) for the Treatment of Intracranial Tumors.

PURPOSE: Laser interstitial thermal therapy (LITT) is an effective minimally invasive treatment option for intracranial tumors. Our group produced plasmonics-active gold nanostars (GNS) designed to preferentially accumulate within intracranial tumors and amplify the ablative capacity of LITT. EXPERIMENTAL DESIGN: The impact of GNS on LITT coverage capacity was tested in ex vivo models using clinical LITT equipment and agarose gel-based phantoms of control and GNS-infused central "tumors." In vivo accumulation of GNS and amplification of ablation were tested in murine intracranial and extracranial tumor models followed by intravenous GNS injection, PET/CT, two-photon photoluminescence, inductively coupled plasma mass spectrometry (ICP-MS), histopathology, and laser ablation. RESULTS: Monte Carlo simulations demonstrated the potential of GNS to accelerate and specify thermal distributions. In ex vivo cuboid tumor phantoms, the GNS-infused phantom heated 5.5× faster than the control. In a split-cylinder tumor phantom, the GNS-infused border heated 2× faster and the surrounding area was exposed to 30% lower temperatures, with margin conformation observed in a model of irregular GNS distribution. In vivo, GNS preferentially accumulated within intracranial tumors on PET/CT, two-photon photoluminescence, and ICP-MS at 24 and 72 hours and significantly expedited and increased the maximal temperature achieved in laser ablation compared with control. CONCLUSIONS: Our results provide evidence for use of GNS to improve the efficiency and potentially safety of LITT. The in vivo data support selective accumulation within intracranial tumors and amplification of laser ablation, and the GNS-infused phantom experiments demonstrate increased rates of heating, heat contouring to tumor borders, and decreased heating of surrounding regions representing normal structures.

Plasmonic nanorod probes' journey inside plant cells for in vivo SERS sensing and multimodal imaging.

Nanoparticle-based platforms are gaining strong interest in plant biology and bioenergy research to monitor and control biological processes in whole plants. However, in vivo monitoring of biomolecules using nanoparticles inside plant cells remains challenging due to the impenetrability of the plant cell wall to nanoparticles beyond the exclusion limits (5-20 nm). To overcome this physical barrier, we have designed unique bimetallic silver-coated gold nanorods (AuNR@Ag) capable of entering plant cells, while conserving key plasmonic properties in the near-infrared (NIR). To demonstrate cellular internalization and tracking of the nanorods inside plant tissue, we used a comprehensive multimodal imaging approach that included transmission electron microscopy (TEM), confocal fluorescence microscopy, two-photon luminescence (TPL), X-ray fluorescence microscopy (XRF), and photoacoustics imaging (PAI). We successfully acquired SERS signals of nanorods in vivo inside plant cells of tobacco leaves. On the same leaf samples, we applied orthogonal imaging methods, TPL and PAI techniques for in vivo imaging of the nanorods. This study first demonstrates the intracellular internalization of AuNR@Ag inside whole plant systems for in vivo SERS analysis in tobacco cells. This work demonstrates the potential of this nanoplatform as a new nanotool for intracellular in vivo biosensing for plant biology.

Optical recognition of constructs using hyperspectral imaging and detection (ORCHID).

Challenges to deep sample imaging have necessitated the development of special techniques such as spatially offset optical spectroscopy to collect signals that have travelled through several layers of tissue. However, these techniques provide only spectral information in one dimension (i.e., depth). Here, we describe a general and practical method, referred to as Optical Recognition of Constructs Using Hyperspectral Imaging and Detection (ORCHID). The sensing strategy integrates (1) the spatial offset detection concept by computationally binning 2D optical data associated with digital offsets based on selected radial pixel distances from the excitation source; (2) hyperspectral imaging using tunable filter; and (3) digital image binding and collation. ORCHID is a versatile modality that is designed to collect optical signals deep inside samples across three spatial (X, Y, Z) as well as spectral dimensions. The ORCHID method is applicable to various optical techniques that exhibit narrow-band structures, from Raman scattering to quantum dot luminescence. Samples containing surface-enhanced Raman scattering (SERS)-active gold nanostar probes and quantum dots embedded in gel were used to show a proof of principle for the ORCHID concept. The resulting hyperspectral data cube is shown to spatially locate target emitting nanoparticle volumes and provide spectral information for in-depth 3D imaging.

Nanoplasmonics Enabling Cancer Diagnostics and Therapy.

In this paper, we highlight several advances our laboratory has developed in the pursuit of cancer diagnostics and therapeutics by integrating plasmonics, photonics, and nanotechnology. We discuss the development and applications of plasmonics-active gold nanostar (GNS), a uniquely shaped nanoparticle with numerous branches that serve to greatly amplify the thermal generation at resonant wavelengths. GNS has also been successfully used in tumor imaging contexts from two-photon fluorescence to surface-enhanced Raman scattering (SERS) sensing and imaging. Finally, GNS has been coupled with immunotherapy applications to serve as an effective adjuvant to immune checkpoint inhibitors. This combination of GNS and immunotherapy, the so called synergistic immuno photo nanotherapy (SYMPHONY), has been shown to be effective at controlling long-lasting cancer immunity and metastatic tumors.

Plasmonic gold nanostars for synergistic photoimmunotherapy to treat cancer.

Cancer is the second leading cause of death and there is an urgent need to improve cancer management. We have developed an innovative cancer therapy named Synergistic Immuno Photothermal Nanotherapy (SYMPHONY) by combining gold nanostars (GNS)-mediated photothermal ablation with checkpoint inhibitor immunotherapy. Our previous studies have demonstrated that SYMPHONY photoimmunotherapy not only treats the primary tumor but also dramatically amplifies anticancer immune responses in synergy with checkpoint blockade immunotherapy to treat remote and unresectable cancer metastasis. The SYMPHONY treatment also induces a 'cancer vaccine' effect leading to immunologic memory and prevents cancer recurrence in murine animal models. This manuscript provides an overview of our research activities on the SYMPHONY therapy with plasmonic GNS for cancer treatment.

Plasmonic assay for amplification-free cancer biomarkers detection in clinical tissue samples.

Developing countries have seen a rise in cancer incidence and are projected to harbor three-quarters of all cancer-related mortality by 2030. While disproportionally affected by the burden of cancer, these regions are ill-equipped to handle the diagnostic caseload. The low number of trained pathologists per capita results in delayed diagnosis and treatment, ultimately contributing to increased mortality rates. To address this issue, we developed a point-of-care (POC) plasmonic assay for direct detection of cancer as an alternative to pathological review. Whereas our assay has general applicability in many cancer diagnoses that involve tissue biopsies, we use head and neck cancer (HNC) as a model system because these tumors are increasingly prevalent in lower-income and underserved regions, due to risk factors such as smoking, drinking, and viral infection. Our method uses surface-enhanced Raman scattering (SERS) to detect unique RNA biomarkers from human biopsy samples without the need for complex target amplification machinery (e.g., PCR), making it time and resource-efficient. Unlike previous studies that required target amplification, this work represents a significant advance for HNC diagnosis directly in clinical samples, using only our SERS-based assay for RNA biomarkers. In this study, we tested our assay on 20 clinical samples, demonstrating the accuracy of the method in the diagnosis of head and neck squamous cell carcinoma. We reported sensitivity of 100% and specificity of 97%. Furthermore, we used a handheld Raman device to read the results in order to illustrate the applicability of our method for POC diagnosis of cancer in low-resource settings.

Plasmonic gold nanostar-mediated photothermal immunotherapy for brain tumor ablation and immunologic memory.

Brain tumors present unique therapeutic challenges and they include glioblastoma (GBM) and metastases from cancers of other organs. Current treatment options are limited and include surgical resection, radiation therapy, laser interstitial thermal therapy and chemotherapy. Although much research has been done on the development of immune-based treatment platforms, only limited success has been demonstrated. Herein, we demonstrate a novel treatment of GBM through the use of plasmonic gold nanostars (GNS) as photothermal inducers for synergistic immuno photothermal nanotherapy (SYMPHONY), which combines treatments using gold nanostar and laser-induced photothermal therapy with checkpoint blockade immunotherapy. In the treatment of a murine flank tumor model with the CT-2A glioma cell line, SYMPHONY demonstrated the capability of producing long-term survivors that rejects rechallenge with cancer cells, heralding the successful emergence of immunologic memory. This study is the first to investigate the use of this novel therapy for the treatment of GBM in a murine model.

Manipulation of the Geometry and Modulation of the Optical Response of Surfactant-Free Gold Nanostars: A Systematic Bottom-Up Synthesis.

Among plasmonic nanoparticles, surfactant-free branched gold nanoparticles have exhibited exceptional properties as a nanoplatform for a wide variety of applications ranging from surface-enhanced Raman scattering sensing and imaging applications to photothermal treatment and photoimmunotherapy for cancer treatments. The effectiveness and reliability of branched gold nanoparticles in biomedical applications strongly rely on the consistency and reproducibility of physical, chemical, optical, and therapeutic properties of nanoparticles, which are mainly governed by their morphological features. Herein, we present an optimized bottom-up synthesis that improves the reproducibility and homogeneity of the gold-branched nanoparticles with desired morphological features and optical properties. We identified that the order of reagent addition is crucial for improved homogeneity of the branched nature of nanoparticles that enable a high batch-to-batch reproducibility and reliability. In addition, a different combination of the synthesis parameters, in particular, additive halides and concentration ratios of reactive Au to Ag and Au to Au seeds, which yield branched nanoparticle of similar localized surface plasmon resonances but with distinguishable changes in the dimensions of the branches, was realized. Overall, our study introduces the design parameters for the purpose-tailored manufacturing of surfactant-free gold nanostars in a reliable manner.

Direct Detection of Unamplified Pathogen RNA in Blood Lysate using an Integrated Lab-in-a-Stick Device and Ultrabright SERS Nanorattles.

Direct detection of genetic biomarkers in body fluid lysate without target amplification will revolutionize nucleic acid-based diagnostics. However, the low concentration of target sequences makes this goal challenging. We report a method for direct detection of pathogen RNA in blood lysate using a bioassay using surface-enhanced Raman spectroscopy (SERS)-based detection integrated in a "lab-in-a-stick" portable device. Two levels of signal enhancement were employed to achieve the sensitivity required for direct detection. Each target sequence was tagged with an ultrabright SERS-encoded nanorattle with ultrahigh SERS signals, and these tagged target sequences were concentrated into a focused spot for detection using hybridization sandwiches with magnetic microbeads. Furthermore, the washing process was automated by integration into a "lab-in-a-stick" portable device. We could directly detect synthetic target with a limit of detection of 200 fM. More importantly, we detected plasmodium falciparum malaria parasite RNA directly in infected red blood cells lysate. To our knowledge, this is the first report of SERS-based direct detection of pathogen nucleic acid in blood lysate without nucleic acid extraction or target amplification. The results show the potential of our integrated bioassay for field use and point-of-care diagnostics.