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Intraoperative Detection and Eradication of Residual Microtumors with Gap-Enhanced Raman Tags Yuanyuan Qiu, Yuqing Zhang, Mingwang Li, Gaoxian Chen, Chenchen Fan, Kai Cui, Jian-Bo Wan, Anpan Han, Jian Ye, and Zeyu Xiao ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02681 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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Intraoperative Detection and Eradication of Residual Microtumors with Gap-Enhanced Raman Tags Yuanyuan Qiu,†,‡,# Yuqing Zhang,¶,# Mingwang Li,†,‡, # Gaoxian Chen,†,‡ Chenchen Fan,†,‡ Kai Cui,†,‡ Jian-Bo Wan,Δ Anpan Han,£ Jian Ye,*,¶,|| Zeyu Xiao*,†,‡,§,※ †Department of Pharmacology and Chemical Biology, and ‡Translational Medicine Collaborative Innovation Center, Institute of Medical Sciences, School of Medicine, ¶State Key Laboratory of Oncogenes and Related Genes, School of Biomedical Engineering, §Institute of Molecular Medicine, and ||Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, School of Medicine, ※Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, P. R. China ΔState Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao, China £DTU Danchip/CEN, Technical University of Denmark, Kgs. Lyngby, Denmark

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ABSTRACT

The inability to intraoperatively diagnose and eliminate microscopic residual tumors represents a significant challenge in cancer surgery. These residual microtumors cause lethal recurrence and metastasis. Herein, we show a crucial example of Raman imaging with gap-enhanced Raman tags (GERTs) to serve as a robust platform for intraoperative detection and eradication of residual microscopic foci, which exist in surgical margins, tumor invasion, and multifocal tumor spread. The GERTs feature gap-enhanced gold core-shell nanostructures, with Raman reporters embedding inside the interior gap junction. This nanostructure elicits highly sensitive and photostable Raman signals for microtumor detection by applying a 785-nm, low-energy laser, and produces hyperthermia effects for microtumor ablation upon switching a 808-nm, high-power laser. In the orthotopic prostate metastasis tumor model, systematic delivery of GERTs enabled precise imaging and real-time ablation of macroscopic malignant lesions around surgical bed, without damaging normal tissues. Consequently, the GERTs-based surgery prevented local recurrence and delivered 100% tumor-free survival. These results suggest the efficiency of theranostic GERTs for precise detection and removal of residual miroctumors, broadening the avenues to apply Raman-based imaging for theranostic precision medicine.

KEYWORDS: nanomedicine, gap-enhanced Raman tag, cancer theranostics, residual microtumors, surface-enhanced Raman spectroscopy


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Despite continuous development in oncosurgery technologies, it remains a great challenge to accurately diagnose and completely eliminate residual microtumors.1 In detail, current clinical strategies mainly rely on imaging technologies, such as positron emission tomography (PET) and magnetic resonance imaging (MRI), to preoperatively determine the location of macroscopic tumors;2, 3 however, the preoperative images of tumors are usually incongruent with their actual locations due to tissues shift during surgery, leading to inaccurate or insensitive detection of microscopic tumor deposits.4, 5 Additionally, in many aggressive or metastasis cancers, tumors are poorly demarcated and indistinctly distributed, and tumor growth infiltrates into surrounding crucial organs or neurological structures.6 In these circumstances, even for an experienced surgeon, it is almost impossible to visualize and resect these microtumors, which comprise of merely tens of tumor cells. These microscopic residual tumor deposits represent a major reason for local recurrence and metastatic spread.7 To remove such microscopic foci, current clinical strategies rely on either intraoperative resection of large margins of normal tissues, or postoperative adjuvant chemo- and radio- therapies;8, 9 nevertheless, these strategies reduce patients’ quality of life, increase morbidity and treatment cost, and elicit severe adverse effects.10, 11 Therefore, there are compelling reasons to establish intraoperative strategies for precise delineation and complete elimination of residual microtumors without causing side effects. Surface-enhanced Raman scattering (SERS)-based imaging offers enormous opportunities to achieve intraoperative microtumor detection with higher specificity and sensitivity, compared to other imaging techniques (e.g., MRI, fluorescence, PET and ultrasound).12-17 Due to the “fingerprint” scattering signals of reporter molecules,18-25 Raman imaging enables specifically distinguish the reporter molecule-contained tumors from the surrounding normal tissues. The Raman scattering of reporter molecules can be massively ampliﬁed by interacting with SERS nanoprobes, allowing for sensitive tumor diagnosis with 10-9–10-12 M limits of detection. Previous research has focused on employing SERS for intraoperative imaging-guided surgical resection of bulk solid tumors with clear boundary,7, 19 while none of investiga-


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tions illustrate its application in intraoperative detection and eradication of unresectable residual microtumors, which are existed after surgical resection of bulk sold tumors or metastasized to adjacent crucial organs. To achieve this goal, the SERS nanoprobes are desirable to possess the following features: (i) ultrahigh sensitivity for delineating the very few residual mictumor cells; (ii) ultrahigh photostability to maintain the Raman signals constant in the harsh tumor environments even after systematic circulation and long-term imaging process, thus enabling accurate detection of sparsely-distributed microtumor lesions; and (iii) simultaneous incorporation of therapeutic functionality without interfering the Raman signals, allowing for intraoperative real-time eradication of the detected residual microtumors. To the best of our knowledge, it remains challenging for currently available SERS nanoprobes to meet these criteria. Herein, we explore a SERS nanoprobe, termed as “gap-enhanced Raman tag (GERT)”, capable of fulfilling these features and achieving intraoperatively precise Raman imaging and complete eradication of residual microtumors. The GERTs are made of Au core-shell particles, embedded with Raman reporters inside the gap junction and coated with a mesoporous silica layer.26-28 The gap junction of GERTs creates a great amount of electromagnetic and chemical hot spots for ultrahigh sensitivity,26, 27 thus enabling mapping tumor cells with a high resolution and contrast. Additionally, different from current SERS nanoprobes that attach Raman reporters on their surface, GERTs build in Raman reporters inside the gap junction, which protects the reporters from the harsh surrounding environments (e.g., blood circulation and tumor microenvironments), leading to their highly stable signals for precise diagnosis of microscopic satellite tumors. Furthermore, we illustrate hereinafter that GERTs elicit strong photothermal transition effects, capable of efficient ablation of detected microtumors in a real-time manner. With all these features, we construct orthotopic prostate metastasis tumor models, and depict systematic delivery of GERTs for intraoperative detection of residual microtumors existing in infiltrative tumor margins and satellite metastases after removing the bulk solid tumors, and real-time eradication of these residual lesions without damaging adjacent critical tissues (Figure 1). We expect this study would broaden the avenues to apply SERS-based bioimaging towards a wide arrange of theranostics-related biomedical applications. 4 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION Synthesis and Characterization of GERTs. The GERTs were composed of plasmonic Au core-shell structures with a nanometer-sized interior gap and an mesoporous silica layer coating on the surface, illustrated by the transmission electron microscopy (TEM) images (Figure 2A and Figure S1). The Raman reporter 1,4-benzenedithiol (BDT) was embedded into the nanometer-sized gap junction. GERTs showed a hydrodynamic diameter of ~97 nm with a narrow mono-dispersed state (Figure 2B). To confirm the SERS photostability, GERTs were exposed in 100% serum at a laser power density of 4.7 × 105 W/cm2 for 60-min continuous irradiation, and SERS spectra were collected every 2 min. Figure 2C illustrated a representative SERS trajectory of GERTs with stationary signal intensities at their characteristic bands at 1055 and 1555 cm-1, respectively.26 Moreover, the Raman signal intensity of GERTs kept constant during 96-h incubation in serum (Figure 2D) and their hydrodynamic size remained invariant in the F-12k cell culture media with 10% serum for 24 h (Figure S2). Meanwhile, we investigated the change of surface properties of GERTs in fresh mouse plasma at 1 min, 4h and 24 h, respectively. As shown in Figure S3, the hydrodynamic size of GERTs increased from 91 nm to 143 nm at 1 min, indicating an immediate biofouling upon injection. After that, the particle size were kept stable at ~ 143 nm till 24 h for efficient tumor accumulation. Similarly, the zeta potential of GERTs changed from -21.5 mV to -11.5 mV, and subsequently recovered to -17.6 mV within 4 h and remained unchanged till 24 h, further confirming the stability of GERTs. In addition, GERTs showed good stability in physiological saline in our previous study.26 It can be also found that the Raman signal intensity of GERTs from different batches was quite uniform (Figure S4). Therefore, the ultrahigh photostability property of GERTs indicates their great potential for real time, repeated and long term intraoperative imaging to detect microscopic residual tumors. By measuring the signals of GERTs with a series of concentrations from 0 ~ 100 pM in the agarose phantom, the Raman detection threshold of GERTs was determined to be approximately 20 fM, at 2.2 × 105 W/cm2 laser power density, 1.86-s exposure time, and 5× objective (Figure 2E). We then performed Raman imaging of ex vivo tumors containing GERTs at increasing depths, and the penetration depth could 5 ACS Paragon Plus Environment

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reach approximately 2.67 mm (Figure 2F). This is sufficient for the diagnostics of microscopic residual tumors in surgical margins that are normally in the range of 1-2 mm depth.1 Delineation of Tumor Cells Using GERTs. To appraise the capability of GERTs for cell-based Raman imaging, PC3 cells were incubated with GERTs for 5 h, washed three times, and then fixed for Raman imaging. As shown in Figure 3, GERTs were extensively internalized and distributed inside the cell, thus generating strong Raman signals to portray the cell boundary. The overlap of Raman and brightfield images further confirmed the coincidence of Raman signals with the distribution of GERTs inside the cell. The representative three Raman spectra showed the different positions inside or outside the cell (Figure 3). Raman spectra obtained from the position of I and II inside the cell revealed pronounced Raman fingerprint spectrum of BDT, with two characteristic bands at 1055 and 1555 cm-1. In contrast, neither of these two peaks appeared in the Raman spectrum from the position III outside the cells. The capability of GERTs for cell imaging was further confirmed by Raman mapping of large number of cells from three random areas (Figure S5). Notably, GERTs are actively internalized into cells rather than binding to cells,29 thus yielding stronger Raman signals. Taken together, GERTs can detect tumor cells with high sensitivity and selectivity, indicating their potential of delineating microscopic residual tumors composed of very few cells. Photothermal Effects of GERTs. Upon continuous laser irradiation (808 nm, 3.6 W/cm2), GERTs elicited decent photothermal transition effects. In Figures 4A and 4B, the temperature of GERTs suspension demonstrated an obvious increase from 25.7 to 72.5 C in 3 min monitored by a thermal camera. In contrast, pure water exhibited a negligible temperature increase under the same experimental condition. Such photothermal heating effect escalated with increasing the concentration and radiant energy of GERTs (Figures S6A and S6B). To evaluate the penetration depth achieved by photothermal effects, we performed laser irradiation on a tissue-mimicking phantom containing GERTs at depths ranging from 0 to 9.63 mm. As shown in Figure S6C, GERTs buried in the phantom demonstrated a depth-dependent 6 ACS Paragon Plus Environment

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temperature increase, while control group without GERTs exhibited a negligible temperature increase. In particular, GERTs were able to induce a temperature increase up to 42 ℃ upon penetrating into 6.88 mm. Thus, tumors invaded within a depth of 6.88 mm may be effectively eliminated via the photothermal effects of GERTs; which is sufficient for eradicating residual microtumors in surgical margins that are normally in the range of 1-2 mm depth. To further investigate the photothermal properties of GERTs, we calculated the photothermal conversion efficiency (η) of the GERTs solution with Roper’s method.30 Briefly, the 808-nm laser was applied to continuously irradiate GERTs solution till reaching a steady temperature, and then was stopped to cool down the solution naturally. The temperature decrease was then monitored to determine the heat transfer rate between the system and the environment during this cooling stage (Figure 4C and 4D). Consequently, the η of GERTs was calculated to be 33.8%.30-32 In addition, GERTs exhibited good stability in photothermal transition, as a negligible change of temperature elevation was observed after five repeated laser on and off cycles (Figure S6D). Notably, phothothermal transition of gold nanoparticles would lead to the migration of outer gold atoms.33 This migration results in the morphological change of some gold nanoparticles (e.g., gold nanostars or gold nanorods) into spherical shape (or “melt” as mentioned in previous reports), thus impairing their photothermal conversion efficiency.34 Distinctly, GERTs are already spherical particles and well protected by mesoporous silica, and thus photothermal transition-induced migration of outer gold atoms would have little effect on their spherical morphology, contributing to the superior photothermal transition stability of GERTs. We investigated the photothermal ablation of GERTs to cancer cells, by a 5 h-incubation of GERTs with PC3 cells and then a subsequent 5-min irradiation by an 808 nm laser at 3.6 W/cm2. GERTs exhibited grievous cell damage under irradiation in a dose-dependent manner, while they showed negligible cytotoxicity without irradiation (Figure 4E). The photothermal transition-based ablation effects of GERTs were further verified by staining live cells with Calcein AM (green color) and labeling dead cells with propidiumiodide (PI, red color). As shown in Figure 4F, cells treated with GERTs and laser irradiation


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suffered a significant cell death, while all the cells kept alive with treatment of GERTs or laser irradiation barely. As such, GERTs could serve as ideal photothermal agents for eradicating tumor cells. Biodistribution and Tumor-Targeted Delivery of GERTs. We then assessed the biodistribution of GERTs in vivo. Mice bearing orthotopic prostate tumors were injected with GERTs via intravenous administration. At different time points after injection, blood samples were collected to determine their Au content with atomic absorption spectrometry. In Figure S7, GERTs showed a blood retention of approximately 2.0 ±0.5% and 0.7 ±0.2% ID/g (percentage of the injected dose per gram of blood) at 8 h and 24 h post-injection, respectively. The corresponding circulation half-life of GERTs was calculated to be 2.3 h. The biodistribution of GERTs was evaluated in orthotopic prostate tumor-bearing mice at 20 h postinjection via intravenous administration. As shown in Figure 5A, GERTs exhibited a good passive targeting capability, due to the enhanced permeability and retention (EPR) effects in tumors with leaky vasculatures.19, 35 Approximately 56.1 ± 7.8 μg/g (Au mass per gram of tissue) of Au was found in the tumor at 20 h post-injection (Figure 5A). The remaining GERTs were taken up predominantly by the liver and spleen, with a Au level of 23.2 ±3.7 and 19.1 ± 3.4 μg/g, respectively. Other organs including heart, lung, kidney and intestine were found with a Au level smaller than 4 μg/g. Subsequently, we investigated tumor-targeted delivery of GERTs. Mice bearing orthotopic prostate tumors were intravenously injected with GERTs. After 20 h, the tumor was resected and fixed for Raman imaging. As shown in Figure 5B, Raman imaging consistently depicted the excised tumor tissue harboring GERTs. Notably, Raman spectrum from the position I of the tumor showed two characteristic Raman bands at 1055 and 1555 cm-1, thus distinguishing tumor from the background (spectrum II) (Figure 5C). Hematoxylin and eosin (H&E) staining further corroborated Raman signal-positive areas to represent prostate tumors, characterized by the existence of prostate tumor nodules, a high nuclear-to-cytoplasmic ratio, cellular crowding, and a necrotic core (Figure 5D). The TEM image confirmed the accumulation of GERTs inside the tumor tissue (Figure 5D). 8 ACS Paragon Plus Environment

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To further illustrate the specificity of GERTs in sketching out prostate tumors rather than surrounding normal tissues, we intravenously injected GERTs into mice without bearing tumor as the control. After 20 h-blood circulation, Raman imaging was performed on the whole prostate and bladder tissues. None of the Raman signals related to GERTs were detected in all the mapping area, suggesting inexistence of GERTs in these normal tissues (Figure S8). Meanwhile, Raman spectra acquired from the prostate (arrow I) and the bladder (arrow II) exhibited negligible characteristic Raman band of GERTs at 1555 cm-1. H&E staining confirmed these areas were normal prostate and bladder tissues. Platform Setup for Intraoperative Raman Imaging-Guided Photothermal Ablation of Residual Microtumors. To establish the surgery platform, we connected the anesthesia apparatus to the 10 cm × 12 cm operation table under the upright objective lens. For intraoperative Raman imaging, we employed an upright confocal Raman microscope with a 785 nm diode excitation laser, which transmits the laser beam through the 5×objective to the operation table. For subsequent photothermal ablation, we fixed the 808 nm laser device adjacent to the 5×objective lens, delivering the laser beam to operation table (Figure 6A). To explore the potential of GERTs for intraoperatively real-time Raman imaging and eradication of residual microtumors, we established orthotopic luciferase-transfected prostate metastasis tumors (PC3M-luc-C6) as a model system to mimic the tumor surgical procedure in a clinical scenario. Human metastatic PC3 cells, which represent the late stage of prostate cancers, were orthotopically implanted into the prostate, reproducing the real organ environment for tumor growth and metastatic spread to adjacent tissues. The mice were injected with GERTs intravenously, allowing 20 h-circulation for sufficient passive accumulation in the tumor tissues due to the EPR effect. Surgery was first performed to remove the primary bulk tumor based on visual inspection. After achieving maximal surgical resection of observable tumors, we performed (i) intraoperative detection of the location of residual microtumors with Raman imaging, and (ii) photothermal ablation of residual microtumors guided by Raman imaging.


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Intraoperative Precise Detection of Residual Microtumors Using GERTs. To evaluate the capability of GERTs for intraoperatively real-time delineation of residual microtumor lesions, we performed Raman imaging on the resection bed and surrounding tissues. In Figure 6B, although no residual tumor tissue could be identified by visual inspection, Raman imaging verified multiple submillimeter foci located at the interface of tumor and normal tissues in the resection bed. Raman spectra acquired from such area (arrow 1) exhibited strong signal intensity at 1555 cm-1, while no signal was from the normal bladder tissue (arrow 3). Impressively, GERTs-based Raman imaging possessed the ability to detect the microscopic satellite metastases (arrow 2), which were at substantial distance (0.2 to 5 mm) from the primary tumors, showing distinguishable Raman signals at 1555 cm-1. H&E-staining confirmed the tissues in Raman signal-positive area (arrow 1) to be tumor lesions (right panel), with differential pathological features from normal bladder tissues (left panel) in Raman signal-negative area (arrow 3). Meanwhile, immunohistochemical (IHC) staining showed the overexpression of NSE (neuron-specific enolase) proteins in Raman signal positive foci (arrow 2), confirming the presence of tumor metastases (Figure S9, arrow 2). TEM results further validated the enrichment of GERTs in the residual tumor tissues. Notably, the morphology of GERTs in the tissue section looks different from in water; which may be due to the low contrast of silica layer to distinguish itself from the tissue background, and the aggregation of GERTs inside the tissue. Taken together, GERTs are capable of precise detection of microscopic tumor deposits in a real time manner. To further substantiate the specificity of GERTs in delineating residual tumors rather than adjacent normal tissues, we collected H&E-stained tissue section at the interface of tumor and normal tissue for Raman scanning. The overlay of Raman image with H&E-stained image illustrated strong Raman signals within the tumors (Figure 7, area T) instead of within adjacent normal tissues (Figure 7, area N).


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Currently, a variety of intraoperative imaging technologies have been developed; however, they demonstrate moderate accuracy, specificity and sensitivity for tumor diagnostics. For example, intraoperative MRI requires repeated injections of imaging agents (e.g., gadolinium chelates), thus resulting in signal inaccuracies due to surgically induced false-positive enhancement. 15, 36 Fluorescence imaging has inadequate selectivity, mainly because of autofluorescence from biological tissues and rapid photobleaching of fluorescent molecules.12, 37 Intraoperative ultrasound-based methods (e.g., photoacoustic imaging and ultrasound microbubbles) are limited by their modest specificity, resulting from the intrinsic property of artifacts induced by biological tissues and the destruction of the contrast agent during imaging.1, 38 Intraoperative PET is not sensitivity enough for the detection of submillimeter microtumors, due to the limited spatial resolution (generally ＞1mm).17, 39 Alternatively, GERTs-based Raman imaging achieves high selectivity and accuracy owing to the “fingerprint” signals of Raman imaging, and attains high sensitivity through the signal amplification by GERTs.40-42 In particular, Raman spectroscopy has been allowed to use in the clinical trial and new Raman imaging devices are continually being developed.14, 43 All of the above, together with clinically validated modalities (e.g., gold nanoparticles and NIR laser), suggest the translational potential of SERS-based Raman imaging for intraoperative medical applications. Intraoperative Eradication of Residual Microtumors Using GERTs. To elucidate whether hyperthermia of GERTs realize intraoperative eradication of residual tumor lesions, we irradiated the Raman signal-positive areas for 5 min using a 808-nm laser at the laser power density of 3.6 W/cm2, and captured the infrared images of whole mice during entire therapy process (Figure 8A). PBS group showed a negligible temperature increment of < 1 °C after the same laser irradiation treatment, revealing that the NIR laser at such power density could not elicit the hyperthermia to harm normal tissues. For GERTs group, the temperature of irradiation area increased from 23.5 to ~43.4 C at 1.5 min, and continuously raised to ~48 C at 5 min, indicating that the accumulated GERTs inside residual tumor lesions can generate sufficient heat for tumor ablation (usually > 42 C).44-46 Notably, we used the thermal imaging camera to 11 ACS Paragon Plus Environment

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monitor the irradiation process, and maintained the temperature in the range of 42 - 48 C to avoid overheating.45 Furthermore, the guidance of Raman imaging helped to apply hyperthermia in a specific and local manner, thus favorably shortening operation time for intraoperative use. Subsequently, the evaluations of therapeutic efficacy and mice survival confirm our assumption. Mice bearing metastatic orthotopic prostate tumors were split into three groups with various treatments (group 1, PBS; group 2, standard surgery; group 3, standard surgery + Raman imaging-guided hyperthermia). After surgery, the wounds were sutured, and tumor recurrence was continuously monitored by measuring the bioluminescence signals for 16 days (Figure 8B and 8C). Mice injected with PBS without surgical resection (group 1) exhibited sustainable growth of tumor volumes, and metastasis to the liver. Mice treated with standard surgery (group 2) demonstrated diminished tumor signals at 3rd day due to the removal of bulk tumors, and a delayed tumor growth in the first six days; however, tumor relapse dramatically occurred afterwards. Comparatively, mice treated with standard surgery and Raman imaging-guided hyperthermia (group 3) displayed complete tumor elimination without any regrowth during 16-day observation; this is mainly due to the efficient laser-elicited hyperthermia of GERTs, leading to obvious damage to residual microtumors. Additionally, body weight curves of the three groups displayed little difference, revealing no obvious toxicity of GERTs (Figure 8D). Survival curves of mice in three treatment groups further vindicated therapeutic effects. GERTs group under laser irradiation achieved complete tumor-free survival for 100% of the animals after 39 days. In contrast, the other two groups had succumbed to disease (Figure 8E). The favorable outcomes are presumably due to complete elimination of residual tumor lesions without damaging the normal tissues. To further access the toxicity of GERTs-based strategy, the main organs (heart, liver, spleen, lung and kidney) of mice in three groups were collected for H&E staining (Figure 8F, Figure S10) at ~28 day postsurgery. Notably, in the PBS group and surgery-without-laser intervention group, residual tumor le-


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sions were observed to metastasize to the livers in mice, and the spleens manifested dramatic inflammation, possibly due to the residual tumor-provoked deterioration. Comparably, no obvious toxicity of harvest organs was displayed in the GERTs-assistant intervention group, substantiating the biosafety of GERTs-based laser surgery. In our strategy, the GERTs achieve tumor-specific imaging and eradication after systematic administration, without requiring the conjugation of tumor-targeting moieties. Such specific tumor accumulation mainly relies on the EPR effects, reported to exist in most of tumor-bearing animal models.47, 48 Additionally, photothermal transition-based hyperthermia with cell-type universality has been under preclinical investigations for the treatment of a variety of tumor types.44, 49 As such, despite using prostate cancer model in our investigation, we expect that the usage of GERTs may be expanded to intraoperative detection and elimination of other types of tumors. CONCLUSIONS In summary, we explored a Raman imaging strategy using GERTs for intraoperative real-time diagnostics and eradication of residual microtumors. Our results showed that the GERTs specifically identified and eliminated the infiltrative tumor margins and microscopic satellite metastases, leading to complete tumor surgery. Impressively, our intraoperative tumor ablation strategy reduced the postsurgical tumor metastasis to the liver. These findings highly facilitate the utilization of GERTs as a robust platform for residual microtumor-complicated surgeries. We believe that GERTs-based intraoperative detection and eradication of residual microtumors would broaden SERS-based Raman technologies towards a wide arrange of theranostics-related in vivo biomedical applications.


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MATERIALS AND METHODS Preparation and Characterization of GERTs. The synthesis of GERTs was followed as our previous work.26, 50 In brief, 25-nm gold core was synthesized using a seed-mediated method, and then cores modified with BDT molecules were utilized as seeds to grow the gold shell. After that, TEOS (50 μL, 5%) was added to gold nanoparticle solution (5 mL, 1 nM) containing NaOH solution (40 μL, 0.1 M) at a 30min interval for three times. After 24 h, the as-synthesized GERTs were washed with ethanol and redispersed in water. The morphology was observed applying a TEM (JEOL-2010, JEOL, Japan) operated at 200 kV. The hydrodynamic diameter was measured using a laser particle size analyzer (Zetasizer Nano ZSP, Malvern, UK). The absorbance was measured with an enzyme-linked immunosorbent assay kit (Synergy 2, Bio-TEK, USA). Raman spectra were collected using a Renishaw inVia Raman microscope equipped with a 785 nm diode laser and a 1020×256 pixels charge-coupled device detector. In Vitro Detection Limit of GERTs. The agarose phantoms containing GERTs were made to test the detection limit of GERTs.51 Briefly, GERTs at different concentrations were mixed with warm liquid agarose (n = 3 samples for each concentration) to form GERTs-agarose solutions at 0, 0.02, 0.2, 0.4, 0.8, 3.125, 6.25, 12.5, 50 and 100 pM. Subsequently, the GERTs-agarose solution (100 µL for each concentration) was separately poured into the wells of 96-well plates. After solidification of the gel, the Raman images of each agarose phantom were acquired using the Renishaw StreamlineTM function (2.2  105 W/cm2, 5× objective) with 785 nm laser, 312 μm step size and 1.86 s exposure time. Approximately 20 min was spent on Raman imaging of each phantom. Ex Vivo Detection of the Penetration Depth of GERTs. The fresh prostate tumor tissues were obtained from the tumor-bearing mice. GERTs solution (0.05 μL, 1 nM) was injected with a microliter syringe (Hamilton, Switzerland) from the top of tumors at the depths of 0.58, 0.76, 1.2, 1.67, 2, 2.35, 2.67, and 3 mm away from the bottom of the tumor. Subsequently, Raman images of tumor tissues were ac-


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quired from the bottom of tumors using the Renishaw StreamlineTM function (2.2  105 W/cm2, 5×objective) with 785 nm laser, 113.4 μm step size and 1.86 s exposure time. The total Raman imaging period was 6–15 min. A tumor without GERTs was used as a control. In Vitro Photothermal Effect and Cytotoxicity. To test the photothermal effect, GERTs solution (1 nM) in 1.5 mL eppendorf tubes were irradiated with a near infrared (NIR) laser (808 nm, GCSLS-057W00, Daheng Science & Technology, Beijing, China) at different laser powers (2.4, 2.7, 3.0, and 3.6 W/cm2) for 5 min, with a spot size of around 6 mm. The temperature of the GERTs suspensions and thermal images were collected with an infrared (IR) thermal imaging camera (DT-980, CEM Co. Ltd, Shenzhen, China) once every 30 s. The luciferase-transfected human metastasis prostate cancer cell line (PC-3M-luc-C6) was obtained from Caliper Life Sciences. The cells were cultured in Ham's F-12K medium at 37 °C in a humidified atmosphere containing 5% CO2. Cells were seeded in 96-well plates at a density of 104 cells/well for 24 h. Cells were washed with phosphate-buffered saline (PBS) and incubated with GERTs solutions at different concentrations (0, 100, 200, 400, 600 pM) for 5 h. The cells were then rinsed twice with PBS to remove unbound GERTs and replaced with fresh Ham's F-12K medium (40 μL). Subsequently, the experimental groups were treated with the NIR laser (808 nm, 3.6 W/cm2) for 5 min and then incubated for 12 h. Similar procedure was performed in control cell groups without laser irradiation. The viabilities of cells were measured following the standard procedure of CCK8 assay. Photostability, and Photothermal Conversion Efficiency. The photostability of GERTs (1 nM, 200 μL) was assessed by using an 808 nm laser (3.6 W/cm2) for 5-repeated laser on and off cycles at 3 min intervals. To test the photothermal conversion efficiency, 808-nm laser was irradiated to the GERTs solutions (100 μL). After continuous irradiation till reaching a steady temperature, the laser was turned off to naturally cool down the solution. The photothermal conversion efficiency was then calculated according to previous reports, 30-32 and detailed procedures were described in the supporting information. 15 ACS Paragon Plus Environment

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In Vitro Cell Raman Imaging. To delineate cells with Raman imaging, the cells seeded on the quartzbottomed plates for 24 h before a 5 h-incubation with GERTs (20 pM). The cells were then rinsed with PBS extensively and fixed with 4% paraformaldehyde for 15 min. After that, the sample was washed with deionized water and air-dried before Raman scanning. The Raman images of cells were measured with a 785 nm laser and 1.86 s exposure time per pixel (8.5 ×104 W/cm2, 1 μm step size, 100×objective, and 912 min imaging time in total). Raman imaging of multiple cells were performed with a 785 nm laser and 1.86 s exposure time per pixel (8.5 × 104 W/cm2, 2 μm step size , 50× objective, and 35-60 min imaging time in total). Animal Studies. All animal studies were conducted in Animal Resource Center of Shanghai Jiao Tong University School of Medicine in accordance with protocols approved by the Animal Care Committee. Establishment of Orthotopic Prostate Tumor Models. Male SCID mice (20−22 g, 6−8 weeks) were used to establish the orthotopic prostate tumor model.52 In brief, the SCID mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (0.1 mL /10 g) under aseptic and pathogen-free condition in a biosafety hood. Subsequently, a small incision was made at the lower abdomen, and the PC-3M-lucC6 cells (107/mouse, in 10 μL PBS) were injected in the prostate lobe of the mice. Tumor growth was monitored by bioluminescence after intraperitoneal injection of 3 mg of D-Luciferin potassium salt in 200 μL PBS with an in vivo imaging system (IVIS-200, Xenogen, USA). Circulation Half-Life and Biodistribution of GERTs. To assess the circulation half-life, tumor-bearing mice (n=4) were intravenously injected with GERTs solution (100 μL, 1nM). At various time points (i.e., 1, 15, 30 min, and 1, 2, 4, 8, 24 h) post-injection, blood samples (50 μL) were harvested from mouse eyes using heparinized capillary tubes and digested with 1 mL of aqua regia to determine the amount of Au with an atomic absorption spectrometry (AAS). To evaluate the biodistribution, tumor-bearing mice were intravenously injected with GERTs, and then sacrificed after 20-h circulation. Tumors and main


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organs (heart, liver, spleen, lung, kidney and intestines) were obtained, weighed and completely digested with aqua regia (4 mL) for quantitative analysis of Au using AAS. Tumor-Targeted Raman Imaging with GERTs. Prostate tumor-bearing mice were intravenously injected with GERTs solution (100 μL, 1 nM). After 20 h circulation, the prostate tumors were collected for Raman imaging. For comparison, mice without tumor bearing were injected with GERTs (100 μL, 1 nM) intravenously, the normal prostate and bladder were exposed for Raman imaging at 20 h post injection. Raman imaging was performed with an inVia Raman microscope (Renishaw) equipped with a 785 nm diode laser and a 1020×256 pixels charge-coupled device detector. Raman scanning was performed in StreamLine high-speed acquisition mode (2.2 × 105 W/cm2, 5× objective) with 113.4 μm step size and 1.86 s exposure time. Characteristic band of GERTs at 1555 cm-1 was selected for image processing. The Raman images were generated and analyzed by a signal to baseline algorithm (WiRE4.2 software, Renishaw). The total time for Raman mapping is 6 to 12 min. Raman Imaging of Tissue Sections. Tissue sections were placed on quartz slides, followed by Hematoxylin and eosin (H&E) staining. Raman imaging and correlating white light images of these tissue sections were acquired using the Renishaw StreamlineTM function (2.2  105 W/cm2, 20×objective) with 785 nm laser,10 μm step size and 2 s exposure time. The total Raman imaging time was 40-50 min. Intraoperative Raman Detection and Photothermal Ablation of the Residual Microtumors. Orthotopic prostate tumor-bearing mice were injected with GERTs (100 μL, 1 nM) intravenously. After 20h circulation for sufficient GERTs accumulation in the tumor tissues, surgery was performed to remove the primary tumor based on visual inspection. After achieving maximal surgical resection of observable tumors, Raman imaging of residual tumor lesions were then performed with an inVia Raman microscope (Renishaw) equipped with a 785 nm diode laser and a 1020×256 pixels charge-coupled device detector. Raman scanning was performed in StreamLine high-speed acquisition mode (2.2 ×105 W/cm2, 5×objective) with 113.4 μm step size and 1.86 s exposure time. Characteristic band of GERTs at 1555 cm-1 was 17 ACS Paragon Plus Environment

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selected for image processing. The Raman maps were generated and analyzed by a signal to baseline algorithm (WiRE4.2 software, Renishaw). The total time required for Raman mapping area of 42~72 mm2 is 15 to 26 min. Subsequently, the residual tumors detected by Raman imaging were irradiated using an 808-nm laser (3.6 W/cm2) for 5 min. A thermal imaging camera was applied to monitor the temperature increase around tumor area. Once the temperature increases to over 48 C, the laser irradiation was intermittently stopped for 5 s, maintaining the temperature in the range of 42–48 C. After treatment, the regrowth of tumors were monitored by in vivo imaging system (IVIS-200, Xenogen, USA) for 16 days, and the body weight and survival of all animals were recorded over 5 weeks. To evaluate the toxicity of the GERTs, major organs (heart, liver, spleen, lungs, and kidneys) were collected after the death of mice during the experimental period, followed by H&E staining.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Experimental details including stability measurements of GERTs, evaluation of the photothermal depths of GERTs, calculating the photothermal transduction efficiency, calcein AM/PI staining and transmission electron microscopy (TEM) images of tumor tissues. Supporting figures including TEM images of a large number of GERTs, the hydrodynamic size of GERTs in cell culture media with 10% FBS, Raman spectra of GERTs in different batches, Raman images of multiple cells, in vitro photothermal ablation test, blood circulation of GERTs, in vivo Raman image of normal prostate and bladder and H&E staining of the liver and spleen of tumor bearing mice in PBS group.


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AUTHOR INFORMATION Corresponding Authors *Email: [email protected] or [email protected] (Z. X.) *Email: [email protected] (J. Y.) ORCID Zeyu Xiao: 0000-0002-3457-5772 Jian Ye: 0000-0002-8101-8362

Author Contributions # Y.Q. and Y.Z. and M. L. contributed equally to this work. The authors declare no conflict of interest.

ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 81471779, 31671003, 81741014, 21375087, 81571763, and 81622026), Thousand Young Talents Program, the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. TP2014028), Shanghai Jiao Tong University (Nos. YG2016MS51 and YG2017MS54) and Shanghai Key Laboratory of Gynecologic Oncology. Q.Y. acknowledges the Doctoral Innovation Fellowship from Shanghai Jiao Tong University School of Medicine (No. BXJ201704). We gratefully acknowledge Jun Cai from Shanghai Jiao Tong University School of Medicine for analyzing pathological images.


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Figure 1. Schematic illustration of gap-enhanced Raman tags (GERTs) for intraoperative detection and eradication of residual microtumors. GERTs are injected intravenously before the surgery, allowing for their sufficient accumulation inside tumor tissues. After removal of observable bulk tumors, operators initially utilized the 785-nm laser to elicit the Raman signals of GERTs for detecting residual tumor lesions, and then manipulated the 808-nm laser to elicit hyperthermia of GERTs for eradicating residual tumors.

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Figure 2. Characterization of GERTs. (A) Schematic diagram and TEM image of GERTs. The inner gap of GERTs is indicated with the yellow arrow. Scale bar is 50 nm. (B) The hydrodynamic diameter size of GERTs. (C) Photostability measurement of SERS spectra of GERTs in 100% FBS with a continuous 60min irradiation. SERS spectra were captured at a 2-min interval with a 785 nm laser, a power density of 4.7 ×105 W/cm2, a 1.86-s exposure time, and a 5× objective. (D) Serum stability measurement of Raman intensity of GERTs during incubation in 100% FBS for 96 h. Measurements for (E) limit of detection of GERTs in agarose phantom and (F) penetration depth of Raman imaging with GERTs in ex vivo tumors (2.2 × 105 W/cm2 laser power density, 1.86 s exposure time and 5× objective).


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Figure 3. Delineation of a PC3 tumor cell using GERTs. Colors are assigned to the characteristic Raman band of GERTs at 1555 cm− 1. Characteristic Raman bands are indicated with black arrow.


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Figure 4. In vitro photothermal properties of GERTs with an 808 nm-laser at the power density of 3.6 W/cm2. (A) Thermal images of GERTs and water with a continuous 180-s laser irradiation. (B) Temperature change curves of GERTs and water with a continuous 180-s laser irradiation. (C) Temperature elevations of GERTs under 180-s irradiation, and subsequent cooling by terminating the irradiation. (D) Time constant (τs) is calculated to be 113.5 s by analyzing the linear time data from the cooling period of (C) versus negative natural logarithm of driving force temperature (-Ln). (E) Cell viability studies of GERTs on PC3 cells. (** p < 0.01, *** p< 0.001) (F) Calcein AM/PI staining to visualize PC3 cell viability treated with GERTs with or without laser irradiation (808 nm, 3.6 W/cm2 for 5 min). Calcein AM stains live cells (green color) and propidium iodide (PI) stains dead cells (red color).


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Figure 5. Biodistribution and tumor-targeted Raman imaging with GERTs. (A) Biodistribution of GERTs at 20 h post-injection. Data are expressed as the Au mass per gram of the tissue (μg/g). (B) Raman imaging of an excised tumor tissue. Colors are assigned to the characteristic Raman band of GERTs at 1555 cm− 1. (C) Raman spectra acquired from position Ⅰ and Ⅱ. Characteristic Raman bands of GERTs are indicated with black arrow. (D) Hematoxylin and eosin (H&E) staining and TEM images acquired from position Ⅰ. H&E staining validated the existence of tumor cells in the GERTs–positive foci. The brown arrow in the TEM image indicated the existence of GERTs in the tumor tissue.


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Figure 6. (A) Platform setup for intraoperative Raman imaging-guided photothermal ablation of residual microtumors. (B) Intraoperative Raman imaging of residual microtumors after surgical resection of primary tumors. Yellow line indicates the primary tumor, and white dotted line indicates the resection bed after surgical removal of the primary tumor. Raman image clearly shows multiple residual mirotumors on the resection bed (arrow 1) or metastasized to the bladder (arrow 2). Characteristic Raman band of GERTs at 1555 cm− 1 is indicated with black arrow. Arrow 3 indicates the signal from normal bladder tissue. GERTs-positive foci are confirmed to be microscopic tumor cell deposits by H&E staining. The brown arrow in the TEM image indicates the accumulation of GERTs in tumor tissue.


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Figure 7. H&E staining and Raman imaging of the tissue section at the interface of the normal tissue and tumor, indicated by the white dashed line. Area N indicates normal tissue, and area T means the tumor. The Raman signals suggest the presence of GERTs inside the tumor rather than inside the adjacent healthy tissue.


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Figure 8. Intraoperative eradication of residual microtumors. (A) Thermal imaging of tumor-bearing mice after surgical resection of bulk tumor with laser irradiation at indicated time points guided by Raman imaging. (B) Bioluminescence monitoring the regrowth of tumors after different treatments. SCID mice were divided into three treatment groups (n = 5 mice/group): PBS, standard surgery, and standard surgery + intraoperative Raman imaging-guided tumor ablation. Images show the five mice per group. (C) Tumor growth profiles of mice with different treatments according to the biological luminescence imaging. (D) Body weight curves of tumor-bearing mice for each group. (E) Animal survival curves of the three treatment groups. (F) H&E staining of major organs. Black arrows indicate the metastatic lesions in the liver. Spleen in the mice of control group shows dramatic inflammation.


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