Multifunctional gap-enhanced Raman tags for preoperative and intraoperative cancer imaging

Abstract

Multi-modality imaging agents are desirable for tumor diagnosis because they can provide more alternative and reliable information for accurate detection and therapy of diseases than single imaging technique. However, most reported conventional imaging agents have not been found to successfully overcome the disadvantages of traditional diagnoses such as sensitivity, spatial resolution, short half-decay time and complexity. Therefore, exploring a multifunctional nanocomposite with the combination of their individual modality characteristics has great impact on preoperative imaging and intraoperative diagnosis of cancer. In our study, mesoporous silica gadolinium-loaded gap-enhanced Raman tags (Gd-GERTs) specifically for preoperative and intraoperative imaging are designed and their imaging capability and biosafety are examined. They exhibit strong attenuation property for computed X-ray tomography (CT) imaging, high T₁ relaxivity for magnetic resonance (MR) imaging capability and surface-enhanced Raman spectroscopy (SERS) signal with good dispersity and stability, which presents CT/MR/SERS multi-mode imaging performance of the tumor of mice within a given time. Furthermore, in vivo biodistribution and long-term toxicity studies reveal that the Gd-GERTs have good biocompatibility and bio-safety. Therefore, Gd-GERTs are of great potential as a multifunctional nanoplatform for accurate preoperative CT/MRI diagnosis and intraoperative Raman imaging-guide resection of cancers.

Statement of Significance

Recent advances in molecular imaging technology have provided a myriad of opportunities to prepare various nanomaterials for accurate diagnosis and response evaluation of cancer via different imaging modalities. However, single bioimaging modality is still challenging to overcome the issues such as sensitivity, spatial resolution, imaging speed and complexity for clinicians. In this work, we designed a kind of unique multifunctional nanoprobes with computed X-ray tomography/magnetic resonance/surface-enhanced Raman spectroscopy (CT/MR/SERS) triple-modal imaging capabilities. Multifunctional nanotags offer the capabilities of preoperative noninvasive CT/MR imaging for identification of tumors as well as intraoperative real-time SERS imaging for guidance of complete resection of tumors. These multifunctional nanoprobes show critical clinical significance on the improvement of tumor diagnosis and therapy.

Introduction

There has been a rapid development in molecular imaging technology in the past decade, which is able to provide the possibility of obtaining physiological and pathological information with high sensitivity and specificity for cancer diagnosis. Noninvasive imaging modalities, such as computed X-ray tomography (CT) [1], magnetic resonance imaging (MRI) [2], [3], [4], ultrasound [5], positron emission tomography (PET) [6,7], and optical imaging (OI) [8], [9], [10], have been applied in the cancer molecular imaging. Signal from each imaging modality has its own unique advantages and intrinsic drawbacks in terms of sensitivity, spatial resolution, imaging speed and complexity, which makes it difficult to obtain accurate and comprehensive information for the diagnosis of cancers just relying on single technique [11]. In addition, the use of multimodal contrast agents is advantageous to the exploitation of diagnostic approaches based on combined platforms and for overcoming the limitations of each single procedure.

Among these imaging modalities, CT technique renders relatively high spatial and density resolution, and provides valuable imaging information of the anatomic structure with three-dimensional visual reconstruction function [12,13]. On the other hand, MR imaging is one of the most powerful non-invasive medical imaging techniques with good spatial resolution and high sensitivity, offering detailed multi-parameter profile of the soft-tissue [14,15]. Surface-enhanced Raman scattering (SERS) imaging is one of OI techniques that provides a highly sensitive and specific spectral signature of the substance interrogated. SERS imaging has attracted great interests due to the advantages of specific vibrational fingerprint-like spectrum, high signal-to-noise ratio, ultra-stable signal, sub-picomolar level sensitivity, good multiplexing capability and single photoexcitation [16], [17], [18]. SERS imaging offers opportunities to achieve intraoperative tumor margin and residual tumor detection for surgical guidance with a higher specificity and sensitivity, compared to other imaging techniques [19], [20], [21]. Recently, some studies have reported that unique multiple-modality nanoprobes have been used to accurately help delineate the margins of tumors intraoperatively [22,23]. The multimodal approach demonstrates its comprehensive imaging capabilities and great potentials for enabling more accurate cancer imaging.

At present, CT and MRI techniques play an important role in early detection and preoperative staging evaluation of tumors [24], [25], [26], [27]. However, the traditional CT and MRI techniques are generally based on small molecular agents, which display severe disadvantages such as fast clearance speed, renal toxicity at a relatively high concentration, repeated injections and non-specificity [28]. Therefore, there are great limitations in the intraoperative detection and treatment of cancers. Intraoperative accurate delimitation of tumor margins is crucial for assisting the surgical resection of malignant tissues while preserving important structures [29,30]. SERS nanoparticles (NPs) can achieve greater Raman signal enhancement, resulting in high spatial resolution and signal specificity with non-invasive utility. A few studies have explored the potential of SERS nanocomposites as a contrast agent to realize the ultrahigh sensitivity and specificity detection in a variety of cancer types in mouse models after intravenous injection [31,32]. The resolution of CT and MR imaging depends on the concentration and retention effect of the contrast agent, so the combination of preoperative and intraoperative imaging is difficult to complete at the same time. While SERS can achieve high sensitivity and specificity detection of the tissue, which can play an important role in the intraoperative treatment. Thus, the development of a nanostructure to combine the CT and MRI technique with SERS imaging capacity without sacrificing their individual characteristics would bring a great improvement towards the preoperative diagnosis and intraoperative imaging of tumors.

Gold (Au) NPs with high atomic number (Z) element have been explored as a CT imaging agent on the basis of the X-ray absorption capacity [33]. Au NPs can provide a high mass attenuation coefficient and energy-absorption coefficient as well as a relatively long circulation time in the cardiovascular system. Gadolinium (Gd)-based agents are efficient proton relaxers. Recently, Gd coated NPs have attracted much attention because of their promising features such as well-defined structure, desirable pharmacokinetics, high in vivo T₁ relaxivity and reduced toxicity compared with the small molecular Gd contrast agents [34], [35], [36]. Mesoporous silica is chemically inert with good biocompatibility, great surface modification, high specific surface area, regular pore structure and adjustable pore size. In addition, silica is transparent for optical signals, and it does not produce magnetic signals that interfere with magnetic nanomaterials, and therefore emerges as a potential role for MRI contrast agent when Gd is connected [20,37]. Recently, we have developed a type of SERS tags termed gap-enhanced Raman tags (GERTs), which are composed of metallic core-shell NPs with an interior nanogap and embedded Raman reporter molecules [38], [39], [40], [41], [42], [43], [44], [45], [46], [47]. These GERTs show high sensitivity and photostability at the near-infrared (NIR) off-resonance condition [48], [49], [50], which are greatly favorable for intraoperative bioimaging [38,39,51]. More recently, we have reported GERTs with petal-like shell structures (P-GERTs for brevity) with an ultrahigh sensitivity down a single-NP level and a fast imaging speed up to 6 s for 50 × 50 pixels (0.7 ms/pixel), as a potential solution towards meeting the clinical intraoperative demands [51,52]. However, the attempt to further modify GERTs as multi-modality imaging contrasts has not been reported previously yet.

In this work, we designed and synthesized multifunctional Gd-loaded P-GERTs (Gd-GERTs for brevity) with CT/MR/SERS triple-modal imaging capabilities (Fig. 1). The GERTs integrated with Raman reporters inside the nanogap junction were coated with a mesoporous silica layer according to our previous study [51,52], and the conjugated Gd ions were loaded in the external mesoporous silica layer. Then the tags were additionally functionalized with a polyethylene glycol (PEG) layer, which is able to provide good water-solubility and biocompatibility. The X-ray absorption and T₁ relaxivity of Gd-GERTs were measured to evaluate the CT and MR imaging potential. The built-in Raman reporters of Gd-GERTs lead to the highly stable signals for SERS intraoperative imaging. Finally, CT, MRI and SERS imaging of mouse tumor model were performed to assess the triple-modal imaging capability. This only involved disposable intravenous injection of Gd-GERTs into the mice. The potential in vivo toxicity and biodistribution of the Gd-GERTs were also investigated. The resulting nanocomposites present strong CT absorbance, enhanced MR imaging performance and stable SERS signals, which show great promise for preoperative imaging and intraoperative therapy of cancer.