Photosensitizer-conjugated magnetic nanoparticles for in vivo simultaneous magnetofluorescent imaging and targeting therapy

Abstract

A major challenge in nanotechnology and nanomedicine is to integrate tumor targeting, imaging, and selective therapy functions into a small single nanoparticle (<50 nm). Herein, photosensitizer-conjugated magnetic nanoparticles with ∼20 nm in diameter were strategically designed and prepared for gastric cancer imaging and therapy. The second generation photosensitizer chlorin e6 (Ce6) was covalently anchored on the surface of magnetic nanoparticles with silane coupling agent. We found that the covalently incorporated Ce6 molecules retained their spectroscopic and functional properties for near-infrared (NIR) fluorescence imaging and photodynamic therapy (PDT), and the core magnetic nanoparticles offered the functions of magnetically guided drug delivery and magnetic resonance imaging (MRI). The as-prepared single particle platform is suitable for simultaneous targeting PDT and in vivo dual-mode NIR fluorescence imaging and MRI of nude mice loaded with gastric cancer or other tumors.

Introduction

Gastric cancer was once the second most common cancer in the world [1]. Up to date, stomach cancer is currently the 14th most common cancer in the United States, and so far 2nd most common cancer in China [2]. Gastric cancer is still the second most common cause of cancer-related death in the world, and it remains difficult to cure, primarily because most patients present with advanced disease. Therefore, how to recognize and track or kill early gastric cancer cells is a great challenge for early diagnosis and therapy of patients with gastric cancer.

According to the report from National Cancer Institute (NCI) in USA, nanotechnology has tremendous potentials to make an important contribution towards cancer prevention, diagnosis, imaging, and treatment [3]. Nanotechnology offers unprecedented capability of not only carrying multiple diagnostic/therapeutic payloads in the same package, but also facilitates the targeted delivery into specific sites and across complex biological barriers [4]. The multifunctional integrated system combines different properties such as tumor targeting, imaging, and selective therapy in an all-in-one system, which will provide more useful multimodal approaches in the battle against cancer [5], [6], [7], [8].

Nanoparticles are general 100- to 10000-fold smaller than cancer cells, they can easily pass through cell barriers and preferentially accumulate at the tumor sites based on the EPR (enhanced permeability and retention) effects [9], [10]. When the diameters of the nanoparticles are reduced to 20–50 nm, the biological pathways in targeted cells can undergo profound changes [11], [12], [13]. Smaller nanoparticles have prolonged circulation in the bloodstream (slow clearance by the liver and spleen) and permeate barriers more rapidly including cell membranes and fenestrated vasculature in cancers [14]. The efficacy of vaccines may be enhanced with ultrasmall 20 nm nanoparticles that can diffuse to the lymph nodes to target resident dendritic cells [15]. Multifunctional ultrasmall paramagnetic iron oxide (USPIO) particles (∼30 nm) have been designed to detect and deliver chemotherapeutic agents directly into prostate cancer cells [16]. Therefore, the development of packing sufficient multifunctionality into smaller nanoparticles (less than ∼50 nm) is a major challenge in nanotechnology and nanomedicine.

In cancer imaging, magnetic resonance imaging (MRI) and optical imaging have attracted extensive attention [17], [18]. MRI can offer high spatial resolution and the capacity to simultaneously obtain physiological and anatomical information based on the interaction of protons with the surrounding molecules of the tissue [17]. Magnetic nanoparticles (MNPs) as a MRI contrast agent exhibit a unique MR contrast enhancement effect that enables noninvasive MRI of cell trafficking, gene expression, and cancer [19], [20], [21]. In addition, MNPs have been recognized as a promising tool for the site-specific delivery of drugs and diagnostics agents by an external magnetic field [22], [23], [24], [25].

Optical imaging involves inference from the deflection of light emitted from (e.g., laser, infrared) source to structure, texture, anatomic and chemical properties of material (e.g., crystal, cell tissue), and allows for rapid screening [26]. Optical imaging probes include various dye molecules, dye-doped silica materials, quantum dots (QDs), lanthanide compounds with up- or down-conversion properties, and near-infrared (NIR) emitting nanomaterials [27], [28], [29], [30]. All those modalities can be coupled with MRI contrast agents. Recently, researchers have focused on designing multimodal nanoparticles that will combine various functionalities, e.g., fluorescence, magnetism, and target ability [31], [32]. QDs have been extensively used as a potential luminescence probes due to their high resistance to photobleaching, narrow emission spectra, broad excitation spectra, and longer fluorescence lifetime [33], [34], [35]. However, potential heavy metal toxicity as a result of leakage constituent ions (e.g., cadmium, mercury, lead, etc.) is still a problem [36]. NIR optical imaging has many advantages over other imaging means, because it can penetrate biological tissues such as skin and blood more efficiently than visible light [37]. Furthermore, optical imaging of live animals has a clear window at wavelengths between 650 and 950 nm, where soft tissue, hemoglobin, and water absorb weakly [38]. So far, few reports are associated with the combinations of NIR optical imaging and MRI [39].

In cancer treatment, photodynamic therapy (PDT) has emerged as an increasingly recognized alternative to classical therapies such as surgery, radiotherapy, and chemotherapy in clinical practice, since it is a minimal invasion and provides painless and repeating treatment advantages for patients [40], [41]. The general procedure for PDT involves the systemic or topical administration of light-sensitive molecules called photosensitizers (PSs) followed by activation with light of appropriate wavelength and power[42]. Upon irradiation, the activated PSs transfers its excited-state energy to surrounding oxygen, resulting in reactive oxygen species (ROS), such as singlet oxygen (¹O₂) or free radicals, which will cause cell death and necrosis of tumor components, with minimal damage to the adjacent healthy tissue [43]. However, PSs are limited in application as a result of prolonged cutaneous photosensitivity, poor water-solubility and inadequate selectivity, which are encountered in clinical applications of numerous chemical drugs [44], [45]. To overcome these limitations, great efforts have been devoted to the development of pharmaceutical formulations for effective delivery of PSs. Many groups have reported the utility of nanoparticles, such as liposome [46], polymer nanoparticles [47], gold nanoparticles [48], quantum dots [49], magnetic nanoparticles [50], [51], [52], [53], silica nanoparticles [54], [55], [56], and so on, for PDT [57], [58]. In our previous studies, magnetic chitosan nanoparticles [22] and silica-based magnetic nanoparticles [23] have been strategically designed and prepared as drug delivery system for targeting PDT. However, those methods are almost relatively complicated for difficult extension in clinical application. Therefore, developing a simple and effective method to prepare PS carrier has become critical to clinical PDT.

To the best of our knowledge, some PSs can emit fluorescence and generate singlet oxygen simultaneously under the irradiation [44]. Therefore, taking advantage of the intrinsic fluorescence of PSs to develop a single photosentizer-conjugated magnetic nanoparticle for simultaneous targeting PDT and dual-mode NIR fluorescence imaging and MRI is very significant. Chlorin e6 (Ce6) is a promising photosensitizer characterized by a high sensitizing efficacy and rapid elimination from the body [59]. Moreover, a chlorin core compound can generate with a high fluorescent emission at longer wavelengths of 660–670 nm, which is in the clear window for optical imaging of live animals [59]. To date, no report is closely with use of chlorin e6-conjugated magnetic nanoparticles (Ce6-MNPs) as imaging and therapy of tumors.

Herein, we selected gastric cancer MGC803 cells as research target, and strategically designed and prepared multifunctional Ce6-MNPs as targeting drug delivery system with the aim of investigating the feasibility of use of prepared Ce6-MNPs as simultaneous targeting PDT and in vivo dual-mode NIR fluorescence imaging and MRI of nude mice loaded with gastric cancer.