Multifunctional magnetic nanoparticles for simultaneous cancer near-infrared imaging and targeting photodynamic therapy

Abstract

Cancer theranostics, the ability to simultaneously diagnose and treat cancer, has become one of the major driving forces in nanobiotechnology. In the present work, a multifunctional system, methylene blue-incorporated folate-functionalized Fe₃O₄/mesoporous silica core–shell magnetic nanoparticles (MNPs), for simultaneous near-infrared (NIR) fluorescence imaging and targeting photodynamic therapy (PDT) has been developed. The core Fe₃O₄ MNPs offers the function of magnetically guided drug delivery, the mesoporous silica shell acts as an efficient drug loaded carrier, the photosensitizer methylene blue (MB) exhibits excellent NIR fluorescence imaging and PDT efficiency, and the folic acid (FA) can effectively enhance the delivery of MB to the targeting cancer cells, which overexpress the folate receptor. The results indicated that the multifunctional system could effectively be used in NIR fluorescence imaging. Moreover, it exhibited a synergistic effect of magnetic targeted PDT of cancer under NIR laser irradiation. Thus, the multifunctional system is promising for simultaneous cancer diagnosis and therapy.

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1. Introduction

Cancer is a devastating disease with an incidence increasing at an alarming rate and survival not improved substantially during the past several decades. Although enormous efforts have been made in early detection and comprehensive treatment for this disease, little or no survival improvement has been obtained, and further development of novel strategies is needed.^1,2 According to the report from the National Cancer Institute (NCI) in USA, nanobiotechnology, which not only carries multiple diagnostic/therapeutic payloads in the same package, but also facilitates the targeted delivery into specific sites and across complex biological barriers,³ has tremendous potential for cancer prevention, diagnosis, imaging, and treatment.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.^5–9

Photodynamic therapy (PDT) is now well established as a technique for cancer treatment.^10–13 In contrast to other conventional medical treatments, PDT doesn't need to release the used drugs and it is based on the concept that photosensitizers (PSs) are able to generate reactive oxygen species (ROS) upon irradiation, such as singlet oxygen (¹O₂) or free radicals, and can irreversibly damage the pathological cells without damaging the adjacent healthy ones.^14–16 Unfortunately, the phototoxicity, hydrophobicity and the low selectivity of the PS agents limit the current applications of PDT in cancer therapy.¹⁷ Therefore, the development of new biocompatible delivery vehicles with stable aqueous dispersion, site-specific and time-controlled delivery abilities is still urgently needed. Among the various delivery vehicles, mesoporous silica nanoparticles (MSNs) hold the promise to be a highly efficient PDT drug delivery platform owing to their attractive features, such as uniform pore size, large surface area and high accessible pore volume, ease of chemical modification, excellent biocompatibility, and avid uptake by cells.^18–22 The porous structure of MSNs not only permits the accommodation of a large quantity of PSs, but also helps to enhance the permeability of oxygen and generate ¹O₂, which is essential for PDT. Furthermore, their surfaces can be modified with special targeting moieties such as antibodies, folate and aptamers for site-specific behavior. However, to our knowledge, the application of multifunctional MSNs as photosensitizing vehicles that provides both MRI and fluorescence imaging diagnosis and photodynamic therapy has not been satisfactorily explored. There are very few reports available on the applications of MSNs as PSs vehicles.

Moreover, the accurate localization of PS-containing nanoparticles in cells or target tissues is very important for effective PDT. It will offer a powerful guidance for site-directed irradiation of target diseased tissues, without causing damage to the healthy tissues. Recently, optical imaging probes have been incorporated into MSNs along with PSs to offer dual capability of imaging and therapy.^23–25 Optical imaging can provide the highest sensitivity and obtain detailed information at subcellular levels, which allow accurate targeting and simultaneous phototherapy treatment. In fact, some PSs can emit fluorescence and generate ¹O₂ simultaneously under irradiation.²⁶ Methylene blue (MB) is a hydrophilic phenothiazinium photosensitizer with promising applications in the PDT due to its high quantum yield of ¹O₂ generation (Φ ∼ 0.5) in the excitation of the therapeutic window (600–900 nm), and low dark toxicity.^27,28 In addition, MB is also the most inexpensive of the commercially available near-infrared (NIR) fluorescent dyes, and has been widely used for bioanalysis.²⁹ Therefore, taking advantage of the intrinsic fluorescence of photosensitizer to develop single photosensitizer-encapsulated nanoparticles for simultaneous in vivo imaging and PDT is significant.

Enhancing tumor accumulation of therapeutic agents by physical forces such as an external magnetic field (MF) has emerged as a new tumor-targeting strategy. During this process, magnetic nanoparticles (MNPs) carrying therapeutics circulating in the bloodstream would be attracted by the MF applied on the tumor, resulting in greatly enhanced enrichment of therapeutic agents in targeted tumor region to improve the cancer treatment efficacy.³⁰ Moreover, MNPs as a magnetic resonance imaging (MRI) contrast agent exhibit a unique magnetic resonance (MR) contrast enhancement effect that enables noninvasive MRI of cell trafficking, gene expression, and cancer.^31,32

Herein, in this paper, we present a core–shell structured nanomaterial, namely, Fe₃O₄@mSiO₂(MB)–FA, which is multifunctional in the fields of NIR fluorescence imaging and targeting PDT. The Fe₃O₄ nanoparticles were prepared via co-precipitation method, and mesoporous silica was coated as shell layer, in which photosensitizer was loaded. Then a layer of poly(ethylene glycol) (PEG) shell and folate receptors were synthesized. Systematic experiments in vitro and in vivo were designed to carefully evaluate the physical and chemical properties, cytotoxicity, cellular uptake, NIR light-induced cell killing, in vivo imaging, as well as targeting PDT of the multifunctional system (Scheme 1).

Scheme 1 The cancer cell targeting PDT mechanism of the multifunctional system.

2. Experimental

2.1. Materials

2.1.1 Reagents and materials. Ferric chloride hexahydrate (FeCl₃·6H₂O), ferrous chloride tetrahydrate (FeCl₂·4H₂O), sodium oleate (C₁₈H₃₃ONa), ammonium hydroxide (25%, aqueous solution), tetraethoxysilane (TEOS), ammonium nitrate (NH₄NO₃), cetyltrimethylammonium bromide (CTAB), 3-amino-propyltriethoxysilane (APTES), methylene blue (MB), folic acid (FA), 1-ethyl-3[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), dimethyl sulfoxide (DMSO) and N-hydroxysuccinimide (NHS) were obtained from Sinopharm Chemical Reagent Co. Ltd., and used without further purification. 2-Methoxy (polyethyleneoxy) propyltrimethoxysilane (PEG–silane) was from Gelest (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4,6-diamidino-2-phenylindole (DAPI) and DPBF were purchased from Sigma-Aldrich (USA). Dulbecco's modified Eagle's medium (DMEM) cell culture medium, penicillin, streptomycin, trypsin, fetal bovine serum (FBS), and heparin sodium were bought from Gibco Invitrogen (USA). All other chemicals and reagents were of analytical grade and used as received.

2.1.2 Cell lines and animal. Human ovarian cancer cells (SK-OV-3 cells), human cervical cancer cells (HeLa cells), mouse sarcoma cells (S-180 cells) and mouse fibroblast cells (NIH 3T3 cells) were purchased from the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Kunming mice of clean grade (female, 4–6 weeks old) and BALB/c mice (female, 4–6 weeks old, 16–18 g of body weight) were purchased from Shanghai SLAC Laboratory Animal Center (Shanghai, China), and were used in accordance with approved institutional protocols established by the Shanghai Department of Experimental Animals Management.

2.2. Methods

2.2.1 Synthesis of Fe₃O₄@mSiO₂(MB)–FA nanoparticles. Fe₃O₄@mSiO₂(MB)–FA nanoparticles were synthesized following the method in our previous report.³³ At first, highly biocompatible monodisperse superparamagnetic Fe₃O₄@mSiO₂ core–shell nanoparticles with mesoporous silica shells were synthesized. Then, these particles were coated with the covalently bonded biocompatible polymer PEG and modified with the cancer targeting ligand FA. Finally, the water-soluble photosensitizer MB was loaded into the mesoporous silica shell.

2.2.2 Characterization of Fe₃O₄@mSiO₂(MB)–FA nanoparticles. The particles were characterized by means of powder X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), Brunauer–Emmett–Teller (BET) analysis, vibrating sample magnetometry (VSM), UV-Vis and fluorescence spectroscopy. High-resolution TEM data were collected on a JEOL model JEM 2100 electron microscope operating at an accelerating voltage of 200 kV. DLS data were obtained on an electrophoretic light scattering spectrophotometer (Beckman Coulter Delsa nano C, USA). XRD data were collected on a Rigaku corporation D/MAX 2550 VB/PC Multi-Purpose X-ray diffractometer with Cu Kα radiation (λ = 0.1542 nm). The specific surface area was calculated by the BET (ASAP 2020-M) method. The pore size distribution was obtained from the Barret–Joner–Halenda (BJH) method. The FT-IR spectra of the nanoparticles were obtained on a Nicolet 6700 spectrometer. Magnetic properties were recorded using a VSM (Lake Shore, USA) instrument. UV-Vis absorption spectra were measured on an UV-Vis spectrophotometer (Evolution 220, Japan). Fluorescence spectra of liquid state were recorded on a Lumina Fluorescence spectrometer. (Thermo scientific, USA). The cellular images were acquired with a confocal laser scanning microscope (CLSM, Nikon AIR).

2.2.3 Detection of singlet oxygen. Commonly, there are two methods for the detection of ¹O₂, first, by using luminescence emission spectra at 1270 nm,^34–36 the second method is based on an indirect method using a chemical ¹O₂ probe.^36,37 In this study, DPBF is used as a probe^34,37 to detect the ¹O₂ quantum yield. DPBF reacts irreversibly with ¹O₂ that causes a decrease in the intensity of the DPBF absorption band at 400 nm. In a typical experiment, 15 mL of DPBF in acetonitrile (5.5 mM) was mixed with 2 mL of Fe₃O₄@mSiO₂(MB)–FA nanoparticles (1.5 mg mL⁻¹) in acetonitrile. The experiment mixed DPBF with free MB dispersed in acetonitrile used as the standard. The solutions were irradiated with a 650 nm laser source at 5 mW cm⁻² and their absorbances at 400 nm were recorded at every 10 seconds, using a UV-Vis spectrophotometer.

2.2.4 Cytotoxicity assessment. The in vitro cytotoxicity was measured by using the MTT assay in SK-OV-3 cells. Cells (1 × 10⁵ well⁻¹) were inoculated into a 96-well cell-culture plate and then incubated at 37 °C in a 5% CO₂-humidified incubator for 24 h. 200 μL of Fe₃O₄@mSiO₂(MB)–FA nanoparticles with different concentrations (10–200 μg mL⁻¹) and DMEM were added to the wells, separately. After incubation for 24 h and 48 h at 37 °C under 5% CO₂, the supernatant was removed. Subsequently, MTT (20 μL, 5 mg mL⁻¹) dissolved in DMEM (200 μL) were added and the plates were incubated at 37 °C for another 4 h. Then supernatant was removed before DMSO was added to each well to dissolve formazan. The absorbance at 492 nm and 630 nm was detected with a spectrophotometric microplate reader (THERMO Multiskan MK3 spectrometer). Each data point was collected as an average of three wells, and the untreated cells were used as controls. The following equation was used to calculate the inhibition of cell viability.

2.2.5 Cellular uptake study. The cellular uptake and distribution of Fe₃O₄@mSiO₂(MB)–FA nanoparticles were performed using flow cytometry and CLSM. For flow cytometry, HeLa cells (1 × 10⁶) that overexpress folate receptors were seeded in 6-well culture plates and cultured for 24 h. Following this, the cells were treated with Fe₃O₄@mSiO₂(MB)–FA nanoparticles (200 μg mL⁻¹) for 2 h and 4 h. After the preset time intervals, the culture medium was discarded, and cells were washed three times with PBS and harvested with trypsinization. The cell pellets were resuspended in PBS and measured for the fluorescence intensity (excitation: 650 nm; emission: 690 nm) on a BD FACSAria flow cytometer (Beckton Dickinson, USA), and Cell Quest software was used to analyze the data.

For CLSM studies, HeLa cells were propagated in DMEM containing FBS (10%) and penicillin/streptomycin (1%). Then, the cells were digested and resuspended in the DMEM medium (without FARs). 1 × 10⁴ cells were transferred into a 6-well tissue culture plates. After 24 h of incubation, the cells were carefully rinsed with PBS (pH 7.4). 2 mL DMEM medium of the Fe₃O₄@mSiO₂(MB)–FA (200 μg mL⁻¹) was added to the Petri dishes and incubated for 2 h, followed by three rinsings with PBS. After fixation with 4% paraformaldehyde in PBS at room temperature for 30 min, cells were treated with 1 mL 0.01% Triton X-100 (Sigma) for 10 min and the nuclei were stained with DAPI (1 μg mL⁻¹, Sigma) for 15 min. Each step discussed above was followed by washing with PBS three times. Then, cover slips containing cells were mounted onto slides and were then observed under CLSM.

2.2.6 In vitro PDT effect. Two 96-well plates were divided into two groups: dark control and experimental group. SK-OV-3 cells were seeded in the 96-well plate at a density of 1 × 10⁴ cells per well for 24 h. Then, DMEM cell medium containing different concentrations of Fe₃O₄@mSiO₂(MB)–FA nanoparticles were added to the wells (200 μL per well, 0, 10, 50, 100, 150, 200 μg mL⁻¹). After incubation for 24 h, the cells were washed three times with 200 μL PBS to remove the unbound nanoparticles. Then, 200 μL PBS was added and the cells were exposed to a 650 nm laser with a power density of 70 mW cm⁻² for 4 min. After irradiation, cells were incubated for another 24 h in a 5% CO₂, 95% air humidified incubator at 37 °C. The dark control group was kept under identical conditions to experimental group but without irradiation. The cell viability was measured by a MTT assay mentioned above and expressed as a percentage of the control.

2.2.7 In vivo imaging of Fe₃O₄@mSiO₂(MB)–FA nanoparticles in animals. The female BALB/c mice were maintained in a pathogen-free condition on arrival. To generate HeLa tumor xenografts, 1 × 10⁷ cells suspended in 200 μL of saline were subcutaneously implanted into the right limb armpits of the mice. Tumors were allowed to grow until the average volume of the xenograft tumors was approximately 50 mm³ (about 10 days). The mice were randomly divided into three groups, with three mice in each group. For group I, the mice were administrated with saline; group II, Fe₃O₄@mSiO₂(MB)–NH₂; and group III, Fe₃O₄@mSiO₂(MB)–FA. 5 mg MB equiv. kg⁻¹ of Fe₃O₄@mSiO₂(MB)–NH₂ and Fe₃O₄@mSiO₂(MB)–FA were administrated through the tail vein in all of the formulations. The entire body in vivo fluorescence imaging of the mice was performed an hour later utilizing a in vivo Imaging System IVIS Lumina II (Coliper, USA). The instrument was equipped with fluorescent filter sets (excitation/emission wavelengths of 650/690 nm).

2.2.8 In vivo anti-tumor properties. Xenograft tumor mouse model. All animal experiments were performed under a protocol approved by the Shanghai laboratory animal center. Mice ascitic tumor models were generated by subcutaneous injection of 1 × 10⁶ S-180 cells in 0.1 mL saline into the right armpit of female Kunming mice (18–22 g, Shanghai SLAC Laboratory Animal Center). The mice were used when the tumor volume reached 50 × 50 mm³.

In vivo PDT. For the in vivo antitumor experiments, the tumor-bearing mice were divided into five groups (n = 5), minimizing the differences of weights and tumor sizes in each group. The mice were administered with (1) saline, (2) magnetic/650 nm laser, (3) Fe₃O₄@mSiO₂(MB)–FA/650 nm laser, (4) Fe₃O₄@mSiO₂(MB)–FA/magnetic, (5) Fe₃O₄@mSiO₂(MB)–FA/magnetic/650 nm laser, (Fe₃O₄@mSiO₂(MB)–FA dose: 5 mg kg⁻¹) in saline were intravenously injected into mice via the tail vein every 2 days, and then the tumor regions were irradiated with a 650 nm laser (70 mW cm⁻², 10 min) at 4 h post-injection. For the purpose of in vivo magnetic targeting, a magnet was glued onto the tumor site of the mice. The mice were observed daily for clinical symptoms and the tumor sizes were measured by a caliper every 24 h.

2.2.9 Statistical analysis. Data were analyzed using the SPSS software package. Quantitative data are expressed as mean ± SD and analyzed by the use of Student's t test. P values < 0.05 were considered statistically significant.

3. Results and discussion

3.1. Synthesis and characterization of Fe₃O₄@mSiO₂(MB)–FA

In our experiments, the multifunctional Fe₃O₄@mSiO₂(MB)–FA nanoparticles were prepared by a multistep process (see Methods section for details in our previous report³⁷). The TEM image displayed in Fig. S1(A)† shows that the prepared hydrophobic Fe₃O₄ are monodispersed nanoparticles with a uniform average diameter of 5 nm. The MB-loaded Fe₃O₄@mSiO₂–FA nanoparticles are discrete and uniform with an average diameter of 70 ± 5 nm, and well-ordered mesopores were also clearly observed (Fig. 1S(B)†). In addition, the DLS data (Fig. S2†) shows Fe₃O₄@mSiO₂(MB)–FA has narrow size distribution with an overall hydrodynamic diameter of 89.8 nm in water without aggregation. The crystallinity of Fe₃O₄ does not change after modification of FA and load of MB (Fig. S3†). Moreover, the uniform mesoporous pore size along with small particle size (<100 nm) are facilitative and favorable for drug delivery applications (Fig. S4†). The representative hysteresis loops of Fe₃O₄@mSiO₂(MB)–FA at ambient temperature are shown in Fig. S5,† and the saturation magnetization of the Fe₃O₄@mSiO₂(MB)–FA is ∼8.46 emu g⁻¹, which suggested the superparamagnetism of the as-prepared nanoparticles.

3.2. Singlet oxygen detection

In this study, the ¹O₂-generation capability of the Fe₃O₄@mSiO₂(MB)–FA nanoparticles was assessed using DPBF, a ¹O₂ chemical probe in acetonitrile under 650 nm laser irradiation (5 mw cm⁻²). DPBF reacts irreversibly with ¹O₂ and the reaction can be followed by recording the decrease in the intensity of the DPBF absorption at around 400 nm. The changes in the absorption spectra of DPBF in the presence of Fe₃O₄@mSiO₂(MB)–FA nanoparticles after different irradiation times are shown in Fig. 5. Control tests were carried out to confirm that the decrease in the absorption of DPBF was induced by ¹O₂ (Fig. 1, inset). In the presence of Fe₃O₄@mSiO₂(MB)–FA nanoparticles, the DPBF absorption at 400 nm dramatically decreases under NIR-laser irradiation (Fig. 1, curve a in the inset), thereby suggesting that these nanoparticles are highly efficient in the generation of reactive ¹O₂. In contrast, there are fewer decreases in DPBF absorbance for free MB (Fig. 1, curve b in the inset). The effective ¹O₂-generating capability of our nanoparticles under NIR light facilitates their applications in NIR-induced PDT.

Fig. 1 Absorption spectra of Fe₃O₄@mSiO₂(MB)–FA in the presence of DPBF after different times of irradiation with a 650 nm laser source at 5 mw cm⁻². Inset: decay curves of absorption of DPBF as a function of time of irradiation. DPBF with dispersion of Fe₃O₄@mSiO₂(MB)–FA (a) and DPBF with MB free (b) in acetonitrile.

3.3. Cellular uptake

The ability to target nanoparticles to specific organelles or receptors is one of the most important factors for their prospective application in bioimaging and drug delivery. Various types of targeting agents, such as antibodies, aptamers and FA, have been developed for the specific identification antigens or receptors on targeting cancer cells. In this study, FA was modified onto the Fe₃O₄@mSiO₂ as the targeting component because folate receptors (FR) are overexpressed in many human cancerous cells.

Flow cytometry and CLSM were used to evaluate the effect of FA on the celluar uptake behavior of Fe₃O₄@mSiO₂(MB)–FA against FR positive HeLa cells. To precisely observe the cellular distributions of the multifunctional nanoparticles, the double fluorescence-labeling experiments and visualized red fluorescence from MB and blue fluorescence from DAPI labeling the nucleus were performed. As described in Fig. S6,† intense red fluorescence of MB loaded Fe₃O₄@mSiO₂–FA is observed in the cytoplasm of FA-positive HeLa and SK-OV-3 cells in comparison with NIH 3T3 cells without folate receptors, whose fluorescence could be negligible. The results confirm that the objective of increasing specificity and sensitivity of Fe₃O₄@mSiO₂(MB)–FA image by labeling cancer cells with over-expression of folate receptors on the surface has been achieved. Moreover, with the increase of the incubation time, the red fluorescence in both cytoplasm and nuclei increased (Fig. 2).

Fig. 2 Confocal laser micrographs of HeLa (D–F) cells incubated with Fe₃O₄@mSiO₂(MB)–FA for 2 h and 4 h. For each panel, from left to right were the cells with unclear staining with DAPI, with MB fluorescence and overlays of images.

The cellular uptake of Fe₃O₄@mSiO₂(MB)–FA into HeLa cells was further quantitatively analyzed with flow cytometry. Fig. 3A shows the flow cytometry histograms of MB fluorescence from HeLa cells incubated with Fe₃O₄@mSiO₂(MB)–FA with the concentration of 200 μg mL⁻¹ for 2 h and 4 h, respectively. Cells without any treatment were used as a negative control to detect autofluorescence. The flow cytometry analysis clearly demonstrated that the changes in fluorescence intensity of MB were observed in the cells after 2 h incubation with Fe₃O₄@mSiO₂(MB)–FA. With the increase of the incubation time, the relative geometrical mean fluorescence intensity of Fe₃O₄@mSiO₂(MB)–FA pretreated cells obviously increased (Fig. 3B).

Fig. 3 Flow cytometry histogram profiles (A) and fluorescence intensity (B) of HeLa cells after incubation with Fe₃O₄@mSiO₂(MB)–FA nanoparticles for 2 h and 4 h.

3.4 Laser induced in vitro PDT effects on cancer cells

In order to assess the dark cytotoxicity of our multifunctional Fe₃O₄@mSiO₂(MB)–FA nanoparticles for in vivo applications, we did an MTT assay using SK-OV-3 cells as model and the results indicate that Fe₃O₄@mSiO₂(MB)–FA displays non-cytotoxicity and good biocompatibility on SK-OV-3 cells within 0–200 μg mL⁻¹ (Fig. S7†). To investigate the PDT efficiency of Fe₃O₄@mSiO₂(MB)–FA, SK-OV-3 cells were first incubated with different concentrations of Fe₃O₄@mSiO₂(MB)–FA for 24 h and then treated with/without laser (650 nm) irradiation. The MTT assay was used to evaluate the cell viabilities. Cell viability was normalized to control cells (no drug and nonirradiated). The combination of 24 h exposure of tumor cells to Fe₃O₄@mSiO₂(MB)–FA followed by laser irradiation-induced dose and light-dependent cytotoxicity on the tumor cells, which was statistically different from the non-irradiation control, are shown in Fig. 4A and Fig. 4B. With the increase of drug dose and light dose, the cell viability gradually decreases. To the irradiated group, 100 μg mL⁻¹ Fe₃O₄@mSiO₂(MB)–FA causes approximately 50% cell viability loss, demonstrating an obvious photodynamic activity. When the concentration of Fe₃O₄@mSiO₂(MB)–FA reaches 200 μg mL⁻¹, the cells are almost dead (about 80% loss of cell viability). The group treated with the drug without light exposure shows that the drug alone has no effects on tumor cells, which coincides with the result of the cytotoxicity assessment.

Fig. 4 Phototoxicity of Fe₃O₄@mSiO₂(MB)–FA to HeLa cells. (A) SK-OV-3 cells were incubated with 0–200 μg mL⁻¹ Fe₃O₄@mSiO₂(MB)–FA for 24 h at 37 °C in the dark prior to irradiation for 4 min with 650 nm laser (70 mW cm⁻²). (B) SK-OV-3 cells were incubated with 150 μg mL⁻¹ Fe₃O₄@mSiO₂(MB)–FA for 24 h at 37 °C in the dark prior to a series of light doses (0, 4.2, 8.4, 12.6, 16.8 and 21 J cm⁻²).

3.5 In vivo imaging

The tumor uptake of Fe₃O₄@mSiO₂(MB)–FA in the mice was determined by NIR fluorescence imaging of cancer tumor model mice after injection of nanoparticles via the tail vein. Fig. 5 shows in vivo near infrared fluorescence imaging of saline (Fig. 5I), Fe₃O₄@mSiO₂(MB)–NH₂ (Fig. 5II) and Fe₃O₄@mSiO₂(MB)–FA (Fig. 5III) in mice 1.5 h, 3 h and 4.5 h post-injection through the tail vein. As expected, from Fig. 5, the fluorescence signal in mouse III (A–C) distributed throughout the whole body, and the intensity of mouse III (A–C) was higher than that of mouse II (A–C) at the tumor site. The results indicated that Fe₃O₄@mSiO₂(MB)–FA exhibited a longer plasma circulation time than others. However, Fe₃O₄@mSiO₂(MB)–NH₂ in blood decreased rapidly after the intravenous administration due to the fact that Fe₃O₄@mSiO₂(MB)–FA reduced the uptake of MB by the reticuloendothelial system (RES) and the mononuclear phagocyte system.²³ In the ex vivo images (Fig. 5D), the high accumulation of nanoparticles was observed in RES organs, including liver, spleen and kidney, possibly caused by the clearance of the RES. A significant enhancement in MB levels was found in tumor site of mouse III, indicating that the FA conjugated nanoparticles are able to be recognized by the FR of tumor cells. The prolonged circulation gave the opportunity for Fe₃O₄@mSiO₂(MB)–FA to accumulate at the tumor site by EPR effect and made FA conjugated nanoparticles contact with tumor cells and internalize into the tumor cell via receptor-mediated endocytosis.

Fig. 5 In vivo optical fluorescence imaging of HeLa tumor xenografted nude mice and their several organs and tumor, which were taken after sacrifice (at 4.5 h post-injection).

3.6 In vivo antitumor efficacy

To investigate in vivo PDT efficacy of Fe₃O₄@mSiO₂(MB)–FA, comparative efficacy studies were conducted. The S-180 tumor-bearing mice were divided into 5 groups and were treated according to protocols as summarized in the method Section 2.2.9. The changes of relative tumor volume as a function of time are plotted in Fig. 6A. After 8 days of treatment, the control group shows a relative tumor volume (V/V₀) of 8.70 ± 0.89, magnet/650 nm laser and Fe₃O₄@mSiO₂(MB)–FA/magnet results in V/V₀ of 9.53 ± 0.96 and 8.79 ± 0.67, the tumor-bearing mice treated with Fe₃O₄@mSiO₂(MB)–FA/650 nm laser and Fe₃O₄@mSiO₂(MB)–FA/magnet/650 nm laser achieves (V/V₀) of 6.11 ± 0.6 and 5.45 ± 0.95. Fe₃O₄@mSiO₂(MB)–FA/magnet/650 nm laser has tumor growth inhibition (TGI) of 62.64%, which is significantly more effective than the other therapeutic groups (p < 0.05). In Fig. 6B and C, it was obvious that compared with control group, for mice treated with Fe₃O₄@mSiO₂(MB)–FA/650 nm laser, the tumor volume is greatly reduced, and this is a successful application in which MB is used as a photosensitizer in PDT to achieve in vivo tumor treatment efficacy. Remarkably enhanced PDT effect is observed for tumor-bearing mice treated by Fe₃O₄@mSiO₂(MB)–FA/magnet/650 nm laser, in comparison to that treated with Fe₃O₄@mSiO₂(MB)–FA/650 nm laser at the same Fe₃O₄@mSiO₂(MB)–FA and light doses. Because of the magnetic targeting property of Fe₃O₄@mSiO₂(MB)–FA, when a magnet is glued to the top of the tumor, more Fe₃O₄@mSiO₂(MB)–FA will go to the tumor site than non-magnetic group, so the PDT efficacy of Fe₃O₄@mSiO₂(MB)–FA with a magnet is higher than that of the non-magnetic group. The growth of tumor tissue is successfully suppressed by Fe₃O₄@mSiO₂(MB)–FA/magnet/650 nm laser. This high therapeutic efficacy originates from the high Fe₃O₄@mSiO₂(MB)–FA accumulation in tumor tissue.

Fig. 6 Antitumor efficacy of Fe₃O₄@mSiO₂(MB)–FA in the Kunming mice bearing S-180 tumors at a dose of 5 mg kg⁻¹; each group was intravenously administered every day for a total of 8 times, error bars were based on SD of five tumors per group. Control: blank; M: magnet; D: drug; L: light. (A): Tumor growth of mice in different treatment groups within 8 days. (B): Quantitative results of tumor weight excised from the tumor-bearing mice sacrificed on day 8. (C): The picture of the tumors in different groups at the end of the experiment.

Conclusions

In summary, a multifunctional system Fe₃O₄@mSiO₂(MB)–FA for simultaneous cancer NIR imaging and targeting PDT has been successfully designed and developed. The prepared Fe₃O₄@mSiO₂(MB)–FA has high water-solubility, non-cytotoxicity, good biocompatibility, and can serve as a powerful PDT agent for in vitro fluorescence imaging and PDT cancer cell killing under NIR light irradiation. In vivo targeting PDT is further demonstrated in our animal experiments, showing excellent tumor ablation therapeutic efficacy by using Fe₃O₄@mSiO₂(MB)–FA as the targeting PDT agent. Consequently, this unique multifunctional system may have great potential application in early diagnosis and therapy of cancer.

Acknowledgements

This research was financially supported by the Science and Technology Commission of Shanghai Municipality (STCSM, contract no. 13ZR1412000 and 12nm0501100).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Additional information includes the characterizations by TGA and TEM of samples; supporting figures of cytotoxicity assessment and cellular uptake. See DOI: 10.1039/c4ra10801a

‡ These authors contributed equally to this work.

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