Iron (III) acetylacetonate [Fe(acac)3 (98%)] and OA (C17H33COOH, 85%) were purchased from Aladdin Chemical Reagent Co. Ltd. Benzyl ether (98%) was purchased from Alfa Aesar. DSPE-PEG2000 (PEG-phospholipids, 99%) was purchased from Shanghai A.V.T. Pharmaceutical Co., Ltd. Cy7 was purchased from Sangon Biotech (Shanghai) Co. Ltd. Paclitaxel (C₄₇H₅₁NO₁₄, 98%) was purchased from Shanghai Yuanye Biotech Co., Ltd. Carbodiimide (EDC) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) were purchased from Sigma-Aldrich (St Louis, MO). The commercially available reagents, including Chloroform (98%) and ethanol (95%), were all purchased from Sinopharm Chemical Reagent Co. Ltd. Monoclonal antibodies for cleaved-Caspase-3 and cleaved-PARP were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). All the chemicals were directly used without any further purification.

RPMI-1640 medium and trypsase were purchased from Gibco-Invitrogen Corp (Carlsbad, CA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) agents were obtained from Sigma-Aldrich (St Louis, MO). Other reagents included paclitaxel (Chemieliva Pharmaceutical Co. China) and Herceptin (Trastuzumab-Herceptin^®, Genetech, San Francisco, CA, USA). High-performance liquid chromatography (HPLC) grade acetonitrile was purchased from Wanqing Chemical Reagent Co., Ltd. (Nanjing, People's Republic of China). Transmission electron microscope (TEM) was of a JEM-200CX model (JEOL Ltd., Akishima, Japan). The laser particle size analyzer and zeta potential analyzer were purchased from Malvern Instruments Ltd. (Worcestershire, UK). Flow cytometry was performed using a FACS Vantage SE model (Becton-Dickson, USA). Another apparatus was an optical microscope (OLYMPUS, CX31, Japan). All reagents used were of analytical grade.

The MNPs-Fe₃O₄ was synthesized and characterized by State Key Laboratory of Bioelectronics (Southeast University, People's Republic of China). MNPs-Fe₃O₄ was prepared by using iron acetylacetonate as the precursor of iron in the form of high temperature thermal decomposition. The selected reaction vessel was a three-neck flask with a capacity of 100 mL, the reaction solvent was dibenzyl ether, and the surfactants were oleic acid and oleylamine. In a specific experiment, the amount of iron acetylacetonate, dibenzyl ether and oleic acid was 2 mmol, 20 mL, and 3.8 mL, respectively. The reaction was placed in a three-necked flask. Nitrogen was blown into the mouth of the left bottle, circulating water condensed and refluxed at the mouth of the right bottle, and a temperature sensor placed in the middle bottle. After the system was successfully constructed, it was heated to 220°C at a heating rate of 3°C/min and held for 1 h. It was then further heated to 290°C at the same heating rate and reacted for 30 min to end the reaction. The heat source was removed with the reaction solution cooling down to room temperature before being poured into a beaker; the solution was then added with ethanol and magnetically separated. It was washed with ethanol three times, and kept in 10 mL of chloroform to a constant volume.

The PEGylated long-circulating lipid DSPE-PEG2000 molecule was modified on the surface of MNPs-Fe₃O₄ to ensure its biocompatibility and water solubility, so that the active functional group COOH could be introduced onto the surface. In a specific experiment, 100 mg DSPE-MPEG2000 powder and 10 mg DSPE-PEG2000-COOH powder were weighed and dissolved in 3 mL of chloroform. Two mL of the above MNPs-Fe₃O₄ (concentration: 6 mg Fe/mL, dispersed in chloroform) were removed. The two were mixed into a 50 mL round bottom flask, and 10 mg of fluorescent dye cy7 and 10 mg of PTX were added simultaneously. It was fully sonicated with an ultrasound system for 5 min, before 3 mL of deionized water was added. It was continued to be sonicated for 3 min to form a milky turbid liquid. It was then rotated and evaporated at 70°C for 15 min to fully evaporate and remove the organic reagents. The sample was filtered through a 220 nm filter and stored in a refrigerator at 4°C.

The 15 mg MNPs-Fe₃O₄@PEG aqueous solution was added to 180 mg of EDC solid powder and 200 mg of NHS solid powder, fully dissolved and shaken on a shaker for 30 min (120 r/min) to ensure -COOH on the nanoparticle surface activated. After activation, it was centrifuged by ultrafiltration and washed three times with deionized water to remove excess EDC and NHS. Next, the above sample was dispersed in 20 mL of borate buffer (0.02 mol/L, pH = 8.0) with a quantitative Herceptin dilution added dropwise, and incubated on a shaker at room temperature for 24 h (100 r/min). Finally, it was centrifuged by ultrafiltration and washed for three times with deionized water to obtain a black translucent Herceptin antibody-coupled magnetic nanoprobe solution, which was filtered through a 220 nm filter and stored at 4°C refrigerator.

The drug loading and encapsulation efficiencies were determined using HPLC with an absorption peak at 230 nm, and were calculated according to the following equations (8):

Transmission electron microscopy (TEM) was used to characterize morphologic size of MNPs-Fe₃O₄ and MNPs-Fe₃O₄@PEG, and dynamic light scattering (DLS) was used for magnetic nanoprobe MNPs-Fe₃O₄@PEG@mAb characterization of hydrodynamic dimensions. A vibrating sample magnetometer (VSM) was used to detect the saturation magnetization of MNPs-Fe₃O₄ to verify the properties of magnetic iron oxide nanoparticles.

The diameter of the NPs was between 1 and 100 nm, and the magnification of the general instrument was not enough to observe its microstructure. The resolution of TEM could reach 0.1–0.2 nm, which was an important instrument for studying NPs. The MNPs-Fe₃O₄ could be directly penetrated by the transmission electron beam when observed by TEM (7, 9). When the sample was prepared, it was only necessary to dilute the nanoparticles in an alcohol solution, and then the sample was picked up with a copper mesh with a carbon film, which was dried and observed with a TEM.

The human breast cancer cell line (SK-BR-3 and MDA-MB-231 cells) was purchased from Shanghai Cell Bank of Chinese Academy of Sciences. SK-BR-3 and MDA-MB-231 breast cancer cells were adherent growth cells. In the experiment, RPMI1640 medium (containing 10% serum and 100 μl of double antibody) was used for cultivation. The incubation conditions were 37°C and 5% CO₂. The cells were observed under a microscope. When the cell fusion rate reached 86% or more, SK-BR-3 and MDA-MB-231 breast cancer cells were subcultured using trypsin digestion solution.

Cell proliferation was measured by MTT assay in vitro. According to our preliminary experiments, inhibitory effects of the drugs were the most significant at 48 h (7). 5 × 10³ SK-BR-3 and MDA-MB-231 breast cancer cells in normal culture medium were seeded into each well of a 96-well plate. The cells were then cultured for 48 h and then washed and collected, following the manufacturer protocol, and then the optical density (OD) value was read at 570 nm using a microplate reader. The inhibition rate of cells was determined as follows: (1-OD of treated group/OD of control group) × 100%, and the cell viability was assessed as follows: OD of treated group/OD of control group × 100% (7).

SK-BR-3 and MDA-MB-231 breast cancer cells were seeded into six-well plates at the density of 4 × 10⁵ cells per well, treated as described in cell cycle analysis, and incubated at 37°C for 48 h. The washed cells were then suspended with 500 μL binding buffer and labeled with 5 μL Annexin V-FITC for 15 min at room temperature in dark. Thereafter, cell apoptosis was determined by FACSCalibur FCM (Becton-Dickinson, Franklin Lakes, NJ, USA) (7).

After treatment, the total proteins were extracted from each group; protein concentration was measured using the Bradford method. Proteins were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride membrane. The membrane was incubated with skimmed milk (5%) as a blocking agent for 1 h, followed by incubation with cleaved-PARP monoclonal antibodies overnight at 4°C. After washing, the membrane was incubated with peroxidase-labeled secondary antibody for 2 h at room temperature. The protein bands were visualized using the ECL system (Amersham, Buckinghamshire, UK) and analyzed using the Gel-Pro32 software (8).

BALB/c-nu mice (aged 6 weeks, weighted 18–22 g, and half male and half female) were purchased from Shanghai National Center for Laboratory Animals (Shanghai, People's Republic of China). They were maintained in specific pathogen-free conditions. Animal care, surgical procedures, and experimental protocols were approved by the Medical Ethics Committee on the Care and Use of Laboratory Animals of Shanghai Jiao Tong University.

The SK-BR-3 cells in the logarithmic growth phase were taken, and centrifuged (1,200 r/min) for 5 min. The cells were then resuspended with the cell density adjusted to 5 × 10⁶ cells/mL. 200 μl of the cell suspension was used to subcutaneously inoculate cells on the right hindlegs of mice and the injected site developed grain-sized tumors. When the tumor size reached 75–150 mm³, a total of 36 nude mice were randomly divided into six groups: Group A, control group, mice were treated with 0.2 mL 0.9% normal saline; Group B, MNPs-Fe₃O₄; Group C, PTX; Group D, PTX-MNPs-Fe₃O₄; Group E, PTX and Herceptin; Group F, PTX-Herceptin-MNPs-Fe₃O₄. These nude mice were injected intravenously with the respective treatment every other day. Based on the drug loading efficiency (7, 10), PTX concentration was 1 mg/kg and Herceptin concentration was 300 ug/kg.

Toxicity symptoms were monitored throughout the entire study period. Length (a) and width (b) of tumors were measured using a digital caliper and tumor volume (V/mm³) was calculated according to the formula V = 1/2 (a × b²), where a and b represented the longest and shortest diameter, respectively (9). Change in tumor size among different groups was recorded as the relative tumor volume (RTV) using the formula RTV = V_X/V₁, where V_X and V₁ represented the volumes on day X and the first day of tumor treatment, respectively. Antitumor inhibition rate was defined as the inhibitory rate, calculated using the formula inhibitory rate (IR) (%): (1-average experimental group RTV/average control group RTV) × 100%. All studies were performed in adherence to the Guidelines for the Care and Use of Laboratory Animals established by the National Institute of Health (11).

Tumor tissue was cut into 5 μm slices, and expression of cleaved-Caspase-3 protein was detected by an immunohistochemical staining SP method. The sections were incubated with anti-cleaved-Caspase 3 antibody (working dilution 1:100) at 4°C overnight. After washing, the sections were reintubated with a secondary anti-mouse biotinylated antibody (1:1,000) in a dark room for 1 h. Positive cells were counted within five randomly selected horizons for each slice at a magnification of 400 × (8, 11).

The German Bruker 7.0T Micro-MR imaging system was used to detect magnetic resonance imaging of tumors in tumor-bearing mice. The inner diameter of the horizontal scanning frame was 10 cm and the mouse coil was used. The mice were anesthetized with 5% isoflurane and placed onto a plexiglass scanning bed. The head of the mouse was fixed. The anesthesia state of the mice was maintained with 1.5% isoflurane air mixed gas. The respiratory rate was adjusted to 40–60 times/min. PTX-Herceptin-MNPs-Fe₃O₄ was injected into the tail vein before (0 h), and 8 h after injection to perform magnetic resonance scanning imaging. The magnetic resonance imaging sequence and parameters were as follows: FLASH-multi-slice T2 ^* (Fast Low Angle Shot, FLASH) sequence: repetition time TR = 333.1 ms, echo time TE = 5.0 ms, flip angle (Flip Angle): 30.0°, 1 repetition time, Field of view (FOV): 4 cm × 4 cm, acquisition matrix 512 × 512, layer thickness 1 mm, interval 0–0.1 mm.

Tumor-bearing mice were treated with local hair removal and cleaned with warm water to minimize the non-specific fluorescence effects on relevant areas. The mice were placed in a gas anesthesia system for isoflurane anesthesia and scanned, followed by PTX-Herceptin-MNPs-Fe₃O₄ injection through the tail vein at 0–8 h for whole-body near-infrared fluorescence distribution imaging. The optical imaging parameters were as follows: excitation wavelength 704 nm, NIR emission filter.

Quantitative data were described as means ± standard deviations (SDs). Intergroup differences were analyzed by F-test. The threshold for significance was P = 0.05. All statistical analyses were conducted using SPSS, Version 15.0 (SPSS Inc., Chicago, IL, USA).

The drug loading efficiencies were 5.5% ± 0.2% for PTX and 3.1% ± 0.1% for Herceptin, and the encapsulation efficiencices were 84.5% ± 2.3% for PTX and 77.6% ± 1.9% for Herceptin, respectively. The molar ratio of PTX to Herceptin was ~2:1 in the NPs, which was an appropriate proportion for targeted efficacy. There were no significant differences in drug loading and encapsulation efficiencies between the repeated batches of MNPs-Fe₃O₄. These results further proved that the formulation process was stable and that the MNPs-Fe₃O₄ could be an effective drug delivery carrier.

Figure 1A displays the findings of the TEM analysis of Fe₃O₄@OA nanoparticles from which we found that most nanoparticles were spherical in shape and uniform in 10-nanometer size. As shown in Figure 1B, the saturation magnetization of Fe₃O₄@OA was 77 emu/g while the coercivity and remanence were zero, indicating that nanoparticles had strong magnetic and superparamagnetic properties.

According to the TEM image (Figure 1C) of negatively stained PTX-Herceptin -MNPs-Fe₃O₄ with 2% phosphotungstic acid, all samples acquired a typical monodisperse core-shell structure (the clear white circles of outer layers). In addition to the core of the magnetic particle, the outer layers of this structure were mainly composed of phospholipid molecules (2 nm). The fluorescent molecules Cy7 and the drug PTX were inserted into the white lipid layer on the surface of the magnetic particles, indicating that the nanoparticles had significant fluorescence properties and drug loading capacities. The DLS results (Figure 1D) identified that the hydrodynamic size of PTX-Herceptin-MNPs-Fe₃O₄ was 28 nm.

The dose-effect curve for the inhibition rate when SK-BR-3 cells were exposed to MNPs-Fe₃O₄ (5–40 μg/mL) for 48 h was shown in Figure 2A (7), and MNPs-Fe₃O₄ with concentrations of <40 μg/mL had no obvious effect on proliferation (P > 0.05). The anticancer efficacy of nanoparticles is reflected by their cytotoxicity effect. PTX enhanced the rate of inhibition of SK-BR-3 cells in a dose-dependent manner; furthermore, PTX-MNPs-Fe₃O₄ increased the cytotoxicity effect on SK-BR-3 cells compared with PTX (Figure 2B).

SK-BR-3 cells have positive HER2 expression, and MDA-MB-231 cells belong to the basal-like subtype due to the negative estrogen receptor (ER), progesterone receptor (PR), and HER2 expressions (10). SK-BR-3 and MDA-MB-231 breast cancer cells were used to detect the targeting effect. We evaluated the combinations of PTX-MNPs-Fe₃O₄ with different concentrations of Herceptin to find the optimal combination targeting SK-BR-3 cells. MTT assays using the SK-BR-3 and MDA-MB-231 cell lines were performed to analyze the potential cytotoxicity of PTX-Herceptin-MNPs-Fe₃O₄. As shown in Figure 2C, the SK-BR-3 cell viability after PTX-Herceptin-MNPs-Fe₃O₄ treatment was negatively correlated with the concentration of Herceptin. As shown in Figure 2D, the PTX-Herceptin-MNPs-Fe₃O₄ exhibited the strongest cytotoxicity among all the groups (control group, MNPs-Fe₃O₄ group, PTX group, PTX-MNPs-Fe₃O₄ group, PTX-Herceptin group, and PTX-Herceptin-MNPs-Fe₃O₄ group) using the SK-BR-3 cell line (P < 0.05). However, there was no significant difference in the viability of MDA-MB-231 cells between the PTX-MNPs-Fe₃O₄ and PTX-Herceptin-MNPs-Fe₃O₄ groups (P > 0.05).

Figure 3A presents no significant difference in apoptosis rate between SK-BR-3 cells treated with MNPs-Fe₃O₄ (4.66% ± 0.51%) and the control group (5.21% ± 0.82%) (P > 0.05). The apoptosis rates of SK-BR-3 cells treated with PTX and PTX-MNPs-Fe₃O₄ were 13.78% ± 0.33% and 20.53% ± 0.42%, respectively. Besides, the rate was 20.22% ± 0.27% for SK-BR-3 cells induced by 0.5 μmol/L PTX-Herceptin, and 31.95% ± 0.64% (P < 0.05) for cells treated by PTX-Herceptin-MNPs-Fe₃O₄, respectively. Among the different treatments (Figure 3B), there was no significant difference in the apoptosis rate of MDA-MB-231 cells between the PTX-MNPs-Fe₃O₄ (16.57% ± 1.28%) and PTX-Herceptin-MNPs-Fe₃O₄ groups (17.19% ± 0.94%) (P > 0.05). The results indicated that PTX-Herceptin-MNPs-Fe₃O₄ had no targeted effect on MDA-MB-231 cells with no expression of HER2 antigen.

As shown in Figure 3C, in comparison with the control group, the levels of cleaved PARP were higher in the PTX, PTX-MNPs-Fe₃O₄, PTX-Herceptin, and PTX-Herceptin-MNPs-Fe₃O₄ groups. The level of cleaved PARP was markedly higher in the PTX-Herceptin-MNPs-Fe₃O₄ group than in the other groups (P < 0.05; Figure 3D).

All mice were injected via tail veins for 14 days and no obvious symptoms of toxicity were observed during the treatment. As shown in Figure 4A, there was no obvious difference in the inhibitory rate between the control group and the MNPs-Fe₃O₄ group (P > 0.05) after treatment. The inhibition rates in the PTX group, PTX-MNPs-Fe₃O₄ group, PTX-Herceptin group, and PTX-Herceptin-MNPs-Fe₃O₄ group were 31.46% ± 2.67%, 51.89% ± 3.58%, 52.68% ± 4.12%, and 70.14% ± 3.63%, respectively. The tumor inhibition rate of the PTX-Herceptin-MNPs-Fe₃O₄ group (70.14% ± 3.63%) was significantly higher than the PTX-Herceptin group (52.68% ± 4.12%) (P < 0.05). These results suggested that PTX-Herceptin-MNPs-Fe₃O₄ had the strongest effect of tumor inhibition on SK-BR-3 xenografts.

Immunohistochemistry was used to detect the expression of cleaved-Caspase-3 in the tumor tissues of nude mice (Figure 4B). Immunohistochemical staining for positive expression was shown as fine brown particles, which were mainly located on the cell membrane and in the cytoplasm of tumor cells, and which exhibited diffuse or focal distribution. The PTX-Herceptin-MNPs-Fe₃O₄ group showed the strongest cleaved-Caspase-3 expression. Compared with the other two groups (control group and MNPs-Fe₃O₄ group), the levels of cleaved-Caspase-3 in the PTX group, PTX-MNPs-Fe₃O₄ group, PTX-Herceptin group, and PTX-Herceptin-MNPs-Fe₃O₄ group were higher with more staining-positive cells. However, there was no significant difference between the control group and the MNPs-Fe₃O₄ group.

We selected mice subcutaneously inoculated with tumors as animal models to examine the in vivo effects of PTX-Herceptin-MNPs-Fe₃O₄ (300 μg Herceptin/kg). We loaded the fluorescent component Cy7 into the hydrophobic inner layers of PEG-modified magnetic nanoprobe, and achieved a dual-modal nanostructure with both optical and magnetic properties. Therefore, we used the near-infrared fluorescence and magnetic resonance imaging (MRI) technology to monitor the enrichment of magnetic nanoprobes in mouse tumors both intuitively and in situ.

During the systemic circulation, magnetic nanoprobe was partially engulfed in liver and spleen due to the unique physiological structure of the tumor tissue (i.e., the permeability and retention (EPR) effect), as well as the surface of the magnetic nanoprobe modified by the PEG molecule with anti-RES phagocytosis and tumor-targeted Herceptin antibody. Another part of the nanoprobe was selectively distributed in tumor tissue and had a long-term enrichment. Fluorescence intensity at the tumor site was greatly enhanced as shown in Figure 5A.

From the MRI images (Figure 5B), the tumor became darker while the signal value of the tumor site reached a peak at 8 h after PTX-Herceptin-MNPs-Fe₃O₄ injection, which proved that PTX-Herceptin-MNPs-Fe₃O₄ had T2 imaging contrast enhancement effect on tumor in tumor-bearing mice. Also, we found that PTX-Herceptin-MNPs-Fe₃O₄ had a considerable amount of enrichment in tumors, laying a foundation for the subsequent treatment with release of anticancer drugs.