Preclinical Assessment of Paclitaxel- and Trastuzumab-Delivering Magnetic Nanoparticles Fe3O4 for Treatment and Imaging of HER2-Positive Breast Cancer

Objective: The purpose of this study was to investigate the anticancer activity and the potential imaging use of the innovative combination of magnetic nanoparticles (MNPs)-Fe3O4, paclitaxel (PTX), and trastuzumab (Herceptin) in HER2-positive breast cancer. Methods: MNPs-Fe3O4 was synthesized and underwent water phase transfer and hydrophobic molecular loading, and its surface was then coupled with Herceptin mono-antibody. The morphological characteristics of MNPs-Fe3O4 were observed under transmission electron microscopy (TEM). Effects of PTX-Herceptin-MNPs-Fe3O4 on breast cancer cells were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide assay and the flow cytometric apoptosis assay. To establish a xenograft model, we injected breast cancer SK-BR-3 cells into the left thighs of nude mice. We measured the effect of PTX-Herceptin-MNPs-Fe3O4 on tumor growth by measuring tumor size and calculating inhibition rate with immunohistochemistry analysis further performed, and analyzed MNPs-Fe3O4 accumulation in tumor lesions using in vivo magnetic resonance imaging and in vivo fluorescence imaging. Results: Most MNPs were in spherical shape of about 10 nm in diameter observed under TEM. PTX-Herceptin-MNPs-Fe3O4 showed greater cytotoxic effects, and induced a higher apoptosis rate of SK-BR-3 cells than all the other groups, with corresponding changes of apoptosis-related proteins. Meanwhile, the in vivo tumor xenograft model showed that tumor inhibition rate in the PTX-Herceptin-MNPs-Fe3O4 group was higher than in the PTX-Herceptin group. Furthermore, PTX-Herceptin-MNPs-Fe3O4 enhanced the T2 imaging contrast enhancement effect on tumors in tumor-bearing mice. Conclusion: The novel PTX-Herceptin-MNPs-Fe3O4 combination may represent a promising alternative breast cancer treatment strategy and may facilitate tumor imaging.


INTRODUCTION
Breast cancer (BC) is one of the most common cancers, and the second leading cause of cancer-related mortality worldwide, representing a grievous threat to women's health and quality of life (1,2). Human epidermal growth factor receptor-2 (HER-2) positive breast cancer accounts for 20-25% of all BC molecular subtypes. Its associations with aggressive tumor growth and inferior prognosis have been well-studied (3). HER-2 is a protooncogene that is negatively or minimally expressed in normal tissues and its overexpression could lead to excessive growth and enhanced invasiveness of tumor cells. HER-2-targeted agents effectively inhibit HER-2 expression, thereby achieving anti-tumor effect. Trastuzumab is a humanized anti-HER-2 monoclonal antibody that was first used in the therapy of HER-2 positive BC with changing its natural biology.
Paclitaxel (PTX) is clinically used as a chemotherapy agent in BC treatment. The efficacy of PTX combined with Herceptin for BC treatment is better than that of Herceptin alone. Several studies pointed out that BC cells exposed to PTX could produce HER2 receptor functional upregulation, making tumors more impressionable to the antiproliferative effects of Herceptin. The combination of Herceptin and PTX is recognized by the international medical community as an effective firstline treatment for patients with metastatic HER-2 positive BC (4). However, chemotherapeutic toxicity affects the application of chemotherapeutical drugs. Therefore, there has been an increasing demand for a more effective targeted drug delivery system addressing the issues of both chemotherapy resistance and toxicity, which has become a critical topic in cancer chemotherapy treatment.
Nanoparticles (NPs) can be used as carriers for transporting endocytosed drugs into tumor cells. Magnetic NPs (MNPs), in particular, offers the following advantages: First, it could offer targeted drug delivery by applying a magnetic field and connecting targeting ligands. Second, MNPs could overcome the limitations of traditional chemotherapy in terms of system distribution. Third, MNPs could generate synergistic effect with certain chemotherapy drugs through increasing the sensitivity of tumor tissues and the concentration of drugs in tumor cells (5,6). With respect to magnetic nanoparticles Fe 3 O 4 (MNPs-Fe 3 O 4 ), it is considered to be one of the most promising nano biomaterials for its dual advantages of nanoparticles and magnetic property, such as MRI response to an external magnetic field (5). MNPs-Fe 3 O 4 with superparamagnetism is a relatively simple preparation process and has an outstanding biocompatibility (6).
Interestingly, MNPs-Fe 3 O 4 is able to accumulate at the tumor site under influence of the magnetic field after entering the human host, thereby causing embolization of tumor blood vessels to make tumor tissues ischemic and necrotic, and then promotes tumor sensitivity to anticancer drugs. Previous evidence demonstrated better outcomes and fewer side effects when MNPs-Fe 3 O 4 served as carriers than traditional nontarget drugs in treatment for lung, pancreatic, and hematological cancers (6,7). We retrieved very few studies reporting the use of MNPs-Fe 3 O 4 in HER-2positive BC therapies.
In this study, we synthesized a new type of nanoparticulate system whose surface was modified with Herceptin, and which consisted of biocompatible and biodegradable MNPs-Fe 3 O 4 . We prepared MNPs-Fe 3 O 4 with superior crystallinity by high-temperature pyrolysis technology. Such high-performance was mainly reflected in size uniformity, regular morphology, higher magnetism and magnetocaloric effects, greater targeting ability, longer systemic circulation, and better biocompatibility. Our synthetic targeted MNPs are as follows. Oil-soluble iron oxide nanoparticles were synthesized using the hightemperature thermal decomposition technology. The surface of the nanoparticles was modified with DSPE-PEG-COOH to make it hydrophilic and the active functional group COOH was then loaded onto the surface. PTX and fat-soluble fluorescent dye Cy7 were loaded into the lipid layer. Herceptin monoclonal antibody (mAb) was attached to the surface of the nanoparticles. The targeted MNPs-Fe 3 O 4 enhanced cellular drug absorption by cancer cells and increased their sensitivity to chemotherapy drugs, suggesting that the targeted MNPs-Fe 3 O 4 might be an optimal nano drug delivery system (NDDS) for the clinical therapy of specific malignancies.

Preparation of MNPs-Fe 3 O 4 (Fe 3 O 4 @OA)
The MNPs-Fe 3 O 4 was synthesized and characterized by State Key Laboratory of Bioelectronics (Southeast University, People's Republic of China). MNPs-Fe 3 O 4 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 threenecked 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 Loading efficiency (LE; %) = (amount of drug in drug − loaded NPs/amount of drug − loaded NPs) × 100%; Encapsulation efficiency (EE; %) = (amount of drug in drug − loaded NPs/initial amount of drug) × 100% 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.

MNPs-Fe 3 O 4 Water Phase Transfer and Hydrophobic Molecular Loading (MNPs-Fe 3 O 4 @PEG)
The PEGylated long-circulating lipid DSPE-PEG2000 molecule was modified on the surface of MNPs-Fe 3 O 4 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-Fe 3 O 4 (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.

MNPs-Fe 3 O 4 Surface Coupled With Herceptin mAb (MNPs-Fe 3 O 4 @PEG@mAb)
The 15 mg MNPs-Fe 3 O 4 @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.

Determination of Drug Loading and Encapsulation Efficiencies
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):

Characterization of Sample Physical and Chemical Properties
Transmission electron microscopy (TEM) was used to characterize morphologic size of MNPs-Fe 3 O 4 and MNPs-Fe 3 O 4 @PEG, and dynamic light scattering (DLS) was used for magnetic nanoprobe MNPs-Fe 3 O 4 @PEG@mAb characterization of hydrodynamic dimensions. A vibrating sample magnetometer (VSM) was used to detect the saturation magnetization of MNPs-Fe 3 O 4 to verify the properties of magnetic iron oxide nanoparticles.

Microscopic Characteristics of Drug-Loaded NPs
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-Fe 3 O 4 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.

Cell Line and Culture
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% CO 2 . 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.

MTT Assay
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 × 10 3 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).

Flow Cytometric (FCM) Apoptosis Assay
SK-BR-3 and MDA-MB-231 breast cancer cells were seeded into six-well plates at the density of 4 × 10 5 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).

Western Blot Analysis
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).

BC Xenograft Model in Nude Mice
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 × 10 6 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 mm 3 , a total of 36 nude mice were randomly divided into six groups: Group A, control group, mice were treated with 0.

Tumor Growth Measurement and Inhibition Rate
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/mm 3 ) was calculated according to the formula V = 1/2 (a × b 2 ), 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 /V 1 , where V X and V 1 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).

Immunohistochemistry Analysis
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 anticleaved-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).

In vivo Magnetic Resonance
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-

In vivo Fluorescence Imaging
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-Fe 3 O 4 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.

Statistical Analysis
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).

Drug Loading and Encapsulation Efficiencies
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-Fe 3 O 4 . These results further proved that the formulation process was stable and that the MNPs-Fe 3 O 4 could be an effective drug delivery carrier. Figure 1A displays the findings of the TEM analysis of Fe 3 O 4 @OA nanoparticles from which we found that most nanoparticles were spherical in shape and uniform in 10nanometer size. As shown in Figure 1B, the saturation magnetization of Fe 3 O 4 @OA was 77 emu/g while the coercivity and remanence were zero, indicating that nanoparticles had strong magnetic and superparamagnetic properties.

Characteristics of MNPs-Fe 3 O 4
According to the TEM image ( Figure 1C) of negatively stained PTX-Herceptin -MNPs-Fe 3 O 4 with 2% phosphotungstic acid, all samples acquired a typical monodisperse coreshell 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-Fe 3 O 4 was 28 nm.

MNPs-Fe 3 O 4 Enhanced the Proliferation-Inhibiting and Cytotoxicity Effects of PTX and Herceptin in vitro
The dose-effect curve for the inhibition rate when SK-BR-3 cells were exposed to MNPs-Fe 3 O 4 (5-40 µg/mL) for 48 h was shown in Figure 2A Figure 3D).

MNPs-Fe 3 O 4 Increased the Tumor Inhibition Capability of PTX and Herceptin in vivo
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-

PTX-Herceptin-MNPs-Fe 3 O 4 had T2 Imaging Contrast Enhancement Effect on Tumor in vivo
We selected mice subcutaneously inoculated with tumors as animal models to examine the in vivo effects of PTX-Herceptin-MNPs-Fe 3 O 4 (300 µg Herceptin/kg). We loaded the fluorescent component Cy7 into the hydrophobic inner layers of PEG-modified magnetic nanoprobe, and achieved a dualmodal 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-Fe 3 O 4 injection, which proved that PTX-Herceptin-MNPs-Fe 3 O 4 had T2 imaging contrast enhancement effect on tumor in tumor-bearing mice. Also, we found that PTX-Herceptin-MNPs-Fe 3 O 4 had a considerable amount of enrichment in tumors, laying a foundation for the subsequent treatment with release of anticancer drugs.

DISCUSSION
Nanoparticles have developed rapidly for cancer treatment as they overcome the limitations of conventional small molecule chemotherapy drugs. Relying on the nano drug carrier system, a meaningful approach should include reducing systemic dose, improving local drug concentration in tumor tissues, and achieving targeted drug delivery (12). As nanomedicine carriers, magnetic nanoparticles have come into focus currently because of their potential on multi-targeting (13,14). First, MNPs could conduct passive targeting by enhanced EPR, while targeting actively by magnetic field effect or surface modification of targeting ligands, thus play its role effectively on antitumor activities (15). Moreover, with the characteristic of tumor cells targeted, MNPs are able to help with the reduction of drug dosage. This is exemplified by the combination of MNPs and paclitaxel, methotrexate, mitoxantrone, and adriamycin, which improve the target specificity (16). Such nanocomposites with magnetic and pH dual-responsive performance provided an outstanding platform for enhanced drug-resistant BC treatment, and achieved not only chemotherapy photodynamic therapy in tumor treatments, but controlled drug release and alleviated side effects (17). Rejinold et al. synthesized MNPs-Fe 3 O 4 with noninvasive radiofrequency (RF) to prolong its circulation time in 4T1 breast cancer cells and enhance apoptosis effects (18).
Compared with other magnetic nanomaterials, MNPs-Fe 3 O 4 is more stable with higher hardness. Because of its strong magnetism, as well as simple preparation and good biocompatibility, Fe 3 O 4 is potentially for magnetically guided drug delivery. Our previous study has proved that Fe 3 O 4 remains stable within the water phase and therefore is able to elongate blood circulation without been rapidly metabolized (7). What we know about MNPs-Fe 3 O 4 is largely based upon studies investigating its use as drug carriers and synergistic chemotherapy drugs against leukemia resistance (6,13). It has been shown in vivo and in vitro and in clinical experiments that MNPs-Fe 3 O 4 has lower toxicity, and better effects in reversing the resistance of synergistic chemotherapy drugs.
In this study, we innovatively synthesized a new type of nanoparticle system (Figure 6), whose surface was modified with Herceptin, and which consisted of biocompatible and biodegradable magnetic nanoparticles (MNPs)-Fe 3 O 4 . We

CONCLUSION
A new targeted nano drug delivery system is constructed using MNPs-Fe 3 O 4 combined with Herceptin and the anti-tumor drug PTX. It effectively delivers anti-tumor drugs to breast cancer cells, and significantly improves the efficacy of chemotherapy. Our findings provide an innovative theoretical basis for clinical application by reducing dose and side effects of chemotherapy drugs on the basis of nanotechnology.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Medical Ethics Committee on the Care and Use of Laboratory Animals of Shanghai Jiao Tong University.
LG: drafting of the manuscript and statistical analysis. HZ, PL, TM, DH, LS, LH, YS, and JZ: critical revision of the manuscript for important intellectual content. YS and JZ: administrative, technical, or material support. All authors contributed to the article and approved the submitted version.