Fabrication of Hybrid Nanostructures Based on Fe3O4 Nanoclusters as Theranostic Agents for Magnetic Resonance Imaging and Drug Delivery

Combining anticancer drugs with inorganic nanocrystals to construct multifunctional hybrid nanostructures has become a powerful tool for cancer treatment and tumor suppression. However, it remains a critical challenge to synthesize compact, multifunctional nanostructures with improved functionality and reproducibility. In this study, we report the fabrication of magnetite hybrid nanostructures employing Fe3O4 nanoparticles (NPs) to form multifunctional magnetite nanoclusters (NCs) by combining an oil-in-water microemulsion assembly and a layer-by-layer (LBL) method. The Fe3O4 NCs were firstly prepared via a microemulsion self-assembly technique. Then, polyelectrolyte layers composed of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) and doxorubicin hydrochloride (DOX) were capped on Fe3O4 NCs to construct the Fe3O4 NC/PAH/PSS/DOX hybrid nanostructures via LBL method. The as-prepared hybrid nanostructures loaded with DOX demonstrated the pH-responsive drug release and higher cytotoxicity towards human lung cancer (A549) cells in vitro and can serve as T2-weighted magnetic resonance imaging (MRI) contrast agents, which can significantly improve T2 relaxivity and lead to a better cellular MRI contrast effect. The loaded DOX emitting red signals under excitation with 490 nm are suitable for bioimaging applications. This work provides a novel strategy to build a Fe3O4-based multifunctional theranostic nanoplatform with T2-weighted MRI, fluorescence imaging, and drug delivery. Electronic supplementary material The online version of this article (10.1186/s11671-019-3026-7) contains supplementary material, which is available to authorized users.


Introduction
In recent years, various multifunctional drug delivery systems have been developed for future diagnosis and therapy in biomedical application [1][2][3][4]. Multifunctional hybrid nanostructures that are integrated favorable properties will possess significant applications such as multimodal imaging and simultaneous diagnosis and therapy [5][6][7][8][9][10][11]. Furthermore, these nanostructures are stimuliresponsive drug delivery systems for improved drug accumulation, enhanced therapeutic efficacy, and/or reduced side effects. Especially, these pH-responsive drug delivery systems have attracted extensive research interest. This is because most human tumors have a more acidic pH value, which provides a possible way to design the controlled release of drug molecules [12][13][14][15][16].
Over the past few decades, various hybrid nanostructures by combining inorganic nanomaterials with organic polymer [17][18][19][20] have been developed, including magnetic particles [21][22][23], upconversion nanoparticles (NPs) [17,24], and mesoporous silica particles [25]. Among those, magnetic hybrid nanostructures based on iron oxides with relatively large magnetization at room temperature have been widely used in the biomedical fields [26][27][28][29]. The functionalization of inorganic nanomaterials coated with polyelectrolyte layers can realize a pH-responsive encapsulation and release of drug molecules [12,17,30]. More recently, the polyelectrolyte layers composed of sodium poly (styrene sulfonate) (PSS) and the polycation poly(allylamine hydrochloride) (PAH) has been widely studied [31][32][33][34][35][36]. Polyelectrolyte layers combined with magnetic and luminescent NPs or drug molecules for multifunctional drug delivery systems have also been recently reported [37][38][39]. Iron oxide (Fe 3 O 4 ) NPs are getting more and more attention in the field of magnetic resonance imaging (MRI) and drug delivery due to their unique superparamagnetic properties, biocompatibility, low-cytotoxicity, and flexibility [9,11,28,29,[40][41][42]. In general, there are two methods to improve the magnetic responsiveness of Fe 3 O 4 NPs. The first one is to synthesize the micrometer-sized magnetite particles. Due to large size, however, they tend to aggregate in aqueous solution, which is not beneficial to biomedical applications. The other approach is to assemble Fe 3 O 4 NPs into nanoclusters (NCs). These Fe 3 O 4 NCs greatly improved the magnetic responsiveness compared to individual Fe 3 O 4 NPs [22,43]. Therefore, if the self-assembled Fe 3 O 4 NCs are adopted as the core to fabricate multifunctional hybrid nanostructures, the MRI performance will be improved by the collective effect of Fe 3 O 4 NPs [43][44][45]. To our knowledge, the self-assembled Fe 3 O 4 NCs functionalized with PAH/PSS multilayers for pH-responsive drug release have rarely been reported.
In this work, a versatile theranostic nanoplatform based on Fe 3 O 4 NPs was built up for MRI and drug delivery. In our approach, Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures were obtained by combining an oil-in-water microemulsion method and a layer-by-layer (LBL) electrostatic adsorption method. It is expected that the packed Fe 3 O 4 NC system can lead to enhanced T 2 relaxivity and imaging contrast, and the large specific surface area of Fe 3 O 4 NC/PAH/PSS hybrid nanostructures allows high loading of anticancer drugs. Moreover, in vitro experiment exhibits that the cellular MRI contrast of human lung cancer (A549) cells incubated with Fe 3 O 4 NC/PAH/PSS/DOX has been significantly enhanced.

Preparation of Ferric Oleate
The synthesis of magnetic NPs started from the synthesis of ferric oleate. FeCl 3 ·6H 2 O (2.59 g), NaOA (14.6 g), C 2 H 5 OH (32 mL), H 2 O (24 mL), and hexane (56 mL) were mixed together in a 150-mL three-neck flask and heated to 70°C for reflux for 4 h to form a transparent ferric oleate complexes solution. After that, the liquid was separated by a separation funnel and the upper oil layer was preserved. Hexane in the liquid evaporated at 70°C by rotating evaporation and dried for 48 h under vacuum. The prepared samples were stored in a vacuum glove box for further use.

Synthesis of Fe 3 O 4 NPs
We synthesized Fe 3 O 4 NPs following previously reported procedures with slight modification [46]. Ferric oleate (7.2 g), OA (1.28 mL), and ODE (50 mL) were mixed together in a 100-mL three-neck flask and heated to 300°C for 40 min under argon protection; after that, the mixture was cooled to room temperature and oxidized in air for more than 12 h. The resultant nanocrystals were precipitated by the addition of isopropanol, centrifuged, and washed twice with an ethanol-water mixture (1:1 v/v). The oleic acid-capped Fe 3 O 4 NPs were finally dispersed in 200 mL cyclohexane, and the supernatant was sealed and stored for the subsequent experiments.

Preparation of Fe 3 O 4 NCs
Fe 3 O 4 NCs were prepared by a facile and straightforward microemulsion self-assembly technique as previously described with modification [47]. Briefly, a 200-μL solution of Fe 3 O 4 nanocrystals in cyclohexane was poured into 4 mL of aqueous solution containing 14 mg of SDBS. The mixed solution underwent ultrasonic treatment for 5 min for 4 times. The formed solid-in-oil-in-water (S/O/W) emulsion was stirred at room temperature for 6 h to evaporate the organic solvent following by the self-assembly of Fe 3 O 4 NPs to form 3D NCs. The final products were washed with deionized water 3 times to remove the excess SDBS, unincorporated nanocrystals, and some possible larger contaminants.

Preparation of Fe 3 O 4 NC/PAH/PSS/DOX Hybrid Nanostructures
The Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures were prepared by electrostatic attractive interactions. The as-prepared Fe 3 O 4 NCs are negatively charged owing to the encapsulation of the anionic surfactants. They were first turned to be positively charged by adsorption of a layer of positively charged polyelectrolyte, poly(allylamine hydrochloride) (PAH, MW 15 000). Specifically, a 300-μL Fe 3 O 4 NC sample was firstly diluted 10 times to 3 mL using deionized water. The Fe 3 O 4 NC mixture was subsequently added dropwise to an aqueous PAH solution (1 mL, 10 g/L, 4 mM NaCl) under vigorous stirring. After the solution was stirred for 24 h, the excess PAH was removed by centrifugation, and the resultant PAH-coated Fe 3 O 4 NCs (Fe 3 O 4 NC/PAH) were redispersed in water (3 mL).
The Fe 3 O 4 NC/PAH were then turned to be negatively charged by adsorption of a layer of negatively charged polyelectrolyte, poly-(sodium 4-styrenesulfonate) (PSS, MW 70 000). Specifically, a 3-mL Fe 3 O 4 NC/PAH sample solution was added dropwise to an aqueous PSS solution (1 mL, 10 g/L, 4 mM NaCl) under vigorous stirring. After the solution was stirred for 24 h, the excess PSS was removed by centrifugation, and the resultant PSS-coated Fe 3 O 4 NC/PAH (Fe 3 O 4 NC/PAH/PSS) were redispersed in water (3 mL).
The DOX aqueous stock solution was first prepared [17]. The concentration was 5.0 mg/mL. The hybrid nanostructure solution was obtained by mixing the Fe 3 O 4 NC/ PAH/PSS solution (3 mL, 32 mg/mL) and the stock DOX solution (60 μL) in a small plastic tube with stirring for 24 h in the darkroom. After centrifugation, the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures were obtained finally.

MRI Measurements
The MRI measurements were performed in an 11.7 T micro 2.5 micro-imaging system (Bruker, Germany). The different amount of the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures were dispersed in 1.2 mL agarose aqueous solution and then loaded into the microtubes for MRI measurements. The final Fe ion concentration were 0 mM, 0.013 mM, 0.026 mM, 0.032 mM, 0.041 mM, 0.052 mM, and 0.065 mM, respectively. The measurement parameters are as follows: repetition time (TR) = 300 ms, echo time (TE) = 4.5 ms, imaging matrix = 128 × 128, slice thickness = 1.2 mm, field of view (FOV) = 2.0 × 2.0 cm, and number of averages (NA) = 2.

Cellular Uptake and MR Imaging
To demonstrate efficient cellular uptake, the A549 cells were seeded on the coverslip in the confocal dish and incubated in a humidified 5% CO 2 atmosphere for 4 h at 37°C. Then, the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures were added into the incubation medium at the different concentration and incubated for 2 h. The final Fe ion concentrations were obtained as 0, 2.2, 4.5, 9.0, and 13.5 μM, respectively. After the medium was removed, the cells were washed twice with PBS (pH = 7.4, 20 mM) and directly used for MR imaging.

Standard Curve of DOX
A suitable quantity of DOX was dissolved in water by oscillation. Then, a series of different concentrations of DOX aqueous solution were prepared (0-0.03 mg/mL). The fluorescence intensity of different concentrations of DOX solution was measured (λ ex = 490 nm). Finally, the standard curve of DOX was determined through the curve fitting of the fluorescence intensity vs the DOX concentration.

DOX Loading and Release
To measure the loading capacities of the where W 0 and W s represent the initial DOX mass and the DOX mass in the supernatants, respectively. For the cumulative DOX release studies in PBS buffer solutions (pH 5.0 and 7.4) with the same NaCl concentration of 0.15 M, the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures were dispersed in 1.0 mL of buffer solution and then transferred into a dialysis bag. Then, it was kept in buffer solution and gently shaken at 37°C in the darkroom. At selected time intervals, 100 μL of solution was withdrawn and analyzed by fluorescence spectrum, and then returned to the original solution.

In Vitro Cytotoxicity of Fe 3 O 4 NC/PAH/PSS/DOX Hybrid Nanostructures
In vitro cytotoxicity of the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures was assessed against A549 cells based on the standard methyl thiazolyltetrazolium (MTT) assay. A549 cells were cultured in APMI 1640 growth medium complemented with 10% fetal bovine serum (FBS), streptomycin at 100 μg/mL, and penicillin at 100 μg/mL. The cells were maintained at 37°C in a humidified atmosphere of 5% CO 2 in air. The assay was performed in triplicate with the same manner. Briefly, A549 cells were seeded into 96well plates at a density of 8 × 10 3 cells per wells in 100 μL of media. After overnight growth, the cells were then incubated at various concentrations of free DOX, Fe 3 O 4 NC/ PAH/PSS, and Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures (0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0 μM) for 24 h. After being incubated for 24 h, the 10 μL 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (5 mg/mL) was then added each well and the cells were further incubated for 4 h at 37°C. After the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution was removed, 150 μL of dimethyl sulfoxide (DMSO) was added to each well and the plate was gently shaken for 10 min to dissolve the precipitated violet crystals. The optical density (OD) was measured at 490 nm using microplate reader (Perkin Elmer, Victor X4). Cell viability was evaluated as a percentage compared to control cells.

Characterization
The sizes and morphologies of Fe 3 O 4 NPs and Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures were examined by a FEI Tecnai G2-F20 transmission electron microscope (TEM) at an accelerating voltage of 200 kV. Dynamic light scattering (DLS) measurements were performed on a particle size and zeta potential analyzer from Malvern (Zetasizer Nano ZS90). The UV-vis absorption spectra were acquired by a Perkin Elmer Lambda-25 UV-vis spectrometer. The fluorescence spectra were recorded using a Hitachi F-4600 fluorescence spectrophotometer. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Agilent 5100) was used to analyze the element Fe concentrations in the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures.

Results and Discussion
The Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures are prepared by self-assembly of primary iron oxide (Fe 3 O 4 ) NPs resulting into densely packed spherical aggregates through a microemulsion self-assembly technique as previously described with modification [17,47], following by a LBL electrostatic adsorption method. Figure 1 illustrates the schematic illustration of synthesis of Fe 3 O 4 NC/PAH/ PSS/DOX hybrid nanostructures. Hydrophobic oleic acidcoated magnetite Fe 3 O 4 NPs were initially produced by the thermal decomposition process in organic solvent [46]. Fe 3 O 4 NPs are spherical and uniform in size with an average particle size of about 15 nm (Additional file 1: Figure S1). For the assembly of magnetic NCs, the OAcoated Fe 3 O 4 NPs were dispersed in cyclohexane and then added dropwise to an aqueous solution containing SDBS. The complex solution was ultrasonically treated to form a stable oil-in-water emulsion. After the evaporation of organic solvent in the emulsion, Fe 3 O 4 NPs were selfassembled to form spherical nanoclusters via hydrophobic interaction. Next, the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures were prepared via a LBL method through electrostatic attractive interactions, which is schematically illustrated in Fig. 1.
The morphologies and sizes of the Fe 3 O 4 NCs and the Fe 3 O 4 NC/PAH/PSS hybrid nanostructures were examined with TEM and DLS, respectively. As shown in Fig. 2a and b, the Fe 3 O 4 NCs demonstrate the quasi-spherical clusters. The average particle size measured by DLS is about 57 nm (Fig. 2e). In previous reports, PAH with positive charge or PSS with negative charge is alternately deposited on the template surface due to their excellent electrostatic properties [48][49][50][51]. To study the formation of each polyelectrolyte layer deposited on the Fe 3 O 4 NCs, the zeta potential experiments were performed. The variation of zeta potential with the polyelectrolyte layer for PSS/PAH and DOX coatings are shown in Additional file 1: Figure S2 Fig. 2c and d. No significant structural and morphology difference is observed after polyelectrolyte coatings. Compared with Fig. 2a and b, the bright contrast can be observed and the size of the Fe 3 O 4 NC/PAH/PSS hybrid nanostructures is increased slightly owing to the coating of PAH and PSS layers. The synthesized magnetic hybrid nanostructures show a nearly mono-dispersed quasi-spherical shape with average size of about 84 nm according to the results of DLS measurement (Fig. 2f ).
To evaluate the potential application of Fe 3 O 4 NC/ PAH/PSS hybrid nanostructures in MRI, the proton transverse relaxation rates (1/T 2 ) as a function of Fe ion concentration were determined using Bruker AVANCE 500WB spectrometer at 11.7 T. A linear relationship between relaxation rates with Fe ion concentration was observed, as shown in Fig. 3b. Furthermore, the transverse relaxation rates (1/T 2 ) increased with increasing concentration of Fe 3 O 4 NCs due to the high degree of aggregation of the Fe 3 O 4 magnetic NPs core, demonstrating that the magnetic hybrid nanostructures could be an effective T 2 -weighted MRI contrast agent (Fig. 3a). Based on the slope of the plot in Fig. 3b, the transverse relaxivity value (r 2 ) was determined to be 651.38 mM −1 S −1 , which is higher than that of the reported work [22]. Compared with commercial T 2 contrast media, the nanoclusters can significantly improve the contrast ability of Fe after the magnetic NPs self-assembled on the basis of collective effect, thus greatly improving the angiographic effect. In the previous work, the assembled magnetite nanocrystals exhibited a higher level of saturation magnetization than individual nanocrystals due to the collective effect of magnetic nanocrystals [43,52].
To evaluate the drug loading capacity of Fe 3 O 4 NC/ PAH/PSS/DOX hybrid nanostructures as drug delivery carriers, a water-soluble anticancer drug (DOX) was chosen as a model drug. The storage of DOX in the hybrid nanostructures with a high efficiency was first revealed by the color change of the solution. The color of the solution of Fe 3 O 4 NC/PAH/PSS and the pure DOX solution was yellowish and red, respectively ( Fig. 4a and b). After forming of the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures, the color of the solution became orange (Fig. 4c). Owing to the presence of Fe 3 O 4 NPs, the DOX-loaded nanostructures in the suspension could be separated by an external magnet, suggesting that the great potential of the obtained hybrid nanostructures for magnetically targeted drug delivery (Fig. 4d). UVvis absorption spectroscopy was used to determine the effective DOX storage capacity. Figure 4e shows the UV-vis absorption spectra of the DOX aqueous solution before and after the interaction with Fe 3 O 4 NC/PAH/ PSS hybrid nanostructures. Compared with free DOX, the similar absorption peak characteristics were observed in the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures, which is the recombination absorption peak of Fe 3 O 4 NCs and DOX. The sample without DOX only shows the absorption peak of Fe 3 O 4 NCs. These data indicate that DOX as a drug can be successfully absorbed onto the surface of the hybrid nanostructures. It is also found that there is an upper limit of adsorption concentration of DOX loaded on the surface of the hybrid nanostructures. Figure 4f shows the PL spectrum of the Fe 3 O 4 NC/PAH/ PSS/DOX hybrid nanostructures after centrifugation when different concentrations of DOX were added into the Fe 3 O 4 NC/PAH/PSS solution. The luminescence intensity of DOX increases as an increase of added DOX until reaching a ceiling (8 mg/mL) with the concentration of Fe 3 O 4 NC/PAH/PSS (1.30 × 10 −2 mg/mL) unchanged. Afterwards, the entrapment amount decreases because of the excess DOX, which cannot be adsorbed on the surface of Fe 3 O 4 NC/PAH/ PSS/DOX hybrid nanostructures. The strongest fluorescence intensity of DOX corresponds to the concentration at 8 mg/mL, and the corresponding sample would be used for further study to perform biomedical experiment. The assured drug loading efficiency of the hybrid nanostructures is crucial for the clinical application. The loading efficiency was calculated by the area integral of DOX fluorescence intensity using the standard curve method of DOX [53,54]. The loading efficiency was calculated up to 24.39% for the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures. Therefore, a theranostic platform has been built up based on the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures due to the effective absorption of antitumor drug DOX. . The released DOX from the hybrid nanostructures was collected and then the release amount of DOX was calculated by fluorescence intensity of the supernatant. At physiological pH 7.4, the observed drug release is a slow release process. About 20 wt% of DOX was released at the initial 5 h, and then entered the stable stage of slow release. At pH 5.0, about 80 wt% of DOX was released from the hybrid nanostructures at the initial 15 h before a release plateau was reached. The plateau percentages of DOX release observed over a period of 30 h were 80 ± 3 wt% and 20 ± 3 wt% at pH 5.0 and 7.4, respectively. It can be seen that the Fe 3 O 4 NC/PAH/PSS/ DOX hybrid nanostructures have been revealed a sustained release profile and a higher DOX release rate at pH 5.0 than at pH 7.4. The low environment pH accelerates the DOX  That is due to the protonation of the -NH 2 group of DOX under acidic conditions, which reduces the electrostatic interaction between DOX and PSS polymers at low pH values [55]. The drug release studies indicate the good stability of electrostatically bound drug molecules in physiological pH and the triggered release at acidic conditions, similar to the reported works [56][57][58]. Therefore, the obtained Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures are the pH-responsive systems for DOX drug delivery and suitable for the specific treatment of solid tumors [59].
The cellular uptake and cytotoxicity are key factors to evaluate the potential of a new drug delivery system. The cellular uptake and cytotoxicity of Fe 3 O 4 NC/PAH/ PSS/DOX hybrid nanostructures on A549 cell lines were studied. The intercellular uptake of Fe 3 O 4 NC/PAH/ PSS/DOX hybrid nanostructures was investigated using optical and fluorescence microscopy, which was mainly realized by monitoring the fluorescence from DOX. The Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures have been proved to be effective in delivering DOX to cancer cells. As shown in Fig. 6, strong red fluorescence from DOX was observed in cancer cells after incubation for 24 h. The hybrid nanostructures were internalized mainly through endocytosis [60]. After cell uptake, the hybrid nanostructures released DOX in the acidic environment around the endosome/lysosomes, in which a sufficiently low pH (4.3) could trigger an effective DOX release (pH 5.0, Fig. 5). The Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures exhibited time-dependent uptake in A549 cancer cells, as shown in Fig. 6. At 0.5 h post incubation, red fluorescence was visible around the cells. The results show that the hybrid nanostructures containing DOX mainly stayed around the A549 cells. However, when the incubation time increased to 24 h, the intercellular fluorescence signal increased from the A549 cells. Obviously, many hybrid nanostructures can enter the cancer cells over time. These results confirm that the Fe 3 O 4 NC/ PAH/PSS/DOX hybrid nanostructures can efficiently transfer DOX into A549 cells. DOX released from the hybrid nanostructures in the cytoplasm passes through the nuclear membrane and eventually accumulates in the nucleus, killing cells by causing changes in DNA conformation [61].
In order to evaluate the pharmacological activity of the Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures, the cytotoxicity to A549 cells in vitro was determined by MTT method. Figure   These results indicate that both free DOX and Fe 3 O 4 NC/PAH/PSS/DOX hybrid nanostructures have dose-dependent cytotoxicity to cancer cells. The cytotoxicity originates from the loaded DOX rather than Fe 3 O 4 NC/ PAH/PSS hybrid nanostructures. Cell uptake of free DOX is faster than that of DOX-loaded hybrid nanostructures. This reason is that small DOX molecules can quickly spread into cells, while Fe 3 O 4 NC/PAH/PSS/ DOX hybrid nanostructures must be endocytosis in order to enter cancer cells. Because of the hypoxiainduced coordinated upregulation of glycolysis, the acidic extracellular environment of solid tumors is stronger than that of normal tissues [62]. At the cellular level, the internalization of most of the hybrid nanostructures will take place through endocytosis. With the increase of DOX concentration, more and more hybrid nanostructures loaded with DOX are endocytosed into cancer cells. After cellular endocytosis, the DOX-loaded hybrid  nanostructures usually enter the early endosomes, then enter the late endosomes/lysosomes, and finally fused with lysosomes. Furthermore, both endosomes (pH 5.0-6.0) and lysosomes (pH 4.5-5.0) have an acidic microenvironment. In our study, the pH-responsive Fe 3 O 4 NC/PAH/ PSS/DOX hybrid nanostructures were more likely to decompose and release drugs in acidic environments, thus effectively reducing side effects, prolonging halflife of drugs, and providing more effective and lasting treatment. Due to the main target of DOX being cell nucleus, DOX can bind to double-stranded DNA to form DNA adducts, inhibit the activity of topoisomerase and induce cell death (apoptosis) [63]. As a result, the released DOX molecules were located in the cell nucleus. Therefore, the obtained Fe 3

Additional file
Additional file 1: Figure S1. TEM image of Fe 3 O 4 nanoparticles.