Macrophage-Laden Gold Nanoflowers Embedded with Ultrasmall Iron Oxide Nanoparticles for Enhanced Dual-Mode CT/MR Imaging of Tumors

The design of multimodal imaging nanoplatforms with improved tumor accumulation represents a major trend in the current development of precision nanomedicine. To this end, we report herein the preparation of macrophage (MA)-laden gold nanoflowers (NFs) embedded with ultrasmall iron oxide nanoparticles (USIO NPs) for enhanced dual-mode computed tomography (CT) and magnetic resonance (MR) imaging of tumors. In this work, generation 5 poly(amidoamine) (G5 PAMAM) dendrimer-stabilized gold (Au) NPs were conjugated with sodium citrate-stabilized USIO NPs to form hybrid seed particles for the subsequent growth of Au nanoflowers (NFs). Afterwards, the remaining terminal amines of dendrimers were acetylated to form the dendrimer-stabilized Fe3O4/Au NFs (for short, Fe3O4/Au DSNFs). The acquired Fe3O4/Au DSNFs possess an average size around 90 nm, display a high r1 relaxivity (1.22 mM−1 s−1), and exhibit good colloidal stability and cytocompatibility. The created hybrid DSNFs can be loaded within MAs without producing any toxicity to the cells. Through the mediation of MAs with a tumor homing and immune evasion property, the Fe3O4/Au DSNFs can be delivered to tumors more efficiently than those without MAs after intravenous injection, thus significantly improving the MR/CT imaging performance of tumors. The developed MA-mediated delivery system may hold great promise for enhanced tumor delivery of other contrast agents or nanomedicines for precision cancer nanomedicine applications.


Introduction
Molecular imaging techniques, based on various functional nanoparticles, have attracted increasing attention in the recent development of cancer nanomedicine which is critical for early tumor therapy guidance [1]. Among them, the development of nanoplatforms integrated with different imaging techniques such as computed tomography (CT) [2], ultrasound (US) [3], photoacoustic (PA) [4], and magnetic resonance (MR) [5] imaging represent a competitive strategy for precision cancer diagnoses. Iron oxide NPs have been adopted as negative contrast agents for MR technology due to the T 2 -weighted effect [6]. Au NPs have been broadly used in CT technology and PTT (photothermal therapy) attributing to their intrinsic advantages of an excellent X-ray attenuation feature as well as a near-infrared (NIR) absorption property, respectively [7].
In order to realize the precision molecular imaging, it was desirable to incorporate both Fe 3 O 4 NPs and Au NPs in one nanoplatform to achieve dual-modal MR/CT imaging [8].
However, to avoid the inaccurate results caused by negative contrast MR images which were hard to be discerned from the tumor site due to dark signal, alternative methods have been explored. Ultrasmall iron oxide (USIO) NPs with a dimension <5 nm have been confirmed and utilized as a promising T 1 -positive contrast agent [9]. Furthermore, the surface modification of USIO with functional moieties could further endow the NPs with targeting or antifouling properties. USIO NPs have also been applied as crosslinkers for alginate nanogels to enhance their r 1 relaxivity [10]. In addition, different shapes of Au NPs such as Au nanostars (NSs) [11], Au nanorods (NRs) [12], and Au NFs [13] have been confirmed to have good biosafety and high photothermal conversion efficiency [14], employed as a wonderful theranostic reagent for PA technology and PTT. In our previous studies, we have demonstrated that USIO NPs can be embedded within Au NFs to form Fe 3 O 4 /Au NFs with a high r1 relaxivity, leading to the feasibility of multimodal MR/CT/PA imaging and combination PTT/radiotherapy (RT) of tumors [15]. However, considering the fact that NPs are easily non-specifically adsorbed by proteins in the blood vessels, and could be removed rapidly by the reticuloendothelial system (RES) [16], construction of a dual-modal imaging nanoplatform which possesses improved tumor penetration and accumulation abilities is still important and urgent.
To increase the payload at the tumor site, two major strategies are applied, which stand as passive targeting and active targeting. For passive targeting, NPs are directed to the enhanced permeability and retention (EPR) taking advantage of the leaky blood vessels of tumors. However, this targeting ability is mainly affected by the biophysicochemical characteristics of NPs [17], and can be largely weakened due to hypoxia and necrosis of central parts of tumors. While for active targeting, various kinds of targeting ligands are modified to recognize and bind to certain receptors specially expressed on tumor cells [18]. However, the previous study has demonstrated that the peak tumor uptake of the NPs with targeting ligand was <2% of the entire dosage [19]. Moreover, one targeting ligand is often limited to identify several certain types of tumors, thus lacking general applicability [20]. It is also noteworthy that the synthesis of the NPs with targeting ligands usually involve unfavorable complex preparation steps and expensive raw materials [21]. In addition, to overcome RES, a "stealth" strategy of NPs has recently been adopted. This strategy has been proven to have the advantage of avoiding clearance by macrophages; however, at the same time, it owns the disadvantage of suppressing the NPs' internalization by target cells [22].
Toward these issues, tumor-directed cell-mediated drug delivery systems have become a major focus in cancer theranostics [23]. Specific cells are able to be used as vehicles to convey NPs across the cancer or along the cancer fringe to improve the accumulation of NPs. For instance, red blood cells [24], mesenchymal stem cells (MSCs) [25], neural stem cells [26], and macrophages (MAs) [27] have been regarded as effective tumor-targeting carriers. In addition, these cell-mediated delivery systems play to their inherent characteristics' strengths, such as having the advantages of non-tumorigenic, low immunogenicity, and being able to cross certain physiological barriers in the body [28]. The FDA has approved a stem cells carrier to be used in clinical trials for glioma treatment. Among these cell vehicles [29], macrophages possess the unique advantages of minimized phagocytosis and extraction from peripheral blood, which is particularly critical in drug delivery. Previously, we have confirmed that MSCs-mediated delivery systems of nanogels with Fe 3 O 4 NPs improved the magnetic MR effect in breast and glioma tumor models in comparison with free nanogels loaded with Fe 3 O 4 NPs [30]. Recently, Guo et al. [31] treated ovarian cancer mice with macrophage-loaded doxorubicine (DOX) and found that macrophages entered the tumor tissue and effectively delivered the drug directly to cancer cells through a tunneling nanotube pathway. Moreover, it was found that the chemokine receptors CCR2 and CCR4 of drug-loaded macrophages were highly expressed, which enhanced the tropism of macrophages. Hence, it is logical to assume that USIO NPs could be loaded onto Au NFs and carried by cancer tropic macrophagocytes to reach tumors for better cancer MR/CT image formation.
In the present study, our team developed a novel MA-laden nanomedicine platform to realize improved cancer MR/CT imaging. First, citrate-stabilized USIO NPs and Amineterminated G5 PAMAM dendrimers-stabilized gold nanoparticles (Au DSNPs) were prepared by a solvothermal route mentioned and self-reduction. Then, Au DSNPs and USIO NPs constituted the seed particles via a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)-mediated covalent reactive process and were used to generate Au NFs. After that, we acetylated the amino group at the terminal of PAMAM to change the surface potential of the

Synthesis of Fe 3 O 4 /Au DSNFs
Firstly, the ultrasmall iron oxide was acquired by a solvothermal route introduced in our past research [30]. Meanwhile, G5 PAMAM Au DSNPs were prepared by the selfreduction method [11]. We obtained Fe 3 O 4 /Au DSNFs through an EDC-mediated covalent reaction with Au DSNPs and EDC-activated USIO NPs based on literature [15]. Briefly, the carboxyl groups of USIO NPs were activated with EDC, then the activated USIO NPs were added dropwise into the solution of Au DSNPs with amino groups on the surface, and finally the Fe 3 O 4 /Au DSNPs was generated through covalent bonding. The terminal amines of the PAMAM were finally treated with acetyl to reduce the positive surface charge of the hybrid nanomaterials. The generated Fe 3 O 4 /Au DSNFs have been characterized by different techniques, such as transmission electron microscopy (TEM), dynamic light scatter (DLS), and UV-vis spectra.

In Vitro Cytotoxicity and Cellular Uptake Assays
Mouse monocyte macrophages (Raw264.7) were incessantly cultivated in the DMEM intermediate. Cytotoxicity of the Fe 3 O 4 /Au DSNFs relative to Raw264.7 cells was detected using a CCK-8 assay according to the literature [27]. The transwell experiment was carried out to verify whether the tumor-tendency of macrophages changed after incubation with

In Vivo MR and CT Imaging of Breast Tumor Model
The mouse breast tumor model was built in male 4~6 weeks old ICR mice for MR and CT imaging. All mice were injected with 2 × 10 6 4T1 cells into the right legs to form the breast cancer models. Until the cancer dimensions of mice registered 0.45~0.75 cm 3

Characterization of Fe 3 O 4 /Au DSNFs
USIO NPs were first synthesized to be used as seed particles. The results of TEM showed that the diameter of formed USIO NPs is 2.6 ± 0.61 nm ( Figure S1a,b). Amineterminated G5 PAMAM dendrimers-stabilized gold nanoflowers were prepared by a self-reduction method, which displayed a size around 20 nm (Figure 1a). Meanwhile, the particles of Fe 3 O 4 /Au DSNPs at the size of 15 nm (Figure 1b) were obtained by EDCactivated USIO NPs which reacted with Au DSNPs by an amide linkage. The size of the Fe 3 O 4 /Au DSNPs measured by TEM is smaller than the individual Au DSNPs, indicating the remarkable interaction between Au DSNPs and USIO NPs, in accordance with the results reported by previous literature [15]. After Fe 3 O 4 /Au DSNPs merge with an Au growth solution, their sizes gradually grow to 98 nm. It is clear to see the nanoflower structure as shown in Figure 1c.
The zeta potential and hydrodynamic size of USIO NPs, Au DSNPs, Fe 3 O 4 /Au DSNPs, and Fe 3 O 4 /Au DSNFs were subsequently tested by DLS. Obviously, the zeta potential of the USIO NPs and Au DSNPs is −24.6 ± 3.1 mV and 23.8 ± 3.5 mV, respectively. The change is supposed to be due to the rich citric acid carboxyl and PAMAM terminal amines on their surface. The surface potentials of the Fe 3 O 4 /Au DSNPs were measured to be 19.2 ± 2.2 mV, because the hybrid reaction neutralized partial terminal amines of Au DSNPs. In order to shield the residual primary amines of G5 dendrimers, the formed Fe 3 O 4 /Au DSNPs were acetylated, leading to a reduction of zeta potential to 11.2 ± 2.7 mV. The hydrodynamic size of USIO NPs, Au DSNPs, seed particles, and Fe 3 O 4 /Au DSNFs is 27.5 ± 2.4 nm, 30.7 ± 7.2 nm, 28.2 ± 3.1 nm, and 265.3 ± 6.9 nm, respectively. Due to the aggregation nature of the nanoparticles in water, an increasing hydrodynamic size was observed in aqueous solutions compared to the data obtained from TEM. However, the hydrodynamic sizes shared a similar variation trend with the TEM results, indicating that the NFs have been successfully prepared.
The structure of Fe 3 O 4 /Au DSNFs was confirmed by UV-vis (Figure 1d). The results showed that the characteristic ultraviolet absorptions of G5 PAMAM gold particles and Fe 3 O 4 /Au DSNFs fall at about 520 nm and 820 nm, respectively. It is proven that the Fe 3 O 4 /Au DSNFs were successfully constructed, which was consistent with the previous literature [15]. In order to assess the steadiness of the Fe 3 O 4 /Au DSNFs, we surveyed the size and polymer dispersity index (PDI) of Fe 3 O 4 /Au DSNFs at diverse concentrations within 14 days. As displayed in Figure S2, no apparent changes could be found, suggesting its good colloidal stability and dispersity.

In Vitro T 1 -Weighted MR Images
As it contains both the radiodense element (Au) and T 1

In Vitro Cytotoxicity and Cellular Uptake Assays
The influence of materials on cell vitality is vital for subsequent in vivo imaging applications. Therefore, the cytotoxicity of Fe 3 O 4 /Au DSNFs was first explored before the in vivo multimode imaging application. The viability of Raw264.7 cells was implemented by CCK-8 assay after a treatment with the Fe 3 O 4 /Au DSNFs for 24 h (Figure 3a). It is obvious that the cell viability gradually decreased with the rise of Au quantity. However, the viability was still higher than 72% when the Au concentration reached 3 mM, implying that the Fe 3 O 4 /Au DSNFs register an acceptable cytocompatibility when the Au concentration ranges from 0 mM to 3 mM. To find the cellular uptake efficiency of Tumor tropism is a typical peculiarity of macrophages because it can be guided by chemokines and cytokines to the inflammation site. The transwell migration experiment was designed to verify whether the functional properties of macrophages have changed after Raw264.7 cells were incubated with the Fe 3 O 4 /Au DSNFs for 18 h (Figure 4a). In this experiment, 4T1 cells were utilized. It can be seen that the presence of 4T1 cells has a significant effect on the migration rate of Raw264.7 cells, which is about three times than that in the absence of 4T1 cells. Although the cell migration rate was slightly reduced after co-incubation with   In addition, we must consider that macrophages are easy to differentiate into various phenotypes that exacerbate or resolve the disease. Previous studies have confirmed that iron can activate the differentiation of macrophages to the M1 phenotype [32]. Therefore, it is necessary to detect the activation states and the biomarkers of macrophage phenotypes after the incubation of Fe 3 O 4 /Au DSNFs. As we know, M1 macrophages could be identified by certain special surface antigens such as CD80 and CD86, while CD206 antigens are expressed in M2 macrophages. Thus, Raw264.7 cells incubated with Fe 3 O 4 /Au DSNFs for 6 h were analyzed by specific M2 and M1 macrophages antigens CD206 ( Figure S4a) and CD80 ( Figure S4b