Introduction

Molecular imaging (MI) provides the means to study in vivo processes that have tremendous potential applications in biomedical research and clinical diagnosis1,2,3. Although each modality imaging has its own merits, no single technique is capable of giving complete information in disease diagnosis due to its intrinsic drawbacks in terms of sensitivity, spatial and temporal resolution, multiplexing capability and response time4,5,6. Among many types of MI technologies, magnetic resonance (MR) imaging has been generally considered as one of the most powerful noninvasive imaging techniques owing to its great spatial resolution and tomographic capabilities7,8,9,10. Computed tomography (CT) affords better spatial and density resolution than other imaging techniques11,12. In addition, photoacoustic (PA) imaging is a non-invasive imaging technique with high resolution and provides fast, quantitative, volumetric measurement with deep tissue penetration capability13,14. However, the spatial resolution of MR imaging is lower than that of CT imaging, while the soft tissue contrast of CT imaging is lower than that of MR imaging. Meanwhile, PA imaging is still at the phases of basic research for now and have not been widely used in clinical applications. Therefore, combination of these three diagnostic modalities is expected to be able to overcome some serious restrictions encountered by each MI technique when used alone, leading to much more accurate disease diagnosis, in particular cancer. Besides precision cancer diagnosis, it is desirable to perform simultaneous treatment of cancer. Among many different cancer therapy approaches, photothermal therapy (PTT) has attracted great interest in recent years due to the advantage that the light-induced heating, as a non-invasive strategy, is able to ablate cancerous cells without damaging surrounding normal tissues15,16,17,18.

Recent advances in nanotechnology exhibit vast potential to generate various platforms that can be used for cancer theranostics19,20,21. Among the used nanomaterials, iron oxide (Fe3O4) nanoparticles (NPs), due to their ability to shorten the T2 relaxation time of their surrounding water protons and biocompatibility, can be used as T2 negative contrast agents for MR imaging10,22. On the other hand, gold (Au) NPs, owning to the higher atomic number of Au than that of iodine for iodinated CT contrast agents (e.g., Omnipaque), have been explored as CT imaging agents23,24. For accurate dual mode MR/CT imaging applications, Fe3O4/Au composite nanoparticles (CNPs) has been designed25,26. For example, Cai et al. synthesized Fe3O4/Au CNPs with the assistance of dendrimers that can be used for MR/CT imaging of animal organs/tissues25 and tumors in vivo27. Zhu et al.20 reported the preparation of Au-Fe3O4 heterostructured NPs for dual mode MR/CT imaging of liver in an intravital model. It is known that Au NPs with a particular shape such as nanostars (NSs)28,29,30, nanorods31,32,33, nanoflowers21,34, or nanocages35,36 are able to exhibit strong localized surface plasmon resonance (SPR) absorbance in near infrared (NIR) region for PTT of cancer cells15,31,37,38. Furthermore, Au NPs with these specific shapes can be used for PA imaging through the absorption of a pulsed light and the detection of the resultant ultrasonic (US) wave profile by special transductor13,14. For example, Nie et al.13 demonstrated that gold NSs were able to be simultaneously used for PTT and PA imaging of tumors. Accordingly, in order to develop a unique platform that can be used for simultaneous multimode imaging and PTT of tumors, it is desirable to integrate Fe3O4 NPs with specific shaped Au NPs.

In our previous study, we have shown that Fe3O4 @ Au NSs can be formed by exposing Fe3O4 @ Ag seed particles to Au growth solution and can be functionalized via polyethyleneimine (PEI)-mediated covalent conjugation reaction for in vivo MR/CT imaging and PTT of tumors via intratumoral administration of the particles39. However, this study was limited to use hyaluronic acid as a targeting ligand and to just use intratumoral injection as an administration route. Furthermore, the PA imaging potential of the developed Fe3O4 @ Au NSs has not been explored. Therefore, it is still challengeable to develop multifunctional Fe3O4 @ Au NSs using different approaches for multimode imaging-guided PTT of tumors.

In this present work, a unique multifunctional nanoplatform based on folic acid (FA) - modified Fe3O4 @ Au NSs were designed for tri-mode MR/CT/PA imaging and PTT of tumors. Fe3O4/Ag composite particles were first synthesized by sodium borohydride (NaBH4) reduction of Ag(I) ions on the surface of citric acid (CA)-coated Fe3O4 NPs formed via a mild reduction route according to the literature10,40,41. The as-prepared Fe3O4/Ag composite particles were utilized as seeds and exposed to the aqueous Au growth solution to induce the formation of Fe3O4 @ Au NSs. Thereafter, the obtained Fe3O4 @ Au NSs were decorated with thiolated PEI (PEI-SH) via Au-S bond. Finally, the PEI-coated Fe3O4 @ Au NSs were sequentially conjugated with FA through a polyethylene glycol (PEG) spacer via the PEI amine-enabled conjugation chemistry. This was followed by acetylation of the remaining PEI amines (Fig. 1a). The formed multifunctional FA-modified Fe3O4 @ Au (Fe3O4 @ Au-PEI.Ac-PEG-FA) NSs were exhaustively characterized. Their hemocompatibility, cytocompatibility, specific recognition to FA receptor (FAR)-overexpressing cancer cells and potential for multi-mode MR/CT/PA imaging and PTT of tumors were evaluated in detail.

Figure 1
figure 1

Synthesis and characterization of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs.

(a) Schematic representation of the synthesis of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs. (b) UV-vis spectra of the aqueous solution of Fe3O4 @ Au-PEI.Ac-PEG-FA NSs before and after laser irradiation (inset shows the digital photo of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs in aqueous solution). (c) TEM image (inset is the high magnification TEM image), (d) size distribution histogram and (e) high-resolution TEM image (only an Au spike is shown) of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs.

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Results

Formation and characterization of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs

According to our previous work10, Fe3O4 NPs were synthesized via a mild reduction route for targeted in vivo MR imaging of tumors. This facile mild reduction strategy enabled the generation of Fe3O4 NPs with ultrahigh r2 relaxivity. In this study, by virtue of the same approach, we synthesized CA-stabilized Fe3O4 NPs (Fe3O4 @ CA) and further created Fe3O4/Ag seed particles by NaBH4 reduction of Ag(I) in the presence of the Fe3O4 @ CA NPs according to the literature41. The formed Fe3O4/Ag seed particles are quite uniform in size (Supplementary Fig. S1a) with an average size of 9.3 nm (Fig. S1b). The apparent absorption peak at 400 nm can be ascribed to the SPR peak of Ag component (Fig. S1c), confirming the formation of Fe3O4/Ag seed particles. Likewise, the existence of both Ag and Fe in the energy dispersive spectrum (Fig. S1d) also confirmed the formation of the Fe3O4/Ag seed particles. The composition of the Fe3O4/Ag seed particles was further quantified using ICP-OES and the molar ratio of Fe3O4/Ag was measured to be 1.9:1.

By exposing the Fe3O4/Ag seeds to the Au growth solution, star-shaped Fe3O4 @ Au were generated. The NIR-absorbance feature of the NSs was validated by UV-vis spectroscopy (Supplementary Fig. S2). By optimizing the concentration of the chemicals, Fe3O4/Au NSs with a desirable NIR-absorption feature were obtained using an Au growth solution containing 2.4 mM HAuCl4, 0.08 mM AgNO3, 5.2 mM AA. The formed CTAB-stabilized Fe3O4 @ Au NSs display unique star-shaped spikes with a narrow size distribution (Supplementary Fig. S3). In order to make the NSs be functionalized, the NSs were copiously washed to remove the surfactant CTAB and modified with PEI-SH via Au-S bond formation according to our previous work39. The PEI-SH synthesized was characterized by 1H NMR to have 15.3 thiol groups per PEI (Supplementary Fig. S4a). The generated PEI-stabilized Fe3O4 @ Au (Fe3O4 @ Au-PEI) NSs with a large amount of primary amines were further grafted with COOH-PEG-FA, which was characterized to have 0.8 FA moieties per PEG by 1H NMR (Fig. S4b). This FA modification onto the NSs via a PEG spacer is expected to endow the NSs with high affinity to cancer cells overexpressing FAR19. Finally, the synthesized Fe3O4 @ Au-PEI-PEG-FA NSs were subjected to an acetylation reaction to neutralize the residual PEI surface amines in order to improve their cytocompatibility42.

Zeta potential and hydrodynamic size of NSs produced in each step were measured to confirm their surface modification (Supplementary Table S1). Clearly, Fe3O4 @ Au-PEI NSs possess a quite positive surface potential (+31.4 mV) due to the surface modification of PEI with a large amount of amines. After the successive modification of COOH-PEG-FA and acetylation reaction, the surface potentials of the Fe3O4 @ Au-PEI-PEG-FA and Fe3O4 @ Au-PEI.Ac-PEG-FA NSs were measured to be +28.3 and +14.4 mV, respectively. The decreased surface potential for both NSs validated the success of the modification of COOH-PEG-FA and acetylation reaction when compared to that of the Fe3O4 @ Au-PEI NSs. Likewise, the hydrodynamic sizes of the Fe3O4 @ Au-PEI-PEG-FA (226.1 nm) and Fe3O4 @ Au-PEI.Ac-PEG-FA (224.2 nm) NSs are larger than that of the Fe3O4 @ Au-PEI NSs (211.3 nm), suggesting the successful surface modification of the NSs. Additionally, the hydrodynamic size of the final Fe3O4 @ Au-PEI.Ac-PEG-FA NSs was also occasionally measured within a time period of 15 days (Supplementary Fig. S5). We show that the hydrodynamic size of NSs does not display any obvious fluctuation, suggesting their laudable colloidal stability. Moreover, the long-term colloidal stability of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs was evaluated by exposing them to different media (water, PBS and DMEM containing 10% FBS, respectively) for at least one month (Fig. S5 and inset). The particles do not precipitate, further confirming their long-term colloidal stability.

To quantify the conjugated PEI and COOH-PEG-FA on the surface of the Fe3O4 @ Au NSs, TGA were performed (Supplementary Fig. S6). At 700 °C, the PEI coating results in a weight loss of 4.8% for the Fe3O4 @ Au-PEI NSs, when compared with the CTAB-free Fe3O4 @ Au NSs. Further conjugation of COOH-PEG-FA affords the Fe3O4 @ Au-PEI-PEG-FA NSs with an increased weight loss of 12.4%. Therefore, the modified COOH-PEG-FA onto the NSs was deduced to be 7.6%.

The optical property of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs was investigated by UV-vis spectroscopy (Fig. 1b). The dark blue color of NSs in aqueous solution was ascribed to the star-shaped Au shell coating on the particle surface (Fig. 1b, inset). An obvious SPR peak in the NIR region at 810 nm can be clearly seen and the NIR absorption feature does not show obvious changes even when the NSs were irradiated with an 808 nm laser at 2.0 W/cm2 for 20 min. This suggests that the NSs display an excellent photothermal stability and also a great potential to use them for PTT of tumors. TEM was employed to observe the morphology and size of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs (Fig. 1c–e). We can clearly see that the NSs with Au shell coating onto the surface of Fe3O4 NPs possess well-defined star shape and a quite uniform size distribution (Fig. 1c). By measuring two maximal margins of the NSs, the mean diameter of NSs was estimated to be 149.6 ± 21.5 nm (Fig. 1d). As depicted in Fig. 1e, high-resolution TEM image reveals the clear lattices of the spike-like Au shell crystals and also a dense polymer coating on the outer surface of the NSs, which is relevant to the PEI modification and COOH-PEG-FA grafting. The elemental composition of Fe and Au in the Fe3O4  @ Au-PEI.Ac-PEG-FA NSs was quantified by ICP-OES and the Fe/Au molar ratio was measured to be 1:53.4.

MR and CT phantom studies

Fe3O4 NPs are generally used as MR contrast agents because of their capacity to shorten the T2 relaxation time of their surrounding water protons. From T2-weighted MR imaging, we can find that the generated Fe3O4 @ Au-PEI.Ac-PEG-FA NSs can gradually decrease the MR signal intensity of water with the increase of Fe concentration (Fig. 2a). By linearly fitting the T2 relaxation rate (1/T2) versus Fe concentration, the r2 relaxivity of the NSs was calculated to be 549.07 mM−1s−1 (Fig. 2b). The ultrahigh r2 relaxivity of the NSs should be due to the nature of the mild reduction approach used to synthesize Fe3O4 NPs, in agreement with the literature10. It seems that the Au shell coating does not appreciably affect the accessibility of water protons to the inner Fe3O4 NPs, due largely to the interstitial spaces between each Au spikes of the NSs.

Figure 2
figure 2

MR relaxometry, X-ray attenuation property, photoacoustic property and photothermal performance of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs.

(a) T2-weighted MR images and (b) linear fitting of 1/T2 of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs as a function of Fe concentration. (c) CT images and (d) linear fitting of CT values (HU) of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs at an Au concentration of 4, 8, 16, 32 and 64 mM, respectively. (e) PA images and (f) linear fitting of PA values of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs at an Au concentration of 0, 0.25, 0.5, 1.0 and 2.0 mM, respectively. (g) Temperature change of water and water containing the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs at varying Au concentrations (1, 2, 4, 10 and 20 mM, respectively) under an 808 nm laser irradiation with the same power density of 1.0 W/cm2 as a function of irradiation time. (h) Temperature change of a water solution containing the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs at an Au concentration of 10 mM under an 808 nm laser irradiation with different power densities (0.25, 0.5, 1.0 and 1.5 W/cm2, respectively) as a function of irradiation time.

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On the other hand, CT phantom studies of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs were carried out to explore their potential for CT imaging (Fig. 2c). It can be clearly seen that, with the increase of Au concentration, the CT image of the NSs-containing aqueous suspension gradually brightens, well matching the quantitative measurement of their CT value (HU) versus Au concentration (Fig. 2d).

Photoacoustic property and photothermal performance of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs

To explore the feasibility to employ the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs for PA imaging, PA phantom studies were first carried out (Fig. 2e). Clearly, with the Au concentration of the NSs, the PA signal intensity gradually enhances, which correlates well with the quantitative PA signal intensity change versus Au concentration (Fig. 2f).

The photothermal behavior of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs was next investigated to unleash their potential to be used for PTT of tumors. The temperature variation of the the NSs-containing aqueous solution versus Au concentration (1–20 mM) was continuously monitored in real time after exposure to an 808 nm laser at 1.0 W/cm2 for 300 s (Fig. 2g). Clearly, the NSs are able to induce a positive temperature enhancement in a concentration-dependent manner. The temperature of the NS suspension can reach 63.3 °C at the Au concentration of 20 mM. For comparison, laser irradiation of pure water under the same conditions does not afford obvious temperature increase. At a given Au concentration of 10 mM, the NS suspension irradiated under an 808 nm laser at different output power densities (0.25–1.5 W/cm2) for 300 s was also monitored to check the temperature change (Fig. 2h). It is evident that the NSs are able to generate heat in a laser output power density-dependent manner. The temperature of the NS suspension rises to 68.5 °C at the highest laser output power density (1.5 W/cm2). These results indicate that the synthesized Fe3O4 @ Au-PEI.Ac-PEG-FA NSs were able to transform NIR laser into heat rapidly under laser irradiation. The photothermal conversion efficiency (η) of the NSs was calculated according to the literature43,44. Details can be seen in Supplementary information. The η of the NSs was calculated to be 88.9%, which is prominently higher than that of other major PTT agents43,44,45,46.

Hemolytic and cytotoxicity assays

For biomedical applications, it is vital to evaluate the hemocompatibility and cytocompatibility of the prepared Fe3O4 @ Au-PEI.Ac-PEG-FA NSs. The hemolytic activity of the NSs was evaluated by hemolytic assay (Supplementary Fig. S8a). When compared to the positive water control, where obvious hemolytic behavior occurs, NSs at different Au concentrations do not display appreciable hemolysis effect, similar to the negative PBS control (inset of Fig. S8a). The hemolysis percentages of HRBCs exposed to NS solution at different Au concentrations were calculated to be 0.39%, 1.79%, 2.51% and 4.39%, respectively, which are all less than the threshold value of 5%, indicating their excellent hemocompatibility in the given Au concentration range47,48.

The cytocompatibility of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs was assessed by MTT assay of viability of HeLa cell (Fig. S8b). Apparently, after cultivation of HeLa cells with NSs at an Au concentration of 0.2, 0.4, 0.8, 1.5 and 2.0 mM, respectively, for 24 h, the cell viability still keeps larger than 80%, suggesting their negligible cytotoxicity19,47. The cytocompatibility of the NSs was further validated by observing the morphology of HeLa cells (Supplementary Fig. S9). Clearly, the morphology of HeLa cells after treatment with the NSs in the given Au concentration range (0.2–2.0 mM) (Fig. S9b–f) is quite similar to that of HeLa cells treated with PBS (Fig. S9a). Taken together, the developed Fe3O4 @ Au-PEI.Ac-PEG-FA NSs display good hemocompatibility and cytocompatibility in the studied concentration range.

In vitro cellular uptake assay and targeted MR and CT imaging of cancer cells

To verify the high affinity of the produced Fe3O4 @ Au-PEI.Ac-PEG-FA NSs to cancer cells overexpressing FAR, ICP-OES was performed to analyze the Au uptake by HeLa cells (Fig. 3a). Obviously, both HeLa-HFAR and HeLa-LFAR cells exhibit gradually enhanced Au uptake with the NS concentration. Under the same Au concentrations, the Au uptake in HeLa-HFAR cells was apparently higher than that in HeLa-LFAR cells (p < 0.01). This implies that the modified FA ligands onto the NSs enable specific targeting of the NSs to FAR-overexpressing cancer cells via FA-mediated pathway49,50,51. The specific uptake of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs was further evaluated by TEM imaging (Fig. 3b,c). Clearly, HeLa-HFAR cells treated with the NSs show the remarkable distribution into the cytoplasm of the cells (Fig. 3b). In contrast, only minimal NSs were able to be detected in HeLa-LFAR cells, which is associated to the nonspecific phagocytosis or diffusion via cell walls (Fig. 3c)52.

Figure 3
figure 3

In vitro cellular uptake assay, targeted MR and CT imaging and photothermal ablation of HeLa cells.

(a) The Au uptake by HeLa-HFAR and HeLa-LFAR cells after treated with the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs at various Au concentrations for 4 h. TEM images of (b) HeLa-HFAR and (c) HeLa-LFAR cells after treated with the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs for 4 h, respectively. (d) T2-weighted MR images, (e) MR signal intensity, (f) CT images and (g) CT value of HeLa-HFAR and HeLa-LFAR cells treated with the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs at varying Fe or Au concentrations for 6 h. (h) MTT assay of HeLa cell viability after treatment with the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs in a given Au concentration range under an 808 nm laser irradiation for 5 min. 1 and 2 represent the HeLa-LFAR and HeLa-HFAR cells, respectively.

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MR and CT imaging were also performed to further validate the specific targeting of the NSs to HeLa-HFAR cells. T2-weighted MR images of both HeLa-HFAR and HeLa-LFAR cells treated with the NSs become darker with the increase of Fe concentration (Fig. 3d). However, HeLa-HFAR cells show a more prominent MR signal intensity decrease under the same Fe concentrations than HeLa-LFAR cells. This was further validated by quantifying the MR signal intensity of HeLa-HFAR and HeLa-LFAR cells treated with the NSs (Fig. 3e). On the other hand, CT images and the quantitative CT values (HU) of the cells treated with the NSs (Fig. 3f,g) reveal that the CT contrast enhancement of HeLa-HFAR cells is much more obvious than that of HeLa-LFAR cells (p < 0.05) under the same Au concentrations, indicating that the FA renders the NSs with a targeting specificity to cancer cells that overexpress FAR. Overall, the developed Fe3O4 @ Au-PEI.Ac-PEG-FA NSs are able to be used for specific MR and CT imaging of FAR-overexpressing cancer cells in vitro.

In vitro photothermal ablation of cancer cells

Inspired by the high-performance photothermal property and FA-enabled targeting specificity, we used the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs for PTT of cancer cells in vitro (Fig. 3h). Apparently, HeLa cells after treatment with the NSs without laser irradiation are quite healthy with viability comparable to those treated with PBS (control). In sharp contrast, the viability of HeLa cells after treatment with the NSs and irradiated under an 808 nm laser (1.0 W/cm2) for 5 min markedly decreases even at the Au concentration as low as 0.1 mM (p < 0.001). With the increase of Au concentration, the NSs exert more prominent ablation effect on cancer cells and 75.3% of HeLa cells can be killed at an Au concentration of 0.8 mM.

The PTT of cancer cells using the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs was further investigated by cell morphology observation (Supplementary Fig. S10). Clearly, the morphologies of HeLa cells treated with PBS (Fig. S10a,f) and the NSs alone at the Au concentrations of 0.1–0.8 mM are quite healthy (Fig. S10b–e). In sharp contrast, after treatment with the NSs plus laser irradiation (Fig. S10g–j), HeLa cells are detached and rounded even at the lowest Au concentration of 0.1 mM, indicating that the cells have undergone apoptosis. The cell morphology observation results corroborate the MTT viability assay data, confirming the excellent performance of the NSs for PTT of cancer cells in vitro.

In vivo MR/CT/PA tri-mode imaging of a xenografted tumor model

Next, the feasibility to use the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs for tri-mode MR/CT/PA imaging of xenografted HeLa tumors in vivo were explored. As shown in Fig. 4a,b, the region of tumor becomes markedly dark at 0.5 h post intratumoral (IT) injection or at 6 h post intravenous (IV) injection. Quantitative MR signal intensity analysis show that the tumor MR signal intensity dramatically decreases from 256.6 to 20.1 and from 362.7 to 247.7 for IT and IV injection, respectively (p < 0.001) (Fig. 4c,d). This validates the use of the NSs for MR imaging of the tumors in vivo. To demonstrate the targeting specificity of the NSs, free FA-blocked HeLa tumor was also imaged by MR after IV injection under the same condition (Supplementary Fig. S11). Clearly, the tumor region displayed similar brightness at 6 h post IV injection to that before injection (Fig. S11a), which can be further confirmed by quantitative MR signal intensity analysis (Fig. S11b).

Figure 4
figure 4

In vivo MR/CT imaging of a xenografted tumor model.

(a,b) T2-weighted MR images, (c,d) MR signal intensity, (e,f) CT images and (g,h) CT value of tumors before and at 0.5 h post IT (a,c,e,g) and at 6 h post IV (b,d,f,h) injection of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs ([Fe] = 1.31 mM, [Au] = 70 mM, 0.1 mL in PBS).

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We then tested the potential to use the NSs for CT and PA imaging of the HeLa tumor. For CT imaging, the brightness of tumor site increases at 0.5 h post IT injection or at 6 h post IV injection (Fig. 4e,f). This can be further validated by collecting the CT value of the tumor region (Fig. 4g,h). Clearly, the CT value of tumor site significantly increases from 32.6 to 364.9 HU at 0.5 h post IT injection and from 30.4 to 43.0 HU at 6 h post IV injection, respectively (p < 0.01). For PA imaging, once the NSs absorb NIR laser and transform it into heat, the ambient environment can be expanded under this heat and generate an ultrasound signal. We then performed both PA and ultrasound imaging of the tumors. As shown in Fig. 5a,c, intense PA signal of tumor area can be easily visualized after IT and IV injection of NSs into tumor-bearing mice. When compared to the PA intensity of tumor region before injection, the PA intensity is approximately 20 folds and 3 folds higher for IT injection (increase from 0.14 to 2.82) and IV injection (increase from 0.14 to 0.41), respectively (p < 0.001, Fig. 5b,d). The enhanced tumor MR/CT/PA imaging should be due to the modification of PEGylated FA onto the particle surfaces. On one hand (particularly for IV injection), the PEGylation modification of NSs enables them to escape from the uptake by the reticuloendothelial system (RES) and to accumulate in the tumor tissue via a passive targeting pathway based on enhanced permeability and retention (EPR) effect53; on the other hand, with the FA-mediated active targeting pathway as demonstrated in the in vitro studies, the NSs are able to specifically target the tumor tissue for highly effective tumor MR/CT/PA imaging.

Figure 5
figure 5

In vivo PA imaging of a xenografted tumor model.

In vivo ultrasound/PA images (a,c) and PA value (b,d) of tumors before and at 0.5 h post IT ([Au] = 100 mM, 0.04 mL in PBS, a,b) and at 6 h post IV ([Au] = 100 mM, 0.1 mL in PBS, c,d) injection of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs.

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In vivo PTT of a xenografted tumor model

Next, the potential to employ the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs for thermal imaging and PTT of tumors were tested (Fig. 6). Intravital thermal imaging of mice was conducted at different laser irradiation time periods using an infrared camera. As shown in Fig. 6a,b, the temperature of Region Ar1 (administrated with the NSs) rapidly increases by 24.7 °C within 150 s and by 30.8 °C after 300 s of irradiation. In contrast, for Region Ar2 (administrated with PBS), a subtle temperature elevation was observed and the temperature increases just approximately 5 °C during the laser irradiation.

Figure 6
figure 6

In vivo photothermal imaging and photothermal ablation of HeLa tumors.

(a) Full-body photothermal images of HeLa tumor-bearing mice after IT injection with 0.1 mL PBS (control, the right mouse, indicated at region Ar2) or Fe3O4 @ Au-PEI.Ac-PEG-FA NSs dispersed in 0.1 mL PBS ([Au] = 20 mM, the left mouse, indicated at region Ar1), followed by exposure to an 808 nm laser with a power density of 1.0 W/cm2 at a time point of 0 min, 2.5 min and 5 min, respectively. (b) The plot of temperature increase in tumor regions (Ar1 and Ar2) as a function of irradiation time. (c) The tumor volume change profiles, (d) body weight change and (e) survival rate of tumor-bearing mice (n = 4) as a function of time posttreatment.

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This high-temperature caused by NSs under laser irradiation is expected to ablate tumors. Next, we examined the tumor volume change of the mice after different treatments (Fig. 6c). The relative volumes of tumors at 24 days posttreatment for the groups of control (PBS), PBS + Laser and NSs are approximately 6–7 times larger than that of the initial tumor. In sharp contrast, the treatment using NSs plus laser is able to completely ablate the tumors at 7 days postinjection of the NSs. This clearly illustrates the possibility to employ the NSs for highly efficient PTT of tumors. The PTT efficacy of tumors can be easily visualized by taking photos of the mice (Supplementary Fig. S12). The mice in the NSs + Laser group maintain healthy at 19 days posttreatment and the completely ablated tumor region does not seem to recur in the studied time period, suggesting the excellent PTT efficiency of the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs. In contrast, for the mice in the groups of Control, PBS + Laser and NSs, the tumors grow bigger and bigger with the time. It should be noted that, during the experimental time period, mice with different treatments are able to maintain their body weights (Fig. 6d), suggesting that the treatments using the laser alone, the NSs alone, or the laser plus NSs are not toxic to the mice. Finally, the PTT efficacy of the tumors was evaluated by monitoring the survival rate of the mice after different treatments (Fig. 6e). Obviously, after treatment with the NSs plus laser, the mean survival rate of mice keeps 100% after 60 days. However, mice treated with PBS (Control group), PBS plus laser and NSs alone survive with an average life-span of 40 days, 43 days and 42 days, respectively.

Histology examinations

The PTT efficacy of tumors were further confirmed by histological examinations using H&E and TUNEL staining of harvested tumor sections (Fig. 7). H&E staining (Fig. 7a) reveals that the tumors either treated with PBS plus laser or the NSs alone exhibit normal HeLa tumor cell morphology, similar to those treated with PBS (control). But for tumors treated with the NSs plus laser, necrosis cells can be clearly seen in the whole section. Likewise, TUNEL staining (Fig. 7b) shows that only a very small amount of positive staining of apoptotic cells can be found in the sections of control, PBS + Laser and NSs groups. In sharp contrast, after treatment with the NSs plus laser irradiation, the tumors display a huge amount of positive staining of apoptotic cells. Quantitative analysis data further reveal that the percentages of apoptotic cells in the tumors of the Control, PBS + Laser, NSs and NSs + Laser groups are 1.5%, 3.3%, 1.3% and 95.0%, respectively (Supplementary Fig. S13).

Figure 7
figure 7

Histological examinations.

(a) H&E and (b) TUNEL staining of tumor sections after the tumor mice were treated under different conditions. The scale bar shown in each panel of (a,b) represents 100 μm.

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Discussion

Generally, Fe3O4/Au CNPs could be synthesized via various routes and utilized for dual mode MR/CT imaging applications10,20,27. On the other hand, star-shaped Au shells with strong SPR band in NIR region enable their applications in PA imaging and PTT of tumors in vivo18. In this work, we designed the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs with targeting specificity to FAR-expressing cancer cells for multi-modal MR/CT/PA imaging and PTT of tumors in vivo. Compared to our previous study utilizing hyaluronic acid as a targeting ligand39, our current work aimed to expand the scope of the designed platform for theranostics of different cancer phenotypes.

Through depositing Au shell on the surface of Fe3O4/Ag seeds (Supplementary Fig. S1), the Fe3O4 @ Au core/shell NSs were formed and then successively decorated with PEI (4.8%) via the formation of Au-S bond and COOH-PEG-FA (7.6%) via the formation of amide bond (Supplementary Fig. S4). After acetylation of the remaining PEI surface amines (Supplementary Table S1), multifunctional Fe3O4 @ Au-PEI.Ac-PEG-FA NSs were formed. The formed NSs have a slightly positive surface potential (+14.4 mV), which doesn’t seem to exert any appreciable in vivo toxicity. This can be confirmed by monitoring the physiological status of mice after IV treatment of NSs for at least 10 days, in agreement with our previous work54. It should be mentioned that the designed Fe3O4 @ Au-PEI.Ac-PEG-FA NSs have a low molar ratio of Fe/Au (1:53.4). Due to the fact that we prepared the Fe3O4 seed NPs using a mild reduction route, the formed Fe3O4 @ Au core/shell NSs have a r2 relaxivity (549.07 mM−1s−1) 3.8 times higher than the Fe3O4 @ Au core/shell NSs (144.39 mM−1s−1) reported in our previous work39. With the higher Au content than other Fe3O4 @ Au NPs27,55 and non-compromised MR imaging sensitivity, the designed NSs should be able to used for sensitive MR and CT imaging. Our results reveal that the NSs display good MR relaxometry, X-ray attenuation, PA property and photothermal performance (Fig. 2), which is due to the co-existence of Fe3O4 core and star-shaped Au shell. The η of the NSs (88.9%, Supplementary Fig. S7) is much higher than that of other major PTT agents reported in the literature43,44,45,46, which is likely ascribed to the particle’s volume, structure and shape56. The FA modification of the particles rendered the NSs with specific affinity to FAR-overexpressing cancer cells in vitro (Fig. 3a–g) and the xenografted tumor model in vivo (Fig. 4b,d and Supplementary Fig. S11). Hence the NSs hold great promise to be used as a nanoprobe for specific MR/CT/PA imaging of a FAR-expressing tumor model in vivo after either IT or IV injection (Figs 4 and 5). The tumor MR/CT/PA signal intensities after IV injection of the NSs are poorer than those of the corresponding imaging signals after IT injection of the NSs. This should be due to the fact that much less NSs were accumulated in tumors via IV injection than via IT injection. Additionally, the NIR-absorption feature of NSs affords their uses for efficient PTT of cancer cells in vitro and the tumor model in vivo (Figs 3h and 6). It should be mentioned that due to the fact that NPs with a size of 50–300 nm regardless of surface modification of targeting ligands have a significant accumulation in the tumor region after IT injection33,44,57, we used IT injection route to evaluate the PTT efficacy of the tumors in vivo using the designed NSs.

To conclude, we presented a novel route to generating Fe3O4 @ Au-PEI.Ac-PEG-FA NSs that can be used as a unique platform for multi-mode MR/CT/PA imaging and PTT of tumors. Via the integration of mild reduction route, seed-mediated growth method and PEI-mediated conjugation, multifunctional Fe3O4 @ Au-PEI.Ac-PEG-FA NSs are able to be formed. The designed NSs possess excellent colloidal stability, good hemocompatibility/cytocompatibility, ultrahigh r2 relaxivity, good X-ray attenuation and PA property and strong NIR absorption feature. These properties afford their uses as a theranostic nanoprobe for multi-mode imaging-guided PTT of tumors. Furthermore, the synthesized FA-modified Fe3O4 @ Au core/shell NSs are likely to be used for theranostics of different types of cancer for further translational medicine applications.

Methods

Materials

All chemicals and materials were from commercial resources and used as received. Details can be seen in Supplementary Information.

Characterization techniques

The intermediate products and Fe3O4 @ Au-PEI.Ac-PEG-FA NSs were thoroughly characterized via hydrodynamic size and zeta potential measurements, 1H NMR spectroscopy, thermal gravimetric analysis (TGA), UV-vis spectroscopy, transmission electron microscopy (TEM), Leeman Prodigy inductively coupled plasma-optical emission spectroscopy (ICP-OES), T2 relaxometry measurements, MR/CT/PA phantom studies and photothermal performance.

Preparation of Fe3O4 @ Au NSs

Fe3O4/Ag seed particles were prepared by a protocol adopted from the literature10,40,41,58. To grow Au NSs onto the surface of Fe3O4 NPs, an Au growth solution with three different proportions of regents (AgNO3, AA and HAuCl4) was used. Only the mixture that changed to blue and exhibited strong NIR-absorption in the UV-vis-NIR spectrum was regarded as the best option of Au growth solution for the formation of Fe3O4 @ Au NSs.

Synthesis of Fe3O4 @ Au-PEI.Ac-PEG-FA NSs

Firstly, PEI-SH and COOH-PEG-FA were synthesized using protocols illustrated in the literature19,39,49,59. Thereafter, the above Fe3O4 @ Au NSs were successively modified with PEI-SH via the formation of Au-S bond and COOH-PEG-FA via the formation amido linkage. After neutralizing the remaining PEI amines, the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs were obtained.

Hemolysis and cytotoxicity assay

Hemolysis assay was performed by exposing human red blood cells (HRBCs) to a phosphate buffered saline (PBS) solution containing Fe3O4 @ Au-PEI.Ac-PEG-FA NSs in the Au concentration range of 0–4 mM for 2 h at room temperature. HeLa cells were routinely cultured and passaged in 25 cm2 plates at 37 °C and 5% CO2 in regular FA-free DMEM with 10% FBS, 1% penicillin/streptomycin. HeLa cells cultured in FA-free medium expressed high-level FAR (denoted as HeLa-HFAR cells)60,61, while HeLa cells grown in the DMEM containing 2.5 μM free FA for 24 h or longer expressed low-level FAR (denoted as HeLa-LFAR). Without specific statement, the term of “HeLa cells” is always deemed to be “HeLa-HFAR cells”. Cytotoxicity assay was conducted by culturing HeLa cells with fresh DMEM containing Fe3O4 @ Au-PEI.Ac-PEG-FA NSs in the tested Au concentration range (0–2 mM) for 24 h. MTT assay and cell morphology observation were used to quantitatively and qualitatively assess the cell viability.

In vitro specific cellular uptake assay

After incubation of both HeLa-HFAR and HeLa-LFAR with Fe3O4 @ Au-PEI.Ac-PEG-FA NSs for 4–6 h, ICP-OES, TEM, MR imaging and CT imaging were performed to determine or demonstrate the specific uptake of the NSs by HeLa-HFAR cells.

In vitro photothermal ablation of HeLa cells

After cultivation with DMEM containing PBS (control) or Fe3O4 @ Au-PEI.Ac-PEG-FA NSs in the studied Au concentration range for 6 h, adherent HeLa cells were then rinsed with PBS and subjected to laser irradiation. MTT viability assay and cell morphology observation were then performed to quantitatively and qualitatively assess the cell viability, respectively.

In vivo MR/CT/PA tri-modal imaging of a xenografted tumor model

All animal experiments were conducted in compliance with institutional guidelines and the policy of the National Ministry of Health and were approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University. A PBS solution (0.1 mL) containing the Fe3O4 @ Au-PEI.Ac-PEG-FA NSs ([Fe] = 1.31 mM) was delivered into each tumor-bearing mouse via an intravenous (IV) or intratumoral (IT) injection route. For MR imaging, tumor 2D MR images were collected before and at 0.5 h post IT injection or at 6 h post IV injection of the NSs by using a Signa HDxt superconductor clinical MR system (1.5 T). To verify the targeting specificity of NSs to FAR-expressing HeLa tumor, IV injection of free FA was first performed to block the FAR expression, followed by IV injection of the NSs. Tumor CT and PA imaging were further carried out with a GE LightSpeed VCT imaging system and the Vevo LAZR PA Imaging System, respectively.

In vivo photothermal ablation of HeLa tumors

Intravital thermal imaging of HeLa tumor-bearing mice was first conducted at different laser irradiation time periods using an infrared camera. For PTT of tumors, the mice were randomly allocated into four groups and subjected to different treatments: Control group (without any treatment), PBS + Laser group (PBS with laser), NSs group (NSs without laser) and NSs + Laser group (NSs with laser). Thereafter, the size of the tumors, body weight and tumor volume were recorded at different time points. More experimental details can be found in Supplementary information.

Additional Information

How to cite this article: Hu, Y. et al. Multifunctional Fe3O4 @ Au core/shell nanostars: a unique platform for multimode imaging and photothermal therapy of tumors. Sci. Rep. 6, 28325; doi: 10.1038/srep28325 (2016).