Introduction

Nasopharyngeal carcinoma (NPC) is an epithelial carcinoma arising from the nasopharyngeal mucosal lining1,2. Recently, cisplatin-based chemoradiotherapy has become a new standard of NPC treatment due to the high sensitivity of NPC to ionizing radiation and cis-platinum (CDDP)3,4. However, the toxicity of systemic therapy remains a pertinent issue due to the low radiosensitivity of solid tumors and the poor targeted effect of free cisplatin5. Although the applications of cisplatin-based nanoparticles for cancer therapy have attracted a great deal of attention6,7,8, the synergistic effect of radiotherapy and chemotherapy has not been substantially improved6. On the one hand, the sensitization ability of cisplatin-based nanoparticles is limited at clinical doses, resulting in a high radiation dose required in clinical use1. On the other hand, most cisplatin-based nanoparticles targeting the tumor site heavily depend on the enhanced permeability and retention (EPR) effect9 and some positive molecular targeting groups, such as RGD peptide10. To date, improving the radiation sensitization and tumor targeting efficiency of nanomedicines is still a major issue in the development of nanomedicines for NPC chemoradiotherapy.

Magnetic nanoparticles (MNPs), such as Co-, Ni-, Mn- and Fe-based nanoparticles11,12, have attracted extensive attention, not only due to their potential radiotherapy sensitization but also due to their outstanding tumor targeting properties in the presence of a magnetic field13. Notably, superparamagnetic iron oxide particle (SPION), one of the most commonly used magnetic nanoparticles, have been widely applied in cancer therapy14,15,16,17. SPION showed a good nanozyme effect for increasing the reactive oxygen species (ROS) level in the tumor microenvironment18,19, thus improving the sensitivity of cancer cells to radiation20,21,22,23,24. Additionally, unlike molecular targeting, magnetic targeting depends on the external magnetic field rather than receptors in the tumor25. By precisely controlling the external magnetic field, nanomedicines can be effectively located in tumors. Therefore, the application of SPION may provide an alternative way to improve the tumor targeting efficiency of nanomedicines for effective cancer treatment, especially for tumors distributed on the mucosa or skin, such as NPC. Moreover, sufficient evidence has suggested that SPION can function as magnetic resonance (MR) contrast agents for image-guided chemoradiotherapy26,27,28,29. Under the guidance of imaging technology, the use of SPION would be ideal for MR-guided chemoradiotherapy of NPC.

Herein, we demonstrate dual-targeted cisplatin-based polymeric nanoparticles that can improve the overall efficacy of NPC. As illustrated in Scheme 1, the SPION@polymer hybrid nanosensitizer (SPHN) have a core-shell structure. The SPION (~5 nm) were first encapsulated inside the core with an amphiphilic block polymer, followed by conjugating CDDP to the carboxyl in the polymer and RGD in the terminus of PEG. During blood circulation, the PEGylated surface endows SPHN with long blood circulation stability. In vitro, these unique nanosensitizers showed good potential in promoting apoptosis by inducing DNA damage when combined with X-rays. In addition, RNA-seq and KEGG enrichment analysis revealed that the PI3K-Akt and TNF signaling pathways were closely related to the molecular mechanism of SPHN in chemoradiotherapy. GSEA revealed that SPHN + X-rays treatment could decrease translation initiation pathways and cytoplasmic translation pathways. Upon reaching tumor sites, the SPHN can substantially increase the tumor accumulation of CDDP in the presence of RGD and an extra magnetic field. Subsequently, the highly accumulated CDDP and SPION in the tumor can act as chemotherapeutics and radiosensitizers for effective chemoradiotherapy. Moreover, coloading SPION can potentially provide high-resolution MR imaging for the precise therapy of NPC.

Scheme 1: Schematic representation of SPHN for effective chemoradiation therapy.
scheme 1

The SPION was encapsulated in the hydrophobic core of SPHN using a self-assembly method, followed by coordinating CDDP with carboxyl groups on the surface of the PLGA core and conjugating RGD to the PEG shell using an amidation and click reaction. SPHN can effectively accumulate in solid tumors via a magnetic-RGD dual-targeting effect for valid CDDP delivery, resulting in a significantly improved antitumor effect with minimal side effects.

Full size image

Materials and methods

Materials

Amino polyethylene glycol-block-poly(lactic-co-glycolic acid) (NH2-PEG-b-PLGA), polyethylene glycol-block-poly(lactic-co-glycolic acid) (PEG-b-PLGA), carboxyl poly(lactic-co-glycolic acid) (PLGA-COOH) and cyclo(-RGDfC) (denoted RGD) were purchased from Guangzhou Tansh-Tech Ltd., China. SPION were purchased from Nanoeast, China.

Preparation of SPHN

For the preparation of SPHN, SPION was encapsulated in the hydrophobic PLGA core, followed by coordinating CDDP with carboxyl groups on the surface of the PLGA core and conjugating RGD in the PEG shell. Briefly, NH2-PEG-b-PLGA (0.5 mg, 1 e.q.), PEG-b-PLGA (2 mg, 4 e.q.), PLGA-COOH (2.5 mg) and SPION (2 mg) were dispersed in 2 mL of tetrahydrofuran (THF). The solution was then added to 20 mL of phosphate buffer solution (PBS, 1 mg/mL) under vigorous stirring, followed by stirring overnight at 35 °C to remove the THF. Then, CDDP (2 mg) was added, and the mixed solution was stirred in the dark at 60 °C for 12 h. RGD was bound to PEG according to our previous work preparing SPHN8. Then, the SPHN solution was dialyzed against PBS for 48 h. For preparation of fluorescence-labeled nanoparticles, Cy5-NHS was employed to label the nanoparticles before RGD conjugation. Briefly, NH2-PEG-b-PLGA (0.5 mg, 1 e.q.), PEG-b-PLGA (2 mg, 4 e.q.), PLGA-COOH (2.5 mg) and SPION (2 mg) were dispersed in 2 mL of tetrahydrofuran (THF). The solution was then added to 20 mL of phosphate buffer solution (PBS, 1 mg/mL) under vigorous stirring, followed by stirring overnight at 35 °C to remove the THF. CDDP (2 mg) was added, and the mixed solution was stirred in the dark at 60 °C for 12 h. Then, 100 µL of Cy5-NHS solution (100 µg/mL) dissolved in DMSO was added and reacted at 37 °C for 2 h. Finally, RGD was bound to PEG according to our previous work for preparing SPHN8. The resulting solution was dialyzed in PBS solution for 48 h.

Physicochemical characterization of SPHN

Dynamic light scattering (DLS) and zeta potential were recorded using a Malvern granulometer. Transmission electron microscopy (TEM) samples were prepared using an SPHN solution (approximately 0.05 mg/mL), and then, the morphology of the SPHN was observed via TEM. The loading efficiencies of SPION and CDDP were recorded by inductively coupled plasma mass spectrometry (ICP‒MS).

In vitro stability assessment

The stability of SPHN (0.5 mg/mL) was assessed in PBS, and the dispersibility over 12 h was recorded using a camera. Then, the SPHN solution was diluted with PBS, and the diameter of the SPHN was recorded using DLS (0, 6 and 12 h).

Drug release

The release of CDDP from SPHN was determined by ICP‒MS. Briefly, 1 mL of SPHN was dialyzed against 9 mL of PBS at pH 7.4, 6.5, and 5.0. Then, 1 mL of dialysate was collected at the set time for ICP‒MS analysis.

In vitro reactive oxygen species (ROS) detection

Human nasopharyngeal carcinoma cells (CNE-1 cells) were plated on confocal dishes and treated with PBS, NPs/CDDP (10 µg CDDP/mL), NPs/SPION (100 µg Fe3O4/mL) and SPHN (10 µg CDDP/mL, 100 µg Fe3O4/mL). Twenty-four hours later, the cells were treated with X-rays (3 Gy), and then, the ROS level in cancer cells was tested via a reactive oxygen species assay kit and observed using confocal laser scanning microscopy (CLSM).

Colony formation assay

CNE-1 cells on 6-well plates were treated with SPHN (5 µg CDDP/mL) for 24 h and exposed to X-ray irradiation (3 Gy). After an additional 7 days of incubation, the colonies were fixed with 4% paraformaldehyde for 15 min. Then, the colonies were stained with 0.1% crystal violet and recorded using an AID vSpot Spectrum plate reader.

Cell apoptosis analysis

CNE-1 cells on 6-well culture plates were treated with PBS, X-rays (3 Gy), SPHN (10 µg CDDP/mL) and SPHN (10 µg CDDP/mL) + X-rays (3 Gy). After an additional 24 h incubation, all cells were collected, washed with PBS three times, stained with an Annexin V-FITC/PI apoptosis kit and then recorded via flow cytometry (FCM).

In vitro combined antitumor effect

The in vitro treatment effect of SPHN on CNE-1 cells was assessed via Cell Counting Kit-8 (CCK-8) kits. CNE-1 cells plated on 96-well plates were treated with SPHN for 12 h with different CDDP concentrations (0-50 μg/mL) and irradiated with X-rays (3 Gy). After an additional 12 h incubation, CNE-1 cells were administered 5% CCK-8 for 2 h. Then, the treatment effect was monitored using a microplate reader.

DNA damage assessment

CNE-1 cells seeded on confocal dishes were treated with SPHN (10 µg CDDP/mL) for 24 h and then irradiated with X-rays (3 Gy). Twelve hours later, the cells were fixed with 4% paraformaldehyde for 15 min, treated with 0.1% Triton X-100 for 20 min, treated with 1% BSA for 1 h, and stained with γH2AX antibody (Alexa Fluor® 488 Mouse anti-H2AX (pS319)) at 4 °C overnight. Finally, the cells were washed with PBS three times, stained with DAPI and then imaged using CLSM.

Western blotting

CNE-1 cells seeded on 6-well culture plates were treated with PBS, X-rays, SPHN and SPHN + X-rays at a CDDP dose of 10 µg/mL and a radiation dose of 3 Gy and incubated for 24 h, washed with PBS three times, lysed with RIPA buffer solution containing phosphatase and protease inhibitors for 0.5 h, and centrifuged at 12,000 rpm at 4 °C for 0.5 h. The proteins were collected, and their concentrations were quantified by a BCA protein assay. Cell lysates were separated by SDS‒PAGE, transferred to a PVDF membrane, and probed with primary antibodies. Membranes were then probed with secondary antibodies against H2AX, γH2AX and β-actin and imaged after enhanced chemiluminescence (ECL) reagents.

Transcriptome sequencing (RNA-Seq)

CNE-1 cells, plated in 6-well culture plates, were treated with PBS, SPHN, X-rays and SPHN + X-rays at a CDDP dose of 10 µg/mL and a radiation dose of 3 Gy. After 24 h of X-rays irradiation, cells were collected, added to 1 ml of TRIzol, and sent to Ige Biotechnology, Ltd. (Guangzhou, China), for RNA extraction and sequencing. An Illumina NovaSeq 6000 System was used for RNA-seq. Fragments per kilobase per million mapped fragments (FPKM) were utilized for gene expression level analysis.

In vivo targeting effect

The in vivo dual-targeting performance of SPHN was assessed in CNE-1 tumor-bearing mice. Groups 1 and 2 were treated with Cy5-labeled non-RGD and SPHN, respectively. Group 3 was injected with Cy5-labeled SPHN and then treated with a magnetic field for 1 h. In vivo imaging was performed in a Kodak IS in vivo FX imaging system at 12 h post-administration. Then, all mice were sacrificed to harvest major organs and tumors for ex vivo imaging. Finally, the tumors were collected to prepare 12 μm-thick paraffin sections, stained with Prussian blue and observed via an optical microscope.

In vivo chemoradiotherapy effect

BALB/c nude mice (4-5 weeks) were inoculated with CNE-1 cells. Ten days later, the mice were treated with PBS, CDDP + X-rays, SPHN, SPHN + X-rays, SPHN+magnetic field (denoted as SPHN(M)) and SPHN(M) + X-rays on Day 1, Day 3 and Day 5 at a CDDP dose of 2 mg/kg. On Days 2, 4 and 6, the CDDP + X-rays, SPHN + X-rays, and SPHN(M) + X-rays groups were treated with X-rays at an irradiation dose of 3 Gy. Tumor size and body weight were recorded for 21 days.

Immunohistochemical staining

The tumors were collected and fixed in 4% paraformaldehyde for 48 h. Then, 4 μm-thick paraffin sections were prepared for HE staining, and the cellular morphologies of tumor tissues were observed via an optical microscope (Leica DMI6000 B). With a similar method, HE staining of the major organs of the SPHN(M) + X-rays-treated groups was performed. Eight micrometer-thick frozen sections were prepared for TUNEL and γ-H2AX staining. After staining with 4′,6-diamidino-2-phenylindole (DAPI, blue fluorescence), the cells were observed using CLSM.

Blood biochemical tests

Blood samples from PBS- or SPHN-treated mice were harvested in heparinized Eppendorf tubes. Key indicators, including alanine transaminase (ALT), aspartate aminotransferase (AST), albumin (ALB), total bilirubin (TBIL), blood urea nitrogen (BUN), and creatinine (Crea), were detected using a fully automatic biochemical analyzer (Chemray 800, China).

In vivo magnetic resonance (MR) imaging

CNE-1 tumor-bearing nude mice were scanned using an MR scanner (Fov: 45×45, thickness: 1 mm, matrix: 224×221, slices: 12, TE: 600 ms, TR: 1200, voxel: 0.2×0.203). With the same method, the acquisition of MRI signals generated by SPHN in the tumor region was performed after SPHN treatments (50 µL, 10 µg SPION/mL).

Results

Preparation and characterization of SPHN

SPHN were prepared using a self-assembly strategy30,31. The morphology and structure of the SPHN were then confirmed by DLS, TEM and zeta potential analysis. As shown in Fig. 1a, the increase in particle size from 6 ± 2 nm to 80 ± 20 nm is consistent with the TEM observations (Fig. 1b), indicating the successful encapsulation of SPION in a hydrophobic PLGA core. The zeta potentials display the weak negative surface charge of SPHN under different pH conditions (pH 7.4, 6.5 and 5.0) (Fig. S1). The contents of Pt and Fe were found to be approximately 5.3% and 13.1% by ICP‒MS, suggesting the successful loading of CDDP and SPION, and the loading efficiency was determined to be approximately 9.06% and 27.07%, respectively. Accordingly, the PEGylated shell coating can endow nanoparticles with improved stability. To evaluate the stability of SPHN, we first observed the in vitro dispersibility in PBS over 12 h. As depicted in Fig. S2, well-dispersed distributions were observed in the SPHN solution, even after standing for 12 h. Moreover, negligible changes in the particle size distribution and intensity were observed in the SPHN PBS solution, which confirmed the good stability of SPHN in vitro. In addition, the release of CDDP was pH-dependent and increased with decreasing pH (Fig. S3).

Fig. 1: Characterization of SPHN and their in vitro therapeutic effect on CNE-1 cells.
figure 1

a, b DLS histogram and TEM images of SPION and SPHN. c CLSM observation of the ability to produce ROS in CNE cells after treatment with PBS, NPs/CDDP (10 μg CDDP/mL), NPs/SPION (29.8 μg SPION/mL), and SPHN (10 μg CDDP/mL, 29.8 μg SPION/mL). All cells were treated with X-rays (3 Gy) 24 h later. The scale bar is 75 µm. d The cytotoxicity of CNE-1 cells treated with SPHN and SPHN + X-rays (3 Gy). e, f The colony formation assay (e) and statistical analysis (f) in CNE-1 cells after treatment with PBS, SPHN (10 μg CDDP/mL), X-rays (3 Gy), and SPHN (10 µg CDDP/mL)+X-rays (3 Gy). **P < 0.01.

Full size image

In vitro therapeutic effect

Accordingly, evidence suggests that CDDP has been extensively used as a radiosensitizer to enhance radiotherapy8. Interestingly, iron oxide nanoparticles also exhibit X-ray-induced radiosensitization effects on cancer cells. Therefore, we next investigated the ROS production ability of CNE-1 cells via CLSM. Compared with that of the PBS-treated group, enhanced mean fluorescence intensity appeared on the cells treated with CDDP-loaded nanoparticles (NPs/CDDP) and SPION-loaded nanoparticles (NPs/SPION). More importantly, the strongest fluorescence appeared in the SPHN-treated mice in the presence of X-rays (3 Gy), which was also verified by CLSM observations (Fig. 1c and Fig. S4), indicating that the combination of SPHN and X-rays can significantly increase the ROS content in cancer cells. Additionally, more G2/M cells were observed in the SPHN and SPHN + X-ray-treated groups (Fig. S5), revealing that our nanomedicines can achieve G2/M cell cycle arrest in CNE-1 cells. Taken together, these results suggest that the codelivery of CDDP and SPION in SPHN, by and large, can improve the radiosensitization effect for enhanced radiotherapy.

Next, we investigated the therapeutic effect of SPHN in CNE-1 cells. As shown, the survival rates of the cells cotreated with SPHN and X-rays (3 Gy) were noticeably decreased in comparison with that of the no irradiation group (SPHN, 0 Gy) (Fig. 1d and Fig. S6). After combination with X-rays, the IC50 of SPHN declined from 26.5 ± 2.9 µg CDDP/mL to 14.1 ± 1.9 µg CDDP/mL (Fig. S7), which suggested that X-ray irradiation can improve the therapeutic effect. To further investigate the potential of SPHN for combined chemoradiation therapy, we evaluated the combined effect using a colony formation experiment. The results indicated that the cell proliferation rate was lower in the SPHN + X-ray-treated cells than in the cells treated with SPHN or radiation alone, confirming that this chemoradiotherapy strategy can significantly inhibit the proliferation of CNE-1 cells (Fig. 1e, f).

Moreover, analysis of cell apoptosis was used to determine the early and late apoptotic cells in CNE-1 cells administered different formulations (Fig. 2a). The percentage of apoptotic cells was 25.29% after SPHN + X-rays treatment and markedly increased compared to that of SPHN or X-rays alone, which were 16.18% and 8.19%, respectively, suggesting that the combination of SPHN and irradiation can accelerate the apoptosis of CNE-1 cells, thus improving the therapeutic effect. To further investigate the antitumor mechanism of SPHN and X-rays, we next observed γ-H2AX, a representative DNA damage marker, by CLSM. As shown in Fig. 2b, the strongest green fluorescence was observed in the cell nucleus of CNE-1 cells treated with SPHN + X-rays. The western blot results also revealed the highest level of γ-H2AX under the combined action of SPHN and X-rays (Fig. 2c), suggesting that the combination of SPHN and X-rays can induce the highest DNA damage and thus improve the anticancer activity for NPC therapy.

Fig. 2: In vitro therapy effect of SPHN in CNE-1 cells.
figure 2

a Apoptosis of CNE-1 cells treated with PBS, X-rays, SPHN, or SPHN + X-rays. b γ-H2AX-stained CNE-1 cells treated with PBS, X-rays, SPHN, or SPHN + X-rays. Scale bar = 50 µm. c Western blot and statistical analysis of γ-H2AX and H2AX in CNE-1 cells after treatment with PBS, X-rays, SPHN, or SPHN + X-rays. *P < 0.05, **P < 0.01, ***P < 0.001.

Full size image

RNA-seq analysis

To determine the cytotoxic mechanisms of SPHN and X-rays, we performed RNA-seq on CNE-1 cells treated with SPHN and X-rays. Venn diagrams visualize the number of common and differentially expressed genes between groups of combinations (Fig. S8). Compared with those of the control group, 332 genes were upregulated (red dots) and 229 genes were downregulated in the X-ray group (Fig. S9a); however, 2513 genes were upregulated and 1137 genes were downregulated in the SPHN group (Fig. S9b). Compared with those of the control group, 2667 genes were upregulated and 1422 genes were downregulated in the SPHN + X-ray group (Fig. 3a). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the genes involved in the tumor necrosis factor (TNF), PI3K-Akt, extracellular matrix (ECM) receptor interaction, NF-kappa B and JAK-STAT pathways were significantly affected by SPHN + X-ray treatment (Fig. 3b). Gene set enrichment analysis (GSEA) was used to further verify the therapeutic mechanism of SPHN in chemoradiotherapy. We performed GSEA based on the Gene Ontology (GO) database, and the top 20 most regulated pathways in the GSEA are shown in Fig. 3c. From the GSEA-GO lollipop chart, several transcriptional and translational signaling pathways were significantly decreased (Fig. 3c). Specifically, translation initiation pathways and cytoplasmic translation pathways were inhibited in the SPHN + X-rays group (Fig. 3d, e). In contrast, immune response pathways were activated after SPHN + X-rays treatment (Fig. 3f). These results demonstrated that SPHN + X-rays treatment might decrease the intracellular translation signaling pathway and thereby trigger apoptosis of cancer cells. Furthermore, the enhancement of the immune response pathway suggested that our therapy may lead to an immune response that further reduces the possibility of tumor recurrence.

Fig. 3: The RNA-seq analysis on CNE-1 cells.
figure 3

a The differentially expressed gene volcano plot comparing the SPHN + X-rays group vs. the control group. Log2 (fold change): log2 value of the fold of difference; Up: upregulated genes, Down: downregulated genes; no-DEGS (no differentially expressed genes): genes with no significant difference. b Bubble chart of KEGG enrichment analysis between the SPHN + X-rays group and the control group. c Lollipop chart of gene set enrichment analysis (GSEA) of the regulated gene pathways with the GO database. d–f Normalized enrichment score (NES) and enrichment map of GSEA plots of differentially expressed pathways in the SPHN + X-rays treatment vs. the nontreated control.

Full size image

In vivo dual targeted effect

To investigate the potential of SPHN for in vivo chemoradiotherapy, we first investigated the targeted effect of SPHN in CNE-1-bearing mice via a biodistribution experiment after administration of non-RGD nanoparticles (denoted NPs) (Fig. 4a(i)), SPHN (Fig. 4a(ii)), and SPHN combined with a magnetic field (denoted SPHN(M) (Fig. 4a(iii)). As shown in Fig. 4b, the strongest fluorescence signal was observed in the mice treated with SPHN(M), and the second strongest fluorescence appeared in the SPHN-treated group, while the weakest signal was displayed in the NP-treated mice. Additionally, consistent results were observed in the ex vivo imaging (Fig. 4c and Fig. S10) and Prussian blue staining experiments (Fig. 4d). These results revealed that RGD in the SPHN, to some extent, can improve the tumor accumulation effect of nanomedicines. After combination with the magnetic targeting of SPION, the targeted effect of SPHN would be significantly enhanced, thus providing an alternative strategy to achieve effective targeted delivery.

Fig. 4: The in vivo targeted effect in CNE-1-bearing mice.
figure 4

a A scheme showing the treatment of tumor-bearing mice. b, c Fluorescence imaging of CNE-1-bearing mice (b) and ex vivo organs (including heart (He), liver (Li), spleen (Sp), lung (Lu), kidney (Ki), and tumor (Tu)) from CNE-1-bearing mice that were treated with Cy5-NPs, Cy5-SPHN and Cy5-SPHN(M) for 12 h (c). d Images (100×) of Prussian blue staining of tumors from CNE-1-bearing mice treated with Cy5-NPs, Cy5-SPHN and Cy5-SPHN(M) for 12 h.

Full size image

In vivo therapeutic effect

Next, the antitumor efficacy of SPHN was evaluated in CNE-1-bearing nude mice (Fig. 5a). Figure 5b-c shows that the mice treated with SPHN + X-rays exhibited significant tumor suppression, with a tumor inhibition rate of 86.17% versus that of PBS treatment over 21 days; this rate was significantly higher than that of the X-ray (49.99%)- and SPHN (75.46%)-treated groups. More importantly, the best tumor suppression with an inhibition rate of 94.49% was found in the SPHN(M) + X-ray-treated group due to the improved tumor accumulation resulting from the magnetic targeting of SPHN. To further demonstrate the chemoradiotherapy effect of SPHN, we conducted H&E and TUNEL staining to evaluate cell apoptosis, and γ-H2AX immunofluorescence staining was conducted to evaluate the DNA damage level. The results displayed the highest level of cell apoptosis in the tumor site and DNA damage in cancer cells after simultaneous administration of SPHN(M) + X-rays (Fig. 5d), which can also be confirmed by further statistical analysis (Fig. 5e, f), suggesting that SPHN + X-rays had the best therapeutic effect compared to other formulations.

Fig. 5: The in vivo therapeutic effect in CNE-1-bearing mice.
figure 5

a Workflow of the in vivo experiment. i Intravenous injection with SPHN. ii Magnetic targeting. iii Radiation with X-rays. b, c The tumor volume growth curves (b) and tumor weight (c) in the mice treated with PBS, X-rays, SPHN, SPHN + X-rays, SPHN(M), and SPHN(M) + X-rays 3 times over 21 days. d Images of HE-, TUNEL- and γ-H2AX-stained tumor slices from CNE-1-bearing mice treated with PBS, X-rays, SPHN, SPHN + X-rays, SPHN(M) and SPHN(M) + X-rays. The scale bar is 100 µm. e, f Quantitative analysis of the TUNEL-positive cells (e) and γ-H2AX density (f) in the tumor tissues after treatment with PBS, X-rays, SPHN, SPHN + X-rays, SPHN(M), and SPHN(M) + X-rays. *P < 0.05, **P < 0.01, ***P < 0.001.

Full size image

Biosafety assessment and MRI

Biosafety is a key determinant in the in vivo application of nanomedicine. To verify the in vivo application potential of SPHN for NPC combined with chemoradiation therapy, we evaluated the biosafety of SPHN by blood biochemistry 21 days post-treatment. According to the results (Fig. 6a–f), the major indicators of liver and kidney function in the mice that were treated with SPHN were within normal limits and showed no significant differences compared to those of the mice treated with PBS. Moreover, the H&E-stained organs of the SPHN + X-ray-treated mice, including the heart, liver, spleen, lung, and kidney, exhibited no obvious histopathological abnormalities, revealing no apparent toxicities caused by SPHN in vivo (Fig. 6g). Image technology is another key determinant for cancer therapy, especially for radiotherapy or chemoradiotherapy. The SPHN are composed of SPION, a widely used MRI contrast agent, and thus show good MRI ability. To investigate the application potential of SPHN in precise NPC treatment, we assessed the in vivo MRI capability of SPHN in CNE-1 tumor-bearing mice. Compared to that of the untreated mice, an obvious T2 signal was observed on the tumors of the SPHN-treated mice (Fig. S11), suggesting that SPHN are likely to act as an MR contrast agent for MR-guided NPC chemoradiation therapy.

Fig. 6: Biosafety assessment of SPHN.
figure 6

a–f The major indicators of liver and kidney function in the mice treated with PBS, X-rays, SPHN, SPHN + X-rays, SPHN(M) and SPHN(M) + X-rays for 21 days. g Images of H&E-stained major organs in the SPHN(M) + X-ray treated mice 3 times over 21 days. Scale bar = 200 µm.

Full size image

Discussion

Nanoparticle-based strategies for NPC chemoradiation therapy have attracted increased attention, but there has been no dramatic improvement in clinical outcomes to date, which is mainly attributed to the poor in vivo targeting ability of traditional nanomedicines. To overcome the challenges mentioned above, we developed SPHN as nanosensitizers for NPC targeted therapy. On the one hand, such nanosensitizers can significantly improve the radiotherapy effect by the radiosensitization of CDDP and SPION. On the other hand, the dual-targeted strategy can significantly improve the tumor-targeting effect of SPHN, thus achieving effective chemoradiotherapy in vivo.

Since the SPHN consist of CDDP and SPION, both of which exhibit X-ray-induced radiosensitization effects for enhancing radiotherapy, we investigated the ROS production ability of SPHN in CNE-1 cells. In Fig. 2a, the results of CLSM and FCM observation suggested that the codelivery of CDDP and SPION in SPHN, by and large, can achieve effective radiosensitization. Next, we investigated the therapeutic effect of SPHN in CNE-1 cells. With the help of X-rays, the SPHN achieved an enhanced therapeutic effect and significantly inhibited the proliferation of CNE-1 cells in vitro. In addition, the results of γ-H2AX staining revealed that the combination of SPHN and X-rays can dramatically induce DNA damage in CNE-1 cells. Altogether, our data suggest that SPHN can achieve an enhanced therapeutic effect in vitro, which is attributed to their good chemoradiation therapy.

The tumor-targeted effect plays an important role in the in vivo application of anticancer nanoagents. As shown in Fig. 4, the RGD-magnetic dual targeting design endows SPHN with outstanding tumor targeting ability. Under a magnetic field, SPHN can significantly accumulate in tumor tissue for effective targeted delivery of antitumor agents. Next, the antitumor effect of SPHN was assessed in CNE-1-bearing nude mice. As shown in Fig. 5, the mice treated with SPHN, X-rays and magnetic fields exhibited the most significant suppression of tumor growth and the highest levels of tumor apoptosis and DNA damage. Since the magnetic field can further improve the tumor accumulation of SPHN, the SPHN can achieve the most effective delivery for CDDP and SPION. In the presence of X-rays, the highest accumulation of CDDP and SPION can induce the best therapeutic effect, thus achieving effective chemoradiotherapy for NPC. In addition, biosafety and therapeutic accuracy are other important factors for nanoparticle-based precise treatment. Here, the in vivo biosafety safety of SPHN was evaluated by blood biochemistry and HE staining of major organs. According to the results (Fig. 6), the indicators of liver and kidney function and the cell morphology of organs were normal after treatment with SPHN, revealing no apparent toxicities caused by SPHN in vivo. Moreover, the in vivo MRI results indicated that SPHN showed good MRI capability (Fig. S11). Altogether, SPHN are likely to achieve a precise therapy for NPC, with no notable side effects.

Conclusion

In summary, we have developed SPHN that can effectively target tumors. With the codelivery of CDDP and SPION, SPHN can induce high-level DNA damage via combined chemoradiotherapy, thus inducing effective apoptosis in CNE-1 cells and significantly inhibiting their proliferation. More importantly, RGD magnetic targeting can endow SPHN with available tumor accumulation for the targeted delivery of antitumor agents in vivo, resulting in a significantly enhanced antitumor effect in CNE-1-bearing mice. Considering their potential in precise treatment, such nanoparticles may also act as an MR contrast agent for MR-guided NPC chemoradiation therapy.