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).

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• Source: https://www.nature.com/articles/s41427-023-00484-x