A multifunctional mesoporous silica drug delivery nanosystem that ameliorates tumor hypoxia and increases radiotherapy efficacy

Materials, cells, and animals

All chemicals were used as received without any additional purification. Ethylene glycol, manganese acetate dihydrate, zinc acetate dihydrate, sodium borohydride, and hydrogen peroxide (H₂O₂) were purchased from Sinopharm Chemical Reagent Co., Ltd. Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES), succinic anhydride, trimethylamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and COOH-PEG-COOH (MW = 5000) were obtained from Sigma‒Aldrich Chemical Company.

H22 hepatocellular carcinoma cells and 4T1 breast cancer cells were obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology. These cells were cultured with RPMI 1640 medium containing 10% FBS and 1% P/S at 37 °C and 5% CO₂. Prior to use, the cells were tested for Mycoplasma contamination, and only Mycoplasma-free cells were used in the experiments.

BALB/c and ICR female mice aged 5–6 weeks were purchased from Shanghai Sippr-BK Laboratory Animal Co., Ltd. (Shanghai, China) and were housed in the specific pathogen-free (SPF) Laboratory Animal Center of the Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School. All animal experimental protocols were approved by the Laboratory Animal Care and Use Committee of the Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School (Approval Number: 2021AE01029).

Synthesis and functionalization of radially-oriented MSNs

Radially-oriented MSNs with nanochannels were synthesized using a surfactant-assembly sol-gel process in a Stöber solution containing CTAB, TEOS, ammonia, and ethanol, as previously described¹¹. To introduce amine groups for functionalization, we utilized APTES as the reagent. The obtained MSNs (300 mg) were then redispersed into a flask with 30 ml of toluene and refluxed for 30 min to ensure optimal nanoparticle dispersion. Subsequently, APTES (300 µl) was added to the refluxing MSN solution, and the mixture was left to react for an additional 10 h. Amine-functionalized MSNs (MSN-NH₂) were thoroughly washed with ethanol. Finally, the surface-bound amine moieties were converted into carboxyl groups by dispersing MSN-NH₂ in 20 ml of DMSO along with succinic anhydride (100 mg) and triethylamine (100 ml), and the resulting solution was stirred at 50 °C for 48 h.

Synthesis and amine functionalization of Mn-doped ZnO₂ (Mn/ZnO₂)

Zinc acetate dihydrate (3 mmol) and manganese acetate dehydrate (0.06 mmol) salts were dissolved in ethanol (80 ml), and the solution was refluxed with vigorous stirring. Subsequently, an ethanolic solution (20 ml) of sodium borohydride (7.5 mmol) was introduced, and following the addition of the borohydride solution, 5 ml of 30% H₂O₂ was added. The addition of H₂O₂ caused the solution to turn milky. The solution was continuously stirred for an additional 15 min under reflux conditions and then left to reach room temperature. The resulting slurry was diluted by adding 200 ml of boiling water and filtered by vacuum, and the resulting filter cake was washed with 50 ml of hot ethanol to remove byproducts. Mn/ZnO₂, as a wet product, was dispersed in anhydrous N,N-dimethylformamide (80 ml). In the next step, 3-aminopropyltriethoxysilane (200 µl) was added to the solution, and the reaction mixture was stirred at 120 °C for 10 min. The resulting amine-functionalized Mn/ZnO₂ precipitate was isolated by centrifugation and washed with DMF. Finally, the amine-functionalized Mn/ZnO₂ nanoparticles were dispersed in water for further use.

Synthesis of MSN@R837-Mn/ZnO₂

For drug loading, carboxyl group-functionalized MSNs (100 mg) were dispersed in 10 ml of a drug solution (2 mg/ml, DMSO) and stirred overnight at room temperature. The resulting R837-loaded MSNs (MSN@R837) were separated by centrifugation to remove any unloaded free drug molecules. To encapsulate the drug within the carboxyl-functionalized MSNs, amine-functionalized Mn/ZnO₂ was used, and EDC chemistry was employed for the sealing process. MSN@R837 was redispersed in water, and then 5 mg of EDC and 5 ml of Mn/ZnO₂ (10 mg/ml) were added and vortexed for 5 min at room temperature. Upon successful generation of the Mn/ZnO_2-sealed MSN@R837 (MSN@R837-Mn/ZnO₂) product, COOH-PEG-COOH was further attached to the surface using EDC chemistry to increase the blood circulation time of MSN@R837-Mn/ZnO₂.

Characterization

Transmission electron microscopy (TEM) was performed using an FEI Tecnai G2 F20 electron microscope operating at 200 kV. Powder RT-ray diffraction (PXRD) patterns were obtained using a Rigaku D/Max 2550 RT diffractometer with Cu-Kα radiation. Fourier transform infrared (FTIR) spectra were collected using a Nicolet Impact 410 FTIR spectrometer within the range of 400–4000 cm⁻¹. Ultraviolet and visible absorption (UV − VIS) spectra were recorded using a Shimadzu UV-3600 plus spectrophotometer. For the elemental analysis of Zn²⁺, a Perkin-Elmer Optima 3300DV ICP‒OES was utilized.

In vitro drug release

The pH-responsive behavior of MSN@R837-Mn/ZnO₂ was investigated by dialysis of a certain amount of pre-made samples in various pH buffer solutions. The drug release profile was determined by periodic sampling using UV/VIS spectroscopy (detected at 319.6 nm). The drug loading efficiency and drug loading capacity were calculated to be 61% and 12.2%, respectively.

In vitro and in vivo MR imaging

To obtain T1-weighted MR images, samples of MSN@R837-Mn/ZnO₂ at different concentrations were placed in a twelve-well plate containing various pH buffer solutions. For the in vivo MRI study, MSN@R837-Mn/ZnO₂ nanoparticles (3.33 mg/ml, 200 µl per mouse, n = 3) were intravenously injected into H22 tumor-bearing mice. MR images were acquired at different times.

Animal experiments

H22 hepatocellular carcinoma cells were subcutaneously injected into the left lower abdomen of ICR mice (2 × 10⁶ cells per mouse, Day 0). Approximately 5 days later, when the tumor size reached almost 100 mm³, the mice were randomly divided into eleven groups: the NS group (200 μl/mouse), MSN group, MSN-Mn/ZnO₂ group, MSN@R837-Mn/ZnO₂ group (200 μg/200 μl/mouse), RT group (5 Gy/mouse), RT + MSN group, RT + MSN-Mn/ZnO₂ group, RT + MSN@R837-Mn/ZnO₂ group, PD-1 group (100 μg/mouse), RT + PD-1 group, and RT + PD-1 + MSN@R837-Mn/ZnO₂ group. The nanoparticle treatment was administered on Day 5 through intravascular injection, followed by RT irradiation 4 h after injection and anti-PD1 antibody treatment was administered on Days 7, 9 and 11. The tumor sizes and body weights were measured once every 2 or 3 days, and the mice were euthanized when the lengths of tumors reached 1.5 cm. On Day 5, 4 h after nanoparticle treatment or RT, the tumors were collected for immunofluorescence staining. On Day 15, the following serum samples were collected to assay the following biochemical indicators: aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (Cr). Hearts, livers, lungs and kidneys were excised for hematoxylin–eosin staining. Spleens, lymph nodes and tumors were also collected for flow cytometry on Day 15.

Similarly, 4T1 breast cancer cells were subcutaneously injected into the left lower abdomen of BALB/c mice (2 × 10⁶ cells per mouse, Day 0). After approximately 5 days, when the tumor size reached almost 100 mm³, the mice were randomly divided into five groups: the NS group (200 μl per mouse), RT group (5 Gy per mouse), RT + MSN group, RT + MSN-Mn/ZnO₂ group, and RT + MSN@R837-Mn/ZnO₂ group (200 μg dissolved in 200 μl of NS per mouse). The nanoparticle treatment was administered on Day 5 through intravascular injection, followed by RT irradiation 4 h after injection. The tumor sizes and body weights were measured once every 2 or 3 days, and the mice were euthanized when the lengths of the tumors reached 1.5 cm.

Therapeutic efficacy of MSN@R837-Mn/ZnO₂ in patient-derived organoids

Two pathological types of human gastric cancer tissues, namely, mucous adenocarcinoma and low-adhesion carcinoma, were obtained from the Pathology Department of the Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School for the culture of patient-derived organoids (PDOs). PDOs were established following previously described culture methods^12,13,14. Organoids were harvested from the Matrigel using 1x TrypLE (Gibco) and dissociated into small clusters for drug response analysis. Subsequently, the organoids were resuspended in 2% Matrigel/organoid culture medium (200–1000 clusters/ml) and dispensed into 384-well plates in triplicate. The dose of MSN@R837-Mn/ZnO₂ used in the experiment was 100 µg/ml. To analyze the drug response, a CellTiter-Glo 3D Cell Viability Assay (Promega) was used according to the manufacturer’s instructions after 14 days of different treatments, and the results were normalized to those of vehicle controls. This research involving human participants was approved by the Research Ethics Committee of the Comprehensive Cancer Center of Drum Tower Hospital (Ethics approval number/ID: 2021–324). All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Prior to participation in the study, written informed consent was obtained from all patients involved.

Immunofluorescence staining

Tumor sections were prepared and incubated with specific fluorescent probes and antibodies to visualize various cellular markers. Zinquin ethyl ester (J&K Scientific, China) was used to stain zinc ions, while DCFH-DA (Sigma‒Aldrich, USA) was used to detect ROS. A Hypoxyprobe Green Kit (HPI Inc., USA) was utilized to label the hypoxic areas. Additionally, an anti-CD31 rat monoclonal antibody (1:200) (Abcam, UK) and an anti-calreticulin rabbit monoclonal antibody (1:200) (Abcam, UK) were applied to detect CD31-expressing blood vessels and ICDs, respectively. The tumor sections were incubated with these probes and antibodies overnight at 4 °C. After thorough washing with PBS three times, the sections were further stained with secondary antibodies to detect the primary antibody-bound targets. Specifically, a goat anti-rat IgG H&L (Cy5, 1:200) (Abcam, UK) and a goat anti-rabbit IgG H&L (Cy3, 1:200) (Abcam, UK) were used as secondary antibodies. DAPI (Sangon Biotech, China) was used as a nuclear counterstain. To preserve the fluorescence signals, the sections were sealed with 50% glycerol. Subsequently, fluorescence images were captured using a confocal microscope (Leica, Germany) to visualize and analyze the specific markers and cellular components within the tumor sections.

Flow cytometry

Single-cell suspensions were prepared as follows: the lymph nodes and spleens were mechanically dissociated, filtered, and suspended in NS (0.5–1 × 10⁶ cells/ml). Tumors were cut into small pieces, incubated with collagenase type IV (1 mg/ml, Sigma, USA) at 37 °C for 3–4 h, filtered, and suspended in NS (0.5–1 × 10⁶ cells/ml). As most tested antigens are expressed on cell membranes, the samples were stained with specific antibodies for 20 min at 4 °C in the dark and then washed before analysis. For Foxp3, which is expressed in the nucleus, the True-Nuclear Transcription Factor Buffer Set (Biolegend, USA) was used. The following monoclonal antibodies obtained from Biolegend were used for flow cytometry: CD11c-FITC (5 μg/ml), CD80-APC (2 μg/ml), CD86-PE (2 μg/ml), CD11b-FITC (5 μg/ml), F4/80-PE-Cy7 (2 μg/ml), CD206-APC (2 μg/ml), CD3-FITC (5 μg/ml), CD4-PE-Cy7 (2 μg/ml), CD8-PE-Cy5 (2 μg/ml), CD44-PE (2 μg/ml), CD62L-APC (2 μg/ml), CD25-APC (2 μg/ml), and Foxp3-PE (2 μg/ml). To measure the levels of IL-6 and IL-10, a BD™ Cytometric Bead Array (CBA), Mouse Interleukin (IL)-6 Flex Set, and Mouse IL-10 Flex Set were used for detection and analysis. By employing these techniques and reagents, we were able to analyze immune cell populations, surface marker expression, and cytokine levels within single-cell suspensions obtained from lymph nodes, spleens, and tumors.

Cytotoxicity assay of mouse splenocytes

H22 hepatocellular carcinoma cells were labeled with CFSE for 10 min at 37 °C in the dark. Subsequently, splenocytes from mice in the four groups (RT group, RT + MSN group, RT + MSN-Mn/ZnO2 group, and RT + MSN@R837-Mn/ZnO2 group) were cocultured with CFSE-labeled H22 hepatocellular carcinoma cells at different effector-to-target ratios (E:T) of 5:1, 10:1, and 20:1. The cocultures were maintained at 37 °C and 5% CO₂. After 6 h, the cells were stained with propidium iodide (PI) for 20 min at 4 °C in the dark and then washed prior to analysis.

Statistical analyses

Unless otherwise specified, all experiments were performed with biological replicates. Paired two-tailed Student’s t tests were used for comparisons between two groups. For comparisons among multiple groups, one-way analysis of variance (ANOVA) or two-way ANOVA was applied. Survival benefit was assessed using the log-rank test. Statistical significance is denoted as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.