A hybrid nanopharmaceutical for specific-amplifying oxidative stress to initiate a cascade of catalytic therapy for pancreatic cancer

Materials

3,5,7-trihydroxy-2-phenyl (Galangin, GAL) were purchased from Desite Biotechnology Co., Ltd. (Chengdu, China). Polyethylene Glycol (PEG, MW 600), ammonia hydroxide (28%) and dimethyl sulfoxide (DMSO) were obtained from TCI Development Co., Ltd. (Shanghai, China). Potassium permanganate (KMnO4), hydrogen peroxide (H2O2, 30%) and ethanol were provided by Chongqing Medical University. Tetraethyl orthosilicate (TEOS), methylene blue (MB) and doxorubicin (DOX) were provided by Aladdin Reagents Company (Shanghai, China). None of the compounds were further purified before usage.

Preparation of SiO2-GAL nanospheres (SG NPs)

The SG NPs were prepared using a previously described procedure, with some alterations [54, 59]. Typically, ethanol (75 mL) and ammonia hydroxide (3 mL) were mixed and stirred for 0.5 h. Then, GAL (20 mg) was added into the liquid before stirring for another 0.5 h. To obtain SG NPs, TEOS (80 µL) as a source of SiO2 was added and stirred for 24 h, centrifuged at 13,000 rpm for 10 min, and washed three times with ethanol and water.

Preparation of SiO2-GAL@MnO2 nanospheres (SG@M NPs)

According to the following equation: 2KMnO4 + H(OCH2CH2)nOH + O2 → MnO2↓ + HO(OCCOOH)n + K2MnO4 + H2O, we selected PEG as the reductant to react with KMnO4 to form the MnO2 shell [60]. Briefly, SG NPs (2 mg) were dissolved in deionized water (10 mL). After that, PEG (500 µg/mL, 200 µL) was added and mixed for 0.5 h to achieve complete fusion. The same volume of aqueous KMnO4 (200 µg/mL, 10 mL) was mixed and stirred for 24 h before centrifugation at 13,000 rpm for 10 min.

Characterization

The morphology of the SG NPs and SG@M NPs was obtained using a Talos F200S TEM (Thermo Fisher Scientific CDLtd, CZ) and the Si and Mn elemental distributions were obtained by energy dispersive X-ray spectroscopy (EDS) using 4 in-column Super-X Detectors. A particle sizer and zeta potential analyzer (NanoBrook Omni, Brookhaven Instruments, UK) was applied to determine the particle size and zeta potential. X-ray diffraction (XRD) analysis was carried out using a D8 Advance X-ray diffractometer (Bruker, Germany). X-ray photoelectron spectroscopy (XPS) measurements of the valence states of Si and Mn were obtained using the K-Alpha XPS System (Thermo Fisher Scientific, Waltham, MA, USA). Fourier transform infrared (FT-IR) spectra of functional group information was captured with a Thermo Scientific Nicolet IS10. All of the samples were freeze-dried to powder form prior to analysis. UV–Vis spectra of MnO2 present in SG@M was detected using a Shimadzu spectrophotometer UV-3600. Each sample was digested using the hydrofluoric acid solution, and then the content of Mn and Si in SG@M were measured using inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer NexION 300X). Loading content of GAL was detected by high performance liquid chromatography (HPLC, Shimadzu LC-20AD) under the following chromatographic conditions: chromatographic column: Ultimate XB-C18 (4.6 × 100 mm 3 µm), mobile phase: 0.1% phosphate solution-methanol (30:70), flow rate: 1.0 mL/min, column temperature: 30 ℃, wavelength: 267 nm.

MnO2 degradation and GSH depletion

SG@M (100 µg/mL, 1 mL) was dispersed in phosphate buffer saline (PBS, pH = 6.5), which included various concentrations of GSH (0, 1, 2, 3, 4, 5 mM). Then, the UV−Vis absorption spectra of these examples were measured, while simultaneously taking digital photos. The absorption spectra of SG@M dispersed in PBS (pH = 7.2) with or without GSH were additionally examined.

Extracellular GSH depletion was discovered using the 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) solution. In summary, SG NPs (100 µg) and SG@M NPs (100 µg) were dissolved in PBS (1 mL) and reacted with GSH (5 mM), respectively. Afterward, the sediment was removed by centrifugation (13,000 rpm, 10 min) for various incubation durations (0, 10, 30 min, 2, 12, 24 h). After adding the supernatant and DTNB solution (3 mg/mL, 10 µL) to a 96-well plate, the absorbance at 412 nm was measured.

·OH generation detection

Methylene blue (MB) identified the production of ·OH by SG@M. First, different concentrations of SG@M (0, 40, 80, 120, 240 µg/mL) were added to a buffer solution containing NaHCO3 (25 mM) and GSH (5 mM) to react for 15 min, and the supernatant was obtained by centrifugation. Second, the supernatant, as the reaction product was mixed with MB (10 µg/mL) and H2O2 (8 mM) and reacted for 30 min at 37 ℃. Finally, the absorbance of MB was measured at 665 nm. Alternately, the absorbance change of MB at 665 nm was also measured after an equivalent amount of SG@M (120 µg/mL) was reacted with different concentrations of GSH (0, 0.5, 1, 2, 5 mM).

Mn2+ and GAL release

To imitate various biological conditions, SG@M (5 mg/mL, 1 mL) was placed into a dialysis bag, which was subsequently placed in 49 mL of PBS (pH 7.2, pH 6.5 with or without GSH (5 mM)). The Mn2+ and GAL were released with continuous shaking at 37 ℃. After that, the solution (6 mL) was taken outside the dialysis bag to measure the Mn2+ concentration by ICP-MS (5 mL) and the drug concentration by HPLC (1 mL) at various times (0, 2, 4, 6, 8, 10, 12, 24 h). After each sampling, the simulated solution (6 mL) was added to keep the volume constant. The cumulative release rate of Mn2+ in 2 h (1, 2, 4, 8, 16, 32, 60, 90, and 120 min) was measured according to the same method.

O2 generation detection

To track O2 production, [Ru(dpp)3]Cl2 (RDPP) functions as a sensitive fluorescence probe that can be quenched by O2 [61]. First, each substance, SG (200 µg/mL, 1 mL) and SG@M (200 µg/mL, 1 mL) suspended in PBS (pH = 6.5) was treated with RDPP (50 µL) and H2O2 (40 µM, 200 µL). Then, the fluorescence intensity was recorded at 615 nm at various times (0, 2, 4, 6, 8, 10, 12 min). As a control, the O2 generation of SG@M (200 µg/mL) and free H2O2 were also measured. Every 6 s, a dissolved oxygen meter (JPBJ-608, Shanghai Oustor Industrial Co.) measured the dissolved oxygen content in the above groups.

Cell culture

Human pancreatic cancer cell (PANC-1) was generously offered by the Stem Cell Bank, Chinese Academy of Sciences, and cultured in Dulbecco's modified eagle medium (DMEM) medium containing 10% fatal bovine serum (FBS) and 1% penicillin–streptomycin at 5% CO2 and 37 °C.

Intracellular ROS generation detection

PANC-1 cells were exposed to free GAL, SiO2, SG, S@M and SG@M for 24 h. After 24 h, each group was introduced to 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (10 µM) for 20 min of incubation before images were captured by laser scanning confocal microscopy (LSCM). Control cells were those that received no treatment. With ImageJ software, the fluorescence intensity was quantitatively analyzed.

Mitochondrial membrane potential (MMP) detection

The changes in MMP in cells were assayed by JC-1 following various treatments. Briefly, PANC-1 cells were seeded in confocal dishes for 24 h, then incubated for another 24 h with either PBS, free GAL, SiO2, SG, S@M or SG@M. All cells were washed with PBS before being stained for 20 min with JC-1 solution (10 µM). LSCM obtained fluorescent images, and flow cytometry measured the ratio of JC-1 aggregates/monomers.

In vitro therapeutic efficacy

For the CCK-8 assay, after PANC-1 cells (1 × 104 cells per well) were seeded into a 96-well plate and incubated for 24 h, the medium was replaced with new medium containing SiO2, SG, S@M and SG@M at varying concentrations (0, 100, 200, 400 µg/mL), and further incubated for 24 h. Then, the cell viability was measured by CCK-8 assay. Cells were treated with different concentrations of S@M (0, 6.25, 12.5, 25, 50, 100, 200, 400 µg/mL) for 24 h and 48 h to further investigate the cytotoxicity of CDT. The cell viability was calculated to follow the Eq. (1) [38], and the additive therapeutic efficiencies of independent treatments were estimated using Eq. (2) [62, 63]:

$$Cell\,viability = \frac}}\,\,\,\times 100\%$$

(1)

$$T_ = \,100 - \left( \times f_ } \right) \times 100,$$

(2)

where f is the fraction of surviving cells after each treatment.

To assess apoptosis, cells (2 × 105 cells per well) were cultured in a 6-well plate for 24 h. Following several treatment methods employing PBS, SiO2, free GAL (22.18 µg/mL), SG (200 µg/mL), S@M (200 µg/mL) and SG@M (200 µg/mL), cells were collected for flow cytometry.

To assess relative Caspase 9 and Caspase 3 activity, a total of 3 × 106 cells per group were coincubated for 24 h with each of the following 6 groups: PBS, SiO2, GAL, SG, S@M, and SG@M. Cell precipitates were obtained through centrifugation and digestion. According to the operating manual of the Caspase 9 Activity Kit and Caspase 3 Activity Kit (Beyotime), Caspase 9 and Caspase 3 activity were calculated for each group.

To assess cell cycle, PBS, free GAL (22.18 µg/mL), SG (200 µg/mL), S@M (200 µg/mL) and SG@M (200 µg/mL) were added to PANC-1 cells (2 × 105 cells per well), which were then incubated for 24 h. For the flow cytometry experiment, cells were collected and dispersed in a solution of PBS (100 µL) and 75% ethanol (900 µL).

Western Blot analysis. PANC-1 cells were digested and centrifuged after 24 h of different treatments, washed 2–3 times with PBS and lysed in RIPA lysis buffer (cat. No. BL504A, Biosharp) with protease inhibitors to extract total proteins. The protein samples were then separated by SDS–PAGE and transferred to PVDF membranes (cat. No. IPVH00010, Millipor). Afterward, the membranes were blocked with 5% BSA solution for 1 h and incubated with primary antibodies against STAT3 (1:1000 v/v, GB11176, Servicebio), p-STAT3 (1:1000 v/v, AF3293, Affinity), p-JAK2 (1:1000 v/v, YP0155, Immunoway), Bcl-2 (1:1000 v/v, BF9103, Affinity), BAX (1:1000 v/v, GB11690, Servicebio), Cyclin B1 (1:1000 v/v, AF6168, Affinity), and GAPDH (1:5000 v/v, AF7021, Affinity) overnight at 4 ℃. After washing with TBST, the membranes were incubated with respective secondary antibodies conjugated with horseradish peroxidase for another 1 h at RT. Finally, the protein signals were recorded and visualized using hypersensitive ECL chemiluminescence reagent (Biosharp).

Animals and tumor models

BALB/c nude mice (male, 5−6 weeks of age, 16−18 g body weight) were obtained from Tengxin Biotechnology Co., Ltd. (Chongqing, China) and housed in the Laboratory Animal Center of Chongqing Medical University. All animal experiments and procedures were approved by Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University. By co-inoculating of PANC-1 cells with the Matrigel mixture, the pancreatic xenograft subcutaneous transplantation tumor model was established. Briefly, PANC-1 cells (1 × 107 cells) were suspended in a PBS (100 µL) and Matrigel mixture (1:1, v/v) and subcutaneously transplanted into the right foreleg axilla of each mouse. After approximately 15 days, the tumor volumes of the tumor-bearing mice reached 100 mm3 for in vivo treatment and imaging. (The tumor volumes were calculated using the formula: 0.5 × length × width2).

US imaging in vitro and in vivo

For in vitro US imaging, various solutions (H2O2, SG + H2O2, SG@M, SG@M + H2O2) were added to a transparent gel tube and then monitored via a Vevo LAZR imaging system (Visual Sonics Inc., Toronto, ON, Canada). For in vivo US imaging, SG@M NPs (10 mg/mL, 100 µL) were intravenously administered into PANC-1 tumor-bearing nude mice (n = 3). Prior to the injection as well as at certain intervals (30 min, 2, 4, 5, 6, and 24 h), B-mode and CEUS images were captured using the US system of EPIQ 7 (Philips, Netherlands).

MR imaging in vitro and in vivo

Both in vitro and in vivo MRI were performed by the Siemens 3 T MRI system at The Second Affiliated Hospital of Chongqing Medical University. In vitro, different concentrations of SG@M (0.0625, 0.125, 0.25, 0.5, 1, 2 mg/mL) were distributed in a PBS solution (pH = 6.5) containing GSH (5 mM), and the samples were subsequently imaged by the MRI system. In addition, the same concentrations of SG@M NPs in PBS solution (pH = 7.2, without GSH) were utilized as a control. In vivo, SG@M NPs (10 mg/mL, 100 µL) were intravenously injected into PANC-1 tumor-bearing nude mice (n = 3), and T1-weighted signals were recorded before and after the injection for 2, 4, 6, 8, 12, and 24 h using the MRI scanner.

Mn accumulation in tumor tissues and blood circulation

A dose of SG@M (10 mg/mL, 100 µL) was injected intravenously into PANC-1 tumor-bearing mice (n = 3). Approximately 20 µL of blood was taken from each tail at various times (2, 4, 6, 8, 12, 24 h) following injection. The mice were killed at the conclusion of the experiment, and the tumors were collected and weighed. ICP-MS was utilized to determine the quantity of Mn in each sample.

Antitumor therapy in vivo

The mice were randomly assigned to 6 groups (n = 6) when tumor sizes reached 100 mm3: control (group 1); SiO2 (group 2); GAL (group 3); SG (group 4); S@M (group 5); SG@M (group 6). In the experimental groups, mice were intravenously injected with GAL (1 mg/mL, 100 µL), equal amounts of SG, S@M and SG@M (10 mg/mL, 100 µL), and the control group only received saline solution. On a regular basis throughout the course of the treatment, the body weight and tumor volume of the mice in each group were noted. On the 18th day, the mice were sacrificed, and the tumor tissues were dissected and stained with hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase dUTP nick end (TUNEL), and Ki-67 antibody. Meanwhile, immunofluorescence staining was performed on the harvested tumor sections to assess the expression using HIF-1α, JAK2, and p-STAT3.

To test the toxicity of SG@M NPs in vivo (n = 3), mice were intravenously injected with SG@M NPs (10 mg/mL, 100 µL). For H&E staining and hematological analysis, the primary organs and blood samples were collected at 0, 2, 4, 10, and 18 days after injection.

Statistical analysis

All data are expressed as the mean ± standard deviation. One-way ANOVA tests and Student’s t-test were used to test the significance of differences among groups. Additionally, the statistical significance difference was shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

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