The O2-dependent chemotherapy usually exhibits limited therapeutic efficiency toward hypoxic solid tumors because of inadequate O2 supply in tumor vasculature. Hence, an O2-elevated nanoplatform for delivering chemotherapeutics and eradicating malignant tumors needs to be urgently developed. In this study, MnO2 nanoenzyme was coated on GSH-sensitive mesoporous silica. Subsequently, an intelligent nanoreactor was constructed after saikosaponin-d (SSD) loading in the mesopore channels and folic acid anchoring on the rough surface. After precise targeting of thyroid tumor cells, the obtained nanoreactor induced the decomposition of tumor endogenous H2O2 to relieve hypoxic tumor microenvironment. In the meantime, the tetrasulfide bonds of silica were broken by cytoplasmic GSH to release the loaded therapeutic molecules. Consequently, a remarkably enhanced chemotherapeutic effect of SSD was achieved both in vitro and in vivo. The mechanism underlying the tumor cell–killing effect was demonstrated to involve massive O2 generation, which facilitated the interruption of the PI3K/Akt signaling pathway. The outstanding biocompatibility of the H2O2/GSH dual-sensitive Mn-based nanoreactor offered an exceptional chemotherapeutic effect against malignant tumors.
Research Article
Dual H 2 O 2 /GSH-Sensitive MnO 2 -Coated Mesoporous Silica Nanoreactor Fabrication with Saikosaponin-d Loading for Hypoxia Alleviation Enhancing Thyroid Carcinoma Chemotherapy
https://doi.org/10.21203/rs.3.rs-3770373/v1
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Chemotherapy enhancement
H2O2 and GSH sensitive
O2 generation
PI3K/Akt signaling pathway
SSD
Papillary thyroid carcinoma (PTC) accounts for about 90% of all thyroid cancers and is characterized by an increasing tendency of thyroid envelope infiltration and compression of surrounding organs and a high rate of diffusion to local lymph nodes [1–4]. Although most PTCs present as unresponsive clinical processes, some of them progress to local or even distant metastases at the time of diagnosis [5]. Currently, the mainstay cancer treatment is surgical resection; radiotherapy and chemotherapy have also been used to effectively eradicate PTC [6]. Among these therapies, chemotherapy is intensively performed in clinics because of its convenient process without any external assistance [7–9]. Natural drugs from traditional Chinese medicine often possess highly diverse activities and functions, thus playing a dominant role in tumor chemosensitivity [10]. In recent years, many natural drugs have been demonstrated to successfully reverse the chemotherapeutic resistance against tumor cells. They have played increasingly important roles in this regard, for example, dauricine [11], quercetin [12], and so forth. However, the mechanism underlying the chemotherapeutic effect is still unclear. Saikosaponin-d (SSD), as a major bioactive triterpenoid saponin extracted from Radix Bupleuri, has been proved to possess anti-inflammatory, anticancer, and anti-oxidative activities [13–16]. Recent studies have shown that SSD can inhibit autophagosome–lysosome fusion and also induce autophagyindependent apoptosis/necrosis in breast cancer cells[15, 17–22]. Nevertheless, the potential clinical translation of SSD to alter the chemotherapeutic efficacy against PTC and its specific underlying mechanism are still largely unexplored[23, 24].
Furthermore, the chemotherapeutic resistance of malignant tumor cells is still a big challenge [25–28]. The high expression of some genes controlling cancerous cell survival are often regulated by tumor microenvironment, especially, hypoxia [29]. This could be directly attributed to the lack of O2 availability and indirectly to the proteome/genome alterations, angiogenesis, and pH changes [30, 31]. Tumor hypoxia has been widely known to negatively affect the therapeutic effects on tumors for decades. A highly influencing factor is known to facilitate tumor cell survival with numerous growth advantages, leading to an aggressive tumor phenotype [32, 33]. As hypoxia implies a low-O2 condition, obviously, increasing the O2 level by reoxygenation should be an effective way to tackle the aforementioned problem [34, 35]. Undoubtedly, hyperbaric oxygen therapy was explored for that purpose. A large number of strategies were developed for hypoxia relief, such as red blood cell/hemoglobin-based O2 delivery [36], perfluorocarbon-based O2 delivery [37], metal–organic framework-based O2 delivery [38], light-triggered O2 delivery systems [39], and nanoenzyme-mediated O2 generation [40]. The catalytic performances of multitudinous nanozymes have been used to overcome the hypoxia barriers during tumor chemotherapy owing to the facile synthesis, high stability, and robustness [41, 42]. As the first nanozyme, manganese oxide (MnO2) nanoparticles have been extensively explored for in situ O2 production under hypoxic intracellular conditions [43]. MnO2 could catalyze the cytoplasmic H2O2 degradation at the physiological pH value to produce O2, similar to the catalase enzyme.
Meanwhile, MnO2-based nanoenzymes could also be slowly decomposed with the simultaneous release of Mn2+ ions at the acidic pH, which was similar to O2 generation by H2O2 degradation.
Consequently, the catalytic property of MnO2 nanoenzymes in the tumor microenvironment could be applied for regulating hypoxia via O2 generation, reducing the intracellular acid condition by consuming protons, and the magnetic resonance imaging contrast agent aginst from released Mn2+ ions. Therefore, a novel nanoplatform needed to be urgently constructed for SSD precise tumor delivery and chemotherapeutic enhancement.
We fabricated a rough-surface MnO2-coated GSH-sensitive mesoporous silica nanoreactor (mSrM) in this study. The SSD molecules were encapsulated (mSrM-S) owing to the stacking pores of MnO2 and mesoporous channels. Then, DSPE-PEG2000-folic acid was functionalized on the MnO2 shell (mSrM-S-FA) to improve the tumor cell–targeting capability. This nanoreactor could release SSD in the cytoplasm after GSH triggering, and the dissolved O2 concentration significantly increased under high-H2O2 conditions. Remarkable tumor cell killing was found after mSrM-S-FA treatment. SSD could induce cell apoptosis by inactivating neurotrophic receptor tyrosine kinase 2 (NTRK2) via the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. In mice bearing subcutaneous thyroid tumors, SSD was effectively delivered after the tail vein injection of mSrM-S-FA, and the solid tumor was significantly inhibited with minimal side effects (Fig. 1). The results demonstrated the anticancer benefits of oxygen generation for the chemotherapeutic enhancement of SSD compared with free SSD and mSrM. The study suggested the advantages of combining an SSD delivery system with hypoxia alleviation and established a novel nanoplatform for highly efficient chemotherapy in clinical practice.
2.1. mSrM-S-FA fabrication
mSN synthesis
In a typical synthesis procedure, [44] triethanolamine (TEA, 0.034 g) and cetyltrimethylammonium p-toluenesulfonate (CTAT, 0.24 g) were added to DI water (11.5 mL). The resulting solution was gently stirred in an oil bath at 80℃ for 30 min. Then, a mixed solution of tetraethyl orthosilicate (TEOS, 1 mL) and bis[3-(triethoxysilyl) propyl] tetrasulfide (BTES, 0.8 mL) was added to the aforementioned CTAT–TEA aqueous solution under vigorous stirring at 80℃ for 16 h. Finally, the prepared products were washed with DI water and ethanol for more than three times (10,000, 10 min). The mSN nanoparticles were dried by vacuum overnight.
mSrM synethesis
100 mg of mSiO2 templates and Mn(NO3)3 ∙ 6H2O (0.9523 g) were added into 60 mL of DI water and moderately stirred (300 rpm) at 90°C in an oil bath for half an hour. Then, 0.105 g of methenamine powder was added. The mixture was vigorously stirred (900 rpm) at 90°C in an oil bath for 2 h. The final mSrM was washed (3000 rpm, 5 min) with DI water several times and dried in an oven at 60°C for more than 12 h.
mSrM-S synthesis
1 mg of SSD was first dissolved in 2.0 mL of DMSO. Then, 25 mg of mSrM was added to the aforementioned SSD solution, and the mixed solution was vigorously stirred at room temperature for 2 days. The SSD molecules could be successfully loaded in the stacking MnO2-based pores and the mesoporous channels of mSiO2. The as-made SSD-loaded mSrM nanoenzymes (mSrM-S) were collected after washing three times (3000 rpm, 10 min). The exact SSD content loading in mSrM was calculated from a standard curve that was measured using UV-vis absorbance.
mSrM-S-FA synthesis
Then, the amino groups were fabricated as follows to anchor FA on the rough surface. For this, 100 mg of mSrM-S was added into 60 mL of ethanol and stirred in an oil bath at 75°C for 2 h. Then, 100 µL of aminopropyltriethoxysilane (APTES) was put into the mixture solution. After 12 h, the final as-prepared mSrM-S-NH2 was obtained after washing with DI water three times. Further, 5 mg of FA was first dissolved in 5 mL of the buffer. The solution containing 2-(N-Morpholino) ethanesulfonic acid (MES) (0.1M, pH 5.5) and then 20 mg of N-(3-dimethylaminopropyl)-N′- ethylcarbodiimide hydrochloride (EDC) and 20 mg of N-hydroxysuccinimide (NHS) were added to the aforementioned buffer and gently stirred (300 rpm) for 2 h. Subsequently, 0.1 mmol of mSrM-S-NH2 was added into the aforementioned mixture under gentle stirring for 12 h. The FA-functionalized mSrM-S-NH2 was washed three times with DI water (12,000 rpm). Finally, mSrM-S-NH2-FA was dispersed in DI water.
2.2. Cellular targeting, O2 generation, and cell-killing effect of mSrM-S-FA
Cell targeting
Thyroid tumor cell line BCPAP cells were seeded in a six-well plate (1 × 105per well) with 1 mL of 1640 fresh medium (10% FBS) for 24-h incubation. Then, the cells were co-cultured with mSrM-FITC and mSrM-FITC-FA (with the same dose, 100 µg/mL). After 4-h incubation, the nuclei were stained using 1 µg/mL DAPI, and the two cell samples were observed using confocal laser scanning microscopy (CLSM) under 488-nm excitation.
O2 generation
Hypoxic cells were cultured at 37°C for 24 h in an incubator with a 5% CO2/2% O2/93% N2 atmosphere. Thyroid tumor cell line BCPAP cells were seeded in a six-well plate (1 × 105per well) with 1 mL of 1640 fresh medium (10% FBS) for 24-h incubation. Then, the cells were pretreated with H2O2 for 2 h, followed by co-culturing with PBS and mSrM-S-FA for 12 h. Finally, DCFH-DA and [Ru(dpp)3]Cl2 were used to detect the intracellular H2O2 and hypoxia conditions, respectively. After the nuclei were stained with 1 µg/mL DAPI, all four cell samples were observed using CLSM.
Cell-killing effect of mSrM-S-FA
Thyroid tumor cell line BCPAP cells were seeded in a six-well plate (1 × 105 per well) with 1 mL of 1640 fresh medium (10% FBS) for 24-h incubation. Then, the cells were co-cultured with mSrM, SSD, and mSrM-S-FA (with 100 µg/mL mSrM and 10 µg/mL SSD) for 12 h. The cells in all three treatment groups were imaged using CLSM after staining using calcein-AM/PI assay. For the apoptosis analysis of antitumor efficiency, all cells were stained using Annexin V-FITC/PI assay, and the percentage of apoptosis + necrosis was evaluated using a flow cytometer after 12-h mSrM and mSrM-S-FA treatments.
RNA sequencing
For RNA sequencing, total RNA was extracted from human undifferentiated thyroid carcinoma cell line 8305c, which was treated with a medium without serum or 10 µM SSD for 48 h. The total RNA of each sample was extracted using the RNeasy Mini Kit (Qiagen, USA), and the quality and quantity of the RNA were assessed using the 2100 Bioanalyzer (Agilent Technologies, CA, USA). RNA libraries were multiplexed and loaded on an Illumina sequencing platform, and the sequences were processed and analyzed by Shanghai OE Biotech Co., Ltd. (Shanghai, China). The transcripts in fasta format were raw reads and required further quality screening. Subsequently, DESeq was employed to estimate gene and isoform expression levels from the paired-end clean data using the file as a reference gene file.
2.3. Antitumor studies of mSrM-S-FA
Chemotherapeutic efficiency evaluation: All animal studies were approved by the Institutional Animal Care and Use Committee of The Seventh People’s Hospital of Shanghai University of Traditional Chinese Medicine (2022-AR-004), which were performed in accordance with all relevant guidelines (Shanghai, China). Female BALB/c nude mice aged 6 weeks were obtained from Shanghai SLAC. Subcutaneous thyroid tumor–bearing mouse models were prepared by transcutaneously injecting TPC-1 tumor cells (1 × 106 cells dispersed in 150 µL of DMEM) into the right armpit. During antitumor ablation studies, when the subcutaneous thyroid tumor volumes were ~ 100 mm3, the thyroid tumor–bearing BALB/c nude mice were divided to four groups (n = 5). The mice received the following intravenous injections: PBS, mSrM, SSD, and mSrM-S-FA (with the same mSrM concentration at 8.2 mg/kg and SSD at 1 mg/kg). The tumor volumes and body weights of mice in all the groups were recorded every 2 days.
Biosafety estimation
At the end of the tumor treatment period, the main organs (heart, liver, spleen, lung, and kidney) were also resected and stained using H&E staining. The blood collected from the mice in all four groups was further used for analyzing the biochemical indexes of the liver and the kidney and their functional evaluation.
3.1. mSrM-S-FA fabrication and in vivo characterization
The synthesis procedure of MnO2-based nanoenzyme mSrM-S-FA is illustrated in Fig. 1. First, silica oxide nanoparticles with dendritic mesoporous and tetrasulfide bonds co-doping < co-doped?> in the framework (mSiO2) were prepared using a biphasic strategy. As shown in Figure S1, uniform and monodispersed GSH-sensitive mSiO2 with a size of ~ 130 nm was found under transmission electron microscopy. Then, MnO2 shells were coated on silica shells via a coprecipitation approach, with Mn(NO3)2·6H2O as the precursor and methenamine as the redundant drug. After 12 h reaction at 90℃ and MnO2 coating, a core-shell nanostructure of size ~ 160 nm, spherical morphology, and a rough surface was obviously observed (mSrM) (Fig. 2A and 2B). Specifically, we found that numerous amorphous MnO2 nanosheets assembled on the mSiO2 surface (Fig. 2A). Besides, the elemental mapping images revealed that the highly homogenous distribution of Mn and O elements evidently overlapped on the silica surface, verifying the successful coating of MnO2 nanoshell (Fig. 2D). As reported in a previous study [45], these building blocks were vulnerable to acidic microenvironment, hinting that our MnO2 nanoenzyme served as a drug delivery system. MnO2 nanogranules (~ 5 nm) were nucleated on the mesoporous silica surface. These nanoparticles could stack and assemble onto a core-shell nanostructure of mSrM. Therefore, the stacking mesopores could be used for SSD loading [46, 47]. As shown in Figure S2, the relatively large specific surface area (~ 500 m2/g) and pore size (~ 8 nm) facilitated the subsequent formation of MnO2 nanoshell. Superior size distribution was observed after immersing mSrM with serum for 48 h, indicating that our nanoplatform had no aggregation behavior in vivo (Fig. S3). SSD could be encapsulated owing to the stacking pores and high surface area of mSrM. Using the standard curve, the loading content of SSD was calculated to be ~ 15.2% and the encapsulation efficiency was determined as ~ 21.75%. Subsequently, the surface modification of animo groups was performed by introducing APTES under an ethanol reflux system at 75℃ for 12 h. Then, the targeting modification on the rough surface was carried out using an electrostatic adsorption–mediated approach (mSrM-S-FA). The final as-prepared nanoreactors exhibited excellent monodispersity without any morphological transformation (Fig. 2C). Furthermore, the rough surface of mSrM-S-FA was also confirmed using scanning electron microscopy (SEM). Homogenous size distribution and virus-like rough surface were observed (Fig. 2E and 2F), implying that this novel morphology could facilitate tumor cellular internalization while evading capture by the reticuloendothelial system.
During mSrM-S-FA stepwise fabrication, the zeta potential value of the synthesized nanoreactors was determined using dynamic laser scanning. As shown in Fig. 3A, mSiO2 displayed negative charges due to -Si-OH. The charge reversal occurred after the surface modification of -NH2, indicating successful APTES hydrolysis on the mSrM surface. Meanwhile, the negative charges decreased owing to the EDC/NHS reaction during FA anchoring. In addition, the size distribution in the presence of different biological buffers was studied before bio-application. An ignorable size fluctuation was detected even after 48-h co-incubation with PBS or cell medium, indicating the superior size stability of mSrM-S-FA (Fig. 3B). Next, we investigated the chemotherapeutic efficacy of mSrM-S-FA as a multifunctional drug delivery system in vitro. We first explored the cytotoxicity of mSrM-S-FA against normal cells (NTHY-ORI 3-1s). The standard CCK-8 result indicated that mSrM-S-FA caused no obvious toxicity to normal cells even at high concentrations of mSrM-S-FA, up to 200 µg/mL (Fig. 3C), implying superior biocompatibility of this novel nanoreactors. We then compared the hypoxia-overcoming capability of nanoreactors by measuring the generated O2 levels in a mixture containing mSrM-S-FA and H2O2 at 37℃. The reaction was carried out inside a homemade semi-sealed chamber under hypoxic conditions, and the dissolved O2 was recorded using a dissolved oxygen meter for 10 h. In the H2O2-treated group, a gradual increase in the concentration of dissolved O2 was observed as a function of incubation time, whereas O2 production was less in the PBS-treated solution, proving the H2O2-sensitive performance of mSrM-S-FA (Fig. 3D). In vitro SSD release from mSrM-S-FA was examined in PBS or 10mM GSH buffer at 37℃. SSD was released from the nanoreactors with an initial rapid release of ~ 65% within 36-h incubation, which was followed by a slow-release behavior. In contrast, SSD displayed a slow-release profile with ~ 40% release even after 3 days (Fig. 3E). This was mainly because GSH could break the tetrasulfide bonds in the silica framework. The results suggested that mSrM-S-FA could achieve anti-hypoxia behavior and sustained release of SSD under the H2O2/GSH tumor microenvironment. MnO2-coated superconversion nanoparticles reacted with H+, H2O2, and GSH simultaneously to relieve hypoxia in tumor tissues [48]. Furthermore, we investigated the SSD release profile of mSrM-S-FA in the presence of GSH + H2O2 buffers. The highest SSD percentage was found in this dual-sensitive solution. The therapeutic drugs were partially loaded in the silica counterpart owing to the dendritic mesopores in this tetrasulfide bond doped mSN. GSH could react with MnO2 nanoshells and silica-based cores, primarily demonstrating that this nanosystem had GSH/H2O2-sensitive drug release behavior.
3.2. In vitro cell–targeting and cell-killing effect via chemotherapy enhancement
Encouraged by the aforementioned O2 generation and SSD release behaviors in bulk solutions, we conducted an in vitro experiment. Less than 12% of normal cells (human umbilical vein endothelial cells, HUVECs) were killed after incubation with mSrM and even with as high as 300 µg/mL mSrM-FA (Fig. S4), tentatively confirming the excellent biocompatibility of this MnO2-coated nanoplatform. We then investigated the cell-targeting and cell-killing abilities of mSrM-S-FA toward a PTC cell line BCPAP. FITC was loaded into mSrM instead of SSD (mSrM-FITC) to label the nanoreactors. The tumor cells were incubated with mSrM-FITC and mSrM-FITC-FA containing 5 µg/mL of equivalent effective FITC for 4 h, followed by rinsing with 1× PBS. Then, the nuclei were stained with DAPI for differentiating cell compartments. CLSM visualized cellular internalization. The images revealed more intensive green signals in the mSrM-FITC-FA-treated group compared with the mSrM-FITC co-cultured group, depicting more effective cell-targeting and accumulation properties of nanoreactors with FA modification (Fig. 4A). We expected that the intracellular H2O2 conversion by MnO2 nanoshells would alleviate the hypoxic condition. We treated BCPAP with the nanoreactors and analyzed H2O2 decomposition and hypoxia reversion to verify the O2 generation in the cytoplasm. As shown in Fig. 4C, the cells treated with mSrM-S-FA + H2O2 exhibited decreased H2O2 degradation (green) compared with control (PBS + H2O2). More pronounced hypoxia relief (red) was found in this group after staining with O2-quenched probe ([Ru(dpp)3]Cl2). The quantitative analysis of the mean fluorescence intensity further confirmed the excellent anti-hypoxic behavior of mSrM-S-FA in the tumor microenvironment (Fig. 4E). Subsequently, the chemotherapeutic efficiency was carefully evaluated. The cancer cell viability was also evaluated using a CCK-8 strategy after incubation with mSrM-S-FA containing different SSD concentrations for 48 h. As shown in Fig. 4B, mSrM-S-FA caused remarkedly higher toxicity, especially at 10 µg/mL SSD concentration, compared with free SSD. In sharp contrast, partial cytotoxicity of mSrM-FA (~ 72%) was found when the concentration was set as 50 µg/mL (SSD, 8 µg/mL), preliminarily demonstrating that GSH depletion could induce cell death to some extent (Fig. S5). Furthermore, live/dead cell discrimination was further investigated using a calcein-AM/PI staining assay. Compared with O2 generation nanoreactors (mSrM-FA) or free SSD, mSrM-S-FA with hypoxia alleviation and SSD precise delivery capabilities were found to be the most effective cancerous cell killing in a chemotherapy enhancement manner (Fig. 4D). Enhanced cell apoptosis + necrosis also revealed ~ 37.8% living cells after mSrM-S-FA treatment, whereas only 84.9% cells with mSrM-FA incubation were alive (Fig. 4F). These results suggested that mSrM-S-FA was expected to improve chemotherapeutic efficiency.
3.3. Underlying chemotherapeutic efficiency of SSD
The tumor cell elimination mechanisms of GSH/H2O2-sensitive nanoplatforms were intensively explored, such as disequilibration of antioxidant balance, O2 generation for hypoxia relief, ROS amplification for oxidative therapy, and so forth. In this study, we made efforts to explore the underlying mechanism of the antitumor capability of SSD. The transcript abundance of every gene was normalized to estimate the significance of gene expression difference between SSD and PBS treatments. As shown in Fig. 5A, the hierarchical clustering of 500 unigenes revealed differential expression in SSD-treated samples. The number of upregulated and downregulated genes is displayed in Figure S6. Comparatively, more downregulated differentially expressed genes (252) were detected. As an international standard classification system for genetic function, Gene Ontology was applied to illustrate the roles of differentially expressed genes in the SSD-stimulated group. The corresponding differential gene expression was divided into three main categories of GO classification: biological process, cellular component, and molecular function. In the biological process category, the most common assignments were the cellular process, single-organism process, and metabolic process. In cellular function categories, the main unigenes were grouped into organelle, organelle’s part, and cellular part. In the molecular function category, genes were primarily sorted into binding, catalytic activity, and transporter subcategories (Fig. 5B). Subsequently, the annotated sequences were mapped according to the reference pathways in a typical KEGG database to identify the exact biological pathways triggered by SSD. As shown in Fig. 5C, the KEGG pathway analysis discovered the top 20 enriched metabolic pathways. The most abundant group was found to be the “PI3K/Akt signal pathway,” demonstrating that SSD could provoke an efficient interruption of this signaling pathway. It is a key regulator of cellular processes, including growth, proliferation, metabolism, survival, and apoptosis. Constitutively active PI3K/Akt signaling pathway blocking provided a new tactic for targeted tumor therapy. Thus, the inhibitors of this novel signaling pathway could act as potential anticancer agents, particularly for cancerous cells whose survival or growth was determined by PI3K/Akt signaling activation. Interestingly, neurotrophic tyrosine kinase receptor type 2 (NTRK2) was capable of activating PI3K, resulting in the secondary messenger PIP3 formation. Then, PIP3 sensitized Akt signaling. Consequently, Akt induced mTOR signaling. So, the mRNA expression of NTRK2 in SSD-treated cells was then performed. As displayed in Fig. 5D, the mRNA expression significantly reduced after SSD co-culture. The result was comparable to the mRNA levels in the NTRK2 gene knockout group (Fig. 5E). All these findings revealed that SSD downregulated NTRK2 levels in tumor cells, followed by PI3K/Akt signaling inactivation, which might be the main reason for the sufficient eradication of cancer cells.
3.4. In vivo antitumor evaluation
Finally, the chemotherapeutic efficacy of mSrM-S-FA was examined with the subcutaneous thyroid carcinoma–bearing BALB/c mice. First, photoacoustic (PA) imaging was used to assess the intratumor O2 in solid tumors via oxygenated hemoglobin measurement after 7 days of treatment. The intravenous injection of mSrM-S-FA could remarkably increase the O2 level compared with mSrM-FA. Negligible O2 signals were found in the SSD group, which were significantly lower than those in both mSrM-FA and mSrM-S-FA groups, further indicating that potent hypoxia relief in solid tumors could substantially improve the chemotherapeutic efficiency (Fig. 6A). In these antitumor studies, all animals were divided into four groups: control (PBS), O2 generation (mSrM-FA), free drugs (SSD), and chemotherapy enhancement (mSrM-S-FA). The tumor volume and body weight were recorded in the following 15 days, 12 h after injecting the aforementioned therapeutic agents (SSD, 1 mg/kg and mSrM-S-FA, 10 mg/kg). As shown in Fig. 6B and 6D, free drug represented some extent of tumor growth–inhibitory effect, likely owing to partial tumor retention of free SSD. Meanwhile, mSrM-FA was more advantageous in terms of the antitumor effect mainly due to the GSH depletion effect of mSiO2 compared with that in the control group. The chemotherapeutic improvement displayed the slowest growth, the smallest tumor volumes, and the lightest weights. This was mainly attributed to the hypoxia relief behavior of mSrM-S-FA. The intratumor O2 production results also verified the outstandingly synergistic effect of mSrM-S-FA for complete tumor elimination. All these results were consistent with the findings of the tumor cell–killing assay in vitro. Simultaneously, no appreciable body weight fluctuation was detected during the whole treatment period (Fig. S7), indicating the superior biosafety of our Mn-based nanoreactors. H&E-stained images of major organs in the control group and groups treated with other therapeutic agents showed no obvious acute and chronic pathological toxicity, further indicating high in vivo biocompatibility of our developed mSrM-S-FA (Fig. S8). The serum biochemistry assay was used to validate that our SSD delivery system had no potential toxicology. The detected indexes were closely related to the hepatic and kidney functions, as shown in Figure S9. No remarkable injury was observed in these two organs compared with the untreated group. In a word, the obtained nanoreactors had no side effects during their antitumor application in clinical practice.
In summary, we introduced nanotechnology into the fabrication of intelligent MnO2 nanoshells coated on GSH-sensitive mesoporous silica with SSD loading and FA modification on the rough surface. After precise targeting of thyroid tumor cells, this novel nanoreactor could be used for GSH/H2O2-responsive chemotherapy enhancement by overcoming hypoxia. O2 production was detected in the cytoplasm of tumor cells based on the catalytic activity of MnO2 nanoenzyme toward H2O2. Meanwhile, the tetrasulfide bonds in the silica framework possessed GSH stimulus–responsive capability, and simultaneous SSD release could be achieved. The tumor cells were significantly killed via O2-elevated chemotherapeutic improvement. The mechanism underlying SSD antitumor effect was demonstrated as PI3K/Akt signaling pathway inhibition via downregulated mRNA expression of NTRK2. The corresponding in vivo results strongly confirmed the unique advantages of mSrM-S-FA in inducing tumor cell death and solid thyroid tumor growth with negligible side effects. At the same time, mSrM-S-FA can be developed as the anti-hypoxia nanoreactor for other therapeutic modalities in clinical practice.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
All animal experiments were approved by the ethics committee of Shanghai University of Traditional Chinese Medicine.
AUTHOR CONTRIBUTIONS
Menghan Wang and Zhongchao Mai, Methodology; formal analysis, Ying Ye and Yanan Song, formal analysis; Miao Zhang, writing; Xinlin Yang, proofreading; Bin Zhao, conceptualization; Wei Xia, project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.
FUNDING
This study was funded by the Natural Science Foundation of China (81873178), the General Project of Shanghai Municipal Health Commission (No. 201840338), the Key Specialty Construction Project of Pudong New Area Municipal Health Commission of Shanghai (PWZxk2022-04), the Science and Technology Development Fund of Shanghai Pudong New Area (No. PKJ2021-Y04), the Major Achievements Cultivation Project of Shanghai University of TCM (2021ZDCG05), and the Talents Training Program of Seventh People’s Hospital of Shanghai University of TCM (QMX2022-04).
DATA AVAILABILITY STATEMENT
All the data pertaining to the discussion are included in the manuscript. Once the study has been completed, the results will be published in separate manuscripts and deposited according to the then-current regulations.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
ACKNOWLEDGMENTS
The authors thank the Shanghai University of Traditional Chinese Medicine and Shanghai Seventh People's Hospital Laboratory for their support.
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Supplementary Figures S1-S9 are not available with this version
No competing interests reported.
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Graphical Abstract Rough MnO2-coated mesoporous silica nanogenerator fabrication with saikosaponin-d loading for thyroid tumor chemotherapy via GSH/H2O2-activated chemotherapy enhancement.
