Cistanche tubulosa is an editable and medicinal traditional Chinese herb and phenylethanoid glycosides are its major components, which have shown various beneficial effects such as anti-tumor, anti-oxidant and neuroprotective activities. However, the anti-obesity effect of C. tubulosa phenylethanoid glycosides (CTPG) and their regulatory effect on gut microbiota are still unclear. In the present study, we investigated its anti-obesity effect and regulatory effect on gut microbiota by 3T3-L1 cell model and obesity mouse model.3T3-L1 adipocytes were used to evaluate CTPG effects on adipogenesis and lipids accumulation. Insulin resistant 3T3-L1 cells were induced and used to measure CTPG effects on glucose consumption and insulin sensitivity. High-fat diet (HFD)-induced C57BL/6 obese mice were used to investigate CTPG effects on fat deposition, glucose and lipid metabolism, insulin resistance and intestinal microorganism.Blautia, Roseburia, Butyrivibrio and Bacteriodes were significantly increased by CTPG treatment.In vitro data showed that CTPG significantly decreased the triglyceride (TG) and non-esterified fatty acid (NEFA) contents of the differentiated 3T3-L1 adipocytes in a concentration-dependent manner without cytotoxicity, and high concentration (100 µg/ml) of CTPG treatment dramatically suppressed the level of monocyte chemoattractant protein-1 (MCP-1) in 3T3-L1 mature adipocytes. Meanwhile, CTPG increased glucose consumption and decreased NEFA level in insulin resistant 3T3-L1 cells. We further found that CTPG protected mice from the development of obesity by inhibiting the expansion of adipose tissue and adipocyte hypertrophy, and improved hepatic steatosis by activating AMPKα to reduce hepatic fat accumulation. CTPG ameliorated HFD-induced hyperinsulinemia, hyperglycemia, inflammation and insulin resistance by activating IRS1/Akt/GLUT4 insulin signaling pathway in white adipose tissue. Moreover, gut microbiota structure and metabolic functions in HFD-induced obese mice was changed by CTPG, especially short chain fatty acids-producing bacteria including CTPG effectively suppressed adipogenesis and lipid accumulation in 3T3-L1 adipocytes and ameliorated HFD-induced obesity and insulin resistance through activating AMPKα and IRS1/AKT/GLUT4 signaling pathway and regulating the composition and metabolic functions of gut microbiota.The online version contains supplementary material available at 10.1186/s12906-022-03743-6. Obesity is mainly caused by an imbalance of energy intake and consumption in white adipose tissue (WAT), and predisposes many metabolic disorders such as type 2 diabetes mellitus (T2DM), hyperlipidemia and cardiovascular diseases , 2. InsuFirmicutes to Bacteroides, suppress some favorable bacteria, while stimulate the potentially harmful bacteria ) + log(Fasting Glucose[microU/mL])).HOMA = (Fasting glucose [mmol/L] x Fasting insulin [microU/mL])/22.5For OGTT, mice were fasted for 6 h after 5 weeks of drug administration, then orally administered with 2 g/kg of glucose. Blood glucose concentrations were measured at 0, 15, 30, 60, and 120 min after glucose treatment. The curves were made and AUC was calculated. For ITT, mice were fasted for 4 h after 6 weeks of drug administration, and then intraperitoneally injected with 0.75 U/kg insulin. Blood glucose levels were measured at 0, 15, 30, 60, and 120 min after insulin injection. The curves were made and the AUC was calculated.After 6 weeks of administration, the mice liver was isolated. For liver lipid content determination, homogenize tissue at the ratio of the weight of the tissue (g): the volume of PBS (ml) = 1:9, then the tissue homogenate was centrifuged with 5,000 rpm for 10 min, and the supernatant was collected. Liver TG and TC content was assessed by TG kit  and TC kit , which was normalized to cell total protein levels using a BCA Protein Assay Kit .Adipose tissues and livers were collected at the end of this study and fixed in 10% formaldehyde solution for 48 h, followed by embedding in paraffin and staining 5 μm-thick sections with hematoxylin and eosin (H&E). The cell sizes of epididymal WAT (eWAT), inguinal WAT (iWAT), and perirenal WAT (pWAT) were measured in H&E stained sections of three individual samples in each group. The sections were imaged by inverted fluorescence microscopy and quantified with Image J.After 6 weeks of administration, eWAT of the mice was isolated. In order to determine the MCP-1 content of eWAT, 1 ml PBS was added per 100 mg frozen eWAT tissue, and samples were centrifuged with 5,000 rpm for 10 min, and the supernatant was collected. eWAT MCP-1 content was assessed by the mouse MCP-1 ELISA kit .The mRNA levels of nuclear receptor CCAAT enhancer-binding protein alpha (C/EBPα), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), preadipocyte factor-1 (Pref-1) and hormone sensitive lipase (HSL) were detected by qRT-PCR. 3T3-L1 cells were treated with 100 μg/ml CTPG for 72 h and the total RNA was extracted by Cell total RNA isolation Kit  according to the manufacturer’s protocol. Reverse transcription and quantitative PCR were performed according to the instructions of RT EasyTM II kit  and RT-qPCR EasyTM (One Step)-SYBR Green I kit , respectively. The gene-specific primers were shown in Table SAt the end of this study, proteins were isolated from liver and eWAT using RIPA lysis buffer  and protein concentrations were measured by BCA Kit . Equal amount of protein in each sample was separated by 10% SDS-PAGE and transferred to PVDF membranes . After blocked with 5% nonfat milk, membranes were incubated with corresponding primary antibodies and secondary antibodies conjugated to horseradish peroxidase, respectively. After washing with PBS contained 0.05% Tween 20, the proteins were detected by EasySee Western Kit , and the blots were analyzed by ImageJ .About 100 mg cecum content from different group mice were used for DNA extraction using a cetyltrimethylammonium bromide (CTAB) protocol . The DNAThe amino acid sequences of representative sequences were made on the basis of KofamKOALA  alignmenhttps://www.genome.jp/kegg/) were summarized, and differential abundances by groups were determined and displayed using R packages.The predicted orthologues based on KEGG database . The variance distribution was equaled using Bartlett’s test. Statistical analyses were performed using a one-way analysis of variance (ANOVA) conditions to the control followed by Dunnett's multiple comparisons test, with a 5% significance level. Cell viability was first examined by MTT assay to determine the optimum concentration of CTPG for further experiments. As shown in Fig. The anti-adipogenic effects caused by CTPG on preadipocyte differentiation into adipocytes were investigated. 3T3-L1 preadipocytes were cultured in adipocyte differentiation media for 8 days in the presence or absence of CTPG. Then, we undertook Oil Red O staining to assess CTPG effects on adịpogenic differentiation at day 8 after the induction of adipocyte differentiation. The quantification of Oil Red O staining demonstrated that showed that 25 to 100 μg/ml of CTPG significantly inhibited lipid droplet accumulations versus that of differentiation mixture (MDI) induced model group in a dose-dependent manner  and lipogenic genes (ACC and FASN), while increased the expression of preadipocytic gene (Pref-1) and lipolysis gene (HSL) in 3T3-L1 cells . Then we investigated the effect of CTPG on IR by measuring insulin tolerance tests (ITT) and HOMA-IR index. The ITT was further tested after 6 weeks of CTPG treatment, the ITT curve increased moderately and the AUC index was smaller in CTPG and MET group  after 5 weeks of CTPG administration. As shown in Fig. The effect of insulin on glucose is regulated by IRS1/Akt signaling pathway and the abnormality of the pathway can lead to the diminishment and impairment of insulin signaling , 19, whiWAT has emerged as a major endocrine organ, producing a variety of adipokines including MCP-1, which affect lipid metabolism and glucose homeostasis , 12. TheProteobacteria and Actinobacteria phyla. Interestingly, Proteobacteria phylum was greatly reduced in mice gut after supplementation with CTPG  orthologs group (KOs) and Carbohydrate-Active enZYmes (CAZy) families showed that the clusters of metabolic pathways were clearly separated among NFD, HFD, and CTPG groups Fig. A&B.Fig. p < 0.05). After CTPG supplementation, the 14 metabolic pathways were reversed at different degree, in which ether lipid metabolism pathway was recovered to normal degree   Effect Size (LEfSe) was used to analyze the metabolism on gut microbial function, and 21 metabolic pathways were screened with the criteria of LDA > 2 Fig. C. Comparree Fig. C. Moreov05) Fig. D. These Alistipes, Helicobacter, Desulfovibrio, Curtobacterium, Brachyspira, Akkermansia were significantly positively correlated with FBG, HbA1c, HDL-C, TC and LDL-C. Curtobacterium, Pseudomonas, Mycolicibacterium, Flintibacter, Flavonifractor and Streptomyces were significantly positively correlated with MCP-1 and FBI. However, Blautia, Anaerostipes, Mediterraneibacter, Hungatella, Butyrivibrio, Roseburia, Lacrimispora, Streptococcus, Parabacteroides, Clostridioides, Lachnoclostridium and Massilistercora were significantly negatively correlated with FBG, HbA1c, HDL-C, TC and LDL-C, and Bacillus, Roseburia, Lacrimispora, Streptococcus, Anaerocolumna were significantly negatively correlated with MCP-1 and FBI.In addition, spearman correlation cluster analysis was conducted for OUT data, and correlation analysis was conducted between the top 50 bacteria with high abundance and MCP-1, FBI, FBG, HbA1c, HDL-C, TC and LDL-C Fig. A. CorrelIn KEGG pathway analysis, gut microbial function was reversed after CTPG supplementation, the lysine biosynthesis, glyoxylate and dicarboxylate metabolism, tryptophan metabolism, lipopolysaccharide biosynthesis proteins, mitochondrial biogenesis were positively correlated with glucose and lipid metabolism. Proteins enriched in AMPK signaling pathway, mitochondrial biogenesis and ether lipid metabolism were also positively correlated with MCP-1 and FBI Fig. B, 10A. SObesity is characterized by increased adipose tissue mass and is often accompanied by a number of other metabolic disorders , 5, 56. Many studies have shown that the mechanisms of anti-obesity are involved in inhibiting the proliferation and differentiation of preadipocytes –59. SimiAdipogenesis is a process that preadipocytes differentiate into mature adipocytes through the regulation of multiple transcription factors and lipogenic genes. C/EBPα is one of the CEBP members and expressed at the early stage of adipogenesis .At the lThe expansion of adipose depots can be driven either by hypertrophy or hyperplasia in obesity . We furtRecent studies strongly support that adipose tissues dysfunctions secreting high levels of free fatty acids (NEFA) and inflammation adipokines under HFD or obese conditions are crucial players in lipotoxicity, glucose metabolism disorder and insulin resistance . MCP-1 aFurthermore, adipose tissue inflammation is also involved in the regulation of the insulin pathway to affect systemic glucose tolerance and insulin sensitivity , 75. InsBacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Verrucomicrobia [Proteobacteria and Actinobacteria phyla, while CTPG supplementation decreased the abundance of Proteobacteria phylum in HFD mice.Changes in the gut microbial composition, structure and function are associated with increased susceptibility to obesity , 78. Themicrobia , which pmicrobia . In thisRoseburia and Lacrimispora are the two genera most closely related to glucose, lipid metabolism and IR, and they might play an important role in the CTPG-mediated improvement of glucose, lipid homeostasis and insulin sensitivity. Studies showed that Roseburia was significantly correlated with body fat percentage and HbA1c [Roseburia was also negatively correlated with HbA1c, LDL-C and FBI. It has been reported that Blautia, Anaerostipes and Roseburia were negatively correlated with fasting blood glucose and insulin resistance index levels [Roseburia and Lacrimispora were significantly negatively correlated with MCP-1. Additionally, the CTPG supplementation significantly increased the abundances of Roseburia and Lacrimispora, which were similar with NFD groups. Roseburia reported as the anti-obesity and anti-inflammatory bacteria in mice [Roseburia might play an important role in the CTPG-mediated anti-inflammatory effects. This is consistent with KEGG analysis of gut microbiota. However, the mechanism remains to be further explored. These findings suggested that CTPG supplementation not only changed the gut microbial structure but also function of gut microbiota in obese mice.Increasing evidence has linked glucose, lipid homeostasis and insulin sensitivity to the gut microbiota composition, which may play an important role in the pathogenesis of metabolic diseases, such as obesity, IR , 81, 82.nd HbA1c . In our x levels , and thi in mice . This suIn conclusion, we demonstrated that supplementation with CTPG significantly suppressed adipocyte differentiation, adipogenesis and inflammation in 3T3-L1 adipocytes, as well as glucose consumption, lipid metabolism and insulin resistance in IR-3T3-L1 cells. Furthermore, CTPG ameliorated HFD-induced obesity by inhibiting hypertrophy and hyperplasia of adipose tissue, inflammation and improving glucolipid metabolism and insulin sensitivity, changing structure and function of gut microbiota in obese C57BL/6 N mice. Our results suggested that CTPG might be a potential candidate for the treatment of obesity and IR.Additional file 1.Additional file 2.


At lower seeding densities , colonies varied in diameter and density, from dense to sparse. In these colonies, the proportion of highly proliferative MSCs increased over time. In contrast, lower proliferative MSCs enlarged more rapidly. Senescent cells were removed using a short detachment treatment. We found that these mechanisms increase the purity of highly proliferative MSCs. Thereafter, we compared MSCs isolated under optimized conditions with a higher density (1.25 × 106 cells/cm2). MSCs under optimized conditions exhibited significantly higher proliferative and differentiation potential into adipocytes and chondrocytes, except for osteocytes. We propose the following conditions to improve MSC quality: (1) optimizing MNC seeding density to form single-cell colonies; (2) adjusting incubation times to increase highly proliferative MSCs; and (3) establishing a detachment processing time that excludes senescent cells.Mesenchymal stem cells (MSCs) hold considerable promise for regenerative medicine. Optimization of the seeding density of mononuclear cells (MNCs) improves the proliferative and differentiation potential of isolated MSCs. However, the underlying mechanism is unclear. We cultured human bone marrow MNCs at various seeding densities (4.0 × 10 Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine because they can be harvested from various tissues, such as bone marrow, adipose, and umbilical cord. They have been proven to be clinically successful in regenerative medicine ,2. Owingp16 and p21, and the regulation of differentiation, such as Wnt signaling and Runx2. As a result, cellular proliferative and differentiation potential decrease. This could result in prolonged manufacturing periods or even production failure. Additionally, known variations in cell quality adversely affect therapeutic efficacy. To solve these problems, a method that stabilizes production must be established.The use of MSCs has resulted in an increase in the number of manufacturing facilities. However, there are several critical points and concerns regarding production . For exaTo accomplish this, we focused on the isolation of MSCs. Previous reports state that MSC yield can be increased by optimizing the seeding density of mononuclear cells (MNCs) ,18,19. HIn this study, we found a new way to improve the purity of MSCs with excellent proliferative and differentiation potential by optimizing MNC seeding density. We aimed to investigate the relationship between seeding density and MSC colony formation, analyze the characteristics of MSCs in each colony, isolate MSCs at higher and lower seeding densities, and compare their proliferative abilities and differentiation potential. Our research findings provide new insights into the MSC isolation process, which will lead to improved MSC manufacturing efficiency and quality.Human BM-MNCs were purchased from Lonza . The MNCs were extracted from the whole bone marrow of healthy donors via Ficoll density gradient separation , which was then frozen. In our study, we used five lots. Donors included HIV/HBV/HCV-negative women and men with an age range of 22–35.2 at 37 °C . The medium was changed every 2–3 d and replaced with Stem Fit for MSC medium  supplemented with 0.2 µg/mL iMatrix-511  on day 7 of culture. When MSC colonies reached a high density, they were washed with Dulbecco’s phosphate buffered saline (Thermo Fisher Scientific) and trypsin (TrypLE Select Enzyme (1×), Thermo Fisher Scientific). Thereafter, cells were detached for 5 min at 37 °C. Detached cells were centrifuged for 5 min at 300× g . Following removal of the supernatant, the pellet was resuspended in fresh medium and seeded into new vessels at 5000 cells/cm2.MNCs were thawed and mixed with Dulbecco’s Modified Eagle’s Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and 5 µg/mL gentamicin . Cells were seeded in culture vessels at various densities and cultured in 5% COMNCs cultured on culture plates were observed every 24 h by multi-area time-lapse imaging microscopy . From time-lapse images, we classified “the fast-growing cells” and “the slow-growing cells” based on the timing at which the MSC reached high density, or doubling time. Doubling time was calculated based on the time taken and the number of cell counts. We counted the number of MSC by using ImageJ software (NIH).Senescence-associated β-galactosidase  staining was used to detect senescent cells. Staining was performed using a commercial kit . Briefly, cells were fixed in formalin. Thereafter, the substrate X-gal and the staining solution were added. Following incubation for 24 h at 37 °C, cells showing SA β-gal activity turned blue. We microscopically observed the stained cells (EVOS).Cells frozen at passage 1 and 2 were thawed, blocked with FcR Blocking Reagent  at 4 °C for 15 min, and labeled with a fluorescent-dyed antibody. Dead cells were stained with 7-AAD  and analyzed using a flow cytometer . The anti-human antibodies  were purchased from BioLegend .4 cells/well. As MSCs were present in 0.001–0.01% of BM-MNCs [MNCs were thawed, mixed with DMEM supplemented with 10% FBS and 5 µg/mL gentamicin, and seeded in 96-well plates  at a density of 1 × 10 BM-MNCs , we assuCells at passages 2 and 3 were seeded at 100 cells in a 60 mm dish (Corning) in DMEM supplemented with 10% FBS and 5 µg/mL gentamicin. After 14 d of culture, colonies were stained with crystal violet and the number of colonies was manually counted.Ki67, PCNA, and β-actin. To calculate relative fold-change values, CT values were standardized using β-actin as an internal control.Total RNA was extracted from cells using a commercial kit . It was reverse-transcribed to cDNA using a commercial kit . Quantitative PCR was performed using FastSYBR™ Green Master Mix  and 1 mL aliquots of the RT reaction. Amplification of samples was carried out in a final volume of 20 µL using the StepOnePlusTM system (Applied Biosystems) with the following program: initial denaturation at 95 °C for 20 s, followed by 40 cycles of annealing at 60 °C for 30 s, and denaturation at 95 °C for 3 s. The following primers were used : Ki67, P5 cells/cm2) and high MNC density (1.25 × 106 cells/cm2). The total RNA was isolated from cells using Maxwell RSC simplyRNA Cell Kit . Ion Ampliseq Transcriptome Human Gene Expression Kit  was used for library creation. An Ion Proton next-generation sequencer library of analysis beads was created, and an Ion 540 Kit  was used for sequencing using an Ion Proton next-generation sequencer. Gene set enrichment analysis (GSEA) were integrated by R programming software. Protein-coding differentially expressed genes (DEGs) in MSCs obtained by lower MNC density (1.25 × 105 cells/cm2) were identified based on a fold change cutoff of 2.0 in comparison with MSCs obtained by high MNC density (1.25 × 106 cells/cm2).For transcriptome analysis, RNA was prepared from the MSC obtained under low MNC density (1.25 × 104 cells/cm2 in 24-well plates (Corning) according to the manufacturer’s instructions . Cells were cultured in differentiation medium for 21 d. The medium used was MEM α (Thermo Fisher Scientific) supplemented with 10% FBS, 1 × addipogenic supplement , 100 U/mL penicillin, and 100 µg/mL streptomycin. Thereafter, they were fixed and stained with fatty acid-binding protein 4 (FABP4) antibody (R&D Systems) and Hoechst 33342 . For osteogenic differentiation, cells at passage 2 were seeded at 4.2 × 103 cells/cm2 in 48-well plates (Corning) and cultured in a commercial differentiation medium  for 21 d. Cells were stained with Alizarin Red and Hoechst 33342. Alizarin Red staining was performed using a commercial kit . For chondrogenic differentiation, cells were seeded at 6.3 × 104 and 1.25 × 105 cells each in 15 mL filter tubes  in a differentiation medium. Spheres were formed and cultured for 21 d. The medium used was D-MEM/F12 medium  supplemented with 10 ng/mL TGF-β1 , 100 nM dexamethasone (Merck), 50 µg/mL ascorbic acid , 100 µg/mL sodium pyruvate , and 1% ITS+ Premix (Corning). After culturing, formalin-fixed paraffin sections were prepared and stained with Alcian blue. Each specimen was microscopically observed (EVOS).For adipogenic differentiation, cells at passage 3 were seeded at 2.1 × 10t-test. Differences were considered statistically significant at p < 0.05.Data are presented as the mean ± standard error. Statistical analysis was performed using JMP Pro software . Data were analyzed using Student’s 3–1 × 106 cells/cm2) have been investigated [4, 1.25 × 105, 2.5 × 105, 6.3 × 105, and 1.25 × 106 cells/cm2) and cultured the cells. First, we stained the colonies with crystal violet to compare the timing of colony formation , there was a notable variation in the number of cells between positions. The differences between positions became more pronounced as time progressed (6 cells/cm2), growth rate of cells per position showed a similar pattern . In contrast, those under the low density condition (1.25 × 105 cells/cm2) were greatly reduced. Upon collection, the percentage of CD45-positive cells was analyzed using fluorescence-activated cell sorting (FACS). The percentage was significantly reduced in the low density condition compared to that in the high density condition  was found to increase the purity of MSCs with high proliferative potential, and 5 min trypsin treatment was found to exclude senescent cells (5 cells/cm2) and high MNC density (1.25 × 106 cells/cm2), which had a clear difference on colony formation. We confirmed that isolated cells in both conditions expressed an MSC-specific marker. In the low MNC density condition, the MSC purity was higher than that in the high MNC density condition  showed that the difference in cell size from position to position was small up to 7 days, but enlargement developed after 9 days, similar to Gnani’s results. We plan to analyze the accumulation of ROS and DNA damage in these cells. Correlation with these factors will help us understand the isolation mechanisms and, thereby, predict the effectiveness of our method for unknown donors. On the other hand, under high density conditions (1.25 × 106 cells/cm2), the distance between colonies became smaller and densified earlier. Thus, we propose that the high density condition made the senescence process invisible.In contrast to high proliferative MSCs, we observed that low proliferative MSCs enlarged and were SA-β-Gal positive under low MNC density condition A,E. Gnan2), the phenotype of senescence, such as flattened morphology and p16 expression, was significantly suppressed under low seeding density conditions (50 cells/cm2) in the late phase of culture. ROS accumulation was especially suppressed under the low density condition. Although the relationship between seeding density and ROS accumulation remains unclear, repeated low seeding densities of isolated MSCs may lead to improved purity of highly proliferative MSCs. In a recent study regarding RECs, the Wnt pathway mediated via FDZ5 suppressed the senescence of MSCs [Repeated low seeding density in isolated MSCs leads to suppression of cellular senescence . Compare of MSCs . The stuIn addition to affecting the properties of MSCs as described above, MNC seeding density also affected the persistence of hematopoietic cells. We hypothesized that high seeding density conditions would enhance the interaction between monocytes or between monocytes and MSCs. Macrophage colony-stimulating factor (M-CSF) is known to be one of the key regulators in the differentiation of macrophages from monocytes . It has This study has some limitations. First, we used commercial MNCs owing to ethical considerations. The purity and proliferative potential of MNCs could vary depending on the manufacturing process and health of the donor. Therefore, it may be necessary to adjust the seeding density depending on the manufacturing procedure. Second, the basic culture conditions were based on our specific manufacturing procedures. The reagents and materials used in each manufacturing facility are different, and this was not considered.In our study, we elucidated the mechanism by which MNC seeding density affects the quality of MSCs. Under low MNC seeding density, the proportion of highly proliferative MSCs increased over time. In contrast, low proliferative MSCs enlarged more rapidly. The senescent cells were removed using a short detachment treatment. This mechanism increases the purity of highly proliferative MSC. Based on our findings, we propose the following conditions to improve MSC quality: (1) optimizing MNC seeding density to form single-cell colonies; (2) adjusting incubation times to increase the purity of high proliferation MSC; and (3) establishing a detachment processing time that excludes senescent cells. Stabilization of proliferation will contribute to increased efficiency and stability of MSC production. We also confirmed improvement in the differentiation potential of the obtained cells. However, the correlation between seeding density and genes related to differentiation needs to be investigated. In the future, we intend to evaluate this in detail and hope that the findings will contribute to tissue engineering. Improving MSC quality is expected to be translated into improved therapeutic efficacy in cell therapy. Thus, future work would focus on verifying the clinical significance of our proposed method.


Our growth assay revealed that none of these (up to 100 µg/mL) negatively affect viability in the budding yeast, Saccharomyces cerevisiae. In contrast, both human thyroid cancer cells (ML-1) and rat medullary thyroid cancer cells (CA77) displayed a significant reduction in cell viability with the treatment of CuO and ZnO. The production of reactive oxygen species (ROS) in these cell lines, when treated with CuO and ZnO, was found to be not significantly altered. However, levels of apoptosis with ZnO and CuO were increased, which led us to conclude that the decreased cell viability is mainly caused by non-ROS-mediated cell death. Consistently, data from our RNAseq studies identified differentially regulated pathways associated with inflammation, Wnt, and cadherin signaling across both cell lines, ML-1, and CA77, after ZnO or CuO MONP treatment. Results from gene studies further support non-ROS-mediated apoptosis being the main factor behind decreased cell viability. Together, these findings provide unique evidence that the apoptosis in response to treatment of CuO and ZnO in these thyroid cancer cells was not mainly due to oxidative stress, but to the alteration of a range of signal cascades that promotes cell death.Metal oxide nanoparticles (MONPs) are widely used in agriculture and food development but there is little understanding of how MONPs, including ZnO, CuO, TiO Metals such as gold, silver, titanium, copper, zinc, and aluminum have all been used to synthesize nanomaterials, as well as their oxides, i.e., silver oxide, copper oxide, magnesium oxide, calcium oxide, and zinc oxide. These common nanomaterials exhibit magnetic, fluorescence quenching, and dielectric properties ,2. In pa2 MONPs of different sizes were all tested on S. aureus, B. subtilis, P. aeruginosa, and E. coli [S. aureus and B. subtilis, while ZnO NPs of 12–15.5 nm caused decreased viability in P. aeruginosa and E. coli [2O3 in a study to understand the potential impact of these MONPs on soil-living bacteria and found that CuO has a more severe negative impact on hydrolytic activity and soil oxidative potential [2O3 used in the study were about the same size , and therefore, this investigation has limited information on the impact of MONP size on cell toxicity. Zhang et al. evaluate TiO2 with several different sizes on human epidermal keratinocytes and found that only TiO2 of 27.5 nm induced modest cytotoxicity compared to larger particles [2O3 and CuO [2O3 and CuO exhibited more toxic effects on cell viability and ROS production than spherical MONPs [Many researchers have uncovered the antibacterial properties of MONPs, which can make an impact on bacterial growth and reproduction . Infecti E. coli . Among t E. coli . Overallotential . Both Cuarticles . A simil and CuO . From thal MONPs . They pral MONPs .2, and SnO2, specifically) impact on human health and the environment [2 on different aquatic cell lines  and tested their cytotoxicity [2 treatment (100 µg/mL and lower) were detected to be normal [2 MONPs. Similarly, SnO2 was reported to have negligible cytotoxic effects [2, concentrations ranging from 10–200 µg/mL, on breast cancer MCF-7 cells [2 alone conveyed no toxic effects but when paired with ZnO nanocomposite (SnO2-ZnO/rGO NPs) the resulting toxicity was greatly increased [2 and SnO2, ZnO NPs are one of the most toxic MONPs. In a 2019 study, HaCaT keratinocytes were treated with ZnO MONPs and showed increased toxicity due to the dissolution of Zn2+ ions, emphasizing that the cytotoxicity is caused by the ability of ions to internalize by endocytosis [In vitro cell culture is a useful tool to gain insight into underlying toxicity mechanisms. Many researchers are concerned with MONPs’  and CA77 (rat medullary thyroid carcinoma), and the budding yeast Saccharomyces cerevisiae to obtain broader understanding to the cytotoxicity of MONPs and their potential environmental implications. Given that CuO and ZnO led to more toxicity over TiO2 and SnO2 had no significant impact on cell viability, thus we decided to focus on investigating the physicochemical properties of CuO and ZnO for the current study. STEM images for each nanomaterial show that the CuO and ZnO nanomaterial sizes are in agreement with that of the data sheet values; 40 nm and 35–45 nm respectively  patterns  of the Z2, and TiO2 on the biological organism Saccharomyces cerevisiae. After analyzing the optical density of MONP-treated cells at 594 nm over a period of 24 h, we found that all MONPs did not reduce the growth of Saccharomyces cerevisiae at concentrations up to 100 µg/mL (2 caused slightly increased growth when compared with non-treated groups (0 µg/mL). These results are expected because budding yeast has a cell wall which is hypothesized to be a protective layer that keeps MONPs from accessing cell cytoplasm. Absorbance-based growth assays were performed to better understand the potential toxicity of ZnO, CuO, SnO00 µg/mL A–H. Rath2, or SnO2, are potentially harmful to ML-1 thyroid cancer cells, an XTT cell viability assay was conducted. ML-1 cells were incubated with concentrations of 4 µg/mL, 20 µg/mL, and 100 µg/mL for each of the four MONPs. After 7 h of incubation with an XTT activation reagent, ML-1 viability correlates to measured absorbance levels. Five groups  showed p < 0.001 as compared with non-treated control (NTC). In particular, at the concentration of 100 µg/mL, both ZnO and CuO nanoparticles had significantly decreased cell viability  of both ZnO and CuO were treated to ML-1 cells (p < 0.01), while 60 µg/mL of CuO had a comparable level of viability reduction to 40 µg/mL of ZnO-treated cells (p < 0.05). Using the AAT Bioquest website, we determined IC50 values of ZnO and CuO for ML-1 cells: 22.8 µg/mL for ZnO and 45.5 µg/mL for CuO.Two MONPs, ZnO and CuO, showed markedly decreased cell viability when treated to ML-1 cells . To dete-1 cells A. After p < 0.05). The next higher concentrations of these MONPs also caused decreased cell viability, with statistically significant p values (p < 0.01). IC50 values were calculated in a manner identical to that with the ML-1 cells: 68.2 µg/mL of ZnO and 72.8 µg/mL of CuO for CA77 cells. CA77 rat medullary thyroid carcinoma cells displayed similar levels of viability loss when compared to that of ML-1 human thyroid cancer. A comparison of these two cell lines can give an insight as to which cell line is more sensitive to MONP treatment. An additional XTT cell viability was conducted on CA77 cells with a similar treatment: 20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL, and 100 µg/mL for both ZnO and CuO B. A concp < 0.05). However, CuO NPs showed similar trends compared to NTC. ML-1 cells were determined to be the most sensitive to ZnO and CuO treatment compared to CA77 cells.Incubation of MONPs with mouse fibroblast cells can help determine if they could be used as an anticancer treatment. An important aspect of cancer therapy we should consider before its application to cancer is if they are able to target only cancer cells. If MONPs also lead to a reduction in fibroblast cell viability, then they would be a poor option for anti-cancer treatment. It was found that fibroblast cells showed decreased cell viability with ZnO treatment  (Reactive oxygen species (ROS) are free radicals that largely contribute to disturbing the cell in stressful environments. Superoxide level was tested in ML-1 and CA77 cells using dihydroethidium (DHE). Cells were treated for 24 h with IC control A–D. In t control A. Median < 0.05) B, but no < 0.05) B. ML-1 c < 0.05) , which p < 0.05) C or a 30 < 0.05) D with Zn < 0.05) E, revealp < 0.01) and CuO (p < 0.001) treatments for 48 h , although ZnO-treated cells were not statistically significantly different from the non-treated control group due to a high standard error (p < 0.0001), while ML-1 cells are under a late apoptotic phase in the same treatment. Together, both cells display an elevated induction of apoptosis within 24 h of treatment with these MONPs.Although ML-1 cells  and CA77 and CuO A,B. ML-1rd error A. Interep-value less than 0.05. Our GO term analysis revealed that the most frequently upregulated pathways include p53, Wnt signaling, CCKR, and Gonadotropin hormone receptor pathway (MT2A and ZNF100) and genes involved in the p53 pathway (MYC and CCNA2). Interestingly, cell-cell adhesion, inflammation, and Wnt signaling pathways were found to be the most frequently downregulated , Ccr5 (inflammation), and Rtkn2 . Downregulated pathways after ZnO NP treatment include integrin signaling pathways, inflammation, and cytoskeletal regulation (Bad (apoptosis), Nlrp10 (immune response), and Bag6 (autophagy). Together, this result suggests that both inflammation and apoptotic mechanisms are not balanced with the ZnO treatment in CA77 cells.CA77 cells treated with ZnO NPs had 256 genes upregulated and 136 genes downregulated. The most frequently upregulated pathways include the thyrotrophin-releasing hormone receptor signaling pathway, inflammation, and Wnt signaling (Aox4 (Xenobiotic function), Dpp4 (collagen binding), and Pdcd1Ig2 (cytokine regulation). In contrast, inflammation mediated by chemokine and cytokine signaling pathways, angiogenesis, and VEGF signaling were the most frequently downregulated pathways affected by CuO treatment , Frat1 , and Pglyrp4 .CA77 cells treated with CuO NPs expressed 118 upregulated genes and 163 downregulated genes. Most of the upregulated genes were not distinctive biological pathways. There were only three main upregulated pathways: TGF-beta signaling, interleukin signaling, and inflammation mediated by chemokine and cytokine signaling pathways  can be a unique discovery in the field. Not only are ML-1 cells rarely studied but comparing toxic effects in human thyroid cells with those in rat thyroid cells can give more insight into which MONPs exert more toxicity. Finally, we used fibroblast cells to check if these NPs can be used as alternative cancer therapy as previously discussed in the literature  One of tsynthase . Interessynthase  through 2, TiO2, ZnO, and CuO), ZnO and CuO only had obvious toxic effects when treated to ML-1 cells, CA77 cells, and mouse fibroblast cells by showing significantly decreased cell viability. Lines of research, in which a range of cancer cells have been used for their research, have suggested MONPs as anticancer therapy agents . Althoug50 values were calculated to be 22.4 µg/mL for the ZnO-treated culture and 45.5 µg/mL for the CuO-treated culture. Although we found that IC50 values calculated in several reports were significantly different depending on the cell lines used, ranging from 10 to 100 µg/mL of ZnO. However, a few reports, including Namvar et al. that used mouse breast cancer (4Ti) treated with ZnO NPs, revealed their corresponding IC50 values very close to the IC50 values we reported here [50 value of 38.41 µg/mL [50 values in ML-1 cells as compared with other sensitive cell lines (4Ti and MOLT4) to ZnO and CuO, we came to the conclusion that ML-1 cells can be categorized as a highly sensitive cell line to these MONPs. ML-1 cells represent human thyroid cancer, which is an excellent model for developing how MONPs function in vitro. ML-1 cells are very rarely researched compared to other cancer cell lines including CA77 cells. In our XTT viability experiment, ML-1 cells showed viability defects with ZnO concentration as low as 40 µg/mL A. CuO-tr41 µg/mL . Given t50 values compared to those of ML-1 cells. After ZnO and CuO treatment, 50% of cells were inhibited in viability at 68.9 µg/mL and 72.9 µg/mL, respectively  after CuO NP treatment, making them very susceptible to MONPs [50 values are expected after the treatment depending on how different cancer cells react. Based on our results, CA77 cells showed decreased cell viability even with a higher concentration of ZnO and CuO than ML-1, indicating that CA77 cells are more resistant than ML-1 to these MONPs.Evaluating the viability of CA77 cells can give further insight into animal models affected by ZnO and CuO treatment. CA77 cells treated with CuO and ZnO have a similar decreased cell viability pattern to ML-1 cells B. Howeveectively B. To comto MONPs . VaryingElevated levels of reactive oxygen species (ROS) can often lead to decreased cell viability as previously shown ,26. ROS 50 values of ZnO and CuO MONPs  in ZnO-treated ML-1 cells, p53 and Wnt signaling pathways were not only frequently  but alsoA damage . Given tA damage , we conjreatment . The uprSMAD5 and CDH1 are implicated in the Wnt signaling function that regulates cell growth, differentiation, and cell death. SMAD5 was found to be a tumor suppressor candidate [CDH1 is a tumor suppressor, too [CDH1 was also upregulated in HaCaT cells after ZnO treatment [In addition, andidate  and CDH1sor, too . Both gereatment . The uprCDH16) and xenobiotic metabolism  is downregulated , and Frat1 . In particular, Frat1 mainly functions as a positive regulator of Wnt signaling and regulates tumor progression [After CuO treatment, the few, most severely downregulated genes shown in gression . Given tCcr5 and Zfp36l1 (Ccr5 and Zfp361l upregulation in response to ZnO treatment takes place to promote inflammation. Genes involved in inflammation were found to be upregulated (more than a 20-fold increase) after ZnO MONP treatment in CA77 cells:  Zfp36l1 . It is kAckr3, associated with inflammation, was downregulated severely after ZnO treatment to CA77 cells  cells were purchased from DSMZ, German Collection of Microorganisms and Cell Cultures . CA77 (rat thyroid cancer) cells were obtained from Jordan Valley Innovation Center (JVIC) from Dr. Paul Durham (Missouri State University). Mouse fibroblast cells were obtained from Dr. Chris Lupfer (Missouri State University). DMEM (Dulbecco’s Modified Eagle Medium), DMSO (Dimethyl Sulfoxide), Dihydroethidium (DHE), CellROX green, FBS , TRIzol, PI (Propidium Iodide), Annexin-V-APC, and XTT kit were obtained from FisherSci . All other reagents and plasticware used in the study were purchased from FisherSci unless otherwise stated.The following MONPs were purchased from US Research Nanomaterials Corporation : ZnO, CuO, SnO−5 Pa was reached. Imaging was conducted in the STEM mode at 30 kV acceleration voltage, and a working distance of 4.4 mm. Images were taken at 25 and 50 kX.These MONPs (1 mg/mL) were received as they are, dispersed in water. Only ZnO and CuO caused a cell viability decrease see , and theX-ray diffraction (XRD) was conducted using a Bruker D2 phaser XRD system  to characterize the crystallinity of the CuO and ZnO nanomaterials. Both nanomaterials were prepared similarly, wherein 10 mg of each was weighed on an Ohaus Discovery scale with 0.01 mg resolution and added to 3 mL of methanol in a 5 mL glass crucible and stirred until a thick paste was formed. For XRD, specific mass/volume of dispersion or paste is not critical if all compared materials are prepared in the same way. The paste was then coated onto a zero-background silicon plate and dried on a hotplate at 75 °C for 10 min. The zero background silicon plate with dried nanomaterial was placed in the XRD chamber. The conditions for the analysis were set to X-ray radiation of 30 kV from a CuKα X-ray source with a theta range of 20–90° (2θ), at 0.02° (2θ) steps and a sample rotation set to 10 rpm.Saccharomyces cerevisiae strain S288C was grown in 2X synthetic-defined glucose (2X SD + Glu) media and placed in a shaking incubator at 30 °C to grow overnight as described previously [2, and SnO2, was independently suspended in water at a concentration of 1000 µg/mL. The budding yeast eviously ,53,54. TFor yeast viability experiments, a round-bottom 96-well plate was utilized to get the most accurate OD reading at 600 nm. A 1:2 serial dilution was performed of the nanoparticle stock solution to provide treatment conditions ranging from 0 µg/mL to 100 µg/mL. Yeast cells (OD of 0.2 at 600 nm) were then added to only the first three rows of the seeded wells leaving the bottom three rows to be a comparative control only containing nanomaterial and 2X SD + Glu media. Each of the treatment concentrations and their corresponding control was seeded in a triplicate manner on the 96-well plate. The seeded plate was then placed in a BioTek ELx808 Absorbance Microplate Reader where the OD was read at 594 nm every 30 min over a period of 24 h. This experiment was conducted multiple times. The data shown  are reprC is the OD value at 594 nm from a culture well with cells and media and ODM is the OD value at 594 from a culture well with media and nanoparticles only.The OD obtained from the treated wells at 594 nm after 24 h incubation was subtracted from the corresponding control wells without cells to obtain a corrected OD .p-value < 0.05). Dunnett’s multiple comparisons were used to visualize the variance in control and treatment groups. The graph bars represent an average of three replicates and the error bars represent the standard deviation. Statistically significant data are represented as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The obtained results were validated by repeating the experiments in triplicate.From each triplicated treatment, we determined the mean corrected OD in order to create growth curves. GraphPad Prism9 was used to perform a one-way ANOVA statistical analysis to determine if the mean OD of treated samples had a statistically significant difference from that of the non-treated samples. Endpoint graphs were made using the OD at the 24 h mark and asterisks were used to identify values significantly different  was used to measure the amount of reduced formazan dye formed in each well. Absorbance values at 450 nm were subtracted by absorbance values at 630 nm to gain a net colorimetric change that most accurately represents the cell viability in the culture.An XTT assay was performed on mouse fibroblast, ML-1, and CA77 cells to explore the impact of ZnO, TiOhttps://www.aatbio.com/tools/ic50-calculator, accessed on 1 July 2022) to determine IC50 values representing the concentration of MONPs at which 50% of cultured cells are inhibited in cell viability. Data from ML-1 cells were inserted into the equation provided on the webpage to determine IC50 values; it was determined that ZnO’s IC50 was 22.8 µg/mL and CuO’s IC50 value was 45.5 µg/mL. The IC50 value was then calculated for CA77 cells from XTT cell viability data. These values were as follows: 67.98 µg/mL for ZnO and 72.88 µg/mL for CuO. The AAT Bioquest website was used  treated at 20%, and the untreated control. An untreated control group and two blanks  were only treated with DMEM. The third day consists of washing treated cells once with 500 µL 1X phosphate-buffered saline (PBS). To detach the cells, 250 µL trypsin + EDTA was added to each well and then placed in the incubator for 15 min. After the incubation period, trypsin+EDTA was neutralized with 500 µL pre-warmed DMEM. The cell suspension in each well was transferred to labeled 2 mL centrifuge tubes to be centrifuged for 10 min at 400× g. During this time, the ROS indicator, DHE (dihydroethidium), was prepared by mixing 26 µL DHE into 26 mL 1X PBS in a 50 mL Falcon tube. Given DHE’s light sensitivity, the 50 mL Falcon tube containing indicator mix was covered with aluminum foil. The supernatant from the centrifuge tubes was removed and 1 mL of indicator mix was added to each tube except the two blanks. Instead, the two blanks received 1 mL 1X PBS. After pipetting up and down, each resuspended mix was transferred to new foil-covered 2 mL centrifuge tubes. The transferred cells were then incubated for 1 h before being analyzed by an Attune NxT Flow Cytometer. The dyed samples were measured at 518/606 nm.Two separate 24-well plates were seeded with 50,000 ML-1 and CA77 cells per well and placed into an incubator for 24 h at 37 °C. The following day, the cells were incubated with either ZnO or CuO at its concentration of IC50 concentration of either ZnO or CuO. Four treatment groups were conducted in triplicate: NTC, ZnO, CuO, and 10% H2O2, where H2O2 was used as a positive control. After treatment, the cells were washed with 100 µL of 1X PBS, and then 5 μM of cellROX green dye solution was applied to each well. Cells were then washed three times with a live cell imaging solution (Fisher) to make sure phenol red from DMEM does not interfere with imaging. Immediately after, cells were visualized with a confocal microscope. We chose 10 cells with detectable fluorescence randomly to evaluate the fluorescence intensity with ImageJ. Three separate 18-well plates  were seeded with 10,000 ML-1 cells per well. Each 18-well plate was incubated for different periods of time, including 24 and 48 h. After 24 h of incubation, all plates were treated with the IC2), and molecular-grade sterile water. A 1x Annexin V binding buffer solution was made by diluting 18 mL of PBS and 2 mL of the 10x Annexin V binding buffer. Initially, 50,000 ML-1 or CA77 cells were seeded per well in a 24-well plate and then incubated for 24 h. On day two, MONPs were treated in concentrations of 22.8 µg/mL of ZnO and 45.5 µg/mL of CuO in triplicate along with DMSO and an NTC group. On day three, each well was washed with 300 µL of 1X PBS and then 250 µL of trypsin  without EDTA was pipetted into each well. The plate was placed in an incubator for 20 min while the cells detached. To neutralize the non-EDTA trypsin, 300 µL of fresh DMEM was added into each well and then pipetted up and down. The treatment groups, NTC, and DMSO groups were resuspended in 100 µL of 1x Annexin V binding buffer solution, 5 µL of Annexin V-APC, and 5 µL of propidium iodide (50 µg/mL), then incubated in the dark for 30 min. After the incubation, an additional 400 µL of Annexin V binding buffer solution was added to each sample. The final analysis of each sample was based on the excitation properties of the dyes. Annexin V emits at 660 nm with a red laser and propidium iodide emits at 617 nm with a blue laser. Apoptosis experiments were conducted in triplicate and repeated three independent times. For this experiment, an Annexin-V binding buffer and two dyes, Propidium Iodide and Annexin V-APC, were used. The 10x Annexin V binding buffer was made by combining 0.1 M of HEPES, 1.5 M sodium chloride (NaCl), 25 mM of calcium chloride . RNA extraction was conducted on CA77 cells as well as ML-1 cells. On the first day, 750,000 CA77 cells were seeded into each well of a 6-well plate. Two 6-well plates were used for a total of nine samples: three NTC, three 68.9 µg/mL of ZnO treated, and three 73 µg/mL of CuO treated. For ML-1 cells, ZnO at 22.8 µg/mL and CuO at 45.5 µg/mL were tested. After 24 h of incubation with the MONPs, total RNA was extracted. For this, 1 mL of TRIzol was added to homogenize the cells. Each sample was incubated for 5 min to complete the dissociation of the nucleoprotein complex. Next, each sample was incubated with 200 µL of chloroform for 2–3 min. After centrifugation, three phases formed: an upper colorless, an intermediate, and a lower red phase. The colorless phase contains RNA. The colorless phase was acquired, and then 100% isopropanol was added in equal parts. After another centrifugation, the supernatant was removed containing RNA, and 1 mL of 75% ethanol was added to each Eppendorf tube. Finally, ethanol was removed, and tubes were placed in the biosafety cabinet until completely dry. Thirty microliters of nuclease-free water was added to each tube to make a total RNA concentration of 2 µg and was shipped to Novogene for the production of cDNA libraries and Nextgen sequencing. Basepair. The spike-in ERCC was used when uploading files to reduce variability and identify errors. There was a linear correlation between the known concentration, expected concentration, and read counts for each of the different probes. Linear correlation indicates good sample quality. The first step in the RNA-seq pipeline was trimming to remove the poor quality of reads as well as adapter sequences. Each read was aligned to STAR, which is the reference genome to accurately identify the source of the gene. After all reads were aligned, Feature Counts were used for expression quantification to look at each gene and how many mapped reads are aligned to that specific gene. Next, the DESeq2 tool was used to conduct statistical analysis in the differential gene expression analysis step to find changes in gene expression between each sample group. After differential expression analysis, a volcano plot displays genes with a log10 p-value and a heat map was used to indicate up- and down-regulated genes. All up- and down-regulated genes were selected with a fold change of +2.01 and −2.01 with a p-adjusted value less than 0.05. The five most up- or down-regulated genes were chosen and investigated with IDTdna and Gorilla. CLC genomics (Qiagen) was used for CA77 cells to identify the differentially regulated genes when treated with ZnO and CuO. Datasets were downloaded, unzipped, then transported into CLC genomics. Fastq files were uploaded and then concatenated in Galaxy. After quality checks, RNA-seq analysis was conducted on each sample and CA77 gene expression was analyzed using CLC genomics. After receiving sequencing files from Novogene, those were uploaded to After analyzing our RNAseq data, many differentially regulated pathways overlapped after ZnO and CuO treatment in both cell lines (ML-1 and CA77). Briefly, both CuO and ZnO MONPs altered inflammation, cadherin, Wnt signaling, and p53 pathways in ML-1 cells. Similarly, CA77 cells exhibited altered Wnt, inflammation, and angiogenesis pathways after both treatments with ZnO or CuO. Therefore, animals and humans have similar pathways altered after ZnO or CuO treatment. Though these altered pathways are similar, the genes differentially regulated varied in each cell line. The biggest common denominators between both cell lines were found to be inflammation and Wnt signaling. These findings further confirm non-ROS-mediated apoptosis being the mechanism behind decreased cell viability in our experiments. Together, ZnO and CuO NPs cause dysregulation in inflammation, which could be the main factor behind increased apoptosis and decreased cell viability.


Two bread wheat lines with similar genetic backgrounds and contrasting in vivo maximum carboxylation activity of Rubisco per unit leaf nitrogen  determined using high-throughput phenotyping methods were selected for detailed study from a panel of 80 spring wheat lines. Detailed phenotyping of photosynthetic traits in the two lines using glasshouse-grown plants showed no difference in Vc,max,25/Narea determined directly via in vivo and in vitro methods. Detailed phenotyping of glasshouse-grown plants of the 80 wheat lines also showed no correlation between photosynthetic traits measured via high-throughput phenotyping of field-grown plants. Our findings suggest that the complex interplay between traits determining crop productivity and the dynamic environments experienced by field-grown plants needs to be considered in designing strategies for effective wheat crop yield improvement when breeding for particular environments.Recognition of the untapped potential of photosynthesis to improve crop yields has spurred research to identify targets for breeding. The CO Wheat plants grown in the glasshouse show different physiological properties compared with plants grown under dynamic field conditions, highlighting the need to consider realistic environmental conditions when breeding for particular environments. Global food demand is expected to double in the next 50 years or so due to the growing world population and dietary changes . Wheat a3 plants is ~4.6%  assembled after screening a range of elite International Maize and Wheat Improvement Center (CIMMYT) spring wheat germplasm , and respiration. After phenotyping photosynthetic traits in this germplasm in the field, two lines that are genetically similar but contrasting for Vc,max per unit leaf nitrogen, yield, and biomass at physiological maturity, were selected and further characterized in glasshouse conditions.It is well known that plant performance is highly affected by environmental conditions. Experiments under controlled or glasshouse conditions are often performed aiming to assess genetic yield potential; however, translation between results obtained under field and controlled conditions is challenging  identify lines in the PStails panel with contrasting photosynthetic traits but similar genetic background under field conditions; and (ii) establish the photosynthetic properties of the two contrasting lines through detailed phenotyping under glasshouse conditions. The lack of correspondence between most of the physiological properties displayed by the two genotypes under a field versus a semi-controlled environment led to a third objective: (iii) to evaluate the correlation for photosynthetic- and yield-related traits determined under glasshouse versus field conditions across the PStails panel. The findings support the need to carefully define aims and design experiments, given the lack of correlation between traits determined in plants of the wheat PStails panel grown under field versus glasshouse conditions.T. aestivum L.) selected from 150 lines of the High Biomass Association Panel  under fully irrigated conditions at the International Wheat Yield Partnership Phenotyping Platform (IWYP-Hub) situated at the International Maize and Wheat Improvement Centre (CIMMYT) Experimental Station Norman E. Borlaug (CENEB) in the Yaqui Valley, near Ciudad Obregon, Sonora, Mexico . Maximum and minimum temperature, and maximum solar radiation (W m–2) during the 2 year field experiments (http://www.siafeson.com/remas/index.php). Experimental design was an alpha-lattice with two replications in raised beds  with two rows per bed (0.24 m between rows) and 4 m long. Seeding rates were 102 kg ha−1. Appropriate weed disease and pest control were implemented to avoid yield limitations. Plots were fertilized with 50 kg N ha−1 (urea) and 50 kg P ha−1 at soil preparation, 50 kg N ha−1 with the first irrigation, and another 150 kg N ha−1 with the second irrigation.The PStails panel is composed of 80 bread wheat lines , leaf nitrogen content per unit dry mass (Nmass), Vc,max,25 per unit leaf nitrogen , electron transport rate [J(HS)], and SPAD (indication for chlorophyll content) were calculated as described in The full PStails panel was screened under field conditions using hyperspectral reflectance. Flag leaves were measured between 11.00 h and 14.00 h at booting stage . Total biomass at physiological maturity was calculated from GY/HI.At physiological maturity, determination of grain yield (GY) and yield components was conducted using standard protocols /Narea , GY, and total biomass, but genetically similar . Pots, each containing one plant, were distributed randomly in the glasshouse, and watered daily to field capacity. Line 51 shows faster development; therefore, seeds from line 64 were sown 12 d before those of line 51, so that plants of the two lines reached booting (Zadoks stage 4.5) and were measured at the same time.Seeds of lines 51 and 64 were sown in 3 litre pots containing commercial compost mix . Twelve replicate plants of each line were grown in a glasshouse at 26/18 °C day/night with a photoperiod of 16 h. Natural light was supplemented with high pressure sodium lamps  when external light was lower than 200 W mACO2 to the intercellular CO2 concentration (ci) combined with the quantum yield of PSII, ΦPSII (Fmʹ–Ft/Fmʹ) from chlorophyll fluorescence (using a multiphase flash) were measured in the mid-section of the flag leaf when the plants reached Zadoks stage 4.3–4.5. For all measurements, leaf temperature was maintained at 25 °C, VPDleaf at ~1.3 kPa, PAR of 1500 µmol m–2 s–1, and flow rate of 500 µmol s–1. Leaves were enclosed in the cuvette and induced to steady state at 43 Pa CO2_r; under this CO2_r a CO2 concentration in the sample analyser (CO2_s) of 40.6 ± 2.8 Pa was obtained, close to the current 41 Pa atmospheric concentration . CO2_r was then stepped down through 35, 27, 20, 15, and 5 Pa, and increased to 43, 48, 53, 58, 63, 68, 73, 79, 85, and 95 Pa. Before the data for each step were logged, the reference and sample gas analyser signals were matched. The minimum and maximum wait time for stability were 60 s and 120 s, respectively.Two LI-6800F portable IRGA systems  were used to assess photosynthetic parameters in the two wheat genotypes. Response curves of ACO2 to ci was modelled as described by ACO2 and [CO2] was described using a version of the FvCB model  and the minimum number of data necessary for each limitation state . The R Language and Environment function optim : gm ~∞ (approximated by setting gm to 1 × 106 μmol m−2 s−1 Pa−1); gm=5.5 μmol m−2 s−1 Pa−1, consistent with gm from the data. Of these, only gm ~∞ both credibly predicted limitation states indicated by ΦPSII (e.g. Rday) >0. Values for VA/ci)c,max,25 was calculated from the ACO2/ci curve . Measurement of leaf width of the frozen sample and the width of any gap between the leaf edge and the perimeter of the freeze-clamp tongs enabled precise calculation of the sampled area  previously harvested and stored at –80 °C by grinding the leaves at 4 °C with an ice-cold pestle and mortar containing 0.8 ml of extraction buffer  binding assay . Subsamples containing 6–8 mg of leaf powder were wrapped into tin capsules and analysed for carbon and nitrogen (%) using an elemental analyser .http://es-websupp.lancs.ac.uk/hazelrigg/) is shown in Addressing the unexpected lack of correspondence between phenotypic properties displayed by the two contrasting genotypes under field versus glasshouse environment conditions, data were analysed for the 80 lines that make up the PStails panel, plus the UK modern spring wheat cultivar Paragon, grown in glasshouse conditions for detailed phenotyping. The ambient conditions in the glasshouses were the same as described in the section ‘Two contrasting lines: glasshouse conditions—plant material and growth’. Four replicates were used, with one plant of each genotype represented in each of four replicate blocks. Due to space constraints, two blocks were grown at the same time in one glasshouse while the other two blocks were planted 17 d later in a second glasshouse set to the same environmental conditions. Maximum and minimum temperature in the two glasshouses during the experimental period are shown in Plants were grown in 3 litre pots containing commercial compost mix . Plants within each block were distributed according to a random design using the Edgar II Experimental Design Generator and Randomiser , and werACO2 to ci were performed in the mid-section of the flag leaf when the plants reached Zadoks stage 4.3–4.5. In all measurements, leaf temperature was maintained at 25 °C, VPDleaf at ~1.3 kPa, PAR of 1500 µmol m–2 s–1, and a flow rate of 200–300 µmol s–1. Leaves were enclosed in the cuvette and induced to steady state at 40 Pa CO2_r. CO2_r was then stepped down through 30, 20, 10, and 7 Pa, and increased to 40, 45, 55, 70, 100, and 120 Pa. After the ACO2/ci response curve, leaves were acclimated back to steady state at 40 Pa CO2_r and PAR of 1800 µmol m–2 s–1; then PAR was stepped down through 1500, 1000, 500, 250, 120, 50, and 25 µmol m–2 s–1. Before data for each step were logged, the reference and sample gas analyser signals were matched; the minimum and maximum wait time for stability were 60 s and 120 s, respectively.Three LI-6400XT portable IRGA systems (LI-COR) were used to assess photosynthetic parameters in the wheat genotypes. Response curves of ACO2 measured in the light response curves at PAR of 500 µmol m–2 s–1 is referred to as the operational photosynthetic rate (Aop)—at similar ambient light to the ambient growth conditions; and at PAR 1800 µmol m–2 s–1 as Asat— saturating light. ACO2/ci response curves were fitted according to the photosynthesis of the FvCB model (VA/ci)c,max,25( and JA/ci) and 50% of anthesis (Zadoks stage 6.5) was recorded for each plant. At the end of the experiment when plants reached physiological maturity (Zadoks stage 8.7), plant height was measured as the length of the main tiller from the soil surface to the tip of the spike excluding the awns. Determination of yield components was conducted using adapted protocols from P<0.05). For phenology, only days after irrigation was used as a covariate. The ANOVA was conducted with the general linear model (GLM) procedure from META R version 6.01  only when its effect was significant  blocks/replications, G, and G×GH. Adjusted means were calculated for each trait using position in the GH as covariate (fixed effect) when its effect was significant. For the gas exchange data, the LI-6400XT (three systems) and time of the day when measurements were performed were used as covariates when their effects were significant.F-test was applied to test for homogeneity in the variances of each set of data (for lines 51 and 64). As no significant difference between the variances were found, the parametric t-test was applied to test the significance of differences between mean values obtained for each trait for the two lines.All figures were prepared in RStudio /Narea. Line 51 showed lower Vc,max,25/Narea at tillering, anthesis, and grain-filling stages, and comparatively lower GY and biomass at physiological maturity than line 64 c,max,25( and JA/ci) regeneration (ci_cJ) occurred at higher ci values for line 51 (38.8 ± 0.6 Pa) than for line 64 (34.4 ± 0.9 Pa). For both lines, this transition was above the operating ci (i.e. that obtained at the current atmospheric level of 41 Pa and PAR of 500 µmol m–2 s–1). Furthermore, line 64 showed consistent limitation by Tp, which was not detected in any biological replicates for line 51  estimated from the ACO2/ci response curve, higher for line 51 than for line 64  than in line 64 , in vivo and in vitro parameters were normalized to N content in the leaves, in order to understand variation in N use efficiency between the lines with contrasting yield. No significant differences were found in VA/ci)c,max,25 , Rubisco25(A/ci) . On the eaf area .ACO2 measured at ambient CO2 and the irradiance experienced by plants in the greenhouse (AQ500) represent a close approximation to the operational photosynthetic rates (Aop). No significant phenotypic variation in AQ500 (P=0.429) or Asat (P=0.669) was observed within the PStails lines c,max,25( and JA/ci). The parameters Asat, Vc,max,25(HS), and J(HS) described above were plotted for the field experiment  and J(HS) were estimated using hyperspectral reflectance. Again, no significant phenotypic variation was found in Vc,max,25(HS) (P=0.719) or J(HS) (P=0.480). On the other hand, Asat was significantly different between the lines (P<0.001) and generally lower for the field-grown than the glasshouse-grown plants.e curves , did notperiment  to show Asat (i.e. ACO2 measured at a PAR of 1800 µmol m–2 s–1) and Vc,max,25(HS) were positively correlated with HI, whilst under glasshouse conditions (JA/ci), and GY also correlated with straw biomass (r=0.68). Interestingly, time to reach booting (Zadoks 4.5) and anthesis (Zadoks 6.5) did not correlate with yield parameters in field-grown plants, but showed positive correlation with GY, total above-ground biomass, and straw biomass in the glasshouse-grown plants. While leaf mass per area (LMA) correlated with Vc,max,25, J, and Vc,max,25/Narea under field conditions, this leaf trait did not correlate with any photosynthetic parameter under glasshouse conditions. While different methods were used in the different environments, these results suggest a different set of limitations to plant productivity in glasshouse and field conditions.Total above-ground biomass correlated strongly and positively with GY in both environments  and 64 (high tail), were selected /Narea (measured at anthesis and the grain-filling stage but not at initiation of booting), total biomass, and GY compared with line 64 (VA/ci)c,max, and the absolute values were similar to those obtained in the field experiments at anthesis and grain-filling stages c,max,25(/Narea (ci (i.e. that obtained at the current atmospheric level of 41 Pa and PAR of 500 µmol m–2 s–1), suggesting that Rubisco activity was limiting photosynthesis in the glasshouse-grown plants. Limitation by Tp was identified in line 64 at ci as low as 49 Pa, but no such effects were found for ci values as high as 70 Pa in line 51. The leaves of line 51 had greater N c,max,25/Narea , some otreater N , chloropreater N , and iWUreater N , especia line 64 . It is ien field  and glasen field  experimeVc,max) is essential to evaluate photosynthetic performance across different elite crops germplasm , another modelled parameter, are more robust than single-point ACO2 measurements to assess photosynthetic performance in C3 plants as they are independent of diurnal variation in gs (/Narea in glasshouse conditions (J(HS)/Narea in field grown-plants  than line 51 (6 ± 2). Plasticity in ear number affects grain yield c,max,25, and J(HS) in the PStails panel grown under field conditions.Fig. S6. gs and ci relationships between glasshouse- and field-grown plants.Fig. S7. Fig. S8. Relationships between time to reach Zadoks stage 6.5 in glasshouse- and field-grown plants.Fig. S9. Relationships between the number of tillers in glasshouse- and field-grown plants.erac096_suppl_Supplementary_Tables_S1-S4_Figures_S1-S9Click here for additional data file.


The resultant nanoparticles showed pH-responsive drug release, and could effectively target tumor cells via the targeting effect of B3int. The presence of CP and Cu2+ permits reactive oxygen species to be generated inside cells; thus, the chemotherapeutic effect of CP is augmented by chemodynamic therapy. In vitro and in vivo experiments showed that the nanoparticles are able to effectively kill tumor cells. An in vivo cancer model revealed that the nanoparticles increase apoptosis in tumor cells, and thereby diminish the tumor volume. No off-target toxicity was noted. It thus appears that the functionalized MSNs developed in this work have great potential for targeted, synergistic anticancer therapies.In this study, a pH-responsive controlled-release mesoporous silica nanoparticle (MSN) formulation was developed. The MSNs were functionalized with a histidine (His)-tagged targeting peptide (B3int) through an amide bond, and loaded with an anticancer drug (cisplatin (CP)) and a lysosomal destabilization mediator (chloroquine (CQ)). Cu Mesoporous silica nanoparticles (MSNs) have been widely used to this end because of their large specific surface area, easy-to-adjust pore size, surface functionalization ability, and biocompatibility, among other properties ,2,3. Howeactions . In otheeactions . In furteactions . 2+ is widely used in CDT because it can produce cytotoxic reactive oxygen species (ROS) via Fenton-like reactions with the high concentrations of H2O2 typically found inside tumor cells. At the same time, excessive glutathione (GSH) in the TME can be consumed by Cu2+, preserving the ·OH radicals produced by CDT [2O2 with the assistance of superoxide dismutase [2+ can help to protect both the chemotherapeutic and CDT activity of drugs such as CP [There are often shortcomings when treating cancer using a single therapy, as a result of which synergistic, multicomponent, strategies have received increasing attention ,24,25,26d by CDT . Cisplatd by CDT . It can ismutase ,33,34. Tismutase ,36. Hencch as CP .2+ in CDT can also offer the opportunity for some degree of passive targeting. Cu2+ can be chelated with a range of pH-sensitive moieties—for instance, the histidine tag (His-tag) [2+ is weakened, and the complex falls apart [The use of CuHis-tag) ,39. In als apart . Howeverls apart ,42,43,44ls apart , with hils apart .2+ and a His-tag , and triethanolamine (TEA) were purchased from Sinopharm Chemical Reagent Co. Ltd. . Hexadecyl trimethyl ammonium bromide (CTAB), o-phenylenediamine (OPD), dimethyl sulfoxide (DMSO), cisplatin (CP), chloroquine (CQ), and doxorubicin (DOX) were provided by Macklin Biochemical Technology Co. Ltd. . 3-Aminopropyltriethoxysilane (APTES), methylene blue (MB), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and pyrocatechol violet (PV) were acquired from Aladdin Biochemical Technology Co. Ltd. . The HtB peptide (Arg-Trp-(d-Arg)-Asn-Arg-His-His-His-His-His-His) and B3int (Arg-Trp-(d-Arg)-Asn-Arg) were obtained from Dangang Biological Co., Ltd. . B16 cells and L929 cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences . RPMI 1640 medium, DMEM high-glucose medium, phosphate-buffered saline (PBS), trypsin, and fetal bovine serum were supplied by Gibco Life Technologies . 3--2,5-diphenyltetrazolium bromide (MTT), 4′,6-diamino-2-phenylindole (DAPI), annexin V–FITC/PI cell apoptosis analysis kits, and lysosome staining kits (green fluorescence) were provided by KeyGEN Bio TECH Co., Ltd. . Calcein acetoxymethyl ester  and propidium iodide (PI) were purchased from Sigma-Aldrich . A reactive oxygen species assay kit (DCFH-DA) was obtained from Beyotime Biotechnology . TUNEL and H&E commercial detection kits were also obtained from Beyotime .Tetraethyl orthosilicate (TEOS), copper chloride (CuCl2O (20 mL) and stirred vigorously at 95 °C. After 1 h, TEOS (1.5 mL) was added to the solution dropwise, and the mixture was stirred for another 1 h. After recovery by centrifugation, the solid product was washed with ethanol several times and dried under vacuum at 60 °C. MSNs were obtained via calcination in a muffle furnace at 550 °C for 3 h.CTAB (2 g) and TEA (150 μL) were sequentially dispersed in H2 NPs.The prepared MSNs (50 mg) were next dispersed in ethanol (50 mL). APTES (3 mL) was added, and the mixture was stirred at room temperature for 24 h. After recovery by centrifugation, the solid product was washed with ethanol several times and dried under vacuum to obtain MSN−NH2 NPs (25 mg) were dispersed in the HtB solution, and the mixture was stirred at room temperature for 24 h. The resultant suspension was dialyzed with deionized water for 48 h (MWCO: 3500 Da) and then lyophilized.HtB peptide (20 mg) was dissolved in PBS  containing EDC (60 mg) and NHS (40 mg), and stirred for 2 h to activate the peptide. Then, the MSN−NHw/v, 10 mL), and then added to a dispersion of MSN−HtB NPs (1 mg·mL−1) in PBS (10 mL). After stirring for 12 h at room temperature, CuCl2 (0.5 g) was added, and the reaction mixture was stirred for a further 24 h. After this, the solid product was dialyzed with deionized water for 48 h (MWCO: 3500 Da) and then lyophilized, resulting in CP/CQ@MSN−HtB/Cu2+ NPs. Control nanoparticles were prepared, in which drug-free nanoparticles were sealed with Cu2+; these were termed MSN−HtB/Cu2+ NPs, and were generated by directly chelating Cu2+ to the MSN−HtB NPs without first drug-loading them. CP@MSN-HtB/Cu2+ NPs were prepared as above but with the omission of CQ from the reaction mixture.CQ (8 mg) and CP (8 mg) were dissolved in an aqueous saline solution  on a JEM-2100UHR instrument . Particle size and zeta potential data were obtained on a Zetasizer Nano ZS90 instrument . Infrared absorption spectra were collected on a Nicolet Nexus 870 spectrometer . UV–Vis absorption spectra were collected from 200 to 1000 nm using a UV 3600 spectrophotometer  at ambient temperature. Thermogravimetric analysis (TGA) was performed on a TGA-101 analyzer . Pore size and specific surface area were quantified on a TriStar 3000 automatic nitrogen adsorption specific surface area analyzer . X-ray photoelectron spectroscopy (XPS) was used to probe the surface chemical composition, with experiments carried out using an ESCALAB 250Xi X-ray photoelectron spectrometer . Elemental composition was determined on a 710-ES inductively coupled plasma–optical emission spectrometer . For the latter, the samples were digested in HNOEE and DL were calculated as follows [To determine the percentage encapsulation efficiency (EE) and drug loading (DL) of CP and CQ in CP/CQ@MSN−HtB at the end of the CP/CQ loading process, the NP dispersion in PBS was centrifuged to precipitate any solid material, and the amount of unencapsulated drug in the supernatant was determined by UV–Vis spectroscopy. The CP and CQ concentrations were quantified with reference to a predetermined calibration curve. In the case of CP, the supernatant was first mixed with an equal volume of o-phenylenediamine (OPD), and heated at 95 °C for 10 min prior to UV–Vis measurements . The EE  follows ,49,50: 2+ with the His-tag was assayed using pyrocatechol violet (PV). HtB peptide (2 mg) and MSN−HtB NPs (5 mg) were dispersed in sodium acetate buffer solution (1 mL) at pH 6.0. The resultant mixture (200 μL) was combined with sodium acetate buffer solution (2.7 mL), PV solution , and CuSO4·5H2O solution , mixed well, and left to stand for 15 min. The chelation of Cu2+ and the His-tag results in insoluble agglomerates [The chelation of Cuomerates  were qua2+ NPs was studied by dispersing CP/CQ@MSN−HtB/Cu2+ NPs (5 mg) in 2 mL of two different buffer solutions (PBS at pH 7.4 or 5.0) placed in a dialysis bag (MWCO: 3500 Da). This was then dialyzed against PBS (20 mL) at the appropriate pH at 37 °C, under stirring. At specific time intervals, the dialysate (2 mL) was removed and replaced with the same amount of preheated PBS. The amount of released CQ was determined by UV–Vis spectroscopy at λ = 343 nm. CP quantification was performed by mixing the release aliquot with OPD  in a 9% w/v aqueous NaCl solution at 95 °C. This mixture was heated for 10 min to form a complex, and analyzed with a UV–Vis spectrophotometer at λ = 705 nm.In vitro release of CP or CQ from the CP/CQ@MSN−HtB/Cu−1); (2) PBS  with MB (10 μg·mL−1) and H2O2 (8 mM); (3) PBS  with MB (10 μg·mL−1) and CP/CQ@MSN−HtB/Cu2+ NPs (0.3 mg·mL−1); (4) PBS  with MB (10 μg·mL−1), H2O2 (8 mM), and CP/CQ@MSN−HtB/Cu2+ NPs (0.3 mg·mL−1); (5) PBS  with MB (10 μg·mL−1), H2O2 (8 mM), and CP/CQ@MSN−HtB/Cu2+ NPs (0.3 mg·mL−1). The suspensions were stirred for 24 h, after which the absorbance at λ = 665 nm was measured.Methylene blue (MB) is a dye that can be degraded by ·OH, and can thus be used as an indicator for the generation of ·OH . Experimv/v) FBS and 1% (v/v) penicillin–streptomycin solution. L-929 cells were cultured in DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin solution. All of the cells were cultured in an atmosphere of 5% CO2 at 37 °C. Negative controls comprised cells treated with an equal volume of PBS (pH 7.4). Three independent experiments were performed, with five replicate wells per experiment.B16 cells were cultured in RPMI 1640 medium containing 10%  and incubated for 24 h at 37 °C before the media was aspirated. Subsequently, the cells were incubated with different concentrations of CP, CQ, and CP/CQ@MSN−HtB/Cu2+ NPs, and cultured in complete 1640 medium for another 12 h. Next, the MTT reagent  was added to each well, and the plate was incubated for 4 h. The media was aspirated, and DMSO (150 μL) added to each well of the plate, which was then incubated for another 20 min to dissolve the formazan crystals. Finally, the absorbance at 570 nm was measured by a microplate reader .The cytotoxicity of the nanoparticles to tumor cells was analyzed by the MTT assay. B16 cells were seeded in 96-well plates  and incubated for 24 h at 37 °C. The medium was then removed and replaced with fresh medium containing the test formulations. The cells were divided into 6 groups—(1) control; (2) free CP; (3) free CQ; (4) MSN−HtB NPs; (5) CP@MSN−HtB/Cu2+ NPs; and (6) CP/CQ@MSN−HtB/Cu2+ NPs—and incubated at an equivalent concentration of Cu2+ (10 μg·mL−1) or CP (30 μg·mL−1) for another 12 h. The cells were then washed with PBS three times and cultured in RPMI for a further 24 h. The cells were subsequently stained with a mixture of calcein-AM/PI solution for 10 min. Living cells (green) and apoptotic cells (red) were observed with an inverted fluorescent microscope .Calcein AM/PI double-labeling was employed to visualize the effects of the NPs on cells. B16 cells were seeded in 6-well plates  and incubated for 24 h at 37 °C. The medium was removed and replaced with fresh medium containing the test formulations. The cells were divided into 6 groups—(1) control; (2) free CP; (3) free CQ; (4) MSN−HtB NPs; (5) CP@MSN−HtB/Cu2+ NPs; and (6) CP/CQ@MSN−HtB/Cu2+ NPs—and incubated for 12 h. Next, the cells were trypsinized (0.25% trypsin without EDTA), collected, and rinsed three times with PBS (pH 7.4). They were re-suspended with binding buffer (500 μL) at a concentration of 1 × 106 cells·mL−1. An annexin V–FITC/PI cell apoptosis analysis kit was used to stain the cells according to the manufacturer’s protocol, and the samples were quantified by flow cytometry .The annexin V–FITC and PI double-staining method was used to quantify cell death. B16 cells were seeded in 6-well plates . B16 and L-929 cells were separately plated on glass-bottomed culture dishes  for 24 h at 37 °C. The media was aspirated, and then replaced with fresh media containing the test formulations. The cells were divided into 4 groups—(1) control; (2) free DOX (0.5 μg·mL−1); (3) DOX@MSN−B3int NPs (DOX: 0.5 μg·mL−1); and (4) DOX@MSN−HtB/Cu2+ NPs (DOX: 0.5 μg·mL−1)—and incubated in complete medium for another 4 h. After removal of the medium and washing with PBS, the cells were stained with a lysosome staining kit for 45 min, fixed with aqueous formaldehyde  at room temperature, stained with DAPI for 3 min, and then imaged by CLSM.In order to study the cellular uptake of NPs, fluorescent DOX was loaded in the MSNs instead of CP and CQ, following the same protocol as detailed above. To check the influence of the His-tag on uptake, control nanoparticles in which B3int was conjugated to the NP surface were also prepared. These were produced by replacing HtB with B3int in the MSN synthesis procedure, and then loading with DOX. The cellular uptake of DOX@MSN−HtB/Cu5 cells·mL−1, 2 mL) and incubated for 24 h at 37 °C. After removing the medium and replenishing it with fresh medium containing the test formulations, five groups of experiments were performed as follows: (1) control; (2) CuCl2; (3) MSN−HtB-Cu2+ NPs; (4) CP/CQ@MSN−HtB NPs; and (5) CP/CQ@MSN−HtB/Cu2+ NPs. The formulations were added to give equivalent concentrations of Cu2+ (10 μg·mL−1) or CP (30 μg·mL−1), and the cells were incubated for another 12 h. Next, the culture medium was replaced with fresh RPMI containing DCFH-DA (10 μM) for another 30 min of incubation. Afterwards, the cells were washed three times with PBS. Finally, the intensity of green fluorescence was observed using an inverted fluorescence microscope.Intracellular total ROS generation in B16 cells was detected using a DCFH-DA ROS assay kit. Cells were seeded in 6-well plates (1 × 106 B16 cells in 100 µL of PBS (pH 7.2–7.4) into the right flank area of each mouse. The mice were randomized into two groups (5 mice each) when the tumor volume (determined as length × width2/2) reached ca. 100 mm3.All animal experiments were undertaken following review and approval by the Experimental Animal Committee of Donghua University (SYXK(SH)2020-0018). C57BL/6 black mice  were acquired from Shanghai Jiesijie Experimental Animal Co., Ltd. . Tumors were implanted by subcutaneous injection of 2 × 102+ NPs or free CP in PBS solution  were injected into the tail vein. The mice were euthanatized after 24 h, and the tumors and major organs  were collected and weighed before being digested with aqua regia. After diluting the digestion solution 10 times with water, the contents of Pt and Cu were determined by ICP–OES.ICP–OES was used to determine the content of Pt and Cu, and to investigate in vivo biodistribution. CP/CQ@MSN−HtB/Cu3 were randomly divided into six groups (n = 5), and injected intravenously through the tail vein with the formulations under test (200 μL). The following treatments were investigated: (1) PBS, (2) free CP, (3) free CQ, (4) MSN−HtB/Cu2+ NPs, (5) CP/CQ@MSN−HtB NPs, and (6) CP/CQ@MSN−HtB/Cu2+ NPs. The amount of CP in each treatment was set at 300 μg·kg−1. During the experiment, the tumor volume and body weight were measured every three days.Tumor-bearing mice with tumor volumes of ca. 100 mmAfter 12 days, mice were euthanatized by cervical dislocation. The tumor tissues and major organs  were excised from the mice and fixed in 4% aqueous formaldehyde. They were then embedded in paraffin and sliced into 5 μm sections. These sections were stained with hematoxylin and eosin (H&E) and studied under an optical microscope. In addition, TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining was used to analyze apoptosis.3 were employed for Kaplan–Meier analysis. These mice were divided into the abovementioned 6 groups (n = 10), and injected intravenously through the tail vein with the formulations under test (200 μL). The survival status was recorded every day for 20 days.Additional tumor-bearing mice with tumor volumes of 100 mmt-test or one-way ANOVA analysis, followed by a Newman–Keuls test; p-values < 0.05 were set as statistically significant findings, and the data were marked with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001.Statistical analyses were performed using either Student’s 2 adsorption–desorption isotherms confirmed the mesoporous structure of the MSNs, and showed that the pore size was ca. 2.6 nm  was performed to analyze the weight loss d. The de2+ was chelated onto the surface to seal the pores. UV spectra are shown in −1 and the content of Pt was 2.2 ± 0.4 mg·g−1  collapses in acidic environments (pH = 4–6) [2+ is reversed in an acidic environment. Thus, the formulations have pH-responsive drug release, and are likely to free more of their cargo in the TME than under normal physiological conditions.The complex of His-tag and CuH = 4–6) . Drug re2+ on the production of ·OH in vitro. ·OH has the ability to destroy the conjugated structure of organic dyes and, thus, cause color fading. Therefore, the production of ·OH can be detected by measuring the change in the absorbance of MB [2O2 for 24 h, the MB solution did not fade, indicating that MB was stable at this concentration of H2O2 [2O2 almost completely faded after adding CP/CQ@MSN−HtB/Cu2+ NPs. This was because Cu2+ released in the acidic environment can react with H2O2 to produce ·OH. In contrast, at pH 7.4, the MB solution faded to a notably lesser degree. This may be due to much of the Cu2+ remaining chelated, with a reduced amount being released into solution. The UV spectra confirm the visual observations  was used as an indicator to explore the effect of CP/CQ@MSN−HtB/Cuce of MB ,56. Afte of H2O2 . As demorvations d. The MBCP and CQ cannot easily be seen by fluorescent microscopy. Hence, the nanoparticles were loaded with the fluorescent drug DOX to detect cellular uptake. The ability of DOX@MSN−HtB NPs to target cancer cells was evaluated in B16 and L-929 cells by CLSM. The resultant images a,b revea2+. Cells treated with CuCl2 or MSN−HtB/Cu2+ produced relatively weak fluorescence, due to some ROS being generated by the Cu2+ present. The CP/CQ@MSN−HtB/Cu2+ group gave markedly enhanced fluorescence, indicating that the NPs successfully released CP and Cu2+ in the acidic environment of the lysosome. The presence of CP and Cu2+ aids Fenton-reaction-mediated production of ROS [Since the DCFH-DA probe can be oxidized to DCF—a green fluorescent molecule—under the action of ROS, it can be used to detect ROS produced in the cell. n of ROS .2+ NPs , confirming that the NPs can effectively transport CP and CQ to the cells and cause cell apoptosis. The cells treated with CP/CQ@MSN−HtB NPs showed stronger green fluorescence (living cells) than those exposed to CP/CQ@MSN−HtB/Cu2+ NPs, indicating that Cu2+ can augment the chemotherapy provided by CP and CQ, proving synergistic chemotherapy/chemodynamic therapy. In addition, the MSN−HtB group had negligible red fluorescence, indicating that MSN−HtB has almost no biological toxicity.Calcein AM/PI double-staining was used to evaluate the apoptosis of B16 cells c. Compar2+ group, 46.5% of the cells were apoptotic. This increased percentage is attributable to the CDT effects induced by Cu2+. The cell survival rate after treatment with MSN−HtB was 88.4%, consistent with the results above, and showing the NPs also have only slight cytotoxicity.Flow cytometry analysis was used to confirm the results of these assays d,e. In t2+ NPs were injected into mice through the tail vein. After 24 h, the contents of Pt and Cu in the heart, liver, spleen, lungs, kidneys, and tumor cells were measured by ICP–OES. Both Pt and Cu could be seen to be concentrated in the tumor  or free CQ treatment groups (6.03 g).The mice were euthanatized and the tumors were excised at day 12 b. The ph weighed , mice tr2+ NPs have good biocompatibility and are well tolerated, with minimal off-target side effects. In addition, the survival time of tumor-bearing mice was evaluated using the Kaplan–Meier test  were barely visible. There was no significant tissue necrosis in the free CP and free CQ treatment groups, which was attributed to their being distributed throughout the body rather than being localized in the tumor. The necrotic area of the tumor tissue increased greatly after the addition of Cu2+. TUNEL staining  loaded with cisplatin and chloroquine. The NPs were functionalized with the targeting ligand His-tag-B3int, and the NP pores were sealed via the chelation of Cu