Zygotic genome activation (ZGA) is essential for early embryonic development. However, the regulation of ZGA remains elusive in mammals. Here we report that a maternal factor TDP-43, a nuclear transactive response DNA-binding protein, regulates ZGA through RNA Pol II and is essential for mouse early embryogenesis. Maternal TDP-43 translocates from the cytoplasm into the nucleus at the early two-cell stage when minor to major ZGA transition occurs. Genetic deletion of maternal TDP-43 results in mouse early embryos arrested at the two-cell stage. TDP-43 co-occupies with RNA Pol II as large foci in the nucleus and also at the promoters of ZGA genes at the late two-cell stage. Biochemical evidence indicates that TDP-43 binds Polr2a and Cyclin T1. Depletion of maternal TDP-43 caused the loss of Pol II foci and reduced Pol II binding on chromatin at major ZGA genes, accompanied by defective ZGA. Collectively, our results suggest that maternal TDP-43 is critical for mouse early embryonic development, in part through facilitating the correct RNA Pol II configuration and zygotic genome activation. Zygotic genome activation is crucial for mammalian embryonic development. Here, the authors find that TDP-43 is indispensable for mouse embryonic development and mediates zygotic genome activation through Pol II configuration. The ZGA is the first transcription event, and is essential for embryonic development. The zygotic genome is activated as two waves, namely minor and major ZGA5. Minor ZGA occurs at the end of the one-cell stage with the expression of dozens of genes6, followed by a major ZGA bursting at the two-cell stages with the expression of thousands of genes for early embryogenesis in mice4. During mouse oocyte to embryo transition, the transcription machinery of RNA Pol II undergoes loading, pre-configuration and elongation for gene expression7. Transiently blocking minor ZGA with a transcription elongation inhibitor DRB leads to a majority of mouse embryos arrested development at two-cell stage7. Timely release of DRB inhibition before major ZGA results in abnormal transcription with the reduced enrichment of Pol II at major ZGA and the residual of Pol II binding at one-cell Pol II target genes, accompanied by the globally reduced expression of major ZGA and ectopic expression of one-cell Pol II targets7. These observations suggest Pol II is tightly regulated during the transition from minor to major ZGA in mice.After fertilization, the mammalian embryo reprograms the terminally differentiated gametes to totipotent blastomeres with the potency to generate a whole organism. During oocyte to embryo transition, the embryo undergoes reprogramming and many key events including maternal RNA degradation and zygotic genome activation (ZGA)9. However, genetic depletion of mouse maternal Pou5f1 was shown to give rise to little effect on early embryonic development10. Recently, several factors, such as Dux, Dppa2, Dppa4 and Nelfa, were identified as the important regulators for ZGA genes in two-cell-like embryonic stem cells (ESCs)17. However, genetic disruption experiments showed that most of these factors are dispensable for both the ZGA and early embryonic development in mice20. Therefore, the regulation of Pol II machinery remains poorly understood during the mammalian oocyte to embryo transition.Due to transcription silence before ZGA, maternal effect genes are anticipated to play dominant roles in the regulation of RNA Pol II machinery during the ZGA. Zebrafish transcription factor Pou5f1 primes at the loci of several hundred ZGA genes and is required for the expression of these genes21. TDP-43 includes several domains composed of an N-terminal nuclear localization sequence (NLS), two RNA binding domains (RRM1 and RRM2), a nuclear export signal (NES), and a C-terminal glycine-rich domain22. Pathogenic aggregates/inclusions of TDP-43 in the cytoplasm of nerve cells are major features of a range of neurodegenerative diseases23. A combination of a toxic gain in cytoplasm and a function loss in nucleus of TDP-43 is believed to be the predominant reason for neuronal death in patients suffering from these diseases24. Since its aggregates have been shown to be related to neurodegenerative diseases, great efforts have been made to explore the roles of TDP-43 in different cells and animals26.TDP-43 (Transactive response DNA binding protein 43 kDa), officially named TARDBP (Tar DNA binding protein), is an evolutionarily conserved DNA/RNA binding proteinTdp43/TDP-43/Tardbp universally expresses and encodes a protein sharing high similarity with its human homolog27. Specific deletion of TDP-43 with Stra8-Cre mice results in a severe loss of male germ cells and male infertile28. The intercrosses of heterozygous (Tdp43+/-) mice could not deliver homozygous (Tdp43-/-) offspring and Tdp43-/- embryos die between embryonic day 3.5 (E3.5) and E8.530. TDP-43 null blastocysts were unable to generate mouse embryonic stem cells (ESCs) due to proliferation defects of inner cell mass30. Thus, zygotic TDP-43 is essential for mouse early embryonic development. Although many mouse models have been developed to investigate TDP-43, the current understanding of its functions and mechanisms remains limited. Whether this multifunctional protein is involved in mouse oocyte to embryo transition remains unknown.Mouse 31. Depletion of TDP-43 in mouse oocytes shows that maternal TDP-43 is required for the ZGA and early embryogenesis. Detailed analyses show that TDP-43 interacts with RNA Pol II and Cyclin T1 of the P-TEFb complex, and is required for proper Pol II configuration and the expression of ZGA genes during mouse oocyte to embryo transition.In this work, we find that TDP-43 translocates from the cytoplasm into the nucleus at the mouse early two-cell stage, a simultaneous occurrence with mouse major ZGATdp43 mRNA in mouse oocytes and preimplantation embryos by analyzing the previous data of RNA-seq and Ribo-seq32. The results showed that the levels of Tdp43 expression were dynamic in mouse oocytes and early embryos  mice . Tdp43oKO mice grew normally to their adulthood. The oocytes from Tdp43oKO females underwent efficient elimination of TDP-43 at both the mRNA and protein levels  that normally produced litters (8.8 ± 2.1 pups/litter), Tdp43oKO females did not deliver any pups after mating with fertile wild type (WT) males for over 3 months , E1.5 and E2.5. We isolated a similar number of 1-cell embryos with pronucleus at E0.5 , MII, 1 C (1-cell embryos), E2C and L2C (early and late 2-cell embryos). Our RNA-seq data were well correlated between the replicates   Fig. . Maternarol Fig. . By control Fig. . About 5Tdp43oKO L2C embryos, we knocked down TDP-43 protein in mouse 1-cell embryos using Trim-away with the specific antibody against TDP-43 and cultured these embryos to the L2C stage 35. TDP-43 was efficiently knock-down in the embryos that were injected with Trim21 mRNA and the antibody against TDP-43 (hereafter KD embryos), but not in the Inj-Contr (injection control with Trim21 mRNA and IgG), probably because of TDP-43 localization in the cytoplasm until early 2-cell stage   were also up-regulated in Tdp43oKO embryos  experiments in mouse ESCs with the antibodies against TDP-43 and Polr2a. Our results showed that the TDP-43 antibody specifically precipitated Polr2a Fig. . Convers38 Fig. . Another38 Fig. . The intyos Fig. . Similaryos Fig. . Altoget7. The Stacc-seq data of TDP-43 were highly reproducible between the replicates, and were distinct between FGO and L2C embryos, with only 8.9%  of TDP-43 peaks overlapping  of TDP-43 peaks co-localized with the Pol II peaks in L2C embryos Fig. , while ~c43 Fig. , indicatTdp43oKO embryos prompted us to investigate whether TDP-43 depletion could affect the transition of Pol II occupancy during ZGA. By applying Stacc-seq7, we found that the change of Pol II occupancy at the promoters and gene bodies was correlated with the expression levels of DEGs in Tdp43oKO L2C embryos  and late 2-cell (L2C), as well as the 1 C and L2C specific cluster. The PoI II enrichment of the 1 C specific cluster became gradually weaker, while the enrichment of the L2C specific cluster became gradually stronger in WT embryos 37. Then, we sought to investigate whether TDP-43 regulates Pol II through the components of the P-TEFb complex in mouse ZGA.Our results suggest that TDP-43 was directly involved in the transcription of ZGA genes through Pol II configuration. As mentioned previously, Pol II enters the elongation for transcript production when it is phosphorylated at its CTD Ser2 (P-Ser2) by P-TEFb  family proteins include six members that highly express during mouse oocyte to embryo transition53. Maternal importin α2 (Kpna7) was reported to be required for mouse early embryonic development and fecundity54. Similar to those lacking maternal TDP-43, depletion of maternal importin α7 (Kpna6) resulted in the arrested embryo development at the two-cell stage and the dramatically decreased expression of ZGA genes in these embryos53. Thus, these importin proteins may interact with TDP-43 and regulate its translocation from the cytoplasm into the nucleus during mouse maternal to zygotic transition. Taking together, the regulations of TDP-43 translocation deserve to be further investigated during mouse maternal to zygotic transition. The investigations of TDP-43 translocation may contribute to the treatment of human neurodegenerative diseases, such as amyotrophic lateral sclerosis and Alzheimer’s disease.Since the discovery of TDP-43 aggregations related to neurodegenerative diseases, the translocation of TDP-43 from the cytoplasm into the nucleus has been broadly investigated in the neuronal cells with cytoplasmic TDP-43 aggregations for the treatment of these diseasesAcrv1  gene during mouse spermatogenesis, in which TDP-43 and Pol II binds the promoter of Acrv1 in mouse spermatocytes and round spermatids, but the expression of this gene is exclusive in the post-meiotic round spermatids22, further supporting this hypothesis. Thus, how the cells control TDP-43 for the active transcription in the specific context deserves to be further investigated. In summary, our study provides strong evidence to support that maternal TDP-43 interacts with Pol II and is required for proper Pol II configuration, ZGA, and early embryogenesis.Although TDP-43 co-localizes with Pol II in mouse full-grown oocytes, the absence of TDP-43 barely affects P-ser2 foci formation and Pol II configuration in these cells. Furthermore, the absence of TDP-43 mildly affects the gene expression in mouse full-grown oocytes. These observations suggest that the regulation of TDP-43 on the active transcription through Pol II is a stage- or context-specific function of ZGA during mouse oocyte to embryo transition. A previous report shows that TDP-43 regulates the expression of a testis-specific Tdp43floxp/floxp mice (No. 017591) that possess loxP sites flanking exon 3 of Tdp43 were obtained from Jackson Laboratory and were mated with Zp3-Cre transgenic mice that express Zp3 promoter driving Cre recombinase33. After obtaining Tdp43+/floxp;Zp3-Cre mice, they were intercrossed to produce Tdp43floxp/floxp;Zp3-Cre and other genotype mice. Tdp43flox/flox females were mated with Tdp43flox/flox;Zp3-Cre males to routinely produce Tdp43floxp/floxp  and Tdp-43floxp/floxp;Zp3-Cre mice. Tdp43floxp/floxp;Zp3-Cre females had the oocytes with TDP-43 deletion, thus designed as Tdp43oKO (Tdp43 oocyte-specific knockout) mice. These mice were maintained in a hybrid background of C57BL/6 J and ICR, and were genotyped using tail DNA or toe DNA as PCR templates with the primers of a previous report (PubMed: 20660762)33.Mice and all animal experiments were manipulated under the guidelines of the Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences (IOZ-IACUC-2021-052). Mice were maintained under the controlled environment conditions, including 12-h alternating light/dark cycles, a temperature controlled at 23-25 °C, and a humidity range of 40–65%. They had ad libitum access to food and water. Oocytes and embryos were isolated in M2 medium from 7–8-week-old female mice that were injected with HCG after the injection of PMSG for 46–48 h or mating with males at the following time points: MII oocytes: 13 h after HCG; PN2 1-cell embryos: 24 h after HCG; PN5 1-cell embryos (Late 1-cell): 30 h after HCG; early two-cell embryos: 36 h after HCG; late two-cell embryos: 48 h after HCG. To obtain embryos, females injected with HCG were mated with normal fertile ICR males. Full-grown oocytes were isolated from the ovaries of 7–8-week-old females after only the injection of PMSG for ~48 h and cultured in M2 medium (Sigma 7167). Embryos were cultured in KSOM medium (Millipore MR-106-D).TM mRNA DIRECTTM Micro Kit (Millipore 61021). mRNA was extracted as the instruction of the manufacturer. Reverse transcription (RT) was performed with Takara PrimeScript RT Reagent Kit according to the instruction of the manufacturer. Quantitative RT-PCR was conducted with Abm EvaGreen 2X qPCR MasterMix-No dye kit (Abm MasterMix-S) with specific primers of TDP-43  and Gapdh . The results were repeated with at least three independent samples. Gapdh was used to normalize the gene expression.Oocytes and embryos were transferred to the lysis buffer of Invitrogen DynabeadsOocytes and embryos were fixed with 4% PFA in PBS for 30 min at 37 °C. These samples were washed with PBSA , permeabilized with 0.5% (V/V) Triton X-100 for 30 min at room temperature. After being washed with PBSA, samples were blocked with 1% BSA for 1 h at room temperature. These samples were incubated with primary antibody overnight at 4˚C . After washed with PBST (0.1% triton X-100 in PBS) three times, oocytes and embryos were incubated with specific fluorescence conjugated secondary antibody for 1 h at room temperature . After washed at least three times, the samples were imaged with Zeiss LSM780 or LSM880 or Airyscan880. For TDP-43 staining (Santa Cruz Sc-376532), the zona pellucida of embryos was removed with Tyrode’s solution (Sigma T1788) before fixation.55. The RNA library was constructed with a kit of Vazyme (TD502) and performed as the protocol of manufacture.Oocytes and embryos were treated gently with Tyrode’s solution for removing the zona pellucida. Twenty oocytes or embryos were collected for each group of RNA extraction. These samples were performed for reverse transcription and PCR pre-amplification as the smart-seq2 steps as previously reported56. The gene expression was calculated with gene annotation from Gencode RefSeq database using HTSeq (v2.2.1), StringTie (v2.1.2) and FeatureCounts (v2.0.1)58. DEGs were identified by DESeq2 (v1.24.0)59, and the threshold of adjusted P-value was 0.05 with fold change ≥2. Heatmaps of the genes with expression changes were clustered with k-means clustering analysis and were visualized with the heatmap package in R. 2D. PCA analysis was performed and visualized by using all expressed genes by factoextra (v.1.0.7). Dot analysis was used to compare the up- or down-regulated expressed genes in the samples and the Spearman’s rank correlation coefficient was used to compare the relevance between different samples. GO term analysis for the enriched biological processes was conducted using clusterProfiler (v.4.6.0) and DAVID (v6.8) with the default parameters60.All RNA-seq data were mapped to the mouse mm9 genome by HISAT2 (v2.2.1) with default parameters after removing the low-quality sequencing reads with Cutadapt (v0.3.8)42. Maternal genes were identified as highly expressed in MII oocytes (FPKM > 5), but down-regulated in late 2-cell embryos (at least 3-fold). Genes with FPKM (MII) < 5 and up-regulation  in either 1-cell embryos or early 2-cell embryos were identified as minor ZGA genes, while those genes up-regulated in late 2-cell embryos were identified as major ZGA genes. Other genes with FPKM (MII) > 5 and highly up-regulated at the late 2-cell stage (over 5-fold) were also included in major ZGA genes.Maternal and ZGA genes were defined with the reference RNA-seq data of mouse oocytes and early embryosTrim21, NLS-TDP43 and Ccnt1 were cloned in the vector Pcs2 that contained a fused-expressing mCherry or EGFP. The cloned genes were PCR with the sequence of SP6 promoter and T3 promoter. The cloned genes were transcripted according to the AM1340 manufacturer’s protocol and the products were purified as the mRNA template for the microinjection of mouse 1-cell embryos before PN2 stage. After injected with NLS-TDP43-mCherry and Ccnt1-EGFP, mouse 1-cell embryos were cultured for these gene expression and fixed for the staining of TDP43 and Ccnt1 with specific antibodies. For Trim-away technology, mCherry-Trim21 mRNA and rabbit anti-TDP-43 antibody  or normal rabbit IgG were simultaneously injected into mouse 1-cell embryos before the PN2 stage, then these embryos were cultured in KOSM to develop into late two-cell stage (pHCG 48 h) for the following analysis.Tdp43floxp/floxp and ER-cre mice, and were treated with Tamoxifen for 72 h for the immunoprecipitation experiments (washing with NP-40 containing 500 nM NaCl). 293t cells were maintained in classical DMEM basic supplement with 10% FBS.ESCs were maintained in classical 2i/lif culture. The induced-knock-out(iKO) ESCs were derived from the blastocysts that were isolated from the mating of Vector and transfection reagent PEI (three times of vector molar volume) were dissolved in isopyknic opti-MEM for 5 min and were mixed. Twenty-five minutes later, the mixture of vectors and PEI was added into 293t cells to incubate for 12 h. Then, the culture medium was replaced with the fresh medium. After 36 h of transfection, these cells were collected with the lysis buffer of Beyotime plus protease inhibitor for the immunoprecipitation (IP) experiments.Mouse ESCs or 293t cells were collected in IP lysis buffer of Beyotime with protease inhibitor, incubated on ice for 15 min. After centrifuging (11833 g for 15 min at 4 °C), the supernatant was collected for Co-IP experiments according to the manufacturer’s protocol of Protein A/G Magnetic Beads (Bimake B23202 or MCE HY-K0202). The antibodies included TDP-43 , Polr2a , Flag , and Myc . We also washed the IP mixture of mouse iKO ESCs with NP-40 containing 500 mM NaCl for the Co-IP experiment of TDP-43. The precipitated products were heated for 5 min at 95 °C for Western blotting.Oocytes and embryos were collected in 10 μl 1X protein loading buffer and heated for 5 min at 95 °C. The heated samples were fractionated at 100 V for 2 h using 10–12% NuPAGE Bis-Tris gels and MOPS SDS running buffer, and were transferred to PVDF membranes for 2 h at 200 mA (3.5 h when Polr2a antibody was blotted). Then, the membranes were blocked with 5% skimmed milk in TBST (M/V) for 1 h at room temperature. After washed with TBST, the membranes were incubated with primary antibodies at 4 °C overnight . After washed with TBST, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. After washed with TBST, the membranes were detected for the targeting proteins according to the instruction of SuperSignal West Dura Extended Duration Substrate and were imaged with a LAS-3000 apparatus.Proximity Ligation Assay was performed with Sigma-Aldrich reagents according to the protocol of Sigma Aldrich Duolink® PLA Fluorescence . The primary antibodies of different host species  were used and diluted as immunofluorescence staining. The embryos were collected as above, and their zona pellucida was removed using Tyrode’s solution. These samples were fixed with 4% PFA for 30 min at 37 °C, and permeabilizated with 0.5% Triton X-100 in PBS for 30 min at room temperature. The embryos were blocked with Sigma-Aldrich Duolink® Blocking Solution in a humidity chamber for 60 min at 37 °C. The samples were transferred into the mixed solution with the primary antibodies in Duolink® Antibody Diluent, and were incubated in a humidity chamber at 37 °C for at least 2 h or at 4 °C overnight. The samples were washed with 1X Duolink® washing buffer A, and were transported to the probe mixture prepared as the manufacturer’s protocol, then were incubated in a humidity chamber at 37 °C for 1 h. After washed with Duolink® washing buffer A, the samples were incubated with the ligation mixture that were prepared as the manufacturer’s protocol in a humidity chamber at 37 °C for 30 min. After washed with 1X Duolink® washing buffer A, the samples were incubated with the amplification mixture containing amplification buffer (1:5) diluted with ultrapure water and amplification polymerase (1:80) in a humidity chamber at 37 °C for 100 min. After washed with 1X Duolink® washing buffer B and 0.01X Duolink® washing buffer B, the samples were incubated with 1X DAPI at room temperature for 30 min or at 4 °C for a long time in a dark condition. After washed with PBSA (0.1% BSA in PBS), the samples were imaged by using a LSM880 or Airyscan880 apparatus.7. Briefly, 1 μl fully dissolved 5% digitonin (Sigma D141) was added to Buffer1 , termed DB1. Then, 0.5 μl PA/G-Tn5 (Vazyme Biotech TD902) and 0.5 μg TDP-43 antibody  or Pol II antibody (Active Motif 102660) were also added to DB1 and the mixture was incubated at 4 °C for 30 min. Meanwhile, mouse oocytes and embryos were treated gently with Tyrode’s solution to remove the zona pellucida, then were picked into a 200 μl low-binding tube containing 6 μl of the DB1. After incubated at 4 °C for 10 min, these samples were added with 35 μl of the mixture of DB1, PA/G-Tn5, the antibody and 12.5 μl of TTBL (Vazyme TD502) for cleaving the chromatin around the binding sites and transposing with adapters. Finally, the DNA was purified and PCR was performed as the Stacc-seq steps. We used at least 150 embryos or oocytes for the high quality data of TDP-43 (200) or Pol II (150) in these experiments.Stacc-seq was performed according to our previous report61. For downstream analysis, read counts were normalized by computing the numbers of reads per kilobase of bin per million of reads sequenced (RPKM) after removing non-uniquely mapped reads, unmapped reads and PCR duplicates. The RPKM was further normalized with Z-score transformation to minimize the batch effect. UCSC genome browser was used to visualize the signals of Stacc–seq62. Repetitive element enrichment was calculated by comparing annotated repeats overlapped by Stacc-seq peaks and randomly shuffled peaks. All the Stacc-seq peaks were called by MACS (v.1.4.2) with the parameters–nolambda–nomodel63, and were compared by using BEDTools (v2.29.2)64. ChIPseeker (v1.26.2) was used to identify and visualize the genomic features of peaks65. Promoters were defined as ±2,5 kb around the transcription starting sites (TSS). Stacc-seq peaks at least 2.5 kb away from TSS were defined as distal peaks by BEDTools (v2.29.2).All paired-end Stacc-seq reads were mapped to mm9 using Bowtie2 (v.2.2.5) after trimming adapters and low quality sequencing readst-test or two-sided Wilcoxon rank-sum to compare the difference between two groups. P-values < 0.05 were considered as a significance. The immunostaining experiments and Western blots were performed at least three independent repetitions with similar results.Quantitative analyses were expressed as the mean ± SD, based on at least three independent biological samples. Statistical analyses were performed using a two-tailed Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Women typically exhibit weaker attentional control ability than men. Lower resting vagally mediated heart rate variability (vmHRV) is thought to reflect the poorer function of the neurophysiological pathways underlying attentional control and thus, poorer attentional control ability. However, existing findings are inconsistent regarding the relationship between vmHRV and attentional control. Gender may be an important moderator.To examine whether gender moderates the relationship between resting vmHRV and attentional control, and to provide neurophysiological evidence for elucidating gender differences in attentional control ability.Two hundred and twenty college students completed the Attentional Control Scale to evaluate their attentional control ability. Resting vmHRV was assessed during a 5 min baseline period using an electrocardiographic amplifier (ECG100C) of the Biopac MP150 physiological recorder.t = 0.498, p > 0.05), but the scores of the attentional shifting dimension of women were significantly lower than those of men ; (2) Resting vmHRV was significantly negatively correlated with attentional control in women, whereas the correlation was not significant in men; (3) Gender significantly moderated the relationship between resting vmHRV and attentional control ; (4) Among participants with lower resting vmHRV, there was no significant difference in attentional control between men and women , but among participants with higher resting vmHRV, men scored significantly higher than women in attentional control .(1) There was no significant difference in the total scores of the Attentional Control Scale between men and women (Gender moderates the relationship between resting vmHRV and attentional control, with higher resting vmHRV in women reflecting a compensatory response to deficits in attentional control. AttentiAccording to the neurovisceral integration model, attentional control is significantly influenced by prefrontal-subcortical inhibitory pathways . The preHeart rate variability (HRV) is a measure of the variability of the interval between two consecutive heartbeats . VagallyHowever, there are inconsistent findings in existing studies regarding the relationship between resting vmHRV and attentional control ability. Although some studies have shown that resting vmHRV is positively correlated with attentional control , 17, othStudies have shown that there are gender differences in attentional control , 19. WomThe differences in attentional control between men and women have a neurophysiological basis. The brain regions involved in attentional control mainly include structures such as the frontal cortex and cingulate gyrus . NeurostA growing body of research has shown that gender is a significant factor in moderating the relationship between resting vmHRV and mood, such as depression, anxiety and stress perception. For example, there are gender differences in the relationship between resting vmHRV and depression . Higher In summary, gender may be an important factors that moderates the relationship between resting vmHRV and attentional control. However, existing studies have not investigated this issue systematically. The current study aimed to examine how gender moderates the relationship between resting vmHRV and attentional control to shed light on the neurophysiological differences in attentional control between men and women.2.2.1.Participants were recruited through two methods. One group of participants was recruited through campus advertisements and received a cash incentive of 15 RMB for participation. The other group of participants was recruited from students enrolled in public psychology courses and received a course credit award. Inclusion criteria included being right-handed and having normal or corrected-to-normal vision. Exclusion criteria included a history of neurological, psychiatric or cardiovascular disease. A total of 220 participants  were included in this study, with a mean age of 19.31 ± 1.17 years. All participants voluntarily signed the informed consent form before the experiment. As sleep quality, physical activity and caffeine intake are important variables influencing HRV , partici2.2.When participants arrived at the laboratory, they naturally sat on a chair to rest, signed the experimental informed consent form, and filled out a series of self-report questionnaires. After the participants felt completely relaxed, they began to collect resting ECG data for 5 min. During the ECG data acquisition process, the participants sat relaxed in a chair, kept their bodies stable, breathed naturally, and looked at the picture of the neutral cup presented on the computer screen. At the end of experiment, participants scored their emotional arousal on a 9-point scale.2.3.The Attentional Control Scale (ACS) is a self-reported measure of attentional control ability . It is w2.4.To assess the subjective emotional experience during the experiment, we instructed the participants to score their emotional arousal  on a 9-point scale .2.5.Cardiac data were collected using the electrocardiographic amplifier (ECG100C) of the Biopac MP150 physiological recorder. Three silver chloride (Ag-AgCL) disposable electrode pads were attached to the participant’s wrist and two ankles in lead II. The filtering range of the Biopac ECG100C amplifier was 0.5–35 Hz with a sampling rate of 1,000 Hz. The Kubios HRV analysis package  was used2.6.t-tests were conducted to analyze the differences between male and female participants on all variables. Pearson correlation was used to analyze the relationship between vmHRV and other important variables. The SPSS macro PROCESS (Model 1) was used to test the moderating effect of gender  on the relationship between attentional control (independent variable) and resting vmHRV (dependent variable) . All tests were two-tailed and significance levels were evaluated using an alpha of 0.05. Independent samples ariable) . Bootstr3.t = 4.133, p < 0.001). There was no significant difference between male and female participants on resting vmHRV . Women’s attentional control scores were slightly lower than men’s, with the difference not reaching a significant level . However, women scored significantly lower than men on the attentional shifting dimension of attentional control .r = −0.233, p < 0.01).B = −3.088(1.189), 95% boot CI , t = −2.598, p = 0.01). Conditional analysis showed a negative correlation between resting vmHRV and attentional control in women (B = −2.120 (0.779), 95% boot CI , p = 0.007), whereas there was no significant correlation between resting vmHRV and attentional control in men (B = 0.968 (0.898), 95% boot CI , p = 0.282). The moderating effect of gender remained significant when analyzed with age, BMI, respiration and emotional arousal as covariates in the moderation analysis (B = −2.897(1.203), 95% boot CI , t = −2.408 p = 0.017), with a negative correlation between resting vmHRV and attentional control in women (B = −2.072 (0.786), 95% boot CI , p = 0.001) and no significant correlation in men (B = 0.825 (0.912), 95% boot CI , p = 0.367). In other words, among women, the difference in attentional control between participants with lower and higher resting vmHRV was significant; whereas among men, the difference in attentional control between participants with lower and higher resting vmHRV was not significant (see B = 2.284 (1.542), 95% boot CI , p = 0.139), but men scored significantly higher in attentional control than women in participants with higher resting vmHRV (B = −3.377 (1.536), 95% boot CI , p = 0.029).cant see . With ge4.This study aimed to investigate whether gender moderates the relationship between resting vmHRV and attentional control, and to provide neurophysiological evidence for elucidating gender differences in attentional control ability. The results showed that there was no significant difference between men and women in the overall scores of attentional control, but women scored significantly lower than men in attentional shifting. Gender was a significant variable in moderating the relationship between resting vmHRV and attentional control. Resting vmHRV was significantly negatively correlated with attentional control in women, whereas it was not significantly correlated with attentional control in men.Although there was no significant difference in the total scores of attentional control between men and women in this study, women had poorer attentional shifting abilities than men, which confirmed the previous findings. Women had weaker attentional shifting ability than men, as evidenced by a lower efficiency in shifting attention across multiple cognitive processing tasks . AttentiMore importantly, the present study showed that gender moderates the relationship between resting vmHRV and attentional control, with resting vmHRV negatively correlated with attentional control in women, whereas the two were not significantly correlated in men. The conditional analysis revealed that among participants with higher resting vmHRV, men had significantly higher attentional control scores than women. This result explains the mechanism of gender differences in attentional control from the perspective of vagal nerve tone. Women with high resting vmHRV were more likely to have difficulty with attentional control, suggesting differences in the neural mechanisms of attentional control reflected by high resting vmHRV in men and women. Consistent with previous studies, we speculated that higher vmHRV in female participants reflects a compensatory mechanism for deficits in attentional control, as an effort by individuals to compensate for dealing with negative emotional responses , 30. DueSome studies support the idea that higher resting vmHRV in women is a compensatory response to inadequate attentional control. Studies have shown that higher resting vmHRV in women is associated with increased activity in the amygdala (the area associated with threat processing), whereas higher resting vmHRV in men is associated with decreased amygdala activity . This coFrom an evolutionary psychology perspective, the tend-and-befriend theory and the parental investment theory could also provide an explanation for the negative correlation between resting vmHRV and attentional control is limited to women , 42. EvoIn general, the results of this study suggest that women have poorer attentional shifting abilities than men, and that gender moderates the relationship between vmHRV and attentional control. The high resting vmHRV levels in men and women may reflect different emotion regulation mechanisms, with higher vmHRV in women reflecting a compensatory response to deficits in attentional control.5.There are several shortcomings in the present study. First, the present study examined the relationship between resting vmHRV and attentional control in a cross-sectional investigation and was unable to determine a causal relationship between the two variables. Future studies could design longitudinal follow-up experiments to examine the neurophysiological basis of gender differences in attentional control. Second, the present study only used the self-report scale to measure attentional control ability, which may not accurately cover all aspects of attentional control . Future The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee of Nanjing University of Chinese Medicine. The patients/participants provided their written informed consent to participate in this study.XZ contributed to the conception and design of the study. YS checked and corrected the scientific issue of the study. XZ and SW conducted the experiment, analyzed the data, and wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by the Research Project of Jiangsu University Philosophy and Social Science (2021SJA0338).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Herein, we have constructed a magnetic graphene field-effect transistor biosensor (MGFETs) for highly sensitive detection of cardiac troponin I (CTNI). Graphene films transferred to ITO conductive glass as conductive channels. CTNI aptamer was immobilized onto the graphene film via 1-pyrene-butanoic acid succinimidyl ester (PBASE) to capture CTNI. Magnetic nanobeads (MBs) modified with CTNI antibody were added to the reaction chamber to form an aptamer/CTNI/antibody/magnetic nanobeads sandwich-type complex. We found that the magnetic force exerted on the complex leads to an impedance change of the graphene film. The reason for this result is that the magnetic field exerts an influence on the MBs, causing CTNI aptamer strand to bend, resulting in a change in the distance between the double conductive layers of the graphene film surface and the test solution. With periodic sampling integration, different concentrations of CTNI can be detected with high sensitivity. Due to the stringent recognition capability and high affinity between the CTNI aptamer and CTNI, MGFETs have the potential to detect various types of proteins. Furthermore, MGFETs also have the potential to be utilized for the detection of DNA or specific cells in the future. Based on statistical data from the World Health Organization (WHO), cardiovascular diseases have emerged as the primary cause of global mortality, with acute myocardial infarction (AMI) being the deadliest among various cardiovascular conditions , 2. The To address these limitations, various cardiac biomarkers have been developed for the diagnosis of acute myocardial infarction (AMI). These include cardiac troponin I (CTNI), CK-MB, cardiac troponin T (CTNT), and myoglobin , 7. CTNIMainly, CTNI detection methods include enzyme-linked immunosorbent assay , radioimGraphene is a single atomic layer of two-dimensional crystals formed by carbon atoms linked by SP2 hybridization. It has good electron mobility and many oxygen-containing groups that can be used to anchor DNA, proteins, and other biomolecules . Many reIn conventional graphene field-effect transistor biosensors (GFETs), applying a gate electric field results in the formation of double conductive layers between the electrolyte solution and the graphene film. Researchers have constructed a capacitive model of a GFETs . The gatHowever, although these techniques have reached extremely high sensitivity through semiconductor analyzers, they are still costly and complicated to use. Additionally, the application of gate voltage through the insertion of Ag/AgCl electrodes into the solution may affect the concentration of the solution, and the gate voltage may cause structural damage to the biomolecules to be measured –27.Therefore, researchers have been exploring alternative methods to improve the performance of GFET biosensors, such as using non-covalent functionalization with biomolecules, which can improve the sensitivity and prevent damage to the biomolecules –30. OtheHerein, we propose a novel approach for CTNI detection using magnetic graphene field-effect transistor biosensors, which eliminates the need for an Ag/AgCl electrode to provide the gate voltage. Firstly, we transfer graphene films onto glass substrates with two indium tin oxide (ITO) electrodes to establish conductive channels . The graThe conductive glass with ITO electrodes was sourced from Hua Nan Xiang Cheng Ltd. Troponin I and mouse anti-troponin I monoclonal antibodies were obtained from Shanghai Linc-Bio Science CO., LTD. 1-Pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) was acquired from Shanghai Aladdin Biochemical Technology Co., Ltd. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide-hydrochloride (EDC) and N-hydroxy succinimide (NHS) were purchased from Sigma-Aldrich. The troponin I aptamer sequence used, (5-NH2-CGT GCA GTA CGC CAA CCT TTC TCA TGC GCT GCC CCT CTT A-3′-6-FAM), was purchased from Sangon Biotech (Shanghai) Co., Ltd. Carboxylated ferric oxide nanoparticles were obtained from Nanjing XFNANO Materials Tech Co., Ltd. Dimethyl sulfoxide (DMSO) was purchased from Sangon Biotech (Shanghai) Co., Ltd. Sodium dodecylbenzene sulfonate (SDS) and sodium dodecyl sulfate phosphate-buffered saline  were obtained from Sigma-Aldrich . Bovine serum albumin (BSA) was purchased from Shanghai Linc-Bio Science CO., LTD. To evaluate the quality of graphene, a Raman microscopic system  was utilized. High-magnification transmission electron micrographs (TEM) were captured using a JEOL JEM 2100F instrument.To obtain a uniformly dispersed magnetic bead solution, the purchased carboxyl-modified magnetic nanobeads were sonicated for 15 min at room temperature. The carboxyl groups on the magnetic nanobeads were activated by adding 5 µL of 0.2 µM NHS and 5 µL of 0.2 µM EDC to 500 µL of 1 mg/mL magnetic nanobeads. Tchenka et al. , followeIn order to deposit the magnetic nanoparticles at the bottom, a permanent magnet was positioned beneath the magnetic nanoparticle test transistor, and the supernatant was removed using a pipette. The resulting pellet was then resuspended in 1 mL of PBS buffer at pH 7.4.To functionalize the magnetic nanobeads, 10 µL of mouse anti-troponin I monoclonal antibodies with a concentration of 1 mg/mL were added to the activated magnetic nanobead solution prepared with NHS and EDC. The mixture was incubated at room temperature for 2 h. Following this, 10 µL of 10% bovine serum albumin (BSA) was introduced into the suspension and incubated for 1 h to block any potential residual sites on the nanobeads.The fixed magnetic nanobeads were obtained by using a permanent magnet underneath the transistor to hold them in place, followed by aspirating the upper layer of unreacted chemicals using a micropipette. The resulting pellet was resuspended in 1 mL of PBS (pH 7.4) supplemented with 1.0% BSA and stored at 4 °C. .The cleaned graphene film was transferred onto a glass substrate with two ITO electrodes, serving as a conductive bridge between the two ITO electrodes Fig. a. The MGThrough the magnetic force between magnetic beads (MBs) and an externally applied magnetic field, the distance between MBs/aptamer conjugates and the graphene film can be dynamically adjusted. This modulation directly influences the conductivity properties of the graphene film, thereby affecting the impedance of the magnetic field effect transistors (MGFETs) with a dual conductive layer Fig. a. In ourID/IG) and the presence of a small D-peak. Furthermore, the intensity of the 2D-peak of single-layer graphene was more significant than that of the G-peak, while the half-peak width of the 2D-peak gradually increased and shifted to a higher wave number as the number of layers increased. These observations led us to conclude that the transferred graphene films were multilayer films [The transferred graphene was characterized using Raman spectroscopy Fig. . The Ramer films –32.Fig. π–π stacking, while the other end can bind to the 5′-NH2 labeled aptamer. We labeled the 3′ end of the CTNI aptamer with a fluorescent group to verify the firm attachment of the aptamer to the graphene film. The . Alternatively, one end of PBASE can be linked to graphene through The Fig.  showed tCarboxyl-modified magnetic nanobeads were activated by NHS and EDC and utilized as a capture probe for binding to CTNI antibodies. The nanobeads were used to form aptamer/CTNI/antibody/MBs sandwich-type immune complexes. Tubes containing 10 µL of CTNI aptamer at a concentration of 2 µM and 100 µL of 10 µM CTNI were incubated for 2 h at room temperature. A fluorescence detection system detected a fluorescence intensity of about 1400 A.U. The mixture of magnetic nanobeads and CTNI antibody binding, which was completed by the aforementioned experiment, was then taken out (100 µL) and added to the test tube. The solution was incubated for 1 h and then separated using a magnetic precipitation method. A part of the supernatant solution was tested using the fluorescence detection system for detection. The results shown in Fig.  indicateAfter the linkage of the CTNI aptamer to the graphene, different concentrations of CTNI solution were added to the reaction chamber of the magnetically controlled MGFET, followed by the addition of an antibody-modified magnetic bead solution for 30 min. The reaction chamber of the MGFET was then washed with PBS to remove any unbound magnetic nanobead and antibody complexes. A permanent magnet was placed at the back of the MGFET to apply a periodic magnetic field, and the impedance change of the MGFET was detected using a laboratory-made device Fig. a.Fig. 7aIn conventional GFETs, the gate electrode applies voltage to the Ag/AgCl electrode, controlling the directional movement of charge carriers between the conductive solution and the graphene film. In MGFETs, the CTNI aptamer can be considered as an elastic model. By applying a periodic magnetic field, the bending degree of the CTNI aptamer chain can be altered, leading to a change in the distance of the double conductive layer between the graphene film and the electrolyte solution. This results in carrier movement and subsequently alter the conductivity between the drain and the source of the MGFET. The variation in the impedance of the MGFET with magnetic field strength was divided into three components Fig. b. When tThe impedance variation of MGFETs with respect to CTNI concentration at a fixed magnetic field strength was measured to determine the sensitivity and detection limit of the MGFETs Fig. a. The MGWe evaluated the specificity and selectivity of MGFETs by detecting various types of proteins. Cardiac troponin T (CTNT), human serum albumin (HSA), CK-MB, bovine serum albumin (BSA), and glucose were added to test the specificity of MGFETs, and the different types of proteins were tested alongside CTNI under the same experimental conditions , 35. TheTo summarize, a graphene-based MGFETs design has been successfully developed as a biosensor for the detection of CTNI. The structure involves laying ITO films on both ends of a glass sheet to serve as the drain-source of the FETs. Then, by transferring a graphene film in the middle, the graphene film was functionalized with an immobilized CTNI aptamer to capture CTNI in solution. A sandwich-type immune complex was formed with CTNI antibody-modified magnetic nanobeads. Finally, a magnetic field was applied to the GFETs by a permanent magnet immobilized under the ITO conductive glass. To reduce noise interference, we utilized the method of periodic sampling and integration to improve the signal-to-noise ratio, achieving high sensitivity detection of CTNI with a detection limit that can reach 10 pM. Our biosensor provides a promising alternative to conventional Ag/AgCl-electrode-based biosensors. Its use of a magnetic field instead of an electric field to control the GFETs not only enables damage-free detection of the biomolecules but also reduces the cost and complexity of the overall detection system.


Exhaled Drug MONitor – EDMON measures exhaled propofol in ppbv every minute based on multi-capillary column – ion mobility spectrometry (MCC-IMS). The MCC pre-separates gas samples, thereby reducing the influence of the high humidity in human breath. However, preliminary analyses identified substantial measurement deviations between dry and humid calibration standards. We therefore performed an analytical validation of the EDMON to evaluate the influence of humidity on measurement performance. A calibration gas generator was used to generate gaseous propofol standards measured by an EDMON device to assess linearity, precision, carry-over, resolution, and the influence of different levels of humidity at 100% and 1.7%  relative humidity (reference temperature: 37°C). EDMON measurements were roughly half the actual concentration without additional humidity and roughly halved again at 100% relative humidity. Standard concentrations and EDMON values correlated linearly at 100% relative humidity (R²=0.97). The measured values were stable over 100min with a variance ≤ 10% in over 96% of the measurements. Carry-over effects were low with 5% at 100% relative humidity after 5min of equilibration. EDMON measurement resolution at 100% relative humidity was 0.4 and 0.6 ppbv for standard concentrations of 3 ppbv and 41 ppbv. The influence of humidity on measurement performance was best described by a second-order polynomial function (R²≥0.99) with influence reaching a maximum at about 70% relative humidity. We conclude that EDMON measurements are strongly influenced by humidity and should therefore be corrected for sample humidity to obtain accurate estimates of exhaled propofol concentrations.The bedside  Propofol is the most used intravenous anaesthetic for induction and maintenance of general anaesthesia and sedation. Anaesthetic effect can be measured by electroencephalographic systems like the Bispectral Index monitor (BIS). However, the electroencephalogram is influenced by opioids and neuromuscular blocking agents, and is subject to internal and external influences such as eye blinking or sensor movement which add noise to the signal –3. An alInappropriate dosing may cause haemodynamic instability, delayed recovery, or (rarely) intraoperative awareness –12. RealReal-time propofol monitoring from exhaled breath is a rapidly developing field of research. Harrison et al. were the first to perform online measurements of exhaled propofol, reported in 2003 . ExhaledHigh humidity in breath influences the underlying technology of ion mobility spectrometry –28. A muThe EDMON monitor is primarily intended to be used in mechanically ventilated patients. During mechanical ventilation, humidity of breath samples varies depending on various factors including use of heat and moisture exchange filter and fresh gas flow . Under n-1 was freshly prepared by dissolving propofol ≥ 97%  in 1% v/v absolute ethanol  in HPLC-grade water or n-hexane ≥ 99%  for all measurements and stored in a 250 mL glass bottle. To enhance accuracy, the exact propofol concentration of the stock solution was calculated according to the weight of the added propofol determined with an analytical scale .Propofol stock solution of 35.9µg mL-1 N2  . The. The2O)nJ mol-1) 44] hamp hamp2O)nJ mol-1) .ref = 37°C), clinical samples will always contain substantial humidity, but may lay under the 70% threshold. Therefor influences on measured propofol concentrations are prone to differ during changing clinical conditions. Such, the amount of humidity in clinical samples is influenced by patient conditions, heat-and-moisture-exchanging filters, ventilation settings, active humidification, oxygen concentration, fresh mixture volume, inspiratory and expiratory settings, lung-protective ventilation, etc. [Relative humidities between 70% and 100% roughly halved the EDMON measured concentrations compared to measurements without additional humidity. Considering the typical humidity of 27g/m³ in the mouth , which con, etc. –50. EDMOJiang et al.  propose v and 23 ppbv, while imprecision over 100 measurements was ≤ 10%. Resolution is comparable for measurements with and without additional humidity whereas carry-over effects are smaller with additional humidity. The influence of humidity was substantial and results in a sigmoidal decrease in measured propofol concentration by up to 50% at relative humidities exceeding 70%. Quantitative propofol measurements must therefore be adjusted for sample humidity on-line, or at least at the beginning of the measurement, for sample humidity if it can be ensured that the humidity remains stable throughout the procedure, preferably above the 70% threshold. The equations we provide suggest an easy way to adjust for sample humidity.In summary, linearity with and without additional humidity was observed for measured concentrations between 1 ppb


Pennyroyal is a species of the Lamiaceae family with potent anti-cancer and antioxidant properties. Combining this antioxidant with chemotherapeutic agents enhances the effectiveness of these agents by inducing more apoptosis in cancerous cells.  Here, methotrexate (MTX) combined with pennyroyal oil based on PEGylated nanostructured lipid carriers (NLCs) was assessed. These nanoparticles were physiochemically characterized, and their anti-cancer effects and targeting efficiency were investigated on the folate receptor-positive human breast cancer cell line (MCF-7) and negative human alveolar basal epithelial cells (A549).P<0.05) for the MCF-7 cells treated with pennyroyal-NLCs and pennyroyal-NLCs/MTX, respectively. Autophagy and reactive oxygen species (ROS) cellular evaluation indicated that treating the cells with pennyroyal-NLCs and pennyroyal-NLCs/MTX could significantly increase their intensity in these cells.  Results showed a mean size of 97.4 ± 12.1 nm for non-targeted PEGylated NLCs and 220.4 ± 11.4 nm for targeted PEGylated NLCs, with an almost small size distribution assessed by TEM imaging. Furthermore, in vitro molecular anti-cancer activity investigations showed that pennyroyal-NLCs and pennyroyal-NLCs/MTX activate the apoptosis and autophagy pathway by changing their related mRNA expression levels. Furthermore, in vitro cellular studies showed that these changes in the level of gene expression could lead to a rise in apoptosis rate from 15.6 ± 8.1 to 25.0 ± 3.2 ( Our results present a new NLCs-based approach to enhance the delivery of pennyroyal and MTX to cancerous breast tissues. Meanwhile, breast cancer is common cancer in women worldwide. The rate of diseases and infections grows with the increasing population. Among the various threats to humans, cancer is responsible for many deaths worldwide, and most of the deaths are due to cancer metastasis.7 Conventional chemotherapy agents are spread non-specifically throughout the body and influence both tumor and healthy tissues. This therapeutic approach leads to excessive toxicity in the body.8 Therefore, new approaches to breast cancer treatment must be developed to target tumor cells. New achievements in nanotechnology have led to the development of novel targeting plans to increase drug concentrations inside the tumor while restricting systemic side effects and toxicity.9 Lipid-based nanoparticles, including liposomes, are highly suitable agent carriers for cancer treatment applications.10 Nanostructured lipid carriers (NLCs) are a promising lipid-based drug delivery system with extensive loading capability, prolonged stability, and the ability to release drugs and medications in a controllable fashion at specified rhythmic intervals.11 While NLCs are suitable carriers for hydrophobic materials and medicines, they do not load well for hydrophilic drugs such as methotrexate (MTX).12 The double emulsion method is a possible way of loading hydrophilic agents in NLCs but causes low drug loading. An excellent approach to this problem, particularly in nanoparticles with a large ratio of surface to mass, is surface ion coordination.12 MTX is one of the drugs used to treat different types of cancers, including breast, skin, lung, and head and neck cancers.13 MTX inhibits folic acid production, which binds to the dihydrofolate reductase enzyme and consequently hinders dihydrofolate from tuning into tetrahydrofolate. MTX acts not only as a drug but also as a ligand due to its structural similarity to folic acid.13 The use of natural ingredients to elevate the synergistic effect of chemotherapy drugs and reduce their side effects is of particular interest. One of the natural ingredients that can inhibit breast cancer cells is pennyroyal oil.14 Despite the progress in various therapeutic methods, the survival rate of patients with breast cancer has not increased significantly.15 Pennyroyal oil contains the volatile oil pulegone and other monoterpenes. As is shown in 16 Antioxidants are like double-edged swords; they can reduce oxidative stress at low concentrations, and on the contrary, increasing their engagement can cause oxidative stress.17 Due to its antioxidant properties, pennyroyal oil can effectively create free radicals in cancer cells and activate the apoptotic pathway.18 Pennyroyal is a natural herbal extract from Mentha pulegium or Hedeoma pulegioides leaves; mint, family plants are European native. This work aimed to decorate PEGylated NLCs with MTX as nanoparticles for the delivery of pennyroyal in a targeted way and assess the anti-cancer effects of this nano-formulation on the apoptosis, proliferation, cell autophagy, reactive oxygen species (ROS), and colony formation of FR expressing MCF-7 human breast cancer cells.® 812) were purchased from Sasol , and Precirol® ATO 5 was acquired from Gattefosse . DSPE‐mPEG 2000 was obtained from Lipoid GmbH (Ludwigshafen Germany). Moreover, pennyroyal oil was obtained from Barij Essence Pharmaceutical , and MTX was provided from Excella GmbH . Also, cell culture materials were obtained from Gibco . Human non-small-cell lung carcinoma cells (A549) and breast cancer cells (MCF-7) were acquired from the Pasteur Institute of Iran . Monodansylcadaverine (MDC), 1,2‐Dioleoyl‐3‐trimethylammonium‐propane (DOTAP), Poloxamer 407, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), DAPI, and 1,2‐dioleoylsn‐glycerol‐3 phosphoethanolamine (DOPE) were purchased from Sigma-Aldrich . Caprylic/capric triglycerides (Miglyol19 The NLCs were prepared by hot-homogenization (HH) method. First, 20 mg of pennyroyal was dissolved in 100 mg of Miglyol. Then, 100 mg of Precirol was added to the oil mixture and heated up to 80 °C. In the second step, 20 mg of DOTAP: DOPE (1: 1) and 0.1 mg of DSPE-mPEG 2000 were added to the hot lipid mixture and stirred with a homogenizer (5000 rpm) until a uniform lipid solution was obtained. Finally, the surfactant solution (20 mg/mL poloxamer 407 dissolved in phosphate buffer 10 mM pH 5.5) was added slowly and dropwise to the lipid phase under high-speed homogenization . The prepared o/w nanoemulsion was allowed to be cooled down rapidly at room temperature in the refrigerator, leading to the recrystallization of the lipid phase. As a result, the pennyroyal-NLCs were prepared.20 To obtain the best conjugation performance in this study, we used both cationic and helper lipids to prepare NLC. According to the previously published studies, MTX was decorated to the prepared positively charged NLCs by electrostatic conjugation, and the optimal ratio of the decoration was determined.13 For electrostatic conjugation of MTX to the surface of the NLCs, 10 mL of pennyroyal-NLCs was added dropwise into 10 mL of MTX solution and mixed without heating at 300 rpm for an hour. Also, to determine the optimal amount of MTX to the NLCs, different ratios of MTX:NLCs  were used. Then, the best MTX: NLCs ratio was selected based on physicochemical characterization results. DOTAP is a well-known cationic lipid, and DOPE is a helper lipid for DOTAP.21 The only difference in all the preparation stages of the above nanoparticles was the replacement of RhB (at the 0.1 mg/mL concentration) with pennyroyal. Also, unloaded RhB was separated from the nanoparticle solution using the ultrafiltration technique . For evaluating the effect of MTX conjugation on the cellular uptake efficacy, red fluorescence-labeled NLCs were prepared by RhB encapsulation into the NLCs. The fluorescence-labeled NLCs were prepared as same as pennyroyal-NLCs.i/Mo × 100 equation, where Mi and Mo indicate the drug weight before and following the separation, respectively. Moreover, the drug loading ratio was computed as the fraction of the weight of the encapsulated drug to the total weight of the carrier, i.e., lipid. To separate the unloaded pennyroyal oil, the prepared nanoparticles were centrifuged  at 5000 rpm for 20 minutes. A sampler collected the surface medium containing the probably unloaded essence. Then, the collected solution was diluted using ethanol. The amount of unloaded pennyroyal was measured using ultraviolet-visible spectroscopy  at the wavelength of 281 nm.11 The encapsulation efficiency (EE) of pennyroyal was evaluated using the M The size, dispersion coefficient, and surface charge of pennyroyal-NLCs and pennyroyal-NLCs/MTX were assessed through dynamic light scattering  at 25 °C. Transmission electron microscopy (TEM) was utilized to examine the core-shell structure of nanocarriers. For TEM imaging, 10-fold diluted NLC sample droplet was located on a carbon copper grid and then dried at room temperature. The images were then recorded by TEM .2 flask and reaching a cell density of over 70%, the cells were detached from the bottom of the flask using 0.25% trypsin, and cell passage was performed on the cells. For cell experiments, third passage cells were used.13 The MCF-7 cells were purchased from the Pasteur Institute of Iran. The cells were defrosted in 7 ml of RPMI-1640 culture medium containing antibiotics and 10% of fetal bovine serum (FBS). By transferring the defrosted cells to a 25 cm22 The prepared samples were put into a DMB sterilization machine at 1.7 bar pressure for 2.5 hours . After finishing the sterilization process. The liquid samples were sonicated in an ultrasonic bath sonicator for 15 minutes to discharge the remaining sterilization gas. We used the ethylene oxide purge technique to sterilize the prepared nanoparticles.23 The MTT assay test was utilized to assess the effect of pennyroyal-NLCs and pennyroyal-NLCs/MTX on cell viability. The cells were seeded in a 96-well plate and incubated for 24 hours. Next, the cells were treated with 100 μL of various concentrations (5-1280 μg/mL) of free pennyroyal, pennyroyal-NLCs, and pennyroyal-NLCs/MTX. The plate media was removed after 24 and 48 hours of drug treatment and washed twice with phosphate-buffered saline (PBS). In the next step, a mixture of 150 μL of fresh medium and 50 μL of MTT solution (2 mg/mL in PBS) was added to each well of plates. After four hours of incubating medium with the mixture, 20 μL of Sorensen’s buffer and 180 μL of DMSO were replaced. Eventually, the absorption of the wells was recorded using the Elisa reader  at the wavelength of 570 nm. The reference wavelength was 630 nm.5 cells per well. After incubation for 48 hours, the cells were treated with IC50 concentrations of free pennyroyal, pennyroyal-NLCs, and pennyroyal-NLCs/MTX, obtained from MTT assay results. After 48 hours of treatment, the cells were washed twice with PBS and fixed with 4% formalin for four hours. After the fixation, the cells were rewashed by PBS and treated with Triton X100 solution (0.01% V/V) for 15 minutes. After Triton X100 treatment, the cells were stained with DAPI solution at the concentration of 1 μg/mL. Finally, the stained cells were imaged and evaluated with a fluorescence microscopy system . DAPI staining were used to evaluate the degree of DNA damage. For this test, the MCF-7 cells were cultured at 12 mm coverslips at a concentration of 2 × 105 cells per well. Following 48 hours of incubation, the culture medium was substituted with 2 ml of fresh medium, including free pennyroyal, pennyroyal-NLCs, and pennyroyal-NLCs/MTX at IC50 concentrations, and incubated again for 48 hours. Then, the cells were isolated and stained with an annexin V/PI staining kit based on the protocols recommended by the manufacturer. The apoptosis rate was obtained through flow cytometry  and analyzed using FlowJo software  in each group.24 Flow cytometry analysis was utilized to assess the cell death (necrosis or apoptosis) pathway. MCF-7 cells were cultured in a 6-well plate at a concentration of 2 × 105 cells per well. Following incubation for 48 hours, the cultured cells were treated with IC50 concentrations of free pennyroyal, pennyroyal-NLCs, and pennyroyal-NLCs/MTX. Following treatment for 48 hours, the cells were washed twice with PBS and stained with 0.05 mM MDC at 37 °C for 10 minutes. The cells were washed three times with PBS to remove excess MDC and assessed using a fluorescence imaging microscope system .11 MDC is a fluorescent marker used to visualize and identify the autophagy process. MDC was utilized to discover the activation of the autophagy pathway in MCF-7 cells. For this purpose, the cells were cultured at 12 mm coverslips at a concentration of 2 × 1025 For evaluating the effect of active targeting on the cells NLC penetration rate, the RhB-loaded fluorescent formulations were used. The MCF-7 and A549 cells were cultured into 12-well plates and then incubated overnight. Next, the cells were treated with RhB-NLCs and RhB-NLCs/MTX with time intervals of 2, 4, 12, 24, and 48 hours. Then, the penetration rate of the prepared formulations into the cell was measured using the flow cytometry instrument, and the obtained data were analyzed using FlowJo software.3 cells per well of MCF-7 cells were cultured in a 6-well plate and then incubated for 15 days; after 15 days of incubation, the cell were treated with IC50 concentration of the samples and incubated for 48 hours. Finally, the colonies were fixed and stained with ten times diluted solution of crystal violet and then photographed with iPhone 8  and counted using ImageJ software.26 The colony formation test was conducted to evaluate the ability of the pennyroyal-NLCs, free pennyroyal, and pennyroyal-NLCs/MTX to inhibit colony formation. For this purpose, 2 × 1050 concentrations of free pennyroyal, pennyroyal-NLCs, and pennyroyal-NLCs/MTX obtained from MTT assay results. H2O2 (100 µM) was used as the positive control. The cells were incubated for 48 hours with these formulations. In the next step, the cells were stained with 500 μL of DCFH-DA (10 μM into PBS) fluorescent dye and incubated for 2 hours. Then, the cells were detached from the plates and washed twice with PBS. Finally, the cells were resuspended into 500 μL of PBS, and their fluorescent intensity was analyzed by flow cytometry using an FL2-H filter.27 The ROS technique was used to evaluate the induction rate of intracellular free radicals by analyzing the intensity of 2-7 dichlorodihydrofluorescein- DCFH-DA diacetate fluorescent dye using FACS flow cytometry. MCF-7 cells were cultured in a 6-well plate and then treated with IC28 Antioxidant activity was evaluated in two ways. In the first step, the antioxidant activity of free pennyroyal and pennyroyal-NLCs was examined by measuring the inhibition rate of free radicals using 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH). In the second, the 2.1 mg/mL concentration was selected to evaluate the antioxidant stability. For this purpose, 5 samples of pennyroyal and pennyroyal-NLCs were prepared at a concentration of 2.1 mg/mL and stored in a dark place during the test. Then, the antioxidant activity of the samples was measured every 15 days up to 60 days by mixing 100 μL of the mentioned samples with 100 μL of DPPH free radical at a concentration of 0.4 mM. The prepared mixtures were poured into a 96-well plate and incubated for 30 minutes in a dark place at room temperature. Finally, the absorbance of the incubated samples was measured at 517 nm (maximum absorption wavelength of DPPH) using a microplate reader.® regent was used. In the first step, the MCF-7 cells at the density of 5 × 104 cells per well were seeded into 6 well plate. After 48 hours of incubation, the cells were treated for 48 hours with IC50 concentrations of free pennyroyal, pennyroyal-NLCs, and pennyroyal-NLCs/MTX. For RNA isolation, the treated cells were harvested and mixed with 1 mL of TRIzol solution and incubated for 20 minutes; next 200 μL of chloroform was added to the TRIzol cocktail and mixed gently. The prepared cocktail was incubated in refrigerators (The temperature was settled at -20 °C) for 20 minutes. For separating the TRIzol/chloroform cocktail layers, the cocktail was centrifuged at 12 000 RPM speed at 4–8 °C for 15 minutes. Finally, colorless upper-phase media was harvested, gently mixed with 500 μL of isopropyl alcohol, and incubated for 10 minutes. For depositing the extracted RNA, the prepared cocktail was centrifuged at 12 000 RPM for 10 minutes. Finally, the formed precipitate, for better drying efficacy, was washed three times with isopropyl alcohol and dried at room temperature. The isolated RNA was dissolved in 20 μL RNase free water, and the RNA concentration was measured by a Nano-Drop spectrophotometer . The complementary cDNA was generated by the QuantiTect Reverse Transcription cDNA synthesis kit . RT-PCR was done via the Power SYBR GREEN Master Mix . β-Actin was amplified as a reference gene. mRNA expression was measured with the 2− ΔΔCT method. 13 For evaluating the molecular mechanism of prepared nanoparticles against MCF-7 cells, the real-time PCR (RT-PCR) gene expression measurement was done. For RNA isolation from cell plates, the TRIzolt test were employed in the statistical analysis, including multiple comparisons between confession data using the Tukey honest noteworthy difference test . A significant P value of less than 0.05 was used, and the FlowJo software package was used to analyze the data obtained by flow cytometry . All the experiments were repeated three times in a row. The two-way analysis of variance (two-way ANOVA) and independent 29 The role of DOPE and DOTAP is to provide cationic charge for lipid nanoparticles,30 leading to the formation of MTX-loaded NLCs through electrostatic conjugation between MTX with a negative charge and nanoparticles with a positive charge.31 Therefore, only MTX was utilized for conjugation onto the surface of the nanoparticles because of cationic lipids, DOPE, and DOTAP. Also, it should be noted to fully benefit from targeting the performance of MTX; we tried to minimize the positive charge of the NLCs to reduce the positive effect of charge in nanoparticle penetration to the surface of negatively charged normal cells. The lipid phase was composed of Precirol, pennyroyal, DOTAP, DSPE-mPEG 2000, and DOPE. The presence of PEG on the surface of lipid nanovesicles extends the circulation lifetime of the vehicle in the body’s bloodstream.in vivo destiny of the prepared nanoparticles might be influenced by multiple physicochemical factors such as surface charge and size. Based on studies examining the effects of nanoparticle size on biological and pharmacokinetic properties, the optimal size of nanoparticles is in the range of 130 to 300 nm for drug delivery purposes.11 Nanoparticles within this size range are more durable in the circulatory system than free drugs since they can efficiently bypass the reticuloendothelial system. It should be noted that nanoparticles larger than 300 nm are eliminated by the reticuloendothelial system.11 The Precirol:DOPE:DOTAP:DSPE-mPEG 2000 was prepared a 10:1:1:0.1 weight ratio, and NLCs:MTX was prepared a 10:1 weight ratio. The optimal proportion of the phospholipids to the drug and the nanoparticles was determined based on our pilot study results. The nanoparticles were produced and described by determining size, the polydispersity index, zeta potential, EE, and drug loading. The  The DLS method was utilized to exhibit zeta and size alterations in different stages of the preparation of nanoparticles. It was also used to confirm the kind of conjugations. Based on the observations, pennyroyal was encapsulated into NLCs successfully, and the zeta potential and average size of the prepared NLCs were + 39.0 ± 6.9 mv and 97.4 ± 12.1 nm, respectively . Also, a We used TEM to examine the shell and core of the nanoparticles . As show50 values for free pennyroyal and pennyroyal-NLCs were 286.1 and 200.8 µg/mL, respectively  resulted in a considerable reduction in cell survivability compared to the control cells. There was a substantial difference between the cytotoxicity of pennyroyal-NLCs and pennyroyal-NLCs/MTX. The pennyroyal-NLCs/MTX showed better cytotoxic features on MCF-7 cells compared to free pennyroyal and pennyroyal-NLCs. However, pennyroyal-NLCs and pennyroyal-NLCs/MTX affected the survivability of MCF-7 cells differently. These results are consistent with the results obtained in the cellular uptake experiment, revealing the increased cellular uptake of MTX-loaded NLCs relative to NLCs on MCF-7 cells. Recent studies have demonstrated superior binding affinity to folate receptors because MTX decorates NLCs. As a result, MTX-loaded NLCs can recognize folate receptors and internalize them in cells through endocytosis with the mediation of receptors.13 Furthermore, NLCs can penetrate cells through nonspecific endocytosis.12 Therefore, it can be concluded that NLCs actively targeted by MTX attachment to folate receptors result in better cytotoxic function and better selectivity toward MCF-7 cells, which is consistent with a recent study.13 We used the MTT test to assess the cytotoxic effects of free MTX, free pennyroyal, NLCs, and NLCs-MTX on the survivability of the MCF-7 cells. The results showed that cell survivability was significantly reduced following 48 hours treatment of the MCF-7 cells. After 48 hours incubation of the MCF-7 cells, the ICectively . The reaP < 0.001)  dendrimers with MTX could significantly increase the cell entrance of HMGA-2 siRNA into MCF-7 cells. Also, their results showed that the surface modification of dendrimers with MTX could actively target the folate receptors of MCF-7 cells. This conclusion was reached when the cell uptake study on A549 cells showed no difference in the fluorescence intensity of non-targeted and MTX-targeted dendrimers.13 The cellular uptake of RhoB-NLCs and RhoB-NLCs/MTX has been assessed quantitatively through flow cytometric analysis on A549 cells (slight folate receptor expression) and MCF-7 cells (overexpressing folate receptors). The corresponding fluorescent intensity of RhoB was evaluated to quantify the number of NLCs exposed to internalization. The findings revealed that the two nanoparticles were internalized into MCF-7 cells successfully in a time-dependent fashion, and the uptake of NLCs/RhoB-MTX was significantly larger than that of RhoB-NLCs (< 0.001) . Cellula< 0.001) . The fin Apoptosis in the MCF-7 cells was quantitatively examined by staining them with annexin V/PI after incubation with free pennyroyal, pennyroyal-NLCs, and pennyroyal-NLCs/MTX for 48 hours. Here, a negative control group was considered to receive blank NLCs treatment. As shown in 12 Eventually, pennyroyal-NLCs targeting via MTX increased the 60.25% apoptosis rate as compared to non-targeted pennyroyal-NLCs (P < 0.05) and free forums of the drugs (P < 0.001). Apoptosis rates were also assessed qualitatively via the DAPI staining test to identify nuclear fragmentation within the processed MCF-7 cells  after 48 hours incubation with free pennyroyal, pennyroyal-NLCs, and pennyroyal-NLCs/MTX. In addition, a negative control group was considered to receive blank NLCs treatment. As shown in 50 values for free pennyroyal and pennyroyal-NLCs were 1.458 mg/mL and 1.136 mg/mL, respectively. In a study by Radbeh et al, the effect of encapsulation on the antioxidant activity of Cornus mas extract was investigated.34 Their findings demonstrated that the encapsulation of the herbal extract could decrease its antioxidant activity. They attributed this phenomenon to heating the extract during the preparation of nanoparticles.34 Also, our previously published research showed that heating could decompose a few amounts of heat-sensitive compounds.11 The DPPH assay was done to assess the effect of encapsulation on the antioxidant activity of pennyroyal. 35 Hence, they may not exhibit their biological effects, such as antioxidant activity, appropriately. pennyroyal encapsulation through NLCs can preserve antioxidants from biodegradation. The DPPH test was conducted to assess antioxidant activity in pennyroyal-NLCs and free pennyroyal throughout 60 days of storage. The results showed a free radical scavenging capacity in a time-dependent manner, but no considerable scavenging activity of NLCs was found  can elevate pennyroyal cellular permeability. As a result, it can reduce the IC All the authors of this article declare that they have no conflict of interest. In the dissemination of our research findings, we uphold the highest standards of publication ethics. This encompasses the transparent disclosure of any potential conflicts of interest and the rightful attribution of prior work and contributions from collaborators. Our paramount commitment revolves around the safety and well-being of researchers, study participants, and the environment. To uphold this commitment, all members of our laboratory team receive comprehensive training in secure laboratory procedures. We rigorously implement safety precautions when handling cell cultures, hazardous substances, or equipment.


MR has the advantages of allowing deep penetration with no radiation; however, its spatial resolution needs to be improved. SERS and MR have complementary imaging features for tumor marker detection. In this study, gold nanorod and ultrasmall iron oxide nanoparticle composites were developed as dual-modal bioprobes for SERS-MRI to detect PD-L1 expression. Anti-PD-L1 (aPD-L1) was utilized to improve the targeting ability and specificity of PD-L1 expression detection. TNBC cells expressing PD-L1 were accurately detected via the SERS imaging mode in vitro, which can image at the single-cell level. In addition, bioprobe accumulation in PD-L1 expression-related tumor-bearing mice was simply and dynamically monitored and analyzed in vivo using MR and SERS. To the best of our knowledge, this is the first time a SERS-MRI dual-modal bioprobe combined with a PD-L1 antibody has been successfully used to detect PD-L1 expression in TNBC. This work paves the way for the design of high-performance bioprobe-based contrast agents for the clinical immunotherapy of TNBC.The efficiency of immunotherapy for triple-negative breast cancer (TNBC) is relatively low due to the difficulty in accurately detecting immune checkpoints. The detection of TNBC-related programmed cell death ligand-1 (PD-L1) expression is important to guide immunotherapy and improve treatment efficiency. Surface-enhanced Raman spectroscopy (SERS) and magnetic resonance (MR) imaging exhibit great potential for early TNBC diagnosis. SERS, an optical imaging mode, has the advantages of high detection sensitivity, good spatial resolution, and “fingerprint” spectral characteristics; however, the shallow detection penetration of SERS bioprobes limits its application  It is noteworthy that ultrasmall IO NPs, with a size distribution of 1–10 nm, have both T1- and T2-enhanced capabilities.in vitro. However, the relatively shallow penetration of SERS technology is an inevitable weakness of in vivo biodetection and bioimaging.Magnetic resonance (MR) imaging, a nonradiative imaging technology, can offer abundant physiological and anatomic information about tumor tissue and is widely utilized for cancer diagnosis. Although gadolinium-based agents are common in vitro with detection sensitivity at the single-cell level. Moreover, PD-L1 expression in positive and negative cancer cells was accurately distinguished through the MR mode of GRm@IO composite bioprobes, and dynamic monitoring of bioprobe aggregation in tumor-bearing mice was realized in vivo via MR. To the best of our knowledge, this is the first time a GR@IO-based bioprobe combined with a PD-L1 antibody has been developed to qualitatively distinguish PD-L1 expression in breast cancer cells based on SERS-MRI dual-modal bioprobes in vitro and in vivo. This study will pave the way for the design of high-performance SERS-MRI bioprobes for tumor PD-L1 expression detection, guide TNBC clinical immunotherapy, and achieve tumor precision medicine.SERS and MRI modalities can complement tumor diagnostic modes and offer complementary bioimaging features with high sensitivity and deep penetration. The development of a SERS–MRI double-mode imaging bioprobe could be a promising method for accurately detecting TNBC-related PD-L1 expression. In this study, GRs with ultrasmall iron oxide (IO) composite bioprobes (GR@IO) were designed to integrate SERS and MR imaging properties, as detailed in Scheme μl, 27.5 μl, 32.5 μl (1 mol/l)]. Broad view transmission electron microscopy (TEM) images of the GR samples are shown in Fig. S1(a). UV-visible absorption spectra and SERS spectra results show that 4MBA molecules adsorbed on GRs (GRm), with the length-to-diameter ratio of 3.72 (GRs-3.72) exhibiting the best SERS enhancement under 785 nm laser illumination [Fig. S1(b) and S1(c)]. The high SERS activity of the GRs-3.72 sample can be explained by the fact that its absorption peak is closer to 785 nm than those of the other two samples. The TEM image of a single GRs-3.72 sample is shown in 2 in the GRs and polyacrylic acid (PAA) on the surfaces of the IO NPs. GRm@IO was prepared based on the amidation reaction between the –COOH of PAA and the -NH2 of SH-PEG-NH2,The design route for the GRm@IO based-bioprobe, which was synthesized via seed growth and nanoprecipitation, is shown in Scheme T1- and T2-weighted images of IO and GRm@IO@PDA-aPD-L1 with different Fe concentrations (0–0.2 μg/ml) are shown in T1 and T2 MR images, indicating they could be used as bimodal MR contrast agents in tumor imaging.To increase the biocompatibility of the GRm@IO composite in solution, a layer of polydopamine (PDA) was wrapped on the surface of the material. aPD-L1 was modified with GRm@IO@PDA to endow the GRm@IO-based SERS bioprobe with high targeting efficiency to PD-L1 expression-related tumor sites. The PDA layer on the GRm@IO composite was observed by TEM  and N/Au/Fe elemental mapping images were observed in the HAADF-STEM images shown in Fig. S4, which indicated the presence of a PDA layer on the SERS bioprobe. In addition, UV-vis absorption spectra  and dynaTo assess the capability of detecting PD-L1 expression with our SERS-MRI dual-modal bioprobes, human breast cancer cell lines of MDA-MB-231 and MCF-7, which have different PD-L1 expression levels, were selected as the research model.1T and 2T signals in TNBC cells than that for MCF-7 cells due to the high uptake and PD-L1 expression level in MDA-MB-231 cells. This is consistent with the results shown in T1 MR images of MDA-MB-231 cells incubated with GRm@IO@PDA-aPD-L1 for 8 h were brighter than those incubated for 24 h, which may due to the degradation of IO over time in these cells.Visual imaging modes based on GRm@IO@PDA-aPD-L1 bioprobes will play a vital role in accurately evaluating average PD-L1 expression in TNBC cells. SERS images of the GRm@IO-based bioprobes co-incubated with MDA-MB-231 and MCF-7 cells for 0, 8, and 24 h are shown in in vivo biocompatibility of GRm@IO@PDA-aPD-L1 was investigated in Institute of Cancer Research (ICR) mice. Fifteen days after tail vein injection of PBS or GRm@IO@PDA-aPD-L1, the blood and major organs of the mice were collected for hematology analysis and hematoxylin-eosin (H&E) staining to study organ pathology . GRm@IO@PDA-aPD-L1 administration had no influence on all indices examined in routine blood tests.in vivo.To further evaluate the potential of our biprobes for PD-L1 expression detection in tumors, the in vivo PD-L1 expression detection by SERS-MRI. To explore the optimum time for maximum bioprobe accumulation, the amount of bioprobes accumulated in TNBC tumor-bearing mice based on PD-L1 expression was directly and dynamically monitored using MRI, as shown in T1- and T2-weighted imaging .in vivo was evaluated in female ICR mice. Mice were divided into three groups and injected with 100 μl PBS or the same volume containing bioprobes with different Fe concentrations (0.1 or 0.2 μg/ml) via the tail vein. After 14 days, mice were killed after cervical dislocation under anesthesia, and blood and major organs were collected for hematological analysis and hematoxylin-eosin (H&E) section staining.The biocompatibility of bioprobes 7 MDA-MB-231 tumor cells on the front of the right leg. After two weeks, the tumor volume was increased to 100 mm3 for subsequent MRI and SERS detection. SERS and MRI experiments were performed on tumor-bearing mice using a LabRAM Odyssey spectrometer (Horiba Scientific) with Labspec 6 software and a MAGNETOM Vida syngo MR XA10 MRI scanner . All mice were then injected with 100 μl of 0.2 μg/ml GRm@IO@PDA-aPD-L1, and the accumulation was observed using MR equipment. MRI images were acquired at different time points. The MRI field of view was selected according to the breast cancer type. According to the MRI results, the SERS spectra of in vivo tumors were acquired 10 h after tail vein injection using LabRAM microscopy.BALB/c-nu mice were subcutaneously implanted with 1 × 10μl of 0.2 μg/ml GRm@IO@PDA-808-aPD-L1 bioprobes and IR 808 via tail vein, followed by real-time monitoring. After IR-808 labeling, GRm@IO@PDA-808-aPD-L1 exhibited fluorescence with excitation at 740 nm and emission at 790 nm.The biodistribution of bioprobes was observed using fluorescence imaging equipment . Tumor-bearing mice were injected with 100 All data were presented as mean ± standard deviation for at least three independent experiments. The measurement data were analyzed statistically using Excel.See the supplementary material for the following details: TEM images of GRm with different aspect ratios and spectra of GRm with different aspect ratios ; zeta potential of the GRm, GRm@IO, GRm@IO@PDA, and GRm@IO@PDA-aPD-L1 ; HRTEM images of GRm@IO@PDA ; HADDF and elemental mapping images for N, Au, and Fe in GRm@IO@PDA ; particle sizes of GRm, GRm@IO, GRm@IO@PDA, and GRm@IO@PDA-aPD-L1 ; CLSM images of GRm@IO@PDA-aPD-L1–488 ; cell viabilities of MCF-7 cells incubated with bioprobes ; CLSM images of MCF-7 cells with Alexa Fluor 488 marked bioprobes ; flowcytometry results of the expression level of PD-L1 in MDA-MB-231cells and MCF-7 cells, and GRm@IO@PDA-aPD-L1–488 bioprobes incubated with MDA-MB-231cells and MCF-7 cells for 24 h ; SERS mapping and MRI images of MCF-7 cells incubated with GRm@IO@PDA-aPD-L1 ; routine analysis of the WBC, RBC, HCT, MCV, HGB, PLT, MCH, and MCHC after injecting bioprobes ; HE results from the lung, liver, spleen, kidney, and heart of ICR white nude mice after injecting the bioprobes ; MRI images of MDA-MB-231 cells bearing mice injected GRm@IO@PDA at different time points ; and fluorescence imaging of MDA-MB-231 tumor in BALB/c female nude mouse at different timepoints .