A polarized-macrophages-based drug delivery system (M1/SLNP) was presented for the cell-chemotherapy of cancer.Polarized-macrophages were used both as therapeutic tool to provide immunotherapy and as delivery vessel to target deliver chemotherapeutic drugs to tumor tissues for chemotherapy simultaneously.M1/SLNP was a multifunctional delivery system with simple structure, excellent safety, and without complex synthesis process.The online version of this article (10.1007/s40820-020-00531-0) contains supplementary material, which is available to authorized users. p < 0.01), which proved M1/SLNP could enhance tumor targeting of SF. An increased ratio of M1-type macrophages to M2-type macrophages, and the CD3+CD4+ T cells and CD3+CD8+ T cell quantities in tumor tissues after treatment with M1/SLNP indicated M1/SLNP could relieve the immunosuppressive tumor microenvironments. The tumor volumes in the M1/SLNP group were significantly smaller than those in the SLNP group (p < 0.01), indicating M1/SLNP exhibited enhanced antitumor efficacy. Consequently, M1/SLNP showed great potential as a novel cell-chemotherapeutic strategy combining both cell therapy and targeting chemotherapy.Cell therapy is a promising strategy for cancer therapy. However, its therapeutic efficiency remains limited due to the complex and immunosuppressive nature of tumor microenvironments. In this study, the “cell-chemotherapy” strategy was presented to enhance antitumor efficacy. M1-type macrophages, which are therapeutic immune cells with both of immunotherapeutic ability and targeting ability, carried sorafenib (SF)-loaded lipid nanoparticles (M1/SLNPs) were developed. M1-type macrophages were used both as therapeutic tool to provide immunotherapy and as delivery vessel to target deliver SF to tumor tissues for chemotherapy simultaneously. M1-type macrophages were obtained by polarizing macrophages using lipopolysaccharide, and M1/SLNPs were obtained by incubating M1-type macrophages with SLNP. Tumor accumulation of M1/SLNP was increased compared with SLNP (The online version of this article (10.1007/s40820-020-00531-0) contains supplementary material, which is available to authorized users. Cell therapy has emerged as a novel immunotherapeutic approach for cancer treatment, by directly transporting therapeutic immune cells, such as T cells, NK cells, and macrophages, to eliminate cancer cells –3. CompaHerein, we hypothesized a “cell-chemotherapy” strategy using drug-loaded therapeutic cells to enhance the antitumor efficacy of therapeutic cells. On the one hand, the cells were used as a therapeutic tool to kill cancer cells or provide immunotherapy; on the other hand, the cell was used as a delivery vessel to target drugs to tumor tissues and achieved chemotherapy. This strategy showed unique synergistic advantages: Therapeutic cells could trigger the antitumor immune response and then kill tumor cells. Also, therapeutic cells could enhance the tumor targeting of chemotherapeutic drugs; chemotherapeutic drugs could directly kill tumor cells and improve the sensitivity of tumor cells to cell therapy.Macrophages are the major tumor-infiltrating immune cells population with a critical role in regulating tumor progression, induced by the tumor microenvironments to differentiate into M1-type macrophages (M1) and M2-type macrophages (M2) –15. M1-tThe method for loading chemotherapeutic drugs into macrophages is another concern in cell-chemotherapy. The ideal method needs to have high drug-loading and appropriate drug-release profiles with low toxicity for macrophages, and does not affect the functions of macrophages or drugs . NanoparIn this study, a polarized macrophage-based treatment and drug delivery system involving M1-type macrophages carrying sorafenib (SF)-loaded lipid nanoparticles (M1/SLNP) were designed for the cell-chemotherapy of hepatocellular carcinoma (HCC). SF is a multityrosine kinase inhibitor that blocks tumor cell proliferation and the first-line drug approved by the FDA in 2007 for the treatment of HCC –42. SF cIn this study, M1/SLNP was prepared successfully. The morphology and particle size of SLNP were investigated. The phenotype, release properties, and deep tumor-penetrating ability of M1/SLNP were studied. In addition, the tumor targeting ability of M1/SLNP was proved in vitro and in vivo. Moreover, the relieved immunosuppressive tumor microenvironments were evaluated by in vivo immunization study. The antitumor efficacy was evaluated in vitro and in vivo. This study provides a potential approach of novel macrophage-based therapy strategy with enhanced antitumor activity.SF was provided by Shanghai Biochempartner Co., Ltd. . Injectable soya lecithin was provided by Shanghai Taiwan Pharmaceutical Co., Ltd. . Coumarin-6 (C6) was bought from Aladdin Chemical Co., Ltd. . DSPE-rhodamine B was purchased from Ruixi Biological Technology Co., Ltd . Methylthiazol tetrazolium (MTT) were purchased from Sigma-Aldrich (US). APC anti-mouse CD3, FITC anti-mouse CD4, PE anti-mouse CD8a, PE anti-mouse CD25, and Alexa Fluor® 647 anti-mouse FOXP3 were purchased from eBioscience. Alexa Fluor ® 488 anti-mouse CD86, PerCP/Cy5.5 anti-mouse F4/80, and APC anti-mouse CD206 were bought from eBioscience. Mouse IL-12p70 Elisa kit, Mouse TGF-β1 Elisa kit, Mouse IL-10 Elisa kit, and Mouse TNF-α ELISA kit were purchased from DAKEWE. All other reagents were of analytical grade and obtained commercially.2.Macrophages  and Hepa1-6 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. All cells were cultured in a 37 °C incubator with 5% COFemale C57BL/6 mice (6–8 weeks old) and female Kunming mice (6–8 weeks old) were provided by the Medical Animal Test Center of Shandong University . All experiments complied with the requirements of the Animal Management Rules of PRC  and of the Laboratory Animal Ethical and Welfare Committee of Cheeloo College of Medicine, Shandong University.−1 and Cy5.5 at a concentration of 500 μg mL−1. The other procedures were similar to that for SLNP.SLNPs were prepared by nanoprecipitation methods. SF was dissolved in 1 mL methyl alcohol. Soya lecithin was dissolved in Tween-80 aqueous solution . The organic phase was added to Tween-80 aqueous solution under constant mechanical agitation using a microsyringe pump . SLNPs were obtained after methyl alcohol evaporation. The optimal formulations were determined by single factor studies. SF/soya lecithin mass ratio and the soya lecithin concentration were investigated. For the preparation of C6-LNP and Cy5.5-LNP, SF was replaced with C6 at a concentration of 600 μg mLWdrug was the drug weight, and Wsoya lecithin was the soya lecithin weight.The morphology of SLNP was characterized by transmission electronic microscopy (TEM). The particle sizes and polydispersity index (PDI) of SLNP were determined by a Zetasizer Nano ZS90 . SF was quantitatively analyzed using high-performance liquid chromatography (HPLC) . Equation (−1), SF solution  were added into dialysis bags (8 to 14 kDa molecular weight cutoff), respectively. The release media for SLNP and SF solution were pH 7.4 phosphate-buffered saline (PBS) included Tween 80  and pH 6.5 PBS included Tween 80 , respectively. The bags were incubated in 10-mL release medium at 37 °C under horizontal shaking. At the predetermined time points, the release medium was obtained and replaced with fresh medium. The released SF was quantitatively analyzed by HPLC. The experiments were carried out in triplicate.In vitro release of SF from SLNP was conducted by the dialysis bag diffusion method. 1 mL of SLNP (15 μg mL−1 for 24 h. Macrophages and M1-type macrophages (1 × 106 cells mL−1) were seeded in a sterile tube and incubated with SLNP (200 µg mL−1) for 2 h at 37 °C. Cells are centrifuged to separate from the SLNP solution and then resuspended in PBS to obtain the M/SLNP and M1/SLNP suspension. To get the total amount of drug loading in cells, SF in SLNP before and after incubating with cells was quantitatively measured using HPLC. The optimal formulations for M/SLNP were determined by single factor study. The concentration of SF and incubation time were investigated. The cytotoxicity of SLNP on macrophages was tested by MTT assay. Macrophages were seeded into 96-well plates (5000/well). A series of doses of SLNP at a SF concentration of 50, 100, 200, 300, and 400 μg mL−1 were added to the wells and incubated for 1, 2, and 4 h. Then, SLNPs were removed, and DMEM medium supplemented with 10% fetal bovine serum was added to each well. After incubating for 48 h, MTT and DMSO was added. The cell viability was measured by a microplate reader .Macrophages carrying SLNP (M/SLNP) and M1/SLNP were obtained by incubating macrophages and M1-type macrophages with SLNP, respectively. M1-type macrophages were obtained by incubating macrophages with LPS at a concentration of 1 µg mL−1) for 2 h at 37 °C. The subsequent procedures were similar to that for M/SLNP. The optimal formulation for M/SF was similar to that for M/SLNP.For preparation of macrophages carrying SF (M/SF), macrophages were incubated with SF solution (200 µg mL5 cells) were cultured overnight in a 12-well plate. M1-type macrophages were obtained by incubating macrophages with LPS at a concentration of 1 µg mL−1 for 24 h. Subsequently, SLNPs were added and incubated with M1-type macrophages for 2 h to obtain M1/SLNP. M1/SLNPs were marked with PerCP/Cy5.5 anti-mouse F4/80, Alexa Fluor® 488 anti-mouse CD86 and APC anti-mouse CD206, and analyzed by FCM. F4/80+CD86+ cells were M1-type macrophages, and F4/80+CD206+ cells were M2-type macrophages. In addition, M1/SLNPs were incubated for 24 h. The level of cytokines in the supernatant secreted from M1-type macrophages was analyzed using the ELISA kit.The in vitro phenotype of macrophages was evaluated by flow cytometry (FCM) assay. Macrophages (2 × 10−1) for 2 h at 37 °C to obtain macrophages carrying C6-loaded lipid nanoparticles (M/C6-LNPs) and M1-type macrophages carrying C6-loaded lipid nanoparticles (M1/C6-LNPs), respectively. M/C6-LNP and M1/C6-LNP were stained with Alexa Fluor ® 647 anti-mouse F4/80 by incubating with Alexa Fluor ® 647 anti-mouse F4/80 (1.5 μg mL−1) for 1 h. After washing with PBS thrice, the double-stained F4/80-M/ C6-LNP and F4/80-M1/ C6-LNP were observed by CLSM. M/SLNP and M1/SLNP were observed using TEM.Confocal laser scanning microscopy (CLSM) and TEM were used to confirm that the SLNP had been successfully loaded into macrophages and M1-type macrophages. Briefly, macrophages and M1-type macrophages were incubated with C6-LNP (20 µg mL5 cells/well) were seeded into 12-well plates and pre-incubated with cytochalasin D (30 mM), genistein (1 μg mL−1), or chlorpromazine (10 μg mL−1). Then, the cells were incubated with C6 or C6-LNP for 1 h and evaluated using FCM.The endocytic pathway of SLNP in macrophages was investigated by FCM. Macrophages (2 × 10 −1 for 24 h. Macrophages or M1-type macrophages were incubated with SLNP (200 µg mL−1) for 2 h at 37 °C and then incubated with fresh DMEM medium supplemented with 10% fetal bovine serum for different periods . At predetermined time points, the released medium from each well was obtained and the amount of the total released SF in the released medium was determined using HPLC. To clarify that SF would be released from M/SLNP or M1/SLNP as SF or SLNP, the released medium was added into a centrifugal filter device (10 K MWCO), followed by centrifugation . The released SF in the filtrate was quantitatively analyzed using HPLC. The amount of the released SLNP (At − Af) was calculated, where At and Af are the amount of the total released SF in the released medium and the released SF in the filtrate, respectively. In addition, the released medium after 24 h was taken and observed using TEM.To evaluate the in vitro release properties of SF from M/SLNP or M1/SLNP, macrophages were cultured overnight in 12-well plates. M1-type macrophages were obtained by incubating macrophages with LPS at a concentration of 1 µg mL−1) for 24 h. C6-LNPs were added and incubated with macrophages or M1-type macrophages for 2 h. After washing by PBS, the cells were incubated with fresh DMEM medium supplemented with 10% fetal bovine serum for different periods . At predetermined time points, the cells were washed with PBS and observed under CLSM. In addition, the cells at 24 h were obtained and visualized under TEM.CLSM and TEM were used to evaluate the stability of SLNP in M/SLNP or M1/SLNP. Briefly, DSPE-rhodamine B and soya lecithin were dissolved in Tween-80 aqueous solution , and the other procedures were similar to that for C6-LNP. Thus, C6-LNP were obtained, in which LNP were fluorescently labeled by rhodamine B. Macrophages were cultured overnight in 12-well plates. M1-type macrophages were obtained by incubating macrophages with LPS (1 µg mL−1) for 24 h. C6-LNPs were added and incubated with macrophages or M1-type macrophages for 2 h to obtain M/C6-LNP and M1/C6-LNP, respectively. The cells were washed with PBS and then incubated with fresh DMEM medium supplemented with 10% fetal bovine serum for 24 h to obtain the released medium. Subsequently, the tumor spheroids were incubated with free C6, C6-LNP, the released medium from M/C6-LNP or the released medium from M1/C6-LNP for 6 h. The tumor spheroids were washed with PBS. The tumor spheroid images were acquired by CLSM.Each well of the 96-well plates was pre-coated with fetal bovine serum-free medium containing sterile agarose. Hepa1-6 cells (5000 cells/well) were seeded into each well and cultured in the medium containing FBS . The tumor spheroids were allowed to grow at 37 °C for 7 days. Macrophages were cultured overnight in 12-well plates. M1-type macrophages were obtained by incubating macrophages with LPS . M/SLNP and M1/SLNP were suspended in DMEM medium and plated in the upper chamber of the Transwell. The lower compartment was filled with fresh DMEM medium or Hepa1-6 conditioned media collected from DMEM medium after culturing Hepa1-6 cells for 48 h. After incubating for 6 h at 37 °C, the cells migrating across the Transwell in the lower chamber were detected under a fluorescence microscope .The chemotaxis of M/SLNP and M1/SLNP was investigated using a Transwell migration assay in vitro  for 2 h at 37 °C to obtain M/C6-SLNP and M1/C6-LNP, respectively. Then, DiI staining macrophages and M1-type macrophages were incubated with M/C6-LNP and M1/C6-LNP at 37 °C for 2 h to obtain DiI-M/C6-LNP and DiI-M1/C6-LNP. The Hepa1-6 tumor-bearing C57BL/6 mice were used as animal models, which were established by inoculating subcutaneously 1 × 107 Hepa1-6 cells at the right axillary. When the tumors grew to approximately 200 mm3, the mice were intravenously injected with DiI-M/C6-LNP or DiI-M1/C6-LNP (5 × 106 cells/mouse). The mice were sacrificed after 24 h, and the tumors were collected and cryo-sectioned at a thickness of 10 μm. The sections were imaged by CLSM.To investigate the tumor targeting capabilities of M/SLNP and M1/SLNP, macrophages and M1-type macrophages were incubated with C6-LNP (20 µg mL−1) at 37 °C for 2 h to macrophages carrying Cy5.5-loaded lipid nanoparticles (M/Cy5.5-LNP) and M1-type macrophages carrying Cy5.5-loaded lipid nanoparticles (M1/Cy5.5-LNP), respectively. Considering that the black hair of C57BL/6 mice might affect the fluorescence signal, the Hepa1-6 tumor-bearing Kunming mice were used as animal models, which were established by inoculating subcutaneously 1 × 107 Hepa1-6 cells at the right axillary. When the tumors grew to approximately 200 mm3, the mice were intravenously injected with free Cy5.5, Cy5.5-LNP, M/Cy5.5-LNP, or M1/Cy5.5-LNP (1 mg kg−1). The mice were anesthetized after 1, 2, 4, 8, 12, and 24 h of injection and observed with the in vivo real-time fluorescence imaging system (IVIS) spectrum . For further ex vivo evaluation, the mice were sacrificed at 12 or 24 h, and tumors or organs were obtained. In addition, to investigate the tumor-penetrating ability in vivo, the tumors were obtained and cryo-sectioned at a thickness of 10 μm, and the tumor sections were labeled with DAPI.The in vivo biodistribution of M/SLNP and M1/SLNP was investigated. Macrophages and M1-type macrophages were incubated with Cy5.5-LNP (30 µg mL5 cells/well) were seeded into 12-well plates. Free C6, C6-LNP, the released medium from M/C6-LNP or the released medium from M1/C6-LNP (100 ng mL−1) were added and further incubated for 1 or 2 h. After washing with PBS, the cells were imaged by fluorescence microscope. In addition, to quantifying the cellular uptake, cells treated with free C6, C6-LNP, the released medium from M/C6-LNP and the released medium from M1/C6-LNP were collected and measured using FCM. The preparation of the release medium from M/C6-LNP and the release medium from M1/C6-LNP were the same as described in the “Tumor-penetrating Ability in vitro” part.Hepa1-6 cells (1 × 10−1) were added and incubated for 48 h. LNP was added at concentration equal to SF in SLNP. Released medium of macrophages (released medium of M) and released medium of M/LNP (released medium of M/LNP) were added at concentration equal to SF released from M/SLNP. Released medium of M1-type macrophages (released medium of M1) and released medium of M1/LNP (released medium of M1/LNP) were added at concentration equal to SF released from M1/SLNP. After incubating for 48 h, MTT and DMSO were added. The cell viability was measured by a microplate reader. The following formula (−1) for 24 h. Macrophages or M1-type macrophages were incubated with SLNP (200 µg mL−1) for 2 h at 37 °C. After washing by PBS, the cells were incubated with fresh DMEM medium supplemented with 10% fetal bovine serum for 24 h. Released medium of M/SLNP and released medium of M1/SLNP were obtained in the supernatant. The preparation of released medium of M and released medium of M/LNP was similar to that of released medium of M/SLNP. The preparation of released medium of M1 and released medium of M1/LNP was similar to that of released medium of M1/SLNP.The cytotoxicity of M1/SLNP in Hepa1-6 cells was investigated by MTT assay. Briefly, Hepa1-6 cells (5000 cells/well) were incubated into 96-well plates. LNP, free SF, SLNP, released medium of M/LNP, released medium of M1/LNP, released medium of M/SLNP, and release medium of M1/SLNP . The mice were intravenously injected with NS, M, M1, free SF, SLNP, M/SLNP, and M1/SLNP  every 4 days for five times. The tumor volume and body weight were measured every other day. At day 19 after the first administration, the mice were sacrificed and tumors were excised, photographed and weighed. The tumor inhibition rate was calculated for different groups, and the following formula .The antitumor efficacy of M1/SLNP was evaluated using the Hepa1-6 tumor-bearing C57BL/6 mice model. The mice were randomly separated into seven groups  and tumors were obtained and then fixed in 4% paraformaldehyde and embedded in paraffin wax for histological analysis. The sections were stained with hematoxylin and eosin (H&E). In addition, the tumor sections were stained with Ki67 to evaluate the cell proliferation.+ cells were the total macrophages; and F4/80+CD86+ cells were M1-type macrophages; and F4/80+CD206+ cells were M2-type macrophages. The following formula  for per mg of tumor in the tumor tissues for different groups:Atotal represents the total number of cells in tumor tissues; PF4/80 represents the percentage of total macrophages (F4/80+ cells) in total cells in tumor tissues; Tweight represents the tumor weights.The in vivo macrophages phenotype was evaluated by FCM. After the in vivo antitumor efficacy study (on day 19 after the first administration), tumor tissues were obtained, and then, tissues were ground, filtered by a copper network. Following centrifugation , the total cells were collected and counted, staining with PerCP/Cy5.5 anti-mouse F4/80, Alexa Fluor® 488 anti-mouse CD86 and APC anti-mouse CD206. Subsequently, the total cells were analyzed by FCM. F4/80After the in vivo antitumor efficacy study (on day 19 after the first administration), the blood serum of the mice was obtained, and the levels of immunogenic cytokines (TNF-α and IL-12) and immunosuppressed cytokines (IL-10 and TGF-β) in serum were measure by the ELISA kit. The levels of cytokines in blood serum were also measured at 48 h post the first administration by the ELISA kit.+CD4+ T cells, CD3+CD8+ T cells and Treg in the tumors was evaluated by FCM. After the in vivo antitumor efficacy study (on day 19 after the first administration), tumor tissues were obtained, and then, tissues were ground, filtered by a copper network. After gradient centrifugated by Percoll, the cells were collected and counted, and stained with corresponding antibody markers for 1 h at 4 °C in dark. Then, the cells were analyzed by FCM. CD3+CD4+ T cells were marked with APC anti-mouse CD3 and FITC anti-mouse CD4; CD3+CD8+ T cells were marked with APC anti-mouse CD3 and PE anti-mouse CD8a, and Treg was marked with FITC anti-mouse CD4, PE anti-mouse CD25 and Alexa Fluor® 647 anti-mouse FOXP3. CD3+ T cells represent the total T cells in tumor tissues. The following formula  for per mg of tumor in the tumor tissues for different groups:Btotal represents the number of cells after centrifugation in tumor tissues; PCD3 represents the percentage of total T cells (CD3+ T cells) in tumor tissues; Tweight represents the tumor weights.The percentage of CD3+ CD4+ T cells, CD3+ CD8+ T cells, and Treg in the tumors were evaluated on day 4 post the first administration, and the method was similar to the evaluation after the in vivo antitumor efficacy study.The macrophages phenotype, the percentage of CD36 cells equivalent for the number of cells in antitumor efficacy study), respectively, and the method to obtain M1-type macrophages is shown in “2.7 Preparation of M/SLNP and M1/SLNP” part. The mice were observed and photographed at 24 h.The C57BL/6 mice and Kunming mice were injected intradermally with 0.1 mL saline, macrophages, and M1-type macrophages  and bovine serum albumin  every other day for four time. The method to obtain M1-type macrophages is shown in “2.7 Preparation of M/SLNP and M1/SLNP” part. The sensitized serum of the mice in each group was collected and was intradermally injected into the back of the mice, respectively. After 24 h, the mice injected with sensitized serum were intravenously injected with saline (negative control group), macrophages and M1-type macrophages  and bovine serum albumin , respectively. After 30 min, the skin on the back of the mice in each group was obtained and photographed.Passive cutaneous anaphylaxis test was carried out. The C57BL/6 mice and Kunming mice were randomly divided into the negative control group, macrophages group and M1-type macrophages group and positive control group, respectively. Mice in each group were sensitized by intravenously injected with saline (negative control group), macrophages, and M1-type macrophages (3 × 10p < 0.05. All results were reported as the mean ± standard deviation (SD).The Student’s t-test was used to analyze the statistical comparisons between two groups, and differences were considered to be statistically significant when −1 were determined as the optimal SF/soya lecithin mass ratio and soya lecithin concentration, respectively. SLNPs were successfully prepared with smaller particle size of 67.63 ± 5.02 nm, higher DL% of 5.58 ± 0.41% and PDI of 0.159 ± 0.018  of M/SF, M/SLNP, and M1/SLNP was 24.46 ± 1.33, 37.43 ± 0.53, and 38.18 ± 0.80 μg/106 cells, respectively  and M/SLNP group   and M/SLNP group   and M/SLNP group . The levels of TNF α were increased in M1 and M1/SLNP groups compared with that in M group  and M/SLNP group . The levels of cytokines were comparable in M1 group and M1/SLNP group, suggesting that the loaded SLNP did not affect the levels of cytokines secreted by M1-type macrophages. Collectively, these results indicated that macrophages were successfully polarized toward M1-type macrophages, and they could secrete cytokines including IL12 and TNFα, providing the theoretical basis for using M1-type macrophages as the therapeutic tool to exert immunotherapeutic antitumor efficacy.To better evaluate the macrophages phenotype and the impact of the loaded SLNP on macrophages phenotype, the proportions of M1-type macrophages and M2-type macrophages, and the ratio of M1-type macrophages to M2-type macrophages (M1/M2) were employed by FCM assay , 34.27% (p < 0.01), and 28.14% (p < 0.01) after treatment with chlorpromazine, genistein, and cytochalasin D, respectively, indicating that clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis were all involved in the internalization process of free C6 in macrophages. For C6-LNP, cellular uptakes in macrophages were inhibited by 54.30% (p < 0.01) and 27.78% (p < 0.01) after treatment with chlorpromazine and genistein, respectively, indicating that the internalization process of C6-LNP in macrophages involved both clathrin-mediated endocytosis and caveolae-mediated endocytosis. Collectively, these results showed that the endocytic pathway of free C6 and C6-LNP was different in macrophages.To investigate the endocytic pathway of free SF and SLNP in macrophages, macrophages were pre-incubated with chlorpromazine, genistein, and cytochalasin D, respectively, before loading them with either free C6 or C6-LNP Fig. lm. The cThe release profiles of SF from SF solution and SLNP are shown in Fig. S3a. SF was sustainably released from SF solution and SLNP in 72 h, respectively. The cumulative release of SF from SF solution at 72 h was 90.2% and 89.5% in pH 6.5 and pH 7.4, respectively. The cumulative release of SF from SLNP at 72 h was 55.8% and 56.8% in pH 6.5 and pH 7.4, respectively.The release profiles of total SF from M/SLNP and M1/SLNP are shown in Fig. The stability of C6-LNP in M1/C6-LNP was evaluated by fluorescence co-localization experiments Fig. e. LipidsWe explored the deep tumor-penetrating ability of SLNP, SLNP released from M/SLNP, and SLNP released from M1/SLNP, respectively, in vitro by constructing a three-dimensional (3D) multicellular tumor spheroid model Fig. g. LNPs wThe in vitro cellular uptake studies for free C6, C6-LNP, C6-LNP released from M/C6-LNP, and C6-LNP released from M1/C6-LNP were studied on Hepa1-6 cells by fluorescence microscopy Fig. a and FCMThe migration ability of M, M1, M/SLNP, and M1/SLNP toward Hepa1-6 cells in vitro is displayed in Fig. p < 0.01, p < 0.01, respectively) and Cy5.5-LNP group . As shown in Fig. p < 0.05, p < 0.01, respectively) and Cy5.5-LNP group . These data indicated that M/Cy5.5-LNP and M1/Cy5.5-LNP could enhance the tumor targeting delivery. As shown in Table S3, the tumor targeting efficiency of M/Cy5.5-LNP group and M1/ Cy5.5-LNP group at 12 h was significantly enhanced compared with free Cy5.5 group  and Cy5.5-LNP group . As shown in Table p < 0.01, p < 0.05, respectively) and Cy5.5-LNP group . The tumor targeting efficiency between M/Cy5.5-LNP group and M1/ Cy5.5-LNP group in the tumor tissues did not have significant deference. These results indicated that M/Cy5.5-LNP and M1/Cy5.5-LNP could target more selectively the tumor compared with free Cy5.5 group and Cy5.5-LNP group, and the tumor targeting ability of M/Cy5.5-LNP and M1/Cy5.5-LNP was comparable.The real-time biodistribution and tumor targeting ability of M1/Cy5.5-LNP, M/Cy5.5-LNP, Cy5.5-LNP, and free Cy5.5 were evaluated in Hepa1-6 tumor-bearing mice, respectively. As shown in Fig. The tumor targeting ability of M/SLNP and M1/SLNP in vivo was further determined using CLSM images of the tumor tissue section after intravenous administration of DiI-M/C6-SLNP or DiI-M1/C6-SLNP for 24 h, respectively Fig. e. Macrop50) of free SF, SLNP, released medium of M1, released medium of M1/LNP, released medium of M/SLNP, and released medium of M1/SLNP was 7.62 ± 0.37, 4.62 ± 0.55, 11.74 ± 0.12, 12.33 ± 0.89, 5.13 ± 0.52, and 2.40 ± 0.23 μg mL−1, respectively (Table 50 of released medium of M1/SLNP was significantly lower compared with SLNP (p < 0.05) and released medium of M/SLNP (p < 0.01), suggesting that M1-type macrophages provided an advantage in improving the cytotoxicity of SLNP and enhancing antitumor efficacy in vitro.The antitumor efficacy was investigated by MTT assay in vitro Fig. a. LNP, rly Table . The IC5p < 0.001), SLNP group (p < 0.001), which could be contributed to the high tumor targeting efficacy of macrophages. M1 significantly inhibited tumor growth compared with M group (p < 0.01), and M1/SLNP showed better antitumor efficacy compared with M/SLNP (p < 0.001), due to the immunotherapeutic antitumor efficacy of M1-type macrophages as therapeutic tool. M1/SLNP significantly inhibited the tumor growth compared with SLNP group (p < 0.01), which exhibited the best antitumor efficiency, indicating that M1/SLNP improved the antitumor efficacy of SLNP. These results could be attributed to both the high tumor targeting efficacy and the immunotherapeutic antitumor efficacy of M1-type macrophages. The tumor growth inhibition rates of M, M1, free SF, SLNP, M/SLNP, and M1/SLNP group were 8.84%, 42.49%, 33.89%, 54.04%, 71.09%, and 85.02%, respectively. Excised tumors were photographed , suggesting the low systemic toxicity of M1/SLNP. The preliminary safety of the carrier was investigated by immunohistochemistry evaluation, dermal sensitivity test, and passive cutaneous anaphylaxis test. Pathological changes or inflammatory infiltrates were not observed in organ tissues , and the ratio of M1/M2 in M1/SLNP group was higher compared with M/SLNP group (p < 0.05)  Fig. , b. The p < 0.05, p < 0.05, respectively). The levels of IL-12 and TNF-α in M1/SLNP group were higher than those in SLNP group . The levels of IL-12 and TNF-α in M1/SLNP group were higher than those in M/SLNP group . Lower levels of IL-10 and TGF-β in M1 group were detected compared with M group . Lower levels of IL-10 and TGF-β in M1/SLNP group were detected compared with SLNP group . Lower levels of IL-10 and TGF-β in M1/SLNP group were detected compared with M/SLNP group . These findings indicated that the immunogenic cytokines increased and immunosuppressed cytokines decreased after the administration of M1-type macrophages. The levels of cytokines  in blood serum were also measured at 48 h post the first administration . Compared with M group, the percentage of Treg in M1 group was lower (p < 0.01). The percentage of CD3+CD4+ T cells and CD3+CD8+ T cells in the M1/SLNP group was higher than that in SLNP group . Compared with SLNP group, the percentage of Treg in M1/SLNP group was lower (p < 0.01). These results indicated that the percentage of CD3+CD4+ T cells and CD3+CD8+ T cells was increased and the percentage of Treg was reduced in M1 group and M1/SLNP group through the immunomodulation of M1-type macrophages. The total number of T cells for per mg of tumor in the tumor tissues for different groups after the in vivo antitumor efficacy study is shown in Fig. S9. Besides, the percentage of CD3+CD4+ T cells, CD3+CD8+ T cells, and Treg in the tumor tissues was analyzed on day 4 post the first administration . M1/SLNP showed a superior antitumor effect with obvious tumor suppression. Overall, M1-type macrophages-based treatment and drug delivery system might provide a new potential strategy for the development of cell therapy.In summary, we developed a M1-type macrophage-based treatment and drug delivery system which promoted the tumor targeting delivery and antitumor efficacy. M1-type macrophages as therapeutic tool displayed immunotherapeutic antitumor efficacy. Meanwhile, M1-type macrophages as drug delivery vessel exhibited tumor targeting ability. Importantly, we demonstrated that M1-type macrophages could significantly increase the accumulation of SF in tumor sites and enhance tumor targeting delivery Below is the link to the electronic supplementary material.


Escherichia coli (resistant and sensitive) and MDR Pseudomonas aeruginosa. The synthesized Cu-MOFs were characterized with DLS, FT-IR, powder X-ray diffraction, scanning electron microscope, and atomic force microscope. They were further investigated for their efficacy against selected bacterial strains. The synthesized ribose-coated Cu-MOFs were observed as spherical shape structure with the particle size of 562.84 ± 13.42 nm. CHL caused the increased inhibition of E. coli and MDR P. aeruginosa with significantly reduced MIC and MBIC values after being encapsulated in ribose-coated Cu-MOFs. The morphological analysis of the bacterial strains treated with ribose-coated CHL-Cu-MOFs showed the complete morphological distortion of both E. coli and MDR P. aeruginosa. Based on the results of the study, it can be suggested that ribose-coated Cu-MOFs may be an effective alternate candidate to overcome the MDR and provide new perspective for the treatment of MDR bacterial infections.The rise in bacterial resistance to currently used antibiotics is the main focus of medical researchers. Bacterial multidrug resistance (MDR) is a major threat to humans, as it is linked to greater rates of chronic disease and mortality. Hence, there is an urgent need for developing effective strategies to overcome the bacterial MDR. Metal–organic frameworks (MOFs) are a new class of porous crystalline materials made up of metal ions and organic ligands that can vary their pore size and structure to better encapsulate drug candidates. This study reports the synthesis of ribose-coated Cu-MOFs for enhanced bactericidal activity of chloramphenicol (CHL) against  Neisseria meningitides, Streptococcus pneumoniae, and Haemophilus influenza . Cu. Cu41]. uum oven .For the encapsulation of CHL in to the CuMOFs, an aqueous mixture of CHL (20 mg) and Cu-MOFs (10 mg) was stirred for 24 h at 200 G. Drug-loaded Cu-MOFs were separated by centrifugation at 10,000 G for 15 min. The drug loading efficiency (DLE) of Cu-MOFs were determined through UV-Vis spectrophotometer . Briefly, a specific amount of CHL-Cu-MOFs was dispersed in water and sonicated for 30 min. The mixture was then centrifuged at 10,000 G for 20 min. Supernatant was collected and analyzed for CHL. The drug was detected at 278 nm and %DLE was determined by using Equation (1) .(1)%DLE4 (0.5 mmol) was added to the reaction mixture and stirred for 3 days at 50 °C. Freshly prepared NaBH4 (0.5 mmol) were added to the reaction mixture after every 12 h.The CHL-Cu-MOFs were further subjected to ribose coating . Briefly3 kPa. The discs of each sample were scanned in a range of 400–4000 cm−1 using an IR spectrophotometer.FT-IR spectra of Cu-MOFs, CHL, CHL-Cu-MOFs, and R-CHL-Cu-MOFs were recorded using IR spectrophotometer . The samples were mixed with KBr and converted into self-supporting disc by applying a high pressure of 1.38 × 10The synthesized samples were diluted properly and subjected for size, PDI, and zeta potential determination through a dynamic light scattering (DLS) instrument  in triplicate at a scattering angle of 90° at 25 °C. Surface morphology of the prepared Cu-MOFs samples was studied using atomic force microscope .Purity and crystallinity of synthesized Cu-MOFs were determined by powder XRD. For the measurement of diffraction patterns, we used an X-ray diffraction instrument  from 5° to 60° (2θ) with Cu-Kα irradiation.In vitro dissolution study was carried out at pH 6.8 using dialysis bag of 12,000 kDa. The ribose-coated CHL-Cu-MOFs were dispersed in water and transferred in the activated dialysis membrane. The membrane was then placed in the release medium of pH 6.8 buffer and subjected for shaking at 100 G. At specific time intervals, 2 mL of dissolution medium was withdrawn to quantify the amount of released drug and refilled with fresh buffer. The samples were then studied on UV-vis spectrophotometer at 278 nm.E.coli ATCC 8739 (sensitive), E. coli ATCC 35,218 (resistant), and Pseudomonas aeruginosa ATCC 10145. Bacterial strains stock culture was kept on Tryptic soya agar  at 4 °C. The microbial strain was sub-cultured on a fresh appropriate agar plate for 24 h prior to antibacterial test. Inocula were prepared by transferring several single colonies of microbes to a sterile Mueller Hinton broth. The microbial cell suspension was mixed to homogeneity to give a final density of 5 × 105 cfu/mL and these were confirmed by viable counts. The infective dose of most microorganisms was 105 cfu/mL.Different strains of the test microorganism were selected for antibacterial assay which included Gram-negative bacterium, E. coli (sensitive/resistant) and P. aeruginosa strains at 5 × 105 cfu/mL. Different concentrations were prepared in Muller Hinton broth ranging from 2.5 to 250 µg of CHL, Cu-MOF, and CHL-Cu-MOFs. This wider range of concentrations was selected so the minimum possible MIC can be determined for any sample. Each concentration (200 µL) was added in triplicate wells and the plates were incubated for 24 h at 37 °C ± 0.5. After that, 50 µL of 3--2, 5-diphenyltetrazolium bromide MTT (0.2 mg/mL) was added in each well followed by incubation at 37 °C for 30 min. As a negative control, DMSO was used while bacterial cells as positive control. The absorbance was measured at 570 nm with a reference wavelength of 650 nm by adding DMSO on a spectrophotometer and the percentage reduction of the dye  was calculated while using values of optical density (O.D) by using Equation (2) [The minimum inhibitory concentration of prepared samples was determined by using tetrazolium microplate assay according to the previously reported method  and the tion (2) .(2)IC50E. coli (sensitive/resistant) and P. aeruginosa strains using the microtiter plate method. The compounds were diluted as mentioned above in a 96-well flat bottom plate . An inoculum containing 5 × 105 CFU ml−1 bacteria was inoculated in each well except the broth control. Plates were incubated overnight at 37 °C and followed by staining to allow the biofilm formation [w/v) crystal violet for 20 min. Stained plates were rewashed and retained crystal violet was dissolved in 30% (v/v) glacial acetic acid. Absorbance was then measured at 590 nm using microplate reader . Percent biofilm inhibition was calculated by using Equation (3):Antibiofilm activity of CHL, Cu-MOFs, and CHL-Cu-MOFs were determined against ormation . Plates E. coli and P. aeruginosa were harvested 37 °C in TSA for 24 h. A freshly cleaved mica slide was loaded with 10 μL of poly-lysine and allowed to dry at room temperature. The slides were then loaded with a drop of diluted cultures of bacterial strain (105 cfu), dried, and analyzed for morphological investigations using AFM. Similarly, the cells incubated with test samples were used for the comparative morphological study. Samples (5–10 μL) at their MIC were withdrawn from microtiter plate and applied to poly-lysine loaded mica slides, dried at room temperature. The slides were then analyzed on AFM for morphological investigations.Morphological changes were studied by AFM and SEM analysis. Selected resistant and sensitive strains of SEM investigation was carried out by incubating the samples at 37 °C for 1 h by using their respective MBIC values. The cells were then fixed for 2 h with glutaraldehyde solution (2%) at 4 °C and dehydrated in a grade series of alcohol. The samples were then coated by gold coater and observed with SEM.All the experiments were carried out in triplicate form and results were expressed as mean ± SEM.−1 (OH and NH stretching). C=O stretching appeared at 1681 cm−1 while C=C stretching observed at 1558 cm−1. Furthermore, the prominent NO2 vibration appeared at 1518 cm−1 with C-Cl stretching at 665 cm−1 ) was observed as 20.44 ± 2.4 and 24.86 ± 2.18 µg/mL, respectively, while for the resistant E. coli (E. coli (R) strain, it was 33.46 ± 3.16 and 44.98 ± 2.86 µg/mL, respectively. The MIC value was decreased (5.96 ± 2.80 µg/mL) for CHL-loaded CHL-Cu-MOFs against E. coli (S), while it was 10.24 ± 1.64 µg/mL against the E. coli (R) strain. The MIC was further decreased to 4.20 ±1.82 µg/mL for R-CHL-Cu-MOFs against E. coli (S) while it was 8.60 ± 3.22 µg/mL for E. coli (R) strains. In the case of P. aeruginosa, CHL showed an MIC value around 20.16 ± 2.22 µg/mL, while the other control, CuSO4, exhibited 39.80 ± 23.22 µg/mL. The MIC value was decreased for CHL-Cu-MOFs and R-CHL-Cu-MOFs which is equivalent to 10.54 ± 1.65 and 7.46 ± 2.17 µg/mL, respectively  and E. coli (R) cells were found to be 48.12 ± 2.14 and 51.86 ± 1.98 µg/mL, respectively. CuSO4 showed comparatively higher IC50 against E. coli (S) and E. coli (R) which is equivalent to 53.12 ± 3.34 and 59.22 ± 2.84 µg/mL, respectively, while the IC50 value of Cu-MOFs was observed as 50.22 ± 1.43 µg/mL against both the strains. CHL-Cu-MOFs showed a decreased IC50 value up to 41.21 ± 2.86 µg/mL while it was further reduced to 39.0 ± 3.10 µg/mL against the sensitive strain after ribose coating. For E. coli (R), CHL-Cu-MOFs showed a decreased IC50 value of 45.22 ± 2.98 µg/mL while it was further decreased to 43 µg/mL upon coating with ribose. IC50 of CHL and Cu-MOFs against P. aeruginosa was found to be 75.04 ± 1.24 and 80.46 ± 3.14 µg/mL, respectively, while CuSO4 exhibited the IC50 92.42 ± 2.14 µg/mL. CHL upon encapsulation in Cu-MOFs significantly reduced the IC50 up until 50.06 ± 2.18 µg/mL which was subsequently decreased to 44 µg/mL after being coated with ribose. Results revealed that ribose coating on CHL-Cu-MOFs significantly reduced the IC50 value of CHL against all tested bacterial strains.Similarly, ICE. coli (S) strain was observed as rods  strains are provided in E. coli (R) control strain  was reduced from 10.16 ± 3.42 µg/mL to 5.14 ± 2.75 µg/mL and 3.21 ± 1.98 µg/mL when encapsulated in Cu-MOFs and ribose-coated Cu-MOFs respectively. MBIC value for CuSO4 was found to be 12.17 ± 2.42 µg/mL. Similarly, for the resistant strain, it was reduced to 5.71 ± 2.44 µg/mL (CHL-Cu-MOFs) and 4.40 ± 1.79 (R-CHL-Cu-MOFs). While the MBIC of CHL and CuSO4 was found to be 12.89 ± 1.12 µg/mL and 14.5 ± 1.42 µg/mL, respectively. On the other bacterial strain P. aeruginosa, the MBIC value of simple CHL was 20.19 ± 2.79 µg/mL which was reduced to 6.60 ± 1.87 µg/mL for CHL-Cu-MOFs and 4.10 ± 1.22 µg/mL for R-CHL-Cu-MOFs, while the MBIC value for the CuSO4 as observed as 11.98 ± 2.24 µg/mL  showed the smooth rod-shaped structure in chain arrangement while when treated with CHL-Cu-MOFs, the smoothness of bacterial cell became disturbed and some of the cells separated from their regular arrangement (E. coli (R) was also observed as rods arranged in chain (P. aeruginosa control revealed the smooth rod-shaped morphology and the smoothness of the cells was affected by the influence of CHL-Cu-MOFs (The results of MBIC study were also confirmed by the investigation of morphological changes of bacterial strains after being treated with test samples. The SEM images of all the bacterial strains are presented in angement c. The E.in chain d and the-Cu-MOFs i.E. coli sensitive and resistant strains as well as MDR pathogen, P. aeruginosa, and significantly reduced their MIC and MBIC values. The results were further authenticated by their morphological investigation using SEM and AFM which showed the complete distortion of bacterial cells upon treatment with R-CHL-Cu-MOFs. The results of the study revealed that ribose-coated Cu-MOFs can significantly overcome the MDR of pathogenic bacteria and represent a promising antibacterial candidate for preclinical application to treat various bacterial infections.This study reported the synthesis of ribose-coated Cu-MOFs for the enhanced antibacterial potential of CHL. The synthesized Cu-MOFs were capable to encapsulate the increased amount of CHL and significantly increase its potential against bacterial population. The ribose-coated CHL-Cu-MOFs showed the promising activity against


Raw materials composed of easily assimilated monosaccharides have been employed as carbon source for production of microbial lipids. Nevertheless, agro-industrial wastes rich in galactose-based carbohydrates have not been introduced as feedstocks for oleaginous yeasts.Aureobasidium namibiae A12 was found to efficiently accumulate lipid from soy molasses and whey powder containing galactose-based carbohydrates, with lipid productions of 5.30 g/L and 5.23 g/L, respectively. Over 80% of the fatty acids was C16:0, C18:0, C18:1, and C18:2. All kinds of single sugar components in the two byproducts were readily converted into lipids, with yields ranging between 0.116 g/g and 0.138 g/g. Three α-galactosidases and five β-galactosidases in the strain were cloned and analyzed. Changes of transcriptional levels indicated GalB and GalC were key α-galactosidases, and GalG was key β-galactosidase. In 10 L fermentor, lipid production from SM and WP achieved 6.45 g/L and 6.13 g/L, respectively. β-galactosidase was responsible for lactose hydrolysis; sucrase and α-galactosidase both contributed to the efficient hydrolysis of raffinose and stachyose in a cooperation manner.In this study, This is a new way to produce lipids from raw materials containing galactose-based carbohydrates. This finding revealed the significance of sucrase in the direct hydrolysis of galactose-based carbohydrates in raw materials for the first time and facilitated the understanding of the efficient utilization of galactose-based carbohydrates to manufacture lipid or other chemicals in bioprocess.The online version contains supplementary material available at 10.1186/s13068-021-02031-8. Aureobasidium, Candida, Cryptococcus, Lipomyces, Rhodotorula, Rhodosporidium, Trichosporon, and Yarrowia [Aureobasidium pullulans, Lipomyces starkeyi, Yarrowia lipolytica, and Rhodosporidium toruloides can accumulate high level of neutral lipids, over 60% of their dry cell weight, from different feedstocks [Microbial lipid can be produced with high efficiency and has no competition from edible oil. Thus, microbial lipid has become important sources for producing biodiesel and other chemicals now , 39. AmoYarrowia . Among tedstocks , 28, 31.At present, the high production cost, especially the cost of carbon source for fermentation, impedes the industrial applications of microbial lipids . In viewOverall, the carbohydrates in these raw materials above mainly rely on glucose or fructose as monosaccharide unit, which can be easily assimilated. However, some agro-industrial by-products contain plenty of galactose-based carbohydrates, such as lactose and raffinose family oligosaccharides (RFOs) . The galSM is a main by-product of the soy protein extraction from soybean. As soybean is one kind of staple crops providing plant proteins and lipid, SM is produced in large amount. Generally, SM has a high carbohydrate content of above 30% (w/v), mainly containing stachyose, raffinose, sucrose, monosaccharides, and other micronutrients , 22, 26.Aureobasidium spp. has recently been evaluated as an excellent lipid producer and reported to efficiently assimilate various monosaccharides [Aureobasidium sp., indicating the ability of assimilating galactose-based carbohydrates [Aureobasidium namibiae strain A12 derived from mangrove was screened to accumulate high content of lipid directly from WP and SM.charides . The gencharides . In our hydrates . In thisL. starkeyi and R. toruloides, with the lipid production of 1.80 g/L and 1.93 g/L, respectively , oleic acid (C18:1), stearic acid (C18:0), palmitoleic acid (C16:1), and palmitic acid (C16:0) in lipid converted from SM were 9.83%, 57.42%, 10.51%, 2.1%, and 20.14%, respectively (Table 18:1) and palmitic acid (C16:0) in lipid of A. namibiae converted from SM account for high proportion (78.94%). The composition of the fatty acids in lipid converted from glucose and WP was rather similar to that converted from SM.Triacylglycerols (TAGs), the reported main components of lipid in oleaginous yeasts, were stored in intracellular lipid droplets: One to three droplets in each cell of strain A12 (data not shown). After being stained by Nile Red, the lipid droplets presented yellow fluorescence which can be observed under a fluorescent microscope with blue excitation light. GC analysis of the fatty acids showed that the content of linoleic acid  [2HPO4, 2.0 g/L KH2PO4, 0.1 g/L MgSO4·7H2O, and 2 g/L corn steep liquor (CSL), with pH 6.0. When other pure carbohydrates were selected as the carbon source, the concentrations of the carbohydrates remained 50 g/L. GPPB medium was used for SIase production and contained 30.0 g/L glucose, 1.0 g/L (NH4)2SO4, 6.0 g/L yeast extract, 2.0 g/L KH2PO4, 3.0 g/L K2HPO4, and 0.1 g/L MgSO4·7H2O with a pH of 6.0 [Four typical oleaginous yeast strains, i.e., extract) . The comH of 6.0 .2 powder which was held for 1 h. Subsequently, the liquid was filtered to remove excess Ca(OH)2 and adjusted to pH 6.0 with H2SO4, which was kept for 1 h. After 10 min of boiling, the solution was centrifuged at 7000 × g, followed by being stored at 4 °C [w/w) glucose, 2.81% (w/w) fructose, 13.42% (w/w) sucrose, 1.73% (w/w) xylose, 3.15% (w/w) raffinose, 14.26% (w/w) stachyose, and 0.78% (w/w) galactose.SM was adjusted to pH 10.0 with Ca(OH) at 4 °C . The preg. The yeast cells were harvested by centrifugation at 5000 × g, washed with sterile water, and dried at 80 ℃ overnight for the biomass detection. Nile Red  was adopted to stain the washed cells (10 μL) which were then observed under an Olympus U-LH100HG fluorescent microscope with blue excitation light [Strain A12 was cultivated in 5 mL YPD medium at 30 ℃ for 20 h which was then switched to 50 mL LP medium at the same temperature. The fermentation proceeded for a total of 120 h, after which the broth was centrifuged at 5000 × on light . In the A. namibiae strain was conducted with the TIANamp Yeast DNA Kit . The DNA polymerase was utilized according to the recommendation by the manufacturer . The transformation of E. coli was realized by the heat shock method of Sambrook et al. [A. namibiae CBS 147.97, and this potential gene in strain A12 was subjected to PCR amplification. The amplified DNA fragments were then transformed into E. coli DH5α . The E. coli transformants were screened on LB agar which contained 100.0 μg/mL ampicillin and play a role in the plasmid amplification and DNA sequencing.The extraction of genomic DNA from the k et al. . The prihttp://www.cbs.dtu.dk/services/SignalP-4.1/) was applied to the signal peptide analysis. The phylogenetic tree was constructed on the basis of reported α-galactosidase and β-galactosidase sequences by virtue of the neighbor-joining method in MEGA version 7.0.The gene protein sequence was compared using HMMER3 based on the CAZy (Carbohydrate-Active enZYmes) database. In this way, the annotation information of carbohydrate-active enzymes became available . The genStrain A12 was inoculated for 36 h at 30 °C in the LP medium containing glucose, SM, and WP, respectively. Total RNA was isolated using TRIzol reagents described by the manufacturer. RNA concentration was analyzed with NanoDrop2000c spectrophotometer  and reversed transcribed into cDNA. Real-time PCR was performed in triplicate using a SYBR green assay kit , with the SYBR real-time PCR master mix (Applied Biosystems). The primers for transcription-quantitative PCR (qRT-PCR) were designed according to the sequences of the genes coding galactosidases; 18S rDNA gene was employed as an internal reference  to act as the substrate for the determination of the optimal reaction pH. pH stability was estimated according to the remaining activity after the incubation at 4 °C for 12 h in buffer at different pH levels. All reactions were allowed to proceed in triplicate.The enzymatic reaction mediated by recombinant galactosidases proceeded in 10 mM glycine–NaOH buffer (pH 6.0) at temperatures ranging from 10 °C to 70 °C, the results of which showed the optimal reaction temperature. To investigate the thermal stability of recombinant galactosidases, the enzyme after purification was first incubated at temperatures ranging from 10 °C to 70 °C for 12 h and then the remaining activity at 40 °C was detected. Tannic acid solutions were prepared with 10 mM buffer at different pH levels  analysis. The carbohydrate concentration was calculated depending on the retention time and peak area. One unit of the α-galactosidase activity (U) was defined as the amount of the enzyme that generated 1 μM p-nitrophenol per minute at 40 °C in buffer at pH 4.5.The α-galactosidase activity was evaluated with pNPG as substrate . The reao-nitrophenyl-β-D-galactopyranoside  in 100 mM citrate buffer (pH 4.0) underwent the incubation at 40 °C for 10 min. The β-galactosidase in the mixture was inactivated by 2.0 mL of 0.5 M Na2CO3 solution. One unit of the β-galactosidase activity (U) was defined as the amount of the enzyme producing 1.0 μM ONP per minute in the conditions of this study.The detection of β-galactosidase activity was carried out by the similar method . The mix2) column .The sucrase activity was detected as reported . The carLarge-scale fermentation was performed in Biostat B 10 L fermentors  for the lipid production from WP and SM, respectively. Strain A12 was first inoculated in YPD medium (600 mL) as a seed culture and then transferred to the fermentor which contained 6 L of lipid production medium. In the next step, the fermentation began and lasted for 120 h under the following conditions: agitation speed: 300 rpm, aeration rate: 50 L/min, temperature: 30 ℃, and pH 6.0. The samples were also taken every 12 h to determine the biomass, lipid yield, and activities of α-galactosidase, β-galactosidase, and sucrase. Besides, the sugar content was monitored as well.Additional file 1: Figure S1. (a) SDS-PAGE analysis of recombinant GalB. Lane M, standard Mw markers; lane 1, purified GalB; lane 2, Bovine albumin. (b) Substrate specificity of GalB (c) The pH activity and stability of GalB. (d) Thetemperature activity and stability of GalB. Figure S2. (a) SDS-PAGE analysis of recombinant GalC. Lane M, standard Mw markers; lane 1, purified GalC; lane 2, Bovine albumin. (b) Substrate specificity of GalB (c) The pH activity and stability of GalC. (d) Thetemperature activity and stability of GalC. Figure S3. (a) SDS-PAGE analysis of recombinant GalG. Lane M, standard Mw markers; lane 1, purified GalG; lane 2, Bovine albumin. (b) The pH activity and stability of GalG. (c) Thetemperature activity and stability of GalG. Table S1. Primers for amplifying genes coding galactosidases. Table S2. Primers used to perform qRT-PCR assays.


APQ reduced SETDB1 activity and H3K9me3 levels in a HD cell line model. In particular, not only APQ reduced H3K9me3 levels in the striatum but it also improved motor function and neuropathological symptoms such as neuronal size and activity in HD transgenic (YAC128) mice with minimal toxicity. Using H3K9me3-ChIP and genome-wide sequencing, we also confirmed that APQ modulates H3K9me3-landscaped epigenomes in YAC128 mice. These data provide that APQ, a novel small molecule SETDB1 inhibitor, coordinates H3K9me-dependent heterochromatin remodelling and can be an epigenetic drug for treating HD, leading with hope in clinical trials of HD.The present study describes evaluation of epigenetic regulation by a small molecule as the therapeutic potential for treatment of Huntington’s disease (HD). We identified 5-allyloxy-2-(pyrrolidin-1-yl)quinoline (APQ) as a novel SETDB1/ESET inhibitor using a combined   GC/MS analyses were performed on Agilent 6890N Network GC system . The analysis of HPLC purity was performed on a Waters Alliance System with UV detector set to at 254 and 280 nm . Samples were injected (10 µl) onto a Waters Sunfire 4.6 × 150 mm, 5.0 µM, C18 column maintained at 25.8 °C. A linear gradient from 30% to 100% B (MeCN) in 20 min was followed by pumping 100% B for another 10 min with A being H2O + 0.1 M NH4OAc (or NH4HCO2). The flow rate was 1.0 ml/min.All reactions were conducted under oven-dried glassware under an atmosphere of nitrogen. All commercially available reagents were purchased and used without further purification. Solvents and gases were dried according to standard procedures. Organic solvents were evaporated with reduced pressure using a rotary evaporator. Reactions were followed by analytical thin layer chromatography (TLC) analysis using glass plates precoated with silica gel (0.25 mm). TLC plates were visualised by exposure to UV light (UV), and then were visualised with a KMnO1  dissolved in DMF (4.0 ml) was slowly added to the round-bottomed flask. The temperature of the reaction mixture was raised to room temperature for 10 min. Ally bromide  was slowly added to the resulting yellow reaction mixture. After confirming the termination of reaction by TLC , H2O was added to the reaction mixture. The reaction mixture was separated into an ethyl acetate layer and an H2O layer using a separatory funnel. After drying the organic layer with anhydrous MgSO4, the solvent was removed by vacuum distillation. The mixture was purified by column chromatography on silica gel (ethyl acetate/hexane = 1:4) to obtain the target compound . 1H NMR  δ 8.90 , 8.62 , 7.70 , 7.59 , 7.37 , 6.85 , 6.22–6.09 , 5.51 , 5.35 , 4.71 . 13C NMR  δ 153.9, 150.5, 149.0, 132.8, 130.7, 129.2, 121.6, 120.8, 120.1, 117.6, 105.3, 69.0. GC/MS: m/z (EI) 185 (M+).After adding sodium hydride  to a round-bottomed flask set to 0 °C using ice and water, DMF (17.4 ml) was slowly added. Then, 5-hydroxylquinoline 2  in dichloromethane (8.0 ml) and then adding mCPBA  at 0 °C, the mixture was stirred at room temperature for 2 h. After confirming the formation of N-oxide by TLC , H2O was slowly added. The reaction mixture was separated into a dichloromethane layer and an H2O layer using a separatory funnel. After drying the organic layer with anhydrous MgSO4, the solvent was removed by vacuum distillation. After dissolving the dried N-oxide mixture in dichloromethane (8.0 ml), POCl3  was slowly added. The reaction mixture was stirred at 50 °C for 7 h. After confirming the termination of reaction by TLC , H2O was slowly added. The reaction mixture was separated into a dichloromethane layer and an H2O layer using a separatory funnel. After drying the organic layer with anhydrous MgSO4, the solvent was removed by vacuum distillation. The crude mixture was purified by column chromatography on silica gel (ethyl acetate/hexane = 1:4) to obtain the target compound 3 . 1H NMR  δ 8.56 , 7.61 , 7.61 , 7.36 , 6.88 , 6.21–6.09 , 5.50 , 5.37 , 4.72 . 13C NMR  δ 154.1, 151.2, 148.8, 134.0, 132.6, 130.4, 122.0, 121.2, 119.4, 118.1, 106.0, 69.3. GC/MS: m/z (EI) 219 (M+).After dissolving 5-quinoline 3  to a vial, pyrrolidine  was slowly added. The reaction mixture was stirred at 140 °C for 12 h. After confirming the termination of reaction by TLC, H2O was slowly added. The reaction mixture was separated into an ethyl acetate layer and an H2O layer using a separatory funnel. After drying the organic layer with anhydrous MgSO4, the solvent was removed by vacuum distillation. The mixture was purified by column chromatography on silica gel (ethyl acetate/hexane = 1:4) to obtain the target compound 4 . 1H NMR  δ 8.30 , 7.39 , 7.31 , 6.67 , 6.54 , 6.20–6.10 , 5.49 , 5.32 , 4.66 , 3.63–3.60 , 2.03–2.00 . 13C NMR  δ 156.1, 154.5, 149.6, 133.5, 131.6, 129.2, 119.0, 117.3, 114.3, 108.9, 101.5, 68.9, 46.8, 25.6. GC/MS: m/z (EI) 254 (M+). HPLC purity: 98.74%.After adding 5--2-chloroquinoline Homology model of the SET domain of SETDB1 (amino acids 792–1291) was taken from our previous studyCells were homogenised with Dounce homogeniser in 500 ml of phosphate-buffered saline containing 0.4 mM sodium butyrate, 5% Triton X-100, 3 mM DTT, 1 mM sodium orthovanadate, 5 mM sodium fluoride, 3 mM PMSF, 3 mM DTT, 0.5 mg/ml leupeptin, and 10 mg/ml aprotinin as previously described,The T-RExTM System -based inducible SETDB1/ESET cell line was established as previously describedHdhQ7/Q7 (wild type) and STHdhQ111/Q111  were generously provided from Dr. Marcy MacDonald ,5 cells were plated onto 48-well cell culture plates 24 h prior to the transfection of 200 ng of ETDB1/ESET promoter construct. DMRIE-C reagent  was used as a transfection reagent and transfections were performed according to the manufacturer’s protocol. Nogalamycin and distamycin were purchased from Sigma-Aldrich .STFluorescence resonance energy transfer (FRET)-based histone H3K9 methylation biosensor was previously describedS-adenosyl-l-homocysteine (SAH) as a product of S-adenosyl-l-methionine (SAM)-dependent transmethylation reaction was used as a non-selective inhibitor of SETDB1. Briefly, SAH and test compound APQ were diluted serially in DMSO and added at a final concentration of 1–100 µM in threefold increment. Total 50 µL mixture of histone methylation transfer buffer  containing 20 µM SAM, each concentration of inhibitors and 1.25 ng/µL SETDB1 enzyme was added to each well of a 96-well plate. The wells were pre-coated with histone H3 peptide substrate. Reactions were allowed to proceed for 1 h 30 min at room temperature. Methylated K9 residue of H3 peptide was detected with anti-methylated H3K9 primary antibody and HRP-conjugated secondary antibody. Chemiluminescence resulting from the reaction between the HRP-conjugated secondary antibody and the added HRP substrate was measured using a SpectraMax M2 plate reader .SETDB1 enzyme activity was analysed using commercially available SETBD1 chemiluminescent assay kit  according to the manufacturer’s instructions. All analyses were performed in duplicates and all reagents used are supplied in the kit. Cell viability was quantified using MTT assay . In brief, cells were cultured in each well of 96-well plates with varying concentrations of compounds for 24 h. Following incubation of cells in each well with MTT (0.5 mg/ml) for 4 h, the culture supernatant was discarded. Cells were washed with PBS three times, and the insoluble formazan product dissolved in DMSO. The optical density (OD) of each culture well was measured using as ELISA reader at 570 nm. The OD570 in control cells was taken as 100% viability.YAC128 mice were bred with females from their background strain (B6CBAFI/J), and offspring were genotyped using PCR as previously describedImmunofluorescence staining and confocal microscopy was used to determine the trimethylated-histone H3 (Lys9) (H3K9me3)  (1:1000). The procedures were performed as previously described,3) and 20 µl of 5 M NaCl. DNA was cleaned by the addition of 500 mM EDTA, 1 M Tris, and proteinase K at 66 °C for 4 h. The DNA solution was extracted with a phenol/chloroform/isoamyl alcohol mixture to remove protein contaminants, followed by precipitation with 100% ethanol. After precipitation, the pellet was washed with 70% ethanol and dissolved in 20 µl DW.ChIP for H3K9me3 binding to DNA was performed using a CHIP assay kit  as described previouslyp values threshold of 0.1 were used for further analyses,p values of ≤.05 and only DMRs in promoter region (≤3 K from transcription start site) were selected. Then genes with the DMRs repressed by H3K9me3 in YAC and derepressed by APQ treatment were defined as seed genes for further analyses.The Model-based Analysis of ChIP-Seq algorithm  was used to identify regions of H3K9me3 enrichment with the default parameters. The peaks that pass a Poisson ,p values of ≤.05. Ingenuity pathway analysis  was used to construct a network model from the seed genes. The network analysis algorithm of IPA was applied to identify networks of regulatory events affected by the seed genes and their interacting genes. The importance of the genes in the network was computed as degree (K) and betweenness (B) centrality using CentiScaPe (v. 2.1), and the non-seed genes with B = 0 or K = 1 were pruned,We performed the over-representation analyses of gene ontology biological processes (GOBPs), Kyoto encyclopedia of genes and genomes (KEGG) pathways, and Reactome pathways using ConsensusPathDBt test; for multiple comparisons, data were analysed by ANOVA followed by Fisher’s protected least significant difference (LSD) test using StatView 4  as described previously. Data were considered significant at a value of p< .05.Data were represented as the mean ± standard error of the mean (SEM). For single comparison, the significance of differences between means was assessed by Student's To identify candidate compounds that possess inhibitory activity against SETDB1, we have employed the previously developed dot blot assay using tetracycline (TET)-inducible SETDB1 cell line 28. When 3 produced 2-chloroquinoline 3. Finally, nucleophilic aromatic substitution (SNAr) of 2-chloroquinoline 3 with pyrrolidine afforded the desired compound APQ in good yield.Among the 129 compounds tested, we have identified 5-allyloxy-2-(pyrrolidin-1-yl)quinoline (APQ) 4 as a lead compound . The strFollowing the discovery of APQ, we tried to obtain some insight as to how APQ could interact with the SETDB1 methyltransferase. In particular, it is crucial to understand whether APQ would fit into substrate binding domain or SAM binding domain for further optimisation of APQ. Thus, APQ molecule docked inside our homology model of SETDB1/ESET domain using the Gold suite-5.2 softwareAPQ has shown several interactions with amino acid residues in the binding site. Quinoline ring intersects with pi-pi-T-shaped interactions with two tyrosine residues Tyr1265 and Tyr832 . AdditioIn order to further verify the effect of APQ on SETDB1 activity, the APQ dose-dependent experiments were performed and analysed H3K9me3 level by Western blot analysis . APQ sigFigure S1a). We prepared a mixture of histone H3 substrate, SAM, and diluted SETDB1 enzyme in buffer solution, which was incubated with increasing concentrations of APQ. The inhibitory activity at each concentration was recorded by detecting chemiluminescence signal after treatment of H3K9 antibody followed by addition of the HRP substrate. The SETDB1 activity was inhibited in a dose-dependent manner although the IC50 value of APQ is only as low as 65 µM. Additionally, the cell viability of APQ was tested by MTT assay . Thus, STHdhQ7/7 (wild type) cells were cultured with varying concentrations of APQ for 24 h. The result indicated that APQ did not show any cytotoxicity at the concentration range of 1–100 µM. Therefore, APQ not only modulates the promoters to reduce the expression level of SETDB1, but also inhibits the enzymatic activity to reduce the trimethylation of H3K9.Next, we evaluated the inhibitory activity of our compound against SETDB1 enzyme by comparing with a well-known methyltransferase inhibitor (substrate) using chemo-luminescence assay .In order to examine the pharmacological properties SETDB1/ESET inhibitor, 10 mg/kg of APQ were intravenously and orally administered to Sprague-Dawley (SD) rats. To assess the effects of APQ, a novel compound targeting SETDB1, on the locomotor activity in HD mouse model, we conducted a rotarod test at 15 months of age . Male YATo further investigate the effects of APQ on epigenetic and neuropathological changes in HD mouse model, we performed immunohistochemistry analysis of coronal brain sections of WT, YAC128, and APQ-treated YAC 128 mice . While HDARPP-32 is a dopamine- and cAMP-regulated neuronal phosphoprotein, which plays an important role in dopamine neurotransmission. Its expression is severely impaired in the striatum of YAC128 mice compared with that of WT mice. Importantly, we observed that APQ administration considerably improved the DARPP-32 expression in in the striatum of YAC128 mice . Similarp values ≤.01 were selected. Then the H3K9me3-enriched sites with 1.5-fold higher in vehicle-treated YAC128 compared to WT and 1.5-fold lower in vehicle-treated YAC128 compared to APQ-treated YAC128 were finally selected. APQ treatment led to a 1.5-fold decrease of H3K9me3 occupancy in a total of 7060 targets in YAC mice , transcription regulation , metabolic process , and signal transduction pathways   . Especia9, etc.) . We furt9, etc.) . As we e9, etc.) .in vitro HD cell (Q111/Q111HTdh) line and in vivo HD transgenic (YAC128) mouse model. Primary screening of a chemical library using the Dot-blot assay rapidly provided APQ as a lead compound. The homology modelling study showed that APQ is predicted to block the methylation process of lysine residue by occupying the binding site of lysine inside the catalytic domain. We observed that APQ down-regulated SETDB1 expression and reduced H3K9 trimethylation. APQ reduced both transcriptional and enzymatic activity of SETDB1 without showing any significant cytotoxicity. Most importantly, APQ restored motor behaviour in the HD mouse model through histone modification by regulation of SETDB1. It is interesting that APQ alters H3K9me3-landscaping in multiple genes that are associated with histone modification and transcription regulation processes that may be involved in the pathogenesis in HD. We have reported that a vicious cycle of H3K9me-dependent heterochromatin condensation by SETDB1 is linked to transcriptional dysfunction, neuropathological processes, and motor behavioural dysfunction in HD animal models. Importantly, the increase of H3K9me3 level directly contributes to the condensation of pericentromeric heterochromatin and it is reversibly regulated by small molecules,,bona fide epigenetic drug, improving motor function in the HD mouse model through the recovery of neuronal activity by reducing SETDB1 activity and H3K9me3-dependent heterochromatin plasticity. Our results show that epigenetic SETDB1 inhibition would be a potential therapeutic target for HD and provide a starting point for lead optimisation into further development of SETDB1 inhibitors for clinical trials.We identified a novel small molecule SETDB1 inhibitor and validated its neuroprotective effect on Click here for additional data file.


The platelet count was positively correlated with air pollutants of 0–6 day moving averages . Additionally, internal dose biomarkers 2-OHNa, 1-OHNa, 2-OHFlu, 2,3-OHPhe, and ∑PAHs were also significantly associated with platelet count in participants. Furthermore, PM2.5 and PM10 are positively linked with methylation of one CpG site at platelet mitochondrial gene CO2 . Both platelet counts and methylation levels returned to their pre-exposure levels after leaving the highly contaminated area. In short, this study investigated the relationship between platelet properties and air pollution exposure, revealing that short-term exposure to air pollution might increase the risk of thrombosis. Our research suggests that platelet count and mitochondrial DNA methylation of mtCO2 site 2 in platelets from healthy adults may be the novel biomarker for acute exposure to air pollution.Air pollution exposure is now considered a growing concern for global public health. RNA or DNA methylation changes caused by air pollution may be related to the development of cardiovascular disease. To investigate the early biomarkers of air pollution exposure, a panel study of eight college students recorded after a business trip from Qingdao to Shijiazhuang and back to Qingdao was performed in this work. The concentration of PM According to the World Health Organization (WHO), air pollution causes approximately 7 million deaths annually , with caMature platelets, 2–4 µm, which survived for 7–10 days after exfoliation from megakaryocytes, act as central hemostasis, and their cross-talk with other blood cells sustains the stability of the circulation system. Moreover, the platelets are involved in different biological processes, including wound healing, angiogenesis, immune regulation, and inflammation, which are all fundamental components of the stress response . Some evRecent studies in environmental epigenetics provide opportunities to understand the mechanistic underpinnings of exposure-related health effects . Epigene2.5 in Shijiazhuang’s air is higher than that in Qingdao (ranging from 45 to 220 μg/m3 vs. ranging from 22 to 92 μg/m3)  . The dif2. Exclusion criteria were as follows: 1) a history of heart disease; 2) asthma symptoms; 3) kidney disease; 4) coagulopathy; and 5) rheumatic disease, or chronic inflammation within the past 6 months. The Institutional Review Board of Qingdao University approved the following study proposal. All research conducted on each participant accepted by themselves through signed an informed consent form after informed of all aspects of the study before starting the study, respectively.Eight healthy young participants from Qingdao University, who had to travel to Shijiazhuang on a six-day business trip for a project, were recruited. Enrollment required each candidate to attend an interview, including a questionnaire on general health and demographic characteristics to ensure that the candidate participants were in a good general state of health of the body. The inclusion criteria were as follows: 1) age >18 years old and 2) body mass index <30 kg/mThe air quality in Shijiazhuang was seriously polluted compared with that in Qingdao . Blood a2.5, PM10, SO2, and other pollutants obtained from the national urban air quality real-time detection data released by the China Environmental Monitoring Center (http://www.cnemc.cn). Thereupon, air quality record time covers the entire process of the scheduled study . To calculate the average daily concentration of each indicator during the study period, we recorded the 24-h value of the entire study period in real time.The air pollution index (API) in both cities was evaluated with various indicators such as PMVenous blood and morning urine samples were collected from the participants. Participants were asked to fast for 8 h before sample collection to reduce the effect of diet on biomarker concentrations. In total, we had 24 EDTA anticoagulated blood samples and 24 urine samples (we collected samples of each person for 3 periods). While some of the blood was tested for routine blood tests , the rest was separated from the plasma and stored at −80°C until platelet mitochondria DNA extraction and pyrosequencing. The platelet parameters involved in this study included platelet count (PLT), platelet hematocrit (PCT), platelet distribution width (PDW), mean platelet volume (MPV), platelet large cell ratio (P-LCR), and methylation of the CpG site at platelet mitochondrial genes. For PAH (polycyclic aromatic hydrocarbon) metabolite assessments, fresh urine samples were collected for the further HPLC-MS/MS evaluation.β-glucosidase were mixed into 2 ml of urine sample, and then the mixture was incubated at 37°C for 8 h in the dark. Eight μL internal standard was added into the urine samples, and then we adjusted the pH value to 5. Subsequently, urine with 4 ml dichloromethane was evaporated to dryness using nitrogen before being dissolved with 10 μL HPLC solvent. Finally, a liquid chromatographic system  and an integrated triple quadrupole mass spectrometer  were used for the analysis. Urinary creatinine, measured using an ELISA kit  according to the manufacturer’s instructions, was used to standardize urinary polycyclic aromatic hydrocarbon metabolite concentrations.The concentrations of PAH metabolites, including 2-hydroxynaphthalene (2-OHNa), 1-hydroxynaphthalene (1-OHNa), 2,3-hydroxyphenanthrene , 1-hydroxypyrene (1-OHP), and 2-hydroxyfluorene (2-OHFlu), in urine were used as internal dose biomarkers and determined using the HPLC-MS/MS method by a blind tester to exposure status. The standards were purchased from Chem Service and Sigma-Aldrich. 1 ml of sodium acetate buffer and 20 μL of TM Kit , the mitochondrial DNA was extracted and transformed into bisulfite-converted mtDNA.To convert mitochondrial DNA into bisulfate for sequencing, plasma samples from 8 participants were used to isolate platelet mtDNA as described before. Briefly, platelet pellets were obtained by centrifugation of 400 μL of plasma samples at 1,400 × g for 15 min at room temperature. To minimize contamination with nuclear DNA, we added 3 μL of DNase I  and 7 μL deionized water and incubated the samples for 3 h at 37°C. The samples were treated with 20 μL proteinase K at 50°C for 1 h and centrifuged at 1,000 × for 10 min for purification, and then the supernatants were retained, respectively. According to the protocol provided by the EZ DNA methylationMT-ATP8  and MT-TL1 , three CpG sites within MT-ND5 , and six CpG sites within MT-CO2 . PCR was performed using 1 µL bisulfite-treated mitochondrial DNA and 25 µL Epiarttm_hs Taq Master Mix . The amplified target genes (5 µL) were taken, and 2 µL loading buffer was added for DNA gel electrophoresis to verify whether the amplification of the target genes was successful. Amplified mtDNA PCR products (10 µL) were then used for pyrosequencing by using PyroMark Q48 Autoprep .The mitochondrial primer sequences were selected as stated in the references , which d2.5, PM10, SO2, NO2, and CO) was calculated based on daily averages over the whole study period. The air pollution metrics entered into the models as continuous variables and each regression coefficient acquired finally provide an estimate of the effect in terms of an interquartile (IQR) increase in each air pollutant exposure. Similar approaches have been taken to assess the effect of PAH with biomarkers. Sensitivity analysis was conducted by excluding a current smoker (n = 1) to test the impact of smoking on the biomarker. For an appropriate supplementary approach to adjust the false discovery rate of the model, the Benjamini–Hochberg method was applied in multiple comparisons to avoid Type I errors. All statistical analyses were performed using SAS software, version 9.4 (site 70239492). All reported p-values less than 0.05 were defined as statistically significant.The participants’ basic information and air quality were expressed as mean ± SD and otherwise by median and range (IQR) depending on whether they were normally distributed demographic and clinical characteristics. Spearman correlation analysis was carried out for various pollutants. Linear mixed-effects models with a random intercept for each participant were used to estimate the associations between air pollutants and biomarkers. Considering that the skewed distribution of exposure indicators may lead to poor model fit, urinary PAH metabolites  of participants were logarithmic-converted to better fit the normality hypothesis. The least absolute redundant selection operator (LASSO) was used to filter variables into the mixed models. The covariates included age, sex, height, BMI, and smoking status. Hence, the interquartile range (IQR) of API , according to the standards of the WHO. One male participant was a current smoker (12.5%). The participants did not receive any medicine and participated in the complete research process.A total of 8 participants were invited to participate in the study . There w2.5, PM10, SO2, NO2, and CO were 76.25 μg/m3, 118.04 μg/m3, 15.95 μg/m3, 61.04 μg/m3, and 1.39 mg/m3 during the period in Shijiazhuang city . There were strong correlations between the pollutants during the whole study period (r ≥ 0.60)  . For exaThe concentration in SJZ was significantly higher than in QD-before for ∑PAHs (390%), 2-OHNa (517%), 1-OHNa (121%), and 2-OHFlu (40%). The concentration of urinary PAHs in the QD-after period was significantly lower than in the SJZ period, and there were no significant differences between the QD-before period and the QD-after period .9/L, 12.7% higher than in the QD-before period (267.14 ± 78.52 × 109/L). PLT in QD-after (258.00 ± 54.15 × 109/L) was 14.3% lower than in SJZ after 6 days of recovery, and the difference also existed after subsequent multiple comparisons (Platelet count (PLT) in the SJZ period was 301.00 ± 62.54 × 10parisons . Mean plparisons . Plateleparisons . The chaparisons .mtCO2 site 2 in the SJZ stage was higher than that in the QD-before stage  . Similarly, the methylation of mtATP8 site 2 increased at first and then decreased throughout the study period  (mtND5 site 1 methylation in SJZ (2.34%) was lower than that in QD-before (2.54%), and the methylation became higher after the recovery stage (2.01%)  . After 6 > 0.05) . The met > 0.05) . In addi (2.01%) .PM2.5 = 88.90, 95% CI: 29.22–148.57; βPM10 = 61.83, 95% CI: 13.61, 110.05; βSO2 = 41.13, 95% CI: 14.46, 67.80; βNO2 = 57.70, 95% CI: 20.28, 95.12; βCO = 62.99, 95% CI: 22.42,103.56, respectively, for an IQR increased) appeared to be stronger than those based on exposure metrics of 0–1 moving averages to 0–5 moving averages. These could indicate that cumulative exposure over multiple days may be able to capture the potential impact of air pollution more robustly than only 1 day. Additionally, 2-OHNa, 1-OHNa, 2-OHFlu, 2,3-OHPhe, and ∑PAHs were significantly associated with PLT in participants. Specifically, IQR increases in 2-OHNa (31.97 μg/μmol Cr) and 1-OHNa (2.84 μg/μmol Cr) were significantly associated with a 24.42 ×109/L  and 5.95 ×109/L  increase in PLT  for an IQR (1.07 μg/μmol Cr) along with increase in 2-OHFlu. An IQR increase in ∑PAHs (43.97 μg/μmol Cr) was significantly associated with a 31.15 ×109/L  increase in the mtCO2 site 2 methylation.Most air pollutants, PAHs, and PLT were positively correlated . In parte in PLT . MoreovemtATP8 site 2 was evaluated. It was found that there was a significant positive correlation between PM2.5, PM10, and mtATP8. . In the same way, this positive correlation was also found in SO2 and NO2 with mtATP8 . The mtCO2 site 2 methylation is positively correlated with the PM2.5 and PM10 . Subsequently, the same positive association was identified in SO2, NO2, and CO . On the other hand, an IQR increase in CO was significantly associated with a 0.33%  decrease in mtCO2 site 2 methylation. We diagnosed significant positive correlations that were seen between 2-OHNa and 1-OHNa with mtCO2 site 2 methylation  and 0.1  with each increase in IQR for 2-OHN (31.97 μg/μmol Cr) and 1-OHN (2.84 μg/μmol Cr). mtCO2 site 2 methylation increases by 0.36  for an IQR (1.07 μg/μmol Cr) increase in 2-OHFlu. An IQR increase in ∑PAHs (43.97 μg/μmol Cr) was significantly associated with a 0.58%  increase in mtCO2 site 2 methylation. There was a positive correlation between 0- to 6-day moving averages of the air pollution index and platelet mitochondrial DNA methylation at mtATP8 site 2 . For mtCO2 site 2 and site 6, the association based on the air pollution index (API) of 0- to 6-day moving averages appeared to be stronger than those based on exposure metrics of 0–1 moving averages to 0–5 moving averages  and 0.49%  increase in platelet count in men and women, respectively  that form the functional core of complex IV. In addition, mutations in the COX gene have been linked to metabolic disorders, mainly affecting high-energy tissues. Among many mitochondrial diseases, the COX dysfunction complex is considered to be the most serious. Recent studies have shown that the lung is vital for platelet biogenesis   . Numerou, 0.048) . Still, us study . Despiteus study , which mus study , and intThe strengths of this research are described in the following. First, various platelet markers were measured, including PLT, MPV, PDW, PCT, and P-LCR, whereas most studies have only measured PLT. Second, this research, unlike previous studies, chose the platelet mtDNA as the research object. It provides more evidence to reveal the link between air pollution and cardiovascular disease. Third, the recovery period of participants was set to investigate whether the platelet changes caused by short-term environmental pollution are reversible. Finally, pyrosequencing, the gold standard of epigenetics, was used to detect the methylation status of specific sites, which improved the reliability of the results. However, several limitations still existed in the current study. First, this natural experiment cannot control all factors, like diet and circadian rhythm, which could influence the outcomes under study. Second, the study included only eight participants in all three phases, although statistically significant differences were found. However, the number of samples may affect the accuracy of the results. Finally, no wearable devices were used to monitor the pollution exposure dose of each participant. Thus, it was impossible to assess individual exposure accurately.mtCO2 site2 in platelets from healthy subjects may be sensitive biomarkers for acute air pollution exposure. The platelet mitochondrial DNA methylation changes may provide a preliminary understanding of the epigenetic regulatory pattern of platelet parameter changes caused by acute air pollution exposure.In summary, this study suggests that acute exposure to air pollution can induce changes in platelet parameters. Platelet count and mitochondrial DNA methylation of


Scientific Reports 10.1038/s41598-021-91283-3, published online 03 June 2021Correction to: The original version of this Article contained errors.In the Discussion and conclusions section,−1 which is equivalent to the power loss of 1–2 kWs45.”“Depending on the size of the wind farm, generally, the annual mean wind speed deficit can reach 2–2.5 msnow reads:−1 which is equivalent to the power loss of 1–2 MW45.”“Depending on the size of the wind farm, generally, the annual mean wind speed deficit can reach 2–2.5 msIn addition, Reference 45 was incorrectly given as:Contract 10.1002/ajmg.10175 (2009).Jonkman, J., Butterfield, S., Musial, W. & Scott, G. Definition of a 5-MW reference wind turbine for offshore system development. The correct reference is listed below:Jonkman, J., Butterfield, S., Musial, W. & Scott, G. Definition of a 5-MW reference wind turbine for offshore system development. United States. 10.2172/947422 (2009).The original Article has been corrected.