Introduction: Diabetes mellitus (DM), a chronic metabolic noncommunicable disease (NCD), has assumed epidemic proportions worldwide. Type 2 diabetes (T2D) is defined as chronic high blood glucose levels due to the deficiency of insulin or resistance to it. Dyslipidemia is one of the major causes of cardiovascular diseases in patients with T2D. It is characterized by elevated plasma triglyceride (TG), low high-density lipoprotein (HDL) cholesterol, and high low-density lipoprotein (LDL) cholesterol, which is mostly present in patients with DM.Methods: We conducted a retrospective cross-sectional study at the King Faisal University (KFU) Health Center in the eastern region of Saudi Arabia. The data of patients from October 2014 to February 2021 were collected. We collected the patients’ data from the KFU Health Center after obtaining approval from the KFU polyclinic administration. Prior ethical approval was taken from the Institutional Review Board of Ethics and Research Committee in the College of Medicine, KFU . We collected patients’ data, including their gender, age, nationality, and blood analysis .Result: This study included 191 patients with T2D, 137 (71.7%) were from Saudi Arabia, and 54 (28.3%) were from other countries. Patients’ age ranged from 21 to 100 years, with a mean age of 56.2 ± 11.8 years. There were 107 (56%) females. Cholesterol levels ranged from 102 to 300 mg/dl with a mean value of 187.3 mg/dl.Conclusion: In the current study, we assessed the association between HbA1c levels and lipid profiles in patients with T2D at the KFU Health Center in the eastern region of the Kingdom of Saudi Arabia. Our results on the adjusted relationship of HbA1c with lipid profile through regression model have demonstrated that HDL alone had significant adjusted relation with HbA1c if other factors are kept constant. We suggest focusing on TC and HDL levels in T2D health management in future studies. Diabetes mellitus (DM), a chronic metabolic noncommunicable disease (NCD), has assumed epidemic proportions worldwide . Type 2 Dyslipidemia is one of the major causes of cardiovascular diseases in patients with T2D. It is characterized by elevated plasma triglyceride (TG), low high-density lipoprotein (HDL) cholesterol, and high low-density lipoprotein (LDL) cholesterol, which is mostly present in patients with DM . The TGsResearch designWe conducted a retrospective cross-sectional descriptive study at the King Faisal University (KFU) Health Center in the eastern region of Saudi Arabia. The data of patients from October 2014 to February 2021 were collected.ProcedureWe collected the patients’ data from the KFU Health Center after obtaining approval from the KFU polyclinic administration. Prior ethical approval was taken from the Institutional Review Board of Ethics and Research Committee in the College of Medicine, KFU . We collected patients’ data, including their gender, age, nationality, and blood analysis .The inclusion criteria for the study were as follows: (i) age > 18 years; (ii) diagnosed with T2D for more than three months, (iii) on a regular follow-up with the KFU Health Center.The exclusion criteria were as follows: (i) age < 18 years; (ii) diagnosed with T2D for less than three months; (iii) type 1 DM patients; (iv) has hypertension, on lipid-lowering drugs, and smoking habit; (v) had no coexisting disease associated with hyperlipidemia .Study populationOne hundred and ninety-one patients, who were suffering from T2D for at least one year, were included in our study.MaterialsAt the KFU Health Center, the cut-off values for patients’ inclusion were as follows: HbA1c level, 4-5.9%; cholesterol, 100-200 mg/dl; HDL, 40-60 mg/dl; LDL, <100 mg/dl; TG, 37-150 mg/dl.Statistical analysesAfter collection, the data were modified, coded, and statistically analyzed using SPSS V22.0 . All statistical analyses were performed using two-tailed tests. A P-value of <0.05 was considered statistically significant. Descriptive analysis was done for all categorical variables , based on the frequency and percent distribution, whereas mean with standard deviation and range described lipid profile and HbA1c level in total and by gender, comparing lipid profile and HbA1c by patients’ gender. Correlation analysis was used to assess the nature and strength of the unadjusted relationship between HbA1c and lipid profile, whereas a multiple linear regression model was used to assess the adjusted relation between HbA1c and lipid profile.This study included 191 patients with T2D, 137 (71.7%) were from Saudi Arabia, and 54 (28.3%) were from other countries. Patients’ age ranged from 21 to 100 years, with a mean age of 56.2 ± 11.8 years. There were 107 (56%) females  patients had high cholesterol levels. HDL levels ranged from 25 to 91 mg/dl ; 40 (22.1%) patients with T2D had high HDL levels. TG values ranged from 56.3 to 616.5 mg/dl ; 64 (33.9%) patients had high levels. LDL values ranged from 33.5 to 195 mg/dl ; 94 (57.7%) patients had high LDL levels. Many prospective studies showed no association between lower HDL levels and the development of T2D in the future, although it affects the control of glycemic level . We can Unlike our results, few studies reported that patients with T2D and an HbA1c value greater or equal 7.0% showed a significant increase in TC and LDL levels without significant changes in TG and HDL level compared with the patients with T2D and an HbA1c value of <7.0% . A studyWe also found a variation in HbA1c and dyslipidemia incidence in different age groups. Cholesterol level was insignificantly higher among patients with T2D aged 40-60 years than among the old age group . HDL level among patients aged 20-39 years was 38.0 mg/dl compared to 52.3 mg/dl for patients aged >60 years with statistically significant difference (P = 0.002). TG was significantly higher among patients aged 20-29 years (210.6 mg/dl) than others aged >60 years (129 mg/dl) with P = 0.001. The average level of HbA1c among young patients (20-29 years) was 7.8% compared to 8.4% for patients aged >60 years (P = 0.186). We explain these findings because of the medications taken by the older age group. This study has a few limitations. First, we could not identify the co-morbidities, medications, and social history of the participants. We suggest further studies in the future on a larger population, considering the limitations in the current study.In the current study, we assessed the association between HbA1c levels and lipid profiles in patients with T2D at the KFU Health Center in the eastern region of the Kingdom of Saudi Arabia. Our results on the adjusted relationship of HbA1c with lipid profile through regression model have demonstrated that HDL alone had significant adjusted relation with HbA1c if other factors are kept constant. We suggest focusing on TC and HDL levels in T2D health management in future studies.


Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1–4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1–3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55–CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55–CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein–coupled receptors.The adhesion G protein–coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF Adhesioimmunity . Furtherimmunity , 6, 7.1,2,5, EGF1,2,3,5, and EGF1–5    and inte0 (Thy1)  α5β1 and0 (Thy1) , which bvia costimulating CD4+ T cells with CD97, resulting in T cell activation and an increase in cell proliferation and cytokine secretion (CD55 is a GPI-linked membrane protein with four short consensus repeat (SCR) domains at the N terminus. CD55 regulates the complement cascade by inactivating the C3 convertases and plays a critical role in inflammation and pathogen defense . The binecretion .1,2,5) has been found to bind to CD55 with a dissociation constant (KD) much lower (>10 fold) than that of CD97   , 21. CryGF1,2,5)  and the  of EMR2  reveal a1–4 domains and CD97 EGF1,2,5 domains were initially purified independently from HEK293 cells. However, the mixture of the two fragments did not yield a stable complex in gel-filtration chromatography, presumably because of the relatively low affinity between the two proteins. Therefore, we designed a 24-residue linker including a tobacco etch virus protease (TEV) site to connect the C terminus of EGF1,2,5 and the N terminus of SCR1–4, enlightened by a head-to-tail docking mode based on NMR titration  (B and C). Cleavage of the linker with TEV generated a size-exclusion profile of two peaks corresponding to the CD55 and CD97 fragments, suggesting each protein within the chimera construct was well folded individually . The small-angle X-ray scattering (SAXS) measurements and alignments with models of the chimera complex and each individual protein indicated the formation of a stable CD55–CD97 complex, mediated by the flexible linker  , 23. TheFig. 1D, . We thenectively .Figure 12 and 748 Å2, named lattice A and lattice B, respectively (A). Previous binding studies of CD55 and CD97 have emphasized the importance of the N-terminal domains of both proteins in recognition, and our designed 24-residue linker was sufficient to connect the 36 Å distance between the C terminus of EGF1,2,5 and the N terminus of SCR1–4 in lattice A but not long enough to fill the 106 Å gap in lattice B. Furthermore, lattice A binding mode can be fitted into a SAXS envelop with a normalized structural difference of 2.74; this is relatively better than the fittings of lattice B and the previously proposed model from NMR titration (B) . I. I2 and E). Structures of CD55 and CD97 in the complex superimposed closely with prior monomeric structures, with a Cα r.m.s.d. of 0.9 to 2.1 Å and 1.1 to 1.2, respectively (E), suggesting no conformational change is needed during binding. Previous structures of CD55 SCR domains have suggested a constant interface of SCR2–SCR3 and very little variation in the interfaces of SCR1–SCR2 and SCR3–SCR4. In our complex structure, the orientation of SCR2–SCR3 is invariant and the other interfaces are within similar ranges of variation as the CD55 molecules of previous studies (E). Similar case in CD97, only slight variation in the EGF1–EGF2 or EGF2–EGF5 interface, is monitored when compared with previous monomeric structures of its homolog protein, EMR2 (E). In CD97 or EMR2, EGF2 and EGF5 each bind a calcium ion in the N-terminal tip, which contributes to rigidity within the interface of EGF1–EGF2 or EGF2–EGF5. These comparisons, together with apparent disorder of the 24-residue linker between the two molecules, indicated the binding mode between CD55 and CD97 is not affected by the protein engineering.The CD55 and CD97 bind roughly antiparallel to each other involving each N-terminal tandem domains E. Structectively E, sugges studies E. Similain, EMR2 E. In CD91,2,5–SCR1–4 interactions was 1920 Å2, an interface area that is above the average for protein–protein interaction  and one N-glycosylation site in CD55 (Asn-95), but none of these carbohydrates are involved in the CD97–CD55 interactions.The total solvent-accessible surface buried by the EGFeraction . All dom1,2,5–SCR1–4 interactions can be divided into three interfaces: the EGF1–SCR2/SCR3 interface, the EGF2–SCR1/SCR2 interface, and the EGF5–SCR1 interface . In addition, EGF1 also forms extensive hydrophobic interactions with SCR2 and SCR3 residues and buries a total surface area of 518 Å2. The preeminent interactions of CD97–CD55 come from the EGF2–SCR1/SCR2 interface in the middle region, which buries a total surface area of 1278 Å2 and contains six pairs of hydrogen bonds (B). At the upper half of this interface, Thr-70 and Glu-86 of EGF2 hydrogen bond to Glu-99 and Gln-111 of SCR2, respectively. In addition, the carbonyl group of Cys-82 in EGF2 forms a hydrogen bond to Asn-117 of SCR2. At the bottom half of the interface, the EGF2–SCR1 interactions are secured by three pairs of side chain–main chain hydrogen bonds between EGF2 residues Asp-79, Asp-80, and Asp-81 and SCR1 residues Asp-77, Lys-71, and Lys-74, respectively. In the third interface of EGF5–SCR1 (C), Val-137 of EGF5 inserts into a joint hydrophobic pocket formed by SCR1 residues Val-60, Lys-76, Val-79, Ile-80, and Leu-82 and EGF2 residue Phe-79. These interfaces are consistent with previous literature, identifying the EGF1,2 and SCR1,2 domains as critical determinants for CD55–CD97 interactions. Moreover, our structure unveils additional interactions contributed by EGF5 of CD97 as well as SCR3 of CD55. Notably, the involvement of EGF5 in the CD55–CD97 interactions may elucidate why the EGF1,2,5 isoform of CD97 shows a higher binding affinity to CD55 than the other two isoforms, EGF1,2,3,5 and EGF1–5.The EGFnterface . In the  of SCR2 A. In adden bonds B. At theGF5–SCR1 C, Val-13KD of 3.2 μM, ∼20-fold higher than the previously reported affinity (A), and in accord with one study demonstrating a statistical correlation between the interface area and binding affinity (−1S−1) and off-rate (1.73 × 10−3S−1). These rates are in line with our structural observation of the antiparallel binding interface that composed by tandem rod-like domains at both sides. The apparent difference in binding kinetics between our measurements and previous reports could be attributed to the differing immobilization techniques. In the previous work, CD55 was randomly covalently attached to the chip via primary amino groups, which may cause a decrease in flexibility and accessibility by CD97 .We further measured affinity of CD97 mutants to confirm the physiological relevance of the binding mode. Within the interface, D63, D81, and E86 of CD97 each hydrogen bonds to a residue in CD55 . Aligned1 and N-terminal portion of SCR2 in CD55 bind to the other side of CD97 with a much smaller interface  is relatively small—and no hydrogen bond is visualized within this interface—this EGF domain may dictate the binding affinity and specificity of the different CD97 isoforms with CD55. In the EGF1,2,3,5 and EGF1–5 isoforms, the EGF3 domain occupies the position of EGF5 and faces the SCR1 domain of CD55. The key EGF5 residue Val-137 is equivalent to a leucine residue in the EGF3 domain. The relative conservation of this position suggests that in the other two isoforms, the EGF3 domain may involve in the binding of CD55 with similar pattern. To further verify the importance of a hydrophobic aliphatic residue in the interface with CD55, we mutated the V137 to an alanine in the EGF1,2,5 fragment and the SPR result showed that the V137A mutant shows an ∼8-fold reduction in binding value (25.3 ± 2.35 μM), confirming the involvement of EGF5 in the binding interface with CD55 (E). The additional methylene group in the EGF3 domain, together with a potential fine-tuning of its orientation compared with the first two EGF domains, may contribute to lower binding affinity of the two longer isoforms  than the EGF1,2,5 isoform.Although the interface involved by EGFith CD55 E. The ad1,2,5 fragment of CD97 and EMR2 is differentiated by only three residues, and previous reference suggested >10-fold lower CD55-binding affinity of EMR2 than CD97 , confirming the involvement of these variant residues in the binding interactions (F). Two of three variant residues are located in the binding interface with CD55, and their side chains can be unambiguously modeled in the CD97–CD55 complex  of the CD97 EGF1 domain encounters the residues Tyr-133, Lys-160, and Phe-182 of CD55. The hydroxyl group of Thr-59 is only ∼4 Å away from Tyr-133 and Lys-160, and this forms a weak polar interaction in the CD97–CD55 interface, whereas similar interaction would be absent in the EMR2–CD55 interface (A and B). The second differing residue is located in a thermal-dynamic loop region in each EGF2 domain. In CD97, the Pro-71 and its preceding residue Thr-70 adopt a cis-peptide conformation, allowing the Thr-70 to flip its backbone and form a hydrogen bond to Glu-99 in the CD55 SCR2 domain (A and C). A cis-peptide conformation is not allowed in EMR2, as the equivalent residue of Pro-71 is Leu-74. Remarkably, in EMR2, the Leu-74 occupies the position of CD97 residue Thr-70, and therefore, no hydrogen bond is formed in this region when it binds to CD55. The third residue difference, Asn-33/Asp-36, is not located in the binding interface, so it may not directly affect the binding of CD97–EMR2 with CD55.The EGFhan CD97 . Althougractions F. Two of complex . The firnterface , A and B2 domain , A and C2–SCR1 interface, indicating substantial specialization in the rodent interface. In line with this specialization, previous references have shown that the CD97–CD55 interaction is species restricted and no cross-reaction is found between human and mouse agents    (27). It module) , 28, 29.1 is immediately different from the other two domains because of its shorter β-sheet (A and D). The EGF1–EGF2 and EGF2–EGF5 interfaces are almost identical, featuring a main chain–main chain hydrogen bond and a van de Waals interaction between a glycine residue (Gly-87/Gly-138) in the β-turn of the EGF2 or EGF5 domain and a conserved aromatic residue (Phe-48/Tyr-97) in the precedent domain (B–D). The aromatic and the non–side-chain glycine residues are also conserved in the EGF3 and EGF4 domains (D), indicating similar domain orientations in the EGF1,2,3,5 and EGF1–5 isoforms. CD55 also contains similar van de Waals interactions between the preceding SCR2 (Gly-132) and SCR3 (Gly-193) domains and their following SCR3 (Phe-182) and SCR4 (Tyr-245) domains (E–G), respectively. Moreover, the van de Waals interactions in the SCR2–SCR3 and SCR3–SCR4 interfaces are each surrounded by additional hydrophobic residues that strengthen each interface. Nevertheless, in the SCR1 and SCR2 domains, the corresponding aromatic residues (Phe-55/Phe-119) are flipped and pointed toward the hydrophobic core of each domain (E–H). Furthermore, the glycine residue is not conserved in SCR1 and its replacement by a serine residue generates a perturbation in the hydrophobic cage, making an orientation similar to SCR2/SCR3 or SCR3/SCR4 energetically unfavorable (H and I). To expose the hydrophilic Ser-68, the SCR1 is tilted about 45° to make contact with SCR2; this is in contrast to roughly straight orientations between the other SCR domains. The SCR1–SCR2 contact buries a total surface area of 560 Å2, the largest among all interdomain contacts in CD55 or CD97.Within the three structured EGF domains of CD97, EGF β-sheet , A and Dt domain , B–D. Th domains D, indica domains , E–G, reh domain , E–H. Fuavorable , H and I2, the tilted SCR1 appears like a hook that holds the body of the CD97 EGF2 domain. The majority of the interactions of CD55 with CD97 come from this SCR1–SCR2 pair, including six of the seven total hydrogen bonds between the two proteins. One example is the Thr-70 bearing loop of CD97 EGF2 domain, which reaches deep into the hook and hydrogen bonds to the Glu-99 of CD55 (2 domain that interacts and complements the first strand of EGF2 in CD97 (I). Adjacent to the hook is a conserved SCR2/SCR3 pair that generates a mostly hydrophobic patch engaging the EGF1 domain. Such domain pairs playing important roles for ligand binding are not uncommon and have been observed in the case of CD46  is retained over a significant force range. The potential capability of withstanding a significant range of mechanical force suggests that the CD55–CD97 may mediate force transmission to the following GAIN domain, which is believed to be important for the function of adhesion GPCRs. For many adhesion GPCRs, the 7TM domain is noncovalently associated with the extracellular region through its very N-terminal fragment  that inserts into a β-sheet module of the extracellular GAIN domain , CD55 (35–285), or chimeric complex gene was inserted into a customed pLEXm (6 tags. In the chimeric construct (named CD97–24a–CD55), a 24-residue linker (GSGENLYFQSGSSSSGWRGGHVGS) was added to link the C terminus of EGF5 and the N terminus of SCR1. HEK293S GnTI− were cultured to 1 to 2 million/ml in suspension and were transiently transfected with DNA: polyethylenimine at 1:3 wt/wt. Culture supernatants were harvested after 3 to 4 days. Purification was with nickel coated NTA affinity followed by Superdex S200 increase gel filtration in 20 mM Tris, pH 7.5, 200 mM NaCl, and 2 mM CaCl2. The three purified proteins were subjected to SAXS analysis, and chimeric complex was concentrated to 16 mg/ml for crystallization trials. In the SPR measurements, the EGF1,2,5 (24–168) fragment of EMR2 (UniProt: Q9UHX3) and the EGF1,2,5 fragment of CD97 were cloned to the same vector as above except a TEV protease site was inserted after each gene sequence to remove the C-terminal His6 tag during purification. Single point-mutation fragments of CD97 were made by overlapping PCR and cloned to the same vector for expression and purification.The CD97 (UniProt: P48960) EGF domains, CD55 (UniProt: P08174) SCR domains, and chimeric complex were expressed and purified as previously described . Brieflyed pLEXm  vector wq = 4π sinθ/λ, where 2θ is the scattering angle] of SAXS experiments was 0.008 to 0.47 Å−1. To reduce the radiation damage, a flow cell made of a cylindrical quartz capillary with a diameter of 1.5 mm and a wall of 10 μm was used. SAXS data were collected as 20 × 1 s exposures, and scattering profiles for the 20 passes were compared at 10 °C using 60 μl sample in 20 mM Tris, pH 7.5, 200 mM NaCl, and 2 mM CaCl2. Measurements were carried out at two different concentrations in all cases using concentrations between 0.5 and 2 mg/ml. The 2D scattering images were converted to 1D SAXS curves through azimuthally averaging after solid angle correction and then normalizing with the intensity of the transmitted x-ray beam, using the software package BioXTAS RAW (P(r) and the maximum sizes Dmax were computed using GNOM  at Shanghai Synchrotron Radiation Facility. The wavelength (λ) of X-ray radiation was set as 0.918 Å. Scattered X-ray intensities were collected using a Pilatus 1M detector (Dectris Ltd). The sample-to-detector distance was set such that the detecting range of momentum transfer [XTAS RAW . Pair diing GNOM . The ab g GASBOR . SAXS daThe concentrated CD97–24a–CD55 complex was set up for crystallization using hanging drop with NT8 (Formulatrix). Diffraction-quality crystals were produced at 18 °C in 0.1 M MES, pH 6.5, and 12% to 15% w/v PEG 20000. Another linker with six repeats of GSGP (GSGPGSGPGSGPGSGPGSGPGSGP) also yielded thin crystals in similar conditions, but we happened to optimize better crystals from the CD97–24a–CD55 construct. In contrast, a shorter linker with 18 residues (GSGGSGGSGGSGGSGGSG) did not crystallize in the same condition. Single crystals were directly frozen in liquid nitrogen. Diffraction data were collected at a wavelength of 0.979 Å at beamline BL17U1 at the Shanghai Synchrotron Radiation Facility and indexed, integrated, and scaled using the automatic XIA2 software package . The str2, and 0.05% Tween 20. The surface was regenerated with 4 M MgCl2 for 4 min at the end of each cycle to restore resource units to baseline. All traces were corrected for refractive index changes by subtraction of a control trace simultaneously recorded from a mock-immobilized channel. Kinetics and affinity analysis were performed with SPR evaluation software .For SPR experiments using Biacore T200 , His-tagged CD55 was immobilized on a nickel coated NTA chips. WT and mutant CD97 (without His-tag) were gel-filtered using Superdex 75 increase to remove aggregates before use. Protein was injected at 50 μl/min for 2 min in a buffer containing 20 mM Tris, pH 7.5, 200 mM NaCl, 2 mM CaClgjsong@bio.ecnu.edu.cn (Gaojie Song).Atomic coordinates and structure factors for the CD97–CD55 structure have been deposited in the Protein Data Bank with identification code 7DO4. Correspondence and requests for materials should be addressed to This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Their structures were elucidated by spectroscopic methods. Compounds 1–11 were evaluated for their potency against Staphylococcus aureus and clinical methicillin-resistant S. aureus (MRSA). Among them, compound 3 was weakly active against S. aureus (MIC = 128 μg/mL), and compounds 6 and 7 exhibited good antibacterial activity against S. aureus and MRSA (MICs = 2–8 µg/mL). A primary mechanism study revealed that compounds 6 and 7 could kill bacteria by destroying bacterial cell membranes. Moreover, compounds 6 and 7 were not susceptible to drug resistance development.Two undescribed ether derivatives of sesquiterpenes, 1-ethoxycaryolane-1, 9 Staphylococcus aureus is one of the most devastating bacterial pathogens that can cause serious infections in both humans and animals +  in the HRESIMS spectrum and exhibited a strong absorption band for hydroxyl groups (3416.6 cm−1) in the IR spectrum. The 13C NMR data revealed 16 carbon signals, consisting of a monoterpene skeleton and a D-glucopyranosyl moiety. The 1H NMR signals of anomeric protons of the D-glucopyranosyl moiety at δH 4.37  are consistent with the configuration for β-D-glucose. The NMR data of aglycone moiety of 5 were similar with those of -p-menthane-1,2,8-triol + .1-Ethoxycaryolane-1, 9β, 9α-diol (3). Colorless gum; + .2-Ethoxyclovane-2α,2α,8-triol-4-O-β-D-glucoside (5). Colorless gum; + .p-Menthane-1Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 25922) were purchased from the American Type Culture Collection. The methicillin-resistant S. aureus (MRSA-1–10) was provided by the First Affiliated Hospital of Zhengzhou University. The bacterial strains were stored in a refrigerator at –80 °C. Before the experiment, the experimental strains were thawed and inoculated on a MHA nutrient agar plate. After being cultured overnight at 37 °C, the strain was picked up into 1 mL MHB culture medium, and incubated in a 37 °C shaker at 200 rpm for 3–5 h.I. simonsii extract was carried out by the disc diffusion method according the literature [The antimicrobial activity of the terature . Briefly1–11 were determined by broth micro-dilution in a Mueller–Hinton broth medium (MHB) according to the guidelines of the Clinical and Laboratory Standards Institute [S. aureus (ATCC 29213), E. coli (ATCC 25922) and clinical MRSA strains (M-1–10) were used in this experiment. Test compounds were dissolved into DMSO, and antibiotic vancomycin was dissolved with sterile Milli-Q water, then these solutions were two-fold serially diluted to a series of gradient concentrations . The results were determined after incubation for 16–18 h at 37 °C. The MIC was defined as the lowest concentration of the compound that produced complete inhibition of visible growth. All MIC determinations were repeated three times.The MIC values of compounds nstitute . S. aure5  and 108  CFU/mL. Then, MASR-3 was challenged with compound 6 , 7  and vancomycin  at 37 °C and 225 rpm. The aliquots were removed at 0 h, 1 h, 2 h, 4 h, 6 h, and 8 h, serial 10-fold dilutions of the sample were made in the diluent (PBS), and a 10 μL aliquot of each dilution was plated out on MHA plates using the surface-spread plate method. The plates were incubated at 37 °C for 24 h to measure viable plate counts (CFU).A time–kill assay was evaluated according to the guidelines of CLSI . BrieflyS. aureus (ATCC 29213) of compounds 6, 7 and control antibiotic norfloxacin was investigated. Briefly, the original MICs of compounds 6, 7 and norfloxacin against S. aureus were determined at first. An S. aureus suspension incubated at the exponential phase at 37 °C was passaged to a fresh MHB containing the test compounds at sub-inhibitory concentration (1/2 MIC), the process was repeated every 20 to 22 h for up to 20 passages, and the MICs for test compounds were assayed at every passage as described above.According to the methods used in previous studies , the pro6 and 7 at the different concentrations  in a 96-well plate (50 μL). Triton X-100 (1%) was used as a positive control. The plate was incubated for 1 h at 37 °C and centrifuged at 3500× g rpm for 5 min. The supernatant (100 μL) was transferred to a fresh 96-well plate. The results are shown as the percentage of hemolysis according to the absorbance measured at 540 nm [Red blood cells (RBCs), isolated from the fresh sterile sheep blood, were resuspended in 1× PBS (5%). A RBCs suspension (150 μL) was then added to solutions of serially diluted compounds t 540 nm .7 were 1/4× MIC, 1/2× MIC, 1× MIC, 2× MIC, 4× MIC, and 8× MIC. The fluorescence reading was monitored for the next 40 min at every 2 min interval at an excitation wavelength of 504 nm and an emission wavelength of 523 nm. Vancomycin and daptomycin were used as negative controls, and melittin was used as a positive control. DMSO  and water were used as blank controls.The bacterial suspension of MRSA-3 was incubated at 37 °C for 6 h and suspended in 1× PBS. Then, the bacterial suspension was incubated with 3 μM of SYTOX green for 20 min at room temperature until the fluorescence readings were stable. The final concentrations of added compound 7 at desired concentrations (2× and 4× MIC). After incubation at 37 °C for 3 h, the bacterial suspension was added and stained with 4′, 6-diamidino-2-phenylindole dihydrochloride  and propidium iodide  for 30 min at 0 °C. Finally, the suspension was observed by a fluorescence microscope with an excitation wavelength of 485 nm. Bacterial no-treatment with 7 was used as a blank control.As described in the literature , the bac


Here, g-C3N4/MoO3 nanohybrid was used for the first time as an adsorbent for the pharmaceutical drug, diclofenac, (an aqueous micropollutant) from water to mitigate its possible environmental toxic effects. Compared to pristine components, the nanohybrid exhibited better adsorptive removal of diclofenac. Adsorption was enhanced with increment in MoO3 content from 1 to 3 wt %; however further increment in MoO3 content resulted in lower adsorption capacity due to agglomeration of MoO3 particles over g-C3N4. 162 mg g−1 adsorption capacity was achieved for 300 mg L−1 diclofenac in solution with 1 g L−1 adsorbent at pH = 6. Adsorption of diclofenac over g-C3N4 /MoO3 followed pseudo 2nd order kinetics. Temkin, Langmuir, Dubinin Radushkevich and Freundlich isotherm models were applied on the experimental results concluding that diclofenac adsorption over g-C3N4/MoO3 followed the Langmuir isotherm. The adsorption mechanism could be explained by the π–π interaction between aromatic rings of diclofenac and g-C3N4/MoO3 (3%) nanohybrid, which is also evident by the FTIR results. This study presents the facile fabrication of a 2nd generation adsorbent for the treatment of diclofenac contaminated water that may as well help achieve the removal of other micropollutants form water.Graphitic carbon nitride modified by molybdenum trioxide (g-C Concentrations such as 380 ng L−1 are documented for German and Spanish groundwaters [−1 diclofenac was detected in the bile of two freshwater fish species, which was approximately 1000 times larger than aqueous concentrations [−1 of diclofenac residues in the kidneys of vultures [−1 in freshwater [Pharmaceuticals are extensively used all over the world to cure, impede, and treat diseases in both humans and animals. Globally, an annual per capita of 15 g of pharmaceutical drugs are consumed, while in industrialized countries this consumption rate is estimated at between 50 and 150 g . Estimattrations . Diclofetrations ,8. Most trations . Oaks anvultures . Owing teshwater . To maineshwater ,15,16,17eshwater  excessiveshwater . Currenteshwater . Despite3N4) exhibiting excellent chemical and thermic stability in ambient environments and without any ecotoxicity [3N4 as an adsorbent can be markedly improved through modification of surface properties by adding chemical groups, surfactants, polymers, etc. Kumar and co-workers oxidized the g-C3N4 to enhance its specific surface area and further functionalized it with polyaniline nanofiber to provide positive surface charge and to bind anionic chromium ions electrostatically [3N4 for adsorptive separation of lead from aqueous solutions. In their study, surface complexation was suggested for the adsorption of lead whereas π–π interaction and hydrogen bonding was responsible for the adsorption of methyl orange onto the functionalized adsorbent [3). MoO3 has been found very useful in numerous research and commercial applications due to its unique catalytic and electrochemical characteristics. However, a limited number of research studies have explored their use as an adsorbent [−1 rhodamine B was achieved by MoO3 within 10 min, which is expressively fast with a maximum removal capacity 9-times higher than commercial activated carbon [3, as a nanosorbent for methylene blue exhibited a removal capacity of 152 mg/g [−1 MB was adsorbed from water in nearly 25 min without the aid of light [3N4 contains numerous “nitrogen pots” as they hold 6 N e− providing functionalities with negative charges. The electrons in the aromatic ring offer metal inclusion in these nitrogen pots [3N4 photocatalyst has been synthesized by Wang and coworkers. The BET results indicated that with the increase in molybdenum concentrations, from 1–5% enhanced the catalyst’s surface area up to 88 m2 g−1. Increase in molybdenum content impeded the crystal growth of g-C3N4, leading the crystalline structure of g-C3N4 towards amorphousness, hence increasing the porosity of the material [3N4 modified with MoO3 was utilized for enhanced adsorption of diclofenac micropollutant.Recently, the application of nano-scale materials as an adsorbent has attracted attention for removing toxic and hazardous contaminants from wastewater . Among ntoxicity . The peratically . Zou’s gdsorbent . Transitdsorbent ,26. For d carbon . Nanocry152 mg/g . Similarof light . Furthergen pots . Molybdematerial . Therefo4)6Mo7O24), hydrochloric acid (HCl 37%), sodium hydroxide (NaOH), ethanol (C2H6O) and diclofenac sodium (C14H10Cl2NNaO2) were purchased from Sigma Aldrich , Urea (CO(NH2)2) was bought from Merck . All chemicals used were of high purity .Ammonium heptamolybdate ((NH3N4 was modified with molybdenum trioxide. Molybdenum trioxide was separately prepared by solid state decomposition method. Ten grams of (NH4)6Mo7O24 was decomposed at 500 °C (for 4 h). The final grey colored powder was washed and then dried (at 60 °C). Nanohybrids of g-C3N4/MoO3 were synthesized by modification in the procedure already described [3  were added to the g-C3N4 in deionized water and then under constant stirring the deionized water was evaporated from the mixture by using hotplate. The slurry thus obtained was dried at 80 °C and ground using a pestle and mortar. Calcination of this ground powder at 300 °C for 3 h in a muffle furnace gave the final g-C3N4/MoO3 nanohybrids.To prepare nanohybrids, the g-Cescribed . Firstly−1 was noted by a FTIR spectrophotometer ).The crystallinity of the synthesized nanomaterials was analyzed by Burker D8 Diffractometer  using Cu Kα radiations (λ = 1.5418 Å) and incident angle (2θ) ranged between 10–80°. XPS measurements were performed using standard Omicron system  equipped with monochromatic Al Kα 1486.7 eV (at 15 keV and energy 100 eV or 20 eV). Morphological studies were conducted by JEOL-2100  Transmission Electron Microscope (TEM). FTIR spectroscopic analysis demonstrated existence of functional groups of as synthesized and used adsorbents. For this purpose, the IR spectrum from 4000 to 400 cmmax of 276 nm. Adsorption experiments for the synthesized nanocomposites were performed for the aqueous pharmaceutical drug, diclofenac sodium, as a model micropollutant. For typical experiments 0.1 g of adsorbent was added to the 100 mL working solution of diclofenac sodium continuously stirred for 3 h at 300 rpm. After regular time gaps 5 mL aliquots were strained through a 0.45 µm syringe filter before storing the samples into amber glass vials. The residual concentrations of diclofenac were quantitatively analyzed by using a UV 3000 spectrophotometer  at a λ3N4 appears at 27.5° which correspond to (002) plane of g-C3N4, pointing out the existence of inter-planar layering of conjugated aromatic carbon nitride units with interlayer spacing of 0.33 nm. The small intensity peak 13.2° is attributed to (100) in-plane repeated heptazine in g-C3N4 structure having in-plane d spacing = 0.67 nm. The orthorhombic phase of MoO3 can be indexed from the XRD patterns which are in accordance with the JCPDS card no 05-0508. The main peaks observed at 12.7°, 23.33°, 25.7°, 27.38° and 38.98° represent (020), (110), (040), (021) and (060) planes, respectively [3 content in the g-C3N4/MoO3 composites from 1 to 5% displayed slight shift in g-C3N4′s characteristic peak at 27.5° towards MoO3′s highest intensity peak at 27.38° indicating deposition of MoO3 did not affect the tri-s-triazine structure of g-C3N4 sheets.The XRD peaks of pristine components and binary nanocomposites are illustrated in ectively . As show3N4 at 807 cm−1, attributed to the vibration of tri-s-triazine ring. Similarly, the 890 cm−1 band could be assigned to N–H deformation mode while 1100–1700 cm−1 band corresponds to stretching mode of bridging C–NH–C units. Vibrations at 1133 cm−1 could be ascribed to C–N stretching of an aromatic amine whereas peaks at 1630 and 1538 cm−1 may correspond to aromatic C=N stretches and peaks at 1312 and 1230 cm−1 attributed to C–N stretching vibrations. The broad bands in the range of the 3000–3300 cm−1 region is assigned to the adsorbed N–H and/or NH2 vibration. Peak intensity at 556–569 cm−1 originated from bending vibration of the Mo–O–Mo unit where oxygen ions are linked to Mo6+; describing deposition of MoO3 onto g-C3N4 [−1 stretching vibrations and larger band adsorption at the 3000–3300 cm−1 region are observed in 3N4/MoO3 (3%) sample indicates π–π stacking of diclofenac onto the g-C3N4/MoO3 nanohybrid. Moreover, the available hydrogen bonding sites (–NH) also tend to increase adsorption of the polar diclofenac molecule [3N4/MoO3 (3%) nanohybrid. The FTIR spectrum in o g-C3N4 . Broaden3N4 appeared to comprise of a few layered nanosheets indicating the aggregation of synthesized nanosheets  nanohybrid analyzed by XPS is provided in 3N4 and g-C3N4/MoO3 (3%) comprising of C, N and C, N, O, Mo, respectively. The N1s spectra of g-C3N4 was resolved into three typical peaks at 398.7, 400.2 and 401.0 eV 3 bonds, respectively [3N4/MoO3 nanohybrid  is shown in 3N4/MoO3 (3%) showed two types of peaks located at 232.4 and 235.6 eV corresponding to Mo3d5/2 and Mo3d3/2 for Mo6+ in MoO3 Mo6+, while other peaks at 231.2 and 234.5 eV correspond to Mo5+ [The resolved spectra of O1s for g-C to Mo5+ ,38. The  to Mo5+ .−1. The best performing nanohybrid was selected by examining the removal efficiency of g-C3N4/MoO3 nanohybrids. The base material, g-C3N4 and MoO3, exhibited 23% and 65% removal within first 15 min of the adsorption process and no further removal was observed for the remaining 175 min. g-C3N4/MoO3 nanohybrids showed prominent adsorptive removal of 44%, 83%, 71% and 68% for 1%, 3%, 5% and 7% MoO3 loaded g-C3N4, respectively. The better adsorption of diclofenac on to different weight percentages of g-C3N4/MoO3 nanohybrids as compared to base materials suggested that MoO3 was completely deposited on g-C3N4 nanosheets. These results are supported by X-ray photoelectron spectroscopy and also by another study suggesting enhanced adsorption shown by the composites may be attributed to the presence of oxygen vacancies, hydroxyl groups, and molecular water on the MoO3 surface [3N4; leading crystalline structure of g-C3N4 towards amorphousness, hence, increasing porosity of the material [3N4/MoO3 (3%) among all adsorbents, it was chosen for further optimization studies.For the adsorptive removal of diclofenac, pristine nanomaterials and their modified forms were scrutinized. All experiments were performed at normal solution pH 6 with Diclofenac 25 mg L surface . Studiesmaterial . Due to 3N4/MoO3 (3%) is influenced by the initial concentrations of diclofenac. As depicted in −1 diclofenac showed an increase in percent removal from 83% to 91%, but further increase in concentration resulted in a decrease in percent of removal efficiency. Initially, at lower starting concentrations, a large number of unoccupied reactive sites were accessible to diclofenac molecules for adsorption, so removal rate was higher. With increased diclofenac concentrations from 200 to 400 mg L−1, inadequate active sites were available for a large sum of molecules. This initiated a competition between excessive molecules of diclofenac over limited binding sites of adsorbent ; consequently, decline in adsorptive removal was seen. However, the adsorption capacity of g-C3N4/MoO3 (3%) enhanced with initial diclofenac concentration. An increment in adsorbate dose from 50 to 300 mg L−1 exhibited enhancement in adsorption capacity from 41.29 to 156.29 mg g−1. Here, rise in initial adsorbate concentration may have acted as a driving force for transfer of mass from surface to the inner pores, therefore increase in adsorption capacity was observed [−1 indicated decrease in adsorption capacity indicating saturation of adsorbent.Pollutant concentration plays a vital role in determining adsorbent’s removal efficiency. Adsorption capacity of g-Cobserved . However3N4/MoO3 (3%). Adsorbent and adsorbate surface chemistry and charge is greatly altered by change in pH of the solution by protonation and deprotonation. In water, diclofenac exists in two species—either neutral or in anionic form. In extreme acidic conditions it exists in neutral form but at pH above its pKa value  anionic species is dominant. As in this study, at pH 4 initial diclofenac concentration in solution of 300 mg L−1 was reduced to 44 mg L−1 while the solution became turbid which indicated insolubility of diclofenac at acidic pH. The higher adsorption capacities of 158 and 132 mg g−1 at pH 6 and 8, respectively showed that diclofenac removal from aqueous solution was not dependent on electrostatic interaction. This appreciable adsorption of diclofenac could be explained by hydrogen bonding and π–π interaction between aromatic rings of diclofenac and g-C3N4/MoO3 (3%) composite [omposite .−1 was optimum for maximum adsorption (3N4/MoO3 (3%) nanohybrid was studied at 15, 25, 35, 45 and 60 °C using initial diclofenac concentration 300 mg L−1. The results illustrated in −1. At higher temperatures, expansion within pores and activation of the adsorbent external surface brings about the availability of a greater sum of active sites, therefore, enhancing the adsorption of micropollutants. Moreover, at elevated temperatures the mobility of adsorbate increases while the viscosity of the solution decreases, thus, resulting in effective removal of the contaminant [An adsorbent dose of 1 g Lsorption a. Furthetaminant .3N4/MoO3 (3%) nanohybrid could be explained by reaction kinetics. The pseudo 1st and 2nd order kinetics were used for better insight of the rate of diclofenac uptake by the 3%MoO3/g-C3N4 nanohybrid (The rate of diclofenac uptake on g-Cnohybrid . The pse1 (min−1) and K2 (g mg−1 min−1) represent rate constants of pseudo 1st and pseudo 2nd order kinetic equations; qe (mg g−1) equilibrium adsorption capacity and qt (mg g−1) refers to temporal adsorption capacity.K2 depends on the operating conditions such as solution adsorbate concentrations, pH, and temperature as evident by 1, and K2 were calculated from the intercept and slope of the kinetic plots and listed in the 2, decreased from 0.0241 min−1 to 0.0062 min−1 with increasing diclofenac concentration from 50 to 300 mg L−1. Furthermore, the calculated adsorption capacities  were in very close agreement to the experimental adsorption capacities. Similarly to the initial concentration, other operating parameters also exhibited trends indicating that the adsorption process favored pseudo 2nd order kinetics.The adsorption rate K−1 by varying initial diclofenac concentrations of 50, 75, 100, 200, 300 and 400 mg L−1 at different temperatures  for each concentration. Four isotherms models, i.e., Langmuir, Temkin, Dubinin–Radushkevich and Freundlich isotherm models were applied to adsorption data for analyzing the nature of adsorption. The Freundlich isotherm model was applied using the following equation: The adsorption isotherms characterize the equilibrium relationship within adsorbate quantity in aqueous and solid phases. Moreover, isotherms determine the adsorbate’s affinity towards adsorbent by evaluating its adsorption capacity . Experimb is Langmuir constant, and Langmuir isotherm model was applied by the Equation (4): Dubinin–Radushkevich is commonly applied to indicate mean free energy distribution on heterogeneous surfaces  and is ee” is saturation adsorption capacity (mg/g), activity coefficient “β” helps in finding the mean free energy and Ɛ is the Polanyi potential. The mean free energy “E” of adsorption per mole of adsorbate and Polanyi potential are given in Equations (6) and (7).Here, “qA” and “B” are binding constant (L g−1) and heat of sorption (J mol−1), respectively. R is the universal gas constant and “T” is absolute temperature. “b” is the Temkin isotherm constant given as:Temkin isotherm model undertakes indirect adsorbate-adsorbate interactions and accounts for linear decrease in heat of adsorption of all molecules in the layer with the increase in surface coverage . Temkin max of g-C3N4/MoO3 (3%) nanohybrid also increased, but temperature above 35 °C displayed decrease in the value of Qmax which suggested that the adsorption performance is enhanced by increasing the operational temperature up to 35 °C. Temkin model indicated that the heat of sorption “b” (J mol−1) decreased with the increase in surface coverage and values of parameter “b” presented in 2) better than all other applied models. The R2 values exhibited by Temkin and Dubinin–Radushkevich model were closer to unity at only 35 and 45 °C, respectively suggesting the optimum temperature range for the adsorption of diclofenac onto the adsorbent. Besides that, adsorption capacities calculated from Langmuir isotherm equation were closer to experimental values and indicating the adsorbent as energetically homogenous where the interaction of diclofenac with g-C3N4/MoO3 (3%) nanohybrid forms monolayer at adsorbent surface. The separation factor RL indicates favorability of adsorption based on the Langmuir equation calculated by:As shown in 3N4/MoO3 (3%) nanohybrid. Additionally, RL were found to be favorable for all the isotherms. 2 values are near to unity. Isotherm results revealed that with rise in temperature, the Qmax of g-C3N4/MoO3 (3%) nanohybrid also increased, but temperature above 35 °C displayed decrease in the value of Qmax which suggested that the adsorption performance is enhanced by increasing the operational temperature up to 35 °C.RL specifies the adsorption type whether it is unfavorable (RL > 1), linear (RL = 1) or favorable (0 < RL < 1). From the above equation, RL value obtained was less than 1 i.e., 0.03 for g-C−1) decreased with the increase in surface coverage and values of parameter “b” presented in 2) better than all other applied models. The R2 values exhibited by Temkin and Dubinin-Radushkevich model were closer to unity at only 35 and 45 °C, respectively suggesting the optimum temperature range for the adsorption of diclofenac onto the adsorbent. Besides that, adsorption capacities calculated from Langmuir isotherm equation were closer to experimental values and indicated that the adsorbent was energetically homogenous where the interaction of diclofenac with the g-C3N4/MoO3 (3%) nanohybrid forms the monolayer at the adsorbent surface.The Temkin model indicated that the heat of sorption “b” (J mol3N4/MoO3 (3%) nanohybrid was assessed up to the four reuse cycles. After each use the g-C3N4/MoO3 (3%) nanohybrid was dissolved in 15 mL deionized water at pH 4, stirred for 10 min at 600 rpm and filtered, the recovered adsorbent was then calcined at 250 °C to remove residual DCF, washed with de-ionized water and dried in air before next use. Upon repeated use, the nanohybrid maintained its efficiency with minimal loss from 158.2 to 142.6 mg g−1 after 4th use, clearly suggesting that the g-C3N4/MoO3 (3%) nanohybrid is very stable and reusable for the removal of diclofenac.The stability and reusability of the spent g-C3N4/MoO3 (3%) nanohybrid, its adsorption capacity for diclofenac removal was 3.95-times higher compared with g-C3N4 at optimized conditions. The highest adsorption capacity of g-C3N4/MoO3 (3%) nanohybrid achieved in this study is compared with previously studies, as shown in 3N4/MoO3 (3%) efficiently removes diclofenac from water. However, organobentonite, porous carbon and magnetic amine-functionalized chitosan showed higher adsorption capacities than the g-C3N4/MoO3 (3%) nanohybrid. The difference in activities may be attributed to different adsorbent dosage and operational conditions.To estimate the efficiency of the g-C3N4/MoO3 showed effective removal of diclofenac contaminated water in comparison to base materials . While using g-C3N4/MoO3 (3%) nanohybrid 162 mg g−1 diclofenac adsorption was achieved at pH 6. The adsorption mechanism could be explained by π–π interaction between aromatic rings of diclofenac and g-C3N4/MoO3 (3%) nanohybrid which is also evident by the FTIR results. Application of different kinetic models revealed that experimental results fit well with pseudo 2nd order kinetics while the Langmuir isotherm explained homogenous adsorption of diclofenac on to g-C3N4/MoO3 (3%) nanohybrid. The linear relation at 45 °C and 35 °C agrees with dubinin-Radushkevich and Temkin isotherms indicated an optimum temperature range of 35–45 °C for the sorption of diclofenac. In this research, the g-C3N4/MoO3 (3%) nanohybrid has appeared as an excellent adsorbent that was well above its environmental concentrations for emerging micropollutants such as diclofenac sodium. This study advocates the application of synthesized adsorbent for the successful elimination of diclofenac micropollutant from aqueous environments to avoid its synergistic toxic effects on human health and environment.g-C


Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.  Azithromycin is a macrolide antibiotic that inhibits protein synthesis by binding to the bacterial 50S ribosomal subunit . It disp+) and proteases into the microresorptive area and decompose inorganic and organic bone tissue components [Osteoblasts and osteoclasts are involved in bone remodeling to maintain the mass and quality of osseous tissue . Osteoblmponents . Imbalanmponents ,11, whermponents . Some evmponents ,14. In vmponents  and the mponents . Sub-antmponents . The premponents . These pThe aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts. Osteoblast-like MC3T3-E1 cells were continuously stimulated with azithromycin and examined for in vitro mineralized nodule formation, ALPase activity, and the expression of collagenous and non-collagenous bone matrix protein. Minimal essential medium α (MEMα) and heat-inactivated fetal bovine serum (FBS) were purchased from Gibco  and HyClone Laboratories , respectively. Azithromycin, dimethyl sulfoxide (DMSO), and penicillin–streptomycin solution were obtained from Sigma .v/v) FBS and 1% (v/v) penicillin–streptomycin solution at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Cells were plated onto an appropriate culture plate at a density of 6.0 × 103 cells/cm2, incubated overnight, then stimulated by the addition of 0.1, 1, or 10 µg/mL azithromycin (solubilized in DMSO), and further incubated for 10 or 14 days. Control cells contained a final concentration of 0.1% DMSO in the culture medium. The medium was changed every 2 days. Murine osteoblastic MC3T3-E1 cells  were seeded on 100-millimeter culture dishes and maintained in MEMα containing 10%  according to the manufacturer’s protocols. ALPase activity was determined by adding 200 µL enzyme assay solution containing 8 mM p-nitrophenyl phosphate [p-nitrophenyl) using a microplate reader . One unit of ALPase activity was defined as the amount of enzyme required for the formation of 1.0 µM p-nitrophenol per minute. Enzyme activity was recorded in milliunits (mU)/104 cells.Cells were plated into 96-well plates at a density of 6.0 × 10hosphate . The abs3 cells/cm2 were cultured in medium with 50 mM β-glycerophosphate and 50 µg/mL ascorbic acid in the presence or absence of azithromycin for 14 days. The conditions of the cells and nodule formation were routinely checked via phase-contrast microscopy  at 100× magnification. Mineralized nodules were detected by staining with alizarin red S  according to a previous method [Cells plated into 24-well plates at a density of 6.0 × 10s method . After c3 cells/cm2 in medium with or without azithromycin for 10 days. Total RNA was isolated from cells harvested after a 7- or 10-day incubation using NucleoSpin® RNA . cDNA synthesis was conducted using 500 ng RNA in a total volume of 10 µL containing 50 pmol random 6 mers, 25 pmol origo dT Primer, 0.5 µL PrimeScript RT Enzyme Mix I, and 2 µL PrimeScript Buffer . Aliquots of 2 µL of the cDNA solution were subjected to quantitative real-time PCR (RT-PCR). Reactions were performed in a total volume of 25 µL containing 2 µL cDNA solution, 12.5 µL TB green® Premix Ex Taq II , and 10 μM forward and reverse primers. Primer sequences were as follows: type I collagen , bone sialoprotein , osteopontin , osteocalcin , and glyceraldehyde 3-phosphate dehydrogenase . PCR was performed using a Thermal Cycler Dice real time system  and analyzed using the instrument’s software. The cycling conditions reported in a previous study were used [Cells were seeded into six-well plates at a density of 6.0 × 10ere used . Culture supernatants were collected from plates on day 10. Phosphoprotein was purified using a phosphoprotein enrichment kit . Osteopontin levels in the crude and the phosphoprotein-purified sample were measured with an ELISA kit  according to the manufacturer’s protocols.p-value <0.05 was considered statistically significant. GraphPad Prism 6.0 software  was used for statistical analyses.Values are reported as means ± standard deviation. Significant differences were determined using a one-way analysis of variance followed by Tukey’s multiple comparison test. A Azithromycin concentrations of 0.1 and 1 µg/mL did not affect osteoblast cell proliferation at all time points, whereas significantly decreased growth was observed on days 5 and 7 following treatment with 10 µg/mL azithromycin compared with untreated cells . There wA previous study reported that DMSO at a concentration of 0.2% or less had no effects, whereas DMSO at a concentration of 0.5% or more increased osteogenic function in MC3T3-E1 cells . Our pilThe osteopontin, osteocalcin, bone sialoprotein, and type I collagen mRNAs were differentially expressed to variable degrees after treatment for 7 and/or 10 days in cells stimulated with azithromycin at 0.1, 1, and 10 µg/mL compared with untreated control cells a–d. OsteOsteopontin levels significantly increased in the crude culture supernatant derived from cells stimulated with 10 µg/mL azithromycin after 7 and 10 days following treatment compared with the untreated control a. PhosphSeveral previous studies reported sustained azithromycin concentration in periodontal tissue following its administration to periodontitis patients ,21,22,23Furthermore, in this study, ALPase activity and mineralized nodule formation in MC3T3-E1 cells were markedly suppressed with 10 µg/mL azithromycin, whereas mRNA expression of type I collagen, bone sialoprotein, osteocalcin, and osteopontin increased. Type I collagen is a crucial scaffold, while bone sialoprotein and osteocalcin are indispensable for the initiation of bone mineralization ,25,26. TIt is well known that azithromycin tends to accumulate in inflamed tissues ,2,3. BlaHigh azithromycin concentration (10 µg/mL) suppressed mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) had no effect on osteogenic function in osteoblastic MC3T3-E1 cells.


Herein, the current study set out to explore the effects of Fe3O4 MNPs on radiosensitivity of lung adenocarcinoma cells.Radiotherapy is the mainstay treatment for lung adenocarcinoma, yet remains highly susceptible to resistance. Fe3O4 MNPs loaded with both negatively-charged small interfering RNA against baculoviral IAP repeat containing 5 (siBIRC5) and oligodeoxynucleotide antisense (AS-ODN) to generate co-delivery NPs, followed by evaluation. Gel retardation assay was further performed to determine the binding ability of Fe3O4 MNPs to AS-ODN/siBIRC5. The radiosensitizing effect of NPs on lung adenocarcinoma cells was determined in the absence or the presence of NPs or radiotherapy. A549 and H460 tumor-bearing mice were established, where tumor tissues were subjected to immunohistochemistry.Fe3O4 MNPs enhanced the uptake of siBIRC5 and AS-ODN by lung adenocarcinoma cells. The presence of NPs under magnetic field reduced the BIRC5 expression and elevated the DR5 expression in lung adenocarcinoma cells. Lung adenocarcinoma cells treated with NPs exhibited inhibited tumor cell migration and increased DNA damage. After magnetic field treatment, tumors were better suppressed in the tumor-bearing mice treated with NPs, followed by radiotherapy.NPs were successfully prepared and characterized. BIRC5 expression levels were augmented in tissues of lung cancer patients. Fe3O4 MNPs-targeted delivery of siBIRC5 and AS-ODN enhances radiosensitivity, providing an innovative solution for the current clinically existing lung adenocarcinoma patients with radiotherapy resistance with a low risk of toxicity.Findings obtained in our study indicated that FeThe online version contains supplementary material available at 10.1186/s12967-021-02971-7. Lung adenocarcinoma is the leading cause of cancer-related mortality, accounting for approximately 1.8 million deaths across the world in 2020, with high mortality rate reported . There h3O4 magnetic nanoparticles (MNPs) are known to exhibit better magnetic particle and magnetic resonance imaging properties, which is designed for precision imaging and cancer therapy [3O4 MNPs may deliver BIRC5 and AS-ODN to tumor cells, which may be applied in radiotherapy, and set out to perform a series of experiments to investigate the molecular mechanism underlying small interfering RNA against BIRC5 (siBIRC5) in lung adenocarcinoma, hoping to discover some promising strategy to increase the radiosensitivity.Nanomaterials (NPs) have been increasingly used in biomedical fields, especially in healthcare, which can be coupled with various antibody and luminescent labeling . Due to  therapy . More in therapy . Meanwhi therapy . On the  therapy . Moreove therapy . Besides therapy , and fur therapy . HoweverDeclaration of Helsinki. Signed informed consents were obtained from all participants prior to specimen collection. Animal experiments were performed in accordance with the standard of the Guide for the Care and Use of Laboratory animals published by the National Institutes of Health. Animal experiment protocols were reviewed and granted by the animal ethics committee of Tongji University School of Medicine. Extensive efforts were made to minimize the suffering of the included animals.The current study was approved by the clinical research Ethics Committee (PR003/13) of Tongji University School of Medicine, and conformed with the guidelines of the 3O4 MNPs were synthesized with the help of an oxidative hydrolysis method. First, to synthesize Fe3O4, FeCl3 deionized aqueous solution  was mixed with FeCl2 4H2O  in deionized water. A black precipitate was observed after the addition of NH4OH solution . The solution was stirred for 30-min to complete the reaction. Next, the solution was placed on a magnet, which allowed the magnetite to be absorbed to the surface. After the reaction solution was poured off, the magnetite dispersion was added with oleic acid solution in hexane (2.5–10 wt% solution in 20 mL hexane) under constant stirring. Concentration was subsequently performed by evaporation of hexane from the dispersion, and a black magnetite concentrate was obtained. Meanwhile, a stannous octoate catalyst was used for the polyethylene glycol lactide (PEG-LAC) copolymerization reactions. A total of 1.00 g lactide and 0.2 g PEG2000 were mixed in 9.00 mL toluene containing 0.005 g stannous octoate. The reaction balloon was then placed in a hot oil bath (140 °C) to react for 6 h. After the reaction mixture was cooled at room temperature, the copolymer was precipitated with icy diethyl ether liquid and dried in a vacuum oven. To cover the magnetite core surface with PEG-LAC layer, 20 mL of magnetite hexane dispersion was added to 20 mL of copolymer aqueous solution (1 wt% copolymer). Afterwards, the mixture was sonicated for 4 h to transfer the particles from the hexane top layer to the aqueous bottom layer. The residual dispersion in the aqueous phase was then dialyzed using a dialysis bag (1200) and lyophilized. To cover the chitosan layer on the surface of the previously synthesized particles, 0.05 g magnetite particles were dissolved in 1 mL dichloromethane, and then dripped onto an aqueous solution containing 0.2 mL acetic acid and 0.05 g chitosan. After overnight stirring, the solution was filtered through a 0.22 mm filter and lyophilized. Additionally, 0.02 g branched polyethyleneimine  was dissolved in 10 mL deionized water, and added with the same amount (0.02 g) of product from the preparation of chitosan layer while maintaining stirring. Finally, the mixture was placed on a stirrer for 24 h.Fe−1.Fourier transform infrared reflection (FTIR) spectroscopy analysis was performed for Characterization of Fe3O4 magnetic materials. The samples (2 mg) and 200 mg potassium bromide were mixed and ground for 3 min. The mixture was then pressed into pellets for measurement. FTIR spectra were recorded using a FTIR spectrophotometer  at the range of 400–4000 cmThe experimental materials for the synthesis of NPs were provided by Dharmacon . Scrambled control FAM-siBIRC5-NC  and FAM-siBIRC5  were synthesized by Integrated DNA Technologies . In addition, FAM-AS-ODN (anti-sense: 5′-CCCAG-CCTTCCAGTCCCTTG-3′) and FAM-AS-ODN-NC (sense: 5′-CAAGGGACTGGAAGGCTGGG-3′) were purchased from Dharmacon .3O4 MNPs loaded with siBIRC5 solution and AS-ODN, the solution of siBIRC5 (100 ng) in Opti-minimum essential medium (MEM) and AS-ODN (200 ng) was added to the Fe3O4 MNPs. Subsequently, NPs were prepared after vortexing the resulting solution for 10 s and a 30-min incubation at room temperature.To synthesize the Fe3O4 MNPs: siBIRC5: AS-ODN) were subjected to 3% agarose gel electrophoresis at 90 mV for 45 min, after which the gels were stained with ethidium bromide solution and observed under a GelDoc imaging system .NPs size and morphology were observed and analyzed with the help of transmission electron microscopy  and scanning electron microscopy . Particle size was analyzed using the ImageJ software. In addition, Zeta potential and hydrodynamic size of NPs were analyzed by dynamic light scattering . NPs prepared with different mass ratios . The expression patterns of BIRC5 were detected by Western blotting.2.Lung adenocarcinoma cells A549  and H460  were cultured in Dulbecco’s modified eagle medium . Meanwhile, human embryonic lung fibroblasts HFL-1  were cultured in Ham’s F-12 . All media were then supplemented with 10% fetal bovine serum , 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine, which were all purchased from Biological industries . Non-essential amino acids  were employed for HFL-1 culture. Afterwards, the cells were cultured in a humidified incubator  at 37 °C with 5% COA549 cells were transfected with the HA-BIRC5 expression vector (pcDNA3-HA-BIRC5) and Myc-DR5 expression vector (DR5 Myc-tag) or corresponding empty vector (pcDNA) following the instructions of the Lipofectamine 2000 transfection reagent . Cells were transfected with 1 μg of pcDNA3-HA-BIRC5 and DR5 Myc-tag or the corresponding empty vector pcDNA. After a 24-h period of transfection, the cells were treated with NPs for subsequent analyses.To detect the expression patterns of related proteins, highly efficient radio immunoprecipitation (RIPA) lysis buffer  was adopted to extract the total protein content from the cells or tissues according to the manufacturer’s instructions. The supernatant was collected after 15-min lysis at 4 °C and 15-min centrifugation at 12,000 rpm. Protein concentration in the samples was subsequently determined using a bicinchoninic Acid (BCA) Kit . Next, the proteins were mixed with sodium dodecyl sulfate (SDS) loading buffer after quantification, and samples were incubated at 10 °C for 5 min and cooled down naturally at room temperature. Samples and protein markers were then separated using SDS–polyacrylamide gel electrophoresis . After electrophoresis, the gel was rinsed with deionized water and then transferred onto a polyvinylidene fluoride membrane. After the transfer, the membranes were blocked with 5% skimmed milk in tris-buffered saline tween (TBST) for 1 h at room temperature. To visualize the proteins on the membranes, the membranes were incubated with mouse anti-BIRC5 , mouse-anti-DR5 , and mouse anti-human caspase-3 antibodies  overnight at 4 °C. The following day, the membranes were then incubated with the horseradish peroxide (HRP) conjugated secondary antibody  for 1 h at room temperature. Afterwards, the membrane was detected using an enhanced chemiluminescence immunoblotting detection kit. In addition, mouse anti-β-actin antibody  was treated as described previously. For absorbance analysis, scanned photographs were quantified using the AlphaEasy FC software . Each experiment was repeated three times to obtain the mean value.Total RNA content was extracted the cells or tissues using RNeasy Mini kits  following the instructions to determine the transcription expression levels of genes. Complementary DNA (cDNA) was then reverse-transcribed with the help of a reverse transcription kits . Subsequently, mRNA expression levels of related factors were determined using an ABI PRISM 7500 sequence detection system . All primer sequences are shown in Additional file 3O4 MNPs loaded siBIRC5 as p-siBIRC5, while Fe3O4 MNPs loaded AS-ODN as p-AS-ODN. Simultaneously, sense-ODN and scrambled siBIRC5 were employed as the negative control (NC).To explore the uptake of siBIRC5 and AS-ODN, the cells were seeded in a 24-well culture plate and incubated for 12 h. A magnet (magnetic field: 0.5 T) was placed under the center position of the culture plate, and then incubated with AS-ODN (concentration of 200 nM) and siBIRC5 (concentration of 100 nM). Sense-ODN and scrambled siBIRC5 were transfected performed using the Lipofectamine 3000 transfection reagent  as a positive control. FAM labeling was employed to observe the cellular uptake of siBIRC5 and AS-ODN with the help of fluorescence microscopy . Fe5 per well) were then seeded into each well. Following treatment with each preparation and magnetic field (0.5 T), the cells were fixed with 4% paraformaldehyde for 20 min, and then permeabilized with 0.2% Triton X-100 for 10 min. Next, the cells were blocked with 3% horse serum for 1 h at room temperature, and incubated with mouse anti-BIRC5 and mouse-anti-DR5 primary antibodies for 1 h. Afterwards, the cells were incubated with the anti-mouse secondary antibody labeled with tetramethylrhodamine for 1 h. Later, the cells were mounted with DAPI  before imaging with a confocal microscope.Five sterile round coverslips (1 cm × 1 cm) were placed on 6-well plates. Cells , the RT group, and the NPs + RT group. After 24 h, the cells were fixed with 4% paraformaldehyde for 20 min. Fixed cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked with 1% bovine serum albumin (BSA) in PBS (blocking solution) for 30 min, and incubated overnight at 4 °C with mouse monoclonal anti-γ-H2AX antibody (dilution ratio of 1: 400 in blocking solution). Later, the cells were incubated in dark conditions with the FITC conjugated anti mouse antibody (dilution ratio of 1: 200 in blocking solution) for 1 h at room temperature and counterstained with 0.1 µg/mL DAPI for 1 min. Fluorescence pictures were taken on a Nikon A1R spectral confocal microscope and cytation 3 imaging multimode plate reader .Cells were seeded in a 96-well plate with transparent black bottom  at 10Cellular uptake efficiency was determined. Following treatment with each preparation and magnetic field (0.5 T), the cells were rinsed twice with cold PBS. Next, the cells were trypsinized and then analyzed using a flow cytometer . The expression patterns of DR5 were determined after cells were treated with each preparation and magnetic field (0.5 T) and trypsinized. Next, the cells were incubated with the PE conjugated anti-human-DR5 (CD262) antibody  for 30 min. The level of DR5 expression on the cell membrane was subsequently reflected by measuring the PE levels using flow cytometry .5 cells in each well. Four groups  of cells were collected and counted. Annexin V-FITC/PI apoptosis detection kits were adopted for testing. Cell pellets were resuspended in 195 μL binding buffer and stained with 5 μL each of annexin V-FITC and PI staining solution for 10 min at room temperature in dark conditions. Flow cytometry was then performed on a FACScan system with CellQuest software. The apoptosis rate was calculated as follows:  × 100%. Three parallels were set for each group to obtain the mean value.Cell apoptosis was measured. Briefly, lung adenocarcinoma cell lines at the logarithmic phase of growth were seeded in 6-well plates at 2.0 × 105 per well) into 6-well plates. After cells were treated with each preparation and magnetic field (0.5 T), cells were treated with 200 µL of 0.5% Triton X-100 lysis buffer for 30 min in an ice bath. Cell lysates were collected, vortexed briefly, and then incubated on ice for an additional 15 min. Cell debris was removed by centrifugation at 2000 g for 5 min, diluted with 1% BSA in PBS and protein concentration was determined by Coomassie blue assay (Bradford method). A 10 mg sample of total protein was added to a 96-well plate precoated with capture antibody, and BIRC5 protein was determined by ELISA Kit .To measure BIRC5 protein, we seeded cells . After 24 h, the cells were irradiated at doses of 0 Gy (no radiation), 2 Gy, 4 Gy, 6 Gy, and 8 Gy, and incubation was continued for another 48 h. The wells were added with a total of 0.5 mg/mL MTT reagent and treated for 1 h, and then with dimethyl sulfoxide . The absorbance of the samples was measured at 570 nm using a synergy HTX microplate reader (BioTek).137Cs light source , the cells were irradiated at a dose rate of 3.66 Gy/min and further incubated for another 6 h. Next, the cells were trypsinized and counted, and then the cells were cultured in drug-free medium to analyze the colony forming ability. After 2 weeks of culture, the cells were fixed and stained with PBS containing 4% formaldehyde and 0.05% crystal violet, and colonies with greater than 50 cells were counted. Three parallels were set for each group.To evaluate cell proliferation, cells with or without NPs  under a magnetic field (0.5 T), which were irradiated at room temperature with γ-ray at doses of 0, 2, 4, 6, and 8 Gy. Subsequently, using a To measure cell migration, cells were added in triplicate in 6-well plates and incubated until cell grew adherent to the wall. Cells were scratched using a 10 μL pipette. Subsequently, the control, the NPs , the radiotherapy (with radiation at 2 Gy dose), as well as the NPs + RT group were set up. Cells were first incubated in the presence or absence of NPs under a magnetic field (0.5 T), and then treated with or without radiation. After a 24-h period of incubation, the samples were observed using a phase contrast microscope.6 cells/100 μL) into the right rear flank of mice, respectively. The experiment was conducted when the tumor volume grew to 100 mm3. Tumor volume = 0.5 × A2 × B .BALB/C female nude mice (aged 5–6 weeks old) were housed in a specific-pathogen-free grade animal room with room temperature of 25 °C and humidity of 70%, 12-h light/dark cycle with ad libitum access to water and food. Animal experiments were performed by subcutaneous injection of A549 and H460 cell suspensions (2 × 103) was measured every other day, and mouse body weight was recorded. Simultaneously, mice in the control group and the RT group were injected with 0.9% NaCl solution alone at the same time point. On the 30th day, the mice were euthanized, and the tumor tissues were removed for photographing and weight measurement.To explore the effects of radiotherapy in vivo, A549 and H460 subcutaneous tumor bearing mice were randomly divided into the following four groups: (a) the control group (P-NC), (b) the NPs injection group, (c) the radiotherapy alone group (RT) and (d) the NPs combined with RT group (NPS + RT). Following intravenous injection of NPs (25 mg/kg/day), a magnet (magnetic field: 0.5 T) was fixed at the tumor site of the mice. After 24 h, radiotherapy (2 Gy/day) was performed for 5 consecutive days. Tumor volume  control (P-NC), (b) NPs injection group, (c) radiotherapy alone group (RT) and (d) NPs combined with RT group (NPS + RT) for RT-qPCR, Western blotting, and immunohistochemical staining.2O2 for 10 min. Next, the sections were blocked with PBS containing 1.5% blocking serum, and then incubated with the primary antibodies rabbit polyclonal anti-DR5, rabbit polyclonal anti-BIRC5, or rabbit monoclonal anti-Ki67 overnight at 4 °C. Later, the sections were added with appropriate amounts of goat anti-rabbit or anti-mouse secondary antibody working solution  for 1 h incubation at 37 °C. Color was developed using diaminobenzidine (DAB) (ZSGB-Bio) for 3–5 min. Afterwards, the sections were counterstained with hematoxylin, while sections without incubation with primary antibody were regarded as the NC. The results were observed microscopically and evaluated by quantifying the staining intensity and the percentage of stained tumor cells. Expressions were analyzed by two independent investigators blinded to clinical data using a multi-headed microscope.For immunohistochemical staining, after deproteinization, hydration, and antigen retrieval, tumor tissue sections were treated with endogenous peroxidase in 0.3% HTUNEL staining was performed according to the manufacturer’s instructions  and previously established methods .To evaluate the NP biocompatibility, healthy BALB/c mice were injected with NPs (25 mg/kg/day), and changes in body weight were observed over 15 days. Another batch of healthy mice was taken, and blood samples were collected for liver and kidney function indexes and blood routine analysis on days 1, 7, and 15 after injection. Mice were euthanized on day 15. Major organs, including heart, lung, kidney, liver, and spleen, were excised, while tissues were fixed using formalin, embedded with paraffin, and then sectioned. Later, the Sections (4 μm) were stained with hematoxylin–eosin for histological examination.t-test. Data comparisons among multiple groups were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Survival was calculated using the Kaplan–Meier method. A value of p < 0.05 was regarded statistically significant.Statistical analyses were performed using the SPSS21.0 software . Measurement data were expressed as mean ± standard deviation, and two group data were compared following an unpaired design with normal distribution and homogeneous variance, using unpaired 3O4 MNPs by means of FTIR spectroscopy  PEG-LAC, (D) Fe3O4-PEG-LAC chitosan, and (E) Fe3O4-PEG-LAC-chitosan-PEI confirmed the successful modification of each component of the MNPs. As shown Additional file 1, the Fe3O4 MNPs spectrum exhibited a characteristic absorption band at 570 cm−1, which was an Fe–O bond, indicating that Fe3O4 MNPs were successfully synthesized. Additional file 2 illustrates the infrared spectra for FTIR PEG-LAC. Absorption bands at 900–1200 cm−1 referred to C–O stretching vibrations, where bands between 1200 and 1500 cm−1 indicated the formation of ester bonds. Meanwhile, the strong band at 175 cm−1 evidenced the presence of carbonyl groups (C = O), while the bands at 3000 cm−1 and 3500 cm−1 were indicative of C–H and O–H stretching vibrations, respectively. The FTIR spectrum of Fe3O4-PEG-LAC-chitosan was shown in Additional file 3, wherein the Fe3O4 characteristic band reappeared at 569 cm−1. The band shown here was the same as in Additional file 2) at 2860 cm−1 and 2925 cm−1. As shown in Additional file 4, though Fe3O4-PEG-LAC-chitosan-PEI presented with the same band, the intensity of the NH2 band was increased, while the intensity of other bands was decreased. We presumed this variation was attributed to the use of PEI for surface coating, which provided a higher positive charge through its amido resulting in a change in the spectrum.Firstly, we verified the surface structure modification of prepared Fe3O4 MNPs, we adopted high positive charge distribution on the surface of Fe3O4 MNPs to load AS-ODN and siBIRC5 to obtain NPs. To further verify that AS-ODN and siBIRC5 could form stable complexes with Fe3O4 MNPs, we performed agarose gel electrophoresis after incubating 1 µg of siBIRC5 and 2 µg of AS-ODN with 1, 5, 10, and 20 µg of Fe3O4 MNPs. As shown in Additional file 3O4 MNPs: siBIRC5: AS-ODN = 5: 1: 2 and above. In addition, the unbound siBIRC5 bands disappeared from the surface of the gel. Thereafter, we used NPs prepared at a mass ratio of 5: 1: 2 (Fe3O4 MNPs: siBIRC5: AS-ODN) for subsequent experiments. We subsequently tested the particle size and zeta potential of the NPs. As shown in Additional file 3O4 MNPs was positively charged (+ 75 mV), which provided the preconditions for the formation of electrostatic nanomaterial NPs loaded with AS-ODN and siBIRC5, whereas the prepared NPs also showed a lower positive charge (+ 3.39 mV), which was favorable for cellular uptake.After successfully preparing Fe3O4 MNPs could enhance the uptake of siBIRC5 and AS-ODN by A549 and H460 cells, we incubated cells under a magnetic field with control PBS, free siBIRC5 mixed with free AS-ODN, Fe3O4 loaded with AS-ODN (P-AS-ODN), Fe3O4 loaded with siBIRC5 (P-siBIRC5), positive control Lipo-NC, and NPs, followed by fluorescence imaging. As shown in Fig. 3O4 MNPs effectively delivered siBIRC5 and AS-ODN to A549 and H460 cells. Meanwhile, the results of flow cytometry demonstrated that the mean fluorescence intensity of NPs treated A540 and H460 cells were about 30 times higher than those of free AS-ODN and siBIRC5 by delivery of Fe3O4 MNPs with AS-ODN and siBIRC5, while the uptake efficiency could reach 80% in both cells  containing sense-ODN and scrambled siBIRC5 exhibited only marginal effects. On the other hand, delivery of AS-ODN (P-AS-ODN) and siBIRC5 (P-siBIRC5) groups alone using Fe3O4 MNPs down-regulated BIRC5 mRNA expression levels by 37% and 30% in A549 cells and by 35% and 31% in H460 cells, respectively. However, the use of NPs simultaneously loaded with AS-ODN and siBIRC5 down-regulated BIRC5 mRNA levels by 60% and 58% in A549 and H460 tumor cells, respectively. As shown in Fig. 3O4 MNPs could also down-regulate BIRC5 protein expression levels in A549 and H460 cells, showing only 36% and 38% BIRC5 positive cells, respectively. However, there were 51% and 58% BIRC5 positive cells in A549 cells and 52% and 60% BIRC5 positive cells in H460 cells treated with P-AS-ODN and P-siBIRC5, respectively. Furthermore, the results of ELISA were consistent with the results of immunofluorescence staining. As shown in Fig. 3O4 MNPs effectively and simultaneously delivered AS-ODN and siBIRC5 to lung adenocarcinoma tumor cells, to reduce the BIRC5 expression in tumor cells via co-inhibition.Both siBIRC5 and AS-ODN could effectively inhibit BIRC5 expression. To verify the whether BIRC5 expression could be co-regulated by the co-delivery of siBIRC5 and AS-ODN by FeFurthermore, we incubated NPs at different concentrations with A549 and H460 cells under a magnetic field, and found that DR5 expression levels in tumor cells were also increased with increasing concentrations of NPs Fig. A, B. As Additionally, we investigated the radiosensitization of NPs in vitro. As shown in Additional file After verifying that the NPs conferred radiosensitizing effects in vitro, we established subcutaneous A549 and H460 tumor xenografts in mice to directly verify whether the NPs loaded with siBIRC5 and AS-ODN could also exhibit radiosensitizing effects in vivo. After the mice were intravenously injected with NPs and maintained under a magnetic field, the tumor bearing mice were subjected to radiotherapy with tumor growth of mice recorded. In addition, the tumor tissues were weighed. It was found that the NPs combined with RT group presented with reduced weight and stronger growth inhibition effects on the subcutaneous tumors of A549 and H460 cells  can also exert certain effects on human lung adenocarcinoma cells A549 [Following the preparation of Feanalysis . Similarcal bond . These ells A549 .3O4 MNPs could reduce the BIRC5 expression in lung adenocarcinoma cells by delivering siBIRC5 and AS-ODN into lung adenocarcinoma cells simultaneously. On the other hand, the expression of DR5, a member of the tumor necrosis factor receptor superfamily (TNFRSF10B), was previously correlated with radiosensitivity in tumor cells [Subsequent experimentation in our study revealed that NPs exerted a suppressive effect on BIRC5 expression in lung adenocarcinoma cells. As a member of the inhibitor of apoptosis protein family, BIRC5 is known to be up-regulated in various malignancies . BIRC5 ior cells . Interesor cells . To our or cells .3O4 MNPs were without obvious side effects for our in vivo models.Additional in vivo experimentation in our study verified that NPs could enhance the radiosensitivity in vivo by down-regulating BIRC5 and up-regulating DR5, as witnessed by reduced tumor growth and down-regulated Ki67 positive expressions. The latter is particularly important as reduced Ki67 levels are correlated with enhanced radiosensitivity of lung adenocarcinoma after MG132 treatment . Neverth3O4 MNPs could co-deliver siBIRC5 and AS-ODN to lung adenocarcinoma cells, which may be used as a potent radiosensitizer for treating lung adenocarcinoma in radiotherapy. Fe3O4 MNPs-targeted delivery of siBIRC5 and AS-ODN enhances radiosensitivity, which provides a novel therapeutic target for lung adenocarcinoma with a low risk of toxicity. Our prepared Fe3O4 MNPs loaded with AS-ODN and siBIRC5 provide a strong basis for enhancing the sensitivity of patients to radiotherapy. We will further investigate the tracer role Fe3O4 MNPs in vivo in our future endeavors, and also confirm the effect of Fe3O4 MNPs/siBIRC5/DR5 on radiotherapy. However, the novel function of Fe3O4 MNP-targeted siBIRC5 and AS-ODN might be a potential radiosensitizer for treating lung adenocarcinoma in the clinic. In order to accelerate the rapid conversion of Fe3O4 MNPs/siBIRC5/DR5 into clinical use, we will further study the metabolism mechanism of Fe3O4 MNPs/siBIRC5/DR5 in vivo and improve the safety data of internal circulation.To summarize, findings obtained in our study indicated that FeAdditional file1:Figure S1. NPs are successfully prepared. A, Schematic diagram of Fe3O4 MNPs preparation. B, FTIR spectra of intermediates at each step of Fe3O4 preparation. B1: Fe3O4 nuclei; B2: Fe3O4-PEG-LAC; B3: Fe3O4-PEG-LAC-chitosan; B4: Fe3O4-PEG-LAC-chitosan-PEI. C, Binding ability among Fe3O4 MNPs, AS-ODN, and siBIRC5 by electrophoresis . D, TEM images of NPs . E, Size distribution of NPs in Figure A. F, SEM image of NPs .Additional file2:Figure S2. Survival rate of tumor cells at different concentrations of NPs and different radiation doses.Additional file3:Table S1. Primer sequences used for RT-qPCR.