Effects and mechanisms of puerarin against neuroblastoma: insights from bioinformatics and in vitro experiments.

BACKGROUND: Neuroblastoma, a prevalent solid tumor in children, often manifests with hidden onset sites, rapid growth, and high metastatic potential. The prognosis for children with high-risk neuroblastoma remains poor, highlighting the urgent need for novel prognostic models and therapeutic avenues. In recent years, puerarin, as a kind of small molecule drug extracted from Chinese medicine Pueraria lobata, has demonstrated significant anticancer effects on various cancer cell types. In this study, through bioinformatics analysis and in vitro experiments, the potential and mechanism of puerarin in the treatment of neuroblastoma were investigated, and a prognostic model was established. METHODS: A total of 9 drug-disease related targets were observed by constructing a database of drug targets and disease genes. Besides, GO and KEGG enrichment analysis was performed to explore the potential mechanism of its therapeutic effect. To construct the prognostic model, risk regression analysis and LASSO analysis were carried out for validation. Finally, the prognostic genes were identified. Parachute test and immunofluorescence staining were performed to verify the potential mechanism of puerarin in neuroblastoma treatment. RESULTS: Three prognostic genes, i.e., BIRC5, TIMP2 and CASP9, were identified. In vitro studies verified puerarin's impact on BIRC5, TIMP2, and CASP9 expression, inhibiting proliferation in neuroblastoma SH-SY5Y cells. Puerarin disrupts the cytoskeleton, boosts gap junctional communication, curtailing invasion and migration, and induces mitochondrial damage in SH-SY5Y cells. CONCLUSIONS: Based on network pharmacology and bioinformatics analysis, combined with in vitro experimental verification, puerarin was hereby observed to enhance GJIC in neuroblastoma, destroy cytoskeleton and thus inhibit cell invasion and migration, cause mitochondrial damage of tumor cells, and inhibit cell proliferation. Overall, puerarin, as a natural medicinal compound, does hold potential as a novel therapy for neuroblastoma.

γ-Cyclodextrin metal-organic frameworks as the promising carrier for pulmonary delivery of cyclosporine A.

γ-Cyclodextrin metal-organic frameworks (CD-MOFs) are considered as a green and biocompatible material with great potential in drug delivery systems. Original CD-MOFs show the poor aerosol properties, which limit the application in pulmonary drug delivery. To improve the in vitro deposition properties, herein, we synthesized CD-MOFs by the vapor diffusion method using a series of modulators to achieve better pulmonary delivery of cyclosporine A (CsA). The results showed that blank CD-MOFs and drug loaded CD-MOFs prepared with different modulators all preserved the cubical shape, and exhibited the similar crystal form, structural characteristics, thermal behaviors and release properties. In addition, drug loaded CD-MOFs prepared with polyethylene glycol 10000 (PEG 10000) as a modulator exhibited better in vitro aerosol performance than those of synthesized using other modulators, and the in vivo pharmacokinetics data demonstrated that the bioavailability of CsA could be significantly enhanced by inhalation administration of drug loaded CD-MOFs compared with oral administration of Neoral®. The repeated dose inhalation toxicity also confirmed the fine biocompatibility of CD-MOFs as the carrier for pulmonary drug delivery. Therefore, the results demonstrated CD-MOFs as the promising carrier could be used for pulmonary drug delivery.

Appraising the role of circulating concentrations of micronutrients in attention deficit hyperactivity disorder: a Mendelian randomization study.

Previous observational researches have discovered a connection between circulating concentrations of micronutrients and attention deficit hyperactivity disorder (ADHD). However, the results may be influenced by confounding factors and reverse causation. This study aims to explore the causal relationship between circulating concentrations of micronutrients and ADHD using Mendelian randomization (MR). In a two-sample MR context, we used summary data from the major European genome-wide association studies (GWAS) for these illnesses to assess the genetically anticipated effects of circulating concentrations of micronutrients on ADHD risk. In order to achieve this, we took single nucleotide polymorphisms (SNPs) from the GWAS that were highly related with concentrations of nine micronutrients. The corresponding data for ADHD were extracted from the Psychiatric Genomics Consortium. Inverse-variance weighted (IVW) method was used as the main MR analysis, and the reliability of the study's conclusions was assessed using sensitivity analyses. Our MR analyses showed that the copper level may be associated with a reduced risk of ADHD. However, the significance of the research results is weak. There were no clear relationships between other micronutrients and ADHD. Our sensitivity studies confirmed the findings of the primary IVW MR analyses. According to this study, there may be some association between copper level and ADHD, but the significance of the research results is weak, and it is recommended that copper level should be used as a long-term monitoring indicator for further research. The results provide a new idea for the further study of ADHD, and provide guidance for the prevention and treatment of ADHD.

Development of Large Hollow Particles for Pulmonary Delivery of Cyclosporine A.

The purpose of this study was to prepare large hollow particles (LHPs) by spray drying for pulmonary delivery of cyclosporine A (CsA), using L-Leucine (LEU) and hydroxypropyl methylcellulose (HPMC) as excipients and ammonium bicarbonate (AB) as a porogen. The prepared LHPs were spherical particles composed of both CsA and LEU on the surface and HPMC on the inner layer. The formulation of CsA-LEU-0.8HPMC-AB as typical LHPs showed excellent in vitro aerodynamic performance with a minimum mass median aerodynamic diameter (MMAD) of 1.15 µm. The solubility of CsA-LEU-0.8HPMC-AB was about 5.5-fold higher than that of raw CsA, and the dissolution of CsA-LEU-0.8HPMC-AB suggested that the drug was released within 1 h. The cell viability of the A549 cell line showed that CsA-LEU-0.8HPMC-AB was safe for delivering CsA to the lungs. In addition, inhalation administration of CsA-LEU-0.8HPMC-AB with the Cmax and AUC0-∞ increasing by about 2-fold and 2.8-fold compared with the oral administration of Neoral® could achieve therapeutic drug concentrations with lower systemic exposure and significantly improve the in vivo bioavailability of CsA. From these findings, the LHPs, with the advantage of avoiding alveolar macrophage clearance, could be a viable choice for delivering CsA by inhalation administration relative to oral administration.

Cyclosporine A-loaded chitosan extra-fine particles for deep pulmonary drug delivery: In vitro and in vivo evaluation.

In this study, the extra-fine dry powder inhalers (DPIs) with chitosan (CS) as carrier were successfully prepared by ionic gel method combined with spray drying technique for deep pulmonary drug delivery of Cyclosporine A (CsA), using sodium hyaluronate (SHA) and sodium polyglutamate (SPGA) as polyanions. The CsA-loaded DPIs of CS-SHA-CsA and CS-SPGA-CsA were spherical particles with wrinkles on the surface, which were more conducive to improving the aerosol properties. The aerodynamic evaluation of CS-SHA-CsA and CS-SPGA-CsA showed that the fine particle fraction (FPF) reached up to 79.22 ± 2.12% and 81.55 ± 0.43%, while the emitted fraction (EF) reached 77.15 ± 1.46% and 78.29 ± 2.10%. In addition, the mass median aerodynamic diameter (MMAD) was calculated as 1.58 ± 0.04 µm and 1.94 ± 0.02 µm for CS-SHA-CsA and CS-SPGA-CsA, indicating that they were all extra-fine particles (d < 2 µm). These in vitro aerodynamic results showed that CS-SHA-CsA and CS-SPGA-CsA could reach the smaller airways, further improving therapeutic efficiency. The cell viability on A549 cell line results showed that CS-SHA-CsA and CS-SPGA-CsA were safe to deliver CsA to lungs. The in vivo pharmacokinetics consequence proved that inhalation administration of CS-SHA-CsA and CS-SPGA-CsA could significantly improve the bioavailability of CsA in vivo compared with oral administration of Neoral®, effectively reducing the risk of a series of adverse effects caused by systemic overexposure. In addition, the safety and compatibility of DPIs using SHA, SPGA, and CS as carriers for pulmonary drug delivery was verified by in vivo repeated dose inhalation toxicity. From these findings, the extra-fine DPIs with CS as carrier could be a viable delivery option for the deep pulmonary drug delivery of CsA relative to orally administered drug.

[Rapid and simultaneous determination of two immunosuppressants in whole blood by high performance liquid chromatography].

Cyclosporine A and sirolimus are immunosuppressants that are widely used in many organ transplantation procedures. They exhibit some complementary mechanisms of action and interact synergistically when used together. However， they are critical-dose drugs and have a narrow therapeutic index. They provide the desired therapeutic effect with acceptable tolerability only within a specific range of blood concentrations. Therefore， the rapid and simultaneous detection of the concentrations of cyclosporine A and sirolimus in whole blood could provide valuable information on planning medicine administration after organ transplantations. In this study， firstly， the chromatographic behaviors of cyclosporine A and sirolimus on a biological liquid chromatography （BioLC） column and traditional liquid chromatography （TraLC） columns were investigated systematically under the same chromatographic conditions. The results suggested that the peak height and peak width of cyclosporine A and sirolimus on the BioLC column， ZORBAX 300SB C8 （250 mm×4.6 mm， 5.0 µm）， were the highest and narrowest， respectively. The number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column increased significantly when the volume ratio of acetonitrile in the mobile phases was greater than 70%. Their retention time on the BioLC and TraLC columns was negligibly affected by the use of formic acid and trifluoroacetic acid as the mobile phases. In the range of the experimental column temperature， the number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column was significantly higher than that on the two TraLC columns. Furthermore， the relationship between the retention factor and column temperature of cyclosporine A on the ZORBAX 300SB C8 column was different from that on the two TraLC columns. Subsequently， a high performance liquid chromatography method based on the ZORBAX 300SB C8 column was established for the rapid separation and determination of cyclosporin A and sirolimus in whole blood. A sample of whole blood with a volume of 50 µL was prepared by protein precipitation with 1 mol/L sodium hydroxide and then extracted into 500 µL of ether-methanol （95∶5， v/v）. After centrifugation at 14000 r/min for 10 min， the organic layer was removed and evaporated under a stream of nitrogen at 50 ℃. The residue was then reconstituted in 200 µL of methanol for use. Cyclosporin A and sirolimus were separated through isocratic elution on the ZORBAX 300SB C8 column. The column temperature was set at 60 ℃. The mobile phase was acetonitrile-water （70∶30， v/v） and the flow rate was 1.0 mL/min. The detection wavelengths were 205 nm for cyclosporine A and 278 nm for sirolimus. The injection volume was 20 µL. The external standard method was used to quantify cyclosporine A and sirolimus. Under the optimized conditions， cyclosporine A and sirolimus were well-separated within 6 min with a resolution of 3.7 at 205 nm. In addition， the endogenous substances in whole blood negligibly interfered in the detection of sirolimus， while two endogenous substances slightly affected the detection of cyclosporine A. Cyclosporine A and sirolimus both showed good linear relationships in their respective concentration （r>0.997）. The limits of detection （LODs） of cyclosporine A and sirolimus were respectively calculated to be 10 ng/mL and 1 ng/mL based on a signal-to-noise ratio of 3， and the limits of quantification （LOQs） were 30 ng/mL and 2 ng/mL based on a signal-to-noise ratio of 10. In the whole blood samples， the recoveries of cyclosporine A and sirolimus at three spiked levels were in the ranges of 83.5%-89.7% and 95.8%-97.8% with relative standard deviations （RSDs） of 3.2%-9.0% and 3.4%-6.7% （n=5）， respectively. The established method is simple in operation， can be performed with a simple mobile phase， has a short analysis time， and provides a wide linear range and high sensitivity； hence， it can be applied to the determination of cyclosporine A and sirolimus in whole blood.

[Separation of budesonide enantiomers with amylose-tris-[(S)-1-phenylethyl carbamate] chiral stationary phase and determination of its contents in pharmaceutical preparations].

The drug budesonide exists as 22R and 22S enantiomers. However, the drug activity of 22R-budesonide is 2-3 times stronger than that of 22S-budesonide. The development of enantiomeric separation and quantitative analysis methods for budesonide can provide an important basis for its drug development and quality control. At present, the enantiomers of budesonide are separated on a reversed C18 solid phase column. However, chiral stationary phases are rarely reported for the separation of the enantiomers of budesonide. In this study, a high performance liquid chromatography (HPLC) method with a chiral stationary phase was developed for the rapid separation and determination of budesonide enantiomers. The effects of the type of chiral stationary phase, mobile phase additives, and column temperature on the resolution of the budesonide enantiomers were also investigated. The results showed that the chiral stationary phase amylose-tris-[(S)-1-phenylethyl carbamate] was more suitable for the separation of budesonide enantiomers. The mobile phase additives used in the experiment had no significant effect on the chromatographic parameters (peak height, peak width, and resolution) of the budesonide enantiomers. However, with an increase in the column temperature, the peak width of the budesonide enantiomers decreased, while the peak height and resolution increased. The optimized HPLC conditions were as follows: column, Chiralpak AS-RH (150 mm×4.6 mm, 5.0 µm); mobile phase, acetonitrile-water (45∶55, v/v); column temperature, 40 ℃; flow rate, 1.0 mL/min; detector, diode array detector (DAD); detection wavelength, 246 nm; injection volume, 10 µL. The external standard method was used to quantify the budesonide enantiomers. Under the optimized conditions, the enantiomers were well separated, and the retention times of 22R-budesonide and 22S-budesonide were 6.40 min and 7.77 min, respectively. The resolution of the enantiomers was 4.64. The linear ranges of 22R-budesonide and 22S-budesonide were 0.16-1000 µg/mL and 0.20-1000 µg/mL, respectively. The peak area of the enantiomers showed a good linear relationship with the corresponding concentration, and the correlation coefficients (R2) were 0.9999. The limits of detection (LODs) of 22R-budesonide and 22S-budesonide were 0.05 µg/mL and 0.07 µg/mL, respectively, based on a signal-to-noise ratio of 3. The limits of quantification (LOQs) were calculated to be 0.16 µg/mL and 0.20 µg/mL, respectively, based on a signal-to-noise ratio of 10. The recoveries at four spiked levels were in the range of 102.63% to 104.17%, with the relative standard deviations (RSDs) of 0.08% to 0.57% (n=6). The budesonide solution was stored in dark at 4 ℃ for 24 h, and no obvious degradation was observed. Finally, the method was applied to determine four actual samples of budesonide suspension for inhalation in a batch. The samples were dissolved in methanol, filtered through a 0.45 µm microporous membrane, and then analyzed. The amounts of 22R-budesonide and 22S-budesonide in the samples were in the ranges of 283.15-284.63 µg/mL and 259.86-261.51 µg/mL, respectively. This method is simple and rapid, in addition to having good repeatability and high accuracy. It can be used for the resolution of budesonide enantiomers and for quality control in budesonide preparations.

Reducing the vulnerability of network by inserting modular topologies.

In this paper, we present a strategy whose purpose is to reduce the vulnerability of a network via inserting modular topologies. The modular topologies are generated as WS small-world random network, which is relatively highly robust. Using betweenness and betweenness centrality as the vulnerability measurement, the strategy searches for remote nodes with low betweenness in the network and sets these nodes to be connected to the modular topologies. We test our strategy on some basis networks and the results show sufficient availability of our strategy. And by comparing with other methods of adding topologies into the network, we show that our strategy is especially efficient in reducing the vulnerability of the critical network components.