Preparation and in vitro evaluation of forsythoside A-loaded exosomes / 中国中药杂志

A drug delivery system of forsythoside A-loaded exosomes(FTA-Exos) with high biocompatibility and low immunogenicity was established to investigate its impact on the migration of human lung epithelial adenocarcinoma A549 cells. The exosomes from A549 cells were extracted and purified by ultra-high speed centrifugation and ultrafiltration. FTA-Exos were prepared by ultrasonic incubation, and characterized by particle size analysis, transmission electron microscopy, and Western blot assay. The uptake of FTA-Exos by A549 cells was observed under the laser confocal microscope, and the impact of FTA-Exos on the migration of A549 cells was investigated by cell scratch assay. The results showed that the average particle size of the prepared FTA-Exos was(138.90±2.37) nm, which increased slightly after drug loading. The PDI was 0.291±0.013, and the average potential was(-10.1±0.66) mV. The FTA-Exos were spheroidal in appearance as observed by transmission electron microscope, with an obvious saucer-like double-layer membrane. Western blot assay indicated that the specific proteins CD63 and Alix were both expressed in exosomes. The laser confocal microscopy suggested that FTA-Exos were taken up by A549 cells and stably maintained in the cell for 4-8 h, and the fluorescence was significantly enhanced at 4 h. The scratch assay showed that the inhibitory effect of FTA-Exos on the migration of A549 cells was significantly stronger than that of forsythoside A(P < 0.05). In conclusion, the drug delivery system of FTA-Exos established in this study had good stability, reliable preparation process, and potent inhibitory effect on the migration of A549 cells in vitro, which can provide an important reference for subsequent in-depth research and application.

Preparation of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes and determination of encapsulation efficiency / 中草药

Objective To develop a method to determine the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes. Methods In this paper, the thin-film rehydration method was used to prepare doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes. Liposomes and free drugs were separated by dialysis, gel microcolumn centrifugation, and ultra-high speed centrifugation. The content of free drugs and drugs in liposomes was determined by HPLC, and the entrapment efficiency of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes was calculated. Results The optimal formulation of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes was DPPC-DSPE-PEG2000-TAIII-DOX with a molar ratio of 5:1:1:1. The liposomes prepared using thin-film rehydration method had a well-defined spherical shape with a size of (55.4 ± 0.40) nm, a PDI of (0.20 ± 0.02), and a weakly negative zeta potential of (-17.4 ± 0.6) mV. The excipients in the liposomal formulation can be well separated from doxorubicin hydrochloride and timosaponin AIII in the selected chromatographic conditions. The calibrated linear curve of doxorubicin hydrochloride was within 24.9-498.0 μg/mL (r = 0.999 9) and that of timosaponin AIII was within 50.55-1 011.0 μg/mL (r = 0.999 6). Free doxorubicin hydrochloride and timosaponin AIII were well separated from liposome by gel microcolumn centrifugation, and the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AIII was (85.12 ± 1.27)% and (76.51 ± 0.46)% respectively. Conclusion The thin-film dispersion- method can be used for the preparation of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes. The method of gel microcolumn centrifugation is accurate, reproducible, simple, and suitable for determination of the encapsulation efficiency of co-loaded liposomes.

Removing Lipemia in Serum/Plasma Samples: A Multicenter Study

BACKGROUND: Lipemia, a significant source of analytical errors in clinical laboratory settings, should be removed prior to measuring biochemical parameters. We investigated whether lipemia in serum/plasma samples can be removed using a method that is easier and more practicable than ultracentrifugation, the current reference method. METHODS: Seven hospital laboratories in Spain participated in this study. We first compared the effectiveness of ultracentrifugation (108,200×g) and high-speed centrifugation (10,000×g for 15 minutes) in removing lipemia. Second, we compared high-speed centrifugation with two liquid-liquid extraction methods—LipoClear (StatSpin, Norwood, USA), and 1,1,2-trichlorotrifluoroethane (Merck, Darmstadt, Germany). We assessed 14 biochemical parameters: serum/plasma concentrations of sodium ion, potassium ion, chloride ion, glucose, total protein, albumin, creatinine, urea, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase, aspartate-aminotransferase, calcium, and bilirubin. We analyzed whether the differences between lipemia removal methods exceeded the limit for clinically significant interference (LCSI). RESULTS: When ultracentrifugation and high-speed centrifugation were compared, no parameter had a difference that exceeded the LCSI. When high-speed centrifugation was compared with the two liquid-liquid extraction methods, we found differences exceeding the LCSI in protein, calcium, and aspartate aminotransferase in the comparison with 1,1,2-trichlorotrifluoroethane, and in protein, albumin, and calcium in the comparison with LipoClear. Differences in other parameters did not exceed the LCSI. CONCLUSIONS: High-speed centrifugation (10,000×g for 15 minutes) can be used instead of ultracentrifugation to remove lipemia in serum/plasma samples. LipoClear and 1,1,2-trichlorotrifluoroethane are unsuitable as they interfere with the measurement of certain parameters.

Study on the Stability Evaluation of 12 Kinds of Submicro Emulsion in Market and the Test Method for the Stability / 中国药房

OBJECTIVE:To evaluate the stability of 12 kinds of submicro emulsion in market,and screen the test method for the stability. METHODS:12 kinds of submicro emulsion in market were selected,high pressure sterilization (121 ℃,30 min), high speed centrifugation(4000 r/min,15 min),accelerated test [placing 6 months under temperature of(40±2)℃,relative hu-midity of (75 ± 5)%] were conducted to investigate the pH,particle size and other indexes,and SPSS 22.0 was used to analyze the distribution variance and chi-square test,and investigate the correlation of 3 evaluation methods. RESULTS:In terms of stabili-ty investigation,the pH value of 12 kinds of submicro emulsion decreased to some extent after accelerated test,average particle size of 6 kinds of submicro emulsion samples were greater than 300 nm,the variance of the particle size distribution of 9 kinds ap-peared in 0.05-0.15,the chi-square test results of 8 kinds distributed below 1. The average particle size of 4 kinds of submicro emul-sions changed more than 10 nm after accelerated test. In terms of stability test method,Pearson chi-square progressive significance of high pressure sterilization and accelerated test was 0.665,which was higher than 0.05,indicating there was no correlation (no significance),the stability results of high pressure sterilization can not represent the results of accelerated test;that of high speed centrifugation and accelerated test was 0.004,which was lower than 0.05,indicating stability results between high speed centrifuga-tion and accelerated test results were significantly correlated. CONCLUSIONS:The submicro emulsion in market can meet the re-quirements of stability. To a certain extent,high speed centrifugation can replace the acceleration test.

Determination of entrapment efficiency of coumarin 6-loaded PLGA nanoparticles by high-speed centrifugation / 中国药学杂志

OBJECTIVE: To establish a method for determination of the entrapment efficiency of coumarin 6-loaded PLGA nanoparticles by high-speed centrifugation. METHODS: The nanoparticle suspensions were diluted by different vehicles, and free drugs were then separated by high-speed centrifugation. The separation effect for free drug and nanoparticles as well as the effect of diluting solvent on nanoparticles morphology were observed by scanning electron microscopy. The method was validated and the determination condition was optimized. RESULTS: The calibration curve for coumarin 6 had good linearity in the range of 0.8-100 ng(r=1.000 0), and the precision were high with RSD≤1.28%. After diluting the nanoparticle suspensions with 1% TPGs, high-speed centrifugation could effectively separate the free drug from nanoparticles. The recovery of free drug was 99.25%-103.00%. The average entrapment efficiencies of coumarin 6-loaded nanoparticles was 88.74% with RSD of 0.65%. CONCLUSION: The method is rapid, accurate and feasible. It can be used to determine the entrapment efficiency of coumarin 6-loaded PLGA nanoparticles.

Research on key parameters of eliminating interference of chyle by highˉspeed centrifugation technique / 国际检验医学杂志

Objective To explore the key parameters of eliminating the interference of chyle by adopting the high-speed centrifu-gation technique.Methods To establish the chylemia by adding chylomicrons to background serum,centrifuge the same model un-der five different parameters,compare the biochemical indicators of lower serum with background by matched t-test,and to analyze the relative bias of the experimental group with statistical differences.Results Compared with the background serum,for ALT, AST,TP,ALB,ALP,GGT and Fe,the correlation coefficient was higher and the differences had no statistical significance(P >0.01 )when centrifuging for 20 min by RCF 29 703 g;for CK and CHE,the correlation coefficient was higher and the differences had no statistical significance(P >0.01)when centrifuging for 15 min by 20 672 g;for Zn,the correlation coefficient was higher and the differences had no statistical significance(P >0.01)when centrifuging for 5 min by 9 168 g.For Glu,LDH,CRE,Urea,UA,Ca and P,all the experimental groups had statistically significant differences(P <0.01),but the relative bias in each group were within the acceptable range.For Ca and P,the correlation coefficient was higher when centrifuging for 15 min by 20 672 g.Conclusion It is recommended to eliminate the interference of chyle on ALT,AST,TP,ALB,ALP,GGT,Fe,Glu,LDH,CRE,Urea and UA by u-sing the centrifugal parameters(29 703 g,20 min),on CK and CHE by using the centrifugal parameters(20 672 g,15 minutes)and on Zn,Ca and P by using the centrifugal parameters(9 168 g,5 minutes).

Neural Stem Cell Harvest and Culture using the High Speed Centrifugation from Rat Brain / 대한마취과학회지

BACKGROUND: During recent two decades of crucial revision of some cornerstone concepts has opened new horizons in neurosciences. Modern basic viewpoints include the idea of high CNS plasticity which means not only rearrangement of neurons and their interconnections, but also the formation of new neural cells in humans and animals during their whole life span. The purpose of this study is to harvest neural stem cell from the adult rat brain using the high speed centrifugation method and study the characteristics of these cell. METHODS: 60 rats (Fisher 344, 150-160 g) brain were saved under inhalation anesthesia and dissect the subventricular zone under the microscope. The brain tissue was digested with enzyme to make a cell suspension. The cell suspension was processed high speed centrifugation to separate the neural stem/progenitor cells according to the buoyancy. After 2 weeks culture, immuno-staining (O4, GFAP, Nestin, beta-tubulin III and DAPI) were performed and replated the cultured cells. RESULTS: The 2 weeks culture cells were positive 92.8% in Nestin, 91.5% in O4 and 87.6% in Gal-C. But only positive 1.4% in beta-tubulin III and 5.5% in GFAP. And replated cell culture shows similar results compared to the primary culture. CONCLUSIONS: With this high speed centrifugation method, authors can harvest neural stem/progenitor cells from the adult rat brain. Although we have many limitations using these cell in clinical trial, but we can afford to next step on neural stem cell research.

The Cell Survival and Differentiation after Transplantation, Which Harvest from Adult Rat Brain by High-speed Centrifugation Method

OBJECTIVE: Many recent reports have shown that the mature mammalian brain harbors multipotent stem cells, rendering the brain capable of generating new neurons and glia throughout life. Harvested stem cells from an adult rat are transplanted in order to evaluate the cell survival and differentiation. METHODS: Using a percoll gradient with a high speed centrifugation method, we isolate neural stem/progenitor cells were isolated from the subventricular zone(SVZ) of a syngeneic adult Fisher 344 rats brain. For 14days expansion, the cultured cells comprised of a heterogeneous population with the majority of cells expressing nestin and/or GFAP. After expanding the SVZ cells in the presence of basic fibroblast growth factor-2, and transplanting then into the hippocampus of normal rats, the survival and differentiation of those cells were examined. For transplantation, the cultured cells were labeled with BrdU two days prior to use. In order to test their survival, the cells were transplanted into the dorsal hippocampus of normal adult Fisher 344 rats. RESULTS: The preliminary data showed that at 7days after transplantation, BrdU+ transplanted cells were observed around the injection deposition sites. Immuno-fluorescent microscopy revealed that the cells co-expressed BrdU+ and neuronal marker beta-tubulin III. CONCLUSION: The data demonstrate that the in vitro expanded SVZ cells can survive in a heterotypic environment and develop a neuronal phenotype in the neurogenic region. However more research will be needed to examine the longer survival time points and quantifying the differentiation in the transplanted cells in an injured brain environment.