In vitro release of paclitaxel derivative liposome by paddle membrane binding assay / 中国药科大学学报

@#The in vitro release is an important index to evaluate the quality of liposome formulation.Currently, there is no evaluation method for the in vitro release of liposome in pharmacopoeia of various countries, which leads to the lack of unified standard and safety guarantee for the quality evaluation of liposome formulation.Taking the self-made paclitaxel derivative liposomes as an example, the paddle membrane binding method established by optimizing external release conditions was used to simulate the complete release of paclitaxel derivative drugs in 12 hours under physiological conditions.The results showed that using 0.5% SDS-HEPES as the release medium and a dialysis bag with a molecular weight cutoff of 1 000 kD to release the liposome solution met the requirements and had discrimination ability, providing a reference for the development of drug-loaded liposomes release methods in vitro.

Preparation of PEOz-modified single-walled carbon nanotube paclitaxel delivery system and evaluation of its anti-tumor effect in vitro / 中草药

Objective: To prepare a PEOz modified single-walled carbon nanotube delivery system (PEOz-SWCNT) with the anti- tumor drug paclitaxel (PTX) as a model drug (PTX@PEOz-SWCNT) and evaluate its physical and chemical properties, in vitro drug release, biocompatibility, and in vitro antitumor effects. Methods: PEOz-SWCNT was synthesized by chemical coupling method, and the products were characterized by UV-Vis spectroscopy (UV-Vis) and infrared spectroscopy (FTIR). The particle size and Zeta potential of PEOz-SWCNT were measured. The drug-loaded complex PTX@PEOz-SWCNT was prepared and the loading efficiency and encapsulation efficiency were measured. The dialysis method was used for in vitro drug release. The safety of the application of PEOz-SWCNT was evaluated by in vitro hemolysis test. The MTT method was used to evaluate the biocompatibility of the material and the growth inhibition rate of the drug-loaded complex on MCF-7 cells. The uptake of Coumarin-6 (C6)-labeled vector in MCF-7 cells was examined by fluorescence inversion microscope. Results: The average particle size of PEOz-SWCNT was (219.8 ± 2.9) nm and the Zeta potential was (-35.23 ± 0.74) mV. The loading efficiency of PTX@PEOz-SWCNT was (38.19 ± 0.74) %, and the encapsulation efficiency was (94.38 ± 0.94)%. The drug release rate was significantly accelerated at pH 5.0, showing obvious pH responsiveness. There was no obvious hemolysis when the concentration of PEOz-SWCNT was below 0.4 mg/mL. The biocompatibility of PEOz-SWCNT on Hela cells was good, and the PTX@PEOz-SWCNT could significantly enhance the cell growth inhibition rate on MCF-7 cells. The in vitro antitumor activity test results showed that the cell uptake of the C6@PEOz-SWCNT was increased compared to C6@SWCNT. Conclusion: PTX@PEOz-SWCNT drug delivery system is promising in tumor-targeted drug delivery.

Development of biodegradation process for Poly(DL-lactic acid) degradation by crude enzyme produced by Actinomadura keratinilytica strain T16-1

Background: Plastic waste is a serious problem because it is difficult to degrade, thereby leading to global environment problems. Poly(lactic acid) (PLA) is a biodegradable aliphatic polyester derived from renewable resources, and it can be degraded by various enzymes produced by microorganisms. This study focused on the scale-up and evaluated the bioprocess of PLA degradation by a crude microbial enzyme produced by Actinomadura keratinilytica strain T16-1 in a 5 L stirred tank bioreactor. Results: PLA degradation after 72 h in a 5 L bioreactor by using the enzyme of the strain T16-1 under controlled pH conditions resulted in lactic acid titers (mg/L) of 16,651 mg/L and a conversion efficiency of 89% at a controlled pH of 8.0. However, the PLA degradation process inadvertently produced lactic acid as a potential inhibitor, as shown in our experiments at various concentrations of lactic acid. Therefore, the dialysis method was performed to reduce the concentration of lactic acid. The experiment with a dialysis bag achieved PLA degradation by weight loss of 99.93%, whereas the one without dialysis achieved a degradation of less than approximately 14.75%. Therefore, the dialysis method was applied to degrade a commercial PLA material (tray) with a conversion efficiency of 32%, which was 6-fold more than that without dialysis. Conclusions: This is the first report demonstrating the scale-up of PLA degradation in a 5 L bioreactor and evaluating a potential method for enhancing PLA degradation efficiency.

Determination of the protein-binding rates of gardenioside, paeoniflorin and naringin in Weiyanling granules in different species of plasma / 国际中医中药杂志

Objective To study the protein binding rates ofgardenioside,paeoniflorin and naringin in Weiyanling granules in bovine serum albumin,rat plasma and human plasma.Methods The equilibrium dialysis method was carried to determine the binding-rates of three components in Weiyanling granules in bovine serum albumin,rat plasma and human plasma.The HPLC method was used to determine the mass concentration of three components in weiyanling granules in inner and outer dialysis and to calculate the plasma protein-binding rates.Results The linear relationship,precision,extraction recovery and stability of gardenoside,paeoniflorin and naringin all conformed to the methodological requirements.Within the low,middle and high concentration (5.084,10.04 and 20.08 g/L) of Weiyanling granules,the protein-binding rates of gardenioside and bovine serum albumin were 23.77％ ± 13.39％,10.75％ ± 1.34％,5.82％ ± 6.53％,respectively;the protein-binding rates of paeoniflorin were 51.81％ ± 6.53％,30.12％ ± 6.61％,21.39％ ± 9.45％,respectively;the protein-binding rates of naringin were 70.21％ ± 3.31％,67.61％ ± 1.31％,56.00％ ± 3.46％,respectively;the protein-binding rates of gardenioside and rat plasma were 71.56％ ± 3.00 ％,43.56％ ± 12.22％,40.63％ ± 4.73％,respectively;the protein-binding rates ofpaeoniflorin and rat plasma were 55.72％ ± 1.60％,47.59％ ± 6.33％,40.07％ ± 0.72％,respectively;the protein-binding rates of naringin and rat plasma were 85.85％ ± 3.53％,85.38％ ± 0.99％,79.99％ ± 2.57％,respectively;the protein-binding rates of gardenioside and human plasma were 13.56％ ± 2.40％,3.02％ ± 2.41％,5.82％ ± 2.08％,respectively;the protein-binding rates of paeoniflorin and human plasma were 9.49％ ± 5.23％,5.01％ ± 1.10％,3.98％ ± 1.54％,respectively;the protein-binding rates of naringin and human plasma were 25.04％ ± 2.61％,26.09％ ± 13.82％,20.20％ ± 2.24％,respectively.Conclusions Within 5.084,10.04 and 20.08 g/L,the protein binding-rate of gardenioside,paeoniflorin and naringin were shown asfollow,rat plasma ＞bovine serum albumin ＞ human plasma.There were significant differences in species.

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.

Optimization of prescription process of cantharidin nanostructured lipid carrier by central composite design-response surface methodology / 中草药

Objective: To integrate the toxic component of cantharidin (CTD) into a novel nanostructured lipid carrier (NLC) and optimize the cantharidin nanostructured lipid carrier (CTD-NLC) formulation process to reduce the toxicity of CTD and enhance its targeting. Methods: CTD-NLC was prepared by emulsified ultrasonic dispersion method. The encapsulation efficiency was determined by dialysis method. The average particle size, particle size distribution (polydispersity index, PDI), Zeta potential, encapsulation efficiency, and drug loading were taken as indicators. Univariate investigation and central composite design-response surface methodology (CCD-RSM) were used to optimize the prescription process of CTD-NLC. Multivariate quadratic fitting was used to evaluate the model equation between indicators and factors. The fitted equation was analyzed by the variance analysis and the optimal prescription was predicted by the resonse surface. Results: The optimized CTD-NLC prescriptions were as follow: mass of total lipid was 453.66 mg, solid to liquid lipid ratio of 1:2, total stable dose of 16.9 mg/mL, ratio of Pluronic F68 to egg yolk lecithin (Lipoid E PC S) of 3.88:1, with ultrasound for 30 min (working 2 s, stopping 2 s). The prepared CTD-NLC was clear clarification in appearance with light blue opalescence, the average particle size was (85.99 ± 0.49) nm, PDI was 0.280 ± 0.002, Zeta potential was (-8.21 ± 0.24) mV, encapsulation efficiency was (98.57 ± 0.05)%, and drug loading was (0.65 ± 0.01)%. Conclusion: The fitting model established by CCD-RSM is accurate and reliable. The optimized CTD-NLC distribution is concentrated, with high encapsulation efficiency and good physical stability. It lays a foundation for the subsequent in vitro and in vivo studies of CTD-NLC.

In vitro release behavior and in situ absorption of evodiamine loaded microemulsion / 中国药理学通报

Aim To study the characteristics of in vitro release and in situ absorption of evodiamine load-ed microemulsion (EDM).Methods EDM was pre-pared, its release in pH 1.2 HCl solution and pH 6.8 phosphate buffer solution were studied by dialysis , and the cumulative release rates were calculated .The sin-gle-pass intestinal perfusion was used to study the ab-sorption of EDM in duodenum , jejunum , ileum and co-lon, the absorption in stomach was also studied , and the absorption rate constant ( Ka ) and effective perme-ability (Pef ) of the drug were calculated.The concen-tration of ED was measured by HPLC .Results The cumulative release rate of EDM and ED in pH 1.2 HCl solution was ( 64.76 ±0.73 )％, ( 13.98 ±0.49 )％, respectively , and that of EDM was 4.63 times of ED . In pH 6.8 phosphate buffer solution the cumulative re-lease rate was ( 91.72 ± 0.51 )％, ( 18.34 ± 0.20)％, respectively, which was 5.01 times of ED. The Ka of EDM was more than 3 times of ED, and Pef was more than 2 times of ED .Conclusion Microe-mulsion can improve in vitro release and in situ absorp-tion of ED.

Determination of Rat Plasma Protein Binding Rate of Mesaconine by UPLC-MS/MS / 中国药学杂志

OBJECTIVE: To establish an analytical method for determination of mesaconine in rat plasma or dialysate, and study the rat plasma protein binding rate of mesaconine. METHODS: The equilibrium dialysis method was applied to determine the in vitro plasma protein binding rate of mesaconine. A UPLC-MS/MS method was established to determine mesaconine in the intra- and extradialysis samples and then the plasma protein binding rate was calculated. RESULTS: The specificity of the established UPLC-MS/MS method was very good. The calibration curve of mesaconine had good linearity over the range of 0.156 3 - 10 μg•mL-1. The intra-day and inter-day precisions and recovery met the requirements of methodology. The plasma protein binding rates of mesaconine were 18.73%, 20.20%, 26.59%, and 23.68% at the concentrations of 0.2, 0.5, 2.0, and 5.0 μg•mL-1, respectively. CONCLUSION: The method is simple, accurate and sensitive, and can meet the requirement for analysis method of biological samples. Mesaconine has a low rat plasma protein binding rate.

Study on preparation and in vivo pharmacokinetics in rats of morellic acid B mPEG liposome / 中草药

Objective: To optimize the preparation process of morellic acid B (MAB) mPEG liposomes (MAB-mPEG-LPS), and to study the in vitro release behavior and pharmacokinetics of MAB-mPEG-LPS in rats. Methods: The analytical method of MAB was established; Encapsulation efficiency and particle size were used as the indexes to optimize the mPEG liposomes by orthogonal test, and the highest encapsulation efficiency of MAB-mPEG-LPS was obtained; Transmission electron microscope was used to observe the surface morphology of MAB-mPEG-LPS, and the stability of MAB-mPEG-LPS was measured in 60 d. Dialysis method was also adopted to study the MAB-mPEG-LPS release in vitro; Male SD rats were injected with MAB (1.50 mg/kg), MAB-LPS (1.50 mg/kg), MAB-mPEG-LPS (1.50 mg/kg) via tail vein, and differences in pharmacokinetics parameters of MAB, MAB-LPS, and MAB- mPEG-LPS were compared. Results: The optimized formula of MAB-mPEG-LPS: HSPC was 128 mg, mPEG was 10 mg, HSPC-CHOL was 8∶1. Encapsulation efficiency of MAB-mPEG-LPS was 83.21%. MAB-mPEG-LPS had uniform particle size and smooth surface; In vitro release results showed that the MAB-mPEG-LPS had slow release and long-term effect. It was stable in 60 d; In vivo study showed that t1/2β of MAB in MAB-mPEG-LPS was 66.925 min, which was 4.43 fold to MAB. MAB-mPEG-LPS was 3.29 fold to MA-LPS; AUC0-∞ of MAB in MAB-mPEG-LPS was 241.372 mg∙min/L, which was 3.64 fold to MAB. MAB-mPEG-LPS was 1.99 fold to MAB-LPS. Conclusion: MAB-mPEG-LPS could prolong the circulation time and increase AUC0-∞ of MAB in rats.

Preparation and pharmacokinetics of honokiol long-circulating liposomes / 中草药

Objective To optimize the preparation process of honokiol long-circulating liposomes (HLCL) and study the in vitro and in vivo release. Methods An orthogonal experiment was designed to optimize the composition of HLCL using entrapment efficiency as evaluation indicator. The liposome surface morphology was observed by transmission electron microscope (TEM), and the liposome release in vitro was studied by dialysis method. The concentration of honokiol in rat plasma was determined by the established LC-MS/MS method, and the differences in pharmacokinetic parameters were compared after honokiol and HLCL (20 mg/kg) were orally administered to SD male rats, respectively. Results The optimal composition of HLCL was 8:1:1 for soya phosphatidyl choline-cholesterol-mPEG2000-DSPE, and 1:10 for honokiol-liposome materials with the ultrasonic time of 12 min. Under the optimized conditions, HLCL was sphere with mean particle size of 121.5 nm and mean Zeta potential of -30.8 mV, the encapsulation efficiency and drug-loading content was 84.7% and 10.4%, respectively. In vitro release results showed that the liposomes could be gently and slowly release with the 24 h cumulative release rate at pH 1.2 and pH 6.9 dissolve medium of 80% and 71%, respectively. Based on the pharmacokinetic results, Cmax, tmax, and t1/2 were (23.29 ± 11.76) ng/mL, (0.13 ± 0.05) h and (10.59 ± 5.72) h for HLCL, and (79.34 ± 56.32) ng/mL, (0.30 ± 0.07) h and (4.44 ± 3.14) h for honokiol, respectively. There was no significant difference about the AUC0-∞ following oral administration of honokiol and HLCL at isodose honokiol (20 mg/kg). Conclusion Compared with honokiol, HLCL was released gently and slowly in vitro, absorpted rapidly and eliminated slowly in vivo.

Determination of plasma protein binding rate of five components in Eucommia ulmoides extract / 中国药理学通报

Aim To determine the plasma protein binding rate of five components of Eucommia ulmoides extract. Methods The equilibrium dialysis method was used to study the plasma protein binding rate. The plasma samples were extracted by protein precipitation with methanol. With the use of puerarin as the internal standard, UPLC-MS/MS was carried out to determine the concentration of the five compounds in and out of the dialysis membrane. Results The average plasma protein binding rates of five compounds on the area of the concentration which was determinate were as fol-lows, respectively: geniposidic acid was ( 25. 77 ± 2. 68 )%, protocatechuic acid was ( 57. 54 ± 3. 79)%, chlorogenic acid was (53. 91 ± 3. 00)%, pinoresinol diglucoside was (24. 15 ± 4. 92)%, and pinoresinol singleglucoside was (49. 78 ± 3. 61)%. Conclusions The results show that the binding percentage of geniposidic acid and pinoresinol diglucoside is relatively low, but the binding rate of the others with rat plasma protein is moderate.

Preparation of paeoniflorin lipid liquid crystalline nanoparticles and its in vitro release / 中草药

Objective: To prepare paeoniflorin lipid liquid crystalline nanoparticles (Pae-LLCN), and to study its in vitro release behavior. Methods: Using encapsulation efficiency (EE) as index, the Pae-LLCN were prepared by spontaneous emulsification and ultrasonic method, and the prescription of Pae-LLCN was optimized by orthogonal design. The in vitro release of Pae-LLCN within 24 h was measured by dialysis method, afterwards its morphology and particle size were studied by transmission electron microscope (TEM). Results: The optimal formulation was poloxamer-glycerol monooleate (1∶10), paeoniflorin inventory (40 mg), and PBS solution (20 mL). The average EE was 73.72%, the average DL was 14.81%, the size of nanoparticles was (170 ± 16) nm, and the 24 h in vitro accumulative release rate was 72.68%. Conclusion: The optimized process is rational and feasible, and the Pae-LLCN has good stability with better sustained release in vitro.

Iron bioavailability of different maize genotypes developed in a breeding program: in vitro and in vivo studies

The objective of this study was to evaluate iron bioavailability of maize genotypes, and analyze the correlation between in vitro and in vivo methods. Dialysable iron was analyzed in 13 genotypes from which 5 were selected for the biological assay. Mean iron content of the genotypes (n=13) was 17.93±2.93 mg kg-1. Phytate varied from 0.77% to 1.03%; phytate: iron molar ratio from 30.64 to 55.41; and soluble iron from 13.17 to 39.63%. The highest value for dialysable iron was 19.14%. In the biological assay, the control group, that received ferrous sulphate, did not present significant difference between the genotypes for Hb gain, Hb gain per gram of iron consumed and HRE. Hb gain did not present a significant correlation with in vitro assay. However, there were positive correlations varying from 0.653 to 0.809. The maize genotypes evaluated presented a good bioavailability since the genotypes showed the same result in hemoglobin gain than control group.

Biodisponibilidade de ferro de diferentes genótipos de milho desenvolvidos em programa de melhoramento genético: estudos in vitro e in vivo. O objetivo deste estudo foi avaliar a biodisponibilidade do ferro de genótipos de milho e analisar a correlação entre métodos in vitro e in vivo. Ferro dialisável foi analisado em13 genótipos, a partir do qual 5 foram selecionados para o ensaio biológico. A média de teor de ferro dos genótipos (n= 13) foi 17,93 ± 2,93 mg kg-1. O teor de fitato variou de 0,77% a 1,03%; razão molar fitato:ferro de 30,64 a 55,41; e ferro solúvel de 13,17 a 39,63%.O valor mais alto para o ferro dialisável foi 19,14%. No ensaio biológico, o grupo controle, que recebeu sulfato ferrso, não apresentou diferença significativa entre os genótipos no ganho Hb, ganho de Hb por grama de ferro consumido e HRE. Ganho de Hb não apresentou correlação significativa com o ensaio in vitro. No entanto, houve correlações positivas variando de 0,653 a 0,809. Os genótipos de milho avaliados apresentaram uma boa biodisponibilidade uma vez que os genótipos apresentaram o mesmo resultado quanto ao ganho de hemoglobina em relação ao grupo controle.

Research on Drug Release in Vitro of Sirolimus Liposome / 中国药房

OBJECTIVE:To investigate release characteristics of sirolimus liposome in vitro.METHODS:The concentration of sirolimus was determined by RP-HPLC.In vitro release rate of sirolimus liposome within 24 h was investigated by the reverse dialysis method with 20% ethanol 500 mL as medium.Release curve of sirolimus was fitted with drug release model equation.RESULTS:The linear range of sirolimus were 0.5～20 ?g.mL-1(r=0.999 8)with an average recovery of 99.42%(RSD=1.23%).At the first 4 hours of release,sirolimus liposome released rapidly with accumulative release rate of 50%.After that release rate of liposome was slowed down with accumulative rate of 80% in 24 h.The in vitro release curve conformed to the first order equation.CONCLUSION:Sirolimus liposome has delayed release capability,and in vitro drug release of sirolimus liposome is in concentration dependant manner.