Intracellular uptake of nanocrystals: Probing with aggregation-induced emission of fluorescence and kinetic modeling

Nanocrystal formulations have been explored to deliver poorly water-soluble drug molecules. Despite various studies of nanocrystal formulation and delivery, much more understanding needs to be gained into absorption mechanisms and kinetics of drug nanocrystals at various levels, ranging from cells to tissues and to the whole body. In this study, nanocrystals of tetrakis (4-hydroxyphenyl) ethylene (THPE) with an aggregation-induced emission (AIE) property was used as a model to explore intracellular absorption mechanism and dissolution kinetics of nanocrystals. Cellular uptake studies were conducted with KB cells and characterized by confocal microscopy, flow cytometry, and quantitative analyses. The results suggested that THPE nanocrystals could be taken up by KB cells directly, as well as in the form of dissolved molecules. The cellular uptake was found to be concentration- and time-dependent. In addition, the intracellular THPE also could be exocytosed from cells in forms of dissolved molecules and nanocrystals. Kinetic modeling was conducted to further understand the cellular mechanism of THPE nanocrystals based on first-order ordinary differential equations (ODEs). By fitting the kinetic model against experimental measurements, it was found that the initial nanocrystal concentration had a great influence on the dynamic process of dissolution, cellular uptake, and exocytosis of THPE nanocrystals. As the nanocrystal concentration increased in the culture media, dissolution of endocytosed nanocrystals became enhanced, subsequently driving the efflux of THPE molecules from cells.

Effect of bioadhesive excipients on absorption of total flavonids from Puerariae Lobatae Radix transporting across Caco-2 cell monolayer / 中草药·英文版

Objective: Pueraria total flavonids (PTF) can treat cardiovascular and cerebrovascular diseases, but it has poor membrane permeability and oral bioavailability. Some excipients, such as carbomer, chitosan, and hydroxypropyl methylcellulose, can improve the oral bioavailability. Traditional in vitro evaluation techniques, including the rat intestinal perfusion and cell line models, cannot evaluate PTF absorption and holistic transporters. Methods: This study evaluated excipients’ adhesiveness and effect on PTF transport across Caco-2 cell monolayer. cDNA microarrays identified gene expression changes in Caco-2 cells exposed to PTF and PTF with excipients, and revealed the mechanism underlying the effect of excipients on PTF absorption. Results: In vitro adhesion and transport experiments across Caco-2 showed that excipients had higher adhesiveness to gastric mucosa and transport efficiency across Caco-2 cells than PTF alone. The interaction of PTF with excipients significantly changed the expression of some genes, which might influence the absorption rate of PTF. Conclusion: Different bioadhesive polymers can improve intestinal absorption of PTF, which was related to some genes affiliated to the ATP-binding cassette (ABC) and solute carrier transporter (SLC) to some extent.

Study on effect mechanism of bioadhesive polymers on panax notoginseng sapo-nins′s absorption by microarray technology / 药学实践杂志

Objective To study the mechanism of bioadhesive materials(Chitosan,Carbomer and hydroxypropyl methyl cellulose(HPMC))on promoting panax notoginseng saponins(PNS)′s oral absorption by microarray.Methods The study was divided into four groups.PNS,PNS-chitosan,PNS-carbomer and PNS-HPMC,microarray were used to investigate the change of genes expression level affiliated to the solute carrier transporter(SLC)and the ATP-binding cassette(ABC)after transpor-ting across Caco-2 cell monolayer,to explore the mechanism of bioadhesive materials′effect on PNS′s absorption.Results Comparing with PNS group ，chitosan and carbomer could significantly increase gene expression level affiliated to SLC transporter ，chitosan could decrease multi-resistant genes and P-gp efflux genes expression level affiliated to ABC transporter . HPMC had no obvious effect on SLC and ABC transporter .Conclusion Chitosan and carbomer increase PNS′s oral due to genes change affiliated to SLC and ABC transporter that could promote absorption and inhibit efflux .The promotion mechanism of HPMC absorption was that it could prolong retention time on absorption site .

Study on absorption activity of five components of Polygonum orientale L. Flower in Caco-2 cell monolayer / 中国药理学通报

Aim To observe the absorption activity of five components of Polygonum orientale L. Flower ex-tract in Caco-2 cell monolayer. Methods The effects of different concentrations, time, temperature, pH and P-glycoprotein inhibitors on Caco-2 cells were investi-gated by UPLC-MS/MS. Results The absorption of five components of Polygonum orientale L. Flower ex-tract presented a concentration-and time-dependent manner, and the uptake of quercetin was reduced in Caco-2 cells after 90 min. There was a highest intake at 37℃,and the uptake of four components was best in acid environment except for the quercetin at pH 6 , which was best at pH5 . The uptake of quercetin and kaempferol was significantly improved after the addition of P-glycoprotein inhibitors of verapamil. Conclusions The cellular uptake mechanisms of the five compo-nents of Polygonum orientale L. Flower extract is man-inly through passive diffusion. P-glycoprotein is in-volved in the uptake of quercetin and kaempferol.

Absorption mechanism of three curcumin constituents through in situ intestinal perfusion method

This study aimed to investigate the absorption mechanism of three curcumin constituents in rat small intestines. Self-emulsification was used to solubilize the three curcumin constituents, and the rat in situ intestinal perfusion method was used to study factors on drug absorption, including drug mass concentration, absorption site, and the different types and concentrations of absorption inhibitors. Within the scope of experimental concentrations, three curcumin constituents were absorbed in rat small intestines through the active transport mechanism.

Progress in oral drug-loaded nano-system / 中国生化药物杂志

Oral drug-loaded nano-system include nano-gel drug delivery system, nano-suspension drug delivery system, nano-particle drug delivery system, liposomes drug delivery system, nano-micelles drug delivery system, alcohol liposoms,nano-framework drug delivery system, nano-emulsions drug delivery system, nano-self assembly drug delivery system.These nano-drug delivery systems can serve as multi-functional drug carriers.They may significantly improve the physicochemical and stabilization and biological properties of the free drug, enhance the therapeutic efficiency and reduce toxic side effects.This paper reviews the recent research progress in oral drug-loaded nano-systems.

Biopharmaceutics classification and absorption mechanisms primary study on four kinds of flavonoids / 中国中药杂志

The solubility and permeability on four kinds of flavonoids (kaempferol, hesperidin, apigenin, genistein) were test according to the theory of biopharmaceutics classification system (BCS), and their absorption mechanism. The solubility was investigated by the method in determination of solubility of "Chinese Pharmacopoeia 2010". To detect appearance permeability of compounds mentioned above, the appropriate concentrations were selected by the MTT method in cell transfer experiments in Caco-2 cell model, which established by in vitro cell culture method. Therefore, these compounds were classified with BCS according to solubility and permeability. In addition, to explore absorption mechanisms, the experiments in three different concentrations of compounds in high, medium and low in bidirectional transformation methods in Caco-2 cell model contacted. The study indicated that all of kaempferol, hesperidin, apigenin, genistein have the characteristics in low solubility and high permeability, which belong to BCSⅡ, and the absorption mechanism of kaempferol was active transportation. Whereas, hesperidin, apigenin, genistein were passive transportation. In this study, it carried out initial explorations on establishment of determination for solubility and permeability in flavonoids, and provided theoretical reference for further research on BCS in traditional Chinese medicine.

Absorption mechanism of neobavaisoflavone in Caco-2 cell monolayer mode / 中国中药杂志

Neobavaisoflavone is one of flavonoids of traditional Chinese medicine Psoralea corylifolial. It has numerous biological properties such as antibacterial, anti-inflammatory, anti-cancer, and anti-osteoporosis effects. This paper aimed to investigate the absorption mechanism of neobavaisoflavone in Caco-2 cell monolayer model. The analyte and osalmide were separated on Thermo Syncronis C18 column with methanol-0.1% formic acid solution (90∶10) as the mobile phase, at a flow rate of 0.2 mL•min⁻¹. The concentration of neobavaisoflavone was determined in eletrospray ionization(ESI) positive ion mode with osalmide as an the internal standard. The effects of time, concentration, P-gp inhibitor verapamil, MRP-2 inhibitor MK-571 and BCRP inhibitor Ko143 on the absorption of neobavaisoflavone were investigated. According to the results, neobavaisoflavone showed a good linearity within the concentration of 10-2 000 μg•L⁻¹, and the results of its specificity, matrix effect, extraction recovery, precision, accuracy and stability all met the requirements. In the Caco-2 cell monolayer model, the transport volume of neobavaisoflavone was correlated positively with the time and concentration. The ER values of 15, 30, 50 μmol•L⁻¹ neobavaisoflavone were 1.64, 1.94,0.99, respectively. As compared with the control group, all of verapamil hyduochloride, MK-571 and Ko143 could promote the transportation of neobavaisoflavone, and the effect was more obvious in verapamil hyduochloride and Ko143. The absorption of neobavaisoflavone may be mainly of active transport in Caco-2 cell monolayer model, and also involve passive transport. Excretion mechanism of intestinal transport protein may be also involved.

Transport characteristics of hydroxysafflor yellow A across Caco-2 cell monolayer model / 中草药

Objective: To investigate the transport characteristics of hydroxysafflor yellow A (HSYA) in Danhong Injection across Caco-2 cell monolayer. Methods: Safe concentration range of HSYA against Caco-2 cell monolayer model was selected by MTT method; The effects of time, drug concentration, temperature, pH, P-gp inhibitor (Verapamil), and energy metabolism inhibitor (sodium azide) on the absorption of HSYA were observed by Caco-2 cell monolayer model; The multidrug resistance (MDR1) gene expression in Caco-2 cells was analyzed by the RT-PCR method. Results: The Papp of HSYA transport from apical (AP) side to basolateral (BL) side was in 2 × 10-6-5 × 10-6 cm/s, which showed a medium absorption. The transport of HSYA was positively correlated with time and concentration. The Papp of HSYA transport at 37°C has significant differences with those at 4 and 25°C (P < 0.01). The Papp of HSYA transport under pH 9.0 has significant differences with those under pH 5.0 and 7.4 (P < 0.01). The gene expression of MDR1 was significantly reduced by Verapamil, but the transport of HSYA was not influenced by Verapamil and sodium azide, the number of Papp(BL→AP)/Papp(AP→BL) was between 1 and 1.5, so the absorption of HSYA was basically in line with the passive diffusion. Conclusion: The transport of HSYA across Caco-2 cell monolayer model is passive diffusion, and is not influenced by the change of P-gp and energy metabolism. Low temperature and alkaline environment are not conducive to the absorption of HSYA.

Transport characteristics of ligustrazine across Caco-2 cell monolayer model and its effect on P-glycoprotein expression / 中草药

Objective: To investigate the transport characteristics of ligustrazine across Caco-2 cell monolayer and the effect on P-glycoprotein (P-gp) expression. Methods: Safe concentration range of ligustrazine against Caco-2 cell monolayer model was selected by the MTT method. The mechanism of ligustrazine bidirectional transport was investigated by Caco-2 cell monolayer model. The influences of time, concentration, and P-gp inhibitor Verapamil on the transport of ligustrazine were studied using apparent permeability coefficient (Papp) as index. P-gp expression in Caco-2 cells was analyzed by the Western blotting method. Results: The Papp of transport from apical (AP) side to basolateral (BL) side was over 10-6 cm/s, which showed a good absorption. In the Caco-2 cell model, the transport amount of ligustrazine was positively correlated with time and concentration, and the transport amount from AP side to BL side was higher than that from BL to AP. The absorption of ligustrazine was not only rejected by P-gp, but also the P-gp expression was inhibited by ligustrazine. Conclusion: The transport of ligustrazine across Caco-2 cell monolayer model is deduced as passive transport, ligustrazine is rejected by P-gp, and there is an inhibition of ligustrazine on the expression of P-gp.

Study on intestinal absorption mechanism and kinetics of dauricine in rats with in situ single-pass perfusion model / 中国药学杂志

OBJECTIVE: To investigate the absorption characteristics of dauricine in rat intestine. METHODS: In situ single-pass perfusion model was used and the concentrations of dauricine in perfusate were determined by HPLC. The effects of perfusion rates, intestinal segments and drug concentrations on the intestinal absorption of dauricine were studied. RESULTS: The absorption rate and absorption degree of dauricine increased with the perfusion rates(0.1, 0.2 and 0.4 mL · min-1)(P0.05); at high, middle and low concentrations, the drug absorption rate constants(Ka) were (2.36±0.0073) × 10-2, (3.73 ± 0.0052) × 10-2 and(5.62 ± 0.0136) × 10-2 min-1, respectively, the apparent permeation coefficients(P.,) were(2.02±0.0002) × 103, (3.10±0.0007) × 10-3 and (5.31±0.0010) × 10-3 cm · min-1, respectively, the absorption percentages(P%) were 8.66%, 10.17% and 19.06%, respectively, and the accumulate absorption amount and accumulate absorption percentages of different concentrations at different time were very low. CONCLUSION: The absorption degree of dauricine increases with perfusion rates; there is no specific absorption site in the whole rat intestinal tract; the absorption of dauricine is very poor and the active transport is involved in the absorption mechanism of dauricine.

Transport mechanism of puerarin and the influence of Angelica dahurica extracts on absorption of puerarin in Caco-2 cell model / 中国药学杂志

OBJECTIVE: To study the transport properties of puerarin across Caco-2 cell membrane and determine the influence of Radix Angelicae Dahuricae extract on the transport of puerarin using the well-characterized, human-based intestinal Caco-2 cell model as a platform. METHODS: The characteristics of the bidirectional transport of puerarin was investigated. The effects of time, drug concentration, pH, P-gp inhibitor and MRP inhibitor on the absorption of puerarin were observed. Then the influence of the extract of Radix Angelicae Dahuricae on the transport of puerarin was studied. Drug concentration was measured by HPLC and the apparent permeability coefficients (Papp) and apparent permeability ratio (PDR) were calculated. RESULTS: Puerarin was better absorbed in weak acid of pH5.5. The Ka and Papp of puerarin was of concentration-dependent, and the transport showed saturation with increasing time and concentration. When P-gp/MRP inhibitors were added to the model, the Papp AP-BL of puerarin in Caco-2 cell increased and the PappBL-AP/PappAP-BL puerarin reduced. The absorption of puerarin was improved when combined with Radix Angelicae Dahuricae. CONCLUSION The intestinal absorption of puerarin in Caco-2 cell monolayer model is mainly a passive diffusion process, and active transportation mediated by P-gp and MRP transporter is also involved. Radix Angelicae Dahuricae can enhance the intestinal absorption of puerarin.

Review on Biosorption of Heavy Metal by Saccharomyces cerevisiae / 中国生物工程杂志

Heavy metal pollution has become one of the most serious environmental problems today. Biosorption, regarded as a cost-effective biotechnology for treating heavy metal of low concentration in wastewater, has not been utilized at large scale successfully. It’s helpful to increase the knowledge of biosorption mechanism and decreasing the costs of biosorbents for the biosorption application. The yeast of Saccharomyces cerevisiae is an ideal biomaterial to be used for exploring the mechanism and for actual utilization because of its unique characteristics in spite of its relatively mediocre capacity of metal uptake to other fungi. The yeast can grow easily in cheap media, and is widely used in food and beverage manufacture. It’s also a safe by-product in large quantity as a waste of the fermentation industry, and easily manipulated at molecular level. The metal uptake specifically by S. cerevisiae was addressed. Firstly, it was discussed to use dead or live cells in biosorption . The yeast can absorb toxic heavy metals (Pb, Hg, Cd, etc), precious metals (Au, Ag, Pd, etc) and radionuclides (U, Am, etc). Secondry, metal-binding capacity of various heavy metals by S. cerevisiae in different conditions were compared. Lead and uranium, for instances, can be effectively removed from dilute solutions, while copper is not easily removed. Thirdly, various mechanism of metal uptake by S. cerevisiae were summarized in details according to the position in which metals are located. Metal uptake process is influenced by the ratio of the initial concentration of metal ions and the concentration of biomass. Cellular wall and its components are important for metal uptake. Functional groups for metallic ion fixation have been identified. Uptake is typically accompanied by ion exchange and complexation, sometimes with precipitation (for Pb) and redox (for Au or Ag). Intracellularly accumulated metal is associated with the cell membrane, vacuole and GSH, but may also be bound to other cellular organelles and biomolecules. The equilibrium and kinetic models used in the metal-yeast biosorption systems were also introduced. In most cases, classic Langmiur model and Freundlich model, widely used to describe single metal biosorption system of equilibrium, fit the experimental data very well. Pseudo-second order equation is often employed to describe biosorption process by S. cerevisiae. Finally, futher researches in metal biosorpiton by S. cerevisiae were proposed.

Caco-2 cell model——an effective tool for the research of drug absorption / 中国药理学通报

The absorption process is an important factor in determining the bioavailability of orally administered drugs. however, the absorption mechanism of many drugs is not clear. Caco-2 cell model is the best in vitro absorption model nowadays. It is widely used in the research on drug absorption process and absorption mechanism, especially in the aspect of traditional Chinese medicine absorption, the use of Caco-2 cell model has become the hot spot recently. Mo- reover, Caco-2 cell model is also applied in the research on drug metabolism. Therefore, Caco-2 cell model will become an important method in the research of drug absorption, and will be helpful to accelerate the process of new drug screening and development.