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In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion.

Zhang YT, Huang ZB, Zhang SJ, Zhao JH, Wang Z, Liu Y, Feng NP - Int J Nanomedicine (2012)

Bottom Line: Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions.Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle.The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity associated with the administration of microemulsion on mouse skin fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, Shanghai University of Traditional Chinese Medicine, Shanghai, The People's Republic of China.

ABSTRACT

Objective: To investigate the cellular uptake of evodiamine and rutaecarpine in a microemulsion in comparison with aqueous suspensions and tinctures.

Materials and methods: A microemulsion was prepared using the dropwise addition method. Mouse skin fibroblasts were cultured in vitro to investigate the optimal conditions for evodiamine and rutaecarpine uptake with different drug concentrations and administration times. Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions. Rhodamine B labeling and laser scanning confocal microscopy (LSCM) were used to explore the distribution of fluorochrome transferred with the microemulsion in fibroblasts. Cellular morphology was also investigated, using optical microscopy to evaluate microemulsion-induced cellular toxicity.

Results: The maximum cellular drug uptake amounts were obtained with a 20% concentration (v/v) of microemulsion and an 8 hour administration time. Drug uptake by mouse skin fibroblasts was lowest when the drugs were loaded in microemulsion. After incubation with rhodamine B-labeled microemulsion for 8 hours, the highest fluorescence intensity was achieved, and the fluorochrome was primarily distributed in the cytochylema. No obvious cellular morphologic changes were observed with the administration of either the microemulsion or the aqueous suspension; for the tincture group, however, massive cellular necrocytosis was observed.

Conclusion: The lower cellular uptake with microemulsion may be due to the fact that most of the drug loaded in the microemulsion vehicle was transported via the intercellular space, while a small quantity of free drug (released from the vehicle) was ingested through transmembrane transport. Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle. The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity associated with the administration of microemulsion on mouse skin fibroblasts.

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Effect of different formulations (A) microemulsion, (B) tincture, and (C) aqueous suspension, on the amount of Evo and Rut uptake by mouse skin fibroblasts.
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f5-ijn-7-2465: Effect of different formulations (A) microemulsion, (B) tincture, and (C) aqueous suspension, on the amount of Evo and Rut uptake by mouse skin fibroblasts.

Mentions: A tincture and an aqueous suspension were used as comparative formulations for further study. Under the same conditions (20% concentration of each formulation, 8 hour incubation time), the results (Figure 5) showed that the uptake amounts for the microemulsion were the smallest and those for the aqueous suspension were the largest. Evo and Rut are small molecules and highly lipid soluble. When dispersed in water, they are likely to be absorbed by cells. However, high amounts of alcohol in the tincture caused massive cellular necrocytosis, which resulted in a considerably lower drug uptake in this group, as compared with the aqueous suspension group. The mechanisms of nanoparticle-cell interaction are still not completely understood. dos Santos et al indicated that the internalization of (nano)particles was highly size dependent for all the cell lines studied, and that (nano)particle uptake may not follow the commonly defined size limits for uptake processes.28 Their results highlight the variability of uptake kinetics for one material in different cell types.28 In general, extracellular matrix proteins have a negative charge, and the particles with a positive charge can adhere to the cell membrane. On the contrary, particles with a positive charge may weaken the adhesivity of the cell.29,30 Other reports31,32 have shown that nanoparticles have a positive surface charge that can attract and combine nonspecific static from the negative charges of glycoprotein, proteoglycan, and phospholipid on the cell surface, and that this effect depends on the quantity of positive charges on the surface of the nanoparticle and on the negatively charged macromolecules from the cytomembrane. For example, liposomes have a positive surface charge that can increase the affinity between nanoparticles and cells.33–35 The microemulsion with the zeta potential −1.02 ± 0.18 was expected to have a negative surface charge on the basis of the presented composition, which may have adversely affected the adhesivity of the cell, further decreasing cellular uptake. Research has also shown that the incubation temperature can influence the cellular uptake of nanoparticles, as the uptake efficiency at 37°C is higher than that at 4°C, which indicates that the uptake process is energy dependent.36 The microemulsion used in this study consisted of droplets with an average size of 74 ± 7.07 nm, but the amount of cellular uptake was lower than that with the aqueous suspension. It is possible that a large distribution of droplet sizes hindered cellular uptake (Table 1).


In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion.

Zhang YT, Huang ZB, Zhang SJ, Zhao JH, Wang Z, Liu Y, Feng NP - Int J Nanomedicine (2012)

Effect of different formulations (A) microemulsion, (B) tincture, and (C) aqueous suspension, on the amount of Evo and Rut uptake by mouse skin fibroblasts.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3367489&req=5

f5-ijn-7-2465: Effect of different formulations (A) microemulsion, (B) tincture, and (C) aqueous suspension, on the amount of Evo and Rut uptake by mouse skin fibroblasts.
Mentions: A tincture and an aqueous suspension were used as comparative formulations for further study. Under the same conditions (20% concentration of each formulation, 8 hour incubation time), the results (Figure 5) showed that the uptake amounts for the microemulsion were the smallest and those for the aqueous suspension were the largest. Evo and Rut are small molecules and highly lipid soluble. When dispersed in water, they are likely to be absorbed by cells. However, high amounts of alcohol in the tincture caused massive cellular necrocytosis, which resulted in a considerably lower drug uptake in this group, as compared with the aqueous suspension group. The mechanisms of nanoparticle-cell interaction are still not completely understood. dos Santos et al indicated that the internalization of (nano)particles was highly size dependent for all the cell lines studied, and that (nano)particle uptake may not follow the commonly defined size limits for uptake processes.28 Their results highlight the variability of uptake kinetics for one material in different cell types.28 In general, extracellular matrix proteins have a negative charge, and the particles with a positive charge can adhere to the cell membrane. On the contrary, particles with a positive charge may weaken the adhesivity of the cell.29,30 Other reports31,32 have shown that nanoparticles have a positive surface charge that can attract and combine nonspecific static from the negative charges of glycoprotein, proteoglycan, and phospholipid on the cell surface, and that this effect depends on the quantity of positive charges on the surface of the nanoparticle and on the negatively charged macromolecules from the cytomembrane. For example, liposomes have a positive surface charge that can increase the affinity between nanoparticles and cells.33–35 The microemulsion with the zeta potential −1.02 ± 0.18 was expected to have a negative surface charge on the basis of the presented composition, which may have adversely affected the adhesivity of the cell, further decreasing cellular uptake. Research has also shown that the incubation temperature can influence the cellular uptake of nanoparticles, as the uptake efficiency at 37°C is higher than that at 4°C, which indicates that the uptake process is energy dependent.36 The microemulsion used in this study consisted of droplets with an average size of 74 ± 7.07 nm, but the amount of cellular uptake was lower than that with the aqueous suspension. It is possible that a large distribution of droplet sizes hindered cellular uptake (Table 1).

Bottom Line: Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions.Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle.The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity associated with the administration of microemulsion on mouse skin fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, Shanghai University of Traditional Chinese Medicine, Shanghai, The People's Republic of China.

ABSTRACT

Objective: To investigate the cellular uptake of evodiamine and rutaecarpine in a microemulsion in comparison with aqueous suspensions and tinctures.

Materials and methods: A microemulsion was prepared using the dropwise addition method. Mouse skin fibroblasts were cultured in vitro to investigate the optimal conditions for evodiamine and rutaecarpine uptake with different drug concentrations and administration times. Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions. Rhodamine B labeling and laser scanning confocal microscopy (LSCM) were used to explore the distribution of fluorochrome transferred with the microemulsion in fibroblasts. Cellular morphology was also investigated, using optical microscopy to evaluate microemulsion-induced cellular toxicity.

Results: The maximum cellular drug uptake amounts were obtained with a 20% concentration (v/v) of microemulsion and an 8 hour administration time. Drug uptake by mouse skin fibroblasts was lowest when the drugs were loaded in microemulsion. After incubation with rhodamine B-labeled microemulsion for 8 hours, the highest fluorescence intensity was achieved, and the fluorochrome was primarily distributed in the cytochylema. No obvious cellular morphologic changes were observed with the administration of either the microemulsion or the aqueous suspension; for the tincture group, however, massive cellular necrocytosis was observed.

Conclusion: The lower cellular uptake with microemulsion may be due to the fact that most of the drug loaded in the microemulsion vehicle was transported via the intercellular space, while a small quantity of free drug (released from the vehicle) was ingested through transmembrane transport. Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle. The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity associated with the administration of microemulsion on mouse skin fibroblasts.

Show MeSH
Related in: MedlinePlus