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Toxicology and drug delivery by cucurbit[n]uril type molecular containers.

Hettiarachchi G, Nguyen D, Wu J, Lucas D, Ma D, Isaacs L, Briken V - PLoS ONE (2010)

Bottom Line: This result suggests that CB[7]-bound drug molecules can be released from the container to find their intracellular target.It demonstrates the uptake of containers by cells and intracellular release of container-loaded drugs.These results provide initial proof-of-concept towards the use of CB[n] molecular containers as an advanced drug delivery system.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT

Background: Many drug delivery systems are based on the ability of certain macrocyclic compounds - such as cyclodextrins (CDs) - to act as molecular containers for pharmaceutical agents in water. Indeed beta-CD and its derivatives have been widely used in the formulation of hydrophobic pharmaceuticals despite their poor abilities to act as a molecular container (e.g., weak binding (K(a)<10(4) M(-1)) and their challenges toward chemical functionalization. Cucurbit[n]urils (CB[n]) are a class of molecular containers that bind to a variety of cationic and neutral species with high affinity (K(a)>10(4) M(-1)) and therefore show great promise as a drug delivery system.

Methodology: In this study we investigated the toxicology, uptake, and bioactivity of two cucurbit[n]urils (CB[5] and CB[7]) and three CB[n]-type containers (Pentamer 1, methyl hexamer 2, and phenyl hexamer 3). All five containers demonstrated high cell tolerance at concentrations of up to 1 mM in cell lines originating from kidney, liver or blood tissue using assays for metabolic activity and cytotoxicity. Furthermore, the CB[7] molecular container was efficiently internalized by macrophages indicating their potential for the intracellular delivery of drugs. Bioactivity assays showed that the first-line tuberculosis drug, ethambutol, was as efficient in treating mycobacteria infected macrophages when loaded into CB[7] as when given in the unbound form. This result suggests that CB[7]-bound drug molecules can be released from the container to find their intracellular target.

Conclusion: Our study reveals very low toxicity of five members of the cucurbit[n]uril family of nanocontainers. It demonstrates the uptake of containers by cells and intracellular release of container-loaded drugs. These results provide initial proof-of-concept towards the use of CB[n] molecular containers as an advanced drug delivery system.

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HEK293 toxicology assays.The assay was performed using CB[7], CB[5], 1, 2, and 3 which indicated high cell tolerance of all containers up to a concentration of 1 mM. MTS (A) and AK assays (B) performed after the cells had been incubated with indicated containers and drugs for two days (UT  =  Untreated, C =  Camptothecin, E =  Erythromycin, EE =  Erythromycin Estolate). AK assay was conducted using supernatant from cells seeded for the MTS assay. This and all other figures are representative of three replicate experiments. Statistical analysis for all figures used unpaired t-test analysis with *P = 0.01–0.05; **P = 0.001–0.01; ***P<0.001.
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pone-0010514-g002: HEK293 toxicology assays.The assay was performed using CB[7], CB[5], 1, 2, and 3 which indicated high cell tolerance of all containers up to a concentration of 1 mM. MTS (A) and AK assays (B) performed after the cells had been incubated with indicated containers and drugs for two days (UT  =  Untreated, C =  Camptothecin, E =  Erythromycin, EE =  Erythromycin Estolate). AK assay was conducted using supernatant from cells seeded for the MTS assay. This and all other figures are representative of three replicate experiments. Statistical analysis for all figures used unpaired t-test analysis with *P = 0.01–0.05; **P = 0.001–0.01; ***P<0.001.

Mentions: The MTS and AK assays for all three cell lines were conducted after two days of incubation with the containers at concentrations of 10 µM, 100 µM, and 1 mM. Relative absorbance and luminescence data was normalized to percent cell viability (MTS) and death (AK). MTS assay for HEK293 cells indicated that camptothecin treatment resulted in approximately 59% decrease in cell viability. Erythromycin, at the highest concentration of 1 mM, produced only a slight reduction in metabolic activity (84% viability) while erythromycin estolate induced an ≈25 fold decrease (4% viability). In sharp contrast containers CB[7], CB[5], and 1–3 at 1 mM dose resulted in 94, 96, 98, 100, and 95% cell viabilities, respectively. Consequently, the cell viability in the presence of the containers was comparable to that of the untreated cell population and was significantly higher than values for camptothecin, erythromycin and erythromycin estolate (Figure 2A). These results suggest that all CB[n]-type containers have good biocompatibility. As a complementary method to assess biocompatibility we used the AK assay that measures cell death through the release of adenylate kinase via a luminescence read-out. The relative luminescence units (RLU) values were then converted to relative cell death scale with camptothecin treatment set at an arbitrary value of 100. Thus, the untreated cell population indicated only 18% cell death and CB[7], CB[5], 1, 2, and 3 at a concentration of 1 mM resulted 9, 11, 10, 14 and 14% cell death. In contrast, at a concentration of 1 mM erythromycin (82%) and erythromycin estolate (246%) presented higher values of cell death comparable to the camptothecin treated population (Figure 2B). Erythromycin estolate showed higher cell death than even the camptothecin control.


Toxicology and drug delivery by cucurbit[n]uril type molecular containers.

Hettiarachchi G, Nguyen D, Wu J, Lucas D, Ma D, Isaacs L, Briken V - PLoS ONE (2010)

HEK293 toxicology assays.The assay was performed using CB[7], CB[5], 1, 2, and 3 which indicated high cell tolerance of all containers up to a concentration of 1 mM. MTS (A) and AK assays (B) performed after the cells had been incubated with indicated containers and drugs for two days (UT  =  Untreated, C =  Camptothecin, E =  Erythromycin, EE =  Erythromycin Estolate). AK assay was conducted using supernatant from cells seeded for the MTS assay. This and all other figures are representative of three replicate experiments. Statistical analysis for all figures used unpaired t-test analysis with *P = 0.01–0.05; **P = 0.001–0.01; ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865549&req=5

pone-0010514-g002: HEK293 toxicology assays.The assay was performed using CB[7], CB[5], 1, 2, and 3 which indicated high cell tolerance of all containers up to a concentration of 1 mM. MTS (A) and AK assays (B) performed after the cells had been incubated with indicated containers and drugs for two days (UT  =  Untreated, C =  Camptothecin, E =  Erythromycin, EE =  Erythromycin Estolate). AK assay was conducted using supernatant from cells seeded for the MTS assay. This and all other figures are representative of three replicate experiments. Statistical analysis for all figures used unpaired t-test analysis with *P = 0.01–0.05; **P = 0.001–0.01; ***P<0.001.
Mentions: The MTS and AK assays for all three cell lines were conducted after two days of incubation with the containers at concentrations of 10 µM, 100 µM, and 1 mM. Relative absorbance and luminescence data was normalized to percent cell viability (MTS) and death (AK). MTS assay for HEK293 cells indicated that camptothecin treatment resulted in approximately 59% decrease in cell viability. Erythromycin, at the highest concentration of 1 mM, produced only a slight reduction in metabolic activity (84% viability) while erythromycin estolate induced an ≈25 fold decrease (4% viability). In sharp contrast containers CB[7], CB[5], and 1–3 at 1 mM dose resulted in 94, 96, 98, 100, and 95% cell viabilities, respectively. Consequently, the cell viability in the presence of the containers was comparable to that of the untreated cell population and was significantly higher than values for camptothecin, erythromycin and erythromycin estolate (Figure 2A). These results suggest that all CB[n]-type containers have good biocompatibility. As a complementary method to assess biocompatibility we used the AK assay that measures cell death through the release of adenylate kinase via a luminescence read-out. The relative luminescence units (RLU) values were then converted to relative cell death scale with camptothecin treatment set at an arbitrary value of 100. Thus, the untreated cell population indicated only 18% cell death and CB[7], CB[5], 1, 2, and 3 at a concentration of 1 mM resulted 9, 11, 10, 14 and 14% cell death. In contrast, at a concentration of 1 mM erythromycin (82%) and erythromycin estolate (246%) presented higher values of cell death comparable to the camptothecin treated population (Figure 2B). Erythromycin estolate showed higher cell death than even the camptothecin control.

Bottom Line: This result suggests that CB[7]-bound drug molecules can be released from the container to find their intracellular target.It demonstrates the uptake of containers by cells and intracellular release of container-loaded drugs.These results provide initial proof-of-concept towards the use of CB[n] molecular containers as an advanced drug delivery system.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT

Background: Many drug delivery systems are based on the ability of certain macrocyclic compounds - such as cyclodextrins (CDs) - to act as molecular containers for pharmaceutical agents in water. Indeed beta-CD and its derivatives have been widely used in the formulation of hydrophobic pharmaceuticals despite their poor abilities to act as a molecular container (e.g., weak binding (K(a)<10(4) M(-1)) and their challenges toward chemical functionalization. Cucurbit[n]urils (CB[n]) are a class of molecular containers that bind to a variety of cationic and neutral species with high affinity (K(a)>10(4) M(-1)) and therefore show great promise as a drug delivery system.

Methodology: In this study we investigated the toxicology, uptake, and bioactivity of two cucurbit[n]urils (CB[5] and CB[7]) and three CB[n]-type containers (Pentamer 1, methyl hexamer 2, and phenyl hexamer 3). All five containers demonstrated high cell tolerance at concentrations of up to 1 mM in cell lines originating from kidney, liver or blood tissue using assays for metabolic activity and cytotoxicity. Furthermore, the CB[7] molecular container was efficiently internalized by macrophages indicating their potential for the intracellular delivery of drugs. Bioactivity assays showed that the first-line tuberculosis drug, ethambutol, was as efficient in treating mycobacteria infected macrophages when loaded into CB[7] as when given in the unbound form. This result suggests that CB[7]-bound drug molecules can be released from the container to find their intracellular target.

Conclusion: Our study reveals very low toxicity of five members of the cucurbit[n]uril family of nanocontainers. It demonstrates the uptake of containers by cells and intracellular release of container-loaded drugs. These results provide initial proof-of-concept towards the use of CB[n] molecular containers as an advanced drug delivery system.

Show MeSH
Related in: MedlinePlus