Limits...
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.

Show MeSH

Related in: MedlinePlus

M. smegmatis treatment using the TB drug, EMB, and CB[7].EMB loaded CB[7] (CB[7]•EMB, white bars) was equally effective in treating M. smegmatis infected RAW264.7 cells as free EMB (patterned bars). RAW264.7cells were incubated with M. smegmatis for two hours and then chased for three days with EMB and CB[7]•EMB. Varying MIC values for EMB and CB[7]•EMB were used: 0.1, 0.4, 0.8, and 1 units. Viable bacteria were quantified using CFU/ml. This figure is representative of two replicate experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2865549&req=5

pone-0010514-g007: M. smegmatis treatment using the TB drug, EMB, and CB[7].EMB loaded CB[7] (CB[7]•EMB, white bars) was equally effective in treating M. smegmatis infected RAW264.7 cells as free EMB (patterned bars). RAW264.7cells were incubated with M. smegmatis for two hours and then chased for three days with EMB and CB[7]•EMB. Varying MIC values for EMB and CB[7]•EMB were used: 0.1, 0.4, 0.8, and 1 units. Viable bacteria were quantified using CFU/ml. This figure is representative of two replicate experiments.

Mentions: Finally, we wanted to assess what effect the loading of a drug into CB[7] would have on the bioactivity of the drug. We used M. smegmatis, a non-virulent mycobacterium, as a model for in vitro infections of macrophages. Ethambutol (EMB) is a widely used antituberculosis drug that has been associated with drug resistance in M. tuberculosis [35], [37], [49]. RAW264.7 cells were infected with M. smegmatis and then treated with EMB and CB[7]•EMB for 3 days and the amount of viable bacteria was determined (Figure 7). At day 3, untreated cells provided high bacterial survival with a CFU of 1.24×107 (±5.4×106) CFU/mL. At a MIC of 0.1 units, cells treated with EMB resulted in 4×105 (±2.3×105) CFU/mL and cells treated with CB[7]•EMB resulted in 8.6×105 (±6×105) CFU/mL. CFU values between EMB and CB[7]•EMB at a MIC value of 0.4 units were found to be 3.6×104 (±1.7×103) CFU/mL and 4.3×104 (±1.7×103) CFU/mL respectively. At a MIC of 0.8 units, EMB was 1×103 (±4×102) CFU/mL and CB[7]•EMB was 6×102 (±3.3×102) CFU/mL. Finally at a MIC of 1 units, CFU values for EMB and CB[7]•EMB were 8×102 (±6.6×102) CFU/mL and 1.4×103 (±1.1×103) CFU/mL respectively. The values of CFUs within each MIC dose for free EMB or CB[7]•EMB were not significantly different from each other (unpaired t-test) indicating no inhibition of the container on the ability of the drug to kill the bacteria (Figure 7).


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)

M. smegmatis treatment using the TB drug, EMB, and CB[7].EMB loaded CB[7] (CB[7]•EMB, white bars) was equally effective in treating M. smegmatis infected RAW264.7 cells as free EMB (patterned bars). RAW264.7cells were incubated with M. smegmatis for two hours and then chased for three days with EMB and CB[7]•EMB. Varying MIC values for EMB and CB[7]•EMB were used: 0.1, 0.4, 0.8, and 1 units. Viable bacteria were quantified using CFU/ml. This figure is representative of two replicate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010514-g007: M. smegmatis treatment using the TB drug, EMB, and CB[7].EMB loaded CB[7] (CB[7]•EMB, white bars) was equally effective in treating M. smegmatis infected RAW264.7 cells as free EMB (patterned bars). RAW264.7cells were incubated with M. smegmatis for two hours and then chased for three days with EMB and CB[7]•EMB. Varying MIC values for EMB and CB[7]•EMB were used: 0.1, 0.4, 0.8, and 1 units. Viable bacteria were quantified using CFU/ml. This figure is representative of two replicate experiments.
Mentions: Finally, we wanted to assess what effect the loading of a drug into CB[7] would have on the bioactivity of the drug. We used M. smegmatis, a non-virulent mycobacterium, as a model for in vitro infections of macrophages. Ethambutol (EMB) is a widely used antituberculosis drug that has been associated with drug resistance in M. tuberculosis [35], [37], [49]. RAW264.7 cells were infected with M. smegmatis and then treated with EMB and CB[7]•EMB for 3 days and the amount of viable bacteria was determined (Figure 7). At day 3, untreated cells provided high bacterial survival with a CFU of 1.24×107 (±5.4×106) CFU/mL. At a MIC of 0.1 units, cells treated with EMB resulted in 4×105 (±2.3×105) CFU/mL and cells treated with CB[7]•EMB resulted in 8.6×105 (±6×105) CFU/mL. CFU values between EMB and CB[7]•EMB at a MIC value of 0.4 units were found to be 3.6×104 (±1.7×103) CFU/mL and 4.3×104 (±1.7×103) CFU/mL respectively. At a MIC of 0.8 units, EMB was 1×103 (±4×102) CFU/mL and CB[7]•EMB was 6×102 (±3.3×102) CFU/mL. Finally at a MIC of 1 units, CFU values for EMB and CB[7]•EMB were 8×102 (±6.6×102) CFU/mL and 1.4×103 (±1.1×103) CFU/mL respectively. The values of CFUs within each MIC dose for free EMB or CB[7]•EMB were not significantly different from each other (unpaired t-test) indicating no inhibition of the container on the ability of the drug to kill the bacteria (Figure 7).

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