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

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.

Figure 1. Structures of the five containers.

CB7, CB5, Pentamer (1), Me-Hexamer (2), and Ph- Hexamer (3) and FITC (4) and Alexa555 (5) conjugates.

Figure 2. HEK293 toxicology assays.

The assay was performed using CB, CB, 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.

Figure 3. HepG2 toxicology assays using various containers.

This assay showed high cell viability and low toxicity. HepG2 MTS (A) and AK (B) assays were conducted using the procedure described in Fig 2.

Figure 4. RAW264.7 toxicology assays using CB.

These results signified high cell tolerance at concentrations of up to 1 mM of CB. RAW264.7cells were incubated with the CB and camptothecin for two days before conducting both MTS (A) and AK (B) assays. Both assays were conducted following the procedure indicated in Fig 2.

Figure 5. CB•4 uptake and intracellular stability of CB in RAW264.7 cells.

Both the dose titration and time course assays used RAW264.7cells incubated with CB•4 for 20 mins prior to analysis. (A) Dose titration assay used CB•4 concentrations of 3.2 (green) and 32 µM (red). (B) Statistical analysis of the percentage of cells positive for fluorescence. (C) Timecourse assay was conducted using 32 µM of CB•4. After incubation with the fluorescent container, cells were chased for 15 (green), 45 (red) and 120 min (blue) (D) Statistical analysis of the percentage of cells positive for fluorescence.

Figure 6. Intracellular localization of CB in RAW264.7 cell.

Cell incubated with Dextran-647 and CB•5 showed intracellular localization of CB through the endosomal pathway. RAW264.7cells were incubated with Dextran-647 (green) overnight and CB•5 (red) for 20 min the following day. Cells were chased for 15 (A), 45 (B) and 120 min (not shown) after incubation with CB•5. Arrows indicate co-localization.

Figure 7. M. smegmatis treatment using the TB drug, EMB, and CB.

EMB loaded CB (CB•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•EMB. Varying MIC values for EMB and CB•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.