Evidence for Drug Release from a Metalla‐Cage Delivery Vector Following Cellular Internalisation

Abstract

One of the main challenges in cancer chemotherapy is to develop drugs that selectively target cancer cells in order to reduce general toxicity and consequently side effects. One such targeting method involves using large carrier compounds that release a drug once inside a cancer cell, since large compounds selectively accumulate in cancer tissue due to the "enhanced permeability and retention effect". [1] Recently, we have shown that the encapsulation of a hydrophobic metallo-drug guest in a water-soluble hexacationic arene ruthenium cage delivery vector produces a synergistic effect, due to the modest cytotoxicity of the cage itself, and the cytotoxicity of the encapsulated guest compound. [2] We have now encapsulated in the cavity of [Ru 6 (p-iPrC 6 H 4 Me) 6 A C H T U N G T R E N N U N G (tpt) 2 -A C H T U N G T R E N N U N G (C 6 H 2 O 4 ) 3 ] 6 + (1) (tpt = 2,4,6-tris(pyridin-4-yl)-1,3,5-triazine), an intrinsically fluorescent pyrenyl compound (pyrene-R = 1-(4,6-dichloro-1,3,5-triazin-2-yl)pyrene), thus giving rise to the hexanuclear metalla-prism [pyrene-R&1] 6 + , in which the pyrenyl derivative occupies the cavity of 1. The fluorescence of pyrene-R is quenched inside the metalla-prism cavity. A study of this new system has provided direct evidence that once inside the cell, the hexaruthenium cage releases the fluorescent guest, thus confirming our initial hypothesis that cage 1 can act as a Trojan horse for cancer cells. [2] In addition, experiments into the uptake mechanism indicate that an assisted diffusion pathway is in operation.

Excellent Correlation between Drug Release and Portal Size in Metalla‐Cage Drug‐Delivery Systems

“…The uptake of ruthenium into the cells was determined for the [pyrene-R&1-3] 6 + systems by inductively coupled plasma mass spectroscopy (ICP-MS), which reveals that all the compounds enter the cells to essentially the same extent (see Figure 9A) and indicates a similar uptake mechanism for the three supramolecular complexes. As previously reported, the fluorescence of pyrene-R is partially quenched inside the ruthenium cage [18] and consequently fluorescence can be used to evaluate the release of free pyrene-R inside the A2780 cells. Interestingly, flow cytometric analysis of cells incubated with [pyrene-R&1-3] 6 + (see Figure 9A) reveals that the ratio between cell fluorescence and the intracellular ruthenium concentration varies as a function of the metalla-cage used to entrap the pyrene-R fluorophore.…”

“…The Gibbs free energies (DG8) for the [pyrene-R&1-3] 6 + systems were then determined from the corresponding stability constants obtained at 21 8C in both CD 3 CN and (CD 3 ) 2 SO from the Gibbs free-energy equation. [25] In contrast to the carceplex system [pyrene-R&Ru 6 (piPrC 6 H 4 Me) 6 A C H T U N G T R E N N U N G (tpt) 2 A C H T U N G T R E N N U N G (dobq) 3 ] 6 + , in which the fluorescence of the pyrene-R guest was totally quenched upon encapsulation, [18] some fluorescence remains in the host-guest systems [pyrene-R&1-3] 6 + (see Figure 7). Indeed, the fluorescence of the pyrenyl part is quenched but the triazin-2-yl group does not entirely lose its fluorescent property.…”

“…Moreover, the [Pd-A C H T U N G T R E N N U N G (acac) 2 &1-3] 6 + systems are more cytotoxic than the corresponding [pyrene-R&1-3] 6 + systems, behaviour that is consistent with other supramolecular drug systems. [18] The IC 50 values indicate that the cage-guest complex acts as a drugdelivery system with the cage ensuring entry of the guest into the cancer cells, with the pyrene-R or PdA C H T U N G T R E N N U N G (acac) 2 acting on cell metabolism following intracellular release.…”

“…[32] A previous microscopy study showed that pyrene-R accumulates in cytoplasmic organelles. [18] In an attempt to better identify these organelles, their fluorescence pattern was compared with that of two known fluorescent reporters, the lysosomal dye Lysotracker Red and the fluid-phase endocytosis marker FITC-dextran (see Figure 10). [33] After 24 h of incubation, the free pyrene-R does not enter lysosomal compartments and does not accumulate in the endocytic compartments that are enriched in FITC-dextran, which suggests that pyrene-R accumulates in endocytic rather than lytic compartments.…”

“…[17] The ability of this host to deliver guest molecules to cells was further confirmed by the encapsulation of a fluorescentlabelled pyrene-R derivative, 1-(4,6-dichloro-1,3,5-triazin-2-yl)pyrene. [18] Fluorescence experiments used to monitor the uptake of pyrene-R into cancer cells demonstrated an uptake of the carceplex [pyrene-R&Ru 6 (p-iPrC 6 H 4 Me) 6 6 + one order of magnitude greater than that of pyrene-R alone. [18] These systems suggest that after internalisation, the cage is destroyed in the cytoplasm and the release of the encapsulated molecule increases the cytotoxicity, thus giving rise to an additive effect.…”