Colloidal aggregation affects the efficacy of anticancer drugs in cell culture

Abstract

Many small molecules, including bioactive molecules and approved drugs, spontaneously form colloidal aggregates in aqueous solution at micromolar concentrations. Though it is widely accepted that aggregation leads to artifacts in screens for ligands of soluble proteins, the effects of colloid formation in cell-based assays have not been studied. Here, seven anticancer drugs and one diagnostic reagent were found to form colloids in both biochemical buffer and in cell culture media. In cell-based assays, the antiproliferative activities of three of the drugs were substantially reduced when in colloidal form as compared to monomeric form; a new formulation method ensured the presence of drug colloids versus drug monomers in solution. We also found that Evans Blue, a dye classically used to measure vascular permeability and to demonstrate the "enhanced permeability and retention (EPR) effect" in solid tumors, forms colloids that adsorb albumin, as opposed to older literature that suggested the reverse.

Figure 1

Transmission electron micrographs of aggregating drugs in phosphate buffer (top row) and 10% FBS (bottom row): (A,D) fulvestrant, (B, E) lapatinib, (C, F) sorafenib. Bars represent 200 nm.

Figure 2

Colloid formation by anticancer drugs in cell culture media. Particle formation was measured by DLS for fulvestrant (A, B), lapatinib (C, D), and sorafenib (E, F) in the absence (A, C, E) and presence (B, D, F) of 0.025% Tween-80 at 0 h (▼), 12 h (◆), and 24 h (●). Media alone (■) and containing standard beads (▲) were measured for comparison. Arrows indicate colloids that are present in cell media. These peaks are disrupted by the addition of Tween-80, indicating the loss of colloidal aggregates.

Figure 3

Cell toxicity was tested for each vehicle formulation. Colloidal formulations contained 0.1% DMSO (□) while monomeric, free drug formulations contained 1% DMSO with 0.025% Tween-80 (■) in media (Columns, mean relative cell proliferation; Bars, standard deviation, n = 6).

Figure 4

Colloidal (□) versus noncolloidal (■) formulations of three anticancer agents, fulvestrant, lapatinib, and sorafenib, were used to measure antiproliferative effects in relevant cell lines (Columns, mean relative cell proliferation; Bars, standard deviation; n = 6, *** denotes p < 0.001).

Figure 5

Transmission electron micrograph of Evans Blue aggregates in phosphate buffer containing 10% FBS. Bar represents 100 nm.

Figure 6

Aggregating dye, Evans Blue, binds albumin via a colloidal mechanism. Thermophoresis was measured at increasing concentrations of Evans Blue in (■) 10 nM BSA, 0.001% Triton X-100, (▲) 10 nM BSA, 0.01% Triton X-100, and (▼) 100 nM BSA, 0.001% Triton X-100. Data represent the mean and range for repeat experiments.