Catechol polymers for pH-responsive, targeted drug delivery to cancer cells

Abstract

A novel cell-targeting, pH-sensitive polymeric carrier was employed in this study for delivery of the anticancer drug bortezomib (BTZ) to cancer cells. Our strategy is based on facile conjugation of BTZ to catechol-containing polymeric carriers that are designed to be taken up selectively by cancer cells through cell surface receptor-mediated mechanisms. The polymer used as a building block in this study was poly(ethylene glycol), which was chosen for its ability to reduce nonspecific interactions with proteins and cells. The catechol moiety was exploited for its ability to bind and release borate-containing therapeutics such as BTZ in a pH-dependent manner. In acidic environments, such as in cancer tissue or the subcellular endosome, BTZ dissociates from the polymer-bound catechol groups to liberate the free drug, which inhibits proteasome function. A cancer-cell-targeting ligand, biotin, was presented on the polymer carriers to facilitate targeted entry of drug-loaded polymer carriers into cancer cells. Our study demonstrated that the cancer-targeting drug-polymer conjugates dramatically enhanced cellular uptake, proteasome inhibition, and cytotoxicity toward breast carcinoma cells in comparison with nontargeting drug-polymer conjugates. The pH-sensitive catechol-boronate binding mechanism provides a chemoselective approach for controlling the release of BTZ in targeted cancer cells, establishing a concept that may be applied in the future toward other boronic acid-containing therapeutics to treat a broad range of diseases.

Figure 1

pH-sensitive polymer–drug conjugates for delivering BTZ selectively into cancer cells. (A) The catechol and the boronic acid structure in BTZ form a stable, covalently-bonded, inactive conjugate at neutral and alkaline pH, but this structure dissociates in acidic environments to release the free active drug. (B) The catechol polymer–BTZ conjugate may dissociate in response to a mildly acidic cancer tissue microenvironment to liberate the free drug, which can be taken up by cancer cells. Alternatively, through the use of a targeting ligand, the catechol polymer–BTZ conjugate may be transported intact into the cell via receptor-mediated endocytosis, whereupon the acidic environment of the endosome induces intracellular drug release.

Figure 2

pH-dependent interactions between BTZ and DA. (A) ¹H NMR spectra of BTZ and DA in deuterated PBS at pH 5.5 and 7.4. DA was chosen as a representative catechol-containing model compound whose conjugate with BTZ is fully soluble in aqueous solutions. Circled in red are proton signals resulting from the formation of a stable BTZ–DA conjugate at pH 7.4, which are not present at pH 5.5. (B) Peak integrals in the pH ranges 7.0–7.2, 6.6–6.8, and 5.5–6.5 ppm were used to estimate the degree of BTZ–DA binding at pH 5.5–8.5.

Scheme 1. Chemical Structures of Polymers 1–5

Figure 3

Selective uptake of fluorescent FITC-terminated PEG molecules by cancer cells (MDA-MB-231 and MCF-10A-H-RasV12) and noncancerous cells (MCF-10A-Vector). All cells showed robust uptake of biotin–PEG–FITC but little uptake of PEG–FITC. Free biotin (0.1 μM) in the culture medium inhibited uptake of biotin–PEG–FITC by noncancerous cells and, to a lesser extent, by the two cancer cell lines, indicating that the cellular entry of FITC-terminated polymers is mediated by biotin receptors on the cell surface and that the expression of these receptors on cancer cell surfaces is very likely higher than on noncancerous cell surfaces.

Figure 4

Time-dependent release of BTZ from the catechol polymers BPC and PC at pH 5.0, 6.5, and 7.4. The fraction of BTZ released was 30% from both polymers at pH 7.4 over 12 h, while >80% of the BTZ was released at pH 5.0 in the same period of time. Drug–polymer conjugates formed between 0.2 mM BTZ and 0.05 mM BPC or PC were used for all measurements of release in 10 mM PBS buffers at 37 °C.

Figure 5

Cytotoxicity of catechol polymer–BTZ conjugates toward breast cancer cells. (A) Proteasome inhibition assays 6 h after treatment of MDA-MB-231 cells with (I) free BTZ, (II) BPC–BTZ, (III) PC–BTZ, (IV) BPC without BTZ, and (V) PC without BTZ. (B) Cell death as determined by fluorescence imaging 48 h after treatment using a commercial cell viability assay kit (calcein AM for live cells and ethidium homodimer-1 for dead cells). (C) IC₅₀ values of BPC–BTZ estimated from MTS assays indicate the enhanced cytotoxicity of BPC–BTZ toward cancer cells relative to noncancer cells.