Journal of the American Chemical Society COMMUNICATION Scheme 1. Chemical Structures of Polymers 1 5 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 environ- ments 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 environ- ment 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. acidic endosomes following cell surface receptor-mediated en- docytosis. In both cases, the acidic environment would result in dissociation of BTZ from the polymer-bound catechol groups, activating its proteasome-inhibiting function (Figure 1). We ﬁrst characterized the pH-dependent reversible binding between BTZ and catechol compounds using 1H NMR spec- troscopy (Figure 2A). At pH 5.5, a mixture of BTZ and dopamine (DA) produced a spectrum similar to that of the two individual compounds superimposed upon each other, indicating the pre- sence of uncomplexed BTZ and DA. However, the same mixture at pH 7.4 gave a spectrum with striking changes in chemical shifts and peak splittings, revealing the formation of a BTZ DA conjugate. To investigate further the pH sensitivity of BTZ catechol dissociation, preformed BTZ DA conjugate [0.1 M in deuterated dimethyl sulfoxide (DMSO-d6)] was diluted to 1 mM in deuterated phosphate buﬀer at pH 5.5 8.5 and then analyzed by NMR spectroscopy (Figure SI 1 in the Supporting In- formation). The peak integrals in the ranges 7.0 7.2, 6.6 6.8, and 5.5 6.5 ppm, corresponding to the H atoms on the phenyl ring of BTZ, the benzene ring of free DA, and the benzene of the BTZ DA complex, respectively, were used to estimate the degree of BTZ DA complexation. As shown in Figure 2B, disso- ciation of the BTZ DA complex increased as the pH decreased from 8.5 to 6.5, and the BTZ DA complex was undetectable at pH 5.5. The pH dependence of the BTZ catechol complex suggested that polymer constructs containing catechols may be useful for pH-triggered release of BTZ. We therefore synthesized several BTZ polymer constructs as pH-responsive carriers of BTZ to cancer cells (Scheme 1). We employed a modular heterobifunc- tional polymer design consisting of a poly(ethylene glycol) (PEG) polymer derivatized with one or more catechols and a cell-targeting moiety. PEG is well-known to reduce nonspeciﬁc interactions of therapeutic molecules with proteins and cells and therefore has been widely applied in the form of drug PEG conjugates that exhibit reduced drug degradation and extended drug circulation time.20 Here we used biotin as a cell-targeting ligand because previous reports have shown that biotinylated polymers can be selectively taken up by cancer cells.21,22 Although the biotin receptors/transporters on cancer cell surfaces have not been well-characterized, biotin has a relatively simple structure and thus can also be used as a tag for cellular tracking of polymer drug carriers, making this molecule an attractive choice for demonstrating cancer cell targeting. We used ﬂuorescence microscopy to examine the uptake of ﬂuorescein isothiocyanate (FITC)-labeled biotinylated PEG (biotin PEG FITC) into two breast cancer cell lines, MDA-MB-231 cells and MCF-10A- H-RasV12 cells, as well as the nontransformed or noncancerous breast epithelial cell line MCF-10A-Vector.23 Figure 3 shows that biotin PEG FITC was rapidly taken up by cells, whereas the cellular uptake of PEG FITC without the biotin moiety was minimal. The uptake of biotinlylated polymers was inhibited by free biotin in a dose-dependent manner (Figure SI 3), demonstrating that polymer uptake by cells is mediated by the cell surface receptor for biotin. Notably, the inhibition of uptake of biotinylated polymer by free biotin was less robust in cancer cell lines (MDA-MB-231 and MCF-10A-H-RasV12) than in noncancer MCF-10A-Vector cells, suggesting greater expression levels of biotin receptors in cancer cells than in noncancer cells. After conﬁrming the cancer-targeting function of the biotin ligand, we synthesized PEG polymers containing four catechol 11851 dx.doi.org/10.1021/ja203077x |J. Am. Chem. Soc. 2011, 133, 11850–11853