Journal of the American Chemical Society COMMUNICATION Figure 3. Selective uptake of ﬂuorescent FITC-terminated PEG mol- ecules 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. groups each (BPC and PC; Scheme 1) in order to take advantage of multiple catechol moieties to increase the drug-loading capacity of the polymeric carrier. Time-dependent release of BTZ from these catechol polymers was carried out in buﬀered solutions at pH 7.4, 6.5, and 5.0, mimicking the physiological pH in normal tissue and blood, the tumor extracellular environment, and subcellular endosome, respectively. Figure 4 shows that at pH 7.4, ∼30% of the BTZ was released from both BPC and PC over a 12 h period, while >80% of the BTZ was released from these catechol polymers at pH 5.0. This further conﬁrmed the pH-dependent BTZ dissociation from the catechol-presenting polymeric delivery vehicles, which could render BTZ non-cell- permeable and inactive in normal tissues but allow the BTZ activity to be recovered in cancer tissue, where the acidity increases. The eﬀects of polymer BTZ conjugates on proteasome inhi- bition and cell viability were ﬁrst evaluated using MDA-MB-231 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. breast cancer cells (Figure 5 A,B). The untargeted (no biotin ligand) PC BTZ conjugate exhibited dramatically decreased proteasome inhibition in comparison to free BTZ, indicating deactivation of BTZ due to formation of the BTZ catechol polymer conjugate. This result is consistent with the previously reported inhibition of BTZ activity by polyphenols,15,16,18 which we speculate may be due to the inability of BTZ catechol complexes to enter cells. In stark contrast, the biotinylated conjugate BPC BTZ retained high proteasome-inhibiting ac- tivity. Cell viability assays 48 h after treatment of cells with the polymer BTZ conjugates demonstrated a similar pattern: the biotin-receptor-targeting BPC BTZ conjugate maintained fairly high cytotoxicity of BTZ, whereas catechol-containing polymers (BPC and PC) without loaded drug exhibited little proteasome inhibition activity or cytotoxicity. These results suggest that cellular uptake of the polymer BTZ conjugate through biotin-receptor-mediated transport can restore the BTZ activity within the cells, possibly via internalization of the polymer BTZ conjugate followed by low-pH-triggered intra- cellular dissociation of BTZ from the polymer in the endosomes. A detailed investigation of the intracellular events responsible for this eﬀect is in progress. Some preliminary evidence for self- assembly of the polymer BTZ construct into nanostructures was observed by cryogenic transmission election microscopy (cryo-TEM) (Figure SI 2). However, the signiﬁcance of these structures in relation to the observed cytotoxic eﬀects requires further study. To evaluate further the selectivity of the polymer drug conjugates for cancer cells over normal cells, we treated immor- talized human mammary epithelial cells (MCF-10A-Vector cells, a noncancer control) and transformed MCF-10A-H-RasV12 cells with BPC BTZ (0, 10, and 25 nM). We found that BPC loaded with BTZ at 25 nM concentration induced 1.7-fold increased cell death in the cancer cell line relative to the control cells (Figure SI 5). Calculated IC50 values for cell growth inhibition by BPC BTZ and free BTZ revealed that cancer cells were 2.2-fold more sensitive to BPC BTZ than noncancer 11852 dx.doi.org/10.1021/ja203077x |J. Am. Chem. Soc. 2011, 133, 11850–11853