Biological evaluation of pH-responsive polymer-caged nanobins for breast cancer therapy

Abstract

A series of doxorubicin-loaded polymer-caged nanobins (PCN(DXR)) were evaluated in vivo in a murine MDA-MB-231 xenograft model of triple-negative breast cancer. The cross-linked polymer cage in PCN(DXR) offers protection for the drug payload while serving as a pH-responsive trigger that enhances drug release in the acidic environments commonly seen in solid tumors and endosomes. Varying the degree of cross-linking in the polymer cage allows the surface potential of PCN(DXR), and thus the in vivo circulation lifetime of the nanocarriers, to be tuned in a facile fashion. Given these design advantages, the present study provides the first in vivo evidence that PCN(DXR) can effectively inhibit tumor growth in a murine model of breast cancer. Importantly, PCN(DXR) was well-tolerated by mice, and drug encapsulation attenuated the toxicity of free doxorubicin. Taken together, this study demonstrates the potential utility of the PCN platform in cancer therapy.

Figure 1

(A) Schematic drawing of click-modifiable, DXR-encapsulated polymer-caged nanobins (PCN_DXR). (B) Schematic drawing of the proposed acid-triggered drug release mechanism. (C-D) Transmission electron microscopy (TEM) image of (C) intact PCNs at pH 7.4 and (D) PCNs after 72 h-incubation in pH 5.0-buffered solution at 37 °C. Both samples are stained with aqueous uranyl acetate (4 wt%) for TEM measurements.

Figure 2

(A) Zeta potentials (ζ) of doxorubicin-loaded bare liposome (BL_DXR) and polymer-caged nanobin (PCN_DXR) with 0, 30, 50, and 70% degree of cross-linking (points, mean ζ; error bars, standard deviation). (B) Hydrodynamic diameters of BL_DXR and PCN_DXR (50% cross-linked) measured by dynamic light scattering (points, mean population; error bars, standard deviation). (C) Cumulative amount of DXR released from PCN_DXR (50% cross-linked) and BL_DXR at 37 °C in fetal bovine serum (FBS) and buffered saline (pH 6.0 and 5.0) (points, mean value; error bars, standard deviation). (D) Standard deviation (SD, σ) for the diameters of BL_DXR and PCN_DXR (50% cross-linked) incubated at pH 7.4 (points, mean value; error bars, standard deviation).

Figure 3

In vitro cytotoxicity of DXR-encapsulated polymer-caged nanobins (PCN_DXR), empty PCN vehicles, or free DXR against (A) MDA-MB-231 and (B) HeLa cells during 48-h (open symbols) and 72-h (closed symbols) incubations. The concentration of the empty PCN vehicle was equivalent to the lipid concentration in the PCN_DXR (points, mean viability; error bars, standard deviation).

Figure 4

Cell death induced by PCN_DXR, was measured by Annexin-V/DAPI staining. In each panel, the lower-left (Annexin-V⁻, DAPI⁻), lower-right (Annexin-V⁺, DAPI^-), and upper-right (Annexin-V⁺, DAPI⁺) quadrants represent the populations of live cells, apoptotic cells, and necrotic/dead cells, respectively. The average %-population in each quadrant is indicated by the numbers at the corners of the panels. This data show that non-transformed MCF-10A breast epithelial cells stably expressing empty vector (i.e., control cells) were less sensitive than MCF-10A cells stably transduced with the H-RasV12 oncogene (i.e., cancerous cells) to PCN_DXR-induced apoptosis.

Figure 5

In vivo antitumor effects of PCN_DXR, empty PCNs, or free DXR as administered by intraperitoneal injection to female nude mice bearing MDA-MB-231 human triple negative mammary tumors (n = 3 mice with bilateral tumors, 6 tumors per group). (A) Mean tumor volume. Pairwise tests were performed to assess statistical significance and were Bonferroni-corrected; * indicates p < 0.05 for PCN_DXR compared with empty PCNs. PCN_DXR was also significantly different from free DXR on weeks 1.5 and 2.5 (not shown in plot). Times of treatments are indicated by arrows (points, raw mean; error bars, standard error). (B) Body weights of each treatment group (points, mean; error bars, standard error).

Figure 6

Non-invasive fluorescent imaging of nude mice with mCherry-labeled MDA-MB-231 mammary tumors treated with empty PCNs, free DXR, or PCN_DXR (scale bar = 1 cm). mCherry fluorescence is pseudo-colored and overlaid over bright-field images. All members in each group are shown (n = 3 mice and 6 tumors).

Figure 7

In vivo antitumor effects of PCN_DXR (2.0, 5.0, or 7.5 mg/kg of DXR) administered by intraperitoneal injection to female nude mice bearing MDA-MB-231 mammary tumors (n = 3 mice with bilateral tumors, 6 tumors per group). (A) Mean tumor volume. Pairwise tests were performed to assess statistical significance and were Bonferroni-corrected; * indicates p < 0.05 for PCN_DXR doses 5.0 and 7.5 mg DXR/kg compared with PBS (points, raw mean; error bars, standard error). PCN_DXR at 2.0 mg DXR/kg dose was also statistically different on days 12 and 15 (not shown in plot). (B) Body weights of mice in each treatment group (points, mean; error bars, standard error).