PEG-polypeptide block copolymers as pH-responsive endosome-solubilizing drug nanocarriers

Abstract

Herein we report the potential of click chemistry-modified polypeptide-based block copolymers for the facile fabrication of pH-sensitive nanoscale drug delivery systems. PEG-polypeptide copolymers with pendant amine chains were synthesized by combining N-carboxyanhydride-based ring-opening polymerization with post-functionalization using azide-alkyne cycloaddition. The synthesized block copolymers contain a polypeptide block with amine-functional side groups and were found to self-assemble into stable polymersomes and disassemble in a pH-responsive manner under a range of biologically relevant conditions. The self-assembly of these block copolymers yields nanometer-scale vesicular structures that are able to encapsulate hydrophilic cytotoxic agents like doxorubicin at physiological pH but that fall apart spontaneously at endosomal pH levels after cellular uptake. When drug-encapsulated copolymer assemblies were delivered systemically, significant levels of tumor accumulation were achieved, with efficacy against the triple-negative breast cancer cell line, MDA-MB-468, and suppression of tumor growth in an in vivo mouse model.

Scheme 1. Synthetic Route to the pH-Responsive Copolymers

Doxorubicin is used as the model cytotoxic drug.

Figure 1

(a) Effect of PEG molecular weight on the hydrodynamic diameter of amine-substituted PEG–polypeptide block copolymer. (b) Effect of serum and salt concentration on the self-assembly of block copolymer 1. (c) Effect of pH on the polydispersity index of amine-substituted PEG–polypeptide assemblies made of block copolymers 2 and 4. (d) TEM image of the vesicles synthesized with copolymer 1 at pH 8.0 with a polymer concentration of 1 mg mL^–1. The scale bar for the TEM image is 200 nm.

Figure 2

Investigation of pH-responsive properties of representative PEG–polypeptide copolymers. (a) pH titration curve of copolymers 1 and 2. (b) Reduction of relative FRET efficiency upon lowering the pH from 7.4 to 4.5. (c) Principle of the FRET experiment carried out by forming a vesicle constituted from the respective copolymer tagged with the Cy5.5 and Cy7 FRET pair.

Figure 3

Cumulative release profile of doxorubicin loaded into vesicles formed from (a) copolymer 3 and (b) copolymer 1 in PBS (pH 7.4) and at acidic pH (5.5).

Figure 4

Confocal fluorescence microscopic images of MDA-MB-468 cells treated with doxorubicin-loaded vesicles of copolymer 1 (upper panel) and cells cotreated with the vesicles and 10 μM chloroquine (lower panel) for 2 h at 37 °C. Doxorubicin fluorescence was visualized by excitation at 480 nm and emission at 560 nm.

Figure 5

Assessment of efficacy of vesicle-loaded doxorubicin in MDA-MB-468 cells. (a) Concentration-dependent inhibition of four different formulations of vesicles on tumor cell growth. Free doxorubicin was used as a control. (b) Cytotoxicity of the empty vesicles. The concentrations of the vesicles were normalized to their corresponding doxorubicin loading in panel a. The experiments were performed in triplicate, and data are presented as the mean ± standard deviation.

Figure 6

(A) Copolymer 1 vesicle stability on the basis of FRET association of the carrier components following systemic administration in non-tumor-bearing BALB/c mice. Top panel corresponds to λ_ex = 640 nm and λ_em = 700 nm (donor channel), and bottom panel corresponds to λ_ex = 640 nm and λ_em = 800 nm (FRET channel). (B) Biodistribution quantitation in necropsied tissue (Li, liver; S, spleen; K, kidneys; H, heart; and Lu, lungs) 24 h following systemic administration in BALB/c mice in both the donor and FRET channels. Fluorescence recovery from harvested tissue includes the background subtraction of tissue from an untreated control for removal of autofluorescence. The resultant quantification is normalized to the injected dose, based on the fluorescence intensity of the injected dose following IVIS imaging of the vial pre-injection.

Figure 7

(A) Biodistribution of tumor-bearing nude mice at 24 h post-systemic administration. The left image corresponds to the luminescence of the tumor cell line (MDA-MB-468). The image in the midpanel corresponds to fluorescence imaging of NPs at λ_ex = 640 nm and λ_ex = 700 nm (donor channel), and the right image corresponds to fluorescence imaging of NPs at λ_ex = 640 nm and λ_ex = 800 nm (FRET channel). (B) Tumor remediation study against MDA-MB-468 xenografts in NCR nude mice, comparing untreated free-doxorubicin-treated and dox-loaded PEG–PPLG systems treated groups. Data normalized to pre-injection tumor luminescence show a temporally resolved fold change in tumor-specific luminescence signal.