Specific targeting of A54 homing peptide-functionalized dextran-g-poly(lactic-co-glycolic acid) micelles to tumor cells

Abstract

The delivery of chemotherapeutics into tumor cells is a fundamental knot for tumor-target therapy to improve the curative effect and avoid side effects. Here, A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran (A54-Dex-PLGA) was synthesized. The synthesized A54-Dex-PLGA self-assembled to form micelles with a low critical micelle concentration of 16.79 μg·mL(-1) and diameter of about 50 nm. With doxorubicin (DOX) base as a model antitumor drug, the drug-encapsulation efficiency of DOX-loaded A54-Dex-PLGA micelles (A54-Dex-PLGA/DOX) reached up to 75%. In vitro DOX release from the A54-Dex-PLGA/DOX was prolonged to 72 hours. The A54-Dex-PLGA micelles presented excellent internalization ability into hepatoma cells (BEL-7402 cell line and HepG2 cell line) in vitro, and the cellular uptake of the micelles by the BEL-7402 cell line was specific, which was demonstrated by the blocking experiment. In vitro antitumor activity studies confirmed that A54-Dex-PLGA/DOX micelles suppressed tumor-cell (BEL-7402 cell) growth more effectively than Dex-PLGA micelles. Furthermore, in vivo biodistribution testing demonstrated that the A54-Dex-PLGA micelles had a higher distribution ability to BEL-7402 tumors than that to HepG2 tumors.

Keywords: doxorubicin; homing peptide; polymeric micelles; tumor-cell targeting.

Figure 1

Flowchart illustrating the preparation and research approach of A54-Dex-PLGA micelles. Abbreviations: A54-Dex-PLGA, A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran; DOX, doxorubicin.

Figure 2

The synthesis route of A54-Dex-PLGA. Abbreviations: A54-Dex-PLGA, A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran; DCC, N,N′-dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine.

Figure 3

¹H-NMR spectra of Dex, PLGA, and Dex-PLGA (A), and A54 peptide and A54-Dex-PLGA (B). Abbreviations: NMR, nuclear magnetic resonance; A54-Dex-PLGA, A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran; DMSO, dimethyl sulfoxide.

Figure 4

Characteristics of blank and DOX-loaded micelles. Notes: (A) Variation of fluorescence intensity ratio for I₁/I₃ against logarithm of Dex-PLGA and A54-Dex-PLGA micelles. The unit of concentration was μg·mL⁻¹. (B) Transmission electron microscopy photographs of Dex-PLGA micelles (1), Dex-PLGA/DOX micelles (2), A54-Dex-PLGA micelles (3), and A54-Dex-PLGA/DOX micelles (4); scale bar 0.1 μm. (C) In vitro drug-release profile of free DOX, Dex-PLGA/DOX micelles, and A54-Dex-PLGA/DOX micelles. Abbreviations: A54-Dex-PLGA/DOX, doxorubicin-loaded A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran; DOX, doxorubicin.

Figure 5

Cellular uptake results of DOX-loaded micelles. Notes: Fluorescence images (A) of DOX after BEL-7402 and HepG2 cells were incubated with A54-Dex-PLGA/DOX micelles for 0.5, 4, and 10 hours. Quantitative analysis results (B) based on images (A) by ImageJ software. Fluorescence images (C) after BEL-7402 cells were incubated with DOX-loaded micelles solution for 2 and 24 hours. Quantitative cellular uptake (D) analyzed based on images (C) by a flow cytometry. Fluorescence images (E) of DOX after BEL-7402 was incubated with Dex-PLGA/DOX micelles, A54-Dex-PLGA/DOX micelles, and their blocking ones for 4 hours. Quantitative cellular uptake (F) analyzed based on images (E) by a flow cytometry (*P<0.05). Abbreviation: A54-Dex-PLGA/DOX, doxorubicin-loaded A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran.

Figure 6

In vitro antitumor activity results of blank and DOX-loaded micelles. Notes: In vitro cytotoxicity of blank (A) and DOX-loaded micelles (B) against BEL-7402 cells. (C) BEL-7402 cells treated with DOX HCl, Dex-PLGA micelles, and A54-Dex-PLGA micelles. Cells were stained with calcein acetoxymethyl ester (green) for visualization of live cells. Abbreviations: A54-Dex-PLGA/DOX, doxorubicin-loaded A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran; DOX HCI, doxorubicin hydrochloride.

Figure 7

In vivo targeting imaging of A54-Dex-PLGA micelles. Notes: Fluorescence images (A) of the mice bearing BEL-7402 and HepG2 cells on left and right sides at different time points after subcutaneous injection of ICG-loaded A54-Dex-PLGA micelles. (B) Ex vivo fluorescence images of dissected organs and tumors at 48 hours postinjection. (C) The accumulation of ICG/A54-Dex-PLGA micelles in tissues was calculated as %ID/g (the percentage of the fluorescent intensity per gram of tissue). Abbreviations: ICG, indocyanine green; A54-Dex-PLGA, A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran.