A Nanosystem of Amphiphilic Oligopeptide-Drug Conjugate Actualizing Both αvβ3 Targeting and Reduction-Triggered Release for Maytansinoid

Abstract

To design a prodrug-based self-assembling nanosystem with both ligand targeting and stimuli-responsive features, and elucidate the superiority of each targeting strategy and the synergistic effect between them, we synthesized four small molecule amphiphilic peptide-drug conjugates (APDCs) using maytansinoid (DM1) as a cytotoxic agent, cRGDfK as a homing peptide, and disulfide (SS) or thioether (SMCC) as linker. Owing to their amphiphilicity, the APDCs could self-assemble into nanoparticles (APDC@NPs) which were evaluated in vitro in three different cell lines and in vivo in tumor-bearing C57BL/6 mice. The RSSD@NPs showed the strongest interaction with αvβ3 integrin, highest cell uptake and intracellular free drug level, and best antitumor efficacy in vitro and in vivo, while it shared the same goodness with other test nanosystems in terms of high drug loading, EPR effect and free of potentially toxic polymers. Especially, the in vivo efficacy of RSSD@NPs was 2 fold of free DM1 which is too cytotoxic to be a drug, while the active targeted APDC@NPs demonstrated acceptable system, tissue and blood compatibility. In αvβ3-positive cells or tumors, the RGD targeting contributed much more than disulfide in anticancer effect. The maximum synergism of the two strategies reached to 22 fold in vitro and 3 fold in vivo. Generally, the active targeting, prodrug and nanosystem could significantly decrease the toxicity of free DM1 and improve its therapy outcome via combining active targeting, prodrug and nanopreparation, especially the dual targeting strategies and their synergism.

Keywords: Amphiphilic peptide-drug conjugate; antitumor therapy.; endocytosis; maytansinoid DM1; reduction-triggered drug release; αvβ3-targeted nanoparticles.

Scheme 1

(A) Chemical structure of small molecule amphiphilic peptide-drug conjugates (APDCs): cRGD-SMCC-DM1 (RCCD), cRGD-SS-DM1 (RSSD), cRPQ-SMCC-DM1 (QCCD), and cRPQ-SS-DM1 (QSSD). (B) Schematic illustrations of the self-assembly of APDC nanoparticles (APDC@NPs), the accumulation of APDC@NPs at the tumor site by the EPR effect, their uptake by tumor cells or tumor angiogenesis endothelial cells by αvβ3 receptor-mediated endocytosis, and the triggered intracellular drug release from APDC@NPs.

Figure 1

(A) The diameter distributions, average hydrodynamic particle sizes (D_RCCD=138.7 nm, D_RSSD=82.2 nm, D_QCCD=104.6 nm, D_QSSD=106.8 nm), and polydispersity indexes (PDI_RCCD=0.142, PDI_RSSD=0.205, PDI_QCCD=0.206, PDI_QSSD=0.247) of the APDC@NPs by DLS analyses. Inset: The digital photographs of APDC@NPs' solution. (B-E) The morphology of RCCD@NPs, RSSD@NPs, QCCD@NPs and QSSD@NPs by TEM images. Scale bars=100nm. (F) Reduction-triggered drug release from the active targeting RCCD@NPs and RSSD@NPs with or without the presence of reducing agent DTT at 37 °C in PBS (pH 7.4, n=3). (G) Reduction-based drug release mechanisms of thioether-linked and disulfide-linked APDC@NPs.

Figure 2

Intracellular concentrations of DM1 determined by HPLC in (A) B16 cells, (B) MCF-7 cells, and (C) HUVEC after treatments with different APDC@NPs at a drug dose of 100 nM for 3 h (n=3), *p < 0.05, p = ns versus the uptake of passive cRPQfK group. (D) Confocal images of B16 cells treated by various APDC@NPs for 3 h after pre-incubation with different inhibitors at 37 °C. Red represents the Cy3.5-loaded APDC@NPs and blue represents the nucleus stained by Hoechst 33258. Scale bars are 50 μm.

Figure 3

Cytotoxicity of free DM1 and various APDC@NPs against (A) B16 cells, (B) MCF-7 cells, and (C) HUVEC cells after 48 h incubation by SRB assay (n=3). The impact of various APDC@NPs with or without pre-treatment of reducing agent GSH-OEt on the cell viability of (D, E) B16 cells, (F, G) MCF-7 cells, and (H, I) HUVEC cells after 48 h incubation by SRB method. Each bar denotes mean ± SD (n=3). (J) The cell cycle of B16 cells, MCF-7 cells, and HUVEC cells after incubation with culture medium, free DM1, RCCD@NPs, RSSD@NPs, QCCD@NPs and QSSD@NPs, respectively, at a dose of 50 nM (calculated by free DM1) for 12 h.

Figure 4

(A) Tumor growth curves of B16 tumor-bearing C57BL/6 mice after intravenous administrations of saline, free DM1, and various APDC@NPs (n=6). All formulations were given every other day for a total of five times via tail vein at the dosage of 400 μg/kg (calculated by free DM1). Black arrows indicate the time for injection after tumor cell inoculation. **p < 0.01 versus the saline control, ^&&p < 0.01 versus the free DM1 group and ^##p < 0.01 versus the passive cRPQfK group. (B) The weight of the excised tumor masses from different treatment groups. Each bar represents mean ± SD (n = 6). *p < 0.05 and **p < 0.01 versus the saline control. (C) The tumor photoprints of different treatment groups after tumors were excised at the end of the test (n=6). Scale bar = 2 cm. (D) Confocal images of TUNEL assay for the detection of apoptotic cells in tumor tissue sections from different treatment groups. DNA strand breaks were labeled with FITC (green), and nuclei were stained with Hoechst 33258 (blue). Apoptotic cells exhibited the co-localization of these two labels.

Figure 5

(A) Fluorescent images of immunostaining with anti-CD31 for detecting tumor angiogenesis in tumor tissue after treatment of B16 tumor-bearing C57BL/6 mice with various APDC@NPs. Red represents the expression of CD31 on a tumor angiogenesis (scale bar = 100 μm). (B) Fluorescent images of proliferating cells in tumor sections from different treatment groups. Tumor sections were immunostained with anti-Ki-67 nuclear antigen for cell proliferation. Green represents the expression of Ki67 on proliferating cells (scale bar = 100 μm).

Figure 6

(A) Body weight changes of B16 tumor-bearing C57BL/6 mice after different treatments during anti-tumor efficacy study (n=6). *p < 0.05 versus the saline control. Black arrows indicate the time for injection after tumor cell inoculation. (B, C) ALT and AST levels in B16 tumor-bearing C57BL/6 mice treated by various APDC@NPs (n=6). *p < 0.05 and **p < 0.01 versus the saline control. (D-F) The counts of blood cells (WBC), red blood cells (WBC) and platelet (PLT) in different treatment groups at the end of test. Each bar represents mean ± SD (n = 6). *p < 0.05 and **p < 0.01 versus the saline control. (G) Histological analysis of major tissues after treatment with various APDC@NPs (200×). Arrows point out the pathologic regions.