Targeted inhibition of human hematological cancers in vivo by doxorubicin encapsulated in smart lipoic acid-crosslinked hyaluronic acid nanoparticles

Abstract

The chemotherapy of hematological cancers is challenged by its poor selectivity that leads to low therapeutic efficacy and pronounced adverse effects. Here, we report that doxorubicin encapsulated in lipoic acid-crosslinked hyaluronic acid nanoparticles (LACHA-DOX) mediate highly efficacious and targeted inhibition of human hematological cancers including LP-1 human multiple myeloma (MM) and AML-2 human acute myeloid leukemia xenografted in nude mice. LACHA-DOX had a size of ca. 183 nm and a DOX loading content of ca. 12.0 wt.%. MTT and flow cytometry assays showed that LACHA-DOX possessed a high targetability and antitumor activity toward CD44 receptor overexpressing LP-1 human MM cells and AML-2 human acute myeloid leukemia cells. The in vivo and ex vivo images revealed that LACHA-DOX achieved a significantly enhanced accumulation in LP-1 and AML-2 tumor xenografts. Notably, LACHA-DOX effectively suppressed LP-1 as well as AML-2 tumor growth and drastically increased mice survival rate as compared to control groups receiving free DOX or PBS. Histological analyses exhibited that LACHA-DOX caused little damage to the major organs like liver and heart. This study provides a proof-of-concept that lipoic acid-crosslinked hyaluronic acid nanoparticulate drugs may offer a more safe and effective treatment modality for CD44 positive hematological malignancies.

Keywords: CD44; Hematological cancer; nanomedicine; reduction-sensitive; targeted chemotherapy.

Figure 1.

Illustration of DOX-encapsulated LACHA for the treatment of CD44 overexpressed hematologic malignancy. (i) LACHA-DOX can actively target to and be efficiently taken up by hematological tumor cells via a CD44-mediated endocytosis mechanism; and (ii) LACHA-DOX is automatically de-crosslinked inside the tumor cell, leading to fast intracellular drug release to the cytoplasm.

Figure 2.

(A) CD44 expression in various blood cancer cells. Human MM cell line (LP-1) and human leukemia cell lines (HL-60, THP-1, K562, NB4, and AML-2) were treated with FITC-labeled antibody against CD44 receptors to determine the receptor levels on the surface of the cells by flow cytometry. (B) Cytotoxicity of bare LACHA nanoparticles in LP-1 and AML-2 cells following 48 h incubation (n = 4). The in vitro proliferative inhibition activity of LACHA-DOX toward LP-1 cells (C) and AML-2 cells (D). The cells were treated with LACHA-DOX for 4 h and then cultured in fresh medium for another 44 h. Free DOX was used as a control. The inhibition experiments were performed by pre-treating cells for 4 h with 5 mg/mL free HA prior to incubation with LACHA-DOX (n = 4). Flow cytometry of LP-1 cells (E) and AML-2 cells (F) following 2 and 4 h incubation with LACHA-DOX (10 μg DOX/mL). Free DOX was used as a control. The competitive inhibition experiments were performed by pre-treating cells with free HA (5.0 mg/mL) for 4 h before adding LACHA-DOX.

Figure 3.

In vivo fluorescence images of nude mice bearing LP-1 human MM (A) and AML-2 human acute myeloid leukemia (B) at different time points following injection of LACHA-DIR. The mouse autofluorescence was removed by spectral unmixing using the Maestro software. Ex vivo DOX fluorescence images of tumor and major organs (1: heart, 2: liver, 3: spleen, 4: lung, 5: kidney, and 6: tumor) from nude mice bearing LP-1 human MM (C) and AML-2 human acute myeloid leukemia (D) at 8 h post injection of LACHA-DOX or free DOX.

Figure 4.

In vivo antitumor effects of LACHA-DOX in LP-1 human MM-bearing nude mice. Inhibition of tumor growth (A), body weight changes (B), and survival rates (C) of mice treated with LACHA-DOX, free DOX and PBS, respectively. The drugs were administrated on day 0, 3, 6, 9, and 12 (dosage: 7.5 mg DOX equiv./kg) (n = 6). ***p < .001 (Student’s t test); (D) H&E-staining of tumor, liver, and heart sections excised from LP-1 human MM-bearing mice on day 24 after different treatments. The scale bar represents 50 μm.

Figure 5.

In vivo antitumor effects of LACHA-DOX in AML-2 human acute myeloid leukemia-bearing nude mice. Inhibition of tumor growth (A), body weight changes (B), and survival rates (C) of mice treated with LACHA-DOX, free DOX and PBS, respectively. The drugs were administrated on day 0, 3, 6, 9, 12 and 15 (dosage: 7.5 mg DOX equiv./kg) (n = 6). ***p < .001 (Student’s t test); Photos of typical tumor blocks (D), and H&E-staining of tumor, liver, and heart sections (E) isolated from different treatment groups on day 15. The scale bar represents 50 μm.