Targeted delivery of HSP90 inhibitors for efficient therapy of CD44-positive acute myeloid leukemia and solid tumor-colon cancer

Abstract

Heat shock protein 90 (HSP90) is overexpressed in numerous cancers, promotes the maturation of numerous oncoproteins and facilitates cancer cell growth. Certain HSP90 inhibitors have entered clinical trials. Although less than satisfactory clinical effects or insurmountable toxicity have compelled these trials to be terminated or postponed, these results of preclinical and clinical studies demonstrated that the prospects of targeting therapeutic strategies involving HSP90 inhibitors deserve enough attention. Nanoparticulate-based drug delivery systems have been generally supposed as one of the most promising formulations especially for targeting strategies. However, so far, no active targeting nano-formulations have succeeded in clinical translation, mainly due to complicated preparation, complex formulations leading to difficult industrialization, incomplete biocompatibility or nontoxicity. In this study, HSP90 and CD44-targeted A6 peptide functionalized biomimetic nanoparticles (A6-NP) was designed and various degrees of A6-modification on nanoparticles were fabricated to evaluate targeting ability and anticancer efficiency. With no excipients, the hydrophobic HSP90 inhibitor G2111 and A6-conjugated human serum albumin could self-assemble into nanoparticles with a uniform particle size of approximately 200 nm, easy fabrication, well biocompatibility and avoidance of hepatotoxicity. Besides, G2111 encapsulated in A6-NP was only released less than 5% in 12 h, which may avoid off-target cell toxicity before entering into cancer cells. A6 peptide modification could significantly enhance uptake within a short time. Moreover, A6-NP continues to exert the broad anticancer spectrum of Hsp90 inhibitors and displays remarkable targeting ability and anticancer efficacy both in hematological malignancies and solid tumors (with colon tumors as the model cancer) both in vitro and in vivo. Overall, A6-NP, as a simple, biomimetic and active dual-targeting (CD44 and HSP90) nanomedicine, displays high potential for clinical translation.

Keywords: Active targeting nanoparticles; Acute myeloid leukemia; CD44; Heat shock protein 90; Solid tumor.

Fig. 1

The chemical structure of G2111

Scheme 1

Schematic illustration on the fabrication of A6-NP and target therapy of acute myeloid leukemia and solid tumor-colon cancer

Fig. 2

Synthesis and characterization of A6-NP. (A) SDS-PAGE of HSA, 2.3%A6-HSA and 5.5%A6-HSA, respectively. (B) MADLI-TOF-MS of HSA, 2.3%A6-HSA and 5.5%A6-HSA, respectively. (C-E) TEM image of NP, 2.3%A6-NP and 5.5%A6-NP, respectively. (F-H) Hydrodynamic size of NP, 2.3%A6-NP and 5.5%A6-NP, respectively. (I) X-ray diffraction patterns: a G2111, b the physical mixture of blank-NP and G2111, c the physical mixture of blank-2.3%A6-NP and G2111, d the physical mixture of blank-5.5%A6-NP and G2111, e NP, f 2.3%A6-NP, g 5.5%A6-NP. (J) In vitro drug release curves (n = 3). (K) The stability of nanoparticles within 7 days at 4 °C

Fig. 3

In vitro cellular uptake. (A) Fluorescence images of MOLM-13 cells incubated with different nanoparticles for 0.5 h; green represents C6 and blue represents nucleus. (B) Fluorescence images of HCT116 cells incubated with different nanoparticles for 0.5 h; green represents C6 and blue represents nucleus. (C) Flow cytometric analyses of MOLM-13 cells incubated with nanoparticles (n = 3). (D) Flow cytometric analyses of HCT116 cells incubated with nanoparticles (n = 3). ^***p < 0.001

Fig. 4

Biocompatibility of A6-NP. (A) Hemolysis test of fabricated nanoparticles. (B) Hemolysis ratio of fabricated nanoparticles. (C) Cell viabilities of blank nanoparticles in HUVECs (n = 3). (D) Cell viabilities of blank nanoparticles in MOLM13 cells (n = 3). (E) Cell viabilities of blank nanoparticles in HCT116 cells (n = 3)

Fig. 5

In vitro anticancer activity of fabricated nanoparticles. (A) Cell viability of MOLM-13 cells treated with different formulations for 48 h (n = 3). (B) Cell viability of HCT116 cells treated with different formulations for 48 h (n = 3). (C) Apoptotic assays of MOLM-13 cells treated with different formulations (n = 3). (D) Western blot assay of CDK-4 in MOLM13 and HCT116 cells treated with NP, 2.3%A6-NP and 5.5%A6-NP, respectively.   ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 6

(A) Overview of the experimental design of in vivo antileukemia activity (n = 5). (B) WBC counts, (C) PLT counts, (D) HGB level of peripheral blood collected from the eye socket of the AML mice on day 9. (E) ALT level in serum. (F) AST level in serum. (G) Body weight changes of mice. (H) Survival curves of mice

Fig. 7

(A) In vivo experimental scheme of leukemia cells infiltrated. (B) Infiltration analysis of leukemia cells in peripheral blood (PB), bone marrow (BM) and spleen determined by flow cytometry (n = 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. (C) Representative views of H&E-stained BM and spleen sections, scale bar 100 μm

Fig. 8

Histological evaluation for tissue damage of the major organs. Representative views for H&E of heart, liver, lung and kidney slices, scale bar 100 μm

Fig. 9

(A) In vivo fluorescence images of HCT116 tumors at different time points after i.v. injection of IR780-loaded nanoparticles. (B) Ex vivo images of main organs and tumors from mice treated with IR780-loaded nanoparticles. (C) Semi-quantitative analysis of fluorescence intensity of tumor and main organs ex vivo. (D) Body weight changes of mice. (E) Tumor volume of each group. (F) Tumor images of each group. (G) Tumor weight of each group (H) H&E, TUNEL and Ki67 staining of tumor sections, scale bar 50 μm. Quantitative results of TUNEL (I) and Ki67 (J).

Fig. 10

Representative views of H&E-stained spleen, heart, liver, lung and kidney sections, scale bar 100 μm