Development a hyaluronic acid ion-pairing liposomal nanoparticle for enhancing anti-glioma efficacy by modulating glioma microenvironment

Abstract

Glioma, one of the most common brain tumors, remains a challenge worldwide. Due to the specific biological barriers such as blood-brain barrier (BBB), cancer stem cells (CSCs), tumor associated macrophages (TAMs), and vasculogenic mimicry channels (VMs), a novel versatile targeting delivery for anti-glioma is in urgent need. Here, we designed a hyaluronic acid (HA) ion-pairing nanoparticle. Then, these nanoparticles were encapsulated in liposomes, termed as DOX-HA-LPs, which showed near-spherical morphology with an average size of 155.8 nm and uniform distribution (PDI = 0.155). HA was proven to specifically bind to CD44 receptor, which is over-expressed on the surface of tumor cells, other associated cells (such as CSCs and TAMs) and VMs. We systematically investigated anti-glioma efficacy and mechanisms in vivo and in vitro. The strong anti-glioma efficacy could attribute to the accumulation in glioma site and the regulation of tumor microenvironment with depletion of TAMs, inhibition of VMs, and elimination of CSCs.

Keywords: CD44; CSCs; Hyaluronic acid; TAMs; glioma.

Figure 1.

Characterization of DOX-HA-LPs. (a) Schematic illustration of DOX-HA-LPs, which is transported into glioma site after tail vein injection, targeting TAMs and CSCs mediated by CD44, resulting in depletion of TAMs, inhibition of VMs and elimination of CSCs; (b) TEM image and schematic diagrams of DOX-HA-LPs. Scale bar, 100 nm; (c) DLS size and zeta-potential measurement of DOX-HA-LPs; (d) the stability of DOX formulation in plasma and PBS for 72 h; DOX release profiles of DOX formulations at pH 7.4 (n = 3, mean ± SD).

Figure 2.

The effect of different formulations on cells. (a) Microscopic image of C6 CSCs spheres. Scale bar, 50 μm; (b) expression of CD44 at C6, C6 CSCs, B16F10 (as a positive control cell line) and HK2 (as a negative control cell line) cells; (c) cellular uptake of DOX formulations after incubation with HK2, C6 cells for 4 h. HA + DOX-HA: cells were incubated with HA solution (10 mg/mL, 100 kDa) in advance. Data represent mean ± SD (n = 3). *p < .05, **p < .01, ****p < .0001; (d) endocytosis inhibition assay on C6 cells. Data represent mean ± SD (n = 3). ***p < .001. ****p < .0001; (e) DOX-HA-LPs selectively targeted IL-4 treated RAW 264.7. Data represent mean ± SD (n = 3). ***p < .001; (f) the destruction of VMs channels after treatment with different DOX formulations. Scale bar, 100 μm.

Figure 3.

DOX-HA-LPs exhibit a different circulating behavior and a glioma-bearing brains concentrated capability. (a) Ex vivo images showed the drug biodistribution of DOX formulations in C6 glioma-bearing mice at 0.5, 1, 2, and 4 h after intravenous administration; (b) the mean concentrations of DOX in different tissues of C6 glioma-bearing mice after intravenous administration of DOX formulations at 0.5, 1, 2, and 4 h. Data represent mean ± SD (n = 5); (c) in vivo fluorescent microscopy images of brain sections from C6 glioma-bearing mice at 1 h post-injection of DOX and DOX-HA-LPs. TAMs were identified by CD206 marker and nuclei stained with DAPI. Scale bar, 50 μm.

Figure 4.

The survival curves of C6 glioma-bearing mice treated with DOX formulations (2 mg/kg DOX) at day 5, 8, 11, and 14 after inoculation. Data represent mean ± SD (n = 10).

Figure 5.

H&E staining of brain glioma site at 24 h after antitumor dosage administration of saline, free DOX and DOX-HA-LPs. Scale bar, 50 μm and 20 μm (small boxes). CD34-PAS dual staining, CD133 staining, CD206 staining on the brain glioma site. Scale bar, 50 μm.