Efficient Delivery of Antibodies Intracellularly by Co-Assembly with Hexahistidine-Metal Assemblies (HmA)

Abstract

Purpose: There has been a substantial global market for antibodies, which are based on extracellular targets. Binding intracellular targets by antibodies will bring new chances in antibody therapeutics and a huge market increase. We aim to evaluate the efficiency of a novel delivery system of His₆-metal assembly (HmA) in delivering intracellular antibodies and biofunctions of delivered antibodies.

Methods: In this study, the physicochemical properties of HmA@Antibodies generated through co-assembling with antibodies and HmA were well characterized by dynamic light scatter. The cytotoxicity of HmA@Antibodies was investigated by Cell Counting Kit-8 (CCK-8). The endocytic kinetics and lysosome escape process of HmA@Antibodies were studied by flow cytometry and fluorescent staining imaging, respectively. Compared to the commercialized positive control, the intracellular delivery efficiency by HmA@Antibodies and biofunctions of delivered antibodies were evaluated by fluorescent imaging and CCK-8.

Results: Various antibodies (IgG, anti-β-tubulin and anti-NPC) could co-assemble with HmA under a gentle condition, producing nano-sized (~150 nm) and positively charged (~+30 eV) HmA@Antibodies particles with narrow size distribution (PDI ~ 0.15). HmA displayed very low cytotoxicity to divers cells (DCs, HeLa, HCECs, and HRPE) even after 96 h for the feeding concentration ≤100 μg mL^-1, and fast escape from endosomes. In the case of delivery IgG, the delivery efficiency into alive cells of HmA was better than a commercial protein delivery reagent (PULSin). For cases of the anti-β-tubulin and anti-NPC, HmA showed comparable delivery efficiency to their positive controls, but HmA with ability to deliver these antibodies into alive cells was still superior to positive controls delivering antibodies into dead cells through punching holes.

Conclusion: Our results indicate that this strategy is a feasible way to deliver various antibodies intracellularly while preserving their functions, which has great potential in various applications and treating many refractory diseases by intracellular antibody delivery.

Keywords: antibody; coordination polymer; intracellular delivery; nanocarrier; peptide assembly.

Figure 1

(A) Schematic illustration of the formation of HmA@Abs particles; (B) Average size, PDI and zeta potential of HmA@Abs particles (HmA@IgG-Cy3, HmA@Anti-β-tubulin and HmA@Anti-NPC); (C) UV-vis spectra of free IgG-Cy3 and HmA@IgG-Cy3; (D) SEM images of HmA@Abs particles (HmA@IgG-Cy3, HmA@Anti-β-tubulin and HmA@Anti-NPC); The scale bars in (D) were 1 μm.

Figure 2

Cell viability of (A) immune cells (DCs), (B) cancer cells (HeLa), and epithelial cells ((C) HCECs and (D) HRPEs) after treatment with different concentrations of the HmA particles (0–100 μg mL⁻¹) for 24 h. (P values: *P < 0.05, all the values are expressed as mean ± SD, n = 3).

Figure 3

(A) Fluorescence images of DCs, HeLa cells, HCECs, and HRPE after treatment with HmA@IgG-Cy3 or free IgG-Cy3 for 6 h; IgG-Cy3 (red); (B) Flow cytometry histogram of the DCs after treatment with HmA@IgG-Cy3 or free IgG-Cy3 for 6 h, 12 h, and 24 h; Endocytosis-positive cell rate graph (C) and average fluorescence intensity graph (D) with HmA@IgG-Cy3 or free IgG-Cy3; *Indicates the differences between the experimental group and the control group: *p < 0.01, and ***p < 0.001. The scale bars were 200 μm.

Figure 4

(A) Fluorescent photos of DCs after treatment with different concentrations of IgG-Cy3-loaded HmA or PULSin for 4 h. IgG-Cy3 (red), the scale bars were 100 μm; (B) Quantified fluorescence intensity graph using Image J. *Indicates the differences between the experimental group and the positive control group: *p < 0.01, and ***p < 0.001.

Figure 5

LSCM images of early endosome (EER)- and lysosome (LyR)-localized receptors in DC cells after treatment with different concentrations of HmA@IgG-FITC particles for 4 h; HmA@IgG-FITC particles (green); EER and LyR (red); DAPI (blue); The scale bars were 20 μm.

Figure 6

(A) LSCM images of anti-β-tubulin immunostained DCs after treating with the same concentration of HmA@Anti-β-tubulin for 6 h, 12 h and 24 h or free anti-β-tubulin (1 μg mL⁻¹) for 24 h; Positive control: LSCM images of anti-β-tubulin immunostained untreated DCs; Anti-β-tubulin (red), DAPI (blue); The white arrow points to the deformed nucleus, the yellow arrow points to the aggregated tubulin; The scale bars were 20 μm; (B) The cell viability of DCs after treating with HmA@Anti-β-tubulin (0 μM, 0.2 μM, 0.5 μM) and HmA@IgG (0.5 μM) at different times. (P values: ***P <0.001, all the values are expressed as mean ± SD, n = 3).

Figure 7

LSCM images of anti-NPC immunostained DCs after treatment with the same concentration of HmA@ Anti-NPC and Free Anti-NPC for 6 h; positive control: the anti-NPC immunostained untreated DCs; anti-NPC antibodies (red), the white arrows indicate the nuclear membrane, and the scale bars were 20 μm.