Enhanced immunocompatibility of ligand-targeted liposomes by attenuating natural IgM absorption

Abstract

Targeting ligands are anticipated to facilitate the precise delivery of therapeutic agents to diseased tissues; however, they may also severely affect the interaction of nanocarriers with plasma proteins. Here, we study the immunocompatibility of brain-targeted liposomes, which inversely correlates with absorbed natural IgM. Modification of long, stable positively charged peptide ligands on liposomes is inclined to absorb natural IgM, leading to rapid clearance and enhanced immunogenicity. Small peptidomimetic D8 developed by computer-aided peptide design exhibits improved immunocompatibility by attenuating natural IgM absorption. The present study highlights the effects of peptide ligands on the formed protein corona and in vivo fate of liposomes. Stable positively charged peptide ligands play double-edged roles in targeted delivery, preserving in vivo bioactivities for binding receptors and long-term unfavorable interactions with the innate immune system. The development of D8 provides insights into how to rationally design immunocompatible drug delivery systems by modulating the protein corona composition.

Fig. 1

Immunogenicity of liposomal formulations. a IgG titrations. Absorbance in the ELISA plate versus serum dilution and antibody titer reported as log (EC50). mPEG2000-DSPE, ^LCDX-PEG3400-DSPE, and ^DCDX-PEG3400-DSPE were used as antigens for sLip, ^LCDX-sLip, and ^DCDX-sLip, respectively. b Absorbance in the ELISA plate for anti-PEG IgM evaluation. mPEG2000-DSPE was used as antigen for all formulations. n = 3, data are means ± s.d. Statistical significances were calculated by Student’s t-test. **p < 0.01, ***p < 0.001

Fig. 2

The uptake of sLip, ^LCDX-sLip, and ^DCDX-sLip by APCs. a The ratio of CD11c⁺ cells in BMDCs. BMDCs were stained with anti-CD11c antibody and counted by flow cytometry. b Microscopy observation of DiI⁺ BMDCs by confocal laser scanning microscopy. BMDCs were cultured with DiI-loaded liposomes for 4 h with serum and the nuclei were stained with DAPI. c The ratio of DiI⁺ BMDCs counted by flow cytometry. d The ratio of DiI⁺ cells in MHC II⁺ APCs. Lymph nodes were isolated 12 h post injection, then suspended in PBS. Cell suspension was incubated with anti-MHC II antibody at 4 °C for 1 h and the ratio of DiI⁺ cells in MHC II⁺ APCs was counted by flow cytometry. e Microscopy observation of DiI⁺ macrophages by confocal laser scanning microscopy. RAW264.7 cells were cultured with DiI-loaded liposomes for 1 h with serum and the nuclei were stained with DAPI. f The normalized fluorescence intensity in RAW264.7 cells by flow cytometry. Scale bar = 20 μm, n = 3, data are means ± s.d. Statistical significances were calculated by Student’s t-test. **p < 0.01, ***p < 0.001

Fig. 3

Pharmacokinetic profile and biodistribution of DiI-loaded sLip, ^LCDX-sLip, and ^DCDX-sLip. a Plasma concentration of DiI determined at 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after intravenous injection in BALB/c mice. b The ABC effect of different liposomal formulations. BALB/c mice were injected with a low dose of empty liposomes (sLip, ^LCDX-sLip, and ^DCDX-sLip, 5 mg HSPC per kg of mouse), followed with a second injection of the normal dose of DiI-loaded liposomes 5 days after the first injection (50 mg HSPC per kg of mouse). c Biodistribution of liposomes in liver and spleen of BALB/c mice. n = 3, data are means ± s.d. Statistical significances were calculated by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Effect of the modified peptides on the composition of protein corona. a Separation of protein corona by SDS-PAGE. IgM (at Mw 72 kDa, circled in the red dashed line) was characterized by nano-LC-MS/MS. b Western blot assay of natural IgM on liposomal surface in vivo 1 and 4 h after injection. c Quantification of absorbed natural IgM by normalizing the gray values. n = 3, data are means ± s.d. Statistical significances were calculated by Student’s t-test. NS indicates not significant, *p < 0.05, **p < 0.01

Fig. 5

Effect of electrostatic interaction on the absorption of natural IgM. a, c Western blot assay of natural IgM on liposomal surface after incubation with serum for 1 h. b, d Quantification of absorbed natural IgM by normalizing gray values. n = 3, data are means ± s.d. Statistical significances were calculated by Student’s t-test. *p < 0.05

Fig. 6

Binding mode of peptides with α7 nAChR. ^DCDX-S8 (green, a) and D8 (brown, b) with α7 nAChR is shown. Subunit A of receptor is shown in white and subunit B is in yellow. Residues involved in binding are represented by sticks, and hydrogen bonds are denoted by black dashed lines

Fig. 7

Characterization of D8. a Stability in mouse serum. D8, ^LCDX, and ^DCDX were dissolved in distilled water (1 mg mL⁻¹) and were incubated with mouse serum. RP-HPLC was used to monitor and quantify peptide hydrolysis at predetermined time points. b ^DCDX and D8 competed the binding of DiI-loaded ^DCDX-Lip with Neuro 2a cells. c Transcytosis efficiency of DiI-loaded sLip, ^DCDX-sLip, and D8-sLip across the primary brain capillary endothelial cell monolayer. d Microscopic observation of brain distribution of liposomes. BALB/c mice was injected with DiI-loaded sLip, ^DCDX-sLip, and D8-sLip via tail vein and brains were dissected, frozen sectioned, and stained with anti-CD31 antibody and DAPI 4 h post injection, blue—nuclei, green—blood vessels, and red—DiI dye, scale bar = 10 μm. e Positive DiI area in three random areas were counted by Image Pro. Scale bar = 20 μm, n = 3, data are means ± s.d. Statistical significances were calculated by Student’s t-test.*p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8

Immunocompatibility of D8-sLip. a, b Western blot assay (a) and quantification (b) of natural IgM on liposomal surface in vivo 1 and 4 h after injection. c Pharmacokinetic profiles of sLip, D8-sLip, and ^DCDX-sLip. d Biodistribution in liver and spleen of BALB/c mice of D8-sLip and ^DCDX-sLip. e IgG titrations. Absorbance in the ELISA plate versus serum dilution and antibody titer reported as log (EC50). D8-PEG3400-DSPE and ^DCDX-PEG3400-DSPE was used as antigens for D8-sLip and ^DCDX-sLip, respectively. f Absorbance in the ELISA plate for anti-PEG IgM evaluation. mPEG2000-DSPE was used as antigen for both formulations. n = 3, data are means ± s.d. Statistical significances were calculated by Student’s t-test. NS indicates not significant, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 9

Relationship between net positive charge of peptide ligands and absorbed natural IgM. n = 3, data are means. Linear regression was performed with ^LCDX-sLip, ^DCDX-sLip, ^DCDX-1-sLip, and ^DCDX-2-sLip