Lipid-insertion enables targeting functionalization of erythrocyte membrane-cloaked nanoparticles

Abstract

RBC membrane-cloaked polymeric nanoparticles represent an emerging nanocarrier platform with extended circulation in vivo. A lipid-insertion method is employed to functionalize these nanoparticles without the need for direct chemical conjugation. Insertion of both folate and the nucleolin-targeting aptamer AS1411 shows receptor-specific targeting against model cancer cell lines.

Fig. 1

Schematic of the preparation of RBC-NPs with targeting ability. Ligand–linker–lipid conjugates are synthesized and then inserted into RBC membrane ghosts. The resulting ligand-functionalized RBC membranes are used to coat polymeric cores to form targeted RBC-NPs.

Fig. 2

Lipid-insertion enables modification of RBC-NPs with FITC. (A) Flow cytometry histograms of plain RBC ghosts (black) and RBC ghosts incorporated with FITC–linker–lipid (green). (B) Fluorescence microscopy visualization of RBC ghosts modified with FITC (green). Scale bar = 8 µm. (C) FITC–linker–lipid was incubated with RBC ghosts derived from 1 mL of mouse blood. The amount of FITC–linker–lipid incorporated onto the RBC ghosts was then quantified after 30 min of incubation and plotted against the initial input. (D) Physicochemical characterizations (size and zeta potential) of both FITC-modified and unmodified RBC-NPs. (E) SEM images of FITC-modified and unmodified RBC-NPs. Insets represent a single particle with a size of ~80 nm. Scale bars = 500 nm. (F) Colocalization of the polymeric core (red) and the FITC-modified RBC membrane shell (green) upon intracellular uptake by KB cells. Cellular nuclei were stained with DAPI (blue). Scale bars = 8 µm.

Fig. 3

Lipid-insertion enables targeting functionalization of RBC-NPs with folate. (A) Schematic representation of folate–linker–lipid. (B) Flow cytometry histograms of KB cells alone (black) and the cells incubated with folate-functionalized RBC-NPs (red), non-targeted RBC-NPs (blue), and folate-functionalized RBC-NPs together with free folate (orange). (C) Quantification of the mean fluorescence intensity of the histograms in (B). (D) Fluorescence microscopy image of KB cells incubated with folate-functionalized RBC-NPs, non-targeted RBC-NPs, and folate-functionalized RBC-NPs together with free folate. A fluorescent probe DiD was loaded inside the RBC-NPs for visualization (red) and cellular nuclei were stained with DAPI (blue). Scale bars = 25 µm.

Fig. 4

Lipid-insertion enables targeting functionalization of RBC-NPs with AS1411 aptamer. (A) Schematic representation of AS1411–linker–lipid. (B) Flow cytometry histograms of MCF-7 cells alone (black) and the cells incubated with AS1411-functionalized RBC-NPs (red), non-targeted RBC-NPs (blue), and AS1411-functionalized RBC-NPs together with the free AS1411 aptamer (orange). (C) Quantification of the mean fluorescence intensity of the histograms in (B). (D) Fluorescence microscopy of MCF-7 cells incubated with AS1411-functionalized RBC-NPs, non-targeted RBC-NPs, and AS1411-functionalized RBC-NPs together with free AS1411. A fluorescent probe DiD was loaded inside the RBC-NPs for visualization (red) and cellular nuclei were stained with DAPI (blue). Scale bars = 25 µm.