Supramolecular Heterodimer Peptides Assembly for Nanoparticles Functionalization

Abstract

Nanoparticle (NP) surface functionalization with proteins, including monoclonal antibodies (mAbs), mAb fragments, and various peptides, has emerged as a promising strategy to enhance tumor targeting specificity and immune cell interaction. However, these methods often rely on complex chemistry and suffer from batch-dependent outcomes, primarily due to limited control over the protein orientation and quantity on NP surfaces. To address these challenges, a novel approach based on the supramolecular assembly of two peptides is presented to create a heterotetramer displaying V_HHs on NP surfaces. This approach effectively targets both tumor-associated antigens (TAAs) and immune cell-associated antigens. In vitro experiments showcase its versatility, as various NP types are biofunctionalized, including liposomes, PLGA NPs, and ultrasmall silica-based NPs, and the V_HHs targeting of known TAAs (HER2 for breast cancer, CD38 for multiple myeloma), and an immune cell antigen (NKG2D for natural killer (NK) cells) is evaluated. In in vivo studies using a HER2+ breast cancer mouse model, the approach demonstrates enhanced tumor uptake, retention, and penetration compared to the behavior of nontargeted analogs, affirming its potential for diverse applications.

Keywords: auto‐assembly; biofunctionalization; nanomedicine; peptide.

Figure 1

Development of the nanoparticle@K3‐E3@V_HHs library. A) Schematic representation of the strategy used to develop the different targeted nanoparticles. B) SDS‐PAGE qualitative analysis of purified E3@V_HHs. C) Flow cytometry study determining the apparent binding affinity of the different E3@V_HHs, E3@HER2 on HCC1954, E3@CD38 on KMS18, and E3@NKG2D on NK‐92 cell lines. D) Thermal shift assays validating the auto‐assembly of the different E3@V_HHs to K3 peptide. E) Fluorescent imaging of the fluorescent dye (FD) Bodipy 493/503@K3‐E3@HER2 on HER2+ (HCC1954) and HER2‐ (MDA‐MB‐231) cell lines. Nucleus is represented in DAPI (blue) and fluorescent dye (FD) in green. Scale bar = 20 µm. F) Flow cytometry study determining the apparent binding affinity of the different FD@K3‐E3@V_HHs; FD@K3‐E3@HER2 on HCC1954, FD@K3‐E3@CD38 on KMS18, and FD@K3‐E3@NKG2D on NK‐92 cell lines. Data are presented as mean ± standard deviation and all experiments were performed in triplicates.

Figure 2

In vitro validation of the different nanoparticles@K3‐E3@V_HHs. A) Cryo‐TEM images of the liposomes and functionalized liposomes (liposomes@K3‐E3@HER2 and liposomes@K3‐E3@eGFP). Scale bar = 50 nm. B) Hydrodynamic diameters of the three liposomes measured by dynamic light scattering. C) Membrane thickness quantified based on the cryo‐TEM images (n = 150 per group). D) Fluorescent correlation spectroscopy measurements confirming the grafting of E3@V_HHs at the surface of the liposomes. E) Fluorescence imaging of liposome@K3‐E3@HER2 and liposome@K3‐E3@eGFP on HCC1954 cells. Scale bar = 20 µm. F) Quantification of the fluorescence intensity for both liposomes (n = 100 per group). G) Cell binding specificity determined by flow cytometry of the liposome@K3‐E3@HER2 and liposome@K3‐E3@eGFP on HER2+ (HCC1954 and SKOV3) as well as on HER2 low (MDA‐MB‐231) cell lines. H) Cell binding specificity determined by flow cytometry of the various nanoparticles@K3‐E3@HER2 and nanoparticles@K3‐E3@eGFP on HER2+ (HCC1954) cell line. I) Cell binding specificity determined by flow cytometry of the liposome@K3‐E3@CD38 and liposome@K3‐E3@eGFP on CD38+ (KMS12‐BM) as well as on CD38‐ (MDA‐MB‐231) cell lines. J) Cell binding specificity determined by flow cytometry of the liposome@K3‐E3@NKG2D and liposome@K3‐E3@eGFP on NKG2D+ (primary NK cells) cell line. P‐value determined by Mann Whitney tests, *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were performed in triplicates.

Figure 3

Pharmacokinetic and biodistribution studies of the targeted liposomes. A) Schematic representation of the in vivo study. B) Pharmacokinetic profiles of the liposome and liposome@K3‐E3@HER2 intraperitoneally (i.p.) administered in healthy balb/c mice (n = 6 per group). C) Comparison of the pharmacokinetic parameters (half‐life – T_1/2; C _max – concentration max. in plasma; AUC_0‐72 h – area under the curves between 0 and 72 h post‐i.p. administration) normalized to liposome results. D) Fluorescent imaging of the liposomes in major organs 72 h after administration. E) Longitudinal quantification of the nanoparticles’ concentration in the major organs (n = 6 per group). Data are presented as mean ± standard deviation. P‐value determined by one‐way ANOVA. All experiments were performed on N = 3 mice per group.

Figure 4

Improved tumor retention of targeted liposomes. A) Longitudinal tumor uptake fluorescence imaging of liposome, liposome@K3‐E3@eGFP, and liposome@K3‐E3@HER2 groups (n = 5 per group) in HER2+ (HCC1954 cells) subcutaneous breast cancer tumors. B) Longitudinal tumor uptake quantification. C) Hematoxylin and eosin (H&E) staining, nanoparticles uptake (fluorescence signal), and segmentation map of the nanoparticle penetration in the tumor. Scale bar = 200 µm. D) Quantification of the liposome and liposome@K3‐E3@HER penetration profile in the tumor. Data are presented as mean ± standard deviation. n.s. denotes nonsignificant; *, P‐value < 0.05, **, P‐value < 0.01, ***, P‐value < 0.001, Mann‒Whitney tests. Studies were performed with N = 6 mice per group.