Biparatopic Protein Nanoparticles for the Precision Therapy of CXCR4+ Cancers.

Olivia Cano-Garrido,Ugutz Unzueta,Esther Vázquez,Eric Voltà-Durán,Aïda Falgàs,Naroa Serna,Laura Sánchez-García,Eloi Parladé,Ramón Mangues,Alejandro Sánchez-Chardi,Patricia Álamo,Isolda Casanova,Antonio Villaverde,Mònica Roldán

doi:10.3390/cancers13122929

Abstract

Simple SummaryAimed at minimizing side toxicities cancer therapies require appropriate functional vehicles at the nanoscale, for receptor-mediated tumor-targeted drug delivery. The aim of the present study was to explore the human peptide EPI-X4 as a CXCR4-targeting agent in self-assembled, protein-only nanoparticles. While the systemic tumor biodistribution of EPI-X4-based materials is modest, this peptide shows potent proapoptotic effects on CXCR4+ cancer cells. Interestingly, the in vivo selectivity of EPI-X4 was dramatically improved, once combined into biparatopic nanoparticles, with a second CXCR4 ligand, the peptide T22. Biparatopic nanoparticles promote a highly selective tumor destruction in a mouse model of human colorectal cancer, probably associated to the CXCR4 antagonist role of EPI-X4. This study not only validates a new human ligand of the tumoral marker CXCR4, but it also offers a novel strategy for the combination, in protein nanoparticles, of dual acting ligands of tumoral markers for highly selective drug delivery. The accumulated molecular knowledge about human cancer enables the identification of multiple cell surface markers as highly specific therapeutic targets. A proper tumor targeting could significantly avoid drug exposure of healthy cells, minimizing side effects, but it is also expected to increase the therapeutic index. Specifically, colorectal cancer has a particularly poor prognosis in late stages, being drug targeting an appropriate strategy to substantially improve the therapeutic efficacy. In this study, we have explored the potential of the human albumin-derived peptide, EPI-X4, as a suitable ligand to target colorectal cancer via the cell surface protein CXCR4, a chemokine receptor overexpressed in cancer stem cells. To explore the potential use of this ligand, self-assembling protein nanoparticles have been generated displaying an engineered EPI-X4 version, which conferred a modest CXCR4 targeting and fast and high level of cell apoptosis in tumor CXCR4+ cells, in vitro and in vivo. In addition, when EPI-X4-based building blocks are combined with biologically inert polypeptides containing the CXCR4 ligand T22, the resulting biparatopic nanoparticles show a dramatically improved biodistribution in mouse models of CXCR4+ human cancer, faster cell internalization and enhanced target cell death when compared to the version based on a single ligand. The generation of biparatopic materials opens exciting possibilities in oncotherapies based on high precision drug delivery based on the receptor CXCR4.

Highlights

A major challenge in cancer therapy is cell-targeted drug delivery intended to increase local drug concentration in tumor tissues, minimize systemic toxicities and improve the therapeutic outcome [1,2]
This size range is ideal for improving enhanced permeability and retention (EPR) effect and cell uptake, and to minimize renal filtration of any associated small molecular weight drugs [32]
While the parental version (EPIX4-GFP-H6) reached an unstable oligomerization in form of nanoparticulate entities of different sizes, the protein version carrying the extra cationic sequence (EPIX4-(RK)-GFP-H6) spontaneously self-assembled as regular NPs of around 40 nm (Polydispersion Index, PdI = 0.343) (Figure 1C,D). Fully supporting these results, field emission scanning electron microscope (FESEM) examinations showed toroid materials with a regular morphometry (Figure 1E), that confirmed the measurements obtained by dynamic light scattering (DLS) and size-exclusion chromatography (SEC)

Summary

Introduction

A major challenge in cancer therapy is cell-targeted drug delivery intended to increase local drug concentration in tumor tissues, minimize systemic toxicities and improve the therapeutic outcome [1,2]. The molecular understanding of cancer-specific cellular processes and the identification of overexpressed surface antigens have rapidly expanded the number of therapeutic targets [3,4]. In this context, tumor cell targeting mediated by surface receptors is a smart strategy to enhance drug accumulation within pathogenic cells, taking advantage of the particular properties of drug carriers with nanoscale size [5,6,7]. The horseshoe crab derivative CXCR4 ligand T22 [15] is one of the few tumor-homing peptides that shows high affinity for CXCR4 in a recombinant form, being capable of promoting selective uptake of macro-molecular complexes and NPs inside CXCR4 overexpressing cells [16]. Its high potential as a precision ligand in the nanomedicine of cancer has been robustly supported, stressing its capability to selectively deliver conventional anticancer drugs [17], imaging agents [18] and proteins with cytotoxic activity, such as pro-apoptotic factors [19] and microbial, plant and animal toxins [20,21,22,23,24]

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