Selective staining and eradication of cancer cells by protein-carrying DARPin-functionalized liposomes

Abstract

Since their discovery, liposomes have been widely employed in biomedical research. These nano-size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand-targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80-90 nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme-containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9-29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub-nanomolar concentrations HER2-positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor-positive cells demonstrated here makes the DARPin-functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy.

Dual Targeting of Cancer Cells with DARPin-Based Toxins for Overcoming Tumor Escape

“…Previously we have shown that liposomes functionalized with HER2-specific DARPin can be an effective vehicle for proteins delivery to cancer cells [ 28 ]. Here we used this method for the preparation of ligand-targeted liposomes comprising large quantities of encapsulated toxic protein for the treatment of HER2-positive cancer in vivo.…”

“…Ribonuclease Bn from B. amyloliquefaciens [ 29 ] was used as a cytotoxic moiety. DARPin and Bn genes were expressed in Escherichia coli BL21(DE3) strain as described by us previously [ 24 , 28 ]. RNAse activity of purified Bn was determined using the acid-insoluble RNA precipitate method [ 30 ].…”

“…Extrusion of the proteolipid mixture through a 100 nm pore size membrane filter yielded unilamelar vesicles containing high quantities of proteins. The spectrum of Bn containing liposomes ( Figure 1 b, blue curve) can be fairly simulated ( Figure 1 b, red curve) by summing spectral curves corresponding to ~1.1 nM liposomes (concentration of the vesicles is 1 mg/ml suspension [ 28 ]) and 6 µM Bn ( Figure 1 b, dotted grey curves). The Bn to liposome molar ratio, which corresponds to the number of protein molecules in a liposome is thus equal to about 5454 (6000 nM/1.1 nM).…”

“…Previously we reported a novel method for preparation of HER2-targeted liposomes (80–90 nm in diameter), each containing thousands of encapsulated protein molecules [ 28 ]. Here we use this approach for the preparation of liposomes loaded with RNAse Bn for anti-HER2 cancer therapy.…”

DARPins: Promising Scaffolds for Theranostics

“…DARPin was also attached to nanostructures via maleimide conjugation. This method was utilized for DARPin 9_29 conjugation with the surface of ETA-containing liposomes functionalized using Trout's reagent [87].…”