A protein nanocontainer targeting epithelial cancers: rational engineering, biochemical characterization, drug loading and cell delivery

Abstract

An original protein nanocontainer for drug vectorization and imaging device open new exciting perspectives in nano-theranostic approaches.

Development of a near infrared protein nanoprobe targeting Thomsen-Friedenreich antigen for intraoperative detection of submillimeter nodules in an ovarian peritoneal carcinomatosis mouse model

“…Those experiments also showed a persistence of the labelling for at least 3 weeks in the peritoneal cavity, a property of the nanoprobe that can be exploited for a post-operative follow up by non-invasive fluorescence imaging. As previously described [48], the A38C protein is also a nanocontainer able to carry a therapeutic molecule, confined in its cavity, to carcinoma cells and deliver it to endosomes and late lysosomes after specific recognition of the TF antigen and subsequent endocytosis. In light of those results, the combination of imaging and targeted drug delivery in a nanotheranostic approach is worth considering with the coinjection procedure.…”

“…In order to perform in vivo detection in small animals, and later on in humans, the probe must be stable over time and easy to use. Biochemical data showed very high temperature stability with Tm in the range of 70°C [48]. We have completed this characterization by evaluating the stability of the protein and its labeling over time.…”

“…Recombinant protein was produced and purified as previously described [44] [48]. After purification, the recombinant A38C protein was labeled with a near infrared emitting dye (Alexa Fluor 647), with an NHS ester covalent bound to -amine lysine residues, using CF®647 SE Protein labeling kit (cat n°#92218, Biotium, USA).…”

“…The A38C variant of the XCL protein was designed by directed evolution as previously described ( Figure 1A) [48]. In order to first study its interaction with cell lines in vitro and later evaluate its efficiency as a nanoprobe in vivo, A38C was labeled with a NIR dye to be followed by micro and macroscopic fluorescence imaging.…”

“…The 3D structure of the XCL protein was resolved by X-ray crystallography and revealed that its homo-tetrameric assembly displayed a complex network of protomer-protomer interfaces and generated a large, hydrated inner cavity of 1 nm 3 [47]. We have recently described the biochemical development and use of this lectin as a nanocontainer enables to specifically deliver in vitro a chemotherapeutic agent [48]. To achieve this objective, it was necessary to control the stability and the dynamic of XCL's quaternary structure by designing XCL variants in which monomers were covalently bonded to each other.…”