Generation of Anti-Epidermal Growth Factor Receptor-2 (HER2) Immunoliposomes Using Microbial Transglutaminase (mTG)-Mediated Site-Specific Conjugated Antibodies

Nanocarriers have found their interests in many fields including drug delivery and labeling of cells with the aim to target and eradicate tumor cells. One of the approaches to specifically address nanocarriers, such as liposomes, to their target is to attach antibodies of interest to their surface. To date, the development of immunoliposomes has been widely explored but has mainly involved chemical and unspecific reactions that could impair antibody stability, integrity, and orientation, thus reducing optimized immunoliposomes generation. In this study, we report the use of the patented COVISOLINK technology and the strain-promoted alkyne-azide cycloaddition (SPAAC) to generate immunoliposomes that target HER2 positive breast cancer with Trastuzumab as the antibody to be coupled. The efficacy of our two-step functionalization strategy and the successful specific coupling of the antibodies were validated by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC), which allowed a precise quantification of antibodies conjugated to liposomes and confirmed by cryo-TEM and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. We also demonstrate by flow cytometry and epifluorescence microscopy that the produced anti-HER2 immunoliposomes were able to interact specifically with their target cells (SK-BR-3) while remaining negative with cells that express HER2 at a low level (MDA-MB-231). Hence, for the first time, our COVISOLINK strategy using microbial transglutaminase (mTG) enables the preparation and production of well-characterized immunoliposomes that could be used in different applications, including therapies.