Nanobody-functionalized PEG-b-PCL polymersomes and their targeting study.

We prepared and characterized polymersomes functionalized with nanobodies (VHHs) on the basis of biocompatible, biodegradable and FDA-approved poly(ethylene glycol)-block-poly(ϵ-caprolactone) (PEG-b-PCL). Fluorescein isothiocyanate (FITC) and N-beta-maleimidopropyl-oxysuccinimide ester were allowed reacting with H2N-PEG-b-PCL to produce FITC and maleimide (Mal) functionalized copolymers, Mal-PEG-b-PCL and FITC-PEG-b-PCL. A mixture of MeO-PEG-b-PCL, Mal-PEG-b-PCL and FITC-PEG-b-PCL was used to prepare polymersomes by thin film hydration and nanoprecipitation methods. Morphological studies by cryogenic transmission electron microscopy (Cryo-TEM) showed that the nanoparticles exhibited predominantly vesicular structures (polymersomes). Their mean diameters measured by dynamic light scattering were around 150 nm and the zeta-potentials around -1 mV at pH 7.4. The nanoparticles were functionalized with either anti-HER2 (VHH1) or anti-GFP (VHH2) nanobodies using maleimide-cysteine chemistry. Their particle size and zeta-potential increased slightly after nanobody-functionalization. The specific binding of VHH-functionalized polymersomes and control nanoparticles towards HER2 positive breast cancer cells was analyzed by flow cytometry and confocal microscopy. The collected results represent the first report which experimentally demonstrates that VHH1-functionalized PEO-b-PCL polymersomes can target specifically breast cancer cells expressing HER2 receptors. The detailed morphological and cell-binding studies described herein pave the way for future in vivo studies to evaluate the feasibility to use such nanoparticles for targeted drug delivery.