Modification of casein micelles by aptamers for targeted drug delivery purposes - biophysical characterization of formulations by size exclusion chromatography

Caseins belong to the group of milk proteins, which gain a lot of interest as a potential biopolymeric platform for creating an affordable and effective drug delivery vehicle [1]. The structural and physicochemical properties of caseins allow their self-assembly into structures called micelles with the possibility to encapsulate hydrophobic bioactive agents and pharmaceuticals including anticancer drugs [2]. Effective transport of loaded material should be targeted – drugs being delivered to the site of their desired therapeutic effect. This can be performed by modification of drug carriers by DNA aptamers [3]. DNA aptamers are short single-stranded oligonucleotides that bind to specific molecular targets due to their three-dimensional structure. In this work, we used sgc8c DNA aptamer that specifically binds to protein tyrosine kinase 7 (PTK7), which is highly expressed in the membranes of the leukemic cells [4].

We have focused on the preparation of β-casein micelles and their modification with fluorescently labeled (Atto542) sgc8c aptamer conjugated by a cholesteryl-TEG linker (Chol-sgc8c-Atto542). The micelles were made from β-casein (2 mg/ml) in Na-phosphate buffer (10 mmol/l, pH 7) in the presence of CaCl₂ (10 mmol/l) or without CaCl₂ under constant stirring for 1.5 h at 25 °C, followed by filtration through a 0.2 μm pore size filter. The filtered micelles were modified with aptamers (2 μmol/l) under constant stirring for 2 h at laboratory temperature. The prepared formulations were analyzed by fluorescence measurements using size exclusion chromatography (SEC-HPLC) on a 30 nm size pore column. The fluorescent signal coming from the internal fluorescence of β-casein (λ_ex = 295 nm, λ_em = 343 nm) and from the Atto542- fluorescence of aptamer (λ_ex = 540 nm, λ_em = 565 nm) were obtained. The fluorescence peak of β-casein eluted at 3.4 min decreased for the complexed sample of β-casein + Chol-sgc8c-Atto542, and the fluorescence signal from the aptamer was partly shifted to this region, indicating a successful modification. Similar results were observed for the complexed sample in the presence of CaCl₂. In both cases, aptamers being not coeluted – signal at later retention times was also observed, indicating the presence of unbound Chol-sgc8c-Atto542, too.

The obtained results can serve for optimization of the experimental protocol and as a base for further studies of the casein micelles modified by aptamers as a targeted drug delivery platform in cancer treatment.