Solid-binding peptide linker for targeted nanocomposites
Solid-binding peptide linker for targeted nanocomposites
E.L.Guryev 1, L. Liang2, A. Care2, A. Sunna2, N.O. Antonova 1, V. A. Vodeneev1, V. Shipunova1,3, S.M. Deyev1,3, A. V. Zvyagin1,2
1Laboratory of Optical Theranostics, N.I. Lobachevsky Nizhny Novgorod State University
2 ARC Centre of Excellence “BioPhotonics at Nanoscale”, Macquarie University, Sydney, Australia
3Laboratory of Molecular Immunology, Shemyakin&Ovchinnikov Institute of Bioorganic Chemistry, Moscow
Targeted constructs for the tumours diagnosis and therapy based on photoluminescent nanoparticles are among the most rapidly developing areas of present-day biomedicine. These particles possess unique photophysical properties and their surface allows to use them as a platform for diverse targeting/therapeutic biomolecules. Upconversion nanoparticles (UCNPs) are one of the most perspective kinds of photoluminescent substances. The UNCPs are capable of efficient conversion of near-infrared (NIR) excitation at the wavelength of 980 nm into the shorter-wavelength infrared and visible spectral range emission. UNCPs are also inherent high spatial-temporal resolution in bioimaging , low cytotoxicity and long lifetimes . Their near-infrared excitation band falls into the "biological tissue transparency window", which allows the excitation light to penetrate deep into the biological tissue .
To minimize the side effects of tumor visualization and treatment UCNP nanocomposites are required to bioconjugate to active tumor-targeting moieties such as peptides  and antibodies . The conventional bioconjugation techniques may reduce the functional activity of the targeting and lead to the particle aggregation. Therefore, new strategies of assembling UCNPs and targeting moieties while maintaining their functionality and selectivity, are highly desirable.
We have developed a facile strategy to construct nanocomposites functionalized for cancer targeting, based on coating of the UCNP with a silica layer and bioconjugation to antibodies (Ab) through a bifunctional fusion protein (Linker-Protein G, LPG). LPG consist of a solid-binding peptide linker (L) genetically fused to Streptococcus Protein G’ (PG). Solid binding peptides are short amino acid sequences that selectively bind to their corresponding solid surfaces with high affinity through a combination of multiple non-covalent interactions (e.g. van der Waals forces, electrostatic, hydrophobic, and π effects) . The solid binding peptide linker used here (with a sequence of (VKTQATSREEPPRLPSKHRPG)4VKTQTAS) is capable of mediating the specific binding of the LPG to silica-coated nanoparticles across a wide pH range (5-9) . The Linker domain of LPG exhibits high binding affinity towards silica surface, and the IgG-binding protein binds to the Fc fragment of IgG antibodies, thereby ensuring the functional display of the conjugated antibodies. Thus, LPG mediates the functionalization of silica-coated UCNPs (UCNP-SiO2) with cancer cell antibodies allowing for specific target recognition and delivery.
The core UCNPs (NaYF4:Yb,Er) were synthesized using a solvothermal decomposition method. An inert shell of NaGdF4 was further deposited onto the core UCNPs. The core-shell UCNPs (NaYF4:Yb,Er/NaGdF4) retained their morphology and dispersion, and had an average diameter of 31 ± 1 nm. Compared to the core UCNPs, the core-shell UCNPs exhibited 1,7-fold enhanced upconversion photoluminescence under continuous-wave 980-nm excitation. A thin layer of silica (SiO2) was coated onto the core-shell UCNPs using a water-in-oil microemulsion method to improve aqueous solubility and stability of UCNPs in physiological environments. The resulting UCNP-SiO2 particles were spherical in shape, with a mean diameter of 43 ± 2 nm.
UCNP-SiO2 were bioconjugated to LPG and monoclonal antibody for epithelial cell adhesion molecules (EpCAM, also known as CD326) via simple mixing and washing steps. EpCAM is a transmembrane glycoprotein that is expressed at low levels in normal epithelia but overexpressed in epithelial cancers (i.e. carcinomas), and is a recognized target for immunotherapy .
The in vitro binding ability and specificity of the functionalized UCNP-SiO2-LPG-Ab nanocomposites were examined using HT-29, an EpCAM-overexpressing human colon adenocarcinoma cell line, and BV2, an EpCAM-negative murine microglia cell line. Each cell line was incubated with 25 μg/mL nanoparticles for 1 h and washed five times before fluorescence imaging. The UCNP nanocomposites selectively labeled the membrane of the EpCAM-positive HT-29 cells. In contrast, negligible UCNP nanocomposites were observed with the EpCAM-negative BV2 cells.
These results confirm that the LPG-mediated bioconjugation approach allows the anti-EpCAM to maintain good targeting capability when conjugated onto the UCNP nanocomposites, and LPG can be successfully used for attachment of targeting antibodies onto silica-coated UCNPs.
This work was supported by grant No. 14.Z50.31.0022.
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