Multifunctional microcapsules for biosensing and delivery applications

Our first area of interest covers a biosensing application of polyelectrolyte microcapsules

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Figure 1. Microcapsule-based sandwich assay for detection of proteins and nucleic acids. 1, 9 – microcapsule; 2 – protein A; 3 – binder antibody, mouse anti-human β2M (Mαhβ2M); 4 – analyte, human β2M (hβ2M); 5 – detector antibody 1, rabbit anti-hβ2M (Rαhβ2M); 6 – detector antibody 2, goat anti-rabbit-AF488 (GαR-AF488); 7, 14 – fluorophore; 8 – streptavidin; 10 – biotin; 11 – anchor oligo; 12 – analyte oligo; 13 – detector oligo.

Modern point-of-care diagnostics requires development of novel assays for detection of soluble analytes such as gene- and protein-based biomarkers in biological fluids. Along with traditional detection methods such as indirect fluorescence immunoassays and ELISA, microbead-based flow cytometry assays are gaining more and more attention these days, since they allow rapid and quantitative analyte measurements with possibilities of multiplexing and automation. In collaboration with Prof. Springer (Jacobs University Bremen) and Prof. Klöck (Hochschule Bremen) we develop new assays for detection of small analytes such as protein biomarkers and nucleic acids [1]. Using layer-by-layer (LbL) assembly, we produce robust chemically crosslinked polyelectrolyte microcapsules with carboxylated surface [2] that is further coated with adaptor proteins such as protein A or streptavidin. Adaptor proteins ensure optimized orientation of binding ligands such as antibodies or biotinylated oligonucleotides, which are highly selective for a particular analyte. The analyte detection is then performed on the capsule surface, when the analyte molecule is sandwiched between a binder and a detector molecule (Figure 1). To quantify the analyte, we use flow cytometry as an optical readout. Recently, we demonstrated the ability of detecting the blood cancer biomarker beta-2 microglobulin (hβ2M) in the fM to pM concentration range with the help of protein A-coated capsules, while streptavidin-coated capsules allowed for detection of nucleic acids in the nM concentration range. The detection limits of our assay are similar to or even below the limits of commercially available bead-based assays and conventional ELISA. The developed assay allows rapid quantitative analyte measurement, while providing high sensitivity and selectivity at very small sample quantities. The assay is universal and can be used for detection of a broad range of analytes, e.g. intermediate or final products of biotechnological processes or multiple analytes in biomedical research.

Our second area of interest covers a delivery application of polyelectrolyte microcapsules.

Figure 2. Selective targeting of MHC class I proteins in living cells with polyelectrolyte microcapsules.

Figure 2. Selective targeting of MHC class I proteins in living cells with polyelectrolyte microcapsules.

Genomics and the recent development in molecular biology have brought a large variety of proteins, peptides, enzymes and nucleic acids that found their application as therapeutic agents in biomedical research, and particularly in the therapy of cancer. However, most proteins or enzymes are fragile, and small conformational changes may reduce their activity. Therefore their stabilization is required. Polyelectrolyte microcapsules are able to load a sufficient amount of therapeutic cargo in their cavity. The cargo then stays protected from hydrolyzing enzymes by separating polymeric shell, thus it can be delivered to the cell in its active form. Adjustability of physicochemical properties is a big advantage of microcapsules over other drug delivery systems, since capsules can be easily chemically modified to meet the needs of individual experimental design [3] [4] [5]. For instance, the cargo can be released into the cytosol in response to well-defined stimuli, e.g. laser light or ultrasound if the capsules are functionalized with nanoparticles, or in the course of biodegradation if the capsules are made of biodegradable materials [6] [7]. Functionalized capsules can be introduced into living cells via electroporation, and then used as in situ reporter or for retrieval of metabolites from cells [8]. On the other hand, microcapsules equipped with targeting molecules such as antibodies, receptor peptides or aptamers have the potential to selectively bind to a certain receptor at the plasma membrane, and thus to target a specific cell type in a particular site of the body. We develop and optimize a protocol of biofunctionalization of polyelectrolyte microcapsules with proteins in order to achieve the best targeting. We vary amount and orientation of targeting ligands on the capsule surface, and then quantify the targeting efficiency by flow cytometry. Recently we have demonstrated that functionalized capsules can target specific allotype of major histocompatibility complex (MHC) class I proteins at the plasma membrane of B cells very selectively (Figure 2) [9]. By selecting cognate antibody-antigen pairs, biofunctionalized capsules have the potential to be used for targeting any receptor at the plasma membrane that is specific for a certain disease, and thus serve as a “smart” delivery tool that could improve the efficiency of the drug.

Grant: Bundesministerium für Bildung und Forschung (BMBF) foundation “Strategieprozess Biotechnologie 2020+: Basistechnologien”, project “Prozessüberwachung in vitro und in vivo mit Polyelektrolyt Nanokapseln” (031A153A-B) in collaboration with S. Springer (coordinator, Jacobs University Bremen) and G. Klöck (Hochschule Bremen).

Related publications:

  1. Verma S.K., Amoah A., Schellhaas U., Winterhalter M., Springer S., Kolesnikova T.A. “To catch or not to catch”: Microcapsule-based sandwich assay for detection of proteins and nucleic acids. Adv. Funct. Mater. (2016) 26, 6015-6024.
  2. Germain M., Grube S., Carriere V., Richard-Foy H., Winterhalter M., Fournier D. Nanocontainer with composite wall: Lipid vesicles coated with several layers of crosslinked polyelectrolytes. Adv. Mater. (2006) 18, 2868-2871.
  3. Laugel N., Betscha C., Winterhalter M., Voegel J.C., Schaaf P., Ball V. Relationship between the Growth Regime of Polyelectrolyte Multilayers and the Polyanion/Polycation Complexation Enthalpy. J. Phys. Chem. B (2006) 110, 19443-19449.
  4. Sukhorukov G.B., Rogach A.L., Garstka M., Springer S., Parak W.J., Munoz-Javier A., Kreft O., Skirtach A., Susha A.S., Ramaye Y., Palankar R., Winterhalter M. Multifunctionalized Polymer Microcapsules: Novel Tools for Biological and Pharmacological Applications. Small (2007) 3(6), 944-955.
  5. Kolesnikova T.A., Skirtach A.G., Möhwald H. Red blood cells and polyelectrolyte multilayer capsules: Natural carriers versus polymer-based drug delivery vehicles. Expert Opin. Drug Deliv. (2013) 10(1), 47-58.
  6. Palankar R., Skirtach A.G., Kreft O., Bédard M., Garstka M., Gould K., Möhwald H., Sukhorukov G.B., Winterhalter M., Springer S. Controlled intracellular release of peptides from microcapsules enhances antigen presentation on MHC class I molecules. Small (2009) 5(19), 2168-2176.
  7. Kolesnikova T.A., Gorin D.A., Fernandes P., Kessel S., Khomutov G.B., Fery A., Shchukin D.G., Möhwald H. Nanocomposite microcontainers with high ultrasound sensitivity. Adv. Funct. Mater. (2010) 20, 1189-1195.
  8. Studer D., Palankar R., Bédard M., Winterhalter M., Springer S. Retrieval of a metabolite from cells with polyelectrolyte microcapsules. Small (2010) 6(21), 2412-2419.
  9. unpublished results