Engineering osteogenic microenvironments by combination of multilayers from collagen type I and chondroitin sulfate with novel cationic liposomes

Y A Brito Barrera; G Hause; M Menzel; C E H Schmelzer; E Lehner; K Mäder; C Wölk; T Groth

doi:10.1016/j.mtbio.2020.100071

Engineering osteogenic microenvironments by combination of multilayers from collagen type I and chondroitin sulfate with novel cationic liposomes

Mater Today Bio. 2020 Jul 31:7:100071. doi: 10.1016/j.mtbio.2020.100071. eCollection 2020 Jun.

Authors

Y A Brito Barrera¹, G Hause², M Menzel³, C E H Schmelzer³, E Lehner⁴, K Mäder⁴, C Wölk⁵, T Groth^{1

6}

Affiliations

¹ Department Biomedical Materials, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Heinrich Damerow Strasse 4, 06120, Halle (Saale), Germany.
² Martin Luther University Halle-Wittenberg, Biocenter, Weinbergweg 22, 06120, Halle (Saale), Germany.
³ Department of Biological and Macromolecular Materials, Fraunhofer Institute for Microstructure of Materials and Systems (IMWS), Walter-Hülse-Strasse 1, 06120, Halle (Saale), Germany.
⁴ Department Pharmaceutical Technology, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Kurt-Mothes Straße 3, 06120, Halle (Saale), Germany.
⁵ Pharmaceutical Technology, Institute of Pharmacy, Faculty of Medicine, Leipzig University, 04317, Leipzig, Germany.
⁶ Interdisciplinary Center of Materials Science, Martin Luther University Halle-Wittenberg, D-06099, Halle (Saale), Germany.

Abstract

Cationic liposomes composed of a novel lipid (N-{6-amino-1-[N-(9Z) -octadec9-enylamino] -1-oxohexan-(2S) -2-yl} -N'- {2- [N, N-bis(2-aminoethyl) amino] ethyl} -2-hexadecylpropandiamide) (OO4) and dioleoylphosphatidylethanolamine (DOPE) possess high amounts of amino groups and are promising systems for lipofection. Moreover, these cationic liposomes can also be used as a polycationic entity in multilayer formation using layer-by-layer technique (LbL), which is a method to fabricate surface coatings by alternating adsorption of polyanions and polycations. Since liposomes are suitable for endocytosis by or fusion with cells, controlled release of their cargo on site is possible. Here, a polyelectrolyte multilayer (PEM) system was designed of chondroitin sulfate (CS) and collagen type I (Col I) by LbL technique with OO4/DOPE liposomes embedded in the terminal layers to create an osteogenic microenvironment. Both, the composition of PEM and cargo of the liposomes were used to promote osteogenic differentiation of C2C12 myoblasts as in vitro model. The internalization of cargo-loaded liposomes from the PEM into C2C12 cells was studied using lipophilic (Rhodamine-DOPE conjugate) and hydrophilic (Texas Red-labeled dextran) model compounds. Besides, the use of Col I and CS should mimic the extracellular matrix of bone for future applications such as bone replacement therapies. Physicochemical studies of PEM were done to characterize the layer growth, thickness, and topography. The adhesion of myoblast cells was also evaluated whereby the benefit of a cover layer of CS and finally Col I above the liposome layer was demonstrated. As proof of concept, OO4/DOPE liposomes were loaded with dexamethasone, a compound that can induce osteogenic differentiation. A successful induction of osteogenic differentiation of C2C12 cells with the novel designed liposome-loaded PEM system was shown. These findings indicate that designed OH4/DOPE loaded PEMs have a high potential to be used as drug delivery or transfection system for implant coating in the field of bone regeneration and other applications.

Keywords: AFM, Atomic force microscopy; C2C12 myoblasts; CLSM, Confocal Laser Scanning Microscopy; CS, chondroitin sulfate; Col I, Collagen I; DLS, Dynamic light scattering; DMEM, Dulbecco’s modified Eagle’s medium; DOPE, dioleoylphosphatidylethanolamine; Dex, Dexamethasone; ECM, Extracellular matrix; GAG, Glycosaminoglycan; LbL, Layer-by-Layer technique; OO4, (N-{6-amino-1-[N-(9Z) -octadec9-enylamino] -1-oxohexan-(2S) -2-yl} -N’- {2- [N, N-bis(2-aminoethyl) amino] ethyl} -2-hexadecylpropandiamide); PBS, Phosphate-buffered saline; PEI, Polyethylenimine; PEM, Polyelectrolyte multilayer; SEM, Scanning electron microscopy; SPR, Surface plasmon resonance; TEM, Transmission electron microscopy; WCA, Water contact angle; cationic lipids; chondroitin sulfate; collagen I; internalization; osteogenic differentiation; polyelectrolyte multilayer system.