Contact-Triggered Lipofection from Multilayer Films Designed as Surfaces for in Situ Transfection Strategies in Tissue Engineering

Abstract

Biomaterials, which release active compounds after implantation, are an essential tool for targeted regenerative medicine. In this study, thin multilayer films loaded with lipid/DNA complexes (lipoplexes) were designed as surface coatings for in situ transfection applicable in tissue engineering and regenerative medicine. The film production and embedding of lipoplexes were based on the layer-by-layer (LbL) deposition technique. Hyaluronic acid (HA) and chitosan (CHI) were used as the polyelectrolyte components. The embedded plasmid DNA was complexed using a new designed cationic lipid formulation, namely, OH4/DOPE 1/1, the advantageous characteristics of which have been proven already. Three different methods were tested regarding its efficiency of lipid and DNA deposition. Therefore, several surface specific analytics were used to characterize the LbL formation, the lipid DNA embedding, and the surface characteristics of the multilayer films, such as fluorescence microscopy, surface plasmon resonance spectroscopy, ellipsometry, zeta potential measurements, atomic force microscopy, and scanning electron microscopy. Interaction studies were conducted for optimized lipoplex-loaded polyelectrolyte multilayers (PEMs) that showed an efficient attachment of C2C12 cells on the surface. Furthermore, no acute toxic effects were found in cell culture studies, demonstrating biocompatibility. Cell culture experiments with C2C12 cells, a cell line which is hard to transfect, demonstrated efficient transfection of the reporter gene encoding for green fluorescent protein. In vivo experiments using the chicken embryo chorion allantois membrane animal replacement model showed efficient gene-transferring rates in living complex tissues, although the DNA-loaded films were stored over 6 days under wet and dried conditions. Based on these findings, it can be concluded that OH4/DOPE 1/1 lipoplex-loaded PEMs composed of HA and CHI can be an efficient tool for in situ transfection in regenerative medicine.

Liposomes Embedded in Layer by Layer Constructs as Simplistic Exosome Transfer Model

“…These previous results indicate that MDA-MB-231 cells will act upon gradual disassembly of the layers and liberation of NR-Liposomes from the substrate. The observed endosomes indicate that the ECM mimic layers enhanced the stability of NR-Liposomes but, similar to LbL lms containing lipoplexes [53], did not obstruct their internalization. On the contrary, they endorse an internalization via endocytosis.…”

Nanoarchitectonics at Interfaces for Regulations of Biorelated Phenomena: Small Structures with Big Effects

“…Wölk and coworkers demonstrated contact‐triggered lipofection using lipid/DNA complexes (lipoplexes) loaded in LbL multilayer films of hyaluronic acid and chitosan. [ 45 ] Surface LbL films promoted attachment of C2C12 cells by exhibiting their sufficient biocompatibility. Although the C2C12 cells are known to be hard to transfect, efficient transfection of the GFP reporter gene from the lipoplex‐loaded LbL films was demonstrated.…”

Investigating 3R In Vivo Approaches for Bio‐Distribution and Efficacy Evaluation of Nucleic Acid Nanocarriers: Studies on Peptide‐Mimicking Ionizable Lipid

“…[ 13 ] Recent studies demonstrate that the CAM is also an effective model to test non‐viral nucleic acid delivery systems in complex tissues. [ 13–18 ]…”

“…[13] Recent studies demonstrate that the CAM is also an effective model to test non-viral nucleic acid delivery systems in complex tissues. [13][14][15][16][17][18] Experiments with zebrafish larvae can be performed within high-throughput screening setups and thus become very promising for nanomedicine research. [19] Key factors for nanomedicine research are transparent zebrafish larvae, fluorescent reporter lines, and sophisticated imaging techniques (confocal or light sheet microscopy).…”

“…Detailed characterization of this complex formation process between the lipid mixture and plasmid DNA (pDNA) as well as stability evaluation of the resulting lipoplexes were published previously. [23,24] The efficient in vitro DNA transfer of OH4:DOPE lipoplexes was demonstrated for various cell lines: A549 (adenocarcinomic human alveolar basal epithelial cells), HELA (human cervical cancer cells), LLC-PK1 (porcine renal tubular epithelial cells), HEK 293T (human embryonic kidney cells), and C2C12 (mouse myoblast cell line), [18,[23][24][25][26] These studies also demonstrate that the most efficient loading degree of lipoplexes can be obtained at an N/P ratio (ratio of primary amines of the lipid formulation to phosphate moieties of DNA, the representative ratio for loading degree) of 4. Nevertheless, currently no information exists, on how the formulation behaves in vivo, despite this information is needed to figure out a clinical application.…”