Lipoplex-Functionalized Thin-Film Surface Coating Based on Extracellular Matrix Components as Local Gene Delivery System to Control Osteogenic Stem Cell Differentiation.

A gene-activated surface coating is presented as a strategy to design smart biomaterials for bone tissue engineering. The thin-film coating is based on polyelectrolyte multilayers composed of collagen I and chondroitin sulfate, two main biopolymers of the bone extracellular matrix, which are fabricated by layer-by-layer assembly. For further functionalization, DNA/lipid-nanoparticles (lipoplexes) are incorporated into the multilayers. The polyelectrolyte multilayer fabrication and lipoplex deposition are analyzed by surface sensitive analytical methods that demonstrate successful thin-film formation, fibrillar structuring of collagen, and homogenous embedding of lipoplexes. Culture of mesenchymal stem cells on the lipoplex functionalized multilayer results in excellent attachment and growth of them, and also, their ability to take up cargo like fluorescence-labelled DNA from lipoplexes. The functionalization of the multilayer with lipoplexes encapsulating DNA encoding for transient expression of bone morphogenetic protein 2 induces osteogenic differentiation of mesenchymal stem cells, which is shown by mRNA quantification for osteogenic genes and histochemical staining. In summary, the novel gene-functionalized and extracellular matrix mimicking multilayer composed of collagen I, chondroitin sulfate, and lipoplexes, represents a smart surface functionalization that holds great promise for tissue engineering constructs and implant coatings to promote regeneration of bone and other tissues.

Extracellular matrix-inspired surface coatings functionalized with dexamethasone-loaded liposomes to induce osteo- and chondrogenic differentiation of multipotent stem cells.

Biomimetic surface coatings can be combined with conventional implants to mimic the extracellular matrix (ECM) of the surrounding tissue to make them more biocompatible. Layer-by-layer technique (LbL) can be used for making surface coatings by alternating adsorption of polyanions and polycations from aqueous solutions without need of chemical reactions. Here, polyelectrolyte multilayer (PEM) systems is made of hyaluronic acid (HA) as polyanion and Collagen I (Col) as polycation to mimic the ECM of connective tissue. The PEM are combined with dexamethasone (Dex)-loaded liposomes to achieve a local delivery and protection of this drug for stimulation of osteo- and chondrogenic differentiation of multipotent stem cells. The liposomes possess a positive surface charge that is required for immobilization on the PEM. The surface properties of PEM system show a positive zeta potential after liposome adsorption and a decrease in wettability, both promoting cell adhesion and spreading of C3H10T1/2 multipotent embryonic mouse fibroblasts. Differentiation of C3H10T1/2 was more prominent on the PEM system with embedded Dex-loaded liposomes compared to the basal PEM system and the use of free Dex-loaded liposomes in the supernatant. This was evident by immunohistochemical staining and an upregulation of the expression of genes, which play a key role in osteogenesis (RunX2, ALP, Osteocalcin (OCN)) and chondrogenesis (Sox9, aggrecan (ACAN), collagen type II), determined by quantitative Real-time polymerase chain reaction (qRT-PCR) after 21 days. These findings indicate that the designed liposome-loaded PEM system have high potential for use as drug delivery systems for implant coatings that can induce bone and cartilage differentiation needed for example in osteochondral implants.

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

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.

A triple chain polycationic peptide-mimicking amphiphile - efficient DNA-transfer without co-lipids.

Non-viral gene delivery in its current form is largely dependent upon the ability of a delivery vehicle to protect its cargo in the extracellular environment and release it efficiently inside the target cell. Also a simple delivery system is required to simplify a GMP conform production if a marketing authorization is striven for. This work addresses these problems. We have developed a synthetic polycationic peptide-mimicking amphiphile, namely DiTT4, which shows efficient transfection rates and good biocompatibility without the use of a co-lipid in the formulation. The lipid-nucleic acid complex (lipoplex) was characterized at the structural (electron microscopy), physical (laser Doppler velocimetry and atomic force microscopy) and molecular levels (X-ray scattering). Stability studies of the lipoplexes in the presence of serum and heparin indicated a stable formation capable of protecting the cargo against the extracellular milieu. Hemocompatibility studies (hemolysis, complement activation and erythrocyte aggregation) demonstrated the biocompatibility of the formulation for systemic administration. The transfection efficiency was assessed in vitro using the GFP assay and confocal laser scanning microscopy studies. With the chorioallantoic membrane model, an animal replacement model according to the 3R strategy (replacement, refinement, and reduction), initial in vivo experiments were performed which demonstrate fast and efficient transfection in complex tissues and excellent biocompatibility.