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.

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

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

Efficient Transfection of Large Plasmids Encoding HIV-1 into Human Cells-A High Potential Transfection System Based on a Peptide Mimicking Cationic Lipid.

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

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.

Nucleic acid carrier composed of a branched fatty acid lysine conjugate-Interaction studies with blood components.

For the development of gene therapeutics for systemic administration several hurdles have to be overcome. In this article we screen the branched fatty acid lysine conjugate T14diLys, a newly designed cationic lipid for lipofection, regarding this problem. The structure and particle size of lipoplexes, prepared with lipid formulations which are based on these lipid as nucleic acid complexing agent, are investigated in absence and presence of serum. Nuclease digestion assays were performed to evaluate the protective characteristics of the lipid formulation for the complexed nucleic acid. Furthermore, the lipid formulation is investigated regarding the interaction with different serum proteins to get first insights into the protein corona formation. Another focus is set on the hemocompatibility using in vitro assays for hemolysis and complement activation and the irritation test at the chorion allantois membrane of the chicken embryo as in vivo model. Finally, preliminary transfection efficiency studies with cell culture models for cells which are assessable via systemic administration are performed to evaluate possibilities for future therapeutic applications of the new lipid formulations. Summarizing, T14diLys with the co-lipid DOPE can be used to prepare a lipoplex formulation which can be applied systemically and can be used to develop gene therapeutics for targeting endothelial cells, macrophages, or leucocytes.

Lysine-based amino-functionalized lipids for gene transfection: 3D phase behaviour and transfection performance.

Based on previous work, the influence of the chain composition on the physical-chemical properties of five new transfection lipids (TH10, TT10, OH10, OT10 and OO10) containing the same lysine-based head group has been investigated in aqueous dispersions. For this purpose, the chain composition has been gradually varied from saturated tetradecyl (T, C14:0) and hexadecyl (H, C16:0) chains to longer but unsaturated oleyl (O, C18:1) chains with double bonds in the cis configuration. In this work, the lipid dispersions have been investigated in the absence and presence of the helper lipid DOPE and calf thymus DNA by small-angle and wide-angle X-ray scattering (SAXS/WAXS) supplemented by differential scanning calorimetry (DSC), attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and Fourier-transform Raman spectroscopy (FTRS). Lamellar and inverted hexagonal mesophases have been observed in single-component systems. In the binary mixtures, the aggregation behaviour changes with an increasing amount of DOPE from lamellar to cubic. The lipid mixtures with DNA show a panoply of mesophases. Interestingly, TT10 and OT10 form cubic lipoplexes, whereas OO10 complexes the DNA sandwich-like between lipid bilayers in a lamellar lipoplex. Surprisingly, the latter is the most effective lipoplex.

A combined FTIR and DSC study on the bilayer-stabilising effect of electrostatic interactions in ion paired lipids.

Investigating lipid ion pair formation is important for understanding the mechanisms of lipid-mediated drug resistance in bacteria. In this study we have used the charged amphiphiles dipalmitoylphosphatidylglycerol (DPPG) and dihexadecyldimethylammonium bromide (DHDAB), as a model to evaluate the formation of ion pairs by a combined Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) analysis. FTIR was employed to study the environment of the DPPC headgroup phosphate and lipid/surfactant alkane chains, in vesicles formed by the two amphiphiles mixed in various molar ratios. An increase of the absorbance ratio of 1221-1201 cm-1 in the asymmetric phosphate stretching mode was found to follow a sigmoidal relationship with the proportion of DHDAB, increasing to a plateau above a DPPG/DHDAB 1:1 molar ratio of, providing evidence that the PG headgroup phosphate is involved in ion pairing. A consistent red shift was measured for the position of the symmetric CH2 stretch band for the lipid/surfactant 1:1 molar ratio mixture, which is indicative of an increased ordering of the hydrophobic chains. The DSC experiments yielded information about the thermotropic and the mixing behaviour of the lipid/surfactant systems. DPPG and DHDAB seem to form an ion pair with cluster compound characteristics at the equimolar ratio. Most interestingly, the DPPG/DHDAB 2:1 molar ratio mixture is characterized by strong intermolecular interactions, which result in a pronounced stabilization of the gel phase, possibly through the formation of a closely-associated ion triplet configuration in which the charges are delocalised across the headgroups.

Overcoming the polycation dilemma - Explorative studies to characterise the efficiency and biocompatibility of newly designed lipofection reagents.

In this explorative study of the novel cationic lipid OO4 in two different formulations the complex formation with DNA, the biopharmaceutical stability of the lipid/DNA complexes in physiological media, and the transfection efficiency were analysed. We investigated liposomes composed of two binary mixtures of OO4 with either DOPE or DPPE as co-lipids in the molar ratio of 1:3. These formulations were compared with regard to their ability to bind the DNA using gel retardation electrophoresis, ethidium bromide exclusion and zeta potential measurements. Colloidal stability of the lipoplexes in foetal bovine serum (FBS) and the protective effect against degradation by endonucleases were studied. Furthermore, the influence of different salt concentrations on the complex formation with DNA was examined. The DOPE mixture was markedly superior compared to the DPPE mixture. Finally, haemocompatibility studies and gene silencing experiments were performed on OO4:DOPE 1:3 (n:n). The experiments demonstrate that the lipoplex formulation OO4:DOPE 1:3 (n:n) at N/P 4 is a promising candidate for systemic application because of the high colloidal stability in serum without PEGylated lipids, high transfection efficiency, superior resistance against nucleases, reproducible complexation independent of ionic effects, and haemocompatibility.