Strategies for the Activation and Release of the Membranolytic Peptide Melittin from Liposomes Using Endosomal pH as a Trigger.

Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high membranolytic activity. However, such lytic peptides also impose a significant safety risk when applied in vivo as they often have similar activity against red blood cells and other nontarget cell membranes. Our aim is to control the membrane-disrupting capacity of these peptides in time and space by physically constraining them to a nanocarrier surface in such a way that they only become activated when delivered inside acidic endosomes. To this end, a variety of chemical approaches for the coupling of lytic peptides to liposomes via functionalized PEG-lipids were explored, including maleimide-thiol chemistry, click-chemistry, and aldehyde-hydrazide chemistry. The latter enables reversible conjugation via a hydrazone bond, allowing for release of the peptide under endosomal conditions. By carefully choosing the conjugation site and by using a pH activated analog of the melittin peptide, lytic activity toward a model membrane is completely inhibited at physiological pH. At endosomal pH the activity is restored by hydrolysis of the acid-labile hydrazone bond, releasing the peptide in its most active, free form. Furthermore, using an analogue containing a nonhydrolyzable bond as a control, it was shown that the activity observed can be completely attributed to release of the peptide, validating dynamic covalent conjugation as a suitable strategy to maintain safety during circulation.

Osteoimmunomodulatory GelMA/liposome coatings to promote bone regeneration of orthopedic implants.

Despite being the most widely used biomaterials in orthopedic surgery, metallic implants do not induce new bone growth because they are bioinert. Surface biofunctionalization of implants with immunomodulatory mediators is a recent approach to promote osteogenic factors that facilitate bone regeneration. Liposomes (Lip) can be used as a low-cost, efficient and simple immunomodulator to stimulate immune cells in favor of bone regeneration. Even though liposomal coating systems have been reported previously, their main disadvantage is their limited ability to preserve liposome integrity after drying. In order to address this issue, we developed a hybrid system in which liposomes could be embedded in a polymeric hydrogel namely gelatin methacryloyl (GelMA). Specifically, we have developed a novel versatile coating strategy using electrospray technology to coat implants with GelMA/Liposome without using adhesive intermediate layer. The two differently charged Lip (i.e., anionic and cationic) were blended with GelMA and coated via electrospray technology on the bone-implant surfaces. The results showed that the developed coating withstood mechanical stress during surgical replacement, and Lip inside GelMA coating stayed intact in different storage conditions for a minimum of 4 weeks. Surprisingly, bare Lip, either cationic or anionic, improved the osteogenesis of human Mesenchymal Stem Cells (MSCs) by inducing pro-inflammatory cytokines, even at a low dosage of Lip released from the GelMA coating. More importantly, we showed that the inflammatory response could be fine-tuned by selecting the Lip concentration, Lip/hydrogel ratio, and coating thickness to determine the timing of the release such that we can accommodate different clinical needs. These promising results pave the way to use these Lip coatings to load different types of therapeutic cargo for bone-implant applications.

Cellular uptake of liposomes targeted to intercellular adhesion molecule-1 (ICAM-1) on bronchial epithelial cells.

Previously, it was demonstrated that immunoliposomes, bearing anti-intercellular adhesion molecule-1 (ICAM-1) antibodies (mAb F10.2), can specifically bind to different cell types expressing ICAM-1. In this study, we have quantified the amount of immunoliposomes binding to IFN-gamma activated human bronchial epithelial cells (BEAS-2B) in vitro and studied the subsequent fate of cell-bound anti-ICAM-1 immunoliposomes. We demonstrate that binding of the immunoliposomes to the epithelial cells depends on the liposome concentration used. After binding to the cell surface, the anti-ICAM-1 immunoliposomes are rapidly internalised by the epithelial cells. Sixty percent of cell-bound immunoliposomes were internalised by the epithelial cells within 1 h of incubation at 37 degrees C. The results indicate that ICAM-1 targeted immunoliposomes may be used as carriers for the intracellular delivery of anti-inflammatory drugs to sites of inflammation characterised by an increased expression of ICAM-1.

Targeting influenza virosomes to ovarian carcinoma cells.

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) have attracted attention as delivery vesicles for cytosolic drug delivery as they possess membrane fusion activity. Here, we show that influenza virosomes can be targeted towards ovarian carcinoma cells (OVCAR-3) with preservation of fusion activity. This was achieved by incorporating poly(ethylene glycol) (PEG)-derivatized lipids into the virosome membrane. This PEG layer serves as shield to prevent interaction of HA with ubiquitous sialic acid residues and as spatial anchor for antibody attachment. Coupling of Fab' fragments of mAb 323/A3 (anti-epithelial glycoprotein-2) to the distal ends of PEG lipids resulted in specific binding of virosomes to OVCAR-3 cells. These antibody-redirected virosomes fused with membranes of OVCAR-3 cells in a pH-dependent fashion.

Liposomes for intravenous drug targeting: design and applications.

Drug targeting with liposomes has been studied for over 25 years and has demonstrated its value in clinical practice. This mini review offers an overview of the design and application of liposomes for i.v. drug targeting. Two approaches are outlined: passive and active targeting. The former approach is based on liposomes with prolonged circulation and selective target localization properties, while in the latter approach specific targeting ligands are coupled to the liposome surface in order to achieve enhanced interaction with target cell membranes.

Fluorescence in situ hybridization to monitor the intracellular location and accessibility of plasmid DNA delivered by cationic polymer-based gene carriers.

Information about the intracellular trafficking of exogenous DNA delivered by nonviral gene delivery systems is of major importance for optimization of such gene carriers. We used fluorescence in situ hybridization (FISH) as a tool to visualize polyplex-delivered pDNA inside cells. This avoids the need to directly label DNA inside the polyplexes, which may influence their cellular behavior and fate. Using FISH the introduced plasmid DNA could be detected in the cytosol and nucleus of different cell lines. The FISH probe itself did not interact with cells nor different polymers used for condensing the DNA. We further demonstrate differences in accessibility of polyplex-delivered DNA when different polymers were used for DNA complexation. Therefore, FISH is a valuable tool to detect location and accessibility of exogenous plasmid DNA delivered in the cell by cationic polymers.

Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells.

PURPOSE: Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake. MATERIALS AND METHODS: The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy. RESULTS: Both pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake. CONCLUSIONS: These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.