Glioblastoma is the most common and aggressive brain tumor. Current treatments do not allow to cure the patients. This is partly due to the blood–brain barrier (BBB), which limits the delivery of drugs to the pathological site. To overcome this, we developed liposomes functionalized with a neurofilament-derived peptide, NFL-TBS.40–63 (NFL), known for its highly selective targeting of glioblastoma cells. First, in vitro BBB model was developed to check whether the NFL can also promote barrier crossing in addition to its active targeting capacity. Permeability experiments showed that the NFL peptide was able to cross the BBB. Moreover, when the BBB was in a pathological situation, i.e., an in vitro blood–brain tumor barrier (BBTB), the passage of the NFL peptide was greater while maintaining its glioblastoma targeting capacity. When the NFL peptide was associated to liposomes, it enhanced their ability to be internalized into glioblastoma cells after passage through the BBTB, compared to liposomes without NFL. The cellular uptake of liposomes was limited in the endothelial cell monolayer in comparison to the glioblastoma one. These data indicated that the NFL peptide is a promising cell-penetrating peptide tool when combined with drug delivery systems for the treatment of glioblastoma.

Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein-lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 °C. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface.

This research highlights the capacity of a newly introduced centrifugation process to form liposomes from water-in-fluorocarbon nano-emulsions stabilized with phospholipids to incorporate macromolecular and sensitive active pharmaceutical ingredients (API). The encapsulation efficiency of the produced liposomes, incorporating fluorescein-sodium, bovine serum albumin and fluorecein isothiocyanate dextran as model APIs, is determined by applying Vivaspin® centrifugation filtration and quantified by UV-Vis spectroscopy. It was found that higher densities of the fluorocarbons used as the hydrophobic phase enable a higher encapsulation efficiency and that an efficiency of up to 98% is possible depending on the used phospholipid. Among the engineering aspects of the process, a comparison between different membrane substances was performed. Efficiency increases with a higher phospholipid concentration but decreases with the addition of cholesterol. Due to the higher bending modulus, liposome formation is slowed down by cholesterol during liposome closure leading to a greater leakage of the model API. The encapsulation of bovine serum albumin and dextran, both investigated under different osmotic conditions, shows that an efflux negatively affects the encapsulation efficiency while an influx increases the stability. Overall, the process shows the potential for a very high encapsulation efficiency for macromolecules and future pharmaceutical applications.

Background: Mistletoe therapy is frequently administered as a supportive treatment in diverse pediatric cancer entities including brain tumors. Medulloblastoma is the most common brain tumor in childhood. Its high risk to metastasize and its long-term sequelae caused by aggressive chemo- or radiotherapies are still challenging. Material and Methods: Effects of a lectin-rich mistletoe extract, abnobaVISCUM Fraxini, were investigated in two medulloblastoma cell lines (Daoy and ONS-76). Responsiveness of tumor cells was assessed by cell viability assays and xCELLigence real-time analyses. Moreover, impacts on proliferation, cell cycle and apoptosis were investigated. Apoptosis was studied by staining of vital mitochondria and assessing the involvement of caspases. In addition, effects on migration and invasion were analyzed. Results: Both medulloblastoma cell lines were more susceptible to treatment with the mistletoe extract than a nontumorigenic fibroblast cell line. In mistletoe-sensitive Daoy cells, reduction of proliferation and induction of caspase-mediated apoptosis were observed upon administration of 0.05 and 0.5 mg/mL abnobaVISCUM Fraxini treatment, respectively. Furthermore, mistletoe extract inhibited migration and invasion properties in Daoy and significantly impaired invasive capabilities of ONS-76 cells. Conclusion: AbnobaVISCUM Fraxini has cell line dependent antitumoral effects in medulloblastoma models. These results call for further investigations, to reveal mechanistic insights into antitumorigenic properties of mistletoe extracts.

Fluorocarbons are novel systems in the fast-growing fields of diverse biomedical applications and fluorocarbon-water emulsions. However, characterization of these systems with modern measuring techniques such as drop profile analysis tensiometry is almost impossible because of practically identical refractive indexes and high-density differences. Due to the material properties of the fluorocarbon-water system, the invasive Du Noüy ring is the most appropriate method to measure interfacial tensions over long times. However, the influence of the ring on a fluorocarbon/water interface packed with phospholipids needs careful analysis. For the proof of methodology, the spinning drop tensiometry was used for comparison as a non-invasive technique to measure interfacial tension between water and perfluoroperhydrophenanthrene (PFPH) covered by 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) proving almost identical results. This demonstrates the validity of the invasive measurement technique for the studied system. The Du Noüy ring method was applied for further measurements of phospholipids with different chain lengths (1,2-dmyristoyl-sn-glycero-3-phostphatidylcholine, DMPC; 1,2-distearoyl-sn-glycero-3-phosphatidylcholine, DSPC) which revealed a difference in interfacial adsorption kinetics and equilibrium tensions. The Du Noüy ring tensiometry is appropriate to examine the slow adsorption kinetics of phospholipids emulsifying fluorocarbons. The results enable functional optimization of fluorocarbon emulsions regarding physical emulsification parameters and the selection of lipids.

Mistletoe lectin-1 (ML1) is a nature-derived macromolecular cytotoxin that potently induces apoptosis in target cells. Non-specific cytotoxicity to normal cells is one of the major risks in its clinical application, and we therefore propose to encapsulate ML1 in a nanocarrier that can specifically release its cargo intratumorally, thus improving the efficacy to toxicity ratio of the cytotoxin. We investigated the encapsulation of ML1 in ultrasound-sensitive liposomes (USL) and studied its release by high-intensity focused ultrasound (HAccessedIFU). USL were prepared by entrapment of perfluorocarbon nanodroplets in pegylated liposomes. The liposomes were prepared with different DPPC/cholesterol/DSPE-PEG2000 lipid molar ratios (60/20/20 for USL20; 60/30/10 for USL10; 65/30/5 for USL5) before combination with perfluorocarbon (PFC) nanoemulsions (composed of DPPC and perfluoropentane). When triggered with HIFU (peak negative pressure, 2–24 MPa; frequency, 1.3 MHz), PFC nanodroplets can undergo phase transition from liquid to gas thus rupturing the lipid bilayer of usl. Small unilamellar liposomes were obtained with appropriate polydispersity and stability. ML1 and the model protein horseradish peroxidase (HRP) were co-encapsulated with the PFC nanodroplets in USL, with 3% and 7% encapsulation efficiency for USL20 and USL10/USL5, respectively. Acoustic characterization experiments indicated that release is induced by cavitation. HIFU-triggered release of HRP from USL was investigated for optimization of liposomal composition and resulted in 80% triggered release for USL with USL10 (60/30/10) lipid composition. ML1 release from the final USL10 composition was also 80%. Given its high stability, suitable release, and ultrasound sensitivity, USL10 encapsulating ML1 was further used to study released ML1 bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging demonstrated its functional activity regarding the interaction with the target cells. We furthermore demonstrated the cytotoxicity of the released ML1 (I.E., After USL were treated with HIFU). The potent cytotoxicity (IC50 400 ng/ml; free ML1 IC50 345 ng/ml) was compared to non-triggered USL loaded with ML1. Our study shows that USL in combination with HIFU hold promise as trigger-sensitive nanomedicines for local delivery of macromolecular cytotoxins.

Asymmetrical lipid nanoparticles are interesting nanocarriers for charged molecules, like nucleic acids. They promise control over inner and outer charge. High charge density on the inside is favourable for efficient condensation and charge neutralisation of highly charged biopharmaceuticals, while a neutral or slightly negative outer layer promotes biocompatibility. The main goal of this work was the development and characterisation of asymmetric liposomes, prepared using water-in-oil (w/o) nanoemulsions of phospholipids (PLs) and squalene in a centrifugal field. This method enables the control over the lipid composition of each monolayer.Liposomes were prepared by passing PL w/o nanoemulsions through an oil–water interface previously saturated with PLs. We used N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-PE) or N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3- phosphocholine (NBD-PC) as a fluorescent marker for either the inner or outer lipid layer and plasmid DNA (pDNA) as nucleic acid payload. The final liposomes had sizes below 200 nm and polydispersity indexes of 0.3 and had a bilayer asymmetry of 70%, thus shielding the charge of positive PLs in the inner bilayer leaflet. Final formulations were examined using negative staining transmission electron microscopy (TEM). Plasmid encapsulation efficiency of the method was 10–15%. Our results indicate that the w/o nanoemulsion-centrifugation method allows the successful production of liposomes with tailored features for encapsulation of nucleic acid therapeutics.