Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization.

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.

Liposomes with asymmetric bilayers produced from inverse emulsions for nucleic acid delivery.

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.

Thermosensitive liposomes for triggered release of cytotoxic proteins.

Lysolipid-containing thermosensitive liposomes (LTSL) are clinically-relevant drug nanocarriers which have been used to deliver small molecule cytostatics to tumors in combination with local hyperthermia (42 °C) to trigger local drug release. The objective of this study was to investigate the feasibility of LTSL for encapsulation and triggered release of macromolecular drugs such as plant-derived cytotoxins. As therapeutic protein we used Mistletoe lectin-1 (ML1) - a ribosome-inactivating protein with potent cytotoxic activity in tumor cells. Model macromolecules (dextrans, albumin) and ML1 were encapsulated in small unilamellar LTSL with varying lipid compositions by the thin film hydration method and extrusion. LTSLs showed molecular weight dependent heat-triggered release of the loaded cargo. The most promising composition, ML1 formulated in LTSL composed of 86:10:4 %mol DPPC:MSPC:DSPE-PEG2000, was further studied for bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging showed uptake of released ML1 after mild hyperthermia at 42 °C, subsequently leading to potent cytotoxicity by LTSL-ML1. Our study shows that LTSL in combination with localized hyperthermia hold promise as local tumor delivery strategy for macromolecular cytotoxins.

Current Trends and Challenges in the Clinical Translation of Nanoparticulate Nanomedicines: Pathways for Translational Development and Commercialization.

The use of nanotechnology in medicine has the potential to have a major impact on human health for the prevention, diagnosis, and treatment of diseases. One particular aspect of the nanomedicine field which has received a great deal of attention is the design and development of nanoparticulate nanomedicines (NNMs) for drug delivery (i.e., drug-containing nanoparticles). NNMs are intended to deliver drugs via various mechanisms: solubilization, passive targeting, active targeting, and triggered release. The NNM approach aims to increase therapeutic efficacy, decrease the therapeutically effective dose, and/or reduce the risk of systemic side effects. In order to move a NNM from the bench to the bedside, several experimental challenges need to be addressed. This review will discuss the current trends and challenges in the clinical translation of NNMs as well as the potential pathways for translational development and commercialization. Key issues related to the clinical development of NNMs will be covered, including biological challenges, large-scale manufacturing, biocompatibility and safety, intellectual property (IP), government regulations, and overall cost-effectiveness in comparison to current therapies. These factors can impose significant hurdles limiting the appearance of NNMs on the market, irrelevant of whether they are therapeutically beneficial or not.

Instability Mechanisms of Water-in-Oil Nanoemulsions with Phospholipids: Temporal and Morphological Structures.

Many food preparations, pharmaceuticals, and cosmetics use water-in-oil (W/O) emulsions stabilized by phospholipids. Moreover, recent technological developments try to produce liposomes or lipid coated capsules from W/O emulsions, but are faced with colloidal instabilities. To explore these instability mechanisms, emulsification by sonication was applied in three cycles, and the sample stability was studied for 3 h after each cycle. Clearly identifiable temporal structures of instability provide evidence about the emulsion morphology: an initial regime of about 10 min is shown to be governed by coalescence after which Ostwald ripening dominates. Transport via molecular diffusion in Ostwald ripening is commonly based on the mutual solubility of the two phases and is therefore prohibited in emulsions composed of immiscible phases. However, in the case of water in oil emulsified by phospholipids, these form water-loaded reverse micelles in oil, which enable Ostwald ripening despite the low solubility of water in oil, as is shown for squalene. As is proved for the phospholipid dipalmitoylphosphatidylcholine (DPPC), concentrations below the critical aggregation concentration (CAC) form monolayers at the interfaces and smaller droplet sizes. In contrast, phospholipid concentrations above the CAC create complex multilayers at the interface with larger droplet sizes. The key factors for stable W/O emulsions in classical or innovative applications are first, the minimization of the phospholipids' capacity to form reversed micelles, and second, the adaption of the initial phospholipid concentration to the water content to enable an optimized coverage of phospholipids at the interfaces for the intended drop size.

Osteogenic poly(ε-caprolactone)/poloxamine homogeneous blends prepared by supercritical foaming.

Homogeneous poly(ε-caprolactone) (PCL) and poloxamines (PLXs) porous blends were prepared using a supercritical carbon dioxide-assisted foaming/mixing (SFM) approach aiming to obtain cytocompatible implantable materials presenting tunable morphologies, bioerosion rates, bioactive molecules release and osteogenic features. Pure PCL, pure PLXs (T908 and T1107 varieties) and three distinct PCL:PLX 75:25, 50:50, 25:75% w/w blends, with and without the osteogenic and angiogenic bioactive molecule simvastatin were processed at constant pressure of 20 MPa and temperature of 40 °C or 43 °C, for T1107 and T908, respectively. Obtained porous blends were characterized applying a wide range of techniques and in vitro methods. Calorimetric analysis showed that hydrophilic T908 and T1107 PLXs are miscible with PCL for all tested compositions. Prepared PCL:PLX porous blends rapidly lost mass when immersed into phosphate buffer pH 7.4 due to PLXs dissolution and then went through slow and almost constant erosion rates for the subsequent weeks due to PCL slow hydrolytic degradation, which explains the rapid initial release of simvastatin and its subsequent sustained release for longer periods of time. PCL and PCL:PLX 75:25% w/w porous blends, containing or not simvastatin, showed a high cytocompatibility with SAOS-2 cells. In addition, prepared biomaterials promoted mesenchymal stem cells proliferation and their differentiation into osteoblasts. Overall, obtained results showed novel possibilities of addressing local treatment of small bone defects/fractures using highly porous PCL:PLX homogeneous blends.