Manganese in Diagnostics: A Preformulatory Study

This investigation aims to find lipid-based nanosystems to be used as tools to deliver manganese for diagnostic purposes in multimodal imaging techniques. In particular, the study describes the production and characterization of aqueous dispersions of anionic liposomes as delivery systems for two model manganese-based compounds, namely manganese chloride and manganese acetylacetonate. Negatively charged liposomes were obtained using four different anionic surfactants, namely sodium docusate (SD), N-lauroylsarcosine (NLS), Protelan AG8 (PAG) and sodium lauroyl lactylate (SLL). Liposomes were produced by the direct hydration method followed by extrusion and characterized in terms of size, polydispersity, surface charge and stability over time. After extrusion, liposomes are homogeneous and monodispersed with an average diameter not exceeding 200 nm and a negative surface charge as confirmed by ζ potential measurement. Moreover, as indicated by atomic absorption spectroscopy analyses, the loading of manganese-based compounds was almost quantitative. Liposomes containing NLS or SLL were the most stable over time and the presence of manganese-based compounds did not affect their size distribution. Liposomes containing PAG and SD were instable and therefore discarded. The in vitro cytotoxicity of the selected anionic liposomes was evaluated by MTT assay on human keratinocyte. The obtained results highlighted that the toxicity of the formulations is dose dependent.

Optimization of Sentinel Lymph Node Imaging Methodology Using Anionic Liposome and Hyaluronidase

The sentinel lymph node (SLN) is the first lymph node into which lymphatic fluid from tumor tissues flows. The development of a highly sensitive probe for detecting SLNs is desired for the lymph node dissection through intraoperative biopsy. We have previously shown that anionic liposomes tend to accumulate in lymph nodes and that macrophage uptake of liposomes contributes to their accumulation. In the present study, we found that among anionic lipids, phosphatidylserine (PS)-containing liposomes were substantially taken up by macrophages. We identified a new lipid composition to improve the SNL-selectivity of liposome accumulation based on Design-of-Experiment. The optimized PS-containing particles were more selectively accumulate to SLN lymph nodes than existing imaging agents indocyanine green. These results indicate the effectiveness of PS-containing anionic particles in SLN imaging.

Antigen Uptake After Intradermal Microinjection Depends on Antigen Nature and Formulation, but Not on Injection Depth

The skin is an attractive alternative administration route for allergy vaccination, as the skin is rich in dendritic cells (DCs) and is easily accessible. In the skin multiple subsets of DCs with distinct roles reside at different depths. In this study antigen (=allergen for allergy) formulations were injected in ex vivo human skin in a depth-controlled manner by using a hollow microneedle injection system. Biopsies were harvested at the injection site, which were then cultured for 72 h. Subsequently, the crawled-out cells were collected from the medium and analyzed with flow cytometry. Intradermal administration of ovalbumin (OVA, model antigen) solution at various depths in the skin did not affect the migration and maturation of DCs. OVA was taken up efficiently by the DCs, and this was not affected by the injection depth. In contrast, Bet v 1, the major allergen in birch pollen allergy, was barely taken up by dermal DCs (dDCs). Antigens were more efficiently taken up by CD14+ dDCs than CD1a+ dDCs, which in turn were more efficient at taken up antigen than Langerhans cells. Subsequently, both OVA and Bet v 1 were formulated in cationic and anionic liposomes, which altered antigen uptake drastically following intradermal microinjection. While OVA uptake was reduced by formulation in liposomes, Bet v 1 uptake in dDCs was increased by encapsulation in both cationic and anionic liposomes. This highlights the potential use of liposomes as adjuvant in intradermal allergy vaccine delivery. In conclusion, we observed that antigen uptake after intradermal injection was not affected by injection depth, but varied between different antigens and formulation.

In Vivo Fluorescence Imaging of Passive Inflammation Site Accumulation of Liposomes via Intravenous Administration Focused on Their Surface Charge and PEG Modification

Nanocarriers such as liposomes have been attracting attention as novel therapeutic methods for inflammatory autoimmune diseases such as rheumatoid arthritis and ulcerative colitis. The physicochemical properties of intravenously administered nanomedicines enable them to target inflamed tissues passively. However, few studies have attempted to determine the influences of nanoparticle surface characteristics on inflammation site accumulation. Here, we aimed to study the effects of polyethylene glycol (PEG) modification and surface charge on liposome ability to accumulate in inflammatory sites and be uptake by macrophages. Four different liposome samples with different PEG modification and surface charge were prepared. Liposome accumulation in the inflammation sites of arthritis and ulcerative colitis model mice was evaluated by using in vivo imaging. There was greater PEG-modified than unmodified liposome accumulation at all inflammation sites. There was greater anionic than cationic liposome accumulation at all inflammation sites. The order in which inflammation site accumulation was confirmed was PEG-anionic > PEG-cationic > anionic > cationic. PEG-anionic liposomes had ~2.5× higher fluorescence intensity than PEG-cationic liposomes, and the PEG-liposomes had ~2× higher fluorescence intensity than non-PEG liposomes. All liposomes have not accumulated at the inflammation sites in healthy mice. Furthermore, cationic liposomes were taken up to ~10× greater extent by RAW264.7 murine macrophages. Thus, PEG-cationic liposomes that have the ability to accumulate in inflammatory sites via intravenous administration and to be taken up by macrophages could be useful.

Multifunctional carriers for controlled drug delivery

In the review we describe a method for concentration of anionic liposomes with encapsulated water-soluble substances within a small volume via electrostatic liposome adsorption on the surface of polymer particles with grafted cationic chains (spherical polycationic brushes), or cationic microgel particles. Dozens of intact liposomes can be bound to each polymer particle, the resulting polymer/liposome complex does not dissociate into the original components in a physiological solution. This allows fabrication of multi-liposomal complexes (MLCs) with a required ratio of encapsulated substances. Two approaches are discussed for the synthesis of stimuli-sensitive MLCs. The first is to incorporate the conformation switch, morpholinocyclohexanol-based lipid, into the liposomal membrane thus forming pH-sensitive liposomes capable of releasing their cargo when acidifying the surrounding solution. These liposomes complexed with the brushes release encapsulated substances much faster than the uncomplexed liposomes. The second is to adsorb liposomes on cationic thermo-responsive microgels. The resulting MLCs contracts upon heating over a volume phase transition temperature from the swollen to the collapsed state of microgel, thus causing the adsorbed liposomes to change drastically their morphology and release an encapsulated substance. Complexation of anionic liposomes with chitosan microgels and polylactide micelles gives MLCs which degrade in the presence of enzymes down to small particles, 10–15 nm in diameter. A novel promising approach suggests that immobilized liposomes can act as a capacious depot for biologically active compounds and ensure their controllable leakage to surrounding solution.