Eleni Markoutsa

Uptake and permeability studies of BBB-targeting immunoliposomes using the hCMEC/D3 cell line.

The targeting potential of OX-26-decorated immunoliposomes was investigated, using the human brain endothelial cell line hCMEC/D3 as a model of the blood-brain barrier (BBB). Immuno-nanoliposomes were prepared by the biotin/streptavidin ligation strategy, and their uptake by hCMEC/D3 cells and permeability through cell monolayers was studied. In order to elucidate the mechanisms of uptake, pH-sensitive fluorescence signal of HPTS was used, while transport was measured using double labeled immunoliposomes (with aqueous and lipid membrane fluorescent tags). PEGylated and non-specific-IgG-decorated liposomes were studied under identical conditions, as controls. CHO-K1 cells (which do not overexpress the transferrin receptor) were studied in some cases for comparative purposes. Experimental results reveal that hCMEC/D3 cells are good models for in vitro screening of BBB-targeting nanoparticulate drug delivery systems. Uptake and transcytosis of immunoliposome-associated dyes by cell monolayers was substantially higher compared to those of control liposomes. HPTS-entrapping OX-26-immunoliposome uptake indicated lysosomal localization and receptor-mediated mechanism. The ratio of aqueous/lipid label transport is affected by pre-incubation with antibody, or use of high lipid doses, suggesting that vesicles are transported intact after lysosome saturation. Co-decoration with a second ligand slightly decreases OX-26-decorated vesicle uptake, but not transcytosis, proving that the biotin-streptavidin technique can be applied for the generation of dual-targeting nanoliposomes.

Innate immunity in insects: surface-associated dopa decarboxylase-dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes

Phagocytosis, melanization and nodulation in insects depend on phenoloxidase (PO) activity. In this report, we demonstrated that these three processes appear to be also dependent on dopa decarboxylase (Ddc) activity. Using flow cytometry, RNA interference, immunoprecipitation and immunofluorescence, we demonstrated the constitutive expression of Ddc and its strong association with the haemocyte surface, in the medfly Ceratitis capitata. In addition, we showed that Escherichia coli phagocytosis is markedly blocked by small interfering RNA (siRNA) for Ddc, antibodies against Ddc, as well as by inhibitors of Ddc activity, namely carbidopa and benzerazide, convincingly revealing the involvement of Ddc activity in phagocytosis. By contrast, latex beads and lipopolysaccharide (LPS) did not require Ddc activity for their uptake. It was also shown that nodulation and melanization processes depend on Ddc activation, because antibodies against Ddc and inhibitors of Ddc activity prevent haemocyte aggregation and melanization in the presence of excess E. coli. Therefore, phagocytosis, melanization and nodulation depend on haemocyte‐surface‐associated PO and Ddc. These three unrelated mechanisms are based on tyrosine metabolism and share a number of substrates and enzymes; however, they appear to be distinct. Phagocytosis and nodulation depend on dopamine‐derived metabolite(s), not including the eumelanin pathway, whereas melanization depends exclusively on the eumelanin pathway. It must also be underlined that melanization is not a prerequisite for phagocytosis or nodulation. To our knowledge, the involvement of Ddc, as well as dopa and its metabolites, are novel aspects in the phagocytosis of medfly haemocytes.

Anti-Aβ-MAb and dually decorated nanoliposomes: effect of Aβ1-42 peptides on interaction with hCMEC/D3 cells.

Anti-Aβ-MAb (Aβ-MAb)-decorated immunoliposomes (LIP) and dually decorated ones (dd-LIP) with OX-26 and Aβ-MAb were constructed. In both cases, the biotin-streptavidin ligation method was applied. All LIP types were characterized for size distribution, zeta potential, and integrity during incubation with serum proteins. Uptake and transcytosis of both LIP types and control vesicles by human brain endothelial hCMEC/D3 cells were measured. All LIP types had mean diameters below 150-200 nm and low polydispersity. Aβ-MAb-LIP uptake was higher than control PEGylated liposomes, while uptake of dd-LIP was similar to that of OX-26-LIP. Aβ-MAb-LIP and dd-LIP uptake increased significantly when cells were pre-incubated with Aβ1-42 peptides; OX-26-LIP uptake was not modulated. Transcytosis of Aβ-MAb-LIP through monolayers was 2.5 times higher when monolayers were pre-incubated with Aβ1-42. Transport of both probes, FITC-dextran and rhodamine-lipid, was equivalent, indicating that Aβ-MAb-LIP are transferred intact through the BBB model. The Aβ peptide-induced increase in binding (and transport) is regulated by the membrane receptors for Aβ1-42 peptides (RAGE), as proven after blocking RAGE by a specific MAb. Aβ1-42 peptides did not modulate the barrier tightness and integrity, as determined by transendothelial resistance and Lucifer Yellow permeability. Additionally, hCMEC/D3 cell viability was not affected by Aβ peptides or by Aβ-MAb-LIP.

Multifunctional LUV liposomes decorated for BBB and amyloid targeting - B. In vivo brain targeting potential in wild-type and APP/PS1 mice

&NA; Multifunctional liposomes (mf‐LIPs) having a curcumin‐lipid ligand (to target amyloids) together with two ligands to target the transferrin, and the low‐density apolipoprotein receptor of the blood‐brain‐barrier (BBB) on their surface, were previously studied (in vitro) as potential theranostic systems for Alzheimers disease (AD) (Papadia et al., 2017, Eur. J. Pharm. Sciences; 101:140–148). Herein, the targeting potential of mf‐LIPs was compared to that of BBB‐LIPs (liposomes having only the two BBB‐specific ligands) in FVB mice (normal), as well as in double transgenic mice (APP/PS1) and their corresponding littermates (WT), by live‐animal (in vivo) and explanted organ (ex vivo) imaging. In FVB mice, the head‐signals of mf‐LIPs and BBB‐LIPs are either similar, or signals from mf‐LIP are higher, suggesting that the co‐presence of the curcumin derivative on the liposome surface does not disturb the functionality of the BBB‐specific ligands. Higher brain / liver + spleen ratios (ex vivo) were calculated post‐injection of mf‐LIP, compared to those found after BBB‐LIP injection, due to the reduced distribution of mf‐LIPs in the liver and spleen; showing that the curcumin ligand increases the stealth properties of liposomes by reducing their uptake by liver and spleen. The later effect is more pronounced when the density of the BBB‐specific ligands on the mf‐LIPs is 0.1 mol%, compared to 0.2%, highlighting the importance of this parameter. When a high lipid dose (4 mg/mouse) is injected in WT and APP/PS1 mice, the head‐signals of mf‐LIPs are significantly higher than those of BBB‐LIPs, but no differences are observed between WT and APP/PS1 mice. However, after administration of a low liposome dose (0.05 mg/mouse) of mf‐LIPs, significant differences in the head‐signals are found between WT and transgenic mice, highlighting the AD theranostic potential of the multifunctional liposomes, as well as the importance of the experimental parameters used in such in vivo screening studies. Graphical abstract Figure. No caption available.

Comparison of Various Types of Ligand Decorated Nanoliposomes for their Ability to Inhibit Amyloid Aggregation and to Reverse Amyloid Cytotoxicity.

Three different amyloid targeting ligands, previously shown to exhibit amyloid specific properties, have been used to develop amyloid -targeted nanoliposomes (AT-NLs. For this a MAb against Aβ-peptides (Aβ-MAb (immobilized on NLs at 0.015 and 0.05 mol %, and two different curcumin-lipid derivatives were attached to the surface of preformed NLs or incorporated in NL membranes during their formation. Following physicochemical characterization, these AT-NLs were studied for their ability to inhibit or delay amyloid peptide aggregation -using the thioflavin-T assay, and for their potential to reverse amyloid-induced (and Zn, or, amyloid + Zn cytotoxicity, on wild type (N2aWT and transformed (N2aAPP neuroblastoma cells, applying the MTT assay. Experimental results reveal that all formulations were found to strongly delay amyloid peptide aggregation (with no significant differences between the different AT-NL types. However, although Aβ-MAb-NLs significantly reversed amyloid-induced cytotoxicity in all cases, both curcumin-NL types did not reverse Zn-induced, nor Zn+Aβ-induced cytotoxicity in N2aWT cells, suggesting lower activity against synthetic-Aβ peptides (compared to endogenous Aβ peptides; perhaps due to different affinity towards different (aggregation stages of peptide species (monomers, oligomers, fibrils, etc. Taken into account that the aggregation stage of amyloid species is an important determinant of their toxicity, the importance of the affinity of each AT-NL type towards specific species, is highlighted.

Multifunctional LUV liposomes decorated for BBB and amyloid targeting. A. In vitro proof-of-concept

ABSTRACT Multifunctional LUV liposomes (mf‐LIPs) were developed, having a curcumin‐lipid ligand (TREG) with affinity towards amyloid species, together with ligands to target the transferrin and the LDL receptors of the blood‐brain‐barrier (BBB), on their surface. mf‐LIPs were evaluated for their brain targeting, on hCMEC/D3 monolayers, and for their ability to inhibit A&bgr;‐peptide aggregation. The transport of mf‐LIP across hCMEC/D3 monolayers was similar to that of BBB‐LIPs, indicating that the presence of TREG on their surface does not reduce their brain targeting potential. Likewise, mf‐LIP inhibitory effect on A&bgr; aggregation was similar to that of LIPs functionalized only with TREG, proving that the presence of brain targeting ligands does not reduce the functionality of the amyloid‐specific ligand. Addition of the curcumin‐lipid in some liposome types was found to enhance their integrity and reduce the effect of serum proteins on their interaction with brain endothelial cells. Finally, preliminary in vivo results confirm the in vitro findings. Concluding, the current results reveal the potential of the specific curcumin‐lipid derivative as a component of multifunctional LIPs with efficient brain targeting capability, intended to act as a theragnostic system for AD. Graphical Abstract Figure. No Caption available.