Ofelia Maniti

Nanocarriers have found their interests in many fields including drug delivery and labeling of cells with the aim to target and eradicate tumor cells. One of the approaches to specifically address nanocarriers, such as liposomes, to their target is to attach antibodies of interest to their surface. To date, the development of immunoliposomes has been widely explored but has mainly involved chemical and unspecific reactions that could impair antibody stability, integrity, and orientation, thus reducing optimized immunoliposomes generation. In this study, we report the use of the patented COVISOLINK technology and the strain-promoted alkyne-azide cycloaddition (SPAAC) to generate immunoliposomes that target HER2 positive breast cancer with Trastuzumab as the antibody to be coupled. The efficacy of our two-step functionalization strategy and the successful specific coupling of the antibodies were validated by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC), which allowed a precise quantification of antibodies conjugated to liposomes and confirmed by cryo-TEM and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. We also demonstrate by flow cytometry and epifluorescence microscopy that the produced anti-HER2 immunoliposomes were able to interact specifically with their target cells (SK-BR-3) while remaining negative with cells that express HER2 at a low level (MDA-MB-231). Hence, for the first time, our COVISOLINK strategy using microbial transglutaminase (mTG) enables the preparation and production of well-characterized immunoliposomes that could be used in different applications, including therapies.

Nanomedicines engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or a short half-life, are targeted towards their cellular destination either passively or through various elements of cell membranes. The differences in the physicochemical properties of the cell membrane between tumor and nontumor cells have been reported, but they are not systematically used for drug delivery purposes. Thus, in this study, a new approach based on a match between the liposome compositions, i.e., membrane fluidity, to selectively interact with the targeted cell membrane was used. Lipid-based carriers of two different fluidities were designed and used to deliver 4(RS)-4-F_4t-Neuroprostane (F_4t-NeuroP), a potential antitumor molecule derived from docosahexaenoic acid (DHA). Based on its hydrophobic character, F_4t-NeuroP was added to the lipid mixture prior to liposome formation, a protocol that yielded over 80% encapsulation efficiency in both rigid and fluid liposomes. The presence of the active molecule did not modify the liposome size but increased the liposome negative charge and the liposome membrane fluidity, which suggested that the active molecule was accommodated in the lipid membrane. F_4t-NeuroP integration in liposomes with a fluid character allowed for the selective targeting of the metastatic prostate cell line PC-3 vs. fibroblast controls. A significant decrease in viability (40%) was observed for the PC-3 cancer line in the presence of F_4t-NeuroP fluid liposomes, whereas rigid F_4t-NeuroP liposomes did not alter the PC-3 cell viability. These findings demonstrate that liposomes encapsulating F_4t-NeuroP or other related molecules may be an interesting model of drug carriers based on membrane fluidity.

Two florescent xanthene‐cyanamide lysosomal trackers emitting strongly at ∼525 nm were prepared from fluorescein and rhodol methyl esters in microwave‐assisted reactions. Both forms named “off” (nonfluorescent lactam) and “on” (strongly fluorescent ring‐opened amide) have been comprehensively characterized out by using a combination of NMR spectroscopy, X‐ray analysis, fluorimetry and confocal microscopy. Known rhodamines bearing electron‐withdrawing groups (EWGs) exhibit an equilibrium between non‐fluorescent (off) and fluorescent (on) depending on the dielectric constant of the medium. Here, cyanamide was introduced as EWG amine into the fluorescein and rhodol framework. Unlike rhodamine‐type dyes, the ring‐opened forms of fluorescein‐ and rhodol‐cyanamides are stable in protic solvents under circumneutral and basic pH conditions. The osteoblastic cell line MC3T3‐E1 from C57BL/6 mouse calvaria was used for confocal imaging where the different organelles and nuclei were distinguished by using an orthogonal combination of fluorescent dyes.

The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This “gel-in” vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant. RA, and to a lesser extent OA, synovial vesicles had altered morphology, corresponding to a “gel-out” situation with vesicles surrounded by a viscous gel, poor mechanical resistance, and poor lubricating qualities. When subjected to inflammatory conditions, healthy cells developed phenotypes similar to that of RA samples, which reinforces the importance of inflammatory processes in the loss of lubricating properties of SF.

The quality of the lubricant between cartilaginous joint surfaces impacts the joint’s mechanistic properties. In this study, we define the biochemical, ultrastructural, and tribological signatures of synovial fluids (SF) from patients with degenerative (osteoarthritis-OA) or inflammatory (rheumatoid arthritis-RA) joint pathologies in comparison with SF from healthy subjects. Phospholipid (PL) concentration in SF increased in pathological contexts, but the proportion PL relative to the overall lipids decreased. Subtle changes in PL chain composition were attributed to the inflammatory state. Transmission electron microscopy showed the occurrence of large multilamellar synovial extracellular vesicles (EV) filled with glycoprotein gel in healthy subjects. Synovial extracellular vesicle structure was altered in SF from OA and RA patients. RA samples systematically showed lower viscosity than healthy samples under a hydrodynamic lubricating regimen whereas OA samples showed higher viscosity. In turn, under a boundary regimen, cartilage surfaces in both pathological situations showed high wear and friction coefficients. Thus, we found a difference in the biochemical, tribological, and ultrastructural properties of synovial fluid in healthy people and patients with osteoarthritis and arthritis of the joints, and that large, multilamellar vesicles are essential for good boundary lubrication by ensuring a ball-bearing effect and limiting the destruction of lipid layers at the cartilage surface.

Matrix vesicles (MVs) are 100–300 nm spherical structures released by mineralization competent cells to initiate formation of apatite, the mineral component in bones. Among proteins present in MVs, annexin A6 (AnxA6) is thought to be ubiquitously distributed in the MVs’ lumen, on the surface of the internal and external leaflets of the membrane and also inserted in the lipid bilayer. To determine the molecular mechanism(s) that lead to the different locations of AnxA6, we hypothesized the occurrence of a pH drop during the mineralization. Such a change would induce the AnxA6 protonation, which in turn, and because of its isoelectric point of 5.41, would change the protein hydrophobicity facilitating its insertion into the MVs’ bilayer. The various distributions of AnxA6 are likely to disturb membrane phospholipid organization. To examine this possibility, we used fluorescein as pH reporter, and established that pH decreased inside MVs during apatite formation. Then, 4‐(14‐phenyldibenzo[a,c]phenazin‐9(14H)‐yl)‐phenol, a vibration‐induced emission fluorescent probe, was used as a reporter of changes in membrane organization occurring with the varying mode of AnxA6 binding. Proteoliposomes containing AnxA6 and 1,2‐Dimyristoyl‐ sn‐ glycero‐3phosphocholine (DMPC) or 1,2‐Dimyristoyl‐ sn‐ glycero‐3phosphocholine: 1,2‐Dipalmitoyl‐ sn‐ glycero‐3‐phosphoserine (DMPC:DPPS 9:1), to mimic the external and internal MV membrane leaflet, respectively, served as biomimetic models to investigate the nature of AnxA6 binding. Addition of Anx6 to DMPC at pH 7.4 and 5.4, or DMPC:DPPS (9:1) at pH 7.4 induced a decrease in membrane fluidity, consistent with AnxA6 interactions with the bilayer surface. In contrast, AnxA6 addition to DMPC:DPPS (9:1) at pH 5.4 increased the fluidity of the membrane. This latest result was interpreted as reflecting the insertion of AnxA6 into the bilayer. Taken together, these findings point to a possible mechanism of AnxA6 translocation in MVs from the surface of the internal leaflet into the phospholipid bilayer stimulated upon acidification of the MVs’ lumen during formation of apatite.

Tethered bilayer lipid membranes (tBLMs) are artificial membranes largely used for the in situ study of biological membranes and membrane-associated proteins. To date, the formation of these membranes was essentially monitored by surface averaging techniques like surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D), which cannot provide both local and real-time information in a single approach. Here, we report an original application of backside absorbing layer microscopy (BALM), a novel white-light wide-field optical microscopy, to study tBLMs. Thanks to the combination of sensitivity and resolution, BALM not only allowed the real-time quantitative monitoring of tBLM formation but also enabled the high-resolution visualization of local fluxes and matter exchanges taking place at each step of the process. Quantitative BALM measurements of the final layer thickness, reproduced in parallel with SPR, were consistent with the achievement of a continuous lipid bilayer. This finding was confirmed by BALM imaging, which additionally revealed the heterogeneity of the bilayer during its formation. While established real-time techniques, like SPR or QCM-D, view the surface as homogeneous, BALM showed the presence of surface patterns appearing in the first step of the tBLM formation process and governing subsequent matter adsorption or desorption steps. Finally, matter fluxes persisting even after rinsing at the end of the tBLM formation demonstrated the lasting presence of dispersed vesicular pockets with laterally fluctuating positions over the final single and continuous lipid bilayer. These new mechanistic insights into the tBLM formation process demonstrate the great potential of BALM in the study of complex biological systems.

Membrane proteins (MPs) are essential for cellular functions. Understanding the functions of MPs is crucial as they constitute an important class of drug targets. However, MPs are a challenging class of biomolecules to analyze because they cannot be studied outside their native environment. Their structure, function and activity are highly dependent on the local lipid environment, and these properties are compromised when the protein does not reside in the cell membrane. Mammalian cell membranes are complex and composed of different lipid species. Model membranes have been developed to provide an adequate environment to envisage MP reconstitution. Among them, tethered-Bilayer Lipid Membranes (tBLMs) appear as the best model because they allow the lipid bilayer to be decoupled from the support. Thus, they provide a sufficient aqueous space to envisage the proper accommodation of large extra-membranous domains of MPs, extending outside. Additionally, as the bilayer remains attached to tethers covalently fixed to the solid support, they can be investigated by a wide variety of surface-sensitive analytical techniques. This review provides an overview of the different approaches developed over the last two decades to achieve sophisticated tBLMs, with a more and more complex lipid composition and adapted for functional MP reconstitution.