Michel Lafleur

Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions

Oxygen depleted hypoxic regions in the tumour are generally resistant to therapies1. Although nanocarriers have been used to deliver drugs, the targeting ratios have been very low. Here, we show that the magneto-aerotactic migration behaviour2 of magnetotactic bacteria3, Magnetococcus marinus strain MC-14, can be used to transport drug-loaded nanoliposomes into hypoxic regions of the tumour. In their natural environment, MC-1 cells, each containing a chain of magnetic iron-oxide nanocrystals5, tend to swim along local magnetic field lines and towards low oxygen concentrations6 based on a two-state aerotactic sensing system2. We show that when MC-1 cells bearing covalently bound drug-containing nanoliposomes were injected near the tumour in SCID Beige mice and magnetically guided, up to 55% of MC-1 cells penetrated into hypoxic regions of HCT116 colorectal xenografts. Approximately 70 drug-loaded nanoliposomes were attached to each MC-1 cell. Our results suggest that harnessing swarms of microorganisms exhibiting magneto-aerotactic behaviour can significantly improve the therapeutic index of various nanocarriers in tumour hypoxic regions.

Characterization of the membrane-destabilizing properties of different pH-sensitive methacrylic acid copolymers

The intracellular delivery of active biomacromolecules from endosomes into the cytoplasm generally requires a membrane-disrupting agent. Since endosomes have a slightly acidic pH, anionic carboxylated polymers could be potentially useful for this purpose since they can destabilize membrane bilayers by pH-triggered conformational change. In this study, five different pH-sensitive methacrylic acid (MAA) copolymers were characterized with respect to their physicochemical and membrane lytic properties as a function of pH. pH-dependent conformational changes were studied in aqueous solution by turbidimetry and spectrofluorimetry. The hydrophobic domains that formed upon a decrease in pH were found to be dependent on copolymers composition. Hemolysis and cytotoxicity assays demonstrated that the presence of the hydrophobic ethyl acrylate monomer and/or sufficient protonation of the carboxylic acid groups were important parameters for efficient membrane destabilization. Excessive copolymer hydrophobicity was not associated with membrane destabilization, but resulted in high macrophage cytotoxicity. Overall, this study gave more insights into the structure-activity relationship of MAA copolymers with membrane bilayers. Gaining knowledge of modulation of the physicochemical properties of copolymers and the optimization of copolymer-lipid interactions may lead to the elaboration of much more efficient drug delivery systems.

Direct Observation of Domains in Model Stratum Corneum Lipid Mixtures by Raman Microspectroscopy

Several studies on intact and model stratum corneum (SC), the top layer of the epidermis, have suggested the presence of crystalline domains. In the present work, we used micro-Raman mapping to detect lipid domains in model lipid mixtures formed by an equimolar mixture of ceramides, cholesterol, and palmitic acid, the three main lipid species of SC. We were able to determine the spatial distribution of the three compounds individually based on the systematic analysis of band areas. As a control, we studied freeze-dried lipid mixtures, and the Raman microspectroscopy reported faithfully the homogeneous distribution of the three compounds. Spectral mapping was then performed on hydrated equimolar mixtures carefully annealed. In this case, clear phase separations were observed. Domains enriched in cholesterol, ceramides, or palmitic acid with a size of a few tens of square microns were detected. These findings constitute the first direct evidence of the formation of heterogeneous domains in the SC lipid models in a bulk phase. Raman microspectroscopy is an innovative approach to characterize the conditions leading to the formation of domains and provides new insights into the understanding of the skin barrier.

MODULATION OF MELITTIN-INDUCED LYSIS BY SURFACE CHARGE DENSITY OF MEMBRANES

Phosphorus NMR spectroscopy was used to characterize the importance of electrostatic interactions in the lytic activity of melittin, a cationic peptide. The micellization induced by melittin has been characterized for several lipid mixtures composed of saturated phosphatidylcholine (PC) and a limited amount of charged lipid. For these systems, the thermal polymorphism is similar to the one observed for pure PC: small comicelles are stable in the gel phase and extended bilayers are formed in the liquid crystalline phase. Vesicle surface charge density influences strongly the micellization. Our results show that the presence of negatively charged lipids (phospholipid or unprotonated fatty acid) reduces the proportion of lysed vesicles. Conversely, the presence of positively charged lipids leads to a promotion of the lytic activity of the peptide. The modulation of the lytic effect is proposed to originate from the electrostatic interactions between the peptide and the bilayer surface. Attractive interactions anchor the peptide at the surface and, as a consequence, inhibit its lytic activity. Conversely, repulsive interactions favor the redistribution of melittin into the bilayer, causing enhanced lysis. A quantitative analysis of the interaction between melittin and negatively charged bilayers suggests that electroneutrality is reached at the surface, before micellization. The surface charge density of the lipid layer appears to be a determining factor for the lipid/peptide stoichiometry of the comicelles; a decrease in the lipid/peptide stoichiometry in the presence of negatively charged lipids appears to be a general consequence of the higher affinity of melittin for these membranes.

A simple FTIR spectroscopic method for the determination of the lower critical solution temperature of N-isopropylacrylamide copolymers and related hydrogels

Linear and crosslinked polymers based on N-isopropylacrylamide (NIPAAm) exhibit unusual thermal properties. Aqueous solutions of poly(N-isopropylacrylamide) (PNIPAAm) phase-separate upon heating above a lower critical solution temperature (LCST), whereas related hydrogels undergo a swelling–shrinking transition at an LCST. A linear copolymer made of NIPAAm/acryloxysuccinimide (98/2 mol/mol) and two hydrogels with different hydrophilicities were prepared. Fourier transform infrared (FTIR) spectroscopy was employed to determine the transition temperature and provide insights into the molecular details of the transition via probing of characteristic bands as a function of temperature. The FTIR spectroscopy method described here allowed the determination of the transition temperature for both the linear and crosslinked polymers. The transition temperatures for PNIPAAm and the gel resulting from the crosslinking with polylysine or N,N′-methylenebisacrylamide (MBA) were in the same range, 30–35 °C. For the gels, the transition temperature increased with the hydrophilicity of the polymer matrix. The spectral changes observed at the LCST were similar for the free chains and the hydrogels, implying a similar molecular reorganization during the transition. The CH stretching region suggests that the N-isopropyl groups and the backbone both underwent conformational changes and became more ordered upon heating above the LCST. An analysis of the amide I band suggests that the amide groups of the linear polymer were mainly involved in hydrogen bonding with water molecules below the LCST, the chain being flexible and disordered in a water solution. During the transition, around 20% of these intermolecular hydrogen bonds between the polymer and water were broken and replaced by intramolecular hydrogen bonds. Similar changes were also observed at the LCST of a gel crosslinked with MBA.

Polymorphism of POPE/cholesterol system: a 2H nuclear magnetic resonance and infrared spectroscopic investigation.

It is well established that cholesterol induces the formation of a liquid-ordered phase in phosphatidylcholine (PC) bilayers. The goal of this work is to examine the influence of cholesterol on phosphatidylethanolamine polymorphism. The behavior of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)/cholesterol mixtures was characterized using infrared and 2H nuclear magnetic resonance (NMR) spectroscopy (using POPE bearing a perdeuterated palmitoyl chain in the latter case). Our results reveal that cholesterol induces the formation of a liquid-ordered phase in POPE membranes, similar to those observed for various PC/cholesterol systems. However, the coexistence region of the gel and the liquid-ordered phases is different from that proposed for PC/cholesterol systems. The results indicate a progressive broadening of the gel-to-fluid phase transition, suggesting the absence of an eutectic. In addition, there is a progressive downshift of the end of the transition for cholesterol content higher than 10 mol %. Cholesterol has an ordering effect on the acyl chains of POPE, but it is less pronounced than for the PC equivalent. This study also shows that the cholesterol effect on the lamellar-to-hexagonal (L(alpha)-H(II)) phase transition is not monotonous. It shifts the transition toward the low temperatures between 0 and 30 mol % cholesterol but shifts it toward the high temperatures when cholesterol content is higher than 30 mol %. The change in conformational order of the lipid acyl chains, as probed by the shift of the symmetric methylene C-H stretching, shows concerted variations. Finally, we show that cholesterol maintains its chain ordering effect in the hexagonal phase.

Comparison between orientational and conformational orders in fluid lipid bilayers.

The orientational order as determined by 2H NMR and the infrared frequencies of the C--H stretching modes of the methylene groups have been measured for several systems (POPC, POPC/cholesterol and POPE), all in the fluid phase, and then were compared; this work reveals an unexpected linear correlation between them. This experimental result shows that both measurements are essentially sensitive to a common motion, most likely trans/gauche isomerisation. This new correlation with those already found in the literature suggest that several measurements related to the hydrophobic core of the fluid bilayer describe different aspects of a universal behavior. The correlation presented here does not extend to the lipid in gel phase where slower motions affect the NMR lineshape.

Characterization of permeability and morphological perturbations induced by nisin on phosphatidylcholine membranes.

Nisin is an antimicrobial peptide used as food preservative. To gain some insights into the hypothesis that its bactericidal activity is due to the perturbation of the lipid fraction of the bacterial plasmic membrane, we have investigated the effect of nisin on model phosphatidylcholine (PC) membranes. We show that nisin affects the PC membrane permeability, and this perturbation is modulated by the lipid composition. Nisin-induced leakage from PC vesicles is inhibited by the presence of cholesterol. This inhibition is associated with the formation of a liquid ordered phase in the presence of cholesterol, which most likely reduces nisin affinity for the membrane. Conversely, phosphatidylglycerol (PG), an anionic lipid, promotes nisin-induced leakage, and this promotion is associated with an increased affinity of the peptide for the bilayer because nisin is a cationic peptide. When the electrostatic interactions are encouraged by the presence of 70 mol% PG in PC, the inhibitory effect of cholesterol is not observed anymore. Nisin drastically modifies the morphology of the dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) multilamellar dispersion without causing a significant change in the gel-to-liquid crystalline phase transition of the lipid. The morphological changes are observed from (31)P and (2)H NMR and cryo-electron microscopy. From the NMR point of view, the interactions giving rise to a broad signal (quadrupolar interactions and chemical shift anisotropy for (2)H NMR and (31)P NMR, respectively) are partly averaged out in the presence of nisin. This phenomenon is interpreted by the formation of curved lipid planes that lead to the lipid lateral diffusion occurring in the intermediate motional regime. By cryo-electron microscopy, large amorphous aggregates containing small dense globular particles are observed for samples quenched from 25 and 50 degrees C. Long thread-like structures are also observed in the fluid phase. A structural description of DPPC/nisin complex, consistent with the experimental observation, is proposed. The presence of 30 mol% cholesterol in DPPC completely inhibits the morphological changes induced by nisin. Therefore, it is concluded that nisin can significantly perturb PC bilayers from both the permeability and the structural points of view, and these perturbations are modulated by the lipidic species in the bilayer.

Influence of the lipid composition on the organization of skin lipid model mixtures: An infrared spectroscopy investigation

The polymorphism of the lipids of the stratum corneum (SC), the top layer of the epidermis, has a fundamental impact on the permeability properties of the skin barrier. In this work, we have examined by infrared spectroscopy the thermal behavior of model mixtures involving ceramide, palmitic acid and cholesterol, the three main components of the SC lipids, to gain a refined description of the participation of the various lipid species in the different phases observed as a function of temperature. The results show that below 40 degrees C ceramide, cholesterol and palmitic acid exist mainly in crystalline domains and the lipidic species show very limited miscibility. Between 40 and 50 degrees C, a transition from the crystalline to a liquid ordered (lo) phase occurs and it involves ceramides, cholesterol and palmitic acid. When the mixture has a high cholesterol content, this lo phase is stable up to 75 degrees C. For low cholesterol content, the mixtures undergo a second transition toward a more disordered phase which is likely not lamellar. The formation of these phases is critically dependent on the lipid composition and, therefore, it is likely that composition changes of SC lipids affect the phase behavior and, consequently, the skin barrier properties.

Melittin-induced leakage from phosphatidylcholine vesicles is modulated by cholesterol: a property used for membrane targeting

Abstract Melittin, an amphiphathic peptide, affects the permeability of vesicles. This can be demonstrated using the dye release technique. Calcein, a fluorescent marker, is trapped in large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) vesicles and melittin-induced leakage of the dye can be monitored directly by increasing fluorescence intensity. First, we characterized the effect of increasing cholesterol content in the membrane on melittin-induced leakage and our results reveal that cholesterol inhibits the lytic activity of the peptide. Using intrinsic fluorescence of the single tryptophan of melittin and 2H-NMR of headgroup deuterated phosphatidylcholine, we demonstrated that the affinity of melittin for phosphatidylcholine vesicles is reduced in the presence of cholesterol; this is associated with the tighter lipid packing of the cholesterol-containing bilayer. This reduced binding is responsible for the reduced melittin-induced leakage from cholesterol-containing membranes. The pathway of release was determined to be an all-or-none mechanism. Finally, we investigated the possibility of achieving specific membrane targeting with melittin, when vesicles of different lipid composition are simultaneously present. Melittin incubated together with vesicles made of pure POPC and POPC containing 30(mol)% cholesterol can empty nearly all the cholesterol-free vesicles while the cholesterol-containing vesicles remain almost intact. Owing to the preferential interaction of melittin with the pure POPC vesicles, we were able to achieve controlled release of encapsulated material from a specific vesicle population.