Thermodynamic and structural properties of lipid-photosensitizer conjugates mixed with phospholipids: Impact on the formation and stability of nano-assemblies.

The photosensitizer Phenalenone (PN) was grafted with one or two lipid (C18) chains to form pure nano-assemblies or mixed lipid vesicles suitable for photodynamic therapy. Mixtures of PN-C18 conjugates with stearoyl-oleoyl phosphatidylcholine (SOPC) form vesicles that disintegrate into bilayer sheets as the concentration of PN-C18 conjugates increases. We hypothesized that PN-C18 conjugates control the thermodynamic and structural properties of the mixtures and induce the disintegration of vesicles due to PN π-π-interactions. Monolayers were analyzed by surface pressure and grazing incidence X-ray diffraction (GIXD) measurements, and vesicles by differential scanning calorimetry and cryo-TEM. The results showed that PN-triazole-C18 (1A) and PN-NH-C18 (1B) segregate from the phospholipid domains. PN-(C18)2 (conjugate 2) develops favorable interactions with SOPC and distearoyl-phosphatidylcholine (DSPC). GIXD demonstrates the contribution of SOPC to the structuring of conjugate 2 and the role of the major component in controlling the structural properties of DSPC-conjugate 2 mixtures. Above 10 mol% conjugate 2 in SOPC vesicles, the coexistence of domains with different molecule packing leads to conjugate segregation, vesicle deformation, and the formation of small bilayer discs stabilized by the inter-bilayer π-π stacking of PN molecules.

Organization of the Interfacial Film of Nanoemulsions Stabilized by Porphyrin Derivatives.

Photodynamic therapies combining the action of a photosensitizer (PS), molecular oxygen, and light make it possible to destroy certain infectious sites and tumors. The incorporation of photosensitizers in nanocarriers allows for better control of their distribution in tissues and increases their concentration in the area that will be then illuminated. Nanoemulsions of glyceryl trioctanoate (GTO) have been designed in which pyropheophobide a (Pyro-A) or its lipid conjugate (Pyro-Lipid) are both stabilizing and photostimulable agents. In this work, we studied by surface pressure measurements and Brewster angle microscopy (BAM) analysis the organization of the interfacial films of nanodroplets. Comparison of preformed porphyrin nanoemulsions and two porphyrin-GTO mixtures, one mimicking the composition of the nanoemulsions and the other that of a porphyrin-rich interfacial film, highlighted the role of GTO and porphyrin derivatives in the formation, organization, and elasticity of the interfacial films in nanoemulsions. Pyro-Lipid and GTO can mix, and some of the GTO molecules remain inserted in the interfacial film at high surface pressures. In contrast, Pyro-A and GTO do not mix well and tend to segregate, leaving Pyro-A alone in the condensed interfacial film. The results of this study demonstrate the importance of characterizing the interfacial properties of porphyrin derivatives and their interaction with the oil to design stable nanoemulsions with well-controlled optical properties.

Photosensitizers incorporation in SOPC films at different hydration levels.

In the present work, two photosensitizing drugs, Temoporfin and Verteporfin have been studied. Both have regular approval in Europe, Temoporfin for the treatment of head and neck cancers and Verteporfin for the treatment of age-related macular degeneration (AMD). The treatment modality, known as "Photodynamic Therapy" (PDT), involves drug activation with visible light in the presence of oxygen and production of reactive oxygen species (ROS) to destroy the pathological tissues. Both drugs are inactive in the absence of light, presenting only few side effects. The incorporation of the two drugs into a SOPC bilayer -used as a model membrane- was studied by ATR-FTIR. An original approach was applied, involving lyotropic transitions and a very slow dehydration rate of the sample. In low water content and dry film, Temoporfin highly affects stretching vibrations of SOPC chains and polar groups, showing that Temoporfin is inserted into the bilayer in both apolar and polar regions. In fully hydrated layers, Temoporfin - SOPC interactions still take place but only impact Temoporfin vibration bands. Verteporfin shows smaller effect on both chain and polar groups' vibrations of SOPC, with the exception of choline group, suggesting that Verteporfin is inserted into the bilayer to a lesser extent and remains at the bilayer polar interface. These results can be used to better understand drugs behavior in biological media.

Novel liposome-like assemblies composed of phospholipid-porphyrin conjugates with photothermal and photodynamic activities against bacterial biofilms.

Phospholipid-Porphyrin (PL-Por) conjugates are unique building blocks that can self assemble into liposome-like structures with improved photophysical properties compared to their monomeric counterparts. The high packing density of porphyrin moieties enables these assemblies to exhibit high photothermal conversion efficiency as well as photodynamic activity. Thus, PL-Por conjugates assemblies can be used for photodynamic therapy (PDT) and photothermal therapy (PTT) applications against resistant bacteria and biofilms. In order to tune the PD/PT properties of such nanosystems, we developed six different supramolecular assemblies composed of newly synthesized PL-Por conjugates bearing either pheophorbide-a (PhxLPC) or pyropheophorbide-a (PyrxLPC) photosensitizers (PSs) for combined PDT/PTT against planktonic bacteria and their biofilms. In this study, the influence of the chemical structure of the phospholipid backbone as well as that of the PS on the photothermal conversion efficiency, the photodynamic activity and the stability of these assemblies in biological medium were determined. Then their antimicrobial efficiency was assessed on S. aureus and P. aeruginosa planktonic cultures and biofilms. The two studied systems show almost the same photothermal effect against planktonic cultures and biofilms of S. aureus and P. aeruginosa. However, PhxLPC vesicles exhibit superior photodynamic activity, making them the best combination for PTT/PDT. Such results highlight the higher potential of the photodynamic activity of PL-Por nanoassemblies compared to their photothermal conversion in combating bacterial infections.

Formation and stabilization of multiple w/o/w emulsions encapsulating catechin, by mechanical and microfluidic methods using a single pH-sensitive copolymer: Effect of copolymer/drug interaction.

Multiple w/o/w emulsions (MEs) are promising systems for protecting fragile hydrophilic drugs and controlling their release. We explore the capacity of a single pH-sensitive copolymer, PDMS60-b-PDMAEMA50, and salts, to form and stabilize MEs loaded with sucrose or catechin by a one-step mechanical process or a microfluidic method. ME cytotoxicity was evaluated in various conditions of pH. Using the mechanical process, the most stable emulsions were obtained with Miglyol®812 N and isopropyl myristate in a final pH range of 8-12 and [0.3 M-1 M] NaCl concentrations. Conversely, with the microfluidic method, isopropyl myristate at pH 3 without salt was more efficient. Catechin strongly affected the formation of droplets by the mechanical process but did not modify the conditions of stability of MEs obtained by the microfluidic method. The antioxidant power of catechin was preserved in the inner droplets, even in emulsions prepared by the mechanical method at pH 8. An incomplete release of sucrose and catechin from the emulsions was observed and attributed to the interaction of molecules with the copolymer through hydrogen bonding. This study highlights some of the barriers to break to formulate multiple emulsions stabilized by a PDMS-b-PDMAEMA copolymer or other polymers which can form hydrogen bonds interaction with encapsulated drugs.

Phospholipid-porphyrin conjugates: deciphering the driving forces behind their supramolecular assemblies.

Phospholipid-porphyrin conjugates (PL-Por) are nowadays considered as a unique class of building blocks that can self-assemble into supramolecular structures that possess multifunctional properties and enhanced optoelectronics characteristics compared to their disassembled counterparts. However, despite their versatile properties, little is known about the impact of the packing parameter of PL-Por conjugates on their assembling mechanism and their molecular organization inside these assemblies. To gain a better understanding on their assembling properties, we synthesized two new series of PL-Por conjugates with different alkyl sn2-chain lengths linked via an amide bond to either pheophorbide-a (PhxLPC) or pyropheophorbide-a (PyrxLPC). By combining a variety of experimental techniques with molecular dynamics (MD) simulations, we investigated both the assembling and optical properties of the PL-Por either self-assembled or when incorporated into lipid bilayers. We demonstrated that independently of the linker length, PhxLPC assembled into closed ovoid structures, whereas PyrxLPC formed rigid open sheets. Interestingly, PyrxLPC assemblies displayed a significant red shift and narrowing of the Q-band indicating the formation of ordered J-aggregates. The MD simulations highlighted the central role of the interaction between porphyrin cores rather than the length difference between the two phospholipid chains in controlling the structure of the lipid bilayer membranes and thus their optical properties. Indeed, while PhxLPC have the tendency to form inter-leaflet π-stacked dimers, PyrxLPC conjugates formed dimers within the same leaflet. Altogether, this work could be used as guidelines for the design of new PL-Por conjugates that self-assemble into bilayer-like supramolecular structures with tunable morphology and optical properties.

Influence of the porphyrin structure and linker length on the interfacial behavior of phospholipid-porphyrin conjugates.

HYPOTHESIS: Phospholipid-porphyrin (Pl-Por) conjugates consist of porphyrin derivatives grafted to a lysophosphatidylcholine backbone. Owing to their structural similarities with phospholipids, Pl-Por conjugates can self-assemble into liposome-like assemblies. However, there is a significant lack of information concerning the impact of the porphyrin type and the length of the alkyl chain bearing the porphyrin on the interfacial behavior of the Pl-Por conjugates. We hypothesized that changing the chain length and the porphyrin type could impact their two-dimensional phase behavior and modulate the alignment between the two chains. EXPERIMENTS: 6 Pl-Por conjugates with different alkyl chain lengths in the sn2 position of C16 lysophosphatidylcholine and coupled to either pheophorbide-a or pyropheophorbide-a were synthesized. Their interfacial behavior at the air/water interface was assessed using Langmuir balance combined to a variety of other physical techniques including Brewster angle microscopy, atomic force microscopy and X-ray reflectometry. FINDINGS: Our results showed that all 6 Pl-Por form stable monolayers with the porphyrin moiety at the air/water interface. We also showed that changing the porphyrin moiety controlled the packing of the monolayer and thus the formation of organized domains. The chain length dictated the structure of the formed domains with no evidence of the alignment between the two chains.

Porphyrin-lipid stabilized paclitaxel nanoemulsion for combined photodynamic therapy and chemotherapy.

BACKGROUND: Porphyrin-lipids are versatile building blocks that enable cancer theranostics and have been applied to create several multimodal nanoparticle platforms, including liposome-like porphysome (aqueous-core), porphyrin nanodroplet (liquefied gas-core), and ultrasmall porphyrin lipoproteins. Here, we used porphyrin-lipid to stabilize the water/oil interface to create porphyrin-lipid nanoemulsions with paclitaxel loaded in the oil core (PLNE-PTX), facilitating combination photodynamic therapy (PDT) and chemotherapy in one platform. RESULTS: PTX (3.1 wt%) and porphyrin (18.3 wt%) were loaded efficiently into PLNE-PTX, forming spherical core-shell nanoemulsions with a diameter of 120 nm. PLNE-PTX demonstrated stability in systemic delivery, resulting in high tumor accumulation (~ 5.4 ID %/g) in KB-tumor bearing mice. PLNE-PTX combination therapy inhibited tumor growth (78%) in an additive manner, compared with monotherapy PDT (44%) or chemotherapy (46%) 16 days post-treatment. Furthermore, a fourfold reduced PTX dose (1.8 mg PTX/kg) in PLNE-PTX combination therapy platform demonstrated superior therapeutic efficacy to Taxol at a dose of 7.2 mg PTX/kg, which can reduce side effects. Moreover, the intrinsic fluorescence of PLNE-PTX enabled real-time tracking of nanoparticles to the tumor, which can help inform treatment planning. CONCLUSION: PLNE-PTX combining PDT and chemotherapy in a single platform enables superior anti-tumor effects and holds potential to reduce side effects associated with monotherapy chemotherapy. The inherent imaging modality of PLNE-PTX enables real-time tracking and permits spatial and temporal regulation to improve cancer treatment.

Assessment of various formulation approaches for the application of beta-lapachone in prostate cancer therapy.

Beta-lapachone (ß-Lap) is an anticancer drug activated by the NAD(P)H:quinone oxidoreductase (NQO1), an enzyme over-expressed in a large variety of tumors. B-Lap is poorly soluble in water and in most biocompatible solvents. Micellar systems, liposomes and cyclodextrins (CDs) have been proposed for its solubilization. In this work, we analyzed the properties and in vitro efficacy of ß-Lap loaded in polymer nanoparticles, liposome bilayers, complexed with sulfobutyl-ether (SBE)- and hydroxypropyl (HP)-ß cyclodextrins, or double loaded in phospholipid vesicles. Nanoparticles led to the lowest drug loading. Encapsulation of [ß-Lap:CD] complexes in vesicles made it possible to slightly increase the encapsulation rate of the drug in liposomes, however at the cost of poor encapsulation efficiency. Cytotoxicity tests generally showed a higher sensitivity of NIH 3T3 and PNT2 cells to the treatment compared to PC-3 cells, but also a slight resistance at high ß-Lap concentrations. None of the studied ß-Lap delivery systems showed significant enhanced cytotoxicity against PC-3 cells compared to the free drug. Cyclodextrins and double loaded vesicles, however, appeared more efficient drug delivery systems than liposomes and nanoparticles, combining both good solubilizing and cytotoxic properties. Ligand-functionalized double loaded liposomes might allow overcoming the lack of selectivity of the drug.

Deciphering the Peculiar Behavior of ß-Lapachone in Lipid Monolayers and Bilayers.

ß-Lapachone (ß-Lap) is a promising anticancer drug whose applications have been limited so far because of its poor solubility and stability. Its encapsulation in liposomes has been proposed to overcome these issues. However, surface pressure measurements show that ß-Lap exhibits atypical interfacial behavior when mixed with lipids. Although the drug does not seem to be retained in lipid monolayers as deduced from the π-A isotherms, small changes in compressibility moduli suggest that ß-Lap actually interacts with lipids, either disorganizing or rigidifying their monolayers. Thermal and structural analyses of lipid bilayers confirm the existence of ß-Lap/lipid interactions and show that the drug inserts between hydrophobic chains, close to the polar headgroup in DPPC bilayers and deeper in the acyl chains in POPC bilayers. Molecular dynamics simulations allow a comprehensive description of the drug position and orientation in DOPC and POPC bilayers in the presence or absence of cholesterol.

Hybrid Lipid Polymer Nanoparticles for Combined Chemo- and Photodynamic Therapy.

Retinoblastoma is a malignant tumor of the retina in infants. Conventional therapies are associated to severe side effects and some of them induce secondary tumors. Photodynamic therapy (PDT) thus appears as a promising alternative as it is nonmutagenic and generates minimal side effects. The effectiveness of PDT requires the accumulation of a photosensitizer (PS) in the tumor. However, most porphyrins are hydrophobic and aggregate in aqueous medium. Their incorporation into a nanocarrier may improve their delivery to the cell cytoplasm. In this work, we designed biodegradable liponanoparticles (LNPs) consisting of a poly(d,l)-lactide (PDLLA) nanoparticle coated with a phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-3-trimethylammonium-propane) bilayer. An anticancer drug, beta-lapachone (ß-Lap) and a PS, m-THPC, were co-encapsulated for combined chemo- and PDT because it has been suggested that they may have a synergistic effect based on the activation of ß-Lap by PDT-induced over-expression of the enzyme NQO1. Using dynamic light scattering measurements, cryogenic transmission electron microscopy, and fluorescence confocal microscopy, we selected the appropriate conditions for the encapsulation of the compounds. LNPs were internalized in retinoblastoma cells within few hours. No obvious synergistic effect related to the activation of ß-Lap by PDT was observed. Conversely, the LNPs were cytotoxic at lower doses of the two encapsulated compounds as compared to the single therapies. Analysis of the combinatorial treatment showed that PDT and chemotherapy had an additive effect on the viability of retinoblastoma cells.

Capillary zone electrophoresis-native mass spectrometry for the quality control of intact therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) are complex glycoproteins and ensuring their safety, efficacy and quality is still challenging. Indeed, during their manufacturing process, they are exposed to several stresses that can lead to their denaturation, misfolding or dimerization. We report here a new method based on capillary electrophoresis coupled to native mass spectrometry (MS) with a sheath liquid interface to analyze an intact therapeutic mAb, Infliximab, under non-denaturing conditions that preserve its conformational heterogeneity as well as self-association without inducing further unfolding / denaturation. For capillary zone electrophoresis (CZE) separation, a triple layer coating using polybrene-dextran sulfate-polybrene was employed. A sheath liquid composed of isopropanol - water - acetic acid with a flow rate of 10 µL min-1 and mild MS conditions allowed optimal signal intensities. A specific mass spectrum was obtained for each Infliximab conformation in a "stressed" formulated preparation. This is the first time that within a single analysis different conformational states, i.e. native and unfolded monomers as well as dimers are simultaneously detected. The results and the lack of analytical bias arising from the CZE-MS conditions were confirmed by using atomic force microscopy (AFM) as an orthogonal technique. A middle-up approach combined to CZE-MS analysis of the stressed samples suggested that the dimer formation involved mostly Fab-Fab interactions.

Relevance of charges and polymer mechanical stiffness in the mechanism and kinetics of formation of liponanoparticles probed by the supported bilayer model approach.

In specific conditions, co-incubation of polymer nanoparticles and phospholipid vesicles leads to the formation of lipid bilayer coated nanoparticles usable as biocompatible drug delivery systems for co-encapsulation of two drugs. In this work, we focused on the preparation and characterization of liponanoparticles obtained by co-incubation of poly(d,l, lactic acid) (PDLLA) and neutral (POPC) or cationic (POPC/DOTAP) liposomes. The comparison of the behavior of the various studied vesicles co-incubated with nanoparticles highlighted the role of electrostatic interactions. Although the bilayer adsorbed at the surface of polymer nanoparticles was not visible by cryoTEM, zeta-potential measurements and fluorescence confocal microscopy showed evidence of the formation of hybrid nanoparticles in the presence of cationic vesicles. Using silicon dioxide and Langmuir-Schaefer transferred polymer layer-coated surfaces, a thorough analysis of the process of formation of a phospholipid bilayer at the surface of a PDLLA film was performed by combining QCM-D and AFM experiments, taking into account the nature and properties of the support, and the concentration and charge of the lipids. Contrarily to POPC vesicles, cationic ones formed a bilayer on the PDLLA layer in water, but their fast rupture on the soft material did not allow complete nanoparticle surface coverage. This work demonstrates the role of charges and polymer mechanical stiffness in the mechanism and kinetics of formation of PDLLA liponanoparticles in pure water.

Photo-triggerable liposomal drug delivery systems: from simple porphyrin insertion in the lipid bilayer towards supramolecular assemblies of lipid-porphyrin conjugates.

Light-responsive liposomes are considered nowadays as one of the most promising nanoparticulate systems for the delivery and release of an active pharmaceutical ingredient (API) in a spatio-temporal manner. Several strategies can be used to design photo-triggered liposomes. One of them consists in the incorporation of a photosensitizer (PS) in the lipid matrix of a liposomal bilayer that induces the release of the cargo either via a photochemical or a photophysical process. Among the described photosensitizers, porphyrin derivatives have appeared as the most potent ones. This review describes the state-of-the-art of photo-triggerable liposomes based on the combination of lipids and porphyrin derivatives either free or conjugated. It focuses on the different light-triggered release mechanisms and the requirements for the development of such systems. It also details the different strategies for the synthesis of lipid-porphyrin conjugates, their self-assembling properties and their biomedical applications.

Bare and Sterically Stabilized PLGA Nanoparticles for the Stabilization of Pickering Emulsions.

Pickering emulsions were formulated using biodegradable and biocompatible poly(lactic- co-glycolic acid) (PLGA) nanoparticles (NPs) prepared without surfactants or any other polymer than PLGA. A pharmaceutical and cosmetic oil (Miglyol) was chosen as the oil phase at a ratio of 10% w/w. These emulsions were then compared with emulsions using the same oil but formulated with well-described PLGA-poly(vinyl alcohol) (PVA) NPs, i.e., with PVA as NP stabilizers. Strikingly, the emulsions demonstrated very different structures at macroscopic, microscopic, and interfacial scales, depending on the type of NPs used. Indeed, the emulsion layer was significantly thicker when using PLGA NPs rather than PLGA-PVA NPs. This was attributed to the formation and coexistence of multiple water-in-oil-in-water (W/O/W) and simple oil-in-water (O/W) droplets, using a single step of emulsification, whereas simple O/W emulsions were obtained with PLGA-PVA NPs. The latter NPs were more hydrophilic than bare PLGA NPs because of the presence of PVA at their surface. Moreover, PLGA NPs only slightly lowered the oil/water interfacial tension whereas the decrease was more pronounced with PLGA-PVA NPs. The PVA chains at the PLGA-PVA NP surface could probably partially desorb from the NPs and adsorb at the interface, inducing the interfacial tension decrease. Finally, independent of their composition, NPs were adsorbed at the oil/water interface without influencing its rheological behavior, possibly due to their mobility at their interface. This work has direct implications in the formulation of Pickering emulsions and stresses the paramount influence of the physicochemical nature of the NP surface into the stabilization of these systems.

Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro.

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of supramolecular structures with multifunctional properties, required for efficient cancer therapy by photodynamic therapy (PDT). The synthesis of two new lipid-porphyrin conjugates coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to either chemically modified lyso-phosphatidylcholine (PhLPC) or egg lyso-sphingomyelin (PhLSM) is reported. The impact of the lipid backbone of these conjugates on their self-assembling properties, as well as on their physicochemical properties, including interfacial behavior at the air/buffer interface, fluorescence and absorption properties, thermotropic behavior, and incorporation rate in the membrane of liposomes were studied. Finally, their photodynamic activity was evaluated on esophageal squamous cell carcinoma (ESCC) and normal esophageal squamous epithelium cell lines. The liposome-like vesicles resulting from self-assembly of the pure conjugates were unstable and turned into aggregates with undefined structure within few days. However, both lipid-porphyrin conjugates could be efficiently incorporated in lipid vesicles, with higher loading rates than unconjugated Pheo-a. Interestingly, phototoxicity tests of free and liposome-incorporated lipid-porphyrin conjugates demonstrated a better selectivity in vitro to esophageal squamous cell carcinoma relative to normal cells.

Retinoblastoma membrane models and their interactions with porphyrin photosensitisers: An infrared microspectroscopy study.

Fourier Transform Infrared (FTIR) microspectroscopy was used to highlight the interactions between two photosensitisers (PS) of different geometries, TPPmOH4 and a glycoconjugated analogous, TPPDegMan, and lipid bilayers modelling retinoblastoma cell membranes. Retinoblastoma is a rare disease occurring in young infants, for whom conservative treatments may present harmful side-effects. Photodynamic therapy (PDT) is expected to induce less side-effects, as the photosensitiser is only activated when the tumour is illuminated. Since efficiency of the treatment relies on photosensitiser penetration in cancer cells, bilayers with three lipid compositions - pure SOPC, SOPC/SOPE/SOPS/Chol (56:23:11:10) and SOPC/SOPE/SOPS/Chol/CL (42:32:9:8:6) - were used as plasma and mitochondria model membranes. FTIR spectra showed that the interaction of the PSs with the lipid bilayers impacted the lipid organization of the latter, causing significant spectral variations. Both studied photosensitisers inserted at the level of lipid hydrophobic chains, increasing chain fluidity and disorder. This was confirmed by surface pressure measurements. Photosensitisers - TPPmOH4 more than TPPDegMan - also interacted with the polar region of the bilayer, forming hydrogen bonds with phosphate groups that induced major shifts of phosphate absorption bands. This difference in PS interaction with moieties in the polar region was more pronounced with the models with complex lipid composition.

Molecular interactions governing the incorporation of cholecalciferol and retinyl-palmitate in mixed taurocholate-lipid micelles.

Cholecalciferol (D3) and retinyl palmitate (RP) are the two main fat-soluble vitamins found in foods from animal origin. It is assumed that they are solubilized in mixed micelles prior to their uptake by intestinal cells, but only scarce data are available on the relative efficiency of this process and the molecular interactions that govern it. The extent of solubilization of D3 and RP in micelles composed of lipids and sodium taurocholate (NaTC) was determined. Then, the molecular interactions between components were analyzed by surface tension and surface pressure measurements. The mixture of lipids and NaTC allowed formation of micelles with higher molecular order, and at lower concentrations than pure NaTC molecules. D3 solubilization in the aqueous phase rich in mixed micelles was several times higher than that of RP. This was explained by interactions between NaTC or lipids and D3 thermodynamically more favorable than with RP, and by D3 self-association.

Effect of high pressure homogenization on the structure and the interfacial and emulsifying properties of ß-lactoglobulin.

The effect of high pressure homogenization (HPH) on the structure of ß-lactoglobulin (ß-lg) was studied by combining spectroscopic, chromatographic, and electrophoretic methods. The consequences of the resulting structure modifications on oil/water (O/W) interfacial properties were also assessed. Moderated HPH treatment (100 MPa/4 cycles) showed no significant modification of protein structure and interfacial properties. However, a harsher HPH treatment (300 MPa/5 cycles) induced structural transformation, mainly from ß-sheets to random coils, wide loss in lipocalin core, and protein aggregation via intermolecular disulfide bridges. HPH-modified ß-lg displayed higher surface hydrophobicity leading to a faster adsorption rate at the interface and an earlier formation of an elastic interfacial film at Cß-lg = 0.1 wt%. However, no modification of the interfacial properties was observed at Cß-lg = 1 wt%. At this protein concentration, the prior denaturation of ß-lg by HPH did not modify the droplet size of nanoemulsions prepared with these ß-lg solutions as the aqueous phases. A slightly increased creaming rate was however observed. The effects of HPH and heat denaturations appeared qualitatively similar, but with differences in their extent.

Comparison of the Micellar Incorporation and the Intestinal Cell Uptake of Cholecalciferol, 25-Hydroxycholecalciferol and 1-α-Hydroxycholecalciferol.

In the context of the global prevalence of vitamin D insufficiency, we compared two key determinants of the bioavailability of 3 vitamin D forms with significant biopotencies: cholecalciferol, 25-hydroxycholecalciferol and 1-α-hydroxycholecalciferol. To this aim, we studied their incorporation into synthetic mixed micelles and their uptake by intestinal cells in culture. Our results show that 1-α-hydroxycholecalciferol was significantly more solubilized into mixed micelles compared to the other forms (1.6-fold and 2.9-fold improvement compared to cholecalciferol and 25-hydroxycholecalciferol, respectively). In Caco-2 TC7 cells, the hydroxylated forms were taken up more efficiently than cholecalciferol (p < 0.05), and conversely to cholecalciferol, their uptake was neither SR-BI(Scavenger-Receptor class B type I)- nor NPC1L1 (NPC1 like intracellular cholesterol transporter 1)-dependent. Besides, the apical membrane sodium-bile acid transporter ASBT (Apical Sodium-dependent Bile acid Transporter) was not involved, at least in vitro, in the uptake of any of the three vitamin D forms. Further investigations are needed to identify the uptake pathways of both 1-α-hydroxycholecalciferol and 25-hydroxycholecalciferol. However, considering its high bioavailability, our results suggest the potential interest of using 1-α-hydroxycholecalciferol in the treatment of severe vitamin D deficiency.