Lipid Dispersions Research Articles

New Insight for Enhanced Topical Targeting of Caffeine for Effective Cellulite Treatment: In Vitro Characterization, Permeation Studies, and Histological Evaluation in Rats

Cellulite (CLT) is one of the commonly known lipodystrophy syndromes affecting post-adolescent women worldwide. It is topographically characterized by an orange-peel, dimpled skin appearance hence, it is an unacceptable cosmetic problem. CLT can be modulated by surgical procedures such as; liposuction and mesotherapy. But, these options are invasive, expensive and risky. For these reasons, topical CLT treatments are more preferred. Caffeine (CA), is a natural alkaloid that is well-known for its prominent anti-cellulite effects. However, its hydrophilicity hinders its cutaneous permeation. Therefore, in the present study CA was loaded into solid lipid nanoparticles (SLNs) by high shear homogenization/ultrasonication. CA-SLNs were prepared using Compritol® 888 ATO and stearic acid as solid lipids, and span 60 and brij™35, as lipid dispersion stabilizing agents. Formulation variables were adjusted to obtain entrapment efficiency (EE > 75%), particle size (PS < 350 nm), zeta potential (ZP < −25 mV) and polydispersity index (PDI < 0.5). CA-SLN-4 was selected and showed maximized EE (92.03 ± 0.16%), minimized PS (232.7 ± 1.90 nm), and optimum ZP (−25.15 ± 0.65 mV) and PDI values (0.24 ± 0.02). CA-SLN-4 showed superior CA release (99.44 ± 0.36%) compared to the rest CA-SLNs at 1 h. TEM analysis showed spherical, nanosized CA-SLN-4 vesicles. Con-LSM analysis showed successful CA-SLN-4 permeation transepidermally and via shunt diffusion. CA-SLN-4 incorporated into Noveon AA−1® hydrogel (CA-SLN-Ngel) showed accepted physical/rheological properties, and in vitro release profile. Histological studies showed that CA-SLN-Ngel significantly reduced mean subcutaneous fat tissue (SFT) thickness with 4.66 fold (p = 0.035) and 4.16 fold (p = 0.0001) compared to CA-gel, at 7th and 21st days, respectively. Also, significant mean SFT thickness reduction was observed compared to untreated group with 4.83 fold (p = 0.0005) and 3.83 fold (p = 0.0043), at 7th and 21st days, respectively. This study opened new avenue for CA skin delivery via advocating the importance of skin appendages. Hence, CA-SLN-Ngel could be a promising nanocosmeceutical gel for effective CLT treatment.Graphical

Cationic lipid-coated bPEI/pDNA complexes: Correlation between physicochemical and morphological properties

Cationic lipid–polymer hybrid nanoparticles (cLPHNPs) complexed with nucleic acids, also named lipopolyplexes, have been developed as an alternative to nonviral vectors. These core/shell structures have been introduced to mitigate the limitations of cationic liposomes and biodegradable polymeric nanoparticles (NPs). The colloidal study of hybrid structures opens interesting perspectives for developing novel carriers for drug and gene delivery applications. Morphology aspects (size, lamellarity, and nucleic acid localization) and physicochemical properties of nonviral vectors influence gene delivery. Unraveling the structure of lipopolyplexes can enable their rational design toward enhanced gene delivery. Herein, we investigated the cationic LPHNPs loading plasmid DNA (pDNA) structure using dynamic light scattering, cryo-TEM, and pDNA accessibility assay. This study used pDNA as a genetic cargo for association with a cationic polymer, branched polyethylenimine (bPEI). It was found that the morphology of cationic LPHNP structures is influenced mainly by the formation process. Cationic LPHNPs formulated with EPC, DOPE, and cationic lipid DOTAP showed various degrees of multilamellarity depending on the production method. The addition of lipid dispersion to bPEI-pDNA anionic polyplex induced multilamellarity, while the preformed cationic liposome induced core/shell-like structures. Also, cLPHNPs showed better mechanical stability than polyplexes in a centrifugal vacuum concentrator (CVC) process. The physicochemical properties of cLPHNPs, formed in the presence of CL, were kept constant without significant changes. We believe these obtained colloidal data may be used to investigate hybrid-gene nanocarriers and their production process.

Structure and Function of Canine SP-C Mimic Proteins in Synthetic Surfactant Lipid Dispersions.

Lung surfactant is a mixture of lipids and proteins and is essential for air breathing in mammals. The hydrophobic surfactant proteins B and C (SP-B and SP-C) assist in reducing surface tension in the lung alveoli by organizing the surfactant lipids. SP-B deficiency is life-threatening, and a lack of SP-C can lead to progressive interstitial lung disease. B-YL (41 amino acids) is a highly surface-active, sulfur-free peptide mimic of SP-B (79 amino acids) in which the four cysteine residues are replaced by tyrosine. Mammalian SP-C (35 amino acids) contains two cysteine-linked palmitoyl groups at positions 5 and 6 in the N-terminal region that override the β-sheet propensities of the native sequence. Canine SP-C (34 amino acids) is exceptional because it has only one palmitoylated cysteine residue at position 4 and a phenylalanine at position 5. We developed canine SP-C constructs in which the palmitoylated cysteine residue at position 4 is replaced by phenylalanine (SP-Cff) or serine (SP-Csf) and a glutamic acid-lysine ion-lock was placed at sequence positions 20-24 of the hydrophobic helical domain to enhance its alpha helical propensity. AI modeling, molecular dynamics, circular dichroism spectroscopy, Fourier Transform InfraRed spectroscopy, and electron spin resonance studies showed that the secondary structure of canine SP-Cff ion-lock peptide was like that of native SP-C, suggesting that substitution of phenylalanine for cysteine has no apparent effect on the secondary structure of the peptide. Captive bubble surfactometry demonstrated higher surface activity for canine SP-Cff ion-lock peptide in combination with B-YL in surfactant lipids than with canine SP-Csf ion-lock peptide. These studies demonstrate the potential of canine SP-Cff ion-lock peptide to enhance the functionality of the SP-B peptide mimic B-YL in synthetic surfactant lipids.

Structure-function relationships in pure archaeal bipolar tetraether lipids.

Archaeal bipolar tetraether lipids (BTLs) are among the most unusual lipids occurring in nature because of their presumed ability to span the entire membrane to form a monolayer structure. It is believed that because of their unique structural organization and chemical stability, BTLs offer extraordinary adaptation to archaea to thrive in the most extreme milieus. BTLs have also received considerable attention for development of novel membrane-based materials. Despite their fundamental biological significance and biotechnological interests, prior studies on pure BTLs are limited because of the difficulty to extract them in pure form from natural sources or to synthesize them chemically. Here we have utilized chemical synthesis to enable in-depth biophysical investigations on a series of chemically pure glycerol dialkyl glycerol tetraether (GDGT) lipids. The lipids self-assemble to form membrane-bound vesicles encapsulating polar molecules in aqueous media, and reconstitute a functional integral membrane protein. Structural properties of the membranes were characterized via small-angle X-ray scattering (SAXS) and cryogenic electron microscopy (cryo-EM). SAXS studies on bulk aqueous dispersions of GDGT lipids over 10-90 °C revealed lamellar and non-lamellar phases and their transitions. Next we asked whether vesicles overwhelmingly composed of a single GDGT species can undergo fusion as it is difficult to conceptualize such behavior with the assumption that such membranes have a monolayer structure. Interestingly, we observed that GDGT vesicles undergo fusion with influenza virus with lipid mixing kinetics comparable to that with vesicles composed of monopolar phospholipids. Our results suggest that GDGT membranes may consist of regions with a bilayer structure or form bilayer structures transiently which facilitate fusion and thus offer insight into how archaea may perform important physiological functions that require dynamical membrane behavior.

Antioxidant properties of bovine liver protein hydrolysates and their practical application in biphasic systems.

The influence of protein hydrolysate produced from bovine liver protein hydrolysate (LPH) by enzymatic hydrolysis, using Alcalase/Protamex (1:1), on lipid dispersions was investigated. LPH production was optimized to maximize the antioxidant activity (at 45, 50, and 55 °C for 12, 18, and 24 h). Different concentrations of LPHs (1, 3, and 5 mg/g) were added to emulsions and to liposomes. Lipid oxidation level and particle size of the lipid dispersions were monitored for 14 days of storage at 25 °C. Radical scavenging activity and reducing power were the highest at 45 °C after 24 h of hydrolysis. Electrophoresis pattern showed that the antioxidant activity was arising from the peptides with molecular weight around 10 kDa. Lipid oxidation occurred more rapidly in samples without LPH during storage. In emulsions, lower thiobarbituric acid-reactive substance and conjugated diene values were measured with increasing concentrations of LPH at day 14. Accordingly, particle size of the samples containing 5 mg/g of LPH was smaller than those of other groups. Phase separation was observed only in lecithin emulsion without LPH at day 14. The use of LPH in liposome limited the lipid oxidation and maintained the size of the particles independently from the concentration. This study highlights the potential applications of animal by-products as natural antioxidants in complex food systems. The results demonstrate that LPH, particularly when hydrolyzed at optimized conditions, can effectively inhibit lipid oxidation. The findings suggest that biphasic systems incorporating LPH have promising prospects for enhancing the stability and quality of food products. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Vesicle‐to‐micelle transition in aqueous mixtures of cationic lipids with Pluronic F127

AbstractAqueous solutions of vesicle‐forming lipids mixed with micelle‐forming copolymers may result in stabilized and biocompatible aggregates. Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) techniques were used here to investigate the effects of the nonionic triblock copolymer Pluronic F127 (F127) on the thermal behavior, structural changes, and hydrodynamic size of cationic vesicles from dioctadecyldimethylammonium chloride (DODAC), dioctadecyldimethylammonium bromide (DODAB), and didodecyldimethylammonium bromide (DDAB) in aqueous dispersion. The DSC thermograms indicate that upon the addition of up to 5.0 mM F127, a vesicle‐to‐micelle transition occurs in three stages. First, there is a predominance of lipid‐F127 mixed vesicles, followed by the co‐existence of mixed vesicles and micelles, and finally, the system is dominated by mixed micelles. DLS results reveal that with the addition of F127 to the lipid dispersions, the size of the assembled structures tends to decrease, also indicating structural changes of vesicles to micelles. These results indicate that the borders between these stages depend on the lipid chain length and counterion type.

Anionic liposomes prepared without organic solvents for effective siRNA delivery.

Currently, organic solvents are necessary for the preparation of anionic liposomes for siRNA delivery. The removal of organic solvent is time-consuming and the residual organic solvent is not only a hidden danger, but also affects the stability of anionic liposomes. Glycerol, which is physiologically compatible and does not need to be removed, is used to promote the dispersion of lipids and the formation of anionic liposomes. Additionally, the preparation process is simple and not time-consuming. The results showed that anionic liposomes, which were typically spherical with a particle size of 188.9nm were successfully prepared with glycerol. And with the help of Ca2+ , siRNA was encapsulated in anionic liposomes. The highest encapsulation efficiency at 2.4mM Ca2+ reached 91%. And the formation of calcium phosphate could promote the endosomal escape of siRNA effectively. The results from cell viability showed that the anionic liposomes had no obvious cytotoxicity. It was also verified that anionic liposomes could improve the resistance of siRNA against degradation. Additionally, siRNA delivered by anionic liposomes could play an effective role in knockout. Therefore, anionic liposomes prepared with glycerol will be a safe and effective delivery platform for siRNA and even other nucleic acid drugs.

Critical point for membrane bilayer formation

Unilamellar liposomes often are employed in investigations of lipid-protein interactions and the delivery of drugs in therapies for disease. Also, related lipid-containing nanoparticles have been developed as elements of a new class of mRNA vaccines. We show that only unilamellar films form in equilibrium lipid dispersions, at temperature values {T*} that depend on the identities of the lipids (e.g., T* ≈ 29 °C for DMPC). Thermodynamic analysis confirms that films at air-water surfaces can be used to monitor the properties of the lipid vesicles that form in the dispersion. When T > T*, critical exponents describing film properties as T approaches T* are μ ≈ 1.4 and ν ≈ 0.7, which are close to values for the interfacial tension and the correlation length of density fluctuations at fluid interfaces. These results, and observations that within the bilayer the lateral diffusion of fluorescent lipid probes demonstrates increases at T*, suggest that unilamellar vesicles at T* are a transition state between two different multilamellar structures. We generalize the thermodynamic arguments to explain the linkage between lipid structures in the surface and bulk dispersion within more complex samples, showing that dispersions containing total lipid extracts of cell membranes have properties similar to those in dispersions containing single lipids. Information from various independent studies indicates that T* noted for bilayer membranes of a population of cells is identical to the temperature at which the growth or gestation of the cells occurs in vivo. Examples include whole-cell lipid extracts obtained from bacteria, and poikilothermic and homeothermic animals.

Extending the Pseudo-Phase Model of Detergent–Lipid Dispersions by a Detergent-Binding Protein

Mixed micellar drug delivery systems for poorly soluble active pharmaceutical ingredients (APIs) are easy to produce and long-term stable, because they represent equilibrium structures. However, their fate after intravenous injection is still largely unknown. Once injected into the bloodstream, they can potentially convert to vesicles or disappear altogether, with both API and excipients being picked up by blood components. Our study aimed at reducing the gap between the good, quantitative understanding of aqueous glycocholate (GC)-lecithin dispersions alone and the highly complex situation in the blood. To this end, we extended the pseudophase model previously established for lipid-detergent dispersions to include the detergent-binding protein albumin as another component. The model predicted a quaternary phase diagram with planar phase boundaries defined by key parameters of the ternary subsystems, which were then determined by isothermal titration calorimetry. They include the aqueous GC concentration upon bilayer-micelle coexistence, 5.2 mM, the GC-to-lipid mole ratios in coexisting bilayers (Resat = 0.2) and micelles (Resol = 0.7), as well as the capacity of the albumin to bind 0.1 GC molecules with a dissociation constant of KD = 0.1 mM and 6 GC molecules with KD = 0.7 mM. Subsequent measurements in the quaternary system showed phase boundaries in good agreement with the model predictions. In addition, the critical micelle concentration of GC shows a minimal value (midpoint of transition) of 9.1 mM at the temperature of 24 °C where the demicellization enthalpy is zero. The demicellization process is accompanied by a heat capacity change of 29 cal/mol K. The model improves the understanding of the mixed micellar drug delivery systems. The success of the approach encourages including even more blood components, like lipoproteins, to a quantitative treatment.

Structural investigation and steric stabilisation of Guerbet glycolipid-based cubosomes and hexosomes using triblock polyethylene oxide-polypropylene oxide-polyethylene oxide copolymers

Aqueous dispersions of inverse nonlamellar liquid crystalline nanostructures namely cubosomes and hexosomes have been used as carriers for drug delivery. The large surface area and internal water channel networks in these nanocarriers make them useful to transport amphiphilic, hydrophilic and hydrophobic active ingredients. However, suitable stabilisers that can maintain the colloidal stability and preserving the internal structures of the complex cubosomes and hexosomes are integral. Herein, Guerbet glycolipids namely 2-hexyl-decyl-β-D-glucopyranoside (β-Glc-OC10C6) and 2-hexyl-decyl-β-D-xylopyranoside (β-Xyl-OC10C6) were investigated for their potential in forming cubosomes and hexosomes respectively using Pluronic copolymers as steric stabilisers. The performance of five different Pluronics was evaluated for stabilisation of the dispersions, with a view to establish a structure-property relationship between the lipids and the stabilisers. The dispersions were assessed through synchrotron small-angle X-ray scattering as a function of temperature and the concentration of stabilisers. The internal structure of dispersed particles of β-Glc-OC10C6 was susceptible to changes upon stabilisation by different Pluronics and with increasing temperature. The lowest molecular weight Pluronic studied, L64, had the greatest propensity to disrupt the structure, suggesting that it is able to penetrate the internal structure to a greater degree than the other Pluronic stabilisers. In contrast, the β-Xyl-OC10C6 counterpart formed a stable inverse hexagonal phase structure that was invariant with the Pluronic used or temperature. The stability of the structure of β-Xyl-OC10C6 suggests it has greater potential for use in drug delivery applications and the studies overall extend the current understanding around the structures formed upon dispersion of Guerbet lipids.

The spatial distribution of the ligand on targeted microbubbles influences the binding efficacy

An important trait of targeted microbubbles is their binding to biomarkers for ultrasound molecular imaging and drug delivery. Using organic solvents in phospholipid-coated microbubble production results in a homogeneous ligand distribution, while dispersion of lipids directly into aqueous medium results in a heterogeneous ligand distribution. In this study, we compared the binding efficacy of ανβ3-targeted microbubbles with a homogeneous or heterogeneous ligand distribution in vitro and in vivo using confocal microscopy. For in vitro studies, human umbilical vein endothelial cells grown statically and under physiological flow were used. For in vivo studies, chicken embryos (day 5) were used. Microbubbles having a homogeneous ligand distribution bound 1.55× more than microbubbles having a heterogeneous ligand distribution in vitro statically, while in vitro under flow this was 1.49× more at 1.25 dyn/cm2 and 1.56× more at 2.22 dyn/cm2; in vivo this was 1.25× more. The dissociation rate in vitro was lower for bound microbubbles with a homogeneous than heterogeneous ligand distribution at low shear stresses (1–5 dyn/cm2). In conclusion, when producing phospholipid-coated targeted microbubbles using organic solvents is preferable over directly dispersing phospholipids in aqueous medium for optimal binding. [Funding by the Phospholipid Research Center (No. KKO-2017-057/1-1) and NWO (VIDI Project No.17543) is gratefully acknowledged.]

A Stepwise Framework for the Systematic Development of Lipid Nanoparticles.

A properly designed nanosystem aims to deliver an optimized concentration of the active pharmaceutical ingredient (API) at the site of action, resulting in a therapeutic response with reduced adverse effects. Due to the vast availability of lipids and surfactants, producing stable lipid dispersions is a double-edged sword: on the one hand, the versatility of composition allows for a refined design and tuning of properties; on the other hand, the complexity of the materials and their physical interactions often result in laborious and time-consuming pre-formulation studies. However, how can they be tailored, and which premises are required for a “right at first time” development? Here, a stepwise framework encompassing the sequential stages of nanoparticle production for disulfiram delivery is presented. Drug in lipid solubility analysis leads to the selection of the most suitable liquid lipids. As for the solid lipid, drug partitioning studies point out the lipids with increased capacity for solubilizing and entrapping disulfiram. The microscopical evaluation of the physical compatibility between liquid and solid lipids further indicates the most promising core compositions. The impact of the outer surfactant layer on the colloidal properties of the nanosystems is evaluated recurring to machine learning algorithms, in particular, hierarchical clustering, principal component analysis, and partial least squares regression. Overall, this work represents a comprehensive systematic approach to nanoparticle formulation studies that serves as a basis for selecting the most suitable excipients that comprise solid lipid nanoparticles and nanostructured lipid carriers.

Effects of high-temperature pressure cooking on cold-grind and blanched soymilk: Physico-chemical properties, in vitro digestibility and sensory quality

The present work investigated effects of high-temperature pressure cooking (HP) on physico-chemical properties and in vitro protein digestibility of cold-grind soymilk (CS) and blanched soymilk (BS). Confocal laser scanning microscopy presented that proteins and lipids in BS were obviously coalesced compared with CS, while HP treatment contributed to homogeneous dispersion of protein and lipid. Particle size of the BS and BSHP were larger than that of CS and CSHP. Tertiary structure of protein suggested that hydrophobic interactions and disulfide bonds in CS and CSHP were main force among protein molecules. Meanwhile hydrophobic interactions were account for 70% of the total bonds in BS and BSHP. The maximum fluorescence excitation wavelength (λmax) of BSHP (349.0 nm) was shown a red shift compared with CS (346.5 nm). For secondary structure of protein, β-sheet was 30.61% in CS, while CSHP, BS and BSHP decreased by 2.65%, 5.73% and 7.77%, respectively. Thus, the protein in BS and BSHP were evidently aggregated, loose and disorder; while the CS and CSHP protein were dense and orderly. Also, HP treatment led to loose and unordered protein structure. Moreover, the in vitro digestibility was BSHP > BS > CSHP > CS. The difference in digestibility of four soymilk was determined by the protein structure. Sensory evaluation showed that blanching treatment effectively reduced beany flavor and HP decreased the diameter of oil bodies of BS, which led to smooth mouth feel and thus the highest global impression of BSHP.

Dispersion and utilization of lipid droplets mediates respiratory syncytial virus-induced airway hyperresponsiveness.

Respiratory viral infections (RSV) can induce acute asthma attacks, thereby destroying lung function and accelerating the progression of the disease. However, medications in the stable phase of asthma are often not effective for acute attacks induced by viral infections. We aimed to clarify the possible mechanism of viral infection-induced asthma through fatty acid metabolism. The airway resistances, inflammatory injuries, and oxidative stress in the RSV-induced animal models were significantly higher than those in the control group at acute phase (7 days) and chronic phase (28 days). Moreover, the concentrations of the medium- and long-chain fatty acids in lung tissue at (28 days) were significantly increased, including 14:0 (myristic acid), 16:0 (palmitic acid, PA), 18:1 (oleic acid, OA), and 18:2 (linoleic acid, LA) using non-targeted metabonomics. Airway epithelial cells treated with RSV showed the reduced expression of FSP27, RAB8A, and PLIN5, which caused the fusion and growth of lipid droplet (LD), and increased expression of the LD dispersion gene perilipin 2. There was also a decrease in PPARγ expression and an increase in the fatty acid catabolism gene PPARα, causing lipid oxidation, free fatty acid releases, and an upsurge in IL-1, IL-2, IL-4, and IL-6 expression, which could be abrogated by GPR40 inhibitor. Treated mice or epithelial cells with C18 fatty acid exhibited inhibition of epithelial proliferation, increases of inflammation, and oxidative damage. RSV promoted lipid dispersion and utilization, causing enlarged oxidative injuries and an upsurge in the pro-inflammatory cytokines, leading to the progression of airway hyperresponsiveness (AHR).

Investigating In Vitro and Ex Vivo Properties of Artemether/Lumefantrine Double-Fixed Dose Combination Lipid Matrix Tablets Prepared by Hot Fusion.

Highly lipophilic antimalarial drugs, artemether and lumefantrine, whilst an effective fixed-dose combination treatment to lower the malarial disease burden, are therapeutically hindered by low aqueous solubility and varied bioavailability. This work investigates the plausibility of directly compressed lipid matrix tablets, their role as lipid-based formulations and their future standing as drug delivery systems. Lipid matrix tablets were manufactured from solid lipid dispersions in various lipid:drug ratios employing hot fusion—the melt mixing of highly lipophilic drugs with polymer(s). Sequential biorelevant dissolution media, multiple mathematical models and ex vivo analysis utilizing porcine tissue samples were employed to assess drug release kinetics and more accurately predict in vitro performance. Directly compressed stearic acid tablets in a 0.5:1 lipid:drug ratio were deemed optimal within investigated parameters. Biorelevant media was of immense value for artemether release analysis, with formulation SA0.5C1 (Stearic Acid:double fixed dose in a 0.5:1 ratio (i.e., Stearic acid 70 mg + Lumefantrine 120 mg + Artemether 20 mg); CombiLac® as filler (q.s.); and 1% w/w magnesium stearate) yielding a higher percentage of artemether release (97.21%) than the commercially available product, Coartem® (86.12%). However, dissolution media lacked the specificity to detect lumefantrine. Nonetheless, stearic acid lipid:drug ratios governed drug release mechanisms. This work demonstrates the successful utilization of lipids as pharmaceutical excipients, particularly in the formulation of lipid matrix tablets to augment the dissolution of highly lipophilic drugs, and could thus potentially improve current malarial treatment regimens.

Preparation of Artificial Red Blood Cells (Hemoglobin Vesicles) Using the Rotation-Revolution Mixer for High Encapsulation Efficiency.

Hemoglobin vesicles (Hb-V) are artificial red blood cells encapsulating highly concentrated hemoglobin (Hb) in liposomes comprising phospholipids, cholesterol, negatively charged lipids, and polyethylene glycol (PEG)-conjugated phospholipids. Safety and efficacy of Hb-V as a transfusion alternative have been extensively studied. For this study, we prepared Hb-V using the kneading method with a rotation-revolution mixer as an alternative to the conventional extrusion method. We optimized the kneading operation parameters to obtain Hb-V with a high yield. Results show that the Hb encapsulation efficiency was increased dramatically up to 74.2%, which is higher than that of the extrusion method (20%) because the kneading method enabled mixing of a highly concentrated carbonylhemoglobin (HbCO) solution (40 g/dL) and a considerably large amount of powdered lipids in only 10 min. The high viscosity of the Hb-lipid mixture paste (ca. 103-105 cP) favorably induces frictional heat by kneading and increases the paste temperature (ca. 60 °C), which facilitates lipid dispersion and liposome formation. During the kneading operation using a thermostable HbCO solution, Hb denaturation was prevented. Hb-V prepared using this method showed no marked changes in particle sizes, Hb denaturation, or Hb leakage from liposomes during two years of long-term storage-stability tests. Collectively, these results demonstrate that the kneading method using a rotation-revolution mixer shows good potential as a new method to produce Hb-V.

Transient Lipid-Protein Structures and Selective Ganglioside Uptake During α-Synuclein-Lipid Co-aggregation.

α-Synuclein is a membrane-interacting protein involved in Parkinson’s disease. Here we have investigated the co-association of α-synuclein and lipids from ganglioside-containing model membranes. Our study relies on the reported importance of ganglioside lipids, which are found in high amounts in neurons and exosomes, on cell-to-cell prion-like transmission of misfolded α-synuclein. Samples taken along various stages of the aggregation process were imaged using cryogenic transmission electron microscopy, and the composition of samples corresponding to the final state analyzed using NMR spectroscopy. The combined data shows that α-synuclein co-assembles with lipids from the ganglioside (GM1)-containing model membranes. The lipid-protein samples observed during the aggregation process contain non-vesicular objects not present at the final stage, thus capturing the co-existence of species under non-equilibrium conditions. A range of different lipid-protein co-assemblies are observed during the time course of the reaction and some of these appear to be transient assemblies that evolve into other co-aggregates over time. At the end of the aggregation reaction, the samples become more homogeneous, showing thin fibrillar structures heavily decorated with small vesicles. From the NMR analysis, we conclude that the ratio of GM1 to phosphatidyl choline (PC) in the supernatant of the co-aggregated samples is significantly reduced compared to the GM1/PC ratio of the lipid dispersion from which these samples were derived. Taken together, this indicates a selective uptake of GM1 into the fibrillar aggregates and removal of GM1-rich objects from the solution.

Cold-Burst Method for Nanoparticle Formation with Natural Triglyceride Oils.

The preparation of nanoemulsions of triglyceride oils in water usually requires high mechanical energy and sophisticated equipment. Recently, we showed that α-to-β (viz., gel-to-crystal) phase transition, observed with most lipid substances (triglycerides, diglycerides, phospholipids, alkanes, etc.), may cause spontaneous disintegration of microparticles of these lipids, dispersed in aqueous solutions of appropriate surfactants, into nanometer particles/drops using a simple cooling/heating cycle of the lipid dispersion (Cholakova et al. ACS Nano 2020, 14, 8594). In the current study, we show that this "cold-burst process" is observed also with natural oils of high practical interest, including coconut oil, palm kernel oil, and cocoa butter. Mean drop diameters of ca. 50-100 nm were achieved with some of the studied oils. From the results of dedicated model experiments, we conclude that intensive nanofragmentation is observed when the following requirements are met: (1) The three-phase contact angle at the solid lipid-water-air interface is below ca. 30 degrees. (2) The equilibrium surface tension of the surfactant solution is below ca. 30 mN/m, and the dynamic surface tension decreases rapidly. (3) The surfactant solution contains nonspherical surfactant micelles, e.g., ellipsoidal micelles or bigger supramolecular aggregates. (4) The three-phase contact angle measured at the contact line (frozen oil-surfactant solution-melted oil) is also relatively low. The mechanism(s) of the particle bursting process is revealed, and on this basis, the role of all of these factors is clarified and discussed. We explain all main effects observed experimentally and define guiding principles for optimization of the cold-burst process in various, practically relevant lipid-surfactant systems.

A supercritical fluid technology for liposome production and comparison with the film hydration method

Liposomes were produced by an innovative method using supercritical carbon dioxide as a dispersing agent. A quality by design strategy was used to find optimal production conditions with specific parameters (lipid concentration, dispersion volume, agitation rate, temperature and pressure) allowing the production of liposomes with predicted physicochemical characteristics (particles size and PdI). Two conditions were determined with specific production parameters. It was shown that these two conditions allowed the production of liposomes of different compositions and that most of the liposome formulations had size and dispersity in accordance with the prediction values. The condition involving the higher lipid concentration showed a higher variability in terms of size and dispersity. However, this variability remained acceptable. This innovative supercritical method allowed the production of liposomes with physicochemical characteristics similar to those obtained by the conventional thin film hydration method. This new supercritical carbon dioxide method easily scalable in GMP conditions is a one-step production method contrarily to conventional methods which generally need an additional step as extrusion to homogenize the size of liposomes.

Cryogenically frozen PEGylated liposomes and micelles: Water penetration and polarity profiles

Poly(ethylene glycol) (PEG)-grafted lipid dispersions are widely investigated in fundamental and biotechnological research for their successful use in drug-delivery. Here, we consider mixtures of the bilayer-forming lipid dipalmitoylphosphatidylcholine (DPPC) with the micelle-forming lipid PEG:2000-phosphatidilethanolamine (PEG:2000-DPPE) fully hydrated in D2O and measured at 77 K. Electron Spin Echo Envelope Modulation and continuous wave Electron Paramagnetic Resonance of chain-labelled lipids are employed to detect the extent of solvent permeation and the environmental polarity, respectively, across the hydrocarbon regions of the lipid assemblies. Sigmoidal water penetration and polarity profiles are described in sterically stabilized liposomes (SSL) formed at submicellar content of PEG:2000-DPPE incorporated in DPPC. Compared to DPPC bilayers, SSL show increased hydrophobicity at both the polar/apolar interface and the chain termini, and a broader transition that is shifted toward the interface. Solvent exposure and polarity decrease on going down the chain in PEG:2000-DPPE micelles. However, compared to SSL, polymer-lipid micelles show higher solvent permeation at any chain segment and the chain termini are accessible to water. In any sample, heterogeneity is found in H-bond formation between the spin-label nitroxide groups and the solvent molecules. The results at cryogenic temperature add new insights into the biophysico-chemical characterization of PEGylated lipid dispersions.