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Formation of high viscous inverse lyotropic liquid crystalline phases in situ upon exposure of low viscous drug-loaded lipid preformulations to synovial fluid provides a promising approach for design of depot formulations for intra-articular drug delivery. Rational formulation design relies on a fundamental understanding of the synovial fluid-mediated dynamic structural transitions occurring at the administration site. At conditions mimicking the in vivo situation, we investigated in real-time such transitions at multiple positions by synchrotron small-angle X-ray scattering (SAXS) combined with an injection-cell. An injectable diclofenac-loaded quaternary preformulation consisting of 72/8/10/10% (w/w) glycerol monooleate/1,2-dioleoyl-glycero-3-phospho-rac-(1-glycerol)/ethanol/water was injected into hyaluronic acid solution or synovial fluid. A fast generation of a coherent drug depot of inverse bicontinuous Im3m and Pn3m cubic phases was observed. Through construction of 2D spatial maps from measurements performed 60 min after injection of the preformulation, it was possible to differentiate liquid crystalline rich- and excess hyaluronic acid solution- or synovial fluid-rich regimes. Synchrotron SAXS findings confirmed that the exposure of the preformulation to the media leads to alterations in structural features in position- and time-dependent manners. Effects of biologically relevant medium composition on the structural features, and implications for development of formulations with sustained drug release properties are highlighted.

Hypothesis: pH-responsive nanocarriers have the potential to provide targeted delivery of antimicrobial peptides (AMPs) to sites of bacterial infection with typically abnormal pH levels in the body. However, the local pH of the infected sites varies substantially among different infection-related diseases, calling for the development of delivery systems capable of targeting local pathological conditions in an adjustable pH range. Experiments: In this study, a highly versatile pH-responsive nanocarrier platform, based on dispersions of oleic acid (OA) and glycerol monooleate (GMO) self-assemblies with the human cathelicidin AMP LL-37, was designed and characterized. Findings: A detailed pH-composition phase diagram was constructed from small angle X-ray scattering and cryogenic transmission electron microscopy data. In addition, the protonation state and apparent pKa of OA embedded in these nano-self-assemblies were investigated by electrophoretic mobility measurements at different pHs and found to be strongly dependent on nanocarrier composition. By varying composition of these nanocarriers, the apparent pKa of embedded OA molecules could be tuned from 7.8 to 6.3, shifting the range of nanocarriers' pH-response. The study advances our fundamental understanding of self-assembly and pH-responsiveness in lipid-peptide systems containing monounsaturated long-chain fatty acids. The results may guide the future design of highly adaptable nanocarriers for patient-optimized pH-targeted AMP delivery.

Pluronic F127-stabilized non-lamellar liquid crystalline aqueous nanodispersions are promising injectable platforms for drug and contrast agent delivery. These nanodispersions, however, trigger complement activation in the human blood, where the extent of complement activation and opsonization processes may compromise their biological performance and safety. Here, we introduce a broad family of nanodispersions from glycerol monooleate (GMO) and oleic acid (OA) in different weight ratios, and stabilized with a plethora of nonionic methoxypoly(ethylene glycol) (mPEG)-lipids of different PEG chain length and variable lipid moiety (monounsaturated or saturated diglycerides or D-α-tocopheryl succinate). Through an integrated biophysical approach involving dynamic light scattering, synchrotron small-angle scattering, and cryo-transmission electron microscopy, we examine the impact of nonionic mPEG-lipid stabilization on size, internal self-assembled architecture, and gross morphological characteristics of nanodispersions. The results show how the nonionic mPEG-lipid type and concentration, and dependent on GMO/OA weight ratio, can variably modulate the internal architectures of nanoparticles. Assessment of complement profiling from selected nanodispersions with diverse structural heterogeneity further suggests a variable modulatory role for the lipid type of the nonionic mPEG-lipid in the extent of complement activation, which span from no activation to moderate to high levels. We comment on plausible mechanisms driving the observed complement activation variability and discuss the potential utility of these nanodispersions for future development of injectable nanopharmaceuticals.

The emergence of nanomedicine as an innovative and promising alternative technology shows many advantages over conventional cancer therapies and provides new opportunities for early detection, improved treatment, and diagnosis of cancer. Despite the cancer nanomedicines’ capability of delivering chemotherapeutic agents while providing lower systemic toxicity, it is paramount to consider the cancer complexity and dynamics for bridging the translational bench-to-bedside gap. It is important to conduct appropriate investigations for exploiting the tumor microenvironment, and achieving a more comprehensive understanding of the fundamental biological processes in cancer and their roles in modulating nanoparticle–protein interactions, blood circulation, and tumor penetration. This review provides an overview of the current cancer nanomedicines, the major challenges, and the future opportunities in this research area.

Microfluidic platforms have become highly attractive tools for synthesis of nanoparticles, including lipid nano-self-assemblies, owing to unique features and at least three important aspects inherent to miniaturized micro-devices. Firstly, the fluids flow under controlled conditions in the microchannels, providing well-defined flow profiles and shorter diffusion lengths that play important roles in enhancing the continuous production of lipid and polymer nanoparticles with relatively narrow size distributions. Secondly, various geometries adapted to microfluidic device designs can be utilized for enhancing the colloidal stability of nanoparticles and improving their drug loading. Thirdly, microfluidic devices are usually compatible with in situ characterization methods for real-time monitoring of processes occurring inside the microchannels. This is unlike conventional nanoparticle synthesis methods, where a final solution or withdrawn aliquots are separately analysed. These features inherent to microfluidic devices provide a tool-set allowing not only precise nanoparticle size control, but also real-time analyses for process optimization. In this review, we focus on recent advances and developments in the use of microfluidic devices for synthesis of lipid nanoparticles. We present different designs based on hydrodynamic flow focusing, droplet-based methods and controlled microvortices, and discuss integration of microfluidic platforms with synchrotron small-angle X ray scattering (SAXS) for in situ structural characterization of lipid nano-self-assemblies under continuous flow conditions, along with major challenges and future directions in this research area., INP grant from the Danish Agency for Science and Higher Education [8073-00022B], AY and OYC acknowledge the INP grant (Ref. no.: 8073-00022B) from the Danish Agency for Science and Higher Education for initiating and exploring new networking and collaboration opportunities between Danish and Turkish research institutions.

Administration of parenteral liquid crystalline phases, forming in-vivo with tunable nanostructural features and sustained release properties, offers an attractive approach for treatment of infections and local drug delivery. It has also a potential use for postoperative pain management after arthroscopic knee surgery. However, the optimal use of this drug delivery principle requires an improved understanding of the involved dynamic structural transitions after administration of low-viscous stimulus-responsive lipid precursors and their fate after direct contact with the biological environment. These precursors (preformulations) are typically based on a single biologically relevant lipid (or a lipid combination) with non-lamellar liquid crystalline phase forming propensity. In relation to liquid crystalline depot design for intra-articular drug delivery, it was our interest in the present study to shed light on such dynamic structural transitions by combining synchrotron SAXS with a remote controlled addition of synovial fluid (or buffer containing 2% (w/v) albumin). This combination allowed for monitoring in real-time the hydration-triggered dynamic structural events on exposure of the lipid precursor (organic stock solution consisting of the binary lipid mixture of monoolein and castor oil) to excess synovial fluid (or excess buffer). The synchrotron SAXS findings indicate a fast generation of inverse bicontinuous cubic phases within few seconds. The effects of (i) the organic solvent N-methyl-2-pyrolidone (NMP), (ii) the lipid composition, and (iii) the albumin content on modulating the structures of the self-assembled lipid aggregates and the implications of the experimental findings in the design of liquid crystalline depots for intra-articular drug delivery are discussed.

Stimuli-responsive nanocarriers based on lipid self-assemblies have the potential to provide targeted delivery of antimicrobial peptides, limiting their side effects while protecting them from degradation in the biological environments. In the present study, we design and characterize a simple pH-responsive antimicrobial nanomaterial, formed through the self-assembly of oleic acid (OA) with the human cathelicidin LL-37 as a model for an amphiphilic antimicrobial peptide. Colloidal transformations from core-shell cylindrical micelles with a cross-sectional diameter of ∼5.5 nm and a length of ∼23 nm at pH 7.0 to aggregates of branched threadlike micelles at pH 5.0 were detected using synchrotron small-angle X-ray scattering, cryogenic transmission electron microscopy, and dynamic light scattering. Biological in vitro assays using an Escherichia coli bacteria strain showed high antimicrobial activity of the positively charged LL-37/OA aggregates at pH 5.0, which was not caused by the pH conditions themselves. Contrary to that, negligible antimicrobial activity was observed at pH 7.0 for the negatively charged cylindrical micelles. The nanocarrier's ability to switch its biological activity "on" and "off" in response to changes in pH could be used to focus the antimicrobial peptides' action to areas of specific pH in the body. The presented findings contribute to the fundamental understanding of lipid-peptide self-assembly and may open up a promising strategy for designing simple pH-responsive delivery systems for antimicrobial peptides.

Lyotropic non-lamellar liquid crystalline (LLC) nanoparticles are attractive nanocarriers for drug delivery, particularly for the solubilization of poorly water-soluble drugs. Due to the reported problems of complement activation and cytotoxicity of most investigated Pluronic F127-stabilized cubosomes and hexosomes, there is an interest in introducing safe stabilizers for these LLC nanodispersions. Citrem appears to be the stabilizer of choice for the colloidal stabilization of these LLC nano-self-assemblies owing to its hemocompatiblity and poor activation of the complement system. This anionic food-grade emulsifier in combination with soy phosphatidylcholine (SPC) can be used to introduce a library of hemocompatible lamellar and non-lamellar liquid crystalline nanodispersions at different lipid compositions. We found that batch-to-batch variability in citrem composition is associated with slight alterations in the size, structural characteristics, and surface charge of the produced citrem/SPC nanoparticles. Further, we report on the temperature-triggered alterations in these nano-self-assemblies at different lipid compositions by using synchrotron small angle X-ray scattering (SAXS). The addition of citrem at different temperatures induces lamellar to non-lamellar structural transitions as evident from the appearance of inverse bicontinuous cubic Pn3m and discontinuous hexagonal (H2) phases, respectively, upon increasing citrem concentration and varying temperature in the range of 5–59 °C. Citrem/SPC nanoparticles are attractive for use in the development of nanocarriers for drug delivery owing to their structural tunability and hemocompatiblity.

Owing to their unique structural features, non-lamellar liquid crystalline nanoparticles comprising cubosomes and hexosomes are attracting increasing attention as versatile investigative drug carriers. Background: Depending on their physiochemical characteristics, drug molecules on entrapment can modulate and reorganize structural features of cubosomes and hexosomes. Therefore, it is important to assess the effect of guest molecules on broader biophysical characteristics of non-lamellar liquid crystalline nanoparticles, since drug-induced architectural, morphological, and size modifications can affect the biological performance of cubosomes and hexosomes. Methods: We report on alterations in morphological, structural, and size characteristics of nanodispersions composed from binary mixtures of glycerol monooleate and vitamin E on thymoquinone (a molecule with wide therapeutic potentials) loading. Results: Thymoquinone loading was associated with a slight increase in the mean hydrodynamic nanoparticle size and led to structural transitions from an internal biphasic feature of coexisting inverse cubic Fd3m and hexagonal (H2) phases to an internal inverse cubic Fd3m phase (micellar cubosomes) or an internal inverse micellar (L2) phase (emulsified microemulsions, EMEs). We further report on the presence of &ldquo, flower-like&rdquo, vesicular populations in both native and drug-loaded nanodispersions. Conclusions: These nanodispersions have the potential to accommodate thymoquinone and may be considered as promising platforms for the development of thymoquinone nanomedicines.

pH-responsive lipid nanocarriers have the potential to selectively target the acidic extracellular pH environment of cancer tissues and may further improve the efficacy of chemotherapeutics by minimizing their toxic side-effects. Here, we present the design and characterization of pH-sensitive nano-self-assemblies of the poorly water-soluble anticancer drug 2-hydroxyoleic acid (2OHOA) with glycerol monooleate (GMO). pH- triggered nanostructural transformations from 2OHOA/GMO nanoparticles with an internal inverse hexagonal structure (hexosomes) at pH around 2.0–3.0, via nanocarriers with an internal inverse bicontinuous cubic structure (cubosomes) at pH 2.0–4.5, to vesicles at pH 4.5–7.4 were observed with synchrotron small-angle X-ray scattering, and cryogenic transmission electron microscopy. ζ-potential measurements highlight that the pH-driven deprotonation of the carboxylic group of 2OHOA, and the resulting charge-repulsions at the lipid–water interface account for these nanostructural alterations. The study provides detailed insight into the pH-dependent self-assembly of 2OHOA with GMO in excess buffer at physiologically relevant pH values, and discusses the effects of pH alterations on modulating their nanostructure. The results may guide the further development of pH-responsive anticancer nanocarriers for the targeted delivery of chemotherapeutics to the local microenvironment of tumor cells.

Improvement of pain management strategies after arthroscopic surgery by multimodal analgesia may include the use of long-acting amide local anesthetics. Among these anesthetics, the low molecular weight local anesthetic agent bupivacaine (BUP) is attractive for use in postoperative pain management. However, it has a relatively short duration of action and imposes a higher risk of systemic toxicity at relatively large bolus doses. Bupivacaine encapsulation in lipid-based delivery systems is an attractive strategy for prolonging its local anaesthetic effect and reducing the associated undesirable systemic side effects. Here, we discuss the potential development of liquid crystalline nanocarriers for delivering BUP by using a binary lipid mixture of citrem and soy phosphatidylcholine (SPC) at different weight ratios. The produced safe-by-design family of citrem/SPC nanoparticles is attractive for use in the development of nanocarriers owing to the previously reported hemocompatibility. BUP encapsulation efficiency (EE), depending on the lipid composition, was in the range of 65–77%. In this study, nanoparticle tracking analysis (NTA) and synchrotron small-angle X-ray scattering (SAXS) were employed to gain insight into the effect of BUP solubilization and lipid composition on the size and structural characteristics of the produced citrem/SPC nanodispersions. BUP loading led to a slight change in the mean sizes (diameters) and size distributions of citrem/SPC nanoparticles. However, we found that BUP accommodation into the self-assembled interiors of nanoparticles, triggers significant structural alterations in BUP concentration- and lipid composition-dependent manners, which involve vesicle–cubosome and vesicle–hexosome transitions. The structural tunability of citrem/SPC nanoparticles and the implications for potential applications in intra-articular BUP delivery are discussed.

There is a need for bio-predictive and well-characterized in vitro release models in the development of intra-articular depot formulations. Here, the commercially-available Scissor system, a membrane-based two-compartment release testing instrument, was applied to characterize the transport and release of the drug diclofenac employing conditions intended to mimic transport in the synovial joint. The fate of hyaluronic acid and human serum albumin, the main bio-relevant components incorporated in the system, was investigated. A promising strategy for providing sustained drug release upon intra-articular administration are lipid-based preformulations forming non-lamellar liquid crystalline phases in situ. The usefulness of the Scissor system for investigating the initial drug release from these delivery systems was evaluated. The diclofenac release rate upon injection of an aqueous solution was influenced by the composition of the injection site matrix, i.e. the hyaluronic acid content. Hyaluronic acid and human serum albumin were found to escape from the donor compartment into the acceptor medium through the employed polycarbonate membrane. Sustained diclofenac release was obtained by formation of highly viscous liquid crystalline phases upon injection of the lipid-based preformulations. The study shows the feasibility and potential of the Scissor system for testing initial release of intra-articular depot formulations of low-molecular-weight drug compounds.

Lung cancer (LC) is the leading cause of cancer-related deaths, responsible for approximately 18.4% of all cancer mortalities in both sexes combined. The use of systemic therapeutics remains one of the primary treatments for LC. However, the therapeutic efficacy of these agents is limited due to their associated severe adverse effects, systemic toxicity and poor selectivity. In contrast, pulmonary delivery of anticancer drugs can provide many advantages over conventional routes. The inhalation route allows the direct delivery of chemotherapeutic agents to the target LC cells with high local concertation that may enhance the antitumor activity and lead to lower dosing and fewer systemic toxicities. Nevertheless, this route faces by many physiological barriers and technological challenges that may significantly affect the lung deposition, retention, and efficacy of anticancer drugs. The use of lipid-based nanocarriers could potentially overcome these problems owing to their unique characteristics, such as the ability to entrap drugs with various physicochemical properties, and their enhanced permeability and retention (EPR) effect for passive targeting. Besides, they can be functionalized with different targeting moieties for active targeting. This article highlights the physiological, physicochemical, and technological considerations for efficient inhalable anticancer delivery using lipid-based nanocarriers and their cutting-edge role in LC treatment.

This article introduces a simple microfluidic device that can be combined with synchrotron small-angle X-ray scattering (SAXS) for monitoring dynamic structural transitions. The microfluidic device is a thiol-ene-based system equipped with 125 μm-thick polystyrene windows, which are suitable for X-ray experiments. The device was prepared by soft lithography using elastomeric molds followed by a simple UV-initiated curing step to polymerize the chip material and simultaneously seal the device with the polystyrene windows. The microfluidic device was successfully used to explore the dynamics of the structural transitions of phytantriol/dioleoylphosphatidylglycerol-based cubosomes on exposure to a buffer containing calcium ions. The resulting SAXS data were resolved in the time frame between 0.5 and 5.5 s, and a calcium-triggered structural transition from an internal inverted-type cubic phase of symmetry Im3m to an internal inverted-type cubic phase of symmetry Pn3m was detected. The combination of microfluidics with X-ray techniques opens the door to the investigation of early dynamic structural transitions, which is not possible with conventional techniques such as glass flow cells. The combination of microfluidics with X-ray techniques can be used for investigating protein unfolding, for monitoring the formation of nanoparticles in real time, and for other biomedical and pharmaceutical investigations. A combination of microfluidics with X-ray techniques has been used to perform dynamic structural studies on nanoparticulate formulations. (Less)

Non-lamellar liquid crystalline aqueous nanodispersions, known also as ISAsomes (internally self-assembled 'somes' or nanoparticles), are gaining increasing interest in drug solubilisation and bio-imaging, but they often exhibit poor hemocompatibility and induce cytotoxicity. This limits their applications in intravenous drug delivery and targeting. Using a binary mixture of citrem and soy phosphatidylcholine (SPC) at different weight ratios, we describe a library of colloidally stable aqueous and hemocompatible nanodispersions of diverse nanoarchitectures (internal self-assembled nanostructures). This engineered library is structurally stable in human plasma as well as being hemocompatible (non-hemolytic, and poor activator of the complement system). By varying citrem to lipid weight ratio, the nanodispersion susceptibility to macrophage uptake could also be modulated. Finally, the formation of nanodispersions comprising internally V2 (inverse bicontinuous cubic) and H2 (inverse hexagonal) nanoarchitectures was achieved without the use of an organic solvent, a secondary emulsifier, or high-energy input. The tunable binary citrem/SPC nanoplatform holds promise for future development of hemocompatible and immune-safe nanopharmaceuticals.

Water-in-oil (W/O) microemulsions based on either refined olive oil (ROO) or sunflower oil (SO), distilled monoglycerides (DMG), and ethanol were used as nisin carriers in order to ensure its effectiveness as a biopreservative. This work presents experimental evidence on the effects of ethanol concentration, hydration, the nature of oil, and the addition of nisin on the nanostructure of the proposed inverse microemulsions as revealed by electrical conductivity measurements, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and electron paramagnetic resonance (EPR) spectroscopy. Modeling of representative SAXS profiles was applied to gain further insight into the effects of ethanol and solubilized water content on the inverse swollen micelles' size and morphology. With increasing ethanol content, the overall size of the inverse micelles decreased, whereas hydration resulted in an increase in the micellar size due to the penetration of water into the hydrophilic core of the inverse swollen micelles (hydration-induced swelling behavior). The dynamic properties of the surfactant monolayer were also affected by the nature of the used vegetable oil, the ethanol content, and the presence of the bioactive molecule, as evidenced by EPR spin probing experiments. According to simulation on the experimental spectra, two populations of spin probes at different polarities were revealed. The antimicrobial effect of the encapsulated nisin was evaluated using the well diffusion assay (WDA) technique against Lactococccus lactis. It was found that this encapsulated bacteriocin induced an inhibition of the microorganism growth. The effect was more pronounced at higher ethanol concentrations, but no significant difference was observed between the two used vegetable oils (ROO and SO).

In the field of drug delivery to the articular cartilage, it is advantageous to apply artificial tissue models as surrogates of cartilage for investigating drug transport and release properties. In this study, artificial cartilage models consisting of 0.5% (w/v) agarose gel containing 0.5% (w/v) chondroitin sulfate or 0.5% (w/v) hyaluronic acid were developed, and their rheological and morphological properties were characterized. UV imaging was utilized to quantify the transport properties of the following four model compounds in the agarose gel and in the developed artificial cartilage models: H-Ala-β-naphthylamide, H-Lys-Lys-β-naphthylamide, lysozyme, and α-lactalbumin. The obtained results showed that the incorporation of the polyelectrolytes chondroitin sulfate or hyaluronic acid into agarose gel induced a significant reduction in the apparent diffusivities of the cationic model compounds as compared to the pure agarose gel. The decrease in apparent diffusivity of the cationic compounds was not caused by a change in the gel structure since a similar reduction in apparent diffusivity was not observed for the net negatively charged protein α-lactalbumin. The apparent diffusivity of the cationic compounds in the negatively charged hydrogels was highly dependent on the ionic strength, pointing out the importance of electrostatic interactions between the diffusant and the polyelectrolytes. Solution based affinity studies between the model compounds and the two investigated polyelectrolytes further confirmed the electrostatic nature of their interactions. The results obtained from the UV imaging diffusion studies are important for understanding the effect of drug physicochemical properties on the transport in articular cartilage. The extracted information may be useful in the development of hydrogels for in vitro release testing having features resembling the articular cartilage.

Hexosomes are nanoparticles enveloping inverted discontinuous hexagonal (HII) liquid crystalline phases that offer exceptional physicochemical characteristics for drug encapsulation and targeted drug delivery. However, the fate of subcutaneously injected hexosomes has rarely been investigated. Here, we have followed the lymphatic fate of a technetium-99 m [99mTc]-labeled hexosome preparation on subcutaneous injection into the rat footpads with single-photon emission computed tomography (SPECT) in combination with CT. Hexosomes were composed of phytantriol and oleic acid and sterically stabilized with Pluronic F127. High tracer loading efficiency was achieved by chelating [99mTc]-pertechnetate to 1,12-diamino-3,6,9-triazadodecane-oleic acid conjugates incorporated within the ordered interior nanocompartments of hexosomes. The preparation was characterized by synchrotron small angle X-ray scattering, cryogenic transmission electron microscopy and nanoparticle tracking analysis. On footpad injection, hexosomes rapidly drained into the lymphatic microvessels and distributed not only to the sentinel (popliteal) lymph node, but also to wider lymph nodes (inguinal and iliac nodes) situated along the pathway of the lymph drainage. The observed hexosome drainage kinetics from the injection site and lymphatic distribution pattern is in line with previously established poly(ethylene oxide) configuration attributes of Pluronic F127 in minimizing nanoparticle aggregation at the injection site, and interaction with the ground substance of interstitium, without interfering with macrophage uptake. Accordingly, we discuss the potential utility of Pluronic F127-stabilized hexosomes for lymphatic targeting and future development of simple, yet effective multifunctional lymphotropic agents and theranostics.

The attractiveness of new omega-3 (ω-3) polyunsaturated fatty acid (PUFA) monoglycerides (MAGs) lies in the amphiphilic nature and the beneficial health effects as PUFA precursors in various disorders including cancer, pulmonary hypertension, and inflammatory diseases. For exploring the potential therapeutic applications of these new amphiphilic lipids, particularly as main lipid constituents in the development of nanocarriers for delivery of drugs and PUFAs, it is of paramount importance to gain insight into their self-assembly behavior on exposure to excess water. This work describes the structural characteristics of self-assemblies based on two newly synthesized MAGs, namely docosahexaenoic acid (MAG-DHA) and docosapentaenoic acid (MAG-DPA) monoglycerides, on exposure to excess water. We found that both lipids tend to form a dominant inverse hexagonal (H2) phase in excess water at 25 °C and a temperature-triggered structural transition to an inverse micellar solution (L2 phase) is detected similar to that recently reported (A. Yaghmur et al., Langmuir, 2017, 33, 14045-14057) for eicosapentaenoic acid monoglyceride (MAG-EPA). An experimental SAXS structural evaluation study on the temperature-dependent behavior of these new monoglycerides is provided, and the effects of unsaturation degree and fatty acyl chain length on the self-assembled structural features in excess water and on the H2-L2 phase transition temperature are discussed. In addition, hexosomes stabilized by using the triblock copolymer F127 and the food-grade emulsifier citrem were investigated to gain insights into the effects of stabilizer and temperature on the internal nanostructure. These nanoparticles are attractive for use in the development of nanocarriers for delivering drugs and/or nutritional compounds as the beneficial health effects of ω-3 PUFA monoglycerides can be combined with those of loaded therapeutic agents or nutraceuticals.

Cisplatin ( cis-diamminedichloroplatinum(II)) is among the most potent cytotoxic agents used in cancer chemotherapy. The encapsulation of cisplatin in lipid-based drug carriers has been challenging owing to its low solubility in both aqueous and lipid phases. Here, we investigated cisplatin encapsulation in nonlamellar liquid-crystalline (LC) nanodispersions formed from a ternary mixture of phytantriol (PHYT), vitamin E (Vit E), and an anionic phospholipid [either phosphatidylglycerol (DSPG) or phosphatidylserine (DPPS)]. We show an increase in cisplatin encapsulation efficiency (EE) in nanodispersions containing 1.5-4 wt % phospholipid. The EE was highest in DPPS-containing nanodispersions (53-98%) compared to DSPG-containing counterparts (25-40%) under similar experimental conditions. Through structural and morphological characterizations involving synchrotron small-angle X-ray scattering and cryogenic transmission electron microscopy, we further show that varying the phospholipid content of cisplatin-free nanodispersions triggers an internal phase transition from a neat hexagonal (H

The delivery of poorly water-soluble antimicrobial peptides (AMPs) that are sensitive to degradation is a major challenge in the pharmaceutical field. In this study, we design and characterize a pH-sensitive nanocarrier with the potential for delivery of AMPs and their protection from degradation. These nanobiointerfaces are prepared through the self-assembly of oleic acid (OA) with the human cathelicidin LL-37 in excess water. Advanced experimental methods including synchrotron small angle X-ray scattering, cryogenic transmission electron microscopy, and dynamic light scattering were used to characterize the OA/LL-37 self-assemblies and their structural alterations in response to changes in pH and composition. Experimental findings reveal colloidal transformations from normal emulsions via micellar cubosomes and hexosomes to vesicles upon increasing the pH from 6.0 to 8.0 at a LL-37 content around 10 wt% relative to OA. Increasing the LL-37 content to 30 wt% in OA led to diminishing of micellar cubosomes and hexosomes in this narrow pH range, favoring the formation of micelles and vesicles of various shapes and sizes. Upon increasing the pH, with the strongest effect around pH 7.5, charge repulsions among the gradually deprotonating carboxylic groups of OA modified the geometric packing of the molecules, significantly affecting the nanostructure. These detailed insights into the formation of this unique family of nanobiointerfaces and their tunable structural features may contribute to the rational design of pH-responsive antimicrobial systems for the delivery of peptides, particularly poorly water-soluble AMPs.

Recent studies demonstrated the potential therapeutic use of newly synthesized omega-3 (ω-3) polyunsaturated fatty acid (PUFA) monoglycerides owing to their beneficial health effects in various disorders including cancer and inflammation diseases. To date, the research was mainly focused on exploring the biological effects of these functional lipids. However, to the best of our knowledge, there is no report on the hydration-mediated self assembly of these lipids that leads to the formation of nanostructures, which are attractive for use as vehicles for the delivery of drugs and functional foods. In the present study, we investigated the temperature-composition phase behaviour of eicosapentaenoic acid monoglyceride (MAG-EPA), which is one of the most investigated ω-3 PUFA monoglycerides, during a heating–cooling cycle in the temperature range of 5–60 °C. Experimental synchrotron small-angle X-ray scattering (SAXS) evidence on the formation of a dominant inverse hexagonal (H2) lyotropic liquid crystalline p...

Lipid exchange among citrem nanoparticles and an ethanol micellar solution containing soy phosphatidylcholine was investigated in situ by coupling small angle X-ray scattering with a microfluidic device. The produced soy phosphatidylcholine/citrem nanoparticles have great potential in the development of hemocompatible nanocarriers for drug delivery.

Triglycerides in food products such as mayonnaise are a vital source of energy and essential for a complete and healthy diet. Their molecular structures consist of a glycerol backbone esterified with fatty acids on the two outer and the middle positions. During the digestion of triglycerides by pancreatic lipase in the small intestine, the ester bonds on the outer positions are hydrolyzed,leading to amphiphilic monoglycerides and free fatty acids as products. Depending on their chain length and degree of saturation, these products can self-assemble into a variety of structures in excess water. In this study, we report the discovery of highly ordered nanostructures inside of the mayonnaise emulsion droplets during in vitro digestion of mayonnaise under simulated in vivo conditions using timeresolved synchrotron small-angle X-ray scattering. The formation of these structures is most likely linked to their function as a carrier and controlled release system for food nutrients, especially poorly water-soluble components, in the aqueous milieu of the digestive tract. This detailed understanding of nanostructure formation during the digestion of triglyceride-containing food products such as mayonnaise may have fundamental implications for the development of foods with improved nutritional and functional properties. Triglycerides in food products such as mayonnaise are a vital source of energy and essential for a complete and healthy diet. Their molecular structures consist of a glycerol backbone esterified with fatty acids on the two outer and the middle positions. During the digestion of triglycerides by pancreatic lipase in the small intestine, the ester bonds on the outer positions are hydrolyzed, leading to amphiphilic monoglycerides and free fatty acids as products. Depending on their chain length and degree of saturation, these products can self-assemble into a variety of structures in excess water. In this study, we report the discovery of highly ordered nanostructures inside of the mayonnaise emulsion droplets during in vitro digestion of mayonnaise under simulated in vivo conditions using timeresolved synchrotron small-angle X-ray scattering. The formation of these structures is most likely linked to their function as a carrier and controlled release system for food nutrients, especially poorly water-soluble components, in the aqueous milieu of the digestive tract. This detailed understanding of nanostructure formation during the digestion of triglyceride-containing food products such as mayonnaise may have fundamental mplications for the development of foods with improved nutritional and functional properties.

Poly(ethylene glycol)-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamines (DSPE-mPEGs) are a family of amphiphilic lipopolymers attractive in formulating injectable long-circulating nanoparticulate drug formulations. In addition to long circulating liposomes, there is an interest in developing injectable long-circulating drug nanocarriers based on cubosomes and hexosomes by shielding and coating the dispersed particles enveloping well-defined internal nonlamellar liquid crystalline nanostructures with hydrophilic PEG segments. The present study attempts to shed light on the possible PEGylation of these lipidic nonlamellar liquid crystalline particles by using DSPE-mPEGs with three different block lengths of the hydrophilic PEG segment. The effects of lipid composition, PEG chain length, and temperature on the morphology and internal nanostructure of these self-assembled lipidic aqueous dispersions based on phytantriol (PHYT) were investigated by means of synchrotron small-angle X-ray scattering and Transmission Electron Cryo-Microscopy. The results suggest that the used lipopolymers are incorporated into the water-PHYT interfacial area and induce a significant effect on the internal nanostructures of the dispersed submicrometer-sized particles. The hydrophilic domains of the internal liquid crystalline nanostructures of these aqueous dispersions are functionalized, i.e., the hydrophilic nanochannels of the internal cubic Pn3m and Im3m phases are significantly enlarged in the presence of relatively small amounts of the used DSPE-mPEGs. It is evident that the partial replacement of PHYT by these PEGylated lipids could be an attractive approach for the surface modification of cubosomal and hexosomal particles. These PEGylated nanocarriers are particularly attractive in designing injectable cubosomal and hexosomal nanocarriers for loading drugs and/or imaging probes.

Identification of new vaccine adjuvants with immunopotentiating properties commonly involves in vitro evaluations of candidate compounds for their ability to stimulate cells of the immune system. Subsequent elaborate experiments are then performed on only the positive candidates. Here we show how this strategy may miss good candidates due to context-dependent supramolecular characteristics of the candidate compounds, since both a specific molecular structure and the correct presentation of specific parts of the compounds are required for successful stimulation of the cells. Nevertheless, the supramolecular structure is rarely evaluated although changes in this structure may have a drastic impact on the presentation of the compounds to the cells. Synthetic analogues of the mycobacterial cell wall lipid monomycoloyl glycerol (MMG) possess immunopotentiating properties, but their biophysical characteristics are largely unresolved and the structural features determining their immunoactivating properties have been poorly explored. In the present study, we demonstrate that the immunostimulatory activity in vitro correlates with the supramolecular characteristics of the self-assembled MMG nanostructures. Thus, a series of MMG analogues displaying different stereochemistry in the hydrophobic moiety and the polar headgroup were designed and synthesized with different alkyl chain lengths. Stimulation of human monocyte-derived dendritic cells in vitro was clearly dependent on the stereochemistry of the hydrophobic part and on the alkyl chain length but not on the stereochemistry of the hydrophilic glycerol moiety. Small-angle X-ray scattering (SAXS) analysis showed that the immunoactivating analogues self-assembled into lamellar phases whereas the biologically inert analogues adopted inverse hexagonal phases. Langmuir monolayers confirmed that analogues with opposite lipid acid configurations displayed different packing modes. These data demonstrate that the biophysical properties and the lipid molecular structure are major determinants for the ability of the MMG analogues to activate antigen-presenting cells. Our findings emphasize the importance of investigating the biophysical and structural properties when assessing the effect of adjuvants in vitro.

A microfluidic platform combined with synchrotron small-angle X-ray scattering (SAXS) was used for monitoring the continuous production of multilamellar vesicles (MLVs). Their production was fast and started to evolve within less than 0.43 s of contact between the lipids and the aqueous phase. To obtain nanoparticles with a narrow size distribution, it was important to use a modified hydrodynamic flow focusing (HFF) microfluidic device with narrower microchannels than those normally used for SAXS experiments. Monodispersed MLVs as small as 160 nm in size, with a polydispersity index (PDI) of approximately 0.15 were achieved. The nanoparticles produced were smaller and had a narrower size distribution than those obtained via conventional bulk mixing methods. This microfluidic platform therefore has a great potential for the continuous production of monodispersed NPs.

Synthetic analogues of the cell-wall lipid monomycoloyl glycerol (MMG) are promising as next-generation vaccine adjuvants. In the present study, the thermotropic phase behavior of an array of synthetic MMG analogues was examined by using simultaneous small- and wide-angle X-ray scattering under excess water conditions. The MMG analogues differed in the alkyl chain lengths and in the stereochemistry of the polar glycerol headgroup or of the lipid tails (native-like versus alternative compounds). All MMG analogues formed poorly hydrated lamellar phases at low temperatures and inverse hexagonal (H2) phases at higher temperatures prior to melting. MMG analogues with a native-like lipid acid configuration self-assembled into noninterdigitated bilayers whereas the analogues displaying an alternative lipid acid configuration formed interdigitated bilayers in a subgel (Lc′) state. This is in contrast to previously described interdigitated phases for other lipids, which are usually in a gel (Lβ) state. All investi...

We present the first report on the effects of hydrostatic pressure on colloidally stabilized lipid nanoparticles enveloping inverse nonlamellar self-assemblies in their interiors. These internal self-assemblies were systematically tuned into bicontinuous cubic (Pn3m and Im3m), micellar cubic (Fd3m), hexagonal (H2), and inverse micellar (L2) phases by regulating the lipid/oil ratio as the hydrostatic pressure was varied from atmospheric pressure to 1200 bar and back to atmospheric pressure. The effects of pressure on these lipid nanoparticles were compared with those on their equilibrium bulk, nondispersed counterparts, namely, inverse nonlamellar liquid-crystalline phases and micellar solutions under excess-water conditions, using the synchrotron small-angle X-ray scattering (SAXS) technique. In the applied pressure range, induced phase transitions were observed solely in fully hydrated bulk samples, whereas the internal self-assemblies of the corresponding lipid nanoparticles displayed only pressure-modulated single phases. Interestingly, both the lattice parameters and the linear pressure expansion coefficients were larger for the self-assemblies enveloped inside the lipid nanoparticles as compared to the bulk states. This behavior can, in part, be attributed to enhanced lipid layer undulations in the lipid particles in addition to induced swelling effects in the presence of the triblock copolymer F127. The bicontinuous cubic phases both in the bulk state and inside lipid cubosome nanoparticles swell on compression, even as both keep swelling further upon decompression at relatively high pressures before shrinking again at ambient pressures. The pressure dependence of the phases is also modulated by the concentration of the solubilized oil (tetradecane). These studies demonstrate the tolerance of lipid nanoparticles [cubosomes, hexosomes, micellar cubosomes, and emulsified microemulsions (EMEs)] for high pressures, confirming their robustness for various technological applications.

Designing efficient colloidal systems for the delivery of membrane active antimicrobial peptides requires in-depth understanding of their structural and morphological characteristics. Using dispersions of inverted type bicontinuous cubic phase (cubosomes), we examine the effect of integrating the amphiphilic peptide LL-37 at different concentrations on the self-assembled structure and evaluate its bactericidal ability against Escherichia coli. Small-angle X-ray scattering, dynamic light scattering, and cryogenic transmission electron microscopy show that LL-37 integrates into the bicontinuous cubic structure, inducing colloidal transformations to sponge and lamellar phases and micelles in a concentration-dependent manner. These investigations, together with in vitro evaluation studies using a clinically relevant bacterial strain, established the composition-nanostructure-activity relationship that can guide the design of new nanocarriers for antimicrobial peptides and may provide essential knowledge on the mechanisms underlying the bacterial membrane disruption with peptide-loaded nanostructures.

The mycobacterial cell-wall lipid monomycoloyl glycerol (MMG) is a potent immunostimulator, and cationic liposomes composed of a shorter synthetic analogue (MMG-1) and dimethyldioctadecylammonium (DDA) bromide represent a promising adjuvant that induces strong antigen-specific Th1 and Th17 responses. In the present study, we investigated the supramolecular structure and in vivo adjuvant activity of dispersions based on binary mixtures of DDA and an array of synthetic MMG-1 analogues (MMG-2/3/5/6) displaying longer (MMG-2) or shorter (MMG-3) alkyl chain lengths, or variations in stereochemistry of the polar headgroup (MMG-5) or of the hydrophobic moiety (MMG-6). Synchrotron small-angle X-ray scattering experiments and cryo transmission electron microscopy revealed that DDA:MMG-1/2/5/6 dispersions consisted of unilamellar and multilamellar vesicles (ULVs/MLVs), whereas a coexistence of both ULVs and hexosomes was observed for DDA:MMG-3, depending on the DDA:MMG molar ratio. The studies also showed that ULVs were formed, regardless of the structural characteristics of the neat MMG analogues in excess buffer [lamellar (MMG-1/2/5) or inverse hexagonal (MMG-3/6) phases]. Immunization of mice with a chlamydia antigen surface-adsorbed to DDA:MMG-1/3/6 dispersions revealed that all tested adjuvants were immunoactive and induced strong Th1 and Th17 responses with a potential for a central effector memory profile. The MMG-1 and MMG-6 analogues were equally immunoactive in vivo upon incorporation into DDA liposomes, despite the reported highly different immunostimulatory properties of the neat analogues in vitro, which were attributed to the different nanostructural characteristics. This clearly demonstrates that optimal formulation and delivery of MMG analogues to the immune system is of major importance and challenges the use of in vitro screening assays with nondispersed compounds to identify potential new vaccine adjuvants.

The present study was designed to evaluate the effect of the negatively charged food-grade emulsifier citrem on the internal nanostructures of oil-free and oil-loaded aqueous dispersions of phytantriol (PHYT) and glyceryl monooleate (GMO). To our knowledge, this is the first report in the literature on the utilization of this charged stabilizing agent in the formation of aqueous dispersions consisting of well-ordered interiors (either inverted-type hexagonal (H(2)) phases or inverted-type microemulsion systems). Synchrotron small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM) were used to characterize the dispersed and the corresponding nondispersed phases of inverted-type nonlamellar liquid-crystalline phases and microemulsions. The results suggest a transition between different internal nanostructures of the aqueous dispersions after the addition of the stabilizer. In addition to the main function of citrem as a stabilizer that adheres to the surface of the dispersed particles, it has a significant impact on the internal nanostructures, which is governed by the following factors: (1) its penetration between the hydrophobic tails of the lipid molecules and (2) its degree of incorporation into the lipid-water interfacial area. In the presence of citrem, the formation of aqueous dispersions with functionalized hydrophilic domains by the enlargement of the hydrophilic nanochannels of the internal H(2) phase in hexosomes and the hydrophilic core of the L(2) phase in emulsified microemulsions (EMEs) could be particularly attractive for solubilizing and controlling the release of positively charged drugs.

In the present study, synchrotron small-angle X-ray scattering (SAXS) combined with a UV light source (in situ SAXS-UV irradiation) was used to determine the structure response of gold nanoparticle (NP)-loaded vesicles. The investigated system consisted of multilamellar vesicles of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) containing hydrophilic gold NPs with a size of 40 A. Our results indicate drastic optothermically induced structural changes in these gold NP-loaded aqueous dispersions, which are accompanied by strong alterations in the phospholipid membrane permeability. A structural mechanism from well-ordered multilamellar vesicles in the fluid lamellar (Lα) phase to an inverted type hexagonal liquid crystalline (H2) phase via the formation of an intermediate phase of uncorrelated fluid bilayers is proposed. In general, these investigations are also crucial for the understanding of the potential of gold NP-loaded model phospholipid systems as efficient drug nanocarriers aiming to impro...

Proteolytically stable α-peptide/β-peptoid peptidomimetics constitute promising cell-penetrating carrier candidates exhibiting superior cellular uptake as compared to commonly used cell-penetrating peptides (CPPs). The aim of the present study was to explore the potential of these peptidomimetics for delivery of small interfering RNA (siRNA) to the cytosol by incorporation of a palmitoylated peptidomimetic construct into a cationic lipid-based nanocarrier system. The optimal construct was selected on the basis of the effect of palmitoylation and the influence of the length of the peptidomimetic on the interaction with model membranes and the cellular uptake. Palmitoylation enhanced the peptidomimetic adsorption to supported lipid bilayers as studied by ellipsometry. However, both palmitoylation and increased peptidomimetic chain length were found to be beneficial in the cellular uptake studies using fluorophore-labeled analogues. Thus, the longer palmitoylated peptidomimetic was chosen for further formulation of siRNA in a dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) nanocarrier system, and the resulting nanoparticles were found to mediate efficient gene silencing in vitro. Cryo-transmission electron microscopy (cryo-TEM) revealed multilamellar, onion-like spherical vesicles, and small-angle X-ray scattering (SAXS) analysis confirmed that the majority of the lipids in the nanocarriers were organized in lamellar structures, yet coexisted with a hexagonal phase, which is important for efficient nanocarrier-mediated endosomal escape of siRNA ensuring cytosolic delivery. The present work is a proof-of-concept for the use of α-peptides/β-peptoid peptidomimetics in an efficient delivery system that may be more generally exploited for the intracellular delivery of biomacromolecular drugs.

In order to improve patient's benefit and safety, comprehensive regulatory guidelines on specificities of Non-Biological Complex Drugs (NBCDs), such as doxorubicin-encapsulated liposomes, and their follow-on versions are needed. Here, we compare Doxil® and its European analog Caelyx® with the two follow-on products DOXOrubicin (approved by the US Food and Drug Administration) and SinaDoxosome (produced in Iran) by cryogenic transmission electron microscopy, dynamic light scattering and Nanoparticle Tracking Analysis, and assess their potential in activating the complement system in human sera. We found subtle physicochemical differences between the tested liposomal products and even between the tested batches of Doxil® and Caelyx®. Notably, these included differences in vesicular population aspect ratios and particle number. Among the tested products, only SinaDoxosome, in addition to the presence of unilamellar vesicles with entrapped doxorubicin crystals, contained empty circular disks. Differences were also found in complement responses, which may be related to some morphological differences. This study has demonstrated an integrated biophysical and immunological toolbox for improved analysis and detection of physical differences among vesicular populations that may modulate their clinical performance. Combined, these approaches may help better product selection for infusion to the patients as well as for improved design and characterization of future vesicular NBCDs with enhanced clinical performance and safety.

Liposomes embedded with gold nanoparticles show light-triggered contents release. We investigated the mechanism of the light-induced changes and functionality of the light-induced release in the cells. The real time small angle X-ray scattering (SAXS) analysis revealed time-dependent phase transitions in distearoylphosphatidylcholine (DSPC)/dipalmitoylphosphatidylcholine (DPPC) liposomes upon heating. Similar changes were observed when gold nanoparticle-embedded liposomes were exposed to the UV light: gold nanoparticles absorb light energy and transfer it to heat, thereby causing lipid phase transition from gel phase to rippled phase, and further to fluid phase. Without UV light exposure the gold nanoparticles did not affect the liposomal bilayer periodicity. The light-triggered release of hydrophilic fluorescent probe (calcein) from the gold nanoparticle-loaded liposomes was demonstrated with fluorescence-activated cell sorting after liposome internalization into the ARPE-19 cells. The liposome formulations did not decrease the cell viability in vitro. In conclusion, the light-triggered release from the liposomes is functional in the cells, and the release is triggered by thermal phase changes in the lipid bilayers.

This work details the in situ dynamical behaviour of local anaesthetic bupivacaine (BUP)-loaded L2 and H2 phase precursors (preformulations of BUP) upon rapid exposure to phosphate buffer under physiological conditions (pH 7.4, 37 °C). The hydration-induced structural transitions are characterized by time-resolved synchrotron SAXS.