Factors Affecting Extracellular Vesicles Based Drug Delivery Systems.

Extracellular vesicles (EVs) play major roles in intracellular communication and participate in several biological functions in both normal and pathological conditions. Surface modification of EVs via various ligands, such as proteins, peptides, or aptamers, offers great potential as a means to achieve targeted delivery of therapeutic cargo, i.e., in drug delivery systems (DDS). This review summarizes recent studies pertaining to the development of EV-based DDS and its advantages compared to conventional nano drug delivery systems (NDDS). First, we compare liposomes and exosomes in terms of their distinct benefits in DDS. Second, we analyze what to consider for achieving better isolation, yield, and characterization of EVs for DDS. Third, we summarize different methods for the modification of surface of EVs, followed by discussion about different origins of EVs and their role in developing DDS. Next, several major methods for encapsulating therapeutic cargos in EVs have been summarized. Finally, we discuss key challenges and pose important open questions which warrant further investigation to develop more effective EV-based DDS.

Sorting sub-150-nm liposomes of distinct sizes by DNA-brick-assisted centrifugation.

In cells, myriad membrane-interacting proteins generate and maintain curved membrane domains with radii of curvature around or below 50 nm. To understand how such highly curved membranes modulate specific protein functions, and vice versa, it is imperative to use small liposomes with precisely defined attributes as model membranes. Here, we report a versatile and scalable sorting technique that uses cholesterol-modified DNA 'nanobricks' to differentiate hetero-sized liposomes by their buoyant densities. This method separates milligrams of liposomes, regardless of their origins and chemical compositions, into six to eight homogeneous populations with mean diameters of 30-130 nm. We show that these uniform, leak-resistant liposomes serve as ideal substrates to study, with an unprecedented resolution, how membrane curvature influences peripheral (ATG3) and integral (SNARE) membrane protein activities. Compared with conventional methods, our sorting technique represents a streamlined process to achieve superior liposome size uniformity, which benefits research in membrane biology and the development of liposomal drug-delivery systems.

Effect of drug molecular weight on niosomes size and encapsulation efficiency.

Encapsulation into nanocarriers, such as niosomes, is a promising way to protect them from degradation, and allow controll and target delivery of bioactive compounds. For biotechnological applications, a tight control of particle size with acceptable encapsulation efficiencies (EE) is a technological challenge, especially for hydrophilic compounds due to its capability to diffuse across biological barriers. Niosomes formulated with mixture of surfactants represent promising nanocarriers due to the advantages of non-ionic surfactants, such as low cost, versatility and enhanced physico-chemical properties. In this work, the effect of both, composition of the hydrating solution and molecular weight of the loaded compound, on the particle size and EE of niosomes prepared by using the thin film hydration method was studied. Particularly, mili-Q water, glycerol solution and PEG-400 solution were tested for niosomes formulated with Span®80-Tween®80 with/without dodecanol as membrane stabilizer. It was found that particle size highly depends on hydration media composition and an interaction with compound MW could exist. Larger vesicles results in an increase in EE, which could be purely related with physical aspects such as vesicle loading volume capacity. The effect of hydration solution composition could be related with their ability to change the bilayer packing and physical properties, as observed by differential scanning calorimetry. Finally, it was possible to compare the suitability of dialysis and gel filtration as purification methods, demonstrating that gel filtration is not an adequate purification method when viscous solutions are used, since they could affect the particle vesicles retention and hence EE measurements would be misrepresentative.

Measurement of Bile Acids as a Marker of the Functionality of iPSC-Derived Hepatocytes.

During the process of differentiation from induced pluripotent stem cells (iPSCs) to hepatocyte-like cells it is crucial to monitor the functionality of cells, in order to avoid an overestimation of the level of maturation. To this end, we propose bile acid profiling as a novel approach which is useful in determining the maturation of hepatocyte-like cells. The main advantages of the method are the simplicity and rapidity of test (i.e., single-step sample preparation followed by 3.5-min analysis), as well as the possibility to localize possible enzyme deficiencies by quantifying the accumulation of specific intermediates involved in the synthetic pathways.

Potential of Continuous Manufacturing for Liposomal Drug Products.

Over the last several years, continuous manufacturing of pharmaceuticals has evolved from bulk APIs and solid oral dosages into the more complex realm of biologics. The development of continuous downstream processing techniques has allowed biologics manufacturing to realize the benefits (e.g., improved economics, more consistent quality) that come with continuous processing. If relevant processing techniques and principles are selected, the opportunity arises to develop continuous manufacturing designs for additional pharmaceutical products including liposomal drug formulations. Liposome manufacturing has some inherent aspects that make it favorable for a continuous process. Other aspects such as formulation refinement, materials of construction, and aseptic processing need development, but present an achievable challenge. This paper reviews the current state of continuous manufacturing technology applicable to liposomal drug product manufacturing and an assessment of the challenges and potential of this application.

Model Phospholipid Liposomes to Study the ß-Amyloid-Peptide-Induced Membrane Disruption.

Model phospholipid liposomes have been utilized widely to study the molecular interactions between peptides and membrane bilayers. In the mechanistic study of Alzheimer's disease (AD), disruption of neuronal cell membranes has been considered as a major contribution for the ß-amyloid (Aß) peptides' neurotoxicity. However, clear interpretation of the Aß-induced cellular membrane at high-resolution level is challenging because of the co-existence of multiple pathways. Here we present the generation of simplified model liposome systems that will facilitate the in-depth mechanistic studies. Protocols for the preparation of model liposomes and the characterization of individual membrane disruption effects will be described.

Preparation, Purification, and Use of Fatty Acid-containing Liposomes.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Deterministic Ratchet for Sub-micrometer (Bio)particle Separation.

Resolving the heterogeneity of particle populations by size is important when the particle size is a signature of abnormal biological properties leading to disease. Accessing size heterogeneity in the sub-micrometer regime is particularly important to resolve populations of subcellular species or diagnostically relevant bioparticles. Here, we demonstrate a ratchet migration mechanism capable of separating sub-micrometer sized species by size and apply it to biological particles. The phenomenon is based on a deterministic ratchet effect, is realized in a microfluidic device, and exhibits fast migration allowing separation in tens of seconds. We characterize this phenomenon extensively with the aid of a numerical model allowing one to predict the speed and resolution of this method. We further demonstrate the deterministic ratchet migration with two sub-micrometer sized beads as model system experimentally as well as size-heterogeneous mouse liver mitochondria and liposomes as model system for other organelles. We demonstrate excellent agreement between experimentally observed migration and the numerical model.

Removal of ligand-bound liposomes from cell surfaces by microbubbles exposed to ultrasound.

Gas-filled microbubbles attached to cell surfaces can interact with focused ultrasound to create microstreaming of nearby fluid. We directly observed the ultrasound/microbubble interaction and documented that under certain conditions fluorescent particles that were attached to the surface of live cells could be removed. Fluorescently labeled liposomes that were larger than 500 nm in diameter were attached to the surface of endothelial cells using cRGD targeting to αvß3 integrin. Microbubbles were attached to the surface of the cells through electrostatic interactions. Images taken before and after the ultrasound exposure were compared to document the effects on the liposomes. When exposed to ultrasound with peak negative pressure of 0.8 MPa, single microbubbles and groups of isolated microbubbles were observed to remove targeted liposomes from the cell surface. Liposomes were removed from a region on the cell surface that averaged 33.1 µm in diameter. The maximum distance between a single microbubble and a detached liposome was 34.5 µm. Single microbubbles were shown to be able to remove liposomes from over half the surface of a cell. The distance over which liposomes were removed was significantly dependent on the resting diameter of the microbubble. Clusters of adjoining microbubbles were not seen to remove liposomes. These observations demonstrate that the fluid shear forces generated by the ultrasound/microbubble interaction can remove liposomes from the surfaces of cells over distances that are greater than the diameter of the microbubble.

A dual-channel endoscope for quantitative imaging, monitoring, and triggering of doxorubicin release from liposomes in living mice.

Doxorubicin (Dox) is approved for use in liposomal form for the treatment of ovarian cancer. We previously developed a long-circulating Dox formulation in liposomes containing small amounts of porphyrin-phospholipid, which enables on-demand drug release with near-infrared irradiation. In this study, we present and evaluate a dual-modal, dual-channel light endoscope that allows quantitative reflectance and fluorescence imaging for monitoring of local Dox concentrations in target areas. The endoscope consists of two flexible imaging fibers; one to transmit diagnostic and therapeutic light to the target, and the other to detect fluorescent and reflected light. Thus, the endoscope serves for imaging, for light delivery to trigger drug release, and for monitoring drug concentration kinetics during drug release. We characterized the performance of this endoscope in tissue phantoms and in an in vivo model of ovarian cancer. This study demonstrates the feasibility of non-invasive, quantitative mapping of Dox distribution in vivo via endoscopic imaging.

Sulfated archaeal glycolipid archaeosomes as a safe and effective vaccine adjuvant for induction of cell-mediated immunity.

Archaeosomes are liposomal vesicles composed of ether glycerolipids unique to the domain of Archaea. Unlike conventional ester-linked liposomes, archaeosomes exhibit high stability and possess strong adjuvant and immunostimulatory properties making them an attractive vaccine delivery vehicle. Traditionally comprised of total polar lipids (TPL) or semi-synthetic phospho-glycerolipids of ether-linked isoprenoid phytanyl cores with varied glycol- and amino-head groups, archaeosomes can induce robust and long-lasting humoral and cell-mediated immune responses against antigenic cargo and provide protection in murine models of infectious disease and cancer. However, traditional TPL archaeosome formulations are relatively complex comprising several lipid species. Semi-synthetic archaeosomes tested previously contain a combination of several phospho-glycolipids (negative and neutral charged) to produce a stable, uniform-sized liposome formulation. Moreover, they involve many synthetic steps to arrive at the final desired glycolipid composition. Herein, we present a novel adjuvant formulation comprising a sulfated saccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA). SLA individually or mixed with uncharged glyolipid (lactosylarchaeol, LA) constituted efficacious carrier vesicles for entrapped antigens (ovalbumin or melanoma associated tyrosinase-related protein 2 [TRP-2]) and induction of strong cell-mediated responses in mice and protection against subsequent B16 melanoma tumor challenge. Thus, semi-synthetic sulfated glycolipid archaeosomes represent a new class of adjuvants that will potentially ease manufacturing and scale-up, while retaining immunostimulatory activity.

High performance liquid chromatography analysis of 100-nm liposomal nanoparticles using polymer-coated, silica monolithic columns with aqueous mobile phase.

Recently, nanoparticles have garnered considerable attention, and the demand for a rapid and simple method for their analysis has increased accordingly. The bimodal pores (few µm- and few tens nm-sized pores) of monolithic columns were thought to be suitable for the separation of nanoparticles and small molecules; however, the residual silanol groups on the column surface resulted in the strong adsorption of liposomes and hindered their analysis. To overcome this problem, we modified the surface of the silica monolith via a two-step process and developed three silica monolithic columns coated with three different polymers: glycidyl methacrylate (GMA), 2-hydroxyethyl methacrylate (HEMA), and N-vinylpyrrolidone (VP). These were used for the analysis of 100-nm liposomal nanoparticles. Since 15% polymer coating prevented the nanoparticle adsorption, liposomes (AmBisome®) and pegylated liposomes (DOXIL®) were eluted rapidly (within 1min) using these columns, without using organic solvents in the mobile phase. Molecular leaching from the liposomes, as well as protein adsorption to the liposomes (corona formation) could be evaluated using the polymer-coated columns, thus illustrating their utility in the rapid and simple analysis of 100-nm liposomal nanoparticles.

Reconstitution of Synaptic SNAREs into Large Liposomes with Reduced Curvature Stress.

Liposomes constitute a convenient biochemical model system to investigate mechanistic aspects of the membrane fusion of synaptic vesicles. The proteins responsible for mediating fusion are the SNAREs that belong to a highly conserved family of transmembrane proteins. Reconstituting SNAREs into liposomes using detergents has become a common approach not only to understand how SNAREs work, but also how fusion is regulated by the vast array of accessory proteins present at the presynapse. However, a concern has been that the high curvature stress of the small liposomes (diameters of ~40 nm) frequently used in many studies renders them prone to spontaneous fusion, bringing into question whether the measurements obtained faithfully represent SNARE-mediated fusion. By systematically varying the detergent concentration and characterizing the SNARE-liposome size distributions by light scattering, we describe a detailed procedure to reconstitute SNAREs into large liposomes with considerably reduced curvature stress.

Archaeal lipid vaccine adjuvants for induction of cell-mediated immunity.

INTRODUCTION: Liposomal vesicles (archaeosomes) composed of total polar lipids (TPL) or semi-synthetic glycerolipids, unique to the domain Archaea, constitute potent vaccine adjuvant and delivery systems. The characteristics of this adjuvant offer a novel prospect for the development of effective vaccines for emerging infections and cancers, which is reviewed in this article. Areas covered: The areas covered in this review include the chemical composition and physical characteristics, various in-vitro and in-vivo pre-clinical immunogenicity and efficacy studies for systemic immunization, induction of mucosal immunity upon modification of the formulation with cations, and the mechanism of adjuvant action following uptake by antigen presenting cells. Expert commentary: The unique features of archaeal lipids confer archaeosomes with many desirable features. With the use of semi-synthetic archaeosomes, highly defined lipids that are safe and robust for induction of cell-mediated immunity may be chosen. These adjuvants function as Toll-like receptor-independent innate immune stimulants.

Quantitative In Vitro Assessment of Liposome Stability and Drug Transfer Employing Asymmetrical Flow Field-Flow Fractionation (AF4).

PURPOSE: In the present study we introduce an efficient approach for a size-based separation of liposomes from plasma proteins employing AF4. We investigated vesicle stability and release behavior of the strongly lipophilic drug temoporfin from liposomes in human plasma for various incubation times at 37°C. METHODS: We used the radioactive tracer cholesteryl oleyl ether (COE) or dipalmitoyl-phosphocholine (DPPC) as lipid markers and (14)C-labeled temoporfin. First, both lipid labels were examined for their suitability as liposome markers. Furthermore, the influence of plasma origin on liposome stability and drug transfer was investigated. The effect of membrane fluidity and PEGylation on vesicle stability and drug release characteristics was also analyzed. RESULTS: Surprisingly, we observed an enzymatic transfer of (3)H-COE to lipoproteins due to the cholesterol ester transfer protein (CETP) in human plasma in dependence on membrane rigidity and were able to inhibit this transfer by plasma preincubation with the CETP inhibitor torcetrapib. This effect was not seen when liposomes were incubated in rat plasma. DPPC labels suffered from hydrolysis effects during preparation and/or storage. Fluid liposomes were less stable in human plasma than their PEGylated analogues or a rigid formulation. In contrast, the transfer of the incorporated drug to lipoproteins was higher for the rigid formulations. CONCLUSIONS: The observed effects render COE-labels questionable for in vivo studies using CEPT-rich species. Here, choline labelled (14)C-DPPC was found to be the most promising alternative. Bilayer composition has a high influence on stability and drug release of a liposomal formulation in human plasma.

Characterization of the physicochemical properties of phospholipid vesicles prepared in CO2/water systems at high pressure.

Phospholipid vesicles were prepared by the nonsolvent method using high-pressure CO2/water systems. The membrane properties of vesicles prepared at different pressures and temperatures were mainly characterized based on analysis of the membrane fluidity and membrane polarity, using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, respectively. The CO2(liquid)/water(liquid) and the CO2(supercritical)/water(liquid) two-phase (heterogeneous) systems resulted in the formation of vesicles with high yield (ca. 85%-88%). The membrane fluidity and polarity of the vesicles were similar to those of liposomes prepared by the conventional method. It is suggested that high-pressure CO2 can be used to form an appropriate hydrophobic-hydrophilic interface where phospholipid molecules as a self-assembled membrane.

Essential oils: from extraction to encapsulation.

Essential oils are natural products which have many interesting applications. Extraction of essential oils from plants is performed by classical and innovative methods. Numerous encapsulation processes have been developed and reported in the literature in order to encapsulate biomolecules, active molecules, nanocrystals, oils and also essential oils for various applications such as in vitro diagnosis, therapy, cosmetic, textile, food etc. Essential oils encapsulation led to numerous new formulations with new applications. This insures the protection of the fragile oil and controlled release. The most commonly prepared carriers are polymer particles, liposomes and solid lipid nanoparticles.

Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing.

Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.

Actividad in vitro contra Leishmania y permeación en piel humana de liposomas ultradeformables de miltefosina / In vitro activity against Leishmania and human skin permeation of miltefosine ultradeformable liposomes

Introducción: los liposomas ultradeformables de miltefosina (LUD-MIL) constituyen una opción para el tratamiento tópico en leishmaniasis cutánea penetrando los estratos de la piel hasta la dermis, sitio donde habita el parásito. Objetivo: diseñar LUD-MIL y determinar su actividad contra L. (Viannia) panamensis y L. (V.) braziliensis y la permeación en piel humana. Métodos: los LUD-MIL, liposomas convencionales de fosfatidilcolina (LConv) y LUD-MIL-fluorescente (LUD-MIL-Fluo) fueron preparados por el método de rehidratación de película lipídica. Se caracterizaron fisicoquímicamente y se determinaron: la liberación en membrana semisintéticas, la retención en las capas de la piel y la permeación en piel humana. La citotoxicidad en THP-1 fue determinada por el ensayo colorimétrico de MTT y la actividad en promastigotes y amastigotes intracelulares por recuento microscópico. Resultados: el tamaño, índice de polidispersión, potencial Z y concentración de fosfolípidos de los LUD-MIL fue de 100,7 nm, 0,147, -12,0 mV y 53,24 mM respectivamente. El flujo de MIL a través de la membrana fue mayor con LUD-MIL que con MIL-libre. El tratamiento con LUD-MIL indujo menor acumulación de la MIL en el estrato corneo y mayor permeación que el tratamiento con MIL libre. Los LUD-MIL y los LConv-MIL mantuvieron la actividad de la MIL en los parásitos y células. Los LUD-MIL fueron más tóxicos para las células que los LConv y la MIL y más activos en amastigotes intracelulares de L. (V.) braziliensis. Conclusión: los LUD-MIL preparados conservaron la actividad anti-Leishmania de la MIL y permitieron la liberación del compuesto en membranas y piel humana. Ensayos en modelos experimentales de leishmaniasis cutánea para evaluar la actividad de estas formulaciones son urgentes de realizar(AU)

Introduction: miltefosine ultradeformable liposomes (MIL-LUD) are an option for the topical treatment of cutaneous leishmaniasis penetrating the skin layers to the dermis where the parasite inhabits. Objective: to design MIL-LUD and determine their in vitro activity against L. (Viannia) panamensis and L. (V.) braziliensis and to determine human skin permeation. Methods: MIL-LUD, phosphatidylcholine liposomes (MIL-LConv) and fluorescent MIL-LUD (MIL-LUD-Fluo) were prepared by lipid film rehydration method. They were physicochemically characterized to determine drug release in semisynthetic membrane, retention in skin layers and permeation on human skin membranes. Cytotoxicity in THP-1 was determined by the MTT colorimetric test and activity in promastigotes and intracellular amastigotes by microscopic counting. Results: the size, the polydispersion index, the Zeta potential and phospholipid content were 100.7 nm, 0.147, -12.0mV and 53.24mM, respectively for MIL-LUD. MIL flow through the semisynthetic membrane was greater with MIL-LUD than MIL-free treatment. MIL-LUD treatment induced lower MIL accumulation in the stratum corneum and increased permeation than MIL free treatment. The MIL-LUD and MIL-Conv maintained MIL activity in parasites and cells. The MIL-LUD was more toxic to cells than MIL-Conv and more active against intracellular amastigotes of L. (V.) braziliensis. Conclusion: prepared LUD -MIL retained the anti-leishmanial activity of the MIL and allowed the compound release in human skin and membranes. Testing of experimental cutaneous leishmaniasis models to evaluate the activity of these formulations are urgently needed(AU)

On the possibility of lipid-induced regulation of conformation and immunogenicity of influenza a virus H1/N1 hemagglutinin as antigen of TI-complexes.

The tubular immunostimulating complex (TI-complex) consisting of cucumarioside A2-2, cholesterol and monogalactosyldiacylglycerol (MGDG) from marine macrophytes is the perspective antigen delivery system for subunit vaccines. MGDG is a lipid matrix for the protein antigen incorporated in the TI-complex. The aim of the present work was to study the influence of MGDGs from different macrophytes on conformation and immunogenicity of the secreted recombinant uncleaved hemagglutinin monomer (HA0S) of influenza A virus H1/N1. Differential scanning calorimetry, fluorescence spectroscopy and circular dichroism showed a dependence of the conformational changes of HA0S on the microviscosity of MGDG. The most viscous MGDG from Zostera marina induced the strongest rearrangements in protein conformation. Immunization of mice with HA0S within TI-complexes comprising different MGDGs resulted in an approximately 2-fold increase of the levels of anti-HA0S antibodies and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared with those induced by HA0S alone. TI-complexes based on MGDG from Z. marina stimulated the maximal production of GM-CSF. However, humoral immune response (anti-HA0S antibodies), unlike cell-mediated immune response (GM-CSF), did not depend on the physicochemical properties of MGDGs. It is assumed that this is due to the different localization and conformational lipid sensitivity of the HA0S regions, which are responsible for these types of immune responses.