Liposome Concentration Research Articles

Liposome-Fe3 O4-Doxorubicin Mediated Treatment of Melanoma Tumors.

Purpose: Magnetic hyperthermia is a treatment method based on eddy currents, hysteresis, and relaxation mechanisms of magnetic nanoparticles (MNPs). MNPs such as Fe3 O4 have the ability to generate heat under an alternating magnetic field. Heat sensitive liposomes (Lip) convert from lipid layer to liquid layer through heat generated by MNPs and can release drugs. Methods: In this study, different groups of doxorubicin (DOX), MNPs and liposomes were evaluated. The MNPs were synthesized by co-precipitation method. The MNPs, DOX and a combination of MNPs and DOX were efficiently loaded into the liposomes using the evaporator rotary technique. Magnetic properties, microstructure, specific absorption rate (SAR), zeta potential, loading percentage of the MNPs and DOX concentration in liposomes, in vitro drug release of liposomes were studied. Finally, the necrosis percentage of cancer cells in C57BL/6J mice bearing melanoma tumors was assessed for all groups. Results: The loading percentages of MNPs and concentration of DOX in the liposomes were 18.52 and 65% respectively. The Lip-DOX-MNPs at the buffer citrate solution, showed highly SAR as the solution temperature reached 42°C in 5 minutes. The release of DOX occurred in a pH-dependent manner. The volume of tumor in the therapeutic groups containing the MNPs significantly decreased compared to the others. Numerical analysis showed that the tumor volume in mice receiving Lip-MNPs-DOX was 9.29% that of the control and a histological examination of the tumor section showed 70% necrosis. Conclusion: The Lip-DOX-MNPs could be effective agents which reduce malignant skin tumors growth and increase cancer cell necrosis.

Glucose Conferred Irinotecan Chemoresistance through Divergent Actions of Pyruvate and ATP in Cell Death and Proliferation of Colorectal Cancer

Altered glucose metabolism is associated with chemoresistance in colorectal cancer (CRC). This study aimed to illustrate the molecular mechanisms of glucose-mediated chemoresistance against irinotecan, a topoisomerase I inhibitor, focusing on the distinct roles of metabolites such as pyruvate and ATP in modulating cell death and proliferation. Four human CRC cell lines, tumorspheres, and mouse xenograft models were treated with various doses of irinotecan in the presence of various concentrations of glucose, pyruvate, or ATP-encapsulated liposomes. In this study, human CRC cell lines treated with irinotecan in high glucose displayed increased cell viability and larger xenograft tumor sizes in mouse models compared to those treated in normal glucose concentrations. Irinotecan induced apoptosis and necroptosis, both mitigated by high glucose. Liposomal ATP prevented irinotecan-induced apoptosis, while it did not affect necroptosis. In contrast, pyruvate attenuated the receptor-interacting protein kinase 1/3-dependent necroptosis via free radical scavenging without modulating apoptotic levels. Regarding the cell cycle, liposomal ATP aggravated the irinotecan-induced G0/G1 shift, whereas pyruvate diminished the G0/G1 shift, showing opposite effects on proliferation. Last, tumorsphere structural damage, an index of solid tumor responsiveness to chemotherapy, was determined. Liposomal ATP increased tumorsphere size while pyruvate prevented the deformation of spheroid mass. Glucose metabolites confer tumor chemoresistance via multiple modes of action. Glycolytic pyruvate attenuated irinotecan-induced necroptosis and potentiated drug insensitivity by shifting cells from a proliferative to a quiescent state. On the other hand, ATP decreased irinotecan-induced apoptosis and promoted active cell proliferation, contributing to tumor recurrence. Our findings challenged the traditional view of ATP as the main factor for irinotecan chemoresistance and provided novel insights of pyruvate acting as an antioxidant responsible for drug insensitivity, which may shed light on the development of new therapies against recalcitrant cancers.

The Bak core dimer focuses triacylglycerides in the membrane

Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis.

Co-assembly of liposomes, Dendrimersomes, and Polymersomes with amphiphilic Janus dendrimers conjugated to Mono- and Tris-Nitrilotriacetic Acid (NTA, TrisNTA) enhances protein recruitment

• Two libraries of Janus dendrimers containing NTA and TrisNTA were synthesized. • Janus dendrimer-NTA and TrisNTA are stable indefinitely at room temperature. • Enhanced his-tagged protein requitement on the hybrid vesicles was demonstrated. • These Janus dendrimers co-assemble with liposomes, dendrimersomes, and polymersomes. Metal-chelating ligands such as nitrilotriacetic acid (NTA) bind to polyhistidine-tagged (His-tagged) proteins. Lipids conjugated to NTA are widely used to decorate the surface of liposomes with proteins in cell biology applications. Multivalent NTA ligands such as tris-nitrilotriacetic acid (TrisNTA) display higher affinities than the monovalent NTA when co-assembled with phospholipids and cholesterol in liposomes. However, there is a limited number of available lipids conjugated to NTA and only few are commercially available. Additionally, their activity diminishes during storage or upon exposure to air. Here we report a library of five amphiphilic Janus dendrimers conjugated to NTA (JD-NTA) and three to TrisNTA (JD-TrisNTA). Both JD-NTA and JD-TrisNTA are indefinitely stable at room temperature in air and preliminary results demonstrate that they co-assemble with phospholipids and cholesterol into liposomes, with Janus dendrimers into dendrimersomes, and with block copolymers into polymersomes. The resulting hybrid liposomes co-assembled with JD-NTA display up to thirty-fold higher activity towards His-tagged fluorescent proteins when compared to lipid-NTAs. Hybrid liposomes co-assembled with JD-TrisNTA exhibit even higher binding affinity to His-tagged proteins and can function at much lower ligand concentration in hybrid liposomes than those containing JD-NTA. These preliminary results demonstrate the power of modular synthesis of JD-NTA or JD-TrisNTA to provide highly efficient new tools for biological reconstitution and synthetic cell biology as well as for nanomedicine.

Zinc Triggered Release of Encapsulated Cargo from Liposomes via a Synthetic Lipid Switch.

Liposomes are effective nanocarriers due to their ability to encapsulate and deliver a wide variety of therapeutics. However, therapeutic potential would be improved by enhanced control over the release of drug cargo. Zinc ions provide exciting new targets for stimuli-responsive lipid design due to their overly abundant concentrations associated with diseased cells. Herein, we report zinc-triggered release of liposomal contents exploiting synthetic lipid switches designed to undergo conformational changes in the presence of this ion. Initially, Nile red leakage assays were conducted that validated successful dose-dependent triggering of release using zinc-responsive lipids (ZRLs). In addition, dynamic light scattering and confocal microscopy experiments showed that zinc treatment led to morphological changes in lipid nanoparticles only when ZRLs were present in formulations. Next, zinc-binding experiments conducted in a solution (NMR, MS) or membrane (zeta potential) context confirmed ZRL-Zn complexation. Finally, polar cargo release from liposomes was achieved. The results from these wide-ranging experiments using four different compounds indicated that zinc-responsive properties varied based on ZRL structure, providing insights into the structural requirements for activity. This work has established zinc-responsive liposomal platforms toward the development of clinical triggered release formulations.

Design of high-performance biomimetic reverse osmosis membranes by introducing loose liposome as an artificial water channel

Superior permeability and high selectivity are crucial performances criterion for reverse osmosis (RO) membranes. Inspired by aquaporin-based biomimetic membranes, the loose liposomes as artificial water channels were prepared by thin-layer hydration method with lyso-phospholipids (C14lyso) and cholesterol followed by incorporating liposomes into the polyamide selective layer via in situ interfacial polymerization to fabricate liposomes-based RO membranes (TFC-C14lyso). Stopped-flow tests verified that C14lyso liposomes possess fast water transportability and the effect of C14lyso liposomes on the intrinsic structure of the polyamide selective layer was comprehensively explored. Meanwhile, the structure and performance of the TFC-C14lyso membranes could be regulated by varying the liposome concentrations. As a result, TFC-C14lyso-2 (the concentration of C14lyso liposomes is 0.25 mg/mL) presented an optimal separation performance, showing high water permeance of 3.80 L m−2h−1 bar−1 and NaCl rejection of 98.6 %. Moreover, the separation mechanism regarding the enhanced permeability of TFC-C14lyso-2 was profoundly revealed via the structure and performance comparison with TFC-DOPC fabricated by incorporating dense DOPC liposomes into its PA selective layer. Building on these studies, our work may provide a facile and versatile pathway for designing advanced membranes in desalination.

Facile Separation of PEGylated Liposomes Enabled by Anti-PEG scFv

PEGylated nanocarriers have gained increasing attention due to reduced toxicity and enhanced circulation compared with free drugs. According to guidances of drug regulatory departments worldwide, it is crucial to determine free and liposomal drug concentrations; however, the conventional used separation methods including dialysis, ultrafiltration, and solid-phase extraction (SPE) have drawbacks of time-consuming, drug leakage, environmental pollution or error bias of trace level drug. Here we developed a facile PEG-scFv-based separation method combined with HPLC to quantify free doxorubicin (DOX) and liposomal DOX in plasma. Anti-PEG single chain variable fragment antibody (PEG-scFv) was adopted to sediment PEGylated liposomes by simple incubation and low speed centrifugation. Compared to SPE, it demonstrated sufficient accuracy and sensitivity to evaluate free and liposomal DOX with intact liposomes. Therefore, it can serve as an alternative approach of SPE, which is suitable for quality assessment and pharmacokinetics evaluation of PEGylated liposomal drugs and possible other PEGylated nanocarriers.

Combination of Carriers with Complementary Intratumoral Microdistributions of Delivered α-Particles May Realize the Promise for 225Ac in Large, Solid Tumors.

α-particle radiotherapy has already been shown to be impervious to most resistance mechanisms. However, in established (i.e., large, vascularized) soft-tissue lesions, the diffusion-limited penetration depths of radiolabeled antibodies or nanocarriers (≤50-80 μm) combined with the short range of α-particles (4-5 cell diameters) may result in only partial tumor irradiation, potentially limiting treatment efficacy. To address this challenge, we combined carriers with complementary intratumoral microdistributions of the delivered α-particles. We used the α-particle generator 225Ac, and we combined a tumor-responsive liposome (which, on tumor uptake, releases into the interstitium a highly diffusing form of its radioactive payload [225Ac-DOTA], potentially penetrating the deeper parts of tumors where antibodies do not reach) with a separately administered, less-penetrating radiolabeled antibody (irradiating the tumor perivascular regions where liposome contents clear too quickly). Methods: In a murine model with orthotopic human epidermal growth factor receptor 2-positive BT474 breast cancer xenografts, the biodistributions of each carrier were evaluated, and the control of tumor growth was monitored after administration of the same total radioactivity of 225Ac delivered by the 225Ac-DOTA-encapsulating liposomes, by the 225Ac-DOTA-SCN--labeled trastuzumab, and by both carriers at equally split radioactivities. Results: Tumor growth was significantly more inhibited when the same total injected radioactivity was divided between the 2 separate carriers than when delivered by either of the carriers alone. The combined carriers enabled more uniform intratumoral microdistributions of α-particles, at a tumor dose that was lower than the dose delivered by the antibody alone. Conclusion: This strategy demonstrates that more uniform microdistributions of the delivered α-particles within established solid tumors improve efficacy even at lower tumor doses. Augmentation of antibody-targeted α-particle therapies with tumor-responsive liposomes may address partial tumor irradiation, improving therapeutic effects.

The Two Sweet Sides of Janus Lectin Drive Crosslinking of Liposomes to Cancer Cells and Material Uptake.

A chimeric, bispecific Janus lectin has recently been engineered with different, rationally oriented recognition sites. It can bind simultaneously to sialylated and fucosylated glycoconjugates. Because of its multivalent architecture, this lectin reaches nanomolar avidities for sialic acid and fucose. The lectin was designed to detect hypersialylation—a dysregulation in physiological glycosylation patterns, which promotes the tumor growth and progression of several cancer types. In this study, the characteristic properties of this bispecific Janus lectin were investigated on human cells by flow cytometry and confocal microscopy in order to understand the fundamentals of its interactions. We evaluated its potential in targeted drug delivery, precisely leading to the cellular uptake of liposomal content in human epithelial cancer cells. We successfully demonstrated that Janus lectin mediates crosslinking of glyco-decorated giant unilamellar vesicles (GUVs) and H1299 lung epithelial cells. Strikingly, the Janus lectin induced the internalization of liposomal lipids and also of complete GUVs. Our findings serve as a solid proof of concept for lectin-mediated targeted drug delivery using glyco-decorated liposomes as possible drug carriers to cells of interest. The use of Janus lectin for tumor recognition certainly broadens the possibilities for engineering diverse tailor-made lectin constructs, specifically targeting extracellular structures of high significance in pathological conditions.

Surface modification with cholesteryl acetyl carnitine, a novel cationic agent, elevates cancer cell uptake of the PEGylated liposomes

The present study aimed to synthesize cholesteryl acetyl carnitine (CAC), and surface modify the PEGylated liposomes with the intention of enhanced cancer cell uptake. For this, CAC synthesis was performed in amine-free esterification conditions and then four liposomal formulations of unmodified, CAC/PEG, and CAC + PEG-modified were prepared by ethanol injection method. Cytotoxicity of the liposomes was investigated in A549 cells, followed by cellular uptake assessments of coumarin 6 (C6)-loaded liposomes. The results of ATR-FTIR, 1HNMR, and 13CNMR demonstrated successful formation of CAC. A molecular docking study showed efficient binding affinities rather than carnitine to the active site of four carnitine transporters. Liposomal formulations possessed spherical morphology with a mean particle size range of 112–138 nm, narrow size distribution, and negative surface charge. All formulations had low cytotoxicity at 0.5 mg/ml, but high cytotoxicity at around 2.5 mg/ml. The lowest IC50 was obtained for CAC modified liposomes. CAC + PEG-modified liposomes had the highest cellular uptake.In conclusion, CAC + PEG modification of liposomes is an effective approach for increasing A549 cellular uptake, with low cytotoxicity at commonly applied liposome concentrations. The elevated uptake may be due to the involvement of the organic cation transporter, cationic structure, and the metabolic preference of CAC in cancer cells.

A solid-supported membrane electrophysiology assay for efficient characterization of ion-coupled transport

Transport stoichiometry determination can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transporter behavior under multiple experimental conditions. Solid-supported membrane electrophysiology (SSME) allows multiple recordings of liposomal or membrane samples adsorbed onto a sensor and is sensitive enough to detect transport currents from moderate-flux transporters that are inaccessible to traditional electrophysiology techniques. Here, we use SSME to develop a new method for measuring transport stoichiometry with greatly improved throughput. Using this technique, we were able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx, reproduce the 1H+:2Cl− antiport stoichiometry of CLC-ec1, and confirm loose proton:nitrate coupling for CLC-ec1. Furthermore, we were able to demonstrate quantitative exchange of internal contents of liposomes adsorbed onto SSME sensors to allow multiple experimental conditions to be tested on a single sample. Our SSME method provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

Tuning lipid layer formation on particle surfaces by using DNA-containing recruiter molecules

Biofunctional interfaces containing DNA-conjugated molecules have been explored for various bioengineering applications. However, there is still a lack of understanding of the interaction between DNA conjugates and surrounding biomolecules. In this study, we prepare DNA-containing recruiter molecules and incorporate them onto DNA immobilized gold nanoparticles through DNA hybridization. Liposomes composed of different phospholipids are then applied to investigate supported lipid layer formation on these recruiter-containing surfaces. We find that the morphology and the amount of lipid layers formed are determined by both the liposome concentration and the type of recruiter molecule. When liposomes are applied in excess above a critical concentration, surface chemistry determines the lipid layers formed, leading to lipid multilayers on hydrophilic DNA recruiter containing surfaces and lipid monolayers on hydrophobic DNA-lipid recruiter containing surfaces. When the liposome concentration is below the critical value, the surface molecules take on a more direct role and recruit lipids through hydrophobic interaction. The total amount of the lipid layers formed is further modulated by the overall charge and the fluidity of the liposomes applied. These results provide quantitative analysis on the interaction of DNA conjugates with lipid molecules and introduce a new approach to fine-tune lipid layer formation behavior.

Single Electrochemical Nano-Impacts of Synthetic Redox Phospholipid Liposomes

Electrochemical discrete nano-impacts at an ultramicroelectrode is a useful technique to detect one at a time single biological entities such as cells, bacteria, macromolecules, viruses, and synthetic or biological vesicles.[1-4] Especially, electrochemical detection of single liposome nano-impacts by recording electron transfer from the ultramicroelectrode to an encapsulated redox species is fully appropriate for studying their membrane permeability.[2,4] Since electron transfer do not readily occur across a bilayer lipid membrane, the electrolysis of the liposome redox active content after collision and membrane rupture or opening at the electrode surface provides insights on the membrane permeation mechanism. The vesicle membrane opening by electroporation is strongly dependent on lipid membrane properties, liposome content, vesicle size, temperature, electrode potential, the nature of the electrode and the concentration of redox species inside and outside the liposome.[2] For example, to observe the current spikes corresponding to the oxidation of ferrocyanide encapsulated inside phospholipid vesicles when they collide with a Pt ultramicroelectrode, the presence of an appropriate concentration of surfactant in solution is required.[4] In the absence of surfactant, we found that impact and adhesion of vesicles at the Pt ultramicroelectrode does not allow the electrolysis of their ferrocyanide content.[4] It is well established that the liposome membrane stability is strongly dependent on its lipid composition and external parameters such as temperature and pH but also depends on interactions with specific molecules able to weaken, permeabilize, and penetrate the lipid bilayer following different pathways. An understanding of these different interactions and especially the mechanism leading to the lipid membrane opening is still an active research field. To this end, chronoamperometry is a useful method to probe the liposomes membrane permeability and to understand vesicle fusion processes onto electrode surface. Especially, the electrochemical detection of single liposome collisions at ultramicroelectrodes is becoming an efficient and complementary tool for analyzing fundamental biological processes related to intracellular and extracellular electron transfers to discrete biological or artificial entities. Electrochemistry of single redox liposome nano-impacts at an ultramicroelectrode is a powerful method which consists in detecting the electrolysis of a redox probe encapsulated inside the liposome when it is released at the ultramicroelectrode after impact (or collision).[4]To explore the factors influencing the vesicles membrane permeability, we investigated the electrochemical and electrocatalytic reaction of different aqueous redox species (potassium ferrocyanide and cobalt(II) nitrate) encapsulated inside synthetic DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) liposomes subjected to different experimental conditions (addition of surfactant, increase of temperature or presence of a redox probe in solution), by single collision detection on 10 μm diameter ultramicroelectrodes (Pt and carbon) as illustrated in Figure.[2] The influence of the presence of surfactant (Triton X-100) and the increase of solution temperature (20-70 °C) on the lipid membrane permeability has been investigated, and the results showed a similar effect of these two parameters with a maximum collision frequency reached for an optimal surfactant concentration (0.2 mM) and temperature (60 °C) respectively, at platinum or carbon ultramicroelectrodes.[2][1] Lebègue, E.; Costa, N.L.; Louro, R.O.; Barrière, F. Communication—Electrochemical Single Nano-Impacts of Electroactive Shewanella Oneidensis Bacteria onto Carbon Ultramicroelectrode. J. Electrochem. Soc. 2020, 167, 105501, doi:10.1149/1945-7111/ab9e39.[2] Lebègue, E.; Barrière, F.; Bard, A.J. Lipid Membrane Permeability of Synthetic Redox DMPC Liposomes Investigated by Single Electrochemical Collisions. Anal. Chem. 2020, 92, 2401–2408, doi:10.1021/acs.analchem.9b02809.[3] Dick, J.E.; Lebègue, E.; Strawsine, L.M.; Bard, A.J. Millisecond Coulometry via Zeptoliter Droplet Collisions on an Ultramicroelectrode. Electroanalysis 2016, 28, 2320–2326, doi:10.1002/elan.201600182.[4] Lebègue, E.; Anderson, C.M.; Dick, J.E.; Webb, L.J.; Bard, A.J. Electrochemical Detection of Single Phospholipid Vesicle Collisions at a Pt Ultramicroelectrode. Langmuir 2015, 31, 11734–11739, doi:10.1021/acs.langmuir.5b03123. Figure 1

The interaction mechanism between liposome and whey protein: Effect of liposomal vesicles concentration

The interaction mechanism between liposomes (Lips) and whey protein isolates (WPI) with different mass ratios was explored in this paper. After binding with different concentration of Lips, the changes in hydrophilic and hydrophobic regions of WPI were investigated with fluorescein isothiocyanate (FITC) and pyrene fluorescence probes. The spatial structure changes of WPI were further characterized by differential scanning calorimetry, Fourier transform infrared spectroscopy, and circular dichroism. The results indicated that the structure of WPI was changed due to binding with Lips in hydrophilic and hydrophobic groups. The binding process might result in the migration, recombination, and alignment of WPI and Lip groups. Moreover, the oil-water interfacial tension with WPI decreased from 9.20 mN/m to 3.29 mN/m upon increasing the Lip-to-WPI ratio. This work suggests that the physiochemical properties of Lip-WPI complexes could be manipulated by adjusting the Lip-to-WPI ratio. This study shed some light on the mechanism explanation of the WPI structural changes due to the interaction with Lips during food processing.

Improvement in storage quality of postharvest tomato fruits by nitroxyl liposomes treatment

Nitroxyl (HNO) has attracted much attention due to its unique biological activity. To investigate the preservation effect of HNO on fruits, a nitroxyl liposome based on 1-nitrosocyclohexyl acetate was prepared and characterized by infrared spectroscopy and transmission electron microscopy. The optimal preparation conditions were explored, and then HNO liposomes were prepared under the optimal conditions to study the effect of HNO liposomes on postharvest quality of tomatoes. The tomato fruits were treated with different concentrations (0, 5, 10, 15 and 20 μmol L-1) of HNO liposomes and stored at room temperature. The results indicated that treatment with HNO liposomes can more effectively delay the browning and slow down the decrease in lightness of tomatoes. Additionally, HNO liposomes can reduce the activity of PPO and POD, inhibit the increase of MDA and total phenol content. These results suggest that treatment with HNO liposomes can effectively preserve the quality of tomatoes.

Engineering motile aqueous phase-separated droplets via liposome stabilisation

There are increasing efforts to engineer functional compartments that mimic cellular behaviours from the bottom-up. One behaviour that is receiving particular attention is motility, due to its biotechnological potential and ubiquity in living systems. Many existing platforms make use of the Marangoni effect to achieve motion in water/oil (w/o) droplet systems. However, most of these systems are unsuitable for biological applications due to biocompatibility issues caused by the presence of oil phases. Here we report a biocompatible all aqueous (w/w) PEG/dextran Pickering-like emulsion system consisting of liposome-stabilised cell-sized droplets, where the stability can be easily tuned by adjusting liposome composition and concentration. We demonstrate that the compartments are capable of negative chemotaxis: these droplets can respond to a PEG/dextran polymer gradient through directional motion down to the gradient. The biocompatibility, motility and partitioning abilities of this droplet system offers new directions to pursue research in motion-related biological processes.

Counterion Phenomena Near Charged Surfaces in Finite Volumes

Lipid-membrane headgroups are invariably charged or zwitterionic. The headgroups can be weak acids (negatively charged when deprotonated), weak bases (positively charged when protonated), or zwitterionic (both types of groups present). Headgroups can also be salts, such as TMA+Cl- in the lipid DOTAP+. The screening of surface charge by dissociated counterions is usually negligible, as dissociated counterions are normally diluted to concentrations far below that of the ambient salt. However for a suspension of charged particles in a finite volume, the mean concentration of dissociated counterions depends on the suspension's surface charge/volume ratio, which for moderately concentrated suspensions of small particles can be quite high. Significant concentrations of dissociated counterions can screen and uncouple electrostatic interactions between like-charged particles. Such effects are readily observed in deionized suspensions where background salt is of order 10−7 M. However in the presence of high salt, surface-dissociated counterions get masked by the salt counterions. Here we determine the maximum background salt concentrations at which dissociated-counterion screening can occur. For example, for 100 nm liposomes containing 20 mol% cationic or weak-acid lipids at 0.1 volume-fraction liposome concentration, dissociated counterions can reach mean concentrations of order 10 mM, or if the counterions are protons, mean pH∼2. Although charge regulation can in some cases reduce the number of dissociated counterions, dissociated-counterion phenomena, i.e. screening-ion numbers proportional to total surface charge, can occur in the presence of surprisingly high values of ambient salt or acid.

Liposome Fusion Mediated by Hydrophobic Magnetic Nanoparticles Stabilized with Oleic Acid and Modulated by an External Magnetic Field.

Membrane fusion is considered relevant in countless scientific areas and biotechnological processes, ranging from vital life events to biomedicine, pharmaceuticals, and materials engineering, among others. In this study, we employed hydrophobic oleic acid (OA)-coated magnetite (Fe3O4) nanoparticles (MNP-OA) as a platform to induce the fusion of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes [large unilamellar vesicles (LUVs)] in a colloidal dispersion. This fusion was monitored through dynamic light scattering, turbidimetry, and fluorescence assay using the well-known Tb/dipicolinic acid (DPA) complex formation assay. MNP-OA have shown to be able to induce fusion with the mixing of liposomal inner content with direct dependence on the nanoparticle concentration added to the LUVs. Moreover, changes in the permeability of the liposome bilayer, upon the addition of MNP-OA to liposomes, were evaluated by studying the leakage of carboxyfluorescein and of the co-encapsulated Tb/DPA complex. These assays allowed us to determine that MNP-OA did not significantly modify liposome permeability during the fusion process. Transmission electron microscopy and confocal microscopy revealed that MNP-OA remained embedded in the lipid bilayer without producing membrane rupture, liposome deformation, or destruction. In addition, we evaluated the effect of applying a low-intensity magnetic field to the LUVs/MNP-OA system and observed that the nanoparticles considerably increased their fusogenic activity under this external stimulus, as well as they are capable of responding to low magnetic fields of around 0.45 mT. These results revealed the potential of hydrophobic magnetic nanoparticles, stabilized with OA, to act as a fusogen, thus representing a valuable tool for biotechnological applications.

LipHosomes: Reporters for Ligand/Anti‐Ligand Assays Based On pH Readout

AbstractA new class of liposomes (LipHosomes) is designed to induce a change of pH upon releasing their content. pH‐readout reports on the number of LipHosomes in the specimen. LipHosomes were prepared by entrapping NaOH or bicarbonate buffer in the intravesicular compartment. The liposomes suspension was purified from unentrapped compounds and brought to pH 7.0. The pH gradient between intra‐ and extra‐liposomal compartments is maintained because the phospholipidic membrane works as a semipermeable membrane thus preventing diffusion of ions across the membrane. The release of the liposomal content triggers a quantifiable variation of the pH of the medium. This feature has been harnessed in analytical assays based on ligand/anti‐ligand molecular recognition by exploiting the biotin‐streptavidin binding scheme. A pH difference of 0.2 units was observed upon the release of the payload from biotinylated LipHosomes bound to streptavidinated plates. The test showed an excellent sensitivity being able to reveal a concentration of bound LipHosomes in the sub‐pM range.

Prolonged distribution of aerosolized PEGylated liposomes in the lungs of mice with bleomycin-induced pulmonary fibrosis

Objective Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic lung disease characterized by abnormal remodeling of the lung parenchyma with subsequent scarring of the alveolar structure. In this study, we examined the distribution characteristics of aerosolized polyethylene glycol (PEG)ylated liposomes in the lungs of mice with bleomycin-induced pulmonary fibrosis. Significance The present study details the utility of aerosolized PEGylated liposomes for improving intrapulmonary pharmacokinetics in fibrotic lungs. Methods Aerosolized PEGylated liposomes were administered to fibrotic mouse lungs using a MicroSprayer. Intrapulmonary pharmacokinetics was evaluated via in vivo imaging, measurement of liposome concentrations in bronchoalveolar lavage fluid (BALF) and alveolar macrophages (AMs), and observation of lung tissue sections. In addition, in vitro accumulation experiments using WI-38, A549, and RAW264.7 cells were performed. Results The decrease of the fluorescence intensity of the PEGylated liposomes was slower than that of the non-modified liposomes. Compared with the non-modified liposomes, the PEGylated liposomes were determined higher in BALF, whereas those in the AMs were lower. Both PEGylated and non-modified liposomes were widely dispersed in fibrotic regions in tissue sections. No difference in accumulation in WI-38 and A549 cells was noted between PEGylated and non-modified liposomes, whereas the PEGylated liposomes exhibited lower intracellular accumulation than non-modified liposomes in RAW264.7 cells. Conclusion Aerosolized drug delivery systems using PEGylated liposomes exhibited prolonged distribution in both healthy and fibrotic mouse lungs. PEGylated liposomes were determined to be efficient drug delivery systems for anti-fibrotic agents targeting lung fibroblasts and alveolar epithelial cells for optimizing the treatment of IPF.