Large Liposomes Research Articles

Activity of Membrane-Permeabilizing Lpt Peptides.

Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin-antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of "lipid pores".

Surfactant-Mediated Assembly of Precision-Size Liposomes.

Liposomes can greatly improve the pharmacokinetics of therapeutic agents due to their ability to encapsulate drugs and accumulate in target tissues. Considerable effort has been focused on methods to synthesize these nanocarriers in the past decades. However, most methods fail to controllably generate lipid vesicles at specific sizes and with low polydispersity, especially via scalable approaches suitable for clinical product manufacturing. Here, we report a surfactant-assisted liposome assembly method enabling the precise production of monodisperse liposomes with diameters ranging from 50 nm to 1 μm. To overcome scalability limitations, we used tangential flow filtration, a scalable size-based separation technique, to readily concentrate and purify the liposomal samples from more than 99.9% of detergent. Further, we propose two modes of liposome self-assembly following detergent dilution to explain the wide range of liposome size control, one in which phase separation into lipid-rich and detergent-rich phases drives the formation of large bilayer liposomes and a second where the rate of detergent monomer partitioning into solution controls bilayer leaflet imbalances that promote fusion into larger vesicles. We demonstrate the utility of controlled size assembly of liposomes by evaluating nanoparticle uptake in macrophages, where we observe a clear linear relationship between vesicle size and total nanoparticle uptake.

Effect of size and pH-sensitivity of liposomes on cellular uptake pathways and pharmacokinetics of encapsulated gemcitabine

To enhance cytoplasmic delivery efficiency, pH-sensitive liposomes (PSL) have been proposed as a novel strategy. To facilitate clinical translation, this study aims to understand the impact of both size and pH-sensitivity on cellular uptake pathways, intracellular trafficking and pharmacokinetics of liposomes. The large liposomes (130-160 nm) were prepared using thin-film hydration method, while small liposomes (∼60 nm) were fabricated using microfluidics, for both PSL and non-pH-sensitive liposomes (NPSL). Cellular uptake pathways and intracellular trafficking was investigated through confocal imaging with aid of various endocytosis inhibitors. Intracellular gemcitabine delivery by various liposomal formulations was quantified using HPLC, and the cytotoxicity was assessed via cell viability assays. Pharmacokinetics of gemcitabine loaded in various liposomes was evaluated in rats following intravenous administration. Larger liposomes had a higher loading capacity for hydrophilic gemcitabine (7% vs 4%). Small PSL exhibited superior cellular uptake compared to large PSL or NPSLs. Moreover, the alkalization of endosomes significantly attenuated the cellular uptake of PSL. Large liposomes (PSL and NPSL) predominantly entered cells via clathrin-dependent pathway, whereas small liposomes partially utilized caveolae-dependent pathway. However, the long circulation of the liposomes, as measured by the encapsulated gemcitabine, was compromised by both pH-sensitivity and size reduction (9.5 h vs 5.3 h). Despite this drawback, our results indicate that small PSL holds promise as vectors for the next generation of liposomal nanomedicine, owing to their superior cytoplasmic delivery efficiency.

Nanoparticles in liposomes: a platform for increased antibiotic selectivity in multidrug resistant bacteria in respiratory tract infections.

Antibiotic resistance is a cause of serious illness and death, originating often from insufficient permeability into gram-negative bacteria. Nanoparticles (NP) can increase antibiotic delivery in bacterial cells, however, may as well increase internalization in mammalian cells and toxicity. In this work, NP in liposome (NP-Lip) formulations were used to enhance the selectivity of the antibiotics (3C and tobramycin) and quorum sensing inhibitor (HIPS-1635) towards Pseudomonas aeruginosa by fusing with bacterial outer membranes and reducing uptake in mammalian cells due to their larger size. Poly (lactic-co-glycolic) acid NPs were prepared using emulsion solvent evaporation and incorporated in larger liposomes. Cytotoxicity and uptake studies were conducted on two lung cell lines, Calu-3 and H460. NP-Lip showed lower toxicity and uptake in both cell lines. Then formulations were investigated for suitability for oral inhalation. The deposition of NP and NP-Lip in the lungs was assessed by next generation impactor and corresponded to 75% and 45% deposition in the terminal bronchi and the alveoli respectively. Colloidal stability and mucus-interaction studies were conducted. NP-Lip showed higher diffusion through mucus compared to NPs with the use of nanoparticle tracking analyzer. Moreover, the permeation of delivery systems across a liquid-liquid interface epithelial barrier model of Calu-3 cells indicated that NP-Lip could cause less systemic toxicity upon in-vivo like administration by aerosol deposition. Monoculture and Pseudomonas aeruginosa biofilm with Calu-3 cells co-culture experiments were conducted, NP-Lip achieved highest toxicity towards bacterial biofilms and least toxicity % of the Calu-3 cells. Therefore, the NP- liposomal platform offers a promising approach for enhancing antibiotic selectivity and treating pulmonary infections.

Development of a novel human stratum corneum mimetic phospholipid ­vesicle-based permeation assay models for in vitro permeation studies

Objectives To develop and evaluate a novel human stratum corneum (SC) mimetic phospholipid vesicle-based permeation assay (PVPASC) model for in vitro permeation studies. Significance Due to the increasing restrictions on the use of human and animal skins, artificial skin models have attracted substantial interest in pharmaceuticals and cosmetic industries. In this study, a modified PVPASC model containing both SC lipids and proteins was developed. Methods The PVPASC model was optimized by altering the lipid composition and adding keratin in the formulation of large liposomes. The barrier properties were monitored by measuring the electrical resistance (ER) and permeability of Rhodamine B (RB). The modified PVPASC model was characterized in terms of the surface topography, solvent influence and storage stability. The permeation studies of the active components in Compound Nanxing Zhitong Plaster (CNZP) were performed to examine the capability of PVPASC in the application of skin penetration. Results The ER and P app values of RB obtained from the optimized PVPASC model indicated a similar barrier property to porcine ear skin. Scanning electron microscope analysis demonstrated a mimic ‘brick-and-mortar’ structure. The PVPASC model can be stored for three weeks at −20 °C, and withstand the presence of different receptor medium for 24 h. The permeation studies of the active components demonstrated a good correlation (r 2 = 0.9136) of P app values between the drugs’ permeation through the PVPASC model and porcine ear skin. Conclusion Keratin contained composite phospholipid vesicle-based permeation assay models have been proven to be potential skin tools in topical/transdermal permeation studies.

Evaluation of New Folate Receptor-mediated Mitoxantrone Targeting Liposomes In Vitro.

<p>Background: Ligand-mediated liposomes targeting folate receptors (FRs) that are overexpressed on the surface of tumor cells may improve drug delivery. However, the properties of liposomes also affect cellular uptake and drug release. <p> Objective: Mitoxantrone folate targeted liposomes were prepared to increase the enrichment of drugs in tumor cells and improve the therapeutic index of drugs by changing the route of drug administration. <p> Methods: Liposomes were prepared with optimized formulation, including mitoxantrone folatetargeted small unilamellar liposome (MIT-FSL), mitoxantrone folate-free small unilamellar liposome (MIT-SL), mitoxantrone folate-targeted large unilamellar liposome (MIT-FLL), mitoxantrone folate-free large unilamellar liposomes (MIT-LL). Cells with different levels of folate alpha receptor (FRα) expression were used to study the differences in the enrichment of liposomes, the killing effect on tumor cells, and their ability to overcome multidrug resistance. <p> The results of the drug release experiment showed that the particle size of liposomes affected their release behavior. Large single-compartment liposomes could hardly be effectively released, while small single-compartment liposomes could be effectively released, MIT-FSL vs MIT-FLL and MIT-SL vs MIT-LL had significant differences in the drug release rate (P<0.0005). Cell uptake experiments results indicated that the ability of liposomes to enter folic acid receptor-expressing tumor cells could be improved after modification of folic acid ligands on the surface of liposomes and it was related to the expression of folate receptors on the cell surface. There were significant differences in cell uptake rates (p<0.0005) for cells with high FRα expression (SPC-A-1 cells), when MIT-FSL vs MIT-SL and MIT-FLL vs MIT-LL. For cells with low FRα expression (MCF-7 cells), their cell uptake rates were still different (p<0.05), but less pronounced than in SPC-A-1 cells. The results of the cell inhibition experiment suggest that MIT-FLL and MIT-LL had no inhibitory effect on cells, MIT-FSL had a significant inhibitory effect on cells and its IC50 value was calculated to be 4502.4 ng/mL, MIT-SL also had an inhibitory effect, and its IC50 value was 25092.1 ng/mL, there was a statistical difference (p<0.05), MIT-FSL had a higher inhibitory rate than MIT-SL at the same drug concentration. Afterward, we did an inhibitory experiment of different MIT-loaded nanoparticles on MCF-7 cells compared to the drug-resistant cells (ADR), Observing the cell growth inhibition curve, both MIT-FSL and MIT-SL can inhibit the growth of MCF-7 and MCF-7/ADR cells. For MCF- 7 cells, at the same concentration, there is little difference between the inhibition rate of MITFSL and MIT-SL, but for MCF-7/ADR, the inhibition rate of MIT-FSL was significantly higher than that of MIT-SL at the same concentration (P<0.05). <p> Conclusion: By modifying folic acid on the surface of liposomes, tumor cells with high expression of folic acid receptors can be effectively targeted, thereby increasing the enrichment of intracellular drugs and improving efficacy. It can also change the delivery pathway, increase the amount of drug entering resistant tumor cells, and overcome resistance.</p>.

Electroformation of giant unilamellar vesicles from large liposomes

AbstractGiant unilamellar vesicles (GUVs) are widely used as model systems for biological membranes to study membrane-related processes in a precisely controlled in vitro environment, owing to their biophysical properties. The classical technique for the formation of giant liposomes starts with the dissolution of lipids or lipid mixtures in an organic solvent, which is then deposited as a thin lipid solution film on a support substrate. In this study, we present a comprehensive analysis investigating the effect of different lipid compositions on the generation of GUVs from preformed liposomes under non-ionic and ionic conditions. For all liposome types tested, the electroformation process, whether performed on indium tin oxide-coated glass slides or platinum wires, consistently produced GUVs that typically ranged in size from 5 to 20 μm. However, the yield of GUVs varied depending on the specific non-ionic or ionic conditions and the lipid composition of the preformed liposomes used. In general, the resulting population of giant vesicles was predominantly characterised by the presence of unilamellar and multivesicular vesicles. These findings have the potential to improve the refinement of protocol parameters for the formation of GUVs containing membrane proteins and for the study of the effects of lipid composition on membrane protein activity.

Histological findings for the absorption of small and large liposomes - the basis of future drug delivery and contrast media systems.

The purpose of our study was to obtain and characterize carrier systems in different sizes that can affect oral absorption, since the mechanisms of liposome absorption are not yet fully understood. From stomach to the small intestine, liposomes can be gradually destroyed. Understanding the factors that affect oral absorption leads to developing safe and effective nanosystems to improve the oral delivery of therapeutics. We determined the efficiency of the absorption of small and large liposomes at the level of gingival mucosa, heart, liver, testicles, kidneys, and lungs, using frozen-section fluorescence microscopy, on rat tissues after liposomes administration. A number of 36 male rats were divided in four groups: control groups, A and C, consisted of six rats each and did not receive liposomes; two other groups, B and D, were the experimental ones, and consisted of 12 male rats each. The animals received small liposomes (75-76 nm) and large liposomes (80-87 nm), respectively, administered either by endogastric tube or intraperitoneal injection. After 24 hours, the animals were sacrificed, and we harvested the organs. We performed frozen sections and analyzed them with fluorescence microscopy. The frozen sections obtained from all organs revealed a higher absorption level of small liposomes in the testicles, liver, and gum, while the large liposomes had a greater affinity for the liver, with variations dependent on the route of administration. Frozen-section fluorescence microscopy is a reliable technique for visualization of liposome absorption. Based on the size of these nanosystems, we revealed significant absorption for small liposomes in testicles, liver, heart, and gum, and for large liposomes mainly in the liver, compared with the control groups. The study advocates for the usage of liposomes for medical purposes, based on their absorption proprieties.

Preparing Size-Controlled Liposomes Modified with Polysaccharide Derivatives for pH-Responsive Drug Delivery Applications.

The liposome particle size is an important parameter because it strongly affects content release from liposomes as a result of different bilayer curvatures and lipid packing. Earlier, we developed pH-responsive polysaccharide-derivative-modified liposomes that induced content release from the liposomes under weakly acidic conditions. However, the liposome used in previous studies size was adjusted to 100-200 nm. The liposome size effects on their pH-responsive properties were unclear. For this study, we controlled the polysaccharide-derivative-modified liposome size by extrusion through polycarbonate membranes having different pore sizes. The obtained liposomes exhibited different average diameters, in which the diameters mostly corresponded to the pore sizes of polycarbonate membranes used for extrusion. The amounts of polysaccharide derivatives per lipid were identical irrespective of the liposome size. Introduction of cholesterol within the liposomal lipid components suppressed the size increase in these liposomes for at least three weeks. These liposomes were stable at neutral pH, whereas the content release from liposomes was induced at weakly acidic pH. Smaller liposomes exhibited highly acidic pH-responsive content release compared with those from large liposomes. However, liposomes with 50 mol% cholesterol were not able to induce content release even under acidic conditions. These results suggest that control of the liposome size and cholesterol content is important for preparing stable liposomes at physiological conditions and for preparing highly pH-responsive liposomes for drug delivery applications.

Selective Endocytic Uptake of Targeted Liposomes Occurs within a Narrow Range of Liposome Diameters.

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes rather than the density of the ligands on their surfaces primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

Intra-articular mm-ii, a novel suspension of large empty multilamellar liposomes, in painful knee osteoarthritis: a 26 week phase 2b placebo-controlled double blind randomized trial

Intra-articular mm-ii, a novel suspension of large empty multilamellar liposomes, in painful knee osteoarthritis: a 26 week phase 2b placebo-controlled double blind randomized trial

Effect of a double-chain surfactant on the stabilization of suspensions of silica and titania particles against agglomeration and sedimentation

Dispersions of vesicles of didodecyldimethylammonium bromide (DDAB) were prepared with magnetic stirring of multilamellar liposomes followed by ultrasonication (SS) in order to generate dispersions that contained only unilamellar vesicles, unlike the stirring-only (S) method which also produces many large liposomes. With the SS method, the vesicles had average diameters ranging from 68 to 80 nm, which were much smaller than the diameters generated with the S method. The new vesicle dispersions had nearly infinite viscosities at very low shear stresses at DDAB weight fractions wD from 0.025 to 0.027, even though they were not close-packed, in contrast to the S dispersions. The new SS vesicle dispersions stabilized completely against sedimentation polydisperse nonspherical crystalline titania particles with sizes ranging from ca. 96–156 nm, as well as suspensions of monodisperse spherical amorphous silica particles with diameters of dsed = 454 nm, 691 nm, and 826 nm. These results establish that DDAB vesicle dispersions can stabilize against sedimentation a broad range of particle types and sizes. In addition, at the low values of wD = 0.009 and 0.018, the effective viscosities, ηeff, of the DDAB dispersions, determined from the sedimentation velocities, ranged from 1.35-1.87 cP and 4.34–5.57 cP, respectively. At higher shear stresses, however, these dispersions were highly shear-thinning and still flowable in a capillary under gravity. This behavior is critical for the practical applications of such dispersions for inkjet printing and possibly paints.

Biosynthetic Gas Vesicles Combined with Focused Ultrasound for Blood-Brain Barrier Opening.

Focused ultrasound (FUS) combined with microbubbles (MBs) has emerged as a potential approach for opening the blood-brain barrier (BBB) for delivering drugs into the brain. However, MBs range in size of microns and thus can hardly extravasate into the brain parenchyma. Recently, growing attention has been paid to gas vesicles (GVs), which are genetically encoded gas-filled nanostructures with protein shells, due to their potential for extravascular targeting in ultrasound imaging and therapy. However, the use of GVs as agents for BBB opening has not yet been investigated. In this study, GVs were extracted and purified from Halobacterium NRC-1. Ultrasound imaging performance of GVs was assessed in vitro and in vivo. Then, FUS/GVs-mediated BBB opening for small molecular Evans blue or large molecular liposome delivery across the BBB was examined. The results showed a good contrast performance of GVs for brain perfusion ultrasound imaging in vivo. At the acoustic negative pressure of 1.5 MPa, FUS/GVs opened the BBB safely, and effectively enhanced Evans blue and 200-nm liposome delivery into the brain parenchyma. Our study suggests that biosynthetic GVs hold great potential to serve as local BBB-opening agents in the development of new targeted drug delivery strategies for central nervous system disorders.

Exposure of the Basophilic Cell Line KU812 to Liposomes Reveals Activation Profiles Associated with Potential Anaphylactic Responses Linked to Physico-Chemical Characteristics.

Lipidic nanoparticles (LNP), particularly liposomes, have been proven to be a successful and versatile platform for intracellular drug delivery for decades. Whilst primarily developed for small molecule delivery, liposomes have recently undergone a renaissance due to their success in vaccination strategies, delivering nucleic acids, in the COVID-19 pandemic. As such, liposomes are increasingly being investigated for the delivery of nucleic acids, beyond mRNA, as non-viral gene delivery vectors. Although not generally considered toxic, liposomes are increasingly shown to not be immunologically inert, which may have advantages in vaccine applications but may limit their use in other conditions where immunological responses may lead to adverse events, particularly those associated with complement activation. We sought to assess a small panel of liposomes varying in a number of physico-chemical characteristics associated with complement activation and inflammatory responses, and examine how basophil-like cells may respond to them. Basophils, as well as other cell types, are involved in the anaphylactic responses to liposomes but are difficult to isolate in sufficient numbers to conduct large scale analysis. Here, we report the use of the human KU812 cell line as a surrogate for primary basophils. Multiple phenotypic markers of activation were assessed, as well as the release of histamine and inflammasome activity within the cells. We found that larger liposomes were more likely to result in KU812 activation, and that non-PEGylated liposomes were potent stimulators of inflammasome activity (four-fold greater IL-1β secretion than untreated controls), and a lower ratio of cholesterol to lipid was also associated with greater IL-1β secretion ([Cholesterol:DSPC ratio] 1:10; 0.35 pg/mL IL-1β vs. 5:10; 0.1 pg/mL). Additionally, PEGylation appeared to be associated with direct KU812 activation. These results suggest possible mechanisms related to the consequences of complement activation that may be underpinned by basophilic cells, in addition to other immune cell types. Investigation of the mechanisms behind these responses, and their impact on use in vivo, are now warranted.

Nanoparticle Fraught Liposomes: A Platform for Increased Antibiotic Selectivity in Multidrug Resistant Bacteria

Increasing antibiotic concentrations within bacterial cells while reducing them in mammalian ones would ultimately result in an enhancement of antibacterial actions, overcoming multidrug resistance, all while minimizing toxicity. Nanoparticles (NPs) have been used in numerous occasions to overcome antibiotic resistance, poor drug solubility, and stability. However, the concomitant increase in antibiotic concentration in mammalian cells and the resultant toxicity are usually overlooked. Without compromising bacterial cell fusion, large liposomes (Lip) have been reported to show reduced uptake in mammalian cells. Therefore, in this work, small NP fraught liposomes (NP-Lip) were formulated with the aim of increasing NP uptake and antibiotic delivery in bacterial cells but not in mammalian ones. Small polylactic-co-glycolic acid NPs were therefore loaded with erythromycin (Er), an antibiotic with low membrane permeability that is susceptible to drug efflux, and 3c, a 5-cyanothiazolyl urea derivative with low solubility and stability. In vitro experiments demonstrated that the incorporation of small NPs into large Lip resulted in a reduction in NP uptake by HEK293 cells while increasing it in Gram-negative bacteria (Escherichia coli DH5α, E. coli K12, and Pseudomonas aeruginosa), consequently resulting in an enhancement of antibiotic selectivity by fourfold toward E. coli (both strains) and eightfold toward P. aeruginosa. Ocular administration of NP-Lip in a P. aeruginosa keratitis mouse model demonstrated the ability of Er/3c-loaded NP-Lip to result in a complete recovery. More importantly, in comparison to NPs, the ocular administration of NP-Lip showed a reduction in TNF-alpha and IL-6 levels, implying reduced interaction with mammalian cells in vivo. This work therefore clearly demonstrated how tailoring the nano-bio interaction could result in selective drug delivery and a reduction in toxicity.

Army liposome formulation containing QS-21 render human monocyte-derived macrophages less permissive to HIV-1 infection by upregulating APOBEC3A

Monocyte-derived macrophages (MDM) are highly permissive to HIV-1 infection potentially due to the downregulation of innate factors during the differentiation process. The environmental milieu and innate anti-viral factors which are modulated during macrophage differentiation, have been associated with their increased permissiveness to HIV-1 infection. Here, we demonstrate that the Army Liposome Formulation containing MPLA, and QS-21 (ALFQ) activated MDM that are normally permissive to HIV-1 infection to generate a proinflammatory environment and upregulated anti-viral factors notably APOBEC3A. Induction of APOBEC3A by ALFQ decreased permissiveness to HIV-1 infection, while knockdown of APOBEC3A with APOBEC3AsiRNA resulted in a significant loss in the restriction of HIV-1 infectivity. The liposome formulation ALF55, with identical lipid composition but lacking QS-21 had no effect. Furthermore, the capacity of ALFQ to modulate MDM permissiveness to HIV-1 infection was predominantly mediated by large ALFQ liposomes. Our findings highlight a relationship between innate immune activation, proinflammatory milieu, and upregulation of anti-HIV proteins. Induction of these responses can switch the HIV-1 permissive MDM into a more refractory phenotype.

Enhancement of cerebroprotective effects of lipid nanoparticles encapsulating FK506 on cerebral ischemia/reperfusion injury by particle size regulation

Delivery of cerebroprotective agents using liposomes has been demonstrated to be useful for treating cerebral ischemia/reperfusion (I/R) injury. We previously reported that intravenous administration of liposomes with diameters of 100 nm showed higher accumulation in the I/R region compared with larger liposomes (>200 nm) by passage through the disintegrated blood-brain barrier, suggesting a size-dependence for liposome-mediated drug delivery. Based on these findings, we hypothesized that regulation of liposomal particle size (<100 nm) may enhance the therapeutic efficacy of encapsulated drugs on cerebral I/R injury. Herein, we prepared lipid nanoparticles (LNP) with particle sizes <100 nm by the microfluidics method and compared their therapeutic potential with LNP exhibiting sizes >100 nm in cerebral I/R model rats. Intravenously administered smaller LNP (ca. 60 nm) exhibited wider accumulation and diffusivity in the brain parenchyma of the I/R region compared with larger LNP (>100 nm). Importantly, treatment with LNP encapsulating the cerebroprotective agent FK506 (FK-LNP) with particle sizes <100 nm showed greater cerebroprotective effects than FK-LNP with sizes >100 nm, and also significantly ameliorated brain injury. These results suggest that particle size regulation of LNP to sizes <100 nm can enhance the therapeutic effect of encapsulated drugs for treatment of cerebral I/R injury, and that FK-LNP could be a promising cerebroprotective agent.

LARGE MULTILAMELLAR NEUTRAL LIPOSOMES FORM A STABLE LAYER ON ARTICULAR CARTILAGE SURFACE UNDER EX-VIVO PHYSIOLOGICAL COMPRESSION

Purpose: Surface active phospholipids (SAPLs) including dipalmitoylphosphatidylcholine (DPPC) play a vital role in the natural boundary lubricant of articular cartilage and are degraded during progression of osteoarthritis (OA). MM-II is a suspension of large multilamellar neutral liposomes composed of two phospholipids, dimyristoylphosphatidylcholine (DMPC) and DPPC which is administered via intra-articular (IA) administration and is currently in a phase 2b clinical study. In a series of ex-vivo cartilage-on-cartilage friction and wear tests, MM-II liposomes were shown to lubricate cartilage and reduce hence friction and wear.

Tissue response and retention of micro- and nanosized liposomes in infarcted mice myocardium after ultrasound-guided transthoracic injection

Different carrier systems have been investigated for myocardial delivery of biopharmaceuticals for heart disease. Here, we aimed to evaluate the heart retention and tissue response of liposomes intended for cardiac drug delivery. Liposomes were produced by the lipid thin film hydration method followed by sonication. Cytocompatibility tests were performed in murine L929 fibroblasts and H2c9 cardiomyocytes using the Alamar Blue assay. In vivo experiments were assessed in a model of myocardial infarction induced by isoproterenol in mice. Cardiac delivery of fluorescent liposomes (rhodamine B-labeled) with different mean sizes (165 nm, 468 nm, 1551 nm and 1954 nm) was performed by ultrasound-guided transthoracic injection. After three days, mice were euthanized for histological evaluation using optical and fluorescence microscopy. No cytotoxic lipid concentrations were determined in the range 9.3 – 120 µM for fibroblasts and cardiomyocytes exposed to liposomes. In vivo, large liposomes induced significant inflammation in myocardium compared with the control group (p < 0.0001). In contrast, heart mice injected with 468 nm-sized liposomes exhibited a lower number of inflammatory cells. Still, a greater tissue retention 72 h post-injection was found. Therefore, this study demonstrated the feasibility of the echocardiography-guided percutaneous injection to deliver liposomes successfully into the myocardium in a minimally invasive manner. In addition, these findings indicate the potential of liposomes as carriers of biopharmaceuticals for myocardial delivery, supporting the development of further research on these delivery systems for heart disease.

Application of Asymmetrical Flow Field-Flow Fractionation for Characterizing the Size and Drug Release Kinetics of Theranostic Lipid Nanovesicles.

Liposome size and in vitro release of the active substance belong to critical quality attributes of liposomal carriers. Here, we apply asymmetric flow field-flow fractionation (AF4) to characterize theranostic liposomes prepared by thin lipid film hydration/extrusion or microfluidics. The vesicles’ size was derived from multi-angle laser light scattering following fractionation (AF4) and compared to sizes derived from dynamic light scattering measurements. Additionally, we adapted a previously developed AF4 method to study zinc phthalocyanine (ZnPc) release/transfer from theranostic liposomes. To this end, theranostic liposomes were incubated with large acceptor liposomes serving as a sink (mimicking biological sinks) and were subsequently separated by AF4. During incubation, ZnPc was transferred from donor to acceptor fraction until reaching equilibrium. The process followed first-order kinetics with half-lives between 119.5–277.3 min, depending on the formulation. The release mechanism was postulated to represent a combination of Fickian diffusion and liposome relaxation. The rate constant of the transfer was proportional to the liposome size and inversely proportional to the ZnPc/POPC molar ratio. Our results confirm the usefulness of AF4 based method to study in vitro release/transfer of lipophilic payload, which may be useful to estimate the unwanted loss of drug from the liposomal carrier in vivo.