Fusogenic Lipid Research Articles

A Mechanical Immune Checkpoint Inhibitor Stiffens Tumor Cells to Potentiate Antitumor Immunity.

Tumor progression is associated with tumor-cell softening. Improving the stiffness of the tumor cells can make them more vulnerable to lymphocyte-mediated attack. Tumor cell membranes typically exhibit higher cholesterol levels than normal cells, making tumor cells soft. Herein, we demonstrate a mechanical immune checkpoint inhibitor (MICI) formulated by cyclodextrin (CD) lipids and fusogenic lipids. Through fusing CD lipids into the tumor cell membrane using a fusogenic liposome formulation, the cholesterol in the plasma membrane is reduced due to the specific host-guest interactions between CD lipid and cholesterol. As a result, tumor cells are stiffened, and the activation of lymphocytes (including NK and cytotoxic effector T cells) is improved when contacting the stiffened tumor cells, characterized by robust degranulation and effector cytokine production. Notably, this treatment has negligible influence on the infiltration and proliferation of lymphocytes in tumor tissues, confirming that the enhanced antitumor efficacy should result from activating a specific number of lymphocytes caused by direct regulation of the tumor cell stiffness. The combination of MICIs and clinical immunotherapies enhances the lymphocyte-mediated antitumor effects in two tumor mouse models, including breast cancer and melanoma. Our research also reveals an unappreciated mechanical dimension to lymphocyte activation.

A Mechanical Immune Checkpoint Inhibitor Stiffens Tumor Cells to Potentiate Antitumor Immunity

Tumor progression is associated with tumor‐cell softening. Improving the stiffness of the tumor cells can make them more vulnerable to lymphocyte‐mediated attack. Tumor cell membranes typically exhibit higher cholesterol levels than normal cells, making tumor cells soft. Herein, we demonstrate a mechanical immune checkpoint inhibitor (MICI) formulated by cyclodextrin (CD) lipids and fusogenic lipids. Through fusing CD lipids into the tumor cell membrane using a fusogenic liposome formulation, the cholesterol in the plasma membrane is reduced due to the specific host–guest interactions between CD lipid and cholesterol. As a result, tumor cells are stiffened, and the activation of lymphocytes (including NK and cytotoxic effector T cells) is improved when contacting the stiffened tumor cells, characterized by robust degranulation and effector cytokine production. Notably, this treatment has negligible influence on the infiltration and proliferation of lymphocytes in tumor tissues, confirming that the enhanced antitumor efficacy should result from activating a specific number of lymphocytes caused by direct regulation of the tumor cell stiffness. The combination of MICIs and clinical immunotherapies enhances the lymphocyte‐mediated antitumor effects in two tumor mouse models, including breast cancer and melanoma. Our research also reveals an unappreciated mechanical dimension to lymphocyte activation.

Sphingolipids containing very long-chain fatty acids regulate Ypt7 function during the tethering stage of vacuole fusion

Sphingolipids are essential in membrane trafficking and cellular homeostasis. Here, we show that sphingolipids containing very long-chain fatty acids (VLCFAs) promote homotypic vacuolar fusion in Saccharomyces cerevisiae. The elongase Elo3 adds the last two carbons to VLCFAs that are incorporated into sphingolipids. Cells lacking Elo3 have fragmented vacuoles, which is also seen when WT cells are treated with the sphingolipid synthesis inhibitor Aureobasidin-A. Isolated elo3Δ vacuoles show acidification defects and increased membrane fluidity, and this correlates with deficient fusion. Fusion arrest occurs at the tethering stage as elo3Δ vacuoles fail to cluster efficiently invitro. Unlike HOPS and fusogenic lipids, GFP-Ypt7 does not enrich at elo3Δ vertex microdomains, a hallmark of vacuole docking prior to fusion. Pulldown assays using bacterially expressed GST-Ypt7 showed that HOPS from elo3Δ vacuole extracts failed to bind GST-Ypt7 while HOPS from WT extracts interacted strongly with GST-Ypt7. Treatment of WT vacuoles with the fluidizing anesthetic dibucaine recapitulates the elo3Δ phenotype and shows increased membrane fluidity, mislocalized GFP-Ypt7, inhibited fusion, and attenuated acidification. Together these data suggest that sphingolipids contribute to Rab-mediated tethering and docking required for vacuole fusion.

Local mRNA Delivery from Nanocomposites Made of Gelatin and Hydroxyapatite Nanoparticles.

Local delivery of messenger ribonucleic acid (mRNA) is increasingly being advocated as a promising new strategy to enhance the performance of biomaterials. While extensive research has been dedicated to the complexation of these oligonucleotides into nanoparticles to facilitate systemic delivery, research on developing suitable biomaterial carriers for the local delivery of mRNA is still scarce. So far, mRNA-nanoparticles (mRNA-NPs) are mainly loaded into traditional polymeric hydrogels. Here, we show that calcium phosphate nanoparticles can be used for both reinforcement of nanoparticle-based hydrogels and the complexation of mRNA. mRNA was incorporated into lipid-coated calcium phosphate nanoparticles (LCPs) formulated with a fusogenic ionizable lipid in the outer layer of the lipid coat. Nanocomposites of gelatin and hydroxyapatite nanoparticles were prepared at various ratios. Higher hydroxyapatite nanoparticle content increased the viscoelastic properties of the nanocomposite but did not affect its self-healing ability. Combination of these nanocomposites with peptide, lipid, and the LCP mRNA formulations achieved local mRNA release as demonstrated by protein expression in cells in contact with the biomaterials. The LCP-based formulation was superior to the other formulations by showing less sensitivity to hydroxyapatite and the highest cytocompatibility.

Engineered hybrid carbon nanohorns-lipid platforms for the delivery of cisplatin to the colorectal cancer

Carbon nanostructures are considered distinctive multi-functional drug delivery platforms for cancer therapy. However, their potential biocompatibility is still a significant challenge and rational design of these carriers is a prospect in the context of efficient cancer therapy. Herein, we report on the synthesis of two bio-inspired carbon nanohorns (CNH) platforms with tumor targeting capabilities for the delivery of cisplatin. Oxidized CNH wrapped with either polyethylene glycol (PEG) and fusogenic lipids (PEG/DSPG CNH-CP) or glutamic acid-cisplatin complex (CNH-GA-CP), revealed nanosized dahlia-like structure approved by dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis. Further structure features analysis by using RAMAN, FTIR spectroscopy and TGA indicated CNH surface modification. Excitingly, the superior nanofeatures of the CNH platforms was due to the reduced hydrophobic interactions, and thus closely linked to the surface modifications. Both platforms preserved cisplatin toxicity in vitro on C26 cells. The PEGylated nanoplatform demonstrated elongated circulation time compared to the free drug, and showed a site-specific delivery to the tumor site, while reducing the accumulation within kidney. Following 3 i.v. doses at 3 mg/kg of cisplatin, PEGylated CNH significantly delayed tumor growth and improved the life span of C26-bearing BALB/c mice. These notable results were further approved by the histopathological data, demonstrating reduced degeneration, necrosis and inflammation in both kidney and liver tissues following PEGylated nanoplatform administration. In conclusion, our findings suggest that the systemic toxicity of cisplatin including nephrotoxicity can be resolved by delivering this chemotherapeutic through a rationally-fabricated surface-modified carbon nanoplatform to improve the cancer therapy outcome.

Fusogenic Lipid Nanovesicle for Biomacromolecular Delivery.

Although biomacromolecules are promising cytosolic drugs which have attracted tremendous attention, the major obstacles were the cellular membrane hindering the entrance and the endosome entrapment inducing biomacromolecule degradation. How to avoid those limitations to realize directly cytosolic delivery was still a challenge. Here, we prepared oligoarginine modified lipid to assemble a nanovesicle for biomacromolecules delivery, including mRNA (mRNA) and proteins which could be directly delivered into the cytoplasm of dendritic cells through subendocytosis-mediated membrane fusion. We named this membrane fusion lipid nanovesicle as MF-LNV. The mRNA loaded MF-LNV as nanovaccines showed efficient antigen expression to elicit robust immuno responses for cancer therapy. What's more, the antigen protein loaded MF-LNV as nanovaccines elicits much stronger CD8+ T cell specific responses than lipid nanoparticles through normal uptake pathways. This MF-LNV represented a refreshing strategy for intracellular delivery of the biomacromolecule.

Control of mitochondrial dynamics by a fusogenic lipid.

Mitochondrial fusion enables cooperation between the mitochondrial population and is critical for mitochondrial function. Phosphatidic acid (PA) on the mitochondrial surface has a key role in mitochondrial fusion. A recent study by Su et al. shows that the nucleoside diphosphate (NDP) kinase NME3 recognizes PA and mediates its effects on mitochondrial dynamics.

Delivery of low-density lipoprotein from endocytic carriers to mitochondria supports steroidogenesis.

The low-density lipoprotein (LDL) is a major cholesterol carrier in circulation and is internalized into cells through LDL receptor (LDLR)-mediated endocytosis. The LDLR protein is highly expressed in the steroidogenic organs and LDL cholesterol is an important source for steroidogenesis. Cholesterol must be transported into the mitochondria, where steroid hormone biosynthesis initiates. However, how LDL cholesterol is conveyed to the mitochondria is poorly defined. Here, through genome-wide small hairpin RNA screening, we find that the outer mitochondrial membrane protein phospholipase D6 (PLD6), which hydrolyses cardiolipin to phosphatidic acid, accelerates LDLR degradation. PLD6 promotes the entrance of LDL and LDLR into the mitochondria, where LDLR is degraded by mitochondrial proteases and LDL-carried cholesterol is used for steroid hormone biosynthesis. Mechanistically, the outer mitochondrial membrane protein CISD2 binds to the cytosolic tail of LDLR and tethers LDLR+ vesicles to the mitochondria. The fusogenic lipid phosphatidic acid generated by PLD6 facilitates the membrane fusion of LDLR+ vesicles with the mitochondria. This intracellular transport pathway of LDL-LDLR bypasses the lysosomes and delivers cholesterol to the mitochondria for steroidogenesis.

V-ATPase modulates exocytosis in neuroendocrine cells through the activation of the ARNO-Arf6-PLD pathway and the synthesis of phosphatidic acid.

Although there is mounting evidence indicating that lipids serve crucial functions in cells and are implicated in a growing number of human diseases, their precise roles remain largely unknown. This is particularly true in the case of neurosecretion, where fusion with the plasma membrane of specific membrane organelles is essential. Yet, little attention has been given to the role of lipids. Recent groundbreaking research has emphasized the critical role of lipid localization at exocytotic sites and validated the essentiality of fusogenic lipids, such as phospholipase D (PLD)-generated phosphatidic acid (PA), during membrane fusion. Nevertheless, the regulatory mechanisms synchronizing the synthesis of these key lipids and neurosecretion remain poorly understood. The vacuolar ATPase (V-ATPase) has been involved both in vesicle neurotransmitter loading and in vesicle fusion. Thus, it represents an ideal candidate to regulate the fusogenic status of secretory vesicles according to their replenishment state. Indeed, the cytosolic V1 and vesicular membrane-associated V0 subdomains of V-ATPase were shown to dissociate during the stimulation of neurosecretory cells. This allows the subunits of the vesicular V0 to interact with different proteins of the secretory machinery. Here, we show that V0a1 interacts with the Arf nucleotide-binding site opener (ARNO) and promotes the activation of the Arf6 GTPase during the exocytosis in neuroendocrine cells. When the interaction between V0a1 and ARNO was disrupted, it resulted in the inhibition of PLD activation, synthesis of phosphatidic acid during exocytosis, and changes in the timing of fusion events. These findings indicate that the separation of V1 from V0 could function as a signal to initiate the ARNO-Arf6-PLD1 pathway and facilitate the production of phosphatidic acid, which is essential for effective exocytosis in neuroendocrine cells.

Application of Cell Penetrating Peptides as a Promising Drug Carrier to Combat Viral Infections.

Novel effective drugs or therapeutic vaccines have been already developed to eradicate viral infections. Some non-viral carriers have been used for effective drug delivery to a target cell or tissue. Among them, cell penetrating peptides (CPPs) attracted a special interest to enhance drug delivery into the cells with low toxicity. They were also applied to transfer peptide/protein-based and nucleic acids-based therapeutic vaccines against viral infections. CPPs-conjugated drugs or vaccines were investigated in several viral infections including poliovirus, Ebola, coronavirus, herpes simplex virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, Japanese encephalitis virus, and influenza A virus. Some studies showed that the uptake of CPPs or CPPs-conjugated drugs can be performed through both non-endocytic and endocytic pathways. Despite high potential of CPPs for cargo delivery, there are some serious drawbacks such as non-tissue-specificity, instability, and suboptimal pharmacokinetics features that limit their clinical applications. At present, some solutions are utilized to improve the CPPs properties such as conjugation of CPPs with targeting moieties, the use of fusogenic lipids, generation of the proton sponge effect, etc. Up to now, no CPP or composition containing CPPs has been approved by the Food and Drug Administration (FDA) due to the lack of sufficient in vivo studies on stability, immunological assays, toxicity, and endosomal escape of CPPs. In this review, we briefly describe the properties, uptake mechanisms, advantages and disadvantages, and improvement of intracellular delivery, and bioavailability of cell penetrating peptides. Moreover, we focus on their application as an effective drug carrier to combat viral infections.

Lipid nanoparticle-based mRNA candidates elicit potent T cell responses.

The induction of a potent T cell response is essential for successful tumor immunotherapy and protection against many infectious diseases. In the past few years, mRNA vaccines have emerged as potent immune activators and inducers of a robust T cell immune response. The recent approval of the Moderna and the Pfizer/BioNTech vaccines based on lipid nanoparticles (LNP) encapsulating antigen-encoding mRNA has revolutionized the field of vaccines. The advantages of LNPs are their ease of design and formulation resulting in potent, effective, and safe vaccines. However, there is still plenty of room for improvement with respect to LNP efficacy, for instance, by optimizing the lipid composition and tuning LNP for specific purposes. mRNA delivery is known to be strongly dependent on the lipid composition of LNPs and the efficiency is mainly determined by the ionizable lipids. Besides that, cholesterol and helper lipids also play important roles in mRNA transfection potency. Here, a panel of LNP formulations was studied by keeping the ionizable lipids constant, replacing cholesterol with β-sitosterol, and changing the fusogenic helper lipid DOPE content. We studied the ability of this LNP library to induce antigen presentation and T cell proliferation to identify superior LNP candidates eliciting potent T cell immune responses. We hypothesize that using β-sitosterol and increasing DOPE content would boost the mRNA transfection on immune cells and result in enhanced immune responses. Transfection of immortal immune cell lines and bone marrow dendritic cells (BMDCs) with LNPs was studied. Delivery of mRNA coding for the model antigen ovalbumin (OVA-mRNA) to BMDCs with a number of LNP formulations, resulted in a high level of activation, as evidenced by the upregulation of the co-stimulatory receptors (CD40 and CD86) and IL-12 in BMDCs. The enhancement of BMDC activation and T cell proliferation induced by the introduction of β-sitosterol and fusogenic DOPE lipids were cell dependent. Four LNP formulations (C12-200-cho-10%DOPE, C12-200-sito-10%DOPE, cKK-E12-cho-10%DOPE and cKK-E12-sito-30%DOPE) were identified that induced robust T cell proliferation and enhanced IFN-γ, TNF-α, IL-2 expression. These results demonstrate that T cell proliferation is strongly dependent on LNP composition and promising LNP-mRNA vaccine formulations were identified.

12]aneN3-modified camptothecin and PEGylated AIEgens co-assembly into core-shell nanoparticles with ROS/NTR dual-response for enhanced cancer therapy.

A novel dual-responsive nanoparticle (NP) system was aimed to be developed for the co-delivery of camptothecin (CPT) and plasmid encoding TNF-related apoptosis-inducing ligand (pTRAIL) DNA in cancer therapy. The combination of the prodrug CPT and the nucleic acid condensing di-(triazole-[12]aneN3) unit with 4-nitrobenzyl ester through alkyl chains resulted in three nitroreductase (NTR) responsive amphiphiles, CNN1-CNN3 (with 5, 8, and 11 carbon chains, respectively). Among them, CNN2 was the most effective in inhibiting the proliferation of HeLa cells in the presence of fusogenic lipid DOPE. The NPs composed of CNN2, pDNA, and DOPE were further co-assembled with ROS-responsive thioketal-linked amphiphilic polymer (TTP) to afford the core-shell NPs (CNN2-DT/pDNA) with an average size of 118 nm, which exhibited high drug-loading capacity, excellent serum tolerance, and good biocompatibility. In the presence of ROS, NTR, and NADH, the core-shell NPs were decomposed, leading to the efficient release of 80% CPT and abundant pDNA. The self-assembly and delivery process of CNN2-DT NPs and DNA were clearly observed through the AIE fluorescent imaging. In vitro and in vivo results demonstrated that the CNN2-DT/pTRAIL NPs synergistically promoted 68% apoptosis of tumor cells and inhibited tumor growth with negligible toxic side effects. This study showed that the combination of prodrug and nucleic acid through dual-responsive core-shell NPs provide a spatially and temporally-controlled strategy for cancer therapy.

Membrane permeabilization can be crucially biased by a fusogenic lipid composition - leaky fusion caused by antimicrobial peptides in model membranes.

Induced membrane permeabilization or leakage is often taken as an indication for activity of membrane-active molecules, such as antimicrobial peptides (AMPs). The exact leakage mechanism is often unknown, but important, because certain mechanisms might actually contribute to microbial killing, while others are unselective, or potentially irrelevant in an in vivo situation. Using an antimicrobial example peptide (cR3W3), we illustrate one of the potentially misleading leakage mechanisms: leaky fusion, where leakage is coupled to membrane fusion. Like many others, we examine peptide-induced leakage in model vesicles consisting of binary mixtures of anionic and zwitterionic phospholipids. In fact, phosphatidylglycerol and phosphatidylethanolamine (PG/PE) are supposed to reflect bacterial membranes, but exhibit a high propensity for vesicle aggregation and fusion. We describe the implications of this vesicle fusion and aggregation for the reliability of model studies. The ambiguous role of the relatively fusogenic PE-lipids becomes clear as leakage decreases significantly when aggregation and fusion are prevented by sterical shielding. Furthermore, the mechanism of leakage changes if PE is exchanged for phosphatidylcholine (PC). We thus point out that the lipid composition of model membranes can be biased towards leaky fusion. This can lead to discrepancies between model studies and activity in true microbes, because leaky fusion is likely prevented by bacterial peptidoglycan layers. In conclusion, choosing the model membrane might implicate the type of effect (here leakage mechanism) that is observed. In the worst case, as with leaky fusion of PG/PE vesicles, this is not directly relevant for the intended antimicrobial application.

The composition of fusogenic lipid mixtures at the air-water modulates the physicochemical properties changes upon interaction with lysicamine

Knowing how a bioactive compound interacts with cell membranes is important to understand its effect at the molecular level. In this sense, this work aimed to study the interaction of lysicamine, an alkaloid with action against lung cancer cell lines, with lipid monolayers as cell membrane models. We employed two lipid mixtures: the first composed of 35% DOPC, 30% DOPE, 20% sphingomyelin, and 15% cholesterol as healthy cell membranes models (MM1), and the second replacing DOPC with DOPS as cancer cells models (MM2). The interaction of lysicamine with the monolayers was evaluated using tensiometry, Brewster angle microscopy (BAM), and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). Lysicamine had interfacial effects in both membrane models. For MM 1, it expanded the lipid monolayer and changed the interfacial rheological properties, increasing the in-plane elasticity of the films. PM-IRRAS spectra suggested a higher conformational disorder of the alkyl chains of the lipids. For MM 2, lysicamine also shifted the isotherms to higher areas, expanding the monolayers, but with no significant alteration in their interfacial rheological properties. PM-IRRAS spectra also suggested higher disorder in the orientation of the lipid alkyl chains upon lysicamine incorporation. For both models, BAM did not show alteration in interfacial aggregation upon drug incorporation. In conclusion, changes in some interfacial properties of membrane models caused by lysicamine depend on the monolayer composition, which can be associated with its bioactivity in cellular membranes.

Intracellular delivery of messenger RNA to macrophages with surfactant-derived lipid nanoparticles

The broad biomedical applications of messenger RNA (mRNA)-based therapeutics rely heavily on the rapid development of mRNA delivery systems. In this study, dual-component lipid nanoparticles (LNPs) were developed for intracellular delivery of mRNA to macrophages. By screening a panel of quaternary ammonium compounds with varying alkyl or aryl side chains, a cationic surfactant containing two hexadecyl tails was found to be effective for mRNA delivery with the assistance of fusogenic lipids. Further investigating structure alterations and optimization of formulation parameters, we found that a surfactant-derived ionizable lipid mixed with a fusogenic lipid was capable of condensing mRNA and self-assembling into LNPs. Without using the PEGylated lipid, the resulting LNPs were able to render the exogenous mRNA resistant to hydrolysis by nucleases and displayed excellent biocompatibility, along with the capacity to deliver mRNA to hard-to-transfect. Overall, the dual-component nanocarrier provides a novel mRNA delivery platform for genetic engineering of macrophages.

Polyphenol-Functionalized Cubosomes as Thrombolytic Drug Carriers.

The safeadministration of thrombolytic agents is a challenge for the treatment of acute thrombosis. Lipid-based nanoparticle drug delivery technologies present opportunities to overcome the existing clinical limitations and deliver thrombolytic therapy with enhanced therapeutic outcomes and safety. Herein, lipid cubosomes are examined as nanocarriers for the encapsulation of thrombolytic drugs. The lipid cubosomes are loaded with the thrombolytic drug urokinase-type plasminogen activator (uPA) and coated with a low-fouling peptide that is incorporated within a metal-phenolic network (MPN). The peptide-containing MPN (pep-MPN) coating inhibits the direct contact of uPA with the surrounding environment, as assessed by an in vitro plasminogen activation assay and an ex vivo whole blood clot degradation assay. The pep-MPN-coated cubosomes prepared with 22 wt% peptide demonstrate a cell membrane-dependent thrombolytic activity, which is attributed to their fusogenic lipid behavior. Moreover, compared with the uncoated lipid cubosomes, the uPA-loaded pep-MPN-coated cubosomes demonstrate significantly reduced nonspecific cell association (<10% of the uncoated cubosomes) in the whole blood assay, a prolonged circulating half-life, and reduced splenic uPA accumulation in mice. These studies confirm the preserved bioactivity and cell membrane-dependent release of uPA within pep-MPN-coated lipid cubosomes, highlighting their potential as a delivery vehicle for thrombolytic drugs.

Lipid nanoparticle-mediated CRISPR/Cas9 gene editing and metabolic engineering for anticancer immunotherapy

Metabolic engineering of the tumor microenvironment has emerged as a new strategy. Lactate dehydrogenase A (LDHA) is a prominent target for metabolic engineering. Here, we designed a cationic lipid nanoparticle formulation for LDHA gene editing. The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein (GFP). The delivery efficiency was affected by the ratios of three components: a cationic lipid, cholesterol or its derivative, and a fusogenic lipid. The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA, yielding the lipoplex, pCas9-sgLDHA/F3. The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells. Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium. pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture. In vivo, combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice. This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.

Optimization and Characterization of a Liposomal Azithromycin Formulation for Alternative Macrophage Activation

Liposomal azithromycin (L-AZM) promotes macrophage polarization toward an M2-like phenotype in the context of myocardial infarction that results in improved cardiovascular outcomes in mice. To improve upon this formulation, we sought to identify optimized formulation, stability, and biological activity parameters necessary to enhance the immunomodulatory activity and efficacy of L-AZM. While our parent formulation contains a mixture of long-chain saturated phosphatidylcholine and phosphatidylglycerol lipids, we evaluated a series of formulations with different amounts of unsaturated lipids and cholesterol with the goal of improving the loading capacity and stability of the formulations. We also introduce fusogenic lipids to improve the cytosolic delivery to enhance the immune modulatory properties of the drug. To achieve these goals, we initially prepared a library of 24 formulations using thin film hydration and assessed the resultant liposomes for size and polydispersity. Five lead formulations were identified based on low polydispersity (<0.3) and stability over time. The lead formulations were then evaluated for stability in serum using dialysis and macrophage polarization activity in vitro as measured by decreased IL-12 expression. Collectively, our data indicate that the formulation components drive the balance between encapsulation efficiency and stability and that all the lead liposomal formulations improve in vitro alternative macrophage activation as compared to free AZM.

Development of a Liver‐Targeted Drug Delivery Platform for Soluble Epoxide Hydrolase Inhibitors

BackgroundMany drugs administered systemically suffer from drawbacks including off‐target effects. One approach to address this limitation is tissue‐ or cell‐targeted drug delivery, which can also improve therapeutic efficacy by increasing the “effective” dose. The goal of the current study was to develop a system to target a soluble epoxide hydrolase (sEH) inhibitor to the liver, since previous studies from our group and others demonstrated that elevated hepatic sEH contributes to the pathogenesis of liver diseases, including alcohol‐associated liver disease (ALD). We tested the hypothesis that encapsulation of an sEH inhibitor in fusogenic lipid vesicles (FLVs) would enable liver targeting and ameliorate liver injury in a mouse model of ALD.MethodsFLVs were constructed with unsaturated lipids 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphate at a 3:2 molar ratio. FLVs were loaded with the sEH inhibitor t‐TUCB at doses ranging from 0‐9 mg/kg, then tagged with the fluorescent tracer DiD. Physical properties including size and zeta potential, as well as encapsulation efficiency were measured using a nanoparticle tracker analyzer and spectroscopy, respectively. To test the efficacy of t‐TUCB‐FLVs in vivo, we used a chronic‐binge mouse model of ALD. Specifically, male C57BL/6 mice were fed a liquid diet containing ethanol for ten days followed by a single ethanol binge nine hours prior to sacrifice. t‐TUCB‐FLVs were administered by intraperitoneal injection two hours prior to the ethanol binge. We measured endpoints related to liver injury, steatosis, inflammation, cell death, and endoplasmic reticulum (ER) stress. One‐way ANOVA was used to determine significance between groups (α = 0.05).ResultsAnalysis of size, zeta potential, and encapsulation efficiency showed favorable physical characteristics for passively targeting t‐TUCB‐FLVs to the liver (129nm diameter, ‐55mV zeta potential, and 92% encapsulation efficiency). In a pilot dose response study, the presence of t‐TUCB‐FLVs in the liver was confirmed by fluorescent imaging and magnetic cell sorting followed by flow cytometry. In an experimental animal model, mice which received ethanol and 3.0 mg/kg t‐TUCB‐FLVs (observed to be the most effective dose) had significantly lower liver injury by plasma ALT levels compared to mice receiving ethanol and empty liposomes alone (47.75 U/L vs. 72.93 U/L, respectively). t‐TUCB‐FLV‐treated mice also had decreased liver parenchymal cell death (by TUNEL staining) and markers of ER stress (Atf4, Atf6, and spliced Xbp1), with no significant effects on steatosis.ConclusionsWe successfully developed a passive liver targeting system to deliver an sEH inhibitor to the liver in mice. In an ALD model, this drug formulation attenuated liver injury, cell death, and ER stress. This novel approach may help avoid clinical pitfalls which cause experimental drugs to fail in clinical trials, such as low efficacy or off‐target side effects. Future research will further evaluate the mechanisms of t‐TUCB‐FLVs in ALD, evaluate potential side effects, and consider an active targeting approach.

Novel Liposomal Rolipram Formulation for Clinical Application to Reduce Emesis.

IntroductionThe phosphodiesterase 4 (PDE4) inhibitor, rolipram, has beneficial effects on tissue inflammation, injury and fibrosis, including in the liver. Since rolipram elicits significant CNS side-effects in humans (ie, nausea and emesis), our group developed a fusogenic lipid vesicle (FLV) drug delivery system that targets the liver to avoid adverse events. We evaluated whether this novel liposomal rolipram formulation reduces emesis.MethodsC57Bl/6J male mice were used to compare the effect of three doses of free and FLV-delivered (FLVs-Rol) rolipram in a behavioral correlate model of rolipram-induced emesis. Tissue rolipram and rolipram metabolite levels were measured using LC-MS/MS. The effect of FLVs-Rol on brain and liver PDE4 activities was evaluated.ResultsLow and moderate doses of free rolipram significantly reduced anesthesia duration, while the same doses of FLVs-Rol had no effect. However, the onset and duration of adverse effects (shortening of anesthesia period) elicited by a high dose of rolipram was not ameliorated by FLVs-Rol. Post-mortem analysis of brain and liver tissues demonstrated that FLVs affected the rate of rolipram uptake by liver and brain. Lastly, administration of a moderate dose of FLVs-Rol attenuated endotoxin induced PDE4 activity in the liver with negligible effect on the brain.DiscussionThe findings that the low and moderate doses of FLVs-Rol did not shorten the anesthesia duration time suggest that FLV delivery prevented critical levels of drug from crossing the blood-brain barrier (BBB) to elicit CNS side-effects. However, the inability of high dose FLVs-Rol to prevent CNS side-effects indicates that there was sufficient unencapsulated rolipram to cross the BBB and shorten anesthesia duration. Notably, a moderate dose of FLVs-Rol was able to decrease PDE4 activity in the liver without affecting the brain. Taken together, FLVs-Rol has a strong potential for clinical application for the treatment of liver disease without side effects.