Liposome Surface Research Articles

Rg3-lipo biomimetic delivery of paclitaxel enhances targeting of tumors and myeloid-derived suppressor cells.

Liposomal drug delivery systems have revolutionized traditional cytotoxic drugs. However, the relative instability and toxicity of the existing liposomal drug delivery systems compromised their efficacy. Herein, we present Rg3-lipo, an innovative drug delivery system using a glycosyl moiety-enriched ginsenoside (Rg3). This system is distinguished by its glycosyl moieties exposed on the liposomal surface. These moieties imitate human cell membranes to stabilize and evade phagocytic clearance. The Rg3-lipo system loaded with paclitaxel (PTX-Rg3-lipo) demonstrated favorable bioavailability and safety in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. With its glycosyl moieties recognizing tumor cells via the glucose transporter Glut1, PTX-Rg3-lipo inhibited gastric, breast, and esophageal cancers in human cancer cell lines, tumor-bearing mice, and patient-derived xenograft models. These glycosyl moieties selectively targeted myeloid-derived suppressor cells (MDSCs) through the glucose transporter Glut3 to attenuate their immunosuppressive effect. The mechanism study revealed that Rg3-lipo suppressed glycolysis and downregulated the transcription factors c-Maf and Mafb overcoming the MDSC-mediated immunosuppressive microenvironment and enhancing PTX-Rg3-lipo's antitumor effect. Taken together, we supply substantial evidence for its advantageous bioavailability and safety in multiple animal models, including nonhuman primates, and Rg3-lipo's dual targeting of cancer cells and MDSCs. Further investigation regarding Rg3-lipo's druggability will be conducted in clinical trials.

Target-Engineered Liposomes Decorated with Nanozymes Alleviate Liver Fibrosis by Remodeling the Liver Microenvironment.

Liver fibrosis is a pathological repair response that occurs after sustained liver damage, and prompt intervention is necessary to prevent liver fibrosis from developing into a potentially life-threatening condition. In long-term liver injury, damaged hepatocytes produce excessive amounts of reactive oxygen species (ROS), which activate hepatic stellate cells (HSCs). This activation leads to excessive accumulation of extracellular matrix proteins in liver tissue. Additionally, liver macrophages contribute to the inflammatory microenvironment in the hepatic fibrotic process, exacerbating liver fibrosis through ROS production and the secretion of pro-inflammatory factors. To address the dysregulation of the hepatic microenvironment associated with liver fibrosis, we developed cerium oxide nanozymes using hyaluronic acid (HA) as a template and decorated them on the surface of liposomes loaded with oleanolic acid (OA). We named this prepared and obtained target-engineered liposome HCOL. The inherent superoxide dismutase (SOD) and catalase (CAT) activities of HCOL enabled it to effectively scavenge ROS in HSCs and alleviate the hypoxic conditions characteristic of fibrotic livers. Furthermore, HCOL reduced the concentrations of ROS in macrophages, promoting a shift in macrophage polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. This transition increased the production of the anti-inflammatory cytokine interleukin 10 (IL-10), which contributed to the mitigation of the inflammatory microenvironment. Consequently, this therapeutic approach proves effective in decelerating the advancement of liver fibrosis.

A Coarse-Grained Molecular Dynamics Perspective on the Release of 5-Fluorouracil from Liposomes.

Liposomes, small bilayer phospholipid-containing vesicles, are frequently used to ensure slow drug release for a prolonged and improved therapeutic effect. Nevertheless, current findings on the membrane affinity and permeability of the anticancer agent 5-fluorouracil (5-FU) are confounding, which leads to a lack of a clear understanding of how lipid composition impacts the distribution of 5-FU within liposomal structures and its delivery. In the current work, we report a comprehensive coarse-grained molecular dynamics (CGMD) investigation on the influence of cholesterol (CHOL) and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) on the partitioning of 5-FU in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) double-bilayer systems, as well as its in vitro release from liposomes with identical lipid compositions. Our results show that 5-FU tends to accumulate at the water-lipid interface, in the vicinity of polar headgroups, without partitioning in the hydrophobic tail region. At the same time, the presence of CHOL proportionally increases the distribution of this drug in the interbilayer aqueous space, decreasing the drug's affinity toward the membrane polar head region, while DOTAP has only a slight effect on drug distribution. Thus, it is expected that 5-FU will be released slower from CHOL-containing DPPC liposomes but not DOTAP-containing vesicles. However, in vitro release studies showed that the release kinetics of 5-FU from DPPC vesicles is not influenced by the presence of CHOL and that the incorporation of 10 mol % DOTAP leads to the best release profile for 5-FU, highlighting the complexity of nanocarrier drug release kinetics. We hypothesize that the initial rapid release seen in dialysis experiments is not related to drug membrane permeability but rather to 5-FU adsorbed on the outer surface of liposomes.

Motor Clustering Enhances Kinesin-driven Vesicle Transport.

Intracellular vesicles frequently travel long distances, despite having few kinesin and dynein motors. By reconstituting liposome motility with kinesin-1 motors, we demonstrate the need for high motor copy numbers for long-range transport when motors are randomly distributed on the liposome surface. We further show that motor clustering reduces the required motor number, emphasizing its potential role in enhancing transport efficiency. Our findings highlight the significance of motor organization in regulating intracellular transport and suggest that motor clustering, such as by scaffolding proteins or lipid domains, influences bidirectional transport outcomes.

Neutrophil Hitchhiking Enhances Liposomal Dexamethasone Therapy of Sepsis.

Sepsis is characterized by a dysregulated immune response and is very difficult to treat. In the cecal ligation and puncture (CLP) mouse model, we show that nanomedicines can effectively alleviate systemic and local septic events by targeting neutrophils. Specifically, by decorating the surface of clinical-stage dexamethasone liposomes with cyclic arginine-glycine-aspartic acid (cRGD) peptides, we promote their engagement with neutrophils in the systemic circulation, leading to their prominent accumulation at primary and secondary sepsis sites. cRGD-targeted dexamethasone liposomes potently reduce immature circulating neutrophils and neutrophil extracellular traps in intestinal sepsis induction sites and the liver. Additionally, they mitigate inflammatory cytokines systemically and locally while preserving systemic IL-10 levels, contributing to lower IFN-γ/IL-10 ratios as compared to control liposomes and free dexamethasone. Our strategy addresses sepsis at the cellular level, illustrating the use of neutrophils both as a therapeutic target and as a chariot for drug delivery.

Electroformation and characterization of soybean protein isolate hydrolysates-modified liposomes

This research aimed to elucidate the application and feasibility of the enhancement of liposome oxidative stability by the interaction of liposomes with soybean protein isolate hydrolysates (SPIHs) during liposome electroformation. It was observed that the degree of hydrolysis, percentage of low-molecular-weight distribution, and solubility of SPIHs after enzymatic hydrolysis increased with the extension of reaction time. The average fluorescence intensity (average gray level) was approximately 200 a.u. for each liposome, indicating a homogeneous distribution of SPIHs on the liposome surface. Moreover, it was found that the advanced structure of SPIHs incorporated with liposomes was altered appreciably. Both hydrophobic forces and hydrogen bonds exhibited an important role in the interaction between SPIHs and liposomes. In addition, morphology observation indicated that the liposomes were spherical and exhibited a more compact structure after coating with SPIHs. Subsequent flow cytometry analysis showed that the diameter and number of vesicles would increase due to vesicles rupturing or reorganizing after phospholipid oxidation during the electroformation of liposomes to produce SPIHs-liposomes. While the phospholipid oxidation level of the original liposomes was 42.13%, the phospholipid oxidation level of SPIHs-liposomes did not exceed 25%, indicating that the optimal balance between electroformation parameters and phospholipid oxidation level was obtained. These results may provide novel insight into the implications of SPIHs-modified liposomes as functional additives.

Cellular uptake of CPX-351 by scavenger receptor class B type 1-mediated nonendocytic pathway

The proper uptake of drugs in liposome formulations into target cells markedly impacts therapeutic efficacy. The protein corona (PC), formed by the adsorption of serum proteins onto the liposome surface, binds to specific surface receptors of target cells, influencing the uptake pathway. We investigated the uptake pathway into leukemia cells based on PC analysis of CPX-351, a liposome containing cytarabine and daunorubicin in a fixed 5:1 synergistic molar ratio. The PC of CPX-351 mixed with fetal bovine serum was analyzed by nanoflow liquid chromatography-tandem mass spectrometry. CPX-351 uptake in HL-60, K562, and THP-1 leukemia cell lines, was measured by flow cytometry using daunorubicin fluorescence. The major components of CPX-351 PC were apolipoproteins A-I and A-II, which bind to scavenger receptor class B type 1 (SR-BI), a nonendocytic pathway that takes up only liposome contents. SR-BI was expressed in each cell, and its expression correlated with CPX-351 uptake. The uptake was significantly decreased by the inhibition of clathrin-mediated endocytosis and macropinocytosis. Additionally, blocks lipid transport-1 (BLT-1), a selective inhibitor of SR-BI, decreased the uptake; however, high-dose BLT-1 addition significantly increased the uptake, which was more strongly inhibited by macropinocytosis suppression compared with clathrin-mediated endocytosis. BLT-1 enhances the binding of SR-BI to liposomes in a dose-dependent manner. These findings indicate that the enhancement of binding between SR-BI and CPX-351 activates different pathways, such as macropinocytosis, distinct from CPX-351 alone. SR-BI may be a biomarker for CPX-351 therapy, and the combination of CPX-351 with high-dose BLT-1 may augment therapeutic efficacy.

Hybridization of Tannic Acid With Technol PG Liposomes

Objective: This study aims to create a complex of Technol PG-based liposomes with tannic acid (TA). Method: Technol PG-based liposome using a gentle hydration method was designed, and the hybridization with TA was achieved by the interaction between the surface of Technol PG-based liposome and TA. Results: Technol PG was highly soluble in methanol and ethanol, and Technol PG-based liposome could be prepared using a gentle hydration method for the film from the ethanol dispersion. Upon addition of TA, it was hybridized with Technol PG liposome, and TA-hybridized Technol PG liposome showed high dispersibility in an aqueous solution. Conclusion: This work would provide a new type of TA-conjugated liposome-based biomaterials.

Panax notoginseng saponins and acetylsalicylic acid co-delivered liposomes for targeted treatment of ischemic stroke

In this study, we aimed to develop brain-targeted co-delivery liposomes for the concurrent delivery of Panax notoginseng saponins (PNS) and acetylsalicylic acid (ASA) for the treatment of ischemic stroke. Within this system, PNS served as a cholesterol substitute, integrating into the phospholipid bilayer of the liposomes, while ASA was encapsulated internally. A poly-2-methacryloyloxyethyl phosphorylcholine (PMPC) polymer was synthesized and incorporated into the liposome surface. This formulation demonstrated an enhanced PNS-loading capacity and facilitated the synchronized delivery of key saponin components. Following PMPC modification, the liposomes exhibited prolonged circulation and improved transport across the blood–brain barrier (BBB) through acetylcholine receptor-mediated pathways. Furthermore, the co-delivery system exhibited enhanced therapeutic efficacy in a rat model of cerebral ischemia–reperfusion injury via the phosphoinositide 3-kinase/protein kinase C pathway. Additional analyses revealed significant effects on the metabolism of neurotransmitters, amino acids, folate, and various other pathways, indicating a multi-faceted therapeutic effect. Overall, this study presents an innovative research strategy for the comprehensive delivery of diverse components in traditional Chinese medicine formulations, highlighting the potential for synergistic treatments that combine traditional Chinese medicine with chemical agents.

Design optimization of Fucoidan-coating Cationic Liposomes for enhance Gemcitabine delivery.

Obstacles facing chemotherapeutic drugs for cancers led scientists to load Gemcitabine (GEM) into nanocarriers like liposomes, known for their nontoxicity profile and targeting capacity. The liposomal nanostructures containing GEM were coated with Fucoidan (FU) due to its anti-tumor properties by targeting cancer cells. Thus four different cationic liposomes formulations were prepared by thin-film hydration method in optimal conditions: DOTAP (formulation A); DPPC/DOTAP (4:1 molar ratio, formulation B), DPPC/DMPC/DOTAP (4:1:1 molar ratio, formulation C) and DPPC/DMPC/DOTAP/DSPE-mPEG2000 (4:1:1:0.1 molar ratio, formulation D). They were studied to identify lipid-compositions offering effective GEM-entrapment and successful coating of FU on the liposome surface. Additional qualitative characteristics, such as particle size, polydispersity index, zeta potential, stability and in vitro drug release were then evaluated. Formulation C gave the best GEM-entrapment efficiency (EE) but formed aggregates when coated with FU, giving non-homogenous large size particles then not suitable for effective delivery. It was the same situation with formulation A and B. Only the formulation D showed a good GEM-EE (> 80%) and affinity by successful coating FU from three different algae species. The PEGylated formulation D coated of FU, with regard to storage stability and drug release studies, revealed to be a promising approach on design of optimal drug delivery system.

Cryo-EM reconstruction of oleate hydratase bound to a phospholipid membrane bilayer

Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20–30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.

A pentavalent peptide vaccine elicits Aβ and tau antibodies with prophylactic activity in an Alzheimer’s disease mouse model

Amyloid-β (Aβ) and hyperphosphorylated tau protein are targets for Alzheimer’s Disease (AD) immunotherapies, which are generally focused on single epitopes within Aβ or tau. However, due to the complexity of both Aβ and tau in AD pathogenesis, a multipronged approach simultaneously targeting multiple epitopes of both proteins could overcome limitations of monotherapies. Herein, we propose an active AD immunotherapy based on a nanoparticle vaccine comprising two Aβ peptides (1–14 and pyroglutamate pE3-14) and three tau peptides (centered on phosphorylated pT181, pT217 and pS396/404). These correspond to both soluble and aggregated targets and are displayed on the surface of immunogenic liposomes in an orientation that maintains reactivity with epitope-specific monoclonal antibodies. Intramuscular immunization of mice with individual epitopes resulted in minimally cross-reactive antibody induction, while simultaneous co-display of 5 antigens (“5-plex”) induced antibodies against all epitopes without immune interference. Post-immune sera recognized plaques and neurofibrillary tangles from human AD brain tissue. Vaccine administration to 3xTg-AD mice using a prophylactic dosing schedule inhibited tau and amyloid pathologies and resulted in improved cognitive function. Immunization was well tolerated and did not induce antigen-specific cellular responses or persistent inflammatory responses in the peripheral or central nervous system. Antibody levels could be reversed by halting monthly vaccinations. Altogether, these results indicate that active immune therapies based on nanoparticle formulations of multiple Aβ and tau epitopes warrant further study for treating early-stage AD.

Molecular Dynamics Simulations Reveal Octanoylated Hyaluronic Acid Enhances Liposome Stability, Stealth and Targeting.

Liposome-based drug delivery systems have been widely used in drug and gene delivery. However, issues such as instability, immune clearance, and poor targeting have significantly limited their clinical utility. Consequently, there is an urgent need for innovative strategies to improve liposome performance. In this study, we explore the interaction mechanisms of hyaluronic acid (HA), a linear anionic polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine connected by alternating β-1,3 and β-1,4 glycosidic linkages, and its octanoylated derivates (OHA) with liposomes using extensive coarse-grained molecular dynamics simulations. The octyl moieties of OHA spontaneously inserted into the phospholipid bilayer of liposomes, leading to their effective coating onto the surface of liposome and enhancing their structural stability. Furthermore, encapsulating liposome with OHA neutralized their surface potential, interfering with the formation of a protein corona known to contribute to liposomal immune clearance. Importantly, the encapsulated OHA maintained its selectivity and therefore targeting ability for CD44, which is often overexpressed in tumor cells. These molecular-scale findings shed light on the interaction mechanisms between HA and liposomes and will be useful for the development of next-generation liposome-based drug delivery systems.

Coating of liposomes with β-glucans from the king oyster mushroom (Pleurotus eryngii)

The aim of this study was to investigate the ability to coat liposomes with β-glucans from Pleurotus eryngii, in order to obtain a suitable formulation for pharmaceutical purposes. After alkali extraction of the fruiting bodies of P. eryngii and solvent fractionation of the raw polysaccharide material, four polysaccharide fractions named PEA2-SS, PEA2-SP, PEA3-SS and PEA3-SP were obtained. Chemical structure analyses by methanolysis, methylation and NMR indicated that all fractions contained two types of β-glucans: one 3-linked with some branching at O-6 and one 6-linked with branches occurring at O-3. The molar mass ranged from 7 – 36 kDa. Fraction PEA2-SP was most suitable for the coating process according to the resulting charge, hydrodynamic diameter (size) and the PDI of this formulation. This was attributed to the higher Mw displayed by this fraction. After enzymatic degradation, the ability of PEA2-SP to coat the liposomes was lost, indicating the requirement of a minimum polymer molar mass, which is suggested to be around 30 kDa. Furthermore, the presence of a positive charge on the liposome surface seemed to be essential. The novel β-glucans coated liposomes were able to activate dectin-1b, a receptor on macrophages, and may have potential as a drug delivery platform.

Kinesin-1-transported liposomes prefer to go straight in 3D microtubule intersections by a mechanism shared by other molecular motors

Kinesin-1 ensembles maneuver vesicular cargoes through the three-dimensional (3D) intracellular microtubule (MT) network. To define how such cargoes navigate MT intersections, we first determined how many kinesins from an ensemble on a lipid-based cargo simultaneously engage a MT, and then determined the directional outcomes (straight, turn, terminate) for liposome cargoes at perpendicular MT intersections. Run lengths of 350-nm diameter liposomes decorated with up to 20, constitutively active, truncated kinesin-1 KIF5B (K543) were longer than single motor transported cargo, suggesting multiple motor engagement. However, detachment forces of lipid-coated beads with ~20 kinesins, measured using an optical trap, showed no more than three simultaneously engaged motors, with a single engaged kinesin predominating, indicating anticooperative MT binding. At two-dimensional (2D) and 3D in vitro MT intersections, liposomes frequently paused (~2 s), suggesting kinesins simultaneously bind both MTs and engage in a tug-of-war. Liposomes showed no directional outcome bias in 2D (1.1 straight:turn ratio) but preferentially went straight (1.8 straight:turn ratio) in 3D intersections. To explain these data, we developed a mathematical model of liposome transport incorporating the known mechanochemistry of kinesins, which diffuse on the liposome surface, and have stiff tails in both compression and extension that impact how motors engage the intersecting MTs. Our model predicts the ~3 engaged motor limit observed in the optical trap and the bias toward going straight in 3D intersections. The striking similarity of these results to our previous study of liposome transport by myosin Va suggests a "universal" mechanism by which cargoes navigate 3D intersections.

Reciprocal Interaction with Neutrophils Facilitates Cutaneous Accumulation of Liposomes.

Liposomes are versatile drug delivery systems in clinical use for cancer and many other diseases. Unfortunately, PEGylated liposomal doxorubicin (sLip/DOX) exhibits serious dose-limiting cutaneous toxicities, which are closely related to the extravascular accumulation of sLip/DOX in the dermis. No clinical interventions have been proposed for cutaneous toxicities due to the elusive transport pathways. Herein, we showed that the reciprocal interaction between liposomes and neutrophils played pivotal roles in liposome extravasation into the dermis. Neutrophils captured liposomes via the complement receptor 3 (CD11b/CD18) recognizing the fragment of complement component C3 (iC3b) deposited on the liposomal surface. Uptake of liposomes also activated neutrophils to induce CD11b upregulation and enhanced the ability of neutrophils to migrate outside the capillaries. Furthermore, inhibition of complement activation either by CRIg-L-FH (a C3b/iC3b targeted complement inhibitor) or blocking the phosphate negative charge in mPEG-DSPE could significantly reduce liposome uptake by neutrophils and alleviate the cutaneous accumulation of liposomes. These results validated the liposome extravasation pathway mediated by neutrophils and provided potential solutions to the devastating cutaneous toxicities occurring during sLip/DOX treatment.

Improved efficacy of cisplatin delivery by peanut agglutinin‑modified liposomes in non‑small cell lung cancer.

Globally, non‑small cell lung cancer (NSCLC) is a significant threat to human health, and constitutes >80% of lung cancer cases. Cisplatin (CDDP), a commonly used drug in clinical treatment, has been the focus of research aiming to mitigate its potent toxicity through encapsulation within liposomes. However, challenges, such as a reduced drug loading efficiency and nonspecific release, have emerged as obstacles. The present study aimed to improve the encapsulation efficiency of CDDP within liposomes by pre‑preparation of CDDP and modifying the liposome surface through the incorporation of peanut agglutinin (PNA) as a ligand [CDDP‑loaded PNA‑modified liposomes (CDDP‑PNA‑Lip)]. This strategy was designed to enhance the delivery of CDDP to tumour tissues, thereby reducing associated side effects. The effect of CDDP‑PNA‑Lip on the proliferation and migration of NSCLC cell lines with high MUC1 expression was elucidated through in vitro studies. Additionally, the capacity of PNA modification to augment the targeted anti‑tumour efficacy of liposomes was assessed through xenograft tumour experiments. The results indicated that in an in vitro uptake assay Rhodamine B (RhB)‑loaded PNA‑modified liposomes were taken up by cells with ~50% higher efficiency compared with free RhB. In addition, CDDP‑PNA‑Lip resulted in a 2.65‑fold enhancement of tumour suppression in vivo compared with free CDDP. These findings suggested that the encapsulation of CDDP within ligand‑modified liposomes may significantly improve its tumour‑targeting capabilities, providing valuable insights for clinical drug development.

Stability enhancement of proanthocyanidin-loaded liposomes via surface decoration with oxidized konjac glucomannan

The stability enhancement of proanthocyanidin-loaded liposomes (PC-Lip) via surface decoration with oxidized konjac glucomannan (OKGM) was investigated. The encapsulation efficiency and drug loading capacity of OKGM-coated PC-Lip (OKGM-PC-Lip) rose significantly. The average size and PDI of OKGM-PC-Lip increased, while the zeta potential decreased compared to those of PC-Lip. PC-Lip membrane fluidity reduced after coating with OKGM. The morphology of OKGM-PC-Lip showed that OKGM “halo layer” was formed on the liposome surface. Hydrogen bonding played an indispensable role in the combination between OKGM and PC-Lip, and the phase transition temperature of PC-Lip slightly increased after coating with OKGM. The retention rate of OKGM-PC-Lip was higher than that of PC-Lip at extreme pH. In vitro release, no significant difference in cumulative release was detected between OKGM-PC-Lip and PC-Lip at gastric stage, while the cumulative release rate of OKGM-PC-Lip was remarkably lower than that of PC-Lip at intestinal stage. The antioxidant activity of OKGM-PC-Lip was notably higher than that of PC-Lip. These results suggested that the resistance of PC-Lip to external influences was fruitfully enhanced after coating with OKGM. Compared with other polysaccharides, OKGM-coated liposomes may be more promising and advantageous in functional foods due to the polysaccharide's benefits to human health.

T cell independent antibody responses with class switch and memory using peptides anchored on liposomes

Vaccines generally require T lymphocytes for B-cell activation and immunoglobulin class switching in response to peptide or protein antigens. In the absence of T cells, limited IgG class switch takes place, germinal centers are short-lived, and the B cells lack memory. Here, immunization of mice with liposomes containing 15mer peptides and monophosphoryl lipid A (MPLA) as adjuvant, induced T-cell independent (TI) IgG class switch within three days, as well as germinal center formation. The antibody responses were long-lived, strictly dependent on Toll-like receptor 4 (TLR4) signaling, partly dependent on Bruton’s tyrosine kinase (BTK) signal transmission, and independent of signaling through T-cell receptors, MHC class II and inflammasome. The antibody response showed characteristics of both TI type 1 and TI type 2. All IgG subclasses could be boosted months after primary immunization, and the biological function of the secreted antibodies was demonstrated in murine models of allergic anaphylaxis and of bacterial infection. Moreover, antibody responses after immunization with peptide- and MPLA-loaded liposomes could be triggered in neonatal mice and in mice receiving immune-suppressants. This study demonstrates T-cell independent endogenous B-cell memory and recall responses in vivo using a peptide antigen. The stimulation of these antibody responses required a correct and dense assembly and administration of peptide and adjuvant on the surface of liposomes. In the future, TI vaccines may prove beneficial in pathological conditions in which T-cell immunity is compromised through disease or medicines or when rapid, antibody-mediated immune protection is needed.

Displaying alphavirus physicochemical consensus antigens on immunogenic liposomes enhances antibody elicitation in mice

Cobalt-porphyrin phospholipid displays recombinant protein antigens on liposome surfaces via antigen polyhistidine-tag (His-tag), and when combined with monophosphorylated lipid A and QS-21 yields the “CPQ” vaccine adjuvant system. In this proof of principle study, CPQ was used to generate vaccine prototypes that elicited antibodies for two different alphaviruses (AV). Mice were immunized with computationally designed, His-tagged, physicochemical property consensus (PCPcon) protein antigens representing the variable B-domain of the envelope protein 2 (E2) from the serotype specific Venezuelan Equine Encephalitis Virus (VEEVcon) or a broad-spectrum AV-antigen termed EVCcon. The CPQ adjuvant enhanced the antigenicity of both proteins without eliciting detectable anti-His-tag antibodies. Antibodies elicited from mice immunized with antigens admixed with CPQ showed orders-of-magnitude higher levels of antigen-specific IgG compared to alternative control adjuvants. The ELISA results correlated with antiviral activity against VEEV strain TC83 and more weakly to Chikungunya virus 118/25. Thus, display of E.coli-produced His-tagged E2 protein segments on the surface of immunogenic liposomes elicits high levels of antigen-specific and AV neutralizing antibodies in mice with vaccination, while facilitating vaccine preparation and providing dose-sparing potential.