Liposome-based Delivery Systems Research Articles

Aptamer-guided siRNA-loaded nanomedicines for systemic gene silencing in CD-44 expressing murine triple-negative breast cancer model

In this study, we describe a liposome-based siRNA delivery system with a core composed of siRNA:protamine complex and a shell designed for the active targeting of CD44-expressing cells using for the first time the anti-CD44 aptamer (named Apt1) as targeting ligand. Among all functions, CD44 is the most common cancer stem cell surface biomarker and is found overexpressed in many tumors making this an attractive receptor for therapeutic targeting. This unique non-cationic system was evaluated for the silencing of the reporter gene of luciferase (luc2) in a triple-negative breast cancer model in vitro and in vivo. We show the possibility of conjugating an aptamer to siRNA-containing liposomes for an efficient gene silencing in CD44-expressing tumor cells in vivo, in the perspective of silencing disease-related genes in tumors.

Carboxylated phytosterol derivative-introduced liposomes for skin environment-responsive transdermal drug delivery system

Transdermal drug delivery systems are a key technology for skin-related diseases and for cosmetics development. The delivery of active ingredients to an appropriate site or target cells can greatly improve the efficacy of medical and cosmetic agents. For this study, liposome-based transdermal delivery systems were developed using pH-responsive phytosterol derivatives as liposome components. Succinylated phytosterol (Suc-PS) and 2-carboxy-cyclohexane-1-carboxylated phytosterol (CHex-PS) were synthesized by esterification of hydroxy groups of phytosterol. Modification of phytosterol derivatives on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes was confirmed by negatively zeta potentials at alkaline pH and the change of zeta potentials with decreasing pH. In response to acidic pH and temperatures higher than body temperature, Suc-PS-containing and CHex-PS-containing liposomes exhibited content release at intracellular acidic compartments of the melanocytes at the basement membrane of the skin. Phytosterol-derivative-containing liposomes were taken up by murine melanoma-derived B16-F10 cells. These liposomes delivered their contents into endosomes and cytosol of B16-F10 cells. Furthermore, phytosterol-derivative-containing liposomes penetrated the 3 D skin models and reached the basement membrane. Results show that pH-responsive phytosterol-derivative-containing DMPC liposomes are promising for use in transdermal medical or cosmetic agent delivery to melanocytes.

Impacts of Salt, Buffer, and Lipid Nature on Molecular Adsorption and Transport in Liposomes As Observed by Second Harmonic Generation

It is of great importance to interrogate the impact of local environment on the transport of small molecules across lipid bilayers, as they are key to the function and capabilities of eukaryotic cells and liposome-based delivery systems. Herein are described real-time studies of the molecular adsorption and transport kinetics of positively charged small-molecule organic dyes at the surface of liposomes under different buffer and salt conditions, made possible by application of second harmonic generation (SHG). The molecular transport of malachite green (MG) within the liposome bilayer is more rapid in 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPG and DOPS, respectively) liposomes in citrate buffer without added salts, whereas no adsorption or transport of MG is observed in trimethyl quinone-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (QPADOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. While the transport rate constant increa...

Identifying the Interaction of Vancomycin With Novel pH-Responsive Lipids as Antibacterial Biomaterials Via Accelerated Molecular Dynamics and Binding Free Energy Calculations.

Nano-drug delivery systems have proven to be an efficient formulation tool to overcome the challenges with current antibiotics therapy and resistance. A series of pH-responsive lipid molecules were designed and synthesized for future liposomal formulation as a nano-drug delivery system for vancomycin at the infection site. The structures of these lipids differ from each other in respect of hydrocarbon tails: Lipid1, 2, 3 and 4 have stearic, oleic, linoleic, and linolenic acid hydrocarbon chains, respectively. The impact of variation in the hydrocarbon chain in the lipid structure on drug encapsulation and release profile, as well as mode of drug interaction, was investigated using molecular modeling analyses. A wide range of computational tools, including accelerated molecular dynamics, normal molecular dynamics, binding free energy calculations and principle component analysis, were applied to provide comprehensive insight into the interaction landscape between vancomycin and the designed lipid molecules. Interestingly, both MM-GBSA and MM-PBSA binding affinity calculations using normal molecular dynamics and accelerated molecular dynamics trajectories showed a very consistent trend, where the order of binding affinity towards vancomycin was lipid4 > lipid1 > lipid2 > lipid3. From both normal molecular dynamics and accelerated molecular dynamics, the interaction of lipid3 with vancomycin is demonstrated to be the weakest (∆Gbinding = -2.17 and -11.57, for normal molecular dynamics and accelerated molecular dynamics, respectively) when compared to other complexes. We believe that the degree of unsaturation of the hydrocarbon chain in the lipid molecules may impact on the overall conformational behavior, interaction mode and encapsulation (wrapping) of the lipid molecules around the vancomycin molecule. This thorough computational analysis prior to the experimental investigation is a valuable approach to guide for predicting the encapsulation ability, drug release and further development of novel liposome-based pH-responsive nano-drug delivery system with refined structural and chemical features of potential lipid molecule for formulation development.

Effects of Membrane PEGylation on Entry and Location of Antifungal Drug Itraconazole and Their Pharmacological Implications.

Itraconazole (ITZ) is an antifungal agent used clinically to treat mycotic infections. However, its therapeutic effects are limited by low solubility in aqueous media. Liposome-based delivery systems (LDS) have been proposed as a delivery mechanism for ITZ to alleviate this problem. Furthermore, PEGylation, the inclusion in the formulation of a protective "stealth sheath" of poly(ethylene glycol) around carrier particles, is widely used to increase circulation time in the bloodstream and hence efficacy. Together, these themes highlight the importance of mechanistic and structural understanding of ITZ incorporation into liposomes both with and without PEGylation because it can provide a potential foundation for the rational design of LDS-based systems for delivery of ITZ, using alternate protective polymers or formulations. Here we have combined atomistic simulations, cryo-TEM, Langmuir film balance, and fluorescence quenching experiments to explore how ITZ interacts with both pristine and PEGylated liposomes. We found that the drug can be incorporated into conventional and PEGylated liposomes for drug concentrations up to 15 mol % without phase separation. We observed that, in addition to its protective properties, PEGylation significantly increases the stability of liposomes that host ITZ. In a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer without PEGylation, ITZ was found to reside inside the lipid bilayer between the glycerol and the double-bond regions of POPC, adopting a largely parallel orientation along the membrane surface. In a PEGylated liposome, ITZ partitions mainly to the PEG layer. The results provide a solid basis for further development of liposome-based delivery systems.

An anti-oxidant, α-lipoic acid conjugated oleoyl-sn-phosphatidylcholineas a helper lipid in cationic liposomal formulations

Development of safe non-viral carrier systems for efficient intra-cellular delivery of drugs and genes hold promise in the area of translational research. Liposome based delivery systems have emerged as one of the attractive strategies for efficient delivery of drugs and nucleic acids. To this end, number of investigations was carried on liposomal formulations using lipids for achieving higher efficiency in transfection with lower cytotoxicities. In our efforts to develop safer and efficient liposomal delivery systems, we synthesized a novel anti-oxidant lipid, α-lipoyl, oleyl-sn-phosphatidylcholine (LOPC) and used as a helper lipid in combination with a cationic amphiphile, Di-Stearyl Dihydroxy Ethyl Ammonium Chloride (DSDEAC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at varying concentrations of LOPC. DNA binding properties of the liposomal formulations (DS, DS LA1, DS LA2 and DS LA3) revealed that increasing the percentage of single aliphatic chain lipid LOPC, did not affect the DNA binding properties. But, transfection profiles of these liposomal formulations in 3 different cell lines (HeLa, HEK 293 and MCF7) showed difference in their efficacies. Results showed that optimal percentage of LOPC i.e. 25% in DSDEAC and DOPC at 1:1 molar ratio (DS LA1) enhanced transfection as compared to DSDEAC:DOPC alone. The endosomal escape studies with NBD labelled lysotracker and Rhodamine labelled liposomal formulations revealed that DS LA1 and DS LA2 facilitated the release of genetic cargo with a better efficiency than their counter parts. Reactive Oxygen Species (ROS), a key modulator of necroptosis were lowered with the treatment of DS LA1 than other liposomal formulations. Here in, we present a novel liposomal formulation using DSDEAC and DOPC at 1:1 molar ratio doped with 25–50% (mole ratio) LOPC as an efficient delivery system for enhanced transfection with quenching of ROS levels compared to formulations without LOPC.

Hesperetin Liposomes for Cancer Therapy.

Hesperetin is a compound from citrus fruit that has previously been found to exert anticancer activity through a variety of mechanisms. However, the application of hesperetin to cancer therapy has been hampered by its hydrophobicity, necessitating the use of toxic solubilizing agents. Here, we have developed the first liposome-based delivery system for hesperetin. Liposomes were fabricated using the thin-layer evaporation technique and physical and pharmacological parameters were measured. The liposomes remained stable for prolonged periods of time in serum and under storage conditions, and displayed anticancer efficacy in both H441 lung cancer cells and MDA-MB-231 breast cancer cells. Furthermore, the anticancer activity was not impaired in cells expressing the multidrug resistance protein 1 (MDR-1). In conclusion, the encapsulation of hesperetin in liposomes does not interfere with therapeutic efficacy and provides a biocompatible alternative to toxic solubilizing agents, thereby enabling future clinical use of this compound for cancer therapy.

Optimization of a cationic liposome-based gene delivery system for the application of miR-145 in anticancer therapeutics.

In order to improve the delivery efficiency of microRNA (miRNA or miR)-145, the present study examined several factors which may affect cationic liposome (CL)-based transfection, including the hydration medium used for the preparation of liposomes, the quantity of the plasmid, the molar ratio of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol (chol), or DOTAP/chol, and the weight ratio of DOTAP/DNA. In order to enhance the transfection efficiency, protamine was selected as a DNA-condensing agent to form liposome‑protamine‑DNA (LPD) ternary complexes. An agarose gel retardation assay was used to examine the DNA binding affinity of the CLs. Following transfection, GFP fluorescence images were captured and flow cytometry was performed to determine the transfection efficiency. Furthermore, an MTT assay was performed to determine the cytotoxicity of the liposome complexes. The final optimal conditions were as follows: 5% glucose as the hydration medium, a molar ratio of DOTAP/chol at 3:1 for the preparation of CLs, a weight ratio of DOTAP/protamine/DNA of 3:0.5:1, with 8 µg plasmid added for the preparation of the LPD complexes. In vitro, the LPD complexes exhibited an enhanced transfection efficiency and low cytotoxicity, which indicated that the presented LPD vector enhanced the transfection efficiency of the CLs. The HepG2 cells were found to have the lowest expression levels of miR‑145 out of the cell lines tested (A549, BGC-823, HepG2, HeLa, LoVo and MCF-7). Following the transient transfection of the HepG2 cells with miR‑145, the results revealed that the overexpression of miR‑145 inhibited the proliferation of the HepG2 cells and downregulated the expression of cyclin-dependent kinase 6 (CDK6), cyclinD1, c-myc, and Sp1 transcription factor (Sp1). In conclusion, in this study, we optimized a liposome‑based delivery system for the efficient delivery of miR‑145 into cancer cells. This may provide a foundation for further research into the use of miR‑145 in anticancer therapeutics.

Preparation, Pharmacokinetic Profile, and Tissue Distribution Studies of a Liposome-Based Formulation of SN-38 Using an UPLC-MS/MS Method.

The application of 7-ethyl-10-hydroxycamptothecin (SN-38) in cancer treatment is limited by its low solubility. This study is to develop a liposome-entrapped formulation of SN-38 (LE-SN38) to solve the obstacle and to evaluate its pharmacokinetic profile in dogs and tissue distribution in mice. LE-SN38 which is more likely to be suitable for large-scale production was prepared by the carrier-deposition method. An UPLC-MS/MS method was used to determinate the concentration of SN-38 in this study. LE-SN38 was cleared rapidly from dog plasma within 1h, and the AUC0-∞ values of three dosages of LE-SN38 indicated an apparent dose-dependent manner. As for the distribution study, the peak of SN-38 levels in most tissues were detected within 10 min after LE-SN38 administration. In addition, concentration of SN-38 in most tissues except kidney and heart in LE-SN38 group was higher than that in irinotecan hydrochloride (CPT-11) group generally, whereas the administrated CPT-11 had 20 times dosage compared to LE-SN38. LE-SN38 was rapidly eliminated from dog plasma and manifested linear dynamics in dose range of 0.411-1.644mg/kg. The distribution behavior of SN-38 is altered in a liposome-based delivery system. At the same time, LE-SN38 has lower toxicity compared to CPT-11 in some degree.

The Targeted-liposome Delivery System of Antitumor Drugs.

The liposome delivery system has been intensively explored as novel drug delivery system (DDS) for antitumor drugs, due to its safety, selective cytotoxicity, long circulation and slow elimination in blood, which is favorable for cancer therapy. The liposome-based chemotherapeutics are used to treat a variety of cancers to enhance the therapeutic index of antitumor drugs. Here, the author reviewed the important targets for cancer therapy and the pharmacokinetic behavior of liposomal drugs in vivo, as well as the application of the targeting liposomal system in cancer therapy. Considering further application for clinical use, the great challenges of the liposome-based delivery system were also proposed as follows: 1) prepare stealth liposome with steric stabilization and further enhance the therapeutic effects and safety; 2) explore more safe clinical targets and complementary or different types of targeting liposome; 3) thirdly, more investment is needed on the research of pharmacokinetics of the elements such as the ligands (antibody), PEG and lipids of liposome delivery system as well as safety evaluation. Considering the complex process of the liposomal encapsulation drugs in vivo, the author inferred that there are maybe different forms of the encapsulation drug to be internalized by the tumor tissues at the same time and space, although there are little reports on it.

Liposome-based delivery systems for ginsenoside Rh2: in vitro and in vivo comparisons

The Ginsenoside Rh2 (Rh2) has been shown to possess anti-cancer properties both in vitro and in vivo. However, the poor bioavailability and fast plasma elimination limit the further clinical applications of Rh2 for cancer treatments. In the present study, three types of Rh2-loaded liposomes including Rh2-loaded normal liposome (Rh2-LP), Rh2-loaded cationic liposome (Rh2-CLP), and Rh2-loaded Methoxy poly(ethylene glycol)-poly(lactide) (mPEG-PLA) liposome (Rh2-PLP) have been optimized and prepared with mean particle size of 80–125 nm. Compared to Rh2-LP, surface modifications with mPEG or octadecylamine significantly improve the physicochemical and biological properties both in vitro and in vivo. Moreover, PLP presented better tumor accumulation of the fluorescent cyanine dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) in HepG2-xenografted nude mice than CLP (1.3-fold) or LP (1.6-fold) and prolong the resident time of DiR in tumor and organs (more than 24 h). The in vivo anti-cancer efficacy assessments indicate that Rh2-PLP presents the most activity on suppressing tumor growth in HepG2-xenografted mice than Rh2-LP and Rh2-CLP and without any significant toxicity. Our results indicate that mPEG-PLA modified liposome should be a potential and promising strategy to enhance the therapeutic index for anti-cancer agents.

Physicochemical and in vitro evaluation of cationic liposome, hyaluronic acid and plasmid DNA as pseudo-ternary complexes for gene delivery

Abstract The association of liposome-based gene delivery systems with different biopolymers has been investigated to provide multifunctional properties for its complexes, such as alternative delivery routes and targeting delivery. Hyaluronic acid (HA) is an anionic mucoadhesive biopolymer that can interact to cell surface receptors. In this study, we explored the colloidal formation of pseudo-ternary complexes based on the electrostatic complexation among plasmid DNA (pDNA), cationic liposomes (CL) and hyaluronic acid of low molecular weight (16 kDa). We used extruded cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (molar proportion of 2:1:1, respectively), pDNA vector model with luciferase as reporter gene, and HA of low molecular weight of 16 kDa. The pseudo-ternary complexes were physicochemically characterized in terms of their hydrodynamic diameter, polydispersity, zeta potential, morphology and accessibility to pDNA probe. These results together indicated that the pDNA/CL complexes are most likely coated by HA molecules with the pDNA incorporated into the liposomal structure. To evaluate the biological viability of the complexes, we performed in vitro transfections of human epitheloid carcinoma (HeLa) cells. The pDNA/CL/HA complexes presented transfection efficiencies similar to the pseudo-binary pDNA/CL complexes for the different HA amounts studied. In addition, we carried out time-lapse imaging in living HeLa cells so we could monitor the kinetics of complexes internalization. We believe that these results may be useful for future developments of multifunctional nanocarriers that explore the association of materials with different net charge properties for gene delivery and vaccine therapy applications.

Mitochondrial delivery of Coenzyme Q10via systemic administration using a MITO-Porter prevents ischemia/reperfusion injury in the mouse liver

We herein report on a mitochondrial therapeutic effect based on the delivery of coenzyme Q10 (CoQ10), an anti-oxidant, to in vivo mitochondria using a MITO-Porter, a liposome-based mitochondrial delivery system that functions via membrane fusion. To evaluate the effects, we used a mouse liver ischemia/reperfusion injury (I/R injury) model, in which mitochondrial reactive oxygen species are overexpressed. We packaged CoQ10 in the lipid phase of a MITO-Porter and optimized the mitochondrial fusogenic activities to produce the CoQ10-MITO-Porter. A histological observation of the carriers in the liver by confocal laser scanning microscopy was done and the accumulation of the carrier labeled with a radio isotope in the liver confirmed that the CoQ10-MITO-Porter was delivered to liver mitochondria via systemic injection. These analytical results permitted us to optimize the compositions of the CoQ10-MITO-Porter so as to permit it to efficiently accumulate in mouse liver mitochondria. Finally, we applied the optimized CoQ10-MITO-Porter to mice via tail vein injection, and hepatic I/R injury was then induced, followed by measuring serum alanine aminotransferase (ALT) levels, a marker of liver injury. We confirmed that the use of the CoQ10-MITO-Porter resulted in a significant decrease in serum ALT levels, indicating that in vivo mitochondrial delivery of the CoQ10via MITO-Porter prevents I/R injury in mice livers. This provides a demonstration of the potential use of such a delivery system in mitochondrial therapies.

Pannexins: the ‘nexus’ between urothelium ATP production and extracellular release

Pannexins: the ‘nexus’ between urothelium ATP production and extracellular release

Liposome-based gene delivery systems containing a steroid derivative: computational and small angle X-ray diffraction study

The structural properties and the phase behaviour of mixed composition liposomes containing a functionalized lipid are investigated with the aim to design neutral liposomes able to coordinate metals and to complex DNA.

Development and in vitro/in vivo Evaluation of Liposomal Gels for the Sustained Ocular Delivery of Latanoprost

Objective: Conventional eye drops commonly used in the treatment of glaucoma suffer from short residence time, which results in frequent administration and poor patient compliance. The objective of this work was to develop a liposome-based delivery system for the sustained ocular delivery of latanoprost, a prostaglandin analog commonly used in the management of glaucoma. Methods: Latanoprost was incorporated into different liposomes that were evaluated using variety of techniques. Selected liposomes were incorporated into different gels and their viscosity and drug release kinetics were evaluated. Optimal liposomal gels were evaluated in vivo in rabbits’ eyes for their irritation potential and ability to reduce intraocular pressure. Results: Fourier transform infrared and differential scanning calorimetry studies confirmed the interaction between the drug and different excipients in the vesicles, which resulted in drug encapsulation efficiency ≥ 90%. Drug encapsulation efficiency increased with the drug/lipid ratio and encapsulation efficiency ~98% was obtained at drug/lipid ratio of 50%. Vesicles incorporated into Pluronic® F127 gel had sustained drug release where ~45% of the encapsulated drug was released in 2 days. Latanoprost liposomal gels had neither irritation nor toxic effects on the rabbits’ eyes. Further, they had a sustained reduction in the rabbit’s intraocular pressure over a period of 3 days, which was significantly longer than that achieved by the commercial latanoprost eye drops. Conclusion: These results confirm the potential of latanoprost liposomal gels as viable alternatives to conventional eye drops for the safe and efficient management of glaucoma.

Liposomes as vaccine delivery systems: a review of the recent advances.

Liposomes and liposome-derived nanovesicles such as archaeosomes and virosomes have become important carrier systems in vaccine development and the interest for liposome-based vaccines has markedly increased. A key advantage of liposomes, archaeosomes and virosomes in general, and liposome-based vaccine delivery systems in particular, is their versatility and plasticity. Liposome composition and preparation can be chosen to achieve desired features such as selection of lipid, charge, size, size distribution, entrapment and location of antigens or adjuvants. Depending on the chemical properties, water-soluble antigens (proteins, peptides, nucleic acids, carbohydrates, haptens) are entrapped within the aqueous inner space of liposomes, whereas lipophilic compounds (lipopeptides, antigens, adjuvants, linker molecules) are intercalated into the lipid bilayer and antigens or adjuvants can be attached to the liposome surface either by adsorption or stable chemical linking. Coformulations containing different types of antigens or adjuvants can be combined with the parameters mentioned to tailor liposomal vaccines for individual applications. Special emphasis is given in this review to cationic adjuvant liposome vaccine formulations. Examples of vaccines made with CAF01, an adjuvant composed of the synthetic immune-stimulating mycobacterial cordfactor glycolipid trehalose dibehenate as immunomodulator and the cationic membrane forming molecule dimethyl dioctadecylammonium are presented. Other vaccines such as cationic liposome-DNA complexes (CLDCs) and other adjuvants like muramyl dipeptide, monophosphoryl lipid A and listeriolysin O are mentioned as well. The field of liposomes and liposome-based vaccines is vast. Therefore, this review concentrates on recent and relevant studies emphasizing current reports dealing with the most studied antigens and adjuvants, and pertinent examples of vaccines. Studies on liposome-based veterinary vaccines and experimental therapeutic cancer vaccines are also summarized.

Physiological roles of asialoglycoprotein receptors (ASGPRs) variants and recent advances in hepatic-targeted delivery of therapeutic molecules via ASGPRs.

The asialoglycoprotein receptor (ASGPR) is a high-capacity C-type lectin receptor mainly expressed on mammalian hepatic cells. The physiological function of ASGPR has not been completely clarified and is thought to be specific binding and internalization of galactose (Gal) or N-acetylgalactosamine (GalNAc)-terminating glycoproteins by hepatocytes. The human ASGPR is comprised of two homologous polypeptides, H1 and H2. ASGPR H1 has two splice variants (H1a and H1b) and ASGPR H2 has three splice variants (H2a, H2b, and H2c). These variants have been discovered to exist both in human liver tissues and in human hepatoma cells. Variant H1b, which has an in-frame deletion of exon 2 resulting in the loss of the transmembrane domain and is secreted as a soluble protein, encodes functional soluble ASGPR (s- ASGPR). Based on our previous results, we proposed the possible physiological function of s-ASGPR, which is well interpreted in the Galactosyl Homeostasis Hypothesis proposed by Weigel. ASGPR is one of the most promising targets for hepatic delivery. In this review, the recent progresses of cationic polysomes and liposomes as effective non-viral delivery system via ASGPR are also presented.

Incorporation of CpG into a Liposomal Vaccine Formulation Increases the Maturation of Antigen-Loaded Dendritic Cells and Monocytes To Improve Local and Systemic Immunity

Liposomal vaccine formulations incorporating stimulants that target innate immune receptors have been shown to significantly increase vaccine immunity. Following vaccination, innate cell populations respond to immune stimuli, phagocytose and process Ag, and migrate from the injection site, via the afferent lymphatic vessels, into the local lymph node. In this study, the signals received in the periphery promote and sculpt the adaptive immune response. Effector lymphocytes then leave the lymph node via the efferent lymphatic vessel to perform their systemic function. We have directly cannulated the ovine lymphatic vessels to detail the in vivo innate and adaptive immune responses occurring in the local draining lymphatic network following vaccination with a liposome-based delivery system incorporating CpG. We show that CpG induces the rapid recruitment of neutrophils, enhances dendritic cell-associated Ag transport, and influences the maturation of innate cells entering the afferent lymph. This translated into an extended period of lymph node shutdown, the induction of IFN-γ-positive T cells, and enhanced production of Ag-specific Abs. Taken together, the results of this study quantify the real-time in vivo kinetics of the immune response in a large animal model after vaccination of a dose comparable to that administered to humans. This study details enhancement of numerous immune mechanisms that provide an explanation for the immunogenic function of CpG when employed as an adjuvant within vaccines.

Physical Characterization of Synthetic Phosphatidylinositol Dimannosides and Analogues in Binary Systems with Phosphatidylcholine

Native phosphatidylinositol mannosides (PIMs) from the cell wall of Mycobacterium bovis (M. bovis) and synthetic analogues have been identified to exert immunostimulatory activities. These activities have been investigated using particulate delivery systems containing native mannosylated lipids or total lipid extracts. Limited work has been carried out examining the incorporation of individual PIM lipids into suitable particulate formulations such as liposomes. The present study explored the possibility of constructing phosphatidylcholine (PC)-based liposomes containing synthetic PIM analogues. A series of six phosphatidylinositol dimannosides (PIM(2)s) and phosphatidylglycerol dimannosides (PGM(2)s) was characterized in this study. Binary Langmuir monolayers are a useful approach for establishing pharmaceutical properties, such as lipid-lipid interactions in mixed monolayers, to facilitate the design of liposome-based delivery systems. In mixed films the phosphoglycolipids were found to be miscible with PC based on evaluation of collapse pressures and deviations of experimental molecular areas from calculated ideal values. Concanavalin A (ConA) agglutination confirmed the presence of mannosylated lipids on the surface of the liposomes. Physicochemical properties of liposomes were affected by the presence of 2% (w/w) of phosphoglycolipids with liposome stability being increased by incorporation of long-chain PIM(2) and PGM2. Overall, while membrane stability of the liposomes was found to be dependent on incorporation of the phosphoglycolipids, all formulations retained proteins in amounts making them suitable for delivery.