Characterization of a Nanovaccine Platform Based on an α1,2-Mannobiose Derivative Shows Species-non-specific Targeting to Human, Bovine, Mouse, and Teleost Fish Dendritic Cells.

Dendritic cells serve as the main immune cells that trigger the immune response. We developed a simple and cost-effective nanovaccine platform based on the α1',2-mannobiose derivative for dendritic cell targeting. In previous work, we have formulated the α1,2-mannobiose-based nanovaccine platform with plasmid DNA and tested it in cattle against BoHV-1 infection. There, we have shown that the dendritic cell targeting using this nanovaccine platform in vivo can boost the immunogenicity, resulting in a long-lasting immunity. In this work, we aim to characterize the α1',2-mannobiose derivative, which is key in the nanovaccine platform. This DC-targeting strategy takes advantage of the specific receptor known as DC-SIGN and exploits its capacity to bind α1,2-mannobiose that is present at terminal ends of oligosaccharides in certain viruses, bacteria, and other pathogens. The oxidative conjugation of α1',2-mannobiose to NH2-PEG2kDa-DSPE allowed us to preserve the chemical structure of the non-reducing mannose of the disaccharide and the OH groups and the stereochemistry of all carbons of the reducing mannose involved in the binding to DC-SIGN. Here, we show specific targeting to DC-SIGN of decorated micelles incubated with the Raji/DC-SIGN cell line and uptake of targeted liposomes that took place in human, bovine, mouse, and teleost fish DCs in vitro, by flow cytometry. Specific targeting was found in all cultures, demonstrating a species-non-specific avidity for this ligand, which opens up the possibility of using this nanoplatform to develop new vaccines for various species, including humans.

Targeting of Micelles and Liposomes Loaded with the Pro-Apoptotic Drug, NCL-240, into NCI/ADR-RES Cells in a 3D Spheroid Model.

PURPOSE: To develop transferrin (Tf)-targeted delivery systems for the pro-apoptotic drug, NCL-240, and to evaluate the efficacy of this delivery system in ovarian cancer NCI/ADR-RES cells, grown in vitro in a 3D spheroid model. METHODS: Tf-targeted PEG-PE-based micellar and ePC/CHOL-based liposomal delivery systems for NCL-240 were prepared. NCI/ADR-RES cells were used to generate spheroids by a non-adhesive liquid overlay technique. Spheroid growth and development were monitored by size (diameter) analysis and H&E staining. The targeted formulations were compared to untargeted ones in terms of their degree of spheroid association and penetration. A cell viability analysis with NCL-240-loaded micelles and liposomes was performed to assess the effectiveness of Tf-targeting. RESULTS: Tf-targeted polymeric micelles and Tf-targeted liposomes loaded with NCL-240 were prepared. NCI/ADR-RES cells generated spheroids that demonstrated the presence of a distinct necrotic core along with proliferating cells in the spheroid periphery, partly mimicking in vivo tumors. The Tf-targeted micelles and liposomes had a deeper spheroid penetration as compared to the untargeted delivery systems. Cell viability studies using the spheroid model demonstrated that Tf-mediated targeting markedly improved the cytotoxicity profile of NCL-240. CONCLUSION: Transferrin targeting enhanced delivery and effectiveness of micelles and liposomes loaded with NCL-240 against NCI/ADR-RES cancer cells in a 3D spheroid model.

In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes.

Dendritic cells (DC) are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6.

Enhanced tumor delivery and antitumor activity in vivo of liposomal doxorubicin modified with MCF-7-specific phage fusion protein.

A novel strategy to improve the therapeutic index of chemotherapy has been developed by the integration of nanotechnology with phage technique. The objective of this study was to combine phage display, identifying tumor-targeting ligands, with a liposomal nanocarrier for targeted delivery of doxorubicin. Following the proof of concept in cell-based experiments, this study focused on in vivo assessment of antitumor activity and potential side-effects of phage fusion protein-modified liposomal doxorubicin. MCF-7-targeted phage-Doxil treatments led to greater tumor remission and faster onset of antitumor activity than the treatments with non-targeted formulations. The enhanced anticancer effect induced by the targeted phage-Doxil correlated with an improved tumor accumulation of doxorubicin. Tumor sections consistently revealed enhanced apoptosis, reduced proliferation activity and extensive necrosis. Phage-Doxil-treated mice did not show any sign of hepatotoxicity and maintained overall health. Therefore, MCF-7-targeted phage-Doxil seems to be an active and tolerable chemotherapy for breast cancer treatment. FROM THE CLINICAL EDITOR: The authors of this study successfully combined phage display with a liposomal nanocarrier for targeted delivery of doxorubicin using MCF-7-targeted phage-Doxil nanocarriers in a rodent model. The method demonstrated improved efficiency and reduced hepatotoxicity, paving the way to future clinical trials addressing breast cancer.

Liposomes for cardiovascular targeting.

Liposome-based pharmaceuticals used within the cardiovascular system are reviewed in this article. The delivery of diagnostic and therapeutic agents by plain liposomes and liposomes with surface-attached targeting antibodies or polyethylene glycol to prolong their circulation time and accumulation at vascular injuries, ischemic zones or sites of thrombi are also discussed. An overview of the advantages and disadvantages of liposome-mediated in vitro, ex vivo and in vivo targeting is presented, including discussion of the targeting of liposomes to pathological sites on the blood vessel wall and a description of liposomes that can be internalized by endothelial cells. Diagnostic liposomes used to target myocardial infarction and the relative importance of liposome size, targetability of immunoliposomes and prolonged circulation time on the efficiency of sealing hypoxia-induced plasma membrane damage to cardiocytes are discussed as a promising approach for therapy. The progress in the use of targeted liposomal plasmids for the transfection of hypoxic cardiomyocytes and myocardium is presented. Stent-mediated liposomal-based drug delivery is also reviewed briefly.

ATP-loaded liposomes for targeted treatment in models of myocardial ischemia.

ATP cannot be effectively delivered to most tissues including the ischemic myocardium without protection from degradation by plasma endonucleotidases. However, it has been established that ATP can be delivered to various tissues by its encapsulation within liposomal preparations. We describe here, the materials needed and methods used to optimize the encapsulation of ATP in liposomes, enhance their effectiveness by increasing their circulation time and target injured myocardial cells with liposomal surface anti-myosin antibody. Additionally, we outline methods for ex vivo studies of these ATP liposomal preparations in an isolated ischemic rat heart model and for in vivo studies of rabbits with an induced myocardial infarction. The expectation is that these methods will provide a basis for continued studies of effective ways to deliver energy substrates to the ischemic myocardium.

Near infrared planar tumor imaging and quantification using nanosized Alexa 750-labeled phospholipid micelles.

A novel highly biocompatible near infrared nanosized contrast agent was developed and used for rapid tumor detection and quantification using planar optical imaging and analysis. With this in mind, the near infrared fluorescent dye Alexa 750 was covalently attached to polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate, and double labeled (with Alexa and rhodamine) PEG-PE micelles were injected into mice and observed using planar optical imaging. Pixel intensity data from the tumor site were normalized versus the autofluorescence of the animal at the same time point and normalized as signal to noise over the scattered light from the various tissues of the mice. The detected signal from the tumor was higher than the background noise allowing for rapid detection of the tumor. The tumor was clearly visible within one hour. Some signal was also detected from the abdomen of the mice. As determined by microscopy analysis, other organs of accumulation were the liver and kidney, which corresponded well to the data from the whole body imaging animal studies.

ATP-loaded liposomes for treatment of myocardial ischemia.

A major obstacle to drug therapy for treatment of potential myocardial infarction is the limited access to the ischemic myocardium by drugs in an active form. Encouraging results have been reported with liposomes loaded with ATP in a variety of in vitro and in vivo models. We describe methods for optimized encapsulation of ATP in liposomes, enhancement of their effectiveness by increasing circulation time, and targeting of injured myocardial cells with surface attached antimyosin. In isolated ischemic rat hearts, ATP-loaded liposomes and ATP-loaded immunoliposomes effectively protected myocardium from ischemia/reperfusion damage as measured by systolic and diastolic functional improvements. In vivo, in rabbits with induced localized myocardial ischemia, liposomal encapsulation of ATP significantly diminished the proportion of ventricular muscle at risk that was irreversibly damaged during reperfusion. Therefore, ATP encapsulated in liposomes can provide an effective exogenous source for in vivo application which can protect ischemically damaged hearts.

Self-assembling micelle-like nanoparticles based on phospholipid-polyethyleneimine conjugates for systemic gene delivery.

With few exceptions, where local administration is feasible, progress towards broad clinical application of gene therapies requires the development of effective delivery systems. Here we report a novel non-viral gene delivery vector, 'micelle-like nanoparticle' (MNP) suitable for systemic application. MNP were engineered by condensing plasmid DNA with a chemical conjugate of phospholipid with polyethylenimine (PLPEI) and then coating the complexes with an envelope of lipid monolayer additionally containing polyethylene glycol-phosphatidyl ethanolamine (PEG-PE), resulting in spherical 'hard-core' nanoparticles loaded with DNA. MNP allowed for complete protection of the loaded DNA from enzymatic degradation, resistance to salt-induced aggregation, and reduced cytotoxicity. MNP also demonstrated prolonged blood circulation and low RES accumulation. Intravenous injection of MNP loaded with plasmid DNA encoding for the Green Fluorescence Protein (GFP) resulted in an effective transfection of a distal tumor. Thus, MNP provide a promising tool for systemic gene therapy.

Protective effect of coenzyme Q10-loaded liposomes on the myocardium in rabbits with an acute experimental myocardial infarction.

PURPOSE: We assessed whether the infusion of Coenzyme Q10-loaded liposomes (CoQ10-L) in rabbits with an experimental myocardial infarction can result in increased intracellular delivery of CoQ10 and thus limit the fraction of the irreversibly damaged myocardium. METHODS: CoQ10-L, empty liposomes (EL), or Krebs-Henseleit (KH) buffer were administered by intracoronary infusion, followed by 30 min of occlusion and 3 h of reperfusion. Unisperse Blue dye was used to demarcate the net size of the occlusion-induced ischemic zone ("area at risk") while nitroblue tetrazolium staining was used to detect the final fraction of the irreversibly damaged myocardium within the total area at risk. RESULTS: The total size of the area at risk in all experimental animals was approx. 20% wt. of the left ventricle (LV). The final irreversible damage in CoQ10-L-treated animals was only ca. 30% of the total area at risk as compared with ca. 60% in the group treated with EL (p < 0.006) and ca. 70% in the KH buffer-treated group (p < 0.001). CONCLUSIONS: CoQ10-L effectively protected the ischemic heart muscle by enhancing the intracellular delivery of CoQ10 in hypoxic cardiocytes in rabbits with an experimental myocardial infarction as evidenced by a significantly decreased fraction of the irreversibly damaged heart within the total area at risk. CoQ10-L may provide an effective exogenous source of the CoQ10 in vivo to protect ischemic cells.

Cytoskeletal-antigen specific immunoliposome-targeted in vivo preservation of myocardial viability.

Preservation of cell viability using Cytoskeletal-antigen Specific ImmunoLiposomes (antimyosin-CSIL) has been demonstrated in cell cultures. The current study utilized the same CSIL intervention for myocardial protection in an in vivo rabbit model of acute myocardial infarction. Rabbit hearts with experimental left ventricular myocardial infarction were treated with CSIL; control liposomes, [(CL), IgG-liposomes (IgG-L) or plain liposomes (PL)], or vehicle (placebo). Mean myocardial infarct size in rabbit hearts treated in vivo with CSIL was 5 times smaller than in those treated with non-specific CL or vehicle. Treatment of ischemic adult myocardium with CSIL results in significant preservation of myocardial viability by dramatically decreasing the infarct size relative to CL or placebo treatment. Immunohistochemical myocardial preservation of CSIL-treated hearts was confirmed by the lack of contraction band necrosis using histological H&E stains relative to controls. Electrocardiographic confirmation of reduction in myocardial injury after CSIL therapy relative to controls was also observed. Application of CSIL technology to non-cardiac tissues would confirm a broader applicability of this cell membrane lesion sealing technology.

ATP-loaded liposomes effectively protect the myocardium in rabbits with an acute experimental myocardial infarction.

PURPOSE: We assessed whether the infusion of ATP-loaded liposomes (ATP-L) can limit the fraction of the irreversibly damaged myocardium in rabbits with an experimental myocardial infarction. METHODS: ATP-L, empty liposomes (EL), or Krebs-Henseleit (KH) buffer were administered by intracoronary infusion, followed by 30 min of occlusion and 3 h of reperfusion. Unisperse Blue dye was used to demarcate the net size of the occlusion-induced ischemic zone (area at risk) and nitroblue tetrazolium staining was used to detect the final fraction of the irreversibly damaged myocardium within the total area at risk. RESULTS: The total size of the area at risk in all experimental animals was approx. 20% wt. of the left ventricle. The final irreversible damage in ATP-L-treated animals was only ca. 30% of the total area at risk as compared with ca. 60% in the group treated with EL (p < 0.009) and ca. 70% in the KH buffer-treated group (p < 0.003). CONCLUSIONS: ATP-L effectively protected the ischemic heart muscle in rabbits with an experimental myocardial infarction as evidenced by a significantly decreased fraction of the irreversibly damaged heart within the total area at risk. ATP-L may provide an effective exogenous source of the ATP in vivo to protect ischemically damaged cells.

PEGylated dextran as long-circulating pharmaceutical carrier.

Dextran-polyethylene glycol (PEG) conjugates were synthesized by activating dextran hydroxy groups with carbonyldiimidazole, introducing amino groups by attaching ethylenediamine, and reacting amino groups with a succinimidyl-activated derivative of PEG. Conjugates with an average of 12 and 21 PEG (5 kDa) residues per single dextran (73 kDa) molecule were prepared. These conjugates have circulation half-lives of 5.3 h and 7.0 h, respectively, compared to 4.0 h for non-PEGylated dextran. The modification of dextran with PEG inhibits the uptake of polymer by the major organ of the reticuloendothelial system, the liver. Dextran-PEG conjugates may represent a convenient platform for long-circulating pharmaceutical preparations.

Increased accumulation of PEG-PE micelles in the area of experimental myocardial infarction in rabbits.

Micelles prepared from polyethyleneglycol/phosphatidyl-ethanolamine conjugates (PEG-PE) with a size of 7-20 nm and zeta-potential of approximately -18 mV were administered i.v. to rabbits with experimental myocardial infarctions. Micelles demonstrated a prolonged circulation in the blood (half-life of 2 h) and accumulated in the infarction zone with efficiency more than 8-fold higher as compared to a non-damaged part of the heart muscle. Obtained results suggest that the enhanced permeability and retention (EPR) effect is the primary mechanism of accumulation of microparticles in the infarct areas, and that drug carriers such as PEG-PE micelles can be used for the delivery of therapeutic or diagnostic agents to an area of myocardial infarction.