Fate of a liposome-associated agent injected into normal and tumour-bearing rodents. Attempts to improve localization in tumour tissues.

Liposomes containing ¹¹¹In-labelled bleomycin were injected intravenously into normal and tumour-bearing rodents and the fate of radioactivity followed. ¹¹¹In levels in tissues retained their maximum values for up to 48h after treatment thereby enabling accurate estimations of tissue participation which with a variety of tumours (Meth ‘A’, 6C3HED, Lewis lung carcinoma and Novikoff hepatoma) in mice and rats was secondary to that of the liver and spleen. Reductions in the size of liposomes decreased liver and spleen participation and increased tumour and kidney involvement. Uptake by lungs, skeletal muscle and brain was also augmented albeit to a lesser extent. Incorporation of anti-Meth ‘A’ cells IgG immunoglobulin into the liposomal carrier led to a modest increase in the uptake of co-entrapped ¹¹¹In by the Meth ‘A’ tumour implanted subcutaneously. Although at the same time, liposomal IgG reduced uptake by the kidney, it effected a drastic increase in hepatic and splenic involvement. This could be prevented by the concurrent administration of excess “empty” liposomes which, however, did not interfere with uptake by tumour tissue.     

Expression of luciferase in selected organs following delivery of naked and formulated DNA to rainbow trout (Oncorhynchus mykiss) by different routes of administration

In the present work, the expression of luciferase in selected organs following administration of DNA delivered as naked, liposome-formulated or chitosan-formulated by different routes of administration (intramuscular, intraperitoneal and intravenous injection, immersion and anal intubation) was studied in rainbow trout (Oncorhynchus mykiss). The different formulations and routes of administration both influenced in which organs luciferase was expressed and the magnitude of expression. The highest expression levels of luciferase in the head kidney and liver were found after an intraperitoneal injection of lipoplex 2. In the spleen, the highest levels were detected after injection of naked DNA (intraperitonal or intramuscular) and lipoplex 2 (intraperitoneal). Following intravenous injection, naked DNA gave higher expression levels in the organs than the formulated plasmids and immersion and anal intubation were not effective routes of delivery as no expression of luciferase could be detected in any of the organs tested. Additionally, PCR using a primer specific for a 600 bp region of the luciferase gene pcDNA3-luc was used to assess the distribution of the plasmid itself after intramuscular and intraperitoneal injection. Positive amplification was obtained in spleen, head kidney, liver and muscle at the injection site following injection of formulated plasmids, while only muscle tissue from the injection site was positive when naked DNA was used.     

Effect of Macrophage Elimination Using Liposome-Encapsulated Dichloromethylene Diphosphonate on Tissue Distribution of Liposomes

Abstract

Effect of macrophage elimination using liposomal dichloromethylene diphosphonate (C1₂MDP)1 on tissue distribution of different types of liposomes was examined in mice. Intravenously administration into mice with CI2MDP encapsulated in liposomes composed of phosphatidylcholine, cholesterol and phosphatidylserine exhibits a temporary blockade of liver and spleen function for liposome uptake. At a low dose of 90 (ig/mouse, the liposome uptake by the liver was significantly decreased. Such decrease was accompanied by an increase in liposome accumulation in either spleen or blood depending on liposome composition and size. Direct correlation between the administration dose of liposomal CI2MDP and the liposome circulation time in blood was also obtained even for liposomes with an average diameter of more than 500 nm. These results indicate that temporary elimination of macrophages of the liver and spleen using liposomal CI2MDP may prove to be useful to enhance the drug delivery efficiency of liposomes.     

Effects of some pesticides on the vital organs of juvenile rainbow trout (Oncorhynchus mykiss)

Gill, trunk kidney, spleen, and liver of rainbow trout (Oncorhynchus mykiss) were examined after exposure to different sublethal concentrations of carbosulfan (25, 50 and 200 μg L⁻¹), propineb (3, 6 and 24 mg L⁻¹), and benomyl (2, 5 and 20 mg L⁻¹) for 14 days. Lesions were observed in gill, trunk kidney, spleen, and liver of rainbow trout exposed to either concentration of pesticides. The most important lesions were determined in the highest concentrations of pesticides. Lamellar fusion, lamellar hyperplasia, epithelial lifting, vacuolization of epithelial tissue, epithelial necrosis, hypertrophy and sloughing of epithelium were observed on fish exposed to carbosulfan, propineb and benomyl. Fish had cell necrosis, degeneration and oedemas in liver, trunk kidney and spleen. None of these lesions were seen in control fish.     

Ontogeny and disease responses of Langerhans-like cells in lymphoid tissues of salmonid fish

The ontogeny and disease responses of Langerhans-like cells within lymphoid tissues of Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, were investigated. These cells were studied in situ with the use of two markers: the ultrastructural presence of Birbeck-like granules and immunohistochemistry with an antibody against human langerin/CD207 that cross-reacts with salmonid tissues. The appearance of Birbeck-like granules was observed in rainbow trout at 2 weeks post-hatch (PH) in the thymus and anterior kidney prior to the development of the spleen. Spleen first appeared at 3 weeks PH in both Atlantic salmon and rainbow trout, and Birbeck-like granules were observed within cells of the newly developed spleens. The cross-reactivity of langerin as seen by immunohistochemistry was not clearly observed in kidney and spleen until 9 weeks PH, when a strong cytoplasmic reaction was observed. To study langerin-positive cells in spleen and kidney during disease, microsporidial gill disease (MGD) in rainbow trout was used as a known disease model inducing a strong cell-mediated adaptive immune response. Langerin-positive cells in healthy fish were seen predominantly in the spleen, and only low numbers were present in the anterior kidney. During MGD, langerin-positive cell numbers were elevated in the anterior kidney and were significantly higher during 5, 6, and 10 weeks post-exposure (PE) compared with healthy control tissue. During MGD, the distribution of langerin-positive cells in the spleen and anterior kidney shifted from having significantly higher numbers of cells in the spleen than in the kidney in controls and at 1 and 4 weeks PE to having a similar distribution of the cells in the two organs at 2, 3, 5, and 6 weeks PE. By 10 weeks PE, significantly higher numbers of langerin-positive cells occurred in the anterior kidney compared with the spleen.     

Pharmacokinetics of the iron chelator desperrioxamine as affected by liposome encapsulation: potential in treatment of chronic hemosiderosis

Desferrioxamine (DF), the chelator of choice for removal of excess stored iron, is limited by its rapid excretion, metabolic breakdown, and low cell uptake. We have encapsulated DF in unilamellar and multilamellar liposomes, and have compared the short-term pharmacokinetics of nonencapsulated and encapsulated ⁵⁹Fe-labeled DF after intravenous administration. Disappearance of ⁵⁹Fe-DF from the plasma was very rapid in mice receiving multilamellar liposome-encapsulated and nonencapsulated drug, but much slower in mice receiving unilamellar liposomes. Between 1 and 24 hours after injection, nonencapsulated ⁵⁹Fe-DF never exceeded 1–5% of the injected dose (ID) in liver or < 0.7% in spleen; whereas after either multilamellar or unilamellar liposomes, the uptake in liver was 30–35% ID, and in spleen was 1–5% ID. Excretion of ⁵⁹Fe-DF was much slower with liposome encapsulation. These results indicate that liposomes can effectively deliver DF to critical organs of iron storage. Thus this drug delivery system is potentially useful for treatment of iron overload.     

Uptake of the fish pathogen, Aeromonas salmonicida, by rainbow trout (Salmo gairdneri L.)

Aeromonas salmonicida was detected in the blood, kidney and spleen of rainbow trout within 2 min of immersion in a suspension containing 10⁴ cells/ml. Uptake into fish was enhanced by culturing the pathogen in low levels of nutrient, i.e., 0.1% (w/v) brain heart infusion (BHI) broth and by the addition of latex particles to the bacterial suspensions. However, there was no apparent difference in the uptake of pathogenic or non-pathogenic isolates. Moreover, the fish did not succumb to clinical signs of disease.     

Proteome Profiles of Head Kidney and Spleen of Rainbow Trout (Oncorhynchus Mykiss)

The head kidney and spleen are major lymphoid organs of the teleost fish. The authors identify proteome profiles of head kidney and spleen of rainbow trout (Oncorhynchus mykiss) using a shotgun proteomic approach. Gene ontology annotation of proteins is predicted using bioinformatic tools. This study represents detailed proteome profiles of head kidney and spleen of rainbow trout, with a total of 3241 and 2542 proteins identified, respectively. It is found that lymphoid organs are equipped with a variety of functional proteins related to defense, receptor, signal transduction, antioxidant, cytoskeleton, transport, binding, and metabolic processes. The identified proteome profiles will serve as a template for understanding lymphoid organ functions in salmonids and will increase the amount of spectra information of rainbow trout proteins in the public data repository PRIDE. This data can be accessed via ProteomeXchange with identifiers PXD008473 and PXD008478.     

Grafting of different glycosides on the surface of liposomes and its effect on the tissue distribution of 125I-labelled γ-globulin encapsulated in liposomes

Different glycosides were grafted on the surface of liposomes containing ¹²⁵I-labelled γ-globulin by two ways: (1) by using glycolipid and (2) by covalent coupling of $\text{p-}\text{aminophenyl-}\text{d}\text{-glycosides}$ to phosphatidylethanolamine liposomes using glutaraldehyde. The distribution of ¹²⁵I-labelled γ-globulin was determined in mouse tissues from 5–60 min after a single injection of these liposomes. The liver uptake of encapsulated ¹²⁵I-labelled γ-globulin was highest from liposomes having galactose and mannose on the surface. Competition experiments and cross-inhibition studies indicate that this uptake are mediated by specific recognition of the surface galactose and mannose residues of liposomes by the receptors present on the plasma membrane of liver cells. Stearylamine-containing liposomes were found to be more efficient in mediating the uptake of ¹²⁵I-labelled γ-globulin by the lung, whereas in the case of spleen, phosphatidylethanolamine liposomes were more efficient. The extent of uptake of ¹²⁵I-labelled γ-globulin from all types of liposome decreases as the amount of given liposomes increases. The uptake of ¹²⁵I-labelled γ-globulin from liposomes containing asialogangliosides depends upon the phospholipid/ glycolipid ratio. These experiments clearly demonstrate that enhanced liposome uptake by liver cells could be achieved by grafting galactose and mannose on the liposomal surface.     

A stromal cell line from rainbow trout spleen, RTS34st, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects of leukocytes

Summary A rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [³H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.     

Effective Skin Cancer Treatment by Topical Co-delivery of Curcumin and STAT3 siRNA Using Cationic Liposomes

The aim of the present study was to evaluate the effectiveness of iontophoretic co-delivery of curcumin and anti-STAT3 siRNA using cationic liposomes against skin cancer. Curcumin was encapsulated in DOTAP-based cationic liposomes and then complexed with STAT3 siRNA. This nanocomplex was characterized for the average particle size, zeta-potential, and encapsulation efficiency. The cell viability studies in B16F10 mouse melanoma cells have shown that the co-delivery of curcumin and STAT3 siRNA significantly (p < 0.05) inhibited the cancer cell growth compared with either liposomal curcumin or STAT3 siRNA alone. The curcumin-loaded liposomes were able to penetrate up to a depth of 160 μm inside the skin after iontophoretic (0.47 mA/cm²) application. The in vivo efficacy studies were performed in the mouse model of melanoma skin cancer. Co-administration of the curcumin and STAT3 siRNA using liposomes significantly (p < 0.05) inhibited the tumor progression as measured by tumor volume and tumor weight compared with either liposomal curcumin or STAT3 siRNA alone. Furthermore, the iontophoretic administration of curcumin-loaded liposome-siRNA complex showed similar effectiveness in inhibiting tumor progression and STAT3 protein suppression compared with intratumoral administration. Taken together, cationic liposomes can be utilized for topical iontophoretic co-delivery of small molecule and siRNA for effective treatment of skin diseases.     

UPTAKE AND INTRACELLULAR PROCESSING OF PEG-LIPOSOMES AND PEG-IMMUNOLIPOSOMES BY KUPFFER CELLS IN VITRO*

Specific targeting of drugs to for instance tumors or sites of inflammation may be achieved by means of immunoliposomes carrying site-specific antibodies on their surface. The presence of these antibodies may adversely affect the circulation kinetics of such liposomes as a result of interactions with cells of the mononuclear phagocyte system (MPS), mainly represented by macrophages in liver and spleen. The additional insertion of poly(ethylene glycol) chains on the surface of the immunoliposomes may, however, attenuate this effect.

We investigated the influence of surface-coupled rat or rabbit antibodies and of PEG on the uptake of liposomes by rat Kupffer cells in culture with ³H-cholesteryloleyl ether as a metabolically stable marker. Additionally, we assessed the effects of surface-bound IgG and PEG on the intracellular processing of the liposomes by the Kupffer cells, based on a double-label assay using the ³H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-¹⁴C-oleate as relative measure of degradation.

Attachment of both rat and rabbit antibodies to PEG-free liposomes caused a several-fold increase in apparent size. The uptake by Kupffer cells, however, was 3–4 fold higher for the rat than for the rabbit IgG liposomes. The presence of PEG drastically reduced the difference between these liposome types. Uptake of liposomes without antibodies amounted to only about 10% (non-PEGylated) or less (PEGylated) of that of the immunoliposomes.

In contrast to the marked effects of IgG and PEG on Kupffer cell uptake, the rate of intracellular processing of the liposomes remained virtually unaffected by the presence of these substances on the liposomal surface.

These observations are discussed with respect to the design of optimally formulated liposomal drug preparations, combining maximal therapeutic efficacy with minimal toxicity.     

Effects of cortisol on gill chloride cell morphology and ionic uptake in the freshwater trout,Salmo gairdneri

Summary Daily intramuscular injection of cortisol (4 mg kg^–1 body weight) in rainbow trout,Salmo gairdneri, for 10 days caused significant increases in the number and individual apical surface area of gill chloride cells per mm² of filament epithelium. Concomitantly, whole body influxes of sodium (Na⁺) and chloride (Cl^–) increased. Acute (3 h) intra-arterial infusion of cortisol did not affect whole body Na⁺ or Cl^– influx. A significant correlation was observed between both Na⁺ and Cl^– influxes and the fractional apical surface area of filament chloride cells in control, sham (saline-injected) and experimental (cortisol-injected) fish. The chloride cells displayed similar ultrastructural modifications in trout undergoing cortisol treatment as in trout transferred to ion-deficient water. These findings suggest the existence of structure/function relationships in which branchial chloride cell morphology is an important determinant of Na⁺ and Cl^– transport capacity. We conclude that chronic cortisol treatment enhances whole body Na⁺ and Cl^– influxes by promoting proliferation of branchial chloride cells. The results of correlation analysis indicate that the chloride cell is an important site of NaCl uptake in freshwater rainbow trout.     

A Novel Liposome-Based Nanocarrier Loaded with an LPS-dsRNA Cocktail for Fish Innate Immune System Stimulation

Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ∼125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health.     

Incorporation of the Lipopolysaccharide and Polysaccharide from Aeromonas Salmonicida Into Liposomes

Abstract

Incorporation of the lipopolysaccharide (LPS) and polysaccharide (PS) from Aeromonas salmonicida into liposomes of varying lipid composition and lamellarity as a function of the LPS and PS concentration was investigated. Positively-charged multilamellar vesicles (MLV) composed of phosphatidylcholine (PC): cholesterol (CH): stearylamine (SA) (6:3:1, mole: mole: mole) incorporated the LPS more readily than negatively-charged liposomes composed of PC: CH: phosphatidylglycerol (PG) in the same molar ratios. Regardless of surface charge, more LPS was incorporated into MLV than into vesicles prepared by relatively mild sonication (SV) or large unilamellar vesicles prepared via extrusion through 200 nm pore size filters (LUVET₂₀₀). In contrast, SV and LUVET₂₀₀ incorporated more PS than did MLV. The total amount of liposomally-incorporated LPS or PS among the three vesicle types was proportional to the concentration of antigen in the hydrating solutions.     

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distributions of ¹⁴C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.     

Detection and Quantification of Flavobacterium psychrophilum-Specific Bacteriophages In Vivo in Rainbow Trout upon Oral Administration: Implications for Disease Control in Aquaculture

The use of bacteriophages in the treatment and prevention of infections by the fish pathogen Flavobacterium psychrophilum has attracted increased attention in recent years. It has been shown recently that phage delivery via the parenteral route resulted in immediate distribution of phages to the circulatory system and the different organs. However, little is known about phage dispersal and survival in vivo in rainbow trout after delivery via the oral route. Here we examined the dispersal and survival of F. psychrophilum phage FpV-9 in vivo in juvenile rainbow trout after administration by three different methods—bath, oral intubation into the stomach, and phage-coated feed—with special emphasis on the oral route of delivery. Phages could be detected in all the organs investigated (intestine, spleen, brain, and kidney) 0.5 h postadministration, reaching concentrations as high as ∼10⁵ PFU mg intestine⁻¹ and ∼10³ PFU mg spleen⁻¹ within the first 24 h following the bath and ∼10⁷ PFU mg intestine⁻¹ and ∼10⁴ PFU mg spleen⁻¹ within the first 24 h following oral intubation. The phages were most persistent in the organs for the first 24 h and then decreased exponentially; no phages were detected after 83 h in the organs investigated. Phage administration via feed resulted in the detection of phages in the intestine, spleen, and kidney 1 h after feeding. Average concentrations of ∼10⁴ PFU mg intestine⁻¹ and ∼10¹ PFU mg spleen⁻¹ were found throughout the experimental period (200 h) following continuous delivery of phages with feed. These experiments clearly demonstrate the ability of the phages to survive passage through the fish stomach and to penetrate the intestinal barrier and enter the circulatory system after oral delivery, although the quantity of phages found in the spleen was 100- to 1,000-fold lower than that in the intestine. It was also shown that phages could tolerate long periods of desiccation on the feed pellets, with 60% survival after storage at −80°C, and 10% survival after storage at 5°C, for ∼8 months. Continuous delivery of phages via coated feed pellets constitutes a promising method of treatment and especially prevention of rainbow trout fry syndrome.     

The effect of elimination of macrophages on the tissue distribution of liposomes containing [H]methotrexate

In the present study the tissue distribution of [³H]methotrexate was studied after intravenous injection of [³H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [³H]methotrexate liposomes in the blood, due to a decreased uptake of [³H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [³H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.     

Effect of turpentine oil on C-reactive protein (CRP) production in rainbow trout (Oncorhynchus mykiss)

The effect of turpentine oil on C-reactive protein (CRP) production was studied in rainbow trout (Oncorhynchus mykiss). Serum CRP concentration was estimated by sandwich enzyme-linked immunosorbent assay using anti-rainbow trout CRP monoclonal antibody (mAb) AC4 and polyclonal antibody. Intracellular CRP was demonstrated by flow cytometry using anti-trout CRP mAb. Hepatocytes, head kidney macrophages, spleen lymphocytes and peripheral blood lymphocytes showed reaction against AC4, but RTG-2 fibroblastic line cells, derived from rainbow trout gonad did not. This is the first report on the detection of intracellular CRP in fish. CRP levels decreased significantly 1 day after intramuscular injection of turpentine oil and remained low for 14 days. Significant decreases in the expression of CRP in hepatocytes, head kidney macrophages and spleen lymphocytes after injection of turpentine oil were found. The reduction of serum CRP concentration after turpentine oil injection may be attributed to decreases in intracellular CRP synthesis.