L. van Bloois

Adhesion molecules: a new target for immunoliposome-mediated drug delivery

The anti‐ICAM‐1 monoclonal antibody F10.2 was conjugated to liposomes to target to cells expressing the cell adhesion molecule ICAM‐1. We demonstrate that F10.2 immunoliposomes bind to human bronchial epithelial cells (BEAS‐2B) and human umbilical vein endothelial cells (HUVEC) in a specific, dose‐ and time‐dependent manner. It appears that the degree of ICAM‐1 expression is the limiting factor in the degree of immunoliposome binding to the cells. These results are a first step in the strategy for specific drug delivery to target sites characterised by increased expression of adhesion molecules.

Intravenous administration of superoxide dismutase entrapped in long circulating liposomes. II. In vivo fate in a rat model of adjuvant arthritis.

Rheumatoid arthritis (RA) is a prevalent and debilitating autoimmune disease that affects the joints. RA is characterized by an infiltration of the affected joint by blood-derived cells. In response to activation, these cells generate reactive oxygen species, resulting in an oxidative stress situation. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. The free radical scavenger enzyme superoxide dismutase (SOD) may be used as a therapeutic agent in rheumatoid arthritis, but its rapid elimination from the circulation is a major limitation. Targeted delivery of SOD may overcome this limitation. In this study, the utility of PEGylated liposomes (PEG-liposomes) for targeting SOD to arthritic sites was explored. The targeting of SOD to arthritic sites following intravenous administration of both PEG-liposomes and positively charged liposomes lacking PEG but containing stearylamine (SA-liposomes) in rats with adjuvant arthritis was studied. At 24 h post injection, the blood levels of long circulating liposomes with a mean size of 0.11 micrometer and 0.20 micrometer were 8- and 3-fold higher, respectively, as compared to the SA-liposomes. The majority of SOD administered in liposomal form remains within the liposomes when they circulate in the bloodstream. The highest target uptake was observed with PEG-liposomes with a mean size of 0.11 micrometer and the lowest uptake with the SA-liposomes. These results demonstrate that SOD can be targeted to inflamed sites most efficiently via small-sized PEG-liposomes. Small-sized PEG-coated liposomes are to be preferred if prolonged circulation and enhanced localization of SOD at arthritic sites are desired.

Cellular uptake of liposomes targeted to intercellular adhesion molecule-1 (ICAM-1) on bronchial epithelial cells

Previously, it was demonstrated that immunoliposomes, bearing anti-intercellular adhesion molecule-1 (ICAM-1) antibodies (mAb F10.2), can specifically bind to different cell types expressing ICAM-1. In this study, we have quantified the amount of immunoliposomes binding to IFN-gamma activated human bronchial epithelial cells (BEAS-2B) in vitro and studied the subsequent fate of cell-bound anti-ICAM-1 immunoliposomes. We demonstrate that binding of the immunoliposomes to the epithelial cells depends on the liposome concentration used. After binding to the cell surface, the anti-ICAM-1 immunoliposomes are rapidly internalised by the epithelial cells. Sixty percent of cell-bound immunoliposomes were internalised by the epithelial cells within 1 h of incubation at 37 degrees C. The results indicate that ICAM-1 targeted immunoliposomes may be used as carriers for the intracellular delivery of anti-inflammatory drugs to sites of inflammation characterised by an increased expression of ICAM-1.

Technetium-99m labelled liposomes to image experimental arthritis

OBJECTIVES Liposomes sterically stabilised with polyethylene glycol (PEG) labelled with technetium-99m were tested for their ability to image adjuvant arthritis in a rat model. METHODS Adjuvant arthritis was induced in the ankle joint of the left hind foot by injection of Mycobacterium butyricum in Freund’s incomplete adjuvant in the foot pad. Seven days later animals received the following radiopharmaceuticals labelled with 99mTc (a) non-PEG-liposomes, (b) PEG-liposomes or (c) non-specific human polyclonal IgG. For each of the radiopharmaceuticals the in vivo distribution of the radiolabel was monitored both scintigraphically as well as by counting the dissected tissues at two, eight, and 24 hours after injection. RESULTS The pharmacokinetics of the radiopharmaceuticals differed considerably (half life in the blood: PEG-liposomes (18 hours) > 99mTc-IgG (3 hours) > non-PEG liposomes (1 hour)). The inflamed focus was visualised with each of the agents. The uptake of each of the radiopharmaceuticals in the inflamed ankle region correlated with their residence time in the blood (inflamed joint uptake: PEG liposomes (1.15% injected dose (ID)/g)>99mTc-IgG (0.35% ID/g)>non-PEG-liposomes (0.05% ID/g)). Quantitative analysis of the images showed that the inflamed ankle to background ratio was highest with the PEG-liposomes (7.5 at 24 hours after injection), while with the other two agents this ratio did not exceed 4. CONCLUSION This study shows that99mTc-labelled PEG-liposomes may be an excellent agent to visualise arthritis. Increased label uptake in the inflamed joint and increased target to background ratios can be obtained with PEG-liposomes because of their long circulating properties. In addition to their use as vehicles for scintigraphic imaging of arthritis PEG-liposomes might also be used for the site specific delivery of antirheumatic drugs.

The Size Reduction of Liposomes with a High Pressure Homogenizer (Microfluidizer™). Characterization of Prepared Dispersions and Comparison with Conventional Methods.

AbstractA high pressure homogenizer (the Microfluidzer™) was tested for its ability to produce liposomal dispersions with a narrow size distribution. For dispersions with 40μmol phospholipid/ml, sonication and high pressure homogenization gave similar average particle diameters. With the Microfluidizer™ it was possible to produce reproducible dispersions with a mean particle size between 0.05 and 0.25μm and a narrow size distribution containing up to 200μmol phospholipid/ml. However, after processing 50 ml batches significant lipid loss was observed and small amounts of metal particles were found in the dispersions.

A DNase Encoded by Integrated Element CJIE1 Inhibits Natural Transformation of Campylobacter jejuni

The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNA-hydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

Preparation and characterization of doxorubicin-containing liposomes. II. Loading capacity, long-term stability and doxorubicin-bilayer interaction mechanism

Doxorubicin loading capacity was determined for negative (phosphatidylcholine-cholesterol-phosphatidylserine, 10:4:1) and positive (phosphatidylcholine-cholesterol-stearylamine 10:4:3) liposomes prepared according to the “film” method with doxorubicin added to the phospholipids before film formation. Doxorubicin association depended on the initial ratio of doxorubicin/phospholipid, the charge and the size. The maximum loading capacity was 60–75 mmol doxorubicin/mol phospholipid for the negative and about 55 mmol doxorubicin/mol phospholipid for positive, non-filtered, multilamellar liposomes. Filtration or ultrasonication/ultracentrifugation reduced the doxorubicin-bilayer interaction. Loss on storage of doxorubicin from the filtered, multilamellar, or unilamellar vesicles decreased in the following order: filtered negative < unilamellar positive < unilamellar negative vesicles. Positive unilamellar vesicles increased in particle size on storage. Negative liposomes were stable. ζ-Potentials of positive or negative liposomes did not depend on the presence of associated doxorubicin. This makes a purely electrostatic mechanism of interaction between the drug and the bilayer unlikely. If doxorubicin was added to the phospholipid film in the hydration buffer a dramatic drop in doxorubicin-bilayer association was observed for positive liposomes. For negative liposomes the loading efficiency was unaffected.

Liposomal encapsulation of dexamethasone modulates cytotoxicity, inflammatory cytokine response, and migratory properties of primary human macrophages

UNLABELLED The encapsulation of drugs into liposomes aims to enhance their efficacy and reduce their toxicity. Corticosteroid-loaded liposomes are currently being evaluated in patients suffering from rheumatoid arthritis, atherosclerosis, colitis, and cancer. Here, using several different fluorophore-labeled formulations, we comprehensively studied the impact of liposome encapsulation of the prototypic corticosteroid dexamethasone on various primary human cells in vitro. Liposomal dexamethasone targeted several primary cell types in a dose and time-dependent manner, but specifically reduced cytotoxicity against human fibroblasts and macrophages in comparison to the solute drug. Furthermore, macrophage maturation and polarization markers were altered. Interestingly, liposomal dexamethasone induced proinflammatory cytokine secretion (specifically TNF, IL1β, IL6) in unstimulated cells, but reduced this response under inflammatory conditions. Monocyte and macrophage migration was significantly inhibited by dexamethasone-loaded liposomes. The findings indicate that the encapsulation of dexamethasone into liposomes modulates their cellular mechanism of action, and provides important indications for follow-up in vivo investigations. FROM THE CLINICAL EDITOR This study investigates mechanism of action of liposomal dexamethason in the treatment of inflammatory conditions. It is concluded that liposomal dexamethasone actually induces proinflammatory cytokine secretion in unstimulated cells, but reduces the same response under inflammatory conditions. Monocyte and macrophage migration was also inhibited. The findings indicate that liposomal dexamethasone may have different mechanisms of action than its native counterpart.

Labelled StealthR liposomes in experimental infection : an alternative to leukocyte scintigraphy?

Indium-111 (111In) and technetium-99m (99Tcm) Stealth liposomes were compared with 111In- and 99Tcm-labelled white blood cells (WBC) in experimental infection in a rabbit model. Preformed polyethylene glycol-coated liposomes and separated WBC were radiolabelled with either 111In-oxine or 99Tcm-hexamethylpropyleneamine oxime (99TcM-HMPAO). After the intravenous administration of one of the four radiopharmaceuticals to rabbits with focal Staphylococcus aureus infection, scintigraphic images were recorded at various time points post-injection and the biodistribution of the radiopharmaceuticals was determined. At 4 h post-injection, uptake of 111In-WBC in the abscess was significantly higher than that of the three other products. AT later time points, 111In-WBC, 111In-liposome and 99Tcm-liposome uptake in the abscess were similar. In contrast, a 20 h post-injection, uptake of 99Tcm-WBC was significantly lower. The abscess-to-background ratios showed a similar pattern to the absolute abscess uptake: initial high values for 111In-WBC, a more gradual increase over time of the liposome preparations to the level of 111In-WBC and persistently low values for 99Tcm-WBC. Clearance from the blood of both labelled WBC preparations was significantly faster and splenic uptake significantly higher compared with those of the labelled liposomes. In conclusion, given the similar in vivo characteristics of labelled liposomes and labelled WBC, labelled liposomes may be an attractive replacement for labelled WBC, providing a continuously available, high-quality, 99Tcm-labelled radiopharmaceutical that can be prepared easily without any need to handle blood.

Preparation and characterization of doxorubicin-containing liposomes: I. Influence of liposome charge and pH of hydration medium on loading capacity and particle size

Abstract Physicochemical characterization of doxorubicin-containing liposomes is essential to obtain reproducible results in in vivo studies. Particle size, loading capacity and release on storage of doxorubicin liposomes were investigated in different stages of liposome preparation as a function of buffer pH (4, 6.3, 7.4, 8.4) and liposome composition: negatively or positively charged. It was found that doxorubicin strongly interacted with both types of liposomes. For the higher pH dispersions non-liposomal structures of unknown composition were suspected. Filtration as sizing procedure was only effective for negative liposomes at low pH. Both positively and negatively charged vesicles could be effectively reduced in size by ultrasonication in the pH range from 4 to 6.3. Release rates of the drug were low under these conditions. For preparation of well-characterized liposome dispersions with doxorubicin this pH range-is to be preferred.