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Biomedical subjects

G Gregoriadis

Publications and source records attributed to G Gregoriadis.

At least 91 records · Page 5Linked to original sources
PubMed1987

Liposomes as adjuvants with immunopurified tetanus toxoid: the immune response.

Immune responses of BALB/c mice to immunopurified tetanus toxoid entrapped in dehydration-rehydration vesicles composed of equimolar egg phosphatidylcholine and cholesterol were compared to those of free toxoid. Animals were injected intramuscularly with the free or liposomal toxoid and identical injections were repeated 4 weeks and, in some experiments, 24 weeks later. Analysis of IgG1, IgG2a, IgG2b, IgG3 and IgM in the sera by an enzyme-linked immunosorbent assay suggested that adjuvanticity of liposomes is reflected in most antibody subclasses and that there is no shift in subclasses compared to the response obtained with the free antigen, thus establishing liposomes as a type I adjuvant. In other, appropriately designed experiments, the relative importance of events following the first and second injections in determining the adjuvant effect of liposomes was investigated. It was found that liposome adjuvanticity is the outcome of events following primary immunization.

Adjuvants, Immunologic↗
PubMed1987

Liposomes as immunological adjuvants: antigen incorporation studies.

Tetanus toxoid was incorporated into liposomes composed of equimolar phospholipid and cholesterol. The toxoid was either passively entrapped into multilamellar vesicles prepared by the dehydration-rehydration procedure (DRV) or covalently coupled by diazotization to the surface of multilamellar vesicles (MLV) prepared by the classical procedure. Up to 82.3% of the antigen used was entrapped in neutral, negatively and positively charged DRV composed of a variety of unsaturated and saturated phospholipids and 63.1% was coupled to MLV composed of egg phosphatidylcholine. After freeze-drying of toxoid-incorporating DRV and MLV and subsequent rehydration, up to 93.5% of the antigen was recovered with liposomes and, in the case of MLV, retained its external localization. Upon freeze-drying in the presence of 0.25 M trehalose, up to 96.1% of the antigen was recovered with the DRV liposomes. In immunization studies using Balb/c mice, DRV composed of equimolar egg phosphatidylcholine and cholesterol were shown to act as immunological adjuvants to the entrapped tetanus toxoid. In addition, there was no difference in immune responses between DRV and MLV of identical composition but bearing the toxoid on their surface. Comparison of immune responses to the toxoid entrapped in DRV made of phospholipids with varying gel to liquid crystalline transition temperature (Tc) revealed a reduction in responses to very low or nil values for DRV made of distearoyl phosphatidylcholine (Tc 54 degrees C).

Adjuvants, Immunologic↗
PubMed1987

Liposomes as adjuvants with immunopurified tetanus toxoid: influence of liposomal characteristics.

The effect of various manipulations of liposomes composed of equimolar phospholipid and cholesterol on immune responses to the incorporated immunopurified tetanus toxoid was investigated in BALB/c mice. In studies designed to establish proper dosage for immunization and to reveal the roles of the liposomal phospholipid to toxoid mass ratio, gel-liquid crystalline transition temperatures (Tc) of the liposomal phospholipids and mode of antigen incorporation into liposomes on immune responses, animals were injected intramuscularly with various amounts of the toxoid, free, entrapped in multilamellar dehydration-rehydration vesicles (DRV) or covalently linked to the surface of multilamellar vesicles (MLV) prepared by the classical procedure. Two identical injections separated by 4 weeks were given and IgG1 and IgG2b antibodies specific for the toxoid assayed in sera by an enzyme-linked immunosorbent assay. Results suggest that the adjuvant effect of liposomes is improved considerably when liposomal phospholipid to toxoid mass ratios are as high as 2049:1; however, adjuvanticity is reduced to reach very low levels for much higher ratios (e.g. 90,361:1); antibody responses are similar for liposomes (phospholipid to toxoid mass ratios: 14.3-33.1) made of a variety of phospholipids with Tcs ranging from -32 degrees to 41.5 degrees but are low or non-existent when liposomes are made of distearoyl phosphatidylcholine (Tc, 54 degrees) (however, see Discussion); there are no differences in antibody responses between liposomes with entrapped and surface-linked toxoid.

Adjuvants, Immunologic↗
PubMed1986

Antibody-coated liposomes. The role of non-specific antibody adsorption.

Incubation of small unilamellar liposomes composed of equimolar phospholipid and cholesterol with mouse IgG or mouse monoclonal IgG1 for up to 24 h resulted in considerable (34-89%) adsorption of the protein onto the liposomal surface. Immunoglobulin remained adsorbed after exposure to mouse plasma and, in the case of monoclonal (anti-HBSAg) IgG1, was able to mediate association of the liposomes with the antigen. Extensive immunoglobulin adsorption suggests caution in interpreting covalent linkage values obtained upon prolonged incubation of chemically modified antibodies with liposomes. It may also be a preferred alternative in preparing targeted liposomes for intravenous use when chemically modified antibodies promote premature clearance of the antibody-liposome complex from the circulation.

Adsorption↗
PubMed1985

Tissue distribution of liposomes exhibiting long half-lives in the circulation after intravenous injection.

The previously established direct relationship between long half-life of uncharged small unilamellar liposomes in the circulation of injected animals and reduced permeability to entrapped solutes in the presence of blood plasma was investigated further. It was found that vesicle size and surface charge override state of membrane permeability in determining rates of vesicle clearance. Thus, half-lives of liposomes that were practically impermeable in plasma were much shorter for larger vesicles or vesicles with a negative charge on their surface. 111In-labelled bleomycin-containing small unilamellar liposomes with long half-lives accumulated in the liver after injection to a much lesser extent (e.g., 26%) than similar liposomes (55% of the dose) exhibiting a shorter half-life. Much of the injected long-lived liposomes (about 35%) were recovered in the carcass of the animals. Scanning of the carcasses revealed quantitative accumulation of radioactivity in the bones, presumably the phagocytic cells of the bone marrow. Long-lived liposomes appear suitable for drug delivery to, or imaging of the bone marrow.

Animals↗
PubMed1984

The use of monoclonal anti-Thy1 IgG1 for the targeting of liposomes to AKR-A cells in vitro and in vivo.

A number of SH-containing proteins or protein derivatives were coupled to small unilamellar liposomes. These were composed of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE) and cholesterol (1:1, phospholipid/cholesterol molar ratio) and activated (DPPE moiety) with the heterobifunctional reagents N-hydroxysuccinimide ester of iodoacetic acid (hydroxysuccinimide iodoacetate), N-succinimidyl-4-(2-bromoacetylamino)benzoate (SBAB) or N-succinimidyl-3-(2-pyridyldithio)proprionate (SPDP). DPPE was activated with the reagents before or after its incorporation into liposomes. Protein coupling values varied widely depending on the reagent and the protein used, but were highest in the case of SPDP-activated liposomes and SPDP-modified immunoglobulin G (IgG). Monoclonal anti-Thy1 125I-IgG1-bearing liposomes (SPDP- or SBAB-activated) containing quenched carboxyfluorescein were incubated under a variety of conditions with mouse AKR-A cells expressing the cross-reactive Thy 1.1 antigen. The following observations were made; (a) binding of intact liposomes to the cells at 4 degrees C reached plateau values after about 1 h with at least 70% of the liposomes used being capable of associating with the target cells; (b) binding of liposomes to AKR-A cells was much more pronounced than when using another cell line (EL4-Tc); (c) binding to AKR-A cells could be effected with as little as 1.3 molecules (average) of IgG1 per vesicle; (d) binding was inhibited only modestly by the presence of 50% mouse plasma; (e) stability of IgG1-bearing liposomes in terms of entrapped solute and IgG1 retention in the presence of plasma at 37 degrees C was maintained quantitatively for at least 5.5 h, and by 24 h, 54% of the IgG1 was still associated with the liposomes. AKR mice were injected intravenously with 99mTc-labelled AKR-A cells and 2.5 min later with anti-Thy1 125I-IgG1-bearing liposomes containing quenched carboxyfluorescein and 111In-Ca-DTPA or with similar liposomes devoid of IgG1. In parallel experiments, AKR mice received either of the liposome preparations without previous injection of cells. On the basis of patterns of quenched carboxyfluorescein, 111In and 125I-clearance from the circulation, of 99mTc levels in the blood and of values of 111In in the liver and spleen, it appeared that IgG1-bearing liposomes were capable of binding to their target cells in the vasculature. Such binding accelerated the clearance of interacting moieties (i.e., AKR-A cells and liposomes). The present results suggest that targeting of liposomes to circulating in vivo is feasible.

Animals↗
PubMed1983

Stability and clearance of small unilamellar liposomes. Studies with normal and lipoprotein-deficient mice.

The effect of high density lipoproteins (HDL) on the stability and clearance of injected liposomes was investigated under conditions of abnormal lipoprotein metabolism in vivo. Small unilamellar liposomes composed of phosphatidylcholine and containing quenched carboxyfluorescein were injected intravenously or intraperitoneally into normal mice or mice previously made lipoprotein deficient with 4-aminopyrazolo[3,4d]pyrimidine (4-APP). As evidenced from quenched carboxyfluorescein values in the blood, levels of stable liposomes in the circulation were increased and clearance rates reduced considerably in lipoprotein-deficient animals indicating increased bilayer integrity. This was confirmed by the demonstration that transfer of liposomal phosphatidylcholine to HDL, occurring in the presence of normal mouse plasma, was virtually abolished in the presence of plasma from lipoprotein deficient mice. The role of other lipoprotein species in destabilizing liposomes was also investigated. Plasma from lipoprotein-deficient mice was supplemented with increasing amounts of HDL, LDL + IDL or VLDL (to cover the physiological range of lipoprotein concentrations in mouse blood) prior to the addition of phosphatidylcholine liposomes, and incubated at 37 degrees C. It was shown that among the lipoprotein species studied only HDL was detrimental to liposomal stability under the conditions employed. Our results indicate that use of liposomal drugs in the treatment of patients must take into account HDL fluctuations in their blood as these could after liposomal membrane permeability to the drugs and thus upset therapeutic efficiency.

Animals↗
PubMed1983

Fate of cholesterol-rich liposomes after subcutaneous injection into rats.

The fate of small (30-60 nm) and large (about 400 nm diameter) liposomes composed of phosphatidylcholine or sphingomyelin and equimolar cholesterol and containing quenched carboxyfluorescein and 111In-labelled bleomycin after footpad injection of rats was investigated. Judging from the values of latent carboxyfluorescein in the plasma, phosphatidylcholine and sphingomyelin small liposomes entered the blood circulation intact, presumably via the lymphatics to reach 3 h after injection a peak of 16.9 and 23.1% of the dose per total blood, respectively. Of the 111In radioactivity given in phosphatidylcholine liposomes, about 32% was recovered in the liver. Hepatic uptake of 111In for sphingomyelin liposomes was lower (about 9%) reflecting their slower rate of clearance. No latent carboxyfluorescein could be detected in the blood after injection with phosphatidylcholine or sphingomyelin large liposomes and levels of 111In in the liver (and spleen) were very low. A proportionally much greater amount of liposomal 111In was intercepted by the popliteal and to a lesser extent, the lumbar lymph nodes. Such uptake was significantly higher (e.g. 463%) for small than for large (e.g. 195% per g popliteal nodes) liposomes. After foot pad injection of large liposomes into Walker 256 tumour-bearing rats, localization of 111In in the popliteal nodes was similar to that seen with control animals. However, 111In localization in the lumbar nodes was augmented more than 5-fold. These results suggest that large liposomes as used in this study, characterized by efficient drug entrapment, inability to reach the liver and spleen and improved localization in the lymph nodes of tumour-bearing animals may be preferable to vesicles of small size for the local treatment of lymphatic metastases.

Animals↗
PubMed1983

The effect of lipid composition of small unilamellar liposomes containing melphalan and vincristine on drug clearance after injection into mice.

Melphalan and vincristine together with their radiolabelled derivatives were entrapped in small unilamellar liposomes of varying cholesterol content and phospholipid composition. After intravenous injection of drug-containing egg phosphatidylcholine liposomes into mice, drug clearance rates from the blood were reduced with increasing cholesterol content. Circulating drugs were partially associated with the carrier and partly free, mostly bound to plasma proteins. The ratio of drug associated with liposomes to that circulating as free was dependent on the type of liposomes used and highest when these were cholesterol-rich. Drug clearance rates were reduced and entrapped: free drug ratios increased further when egg phosphatidylcholine in cholesterol-rich liposomes was replaced by sphingomyelin. Drug-containing cholesterol-rich liposomes injected intraperitoneally were found capable of entering the periphery intact and quantitatively to assume clearance rates similar to those observed after intravenous treatment. Such manipulations in liposomal lipid composition can alter pharmacokinetics in ways that could provide optimal conditions for drug distribution into tumours and a therapeutic effect.

Animals↗
PubMed1983

Toxicity of sphingomyelin-containing liposomes after chronic injection into mice.

Chronic treatment (10 i.p. injections over 20 days) of Balb/c mice with SM liposomes led to 50 and 300% enlargement of the liver and spleen respectively. No such effect was observed after similar treatment with PC liposomes. Biochemical analysis of the enlarged tissues showed no significant changes in the concentrations of glycolipid, phospholipid and certain hydrolytic enzymes. However, the increase in tissue size was paralleled by an increase in protein content. Light and electron microscopy studies of the enlarged tissues revealed an increase in the number of Kupffer cells and collections of inflammatory cells in the liver and widespread granulomatous inflammation in the spleen. We conclude that SM liposomes, although probably toxic for use as a drug carrier, may serve as a model agent in the study of tissue granulomatous inflammation.

Animals↗