Tan, Lloyd Seng Kee; (1990) The applications of liposomes in immunoprophylaxis and immunotherapy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Liposomes have proven to be useful tools in numerous areas of biological research. The aims of this thesis are to further investigate their role and effectiveness in two different but well-established immunological applications, namely as adjuvants to bacterial and viral components and as antibody-mediated targeted carriers of entrapped drugs. Dehydration-rehydration vesicles (DRV) were found to be capable of entrapping relatively large reconstituted influenza virus envelopes (RIVE) with high efficiency and reproducibility. Balb/c mice inoculated with DRV containing RIVE exhibited primary and secondary IgG1 responses significantly higher as compared to mice inoculated with equal doses of unencapsulated RIVE. Similar responses were observed in the IgG2a , IgG2b and IgG3 subclasses. The adjuvanticity of liposomes on poorly immunogenic synthetic virus subunit peptides was demonstrated using DRV incorporating the peptides in their internal space and liposomes with covalently surface-linked peptides. It was found that liposome association boosted the primary and secondary IgG1 responses against the peptides as compared to controls in which free peptides were administered. Surface-linked peptide gave an initially more rapid rise in antibody levels as compared to entrapped peptide but with no immunological memory whereas one of the encapsulated peptides elicited a milder primary response but later exhibited a strong anamnestic response. The second part of the thesis relates to the behaviour of liposomes as targeted carriers of drugs. The influence of charge on the electrophoretic mobility of MLV in vitro and on the clearance from the circulation of SUV after intravenous administration was studied. It was found that positively-charged lipids (stearylamine and BisHOP) incorporated into DSPC SUV were cleared from the circulation in mice at the same rate as negatively-charged ones incorporating phosphatidic acid (PA), and in both groups, more rapidly than neutral SUV. However, stearylamine PC SUV were cleared more slowly than similarly positively-charged BisHOP PC SUV and negative PA PC SUV. This suggests that positively and negatively-charged SUV made by adding charged lipids to neutral phospholipids are cleared at the same rates if the charged lipid is confined in a rigid bilayer (eg. in DSPC SUV) or if the acyl chain of the charged lipid is saturated and long enough to prevent its removal from a more "fluid' bilayer (eg. in PC SUV). A study of the effect of pH on the electrophoretic mobility of charged MLV in vitro revealed that the net surface charge on liposomes is markedly influenced by changes in pH. These findings may have important implications for the clinical use of liposomes containing charged lipid, ligands or drugs and in states of acidosis or alkalosis. For use as targeting ligands, monoclonal antibodies that bound to 3 human hepatocellular carcinoma cell lines and snap-frozen sections of a patient's tumour were raised by the novel method of immunizing mice with PLC/PRF/5 membrane preparations. The monoclonals designated RF-HCC 1 and RF-HCC 2 were of IgG1 and IgM subclasses respectively. A simple method for the coating of liposomes with proteins was also developed. Several proteins (eg. BSA, pig gammaglobulins and tetanus toxoid) could be made to adhere rapidly and efficiently to both MLV and SUV after treatment with HC1 and NaNO2. The reaction is complete within four hours and almost all of the protein is retained on the liposomes after storage for 2 weeks at 4°C. CF-containing SUV coated with passively-adsorbed monoclonal antibody (RF-HCC1 and MOPC 21 IgG1) were used in in vitro studies for targeting to Mahlavu cells. Significantly greater amounts of the marker entrapped in antibody-coated SUV were associated with the target cells after incubation for one and a half hours at 4°C as compared to plain SUV. These results demonstrate that passively adsorbed antibodies represent a rapid, convenient and effective method of ligand-mediated targeting of SUV to cells in vitro.

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