EFFECTS OF PHOSPHOLIPID COMPOSITION ON ADJUVANT EFFICIENCY OF LIPOSOMES

Hampl J., J. Franz, K. Jordanova, J. Stepanek:EffectsojPhospholipidComposition on Adjuvant Efficiency oj Liposomes. Acta vet. Brno 1995, 64: 163-164. Phospholipids with various transition temperatures, including egg phosphatidyl lecithine (EPL), hydrogenated egg phosphatidyl lecithine (HEPL), dimyristoyl phosphatidylcholine (DMPC) and dipalmitoyl phosphatidylcholine (DPPC), and cholesterol (C), stearylamine (SA) and dicetylphosphate (DCP) were used in various molar proportions for the preparation of multilamellar liposomes by the dehydration-rehydration method. BALB/c mice were then immunized and reimmunized with BSA entrapped in the liposomes. No significant effects of transition temperatures of the phospholipids or of surface charge on the intensity of antibody responses were demonstrable. The strongest antibody responses were found in mice immunized with multilamellar liposomes containing in their structures EPL, HEPL or DMPC combined with C, DCP and/or SA, which also showed a high entrapment stability when incubated in vitro in blood serum. Liposomes, BSA, antibody response, transition temperature, sUrface charge, stability Advances in biochemical and biotechnological methods have provided a basis for the development of new generations of biologicals including subunit vaccines and vaccines containing highly purified, inactivated microorganisms. However, the immunogenicity of this class of vaccines is often insufficient. One of the ways how to overcome this drawback is simultaneous administration of effective adjuvants or immunomodulants. It has been well established that liposomes can enhance immune responses to a number of microbial antigens. Currently, they are becoming one of the few non-toxic, biologically degradable adjuvants with prospective uses in human and veterinary medicine (A I v i n g andRichards 1990; Gregoriadis 1990; Friede et al. 1994).Thereexistseveral ways of modulating of the adjuvant activity of liposomes, including modifications of phospholipid composition allowing the introduction of surface charge or influencing the fluidity of the phospholipid bilayer, or control of morphological structure by various preparative procedures (Van H 0 ute et al. 1981; G reg 0 ria dis et al. 1987; Gregoriadis and Panagiotidi 1989; Gabizon and Papahadjopoulos 1992; B a k k e r W 0 u den b erg et al. 199·3a; For tin and The r i e n 1993; C h a r I e s et al. 1994). The object of our investigations were immune responses in mice treated with BSA as a model antigen entrapped in multilamellar liposomes prepared from phospholipids with various transition temperatures (Tc) and different surface charges. Materials and Methods Chemicals Egg phosphatidy llecithine (E 80) EPL and hydrogenated egg phosphatidyllecithine HEPL supplied by Lipoid K.G.; dimyristoyl phosphatidylcholine DMPC, dipalmitoyl phosphatidylcholine DPPC, stearylamine SA, dicetylphosphate DCP, and tetramethyl benzidine supplied by Sigma; cholesterolC and bovine serum albumin BSA supplied by Flow and DSOL, Prague, respectively.

Advances in biochemical and biotechnological methods have provided a basis for the development of new generations of biologicals including subunit vaccines and vaccines containing highly purified, inactivated microorganisms.However, the immunogenicity of this class of vaccines is often insufficient.One of the ways how to overcome this drawback is simultaneous administration of effective adjuvants or immunomodulants.
It has been well established that liposomes can enhance immune responses to a number of microbial antigens.Currently, they are becoming one of the few non-toxic, biologically degradable adjuvants with prospective uses in human and veterinary medicine (A I v i n g andRichards 1990 ;Gregoriadis 1990;Friede et al. 1994).Thereexistseveral ways of modulating of the adjuvant activity of liposomes, including modifications of phospholipid composition allowing the introduction of surface charge or influencing the fluidity of the phospholipid bilayer, or control of morphological structure by various preparative procedures (Van H  The object of our investigations were immune responses in mice treated with BSA as a model antigen entrapped in multilamellar liposomes prepared from phospholipids with various transition temperatures (Tc) and different surface charges.

Chemicals
Egg phosphatidy llecithine (E 80) -EPL and hydrogenated egg phosphatidyllecithine -HEPL supplied by Lipoid K.G.; dimyristoyl phosphatidylcholine -DMPC, dipalmitoyl phosphatidylcholine -DPPC, stearylamine -SA, dicetylphosphate -DCP, and tetramethyl benzidine supplied by Sigma; cholesterol-C and bovine serum albumin -BSA supplied by Flow and DSOL, Prague, respectively.respectively.Each of the phospholipids was used alone or in compositions with C at molar ratios 0.8:0.2 or 0.5:0.5, or with SA and DCP at the molar ratio 0.45:0.45:0.1.The initial weight ratio of the phospholipid composition and BSA was invariably 100: I.The preparation of liposomes from phospholipids with above-zero Tc was made at temperatures by 2 to 3 °C higher than was the declared Tc.BSA labelled with 125 1 by the oxidative method with chloramine T (H u n t e r and G r e e n woo d 1962) was used as a tracer for the determination of the rate of entrapment of BSA into ML V and in entrapment stability tests.

Entrapment stability tests
The following compositions with the highest BSA entrapment rates and surface charges were selected from each of the ML V variants to be tested for the entrapment stability: Variant I : EPL-C-DCP Variant 2: HEPL-C-SA Variant 3: DMPC-C-SA Variant 4 : DPPC-C-SA All tests were made at 22°C in triplicates.The liposomal suspensions were incubated in PBS, goat serum diluted 1:1 with PBS, or isotonic glycerol solution for 24 hours and centrifugated (Beckman, rotor SW 55 Ti, 40,000 r.p.m., 30 min) after 2, 4 and 24 hours of incubation.Radioactivity of the 1251-labelled BSA was measured in aliquots of supernatants, the aliquots were then quantitatively returned to the respective centrifugation vessels and the liposomal pellets were resuspended after each centrifugation (H amp I et al. 1994).

Immunization
Seventy BALBIc mice were divided into four experimental and one control groups.Each experimental group was treated with one of the variants of ML V showing the highest entrapment rate and surface charge (see above).The uniform dose was 2 ug BSA intraperitoneally per animal for both the primary immunization and reimmunization made after 3 weeks.
The immunization scheme was as follows:

Serology
Antibodies to BSA were determined by the conventional indirect ELISA using polystyrene microtitre plates, porcine antibodies to murine IgG purified by affinity chromatography and labelled with horse-radish peroxidase as the conjugate, and hydrogen peroxide and tetramethyl benzidine as the substrate.All the tests were made in duplicates in dilution series starting from I: 100.

Results
The effects of various phospholipid compositions on the entrapment of BSA into ML V are summarized in Table 1.It is evident that the highest entrapment rate (57 %) was obtained with the composition HEPL-C-SA = 0.45:0.45:0.1.Stearylamine, providing the liposomes with surface charge, increased also the rate of entrapment into liposomes prepared from synthetic saturated phospholipids (DMPC 36%; DPPC 38%).Up to three -and fivefold differences between the minimal and the maximal entrapment rates were found in liposomes prepared from DMPC or DPPC and HEPL, respectively.The lowest entrapment rates were found invariably in liposomes prepared from the basic phospholipids alone.DCP and C had favourable effects on entrapment rate also in liposomes prepared from EPL.
Results of BSA entrapment stability tests are presented in Tables 2 through 4. The highest stability was found in HEPL-based liposomes (Variant 2) irrespective of the incubation medium.On the other hand, the lowest stability and highest release of BSA into the medium were found in the DPPC-based liposomes (Variant 4).Isotonic glycerol solution and PBS proved to be suitable media for the preparation and maintenance of liposomal suspensions reconstituted from freeze-dried staff at least four hours.The release of BSA did not exceed 6 % in any of the compositions.Immunization experiments were conducted with ML V with various phospholipid compositions tested for entrapment stability.All the experimental groups were treated with the uniform dose 2 p,g BSA.Control mice received the same dose of free BSA.Expectably, the weakest antibody responses were recorded at each sampling in the control group.No significant differences in antibody titres were observed among the Groups 1 -3.
The primary antigenic stimulation was followed by an increase of antibody titres in all the groups including controls.The reimmunization resulted in a further marked increase in Groups 1 -3, but was followed by a decrease in controls.Low antibody levels were demonstrable in all the experimental groups at the end of the observation period, i.e. on Day 84 (Fig. 1).

Discussion
It is well known that liposomes can be used as excipients for various macromolecular substances, such as proteins, nucleic acids, enzymes or hormones, as well as for drugs, including antibiotics (S ato and S unamoto 1992; Gregoriadi s and Florence 1993; B a k k e r -W 0 u den b erg et al. 1993b).
The entrapment rate can be increased and the release into the environment modulated by modifying the composition, fluidity, morphology and surface properties of the phospholipid bilayerofliposomes (Kirb y and Gre g oriadi s 1984; Gre g ori adi s 1990; Therien et al. 1991).
The entrapment of BSA as a model antigen into multilamellar liposomes prepared from saturated phospholipids or one phospholipid containing in its structure unsaturated fatty acids was tested in our experiments.Transition temperatures of the phospholipids were chosen to exceed (DPPC, HEPL) or to be lower (EPL, DMPC) than body temperature of the immunized animals.However, the actual Tc were different owing to the inclusion of cholesterol, dicetyl phosphate and/or stearylamine into the respective phospholipid composition.
It is evident from the results of the entrapment experiments that the rate ofBSA entrapment depends rather on surface charge and the presence of cholesterol than on Tc of phospholipids.Although the highest entrapment rates of individual variants of multilamellar liposomes ranged between 36 and 57 %, it is evident that positive surface charge is decisive in saturated phospholipids.
Similarly as other authors, we could demonstrate favourable effects of higher molar proportions of cholesterol on the entrapment rate and also confirm the direct proportionality between the length of fatty acid chain in the phospholipid molecule and the rate of entrapment of hydrophilic substances as reported by Bet age r i (1993).
Our immunization experiment with ML V were made to reveal possible dependence of antibody responses on Tc or surface charge.Available data concerning this dependence are controversial.Thus Gre g ori adi setal.(1987) andD a vi s and Greg ori adi s (1987) reported strong and negligible antibody responses in mice immunized with tetanus toxoid entrapped in liposomes prepared from phospholipids with low (EPC) and high (DSPC, 54°C) Tc, respectively.In another of their papers, G reg 0 ria dis et al. (1992) reported similar antibody responses to influenza membrane antigen entrapped in liposomes prepared from EPC or DSPC.The same conclusion was arrived at by G reg 0 ria dis and Pan agioti di (1989) who immunized mice withBSA.Ki n sky (1978) and B akou che and G e r 1 i e r (1986), who used other membrane antigens, found stronger immune responses in mice treated with liposomes prepared from phospholipids with higher Tc.
A I par et al. (1992) confirmed that adjuvant effects ofDSPC-C liposomes were superior to those of aluminium hydroxide in guinea pigs immunized with tetanus toxoid.
C I ark e and S to k e s (1992), who immunized mice intraperitoneally with egg albumin entrapped in DPPC-C-DCP or EPC-S-DPC liposomes found stronger antibody responses in the former.However, the latter liposomes were more effective when administered orally.
Antibody responses depend apparently rather on the properties of antigen and mode of its entrapment in liposomes than on Tc of phospholipids and fluidity of the liposomal bilayer (S h a hum and The r i e n 1994).The effects of fluidity on liposome degradation was demonstrated, among others, by Nag a y a suet al. (1994) who administered daunorubicin, entrapped in EPC-C-DCP or HEPC-C-DCP liposomes to rats affected by Yoshida sarcoma and observed therapeutic effects only in those treated with HEPC liposomes.
In our experiments, the antibody responses in mice treated with the EPL-, HEPL-or DMPC-based liposomes were similar and no effects of Tc were demonstrable.Thereby it was also confirmed that surface charge is decisive.While the HEPL-and DMPC-based liposomes carried positive surface charge as a results of the presence of SA, DCP provided the EPL-based liposomes with negative surface charge.
The entrapment stability tests, which were made by 24-hour incubation in PBS, isotonic glycerol solution or blood serum, yielded the highest values of 96 to 98 % in HEPL-based liposomes and the lowest ones of 70 to 87 % in DPPC-based liposomes.In both cases, the lower values pertained to the incubation in blood serum as a biological medium.This fact apparently influenced the results of the immunization experiments.DPPC-based liposomes, which, in a biological environment, release rapidly the entrapped antigen, do not fulfil their depot function in the optimal manner, as confirmed also by the weakest responses to both the first and the repeated treatments in mice.
0 ute et al. 1981; G reg 0 ria dis et al. 1987; Gregoriadis and Panagiotidi 1989; Gabizon and Papahadjopoulos 1992; B a k k e r -W 0 u den b erg et al. 199•3a; For tin and The r i e n 1993; C h a r I e s et al. 1994).
Preparation of liposomes and entrapment of BSA Multilamellar liposomes (ML V) were prepared as described by K i r b y and G reg 0 ria dis (1984) in four variants based on EPL (Tc approx.-15°C), HEPL (Tc = 54°C), DMPC (Tc = 23°C), and DPPC (Tc = 45°C), EPL-C-DCP liposomes BSA entrapped in HEPL-C-SA liposomes BSA entrapped in DMPC-C-SA liposomes BSA entrapped in DPPC-C-SA liposomes free BSA in PBS Three mice of each group were sacrificed on experimental days 21, 35, 49, and 63 and blood was collected for serological examinations.

Fig. 1
Fig. 1 Antibody response of mice after immunization and reimmunizatoin with BSA liposomes Legend: Immunization Day 0 Reimmunization Day 21

Table 1
Entrapment of BSA into liposomes

Table 3
Release of BSA from Iiposomes during incubation in glycerol Percentage of entrappe d BSA