Effects of gamma irradiation on solid and lyophilised phospholipids

Radiation Physics and Chemistry 56 (1999) 611±622

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E€ects of gamma irradiation on solid and lyophilised phospholipids G. Stensrud a,*, K. Redford b, G. Smistad a, J. Karlsen a a

School of Pharmacy, Department of Pharmaceutics, University of Oslo, P.O. Box 1068, Blindern, N-0316, Oslo, Norway b Sintef Materials Technology, P.O. Box 124, Blindern, N-0314, Oslo, Norway Received 9 November 1998; accepted 27 March 1999

Abstract The e€ects of gamma irradiation (25 kGy) as a sterilisation method for phospholipids (distearoylphosphatidylcholine and distearoylphosphatidylglycerol) were investigated. 31P-NMR revealed minor chemical degradation of the phospholipids but lower dynamic viscosity and pseudoplasticity, lower turbidity, higher di€usion constant, smaller size, more negative zeta potential and changes in the phase transition behaviour of the subsequently produced liposomes were observed. The observed changes could to some extent be explained by the irradiation-induced degradation products (distearoylphosphatidic acid, fatty acids, lysophospholipids). # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Phospholipids; Liposomes; Gamma irradiation; Chemical stability; Physical stability

1. Introduction Gamma irradiation is an approved sterilisation technique for some pharmaceuticals (Woods and Pikaev, 1994; Reid, 1995) and has turned out to be an interest-

Abbreviations: DPPC, dipalmitoylphosphatidylcholine; DPPG, dipalmitoylphosphatidylglycerol; DSC, di€erential scanning calorimetry; DSPA, distearoylphosphatidic acid; DSPC, distearoylphosphatidylcholine; DSPG, distearoylphosphatidylglycerol; FTIR, Fourier transform infrared; GC, gas chromatography; HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; LPC, lysophosphatidylcholine; LPG, lysophosphatidylglycerol; mlv's, multilamellar vesicles; PCS, photon correlation spectroscopy; 31P-NMR, phosphorous-31 nuclear magnetic resonance; SA, stearic acid; TGA, thermogravimetric analysis. * Corresponding author. Tel.: +4722857902; fax: +4722854402. E-mail address: [email protected] (G. Stensrud)

ing and promising technique also for the sterilisation of liposomes. Zuidam et al. (1996a) have published a review of the di€erent sterilisation techniques for liposomes. They concluded that before gamma irradiation can be accepted as a safe and convenient sterilisation technique for liposomes more studies are necessary. Stark (1991) and Albertini and Rustichelli (1993) presented reviews summarising the e€ects of gamma irradiation on the lipids and the liposomal structure. Unfortunately, chemical degradation of the phospholipids takes place during gamma irradiation. Peroxidation of unsaturated phospholipids and formation of lysophospholipids, free fatty acids, phosphatidic acid and di€erent hydrocarbon compounds have been observed (Tinsley and Maerker, 1993; Zuidam et al., 1995, 1996b; Stensrud et al., 1996). E€orts have been done to reduce the degradation by addition of radical scavengers or by freezing and lyophilisation (Zuidam et al., 1995; Stensrud et al., 1996; Samuni et al., 1997).

0969-806X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 6 X ( 9 9 ) 0 0 2 9 7 - 2

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The physical properties of gamma irradiated liposomes (size, bilayer rigidity, permeability) are less sensitive to changes than the chemical structure (Zuidam et al., 1995, 1996b; Stensrud et al., 1997). Interestingly, there has been reported an increased physical stability due to a resulting increased electrostatic repulsion between the liposomes preventing especially the neutral liposomes from aggregation and fusion (Stensrud et al., 1997). The toxicity of gamma irradiated liposomes in vitro was recently evaluated (Stensrud et al, 1999a,b). Depending upon the type and concentration of the irradiated liposomes hemolysis of erythrocytes, reversible platelet aggregation and disturbance of the coagulation cascade were seen. The cytotoxicity test and growth inhibition test revealed toxic e€ects of liposomes composed of unsaturated phospholipids. Liposomes composed of saturated phospholipids were non-toxic for the cells and irradiation did not a€ect their drug delivery properties. However, gamma irradiated liposomes (15 kGy) made of egg yolk lecithin, cholesterol and stearylamin (4:3:1) have been given intravenously in large volumes to patients without serious side e€ects (Coune et al., 1983; Sculier et al., 1986). In spite of the minor physical changes and the promising toxicological results, the chemical degradation and the subsequent presence of high amounts of degradation products might restrict the use of gamma irradiation as a sterilisation method for aqueous liposome preparations. The alternative to this is to sterilise the solid phospholipids prior to liposome production or to sterilise the lyophilised product prior to hydration (Anderson et al., 1994; Zuidam et al., 1995). Little work has so far been done in this ®eld and these options need therefore to be further evaluated. In this study the e€ects of gamma irradiation on phospholipids (DSPC and DSPG) both as solids and as lyophilisates are investigated in more detail. Chemical/physical analysis of the solids/lyophilisates (pH, FTIR, 31P-NMR, TGA, DSC, X-ray) are followed by physical characterisation of the subsequent produced liposomes (zeta-potential, size/di€usion constant, turbidity, viscosity, phase transition behaviour). Furthermore, known irradiation induced degradation products are incorporated in the liposomes in order to simulate and explain the observed physical changes. 2. Experimental 2.1. Materials Distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG) were kindly provided

by Nattermann Phospholipids, KoÈln, Germany. Lysophosphatidylcholine (LPC), lysophosphatidylglycerol (LPG), stearic acid (SA) and distearoylphosphatidic acid (DSPA) were obtained from Sigma Chemical Co. (St Louis, USA). All other chemicals were of analytical grade. 2.2. Preparation of liposomes and irradiation Liposome samples were prepared in three di€erent ways: (1) Liposomes were prepared from solids by the ®lm method as follows; the phospholipids were dissolved in chloroform:methanol (2:1) and evaporated to dryness. The phospholipid ®lm was then hydrated above the phase transition temperature for two hours (Stensrud et al., 1997). (2) Lyophilised liposomes were then prepared using an Alpha 2-4 freeze dryer with a LDC-1 controller (Christ, Osterode am Harz, Germany). The lyophilised liposomes were re-hydrated above the phase transition temperature. (3) Alternatively, samples were made by vortexing the solid lipid in bu€er followed by hydration above the phase transition temperature for 2 h. 13 mM phosphate bu€er was used as hydration bu€er to give a phospholipid concentration of 10 mg/ml (50 mg/ml for the DSC analysis). These three production methods are further referred to as ``®lm'', ``lyophilisation'' and ``directly hydration'', respectively. The liposomes were extruded (Lipex extruder) using 0.1 mm polycarbonate membranes (Nucleopore) for the last extrusion step. Samples were exposed to a dose of 25 kGy (15 kGy/ h, 60Co source) at ambient temperature. The irradiation was performed on solid and lyophilised substances prior to liposome production/hydration. 2.3. Chemical characterisation 2.3.1. 31P-NMR Spectra were recorded using a NMR-Spectrometer AC-P 300 (Bruker, Karlsruhe, Germany) equipped with a QNP-head (121.496 MHz). 50 mg of the phospholipids was dissolved in 1.5 ml of CDCl3/MeOH/CsEDTA (0.2 M, pH 8.5). Number of scans 256, sweep width 5747.13 Hz, pulse width 4.0 ms, acquisition time 2.851 s. 2.3.2. FTIR Spectra of the solid and lyophilised samples in KBr disks were obtained using a Nicolet Magna-IR 550. 2.3.3. TGA Residual moisture content of the lyophilised liposomes was measured with a Perkin±Elmer TGA 7. The samples were investigated at 20±2008C with a heating rate of 208C/min.

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2.4. Physical characterisation 2.4.1. Size/di€usion constant The mean diameter of the extruded liposomes was measured at 908 (258C) by photon correlation spectroscopy (PCS) using a Coulter N4 MD. When the diffusion constant (D ) was measured for the nonextruded liposomes (mlv's), the 118 angle was utilised. The refractive index and viscosity of water were used as calculation parameters. 2.4.2. Turbidity Absorbance (turbidity) of the samples (1 mg/ml) was measured at ambient temperature using a Shimadzu UV-2101PC UV-VIS scanning spectrophotometer at 450 nm. When necessary, sucrose was included in the dilution bu€er to avoid sedimentation. 2.4.3. Viscosity Kinematic viscosity of the liposomes in the range of 0.1±1.0 % w/v was measured at 208C in a microOstwald viscosimeter (type No. 51610/1; Schott-GeraÈte GmbH, Germany). The intrinsic viscosity was determined by means of linear regression. Dynamic viscosity (4 mg/ml samples) was calculated from the kinematic viscosity by multiplying with the corresponding density. The density was measured with a Mohr± Westphals balance (Pharmacopoea Nordica, 1963). Step stress tests were performed with a dynamic stress rheometer (Rheometrics DSR) at 258C. Parallel plates with a diameter of 40.0 mm and a gap of 0.300 mm were used. The shear stress was programmed from 0.1 to 2 Pa and the shear times from 180 to 360 s. 2.4.4. Zeta-potential Zeta-potential of the liposomes was calculated from the results of electrophoretic mobility measurements on a Coulter DELSA 4401 (Doppler-Electrophoretic Light Scattering Analyser) as described earlier (Stensrud et al., 1997). 2.4.5. DSC The analysis were performed with scanning rates of 2.58C/min (liposomes) or 108C/min (solids) with a Perkin±Elmer DSC 7 equipped with Intracooler I (cooling rate; 2008C/min). 2.4.6. X-ray di€raction Samples were exposed to CuKa radiation (40 kV  40 mA) in a Simens D5000 di€ractometer. A 18 divergence slit and a 0.38 antiscatter slit were used. The diffractometer was operated in the step-scan mode with a step time of 18 and a step size of 0.028 from 382y to 6082y.

613

Table 1(a) Levels of the factors in the 23 factorial design applied to study the e€ects of irradiaton, lipid class and treatment on the pH and the phase transition behaviour of the subsequent produced liposomes. aThe treatment was extended with ``directly hydratation'' (mixed levels) in the design for analysis of the e€ects on the dynamic viscosity, turbidity, size and zeta-potential. bIn the analysis of the melting behaviour of the phospholipids, the levels of the treatment factor were ``solid'' and ``lyophilised'' phospholipids as the analysis was performed prior to liposome production/hydration Factor

Level

(A) Irradiation (kGy) 0 25 (B) Lipid class DSPG DSPC (C) Treatmentb Film Lyophilisation Directly hydrationa

2.5. Statistics A 23 full factorial design was used to study the e€ects of irradiation (irr), lipid class (li) and treatment (tr) on the pH and the phase transition behaviour of the subsequent produced liposomes. The treatment was extended with ``directly hydration'' (mixed levels) in the design for analysis of the e€ects on the dynamic viscosity, turbidity, size and zeta-potential. The levels of the investigated factors are given in Table 1(a). In the analysis of the melting behaviour of the phospholipids, the levels of the treatment factor were ``solid'' and ``lyophilised'' phospholipids as the analysis was performed prior to liposome production/hydration. A two factor full factorial design (mixed levels) was applied to study the e€ects of irradiation and treatment on the di€usion constant (D ) of DSPG-liposomes (Table 1(b)). Experiments were run in triplicate to allow estimation of the experimental error. The estimated e€ects of increasing the factors from a low to a high level were tested for signi®cance by analysis of variance (P < 0.05) (MODDE software, UMETRI, UmeaÊ, Sweden). Since the topic of this work was to evaluate possible e€ects of gamma irradiation on phospholipids, the results and the discussion is mainly restricted to the irradiation factor and its related interactions. The e€ects of incorporation of di€erent

Table 1(b) Levels of the factors in the two factor full factorial design (mixed levels) applied to study the e€ects of irradiation and treatment on the di€usion constant (D ) of DSPG-liposomes Factor

Level

(A) Irradiation (kGy) 0 25 (B) Treatment Film Lyophilisation Directly hydration

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Table 2 The phospholipid distribution (mol%) analysed by Phospholids DSPG

Treatment Solid Lyophilised

DSPC

31

Solid Lyophilised

P-NMR DSPG/DSPC (mol%)

Lyso-PG/PC (mol%)

DSPA (mol%)

99.7 98.8 99.4 94.7 99.6 98.8 99.6 98.2

0.1 0.2 0.4 0.9 0.3 0.6 0.2 0.6

0.2 0.7 0 2.5 0 0.3 0 0.7

Non-irradiated Irradiated Non-irradiated Irradiated Non-irradiated Irradiated Non-irradiated Irradiated

amount of degradation products in the liposomes were evaluated by analysis of variance (P < 0.05). 3. Results A saturated model was employed to evaluate the signi®cance of the various e€ects and interactions. 3.1. E€ects on solid and lyophilisates 3.1.1. Chemical structure 31 P-NMR analysis revealed irradiation induced degradation of DSPC and DSPG (solid and lyophilisate) (Table 2). Two main components were identi®ed as lyso-PC/lyso-PG and DSPA. The fatty acid chains remained intact after irradiation. There were other phosphorous containing molecules detectable in amounts smaller than 0.1 mol%. The structures of these molecules are not known. In general, lyophilised phospholipids seem to be more unstable than solid phospholipids and DSPG more unstable than DSPC.

Fig. 1. X-ray di€raction pattern of non-irradiated and irradiated DSPG-lyophilisate.

The FTIR spectra for the non-irradiated and irradiated samples showed no signi®cant di€erences. The water content of the lyophilisate did not change after irradiation and was 0.7% (w/w) in the DSPC-lyophilisate and less than 0.1% (w/w) in the DSPG-lyophilisate (n=3). 3.1.2. Physical structure No di€erences were observed in the X-ray patterns of the crystalline solid substances of DSPC and DSPG after irradiation. For the lyophilisates, a slight change was observed in the crystalline/amorphous structure of DSPG (2y=7.68 and 2y=21.48) (Fig. 1). This change was further con®rmed by the DSC-scans with the appearance of a solid phase-transition before the ®nal melting (Fig. 2(A)). The merged endotherm and exotherm represent a combined melt/re-crystallisation event. The endotherm was reversible on cooling and heating The endotherm could be decreased with a slower heating rate (2.58C/min) or by holding the sample isothermally for 1 h at 708C followed by a second run. In the non-irradiated sample, exothermic recrystallisation peaks appeared before the ®nal melting (Fig. 2(B)). In the irradiated samples of solid DSPG, a small endotherm and a deep exotherm appeared in the second run (Fig. 3). This was associated with a solid phase-transition similar to the one observed in the irradiated lyophilisate. In the solid and lyophilised DSPC only the melting properties changed following irradiation. The melting peaks appearing in all scans were very sensitive towards gamma irradiation. The irradiated samples showed signi®cantly lower melting temperatures (DTonset, main e€ect second run: ÿ2.78C) and associated enthalpies (DH, main e€ect second run: ÿ10.8 J/g,). The e€ects of the phospholipid class and treatment was signi®cant as DSPC and lyophilisation both gave lower melting temperatures (ÿ19.18C and ÿ1.18C) and associated enthalpies (ÿ43.2 J/g and ÿ7.1 J/g). All the interactions except trli were signi®cant but considerably smaller with regards to the melting point.

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615

Fig. 2. Representative DSC-thermograms of DSPG-lyophilisate, (A) Irradiated, (B) Non-irradiated. The peaks are the endotherms. Scan rate 108C/min.

Fig. 3. Typical DSC-curves of solid DSPG. The peaks are the endotherms. Scan rate 108C/min.

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Fig. 4. (A) Representative DSC-thermograms of the second run of solid DSPG. The ®rst run was immediately followed by an isothermic run at 1508C for di€erent periods (0, 10, 30 min) before the second run. (B) Representative DSC-thermograms of DSPGlyophilisate containing 2.5 mol% DSPA and 0.9 mol% LPG. The peaks are the endotherms. Scan rate 108C/min.

The lower melting temperature and the broader melting peak seen in the second run was due to the temperature treatment of the samples as con®rmed by holding the solid DSPG at 1508C at various time intervals followed by a second run (Fig. 4(A)). By this treatment, the endotherm associated with a solid phase-transition so far observed only in the irradiated samples of DSPG could be obtained (Figure 2A)).

Especially solid DSPC showed a considerable reduction in enthalpy (ÿ52.6 J/g on average) and decrease in melting temperature (ÿ14.98C on average) from the ®rst to the second run. For all the samples, the third run was similar to the second. The endotherm observed in the DSC-thermograms of irradiated DSPG-lyophilisate was also obtained in DSPG-lyophilisate after the addition of 2.5 mol%

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617

Table 3 E€ects of irradiation, lipid class and treatment on the pH (10 mg/ml phospholipids in distilled water) and phase transition behaviour of the liposomes (mlv's, phosphate bu€er pH 7.4) obtained in the 23 factorial design pH ÿ0.97e ÿ1.36e ÿ0.18e ÿ0.67e ÿ0.15e ÿ0.12e ÿ0.21e 0.03

Irradiation (irr) Lipid (li) Treatment (tr) irrli intr litr irrlitr Conf.interval (+/ÿ)

Tona (8C)

Tmb (8C)

DHc (J/g)

DT1/2d (8C)

ÿ0.26e 0.34e ÿ0.06 ÿ0.04 0.03 ÿ0.12e ÿ0.22e 0.09

0.17e 0.47e 0.01 0.17e 0.11e 0.01 ÿ0.10 0.11

ÿ2.5 ÿ23.7e ÿ6.6e ÿ1.5 ÿ7.9e 0.7 4.1e 2.9

0.39e 0.26e 0.13e 0.17e 0.10e 0.09e 0.04e 0.03

a

Temperature at the onset of the main phase transition. Temperature at the main phase transition. c Entalphy of the main phase transition. d Width at half height for the main phase transition. e Signi®cant e€ects (nr3). b

DSPA and 0.9 mol% LPG (the same amount as in irradiated DSPG-lyophilisate) (Fig. 4(B)). No changes were seen in the DSC-scans after 8 months storage at ÿ208C for non-irradiated and irradiated DSPG (solid and lyophilised). 3.2. E€ects on the subsequently produced liposomes 3.2.1. pH and phase transition behaviour After irradiation of the solid substances and the lyophilisates, the pH of the subsequently produced liposomes (10 mg/ml) in distilled water decreased

signi®cantly (Table 3). In general, the pH was signi®cantly lower for the DSPC-liposomes than for the DSPG-liposomes and the irradiation induced pH decrease was also most pronounced for the DSPC-liposomes. The other interaction terms were less signi®cant. The main phase transition (Tm) appeared around 54.98C for the DSPG-liposomes and around 55.28C for the DSPC-liposomes (mlv's). Changes were seen in the main phase transition behaviour of the liposomes after irradiation (Table 3). The pre-transition was only evident in liposomes prepared from non-irradiated DSPC.

Table 4 Experimental results obtained in the factorial designs (mixed levels). The dynamic viscosity (4 mg/ml), di€usion constant (4 mg/ ml), turbidity (1 mg/ml), size and zeta-potential of di€erent liposome-preparations in phosphate bu€er pH 7.4 were investigated (n=3). The turbidity-results are related to liposomes (DSPC/DSPG) made by the ®lm method (100%) Phospholipids DSPG

Treatment Film Lyophilisation Directly hydration

DSPC

Film Lyophilisation Directly hydration

SD error a b

Mlv-liposomes. Liposomes extruded 100 nm.

Non-irradiated Irradiated Non-irradiated Irradiated Non-irradiated Irradiated Non-irradiated Irradiated Non-irradiated Irradiated Non-irradiated Irradiated

Dyn. viscositya (mPa.s)

Di€. Constanta (10ÿ9, cm2/s)

Turbiditya (%)

Sizeb (nm)

Zeta potentialb (mV)

1.65 1.41 2.03 1.51 2.43 1.70 1.04 1.08 1.04 1.05 1.04 1.05 0.06

3.8 5.2 2.3 4.3 1.5 3.0 nd nd nd nd nd nd 0.2

100 76 104 99 110 103 100 90 85 81 90 82 2

97 86 105 98 106 100 93 81 137 95 177 98 4

ÿ53 ÿ68 ÿ56 ÿ66 ÿ55 ÿ66 ÿ2 ÿ8 ÿ1 ÿ5 ÿ1 ÿ7 1

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Fig. 5. Flow curves for DSPG-liposomes (10 mg/ml) prepared from non-irradiated (Q) and irradiated (w) solids by directly hydration in phosphate bu€er pH 7.4. Error bars are the maximum and minimum values (n=3).

3.2.2. Physical properties DSPG-liposomes (mlv's) showed a signi®cant higher dynamic viscosity than DSPC-liposomes (4 mg/ml) (Table 4). The dynamic viscosity of DSPG-liposomes was signi®cantly lowered after irradiation. The dynamic viscosity was also dependent upon the preparation method as the highest viscosity was achieved

for DSPG-liposomes made by directly hydration and the lowest made by the ®lm method. The decrease in dynamic viscosity was most pronounced for liposomes made of the irradiated substance by directly hydration and least for liposomes made by the ®lm method. Also the interaction term litrirr was signi®cant. The ¯ow curves of liposomes composed of non-irradiated and irradiated solid DSPG (10 mg/ml, phosphate bu€er pH 7.4, directly hydration) are presented in Fig. 5. These liposome-suspensions showed sheardependent viscosity and a pseudoplastic ¯ow. The apparent dynamic viscosity decreased from 21.3 to 6.4 mPa.s after irradiation and the ¯ow became less pseudoplastic (nearer to newtonian) as the index of pseudoplasticity (n ) decreased from 2.4 to 1.4 (no overlap in the estimated 95% con®dence intervals of the ®tted parameters). The intrinsic viscosity of DSPG-liposomes composed of non-irradiated and irradiated phospholipids was the same (Fig. 6). The relationship between Zsp/C (reduced viscosity) against C (Zsp denotes speci®c viscosity and C denotes concentration of phospholipids) was best ®tted to a logarithmic relationship for these DSPG-liposomes. The reduced viscosity varied with the concentration due to molecular interactions. Interestingly, the curve was signi®cantly steeper (no overlap between the corresponding 95% con®dence intervals) for liposomes prepared from non-irradiated DSPG compared with irradiated DSPG. Similar e€ects

Fig. 6. The relationship between log Zsp/C(reduced viscosity) against C where the intercept gives the intrinsic viscosity ([Z ]). DSPGliposomes prepared from non-irradiated (Q) and irradiated (w) solids by directly hydration in phosphate bu€er pH 7.4. Error bars are the maximum and minimum values (n=3).

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619

Table 5 The dynamic viscosity (4 mg/ml), di€usion constant (4 mg/ml), turbidity (1 mg/ml) and zeta-potential of DSPG-liposomes (mlv's, phosphate bu€er pH 7.4) including di€erent amounts of known degradation products prepared by the ®lm method (n=3) Phospholipids DSPG ref LPG 1 mol% 2 mol% 5 mol% LPG+SA 1 mol% 2 mol% 5 mol% DSPA 1 mol% 2 mol% 5 mol% SD error LPG SD error LPG+SA SD error DSPA

Dyn. viscosity (mPa.s)

Di€. constant (10ÿ9, cm2s)

Turbidity (%)

Zeta potential (mV)

1.71

4.1

100

ÿ56

1.67 1.67 1.64

4.2 4.2 5.0

83 71 57

ÿ55 ÿ56 ÿ57

1.67 1.64 1.58

4.2 4.2 6.1

77 61 32

ÿ56 ÿ58 ÿ64

1.69 1.68 1.68 0.01 0.01 0.01

4.2 4.1 4.3 0.5 0.7 0.4

101 99 97 4 4 4

ÿ64 ÿ67 ÿ74 3 3 1

were observed for the lyophilised liposomes. The DSPC-liposomes behaved as an ideal solution as the reduced viscosity was almost independent of the concentration. Again, the same intrinsic viscosity was measured for liposomes prepared from non-irradiated and irradiated DSPC. Liposomes prepared from irradiated DSPG (mlv's) showed a signi®cant higher di€usion constant (D ) compared to liposomes prepared from non-irradiated DSPG (Table 4). The highest di€usion constant was measured for liposomes made by the ®lm method and the lowest for liposomes made by directly hydration. The interaction between irradiation and treatment was not signi®cant. Unfortunately, it was not possible to measure the di€usion constant for the DSPC-liposomes due to sedimentation. However, when these liposomes were stored and manually examined, the sedimentation proceeded much faster in the liposomes prepared from non-irradiated phospholipids than in the liposomes prepared from irradiated phospholipids. Irradiation had a signi®cant e€ect on the turbidity of the di€erent liposome-preparations (mlv's) and the decrease in turbidity was especially pronounced for the DSPG-liposomes, (Table 4). The turbidity was highest for liposomes made by direct hydration of solid substance and least for liposomes prepared from solid substance by the ®lm method. The interaction between irradiation and treatment was also signi®cant. The decrease in turbidity was most pronounced for liposomes prepared from irradiated phospholipids by the ®lm method followed by directly hydration of solid and least for liposomes prepared from lyophilised substance. The interaction litrirr was also signi®cant.

3.2.3. Physical properties of extruded liposomes Signi®cantly smaller sizes were obtained with DSPGliposomes compared to DSPC-liposomes after extrusion (100 nm ®lter) (Table 4). Smaller liposomes were also achieved when the phospholipids were irradiated prior to liposome production. This behaviour was especially pronounced when liposomes were prepared from irradiated DSPC. The size was also greatly dependent upon the production method as the smallest liposomes were achieved by the ®lm method and the largest liposomes by directly hydration of solid substances. Also the interaction between treatment and irradiation was signi®cant. The size reduction was most pronounced for liposomes prepared from irradiated phospholipids by directly hydration and least for liposomes prepared by the ®lm method. The interaction litrirr was also signi®cant. Irradiation of the phospholipids signi®cantly decreased the zeta-potential of the subsequently produced liposomes (more negatively charged) (Table 4). Irradiation of DSPG gave a more pronounced decrease in the zeta-potential of the corresponding liposomes than irradiation of DSPC. The other interaction terms were less signi®cant. 3.3. E€ects of incorporation of di€erent degradation products By incorporation of di€erent degradation products in the DSPG-liposomes made by the ®lm method (mlv's), similar e€ects previously observed with the irradiated liposomes appeared (Table 5). Over the concentration range 1±5 mol% lysophospholipids in¯u-

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enced signi®cantly the dynamic viscosity and the turbidity. The di€usion constant was only increased by incorporation of 5 mol% LPG. Degradation of DSPG forms stearic acid together with LPG. The observed e€ects were even greater with the simultaneous presence of both degradation products. As fatty acids present in the membrane are partially charged at pH 7.4, they might also in¯uence the zeta potential. In combination with LPG, stearic acid in¯uenced the zeta-potential signi®cantly. DSPA showed less e€ect on the viscosity, but as this degradation product contains two negatively charged groups a signi®cantly lower (more negative) zeta-potential was measured. The di€usion constant and turbidity were not changed by the inclusion of DSPA in the DSPG-liposomes. Degradation products were also responsible for the changes in the main phase transition behaviour for liposomes prepared from irradiated phospholipids. Incorporation of the phospholipid degradation product LPC (1±5 mol%) in DSPC-liposomes prepared by the ®lm method (mlv's) decreased the onset temperature, whereas incorporation of DSPA and SA gave signi®cantly broader phase transitions and the peak shifted to higher temperatures. 4. Discussion 3.3. E€ects on solids and lyophilisates So far no reports have proved irradiation-induced degradation of solid/frozen/lyophilised saturated phospholipids. The methods which have been used are GC, HPLC, HPTLC and IR (Anderson et al., 1994; Zuidam et al., 1995; Stensrud et al., 1996; Samuni et al., 1997). However, with 31P-NMR we were able to prove irradiation induced degradation of the saturated phospholipids. Due to the structure and the presence of residual water the lyophilisates were more prone for indirect radical attack. It has been earlier observed that liposomes composed of DSPG are more sensitive towards irradiation than DSPC-liposomes (Tinsley and Maerker 1993; Zuidam et al., 1995, 1996b; Stensrud et al., 1996). Irradiation resulted in a new form/modi®cation of the DSPG-lyophilisate which was con®rmed both by DSC and X-ray di€raction. Irradiation induced degradation products proved to be responsible for this. In the solid DSPG, the degradation was only 0.9 mol% and this may explain the hardly evident solid phasetransition in this sample. Measurements of the melting point depression of a substance are often used to determine its purity. In the present case, the melting peak proved to be very sensitive towards the presence of degradation products as the melting point and entalphy of fusion were signi®-

cantly lowered with <1 mol% degradation. DSPG proved to be the most unstable phospholipid against gamma irradiation and showed also the greatest melting point depression. The lower melting points and enthalpies measured for the lyophilisates are caused by the amorphous state of the freeze-dried cakes. 4.2. E€ects on the subsequently produced liposomes 4.2.1. pH and phase transition behaviour The lower pH measured after irradiation of solid and lyophilised substances is caused by degradation of the phospholipids and the formation of acidic degradation products such as DSPA and fatty acids (Zuidam et al., 1995, 1996b; Stensrud et al., 1996, 1997). Lyophilised samples are more prone to irradiation induced degradation. This was also re¯ected by the extensive pH decrease. DSPC showed less degradation than DSPG, but the irradiation induced pH decrease was still more pronounced. At the moment, we are not able to explain this observation. The change in the main phase transition behaviour for liposomes prepared from irradiated phospholipids also re¯ects the presence of degradation products in the membrane (Biltonen and Lichtenberg, 1993; Zuidam et al., 1995, 1996b, Stensrud et al., 1996, 1997). This was con®rmed by incorporation of di€erent degradation products in the liposomes. DSPG was more sensitive to the irradiation than DSPC. However, as the changes in the main phase transition behaviour was most pronounced for liposomes prepared from irradiated DSPC other factors like di€erent molecular packing in the phospholipid bilayers also seem to be important. The greater sensitivity of lyophilised phospholipids against irradiation was also seen from the changes in the phase transition behaviour. DSPC-liposomes showed a higher main phase-transition due to the choline-headgroup. The pre-transition is known to be particularly sensitive to the e€ect of small amounts of degradation products as also observed in this study (Biltonen and Lichtenberg, 1993). 4.2.2. Physical properties The DSPG-liposomes displayed at higher concentrations a gel-like behaviour. This can be explained by multiple contacts between the particles due to steric factors or by an extended network permeated by the solvent (semi-permanent bonds; hydrogen bonds). In the network, the kinetic independence of the particles is partially lost and these gels exhibit yield behaviour. At lower concentration (4±10 mg/ml) or for liposomes composed of irradiated phospholipids, this gel-like behaviour was less pronounced. The signi®cant reduction in dynamic viscosity of the liposomes composed of irradiated DSPG was closely connected to the

G. Stensrud et al. / Radiation Physics and Chemistry 56 (1999) 611±622

decrease in turbidity and the higher di€usion constants. Higher di€usion constants indicate smaller particles or fewer interactions between the particles. Lower turbidities con®rm the presence of smaller liposomes. Einstein has explained the correlation between viscosity and particle sizes (Einstein, 1906, 1911). Spherical particles show the same intrinsic viscosity ([Z ]) independent of size. The di€erent ¯ow behaviour of the samples is likewise most probably due to the di€erent particle sizes in the samples, but di€erent particle interactions may also be responsible for this behaviour. In addition, as the size increases, the liposomes become more multilamellar and thereby more sti€. DSPC-liposomes displayed lower viscosities compared to DSPG-liposomes due to poor interaction with water. The viscosity did not increase considerably with increased concentration. This indicates fewer interactions between the DSPC-liposomes compared to DSPG-liposomes. No changes in the viscosity were seen after irradiation, but as the dynamic viscosity of the DSPC-liposomes are close to water, eventually changes are dicult to detect. The lower turbidities measured after irradiation still indicates the presence of smaller liposomes or de-aggregation. Smaller liposomes are most probably due to the presence of micelle-forming degradation products (lysophospholipids, SA) which result in di€erent packing of the liposomal bilayer. However, as a negative charge favours the formation of smaller liposomes the presence of charged degradation products (DSPA, SA) have to be taken into account. Liposomes with a neutral charge are known to aggregate below the main phase-transition temperature (gel-state). The presence of a negative charge in the liposomes composed of irradiated phospholipids lead to repulsion of the liposomes and de-aggregation. The same e€ect was also observed in an earlier study where neutral DPPC-liposomes where irradiated (Stensrud et al., 1997). Aggregation might also explain the lower turbidities measured for non-irradiated DSPC-liposomes made from freeze-dried substance and from solid phospholipids by directly hydration compared to liposomes made by the ®lm method. By the ®rst two methods larger vesicles/aggregates were formed which most probably gave sedimentation despite the addition of sucrose in the dilution medium. 4.2.3. Physical properties of extruded liposomes The smaller sizes measured for all the liposomes composed of irradiated phospholipids after extrusion are likely caused by the degradation products. A negative charge favours the formation of smaller liposomes and results in de-aggregation and explains the great size-reduction for liposomes with originally a neutral charge (DSPC). Interestingly, no change in the liposome size was observed when egg PC, egg PC/PG or

621

DPPC/DPPG liposomes were prepared and then irradiated (Zuidam et al., 1995, 1996b; Stensrud et al., 1997). In these experiments the phospholipid degradation was pronounced (up to around 20% for PC) and the net negative charge increased, but as these liposomes did not go through a gel-to-liquid phase transition no change in size was seen. This was further con®rmed in a study of Zuidam et al. (1995), where no change in size were seen when gamma irradiated freeze-dried saturated liposomes were reconstituted by addition of water at ambient temperature (below Tm). The more negative zeta-potential measured for liposomes composed of irradiated phospholipids compared to non-irradiated phospholipids is most probably due to the presence of the negatively charged degradation products DSPA and SA. The decrease in the zeta-potential was most pronounced for the DSPG-liposomes re¯ecting the higher amounts of degradation-products, especially DSPA, in these liposomes.

5. Conclusion Gamma irradiation of phospholipids as solids and as lyophilisates resulted in only minor chemical degradation. From this point of view, gamma irradiation seems to be a suitable sterilisation technique for solid and lyophilised saturated phospholipids. However, changes were seen in the physical behaviour of the subsequent produced liposomes as smaller liposomes were formed with a further possibility to in¯uence the dynamic viscosity of the suspensions. The known phospholipids degradation products could to a certain degree explain these results.

Acknowledgements The authors wish to thank Institute for Energy Technology, Kjeller, Norway for performing the irradiation. Special thanks to Mr E. Ho€, Nattermann Phospholipids, Kùln, Germany for providing the phospholipids and performing the 31P-NMR analysis. We are grateful to Dr J.E. Roots, University of Oslo, Norway for many helpful discussions. Thanks to Ms T.L. Rolfsen, Sintef, Oslo, Norway for help with the X-ray and TGA analysis. We would also like to thank Dr S.A. Sande and Ms T. Larsen, University of Oslo, Norway for their contribution to this work.

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