journal of ELSEVIER
Journal of Controlled Release31 (1994) 73-87
controlled release
Phase transition temperature reduction and glass formation in dehydroprotected lyophilized liposomes W. Cary Mobley a, Hans Schreier "'b'* aDepartment of Pharmaceutics, Universityof Florida College of Pharmacy, Gainesville, FL 32610-0494, USA bThe Centerfor Lung Research, Vanderbilt University School of Medicine, B 1308 MCN, Nashville, TN 73232-2650, USA
Received 11 December1993; acceptedin revisedform7 January 1994
Abstract
Two prevailing theories for dry membrane preservation are the water replacement hypothesis and glass formation. A manifestation of the water replacement hypothesis is the ability of sugars to depress dry membrane main phase transition temperatures (Tin). Differential scanning calorimetry (DSC) was employed to test the effects of sugars (trehalose, a-lactose, maltose and glucose) on the Tins of slowly frozen, lyophilized liposomes prepared from hydrogenated egg phosphatidylcholine [HEPC], dipalmitoylphosphatidylcholine [DPPC] and palmitoyloleoylphosphatidylcholine [POPC]. For each lyophilized phospholipid membrane, the disaccharides caused significant Tm reduction to at least 14°C below the hydrated membrane Tins. The Tinreduction was achieved by heating the lyophilized product: an annealing process that included a membrane phase transition and a disaccharide glass transition. Thermogravimetric analysis (TGA) showed residual water loss (3-6%) during annealing and FTIR spectra suggested an annealing-induced disaccharide/phospholipid-carbonyl interaction. Scanning electron microscopy (SEM) showed an amorphous appearance of the lyophilized trehalose/HEPC matrix, which was confirmed by DSC to be glassy, and which remained intact after annealing. Also observed with SEM were membrane infolding, fusion of unprotected liposomes, and matrix porosity. Discussed are the potential implications of annealing, dry membrane Tin-reduction and glass formation for liposome dehydroprotection. Keywords: Liposome;Lyophilization;Sugar; Glass formation;Phasetransition
1. Introduction
Lyophilization (freeze-drying) is considered a promising means of extending the shelf-life of liposomes. However, both freezing and drying can induce structural and functional damage to liposomes. Freezing may induce membrane damage from osmotic stress, phase changes, or from freeze-dehydration [1-5]. Membrane dehydration during freezing and drying * Correspondingauthor: The Centerfor Lung Research,Vanderbilt UniversitySchoolof Medicine, B 1380MCN, Nashville,TN 732322650, USA. 0168-3659/94/$07.00 © 1994 ElsevierScienceB.V. All rights reserved SSDIO168-3659(94)OOOO6-G
induces lateral stresses that may lead to membrane deformation, lateral phase separations, gel/liquid crystalline phase transitions and lamellar to hexagonal phase transitions [1,5]. Liposome fusion and loss of vesicle contents are potential consequences that may compromise the pharmaceutical advantages of the formulation. The liposome damages and those stresses that induce them may be minimized by including protective excipients in the formulation. Disaccharides are often the chosen excipients because of their capacity for both cryoprotection and dehydroprotection.
74
W (7. Mobley. H. S~ hreier / Journal of Controlled Release 31 (I994) 73--87
The membrane-protective ability of disaccharides is thought to be due, in part, to the behavior of aqueous solutions of sugars at high concentration. Sugars at high concentration can reduce water activity and increase solution volume, and can increase solution viscosity to the point of vitrification [5]. Vitrification (glass formation) is thought to be important for dry membrane preservation, because the lack of long range motion in the glassy state limits diffusion-dependent degradation processes [6,7]. In addition to glass formation, disaccharides are postulated to preserve dry membranes via a specific interaction at the membrane interface. This theory, termed the 'water replacement hypothesis', attributes the membrane stabilizing effects of sugars to their ability to hydrogen-bond to phospholipid headgroups, thus supplanting water as a membrane stabilizer [8]. One manifestation of membrane interactions is the ability of some disaccharides to reduce the gel to liquid crys-
talline phase transition temperature (T,,) of dry membranes, presumably by increasing phospholipid spacing [9-12]. Most studies of the effects of sugars on dry membrane Tins have been performed on membranes (or lipid/sugar mixtures in organic solvent [13,14] ) frozen rapidly in liquid nitrogen, followed by sublimation [ 10,11,14,15] ; or on membranes desiccated without freezing [ 10,12]. In this study, we examined the effects of sugars on liposomes that were slowly ( = 0.7°C/min) shelf-frozen, followed by primary and secondary drying. For examining dry sugar/membrane interactions, differential scanning calorimetry (DSC) was employed to measure the effect of the disaccharides trehalose, c~-lactose, maltose and the monosaccharide glucose on the dry membrane Tm of lyophilized HEPC, DPPC and POPC liposomes. Lyophilized lipsomes were examined with DSC to measure glass formation,
Fig. 1. SEM micrographsof lyophilizedunextrudedHEPCliposomes.(a) Infoldedliposome.
w . c Mobley, H. Schreier/ Journal of Controlled Release 31 (1994) 73-87
with thermogravimetric analysis (TGA) to measure water loss, with Fourier-transform infrared spectroscopy (FTIR) to examine disaccharide/membrane interactions, and with scanning electron microscopy (SEM) to visualize the lyophilized product. The experiments were performed with unextruded and extruded liposomes. Initial calorimetric and microscopic experiments were performed on more rapidly prepared unextruded liposomes in water, in order to study the effect of sugars on the lipid membrane in its most relaxed state and without the possible interference of buffer ions with sugar/membrane interactions. Potential pharmaceutical liposome formulations are generally sized (in the 0.1-0.2/zm diameter range for parenteral applications) and contain drugs and buffer salts. Therefore, a series of experiments was also performed on liposomes prepared with phosphate buffer and extruded through a 0.1 /xm polycarbonate
75
membrane, and the results of the unextruded and extruded liposome experiments were compared.
2. Materials and methods 2.1. Materials
The following materials were used as purchased: hydrogenated egg phosphatidylcholine (HEPC) and 1palmitoyl-2-oleoyl phosphatidylcholine (POPC) in chloroform and dipalmitoylphosphatidylcholine (DPPC) powder from Avanti Polar Lipids (Alabaster, AL, USA); D-glucose (anhydrous, certified A.C.S.) and maltose ( monohydrate, reagent grade) from Fisher Scientific (Fair Lawn, NJ, USA); D( + )-trehalose (dihydrate, from Saccharomyces cerevisiae) and alactose (monohydrate, 2%/3 content, substantially glu-
Fig. 1(b). Membranedisintegrationof a liposomefromthe samepreparationas in (a), 20 dayslater.
76
W. (2 Mobley, H. &:hreier /Journal t~f Controlled Release 31 (1994) 73~'¢7
Fig. 1(c). Fused liposomes.
cose free) from Sigma Chemical Co. (St. Louis, MO, USA); and t-butanol (puriss. p.a.) from Fluka (Ronkonkoma, NY, USA).
2.2. Methods Liposome preparation The phospholipids were dried from chloroform in a round bottom flask via rotary evaporation, dissolved in t-butanol, then lyophilized to generate an optimal surface area for hydration. Multilamellar liposomes (50--60 mg/ml phospholipid) were prepared by the addition of sugar solutions (in deionized water) of varying concentrations to a constant amount of the lyophilized phospholipids, followed by vigorous vortexing. The concentrations of the sugar solutions were varied to achieve a 0-5 molar ratio of sugar to phospholipid.
Sized liposomes (40 mg/ml phospholipid) were prepared by the extrusion of multilamellar liposomes, prepared in phosphate buffer (8.1 mM Na2HPO4 and 1.5 mM KH2PO4, pH=7.5). The liposomes were extruded by passing the multilamellar suspension back and forth >31 times between two glass syringes through one 0.1 /zm polycarbonate membrane (Poretics Corp., Livermore, CA), which was placed in a central stainless-steel filter housing (LiposofastO~;Avestin, Ottawa, Canada) [ 16]. HEPC and DPPC liposomes were extruded above their membrane phase transition temperatures by wrapping Liposofast® with a temperature-controllable heating blanket. Volume-weighted liposome size distributions were determined by dynamic light scattering measurements using a Nicomp Model 370 Submicron Particle Size Analyzer (Particle Sizing Systems, Santa Barbara, CA).
W.C. Mobley, H. Schreier / Journal of Controlled Release 31 (l 994) 73--87
77
Fig. 1(d). Ruptured liposome.
Table 1 Gel to liquid crystalline phase transition temperatures (Tin) of hydrated liposomes and lyophilized liposomes (with and without dehydroprotection) Lipid
Hydrated
Lyophilized without sugar
11.25'
51.5°C (4)
105 (3)
DPPC
42 (2)
105 (3)
POPC
- 3 . 5 (2)
40 (2)
/i
with disaccharidesa
HEPC
15
34 to 38 (67) c 35 to 37 (29) d 22 to 26 (43) c 22 to 25 (23) ~ - 2 5 to - 2 0 (28) c - 2 5 to - 2 1 (20) d
HEPC
glucoseb 52 (2)
~7.5 u.
DPPC~ 3.75
~ /
POPC
42 (2) i
- 2 5 (3)
Numbers of samples in parentheses. aannealed; bannealing not essential; Cunextruded liposomes; dextruded liposomes.
-30
i
-10
i
10
i
30
i
50
~
70
i
90
110
Temperature ['C]
Fig. 2. Initial and repeated DSC thermograms of lyophilized unextraded trehalose/liposomes [3:1 molar ratio]. (solid lines, initial scan; dotted lines, repeated scan).
78
W.C. Mobley, H. Schreier / Journal +?/ControlledRelease ~I (1994) 73-,~7
Lyophilization Fifty microliters of the sugar/liposome suspensions and sugar solutions were placed in flat aluminum DSC pans in 2 ml lyophilization vials. The samples were shelf-frozen at a rate of =0.7°C/min in an Edwards Model 12K Supermodulyo freeze-dryer (Edwards High Vacuum, West Sussex, England) to = - 4 0 ° C where they were held for = 1.5 h. Vacuum was drawn and lyophilization commenced with a cycle of 12-15 h of primary drying at - 3 5 ° C (at =0.07 mbar) followed by = 5 h of secondary drying at 25°C. The samples were stoppered under vacuum and stored at ambient temperature until calorimetric analysis (generally within one week of lyophilization). Differential scanning calorimetry ( DSC) The samples were analysed with a Perkin Elmer DSC7 Differential Scanning Calorimeter (PerkinElmer, Norwalk, CT, USA) fitted with an Intracooler II refrigeration unit. The calorimeter was calibrated at 10°C/min with indium (To = 156.6°C) and sterile water for injection, U.S.P.. An empty pan served as reference for all DSC scans. Samples were ramped at 20°C per min to the following starting temperatures: 0°C (HEPC), - 1 0 ° C (DPPC) and - 5 0 ° C (POPC). Initial DSC scans of lyophilized liposomes were performed at 20°C per min and repeated scans at 10°C per min. Prior to repetition, initial scans were held at scan completion temperature ( = 90°C) until cessation o f the decline in the power input of the calorimeter ( = 2 4 min) ; hereafter referred to as the annealing process. The gel to liquid crystalline phase transition temperatures (T,,) were measured as the endotherm peaks.
60 50
,!
40 + .~ 30-
+~
: ++ . . . . . . .
:
+,
20-
extruded liposornes unextruded flpos(xnes
- 10
......
0
0
05
1
15 2 25 3 35 4 Sugar/Phospholipid Molar Ratio
4.5
5
5.5
41s
,~
5,5
415
5
2 0 -
.z3~
15
//
10-
[11 O+
0
O
O
'
/
0 i
015
1
i 15
] 2
i 25
i 3
i 3.5
i 4
$ugar/Phospholipid Molar Ratio 45 40
O
35 30 25 20
~3.75
Mole Ratio Trehalose/H E PC
//'\ /-
\
ft. 2.5
0 --
~
~
.
.
.
.
.
~
~
--
~
1.25
~
1.5
-
/
3'0
i
so
0
X --
zb
ols
i
1
i
,5
i
2
21s
i
3
31s
F
4
[
5.5
Sugar/Phospholipid Molar Ratio
' ~--~0.5 0.3 0
°1o
10 5
o
T
15
~o
/'~
IL___ i
i;o
Temperature ['C]
Fig. 3. Repeated DSC thermograms of lyophilized unextruded HEPC liposomes with increasing amounts of trehalose.
Fig. 4. Effect of disaccharide content on the low temperature transition enthalpy (AHk,w) of annealed lyophilized liposomes: (a) HEPC (extruded, solid lines; unextruded, dotted lines); (b)POPC (extruded) and (c) DPPC (extruded). n = 2-4 for unextruded HEPC and n = I for all extruded preparations ( 4, trehalose; C), maltose; [i], lactose).
79
W.C Mobley, H. Schreier / Journal of Controlled Release 31 (1994) 73--87
Enthalpy readings of the endotherms were corrected for phospholipid masses, which were determined after DSC analyses.
Phospholipid quantitation The phospholipid content of the DSC samples was quantified by a modification of an assay by Stewart [ 17]. The freeze-dried samples were rehydrated with a quantity of deionized water sufficient to achieve a phospholipid concentration = 6 mg/ml. A 0.050 ml aliquot of the rehydrated liposome suspension was added to a polypropylene test tube followed by 3 ml of ammonium ferrothiocyanate solution (containing 0.1 M ferric chloride hexahydrate and 0.4 M ammonium thiocyanate). After shaking for 15 min, 4 ml of chloroform was added to the complex suspension followed by 10 s of vigorous vortexing for extraction of the complex into chloroform. The aqueous layer was removed and absorbances of the complex solution were monitored at 475 nm for DPPC and POPC, and at 477 nm for HEPC.
Scanning electron microscopy (SEM) Samples of unextruded HEPC liposomes were lyophilized on either polylysine-coated round glass microscope coverslips or aluminum DSC pan tops and examined with a Hitachi S - 4000 Field Emission scanning electron microscope. The samples were coated (either gold or Pt/C) prior to analysis.
rier-Transform Infrared Spectrophotometer running eight scans with a resolution of 4 c m - ~. 3. Results
SEM micrographs of unprotected unextruded lyophilized HEPC liposomes are shown in Fig. 1.Membrane infolding (Fig. la) and liposome fusion (Fig. lc) were commonly seen. Small surface holes were also common, but occasionally, liposome rupturing occurred (Fig. ld). Membrane disintegration was a consequence of aging the dried unprotected liposomes (Fig. lb; taken from the same preparation as in Fig. la, but aged 20 days at room temperature).
3.1. Effect of sugars on dry membrane Tm The removal of water (by lyophilization) from HEPC, DPPC and POPC liposomes caused at least a a= trehalose/HEPC b= trehalose/DPPC c= lactose/DPPC d= maltose/DPPC e= trehalose/POPC
105
o~o 101,3
a
97.5
93.7!
90
4=0
50
Thermogravimetric analyses were performed on lyophilized cakes using a Perkin Elmer TGA 4 Thermogravimetric Analyzer, calibrated with a 2 mg standard and with the Curie points of alumel (163°C) and nickel (354°C) at a rate of 20°C/min. The samples were scanned at 20°C/min from 30 to 200°C using dry nitrogen as the purge gas.
60
70
80
90
1'00 ;10 ;20 1'30
Temperature['C]
Thermogravimetric analysis (TGA )
Fig. 5. TGA thermograms of lyophilized extruded disaccharide/ liposomes [4:11.
~o.6 0.8
E
it. 0.4
Fourier-transform infrared spectroscopy (FT1R) Twenty-five microliter aliquots of 40 mg/ml extruded liposome suspensions in deionized water (yielding 1 mg of phospholipid) were lyophilized, then ground with 100 mg of IR-grade potassium bromide (Fisher Scientific) with a mortar and pestle. Transparent pellets, prepared from the mixtures via mechanical compression, were analysed with a Perkin-Elmer Fou-
-10.2
0 40
I ~A_,
50
' 60 ~0 80 Ts Temperature ['C]
,_
90
100
Fig. 6. Repeated DSC thermograms of lyophilized unextruded trehalose/liposomes [3:1 ] (enlargement of Fig. 2).
80
w. (~ Mobley, H. Schreier / Journal ~ Controlled Release 31 (1994) 7 3 ~ 7
40°C increase in their phase transition temperatures (Tin) (Table 1). However, when lyophilized in the presence of disaccharides (trehalose, maltose, or alactose) and heat-annealed (as described in Materials and Methods), the dry membrane Tms were reduced to at least 14°C below those of the hydrated membranes. The monosaccharide glucose reduced the dry POPC Tm to 22°C below the hydrated membrane Tm, but reduced dry DPPC and HEPC Tins only to hydrated membrane levels. Annealing was not an absolute requirement for the maximal Tm-reducing effects of glucose: the Tins of the annealed samples were about 2-3°C less than the nonannealed samples. In contrast, for disaccharide/liposomes, the Tins of the annealed samples were markedly less than the non-annealed. DSC thermograms (Fig. 2) illustrate this fact, as well as the thermal events that occur during the annealing process. In Fig. 2, the initial and repeated (post-annealed) DSC thermograms are shown for 3:1 mixtures of lyo-
philized, unextruded trehalose/HEPC, trehalose/ DPPC and trehalose/POPC liposomes. Initial scans show complex thermotropic behavior characterized by the appearance of a narrow endotherm in the midst of a broader endotherm. Repeated thermograms show only a single, narrow endotherm occurring at the temperatures reported in Table 1 for annealed disaccharide/liposome preparations. The reduced temperature endotherms of the repeated scans occurred with all lyophilized disaccharide/liposome formulations over the examined range of 0.3:1-5:1 disaccharide/phospholipid molar ratio. However, the enthalpy of the reduced temperature endotherm changed significantly over that range. Fig. 3 shows the general trend in liposome membrane Tm reduction for annealed, unextruded HEPC liposomes as a function of trehalose content; this trend was similar for all lyophilized disaccharide/liposome mixtures, including extruded preparations. At low tre-
Fig. 7. SEM micrographsof the surfacesof the lyophilizationmatricesof: (a) non-annealed.
w.C. Mobley, H. Schreier/ Journal of Controlled Release 31 (1994) 73--87
halose/HEPC molar ratio [0.3:1 ], a relatively strong endotherm appears at ~ 75°C along With a barely perceptible endotherm occurring at 35°C. As the trehalose content was increased, the high temperature ( ~ 75°C) endotherm enthalpy diminished as the low temperature (35°C) endotherm enthalpy (,AHlow) increased. When plotted against disaccharide/phospholipid molar ratio, AHlow of the annealed disaccharide/liposomes rose (with varying slopes) to a maximum. As Fig. 4a illustrates, the dJ-/lowplots may vary according to the type of liposome preparation: the plots for extruded HEPC liposomes (mean diameter = 111 nm) plateaued at lower sugar contents than for unextruded liposomes. Plots for extruded disaccharide/POPC (mean diameter= 152 nm) a n d / D P P C (mean diameter = 124 nm) liposomes are shown in Fig. 4b and c. 3.2. Examination o f the annealing process
In addition to the membrane phase transitions that were apparent on the initial DSC thermograms (Fig.
81
2), possible events in the annealing process were further examined with DSC, TGA and SEM. Potential annealing-induced molecular changes were examined with FTIR spectroscopy. Thermogravimetric analyses of lyophilized extruded disaccharide/liposome [4:1 ] mixtures (performed at the same scan rate as the initial DSC scans) showed a mass loss of approximately 3-6% over a 30--125°C temperature range (Fig. 5). Differences in magnitude and kinetics of weight loss were observed, but were not extensively studied. However, in general mass loss tbr disaccharide/POPC liposomes was greater and occurred faster than for disaccharide/DPPC or/HEPC liposomes. The repeated DSC thermograms (Fig. 2) also show, in the same temperature region of the broad endotherm of the initial scans, weakly perceptible discontinuous heat capacity elevations that are characteristic of glassrubber transitions in DSC heating scans. An enlarge-
Fig. 7(b). Annealedunextrudedtrehalose/HEPCliposomes [3:1].
~2
0.2586
W.C. Mobley, H. Schreier / Journal of Controlled Release 31 (1994) 7 3 ~ 7
//'°°
number (not shown), but annealing was not required for the shift.
",\
/" HEPC/trehalose [1:2] ' \
4. Discussion HEPC
\\
\,.
0.057~1750
1745
1740 1735
1730
1725
1 7 2 0 1715
Wave Number [era"1 ]
Fig. 8. FT1R spectra of the carbonyl region lyophilized extruded HEPC liposomes.
ment of this region more readily shows the glass transitions (Fig. 6). Glass transition temperatures (Tg) were measured in these repeated thermograms as the midpoint of the heat capacity increase and they were as follows: POPC/trehalose, 560(2; DPPC/trehalose, 79°C; HEPC/trehalose, 82°C. The measured glass transition temperatures for annealed maltose/ and a-lactose/liposomes paralleled the Tgs for trehalose/ liposomes; all were greater than 50°C. The glass transitions for non-annealed formulations were not often discernible, as they were possibly obscured by the broad endotherms. However, glass transitions were discernible in the initial glucose/liposome DSC thermograms and they generally occurred at or below 25°C. SEM micrographs of the surfaces of annealed and non-annealed lyophilized, unextruded trehalose/ HEPC liposomes [3:1 ] are shown Fig. 7. Two salient features in both micrographs are the amorphous appearance and matrix porosity. FTIR spectra of the carbonyl region of HEPC in lyophilized trehalose/HEPC [2:1 ] liposomes showed that annealing caused an intensity increase of the 1720 cm-~ band relative to the 1737 cm -I band (Fig. 8). Lyophilized unextruded (multilamellar) trehalose/ HEPC [ 2:1 ] liposomes were later examined (spectra not shown) and the same general effect of the annealing-induced increase in low frequency band intensity was found, but in this case the low frequency band appeared at 1728 cm -~. Trehalose also shifted the phosphate stretch band ( 1240 cm- 1) to a lower wave
The SEM micrographs of lyophilized liposomes showed the potentially deleterious effects of the freezing and dehydration stresses of the lyophilization process. These stresses led to membrane infolding (Fig. 1a), liposome fusion (Fig. lc) and membrane rupture (Fig. ld). Membrane infolding indicates that slow freezing caused water efflux from the liposomes, despite the fact that the liposomes were formulated in deionized water. Therefore, the potential gradient down which water escaped from the liposome, was not of osmotic origin, but, likely, resulted from the vapor pressure gradient between internal water and external ice, as described by Mazur for freezing cells [ 18,19]. Similarly, liposome rupture was probably not of osmotic origin, but was possibly caused by the adhesion of ice to phospholipid headgroups. Olien and Smith [ 20] postulated that adhesive forces develop from the competition for liquid water between the growing ice dendrites and hydrophilic substances, including the hydrophilic portion of membranes. Another possible cause of membrane rupture was the coalescence of dissolved gas within the liposome to a point where a bubble was expelled; gas bubbles are known to escape from a freezing solution [21]. Liposome aggregation and fusion, which was ubiquitous in electron micrographs of unprotected liposomes, was expected for two reasons: lyophilization removed the hydration barrier to fusion and the propagating ice front concentrated the liposomes, thus increasing the chances of membrane apposition. The concentration effect of freezing, which has been described for cells and particles by Krrber [ 21 ], was evident in SEM micrographs (not shown) of the lyophilization matrices of unprotected liposomes, which showed extensive aggregation. Perhaps the most important effect of lyophilization, in terms of long-term dry liposome preservation, was illustrated in Fig. lb, which showed membrane disintegration; the expected consequence when the membrane stabilizing role of water is lost to the liposome.
w.C. Mobley, H. Schreier / Journal of Controlled Release 31 (1994) 73-87
With the combined results from the analytical methods ofDSC, SEM, TGA and FTIR, we hypothesize that when lyophilized disaccharide-containing liposomes are heated beyond their membrane phase transition and sugar glass transition temperatures (Figs. 2 and 6), an annealing occurs whereby an increased molecular mobility of the rubbery sugar and liquid crystalline membrane allows the disaccharide to supplant lost water (Fig. 5) at the carbonyl region of the membrane interface (Fig. 8), causing a reduction in dry membrane Tm (Fig. 2 and Table 1). Thus, this study provides evidence for the two prominent theories of membrane dehydroprotection: the water replacement hypothesis and glass formation. The membrane interactive role of dehydroprotectants, in accordance with the water replacement hypothesis, was manifested as a dehydroprotectant-induced dry membrane Tm reduction in the DSC analyses. Previous studies of dry iiposomes composed of trehalose/ DPPC [ 10,11], sucrose/DPPC [ 12] and trehalose/ POPC/POPS [ 14,15], have found that trehalose and sucrose reduced the dry membrane TmS. However, the reported magnitudes of Tm reduction have varied widely. This study generalizes the Tm-reducing phenomenon to include the disaccharides a-lactose and maltose and the phospholipid HEPC. In addition, it was found that a-lactose and maltose had essentially equivalent Tin-reducing abilities to trehalose, indicating that trehalose is not unique in this regard. Common to all of the disaccharide/liposome formulations in this study was a disaccharide-induced dry membrane T~ reduction to at least 14°C below the hydrated membrane T~, which suggests that phospholipid spacing is greater in the annealed/dehydroprotected dry membrane than in its hydrated counterpart. The assertion of an annealing-induced phospholipid spreading is supported by the FTIR spectra of the carbonyl-region of the phospholipid (Fig. 8). Using FTIR, Crowe et al. [22] established that trehalose interacts with the phosphate group of membrane phospholipids, as manifested by a red-shift of the asymmetric phosphate stretch band of DPPC. A similar band-shift was observed here for trehalose/HEPC [2:1 ] liposomes (spectra not shown) and annealing did not affect the position. However, annealing did appear to influence the carbonyl stretch bands manifested as an increased relative intensity of the low frequency band (Fig. 8), suggesting that it caused
83
trehalose to penetrate deep into the membrane interface and possibly hydrogen bond with one of the phospholipid carbonyl groups. In an IR study of the potential binding sites of water to 1,2-diacyl phospholipids, Wong and Mantsch [ 23 ] assigned (to DPPC) the high wave number (1737.5 cm- 1) band to the free sn-2 C=O stretch and the low wave number (1728.2 cm - t ) band to hydrogenbonded sn-2 C--O stretch. In that study, IR spectra of fully hydrated DPPC showed an increase in relative intensity (vs. nearly anhydrous DPPC) of the lower wave number band. Thus, it may be postulated that the annealing-induced increase in relative intensity of the low wave number band for HEPC ( 1720 cm- 1) in the present study is due to trehalose/sn-2 carbonyl hydrogen bonding. The discrepancy in the position of the low wave number bands in the present study ( 1720 cm- 1) and in the study of Wong and Mantsch (1728.2 cm-1) possibly lies in the type of liposomes examined. In that study, multilamellar liposomes and lipids lyophilized from solvent were examined. The formulations that showed a band at 1720 cm- 1 in the present study were extruded. However, annealed multilamellar trehalose/ HEPC [2:1] liposomes showed a low frequency band at a nearly identical position [ 1728 cm -1 ] to that of hydrated multilamellar DPPC liposomes found by Wong and Mantsch. The ultimate cause of the spectral differences between the extruded and multilamellar liposomes is uncertain and outside the scope of this study, but it may reflect the particular subgel phase of the membrane; different subgel phases of hydrated liposomes have been shown by Lewis and McElhaney [24] to give different IR spectra. The important finding for this study is that annealing lyophilized trehalose/HEPC [ 2:1 ] liposomes caused an increase in relative intensity of the low frequency band, regardless of the two preparation methods. Since maltose and a-lactose had similar Tin-effects to trehalose for both preparation methods, it may be inferred that annealing enables all three disaccharides studied to hydrogen-bond to the sn-2 carbonyl group of phospholipids. However, this is a tentative hypothesis, as the assignment of bands in the carbonyl region of phospholipids is controversial. It has been asserted that hydration-induced spectral differences are due to
84
W.C. Mobley. H. Schreier / Journal of Controlled Release 31 (I994) 7 3 ~ 7
conformational and packing changes rather than hydrogen bonding [ 25 ]. The inconsistent Tin-reducing behavior of glucose among the various liposome formulations indicates a phospholipid-dependent membrane interactive ability of the monosaccharide. The significant Tin-reduction (22°C below the hydrated membrane Tm) by glucose for POPC liposomes suggests that their inherent greater phospholipid spacing (vs. DPPC and HEPC) allows the depth of interaction necessary for such a Tm reduction. However, for DPPC and HEPC, glucose induced Tm-reduction to only the hydrated membrane level, suggesting a fundamentally different interaction with those membranes, which may partially explain the reported relative deficiency of glucose as a membrane dehydroprotectant [ 9 ]. The lack of a requirement for an annealing process for Tm-reduction by glucose may be explained by the finding that lyophilized glucose/liposome preparations were generally in the rubbery phase at room temperature (Tg < 25°C), thus possessed the required molecular mobility for the interaction with the phospholipid headgroup. A glass transition below ambient conditions would mean that glucose would also be an insufficient dehydroprotectant according to the glass formation theory for membrane dehydroprotection. The importance of glass formation in membrane dehydroprotection lies in the fact that long range motion is inhibited in the glass, thus presumably, so are degradation processes. A Tg above ambient conditions (a central element of the glass formation theory for membrane dehydroprotection [7]) was observed for all of the disaccharides studied, but not glucose. Glucose, because of its low Tg, would therefore be expected to be a deficient protectant, and a lyophilized glucose/liposome cake would be expected to collapse over time and become unreconstitutable. In contrast, the SEM micrograph of the lyophilization matrix of unextruded trehalose/HEPC [3:1] liposomes (Fig. 7a) showed an amorphous and stable, porous cake that was not destabilized by annealing (Fig. 7b). DSC thermograms (Figs. 2 and 6) confirmed that the amorphous structure seen with SEM was that of a glass, with a Tg well above ambient temperatures. Glass formation occurs during the freezing stage of iyophilization as the crystallization of water results in an increasingly concentrated solution (referred to as the concentrated amorphous solute phase [26]), ulti-
mately reaching a point where no further water crystallization occurs, leaving a glassy solute/unfrozen water mixture that essentially determines the ullimate product morphology. A portion of the unfrozen water is removed during the secondary drying (desorption) stage of lyophilization and the ultimate Tg of the lyophilized product, all else being equal, will be a function of the residual water content. Further reductions in water content brought about by annealing should be expected to increase Tg, provided that the sample remains a glass. Thermogravimteric analyses (Fig. 5) showed that water evaporated during the annealing process and, in those samples where a glass transition was discernible before and after annealing, an increase in Tg was observed. For example, Fig. 2 showed an apparent glass transition at = 53°C in the initial thermogram of trehalose/HEPC. Upon annealing, the Tg increased to 82°C (Fig. 6). Although the thermogravimetric analyses reported here were not extensive, it was apparent that the residual water content did not vary greatly between the disaccharides. However, the greater and faster water loss observed for disaccharide/POPC liposomes indicated that water-retention may be phospholipiddependent. A more comprehensive TGA study may yield valuable information, such as the relationship of water-loss kinetics to strength of sugar-water bonding in the glass. The facts that all of the disaccharides reduce the dry membrane Tins similarly and endow the final products with Tgs above normal storage temperatures (e.g., room temperature) demonstrate that these parameters may not be the best measures of the evolutionary uniqueness of trehalose as a membrane dehydroprotectant. Certainly, the nonreducing nature of trehalose must have importance, but an additional distinguishing feature may be the relative strength of the sugar/ membrane interaction. It is plausible that an equilibrium exists between sugar/sugar and sugar/membrane interactions and that the equilibrium for trehalose/membrane interactions is more favorable than other disaccharide/membrane interactions. The plots of ~/low VSdisaccharide content indicate that such a distinction may be made for trehalose. In all of the plots, the rise to maximum is steepest for trehalose/liposome formulations. If these plots are
W.C. Mobley, H. Schreier / Journal of Controlled Release 31 (1994) 73--87
indicative of the degree of membrane/disaccharide complexation, then the steepness of the rise could be a measure of interaction equilibria, which appear to favor trehalose/membrane interactions in the present study. However, replicate experiments are necessary to support this hypothesis. The fact that the AH~ow plots were less steep for unextruded disaccharide/HEPC liposomes than for their extruded counterparts is possibly due to a greater access of the sugar to the phospholipid headgroup, afforded by the mixing during the extrusion process. In addition, liposomes extruded through 100 nm pore size membranes will have a large population of unilamellar vesicles, with greater access of the sugars to both sides of the liposome membrane. In contrast, unextruded liposomes will consist of subpopulations of multilamellar liposomes with tightly packed, concentrically aligned lamellae that may be restrictive to headgroup access by the sugars. The increase in the low temperature transition enthalpy with increased disaccharide content likely correlates with the degree of saturation of the membrane with the disaccharide. The existence of a high temperature transition at low sugar amounts in the DSC thermograms (Fig. 3) may be due to a transition of only one side of the membrane, a hypothesis put forth by Crowe et al. [27]. Its disappearance at higher sugar amounts may indicate saturation of both sides of the membrane. Two additional important points to consider are the operating conditions of lyophiligation and of DSC analyses. The products were frozen at a slow rate ( = 0.7°C per min) which can have the following consequences. (1) The amount of freezable water that freezes will be maximized, resulting in a more concentrated amorphous solute possessing the highest Tg obtainable by freezing [26], which, in turn, can help to minimize lyophilization costs (due to an increased collapse temperature). The removal of the frozen water via sublimation forms the porous network of the final product (Fig. 7). (2) Slow freezing may also cause vesicular dehydration which leads to the stresses discussed in the Introduction; however, these stresses are minimized by the dehydroprotective solutes. The overall effects of freezing on liposome lyophilization are crucial and have been studied in detail elsewhere [28-30] ; but they are beyond the scope of this paper.
85
The final freezing temperature of the lyophilization process was limited by the lyophilizer to between - 3 7 ° C and -40°C, which is perilously close to the reported aqueous Tgs of the disaccharides studied: - 36 to - 2 8 ° C [31,32]. Although the lyophilized products in this study were found to be in the glassy state, a lower final freezing point would be preferable to assure glass formation during freezing. DSC and TGA were performed under a nitrogen atmosphere which can facilitate water removal during heating which, in turn, may be vital for annealinginduced dry membrane Tin-reduction. Preliminary studies indicate that the Tms of HEPC/disaccharide samples, heated under ambient humidity, are not reduced to below the hydrated membrane levels, although the TmS for disaccharide/POPC liposomes a re .
5. Conclusions It remains to be seen whether or not the annealinginduced dry membrane Tm reduction is important for long term membrane dehydroprotection. Tm reduction should only be important for those formulations that can undergo a phase transition during reconstitution; membranes are known to be leaky during phase transitions [33]. However, if the Tm reduction is an accurate measure of dehydroprotectant/phospholipid membrane complexation (as has been hypothesized), and if this complexation is valuable for dry membrane preservation (e.g., to supplant the role of water), then the annealing process could prove beneficial for long term dry liposome storage. The maintenance of the lyophilized product in the glass form may be essential. It would thus be important to assure dehydroprotectant/membrane annealing does not cause sugar crystallization. The studies here indicated that the glass form is maintained in the annealed product. However, in the development of lyophilization and annealing protocols, one must be cognizant that the conversion of the metastable glassy state to the stable crystalline state is a sugar-specific function of moisture- and heat-induced molecular mobility [26] and can be altered by excipients [26,34,35] ; therefore such techniques as DSC and SEM may be required to confirm a glassy product.
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Acknowledgements Scanning electron microscopy was performed at the Electron Microscopy Laboratory of the Interdisciplinary Center for Biotechnoiogy Research at the University of Florida with valuable input from Dr. Greg Erdos. Support was provided by NIH grants AI26339 and GM 46922 and by an unrestricted research grant from Glaxo, Inc. (Research Triangle Park, NC, USA)
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[32] Y. Roos and M. Karel, Nonequilibrium ice formation in carbohydrate solutions, Cryo-Lett., 12 ( 1991 ) 367-376. [33] J.H. Crowe, L.M. Crowe and F.A. Hoekstra, Phase transitions and permeability changes in dry membranes during rehydration, Bioenerg. Biomemb., 21 (1989) 77-91.
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[34] F. Franks, Solute-water interactions: do polyhydroxycompounds alter the properties of water?, Cryobiology, 20 (1983) 335-345. [35] D.J. Korey and J.B. Schwartz, Effects of excipients on the crystallization of pharmaceutical compounds during lyophilization, J. Parenter. Sci. Technol., 43 (1989) 80-83.