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Chemical Hydrolysis of Phospholipids
Chemical Hydrolysis of Phospholipids N. J. ZUIDAM~ AND D. J. A. CROMMELIN Received November 21, 1994, from the Depatfment of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Groningen Utrecht Institute for Drug Exploration, Utrecht University, P. 0. Box 80.082. 3508 TB Utrecht, The Netherlands. Accepted for publication June 2, 1995@. Abstract 0 Hydrolysis kinetics of phospholipids in liposomes composed of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholinel cholesterol (DPPC/CHOL) 10/4 (molar ratio) and egg phosphatidylcholine (EPC) at pH 4.0 and different temperatures could be described by Arrhenius curves without breaks. However, the Arrhenius curves for the hydrolysis of liposomal DPPC and distearoylphosphatidylcholine (DSPC) under the same conditions were biphasic. A break was observed in the curves extending over a broad range before and after the known Tm of each of these phospholipids in liposomes (42 and 56 "C,respectively). The activation energy (Ea)for the hydrolysis of liposomal DPPC and DSPC below the Tm was substantially larger than the Eafor liposomal DMPC, DPPCICHOL 10/4, and EPC and decreased when DPPC was mixed with CHOL in a 1014 molar ratio. Hardly any influence of the presence of a-tocopherol, cryoprotectants (glucose, trehalose, sucrose, and propylene glycol), and the major hydrolysis products lysophospholipids and fatty acids or of the absence of sodium chloride on the hydrolysis kinetics of DPPC at pH 4.0 and 30 "C was observed. Changes in fatty acid chains and size did not influence the hydrolysis rate constant (kbS) of liposomal phospholipids at pH 4.0 and 30 "C either. The only effects of uncharged compounds on the k b s of liposomal DPPC at pH 4.0 and 30 "C were found upon mixing with a high concentration of the detergent Triton X-100 or palmitic acid. However, major effects on the hydrolysis kinetics of liposomal DPPC andlor dipalmitoylphosphatidylglycerol (DPPG) were observed by incorporation of charged molecules into liposomal bilayers composed of DPPC and DPPG or steatylamine or dicetyl phosphate, at pH 4.0 and 30 "C. A relation between the logarithmic values of kbs and surface potential was presented in this study. The influence of the phospholipid head group on k b s is small (when corrected for charge); under the chosen conditions there is a tendency toward faster degradation in the order dipalmitoylphosphatidylethanolamine (DPPE) < DPPC < DPPG.
kinetics of liposomal phospholipids.6 Until now, no systematic study has been performed on the effect of chain length and head group on the hydrolysis of phospholipids in liposome dispersion^.^ Also the effect of the organization of the phospholipid state (bilayers, micelles, gel phase, liquid phase, etc.) on the hydrolysis process is still unclear. Frakjaer et al. found a higher activation energy in distearoylphosphatidylcholine (DSPC) liposomes at pH 6.5 for bilayers in the gel state than in the liquid crystalline state.3 However, other studies could not confirm this ~ b s e r v a t i o n .Furthermore, ~~~ possible effects of different types of water soluble and lipophilic additives t o liposome dispersions on the hydrolysis kinetics of phospholipids have never been investigated in an appropriate way, except for those few referred to above. Therefore, the objectives of this study are to assess in more detail factors which influence hydrolysis kinetics of phospholipids. Liposomes composed of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine(DPPC), DSPC, egg phosphatidylcholine (EPC), or DPPC/cholesterol (CHOL) 10/4 (molar ratio) were stored at pH 4.0 and a t different temperatures and their hydrolysis kinetics were monitored. Moreover, to obtain additional information on the effect of chain length on the hydrolysis kinetics of PC, hydrolysis of dicaproylphosphatidylcholine (DCPC) and dilauroylphosphatidylcholine (DLPC) a t pH 4.0and 30 "C was determined. As it is known that additives can affect the bilayer characteristics, experiments were also performed to analyze the influence of additives on the hydrolysis kinetics of DPPC a t pH 4.0 and a t 30 "C, a temperature just below the pretransition of DPPC bilayers. The pH of 4.0 was chosen, because the hydrolysis rate of liposomal phospholipids is relatively fast and because fatty acids and lysophosphatidylcholine (the major degradation products) and phosphatidylcholine are uncharged at this PH.
Experimental Section
Introduction Insight into the chemical hydrolytic process of liposomal phospholipids is important for the design of liposomal dispersions to be used as drug carriers. In an aqueous liposome dispersion, the liposomal phospholipids can hydrolyze to free fatty acids and 8-acyl and 1-acyl lysoph~spholipids.~-~ Further hydrolysis of both lysophospholipids results in glycerophospho compounds. Glycerophosphorate acid is the usual end product after further, relatively slow degradation of the ester bond between the phosphate and the head group of the glycerophospho compound.lOJ1 The hydrolysis of liposomal phospholipids is catalyzed by protons and hydroxyl ions and reaches a minimum at pH 6.5; a V-shaped pH profile for the hydrolysis process is obtained upon plotting the logarithmic of the hydrolysis rate constants against the pH.2,3,5-9 The hydrolysis rate of the phospholipids is not only influenced by pH, but also by temperature (following Arrhenius kinetic~l-3+-~), charge of the lipo~omes,~ and buffer specie^.^,^^^ Cholesterol and ionic strength do not affect the hydrolysis @
Abstract published in Advance ACS Abstracts, July 1, 1995.
0 1995, American Chemical Society and American Pharmaceutical Association
Materials-DCPC, DLPC, DMPC, DPPC, DSPC, and dipalmitoylphosphatidylglycerol (DPPG) were gifts from Nattermann Phospholipid (Cologne,FRG). EPC and dipalmitoylphosphatidylethanolamine (DPPE) were gifts from Lipoid K. G. (Ludwigshafen, FRG). According to the manufactures the purity of the phospholipids was a t least 99%. CHOL ('99% pure), palmitic acid (PA, purity about 99%), dicetyl phosphate (DCP, > 99% pure), and trehalose were purchased from Sigma (St. Louis, MO). Monopalmitoylphosphatidylcholine (LPC, >99% pure) was obtained from Avanti Polar Lipids (Pelham, AL). Stearylamine (SA, '99% pure) was purchased from Janssen Chimica (Beerse, Belgium). a-Tocopherol (> 98% pure) was obtained from Fluka (Buchs, Switzerland). Sucrose and glucose were obtained from Merck (Darmstadt, FRG). Propylene glycol was purchased from OPG (Utrecht, The Netherlands). Triton X-100 was obtained from BDH (Poole, England). These and all other chemicals were of analytical grade. The water was doubly distilled before use. Preparation of Liposome Dispersions-Liposomes were prepared by the "film" method. Appropriate mixtures of the (phospho)lipids were dissolved in chloroforndmethanol(1:l)in a round-bottom flask. The organic solvent was removed under vacuum by rotary evaporation. The thin film obtained was dried for a t least 3 h under reduced pressure. Then, the film was hydrated with 50 mM acetate buffer (pH 4.0) and 0.12 M sodium chloride. Most of the time, the
0022-3549/953184-1 1 13$09.00/0
Journal of Pharmaceutical Sciences / 1I13 Vol. 84, No. 9, September 1995
liposomes used were unextruded. If necessary, the liposome dispersions were sized with an extrusion system (Sartorius, Gdttingen, FRG) once through 0.6 pm and three times through 0.2 pm pore size filters (Nuclepore, Costar Corp., Cambridge, MA). At the storage temperature the pH of the dispersion was measured and adjusted before and after extrusion, if necessary. The liposome dispersions were stabilized in the refrigerator overnight. Then, the pH of the dispersion was measured again at the storage temperature and adjusted, if necessary. The final phospholipid concentration was about 20 mM. Hydrolysis Experiments-The prepared liposome dispersions were filled into ampules under a nitrogen atmosphere and sealed. Ampules were stored in a constant temperature water bath (15-80 "C) or in a constant temperature room (4"C). Samples ( n 2 6) were taken after appropriate time intervals and stored in the freezer (-20 "C) until analysis. Analytical Methods-Phospholipids were analyzed by HPLC as described earlier.12 Samples for the HPLC analysis were prepared by the Bligh and Dyer e ~ t r a c t i 0 n . lThe ~ phospholipids were collected in the chloroform phase. After dilution of the chloroform phase in methanol, aliquots of 100 pl were directly injected into the column. The HPLC system consisted of a type 400 solvent delivery system (Kratos, Ramsey, NJ), a Kontron sampler MSI 660 (Kontron AG, Zurich, Switzerland), and a Waters 410 RI detector (Waters Associates, Milford, MA). Peak areas were measured with a computer controlled integrator-based data system (WOW, Thermo Separation Products, Fremont, CA). The separation of the phospholipids was carried out on a Zorbax aminophase column (25 cm x 4.6 mm, i.d., 5 pm particle size, Du Pont Co., (Wilmington, DE) a t 35 "C. An Adsorbosphere NH2 5 pm guard column (Alltech Associates, Deerfield, IL) was connected before the Zorbax aminophase column. The mobile phase consisted of a mixture of acetonitrile/methanoY5 mM ammonium dihydrogen phosphate solution, pH 4.8. Because of columnto-column variations, the ratio of the solvents used in the mobile phase was different for the different columns used. A typical example of a mobile phase composition used is 57/38/4 (v/v). The flow rate was 1.5 mllmin. The 2-average particle size and polydispersity index (pd) a t 25 "C were determined by dynamic light scattering with a Malvern 4700 system using a 25 mW He-Ne laser (NEC Corp., Tokyo, Japan) and the automeasure version 3.2 software (Malvern Ltd, Malvern, UK). See elsewhere for further details.14 For viscosity and refractive index, the values of pure water were used. The pd is a measure of the width of the particle size distribution and ranges from 0.0 for a n entirely homogeneous size distribution to 1.0 for a completely heterogeneous one. Statistics-Significance tests on a mean were performed by using the Student t-test assuming equal variances (two-sided). Differences in slopes were compared by using the test for homogeneity of the lines. Differences in intercepts were tested by using Analysis of Covariance (ANCOVA). The value for p is given or else a value of 0.05 for a was assumed in all the tests. See elsewhere for details about the statistical tests.l5
Results and Discussion Order of Reaction and Influence of Temperature and Organization of the Phospholipid Assembly-The disappearance of phospholipids in buffered dispersions follows pseudo-first-order In this study this observation was confirmed for most of the phospholipids. As an example, semilogarithmic plots for the hydrolysis of DPPC in acetate buffer (pH 4.0) a t different temperatures are shown in Figure 1. From such straight lines, observed pseudo-first-order rate constants ( k o b s ) were obtained. The kobs is the sum of the firstorder rate constant for the hydrolysis in water only (ko) and of the second-order rate constants for proton (kH+), hydroxyl ion ( k O H - ) and buffer component (kbuffer) catalyzed hydrolysis, multiplied by the concentrations of these specific compounds. The effect of temperature on the k o b s can be described by the Arrhenius equation.1-3,6-9 In this study, the validity of Arrhenius kinetics was investigated for the hydrolysis of phospholipids in nonsized liposomes composed of DMPC, DPPC, DSPC, EPC, or DPPC/ 1114 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
I00
170ic
i5O.c
- - -o l 0
100
50 Time (days)
Figure 1-Typical examples of semilogarithmic, apparent first-order plots for the hydrolysis of DPPC in 50 mM acetate buffer (pH 4.0) and 0.12 M NaCl at the indicated temperatures. More determinations (n = 7-8) were made at 4 and 20 "C,as indicated by the extrapolation beyond the last experimental determinations. However, these determinations are not shown here so the curves at the higher temperatures can be seen properly.
CHOL 10/4 (molar ratio). These liposomes were stored a t pH 4.0 in the temperature range from 4 to 80 "C to determine the effect of the organization of the phospholipid assembly (gel phase, liquid phase, and condensing effect of cholesterol) and chain length on the hydrolysis kinetics of the phospholipids. The results are shown in Figure 2 and in Table 1.The hydrolysis of liposomal EPC could be adequately described by an Arrhenius curve without breaks (see Figure 2E and Table 1). A break in the Arrhenius curve was expected when the liposome dispersion contained a gel-to-liquidphase transition in the experimental range (in the case of DMPC, DPPC, and DSPC liposomes, see Table 1 and elsewherei6 for further information), because the effect of temperature on the hydrolysis of phospholipids with such a phase transition has been described by biphasic Arrhenius curves b e f ~ r e . ~In ,~,~ this study statistical tests were performed to analyze if such breaks could be statistically proven and if differences in slopes describing hydrolysis kinetics of phospholipids above and below T , were statistically significant as well. Theoretically, one straight line was fitted through data points above the T, and another line was fitted through points below the T,. Both the slopes and the intercepts of these lines were compared by using the test for the homogeneity of the slopes and by using analysis of covariance (ANCOVA), respectively. A break in the Arrhenius curve will a t least result in a difference in intercepts. The results of the statistical tests are also shown in Table 1. No break in the Arrhenius curve describing the hydrolysis kinetics for liposomal DMPC was observed. However, breaks in the Arrhenius curves for both DPPC and DSPC occurred, because the slopes of the lines above and below the T, were statistically different. Thus, the activation energy (E,) in the fluid and E , in the gel phase were different for both DPPC and DSPC liposomes. The kobs of DSPC liposomes a t 50 "C deviated from the biphasic Arrhenius plot (see Figure 2C). This temperature is just above the temperature of the pretransition of DSPC liposomes, 49 'C.I6 The slope of the line calculated by linear regression through the k o b s values at 4, 20, 30 and 50 "C and the slope through these k o b s values without the 50 "C value are statistically different (p = 0.027). Therefore, the Arrhenius curve has been fitted without the kobs values a t 50 "C (see Figure 2C and Table 1). Cholesterol has a condensing effect on DPPC liposomes, resulting in a disappearance of the phase transitions of DPPC in the experimental range above 27 mol%.17 As expected, no break in the Arrhenius curve for the hydrolysis of DPPC in DPPC/ CHOL 10/4 liposomes occurred (no difference in the slopes of the lines above and below T, of DPPC was observed). However, the homogeneity of the intercepts of these two lines is not clearly significant; the p-value is relatively low (0.046). A difference in intercepts is theoretically hard to explain.
DMPC
loo
DPPC
10
A
70 60 50 I
0.0028
1
I
I
30
20
1
I(
0.0032
B
70 60 50
4'C
;
0.0028
0.0036 l/T (1K)
30
0.0032
4 C
20
lo4 0.0028
0.0036
70 60 50
i
0.0028
'
30 I
'
20
0.0036 1IT (1K)
EPC
E
D
'
0.0032
1/T (1iK)
DPPCICHOL 1014
loo
7
'o-j8; '0 70 30
4 T
70 60 50
30 20
4°C
1'
0.0032
0.0036 1/T (1IK)
0.0028
0.0032
0.0036 1/T (1K)
Figure 2-Arrhenius plots describing the effect of temperature on the hydrolysis of liposomal DMPC (A), DPPC (B), DSPC (C), DPPCiCHOL 1014 (D), and EPC (E) in 50 mM acetate buffer (pH 4.0) and 0.12 M NaCI. Vertical bars indicate the SD of three determinations. When no bars are shown, the SD fell within symbol dimensions. Table 1-Results of the Arrhenius Curves for the Hydrolysis of Liposomal Phosphatidylcholine in 50 mM Acetate Buffer (pH 4.0) and 0.12 M NaCl As Shown in Figure 2a
p value Tm
Liposomes DMPC DPPC DSPC
("C)
Phase
Eaf SE A homogeneity analysis of (kJ/mol) (days-') of slopes covariance
+
24 Total range 61 1 3 x lo* 42 Above T, 67+3 3x lo9 Below Tm 91 + 3 5 x I O l 3 56 Above T,, 48+3 4 x lo6 Below T, 104+4 1 x 1OI6 c Total range 64+1 1 x109
DPPC/CHOL 10/4 EPC -10 Total range
0.391 0.011
0.686 d
0.000
d
0.292
0.046
61 k 2 3 x lo8
aThe statistical analysis is based on at least nine (3 x 3) independent kbs values above the gel-to-liquid phase transition temperature ( Tm)or below Tm. "Total range" implies that no break in the Arrhenius curve was observed around T,. €, is the activation energy and A is the frequency factor. bThe values of Tm were obtained from the Phospholipids Handbook.16 CAbove 27 mol % cholesterol liposomes composed of DPPClCHOLdo not contain a gel-to-liquidphase transition anymore.I7 If the slopes are inhomogeneous, the analysis of covariance cannot be performed anymore.
The E, of DMPC, DPPCKHOL 1014, and EPC liposomes did not differ from the E , of DPPC and DSPC liposomes in the fluid state, as was determined by testing the homogeneity of the slopes (p = 0.277). However, the small differences in intercepts of these lines were statistically significant (ANCOVA, p = 0.000). The differences in E , of DMPC, DPPC/ CHOL 10/4, and EPC liposomes and in E, of DPPC and DSPC liposomes in the gel state were significant ( p = 0.026). Thus, for bilayers in the fluid state or when CHOL is present in the bilayer, the E, for the hydrolysis of all phospholipids studied is the same. However, when the DPPC and DSPC bilayers convert in the gel state a t lower temperatures, the E, for the hydrolysis increases in the following order: DMPC % EPC % DPPCKHOL 10/4 < DPPC < DSPC. Apparently, the packing of the phospholipids has an influence on the hydrolysis kinetics. It has been reported that the
boundary between the hydrophobic and hydrophilic part in phospholipid bilayers is located at the first CH2 group in the hydrocarbon chains.18It has also been shown that the degree of hydration is dependent on the level of acyl chain unsaturation and that lengthening of the acyl chain length results in tighter packing.18 One might suggest that these intrabilayer forces among the hydrocarbon chains (such as Van der Waals forces) affect the hydrolysis kinetics by providing more effective steric hindrance to the approach of protons t o the ester bonds of phospho1ipids.l Influence of Chain Length-dispersions of PC with different fatty acid chains were prepared and hydrolyzed at pH 4.0 and 30 "C. The results are shown in Table 2. All phospholipids formed bilayers, except DCPC, which is present as monomers and micelles (critical micelle concentration is 14 mM at neutral pH19). A tendency in kobs to increase was found upon lengthening of the acyl chain length. The reason for this is not known. Influence of the Neutral Additives a-Tocopherol, LPC, Palmitic Acid, Sodium Chloride, Cryoprotectants, and Triton X-100-In Table 2 the kobs of liposomes composed of DPPC and/or a-tocopherol a t pH 4.0 and 30 "C are shown. Villalain et al. suggested a n interaction between the hydroxyl group of a-tocopherol and the polar region of DPPC, but it was not fully clear whether this bond was between the phosphate group or with the carbonyl group of the DPPC molecule.20 The same group observed a small decrease in LPCPC ratio upon storage of liposomes composed of EPC and a-tocopherol a t pH 7.4 and a t 4 "C or ambient temperature, suggesting that a-tocopherol could decrease the kobs of liposoma1 phospholipids.21 In the present study, we could not confirm this observation and, therefore, do not provide support for the proposed interaction between a-tocopherol and the carbonyl group of DPPC. Apparently, the presence of both hydrolysis products lysophospholipids and fatty acids in the bilayer does not influence the kobs of liposomal phospholipids as indicated by the straight Journal of Pharmaceutical Sciences / 1115 Vof. 84, No. 9, September 1995
Table
2-kbs of PC in Neutral Liposomes or Micelles in 50 mM Acetate (pH 4.0) and 0.12 M NaCl at 30 ‘CB kobs
Parameter of Investigation Length of fatty acid chain
Effect of a-tocopherol Effect of hydrolysis products
Effect of sodium chloride Effect of cryoprotectant
Effect of Triton X-100
Composition of Liposomes
Remark
DPPC DCPC DLPC DMPC DSPC EPC DPPClatocopherol 10/2 DPPCia-tocopherol 10/4 DPPClLPC 1/1 DPPC/PA 1011 DPPC/PA 2/1 DPPClPA 1/1 DPPC without sodium chloride DPPC + 10% glucoseb DPPC + 10% trehaloseb DPPC + 10% sucroseb DPPC + 10% propylene glycolb DPPCnriton X-100 111 DPPCnriton X-100 1/8
diCls-PC dlC6-PC diCip-PC diC14-PC diCls-PC diC14-22-PC
(10-2days-1) 1.0io.2 0.7 i0.1 0.8i0.1 1.O ? 0.3 1.1 * O . l 0.8 If- 0.1 1.2 i 0.1 1.1 k 0.2 1.o i0.1 0.9 If- 0.1 0.9 i0.1 0.6 k 0.1 1.o i0.2 0.9 i0.1
l.oio.l
1.1 i o . l l.oio.l 0.9 i0.1c 0.07 k 0.06d
a Experimental data represent the mean k SD of at least three determinations. The concentration of PC was 20 mM. The liposornes were nonsized. Ir The 10% cryoprotectants are in weightlvolume. After 45% hydrolysis, the hydrolysis rate decreased suddenly. T’he hydrolysis rate shown in this table has been derived from the values before this deviation. See also the text. Hardly any decrease was found in the experimental time period. Therefore the SD is relatively large here.
lines (see Figure 1). To examine if this phenomenon is a combined, counteracting effect of both hydrolysis products or not, DPPC liposomes were made in the presence of a n equal molar ratio of monopalmitoylphosphatidylcholine (LPC) or different molar ratios of palmitic acid (PA) and stored at pH 4.0 and 30 “C. The addition of PA in DPPC bilayers in a n acidic environment leads to disappearance of the pretransition and subsequently broadening and shifting to higher temperatures (50-60 “C) of the main phase transition.2z The addition of 50 mol % LPC to DPPC results in almost complete formation of micelles instead of liposomes, while the main phase transition is hardly affected.23 The results of the hydrolysis experiments are shown in Table 2. No effect of LPC on kobs of liposomal DPPC was observed. However, a small effect on kobs of liposomal DPPC was found upon mixing with PA. It was only statistically significant for a molar ratio of 1/1, but not in a molar ratio of 10/1 or 2/1. Both hydrolysis products alone do not or only slightly influence the hydrolysis rate of DPPC and that explains why their presence does not affect the kobs of liposomal phospholipids. Studies were also done to analyze the effect of sodium chloride, cryoprotectants (glucose, trehalose, sucrose, and propylene glycol) and the detergent Triton X-100 on the hydrolysis of liposomal DPPC a t pH 4.0 and at 30 C. The results are shown in Table 2. Sodium chloride (as reported before”*)and cryoprotectants did not affect the hydrolysis rate of liposomal DPPC. It has been reported that sugars interact with liposomal bilayers, probably via hydrogen bindings of hydroxyls of the sugar with the head group of the phosphol i p i d ~ .Apparently, ~~ this interaction does not influence the hydrolysis rate of phospholipids in aqueous liposome dispersions. Mixing DPPC with Triton X-100 results in a micellar solution. Kensil and Dennis observed that both EPC and DPPC hydrolyzed faster in the presence of Triton X-100 at pH 12.7 and a t 40 “C.’ However, this observation was not confirmed in this study. On the contrary, the kobs of DPPC decreased upon mixing with Triton X-100 (see Table 2). At the moment, one can only speculate about the reason for the discrepancy. Influence of Head Group, Charge and Size-To analyze the effect of the head group on the kobs of liposomal phospholipids, liposomes were prepared with different (phospho)lipid compositions as shown in Table 3. Attempts to prepare 1116 /Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
liposomes consisting of only DPPE failed, because irregular aggregates were observed visually following the preparation procedure as described in Material and Methods. The results of the hydrolysis experiments a t pH 4.0 and 30 “C are shown in Table 3 and in Figure 3. The kobs of DPPC in pure DPPC liposomes did not differ from the hobs of DPPC in the uncharged DPPCDPPE 10/1 liposomes. The kobs of liposomal DPPE was only slightly smaller than the kob, of DPPC in the DPPCDPPE 10/1 liposomes. However, under the chosen conditions, addition of DPPG to DPPC liposomes dramatically increased both the kobs of DPPC and the hobs of DPPG. Interestingly, the hobsof DPPG in the liposomes composed of mixtures of DPPC and DPPG was always slightly larger than the hobsof liposomal DPPC. At high liposomal DPPG content in these liposomes deviation from pseudo-first-order kinetics was observed (see Figure 3). Initially, the kobs is faster than in later stages of the experiment, when more hydrolysis products are present. This might be due to the gradual reduction of negative charge density from the liposomal bilayers. Hydrolysis of liposomal phospholipids is highly charge dependent (see below). Hydrolysis of DPPG resulted in formation of uncharged PA and negatively charged lysophosphatidylglycerol, both of which remain associated with the lipid bilayer. This will not change the charge on the surface. However, further hydrolysis of LPG will decrease the negative charge density on the surface, because now not only the lipophilic PA is formed but also the water soluble, negatively charged degradation product glycerophospatidylglycerol. In this study, the influence of this hypothezised surface charge density change is minimized by obtaining the value for hobsfrom the data where hydrolysis is less than 50%. Incorporation of CHOL into the liposomes resulted in a minor decrease of both the kobs of DPPC and the kob? of DPPG (compare the results of DPPCDPPG 10/1liposomes with those composed of DPPC/DPPG/CHOL 10/1/4). Only charged DPPC/ DPPG 10/1 liposomes were sized to 0.19 pm (pd f 0.15) and hydrolyzed t o determine if the degradation process was influenced by size as we found upon y i r r a d i a t i ~ n .This ~ ~ was not the case; tbe kobs of DPPC and DPPG in the multilamellar, nonsized liposomes was the same as for the sized liposomes (see Table 3). Upon hydrolyzing liposomes composed of partially hydrogenated EPC and “natural” EPG, Grit and Crommelin showed
Table 3 - b s of Phospholipids in Charged Liposomes in 50 mM Acetate Buffer (pH 4.0) and 0.12 M NaCl at 30 "Cf
kbs(10-*
Liposome Composition and Phospholipid Concentration
DPPC
days-') DPPG or DPPE
*
1.o 0.2 2.1 f O . l 2.2 0.1 1.8 f 0.1 8.4 ? 0.7 8.4? 0.1
20 mM DPPC 22 mM DPPC/DPPG 10/1 22 mM DPPClDPPG 10/1 (sized)a 30 mM DPPC/DPPG/CHOL ION4 30 mM DPPC/DPPG 40 mM DPPUDPPG 10/lOb 20 mM DPPGb 22 mM DPPC/DCP 1O/Ic 22 mM DPPC/DPPE 10/1 22 mM DPPClSA 10/1
*
3.0 ? 0.2 1.o i 0.1 0.35 i0.04
2.6 i0.1 2.8 f 0.3 2.3 ? 0.1 9.0 0.8 9.9 f 0.5 17f2
+
0.7 f 0.1
ael (C/m2)
ueff(C/m2)
0 -0.028 -0.028 -0.023 -0.105 -0.160 -0.334 -0.029 0 0.029
0 -0.023 -0.023 -0.01 9 -0.058 -0.073 -0.271 -0.029 0 0.029
pH at bilayer surfacee 0 -0.027 -0.027 -0.023 -0.059 -0.069 -0.134 -0.033 0 0.034
4.0 3.6 3.6 3.6 3.0 2.9 1.8 3.5 4.0 4.6
~____
The size of these liposomes was 0.19 p m with a pd of about 0.15. Other liposomes were nonsized. *The kb~ of these I F o m e s with a relatively high DPPG content has been derived from the values above 50% hydrolysis. Also see the text. DCP and SA were analyzed by the HPLC method used and did not degrade in the experimental time period. Hauser et a P and Maitani et a/?' reported an apparent pK, for DCP in EPC of 4.7 and 4.5, respectively. In the present study we did not find any indication for the protonation of DCP. dThe buffer used to maintain the pH at 4.0 at 30 "C consisted of 39 mM acetic acid, 11 mM sodium acetate, and 0.12 M sodium chloride. Therefore, the values used in the calculations of Y and aenwere 0.131 for ionic strength, 0.131 M for "a+], and 8.3 x m for 1.eThe surface pH can be calculated by using the Boltzmann equation for a given p~tential:~'4.0 + ApH = 4.0 + (eY)/(2.303kT). Experimental data represent the mean f SD for three determinations. a
as
0
10
20
Here kobs(y=")is the kobs of phospholipids in uncharged liposomes, z is the valency of the counterion (i.e. +1for hydrolysis catalyzed by protons), e is the electron charge, k is the Boltzmann constant, and T is the absolute temperature. Y can be calculated according to the Gouy-Chapman-Stern mode129+30 and is given by
30 40 50 Time (days)
Figure 3-Typical example of the disappearance of the phospholipids of DPPC and DPPG liposomes and of liposomes composed of different molar ratios of DPPC and DPPG (lO/i, 10/5, or 10/10) in 50 mM acetate buffer (pH 4.0) and 0.12 M NaCl at 30 "C. In the case of hydrolysis of liposomes composed of only DPPC, more determinations were made as indicated by the extrapolation beyond the last experimental determinations. However, these determinations are not shown here so the other curves can be seen properly. Key: A, 100% DPPC; 0, DPPC in DPPClDPPG lO/l; 0 ,DPPG in DPPCiDPPG 1011; 0 ,DPPC in DPPClDPPG lo/$ I, DPPG in DPPC/ DPPG 10/5; 0,DPPC in DPPC/DPPG 10/10; DPPG in DPPC/DPPG 10/10; A, 100% DPPG.
+,
that the kobs was a function of the surface pH and not of the bulk P H . ~This surface pH deviation from bulk pH depended on surface charge density and composition of the aqueous medium. To confirm that this phenomenon is indeed surface charge dependent, in the present study hydrolysis experiments were also done with positively charged DPPC/stearylamine (SA) 10/1 liposomes. Indeed, the k& of DPPC decreased in the presence of SA (see Table 3). A similar influence of surface charge on hydrolysis was also described by Davies and Rideal upon hydrolyzing monolayers of monocetyl succinate ions (negatively charged) and upon hydrolyzing monolayers of octadecylacetate or cholesterol formate, both mixed with a long-chain quaternary amine (positively charged).28 Davies and Rideal used the Boltzmann relationship between the bulk and surface concentrations of ions for a given surface potential (Y) to describe the rate equation for proton- or hydroxyl-catalyzed hydrolysis. Similar equations can be used in this study to describe the influence of charge on hydrolysis of phospholipids in liposomes. At nonneutral pH (where the (noncatalyzed) hydrolysis in water only can be neglected) the pseudo-first-order rate equation can be rearranged to
In(% remaining) = -kobs(y=O) exp(-ze@/kT)t
(1)
Y = 2(kT/ze) sinh-l(zeuA/2c0c$T)
(3)
where G is the surface charge density, k is the Debye screening length, EO is the relative permittivity of free space, and E~ is the dielectric constant of water. The value of er does not have a constant value at the waterAipid interface (see elsewhere for a detailed discussion31). Here a value of 76 for cr has been used. G values of the liposomes were calculated taking a molecular surface area of 0.52 nm2 for DPPC,32of 0.48nm2 for DPPG,33of 0.30 nm2 for CHOL (deduced from refs 34 and 35)of 0.25 nm2 for SA (assumption) and of 0.40nm2for DCP.26 k is given by
A = (coc&T/Ne2~cciz,2)1i2
(4)
where N is the Avagadro constant and c; is the concentration of all individual ions (in mol/m3). Not only protons (in the case of negatively charged layers) or hydroxyl ions (in the case of positively charged layers) are attracted to the surface but also other positive or negative ions (in this study Na' or C1and negatively charged acetate ions, respectively). Therefore, the surface potential must be corrected (then called effective surface potential) by calculating first the effective surface charge density (u,~). To account for the effect of ion binding, the following equation, derived by combining the equation for the Langmuir isotherm and the Boltzmann equation, was ~sed29,30,36
oeff= 4 1 + Kc expke@lkT)l where K is the ion-lipid binding constant for the cation or anion and here c is the concentration of the individual interacting cation or anion. The solutions to eqs 3 and 5 are found iteratively. For the calculations, a value of 6.5 mol/m3 for K of Na+-DPPG was taken.29 The K of Na+-DCP and Journal of Pharmaceutical Sciences / 1117 Vol. 84, No. 9, September 1995
-0.15
-0 1
-0.05 YJ
0
0.05
(V)
DPPC and only slightly larger than the kobs for DPPE (see Table 3). It was also found that DPPG always hydrolyzes slightly faster than DPPC in liposomes composed of both phospholipids, which might be an indication for local inhomogeneities in terms of surface pH and ionic distributions a t the bilayer surface (see Table 3) and that DPPC hydrolyzes slightly faster than DPPE in liposomes composed of both phospholipids. From these considerations, we conclude that the influence of the phospholipid head group on kobs is small if corrections are made for surface charge effects. Under the chosen conditions, there is a tendency toward faster degradation in the order DPPE < DPPC < DPPG.
Figure 4-The values for kbsof liposomal DPPC (0)and DPPG (A) from Table 3 plotted against Y o f liposome dispersions stored in 50 mM acetate buffer (pH 4.0) and 0.12 M NaCl at 30 "C.
the K of the anions C1- and acetate ions binding to stearylamine in liposomes are not known, to our knowledge. Therefore, the calculations mentioned above were done without correcting for these ion-lipid interactions (see Table 3). No unusual results were found. In accordance with theory, Figure 4 shows linear relationships (r > 0.97) when the logarithmic of the kobs values of liposomal DPPC and DPPG from Table 3 are plotted against Y. Only the kobs of DPPG in pure liposomal DPPG deviates from the straight line. This might be explained by the low value for the surface pH (1.8, see Table 3) which is almost similar to the intrinsic pK, of the phosphate group of DPPG, 1.6-1.7.37 Under those conditions DPPG is no longer fully deprotonated and this affects cr,~,and thus Y, dramatically. Consequently, the values for the kobs of DPPG in pure liposomal DPPG have been left out of the calculations below. According to eq 2, the slopes of the lines in Figure 4 should be equal to 16.6 V-l [=e/(kT ln(10))l. However, the slopes of the experimental lines are different: 14.1 f 0.4 V-' for DPPC and 14.3 f 0.8 V-l for DPPG (mean f SE). This deviation might be the result of overestimation of the calculated Y of liposomes when using the Gouy-Chapman-Stern model (see elsewhere for detailed discussion^^^^^^^^^). Another reason for the deviation observed in Figure 4 might be the fact that hydrolysis of liposomal phospholipids does not follow exactly the rate-pH profile of a general acid-base reaction. It was found before that the hydrolysis rate of phospholipids reached a minimum a t pH 6.5; a V-shaped hydrolysis profile was obtained upon plotting the logarithmic values of kobs against the pH.2,3,5-9Theoretically, the acidic branch of a V-shaped hydrolysis profile of a general acid-base reaction should have a slope of -l.0,39 but experimentally lower values were obtained (between about -0.5 and -0.82,3,5-9).One explanation for such a deviation might be catalysis by buffer compon e n t ~ However, .~~ even after correction for buffer effects, the slope of the acidic branch of the hydrolysis profile for liposomal phospholipids still differs from -1.0 (about -0.73). The reason for this phenomenon is unknown. In the present study, the value for the slope of the acidic branch of the hydrolysis profile for liposomal DPPC was -0.62 f 0.07 (mean f SE) as determined after hydrolysis of DPPC liposomes in acetate buffer and NaCl a t pH 3.5, 4.0, or 4.5 and a t 30 "C. Interestingly, when the logarithmic values of kobsof DPPC in charged liposomes were plotted against the calculated surface pH, a slope of -0.85 & 0.03 (mean f SE) was found, which was statistically different from the -0.62 slope mentioned above (p = 0.001). We did not further pursue this discrepancy between theory and experimental observations. From the lines correlating the logarithmic values of kobs of liposomal DPPG with Y (see Figure 41, it was calculated that the kobs of (theoretically) uncharged DPPG (Y = 0 V) a t pH f 0.1 x 4.0 and 30 "C was 1.1 x days-' (mean f SE). This value is not significantly different from the kobsfor 1118 / Journal of Pharmaceutical Sciences
Val. 84, No. 9, September 1995
References and Notes 1. Kensil, C. R.; Dennis, E. A. Biochemistry 1981,20,6079-6085. 2. Frokjaer, S.; Hjorth, E. L.; Worts, 0. In Optimization of Drug Deliuery; Bundgaard, H.; Bagger Hansen, A.; Kofod, H., Eds.; Munkseaard: Cooenhaeen. 1982: DD 384-404. 3. Grit, M ;: De Smidt, J . H ; Struijke,-A.; Crommelin, D. J . A. Int. J . Pharm. 1989, 50, 1-6. 4. Grit, M.; Crommelin, D. J. A. Chem. Phys. Lipids 1992,62,113,no
LA&.
,
5. Grit, M.; Crommelin, D. J. A. Biochim. Biophys. Acta 1993,1167, 49-55. 6. Grit, M.; Zuidam, N. J.; Underberg, W. J . M.; Crommelin, D. J . A. J. Pharm. Pharmacol. 1993, 45, 490-495. 7. Grit, M.; Underberg, W. J. M.; Crommelin, D. J. A. J . Pharm. Sci. 1993, 82 (41, 362-366. 8. Grit, M.: Zuidam, N. J.; Crommelin, D. J . A. In Liposome Technology, 2nd ed.; Gregoriadis, G., Ed.; CRS Press, Inc.: Boca Raton, FL, 1993; Vol I, pp 455-487. 9. Grit, M.; Crommelin, D. J . A. Chem. Phys. Lipids 1993,64,3-18. 10. Deuel, H. J. In The Lipids, their Chemistry and Biochemistry Volume I: Chemistry; Interscience Publisher: New York, 1951; pp 408-439. 11. Hanahan, D. J . In Lipide Chemistry; John Wiley & Sons: New York, 1960; chapter 3. 12. Grit, M.; Crommelin, D. J . A.; Lang, J. K. J . Chrornatogr. 1991, 585, 239--246. 13. Bligh, E. G.; Dyer, W. J. Can. J . Biochem. Physiol. 1959,37 (8 ), 911-917. 14. Schurtenberger, P., Hauser, H. In Liposorne Technology, 2nd ed.; Gregoriadis, G., Ed.; CRS Press, Inc.: Boca Raton, FL, 1993; Vol. I, pp 253-270. 15. Armitage, P.; Berry, G. Statistical Methods in Medical Research; 2nd ed.; Blackwell Scientific Publications: Oxford, Great Britain, 1991. 16. Cevc, G. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker, h c . : New York, 1993; pp 939-956. 17. Singer, M. A.; Finegold, L. Biophys. J . 1990, 57, 153-156. 18. Scherer, J. R. Biophys. J. 1989,55, 957-964. 19. Tausk, R. J . M.; Karmiggelt, J.; Oudshoorn, C.; Overbeek, J. Th.G. Biophys. Chem. 1974,1, 175-183. 20. Villalain, J.; Aranda, J . A.; G6mez-Fernandez, J. C. Eur. J . Biochern. 1986,158, 141-147. 21. Hernandez-Caselles, T.; Villalain, J.; Gomez-Fernandez, J . C. J . Pharm. Pharmacol. 1990,42, 397-400. 22. Schullery, S. E.; Seder, T. A.; Weinstein, D. A.; Bryant, D. A. Biochemistry 1981,20, 6818-6824. 23. Van Echteld, C. J. A.; De Kruijff, B.; De Gier, J. Biochim. Biophys. Acta 1980, 595, 71-81. 24. Crowe, J. H.; Crowe, L. M. In Liposome Technology, 2nd ed.; Gregoriadis, G., Ed.; CRC Press, Inc.: Boca Raton, FL, 1993; pp 229-252. 25. Zuidam, N. J . In Stability of liposomes. Chemical and physical characterisation of liposomes upon autoclaving, gamma-irradiation and storage; Ph.D. Thesis; Utrecht University, The Netherlands, 1994. 26. Hauser, H.; Darke, A,; Phillips, M. C. Eur. J. Biochem. 1976, 62. 335-344. 27. Maitani, Y.; Nagasaki, M.; Nagai, T. Int. J . Pharm. 1990, 64, 89-98. 28. Davies, J. T.; Rideal, E. K. In Znterfacial phenomena; Academic Press: New York, 1961; Chapter 6. 29. Cevc, G. Biochim. Biophys. Acta 1990,1031, 311-382. 30. Cevc, G. Chem. Phys. Lipids 1993, 64, 163-186. 31. Tocanne, J. F.; TeissiB, J . Biochim. Biophys. Acta 1990, 1031, 3 -1 -1 -1 -1 - 6_-. 32. Cevc, G.; Seddon, J. M.; Marsh, D. Faraday Discuss. Chem. SOC. 1986, 81, 179-189.
33. Watts, A., Harlos, K.; Marsh, D. Biochim. Biophys. Acta 1981, 645,91-96. 34. Rand, R. P. Annu. Rev. Biophys. Bioeng. 1981, 10, 277-314. 35. Cruzeiro-Hansson, L.; Ipsen, J. H.; Mouritsen, 0. G. Biochim. Biophys. Acta 1989,979, 166-176. 36. Nir, S.; Bentz, J. J. Colloid Interface Sci. 1978, 65, 399-414. 37. Tatulian, S. A. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker, Inc.: New York, 1993; pp 511-552. 38. Cevc, G.; Watts, A.; Marsh, D. FEBS Lett. 1980, 120 (21, 267270. 39. Martin, A.; Swarbrick, J.; Cammarata, A. Physical Pharmacy; Lea & Febiger: Philadelphia, PA, 1983; pp 380-381.
Acknowledgments We gratefully acknowledge the financial support provided by Bayer (Leverkusen, FRG) and the gifts of phospholipids by Nattermann Phospholipid and Lipoid K.G. We also would like to thank Prof. Dr. G. Cevc (Technical University of Munchen, FRG) for his help with the Gouy-Chapman-Stern calculations, Dr. W. J. M. Underberg (Analytical Department of our faculty) for stimulating discussions and Dr. H. J. A. Wijnne (Centre for Biostatistics of our university) for advice on the statistical methods used in this study. JS940647F
Journal of Pharmaceutical Sciences / 1119 Vol. 84, No. 9, September 1995
kinetics of liposomal phospholipids.6 Until now, no systematic study has been performed on the effect of chain length and head group on the hydrolysis of phospholipids in liposome dispersion^.^ Also the effect of the organization of the phospholipid state (bilayers, micelles, gel phase, liquid phase, etc.) on the hydrolysis process is still unclear. Frakjaer et al. found a higher activation energy in distearoylphosphatidylcholine (DSPC) liposomes at pH 6.5 for bilayers in the gel state than in the liquid crystalline state.3 However, other studies could not confirm this ~ b s e r v a t i o n .Furthermore, ~~~ possible effects of different types of water soluble and lipophilic additives t o liposome dispersions on the hydrolysis kinetics of phospholipids have never been investigated in an appropriate way, except for those few referred to above. Therefore, the objectives of this study are to assess in more detail factors which influence hydrolysis kinetics of phospholipids. Liposomes composed of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine(DPPC), DSPC, egg phosphatidylcholine (EPC), or DPPC/cholesterol (CHOL) 10/4 (molar ratio) were stored at pH 4.0 and a t different temperatures and their hydrolysis kinetics were monitored. Moreover, to obtain additional information on the effect of chain length on the hydrolysis kinetics of PC, hydrolysis of dicaproylphosphatidylcholine (DCPC) and dilauroylphosphatidylcholine (DLPC) a t pH 4.0and 30 "C was determined. As it is known that additives can affect the bilayer characteristics, experiments were also performed to analyze the influence of additives on the hydrolysis kinetics of DPPC a t pH 4.0 and a t 30 "C, a temperature just below the pretransition of DPPC bilayers. The pH of 4.0 was chosen, because the hydrolysis rate of liposomal phospholipids is relatively fast and because fatty acids and lysophosphatidylcholine (the major degradation products) and phosphatidylcholine are uncharged at this PH.
Experimental Section
Introduction Insight into the chemical hydrolytic process of liposomal phospholipids is important for the design of liposomal dispersions to be used as drug carriers. In an aqueous liposome dispersion, the liposomal phospholipids can hydrolyze to free fatty acids and 8-acyl and 1-acyl lysoph~spholipids.~-~ Further hydrolysis of both lysophospholipids results in glycerophospho compounds. Glycerophosphorate acid is the usual end product after further, relatively slow degradation of the ester bond between the phosphate and the head group of the glycerophospho compound.lOJ1 The hydrolysis of liposomal phospholipids is catalyzed by protons and hydroxyl ions and reaches a minimum at pH 6.5; a V-shaped pH profile for the hydrolysis process is obtained upon plotting the logarithmic of the hydrolysis rate constants against the pH.2,3,5-9 The hydrolysis rate of the phospholipids is not only influenced by pH, but also by temperature (following Arrhenius kinetic~l-3+-~), charge of the lipo~omes,~ and buffer specie^.^,^^^ Cholesterol and ionic strength do not affect the hydrolysis @
Abstract published in Advance ACS Abstracts, July 1, 1995.
0 1995, American Chemical Society and American Pharmaceutical Association
Materials-DCPC, DLPC, DMPC, DPPC, DSPC, and dipalmitoylphosphatidylglycerol (DPPG) were gifts from Nattermann Phospholipid (Cologne,FRG). EPC and dipalmitoylphosphatidylethanolamine (DPPE) were gifts from Lipoid K. G. (Ludwigshafen, FRG). According to the manufactures the purity of the phospholipids was a t least 99%. CHOL ('99% pure), palmitic acid (PA, purity about 99%), dicetyl phosphate (DCP, > 99% pure), and trehalose were purchased from Sigma (St. Louis, MO). Monopalmitoylphosphatidylcholine (LPC, >99% pure) was obtained from Avanti Polar Lipids (Pelham, AL). Stearylamine (SA, '99% pure) was purchased from Janssen Chimica (Beerse, Belgium). a-Tocopherol (> 98% pure) was obtained from Fluka (Buchs, Switzerland). Sucrose and glucose were obtained from Merck (Darmstadt, FRG). Propylene glycol was purchased from OPG (Utrecht, The Netherlands). Triton X-100 was obtained from BDH (Poole, England). These and all other chemicals were of analytical grade. The water was doubly distilled before use. Preparation of Liposome Dispersions-Liposomes were prepared by the "film" method. Appropriate mixtures of the (phospho)lipids were dissolved in chloroforndmethanol(1:l)in a round-bottom flask. The organic solvent was removed under vacuum by rotary evaporation. The thin film obtained was dried for a t least 3 h under reduced pressure. Then, the film was hydrated with 50 mM acetate buffer (pH 4.0) and 0.12 M sodium chloride. Most of the time, the
0022-3549/953184-1 1 13$09.00/0
Journal of Pharmaceutical Sciences / 1I13 Vol. 84, No. 9, September 1995
liposomes used were unextruded. If necessary, the liposome dispersions were sized with an extrusion system (Sartorius, Gdttingen, FRG) once through 0.6 pm and three times through 0.2 pm pore size filters (Nuclepore, Costar Corp., Cambridge, MA). At the storage temperature the pH of the dispersion was measured and adjusted before and after extrusion, if necessary. The liposome dispersions were stabilized in the refrigerator overnight. Then, the pH of the dispersion was measured again at the storage temperature and adjusted, if necessary. The final phospholipid concentration was about 20 mM. Hydrolysis Experiments-The prepared liposome dispersions were filled into ampules under a nitrogen atmosphere and sealed. Ampules were stored in a constant temperature water bath (15-80 "C) or in a constant temperature room (4"C). Samples ( n 2 6) were taken after appropriate time intervals and stored in the freezer (-20 "C) until analysis. Analytical Methods-Phospholipids were analyzed by HPLC as described earlier.12 Samples for the HPLC analysis were prepared by the Bligh and Dyer e ~ t r a c t i 0 n . lThe ~ phospholipids were collected in the chloroform phase. After dilution of the chloroform phase in methanol, aliquots of 100 pl were directly injected into the column. The HPLC system consisted of a type 400 solvent delivery system (Kratos, Ramsey, NJ), a Kontron sampler MSI 660 (Kontron AG, Zurich, Switzerland), and a Waters 410 RI detector (Waters Associates, Milford, MA). Peak areas were measured with a computer controlled integrator-based data system (WOW, Thermo Separation Products, Fremont, CA). The separation of the phospholipids was carried out on a Zorbax aminophase column (25 cm x 4.6 mm, i.d., 5 pm particle size, Du Pont Co., (Wilmington, DE) a t 35 "C. An Adsorbosphere NH2 5 pm guard column (Alltech Associates, Deerfield, IL) was connected before the Zorbax aminophase column. The mobile phase consisted of a mixture of acetonitrile/methanoY5 mM ammonium dihydrogen phosphate solution, pH 4.8. Because of columnto-column variations, the ratio of the solvents used in the mobile phase was different for the different columns used. A typical example of a mobile phase composition used is 57/38/4 (v/v). The flow rate was 1.5 mllmin. The 2-average particle size and polydispersity index (pd) a t 25 "C were determined by dynamic light scattering with a Malvern 4700 system using a 25 mW He-Ne laser (NEC Corp., Tokyo, Japan) and the automeasure version 3.2 software (Malvern Ltd, Malvern, UK). See elsewhere for further details.14 For viscosity and refractive index, the values of pure water were used. The pd is a measure of the width of the particle size distribution and ranges from 0.0 for a n entirely homogeneous size distribution to 1.0 for a completely heterogeneous one. Statistics-Significance tests on a mean were performed by using the Student t-test assuming equal variances (two-sided). Differences in slopes were compared by using the test for homogeneity of the lines. Differences in intercepts were tested by using Analysis of Covariance (ANCOVA). The value for p is given or else a value of 0.05 for a was assumed in all the tests. See elsewhere for details about the statistical tests.l5
Results and Discussion Order of Reaction and Influence of Temperature and Organization of the Phospholipid Assembly-The disappearance of phospholipids in buffered dispersions follows pseudo-first-order In this study this observation was confirmed for most of the phospholipids. As an example, semilogarithmic plots for the hydrolysis of DPPC in acetate buffer (pH 4.0) a t different temperatures are shown in Figure 1. From such straight lines, observed pseudo-first-order rate constants ( k o b s ) were obtained. The kobs is the sum of the firstorder rate constant for the hydrolysis in water only (ko) and of the second-order rate constants for proton (kH+), hydroxyl ion ( k O H - ) and buffer component (kbuffer) catalyzed hydrolysis, multiplied by the concentrations of these specific compounds. The effect of temperature on the k o b s can be described by the Arrhenius equation.1-3,6-9 In this study, the validity of Arrhenius kinetics was investigated for the hydrolysis of phospholipids in nonsized liposomes composed of DMPC, DPPC, DSPC, EPC, or DPPC/ 1114 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
I00
170ic
i5O.c
- - -o l 0
100
50 Time (days)
Figure 1-Typical examples of semilogarithmic, apparent first-order plots for the hydrolysis of DPPC in 50 mM acetate buffer (pH 4.0) and 0.12 M NaCl at the indicated temperatures. More determinations (n = 7-8) were made at 4 and 20 "C,as indicated by the extrapolation beyond the last experimental determinations. However, these determinations are not shown here so the curves at the higher temperatures can be seen properly.
CHOL 10/4 (molar ratio). These liposomes were stored a t pH 4.0 in the temperature range from 4 to 80 "C to determine the effect of the organization of the phospholipid assembly (gel phase, liquid phase, and condensing effect of cholesterol) and chain length on the hydrolysis kinetics of the phospholipids. The results are shown in Figure 2 and in Table 1.The hydrolysis of liposomal EPC could be adequately described by an Arrhenius curve without breaks (see Figure 2E and Table 1). A break in the Arrhenius curve was expected when the liposome dispersion contained a gel-to-liquidphase transition in the experimental range (in the case of DMPC, DPPC, and DSPC liposomes, see Table 1 and elsewherei6 for further information), because the effect of temperature on the hydrolysis of phospholipids with such a phase transition has been described by biphasic Arrhenius curves b e f ~ r e . ~In ,~,~ this study statistical tests were performed to analyze if such breaks could be statistically proven and if differences in slopes describing hydrolysis kinetics of phospholipids above and below T , were statistically significant as well. Theoretically, one straight line was fitted through data points above the T, and another line was fitted through points below the T,. Both the slopes and the intercepts of these lines were compared by using the test for the homogeneity of the slopes and by using analysis of covariance (ANCOVA), respectively. A break in the Arrhenius curve will a t least result in a difference in intercepts. The results of the statistical tests are also shown in Table 1. No break in the Arrhenius curve describing the hydrolysis kinetics for liposomal DMPC was observed. However, breaks in the Arrhenius curves for both DPPC and DSPC occurred, because the slopes of the lines above and below the T, were statistically different. Thus, the activation energy (E,) in the fluid and E , in the gel phase were different for both DPPC and DSPC liposomes. The kobs of DSPC liposomes a t 50 "C deviated from the biphasic Arrhenius plot (see Figure 2C). This temperature is just above the temperature of the pretransition of DSPC liposomes, 49 'C.I6 The slope of the line calculated by linear regression through the k o b s values at 4, 20, 30 and 50 "C and the slope through these k o b s values without the 50 "C value are statistically different (p = 0.027). Therefore, the Arrhenius curve has been fitted without the kobs values a t 50 "C (see Figure 2C and Table 1). Cholesterol has a condensing effect on DPPC liposomes, resulting in a disappearance of the phase transitions of DPPC in the experimental range above 27 mol%.17 As expected, no break in the Arrhenius curve for the hydrolysis of DPPC in DPPC/ CHOL 10/4 liposomes occurred (no difference in the slopes of the lines above and below T, of DPPC was observed). However, the homogeneity of the intercepts of these two lines is not clearly significant; the p-value is relatively low (0.046). A difference in intercepts is theoretically hard to explain.
DMPC
loo
DPPC
10
A
70 60 50 I
0.0028
1
I
I
30
20
1
I(
0.0032
B
70 60 50
4'C
;
0.0028
0.0036 l/T (1K)
30
0.0032
4 C
20
lo4 0.0028
0.0036
70 60 50
i
0.0028
'
30 I
'
20
0.0036 1IT (1K)
EPC
E
D
'
0.0032
1/T (1iK)
DPPCICHOL 1014
loo
7
'o-j8; '0 70 30
4 T
70 60 50
30 20
4°C
1'
0.0032
0.0036 1/T (1IK)
0.0028
0.0032
0.0036 1/T (1K)
Figure 2-Arrhenius plots describing the effect of temperature on the hydrolysis of liposomal DMPC (A), DPPC (B), DSPC (C), DPPCiCHOL 1014 (D), and EPC (E) in 50 mM acetate buffer (pH 4.0) and 0.12 M NaCI. Vertical bars indicate the SD of three determinations. When no bars are shown, the SD fell within symbol dimensions. Table 1-Results of the Arrhenius Curves for the Hydrolysis of Liposomal Phosphatidylcholine in 50 mM Acetate Buffer (pH 4.0) and 0.12 M NaCl As Shown in Figure 2a
p value Tm
Liposomes DMPC DPPC DSPC
("C)
Phase
Eaf SE A homogeneity analysis of (kJ/mol) (days-') of slopes covariance
+
24 Total range 61 1 3 x lo* 42 Above T, 67+3 3x lo9 Below Tm 91 + 3 5 x I O l 3 56 Above T,, 48+3 4 x lo6 Below T, 104+4 1 x 1OI6 c Total range 64+1 1 x109
DPPC/CHOL 10/4 EPC -10 Total range
0.391 0.011
0.686 d
0.000
d
0.292
0.046
61 k 2 3 x lo8
aThe statistical analysis is based on at least nine (3 x 3) independent kbs values above the gel-to-liquid phase transition temperature ( Tm)or below Tm. "Total range" implies that no break in the Arrhenius curve was observed around T,. €, is the activation energy and A is the frequency factor. bThe values of Tm were obtained from the Phospholipids Handbook.16 CAbove 27 mol % cholesterol liposomes composed of DPPClCHOLdo not contain a gel-to-liquidphase transition anymore.I7 If the slopes are inhomogeneous, the analysis of covariance cannot be performed anymore.
The E, of DMPC, DPPCKHOL 1014, and EPC liposomes did not differ from the E , of DPPC and DSPC liposomes in the fluid state, as was determined by testing the homogeneity of the slopes (p = 0.277). However, the small differences in intercepts of these lines were statistically significant (ANCOVA, p = 0.000). The differences in E , of DMPC, DPPC/ CHOL 10/4, and EPC liposomes and in E, of DPPC and DSPC liposomes in the gel state were significant ( p = 0.026). Thus, for bilayers in the fluid state or when CHOL is present in the bilayer, the E, for the hydrolysis of all phospholipids studied is the same. However, when the DPPC and DSPC bilayers convert in the gel state a t lower temperatures, the E, for the hydrolysis increases in the following order: DMPC % EPC % DPPCKHOL 10/4 < DPPC < DSPC. Apparently, the packing of the phospholipids has an influence on the hydrolysis kinetics. It has been reported that the
boundary between the hydrophobic and hydrophilic part in phospholipid bilayers is located at the first CH2 group in the hydrocarbon chains.18It has also been shown that the degree of hydration is dependent on the level of acyl chain unsaturation and that lengthening of the acyl chain length results in tighter packing.18 One might suggest that these intrabilayer forces among the hydrocarbon chains (such as Van der Waals forces) affect the hydrolysis kinetics by providing more effective steric hindrance to the approach of protons t o the ester bonds of phospho1ipids.l Influence of Chain Length-dispersions of PC with different fatty acid chains were prepared and hydrolyzed at pH 4.0 and 30 "C. The results are shown in Table 2. All phospholipids formed bilayers, except DCPC, which is present as monomers and micelles (critical micelle concentration is 14 mM at neutral pH19). A tendency in kobs to increase was found upon lengthening of the acyl chain length. The reason for this is not known. Influence of the Neutral Additives a-Tocopherol, LPC, Palmitic Acid, Sodium Chloride, Cryoprotectants, and Triton X-100-In Table 2 the kobs of liposomes composed of DPPC and/or a-tocopherol a t pH 4.0 and 30 "C are shown. Villalain et al. suggested a n interaction between the hydroxyl group of a-tocopherol and the polar region of DPPC, but it was not fully clear whether this bond was between the phosphate group or with the carbonyl group of the DPPC molecule.20 The same group observed a small decrease in LPCPC ratio upon storage of liposomes composed of EPC and a-tocopherol a t pH 7.4 and a t 4 "C or ambient temperature, suggesting that a-tocopherol could decrease the kobs of liposoma1 phospholipids.21 In the present study, we could not confirm this observation and, therefore, do not provide support for the proposed interaction between a-tocopherol and the carbonyl group of DPPC. Apparently, the presence of both hydrolysis products lysophospholipids and fatty acids in the bilayer does not influence the kobs of liposomal phospholipids as indicated by the straight Journal of Pharmaceutical Sciences / 1115 Vof. 84, No. 9, September 1995
Table
2-kbs of PC in Neutral Liposomes or Micelles in 50 mM Acetate (pH 4.0) and 0.12 M NaCl at 30 ‘CB kobs
Parameter of Investigation Length of fatty acid chain
Effect of a-tocopherol Effect of hydrolysis products
Effect of sodium chloride Effect of cryoprotectant
Effect of Triton X-100
Composition of Liposomes
Remark
DPPC DCPC DLPC DMPC DSPC EPC DPPClatocopherol 10/2 DPPCia-tocopherol 10/4 DPPClLPC 1/1 DPPC/PA 1011 DPPC/PA 2/1 DPPClPA 1/1 DPPC without sodium chloride DPPC + 10% glucoseb DPPC + 10% trehaloseb DPPC + 10% sucroseb DPPC + 10% propylene glycolb DPPCnriton X-100 111 DPPCnriton X-100 1/8
diCls-PC dlC6-PC diCip-PC diC14-PC diCls-PC diC14-22-PC
(10-2days-1) 1.0io.2 0.7 i0.1 0.8i0.1 1.O ? 0.3 1.1 * O . l 0.8 If- 0.1 1.2 i 0.1 1.1 k 0.2 1.o i0.1 0.9 If- 0.1 0.9 i0.1 0.6 k 0.1 1.o i0.2 0.9 i0.1
l.oio.l
1.1 i o . l l.oio.l 0.9 i0.1c 0.07 k 0.06d
a Experimental data represent the mean k SD of at least three determinations. The concentration of PC was 20 mM. The liposornes were nonsized. Ir The 10% cryoprotectants are in weightlvolume. After 45% hydrolysis, the hydrolysis rate decreased suddenly. T’he hydrolysis rate shown in this table has been derived from the values before this deviation. See also the text. Hardly any decrease was found in the experimental time period. Therefore the SD is relatively large here.
lines (see Figure 1). To examine if this phenomenon is a combined, counteracting effect of both hydrolysis products or not, DPPC liposomes were made in the presence of a n equal molar ratio of monopalmitoylphosphatidylcholine (LPC) or different molar ratios of palmitic acid (PA) and stored at pH 4.0 and 30 “C. The addition of PA in DPPC bilayers in a n acidic environment leads to disappearance of the pretransition and subsequently broadening and shifting to higher temperatures (50-60 “C) of the main phase transition.2z The addition of 50 mol % LPC to DPPC results in almost complete formation of micelles instead of liposomes, while the main phase transition is hardly affected.23 The results of the hydrolysis experiments are shown in Table 2. No effect of LPC on kobs of liposomal DPPC was observed. However, a small effect on kobs of liposomal DPPC was found upon mixing with PA. It was only statistically significant for a molar ratio of 1/1, but not in a molar ratio of 10/1 or 2/1. Both hydrolysis products alone do not or only slightly influence the hydrolysis rate of DPPC and that explains why their presence does not affect the kobs of liposomal phospholipids. Studies were also done to analyze the effect of sodium chloride, cryoprotectants (glucose, trehalose, sucrose, and propylene glycol) and the detergent Triton X-100 on the hydrolysis of liposomal DPPC a t pH 4.0 and at 30 C. The results are shown in Table 2. Sodium chloride (as reported before”*)and cryoprotectants did not affect the hydrolysis rate of liposomal DPPC. It has been reported that sugars interact with liposomal bilayers, probably via hydrogen bindings of hydroxyls of the sugar with the head group of the phosphol i p i d ~ .Apparently, ~~ this interaction does not influence the hydrolysis rate of phospholipids in aqueous liposome dispersions. Mixing DPPC with Triton X-100 results in a micellar solution. Kensil and Dennis observed that both EPC and DPPC hydrolyzed faster in the presence of Triton X-100 at pH 12.7 and a t 40 “C.’ However, this observation was not confirmed in this study. On the contrary, the kobs of DPPC decreased upon mixing with Triton X-100 (see Table 2). At the moment, one can only speculate about the reason for the discrepancy. Influence of Head Group, Charge and Size-To analyze the effect of the head group on the kobs of liposomal phospholipids, liposomes were prepared with different (phospho)lipid compositions as shown in Table 3. Attempts to prepare 1116 /Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
liposomes consisting of only DPPE failed, because irregular aggregates were observed visually following the preparation procedure as described in Material and Methods. The results of the hydrolysis experiments a t pH 4.0 and 30 “C are shown in Table 3 and in Figure 3. The kobs of DPPC in pure DPPC liposomes did not differ from the hobs of DPPC in the uncharged DPPCDPPE 10/1 liposomes. The kobs of liposomal DPPE was only slightly smaller than the kob, of DPPC in the DPPCDPPE 10/1 liposomes. However, under the chosen conditions, addition of DPPG to DPPC liposomes dramatically increased both the kobs of DPPC and the hobs of DPPG. Interestingly, the hobsof DPPG in the liposomes composed of mixtures of DPPC and DPPG was always slightly larger than the hobsof liposomal DPPC. At high liposomal DPPG content in these liposomes deviation from pseudo-first-order kinetics was observed (see Figure 3). Initially, the kobs is faster than in later stages of the experiment, when more hydrolysis products are present. This might be due to the gradual reduction of negative charge density from the liposomal bilayers. Hydrolysis of liposomal phospholipids is highly charge dependent (see below). Hydrolysis of DPPG resulted in formation of uncharged PA and negatively charged lysophosphatidylglycerol, both of which remain associated with the lipid bilayer. This will not change the charge on the surface. However, further hydrolysis of LPG will decrease the negative charge density on the surface, because now not only the lipophilic PA is formed but also the water soluble, negatively charged degradation product glycerophospatidylglycerol. In this study, the influence of this hypothezised surface charge density change is minimized by obtaining the value for hobsfrom the data where hydrolysis is less than 50%. Incorporation of CHOL into the liposomes resulted in a minor decrease of both the kobs of DPPC and the kob? of DPPG (compare the results of DPPCDPPG 10/1liposomes with those composed of DPPC/DPPG/CHOL 10/1/4). Only charged DPPC/ DPPG 10/1 liposomes were sized to 0.19 pm (pd f 0.15) and hydrolyzed t o determine if the degradation process was influenced by size as we found upon y i r r a d i a t i ~ n .This ~ ~ was not the case; tbe kobs of DPPC and DPPG in the multilamellar, nonsized liposomes was the same as for the sized liposomes (see Table 3). Upon hydrolyzing liposomes composed of partially hydrogenated EPC and “natural” EPG, Grit and Crommelin showed
Table 3 - b s of Phospholipids in Charged Liposomes in 50 mM Acetate Buffer (pH 4.0) and 0.12 M NaCl at 30 "Cf
kbs(10-*
Liposome Composition and Phospholipid Concentration
DPPC
days-') DPPG or DPPE
*
1.o 0.2 2.1 f O . l 2.2 0.1 1.8 f 0.1 8.4 ? 0.7 8.4? 0.1
20 mM DPPC 22 mM DPPC/DPPG 10/1 22 mM DPPClDPPG 10/1 (sized)a 30 mM DPPC/DPPG/CHOL ION4 30 mM DPPC/DPPG 40 mM DPPUDPPG 10/lOb 20 mM DPPGb 22 mM DPPC/DCP 1O/Ic 22 mM DPPC/DPPE 10/1 22 mM DPPClSA 10/1
*
3.0 ? 0.2 1.o i 0.1 0.35 i0.04
2.6 i0.1 2.8 f 0.3 2.3 ? 0.1 9.0 0.8 9.9 f 0.5 17f2
+
0.7 f 0.1
ael (C/m2)
ueff(C/m2)
0 -0.028 -0.028 -0.023 -0.105 -0.160 -0.334 -0.029 0 0.029
0 -0.023 -0.023 -0.01 9 -0.058 -0.073 -0.271 -0.029 0 0.029
pH at bilayer surfacee 0 -0.027 -0.027 -0.023 -0.059 -0.069 -0.134 -0.033 0 0.034
4.0 3.6 3.6 3.6 3.0 2.9 1.8 3.5 4.0 4.6
~____
The size of these liposomes was 0.19 p m with a pd of about 0.15. Other liposomes were nonsized. *The kb~ of these I F o m e s with a relatively high DPPG content has been derived from the values above 50% hydrolysis. Also see the text. DCP and SA were analyzed by the HPLC method used and did not degrade in the experimental time period. Hauser et a P and Maitani et a/?' reported an apparent pK, for DCP in EPC of 4.7 and 4.5, respectively. In the present study we did not find any indication for the protonation of DCP. dThe buffer used to maintain the pH at 4.0 at 30 "C consisted of 39 mM acetic acid, 11 mM sodium acetate, and 0.12 M sodium chloride. Therefore, the values used in the calculations of Y and aenwere 0.131 for ionic strength, 0.131 M for "a+], and 8.3 x m for 1.eThe surface pH can be calculated by using the Boltzmann equation for a given p~tential:~'4.0 + ApH = 4.0 + (eY)/(2.303kT). Experimental data represent the mean f SD for three determinations. a
as
0
10
20
Here kobs(y=")is the kobs of phospholipids in uncharged liposomes, z is the valency of the counterion (i.e. +1for hydrolysis catalyzed by protons), e is the electron charge, k is the Boltzmann constant, and T is the absolute temperature. Y can be calculated according to the Gouy-Chapman-Stern mode129+30 and is given by
30 40 50 Time (days)
Figure 3-Typical example of the disappearance of the phospholipids of DPPC and DPPG liposomes and of liposomes composed of different molar ratios of DPPC and DPPG (lO/i, 10/5, or 10/10) in 50 mM acetate buffer (pH 4.0) and 0.12 M NaCl at 30 "C. In the case of hydrolysis of liposomes composed of only DPPC, more determinations were made as indicated by the extrapolation beyond the last experimental determinations. However, these determinations are not shown here so the other curves can be seen properly. Key: A, 100% DPPC; 0, DPPC in DPPClDPPG lO/l; 0 ,DPPG in DPPCiDPPG 1011; 0 ,DPPC in DPPClDPPG lo/$ I, DPPG in DPPC/ DPPG 10/5; 0,DPPC in DPPC/DPPG 10/10; DPPG in DPPC/DPPG 10/10; A, 100% DPPG.
+,
that the kobs was a function of the surface pH and not of the bulk P H . ~This surface pH deviation from bulk pH depended on surface charge density and composition of the aqueous medium. To confirm that this phenomenon is indeed surface charge dependent, in the present study hydrolysis experiments were also done with positively charged DPPC/stearylamine (SA) 10/1 liposomes. Indeed, the k& of DPPC decreased in the presence of SA (see Table 3). A similar influence of surface charge on hydrolysis was also described by Davies and Rideal upon hydrolyzing monolayers of monocetyl succinate ions (negatively charged) and upon hydrolyzing monolayers of octadecylacetate or cholesterol formate, both mixed with a long-chain quaternary amine (positively charged).28 Davies and Rideal used the Boltzmann relationship between the bulk and surface concentrations of ions for a given surface potential (Y) to describe the rate equation for proton- or hydroxyl-catalyzed hydrolysis. Similar equations can be used in this study to describe the influence of charge on hydrolysis of phospholipids in liposomes. At nonneutral pH (where the (noncatalyzed) hydrolysis in water only can be neglected) the pseudo-first-order rate equation can be rearranged to
In(% remaining) = -kobs(y=O) exp(-ze@/kT)t
(1)
Y = 2(kT/ze) sinh-l(zeuA/2c0c$T)
(3)
where G is the surface charge density, k is the Debye screening length, EO is the relative permittivity of free space, and E~ is the dielectric constant of water. The value of er does not have a constant value at the waterAipid interface (see elsewhere for a detailed discussion31). Here a value of 76 for cr has been used. G values of the liposomes were calculated taking a molecular surface area of 0.52 nm2 for DPPC,32of 0.48nm2 for DPPG,33of 0.30 nm2 for CHOL (deduced from refs 34 and 35)of 0.25 nm2 for SA (assumption) and of 0.40nm2for DCP.26 k is given by
A = (coc&T/Ne2~cciz,2)1i2
(4)
where N is the Avagadro constant and c; is the concentration of all individual ions (in mol/m3). Not only protons (in the case of negatively charged layers) or hydroxyl ions (in the case of positively charged layers) are attracted to the surface but also other positive or negative ions (in this study Na' or C1and negatively charged acetate ions, respectively). Therefore, the surface potential must be corrected (then called effective surface potential) by calculating first the effective surface charge density (u,~). To account for the effect of ion binding, the following equation, derived by combining the equation for the Langmuir isotherm and the Boltzmann equation, was ~sed29,30,36
oeff= 4 1 + Kc expke@lkT)l where K is the ion-lipid binding constant for the cation or anion and here c is the concentration of the individual interacting cation or anion. The solutions to eqs 3 and 5 are found iteratively. For the calculations, a value of 6.5 mol/m3 for K of Na+-DPPG was taken.29 The K of Na+-DCP and Journal of Pharmaceutical Sciences / 1117 Vol. 84, No. 9, September 1995
-0.15
-0 1
-0.05 YJ
0
0.05
(V)
DPPC and only slightly larger than the kobs for DPPE (see Table 3). It was also found that DPPG always hydrolyzes slightly faster than DPPC in liposomes composed of both phospholipids, which might be an indication for local inhomogeneities in terms of surface pH and ionic distributions a t the bilayer surface (see Table 3) and that DPPC hydrolyzes slightly faster than DPPE in liposomes composed of both phospholipids. From these considerations, we conclude that the influence of the phospholipid head group on kobs is small if corrections are made for surface charge effects. Under the chosen conditions, there is a tendency toward faster degradation in the order DPPE < DPPC < DPPG.
Figure 4-The values for kbsof liposomal DPPC (0)and DPPG (A) from Table 3 plotted against Y o f liposome dispersions stored in 50 mM acetate buffer (pH 4.0) and 0.12 M NaCl at 30 "C.
the K of the anions C1- and acetate ions binding to stearylamine in liposomes are not known, to our knowledge. Therefore, the calculations mentioned above were done without correcting for these ion-lipid interactions (see Table 3). No unusual results were found. In accordance with theory, Figure 4 shows linear relationships (r > 0.97) when the logarithmic of the kobs values of liposomal DPPC and DPPG from Table 3 are plotted against Y. Only the kobs of DPPG in pure liposomal DPPG deviates from the straight line. This might be explained by the low value for the surface pH (1.8, see Table 3) which is almost similar to the intrinsic pK, of the phosphate group of DPPG, 1.6-1.7.37 Under those conditions DPPG is no longer fully deprotonated and this affects cr,~,and thus Y, dramatically. Consequently, the values for the kobs of DPPG in pure liposomal DPPG have been left out of the calculations below. According to eq 2, the slopes of the lines in Figure 4 should be equal to 16.6 V-l [=e/(kT ln(10))l. However, the slopes of the experimental lines are different: 14.1 f 0.4 V-' for DPPC and 14.3 f 0.8 V-l for DPPG (mean f SE). This deviation might be the result of overestimation of the calculated Y of liposomes when using the Gouy-Chapman-Stern model (see elsewhere for detailed discussion^^^^^^^^^). Another reason for the deviation observed in Figure 4 might be the fact that hydrolysis of liposomal phospholipids does not follow exactly the rate-pH profile of a general acid-base reaction. It was found before that the hydrolysis rate of phospholipids reached a minimum a t pH 6.5; a V-shaped hydrolysis profile was obtained upon plotting the logarithmic values of kobs against the pH.2,3,5-9Theoretically, the acidic branch of a V-shaped hydrolysis profile of a general acid-base reaction should have a slope of -l.0,39 but experimentally lower values were obtained (between about -0.5 and -0.82,3,5-9).One explanation for such a deviation might be catalysis by buffer compon e n t ~ However, .~~ even after correction for buffer effects, the slope of the acidic branch of the hydrolysis profile for liposomal phospholipids still differs from -1.0 (about -0.73). The reason for this phenomenon is unknown. In the present study, the value for the slope of the acidic branch of the hydrolysis profile for liposomal DPPC was -0.62 f 0.07 (mean f SE) as determined after hydrolysis of DPPC liposomes in acetate buffer and NaCl a t pH 3.5, 4.0, or 4.5 and a t 30 "C. Interestingly, when the logarithmic values of kobsof DPPC in charged liposomes were plotted against the calculated surface pH, a slope of -0.85 & 0.03 (mean f SE) was found, which was statistically different from the -0.62 slope mentioned above (p = 0.001). We did not further pursue this discrepancy between theory and experimental observations. From the lines correlating the logarithmic values of kobs of liposomal DPPG with Y (see Figure 41, it was calculated that the kobs of (theoretically) uncharged DPPG (Y = 0 V) a t pH f 0.1 x 4.0 and 30 "C was 1.1 x days-' (mean f SE). This value is not significantly different from the kobsfor 1118 / Journal of Pharmaceutical Sciences
Val. 84, No. 9, September 1995
References and Notes 1. Kensil, C. R.; Dennis, E. A. Biochemistry 1981,20,6079-6085. 2. Frokjaer, S.; Hjorth, E. L.; Worts, 0. In Optimization of Drug Deliuery; Bundgaard, H.; Bagger Hansen, A.; Kofod, H., Eds.; Munkseaard: Cooenhaeen. 1982: DD 384-404. 3. Grit, M ;: De Smidt, J . H ; Struijke,-A.; Crommelin, D. J . A. Int. J . Pharm. 1989, 50, 1-6. 4. Grit, M.; Crommelin, D. J. A. Chem. Phys. Lipids 1992,62,113,no
LA&.
,
5. Grit, M.; Crommelin, D. J. A. Biochim. Biophys. Acta 1993,1167, 49-55. 6. Grit, M.; Zuidam, N. J.; Underberg, W. J . M.; Crommelin, D. J . A. J. Pharm. Pharmacol. 1993, 45, 490-495. 7. Grit, M.; Underberg, W. J. M.; Crommelin, D. J. A. J . Pharm. Sci. 1993, 82 (41, 362-366. 8. Grit, M.: Zuidam, N. J.; Crommelin, D. J . A. In Liposome Technology, 2nd ed.; Gregoriadis, G., Ed.; CRS Press, Inc.: Boca Raton, FL, 1993; Vol I, pp 455-487. 9. Grit, M.; Crommelin, D. J . A. Chem. Phys. Lipids 1993,64,3-18. 10. Deuel, H. J. In The Lipids, their Chemistry and Biochemistry Volume I: Chemistry; Interscience Publisher: New York, 1951; pp 408-439. 11. Hanahan, D. J . In Lipide Chemistry; John Wiley & Sons: New York, 1960; chapter 3. 12. Grit, M.; Crommelin, D. J . A.; Lang, J. K. J . Chrornatogr. 1991, 585, 239--246. 13. Bligh, E. G.; Dyer, W. J. Can. J . Biochem. Physiol. 1959,37 (8 ), 911-917. 14. Schurtenberger, P., Hauser, H. In Liposorne Technology, 2nd ed.; Gregoriadis, G., Ed.; CRS Press, Inc.: Boca Raton, FL, 1993; Vol. I, pp 253-270. 15. Armitage, P.; Berry, G. Statistical Methods in Medical Research; 2nd ed.; Blackwell Scientific Publications: Oxford, Great Britain, 1991. 16. Cevc, G. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker, h c . : New York, 1993; pp 939-956. 17. Singer, M. A.; Finegold, L. Biophys. J . 1990, 57, 153-156. 18. Scherer, J. R. Biophys. J. 1989,55, 957-964. 19. Tausk, R. J . M.; Karmiggelt, J.; Oudshoorn, C.; Overbeek, J. Th.G. Biophys. Chem. 1974,1, 175-183. 20. Villalain, J.; Aranda, J . A.; G6mez-Fernandez, J. C. Eur. J . Biochern. 1986,158, 141-147. 21. Hernandez-Caselles, T.; Villalain, J.; Gomez-Fernandez, J . C. J . Pharm. Pharmacol. 1990,42, 397-400. 22. Schullery, S. E.; Seder, T. A.; Weinstein, D. A.; Bryant, D. A. Biochemistry 1981,20, 6818-6824. 23. Van Echteld, C. J. A.; De Kruijff, B.; De Gier, J. Biochim. Biophys. Acta 1980, 595, 71-81. 24. Crowe, J. H.; Crowe, L. M. In Liposome Technology, 2nd ed.; Gregoriadis, G., Ed.; CRC Press, Inc.: Boca Raton, FL, 1993; pp 229-252. 25. Zuidam, N. J . In Stability of liposomes. Chemical and physical characterisation of liposomes upon autoclaving, gamma-irradiation and storage; Ph.D. Thesis; Utrecht University, The Netherlands, 1994. 26. Hauser, H.; Darke, A,; Phillips, M. C. Eur. J. Biochem. 1976, 62. 335-344. 27. Maitani, Y.; Nagasaki, M.; Nagai, T. Int. J . Pharm. 1990, 64, 89-98. 28. Davies, J. T.; Rideal, E. K. In Znterfacial phenomena; Academic Press: New York, 1961; Chapter 6. 29. Cevc, G. Biochim. Biophys. Acta 1990,1031, 311-382. 30. Cevc, G. Chem. Phys. Lipids 1993, 64, 163-186. 31. Tocanne, J. F.; TeissiB, J . Biochim. Biophys. Acta 1990, 1031, 3 -1 -1 -1 -1 - 6_-. 32. Cevc, G.; Seddon, J. M.; Marsh, D. Faraday Discuss. Chem. SOC. 1986, 81, 179-189.
33. Watts, A., Harlos, K.; Marsh, D. Biochim. Biophys. Acta 1981, 645,91-96. 34. Rand, R. P. Annu. Rev. Biophys. Bioeng. 1981, 10, 277-314. 35. Cruzeiro-Hansson, L.; Ipsen, J. H.; Mouritsen, 0. G. Biochim. Biophys. Acta 1989,979, 166-176. 36. Nir, S.; Bentz, J. J. Colloid Interface Sci. 1978, 65, 399-414. 37. Tatulian, S. A. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker, Inc.: New York, 1993; pp 511-552. 38. Cevc, G.; Watts, A.; Marsh, D. FEBS Lett. 1980, 120 (21, 267270. 39. Martin, A.; Swarbrick, J.; Cammarata, A. Physical Pharmacy; Lea & Febiger: Philadelphia, PA, 1983; pp 380-381.
Acknowledgments We gratefully acknowledge the financial support provided by Bayer (Leverkusen, FRG) and the gifts of phospholipids by Nattermann Phospholipid and Lipoid K.G. We also would like to thank Prof. Dr. G. Cevc (Technical University of Munchen, FRG) for his help with the Gouy-Chapman-Stern calculations, Dr. W. J. M. Underberg (Analytical Department of our faculty) for stimulating discussions and Dr. H. J. A. Wijnne (Centre for Biostatistics of our university) for advice on the statistical methods used in this study. JS940647F
Journal of Pharmaceutical Sciences / 1119 Vol. 84, No. 9, September 1995