Association and release of prostaglandin E from liposomes

Biohimia et Biophysia Ata 1327 1997 97 106 Assoiation and release of prostaglandin E from liposomes 1 Sharon M.K. Davidson, Donna Cabral-Lilly, Frank P. Maurio, J. Craig Franklin, Sharma R. Minhey, Patrik L. Ahl, Andrew S. Janoff ) The Liposome Company, In., 1 Researh Way, Prineton, NJ 08540, USA Reeived 15 Otober 1996; revised 11 February 1997; aepted 13 February 1997 Abstrat PGE1 lipid interations were studied in several liposome systems. Data from both irular dihroi Ž CD. measurements and differential sanning alorimetry Ž DSC. indiated that PGE1 in the protonated form seeks the less polar environment of the lipid bilayer. CD measurements made on PGE1 in solution showed that the wavelength of maximum absorbane red shifted approximately 8 nm with dereasing solvent polarity. The CD spetrum of liposomal PGE1 prepared in ph 4.5 but not ph 7.2 buffer was also red shifted. There was no red shift in the CD spetrum of PGE1 deteted at ph 4.5 in the absene of phospholipid. DSC measurements on DSPC bilayers prepared with 5 mol% PGE1 at ph 4.5 but not ph 7.2 revealed an almost omplete loss of the pre-transition as well as broadening of the main phase transition. The amount of 3 H-PGE1 initially assoiated with EPC, POPC or DSPC liposomes was determined using size exlusion filters and entrifugation. This amount was found to be dependent on the ph of the buffer Ž ph 4.54pH 7.2. and fluidity of the bilayer Ž EPC s POPC) DSPC., but independent of the lamellarity of the liposome. In all ases, addition of holesterol redued the amount of PGE1 assoiated with the liposome. The time-dependent release of PGE1 from the liposomes was determined by rapidly diluting the sample 100-fold into ph 7.2 buffer. Lipid saturation was a key fator influening this release. Gel-phase liposomes of DSPC showed a rapid initial release Ž t - 2 min. 1r2 of PGE1, orresponding to the amount in the outer monolayer, followed by a very slow, almost negligible release of the remaining PGE1. A rapid initial release also ourred in fluid-phase membranes, followed by a more gradual release of the remaining PGE1 over several hours. This release rate ould be slowed by inreasing the lamellarity of these liposomes, or adding holesterol to derease the fluidity of the membrane. q 1997 Elsevier Siene B.V. Keywords: Prostaglandin E ; Phospholipid; Vesile; Drug delivery; Inflammation 1 Abbreviations: EPC, egg phosphatidylholine; POPC, 1-palmitoyl-2-oleoyl-sn-glyero-3-phosphoholine; DSPC, 1,2-distearoyl-sn-glyero-3-phosphoholine; DSPE-GA, 1,2-distearoyl- x; DOPE-GA, 1,2- sn-glyero-3-phosphoethanolamine-n-wglutaryl dioleoyl-sn-glyero-3-phosphoethanolamine-n-wglutaryl x; PGE1, prostaglandin E ; Hepes, 4-Ž 2-hydroxyethyl. 1-1-piperazineethanesulfoni aid; MLV, multilamellar vesile; LUV, large unilamellar vesile; SPLV, stable plurilamellar vesile ) Corresponding author. Fax: q1 609 520 8250. E-mail: ajanoff@lipo.om 1. Introdution Prostaglandins are a family of yli, oxygenated fatty aids that exert diverse and potent effets on ell funtion in many organ systems. In partiular, prostaglandins E1 and E2 have a well doumented role in the series of events that enompass the inflamw1,2 x. Sine both pro- and matory asade anti-in- 0005-2736r97r$17.00 q 1997 Elsevier Siene B.V. All rights reserved. PII S0005-2736 97 00049-7

98 ( ) S.M.K. DaÕidson et al.rbiohimia et Biophysia Ata 1327 1997 97 106 flammatory ations w3 11x have been attributed to these moleules, it is likely that some therapeuti uses ould be expanded but these would undoubtedly depend on the timing, dose and targeting of prostaglandins to their sites of ation. A major problem, however, exists regarding some linial uses of prostaglandin E Ž PGE1.: When administered intravenously, this moleule is rapidly metabolized during irulation through the lungs, whih results in an in vivo half-life of approxiw12 x. In order to irumvent these mately 1 min diffiulties, onsiderable efforts have been made in designing delivery vehiles for PGE1 that might prolong its in vivo half-life and deliver the moleule to its site of ation w13 18 x. One area of researh has been in the design of lipid emulsions to improve the hemial stability and retention of PGE1 w14,16 x. Suh formulations are reported to exhibit inreased biologial ativity and redued side effets as ompared to the free drug in the treatment of peripheral vasular diseases and diabeti neuropathy w19 21 x. Here we desribe the physial harateristis of various liposomal formulations as potential PGE1 delivery systems. This investigation has been fuelled by enouraging prelinial w22 26x and linial w27,28x results using liposomal PGE1 in the treatment of a number of inflammatory diseases. The formulation used in these studies onsists of extruded Ž0.1 mm. liposomes prepared from EPC and PGE1 in a 200:1 molar ratio. At physiologi ph, the PGE1 rapidly dissoiates from these vesiles. This ombination of liposomes with PGE1 has been shown to toggle the ativity of PGE1 from pro- to anti-inw22,23x and has also flammatory in animal models shown signifiant effet in linial trials w27,28 x. Potentially, the preparation of liposomal PGE1 with inreased drug retention ould provide aess to the treatment of other disease indiations. In this report we desribe the assoiation and release harateristis of PGE1 in various liposomal formulations. Fators suh as ph, dilution, lipid omposition and vesile lamellarity were investigated. 1 2. Materials and methods 2.1. Chemials EPC, POPC, DSPC, DOPE-GA, DSPE-GA and holesterol were purhased from Avanti Polar Lipids Ž Alabaster, AL.. w5,6ž n. - 3 HxPGE1 was obtained from Amersham Life Siene Ž Arlington Heights, IL. and PGE1 from Chinoin Pharm. and Chemial Works Ž Budapest, Hungary.. HPLC grade solvents, methylene hloride, hloroform and methanol were purhased from Baxter Ž MGaw Park, IL. and sterile 0.9% saline from Abbott Laboratories ŽNorth Chiago, IL.. Aeti aid was obtained from Aldrih Chemial Co. Ž Milwaukee, WI. and Hepes from Sigma Chemial Co. Ž St. Louis, MO.. All lipids, hemials and solvents were used without further purifiation. 2.2. Liposome preparation Stable plurilamellar vesiles Ž SPLVs. of EPC, POPC or DSPC and PGE1 were prepared by a variaw29 x. Briefly, the tion of the method of Gruner et al. phospholipid and PGE1 were mixed in organi solvent Ž CHCl, CH Cl, ethanol. 3 2 2 and 3% of the total buffer volume Ž50 mm sodium aetate, ph 4.5, 0.9% saline. was added. The solvent was evaporated under a ontinuous stream of nitrogen until a wet lipid paste formed. This paste was then hydrated with buffer to the desired volume. Typial onentrations were 10 mgrml phospholipid and 25 mgrml PGE1. The SPLVs were then extruded under pressure 10 times through 14 mm polyarbonate filters using a Lipex extruder ŽLipex Biomembranes Corp., Vanouver, BC.. For DSPC SPLVs, the hydration and extrusion steps were performed in 10 mm sodium aetate, ph 4.5, 0.9% saline at 708C. Samples were radioatively 3 labeled by adding small quantities of H-PGE1 Ž0.4 mcirml. in ethanol to the organi phase before evaporation. For samples ontaining either holesterol Ž 10, 30 or 40 mol%., DOPE-GA Ž 10 mol%. or DSPE-GA Ž 10 mol%., these lipids Ž in CHCl. 3 were added to the organi solvent phase and the POPC or DSPC molar onentration was redued appropriately. All liposome preparations used in the release assays had a molar ratio of total lipid to PGE1 of 200:1. To determine the apparent partition oeffiient

( ) S.M.K. DaÕidson et al.rbiohimia et Biophysia Ata 1327 1997 97 106 99 of liposomal and aqueous PGE1 at ph 4.5, samples were made at 0.5 8 mol% PGE1 by holding the PGE1 onentration onstant at 25 mgrml and dereasing the lipid onentration. Unilamellar liposomes of DSPC were made by ombining phospholipid and PGE1 in organi solvent then drying by rotary evaporation. Multilamellar vesiles were formed from this film by adding buffer Ž10 mm sodium aetate, 0.9% saline, ph 4.5. and inubating at 708C for 30 min, vortexing periodially. Typial onentrations were 4 mgrml phospholipid and 10 mgrml PGE1. After 10 freezerthaw yles, the sample was extruded 10 times through two-staked 0.1 mm polyarbonate filters at 708C. Unilamellar liposomes of EPCrPGE1 were made in a similar way, exept the lipid film was hydrated in 50 mm sodium aetate, and all steps were arried out at room temperature. Vesile size was onfirmed by quasielasti light sattering using a Niomp Model 270 submiron partile sizer ŽNiomp Instruments, In., Goleta, CA.. 2.3. Cirular dihroi measurements of PGE1 Cirular dihroi measurements of PGE1 were made using a JASCO J-710 Spetropolarimeter ŽJapan Spetrosopi Co., Tokyo.. Spetra were obtained between 250 nm and 350 nm in order to monitor the n p ) transition of the arbonyl group of PGE1. For these experiments, liposomes omposed of EPC Ž 2mgrml. and PGE1 Ž 50 mgrml. were prepared via extrusion through 50 nm polyarbonate filters. Small extruded liposomes were used for these experiments in order to minimize light sattering. Samples were prepared in 50 mm sodium aetate, 0.9% saline ŽpH 4.5., as well as in 10 mm Hepes, 0.9% saline ŽpH 7.2.. CD measurements were made at room temperature using a 10 mm path length quartz ell and a sanning speed of 50 nmrmin. A minimum of eight sans was olleted for eah sample. Appropriate blanks were subtrated before reporting molar iru- Ž 2 lar-dihroi absorbane m rmmol.. 2.4. Differential sanning alorimetry Phase transition temperatures of multilamellar vesiles were determined using a differential sanning alorimeter ŽMiroal MC-2 with DA-2 data aquisition software, Northampton, MA.. Multilamellar vesiles were prepared by dispersing a thin film of DSPC Ž 100 mg. in the absene and presene of PGE1 Ž 2.5 mg. in 25 ml of 10 mm aetate buffer Ž150 mm NaCl, ph 4.5.. The lipid was hydrated at 658C for 0.5 h. Their melting behavior was measured using that same buffer solution as a referene. Heating and ooling sans were reorded from 108C to658cata rate of 208Crh. Samples were equilibrated at the starting temperature for at least 1 h prior to eah san. 2.5. NMR spetrosopy Measurements were ompleted on a Bruker AC300 spetrometer operating at 121.5 MHz. In a typial experiment, the 31 P signals were olleted using a 908 pulse of 15 ms for the total 3000 transients. The gated proton deoupler was used to suppress phosphorus proton ouplings with exponential line broadening of 50 Hz applied to all FIDs. EPC Ž50 mgrml. rpge1 Ž 125 mgrml. SPLVs were made as desribed above. Samples were then extruded 10 times through a 14 mm or 0.8 mm polyarbonate filter. In addition, one sample was frozen and thawed 10 times then extruded through a 0.1 mm filter. The lamellarity of the liposomes was determine by titrat- 2q ing the relaxation agent Mn Ž as MnCl. 2 into eah sample w30 x, and reording spetra at eah titration point. The integrated area of the EPC peak was determined relative to an external triphenyl phosphite standard. The perent of total lipid exposed to the bulk aqueous phase was alulated from the ratio of EPC area at saturating Mn 2q to EPC area at 0 mm Mn 2q. 2.6. Measurement of initial assoiation and release of PGE1 from liposomes The amount of PGE1 initially inorporated into the liposomes was determined using 3 H-PGE1. Liposomes and assoiated PGE1 were separated from bulk buffer and unassoiated PGE1 by entrifugation using Centrion-30 miroonentrators ŽAmion, Beverly, MA. in a fixed-angle rotor at 3000 5000 = g for 15 60 min at 238C. Aliquots of the starting liposome preparation and the lipid-free filtrate were ounted on a LS6800 liquid sintillation ounter ŽBe-

100 ( ) S.M.K. DaÕidson et al.rbiohimia et Biophysia Ata 1327 1997 97 106 kman Instruments, Irvine, CA.. The perentage of PGE1 assoiated with the liposome was alulated from the radioativities in the filtrate and the starting sample. The time-dependent release of PGE1 from liposomes after dilution was determined by rapidly mixing 1 ml of the tritiated sample with 100 ml of 10 mm Hepes, 0.9% saline, ph 7.2. PGE1 is monomeri under neutral Ž0 0.4 mm, the highest onentration is a saturated solution. as well as basi Ž 0 45 mm. onditions w31 x. All experiments were performed at room temperature, exept those using DPPC MLVs at 558C, as noted. Aliquots were removed at various time points ranging from 3 min to 20 h. The liposome-assoiated and non-assoiated PGE1 were separated by entrifugation in a fixed-angle rotor at 5000 8000= g for 1 2 min using either Centrion- 30 or Miroon-100 miroonentrators ŽAmion, Beverly, MA.. No differene was found between the two types of onentrators or speed of entrifugation. The miroonentrators were also tested using radiolabeled liposomes to ensure that the filtrate did not ontain PGE1 that was still liposome-assoiated. The perentage of PGE1 that remained assoiated with the liposomes was alulated as desribed above for initial inorporation. Control experiments were also performed in order to ensure that the PGE1 release profiles were indiative of the kinetis of PGE1 release, not PGE1 equilibration. A 50-fold and a 250-fold dilution were substituted for the 100-fold dilution. Under both of these onditions, the same release profiles were obtained. Fig. 1. Cirular dihroi spetra of free and liposomal PGE1. Ž. a Cirular dihroi spetra of PGE1 Ž 50 mgrml. dissolved in ethanol Ž - - -., hexane Ž PP. and aqueous buffers at ph 4.5 and ph 7.2 Ž PPP.. The vibrational fine struture evident in the CD spetrum of PGE1 dissolved in hexane has been observed for other arbonyl ompounds in hydroarbon solvents w32 x. Ž. b Cirular dihroi spetra of liposomal PGE1 in aqueous buffers at ph 4.5 and ph 7.2 Ž PPP.. In both ases, the LUV dispersion was prepared by extrusion through 50 nm polyarbonate filters and onsisted of EPC Ž 2 mgrml. and PGE1 Ž 50 mgrml.. 3. Results and disussion 3.1. PGE1 assoiates with lipid membranes at ph 4.5 Cirular dihroi Ž CD. measurements of optially ative arbonyl ompounds, suh as PGE1, are known to be sensitive to solvent environment w32 x. Speifially, the n p ) transition of the arbonyl group is red shifted with dereasing solvent polarity. The absene of hydrogen bonding in nonpolar solvents raises the energy level of the n orbital, thus dereasing the n p ) transition energy. We hypothesized that this property ould be used as an indiator of liposomal PGE1 environment Žsurfae vs. intra-membrane asso- iation.. CD spetra of free and liposomal PGE1 were obtained between 250 nm and 350 nm, as shown in Fig. 1. As expeted, the wavelength of maximum absorbane of free PGE1 red shifted approximately 8 nm with dereasing solvent polarity. The magnitude of this shift is typial of that seen with other arbonyl ompounds w32 x. The wavelength of maximum absorbane of free PGE1 in aqueous buffer remained unhanged at ph 4.5 or ph 7.2. In ontrast, the CD spetra of liposomal PGE1 Ž Fig. 1b., was dependent on the ph of the buffer used to prepare the sample. The spetrum in ph 7.2 buffer was very similar to that of free PGE1 in buffer at ph 7.2 or ph 4.5. In this ase, very little 9% of the

( ) S.M.K. DaÕidson et al.rbiohimia et Biophysia Ata 1327 1997 97 106 101 PGE1 was liposome assoiated Ždeteted using tritiated PGE1.. At ph 4.5 however, where approximately 80% of the PGE1 was found to be assoiated with the liposome, the spetrum was red shifted. This data indiates that, at ph 4.5, PGE1 loates within the lipid bilayer. In these fluid-phase membranes the bulk of the PGE1 moleule likely resides primarily in the head group region with the hydrophobi C 20 end penetrating to approximately the C 2 C5 region of the bilayer. The pka of PGE1 in water is reported to be 4.8, as determined by methanol water titrations w33 x. Thus, at ph 7.2 PGE1 exists predominantly as the arboxylate anion. Signifiant liposome assoiation is only ahieved at lower ph where a larger perentage of PGE1 is protonated and inorporation into the lipid membrane of the less water-soluble speies beomes thermodynamially favorable. Further evidene of PGE1 membrane assoiation was provided from differential sanning alorimetry experiments gathered from multilamellar vesiles omposed of DSPC Ž 4 mgrml. prepared in 10 mm sodium aetate Ž ph 4.5. in 0.9% saline Ž Fig. 2.. PGE1 dereased the main phase transition temperature of DSPC by 0.58C and inreased the peak width at half-height by five-fold. The pre-transition was almost ompletely obliterated. This is typial of hanges that an our when a perturbing moleule is present within the bilayer w34 37 x. When we performed the same experiments using liposomes prepared in ph 7.2 Hepes buffer Ž data not shown., the main phase transition of DSPC was unaffeted but Fig. 3. PGE1 partitions between aqueous and organi Ž lipid. phases. Plot of the log of the partition ratio as a funtion of PGE1 onentration in the aqueous phase. EPCrPGE1 SPLVs in 50 mm sodium aetate in 0.9% saline Ž ph 4.5. were prepared suh that the lipid onentration varied from 10 to 0.6 mgrml while the PGE1 onentration was held onstant at 25 mgrml. Eah preparation was labeled with tritiated PGE1 in order to determine the extent of liposome assoiation Ž as desribed in Setion 2.. This data is shown in the inset hart. The apparent partition oeffiient, K app, was determined for eah preparation using the onentration of PGE1 found in the aqueous and lipid phases. the pre-transition was lowered slightly Ž 18C.. Using tritiated preparations, we found that in these ases only approximately 10% of the PGE1 was liposomeassoiated. This low level of assoiation was either insuffiient to ause observable endotherm broadening or the deprotonated moleule bound only to the interfaial regions of the membrane ausing no disruption of ayl-hain paking. The partitioning behavior of PGE1 between immisible organi Ž lipid. and aqueous phases is shown in Fig. 3 for EPCrPGE1 SPLVs in 50 mm sodium aetate in 0.9% saline Ž ph 4.5.. Partition oeffiients measured for PGE1 in several liposomal preparations are tabulated in Table 1. In eah ase the lipid onentration varied from 10 to 0.6 mgrml while the Table 1 PGE1 apparent partition oeffiients in several liposomal formulations Fig. 2. High-sensitivity exess heat apaity profiles of DSPC bilayers in the absene and presene Ž - - -. of PGE1. MLVs were prepared in 10 mm aetate buffer Ž150 mm NaCl, ph 4.5.. The sub-main phase transition evident in both DSC traes is aused by a salt effet w55,56 x. DSPC-MLV 7.4=102 Liposome formulation K app DSPCrCHOLŽ 90:10. -MLV 2 3.1=10 EPC-SPLV 1.5=10 3 EPC-MLV 2.4=10 3 EPCrCHOLŽ 90:10. -MLV 3 1.6=10

102 ( ) S.M.K. DaÕidson et al.rbiohimia et Biophysia Ata 1327 1997 97 106 PGE1 onentration was held onstant at 25 mgrml. Eah preparation was labeled with tritiated PGE1 in order to determine the extent of liposome assoiation. The apparent partition oeffiient: K s PGE1 r PGE1 app lipid was determined for eah preparation using the onentration of PGE1 found in the aqueous and lipid phases. These alulations were made using values of 0.983 mlrg and 0.952 mlrg for the speifi volume of EPC w38x and DSPC w39 x, respetively, and a value of unity for the ativity oeffiient. The apparent partition oeffiients measured for fluid-phase bilayers omposed of EPC or EPCrholesterol agreed well with the otanol water partition oeffiient of PGE1 measured at ph 4.5 by Avdeef et al. w33 x. A signifiant derease Ž 3 8-fold. in PGE1 partitioning was observed in DSPC or DSPCrholesterol gel-phase bilayers. This derease is a likely onsequene of the inreased surfae density of the bilayer hains, whih leads to solute exlusion w40,41 x. 3.2. The assoiation of PGE1 with liposomes prepared in ph 4.5 buffer is dependent upon the lipid omposition As predited by the apparent partition oeffiients measured for PGE1 in fluid- and gel-phase membranes, the degree of assoiation of PGE1 with liposomes prepared in ph 4.5 buffer was found to depend upon lipid omposition Ž Table 2.. PGE1 was more highly inorporated into fluid unsaturated ŽEPC or POPC. than gel-phase saturated Ž DSPC. bilayers. It is not surprising that fluid-phase lipid membranes with a higher degree of alkyl-hain disorder aommodate the funtionalized ylopentane ring of PGE1 more effetively than tightly paked and highly ordered gel-phase membranes. Cholesterol redued PGE1 assoiation only slightly in fluid-phase bilayers but quite signifiantly in gelphase bilayers Žnote the 2-fold derease in Kapp for DSPCrholesterol bilayers as ompared to DSPC bilayers in Table 1.. We suspet that the fused-ring struture of holesterol in ombination with gel-phase lipid is highly dimensionally unomplimentary to that of PGE1. The fluidizing effet that holesterol has on gel-phase lipid w42x was overshadowed by these geometri onstraints. aq Table 2 Perentage of PGE1 initially assoiated with various liposome formulations a Liposome formulation b DSPC-LUV 68"1 DSPC-SPLV 84"1 b DSPCrDSPE-GA 90:10 -LUV 72"1 b DSPCrCHOL 90:10 -LUV 45"1 b DSPCrCHOL 60:40 -LUV 7"2 EPC-LUV 94"3,d EPC-SPLV 94"1,e EPC-SPLV 94"1 POPC-SPLV 95"2 POPCrDOPE-GA 90:10 -SPLV 93"3 POPCrCHOL 90:10 -SPLV 93"2 POPCrCHOL 70:30 -SPLV 78"3 PGE1 assoiated % a Formulations at total lipid:pge1 mol ratio of 200:1. b 10 mgrml PGE1. 25 mgrml PGE1. d EPC SPLV with 10% of total phospholipid loated in the outer leaflet. e EPC SPLV with 20% of total phospholipid loated in the outer leaflet. The effet of negative surfae harge on PGE1 inorporation was investigated by the addition of the phospholipid derivatives DSPE-GA or DOPE-GA into liposomes prepared from saturated and unsaturated lipids, respetively. These preparations were of interest to us from an experimental as well as a pratial point of view beause these lipid derivatives are known to inrease the irulation lifetime of lipow43,44 x. As shown in Table 2, the somes in vivo addition of 10 mol% of these negatively harged lipid derivatives had no effet on the degree of PGE1 inorporation. Assoiation was also independent of liposome lamellarity, as shown by the idential inorporation of PGE1 into EPC LUVs and SPLVs. A slightly higher PGE1 assoiation is reported for DSPC SPLVs than LUVs beause the SPLVs were prepared at a higher onentration. 3.3. The dissoiation of PGE1 from liposomes at physiologi ph is dependent upon liposome lamellarity and lipid omposition The release of PGE1 from liposomes prepared in either 10 or 50 mm sodium aetate and 0.9% saline

( ) S.M.K. DaÕidson et al.rbiohimia et Biophysia Ata 1327 1997 97 106 103 Ž ph 4.5. was measured using a method that relies upon a 100-fold dilution of the sample into a ph 7.2 Hepes buffer. These onditions were hosen in order to mimi the ph hange and rapid sample dilution that would our in vivo. Results from these experiments are shown in Fig. 4. We studied the release of PGE1 from EPC LUVs that were prepared by extrusion through 0.1 mm filters. Quasi-elasti light sattering measurements revealed them to have a mean diameter of approximately 91 nm. The integration of 31 P-NMR spetra in the absene and presene of Mn 2q revealed approximately 43% of the total phospholipid was loated in the outer leaflet. Release of PGE1 from these single lamellar liposomes was rapid and essentially omplete within 3 min, whih was the first onvenient measurement point. Upon dilution into ph 7.2 buffer, the PGE1 putatively loated in the outer leaflet was rapidly deprotonated, beoming muh more soluble in the aqueous phase. PGE1 Fig. 4. Dissoiation of PGE1 from liposomes at physiologi ph depends upon liposome lamellarity and lipid omposition. PGE1 release profiles from DSPC SPLVs Ž `. and LUVs Ž I., DPPC MLVs Ž rt. Ž i. and 558C Ž d., EPC SPLVs with 10% Ž '. and 20% Ž v. of total phospholipid loated in the outer leaflet, EPC LUVs with 43% Ž l. of total phospholipid loated in the outer leaflet. Lamellarity alulations are based on the integration of 31 P-NMR spetra in the absene and presene of Mn 2q. In all ases, 1 ml of the preparation was rapidly diluted with 100 ml of 10 mm Hepes in 0.9% saline Ž ph 7.2.. Aliquots were removed at various time points Ž 3 min to 20 h. and the liposome-assoiated and non-assoiated PGE1 were separated by entrifugation using miroonentrators. Data is plotted as the perentage of PGE1 remaining assoiated with the liposome as a funtion of time. The graphs are not normalized; 100% retention is equal to the total amount of PGE1 initially present Žliposome assoiated and nonassoiated. in eah preparation. In eah ase, the perentage of PGE1 initially assoiated with the liposome was 94%, exept where noted in parentheses. loated in the inner leaflet was also rapidly released from the vesile, whih indiates that in this situation there was virtually no barrier to PGE1 flip-flop aross the membrane. Similar rapid release of PGE1 from 200 nm lipid mirospheres Ž Lipo-PGE1. has been reported w45 x. Lipo-PGE1 is a 10% oil in water Ž ph 4.5 6.0. emulsion prepared from soyabean oil, EPC and other omponents. The release of PGE1 from these mirospheres was determined by ultrafiltration in a similar manner to the method we report here and was found to be over 90%. Confliting results using a dialysis method to measure PGE1 release from Lipo-PGE1 have also been reported w13,46 x. In this ase, Lipo- PGE1 was enlosed in ellulose tubing and immersed into ph 7.4 buffer Ž 10-fold inrease in volume. at 208C. In these studies only 50% of the PGE1 was released after 2 h but the rate-limiting step was probably diffusion through the ellulose membrane not release of PGE1 from the lipid mirospheres. We have found that equilibration of free PGE1 aross dialysis membranes requires extended time frames Ž t)60 min.. PGE1 release kinetis from DSPC LUVs were in marked ontrast to the release profile from EPC LUVs Ž Fig. 4.. LUVs prepared from DSPC released only approximately 50% of the initially assoiated PGE1. After this rapid release phase the remaining fration of PGE1 Ža. 48% of initially assoiated PGE1. remained assoiated for ) 5.5 h. Presumably, PGE1 exposed to the ph 7.2 buffer in the outer leaflet was rapidly deprotonated and released from the membrane. PGE1 loated in the interior leaflet was rigidly embedded in the tightly paked gel-phase lipid and transbilayer movement Ž flip-flop. aross the membrane is essentially abolished. The release kinetis of PGE1 from multilamellar fluid- and gel-phase membranes are also shown in Fig. 4. Release profiles of PGE1 from EPC SPLVs with 10% or 20% of total phospholipid loated in the 31 outer leaflet Ždata from P-NMR. showed a rapid initial release of PGE1, followed by a more gradual release over a period of several hours. ŽDSPC SPLVs showed long-term retention of the bulk of the initially assoiated PGE1.. Clearly, liposome lamellarity plays a signifiant role in the retention of PGE1. For multilamellar vesiles, the majority of PGE1 is loated within internal lipid membranes. Release of the

104 ( ) S.M.K. DaÕidson et al.rbiohimia et Biophysia Ata 1327 1997 97 106 drug requires rossing multiple aqueous ompartments and lipid bilayers, whih greatly attenuates its release from fluid-phase systems. For gel-phase systems the fration of PGE1 loated in internal lamellae is apparently unable to gradually migrate through suessive bilayers, only material in the outer leaflet is released. This onlusion is supported by data olleted on the release of PGE1 from DPPC MLVs at room temperature and 558C Ž Fig. 4.. Below the phase transition these liposomes showed long-term retention of the bulk of the initially assoiated PGE1, similar to the DSPC SPLVs. However, above the phase transition a rapid release phase is followed by a gradual release over a period of several hours, similar to fluid-phase EPC multilamellar membranes. These results strongly support our onlusion that the rate of transbilayer movement of PGE1 is greatly influened by the physial state of the membrane. The effet of lipid omposition on PGE1 dissoiation was further investigated for POPC and DSPC liposomes that inorporated holesterol or negative surfae harge. The addition of inreasing amounts of holesterol to POPC SPLVs dereased the PGE1 release rate, partiularly the initial rapid release phase, from these systems Ž Fig. 5a.. The effet of holesterol on lipid bilayers has been extensively studied w47 53 x. In a membrane above its phase transition, holesterol redues the freedom of the lipid ayl hains. This results in bilayers that are more tightly paked and less fluid, more like a membrane omposed of lipids that are in the gel phase. Dissoiation of PGE1 from this type of membrane is more diffiult, as previously determined for gel-phase DSPC bilayers. On the other hand, addition of inreasing amounts of holesterol to DSPC LUVs had a profound effet on the ability of PGE1 to be inorporated into these membranes Ž Table 2. but no effet on release ŽFig. 5b.. The release profiles were idential, after onsidering the amount of PGE1 initially assoiated in eah liposome preparation. In all ases approximately 50% of the PGE1 was rapidly released and the remaining fration had long-term retention. Of ourse, in DSPCrholesterol Ž 60r40. LUVs, virtually no PGE1 was initially assoiated with this membrane. Inorporation of DOPE-GA or DSPE-GA into liposomes had only a slight effet on the rate of PGE1 release Ž data not shown.. Both POPC SPLVs, pre- Fig. 5. Effet of holesterol on PGE1 release. Ž. a PGE1 release profiles from POPC SPLVs prepared with 0% Ž l., 10% Ž B., and 30% Ž '. holesterol. Ž b. PGE1 release profiles from DSPC LUVs prepared with 0% Ž l., 10% Ž B., and 40% Ž '. holesterol. Experimental onditions are the same as desribed in Fig. 4. The value in parentheses is the perentage of PGE1 initially assoiated with the liposome. pared with 10 mol% DOPE-GA, and DSPC LUVs, prepared with 10 mol% DSPE-GA, showed a slight inrease in the initial rate of release of PGE1 from the membrane as ompared to POPC SPLVs or DSPC LUVs prepared without these lipid derivatives. In both ases, approximately 35% of the initially assoiated PGE1 remained with the liposome at the first time point measured Ž 3 min.. After this rapid initial release the data were idential to the underivatized liposomes in both ases. This small inrease in the initial rate of PGE1 release was likely due to harge repulsion or the hange in liposome morphology produed by the inorporation of these moleules w54 x. 4. Conlusions We have shown that protonated PGE1 assoiates with lipid membranes and the degree of this assoiation is highly ompositionally dependent. The disso-

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