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avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /e jps

Long-circulating, pH-sensitive liposomes stericallystabilized by copolymers bearing short blocks oflipid-mimetic units

Denitsa Momekovaa,∗, Stanislav Rangelovb, Stanislav Yanevc, Elena Nikolovad,Spiro Konstantinove, Birgit Rombergf, Gert Stormf, Nikolay Lambova

a Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Medical University-Sofia,2 Dunav Street, 1000 Sofia, Bulgariab Institute of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgariac Laboratory of Drug Toxicology, Institute of Physiology, Bulgarian Academy of Sciences, Sofia, Bulgariad Institute of Morphology and Anthropology, Bulgarian Academy of Sciences, Sofia, Bulgariae Laboratory of Experimental Chemotherapy, Department of Pharmacology and Toxicology, Faculty of Pharmacy, MedicalUniversity-Sofia, Sofia, Bulgariaf Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, The Netherlands

a r t i c l e i n f o

Article history:

Received 24 October 2006

Received in revised form

28 June 2007

Accepted 27 August 2007

Published on line 4 September 2007

Keywords:

pH-sensitive liposomes

DOPE

Steric stabilization

PEG-lipids

Block copolymers

a b s t r a c t

A major hurdle towards in vivo utilization of pH-sensitive liposomes is their prompt seques-

tration by reticuloendothelial system and hence short circulation time. Prolonged circulation

of liposomes is usually achieved by incorporation of pegylated lipids, which have been fre-

quently reported to deteriorate the acid-triggered release. In this study we evaluate the

ability of four novel nonionic copolymers, bearing short blocks of lipid-mimetic units to

provide steric stabilization of DOPE:CHEMs liposomes. The vesicles were prepared using the

lipid film hydration method and extrusion, yielding liposomes of 120–160 nm in size. Their

pH-sensitivity was monitored via the release of encapsulated calcein. The incorporation of

the block copolymers at concentration up to 10 mol% did not deteriorate the pH-sensitivity

of the liposomes. A selected formulation was tested for stability in presence of 25% human

plasma and proved to significantly outclass the plain DOPE:CHEMs liposomes. The abil-

ity of calcein-loaded liposomes to deliver their cargo inside EJ cells was investigated using

fluorescent microscopy and the results show that the surface-modified vesicles are as effec-

tive to ensure intracellular delivery as plain liposomes. The pharmacokinetics and organ

distribution of a selected formulation, containing a copolymer bearing four lipid anchors

was investigated in comparison to plain liposomes and PEG (2000)–DSPE stabilized lipo-

somes. The juxtaposition of the blood clearance curves and the calculated pharmacokinetic

parameters show that the block copolymer confers superior longevity in vivo. The block

copolymers utilized in this study can be consider as promising sterically stabilizing agents

for pH-sensitive liposomes.

© 2007 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +359 2 9236 528; fax: +359 2 9879 874.E-mail address: dmomekova@yahoo.com (D. Momekova).

0928-0987/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejps.2007.08.009

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1. Introduction

Since their discovery (Bangham et al., 1965) and recognitionof the structure and basic properties, liposomes have beenconsidered as almost universal carriers for drugs and diag-nostic agents as they can accommodate both hydrophilic andhydrophobic agents (Allen, 1998). However, the clinical use-fulness of liposomes has been up to recently largely limiteddue to two major obstacles: (i) the prompt recognition andsequestration by the reticuloendothelial system (RES) uponintravenous administration, yielding short circulation half-lifeand (ii) their propensity to accumulate in certain sub-cellularcompartments, mainly lysosomes, where the encapsulateddrug is retained for relatively long period of time and oftendegraded, thus limiting its availability at the cytosolic targetsite (Straubinger et al., 1983; Huakg et al., 1983; Dijkstra et al.,1984). The latter problem is a serious hurdle in the develop-ments of liposome-based carriers for intracellular delivery ofpeptides, proteins, nucleic acids and certain polar anticancerdrugs, which are characterized by both low cellular perme-ation ability and chemical/enzymatic instability. An attractiveapproach to avoid lysosomal sequestration and degradationof entrapped materials is the use of pH-sensitive liposomes.They exhibit considerable fusogenic activity and ability toundergo pH-dependent phase transitions (Drummond et al.,2000; Venugapalan et al., 2002; Simoes et al., 2001). These fea-tures ascertain improved cytosolic availability of encapsulatedmaterials (Simoes et al., 2001; Gazzola et al., 1997). As thepH-sensitive liposomes designed as drug carriers for systemictreatment should also display long circulation times, there hasbeen considerable interest in formulations combining bothprolonged circulation time and pH-responsiveness.

Typically, prolonged circulation of liposomes is achievedusing poly(ethylene glycol) (PEG) covalently connected to alipid residue which is incorporated into the liposome bilayer(Lasic et al., 1999; Woodle, 1995). The polymer chains cre-ate a repulsive barrier around the liposomes, which reducesthe interactions with blood components and consequentlyincreases the circulation times of liposomes in the blood.These steric stabilizers, referred to as PEG-lipids, are commer-cially available in PEG molecular weights ranging from 350to 5000 and diverse lipid anchors. Usually, PEG-lipids of PEGmolecular weight of 2000 and contents of 5–8 mol% are usedfor formulations of sterically stabilized liposomes (Woodle,1995). By increasing PEG-lipid content the density and thick-ness of the repulsive barrier can be increased and supposedlythe liposome circulation life prolonged. However, at a cer-tain critical content referred to as saturation limit, whichdepends on lipid composition and PEG-molecular weight, thePEG-lipids induce a transition from bilayers to a micellar phase(Hristova et al., 1995; Edwards et al., 1997; Belsito et al., 2000;Marsh et al., 2003). This transition may occur via intermediatestructures, such as open or perforated lipid bilayer aggre-gates, which are useless as far as encapsulation and deliveryof active substances are concerned (Edwards et al., 1997).Furthermore, the PEG-lipids whose macromolecules can beapproximated to cone provide lower saturation limits thanthose provided by more cylindrical molecules. It has been sug-gested earlier that copolymers containing short sequences of

lipid-mimetic units would provide stronger anchoring in theliposome bilayer (Rangelov et al., 2001). The larger hydrocar-bon volume and hence the more cylindrical macromolecules,compared to those of the conventional PEG-lipids have beenreported to destabilize the bilayer structure upon the incorpo-ration of considerably larger quantities of these copolymers(Rangelov et al., 2003). The sterically stabilized pH-sensitive-liposomes face another dilemma. On one hand, the PEG-lipidshave been shown to facilitate the liposome formation fromlipids that prefer inverted non-lamellar, in particular hexago-nal, phases such as phosphatidylethanolamine lipids, whichare the main constituents of the pH-sensitive liposomes. Onthe other hand however, in a number of studies (Slepushkinet al., 1997; Zignani et al., 2002; Hong et al., 2002) reductionand even inhibition of the liposomal pH-sensitivity in vitrohas been reported.

In the present paper we report on the development ofpH-sensitive liposomes based on DOPE:CHEMs sterically sta-bilized by a number of nonionic copolymers that differ inPEG-molecular weight and number of lipid-mimetic anchorsper copolymer chain. The chemical structures of the lipidicmoieties are depicted in Figs. 1 and 2. The copolymercompositions are given in Table 1. The liposomes are physico-chemically characterized and the ability to release their cargoin a pH-dependent manner investigated. In vivo experimentswere carried out to assess the pharmacokinetic propertiesof a selected formulation in comparison to non-stabilizedDOPE:CHEMs liposomes and liposomes stabilized by a com-mercially available PEG-lipid.

2. Materials and methods

2.1. Materials

Dioleoylphosphatidylethanolamine (DOPE), cholesterylhemisuccinate (CHEMs), calcein, Triton X-100, HEPES buffer,chloroform, RPMI 1640, fetal calf serum were purchasedfrom Sigma Chemical Corporation (USA), 3H-cholesteryloleoyl ether was supplied from Amersham Biosciences (UK)and poly(ethylene glycol) (2000)–distearoyl phosphatidylethanolamine (PEG (2000)–DSPE) was purchased from LipoidGmbH (Germany). Ultima GoldTM scintillation cocktail waspurchased from Canberra Packard Central Europe GmbH(Austria). The PEG-based copolymers used in this study(Figs. 1 and 2, Table 1) were synthesized as describedelsewhere (Rangelov et al., 2002a,b).

2.2. Methods

2.2.1. Preparation of liposomesChloroform solutions of DOPE and CHEMs (3:2 M ratio;20 �mol total lipid/ml) and the respective copolymer in adefined copolymer/lipid molar ratio were placed into glasstubes and the solvent was evaporated under a stream of argon.For the in vivo tested liposomes during the preparation step10 �l of 3H-cholesteryl oleoyl ether were added to each testedformulation as a non-degradable lipid-phase marker priorto the evaporation of the organic solvent. Traces of chloro-form were removed under vacuum overnight. HEPES-buffered

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Fig. 1 – Chemical structures of (a) (DDGG)4(EO)114 and (b) PEG (2000)–DSPE.

saline (pH 7.4) was added to the dry lipid/copolymer filmand the resulting dispersions were extruded 30 times throughpolycarbonate filters of pore size 100 nm using a LiposoFasthandle type extruder (Avestin Inc., Canada). For the in vitroleakage and cellular interaction studies the hydration stepwas performed with 80 mM buffered solution of calcein (pH7.4). The unentrapped dye was removed by passing through aSephadex G50 column (Pharmacia, Uppsala, Sweden), equili-brated with HEPES-buffered saline (pH 7.4).

2.3. Physicochemical characterization of liposomes

2.3.1. Dynamic light scattering (DLS)

The mean size and size distribution of the liposomeswere determined using a photon correlation spectrometer(Brookhaven Instruments Co., USA). The values of the hydro-dynamic diameters d90 were evaluated from measurementsat an angle of 90◦ and calculated using the Stokes–Einsteinequation:

d90 = kT

3��D90

Fig. 2 – Structural formulas of the lipid-mimetic units: (top)1,3-didodecyloxy-propane-2-ol, DDP; (bottom)1,3-didodecyloxy-2-glycidyl glycerol, DDGG.

where kT is the thermal energy factor, � the temperaturedependent viscosity of water and D90 is the diffusion coeffi-cient at 90◦. The measurements were made at 25 and 37 ◦C.Each measurement was performed five times.

2.3.2. �-PotentialThe �-potential was determined using Zetasizer 2000 fromMalverns Instruments Ltd., Spring Lane South, Malvern, UK.The measurements were performed at 25 ◦C in HEPES-bufferedsaline of pH 7.4.

2.3.3. Leakage assaysTwenty microliters of 80 mM calcein-loaded liposomes, sep-arated from non-encapsulated dye, were added to 2 ml ofbuffered solutions of pH ranging from 4.5 to 7.4. At concentra-tions of 80 mM and above the fluorescense intensity of calceinis self-quenched. As the dye leaks out from the liposomes,it becomes diluted and the fluorescense intensity increases.After 10 min incubation at 37 ◦C fluorescence was assessed at520 nm after excitation at 490 nm, before and after the addi-tion of 100 �l of a 10 wt.% solution of Triton X-100 using aPerkin-Elmer fluorometer. The percentage of calcein leakage

Table 1 – Composition and nominal molecular weightsof the investigated copolymers

Composition Molecular weight

DDP(EO)52 2715DDP(EO)92 4475(DDGG)2(EO)115 6028(DDGG)4(EO)114 6808

See Fig. 1 for the abbreviations DDP and DDGG. EO denotes ethyleneoxide monomer units of the PEG chains.

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was calculated according to the following equation:

leakage (%) = IpH − I7.4

It − I7.4× 100

IpH is the corrected intensity at acidic pH before the addi-tion of Triton X-100, I7.4 the fluorescense at pH 7.4 and It is thetotal fluorescense intensity evaluated after the destruction ofliposomes.

2.4. Cells and culture conditions

The human urinary bladder carcinoma-derived cell line EJwas used in this study. It was obtained from the Ameri-can Type Culture Collection (Rockville, MD, USA). The cellswere maintained as monolayer culture in a controlled envi-ronment: RPMI-1640 medium supplemented with 10% fetalcalf serum and 2.5 mg/ml l-glutamine in an incubator with5% CO2 humidified atmosphere at 37 ◦C. The cells werekept in log-phase by trypsinization and consequent sup-plementation with fresh medium, two to three times perweek.

2.5. Cellular interactions of liposomes

The interactions of the tested pH-sensitive liposomes withEJ cells were evaluated using fluorescence microscopy. Expo-nentially growing EJ cells (passage number <7) were detachedfrom the cultivation flask via trypsinization and were platedin sterile petri dishes at a density of 3 × 105 cells per dish andallowed to adhere for 48 h at 37 ◦C. Thereafter the cell cultureswere treated with calcein-loaded liposomes (at a final lipidconcentration of 100 �g/ml) or with equivalent concentrationof free calcein and incubated at 37 ◦C for 1 h. Subsequently,the medium was removed, the cells were washed thrice withphosphate buffered saline (PBS) and the samples were ana-lyzed with fluorescent microscopy.

2.6. Animals

Male outbred Wistar rats of approximately 250 g body weightwere obtained from Harlan Nederland (Horst, The Nether-lands). All experiments were carried out in accordance withthe local Animal Welfare Committee. All painful and inva-sive procedures (i.v. treatment, blood sampling, etc.) wereperformed under isofluorane anaesthesia.

2.7. In vivo biodistribution and pharmacokineticstudies of liposomes

The pharmacokinetic behavior and the biodistribution ofliposomes coated with 5 mol% (DDGG)4(EO)114, 5 mol% PEG(2000)–DSPE and plain DOPE:CHEMs liposomes were evaluatedin rats. The animals were injected at a dose of 20 �mol lipid/kgin cadual vein. At different time points post-injection theanimals were anaesthetized with isofluorane and blood sam-ples of 150 �l were collected. Blood samples were solubilizedwith solvable tissue solubilizer and bleanched with 35% H2O2

overnight. Scintillation activity of the samples was deter-mined in Ultima GoldTM liquid scintillation cocktail with aPackard Tricarb 2200 CA liquid scintillation counter. The ani-

mals were sacrificed 48 h post-injection and liver, spleen,lungs and kidneys were dissected and weighed. The organswere homogenized and after the solubilizing and bleachingthey were diluted with Ultima GoldTM scintillation cocktailand counted as described above. Besides tissue and bloodsamples also radioactivity of the injected dose was counted.The concentration of the radioactive label was calculated aspercentage of the injected dose and the data were fitted toconcentration–time curves. The plasma concentration datawere fitted using Kinetica® software for PC and the areaunder the curve (AUC), plasma clearance (CLp), mean res-idence time (MRT) and volume of distribution (Vdss) werecalculated.

3. Results

3.1. Liposomes characterization by DLS

Representative examples of liposome size distribution mea-sured at 25 and 37 ◦C are shown in Fig. 3. The distributions aremonomodal and, importantly, the monomodality is preservedat 37 ◦C as well. This indicates that the increase of temperatureto the physiologically relevant level is not related to significant

Fig. 3 – Size distributions of DOPE:CHEMs liposomescontaining 5 mol% of (DDGG)4(EO)114 at 25 ◦C (a) and 37 ◦C(b), measured at an angle of 90◦.

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Fig. 4 – Variations of the hydrodynamic diameters at anangle of 90◦, d90, of series of DOPE:CHEMs liposomesstabilized by DDP(EO)52 (circles), DDP(EO)92 (triangles),(DDGG)2(EO)115 (inverted triangles) and (DDGG)4(EO)114

(diamonds) as a function of copolymer content at 25 ◦C.Each data point represents the arithmetic mean of fiveseparate measurements.

structural alterations of the liposomes, such as vesicle fusionor aggregation.

The apparent hydrodynamic diameters of the present lipo-somes range between 120 and 160 nm. The variations of thehydrodynamic diameters with copolymer composition andcopolymer content in the formulations at 25 ◦C are depictedin Fig. 4.

As seen, at lower copolymer contents the liposomes aresomewhat larger than the control (i.e., non-coating copoly-mer) DOPE:CHEMs liposomes. With a further increase of thecopolymer content the liposomes tend to decrease in size,especially at 10 mol%. The effect of the copolymer compo-sition is hardly reflected in the size although the liposomescontaining (DDGG)2(EO)115 and (DDGG)4(EO)114 are typicallyslightly larger than those stabilized by the other two copoly-mers. At 37 ◦C the liposomes typically increase in size (Fig. 5).The increase in size is consistent with the chain melting tem-perature. For DOPE it is around 40 ◦C. When this temperatureis approached the chains transform to a more fluid and, conse-quently, more expanded state. The effect of the temperature ismore pronounced for the formulations containing DDP(EO)52

and DDP(EO)92 especially at lower copolymer content (Fig. 5).The value of the �-potential, which is known to affect the

adsorption and permeability of charged species (Flewellingand Hubbell, 1986) of the pure DOPE:CHEMs liposomes wasfound to be −36.9 mV measured in HEPES-buffered saline.Upon the incorporation of the 5 mol% of the PEG (2000)–DSPEthe �-potential attains a more positive value of −16.4 mV. Suchan observation has been reported earlier by Yoshida et al.(1999), who observed an increase in the surface potential, i.e.,more positive, on inclusion of PEG-lipids into anionic lipo-somes. These findings can be explained in terms of screeningeffects of the lengthy PEG chains on electric charges (Yoshidaet al., 1999; Cevc, 1990). In line with these expectations a valueas high as −5 mV was measured when a nonionic copolymer

Fig. 5 – Histogram representation of d90 at 25 ◦C (black) and37 ◦C (grey) of formulations containing 2.5 mol% (top) and10 mol% (bottom) of the copolymers studied. The size of thepure DOPE:CHEMs liposomes are given for comparison.

of a higher PEG-molecular weight (DDGG)4(EO)114 was incor-porated in the membrane.

3.2. Leakage assays

The effects of the copolymer on the pH-induced leakageof DOPE:CHEMs liposomes was investigated using calcein at37 ◦C. The leakage as a function of pH is given as percentageof released calcein. The leakage profiles for all formulationsstudied are presented in Fig. 6.

A common feature for all formulations is that the pH-sensitivity is preserved as evidenced by the well-pronouncedpatterns of an increase of the calcein leakage with decreasingpH. The pH-sensitivity is not eliminated even at copolymerconcentrations as high as 10 mol%. These data contrast thewell established propensity of PEG–DSPE-lipids, even whenintroduced in very small amounts (e.g., 1 mol%) to hamperthe transition from a lamellar to inverted hexagonal phase atlow pH and, consequently, to inhibit the pH-triggered release(Slepushkin et al., 1997; Holland et al., 1996; Koynova et al.,1999).

The pH-sensitivity is useless if the liposomes are not able toretain their cargo in the blood. To evaluate the extent to whichthe membrane permeability is affected by the incorporationof 5 mol% of (DDGG)4(EO)114 a leakage assay in 25% humanplasma was carried out (Fig. 7). As evident from the presenteddata the ability of copolymer-modified liposomes to retain thefluorophore was superior: the release was less than ca. 20%,while the plain liposomes were found to be more leaky.

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Fig. 6 – Spontaneous pH-dependent leakage at 37 ◦C of calcein from DOPE:CHEMs (3:2) liposomes (open squares) stabilizedwith DDP(EO)52 (a), DDP(EO)92 (b), (DDGG)2(EO)115 (c) and (DDGG)4(EO)114 (d), at contents of 2.5 mol% (circles), 5 mol%(triangles), 7.5 (inverted triangles) and 10 mol% (diamonds). Data points represent the mean values of four independentexperiments.

3.3. Cellular interactions of liposomes

Typically the cell internalisation of liposomes requires closecontacts between the liposomal bilayer and the cellular mem-brane, hence the hydrophylic coating by grafted copolymerscould hinder the membrane interactions and could compro-mise the cellular uptake of liposomes. On this ground we

Fig. 7 – Leakage of calcein from DOPE:CHEMs (opensquares) liposomes and DOPE:CHEMs liposomes stabilizedby 5 mol% of (DDGG)4(EO)114 (diamonds) as a function oftime at 37 ◦C in 25% human plasma. Data points representmean values of three separate experiments.

sought to determine whether the incorporation of 5 mol% ofthe tested copolymers is detrimental for the cellular uptakeof the vesicles and to the effective cytosolic delivery of theircargo. To address this issue in vitro experiments with calcein-loaded DOPE-based liposomes varying in type and contentof steric stabilizers were carried out. The cellular interac-tions were evaluated by means of fluorescence microscopy.Representative fluorescence and phase contrast images areshown in Fig. 8. After 1 h of exposure of liposomes to EJ carci-noma cells a uniform cytoplasmic fluorescence was observed,which is indicative for effective cytosolic delivery of the mem-brane non-permeable calcein as judged from a large collectionof images of pure and copolymer-coated DOPE:CHEMs lipo-somes. This implies that the presence of a bulky hydrophilicPEG layer in copolymer-modified liposomes does not compro-mise their ability to ensure cytosolic delivery of their cargoinside cells. The intracellular fluorescence was incomparablyweaker when EJ cells were exposed to a calcein solution (notshown).

3.4. In vivo biodistribution and pharmacokinetic studyof liposomes

The clearance from the blood circulation of DOPE liposomescontaining 5 mol% of (DDGG)4(EO)114 as well as non-coatedand PEG (2000)–DSPE coated liposomes is illustrated in

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Fig. 8 – Fluorescence (left panel) and phase contrast (right panel) photomicrographs of EJ carcinoma cells, following exposureto pH-sensitive-liposomes (100 �g/ml total lipid) at 37 ◦C for 1 h. Non-coated DOPE:CHEMs liposomes (top), DDP(EO)92-coatedliposomes (middle), (DDGG)4(EO)114-coated liposomes (bottom). Copolymer content in the formulations is 5 mol%.

Fig. 9 as blood concentration versus time curves. For betterillustration of the different behavior of the three formu-lations, pharmacokinetic parameters were calculated andsummarized in Table 2. The liposomal clearance for all for-mulations studied was nearly single-exponential. The curvesclearly show that only the 5 mol% (DDGG)4(EO)114-modifiedliposomes are characterized with drastically prolonged cir-culation time. While the non-coated liposomes were clearedfrom the circulation within 1 h post-injection, the PEG(2000)–DSPE-coated liposomes retained 15.9% and 1.3% ofthe initial concentration after 8 and 48 h, respectively. Theliposomes containing (DDGG)4(EO)114 exhibit even slowerelimination as 15.1% of the initial dose are still in cir-culation at 48 h after the administration. Moreover theirpharmacokinetic parameters are superior compared to thoseof the two reference formulations. They were found tochange favorably by factors of 3.31 (AUC), 1.4 (Vdss) to4.5 (CLp) compared to the PEG (2000)–DSPE-stabilized onesand by a factor of 30.6 (AUC) versus the non-coatedliposomes.

The results are in line with the organ accumulation datagiven in Table 3. Compared to the plain liposomes, the RES-accumulation is reduced by a factor of 1.3 and 2.2 for the PEG

Fig. 9 – Blood concentration (percentage of injected dose)vs. time curves of DOPE:CHEMs liposomal formulationsfollowing intravenous injection in rats: non-coatedliposomes (open squares), liposomes containing 5 mol% ofPEG (2000)–DSPE (circles) and (DDGG)4(EO)114 (diamonds).Each data point represents the arithmetic mean ± S.D.(n = 3).

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Table 2 – Pharmacokinetic parameters for the three evaluated DOPE:CHEMs-based formulations following in vivoapplication in healthy rats

Parameter Non-coated PEG (2000)–DSPE coated (DDGG)4(EO)114 coated

AUC0–48 (% dose h/ml) 49.29 456.76 1510.12MRT (h) n.d. 11.63 37.02Lz (h−1) n.d. 0.059 0.026Vdss (ml) 47.61 2.44 1.77CLp (ml/h) 1.585 0.210 0.047

n.d.: not detected.

(2000)–DSPE and (DDGG)4(EO)114 coated liposomes, respec-tively.

Apparently the presence of four lipid anchors in(DDGG)4(EO)114 versus only one in the conventional coat-ing agent results in its superior ability to confer stericstabilization, avoidance of RES-uptake and consequentlyblood circulation time prolongation.

4. Discussion

Sterically stabilized pH-sensitive liposomes have been inves-tigated in attempts to develop compositions that simulta-neously avoid the excessive phagocytosis during circulationand provide enhanced intracellular accumulation of encapsu-lated hydrophilic compounds such as genes, oligonucleotidesand antineoplastics. To the best of our knowledge, the for-mulations proposed so far typically compromise betweenthe enhanced circulation longevity and pH-sensitivity. In thepresent study we evaluated the possibilities to develop long-circulating pH-sensitive-liposomes via grafting them withcopolymers bearing short blocks of lipid-mimetic units. Inthe liposomes composed of DOPE and CHEMs, amounts ofup to 10 mol% of the above copolymers were incorporatedusing a conventional method, i.e., freeze thaw and extrusionof hydrated lipid/copolymer mixtures. The light scatteringresults show that the monomodal distribution and the ther-apeutically optimal size for intravenous administration arepreserved at 37 ◦C. Similarly to the commercially availablePEG-lipids the copolymers increase the spontaneous curva-ture of the lipid mixtures, thus facilitating the formationof a lamellar phase. A frequently reported drawback of thePEG-lipids is that they may significantly influence the pH-dependent release of liposomal payload, which makes them oflittle use to achieve steric stabilization and, at the same time,maintain the pH-sensitivity (Slepushkin et al., 1997). The pH-responsiveness of the DOPE-based liposomes is based on the

proton-induced lamellar to inverted hexagonal phase transi-tion (Venugapalan et al., 2002; Simoes et al., 2001). The impactof conventional PEG-lipids on pH-sensitivity of DOPE:CHEMsliposomes could be explained in two ways: (i) because of thestructural and geometrical properties of PEG–DSPE molecules.It is well known fact that DOPE in aqueous medium at nearneutral or acidic pH, where its molecules are zwitterionic forman inverted micelles (Siegel and Epand, 1997). On the otherhand poly(ethylene glycol) derivatized phospholipids formspherical micelles in diluted aqueous solutions (Kenworthy etal., 1995; Edwards et al., 1997). It is clear that PEG-lipids shouldexhibit a complementary shape to DOPE, which prefers aninverted lipid phase. It was reported earlier that the additionof only 2.5 mol% PEG (2000)–DSPE stabilizes the lamellar phaseof DOPE-based liposomes even in acidic pH (Johnsson andEdwards, 2001). And (ii) the protonation of the head group ofDOPE and, hence, the acid-driven transition (Hong et al., 2002),might by hindered by ionization of the phosphate or carba-mate linkages introduced by the incorporation of conventionalPEG-lipids in the bilayer membrane. In the herein-describedcopolymers the lipid anchors are linked to the PEG moiety vianon-ionizable ether groups, which could be a possible expla-nation for the preserved pH-sensitivity of the liposomes inthis study. Another possible explanation is that the testedcopolymers especially those bearing two or four lipid-mimeticanchor represent more cylindrical macromolecules because ofthe greater hydrophobic part and hence lower complemen-tary shape to the inverted cone shaped molecules of DOPE ascompared with conventional PEG-lipids.

Apart from the size distribution and pH-sensitivity anotherissue addressed in this study was the influence of the coat-ing on the �-potential of the liposomes. The latter is ofgreat importance as the overall charge of colloidal carri-ers is crucial for their in vivo fate and biodistribution. The�-potential of PEG (2000)–DSPE and (DDGG)4(EO)114 coatedliposomes were compared versus plain DOPE:CHEMs lipo-somes. In both sterically stabilized formulations an increase in

Table 3 – Organ distribution of liposomes 48 h after injection

Formulation % of initial dose recovered at 48 h post-injectiona

Liver Spleen Liver and Spleen Lung Kidney

Non-coated 68 ± 4 6 ± 0.2 74 ± 4 0.1 ± 0.0 0.4 ± 0.1PEG-2000-coated 31 ± 1 26.9 ± 1.3 57 ± 1 0.2 ± 0.1 0.8 ± 0.2(DDGG)4(EO)114-coated 19 ± 3 15.1 ± 1 34 ± 3 0.4 ± 0.1 0.3 ± 0.1

a Each data point represents the arithmetic mean ± S.D. (n = 3).

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�-potential was observed being more pronounced for the for-mulation with (DDGG)4(EO)114. The more pronounced effectof (DDGG)4(EO)114 on the �-potential as compared with con-ventional PEG-lipid is probably an outcome of its specificproperties, namely, lack of ionisable linkages in its macro-molecules. Furthermore, the observed uniform fluorescenceof treated EJ carcinoma cells implies that the intracellulardelivery is not affected by the incorporation of the copoly-mers. However, the detailed mechanistic aspects of thecellular uptake are beyond the scope of the present paper.Nevertheless, the copolymers seem to be promising can-didates to develop liposomal formulations with combinedproperties of pH-sensitivity and prolonged circulation. More-over, preliminary experiments, the results of which willbe presented separately, show no significant cytotoxicityof the copolymers and corresponding liposomal formula-tions.

With a formulation containing (DDGG)4(EO)114 plasma sta-bility tests and pharmacokinetic studies were carried out.(DDGG)4(EO)114 was selected because of its greater dissimilar-ities with conventional PEG-lipids and furthermore becauseits anchoring strength was supposedly highest due to the fourlipid-mimetic units per copolymer chain, which would allowbetter incorporation in the membrane bilayer (Rangelov etal., 2001). This formulation exhibits considerably lower cal-cein leakage in plasma compared to the control DOPE:CHEMsliposomes, which indicates that the grafted PEG chains hin-der the detrimental interactions with the plasma proteinsApart from the demonstrated plasma stability and preservedpH-sensitivity, the most important property of this particu-lar formulation is its excellent blood circulation versus bothplain and PEG (2000)–DSPE stabilized DOPE:CHEMs vesicles.These liposomes are obviously able to avoid the reticuloen-dothelial system, localized in the liver and spleen, to a largerextend compared to DOPE:CHEMs liposomes stabilized by con-ventional PEG (2000)–DSPE.

In conclusion, this study shows that the utilized blockcopolymers bearing more than one lipid-mimetic anchorcan be considered as promising sterically stabilizing agentsfor the development of pH-sensitive-liposomes. We showthat the copolymers condition the formation of DOPE:CHEMsliposomes, which are stable at physiological temperatureas no significant alteration of the physicochemical param-eters related to structural changes, fusion or aggregationwere observed. An important advantage of the copolymersis that their incorporation in the DOPE:CHEMs membranedoes not significantly interfere with the pH-sensitivityof the formulation and even in some cases the acid-triggered calcein leakage was optimized. In addition, theability of the formulations to deliver calcein intracellu-larly remains unaltered, which implies that the fusogenicactivity of the liposomes is not influenced upon the incor-poration of the copolymers. Excellent blood circulationtime and ability to avoid excessive accumulation in theRES organs were achieved with liposomes stabilized with(DDGG)4(EO)114, that is the copolymer containing the largestnumber of lipid-mimetic anchors per chain. The formula-tions containing this particular copolymer were superior tothose containing commercially available steric stabilizer PEG(2000)–DSPE.

Acknowledgments

The present study received financial support from the MedicalScience Council at the Medical University-Sofia. The authorsare indebted to Eng. Cor Snell (UIPS, Utrecht, The Netherlands)for his excellent technical support for the in vivo studies.

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