JOURNAL OF LIPOSOME RESEARCH, 11(1), 55–71 (2001)

LIPOSOMES CONTAINING DOPAMINEENTRAPPED IN RESPONSE TO

TRANSMEMBRANE AMMONIUM SULFATEGRADIENT AS CARRIER SYSTEM FOR

DOPAMINE DELIVERY INTO THE BRAIN OFPARKINSONIAN MICE

I. V. Zhigaltsev,1,2 A. P. Kaplun,1,* V. G. Kucheryanu,2 G. N.Kryzhanovsky,2 S. N. Kolomeichuk,1 V. I. Shvets,1

and V. V. Yurasov2

1M. V. Lomonosov Institute of Fine Chemical Technology,Pr. Vernadskogo 86, 117571, Moscow, Russia

2Institute of General Pathology and Pathophysiology, Academy ofMedical Sciences, Baltiyskaya 8, 125315, Moscow, Russia

ABSTRACT

The active loading of liposomes with dopamine in response to an ammoniumsulfate gradient was studied. This method can be regarded as a mean to moreefficiently improve the liposomal dopamine/lipids ratio in comparison to con-ventional methods of liposome preparation. Trapping efficiency of dopamineinto liposomes exhibiting a transmembrane ammonium sulfate gradient wasshown to be dependent on liposome lipid composition, lipid concentration andtemperature. Dopamine-containing liposomes with α-tocopherol in the lipidbilayer were shown to be stable at least for three weeks. It has been foundthat intraperitoneal (i.p.) administration of conventionally prepared dopamine-containing liposomes as well as liposomes with increased dopamine/lipid ratiomay efficiently suppress the expression of parkinsonian symptoms in C57BL/6 mice with experimental parkinsonian syndrome. On the other hand, onlythrough increasing of liposomal dopamine/lipid ratio the complete compensa-tion of dopamine deficiency in the mice brain was achieved. The obtained

*Corresponding author. Fax: (095)434-87-11; E-mail: alex.kaplun@mtu-net.ru

55

Copyright 2001 by Marcel Dekker, Inc. www.dekker.com

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56 ZHIGALTSEV ET AL.

data may be considered as biochemical evidence in favor of liposomes’ abilityto act as a carrier system for the delivery of dopamine into the brain.

Key Words: Active liposome loading; Ammonium sulfate gradient; Dopa-mine; Liposomes; Parkinsonian syndrome.

INTRODUCTION

Parkinsonian syndrome is a clinical syndrome characterized by akinesia,muscular rigidity and tremor. The neurochemical hallmark of parkinsonian syn-drome is the reduction of dopamine levels in the corpus striatum due to the lossand damage of dopaminergic neurons in the substantia nigra. Recovery of dopa-mine neurotransmission has become the central goal of the pharmacological ther-apy of parkinsonian syndrome [1]. However, striatal dopamine levels are not af-fected by systemic administration of dopamine because of its inability to cross theblood-brain barrier. Thus as the mainstream of current treatment of parkinsoniansyndrome remains the use of biochemical precursor of dopamine–L-DOPA [1].Earlier we have demonstrated that long-term administration of liposome-encapsu-lated L-DOPA at a dose 10-fold lower than its traditional therapeutic dose resultsin more effective compensation of behavioral disorders in parkinsonian mice thanin case of non-liposomal L-DOPA [2]. Taking into consideration the fact thatbrain-penetrating properties of many drugs can be dramatically improved by ad-ministration in a liposomal form [3-7], we supposed that liposomally encapsulateddopamine would be able to reveal some antiparkinsonian activity in contrast tonon-liposomal dopamine. In order to confirm this assumption, we examined lipo-somally encapsulated dopamine for its therapeutic activity. Our results indicatedthat, indeed, liposomally encapsulated dopamine but not dopamine alone may par-tially restore the biochemical and behavioural deficiencies in mice with experi-mental parkinsonian syndrome [8]. Obviously, such application requires a suffi-cient loading of the drug into the liposomes. At the same time, we found thatconventional liposome preparation techniques (sonication or extrusion methods)do not provide sufficient levels of dopamine uptake into small and intermediate-sized (up to 100 nm) unilamellar liposomes due to small inner volume and drugleakage [9]. Thus, the increasing of liposomal drug-to-lipid ratio leads to unjusti-fied drug expense and, therefore, is not cost-effective. One can imagine that thisdrawback may be overcome by using transmembrane ionic gradients as a drivingforce for the liposome loading. It has been shown that many drugs including sev-eral biogenic amines can be accumulated by liposomes in response to an imposedproton gradient [10–16]. The pH-based active loading provides much higher drug-to-lipid ratio in comparison to conventional methods, but, as a rule, the pH-gradi-ents are unstable over long periods, and this results in the considerable drug leak-age. Moreover, application of this approach for catecholamines is questionable,since they can be rapidly oxidised in the alkaline medium.

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DOPAMINE AND AMMONIUM SULFATE GRADIENT 57

Stable ionic gradients suitable for loading of amphipathic weak bases canbe created on the basis of inorganic ammonium salts. In this case, a gradient of thesalt concentration between two aqueous compartments of a liposomal suspensionseparated by a lipid bilayer (inside and outside the liposomes) may be created byremoving the salt from the outside medium. Gradients of ammonium sulfate andammonium chloride were successfully used for the loading of liposomes withdoxorubicin and other anthracyclines [17–20]. Haran et al. [17] also showed thatthe ionic gradient of ammonium sulfate is stable for a long time, requires no pHchange in the outer medium, and is suitable for liposomes exhibiting a wide varietyof lipid compositions. Thus, this approach might be considered the most promisingfor the preparation of liposomes with dopamine as the functioning component,since it helps perform the liposome loading with high efficiency and using safepH values. For this reason, we have employed this method as a means to obtainthe efficient uptake of dopamine into the liposomes and improve drug retentionproperties. Our hypothesis is that alteration in the dopamine/lipids ratio wouldhave a crucial effect on the antiparkinsonian activity of dopamine-containing lipo-somes. For this reason, we studied some physic-chemical and therapeutical proper-ties of liposomal systems with improved dopamine/lipids ratios.

MATERIALS AND METHODS

Materials

Cholesterol, dopamine, EDTA, 2-thiobarbituric acid, trichloracetic acid, so-dium deoxycholate and VISKING dialysis bags were purchased from Serva (Ger-many); D,L-α-tocopherol, sodium metabisulfite and sucrose were purchased fromMerck (Germany); ammonium sulfate, chloroform, NaCl were purchased fromKhimmed (Russia); ePC was obtained from Biolek (Ukraine); hsPC and DMPCwere obtained as a gift from Lipoid GmbH (Germany). The neurotoxin MPTPused in this study was synthesized at the Institute of Pharmacology, Moscow,Russia.

Liposome Preparations

Unilamellar vesicles containing saline or passively trapped dopamine wereprepared according to the sonication method [9] using a probe sonicator (UZDN-2T, 44 kHz, 100 W (‘‘Techpribor’’, Sumy, USSR)). Briefly, dried lipid films con-sisting of ePC and cholesterol (7:3 mol/mol) were dispersed in saline or dopaminesolution to final lipid concentration of 50 mg/ml and subjected to 5 min longsonication at cooling on ice; the sonication/relaxation time ratio was 30s/30s.Vesicle size distribution (60-100 nm) was determined using JEM100-SX (JEOL,Japan) electron microscope [9], and by photon correlation spectroscopy, using the

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58 ZHIGALTSEV ET AL.

Malvern 4700 Automeasure laser light scattering spectrometer system (MalvernInstruments, UK).

All liposome preparations exhibiting a transmembrane ammonium sulfategradient were performed using phosphatidylcholine (ePC, hsPC or DMPC) andcholesterol (7:3 mol/mol). The dry lipid films formed by evaporation from chloro-form solution were hydrated with 120 mM ammonium sulfate solution with finallipid concentrations of 20, 50, and 100 mg/ml. Multilamellar vesicles were thendownsized to form unilamellar vesicles (size range 60-100 nm) using sonicationmethod (see above). Obtained suspension was then placed into dialysis bags (3ml in a bag). To establish a transmembrane ammonium sulfate gradient two conse-quent dialysis exchanges against 10% sucrose (2 � 250 ml) were used [17]. Afterthe creation of the gradient liposomes were incubated with the drug (0.5 or 1 mg/ml). Before incubation, sodium metabisulfite (1 mg/ml) was added to liposomalpreparations in order to slow down the dopamine oxidation. At various times thealiquots (0.1 ml) of the mixture were taken and vesicles separated from unen-trapped drug using a Sephadex G-50 column preequilibrated with NaCl solution(isoosmotic with the ammonium sulfate solution). Drug concentrations in liposo-mal suspensions were assayed by UV-spectroscopy at 280 nm following solubili-sation of the vesicles in sodium deoxycholate. The drug loading efficiency (%)was calculated according to the following formula:

% loading efficiency � D1 � V1 � 100/(D1 � V1 � D2 � V2)

where D1 and D2 are optical densities of the liposomal fraction and the free drugfraction (both treated with sodium deoxycholate) and V1 and V2 are the totalvolumes of the corresponding fractions.

The Determination of Ammonium Ion Concentration in theDialysis Medium

The concentration of ammonium ions was determined by measurement ofsolution potential by means of an NH4

�-selective electrode (EV-74, Russia). Thecalibration curve in coordinates EMF (mV) vs. �log[NH4

�] was obtained frommeasurements of 100, 10, 1 and 0.1 mM solutions of ammonium sulfate in 10%sucrose.

The Dynamics of Accumulation of LPO Products and Oxidized Formof Dopamine in Dopamine-Containing Liposomes

Antioxidant activity of α-tocopherol (0.005 mol/mol lipids, 0.01 mol/mol lip-ids and 0.05 mol/mol lipids) in dopamine-containing EPC/cholesterol liposomes(lipid and dopamine concentrations 20 mg/ml and 1 mg/ml, correspondingly) wasstudied. The liposomes were stored at 4°C for 21 days and samples were takenevery 24 h. To establish the level of LPO products, reaction with 2-thiobarbituric

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DOPAMINE AND AMMONIUM SULFATE GRADIENT 59

acid was used. Before incubation, EDTA up to a final concentration of 1 mM, 25%trichloroacetic acid (1.3 ml) and 0.5% 2-thiobarbituric acid (1.5 ml) were added todiluted (1%) liposome suspension (0.2 ml). The samples were then incubated for10 min in a boiling water bath, and the optical density of the solutions was measuredat 532 and 580 nm. To account for unspecific absorption in the long-wave area,the value ∆A � A532 � A580 was used [21]. Detection of oxidized form of dopaminewas carried out by absorption measurement at 390 nm [21].

Experimental Model of Parkinsonian Syndrome and LiposomesAdministration Scheme

In this study MPTP-induced model of parkinsonian syndrome [22–24] wasused. Experimental parkinsonian syndrome was induced in 6-months-old maleC57BL/6 mice by systemic i.p. injection of MPTP (20 mg/kg, twice daily, every12 h for 14 days). Animals were divided into following experimental groups, eachof 12 mice: (I) mice treated i.p. with saline instead of MPTP; (II) mice injectedi.p. with MPTP; (III) mice injected i.p. with MPTP and saline-containing lipo-somes; (IV) mice injected i.p. with MPTP and conventionally prepared dopamine-containing liposomes; (V) mice injected i.p. with MPTP and dopamine-containingliposomes with increased dopamine/lipid ratio prepared using ammonium sulfategradient. Both saline- and dopamine-containing liposomes were administered as5% lipid suspension (500 mg/kg, twice daily, every 12 h for 14 days (III, IV)and 250 mg/kg, twice daily, every 12 h for 7 days (V)). Dopamine in liposomeswas used at a dose of 3.5 mg/kg for conventionally prepared dopamine-containingliposomes or 2.5 mg/kg for liposomes with increased dopamine/lipid ratio. Thestarting parameters of motor activity in each group did not differ from one anotherand, therefore, animals of I-III groups were used for comparing of liposome-encapsulated dopamine effects on the development of parkinsonian syndrome.

Assessment of the Expression of Parkinsonian Syndrome

The development of parkinsonian syndrome was assessed by oligokinesiaestimated by changes in the motor activity. Motor activity was monitored by Auto-Track test using Opto-varimex-3 system (Columbus Instruments, USA) before and1, 7, 14 and 15 days after the beginning of MPTP administration. Using HPLC/ED(LC-4B, BAS, West Lafayette, USA) dopamine, DOPAC and HVA in mice stria-tum were analysed ex vivo.

RESULTS AND DISCUSSION

Formation of Ammonium Sulfate Gradient

First, we compared different procedures for salt removal from the outer me-dium. Unilamellar vesicles (size range 60-100 nm) were prepared in 120mM am-

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60 ZHIGALTSEV ET AL.

monium sulfate by the sonication method from a suspension of multilamellar lipo-somes made from phosphatidylcholine (ePC, hsPC or DMPC):cholesterol mixture(7:3 mol/mol). We believe that gel-exclusion chromatography is impractical forthe removal of ammonium sulfate due a significant (40-fold) dilution of the start-ing liposomal suspension. The best result was obtained when the ammonium sul-fate from outer medium was removed by twofold dialysis for 24 h against 10%sucrose solution. The concentration gradient of ammonium sulfate is characterizedby the difference in intraliposomal concentration of this salt and its concentrationin extraliposomal medium ([(NH4)2SO2]in � [(NH4)2SO4]out). The efficiency ofloading obviously depends on the value of this gradient. After 10- and 24-h dialy-ses, the ammonium ion concentration was measured using a NH4

� -selective elec-trode, whose response was preliminarily calibrated by standard (100–0.1 mM)(NH4)2SO4 solutions. We showed that twofold dialysis for 10 h and one day re-sulted in 1.2 and 0.18 mM concentrations of ammonium ions dialysis medium,respectively. Thus, an ammonium ion gradient [(NH4)2SO4]out/[(NH4)2SO4]in wasquantified as � 0. 001.

Haran et al. [17] offered the following explanation for the mechanism ofliposome loading using the ammonium sulfate gradient. The driving force of thegradient (the dissociation of (NH4)2SO4 is represented by equation 1 in Fig. 1) isassociated with a significant difference in coefficients of permeability through thelipid bilayer (P) of different compounds and ions: P(NH4)2SO4 � PSO4

2� PNH4� �� PH�

��� PNH3. A very high permeability coefficient for the ammonia molecule (PNH3

� 0.13 cm/s) results in a rapid diffusion of neutral NH3 molecules that are formedthrough the deprotonation of ammonium cations inside liposomes (Fig. 1, equa-tion 2). Such rapid ammonia diffusion from liposomes along with the concentra-tion gradient (the NH3 concentration outside liposomes is negligibly small in theinitial moment) shifts the equilibrium of reaction 2 to the right; i.e., pH valuesinside liposomes are decreased. In their turn, catecholamines in neutral form arecapable of penetrating through the bilayer. However, they acquire positive chargein the acidic medium of the inner liposome volume and almost completely losethis penetration ability, since the permeability coefficients for their charged formsare substantially less than those for neutral ones (Fig. 1, equations 3–6). Therefore,the influx rate for dopamine would be higher than the efflux rate until concentra-tion of its neutral forms inside and outside liposomes became equal.

Liposome Loading with Dopamine

Secondly, the influence of the starting lipids and dopamine concentrationsand incubation temperature on the dopamine loading into liposomes was studied.Dopamine hydrochloride was dissolved in the liposomal suspension after dialysis,and sodium metabisulfite was added in order to slow down the dopamine oxida-tion. The loading was terminated at the desired time and the efficiency of dopa-mine loading into liposomes (i.e., the ratio of dopamine loaded and primarily

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DOPAMINE AND AMMONIUM SULFATE GRADIENT 61

Figure 1. Scheme of liposome loading with dopamine by means of a transmembrane ammoniumsulfate gradient (DA, dopamine and DAH�, protonated dopamine). See text for the further details.

added) was determined by dopamine separation from the outer medium using aSephadex G-50 column. The most rapid loading of dopamine was observed duringthe first half an hour of incubation, followed by a slower phase (Fig. 2). Theaccumulation of the drug was completed after 24 h-long incubation. The achievedextent of dopamine loading did not change for at least two weeks for preparationsstored at 4°C.

It has been shown that the rate and extent of dopamine loading are tempera-ture-dependent. For ePC/cholesterol liposomes, 37% entrapment is achievedwithin 1 h at 4°C. However, at 20°C 32%, and at 37°C only 28% entrapment isachieved (Fig. 2). This fact indicates that the leakage of gradient-constitutive spe-cies (NH4

� and H�) from ePC/cholesterol liposomes increases with increasing in-cubation temperature, reducing the magnitude of ammonia gradient. In this case,some loss of the loading efficiency occurs.

It has been demonstrated also that different lipid compositions lead to differ-ent drug uptake behaviour. Table 2 shows a comparison of dopamine loading into

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62 ZHIGALTSEV ET AL.

Figure 2. Effect of incubation temperature on uptake rates and loading efficiencies of dopamine,accumulated into ePC/cholesterol liposomes exhibiting a transmembrane ammonium sulfate gradi-ent. Dopamine was added to liposomes equilibrated at 4°C (�), 20°C (�), and 37°C (�). Initiallipid and dopamine concentrations were 20 mg/ml and 1 mg/ml, correspondingly (initial drug/lipidratio � 214 mmol/mol). The loading efficiency was calculated as the ratio of dopamine loaded andprimarily added.

3 types of liposomes. The presence of DMPC instead of ePC reduces the loadingefficiency of dopamine to 24% within 24 h at 4°C. For hsPC/cholesterol liposomes18% entrapment is achieved at 4°C, and at 60°C 35% (Table 1). Thus, in hsPC/cholesterol and DMPC/cholesterol liposomes, the loading efficiency increaseswith increasing temperature. In this case, viscosity of lipid bilayer becomes a mainlimiting factor and, therefore, factors as increased acyl chain length or saturationresult in lower dopamine loading efficiencies.

As expected, the higher the initial dopamine/lipid ratio, the greater the intra-liposomal dopamine/lipid ratio (Table 2). It is also quite obvious that the efficiencyof dopamine loading into liposomes directly depends on the inner to outer volumeratio of liposomal suspension. A gradual increase in the lipid concentration and,

Table 1. Effect of Lipid Composition on the Loading Efficiency ofDopamine

Temperature, °C

Lipid composition Phase transition Incubation Loading(molar ratio) temperature of PC temperature efficiency, %

ePC/cholesterol �5 4 497:3 20 39

37 28DMPC/cholesterol 23 4 247:3 25 36hsPC/cholesterol �59 4 187:3 60 35

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DOPAMINE AND AMMONIUM SULFATE GRADIENT 63

Table 2. Characterisation of ePC/Cholesterol Liposomes Loaded withDopamine by the Ammonium Sulfate Gradient

Initial concentration, Dopamine/lipids Loading efficiencymg/ml ratio, mmol/mol within 24 h

Lipids Dopamine Initial Liposomal (14 days), %

50* 2.5 214 20 10 (8)20 0.5 107 53 49 (49)20 1 214 79 37 (39)50 0.5 42 28 66 (67)50** 1 85 44 52 (56)100 0.5 21 19 87 (89)100 1 42 33 79 (81)

* Liposomes prepared using conventional technique (sonication of multi-lamellar liposomes) tested for their antiparkinsonian activity** Liposomes with increased dopamine/lipid ratio tested for their antipar-kinsonian activity

therefore, in the general aqueous volume of liposomes led to a concomitant in-crease in the dopamine loading into liposomes. However, the increased lipid con-centration decreased the intraliposomal dopamine/lipid ratio. At the same time,it is desirable that the drug/carrier ratio be as great as possible for liposomes thatare embedded with a physiologically active substance and are potential medicines.Therefore, one should find those starting concentrations of dopamine and lipidsthat help reach the maximal dopamine/lipid ratio even at the sacrifice of somedecrease in the loading efficiency. Our results show that applied method providedan increase of intraliposomal dopamine/lipid ratio up to 79 mmol/mol (seeTable 2).

In addition, the efficiency of loading of some substances into liposomes maybe increased when insoluble salts of these substances are formed in the innervolume of liposomes. In this case, any additional amounts of such a substance ina liposome cannot increase its intraliposomal concentration. This phenomenonwas demonstrated for liposomal preparations of doxorubicin [18]. We could notmanage to obtain the saturated dopamine solution in the inner liposomal volumeby using our procedure. Nevertheless, this procedure provided almost a 90%-effi-ciency of dopamine loading into liposomes at the starting dopamine/lipid ratioof 21 mmol/mol (Table 2). Thus, we showed that our procedure helps to obtainliposomal preparations with high dopamine content, which were stable for at least14 days.

Antioxidant Activity of �-Tocopherol in Dopamine-Containing Liposomes

We had previously found [21] that dopamine loaded into liposomes in theform of 2.5 mg/ml solution slowed down the rate of ePC oxidation almost fivefold.

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64 ZHIGALTSEV ET AL.

Figure 3. Antioxidant activity of α-tocopherol (0.005 mol/mol lipids (�), 0.01 mol/mol lipids(�) and 0.05 mol/mol lipids (�)) in dopamine-containing EPC/cholesterol liposomes (lipid anddopamine concentrations 20 mg/ml and 1mg/ml, correspondingly). The level of LPO products inliposomes without α-tocopherol ( ) was taken as 100%. To establish the levels of LPO products,liposomes were incubated with EDTA (1 mM), 25% trichloracetic acid (1.3 ml) and 0.5% 2-thiobar-bituric acid (0.2 ml) for 10 min in a boiling water bath, and the optical density of the solutions wasmeasured at 532 and 580 nm. To account for unspecific absorption in the long-wave area, the value∆A � A532 � A580 was used.

However, a considerable increase in the LPO level was observed in liposomesloaded by an ammonium sulfate gradient to higher dopamine/lipid ratio. The LPOproducts usually destabilize liposomes, which became more permeable. Therefore,an efficient antioxidant was required to stabilize the LPO level. We tested α-tocopherol for this purpose and found that levels of LPO products in liposomescontaining dopamine and α-tocopherol (molar ratio 0.005, 0.01 and 0.05 mol/mol lipids) were reduced by 15%, 30% and 40%, respectively when compared toantioxidant-free liposomes (the level of LPO products in liposomes without α-tocopherol was taken as 100%). Moreover, the presence of α-tocopherol main-tained the starting level of LPO products in the lipid bilayer for at least 3 weeks.We also noticed that the intraliposomal dopamine was stable for two weeks inthe presence of α-tocopherol, whereas, without the antioxidant, oxidized forms ofdopamine were already detected 48 h after liposome preparation by means of spec-trophotometry at 390 nm. These data suggest that protective role of liposome-incorporated α-tocopherol extends both on lipids and encapsulated dopamine. Be-sides, no change in the dopamine/lipid ratio was observed in comparison withcontrol samples containing no α-tocopherol. Thus, the presence of α-tocopherolat the aforementioned concentrations produced no noticeable change in the bilayerpermeability for dopamine.

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DOPAMINE AND AMMONIUM SULFATE GRADIENT 65

Antiparkinsonian Effect of Dopamine-Containing Liposomes

Intermediate-sized (60–100 nm) unilamellar liposomes loaded with dopa-mine were applied by us in animal studies using mice with experimental parkinso-nian syndrome. Parkinsonian syndrome was induced in C57BL/6 mice by sys-temic i.p. injection of MPTP (20 mg/kg, twice daily, every 12 h). In MPTP-treatedmice the maximal decrease in the motor activity (down to 12–15% of saline-treated mice) was observed on day 7 after the beginning of MPTP injections andpersisted up to the end of experiment.

Administration of dopamine solution (i.p., 50 mg/kg) had not any effect onmotor activity of MPTP-treated mice. Administration of conventionally preparedePC/ cholesterol dopamine-containing liposomes (dopamine 3.5 mg/kg, lipids500 mg/kg, i.p., every 12 h for 14 days) resulted in increased levels of motoractivity only on day 14 after the beginning of liposomes injections (data notshown). Achieved antiparkinsonian effect remained for 5–7 h after the last lipo-some injection. However, administration of liposomes with increased dopamine/lipid ratio but in lower dose (dopamine 2.5 mg/kg, lipids 250 mg/kg, i.p., every12 h for 7 days) significantly increased the motor activity as early as on day 7after the beginning of liposomes injections (Fig. 3); and antiparkinsonian effectremained for 24 h after the last liposome injection (Fig. 4).

It has been shown also that, as expected, chronic administration of dopaminesolution into parkinsonian mice had not any effect on dopamine and its metaboliteslevels in striatum. Administration of conventionally prepared ePC/cholesterol do-pamine-containing liposomes (dopamine 3.5 mg/kg, lipids 500 mg/kg, i.p., every12 h for 14 days) elevated the striatal dopamine levels by 40%. The striatal concen-trations of DOPAC (the hallmark of intracellular dopamine metabolism) and HVA(the criteria of postsynaptic dopamine biotransformation) after 14 day long admin-istration increased in 2.6 times and by 70%, correspondingly (data not shown).In contrast, administration of liposomes with increased dopamine/lipid ratio (do-pamine 2.5 mg/kg, lipids 250 mg/kg, i.p., every 12 h for 7 days) results in com-plete compensation of the biochemical deficiency of dopamine in the striatum(Fig. 5, A). The striatal concentrations of DOPAC and HVA after only 7 day longadministration increased in 18 and 8.5 times, correspondingly (Fig. 5, B). Thisdata suggest that 2-fold increase of liposomal dopamine/lipid ratio results in morethan 4-fold increase of the striatal dopamine levels (Fig. 6).

At the same time, it was found that saline-containing liposomes showedsome beneficial effects on parkinsonian mice, including some increase of motoractivity (Fig. 4) and, probably, modulation of dopamine release from survivedneurons (Fig. 5). This phenomenon was more minutely described by us earlier[25].

In conclusion, the results of present study indicate that increase of liposomaldopamine/lipid ratio allowed considerable diminish lipid as well as dopaminedosage required for suppression of parkinsonian symptoms. It has been foundthat intraperitoneal administration of conventionally prepared dopamine-con-

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66 ZHIGALTSEV ET AL.

Figure 4. Effect of i.p. administered ePC/cholesterol liposomes with increased dopamine/lipidratio (dopamine 2.5 mg/kg, lipids 250 mg/kg, every 12 h for 7 days) on motor activity of MPTP-treated mice. Abscissa, test periodicity (1–before MPTP administration, 7–after 7 day long MPTPadministration (the day when liposomes were administered for the first time), 14–after 7 day longliposomes administration, 15–after 24 h later last liposomes administration). Ordinate, motor activity(distance traveled, cm). Data are shown as mean and SEM. Statistical analysis was performed usingANOVA-test followed by Neumar’s t-test. Differences considered to be statistically significant areindicated as follows: *, as compared with the saline-treated animals (p � 0.05); �, as comparedwith MPTP-treated animals (p � 0.05). Groups of animals (each of 12):�-mice treated i.p. with saline;�-mice injected i.p. with MPTP and saline;

-mice injected i.p. with MPTP and saline-containing liposomes;-mice injected i.p. with MPTP and dopamine-containing liposomes with increased dopamine/lipid

ratio.

taining liposomes as well as liposomes with increased dopamine/lipid ratio mayefficiently suppress the expression of parkinsonian symptoms in C57BL/6 micewith MPTP-induced experimental parkinsonian syndrome. On the other hand,only through increasing of liposomal dopamine/lipid ratio the complete compen-sation of dopamine deficiency in the mice brain was achieved. Furthermore, 2-fold increase of dopamine content in the liposomes permitted to shorten the termof liposomes administration from 14 to 7 days and prolong a therapeutic effectup to 24 h.

Taken together, the obtained data may be considered as biochemical evi-dence in favor of liposomes’ ability to act as a carrier system for the delivery ofdopamine into the brain. These findings lead to possibility that there are somespecific transport mechanisms which facilitate permeation into the brain for lipo-somally encapsulated substances.

The mechanism of brain uptake of liposomes is still unclear; there are twomost probable ones: endothelial tight junctions opening or endothelial transcytosis

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DOPAMINE AND AMMONIUM SULFATE GRADIENT 67

Figure 5. Levels of dopamine (A) and its metabolites: HVA and DOPAC (B) in the striatum ofMPTP-treated mice. Dopamine, DOPAC and HVA were analysed ex vivo using HPLC/ED after 7day long administration of liposomes with increased dopamine/lipid ratio. Data are shown as meanand SEM. Statistical analysis was performed using ANOVA-test followed by Neumar’s t-test. Dif-ferences considered to be statistically significant are indicated as follows: *, as compared with thesaline-treated animals (p � 0.05); �, as compared with MPTP-treated animals (p � 0.05). Groupsof animals (each of 12):�-mice treated i.p. with saline;�-mice injected i.p. with MPTP and saline;

-mice injected i.p. with MPTP and saline-containing liposomes;-mice injected i.p. with MPTP and dopamine-containing liposomes with increased dopamine/lipid

ratio.

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68 ZHIGALTSEV ET AL.

Figure 6. Comparison of striatal dopamine levels in MPTP-treated mice after 14 day long adminis-tration of conventionally prepared dopamine-containing liposomes (dopamine/lipid ratio 20 mmol/mol lipids) and 7 day long administration of liposomes with increased dopamine/lipid ratio (44mmol/mol lipids). The level of dopamine in mice injected with saline was taken as 100%. Groupsof animals:�-mice treated i.p. with saline instead of MPTP;�-mice injected i.p. with MPTP and saline;

*-mice injected i.p. with MPTP and conventionally prepared dopamine-containing liposomes;**-mice injected i.p. with MPTP and dopamine-containing liposomes with increased dopamine/

lipid ratio.

[26]. Opening of the blood-brain barrier can be obtained by a local or systemicincrease of the intravascular pressure or by intracarotid injection of hyperosmolarsolutions. According to current opinions, endothelia of brain capillaries have areduced ability to endocytosis. However, it was found recently that liposomes aswell as nanoparticles seem to be able to cross the blood-brain barrier by trans-cytosis [27–32].

In case of dopamine-loaded liposomes administration the opening of theblood-brain barrier owing to increase of blood pressure is quite possible, becausedopamine is well known as blood pressure increasing agent. Moreover, it is possi-ble that liposomal encapsulation helps to prevent untimely biotransformation ofdopamine due to depot effect and thus contribute to the general therapeutic effect.But some data, for example, magnification of biochemical effect in 4 times bymeans of 2 times increase of liposomal drug loading persuade us to assume thatin this case, most probably, transcytosis takes place.

Further investigation of these mechanisms would be worthwhile for moreprecise design of new drug forms.

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DOPAMINE AND AMMONIUM SULFATE GRADIENT 69

ABBREVIATIONS

EDTA–ethylenediaminetetraacetic acid, EMF–electromotive force, ePC–egg phosphatidylcholine, DMPC–dimyristoylphosphatidylcholine, DOPAC–3,4-dihydroxyphenylacetic acid, HPLC/ED–high-pressure liquid chromatographywith electrochemical detection, hsPC–hydrogenated soybean phosphatidylcho-line, HVA–homovanillic acid, i.p.–intraperitoneal, L-DOPA–L-3,4-dihydroxy-phenylalanine, LPO–lipid peroxidation, MPTP–1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine, PC–phosphatidylcholine.

ACKNOWLEDGMENT

This work is supported in part by the grant 05.04.05 from MINVUZ ofRussia.

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