a new class of drug carriers: micelles of poly(oxyethylene)-poly(oxypropylene) block copolymers as...

Jou*ndofConIralledRele~e, 22 (1992) 141-I 58 @ 1992 Elrwier Science Publishers B.V. All rights reserved 0168-3659/92/$05.00

COREL 00770

A new class of drug carriers: micelles of poly (oxyethylene)- poly (oxypropylene) block copolymers as microcontainers for

drug targeting from blood in brain’

Alexander V. KabanoP, Elena V. Batrakova”, Nikolai S. Melik-Nubarovs, Nikolai A. Fedoseev’, Tatiyana Yu. Dorodnich”, Valery Yu. Alakho+, Vladimir P. Chekhoninb, Irina

R. NaaarovaC and Victor A. KabanovC

(Received I4 November 1991; accepted in revised form lOJune 1992)

A new concept of design of drug delivery systems based on using self-assembling supramacromole- cular complexes is formulated. Microcontainers for drug targeting were prepared using polymeric surfactant poly(oxyethylene)-poly (oxypropylene) block copolymer (pluronic). Molecules of a drug arc solubilized in a pluronic micelle being incorporated into its inner hydrophobic core, formed by poly(oxypmpylene) chain blocks. The outer hydrophilic shell of such micelles is formed by nontoxic and nonimmunogenic poly(oxyethylene) blocks. Solubilization of low molecular weight compounds (fluorescein isothiocyanate (FITC), halopetidol etc.) in pluronic micellcs was studied using fluorcs- cence and ultracentrifugation. The dimensions of the aggregates formed in the solutions of various pluronics (PS5, F64, L68, LlOl ) and its mixtures were determined using quasielastic light-scattering technique. In a majority of cases the diameter of pluronic micelles (including those containing solubilized compounds ] was in the range of 12-36 nm. For targeting of such microcontainers to a certain cell the pluronic molecules were conjugated with antibodies against a target-specific antigen or with protein ligands selectively interacting with target cell receptors. The obtained conjugates were then incorporated into the drug-containing micelles by simple mixing of the corresponding components. It was found that solubilization of FITC in pluronic micelles considerably influences its distribution in animal (mouse) tissues resulting, in particular, in the drastic increase of FITC fluorescence in lung. Con- jugation of FITC-containing micelles with insulin vector results in increase of FITC penetration in all tissues including the brain. The specific targeting of the solubilixed FITC in brain was observed in the case when the pluronic conjugate with antibodies to the antigen of brain glial cells (curglycoprotein) was incorporated into micelles. Under these conditions the considerable increase of FITC fluorescence in the brain and decrease of its fluorescence in the lungs has been registered. Possibility of using micellar microcontainers for targeting of solubilixcd neuroleptics (haloperidol) in brain was studied. Incor- poration of antibodies to cY,-glycoprotein into haloperidol-containing micelles results in a drastic in-

Correspondence IO: A.V. Kabanov, Dept. of Bioregulation. Research Center of Molecular Diagnostics and Therapy. Simphew polskii bd. 8, Moskow 133149, Russian Federation. ‘Paper presented at the Is1 ChinaJapan-USSR Joint Symposium on Advanced Polymers, Moscow, September I S-21.1991.

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crease of drug effect. This result indicates that vector-containing pluronic micelles provide an effective transport of solubihzed neuroleptics across blood-brain barrier.

Key words: Blood-brain barrier: Drug targeting; Polymeric micelles; Neuroleptics

Introduction

In the middle of seventies many researchers hoped to End a possibility to use liposomes as universal containers for the targeted transport of various biologically active substances in an organism [ I 1. Studies during the next decade have revealed serious limitations of this technology. At present, we knoa .~.ly about a few examples of successful application of the liposomal method of drug introduction mainly for the therapy of diseases of the reticuloendocytic system [2].

The matter is that some intrinsic properties of liposomes principally restrict the possibilities of their application for drug targeting. Being ther- modynamically unstable, liposome systems cannot therefore be stored for a long time. The efficiency of incorporation of many biologically active substances into liposomes is relatively low. Liposomes introduced into the blood stream do not penetrate very well in tissues through capillary walls and are mostly taken up by the cells and organs of the reticuloendothelial system. And, finally, liposomes are recognized by the immune system of the organism and are in this way removed from circulation [ 21.

One of the possible approaches to increase the stability of liposomes is to use polymerizing lip- ids for their production [ 31. Thus it cannot be excluded that in some cases, polymeric lips somes may become a prospective tool for drug targeting. At the same time, high stability of polymeric liposomes may be a negative factor suf- ficiently hindering their removal from the organism.

We assume that most of the above mentioned problems can be solved using another basic concept, namely, creating drug targeting systems as self-assembling supramolecular complexes. Such complexes should meet the following major requirements:

(i) The complex should form spontaneously from drug molecules, components providing for the recognition of target calls and other components that are necessary for its functioning. (ii) For effective penetration in tissues, the size of the complex species should not exceed the size of viruses. (iii) Before reaching the target cell the complex should be stable and biologically inert (nontoxic, not recognizable by the immune system, not degradable, not affecting nontarget ceils of the organism, etc.); a drug release should pro- ceed only as a result of interaction of the complex with a target cell (in case of necessity such interaction should be accompanied by effective intracellular translocation of the drug). (iv) After interaction with a target, it should be possible to remove the components of the complex easily from the organism.

Most of the above enumerated requirements to organization and functioning of the supramolecular complexes are characteristics of viral species.

The phenomenon of spontaneous assemblage of molecules in supramolecular complexes with strictly controlled composition and structure is characteristic of most micellar colloid systems. In such systems aggregates with various packing of surfactant molecules are formed, the parame- ters of which (geometric dimensions in particular) may be purposefully varied in the wide range [4]. Within the framework of the strategy proposed, it is of principal importance whether the mixed micelles can be assembled from several different micelle-forming components and whether those micellar systems are able to solu- bilize, i.e., spontaneously incorporate the non- micelle-forming molecules being poorly dissolved in bulk solvents. Exactly these properties of micelles make it possible to use them as microcontainers for drug targeting.

Two major approaches to the creation of systems for drug targeting on the basis of surfactant micelles have been suggested (Fig. 1). One of them implies covalent bonding of a drug to the micelleforming surfactant molecules [ 5 1. This method (Fig. 19) permits to increase in vitro and in viva stability of the antitumor drug (adriamycin) and to diminish its toxicity, keeping constant its chemotherapeutic effect (61. An- other approach is based on noncovalent incorporation of a drug in micelles [ 71. Recently the possibility of application of this approach for in viva drug targeting using the micelles of polymeric surfactant-pluronic (poly(oxyethylene)- poly(oxypropylene) block copolymer) has been successfully demonstrated [ 8 1. Being solubilized in the micelles, drug molecules incorporate into the micelle hvdronhobic core. formed bv poly (oxypropylenk) chain blocks. The outer hi- drophilic shell of such micelle is formed by nontoxic and nonimmunogenic poly (oxyethylene) blocks. For targeting of micellar microcontainers to a certain cell, micelle-forming pluronic molecules are conjugated with antibodies against a

target-specific antigen or with protein l&and selectively interacting with target cell receptors. The obtained conjugates are then incorporated into the drug-containing micelles by simple mixing of the corresponding components (Fig. 1 A).

We have demonstrated [8] that incorporation of the neuroleptic (haloperidol) into pluronic microcontainers, conjugated with brain-specific antibodies or insulin significantly increases the biological action of the drug. The possibilities of using pluronic micelles for targeted delivery of a drug from blood stream into brain are analyzed in this paper. The paper consists of three parts. In the first part we characterize the micelles of various pluronics and study the solubilization of low-molecular compounds into them. The second part is devoted to the study of in viva distribution of a fluorescent dye (model drug), incorporated into pluronic micellcs, upon its introduction into mouse organism. The possibility of using the above described technique for targeted delivery of this dye into the brain is demonstrated. In the third part the data on the biological activity of haloperidol solubilized in

Fig. I. Schematic representation of mirellar microcontainers for targeted delivery of drugs (A) on the basis of plumnic PBS surfactant [g] and (8) of adriamycin conjugate with poly(oxyethylene)-paly(arpaRic acid) block copolymer [6,7]. Sizes of

micelles measured by quasielastic light scatteringare presented.

144

pluronic micelles, including those containing brain-specilic antibodies and insulin, are presented.

Experimental part

Material characterization

Pluronics (synperonics, poly(oxypropylene)- poly(oxyethylene) block cv:~lymers) were sup- plied by Serva and used witnout further pttriti- cation. Main characteristics of these compounds are presented in Table I.

Polyclonal antibodies (Ab) against human brain ol,-glycoprotein (anti-a&P Ab) were obtained from Shinshilia rabbits, immunized with purified preparations of corresponding antigens [9-l I]. Specific antibodies were purified by im- munosorption using the antigen immobilized at the CNBr-activated Sepharose 4B [ 121. Mono- clonal mouse Ab against human alcoholdehy- dmgenase (anti-ADH Ab) were provided by Dr. P.G. Sveshnikov (Research Center of Molecular Diagnostics and Therapy, Moscow). The clone ADHIES producing these Ab has been derived from hybridization of Sp 2/O myeloma cells of a BALB/c mouse immunized with purified ADH.

Iatraductlon of sldehyde groups in plumnic molecules

Terminal hydroxyl groups of plurcmic P85 molecules were oxidized by tert-butylhypochlor- ide (TBHC) in fert-butyl alcohol (TBA):

TABLE I

Plumnic (synperonic) chsracwislics

HO-(EtO),,&PrO).-(EtO),,,H+t-BuOOCl-+

-tHO-(EtO),,JPrO).-(EtO),,,z,.,-CHrCHO

+t-BuOH+H,O

TBHC was obtained by treatment of TBA (Reakhim, Russian Federation) with dry chlo- rine at 20°C [ 141. Five hundred and twenty mil- ligrams of pluronic P85 were dissolved in 15 ml of TBA. One hundred and twenty microliters of TBHC were added to this solution during stirring of the latter. The reaction mixture obtained was incubated at 20°C in dark during 20 h. The precipitate formed (containing modified pluronic P85) was tiltrated and dried in vacua at 10°C.

The content of aldehyde groups in pluronic molecules was determined spectrophotometri- tally at 460 nm using the reaction of oxidized phtronic P85 with excess of dinitrophenylhydra- zinc in 50 mM HsP04, pH 0.56. The content of aldehyde groups depended on the concentration of TBHC and on the duration of incubation of reaction system. In the above described example the average content of aldehyde groups was I. 15 per 1 pluronic molecule.

Synthesis of FITC-plaronic majugate

In order to introduce fluorescent label in pluronic molecule the oxidized plumnic P85 containing aldehyde groups was modified with hexa- methylenediamine (HMDA) and then with fluorescein isothiocyanate (FITC). Twenty-fold

Code General formula’ Block length Molecularmasa Hydrophilic-li- pophilicbslan-

(n) (m) ce (HLl3)

F68

PRS L64 LIOI

HO-(EtO),,r(PrO),- (EtOl,,,H 30 160 8800 29

39 51 4500 I6 30 21 2950 15 55 1 3500 I

*EtO=-CHrCH@-,PrG=-CH>-CH(CH,)O-

145

Micellar solution preparation

Bid&i&d water with par&i conductivity 0.056 &cm purified by Milii-Q device (MiIIi- pore) was used for preparation of all miceliar solutions. From 1% to 10% piuronic solutions in water (or in aqueous buffer) were used for solu- biiization of Sudan, FITC, or halope~dol. (FITC was incubated during 2 h in 1.5 M aqueous solution of 2-amino-2(hydroxymethyl)-1,3-pro- pancdioi (TRIS) at pH 6.8 before sob&&&ion in piuronic mice&s.) In a number of cases, the concentrated solutions of Ins, Ab or their conjugates with pluronic P85 were added to the miceliar systems. In all cases micellar systems were incubated for 1 h at 37°C just before using in experiments described below.

Determination of critical micelle cmwentration (CMC)

The values of the CMC of pluronics (Table 1) in aqueous solutions were determined in the course of the study of solubilization of water in- soluble dye-Sudan. The dry Sudan (Reakhim, Russian Fede~tion) was added to pluronic so- lutionsof various surfactant concentrations. The mixtures were shaken during 5 h at 37’C and then centrifuged at 7000 rpm for separation of the aqueous phase from Solid Sudan particles. The con~nt~tion of a soiubiiizcd Sudan in the micellar phase was determined spcctrophotometri- tally at 540 nm.

Fiuoresccnce spectra of the micellar solutions were obtained at 37OC using a Hitacid F 4000 spectrofluorimeter with a thermostatic cell unit equipped with a stirring device. Absorption spectra were obtained at 37°C using the Hitachi 150/20 spectrophotometer.

The I-average translational diffusion co&- cients and the hydrodynamic diameters of pluronic micelles were measured at 37°C by the

molar excess of HMDA (Sigma) and 50-fold molar excess of sodium cyanoborohydride (Sigma) were added to the solution of 300 rag of oxidized piuronic P85 (containing 0.73 aI&- hyde groups per 1 surfactant molecule) in 10 ml of 0.1 M H,B04 buffer (pH 9.5). After 2 h incubation at 20°C 300 mg of FITC on celite (Sigma) were added to the reaction mixture. After I h incubation at 20°C the reaction mixture was filtrated and purified by J-step dialysis against water, 50% and 90% isopropanol in water. The conjugate obtained was purified 2 times by gel-filtration on Sephadex LH-20 in 90% isopropanol media. According to the data of gel-filtration on Uitragei A44 the sample obtained did not contain free FITC molecules.

Pluronic cnnjugation with proteins

Proteins were conjugated with oxidized pluronic P85 molecules using the reductive alkyia- tion reaction [ i 5 1, Two-fold molar excess of insulin (Sigma) and 100-fold excess of sodium ~~o~robyd~de were added to the solution of 10 mg of oxidized pluronic P85 (containing 0.8 aldehyde groups per 1 surfactant molecule) in I ml of 0.1 M H,BOS buffer (pH 9.5). After 4 h incubation at 20°C the reaction mixture was dialyzed against I mM ammonium solution in water and lyophilized. The conjugate obtained was puriiied by geitiltration on Ultragei A-202.

One thousand two hundred micrograms of Ab (ant&-GP Ab, anti-ADG Ab) were incubated during 4 h at 20°C with iOO-fold molar excess of oxidized piuronic P85 (containing 0.8 aldehyde groups per 1 surfactant molecule) and lOO-fold molar excess of sodium ~anoborohyd~de in 0.1 M H3BO3 (PH 8.5). The reaction mixture was dialyzed and lyophiiized as described above. The conju@es were purified using gel-filtration on Ultragei A-44. The Ab activity was determined by indirect enzyme immunoassay using peroxidase-labelled antispecies Ab. After conjugation with piuronic molecules the Ab retained from 50 to 70% of their initial titer.

quasielastic light-scattering method using Auto- sizer 2c (M&em) small-angle laser photometer with 10 mW He-Ne laser (633 nm). The auto- correlation function of the fluctuations of’ the scattering intensity was determined using Cor- relator K 7032-05 (Malvern). The z-average coefficients of translational diffusion of scattering species were calculated by cumulants method. The diameters of equivalent hydrodynamic spheres ofthe micelles were calculated on the basis of the obtained values of the translational diffusion coefficients.

Sedimentation coefficients of the micelles in l-2% pluronic P85 solutions were measured at 37°C either in scanning or in schliren modes. In the first case (scanning mode) the analytical ul- tracentrifuge Beckman E equipped by photoe- lectri~dl scanning device with monocromator and multiplexor was used. In the second case (schliren mode) we used the Beckman E ultracentri- fuge with schliren-optic registration system. The rotation rates of An-D rotor with 12 nm mono- sector cell were equal either to 48000 rpm (scanning mode) or 56000 rpm (schliren mode). The scanning was carried out at 280 nm.

Sedimentation coefftcients were calculated from experimental data using the Nina1 program.

The molecular mass (M) of pluronic P85 micelles at 37°C was determined using the Sved- berg equation:

M=s”RT’fDo( 1 -up),

where so and D” are ihe sedimentation and translational diffusion coefficients at infinite dilutions, o the partial specific volume of the micelle, Tthe temperature, R the universalgas constant and p the solvent density. The so and D” values, determined using the extrapclation of the sedimentation and translational diffusion coefficients to infinite dilutions were equal to 6S=6* lO’-‘s and 3.7.10-’ cm2/s respectively.

The a value, determined using the pycnometer, was equal to 0.84 cm3/g.

FITC distribution in mause organism

The experiments were conducted using male DBA-2 mice (20-25 g body weight) obtained from Kriukovo department of the nursery of the Russian Academy of Medical Sciences. From 1 to 5 tug/ ml FITC solutions in aqueous buffer (2 mM TRIS, pH 9.0) or in pluronic micelIar sys- tams ( 10% pluronic solution in 2 mM TRIS, pH 9.0) were intraperitoneally or intravenously introduced in mice organism in the dose 200 $1 per 20 g body mass. In the case when the distribution of RTC-pluronic PSS conjugate was studied micellar solutions introduced into mice contained 0.5 mg/ml of the conjugate and 10% of pluronic P85. All micellar solutions were incubated during 1 hour at 37°C before introduction in mice.

One or two days after FITC introduction mice were killed by cervical dislocation and the Sam- ples of lungs, liver, brain, heart and kidneys were collected for histological studies. These samples were rapidly frozen and used for the preparation of the 10 m cryostatic slices which were ana- lysed and photographed using a fluorescent Op ton microscope.

Bioiogical activity of solubilized haloparidol

In viva biological action of sohtbilized haloperidol was studied as described [8]. Briefly, haloperidol (6 mg/ml) was dissolved in micellar solutions of various compositions or in dis- tilled water (pH 6.0). Micellar solutions were incubated at 37°C for 1 h, and then intraperitoneally introduced to the groups of 10 male DBA- 2 mice in the dose approximately equal to LDso. The control groups of mice were treated with the physiological solution or with corresponding micellar systems without haloperidol. The kinetics of mouse death in the group was determined.

The development of specific neuroleptic symptoms (closing symptom, extremity trac- tions, other extrapyramidal disorders, seizures)

was registered according the previously described criteria [ i 6 1.

The quantitative estimation oftbe changes of mouse behavior reactions resutting from haloperidol introduction was nerformed. The arouns bf 10 DBA-2 male mice with similar charaaer- istics of moving activity were formed. Haloperi- dol, dissoived either in micellar solutions or in physiological solution was intrdpcritoneally introduced in mice body in the dose 2 mg/k8 body web&t. Mouse vertical and horizontal mobility and grooming characteristics were registered for each group with 30 min intervals during 15 h using the Rhema Labortechnik device.

Characteristics of micellar microrontainers

The values of the critical micelle concentration (CMG) of pluronics in aqueous solution, presented in the literature usually differ considerably, depending on the procedure used for their determination [ 171. We have estimated the CMC for piuronics P85 and L64 in the course of me study of the water-insoIubIe dye (Sudan) so- hbiiization in pluronic solutions. At 37°C thus obtaffled CMC were equal to 0.01% (PBS) and 0.05% (L64). These vahzs are in good agree ment with the ones determined eadier using Ross method and surface tension measurements [ 181. All fulzher experiments described in this section were performed at 37°C: using from I to 10% pluronic solutions, in the conditions where ae- cording to the literature data f 17,181 and our own observations pluronic molecules form micellar associates.

Tbe dimensions of pluronic associates were determined using quasielastic ~~~ts~tte~ng [ 191. As can be seen in Table 2 large surfactant species with diameters lying in the micrometer range are formed in the solutions of pluronic MS and of the mixture of phuonics P85 and LlOl. The sizes of the micelles formed in all other systems studied lie in the range from 15 nm [for pluronic P85) to 33 nm (for the mixture of pluronics F68 and L64). it is exactly those size val-

10% plumnic F68 Fmm i fo 10% pbuonie P85 10% plumnic L64 10% pluronic mixtures? PS5+L64(1:1.5) P85+L101 (I :0.4) F68+L64(1:2.5) Fmm I to i% pi&& 5’85, containing 3% of BSA 1% pluronic PBS. containine 0.7kglmlAb ” 198 pifuro~ic P85, containing 0.6 mg/ml insulin 10% plumnic PG. containing: 0.05 mg/ml Sudan I mg/ml FITC 6 me/ml baiooeridol

720 From 11 to IS

20

17 5700

33 Fran I3 to 14

Fwm 14 to 16

36

17 12 1s

ues that are characteristic for the dimensions of viruses.

The molecular mass of pluronic IV.5 mice!@ determined using the extrapolation of the zbv- erage translational diffusion and sedimentation coeficients to infinite dilutions was equal to 260 kDa which yields an aggregation number of 58. This value lies in the range of the molecubu masses f 80 to 680 kDa) and aggregation num- bers (25 to 70) described earlier for micelles of various pluronics (L64, F68 etc.) under similar experimental conditions (surfactant concentration, temperature) [ lS,lS]. The radius of the micelle poly(oxypropyIene) core and the thick- ness of its poly( oxyethylene) shell estimated using the obtained value of the aggregation number ( = 58 ) as described [ 191 were equal to 3.7 nm and 2.1 nm respectivefy.

Micelle sizes do not change significantly in the presence of Ab, insulin and even 3% of bovine serum albumin, which represent the major pro-

148

tein existing in blood serum. Moreover, the sedimentation coefficients ofpluronic micelles do not change in the presence of Ab or BSA (data not shown). This is in agreement with the earlier statement that water soluble pluronics do not interact with plasma proteins (in particular with serum albumins) [11X,20].

Introduction in the mice&u systems of the pluronic-FITC conjugate as *veil as low molecular compounds (FITC, Sudan and haloperidol) does not result in considerable change of micelle sizes (Table 2). At the same time changes of fluorescence spectra of PITC and haloperidd, which take place in the presence of pluronic in solution (Fig. 2 ), are indicative of incorporation of these compounds in the micelle content.

The direct evidence of the solubilization of low-molecular compounds under study in pluronic micelles was obtained in course of ultra- centrifugaiion performed in schliren and scanning modes. The sedimentation coefficients of pluronic micelles in aqueous solution are deter-

k 350

Fig. 2. Fluorescence emission spectra of (A) FITC and (B) haloperidol measured either in regular water solulion (pH 9.0) or in 296solutionofpluronicP85 (QH 9.0). Experimen- tal conditions: (A) [FITC]=O.OS mglml, lex=486 nm; (9) [Haloperidal]=2.0 mglml. lex=338 nm;

mined using schliren technique. The micelles do not absorb light in the UV-range, hence, their sedimentation can not be followed in scanning mode. At the same time addition of a low moles- ular weight dye (FITC or haloperidol) permits to “visualize” the pluronic micelles under these experimental conditions. The border corresponding to the sedimenting micelles appears at the sedimentation curve in the case scanning is performed at the absorption wavelength of the dye. The sedimentation coefficients of pluronic micelles determined in this case are approximately equal to the ones measured using schliren technique (Table 3).

Though the question about the distribution of solubihzed compounds among pluronic micelles needs further study, the shape of the sedimentation curves indicates that this distribution is rather uniform and the solubilization at least does not result in the formation of a new microphase.

The incorporation of the conjugates of pluronic P85 with FITC and Ins in the micelles was proved in the course of gel-filtration experiments using Ultragel A44 and A202 resins respectively (data not shown in Figures). The pluronic P85 micelles do not remain in the gels and elute in the void volume. This permits separat- ing the micelles and unsolubilized compounds with lower molecular mass. Unlike the free FITC (unsolubilized part) and Ins, remaining in the gels, the corresponding conjugates elute in the void volume with the micelles. This method was unsuitable for the micelles and Ab separation since they are characterized by similar retaining volumes. However, the data obtained for some

TABLE 3

Sedimentation coeiticientr of pluronic P85 micelles ar 37-C ---

Micellar system studied Sedimentation coefficient (S)

Schliren mode Scanning mode

10% pluronic P85 3.1 10% phwonic, containing

0.05 mg/ml Sudan 5.0 5.0 I mg/ml FITC 3.0 3.0 2 mg/ml haloperidol 3.0 3.0

other proteins (peroxidase, Staphylococcus au- rats enterotoxin B) permit the conclusion that protein conjugation with pluronic P85 using the above described method results in its incorporation in the micelle content.

In rive distribution of FITC incurporated iGo micellar microcontainers

In order to study in viva distribution of a drug after its administration in mice we used FITC as a model drug compound. The free FITC or its micellar solutions were administrated either intraperitoneally or intravenously. Using the examples of free and solubilized in pluronic P85

149

micelles FITC we proved that the results obtained in the cases of intraperitoneal and intra- venous administration are similar. The fluorescent micmphotographs of the slices of mouse organs corresponding to the case of intraperitoneal administration are presented in Figs. 3-6. Since major differences in distribution of FITC were observed in the cases of lungs, liver and brain, only photographs of these organs are shown. FITC distribution pictures obtained one or two days after FITC introduction in mice were similar. However, the maximal fluorescence of FITC in mouse tissues was observed one day after administration. The experimental data corre-

Fig. 3. Fluorescent micropholographs oflung slices of (A) comrol mowe (FITC was not introduced). (B) mouse lrcated with 5 mg/ml FlTC in regular aqueow soludon and mice treated with 5 mglml FITC in micellar solutions containing: (C) 1046 pluronic F68, (D) lO%pluronic P85, (E) 10% plumnic L64, (F) IO% pluronie P85+L44 (1: 1.5) mixture, (0) 10% Pluronic

P85+LlOl (I :0.4) mixture. (H) 10% pluronic F68 t L64 ( 12.5) mixture. Ma@icatian X200.

Fig. 4. Fluorescent microphatographsofliverslicerofmice treated with 5 mglml FITC in micellarsolutionsof (A) IO%pluranic P85 and (Et) Io%pluronic P85+LIOI (I :0.4) mixture. Magnification X200.

sponding to this case are presented below (Figs. 3-6).

FITC solubilization in pluronic micelles results in a drastic change of distribution pictures observed. The fluorescence of organ cuts increases in the sequence: brain, heart, kidneys, liver and is maximal in lungs, when FITC micellar solutions are administered (data not shown in Figures). At the same time it is known that being introduced in regular aqueous solutions the majority of low-molecular compounds (including sodium fluorescein) are accumulated mainly in kidney and liver [ 2 L 1.

The distribution picture observed for micellar solutions is strongly influenced by the pluronic chemical constitution and micelle system composition. This phenomenon being observed for all tissues studied, is most pronounced in lungs and liver.

As seen in Fig. 3, fluorescence in lungs is considerably higher when FITC micellar solutions have been administered if compared with ho- mogeneous ones. While solubilized in pluronic

micelles, FITC penetrates basal capillary membranes surrounding the bronchioles, alveolar paths and alveoli and contrasts lung structural units. The minimal penetration in tissues was observed for the micelles of hydrophilic phwonic F68. The penetration er&iency increases with increasing pluronic hydrophobicity in the series: F68, P85, L64 (Fig. 3 C-E).

The reason for such behaviour may be rather complicated. On the one hand it cannot be excluded that the efficiency of FITC penetration in tissues depends on the membranetropic properties of pluronic which may correlate with HLB of the latter (see [ 22: ). Meanwhile, it is also probable that the picture observed is affected by FlTC partitioning between the aqueous and micellar phases which may depend on the surfactant HLB.

On the other hand it is reasonable to expect that the micelle size is an essential factor influ- encing the distribution picture. In fact, the fluorescence of liver cuts is much stronger in the case of pluronic P85+LlOI mixture than that ob-

served in ah other cases studied, for example, for pluronic P85 itself (Pig. 4). It is Iikely that large

change of the size of the latter (Table 2). The in

PBS +LlOl micelies, of which the size lies in the viva distribution of conjugated and solubilimd

micrometer range (Table Z), am captured in liver (~on~ovalen~y inw~mted in the micelIe)

tissues more effectively than other significantly FITC molecules was compared (Fig 5). The flu-

smaller micel!ar species. At the same time we orescence of heart and kidney cuts in these two

have not observed any striking differences in cases was found to be identically low. At the same

FfTC fluorescence in lungs for mixtures of var- time the iluorescence of the conjugated FITC was

ious pluronics (Pig. 3 F-H), which diRer both ~nsidem~y lower in lungs aad higher in kidney and brain than that observed in the case of the

in HLB (Table 1) and in the size of aggregates formed (Table 2).

In order to study the pluromc ~~~tiun after its administration in mice the covalent FITC- pluronic P85 conjugate was synthesized. As was mentioned above thus labelled surfactant mole cules incorporate in phtronic miceiies without

solubilized dye (Fig. 5). It is highly probable that after “releasing” of the solubilieed FITC in lungs the mi~ell~fo~ing surfactant molecules can contimte their advancement is mouse organism, finally being collected in liver and brain.

The basic question arises in this connection: is it possible to address a schtbihzed drug to a cer-

Fig. 6. Fluorescent microphamgraphsoi (A-D) bmgand (E-H) brain slicesaimice, treated with I mg/ml FlTCin (A,E) vector- irec IO% pluronic P85 solution or in the same zalufion containing: (B,F) 0.4 mglml Ins-pluronic P85 conjugate, (C,G) 2.5 mgl ml antia,GP Ab-pluranic P8.5 conjugate and (D.H) both 0.4 mg/ml Ins-pluronic P&5+*.5 mg,ml antia,CiP Ab-pluronic

P85 conjugatn Magnification x600.

tain cell or organ in the organism? Two types of “vector” molecules, namely Ins and Ab to neu- rospecitic protein (anti-a,-GP Ab) were used for targeting of drug-containing plumnic micelles in mouse brain. Ins is known to be capable of specific binding with receptors of cells of various types 1231, presumably with the ones in the blood brain barrier (BBB). According to the literature data it may possess brain convulsion activity [24] and may be capable of transcytosis across BBB [ZS]. lmmunoaffinity purified Ab to a+P (specific antigen of brain glial cells [9,10] ) represented much more selective veo tom. The immunofhtorescence study, performed using cuts of various mouse organs, revealed that

anti-az-GP Ab interact only with brain tissues (data not shown).

Vector molecules were conjugated with pluronic P85 using the procedure described under Experimental. These conjugates were incorporated in the content of plumnic P85 micelles by mixing corresponding components.

In vivo distribution of FITC solubilized in thus obtained micellar microcontainers was studied. The increase of FITC fluorescence in all organs under study was observed when Ins-conjugated micelles were administered instead of vector-free system. At the same time the general regularities of FITC distribution in various organs were not changed (Fig. 6 A,B,E,F). We suppose that the

153

Ins moiety exposed on the surface of micelles can bind with Ins receptors, thus enhancing penetration of the sohtbilixed FITC in ah tissues,

The FlTC distribution picture changes dmsti- tally when anti-crs-GP Ab are used as a vector. lo this case the amount of FITC captured in lungs is considerably decreased (Fig. 6C), while the fluorescence of the brain cuts increases (Fig. 6G). As seen in Fig. 6G along with capillary staining the diffusive fluorescence of tissues, separated from capillaries, is observed which is indicative of FITC penetration across capillary basal membranes in brain.

Administration of micelles containing simul- taneously Ins and anti-cY*-GP Ab vectors results in appearance of FITC fluorescence both in lung and brain slices (Fig. 6D,H).

The data obtaiaed do not bring the direct in- dication on the mechanism of the targeted transport of Ab-containing micelles in the brain. It is to be expected that interaction of Ab with the antigens of brain @ai cells [S,lO] is only possible after penetration of the former through the BBB. It cannot be excluded that micelles themselves can penetrate BBB, e.g. via transcytosis (see in this respect the data on FITC-pluronjc P85 con- jugatepenetration in brain tissues (Pig. 5F)). In this case the accumulation of solubilimd FITC in tite brain may result from interaction of micelle-itmorporated Ab with antigens in glia ceils. At the same time it should be taken into consid- eration that we use in this work polyclonal Ab that are probably capable of interacting with antigens located on the external side of BBB (e.g. in the sites of endothel~al cell contacts). We have earlier described the phenomenon of targetiug from blood in brain of fatty acylated Ab to a*- GP and GFA (glial fibrillar acid antigen) [26], which like the effect discussed in this paper may result from Ab binding with the external side of BBB.

Eiohtgicai activity of solubilimd haloperidol

To8ether with neuroleptic action haloperidol produces toxic effect on mice, which can be qusntitatively estimated by measuring LX&. It has been previously reported [ 8 f that solubili-

-of

Dead MC.3

lo T--- _.-

8

0

a&ini&tion in mice i~re+plar aqueous &ution, in 109b ptumnic FE5 solution and in 10% plumnic P85 solution containing 0.4 a&/ml Ins-pluronic conjugate. Walaperidol ~8 administrated to smups of 10 mice in doses approximately equat to LD,,,: 55 mg/kgiwdyweigbt (reguiarsoiution): 42.4 m~kgb~y weight (~I~r~~rni~lle5) ~4.5 mg/kg (1~s~ containing miceller). The doses of pluronic P85 admink trated with the drug are equal to 700 mg/kp body weight (vector-free micelIes) or 76 mglkg body weight (Ins-con- faioing micelles). Administration of haloperidot-free I@% plumnieP85soluiioninthesamedosesdoesn~~lnoure

death.

zation of haloperidol in pluronic P85 micelles

results in minor increase of the neuroleptic toxicity. Under these ~nditions the h~~~doI*f~ micelles are nontoxic: in the doses up to I g per

kg body weight pluronic P85 does not produce any toxic effect on mice for at least 14 days after its admi~stmtion. This is in agreement with known data about low toxicity of water soluble pluronics [ 27 1.

Incorporation into the micelles of Ins cova- lently linked to the surfactant molecule was fob

i 1

lowed by much stronger increase (up to 25-fold) in the drug efkct. M~nwbiIe the nonmodi~ed Ins, which is not incorporated into tbe mice&s, does not affect the toxicity of haloperidol. These data [8] correlate with the above result of FITC distribution study indicating that Ins vector enhances penetration of a solubilized drug in aJJ tissues including brain.

Along with the incmase of the toxicity the change of the dynamics of halopetidoi biological action upun its incorporation in pluronic micelles was observed l-3 days after the drugintro- duction in mice in doses appmximately equal to LD,,. As seen in Fig. 7 in the case of adminJstra- tion of reguJar aqueous solution of haloperidol about 40% of n&e die during the first day. The biaJogicuJ action of haloperidol sohtbilized in the

micelles of pluronic P85 fincludbtg those containing Ins vector) is deveJoped much more slowly: its toxicity is manifested only 3 days after administration. When pluronic P85 and haloperidol are introduced separately (with au inter- val of 15-30 mitt), the delay in the action of the neuroleptie is not observed. The effect of the psolo~ed action ofthe solub~z~ form of hakr- peridol is evidently connected with the differ- ence in its pathway and mechanism of reaching the biological targets.

The study of animal behavior reactions, per- mitting to estimate precisely the pharmacolo- gical activity of dopamine~~ ~rn~unds [ 271 was performed. The mouse behaviour reactions, namely its mobility (the number of mouse mi- gcations in acell) and grooming (characterizing

155

TABLE 4

Effect of pluronic PBS on mouse behaviaur reaction

Behaviourcharacteristic studied. Measurement petformed on

Tested system

Physiological solution

10% pluronic Pg5b

Horizontal mobility 1st day (arb. units) 2nd day

72k22 66+_30 77*17 71*41

Vertical mobility (arb. units)

Grooming (arb. units)

36 day’ 1st day 2nd day 3d day tst day 2nd day 3d day

65i13 70f34 40229 38234 36f25 30f28 3c+1a 24516

329?17 33or97 369569 3942 58 324?105 394*57

aValuesaremeanfSD (Pc!l.OS). bPluronic PBS dose equals to SO mg/kg body weight.

the rate of animal adaptation to unknown conditions) were determined as described under Ex- perimental. IO these experiments halopeddol was introduced intraperitoneally in the doses approximately 25-fold lower than its LDra in aqueous solution.

As seen in Fig. 8 (Series I) haloperidol solubilization in pluronic micelles results in minor decrease of mouse mobility and more pronounced decrease of grooming. Meanwhile haloperidol-free phtmnic solution does not produce any effect on mouse behaviour (Table 4). Incor- poration of Ins-plumnic I’85 conjugate in haloperidd containing micelles considerably increases the neuroleptic effect [Fig. 8 (Series 1) 1.

Fig. 8 (Series II) illustrates the effect of anti- or,-GP Ab vector on the biological activity of solubilized haloperidol. Incorporation of Ab-pluronic P85 conjugate in the micelles results not only in drastic decrease of mouse mobility and grooming but in some cases in almost complete termination of moving activity, which then results in mouse death.

This result is in agreement with the above ob- servation that anti-+GP Ab may serve as the vector for targeting of the sohtbiiized FITC in brain. At the same time it correlates with the results of our previous work [X] demonstrating that incorporation of Ab to neurospeciiic pro-

tein (GFA) in haloperidol-containing pluronic P85 micelles leads to the drastic increase (up to SIO-fold) in the toxicity and neumleptic effect of the drug.

The data obtained give evidence that haloperidol incorporation into vector containing phtmnic micelles increases the efficiency ofthe neu- role& transport across the BBB in brain and enhances its action at brain dopamine receptors.

Conclusion

Several attempts have been made over the last years to develop the technique for drug targeting across undamaged BBB [ 26,29-3 I 1. In this paper we demonstrate that the possible solutions of the problem can be based on the above formulated concept of drug targeting using micellar microcontainers. We believe that one of the key trends of future research in this area should be the elucidation of the mechanism of transbarrier transfer of a solubilized drug.

At the same time the study of the possibilities of micege delivery not only in brain, but in other tissues towards various targets (e.g. tumors, in- fected cells, etc.) is of great importance for de- veloping the proposed concept and subsequent design of new drug forms.

We have recently demonstrated that low-mo-

156

lecular compound (ATP) being solubilized in pluronic micelles acquires the ability to penetrate within an intact cell in vitro [ 321. The incorporation in the pluronis micclles of the protein vector, capable of receptor-mediated endocvtosis significantly enhances the cell loading with the soiubiliscd fluorescent dye [33]. These results permit to expect that pluronic micelles may become an effective tool for targeting into a cell of various bioactive compounds.

Acknowledgements

This work was supported by the grant “New Methods of Bioengineering” of the Russian State Committee of Science and Technology. The au- thors want to thank Profs. S.A. Arzhakov, A.V. Levashov and ES. Severin for fruitful discus- sions, Dr. PG. Sveshnikov for donating anti- ADG Ab, Dr. O.V. Makarova for the help in FITC distribution study, Dr. N.Y. Lebedeva for synthesizing oxidized pluronic, and T.S. Grigo- rieva for help in preparation of this manuscript.

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a new class of drug carriers: micelles of poly(oxyethylene)-poly(oxypropylene) block copolymers as...

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