Liposome-polymer systems. Introduction of liposomes into a polymer gel and preparation of the polymer gel inside a liposome

Liposome-polymer systems

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14. P. De GENNES, Macromolecules 9: 587, 1976 15. V. P. BUDTOV, Vysokomol. soyed. A25: 477, 1983 (Translated in Polymer Sci. U.S.S.R. 25: 3, 555, 1983) 16. KLEIN, Macromolecules 11: 852, 1978 17. M. DAUOD and P. De GENNES, J. Polymer Sci. Polymer Phys. Ed. 17: 1971, 1979 18. T. CALLAGHAM and D. PINDER, Macromolecules 18: 373, 1985 19. B. SMITH, S. MUMBY, E. SAMULSKY and P. YU, Macromolecules 19: 470, 1986 20. J. MANNER, K. LIU and J. ANDERSON, Macromolecules 44: 5, 686; 6, 748, 1971; Amer. Chem. Soc. Polymer Preprints 16: 116, 1975 21. P. GREEN, P. MILLS, C. PALMSTROM, J. MAYER and E. KRAMER, Phys. Rev. Letters 53: 2246, 1984 22. M. ANTONIETTI, J. COUNTANDINI and H. SILLESCU, Macromolec. Chem. Rapid Commun. 5: 525, 1984; Macromolecules 19: 793, 1986

Polymer Science U.S.S.R. Vol. 30, No. 10, pp. 2307-2312, 1988 Printed in Poland

0032-3950/88 $10.00+ .00 © 1990 Pergamon Press plc

LIPOSOMF~POLYMER SYSTEMS. INTRODUCTION OF LIPOSOMES INTO A POLYMER GEL A N D PREPARATION OF THE POLYMER GEL INSIDE A LIPOSOME* V. P. TORCHILIN, B. SCHLARB, A. L. KLIBANOV, N. N. IVANOV, H. RINGSDORF (

Institute of Experimental Cardiology All-Union Cardiological Science Centre, U.S.S.R. Academy of Medical Sciences Institute of Organic Chemistry, Gutenberg University, Mainz, West Germany

(Received 20 May 1987) The evolution of liposomes after their introduction into a model polymer gel based on acrylamide is studied. Liposomes containing the polymerizable monomer in the internal aqueous phase are obtained. This monomer has been polymerized with formation of microsphere polymer particles coated with a phospholipid double layer. Both types of liposomal polymer preparations obtained can be useful for the creation of new systems of controlled drug release and new types of immunological aids.

ONE possible m e t h o d of using liposomes as carriers for medicinal substances or i m m u nological aids is i n t r a m u s c u l a r or s u b c u t a n e o u s i n t r o d u c t i o n [1]. U n f o r t u n a t e l y , the fairly r a p i d d e s t r u c t i o n of the liposomal m e m b r a n e u n d e r physiological c o n d i t i o n s prevents e s t a b l i s h m e n t of a long lasting medicinal aid by this method. O n the other * Vysokomol. soyed. A30: No. 10, 2160-2164, 1988.

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V.P. TORCI-tiLINet al.

hand, many attempts have been made to establish medicinal substances based on polymer materials [2] containing medicinal preparations, and introduced subcutaneously or implanted in muscle tissue. There are two complex problems to be solved when these methods are used: the bio-utilization of the polymer material when it has lost its therapeutic action, and the possibility of including low molecular weight medicinal substances in the polymer gels, for which the rate of diffusion from the polymer carrier is too high. The establishment of carriers combining polymer stability and the low permeability of liposomes could be a solution to this problem. The inclusion of the appropriate medicinal substance in the liposome and subsequent inclusion of the liposome containing the medicinal substance in a polymer gel obtained from biologically degraded polymers, for example from polysaccharides, polyamino acids, or synthetic polymers with biologically degraded bonds [3, 4] is a natural method of obtaining such preparations. In this case diffusion of the medicament from the preparation takes place in two stages, i.e. slow separation of the liposome from the polymer, followed by quicker degradation of the liposomes and exit of the free medicament from them. ff it were possible to control the properties of liposomes and polymer materials over very wide limits, it would be possible to obtain a wide variation of the parameters of the preparatioms obtained. The first mention of the inclusion of liposomes in polymer gels is contained in papers by Weiner et al. [4] and Popescu et al. [5]. Polymerization in the aqueous phase inside liposomes is of interest for several reasons. Firstly, polymerization of counter-ions on the internal side of a liposomal membrane without polymerization within the whole of the internal volume of the liposome provides a means of establishing structures simulating to a specific extent the cyto-skeletal protein structures adjacent to the cell membrane on the internal side; in particular, a model of erithrocyte spectrin cells developed by Ringsdorf et al. [6, 7]. Secondly, polymerization in the whole volume of the intra-liposomal aqueous phase provides a means of obtaining nano-particles with a fairly narrow particle size range. These nanoparticles can be easily isolated by treatment with a detergent or an organic solvent. Various physiologically active compounds can easily be included in the phospholipid polymer particles, the rate of release of which is determined by two diffusion processes, i.e. diffusion through the polymer gel and diffusion through the phospholipid double layer, and can be controlled over very wide limits. Furthermore, since the phospholipid composition and the physicochemical properties of the liposomal membrane can be controlled, a path is opened for establishing microspherieal preparations having different degrees of effectiveness on interaction with various cells and tissues. Such preparations are suitable for various medico-biological studies, including establishment of new active substance controlled release systems. In this work an attempt was made to study the fate of liposomes on their inclusion in a model polymer gel based on acrylarnide, and also to obtain liposomes containing a polymerizable monomer in the internal aqueous phase, and subsequent polymerization of this monomer with formation of microspherical polymer particles coated with a phospholipid double layer.

Liposome-polymer system~

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~luorescent liposomes were obtained by phase inversion [7] from a mixture of lecithin (Kharkov Factory for Bacterial Preparations, U.S.S.R.) and cholesterol ("Sigma"), in 2 : 1 weight ratio at a total concentration of 10 mg/ml in 0.15 molar of buffered physiological solution (BPS) (0.15 mmole/l. NaCI, 10 mmole/1, phosphate), pH 7"4, in the presence of 1 mmole/l, of the fluorescent dye eosin ("Merk") and 0'5 or 1.5 mole of glucose solution (or subsequent inclusion in 3 and 10% polymer gels respectively). With this in view, 20 mg of egg lecithin and 10 mg of cholestrol were dissolved in 9 ml of anhydrous ether, and the solution obtained was mixed with 3 ml of 0"15 molar BPS, containing one rumple/1, eosin and the required amount of glucose. The mixture was treated for a short time on a "Branson" ultrasonic disintegrator, and the emulsion obtained was evaporated on a rotary evaporator at a pressure of 30 mbar to produce a gel, and the flask containing the gel was shaken on a "Vorteks" instrument, after which the evaporation of the mixture was continued at a pressure of 40 mbar until no ether odour could be detected, and an opalescent liquid was formed. The liposomes were separated from the unused dye by gel filtration on a PD-10 column in a solution containing 6 g acrylamide and 0"3 g methytene-bis-acrylamide (both from "Merk") or in a solution of 20 g acrylamide and 1 g methylene-bis-acrylamide in 200 ml of BPS, for subsequent production of the 3 and I 0 ~ polyacrylamide gel respectively. In both cases the column was previously equilibrated by an appropriate monomer solution, and the dye was separated in this solution. Inclusion of appropriate concentrations of glucose in the liposome is necessary in order to avoid hyperosmotic degradation of the liposomes. After gel filtration, 1 ml each of a liposome suspension (liposome with. 0.5 mole of glucose in a 3 ~ monomer solution and liposome with 1"5 mole glucose in a 10~ monomer solution were mixed with 2 pl of tetramethylethylenediamine and 15/al of a 10 ~ solution of sodium persulphate in BPS. The specimens were then polymerized by heating at 50°C at 30 min in a water bath. To obtain liposomes containing monomer inside them, the phase inversion method [7] was also used. The aqueous phase was 3 ml of BPS, containing 0"2 g acrylamide, 0.02 g methylene-bisacrylamide, 3 pl tetramethylethylenediamine, and 1 mg of the polymerization initiator 4,4-azo-bis(4-cyanopentanic) acid. In the next stage, 2.5 ml of the liposome suspension obtained were applied to the BD-10 column ("Pharmacia"), and the 2.5 ml was collected in up to 6 ml eluant. The column was equilibrated with a 2 molar glucose solution in BPS to avoid osmolysis of the liposomes, and 3"5 ml of this solution were used for elution. The liposome fraction was separated into two equal parts, one of which was used as a control, and the other was polymerized in a thin-walled test tube by irradiation from a mercury lamp over 30 min. The state of the liposomes in the gels was controlled visually, using a "Zeiss IM-35" fluorescence microscope. The liposome solution or fine pieces of polymer gel were placed on the microscope stage between glass plates. The specimens were photographed with the screen and microscope connected to a "Sony" videomagnetic recorder. Liposome formation and the formation of polymer granules inside the liposomes was controlled by dynamic light scattering with a "Nanosaizer" instrument (West German), on an optical microscope under phase contrast conditions (Zeiss 1-35), and by means of a scanning electron microscope [8]. F l u o r e s c e n t liposomes were used to study the feasibility of including liposomes i n a n acrylamide p o l y m e r gel. These liposomes c a n be easily separated f r o m ' t h e excess, u n used dye by gel filtration, a n d r e m a i n intact (no separation of dye) at least for several hours (Fig. l a shows a typical picture). P o l y m e r i z a t i o n does n o t affect the intactness of the liposomes, a n d they cart be clearly seen in 3 a n d 1 0 ~ p o l y a c r y l a m i d e gel, a l t h o u g h in this case gel f o r m a t i o n results to some extent in their aggregation (Fig. lb).

2310

V.P. TORCI-IILINet

aL

Intact liposomes can be observed in the gel specimens even after 2 weeks of storage, although in both cases the gels acquire some degree of fluorescence over the whole volume, which indicates gradual exit of the dye from the liposomes (Fig. 2).

FIG. 1. Liposomes containing eosin in unpolymerized lOyo acrylamide solution (a) and in the same solution after polymerization (b). Addition of excess 0.2 ~ of a solution of the detergent X-100 to polyacrylamide gels containing liposomes results in rapid degradation of all the liposomes in 3 Yo gel and uniform distribu.tion of the fluorescence over the whole specimen volume, whereas in 10 ~o polyacrylamide, evert after several hours, individual liposomes cart be distinguished, or in each case, local particles with increased fluorescence (Fig. 3). According to electron microscope data (Fig. 4) and dynamic light scattering data (Table 1), ort polymerization in the internal aqueous liposome phase microspherical particles of size about 600 nm are formed. The conditions of isolation and polymerization do not affect the external form and size of the particles. Addition of a 10~o solution

FIG. 2

FIG. 3

FIG. 2. Liposomes containing eosin in 3 % polyacrylamide gel after 2 weeks storage. FIo. 3. 10~ gel with liposomes containing eosin, 4 hr after treatment with Triton X-100.

iAposorde-poiymer systerds

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DIMENSIONSOF PARTICLESIN NM (RESULTSOF DYNAMICLASERLIGHTSCATTERING) Specimen, No.

Specimen characteristics Whole mixture after preparing liposomes Specimen 1 +Triton X-100 Liposomes, separated from monomer not included by gel-filtration Specimen 3+Triton X-100 Liposomes after UV irradiation Specimen 5 +Triton X-100 ~f:

[lipid] = 6 mg/ml 587, 624, 671

Particle size [lipid] = 10 mg/ml 688, 638, 643

681, 595, 646

Particles not observed 654, 678, 646

615, 630, 684 554, 567, 493

Particles not observed 707, 767, 671 508, 575, 564

of triton X-100 to a specimen of unpolymerized liposome results in fluorescence of the specimens and the disappearance of particles, which can be recorded microscopically or by means of dynamic light scattering. On the other hand, the detergent does not affect the particles in the polymerized specimen. The formation in this ease of phase-separated polymeric nanoparticles also indicates the preservation of intactness by the liposomes during their manipulation and that the monomer is not ejected into the medium. In both cases the action of the detergent should result in complete solubiliza-

FIG. 4. Polymerized liposomes before (a) and after (b) addition of Triton X-100 solution. Scale 1/2m. tion of the liposomes, but whereas in the case of the unpolymerized l!posomes this produces distribution of the intra-liposomal aqueous phase (monomer solution) over the whole specimen volume, in the case of the polymerized liposomes the detergent only removes the external phospholipid double layer and does not affect the polymeric nanoparticles formed inside the liposomes. Accordingly, methods of forming a polymeric gel in the internal aqueous liposome phase, mad also inclusion of liposomes in a polymer gel have been considered. The liposomes included in the gel can be preserved in it for a long period, and release their

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A.V. YEHMOVet aL

c o n t e n t s only slowly. T h e extent to which the l i p o s o m e s are affected by external d a m aging factors c a n be c o n t r o l l e d b y c h a n g i n g the gel con0entration. B o t h types o f l i p o s o m a l p o l y m e r i c p r e p a r a t i o n s o b t a i n e d c a n evidently be useful f o r establishing new systems f o r c o n t r o l l e d release o f a drug, a n d also for establishing new t y p e s o f i m m u n o l o g i c a l aids.

Translated by N. STANDEN REFERENCES 1. Liposomes and Immunobiology (ed. by V. N. Tom and H. R. Six), New York, 1980 2. Controlled Drug Delivery I-1I (ed. by S. D. Bruck)~ Boca Roton, 1982 3. J. KOPECEK and P. REJMANOVA, Controlled Drug Delivery, Vol. 1 (ed. S. Bruck), Boca Raton, 1982 4. A . L . WEINER, S. S. CARPENTER-GREEN, E. C. SOEHNGEN, R. P. LENK and M. C. POPESCU, J. Pharm. Sci. 74: 922, 1985 5. M. C. POPESCU, A. L. WEINER and S. S. CARPENTER-GREEN, PST Intern. Appl. WO 85 03 640, 1985 6. H. BADER, K. DORN, B. HUPFER and H. RINGSDORF, Advances Polymer Sci. 64: 1, 1985 7. F. SZOKA and D. PAPAHADJAPOULOS, Proc. Nat. Acad. Sci. USA 75: 4194, 1978 8. E. I. CHAZOV, A. V. ALEXEEV, A. S. ANTONOV, V. E. KOTELIANSKY, V. L. LEYTIN, E. V. LYUBIMOVA, V. S. REPIN, D. D. SVIRIDOV, V. P. TORCHILIN and V. N. SMIRNOV, Proc. Nat. Acad. Sci. USA 78: 5603, 1981

Polymer Science U.S.S.R. VoL 30, No. 10, pp. 2312-2318, 1988 Printed in Poland

0032-3950/88 $10.00+ .00 O 1990 Pergamon Press plc

RELATION BETWEEN CRAZE FORMATION AND SHEAR STRAIN ON STRETCHING OF CROSSLINKED HIGH DENSITY POLYETHYLENE IN A LIQUID MEDIUM* A. V. YEFIMOV, N . N . VALIOTTI, F. F. SUKHOV, V. I. DAKIN a n d N. F. BAKEYEV M. V. Lomonosov State University, Moscow

(Received 22 May 1987) The effect of irradiation induced chemical crosslinking on the strain produced in high density polyethylene specimens in a liquid medium is studied. The efficiency of action of the liquid medium, as evaluated from the decrease in the forced rubber-like elasticity limit, is inversely related to the irradiation dose. This effect is due ~o the decrease of the craze formation contribution to the high pressure polyethylene strain with increase in crosslinking density. •* Vysokomol. soyed. A30: No. 10, 2165-2169, 1988.