- Email: [email protected]
Biotinylated Stealth® magnetoliposomes
Chemistry and Physics of Lipids 120 (2002) 75 /85 www.elsevier.com/locate/chemphyslip
Biotinylated Stealth† magnetoliposomes Michael Hodenius a, Marcel De Cuyper b,*, Linda Desender b, Detlef Mu¨llerSchulte a, Alois Steigel c, Heiko Lueken a b
a Rheinisch-Westfa¨lische Technische Hochschule Aachen, Prof. Pirlet-Straße 1, D-52074 Aachen, Germany Interdisciplinary Research Centre, Katholieke Universiteit Leuven-Campus Kortrijk, B-8500 Kortrijk, Belgium c Heinrich-Heine-Universita¨t Du¨sseldorf, Universita¨tsstraße 1, D-40225 Du¨sseldorf, Germany
Received 7 January 2002; received in revised form 17 June 2002; accepted 22 July 2002
Abstract Dimyristoylphosphatidylethanolamine (DC14:0PE) and the dioleoyl analogue (DC18:1cis PE) were mixed with abiotinylamido-v-N -succinimidoxycarbonyl-poly(ethylene glycol) (NHS-PEG-biotin) and quantitatively converted to abiotinylamido-v-(dimyristoylphosphatidylethanolamino-carbonyl)polyethylene glycol (DC14:0PE-PEG-biotin) and the dioleoyl analogue DC18:1cis PE-PEG-biotin, respectively. As shown by thin-layer chromatography and 1H NMR spectroscopy, PEGylation of both phosphatidylethanolamine types went to completion if the reaction was performed in organic solvent in the presence of triethylamine. The resulting derivatives were successfully incorporated into both classical phospholipid vesicles and a phospholipid bilayer surrounding nanometer-sized magnetite cores. In the latter case, the so-called activated Stealth† 1 magnetoliposomes were produced which very efficiently immobilized streptavidinylated alkaline phosphatase. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Magnetoliposomes; Phospholipid /protein conjugates; Phospholipid vesicles; Poly(ethylene glycol)-biotin derivatized phospholipids; Streptavidinylated enzymes; Stealth† magnetoliposomes
1. Introduction
* Corresponding author. Tel.: /32-56-246221; fax: /32-56246997 E-mail address: [email protected] (M. De Cuyper). 1 Stealth † is a registered trademark of SEQUUS Pharmaceutical, Inc. The term ‘Stealth’ was proposed for these long-circulating, sterically stabilized liposomes by T.M. Allen, 1989. In: Lopez-Bernstein, G., Fidler, I. (Eds.), Liposomes in the Therapy of Infectious Diseases and Cancer, New Series 89. Alan R. Liss, New York, pp. 405 /415.
Liposomes are colloidal structures consisting of phospholipid bilayers, encapsulating an aqueous space. In recent years, these structures have been proven to have great potential in the medical field, e.g. as promising homing devices for selective delivery of drugs to diseased cells (Baldeschwieler and Schmidt, 1997). In general, the size and type of liposome depend on the phospholipid composition and the preparation procedure (New, 1997). For instance, small unilamellar vesicles (SUVs), which are used in this work, consist of only a single
0009-3084/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 0 2 ) 0 0 1 0 5 - 6
76
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
bilayer and have a diameter of about 30 nm. Unfortunately, a main drawback with regard to the medical use of these classical liposomes is their limited blood circulation time due to their rapid uptake by the reticuloendothelial system (RES) in the liver and spleen. However, Jones (1995), Lasic et al. (1991), Papahadjopoulos et al. (1991), Trubetskoy and Torchilin (1995) found that the blood clearance rate can be significantly reduced by grafting a limited number of water-soluble, flexible polymer chains, such as poly(ethylene glycol) (PEG), onto the surface of SUV. The continual conformational change of the polymer, indeed, hinders recognition by the RES and, thus, increases the blood halflife (Blume and Cevc, 1993). This approach is used to produce the socalled Stealth† liposomes. Specific targeting of these particles to a malignant tissue can be achieved by linking a specific monoclonal antibody to the particle surface (Allen et al., 1994), e.g. by covalently coupling a relevant monoclonal antibody to the PEG terminus of a phospholipid /PEG complex (Holmberg et al., 1989; Mori et al., 1991; Allen et al., 1995). In this context, the so-called post-insertion technique of Zalipsky et al. (1997), Moreira et al. (2002) is worth mentioning. These authors first synthesized phospholipid /PEG /oligopeptide conjugates and subsequently incubated them as micellar aggregates with preformed Stealth† liposomes. Ultimately, the tailor-made complexes were embedded in the outer, surface-grafted polymeric ‘‘brush’’. In applying this strategy, however, a new derivative must be synthesized for each application (Viitala et al., 1998). Fortunately, this cumbersome work can be circumvented using a universal precursor complex that can bind a wide variety of antibodies. A phospholipid /PEG /biotin complex is a good candidate for achieving this goal, since it can interact with a broad scale of commercially available (strept)avidinylated antibodies. To further enlarge the number of potential applications, in this study, we chose to work with Stealth† liposomes in which the inner aqueous space is completely filled with magnetizable iron oxide, magnetite (Fe3O4). As a result, these structures can easily be captured in a high-gradient magnetic field, thus allowing quantitative removal of non-
membrane-adsorbed material from the reaction mixture in a rapid and elegant way. In this study, we first investigated the optimal conditions for the synthesis of the phosphatidylethanolamine (PE)-PEG-biotin derivative, starting from dimyristoylPE (DC14:0PE) or dioleoylPE (DC18:1cis PE) and a-biotinylamido-v-N -succinimidoxycarbonyl-poly(ethylene glycol) (NHS-PEGbiotin), and verified the degree of adduct formation by thin-layer chromatography (TLC) and 1H NMR spectroscopy. Subsequently, the ability of the conjugate to be incorporated into phospholipid vesicles and magnetoliposomes was checked. Finally, using streptavidinylated alkaline phosphatase (SAP) as a model protein, we proved that the membrane-linked PE conjugate was able to strongly bind streptavidinylated biomolecules. The binding of a streptavidinylated enzyme to biotin-grafted Stealth† magnetoliposomes is illustrated in Scheme 1.
2. Experimental procedures 2.1. Materials Dimyristoylphosphatidylglycerol (DC14:0PG) and DC14:0PE, both stored at /20 8C as lyophilized powder, and DC18:1cis PE (stored in CHCl3) were supplied by Avanti Polar Lipids (Birmingham, AL). NHS-PEG-biotin (lyophilized powder) with an average molecular mass of approximately 3400 was supplied by Shearwater Polymers Europe (Enschede, The Netherlands). SAP, used as a suspension containing 1000 U/ml, was from Boehringer GmbH (Mannheim, Germany). The 1H NMR-solvent, chloroform-d (CDCl3), was purchased from Cambridge Isotope Laboratories, Inc. (Andover, USA). p -Nitrophenyl phosphate (disodium salt) was from Sigma (Deisenhofen, Germany). All solvents, buffer substances, and chemicals used for TLC were of analytical grade and were purchased from Sigma or Merck-Eurolab (Darmstadt, Germany). Analytical silicagel 60 plates and Kieselgel 60 (0.040 /0.063 mm), used as the matrix for silica gel adsorption chromatography (henceforth referred to as ‘‘flash chromatography’’), were also from Merck-Eurolab.
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
77
Scheme 1. Binding of streptavidinylated enzyme to biotin-PEG-grafted magnetoliposomes.
Sephacryl S-300 and the solvent-resistant column for size exclusion chromatography were purchased from Pharmacia Biotech (Freiburg, Germany). 2.2. Phospholipid and Fe3O4 determinations The phospholipid and Fe3O4 contents of magnetoliposomes were measured spectrophotometrically on a Uvikon 933 instrument. The determination of phospholipid content according to the method of Vaskovsky et al. (1975) involved a prior, complete thermal digestion at 180 8C for 45 min of the phospholipid sample in the presence of 70% perchloric acid; the resulting inorganic orthophosphate was then reacted with acidic molybdenum to form a blue complex with an absorption maximum at 820 nm. The Fe3O4 content was determined by dissolving the magnetite core in 37% hydrochloric acid/65% nitric acid (3/1, v/v), chelating the Fe3 with Tiron (De Cuyper and Joniau, 1992), and measuring the absorption at 480 nm. 2.3. Preparation and fractionation of phospholipid vesicles Phospholipid vesicles were prepared by codissolving DC14:0PG and the PE-PEG-biotin complex in chloroform at the concentrations indicated. After evaporation of the organic solvent in a stream of nitrogen, the residue was taken up in 2 ml of 5 mM N -Tris[hydroxymethyl] methyl-2aminoethanesulfonic acid (TES) buffer, pH 7.0. To obtain a clear homogeneous dispersion, the lipid /buffer mixture was then sonicated for 4 min using a probe-tip sonicator (MSE Ultrasonic 150W disintegrator, equipped with a 3/8 in. probe) set at the maximal power setting at which no
frothing occurred. The resulting vesicles were then centrifuged for 10 min at 10,000 rpm/min (Sorvall RC-5B equipped with a SS-34 rotor) to remove titanium particles produced by abrasion of the probe. Occasionally, the vesicles (2 ml at a phospholipid concentration of 4.69 mol/ml) were loaded onto a Sephacryl S-300 column (46 /1 cm2) and eluted with 5 mM TES buffer, pH 7.0, at a rate of 16 ml/h. Fractions (1 ml) were collected and examined for light scattering at 260 nm and for the presence of phospholipid by phosphate determination (see above). Fractions of interest were pooled and analyzed by 1H NMR spectroscopy (see below). 2.4. Magnetoliposome preparation Magnetoliposomes were produced according to a previously described protocol (De Cuyper and Joniau, 1988). In short, nanometer-sized magnetite grains (diameter about 15 nm) were first stabilized by a cushion of laurate molecules and then incubated and dialyzed in the presence of preformed sonicated vesicles. During this step, the fatty acid molecules were gradually exchanged for a bilayer of phospholipid molecules. The practical details were as follows: a mixture of 9 ml of SUVs with a phospholipid concentration of 8.41 mmol/ml (DC14:0PG/PE-PEG-biotin molar ratio equal to 9/ 1) and 0.177 ml of the magnetic fluid stock solution (concentration: 61.08 mg Fe3O4/ml) was dialyzed for 72 h at 37 8C in Spectra/Por dialysis tubings (molecular weight cut-off, 12,000 /14,000) (Spectrum medical Industries, Los Angeles, CA). The dialysis buffer, which was changed at regular intervals, consisted of 5 mM TES, pH 7.0. Excess vesicles were removed by high-gradient magneto-
78
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
phoresis. Fourteen aliquots of 0.643 ml each were simultaneously pumped at a uniform rate of 6 ml/h through tubing (inner diameter: 0.078 in.), plugged with loosely packed steel wool (/50 mg; Type 430; Bekaert Steel Corporation, Zwevegem, Belgium), placed in a 3 mm gap between the two pole faces of a water-cooled electromagnet (Bruker, Type BE-15, Karlsruhe, Germany). The electromagnet was operated at 30 A and 80 V. Under these conditions, but in the absence of the magnetic filter matrix, the magnetic field between the two poles is about 2.3 T. After separation, each retentate was thoroughly washed with 2.9 ml of TES buffer to remove any nonadsorbed phospholipids remaining in the capillary liquid surrounding the iron fibers. Then the magnetic field was turned off and the retentates were recovered from the filter matrix by a high-speed buffer stream (500 ml/ h) of 0.643 ml of buffer per aliquot. The retentates were then checked for phospholipid and Fe3O4 content. The magnetoliposomes which contained the a-biotinylamido-v-(dimyristoylphosphatidylethanolaminocarbonyl)polyethylene glycol (DC14:0PE-PEG-biotin; 1) conjugate had a mmol phospholipid/g Fe3O4 ratio of 0.69, while magnetoliposomes prepared using the dioleoylanalogue of the phospholipid derivative (DC18:1cis PE-PEGbiotin; 2) had a ratio of 0.61. These values are indicative of the presence of a complete bilayer enwrapping the iron oxide core (De Cuyper, 1996). Transmission electron microscopy (Zeiss EM 10C) further revealed a uniform diameter of 25 nm for the magnetoliposome colloids. 2.5. Purity tests: TLC and 1H NMR analyses TLC was carried out in CHCl3/CH3OH/25% NH3 (70/25/4, v/v). After drying, the plates were sprayed with molybdenum blue reagent (Dittmer and Lester, 1964) and ninhydrin solution in acetone (Christie, 1982) to detect phosphate and free NH2 groups, respectively. NHS-PEG-biotin was detected using iodine vapor. 1 H NMR spectra were recorded on a Varian MERCURY 200, INOVA 400 or UNITY 500 spectrometer. Chemical shifts are expressed in ppm downfield of the internal standard, tetra-
methylsilane. The test molecules under investigation were dissolved in CDCl3. The following data were obtained for NHSPEG-biotin by TLC and 1H NMR: TLC (silica gel) CHCl3/CH3OH/25% NH3 (70/25/4, v/v) */ Rf :/0.8. 1H NMR (400 MHz, 8.9 mmol in 0.7 ml of CDCl3): d 1.45 (2H, quintet, J/7.7, CH2CH2CH2), 1.63 /1.79 (4H, m, 2 /CH2), 2.20 /2.26 (2H, t, SCH(CH2)3CH2), 2.74 (1H, d, J/12.6, SCHaCHb), 2.84 (4H, s, N(COCH2)2), 2.92 (1H, dd, J /5.0, 12.8, SCHaCHb), 3.15 (1H, dt, J /4.6, 7.2, SCH ), 3.44 (2H, q, NHCH2), 3.56 (2H, t, J/5.1, NHCH2CH2O), 3.60 /3.70 (300 H, m, (CH2CH2O)75), 4.33 (1H, ddd, J /7.7, 4.6, / 1.2, SCHCH NH), 4.46 (2H, t, COOCH2), 4.50 (1H, ddt, J /7.7, 5.1, /1.1, SCH2CH), 5.06 (1H, br, SCHCHNH ), 5.82 (1H, br, SCH2CHNH ), 6.71 (1H, br t, J /5.1, CH2CONH CH2) ppm. The characteristics for the purified reaction product 1 were: TLC (silica gel) CHCl3/CH3OH/ 25% NH3 (70/25/4, v/v): Rf :/0.8. 1H NMR (500 MHz, 5.3 mmol in 0.7 ml of CDCl3). d 0.88 (6H, t, J/7.0, CH3 /2), 1.20 /1.35 (40H, m, CH3(CH2)10 /2), 1.45 (2H, quintet, J/7.6, CH2CH2CH2), 1.58 (4H, m, CH3(CH2)10CH2 / 2), 1.63 /1.79 (4H, m, CH2 /2), 2.23 (2H, t, SCH(CH2)3CH2), 2.28 (4H, m, CH3(CH2)11CH2 /2), 2.75 (1H, d, J/12.8, SCHaHb), 2.91 (1H, dd, J/12.8, 4.9, SCHaHb), 3.16 (1H, dt, J /4.6, 7.3, SCH), 3.37 (2H, br q, OC(O)NHCH2), 3.44 (2H, q, J/5.1, NHCH2), 3.56 (2H, t, J /5.0, NHCH2CH2O), 3.60 /3.70 (300H, m, (CH2CH2O)75), 3.86 /4.01 (4H, m, POCH2CH2, POCH2CH2), 4.16 (1H, dd, J/7.0, 11.9, CHCHaHbOCO), 4.21 (2H, br t, COOCH2CH2), 4.32 (1H, br dd, J /4.9, 7.6, SCHCHNH), 4.38 (1H, dd, J /2.9, 12.1, CHCHaHbOCO), 4.50 (1H, br dd, J /5.1, 7.8, SCH2CH), 5.14 /5.24 (2H, m, OCH , SCHCHNH), 5.82 (1H, br, SCH2CHNH ), 6.3 / 7.1 (br) and 6.80 (br t) (2H, OCONH and CH2CONH CH2, respectively) ppm. 2.6. Determination of phosphatase activity Alkaline phosphatase activity was monitored spectrophotometrically at 410 nm for 5 min with p-nitrophenyl phosphate as substrate (Forstner et
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
al., 1968). Typically, the reaction mixture consisted of 1 ml of substrate (concentration of the stock solution: 0.354 mmol/l), 1.5 ml of 100 mM borate buffer, pH 10, and 100 ml of the enzyme solution. The hydrolysis rate was described quantitatively using first-order reaction mathematics. The firstorder rate constant, k1, was calculated from the formula k1 (t2 t1 ) ln(At At2 )ln(At At1 ); where At1 ; At2 and At refer, respectively, to the absorption at 410 nm at time t1 and t2 and after complete conversion of the substrate.
3. Results and discussion 3.1. 1H NMR characterization of NHS-PEG-biotin Besides all the expected proton resonances, the H NMR spectrum of NHS-PEG-biotin showed large 13C satellite signals (d /3.46 and 3.82 ppm, 1 JH13 C/ /141 Hz) of the dominant PEG resonance, which showed up as triplet-like signals, symmetrical to the 1H resonance of the PEG chain protons (OCH2CH2)75) at d/3.60 /3.70 ppm. Since, with the size of the PEG chain used, the integrals of these satellite signals were both very close to 2, they could easily be mistaken for methylene groups in an ethylene fragment. Furthermore, a singlet at d/2.72 ppm was observed. The latter was assigned to a small amount of N -hydroxysuccinimide which, the supplier states, is present as an impurity. 1
3.2. Synthesis of PE-PEG-biotin conjugates An outline of the synthesis protocol for the PEPEG-biotin conjugates (compounds 1 and 2) is given in Scheme 2. Mechanistically, the reaction involves a nucleophilic attack by the PE amino group on the activated NHS carbonate group, present at the extremity of the PEG-biotin conjugate. In practice, in a glass vial sealed with a Teflon-coated cap, a mixture of 4.7 mmol of PE (3.0 mg DC14:0PE; 3.5 mg DC18:1cis PE) and a slight excess of NHS-PEGbiotin (5.64 mmol, equivalent to 19.2 mg) was
79
stirred for 3 h at room temperature in 0.75 ml of CHCl3/CH3OH (8/2, v/v) in the presence of 1 ml of N(C2H5)3 (final concentration: 0.15%). Then, the mixture was analyzed by TLC. In Fig. 1, the chromatograms obtained with DC14:0PE as the parent PE type are shown as an example, but with DC18:1cis PE the same TLC patterns were seen. After spraying with molybdenum reagent (left panel), it could be seen that the spot with an Rf of 0.3, corresponding to the original PE, had completely disappeared and that a new spot with an Rf of :/0.8 had appeared. Staining with ninhydrin (central panel) further proved that free NH2 groups were no longer detectable in the reaction mixture. To achieve such a high quantitative conversion, both the organic medium and the presence of N(C2H5)3 were necessary. Omitting N(C2H5)3 drastically reduced PE conversion, since, after 3 h of reaction and even with a fourfold excess of NHS-PEG-biotin over PE, only a 60% conversion was obtained, whereas in aqueous medium (pH 6.0 or 8.0), no reaction at all occurred, probably because of hydrolysis of the PEG-NHS carbonate, releasing NHS and CO2 and regenerating the underivatized PEG hydroxyl (Hermanson, 1996). 3.3. Purification and 1H NMR characterization of 1 Although the TLC analyses showed that in the reaction mixture the original PE had completely disappeared, unfortunately, NHS-PEG-biotin and reaction product 1 had the same Rf-values (Fig. 1, right panel). This seriously hampers a further purification and consequently, hinders an unambiguous characterization of the reaction product. To overcome this problem, the reaction was performed using an excess of DC14:0PE. Furthermore, to speed up the purification step and to scale up the synthesis to a semi-preparative level, fractionation was performed by the so-called ‘‘flash chromatography’’ on a column, rather than on preparative TLC plates. The details were as follows: NHS-PEG-biotin (44.1 mmol, 150.0 mg) and 66.2 mmol (42.1 mg) of DC14:0PE, corresponding to an NHS-PEG-biotin/ PE ratio of 0.67, were mixed in 7.0 ml of CHCl3/
80
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
Scheme 2. Synthesis of PE-PEG-biotin conjugates.
Fig. 1. TLC of DC14:0PE (lane 1), NHS-PEG-biotin (lane 2), and the reaction mixture (DC14:0PE/NHS-PEG-biotin starting molar ratio of 0.83) incubated for 3 h at room temperature (lane 3). The plates were stained by spraying with molybdenum blue (left) or ninhydrin (middle) or by putting the plate in an I2 atmosphere (right). In lanes 1 and 3, 3 l of solution containing about 20 nmol of phospholipid was applied.
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
CH3OH (8/2, v/v), and 113.5 mmol (15.7 ml) of N(C2H5)3 was added. After incubation for 3 h at room temperature, the volume of the reaction mixture was reduced to 2 ml by evaporation under a stream of nitrogen, the residue applied to a 18/ 2 cm2 solvent-resistant column filled with silica gel, and 0.4 bar argon pressure was applied to increase the flow rate of the mobile phase to 7 ml/min. As shown by TLC, the desired lipid derivative (1) eluted in the first 350 ml of CHCl3/CH3OH (8/2, v/ v); apparently, under the experimental conditions used, the starting DC14:0PE did not elute. The column fractions containing the conjugate 1 were pooled and the organic solvent removed under reduced pressure, yielding 57.7 mg of purified product. This corresponds to a 33% recovery with respect to the theoretical value, calculated on the basis of the starting amount of NHS-PEGbiotin present. The moderate yield obtained using this fractionation method probably results from an instability of conjugate 1 on the silica matrix. From literature (Boden et al., 1998), indeed, it is known that, using this purification method, decomposition of lipid-polyoxyethylene conjugates occurs. In our experiments, we also observed that, upon applying a lower flow rate regime (1 ml/h, i.e. without applying any additional pressure), the product was converted to an as yet unidentified compound, which we could reproducibly isolate in small quantities. Its 1H NMR spectrum in CDCl3 showed the absence of the biotinylamido-PEG moiety, and only contained broad, but distinctive, signals with signal positions and integrations similar to those of DC14:0PE. However, in contrast to pure DC14:0PE, it was soluble in CDCl3 and, moreover, did not give a positive signal with ninhydrin. Excellent spectroscopic data were obtained for ‘‘flash chromatography’’-purified product 1 (see Section 2). Analogous to the spectrum of NHSPEG-biotin, triplet-like 13C satellites at d -values of 3.46 and 3.82 ppm (/1 JH13 C/ /141 Hz) were observed. Furthermore, the spectrum revealed the absence of the characteristic triethylammonium multiplets at d equal to 1.33 ppm (triplet) and 3.06 ppm (quartet), which suggests that 1 was isolated in the acid form.
81
Another remarkable feature of the conjugate spectrum was the significant broadening and downfield shift of the urethane signal (OCONH ) on decreasing the concentration of 1 from 9.2 to 5.5 mmol per 0.7 ml of CDCl3. In addition, at the highest concentration investigated (9.2 mmol 1 per 0.7 ml of CDCl3), the integration of the very broad urethane signal at d /6.5 ppm was about twothird of the theoretical value expected for a single proton. These observations can be explained by assuming the existence of an equilibrium between urethane cis - and trans -configurational isomers (Scheme 3). In this respect, the works of Bats et al. (1980), Branik and Kessler (1974, 1975), De Wal and Zeeghers-Huyskens (1997), dealing with equilibria between the urethane cis - and trans -isomers of t-butyloxycarbonyl-protected amino acids, are worth mentioning. NMR spectra of these protected amino acids in CDCl3, taken at various temperatures, show a shift in the equilibrium to the trans -isomer as the temperature decreases. Most probably, this shift is favored by the stabilization of the trans -form in a seven-membered ring, which contains a H-bond between the urethane carbonyl and the carboxyl-OH group. The chemical shifts of the urethane 1H resonances are 4.8 /5.4 and 6.5 /7.7 for cis - and trans -configurated amino acids, respectively (Branik and Kessler, 1974, 1975). Application of these data to the NMR characteristics of compound 1, too, suggests an equilibrium in favor of the trans isomer. The small broad signal at d equal to 4.9 ppm probably corresponds to the 1H urethane resonance of the cis -isomer. At the highest concentration of 1 (9.2 mmol in 0.7 ml CDCl3), the integration of the signal corresponded to approximately 20% of the theoretical value, expected on the basis of one proton. A further indication of a dynamic equilibrium between a cis - and a trans -
Scheme 3. Equilibrium of the cis - and the trans -isomer of compound 1.
82
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
isomer of compound 1 is the broad ‘‘feet’’ signals, located up- and downfield of the broadened OC(O)NHCH2 (d /3.37 ppm) and COOCH2CH2 (d /4.21 ppm) resonances, respectively. Whereas increasing concentrations of 1 gave rise to an upfield shift of the urethane 1H resonance (trans -isomer), downfield shifts were observed for the ureid resonances in the biotin residue (SCHCHNH and SCH2CHNH ). A similar behavior has been described by Crisp and Gore (1997), who explained the observed downfield shift of the ureid resonances seen with increasing concentrations of biotin-undec-10-yn-1-ol amide, by a concentration-dependent, intermolecular H-bonding of the ureid-protons (NH). In general, the formation of H-bonds causes downfield shifts of NMR resonances. Thus, to explain the shifts observed, we assume that mainly intramolecular interactions occur at low concentrations of 1, i.e. the formation of a seven-membered ring with H-bonding between the urethane (N /H) and the phosphoryl (P /O) group, whereas at high concentrations of compound 1 presumably intermolecular H-bondings between the ureid NHCONH moiety and the phosphate HO /P /O atoms may dominate. 3.4. Size exclusion chromatography of 1-containing SUVs The presence of excess NHS-PEG-biotin in the reaction mixture, which hindered the structural characterization of 1 (see above), is not an insurmountable problem if the phospholipid derivative is subsequently used as a (minor) building stone in the so-called phospholipid vesicles or liposomes. The differences in the dimensions of these biocolloidal structures (e.g. the molecular weight of SUVs is about 2000 kDa; De Cuyper and Joniau, 1988; New, 1997) and of NHS-PEGbiotin (3.4 kDa) are so great that they can be easily separated by gel permeation chromatography, e.g. on a Sephacryl S-300 matrix. This is clearly illustrated using intramembranously mixed vesicles made up of DC14:0PG and compound 1 at a molar ratio of 9/1 (Fig. 2). The eluted vesicles, present in fractions 13 /21, were well separated from NHS-PEG-biotin (maximum at fraction 28). In a similar experiment (not shown) in which the
Fig. 2. Sephacryl S-300 gel permeation chromatography on vesicles made up of DC14:0PG and 1 in a molar ratio of 9/1: (m) the absorption signal at 260 nm; (j) the A820 nm-values, after treating the samples with phosphomolybdate reagent.
vesicles were prepared from equimolar amounts of DC14:0PG and 1, the phospholipid-containing fractions were pooled, the phospholipids extracted three times with five volumes of CHCl3/CH3OH (15/5, v/v), and the organic phases combined and dried over MgSO4 before removing the organic solvent under reduced pressure. 1H NMR spectroscopy (200 MHz, CDCl3) of the product obtained showed that the characteristic signals of C14:0 fatty acyl chain- and PEG-chain-protons were present. The integration ratio for the signals at d /1.15 / 1.30 (40 H, m, CH3(CH2)10 /4) and d /3.60 /3.70 ppm (300 H, m, (CH2CH2O)75) was equal to 3.86, in good agreement with the theoretical value of 3.75 for an equimolar mixture of 1 and DC14:0PG. Although complete separation of NHS-PEGbiotin and phospholipids is also feasible in case solely 1 is used to construct lipid dispersions, we have not been able to use this material to successfully construct vesicles (see also Kenworthy et al., 1995a,b; Koynova et al., 1999; Yoshida et al., 1999), the reason being the huge enlargement of the hydrophilic part in the phospholipid derivative, which influences the packing of these molecules in an aqueous environment. Similar observations have been made by Armen et al. (1998), Israelachvili et al. (1977, 1980), who found that, for the phospholipid molecules to be able to adopt a classical bilayer configuration, the polar/ apolar volume ratio can only vary over a rather
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
83
limited range. Thus, to relieve the mutual stress exerted by the bulky headgroup of the PE derivatives within a membrane, 1 must be mixed with ‘‘classical’’ membrane-forming phospholipids, such as DC14:0PG (see above). For most biomedical applications, however, this will not cause a problem, since, to avoid uptake by the liver, the optimal content of PEGylated phospholipid in the vesicle membrane is only of the order of 5% (Gabizon et al., 1994; Jones, 1995; Zalipsky, 1995). 3.5. Binding capacity of Stealth† magnetoliposomes for SAP Magnetoliposomes, built up of DC14:0PG and 1 or 2 (9/1 molar ratio) were prepared as described in Section 2. The ability of the membrane-embedded PE-PEG-biotin conjugate to interact with streptavidinylated biomolecules was checked with alkaline phosphatase as a model. Increasing volumes (8, 16, 33, 66 and 134 l) of SAP stock suspension were added to five separate tubes, each containing an identical amount of purified Stealth† magnetoliposomes (1.34 ml, 0.30 mol phospholipid/ml, 434.7 g Fe3O4/ml). Then, after a 100 min incubation period, 1.2 ml of each mixture was subjected to a high-gradient magnetic fractionation. To ensure that no non-fixed enzyme remained between the magnetic filter fibers, they were thoroughly washed with 9 ml of buffer. An idea of the amount of bound/non-bound enzyme was obtained by measuring the catalytic activity in the different retentates and eluates and also in the original Stealth† magnetoliposome /SAP mixtures. Fig. 3 shows the results for 2-containing magnetoliposomes. The enzymatic activities of the unfractionated mixture (j), eluates (') and retentates (m) are expressed as first-order rate constants. As shown, in the presence of relatively lower amounts of SAP, the enzyme was entirely associated with the magnetic colloid, whereas, at higher amounts of SAP, saturation of the magnetoliposome surface occurred. This behavior is in concert with the high affinity constant reported for the biotin /(strept)avidin interaction (Kass /1015) (Bayer and Wilchek, 1979; Chu et al., 1997). To exclude the possibility that SAP spontaneously associated with membrane structures, we also
Fig. 3. Binding of SAP to 2/DC14:0PG (1/9 molar ratio) Stealth† magnetoliposomes. The x -axis shows the volume of SAP stock solution added per ml of magnetoliposome. The y axis shows the SAP activity present in the unfractionated mixture (j), the eluate ('), and the retentate (m).
confronted SAP with magnetoliposomes, which were devoid of the biotin-containing PE derivative. In this case, after high-gradient magnetophoresis, no activity could be detected in the retentate fraction.
4. Outlook and perspectives for applications The present results highlight that the special type of Stealth† magnetoliposome described here can bind streptavidinylated biomolecules very strongly. We have taken SAP as a model protein because it can be readily monitored spectrophotometrically in a very sensitive assay. Undoubtedly, other streptavidinylated molecules will interact with the colloidal particle with a similar high efficiency (Powers et al., 1989). This approach opens very promising routes for applications in the biomedical field, e.g. in the development of targeting systems involving monoclonal antibodies. Indeed, tailoring of Stealth† magnetoliposomes to a variety of patient’ disease profiles is no longer time-consuming. Repeated administration of magnetoliposome-PEG-biotin-streptavidinylated-monoclonal antibody complexes, however, may possibly elicit a gradual engendered immune response towards the biotin-streptavidin moiety (Harding et al., 1997). Although many studies suggest the benefit of having more ligand mole-
84
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
cules for targeting, further investigations are needed to pinpoint the optimal ligand concentration at which elimination caused by immune recognition is balanced by the benefit of targeting. With respect to the targeting features of Stealth† magnetoliposomes, one must realize that the internal space is completely stuffed with an iron oxide core. Consequently, their applicability for targeting purposes will be mainly restricted to apolar molecules that are embedded in the phospholipid bilayer, though appending of water-soluble drugs at the external liposome surface should be feasible by chemical means. A unique and unequivocal property of targetable Stealth† magnetoliposomes, however, is that once specifically fixed at a malignant tissue, the presence of the magnetizable iron oxide core can be further exploited to localize the particles by magnetic resonance imaging (Bulte et al., 1999), and/or to induce hyperthermia by application of an alternating magnetic field to the tissue envisaged (Jordan et al., 1997; Shinkai et al., 1999).
Acknowledgements We would like to thank Mr. W. Noppe for his help in the experimental work. The financial support of the Nationaal Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Contract No. G.0170.96) is gratefully acknowledged.
References Allen, T.M., Agrawal, A.K., Ahmad, I., Hansen, C.B., Zalipski, S., 1994. Antibody-mediated targeting of longcirculating (Stealth) liposomes. J. Liposome Res. 4, 1 /26. Allen, T.M., Brandeis, E., Hansen, C.B., Kao, G.Y., Zalipsky, S., 1995. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim. Biophys. Acta 1237, 99 /108. Armen, R.S., Uitto, O.D., Feller, S.E., 1998. Phospholipid component volumes: determination and application to bilayer structure calculations. Biophys. J. 75, 734 /744. Baldeschwieler, J.D., Schmidt, P.G., 1997. Liposomal drugs: from setbacks to success. Chemtech 27, 34 /42. Bats, J.W., Fuess, H., Kessler, H., Schuck, R., 1980. tert Butoxycarbonyl-L-a-phenylalanine: crystal structure and
conformational changes in solution. Chem. Ber. 113, 520 / 530. Bayer, E.A., Wilchek, M., 1979. The use of the avidin /biotin complex as a tool in molecular biology. Meth. Biochem. Anal. 26, 1 /45. Blume, G., Cevc, G., 1993. Molecular mechanism of lipid vesicle longevity in vivo. Biochim. Biophys. Acta 1146, 157 / 168. Boden, N., Bushby, R.J., Liu, Q., Evans, S.D., Toby, A., Jenkins, A., Knowles, P.F., Miles, R.E., 1998. N ,N ?disuccinimidyl carbonate as a coupling agent in the synthesis of thiophospholipids used for anchoring biomembranes to gold surfaces. Tetrahedron 54, 11537 /11548. Branik, M., Kessler, H., 1974. Zur konformation geschu¨tzter aminosa¨uren. Tetrahedron 30, 781 /786. Branik, M., Kessler, H., 1975. Conformation of protected amino acids: NMR and IR investigations of Boc-v-amino acids. Chem. Ber. 108, 2722 /2727. Bulte, J.W.M., De Cuyper, M., Despres, D., Brooks, R.A., Frank, J.A., 1999. Short- vs long-circulating magnetoliposomes as bone marrow-seeking MR contrast agents. J. Magn. Reson. Imaging 9, 329 /335. Christie, W.W., 1982. The analysis of complex lipids. In: Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids, 2nd ed.(Chapter 7). Pergamon Press, Oxford, UK, p. 120. Chu, V., Freitag, S., Le Trong, I., Stenkamp, R.E., Stayton, P.S., 1997. Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin-biotin system. Protein Sci. 7, 848 /859. Crisp, G.T., Gore, J., 1997. Biotin derivatives as gelators of organic solvents. Synth. Commun. 27, 2203 /2215. De Cuyper, M., 1996. Applications of magnetoproteoliposomes in bioreactors operating in high-gradient magnetic fields. In: Barenholz, Y., Lasic, D.D. (Eds.), Handbook of Nonmedical Applications of Liposomes */From Design to Microreactors, vol. III(Chapter 18). CRC Press, Boca Raton, FL, pp. 325 /342. De Cuyper, M., Joniau, M., 1988. Magnetoliposomes: formation and structural characterization. Eur. Biophys. J. 15, 311 /319. De Cuyper, M., Joniau, M., 1992. Binding characteristics and thermal behavior of cytochrome c -oxidase inserted into phospholipid-coated, magnetic nanoparticles. Biotechnol. Appl. Biochem. 16, 201 /210. De Wal, K., Zeegers-Huyskens, Th., 1997. Fourier transform IR studies of the self-association of N -tert -butyloxycarbonyl-a-amino acids. Biopolymers 41, 205 /212. Dittmer, J.C., Lester, R.L., 1964. A simple, specific spray for the detection of phospholipids on thin layer chromatograms. J. Lipid Res. 5, 126 /127. Forstner, G.G., Sabesin, S., Isselbacher, K.J., 1968. Rat intestinal microvillus membranes: purification and biochemical characterization. Biochem. J. 106, 381. Gabizon, A., Catane, R., Uziely, B., Kaufman, B., Safra, T., Cohen, R., Martin, F., Huang, A., Barenholz, Y., 1994. Prolonged circulation time and enhanced accumulation in
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85 malignant exudates of doxorubicin encapsulated in polyethylene glycol-coated liposomes. Cancer Res. 54, 987 /992. Harding, J.A., Engbers, C.M., Newman, M.S., Goldstein, N.I., Zalipsky, S., 1997. Immunogenicity and pharmacokinetic attributes of poly(ethylene glycol)-grafted immunoliposomes. Biochim. Biophys. Acta 1327, 181 /192. Hermanson, G.T., 1996. Modification with synthetic polymers. In: Bioconjugate Techniques, 3rd ed.(Chapter 15). Academic Press, San Diego, CA, p. 611. Holmberg, E., Maruyama, K., Litzinger, D.C., Wright, S., Davis, M., Kabalka, G.W., Kennel, S.J., Huang, L., 1989. Highly efficient immunoliposomes prepared with a method which is compatible with various lipid compositions. Biochem. Biophys. Res. Commun. 165, 1272 /1278. Israelachvili, J.N., Mitchell, D.J., Ninham, B.W., 1977. Theory of self-assembly of lipid bilayers and vesicles. Biochim. Biophys. Acta 470, 185 /201. Israelachvili, J.N., Marcelja, S., Horn, R.G., 1980. Physical principles of membrane organization. Q. Rev. Biophys. 13, 121 /200. Jones, M.N., 1995. The surface properties of phospholipid liposome systems and their characterisation. Adv. Colloid Interf. Sci. 54, 93 /128. Jordan, A., Scholz, R., Wust, P., Fa¨hling, H., Krause, J., Wlodarczyk, W., Saunder, B., Vogl, Th., Felix, R., 1997. Effects of magnetic fluid hyperthermia (MFH) on C3H mammary carcinoma in vivo. Int. J. Hyperthermia 13, 587 / 605. Kenworthy, A.K., Simon, S.A., McIntosh, T.J., 1995. Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1903 /1920. Kenworthy, A.K., Hristova, K., Needham, D., McIntosh, T.J., 1995. Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1921 /1936. Koynova, R., Tenchov, B., Rapp, G., 1999. Effect of PEG-lipid conjugates on the phase behavior of phosphatidylethanolamine dispersions. Colloids Surf. A 149, 571 /575. Lasic, D.D., Martin, F.J., Gabizon, A., Huang, S.K., Papahadjopoulos, D., 1991. Sterically stabilized liposomes: a hypothesis on the molecular origin of extended circulation times. Biochim. Biophys. Acta 1070, 187 /192. Moreira, J.N., Ishida, T., Gaspar, R., Allen, T.M., 2002. Use of the post-insertion technique to insert peptide ligands into pre-formed Stealth liposomes with retention of binding activity and cytotoxicity. Pharm. Res. 19, 265 /269.
85
Mori, A., Klibanov, A.L., Torchilin, V.P., Huang, L., 1991. Influence of the steric barrier activity of amphipatic poly(ethylene glycol) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS Lett. 284, 263 /266. New, R.R.C., 1997. Liposomes */a practical approach. In: Rickwood, D., Hames, B.D. (Eds.), The Practical Approach Series. IRL Press at Oxford University Press, Oxford, UK, pp. 1 /161. Papahadjopoulos, D., Allen, T.M., Gabizon, A., Mayhew, E., Matthay, K., Huang, S.K., Lee, K.-D., Woodle, M.C., Lasic, D.D., Redemann, C., Martin, F.J., 1991. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. USA 88, 11460 /11464. Powers, J.D., Kilpatrick, P.K., Carbonell, R.G., 1989. Protein purification by affinity binding to unilamellar vesicles. Biotechnol. Bioeng. 33, 173 /182. Shinkai, M., Yanase, J.M., Suzuki, M., Honda, H., Wakabayashi, T., Yoshida, J., Kobayashi, T., 1999. Intracellular hyperthermia for cancer using magnetite cationic liposomes. J. Magn. Magn. Mater. 194, 176 /184. Trubetskoy, V.S., Torchilin, V.P., 1995. Use of polyoxyethylene-lipid conjugates as long-circulating carriers for delivery of therapeutic and diagnostic agents. Adv. Drug Delivery Rev. 16, 311 /320. Vaskovsky, V.E., Kostetsky, E.Y., Vasendin, I.M., 1975. A universal reagent for phospholipid analysis. J. Chromatogr. 114, 129 /141. Viitala, T., Albers, W.M., Vikholm, I., Peltonen, J., 1998. Synthesis and Langmuir film formation of N -(o-maleimidocaproyl)(dilinoleylphosphatidyl)ethanolamine. Langmuir 14, 1272 /1277. Yoshida, A., Hashizaki, K., Yamauchi, H., Sakai, H., Yokoyama, S., Abe, M., 1999. Effect of lipid with covalently attached poly(ethylene glycol) on the surface properties of liposomal bilayer membranes. Langmuir 15, 2333 /2337. Zalipsky, S., 1995. Polyethylene glycol-lipid conjugates. In: Lasic, D.D., Martin, F. (Eds.), Stealth Liposomes(Chapter 9). CRC Press, Boca Raton, FL, pp. 93 /102. Zalipsky, S., Mullah, N., Harding, J.A., Gittelman, J., Guo, L., DeFrees, S.A., 1997. Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains. Bioconjugate Chem. 8, 111 /118.
Biotinylated Stealth† magnetoliposomes Michael Hodenius a, Marcel De Cuyper b,*, Linda Desender b, Detlef Mu¨llerSchulte a, Alois Steigel c, Heiko Lueken a b
a Rheinisch-Westfa¨lische Technische Hochschule Aachen, Prof. Pirlet-Straße 1, D-52074 Aachen, Germany Interdisciplinary Research Centre, Katholieke Universiteit Leuven-Campus Kortrijk, B-8500 Kortrijk, Belgium c Heinrich-Heine-Universita¨t Du¨sseldorf, Universita¨tsstraße 1, D-40225 Du¨sseldorf, Germany
Received 7 January 2002; received in revised form 17 June 2002; accepted 22 July 2002
Abstract Dimyristoylphosphatidylethanolamine (DC14:0PE) and the dioleoyl analogue (DC18:1cis PE) were mixed with abiotinylamido-v-N -succinimidoxycarbonyl-poly(ethylene glycol) (NHS-PEG-biotin) and quantitatively converted to abiotinylamido-v-(dimyristoylphosphatidylethanolamino-carbonyl)polyethylene glycol (DC14:0PE-PEG-biotin) and the dioleoyl analogue DC18:1cis PE-PEG-biotin, respectively. As shown by thin-layer chromatography and 1H NMR spectroscopy, PEGylation of both phosphatidylethanolamine types went to completion if the reaction was performed in organic solvent in the presence of triethylamine. The resulting derivatives were successfully incorporated into both classical phospholipid vesicles and a phospholipid bilayer surrounding nanometer-sized magnetite cores. In the latter case, the so-called activated Stealth† 1 magnetoliposomes were produced which very efficiently immobilized streptavidinylated alkaline phosphatase. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Magnetoliposomes; Phospholipid /protein conjugates; Phospholipid vesicles; Poly(ethylene glycol)-biotin derivatized phospholipids; Streptavidinylated enzymes; Stealth† magnetoliposomes
1. Introduction
* Corresponding author. Tel.: /32-56-246221; fax: /32-56246997 E-mail address: [email protected] (M. De Cuyper). 1 Stealth † is a registered trademark of SEQUUS Pharmaceutical, Inc. The term ‘Stealth’ was proposed for these long-circulating, sterically stabilized liposomes by T.M. Allen, 1989. In: Lopez-Bernstein, G., Fidler, I. (Eds.), Liposomes in the Therapy of Infectious Diseases and Cancer, New Series 89. Alan R. Liss, New York, pp. 405 /415.
Liposomes are colloidal structures consisting of phospholipid bilayers, encapsulating an aqueous space. In recent years, these structures have been proven to have great potential in the medical field, e.g. as promising homing devices for selective delivery of drugs to diseased cells (Baldeschwieler and Schmidt, 1997). In general, the size and type of liposome depend on the phospholipid composition and the preparation procedure (New, 1997). For instance, small unilamellar vesicles (SUVs), which are used in this work, consist of only a single
0009-3084/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 0 2 ) 0 0 1 0 5 - 6
76
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
bilayer and have a diameter of about 30 nm. Unfortunately, a main drawback with regard to the medical use of these classical liposomes is their limited blood circulation time due to their rapid uptake by the reticuloendothelial system (RES) in the liver and spleen. However, Jones (1995), Lasic et al. (1991), Papahadjopoulos et al. (1991), Trubetskoy and Torchilin (1995) found that the blood clearance rate can be significantly reduced by grafting a limited number of water-soluble, flexible polymer chains, such as poly(ethylene glycol) (PEG), onto the surface of SUV. The continual conformational change of the polymer, indeed, hinders recognition by the RES and, thus, increases the blood halflife (Blume and Cevc, 1993). This approach is used to produce the socalled Stealth† liposomes. Specific targeting of these particles to a malignant tissue can be achieved by linking a specific monoclonal antibody to the particle surface (Allen et al., 1994), e.g. by covalently coupling a relevant monoclonal antibody to the PEG terminus of a phospholipid /PEG complex (Holmberg et al., 1989; Mori et al., 1991; Allen et al., 1995). In this context, the so-called post-insertion technique of Zalipsky et al. (1997), Moreira et al. (2002) is worth mentioning. These authors first synthesized phospholipid /PEG /oligopeptide conjugates and subsequently incubated them as micellar aggregates with preformed Stealth† liposomes. Ultimately, the tailor-made complexes were embedded in the outer, surface-grafted polymeric ‘‘brush’’. In applying this strategy, however, a new derivative must be synthesized for each application (Viitala et al., 1998). Fortunately, this cumbersome work can be circumvented using a universal precursor complex that can bind a wide variety of antibodies. A phospholipid /PEG /biotin complex is a good candidate for achieving this goal, since it can interact with a broad scale of commercially available (strept)avidinylated antibodies. To further enlarge the number of potential applications, in this study, we chose to work with Stealth† liposomes in which the inner aqueous space is completely filled with magnetizable iron oxide, magnetite (Fe3O4). As a result, these structures can easily be captured in a high-gradient magnetic field, thus allowing quantitative removal of non-
membrane-adsorbed material from the reaction mixture in a rapid and elegant way. In this study, we first investigated the optimal conditions for the synthesis of the phosphatidylethanolamine (PE)-PEG-biotin derivative, starting from dimyristoylPE (DC14:0PE) or dioleoylPE (DC18:1cis PE) and a-biotinylamido-v-N -succinimidoxycarbonyl-poly(ethylene glycol) (NHS-PEGbiotin), and verified the degree of adduct formation by thin-layer chromatography (TLC) and 1H NMR spectroscopy. Subsequently, the ability of the conjugate to be incorporated into phospholipid vesicles and magnetoliposomes was checked. Finally, using streptavidinylated alkaline phosphatase (SAP) as a model protein, we proved that the membrane-linked PE conjugate was able to strongly bind streptavidinylated biomolecules. The binding of a streptavidinylated enzyme to biotin-grafted Stealth† magnetoliposomes is illustrated in Scheme 1.
2. Experimental procedures 2.1. Materials Dimyristoylphosphatidylglycerol (DC14:0PG) and DC14:0PE, both stored at /20 8C as lyophilized powder, and DC18:1cis PE (stored in CHCl3) were supplied by Avanti Polar Lipids (Birmingham, AL). NHS-PEG-biotin (lyophilized powder) with an average molecular mass of approximately 3400 was supplied by Shearwater Polymers Europe (Enschede, The Netherlands). SAP, used as a suspension containing 1000 U/ml, was from Boehringer GmbH (Mannheim, Germany). The 1H NMR-solvent, chloroform-d (CDCl3), was purchased from Cambridge Isotope Laboratories, Inc. (Andover, USA). p -Nitrophenyl phosphate (disodium salt) was from Sigma (Deisenhofen, Germany). All solvents, buffer substances, and chemicals used for TLC were of analytical grade and were purchased from Sigma or Merck-Eurolab (Darmstadt, Germany). Analytical silicagel 60 plates and Kieselgel 60 (0.040 /0.063 mm), used as the matrix for silica gel adsorption chromatography (henceforth referred to as ‘‘flash chromatography’’), were also from Merck-Eurolab.
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
77
Scheme 1. Binding of streptavidinylated enzyme to biotin-PEG-grafted magnetoliposomes.
Sephacryl S-300 and the solvent-resistant column for size exclusion chromatography were purchased from Pharmacia Biotech (Freiburg, Germany). 2.2. Phospholipid and Fe3O4 determinations The phospholipid and Fe3O4 contents of magnetoliposomes were measured spectrophotometrically on a Uvikon 933 instrument. The determination of phospholipid content according to the method of Vaskovsky et al. (1975) involved a prior, complete thermal digestion at 180 8C for 45 min of the phospholipid sample in the presence of 70% perchloric acid; the resulting inorganic orthophosphate was then reacted with acidic molybdenum to form a blue complex with an absorption maximum at 820 nm. The Fe3O4 content was determined by dissolving the magnetite core in 37% hydrochloric acid/65% nitric acid (3/1, v/v), chelating the Fe3 with Tiron (De Cuyper and Joniau, 1992), and measuring the absorption at 480 nm. 2.3. Preparation and fractionation of phospholipid vesicles Phospholipid vesicles were prepared by codissolving DC14:0PG and the PE-PEG-biotin complex in chloroform at the concentrations indicated. After evaporation of the organic solvent in a stream of nitrogen, the residue was taken up in 2 ml of 5 mM N -Tris[hydroxymethyl] methyl-2aminoethanesulfonic acid (TES) buffer, pH 7.0. To obtain a clear homogeneous dispersion, the lipid /buffer mixture was then sonicated for 4 min using a probe-tip sonicator (MSE Ultrasonic 150W disintegrator, equipped with a 3/8 in. probe) set at the maximal power setting at which no
frothing occurred. The resulting vesicles were then centrifuged for 10 min at 10,000 rpm/min (Sorvall RC-5B equipped with a SS-34 rotor) to remove titanium particles produced by abrasion of the probe. Occasionally, the vesicles (2 ml at a phospholipid concentration of 4.69 mol/ml) were loaded onto a Sephacryl S-300 column (46 /1 cm2) and eluted with 5 mM TES buffer, pH 7.0, at a rate of 16 ml/h. Fractions (1 ml) were collected and examined for light scattering at 260 nm and for the presence of phospholipid by phosphate determination (see above). Fractions of interest were pooled and analyzed by 1H NMR spectroscopy (see below). 2.4. Magnetoliposome preparation Magnetoliposomes were produced according to a previously described protocol (De Cuyper and Joniau, 1988). In short, nanometer-sized magnetite grains (diameter about 15 nm) were first stabilized by a cushion of laurate molecules and then incubated and dialyzed in the presence of preformed sonicated vesicles. During this step, the fatty acid molecules were gradually exchanged for a bilayer of phospholipid molecules. The practical details were as follows: a mixture of 9 ml of SUVs with a phospholipid concentration of 8.41 mmol/ml (DC14:0PG/PE-PEG-biotin molar ratio equal to 9/ 1) and 0.177 ml of the magnetic fluid stock solution (concentration: 61.08 mg Fe3O4/ml) was dialyzed for 72 h at 37 8C in Spectra/Por dialysis tubings (molecular weight cut-off, 12,000 /14,000) (Spectrum medical Industries, Los Angeles, CA). The dialysis buffer, which was changed at regular intervals, consisted of 5 mM TES, pH 7.0. Excess vesicles were removed by high-gradient magneto-
78
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
phoresis. Fourteen aliquots of 0.643 ml each were simultaneously pumped at a uniform rate of 6 ml/h through tubing (inner diameter: 0.078 in.), plugged with loosely packed steel wool (/50 mg; Type 430; Bekaert Steel Corporation, Zwevegem, Belgium), placed in a 3 mm gap between the two pole faces of a water-cooled electromagnet (Bruker, Type BE-15, Karlsruhe, Germany). The electromagnet was operated at 30 A and 80 V. Under these conditions, but in the absence of the magnetic filter matrix, the magnetic field between the two poles is about 2.3 T. After separation, each retentate was thoroughly washed with 2.9 ml of TES buffer to remove any nonadsorbed phospholipids remaining in the capillary liquid surrounding the iron fibers. Then the magnetic field was turned off and the retentates were recovered from the filter matrix by a high-speed buffer stream (500 ml/ h) of 0.643 ml of buffer per aliquot. The retentates were then checked for phospholipid and Fe3O4 content. The magnetoliposomes which contained the a-biotinylamido-v-(dimyristoylphosphatidylethanolaminocarbonyl)polyethylene glycol (DC14:0PE-PEG-biotin; 1) conjugate had a mmol phospholipid/g Fe3O4 ratio of 0.69, while magnetoliposomes prepared using the dioleoylanalogue of the phospholipid derivative (DC18:1cis PE-PEGbiotin; 2) had a ratio of 0.61. These values are indicative of the presence of a complete bilayer enwrapping the iron oxide core (De Cuyper, 1996). Transmission electron microscopy (Zeiss EM 10C) further revealed a uniform diameter of 25 nm for the magnetoliposome colloids. 2.5. Purity tests: TLC and 1H NMR analyses TLC was carried out in CHCl3/CH3OH/25% NH3 (70/25/4, v/v). After drying, the plates were sprayed with molybdenum blue reagent (Dittmer and Lester, 1964) and ninhydrin solution in acetone (Christie, 1982) to detect phosphate and free NH2 groups, respectively. NHS-PEG-biotin was detected using iodine vapor. 1 H NMR spectra were recorded on a Varian MERCURY 200, INOVA 400 or UNITY 500 spectrometer. Chemical shifts are expressed in ppm downfield of the internal standard, tetra-
methylsilane. The test molecules under investigation were dissolved in CDCl3. The following data were obtained for NHSPEG-biotin by TLC and 1H NMR: TLC (silica gel) CHCl3/CH3OH/25% NH3 (70/25/4, v/v) */ Rf :/0.8. 1H NMR (400 MHz, 8.9 mmol in 0.7 ml of CDCl3): d 1.45 (2H, quintet, J/7.7, CH2CH2CH2), 1.63 /1.79 (4H, m, 2 /CH2), 2.20 /2.26 (2H, t, SCH(CH2)3CH2), 2.74 (1H, d, J/12.6, SCHaCHb), 2.84 (4H, s, N(COCH2)2), 2.92 (1H, dd, J /5.0, 12.8, SCHaCHb), 3.15 (1H, dt, J /4.6, 7.2, SCH ), 3.44 (2H, q, NHCH2), 3.56 (2H, t, J/5.1, NHCH2CH2O), 3.60 /3.70 (300 H, m, (CH2CH2O)75), 4.33 (1H, ddd, J /7.7, 4.6, / 1.2, SCHCH NH), 4.46 (2H, t, COOCH2), 4.50 (1H, ddt, J /7.7, 5.1, /1.1, SCH2CH), 5.06 (1H, br, SCHCHNH ), 5.82 (1H, br, SCH2CHNH ), 6.71 (1H, br t, J /5.1, CH2CONH CH2) ppm. The characteristics for the purified reaction product 1 were: TLC (silica gel) CHCl3/CH3OH/ 25% NH3 (70/25/4, v/v): Rf :/0.8. 1H NMR (500 MHz, 5.3 mmol in 0.7 ml of CDCl3). d 0.88 (6H, t, J/7.0, CH3 /2), 1.20 /1.35 (40H, m, CH3(CH2)10 /2), 1.45 (2H, quintet, J/7.6, CH2CH2CH2), 1.58 (4H, m, CH3(CH2)10CH2 / 2), 1.63 /1.79 (4H, m, CH2 /2), 2.23 (2H, t, SCH(CH2)3CH2), 2.28 (4H, m, CH3(CH2)11CH2 /2), 2.75 (1H, d, J/12.8, SCHaHb), 2.91 (1H, dd, J/12.8, 4.9, SCHaHb), 3.16 (1H, dt, J /4.6, 7.3, SCH), 3.37 (2H, br q, OC(O)NHCH2), 3.44 (2H, q, J/5.1, NHCH2), 3.56 (2H, t, J /5.0, NHCH2CH2O), 3.60 /3.70 (300H, m, (CH2CH2O)75), 3.86 /4.01 (4H, m, POCH2CH2, POCH2CH2), 4.16 (1H, dd, J/7.0, 11.9, CHCHaHbOCO), 4.21 (2H, br t, COOCH2CH2), 4.32 (1H, br dd, J /4.9, 7.6, SCHCHNH), 4.38 (1H, dd, J /2.9, 12.1, CHCHaHbOCO), 4.50 (1H, br dd, J /5.1, 7.8, SCH2CH), 5.14 /5.24 (2H, m, OCH , SCHCHNH), 5.82 (1H, br, SCH2CHNH ), 6.3 / 7.1 (br) and 6.80 (br t) (2H, OCONH and CH2CONH CH2, respectively) ppm. 2.6. Determination of phosphatase activity Alkaline phosphatase activity was monitored spectrophotometrically at 410 nm for 5 min with p-nitrophenyl phosphate as substrate (Forstner et
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
al., 1968). Typically, the reaction mixture consisted of 1 ml of substrate (concentration of the stock solution: 0.354 mmol/l), 1.5 ml of 100 mM borate buffer, pH 10, and 100 ml of the enzyme solution. The hydrolysis rate was described quantitatively using first-order reaction mathematics. The firstorder rate constant, k1, was calculated from the formula k1 (t2 t1 ) ln(At At2 )ln(At At1 ); where At1 ; At2 and At refer, respectively, to the absorption at 410 nm at time t1 and t2 and after complete conversion of the substrate.
3. Results and discussion 3.1. 1H NMR characterization of NHS-PEG-biotin Besides all the expected proton resonances, the H NMR spectrum of NHS-PEG-biotin showed large 13C satellite signals (d /3.46 and 3.82 ppm, 1 JH13 C/ /141 Hz) of the dominant PEG resonance, which showed up as triplet-like signals, symmetrical to the 1H resonance of the PEG chain protons (OCH2CH2)75) at d/3.60 /3.70 ppm. Since, with the size of the PEG chain used, the integrals of these satellite signals were both very close to 2, they could easily be mistaken for methylene groups in an ethylene fragment. Furthermore, a singlet at d/2.72 ppm was observed. The latter was assigned to a small amount of N -hydroxysuccinimide which, the supplier states, is present as an impurity. 1
3.2. Synthesis of PE-PEG-biotin conjugates An outline of the synthesis protocol for the PEPEG-biotin conjugates (compounds 1 and 2) is given in Scheme 2. Mechanistically, the reaction involves a nucleophilic attack by the PE amino group on the activated NHS carbonate group, present at the extremity of the PEG-biotin conjugate. In practice, in a glass vial sealed with a Teflon-coated cap, a mixture of 4.7 mmol of PE (3.0 mg DC14:0PE; 3.5 mg DC18:1cis PE) and a slight excess of NHS-PEGbiotin (5.64 mmol, equivalent to 19.2 mg) was
79
stirred for 3 h at room temperature in 0.75 ml of CHCl3/CH3OH (8/2, v/v) in the presence of 1 ml of N(C2H5)3 (final concentration: 0.15%). Then, the mixture was analyzed by TLC. In Fig. 1, the chromatograms obtained with DC14:0PE as the parent PE type are shown as an example, but with DC18:1cis PE the same TLC patterns were seen. After spraying with molybdenum reagent (left panel), it could be seen that the spot with an Rf of 0.3, corresponding to the original PE, had completely disappeared and that a new spot with an Rf of :/0.8 had appeared. Staining with ninhydrin (central panel) further proved that free NH2 groups were no longer detectable in the reaction mixture. To achieve such a high quantitative conversion, both the organic medium and the presence of N(C2H5)3 were necessary. Omitting N(C2H5)3 drastically reduced PE conversion, since, after 3 h of reaction and even with a fourfold excess of NHS-PEG-biotin over PE, only a 60% conversion was obtained, whereas in aqueous medium (pH 6.0 or 8.0), no reaction at all occurred, probably because of hydrolysis of the PEG-NHS carbonate, releasing NHS and CO2 and regenerating the underivatized PEG hydroxyl (Hermanson, 1996). 3.3. Purification and 1H NMR characterization of 1 Although the TLC analyses showed that in the reaction mixture the original PE had completely disappeared, unfortunately, NHS-PEG-biotin and reaction product 1 had the same Rf-values (Fig. 1, right panel). This seriously hampers a further purification and consequently, hinders an unambiguous characterization of the reaction product. To overcome this problem, the reaction was performed using an excess of DC14:0PE. Furthermore, to speed up the purification step and to scale up the synthesis to a semi-preparative level, fractionation was performed by the so-called ‘‘flash chromatography’’ on a column, rather than on preparative TLC plates. The details were as follows: NHS-PEG-biotin (44.1 mmol, 150.0 mg) and 66.2 mmol (42.1 mg) of DC14:0PE, corresponding to an NHS-PEG-biotin/ PE ratio of 0.67, were mixed in 7.0 ml of CHCl3/
80
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
Scheme 2. Synthesis of PE-PEG-biotin conjugates.
Fig. 1. TLC of DC14:0PE (lane 1), NHS-PEG-biotin (lane 2), and the reaction mixture (DC14:0PE/NHS-PEG-biotin starting molar ratio of 0.83) incubated for 3 h at room temperature (lane 3). The plates were stained by spraying with molybdenum blue (left) or ninhydrin (middle) or by putting the plate in an I2 atmosphere (right). In lanes 1 and 3, 3 l of solution containing about 20 nmol of phospholipid was applied.
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
CH3OH (8/2, v/v), and 113.5 mmol (15.7 ml) of N(C2H5)3 was added. After incubation for 3 h at room temperature, the volume of the reaction mixture was reduced to 2 ml by evaporation under a stream of nitrogen, the residue applied to a 18/ 2 cm2 solvent-resistant column filled with silica gel, and 0.4 bar argon pressure was applied to increase the flow rate of the mobile phase to 7 ml/min. As shown by TLC, the desired lipid derivative (1) eluted in the first 350 ml of CHCl3/CH3OH (8/2, v/ v); apparently, under the experimental conditions used, the starting DC14:0PE did not elute. The column fractions containing the conjugate 1 were pooled and the organic solvent removed under reduced pressure, yielding 57.7 mg of purified product. This corresponds to a 33% recovery with respect to the theoretical value, calculated on the basis of the starting amount of NHS-PEGbiotin present. The moderate yield obtained using this fractionation method probably results from an instability of conjugate 1 on the silica matrix. From literature (Boden et al., 1998), indeed, it is known that, using this purification method, decomposition of lipid-polyoxyethylene conjugates occurs. In our experiments, we also observed that, upon applying a lower flow rate regime (1 ml/h, i.e. without applying any additional pressure), the product was converted to an as yet unidentified compound, which we could reproducibly isolate in small quantities. Its 1H NMR spectrum in CDCl3 showed the absence of the biotinylamido-PEG moiety, and only contained broad, but distinctive, signals with signal positions and integrations similar to those of DC14:0PE. However, in contrast to pure DC14:0PE, it was soluble in CDCl3 and, moreover, did not give a positive signal with ninhydrin. Excellent spectroscopic data were obtained for ‘‘flash chromatography’’-purified product 1 (see Section 2). Analogous to the spectrum of NHSPEG-biotin, triplet-like 13C satellites at d -values of 3.46 and 3.82 ppm (/1 JH13 C/ /141 Hz) were observed. Furthermore, the spectrum revealed the absence of the characteristic triethylammonium multiplets at d equal to 1.33 ppm (triplet) and 3.06 ppm (quartet), which suggests that 1 was isolated in the acid form.
81
Another remarkable feature of the conjugate spectrum was the significant broadening and downfield shift of the urethane signal (OCONH ) on decreasing the concentration of 1 from 9.2 to 5.5 mmol per 0.7 ml of CDCl3. In addition, at the highest concentration investigated (9.2 mmol 1 per 0.7 ml of CDCl3), the integration of the very broad urethane signal at d /6.5 ppm was about twothird of the theoretical value expected for a single proton. These observations can be explained by assuming the existence of an equilibrium between urethane cis - and trans -configurational isomers (Scheme 3). In this respect, the works of Bats et al. (1980), Branik and Kessler (1974, 1975), De Wal and Zeeghers-Huyskens (1997), dealing with equilibria between the urethane cis - and trans -isomers of t-butyloxycarbonyl-protected amino acids, are worth mentioning. NMR spectra of these protected amino acids in CDCl3, taken at various temperatures, show a shift in the equilibrium to the trans -isomer as the temperature decreases. Most probably, this shift is favored by the stabilization of the trans -form in a seven-membered ring, which contains a H-bond between the urethane carbonyl and the carboxyl-OH group. The chemical shifts of the urethane 1H resonances are 4.8 /5.4 and 6.5 /7.7 for cis - and trans -configurated amino acids, respectively (Branik and Kessler, 1974, 1975). Application of these data to the NMR characteristics of compound 1, too, suggests an equilibrium in favor of the trans isomer. The small broad signal at d equal to 4.9 ppm probably corresponds to the 1H urethane resonance of the cis -isomer. At the highest concentration of 1 (9.2 mmol in 0.7 ml CDCl3), the integration of the signal corresponded to approximately 20% of the theoretical value, expected on the basis of one proton. A further indication of a dynamic equilibrium between a cis - and a trans -
Scheme 3. Equilibrium of the cis - and the trans -isomer of compound 1.
82
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
isomer of compound 1 is the broad ‘‘feet’’ signals, located up- and downfield of the broadened OC(O)NHCH2 (d /3.37 ppm) and COOCH2CH2 (d /4.21 ppm) resonances, respectively. Whereas increasing concentrations of 1 gave rise to an upfield shift of the urethane 1H resonance (trans -isomer), downfield shifts were observed for the ureid resonances in the biotin residue (SCHCHNH and SCH2CHNH ). A similar behavior has been described by Crisp and Gore (1997), who explained the observed downfield shift of the ureid resonances seen with increasing concentrations of biotin-undec-10-yn-1-ol amide, by a concentration-dependent, intermolecular H-bonding of the ureid-protons (NH). In general, the formation of H-bonds causes downfield shifts of NMR resonances. Thus, to explain the shifts observed, we assume that mainly intramolecular interactions occur at low concentrations of 1, i.e. the formation of a seven-membered ring with H-bonding between the urethane (N /H) and the phosphoryl (P /O) group, whereas at high concentrations of compound 1 presumably intermolecular H-bondings between the ureid NHCONH moiety and the phosphate HO /P /O atoms may dominate. 3.4. Size exclusion chromatography of 1-containing SUVs The presence of excess NHS-PEG-biotin in the reaction mixture, which hindered the structural characterization of 1 (see above), is not an insurmountable problem if the phospholipid derivative is subsequently used as a (minor) building stone in the so-called phospholipid vesicles or liposomes. The differences in the dimensions of these biocolloidal structures (e.g. the molecular weight of SUVs is about 2000 kDa; De Cuyper and Joniau, 1988; New, 1997) and of NHS-PEGbiotin (3.4 kDa) are so great that they can be easily separated by gel permeation chromatography, e.g. on a Sephacryl S-300 matrix. This is clearly illustrated using intramembranously mixed vesicles made up of DC14:0PG and compound 1 at a molar ratio of 9/1 (Fig. 2). The eluted vesicles, present in fractions 13 /21, were well separated from NHS-PEG-biotin (maximum at fraction 28). In a similar experiment (not shown) in which the
Fig. 2. Sephacryl S-300 gel permeation chromatography on vesicles made up of DC14:0PG and 1 in a molar ratio of 9/1: (m) the absorption signal at 260 nm; (j) the A820 nm-values, after treating the samples with phosphomolybdate reagent.
vesicles were prepared from equimolar amounts of DC14:0PG and 1, the phospholipid-containing fractions were pooled, the phospholipids extracted three times with five volumes of CHCl3/CH3OH (15/5, v/v), and the organic phases combined and dried over MgSO4 before removing the organic solvent under reduced pressure. 1H NMR spectroscopy (200 MHz, CDCl3) of the product obtained showed that the characteristic signals of C14:0 fatty acyl chain- and PEG-chain-protons were present. The integration ratio for the signals at d /1.15 / 1.30 (40 H, m, CH3(CH2)10 /4) and d /3.60 /3.70 ppm (300 H, m, (CH2CH2O)75) was equal to 3.86, in good agreement with the theoretical value of 3.75 for an equimolar mixture of 1 and DC14:0PG. Although complete separation of NHS-PEGbiotin and phospholipids is also feasible in case solely 1 is used to construct lipid dispersions, we have not been able to use this material to successfully construct vesicles (see also Kenworthy et al., 1995a,b; Koynova et al., 1999; Yoshida et al., 1999), the reason being the huge enlargement of the hydrophilic part in the phospholipid derivative, which influences the packing of these molecules in an aqueous environment. Similar observations have been made by Armen et al. (1998), Israelachvili et al. (1977, 1980), who found that, for the phospholipid molecules to be able to adopt a classical bilayer configuration, the polar/ apolar volume ratio can only vary over a rather
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
83
limited range. Thus, to relieve the mutual stress exerted by the bulky headgroup of the PE derivatives within a membrane, 1 must be mixed with ‘‘classical’’ membrane-forming phospholipids, such as DC14:0PG (see above). For most biomedical applications, however, this will not cause a problem, since, to avoid uptake by the liver, the optimal content of PEGylated phospholipid in the vesicle membrane is only of the order of 5% (Gabizon et al., 1994; Jones, 1995; Zalipsky, 1995). 3.5. Binding capacity of Stealth† magnetoliposomes for SAP Magnetoliposomes, built up of DC14:0PG and 1 or 2 (9/1 molar ratio) were prepared as described in Section 2. The ability of the membrane-embedded PE-PEG-biotin conjugate to interact with streptavidinylated biomolecules was checked with alkaline phosphatase as a model. Increasing volumes (8, 16, 33, 66 and 134 l) of SAP stock suspension were added to five separate tubes, each containing an identical amount of purified Stealth† magnetoliposomes (1.34 ml, 0.30 mol phospholipid/ml, 434.7 g Fe3O4/ml). Then, after a 100 min incubation period, 1.2 ml of each mixture was subjected to a high-gradient magnetic fractionation. To ensure that no non-fixed enzyme remained between the magnetic filter fibers, they were thoroughly washed with 9 ml of buffer. An idea of the amount of bound/non-bound enzyme was obtained by measuring the catalytic activity in the different retentates and eluates and also in the original Stealth† magnetoliposome /SAP mixtures. Fig. 3 shows the results for 2-containing magnetoliposomes. The enzymatic activities of the unfractionated mixture (j), eluates (') and retentates (m) are expressed as first-order rate constants. As shown, in the presence of relatively lower amounts of SAP, the enzyme was entirely associated with the magnetic colloid, whereas, at higher amounts of SAP, saturation of the magnetoliposome surface occurred. This behavior is in concert with the high affinity constant reported for the biotin /(strept)avidin interaction (Kass /1015) (Bayer and Wilchek, 1979; Chu et al., 1997). To exclude the possibility that SAP spontaneously associated with membrane structures, we also
Fig. 3. Binding of SAP to 2/DC14:0PG (1/9 molar ratio) Stealth† magnetoliposomes. The x -axis shows the volume of SAP stock solution added per ml of magnetoliposome. The y axis shows the SAP activity present in the unfractionated mixture (j), the eluate ('), and the retentate (m).
confronted SAP with magnetoliposomes, which were devoid of the biotin-containing PE derivative. In this case, after high-gradient magnetophoresis, no activity could be detected in the retentate fraction.
4. Outlook and perspectives for applications The present results highlight that the special type of Stealth† magnetoliposome described here can bind streptavidinylated biomolecules very strongly. We have taken SAP as a model protein because it can be readily monitored spectrophotometrically in a very sensitive assay. Undoubtedly, other streptavidinylated molecules will interact with the colloidal particle with a similar high efficiency (Powers et al., 1989). This approach opens very promising routes for applications in the biomedical field, e.g. in the development of targeting systems involving monoclonal antibodies. Indeed, tailoring of Stealth† magnetoliposomes to a variety of patient’ disease profiles is no longer time-consuming. Repeated administration of magnetoliposome-PEG-biotin-streptavidinylated-monoclonal antibody complexes, however, may possibly elicit a gradual engendered immune response towards the biotin-streptavidin moiety (Harding et al., 1997). Although many studies suggest the benefit of having more ligand mole-
84
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85
cules for targeting, further investigations are needed to pinpoint the optimal ligand concentration at which elimination caused by immune recognition is balanced by the benefit of targeting. With respect to the targeting features of Stealth† magnetoliposomes, one must realize that the internal space is completely stuffed with an iron oxide core. Consequently, their applicability for targeting purposes will be mainly restricted to apolar molecules that are embedded in the phospholipid bilayer, though appending of water-soluble drugs at the external liposome surface should be feasible by chemical means. A unique and unequivocal property of targetable Stealth† magnetoliposomes, however, is that once specifically fixed at a malignant tissue, the presence of the magnetizable iron oxide core can be further exploited to localize the particles by magnetic resonance imaging (Bulte et al., 1999), and/or to induce hyperthermia by application of an alternating magnetic field to the tissue envisaged (Jordan et al., 1997; Shinkai et al., 1999).
Acknowledgements We would like to thank Mr. W. Noppe for his help in the experimental work. The financial support of the Nationaal Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Contract No. G.0170.96) is gratefully acknowledged.
References Allen, T.M., Agrawal, A.K., Ahmad, I., Hansen, C.B., Zalipski, S., 1994. Antibody-mediated targeting of longcirculating (Stealth) liposomes. J. Liposome Res. 4, 1 /26. Allen, T.M., Brandeis, E., Hansen, C.B., Kao, G.Y., Zalipsky, S., 1995. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim. Biophys. Acta 1237, 99 /108. Armen, R.S., Uitto, O.D., Feller, S.E., 1998. Phospholipid component volumes: determination and application to bilayer structure calculations. Biophys. J. 75, 734 /744. Baldeschwieler, J.D., Schmidt, P.G., 1997. Liposomal drugs: from setbacks to success. Chemtech 27, 34 /42. Bats, J.W., Fuess, H., Kessler, H., Schuck, R., 1980. tert Butoxycarbonyl-L-a-phenylalanine: crystal structure and
conformational changes in solution. Chem. Ber. 113, 520 / 530. Bayer, E.A., Wilchek, M., 1979. The use of the avidin /biotin complex as a tool in molecular biology. Meth. Biochem. Anal. 26, 1 /45. Blume, G., Cevc, G., 1993. Molecular mechanism of lipid vesicle longevity in vivo. Biochim. Biophys. Acta 1146, 157 / 168. Boden, N., Bushby, R.J., Liu, Q., Evans, S.D., Toby, A., Jenkins, A., Knowles, P.F., Miles, R.E., 1998. N ,N ?disuccinimidyl carbonate as a coupling agent in the synthesis of thiophospholipids used for anchoring biomembranes to gold surfaces. Tetrahedron 54, 11537 /11548. Branik, M., Kessler, H., 1974. Zur konformation geschu¨tzter aminosa¨uren. Tetrahedron 30, 781 /786. Branik, M., Kessler, H., 1975. Conformation of protected amino acids: NMR and IR investigations of Boc-v-amino acids. Chem. Ber. 108, 2722 /2727. Bulte, J.W.M., De Cuyper, M., Despres, D., Brooks, R.A., Frank, J.A., 1999. Short- vs long-circulating magnetoliposomes as bone marrow-seeking MR contrast agents. J. Magn. Reson. Imaging 9, 329 /335. Christie, W.W., 1982. The analysis of complex lipids. In: Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids, 2nd ed.(Chapter 7). Pergamon Press, Oxford, UK, p. 120. Chu, V., Freitag, S., Le Trong, I., Stenkamp, R.E., Stayton, P.S., 1997. Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin-biotin system. Protein Sci. 7, 848 /859. Crisp, G.T., Gore, J., 1997. Biotin derivatives as gelators of organic solvents. Synth. Commun. 27, 2203 /2215. De Cuyper, M., 1996. Applications of magnetoproteoliposomes in bioreactors operating in high-gradient magnetic fields. In: Barenholz, Y., Lasic, D.D. (Eds.), Handbook of Nonmedical Applications of Liposomes */From Design to Microreactors, vol. III(Chapter 18). CRC Press, Boca Raton, FL, pp. 325 /342. De Cuyper, M., Joniau, M., 1988. Magnetoliposomes: formation and structural characterization. Eur. Biophys. J. 15, 311 /319. De Cuyper, M., Joniau, M., 1992. Binding characteristics and thermal behavior of cytochrome c -oxidase inserted into phospholipid-coated, magnetic nanoparticles. Biotechnol. Appl. Biochem. 16, 201 /210. De Wal, K., Zeegers-Huyskens, Th., 1997. Fourier transform IR studies of the self-association of N -tert -butyloxycarbonyl-a-amino acids. Biopolymers 41, 205 /212. Dittmer, J.C., Lester, R.L., 1964. A simple, specific spray for the detection of phospholipids on thin layer chromatograms. J. Lipid Res. 5, 126 /127. Forstner, G.G., Sabesin, S., Isselbacher, K.J., 1968. Rat intestinal microvillus membranes: purification and biochemical characterization. Biochem. J. 106, 381. Gabizon, A., Catane, R., Uziely, B., Kaufman, B., Safra, T., Cohen, R., Martin, F., Huang, A., Barenholz, Y., 1994. Prolonged circulation time and enhanced accumulation in
M. Hodenius et al. / Chemistry and Physics of Lipids 120 (2002) 75 /85 malignant exudates of doxorubicin encapsulated in polyethylene glycol-coated liposomes. Cancer Res. 54, 987 /992. Harding, J.A., Engbers, C.M., Newman, M.S., Goldstein, N.I., Zalipsky, S., 1997. Immunogenicity and pharmacokinetic attributes of poly(ethylene glycol)-grafted immunoliposomes. Biochim. Biophys. Acta 1327, 181 /192. Hermanson, G.T., 1996. Modification with synthetic polymers. In: Bioconjugate Techniques, 3rd ed.(Chapter 15). Academic Press, San Diego, CA, p. 611. Holmberg, E., Maruyama, K., Litzinger, D.C., Wright, S., Davis, M., Kabalka, G.W., Kennel, S.J., Huang, L., 1989. Highly efficient immunoliposomes prepared with a method which is compatible with various lipid compositions. Biochem. Biophys. Res. Commun. 165, 1272 /1278. Israelachvili, J.N., Mitchell, D.J., Ninham, B.W., 1977. Theory of self-assembly of lipid bilayers and vesicles. Biochim. Biophys. Acta 470, 185 /201. Israelachvili, J.N., Marcelja, S., Horn, R.G., 1980. Physical principles of membrane organization. Q. Rev. Biophys. 13, 121 /200. Jones, M.N., 1995. The surface properties of phospholipid liposome systems and their characterisation. Adv. Colloid Interf. Sci. 54, 93 /128. Jordan, A., Scholz, R., Wust, P., Fa¨hling, H., Krause, J., Wlodarczyk, W., Saunder, B., Vogl, Th., Felix, R., 1997. Effects of magnetic fluid hyperthermia (MFH) on C3H mammary carcinoma in vivo. Int. J. Hyperthermia 13, 587 / 605. Kenworthy, A.K., Simon, S.A., McIntosh, T.J., 1995. Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1903 /1920. Kenworthy, A.K., Hristova, K., Needham, D., McIntosh, T.J., 1995. Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1921 /1936. Koynova, R., Tenchov, B., Rapp, G., 1999. Effect of PEG-lipid conjugates on the phase behavior of phosphatidylethanolamine dispersions. Colloids Surf. A 149, 571 /575. Lasic, D.D., Martin, F.J., Gabizon, A., Huang, S.K., Papahadjopoulos, D., 1991. Sterically stabilized liposomes: a hypothesis on the molecular origin of extended circulation times. Biochim. Biophys. Acta 1070, 187 /192. Moreira, J.N., Ishida, T., Gaspar, R., Allen, T.M., 2002. Use of the post-insertion technique to insert peptide ligands into pre-formed Stealth liposomes with retention of binding activity and cytotoxicity. Pharm. Res. 19, 265 /269.
85
Mori, A., Klibanov, A.L., Torchilin, V.P., Huang, L., 1991. Influence of the steric barrier activity of amphipatic poly(ethylene glycol) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS Lett. 284, 263 /266. New, R.R.C., 1997. Liposomes */a practical approach. In: Rickwood, D., Hames, B.D. (Eds.), The Practical Approach Series. IRL Press at Oxford University Press, Oxford, UK, pp. 1 /161. Papahadjopoulos, D., Allen, T.M., Gabizon, A., Mayhew, E., Matthay, K., Huang, S.K., Lee, K.-D., Woodle, M.C., Lasic, D.D., Redemann, C., Martin, F.J., 1991. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. USA 88, 11460 /11464. Powers, J.D., Kilpatrick, P.K., Carbonell, R.G., 1989. Protein purification by affinity binding to unilamellar vesicles. Biotechnol. Bioeng. 33, 173 /182. Shinkai, M., Yanase, J.M., Suzuki, M., Honda, H., Wakabayashi, T., Yoshida, J., Kobayashi, T., 1999. Intracellular hyperthermia for cancer using magnetite cationic liposomes. J. Magn. Magn. Mater. 194, 176 /184. Trubetskoy, V.S., Torchilin, V.P., 1995. Use of polyoxyethylene-lipid conjugates as long-circulating carriers for delivery of therapeutic and diagnostic agents. Adv. Drug Delivery Rev. 16, 311 /320. Vaskovsky, V.E., Kostetsky, E.Y., Vasendin, I.M., 1975. A universal reagent for phospholipid analysis. J. Chromatogr. 114, 129 /141. Viitala, T., Albers, W.M., Vikholm, I., Peltonen, J., 1998. Synthesis and Langmuir film formation of N -(o-maleimidocaproyl)(dilinoleylphosphatidyl)ethanolamine. Langmuir 14, 1272 /1277. Yoshida, A., Hashizaki, K., Yamauchi, H., Sakai, H., Yokoyama, S., Abe, M., 1999. Effect of lipid with covalently attached poly(ethylene glycol) on the surface properties of liposomal bilayer membranes. Langmuir 15, 2333 /2337. Zalipsky, S., 1995. Polyethylene glycol-lipid conjugates. In: Lasic, D.D., Martin, F. (Eds.), Stealth Liposomes(Chapter 9). CRC Press, Boca Raton, FL, pp. 93 /102. Zalipsky, S., Mullah, N., Harding, J.A., Gittelman, J., Guo, L., DeFrees, S.A., 1997. Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains. Bioconjugate Chem. 8, 111 /118.