Bioconjugate Chem. 1995, 6,242-248
242
Synthesis and Applications of a New Poly(ethy1ene glycol) Derivative for the Crosslinking of Amines with Thiols Thomas Haselgriibler, Alexandra Amerstorfer, Hansgeorg Schindler, a n d H e r m a n n J. Gruber” Institute of Biophysics, J. Kepler University, Altenberger Str. 69, A-4040 Linz, Austria. Received July 22, 1994@
A heterobifunctional crosslinker was synthesized from a diamine derivative of poly(ethy1ene glycol) (PEG, average molecular weight 800 Da), with the functional groups 2-(pyridyldithio)propionyl(PDP) and N-hydroxysuccinimide ester (NHS). The crosslinker can be used for linkage of two different proteins for which a suitable protocol is presented, explified by crosslinking of two antibodies with 50% yield. In a second application the crosslinker is used to generate immunoliposomes. The NHS group was reacted with an aminolipid for liposome anchorage, and antibodies were bound to the PDP group via disulfide bonds. Loading of liposomes with antibodies was easily adjustable, even down to only a few per liposome. This crosslinker with its particular length appears especially suited for the flexible anchorage of biomembranes, opening new perspectives in membrane research as discussed.
INTRODUCTION
Covalent attachment of poly(ethy1ene glycol) (PEG’ 1 to biological or biocompatible materials has become of great interest in clinical research (Merrill, 1992; Ouchi et al., 1987; Topchieva, 1990; Zalipsky et al., 1983). As a result of PEG conjugation, proteins no longer elicit strong immunogenic responses (Dreborg and Akerblom, 1990; Pool, 1990; Veronese et al., 1992; Zalipsky and Lee, 1992) and the circulation times of liposomes in blood can be sufficiently extended to be suitable for the requirements of clinical use (Blume and Cevc, 1990; Klibanov et al., 1991; Papahadjopoulos et al., 1991; Senior et al., 1991; Woodle et al., 1992). In all these cases monofmctional PEG derivatives with a nonreactive methoxy terminus and a n amine-reactive group on the other end were used for PEG attachment. Homobifunctional PEG derivatives with two aminereactive end groups have been used to attach antibodies to liposomes via long, flexible spacers ( B l u e et al., 1993). Hetero-bifunctional PEG derivatives with two different reactive groups have found little application so far although several such crosslinkers and important asymmetric precursors for a larger variety have been synthesized (Yokoyama, 1992; Zalipsky and Barany, 1986; Zalipsky and Barany, 1990; Zalipsky, 1993). The present study reports on the synthesis of a heterobifunctional PEG derivative with the coupling
* To whom correspondence should be addressed. Tel.: +43 (732) 2468-9271. Fax: +43 (732) 2468-822. Abstract published in Advance ACS Abstracts, March 15, 1995. Abbreviations: AFM, atomic force microscopy; DCC, N,”dicyclohexylcarbodiimide; DMPE, 1,2-bis(myristoylphosphatidyllethanolamine; DTNB, 5,5’-dithiobis(2-nitrobenzoicacid); DTT, dithiothreitol; eggPC, egg yolk phosphatidylcholine; FLUOS, 5-(and-6)-carboxyfluorescein,succinimidyl ester; HSA, human serum albumin; NH2-PEG-NH2, O,O-bis(2-aminopropyl)poly(ethylene glycol) 800 (see Scheme 1); NHS, N-hydroxysuccinimide or N-hydroxysuccinimidyl residue (connected to NH2-PEGNHz by a glutaryl residue); PDP, 2-pyridyldithiopropionyl residue; PE, phosphatidylethanolamine; PEG, poly(ethy1ene glycol) (in the Experimental Procedures and Results sections the term “ P E G is used as defined in Scheme 1); POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; rt, room temperature; SATP, N-succinimidyl-3-(S-acetylthio)propionate; SBL, soy bean lipids (asolectin); SPDP, N-succinimidyl-3-(2-pyridyldithio)propionate; VETzw, vesicles produced by extrusion through a double layer of 200 nm Nuclepore membranes. @
functions NHS and PDP (see Scheme l), particularly chosen for purposes of biomolecular coupling. Two examples are included in the Results, the conjugation of two soluble proteins and the preparation of immunoliposomes. Particular impact is expected from its use in methods of membrane research as outlined in the Discussion. EXPERIMENTAL PROCEDURES
Materials. SBL (asolectin) were purified from soy bean lecithin (Sigma type 11-S)by a standard method (Kagawa and Racker, 1971). EggPC was prepared chromatographically from Ovothin 200 (already 99% pure egg PC, Lucas Meyer, Hamburg, Germany) as described before (Gruber and Schindler, 1994). Anti-HSA (polyclonal sheep antibody, purified by immunsorption) was the generous gift of Dr. K. Hallermayer from Boehringer Mannheim, Penzberg, Germany. All other proteins were obtained from Sigma, and the same applies to DMPE, POPC, SATP, and to all other reagents unless specified. All solvents, aqueous buffer components, and inorganic salts were p.a. grade and purchased from Merck. DCC and ninhydrin were from the same source. NH2-PEGNH2 and triethylamine were obtained from Fluka. FLUOS was obtained from Lambda Probes & Diagnostics, Graz, Austria. Argon (Linde) was 99.998% ( 1 3 ppm 0 2 ) . SPDP was prepared as described (Ofitserov et al., 1985; Shval’e et al., 1985). Methods. Thin Layer Chromatrography. Plastic sheets, precoated with 0.2 mm silica gel 60 without fluorescent indicator (Merck) were used. Eluents I and I1 contained chloroform and methanol a t a ratio of 90110 and 80120, respectively. Eluents I11 and IV contained chlorofordmethanolcetic acid a t volume ratios of 851 1510.1 and 10013012, respectively. Eluent V was a mixture of chlorofordmethanolater (8012011). Amino groups were detected by spraying with 0.1% ninhydrin in 2-propanol and heating to 100 “C (SchuurmansStekhoven et al., 1992). Free or PEG-bound phospholipids were also visualized by spray reagents (Dittmer and Lester, 1964). By spraying with 2,6-dichlorophenolindophenol (0.1% sodium salt in ethanol; Passera et al., 1964) only the COOH of glutaric acid could be specifically detected, while all PEG derivatives with and without COOH groups gave intense dark blue spots. Determination of PDP Group Contents of PEG Derivatives. Typically, 200 pL of an aqueous sample
1043-1802/95/2906-0242$09.00/00 1995 American Chemical Society
Synthesis of a New Poly(ethy1ene glycol) Derivative
Bioconjugafe Chem., Vol. 6,No. 3, 1995 243
Scheme 1
NHz-PEG-NHz
I
SPDP
PDP-NH-PEG -NH2
atmosphere. A solution of 600 mg (1.92 mmol) of SPDP in 30 mL of CHC4 was added dropwise over a period of 15 min. The solution was stirred for a n additional 15 min, 1mL of acetic acid was added, and the solvent was evaporated. Residual acetic acid was azeotroped with toluene. The residue was dissolved in 10 mL of CHC13 and subjected to chromatography on 50 g of silica gel. The product was eluted by first applying 250 mL of CHCldMeOWAcOH = 90/10/0.1 and then 300 mL of 70/ 3 0 6 Fractions containing the product were pooled, evaporated, and azeotroped with toluene. The residue was again dissolved in CHCl3, filtered, and evaporated to yield 750 mg (0.64 mmol, 29%) of highly viscous, yellowish liquid PDP-NH-PEG-NH-NH3 acetate: RrJ" (product) = 0.17-0.47, R,J"(bis(pyridyldithio1-) = 0.570.72, R,J"(diamine) = 0-0.1. IH-NMR (200 MHz, CDC13) 6: 1.05-1.25 (m, CH3, 2-aminopropyl), 1.97 (CH&OOH), 2.56 (2H, t, J = 7.1 Hz, -COCHzCHz-), 2.6 (2H, S, NHz), 3.05 (2H, t, J = 7 Hz, -SCH&Hz-). 3.64 (broad s. PEG polymer signal), 7-8.5 (4H, 3m, p-yridyl), 8.75 (lH,broad S, CH3COOH). Preparation of PDP-NH-PEG-NH-glu. To 700 mg (0.64 mmol) of PDP-NH-PEG-NHs+ acetate and 110 mg (0.96 mmol) of glutaric anhydride was added 7 mL of pyridine. The mixture was stirred overnight. Pyridine was evaporated and the residue azeotroped with toluene. Ten mL of water was added, and the mixture was stirred for 1 h. Water was evaporated, and the residue was again azeotroped with toluene and subjected to chromatography on silica gel. Elution with 100 mL of CHCld MeOH 92/8 and 300 mL of CHClfleOH 90/10 gave 362 mg (0.296 mmol, 46%) of highly viscous liquid PDP-NHPEG-NH-glu which was free of glutaric acid: R,P (product) = 0.35-0.5, RJ' (side product) = 0.67, R;' (glutaric acid) = 0.35-0.5. 'H-NMR (200 MHz, CDC13) 6: 1.11.3 (m, CH3,2-aminopropyl), 1.97 (2H, m, -COCHzCH2CHz-), 2.27 (2H, t, J = 7 Hz, -COCHzCHz-), 2.38 (2H, t, J = 6.8 Hz, -CHzCHzCOOH), 2.58 (2H, t, J = 6.9 Hz, -COCHzCHz-), 3.06 (2H, t, J = 6.9 Hz, -SCHzCHZ-), 3.63 (broad s, PEG polymer signal), 7.0-8.5 (4H, 3m, pyridyl), no proton signal was detected for the carboxylic group. IR (CHCl3) v (cm-l): 3436, 3029, 1711, 1657. Preparation of NHS-NH-PEG-NH-PDP.To a solution of 362 mg (0.296 mmol) of PDP-NH-PEG-NH-glu in ethyl acetate were added 38 mg (0.33 mmol) of NHS and 67 mg (0.33 mmol) of DCC. The reaction mixture was stirred overnight. Dicyclohexylurea was removed by filtration. The filtrate was evaporated, and the residue obtained was used without further purification. As for NMR and IR measurements, 100 mg of the residue was subjected to rapid chromatography on 10 g of silica gel (2 cm column diameter). Elution with 100 mL of CHCld MeOH 911 yielded 35 mg of highly viscous liquid NHSNH-PEG-NH-PDP: R,Z1(product) = 0.62, RJ' (SPDP) = 0.74. lH-NMR (200 MHz, CDC13) 6: 1.05-1.25 (m, CH3, 2-aminopropyl), 2.55 (2H, t, -COCHZCHZ-), 2.84 (4H, s, -CHzCHz-, succinimidyl), 3.06 (2H, t, J = 7 Hz, -SCHzCHz-), 3.61 (broad s, PEG polymer signal), 7.08.5 (4H, m, pyridyl). 13C-NMR(200 MHz, CDC13)6: 70.3 (PEG); C(1) 159.7, C(2) 120.8, C(3) 137.0, C(4) 119.8, C(5) 149.6 (pyridyl). IR (CHC13) v (cm-l): 3446, 3030, 1822, 1793,1742 , 1662. Pyridyldithio group content (without solvent correction): 0.66 mequivlg (87% of the theoretical value). Preparation of DMPE-NH-PEG-NH-PDP.Thirty mg (0.047 mmol) of DMPE and 10 pL of triethylamine were added to a solution of 95 mg (0.072 mmol) of NHSNH-PEG-NH-PDP in 2 mL of CHC13, and the reaction mixture was stirred for 2 h. The solvent was evaporated, and the residue was dissolved in 5 mL of CHC13 and
+
1
glutaric anhydride
PDP-NH-PEG -NH -glU NHS/DCC
PDP-NH-PEG-NH-NHS
IDMPE b0
NH
5 0
0
PDP-NH-PEG-NH-DMPE
containing ~ 0 . 8mM PDP groups were diluted with 1mL of 0.1 M NaHzP04, titrated to pH = 8.0 with NaOH. Insoluble compounds like DMPE-NH-PEG-NH-PDPwere solubilized by inclusion of Triton X-100 (up to 1%final concentration). One mL of this mixture was pipetted into a microcuvette, and A343 was determined both before and after thorough mixing with 100 pL of DTT or glutathione (0.2 M in buffer A, readjusted to pH 7.5 with NaOH; see below for buffer A). The PDP group concentration for the original 200 pL sample was calculated as [PDP] = 6/t343 (1.lA34i- A343), whereby A343 and A34i denote the absorption values before and after glutathione addition, respectively, and ~ 3 of ~ the 3 released thiopyridone is known to be 8080 cm-l M-l (Leserman et al., 1984). In this way background levels of absorption (e.g., from Triton) were best corrected for. Glutathione was equally effective as DTT and was much preferred in the case of large sample series to avoid the severe irritation caused by DTT. Preparation of PDP-NH-PEG-NH3 Acetate. A solution of 2 g (2.19 mmol) of NHz-PEG-NH~ (see Scheme 1for abbreviations of molecular structures) in 30 mL of CHC13 was stirred a t room temperature under an argon
+
244 Bioconjugate Cbem., Vol. 6,No. 3, 1995
subjected to chromatography on 20 g of silica gel. Elution with 100 mL of CHClmeOH 90/10 and 150 mL of 70/30 provided 48 mg (0.026 mmol, 55%) of DMPE-NHPEG-NH-PDP: R t l (product) = 0.3-0.39, RfV(product) = 0.35-0.49; Rtr (crosslinker) = 0.62, RfV (crosslinker) = 0.71, RF' (DMPE) = 0.1. 'H-NMR: (200 MHz, CDC13) 6: 0.88 (6H, t, J = 6. 8 Hz, CH3, myristoyl), 1.05-1.25 (m, CH3, 2-aminopropyl), 1.26 (5, CH2, myristoyl), 2.28 (4H, t, CH2C=O), 3.64 (broad s, PEG polymer signal), 3.94 (m, CHZ glycerol and -CHzCH20P-), 4.17 (dd, J = 7, 12 Hz, -CHzOP-), 4.39 (dd, J = 3, 12 Hz, -CHzOC-01, 5.2 (m, CH glycerol, lH), 7-8.5 (4H, 3m, pyridyl). 13C-NMR (200 MHz, CDC13) 6: 70.3 (PEG); pyridyl: C(1) 159.7, C(2) 120.8, C(3) 137.0, C(4) 119.8, C(5) 149.6; DMPE moiety: 14.1 (CH3), 22.7 (CHZCH~), 24.9 (CH~CHZC-O),29.6 (polyCHz),31.9 (CHzCHzCH31, 34.0 and 34.2 (2 CHZC=O), 45.5 (HNCHZCHz),62.8 (CH2OC-O), 69.5 (CHOC=O), 173.1 and 173.5 (C=O of two esters). IR (CHC13) Y (cm-I): 3431, 2930, 2869, 1740, 1657,1100. Pyndyldithio group content (without solvent correction): 0.47 mequivlg (87.5% of the theoretical value). Preparation of SATP Derivatives of Antibodies. A modification of published procedures was used (Duncan et al., 1983; Jones and Hudson, 1993). Antibodies were dissolved a t 65 pM protein concentration in buffer A (100 mM NaC1, 50 mM NaH2P04, 1 mM EDTA, pH = 7.5 adjusted with NaOH), and proper amounts of 16-18 mM stock solutions of SATP in pure DMSO were added to achieve different initial molar ratios of reagent'protein. After 30 min incubation under argon a t rt IgG was separated from the reagents by gel filtration in buffer A (PD-10 column, Sephadex G-25 M, Pharmacia). For the determination of newly-introduced, protected SH groups 1.2 mL from the PD-10 peak fraction were treated with 50 pL NHzOH reagent (500 mM NHzOHaHCl, 25 mM EDTA, pH = 7.5 adjusted with solid Na2C03) under argon for 60 min a t rt, and deprotected SH groups were assayed using DTNB by the Ellman method (Ellman, 1959). A linear dose response with a constant yield (37 %) of covalent SATP incorporation into IgG was found for up to four covalently bound SATP per IgG. This simple relation allowed an adjustment of the number average of SATP groups per antibody even if direct determination was not possible, as in the presence of FLUOS labels which interfered with the Ellman test. Double Labeling of Anti-HSA with FLUOS and SATP. As outlined in the instructions from Molecular Probes, 5 pL of 16.7 mM FLUOS in DMSO was added to 500 pL of 10 mglmL anti-HSA in buffer C (100 mM NaC1, 35 mM H3B03, pH = 8.6 adjusted with NaOH) and stirred for 90 min a t rt in the dark. Subsequently the pH was lowered to 7.5 by the addition of 4.2 pL of 1M NaHzP04, and 5 pL of 33 mM SATP in DMSO were added. After 20 min of stirring a t rt in the dark the mixture was loaded on a PD-10 column and eluted with buffer A. Aliquots of derivatized anti-HSA were immediately frozen and stored at -70 "C. Crosslinkingof Bovine IgG to FLUOS- and SATPLabeled Anti-HSA In the first step bovine IgG was derivatized with NHS-NH-PEG-NH-PDP by mixing 1450 pL of buffer A containing 100 nmol of bovine IgG with 50 pL of DMSO containing the 1.5- to 12-fold amount of crosslinker (determined by PDP group content) and incubating a t 19 "C for 70 min in the dark. The reaction was terminated by freezing and immediately after thawing IgG with bound crosslinker was separated from free crosslinker by gel filtration on Sephadex G-100 (1 x 26 cm column, flow rate 0.25 m u m i n ) in buffer B (100 mM NaC1,20 mM CHSCOOH,pH = 5.5 adjusted with NaOH).
Haselgrubler et ai.
Fractions (4 min) were assayed for protein contents by measuringAZT8(€278 = 174 000 M-l cm-l was determined for bovine IgG) and for PDP group contents as described above. In a control run 50 pL of DMSO with 300 nmol of crosslinker were first mixed with 450 pL of 10 mM ethanolamineHC1 in buffer C (pH = 8.6) and incubated a t 37 "C for 15 min in order to destroy all NHS ester groups before 1mL of buffer A with 100 nmol of IgG was added and incubated for 70 min as usual. For the second step the three peak fractions from the Sephadex G-100 column were combined which together contained 20.4 pM bovine IgG with 113 pM covalently bound crosslinker (determined from PDP group contents). Twenty pL of this derivatized IgG was adjusted from pH = 5.5 to pH = 7.5 by the addition of 10 pL of buffer D (50 mM NaC1, 100 mM NaH2P04, pH = 7.66 adjusted with NaOH) and mixed with 20 pL of FLUOS- and SATPmodified anti-HSA which had been thawed immediately before use and diluted to a concentration of 10 pM. The tube was sealed with a rubber septum, and the gas phase was perfused with argon via hypodermic needles while vortexing for 2 min. Crosslinking was triggered by injecting 2 pL of degassed NH20H reagent (see above) and allowed to proceed for 60 min a t 25 "C in the dark. In a control experiment the newly-formed disulfide bonds were cleaved by addition of 8.2 pL of 0.2 M glutathione (in buffer A, readjusted to pH = 7.5 with NaOH) and 20 min incubation a t 37 "C. Formation of Liposomes Containing DMPE-NHPEG-NH-PDP. Lipids were mixed with the indicated amounts of DMPE-NH-PEG-NH-PDPin CHC13 and dried by evaporation and 2 h evacuation a t mbar. Buffer B was added to give 50 mg/ml of lipid concentration. The lipid was completely suspended by repeated vortexing, and VETZOO were prepared by freeze-thaw cycles and repeated extrusion according to established procedures (Mayer et al., 1986; Hope et al., 1985) with slight modification (Gruber and Schindler, 1994) a t a final lipid concentration of 20 mg/mL. The pH 5.5 was chosen in order to extend PDP group stability (Carlsson et al., 1978). Coupling of Bovine IgG to Liposomes via DMPENH-PEG-NH-PDP. Bovine IgG was derivatized with SATP to give 1.0 protected SH group per protein. A 1.2 mL portion of derivatized IgG in buffer A was deoxygenated with argon and treated with 50 pL of NHzOH reagent (see above) for 60 min a t rt while maintaining an argon atmosphere. Meanwhile the vesicles (consisting of eggPC and the indicated amount of DMPE-NH-PEGNH-PDP) were thawed from the last freeze-thaw cycle and extruded as described above. After the pH was adjusted to 7.5 with NaOH and deoxygenation with argon 1 mL of degassed vesicles was injected into the IgG solution. After 2 h of incubation under argon at rt vesicles with bound protein were separated from free protein by flotation in a sucrose gradient: Linear density gradients were formed from 6.5 mL of 15% sucrose (w/v in buffer A, the same applies to all other sucrose or trehalose solutions) and 5.5 mL of 20% sucrose, and 300 pL portions of lo%, 5%, and 2% sucrose were layered on top. A 910 pL portion of liposome-IgG mixture was mixed with either 90 pL of buffer A or 90 pL of 200 mM DTT in buffer A (control for noncovalent protein adsorption to the liposomes; compare Leserman et al., 1984) and with 330 pL of 100% sucrose. The resulting dense liposome mixtures were carefully injected below the linear sucrose gradients. &r centrifugation a t 95000gm, for 3 h a t 20 "C tubes were pierced from the side and 0.9 mL fractions were harvested. The remaining bottom layer (about 3.5 mL each) was mixed and termed fraction
Synthesis of a New Poly(ethy1ene glycol) Derivative
Bioconjugate Chem., Vol. 6,No. 3, 1995 245
1. All gradient fractions were analyzed for lipid (Stewart, 1980) and protein contents (Schaffner and Weissmann, 1973).
Coupling of Fluorescent Anti-HSA to Liposomes via DMPE-NH-PEG-NH-PDP.VETZOO were prepared from a lipid mixture of POPC/SBL/DMPE-NH-PEG-NHPDP (75/25/0.5, molar ratios) a t 20 mg/mL final lipid concentration. 80 pL portions of these liposomes in buffer B were adjusted to pH = 7.5 by the addition of 40 pL buffer D (50 mM NaC1, 100 mM NaH2P04, pH = 7.66 adjusted with NaOH), followed by the addition of 80 pL of freshly-thawed, double-labeled anti-HSA (17 pM, same batch as above) in buffer A. Tubes were closed with rubber septa, and through hypodermic needles the gas phase was perfused with argon for 5 min, with frequent vortexing. Coupling was initiated by injecting 5 pL of deoxgenated NHzOH reagent (see above). Samples were incubated a t rt for 2 h in the dark before vesicles with bound protein were separated from free protein by flotation in trehalose gradients, in close analogy to the sucrose gradients described above. Gradient fractions were analyzed for lipid concentration with a n enzymatic method (Menagent Phospholipids from Menarini Diagnostics, Florence, Italy). Protein was determined indirectly via fluorescein labels after solubilizing liposomes with excess Triton X-100.
elution volume (ml) 5
10
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20
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RESULTS
Synthesis of NHS-NH-PEG-NH-PDP.As can be seen from Scheme 1, a diamine derivative of PEG (NH2PEG-NH2, MW = 800 Da) served as starting material for the heterobifunctional crosslinker. SPDP, rather than usual amine protection, was used to introduce end group asymmetry in the first step, for the following reasons: First, the resulting asymmetric product was easy to isolate from the statistical mixture of unreacted diamine and symmetric product. Second, the ever-occurring impurity PDP-OH (hydrolyzed SPDP) could be removed in this first step only when the other end group was still a NH2 group, rather than a COOH group (i.e., it was not possible to synthesize NH2-PEG-NH-glu first and then to react with SPDP). Third, the PDP group was sufficiently stable during all subsequent steps. These reasons dictated the svnthetic route. going from PDPNH-PEG-NH~via PDP-NH-PEG-NH-~~U to NHS-NHPEG-NH-PDP. All purification steps were by silica gel chromatography. The NHS ester derivative of PEG, however, was regularly used without further purification since silica gel chromatography resulted in extensive hydrolysis. The alternative method of selective precipitation of PEG derivatives (Zalipsky, 1993) was inapplicable, as well, since precipitation only occurred if the PEG chains had a molecular weight of a t least 2000 Da. Crosslinking of T w o Different Soluble Proteins via NHS-NH-PEG-NH-PDP.The crosslinking properties of NHS-NH-PEG-NH-PDP were similar to that of its short analogue SPDP (Carlsson et al., 1978) but after the first reaction step the excess of unreactedhydrolyzed crosslinker could not be removed by simple "desalting" methods. Chromatography on Sephadex G-100, however, gave complete separation of crosslinker-modified protein (bovine IgG, Figure 1A) from free crosslinker. The control experiment in Figure 1B proved that the PEGderived crosslinker does not adsorb to IgG in a nonspecific way. The dose dependence of IgG derivatization is summarized in Figure 1C. It is important to note that the crosslinker itself is well soluble in water but DMSO was used to prepare appropriate stock solutions because no hydrolysis of the NHS ester function occurred in this
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solvent, even after several months of storage a t -20 "C and repeated thawing. In the second coupling step the PDP groups at the outer ends of the IgG-bound crosslinkers were reacted with free SH groups on another type of antibody. This second antibody (anti-HSA) was derivatized with both FLUOS (1.6 per protein) and SATP (1.8 per protein). Deprotection of bound SATP groups by NHzOH provided the free SH groups necessary for disulfide bond formation. Figure 2 shows the HPLC gel filtration chromatogram aRer coupling of fluorescent anti-HSA to bovine IgG with an average number of 5.5 covalently bound crosslinkers (trace in the middle of Figure 2). Here a considerable fraction of fluorescent anti-HSA appeared in the void volume (>lo6 Da) and also in subsequent fractions corresponding to molecular weights larger than that of one antibody. This result meets the expectation for a high degree of crosslinking under the conditions chosen. The residual peak a t 10 mL coincides with the position of unmodified IgG from the Bio-Rad standard (triangle with number 3 in Figure 2) as well as with the major fluorescence peak of FLUOS- and SATP-labeled anti-HSA
Haselgrubler et al.
246 Bioconjugate Chem., Vol. 6,No. 3, 1995
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elution volume (ml) Figure 2. Crosslinking of bovine IgG with FLUOS- and SATPlabeled anti-HSA via NHS-NH-PEG-NH-PDP. Bovine IgG with 5.5 crosslinkers per protein (compare Figure 1C) was coupled to FLUOS- and SATP-modified anti-HSA by disulfide bond formation. Crosslinking was examined by HPLC gel filtration on a Bio-Si1 TSK-400 column (Bio-Rad). The concentration of anti-HSA in the eluent was monitored indirectly via FLUOS label concentration. Triangles on top indicate the position of marker proteins (Bio-Rad gel filtration standard): 1 = void volume (aggregates), 2 = thyroglobulin (670 kDa), 3 = IgG (150 kDa), 4 = ovalbumin (44kDa), 5 = myoglobin (17 kDa), and 6 = cyanocobalamine (1.35 kDa). Upper trace: control in the the absence of the crosslinker-modified bovine IgG. Middle trace: after crosslinking of bovine IgG with anti-HSA. Bottom trace: after cleavage of intermolecular disulfide bonds with glutathione.
if the crosslinking procedure was performed in the absence of crosslinker-modified bovine IgG (upper trace in Figure 2). Crosslinking was demonstrated to occur by disulfide bond formation and not by mere adsorption since 10 mM glutathione fully reversed the effect of NH2OH-induced crosslinking (bottom trace in Figure 2). Heterobifunctional Coupling of Antibodies to Liposomes. The crosslinker NHS-NH-PEG-NH-PDP was first reacted with aminolipid (DMPE) in organic solvent, and the product was isolated by silica gel chromatography. Liposomes were then formed from dry films of phospholipid containing between 0.2 and 2 mol % of DMPE-NH-PEG-NH-PDP; freeze- thaw and extrusion was applied to ensure reproducible vesicle morphology and near unilamellarity. In the second coupling step disulfide bonds were formed between the PDP groups on the liposome surface and free SH groups on antibodies. As above, coupling was triggered when IgG-bound SATP groups were deprotected with NHzOH reagent. In a proper control experiment with liposomes prepared from highly purified eggPC this NHzOH treatment was shown to cause absolutely no breakdown of phospholipid. Liposomes with bound IgG were separated from free IgG by flotation in sucrose gradients (Figure 3) which does not alter vesicle morphology (H. J. Gruber et al., submitted for publication). Thirty-six percent of total IgG comigrated with the liposomes to the top of the gradient (Figure 3A), and all of it was specifically coupled by disulfide bonds, as judged from a parallel control experiment in which DTT had been included (Figure 3B). The relatively low yield of IgG coupling was certainly not due to lack of reactive PDP groups because the ratio of externally accessible PDP groups over IgG was around 16 (the external surface of this type of VETZOO is invariably close to 40%; H. J. Gruber et al., submitted for publication). The lateral density of antibodies on the
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sucrose gradient (ml from bottom) Figure 3. Flotation of liposomes after covalent coupling of SATP-derivatized bovine IgG. IgG with one SATP per protein was coupled to VET200 (eggPC/DMPE-NH-PEG-NH-PDP= 100/ 5, molar ratio) by disulfide bond formation and liposomes with bound protein were separated from free protein by flotation in a sucrose gradient (panel A). For a control, excess DTT was included in a parallel gradient (panel B).
external liposome surface in this experiment (2200 external lipids per bound IgG) was also far from maximal (86 lipids per IgG, Tamm and Bartoldus, 1988). Interestingly, the yield of IgG coupling dropped very little (from 36% to 28%) when the liposomes contained a 10-fold lower percentage of SH-reactive lipids. The incompleteness of IgG binding must, therefore, be attributed to the presence of underivatized IgG, as well a s to premature deprotection and oxidation of SH groups. Figure 4 shows that coupling of fluorescent anti-HSA to liposomes with a low percentage of SH-reactive lipids could be controlled very well by variation of the protein concentration. At infinite dilution the maximal "bindability" of the protein was extrapolated to be 65% (Figure 4, ordinate axis on the right). Coupling yield with respect to antibody dropped significantly a t higher protein concentration-although the ratio of externally accessible PDP groups over anti-HSA was still 3 a t the highest protein concentration. Obviously very low numbers of antibodies per vesicle (Figure 4,ordinate axis on the left) can be fine-adjusted in a reliable way by the presented method. DISCUSSION
In this study we report on the synthesis of a n SPDPlike heterobifunctional crosslinker containing a PEG spacer with a chain length of 18 ethylene glycol units (MW = 800 Da). In fact, the synthetic route was found to work also for a longer chain length of PEG (MW = 2000 Da, data not included). Our particular interest in PEGBOO with two different reactive groups relates to membrane research. This type of crosslinker appears to be required for application of emerging techniques of molecular microscopy allowing for resolution of single functional components in biomem-
Synthesis of a New Poly(ethy1ene glycol) Derivative
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Bioconjugate Chem., Vol. 6, No. 3, 1995 247 J
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offered mol IgG / mol PDP group Figure 4. Coupling of low numbers of antibodies per vesicle. Anti-HSA derivatized with both FLUOS and SATP (same batch as in Figure 2) was coupled to VET200 (containing 0.5 mol % DMPE-NH-PEG-NH-PDP) by disulfide bond formation, and vesicles with bound fluorescent anti-HSA were separated by flotation in trehalose gradients. Lipid recovery in the top part of the gradient was 95 & 1%.Fluorescence comigrating with the liposomes was classified as “specifically bound antibody” since in the presence of DTT all fluorescence remained in the bottom of the gradient. The average vesicle was assumed to have a diameter of 200 nm and to contain 4 x lo5 lipids (0.65 nm2 area per lipid) and 2 x lo3 DMPE-NH-PEG-NH-PDP.
branes. Such techniques are atomic force microscopy (AFM, for a first example see Florin et al. (1994)) and single particle tracking using fluorescence imaging (Fein et al., 1993; Ghosh and Webb, 1994). Membrane anchorage to a flat surface is indispensable in these methods. Optimally, anchorage should not perturb membrane structure, dynamics, and functions. The PEGBOO crosslinker was designed to fulfill this need: (i) It is flexible and is inert towards biomolecules. (ii) The use of lipidconjugated PEG800 guarantees anchorage of membranes or cells a t nonadsorbing conditions and a t appropriate distances. I t allows for a suffkiently thick water layer (required for integrity of membranes) and for free mobility of typical membrane components, in contrast to presently applied adsorption techniques in fluorescence microscopy methods (Hinterdorfer et al., 1994). (iii) Additional selective anchorage of particular membrane proteins via PEG80~-conJugatedsensor molecules (antibodies, peptide toxins, or other specific ligands) is one prerequisite to image the surface topology of intact membrane proteins by AFM techniques. (iv) Conjugation of AFM measuring tips with sensor molecules via PEGBOO allows to assign observed surface topologies to known functional components. We were able to realize this measuring principle in observing single antibody-antigen recognition events, including measurements of antibodyantigen unbinding forces (1 nN; P. Hinterdorfer et al., manuscript in preparation). The use of PEG800 linkage renders possible not only AFM studies but also applications of other current methods to nonperturbed, but anchored membranes. Besides these and other uses in surface science (Egger et al., 1990; Muller et al., 1993), the above-presented crosslinker may be instrumental in creating flexible protein-protein linkages or to attach homing devices to liposomes, the examples chosen in this study to evidence crosslinker functionality. In the latter application it should be noted that heterobifunctional crosslinking with a n SPDP analogue provides a convenient alternative to
homobifunctional coupling methods (Blume et al., 19931, and it is of particular interest for the flexible coupling of two different proteins to the same batch of liposomes (compare Vingerhoeds et al. (1993)). ACKNOWLEDGMENT
We are indebted to Prof. Dr. H. Falk and his staff for helpful advice and use of spectrometer facilities, to Dr. K. Hallermayer for providing anti-HSA, to Dr. M. Sanger for specific information with respect to manuscript preparation, and to S. Buchegger and B. Kenda for technical assistance. We are very grateful for helpful criticism by both reviewers. This work was supported by the Austrian Research Funds (project S-6607 to H.S.). LITERATURE CITED Blume, G., and Cevc, G. (1990) Liposomes for the sustained drug release in vivo. Biochim. Biophys. Acta 1029, 91-97. Blume, G., Cevc, G., Crommelin, M. D. J. A., Bakker-Woudenberg, I. A. J. M., Kluft, C., and Storm, G. (1993) Specific targeting with poly(ethy1ene glycol)-modified liposomes: coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times. Biochim. Biophys. Acta 1149, 180-184. Carlsson, J., Drevin, H., and k e n , R. (1978) Protein thiolation and reversible protein-protein conjugation: N-succinimidyl 3-(2-pyridyldithio)propionate,a new heterobifunctional reagent. Biochem. J . 173, 723-737. Dittmer, J. C., and Lester, R. L. (1964) A simple, specific spray for the detection of phospholipids on thin-layer chromatograms. J . Lipid Res. 5, 126-127. Dreborg, S., and Akerblom, E. B. (1990) Immunotherapy with monomethoxypolyethylene glycol modified allergens. Crit. Rev. Ther. Drug Carrier Syst. 6, 315-365. Duncan, R. J. S., Weston, P. D., and Wrigglesworth, R. (1983) A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem. 132, 68-73. Egger, M., Heyn, S. P., and Gaub, H. E. (1990) Two-dimensional recognition pattern of lipid-anchored fab‘ fragments. Biophys. J . 57, 669-673. Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77. Fein, M., Unkeless, J.,Chuang, F. Y. S., Sassaroli, M., da Costa, R., Vaanaen, H., and Eisinger, J. (1993) Lateral mobility of lipid analogues and GPI-anchored proteins in supported bilayers determined by fluorescent bead tracking. J. Membrane Biol. 135, 83-92. Florin, E.-L., Moy, V. T., and Gaub, H. E. (1994)Adhesion forces between individual ligand-receptor pairs. Science 264, 415417. Ghosh, R. N.,and Webb, W. W. (1994) Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules. Biophys. J. 66, 1301-1318. Gruber, H. J., and Schindler, H. (1994) External surface and lamellarity of lipid vesicles: a practice-oriented set of assay methods. Biochim. Biophys. Acta 1189, 212-224. Hinterdorfer, P., Baber, G., and Tamm, L. K. (1994) Reconstitution of membrane fusion sites. J . Biol. Chem. 269, 2036020368. Hope, M.J., Bally, M. B., Webb, G., and Cullis, P. R. (1985) Production of large unilamellar vesicles by a rapid extrusion procedure: Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812, 55-65. Jones, M. N., and Hudson, M. J. H. (1993) The targeting of immunoliposomes to tumor cells (A431) and the effects of encapsulated methotrexate. Biochim. Biophys. Acta 1152, 231-242. Kagawa, Y.,and Racker, E. (1971) Partial resolution of the enzymes catalyzing oxidative phosphorylation: XW. Reconstitution of particles catalyzing 32P,-adenosine triphosphate exchange. J . Biol. Chem. 246, 5477-5487. Klibanov, A. L., Maruyama, K., Beckerleg, A. M., Torchilin, V. P., and Huang, L. (1991) Activity of amphipathic poly-
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