Development of EGF-Conjugated Liposomes for Targeted Delivery of

Erika Bohl Kullberg,*,† Nill Bergstrand,‡ Jörgen Carlsson,† Katarina Edwards,‡ Markus Johnsson,‡. Stefan Sjöberg,§ and Lars Gedda†. Div...
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Bioconjugate Chem. 2002, 13, 737−743

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Development of EGF-Conjugated Liposomes for Targeted Delivery of Boronated DNA-Binding Agents Erika Bohl Kullberg,*,† Nill Bergstrand,‡ Jo¨rgen Carlsson,† Katarina Edwards,‡ Markus Johnsson,‡ Stefan Sjo¨berg,§ and Lars Gedda† Division of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala University. S- 751 85 Uppsala, Sweden. Received September 21, 2001; Revised Manuscript Received January 23, 2002

Liposomes are of interest as drug delivery tools for therapy of cancer and infectious diseases. We investigated conjugation of epidermal growth factor, EGF, to liposomes using the micelle-transfer method. EGF was conjugated to the distal end of PEG-DSPE lipid molecules in a micellar solution and the EGF-PEG-DSPE lipids were then transferred to preformed liposomes, either empty or containing the DNA-binding compound, water soluble acridine, WSA. We found that the optimal transfer conditions were a 1-h incubation at 60 °C. The final conjugate, 125I-EGF-liposome-WSA, contained approximately 5 mol % PEG, 10-15 EGF molecules at the liposome surface, and 104 to 105 encapsulated WSA molecules could be loaded. The conjugate was shown to have EGF-receptor-specific cellular binding in cultured human glioma cells.

INTRODUCTION

Liposomes have gained considerable interest as drug delivery tool for therapy of cancer and infectious diseases. They have therapeutic advantages, such as the ability to deliver large amounts of drugs to specific sites, spare healthy tissue from toxic effects, and increase the systemic circulation time of the drug (1-6). However, one problem with liposomes is their poor stability in the circulatory system. To improve this, poly(ethylene glycol), PEG, is frequently used as a component in the lipid bilayer. PEG stabilizes the liposomes sterically and also effectively protects the liposomes from degradation by the reticuloendothelial system (RES) (7-9). Specific accumulation of liposomal substances in tumor cells may be achieved by attachment of targeting agents to the liposomal surface. One way to gain both stability and specificity is to use PEG as a spacer and conjugate the targeting agent to the terminus of the polymer (10). We chose this approach and developed a conjugate of epidermal growth factor (EGF) attached to PEG-stabilized liposomes. EGF can be used as a tumor-seeking agent since the EGF receptor is overexpressed in many tumor cells including gliomas and squamos carcinomas (11). EGF is a stable protein with well-defined reaction sites for conjugation. It is also known that EGF undergoes receptor-mediated endocytosis, which brings the ligand-receptor complex inside the cell. The compound Water-Soluble Boronated Acridine-1 (WSA) is a candidate drug for boron neutron capture therapy, BNCT (12), and can be actively loaded into liposomes using a pH-gradient procedure (13). WSA consists of a boron cage with 10 boron atoms coupled to a DNA intercalating acridine analogue and spermidine * To whom correspondence should be addressed. Erika.Bohl@ bms.uu.se, Fax +46 18 471 3432. † Division of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology. ‡ Department of Physical Chemistry. § Department of Organic Chemistry.

Figure 1. Schematic drawing of the micelle-transfer method. EGF is attached to DSPE-PEG-maleimide lipids in micelles via a thiol linkage. The EGF lipids, in the form of micelles, are mixed with preformed liposomes, and the EGF-PEG-DSPE molecules are thereby incorporated into the liposome membranes

to achieve high water solubility. The compound is primarily developed for BNCT, and the stable nuclide, 10B, produces a helium and a lithium ion after neutron capture-induced fission (14). Healthy tissue can potentially be saved since only the cells that take up the compound are affected by the ion particles (15). We investigated the possibility to attach EGF to DSPC/ cholesterol/DSPE-PEG liposomes, empty or loaded with the drug WSA, using the micelle transfer method (Figure 1). EGF was coupled to maleimide-PEG-DSPE molecules in micelles to achieve a protocol that can easily be adjusted to new targeting agents and new drugs (16). The EGF-conjugated micellar lipids were then incorporated into preformed liposomes (17). This approach is appealing since the development of new formulations can be reduced to only redesigning the conjugation or the loading step. Optimizations of the micelle transfer conditions, i.e., time, temperature, and concentration, were performed. We studied the stability of the formulation regarding

10.1021/bc0100713 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/10/2002

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ligand attachment and leakage of the encapsulated compound. The receptor specificity was analyzed by incubating the EGF liposome conjugate with U-343 MGa glioma cells (18) with either blocked or nonblocked receptors. MATERIALS AND METHODS

Murine EGF, obtained from Chemicon International , was labeled with 125I from Amersham Pharmacia Biotech, Uppsala, Sweden. The protein was modified with 2-iminothiolane (Traut’s Reagent from Sigma, St. Louis, MO) to obtain thiol groups for conjugation. The Sephadex G-25 columns (NAP-5 columns), the Sephadex G-150 and Sepharose CL-4B gels used for separation, and the empty PD-10 columns were purchased from Amersham Pharmacia Biotech, Uppsala, Sweden. NHS-PEG(3400)-maleimide was purchased from Shearwater Polymers, Huntsville, AL, and 1,2-disteaoryl-sn-glycero-3-phosphatidylethanoleamine (DSPE) was obtained from Avanti Polar Lipids, Alabaster, AL. The lipids forming the liposomes 1,2 disteaoryl-sn-glycero-3-phosphatidylcholine (DSPC) and 1,2-disteaoryl-sn-glycero-3-phosphatidylethanolamineN-[poly(ethylene glycol)-2000] (DSPE-PEG) were purchased from Avanti Polar Lipids. Cholesterol (Cho) was obtained from Sigma Aldrich (Stockholm, Sweden). The DNA binding compound, 1,8-diamino-4-N-3-[12-(N-9acridinyl-3-aminopropyl)-p-carborane-1-yl]propyl-4-azooctane hydrogen chloride (WSA, Water-Soluble Acridine Derivative) was produced as described (12). The cell line, U-343 MGaCl2:6 (a subclone of U-343MG, hereafter referred to as U-343 MGa), was grown in Ham’s F-10 medium supplemented with 10% FCS and PEST (penicillin 100 IU/mL and streptomycin 100 µg/mL), L-glutamine (2 mM), and amphotericin B (2.5 µg/mL) (all from Biochrom KG, Berlin, Germany). The cells were incubated at 37 °C in humidified air containing 5% CO2. Micelles. The micelles were composed of DSPEPEG-maleimide lipids. NHS-PEG(3400)-maleimide was coupled to the phospholipid DSPE as described (19) and dried to form a lipid film. The micelles were formed by hydrating the dried lipid DSPE-PEG-maleimide film in Hepes buffer (20 mM with 150 mM NaCl pH 7.4) at a concentration above the critical micelle concentration, approximately 5 µM (20). Liposomes. The liposomes were composed of DSPC: Cho (60:40 molar ratio), and they contained additionally 0, 3, or 5 mol % DSPE-PEG compared to total lipid. The liposomes were prepared by freeze thawing and extrusion (13). Mostly unilamellar liposomes with 100 nm diameter were produced. Liposomes loaded with the DNA binding agent WSA were identical to the above. The loading of WSA was performed using a pH gradient as described (13). Briefly, liposomes with an acidic interior (citrate buffer, 300 mM pH 4) were created, and the pH on the outside of the liposomes was raised to 7.8 with sodium carbonate. This pH gradient caused the WSA molecules to enter the liposomes actively and be trapped inside with a concentration up to 300 mM, which gives approximately 105 to 106 boron atoms per liposome (13). Liposomes with an acidic interior, but without WSA, were used in a control experiment. Radiolabeling of EGF. EGF, diluted in 0.1 M sodium phosphate buffer (0.1 M NaCl, pH 7.5) (25-75 µL, 1 µg EGF/µL) was labeled with 2-7 MBq 125I by the chloramine-T method. The desired amount of 125I (1-5 µL) was added to the EGF solution and 10 µL chloramine-T (2 µg/µL in 0.1 M sodium phosphate buffer 0.1 M NaCl, pH 7.5) was added and mixed for 1 min. The reaction was

Figure 2. Scheme of the reactions of thiolation of EGF with Traut’s reagent (2-iminothiolane) and the thiol conjugation of EGF-SH to maleimide-PEG-DSPE.

stopped by 25 µL sodium metabisulfite 25 µL (2 µg/µL in 0.1 M sodium phosphate buffer 0.1 M NaCl, pH 7.5). The labeled protein was separated from reagents by gel filtration on a Sephadex G-25 column (NAP-5) and eluted in 500-600 µL with Traut’s buffer (50 mM triehanolamine 0.15 M NaCl, 1 mM EDTA, pH 8). Conjugation of 125I-EGF to Maleimide-PEGDSPE in Micelles (Figure 2). The labeled protein was modified with an excess of Traut’s reagent (300-700 µg) for 1h in room temperature (20-23 °C) under argon. Excess reagent was removed by gel filtration on a NAP-5 column eluted with 0.1 M sodium phosphate buffer (0.1 M NaCl, pH 7.5). The thiolated 125I-EGF was mixed with the micelle solution at room-temperature overnight (1624 h) at either 1:100 or 1:30, EGF: phospholipid molar ratio (PL concentration 0.35 to 0.12 mM). To remove unbound EGF, the sample was separated by gel filtration on a small (PD-10 size) column with Sephadex G-150 gel and eluted with HEPES buffer in 600 µL fractions. The fractions were measured on a gamma counter (1480 Wizard 3, Wallac, Turku, Finland), and the fraction of EGF conjugated to the micellar lipids was determined. Transfer of 125I-EGF-PEG-DSPE to Liposomes. The influence of various parameters on the rate and the amount of incorporated lipid was studied. The parameters were temperature (room temperature (RT), 37 °C, and 60 °C), lipid concentration (0.5-2.5 mM total lipid), the ratio of liposomal lipid to micellar lipid (1-5% micellar lipid:liposomal lipid), and concentration of PEGDSPE in the preformed liposomes (0%, 3%, and 5% PEG). The amount of 125I-EGF-PEG-DSPE incorporated was studied after 1, 4, and 24 h, and the same procedure was used for all experiments: 125I-EGF-PEG-DSPE micelles were mixed with the liposome solution under the varying conditions and for the desired time. The transfer was performed in a heating block. After, EGF-liposomes were purified by gel filtration on a small column (PD-10 size) with Sepharose CL-4B gel, eluted with HEPES buffer. The fraction of 125I-EGF-PEG-DSPE incorporated into the liposomes was determined by measuring the eluted fractions (0.5 mL) on a gamma counter. The final conjugates are hereafter referred to as 125I-EGF-liposome-WSA for the loaded liposomes and 125I-EGFliposome for the unloaded. Stability and Leakage. The stability of the conjugate was tested by incubation of the 125I-EGF-liposome conjugate diluted in culture medium for up to 3 weeks in 4 °C or 1 week in 37 °C. After incubation, the sample was separated by gel filtration on a CL-4B column. The amount of 125I found in the liposome fraction was determined and was assumed to constitute intact conjugate, and a comparison was made with the total amount of 125I eluted from the column. The leakage of the

EGF−Liposomes with Boronated DNA Binding Agents

encapsulated compound was determined as described (13). Briefly, the liposomal sample was separated on a gel filtration column (NAP-5), and the absorbance of lysed separated sample was compared with the absorbance of the lysed initial sample. Stability of EGF at Different Temperatures and Times. EGF (2.5 µg) was labeled with 10 MBq 125I (as described above) and after purification incubated in HEPES buffer at RT, 37 °C, and 60 °C for 1 and 24 h. The binding activity was examined with U-343 MGa cells grown in small cell dishes (3 cm) with approximately 600000 cells in each dish. The cell dishes were first washed with serum-free medium and the 125I-EGF samples were added (approximately 10 ng/dish). As a control, preincubated dishes with an excess of unlabeled EGF were used. After 30 min, the incubation media was removed, and the cells were washed 6 times with serumfree medium. The cells were trypsinized for 15 min and then resuspended in medium to a total volume of 1.5 mL per cell dish. Of this, 0.5 mL was used for cell counting and 1 mL was measured in a gamma counter to determine the bound radioactivity. The amount of bound ligand was determined and compared to untreated 125IEGF. Displacement Test. A displacement study was made to analyze the specificity of the EGF-liposome conjugate. U-343 MGa cells were grown in 3-cm cell dishes, approximately 106 cells per dish. Loaded liposome conjugate, 0.5 mL (125I-EGF-liposome-WSA), or unloaded liposome conjugate (125I-EGF-liposome) was added to dishes with 0.5 mL of nonradioactive EGF at the concentration 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000, or 10,000 ng/mL. The cells were incubated with the liposomes for 4 h, the incubation media was removed, and cells were washed six times with cold serum-free medium, trypsinized (trypsin EDTA), and resuspended (total volume 1.5 mL/dish). A volume of 0.5 mL was used for cell counting, and 1 mL was measured in the gamma counter to determine the amount of liposome conjugate bound to the cells. In the displacement study, the liposomal compounds were prepared as described above, and the following composition was used: 3% DSPE-PEG liposomes, 25 µg of EGF labeled with 50 MBq 125I, conjugation overnight, transfer of micellar lipids at 60 °C for 16 h, 3% micellar lipid:liposomal lipid ratio for the unloaded liposomes, 5% micellar lipid:liposomal lipid ratio for the loaded liposomes (to compensate for the lower incorporation level, see Results). RESULTS

Conjugation of 125I-EGF to Maleimide-PEGDSPE in Micelles. The coupling efficiency was determined by separating the 125I-EGF micelle conjugate from free 125I-EGF by gel filtration on a Sephadex G-150 column (Figure 3). Approximately 30% of the added 125IEGF was coupled to the micelles for both 1:100 and 1:30 molar ratio. The figure shows the 1:30 case; a similar pattern was obtained for the 1:100 case. This gives approximately 1 EGF /100 lipids for the highest molar ratio. The conjugation of thiolated 125I-EGF to the maleimide groups on the lipids was shown to be thiol-specific (Figure 3). Transfer of 125I-EGF-PEG-DSPE to Liposomes. The 125I-EGF-PEG-DSPE transfer efficiency into both WSA-loaded and -unloaded liposomes was examined during the conditions described below. Temperature and Time. Three different temperatures were studied: RT, 37 °C, and 60 °C (Figure 4). We found

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Figure 3. Gel filtration chromatogram for purification of 125IEGF-PEG micelles. The peak in fraction 4-8 represents the 125I-EGF-PEG-lipid micelles. Fraction 10-25 is 125I-EGF and 125I. The solid line is the elution curve for 125I-EGF modified with Traut’s reagent to obtain a thiol group for conjugation to the maleimide group of the maleimide-PEG-DSPE lipid. The dotted line is the elution curve for unmodified 125I-EGF and only a limited unspecific attachment (see fractions 6 and 7) to the maleimide group is seen.

that 60 °C was the best of the tested temperatures for the incorporation of EGF lipids into liposomes. At this temperature no further increase in the amount of incorporated 125I-PEG-DSPE could be seen after 1 h. For RT and 37 °C, the amount incorporated increased with time, but was after 24 h still not as much as 60 °C at 1 h. A smaller amount of lipid was incorporated into the liposomes when the liposomes were loaded with the DNA binding compound WSA (Figure 4). After 1 h at 60 °C, about 30% of the added micellar lipid (Figure 4D) had entered in WSA-loaded liposomes whereas in empty liposomes, with the same PEG amount, this value was about 60% (Figure 4B). To test whether the acidic interior, pH 4, of the loaded liposomes affected their ability to incorporate EGF-lipids, unloaded liposomes with a similar acidic interior were used in control experiments. The level of incorporation at 60 °C (50%) was about the same as for nonacidic unloaded liposomes (data not shown), indicating that the lower level of incorporation in the loaded liposomes was not due to their acidic interior. Different PEG-Lipid Concentrations in the Liposomes. The PEG-lipid ratio in the liposomes before transfer did not seem to affect the incorporation of 125I-EGF-DSPE at 60 °C or RT (Figure 4A,B,C). Somewhat lower incorporation was seen at 60 °C for 1 and 4 h with 5% PEG, but this increased with time (Figure 4C). Transfer at 37 °C with 0% PEG gave lower incorporation than with 3% and 5% PEG during all studied times (Figure 4A,B,C). The overall transfer with WSA-loaded liposomes was much lower than for liposomes with no load (Figure 4D), as mentioned above. For the loaded liposomes, we chose to continue with 3% PEG since PEG-lipid is needed for the stability and at least after shorter transfer times, the larger amount, 5%, might reduce the incorporation of 125IEGF-PEG-DSPE. Concentration Effects. The incorporation of 125I-EGFDSPE was analyzed as a function of the amount of added 125 I-EGF-DSPE. Three different concentrations of 125IEGF-DSPE were applied. A saturation effect could be seen since 3% and 5% 125I-EGF-DSPE to liposomal lipid gave about the same incorporation, about twice the

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Figure 4. Incorporation ability of 125I-EGF-PEG-DSPE lipid molecules into preformed liposomes. The percentage of added lipid incorporated in the liposome membrane was measured using the 125I-label on EGF. The amount of added EGF lipid was 2.7% to total liposomal lipid. A: 0% PEG in the preformed liposomes, B: 3% PEG-DSPE to total lipid, C: 5% PEG-DSPE to total lipid, D: WSA loaded liposomes with 3% PEG-DSPE, to total lipid. Error bars represent maximal errors from double experiments. Table 1. Micelle Transfer to Liposomes as a Function of the Percentage Micellar EGF-Lipid, 125I-EGF-PEG-DSPE, to Liposome Lipida 125I-EGF-PEG-DSPE

to liposome lipid, ratio

% incorporated

nmol EGF/ mmol lipid

0.01 0.03 0.05

66 ( 1 52 ( 1 35 ( 2

19 ( 1 42 ( 2 46 ( 1

a Incorporation was performed for 1 h in 60 °C and with an initial concentration of 3% PEG in the liposomes.

Table 2. Stability of the 125I-EGF-Liposome-WSA Conjugate after Storage in Complete Culture Medium at Either 4 or 37 °Ca time

4 °C

37 °C

2 days 5 days 1 week 3 weeks

97% nd nd 90%

90% 85% 79% nd

a

The result is given as the percentage of total radioactivity that remained in the liposome fraction. nd ) not determined.

amount of EGF incorporated compared to 1% (Table 1). The total lipid concentration in the reaction tube did not affect the incorporation of micelles when 3% 125I-EGFDSPE to liposomes was applied. No difference regarding the transfer could, in this case, be seen for 2.5 mM, 1 mM, and 0.5 mM total lipid (data not shown). Stability and Leakage. The stability was rather good since 79% of the radioactivity remained in the liposome fraction after 1 week of incubation at 37 °C with 125IEGF-liposome-WSA diluted in complete culture medium (Table 2). Samples incubated for 3 weeks at 4 °C had 90% radioactivity in the liposome fraction. The leakage of WSA from the liposomes was about 12% at 60 °C after 1 h. At 37 °C, the leakage was about 10% after 24 h. No extra leakage due to the transfer process was observed for either temperature.

Stability of EGF at Different Temperatures and Times. The cell tests showed that the binding ability and receptor specificity of 125I-EGF was almost unaltered for all studied times and temperatures during the micelle transfers. A slight decrease was however detected for 24 h in 60 °C (Figure 5). Displacement Test. By adding high concentrations of EGF, it was possible to displace the binding of 125IEGF-liposomes almost completely (Figure 6) proving receptor specific binding. Specificity was also seen for 125IEGF-liposome-WSA. However, the fraction of nonreceptor specific binding of radioactivity increased to about 30% in the latter case. The reason for this increase in nonspecific binding is at present not clear, but it appears likely that the WSA in some way modifies the properties of the liposomes. DISCUSSION

Here, we showed that EGF can be coupled to PEGstabilized liposomes using the micelle transfer method. A transfer temperature of 60 °C produces higher amounts of liposome incorporated 125I-EGF-PEG-DSPE than at room temperature or 37˚C. This is not unexpected since the bilayer fluidity increases with increasing temperature. EGF loses nearly no biological activity at 60 °C. The leakage of WSA at 60 °C was higher than at 37 °C, but it was not severe enough to defend the use of 37 °C as transfer temperature. The optimization studies included investigations of how the amount of PEG-lipid in the initial liposome preparation affected the incorporation of EGF-conjugated PEGlipid. Three different PEG-lipid concentrations were tested in the preformed liposomes, and only small differences were observed between them. At short time scales, a somewhat lower level of incorporation was observed for the preparation containing the highest amount of PEG-lipid, i.e., 5 mol %. The incorporated amount increased, however, with time and reached after

EGF−Liposomes with Boronated DNA Binding Agents

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Figure 5. Test of the binding ability of temperature pretreated 125I-EGF to cultured glioma cells. Binding was also tested on blocked receptors to ensure that receptor specificity did not change. An excess of unlabeled EGF was used to block the receptors in those cases. The cells were incubated with EGF for 30 min. Each bar corresponds to three cell dishes. Error bars represent maximal errors.

Figure 6. Test of binding specificity of 125I-EGF-liposomeWSA conjugate and 125I-EGF-liposome conjugate. The added amount of conjugate was the same, and increasing concentrations of unlabeled EGF displaced the binding to the EGF receptor. The incubation time was 4 h for both conjugates. For all studies the amount bound without blocking was normalized to unity. Error bars represent maximal errors; 125I-EGFliposome-WSA, n ) 6, 125I-EGF-liposome, n ) 3.

24 h the same level as observed for 0 mol % and 3 mol %. Since an initial PEG-lipid concentration of 3 mol % gave the preferred final PEG-lipid content, we chose this concentration for our further experiments. A clear difference in the transfer was observed between empty and WSA-loaded liposomes. The amount of incorporated EGF-conjugated PEG-lipid was significantly lower for liposomes containing WSA than for unloaded liposomes with the same PEG-lipid content. A similar study published by Ishida et al. (16) reported that the amount of DSPE-PEG in the preformed liposomes affects the ability of lipids to incorporate into liposomes. Ishida et al. also studied coupling of antibody to Caelyx/ Doxil liposomes with the micelle transfer method. The results indicated a much lower transfer for liposomes loaded with drug than for empty liposomes with slightly lower PEG-lipid content (4% PEG-lipid vs 5% PEGlipid). We do not rule out the possibility that the PEG-

lipid concentration in the preformed liposomes may influence the amount of incorporation but believe that, at least in our case, the decrease in the transfer is mainly due to the content of the liposomes. A final concentration of about 5 mol % PEG-lipid is desired for prolonged liposome circulation in vivo (21). With the unloaded liposomes, this can be achieved by using 3 mol % PEG-lipid in the liposomes before transfer and 3% micellar lipid to total lipid the transfer. With the loaded liposomes, we obtained about 4 mol % total PEGlipid in the final conjugate. One way to increase the final PEG-lipid amount, and also the EGF amount, is to slightly increase the micelle lipid to total lipid ratio. We studied liposomes with a radius of about 100 nm and such liposomes contain approximately 75000-80000 phospholipids per liposome (19). For these liposomes we managed to obtain about 10-15 EGF molecules /liposome. This amount is assumed to be enough to achieve a satisfactory cellular uptake of antibody-targeted liposomes (10, 19). However, the optimal amount of EGF molecules on the liposome surface needed for a high cell uptake, and desirable clearance from the systemic circulation, has to be studied in future in vitro and in vivo experiments. In two earlier studies, direct coupling of EGF to phospholipids via disulfide bonds was used (22, 23). This approach would for our PEG-stabilized, long-circulating liposomes probably lead to lower binding to tumor cells since the ligand would be “buried” within the PEG-layer (24). Instead, we conjugated EGF to the PEG-part of the lipids, ending up with the targeting agent outside the PEG-layer of the liposomes. We chose to conjugate EGF to maleimide-PEG-DSPE, thereby also avoiding the problem of unstable disulfide bonds within the circulation. The stability of the125I-EGF-liposome-WSA conjugate was good; besides, it could be stored at 4 °C for weeks. The stability at 37 °C was also acceptable and high enough for us to be sure that no significant spontaneous degradation occurred during the in vitro tests with cultured tumor cells. The 125I-EGF-liposome conjugate showed mainly EGFreceptor-specific binding to cultured glioma cells, as proven by displacement of the conjugate with increasing

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Figure 7. Cryo-Tem analysis picture of liposomes loaded with WSA. Bar represents 100 nm.

amounts of nonradioactive EGF. We cannot, at present, explain why the nonspecific binding of conjugates loaded with WSA was higher than that of the conjugate without WSA. There seems to be no disturbance of the liposome bilayer due to the load as shown in previous studies based on Cryo-TEM(13). The Cryo-TEM analyses showed intact liposomes with WSA crystals in the middle (Figure 7). More studies on the cellular uptake, retention kinetics, and fate of the load are currently in progress. CONCLUDING REMARKS

Although the problem with high uptake of liposomes by the RES system has been widely addressed during the last years (7-9), the dose-limiting organ for radionuclide therapy of malignancies using liposomes is still thought to be the liver (25). Further improvements are needed and one important factor is the tumor cell specificity of the liposomes. An active tumor targeting approach using antibodies, antibody fragments, or receptor ligands coupled to liposomes can hopefully increase the tumor specificity and decrease the dose to the liver. A binary system, such as BNCT, would give the opportunity to choose the area of neutron activation of boron and thereby further decrease the liver dose. Earlier reports on liposomes for BNCT lack specific tumor targeting ability (3, 27-30). Here, we presented a liposomal delivery vehicle for specific tumor targeting. We loaded about 105 to 106 boron atoms in each liposome, which is enough to make the liposomes interesting for therapy. It is known that 108 to 109 boron atoms are needed per tumor cell for therapeutic effect (26), and with the achieved number of boron atoms per liposome, it is realized that only 102 to 104 receptor interactions are needed per cell. The targeting ability will hopefully give the liposomes a much higher potential to find, bind, and inactivate tumor cells. The procedure of the micelle-transfer method is attractive since the load and the targeting agent can be easily exchanged. We are currently developing DNAbinding compounds for halogen labeling, such as 211At and 125I, which may be exchanged for WSA used in this study. The use of EGF as a targeting molecule is at present being evaluated at our laboratory in a clinical study on bladder cancer patients. In this study, EGFdextran (31) is used, but EGF-labeled liposomes might be applied if the penetration properties are found to be satisfactory. ACKNOWLEDGMENT

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