Exchange Kinetics of Protein-Functionalized Micelles and Liposomes

Apr 16, 2010 - Emission spectra (a) are shown after 0 min (black), 60 min (dark gray), ... The solid line represents a fit to eq 1 using a rate consta...
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Bioconjugate Chem. 2010, 21, 860–866

Exchange Kinetics of Protein-Functionalized Micelles and Liposomes Studied by Förster Resonance Energy Transfer Sanne W. A. Reulen and Maarten Merkx* Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. Received September 13, 2009; Revised Manuscript Received April 2, 2010

Protein-functionalized micelles and liposomes are attractive delivery systems for applications ranging from targeted drug delivery to molecular imaging. In particular, systems that use pegylated phospholipids have become popular, but little is known about the stability of these lipid-functionalized proteins toward exchange. In this study, Förster resonance energy transfer (FRET) between the fluorescent proteins ECFP and EYFP was used to investigate the lipid exchange behavior of protein-functionalized liposomes and micelles. Native chemical ligation was used as an efficient method to site-specifically couple varying amounts of proteins to pegylated phospholipids. No exchange was observed between protein-functionalized phospholipids in sterically stabilized liposomes. In micelles, however, protein-functionalized lipids were found to exchange with a half-time of exchange ranging from almost 2 h at room temperature to 4 min at 37 °C. These pegylated micelles remained intact at lipid concentrations down to 0.15 µM, indicating that they are even more stable than previously assumed. The results obtained in this study provide a useful frame of reference for assessing the potential role of protein exchange in biomedical applications of these lipid-based nanoparticles.

INTRODUCTION Lipid-based nanoparticles such as micelles and liposomes are attractive delivery systems in a variety of biomedical applications including targeted drug delivery and molecular imaging (1, 2). An important development in this area has been the use of poly(ethylene glycol) (PEG) modified phospholipids. Incorporation of PEGmodified phospholipids in liposomes was shown to improve their biodistribution and half-time of circulation (3), most likely because the PEG shell prevents adsorption of proteins and cells. Pegylated phospholipids are also known to form relatively stable and welldefined micelles, allowing applications ranging from targeted delivery of hydrophobic drugs to their use as relatively small, macromolecular MRI contrast agents (4-6). Active targeting of micelles and liposomes has been accomplished by coupling targeting ligands such as proteins (4, 7, 8), peptides (9, 10), and carbohydrates (11-13) to the end of the PEG-chain, ensuring good accessibility of the targeting ligand. Typically, peptides and proteins are coupled to pegylated phospholipids by reaction of lysine or cysteine side chains to active esters or maleimide functionalities. However, the limited control of these classical bioconjugation reactions often yields heterogeneous conjugates and sometimes leads to protein inactivation. An important property of protein-functionalized micelles and liposomes that has received little attention is their stability toward exchange of lipidated proteins. Successful therapeutic application of liposomes and micelles requires that the proteinfunctionalized phospholipids remain associated with the nanoparticle within the time frame of the experiment. While exchange of lipidated proteins has been studied previously for vesicles and other biologically relevant bilayer systems (14-16), such studies have not been reported for protein-functionalized micelles or sterically stabilized liposomes. Work by Silvius et al. * Corresponding author. Maarten Merkx, Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. E-mail: [email protected]. Fax: (+31) 40-245-1036; Tel: (+31) 40-2474728.

showed that derivatization of phospholipids with PEG chains or protein domains can substantially enhance phospholipid transfer rates between egg phosphatidyl choline vesicles (14). However, phospholipid exchange was found to be much slower in the liposome preparations that are typically used for molecular imaging applications, which are stabilized by the incorporation of PEG-modified phospholipids and high concentrations of cholesterol (>30%). Exchange rates were also found to be highly dependent on the acyl chain length, with Li and co-workers reporting essentially no exchange for PEG2000-DSPE (17). The latter study used biotinylated PEG2000-DSPE, however, and the effect of protein conjugation was not taken into account. Desorption rates of pegylated phospholipids in PEG2000-DSPE micelles have recently been shown to be highly temperature dependent, with half-lives ranging from several days at 5 °C to less than 1 h at 30 °C (6). We and others have recently explored the use of native chemical ligation (NCL) as an attractive chemoselective conjugation reaction to couple proteins site-specifically via their C-terminus to supported lipid bilayers, micelles, and liposomes (18-20). In this study, we applied Fo¨rster resonance energy transfer (FRET) between donor and acceptor fluorescent proteins (ECFP and EYFP, respectively) as a tool to study protein exchange in protein-functionalized micelles and liposomes. Micelles and liposomes with high and tunable protein/particle ratios were obtained by NCL of fluorescent proteins to cysteine-functionalized PEG2000-distearoylphosphatidyl-ethanolamine (DSPE) phospholipids (Figure 1a). The availability of these well-defined protein-functionalized micelles and liposomes allowed an in-depth characterization of the exchange kinetics of lipidated proteins under a variety of conditions (Figure 1b). Micelles containing a 1:1 ratio of ECFP and EYFP also provided an opportunity to investigate structural properties of micelles such as aggregation number and stability (Figure 1c,d).

EXPERIMENTAL PROCEDURES General. Unless stated otherwise, all reagents and chemicals were obtained from commercial sources and used without further purification. 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-

10.1021/bc900398p  2010 American Chemical Society Published on Web 04/16/2010

Protein Exchange in Micelles and Liposomes

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Figure 1. Fo¨rster resonance energy transfer (FRET) between donor (ECFP) and acceptor (EYFP) fluorescent domains allows characterization of protein transfer kinetics and various other structural aspects of protein functionalized micelles and liposomes. (a) Preparation of fluorescent protein micelles and liposomes by native chemical ligation of fluorescent proteins with a C-terminal MESNA-thioester to cysteine-functionalized micelles and liposomes. (b) The kinetics of lipidated protein exchange can be studied by monitoring the increase in FRET upon mixing ECFP- and EYFP micelles or liposomes. (c) Dilution of mixed ECFP-EYFP micelles with increasing amounts of nonfunctionalized pegylated phospholipids informs about the average aggregation number. (d) Determination of critical micelle concentration by stepwise dilution of mixed ECFP-EYFP-micelles in buffer.

N-(Lissamine rhodamine B sulfonyl) (rhodamine-DPPE), 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3benzoxadiazol-4-yl) (NBD-DPPE), and 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))2000] (PEG2000-DSPE) were purchased from Avanti Polar Lipids. Cysteine-functionalized 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[amino(poly(ethylene glycol))2000] (CysPEG2000-DSPE) was synthesized as described previously (19). 4-(Carboxylmethyl) thiophenol (MPAA), Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), and sodium 2-mercaptoethanesulfonate (MESNA) were purchased from Sigma. Protein Expression and Purification. ECFP and EYFP with a C-terminal thioester were expressed using the IMPACT system (New England Biolabs) as previously described (21). Briefly, proteins were expressed in E. coli BL21 (DE3) cells using IPTG induction. After protein expression, the cell pellet was harvested and proteins were extracted using the BugBuster protocol

(Novagen). The proteins were obtained with C-terminal MESNA thioester using chitin affinity chromatography, followed by MESNA-induced cleavage of the protein-intein bond. The concentrations of ECFP with a C-terminal MESNA thioester (ECFP-MESNA) and EYFP with a C-terminal MESNA thioester (EYFP-MESNA) were determined by UV-vis using ε434 nm ) 29 000 M-1 cm-1 (22) and ε514 nm ) 84 000 M-1 cm-1 (23), respectively. Cysteine-Liposome Preparation. Liposomes were prepared by lipid film hydration as described previously (19). A mixture of DSPC (37 µmol), Gd-DTPA-bis(stearylamide) (25 µmol), cholesterol (33 µmol), PEG2000-DSPE (2.5 µmol), and CysPEG2000-DSPE (2.5 µmol) was dissolved in CHCl3/MeOH 4:1 (v/v) and concentrated under reduced pressure at room temperature. The obtained lipid film was hydrated in 2.5 mL HBS (10 mM HEPES, 135 mM NaCl, pH 7.4). This dispersion was extruded five times at 65 °C through 200 nm polycarbonate

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membrane filters. Cysteine-liposomes were stored at 4 °C before further modification. Synthesis of Protein-Functionalized Liposomes. Native chemical ligation of EYFP-MESNA (97 µM) to cysteineliposomes (581 µM Cys-PEG2000-DSPE) was done for 24 h at 20 °C in HBS containing 50 mM MPAA and 10 mM TCEP and pH of 6.2. Similar conditions were used to ligate ECFPMESNA (103 µM) to cysteine-liposomes (620 µM CysPEG2000-DSPE). The amount of lipidated protein was determined from SDS-PAGE analysis by integration of the bands of unreacted and lipidated protein. The liposome concentration was calculated from the lipid concentration that was determined via phosphate analysis, using the assumption of unilamellar liposomes and a lipid surface area of 0.6 nm2 (24, 25). The number of proteins per liposome was then obtained by dividing the concentration of lipidated protein by the liposome concentration. The protein-functionalized liposomes were separated from nonreacted protein by ultracentrifugation for 1 h at 270 000 g and 20 °C using a Kontron Centrikon T-2060 ultracentrifuge with a TFT 70.38 rotor. The liposomal pellet was resuspended in HBS pH 7.4, and pellet and supernatant were analyzed using SDS-PAGE to confirm the separation between lipidated and unreacted protein. Protein-functionalized liposomes were stored at 4 °C. Synthesis of Protein-Functionalized Micelles. 0.3 µmol Cys-PEG2000-DSPE was dissolved in chloroform and placed in a glass vial. After chloroform evaporation, the obtained lipid film was rehydrated in 0.1 M sodium phosphate pH 8.5 with 50 mM MPAA and 10 mM TCEP, vortexed for 2 min, followed by 5 min sonication. ECFP-MESNA and EYFP-MESNA were added in varying amounts depending on the required protein load per micelle and the pH was adjusted to ∼6.5 with 1 M sodium phosphate pH 9.2. After overnight incubation at room temperature, the protein micelles were analyzed using SDSPAGE to check for complete conversion to the lipidated protein. Upon complete conversion, the average amount of proteins per micelle was calculated by simply dividing the protein concentration over the micelle concentration. The latter was calculated from the total amount of lipids and the previously reported aggregration number of 90 lipids per micelle. Protein-functionalized micelles were stored at 4 °C. Preparation of NBD and Rhodamine-Containing Micelles. 0.9 µmol of Cys-PEG2000-DSPE and 0.02 µmol NBD-DPPE or 0.02 µmol rhodamine-DPPE in chloroform were placed in a vial. After chloroform evaporation, the obtained lipid film was rehydrated in HBS with 50 mM MPAA and 10 mM TCEP and vortexed for 2 min, followed by 5 min sonication. Micelles were stored at 4 °C. Fluorescence Spectroscopy. Fluorescence emission spectra were recorded on a Varian Cary Eclipse photoluminescence spectrometer, equipped with a cuvette holder that was temperature controlled by a Peltier element. Liposomes and micelles were diluted separately and prewarmed in cuvettes. The liposomes or micelles were mixed in a 1:1 ratio, and protein-lipid or dye-lipid transfer was monitored by exciting the donor and monitoring the emission of the donor and acceptor for a certain time period. All spectra were corrected for wavelengthdependent efficiency of the instrument. The observed rates of exchange were obtained by fitting the emission ratio as a function of time using eq 1 ratio(t) ) (ratioend - ratiostart) × (1 - exp-kt) + ratiostart

(1)

RESULTS AND DISCUSSION Protein-Phospholipid Exchange between Liposomes. Liposomes were prepared containing 2.5% Cys-PEG2000-DSPE, 2.5% PEG2000-DSPE, 25% Gd-DTPA-bis(stearylamide), 37%

Figure 2. Characterization of protein-phospholipid transfer between ECFP-liposomes and EYFP/liposomes using steady-state fluorescence spectroscopy. All measurements were performed at 37 °C in 10 mM HEPES, 135 mM NaCl, pH 7.4, with a total protein concentration of 2.3 µM ([liposome] ) 1.9 nM). Emission spectra were obtained using an excitation wavelength of 420 nm with a slit width of 10 nm, monitoring the emission from 450 to 600 nm with a slit width of 5 nm. The plot with the black squares shows a summation of the emission spectra of ECFP and EYFP liposomes measured before mixing (black squares). The spectrum labeled with open squares shows the emission spectrum obtained 23 h after mixing, while the emission spectrum labeled with gray triangles is from liposomes that contained approx 630 copies of ECFP and 630 copies of EYFP per liposome. All spectra were normalized to ECFP emission at 475 nm.

DSPC, and 33% cholesterol by lipid film hydration as described previously (19). These sterically stabilized liposomes have previously been thoroughly characterized using both DLS and cryoTEM, showing very stable liposomes with an average diameter of 200 nm (26). The liposomes obtained in this manner were subsequently functionalized with thioester-modified ECFP or EYFP using native chemical ligation in the presence of the thiol MPAA (27) as a catalyst. After removal of unreacted proteins via ultracentrifugation, protein-functionalized liposomes were obtained containing ∼1300 ECFP/liposome or ∼1000 EYFP/liposome (Supporting Information Figure S1). ECFP and EYFP liposomes were mixed at a 1:1 ratio, and emission spectra were monitored in time (Figure 2). Even after 24 h of incubation at 37 °C, the emission spectrum remained identical to the summation of the emission spectra of the separate ECFP and EYFP liposomes, consistent with the absence of any protein exchange between the liposomes under these conditions. Experiments at higher temperatures (55 °C) were hampered by the instability of the ECFP domain under these conditions, which prevented reliable measurements over prolonged periods of time. To establish that the surface density of proteins used here was sufficient to allow significant energy transfer between ECFP and EYFP when present on the same liposome, we also prepared liposomes that were reacted with a 1:1 mixture of ECFP- and EYFP-thioester. The emission spectrum of this control liposome, which contained approx 630 copies of ECFP and 630 copies of EYFP, indeed showed an intense EYFP peak at 527 nm when excited at 420 nm, providing clear evidence for energy transfer between ECFP and EYFP (Figure 2). Li et al. previously also did not observe any lipid exchange when monitoring the reaction at 37 °C for up to 25 h for biotinylated PEG2000-DSPE using a more indirect assay in the same type of liposomes (17). We thus conclude that in these liposomes protein functionalization does not result in an enhanced exchange of PEG2000-DSPE, at least not under the conditions and the time scale that are relevant for molecular imaging applications. Protein-phospholipid Exchange between Micelles. Several studies have reported efficient transfer of pegylated phospholids from micelles to liposomes (17, 28, 29). In addition, Tirrell and

Protein Exchange in Micelles and Liposomes

Figure 3. Monitoring protein exchange between ECFP-micelles and EYFP-micelles at 20 °C using steady-state fluorescence spectroscopy. All measurements were performed in HBS pH 7.4 with a protein-lipid concentration of 4.8 µM and a total lipid concentration of 40 µM. Emission spectra were obtained using an excitation wavelength of 420 nm with a slit width of 5 nm. Emission spectra (a) are shown after 0 min (black), 60 min (dark gray), 120 min (medium gray), 178 min (gray), and overnight incubation (light gray). (b) Plot showing the increase in emission ratio R527/475 as a function of time. The solid line represents a fit to eq 1 using a rate constant kobs ) 1.0 × 10-4 s-1.

co-workers recently showed a desorption rate of ∼3 h-1 at 30 °C for PEG2000-DSPE micelles (6). However, as far as we know exchange of pegylated phospholipids between micelles has not been studied for protein functionalized systems. Micelles containing an average of 11 copies of EYFP or ECFP were prepared via native chemical ligation with MPAA as the catalyst (20). SDS-PAGE analysis showed essentially complete conversion of ECFP and EYFP to their lipidated forms (Supporting Information Figure S2). Structural characterization of these preparations using dynamic light scattering (DLS) showed the presence of well-defined protein micelles with an average diameter of 14 nm and only a very small amount of larger aggregates (Supporting Information Figure S3). These ECFPand EYFP-functionalized micelles are thus similar to the previously characterized CNA35-micelles, and do not show a higher tendency to form other aggregates (20). Protein exchange was observed after mixing the ECFP and EYFP micelles at room temperature as evidenced by the decrease in ECFP emission at 475 nm and the concomitant increase in EYFP emission at 527 nm (Figure 3). However, at 20 °C the exchange is still slow. A fit of the emission ratio as a function of time yielded an observed rate of 1.0 × 10-4 s-1, corresponding to a half-life of almost 2 h at room temperature (Table 1). Since protein micelles are typically used at higher temperatures, we also monitored the exchange reaction at 28 and 37 °C (Figure 4). Protein exchange was strongly dependent on the temperature, showing a half-life of only 4 min at 37 °C. Please note that the final emission ratio that is reached at equilibrium is different at various temperatures, probably reflecting differences in temperature-dependent fluorescent properties of ECFP and EYFP. Exchange experiments were also performed at lower protein functionalization levels to exclude the possibility that interactions between donor and acceptor proteins influenced the exchange kinetics. Similar

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exchange rates were observed for micelles carrying on average 5 and 2 proteins per micelle (Table 1), suggesting that protein-protein interactions did not affect the rate-limiting step in the exchange reaction. (Supporting Information Figure S4). To understand the contribution of the protein and the PEG chain on the protein-phospholipid transfer rate, we also studied the exchange of phospholipids that contained nitrobenzoxazole (NBD) and rhodamine as donor and acceptor dyes directly attached to the phosphate ester headgroup. Cys-PEG2000-DSPE micelles were prepared containing either 2.5 mol % of rhodamineDPPE or NBD-DPPE and exchange was monitored at 37 and 55 °C (Supporting Information Figure S5). At 37 °C, the exchange of the dye-labeled phospholipids was observed to be 50× slower than the exchange of the protein-PEG2000-DSPE conjugates under otherwise similar conditions (Table 1). Very similar exchange rates were observed for micelles containing PEG2000-DSPE lacking the cysteine functionality, thus excluding the possibility that the slow exchange rate was affected by possible disulfide bond formation. Direct comparison of the exchange rates between the dye-lipid system and the proteinPEG2000-lipid system is difficult because the acyl tail in the dye-lipid system is two carbon atoms shorter. Since lipid exchange rates are highly dependent on the acyl length of the lipid tail and exhibit slower transfer rates upon increasing tail lengths (30), we expect that the half-time of exchange of dye-DSPE lipids would be even slower. This in turn implies that introduction of protein and PEG chain together enhanced the rate of phospholipid exchange more than 50-fold. Concentration Dependence of Protein-Phospholipid Transfer between Micelles. Dissociation of amphiphiles from micelles into solution is generally considered to be the rate limiting step in phospholipid exchange (31). However, some studies on mixed micelles have found an increase in exchange rates at high micelle concentrations, suggesting a role for micelle collisions in the transfer reaction (32). To investigate the role of such collisions on the exchange rate of protein-phospholipid in micelles, additional exchange experiments were performed at higher lipid concentrations. Micelles containing an equimolar amount of ECFP and EYFP were first prepared by mixing ECFP and EYFP micelles at 37 °C until full exchange was reached. Next, a large excess of nonfunctionalized PEG2000-DSPE micelles was added and the transfer of fluorescent proteins was monitored by the decrease in FRET. Figure 5 shows the emission ratio as a function of time after adding PEG2000-DSPE micelles (594 µM PEG2000-DSPE) to the mixed ECFP-EYFP micelles (40 µM Cys-PEG2000-DSPE) at 20 °C, 28 °C, and 37 °C. The exchange rates observed at these high PEG2000-DSPE concentrations are similar to the rates obtained in the initial experiments where the formation of mixed micelles was monitored at 15-fold lower PEG2000-DSPE concentrations (Table 1). This observation shows that collisions between micelles do not play a significant role in protein exchange, rendering dissociation of the proteinPEG2000-DSPE from the micelle the most likely rate-limiting step in the transfer reaction. In addition, the exchange rates observed in this study for ECFP- and EYFP-functionalized CysPEG2000-DSPE micelles are similar to the desorption rates reported by Tirrell and co-workers for PEG2000-DSPE micelles at 20 and 28 °C (6). This finding thus confirms that desorption of protein-functionalized PEG2000-DSPE is the rate-limiting step in the exchange process and suggests that protein functionalization does not significantly increase exchange rates for these pegylated phospholipids. Structural Properties of Protein-Functionalized Micelles. Micelles functionalized with ECFP and EYFP not only provide a straightforward manner to study the kinetics of protein exchange, but they also offer an opportunity to probe structural properties of these micelles. The protein-micelle ratios reported

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Table 1. Characteristics of the Transfer Process between Micelles Using ECFP and EYFP or Rhodamine and NBD as Probes

a

mixing process between

[total lipid] (µM)

T (°C)

kobs (s-1)a

t1/2 (min)b

ECFP (n ) 11) + EYFP (n ) 11) micelles ECFP (n ) 11) + EYFP (n ) 11) micelles ECFP (n ) 11) + EYFP (n ) 11) micelles ECFP (n ) 5) + EYFP (n ) 5) micelles ECFP (n ) 2) + EYFP (n ) 2) micelles rhodamine + NBD micelles rhodamine + NBD micelles ECFP-EYFP micel + unfunctionalized micelles ECFP-EYFP micel + unfunctionalized micelles ECFP-EYFP micel + unfunctionalized micelles

40 40 40 40 40 40 40 627 627 627

20 28 37 37 37 37 55 20 28 37

1.0 × 10-4 ( 1.5 × 10-6 6.0 × 10-4 ( 8.1 × 10-6 2.9 × 10-3 ( 6.9 × 10-5 1.7 × 10-3 ( 5.2 × 10-5 2.2 × 10-3 ( 2.6 × 10-4 5.6 × 10-5 ( 1.3 × 10-6 8.8 × 10-4 ( 2.1 × 10-5 1.7 × 10-4 ( 3.8 × 10-6 4.7 × 10-4 ( 2.9 × 10-6 2.6 × 10-3 ( 2.6 × 10-5

115.5 19.3 4.0 6.8 5.3 206.3 13.1 68.0 24.6 4.5

The observed rate constants (kobs) were calculated using eq 1. b The half-times of decay (t1/2) were calculated using the relation ln 2/kobs.

Figure 4. Comparison of protein exchange between ECFP-micelles and EYFP-micelles at 20 °C (light gray), 28 °C (dark gray), and 37 °C (black) using steady-state fluorescence spectroscopy. All measurements were performed in HBS pH 7.4 with a protein-lipid concentration of 4.8 µM and a total lipid concentration of 40 µM. The exchange was monitored by exciting ECFP at 420 nm and monitoring the EYFP to ECFP ratio (527 nm/475 nm) over time with excitation and emission slit widths of 5 nm. Plots of the emission ratio as a function of time were fitted to eq 1 (black lines).

Figure 5. Exchange of ECFP-EYFP micelles with high concentrations of unfunctionalized PEG2000-DSPE micelles monitored using steadystate fluorescence spectroscopy. All measurements were performed in HBS pH 7.4 with a protein-lipid concentration of 4.0 µM and a total lipid concentration of 627 µM. The exchange was monitored by exciting ECFP at 420 nm and monitoring the EYFP to ECFP ratio (R527 nm/ 475 nm) over time at 20 °C (light gray), 28 °C (gray), and 37 °C (black). Plots of the emission ratio as a function of time were fitted to eq 1 (solid lines).

here were calculated assuming an aggregation number (Na) of 90 lipids per micelle, a number that is based on previous SANS measurements on (nonfunctionalized) PEG2000-DSPE micelles (33). Since the amount of energy transfer is correlated to the presence of ECFP and EYFP on a single micelle, titration of PEG2000-DSPE to mixed ECFP-EYFP micelles should result in a decrease in emission ratio until a regime is reached where the average protein-micelle ratio is less than two. Figure 6A shows the results of such a titration experiment in which increasing concentrations of PEG2000-DSPE were added to ECFP-EYFP micelles. Indeed, a strong decrease in FRET is

Figure 6. Structural properties of mixed ECFP-EYFP micelles studied using FRET. Mixed ECFP-EYFP micelles were prepared by mixing ECFP-micelles and EYFP-micelles in a 1:1 ratio yielding a starting concentration of 2.4 µM ECFP-PEG2000-DSPE, 2.4 µM EYFPPEG2000-DSPE, and 35 µM Cys-PEG2000-DSPE. All measurements were performed in HBS pH 7.4 at 37 °C. (A) Decrease of FRET observed in ECFP-EYFP micelles by the addition of increasing concentrations of PEG2000-DSPE. Emission spectra were measured after 20 min incubation at 37 °C to allow complete exchange of lipidated proteins. (B) Determination of the CMC by stepwise dilution of mixed ECFP-EYFP-micelles in buffer. After each dilution step, micelles were left to equilibrate for 30 min before measuring an emission spectrum using an excitation wavelength of 430 nm. The arrow indicates the appearance of a Raman scatter band that increases in relative intensity at low protein concentrations.

observed up to a total PEG2000-DSPE concentration of 150-200 µM, after which the ratio stabilizes. The observation of an inflection point around 200 µM is consistent with the aggregation numbers of 72-90 that were reported in previous studies, suggesting that protein functionalization did not significantly alter the aggregation number (33-35). Micelles functionalized with a combination of donor and acceptor fluorescent proteins also provide an alternative method to assess the stability of protein-functionalized micelles by monitoring FRET upon dilution of these micelles in buffer. The critical micelle concentration is typically measured by employing methods that rely on solubilization of fluorescent dyes in the micellar interior, but it has been argued that these methods could be less reliable for stable micelles (36). The ratio of EYFP and

Protein Exchange in Micelles and Liposomes

ECFP emission was determined as a function of Cys-PEG2000DSPE concentration after time was taken to allow equilibration at 37 °C. Figure 6B shows the emission spectra normalized for dilution up to a Cys-PEG2000-DSPE concentration of 0.15 µM. Surprisingly, no significant change was observed in the ratio between EYFP emission and ECFP emission, showing that micelles remained intact under these conditions even at 0.15 µM PEG2000-DSPE. Unfortunately, no reliable measurements could be obtained at concentrations below lipid concentrations of 0.15 µM (corresponding to 10 nM ECFP and EYFP) due to the increasing intensity of a Raman scatter at 500 nm. Nonetheless, the CMC determined in this manner is at least 10-fold lower than the 1-10 µM CMC values that were previously reported for (protein functionalized) PEG2000-DSPE micelles (20, 33, 37). It is unclear whether this difference is being caused by the more direct measurement of the CMC or reflects a possible stabilization of the micelles induced by the presence of ECFP and EYFP.

CONCLUSION Functionalization of pegylated phospholipids with donor and acceptor fluorescent domains provides a straightforward approach to study the stability of protein-functionalized liposomes and micelles with respect to protein-lipid transfer. Even after incubation for 24 h at 37 °C, no protein exchange is observed for sterically stabilized liposomes. Protein exchange is relatively fast for PEG2000-DSPE micelles at 37 °C, but much slower at room temperature. The similar exchange rates obtained for protein-functionalized PEG2000-DSPE found here and nonfunctionalized PEG2000-DSPE micelles reported previously suggest that protein functionalization does not result in an additional enhancement of lipid-exchange rates. Nonetheless, the short half-life of 4 min observed at 37 °C shows that these micellar systems are kinetically unstable under physiological conditions and might rapidly exchange their protein-functionalized lipids with other lipid pools such as cellular membranes.

ACKNOWLEDGMENT The authors thank Wouter Habraken for his help with DLS, Marcel Rooijackers for initial exploratory research with the fluorescent protein micelles, Ilmar Kruis for exploratory work with the fluorescent protein liposomes, Erik Sanders for liposome preparation, and Bert Meijer and Luc Brunsveld for support and useful discussions. This work was supported by an NWO VIDI grant 700.56.428 to M.M. and the BSIK program entitled Molecular Imaging of Ischemic Heart Disease (project number BSIK03033). Supporting Information Available: SDS-PAGE analysis of protein conjugation to liposomes and micelles (Figures S1-S2), DLS characterization of the ECFP-and EYFP micelles (Figure S3), the effect of protein-loading on protein exchange kinetics of the protein-micelles (Figure S4) and exchange between NBDDPPE and Rhodamine-DPPE in Cys-PEG2000-DSPE micelles (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

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