Avidin−Biotin−PEG−CPA Complexes as Potential ... - ACS Publications

Bioconjugate Chem. 2007, 18, 1644−1650

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Avidin-Biotin-PEG-CPA Complexes as Potential EPR-Directed Therapeutic Protein Carriers: Preparation and Characterization Shan Ke,†,‡ John C. Wright,† and Glen S. Kwon*,‡ Department of Chemistry, University of WisconsinsMadison, 1101 University Avenue, Madison, Wisconsin 53706-1322, and Division of Pharmaceutical Sciences, School of Pharmacy, University of WisconsinsMadison, 777 Highland Avenue, Madison, Wisconsin 53705-2222. Received May 21, 2007; Revised Manuscript Received July 10, 2007

Bovine carboxypeptidase A (CPA) conjugated with biotinylated poly(ethylene glycol) (PEG) has been synthesized and characterized in terms of stoichiometry and half-life of the avidin-biotin-PEG(s)-CPA complex. The halflives for dissociation are 3.34 days for the avidin-biotin-PEG(3400)-CPA 1:1 complex, 3.65 days for the avidinbiotin-PEG(5000)-CPA 1:1 complex, 3.91 days for the avidin-biotin-PEG(3400)-CPA-PEG(2000) 1:1 complex, and 2.74 days for the avidin-biotin-PEG(5000)-CPA-PEG(2000) 1:1 complex. The slow dissociation demonstrates the stability of complexes using a PEGylated biotin terminus as a linker with avidin. The stoichiometry of the biotin-PEGylated CPA with avidin was determined by the 2,6-ANS method, and the results are consistent with measurements of the stoichiometry using size exclusion chromatography. The stoichiometries are 1:2 for the avidin-biotin-PEG(3400)-CPA complex and the avidin-biotin-PEG(3400)-CPA-PEG(2000) complex, 1:1 for the avidin-biotin-PEG(5000)-CPA complex, and 1:4 for the avidin-biotin-PEG(5000)-CPA-PEG(2000) complex. These findings stress both the importance of the length of a PEG chain as an appropriate spacer between the biotin terminus and a functional group, and the great potential of the avidin-biotin-PEGylated-protein complex as a therapeutic protein delivery system for solid tumor prodrug targeting.

INTRODUCTION Successful applications of chemotherapy for clinical cancer treatments are limited by deficient drug cytotoxic concentration and systemic toxicity arising from low selectivity for tumors over the normal cells (1). Antibody-directed enzyme prodrug therapy (ADEPT) has been explored as an answer to the challenge of delivering sufficient cytotoxic reagents to tumor tissues in the monoclonal antibody therapies for solid tumors (2-4). The benefit from the ADEPT is based on a catalytic conversion of a large number of prodrug molecules into cytotoxins by each targeted enzyme. However, even though monoclonal antibodies recognize certain proteins that are found on the surface of some cancer cells, so far the most striking understanding in monoclonal antibody therapy is that only 1 to 10 parts per 100 000 of the administered monoclonal antibodies can reach the target in vivo (1, 5). The enhanced permeability and retention (EPR) effect is another important method in antitumor drug delivery. This method exploits the ineffective tumor lymphatic drainage, and the defective vessels within and between the endothelium and basement membranes of various tumors, where the macromolecules can extravasate more easily and localize in the tissue intrastitial space. Through the EPR effect, the prolonged plasma circulation of macromolecules such as the polymer-conjugated drugs leads to significant passive tumor targeting (6-8). We propose a new EPR-directed enzyme prodrug therapy (EDEPT) based on the EPR effect of macromolecules. Bovine carboxypeptidase A (CPA) was chosen as the prototype of our series of targeted enzymes. The capacity of CPA to hydrolyze the first peptide or amide bond at the carboxyl or C-terminal end of proteins and peptides offers an important way to generate * Corresponding author. Tel: 608-265-5183; fax: 608-262-5345; e-mail: [email protected]. † Department of Chemistry. ‡ Division of Pharmaceutical Sciences.

the active methotrexate (MTX) from the inactive prodrug of MTX, such as Phe-MTX (9, 10). Ideally, the release of the cytotoxic MTX only occurs in the vicinity of tumor cells, thereby sparing normal cells from concomitant destruction. This type of drug release might be accomplished by pretargeting CPA to the tumors through the EPR effect. In the field of polymer-conjugated drugs, poly(ethylene glycol) (PEG) is a highly investigated polymer. PEG’s importance in pharmaceutical and biotechnical applications originates from its modification of biological macromolecules, such as peptides and proteins. The potential benefits include shielding these macromolecules from being uptaken by the reticuloendothelial system (RES), decreasing the protein immunogenicity, and preventing recognition and degradation by proteolytic enzymes (11-13). Moreover, PEG modification also increases the apparent size of an enzyme and thus reduces the renal filtration of the enzyme (14). Previous research on PEGylated CPA has demonstrated that CPA conjugated with two PEG chains has a much higher accumulation in a tumor than CPA conjugated with one PEG chain because the former’s plasma circulation time is longer (15). Also, positive tumor accumulation of a non-biodegradable poly(vinylpyrrolidone) polymer occurs when it is administered using a molecular weight greater than the glomerular filtration threshold of ∼87 000 Da (16, 17). Therefore, we attempted to increase the molecular weight of CPA above the glomerular filtration threshold by PEGylation to prolong the plasma circulation time. However, to achieve a high tumor-to-blood ratio, the concentration of a PEGylated CPA in the circulation system must be kept as low as possible. Since the removal of PEG or PEGylated proteins depends primarily on the renal filtration, it is necessary to keep the molecular weight of PEGylated CPA smaller than the glomerular filtration threshold. To solve this dilemma, we propose using an avidin-biotinPEG-CPA complex system as a potential drug delivery system (DDS) for EDEPT. We hypothesize that the ideal avidin-

10.1021/bc700182t CCC: $37.00 © 2007 American Chemical Society Published on Web 08/18/2007

Potential EPR-Directed Therapeutic Protein Carriers

biotin-PEG-CPA complex with a molecular weight higher than the glomerular filtration threshold might dissociate avidin and biotin-PEG-CPA in the circulation system and be cleared by the renal system, but also that the half-life of the avidinbiotin-PEG-CPA complex might be sufficiently long for significant tumor accumulation. The avidin-biotin system has an extremely high binding affinity and has been explored as a molecular linker apparatus in biosensors, purifications, and syntheses of important molecules for biotechnology (18, 19). Yet such strong avidin-biotin binding interaction also limits the applications of the system in some fields requiring facile capture and release (20). Research on the effects of biotinylated derivatives on the binding constant has been reported (21). For example, PEGylated biotin has a lower affinity with avidin than biotin (22). Our previous research on intermolecular interactions of avidin and PEGylated biotin demonstrates that a PEG chain can alter not only the affinity but also the stoichiometry of the avidin-biotin complex system. Following these findings, in this work we focused our research on (i) increasing the molecular weight of CPA by biotinPEGylation and successive formation of the avidin-biotinPEG-CPA complexes, and (ii) adjusting the stoichiometry and the half-life of the avidin-biotin-PEG-CPA complex systems by the PEG chains. The aim is for novel, reversible, and nonimmunogenic complexes as a DDS for EDEPT.

EXPERIMENTAL PROCEDURES Materials. Commercially available analytical grade or higher materials were used, except for the aqueous crystalline suspension of bovine carboxypeptidase A (CPA) and the PEGylated biotins. Bovine CPA, D-biotin, and hippuryl-L-phenylananine (hipp-L-phe) were obtained from Sigma (St. Louis, MO). Avidin was purchased from Pierce (Rockford, IL). PEG(2000)-SPA, biotin-PEG(3400)-NHS, and biotin-PEG(5000)-NHS were obtained from Nektar (Huntsville, AL). 2,6-Anilinonaphthalenesulfonate (2,6-ANS) was obtained from Molecular Probes (Carlsbad, CA). PEGylation of CPA. CPA was obtained as an aqueous crystalline suspension and purified as described elsewhere (23). Briefly, the crystals were washed with Millipore pure water and centrifuged three times. The protein pellet was collected. Bicarbonate buffer with a high salt concentration (0.5 M NaHCO3/0.5M NaCl, pH 8.0) was added until the entire protein pellet dissolved. This CPA bicarbonate buffer solution was then sterile-filtered with a 0.4 µm filtration disc from Millipore to remove the insoluble impurities. The concentration of the CPA solution was determined by the absorption at 278 nm using the molar absorptivity of 6.41 × 104 M-1 cm-1. The CPA solution was stored at 4 °C until use. Biotin-PEG(3400)-CPA was prepared by conjugating CPA with the N-hydroxysuccinamide ester of biotinylated PEG 3.4 kDa (biotin-PEG(3400)-NHS). Biotin-PEG(3400)-NHS solution was prepared in the same bicarbonate buffer and immediately added dropwise into a 0.20 mM stirred CPA solution, which was cooled by incubation in an ice bath. A sufficient yield of biotin-PEG(3400)-CPA was achieved using a 1.25:1 reaction molar ratio of biotin-PEG(3400)-NHS to CPA with a 120 min PEGylation time. PEGylation of CPA with biotinPEG(5000)-NHS was performed using the same reaction conditions. Biotin-PEG(3400)-CPA-PEG(2000) and biotin-PEG(5000)-CPA-PEG(2000) were prepared in two steps. First, CPA was PEGylated using a succinimidyl propionate derivative of PEG, PEG(2000)-SPA, by the same PEGylation conditions described above. After CPA-PEG(2000) was separated and collected through SEC (the concentration of CPA-PEG(2000) decreased to ∼10-6 M), we PEGylated CPA-PEG(2000)

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immediately with biotin-PEG(3400)-NHS or biotin-PEG(5000)-NHS at a 2:1 reaction molar ratio of PEG-NHS/CPAPEG. Purification of Biotin-PEGylated CPA. The purification of biotin-PEGylated CPA was achieved by size-exclusion chromatography (SEC) on an A ¨ KTA fast protein liquid chromatograph (FPLC) system (Pharmacia, Piscataway, NJ). The PEGylation mixtures were loaded directly into a Superose 12/ 30 HR column preequilibrated by PBS buffer. The elution buffer was also PBS buffer (pH 7.4) with a flow rate 0.50 mL/min, and the UV detector measured the absorbance at 280 nm. The fractions of the targeted biotin-PEGylated CPAs were collected and stored at 4 °C until use. Because the ultraviolet-visible spectra of biotin-PEG(3400)-CPA, biotin-PEG(5000)-CPA, biotin-PEG(3400)CPA-PEG(2000), and biotin-PEG(5000)-CPA-PEG(2000) are the same as that of native CPA, the concentrations of these four biotin-PEGylated CPAs were first roughly determined by the absorption at 278 nm using the molar absorptivity of native CPA. After determining the stoichiometry of the binding of their avidin complexes, their concentrations were measured using a displacement titration where a standard D-biotin or a biotinPEGylated CAP titrant displaced weakly bound 2,6-ANS from the fluorescent 2,6-ANS-avidin complex. The molecular weight and the degree of the PEGylation of the purified biotin-PEGylated CPA were determined by MALDI-TOF mass spectroscopy using a Bruker REFLEX II (Billirica, MA), equipped with a reflectron, a 337 nm nitrogen laser, and delayed extraction. Native CPA was used as the external calibrant, and the matrix chosen for this study was a freshly prepared, saturated sinapic acid solution in acetonitrile/ water (50% V/V). Briefly, the sample solutions were prepared before measurement with a typical concentration of 1 mg/mL. A 1 µL amount of the matrix and sample were mixed in a spot plate, and then a 0.5 µL sample of the mixture was deposited on the target. Analysis was performed using the reflectron mode, and the results were averaged over at least 50 shots with the laser power just above the threshold of ion formation/desorption. In particular, the samples of biotin-PEGylated CPAs for MALDI-TOF mass spectroscopy were specially purified by SEC using the volatile ammonium acetate buffer as the elution solution (24). Quantitative Assay for Stoichiometry of Biotin-PEGylated CPA with Avidin. Two biotin-binding assays were exploited to determine the stoichiometry of the binding of biotin-PEG(s)-CPA with avidin. The first was based on the use of the dye 2,6-anilinonaphthalene sulfonate (ANS), which binds to avidin and can be removed by the addition of biotin or biotinylated derivatives (25). D-Biotin solution was prepared as the primary standard to titrate the avidin solution. The standardized avidin solution was then used as the secondary standard to titrate the unknown biotin-PEG(s)-CPA solutions. Generally, we added the primary standard D-biotin solution or the unknown biotin-PEGylated CPA solution in increments to 20 mL of a PBS buffer containing about 0.50 nmol of avidin and 10 nmol of 2,6-ANS. After each addition, the fluorescence was excited at 328 nm (bandwidth of 10 nm) and measured at 408 nm (bandwidth of 10 nm), using a Shimadzu RF-1501 spectrofluorophotometer (Shimadzu, Tokyo 101-8448, Japan). A typical titration required ∼1.5 h. The second assay was based on the SEC chromatography of the avidin-biotin-PEG-CPA complexes. Briefly, 0.50 mL 1 × 10-6 M avidin PBS solutions were added with various amounts of biotin-PEG(s)-CPAs that the ratios of the two mixture components ranged from 0.5 to 6. After 24 h incubation at room temperature to ensure the equilibrium, these mixture solutions were loaded into a Superose 12/30 HR column in the

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A ¨ KTA FPLC system to perform the SEC chromatography. A quantitative evaluation of the biotin/avidin stoichiometry was achieved from the presence of the peaks of 1:1, 1:2, 1:3, or 1:4 avidin-biotin-PEG(s)-CPA complexes in the SEC chromatography against the molar ratio of avidin/biotin-PEG-CPA. PBS buffer (pH 7.4) was employed as the elution buffer with a flow rate 0.50 mL/min and the absorbance was measured at 280 nm. Quantitative Assay for Half-Life of Avidin-Biotin-PEG(s)-CPA Complex. Of the four avidin-biotin-PEG(s)-CPA complexes, the 1:1 avidin-biotin-PEG(s)-CPA complex has the longest half-life and was chosen for our dissociation rate constant investigation. The 1:1 avidin-biotin-PEG(s)-CPA complex was purified and collected by SEC separation after biotin-PEG(s)-CPA and avidin were mixed with a 1:1 molar ratio in PBS buffer for ∼2 h at room temperature. Once a 1:1 complex was collected, the kinetic dissociation rate constant koff was immediately analyzed from the active ligand displacement by adding D-biotin (∼500 mol/mol of avidin) to 30 mL ∼5 × 10-7 M 1:1 avidin-biotin-PEG(s)-CPA complex solution. Since a large excess of D-biotin prevented reassociation of biotin-PEG(s)-CPA with avidin, the dissociation became first order with respect to the concentration of the 1:1 avidinbiotin-PEG(s)-CPA complex. Aliquots of 300 µL mixture were loaded to a Superose 12/30 HR column in the FPLC A ¨ KTA system to monitor the relative concentrations of 1:1 avidin-biotin-PEG(s)-CPA complexes after the indicated incubation times (recorded from the introduction of excess D-biotin) at room temperature. The hydrolysis product of PEG(3400)-NHS or NHS was utilized as the internal standard to remove any effects of drift in the FPLC system during the long measurement period. The natural logarithm of the relative peak height of the avidin-biotin-PEG(s)-CPA complex compared with the internal standard was plotted against the incubation times. From the slope of the plot, koff and the half-life were determined. Since the peaks of 1:1 avidin-biotin-PEG(s)CPA complexes overlap with the peaks of avidin and biotinPEG(s)-CPAs in SEC chromatography, OriginPro 7.0 Gaussian peak fitting software was applied to deconvolute these overlapped peaks. Surface Plasmon Resonance (SPR) for Half-Life of Streptavidin-Biotin-PEG(s)-CPA Complex. SPR analysis was performed using a Biacore 2000 with a streptavidin (SA) sensor chip. The SA sensor chips were docked into the instrument and were preconditioned by applying three consecutive 1-min injections of 1.0 M NaCl in 50 mM NaOH solution with running PBS buffer at 20 µL/min. After that, the flow rate was adjusted to 10 µL/min, and 10 µL of biotin-PEGylated CPA solution with the concentration on the order ∼10-6 M in PBS buffer was injected. SPR measurements were made using standard procedures. Previous research shows that the binding between avidin and PEGylated biotin is temperature dependent (26), and all data in the present study were specifically collected at 24 °C.

RESULTS PEGylation of CPA. Because none of both the fifteen lysine groups and C-terminal carboxylic acid on CPA is involved in its enzymatic hydrolysis of the peptide bond, PEGylation was performed using biotin-PEG-NHS to conjugate lysine -NH2 groups or C-terminal carboxylic acid on CPA (27). PEGylated CPAs with one biotin terminus were chosen for study to delete the unwanted cross-linked products when we formulated the avidin-biotin-PEG(s)-CPA complexes. After various reactions were tested, the PEGylation conditions to yield biotinPEG-CPA with one biotin terminus were optimized as 1.25: 1.0 molar ratio of biotin-PEG-NHS per CPA in 0.50 M

Ke et al.

Figure 1. (a) Fractionation of biotin-PEG(3400)-CPA conjugates after PEGylation, using a Superose 12/30 HR column on an A ¨ KTA FPLC system. Peak I is from the tri- and di-biotin-PEG(3400)-CPA mixture, Peak II is from mono-biotin-PEG(3400)-CPA and Peak III is from unmodified native CPA. The elution buffer was PBS buffer (pH 7.4), with a flow rate of 0.50 mL/min. (b) SEC chromatogram of Fraction II. (c) MALDI-TOF mass spectroscopy of Fraction II.

NaHCO3/0.50 M NaCl buffer (pH 8.0) with 2 h reaction time in the ice bath. The PEGylation reaction stoichiometry might increase to 2:1 of PEG-NHS/CPA because of self-hydrolysis of PEG-NHS with water during storage. Although it was not necessary to site-specifically PEGylate the lysine groups on CPA, PEGylation generated a highly heterogeneous distribution of m-biotin-PEG-CPA products where m indicates the number of PEG chains coupled on CPA such as di-biotin-PEG-CPA and tri-biotin-PEG-CPA (Figure 1). Purification of biotinPEG-CPA was necessary and was achieved through SEC chromatography. Hydrophobic interaction chromatography was also tried but worked poorly for this application because PEGs also bind to chromatography media that have hydrophobic interactions (28). Previous research shows that di- or tri-PEG-CPA has a longer circulatory half-life than mono-PEG-CPA (15). We prepared biotin-PEG-CPA-PEG′, in which there are two conjugated PEG chains to protect CPA during circulation and only one of them has a biotin terminus that acts as a linker and a spacer with avidin. Such PEGylation was achieved by two steps as shown in Figure 2. The PEGylation conditions and purification procedure were the same as those for the biotinPEG-CPA preparation, except that PEG-SPA(2000) was used for the first PEGylation and that the reaction stoichiometry increased to 2:1 of PEG-NHS/PEG-CPA in the secondary PEGylation. Stoichiometry of Avidin-Biotin-PEG-CPA Complex. Although avidin has four binding sites for biotin/biotinylated derivatives, PEG can alter the stoichiometry. For example, a 1:1 stoichiometry was found for the avidin-biotin-PEG(5000) complex in our previous work. The 2,6-ANS fluorometric analysis was performed to measure the stoichiometry of biotinPEGylated CPA with avidin and the results are shown in Figure 3. Biotin-PEG(3400)-CPA and biotin-PEG(3400)-CPAPEG(2000) accessed two sites on avidin while biotin-PEG(5000)-CPA accessed only one. In contrast, it was surprising to find that biotin-PEG(5000)-CPA-PEG(2000) accessed all

Potential EPR-Directed Therapeutic Protein Carriers

Figure 2. Scheme for the synthesis of biotin-PEG-CPA-PEG′.

four sites on avidin. It appears that biotin-PEG(5000)-CPA bound on avidin sterically hinders the binding to both the adjacent and the oppositely located biotin binding sites on avidin, while biotin-PEG(5000)-CPA-PEG(2000) does not have such steric hindrance. To obtain independent evidence of the binding stoichiometry, we also investigated the avidin-biotin-PEG-CPA complex system using size exclusion chromatography (SEC). The peak positions in SEC indicate the size of the complex and therefore the stoichiometry. As shown in Figure 4a, three identifiable

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peaks corresponding to avidin (Peak A), biotin-PEG(5000)CPA (Peak B), and the avidin-biotin-PEG(5000)-CPA complex (Peak C) were present in the chromatogram. No free biotin-PEG(5000)-CPA was seen when it was mixed with avidin at molar ratio 0.5:1, while excess avidin appeared as Peak A and the avidin-biotin-PEG(5000)-CPA complex appeared as Peak C. When the molar ratio was increased to 1:1, Peak A almost vanished whereas the area of Peak C doubled. After mixing avidin with a 2-fold and 4-fold molar excess of biotinPEG(5000)-CPA, the elution position and the area of Peak C did not change significantly, except that Peak B from excess biotin-PEG(5000)-CPA began to appear. On the basis of this information, we conclude the avidin-biotin-PEG(5000)-CPA complex stoichiometry is 1:1. In contrast, except for the excess avidin and biotin-PEG(5000)-CPA-PEG(2000) that appeared as peaks A and B in Figure 4b, there were four other peaks labeled C, D, E, and F. If the biotin-PEG-CPA/avidin ratio increased from 1:1.5 to 2:1, the areas of Peak E and F increased while the areas of Peak A, C, and D decreased. When the ratio was larger than 4:1, the A, C, D, and E peaks disappeared while Peak B appeared. Peak F in the 6:1 mixture did not change significantly compared with that from the 4:1 mixture. Accordingly, the stoichiometry for the avidin-biotin-PEG(5000)CPA-PEG(2000) complex must be 1:4, and peaks C, D, E, and F must correspond to the 1:1, 1:2, 1:3, and 1:4 avidinbiotin-PEG-CPA complexes, respectively. The same strategy was applied for the avidin-biotin-PEG(3400)-CPA complex and the avidin-biotin-PEG(3400)-CPA-PEG(2000) complex, and the stoichiometries for both systems were determined to be 1:2.

Figure 3. Titration curves of 2,6-ANS fluorometric assay for stoichiometry of the avidin-biotin-PEG-CPA complex are (a) biotin-PEG(3400)-CPA, (b) biotin-PEG(5000)-CPA, (c) biotin-PEG(3400)-CPA-PEG(2000), and (d) biotin-PEG(5000)-CPA-PEG(2000) with avidin/ 2,6-ANS in PBS buffer (pH: 7.4).

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Ke et al. Table 1. Stoichiometry and Half-Life of Avidin-Biotin-PEG-CPA Complexes at 24 °C stoichiot1/2 t1/2 metry MW (day)b (day)c with avidin (kDa) from SEC from SPR biotin-PEG(3400)-CPA biotin-PEG(5000)-CPA biotin-PEG(3400)-CPA-PEG(2000) biotin-PEG(5000)-CPA-PEG(2000)

1:2 1:1 1:2 1:4

142.3 105.7 146.3 233.0

3.34 3.65 3.91 2.74

2.09 2.60 4.14 1.26

a MW, molecular weight of the avidin-biotin-PEG-CPA complex with the maximum stoichiometry. b t1/2, half-life of the 1:1 avidin-biotin-PEGCPA complex from SEC chromatography analysis. c t1/2, half-life of the 1:1 avidin-biotin-PEG-CPA complex from SPR analysis.

Figure 4. SEC elution profiles of (a) the avidin-biotin-PEG(5000)CPA complex vs molar ratio of avidin to biotin-PEG(5000)-CPA, i, 1:0.5; ii, 1:1; iii, 1:2; iv, 1:4; and (b) the avidin-biotin-PEG(5000)CPA-PEG(2000) complex vs molar ratio of avidin to biotin-PEG(5000)-CPA-PEG(2000), i, 1.5:1; ii, 1:2; iii, 1:4; iv, 1:6. Chromatograms were obtained using a Superose 12/30 HR column on an A ¨ KTA FPLC system. Eluting buffer was PBS buffer (pH 7.4) with a flow rate of 0.50 mL/min.

Half-Life of Avidin-Biotin-PEG(s)-CPA Complex. Because the tailing factors of avidin, biotin-PEG-CPA and their complexes are almost equal to one in SEC, their overlapped peaks were deconvoluted with the OriginPro 7.0 Gaussian peak fitting illustrated in Figure 5. The natural logarithm of the relative peak height of the 1:1 avidin-biotin-PEG(5000)-CPA complex decreased linearly with time so the dissociation of the biotin-PEG(5000)-CPA from avidin followed pseudo-firstorder kinetics (29). The pseudo-first-order dissociation rate constants (koff) are the slopes of the temporal decay and have the following values: 1.32 × 10-4 min-1 for the 1:1 avidinbiotin-PEG(5000)-CPA complex, 1.44 × 10-4 min-1 for the 1:1 avidin-biotin-PEG(3400)-CPA complex, 1.76 × 10-4 min-1 for the 1:1 avidin-biotin-PEG(5000)-CPA-PEG(2000) complex, and 1.23 × 10-4 min-1 for the 1:1 avidin-biotinPEG(3400)-CPA-PEG(2000) complex. The corresponding half-lives are 3.6 days, 3.3 days, 2.7 days, and 3.9 days. The dissociation rate constant (koff) of biotin-PEGylated CPA on the SA sensor chip was determined to be on the order of 10-6 s-1 from SPR analysis. The data, together with the stoichiometries, are summarized in Table 1

Figure 5. Elution profile of dissociation of the 1:1 avidin-biotin-PEG(5000)-CPA complex using a Superose 12/30 HR column on an A ¨ KTA FPLC system. (a) incubation time ) 0; (b) incubation time ) 7059 min; (c) incubation time ) 10276 min; (d) 1:1 avidin-biotin-PEG(5000)CPA complex dissociation rate at 24 °C. The peak at elution position ∼11.45 mL is from the 1:1 avidin-biotin-PEG(5000)-CPA complex and A in Ln(A)is the relative peak height of the complex in SEC chromatography. The elution buffer was PBS buffer (pH 7.4) with a flow rate of 0.50 mL/min. The dash lines (-----) stand for the deconvoluted peaks using OriginPro 7.0 Gaussian peak fitting.

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Potential EPR-Directed Therapeutic Protein Carriers

DISCUSSION Stoichiometry. The stoichiometries of avidin binding with biotin-PEG(3400)-CPA (1:2), biotin-PEG(5000)-CPA (1: 1), and biotin-PEG(3400)-CPA-PEG(2000) (1:2) support our previous conclusion that, as a spacer, PEG can block the further binding of biotin derivatives with avidin. But it was also surprising to find that biotin-PEG(5000)-CPA-PEG(2000) accesses all four binding sites on avidin. While the avidin-biotin-PEG(3400) complex has a 1:4 stoichiometry, biotin-PEG(3400)-CPA only accesses two oppositely located biotin binding sites on avidin. This 1:2 stoichiometry is attributed to the increased steric hindrance from PEG and CPA, as well as to the electrostatic repulsion among CPAs (9). The hindrance and repulsion also result in a 1:2 stoichiometry for the avidin-biotin-PEG(3400)-CPA-PEG(2000) complex. The 1:1 stoichiometry with avidin shows that the biotinPEG(5000)-CPA hinders the binding to not only adjacent but also oppositely located biotin binding sites on avidin, as observed in biotin-PEG(5000) (to be published). We speculate that the attractive interaction between PEG and avidin should be stronger than that between PEG and CPA; thus, the PEG(5000) chain surrounds avidin instead of CPA, even though CPA is first conjugated with PEG(5000). It is also possible that PEG(5000) is large enough to surround avidin while it is not for CPA. PEG(5000) has a Flory radius of 60 Å (30), avidin has dimensions of approximately 56 Å × 50 Å × 40 Å (31), and CPA has dimensions of approximately 51 Å × 60 Å × 47 Å (32). In contrast, avidin is not surrounded by the PEG chain in the avidin-biotin-PEG(5000)-CPA-PEG(2000) complex since biotin-PEG(5000)-CPA-PEG(2000) is able to access all four binding sites of avidin. This inability to surround avidin may occur because the PEG-PEG repulsion between PEG(2000) and PEG(5000) prevents PEG(5000) from surrounding avidin. Moreover, the release of biotin-PEG(5000)-CPA from avidin by introduction of biotin also indicates the flexibility of such a surrounding. The observed 1:2 stoichiometry of the avidinbiotin-PEG(3400)-CPA-PEG(2000) complex and the 1:4 stoichiometry of the avidin-biotin-PEG(5000)-CPA-PEG(2000) complex may mean that the longer PEG can relieve more electrostatic repulsion among CPAs and that the electrostatic repulsion among CPAs may be stronger than PEG-PEG steric repulsion. Half-Life. The duration of efficacy of a protein ligand complex cannot be determined by the in vitro measured equilibrium dissociation constant but rather depends on the complex dissociation rate constants (koff) or half-life (33). As we proposed, the ideal avidin-biotin-PEG(s)-CPA complexes would dissociate but still have a prolonged plasma circulation for the sufficient accumulation in tumor tissues through the EPR effect. Optimization of the half-life of the complex is therefore crucial for such a drug delivery. The results show that the dissociation half-life and the stoichiometry of the complex can be adjusted by the lengths of the PEG chains, and the halflives of these 1:1 complexes are about 3-4 days, which is very promising (Table 1). In addition, since the 1:4 complex dissociates to 1:3 and 1:2 and finally 1:1, the 1:4 complex can be explored for a drug release with a relatively constant concentration over a long period. We also obtained the dissociation rate constant (koff) of these biotin-PEGylated CPAs with streptavidin using the surface plasmon resonance (SPR) analysis. A streptavidin sensor chip was used because an avidin chip was not available through Biacore. The dissociation rate constants determined from the SPR analysis are different than those determined by the solutionbased method (see Table 1). This result reflects the difference

between avidin and streptavidin, as well as a variety of potential artifacts in surface-based measurements (34, 35). It signals the advantage of the solution-based method, which is closer to the physical environment involved in drug delivery applications. Although it is simpler to obtain the data by SPR, we use the data from the solution-based method in all of our work. It was reported that PEG could create attractive interactions among proteins and cause protein crystallization or aggregation (36). To investigate the stability of our system, we kept biotinPEG(3400)-CPA, biotin-PEG(3400)-CPA-PEG(2000), biotin-PEG(5000)-CPA, biotin-PEG(5000)-CPA-PEG(2000), avidin-biotin-PEG(3400)-CPA complex, avidin-biotinPEG(5000)-CPA complex, avidin-biotin-PEG(3400)-CPAPEG(2000) complex, and avidin-biotin-PEG(5000)-CPAPEG(2000) complex in a refrigerator at 4 °C. We did not find any precipitation or any obvious SEC peak from the aggregated proteins over three weeks. This observation means that these complexes can be kept at least for three weeks without precipitation and aggregation. The study on PEGylated CPA shows that the attachment of up to three PEG to CPA improves CPA catalytic properties (15). Following the same strategy using hipp-L-phe, we simply evaluated the enzymatic hydrolysis capacity of CPA in a 1:4 complex of avidin-biotin-PEG(5000)-CPA-PEG(2000) and in a 1:2 complex of avidin-biotin-PEG(3400)-CPA-PEG(2000) by comparison with native CPA. It appears that CPA in both complexes has similar catalytic properties to those of native CPA. Even though these preliminary findings show that CPA does not lose its enzymatic capacity during the PEGylation and formation of the avidin-biotin-PEG(s)-CPA complexes, the systemic investigation of the enzymatic ability, bioactivity, and pharmacokinetic properties of these complexes will definitely be our next approach in this field.

CONCLUSIONS The aim of the present study is to investigate the practical potential of the avidin-biotin-PEG-CPA complex as a novel DDS for MTX prodrug EPR-directed pretargeting. Basic steps include synthesis of biotin-PEG(s)-CPA, formation of the avidin-biotin-PEG(s)-CPA complexes, and analytical procedures to measure the stoichiometries and half-lives of the complexes. The results show (i) the molecular weights of the avidin-biotin-PEG-CPA complexes are in the same order as that of an antibody, (ii) the stoichiometries of avidin-biotinPEG-CPA complexes are 1:4 for avidin-biotin-PEG(5000)CPA-PEG(2000), 1:2 for avidin-biotin-PEG(3400)-CPA and avidin-biotin-PEG(3400)-CPA-PEG(2000), and 1:1 for avidin-biotin-PEG(5000)-CPA, and (iii) the half-lives of these 1:1 complexes are around 3-4 days at 24 °C so the dissociation kinetics are slow enough for typical tumor accumulations through EPR. On the basis of the half-life and stoichiometry, the avidin-biotin-PEG-CPA complexes or their analogues have potential as a new protein drug delivery system, in which the avidin-biotin-PEG-CPA complex acts as both the transporting device and the targeting device, while CPA is the drug moiety for the prodrug pretargeting strategy. In addition, the data in the present study provides information on four interactions that can alter the binding of avidin with biotin in avidin-biotin-PEG(s)-CPA complexes: (i) the steric repulsion between PEG-PEG; (ii) the interaction among the attached functional groups on the second terminus of PEG, such as the electrostatic repulsion among the conjugated CPAs; (iii) the interaction between the attached functional group and PEG, such as that PEG chains relieve the interaction among CPAs; (iv) the surrounding of PEG around avidin. Both the length of a PEG chain and the interactions of the attached functional

1650 Bioconjugate Chem., Vol. 18, No. 5, 2007

groups on PEG should be considered when a PEG chain is chosen as an appropriate spacer.

ACKNOWLEDGMENT We thank Dr. McClain for his assistance in fluorometric assay for the stoichiometry quantitative evaluation and helpful discussion of the paper.

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