Bioconjugate Chem. 2007, 18, 791−799
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Synthesis, Characterization, and in Vitro Activity of Dendrimer-Streptokinase Conjugates Xiangtao Wang,†,§ Rajyalakshmi Inapagolla,‡ Sujatha Kannan,*,† Mary Lieh-Lai,† and Rangaramanujam M. Kannan‡ Critical Care Medicine, Department of Pediatrics, Children’s Hospital of Michigan/Wayne State University, Detroit, Michigan 48201, and Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202. Received October 13, 2006; Revised Manuscript Received January 30, 2007
Dendrimer conjugation with low molecular weight drugs has been of increasing interest recently for improving pharmacokinetics, targeting drugs to specific sites, and facilitating cellular uptake. Opportunities for increasing the performance of relatively large therapeutic proteins such as streptokinase (SK) using dendrimers are being explored in this study. Using the active ester method, a series of streptokinase-poly(amido amine) (PAMAM) G3.5 conjugates were synthesized with varying amounts of dendrimer-to-protein molar ratios. Characterization of these conjugates by GPC, IEC, and native-PAGE suggested that the conjugation reaction was successful, resulting in relatively pure SK-dendrimer conjugates. The conjugate made with an equimolar ratio of dendrimer to streptokinase (1:1) exhibited the highest enzymatic activity retention (∼80% retained) that has been reported so far for conjugated streptokinase with macromolecules such as PEG or dextran. SK conjugates with higher streptokinase-to-dendrimer molar ratios (1:10 and 1:20) exhibited lower initial enzymatic activities. However, these conjugates showed sustained thrombolytic activity in plasma, perhaps due to the release of SK from the conjugate. All of the SK conjugates displayed significantly improved stability in phosphate buffer solution, compared to free SK. The high coupling reaction efficiencies and the resulting high enzymatic activity retention achieved in this study could enable a desirable way for modifying many bioactive macromolecules with dendrimers.
INTRODUCTION Acute thromboembolic events such as stroke, myocardial ischemia, pulmonary embolism, and deep vein thrombosis are major causes of increased morbidity. Of these, coronary artery disease and stroke account for 40% of all annual deaths in the U.S.A. Timely therapy is crucial for achieving adequate reperfusion to salvage the affected organ. Local or systemic infusion of thrombolytic agents is the most common treatment for clot dissolution. A number of clinical trials have demonstrated the benefits of thrombolytic treatment, especially in myocardial ischemia and stroke. Streptokinase (SK), a 47 kDa single-chain protein, was one of the first clinically used intravenous thrombolytic agents. The initial studies comparing streptokinase to a placebo in the 1980s were extremely influential in proving the benefits of the use of thrombolytic agents in myocardial ischemia. Streptokinase interacts with the circulating or clot-bound plasminogen to form a noncovalent, 1:1 stoichiometric complex capable of converting another plasminogen molecule to active plasmin (1-4). The latter degrades fibrin, the main component and skeleton of a clot network, leading to rapid lysis of intravascular clots. Though SK is a relatively inexpensive and effective thrombolytic agent, its clinical use is limited by its short in ViVo half-life, immunogenicity, and high incidence of systemic bleeding, undermining its therapeutic efficacy (5). Due to the short plasma half-life of SK (30 min) (6), higher doses and continuous infusion of SK are often required for clinical treatment; this in * Corresponding author. Dr Sujatha Kannan, Department of Pediatrics, Critical Care Medicine, Children’s Hospital of Michigan, 3901 Beaubien Detroit, Michigan, Detroit, MI-48201 (USA). Phone: 313745-6109. Fax: 313-966-0105. E-mail:
[email protected]. † Department of Pediatrics. ‡ Department of Chemical Engineering and Materials Science. § Currently with the Institute for Medicinal Plant Development, Chinese Academy of Medical Sciences, Republic of China.
turn increases the risk of bleeding and immune reactions. Over the past decade, a number of studies have concentrated on improving the efficacy, potency, ease of administration, and duration of action of thrombolytic agents, while trying to decrease the side effects. Development of new or modified thrombolytic agents with higher clot specificity, longer circulation time, and lower antigenicity still remains a challenge. Strategies that have been attempted to improve the therapeutic efficacy of thrombolytic agents include attachment of polymers such as poly(ethylene glycol) (PEG) or polysaccharides (dextran) (7-9) and encapsulation in liposomes or microsomes (10). Attachment of SK to PEG appeared to decrease SK’s antigenicity, thereby increasing its circulation time, but resulted in a 67% loss in its enzymatic activity. Conjugation of SK with dextran (MW 35-50 kDa) increased its circulation time to over 3 days, with a reduction in the hemorrhagic complications and allergic reactions associated with SK use (8, 11, 12). This conjugate had a 50% loss in enzymatic activity (8) and was less efficient than free SK in achieving fibrinolysis (13). Liposomal entrapment suffered from drawbacks such as poor stability, low entrapment efficiency (30% to small liposomes and up to 60% to large liposomes) (14, 15), and a great loss in protein activity (5-15% activity retained), thus limiting its further application. Recently, there has been a significant thrust to explore dendrimers as potential drug delivery vehicles. Dendrimers are monodisperse, branched polymers with highly reactive end functionalities (Figure 1) that can be used for covalent conjugation of drugs, ligands, and/or antibodies for targeted delivery of the drug to the site of action (16-27). Dendrimers have predominantly been conjugated to small molecules, rather than biological macromolecules. The few previous attempts to conjugate dendrimers with proteins have mostly focused on antibodies (28-36) using an amine-terminated dendrimer and relatively lengthy coupling procedures to introduce sulfhydryl
10.1021/bc060322d CCC: $37.00 © 2007 American Chemical Society Published on Web 04/13/2007
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Wang et al. Scheme 1. Protocola
Schematic Representation of the Experimental
Figure 1. Dendritic structure with the core and branching units.
groups onto the dendrimer/protein. Nearly all of the conjugates maintained, to varying degrees, the binding specificity of the original antibody (28). Unlike linear polymers such as PEG, we hypothesize that dendrimers, with their compact structure, may not wrap around proteins to mask the enzyme’s active sites but might still increase the protein stability by occupying the antigen recognition sites. Therefore, conjugating SK with dendrimer may not only retain the enzymatic activity of the drug but also improve its stability by increasing its circulation time. Anionic dendrimers have been shown to have lower toxicity with a longer clearance time from the blood than cationic dendrimers (37-40). Hence, the commercially available starburst poly(amidoamine) (PAMAM) dendrimer of generation G3.5 (Figure 1) with an ethylenediamine core and peripheral carboxyl groups was used to attach SK (30). Our long-term goal is to utilize this novel dendritic nanodevice to achieve effective clot lysis while minimizing immunogenicity and the risk of bleeding. As a first step toward this goal, dendrimer-SK conjugates with varying SK-dendrimer molar ratios were prepared and characterized to determine coupling efficiency, and in Vitro enzymatic activity was tested and compared against free streptokinase.
EXPERIMENTAL PROCEDURES Materials. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDC-methiodide), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), tris (hydroxymethyl)aminomethane hydrochloride, and agarose (for routine use, gelling temperature 36 ( 1.5 °C) were purchased from Sigma. Generation 3.5 PAMAM COOH dendrimer was purchased from Dendritech, Inc., MI, and dialysis membranes (MWCO 3500 Da) from Spectra Pore, CA. Recombinant streptokinase and SK activity assay kit were purchased from Shanghai SIIC-SMU Biotech Com, Ltd. (Shanghai, PR China). 125I-fibrinogen was obtained from Amersham Corp. (Arlington Heights, IL). Ultracel YM3, YM-10, and YM-30 Centricon centrifugal filters (regenerated cellulose 3000 Da MWCO, 10 000 Da MWCO, and 30 000 Da MWCO) were purchased from Millipore Corporation (Bedford, MA). Superdex 75 prep grade (34 µm), Superdex G-25 PD-10 Column, and Mono QTM 4.6/100 PE column were purchased from Amersham Bioscience AB (SE-751 84 Uppsala Sweden). 4-20% Tris-glycine gradient precast polyacrylamide minigels were purchased from ISC BioExpress (Kaysville, UT). All other reagents were of analytical grade or HPLC grade. Purification of PAMAM G3.5 Dendrimer. A BioSuite 250 HR SEC column (5 µm, 7.8 × 300 mm) connected to a Waters HPLC system was used for PAMAM dendrimer purification. The column was eluted with orthophosphate buffer (0.1 M, pH 7.00) at a flow rate of 0.5 mL/min. UV absorbance of the eluent
a Three major steps: 1. Synthesis (1a) of the protein-dendrimer conjugates (EDC as coupling agent) and purification using a Superdex 75 preparative column (1b). 2. Characterization of the conjugates using GPC (2a), Anion exchange column (2b), and native-PAGE (2c). 3. In Vitro performance of the conjugates using a fibrin gel (3a) and clot (3b) lysis assays.
was detected at both 280 and 210 nm. Fractions containing pure G3.5 peak were collected, concentrated by Ultracel YM-3 Centricon centrifugal filter (MWCO 3000 Da), dialyzed against water for 24 h (dialysis membrane MWCO 3500 Da, under perfect sink conditions), and lyophilized to obtain solid purified G3.5 (50-60% in yield). SK-G 3.5 Conjugation. SK (13.5 mg, lyophilized powder) containing mannitol as the stabilizing agent was reconstituted with deionized water. A PD-10 column was used to remove mannitol from the reconstituted SK by eluting with borate buffer (0.1 M, pH 8.50). The carboxyl groups of PAMAM G3.5 dendrimer were activated in aqueous solution (pH 2.0) at ambient temperature for 10 min using EDC-methiodide and s-NHS as the coupling reagents (G3.5/EDC/sulfo-NHS ) 1:64: 64 molar ratio). The activated dendrimer solution was added to SK solution (in borate buffer, 0.1 M, pH 8.50) at different SK/ G3.5 molar ratios (1:1, 1:5, 1:10, and 1:20) and gently stirred at ambient temperature for 2 h (Scheme 1). Purification of SK-G3.5 Conjugates. The experimental protocol followed in this study is depicted in Scheme 1. The reaction solution was initially purified by gel permeation chromatography (GPC) using a Superdex 75 column (40 × 1.6 cm i.d.; 50 mL Superdex 75, preparative grade, 34 µm) connected to a Waters HPLC system. The column was eluted with orthophosphate buffer (0.1 M, pH 7.00) as the mobile phase (flow rate 0.6 mL/min), and UV absorbance was measured at 210 and 280 nm. Fractions containing SK conjugate and possibly free SK were collected and concentrated by Ultracel YM-30 Centricon centrifugal filter (MWCO 30 000 Da). The concentrated fractions were further purified by ion exchange chromatography (IEC) to remove free protein from the conjugate. Chromatographic Characterization of SK-G3.5 Conjugates by GPC and IEC. Analytical gel permeation chromatography (GPC) was used to analyze the obtained SK-G3.5 conjugates using a BioSuite 250 HR SEC column and ortho-
Dendrimer−Streptokinase Conjugates
Figure 2. Agar gel plate and the fibrin lysis cycles, scanned against black background.
phosphate buffer (0.1 M, pH 7.00) as eluent at 0.6 mL/min. The progress of the coupling reaction was monitored by analyzing the diluted reaction solution using GPC. For IEC examination, a prepacked Mono Q 4.6/100 PE column was preequilibrated with Tris-HCl buffer (20 mM, pH 6.50) containing 0.15 M sodium chloride. After sample injection, the column was eluted at 0.9 mL/min with a gradient pattern: 0-4 min, Tris-HCl buffer (20 mM, pH 6.50) containing 0.15 M NaCl; 4-16 min, Tris-HCl buffer with NaCl concentration (from 0.15 to 1 M). UV absorbance was measured at 210 and 280 nm. Native PAGE. Native PAGE conditions used for the analysis of SK-dendrimer conjugates were similar to those described previously for dendrimers (41, 42). The free protein, free dendrimer, and protein-dendrimer conjugates (1:1, 1:5, 1:10, and 1:20; SK/G3.5 molar ratio) were analyzed using 4-20% Tris-glycine gradient express gel under native conditions. The running buffer used was Tris-glycine native buffer (2.5 mM Tris, 19.2 mM glycine, pH 8.3). A sample buffer containing 50% sucrose and 1% methylene blue was mixed with an equal volume of salt-free sample solution, and 10 µL of the mixture was loaded into each well. The gel was run at low voltage on a Bio-Rad Mini PROTEAN III vertical gel electrophoresis system at 4 °C and then stained overnight with 2.5% Coomassie Blue-R250 solution (in 10% acetic acid and 50% methanol). The destaining solution used was a 10% acetic acid solution with 50% methanol. Both staining and destaining were performed at 4 °C. Activity Assay Based on Fibrin Gel Lysis. A modified fibrinolytic activity assay method based on that described by Li (43) was used to determine the in Vitro biological activity of SK. 250 mg of agarose was dissolved in 20 mL normal saline at 60 °C and mixed with human fibrinogen (100 mg, in 10 mL normal saline), thrombin (0.1 mL, 10 U/mL), and plasminogen (120 µg, 1.2 µg/mL). This mixture was then quickly poured into a 12.5 × 8 × 0.5 cm3 plastic plate to form an even, solidified, fibrin gel. Wells of 2 mm in diameter were made in the solidified fibrin gel, and 10 µL of free SK or conjugates (400 IU/mL of SK calculated from the amount used for the conjugation reaction) were loaded in each well in triplicate. The plate was then sealed and incubated at 37 °C. Streptokinase loaded into the wells converted plasminogen in the gel to plasmin, which lyses the fibrin, leading to a transparent ring around the well. After 8 h, the plate was scanned to record the size of the lytic ring (Figure 2). The area and the diameter of the lytic rings in the gel were measured using Image J software. The regression of the average diameter of the lytic rings against the logarithm of the activity was calculated to get a calibration curve from standard SK. The activities of the test samples were determined from this calibration curve based on the average diameter of the corresponding rings. Lysis of 125I-Fibrinogen Clots in Plasma. 125I-labeled clots were prepared from human plasma as previously described (44,
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45). 125I-fibrinogen containing plasma (0.68 µCi/mL) was quickly mixed with CaCl2 (20 mM) and thrombin (1 U/mL). The mixture was aliquoted (100 µL each) into 1.5 mL Eppendorf tubes to form clots and then incubated at 37 °C for 2 h. The obtained clots were washed four times with PBS to remove free 125I or unconverted 125I-fibrinogen. The clots were then suspended in 1 mL of plasma containing free SK or SK-G3.5 conjugates (400 U/mL) and incubated at 37 °C. The negative control sample contained clot suspended in plasma without SK. At different time intervals, 25 µL of supernatant was collected with a corresponding amount of plasma supplementation. The radioactivity was measured using Packard Cobra II Auto Gamma Counters (Canberra Packard, Meriden, CT 06450, U.S.A.). The overall radioactivity in the supernatant was calculated and divided by the original radioactivity of the clot after accounting for time decay in radioactivity to obtain the percent clot lysis.
RESULTS AND DISCUSSION Purification of PAMAM G3.5 Dendrimer and SK. The GPC analysis of commercially available PAMAM G3.5 displayed a single peak at 210 nm and three to four peaks at 280 nm, indicating some impurities (Figure 3). After purification, the dendrimer displayed a single peak at both 210 and 280 nm (Figure 3). BSA, glutamine, glycine, and mannitol are the widely used stabilizing agents for SK to maintain its enzymatic activity during lyophilization and storage (46, 47). However, the functional groups (amine and hydroxyl groups) on these additives can compete with SK for reacting with the carboxyl groups of PAMAM G3.5 dendrimer, leading to reduced coupling efficiency, heterogeneous conjugates, or conjugation failure. Therefore, SK needs to be purified before reaction. The mannitol in the received SK product was removed using a PD-10 column. Preparation of SK-G3.5 Conjugates. Water-soluble EDC-methiodide and sulfo-NHS were used to activate the carboxyl groups of PAMAM G3.5 dendrimer in aqueous solution. The activated ester of the dendrimer would be expected to react with the amine groups of SK to form an amide bond. We found that the activation step played a key role in the bioconjugation. Since active ester is unstable and can be easily hydrolyzed by water, the activated dendrimer should be reacted immediately with SK. However, if the activation time is too short, the active coupling reagents may cause SK cross-linking. Activation times of 10, 30, and 60 min were tried, and a 10 min activation time was found to be optimal, resulting in high coupling efficiency. Also, no obvious protein cross-linking was suggested by chromatographic analysis (data not shown). Therefore, a 10 min activation time was used for all the conjugates. Since carboxyl groups are easily activated in neutral to slightly acidic conditions, the purified G3.5 dendrimer was dissolved in water (pH 2) to reach a final pH of 4.0 activation (48). After activation, SK in borate buffer (0.1 M, pH 8.5) was immediately added, and the final pH of the reaction solution ranged between 8.2 and 8.5, at which SK is stable. Several reaction times (2, 4, and 6 h) and SK/G3.5 molar ratios (1:1, 1:5, 1:10, and 1:20) were investigated. The conjugates synthesized with reaction times of 4-6 h displayed nearly identical chromatographic profiles to those synthesized in 2 h (Figure 4). Since shorter reaction times are desirable to preserve the stability of protein, we used a 2 h reaction time for all the conjugates. We have shown here that relatively mild conjugation conditions involving dendrimer activation at a pH of 4.0 for 10 min, followed by a 2 h reaction with SK in borate buffer, were effective in achieving PAMAM G3.5-SK conjugates. Purification and Characterization of SK-G3.5 Conjugates. Purification of protein-dendrimer conjugates is challenging because attaching a polymer usually widens the protein peak, thus reducing the separation resolution (49). For this
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Figure 3. Comparison of GPC profiles of crude PAMAM G3.5 dendrimer and purified PAMAM G3.5 dendrimers (A, 210 nm; B, 280 nm). After purification by GPC, the PAMAM dendrimer displayed as single peaks at both 210 nm and 280 nm.
Figure 4. GPC comparisons of SK-G3.5 conjugation solutions of different starting SK-G3.5 molecular ratios (reaction solution, 2 h, at 280 nm): A for free SK, B for free PAMAM, C for SK-G3.5 (1:1), D for SK-G3.5 (1:5), E for SK-G3.5 (1:10), F for SK-G3.5 (1:20).
purpose, a preparative GPC column was used with Superdex 75 (34 µm) as packing material. This column has a Mr
fractionation range of 3 × 103 to 7 × 104 Da, suitable for separating SK-G3.5 conjugates (theoretical MW > 6 × 104
Dendrimer−Streptokinase Conjugates
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Figure 5. Chromatographic profile of separation of SK-G3.5 (1:5) conjugate using Superdex 75 column. Peak A: conjugate (plus free SK if there was some left in the reaction solution). Peak B: unreacted PAMAM G3.5 dendrimer. Peak C: coupling reagents.
Figure 6. IEC comparison (280 nm) of SK-G3.5 (1:1, 1:5, 1:10, 1:20, molar ratio) conjugates with free SK and free dendrimer: A for free SK (RT ) 4.041 min), B forfree PAMAM G3.5 dendrimer (RT ) 11.323 min), C for SK-G3.5 (1:1), D for SK-G3.5 (1:5), E for SK-G3.5 (1:10), and F for SK-G3.5 (1:20). (Only the sections of the original IEC chromatograms containing the SK-G3.5 conjugate peak are shown here; however, the complete IEC profiles were similar to those presented in Figure 7, with no free SK or free dendrimer observed.)
Da) from unreacted PAMAM G3.5 dendrimer (MW 12 931 Da) and coupling reagents (EDC-methiodide and sulfo-NHS, MW < 400). Figure 5 depicts the chromatographic profile of SKG3.5 conjugate solution, where the dendrimer-SK conjugate along with possibly free SK was separated from unreacted dendrimer and coupling agents (retention times around 34, 45, and 74 min, respectively). Fractions containing SK conjugate and possibly some free SK (peak at 34 min in Figure 5) were collected. Free SK in the reaction solution cannot be clearly separated from the conjugate by GPC. However, using IEC, a separation technique based on surface charge density of molecules, a clear
separation of free SK (RT ) 4.0 min) and free dendrimer (RT ) 11.3 min) from SK conjugates (RT ) 12.5 to 15.6 min) was achieved (Figure 6 and Table 1). PAMAM G3.5 is a highly branched polymer with 64 carboxyl groups on the surface carrying a high density of negative charge. When conjugated to SK, the high negative charge of the dendrimer will lead to a significant change in the surface charge density of SK. We expect that attaching more dendrimer molecules leads to a greater negative charge and hence longer IEC retention time for the conjugates. Thus, desirable separation was achieved using anion IEC. We observed no free SK peak in IEC analysis of the SK conjugates (Figure 7), suggesting that the conjugation
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Table 1. Comparison of IEC Retention Time of SK-G3.5 Conjugates with That of Free PAMAM G3.5 Dendrimer (280 nm)
free SK purified G3.5 SK-G3.5 (1:1)-2 h SK-G3.5 (1:5)-2 h SK-G3.5 (1:10)-2 h SK-G3.5 (1:20)-2 h a
RT (min)
∆RTa (min)
4.041 11.340 12.478 14.776 15.458 15.597
1.138 3.436 4.118 4.257
∆RT was the retention time shift using free dendrimer as reference.
reaction was effective. We would expect the retention time of the conjugate to increase with an increase in the molar ratios of dendrimer used (1:1 < 1:5 < 1:10 < 1:20), as the charge of the conjugate becomes more negative when more dendrimer molecules are attached to SK (Figure 6 and Table 1). However, it was observed that the increase in retention time of the 1:20 conjugate was not appreciably different from that of the 1:10 conjugate. This may be ascribed to the steric hindrance of the attached dendrimer that prevents more dendrimer molecules from approaching the amine groups of the SK molecule, leading to saturation in the number of dendrimer molecules that could be attached to SK. After purification by GPC, the product displayed a single peak for the SK conjugate. There was no peak observed at retention times less than 5 min, indicating the absence of unreacted or cross-linked SK in the final product (Figures 6 and 7). This also suggests that our coupling efficiency was high. These purified conjugates were then used for the in Vitro studies. The possibility of formation of the SK-G3.5 complex was excluded on the basis of the observation that a physical mixture of PAMAM G3.5 and SK (at similar molar ratios in borate buffer)
demonstrated only individual peaks for dendrimer and SK, by both GPC and IEC. The conjugates were also characterized by native PAGE, where the SK conjugates migrated differently from that of free SK (Figure 9). The migration distance in native PAGE is dependent upon the charge and molecular weight of the protein. The attachment of a negatively charged dendrimer to SK results in a conjugate that is more negatively charged than free SK and thus has a longer migration distance (Figure 9). The purified conjugates did not demonstrate any bands in the regions corresponding to either free SK or free dendrimer. The dendrimer-protein conjugations that have been previously attempted used high amounts of NH2-terminated dendrimer with expensive heterobifunctional cross-linking agents and lengthy procedures of introducing sulfhydryl groups on the dendrimer or protein before conjugation, leading to reduced overall yield and loss of protein activity (33-35, 40). This is the first study reporting conjugation of a therapeutic protein with dendrimer with high coupling efficiency. The inherent properties of this negatively charged PAMAM dendrimer, such as low cytotoxicity and longer blood circulation times, together with mild reaction conditions and high coupling efficiency as demonstrated here, makes it suitable for conjugating dendrimer to various biologically active proteins and polypeptides. Previous attempts at conjugating polymers such as PEG to SK have involved long reaction times and use of large amounts of polymer, resulting in low yield of conjugates (9, 50). However, in this study, even at a molar ratio of 1:1 (SK/G3.5), most of the SK was attached to PAMAM G3.5 dendrimer within 2 h. This may be because of the remarkably higher reactivity of the dendrimer functional groups (36, 51). This unusual
Figure 7. IEC examination of the purity of the SK-G3.5 (1:1)-2 h conjugate (A, 210 nm; B, 280 nm) fraction collected from GPC. Free SK was usually eluted out at about 4 min under the same condition, and free dendrimer around 11.2 min. IEC chromatograms of other conjugates were similar, with no peak observed between 0 and 10 min (also see Figure 6).
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Figure 8. GPC of purified SK-G3.5 conjugates (A for SK-G3.5 (1:1), B for SK-G3.5 (1:10), C for SK-G3.5 (1:20).
Figure 9. Native-PAGE of SK-G3.5 conjugates. Lane 1 for free SK, lane 2 for SK-G3.5 (1:1), lane 3 for SK-G3.5 (1:5), lane 4 for SKG3.5 (1:10), lane 5 for SK-G3.5 (1:20), lane 6 for free dendrimer. Table 2. SK Activity Measured for Different Conjugates (mean ( SD) (n ) 3)a SK-G3.5 conjugate 1:1-2 h 1:5-2 h 1:10-2 h 1:20-2 h
average activity (U/mL)
% activity retention
319.40 ( 2.73 94.52 ( 2.38 62.66 ( 0.75 40.55 ( 1.63
79.8 ( 0.7 23.6 ( 0.6 15.7 ( 0.2 10.14 ( 0.4
a Theoretical activity of all the SK conjugates was 400 IU/mL based on calculation.
reactivity appears to allow coupling reactions under mild reaction conditions and with high efficiency. Fibrin Gel Lysis. The activity of the SK-dendrimer conjugates, as measured by fibrin gel lysis, decreased as the molar ratio of dendrimer used for conjugation increased (Table 2). The 1:1 conjugate (molar ratio, SK/dendrimer) retained almost 80% of the original activity, while the 1:5, 1:10, and 1:20 conjugates retained only 24%, 16%, and 10% respectively. Similar results have also been recently reported for SKpolyglycerol conjugates (52). The problem of significant loss (sometimes even 95%) in biological activity during conjugation is one of the major concerns with protein modification (53). Attaching a polymer conveys many advantages to proteins, e.g., increased blood circulation, improved stability, and reduced immunogenicity. However, the same mechanism that prevents the approach of proteolytic enzymes or antibodies to modified protein can also hinder a substrate from approaching the active site of the protein,
Figure 10. Radiation clot lysis of SK conjugates of different SKG3.5 molar ratios in comparison with free SK (within 8 h).
especially when the substrate has high molecular weight. It is also possible that any nonspecific attachment of polymer to SK may directly occupy or shield SK’s active sites, especially when a large amount of polymer is used. Hence, less modification would lead to higher activity in the SK conjugates. However, for SK conjugates with a higher degree of modification, the activity may be recovered once the dendrimers are detached, which could result in sustained release of SK and a longer duration of action. This is further detailed in the in Vitro clot lysis assay. Lysis of 125I-Fibrinogen Clots. The thrombolytic activity of the conjugates compared to that of free SK over 8 h is depicted in Figure 10. The activities of free SK and SK-G3.5 (1:1) conjugate were similar to each other with 100% clot lysis within 3 h, whereas SK-G3.5 (1:5), SK-G3.5 (1:10), and SK-G3.5 (1:20) conjugates showed much less activity (62%, 22%, and 13% clot lysis, respectively) even at 8 h. However, when thrombolytic activities of the conjugates were compared over a prolonged period of time (Figure 11, up to nearly 48 h), a rapid increase in the rate of clot lysis was noted for SK-G3.5 (1:10) and SK-G3.5 (1:20) conjugates after the initial slow phase of thrombolysis. This variation in the thrombolytic profiles of the conjugates with greater protein modification may be due to a difference in the release of SK or active SK fragments from the conjugates over time. Although amide bonds between SK and dendrimer are generally considered to be stable in plasma (12), it is possible that proteolytic enzymes in plasma may cause degradation of the SK conjugate with prolonged incubation, leading to a release of free SK and/or of active SK fragments. Since SK has to bind to plasminogen to form the activator
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due to the masking of active sites of SK by dendrimer. The SK-G3.5 (1:1) conjugate retained up to 80% of protein activity and exhibited quick in Vitro clot lysis (comparable to that of free SK). The in Vitro stability study indicates that attachment of dendrimer improved SK stability. Along with the increasing dendrimer/SK ratio, the stability increased. Hence, it could be expected that all of the SK conjugates would have much better stability and longer circulation times than free SK in ViVo. SKG3.5 (1:1) conjugate could also be further conjugated to thrombus-specific antibodies or ligands to realize thrombustargeted SK delivery and targeted thrombolysis.
ACKNOWLEDGMENT Financial support from the Children’s Research Center of Michigan, Wayne State University Presidential Research Enhancement Fund, Pfizer Scholars grant, and NIH-Pediatric Pharmacologic Research Unit supplemental funding (NIH 3U01HD-37261-04SI) are gratefully acknowledged.
LITERATURE CITED Figure 11. Radiation clot lysis of SK conjugates of different SKG3.5 molar ratios over 48 h and the possible activity release.
complex, it is possible that the plasminogen binding sites are not readily accessible in the SK conjugates containing more dendrimer molecules. As the dendrimer gets released over time, the SK molecule may become more accessible. Once plasminogen binds with SK to form the activator complex, the thrombolytic activity rapidly increases leading to 100% clot lysis. In order to assess the release of SK and SK fragments from the conjugates, the stability of these conjugates was studied in PBS at 37 °C. While free SK was completely degraded at 34 h, the SK-G3.5 conjugates degraded slowly, with only 4% of the 1:1 conjugate, 11% of the 1:5, 15% of the 1:10, and 34% of the 1:20 conjugate remaining intact even at 94 h. This indicates that attaching dendrimer greatly improved SK stability, and this improvement was dependent on the degree of modification of SK conjugates, with higher modification leading to higher stability. The thrombolytic action regained from the SK-G3.5 (1:10) and SK-G3.5 (1:20) conjugates demonstrated that their initial reduced activity was not completely due to the real activity loss during conjugation but at least partly due to temporary coverage of the SK active sites by dendrimers. Through careful tailoring of the linkage between SK and dendrimer (for example, ester bond, Schiff base bond, or stimuli-sensitive spacer), the highly modified SK conjugates could offer a prodrug model to provide a sustained release or long-term effectiveness for a large number of bioactive proteins.
CONCLUSIONS Dendrimers are being used for drug targeting, imaging, and many other biomedical applications. This paper describes a useful method for effective attachment of dendrimer to various proteins, peptides, and other amine-containing hydrophilic molecules. Through a simple active ester reaction, carboxyl groups of the PAMAM G3.5 dendrimer were reacted with the amino groups of SK under mild aqueous conditions. A series of conjugates with increasing amounts of dendrimer to protein molar ratios have been synthesized. Optimal activation conditions (pH and activation time) led to rapid conjugation (2 h) with high coupling efficiency, as verified by GPC, IEC, and PAGE. The SK-dendrimer molar ratio had a major effect on the activity retention of SK conjugates. With an increase in the number of dendrimer molecules attached to SK, the activity (at shorter time points) decreases for the conjugate. This is possibly
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