Targeted Delivery of Dendritic PolyglycerolâDoxorubicin Conjugates

Article pubs.acs.org/Biomac

Targeted Delivery of Dendritic Polyglycerol−Doxorubicin Conjugates by scFv-SNAP Fusion Protein Suppresses EGFR+ Cancer Cell Growth Ahmad Fawzi Hussain,†,○ Harald Rune Krüger,‡,○ Florian Kampmeier,§ Tim Weissbach,∥ Kai Licha,⊥ Felix Kratz,# Rainer Haag,‡ Marcelo Calderón,*,‡ and Stefan Barth*,∥,▽ †

Department of Gynecology and Obstetrics, University Hospital RWTH Aachen, Pauwelsstrasse 30, 52074, Aachen, Germany Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195, Berlin, Germany § Department of Imaging Sciences and Biomedical Engineering, King’s College London, Westminster Bridge Road London SE1 7EH, U.K. ∥ Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstrasse 20, 52074, Aachen, Germany ⊥ mivenion GmbH, Robert-Koch-Platz 4, 10115, Berlin, Germany # Tumor Biology Center and Proquinase GmbH, Breisacher Strasse 117, 79106, Freiburg, Germany ▽ Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, 52074, Aachen, Germany ‡

S Supporting Information *

ABSTRACT: Development of effective polymer-based nanocarriers for the successful application in cancer therapy still remains a great challenge in current research. In the present study we present a dendritic polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing epidermal growth factor receptor (EGFR). The nanocarrier was provided with a dendritic core as a multifunctional anchoring point, doxorubicin (Doxo) coupled through a pH-sensitive linker, a fluorescence marker, poly(ethylene glycol), as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-Tag technology. The study provides the proof of principle that SNAP-tag technology can be used to generate drug-carrying nanoparticles efficiently modified with singlechain antibodies to specifically target and destroy cancer cells.

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INTRODUCTION Tumor targeting therapy has emerged as a strategy to overcome the lack of specificity of traditional therapeutic agents. Several classes of disease-specific ligands have been used to reduce unspecific toxicity of therapeutic agents by their specific delivery to target cells including folic acid,1,2 transferrin,3 peptides,4−6 hormones,7 vitamins,8 aptamers,9,10 monoclonal antibodies and their fragments.11−13 Two main approaches have been described to conjugate therapeutic agents to these ligands. In the first approach, the therapeutic agent is conjugated directly to the ligand and in the second approach a ligand is conjugated to a vehicle such as nanoparticles and soluble polymers, which serves as a carrier for a therapeutic agent.14−17 These disease-specific nanoparticles can increase the efficacy and achieve a satisfactory safety of conventional small molecule drugs by increasing accumulation in the target tissue, through the “enhanced permeation and retention” (EPR) effect, slower blood clearance, as well as improving drug solubility and stability. Furthermore, nanoparticles can be used to combine a © 2013 American Chemical Society

therapeutic agent with an imaging agent thus allowing the direct monitoring of drug delivery.18 Several types of nanoparticles such as metal-, polymer-, and lipid-based nanoparticles have been developed to deliver therapeutic agents, including chemotherapeutic agents, radionuclides, photosensitizers, and siRNA.19,20 Owing to the special chemical, physical, and biological features of dendritic polymers, an extensive effort has been pushed forward to utilize these unique molecules in therapeutic and diagnostic purposes for the treatment of cancer.21−26 The unique advantages offered by a dendritic molecule over conventional macromolecules and polymers are the presence of multiple functional groups and their amenability to further chemical modification, extremely low polydispersity, low solution viscosity, reduced molecular entanglement, the presence of nanocavities, and scalable size within the range of Received: November 16, 2012 Revised: June 7, 2013 Published: June 19, 2013 2510

dx.doi.org/10.1021/bm400410e | Biomacromolecules 2013, 14, 2510−2520

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Scheme 1. Schematic Pathway for the Synthesis of Doxorubicin-Polyglycerol Derivatives in Phosphate/EDTA Buffer, pH 7.0, Room Temperaturea

a

(1) Polyglycerolamine (MW 10 kDa, 20% amine functionalization); (2) thiolated polyglycerol; (3) (6-maleimidocaproyl-(hydrazone) derivative of doxorubicin (Doxo-EMCH), a thiol-reactive doxorubicin prodrug bearing a maleimide moiety as the thiol-binding group and a hydrazone bond as a pH-sensitive linker; (4a) BG-PEG-Mal, 2 kDa; (4b) BG-Mal; (5) PEG-Mal, 2 kDa; (6) IDCC dye as fluorescence label (7a) PG-Doxo-PEG-BG; (7b) PG-BG-Doxo-PEG.

1−10 nm.27 Drug molecules can either be encapsulated within the dendritic structure or conjugated covalently using engineered functional groups at the surface of the dendritic polymers. The latter approach involves the utilization of Ringsdorf’s concept of drug−polymer conjugate using the dendritic scaffold.28,29 The idea involves the attachment of bioactive molecules directly or via spacer molecules to terminal groups of the polymer. The covalent bonds are intracellularly degradable or hydrolyzable, and in most cases ester, hydrazone, ketals, or amide bonds are employed. Due to the feature of having multiple functional groups it is possible to attach several copies of a drug or imaging agent or combinations of different drugs to the same carrier molecule. Targeting ligands/moieties such as peptides, antibodies or antibody fragments can be attached to the dendritic scaffold to improve therapeutic efficacy. In an attempt to increase specificity and decrease off-target effects, dendritic polymers have been conjugated to monoclonal antibodies or antibody fragments to deliver them specifically to target cells.30−32 However, the limitation arising from the wide distribution of conjugates with different numbers of ligands, which is further exacerbated when multiple functions are introduced, still needs to be addressed. Recombinant antibody fragments have several advantages for use as targeting ligands, including small size combined with high affinity and specificity, lack of immune effector function, and ease of production. They can further be engineered to facilitate site-specific conjugation to a polymer or particle surface. We have previously described a method for the sitespecific modification of antibody fragments by fusing them to an engineered version of the human DNA-repair enzyme O6alkylguanine DNA alkyltransferase (AGT), known as SNAPTag.33−36 We have shown that the genetic fusion of single-

chain antibody fragments to the SNAP-Tag results in a selfcoupling antibody species that can be modified with small effector molecules as well as coupled site specifically to the surface of nanoparticles. The use of self-labeling proteins such as SNAP, CLIP, and Halo tags can provide a unique conjugation site on the antibody, allowing the production of a homogeneous conjugate preparation.37 The SNAP-Tag is allowing covalent attachment of O6-benzylguanine (BG) substrates via irreversible transfer of an alkyl group to a cysteine residue.38 This technology provides an easy, fast, site specific, and flexible method for labeling proteins with various BG-modified molecules and has been used in different approaches, including protein labeling in living cells,39,40 facilitating the plasmon resonance and protein microarray surface immobilization with SNAP tag fusion proteins,41−43 in vivo imaging,44 and therapeutic agent delivery.12 In this study we used BG-modified dendritic polyglyceroldoxorubicin conjugates (PG-Doxo) and coupled them via the SNAP-tag to an antibody fragment directed against epidermal growth factor receptor (EGFR) to investigate the effects of receptor specific targeting on in vitro toxicity.45−47

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EXPERIMENTAL PROCEDURES

Materials. All chemicals were of analytical grade and purchased from Fluka (Germany), Aldrich (Germany), and Merck (Germany), respectively. Polyglycerol (MW = 10 kDa, PDI∼1.6) was prepared according to published procedures.48 Polyglycerol amine with 20% of the total hydroxyl groups bearing amino groups was prepared as previously described (ca. 110 OH and 27 NH2 groups per PG scaffold).49 Briefly, polyglycerol amine was prepared by a three-step protocol starting from hyperbranched polyglycerol, conversion of OH groups into mesyl (Ms) groups followed by transformation of Ms 2511


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Figure 1. Idealized structure from the PG-Doxo derivatives. PG-Doxo-PEG-BG (7a) bears the BG moiety in the shell of the construct, while PGBG-Doxo-PEG (7b) bears it in the core. PG-Doxo-PEG (7c) was used as the nontargeted control. groups into azide (N3) functionalities, and finally reduction of the N3 groups to primary amino (NH2) groups using triphenylphosphine as a reducing agent (Scheme S1 in the Supporting Information (SI)). Maleimido-poly(ethylene glycol) (Maleimido-PEG) with MW = 2 kDa was purchased from Rapp Polymer, Germany. The (6maleimidocaproyl) hydrazone derivative of doxorubicin (DoxoEMCH·HCl) was prepared as described previously.50 O6-(4Aminomethyl)benzylguanine (BG-amine) and maleimide derivative (BG-mal) were purchased from New England Biolabs, United States. Maleimido-PEG-succinimidyl ester was purchased from Celares, Germany. Indodicarbocyanine (IDCC) maleimide dye was obtained from mivenion GmbH.51 Water of Millipore quality (resistivity ∼ 18 MΩ cm−1) was used in all experiments and for preparation of all samples. If not otherwise specified, sodium phosphate buffer (10 mM) was used for the pH range of 7.4−5.8, for acidic pH values 50 mM sodium acetate buffer was employed. All measurements were carried out with freshly prepared solutions at 25 °C. pH values were measured with a Piccolo Plus ATC pH/C-meter at 25 °C. Thin layer chromatography (TLC) was conducted on Merck silica gel 60 RP18 F-254s plates. Spots were visualized by UV light. In order to determine the UV/vis absorption a Scinco S-100 spectrometer was used. Electrospray ionization time-of-flight (ESIToF) analyses were performed on an Agilent 6210 ESI-ToF, Agilent Technologies, Santa Clara, CA, USA. Synthesis of Benzylguanine−Poly(ethylene glycol) Maleimide (BG-PEG-maleimide). To synthesize the benzylguanine precursor, 1 mg of benzylguanine amine (2.0 μmol) and 3.72 mg of maleimido-poly(ethylene glycol) succinimidyl ester (NHS-PEGmaleimide) (2.6 μmol) were dissolved in 1 mL of anhydrous DMF. After the addition of 0.56 μL TEA (4.0 μmol) the solution was stirred overnight at room temperature. The full conversion of benzylguanine educt was monitored by chromatography on reverse-phase TLC (70% CH3CN/H2O) and MS. The reaction mixture was evaporated to dryness with a rotation evaporator and the residue was redissolved in water. The suspension was filtered and the solvent evaporated to dryness to deliver the crude product as colorless viscous oil. The product (BG-PEG-mal) was used without further purification due to the very small amount of product and due to the fact that possible free PEG-mal does not disrupt with the synthetic strategy. The structure was confirmed by MS (ESI-ToF): m/z 1767.9428 [M + H]+ and is disclosed with its reaction scheme as Figure S2 in the SI. Synthesis of PG-Doxo Conjugates. A series of compounds was employed, including dendritic polyglycerolamine (1) (PG 10 kDa and 20% of amine loading), the (6-maleimidocaproyl) hydrazone derivative of doxorubicin (3) (Doxo-EMCH), maleimido-poly(ethylene glycol) (5) (PEG-mal 2 kDa), IDCC-mal (6), and benzylguanine PEG maleimide (4a) (BG-PEG-mal) or benzylguanine maleimide (4b) (BG-mal). Dendritic polyglycerolamine (1) was thiolated using 2-iminothiolane as a first step to yield the compound (2). Sequential thiol−ene reactions were performed to couple each of the maleimido-bearing components to the thiolated polyglycerolamine (2) according to the pathways described in Scheme 1. Three PG-Doxo derivatives were prepared, namely, PG-Doxo-PEG-BG and PG-BGDoxo-PEG (Figure 1, 7a and 7b respective shell and core BG functionalized), and PG-Doxo-PEG as control (Figure 1, 7c).

The conjugation reaction was performed at room temperature with vigorous stirring for 150 min. To four different flasks, containing 0.5 mL of a solution of polyglycerolamine (10 mg/mL, 0.5 μmol) in 50 mM sodium phosphate (pH 7.4) containing 5 mM EDTA 1.25 mL of a solution of 2-iminothiolane (2 mg/mL in the same solvent system, 36 μmol) was added. After 20 min, the reaction mixture was directly applied on a Sephadex G-25 superfine column and eluted with 50 mM phosphate buffer (pH 7.4) for prepurification before conjugation. To the obtained solution 360 μL solution of Doxo-EMCH (5 mg/mL in 10 mM sodium phosphate buffer, pH 5.8, 4.5 μmol) was added. After 10 min, a solution of the respective BG derivative (20 mg/mL, 4.5 μmol) in 50 mM sodium phosphate (pH 7.4) containing 5 mM EDTA or DMF (in case of BG-mal) was added (for PG-Doxo-PEG-BG: 0.2 mL and for PG-BG-Doxo-PEG: 11 μL). After 10 min, 0.2 mL (2.7 μmol) of a solution of 10 mg/mL of IDCC-mal in 50 mM sodium phosphate (pH 7.4) containing 5 mM EDTA was added in each vial. After 10 min, 2.28 mL (45 μmol) of a solution of 20 mg/mL of 2 kDa PEG-mal in 50 mM sodium phosphate (pH 7.4) containing 5 mM EDTA were added in each vial and the resulting solutions were stirred for 60 min. The solutions were concentrated with CENTRIPREP-10concentrators from Amicon, FRG (20 min at 4 °C and 4000 rpm) to a volume of approximately 1 mL. The PG-doxorubicin derivatives were purified by gel filtration using a Sephacryl S-100 column (Amersham) eluted with 10 mM sodium phosphate buffer (pH 7.0) yielding 10−20 mL of a blue solution. Finally the conjugates were lyophilized at −60 °C for 16 h to yield a blue powder. Yields obtained (between 54% and 64%), weight composition, and the idealized structures of each conjugate are presented in Table S1 (SI). Conjugate formation was confirmed by Ellmans test (see Figure S1 for detailed description), chromatography on reverse phase TLC (70% CH3CN/20 mM sodium phosphate pH 7), and appearance of a faster band on a Sephadex G-25 column. Physical encapsulation was ruled out by performing the same coupling procedure without the addition of 2-iminothiolane. The nontargeted control probe was synthesized using the same synthetic method, but without addition of a BG moiety. Drug Release Profile Determination. The prodrug doxorubicinEMCH contains a pH labile hydrazone linkage, which is selectively cleaved at pH below 6 and sustains good hydrolytically stability at pH 7.4. To investigate the pH cleavability of the coupled drug, 150 μL of a 650 μM solution of the PG-Doxo-PEG-BG conjugate (concentration stated in doxo equivaltens) with a loading of 1.8 w% was incubated at room temperature in the respective buffer system (PB 7.4 and acetate buffer pH 4). Every hour a sample of 20 μL was taken, and the conjugate was separated from the free drug using a short Sephadex G15 column eluted with PB pH 7.4 to avoid further cleavage during analysis. The free drug band was collected into a graduated flask which was filled up to 2 mL with PB pH 7.4. The amount of doxorubicin in the solution was directly determined by UV/vis spectroscopy at 495 nm (ε495 = 10645 M−1cm−1) and plotted in correlation to the maximal amount of doxo loaded to the conjugate (%released) against the time. The experiment was performed in duplicate. Cell Culture. The EGFR+ human epidermal carcinoma cell line A431 (ATCC no.: CRL-259), EGFR− Chinese hamster ovary cell line CHO-K1 (ATCC: CCL-6), and human embryonic kidney cell line HEK-293T cells (ATCC: CRL-11268) were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine 2512


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serum (FBS) and 100 U/mL penicillin-streptomycin. The EGFR+ human breast carcinoma cell lines MDA-MB-468 (ATCC: HTB-132) and EGFR+ human pancreatic carcinoma cell line Panc-1 (ATCC: CRL-1469) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (v/v) FBS and 100 U/mL penicillin− streptomycin. All cells were incubated at 37 °C in a 5% CO2 atmosphere. All media and additives were obtained from Invitrogen, Darmstadt, Germany. Protein Expression and Purification. The scFv-425-SNAP tag fusion protein was expressed in HEK-293T cells as described before.12 Briefly, HEK-293T cells were transfected with the eukaryotic expression vector pMS-scFv-425-SNAP, containing a N-terminal binding ligand (scFv-425), a C-terminal SNAP-Tag sequence, and His6 tag sequence, using RotiFect transfection reagent (Carl Roth GmbH, Karlsruhe, Germany) following the manufacturer instructions. Proteins were purified from culture supernatants by Ni-NTA affinity chromatography using an Ä kta FLPC system with a 5 mL Ni-NTA Superflow cartridge (Qiagen, Hilden, Germany). Purified protein was dialyzed against phosphate-buffered saline (PBS) containing 1 mM dithiothreitol (DTT). Before storage at −20 °C, ectoine was added as a cryopreservative to a final concentration of 50 mM.52 Conjugation of scFv-425-SNAP-Tag Fusion Protein to PGDoxo Derivatives. The BG modified polyglycerol derivatives were conjugated to scFv-425-SNAP by incubating them for 2 h at room temperature in a 3:1 molar ratio. Conjugated protein was purified from unconjugated protein and free polyglycerol derivatives by size exclusion chromatography using a 24 mL Superdex 75 column (GE Healthcare München, Germany) and PBS as running buffer. Runs were monitored at 280 nm and 650 nm to visualize protein and the IDCC-modified polyglycerol. Flow Cytometric Analyses. Flow cytometry was used to evaluate binding of scFv-425-SNAP-PG-Doxo-PEG to target and nontarget cells (EGFR+: A431, MDA-MB-468, Panc-1; EGFR−: CHO-K1). Cells (4 ×105) were incubated in 200 μL of PBS containing 0.01 μM of IDCC equivalent concentration of conjugated protein or PGDoxo-PEG derivatives for 30 min on ice or 37 °C. To confirm the binding specificity, EGFR was blocked by incubating the cells with 10 μg anti-EGFR scFv-425-SNAP-Vista green for 30 min on ice. The cells were then washed twice with 1.8 mL of PBS in a conventional cell washer and analyzed by a FACSCalibur device and CellQuest software (Becton & Dickinson, Heidelberg, Germany) using a 633 nm laser source. Confocal Microscopy. Binding and internalization properties of the conjugates were further confirmed by confocal microscopy (TCS SP5 confocal microscope, LEICA Microsystem, Wetzlar, Germany). Cells were prepared as described for flow cytometry and analyzed after 30 min incubation with scFv-425-SNAP-PG-Doxo-PEG at 4 or 37 °C. STYO9 Green is used as cell counterstain. Cell Cytotoxicity. Cell viability was analyzed using a cell proliferation kit based on 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide (XTT) (Roche, Mannheim Germany) as described recently.53 Briefly, 2 × 104 cells of EGFR+ A431, MDAMB-468, Panc-1 or EGFR− CHO-K1 were seeded in 96-well plates in triplicates and incubated for 24 h at 37 °C. The cells were treated with different concentrations (0, 100, 200, 400, 800, and 1600 nM) of scFv425-SNAP-PG-Doxo-PEG, PG-Doxo-PEG, and free doxorubicin followed by incubation for 48 h at 37 °C. As a positive control, 5 μg/mL Zeocin (Invitrogen) was used. After incubation, cells were treated for 4 h with XTT solution (1 mg/mL) at 37 °C. Cell viability was evaluated by colorimetric monitoring of the reduction of XTT to formazan at an absorbance wavelength of 450 nm and a reference wavelength of 630 nm using an ELx808 microtiter plate reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany). Data Analysis. GraphPad Prism software (GraphPad software) was used for statistical analysis. Data represent the average of triplicates ± standard error of the mean (SEM). Student’s t test and two-way analyses of variance were used to evaluate the significance of independent experiments, with P < 0.05 as the threshold for statistical significance.

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RESULTS

Synthesis of Multicomponent PG-Doxo-PEG-BG Derivatives. In order to deliver PG-Doxo conjugates specifically to EGFR expressing cells, pegylated polyglycerol scaffolds containing five equivalents of doxorubicin per particle were modified with BG at either the surface or the core. The BG moiety enables the conjugation of the PG-Doxo-PEG derivatives to the EGFR specific antibody fragment scFv-425SNAP. To synthesize the two different multicomponent BG bearing PEGylated PG conjugates (i.e., BG in the core or shell) a multistep approach in a one-pot process was followed according to Scheme 1. The first step comprised the thiolation of the dendritic polyglycerol amine (1) (MW 10 kDa, 20% amine functionalization), previously synthesized using reported procedures.49 The reaction of the polymer with 2-iminothiolane was followed by a selective Michael addition between the maleimide group of the doxorubicin prodrug (Scheme 1) (3) and the sulfhydryl groups from thiolated polyglycerol (2) in PBS solution at pH 7.4. The thiol group adds across the double bond of the maleimide group in a fast and selective reaction at room temperature forming a stable thioether bond.54 After purification by size exclusion chromatography (SEC) using Sephadex G-25, solutions of IDCC-mal, BG-mal derivative (4a or 4b), and PEG-mal (5) were sequentially added to the reaction mixture. The compound 4a was previously synthesized via amide bond formation using a NHS-activated PEG maleimide and an amino bearing BG derivative (see Figure S2 in the SI). The final reaction mixture was stirred for 2 h followed by a reduction of the volume by ultracentrifugation using a Centriprep. The residual volume of reaction mixture was directly applied to a SEC column packed with Sephacryl S100 gel. The polymeric fraction was collected and lyophilized to give the derivatives PG-Doxo-PEG-BG (7a) and PG-BG-DoxoPEG (7b). A nontargeted control was synthesized using the same synthetic method without the addition of BG to the reaction mixture to yield the PG-Doxo-PEG (7c). Conjugate formation was confirmed by an Ellmans test (see Figure S1 for detailed description), chromatography on reverse phase TLC, and the appearance of a faster band on a Sephadex G-25 column. Yield after purification ranged from 55% to 65%. Figure 1 shows idealized structures from the PG-Doxo-PEG derivatives that have been prepared. The drug concentration of the conjugates was determined photometrically at 495 nm (ε495 = 10645 M−1 cm−1) for the doxorubicin conjugates after reconstitution of the lyophilized samples (see Table S1 for details, SI) with PB pH 7.4. The ratio of polymer to Doxo and BG was set to 5 and 1, respectively. The in vitro stability studies performed by SEC showed that the release of doxorubicin was minimal at pH 7.4 after 24 h (less than 15%), while at acidic pH half-life was below 2 h allowing doxorubicin to be released in the slightly acidic intracellular environment of tumor cells (e.g., pH 5−6 in endosomes and down to pH 4−5 in lysosomes) (Figure S3, SI). The properties of the three different polymer conjugate precursors are summarized in Table 1. Coupling of SNAP-Tag Fusion Proteins to PG-Doxo Conjugates. The polyglycerol-based compounds were conjugated to the scFv using the SNAP-Tag technology.36 The conjugated protein was purified from unconjugated protein and free PG-Doxo derivatives by SEC. The coupling efficiency was determined by integrating the protein and conjugate peaks in 2513


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Importantly, preincubating the cells with 10 μg of scFv-425SNAP fusion protein abrogated the binding and internalization activities of scFv-425-SNAP-PG-Doxo-PEG at both 4 and 37 °C, whereas a strong shift was observed when cells were incubated with PG-Doxo-PEG dendrimer alone at 37 °C (Figure 3). Internalization of scFv-425-SNAP-PG-Doxo-PEG. Confocal microscopy was used to further analyze binding and internalization of targeted and nontargeted PG-Doxo-PEG-BG. Indeed, homogeneous and specific membrane staining on A431, MDA-MB-468, and Panc-1 cells was shown upon incubation with scFv-425-SNAP-PG-Doxo-PEG (Figure 4), while no signal was detected on EGFR− CHO-K1 negative control cells after an incubation of 30 min at 4 °C (Figure 4). The conjugated fusion proteins were specifically and efficiently taken up by target cell lines after an incubation of 30 min at 37 °C but not at 4 °C (Figure 4). Cytotoxicity of scFv-425-SNAP- PG-Doxo conjugates. The cytotoxic effects of PG-Doxo-PEG-BG conjugated to scFv425-SNAP and unconjugated PG-Doxo-PEG were evaluated using an XTT-based colorimetric cell proliferation assay with the EGFR+ cell lines (A431, MDA-MB-468, and Panc-1) and CHO-K1 cells again as negative control. The viability of A431, MDA-MB-468, and Panc-1 cells upon treatment with PGDoxo-PEG-BG conjugated with scFv-425-SNAP fusion protein was found to decrease significantly in a concentrationdependent manner after an incubation of 48 h at 37 °C. The IC50 values were 440 nM (A431), 424 nM (MDA-MB-468), and 510 nM (Panc-1) (Figure 5A-C: black squares). CHO-K1 cells remained unaffected even when exposed to 1600 nM of the conjugated fusion proteins (Figure 5D: black squares). In contrast, unconjugated PG-Doxo-PEG and free doxorubicin were toxic toward all cell lines. The IC50 values for PG-Doxo-

Table 1

compound

MW of PG corea

reactive groups per PGb

doxorubicin loading in weight %c

dye loading in weight %d

theoretical MW PEGylated particlee

7a 7b 7c

10 kDa 10 kDa 10 kDa

27 NH2 27 NH2 27 NH2

1.80 1.36 1.57

0.35 0.27 0.30

52 kDa 50 kDa 52 kDa

a

Determined by GPC including MALS detector (PDI = 1.6). Determined by 1H NMR spectroscopy. cDetermined by UV/visspectrophotometry and absorption coefficient of doxorubicin (ε495 = 10645 M−1 cm−1). dDetermined by UV/vis spectrometry and absorption coefficient of IDCC (ε682 = 240000 M−1 cm−1). eEstimated by feed ratio (Table S1, SI). b

the FPLC chromatogram. More than 80% of scFv-425-SNAP was coupled to PG-Doxo-PEG-BG (Figure 2). The conjugates bearing BG at the core of the dendritic conjugate did not show any coupling to the scFv-425-SNAP protein. Flow Cytometric Binding Analyses of scFv-SNAP Conjugated to Polyglycerol Dendrimer. The binding activity of the scFv-425-SNAP-PG-Doxo-PEG conjugates was confirmed by flow cytometry using A431, MA-MB-468, and Panc-1 cell lines that express EGFR and CHO-K1 cell line which is lacking this antigen. Here, a rapid and efficient binding of scFv-425-SNAP-PG-Doxo-PEG to EGFR+ target cell lines was demonstrated, whereas no significant binding signal was detected on EGFR− cells (CHO-K1) (Figure 3). Furthermore, no significant signals were observed when PGDoxo-PEG particles were incubated with all cell lines at 4 °C, whereas fluorescence was detected when the cell lines were incubated with the PG-Doxo-PEG particles at 37 °C, indicating unspecific uptake of the PG-Doxo-PEG particles by treated cells.

Figure 2. Purification profile of scFv-425-SNAP-PG-Doxo-IDCC using SEC. The conjugated protein was purified from unconjugated protein and free PG-Doxo-PEG-BG using a Superdex 75 column. The purification profile was monitored at 280 (blue curve) and 650 nm (red curve). 2514


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Figure 3. Binding activity of scFv-425-SNAP-PG-Doxo-PEG to target and control cell lines. Flow cytometry was carried out after incubating 4 × 105 cells (filled gray curves) with scFv-425-SNAP fusion protein conjugated to PG-Doxo-PEG derivatives (gray curve), PG-Doxo-PEG (bold black curve). In competition experiments scFv-425-SNAP-PG-Doxo-PEG (black curve) and PG-Doxo-PEG (dotted black curve) were added to cells preincubated with excess scFv-425-SNAP fusion protein at 4 °C (upper panel) and 37 °C (lower panel) for 30 min in PBS.

PEG were 282 nM (A431), 339 nM (MDA-MB-468), 349 nM (Panc-1) and 866 nM (CHO-K1), whereas the IC50 values for free doxorubicin were 261 nM (A431), 290 nM (MDA-MB468), 293 nM (Panc-1), and 644 nM (CHO-K1) (Figure 5). All concentrations used in this experiment are doxorubicin equivalent concentration.

One way of achieving site-specific conjugation is to fuse the protein of interest to reactive tag sequences. These protein tags can specifically react with chemical groups and mediate effector molecule conjugation, leading to a highly homogeneous product. For example, the SNAP tag fusion protein reacts specifically with benzylguanine (BG)-modified molecules, the CLIP tag fusion protein with benzylcytosine (BC)-modified molecules, the HALO tag fusion protein reacts with chloroalkane-modified molecules and tetracysteine (TC) tag with biarsenical compounds like FlAsH or ReAsH.37 The SNAP-Tag is a derivative of the O6-alkylguanine-DNA alkyltransferase (AGT), which has the ability to couple BGmolecules depending on its folding pattern.33,61 The use of SNAP-Tag for conjugation of effector molecules to the protein of interest provides several exploitable features like (1) high selectivity of the conjugation (reacts only with BG molecules), (2) speed of the reaction (1−2 h for efficient conjugation), (3) simplicity of the reaction conditions (PBS can be used without adding any activating agents), (4) flexibility of selecting the expression host, (5) availability of a wide range of BG-modified molecules, (6) lack of activity against any other cellular substrates, and (7) a 1:1 stoichiometry (one SNAP-Tag reacts with one BG modified substrate).38,62−64 We have recently developed a platform comprising dendritic PG as a multifunctional core and PEG as a decorating agent. The linear monohydroxy and terminal dihydroxy functionalities of PG scaffolds can easily be modified or functionalized following classical hydroxyl group chemistry to render a broad spectrum of products.49,65,66,68 High loading capacity, water solubility, and ease of purification of the product make PGs attractive architectures for carrying out postpolymerization modifications. A substantial amount of research has been directed to design different architectures by modification of PG hydroxyl groups into different functionalities. These architectures have already demonstrated their usefulness in therapeutic approaches related to multivalency, given by the synergy between the nanosized dimensions combined with the high density of functional groups.65,67−78 In a recent communication, we reported the use of the dendritic PG scaffold for conjugation to maleimide-bearing prodrugs of doxorubicin or

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DISCUSSION Polymeric nanocarriers have been studied throughout the last decades as a strategy to increase the concentration of therapeutic agents, especially chemotherapeutic drugs, in malignant tissue. These systems can overcome problems related to the conventional treatment by increasing the drug stability and solubility, decreasing the clearance rate, and reducing the systemic side effects. 55,56 Improving the pharmacological/pharmacokinetic properties of nanoparticles is achieved by modifying their surface and size.14,20 Selective accumulation of polymers can be achieved by passively targeting malignant tissue using the distinct pathophysiological characteristics of solid tumor vessels and the properties of microenvironments surrounding the tumors.55,56 Conjugation of chemotherapeutic drugs to polymeric/nanocarriers has improved their safety and efficacy.14,37 To avoid the limitations discussed above, several approaches have been developed to conjugate therapeutic nanoparticles to antibodies, homing peptides, or ligands.6,57,58 However, there are serious limitations to be taken into consideration to develop an effective antibody-based delivery system. One of these challenges is the conjugation of the antibody to the effector molecule. In general, conjugation is done using either the amino groups of lysine side chains or by reducing the sulfhydryl group of cysteine residues. These methods yield heterogeneous products, presenting a mixture of different conjugation sites and ratios, resulting in different pharmacokinetic, therapeutic efficacy, and safety profiles.59 A variety of site specific conjugation methods for proteins has been developed in the recent years, including the selective incorporation of nonnatural amino acids, engineering of unpaired cysteins, protein tags with unique reactivities, and different enzyme base modification strategies, all with their own advantages and limitations.37,57,60 2515


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Figure 4. Confocal microscopy analysis of scFv-425-SNAP conjugated to PG-Doxo-PEG-BG. Confocal images were obtained for the EGFR+ cell lines (A431, MDA-MB-468, Panc-1) and for the EGFR− cell line (CHO-K1). The cells were incubated with 0.5 μg scFv-425-CAT-PG-Doxo-PEG, for 30 min at 4 or 37 °C. Thereafter, the cells were incubated with STYO9 Green which used as a counterstain. (1) STYO9 Green fluorescence signal; (2) IDCC fluorescence signal; (3) overlay of fluorescence signal and transmitted light.

methotrexate, which incorporate either an enzymatically- or a pH-sensitive linker.79,80 Such modular approach proved to be flexible for coupling different drugs, solubilizing agents, as well as imaging, and targeting moieties.66,80 We explored this methodology to prepare PG doxorubicin prodrugs that were flexible for drug loading by using an acid-sensitive hydrazone linker and further postmodification with a PEG shell. The resulting polymer drug conjugates showed optimal properties for in vitro and in vivo applications because of their high water

solubility, an appropriate size for passive tumor targeting, a high stability at physiological conditions, pronounced acid-sensitive properties, cellular internalization, and a favorable toxicity profile. PG-Doxo conjugates with a high drug-loading ratio showed clearly improved antitumor efficacy over the free drug in an ovarian xenograft tumor model (A2780).81 The conjugate induced transient complete remissions and thus demonstrated its potential for development of an efficient multifunctional dendritic drug delivery system.79,82−84 2516


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Figure 5. Cytotoxic effects of 425-SNAP-PG-Doxo-PEG bioconjugates on target and nontarget cells. Cell viability was analyzed after incubation with different equivalent doxorubicin concentrations (0 nM, 100 nM, 200 nM, 400 nM, 800 nM, and 1600 nM) of targeted PG-Doxo-PEG (■) nontargeted PG-Doxo-PEG derivative (▲), and free doxorubicin using an XTT based assay (●). (A) A431 (EGFR++), (B) MDA-MB-468 (EGFR+ +), (C) Panc-1 (EGFR+), and (D) CHO-K1 (EGFR−).

analysis. The selectivity for these cells was confirmed by revealing a strong and specific binding of conjugated particles to targeted cancer cells, while no significant unspecific binding activity was observed. Interestingly, an EGFR-negative cell line as well as cell line expressing moderate levels of EGFR (Panc3) showed lower PG-Doxo-PEG uptake than the cell lines expressing high levels of EGFR. This could be due to the change in overall charge of cell surface by the extraordinary high numbers of EGFR (isoelectronic point of EGFR 6.26). However, the relation between EGFR expression and cell surface charge needs further investigation. Using confocal microscopy, it was confirmed that the scFv425-SNAP-PG-Doxo-PEG conjugates were able to specifically target EGFR+ cancer cells and were internalized in less than 30 min at 37 °C. This is most probably caused by binding of scFv425-SNAP-PG-Doxo-PEG to EGFR, resulting in receptormediated endocytosis, whereas no significant unspecific internalization was detected in the negative control cells. These results revealed that conjugating PG-Doxo-PEG to scFvSNAP fusion protein abolishes nonspecific internalization, which is of crucial importance in order to reduce undesired accumulation in healthy tissues. Similar effects were described for mAb425 (anti-EGFR) antibody-conjugated PAMAM dendrimer, which could efficiently bind and internalize to the target cell.31 Moreover, scFv-425-SNAP-PG-Doxo-PEG was found to selectively kill EGFR+ (A431, MDA-MB-468, and Panc-1) cell lines in a dose-dependent manner after 48-h incubation. The cytotoxicity of scFv-425-SNAP-Ce6 was found to be dependent on the presence of EGFR, and toxicity was most potent in A431 and MDA-MB468 cells, which express the largest amount of the receptor (1−1.3 × 106 receptors/ cell),85,86 while the cell viability of Panc-1, which expresses 4 × 105,87 was comparably reduced. Of important notice, no toxicity

However, this promising passively targeted approach showed limitations regarding cell specificity that restricts the broader use of PG-Doxo-PEG in vitro and in vivo. Therefore, the present work deals with the development of a multifunctional PG derivative that bears (1) doxorubicin as cytostatic agent, (2) IDCC as a fluorescent probe that enables intracellular tracking, (3) PEG as solubilizing and shielding agent, and (4) BG as anchoring group for the further bioconjugation to antibodies. The antibody will render a selective cellular targeting of the multifunctional prodrug, which should lead to a decrease of the inherent toxicity of the PG-Doxo derivative while increasing its anticancer efficacy. Hence, we investigated the use of SNAPTag technology to provide a unique conjugation site on the scFv antibody, allowing the conjugation to PG that is covalently linked to Doxo through a pH labile hydrazone linker. This linker showed high stability in physiological conditions, with less than 15% of doxorubicin released after 24 h incubation at pH 7.4, while maximum release occurred at acidic conditions (Figure S3, SI).45 In addition, linear PEG was conjugated to PG-Doxo in order to improve aqueous solubility and to reduce immunogenicity. BG was either attached to the core or to the surface of the pegylated PG-Doxo derivative, and its accessibility was assessed through bioconjugation experiments. Furthermore, all the compounds prepared were fluorescently labeled with a IDCC fluorophore, which enabled the detection of the conjugates in the imaging analysis. scFv-425 genetically fused to the SNAP-Tag fragment allowed site-specific conjugation of BG-modified PG-DoxoPEG derivatives. The conjugation reaction showed to be efficient for the derivative that bears the BG at the surface (PGDoxo-PEG-BG), which is more accessible for proteins than the BG groups located in the dendritic core. The antibody conjugate scFv-425-SNAP-PG-Doxo-PEG has been investigated regarding its specific targeting to EGFR+ tumor cells using flow cytometry and confocal microscopy 2517


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was observed when EGFR− cells were incubated with the same conjugate. The toxic effect of PG-Doxo-PEG particles compared to free doxorubicin on target cells was observed to be 1.5−2 times higher than scFv-425-SNAP-PG-Doxo-PEG conjugate, which is similar to the toxicity range previously described against a human ovarian cancer A2780 cell line.45 Significant in vitro toxicity differences have been reported between unconjugated drug that enter the cells via cell diffusion and a drug nanoparticle conjugated with scFv, which is internalized by receptor mediated endocytosis pathway.88 Although the toxicity of scFv-425-SNAP-PG-Doxo-PEG was lower compared to the unconjugated PG-Doxo-PEG derivative, the specificity of scFv425-SNAP-PG-Doxo-PEG was found to be higher, as no toxic effect was observed when the negative cells were treated with the conjugated particles. This warrants future studies on combinations of scFv-SNAP fusion proteins with multifunctional polyglycerol to prepare preclinical in vivo applications.

Author Contributions ○

Ahmad Fawzi Hussain and Harald Rune Krüger as well as Marcelo Calderón and Stefan Barth contributed equally to this manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge the support from the FU Focus area “Nanoscale” and the NanoFutur award to F.K. and R.H. as well as the German Academic Exchange Service (DAAD) for the financial support of A.F.H.

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(1) Chen, H.; Ahn, R.; Van den Bossche, J.; Thompson, D. H.; O’Halloran, T. V. Mol. Cancer Ther. 2009, 8 (7), 1955−1963. (2) Pinhassi, R. I.; Assaraf, Y. G.; Farber, S.; Stark, M.; Ickowicz, D.; Drori, S.; Domb, A. J.; Livney, Y. D. Biomacromolecules 2010, 11 (1), 294−303. (3) Vaidya, B.; Vyas, S. P. J. Drug Target. 2012, 20, 372−380. (4) Kim, Y.; Pourgholami, M. H.; Morris, D. L.; Stenzel, M. H. Macromol. Biosci. 2011, 11 (2), 219−233. (5) Qin, J.; Chen, D.; Hu, H.; Cui, Q.; Qiao, M.; Chen, B. Chem. Pharm. Bull. 2007, 55 (8), 1192−1197. (6) Su, W.; Wang, H.; Wang, S.; Liao, Z.; Kang, S.; Peng, Y.; Han, L.; Chang, J. Int. J. Pharm. 2012, 426 (1−2), 170−181. (7) Dharap, S. S.; Wang, Y.; Chandna, P.; Khandare, J. J.; Qiu, B.; Gunaseelan, S.; Sinko, P. J.; Stein, S.; Farmanfarmaian, A.; Minko, T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (36), 12962−12967. (8) Chen, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Wong, S. S.; Ojima, I. Bioconjug. Chem. 2010, 21 (5), 979−987. (9) Ferreira, C. S.; Cheung, M. C.; Missailidis, S.; Bisland, S.; Gariepy, J. Nucleic Acids Res. 2009, 37 (3), 866−876. (10) Neff, C. P.; Zhou, J.; Remling, L.; Kuruvilla, J.; Zhang, J.; Li, H.; Smith, D. D.; Swiderski, P.; Rossi, J. J.; Akkina, R. Sci. Transl. Med. 2011, 3 (66), 66ra6. (11) Doronina, S. O.; Toki, B. E.; Torgov, M. Y.; Mendelsohn, B. A.; Cerveny, C. G.; Chace, D. F.; DeBlanc, R. L.; Gearing, R. P.; Bovee, T. D.; Siegall, C. B.; Francisco, J. A.; Wahl, A. F.; Meyer, D. L.; Senter, P. D. Nat. Biotechnol. 2003, 21 (7), 778−784. (12) Hussain, A. F.; Kampmeier, F.; von Felbert, V.; Merk, H. F.; Tur, M. K.; Barth, S. Bioconjugate Chem. 2011, 22 (12), 2487−2495. (13) Kelly, R. K.; Olson, D. L.; Sun, Y.; Wen, D.; Wortham, K. A.; Antognetti, G.; Cheung, A. E.; Orozco, O. E.; Yang, L.; Bailly, V.; Sanicola, M. Eur. J. Cancer 2011, 47 (11), 1736−1746. (14) Cho, K.; Wang, X.; Nie, S.; Chen, Z. G.; Shin, D. M. Clin. Cancer Res. 2008, 14 (5), 1310−1316. (15) Ducry, L.; Stump, B. Bioconjugate Chem. 2010, 21 (1), 5−13. (16) She, W.; Luo, K.; Zhang, C.; Wang, G.; Geng, Y.; Li, L.; He, B.; Gu, Z. Biomaterials 2013, 34 (5), 1613−23. (17) Yuan, H.; Luo, K.; Lai, Y.; Pu, Y.; He, B.; Wang, G.; Wu, Y.; Gu, Z. Mol. Pharmaceutics 2010, 7 (4), 953−62. (18) Sinha, R.; Kim, G. J.; Nie, S.; Shin, D. M. Mol. Cancer Ther. 2006, 5 (8), 1909−1917. (19) Andreev, O. A.; Engelman, D. M.; Reshetnyak, Y. K. Mol. Membr. Biol. 2010, 27 (7), 341−352. (20) Chrastina, A.; Massey, K. A.; Schnitzer, J. E. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2011, 3 (4), 421−437. (21) Quadir, M. A.; Calderón, M.; Haag, R. In Drug Delivery in Oncology; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2011; pp 513−551. (22) Reichert, S.; Calderón, M.; Licha, K.; Haag, R. In Multifunctional Nanoparticles for Medical Applications: Imaging, Targeting, and Drug Delivery; Svenson, S., Prud’homme, R. K., Eds.; Springer: New York: 2012; pp 315−344. (23) Khandare, J.; Calderon, M.; Dagia, N. M.; Haag, R. Chem. Soc. Rev. 2012, 41 (7), 2824−2848.

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CONCLUSIONS In summary, we have developed a dendritic polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing EGFR. The synthetic modular approach allowed to use dendritic PG as a multifunctional anchoring point for the coupling of doxorubicin through a pH-sensitive linker, IDCC as a fluorescence marker, PEG as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-Tag technology. The SNAP tag technology provided an easy, fast, and sitespecific protein modification, which could be used to conjugate a scFv antibody to the surface of the PG-Doxo-PEG-BG derivatives, yielding a homogeneous conjugate. The study provides the proof of principle that SNAP-tag technology can be used to conjugate nanoparticles to recombinant antibody fragments and hence to target cancer cells. The superior cell targetability of the scFv-425-SNAP-PGDoxo-PEG conjugate, the distinct increase in tolerability and antitumor activity of the PG-Doxo-PEG derivatives,45 along with the previous in vivo accumulation results of scFv-425SNAP,44 suggest a great synergetic potential of the developed bioconjugates for further in vivo applications.

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ASSOCIATED CONTENT

S Supporting Information *

Details about the synthesis of polyglycerolamine, BG-PEG-mal, PG-Doxo-PEG-BG conjugates, and proof of maleimide coupling by Ellman’s test are available in the Supporting Information. In addition, the idealized structure of the conjugates and details about drug content, release kinetics, and yields are disclosed. This material is available free of charge via Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Corresponding author on dendritic polymer technology: Marcelo Calderón, Tel. +49-30-8385-2472, Fax. +49-30-83853357, E-mail: [email protected]. Corresponding author on SNAP tag technology: Stefan Barth, Tel. +49- 241-6085-11060/1, Fax. +49-241-6085-10000, E-mail: [email protected]. 2518


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