A Novel Method for the Assessment of Targeted PEI-Based

Article pubs.acs.org/ac

A Novel Method for the Assessment of Targeted PEI-Based Nanoparticle Binding Based on a Static Surface Plasmon Resonance System Sabrina Höbel,†,‡ Doru Vornicescu,§ Marius Bauer,⊥ Dagmar Fischer,⊥ Michael Keusgen,§ and Achim Aigner*,†,‡ †

Rudolf-Boehm-Institute of Pharmacology and Toxicology, Clinical Pharmacology, Faculty of Medicine, University of Leipzig, Härtelstrasse 16-18, D-04107 Leipzig, Germany ‡ Institute of Pharmacology, Faculty of Medicine, Philipps-University Marburg, Karl-von-Frisch-Strasse 1, D-35032 Marburg, Germany § Institute for Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps-University Marburg, Marbacher Weg 6, D-35032 Marburg, Germany ⊥ Institute of Pharmacy, Department of Pharmaceutical Technology, Faculty of Biology and Pharmacy, Friedrich-Schiller-University Jena, Otto-Schott-Strasse 41, D-07745 Jena, Germany S Supporting Information *

ABSTRACT: The delivery of nucleic acids is a major hurdle in gene therapy or therapeutic gene knockdown, and the development of intelligent and safe nanoparticles as carrier systems is thus under intense investigation. The introduction of ligands for their targeted delivery is of major interest. Here, we describe a novel approach for the analysis of the binding properties of antibody-functionalized nanoparticles, using surface plasmon resonance (SPR) in a static cuvette system. By chemical coupling of the Epidermal Growth Factor Receptor (EGFR)-specific antibody cetuximab to poly(ethylene imine) (PEI) via a PEG-spacer and subsequent DNA or siRNA complexation, we generated targeted nanoplexes with low surface charge. Antibody-mediated uptake into EGFR overexpressing cells was observed. SPR measurements with use of a novel, protein A-based sandwich system for the immobilization of the target receptor in its correct steric orientation allowed the analysis of the specific PEI-PEG-cetuximab binding to EGFR and the determination of binding affinities. Importantly, our cuvette-based SPR assay system was also suitable for the monitoring of ligand-mediated nanoparticle binding, without convection or shear stress. We conclude that our SPR sandwich system allows the precise analysis of the binding of ligand-functionalized nanoparticles in real-time, and we thus establish SPR for the in vitro evaluation of ligand modifications for generating targeted nanoparticles.

G

degradation. After cellular uptake, PEI/nucleic acid complexes are able to escape from the lysosome due to the so-called “proton-sponge-effect” of PEI.6 Once in the cytosol, siRNA molecules can be incorporated into RISC (RNA-induced silencing complex) to induce RNAi, and DNA can enter the nucleus for gene expression. We have introduced previously the low molecular weight branched PEI F25-LMW7 as a powerful system for the transfection of nucleic acids in vitro8 and for the delivery of small RNAs in vivo,9−11 based on its low cytotoxicity and high biological activity. However, the ultimate goal is the ligandmediated, targeted delivery of nucleic acids in vivo. In this context, the ligand-mediated, specific cellular uptake by the target tissue (e.g., tumor cells), which also relies on reducing the interaction of the particles with nontarget tissues, poses a

ene therapy approaches aiming at the induction of RNA interference (RNAi) or gene replacement are promising strategies for the treatment of various diseases including cancer. RNAi offers an approach for the specific knockdown of diseaserelated genes,1 based on the rational design of highly specific synthetic RNA molecules (small interfering RNAs, siRNAs) directed against any gene of interest.2 However, the delivery of small RNA molecules like siRNAs or microRNAs (miRNAs) poses a major limitation so far, particularly in vivo, and this evolves as a major bottleneck for efficient gene therapy.3 Likewise, the therapeutic exploration of DNA molecules requires formulation strategies, especially when aiming at tissue specificity. The delivery of nucleic acids can be achieved by different nonviral approaches, and various systems have been investigated for efficient and cell-specific delivery. Among cationic polymers, poly(ethylene imine) (PEI) is one of the most efficient compounds for nucleic acid delivery.4,5 PEI is able to condense DNA and small RNA molecules and to build nucleic acid complexes, thereby protecting them from nuclease © XXXX American Chemical Society

Received: July 2, 2013 Accepted: November 5, 2013

A

dx.doi.org/10.1021/ac402001q | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

■

major challenge for intelligent drug delivery systems. Since antibody−antigen binding affinities are among the strongest intermolecular interactions observed in nature, the coupling of antibodies directed against characteristic cellular structures, e.g., extracellular domains of receptor tyrosine kinases that are overexpressed on tumor cells, is of high interest for the development of targeted nanoparticulate delivery platforms.12 However, this requires the thorough analysis of specific ligand− receptor binding properties of the given nanoparticle, preferentially in vitro. Surface plasmon resonance (SPR) offers a standard tool for the measurement of adsorption processes onto a surface, and is thus a well-known method for exploring biokinetic reactions at surfaces as well as real-time observation of interactions between biomolecules. This offers a wide range of applications, including the determination of specificities, concentrations, kinetic data, and affinity or equilibrium constants of binding, membrane, and membrane-receptor studies, structure/function relationships in direct, competitive sandwich and displacement assays, and its use in screening or in immunoassays. Surface plasmons are collectively oscillating electrons in thin noble metal films (about 50 nm) coating the surface of a waveguide that can be excited with p-polarized light. The excitation leads to resonance depending on wavelength, angle of incidence from the polarized light, and refraction index of a biological layer placed on the metal film surface. This biological layer consisting of molecules that bind to or desorb from receptors which are immobilized on the chemically functionalized metal surface cause a change of surface plasmons and consequently a change in refractive index. Thus, real time observation of the change of refractive index yields information about the kinetics of the ligand−receptor interaction. Furthermore, SPR is a rapid procedure that can be automated, it allows one to work with extremely small reagent amounts, it is more sensitive than other label-free kinetic measurements like impedance spectroscopy or quartz crystal microbalances, and it can be easily standardized.13,14 While SPR is widely used for the analysis of molecular interactions, its application in the characterization of the binding properties of targeted nanoparticles has been barely explored so far. Also, a static cuvette system avoiding shear stress rather than a fluidic SPR system is more appropriate for simulating the fluid dynamics in the in vivo situation at the site of nanoparticle binding, which would be a solid tumor for the PEI-PEG-antibody conjugate developed here. In this paper, we describe the development of a novel SPR sandwich assay system to mimic on-chip the target site in vivo, and we perform ligand−receptor binding studies based on SPR by analyzing cetuximab-modified polymeric nanoparticles. To this end, we generated novel ligand-modified, shielded nanoparticles for the targeted delivery of nucleic acids, i.e., antibody-modified, PEGylated PEI conjugates, by chemical coupling of the anti-EGFR antibody cetuximab to PEI via PEGspacer molecules. Beyond the grafted polymers, we extended our studies to the analysis of cetuximab-modified PEI/nucleic acid complexes. Their specific binding as analyzed in SPR experiments was found to correlate with ligand-mediated uptake and enhanced biological activity of the conjugates in cultivated EGFR-overexpressing cells. We thus establish SPR that uses a static cuvette system as a powerful system for the sensitive, label-free, and real-time assessment of targeted PEIbased nanoparticle binding.

Article

EXPERIMENTAL SECTION

Chemicals and Nucleic Acids. The 25 kDa branched PEI, protein A, and human IgG were from Sigma-Aldrich (Taufkirchen, Germany). Cetuximab (Erbitux), trastuzumab (Herceptin), and bevacizumab (Avastin) were purchased from Roche (Basel, Switzerland). Functionalized PEG molecules, MS(PEG)24 (Methyl-PEG24-NHS Ester), and SM(PEG)24 (NHS-PEG24-Maleimide) were obtained from Pierce/Thermo Fisher (Rockford, IL). SPDP, N-succinimidyl 3-(2pyridyldithio)propionate, and BSA were from Calbiochem/ Merck (Darmstadt, Germany). DC Protein-Assay and MacroPrep High S were purchased from BioRad (Hercules, CA). Sephadex G50 fine resin and Sephadex G25 fine resin were from Amersham Biosciences (Freiburg, Germany). Recombinant human EGFR/ErbB1 Fc Chimera was purchased from R&D Systems (Wiesbaden, Germany). Chemically synthesized siRNA duplexes (see the Supporting Information for sequence details) directed against luciferase, siGL3 (specific), and siGL2 (nonspecific), were from MWG (Ebersberg, Germany), and Alexa Fluor 488 unrelated siRNA was from Qiagen (Hilden, Germany). Standard chemicals were of analytical grade. Preparation of Low Molecular Weight Polyethylenimine (PEI F25-LMW) and PEI Conjugates. Branched low molecular weight (∼4−10 kDa) polyethylenimine, PEI F25LMW, was prepared as described previously.7 PEI concentrations were determined by mixing 20 μL of the PEI solution (e.g., a test sample from an eluate fraction) or a PEI standard of known concentration with 100 μL of 0.02 M cupric acetate in 5% (w/v) potassium acetate (pH 5.5) in a 96-well plate. The absorption at 630 nm was measured with use of an ELISA reader (Bio-Tek Instruments, Winooski, VT), and concentrations were determined by comparison with the standard curve. For PEGylation, 5 mg of PEI F25-LMW (5 mg/mL in 0.15 M NaCl) was adjusted to pH 7.5 with HCl and 6 mg of MS(PEG)24 (100 mg/mL in DMF) was added to the PEI solution. After incubation at RT for 30 min on a rocking shaker, excess MS(PEG)24 and the NHS leaving group were removed by gel permeation chromatography (GPC), using Sephadex G25 fine resin with 0.15 M NaCl as flow buffer. Fractions of 0.5 mL were collected, the PEI and the protein concentrations (A280) were measured, and PEI-containing fractions were pooled and concentrated in an Amicon stirred cell. For coupling of the antibodies to PEI F25-LMW, a bifunctional PEG spacer was used. A sample of 5 mg of PEI F25-LMW was PEGylated as described above, using 6 mg of SM(PEG)24 (100 mg/mL in DMF). Excess PEG and NHS leaving groups were removed by GPC. In parallel, 10 mg of antibody (cetuximab or trastuzumab) was activated by addition of a 4× molar excess of the heterobifunctional cross-linker SPDP (solubilized in ethanol) and incubated for 30 min on a rocking shaker. After reduction of SPDP-functionalized antibodies by addition of a 20× molar excess of DTT (relating to SPDP used) for 30 min at RT, the thiol-functionalized antibody conjugate was again separated from low molecular weight reaction products by GPC and mixed with PEGylated PEI for reaction with the maleimide groups of SM(PEG)24. PEGylated PEI and thiol-functionalized antibody were allowed to react overnight at 4 °C while shaking. The coupling product “PEI-PEG-antibody” was separated from antibody molecules by cation exchange chromatography Macro-Prep High S (BioRad, Hercules, CA, USA). After binding to the resin, four 0.5-mL fractions were collected by stepwise increase of the salt concentration from 0.5 M NaCl to B



Article

Figure 1. Schematic overview of the layer composition for binding studies on functionalized SPR prisms. The C:18-functionalized gold surface (silane with C:18 chains) of the SPR prisms was first coated with protein A, followed by the saturation of free nonspecific binding sites with BSA. The orientated immobilization of recombinant EGFR-Fc-fusion protein was achieved by binding of the Fc domain to the immobilized protein A molecules, followed by the saturation of free Fc-binding sites with human nonspecific IgG. The schemes illustrate the specific binding of PEI-PEGcetuximab (A) or cetuximab-coupled polyplexes (B) to the immobilized EGFR, and absence of specific binding when using PEI-PEG-trastuzumab (C) or trastuzumab-coupled polyplexes (D).

were rinsed with double-distilled water and then subjected to a long chain alkylation, referred to as “C18-functionalization” and performed according to the established method described previously, resulting in stable and reproducible results.15,16 To this end, the activated SPR prisms were incubated in an octadecyltrimethoxysilane solution (1% (w/w) in toluene) for 6 h at room temperature on a shaker (350 rpm) in order to obtain a uniform “grafted” gold surface with C:18 chains. The approximate volume of the solution used for each prism was 6 mL. Subsequently, the prisms were rinsed three times each in toluene, methanol, and double-distilled water, air dried, and stored under vacuum until use. SPR Assay Setup. The coating scheme and the sequence of the incubation steps were as outlined in Figures 1−3. More specifically, the C:18-functionalized gold surface was first coated with protein A (2 mg/mL). After blocking of the remaining free binding sites with BSA (1 mg/mL), the EGFR/ Fc-fusion protein (0.2 mg/μL) was bound to immobilized protein A, with subsequent saturation of free Fc-binding sites on the protein A layer by addition of IgG (1.8−2 mg/mL, human IgG/bevacizumab). Specific or nonspecific antibodies (1 mg/mL cetuximab, bevacizumab), polymer conjugates PEIPEG-cetuximab (corresponding to 0.35 or 1.35 mg/mL cetuximab) and PEI-PEG-trastuzumab (corresponding to 0.23 mg/mL trastuzumab), or complexes (PEI-PEG-cetuximab/ siRNA corresponding to 1.35 mg/mL cetuximab; PEI-PEGtrastuzumab/siRNA corresponding to 0.23 mg/mL trastuzumab) were added as indicated in the text and figures. In additional experiments, the interaction of PEI F25-LMW (0.4 mg/mL) and PEI-PEG (equivalent to 0.4 mg/mL PEI) with protein A was investigated. For the determination of binding affinities to EGFR, cetuximab and PEI-PEG-cetuximab were

3 M NaCl in 0.5 M steps. For each fraction, PEI and protein concentrations were determined and fractions containing PEI as well as antibody were pooled, adjusted to 0.15 NaCl by dilution with water, and concentrated in an Amicon stirred cell as described above. The PEI-PEG-antibody conjugates were stored at 4 °C until use. The antibody coupling to PEI was also analyzed by SDS-polyacrylamide gel electrophoresis. To this end, PEI-PEG-antibody conjugates (containing 3 μg antibody), and free antibody for comparison, were loaded onto a denaturing 12% polyacrylamide gel, and bands were stained at 4 °C overnight with Coomassie Brilliant Blue R-250 (SigmaAldrich, St. Louis, MO, USA) in 10% (v/v) acetic acid and 30% (v/v) ethanol. Preparation PEI/siRNA Complexes for SPR Measurements. Samples of 10 μg of siRNA and 50 μg of PEI-PEGantibody were each dissolved in 50 μL of 0.015 M NaCl, pH 7.4. After incubation for 5 min at room temperature, the PEIPEG-antibody solution was added to the siRNA-containing vial and mixed by vortexing. Complex formation is based on electrostatic interactions between the positively charged PEI and the negatively charged siRNAs. For complete complexation, the complexes were incubated at room temperature for 30 min and vortexed again prior to use. Surface Coating of SPR Prisms and Assay Setup. C:18Functionalization of the SPR Prisms. The SPR instrument used for the present work (Plasmonic) takes advantage of a prism to excite the surface plasmons (Kretschmann configuration), and in this system biomolecules are immobilized directly on the prism surface. Prior to surface functionalization, the SPR prisms were cleaned in acetone (5−10 min), followed by a mixture of 0.1 M potassium hydroxide (KOH) and 30% hydrogen peroxide (H2O2) (1:1, v/v) for 20 min. The prisms C



Article

Figure 2. SPR-based binding studies of antibody-functionalized PEG-PEIs and determination of binding affinities. Open arrowheads indicate initiation of washing steps; black arrows indicate the position of relevant detection signal levels used for quantitation (compare distances between the levels/arrows). Specific binding of PEI-PEG-cetuximab to immobilized EGFR (A) and absence of specific binding of PEI-PEG-trastuzumab (B). Binding of the specific ligand cetuximab to immobilized EGFR (C), but not of the unrelated antibody bevacizumab (D). Saturation of all Fc-binding sites of protein A (E). Absence of electrostatic interactions between the SPR surface and PEGylated PEI (F) or nongrafted PEI (G). Determination of the binding affinities of cetuximab and the antibody-coupled polymer conjugate PEI-PEG-cetuximab to immobilized EGFR (H).

added at different concentrations in the range of 0.005−0.05 mg/mL, respectively. Between the addition of the different components of the SPR sandwich, the cuvette was extensively washed with several volumes of PBS. Since 10 μL solutions were added to 10 μL of buffer remaining from the last washing step, a 1:1 dilution of the initially added solutions was obtained as the final concentration. The value of the angle shift that resulted from the association of each component (in arbitrary units, AU) was documented as readout and used for analysis. All components, except the PEI-based complexes (“polyplexes”), were diluted in PBS (PAA, Cölbe, Germany). Polyplexes were prepared as described above and used without

further dilution. A schematic overview of the layer composition on the SPR-prism is available as Supporting Information, Figure S1. SPR Device and Experimental Conditions. Based on the principle of surface plasmon resonance (SPR),20 the Plasmonic biosensor (Plasmonic Biosensor AG, Wallenfels, Germany) belongs to the category of fan-shaped beam instruments. It uses rectangular glass prisms coated on their upper surface with a 50 nm gold layer as disposables (chips). Every assembled chip has eight measuring channels, each of them corresponding to a cuvette that can sustain a volume of 20 μL. Monochromatic diverging light (785 nm) from a diode laser reflects onto the D



Article

2A) and the final plateau reached after washing determined the final level of the detection signal (relevant plateaus are indicated by black arrows). For additional clarity, the individual components added to the functionalized SPR prisms are indicated in the lower part of the diagrams in Figure 2, panels A−E, and final layer compositions of the binding assays are shown in the insets on the upper right of each graph. While both PEI-PEG-conjugates caused an angle shift of the detected light, the binding curves showed major differences in shape, indicating that cetuximab-PEG-PEI specifically bound to EGFR while trastuzumab-PEG-PEI conjugates did not (compare panels A and B of Figure 2). More specifically, PEI-PEGcetuximab showed substantial binding to immobilized EGFR, as indicated by the large shift in the detection signal (4900−6500 s time interval in Figure 2A; see arrow). Importantly, the addition of free ligand did not lead to further binding (see wash plateau after 7500 s). In contrast, a different shape of the binding curve and a smaller shift (see arrows) was observed in the case of PEI-PEG-trastuzumab, while substantial specific binding to the receptor was detected upon subsequent addition of free cetuximab (Figure 2B). Notably, the height of the binding signal after addition of cetuximab to EGFR was found to be 35 arbitrary units, which was precisely the same height as observed in Figure 2C where cetuximab was added directly after immobilization of the receptor (Figure 2C and data not shown). This demonstrates that the nonspecific PEI-PEGtrastuzumab conjugate neither interferes with cetuximab binding sites of EGFR, nor elicits sterical hindrance since the EGFR binding sites for cetuximab are completely free to interact with the ligand. Furthermore, this result was in good agreement with the observation that reducing the amounts of PEI-PEG-cetuximab allowed the subsequent binding of free cetuximab (data not shown), indicating incomplete saturation of the immobilized EGFR by substoichiometric amounts of the conjugate. The maximal signal for the binding of cetuximab to EGFR detected under our experimental conditions was 35 arbitrary units and was reached when adding 1 mg/mL cetuximab. This was also seen in control experiments demonstrating the complete protein A saturation and the binding specificity (Figure 2C−E). Since in theory the chemical antibody coupling procedure to the PEI-PEG may lead to some antibodies being bound in an incorrect orientation with exposure of the Fc domain, it was imperative to exclude false positive results based on the binding of the Fc parts of cetuximab or trastuzumab to free binding sites on protein A. Therefore, protein A saturation is an indispensable prerequisite, and all experiments were controlled by the parallel analysis of the nonspecific antibody trastuzumab. Complete saturation of Fc binding sites on the protein A layer was ensured by determining the amount of IgG molecules needed to cover the surface, with 2 mg/mL IgG being identified as sufficient. More specifically, while the saturation step still left the immobilized receptor exposed and available for the specific binding of cetuximab (Figure 2C), no additional binding of antibodies to protein A via their Fc domain was observed, as determined by the absence of a signal shift when adding, e.g., the unrelated anti-VEGF antibody bevacizumab (Figure 2D). Similar results were obtained in binding studies performed in the absence of EGFR-Fc, with the absence of a signal shift confirming that the binding of cetuximab relied on the presence of the immobilized EGFR (Figure 2E). Since some nonspecific interactions between the nontargeted PEI-PEG-trastuzumab conjugate and the functionalized chip surface were observed (see Figure 2B),

chemically functionalized gold-coated plane of the prism. The reflected light reaches the detector (CCD camera) at different positions depending on the reflection angle. The detector records the light intensity for each channel, and the timeresolved signal for the minimum of the reflected light gave the SPR sensogramm. The evanescent field changes when biomolecules interact with the receptors immobilized on the functionalized gold surface (“sensor chip surface”) inside the cuvette. Thus, the refractive index on the gold surface changes and the SPR dip moves over the sensor chip allowing the in vitro observation in real time of interactions between biomolecules. The sample handling performs via a pipet tip system preventing any kind of cross-contamination. Calculation of binding affinities was performed as described in the Results section, using the ORIGIN8.0 (OriginLab Corporation, Northampton, MA) software. Confocal Microscopy. Visualization of PEI-PEG-cetuximab/ siRNA complexes was performed by confocal laser scanning microscopy. To this end, PEI complexes containing Alexa Fluor 488 labeled siRNA or unlabeled Luciferase siRNA GL2 were prepared as described above. After SPR measurements of PEIPEG-cetuximab/siRNA polyplexes, the remaining PBS was removed from the cuvette system. The polyplexes were embedded in fluorescence mounting medium (Dako, Glostrup, Denmark) and coverslipped prior to microscopy. Polyplexes bound to the surface of the SPR prism were visualized with a LSM 510 META (Carl Zeiss GmbH, Oberkochen, Germany) by excitation at a wavelength of 488 nm with an argon laser, and a 505−550 nm BP emission filter was used for detection of the fluorescence signals.

■

RESULTS Surface Coating of the SPR Prism and Setup of Immunosandwiches. Immunosandwiches were set up as outlined in Figure 1. More specifically, antibodies cetuximab or, as negative control, trastuzumab were coupled to the low molecular weight PEI F25-LMW via a bifunctional PEG spacer to generate ligand-modified polymers. In a first series of SPR measurements, we analyzed the specific binding of the cetuximab-modified PEG-PEIs to an immobilized EGFR-Fc fusion protein. To this end, a sandwich was built as described in Figure 1A and the Experimental Section, which allowed the controlled, sterically correct exposure of the extracellular domain of the receptor to the chip surface. Since EGFR was used in substoichiometric amounts, free Fc binding sites were saturated by subsequent addition of human IgG molecules. Notably, beyond the polymers it was also possible to analyze the specific binding of cetuximab-functionalized PEI/siRNA complexes to the EGFR (Figure 1B), thus also demonstrating that after complex formation antibodies are exposed to the outside of the nanoparticle. The antibody-mediated specific binding was controlled by the parallel analysis of PEI-PEGtrastuzumab conjugates (Figure 1C) and of complexes exposing this antibody which, due to its specificity for the human epidermal growth factor receptor 2 (HER2), do not bind to EGFR (Figure 1D). Analysis of the Binding Specificity of CetuximabModified PEI Conjugate and Comparison of the Affinities of Cetuximab and PEI-PEG-Cetuximab to EGFR. Incubation steps were performed as indicated in Figure 2, each followed by three washing steps with PBS. The beginning of the wash at ∼2/3 of each interval is visible in the curves of the SPR signal (see also open arrowheads in Figure E



Article

Figure 3. SPR-based measurements of antibody-conjugated PEI/siRNA complexes. Ligand-mediated specific binding of PEI-PEG-cetuximab/siRNA complexes to EGFR (A). Qualitative proof of the binding of intact nanoparticles onto the SPR prism by confocal microscopy, as shown by two representative samples of Alexa 488 labeled PEI/siRNA complexes (“Alexa 488”) vs the corresponding unlabeled negative control (“GL2”). (B). Absence of specific binding of PEI-PEG-trastuzumab/siRNA complexes to immobilized EGFR (C). Black arrows indicate the position of relevant detection signal levels used for quantitation (compare distances between the levels/arrows).

affinity of cetuximab chemically coupled to PEI (Figure 2H). Reactions between an analyte A and a ligand L can be described by the association and dissociation rate constants kass and kdiss, respectively, with the dissociation equilibrium constant KD = kdiss/kass. While conventional analytical methods are mainly focused on the determination of KD, SPR is an excellent optical method for direct evaluation of kass and kdiss. For the kinetic analysis we adopted the “initial rate analysis” model from Edwards and Leatherbarrow.17 This method to evaluate the binding kinetics differs from well-established approaches only by taking into account the initial rate (linear domain) of binding and was developed especially on a SPR-based cuvette system. It is predicted on an exponential equation for the biosensor signal:

we further analyzed nonspecific binding effects to the SPR surface. Due to its partial protonation at physiological pH, PEI is positively charged and ζ-potential measurements upon complexation revealed that the PEGylation shielded the positive charge only partially (see below; Supporting Information, Figure S1A, right). Thus, electrostatic interactions, as well as dipole−dipole interactions or hydrogen bonds between the grafted polymer and the surface, may in theory account for nonspecific binding. Notably, however, in the SPR measurements no signal shift was observed in the case of PEIPEG, as indicated by identical plateau levels of the washing steps before and after PEI-PEG addition (Figure 2F; compare very left and right of the curve). The irrelevance of these interactions in our experimental SPR setting was further confirmed by the absence of nonspecific binding even in the case of nongrafted PEI with a still higher positive charge density (Figure 2G). Thus, these data indicate that the nonspecific binding observed in Figure 2B is dependent on the presence of the protein ligand rather than an effect of interactions of the polymer or the copolymer. To push the system further, the binding affinity in terms of association and dissociation constants of the free ligand cetuximab was determined and compared to the binding

R t = Coeff. (1 − e(−kass[L] + kdiss)t )

where Coeff. is Rmax[L]2/{(Kdiss/Kass) + [L]}, Rmax is the maximum capacity of the immobilized ligand, [L] is the ligate concentration, and kass and kdiss are the association and dissociation constants, respectively. Thus, Edwards and Leatherbarrow presumed that in the case of optical biosensors, the kinetics of all binding events involving interactions between an immobilized ligand and a ligate (analyte) consists of two F



Article

was absent in the unlabeled complex control (Figure 3B, “GL2”) and confirms the presence of siRNA, and thus the binding of the whole complex, to the immobilized receptor.

phases. A fast initial linear response (R) phase and a slower second nonlinear phase. In the case of SPR both these phases are recorded in real time on a sensorgram. The initial binding rates of the linear region of the binding curves for each concentration of the analyte are obtained by linear regression. The concentration of the immobilized ligand (EGFR) is kept constant. Thus, a plot of the binding rate (dR/dt) vs concentration of the analyte results in a straight line. The slope of this line, according to the equation, is the product of the association rate constant (kass) and the maximum binding capacity of the immobilized ligand (Rmax). For the calculation of the dissociation rate constant (kdiss), the dissociation phase data of the sensorgram obtained at the saturation concentration of the analyte were used. In the dissociation phase, the response depending on time decreases exponentially with kdiss. From the plot of ln(R0/Rt) on the y-axis vs time (t), the slope corresponds to kdiss. The equilibrium dissociation (KD) constant is then calculated by using the calculated values of kass and kdiss. For the determination of the binding affinity of cetuximab and the PEI-PEG-cetuximab conjugate to the immobilized EGFreceptor, different ligand concentrations were used, corresponding to 5−50 μg/mL of cetuximab. Molecular weights for the calculation are 145 kDa for cetuximab and 157 kDa for PEIPEG-cetuximab, respectively. Notably, the calculation of the equilibrium dissociation (KD) constants revealed that the native antibody and the antibody-modified conjugate have very similar affinities toward immobilized EGFR, with 90.2 ± 9.5 and 183.3 ± 26.3 nmol/L, respectively. SPR-Based Analysis of Specific Cetuximab-PEG-PEI/ siRNA-Complex Binding. While the SPR results shown above demonstrate the ligand-mediated binding of the antibody-PEG-PEI conjugates to the immobilized receptor, the ultimate goal is the binding analysis of the whole complexes. To this end, antibody-modified nanoparticles, consisting of siRNA molecules complexed with PEI-PEGantibody conjugates, were measured by SPR. Comparable to PEI-PEG-cetuximab, cetuximab-decorated polyplexes specifically bound to immobilized EGFR (Figure 3A). The specificity of the binding to the EGFR was confirmed by the subsequent addition of free ligand (cetuximab). The absence of a further increase in detection signal demonstrates that all immobilized receptor molecules were saturated by the antibody-mediated binding of the complexes. In contrast, the nontargeted, trastuzumab-modified polyplexes showed only nonspecific interaction with the surface, leaving the immobilized EGFR available for subsequent binding of free cetuximab (Figure 3C). Thus, these results are in agreement with the binding characteristics of the conjugates and establish our SPR measurements as a method for the analysis of complex binding properties. It is known that PEI-based polyplexes can be accompanied by free polymer.18 To confirm that the SPR signal observed upon addition of the nanoparticles is indeed due to the binding of the intact polyplexes to the EGFR and does not merely rely on some noncomplexed PEI-PEG-cetuximab conjugates, we complexed fluorescently labeled siRNA and performed confocal microscopy studies. The SPR binding curves of PEI-PEG-cetuximab/siRNA polyplexes were identical with Figure 3A (data not shown). Importantly, confocal microscopy of the SPR prism clearly revealed a fluorescence signal in the case of the polyplexes containing labeled siRNA (Figure 3B, “Alexa488” panels showing two representative sections with the minimum and maximum intensities observed). This signal was specific for the Alexa488 since it

■

DISCUSSION Ligand-decorated nanoparticles are promising delivery systems for drug targeting approaches.3,5,19−21 Since the ultimate goal of targeted nanoparticles is a tissue- or cell-specific binding and uptake, their physicochemical and biological properties have to be analyzed extensively in vitro prior to testing in animals. A very straightforward method to investigate the binding characteristics of a nanoparticle should be surface plasmon resonance spectroscopy (SPR). Since it is a highly sensitive method to study biomolecular interactions based on the change of the refractive index within an evanescent field, SPR has indeed become increasingly important for studying ligand− receptor interactions,22 and it has been successfully employed in the biomedical field, e.g., for biosensoring biological markers and toxins.23−26 Some years ago, it was hypothesized that SPR might also become a valuable tool to study gene delivery systems,27 which has become true.28−32 In fact, SPR offers several advantages over other methods for analyzing biomolecular interactions. Among them is the possibility to work without labeling and to monitor the binding process in realtime. While other techniques like impedance spectroscopy or quartz crystal microbalances also have the advantage of labelfree kinetic measurements, they are in general less sensitive as compared to SPR. Furthermore, only small amounts of the reagents are needed in SPR, especially when working with a cuvette system. Since the validity of SPR-based measurements depends not only on the quality of the binding partners but to a great extent also on the quality of the gold surface in terms of coating, receptor immobilization, and saturation of nonspecific binding sites, we put considerable effort into the optimization of the EGFR immobilization and the design of the assay sandwich. The modification of the gold surface with hydrophobic moieties allowed the efficient coating with protein A via hydrophobic interactions, as indicated by the very low binding signals of the blocking reagent BSA to the chemically modified gold surface. Furthermore, the protein A coating of the prism proved to be an excellent basis for the immobilization of the EGFR as EGFR-Fc fusion protein exclusively in the correct orientation and without the need of any further chemical modification of the receptor.33−35 The oriented immobilization allows the use of lower receptor amounts and results in a more sensitive sensor surface.33 Other attempts to control the correct sterical orientation of surface ligands during immobilization include NTA (nitrilotriacetic acid) sensor chips binding Histagged proteins, coupling of antibodies against immobilized tags such as GST (glutathione S-transferase), FLAG or His (poly histidine),35 the biotin-streptavidin system, or oxime ligation.36 Since all these approaches include major chemical modifications, they may come with issues regarding accuracy and sensitivity. Our immobilization strategy and thus the quality of our biosensor system were strongly supported by the high reproducibility of the experiments and the broad applicability of our SPR platform. In this context, it should also be noted that no sterical hindrance was observed, even when using the rather bulky nanoparticles. Importantly, the SPR system allows the analysis of the specific binding of whole nanoparticles which has been barely attempted so far. Here, we employed polyethylenimine-based complexes, which are efficiently used for the nonviral delivery of nucleic G



■

acids in vitro and in vivo.11 Our low molecular weight PEI F25LMW-based polyplexes have been shown previously to exhibit improved properties for the therapeutic delivery of nucleic acids in vitro as well as in different in vivo tumor models.9−11 Of note, however, the nonspecific uptake of cationic nanoparticles facilitated by electrostatic interactions with the negatively charged proteoglycans at the cell surface (“adsorptive endocytosis”) may compete with specific uptake mechanisms mediated by ligand−receptor interactions.37−40 Thus, we combined ligand coupling to PEI with PEG grafting (PEGylation) to abolish nonspecific electrostatic interactions of the polyplexes with several biological structures. In fact, PEGylated immunonanoparticles are considered to be among the most promising nanocarriers for the targeted delivery of drugs after systemic administration.41−43 Our data, in line with results from other groups,44−47 support the notion that PEGylation markedly reduces, or even abolishes the nonspecific uptake of PEI-based polyplexes and thus provides the basis for the ligand-mediated specific cellular uptake. To generate polyplexes with tumor-targeting properties, we chose the antiEGFR antibody cetuximab that is already in clinical use for the treatment of EGFR overexpressing tumors. Ligand-specific binding to the receptor could be analyzed precisely with our SPR method. Thus, this method allows in general the direct comparison of the binding affinities of free vs the conjugated ligand (i.e., antibody) to the immobilized receptor. Notably, the similar KD values of cetuximab and the PEI-PEG-cetuximab conjugates determined in our experiments reveal that the polymer coupling did not impair the binding affinity of the antibody. Importantly, the favorable physicochemical properties of our antibody-modified polyplexes in terms of particle size and surface charge allow specific ligand-mediated cellular uptake in vitro resulting in transgene expression and gene knockdown strongly supporting their biological activity (see the Supporting Information Figure S1). To our knowledge, this is the first study to describe an SPR analysis of the binding of ligand-modified PEI-based polyplexes. We thus present PEGylated PEI-based immunonanoparticles targeted against EGFR overexpressing cells and tissues as promising formulations for the delivery of therapeutic small RNA molecules for gene therapy approaches for cancer, and we show that their ligand-mediated binding can be analyzed by our SPR method.

Article

ASSOCIATED CONTENT

S Supporting Information *

Additional methods and results as noted in text, and a comprehensive list of abbreviations used. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +49 341 9724661. Fax: +49 341 9724669. E-mail: achim. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by grants from the Deutsche Forschungsgemeinschaft (German Research Foundation; Forschergruppe ‘Nanohale’ AI 24/6-1 and DFG single grant AI 24/ 9-1) and the Deutsche Krebshilfe (German Cancer Aid). S.H. and D.V. contributed equally to this work.

■

REFERENCES

(1) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Nature 1998, 391, 806−811. (2) Tuschl, T.; Borkhardt, A. Mol. Interventions 2002, 2, 158−167. (3) Aigner, A. Appl. Microbiol. Biotechnol. 2007, 76, 9−21. (4) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347−360. (5) Basarkar, A.; Singh, J. Int. J. Nanomed. 2007, 2, 353−360. (6) Behr, J. P. Chimia 1993, 1, 34−36. (7) Werth, S.; Urban-Klein, B.; Dai, L.; Höbel, S.; Grzelinski, M.; Bakowsky, U.; Czubayko, F.; Aigner, A. J. Controlled Release 2006, 112, 257−270. (8) Höbel, S.; Prinz, R.; Malek, A.; Urban-Klein, B.; Sitterberg, J.; Bakowsky, U.; Czubayko, F.; Aigner, A. Eur. J. Pharm. Biopharm. 2008, 70, 29−41. (9) Höbel, S.; Koburger, I.; John, M.; Czubayko, F.; Hadwiger, P.; Vornlocher, H. P.; Aigner, A. J. Gene Med. 2010, 12, 287−300. (10) Ibrahim, A. F.; Weirauch, U.; Thomas, M.; Grünweller, A.; Hartmann, R. K.; Aigner, A. Cancer Res. 2011, 71, 5214−5224. (11) Hö bel, S.; Aigner, A. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2013, 5, 484−501. (12) Allen, T. M. Nat. Rev. Cancer 2002, 2, 750−763. (13) Malmqvist, M. Nature 1993, 361, 186−187. (14) Keusgen, M. Naturwissenschaften 2002, 89, 433−444. (15) Barlen, B.; Mazumdar, S. D.; Keusgen, M. Phys. Status Solidi A 2009, 3, 409−416. (16) Merkl, S.; Vornicescu, D.; Dassinger, N.; Kehrel, M.; Harpel, S.; Keusgen, M. Phys. Status Solidi A 2012, 5, 864−870. (17) Edwards, P. R.; Leatherbarrow, R. J. Anal. Biochem. 1997, 246, 1−6. (18) Boeckle, S.; von Gersdorff, K.; van der Piepen, S.; Culmsee, C.; Wagner, E.; Ogris, M. J. Gene Med. 2004, 6, 1102−1111. (19) Nel, A. E.; Mädler, L.; Velegol, D.; Xia, T.; Hoek, E. M.; Somasundaran, P.; et al. Nat. Mater. 2009, 8, 543−557. (20) Wagner, E.; Kircheis, R.; Walker, G. F. Biomed. Pharmacother. 2004, 58, 152−161. (21) Aigner, A. J. Mol. Med. 2011, 89, 445−457. (22) Rich, R. L.; Myszka, D. G. J. Mol. Recognit. 2010, 23, 1−64. (23) Meyer, M. H.; Hartmann, M.; Keusgen, M. Biosens. Bioelectron. 2006, 21, 1987−1990. (24) Vornholt, W.; Hartmann, M.; Keusgen, M. Biosens. Bioelectron. 2007, 22, 2983−2988. (25) Mazumdar, S. D.; Barlen, B.; Kramer, T.; Keusgen, M. J. Microbiol. Methods 2008, 75, 545−550. (26) Kuziemko, G. M.; Stroh, M.; Stevens, R. C. Biochemistry 1996, 35, 6375−6384. (27) Gambari, R. Am. J. PharmacoGenomics. 2001, 1, 119−135.

■

CONCLUSIONS The SPR system described here, based on convection-free cuvettes and a protein A sandwich for the immobilization of the receptor in the correct steric orientation, allows the precise assessment of the binding of ligand-functionalized nanoparticles in real-time, as shown here for PEI-PEG-cetuximab-based complexes. SPR results are confirmed by the analysis of physicochemical and biological properties of the nanoplexes. We also describe immunonanoparticles based on a branched, PEGylated low molecular weight PEI for the targeted delivery of therapeutic nucleic acids as a promising platform for nonviral gene therapy approaches, especially for the therapeutic delivery of small RNA molecules. To our knowledge, this is the first study to assess the binding of ligand-modified PEI-based nanoplexes by SPR measurements. We thus establish SPR for the in vitro quality control of targeted nanoparticles with regard to their ligand-mediated binding affinities, and introduce this method as a valuable tool for the qualitative and quantitative exploration of binding affinities and characteristics. H



Article

(28) Hong, S.; Leroueil, P. R.; Majoros, I. J.; Orr, B. G.; Baker, J. R., Jr.; Banaszak Holl, M. M. Chem. Biol. 2007, 14, 107−115. (29) Tassa, C.; Duffner, J. L.; Lewis, T. A.; Weissleder, R.; Schreiber, S. L.; Koehler, A. N.; Shaw, S. Y. Bioconjugate Chem. 2010, 21, 14−19. (30) Brambilla, D.; Verpillot, R.; Taverna, M.; De Kimpe, L.; Le Droumaguet, B.; Nicolas, J.; Canovi, M.; Gobbi, M.; Mantegazza, F.; Salmona, M.; Nicolas, V.; Scheper, W.; Couvreur, P.; Andrieux, K. Anal. Chem. 2010, 82, 10083−10089. (31) Debotton, N.; Zer, H.; Parnes, M.; Harush-Frenkel, O.; Kadouche, J.; Benita, S. Eur. J. Pharm. Biopharm. 2010, 74, 148−156. (32) Gobbi, M.; Re, F.; Canovi, M.; Beeg, M.; Gregori, M.; Sesana, S.; Sonnino, S.; Brogioli, D.; Musicanti, C.; Gasco, P.; Salmona, M.; Masserini, M. E. Biomaterials 2010, 31, 6519−6529. (33) Ko, S.; Park, T. J.; Kim, H. S.; Kim, J. H.; Cho, Y. J. Biosens. Bioelectron. 2009, 24, 2592−2597. (34) Lee, W.; Oh, B. K.; Min Bae, Y.; Paek, S. H.; Hong Lee, W.; Choi, J. W. Biosens. Bioelectron. 2003, 19, 185−192. (35) Blanchette, C. D.; Fischer, N. O.; Corzett, M.; Bench, G.; Hoeprich, P. D. Bioconjugate Chem. 2010, 21, 1321−1330. (36) Dettin, M.; Muncan, N.; Bugatti, A.; Grezzo, F.; Danesin, R.; Rusnati, M. Bioconjugate Chem. 2011, 22, 1753−1757. (37) Rhaese, S.; von Briesen, H.; Rübsamen-Waigmann, H.; Kreuter, J.; Langer, K. J. Controlled Release 2003, 92, 199−208. (38) Fischer, D.; Bieber, T.; Li, Y.; Elsässer, H. P.; Kissel, T. Pharm. Res. 1999, 16, 1273−1279. (39) Duncan, R.; Pratten, M. K.; Lloyd, J. B. Biochim. Biophys. Acta 1979, 587, 463−475. (40) Mislick, K. A.; Baldeschwieler, J. D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12349−12354. (41) Avgoustakis, K.; Beletsi, A.; Panagi, Z.; Klepetsanis, P.; Livaniou, E.; Evangelatos, G.; Ithakissios, D. S. Int. J. Pharm. 2003, 259, 115− 127. (42) Tobío, M.; Sánchez, A.; Vila, A.; Soriano, I.; Evora, C.; Vila-Jato, J. L.; Alonso, M. J. Colloids Surf., B 2000, 18, 315−323. (43) Soppimath, K. S.; Aminabhavi, T. M.; Kulkarni, A. R.; Rudzinski, W. E. J. Controlled Release 2001, 70, 1−20. (44) Pavey, K. D.; Olliff, C. J. Biomaterials 1999, 20, 885−890. (45) Kircheis, R.; Blessing, T.; Brunner, S.; Wightman, L.; Wagner, E. J. Controlled Release 2001, 72, 165−170. (46) Schiffelers, R. M.; Ansari, A.; Xu, J.; Zhou, Q.; Tang, Q.; Storm, G.; Molema, G.; Lu, P. Y.; Scaria, P. V.; Woodle, M. C. Nucleic Acids Res. 2004, 32, e149. (47) Kichler, A. J. Gene Med. 2004, 6, S3−S10.

I


A Novel Method for the Assessment of Targeted PEI-Based

Recommend Documents