Molecularly Imprinted Nanocavities Capable of Ligand-Binding

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Molecularly Imprinted Nanocavities Capable of LigandBinding Domain and Size/Shape Recognition for Selective Discrimination of Vascular Endothelial Growth Factor Isoforms Yuri Kamon, and Toshifumi Takeuchi ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00622 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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Molecularly Imprinted Nanocavities Capable of Ligand-Binding Domain and Size/Shape Recognition for Selective Discrimination of Vascular Endothelial Growth Factor Isoforms

Yuri Kamon and Toshifumi Takeuchi* Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan

Corresponding author: Toshifumi Takeuchi TEL/FAX: +81-78-803-6158, E-mail: [email protected]

KEYWORDS ： Molecular imprinting, Vascular endothelial growth factor, Ligand-protein interaction, Surface initiated atom transfer radical polymerization, Protein sensing

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ABSTRACT Vascular endothelial growth factor 165 (VEGF165) is known to be predominantly expressed in the first stage of vascularization; therefore, the detection of VEGF165 is important in the stage diagnosis of cancers. Molecularly imprinted nanocavities, capable of the selective discrimination of VEGF165 from other VEGF isoforms, were prepared by surface-initiated atom transfer radical polymerization. VEGF165 was immobilized on a gold-coated glass substrate by anchored heparin moieties, where the immobilized heparin was able to capture VEGF165 by binding with the heparin-binding domain (HBD) on VEGF165. Molecular imprinting was conducted on the immobilized VEGF165 by using methacrylic acid (MAA) as a functional monomer to interact with basic amino acids outside of the HBD of VEGF165 by electrostatic interaction. After the removal of VEGF165 from the obtained polymer thin layer (ca. 7 nm), VEGF165-imprinted nanocavities remained, in which the heparin moiety and MAA residues were located in suitable positions for VEGF165 recognition. The molecularly imprinted polymer (MIP) thin layer showed a binding affinity for VEGF165 (dissociation constant: 3.4 nM) that was ten times higher than that of the substrate before polymerization (heparin-immobilized substrate). A much lower binding affinity for VEGF121, which contains no heparin-binding domain, was observed. Moreover, the MIP thin layer distinguished VEGF165 from VEGF189, which possesses a larger molecular size than VEGF165, an amino acid sequence homology of 87%, and contains HBDs, 2 ACS Paragon Plus Environment

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whereas the heparin-immobilized substrate showed almost no selectivity. These results suggested that the heparin moiety within the nanocavity provided HBD selectivity and the polymer matrix comprising the molecularly imprinted nanocavity provided size/shape selectivity, which resulted in the highly selective discrimination of VEGF isoforms.


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Proteins perform important functions, such as the transportation of molecules, the transduction of stimuli-responsive signals, and the mediation of catalysis, to maintain various biological systems through interactions with specific molecules, owing to their unique binding domains, such as ligand-binding domains (LBDs). The selective recognition of a specific protein from highly homological protein isoforms with common LBDs is of great importance when each isoform plays different roles and functions in biological systems. Vascular endothelial growth factor (VEGF)-A is a promoter of angiogenesis induced by its binding to VEGF receptors-1, 2, or 3 on cell membranes via the common receptor binding domain of VEGFs [1]. VEGF-A has three main alternatively spliced isoforms: VEGF165, VEGF189, and VEGF121. VEGF165 and 189 possess high sequence homology (87%) and contain the same LBD for heparin (the heparin-binding domain, HBD [2]), whereas VEGF121 contains no HBD [3]. As VEGF isoforms are produced depending on tissues and precede the occurrence of cancers, the sensitive and selective detection of these VEGF isoforms is an important requisite for the early diagnosis of various types of cancers [4]. Bio-based recognition materials, including monoclonal antibodies, have been frequently used to detect specific protein isoforms as they exhibit binding activity with selective recognition towards the slightly different domains found in the isoforms [5]. DNA/peptide aptamers also have similar binding properties for the recognition of target proteins [6]. However, there are some 4 ACS Paragon Plus Environment

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drawbacks, such as the large period of time required for their discovery and the production with higher specificity and affinity towards target proteins compared with off-target protein families and isoforms. Synthetic polymer materials with molecular recognition activity have attracted a great deal of attention as they can be designed and synthesized by the copolymerization of diverse monomers containing groups that interact with the target proteins in an optimal combination, in order to achieve the recognition of highly homologous proteins, such as the VEGF isoforms of VEGF165, 189, and 121. It is important to create artificial binding domains in polymeric materials composed of specific HBD interaction sites within a binding nanocavity complementary in size and shape, which may enable us to discriminate slightly different protein isoforms. Heparins are known as common specific ligands for the VEGF isoforms; VEGF165 and 189 possess HBDs [7], whereas no HBD is found in VEGF121, which suggests that the use of heparins as interaction sites can facilitate the reduced recognition of VEGF121 compared with VEGF165 and 189, as we have already demonstrated that protein-ligand interaction occurs in specific ligand-immobilized polymeric materials [8]. To distinguish between VEGF165 and 189, recognition of their sizes and shapes may be necessary in addition to their heparin-HBD interactions. Molecular imprinting has been recognized as a promising technique to create binding nanocavities for target proteins 5 ACS Paragon Plus Environment

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complementary in size, shape, and amino acid residues that interact with the functional monomers. Molecularly imprinted polymers (MIPs) can be prepared by the copolymerization of comonomer(s) and a crosslinker in the presence of template protein-functional monomer complexes formed covalently or non-covalently. The subsequent removal of the template proteins results in imprinted nanocavities that contain binding sites derived from the functional monomers, which can act as interaction sites for the target proteins [9]. In this study, we demonstrated the precise recognition of VEGF isoforms in the VEGF-A family by the means of protein-ligand interaction-based molecular imprinting. VEGF165 was selected as the model VEGF isoform, since VEGF165 is known to be predominantly expressed in the first stage of vascularization, especially in rectal cancers [4]. Nanometer-sized thicknesses of VEGF165-targeted MIP thin layers were prepared on a heparin-immobilized gold-coated substrate in a bottom-up manner, in which immobilized heparin works both to bind VEGF165 before polymerization and as an HBD recognition site after polymerization. Further, the VEGF165-imprinted nanocavity is complementary in size and shape to VEGF165, which enhances the recognition between VEGF165 and 189. Methacrylic acid (MAA) was used as a functional monomer that can interact with the HBD-outside of basic amino acid residues on VEGF165 during polymerization to enhance the size/shape recognition on VEGF165. Surface-initiated atom transfer radical polymerization using activators generated by electron 6 ACS Paragon Plus Environment

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transfer (SI-AGET ATRP) [10] was conducted on the VEGF165-immobilized substrate using 2-methacryloyloxyethyl phosphorylcholine (MPC) as a biocompatible comonomer [11] and N,N’-methylenebisacrylamide (MBAA) as a crosslinker, in which the polymer thickness can be optimized by polymerization time to achieve high sensitivity and selectivity [8, 12]. This study has demonstrated the effectiveness of protein-ligand interaction-based MIPs with precisely controlled polymer thickness to recognize structurally and functionally related protein isoforms that contain common LBDs and high amino acid sequence homology.

EXPERIMENTAL SECTION Preparation of heparin-immobilized substrates bearing surface bromo groups. Gold-coated surface plasmon resonance (SPR) sensor chips and gold-coated glass substrates were cleaned by UV-O3 treatment (20 min). The cleaned substrates were immersed in an EtOH solution of 0.5 mM 11-sulfanyloundec-1-yl 2-bromo-2-methylpropionate and 0.5 mM (11-mercaptoundecyl)tetra(ethylene glycol) for 24 h at 30°C (Scheme 1a). Next, the mixed SAM-formed substrates were washed with ethanol and distilled water, and then dried with N2 gas. The substrates were then immersed in distilled dichloromethane containing 5 mM N-hydroxysuccinimidyl-15-(3-maleimidopropionyl)-amido-4,7,10,13-tetraoxapentadecanoate (MAL-dPEG4-NHS ester) ester overnight at 25°C to introduce maleimide groups (Scheme 1b), 7 ACS Paragon Plus Environment

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washed with dry dichloromethane and ethanol, and then dried with N2 gas.

Scheme 1. Preparation of MIP thin layer on a gold substrate.

The maleimidated substrates were treated with 10 mM phosphate buffer (pH 7.0) containing the thiolated heparin (40 µM) at 25°C for 2 h (Scheme 1c), and the obtained heparin-immobilized substrates were washed with 10 mM phosphate buffer (pH 7.0). In order to cap the unreacted maleimide

groups,

the

substrates

were

incubated

with

0.5

mM

of

2-{2-[2-(2-mercaptoethoxy)ethoxy]ethoxy}ethanol in 10 mM phosphate buffer (pH 7.0) at 25°C


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for 1 h (Scheme 1d), followed by washing with 10 mM phosphate buffer (pH 7.0) to obtain the heparin-immobilized substrates bearing bromo groups on the surface. Preparation of MIP thin layers by SI-AGET ATRP. VEGF165 (10 µg/mL) dissolved in 10 mM phosphate buffer (pH 7.4) was dropped on the heparin-immobilized substrates (Scheme 1e), incubated at 25°C for 1 h, and washed with 10 mM phosphate buffer (pH 7.4). A pre-polymer mixture (120 µL) containing MAA (1 mM) as a functional monomer, MPC (180 mM) as a biocompatible comonomer, and MBAA (20 mM) as a hydrophilic crosslinker, CuBr2 (10 mM) as a catalyst, and 2,2'-bipyridyl (20 mM) as a ligand for Cu ions were prepared in 10 mM Tris-HCl buffer (pH 7.4). SI-AGET-ATRP was then performed at 25 °C for 4 h, initiated by the addition of L-ascorbic acid (30 µL, final concentration: 5 mM) (Scheme 1f). After polymerization, the substrates were incubated with a 1 M EDTA-4Na aqueous solution overnight to remove Cu ions and washed with pure water. In order to remove VEGF165 and leave VEGF-imprinted nanocavities, the substrates were incubated consecutively with 2 M NaCl aqueous solution for 6 h and 0.5 w% Triton X-100 aqueous solution for 3 h, and washed with pure water (Scheme 1g). X-ray photoelectron spectroscopy (XPS) and X-ray reflectometry (XRR) were performed to examine the polymer contents and thickness of the obtained MIP layer, respectively. MIP without MAA thin layer was prepared without the addition of MAA, and non-imprinted 9 ACS Paragon Plus Environment

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polymer (NIP) was prepared on the heparin-immobilized substrate without the addition of VEGF165

and MAA. NIP without heparins was prepared on

the tetra(ethylene)

glycol-immobilized substrate without the addition of VEGF165 and MAA.

VEGFs binding tests by SPR measurements Binding isotherms were drawn by ∆ resonance unit (∆RU) values obtained by using an SPR sensor BIACORE 3000 at a flow rate of 20 µL/min 10 mM phosphate buffer (pH 7.4). An injection volume was 20 µL. The amounts of bound proteins were plotted by ∆RU at 150 s after injection. The curve fitting analysis was conducted by using Delta Graph 5.4.5v. to estimate the dissociation constants of VEGF 165 towards the prepared polymer thin layers (SI Equation S3). The regeneration solutions were selected appropriately from the following candidates: 1 M NaCl aqueous solution, 0.1% Triton X-100 aqueous solution, and mixed aqueous solutions containing 1 M NaCl + 0.1% Triton X-100, 1 M NaCl + 0.05% Triton X-100, and 1 M NaCl + 0.01% Triton X-100. The optimal regeneration solutions were injected once or twice for 18, 30, or 60 s until the ∆RU of the bound proteins reaches 0. All binding experiments were repeated three times. For the selectivity test, each VEGF isoform (100 ng/mL) was dissolved in 10 mM phosphate buffer (pH 7.4) and the binding experiments were conducted in the same manner.


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RESULTS AND DISCUSSION Preparation of MIP thin layers. In order to immobilize the heparins on the substrate, the thiolated heparins were prepared by coupling p-aminothiophenol with the reductive terminal of heparins (SI Scheme S1) [13]. The thiolation ratio was estimated to be approximately 50% by Ellman’s assay and 1H-NMR (SI Equations S1 and S2). From the UV-vis spectra and CD spectra (SI Figure S2), the absorption maximum wavelength at approximately 260 nm was derived from p-aminothiophenol, and the thiolated heparins induced a negative Cotton effect peak at approximately 210 nm; these peaks confirmed that the helical structure was maintained, which is important for binding to the HBD [14]. A mixed self-assembled monolayer (SAM) was formed on a gold-coated glass substrate to introduce bromo groups as initiators for the SI-AGET ATRP and oligo ethylene glycol groups to connect the thiolated heparins (Scheme 1a). XPS showed the S 2p peak at 162 eV and the Br 3d peak at 68 eV after the mixed SAM formation (SI Figure S3a, b). The C 1s peaks can be deconvoluted into the C-O peak at 286.5 eV, which was mainly derived from the oligo ethylene glycol, and the C=O peak at 288 eV, which was derived from the initiator groups (SI Figure S3c, 3d). These results suggested that the designed mixed SAM was successfully formed on the substrate, as expected. The maleimide groups were then coupled with hydroxy groups via ester bonds (Scheme 1b), and the thiolated heparins were attached to the maleimide groups by Michael


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addition reaction (Scheme 1c) [15]. After unreacted maleimide groups were capped with thiolated tetra(ethylene glycol) (Scheme 1d), the heparin-immobilized substrate was obtained. The binding activity of the heparin-immobilized substrate toward VEGF165 was examined by SPR measurements using 10 mM phosphate buffer (pH 7.4) as a running buffer at 25 °C, and the dissociation constant was estimated to be 72 nM by curve fitting analysis (SI Figure S4 and Figure S7a). To prepare the MIP thin layer, MAA was used as a functional monomer to interact with the basic amino acid residues outside the HBD during SI-AGET ATRP, because positively charged HBD was already occupied by the immobilized heparins, which enhanced the size/shape recognition on VEGF165. From a ∆RU value of around 10 µg/mL (SI Figure S4), the immobilized amount of VEGF165 was roughly estimated to be 40 fmol/mm2 in the MIP preparation conditions, where 1 RU is converted into the commonly used value of 1 pg/mm2. As the number of lysine and arginine residues outside the HBD is 14 and 7, respectively (total amount: ca 840 fmol/mm2); therefore, the amount of MAA added (150 nmol on a polymerization area of 56 mm2: 2.7 nmol/mm2) was enough to complex MAA with basic amino acid residues in VGEF165 during SI-AGET ATRP. With regard to the crosslinker content, 10 mol% of MBAA was previously reported to be preferable to prevent the non-specific adsorption of off-target proteins [12a]; therefore, the crosslinker content of 10 mol% was adopted in this study. After the 12 ACS Paragon Plus Environment

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template VEGF165 was removed by washing with 2 M NaCl [16] and 0.5 wt% Triton X-100 to weaken electrostatic and hydrophobic interactions, the imprinted nanocavities remained, in which both the heparins and MAA residues were located as orthogonal interaction sites. Based on our previous work on immobilized protein-based MIPs, polymer thickness can be controlled by changing the polymerization time [8, 12b]. When SI-AGET ATRP was conducted for 4 h at 25 °C, the thickness of the obtained MIP thin layer was estimated to be 7 nm by XRR (SI Figure S5, Table S1), which was similar to the average diameter of VEGF165 (approximately 6.4 nm), which suggested that the imprinted nanocavities may not be buried in the polymer matrix. The XPS analysis showed a P 2p peak at 134 eV derived from the phosphorylcholine group of MPC (SI Figure S6), which confirmed the copolymerization of MPC on the gold-coated glass substrate.

Imprinting effects. The binding activity of the obtained MIPs for VEGF165 was investigated by SPR measurements. MIP showed a saturable binding behavior at nanomolar concentrations of VEGF165 (Figure 1a). The dissociation constant was estimated to be 3.4 nM by curve fitting analysis with a Langmuir binding model (SI Figure S7b), which was comparable with those of general antibodies [17]. To confirm the imprinting effect and the synergistic effect that resulted from the HBD 13 ACS Paragon Plus Environment

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binding activity, basic amino acid residue recognition ability by carboxyl groups, and the size/shape selectivity that simultaneously occurred in an imprinted nanocavity for VEGF165, three reference polymer thin layers were prepared: MIP without MAA (Figure 1b), which possessed only immobilized heparins as interaction sites within a nanocavity; non-imprinted polymer (NIP) prepared without VEGF165 and MAA (Figure 1c), which possessed no VEGF165-imprinted nanocavity; and NIP without heparin, which was prepared without VEGF165, MAA and heparin. In addition to the above three, the heparin-immobilized substrate (Figure 1d), with no cavities and no polymeric thin layer, was also examined to confirm the imprinting effect. MIP without MAA showed a linear profile similar to that of the heparin-immobilized substrate in the nanomolar concentration range of VEGF165. A dissociation constant of MIP without MAA was estimated to be approximately 40 nM (SI Figure S7c), which was 10 times higher than that of MIP. These results suggested that a synergistic effect of the polymer matrix and the MAA residues was induced by the imprinting process. The dissociation constant of MIP without MAA was 1.8 times lower than that of the heparin-immobilized substrate. This may be a result of less non-specific binding of the MPC-based polymer matrix as previously reported [10h, 11]. NIP showed no significant VEGF165 binding, which was almost equivalent to NIP without heparins (SI Figure S8), suggesting that the immobilized heparin in NIP may be buried within the MPC-based polymer matrix. 14 ACS Paragon Plus Environment

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The association rate constants of MIP and MIP without MAA at 2.2 nM VEGF165 (100 ng/mL), estimated from the sensorgrams (SI Figure S9) by curve fitting analysis, appeared to be 1.0 × 107 M-1 s-1, whereas that of the heparin-immobilized substrate was 1.3 × 107 M-1 s-1, which suggested that the polymer matrix affects the binding kinetics and reduces the accessibility to the immobilized heparin. With regard to the dissociation rate constants, although it is difficult to estimate owing to their extremely slow kinetics, it may be anticipated from the corresponding dissociation constants that the order of dissociation rates was MIP < MIP without MAA < heparin-immobilized substrate, as a dissociation constant can be calculated by a dissociation rate constant divided by an association rate constant.


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Figure 1. Binding isotherms of VEGF165 towards MIP (a), MIP without MAA (b), NIP (c), and heparin-immobilized substrate (d). Running buffer: 10 mM phosphate buffer at pH 7.4 (20 µL/min, 25°C); Injection volume: 20 µL.


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Binding selectivity for VEGF isoforms. In order to examine the selectivity of MIP for VEGF165, the binding behaviors of VEGF isoforms in MIP, MIP without MAA, NIP, and the heparin-immobilized substrate were investigated with two reference VEGF isoforms, VEGF189 (bearing HBD) and VEGF121 (no HBD). The selectivity factor (α), which is a ratio of a bound amount of VEGF189 or 121 to that of VEGF165 at 100 ng/mL (2.2 nM), was employed as an index of the selectivity for VEGF165. MIP and MIP without MAA clearly distinguished VEGF165 from VEGF121, with α values of 0.05 (p < 0.0005) and 0.07 (p < 0.05), respectively (Figure 2a, b). This may be a result of the presence of the immobilized heparin working with the specific ligand for the HBD in the two MIP thin layers. With respect to the cross-reactivity of VEGF165 and VEGF189, MIP recognized the difference between VEGF165 and VEGF189 (α = 0.54, p < 0.05), whereas MIP without MAA resulted in almost no recognition (Figure 2a, b). This may be attributable to the positions of the MAA residues in the imprinted nanocavity induced by the imprinting effect, which were complementary to the positions of the basic amino acid residues of VEGF165, thereby resulting in favorable binding to VEGF165. In addition to the effect of MAA-based electrostatic interaction, MAA is expected to induce better template-oriented shape/size matching for VEGF165 during polymerization, because a polymerizable group of MAA can exist close to the surface of VEGF165 owing to electrostatic interaction. The heparin-immobilized substrate was unable to recognize VEGF165 from 17 ACS Paragon Plus Environment

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VEGF189 as expected, and showed higher binding for VEGF121 (Figure 2c) than MIP and MIP without MAA. As there was no MPC-based polymer thin layer on the heparin-immobilized substrate, relatively high non-specific binding, based on hydrophobic interactions with the mixed SAM layer and hydrophobic regions such as tyrosine-25, tyrosine-21, and phenylalanine-17, which are important for the binding of VEGF receptors [18], may occur. As indiscrete heparins were used in this study, the isoform selectivity may change if more precisely defined heparins are utilized. For the NIP thin layer, the binding was too weak to obtain statistically significant α values. From these results, the fabrication of the imprinted nanocavity fit to VEGF165 in size and shape, in which MAA residues were positioned suitably for the interaction with basic amino acid residues on VEGF165 and the immobilized heparins were grafted on the bottom as the specific ligands for the HBD of VEGF165, is of significant importance in the development of isoform selectivity. It should be noted that the isoelectric points of VEGF165 and VEGF189 are 7.9 and 9.3, respectively [19]. Although VEGF189 is more positively charged in 10 mM phosphate buffer at pH 7.4, its binding was suppressed in the MIP thin layer, which revealed that the negatively charged MIP contained heparins and methacrylic acid residues at pH 7.4, showed the unique characteristics of preferential binding to the less positive VEGF165, unlike normal cation exchange media. This also provided evidence that the imprinting effect was attributable to the development of VEGF isoform selectivity, realizing the changes in the intrinsic property of 18 ACS Paragon Plus Environment

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base-polymer matrix into target-specific binding properties.

Figure 2. Comparison of binding selectivity of VEGF isoforms, including VEGF165 and 189, which contain HBDs, and VEGF121, which contains no HBD, towards MIP (a), MIP without MAA (b), and heparin-immobilized substrate (c). The selectivity factor (α) is defined as relative bound amounts of reference VEGFs towards the target VEGF165 at 100 ng/mL VEGFs. Running buffer: 10 mM phosphate buffer at pH 7.4 (20 µL/min, 25°C); Injection volume: 20 µL. *: p < 0.05, **: p < 0.0005 (n = 3).


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CONCLUSION Abiotic polymeric materials were first demonstrated to show VEGF isoform selectivity, even when the isoforms have high amino acid sequence homology (87%). These materials were prepared by using a protein-ligand interaction-based molecular imprinting method to construct the binding nanocavity containing three orthogonal binding modes, i.e. HBD-binding activity, electrostatic interaction, and size/shape selectivity. Based on the binding characteristics of MIPs, NIP, and the heparin-immobilized substrates for VEGF isoforms, the imprinting effect was found to play a significant role in the development of nanomolar affinity and isoform selectivity. Although further optimization of the heparins used, the MAA content, and the polymerization conditions should be addressed, the proposed method can provide a means for preparation of natural antibody-comparable abiotic materials to emerge as a new platform for the development of molecular recognition-based synthetic sensing materials useful in life science applications.


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Supporting Information Supporting Information Available: The following files are available free of charge on the ACS Publications website at DOI: xxxx. Materials, Apparatus, Synthetic procedures and characterization, Polymer characterization by XPS, XRR, and binding behaviors, and Curve fitting for the estimation of dissociation constants (PDF) Author Information Corresponding Author Toshifumi Takeuchi ORCID: 0000-0002-5641-2333 Author Contributions T.T. conceived the study and supervised the experiments. Y.K. designed and conducted the experiments. The manuscript was written through contributions of all authors. Acknowledgment This work was partially supported by Grant-in-Aid for Scientific Research (grant no.15K14943 and 17K14093) and Research Fellowship of JSPS for Young Scientists (JSPS KAKENHI 15J04455) from the Japan Society for the Promotion of Science (JSPS).


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Molecularly Imprinted Nanocavities Capable of Ligand-Binding

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