Article pubs.acs.org/JPCC
Self-Assembly of an Europium-Containing Polyoxometalate and the Arginine/Lysine-Rich Peptides from Human Papillomavirus Capsid Protein L1 in Forming Luminescence-Enhanced Hybrid Nanoparticles Teng Zhang, Hong-Wei Li, Yuqing Wu,* Yizhan Wang, and Lixin Wu* State Key Laboratory of Supramolecular Structure and Materials, Jilin University, No. 2699, Qianjin Street, Changchun 130012, P. R. China S Supporting Information *
ABSTRACT: Through a self-assembly of arginine/lysine-rich peptide from human papillomavirus (HPV) capsid protein and an Eu-containing polyoxometalate (POM), Na9[EuW10O36]·32H2O (EuW10), the formation of well-defined hybrid nanospheres in aqueous solution is presented, showing large luminescence enhancement of POM and use as a potential “turn-on” fluorescence probe in biology. The binding mechanisms between them have been explored at the molecular level by using transmission electron microscopy (TEM), scanning electron microscopy (SEM), fluorescence spectra, isothermal titration calorimetry (ITC), ζ-potential, and nuclear magnetic resonance (1H NMR) titration spectra. ITC study confirmed the assembly was completely enthalpy driven, and ζ-potential proved that the driving force was governed mainly by the electrostatic interaction. 1H NMR spectroscopy indicated changes in hydrogen bond of EuW10 and the peptide segment, and the binding model was clarified. Our design constructed the self-assembly fabrication of well-defined nanoparticles by using inorganic POM and bioapplicable peptide combined with strong fluorescence characterization together. The enhanced luminescence and specific targeted-HPV peptide ability would be important and useful in the detection of HPV capsid protein and/or HPV genotypes, and such a protocol could be extended to another virus once using the corresponding peptides. Therefore, the present report will be helpful to promote the development of antivirus agents in the future.
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INTRODUCTION As a family of nonenveloped viruses, papillomavirus (PVs) can easily infect human epithelial cells and have attracted more and more attention recently because of their high hazard to human health.1,2 The related studies indicate that some pre- and malignancies as head−neck, anogenital, and especially cervical cancer are closely related to the infection of high-risk HPV types.3−7 Therefore, possible biomarkers should be developed for HPV detection, and several ones have been suggested, including HPV genotype8 and capsid protein L1.9 HPV L1 is the major protein comprising the viral capsid and can assemble virus-like particles (VLPs) that are associated with immune responses.10−12 Recently, a prospective study indicated that the detection HPV L1 could be used to discriminate efficiently cervical precancer from transient HPV infection.13 Therefore, the presence of L1 could be used as a reliable biomarker and would be used in future clinical diagnosis and treatment.14 However, efficient, cost-effective, easily handled, and widely applicable methods or agents are short in supply currently for HPV L1 detection, of which there is an ever-increasing demand to be developed. Well-defined hybrid self-assembly, especially those constructed from bio- and inorganic compositions, becomes more popular and useful in integrating the function of each component and beyond.15,16 Construction of hybrid materials based on peptide and inorganic components has proven to be an effective strategy to obtain biofunctions. As a class of metal− © 2015 American Chemical Society
oxygen−anion clusters, polyoxometalates (POMs) have already shown versatile structural and biofunctional applications in the past decade.17−20 New progress in fabricating well-defined selfassemblies by using POMs as construction units has been reported, especially for three-dimensional vesicles15,21−23 and nonospheres24−26 with multifunctions. However, the exact binding model and interactions between these building blocks are still not robust and specific enough, needing to be further clarified and improved. The high-affinity interactions of basic peptides, called also nuclear localization signals (NLS), from the capsid proteins (L1 and L2) of HPV-16 and HPV-18 with negatively charged cell surface receptor heparan/heparin have been reported, which paved an important pathway to understanding the infection of virus particles to host cells.27,28 In the present study a biLindqvist-type POM linked by europium, Na9[EuW10O36]· 32H2O(EuW10), is used to assemble with these arginine/ lysine-rich peptides from two high-risk subtypes of HPV major capsid protein, HPV-18 and -16 L1 (Table 1). The well-defined nanoparticle assembled from EuW10 and peptide is illustrated as model, which opens a way to fabricate multifunctional fluorescence bioinorganic materials. Furthermore, the clarification of binding mechanism between EuW10 and the peptides at Received: January 2, 2015 Revised: March 26, 2015 Published: March 26, 2015 8321
DOI: 10.1021/acs.jpcc.5b00032 J. Phys. Chem. C 2015, 119, 8321−8328
Article
The Journal of Physical Chemistry C
transmission electron microscopy images were taken by using a JEOL JEM-2100F instrument. For the measurements by scanning electron microscope, the above solution was placed onto a silicon substrate. Before sputtering a thin gold film on sample the excess fluid should be removed to dry. The SEM images were measured by an S-4500 instrument. A spectrometer of VERTEX 80 V IR (Bruker) was used to record the corresponding IR spectrum. For that, the sample was prepared from a solution containing 30.0 μM POM and 30.0 μM in forming the colloidal spheres, which were coated on a KBr plate, and after the aqueous solution was evaporated, an IR spectrum was recorded. An X-ray diffractometer from Rigaku (Japan) was used to obtain the diffraction patterns, with graphite monochromatized Cu Kα radiation (k = 1.542 Å), and the step size was set at 0.01°. Each measurement was repeated three times, and the representative displayed is one of them. Fluorescence Spectral Methods. A fluorescence spectrophotometer (SHIMADZU RF-5301) was used for fluorescence spectral measurement, and each was repeated three times. To get a reliable spectrum, the excitation lamp should be kept on for 0.5 h and the samples should be aged for 2 h before the spectral recording. Quartz cuvettes (1.0 × 1.0 cm) were used to put sample, and 265 nm was fixed as the excitation wavelength for the luminescence record of EuW10. All the fluorescence measurements were performed at 25 °C in a buffer solution of MES−NaOH (10 mM, pH 6.0). Time-resolved fluorescence spectra were recorded by using an analytical instrument of FLS980 (Edinburgh). Each sample was repeated three times to obtain more reliable lifetimes. The sample was prepared in the buffer solution of MES−NaOH (10 mM, pH 6.0) at required concentration. To obtain the fluorescence decay curve, the excitation wavelength was
Table 1. Sequence and Origination of the HPV L1 Peptides peptide
amino acid sequence
position in protein
HPV18Ctb HPV16Ctb
SSKPAKRVRVRARK SSTSTTAKRKKRKL
555−568 of HPV18 L1 492−505 of HPV16 L1
the molecular level would promote understanding of the biological activity of inorganic chemicals to promote design of multifunctional targeting agents for future virus treatment.
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EXPERIMENTAL METHODS Materials. The peptides of HPV18Ctb and HPV16Ctb were purchased from Shanghai Apeptide Co., Ltd. (Shanghai, China); their purity is 99.11% as confirmed by HPLC. As illustrated in Table 1, HPV18Ctb is 14 amino acids in Cterminal (from 555 to 568) of HPV-18 L1, while HPV16Ctb is from 492 to 505 of HPV-16 L1. The procedures to synthesize and characterize the Na9[EuW10O36]·32H2O (EuW10) are similar to previously reported procedures.29 All the chemicals were used as obtained without further treatment. Distilled water (ρ = 18.2 MΩ cm, 25 °C) was obtained from a Millipore Milli-Q water purification system. The stock solution of EuW10 was prepared for 2 mM in aqueous solution and placed under dark conditions (4 °C). Then, it was diluted to the required concentrations according to different experiments. Fabrication and Characterization of the Nanospheres. The fabrication of the nanospheres was similar to previous fabrication,26 which could be summarized as follows. 2 mM HPV18Ctb aqueous solution (7.5 μL) was added to 492.5 μL of a 30.5 μM EuW10 solution with stirring at 25 °C. After aging (6 h), a drop of solution was put onto a holey carbon grid for transmission electron microscopy measurements. Before drying under vacuum, the excess liquid should be removed. The
Figure 1. Hybrid nanospheres obtained by mixing HPV18Ctb (30.0 μM) and EuW10 (30.0 μM) in the pure water (pH 6.5). (A) SEM image, at a scale bar of 1 μm. (B) TEM image, at a scale bar of 200 nm. (C) TEM image, at a scale bar of 100 nm; inset in (C) is the locally amplified TEM image. (D) FT-IR spectra measured for the hybrid nanospheres and the two constructed materials. 8322
DOI: 10.1021/acs.jpcc.5b00032 J. Phys. Chem. C 2015, 119, 8321−8328
Article
The Journal of Physical Chemistry C
Figure 2. Fluorescence spectra of EuW10 (30 μM) in MES−NaOH buffer solutions (10 mM, pH 6.0) upon the titration of (A) HPV18Ctb and (B) HPV16Ctb. The corresponding (C) band intensities at 591 nm and (D) intensity ratios of 591 to 618 nm (I591/I618) along the gradually addition of peptide.
presaturation.30 Samples for NMR measurements were dissolved in an aqueous solution containing 10% D2O. The concentration of peptide was 1.2 mM, and those of EuW10 were fixed at 12, 24, 60, and 120 μM as perturbations; the sample volume in the Shigemi tube for measurement was 600 μL.30
selected as 265 nm and the intensity at 591 nm was collected. The fluorescence lifetime and the percentage contribution of each component were calculated by exponentially fitting the fluorescence decay curve with the software included in the FLS980 Edinburgh Instruments. Measurements of Isothermal Titration Calorimetry. A MicroCal ITC200 (GE) was used to measure the ITC curves at several fixed temperatures, and the procedure was modified based on a previous report.28 All the experiments were repeated, and the displayed curve was a representative other than the averaged one. Either peptide or EuW10 was prepared in the buffer solution of MES-NaOH (10 mM, pH = 6.0) in a concentration of 450 and 50 μM, respectively. For that, each 0.7 μL solution of peptide was injected into a calorimeter cell (Vcell = 200 μL) containing a solution of EuW10, and the record was performed under stirring (1000 rpm) with an interval of 180 s. Thermodynamic parameters were obtained by fitting the data to a suitable binding model. A program of Origin 7.0 updated by the ITC-200 instrument was used for the nonlinear least-squares fitting. Measurement of ζ-Potential. A Zetasizer Nano ZS apparatus (Malvern) was used to perform the ζ-potential measurements. A backscattering detection at a constant scattering angle of 173°, equipped with a He−Ne laser (k = 632.8 nm), was used to perform the measurement at 25 °C. In addition, the capillary cells of DTS 1060 from the same company were used for the experiments, and the software loading with instrument was utilized to process the obtained data. Measurements of 1H NMR Spectroscopy. The 1H NMR spectra were measured on a Bruker spectrometer (Avance 600 MHz) at T = 25 °C. All spectra were recorded by standard pulse sequences with suppression of the water resonance by
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RESULTS AND DISCUSSION Fabrication and Characterization of the Hybrid Nanospheres. The assembly of POM and peptide was performed by adding EuW10 solution to that of HPV18Ctb under stirring. To fully finalize the self-assembly, the solution was aged for 6 h after mixing. After that, the shape and size of the produced nanoparticles were characterized by using either SEM or TEM, respectively. Spherical nanoparticles with diameters between 250 and 350 nm were clearly observed in the SEM image of Figure 1A. The images of TEM in Figure 1B also illustrate the existence of regular particles with an average size of ∼300 nm. The enlarged TEM showed that the nanospheres contained a large number of dark spots (being attributed to the clusters of POM), in surrounding with the gray regions might be the peptide (Figure 1C). Then, the energy dispersive X-ray spectroscopy (EDX) connected with TEM (Figure S1A) and SEM (Figure S1B) indicated the appearance of tungsten, europium, oxygen, and nitrogen as well as carbon inside a particle, illustrating the particle was indeed constructed by both peptides and EuW10. In addition, Figure S2 shows the X-ray diffraction (XRD) results both of POM and the constructed nanospheres, illustrating EuW10 itself had well-defined structure. In addition, a new reflection was observed for the nanospheres at 2θ = 11.35° accompanying the decrease of POM reflection, confirming the ordered structures in nanospheres (Figure S2). 8323
DOI: 10.1021/acs.jpcc.5b00032 J. Phys. Chem. C 2015, 119, 8321−8328
Article
The Journal of Physical Chemistry C
I591/I618 was calculated to be 0.58, whereas for the mixed solution it increased to 1.72 and 1.61 at the final concentration for the two peptides, respectively. As higher surface charge generally induced stronger electrostatic interaction, it was reasonable to see the one of HPV18Ctb with more surface charges induced stronger binding with EuW10. In addition, the more dispersive surface charges of HPV18Ctb might be matching better in size and/or distance of the binding sites of EuW10, which consequently pushed higher binding affinity and finally a higher luminescence enhancement. Therefore, in following, the intrinsic nature of the size/distance matching of the binding sites between EuW10 and peptides would be explored by using other techniques. Lifetime Changes of EuW10 upon Binding with Peptides. For deep insight into the origination of luminescence enhancement of EuW10 in binding with peptides, the lifetimes of the EuW10 before and after binding with peptide were measured by the time-resolved fluorescence spectrum. Figure 3 illustrates decay curves of the fluorescence
In addition, FT-IR spectra of POM@Pep, POM, and HPV18Ctb were measured and are shown in Figure 1D. Bands between 1000 and 650 cm−1, attributed to the vibrations of EuW10,30 are observed easily, indicating EuW10 kept its structure well in the assembly. In addition, bands appeared at 1530, 1200, and 1135 cm−1 are attributed to the stretching models of amide group from HPV18Ctb, illustrating the incorporation of peptide in the assembled nanospheres (Figure 1D). Furthermore, the enlarged IR spectra indicate that in the assembly of peptide and EuW10 band shifts for the stretching models of W = Od, W−Ob−W, and W−Oc−W were observed,31 illustrating powerful interactions of HPV18Ctb with EuW10 (Figure S3). Recently, a combined assembly of a Keggin-type POM with a cationic dipeptide (CDP) in forming a new hybrid material was reported by Li et al.25 The assembly of a short peptide from Aβ and a Wells−Dawson-type POM was demonstrated by another group26 for the hybrid nanospheres in aqueous solution, proposed to be dominated by the electrostatic interactions. Being positively charged at neutral condition, it is expected that the peptide of HPV18Ctb can behave as a cationic surfactant to form nanoclusters with EuW10 through strong charge binding between them. Subsequently, such an initial assembly can further stack and form larger networks via electrostatic interaction, hydrogen bonding, and/or other weak interactions, which will be assayed in detail in following by ITC. An increase of HPV18Ctb to POM from 0:1 to 1:1 would obviously enlarge the particle size (Figure S4A−D) and further increase of peptide to 2:1 induce cross-linking and/or aggregation of the particles (Figure S4E,F). In addition, the TEM images for the assembly of EuW10 and an identical peptide from another high-risk subtype of HPV16 L1, HPV16Ctb, showed similar spontaneous assembly of nanoparticles, although the size distributions of particles are relatively broader in range (Figure S5). It illustrated the universality to form the nanospheres between EuW10 and the Arg/Lys-rich peptide from HPV capsid protein, which could be extended to other subtypes and/or other segment of protein to fabricate hybrid nanoparticles with different functions. Luminescence Enhancement of EuW10 Induced by Peptides Binding. In order to clarify the direct binding between POM and peptide at molecular level in detail, we then investigated the interaction of EuW10 and the Arg/Lys-rich peptide of HPV L1, HPV18Ctb and HPV16Ctb, by using fluorescence titration spectra (Figure 2). Under slightly acidic buffer conditions (pH = 6.0), EuW10 displayed two emission bands at 591 and 618 nm, which were assigned to the transition of 5D0 → 7F1 (591 nm) and the 5D0 → 7F2 transition (618 nm) of Eu3+, respectively.32 During the titration process of peptide to POM, the luminescence of EuW10 was enhanced and reached a maximum when the concentration of peptides was reached to ∼86 μM for HPV18Ctb, but being saturated at a much higher concentration of ∼120 μM for HPV16Ctb. Such differences suggest the affinity gap between HPV18Ctb and HPV16Ctb in binding with EuW10 in solutions. The much higher luminescence enhancement for HPV18Ctb than that for HPV16Ctb (Figure 2C, 35.8-fold vs 20.2-fold) suggests a stronger binding ability between EuW10 and HPV18Ctb than HPV16Ctb. In addition, the microenvironment of Eu3+ in binding with HPV18Ctb and HPV16Ctb was further assayed by using the intensity ratio of I591/I618 (Figure 2D); dramatic changes clearly showed upon addition of a little amount of peptides (
