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Jan 30, 2018 - VLP-based vaccines are efficacious in preventing infections and precancerous lesions caused by HPV vaccine-related types.28. However ...
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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 6137−6145

Hybrid Assembly toward Enhanced Thermal Stability of Virus-like Particles and Antibacterial Activity of Polyoxometalates Ding-Yi Fu,† Simin Zhang,† Zhiyu Qu,† Xianghui Yu,‡ Yuqing Wu,*,† and Lixin Wu*,† †

State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, and ‡State Engineering Laboratory of AIDS Vaccine, Jilin University, No. 2699, Qianjin Street, Changchun 130012, China S Supporting Information *

ABSTRACT: In an effort to improve both the stability of virus-like particles (VLPs) and the medical activity of polyoxometalates (POMs), a new hybrid assembly system between human papillomavirus (HPV) capsid protein L1 and a europium-containing POM (EuW10) has been constructed, for the first time, via the electrostatic interactions between them. The co-assembly of EuW10 and HPV 16 L1-pentamer (L1-p) in buffer solution resulted in the encapsulation of POMs in the cavity of VLPs, which was further confirmed by cesium chloride (CsCl) gradient ultracentrifugation, SDS-PAGE, dynamic light scattering, and transmission electron microscopy, whereas the post-assembly of EuW10 with the as-prepared VLPs leads to the adsorption of POMs only on the external surface of particles, and both cases improved the thermal and storage stabilities of VLPs obviously. Particularly, the encapsulation of POMs in VLPs largely improved the antibacterial activity of EuW10, and thereby, the present study will be significant for both the stability improvement of protein vaccines and the development of POM medicine. KEYWORDS: hybrid assembly, virus-like particles, polyoxometalates, stability, antibacterial activity

1. INTRODUCTION Polyoxometalates (POMs) are kinds of negatively charged clusters of transition metals (mainly tungsten, molybdenum, and vanadium) with oxygen, which have shown potential applications in catalysis,1 material science,2 medicine,3 bio- and nanotechnology,4 and macromolecular crystallography.5 Because of their specific three-dimensional structure and high negative charges, POMs selectively bind to the positive regions of proteins.5 Recent reports reveal that POMs are effective in the treatment of cancer,6 diabetes,7 and infections associated with bacteria8 and viruses.9 Particularly, POMs have illustrated excellent potentials for the treatments of many types of cancers including pancreatic cancer,10 leukemia,11 hepatocellular carcinoma,12 colon carcinoma,13 and ovarian14 and gastric cancer.15 However, unfortunately, up to now, POMs have not been actually used as medicine, mainly because of the fact that most POMs are not sufficiently stable at physiological pH and easy to be degraded into a mixture of inorganic products.16 One of the best ways to overcome the bottleneck is encapsulating them into biosystems such as starch,17 lipid,18 or liposome19 as well as particular nanospheres,20 and several successful examples have been reported. Such performances created new systems as drug carriers, which have gained more promising © 2018 American Chemical Society

therapeutic applications because of the newly introduced biocompatibility, biodegradability, and physical stability of these covers. Viral capsids have attracted great attention recently in the fields of both nanotechnology and nanobiology, essentially because of their nanoscale size, uniform and symmetrical structural organization, loading capacity, easily controllable selfassembly, and modification.21 Therefore, hybrid structures of viral capsids and nanoparticles (NPs), which combine the bioactivities of viruses with the functions of NPs, are a new class of bio-nanomaterials that have shown great potentials as therapeutic and diagnostic vectors, imaging agents, and advanced nanosynthesis reactors.22,23 Since the initial study describing the capture of NPs by the self-assembly of brome mosaic virus capsid proteins,24 large amount of studies focused on constructing virus-like particles (VLPs) containing Au NPs,25 quantum dots,26 and magnetic cores27 have been reported albeit the functionalized inorganic NP cargos are very limited.24 In addition, for human papillomavirus (HPV), the L1 Received: November 9, 2017 Accepted: January 30, 2018 Published: January 30, 2018 6137

DOI: 10.1021/acsami.7b17082 ACS Appl. Mater. Interfaces 2018, 10, 6137−6145

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sample loading, all samples and solvents were first treated by filtration through a 0.22 μm filter. The sample was finally eluted by using buffer L at a flow rate of 4 mL/min and detected at a wavelength of 280 nm to determine the protein concentration. All steps for above purification were conducted at 4 °C. The target L1 protein from SEC was then identified by SDS-PAGE with Coomassie blue, which was conducted using 12% polyacrylamide gels. Prior to that, the samples were denatured by boiling at 95 °C in Laemmli buffer (12 mM Tris−HCl, 0.4% SDS, 2.88 mM mercaptoethanol, 5% glycerol, and 0.02% bromophenol blue). 2.3. Preparation of Na9[EuW10O36]·32H2O (EuW10). EuW10 was synthesized and characterized according to a published procedure.34 Briefly, 8.3 g of Na2WO4·2H2O was dissolved in 20 mL of water, and the pH of the solution was adjusted to 7.0−7.5 with CH3COOH. An aqueous solution (2 mL) containing 1.1 g of Eu(NO3)3·6H2O was added dropwise to the abovementioned solution with stirring at 80− 90 °C. Cooling the solution at room temperature yielded colorless crystals of EuW10, which were characterized by X-ray diffraction after being filtered off and dried in air. 2.4. Hybrid Assembly Monitoring of EuW10 and HPV 16 VLPs. For the in vitro hybrid assembly of L1-p and EuW10, two different protocols were used. One was the coassembly, where the final concentration of L1-p was fixed at 2 μM, after adding the stock solution of EuW10 (1 mM) to get a 10 equiv concentration (20 μM). The mixture was incubated for 3 h before dialyzing against 1 L of assembly buffer (1 M NaCl, 50 mM H2PO4−/HPO42−, pH = 5.4), which was performed at 4 °C for 24 h to get full assembly. In this case, the coassembly product between EuW10 and HPV 16 L1-p was defined as EuW10@VLPs. The second protocol was termed post-assembly, where the HPV 16 VLPs were formed first by dialyzing 2 μM L1-p alone in the assembly buffer for 24 h (4 °C), then 10 equiv of EuW10 was added, and the solution was kept at 4 °C for another 24 h to get full assembly. The postassembly product between EuW10 and HPV 16 VLPs, in this case, was defined as VLPs@EuW10. 2.5. Dynamic Light Scattering Measurement. The dynamic light scattering (DLS) measurement was used to monitor the size distribution of particles in solution. The concentration of HPV L1-p was 2 μM in buffer L, and the concentration of EuW10 was 4 μM in assembly buffer solution. First, the big dust particles in sample were removed through a 0.22 μm filter unit, then an analyzer (Malvern Zetasizer Nano-ZS 90) for particle size with a 4 mL cuvette and the Dispersion Technology Software (DTS, V6.01) were used to collect and analyze the data. 2.6. Transmission Electron Microscopy Measurements and Energy-Dispersive X-ray Analysis. For the transmission electron microscopy (TEM) measurement, sample (5 μL) was spotted on the carbon- and formvar-coated copper grids and kept there for 2 min before drying completely. Then, it was negatively stained with 2% phosphotungstic acid (5 μL) for 2 min. The excess amount of staining solution was carefully removed by filter paper and air-dried again for TEM measurement. The stained samples were examined using a H7650 transmission electron microscope (Hitachi Japan), with an accelerating voltage of 80 kV. A magnification of 40 000× was used because pictures at this magnification were observed to include a sufficient number of particles for statistical validity. For comparison, TEM measurement was also performed in parallel on the samples without negative staining. The sample was just spotted on the carbon- and formvar-coated copper grids and kept there for 2 min before drying. The objective to do that was to show the location of EuW10 in the hybrid assembly precisely. In this case, the samples were examined using a JEM-2200FS transmission electron microscope (JEOL Ltd. Japan), with an accelerating voltage of 200 kV. For this examination, a magnification of 100 000× was used to observe the particles more clearly. For electron-dispersive X-ray analysis, individual particles were collected by the energy-dispersive X-ray (EDX) system (INCA-Oxford) attached with the TEM. 2.7. Purification of the Hybrid Assembly by CsCl Density Gradient Ultracentrifugation. Ultracentrifugation using CsCl density gradient was used to purify the VLPs based on its buoyant specific density.35 A layer aqueous phase onto the CsCl gradient was

VLP-based vaccines are efficacious in preventing infections and precancerous lesions caused by HPV vaccine-related types.28 However, a vaccine with limited valency may possibly permit less common subtypes to proliferate, potentially allowing persistence of infection and progression of precancerous lesions.29 Being type-specific and expensive and requiring cold storage for transportation, these vaccines have not been widely used in developing countries.30 Therefore, there is an urgent need to develop new ways of improving the stability and lowering the cost of VLPs. The use of HPV VLPs as a vaccination platform is already a medical evidence; however, studies on VLPs as drug delivery vectors are in their infancy, although VLPs feature cell-specific targeting, efficient cell entry, lack of endosomal sequestering, multivalency, and biocompatibility for drug delivery.31 Therefore, in the present study, we will perform the hybrid assembly of the capsid protein of HPV and POMs, aiming to improve both the stability of VLPs and the medical activity of POMs. The VLP has a T = 7 icosahedral lattice structure, which is composed of 72 L1-pentamers (L1-p), and each L1-p is composed of five L1. As Eu-containing POM (EuW10) has a negatively charged surface, it will be possible to construct a hybrid assembly with HPV 16 L1-p via the electrostatic interactions. The binding of EuW10 either inside the cavity or at the external surface of HPV VLPs indeed largely improved the functions of the constitution dually: (1) the antibacterial ability of EuW10 was improved after being encapsulated in HPV 16 VLPs and (2) the thermal and storage stabilities of VLPs were largely improved after the participation of EuW10. It is a new approach to improve the stability of HPV capsid protein and would be significant to improve the protein vaccine. Therefore, the binding of EuW10 will be significant for both the improvement of protein vaccines and the development of POM medicine.

2. MATERIALS AND METHODS 2.1. Reagents and Materials. Tryptone and yeast extract were from OXOID (OXOID Ltd, Basingstoke, UK). Isopropyl-β-Dthiogalactopyranoside (IPTG), ethylenediaminetetraacetic acid (EDTA), DL-dithiothreitol (DTT), sodium dodecyl sulfonate (SDS), Tris, Coomassie blue G-250, and cesium chloride (CsCl) were bought from GENView (GENView Scientific Inc., USA). Sodium chloride (NaCl), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were purchased from Beijing Chemical Factory. All chemicals are of analytical reagent grade and were used without further purification. Distilled water was obtained from a Millipore Milli-Q water purification system. Sterile filtration membranes (0.22 and 0.45 μm), used for filtering large particles, were bought from Millipore. 2.2. Protein Expression and Purification. The HPV 16 L1 coding sequences, lacking four amino acids at the N-terminus (residues 1−4) and 29 amino acids at the C-terminus (residues 477−495), were employed for better expression. The Escherichia coli strain BL21 carrying pGEX-6p-1 was grown at 37 °C to get an OD600 of 0.8 and then induced with 1 mM IPTG at 28 °C overnight. Actually, the expression and purification of protein were carried out essentially as described previously.32,33 Briefly, cells from a 1 L culture were resuspended in buffer L (50 mM Tris−HCl, 0.2 M NaCl, 1 mM DTT, 1 mM EDTA, pH = 8.0) and then were lysed by sonification for 30 min. The lysate was separated by centrifugation at 24 041g for 30 min. The obtained GST-L1 in the supernatant was first purified using the glutathione affinity column. Then, the column was washed with 20 bed volumes of buffer L to remove the contaminants. Then, HPV 16 L1 was cleaved from the GST fusion by adding 200 U PreScission Protease into the column and maintaining at 4 °C for 14−16 h. After digestion, the elution was collected and further purified by using sizeexclusion chromatography (SEC, Superdex 200, 26/60). Before 6138

DOI: 10.1021/acsami.7b17082 ACS Appl. Mater. Interfaces 2018, 10, 6137−6145

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Figure 1. (A) Photographs of HPV 16 VLPs and EuW10@VLPs after CsCl gradient ultracentrifugation in tubes. (B) SDS-PAGE of the corresponding components extracted from the positions of F1/F1′, F2/F2′, and F3/F3′. (C) DLS plots of EuW10@VLPs (top) and HPV 16 VLPs only (bottom) after CsCl gradient centrifugation, extracted from the positions of F1/F1′, F2/F2′, and F3/F3′. prepared as follows: CsCl samples with densities of 1.5 g/mL × 1 mL, 1.35 g/mL × 3 mL, and 1.25 g/mL × 4 mL were prepared and then consequently loaded into tubes; finally, the sample was added on the top layer. The concentration of VLPs was 4 μM in assembly buffer (1 M NaCl, 50 mM H2PO4−/HPO42−, pH = 5.4). Tubes containing samples were spun for 3 h at 226 000g, using a Beckman Optima XPN80 ultracentrifuge. To collect the target bands, either (1) puncturing the tubes with a 5 mL syringe and a needle below the band of interest and slowly drawing the sample into the syringe or (2) slowly removing the liquid using a pipettor from the top of the tube until the band of interest is reached was performed. After that, the potential HPV 16 VLPs as well as the counterparts were loaded into a tube for 24 h of dialysis to remove CsCl. 2.8. UV Cloud Point Temperature Determination and DLS Measurement of the Stability of Protein. The thermal stabilities of HPV 16 VLPs and the assemblies of them with EuW10 were evaluated with a UV cloud point temperature (UV CP-Temp) assay, which was carried out by using a UV-2450 spectrophotometer (Shimadzu, Japan) equipped with a DC-0506 low-constant temp bath temperature control system (Hangping, China). The UV CP-Temp determination was performed by observing the ultraviolet absorption signals at 350 nm for the solution of HPV 16 L1. The concentration of VLPs was diluted to 0.8 μM from 4 μM in assembly buffer (1 M NaCl, 50 mM H2PO4−/HPO42−, pH = 5.4). All samples were measured at a scan rate of 0.8 °C/min at a scan temperature ranging from 37 to 77 °C to monitor the thermal denaturation and/or aggregation of HPV 16 L1 in versatile VLP assemblies in solution. In addition, DLS measurements (Malvern Zetasizer Nano-ZS 90) of HPV 16 VLPs, EuW10@VLPs, and VLPs@EuW10 in solution were performed to determine the hydrodynamic size distribution of the particles to monitor the storage stability of them over a period of 28 days, in an interval of 1 day at 4 °C. 2.9. Effect of EuW10 and EuW10@VLPs on E. coli Growth. E. coli bacteria seeds from the glycerine stock tube were inoculated and cultured in 5 mL of Luria−Bertani (LB) medium (consisting of 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl), they were incubated at 37 °C with shaking overnight, and then 10 μL of bacterial culture was transferred to 4× 5 mL of fresh LB medium for further experiments. The EuW10, VLPs@EuW10, and EuW10@VLPs in

different molar ratios were added into the medium. For VLPs@ EuW10 and EuW10@VLPs, the fraction ratios of EuW10/VLPs were different, but the concentrations of VLPs in each sample were the same. Meanwhile, the HPV 16 VLPs with the same concentration as EuW10@VLPs were added as a control sample. The original concentration of particles was 8 μM in assembly buffer. The cultures were kept in an incubator at 37 °C for 24 h, and 200 μL of each sample was taken out every 2 h and was monitored by a BioPhotometer (Eppendorf AG 22331, Hamburg, Germany) to observe the OD600 value. In addition, after 24 h, the sample was prepared by transferring a drop of solution onto a silicon wafer and airdried. Scanning electron microscopy (SEM) image was then taken for each sample using a JEOL JSM 6700F field emission equipment with a primary electron energy of 3 kV, and the sample was sputtered with a layer of Pt prior to imaging to improve the conductivity.

3. RESULTS AND DISCUSSION 3.1. Encapsulation of EuW10 in HPV 16 VLPs. The fast protein liquid chromatography elution profile showed an intense peak at ∼265 kDa for HPV 16 L1, which is an indication of the L1-p, whereas a weak peak corresponding to ∼53 kDa was attributed to the L1 monomer (L1-m) (Figure S1A), which is well consistent with our previous reports.33,36,37 The synchronous SDS-PAGE test (Figure S1A, top) identified the components in the intense peak, where the band at ∼53 kDa belonged to one L1-m. The size and distributions were then determined by DLS and TEM. The column bar of the hydrodynamic diameter distributions for L1-p (Figure S1B, black line) indicated an averaged central size of 12.48 nm. In addition, the determination for HPV 16 VLPs by DLS was performed after controlled assembly of L1-p in buffer solution, which indicated an average diameter of 55.29 nm (Figure S1B, red line), which is typical for the full-sized HPV VLPs.32,33 Then, the negatively stained TEM analysis confirmed that the pentamer appeared as five-pointed stars, with an average size of ∼12 nm (Figure S2A). They underwent a controlled assembly 6139

DOI: 10.1021/acsami.7b17082 ACS Appl. Mater. Interfaces 2018, 10, 6137−6145

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Figure 2. (A) TEM image of EuW10@VLPs after CsCl gradient ultracentrifugation in tubes, extracted from the position of F2. (B) Size distributions of EuW10@VLPs, statistic from 200 particles from TEM images.

to form HPV 16 VLPs with a statistic central size of ∼55 nm (Figure S2B). These results confirmed that the purified HPV 16 L1 protein was able to assemble into HPV 16 VLPs easily, being ready for the subsequent hybrid assembly with EuW10. The previous study38 suggested that pH 5.4 of assembly buffer solution leads to the formation of stable capsidlike assemblies, so we tried this condition for the encapsulation of EuW10 by HPV 16 VLPs. After assembly monitoring, both the finalized HPV 16 VLPs (used as a control group) and the potential coassembly of EuW10 and HPV 16 VLPs (defined as EuW10@VLPs here) were concentrated by using CsCl gradient ultracentrifugation, and the results are shown in Figure 1A. A visible narrow light blue band was observed in the gradient centrifugation tubes, the position of which is well consistent with a previous report for the density of CsCl fraction containing the HPV 16 VLPs,39 making it our possible target protein. Then, the band-related regions were divided into three parts, which were labeled as F1/F1′−F3/F3′ for two tubes according to the position of the candidate protein band. Then, each was extracted out with a syringe for further characterization and confirmation of the involved component. The SDS-PAGE assay was performed first on the extracted sample, which confirmed that only fractions F2 and F2′ contained the target HPV 16 L1 protein (Figure 1B), whereas no sign of it was observed in the other two parts. Moreover, the DLS results showed a similar particle size at ∼55 nm for the fractions of F2 and F2′ (Figure 1C), which is the typical one of HPV 16 VLPs in solution.38,40 However, almost no signal was observed in the DLS test for the fractions F1/F1′ or F3/F3′. This confirmed that the HPV 16 VLPs were concentrated only in a narrow region around F2/F2′ in the tubes. Of note, the light blue band for HPV 16 VLPs in the tube was relatively wider in the presence of EuW10 (F2′) in comparison to that for HPV 16 VLPs alone (F2), although both were presented in a horizon level. This suggested that EuW10@VLPs had a more uniform density than the corresponding empty HPV 16 VLPs. Up to this stage, taken all results together, it suggested that, at least, the incorporation of EuW10 to L1-p solution did not retard the assembly of HPV 16 VLPs. However, in regard to whether EuW10 was assembled together with HPV 16 VLPs and has it been encapsulated inside the HPV 16 VLPs or just bound at the particle surface, further experiments should be conducted. 3.2. Characterization of the Coassembly of EuW10@ VLPs. To further determine the component and morphology features of fraction F2′, TEM was used for characterization. Albeit the sample was not negatively stained with phospho-

tungstic acid, black dots were still observed clearly in the TEM image (Figure 2A). Therefore, this suggested that EuW10 might be involved in the particles to give a nontransparent image at the present condition. Furthermore, the corresponding EDX analysis based on the TEM image detected two kinds of elements, tungsten and europium, within one particle (Figure S3), proving that EuW10 was indeed involved in the fraction F2′, together with L1. The copper element was from the copper grids rather than the contamination. Furthermore, the morphology of the particles was observed to berelatively uniform, and the distributions of particle sizes were evaluated to focus on ∼34 nm (Figure 2B). Of note, such a size was obviously smaller than the outer diameter of HPV 16 VLPs (∼55 nm) but fitted well with the inner diameter of the VLP cavity as reported previously.41 Therefore, we concluded that the TEM images observed here were essentially originated from EuW10, being confined within the cavity of HPV 16 VLPs. In the TEM images of the coassembly (EuW10@VLPs), we did not see POMs being attracted on the external surface under the current concentration of 20 μM EuW10. However, if we continue to increase the concentration to much excessive, it might be possible for POMs to be partially adsorbed on the external surface. In the following, the negatively stained TEM image was obtained on the same batch of sample to perform further morphology characterization on it. The particles were also observed to be uniform, and the actual sizes were evaluated to be ∼55 nm, which is similar to that reported in the literature for the full-sized HPV VLPs38 and those we tested for empty HPV 16 VLPs (Figure S2B). However, large differences were also observed clearly between Figures 3A and S2B. The particles for EuW10@VLPs appeared as a dark center covered by white protein shells, which is very similar to that reported in the

Figure 3. Morphologies of (A) HPV 16 EuW10@VLPs and (B) VLPs@EuW10 with negative staining. 6140

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Figure 4. (A) TEM image of HPV 16 VLPs-EuW10 without negative staining, which was prepared by the postassembly process by mixing EuW10 with the as-formed HPV 16 VLPs; (B) DLS plots of the corresponding particles; and (C) corresponding EDX spectroscopy of the representative particle.

Figure 5. UV cloud point measurements with temperature ramping (λex = 350 nm) of (A) HPV 16 VLPs (0.75 μM, black), EuW10@VLPs (20 μM, red), and VLPs@EuW10 (20 μM, blue) and (B) the coassembled HPV 16 VLPs (0.75 μM) constructed from HPV 16 L1-p and EuW10 in different concentrations (10−30 μM), showing the thermal stability differences between them. ΔTm is an average value of three repeat experiments, and each displayed plot is only a representative one among three repeated measurements.

literature for AuNPs encapsulated in simian virus 40 capsids.42 The basic residues of protein provide positive charges to the interior surface of the cavity for the encapsulation of negatively charged species.43 Therefore, this confirmed that EuW10 was indeed encapsulated into the cavity of HPV 16 VLPs, forming EuW10@VLPs under the concentration of 20 μM EuW10 in the present study. That is, the encapsulation of EuW10 in HPV 16 VLPs was successfully performed via the coassembly. Meanwhile, we tried to use the postassembly approach for the binding of EuW10 and HPV 16 VLPs, and the product was termed VLPs@EuW10 to distinguish from that of coassembly, EuW10@VLPs. For that, empty HPV 16 VLPs were formed first, and then 10 equiv of EuW10 was added to either the dialysis bag containing HPV 16 VLPs or the buffer solution outside the dialysis bag. For the first case, the product was extracted and put forward for the TEM measurement after further dialysis for 24 h. After staining with 2% phosphotungstic acid, the normal morphology and size (∼53 nm) of HPV 16 VLPs were observed clearly (Figure 3B), although they looked relatively darker, especially in the region among pentamers. For the second case, the same concentration of EuW10 (20 μM) was presented in the assembly buffer to exclude the possibility of EuW10 preaggregation in the dialysis bag. The TEM image showed results (data not shown) identical to those obtained in the first case, confirming that the state of EuW10 even in the dialysis bag was dispersive. Particularly, without negative staining, both of them showed clear TEM images (Figure 4A) with an average size of ∼53 nm, which was close to those

evaluated by DLS (Figure 4B). In addition, TEM image-based EDX analysis on the particles indicated the appearance of both tungsten and europium (Figure 4C). Therefore, we concluded that EuW10 was combined with HPV 16 VLPs through such a postassembly monitoring. However, considering the size difference revealed between Figures 2 and 4, we preferred a conclusion that EuW10 was assembled at the external surface other than the cavity of HPV 16 VLPs in this way. As shown in Scheme S1, the dispersive EuW10 in aqueous solution appears as two Lindqvist-type POMs linked by an europium ion with a scale of 1 to 2 nm, according to the size of the EuW10 clusters determined by crystal structure parameters.38 Meanwhile, L1-p consists of five monomers, displaying a typical pentamer similar to “donuts”. On the basis of the crystal structure of HPV 16 L1p (PDB: 2R5H), the DE loop was located on the inner channel ridge of the L1-p. In theory, the diameter of the pentamer channel is ∼1.8 nm for HPV 16 L1.32 Therefore, it was just on the edge size for EuW10 to access into the cavity of HPV 16 VLPs, and it would be little bit difficult to be encapsulated in the cavity of HPV 16 VLPs. 3.3. Encapsulation of EuW10 Improved the Thermal and Storage Stabilities of HPV 16 VLPs. The question then is whether an increase in stability could be achieved through the incorporation of a solid template into the cavity of HPV 16 VLPs. Furthermore, the thermal stabilities of HPV 16 VLPs, EuW10@VLPs, and VLPs@EuW10 were assayed by using UV cloud point measurements. The experimental traces and the corresponding transition temperature (Tm, Figure 5A) revealed 6141

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ACS Applied Materials & Interfaces clear differences of the thermal stabilities between them. A reasonable Tm value of 68.83 °C was obtained for HPV 16 VLPs, which is close to that reported previously.44 However, a higher value of Tm was shown at 70.30 and 69.53 °C, respectively, for EuW10@VLPs and VLPs@EuW10. These difference values indicated that an anionic NP binding is sufficient to promote the thermal stability of HPV 16 VLPs; consequently, the thermal stabilities of both EuW10@VLPs and VLPs@EuW10 were improved obviously in comparison to that of pure HPV 16 VLPs. In a previous report, the interaction between EuW10 and HPV 16 L1-p in MES buffer solution was directly confirmed by using isothermal titration calorimetry, where the fitting of the isotherm plot revealed ΔH = −9.84 ± 1.83 kcal mol−1, ΔG = −6.42 ± 1.83 kcal mol−1, n = 2.33 ± 0.33, and a binding constant of Kb = (1.09 ± 0.45) × 105 M−1.45 These results, together with the binding between EuW10 and Arg-/Lys-rich cationic peptides from HPV capsid proteins, revealed that the electrostatic interaction between them played a crucial role. Such a conclusion was supported by other report, where nucleic acids such as tRNA or simply polyanions such as dextran sulfate serve to provide a reservoir of negative charges to attract coat protein subunits to their surface through electrostatic interactions with amino terminal tails for driving the assembly.46 Therefore, the charge neutralization was supposed to be necessary to stabilize the HPV 16 VLPs. In addition, the thermal stability of EuW10@VLPs was further optimized by changing the concentration of EuW10 when the concentration of L1-p was fixed in the assembly. It was found that electrostatically driven EuW10@VLP association occurred regardless of the concentration of POMs, as all cases improved Tm obviously (Figure 5B). Among the tested concentrations, a maximum Tm at 70.78 °C was obtained in the presence of 30 μM EuW10, resulting in a ΔTm of 1.95 ± 0.13 °C than that of HPV 16 VLPs itself. In this case, the assembly protocol yielded spherical capsids, which are very similar in both shape and size to those of wild-type HPV 16 VLPs. As a higher concentration will lead to the selfaggregation of EuW10 in assembly buffer, for 0.75 μM HPV 16 L1-p, 30 μM EuW10 was proven to be the best one to stabilize the virus particle. In addition, a recent research about the encapsulation of DNA-protected silver nanoclusters (AgNCs@dsDNA) in HPV VLPs revealed that the thermal stability of VLPs was improved. However, the maximum ΔTm was achieved only at 1.22 ± 0.16 °C (figure not shown). In comparison to that, the encapsulation of EuW10 in VLPs improved the stability of protein capsid better. In the following, DLS was used to compare the stability of either EuW10@VLPs or VLPs@EuW10 to that of the empty HPV 16 VLPs by monitoring for 28 days (Figures 6 and S4). The hydrodynamic diameters of EuW10@VLPs and VLPs@ EuW10 remained almost the same throughout the entire duration of test in 18 and 24 days, respectively, whereas a drift toward larger average diameters was observed for the empty HPV 16 VLPs after 3 days. They started to aggregate, showing a quick increase of the DLS intensity, as the appearance of eye-view aggregation DLS cannot give further report after the fifth day. For the postassembled VLPs@EuW10, the DLS intensity increased a little bit from the 15th day and then it jumped at the 18th day, illustrating that the aggregation started to form since then. The particle size became larger and larger and similar to that of the empty HPV 16 VLPs; finally, the eye-view aggregation was shown. However, the coassembled EuW10@VLPs were quite stable in solution as they

Figure 6. Storage stability of HPV 16 VLPs, EuW10@VLPs, and VLPs@EuW10 as a function of time at 4 °C, as indicated by the changes in the position of the peak of size distribution based on the DLS measurements performed over a period of 28 days in an interval of 1 day.

started to aggregate from the 24th day. Therefore, the assembly with EuW10, either in the cavity or at the external surface, greatly improved the stability of HPV 16 VLPs upon storage. Such a result opened a new way to improve the stability of HPV VLPs through the assembly with POMs and would be significant in protein vaccines. 3.4. Effect of EuW10 and EuW10@VLPs on E. coli Growth. Recent reports reveal that POMs are effective in the treatment of infections associated with bacteria, so we chose an E. coli system as a model to monitor the medical function of EuW10. The growth of E. coli was first monitored by using a time-dependent curve of OD600 over 14 h (37 °C) in the absence and presence of HPV 16 VLPs, EuW10, and different molar ratios of EuW10 to HPV 16 VLPs in EuW10@VLPs. It showed that the presence of HPV 16 VLPs lead very close OD600 value (Figure 7, red line) to that of E. coli itself as control

Figure 7. Effect of HPV 16 VLPs, EuW10, and EuW10@VLPs on E. coli growth based on the OD at 600 nm, where differences were observed between the tests (red, blue, green, and pink) and the control group (black) during 14 h.

being cultured at identical conditions (Figure 7, black line), indicating that empty HPV 16 VLPs hardly affect the growth of E. coli during the process. However, in the presence of different concentrations of EuW10 and EuW10@VLPs, the inhibition of E. coli growth was obvious albeit differences existed between them. With the increase of EuW10, the OD600 value of E. coli (Figure 7, solid line) was becoming lower and lower in comparison to that of the control of E. coli being cultured at identical conditions, suggesting that EuW10 held a concen6142

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blocked their medical usage to some extent.17 Therefore, biological drug carriers as microsphere-like virus capsids are necessary to extend the biomedical applications of POMs. Moreover, the highly charged surface of POMs should make it difficult for them to pass through the hydrophobic-prone cell membrane. It is well known that the capsid protein of virus generally display remarkable cell entry propensity being attributed the fact that originally they are responsible for cell penetration for the corresponding virus.47 They utilize endocytosis as an essential way to transport the drug into the cells and then liberate it into the cytoplasm. Therefore, it would be interesting to know if the coassembly, EuW10@VLPs, is able to improve the entry ability of EuW10 into cells and alter their biological functions efficiently. The above results indeed showed an improved antibacterial activity of EuW10 after coassembly with HPV 16 VLPs. It should be the cell entry ability of the virus capsid that promoted greatly the cell penetration of POMs. In addition, as the capsid proteins can be slowly removed from the cell thanks to the proteolytic mechanisms, they are biocompatible and would be ideal materials for drug carriers. Although HPV 16 VLPs can induce immune response, the as-mentioned advantages may compensate this deficiency greatly. Therefore, HPV 16 VLPs could be used for POM delivery in the medicinal area in future.

tration-dependent inhibition on the growth of E. coli. On the other hand, in the presence of the EuW10@VLP assembly, the OD600 values (Figure 7, dash line) were even lower in comparison to that of corresponding EuW10, suggesting that the inhibition of E. coli growth was promoted by the coassembly. At the same time, we also tried to test the antibacterial ability of VLPs@EuW10 for comparison (Figure S5). However, in this case, EuW10 at the VLP surface was easy to be released to the medium. Therefore, this showed that the presence of VLPs@EuW10 hardly affect the growth of E. coli during the test. Considering that no antibacterial activity was shown for empty HPV 16 VLPs, the largest gap observed between 30 μM EuW10 and 30 μM EuW10@VLPs should be originated from the coassembly of EuW10 and HPV 16 VLPs. It was the encapsulation of EuW10 in HPV 16 VLPs that enhanced the antibacterial ability of POMs. Figure 8 shows the SEM micrographs of E. coli that was cultured for 24 h in the presence of EuW10, empty HPV 16

4. CONCLUSIONS With an objective to improve both the stability of HPV 16 VLPs and the medical activity of POMs, new hybrid assembly systems of HPV viral capsid protein and a europium-containing POM (EuW10) have been constructed for the first time. The co-assembly of EuW10 and HPV 16 L1-p in buffer solution resulted in the encapsulation of POMs in the cavity of HPV 16 VLPs, whereas the postassembly of EuW10 with the as-prepared HPV 16 VLPs leads to the adsorption of POMs only on the particle surface, and both improved the thermal stability and storage stability of HPV 16 VLPs obviously. In addition, the encapsulation of POMs in HPV 16 VLPs largely improved the antibacterial activity of EuW10. Therefore, the present study will be significant for both the improvement of protein vaccines and the development of POM medicine.

Figure 8. SEM micrographs of E. coli exposed to (A) control; (B) HPV 16 VLPs (0.75 μM); (C) EuW10 (20 μM); and (D) VLPs-EuW10 for 24 h.

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VLPs, or EuW10@VLPs. For the control group in Figure 8A, essentially bacillary form of the bacterial cells was observed, indicating their well growth at present conditions. However, their exposure to either EuW10 (Figure 8C) or EuW10@VLPs (Figure 8D) gave rise to a morphological change from the bacillary forms to shortened bars or even balls; particularly, in the presence of EuW10@VLPs, the case was worse where almost nothing was observed. The difference of the morphological change between EuW10 and the coassembly should be attributed to the enhanced inhibition by EuW10@ VLPs on E. coli growth. The surface charges of POMs strongly affected their biological functions. The highly negative charge of them often stimulates bacterial morphology changes from the bacillary form to the dot one, reflecting the bacterial death. In addition, the particle size is highly important for the determined particles, and a particle size ranging from 5 to 200 nm is reported to be highly efficient to enter a specific organ.6 Although the particle size of dispersive EuW10 in dilute solution is around 1−2 nm, a high concentration on the order of micromoles often leads to the aggregation of POMs into larger particles and makes it possible for them to enter cells.19 However, direct toxicity of POMs was often observed, which

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b17082. Crystal structure of EuW10; purification and characterization of HPV 16 L1-p and HPV 16 VLPs, EDX spectrum of EuW10@VLPs; DLS size distribution curves of HPV 16 VLPs, EuW10@VLPs, and VLPs@EuW10; and effect of HPV 16 VLPs, EuW10, and EuW10@VLPs on E. coli growth based on the OD at 600 nm (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.W.). *E-mail: [email protected] (L.W.). ORCID

Yuqing Wu: 0000-0003-4883-5982 Lixin Wu: 0000-0002-4735-8558 Notes

The authors declare no competing financial interest. 6143

DOI: 10.1021/acsami.7b17082 ACS Appl. Mater. Interfaces 2018, 10, 6137−6145

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ACS Applied Materials & Interfaces

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ACKNOWLEDGMENTS The present work was supported by the National Natural Science Foundation of China (NSFC) (nos. 91027027 and 21373101) and the Innovation Program of State Key Laboratory for Supramolecular Structure and Materials (no. Cx2017apply-05).

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