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Hybrid-Assembly toward Enhanced Thermal Stability of Viruslike Particles and Antibacterial Activity of Polyoxometalates Ding-Yi Fu, Simin Zhang, Zhiyu Qu, Xianghui Yu, Yuqing Wu, and Lixin Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17082 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018

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ACS Applied Materials & Interfaces

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, Jilin University, No.2699, Qianjin Street, Changchun, 130012, China. ‡ State Engineering Laboratory of AIDS Vaccine, Jilin University, No. 2699, Qianjin Street, Changchun, 130012, China. KEYWORDS: Hybrid-assembly, virus-like particles, polyoxometalates, stability, antibacterial activity

ABSTRACT: In an effort to improve both the stability of virus-like particles (VLPs) and 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 (DLS) and transmission electron

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microscopy (TEM); while the post-assembly of EuW10 with the as-prepared VLPs lead only the adsorption of POMs on the external surface of particle, and both cases improved the thermal and storage stability of VLPs obviously. Particularly, the encapsulation of POMs in VLPs largely improved the antibacterial activity of EuW10, and thereby, the present study will be significance both for the stability improvement of protein vaccines and the development of POM medicine.

INTRODUCTION Polyoxometalates (POMs) are kinds of negatively charged clusters of transition metals (mainly tungsten, molybdenum and vanadium) with oxygen that have shown potential applications in catalysis,1 material science,2 medicine,3 bio- and nanotechnology,4 and macromolecular crystallography.5 Due to their specific three-dimensional structure and high negative charges, POMs selectively bind to the positive regions of proteins.5 Recent reports reveal POMs are effect on the treatment of cancer,6 diabetes7 and infections associated with bacteria,8 viruses,9 respectively. 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 ovarian14 and gastric cancer.15 However, unfortunately, up to now POMs has not been actually used as medicine yet which mainly because of most POMs are not sufficiently stable at physiological pH and easily to be degraded into a mixture of inorganic products.16 One of the best way to overcome the bottle neck is encapsulating them into bio-systems such as starch,17 lipid18 or liposome19 as well as particular nanospheres20, while several successful examples have been reported. Such performances created new systems as drug carrier that have gained more promising therapeutic applications due to the newly introduced biocompatibility, biodegradability and physical stability of these covers.


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Viral capsids have attracted great attention recently in the fields both of nano-technology and biology essentially because of their nanoscales size, uniform and symmetrical structural organization, loading capacity, easily controllable self-assembly and modification.21 Therefore, the hybrid structures of viral capsid and nanoparticle, which combine the bio-activities of virus with the functions of nanoparticles, are a new class of bio-nanomaterials that have shown great potentials as therapeutic and diagnostic vectors, imaging agents, and advanced nano-synthesis reactors.22, 23 Since the initial study describing the capture of nanoparticles (NPs) by the selfassembly of brome mosaic virus (BMV) capsid proteins,24 large amount of studies focused on constructing virus-like particles (VLPs) containing Au nanoparticles,25 quantum dots26 and magnetic cores27 have been reported albeit the functionalized inorganic nanoparticle cargos are very limited.24 In addition, for human papillomavirus (HPV), the L1 VLP-based vaccines are efficacious at preventing infections and pre-cancerous 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, expensive, and requiring cold storage for transportation, these vaccines have not been widely used in developing countries.30 Therefore, urgent needs to develop new ways improving the stability and lowing the cost of VLPs. The use of HPV VLPs as a vaccination platform is already a medical evidence, however, the studies on VLPs as drug delivery vectors are in their infancy although the VLPs features for drug delivery are cellspecific targeting, efficient cell entry, lack of endosomal sequestering, multi-valency and biocompatibility.31 Therefore, in the present study we will perform the hybrid-assembly of the capsids protein of human papillomavirus (HPV) and POMs, aiming to improve both the stability of VLPs and


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medical activity of POMs. The VLP has a T=7 icosahedral lattice structure, being composed of 72 L1-pentamer (L1-p), and each L1-p composed 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 from both sides: 1) the antibacterial ability of EuW10 was improved after being encapsulated in HPV 16 VLPs; 2) the thermal and storage stability 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 significance to improve the protein vaccine. Therefore the binding of EuW10 will be significance both for the improvement of protein vaccines and the development POM medicine.

MATERIALS AND METHODS 1. Reagents and materials. Tryptone and yeast extract were from OXOID (OXOID Ltd, Basingstoke, UK). Isopropyl-β-D-thiogalactopyranoside (IPTG), ethylene diaminetetraaceticacid (EDTA), DL-Dithiothreitol (DTT), sodium dodecyl sulfonate (SDS), Tris, Coomassie blue G250, and Caesium Chloride (CsCl) were bought from Genview (Gen-view scientific Inc., USA). Sodium chloride (NaCl), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Beijing Chemical Factory. All chemicals are analytical reagent and used without further purification. Distilled water is obtained from a Millipore Milli-Q water purification system. 0.22 µm and 0.45 µm sterile filtration membranes, used for filtering large particle, were bought from Millipore. 2. Protein Expression and Purification. The HPV 16 L1 coding sequences, in lacking of 4 amino acids at the N-terminus (residue 1-4) and 29 amino acids at the C-terminus (residue 477-


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495), were employed for better expression. The E.coli strain BL21 carrying pGEX-6p-1 was grown at 37 ºC to get an OD600 of 0.8, and then induced with 1 mM IPTG at 28 ºC overnight. Actually, the expression and purification of protein were carried out essentially as described previously.32. 33 Briefly, cells from one liter culture were re-suspended in buffer L (50 mM TrisHCl, 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 24,041 g 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 column and keeping at 4 ºC for 14-16 h. After digestion, the elution was collected and further purified by using sizeexclusion chromatography (SEC, Superdex 200, 26/60). Before 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 protein concentration. All the steps for above purification were conducted at 4 ºC. 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 ºC in Laemmli buffer (12 mM Tris-HCl, 0.4% SDS, 2.88 mM mercaptoethanol, 5% glycerol, and 0.02% bromophenol blue). 3. Preparation of Na9[EuW10O36]·32H2O (EuW10). EuW10 was synthesized and characterized according to a published procedure.34 Briefly, 8.3 g of Na2WO42H2O was dissolved in 20 ml water and the pH in solution was adjusted to 7.0-7.5 with CH3COOH. An aqueous solution (2 ml) containing 1.1 g of Eu(NO3)36H2O was added dropwise to the above-mentioned solution


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with stirring at 80-90 ºC. 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. 4. Hybrid-assembly Monitoring of EuW10 and HPV 16 VLPs. For the in vitro hybridassembly of L1-p and EuW10, two different protocols were used. One was the co-assembly where the final concentration of L1-p was fixed at 2 µM, after adding the stock solution of EuW10 (1 mM) to get a ten equivalents concentration (20 µM). The mixture was incubated for 3h before dialyzing against one liter assembly buffer (1 M NaCl, 50 mM H2PO4-/HPO42-, pH=5.4), being performed at 4 ºC for 24 h to get fully assembly. In this case, the co-assembly product between EuW10 and HPV 16 L1-p was defined as EuW10@VLPs. The second protocol was termed as post-assembly where the HPV 16 VLPs were formed firstly by dialyzing 2 µM L1-p alone in the assembly buffer for 24 h (4 ºC), and then ten equivalents EuW10 was added and the solution was kept at 4 ºC for another 24 h to get fully assembly. The post-assembly product between EuW10 and HPV 16 VLPs, in this case, was defined as VLPs@EuW10. 5. Dynamic Light Scattering (DLS) Measurement. The DLS measurement was used to monitor the size distribution of particles in solution. The concentration of HPV L1-p is 2 µM in buffer L and the concentration of EuW10 is 4 µM in assembly buffer solution. Firstly, the big dust particles in sample were removed through a 0.22 µm filter unit, then the 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. 6. Transmission Electron Microscopy (TEM) Measurements and EDX Analysis. For the 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%


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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 H-7650 transmission electron microscope (Hitachi Japan), with an accelerating voltage at 80 kV. A magnification of 40,000× was used, since 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 stain. 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 JEM-2200FS transmission electron microscope (Jeol Ltd. Japan), with an accelerating voltage at 200 kV. For this examination, a magnification of 100,000× was used to observe the particles more clearly. And for electron dispersive X-ray analysis, individual particle was collected by the EDX-system (INCA-Oxford) attached with TEM. 7. Purification of the Hybrid-assembly by CsCl Density Gradient Ultracentrifugation. Ultracentrifugation using CsCl density gradient is used to purify the virus-like particles based on its buoyant specific density.35 Layer aqueous phase onto CsCl Gradient was performed as follows: CsCl density of 1.5 g/mL × 1 mL, 1.35 g/mL × 3 mL, and 1.25 g/mL × 4 mL were prepared and then loaded into tube consequently; finally, the sample was added on the top layer. The concentration of virus-like particles 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 226,000 g, using a Beckman Optima XPN-80 ultracentrifuge. To collect the target bands, either (1) puncture tube with a 5 mL syringe and a needle below the band of interest, and slowly draw the sample into the syringe; or (2) slowly remove liquid using a pipettor from the top of the tube until the band of


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interest is reached, was performed. After that, the potential HPV 16 VLPs as well as the counterparts were loaded into tube for 24 h dialysis to remove CsCl. 8. The UV-cloud Point-temperature Determination and DLS Measurement of the Stability of Protein. The thermal stability 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 LOWCONSTANTTEMP BATH temperature control system (Hangping, China). The UV CP-Temp determination was performed by observing the ultraviolet absorption signals as the optical density at 350 nm of the solution of HPV 16 L1. The concentration of virus-like particles is 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 ºC/min with the scan temperature ranged from 37-77 ºC to monitor the thermal denaturation and/or aggregation of HPV 16 L1 in versatile VLP assemblies in solution. In addition, DLS (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 ºC. 9. Effect of EuW10 and EuW10@VLPs on E. coli growth. Inoculating the E. coli bacteria seeds from the glycerine stock tube being cultured in 5 mL Luria-Bertani (LB) medium (consisting of 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L NaCl) and incubating them at 37 ºC with shaking overnight and then transferring 10 µL bacterial culture to 4×5 mL fresh LB medium for further experiments. The EuW10, VLPs@EuW10 and EuW10@VLPs in different molar ratio was


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respectively added into the medium. For VLPs@EuW10 and EuW10@VLPs, the fraction ratio of EuW10/VLPs was different but where the concentration of VLPs in each sample was same. Meanwhile, the HPV 16 VLPs with the same concentration as EuW10@VLPs were added as a control sample. The original concentration of particles is 8 µM in assembly buffer. The cultures were kept in incubator at 37 ºC for 24 h. Along with the growth going on each sample was taken out every 2 h in 200 µL and was monitored by 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 silicon wafer and air-dried. SEM image was then taken for each sample using a JEOL JSM 6700F field emission equipment with the primary electron energy of 3 kV, and the sample was sputtered with a layer of Pt prior to imaging to improve conductivity.

RESULTS AND DISCUSSION 1. Encapsulation of EuW10 in HPV 16 VLPs. The FPLC elution profile showed an intense peak at ~265 kDa for HPV 16 L1, being an indication of the L1-p; while a weak peak corresponding to ~53 kDa was attributed to the L1 monomer (L1-m) (Figure S1A), being consistent well with we reported before.33,

36, 37

The

synchronous SDS-PAGE test (Figure S1A, upside) identified the components in the intense peak, where the band at~53 kDa belonged to one L1-m. The size and distributions was then determined by dynamic light scattering (DLS) and transmission electron microscopy (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


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diameter of 55.29 nm (Figure S1B, red line), being typical for the full-sized HPV VLPs.32, 33 Then the negative stained TEM analysis confirmed that the pentamer was shown as five-pointed stars, with an average size of ~12 nm (Figure S2A). They underwent a controlled assembly to form HPV 16 VLPs with a statistic central-size of ~55 nm (Figure S2B). These results confirmed 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 in assembly buffer solution lead to the formation of stable capsid-like assemblies, so we tried this condition for the encapsulation of EuW10 by HPV 16 VLPs. After assembly monitoring, both the finalized HPV 16 VLPs (being used as a control group) and the potential co-assembly of EuW10 and HPV 16 VLPs (being defined as EuW10@VLPs here) were concentrated by using CsCl gradient ultracentrifugation, and the results were shown in Figure 1A. A visible narrow light-blue band was observed in the gradient centrifugation tubes, the position of it consisted well with a previous report for the density of CsCl fraction containing the HPV 16 VLPs,39 being possible to be our target protein. Then the band-related regions were divided into three parts, being labeled as F1/F1′-F3/F3′, respectively, for two tubes according to the position of 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 firstly on the extracted sample, which confirmed that only fraction F2 and F2′ contained the target HPV 16 L1 protein (Figure 1B); while no sign of it in the other two parts. Moreover, DLS results showed similar particle size at ~55 nm for the fractions of F2 and F2′ (Figure 1C), being the typical one of HPV 16 VLPs in solution.38, 40 However, there was almost no signal was observed in DLS test for the fraction F1/F1′or F3/F3′.


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It 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 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. It suggested that EuW10@VLPs had a more uniform density than the corresponding empty HPV 16 VLPs. Up to this stage, taken all the results together it suggested, at least, the incorporation of EuW10 to L1-p solution did not retarded the assembly of HPV 16 VLPs. However, in regard to whether the EuW10 was assembled together with HPV 16 VLPs, or has it been encapsulated inside the HPV 16 VLPs or just bound at particles surface, further experiments should be conducted.

Figure 1. (A) The photographs of HPV 16 VLPs and EuW10@VLPs after CsCl gradient ultracentrifugation in tubes. (B) SDS-PAGE of the corresponding components extracted from the position of F1/F1′, F2/F2′ and F3/F3′, respectively. (C) DLS plots of EuW10@VLPs (upper) and HPV 16 VLPs only (bottom) after CsCl gradient centrifugation, being extracted from the position of F1/F1′, F2/F2′ and F3/F3′, respectively. 2. Characterization of the co-assembly of EuW10@VLPs.


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To further determine the component and morphology features of fraction F2′, transmission electron microscopy (TEM) was used to characterize. Albeit the sample was not negatively stained with phosphotungstic acid, black dots still were shown clearly in TEM image (Figure 2A). Therefore, it suggested that EuW10 might be involved in the particles to give nontransparent image at present condition. Furthermore, the corresponding EDX analysis based on TEM image detected two kinds of elements, tungsten and europium within one particle (Figure S3), proving 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 shown in relative uniform and the distributions of particle sizes were evaluated to focus on ~34 nm (Figure 2B). Of note, such size was obvious smaller than the outer diameter of HPV 16 VLPs (~55 nm) but fitted well with the inner diameter of VLPs cavity as reported previously.41 Therefore, we concluded that the TEM images observed here were essentially originated from the EuW10, being confined within the cavity of HPV 16 VLPs. Based on the TEM images of the co-assembly (EuW10@VLPs), we did not see POMs being attracted on the external surface under the current concentration of 20 µM EuW10. If we continue to increase the concentration until much excessive, it might be possible for POMs to be adsorbed on the external surface partially.


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Figure 2. (A) The TEM image of EuW10@VLPs after CsCl gradient ultracentrifugation in tubes, being extracted from the position of F2. (B) Size distributions of EuW10@VLPs, being statistic from 200 particles from TEM images. In following, the negative-stained TEM image was performed on the same batch of sample to give further morphology characterization on them. The particles were also shown in uniform and the actual sizes were evaluated to be ~55 nm, being similar as 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 shown clearly between Figure 3A and S2B. The particles for EuW10@VLPs appeared as dark center covered by white protein shells, being very similar as that reported in the literature for gold nanoparticles (AuNPs) being encapsulated in simian virus 40 capsids.42 The basic residues of protein provide positive charges to the interior surface of cavity for the encapsulation of negatively charged species.43 Therefore, it confirmed that EuW10 were indeed encapsulated into the cavity of HPV 16 VLPs, forming the EuW10@VLPs under the concentration of 20 µM EuW10 in the present study. That was, the encapsulation of EuW10 in HPV 16 VLPs was successfully constructed via the co-assembly.


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Figure 3. The morphologies (A) HPV 16 EuW10@VLPs and (B) VLPs@EuW10 with negative stain. Meanwhile, we either tried to use post-assembly approach for the binding of EuW10 and HPV 16 VLPs, and the product was termed as VLPs@EuW10 to distinguish from that of co-assembly, EuW10@VLPs. For that, empty HPV 16 VLPs were formed firstly, and then ten equivalents of EuW10 were 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 pre-aggregation in dialysis bag. TEM image showed identical results (data not shown) to that obtained in the first case, confirming the state of EuW10 even in dialysis bag was dispersive. Particularly, without negative staining both of them either showed clear TEM images (Figure 4A) with the average size of ~53 nm, which were close to those evaluated by DLS (Figure 4B). In addition, TEM image-based EDX analysis on the particles indicated the


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appearance both of tungsten and europium were shown there (Figure 4C). Therefore, we concluded that the EuW10 were either combined with HPV 16 VLPs through such a postassembly monitoring. However, in considering the size difference revealed between Figure 2 and Figure 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 POM linked by europium ion with 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 typical pentamer similar as “donuts”. Based on the crystal structure of HPV 16 L1-p (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 a little bit difficulty to be encapsulated in the cavity of HPV 16 VLPs.

Figure 4. (A) The TEM image of HPV 16 VLPs-EuW10 without negative stain, which were prepared by the post-assembly process by mixing EuW10 with the as-formed HPV 16 VLPs; (B) DLS plots of the corresponding particles; (C) The corresponding energy dispersive X-ray spectroscopy (EDX) of the representative particle.


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3. The Encapsulation of EuW10 improved the thermal and storage stability of HPV 16 VLPs. The following question then is whether an increase in stability could be achieved through the incorporation of a solid template into the cavity of HPV16 VLPs. Furthermore, the thermal stability of HPV 16 VLPs, EuW10@VLPs and VLPs@EuW10 was assayed respectively by using UV-cloud point measurements. The experimental traces and the corresponding transition temperature (Tm, Figure 5A) revealed clear differences of the thermal stabilities between them. A reasonable Tm value of 68.83 ºC was exhibited for HPV 16 VLPs, being closed to that reported previously.44 However, a higher value of Tm was shown at 70.30 ºC and 69.53 ºC, respectively, for EuW10@VLPs and VLPs@EuW10. These difference values indicated that an anionic nanoparticle binding is sufficient to promote the thermal stability of HPV 16 VLPs, consequently the thermal stabilities both of 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 ITC, where the fitting of the isotherm plot revealed ∆H =-9.84 ± 1.83 kcalmol-1, ∆G= -6.42 ± 1.83 kcalmol-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 the electrostatic interaction between them played a crucial role. Such conclusion was either supported by other report, where the nucleic acids such as tRNA or simply polyanions such as dextran sulfate, serve to provide a reservoir of negative charge to attract coat protein subunits to its surface through electrostatic interactions with amino terminal tails for driving assembly.46 Therefore, the charge neutralization was supposed to be necessary to stabilize the HPV 16 VLPs.


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In addition, the thermal stability of EuW10@VLPs was further optimized by changing the concentration of EuW10 when fixed the concentration of L1-p in the assembly. It was found electrostatically driven EuW10-VLP association occurred regardless much the concentration of POMs, as all cases improved the Tm obviously (Figure 5B). Among the tested concentrations, maximum Tm at 70.78 °C was obtained in the presence of 30 µM EuW10 resulting the biggest

∆Tm of 1.95±0.13 °C from that of HPV 16 VLPs itself. In this case, the assembly protocol yielded spherical capsids being very similar both in shape and size as those of wild type (wt) HPV 16 VLPs. As higher concentration will lead the self-aggregation of EuW10 in assembly buffer, for 0.75 µM HPV16 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 the thermal stability of VLPs was either improved. However, the maximum ∆Tm was achieved only at 1.22±0.16 ˚C (Figure not shown). In comparison to it, the encapsulation of EuW10 in VLPs improved the stability of protein capsid better.

Figure 5. The 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),


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and (B) and the co-assembled HPV 16 VLPs (0.75 µM) constructed from HPV 16 L1-p and EuW10 in different concentrations (10-30 µM), which shows the thermal stability differences between them. ∆Tm was an average value of three repeat experiments, and each displayed plot was only a representative one among three repeated measurements. In following, DLS was used to compare the stability of either the EuW10@VLPs or VLPs@EuW10 to that of the empty HPV 16 VLPs as a monitoring during 28 days (Figure 6 and Figure S4). The hydrodynamic diameter of either EuW10@VLPs or VLPs@EuW10 remained almost the same throughout the entire duration of test in 18 and 24 days, respectively; while the drift toward larger average diameters was observed for the empty HPV 16 VLPs after 3 days. They started to aggregate in showing quick increase of DLS intensity, as the appearance of eyeview aggregation DLS cannot give further report after 5th-day. For the post-assembled VLPs@EuW10, the DLS intensity increased a little bit from 15th-day and then it jumped from the 18th-day, illustrating the aggregation started to form since then. The particle size became larger and larger, and being similarly as the empty HPV 16 VLPs, finally the eye-view aggregation was shown. However, the co-assembled EuW10@VLPs was quite stable in solution as the aggregation of it started to form 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 assembling with POMs, and would be significant in protein vaccines.


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Figure 6. Storage stability of HPV 16 VLPs, EuW10@VLPs and VLPs@EuW10 as a function of time at 4 ºC , as indicated by 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. 4. Effect of EuW10, and EuW10@VLPs on E. coli growth. Recent reports reveal POMs are effect on the treatment of infections associated with bacteria, so we chose E. coli system as a model to monitor the medical function of EuW10. The growth of E. coli was firstly monitored by using time-dependent curve of OD600 over 14 h (37 ºC) in the absence and presence of HPV 16 VLPs, EuW10 and different molar ratios of EuW10 to HPV 16 VLPs in EuW10@VLPs, respectively. It showed that the presence of HPV 16 VLPs lead very close OD600 values (Figure 7, red line) as the control of E. coli itself at identical condition (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, their inhibitions on E. coli growth were obvious albeit differences existed between them. With the increase of EuW10, the OD600 value of E. coli (Figure 7, solid line) were becoming lower and lower in comparison to the control of E. coli itself being cultured at identical condition, suggesting EuW10 held the concentration-dependent inhibition on the growth


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of E. coli. While in the presence of EuW10@VLPs assembly, the OD600 values (Figure 7, dash line) were even lower in comparison to that of corresponding EuW10, suggesting the inhibition on E. coli growth was promoted by the co-assembly. At the same time, we also tried to test the antibacterial ability of VLPs@EuW10 for comparison (Figure S5). However, in this case the EuW10 at VLPs surface was easily to be released to medium. Therefore, it showed that the presence of VLPs@EuW10 hardly affect the growth of E. coli during test. In considering 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 co-assembly of EuW10 and HPV 16 VLPs. It was the encapsulation of EuW10 in HPV 16 VLPs enhanced the antibacterial ability of POMs.

Figure 7. Effect of the HPV 16 VLPs, EuW10 and EuW10@VLPs on E. coli growth basing on the optical density (OD) at 600 nm, where differences were observed between the tests (red, blue, green and pink) and control group (black) during 14 h. At the same time, Figure 8 showed scanning electron micrographs (SEM) of E. coli that was cultured for 24h in the presence of EuW10, empty HPV 16 VLPs or EuW10@VLPs, respectively.


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Being as a control group in Figure 8A, essentially bacillary form of the bacterial cells were shown, indicating the well growth of them at present conditions. However, the exposure of them to rather EuW10 (Figure 8C) or EuW10@VLPs (Figure 8D) gave rise to the morphological change from the bacillary forms to shorted bars or even balls; particularly, in the presence of EuW10@VLPs the case was worse where almost nothing was shown. The difference of the morphological change between EuW10 and the co-assembly should be attributed to the enhanced inhibition of EuW10@VLPs on E. coli growth. The surface charges of POMs strongly affected their biological functions. The highly negative charge of them often stimulate the bacterial morphology changes from bacillary form to dots one, reflecting the bacterial death. In addition, the particle size is either highly important for the determined particles, while a particle size ranging of 5~200 nm is reported highly efficient to enter a specific organ.6 Although the particle size of dispersive EuW10 in dilute solution is around 1~2 nm, high concentration of micromole often lead POMs to be aggregated into larger particles and become possible to enter cell.19 But direct toxicity of POMs was often shown which blocked their medical usage at some extent.17 Therefore, biological drug carriers as microspheres like virus capsids are necessary to extend the biomedical application of POMs. Moreover, the highly charged surface of POMs should lead them difficulty to go through the hydrophobicprone cell membrane. It is well known that the capsid of virus generally display remarkable cell entry propensity being attributed the fact that they are responsible for cell penetration by the corresponding virus.47 It utilize endocytosis as an essential way to transport the drug into the cells and then liberate them into the cytoplasm. Therefore, it would be interesting to know if the co-assembly, EuW10@VLPs, is able to improve the entry ability of EuW10 into cells and alter their biological functions efficiently. The above results indeed showed improved antibacterial


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activity of EuW10 after co-assembly with HPV 16 VLPs. It should be the cell entry ability of virus capsid promoted greatly the cell penetration of POMs. In addition, as the capsid protein can be slowly removed from the cell thanks to proteolytic mechanisms, they are biocompatible and would be the ideal materials for drug carrier. 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 POMs delivery in the medicinal area in future.

Figure 8. Scanning electron 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.

CONCLUSION In an objective to improve both the stability of HPV 16 VLPs and medical activity of POMs, the 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; while


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the post-assembly of EuW10 with the as-prepared HPV 16 VLPs lead only the adsorption of POMs 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 significance both for the improvement of protein vaccines and the development of POM medicine.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. The crystal structure of EuW10; the purification and characterization of HPV 16 L1-p and HPV 16 VLPs, the EDX spectrum of EuW10@VLPs; the DLS size-distribution curves of (A) HPV 16 VLPs, (B) EuW10@VLPs and (C) VLPs@EuW10; effect of the HPV 16 VLPs, EuW10 and EuW10@VLPs on E. coli growth basing on the optical density (OD) at 600 nm (PDF file) AUTHOR INFORMATION Corresponding Author *Email: [email protected], [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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The present work was supported by the National Natural Science Foundation of China (NSFC) (Nos. 91027027, 21373101) and the Innovation Program of State Key Laboratory for Supramolecular Structure and Materials (No. Cx2017apply-05). REFERENCES (1) Proust A.; Thouvenot R.; Gouzerh P. Functionalization of polyoxometalates: towards advanced applications in catalysis and materials science. Chem. Commun. 2008, 39, 1837– 1852. (2) Coronado E.; Gomez-Garcia J. Polyoxometalate-Based Molecular Materials. Chem. Rev. 1998, 98, 273–296. (3) Rhule J. T.; Hill C. L.; Judd D. A. Polyoxometalates in Medicine. Chem. Rev. 1998, 98, 327– 357. (4) Long D.-L.; Burkholder E.; Cronin L. Polyoxometalate clusters, nanostructures and materials: From self assembly to designer materials and devices. Chem. Soc. Rev. 2007, 36, 105–121. (5) Bijelic A.; Rompel A. The use of polyoxometalates in protein crystallography – An attempt to widen a well-known bottleneck. Coordin. Chem. Rev. 2015, 299, 22–38. (6) Shah H. S.; Joshi S. A.; Haider A.; Kortz U.; Rehman N.; Iqbal J. Synthesis of chitosancoated polyoxometalate nanoparticles against cancer and its metastasis. RSC Adv. 2015, 5, 93234–93242. (7) Balici S.; Susan S.; Rusu D.; Nicula G. Z.; Soritau O.; Rusu M.; Biris A. S.; Matei H. Differentiation of stem cells into insulinproducing cells under the influence of nanostructural polyoxometalates. J. Appl. Toxicol. 2016, 36, 373–384. (8) Inoue M.; Segawa K.; Matsunaga S.; Matsumoto N.; Oda M.; Yamase T. Antibacterial activity of highly negative charged polyoxotungstates, K27[KAs4W40O140] and K18[KSb9W21O86], and Keggin-structural polyoxotungstates against Helicobacter pylori. J. Inorg. Biochem. 2005, 99, 1023–1031. (9) Shigeta S.; Mori S.; Yamase T.; Yamamoto N.; Yamamoto N. Anti-RNA virus activity of polyoxometalates. Biomed. Pharmacother. 2006, 60, 211–219. (10) Ogata A.; Yanagie H.; Ishikawa E.; Morishita Y.; Mitsui S.; Yamashita A.; Hasumi K.; Takamoto S.; Yamase T.; Eriguchi M. Antitumour effect of polyoxomolybdates: induction of apoptotic cell death and autophagy in in vitro and in vivo models. Br. J. Cancer 2008, 98, 399–409. (11) Thomadaki H.; Karaliota A.; Litos C.; Scorilas A. Enhanced Antileukemic Activity of the Novel Complex 2,5-Dihydroxybenzoate Molybdenum(VI) against 2,5-


24 / 28

Page 25 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Dihydroxybenzoate, Polyoxometalate of Mo(VI), and Tetraphenylphosphonium in the Human HL-60 and K562 Leukemic Cell Lines. J. Med. Chem. 2007, 50, 1316–1321. (12) Dong Z.; Tan R.; Cao J.; Yang Y.; Kong C.; Du J.; Zhu S.; Zhang Y.; Lu J.; Huang B. Discovery of polyoxometalate-based HDAC inhibitors with profound anticancer activity in vitro and in vivo. Eur. J. Med. Chem. 2011, 46, 2477–2484. (13) Wang L.; Yu K.; Zhou B. B.; Su Z. H.; Gao S.; Chu L. L.; Liu J. R. The inhibitory effects of a new cobalt-based polyoxometalate on the growth of human cancer cells. Dalton Trans. 2014, 43, 6070–6078. (14) Zhai F.; Wang X.; Li D.; Zhang H.; Li R.; Song L. Synthesis and biological evaluation of decavanadate Na4Co(H2O)6V10O2818H2O. Biomed. Pharmacother. 2009, 63, 51–55. (15) Wang L., B. B. Zhou, K. Yu, Z. H. Su, S. Gao, L. L. Chu, J. R. Liu and G. Y. Yang, Novel Antitumor Agent, Trilacunary Keggin-Type Tungstobismuthate, Inhibits Proliferation and Induces Apoptosis in Human Gastric Cancer SGC-7901 Cells. Inorg. Chem. 2013, 52, 5119–5127. (16) Menon D.; Thomas R. T.; Narayanan S.; Maya S.; Jayakumar R.; Hussain F.; Lakshmanan V.-K.; Nair S. A novel chitosan/polyoxometalate nano-complex for anti-cancer applications. Carbohydr. Polym. 2011, 84, 887–893. (17) Zhai F.; Li D.; Zhang C.; Wang X.; Li R. Synthesis and characterization of polyoxometalates loaded starch nanocomplex and its antitumoral activity. Eur. J. Med. Chem. 2008, 43, 1911–1917. (18)

Noguchi T.; Chikara C.; Kuroiwa K.; Kaneko K.; Kimizuka N. Controlled morphology

and photoreduction characteristics of polyoxometalate(POM)/lipid complexes and the effect of hydrogen bonding at molecular interfaces. Chem. Commun. 2011, 47, 6455–6457. (19) Wang X.; Li F.; Liu S.; Pope M. T. New liposome-encapsulated-polyoxometalates: synthesis and antitumoral activity. J. Inorg. Biochem. 2005, 99, 452–457. (20) Neves C. S.; Granadeiro C. M.; Cunha-Silva L.; Ananias D.; Gago S.; Feio G.; Carvalho P. A.; Eaton P.; Balula S. S.; Pereira E. Europium Polyoxometalates Encapsulated in Silica Nanoparticles – Characterization and Photoluminescence Studies. Eur. J. Inorg. Chem. 2013, 2013, 2877–2886. (21) Aniagyei S. E.; DuFort C.; Kao C. C.; Dragnea B. Self-assembly approaches to nanomaterial encapsulation in viral protein cages. J. Mater. Chem. 2008, 18, 3763–3774. (22) Kwak M.; Minten I. J.; Anaya D.-M.; Musser A. J.; Brasch M.; Nolte R. J. M.; Mullen K.; Cornelissen J. J. L. M.; Herrmann A. Virus-like Particles Templated by DNA Micelles: A General Method for Loading Virus Nanocarriers. J. Am. Chem. Soc. 2010, 132, 7834–7835. (23) Bronstein L. M. Virus-Based Nanoparticles with Inorganic Cargo: What Does the Future Hold? Small, 2011, 7, 1609–1618.


25 / 28


Page 26 of 28

(24) Liu Z.; Qiao J.; Niu Z.; Wang Q. Natural supramolecular building blocks: from virus coat proteins to viral nanoparticles. Chem. Soc. Rev. 2012, 41, 6178–6194. (25) Goicochea N. L.; De M.; Rotello V. M.; Mukhopadhyay S.; Dragnea B. Core-like Particles of an Enveloped Animal Virus Can Self-Assemble Efficiently on Artificial Templates. Nano Lett. 2007, 7, 2281–2290. (26) Dixit S. K.; Goicochea N. L.; Daniel M.-C.; Murali A.; Bronstein L.; De M.; Stein B.; Rotello V. M.; Kao C. C.; Dragnea B. Quantum Dot Encapsulation in Viral Capsids. Nano Lett. 2006, 6, 1993–1999. (27) Huang X.; Bronstein L. M.; Retrum J.; Dufort C.; Tsvetkova I.; Aniagyei S.; Stein B.; Stucky G.; McKenna B.; Remmes N.; Baxter D.; Kao C. C.; Dragnea B. Self-Assembled Virus-like Particles with Magnetic Cores. Nano Lett. 2007, 7, 2407–2416. (28) Paavonen J.; Naud P.; Salmeron J.; Wheeler C. M.; Chow S. N.; Apter D.; Kitchener H.; Castellsague X.; Teixeira J. C.; Skinner S. R.; Hedrick J.; Jaisamrarn U.; Limson G.; Garland S.; Szarewski A.; Romanowski B.; Aoki F. Y.; Schwarz T. F.; Poppe W. A. J.; Bosch F. X.; Jenkins D.; Hardt K.; Zahaf T.; Descamps D.; Struyf F.; Lehtinen M.; Dubin G. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lancet 2009, 374, 301–314. (29) Pomfret T. C.; Gagnon J. M.; Gilchrist A. T. Quadrivalent human papillomavirus (HPV) vaccine: a review of safety, efficacy, and pharmacoeconomics. J. Clin. Pharm. Ther. 2011, 36, 1–9. (30) Luciani S.; Jauregui B.; Kieny C.; Andrus J. K. Human papillomavirus vaccines: new tools for accelerating cervical cancer prevention in developing countries. Immunotherapy 2009, 1, 795−807. (31) Zdanowicz M.; Chroboczek J. Virus-like particles as drug delivery vectors. Acta Biochim. Pol. 2016, 63, 469−473. (32) Bishop B.; Dasgupta J.; Klein M.; Garcea R. L.; Christensen N. D.; Zhao R.; Chen X. S. Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins. J. Biol. Chem. 2007, 282, 31803−31811. (33) Fu D.-Y.; Jin S.; Zheng D.-D; Zha X.; Wu Y. Peptidic Inhibitors for in Vitro Pentamer Formation of Human Papillomavirus Capsid Protein L1. ACS Med. Chem. Lett. 2015, 6, 381– 385. (34) Sugeta M.; Yamase T. Crystal Structure and Luminescence Na9[EuW10O36]32H2O. Bull. Chem. Soc. Jpn. 1993, 66, 444–449.

Site

of

(35) James K. T.; Cooney B.; Agopsowicz K.; Trevors M. A.; Mohamed A.; Stoltz D.; Hitt M.; Shmulevitz M. Novel High-throughput Approach for Purification of Infectious Virions. Sci. Rep. 2016, 6, 36826.


26 / 28

Page 27 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(36) Zheng D. D.; Pan D.; Zha X.; Wu Y.; Jiang C.; Yu X. In vitro monitoring of the formation of pentamers from the monomer of GST fused HPV 16 L1. Chem. Commun. 2013, 49, 8546−8548. (37) Jin S.; Pan D.; Yu X.; Wu Y.; Liu Y.; Yin F.; Chen X. S. The critical residues of helix 5 for in vitro pentamer formation and stability of the papillomavirus capsid protein, L1. Mol. BioSyst. 2014, 10, 724−727. (38) Chen X. S.; Casini G.; Harrison S. C.; Garcea R. L. Papillomavirus Capsid Protein Expression in Escherichia coli: Purification and Assembly of HPV11 and HPV16 L1. J. Mol. Biol. 2001, 307, 173−182. (39) Kirnbauer R.; Booy F.; Cheng N.; Lowy D. R.; Schiller J. T. Papillomavirus Li major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc. Nati. Acad. Sci. USA 1992, 89, 12180−12184. (40) Xie X.; Liu Y.; Zhang T.; Xu Y.; Bao Q.; Chen X.; Liu H.; Xu X. Human papillomavirus type 58 L1 virus-like particles purified by two-step chromatography elicit high levels of long-lasting neutralizing antibodies. Arch. Virol. 2013, 158, 193−199. (41) Baker T. S.; Newcomb W. W.; Olson N. H.; Cowsert L. M.; Olson C.; Brown J. C. Structures of bovine and human papillomaviruses Analysis by cryoelectron microscopy and three-dimensional image reconstruction. Biophys. J. 1991, 60, 1445−1456. (42) Wang T.; Zhang Z.; Gao D.; Li F.; Wei H.; Liang X.; Cui Z.; Zhang X. Encapsulation of gold nanoparticles by simian virus 40 capsids. Nanoscale 2011, 3, 4275−4282. (43) Douglas T.; Young M. Virus Particles as Templates for Materials Synthesis. Adv Mater. 1999, 11, 679−681. (44) Tallec D. L.; Doucet D.; Elouahabi A.; Harvengt P.; Deschuyteneer M.; Deschamps M. CervarixTM, the GSK HPV-16/HPV-18 AS04-adjuvanted cervical cancer vaccine, demonstrates stability upon long-term storage and under simulated cold chain break conditions. Human Vaccines 2009, 5, 467−474. (45) Zhang T.; Fu D.-Y.; Wu Y.; Wang Y.; Wu L. A fluorescence-enhanced inorganic probe to detect the peptide and capsid protein of human papillomavirus in vitro. RSC. Adv. 2016, 6, 28612−28618. (46) McPherson A. Micelle formation and crystallization as paradigms for virus assembly. BioEssays 2005, 27, 447−458. (47) Horvath C. A. J.; Boulet G. A. V.; Renoux V. M.; Delvenne P. O.; Bogers J.-P. J. Mechanisms of cell entry by human papillomaviruses: an overview. Virol. J. 2010, 7, 11.


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