Sialylglycan-Assembled Supra-Dots for Ratiometric Probing and

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Sialylglycan-Assembled Supra-dots for Ratiometric Probing and Blocking of Human-Infecting Influenza Viruses Chang-Zheng Wang, hai-hao han, Xin-Ying Tang, Dong-Ming Zhou, Changfeng Wu, Guo-Rong Chen, Xiao-Peng He, and He Tian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07485 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

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ACS Applied Materials & Interfaces

Sialylglycan-Assembled Supra-dots for Ratiometric Probing and Blocking of Human-Infecting Influenza Viruses

Chang-Zheng Wang,1 Hai-Hao Han,1 Xin-Ying Tang, Dong-Ming Zhou,* Changfeng Wu, Guo-Rong Chen, Xiao-Peng He,* and He Tian

a

Key Laboratory for Advanced Materials & Institute of Fine Chemicals, School of Chemistry and

Molecular Engineering, East China University of Science and Technology, 130 Meilong Rd., Shanghai 200237, PR China b

Vaccine Research Center, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur

of Shanghai, Chinese Academy of Sciences, Shanghai 200031, PR China c

Department of Biomedical Engineering, Southern University of Science and Technology, Shen-

zhen, Guangdong 510855, PR China 1

Equal contribution

ABSTRACT: The seasonal outbreak of influenza causes significant morbidity and mortality worldwide since a number of influenza virus (IV) strains have been shown to infect and circulate in humans. Development of effective means to timely monitor as well as block IVs is still a challenging task. While conventional fluorescence probes rely on a fluorimetric change upon recognizing IVs, here we developed simple “Supra-dots” that are formed through the aqueous supramolecular assembly between a blue-emitting polymer dot and red-emitting sialylglycan probes

ACS Paragon Plus Environment


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for the ratiometric detection of IVs. Tuning the Förster resonance energy-transfer from polymer dots to glycan-probes by selective sialylglycan-virus recognition enables the fluorescence ratiometric determination of IVs, whereas the presence of unselective, control viruses quenched the fluorescence of the Supra-dots. Meanwhile, we show that the Supra-dots can effectively inhibit the invasion of a human-infecting IV towards a human cell line, thereby making possible a unique bifunctional, supramolecular probe for influenza theranostics. KEYWORDS: Influenza, Virus, Supramolecular, Polymer dot, Probe

Introduction Influenza virus (IV) is a segmented RNA virus that can cause severe respiratory tract infection. It evolves rapidly by antigen drift and antigen shift, and thus results in seasonal epidemics or frequent outbreaks. It is estimated that IV infection leads to 3-5 million of severe cases and about 250,000-500,000 deaths annually,1 posing huge threats to public health worldwide. Especially in recent years, the emerging cross-species infection of avian influenza viruses presents significant risks, leading to increased morbidity and mortality.2 To reduce the impact of influenza infection on clinic and socio-economics, simple and effective methods for rapid detection of the subtypes of IV are urgently needed for making timely treatment decision. Although various techniques have been established in the clinic for diagnosis of IV infection, such as polymerase chain reaction (PCR), enzyme linked immunosorbent assay (ELISA) and virus isolation assay, these methods are complicated, time-consuming, laborious, costly, or are not suitable for field test.3,4 Therefore, development of effective means towards the timely identification of IV with, simultaneously, the function of blocking IV infection is still a challenging task.


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Emissive organic nanoparticles that are consisted of conjugated polymers have been increasingly employed as the material of choice in a variety of research areas, because of their high loading capability, fluorescence brightness, structural diversity and photo-stability. The poly(9,9dioctylfluorene) (PFO) nanoparticles (P-dots) have extraordinary blue-fluorescence brightness with an outstanding hole mobility and good biocompatibility for cell imaging, imaging-guided drug delivery and theranostics.5-15 These merits render the P-dots promising for biomedical applications. Despite their numerous elegant applications, the use of P-dots for the simultaneous detection and blocking of highly infectious viruses has been elusive.

DCM23

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Figure 1. Structure of the sialylglycan probes, DCM23 and DCM26 (where DCM is dicyanomethylene-4H-pyran), and the polymers, PFO (poly(9,9-dioctylfluorenyl-2,7-diyl) and PSMA (styrene-co-maleic anhydride) for the construction of the Supra-dots. 3



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x

Figure 2. (a) Schematic illustration of the stepwise supramolecular construction of the Supradots by the folding of PFO/PSMA and aqueous self-assembly of the resulting P-dot with sialylglycan probes. (b) Schematic illustration of the ratiometric probing of influenza viruses that express hemagglutinin as a result of sialylglycan-hemagglutinin recognition, and the inhibition of viral invasion through blocking of the interaction between human-infecting virus and glycoconjugates on the surface of human cells.

We have previously developed fluorogenic IV probes based on a glycofoldamer motif, which showed an enhanced fluorescence after binding to a selective IV.16 Whereas a fluorimetric signal 4


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(the intensity change of fluorescence) can be influenced by the change of detection environment, the development of ratiometric probes (i.e. the fluorescence emission peak shifts upon analyte sensing) has been viewed as a more specific approach for biosensing.17,18 Here, we developed a unique type of P-dot-based supramolecular nanoparticles (Supra-dots) for the selective, ratiometic probing of human and bird-infecting IVs. Aqueous supramolecular assembly between Pdots and glycoprobes produces the Supra-dots (Fig. 1 and Fig. 2a) with a ratiometric fluorescence response towards IVs, since the sialylglycans of the material can be recognized by the hemagglutinins (HAs) on the virus surface. In addition to the ratiometric sensing, these Supra-dots have also proven to be amenable for competitive blocking the infection of a human-infecting IV towards a human cell line with IV invasion sites (Fig. 2b).19,20

Results and Discussion The P-dots were prepared by mixing PFO with poly(styrene-co-maleic anhydride) (PSMA, 20% w/w) in THF, followed by a quick transfer to an aqueous solution under ultrasound. The solution was then filtrated through a 0.22 μm filter to produce the nanosized, blue-emitting P-dots.21,22 The red-emitting sialylglycan-attached dicyanomethylene 4H-pyran (DCM) probes (DCM23 and DCM26) were synthesized by conjugation of Neu5Acα2,3Gal-β1,4Glc (an avian-specific glycan) and Neu5Acα2,6Gal-β1,4Glc (a human-specific glycan) sialylglycans with an amino DCM according to our previously developed protocol.23 The well-overlapping emission spectrum of P-dots with the absorbance spectrum of DCM sets the basis for the Förster resonance energy transfer between the two species (Fig. S1).24

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Figure 3. Fluorescence titration of (a) P-dot (0.125 ppm) with increasing DCM23 (from bottom to top: 0-16 μM) and (d) P-dot (0.125 ppm) with increasing DCM26 (from bottom to top: 0-16 μM) in Tris-HCl (0.01 M, pH 7.4; excitation wavelength = 380 nm). Dynamic light scattering of (b) Pdot (8 ppm) and Supra-dot-23 (P-dot/DCM23: 8 ppm/4 μM) and (e) P-dot (8 ppm) and Supradot-26 (P-dot/DCM26: 8 ppm/4 μM). Zeta potential of (c) P-dot (0.125 ppm), DCM23 (1 μM) and Supra-dot-23 (P-dot/DCM23: 0.125 ppm/1 μM) and (f ) P-dot (0.125 ppm), DCM26 (1 μM) and Supra-dot-26 (P-dot/DCM26: 0.125 ppm/1 μM).

The Supra-dots were formed by a simple aqueous self-assembly between P-dots and sialylglycan DCM probes probably by the hydrophobic interactions between DCM and the PFO core.22,25 Fluorescence spectroscopy was first used to characterize the Supra-dots. Using an excitation wave6


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length of 380 nm, we determined that the blue emission of P-dots gradually decreased with increasing DCM23 (Fig. 3a) and DCM26 (Fig. 3d), whereas the red emission of DCM gradually enhanced. This concentration-dependent, ratiometric fluorescence-emission shift preliminarily suggests a FRET process from P-dots (donor) to DCM (acceptor).24 To further prove the FRET, time-resolved fluorescence spectroscopy was used. The decreased excited-state lifetime of P-dots in the presence of DCM23 or DCM26 suggests the energy transfer from the nanomaterial to the DCM molecules (Fig. S2).26 A series of other techniques were also used to characterize the formation of Supra-dots. Dynamic light scattering showed that the particle size of both Supra-dot-23 (Fig. 3b) and Supradot-26 (Fig. 3e) increased slightly with respect to P-dots alone. The zeta potential of the Supradots also decreased comparing to P-dots, suggesting the assembly between the DCM probes and the polymer. In addition, the blue-shifted absorbance band of Supra-dots with respect to P-dot alone might suggest a change of the conjugate structure of DCM upon hydrophobic binding with the polymer dot (Fig. S3).27 These data corroborate the formation of Supra-dots. In addition, the Supra-dots showed good stability with a wide range of pH and salt strength (Fig, S4), implying the suitability of the materials for selective IV detection. Next, we used fluorescence spectroscopy to analyze the ratiomertic sensing of the Supra-dots for IVs. It is reported that HA, a major surface protein of IVs, can have different glycan receptor preferences. The avian-adapted HA prefers the α2,3-glycans commonly found in birds and the human-adapted HA preferentially binds to α2,6-glycans.28 We primarily used two known IV strains, H3N2 (A/Beijing/353/89, a human-infecting virus, which has a perference for α2,6-glycans) and H10N8 (Lake/Hunan/3-9/2007, a bird-infecting virus, which has a perference for α2,37



glycans)16 to interact with the Supra-dots. We determined that, with increasing H3N2 (humaninfecting), while its red emission gradually decreased, the blue emission of Supra-dot-26 (with human-sialylglycan) enhanced in a concentration-dependent manner (Fig. 4a). Likewise, the red emission of Supra-dot-23 (with avian-sialylglycan) decreased with increasing H10N8 (birdinfecting), with a gradual increase in the blue emission (Fig. 4e). In contrast, the emission of Supra-dot-26 (Fig. 4b) and Supra-dot-23 (Fig. 4e) decreased with H10N8 and H3N2, respectively. These data suggest that the Supra-dots can selectively respond to a specific virus strain (i.e. Supra-dot-26 to H3N2 and Supra-dot-23 to H10N8) with a ratiometric signal, whereas their emission is quenched with an unselective virus. The fact that incubation of P-dot alone with the viruses similarly led to the fluorescence decrease of the polymer (Fig. S5) suggests that the fluorescence quenching was probably a result of the interaction of P-dots with viruses.

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Figure 4. Fluorescence titration of (a) Supra-dot-26 (P-dot/DCM26: 0.125 ppm/2 μM) and (d) Supra-dot-23 (P-dot/DCM23: 0.125 ppm/2 μM) with increasing H3N2 (A/Beijing/353/89, 0-256 HAU 50 μL-1). Fluorescence titration of (b) Supra-dot-26 (P-dot/DCM26: 0.125 ppm/2 μM) and (e) Supra-dot-23 (P-dot/DCM23: 0.125 ppm/2 μM) with increasing H10N8 (Lake/Hunan/39/2007, 0-256 HAU 50 μL-1). Fluorescence titration of (c) Supra-dot-26 (P-dot/DCM26: 0.125 ppm/2 μM) and (f ) Supra-dot-23 (P-dot/DCM23: 0.125 ppm/2 μM) with increasing H7N9 (A/Anhui/1/2013 derived HA and NA in the background of A/Puerto Rico/8/1934, 0-256 HAU 50 μL-1). All fluorescence spectra were measured in Tris-HCl (0.01 M, pH 7.4) with an excitation wavelength of 380 nm.

To further rationalize the fluorescence variation, we used several other viruses to treat with the Supra-dots. Incubation of H7N9 (A/Anhui/1/2013 derived HA and NA in the background of A/Puerto Rico/8/1934; human and bird-infecting)29 with Supra-dot-26 (Fig. 4c) and Supra-dot23 (Fig. 4f ) led to the ratiometric fluorescence change of both probes. This is in accordance with the human and bird-dual-infecting property of H7N9.16,23,29 We also observed that the treatment of two other unselective adenoviruses (the simian adenovirus AdC68 and the human adenovirus AdHu7) with the Supra-dots similarly led to their fluorescence quenching (Fig. 5 and Fig. S6). Since HA is also expressed in other viruses including influenza B,30 we used a virus strain (B/Phuket/3073/2013) known to infect humans to treat with the probes. The result showed that while Supra-dot-26 exhibited a ratiometric fluorescence change with the virus, the fluorescence of Supra-dot-23 was quenched upon incubation with B/Phuket/3073/2013 (Fig. 5 and Fig. S6).

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This is in good agreement with the HA selectivity of the probes. These additional evidence corroborate that the ratirometric signal is specific for selective HAs on the virus surface (Fig. 5).

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Figure 5. Fluorescence ratiometric change (where I400 and I600 are the fluorescence emission intensity of the probe at 400 and 600 nm, respectively) of (a) Supra-dot-23 (P-dot/DCM23: 0.125 ppm/2 μM) and (b) Supra-dot-26 (P-dot/DCM26: 0.125 ppm/2 μM) with different viruses including H3N2 (A/Beijing/353/89, 256 HAU 50 μL-1), H10N8 (Lake/Hunan/3-9/2007, 256 HAU 50 μL-1), H7N9 (A/Anhui/1/2013 derived HA and NA in the background of A/Puerto Rico/8/1934, 256 HAU 50 μL-1), AdC68 (simian adenovirus, 3 × 109 virus particles mL-1), AdHu7 (human adenovirus, 3 × 109 virus particles mL-1) and B/Phuket/3073/2013 (256 HAU 50 μL-1). The fluorescence was measured in Tris-HCl (0.01 M, pH 7.4) with an excitation wavelength of 380 nm.

Having demonstrated the ratiometric sensing ability of the Supra-dots for IVs, we tested whether the materials can also inhibit the infection of the viruses towards human cells. Since 10


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human-infecting IVs such as H3N2 are known to infect humans through binding to the α2,6sialylglycans that exist on the surface of human cells, we used a human lung cancer cell line (A549), which is known to express α2,6-sialylglycans on the cell surface,19,20 for the inhibition assay. The virus (H3N2) was pre-labeled with a known membrane-staining dye (DiL) for fluorescence tracking of viral infection towards A549. We first observed that incubation of DiL-labeled H3N2 with A549 largely enhanced the fluorescence of the cells, whereas use of the bird-infecting H10N8 did not enhance the cell fluorescence (Fig. S7). This suggests that the human-infecting H3N2 rather than bird-infecting H10N8 can effectively infect A549. Next, we used the Supra-dots and P-dot (as control) to treat with H3N2 prior to cell incubation. The result showed that whereas treatment of increasing P-dot (Fig. 6a and Fig. 6d) and Supra-23 (Fig. 6b and Fig. 6d) with the virus led to a slight fluorescence decrease of the cells, the incubation of increasing Supra-dot-26 (Fig. 6c and Fig. 6d) with H3N2 caused a much stronger fluorescence suppression. This suggests that the selective interaction between Supra-dot-26 and H3N2 could block virus infection towards human lung cells. The materials also did not show cytotoxicity towards A549 with concentrations well-above that used for virus blocking (Fig. S8). These preliminary results suggest the possibility of using supramolecular glycomaterials for IV inhibition.

Conclusions To summarize, we have developed a bifunctional supramolecular probe based on the simple aqueous self-assembly between sialylglycan DCM probes and emissive polymeric dots for the simultaneous sensing and blocking of IVs. Tuning the FRET process between P-dots and DCM probes through the selective recognition between sialylglycans and viruses enables a unique rati11



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ometric rationale for IV detection. In addition, the selective binding of Supra-dots comprising an α2,6-sialylglycan with a human-infecting IV largely inhibits the viral invasion towards a human cell line. This research paves the way for the simple construction of bifunctional supramolecular materials for the ratiometric sensing as well as blocking of acutely infectious viruses. As regards the virus specificity, note that while HA is also expressed in other viruses in addition to IV, the different HA proteins can mediate the viral entry via distinct receptors in host cells. For example, the HA of measles virus binds to CD150/SLAM, Nectin, and CD46 receptors on host cells,31 while rinderpest virus uses CD150/SLAM as the receptor.32 In contrast, IV initiates its infection by binding to α2,6- or α2,3-sialylglycans. This unique feature makes the probes developed particularly useful for blocking IV invasion towards human cells.

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Figure 6. Fluorescence imaging of A549 (human lung cancer cells) pretreated with DiL (1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-labeled (5 μM) H3N2 (8 HAU 50 μL-1) with (a) increasing P-dots (0-7 ppm), (b) increasing Supra-dot-23 (P-dot/DCM23: 0-7 ppm/10 μM) and (c) increasing Supra-dot-26 (P-dot/DCM26: 0-7 ppm/10 μM). The cell nuclei were stained by Hoechst 33342 (5 μg mL-1). (d) Fluorescence quantification of A549 cells with the different materials. The fluorescence images were recorded using an Operetta high content imaging system (Perkinelmer, US) at the excitation wavelengths of 360-440 nm and 520-550 nm and the emission wavelengths of 410-480 nm and 560-630 nm for Hoechst 33342 and DiL, respectively. The fluorescence of 560-630 nm was quantified and plotted by columbus analysis system (Perkinelmer, US). 13



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ASSOCIATED CONTENT Supporting Information. Additional figures (Fig. S1-S8) and experimental section including details for solution based tests and cell assays. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected] (X.-P. He) [email protected] (D.-M. Zhou)

ACKNOWLEDGMENT This research is supported by the 973 project (2013CB733700), the Natural Science Foundation of China (21572058 and 21576088), the Fundamental Research Funds for the Central Universities (222201717003) and the Shanghai Rising-Star Program (16QA1401400). Profs. Jia Li and Yi Zang at SIMM, CAS are warmly thanked for their help in cell imaging.

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TOC only

Internalization/ Replication

Recognition

Blocking FRET cancelled x

Binding/ Fusion x

x

Transmembrane glycoconjugates

Influenza virus Inhibited

x

Hemagglutinin

x

x

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x

19


Sialylglycan-Assembled Supra-Dots for Ratiometric Probing and

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