Targeting and Enrichment of Viral Pathogen by Cell Membrane

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Targeting and Enrichment of Viral Pathogen by Cell Membrane Cloaked Magnetic Nanoparticles for Enhanced Detection Hui-Wen Chen, Zih-Syun Fang, You-Ting Chen, Yuan I Chen, Bing-Yu Yao, Jui-Yun Cheng, Chen-Ying Chien, Yuan-Chih Chang, and Che-Ming J. Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09931 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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ACS Applied Materials & Interfaces

Targeting and Enrichment of Viral Pathogen by Cell Membrane Cloaked Magnetic Nanoparticles for Enhanced Detection Hui-Wen Chen2,4*, Zih-Syun Fang1,2, You-Ting Chen2, Yuan-I Chen1, Bing-Yu Yao1, Jui-Yun Cheng1,2, Chen-Ying Chien1, Yuan-Chih Chang3, and Che-Ming J. Hu1,4* 1. Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan 2. Department of Veterinary Medicine, National Taiwan University. 1 Sec. 4, Roosevelt Road, Taipei 10617, Taiwan 3. Institute of Cellular and Organismic Biology, Academia Sinica, No. 128, Sec. 2, Taipei 11529, Taiwan 4. Research Center for Nanotechnology and Infectious Diseases, Taipei, Taiwan.

E-mail: [email protected] and [email protected]

KEYWORDS: cell membrane cloaked nanoparticles, influenza virus, host-pathogen interaction, superparamagnetic iron oxide nanoparticles, membrane corona

ACS Paragon Plus Environment

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ABSTRACT

Attachment to cellular surfaces is a major attribute among infectious pathogens for initiating disease pathogenesis. In viral infections, viruses exploit receptor-ligand interactions to latch onto cellular exterior prior to subsequent entry and invasion. In light of the selective binding affinity between viral pathogens and cells, nanoparticles cloaked in cellular membranes are herein employed for virus targeting. Using influenza virus as a model, erythrocyte membrane cloaked nanoparticles are prepared and modified with magnetic functionalities (RBC-mNP) for virus targeting and isolation. To maximize targeting and isolation efficiency, density gradient centrifugation and nanoparticle tracking analysis were applied to minimize presence of uncoated particles and membrane vesicles. The resulting nanoparticles possess a distinctive membrane corona, a sialylated surface, and form colloidally stable clusters with influenza viruses. Magnetic functionality is bestowed to the nanoparticles through encapsulation of superparamagnetic ironoxide particles, which enable influenza virus enrichment via magnetic extraction. Viral samples enriched by the RBC-mNPs result in significantly enhanced virus detection by multiple virus quantification methods, including qRT-PCR, immunnochromatographic strip test, and cell-based titering assays. The demonstration of pathogen targeting and isolation by RBC-mNPs highlights a biologically inspired approach towards improved treatment and diagnosis against infectious disease threats. The work also sheds light on the efficient membrane cloaking mechanism that bestows nanoparticles with cell mimicking functionalities.


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1.

INTRODUCTION Plasma membranes of cells are a major biological interface that separates living systems

from the external environment. This dividing membrane barrier deters infectious threats from accessing cellular interior, and, as a result, many pathogens—such as parasites, bacteria, and viruses—have evolved to target cellular membranes, exploiting membrane-bound moieties for invasion, colonization, and dissemination1. The common host cell binding phenotype in hostpathogen interactions makes cell membranes an intriguing tool for designing therapies and diagnostics against pathogenic threats1. Recent advances in nanomaterials engineering have made possible the integration of cell membranes and synthetic nanoparticles through a nondisruptive cloaking approach. Cell membrane cloaked nanoparticles display multiple desirable characteristics, including unilamellar and right-side-out membrane coverage2-3, retention of membrane proteins4-6, and enhanced particle stability7-8. Prior works have shown utility of these biomimetic nanoparticles in targeting toxins9-12, immunoglobulin13-14, bacteria3, and cancer cells6, 15-19

. Given the role of host cell membrane binding in viral pathogenesis, we herein apply cell

membrane cloaked nanoparticles for virus targeting. Adaptation of the biomimetic platform with magnetic functionality was accomplished to facilitate pathogen isolation. Successful demonstration of virus targeting and isolation has practical implications in advancing antiviral therapies and diagnosis. As the cause of influenza disease, influenza viruses are negative-sense, single-stranded RNA viruses with well-studied interaction mechanisms against host cell membranes. Displayed by


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different types and subtypes of influenza viruses are variants of hemagglutinin proteins, which are viral glycoproteins with affinity to sialic acid residues20. In nature, sialic acids are abundantly expressed on the surfaces of cells as these sugar moieties serve a major role in modulating cells’ stability and viability21. The affinity between hemagglutinin and sialic acids give rise to many of influenza viruses’ attributes, including their tropism, infectivity, and their ability to agglutinate red blood cells (RBCs)22-23. Such interaction has inspired development of sialic acid-conjugated nanoparticles for virus targeting23-27. To demonstrate biomembrane-mediated nanoparticle/virus binding, sialic acid-rich RBC membrane is herein utilized for nanoparticle functionalization. We first developed a protocol to ensure the purity of RBC membrane cloaked nanoparticles (RBCNPs) with minimal uncoated particles and cell membrane vesicles. The resulting RBC-NPs were examined for surface sialyl moieties using immunogold staining and for influenza virus interactions using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Magnetic functionality was incorporated into the nanoparticles (RBC-mNPs) via the encapsulation of superparamagnetic iron oxide nanoparticles (SPIONs) for the isolation of the targeted pathogen (Figure 1). Lastly, utility of the RBC-mNP was shown via enhancement of virus detection with multiple diagnostic assays. In addition to demonstrating nanoparticlemediated virus targeting, the study offers mechanistic insights into the efficient and ordered assembly of the cell membrane cloaked nanoparticles. The energetic basis behind the cell membrane cloaking is discussed.

2. EXPERIMENTAL METHODS 2.1. Preparation of red blood cell membrane vesicles. RBC membrane was derived from blood of BALB/c mice. To isolate RBCs, EDTA-anticoagulated blood was centrifuged at 800 ×


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g for 5 min at 4°C, following which the serum and the buffy coat were removed. The collected RBCs were resuspended in 0.25X PBS for 3 min, following which the solution was adjusted to 1X PBS and sample was centrifuged at 800 × g for 5 min for membrane ghost isolation. The purification process was repeated 3 times to remove intracellular proteins. The resulting membrane ghosts were sonicated using an FS30D bath sonicator (Fisher Scientific) at a frequency of 42 kHz and a power of 100 W for 5 min to generate membrane vesicles. Size, zeta potential, and particle number of the membrane vesicles were characterized using dynamic light scattering (ZEN 3600 Zetasizer, Malvern) and nanoparticle tracking analysis (NanoSight NS500, Malvern). 2.2. Preparation of PLGA nanoparticles, PEG-PLGA nanoparticles, and iron-oxide loaded PLGA nanoparticles. PLGA nanoparticles were prepared using 0.67 dL/g carboxy-terminated 50∶50

poly(DL-lactide-co-glycolide)

(LACTEL

Absorbable

Polymers).

PEG-PLGA

nanoparticles were prepared using poly(ethylene glycol) methyl ether-block-poly(lactide-coglycolide) (PEG average Mn 5000, PLGA Mn 7000; Sigma-Aldrich). For 120 nm PLGA cores, the PLGA polymer was dissolved in acetone at a 10 mg/mL concentration. To prepare the desired PLGA cores via nanoprecipitation, 1 mL of the solution was added rapidly to 3 mL of water. The mixture was stirred in open air for 2 h. For 180 nm PLGA cores and PEG-PLGA nanoparticles, a single emulsion process was used. 10 mg of either the PLGA or the PEG-PLGA were dissolved in 1 mL of dichloromethane. The polymer solution was added to 3 mL of water and sonicated at 80% amplitude for 20 seconds using a probe sonicator (Q500 Sonicator, Qsonica Sonicators). For SPION incorporation, 1 mg of 10 nm iron oxide nanoparticles (Sigma Aldrich) was dissolved with 10 mg of PLGA in 1 mL of dichloromethane prior to the single emulsion process. The resulting emulsions were left in open air under gentle stirring for 3 h for


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solvent evaporation. Nanoparticles were washed using Amicon Ultra-15 Centrifugal Filter Units (Millipore) with a 100,000 kDa MWCO prior to cloaking with cellular membranes. 2.3. Cell membrane cloaking on PLGA nanoparticles. RBC membrane cloaking was accomplished by dispersing and fusing RBC membrane vesicles with PLGA particles via sonication using a bath sonicator at a frequency of 42 kHz and a power of 100 W for 2 min. Excess RBC membrane as verified by NTA was used to ensure complete particle coverage. Following nanoparticle cloaking, excess membrane was removed via centrifugation at 30,000 × g for 5 min with the sample solution layered above a 40 wt% sucrose solution. The resulting pellet was collected and dispersed for size and the surface zeta potential characterizations using DLS and NTA. 2.4. Influenza virus preparation. A/PuertoRico/8/34(H1N1) was a gift from Dr. Shin-Ru Shih at the Chang-Gung University, Taoyuan, Taiwan. The virus was propagated in 10-day-old specific-pathogen-free (SPF) chicken embryos (JD-SPF Biotech, Miaoli, Taiwan) via the allantoic route as previously described28. The virus-containing allantoic fluid (AF) was concentrated by ultra-centrifugation and purified through 20%-50% sucrose gradient solution to derive the native virions. 2.5. Transmission electron microscopy. Negative staining was performed with 1 wt% uranyl acetate for structural examination of nanoparticles and nanoparticle/virus clusters. Immunogold staining was performed using biotin-labeled Sambucus Nigra lectin (Vector Laboratories) and streptavidin-conjugated 5 nm gold nanoparticle (cytodiagnostics) to visualize surface sialyl groups on nanoparticles. Briefly, a drop of the RBC-NP solution (1 mg/mL) was deposited onto a glow-discharged grid, which was subsequently blocked with 1% BSA for 15 min. The grid was


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then stained with 0.1 mg/mL biotin-labeled Sambucus Nigra lectin for 1 h. Following rinsing with 1% BSA, the sample grid was stained with streptavidin-conjugated 5 nm gold nanoparticles. All the staining steps were performed at room temperature. Negatively stained and immunogold stained samples were visualized using an FEI 120 kV Sphera microscope (FEI Tecnai F20). Cryo-EM images were obtained from frozen hydrated sample of RBC-NP/virus mixture. 3 µL of the sample was added to a glow-discharged holey carbon grid, and the sample grid was plunged into liquid ethane. The sample was imaged using a Tecnai F20 transmission electron microscope (FEI Company, the Netherlands) operating at an acceleration voltage of 200 kV. 2.6. Influenza virus binding and isolation by RBC membrane cloaked nanoparticles. For qualitative assessment of nanoparticle binding with influenza virus, nanoparticles and native virions were incubated at a 1:1 molar ratio. 1 mL of PBS solution containing 3 × 1011 of nanoparticles (1 mg) and influenza viruses was incubated for 30 min at room temperature prior to sample evaluation by DLS, NTA, and TEM. For virus isolation by RBC-mNP in PBS, 5 × 1011 virions in 1 mL PBS were mixed with 0.15, 0.5, 1, 2, and 5 mg of RBC-mNPs in roundbottomed polypropylene tubes (Fisher Scientific) for 30 min at 4°C prior to magnetic separation. The tubes were subsequently placed in EasySep Magnet (Stemcell Technologies) for 30 min at 4°C, following which the supernatant was removed and the sample was rinsed gently with PBS prior to resuspension. The washed extract was then resuspended in PBS for analysis by qRTPCR and TEM. For virus enrichment studies, plasma was first derived from BALB/c mice and spiked with influenza virus. 1 mL of plasma samples containing 1 × 1011 virions were incubated with 5 mg of RBC-mNPs in a round-bottomed polypropylene tube for 30 min at 4°C. Subsequently, the samples underwent magnetic isolation in EasySep Magnet for 30 min at 4°C. The extracted materials were resuspended in 50 µL of RNA extraction buffer for qRT-PCR, in


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PBS buffer for virus titration studies, or in lysis buffer for the immunochromatographic strip test. Plasma samples containing non-enriched viruses and RBC-mNPs only were prepared as controls. Non-enriched virus samples were prepared by directly withdrawing 50 µL samples from the 1 mL virus-spiked mouse plasma prior to processing for the virus quantification assays. 2.7. Quantitative RT-PCR of influenza virus. The quantitative RT-PCR of influenza virus was conducted as previously described29 with some modifications. Briefly, viral RNA was extracted using TriSolution Reagent Plus (GMbiolab, Taipei, Taiwan) according to the manufacturer's manual. Then cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) and random hexamer (Invitrogen), and the transcribed cDNA was used for quantitative RT-PCR. Real-time qPCR was performed with Prime Time 2X master mix (Integrated DNA Technologies) using previously published primers and probes that target the M1 gene of influenza A virus30. The reaction mixture was heated at 95°C for 3 min and underwent 45 thermal cycles of 95°C for 15 s and 60°C for 1 min on the CFX Connect Real-Time detection system (Biorad). All reactions were set up in duplicate and Ct values were obtained. 2.8. Immunochromatographic strip test. The viral antigen in mouse plasma samples was detected by the Quick-Navi-Flu strip test (Denka Seiken, Japan) according to the manufacturer’s protocol. 2.9. Influenza virus titration and plaque assays. The virus titers were determined in MardinDarby canine kidney (MDCK) cells and expressed as 50% tissue culture infectious dose (TCID50), and plaque assays were performed as previously described31. Briefly, MDCK cells (CCL-34, ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (all reagents were from Invitrogen) at 37°C with


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5% CO2. For TCID50 determination of virus, enriched and non-enriched virus samples were 10fold serially diluted in the medium for viral infection (DMEM containing 0.2% BSA, 2 µg/ml TPCK-treated trypsin, and 25 mM HEPES buffer) and incubated with MDCK cells for 1 hr. After washes, cells were further incubated for 3 days. The Reed-Muench method was used to determine the 50% endpoint by performing a hemagglutination assay for each culture supernatant32. For plaque assays, MDCK cells were infected with 10-fold serially diluted virus samples for 1 hr. After washes, the cells were overlaid with 1% agarose solution (Agarose B, Bio Basic) and incubated for 3 days. Cell monolayers were fixed with 10% formalin, and plaque formation was visualized following staining with 1% crystal violet. 3. RESULTS Cell membrane cloaked nanoparticles are formed via coupling of two similarly sized nanoparticulates, namely PLGA cores and cell membrane vesicles. The resulting sample may thus contain three distinctive nanoparticle populations. As impurities could compromise sample performance and complicate experimental analysis, a protocol was developed to improve the purity of the cell membrane cloaked nanoparticle formulation. To ensure a sufficient membrane to polymer ratio for complete particle cloaking, RBC membrane vesicles in excess of the total surface area of PLGA nanoparticles were used. Following preparation of PLGA cores and RBC membrane vesicles, the two components were quantified using NTA prior to the cloaking process. In the example shown in Figure 2A, 200 µg of PLGA and membrane vesicles derived from 50 µL of mice blood were used to prepare 120 nm nanoparticles and 150 nm membrane vesicles. The starting materials yielded 2.17±0.14 × 1011 nanoparticles and 3.34±0.09 × 1011 membrane vesicles, corresponding to a membrane to particle ratio of approximately 2:1 in terms


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of membrane to particle surface area ratio. The total particle number was examined throughout the particle preparation, and a reduction in total particle number was observed in the particle/membrane vesicle mixture following ultrasonic dispersion (5.18±0.23 × 1011 before sonication and 4.29±0.14 × 1011 after sonication) (Figure 2A). This change in particle number indicates fusion between the two particle populations and highlights the role of the dispersion process in providing the needed energy for the fusion process. The sonicated mixture then underwent purification by centrifugation in 40% sucrose, the density of which (1.18 g/cm3) is in between that of plasma membrane (1.05 g/cm3) and PLGA particles (1.27 g/cm3). Free membrane vesicles layered above the sucrose layer was removed. Quantification of purified RBC-NPs yielded 1.91±0.08 × 1011 particles, corresponding to 90% recovery of the starting PLGA nanoparticle sample. Particle analysis by dynamic light scattering (DLS) showed that the collected RBC-NPs (140.4±4.2 nm; -24.6±3.1 mV) were 19.4 nm larger in diameter compared to the bare NP (121.0±3.0 nm; -48.3±1.9 mV) and adopted a less negative zeta potential (Figure 2B). RBC-NPs were further compared to bare NPs and RBC membrane vesicles under NTA, in which particle diameter and zeta potential were measured simultaneously on a particle-byparticle basis. Visualization of particle distribution under NTA showed that the RBC-NP population was distinct from the RBC membrane vesicles and bare NPs (Figure 2C,D). Both RBC-NPs and bare NPs possessed sharper size distributions as compared to the membrane vesicles, reflecting the monodisperse nature of the polymeric cores. Between the RBC-NPs and the bare NPs, the RBC-NPs had an elevated overall zeta potential, which is indicative of shielding of the anionic PLGA surfaces by the cell membrane cloak. Characterization by NTA (Figure 2C,D) offers improved resolution on the particle purity as compared to the conventional DLS measurements (Figure 2B).


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To verify cell membrane cloaking and presence of surface sialyl groups on RBC-NPs, transmission electron microscopy (TEM) was performed on both bare NPs and RBC-NPs using negative staining and immunogold staining against sialic acids. Under negative staining, RBCNPs revealed a clear core-shell structure as compared to bare NPs (Figure 2E; top panel), indicating successful surface cloaking by erythrocyte membranes. Under immunogold staining, multiple electron-dense gold particles were observed to cluster around RBC-NPs, and very few immunogold particles were found on bare NPs (Figure 2E; bottom panel). The TEM images indicate that the RBC-NPs had a glycan-rich exterior, which offer mechanistic insights behind the dynamics of the membrane cloaking. As the glycan layer consists of hydrophilic polysaccharides that serve as a major stabilizing component among cells and proteins33, the membrane cloak may provide a stabilizing effect that renders nanoparticles more energetically favorable. This stabilizing glycan layer helps explain the efficient cloaking process and the membrane-mediated nanoparticle stabilization that have been consistently observed in prior studies with the cell membrane cloaked nanoparticles13,

34

. The right-side-out membrane

orientation on these nanoparticles can also be explained by the presence of the membrane-bound glycans2-3, which are located asymmetrically on the exoplasmic side of cellular membranes. Considering the steric effect, hydrophilicity, and electrostatic properties of these polysaccharides, an inside-out membrane cloak exposing the glycan layer inward against the PLGA nanoparticle substrate would be energetically unfavorable. It may thus be reasoned that these surface glycans play an important role in facilitating proper membrane cloaking, enabling cell-mimetic nanoparticle biointerfacing towards pathogen targeting. To assess nanoparticle binding with influenza viruses, similarly sized RBC-NPs (D= 181.2 nm; PDI=0.072) and PEG-coated nanoparticles (PEG-NPs) (D=176.3 nm; PDI=0.111)


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were prepared. The nanoparticles were mixed with influenza viruses (D=140.3 nm; PDI=0.102) at a 1:1 particle-to-particle ratio, and the resulting samples were analyzed using both DLS and NTA. Under DLS, the mixture with RBC-NPs and influenza viruses yielded an average diameter of 200.0 nm with a PDI of 0.202. This increase in size indicated association between the viral particles and RBC-NPs. In contrast, PEG-NP/virus mixture had a diameter of 159.7 nm with a PDI of 0.098 (Figure 3A). NTA also revealed selective binding between RBC-NPs and influenza viruses. By examining the proportion of particles that were over 200 nm, it was observed that the RBC-NPs, influenza viruses, and their mixture had 4.8%, 3.7%, and 27.8% of the total particle population over the threshold, respectively. The mixture of PEG-NP and influenza viruses, on the other hand, had 6.8% of its population over the threshold (Figure 3B). No visible particle aggregation was observed in any of the samples. To further examine the interactions between RBC-NPs and influenza viruses, TEM was performed. Upon negative staining, influenza viruses with their characteristic spiky surfaces were found around the core-shell structures of RBC-NPs. Close contacts between viruses and RBC-NPs were observed, suggesting adhesive interactions between the two particles (Figure 3C; top panel). Both monovalent and polyvalent virus attachments were observed, reflecting the heterogeneity behind the clustering dynamics. To rule out non-specific particle clustering as a consequence of sample drying, cryo-EM was further applied to examine the virus/particle mixture. The cryo-EM images are consistent with the TEM observations, revealing clustering of multiple influenza viruses around nanoparticles (Figure 3C; bottom panel). Close scrutiny of the cryo-EM images shows some of the influenza viruses adjacent to nanoparticles had induced surface curvatures, which suggest attractive interactions and multivalent binding. In contrast, mixtures containing influenza viruses and PEG-NPs did not yield virus/particle clusters


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(Supplementary Figure 1). It should be noted that no discernible core-shell structure was observed for RBC-NPs under cryo-EM. The lack of visible structural feature may be attributed to the close electron density between cellular membranes and PLGA, the small spacing of the membrane/particle interface, and the limited resolution of the cryo-EM setup (~10 nm). RBCNPs also appear bigger under negative staining as compared to cryoEM, which can be attributed to staining artifact and particle collapse under sample drying. Based on the results from DLS, NTA, TEM, and cryo-EM, it can be concluded that the cell membrane functionalization enabled RBC-NPs to form colloidally stable clusters with influenza viruses. To bestow RBC-NPs with magnetic functionalities, SPIONs were incorporated inside the PLGA particles via a single emulsion process for the preparation of RBC-mNPs. DLS characterization showed that the SPION-loaded PLGA nanoparticles (magnetic NP; mNP) underwent similar transformation as bare PLGA particles following cell membrane cloaking, adopting an increased particle size (194.4±4.2 nm vs. 227.0±7.0 nm) and a less negative zeta potential (-48.6±3.1 mV vs. -34.3±2.9 mV) (Figure 4A). Under TEM, both mNPs and RBCmNPs showed dark punctates in the particle interior, indicating successful encapsulation of the electron-dense SPIONs inside the PLGA matrix (Figure 4B). SPION incorporation did not affect the membrane-particle cloaking dynamics as a distinctive membrane corona was observed on the RBC-mNPs. The magnetic property of the resulting RBC-mNPs was assessed by evaluating particle extraction upon application of a magnetic field. Magnetic separation of RBCmNPs was visually confirmed upon placement of a magnet next to the particle solution (Figure 4C). NTA was further applied to evaluate the quality of the RBC-mNPs. Following 30 min of particle extraction in a commercial magnetic base, solutions with different particle concentrations were separated into extracted particle pellets and supernatants containing non-


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extracted particles. For 1 mL solutions containing 1, 3, and 5 mg of RBC-mNPs, the extracted fractions contained 2.7 × 1011, 8.4 × 1011, and 13.9 × 1011 particles, and the supernatants had 1.0 × 1010, 3.6 × 1010, and 3.1 × 1010 particles, respectively (Figure 4D). The quantification result indicates more than 95% of the total RBC-mNPs had magnetic functionality, thereby confirming uniform SPION incorporation in the sample. The high percentage of magnetic RBC-NPs is important towards isolation of desired samples as presence of non-magnetized particles would reduce sample extraction efficiency. To quantitatively assess RBC-mNPs’ ability to bind and extract influenza viruses, a fixed number of influenza viruses (5.0 × 1011) were mixed with different concentrations of RBC-mNPs. At 0.15, 0.5, 1, 2, and 5 mg/mL of RBC-mNPs, the particle to virus ratios were 0.075, 0.25, 0.5, 1, and 2.5, respectively. Following magnetic isolation using a magnetic base, total viral RNA was quantified using qRT-PCR and virus extraction efficiency was calculated by comparing the extracted viral RNA to the initial input. The addition of RBC-mNPs did not influence the qRTPCR readings in virus-positive and virus-negative samples (Supplementary Figure 2). With higher concentrations of RBC-mNPs, virus extraction efficiency increased proportionally as indicated by the fluorescence levels in the real-time PCR result (Figure 4E). Near complete virus extraction was achieved with 5 mg/mL of the RBC-mNPs (Figure 4F). Extraction of influenza viruses was further confirmed via TEM visualization of isolated samples. Clusters of virions and RBC-mNPs were observed, indicating that both the nanoparticles and viruses remained intact in structure through the sample isolation process (Figure 4G). These results demonstrate successful targeting and isolation of influenza viruses by RBC-mNPs, which integrate magnetic functionalities with cell membrane interface to exploit the host cell binding phenotype for pathogen extraction.


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Virus enrichment in biological samples for downstream viral identification was further demonstrated with the RBC-mNPs using multiple detection assays based on viral RNA, viral antigen, and viral infectivity. Mouse plasma samples containing a low titer of influenza virus were examined for virus enrichment upon incubation and magnetic isolation with RBC-mNPs. Comparing to non-enriched samples, virus-enriched samples revealed 4.2-fold (p=0.0004) higher level of viral RNA (Figure 5A), and visible positive signal in the rapid viral antigen test (Figure 5B). Notably, enriched viral samples showed a 14.8-fold enhancement in viral titer (p=0.0238) (Figure 5C) and enhanced plaque formation (Figure 5D) in cell-based assays. Plasma samples containing RBC-mNPs without viruses did not yield any positive detection results. Also, plasma samples containing RBC-mNPs and viruses without magnetic enrichment did not improve the virus quantification in these assays (data not shown). The different viral features and functionalities examined by the assay tests collectively highlight the non-disruptive nature of the RBC-mNP-mediated virus enrichment. The qRT-PCR assay revealed that the captured viruses retained their viral RNA for amplification and quantification; the rapid strip test showed that the enriched viruses preserved viral antigens for affinity binding; the viral titering and plaque assay indicated that the captured viruses kept their infectivity. These results have practical implications in virology research and clinical diagnostics, in which enrichment of non-disrupted pathogen samples is valuable towards downstream analysis.

4. DISCUSSION Virus attachment to cellular surfaces is an indispensable mechanism through which viruses gain entries into target cells; this mechanism offers a design inspiration towards


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engineering virus-targeted formulations. It was previously reported that targeting of viral proteinexpressing cells could be enhanced using cell membrane derived vesicles35. The present study expands upon the concept using cell membrane cloaked nanoparticles, which incorporates polymeric cores for versatile cargo encapsulation. Nanoparticles outfitted with an erythrocyte membrane coat were demonstrated to target influenza viruses, and SPIONs were incorporated inside the RBC-NPs for virus isolation via magnetic extraction. In addition to the multiple assays that are demonstrated to be compatible with the particle-enriched sample, the biomimetic magnetic particles may also be adapted for rapid pathogen detection using magnetic relaxation switching for monitoring pathogen-induced particle clustering36. Given the functionalizability of the cell membrane cloaked nanoparticles, extension of the present work with alternative membrane cloaks and theranostic cargoes for different pathogen-targeting applications can be envisioned. As numerous viral threats, such as human immunodeficiency virus, MERS-CoV, and dengue virus, display preferential binding affinity to membrane-bound receptors37-38, the present work may be adapted for different antiviral applications. The bioinspired approach of targeting the membrane-binding phenotype rather than specific epitopes may be less susceptible to antigenic drifts and variations among viral strains. The present platform may also be adapted to enrich other biological targets of interest, including chemicals, proteins, bacteria, or cells with known affinity to specific membrane types. Influenza virus infection is a continuously evolving public health threat that has prompted ongoing technology development for disease treatment and diagnosis. Among nanoparticle-based anti-influenza strategies, sialic acid functionalized nanoparticles, such as sialylated dendrimers2627

and gold nanoparticles23-25, 39-40, have been prepared for virus inhibition and detection. In the

present work, biomimetic glycosylation on nanoparticles was accomplished using a membrane


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cloaking technique. The use of RBC membranes yielded highly sialylated PLGA nanoparticles for influenza virus targeting. Virus-containing samples enriched by the RBC-mNPs retain viruses’ morphology and infectivity, thereby permitting downstream analysis. The lack of inhibitory effect of the RBC-mNPs is consistent with a recent report on virus-inhibiting glycoconjugates, which shows that a small particle geometry (4.5 nm in diameter) is needed to avoid the hydrolytic activity of virus-bound neuraminidases responsible for sialic acid cleavage26. As the RBC-mNPs are over 100 nm in diameter, virus dissociation due to neuraminidase activity likely accounts for the retained viral infectivity in the enriched samples. Towards future platform development for virus inhibition, incorporation of neuraminidase-inhibiting compounds (eg. oseltamivir) with the RBC-NPs may be considered. The prospect of enhancing antiviral efficacy via the biomimetic platform presents an intriguing anti-influenza approach that warrants future investigations. The cell membrane coating approach adopted in the present study is an emerging functionalization strategy that has bestowed nanocarriers with immunocompatibility and targeting capabilities41. Preparation and purification of the resulting nanoparticles, however, can be encumbered by the need to distinguish three physicochemically similar nanoparticulates that include the desired biomimetic nanoparticles, uncoated nanoparticles, and membrane vesicles. By employing density gradient centrifugation and NTA, improved quality control of cell membrane coated nanoparticles was demonstrated. Enumerating both nanoparticle cores and membrane vesicles prior to the cloaking process ensures sufficient cell membrane content for complete nanoparticle coverage. This approach can be particularly useful to formulations involving nucleated cells as their derived plasma membranes are difficult to quantify6, 15, 42-43. Upon nanoparticle cloaking, RBC-NPs were purified using sucrose gradient centrifugation. The


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purification step removes excess membrane vesicles that would otherwise compete with the cell membrane cloaked nanoparticles for their intended targets. This step is critical to applications involving sample enrichment as impurity would significantly compromise isolation yield. Lastly, quality assurance of the RBC-NPs was performed via coordinated measurements of particle size and zeta potential. The particle-by-particle analysis offers enhanced resolution over particle populations, allowing for better identification of specific nanoparticle types. These techniques serve to overcome multiple technical hurdles in developing cell membrane cloaked nanoparticles. While cell membrane cloaked nanoparticles have been applied in multiple biomedical applications41, few studies have discussed the mechanisms and energetics behind the membrane cloaking process. In the case of the PLGA nanoparticles in the present study, it may seem counterintuitive that the negatively charged particles can associate efficiently with the likecharged membrane vesicles. Yet membrane cloaking on these particles has been shown to be ordered and efficient, leading to a high yield of biomimetic nanoparticles with unilamellar, rightside-out membrane cloaks2-3. These observations suggest that the cloaking process is not a simple, passive membrane encapsulation but rather an enthalpy driven process. Visualization of the surface glycans on the RBC-NPs (Figure 2E) sheds light on one of the driving forces behind the membrane cloaking. The carbohydrates on the exoplasmic side of the cell membranes are hydrophilic polymers that can enhance stability of colloids in suspension. This glycan layer provides a stabilizing effect and favors right-side-out membrane coverage upon association with nanoparticles. For the nanoparticles to efficiently couple with cell membranes, the polymeric cores must be of lesser stability, or possess higher surface free energy in other words, than the glycosylated membranes. Upon application of a dispersive force that exposes the endoplasmic


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side of the membrane vesicles, a membrane cloak spontaneously forms around the nanoparticle, releasing free surface energy in the process. A parallel can be drawn between the membrane cloaking mechanism and the well-known phenomenon of protein corona formation44-46. Nanoparticles have been shown to rapidly associate with biomolecules owing to a combination of interaction forces. As a result, through a process that relieves surface free energy, nanoparticles in complex biological media are encased in a layer of proteins that dictate the particles’ biological identity. The RBC-NPs and other cell membrane cloaked nanoparticles can be pictured as particles with “membrane coronas” that provide enhanced colloidal stability and cell-like functionalities. It should be noted that much like protein coronas can be influenced by nanoparticle size, charge, rigidity, and chemical functionalization47, membrane coronas can likewise be affected by these factors. For instance, inside-out and multilamellar cell membrane coatings have been observed on particles with varied compositions and surface chemistries43, 48, indicating not all cell membrane/particle complexations are created equal. Currently, a multitude of inorganic and organic nanomaterials, including iron oxide, silica, gold, PLGA, and gelatin, have been formulated with cell membranes for functionalization3, 7, 19, 43, 49. As scientists continue to unravel the biophysicochemical interactions at the nano-bio interface, formation of cell membrane coronas on different nanomaterials may offer an intriguing approach towards elucidating the surface dynamics of synthetic materials. Through information including membrane sidedness, lamellarity, fluidity, and protein functionalities, these membrane coronas may help delineate the intricate forces surrounding a nanoparticle. These understandings would further contribute to the platform development towards innovative theranostics designs.

5.

CONCLUSIONS


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We have demonstrated a bioinspired nanoparticle-based approach targeting and enrichment of viral pathogen. By integrating cell membrane, PLGA polymers, and SPIONs using a refined nanoparticle preparation and characterization protocol, purified RBC-mNPs were prepared. With a sialic acid rich exterior and magnetic functionality, the nanoparticles enable for influenza virus targeting and isolation. These nanoparticles exploit host-pathogen interactions for virus binding, enabling extraction and enrichment of non-disrupted viral samples. Extracted virus remained infective and could be analyzed using conventional diagnostic assays. The work highlights cell membrane cloaked nanoparticles’ unique function in exploiting pathogens’ membrane binding phenotype for targeting. Adaptation of this pathogen-targeting concept is expected to pave ways to novel treatments and diagnostics against infectious diseases. The study also offers methodologies and insights towards understanding and improving the preparation of the cell membrane cloaked nanoparticle platform. Examination of membrane coronas is herein proposed as an alternative approach towards elucidating and defining nanoparticle surface dynamics.

Supporting Information. Additional figures.


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FIGURES

Figure 1. Schematic illustration of erythrocyte membrane cloaked magnetic nanoparticles (RBC-mNPs) for influenza virus isolation. (A) The RBC-mNPs are comprised of SPIONloaded PLGA nanoparticles cloaked in erythrocyte membranes. The nanoparticle surface is rich in sialic acids for influenza virus binding. Magnetic functionality facilitates virus isolation and enrichment via magnetic extraction. (B) Virus binding and enrichment are accomplished by mixing RBC-mNPs with viral samples followed by magnetic extraction.


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Figure 2. Preparation and evaluation of purified RBC-NPs. (A) Enumeration of particles in different reaction mixtures throughout the RBC-NP preparation by NTA. (B) Size and zeta potential characterizations of bare nanoparticles, RBC membrane vesicles, and RBC-NPs by DLS. (C) Particle-by-particle analysis with coordinated measurements of size and zeta potential by NTA highlights the distinctive populations for the bare nanoparticles, RBC membrane vesicles, and RBC-NPs. (D) A 3D visualization of the distinctive populations of bare nanoparticles, RBC membrane vesicles, and RBC-NPs. (E) Visualizations of bare nanoparticles and RBC-NPs under TEM following negative staining and immunogold staining against sialic acids. The negative staining and immunogold staining reveal a unilamellar membrane corona and a sialic-acid-rich surface on the RBC-NP. Scale bars = 50 nm.


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Figure 3. Evaluation of nanoparticle binding to influenza virus. (A) Size measurements of RBC-NPs (left) and PEG-NPs (right) before and after mixing with influenza viruses as measured by DLS. (B) Size measurements of RBC-NP (left) and PEG-NP (right) before and after mixing with influenza viruses as measured by NTA. (C) TEM (top) and cryo-EM images (bottom) validate binding interactions between RBC-NPs and influenza viruses. Scale bars = 100 nm.


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Figure 4. Preparation of RBC-mNPs and magnetic extraction of influenza virus. (A) Size and zeta potential of bare mNPs and RBC-mNPs analyzed by DLS. (B) TEM images validates SPION encapsulation in the polymeric cores and a unilamellar membrane corona on RBC-mNPs (right). (C) Magnetic property of RBC-mNPs was visually confirmed upon placement of a magnet to the particle solution. (D) Particle enumeration shows that >95% of the RBC-mNPs are magnetically extractable in a commercial magnetic isolation base. (E) qRT-PCR analysis of virus content shows a positive correlation between virus extraction efficiency and RBC-mNP content. Arrow indicates increasing concentrations of RBC-mNPs ranging from 0, 0.15, 0.5, 1, 2, and 5 mg/mL. (F) Virus extraction efficiency by different RBC-mNP content was quantified via qRTPCR analysis. (G) TEM images of virus samples extracted by RBC-mNPs shows clusters of influenza viruses and RBC-mNPs. Scale bars = 100 nm.


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Figure 5. Enrichment of influenza virus by RBC-mNPs and analysis by influenza diagnostic assays. Mouse plasma containing a low titer of influenza virus was subjected to RBC-mNP enrichment. (A) qPCR analysis shows increased viral RNA content following enrichment by RBC-mNPs. (B) A strip-based rapid flu testing shows that sample enrichment by RBC-mNPs improves virus detection as evidenced by the enhanced signal for the presence of influenza type A viral antigen (arrowed). The samples were prepared from 1 mL of mouse serum containing 4x108 virions (0.66 pM). 50 µL of sample solutions were taken before and after nanoparticle enrichment for the rapid strip test. The presence of the blue line indicates a valid lateral flow assay. (C) TCID50 assay shows an increased infectious titer of influenza virus following RBC-mNP enrichment. (D) Plaque assay demonstrates that, following RBC-mNP enrichment, the viral sample generated increased plaque formation than non-enriched sample under the same dilution factors. (10X and 100X sample dilutions are shown in the representative figures).


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AUTHOR INFORMATION Corresponding Author *Correspondence should be addressed to [email protected] and [email protected]

Author Contributions H.-W. C., Z.-S. F., and C.-M.J.H. conceived and designed the experiments; H.-W.C., Z.-S. F., Y.-T.C., Y.-I.C., B.-Y.Y., J.-Y.C., C.-Y.C., Y.-C.C., and C.-M.J.H. perform all the experiments. The manuscript was written through contributions H.-W.C., Z.-S. F., and C.-M.J.H. All authors have given approval to the final version of the manuscript. Funding Sources The authors acknowledge funding support by the Ministry of Science and Technology, Taiwan (105-2119-M-001-014), Academia Sinica Career Development Award, and the National Taiwan University (106R104515).

ACKNOWLEDGMENTS The authors acknowledge support from the Imaging Core Facilities at the Institute of Cellular and Organismic Biology of Academia Sinica for assistance on TEM image acquisition.


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BRIEFS Cell-mimetic magnetic nanoparticles exploit viruses’ host-cell binding phenotype for virus capture and enhanced detection.


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Table of Contents graphic

Non-enriched

Enriched

Magnetic Isolation

Biomimetic Virus Targeting

Virus Capture

Enhanced Detection


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Targeting and Enrichment of Viral Pathogen by Cell Membrane

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