Glycosaminoglycans-Specific Cell Targeting and Imaging Using

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Glycosaminoglycans-Specific Cell Targeting and Imaging Using Fluorescent Nanodiamonds Coated with Viral Envelope Proteins Minh D. Pham, Chandra Prakash Epperla, Chia-Lung Hsieh, Wen Chang, and Huan-Cheng Chang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 26 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017

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Analytical Chemistry

Glycosaminoglycans-Specific Cell Targeting and Imaging Using Fluorescent Nanodiamonds Coated with Viral Envelope Proteins

Minh D. Pham,1,2 Chandra Prakash Epperla,1,3,4 Chia-Lung Hsieh,1 Wen Chang,5 and Huan-Cheng Chang*,1,3,6

1

Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan

2

Institute of Biotechnology, Vietnam Academy of Science and Technology, 18-Hoang Quoc

Viet, Cau Giay, Ha noi, Vietnam 3

Taiwan International Graduate Program – Molecular Science and Technology, Academia

Sinica, Taipei 115, Taiwan 4

Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan

5

Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan

6

Department of Chemical Engineering, National Taiwan University of Science and

Technology, Taipei 106, Taiwan

ABSTRACT Understanding virus-host interactions is crucial for vaccine development.

This study

investigates such interactions using fluorescent nanodiamonds (FNDs) coated with vaccinia envelope proteins as the model system. To achieve this goal, we noncovalently conjugated 100-nm FND with A27(aa 21–84), a recombinant envelope protein of vaccinia virus, for

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glycosaminoglycans (GAGs)-specific targeting and imaging of living cells.

Another

recombinant protein A27(aa 33–84) that removes the GAGs-binding sequences was also used for comparison. Three types of A27-coated FNDs were generated, including A27(aa 21–84)FND, A27(aa 33–84)-FND, and hybrid A27(aa 21–84)/A27(aa 33–84)-FND. The specificity of these viral protein-FND conjugates toward GAGs binding was examined by flow cytometry, fluorescence microscopy, and gel electrophoresis. Results obtained for normal and GAGs-deficient cells showed that the recombinant proteins maintain their GAG-targeting activities even after immobilization on the FND surface.

Our studies provide a new

nanoparticle-based platform not only to target specific cell types, but also to track the fates of these immobilized viral proteins in targeted cells as well as to isolate and enrich GAGsassociated proteins on cell membrane.

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INTRODUCTION Viruses are known for their ability to specifically identify suitable target cells for infection initiation and propagation in a host. This is established mostly via the specific interactions between viral and host cell surface components that are under constant selection pressure in natural evolution.1 Understanding the virus-host interactions is crucial for the development of cell-specific delivery vehicles such as viral-like particles (VLPs) to mimic virus entry strategies in order to deliver drugs, DNAs, or RNAs in a targeted manner.2,3 Viral envelope proteins that mediate virus entry steps are particularly interesting from a chemist’s perspective because they are easier to produce than VLPs, retain high binding specificity to host cells, and are biologically safe with no infectivity. Thus, we rationalize that viral envelope protein-coated synthetic nanoparticles may provide an appealing alternative to VLPs and are worthy of studying in great detail. Fluorescence imaging is a commonly used technique to track the viral infection process.4 However, rapid photobleaching of fluorescent labels, which mainly consist of organic dye or fluorescent protein molecules, hampers their long-term observations. An elegant way to overcome this obstacle is to coat quantum dots (QDs) with viral capsid proteins.5-7 Dragnea and coworkers have explored several strategies to incorporate QDs into brome mosaic virus capsids and found that particles functionalized with poly(ethylene glycol) can be readily encapsulated by self-assembly of the capsid proteins in three dimensions.5 They proposed that electrostatic interactions first lead to the formation of structurally disordered protein-QD complexes, followed by a crystallization phase in which a regular capsid is formed through protein-protein interactions. Other nanoparticles that have been successfully encapsulated in the viral capsids include gold and iron oxide nanobeads.8,9 However, whether these nanoparticle-conjugated viral proteins retain their targeting and penetration abilities is an open question and deserves further investigation.10 3



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Fluorescent nanodiamond (FND) is a carbon-based nanoparticle that has found a wide range of applications in physics, chemistry, biology, and medicine.11-14 Containing nitrogenvacancy centers as atom-like fluorophores, the nanomaterial is highly biocompatible and perfectly photostable, well suited for long-term tracking and imaging of biological processes in living cells.15,16 Our previous studies have shown that diamond nanoparticles (including FND) after strong oxidative acid treatment have an unusually high affinity for proteins.17-19 This characteristic allows one-step loading of a high amount of water-soluble protein molecules on their surface by physical adsorption. Remarkably, native membrane protein complexes solubilized in detergent micelles can also be captured by the particles, likely due to the intrinsic hydrophobicity of the carbon-based nanomaterial.20,21 For FNDs of 100 nm in diameter, more than 1000 globular protein molecules can be loaded on the particles at full coverage.17,19 Here, we propose to use FND as a unique nanoparticle platform for cell-type or tissue specific targeting after one-step surface modification with viral envelope proteins to elucidate the nature of virus-host interactions. The cell surface receptors of particular interest in this study are glycosaminoglycans (GAGs), which are unbranched polysaccharides consisting of repeating disaccharide units and are widely exploited by viruses during cell entry.22 To target the receptors, we chose to use the recombinant A27 proteins from vaccinia virus (VacV) to functionalize the FND surface. A27 is one of more than 20 envelope proteins and abundantly expressed on mature virion (MV) particles.23,24 The protein has multiple functions in VacV life cycle25 and is a critical target of neutralizing antibodies against pathogenic poxvirus infection in humans.26-28 During virus entry, A27 mediates the attachment of MV to heparan sulfates (a type of GAGs) on host cell surface through its N-terminal domain of aa 21–34 that contains a turn-like KKPE segment.29,30 The protein is also essential for the egress of MV from infected cells during virion morphogenesis.25 Crystal structure analysis revealed that 4


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the A27 protein forms disulfide-linked trimers and higher oligomers.31 In vitro mutagenesis studies also showed that the protein forms complexes with another viral envelope protein A26 and thus indirectly regulates membrane fusion.31,32 This work aims to combine the unique biochemical properties of viral surface proteins with the exceptional physicochemical properties of FND for GAGs-specific targeting, cell labeling, as well as high resolution fluorescence imaging and tracking of the nanoparticle bioconjugates in living cells. The advantages of using A27 as the model protein are manyfold: (1) VacV is safer to handle than many other mammalian viruses, (2) wild-type recombinant A27 protein (containing aa 21–84) is stable, soluble, and easily to be produced in a relative large quantity, and (3) DA27, a mutant recombinant A27 protein (containing aa 33–84) defective for binding to heparan sulfates,30,33 is available for use as a negative control. We first synthesized the nanoparticle bioconjugates by noncovalent interactions of FNDs with the recombinant A27 and DA27 (denoted as rA27 and rDA27, respectively) overexpressed in E. coli. Three types of viral protein-FNDs were generated, including rA27-FND and rDA27-FND with high and low GAGs-specific affinity, respectively, and rA/DA27-FND (a mixture of rA27 and rDA27 with FND) with tunable GAGs specificity. The GAGsspecific targeting abilities of these particles were characterized by flow cytometry, fluorescence microscopy, and gel electrophoresis.

EXPERIMENTAL SECTION FND production and characterization. FNDs were produced by ion irradiation of type Ib diamond powders, followed by thermal annealing, air oxidation, and acid treatments as detailed elsewhere.34 Hydrodynamic sizes and surface charges of the final products were measured with a combined particle size and zeta potential analyzer (Delsa Nano C, BeckmanCoulter). 5



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Viral protein expression and purification. Plasmid constructs expressing recombinant A27(aa 21–84), A27(aa 33–84), and A27(aa 21–84, C71/72A) with the point mutations C71A/C72A have been previously described.31 All the recombinant proteins were overexpressed in Escherichia coli and purified by using His-tag affinity chromatography. Purified recombinant A27 proteins were dialyzed in phosphate-buffered saline (PBS) and stored at –80 °C until use. The experiments were carried out in a Biosafety Level 2 (BSL-2) laboratory.

Synthesis of viral protein-FNDs.

FNDs of ~100 nm in size were thoroughly

suspended in distilled deionized water (DDW) at the particle concentration of 1 mg/ml by sonication for 30 min. The FND suspension was then mixed with the protein solution in PBS diluted 10 folds with DDW (i.e. 0.1X PBS).

To find the best conditions for protein

conjugation, the weight ratio of protein:FND was varied from 5:1, 1:1, and 1:5, and the mixing time was varied from 1, 3, and 16 h. For the production of FNDs with tunable GAGsbinding affinity, the rA27 and rDA27 proteins were pre-mixed at the ratio of 1:1 and 1:2 (w/w) prior to coating on the FND surface.

Characterization of viral protein-FNDs. Hydrodynamic sizes and surface charges of viral protein-FNDs were characterized by dynamic light scattering (DLS) and zeta-potential analysis, while the quantity and stability of the viral proteins on FNDs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as detailed elsewhere17-21 and in Supporting Information.

Cell culture and labeling. Human HeLa cells and mouse L and Sog9 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 µg/ml penicillin, and 100 µg/ml streptomycin at 37 °C with 5% CO2 in a humidified air incubator with the culture periodically screened for mycoplasma infection. 6


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Prior to labeling, cells were seeded at a density of 2 × 105 cells per 35-mm dish and cultured for 24 h. These cells were then incubated with 10 µg/ml or 30 µg/ml rA27-FND, rDA27FND, and rA/DA27-FND suspended in the culture medium. After incubation at 37 °C for 3.5 h, cells were detached from the culture plates by 0.05% trypsin solution and collected by centrifugation for both flow cytometry and fluorescence microscopy analysis.

Flow cytometry.

A flow cytometer (FACSArray Bioanalyzer, BD Biosciences)

equipped with a 532 nm laser was used to measure the uptake levels of rA27-FND, rDA27FND, and rA/DA27-FND by HeLa, Sog9, or L cells. Typically, 5000 cells were examined in each measurement and the fluorescence signals of FNDs were detected at the emission wavelength of >590 nm.

Endosomal labeling. CellLight BacMam 2.0 reagents were used to transfect cells with green fluorescent proteins (GFPs) for labeling of early endosomes, late endosomes, and lysosomes, following the manufacture’s protocols. Briefly, early endosomes-GFP fusion constructs (10 µg/ml) were added to cells one day prior to the experiment and left overnight. Cells were then washed with PBS twice before the addition of the FND labeling solution. The same procedures were followed to label late endosomes and lysosomes by using targeting reagents from the same manufacture but in different cell plates.

Confocal fluorescence microscopy. Fluorescence images were acquired by using a confocal laser scanning microscope (TCS SP8, Leica) equipped with a white light laser (470 – 670 nm). The GFP-labelled endosomes/lysosomes and internalized FNDs were excited by 488 nm and 561 nm light, respectively. The corresponding fluorescence was collected at 500 – 530 nm and 650 – 800 nm through an oil immersion objective (63×, NA 1.4) and detected with a photomultiplier tube (PMT) for GFP and a hybrid detector (HyD) for FND, respectively.

The microscope was also equipped with an on-stage incubator, allowing

confocal fluorescence imaging of live cells at 37 °C with 5% CO2. 7



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RESULTS AND DISCUSSION Synthesis and characterization of viral protein-FNDs. Vaccinia A27 is a protein consisting of 110 amino acid residues with a theoretical molecular weight of 12572 Da.31 It does not contain a transmembrane domain but interacts with the integral membrane protein A17 during MV morphogenesis.35,36 Table 1 presents the amino acid sequences of the recombinant A27 proteins, rA27 and rDA27, along with their molecular weights (Mw) and theoretical isoelectric points (pI). Each recombinant protein has a hexahistidine tag at its Cterminus. The net charge of the rA27 monomer is positive at neutral pH, with a theoretical pI of 8.60. The pI value decreases to 6.28 for the rDA27 monomer due to the lack of the GAGsspecific binding domain:

21

STKAAKKPEAKR32. MALDI-TOF MS analysis showed that

both rA27 and rDA27 form trimers in aqueous solution (Figure S1). The trimeric form is stabilized by the disulfide bonding between two cysteine residues (C71 and C72) in each monomer. The first step in this GAGs-specific cell targeting experiment is to synthesize a new type of FNDs surface-coated with rA27. The synthesis took advantage of the fact that the acid-treated FND has an exceptionally high affinity for proteins (including membrane proteins) owing to the presence of a variety of oxygen-containing groups (including -C=O, C-OH, and -COOH) on surface.17,19 By simply mixing the protein molecules with FNDs in DDW, PBS, or their mixtures (such as 0.1X PBS), the nanoparticle bioconjugates rapidly formed in the solution. Figure 1a shows the average hydrodynamic sizes of FNDs before and after noncovalent conjugation with rA27, rDA27, or a mixture of 50% rA27/50% rDA27 (w/w) at the weight ratio of protein:FND = 1:1 in PBS. When suspended in PBS at 30 µg/ml, which is the typical concentration used in cell labeling, all three samples exhibited good dispersibility in the buffer for 5 h at room temperature. The result demonstrates that the 8


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coating with viral envelope proteins can effectively avoid aggregation and/or precipitation of FNDs in PBS and cell medium, an important characteristic that makes the nanoparticle bioconjugates useful for GAGs-specific targeting.

However, it was noticed that the

dispersibility and stability of these nanoparticle bioconjugates in PBS strongly depended on the coating conditions and the nature of the protein molecules. We found that the coating at the protein/FND ratio of 1/1 (w/w) in DDW for 3 h with frequent sonication produced best results. The typical size of the viral protein-FNDs is in the range of 150 nm. Another indication for the successful coating of FNDs with the viral envelope proteins is the change of the surface charge states of the nanoparticles. Experimentally, we found that the zeta potential of bare FND drastically changed from –43 mV to +23 and +38 mV after coating with rDA27 and rA27, respectively (Figure 2a). It evidences that the surface of the nanoparticles has been covered with a significant amount of the protein molecules. To determine the quantity, we performed SDS-PAGE analysis. Figure 2b shows results of the analysis for both rA27-FND and rDA27-FND after extensive washing with PBS and separation by centrifugation. The appearance of the two absorption bands at ~10 kDa proved successful conjugation of the viral proteins with the surface-oxidized FNDs. Additionally, it indicated that the binding of rA27 and rDA27 with FND is so strong that they can sustain extensive washes by high salt solutions.

However, such noncovalent bonding can be

disrupted by using SDS-PAGE buffers that contain a high concentration of detergents (3% SDS) to denature the adsorbed protein molecules.18 From the SDS-PAGE analysis (Figure

S2), we determined the amount of rA27 and rDA27 loaded on FNDs at full coverage to be rA27:FND = 1:20 and rDA27:FND = 1:25 (w/w). These weight ratios are similar to the previously observed for other proteins like myoglobin and lysozyme.17,19 We next characterized the structure of the viral envelope proteins immobilized on FND by MALDI-TOF MS. The analysis was conducted by mixing directly the nanoparticle 9



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bioconjugates with the MALDI matrix (typically 2,5-dihydroxybenzoic acid) for laser desorption and ionization without preseparation of the protein molecules.17 Results presented in Figure 3 clearly show the presence of homogeneous trimers of these two proteins (rA27 and rDA27) on the FND surface. The similarity between the mass spectra of them and the corresponding protein molecules (Figure S1) suggests that the trimeric structures are highly stable and their functionality is likely preserved even after attachment to FND. As for the case of rA27(C71/72A), where both the cysteine residues in rA27 were replaced by alanine, only the monomeric peaks were found in the mass spectra. These point mutations (C71/72A), surprisingly, can alter the dispersibility of the rA27-FND conjugates in PBS. As shown in

Figure 1b for the DLS result, the particle size of rA27(C71/72A)-FND drastically increased from ~150 nm to more than 1000 nm after 5 h incubation in the high salt solution. We attribute the result to the fact that rA27(C71/72A) interacts with FND in its monomeric form and the formation of such a thin protein layer on the FND surface is insufficient to stabilize the colloids in PBS or cell medium. The result has important implication for the synthesis of virus-like FNDs and other nanoparticles in practical applications.

Cellular uptake of viral protein-FNDs. Previous studies of vaccinia MV infections in HeLa cells have identified several steps of virus entry into host cells: vaccinia MV envelope proteins H3 and A27 binding to cell surface heparan sulfates,37-39 viral D8 protein binding to chondroitin sulfates,38 and viral A26 binding to laminin.38 Attachment of MV particles triggers actin-mediated internalization as the major route mediating VacV entry into the cells.40,41 Among these four viral envelope proteins, A27 is best studied for its binding specificity toward heparan sulfates of host cells. We therefore tested whether the rA27-FND conjugate can act as a virus-mimic vehicle that remains competent to specifically recognize heparan sulfates on HeLa cells and be delivered into cytoplasm by endocytosis.

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The experiment was conducted by feeding HeLa cells with rA27-FND and rDA27-FND suspended in cell medium and analyzing how the cells endocytosed them by flow cytometry.

Figure 4 shows flow cytometric analysis of the cellular uptake of rA27-FND and rDA27FND at the particle concentration of 10 µg/ml. Thanks to the high fluorescence brightness of the 100-nm FND particles, we were able to observe a distinct change in the fluorescence intensity of rA27-FND-fed cells at the Far-Red channel (wavelength > 590 nm). Compared with that of the control cells without feeding, the fluorescence intensity of the rA27-FND-fed cells is more than 30-fold higher, suggesting that a significant amount of the nanoparticle bioconjugates has been taken up by the cells. In contrast, the rDA27-FND-fed cells show almost the same far-red fluorescence intensity as the control cells.

Since the feeding

conditions are identical for both rA27-FND and rDA27-FND, the result strongly implies that the GAGs-binding activity of rA27 is responsible for the effective targeting of HeLa cells by rA27-FND. To confirm that rA27-FNDs are indeed internalized by the cells, we applied confocal fluorescence microscopy to identify the endocytosed FNDs.

Figure 5 displays the

fluorescence images of HeLa cells incubated with 25 µg/ml of either rA27-FND or rDA27FND for 2 h. Evidently, there is considerably more cellular uptake of rA27-FND than rDA27-FND. Z-section images verified that most of the rA27-FND particles are inside the cells and the uptake process could be readily observed at low particle concentrations (~5 µg/ml) for just 30-min incubation. Additionally, the rA27-FND can be detected individually in the cytoplasm and tracked continuously over a long period of time (more than 1 min) without photobleaching.16 Such resistance to photobleaching is not feasible with VacV either fluorescently labeled with organic dyes or transfected with fluorescent proteins due to their lack of photostability. Using the same technique, we also compared the uptake of rA27FNDs with those of commonly used FND particles (bare FNDs and FNDs coated with bovine 11



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serum albumin) for cell labeling. As shown in Figure S3, the rA27-FND has the highest cellular uptake, likely due to its better interactions with cell surface. The data suggest the potential use of rA27-FND as an effective cell labeling agent. VacV is a large enveloped virus, having dimensions of roughly 360 × 270 × 250 nm.42 The virus is endocytosed into cells through fluid phase endocytosis or micropinocytosis.43,44 Previous studies have shown that, after endocytic internalization, vaccinia MV inside macropinosomes first travels to Rab5-positive endosomes40,41 and is subsequently sorted to Rab11-positive Rab22-positive recycling compartments prior to membrane fusion.40

To

explore whether rA27-FND also follows a similar trafficking route, we examined the colocalization of these FND particles with the fluorescent markers of various cellular vesicles in HeLa cells after incubation for 1 h.

In this particular experiment, we kept the

concentration of rA27-FND below 10 µg/ml to ensure the particles to be internalized in isolated forms. Figure 6 displays cross-sectional confocal fluorescence images of the cells whose endosomal/lysosomal vesicles were labeled with GFP.

While some of the early

endosomes (first row) showed colocalization, many of the viral protein-FND conjugates were found in other vesicles including late endosomes and lysosomes (second and third rows). The results suggest that, similar to VacV, the viral protein-FNDs first bind to the cell surface GAGs, followed by actin-mediated endocytosis into cells. These particles entered early endosomes; however, unlike vaccinia MVs, they could not escape from the endosomal vesicles due to the lack of membrane fusion and consequently were sent to late endosomes/lysosomes for cargo degradation.

GAGs-specific cell labeling and targeting. The preferential uptake of rA27-FND over rDA27-FND by HeLa cells in Figure 4 and 5 demonstrates that rA27 maintains its GAGtargeting activity even after immobilization on the FND surface. To further investigate the specific rA27-GAGs interactions, we compared the rA27-FND uptake by using two mouse 12


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cell lines that express different levels of surface GAGs: L cells and Sog9 cells. The mouse Sog9 cells were derived from the L cells and are known to lack cell surface GAGs.45 Not surprisingly, the cellular uptake of rA27-FND by the GAGs-deficient Sog9 cells is ~10 folds less, compared with that of the L cells (Figure 7a and 7b). More rA27-FNDs were engulfed by the L cells at higher particle concentrations, resulting in an increase in florescence intensity, whereas there was almost no change in the intensity for the GAGs-deficit Sog9 cells (Figure 7c). Such a distinct difference in the uptake behavior between these two types of cell lines is in line with the aforementioned flow cytometric analysis for HeLa cells (Figure 4). It supports the suggestion that the interaction between rA27 and GAGs is crucial for the attachment and subsequent internalization of the viral protein-coated FNDs by the cells. Given that there is almost no binding between rDA27-FND and GAGs, we produced viral protein-FNDs with tunable GAGs-binding affinity. The particles, denoted as rA/DA27, were synthesized by mixing rA27 with rDA27 at different weight ratios before coating on the FNDs. Indeed, we observed different levels of cellular uptake for three different types of nanoparticles with rA27:rDA27 = 1:2, 1:1, and 1:0 after feeding to the L cells (Figure 8). In accord with our expectation, the rA/DA27-FND with a higher weight ratio of the GAGsbinding protein showed a higher level of cellular uptake. The facile synthesis of these hybrid nanoparticle bioconjugates opens the door to fabricate multi-functional, virus-like FNDs using this simple approach. These virus-like FNDs are potentially useful for targeting and labeling of other cell types as well. Despite that the interaction between A27 and heparan sulfates on host cell surface has been recognized for vaccinia MV infection, it remains unclear whether a particular core protein of these GAGs-containing proteoglycans participates in the MV entry. In an attempt to shed some light on the interaction, we used the technique previously developed for 13



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membrane protein extraction21 and applied the rA27-FND nanoparticles as a probe to isolate and enrich host cell membrane protein(s) that interact directly with rA27. A preliminary result is shown in Figure S4, where rA27-FND was used as a specific “bait” to “fish” (isolate and enrich) rA27-binding membrane protein(s) from the cell membrane fractions of HeLa cells. Both rDA27-FND and bare FND served as the controls in this SDS-PAGE analysis. In accord with our previous observations21 and a recent study,46 the bare FND pull down many proteins effectively. A comparison of the results of the control and treatment groups revealed that some membrane proteins did co-precipitate with rA27-FND, but not with rDA27-FND. The most prominent one is the band at the molecular weight of >250 kDa. The results indicate that only a few proteins or protein complexes of HeLa cell membrane may interact specifically with the GAGs-binding site of rA27. It suggests that the viral protein-FNDs may be useful as a specific targeting nanoprobe for proteomics research. While there have been several solid-phase extraction supports available commercially for high-throughput and largescale proteomic analysis,47 the major advantage of the FND-based nanoparticle platforms is that the activity and specificity of the proteins being extracted can be directly examined by fluorescence assays at the single cell level.

CONCLUSION We have characterized in detail the virus-like nanoparticles consisting of FNDs noncovalently conjugated with the recombinant envelope proteins of VacV by light scattering, flow cytometry, fluorescence microscopy, and gel electrophoresis. Our results indicate that the conjugation does not significantly alter the protein’s structure and function after attachment to the FND surface. The method is simple, general, and can be readily extended to synthesize new types of nanoparticle bioconjugates using other viral proteins including both water-soluble proteins and membrane proteins.

The FND-based nanoparticle

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bioconjugates are highly biocompatible, brightly fluorescent, and perfectly photostable, well suited for specific cell targeting and long-term cell tracking applications. Through further optimization of these properties, it is anticipated that the virus-like nanoparticles will find broad applications such as the production of antibodies against membrane proteins and nanovaccine development.

SUPPORTING INFORMATION Experimental procedures for the characterization of viral protein-FNDs by DLS, MALDITOF MS, and SDS-PAGE; methods and results of isolation and enrichment of rA27-binding membrane proteins.

AUTHOR INFORMATION Corresponding Author *Address: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan. E-mail: [email protected]

ACKNOWLEDGEMENTS This work was supported by Academia Sinica and the Ministry of Science and Technology, Taiwan, with Grant No. 105-2811-M-001-111. M. D. Pham was partially supported by the research grant for “Study on development of nanodiamonds for enrichment, fractionation and mass spectrometric/proteomics analysis of membrane proteins” from the Vietnam Academy of Science and Technology (VAST). We thank C.-C. Han for access to Mascot server and SDS-PAGE analysis.

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Table 1. Amino acid (aa) sequences, theoretical molecular weights (Mw), theoretical isoelectric points (pI) of recombinant A27 and DA27 proteins

Amino acid sequencea

rA27

rDA27

MASMTGGQQM

MASMTGGQQM

GRGSSTKAAK

GRGSEAIVKA

KPEAKREAIV

DEDDNEETLK

KADEDDNEET

QRLTNLEKKI

LKQRLTNLEK

TNVTTKFEQI

KITNVTTKFE

EKCCKRNDEV

QIEKCCKRND

LFRLENKLAA

EVLFRLENKL

ALEHHHHHH

AAALEHHHHH H Theoretical Mwb

10380.76

9084.22

Theoretical pIb

8.60

6.28

a

Italic fonts represent the His-T7 tag plus aa sequences coming from restriction sites in the

vector and bold fonts represent the GAGs-specific binding domain. b

Mw

and

pI

are

calculated

with

the

(http://web.expasy.org/compute_pi/).

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Compute

pI/Mw

tool

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Figure 1. Hydrodynamic sizes of bare and viral-protein-coated FND particles in PBS, determined by dynamic light scattering. Note that bare FNDs rapidly agglomerated in the high-salt solution (a) and the point mutation at C71/72A in rA27 markedly reduced the dispersibility of the rA27(C71/C72A)-FND conjugates in the same solution (b). Experiments were repeated in triplicate and error bars represent one standard deviation of uncertainty.

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Figure 2. (a) Zeta potentials of FNDs before and after conjugation with rA27 (rA27-FND), rDA27 (rDA27-FND), or a mixture (50%/50%, w/w) of these two proteins (rA/DA27-FND). (b) SDS-PAGE analysis to quantify the amounts of rA27 and rDA27 loaded on the surface of FNDs by physical adsorption. Mk: Molecular weight markers.

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Figure 3. MALDI-TOF MS of rDA27-FND, rA27-FND, and rA27(C71/72A)-FND. The trimeric forms of both rA27 and rDA27 on the particles can be readily identified in the mass spectra of rA27-FND and DA27-FND. Only monomeric ions appear in the spectrum of rA27(C71/72A)-FND due to the two-point mutation at (C71/72A) in rA27.

The peaks

denoted by M+, M2+, M3+ correspond to monomeric, dimeric, and trimeric ions of rA27 and rDA27, respectively, and the asterisk denotes fragment ions.

The mass spectrum of rDA27-

FND is shifted vertically for clarity.

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Figure 4. Flow cytometric analysis of the cellular uptake of FNDs conjugated with rA27 and rDA27 in comparison with HeLa cells only. About 75% of the rA27-FND-fed cells showed far-red emission, compared with 5% of the rDA27-FND-fed cells. The particle concentration used in both measurements was 10 µg/ml.

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Figure 5. Confocal fluorescence microscopy analysis of the cellular uptake of (a) rA27FNDs and (b) rDA27-FNDs in HeLa cells. The fluorescence of FNDs is in red and the cell nuclei are stained with Hoechst 33342 in blue. Scale bar: 25 µm.

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Figure 6. Confocal fluorescence imaging of rA27-FNDs internalized by HeLa cells. The green color is from GFP-labeled early endosomes, late endosomes, and lysosomes markers and the red color is from FNDs. Yellow spots are the colocalizations of FNDs with GFPlabeled vesicles. Enclosed in the white boxes are the zoom-in images. Scale bar: 10 µm.

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Figure 7. Flow cytometric analysis of the cellular uptake of rA27-FNDs by (a) mouse Sog9 and (b) mouse L cells expressing different levels of surface GAGs. About 86% of the L cells showed far-red emission, compared with 1.5% of the Sog9 cells. The particle concentration used in both measurements was 10 µg/ml. (c) Dose dependence of the cellular uptake of rA27-FND at 0, 10, and 30 µg/ml for both cells.

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Figure 8. Flow cytometric analysis of the cellular uptake of rA/DA27-FNDs by mouse L cells as a function of the rA27:rDA27 ratio. The particle concentration was 30 µg/ml in all measurements.

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Glycosaminoglycans-Specific Cell Targeting and Imaging Using

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