Universal Cell Surface Imaging for Mammalian, Fungal, and Bacterial

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Universal Cell Surface Imaging for Mammalian, Fungal, and Bacterial Cells Hong-Yin Wang, Xian-Wu Hua, Hao-Ran Jia, Chengcheng Li, Feng-ming Lin, Zhan Chen, and Fu-Gen Wu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00130 • Publication Date (Web): 05 May 2016 Downloaded from http://pubs.acs.org on May 7, 2016

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ACS Biomaterials Science & Engineering

Universal Cell Surface Imaging for Mammalian, Fungal, and Bacterial Cells

Hong-Yin Wang,†,# Xian-Wu Hua,†,# Hao-Ran Jia,† Chengcheng Li,† Fengming Lin,† Zhan Chen,*,‡ and Fu-Gen Wu*,†

†

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering,

Southeast University, Nanjing 210096, P. R. China ‡

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor,

Michigan

1

ACS Paragon Plus Environment


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ABSTRACT Owing to the distinct surface structures of different cells (mammalian cells, fungi, and bacteria), surface labeling for these cells requires a variety of fluorescent dyes. Besides, fluorescent dyes (especially the commercial ones) for staining Gram-negative bacterial cell walls are still lacking. Herein, a conformation-adjustable glycol chitosan (GC) derivative (GC-PEG cholesterol-FITC) with “all-in-one” property was developed to realize universal imaging for plasma membranes of mammalian cells (via hydrophobic interaction) and cell walls of fungal and bacterial cells (via electrostatic interaction). By comparing the different staining behaviors of GC-PEG cholesterol-FITC and three other analogs (GC-PEG-FITC, GC-FITC, and cholesterol-PEG-FITC), we have elucidated the different roles the hydrophobic and electrostatic interactions play in the staining performance of these different cells. Such a simple, non-cytotoxic, economic, and universal cell surface staining reagent will be very useful for investigating cell surface-related biological events and advancing cell surface engineering of various types of cells.

KEYWORDS: cell surface–biomaterial interaction, bioimaging, glycol chitosan, cell surface engineering, hydrophobic interaction, electrostatic interaction

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INTRODUCTION Mammalian, bacterial, and fungal cells are among the most frequently studied cell types due to their important research values in biomedical fields. The surfaces of these cells, including the plasma membrane of mammalian cells and the cell walls of fungi and bacteria, are important interfaces between the cells and their surroundings. Cell surface provides each cell with an isolated environment, regulates the ways the cell communicates with extracellular substances (such as the nutrients and drugs), and influences many significant cellular behaviors such as signal transduction, endocytosis, adhesion, division, and motility. Knowledge on the interactions between cell surfaces and materials1–3 as well as drugs4–7 has significantly promoted many important developments in fields such as tissue engineering,8,9 cell surface engineering,10–14 and drug discovery/delivery.15–18 To precisely control the cellular behaviors in these applications, an improved understanding of the physicochemical properties of cell surfaces as well as their interactions with materials is required. Fluorescence labeling is a powerful tool for visualizing the cell surface structures and their time-dependent changes when cells interact with various materials. There are many commercially available cell surface labeling dyes, which can mainly be classified into two groups by how they interact with the cell surfaces: (1) hydrophobic/nonspecific interaction-based dyes and (2) specific recognition-based dyes. The hydrophobic interaction-based dyes are frequently used for staining cell membranes, especially the plasma membranes of mammalian cells. Typically, these dyes are hydrophobic molecules like those in DiD (such as DiO, DiL, and DiA) and FM (including FM 4-64 and FM 1-43) families. After incubation with cells, these small molecules insert into the lipid membranes through

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hydrophobic interaction and diffuse rapidly in the lipid bilayer, thus effectively label the plasma membranes. Although the plasma membranes of bacterial and fungal cells can also be stained with some of these lipophilic dyes (like Nile Red,19 DiI,20 and FM 4-6421–24), in most cases the exterior cell walls cannot be labeled (Gram-negative bacteria are exception since their outermost surfaces are the outer membranes, which might be stained by some lipophilic dyes). Moreover, these dyes in yeast cells are usually rapidly internalized from plasma membrane to vacuolar membrane, leading to the failure of plasma membrane labeling after a short period of time.25 Specific recognition-based dyes can label cell surfaces through selective binding to their target ligands. For instance, wheat germ agglutinin (WGA, a lectin) can specifically bind to N-acetylglucosamine (sialic acid) and N-acetylneuraminic acid residues on cell surfaces.26 Thus fluorescence-labeled WGA (WGA conjugates), such as WGA-fluorescein isothiocyanate (FITC),27 WGA-quantum dots (QDs),28 WGA-647,29,30 and WGA-48831 were used to stain mammalian cell plasma membranes (containing N-acetylneuraminic acid residues of glycoproteins) as well as bacterial cell walls (containing N-acetylglucosamine residues of peptidoglycan).32 However, for mammalian cell labeling, WGA conjugates could not stain effectively for all cell types because different cell types may have varied cell surface sugars. For bacteria staining, the cell wall of Gram-positive bacteria contains many layers of peptidoglycan which can be directly accessible to WGA molecules, while the peptidoglycan layer in Gram-negative bacteria is shielded by the outer membrane, which contains lipopolysaccharides (LPS) and lipoproteins that cannot be recognized by WGA. Therefore, WGA conjugates can selectively stain Gram-positive but not Gram-negative bacteria.33–35 In some recently reported cases, although WGA-conjugates were

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successfully used to label Escherichia coli (E. coli, a Gram-negative bacterium) cell walls with unknown reasons,36,37 these dyes still cannot be used to effectively label many different Gram-negative bacterial cell walls. Therefore, fluorescent dyes for effectively staining Gram-negative bacterial cell walls are still urgently needed. Additionally, although the fungal cell wall is constituted mainly by chitin molecules, different from bacterial cells which have peptidoglycans, the above mentioned WGA conjugates can also stain fungal cell walls via binding to the chitin molecules on fungal surfaces.38,39 Even so, the classic and widely used fluorescent dye for fungal cell wall imaging is Calcofluor White (CFW),40,41 a fluorescent brightener that can bind to β-linked fibrillar polymers such as chitin and cellulose through hydrogen bonding.42 Since the proper fluorescent dyes (especially the commercial ones) for labeling Gram-negative bacterial cell walls are still lacking, and using different dyes with varied protocols for plasma membrane (mammalian cell) and cell wall (bacteria or fungi) imaging can be costly and complicated, the development of a simple, economic, and universal cell surface staining reagent is desirable. In this work, we discovered that our previously reported fluorescent polymer, glycol chitosan (GC)-polyethylene glycol (PEG) cholesterol-FITC,43 could stain not only the plasma membranes of mammalian cells, but also the cell walls of bacterial and fungal cells in their respective physiological media. This staining reagent was designed based on a multisite anchoring strategy with GC serving as the backbone, PEG-cholesterols and FITC molecules conjugated on the backbone serving as the cell surface anchors and fluorophores, respectively (Scheme 1A). GC was chosen since it is highly biocompatible and is soluble in aqueous solution at a wide pH range.43,44 The PEG segments

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ensure the excellent water solubility of the staining reagent, and prevent it from self-assembling into large nanoparticles (100–400 nm).43 This GC-PEG cholesterol-FITC molecule is soluble in all physiologically relevant solutions and can interact with various cell surfaces either via hydrophobic interaction between the cholesterol moiety and the plasma membrane (for mammalian cells), or via electrostatic interaction between the positively charged GC molecules and the negatively charged cell walls (for bacterial and fungal cells), making it a “smart” reagent with “all-in-one” property that can universally stain all the surfaces of mammalian, fungal, and bacterial cells. To elucidate the underlying mechanism of such universal staining, we also compared the staining effect of this reagent with three other related derivatives: GC-PEG-FITC, GC-FITC, and cholesterol-PEG-FITC (Scheme 1B, C, and D). The molecular structures of the four reagents listed in Scheme 1 are shown in Figure S1. The different staining behaviors of these reagents can shed new light on the interaction mechanisms between cell surfaces and biomaterials, which would promote the advancement of cell surface-related studies.

Scheme 1. Schematics of the four staining reagents. (A) GC-PEG cholesterol-FITC (multisite), (B) GC-PEG-FITC, (C) GC-FITC, and (D) cholesterol-PEG-FITC (single-site).

MATERIALS AND METHODS Materials. Glycol chitosan (G7753, its molecular weight and degree of deacetylation is 67 kDa and 88%, respectively45) was purchased from Sigma-Aldrich (St. Louis, MO). FITC was obtained from 6


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Fanbo

Biochemicals

Co.

Ltd.

(Beijing,

China).

N-hydroxysuccinimide-polyethylene

glycol2000-cholesterol (NHS-PEG2k-cholesterol), NHS-PEG2k-OMe and cholesterol-PEG2k-FITC were purchased from Nanocs, Inc. (New York, NY). Dialysis membranes (Spectra/Por®6) were purchased from Spectrum Labs (Rancho Dominguez, CA). Potato dextrose agar (PDA) and lysogeny broth (LB) were ordered from Beijing Land Bridge Technology (Beijing, China). Potato dextrose broth (PDB), N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) were purchased from Aladdin Reagent Company (Shanghai, China). Deionized water (18.2 MΩ·cm) was obtained from a Milli-Q synthesis system (Millipore, Billerica, MA). U14 cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Bacterial cells including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Proteus sp., Staphylococcus aureus (S. aureus), and Micrococcus luteus (M. luteus), and fungal cells including Saccharomyces cerevisiae (S. cerevisiae) yeast cells and Trichoderma reesei (T. reesei, Rut C-30 strain) were obtained from China Center of Industrial Culture Collection (CICC, Beijing, China). Lactobacillus plantarum (L. plantarum) bacteria were isolated in our own lab. Syntheses

of

GC-PEG

cholesterol-FITC,

GC-PEG-FITC,

and

GC-FITC.

GC-PEG

cholesterol-FITC was synthesized according to our previously reported method (here we synthesized GC-10% PEG cholesterol-2% FITC, with 10% and 2% of the repeating units of GC conjugated with NHS-PEG2k-cholesterol and FITC, respectively).43 In brief, 5.0 mg NHS-PEG2k-cholesterol and 4.0 mg glycol chitosan (GC) were separately dissolved in 1.0 mL phosphate buffered solution (PBS, pH 7.4, 50 mM). Then, they were mixed together to react for 4 h under stirring at room temperature. After dialyzing (MWCO 10K) the mixture solution against deionized water for 3 days, the obtained GC-PEG cholesterol compound was lyophilized for 24 h. Then 1.0 mg of the GC-PEG cholesterol compound

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was dissolved in 1 mL PBS (pH 7.8, 50 mM) and 16 µL FITC (1 mg/mL in DMF) was added into the solution. The mixture was allowed to react at room temperature overnight. After dialyzing (MWCO 10K) the mixture solution against deionized water for 3 days and freeze drying for 24 h, GC-PEG cholesterol-FITC reagent was finally obtained. All the procedures were carried out in dark. For the synthesis of GC-PEG-FITC, the experimental procedures were similar to those for GC-PEG cholesterol-FITC except that the above used NHS-PEG2k-cholesterol was replaced with NHS-PEG2k-OMe. For the synthesis of GC-FITC, FITC molecules were directly conjugated on glycol chitosan backbone in PBS (pH 7.8, 50 mM). Cell Culture. Mammalian cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin-streptomycin at 37 °C in a humid atmosphere with 5% CO2. Bacterial cells including E. coli, P. aeruginosa, Proteus sp. and S. aureus were cultured in LB medium under shaking for 12 h at 37 °C. The M. Luteus was cultured in LB medium under shaking for 12 h at 30 °C. L. plantarum was grown in MRS broth without shaking for 24 h at 30o. For fungal cells, S. cerevisiae was cultured in PDB under shaking for 12 h at 28 °C. T. reesei was cultured on PDA slants for 5 days at 28 °C. Then the spores were harvested and washed with sterilized water and cultured in PDB for 72 h at 28 °C. Zeta Potential Measurements. Zeta potential of different cell lines (U14, E. coli, S. aureus, S. cerevisiae, and T. reesei) as well as the four staining reagents (GC-PEG cholesterol-FITC, GC-PEG-FITC, GC-FITC, and cholesterol-PEG-FITC) were measured using a Zetasizer instrument (Malvern Instruments, Nano ZS, United Kingdom) in four different solutions including: cell PBS (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.4), 0.9% NaCl, 10 mM NaCl, and deionized water. For microbial cell characterization, E. coli, S. aureus, S. cerevisiae, and T. reesei cells

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were washed and prepared in the above mentioned four different solutions, respectively. The cell concentration used for these measurements was 107 cells/mL. For U14 cells, the zeta potential measurements were only conducted in cell PBS and 0.9% NaCl solutions, and the cell concentration was 107 cells/mL. For reagent characterization, samples of GC-PEG cholesterol-FITC, GC-PEG-FITC, and GC-FITC were prepared with a concentration of 0.3 mg/mL, while the concentration of cholesterol-PEG-FITC was 0.1 mg/mL. Cell Staining and Confocal Imaging. Confocal images of the stained cells were taken using a confocal microscope TCS SP8 (Leica, Germany) with a 63× oil immersion objective for mammalian and fungal cells and a 100× oil immersion objective for bacterial cells. The 488 nm laser was selected to excite the samples and the fluorescence emission was detected between 500–550 nm. For mammalian cells, the cells were seeded at 5 × 104 cells/well in 35 mm confocal dishes and incubated at 37 °C for 24 h. After washing with PBS, the attached cells were incubated with the staining reagent (typically 100 µg/mL for GC derivatives and 10 µg/mL for cholesterol-PEG-FITC) in cell PBS for 10 min at 37 °C. Then, the cells were washed twice with PBS and subsequently observed using the confocal microscope. For bacterial and fungal cells, the cells were collected in mid-exponential growth phase through centrifuging at 8000 rpm for 5 min. Then, the cell pellet was re-suspended in the staining solution and incubated at 37 °C for 10 min in dark. After washing by centrifuging at 8000 rpm for 5 min, the stained cells were re-suspended and observed under the confocal microscope. The washing solution and working solution (cell PBS, 0.9% NaCl, 10 mM NaCl, or deionized water) used in one independent experiment were the same as that used for dissolving the staining reagent. Flow Cytometry. The stained mammalian cells in the 35 mm confocal dishes were washed with PBS, digested with trypsin, collected in RPMI 1640 medium and analyzed using a flow cytometer (ACEA

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Bioscience, NovoCyte, SD). The stained bacterial and yeast cells were maintained in corresponding washing/staining solution and analyzed using the cytometer. Channel used for analyses was FITC with the excitation at 488 nm. Cytotoxicity Evaluation. The cytotoxicity of GC-PEG cholesterol-FITC to U14 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded at 5×103 cells/well in 96-well plate and cultured for 24 h when the cells grew to 80% confluence. Then, the medium in each well was replaced with 100 µL fresh medium containing GC-PEG cholesterol-FITC with various concentrations and the treated cells were further cultured for 24 h. Then, 10 µL MTT solution (5.0 mg/mL in PBS) was added into each well directly and the cells were incubated for an additional 4 h at 37 °C. After that, the medium was carefully aspirated and 150 µL DMSO was added to dissolve the produced formazan crystals, and then absorbance at 492 nm was measured using a microplate reader (Thermo Scientific, Multiskan FC, USA). The cytotoxicity of GC-PEG cholesterol-FITC to bacterial cells was measured via microplate assay due to the good correlation between the turbidity of the bacterial suspension and the amounts of the bacteria. In brief, the log phase bacteria were inoculated 1:10 into 150 µL fresh LB medium containing 100 µg/mL GC-PEG cholesterol-FITC and seeded into each well of the 96-well plate and cultured under shaking for 24 h at 37 °C. During the culturing process, OD600 of each well was monitored at different time intervals. The cytotoxicity of GC-PEG cholesterol-FITC to yeast cells was measured via colony forming unit (CFU) counting method, rather than the above mentioned OD600 monitoring approach due to the low correlation between the turbidity of the yeast cell suspension with the yeast cell number. In brief, the log phase yeast cells were cultured in PDB medium containing GC-PEG cholesterol-FITC with various

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concentrations for 24 h at 28 °C. After that, each culture was diluted (with a dilution factor of 105) using the PDB medium and plated in triplicate on PDA plates and incubated for 48 h at 28 °C. Then, the cell colonies formed were counted and recorded. The cytotoxicity was evaluated by comparing the number of CFUs in treated groups to that in control group. Size Characterization of GC-PEG cholesterol-FITC and GC-PEG-FITC. The hydrodynamic diameters of GC-PEG cholesterol-FITC (1.0 mg/mL) and GC-PEG-FITC (1.0 mg/mL) were measured by dynamic light scattering (DLS) using a Zetasizer instrument (Malvern Instruments, Nano ZS, United Kingdom) in 0.9% NaCl solutions at 25 °C.

RESULTS Surface Charge of Staining Reagents and Cells. The cell surface–material interaction is dictated by the physicochemical properties of the cell–material interface. In our study, there are two major driving forces involved in cell surface labeling: (1) The hydrophobic insertion of cholesterol moieties into the lipophilic plasma membrane and (2) the electrostatic interaction between the positively charged GC polymer and the negatively charged cell walls. For the GC derivatives we used, it was reported that the isoelectric point (IEP) was around pH 8.8.46 Thus, at the physiological pH of 7.4, the GC molecule should be positively charged. To confirm this, zeta potential measurement was conducted for GC derivatives (including GC-FITC, GC-PEG-FITC, and GC-PEG cholesterol-FITC) in solutions with varied pH values and ionic strengths, including a cell PBS solution (commonly used for mammalian cell treatment), a 0.9% NaCl solution (commonly used for microorganism treatment), a 10 mM NaCl solution, and deionized water. Cell PBS and 0.9% NaCl solutions have a similar ionic

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strength (around 150 mM) but different pH values (7.4 and 6.0–6.5, respectively), while the 0.9% NaCl solution, 10 mM NaCl solution, and water have almost the same pH but different ionic strengths. As revealed in Figure 1A, although GC with the IEP at pH 8.8 should be positively charged under physiological condition (pH 7.4), the zeta potential of GC derivatives in cell PBS was measured to be around zero, indicating that the relative large ionic strength of the cell PBS solution likely influences the GC surface charge. This result indicates that when these GC derivatives interact with cells in cell PBS solutions, the electrostatic interaction may be insignificant. As the decrease of the solution pH (from 7.4 in a cell PBS solution to 6.0–6.5 in a 0.9% NaCl solution, or a 10 mM NaCl solution, or deionized water) and of the ionic strength (from 150 mM in a cell PBS solution to 10 mM in a 10 mM NaCl solution to 0 mM in deionized water), the zeta potential values of these GC derivatives became positive and significantly increased to above 40 mV, which would enable the electrostatic interaction between these molecules and negatively charged cell surfaces. In contrast, the zeta potential value of cholesterol-PEG-FITC (without GC) remained at around 0 mV in almost all the solutions (except the deionized water), suggesting that this molecule might only have hydrophobic interactions with the cells.

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Figure 1. Zeta potential of (A) four staining reagents (GC-FITC, GC-PEG-FITC, GC-PEG cholesterol-FITC, and cholesterol-PEG-FITC) and (B) five different cells (E. coli, S. aureus, S. cerevisiae, T. reesei, and U14) in different solutions (cell PBS solution, 0.9% NaCl solution, 10 mM NaCl solution, and deionized water). The reagent concentrations were 100 µg/mL for GC derivatives and 10 µg/mL for cholesterol-PEG-FITC. The cell concentration was 107 cells/mL.

It is well known that the plasma membrane of mammalian cells as well as the cell walls of bacteria or fungi are all negatively charged.47–49 For mammalian cells, the negative charge is mainly contributed by the anionic phospholipids in the cell membrane, such as phosphatidylserine (PS) and phosphatidylinositol (PI).50 For bacterial and fungal cells, besides the negatively charged plasma membranes, the cell walls are also negatively charged due to the presence of negative charge-carrying glycans. Specifically, for Gram-positive bacteria, the negative charge is derived from the wall- and lipo-teichoic acids (phosphate carrying) on the cell wall. For Gram-negative bacteria, the negative charge is contributed by the outer membrane-resided LPS which are also phosphate-containing molecules.51 As for fungi, the negatively charged cell wall is attributed to the mannosylphosphate (phosphate carrying) or pyruvylated galactose (pyruvate carrying) molecules.52,53

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The surface charge of one mammalian cell (U14), two bacterial cells (E. coli and S. aureus), and two fungal cells (S. cerevisiae and T. reesei) were characterized through zeta potential measurements. As expected, all the bacterial and fungal cells are negatively charged in all the four solutions (Figure 1B). Moreover, we can see that bacterial cells are more negatively charged as compared to the fungal cells at the same condition. However, their charge variation trend in the four solutions is similar: as pH decreases from 7.4 in a cell PBS solution to 6.0–6.5 in a 0.9% NaCl solution, the negative charge shows a slight decline due to the protonation of the anionic groups (such as phosphate and carboxyl) on the cell surface. As the ionic strength decreases (from 150 mM in a 0.9% NaCl solution to 10 mM in a 10 mM NaCl solution to zero in deionized water), the absolute value of the negative charge increases and reaches the highest level in deionized water. These results suggest that the cells would be more negatively charged in solutions with lower ionic strengths. Since mammalian cells do not contain the more rigid cell wall structures and are highly vulnerable to hypotonic solutions such as a 10 mM NaCl solution or deionized water, the zeta potential of U14 mammalian cells were measured only in cell PBS and a 0.9% NaCl solution, and the values were around −9.8 and −7.5 mV, respectively, also confirming the negative charge property of the cells. Staining of Mammalian Cells. Since cell PBS solution is the most commonly used solution for mammalian cell treatment, we investigated the staining effect of the four reagents in this solution. As discussed above, the GC derivatives in cell PBS solution were not positively charged, and thus electrostatic interaction was negligible, making hydrophobic interaction a determining factor. Using U14 mammalian cell as an example, GC-PEG cholesterol-FITC

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could effectively stain the plasma membrane through the hydrophobic insertion of the cholesterol moieties into the lipid membrane (Figure 2A). In contrast, the two other GC derivatives, GC-PEG-FITC and GC-FITC, failed to label plasma membranes (Figure 2B and 2C) since they do not contain the cholesterol moieties. Consistent results could also be seen in other mammalian cells. We observed in our previous work that GC-PEG-FITC and GC-FITC could barely or not stain the KB cells, while the multisite anchoring reagent GC-30% PEG cholesterol-2% FITC could effectively label several mammalian cells (such as KB, AT II, A549, MDA-MB-231, and HepG2).43 Furthermore, the single-site anchoring reagent, cholesterol-PEG-FITC, could also stain U14 cells due to the membrane anchoring ability of its cholesterol moiety (Figure 2D).

Figure 2. Confocal images (A–D) and flow cytometry analyses (E) of U14 cells stained with four different reagents in cell PBS solution. (A) GC-PEG cholesterol-FITC, (B) GC-PEG-FITC, (C) GC-FITC, and (D) cholesterol-PEG-FITC.

Besides the confocal imaging studies, the staining performance of these four reagents was also evaluated through flow cytometry. As shown in Figure 2E, only GC-PEG cholesterol-FITC and cholesterol-PEG-FITC effectively labeled U14 cells (with the 15



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fluorescence peak at around 105 and above 106, respectively), while GC-PEG-FITC and GC-FITC failed to label the cells (with the fluorescence peaks at around 104, more or less similar to that of the control group). The reason for the brighter fluorescence of cholesterol-PEG-FITC stained cells compared to that of GC-PEG cholesterol-FITC is because of the higher FITC content in the single-site anchoring reagent. Taken together, these results suggest that the hydrophobic interaction between the reagents and the plasma membrane is the main drive force for mammalian cell surface labeling. Staining of Bacterial Cells. For bacterial culture and treatment, the 0.9% NaCl (physiological saline) solution is predominantly used, and thus the bacteria staining was conducted in this solution. Although hydrophobic interaction plays a vital role in mammalian cell membrane staining, we have found that it is not the key factor for bacterial cell wall staining. As shown in Figure 3D, cholesterol-PEG-FITC with the cholesterol moiety cannot stain the E. coli bacteria. This is because that the plasma membrane of the bacterium is buried by one layer of hydrophilic cell wall (acting as a barrier here),54 making the hydrophobic insertion of cholesterol into the bacterial plasma membrane difficult. In contrast, the cholesterol-free reagent GC-FITC clearly stained the cell wall of E. coli with green fluorescence (Figure 3C). As shown in Figure 1, in a 0.9% NaCl solution, GC-FITC was positively charged with a zeta potential value of 4 mV, while the E. coli bacteria were negatively charged with a zeta potential of −8 mV. Therefore the successful staining of E. coli with GC-FITC can be attributed to the electrostatic interaction between the negatively charged cell wall and the positively charged GC polymer. Taken together, the above results indicate that it is the electrostatic interaction that ensures the attachment of the staining

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reagent to the bacterial cell surface.

Figure 3. Confocal images (A–D) and flow cytometry analyses (E) of E. coli bacteria stained with four different reagents in 0.9% NaCl solutions. (A) GC-PEG cholesterol-FITC. (B) GC-PEG-FITC. (C) GC-FITC. (D) Cholesterol-PEG-FITC.

Next, we incubated the E. coli bacteria with another reagent, GC-PEG-FITC, which has additional PEG segments compared to GC-FITC. Since GC-PEG-FITC has the similar zeta potential-variation behavior as GC-FITC (Figure 1A), it was supposed to have similar electrostatic interaction with the negatively charged cell wall. However, it turned out that GC-PEG-FITC failed to stain the bacteria (Figure 3B). This negative result could be explained by the steric hindrance effect of the PEG segments, which was frequently used to endow the surface or interfaces with anti-fouling/anti-adsorption properties.55–57 The bulky PEG segments would increase the distance between the negatively-charged cell wall and the positively charged GC backbone, and the steric hindrance of PEG would further reduce the electrostatic attractive force between the cell wall and the GC backbone, leading to the poor labeling performance of GC-PEG-FITC. Interestingly, when conjugating hydrophobic

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cholesterol molecules to the end of PEG segments on GC-PEG-FITC, the resultant GC-PEG cholesterol-FITC (multisite anchoring reagent) exhibited the cell wall staining capability (Figure 3A). Besides the confocal imaging studies, flow cytometry experiments were also conducted to further evaluate the staining performance of the four reagents. The results matched the confocal imaging data quite well. As shown in Figure 3E, the fluorescence intensity of cholesterol-PEG-FITC stained cells is almost the same as that of the untreated cells (102–103), demonstrating that the single-site anchoring reagent could not stain the bacterial cell wall. In contrast, the fluorescence intensity peaks for GC-FITC and GC-PEG cholesterol-FITC locate at 104–105, two-orders of magnitude larger than that of the unlabeled cells. While for GC-PEG-FITC, the fluorescence intensity (103–104) is higher than that of the control sample but much lower than that of the other two molecules which can effective stain the cell walls, suggesting that it could not stain the bacterial cells effectively, although some molecules could attach to the cell wall through weak electrostatic interactions. Collectively, the results presented here confirmed that only GC-FITC and GC-PEG cholesterol-FITC could stain the bacterial cell wall. Notably, the successful bacterial cell wall staining by GC-FITC and GC-PEG cholesterol-FITC were achieved in 0.9% NaCl solution, but not in cell PBS solution (data not shown). This is because the surface charges of these two GC derivatives are around zero in cell PBS solution (as discussed above on the observations shown in Figure 1). Therefore, GC-FITC and GC-PEG cholesterol-FITC can only stain the bacterial cell walls in 0.9% NaCl solution, 10 mM NaCl solution, and deionized water where these two molecules are positively

18


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charged. Since all the bacterial cell walls are negatively charged, we hypothesize that the interactions between other bacteria and the four staining reagents should be the same as for E. coli. We have tested and validated this hypothesis by incubating two other Gram negative bacteria (P. aeruginosa and Proteus sp.) and three Gram positive bacteria (S. aureus, L. Plantarum, and M. luteus) with these four reagents. As expected, all of these bacterial cells could

be

labeled

with

GC-FITC

and

GC-PEG

cholesterol-FITC,

but

not

cholesterol-PEG-FITC and GC-PEG-FITC. Since our aim for this research was to develop the multisite GC-PEG cholesterol-FITC molecule into a universal staining reagent, here we only present the staining performance of this reagent. Figure 4 shows that all the Gram-positive and Gram-negative bacteria could be clearly stained by GC-PEG cholesterol-FITC regardless of their shapes (bacillus/coccus). The results indicate that this cell wall imaging reagent can stain many if not all bacterial cells. This is especially important for Gram-negative bacterial cell wall imaging, which is hard to achieve via commercial dyes such as WGA conjugates as discussed above. Figure 4 also shows clearly that the imaging reagents could stain not only the cell walls, but also the formed septum structures (indicated by the red arrows). Therefore we believe that this reagent is suitable for cell division-related studies as well.

19



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Figure 4. Confocal images of (A) P. aeruginosa (Gram-negative or G–, rod-shaped/bacillus), (B) Proteus sp. (G–, rod-shaped/bacillus), (C) S. aureus (Gram-positive or G+, spherical/coccus), (D) L. plantarum (G+, rod-shaped/bacillus), and (E) M. luteus (G+, spherical/coccus) bacteria stained with GC-PEG cholesterol-FITC in 0.9% NaCl solutions.

Staining of Fungal Cells. Since the cell walls of fungi are also negatively charged, we would also like to test whether the four reagents can also image fungal cells. Unfortunately, none of these four reagents could stain yeast cell (S. cerevisiae) in 0.9% NaCl solution. However, by decreasing the ionic strength of the solution to 10 mM (using a 10 mM NaCl solution) or 0 mM (using deionized water), the cell wall of yeast could be labeled with GC-FITC and GC-PEG cholesterol-FITC, but still not with cholesterol-PEG-FITC and GC-PEG-FITC (Figure 5), in good agreement with the observations obtained from bacterial cell staining experiments presented above. This could be explained by the different zeta potential values of fungal and bacterial cells in different solutions. As revealed in Figure 1B, in a 0.9% NaCl 20


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solution, the yeast cells (zeta potential: −3 mV) are less negatively charged compared to E. coli and S. aureus bacteria (zeta potential: −8 and −14 mV, respectively), thus the yeast cell wall could not be stained by the positively charged GC derivatives due to the weaker electrostatic interaction. However, the yeast cells became more negatively charged (zeta potential: −7 mV) in a 10 mM NaCl solution, resulting in the successful staining of yeast cell wall with GC-FITC and GC-PEG cholesterol-FTIC (Figure 5), because now the electrostatic attractions between the yeast cell walls and these two molecules are strong enough. The successful staining of yeast cells could also be achieved in deionized water since the zeta potential of yeast in water is more negative (at around −20 mV) (data not shown). The staining results of yeast cells further validate the conclusion that the cell wall imaging is achieved mainly through the electrostatic interaction. Since the fungal cell culture medium (PDB, consisting of only 4.0 g/L potato extract and 20.0 g/L dextrose) has a very low salinity (correspondence to 30 mM NaCl as we measured), the short staining time (within 10 min) conducted in a 10 mM NaCl solution should not be harmful to the cells. Additionally, fungal cell staining was also carried out in a 30 mM NaCl solution. The results show that the yeast cell walls could still be clearly labeled by GC-PEG cholesterol-FITC, although the fluorescence intensity slightly decreases compared to that observed in a 10 mM NaCl solution (data not shown).

Figure 5. Confocal images of yeast cells stained with four different reagents in 10 mM NaCl 21



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solutions. (A) GC-PEG cholesterol-FITC, (B) GC-PEG-FITC, (C) GC-FITC, and (D) cholesterol-PEG-FITC.

T. reesei, another fungal cell commonly used in bioengineering, was also negatively charged, and its zeta potential values were similar with those of the yeast cell in the four different solutions (Figure 1B). Interestingly, successful labeling of T. reesei cell wall could be achieved even in a 0.9% NaCl solution, not strictly restricted in a 10 mM NaCl solution (Figure 6). We believe that this may indicate that for some special fungal cells, besides the electrostatic interactions, other factors may also play a role. Nevertheless, we believe that as long as we lower the ionic strength of the staining solution, the cells will be more negatively charged, and thus all the fungal cells would be stained by the multisite anchoring reagent GC-PEG cholesterol-FITC. We also noticed that not only the cell walls, but also the septum structures of T. reesei (marked by arrows in Figure 6) were stained, making the reagent applicable for septum-related studies.

Figure 6. Confocal images of T. reesei stained with GC-PEG cholesterol-FITC. (A) Bright field. (B) Fluorescence image (only the cells in the focal plane were imaged). Red arrows indicate the septum structures.

Optimization of Staining Concentration. To optimize the working concentrations during the

22


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staining procedures, we investigated the effects of reagent concentration on the staining performances of four types of cells (E. coli, S. aureus, S. cerevisiae, and U14). The flow cytometric results shown in Figure S2−4 reveal that the staining performances of bacterial cells (E. coli and S. aureus) and fungal cells (S. cerevisiae) all exhibit the concentration-dependent manner: the fluorescence intensity increases with the increase of the reagent concentration from 0.2 to 100 µg/mL. For bacterial and fungal cell staining, the fluorescence intensity almost reaches a plateau at a concentration of 20 or 50 µg/mL, suggesting that for microbial labeling, 20 or 50 µg/mL can already realize high-quality staining. While for mammalian cells, the results shown in Figure S5 suggest that 100 µg/mL is required to achieve excellent staining performance. Cytotoxicity Evaluation. Since some positively charged molecules like cationic antimicrobial polymers and peptides are reported to be toxic to some cells,58–61 it is necessary to evaluate the cytotoxicity of this staining reagent. Figure 7 shows that even after 24 h of incubation with 100 µg/mL GC-PEG cholesterol-FITC, all the four types of treated cells, including U14 mammalian cells, yeast fungal cells, and E. coli and S. aureus bacterial cells, exhibited the same viability as the untreated cells. The non-cytotoxicity of GC-PEG cholesterol-FITC is attributed to its biocompatible components (GC, PEG, and cholesterol), ensuring that it can be used as a “safe” fluorescent dye to stain various types of cells.

23



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Figure 7. Cytotoxicity evaluation of GC-PEG cholesterol-FITC to mammalian cells, bacteria, and fungi. (A) MTT assay for U14 mammalian cells. (B) Colony unit forming counting assay for yeast cells. (C) and (D): Real-time cell growth monitoring of E. coli and S. aureus bacteria (with or without the presence of 100 µg/mL GC-PEG cholesterol-FITC), respectively.

DISCUSSION If we analyze the staining behaviors of the four reagents discussed here, only the multisite anchoring reagent, GC-PEG cholesterol-FITC, has the ability to stain all the surfaces of mammalian, bacterial, and fungal cells (as summarized in Table 1). This can be explained by the fact that this molecule contains both the hydrophobic cholesterol moieties and the positively charged amine-bearing GC backbone. The hydrophobic cholesterol moieties can insert into the plasma membranes of mammalian cells, while the positively charged GC

24


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backbone can have electrostatic interaction with the negatively charged cell walls of bacteria and fungi. However, in terms of cell wall staining, GC-PEG-FITC cannot stain cell walls due to the reduced electrostatic interactions induced by the steric hindrance effect of its PEG segments (as discussed above). While interestingly, GC-PEG cholesterol-FTIC, with additional cholesterol moieties compared to GC-PEG-FITC, regains the ability to stain cell walls. We believe that the end cholesterol moieties could induce the self-assembly of GC-PEG cholesterol-FITC molecules into core-shell structured nanoparticles, with the hydrophobic cholesterol moieties buried in the inner core and the PEG segments attached to the outer GC backbone as the shell. This assumption can be supported the DLS results (Figure 8) which reveal that GC-PEG cholesterol-FITC has a smaller hydrodynamic diameter (around 17 nm) compared to that of GC-PEG-FITC (around 25 nm), suggesting that the former has a relatively tighter packing than the latter. Such a self-assembled conformation likely reduces the steric hindrance effect of PEG segments and also results in the exposure of the positively charged amine groups on the GC backbone, leading to the much stronger electrostatic interaction between GC-PEG cholesterol-FITC and the bacterial/fungal cell walls. Thus the cell wall staining can be achieved by GC-PEG cholesterol-FITC.

Table 1. Staining performance

of

the four compounds (GC-PEG cholesterol-FITC,

GC-PEG-FITC, GC-FITC, and cholesterol-PEG-FITC) on mammalian, bacterial, and fungal cells. Here “√” represents successful staining while “×” represents failure in staining.

Compound GC-PEG cholesterol-FITC

Mammalian

Bacterial

Fungal

cells

cells

cells

√

√

√

25



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GC-PEG-FITC

×

×

×

GC-FITC

×

√

√

√

×

×

cholesterol-PEGFITC

Page 26 of 37

Figure 8. DLS results of (A) GC-PEG cholesterol-FITC (1.0 mg/mL in a 0.9% NaCl solution) and (B) GC-PEG-FITC (1.0 mg/mL in a 0.9% NaCl solution).

Scheme 2 illustrates the possible interaction mechanisms of GC-PEG cholesterol-FITC with mammalian, bacterial, and fungal cells. GC-PEG cholesterol-FITC can self-assemble into nanoparticle in aqueous solution due to its amphiphilic nature. When interacting with mammalian cells, GC-PEG cholesterol-FITC changes from a self-assembled nanoparticle to a more stretched polymer through disassembly, exposing the interior hydrophobic cholesterol molecules which can subsequently anchor the whole polymer to the plasma membranes. When interacting with bacterial and fungal cells, GC-PEG cholesterol-FITC maintains its nanoparticulate state and its positively charged amine groups on the surface GC backbones mediate the direct adsorption of the whole molecule onto the negatively charged cell walls via electrostatic

interaction.

The

conformation-adjustable

property

26


makes

GC-PEG

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cholesterol-FITC a “smart” reagent with the “all-in-one” property that can universally stain all the surfaces of mammalian, fungal, and bacterial cells.

Scheme

2.

Schematic

showing

the

possible

interaction

mechanisms

of

a

conformation-adjustable amphiphilic polymeric construct, GC-PEG cholesterol-FITC, with mammalian, bacterial, and fungal cells. This amphiphilic polymer self-assembles into a nanoparticle in aqueous solution and upon interaction with mammalian cells, it will disassemble and anchor to the plasma membranes via cholesterol insertion. When interacting with the negatively charged bacterial and fungal cells, the polymeric construct retains its spherical shape with the exposure of surface positive charges, and directly adsorbs onto the surfaces of bacteria and fungi.

To summarize, the staining reagent GC-PEG cholesterol-FITC has the following advantages: (1) Universal cell surface labeling. The surfaces of all mammalian cells can be stained due to the nonspecific hydrophobic interaction between the reagent and the plasma membranes. Thus this reagent can be used for plasma membrane-related studies such as cell division, endocytosis, and apoptosis. For microbial cells, due to their negative charge-carrying nature, all the microbial cell walls (including the septum structures) can be labeled. Thus this reagent can be used for cell wall/septum-related studies such as cell cycle and proliferation; (2) 27



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Gram-negative bacterial cell wall staining which cannot be realized by common commercial dyes. This technique can be used as an alternative of GFP labeling which is complicated and time-consuming; (3) Insight into biomaterial–cell surface interactions. Through elucidating different staining behaviors of four relevant reagents, a better understanding of the interaction mechanism between biomaterials and different cell surfaces can be obtained; (4) Cell surface engineering. The universal labeling strategy has the potential to manipulate cell behaviors of various types of cells in the field of cell surface engineering. CONCLUSION In this research, we developed a universal staining reagent, GC-PEG cholesterol-FITC, which could realize high-quality imaging of the plasma membranes of mammalian cells as well as the cell walls of bacterial and fungal cells. Compared with the staining effects of three other analogs (GC-PEG-FITC, GC-FITC, and cholesterol-PEG-FITC), GC-PEG cholesterol-FITC was proved to be able to interact strongly with the plasma membrane through hydrophobic interaction and the cell wall through electrostatic interaction. This method of cell surface labeling is simple, fast (within 10 min), economic, and safe (not cytotoxic). Besides, it can easily realize successful Gram-negative bacterial cell wall staining which is difficult to achieve by other commercial dyes. Some finer structures (such as the septum structures) of bacteria and fungal cells could also be imaged, which could greatly impact the septum-related biological studies such as cell division and growth studies. By using different fluorescent molecules, we can fabricate various GC-based cell surface staining reagents which emit different fluorescent colors. We should mention that besides the universal cell surface imaging ability, the GC derivative also represents a universal reagent for cell surface

28


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engineering of various cell types, where the fluorophores can be replaced by functional molecules (such as photosensitizers, which can realize cell surface-based photodynamic therapy). Moreover, the different staining behaviors of the four reagents revealed by this work will shed new light on the interaction mechanisms of biomaterials with cell surfaces.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Molecular structures of the four staining reagents (GC-PEG cholesterol-FITC, GC-PEG-FITC, GC-FITC, and cholesterol-PEG-FITC) (Figure S1), the effect of reagent (GC-PEG cholesterol-FITC) concentration on the staining performances of E. coli (Figure S2), S. aureus (Figure S3), yeast (Figure S4), and U14 (Figure S5) cells evaluated by flow cytometry or confocal microscopy (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected]. Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

29



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This work was supported by grants from the National High Technology Research & Development Program of China (2015AA020502), National Natural Science Foundation of China (21303017), Natural Science Foundation of Jiangsu Province (KB20130601), Fundamental Research Funds for the Central Universities (2242015R30016), Six Talents Peak Project in Jiangsu Province (2015-SWYY-003), Scientific Research Foundation of Graduate School of Southeast University (YBPY1508), and Graduate Students’ Scientific Research Innovation Project of Jiangsu Province Ordinary University (CXZZ13_0122).

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For Table of Contents Use Only.

Universal Cell Surface Imaging for Mammalian, Fungal, and Bacterial Cells Hong-Yin Wang, Xian-Wu Hua, Hao-Ran Jia, Chengcheng Li, Fengming Lin, Zhan Chen,* and Fu-Gen Wu*

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Universal Cell Surface Imaging for Mammalian, Fungal, and Bacterial

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