Article
pubs.acs.org/journal/abseba
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, United States
S Supporting Information
*
ABSTRACT: Because of 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 cholesterolFITC) 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-PEGFITC, 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, noncytotoxic, 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
■
INTRODUCTION
Mammalian, bacterial, and fungal cells are among the most
frequently studied cell types because of 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© 2016 American Chemical Society
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 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
Received: March 7, 2016
Accepted: May 5, 2016
Published: May 5, 2016
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Scheme 1. Schematics of the Four Staining Reagents: (A) GC-PEG Cholesterol-FITC (Multisite), (B) GC-PEG-FITC, (C) GCFITC, and (D) Cholesterol-PEG-FITC (Single-Site)
bind to N-acetylglucosamine (sialic acid) and N-acetylneuraminic acid residues on cell surfaces.26 Thus, fluorescencelabeled WGA (WGA conjugates), such as WGA-fluorescein
isothiocyanate (FITC),27 WGA-quantum dots (QDs),28 WGA647,29,30 and WGA-48831 are 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 are 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
Because 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
because it is highly biocompatible and is soluble in aqueous
solution at a wide pH range.43,44 The PEG segments 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 was soluble in all
physiologically relevant solutions and could 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−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.
■
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 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/
Por6) were purchased from Spectrum Laboratories (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
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(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 GC-PEG cholesterol compound was dissolved in 1 mL of PBS
(pH 7.8, 50 mM) and 16 μL of 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-PEG2kOMe. For the synthesis of GC-FITC, we directly conjugated FITC
molecules 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 30°. 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. The spores were then 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 were
washed and prepared in the above-mentioned four different solutions,
respectively. The cell concentration used for these measurements was
1 × 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 1 × 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 midexponential growth
phase through centrifuging at 8000 rpm for 5 min. Then, the cell pellet
was resuspended 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 resuspended 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
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 of fresh medium containing GC-PEG cholesterol-FITC
with various concentrations and the treated cells were further cultured
for 24 h. Then, 10 μL of 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 of 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
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. The cell
colonies formed were then 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 GCPEG-FITC. The hydrodynamic diameters of GC-PEG cholesterolFITC (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 polymer we used, it
was reported that the isoelectric point (IEP) is 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 GCFITC, 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 strength (around 150 mM) but different pH values
(7.4 and 6.0−6.5, respectively), whereas the 0.9% NaCl
solution, 10 mM NaCl solution, and water have almost the
same pH but different ionic strengths.
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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 1 × 107 cells/mL.
Figure 2. (A−D) Confocal images and (E) flow cytometry analyses of U14 cells stained with four different reagents in cell PBS solution. (A) GCPEG cholesterol-FITC, (B) GC-PEG-FITC, (C) GC-FITC, and (D) cholesterol-PEG-FITC.
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.
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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Figure 3. (A−D) Confocal images and (E) flow cytometry analyses 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.
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. Because 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 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, C)
because 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).
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 cholesterolFITC and cholesterol-PEG-FITC effectively labeled U14 cells
(with the fluorescence peak at around 1 × 105 and above 1 ×
106, respectively), whereas GC-PEG-FITC and GC-FITC failed
to label the cells (with the fluorescence peaks at around 1 ×
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 bacteria 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
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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.
could not stain the bacterial cell wall. In contrast, the
fluorescence intensity peaks for GC-FITC and GC-PEG
cholesterol-FITC locate at 1 × 104 to 1 × 105, two-orders of
magnitude larger than that of the unlabeled cells. While for GCPEG-FITC, the fluorescence intensity (1 × 103 to 1 × 104) is
higher than that of the control sample but much lower than that
of the other two molecules which can successfully 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 GCPEG cholesterol-FITC could stain the bacterial cell wall.
Notably, the successful bacterial cell wall staining by GCFITC 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 (see 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 charged.
Because 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 GCPEG-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).
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 reagent
to the bacterial cell surface.
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 GCPEG-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 antifouling/antiadsorption 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 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 (1 × 102 to
1 × 103), demonstrating that the single-site anchoring reagent
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Figure 5. Confocal images of yeast cells stained with four different reagents in 10 mM NaCl solutions. (A) GC-PEG cholesterol-FITC, (B) GCPEG-FITC, (C) GC-FITC, and (D) cholesterol-PEG-FITC.
fluorescence intensity slightly decreases compared to that
observed in a 10 mM NaCl solution (data not shown).
T. reesei, another fungal cell commonly used in bioengineering, was also negatively charged, and its zeta potential values
were similar to 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
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.
Staining of Fungal Cells. Because 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 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), and thus the yeast cell
wall could not be stained by the positively charged GC
derivatives because of 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 GCPEG 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 because 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.
Because 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
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.
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 will 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.
Optimization of Staining Concentration. To optimize
the working concentrations during the staining procedures, we
investigated the effect of reagent concentration on the staining
of four types of cells (E. coli, S. aureus, S. cerevisiae, and U14).
The flow cytometric results shown in Figures S2−S4 reveal that
the staining 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. For mammalian cells, the
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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, 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.
results shown in Figure S5 suggest that 100 μg/mL is required
to achieve excellent staining performance.
Cytotoxicity Evaluation. Because 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 cells, and E. coli and S. aureus
bacterial cells, exhibited the same viability as the untreated cells.
The noncytotoxicity 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.
charged amine-bearing GC backbone. The hydrophobic
cholesterol moieties can insert into the plasma membranes of
mammalian cells, while the positively charged GC 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 because of the
reduced electrostatic interactions induced by the steric
hindrance effect of its PEG segments (as discussed above).
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 by 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.
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 cholesterolFITC 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
■
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
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 Cellsa
compound
mammalian cells
bacterial cells
fungal cells
GC-PEG cholesterol-FITC
GC-PEG-FITC
GC-FITC
cholesterol-PEG-FITC
√
×
×
√
√
×
√
×
√
×
√
×
“√” represents successful staining, whereas “ × ” represents failure in
staining.
a
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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).
dyes. (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.
Scheme 2. Schematic Showing the Possible Interaction
Mechanisms of a Conformation-Adjustable Amphiphilic
Polymeric Construct, GC-PEG Cholesterol-FITC, with
Mammalian, Bacterial, and Fungal Cellsa
■
CONCLUSION
In this research, we developed a universal staining reagent, GCPEG 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, GCFITC, 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 Gramnegative 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 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.
a
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.
interacting with bacterial and fungal cells, GC-PEG cholesterolFITC 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 makes GC-PEG cholesterol-FITC a
“smart” reagent with the “all-in-one” property that can
universally stain all the surfaces of mammalian, fungal, and
bacterial cells.
To summarize, the staining reagent GC-PEG cholesterolFITC 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) Gram-negative bacterial cell
wall staining which cannot be realized by common commercial
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00130.
Molecular structures of the four staining reagents (GCPEG cholesterol-FITC, GC-PEG-FITC, GC-FITC, and
cholesterol-PEG-FITC) (Figure S1), the effect of reagent
(GC-PEG cholesterol-FITC) concentration on the
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staining 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].
*E-mail: [email protected].
Author Contributions
§
H.-Y.W. and X.-W.H. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
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). Z.C. thanks the University of
Michigan for supporting his sabbatical.
■
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DOI: 10.1021/acsbiomaterials.6b00130
ACS Biomater. Sci. Eng. 2016, 2, 987−997