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Mannose-modificated Polyethyleneimine: A Specific and Effective Antibacterial Agent against Escherichia coli Mei Liu, Jiao Li, and Baoxin Li Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03556 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 7, 2018
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Mannose-modificated Polyethyleneimine: A Specific and Effective
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Antibacterial Agent against Escherichia coli
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Mei Liu1, Jiao Li1, Baoxin Li 2
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1
5
of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi'an 710119,
6
China
7
Phone: 86-29-85310517, Fax: 86-29-85310517
8
E-mail:
[email protected] 9
2
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, College
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School
10
of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710119,
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China.
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Phone: 86-29-81530726, Fax: 86-29-81530727
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E-mail:
[email protected] 14 15
Abstract
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Polyethyleneimine (PEI) has antimicrobial activity against Gram-positive
17
(Staphylococcus aureus, S. aureus) and Gram-negative (Escherichia coli, E.
18
coli), but it possesses highly cytotoxic and the selective antimicrobial
19
activity against S. aureus is obviously better than E. coli. To reduce the
20
cytotoxicity and improve the antibacterial activity against E. coli, we
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modified PEI by D-mannose through nucleophilic addition between primary 1
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amine and aldehyde group to get mannose-modificated polyethyleneimine
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copolymer particles (Man-PEI CPs). The use of mannose may provide good
3
targeting ability towards E. coli pili. The antibacterial activity of Man-PEI
4
CPs was investigated. Man-PEI CPs shows the specific and very strong
5
killing capability against E. coli at the concentration of 10 µg/mL, which is
6
the highest antimicrobial efficiency compared with unmodified PEI (220
7
µg/mL). The antibacterial mechanism demonstrated that the enhancement in
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antibacterial activity is due to specific recognition of the mannose and
9
destroying the cell wall of the bacteria by PEIs. Importantly, the Man-PEI
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CPs show less cytotoxicity and excellent biocompatibility. The results
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indicate that Man-PEI CPs have great potential as novel antimicrobial
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materials to prevent bacterial infections and provide specific application for
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killing E. coli.
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KEYWORDS:
15
particles, mannose, antibacterial activity, specific recognition
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1. INTRODUCTION
mannose-modificated
polyethyleneimine
copolymer
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The prevalence of bacterial infectious diseases is one of the most common
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causes of morbidity in patients. After the emergence of antibiotics, it plays
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very important role in promoting the way for medical and social
20
developments.1,2 However, multiple antibiotics-resistant bacteria has widely 2
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emerged among many species of pathogenic bacteria which led to some
2
antibiotics no longer effective in controlling of infectious diseases.3 To
3
discover and design new efficient antibacterial materials is a considerable
4
attention for the treatment of microbial disease.4-6
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Among the antibacterial materials investigated, cationic polymers have
6
emerged as a potential antibacterial material and have some obvious
7
advantages,
8
antibacterial features and better biocompatibility compared with small
9
molecular antibiotics.7-9 Kuroda groups10 have investigated the antibacterial and
including
sustained
cytotoxicity
broad-spectrum
activity
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polyethyleneimine polymers (PEIs). They have found that PEIs antibacterial
12
activity against E. coli and S. aureus depended on both the PEIs architecture
13
and molecular weight (MW). Furthermore，the low MW PEIs are less
14
cytotoxic to human cells than others, but the unmodified PEIs displayed
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selective activity against S. aureus over E. coli. This property of PEIs
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restricts its broad-spectrum antibacterial features, especially the antibacterial
17
activity against E. coli. At present, a large number of studies have focused
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on improving the antibacterial activity of PEIs. The ways mainly recurs to
19
modify PEIs by covalent linkage with other components, such as
20
surface-grafted quarternized PEI,11 functional groups modified PEI
21
microgels,12
functionalized
conventional
effect,
10
PEI
of
inhibitory
silver
unmodified
nanoparticles,13
3
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cationic
phenylalanyl
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integrated PEI.14 Although there is some progress in enhancing the
2
antibacterial activity of PEIs, these works do not involve the selective
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antibacterial activity and the cytotoxicity to cells of modified PEIs.
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On the other side, PEIs have been utilized as drug carriers in biomedical
5
application because of their ability to give high gene transfection efficiency,
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but it is highly cytotoxic.15-17 There are some efforts on how to abate the
7
toxic effects of PEIs so as to provide their potential applications in gene
8
delivery. Various modifications of PEI have been introduced to alter the
9
surface charge characteristics of PEI.18 Such as grafting PEI with
10
poly(ethylene glycol),19 hyaluronic acid-PEI particles,20 chitosan-PEI,21
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galactosylated PEI22 and mannose PEI.23 However most of these
12
modifications were tedious without controlling over composition and
13
molecular structure, and achieved variable success. To the best of our
14
knowledge, the investigation on how to lower the cytotoxicity of PEIs and
15
meanwhile improve specific antibacterial features is still limited. Therefore,
16
there is a need to modify PEIs in an easy and well controlled manner to
17
achieve that low MW PEIs are likely to be less toxic while still exhibiting
18
antibacterial behavior especially against E. coli.
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E.coli is a typical pathogenic bacterium, especially problematic because it
20
only takes as few as 10 cells to infect humans and cause serious illnesses.24
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It is worthy of investigation for the purpose of protecting humans and the 4
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environment. In this study, we examined the sterilization potential of
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Man-PEI CPs which was modified by D-mannose through facile
3
nucleophilic addition chemistry between primary amine and aldehyde group.
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The modification of PEIs resulted in a decrease in the cytotoxicity of PEI as
5
that the primary amine groups of PEI was substituted and exhausted by the
6
carbohydrate. Furthermore, the use of mannose may also provide targeting
7
ability towards E. coli through specific and multivalent interactions between
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the mannose on Man-PEI CPs and FimH lectin pili on the surface of E.
9
coli.25 Man-PEI CPs exhibit good biocompatibility, low cytotoxicity and
10
efficient antibacterial ability, demonstrating safe antibacterial property in the
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application of healthcare.
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2. MATERIALS AND METHODS
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Materials and Measurements. Branched PEI (MW = 600, 1800, 10 000,
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99%), D-mannose and ascorbic acid were purchased from Aladdin Ltd.
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(Shanghai,
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bromide (MTT) was purchased from Sigma Chemical Company. The
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propidium iodide (PI) was purchased from Solarbio Ltd. (Beijing, China).
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The bacteria medium components and phosphate buffer saline (PBS, pH 7.4)
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were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The
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DMEM medium was purchased from HyClone Thermofisher (Beijing,
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China). E. coli K-12 and Staphylococcus aureus were purchased from the
China).
3-(4,5-dimethylthiazol-2-yl)-2,5–diphenyltetrazolium
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China General Microbiological Culture Collection Center (Beijing, China).
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Human cervix adenocarcinoma cells (HeLa) were purchased from KeyGen
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Biotech. Co. Ltd. (Nanjing, China). All other chemicals were of analytical
4
reagent grade and used without further purification.
5
UV-vis adsorption spectra were recorded on a U-3900H UV-Vis
6
Spectrophotometer (Hitachi, Japan). Fluorescence spectra were measured on
7
an F-4600 Spectrometer (Tokyo, Japan). Scanning electron microscope
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(SEM) measurements were performed with a FEI Quanta 200 scanning
9
electron microscope (FEI, America). The nuclear magnetic resonance (NMR)
10
spectra were collected on a Bruker AVANCE III 600 (600 MHz) (Bruker,
11
Germany) with the freeze-dried product dissolved in D2O. Fluorescence
12
images were recorded on a fluorescence microscope (Olympus, FV1200).
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Synthesis of Man-PEI Copolymer Particles.The synthetic method of
14
Man-PEI CPs was carried out according to a previously reported procedure
15
with slight adjustments.26 1 mL of PEI (0.1 g mL-1) was first dissolved in 7
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mL of PBS buffer (10 mM, pH 7.4) by stirring for about 1 min, and then 2
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mL of mannose (0.1 M) was added. Subsequently, after vigorous stirring for
18
1 min, the mixture was heated at 90 ℃ for 40 min via hydrothermal
19
treatment. Then the Man-PEI CPs solutions were dialyzed against ultrapure
20
water for 24 h through dialysis bag (MWCO = 500 Da). The products inside
21
the dialysis bag were collected to further study. In addition, PEI was 6
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modified with ascorbic acid to construct AA-PEI CPs according to the same
2
method.
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Bacterial Cultivation. E.coli bacterial samples were transferred from
4
-80 ℃ refrigerator onto agar slants (25 g of Lysogeny Broth and 15 g agar
5
were dissolved into 1 L water) and incubated at 37 ℃for 16 h, then held at 4 ℃
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for up to 2 weeks. A single colony from the slants was cultured overnight in
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20 mL sterile medium for 14 h in a shaker at 37 ℃. After growth, the original
8
E. coli was washed with 0.9 % sodium chloride solution twice to remove the
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medium. After centrifugation at 6000 rpm for 2 min, the remaining E.coli
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was redispersed in 0.9 % sodium chloride solution.
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Antibacterial activity experiments. The antibacterial activity of PEI,
12
Man-PEI CPs was respectively investigated by incubation with bacterial
13
cells suspensions in PBS buffer (10 mM, pH 7.4). E.coli with a
14
concentration of 3×104 cells·mL-1 was mixed with different concentration
15
PEI or Man-PEI CPs. After incubating at 37 ℃ for 3 h, 100 µL of the
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bacterial suspension was spread onto the solid LB agar plate. The colonies
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formed units (CFU) were counted after 16 h incubation at 37 ℃. The
18
sterilization rate was determined by the following formula.
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Sterilization Rate % = (C0 - C)/C0 ×100%
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Where C is the CFU of the experimental group treated with PEI or Man-PEI
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CPs, and C0 is the CFU of the control group without any treatment. 7
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Fluorescent microscope measurements. The antibacterial efficiency of
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PEI or Man-PEI CPs was also proved by fluorescence microscopy. After
3
treating suspensions of E. coli with PEI or Man-PEI CPs (the final
4
concentration, 10 µg mL-1) at 37 ℃ for 3 h and staining them with PI for 15
5
min, the bacteria were separated by centrifugation at 6000 rpm for 10 min,
6
then precipitate was re-suspended in 20 µL PBS buffer (10 mM, pH 7.4).
7
Fluorescent microscope samples were obtained by adding 10 µL of the
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pre-prepared mixed suspensions to clean glass slides and covering them with
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coverslips for immobilization. The color of PI is red and the type of light
10
filter is BP 540−585 nm exciter, and DM 595 nm emitter. Magnification of
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object lens is 40×.
12
Cell viability assay. Cytotoxicity against HeLa cells was evaluated
13
according
to
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
14
bromide (MTT) method.27 HeLa cells were seeded into a 96-well plate at a
15
density of 1.0 × 103 cells per well in 100 µL of Dulbecco’s Modified Eagle’s
16
Medium (DMEM) supplemented with 10% fetal bovine serum and incubated
17
for 12 h at 37 ℃ in 5% CO2. The PEI and Man-PEI CPs were diluted with
18
DMEM and added to wells at final concentrations of 10, 50, 100, 250, 500
19
µg mL-1, with three replicates of each concentration. After culturing for 24 h,
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the cells were washed with PBS, and a 20 µL aliquot of MTT was then
21
added into each well to remove PEI and Man-PEI CPs. Finally, the MTT 8
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was removed, then any formazan that formed was dissolved with dimethyl
2
sulfoxide (DMSO) after 15 min of shaker. Absorption was measured at a
3
wavelength of 490 nm.
4
3. RESULTS AND DISCUSSION
5
Preparation and Characterization of Man-PEI CPs. The preparation of
6
Man-PEI CPs was conveniently accomplished by one step process.
7
Following the previously reported protocol,26 we synthesized Man-PEI CPs
8
through facile nucleophilic addition chemistry between primary amine on
9
PEI and aldehyde group on D-mannose. The Man-PEI CPs were soluble in
10
aqueous solution without the need of further modification. The absorption
11
spectra of Man-PEI CPs, PEI and mannose in water are respectively shown
12
in Figure 1. It shows that the Man-PEI CPs solution has a new absorption
13
peak at 352 nm, whereas PEI and mannose have nearly no absorption at
14
above 250 nm. On the other hand, Man-PEI CPs display an intense
15
fluorescence at 460 nm. The intrinsic fluorescence emission closely
16
resembles those of PAMAM and methylated PEI.28-30 And the inset in Figure
17
1 displays the optical property of Man-PEI CPs exhibits faint yellow under
18
daylight and bright blue fluorescence under ultraviolet lamp (365 nm).
19
SEM was employed to characterize the morphological and structural
20
characteristics of the obtained composite materials (Figure 2). Man-PEI CPs
21
are monodisperse and exhibit rough spheres particles with a diameter of 9
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about 20 µm, which demonstrated that the copolymer particles were
2
successfully prepared. To further verify the formation of a schiff base
3
between PEI and mannose, we utilized Fourier transform infrared (FT-IR)
4
spectroscopy and 1H NMR spectroscopy to investigate. As shown in Figure
5
3, curves a, b, and c represent FT-IR spectra of PEI, mannose, and Man-PEI
6
CPs, respectively. The raw PEI has absorption peaks at 2942, 2831, and
7
1471 cm-1 corresponding to the stretching vibration and bending vibration of
8
CH2 bonds, and characteristic absorptions at 3284 and 1577 cm-1 belong to
9
the N-H bond. Compared to the spectrum of PEI and mannose, the bending
10
vibration of the C=O groups of mannose at 1597 cm-1 disappeared and an
11
obvious new peak at 1632 cm-1 was observed in the Man-PEI CPs spectrum,
12
which can be attributed to the C=N bond.26,31,32 In addition, the absorption
13
bands at 3437 and 1385 cm-1 are associated with the stretching vibration and
14
bending vibration of O-H, respectively, and the stretching vibration of C-O
15
is located at 1084 cm-1, which reveals the presence of C-OH. In addition, the
16
1
H NMR spectra (Figure S1) of Man-PEI CPs has a new peak at 8.40 ppm
17
belonging to H2C=N-protons,33 while PEI is no signal at this location. The
18
results well confirm the favorably synthesis of the Man-PEI CPs.
19
Evaluation of Antibacterial Activity.The molecular weight of PEI had a
20
significant impact on antibacterial activity,10 so the antibacterial activity of
21
Man-PEI CPs prepared by PEI with different molecular weight was 10
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investigated. The antibacterial activity against Gram-negative bacteria, E.
2
coli K-12, was evaluated by colony counting. As shown in Figure 4, the
3
antibacterial activity of PEI depended on its molecular weight. Increasing
4
the molecular weight resulted in an increase in the antibacterial performance.
5
The antibacterial activity of corresponding Man-PEI CPs was all improved
6
compared with unmodified PEI. Importantly, when the molecular weight of
7
PEI was 600, the antibacterial activity increased significantly (Figure 5a).
8
Therefore, PEI with molecular weight of 600 was selected as the candidate
9
to prepare Man-PEI CPs for next experiment.
10
In addition, we further conducted fluorescence imaging tests to prove
11
excellent bactericidal effect of Man-PEI CPs. After antibacterial experiments,
12
PI was added to suspensions of E. coli with PEI and Man-PEI CPs for 15
13
minutes, which can specifically stain damaged or dead bacteria. Figure 5b
14
shows the fluorescence images of E. coli suspensions and the merged images
15
under phase contrast bright-field and fluorescence filed. After incubation
16
with Man-PEI CPs, all cells emit red fluorescence that means cells are killed,
17
whereas less red fluorescence is observed when the bacterial cells were
18
incubated with PEI. Man-PEI CPs exhibited highly efficient killing
19
capability against bacteria than PEI at the same dosage concentration,
20
implying that the antibacterial activity was obviously enhanced when PEI
21
was modified with mannose. These results indicate that mannose played a 11
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vital role in the excellent bactericidal effect of Man-PEI CPs.
2
In order to discuss the effect of mannose on the antibacterial activity, the
3
antibacterial activity of Man-PEI CPs with different mass ratio (PEI :
4
mannose) was evaluated by colony counting. As shown in Figure 6a, the
5
sterilization rate of PEI (100 : 0 mg mL-1) only exhibited 9.29% ± 2.27%.
6
After grafting with mannose, the antibacterial activity enhanced as the
7
amount of mannose increased. At a mass ratio of 100 : 36 mg mL-1, the
8
sterilization rate was 99.45% ± 0.6%. These observations were further
9
demonstrated by the agar plates of E. coli (Figure 6b). In comparison with
10
control plate, where a large number of E. coli colonies were observed,
11
however, no colonies were found by treatment with Man-PEI CPs (100 : 36
12
mg mL-1). These clearly demonstrated that the outstanding bactericidal effect
13
of Man-PEI CPs is relevant to high surface mannose ligand content. The
14
phenol-sulfuric acid method
15
Table S2, Table S3). It can be seen from the result that the suitable
16
surface-grafting degree of Man-PEI was achieved under the preferential
17
molecular weight and mass ratio.
34
was used to quantify sugars on PEI (seen in
18
The antibacterial performance of the antibacterial agent is usually
19
determined by the minimum bactericidal concentration (MBC). MBC is the
20
minimum concentration of killing all bacteria with antibacterial agent. It is
21
can be observed from Figure S2 that 99.9% sterilization rate was recorded 12
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for E. coli K-12 with 220 µg mL-1 PEI, whereas a same sterilization rate
2
could be readily obtained with 10 µg mL-1 Man-PEI CPs. Furthermore, the
3
MBC of Man-PEI CPs was markedly lower than some antibiotics and
4
recently reported antibacterial polyethyleneimine materials (Table S1).
5
All of above results indicate that the high antibacterial capability of
6
Man-PEI CPs is resulted from the presence of mannose. So, we speculated
7
that mannose was closely related to the adherence of PEI on the bacterial
8
surface.
9
Mechanism of Antibacterial Activity. The majority of E. coli strains
10
possess a lot of fimbriae with different structure and function, which can be
11
mediated bacteria on the target cell adhesion and infection. Type I fimbriae
12
is the most common fimbriae of E. coli, consisting of four different subunits
13
of FimA, FimF, FimG and FimH. It is well known that FimH is the
14
determinant of the protein’s mannose-specific binding property; it possesses
15
carbohydrate recognition sites, which produces a strong affinity for
16
mannose.35-37 So, it is assumed that the excellent bactericidal effect of
17
Man-PEI CPs is relevance to mannose, which improves the adherence of
18
PEI on the bacterial surface.
19
In our work, the Man-PEI CPs were incubated with E. coli for
20
fluorescence-based agglutination assay to determine if the mannose
21
molecules attached to Man-PEI CPs still retained their ability to bind with 13
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the FimH proteins of the pili in E. coli. From Figure S3, it can be shown that
2
Man-PEI CPs can interact with the FimH proteins of the pili in E. coli by the
3
better bacteria agglutination behavior. As a control experiment, PEI without
4
mannose was also incubated with E. coli. PEI exhibited very little
5
nonspecific binding to E. coli. These results show that the binding of
6
Man-PEI CPs with E. coli is due to the interaction between the mannose and
7
the FimH proteins rather than to nonspecific absorption to the Man-PEI
8
CPs.38,39 The aldehyde group of mannose may consume the amino group of
9
PEI and further reduce the nonspecific binding of Man-PEI CPs with E. coli.
10
The enhancement of antibacterial ability can be attributed to the specific
11
adherence of PEI on the surface of E. coli.
12
To demonstrate the positive effect of mannose in Man-PEI CPs, we
13
modified PEI with ascorbic acid to acquire AA-PEI CPs, which was selected
14
as a model to make the control experiment. AA-PEI CPs could not
15
specifically bind to E. coli as that there is no specific recognition between to
16
ascorbic acid and E. coli. It can be seen from Figure S4, AA-PEI CPs hardly
17
had antibacterial activity, whereas Man-PEI CPs exhibit excellent
18
bactericidal effect. In the meantime, the antibacterial activity of PEI and
19
Man-PEI CPs against Gram-positive bacteria, S. aureus, was studied at
20
varying concentrations by colony counting. As shown in Figure S5, the
21
activities of PEI and Man-PEI CPs against S. aureus showed 14
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concentration-dependent. However, compared with the unmodified PEI, the
2
antibacterial activity of Man-PEI CPs did not exhibit any improvement. The
3
major causes for this result are that Man-PEI CPs has nonspecific binding to
4
S. aureus.
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These observations were demonstrated that the antibacterial ability of
6
Man-PEI CPs mainly depend on electrostatic action of the positive charge
7
PEI onto the negative charge bacterial surface, which causes the destruction
8
of the cell wall and the leakage of the cytoplasmic constituents, thereby
9
leading to the death of bacteria.40 The enhancement of antibacterial ability is
10
due to specific recognition between the mannose and FimH, which improves
11
the specific adherence of PEI on the surface of E. coli. Moreover, previous
12
studies had shown that the schiff base ligands are used in antibacterial and
13
antifungal material.41,42 Similarly, the 1H NMR spectra (Figure S1) had
14
confirmed the formation of schiff base between PEI and mannose that is
15
conducive to the enhancement of antibacterial activity.
16
Cytotoxicity of PEI and Man-PEI CPs. The cell cytotoxicity of the PEI
17
and Man-PEI CPs was tested using MTT assay against HeLa cells. As shown
18
in Figure 7, Man-PEI CPs do not exhibit obvious cytotoxicity under the
19
antibacterial condition (10 µg mL-1). The cell viability remained at ~75%
20
after 24 h incubation even if the concentration of Man-PEI CPs was
21
increased to 500 µg mL-1. However, the PEI exhibits certain cytotoxicity 15
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under the antibacterial condition (220 µg mL-1). The cell viability decreased
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to 56% at 500 µg mL-1 PEI. Therefore, the characteristic of low cell
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cytotoxicity indicates that Man-PEI CPs has great potential application as an
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antibacterial agent.
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4. CONCLUSION
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In this study, we have successfully modified PEI with mannose to
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construct Man-PEI CPs. By contrast with unmodified PEI, Man-PEI CPs
8
possess low cytotoxicity and excellent antibacterial activity on E. coli by the
9
specific recognition between FimH and mannose. Therefore, as an efficient
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antibacterial agent, Man-PEI CPs efficiently broaden the antibacterial
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spectrum of PEI. In addition, the synthesis of Man-PEI CPs is simple, rapid
12
and cost-efficient without complicated chemical modification. Given the
13
above advantages, Man-PEI CPs provide promising applications for
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combating multiple bacteria and also can be used as special agent for killing
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E. coli with low cytotoxicity.
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ASSOCIATED CONTENT
17
Supporting Information
18
One table listing about antibacterial activity of antibiotics and PEI
19
nanomaterials, 1H NRM spectra of PEI and Man-PEI CPs, phenol-sulfuric
20
acid method to quantify sugars on PEI, fluorescence-based bacterial
21
aggregation assay, sterilization rate of PEI, Man-PEI CPs and AA-PEI CPs 16
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against E. coli and S. aureus.
2
AUTHOR INFORMATION
3
Corresponding Authors
4
*(M.L.)
5
[email protected] 6
*(B.L.)
7
[email protected] 8
Notes
9
The authors declare no competing financial interest.
10
Phone:
Phone:
+86-29-85310517;
+86-29-81530726;
Fax:
+86-29-85310517;
E-mail:
Fax:
+86-29-81530727;
E-mail:
ACKNOWLEDGMENTS
11
This work was supported by the National Natural Science Foundation of
12
China (No. 21405101) and the Shaanxi Science and Technology Plan
13
Projects (No. 2017NY-121).
14
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Figure captions
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Scheme 1. Schematic illustration of PEI and Man-PEI CPs antibacterial
5
strategy.
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Figure 1. UV-vis absorption spectra of PEI, mannose, Man-PEI CPs and
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fluorescence emission spectra of Man-PEI CPs.
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Figure 2. The SEM images of (a) PEI and (b) Man-PEI CPs.
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Figure 3. FT-IR spectra of (a) PEI, (b) mannose, and (c) Man-PEI CPs.
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Figure 4. Sterilization rate of PEI and Man-PEI CPs at 10 µg mL-1
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according to the number of E. coli colonies on agar plates.
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Figure 5. (a) Agar plates of E. coli at a density of 3 × 104 treated with PEI
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and Man-PEI CPs for 3 h at 10 µg mL-1; plates were then incubated at 37 °C
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for 16 h.
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(b) Fluorescence microscope images of E. coli with PEI and Man-PEI CPs at
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10 µg mL-1 stained by PI after sterilization for 3 h. Unstained cells indicate 24
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live bacteria while red staining indicates dead bacteria. Scale bar, 50 µm.
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Figure 6. (a) Sterilization rate of 10 µg mL-1 Man-PEI CPs prepared by
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different mass ratio (PEI:mannose) according to the number of E. coli
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colonies on agar plates.
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(b) Agar plates of E. coli at a density of 3 × 104 treated with 10 µg mL-1
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Man-PEI CPs for 3 h; plates were then incubated at 37 °C for 16 h.
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Figure 7. Cell viability of PEI and Man-PEI CPs against HeLa cells at
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different concentrations for 24 h. The error bars represent the standard
11
deviations of three parallel measurements.
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