Thin Solid Films 520 (2011) 1120–1124

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Preparation and characterization of chemically-crosslinked polyethyleneimine filmson hydroxylated surfaces for stable bactericidal coatings

Bing Xia a,b,⁎, Chen Dong a,1, Ye Lu a, Ming Rong b, Yun-zhou Lv a, Jisen Shi a,⁎⁎a Key Laboratory of Forest Genetics & Biotechnology (Ministry of Education of China), Nanjing Forestry University, Nanjing 210037, PR Chinab College of Science, Nanjing Forestry University, Nanjing 210037, PR China

⁎ Correspondence to: B. Xia, College of Science, Nanjin210037, PR China. Tel.: +86 25 85428960.⁎⁎ Correspondence to: J. Shi, Key Laboratory of For

(Ministry of Education of China), Nanjing Forestry UChina.

E-mail address: xiabing@njfu.edu.cn (B. Xia).1 Equal work with first author.

0040-6090/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.tsf.2011.09.056

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 February 2011Received in revised form 22 September 2011Accepted 23 September 2011Available online 29 September 2011

Keywords:PolyethyleneimineGlutaraldehyde crosslinkingLong-term stabilityAntibacterial activity

Polyethyleneimine (PEI) thin films had been built up on aminosilanized glass surfaces via a layer-by-layerprocess of glutaraldehyde crosslinking, which was stepwise characterized by means of multi-surface analysisincluding Fourier transform infrared spectroscopy (FT-IR), atomic force microscope, ellipsometer and contactangle measurements. In phosphate buffered saline (PBS) solution, the degradation process of PEI thin filmswas monitored by FI-IR at 37 °C. As compared with aminosilanized surfaces alone (~80% loss after 2 days),chemically-crosslinked PEI films had a good long-term stability (only ~10% loss after 30 days). The antibacterialtests futher revealed that these PEI films could fast kill Gram-positive or Gram-negative bacteria (~100%) within~10 min. The killing efficiency of the film with five PEI layers treatment was 1.74×105 units/cm2, and after30 days of incubation in PBS solution, it still retained the antibacterial activity of 1.72×105 units/cm2.

g Forestry University, Nanjing

est Genetics & Biotechnologyniversity, Nanjing 210037, PR

rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

In the years to come, food industries, hospitals or community set-tings acquired bacterial infection will pose an increasing challenge tohealth care systems. Microbial contamination and the associated risk ofinfection lead to the needs of the rapid developments of microbicidalcoatings. Bacterial infection mainly occurs via bacterial adhesion, surfacecolonization, and subsequently biofilm formation, which always triggerpathogenic changes in the surrounding tissues [1,2].Microbicidal coatingsfunctionalized with polycations, antimicrobial peptide, antibiotics, en-zyme, and nanomaterials (e.g., Ag, TiO2, SiO2, MgO, CuO, ZnO, CdSe/CdTe nanoparticles, and single-walled carbon nanotubes) can activelyfight the spread of bacterial infections by repelling bacterial adhesion, kill-ing the adherent bacteria, or inhibiting biofilm formation [3–12]. General-ly, two approaches to designmicrobicidal coatings are based on released-and non-released antimicrobial systems. For the releasing coatings, thefast exhaustion of the antibiotics and antiseptics and the necessity of fre-quent loading of the antimicrobial agent are some of the disadvantages.On the other hand, the degradation of non-released coatings under phys-iological environment can also hinder their applications on surgical in-struments and biomedical devices [3]. Therefore, the development ofantibacterial coatings with long-term stability is critical to resolve the

problem of microbial infections occurred on food industries, hospitals orcommunity settings.

Among various antimicrobial agents, polyethyleneimine (PEI)containing polycationic moieties have been widely used to modifydifferent types of substrates due to (1) their long-term antimicrobial ac-tivity with no resistance developed, (2) minimal cytotoxicity to mam-malian cells, (3) regeneration upon loss of activity, and (4) biocidalability for a broad spetrum of pathogenic microorganisms in brieftimes of contact [13–29]. Coatings that physically entrap PEI by air-dry[27], or electrostatic attraction [28] easily lose their antibacterial abilityas the biocidal substance gets released into the surrounding environ-ment or solution. In contrast, surfaces with covalently attached PEI canbe bactericidal through a contact mechanism [19–25]. Since the func-tional groups are covalently attached, the surfaces can retain its antimi-crobial property even after multiple uses. Herein, we adopted onespecific chemically-crosslinking methodology to fabricate PEI thin filmson hydroxylated surfaces (shown in Fig. 1). The bactericidal ability ofthese PEI films was subsequently investigated, which indicated thatthey could provide long-term, fast disinfecting and wide-spectrum anti-bacterial ability.

2. Experimental details

2.1. Materials

Glass slides (25.4×27.6×1.2 mm, optical microscope slides, Sailbrand, China), (3-aminopropyl) triethoxysilane (APTES) (Sigma,USA), glutaraldehyde (Aldrich, USA), sodium borohydride (NaBH4)(Aldrich, USA), branched PEI (Mw 60000, Sigma, USA), LIVE/DEAD Bac-Light stainingTM bacterial viability kit (Invitrogen, USA), Luria-Bertani

APTES

( PEI )NH2

NH2

H2N

H2N

NH2

NH2

Glass Surface

N

NH2

NH2

H2N NH2

H2N

N

NaBH4

STEP 1 STEP 2

STEP 4

STEP 3

APTES-Glass

G1.0-PEI-Glass

NH2 N

O

N

O

N

O

NH2NH2 OO

N

N

NH2

NH2

H2N NH2

H2N

H2N

NH2

NH2N NH2

H2N

N

NH

NH2

NH2

H2N NH2

H2N

NHNH

HN

NH2

NH2

H2N NH2

H2N

H2N

NH2

NH

H2N NH2

H2N

NH

Fig. 1. The growth of G1.0-PEI films on glass surface.

1121B. Xia et al. / Thin Solid Films 520 (2011) 1120–1124

(LB) medium (1% bacto tryptone, 0.5% yeast extract, 1% NaCl solution,pH=7.2), proteose peptone-beef extract medium (0.5% bacto beef ex-tract, 1% bacto tryptone, 0.5% NaCl, pH=7.2), phosphate buffered sa-line (PBS) solution (deionized (DI) water (resistanceN18 MΩ cm)supplemented with 8.0 g/L NaCl, 0.2 g/L KCl, 1.2 g/L Na2HPO4 and0.2 g/L KH2PO4, pH=7.2), L-agar plate (1% (w/w) agar in LBmediumwas sterilized, and then incubated in culture dishes).

2.2. Instrumentations

(1) Transmission Fourier transform infrared spectroscopy (FT-IR)spectra were recorded using Bruker Vertex 70 spectrometer at0.25 cm−1 resolution. Typically, 128 interferograms were acquiredper spectrum. The samples were mounted in a purged sample cham-ber. Background spectra were obtained using bare glass slides. (2)Contact angle measurements of different layers on glass surfaceswith DI water were carried out in air by the sessile-drop methodwith Drop Shape Analyzer (Krüss DSA 100). Readings were takenafter the angles were observed to be stable with time. Reporteddata are an average of the readings taken at five different spots foreach sample. (3) Atomic force microscope (AFM) images wereobtained using Seiko SPI 3800 N in dynamic mode. A Si3N4 probewith a resonance frequency of 27 KHz was used. (4) An ellipsometer(Tuopu Instrument, Auto TPY-2), operated with a 632.8 nm He–Nelaser at an incident angle of 0–180°, was employed for thickness mea-surement. We use the value of 1.53 as the refractive index of PEI films.At least three measurements were taken for each sample, and themean values were reproducible within ±1 Å. (5) The green fluores-cent live stain SYTO 9 and the red fluorescent dead stain propidium io-dide (PI) were visualized separately with a green (excitation/emissionmaxima, 480/500 nm) and red (excitation/emission maxima,490/635 nm) bandpass filter set of laser confocal scanning microscope(LSCM) (Leica TCS SL, Heidelberg, Germany), respectively. (6) Bacteriacells were fixed by 4% glutaraldehyde in PBS solution for 4 h, washed 3times by PBS. And then these sampleswere soaked in a serials of ethanolsolutions (a step gradient of 30%, 50%, 70%, 90% in water for 15 min perstep), ending with 100% ethanol. And dehydrated, the samples weredried in a critical point dryer (Emitech K850), coated with gold

(15 mA, 90 s, Hitach E-1010), and then imaged with scanning electronmicroscope (SEM) (FEI QUANTA 200).

2.3. APTES modification on glass surfaces

Glass slides were cleaned in piranha solution (3:1 (v/v) concen-trated H2SO4/30% H2O2) for 15 min at 80 °C, removed and then rinsedcopiously with DI water. The clean slides were immersed in APTESaqueous solution (1%, v/v, pH=5.5) for 1 h at room temperature.These slides were taken out, washed with DI water to remove physi-sorbed silane molecules, and then dried by high-purity N2. Finally, thesamples were incubated in an oven at 110 °C for 30 min to stableAPTES-glass modification.

2.4. The growth-up of Gn-PEI films on glass surfaces

APTES-glass samples were immersed in glutaraldehyde aqueoussolution (5%, v/v, pH=9.5) for 2 h at room temperature (step 2 inFig. 1). After DI water rinsing, the samples were then incubated inPEI aqueous solution (1%, v/v, pH=9.5) for 2 h at room temperature(step 3 in Fig. 1). After rinsing with DI water and ethanol, the sampleswere subsequently immersed in NaBH4 aqueous solution (1 mol/L)for 2 h at room temperature, followed by washing with DI water,and drying with high-purity N2 (step 4 in Fig. 1). G1.0-PEI-glass hadbeen achieved by steps 2, 3, 4, and Gn-PEI-glass could be preparedby repeating n reaction cycles of steps 2, 3, 4.

2.5. Bacteria culture

At 37 °C, Escherichia coli (E. coli) JM109 strains were grown with250 rpm shaking in LB growth medium supplemented with appropri-ate antibiotics (50 μg/mL ampicillin) overnight. And the cultures wereadjusted to ~5×108 colony forming units (cfu)/mL (OD600=~0.15)by PBS solution. Bacillus subtilis (B. subtilis) strain 168 were grownwith 250 rpm shaking in proteose peptone-beef extractmedium supple-mented with appropriate antibiotics (50 μg/mL ampicillin) overnight.And the cultures were adjusted to ~5×108 cfu/mL (OD600=~0.15) byPBS solution.

2.6. Fluorescence microscopy

The viability of E. coli or B. subtilis was tested using the live/deadbacterial kits containing the two nucleic acid stains SYTO 9 and PI. Be-fore applying to modified glass surfaces, a 100 μL mixture of SYTO 9and PI (v/v 1:1) was added into 100 μL bacteria suspension(~5×107 cells/mL) for 15 min at 37 °C in the dark, and then viewedby LSCM under a green filter or a red filter.

2.7. Antimicrobial activities determination

Antimicrobial testing was performed using a modified ASTM stan-dard: E2149-01 Standard Test Method for Determining the Antimi-crobial Activity of Immobilized Antimicrobial Agents under DynamicContact Conditions [30]. E. coli cells were diluted with PBS to the de-sired concentration. The actual number of cells used for a given ex-periment was determined by standard serial dilution. Gn-PEI-glassslides (1.5×2.5 cm2) were immersed in 5 mL of cell suspension(7×105 cfu/mL in a 50 mL conical tube (Falcon) at 37 °C and250 rpm). Bare glass slides were used as a control. A certain volumeof bacterium suspension was taken after 1 h, diluted appropriately,and plated on L-agar plates. Each viable bacterium developed into abacterial colony that was identified using magnifying glasses andcounted. The antibacterial activity of a modified surface was deter-mined by using Eq. (1). Ncontrol and Nsample correspond to the colonieson the L-agar plates of the control and the sample, respectively, whileFcontrol and Fsample represent the dilution factor of the control and

3600 3400 3200 3000 2800 26000.95

0.96

0.97

0.98

0.99

1.00

(c)

2851 cm-12933 cm-1

3282 cm-1

3369 cm-1

(d)

(b)

(a)T

ran

smit

tan

ce (

%)

Wavenumber (cm-1)

Fig. 2. FT-IR spectra of (a) APTES-glass, (b)G1.0-PEI-glass, (c)G5.0-PEI-glass, and (d) PEI-glassby spin coating.

1122 B. Xia et al. / Thin Solid Films 520 (2011) 1120–1124

experiment, respectively. All experiments were performed in tripli-cate.

N ¼ NcontrolFcontrol−NsampleFsample ð1Þ

3. Results and discussion

3.1. Characterizations of Gn-PEI films

The detailed modification route of Gn-PEI films on glass surfaceswas in Fig. 1. Glutaraldehyde is themost popular biofunctional crosslin-ker with amine groups by schiff base formation. PEI thin films could bebuilt up on glass surfaces through schiff base reaction between amines

Fig. 3. AFM topographic images of (a) bare glass, (b) APTES-glass, (c) G1.0-PEI-glass,and (d) G5.0-PEI-glass.

and aldehydes. In additional, stable secondary linkages of C―N bondhad been yielded by NaBH4 reduction to avoid the cleavage of imide(―C;N―) bonds. To confirm the grafting of Gn-PEI films on glass sur-faces, we collected the FT-IR spectra of APTES-glass, G1.0-PEI-glass,G5.0-PEI-glass and PEI-glass by spin coating, which were all showedin Fig. 2a–d. In the spectra, vibrations of ―NH2 and ―CH2― of PEIwere all characterized by the νas stretching mode of N–H at3369 cm−1, νs stretching mode of N―H at 3282 cm−1, νas stretchingmode of C―H at 2933 cm−1 and νs stretching mode of C―H at2851 cm−1, which could be found in the spectrum of PEI-glass byspin coating (in Fig. 2d). FT-IR measurements demonstrated thatPEI films had been covalently immobilized onto glass surfaces viaglutaraldehyde crosslinking. Siloxane bonding (Si―O―Si) alone easilyhydrolyzedunder alkaline conditions, however, upper PEIfilms via stablelinkages of C―N or C―C bonds could protect the siloxane bondingunderlayer to avoid hydrolysis.

The growth of Gn-PEI-glass was also characterized by contactangle measurements, ellipsometer and AFM. The water contact anglesfor APTES-glass (54º), G1.0-PEI-glass (63º), G5.0-PEI-glass (75º) exhib-ited a slight increase, due to the grafting amount of PEI increasing. Tofurther investigate the surface morphology, AFM images of the bareglass, APTES-glass, G1.0-PEI-glass and G5.0-PEI-glass were taken(shown in Fig. 3a–d). Compared to the clean and smooth surfaces ofthe bare glass, there were embossed nanodomes homogeneously dis-persed on the surfaces of G1.0-PEI-glass (Fig. 3c) and G5.0-PEI-glass(Fig. 3d). The surface RMS roughness (the standard deviations ofthe Z values within the same area) of four substrates was 1.6, 2.1,9.0, and 12.2 nm, respectively, which revealed that overall surfaceroughness was dominated by these nanodomes. On G1.0-PEI-glasssurface, the domes were 130±40 nm in diameter and 32 nm inheight. However, after five PEI layers grafting, the domes were enlargedto 160±60 nm in diameter and 64 nm in height. The increasing of the

3100 3000 2900 2800 27000.95

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0.33b)

IR d

egre

e o

f g

raft

ing

PE

I

Incubation Time (day)

Fig. 4. (a) FT-IR spectra of G5.0-PEI-glass surfaces incubated in PBS solution, and (b) therelationship between integrated areas of ―CH2― groups and incubation time (νas(-■-) and νs (-○-) stretching mode).

Fig. 5. LSCM images of E. coli deposition on bare glass surfaces under green filter (a) and under red filter (b), E. coli deposition on APTES-glass surfaces under green filter (c) andunder red filter (d), E. coli deposition on G1.0-PEI-glass surfaces under green filter (e) and under red filter (f), E. coli deposition on G5.0-PEI-glass surfaces under green filter (g) andunder red filter (h), B. subtilis deposition onG5.0-PEI-glass surfaces under greenfilter (i) and under redfilter (j). SEM images of E. coli deposition on bare glass surfaces (k), andG5.0-PEI-glasssurfaces (l).

1123B. Xia et al. / Thin Solid Films 520 (2011) 1120–1124

size and density indicated these nanodomes were formed through thelocalized growth of hyperbranched PEI. Finally, the film thicknesses ofAPTES-glass (4.3±0.5 nm) G1.0-PEI-glass (28.2±0.8 nm) and G5.0-PEI-glass (55.0±1.2 nm) were also measured, which revealed thatGn-PEI films could be grafted onto glass surfaces by a layer-by-layerprocess.

3.2. Stability tests of Gn-PEI films

G5.0-PEI-glass samples had been incubated in PBS solution(pH=7.2) for 30 days, and the degradation process of G5.0-PEI filmswas monitored by FT-IR (shown in Fig. 4a). To quantify the surfacecoverage of PEI, we defined the integrated peak area of 2700–2885or 2885–3100 cm−1 (νs and νas stretching mode of―CH2―) as IR de-gree of PEI grafting. As seen in Fig. 4b, a loss of ~5% occurred in thefirst day due to physical absorption of PEI, followed by a continuousloss of about 5% over one month. However, silane layers alone easilyhydrolyzed in water and a common loss of ~80% occurred after onemonth [31]. The stability of chemically-crosslinked PEI films couldbe significantly improved under physiological environment, whichalso demonstrated that stable PEI films could effectively protect silanelayers against hydrolysis.

3.3. Bacteria viability assay on Gn-PEI-glass surfaces

To confirm that surface-grafted PEI polymer kills bacteria, a live/deadtwo-color fluorescence method was adopted with a mixture of SYTO 9green-fluorescent nucleic acid stain, and the red-fluorescent nucleicacid stain (PI). The SYTO 9 stain generally labels all bacteria in a popula-tion including those with intact membranes and those with damagedmembranes. On the other hand, red PI stain only penetrates bacteria

with damaged membranes. The dye mixture was incubated with thebacterial suspension before applying to various substrates. After staining,100 μL of cell suspensionwas dropped onto bare glass, APTES-glass, G1.0-PEI-glass and G5.0-PEI-glass surfaces, and then observed by LSCM. Asseen in Fig. 5a,b, almost all E. coli cells (Gram-negative bacteria) gaveonly green fluorescence after 30 min of exposure to the bare glass as acontrol, indicating that all bacteria had intact cell membranes. In con-trast, Fig. 5e–h revealed that almost all E. coli cells (~100%) turned reddue to a loss of cell membrane integrity after 10 min of exposure toG1.0-PEI-glass or G5.0-PEI-glass surfaces. G1.0 (or G5.0)-PEI-glass surfaceswere also found to be active against Gram-positive bacteria such as B.subtilis (shown in Fig. 5i,j), which thus exhibited that PEI thin films hadwide-spectrum antibacterial properties. Compared to inactive APTES-glass in Fig. 5c,d (only few bacterial cells was killed after 10 min of expo-sure), the results further demonstrated that PEImolecules grafted are theactive species for fast killing bacteria cells. Gn-PEI films bearing aminegroups appear to act by inducing an ion exchange between the positivecharges and cationswithin themembrane, disrupting negatively chargedbacterial cell membrane to lead to a loss of cell membrane integrity, fol-lowed by release of K+ ions and other cytoplasmic constituents, andresulting in immediate death of the bacterial cell [7,9,29]. The hypothesiswas further supported by SEM images (shown in Fig. 5k,l),which indicat-ed that the dead bacterial cells seem shrinking due to the leakage of cy-toplasmic constituents.

3.4. Antibacterial assessment of Gn-PEI films

According to the ASTM standard above described, the effects of PEIamount on the antibacterial activities were systematically investigated.Fig. 6a–d showed a representative set of plates for bare glass, APTES-glass, G1.0-PEI-glass and G5.0-PEI-glass, and live bacteria (cfu's) on plates

0.05 0.10 0.15 0.20 0.25 0.301.64

1.66

1.68

1.70

1.72

1.74 e)G5.0G4.0

G3.0

G1.0

G2.0

Nu

mb

er o

f K

illed

E. c

oli

(105

un

its/

cm2 )

IR degree of grafting PEI

a) b)

c) d)

Fig. 6. Representative optical images of agar plates for (a) bare glass, (b) APTES-glass,(c) G1.0-PEI glass, and (d)G5.0-PEI glass samples after theE. coli challenge. (e) The relationshipbetween IR degree of grafting PEI and the antibacterial activities.

1124 B. Xia et al. / Thin Solid Films 520 (2011) 1120–1124

can be counted to obtain the number of killed bacteria. Compared toAPTES-glass (8.93×104 units/cm2), the number of E. coli killed on G1.0-PEI-glass increased to 1.65×105 units/cm2, which suggested that graft-ing PEI could effectively improve the antibacterial activities. The absor-bance areas from 2885 to 3100 cm−1 (νas stretching mode of ―CH2―)of Gn-PEI-glass were integrated as IR amount of grafting PEI, and the re-lationship between grafting PEI amount and antibacterial activities wasfurther shown in Fig. 6e. The number of killed E. coli of Gn-PEI-glass in-creased from ~1.65×105 to ~1.74×105 units/cm2 with PEI generationincreasing, until the saturation reached after three PEI layers treatment.By comparing G3.0-PEI with G5.0-PEI-glass, ~ 40% of PEI amount hadbeen reduced, but antibacterial activities of these two samples were sig-nificantly unchanged. After 30 days incubation in PBS solution, G5.0-PEI-glass still retained the antibacterial activity of 1.72×105 units/cm2,which was attributed to only ~10% loss of PEI amount. These resultsthus demonstrated a good long-term antibacterial activity of G5.0-PEI-glass, which gives hopes to combating bacterial infections of biomedicaldevices.

4. Conclusions

In conclusions, we described a general strategy to fabricatechemically-crosslinked PEI films with glutaraldehyde on hydroxylated

surfaces. The antibacterial investigations demonstrated that PEI filmscould fast kill Gram-positve or Gram-negative bacteria within ~10 min,and the killing efficiency of glass surfaces with five PEI layers treatmentwas 1.74×105 units/cm2. Moreover, PEI thin films had a good long-termstability (only ~10% loss) after 30 days incubation in PBS solution, whichstill retained the antibacterial activity (1.74×105 units/cm2). In oneword,these PEI films are long-term stable, fast disinfecting and wide-spectrumantibacterial coatings, which would be widely applied on surgical instru-ments and biomedical devices.

Acknowledgments

This work was funded by the National Natural Science Foundationof China (nos. 30930077, 31000164). Thanks to Dr. Yaqing Chen forhis help in the AFM measurements and Dr. Jing Yang for her help inthe SEM measurements.

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