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Quaternary Ammonium Salt based Cross-Linked Micelles to Combat Biofilm
Fangqin Liu, Dengfeng He, Yunlong Yu, Lei Cheng, and Shiyong Zhang
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00010 • Publication Date (Web): 06 Feb 2019
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7 Quaternary Ammonium Salt based Cross-Linked Micelles to
8 Combat Biofilm
9
10 Fangqin Liu,† Dengfeng He,† Yunlong Yu,† Lei Cheng,‡ and Shiyong Zhang*,†
11
12 † National Engineering Research Center for Biomaterials, and College of Chemistry, Sichuan University, 29 Wangjiang Road,
13 Chengdu 610064, China.
14 ‡ State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, China

15
ABSTRACT: Due to self-produced extracellular polymeric substances (EPS), biofilms are hard to be eradicated by common
16
antimicrobials. Herein, a new quaternary ammonium salt based cross-linked micelle (QAS@CM) was created to combat biofilms.
17 The QAS@CM adsorbed first onto the biofilm surface through multi-charged interaction, then penetrated the EPS in the form of
18 nanoparticles and diffused throughout the films. By responding to biofilm acid/lipase microenvironment, these nanoparticles would
19 further break into quaternary ammonium oligomers and acted as the polyvalent inhibitors to effectively destroy the established
20 biofilm and kill the corresponding bacteria within it.
21
22
23
24 The biofilm is complex multicellular communities of bacterial cells via the electrostatic interaction, then the
bacteria embedded in self-produced extracellular polymeric hydrophobic chains penetrate the cell wall, disrupt the
25
substances (EPS) composed of polysaccharides, proteins, cytoplasmic membrane, release the cytoplasmic constituents,
26 lipids, and extracellular DNA. The EPS provides not only a and cause the cell death.34 This antimicrobial peptide-like
27 suitable microenvironment for microbial growth, but also antibacterial mechanism endows the QAS antimicrobials with
28 decreases antibiotics permeability and deactivates antibiotics wide application potential.35 Unfortunately, when the QAS
29 so that the biofilms are extremely recalcitrant to conventional interfaces with biofilm, it would be retarded by binding to the
30 antimicrobials.1,2 Since more than 80% of the clinical negatively charged EPS matrices. This causes that the
31 infections are caused by biofilms,3 the biofilm resistance is an common QASs, which effectively eradicate planktonic
32 urgent problem remained to be resolved. bacteria, might be up to 200-fold less potent against biofilm
33 The nanoparticles, benefited from the high surface to bacteria.36 Besides, the biocompatibility of QAS is also a big
34 volume ratio and controllable surface property, have shown concern. The high hemolysis makes the QAS difficult to be
good ability to penetrate EPS and are believed to be one of the applied in clinic.37,38
35
36 most promising platforms to combat biofilms.4-6 The current Scheme 1. Quaternary ammonium salt based cross-linked
37 anti-biofilm nanoparticles can be broadly classified into two micelle (QAS@CM) to combat biofilm.
groups. The first group is the metallic/inorganic
38
nanocomposites, such as metal (Gold, Silver),7-10 metal
39 oxides,11-13 and others.14-17 Although these metal/inorganic
40 nanoparticles have demonstrated good anti-biofilm properties,
41 the high toxicity limits their applications.18-20 The other group
42 is various nanocarriers, such as polymeric micelles,21-24
43 liposomes,25-28 and silica nanoparticles.29,30 These nanocarriers
44 can not only load antimicrobial agents to penetrate EPS but
45 also provide on-demand antimicrobial activity by responding
46 to biofilm microenvironment. Although highly promising, a
47 satisfactory nanocarrier usually requires tedious design and/or
synthesis to meet the stringent requirements of size, stability
48
and loading capacity. What’s more, these nanocarriers
49 themselves do not have antimicrobial effect. Once the delivery
50 task is finished, they become redundant substances and cause
51 easily the cumulative toxicity such as inflammation and Following our continuous interest in covalent capture of
52 carcinogenesis.31-33 In this regard, development of new anti- self-assemblies,39-43 we herein report a new quaternary
53 biofilm nanoparticles which could overcome above problems ammonium salt based cross-linked micelle (QAS@CM) to
54 would be highly valuable. combat biofilm. As shown in Scheme 1, the simple cross-
55 linking gave the micelles excellent stability, which was not
As an excellent amphiphilic antimicrobial, the quaternary
only advantageous for them to be adsorbed onto the biofilm
56 ammonium salt (QAS) first absorbs onto the surface of
surface through multi-charged interaction, but also
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advantageous for them to penetrate the EPS in the form of (Figure 1d) or under neutral condition (Figure 1e). However,
1 stable nanoparticles and reach the bottom of the biofilms. when the lipase was involved or the pH was adjusted to 5.0,
2 Notably, by responding to the biofilm acid/lipase both size and count rate dropped quickly, suggesting the
3 microenvironment, the QAS@CM broke into quaternary dissociation of the cross-linked nanoparticles. Interestingly, by
4 ammonium oligomers, which acted as the polyvalent inhibitors analyzed the residues after cleavage, it was found that the
5 to achieve a much better antimicrobial ability than that of QAS@CM was not dissociated into QAS monomers but
6 monomers. Moreover, owing that the long hydrophobic alkyl oligomers such as dimer and trimer (Figure S4). This finding
chains were buried inside the cross-linked micelle, the is important since the oligomers might act as the polyvalent
7
concentration of QAS@CM causing hemolysis was measured inhibitors to achieve a much better antimicrobial ability than
8 to be over ~160 times higher than that of free QAS. This that of monomers.46,47
9 greatly improved biocompatibility makes the QAS@CM a The antimicrobial superiority of QAS@CM was first tested
10 promising anti-biofilm candidate for clinical use. in the lipase secreting bacterium Staphylococcus aureus (S.
11 The QAS@CM was quickly prepared by dissolving the aureus, see Supporting Information for details). As shown in
12 QAS 1 in water above its critical micelle concentration (CMC, Figures 2a and b, both the free QAS and QAS@CM showed
13 63.6 μM, Figure S1) and cross-linking the acrylate groups in dose-dependent toxicity on S. aureus, in which the
14 the presence of dithiothreitol (DTT, See Scheme 1 and concentration of the free QAS inhibiting the bacterial growth
15 Supporting Information for details). The successful cross- (minimum inhibitory concentration, MIC) after 24 h was 69.6
16 linking was confirmed by the broadened proton signals and μM, while the MIC of QAS@CM was measured to be 49.7
17 disappearance of the alkenyl group in 1H NMR spectra μM, a value below its CMC (63.6 μM).
18 (Figure S2). The dynamic light scattering (DLS)
19 measurements revealed the QAS@CM with a hydrodynamic
diameter of 33.3 nm and zeta potential of 34.4 mV (Figure 1a
20
and b). Consistent with the DLS assay, the TEM image
21 obtained from the aqueous emulsion of QAS@CM showed the
22 formation of distinct spherical aggregates with a mean
23 diameter of ~32.5 nm (Figure 1c). The further stability test
24 demonstrated that the QAS@CM could maintain constant size
25 and particle dispersion index (PDI) upon dilution below CMC
26 (Figure S3), suggesting its good structural stability coming
27 from the cross-linking.
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Figure 2. Dose-dependent cell viability of S. aureus in the
35 presence of free QAS (a, c) and QAS@CM (b, d) at different pH
36 over time. The data showed that the MICs after 24 h are 69.6 μM,
37 49.7 μM, 69.6 μM, 39.8 μM for a-d, respectively. Data are
38 presented as the average ± the standard deviation (n = 3).
39 It has been reported that the antimicrobial effect of QASs
40 strongly depends on their ability to form micelles, which
41 enables them to contact with the bacteria surface through
42 multi-charge interaction to achieve the effective
Figure 1. Characterization of QAS@CM. (a) Hydrodynamic
43 diameter and (b) zeta potential determined by dynamic light antibacterial.48 This below CMC’s MIC of QAS@CM is
44 scattering. (c) TEM micrograph. The sample was stained with an significant since it is suggested that the cross-linked micelles
45 aqueous solution of 2% phosphotungstic acid. (d) Dissociation of could contact with bacteria by multivalent action without the
46 QAS@CM in the absence and presence of lipase at 37 °C over limitation of CMC. This superiority, together with the
time. (e) Dissociation of QAS@CM in the media of pH = 7.4 oligomers after cleavage, takes in charge of the high
47
(PBS buffer) and pH = 5.0 (acetate buffer) at 37 °C over time. antibacterial activity of QAS@CM. It is worth mentioning that
48 when the pH of culture medium was adjusted from 7.4 to 5.0,
Data are presented as the average ± the standard deviation (n = 3).
49 the MIC of QAS@CM further reduced to 39.8 μM (Figure
50 The design of QAS@CM produces a large number of β- 2d), while the free QAS remained unchanged (Figure 2c),
51 thiopropionate bonds on the particle surface, which would be indicating that the dual sensitivity would speed up the
52 easily cleaved under the conditions of lipase and/or mild breakage of the β-thiopropionate bonds and get better
53 acid,41 two common substances overexpressed by bacteria and antibacterial activity. To further investigate the broad-
biofilms.44,45 To test the sensitivity, the performance of spectrum activity of QAS@CM, another lipase secreting
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QAS@CM under different lipase and acidic environments bacterium Pseudomonas aeruginosa (P. aeruginosa) was
55 were then determined. As expected, the QAS@CM kept
56 employed. As expected, the QAS@CM demonstrated also a
constant size and count rate overtime in the absence of lipase better antimicrobial activity to P. aeruginosa (MIC: 248.7 μM)
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than that of free QAS (MIC: 348.1 μM); Under the acidic cross-linking was indeed an effective approach for QASs to
1 condition, the antimicrobial activity of QAS@CM enhanced efficiently penetrate EPS and diffuse into the deep layer of the
2 further (MIC: 198.9 μM) compared to the no change of the biofim matrix.
3 free QAS (Table S1). By contrast, for lipase non-secreting With the promising penetration result, we then tested the
4 bacterium Escherichia coli (E. Coli.), it was found that the free therapeutic behavior of the QAS@CM against maturely
5 QAS exhibited reasonable antibacterial activity with the MIC formed biofilms. Based on crystal violet staining, it was
6 of 298.4 μM, while the cross-linked nanoparticles exhibit no observed that the free QAS presented an 80% biofilm mass
activity even above the concentration of 497.3 μM (Table S1). disruption at the concentration of 248.7 μM, while for the
7
This on one hand suggested the on-demand antibacterial QAS@CM, only 99.5 μM was needed to achieve the same
8 property of cross-linked nanoparticles, on the other hand
9 disruptive efficiency (Figure 4a), suggesting an obviously
hinted the necessity of long alkyl chains in QAS sterilization. enhanced removal ability compared with free QAS.
10 Notably, although the QAS@CM cannot exert the
11 To further evaluate the bactericidal effect, the live/dead
antimicrobial activity for lipase non-secreting bacteria under
stain assay was conducted, in which the live bacteria with
12 the neutral condition, it can break into fragments under acidic
continuous membranes would be stained with green while the
13 condition and achieve antimicrobial activity better than that of
dead bacteria with damaged membranes would be stained with
14 free QAS (MIC: 248.7 μM, Table S1), thus suggesting its
red (See Supporting Information for details). As shown in
15 broad-spectrum capability against biofilms.44
Figure 4b, in the biofilm treated without materials, the CLSM
16 As mentioned in the beginning, biofilms produce EPS that image showed that majority of the bacteria were stained green.
17 prevent effective penetration of therapeutics. Having When the biofilm incubated with free QAS at the
18 established that the QAS@CM was capable of inhibiting the concentration of 99.5 μM, the bacteria showed partial green
19 growth of planktonic bacteria below its CMC, we set out to fluorescence and partial red fluorescence, hinting some
determine whether these nanoparticles could effectively bacteria were killed. However, upon the biofilm incubated
20
penetrate into biofilms. Briefly, biofilms formed by S. aureus with QAS@CM (99.5 μM), the bacteria which mostly stained
21 (green-fluorescence) were exposed to the suspensions of Nile
22 with red were observed and the biofilm became obviously less
red loaded QAS@CM and imaged using confocal laser dense compared to the control and the free QAS treated ones.
23 scanning microscopy (CLSM), and the free QAS was set as a This result verified that the QAS@CM could not only have an
24 control (See Supporting Information for details). enhanced efficacy to destroy the established biofilm but also
25 As expected, the QAS@CM readily penetrated and kill the corresponding bacterial cells within it. Consistent with
26 dispersed throughout the biofilm with fluorescence the live/dead stain assay, the scanning electron microscope
27 colocalizing with enclosed bacteria (Figure 3a), whereas the (SEM) showed that the biofilm treated with QAS@CM
28 free QAS demonstrated only limited penetration (Figure 3b). presented seriously structural destruction, and the bacteria in it
29 The further quantitative penetration profiles showed that the decreased significantly as well (Figure 4c, right), while the
30 QAS@CM accumulated preferentially in ~12.0-17.0 μm of biofilm treated by equivalent free QAS still showed obvious
31 biofilm depth, closing to the bottom of the biofilms, while the intercellular adsorption and the large bacterial aggregates
32 free QAS was more concentrated around 1.0-4.0 μm, near the coated by EPS were clearly visible (Figure 4c, middle).
top of the films (Figure 3c). These data demonstrate that the
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42 Figure 3. CLSM micrographs of S. aureus biofilms after 1 h treatment with QAS@CM (a) and free QAS (b). The upper and lower panels
43 are 3D and sectional images, respectively. (c) Penetration profile of S. aureus biofilms after 1 h treatment with QAS@CM and free QAS.
44 The x-axis is the depth of penetration of biofilms, and the y-axis is normalized intensity of red channels.
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53 Figure 4. (a) Percentage of S. aureus biofilms biomass as a function of concentrations of free QAS and QAS@CM. The samples treated
54 with deionized water as a control. Data are presented as the average ± the standard deviation (n = 3). (b) CLSM images of S. aureus
55 biofilms stained by PI & SYTO9 treated from left to right by deionized water, free QAS (99.5 μM) and QAS@CM (99.5 μM) for 24 h. (c)
56 SEM images of S. aureus biofilms treated from left to right by deionized water, free QAS (99.5 μM) and QAS@CM (99.5 μM) for 24 h.
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In addition to the anti-biofilm superiority, the QAS@CM Foundation of Sichuan Province (No. 2016JQ0028), and the
1 was characterized by good biocompatibility. It was reported Applied Basic Research Project of Sichuan Province (No.
2 that the QASs could also pierce the membrane of eukaryotic 15JC0440). We thank the Center of Testing and Analysis, Sichuan
3 cells by their hydrophobic alkyl chains and result in severe University, for NMR, MS and TEM measurements.
4 hemolysis.37 For example, the free QAS 1 showed significant
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* E-mail: szhang@scu.edu.cn
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