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Block copolymer nanoparticles remove biofilms of drug-resistant Gram-positive bacteria by nanoscale bacterial debridement Jianghua Li, Kaixi Zhang, Lin Ruan, Seow Fong Chin, Nirmani Wickramasinghe, Hanbin Liu, Vikashini Ravikumar, Jinghua Ren, Hongwei Duan, Liang Yang, and Mary B Chan-Park Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b01000 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 15, 2018
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Block copolymer nanoparticles remove biofilms of drug-resistant Gram-
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positive bacteria by nanoscale bacterial debridement
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Jianghua Li,1 Kaixi Zhang,1 Lin Ruan,1 Seow Fong Chin,3 Nirmani Wickramasinghe, 3 Hanbin
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Liu, 1 Vikashini Ravikumar,3Jinghua Ren,4 Hongwei Duan,1 Liang Yang,*3 Mary B. Chan-
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Park*1,2
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1. Centre for Antimicrobial Bioengineering, School of Chemical and Biomedical Engineering,
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Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore
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2. Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive
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Singapore 636921, Singapore
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3. Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang
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Technological University, 60 Nanyang Drive, SBS-01N-27, Singapore 637551, Singapore
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4. Cancer Center, Union Hospital, Huazhong University of Science & Technology, Wuhan
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430022, China
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Keywords: biofilm, infection, antibiofilm, cationic copolymer nanoparticles, non-hemolytic,
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biocompatibility
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Abstract: Biofilms and rapid evolution of multi-drug resistance complicate the treatment of
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bacterial infections. Antibiofilm agents such as metallic/inorganic nanoparticles or peptides act
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by exerting anti-bacterial effects and hence do not combat biofilms of antibiotics-resistant strains.
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In this paper we show that the block copolymer DA95B5, dextran-block–poly((3-
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acrylamidopropyl) trimethylammonium chloride (AMPTMA)-co-butyl methacrylate (BMA)),
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effectively removes preformed biofilms of various clinically relevant multi-drug resistant Gram-
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positive bacteria including Methicillin-resistant Staphylococcus aureus, Vancomycin-Resistant
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Enterococci (VRE V583) and Enteroccocus faecalis (OG1RF). DA95B5 self-assembles into
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core-shell nanoparticles with a non-fouling dextran shell and a cationic core. These nanoparticles
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diffuse into biofilms and attach to bacteria but do not kill them; instead, they promote the gradual
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dispersal of biofilm bacteria probably because the solubility of the bacteria/nanoparticle complex
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is enhanced by the nanoparticle dextran shell. DA95B5 when applied as a solution to a hydrogel
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pad dressing shows excellent in vivo MRSA biofilm removal efficacy of 3.7 log reduction in a
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murine excisional wound model, which is significantly superior to vancomycin. Further,
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DA95B5 has very low in vitro hemolysis and negligible in vivo acute toxicity. This new strategy
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for biofilm removal -- nanoscale bacterial debridement -- is orthogonal to conventional rapidly
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developing resistance traits in bacteria so that it is as effective towards resistant strains as
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towards sensitive strains and may have widespread applications.
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Bacteria have developed resistance towards almost all classes of antibiotics, with serious
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consequences for anti-infection therapy. Further, bacterial infections often occur in biofilm form
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in which bacteria are protected by extracellular polymeric substances (EPS);1,
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antibiotics, which typically eradicate metabolically active planktonic bacteria, may be as much as
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1000-fold less potent against biofilm bacteria.3 The combination of multi-drug resistance and the
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protective character of biofilms is particularly worrisome from the perspective of therapy.
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common
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There has been much recent effort to develop new antibiofilm agents.4 Small molecules,5-7
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such as bromophenazine,5 have been found to eradicate biofilm formed by Gram-positive
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bacteria such as S. aureus. Antimicrobial peptides (AMPs) tend to get trapped in anionic biofilms
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and also suffer enzymatic degradation in biofilms.8-10 A few AMPs such as IDR-1018 have
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shown efficacy for removal of pre-established biofilm by downregulation of genes involved in
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biofilm formation, but this may be prone to resistance evolution.11-13Small molecules and AMPs
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commonly have biocompatibility issues in terms of acute toxicity and/or hemolysis.14,
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Surfactants16 and surfactant-like molecules17 have also shown the ability to remove biofilm.
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Cetyltrimethylammonium bromide (CTAB)18, sodium dodecyl sulfate (SDS)
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soluble modulins (PSMs)17 have shown antibiofilm effect. However, their hemolytic properties
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limit their applications.20, 21 Nanoparticles (NPs) are an alternative class of antibiofilm agents22
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and many metallic nanocomposites, such
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nanoparticles(AuNPs),24 magnetic iron oxide NPs25, 26 and other metal complex NPs,27 have been
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demonstrated to have antibiofilm effects. However, the toxicity of these metal/inorganic NPs
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remains a concern.28 Polymeric micelles which are themselves not effective in dispersing biofilm
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but can function as nanocarriers of antibiofilm agents have been shown to improve biofilm
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dispersal efficacy29 and may possess good biocompatibility.30-34 Again, these antibiofilm NPs
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and phenol-
as silver nanoparticles (AgNPs),23 gold
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remove biofilms through bactericidal action. There are few previous research reports on
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antibiofilm agents which are not antibacterial
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are of great interest because they are not affected by the problem of conventional antibiotics
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resistance.
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and such non-bactericidal antibiofilm agents
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Herein, we report novel polymeric NPs that can effectively remove biofilms of multi-drug
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resistant Gram-positive bacteria. The weakly amphiphilic cationic block copolymer of dextran
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and
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(P(AMPTMA-co-BMA)) (hereafter called DA95B5) self-assembles into NPs with a thin
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polysaccharide shell and a cationic core. These NPs diffuse through biofilms of Gram-positive
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bacteria to electrostatically complex with bacteria surfaces without killing the bacteria. Instead,
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the NPs cause the gradual removal of biofilms by weakening the attachment of the bacteria to the
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biofilm and exhibit biofilm removal efficacy comparable or superior to current standard
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antibiotics. Specifically, DA95B5 effectively removes biofilms of Methicillin-resistant
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Staphylococcus aureus (MRSA), Vancomycin-Resistant Enterococci (VRE) and also
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Enterococcus faecalis OG1RF which is implicated in catheter-associated infections. In vivo data
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(using a murine excisional wound model) also shows that DA95B5 solution when soaked into a
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hydrogel pad dressing can remove MRSA biofilm by 3.7 log reduction, compared to 2.1 log
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reduction achieved by vancomycin. The NPs are also non-hemolytic in vitro and have low in
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vivo cytotoxicity. This is the first report of polymeric NPs with a new biofilm removal
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mechanism, which we term “nanoscale bacterial debridement,” that is orthogonal to bactericidal
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activity and antibiotics resistance. This new class of agent has good Gram-positive biofilm
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removal efficacy and is as effective in biofilm removal of multi-drug resistant Gram-positive
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bacteria as is it for drug-sensitive strains.
poly((3-acrylamidopropyl)trimethylammonium
chloride)-co-(butyl
methacrylate)
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Four (co)polymers were synthesized based on the hydrophilic cationic (3-acrylamidopropyl)
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trimethylammonium chloride (AMPTMA) monomer (A, Scheme 1a) with incorporation of the
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non-fouling dextran block (D) and/or hydrophobic butyl methacrylate (B). The four
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(co)polymers investigated were: (i)A100, a homopolymer made of AMPTMA; (ii)A95B5,
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poly(AMPTMA(95%)-co-butyl
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poly(AMPTMA) copolymer (Dextran Mw is 6000 Daltons); and (ii) DA95B5, a dextran-block-
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poly(AMPTMA(95%)-co-butyl
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Information Figure S1-S4) and GPC (Supporting Information Table S1) confirmed the
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successful syntheses of these (co)polymers.
methacrylate(5%));
methacrylate(5%)).
(iii)
The
DA100,
NMR
a
spectra
dextran-block-
(Supporting
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Dynamic light scattering (DLS) (Table 1) analysis revealed that all the (co)polymers, except
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DA95B5, existed in DI water as individual molecules with hydrodynamic radius (Rh) less than
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10 nm at the concentration of 512 µg/mL. However, DA95B5 self-aggregated into NPs with Rh
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of 75.2±3.1 nm and had a critical micelle concentration (CMC) of around 32 µg/mL, which was
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determined using pyrene as a fluorescent probe (Supporting Information Figure S5a). The Rg
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(radius of gyration) was also measured; the Rg/Rh ratio of DA95B5 NPs in DI water was around
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0.4 (Table 1 and Supporting Information Table S2), indicating that the copolymer self-
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aggregated in DI water into core-shell NPs.37 The average diameter of DA95B5 NPs determined
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by transmission electron microscopy (TEM) was found to be between 20 and 30 nm
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(Supporting Information Figure S5b), corroborating the DLS results. We also found that
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DA95B5 NPs in PBS have size and Rg/Rh (~0.4) similar to that of particles in DI water
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(Supporting Information Table S2). DA95B5, which contains the hydrophobic butyl
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methacrylate constituent, reduced the solution surface tension (Table 1) from around 70 mN/m
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to about 45 mN/m. The zeta potential values of all the (co)polymers in DI water were in the
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range 36 to 41 mV.
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We determined the minimum inhibitory concentrations (MICs) of our (co)polymers against
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both Gram-positive and Gram-negative bacteria (Table 2). The (co)polymers A100 and DA100
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had excellent antimicrobial activity (8-16 µg/mL) against Gram-positive bacteria (S. aureus,
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including MRSA (BAA40 and USA300)), moderate efficacy (128-512 µg/mL) against the Gram-
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negative species E. coli and poor antibacterial activity against E. faecalis strains (VRE and
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OG1RF). For A95B5, which contains a small proportion of the hydrophobic BMA, the
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antibacterial activity against all the Gram-positive bacteria (S. aureus, MRSA, VRE and OG1RF)
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was similar to that of A100 or DA100, but the killing of E. coli was significantly improved (MIC:
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32-64 µg/mL). However, the terpolymer DA95B5 had much higher MICs (≥512 µg/mL) against
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all of the planktonic pathogens tested, probably because it forms micelles with the bactericidal
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cationic and hydrophobic components of AMPTMA and BMA hidden in the core (Scheme 1b),
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beneath a non-fouling and non-toxic dextran corona.
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Using the MBECTM assay based on ASTM E2799-17,38,
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we observed that DA95B5
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effectively reduced bacteria counts in preformed biofilms of Gram-positive bacteria (Figure 1a
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and Table 3). We studied the effect of DA95B5 copolymer on preformed biofilms of five multi-
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drug resistant/clinically relevant Gram-positive bacteria (MRSA BAA40, MRSA USA300,
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MRSA KKH5, Vancomycin-Resistant E. faecalis V583 (VRE) and E. faecalis OG1RF) and one
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drug-sensitive Gram-positive strain (S. aureus SA29213). DA95B5 showed reduction in cell
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counts of biofilm bacteria of all Gram-positive bacteria tested in a dose-dependent manner with
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efficacy much better than or similar to that of current standard antibiotics. For MRSA BAA40
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(Figure 1a and Table 3), DA95B5 reduced the biofilm bacterial cell counts by up to 2.0 log 6
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reduction after a single polymer treatment at 32 µg/mL; these reductions were higher than those
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of first-line MRSA antibiotics (oxacillin, doxycycline and linezolid) as well as that of the last
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resort antibiotic vancomycin. DA95B5 also reduced the biofilm cell counts of other MRSA and
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SA strains: it achieved generally higher log reductions (i.e. 1.1, 1.7 and 1.2) against MRSA
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USA300, MRSA KKH5 and SA29213 biofilm bacteria respectively compared to those of
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standard antibiotics (Table 3 and Supporting Information Figure S6). Against VRE biofilm
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bacteria, DA95B5 achieved 2.5 log reduction, albeit with a higher concentration of 512 µg/mL,
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which is not achievable by any of the standard antibiotics investigated that only achieved
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maximum log reduction of 1.3 with ampicillin antibiotic. With a lower concentration of 128
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µg/mL, DA95B5 showed 0.8 log reduction of VRE biofilm bacteria which was still better than
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the standard antibiotics linezolid and nitrofurantoin (with 0.7 and 0.1 log reductions, respectively)
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(Table 3) but worse off compared to ampicillin (with 1.8 log reduction). With the clinically
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relevant bacteria OG1RF, DA95B5 (128 µg/mL) reduced the biofilm bacteria by 0.8 log
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reduction, which was superior or comparable to the current standard antibiotics (0.2 to 0.8 log
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reduction) (Table 3). The FESEM images (Figure 1b) corroborated that the cell densities of
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Gram-positive biofilm bacteria clearly declined after treatment with a single dose (128 µg/mL)
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of DA95B5. However, DA95B5 showed no/poor reduction of biofilm bacteria of the two Gram-
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negative strains (E. coli EC8739 and E. coli K12) tested (Supporting Information Figure S7).
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We also investigated the efficacy of DA95B5 in removing more matured biofilms. We found
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that DA95B5 at the same concentration (i.e. 32 µg/mL) can also remove the longer-term 3-day
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biofilm by around 2.0 log reduction, so that its efficacy was much higher than that of
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vancomycin (1.0 log reduction). With even longer-term (7-day) biofilm, DA95B5 at higher
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concentration (i.e. 512 µg/mL) reduced the biofilm by 1.7 log reduction as opposed to around 0.1 7
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log reduction with vancomycin, again corroborating the superior efficacy of DA95B5 in MRSA
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BAA40 biofilm removal (Supporting Information Figure S8).
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We also tested the other three (co)polymers (A100, DA100 and A95B5) against one Gram-
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positive strain (SA29213) and one Gram-negative strain (EC8739) using the same MBEC™
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assay. These 3 (co)polymers show no clear reduction of the viability of the Gram-positive
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biofilm bacteria (SA291213) (Supporting Information Figure S9). (For A100 against
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SA29213, clear reduction was observed only at the highest polymer concentration tested (512
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µg/mL)). For the Gram-negative strain (EC8739), no clear biofilm reduction was found for these
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3 (co)polymers (A100, DA100 and A95B5), as well as DA95B5, at all concentrations tested
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(Supporting Information Figure S10).
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We also assessed the biomass on the MBEC™ pegs by crystal violet staining. Treatment with
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DA95B5 reduced, compared with untreated controls, biofilm biomass of Gram-positive strains
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(Supporting Information Figure S11) but not Gram-negative strains. The crystal violet results
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were consistent with the bacterial counting results (Supporting Information Figure S6 and S7).
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In summary, a) DA95B5 but not the other (co)polymers, reduced biofilms of the Gram-positive
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bacteria and b) the antibiofilm action of DA95B5 was not the result of bactericidal effects.
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We further demonstrated the superior in vivo antibiofilm efficacy of a hydrogel dressing
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soaked with DA95B5 solution using a murine excisional wound model (Figure 1c). An
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excisional wound was created and 103 CFU MRSA BAA40 bacteria was inoculated to the wound
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site. After 24-hour development, the bacteria in each wound greatly multiplied to 108~109 CFU,
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establishing severely infected wounds with biofilms. A porous hydrogel40 dressing soaked with
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DA95B5 solution (5 mg/kg) was applied three times to fully cover the wound site, with 4-hour
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interval between each treatment. PBS and vancomycin-soaked (2.5 mg/kg) hydrogels were used 8
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as infection and antibiotic controls respectively. Based on the results in Figure 1d, the DA95B5
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treatment effectively reduced the biofilm bacteria on the wound site (p512 8
MIC: µg/mL HC10 S. S. aureus VRE E. E. coli E. (µg/mL) RBC aureus (USA300) faecalis (ATCC coli (ATCC OG1RF 8739) K12 29213) 16 16 16 512 >512 8
8-16 8-16 16 >512 >512 8
>512 >512 >512 >512 >512 16
>512 >512 >512 >512 >512 16
128 128 32 512 64 32
256 256 64 512 64 32
>20000 >20000 >20000 >20000 >500
