A Membrane-Intercalating Conjugated Oligoelectrolyte with High-Efficiency Photodynamic Antimicrobial Activity

Article abstract of DOI:10.1002/anie.201701146

A membrane-intercalating conjugated oligoelectrolyte (COE), PTTP, was designed and synthesized with the goal of providing red-shifted absorption spectra relative to previously synthesized COE analogs. Specifically, electron-rich and electron-poor subunits were introduced in the conjugated backbone to modulate the band gap. PTTP exhibits maxima of absorption at 507 nm and of emission at 725 nm. PTTP can also efficiently function to generate singlet oxygen in situ (Φ_Δ≈20 %) and has appropriate topology and dimensions to interact with lipid membranes. The resulting rapid membrane insertion and sensitizing ability provide PTTP with a highly efficient antibacterial capability under a low light dose (0.6 J cm^?2) toward Gram-negative bacteria E. coli, making it a remarkably efficient optically mediated antimicrobial agent.

Full text of DOI:10.1002/anie.201701146

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201701146
German Edition: DOI: 10.1002/ange.201701146
Biocidal Agents
A Membrane-Intercalating Conjugated Oligoelectrolyte with
High-Efficiency Photodynamic Antimicrobial Activity
Bing Wang, Ming Wang, Alexander Mikhailovsky, Shu Wang, and Guillermo C. Bazan*
Abstract: A membrane-intercalating conjugated oligoelectro-
lyte (COE), PTTP, was designed and synthesized with the goal
of providing red-shifted absorption spectra relative to previ-
ously synthesized COE analogs. Specifically, electron-rich and
electron-poor subunits were introduced in the conjugated
backbone to modulate the band gap. PTTP exhibits maxima of
absorption at 507 nm and of emission at 725 nm. PTTP can
also efficiently function to generate singlet oxygen in situ (FD
It seemed appropriate to combine two functions (mem-
1
brane accumulation and O generation) into the design of
2
a
COE structure specific to the required properties
(Scheme 1). It is also worth considering that the light
penetration increases and light toxicity decreases with
ꢀ
20%) and has appropriate topology and dimensions to
interact with lipid membranes. The resulting rapid membrane
insertion and sensitizing ability provide PTTP with a highly
efficient antibacterial capability under a low light dose
À2
(
0.6 Jcm ) toward Gram-negative bacteria E. coli, making it
a remarkably efficient optically mediated antimicrobial agent.
M
embrane-intercalating conjugated oligoelectrolytes
MICOEs) typically consist of a linear p-conjugated core
and terminal side chains with functionalities capable of
(
[1]
ionizing in aqueous media. Certain MICOEs lead to
increases in the current generation of bioelectrochemical
[
2]
[3]
systems, serve as cell staining methods, and provide the
molecular basis for membrane permeation and antimicrobial
[
1a,4]
function.
All these properties are known to be dependent
on the specific chemical structure of the MICOE.
Certain COEs have been developed as antimicrobial
agents that function by virtue of their ability to photosensitize
1
oxygen into singlet oxygen ( O ), however their design did not
2
[5]
Scheme 1. Chemical structure of PTTP, cartoon representation of the
include specificity for accumulation in lipid membranes. The
1
insertion into a cell membrane, and proposed mechanism of PDAT
protection by the outer membrane and limitations of O2
1
through photogeneration of O .
2
diffusion make Gram-negative bacteria less sensitive to
photodynamic antimicrobial therapy (PDAT), relative to
[6]
[10]
Gram-positive bacteria. Photosensitizers that bind to bac-
teria by electrostatic interactions, or via agents that increase
membrane permeability, have been reported to increase the
longer wavelengths. Most MICOEs absorb blue-UV light,
[
1a,4b,d]
which limits their use in PDAT.
see Scheme 1. Key structural elements include a) electron-
rich (thieno[3,2-b]thiophene) and electron-poor
([1,2,5]thiadiazolo[3,4-c]pyridine) subunits that give rise to
We thus designed PTTP,
[
7]
killing efficiency of Gram-negative cells. The motivation
behind improving PDAT stems from reports of antibiotic
[8]
[5d,11]
resistant strains, and the effort has led to improved agents,
red shifted charge transfer excited states,
and molecular length that are commensurate for insertion
b) a topology
[
9]
many of which are based on classical chromophores.
[4d,11a]
into lipid membranes,
and c) six cationic pendant groups
[
*] Dr. B. Wang, Dr. M. Wang, Dr. A. Mikhailovsky, Prof. G. C. Bazan
Center for Polymers and Organic Solids, Department of Chemistry
and Biochemistry, University of California
Santa Barbara, CA 93106 (USA)
E-mail: bazan@chem.ucsb.edu
for solubility in aqueous media.
Details on the synthesis of PTTP are in the Supporting
Information. The absorption and emission spectra of PTTP
were studied in different solvents. For solubility reasons,
PTTP-Cl was used in toluene and chloroform. Relevant
spectra and data are shown in Figure 1 and Table 1. Absorp-
tion maxima (labs) are in the range of 505–535 nm, with
a progressive blue shift as the polarity of the medium
Prof. S. Wang
Beijing National Laboratory for Molecular Science, Key Laboratory of
Organic Solids, Institute of Chemistry, Chinese Academy of Sciences
Beijing 100190 (P.R. China)
increases. Fluorescence maxima (l ) are in the 660–725 nm
range; more polar media lead to a red shift, consistent with an
excited state with intramolecular charge transfer character-
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201701146.
em
Angew. Chem. Int. Ed. 2017, 56, 1 – 5
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

Angewandte
Communications
Chemie
Figure 1. a) Normalized absorption and b) fluorescence spectra of
PTTP-Cl in toluene and chloroform, and PTTP in methanol and water.
The excitation wavelength is 530 nm for fluorescence spectra.
Figure 2. a) The fluorescence intensity of SOSG at 525 nm in the
4À
presence of 2.5 mm PTTP, RB, H TPPS and 25 mm MB, respectively,
2
as a function of irradiation time using light with a fluence of
À2
5
mWcm . RB in PBS (pH 7.4) was used as reference. b) The
1
4À
emission of O generated by PTTP, RB and H TPPS in DPBS when
2
2
excited at 520 nm.
Table 1: Absorption maxima, molar extinction coefficients, emission
maxima and quantum yields of PTTP-Cl and PTTP at 10 mm.
[
a]
[a]
Toluene
CHCl3
CH OH
H O
2
PBS
3
E. coli K-12 was used as a Gram-negative bacteria model.
Cell association of PTTP on E. coli cells (OD600 = 1.0) in
l
e
[nm]
534
6.0
662
44
529
5.9
671
29
516
4.4
713
4
507
3.4
725
0.2
535
2.9
785
0.5
abs
b]
[
5
0 mm PBS buffer was examined by monitoring the absorp-
lem [nm]
F [%]
tion change of PTTP in the staining medium after treatment
[
c]
F
[16]
and removal of the cells. These affinity studies (Figure 3)
À1
À1
4
[
[
a] PTTP-Cl in non-polar organic solvents; [b] Lmol cm ꢀ10 .
c] Quantum efficiency (F ) values were measured relative to Oxazine
show incorporation of PTTP within 5 min at concentrations
below 10 mm. Interestingly, the rates of association are slower
in 150 mm PBS buffer (Figure S4). The fluorescence spectra
of PTTP in E. coli were then measured. Relative to spectra of
PTTP in PBS, blue-shifted fluorescence (lem = 740 nm) in the
presence of E. coli was observed (Figure S1b), which is
consistent with previous reports for MICOEs embedded in
F
1
70 standard in methanol.
[12]
istics.
Fluorescence quantum efficiencies (F ) were also
F
measured in different solvents. As shown in Table 1, FF
decreases with increasing solvent polarity, from 44% in
toluene to 0.2% in water.
PTTP was also examined in phosphate-buffered saline
solution (PBS, pH 7.4) in anticipation of the media relevant
for microbial environments (Figure S1a in the Supporting
Information). Both absorption (labs = 535 nm) and fluores-
cence (lem = 785 nm) spectra red shift in PBS vs. water
[1d]
a lipid environment.
The sensitizing ability of PTTP in
bacteria membrane was not significantly affected (Fig-
ure S2b).
(
Table 1), which suggests possible molecular aggregation. Our
current thinking is that the salt content in PBS decreases
electrostatic repulsion between PTTP units, and encourages
formation of aggregates in which PTTP···PTTP interactions
[
13]
cause the observed spectral shifts.
1
The O quantum efficiencies (F ) were determined using
Singlet Oxygen Sensor Green (SOSG)
2
D
[14]
and rose bengal
(
RB, F ꢀ 75%) as the standard. Methylene blue (MB, F
D
D
ꢀ
39%) and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphine
4
À
[15]
(
H TPPS , F ꢀ 60%), were included for verification.
2
D
Figure 3. PTTP associated with E. coli cells as a function of staining
concentration for a series of time in 50 mm PBS.
Figure 2a provides the emission intensity of SOSG at
4
À
5
25 nm vs. time for RB, MB, H TPPS and PTTP. From
2
4
À
Figure 2a the FD values of MB, H TPPS , and PTTP are
2
3
3%, 58% and 19%, respectively. We also examined PTTP in
the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine
lipid membranes. These conditions lead to a blue shift in lem
E. coli cells stained with PTTP were imaged by using
confocal laser scanning microscopy (CLSM), see Figure 4.
DAPI (4’,6-diamidino-2-phenylindole) was used to stain the
interior of the cells. One observes “halo” patterns around the
edges of the cells from the emission of PTTP. The DAPI
fluorescence was predominantly located within the cells.
PTTP therefore preferentially accumulates on the outer
(
Figure S1b), consistent with membrane insertion, and do not
impact sensitizing ability; see Figure S2a. Further determi-
1
nations of FD by measuring the O2 phosphorescence in
deuterated PBS (DPBS), see Figure 2b and Figure S3,
4
À
provide values for PTTP (17%) and H TPPS (61%).
2
1
[11a]
Thus, excitation of PTTP generates O2 in a variety of
portion of the cells.
environments with moderate efficiency, a necessary condition
for the proposed mechanism in Scheme 1.
The photodynamic antibacterial activity against E. coli
was assessed by irradiation (540 Æ 60 nm) with a total light
2
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 5
These are not the final page numbers!

Angewandte
Communications
Chemie
ure S11). It is worth noting
that the light dose needed to
kill E. coli cells with PTTP is
excellent when contrasted
against porphyrin systems
described
in
literature
Also for
a cationic
À2 [18]
(30–266 Jcm ).
comparison,
poly(p-phenylenevinylene)
and polythiophene/porphy-
rin complexes were reported
to exhibit ca. 70% anti-
bacterial capability, with
Figure 4. CLSM images of E. coli cells treated with 10 mm PTTP in 50 mm PBS. The false colors of PTTP and
DAPI (nucleus dye) are red and green, respectively. The merged color should be yellow. Scale bars are 5 mm.
light intensity and doses
À2
of
75 mWcm
and
À2
À2 [19]
dose of 9 Jcm . A traditional plate colony counting method
27 Jcm ,
while a cationic oligo(thiopheneethynylene)
À2 [20]
was used to determine the percentage of live cells. Figure 5a
shows the percentage of colony forming units (CFU) vs.
PTTP], with non-irradiated cells as control group. From
derivate exhibits high efficiency at > 150 Jcm . The high
efficiency of PTTP therefore suggests a promising potential
for improving PDAT performance through optimization of
MICOE structures.
In conclusion, we report the design of a new MICOE
structure with a red-shifted absorption profile, namely PTTP.
Of note is the conjugated backbone containing a sequence of
electron-poor/electron-rich/electron-poor structural units,
which leads to charge transfer characteristics and a desirable
tuning of optical characteristics. PTTP is an efficient sensi-
[
Figure 5a, one first notes no obvious toxicity without
illumination, at least within the concentrations tested in the
study. Biocompatibility was independently confirmed through
minimum inhibitory concentration tests (Figure S5). How-
ever, the percentage of live cells significantly decreased under
light exposure (Figure 5a and S6). Approximately 99.5% of
the cells were killed under irritation when [PTTP] ꢁ 2 mm.
tizer for the generation of singlet oxygen with F ꢀ 20%. Cell
D
association and CLSM studies indicate that PTTP inserts into
membranes similarly to previously reported MICOEs with
oligo(phenylenevinylene) backbones. Benefiting from the
membrane interactions and sensitizing ability, PTTP exhibits
highly efficient antibacterial capability under a very low light
À2
dose (1.2–0.6 Jcm ) to E. coli. The results described here
provide evidence for the promising potential of MICOEs as
sensitizers for antimicrobial photodynamic inactivation. Fur-
ther advances in molecular design are anticipated to yield
improvements with respect to concentration levels and light
Figure 5. a) Biocidal activity of PTTP towards E. coli in the dark and
under light irradiation as a function of concentration (5 min in 50 mm
[
4b,d]
dose intensity.
Achieving absorption beyond 650 nm
À2
PBS) by analyzing CFUs on an LB plate. The light dose was 9 Jcm
would allow activation deeper into tissue, while side chain
modifications open the opportunity to interact preferentially
À2
(
irradiation for 15 min at a fluence of 10 mWcm ). b) Biocidal activity
of PTTP as a function of light dose. E. coli were stained with 3 mm
[10d,19a]
with bacteria relative to mammalian cells.
PTTP for 5 min and then irradiated for different durations under light
À2
with a fluence of 10 mWcm
.
Acknowledgements
Photodynamic antibacterial activity was also tested in 150 mm
PBS, with 2 h as the staining time for PTTP association. After
the same irradiation process, more than 99.5% of the cells
were also killed when [PTTP] ꢁ 5 mm (Figure S7 and S8).
Scanning electron microscopy (SEM) was utilized to
obtain insight on cell morphology (Figure S9 and S10).
Controls show smooth outer surfaces for E. coli, E.coli
This work was supported by the Institute for Collaborative
Biotechnologies through grant W911NF-09-D-0001 from the
U.S. Army Research Office and made use of MRL Central
Facilities (NSF DMR 1121053).
treated with PTTP in the dark or after irradiation. For the Conflict of interest
group treated with PTTP and light exposure, the bacterial
wall is collapsed, thereby illustrating the impact of localized
generation of singlet oxygen.
The authors declare no conflict of interest.
[
17]
The PDAT effect was further examined by varying the
irradiation dose. As shown in Figure 5b, a dose of 1.2 Jcm
Keywords: biocidal activity · conjugated oligoelectrolyte ·
membrane intercalating · photodynamic antimicrobial therapy
À2
proved sufficient to kill more than 99.9% of E. coli, while
À2
0
.6 Jcm exhibited a 98% kill efficiency (see also Fig-
Angew. Chem. Int. Ed. 2017, 56, 1 – 5
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

Angewandte
Communications
Chemie
[
9] L. Jiang, C. R. R. Gan, J. Gao, X. J. Loh, Small 2016, 12, 3609 –
644.
3
[
10] a) J. G. Peak, M. J. Peak, Photochem. Photobiol. 1995, 61, 484 –
487; b) B. F. Godley, F. A. Shamsi, F. Q. Liang, S. G. Jarrett, S.
Davies, M. Boulton, J. Biol. Chem. 2005, 280, 21061 – 21066;
c) R. B. Setlow, E. Grist, K. Thompson, A. D. Woodhead, Proc.
Natl. Acad. Sci. USA 1993, 90, 6666 – 6670; d) K. Konig, J.
Microsc. 2000, 200, 83 – 104.
[
1] a) Y. Wang, E. Y. Chi, K. S. Schanze, D. G. Whitten, Soft Matter
012, 8, 8547 – 8558; b) A. W. Thomas, C. Catania, L. E. Garner,
2
G. C. Bazan, Chem. Commun. 2015, 51, 9294 – 9297; c) L. E.
Garner, J. Park, S. M. Dyar, A. Chworos, J. J. Sumner, G. C.
Bazan, J. Am. Chem. Soc. 2010, 132, 10042 – 10052.
[
[
2] H. J. Yan, C. Catania, G. C. Bazan, Adv. Mater. 2015, 27, 2958 –
[11] a) A. W. Thomas, Z. B. Henson, J. Du, C. A. Vandenberg, G. C.
Bazan, J. Am. Chem. Soc. 2014, 136, 3736 – 3739; b) P. M.
Beaujuge, C. M. Amb, J. R. Reynolds, Acc. Chem. Res. 2010,
43, 1396 – 1407; c) S. Cekli, R. W. Winkel, E. Alarousu, O. F.
Mohammed, K. S. Schanze, Chem. Sci. 2016, 7, 3621 – 3631.
[12] Y. Y. Wu, Y. Chen, G. Z. Gou, W. H. Mu, X. J. Lv, M. L. Du,
W. F. Fu, Org. Lett. 2012, 14, 5226 – 5229.
2973.
3] P. Gwozdzinska, R. Pawlowska, J. Milczarek, L. E. Garner,
A. W. Thomas, G. C. Bazan, A. Chworos, Chem. Commun. 2014,
50, 14859 – 14861.
[
4] a) Y. Wang, Y. L. Tang, Z. J. Zhou, E. Ji, G. P. Lopez, E. Y. Chi,
K. S. Schanze, D. G. Whitten, Langmuir 2010, 26, 12509 – 12514;
b) Y. Wang, K. S. Schanze, E. Y. Chi, D. G. Whitten, Langmuir
[
13] a) H. Maeda, S. Muroi, R. Kakehashi, J. Phys. Chem. B 1997,
101, 7378 – 7382; b) E. H. Hill, D. Sanchez, D. G. Evans, D. G.
Whitten, Langmuir 2013, 29, 15732 – 15737.
2013, 29, 10635 – 10647; c) J. Hinks, W. H. Poh, J. J. H. Chu,
J. S. C. Loo, G. C. Bazan, L. E. Hancock, S. Wuertz, Appl.
Environ. Microbiol. 2015, 81, 1949 – 1958; d) H. J. Yan, Z. D.
Rengert, A. W. Thomas, C. Rehermann, J. Hinks, G. C. Bazan,
Chem. Sci. 2016, 7, 5714 – 5722; e) C. Catania, C. M. Ajo-
Franklin, G. C. Bazan, RSC Adv. 2016, 6, 100300 – 100306.
5] a) H. X. Yuan, H. Chong, B. Wang, C. L. Zhu, L. B. Liu, Q. Yang,
F. T. Lv, S. Wang, J. Am. Chem. Soc. 2012, 134, 13184 – 13187;
b) Z. J. Zhou, T. S. Corbitt, A. Parthasarathy, Y. L. Tang, L. F.
Ista, K. S. Schanze, D. G. Whitten, J. Phys. Chem. Lett. 2010, 1,
[14] a) H. Y. Lin, Y. Shen, D. F. Chen, L. S. Lin, B. C. Wilson, B. H.
Li, S. S. Xie, J. Fluoresc. 2013, 23, 41 – 47; b) S. Clement, M.
Sobhan, W. Deng, E. Camilleri, E. M. Goldys, J. Photochem.
Photobiol. A 2017, 332, 66 – 71; c) L. Xiao, L. Gu, S. B. Howell,
M. J. Sailor, ACS Nano 2011, 5, 3651 – 3659.
[
[
15] F. Wilkinson, W. P. Helman, A. B. Ross, J. Phys. Chem. Ref. Data
1993, 22, 113 – 262.
[
16] C. Catania, A. W. Thomas, G. C. Bazan, Chem. Sci. 2016, 7,
2023 – 2029.
3
207 – 3212; c) Y. L. Tang, T. S. Corbitt, A. Parthasarathy, Z. J.
Zhou, K. S. Schanze, D. G. Whitten, Langmuir 2011, 27, 4956 –
962; d) A. Parthasarathy, S. Goswami, T. S. Corbitt, E. Ji, D.
[17] A. W. Girotti, J. Photochem. Photobiol. B 2001, 63, 103 – 113.
[
18] a) S. Ferro, G. Jori, S. Sortino, R. Stancanelli, P. Nikolov, G.
4
Dascier, D. G. Whitten, K. S. Schanze, ACS Appl. Mater.
Interfaces 2013, 5, 4516 – 4520.
Tognon, F. Ricchelli, A. Mazzaglia, Biomacromolecules 2009, 10,
2592 – 2600; b) S. Banfi, E. Caruso, L. Buccafurni, V. Battini, S.
[
[
6] a) M. Wainwright, J. Antimicrob. Chemother. 1998, 42, 13 – 28;
b) T. A. Dahl, W. R. Midden, P. E. Hartman, J. Bacteriol. 1989,
Zazzaron, P. Barbieri, V. Orlandi, J. Photochem. Photobiol. B
2006, 85, 28 – 38; c) E. Feese, H. Sadeghifar, H. S. Gracz, D. S.
Argyropoulos, R. A. Ghiladi, Biomacromolecules 2011, 12,
3528 – 3539.
171, 2188 – 2194.
7] a) G. Bertoloni, B. Salvato, M. Dallacqua, M. Vazzoler, G. Jori,
Photochem. Photobiol. 1984, 39, 811 – 816; b) F. F. Sperandio,
Y. Y. Huang, M. R. Hamblin, S. C. O. M. de Sousa, Pat. Anti-
infect. Drug Discov. 2013, 8, 108 – 120.
8] a) J. M. A. Blair, M. A. Webber, A. J. Baylay, D. O. Ogbolu,
L. J. V. Piddock, Nat. Rev. Microbiol. 2015, 13, 42 – 51; b) T.
Maisch, Photochem. Photobiol. Sci. 2015, 14, 1518 – 1526;
c) M. R. Hamblin, Curr. Opin. Microbiol. 2016, 33, 67 – 73;
d) WHO, Antimicrobial resistance: global report on surveil-
lance, 2014.
[
19] a) C. L. Zhu, Q. O. Yang, L. B. Liu, F. T. Lv, S. Y. Li, G. Q. Yang,
S. Wang, Adv. Mater. 2011, 23, 4805 – 4810; b) C. F. Xing, Q. L.
Xu, H. W. Tang, L. B. Liu, S. Wang, J. Am. Chem. Soc. 2009, 131,
13117 – 13124.
[
[20] Q. Zhao, J. T. Li, X. Q. Zhang, Z. P. Li, Y. L. Tang, ACS Appl.
Mater. Interfaces 2016, 8, 1019 – 1024.
Manuscript received: February 1, 2017
Final Article published: && &&, &&&&
4
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Biocidal Agents
Light on, bugs out: A membrane-interca-
lating conjugated oligoelectrolyte (COE),
PTTP, was designed to exhibit red-shifted
absorption spectra relative to previously
synthesized COEs. PTTP accumulates in
the membrane of E. coli and is efficient
toward singlet-oxygen photogeneration.
This combination of properties gives rise
to excellent optically mediated antibacte-
rial capability under low light dosage.
B. Wang, M. Wang, A. Mikhailovsky,
S. Wang, G. C. Bazan*
&&&& — &&&&
A Membrane-Intercalating Conjugated
Oligoelectrolyte with High-Efficiency
Photodynamic Antimicrobial Activity
Angew. Chem. Int. Ed. 2017, 56, 1 – 5
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!