A Multifunctional Subphthalocyanine Nanosphere for Targeting, Labeling, and Killing of Antibiotic-Resistant Bacteria

Article abstract of DOI:10.1002/anie.201507140

Developing a material that can combat antibiotic-resistant bacteria, a major global health threat, is an urgent requirement. To tackle this challenge, we synthesized a multifunctional subphthalocyanine (SubPc) polymer nanosphere that has the ability to target, label, and photoinactivate antibiotic-resistant bacteria in a single treatment with more than 99 % efficiency, even with a dose as low as 4.2 J cm^-2 and a loading concentration of 10 nM. The positively charged nanosphere shell composed of covalently linked SubPc units can increase the local concentration of photosensitizers at therapeutic sites. The nanosphere shows superior performance compared to corresponding monomers presumably because of their enhanced water dispersibility, higher efficiency of singlet-oxygen generation, and phototoxicity. In addition, this material is useful in fluorescence labeling of living cells and shows promise in photoacoustic imaging of bacteria in vivo.

Full text of DOI:10.1002/anie.201507140

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Angewandte
Communications
International Edition: DOI: 10.1002/anie.201507140
German Edition: DOI: 10.1002/ange.201507140
Nanomaterials
A Multifunctional Subphthalocyanine Nanosphere for Targeting,
Labeling, and Killing of Antibiotic-Resistant Bacteria
Indranil Roy, Dinesh Shetty, Raghunandan Hota, Kangkyun Baek, Jeesu Kim, Chulhong Kim,
Sandro Kappert, and Kimoon Kim*
Dedicated to Professor Nagao Kobayashi
Abstract: Developing a material that can combat antibiotic-
resistant bacteria, a major global health threat, is an urgent
requirement. To tackle this challenge, we synthesized a multi-
functional subphthalocyanine (SubPc) polymer nanosphere
that has the ability to target, label, and photoinactivate
antibiotic-resistant bacteria in a single treatment with more
than 99% efficiency, even with a dose as low as 4.2 JcmÀ2 and
a loading concentration of 10 nm. The positively charged
nanosphere shell composed of covalently linked SubPc units
can increase the local concentration of photosensitizers at
therapeutic sites. The nanosphere shows superior performance
compared to corresponding monomers presumably because of
their enhanced water dispersibility, higher efficiency of singlet-
oxygen generation, and phototoxicity. In addition, this material
is useful in fluorescence labeling of living cells and shows
promise in photoacoustic imaging of bacteria in vivo.
(ROS) and has been proposed as an alternative to controlling
bacterial infections.[3] In most cases, although Gram-positive
bacteria are susceptible to the photosensitizing action of
a variety of sensitizers, Gram-negative bacteria exhibit
a remarkable resistance to negatively charged or neutral
agents.[4] However, studies shown that cationic photosensi-
tizers can cause direct photoinactivation of Gram-negative
bacteria even in the absence of additives.[5] Another promis-
ing approach to combating such bacteria is based on nano-
materials, in which the higher number of functional sites
compared to any given small molecule enhances the inter-
action with a given microbe.[6] In spite of progress in this field,
however, existing materials have several shortcomings, such
as poor water dispersibility and/or nonspecific accumulation
of photosensitizers.[7,8] Selective targeting of photosensitizers
in microorganisms may solve the problem of nonspecific
accumulation and may also reduce the dosage required for
PDT. Considering all these aspects, developing a material that
possesses the above-mentioned critical criteria would be
a great addition to the ongoing fight against the major medical
problem of antiobiotic-resistant bacteria.
Subphthalocyanines (SubPcs) have interesting photophys-
ical features, such as intense fluorescence and photosensitiz-
ing properties, stemming from their cone-shaped structure
and 14-p-electron aromatic conjugated system.[9] In partic-
ular, SubPcs have a longer triplet excited-state lifetime
compared to other well-known classes of photosensitizers,
including porphyrins (Ps) and phthalocyanines (Pcs), which
results in a higher quantum yield for the generation of singlet
oxygen (0.67 for SubPcs, 0.44 for Ps, and 0.52 for Pcs).[9,10,11b]
Additionally, SubPcs show more intense fluorescence signals
compared to Ps or Pcs, which may be exploited in efficient
fluorescent labeling of living cells. As a result of these merits
along with their high phototoxicity and intense light absorp-
tion in the therapeutic window (l = 600–900 nm), SubPcs
have been explored as photosensitizers in PDT.[9] However,
these molecules have some serious limitations including
limited photostability as well as poor dispersibility in water
or phosphate-buffered saline (PBS).[8] To overcome these
limitations, we decided to develop well-defined, water-
dispersible SubPc-based nanomaterials that can target,
label, and photoinactivate antibiotic-resistant bacterial cells.
Our strategy involves: 1) synthesis of a hollow nanosphere
composed of covalently linked SubPc molecules utilizing the
template-free, covalent self-assembly method recently devel-
oped by us,[11] which can increase the local concentration of
R
esistance of bacteria to multiple antibiotics has increased
dramatically over the past few years and is currently
recognized as a major medical challenge in most healthcare
settings.[1] The Center for Disease Control and Prevention has
recently issued an assessment that we are very close to
entering the “post-antibiotic era”,[2] which highlights the need
to find innovative and creative solutions to inhibit bacterial
growth. Photodynamic therapy (PDT) combines three intrins-
ically nontoxic components, namely a photosensitizer, light,
and oxygen, to generate cytotoxic reactive oxygen species
[*] I. Roy, Dr. D. Shetty, Dr. K. Baek, S. Kappert, Prof. Dr. K. Kim
Center for Self-assembly and Complexity (CSC)
Institute for Basic Science (IBS)
Pohang, 790-784 (Republic of Korea)
E-mail: kkim@postech.ac.kr
Homepage: http://csc.ibs.re.kr/
I. Roy, Dr. R. Hota, Prof. Dr. K. Kim
Department of Chemistry, Pohang University of Science and
Technology, Pohang, 790-784 (Republic of Korea)
Prof. Dr. K. Kim
Division of Advanced Materials Science
Pohang University of Science and Technology
Pohang, 790-784 (Republic of Korea)
J. Kim, Prof. Dr. C. Kim
Department of Creative IT Engineering and Electrical Engineering
Pohang University of Science and Technology
Pohang, 790-784 (Republic of Korea)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201507140.
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photosensitizers at therapeutic sites; and 2) introduction of
positive charges on the surface of the nanosphere, which
enhances their water dispersibility as well as their ability to
target negatively-charged bacterial membranes (Figure 1).
For in vitro studies, water-dispersible nanosphere 3 was
synthesized by treating 2 with methyl iodide followed by
dialysis, which resulted in partial alkylation of the thioether
units of 2 to convert them into sulfonium groups (Figure 1).
There was no significant change in the hydrodynamic size
(average diameter 150 Æ 30 nm) of 2 even after introducing
positive charges onto the surface of the nanosphere. How-
ever, the zeta potential of 3 increased to 23 Æ 4 mV from 1 Æ
3 mV of 2 (Figure S7,S8). This observation indicates that the
resulting sulfonium groups are mainly distributed over the
surface of 3. Scanning transmission electron microscopy
(STEM) images of 3 showed a very thin shell with an average
thickness of 1.0 Æ 0.2 nm (Figure 2b,c), indicating that the
Figure 1. Direct synthesis of SubPc nanosphere 2 from SubPc mono-
mer 1 and its conversion into water-dispersible SubPc nanosphere 3.
The alkylated monomer 1’ was synthesized for control experiments.
DMSO=dimethyl sulfoxide.
Figure 2. Electron microscopy images of SubPc nanospheres 3.
a) SEM image. Inset in (a): low-resolution TEM image after uranyl
acetate staining. b),c) STEM images. d) Absorption and emission
spectra of 1’ and 3 in DMSO. e) In vitro photoacoustic properties:
spectroscopic photoacoustic signal amplitude (PASA) of 1’ and 3.
Herein, we report a stable, water-dispersible, hollow SubPc
nanosphere, which is capable of targeting and killing anti-
biotic-resistant bacteria with exceptional efficiency even at
a low dosage upon visible light irradiation. In addition, this
nanomaterial is also useful for fluorescent labeling of living
cells and shows great promise in photoacoustic imaging of
bacteria in vivo.
shell is almost one monomer thick (height of the SubPc core =
0.8 nm). The spherical shape of 3 along with the cone-shaped
SubPc units effectively prevents aggregation; thus it remains
well dispersed in water even after several months (Figure S9).
A molecular-mechanics modeling study suggests that the shell
of a SubPc nanosphere with a diameter of 150 nm is composed
of approximately 18000 SubPc units (Figure S10). Addition-
ally, alkylated SubPc monomer (1’) was also synthesized for
control experiments (Figure 1).
The absorption and emission spectra of 1’ and 3 are shown
in Figure 2d. The absorption spectra of both 1’ and 3 display
typical Q bands between l = 500 and 630 nm with an absorp-
tion maximum at lmax = 600 nm, characteristic of SubPc. The
Q band of nanosphere 3 is somewhat broader compared to
monomer 1’ with a lower molar extinction coefficient but
essentially the same lmax value, suggesting that photophysi-
cally the SubPc units in 3 behave almost like a SubPc
monomer. The quantum yields of singlet oxygen generation
(SOG) for 3 and 1’ in DMSO are similar (0.7 and 0.68,
respectively; measured by using a reported procedure),[12]
suggesting that most of the SubPc units of 3 behave as
The SubPc nanosphere 2 was synthesized by an olefin
cross-metathesis reaction of monomer 1 without using any
templates (Figure 1). In a typical experiment, reaction of
1 (synthesized by means of a seven-step process; see
Scheme S1 in the Supporting Information) in DMSO in the
presence of 5 mol% of Grubbs catalyst (second generation)
at room temperature, followed by dialysis, produced polymer
nanosphere 2 (71% yield based on 1). A combination of
scanning electron microscopy (SEM), transmission electron
microscopy (TEM), and light-scattering studies confirmed the
formation of 2 with an average diameter of 150 Æ 30 nm
(Figure S4). Furthermore, the FTIR, UV/Vis, and fluores-
cence spectra of 2 showed characteristic signals of SubPc,
confirming that the SubPc core of 1 remain intact in the
nanosphere network (Figure S5,S6). We note that the sponta-
neous formation of a hollow nanosphere from a cone-shaped
monomer was a pleasant surprise as covalent self-assembly
had previously been achieved only with monomers containing
a rigid, flat core.[11]
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monomers. However, 3 shows an approximately nine times
higher SOG efficiency than 1’ in PBS buffer (0.61 for 3 and
0.07 for 1’), indicating a much lower propensity for 3 to
aggregate in aqueous media.
Several antibiotic-resistant bacterial strains were
employed as model biological targets in our studies. Nano-
sphere 3 with a cationic surface was used to efficiently label
E. coli, as seen in the microscopy images (Figure 3b). Live-
cell imaging was conducted using a fluorescence microscope,
efficiency (ca. 200 times) compared to 1’ for all of the tested
bacterial cells at a loading concentration for 3 or 1’ of 10–
20 nm and a radiation dose of 4.2 JcmÀ2. Furthermore, no
significant cell death was detected either upon treatment of
the cells with 3 without laser irradiation (dark toxicity;
Figure 4b, Ctrl 1), or in the absence of 3 but with laser
irradiation (Ctrl 2). These results suggest that 3 can efficiently
photoinactivate bacterial cells (Figure S13–S15). Remarkably,
the dosage used in our studies for efficient photoinactivation
is significantly lower (4.2 JcmÀ2; 60 s)
compared to other reported nanoma-
terials (27 JcmÀ2; 2 h),[6c] which may
have significant clinical implications.
We believe that the much higher photo-
toxicity of 3 compared to 1’ or other
nanomaterials is likely due to more
efficient interactions with bacterial
membranes and a much higher local
concentration of 1O2 generated by
Figure 3. a) Bright-field and b) fluorescence microscopy images of E. coli DH5a bacterial cells
treated with 3. c) STEM image (with enlarged portion of the image inset) of E. coli DH5a treated
with 3 showing the interaction between 3 and the surfaces of the bacterial plasma membranes.
using 3.
Interestingly, in addition to the
above-described fluorescence signals,
strong photoacoustic signals were
detected for both 3 and 1’ at l =
with the laser employed for excitation directly into the
Q band of 3. No additional dye molecules were required to
achieve labeling of the E. coli cells, confirming that 3 could be
used as a fluorescent labeling agent for bacteria. The STEM
images of E. coli bacteria treated with 3 confirm the adhesion
of the nanospheres to the surface of bacterial cells (Fig-
ure 3c). Moreover, complete rupture of the bacterial mem-
brane was detected after laser irradiation on the nanomate-
rial-incubated samples (Figure S11). These results suggest
that the positively charged surface of 3 promotes its adhesion
to the negatively charged bacterial membranes. The subse-
quent efficient local generation of singlet oxygen destroys the
bacterial membranes leading to bacterial cell death.
680 nm in PBS (Figure 2e). Moreover, the stronger photo-
acoustic signals generated from 3 than those from 1’ indicated
that the heat conversion efficiency of the SubPc nanosphere is
higher than that of the SubPc monomer. To explore these
To assess the photobiological activity of 1’ and 3 in more
detail, an in vitro study was carried out with different Gram-
negative (E. coli DH5a and E. coli K-12), Gram-positive (S.
aureus (SA)), and antibiotic-resistant bacterial cells (ampi-
cillin-resistant E. coli DH5a (E. coli DH5aAmp) and methicil-
lin-resistant S. aureus (MRSA)). The different bacterial
strains were incubated separately with materials 1’ or 3
dispersed in PBS buffer for 30 minutes and the samples were
irradiated at lex = 600 nm for a time range between 20 and
120 s (irradiance of 1.4 JcmÀ2 to 8.4 JcmÀ2). The inactivation
of bacterial cells was evaluated by counting the number of
colony forming units (CFU) on the Luria broth agar plate
(Figure S12). The results of photodynamic inactivation of
E. coli DH5a and the ampicillin-resistant E. coli DH5a after
laser irradiation for 60 s are shown in Figure 4. Nanosphere 3
can photoinactivate antibiotic-resistant E. coli in a single
treatment with more than 99% efficiency even with a dose as
low as 4.2 JcmÀ2 and a loading concentration of 10 nm
(Figure S13). The minimal bactericidal concentration
(MBC) values for 3 and 1’, which provide valuable informa-
tion on the potential action of antibacterial agents in vitro, are
recorded in Table 1. Interestingly, 3 shows much higher
Figure 4. a) Representation of bacterial photoinactivation in the pres-
ence of 3. b) Histogram showing the photodynamic inactivation of
E. coli DH5a and the ampicillin-resistant strain E. coli DH5aAmp. Initial:
original bacteria count. Ctrl 1: dark toxicity experiments; the bacterial
suspension was incubated in the dark with 1’ or 3 dispersed in PBS
overnight. Ctrl 2: the bacterial suspension was irradiated with a laser
in the absence of photosensitizer. Irr20s, Irr40s, and Irr60s: the
bacterial suspension was incubated with 1’ or 3 dispersed in PBS for
30 min and was then irradiated with a laser for 20 s, 40 s, or 60 s,
respectively. (An aliquot of 50 mL of a 10 nm solution of 1’ or 3 in PBS
was used to inactivate a 150 mL bacterial suspension (108 CFUmLÀ1)).
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Table 1: Minimal bactericidal concentration (MBC) values of 3 and 1’.[a]
Dr. P. Aich and J. Park for useful discussions related to in vitro
experiments.
E. coli K12
E. coli DH5a
E. coli DH5aAmp
SA
MRSA
3
1’
10 nm
2 mm
10 nm
2 mm
10 nm
2 mm
20 nm
4 mm
20 nm
4 mm
Keywords: antibiotic resistance · nanostructures ·
photodynamic therapy · self-assembly · subphthalocyanines
[a] Radiation dose used 4.2 JcmÀ2 (60 s).
How to cite: Angew. Chem. Int. Ed. 2015, 54, 15152–15155
Angew. Chem. 2015, 127, 15367–15370
properties for bacterial imaging we carried out a preliminary
in vivo test using 3. An aqueous solution of 3 (0.4 mm, 50 mL)
was intradermally injected into the bacteria-infected thigh of
a rat and photoacoustic images were acquired before and
after (60 min) the injection of 3 at l = 680 nm. After the
injection, distributions of 3 in the bacteria-infected region and
bleeding regions caused by the needle insertion were clearly
delineated. Most importantly, photoacoustic signals were
much stronger in the bacteria-infected region (Figure S18).
Similar results were obtained from fluorescence imaging
experiments. The fluorescence signal intensity at the infected
area after the injection of 3 is significantly higher than that at
a control area (Figure S19). Even though further detailed
studies are required to confirm the target specificity of 3,
these results suggest another exciting application of 3, namely
in vivo bacterial imaging.[13]
In summary, we have developed a highly stable, water-
dispersible, multifunctional SubPc-based nanomaterial that
can target, label, and inactivate antibiotic-resistant bacterial
cells in a single treatment with more than 99% efficiency. This
SubPc hollow nanosphere can be readily synthesized by
template-free, covalent self-assembly followed by simple
postsynthetic modification. The superior performance of the
nanospheres compared to corresponding monomers in the
photoinactivation of antibiotic-resistant bacteria is due pri-
marily to its enhanced water dispersibility, higher SOG
efficiency, and phototoxicity. The nanosphere is metal free
(and thus may have lower toxicity) and can address several
other key shortcomings often associated with existing photo-
active materials for PDT, such as poor water dispersibility,
high dosage requirements, and skin sensitivity.[8,14] Apart from
the photodynamic therapeutic benefits, the multifunctional
nature of this material may be useful in bacterial imaging
in vivo. Incorporation of a covalently linked targeting ligand
at the axial position of SubPc units may enhance the chances
of target-specific imaging of bacteria. In particular, the
photoacoustic properties of our material may render it
useful in photoacoustic tomography (PAT) imaging.[15]
These results pave the way towards new possibilities for the
photodynamic treatment of infectious diseases and the design
of next-generation photosensitizers.
[1] a) U. Theuretzbacher, J. Glob. Antimicrob. Resist. 2013, 1, 63 –
69; b) World Health Organization, 2014, available at http://www.
who.int/mediacentre/news/releases/2014/amr-report/en/.
[2] T. Frieden, Center for Disease Control, 2013, available at http://
www.cdc.gov/drugresistance/threat-report-2013/.
[3] E. Alves, M. A. Faustino, M. G. Neves, A. Cunha, J. Tome, A.
Almeida, Future Med. Chem. 2015, 7, 1221 – 1224.
[4] M. R. Hamblin, T. Hasan, Photochem. Photobiol. Sci. 2004, 3,
436 – 450.
[5] a) D. Lazzeri, M. Rovera, L. Pascual, E. N. Durantini, Photo-
chem. Photobiol. 2004, 80, 286 – 293; b) S. Banfi, E. Caruso, L.
Buccafurni, V. Battini, S. Zazzaron, P. Barbieri, V. Orlandi, J.
Photochem. Photobiol. B 2006, 85, 28 – 38.
[6] a) E. Navarro, F. Piccapietra, B. Wagner, F. Marconi, R. Kaegi,
N. Odzak, L. Sigg, R. Behra, Environ. Sci. Technol. 2008, 42,
8959 – 8964; b) C. N. Lok, C. M. Ho, R. Chen, Q. Y. He, W. Y.
Yu, H. Sun, P. K. Tam, J. F. Chiu, C. M. Che, J. Proteome Res.
2006, 5, 916 – 924; c) C. A. Strassert, M. Otter, R. Q. Albuquer-
que, A. Hone, Y. Vida, B. Maier, L. De Cola, Angew. Chem. Int.
Ed. 2009, 48, 7928 – 7931; Angew. Chem. 2009, 121, 8070 – 8073;
d) K. Liu, Y. Liu, Y. Yao, H. Yuan, S. Wang, Z. Wang, X. Zhang,
Angew. Chem. Int. Ed. 2013, 52, 8285 – 8289; Angew. Chem.
2013, 125, 8443 – 8447.
[7] L. B. Josefsen, R. W. Boyle, Br. J. Pharmacol. 2008, 154, 1 – 3.
[8] M. B. Spesia, E. N. Durantini, Dye. Pigment. 2008, 77, 229 – 237.
[9] C. G. Claessens, D. Gonzµlez-Rodríguez, T. Torres, Chem. Rev.
2002, 102, 835 – 853.
[10] A. Blum, L. I. Grossweiner, Photochem. Photobiol. 1985, 41, 27 –
32.
[11] a) K. Baek, I. Hwang, I. Roy, D. Shetty, K. Kim, Acc. Chem. Res.
2015, 48, 2221 – 2229; b) R. Hota, K. Baek, G. Yun, Y. Kim, H.
Jung, K. M. Park, E. Yoon, T. Joo, J. Kang, C. G. Park, S. M. Bae,
W. S. Ahn, K. Kim, Chem. Sci. 2013, 4, 339 – 344; c) D. Kim, E.
Kim, J. Kim, K. M. Park, K. Baek, M. Jung, Y. H. Ko, W. Sung,
H. S. Kim, J. H. Suh, C. G. Park, O. S. Na, D. K. Lee, K. E. Lee,
S. S. Han, K. Kim, Angew. Chem. Int. Ed. 2007, 46, 3471 – 3474;
Angew. Chem. 2007, 119, 3541 – 3544.
[12] W. Spiller, H. Kliesch, D. Wohrle, S. Hackbarth, B. Roder, G.
Schnurpfeil, J. Porphyrins Phthalocyanines 1998, 2, 145 – 158.
[13] a) X. Ning, S. Lee, Z. Wang, D. Kim, B. Stubblefield, E. Gilbert,
N. Murthy, Nat. Mater. 2011, 10, 602 – 607; b) M. van Oosten, T.
Schäfer, J. A. C. Gazendam, K. Ohlsen, E. Tsompanidou, M. C.
de Goffau, H. J. M. Harmsen, L. M. A. Crane, E. Lim, K. P.
Francis, L. Cheung, M. Olive, V. Ntziachristos, J. M. van Dijl,
G. M. van Dam, Nat. Commun. 2013, 4, 2584 – 2591.
[14] a) T. S. Mang, T. J. Dougherty, W. R. Potter, D. G. Boyle, S.
Somer, J. Moan, Photochem. Photobiol. 1987, 45, 501 – 506; b) B.
Petersen, S. R. Wiegell, H. C. Wulf, Br. J. Dermatol. 2014, 171,
175 – 178.
Acknowledgements
[15] C. Kim, C. Favazza, L. V. Wang, Chem. Rev. 2010, 110, 2756 –
2782.
This work was supported by the Institute for Basic Science
(IBS) (IBS-R007-D1). We thank Prof. Dr. W. J. Kim for
permitting us using the culture room facility. We also thank
Received: July 31, 2015
Published online: October 23, 2015
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