Therapeutic nanoplatforms with bacteria-specific activation for directional transport of antibiotics

Article abstract of DOI:10.1039/c8cc06398b

An entirely new strategy is explored for directional transport delivery of antibiotics to bacteria utilizing a bacteria-activated nanoplatform. The nanoplatform can effectively prevent the premature leakage of the therapeutic payload, but release was triggered when the nanoplatforms adhere to bacteria, promising potential applications for the delivery of a wide-range of antimicrobials.

Full text of DOI:10.1039/c8cc06398b

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Therapeutic nanoplatforms with bacteria-specific
activation for directional transport of antibiotics†
Cite this: Chem. Commun., 2018,
54, 12754
Yunjian Yu, Tianqi Zhang, Xijuan Dai, Xiaomei Dai, Xiaosong Wei, Xinge Zhang
and Chaoxing Li
*
Received 6th August 2018,
Accepted 11th October 2018
DOI: 10.1039/c8cc06398b
rsc.li/chemcomm
An entirely new strategy is explored for directional transport delivery pharmacokinetic properties of antibiotics with preferable anti-
of antibiotics to bacteria utilizing a bacteria-activated nanoplatform. bacterial behaviour and little side effects to normal tissues.
The nanoplatform can effectively prevent the premature leakage Compared with traditional therapy, the above anti-virulence
of the therapeutic payload, but release was triggered when the nano- approaches exhibit less selective pressure, which overwhelms
platforms adhere to bacteria, promising potential applications for the the development of bacterial resistance.10 Nevertheless, a single
delivery of a wide-range of antimicrobials.
responsive channel can only achieve the on-demand release of
antibiotics irrespective of precise administration due to the
Bacterial infections pose a serious threat to human health with lack of direct bacteria targeting.11 Few investigations have
significant mortality and morbidity worldwide in spite of the involved the construction of biologically responsive drug vectors
widespread use of antibiotics.1 Furthermore, the emergence of with uncomplicated but innovative material designs that respond
drug-resistant bacterial strains weakens the therapeutic effect to bacteria directly instead of the microenvironment of the bacterial
due to the abuse of antibiotics, which has become a public infection sites to achieve drug targeting delivery.
issue of global concern.2 Bacteria can also be protected by
Herein, we explored an entirely new concept for directional
cells escaping from the attack of the immune system, thus transport of antibiotics to bacterial infection sites utilizing
making the action of antibiotics in vain, leading to infection bacteria-activated copolymeric nanoparticles. Different from the
recurrence.3 Moreover, the inherent defects of antibiotics such common bioresponsive drug delivery systems, we took advantage
as side effects, low bioavailability and little potency to biofilm- of the dynamic boronate esters reversibly formed by PBA and
associated infections limit their application.4 Therefore, it is of diols on the bacteria surface to respond to bacteria of interest
paramount urgency to develop new antimicrobial strategies directly instead of the microenvironment of the bacterial infec-
which can reduce side effects and the possibility of drug tion sites, triggering the fast release of therapeutic agents and
resistance emerging with a high local drug concentration to subsequently killing the bacteria. The synthesized nanoparticles
displace the convenient antibiotic treatment.
were found to show superior performance of bacteria binding
The selective delivery of therapeutic payloads to the lesion with little impact on normal cells, and we systematically optimized
sites has attracted widespread attention in the treatment of the copolymeric composition and construction to single out the
tumor or bacteria-related diseases.5 The bioresponsive poly- one with the best behaviour of bacteria binding. We further
meric nanocarriers can be functionalized by special moieties evaluated the bacteria-activated therapeutic nanoparticle system
that respond to the unique microenvironment of the bacterial for specific ablation of bacteria.
infection sites and selectively release the loaded cargo via the
For the fabrication of copolymers, 3-acrylamidophenylboronic
triggers of bacteria-associated secondary biological cues including acid (AAPBA) and 2-acrylamido glucopyranose (AGA) were utilized
local acidification,6 toxins,7 enzymes,8 reducibility,9 and so on. as monomers to prepare p(AAPBA-r-AGA) or p(AAPBA-b-AGA) via
These innovative bioresponsive nanocarriers for ‘‘on demand’’ RAFT polymerization using boron-dipyrromethene (BODIPY)-
drug delivery have been demonstrated to significantly improve conjugated dithioester as a RAFT agent (see Scheme S2, Fig. S1,
S2 and Table S1, ESI†). It is well-known that phenylboronic acid
(PBA) exists in equilibrium between the neutral form and a
hydroxyboronate anion after complexation with a hydroxide
ion in the aqueous solution, and the latter is easily combined
Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute
of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071,
China. E-mail: zhangxinge@nankai.edu.cn
with saccharides containing a diol group to form a stable
† Electronic supplementary information (ESI) available: Detailed experimental
methods and additional data. See DOI: 10.1039/c8cc06398b
boronate ester12 (Scheme S1, ESI†). Thus, the PBA groups in
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PBA-based copolymers to improve the cell compatibility.
As shown in Fig. S3A and B (ESI†), it was observed that the
relative cell proliferation rates were more than 80% after
incubation with various concentrations of RCN and BCN for
48 h. A hemolysis assay was further employed to evaluate the
adverse impact of nanoparticles on human red blood cells. As
shown in Fig. S3C and D (ESI†), no obvious hemolysis was
found for both RCN and BCN, even if the concentration was up
to 500 mg mLÀ1. The results of cell viability and hemolysis
assays suggested that the introduction of AGA effectively
reduced the cytotoxicity of the copolymers, implying that our
nanovehicles have a potential application in in vivo drug
delivery.
PBA can reversibly form stable boronate esters with diols of
carbohydrates. And when there is a more stable diol bound to
PBA, exchange can be accomplished rapidly to form a more
stable complex.13 Taking into account the presence of sugar-
based structures on the surface of bacteria,14 it is speculated
that RCN and BCN have the ability to bind to a variety of
bacteria surfaces to achieve the performance of clustering
bacteria. In addition, the globular form allows an easier access
to a binding site by virtue of the spherical micelles having
multiple AAPBA binding sites, resulting in the enhancement of
adhesion properties via the ‘‘polyvalent effect’’. So we evaluated
the adhesion effectiveness of copolymeric nanoparticles toward
Staphylococcus aureus (S. aureus), Bacillus amyloliquefaciens
(B. amyloliquefaciens), and Escherichia coli (E. coli), Pseudomonas
aeruginosa (P. aeruginosa) by co-culturing bacteria with RCN
or BCN with various monomer compositions, and the bacterial
assemblages were observed via confocal laser scanning micro-
scopy (CLSM).
Scheme 1 Self-assembly of RCN-T and BCN-T as well as the bacteria-
triggered drug release.
the copolymer are expected to interact with 4-OH or 5-CH2OH of
glucopyranose moieties, leading to the formation of a stable
intramolecular or intermolecular crosslinked structure, which
promotes the copolymer self-assembly (Scheme 1).
The morphology and size of both random copolymeric nano-
particles (RCN) and block copolymeric nanoparticles (BCN)
observed using a transmission electron microscope (TEM)
presented a spherical shape with good dispersion and distribution
of B160 nm in diameter (Fig. 1A and B), which was in accordance
with the results determined by dynamic light scattering (DLS) (the
inset of Fig. 1A and B and Table S2, ESI†). The UV maximum
absorption wavelength of RCN and BCN dispersed in PBS located
at around 260 nm (Fig. 1C). The inset of Fig. 1C exhibited the
macroscopic morphology of nanoparticles which had been stood
at 4 1C for 5 days, demonstrating their superior stability.
As BODIPY molecules with high fluorescence quantum yields
and light stability were conjugated to the copolymers, both RCN
and BCN showed strong green fluorescence in PBS buffer (pH 7.4)
(the inset of Fig. 1D). The maximum excitation wavelength in PBS
located at 503 nm for both nanoparticles, while their fluorescence
spectra exhibited strong emission around 513 nm. The nano-
particles with excellent fluorescent features display great potential
applications in the field of bacterial cell imaging.
The CLSM results in Fig. 2 and Fig. S4 (ESI†) showed that the
size of bacterial aggregates increased as the content of PBA
moieties in the copolymer augmented, demonstrating the
sequence of adhesion capacity of copolymeric nanoparticles
with different monomer compositions to a certain bacterium
Considering that boric acid and its derivatives are toxic
to multiple cell lines,13 we here introduced AGA groups into
to be the following: pAAPBA60
4 p(AAPBA45-AGA15) 4
p(AAPBA30-AGA30) 4 p(AAPBA15-AGA45) 4 pAGA60, which was
Fig. 1 TEM photographs of (A) BCN and (B) RCN; (C) UV-visible absorp-
tion spectra of RCN and BCN, the insets are the visible images of the
nanoparticles in PBS solution (pH 7.4); (D) fluorescence excitation and
emission spectra of BODIPY-based RCN and BCN.
Fig. 2 Adhesion activity to bacteria observed via CLSM when treated with
pAGA60, RCN3 and BCN3 for 2 h at 37 1C, respectively.
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in agreement with our above-mentioned deduction that the adhe- and block copolymeric nanoparticles (BCN3-T) both increased by 20
sion of copolymeric nanoparticles to bacteria was derived from the to 35 nm with a narrow size distribution (Fig. S7 and Table S4,
interaction of PBA with bacteria. Meanwhile, slightly weak green ESI†). The loading capacity (LC) of RCN3 and BCN3 was up to
fluorescence of materials in dispersed or thinly clustered states 20.0 Æ 1.2% and 18.7 Æ 1.5%, and the drug encapsulation
was found in the micrographs of bacteria co-cultured with homo- efficiency (EE) was 83.3 Æ 0.9% and 76.7 Æ 1.3%, respectively.
polymer pAGA60, demonstrating the specific multivalent interac- The in vitro release behaviour of triclosan from RCN3-T and
tions of polymeric carbohydrates with bacterial cell walls, especially BCN3-T was investigated in PBS buffer at pH 7.4 (a simulated
E. coli.15 Furthermore, the adhesion capacity of copolymeric nano- physiological environment) and pH 6.5 (a simulated weak acidic
particles toward S. aureus and B. amyloliquefaciens was stronger environment of the bacterial infection site) either with or without
than that toward E. coli and P. aeruginosa (Fig. S5, ESI†). This might the presence of bacteria. As shown in Fig. S8 (ESI†), for both
be attributed to the fact that different bacterial cell walls contain RCN3-T and BCN3-T, the cumulative release of triclosan in the
different carbohydrates with various affinity constants to PBA absence of bacteria was approximately 38% and 15% at pH 7.4,
groups,16 which could potentially impact the adhesion perfor- respectively. After the treatment of S. aureus or B. amyloliquefaciens,
mance of copolymer nanocomposites to different kinds of bacteria. however, both types of nanoparticles displayed an unambiguous
Nevertheless, considerably strong green fluorescence derived from burst release, up to 86% for S. aureus and 83% for B. amylolique-
BODIPY-based copolymeric nanoparticles can be observed both in faciens, respectively. The results confirmed that the presence of
the CLSM images of E. coli and P. aeruginosa despite few large bacteria indeed accelerated the release of the drug from copoly-
aggregates being formed, indicating that the copolymer could still meric nanoparticles, which was labelled as the bacteria-triggered
strongly bind and image these bacteria. As a result, the obtained drug release. The same phenomenon was also observed in the case
PBA-based glycopolymers were able to cross-link and agglutinate of pH 6.5. We found that the total release amount of triclosan was
bacteria through strong polyvalent cooperative interactions higher at pH 6.5 than that at pH 7.4 whether there were bacteria or
between PBA moieties and bacterial cell walls, forming highly not, which was attributed to the dissociation of nanoparticles at low
fluorescent bacterial clusters.
pH, leading to faster drug release (additional discussions about
We further investigated the binding capability and aggregation weak acid sensitivity are present in the ESI,† Table S3 and Fig. S9).
rate of RCN3 and BCN3 towards both Gram-positive and Gram- In addition, comparing the release profiles of triclosan between
negative bacteria based on the solution-turbidity method (Fig. S6, RCN3-T and BCN3-T, the rate and the cumulative amount of the
ESI†). Upon incubation of bacteria (OD 600 = B1.2) with RCN3 triclosan release of RCN3-T were higher than that of BCN3-T
or BCN3 at a concentration of 0.5 mg mLÀ1 in sterile PBS whether at pH 7.4 or 6.5, which was caused by the compact
(pH 7.4), it can be observed that Gram-positive S. aureus and structure of the block copolymeric nanoparticles when compared
B. amyloliquefaciens were rapidly agglutinated within 10 min and with random ones. Since the bacterial infection microenvironment
consequently sedimented to the bottom of the centrifuge tube, so is weakly acidic, bacteria-derived dissociation of RCN3-T and
the OD600 value of the supernatant declined dramatically from BCN3-T can effectively promote directional transport of antibiotics
B1.2 to B0.5 within 20 min. Although Gram-negative E. coli and to bacteria in the bacterial infection sites, resulting in enhanced
P. aeruginosa displayed a slightly slower agglutination rate than bacterial ablation and decreased damage to normal tissues.
Gram-positive bacteria species, it ultimately reached up to 60–70%
The LIVE/DEAD cell viability assays were used to evaluate the
of bacteria sedimentation at 2 h. In contrast, agglutination antibacterial activity of RCN3-T and BCN3-T. Through CLSM,
and sedimentation phenomena were not observed for untreated the green fluorescence belonged to the BODIPY-based copolymeric
bacteria under the same conditions. In addition, there was a subtle nanoparticles that adhere to bacteria, while the red points were
distinction between the adhesion behaviour of RCN and BCN derived from the dead bacterial cells stained by EB. As shown in
toward various kinds of bacteria, which might be a result of the Fig. 3A and Fig. S10 (ESI†), the untreated bacterial cells exhibited
construction difference between the two types of copolymeric no red fluorescence, indicating that they were alive. After treatment
nanoparticles. As RCN3 and BCN3 possessed the best adhesion with RCN3 and BCN3 for 2 h, although the bacteria gathered driven
properties towards S. aureus and B. amyloliquefaciens, RCN3, BCN3 by nanoparticles into clusters which exhibited strong green fluores-
and S. aureus, B. amyloliquefaciens were selected for further cence, almost no red fluorescence was observed, suggesting that
investigations.
RCN3 and BCN3 did not result in the death of S. aureus and
According to the above-mentioned mechanism analysis, the B. amyloliquefaciens, which is suggestive of no possibility for the
adhesion of the copolymeric nanoparticles to bacteria is accom- nanoparticles to induce bacterial resistance. By contrast, strong
panied by the hydrolysis of the borate linkage formed by AAPBA green fluorescence and red fluorescence were observed at the same
and AGA in the nanoparticles, leading to the dissociation and time which was highly coincident with the aggregation of bacteria,
looseness of the nanoconstruction, which could possibly accelerate demonstrating that the bacteria were efficiently killed by the
the release of the loaded cargo. Thus, we encapsulated the model released drug via the bacteria-triggered release pattern.
antibiotic triclosan to the nanoparticle’s hydrophobic core and
To further verify membrane disruption mediated by the nano-
attempted to validate the bacteria-activated and weak acid- particles, morphological changes of S. aureus and B. amyloliquefaciens
triggered drug release as well as the antibacterial activity against isolates were observed via scanning electron microscopy (SEM).
S. aureus and B. amyloliquefaciens. When encapsulated with As shown in Fig. 3C and Fig. S11 (ESI†), the untreated bacteria with
triclosan, the size of random copolymeric nanoparticles (RCN3-T) a round or rod shape displayed a smooth and complete cell wall.
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The obtained nanoplatforms were firstly found to exhibit specific
interactions with bacteria and possessed a superior activity of
bacteria binding for Gram-positive bacteria with little impact on
normal cells. The nanoparticles directly transported the drug into
bacteria and efficiently released the drug to eliminate bacteria.
Considering that the bacteria-triggered drug delivery system was
unique in responding to bacteria directly instead of certain bacteria
secretions followed by programmed cargo release at the surface of
bacteria, minimizing the possible off-target effect of the drug, we
believe that this strategy can be broadly applied for the selective
delivery of various antimicrobials to treat a variety of extracellular or
intracellular bacterial infection diseases with a decreased dose of
antibiotics and avoiding the occurrence of bacterial resistance.
We gratefully acknowledge the support from the National
Natural Science Foundation of China (Grant No. 21474055,
21774062 and 51673102) and the Natural Science Foundation
of Tianjin, China (Grant No. 18JCYBJC29300).
Conflicts of interest
There are no conflicts to declare.
Fig. 3 Antibacterial activity observed via CLSM of (A) S. aureus, and
(B) B. amyloliquefaciens when treated with PBS, RCN3 and RCN3-T,
respectively; (C) SEM images of S. aureus, and B. amyloliquefaciens when
treated with PBS, RCN3 and RCN3-T, respectively; the MIC of RCN3-T
and BCN3-T as well as free triclosan against (D) S. aureus and
(E) B. amyloliquefaciens. Bacteria treated with PBS were set as negative
controls (À). Each sample was analyzed in triplicate and results were
reported as mean Æ standard deviation (n = 3).
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gathered into clusters, most of which maintained an intact
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or BCN3-T. The results indicated that the copolymeric nanoparticles
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triclosan to efficiently kill the bacterial cells.
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