A β-Lactamase-Imprinted Responsive Hydrogel for the Treatment of Antibiotic-Resistant Bacteria

Article abstract of DOI:10.1002/anie.201600205

Antibiotics play important roles in infection treatment and prevention. However, the effectiveness of antibiotics is now threatened by the prevalence of drug-resistant bacteria. Furthermore, antibiotic abuse and residues in the environment cause serious h

Full text of DOI:10.1002/anie.201600205

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201600205
German Edition: DOI: 10.1002/ange.201600205
Molecular Imprinting
A b-Lactamase-Imprinted Responsive Hydrogel for the Treatment of
Antibiotic-Resistant Bacteria
Wen Li, Kai Dong, Jinsong Ren, and Xiaogang Qu*
Abstract: Antibiotics play important roles in infection treat-
ment and prevention. However, the effectiveness of antibiotics
is now threatened by the prevalence of drug-resistant bacteria.
Furthermore, antibiotic abuse and residues in the environment
cause serious health issues. In this study, a stimuli-responsive
imprinted hydrogel was fabricated by using b-lactamase
produced by bacteria for deactivating antibiotics as the
template molecule. The imprinted hydrogel could initially
trap b-lactamase excreted by drug-resistant bacteria, thus
making bacteria sensitive to antibiotics. After the bactericidal
treatment, the “imprinted sites” on the hydrogel could be
reversibly abolished with a temperature stimulus, which
resulted in the reactivation of b-lactamase to degrade antibiotic
residues. We also present an example of the use of this
antibacterial design to treat wound infection.
ance to them.[7] The expression of b-lactamase is the most
pervasive resistance mechanism employed by bacteria.[8] This
enzyme can hydrolyze the b-lactam ring to deactivate these
antibiotics.[9] To inhibit b-lactamase, several molecules with
the b-lactam core structure have been introduced.[10] Unfortu-
nately, such inhibitors usually upregulate the expression of b-
lactamase. Some non-b-lactam inhibitors, including boronic
acid derivatives and phosphonates, have also been
designed.[10] Although interesting, these small-molecule-
based inhibitors often have poor selectivity,[11] and none of
them could resolve the problems of antibiotic resistance and
antibiotic residues simultaneously.
Molecular imprinting has attracted wide interest for the
fabrication of artificial receptors.[12] During the imprinting
process, the target template and functional monomers are first
polymerized in place.[13] After removal of the template, tailor-
made recognition sites exist, which can rebind the target with
high affinity and selectivity.[14] As compared to antibodies,
molecularly imprinted polymers are straightforward to pre-
pare, cost-effective, and highly robust.[14,15] These properties
make them attractive for separations[16] and sensing,[17] as
enzyme inhibitors[18] and protein mimics,[19] and for toxin
clearance[20] and cell/tissue imaging.[21] More interestingly,
intelligent imprinted polymers have been developed recently,
in which the recognition sites can be switched on and off by
triggers.[22]
Inspired by these achievements, we have now developed
a stimuli-responsive imprinted hydrogel to resolve the
demanding challenges faced by antibiotic therapy
(Figure 1). The intelligent imprinted hydrogel was fabricated
by using b-lactamase as the template and N-isopropylacryl-
amide (NIPAAm) as the temperature-responsive mono-
mer.[23] It could recognize and trap the b-lactamase produced
by bacteria, thus attenuating the bacterial resistance to
antibiotics. After the killing of bacteria, the b-lactamase
bound on the imprinted-polymer (IP) hydrogel could be
reversibly activated by a temperature stimulus to degrade
antibiotic residues. Accordingly, the imprinted hydrogel could
act as an intelligent gate for b-lactamase to overcome
antibiotic resistance and residues during different stages.
Although imprinted polymers have exhibited some expanded
bio-applications, this study is the first demonstration of
imprinting b-lactamase for multifunctional antibacterial ap-
plication.
D
espite significant improvements in biomedical technology,
the global burden of infectious diseases remains high.[1] Since
the first introduction of penicillin, it was believed that
antibiotics would put an end to bacterial infections.[2] How-
ever, the effectiveness of antibiotics is threatened by the
prevalence of antibiotic resistance.[3] Nowadays, antibiotic-
resistant pathogenic bacteria account for numerous treatment
failures and mortality in the clinic. Another risk associated
with antibiotics is their residues in the environment and
animal products.[4] These residues may cause health hazards
and promote bacterial resistance. Therefore, the development
of effective strategies to combat antibiotic resistance and
residues is urgent for infection treatments.
The design of new antibiotics may address the urgent
threat of drug resistance. However, the development of novel
antibiotics is relatively slow,[5] and the inevitable rise of
resistance will ruin new antibiotics introduced in the near
future. As an alternative, the suppression of bacterial
resistance and restoration of the efficacy of conventional
antibiotics is appealing. Among conventional antibiotics, b-
lactam antibiotics are the most widely used and have a long
history.[6] However, bacteria have gradually developed resist-
[*] W. Li, K. Dong, Prof. J. Ren, Prof. X. Qu
Laboratory of Chemical Biology and State Key Laboratory of Rare
Earth Resource Utilization, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences
Changchun, Jilin 130022 (China)
We first synthesized the b-lactamase-imprinted responsive
hydrogel (IP hydrogel). NIPAAm was chosen as the thermo-
responsive monomer in combination with acrylamide as the
hydrogen-bonding monomer. Furthermore, to mediate the
polymerization in close proximity to the enzyme,[18,24]
a known small-molecule inhibitor of b-lactamase, 3-amino-
E-mail: xqu@ciac.ac.cn
W. Li, K. Dong
University of Chinese Academy of Sciences
Beijing, 100039 (China)
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201600205.
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temperature on the rebinding pro-
cess. The affinity between the IP
hydrogel
and
b-lactamase
decreased at 208C (Figure 2c).
The pNIPPAm-based IP hydrogel
exhibited temperature-responsive
behavior. It became swollen at low
temperatures (Figure 2d). During
this process, the imprinted cavities
in the hydrogel were no longer
complementary to b-lactamase,
thus weakening their interactions.
We next studied the temperature-
dependent release of b-lactamase.
After being loaded with b-lacta-
mase at 378C, the IP hydrogel was
immersed in a fresh solution at
208C. The release of b-lactamase
was clearly observed on the basis
of the absorption change of the
surrounding solution (see Fig-
ure S3). However, the release of
b-lactamase at 378C was much
slower. The NP hydrogel showed
serious leakage of b-lactamase at
both 37 and 208C (see Figure S4).
These results proved that the tem-
perature-responsive recognition
sites on the IP hydrogel could
control the binding and release of
b-lactamase.
Figure 1. a) Fabrication of a temperature-responsive imprinted hydrogel with b-lactamase as the
template. b) Bacteria could express b-lactamases to hydrolyze b-lactam antibiotics. The imprinted
hydrogel bound b-lactamase and protected antibiotics from enzymatic degradation. After bactericidal
treatment, the b-lactamase trapped in the hydrogel was released by a temperature stimulus and could
then degrade antibiotic residues. The residual b-lactamase in solution could be rebound by the IP
hydrogel to decrease their health risk.
phenylboronic acid (3-APBA),[25] was used as the anchoring
ligand. It was rendered polymerizable by coupling with
acryloyl chloride. These functional monomers were first
assembled with b-lactamase at 378C and then polymerized
between two glass plates. The b-lactamase templates and
unreacted monomers were finally removed. A non-imprinted
control hydrogel (NP hydrogel) was also prepared in an
identical manner but without the addition of a template.
The ability of the IP hydrogel to rebind b-lactamase was
investigated by a batch binding approach at 378C. As
presented in Figure 2a, the adsorption capacity of the IP
hydrogel increased as the initial b-lactamase concentration
increased. In comparison with the NP hydrogel, the IP
hydrogel clearly showed a higher affinity toward b-lactamase.
The two types of hydrogels had the same monomer compo-
sition (see Figure S1 in the Supporting Information). Little
difference in their macrostructure was observed by scanning
electron microscopy (see Figure S2), thus excluding the
possibility that differences in physical adsorption might
result in the different binding capacity of the two hydrogels.
To study binding selectivity, we used five non-template
proteins as controls: cytochromec (Cyt), bovine serum
albumin (BSA), urease (Ure), lysozyme (Lys), and ovalbumin
(OVA). Relatively low binding affinity of the IP hydrogel
toward these control proteins was observed (Figure 2b).
After confirming the efficient recognition of b-lactamase
by the IP hydrogel at 378C, we studied the influence of
Figure 2. a) Binding isotherms of the IP (black line) and NP hydrogel
(red line) toward b-lactamase at 378C. b) Selective adsorption of b-
lactamase by the IP (black) and NP hydrogel (red). c) Binding iso-
therms of the IP and NP hydrogel at 208C toward b-lactamase.
d) Weight change of the IP hydrogel at different temperatures. Inserts
are photographs of the hydrogel at 20 and 378C.
Next, the effect of the IP hydrogel on b-lactamase activity
was investigated. Nitrocefin, a b-lactam antibiotic, was used
to indicate the activity of b-lactamase with color readout. The
solution of nitrocefin typically appeared yellow with an
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absorption peak at 390 nm. After hydrolysis by lactamase, the
nitrocefin solution gradually turned red with a new absorption
band near 486 nm (see Figure S5). However, upon co-
incubation with b-lactamase and the IP hydrogel, the nitro-
cefin solution was barely hydrolyzed at all and retained its
original yellow color. The results confirmed that the IP
hydrogel could inhibit b-lactamase activity, which was con-
sistent with its binding capacity toward b-lactamase. Once
bound to the IP hydrogel, the active site of the enzyme was
presumed to be masked, thus decreasing its accessibility to the
substrate. An increase in the amount of the IP hydrogel
resulted in more efficient inhibition of b-lactamase (Fig-
ure 3a). At equivalent doses, the NP hydrogel clearly
exhibited a lower inhibition level (Figure 3b). Furthermore,
Figure 4. a) Live–dead fluorescence imaging of bacteria samples.
Living cells showed a green signal, and dead cells showed a red signal.
Scale bars: 20 mm. b) Viability analysis of bacteria samples. c) SEM
images of bacteria samples. Scale bars: 2 mm. The bacteria samples 1–
5 were prepared by incubating bacteria with phosphate-buffered saline
(PBS), the IP hydrogel, penicillin G, the NP hydrogel+penicillin G, and
the IP hydrogel+penicillin G, respectively. d) Degradation of antibiotic
residues after the b-lactamase-loaded IP hydrogel was cooled to 208C.
b-lactamase to destroy the antibiotic. However, in the
presence of the IP hydrogel, the efficacy of the antibiotic
was greatly enhanced, and the bacterial viability decreased by
nearly 80% (Figure 4b). As a control, the IP hydrogel alone
without an antibiotic exhibited little toxicity. This result
implied that the IP hydrogel did not directly act as an
antibacterial agent but rather as an adjuvant to enhance the
efficacy of the antibiotic. It could trap and inhibit b-lactamase
excreted by bacteria, thus making bacteria sensitive to
conventional antibiotics. Notably, although the free APBA
monomers showed some inhibitory effect, the inhibition
capability of the IP hydrogel was mainly derived from the
imprinting effect, not direct inhibition by APBA (see Fig-
ure S9). As compared with the IP hydrogel, the NP hydrogel
with the same monomer composition caused some but
a significantly lower decrease in the bacterial resistance.
Besides viability analysis, the growth of bacteria was eval-
uated with a disk diffusion assay (see Figure S10). Only in the
presence of the IP hydrogel could the antibiotics dramatically
inhibit the bacterial growth. Morphology changes of the
bacteria were investigated by SEM (Figure 4c). In the
untreated and IP-hydrogel-treated samples, bacteria typically
had a round shape with a smooth cell wall. After antibiotic
treatment, only a small amount of cells were deformed.
However, most of the cells were seriously deformed and some
were even lysed upon co-incubation with the IP hydrogel and
the antibiotic. These results showed that the IP hydrogel
could improve the antibacterial efficiency of b-lactam anti-
biotics. Control protein-imprinted hydrogels were synthesized
Figure 3. a,b) UV/Vis absorption of solutions of nitrocefin after treat-
ment with different b-lactamase samples: a) b-lactamase was preincu-
bated with (or without) the IP hydrogel at a concentration of 0
(sample 1), 2 (sample 2), 5 (sample 3), 15 mgmLÀ1 (sample 4) at
378C for 1.5 h; b) b-lactamase was preincubated with the equivalent
dose of the NP hydrogel at 378C. Inserts are the corresponding
photographs of the nitrocefin solutions. c) Inhibition of b-lactamase
activity by different amounts of the IP (black) or NP hydrogel (red).
d) Inhibition and reactivation of b-lactamase by the IP hydrogel at 37
and 208C.
when the hydrogels were synthesized without the anchoring
monomer, they showed less effective b-lactamase binding and
inhibition (see Figure S6). The activity of the enzyme was
turned on when the IP hydrogel was cooled to 208C (see
Figure S7). b-Lactamase was released from the swollen IP
hydrogel and hydrolyzed nitrocefin to give a red color. When
the temperature was alternated between 37 and 208C, the
activity of b-lactamase in the presence of the IP hydrogel was
inhibited, then restored, and then inhibited again (Figure 3d).
The potential of the IP hydrogel for antibacterial appli-
cations was then evaluated. Methicillin-resistant staphylococ-
cus aureus (MRSA) bacteria were used as the model bacteria.
Their viability was analyzed by a live/dead assay. The b-
lactam antibiotic penicillin G, which is commonly used in the
clinic, exhibited low inhibition of MRSA (Figure 4a). This
result was consistent with previous studies and our nitrocefin
tests (see Figure S8), which proved that MRSA could produce
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to verify the effect of b-lactamase imprinting on the
antibacterial process. The sizes of control template proteins
were larger than, or smaller than, or similar to that of b-
lactamase. However, these control hydrogel-based systems
showed lower antibacterial efficiency than that of the b-
lactamase-imprinted hydrogel (see Figure S11; for the batch-
to-batch performance of the IP hydrogel, see Figure S12).
In addition to suppressing antibiotic resistance, the IP
hydrogel-based system could be further utilized to degrade
antibiotic residues (Figure 1). After the killing of bacteria, the
antibacterial system containing the IP hydrogel and the
antibiotic was cooled to 208C. The residual antibiotic in
solution was monitored by a microbiological method. About
80% of the antibiotic was gradually degraded within 2 h
(Figure 4d). At low temperature, the b-lactamase bound by
the IP hydrogel was released into solution and could hydro-
lyze the residual antibiotic molecules. Although the direct
addition of b-lactamase has been reported for the elimination
of antibiotic residues, residual b-lactamase is also a health
risk.[26] In our system, after the degradation of the antibiotic,
the b-lactamase in solution could be conveniently rebound
into the IP hydrogel at 378C, which avoided the side effect of
b-lactamase. Thus, the IP hydrogel provided an efficient and
safe method to eliminate antibiotics. It could be used
repeatedly for further cycles after washing.
Finally, we present an example of the use of this
antibacterial design to treat wound infection.[27] As heavily
hydrated materials, hydrogels can protect wounds in a suitably
moist healing environment. First, the biocompatibility of our
IP hydrogel was studied. More than 95% of cells cultured on
the IP-hydrogel surface were alive after 24 h (see Figure S13).
We then tested the IP hydrogel in serum to confirm its
feasibility for use in biological samples (see Figure S14). For
infection treatment, the backs of mice were slashed and
injected with MRSA to build the infected-wound model. The
wounds of the mice were treated with PBS buffer, with the IP
hydrogel alone, with penicillin G alone, with the NP hydro-
gel + penicillin G, or with the IP hydrogel + penicillin G.
During the whole therapeutic process, the wounds treated
with the IP hydrogel + antibiotics did not show erythema, and
they formed scabs after therapy. The wounds in the other four
groups had different levels of erythema (Figure 5a). To assess
the bactericidal effect, we excised the wound tissues to
quantify the number of bacteria on them (Figure 5b,c). From
the grown colonies, we could see that the combination of the
IP hydrogel and the antibiotic resulted in the most effective
wound antibacterial therapy. The control protein-imprinted
hydrogels were also tested for infection treatment. As
compared with the b-lactamase-imprinted hydrogel, these
control hydrogels showed an unsatisfactory therapeutic effect
(see Figure S15). In this study, the imprinted polymers were
prepared in the form of hydrogel layers for treating wound
infection. They could also be prepared readily in other forms,
such as nanoparticles. Nanostructured imprinted materials
have a high surface-to-volume ratio, and the binding capacity
and kinetics would be further improved.
Figure 5. a) Wounds of mice after 1 and 3 days of therapy. The mice
samples 1–5 were treated with PBS buffer, the IP hydrogel, penicillin G,
the NP hydrogel+penicillin G, and the IP hydrogel+penicillin G,
respectively. b) The bacteria separated from wound tissue were cul-
tured on agar plates. Inserts are the wound tissue. c) Number of
bacteria in the wound tissue of each sample.
drug-resistant bacteria, thus making the bacteria sensitive to
antibiotics. It could thus act as an adjuvant to enhance the
efficacy of conventional antibiotics against drug-resistant
bacteria. After the killing of bacteria, b-lactamase bound on
the IP hydrogel could be reactivated by a temperature
stimulus for the degradation of antibiotic residues. With
thermoresponsive “b-lactamase-recognition sites”, the IP
hydrogel might resolve two challenging issues in antibiotic
therapy: the developed drug resistance and antibiotic resi-
dues. We hope our study will guide the use of smart imprinted
polymers for multifunctional biomedical applications.
Acknowledgements
This research was supported by the 973 Project
(2012CB720602, 2011CB936004) and the NSFC (21210002,
21431007, 21533008).
Keywords: antibiotics · antibiotic resistance · enzymes ·
molecular imprinting · responsive hydrogels
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In summary, we have prepared a b-lactamase-imprinted
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cations. The IP hydrogel could trap b-lactamase excreted by
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Received: January 7, 2016
Revised: March 10, 2016
Published online: && &&, &&&&
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Communications
Molecular Imprinting
W. Li, K. Dong, J. Ren,
X. Qu*
&&&& — &&&&
A b-Lactamase-Imprinted Responsive
Hydrogel for the Treatment of Antibiotic-
Resistant Bacteria
New hope for old drugs: b-Lactamase
was used as a template molecule to
fabricate a stimuli-responsive imprinted
hydrogel (see picture). The hydrogel
could trap b-lactamase excreted by drug-
resistant bacteria, thus making the bac-
teria sensitive to conventional antibiotics.
The thermoresponsive “binding sites” on
the hydrogel could then be abolished to
release b-lactamase for the degradation
of antibiotic residues.
6
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Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!