Enzyme-Responsive Polymeric Vesicles for Bacterial-Strain-Selective Delivery of Antimicrobial Agents

Article abstract of DOI:10.1002/anie.201509401

Antimicrobial resistance poses serious public health concerns and antibiotic misuse/abuse further complicates the situation; thus, it remains a considerable challenge to optimize/improve the usage of currently available drugs. We report a general strategy

Full text of DOI:10.1002/anie.201509401

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International Edition: DOI: 10.1002/anie.201509401
German Edition: DOI: 10.1002/ange.201509401
Polymeric Vesicles
Enzyme-Responsive Polymeric Vesicles for Bacterial-Strain-Selective
Delivery of Antimicrobial Agents
Yamin Li, Guhuan Liu, Xiaorui Wang, Jinming Hu, and Shiyong Liu*
Abstract: Antimicrobial resistance poses serious public health
concerns and antibiotic misuse/abuse further complicates the
situation; thus, it remains a considerable challenge to optimize/
improve the usage of currently available drugs. We report
a general strategy to construct a bacterial strain-selective
delivery system for antibiotics based on responsive polymeric
vesicles. In response to enzymes including penicillin G amidase
block copolymers can encapsulate both hydrophobic and
hydrophilic payloads ranging from low to ultrahigh molecular
weight species (such as antibiotics, therapeutic peptides/
[
3]
proteins, and bacteriophage). Stimuli-responsive polymeric
vesicles with tunable permeability possessing a “sense and
act” module for on-demand drug release in a spatiotemporal
and dosage-controlled manner are highly advantageous for
[4]
(
PGA) and b-lactamase (Bla), which are closely associated
antibiotic delivery. As for triggering motifs, enzymes
provide the most promising ones as enzymatic reactions are
highly selective and efficient under mild conditions, and
involved in all biological, metabolic, and pathological pro-
with drug-resistant bacterial strains, antibiotic-loaded poly-
meric vesicles undergo self-immolative structural rearrange-
ment and morphological transitions, leading to sustained
release of antibiotics. Enhanced stability, reduced side effects,
and bacterial strain-selective drug release were achieved.
Considering that Bla is the main cause of bacterial resistance
to b-lactam antibiotic drugs, as a further validation, we
demonstrate methicillin-resistant S. aureus (MRSA)-triggered
release of antibiotics from Bla-degradable polymeric vesicles,
in vitro inhibition of MRSA growth, and enhanced wound
healing in an in vivo murine model.
[
5]
cesses. Most importantly, specific enzymes are actively
produced by resistant pathogens (for example, penicillin G
[6]
amidase (PGA) and b-lactamase (Bla)), and Bla-catalyzed
hydrolysis is mainly responsible for drug resistance towards
a series of b-lactam antibiotic drugs, such as penicillin,
cephalosporin, and carbapenem.
Note that enzyme-responsive polymeric vesicles have
[
7]
been far less explored. The only two reports concerning
bacterial enzyme-responsive polymeric vesicles focused on
fluorescent bacteria detection under simulated conditions,
and none of these enzymes are relevant to the acquisition of
S
ince their commercial introduction in 1940s, antibiotics
have substantially reduced human morbidity and mortality.
Unfortunately, pathogens can develop antibiotic resistance
through de novo gene mutation or horizontal gene transfer.
Furthermore, antibiotic abuse/misuse has produced more
virulent and resistant strains, which pose huge threats to
[7e,f]
bacterial resistance.
Herein, we aim to establish a general
strategy to construct bacterial strain-selective delivery nano-
carriers for antimicrobial agents based on block copolymer
vesicles, which are responsive to enzymes relevant to
clinically important bacterial strains (Scheme 1). PGA and
Bla-responsive polymeric vesicles were self-assembled from
amphiphilic diblock copolymers consisting of hydrophilic
poly(ethylene glycol) (PEG) block and hydrophobic block
containing enzyme-cleavable self-immolative side linkages.
During vesicle formation, antimicrobial agents were loaded
into either hydrophobic bilayers or aqueous interiors. Upon
incubation with specific enzymes, the chemical structural
transformation, morphology transition, drug release profile,
and antibacterial activities of polymeric vesicles were inves-
tigated.
[
1]
global health. Therefore, it is necessary to screen for next-
generation antibiotics and explore therapeutically effective
strategies. The invention of antibiotics by high-throughput
screening of natural products and chemical libraries was
successful before 1970s, but the progress has been extremely
slow since then. On the other hand, nanoparticle-based drug
delivery strategies aiming to improve therapeutic efficacy of
currently available antibiotics provides an alternate solu-
[
2]
tion.
Among various inorganic and polymeric drug nanocar-
riers, polymeric vesicles self-assembled from amphiphilic
In previously reported systems concerning enzyme-
responsive polymeric nanoparticles, an enzymatic backbone
[
*] Y. Li, Dr. G. Liu, X. Wang, Dr. J. Hu, Prof. Dr. S. Liu
CAS Key Laboratory of Soft Matter Chemistry
Hefei National Laboratory for Physical Sciences at the Microscale
iChem (Collaborative Innovation Center of Chemistry
for Energy Materials)
Department of Polymer Science and Engineering
University of Science and Technology of China
Hefei, Anhui Province 230026 (China)
[
7]
scission mechanism was solely involved. However, the
diversity in enzymes and substrates leads to drastically
different degradation kinetics and release profiles. In this
work, we attempted to introduce side chain enzymatic
cleavage mechanism, and chose PGA and Bla as target
enzymes, considering that both enzymes are associated with
bacterial resistance. As shown in Scheme 1, side chain
moieties in hydrophobic blocks of PEG-b-PP and PEG-b-
PC are subjected to enzymatic uncapping of terminal groups
E-mail: sliu@ustc.edu.cn
Supporting information, including experimental details, character-
ization methods, and all relevant characterization data, and
ORCID(s) from the author(s) for this article are available on the
WWW under http://dx.doi.org/10.1002/anie.201509401.
(phenylacetic amide and cephalosporin) and subsequent self-
immolative cleavage (spontaneous rearrangement through
1
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Figure 1. TEM (left and middle columns) and SEM (right column)
images of: a–c), PP2 LCVs; d–f), PP4 LCVs; g–i), PC1 vesicles.
Scheme 1. Enzyme-responsive polymeric vesicles for bacterial strain-
selective delivery of antibiotics. Polymeric vesicles self-assembled from
PEG-b-PP and PEG-b-PC are subjected to side chain cleavage and
microstructural transformation in response to penicillin G amidase
(
PGA), and b-lactamase (Bla), respectively. This process is accompa-
positions and hydrophobic block lengths, the general trend of
vesicles and LCVs formation (Table S1) can be ascribed to
synergistic and cooperative effects from hydrophobic, hydro-
gen bonding, and p-p stacking interactions between carba-
nied with sustained release and bioactivity recovery of antimicrobial
agents encapsulated within vesicles.
[8d]
1
,6-elimination of p-hydroxybenzyl alcohol or p-aminobenzyl
mate-containing hydrophobic blocks.
alcohol), both affording poly(2-aminoethyl methacrylate)
Next, the colloidal stability of polymeric vesicles was
examined by fluorescence and DLS measurements. In the
absence of PGA, both fluorescence resonance energy transfer
(FRET) ratios (I575/I505) of DiO/DiI co-loaded PP2 LCVs and
emission intensities of ESICT (excited state intramolecular
charge transfer; 451 nm) and ESIPT (excited state intra-
molecular proton transfer; 537 nm) bands of 3-HF-labeled
PP6 LCVs exhibited negligible changes (Figures 2a; Sup-
porting Information, Figure S14). The long-term stability of
PP2 LCVs and PC1 vesicles were also confirmed by DLS
(Figures 2b; Supporting Information, Figure S15a). More-
[
8]
scaffolds. By involving side chain degradation triggered by
drug-resistant enzymes and final nanostructures with com-
parable chemical compositions, we aim to achieve controlled
degradation kinetics, predictable payload release profiles, and
targeted antibiotic delivery.
PGA- and Bla-responsive monomers, PBC and CBC,
were first synthesized (Supporting Information, Figures S1–
S3). Reversible addition-fragmentation chain transfer
(
RAFT) polymerization of PBC using PEG45 macroRAFT
agent (PEG -CTA) afforded PGA-responsive PEG-b-PP
4
5
(
PP1-PP5) diblock copolymers (Figures S4-S5 and Table S1).
over, both hydrodynamic diameters (hD i) and polydisper-
h
2
However, CBC cannot be polymerized under the same
conditions, which was likely due to the relatively high steric
hindrance. Alternatively, a polymerizable precursor bearing
latent isocyanate functionality, BBC, was synthesized (Figur-
es S1 and S6). RAFT polymerization of BBC using PEG -
CTA afforded PEG-b-PB (PB1-PB2) (Figures S4 and S7).
After reacting with PHCC and deprotection of ester linkages,
Bla-responsive PEG-b-P(C-co-B) (PC1-PC2) block copoly-
mers were obtained (Figures S4 and S8, Table S1). Polarity-
sensitive probe, 3-hydroxyflavone (3-HF)-labeled and PGA-
responsive PP6 and enzyme-inert PEG-b-polystyrene (PEG-
b-PS, PS1) were also synthesized (Figures S9 and S10a,
Table S2).
Self-assembly of above diblock copolymers was con-
ducted using the cosolvent approach. Assemblies with uni-
form morphologies, such as spherical micelles, vesicles, large
compound vesicles (LCVs), and large compound micelles
(
These nanostructures were characterized by dynamic light
scattering (DLS), transmission electron microscopy (TEM),
scanning electron microscopy (SEM), and atomic force
microscopy (AFM) (Figure 1; Supporting Information, Fig-
ures S11–S13). In spite of varying chemical structures/com-
sities (m /G ) of PP2 LCVs showed no obvious changes when
2
dispersed in PBS or 20% fetal bovine serum (FBS; Fig-
ure S15b).
The loading capability of polymeric vesicles was then
examined by fluorescence measurements and confocal laser
scanning microscopy (CLSM), using hydrophilic/hydrophobic
small molecule dyes (calcein and Nile red (NR)) and
fluorescent macromolecules (FITC-parasin I) as model pay-
loads (Figure S16). Moreover, the concurrent loading of
calcein/NR or FITC-Parasin I/NR was verified by CLSM
technique (Figure 2 f; Supporting Information, Figure S17).
Encapsulating antibiotics and therapeutic proteins into poly-
meric vesicles is expected to enhance their structural stabil-
ities, as bioactive molecules are often unstable when exposed
to elevated temperature, ionic strengths, and enzymes. To
confirm this, chlorophenol red-b-d-galactopyranoside
(CPRG, as a small molecular antibiotic model) and myoglo-
bin (Myo, as a therapeutic protein model) were loaded into
PP2 LCVs, respectively (Figure S18). Free CPRG can be
readily cleaved by b-galactosidase to generate chlorophenol
red (CPR), whereas no colorimetric reaction occurred when
CPRG was encapsulated within PP2 LCVs (Figure S19).
Furthermore, although the protein activity of free Myo
45
[
9a]
[9]
LCMs), were obtained (see the Supporting Information).
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Figure 3. Enzyme-triggered degradation of polymeric vesicles. a) Per-
centage degradation of PP2 LCVs against incubation duration in the
absence and presence of PGA. b) Fluorescence analysis of PP2 LCVs in
the absence and presence of fluorescamine (FA) with or without PGA
degradation. c) Time-dependent evolution of PGA-digested PP2 LCVs.
TEM images of d) PGA-treated PP2 LCVs diluted with DMF and e) Bla-
treated PC1 vesicles.
Figure 2. Time-dependent evolution of a) fluorescence emission spec-
tra of DiO/DiI co-loaded PP2 LCVs in the absence of PGA, and
2
b) hydrodynamic diameters (hD i) and polydispersities (m /G ) of PC1
h
2
vesicles in the absence of Bla. c) Enhanced stability of myoglobin
(Myo) encapsulated in PP2 LCVs. d,e) Reduced side effects of CPFX-
loaded PP2 LCVs against HeLa cells and SAMP1-loaded PC1 vesicles
against human red blood cells. f) CLSM images of FITC-parasin I/NR
co-loaded PP2 LCVs (scale bars=10 mm); *p<0.01, student’s t-test.
that in water ( ꢀ 68 nm), and much larger than molecularly
dissolved monomers. The morphology transition of vesicles
was then monitored by TEM (Figure 3c). Upon PGA
incubation for 4 h, coexistence of PP2 LCVs with spherical
nanoparticles were observed; at 12 h incubation, LCVs
almost disappeared, accompanied with the emergence of
nanoparticles and hollow nanostructures. After 24 h, only
spherical nanoparticles were observed. Moreover, both TEM
and AFM revealed the retention of spherical micelles when
the final dispersion was diluted into DMF (Figures 3d;
Supporting Information, Figure S22d). When PGA-treated
gradually decayed within 2 days, PP2 LCVs encapsulating
Myo showed much less bioactivity loss, as verified by the
guaiacol oxidation assay (Figure 2c).
On the other hand, antibiotics and macromolecular
antimicrobial agents typically incur adverse effects (e.g.
nephrotoxicity for vancomycin, cytotoxicity for ciprofloxacin
(
CPFX), and hemolysis for antimicrobial polymers). Loading
of them within polymeric vesicles could attenuate side effects
Figure S20). Although free CPFX showed cytotoxicity
(
PP2 LCVs were dialyzed against water, lyophilized, and
1
against HeLa cells, PP2 LCVs encapsulated CPFX exhibited
negligible effects on cell viabilities (Figure 2d). Additionally,
loading antimicrobial polymer SAMP1 (Figure S9) into PC1
vesicles could significantly reduce hemolysis against human
red blood cells (hRBCs; Figure 2e).
dispersed into D O, H NMR analysis only revealed signals
2
characteristic of PEG (Figure S21c). Taken together, we
concluded that PP2 LCVs transformed into core-crosslinked
(CCL) spherical micelles upon PGA degradation.
Previous reports established that enzymatic degradation
of polymeric assemblies can proceed by the monomer–
The enzymatic degradation process was then studied.
Upon PGA incubation, ꢀ 80% of side chain moieties of PP2
were digested within 36 h, as monitored by the emergence of
[5f]
aggregate exchange pathway; whereas in other cases the
interior of polymeric assemblies is directly accessible to
1
[10]
H NMR signals of phenylacetic acid and p-aminobenzyl
enzymes. As for PP2 LCVs, considering the relatively high
alcohol (Figures 3a; Supporting Information, Figure S21a). In
contrast, no apparent PP2 LCV degradation can be discerned
in the absence of PGA. The generation of primary amine
moieties (Figure S21b) was verified by amine-induced emis-
sion turn-on of fluorescamine (FA; Figure 3b). DLS meas-
CAC (critical aggregation concentration; Figure S11a and
Table S3), PGA could digest monomer chains, which are in
equilibrium with supramolecular aggregates in solution.
Cascade reactions (cleavage of terminal groups, release of
self-immolative linkers, and generation of primary amines)
will shift the monomer-aggregate equilibrium and alter the
hydrophilic/hydrophobic balance, resulting in the formation
of spherical micelles (Figure S23). Enzymatically generated
primary amines will undergo inter/intra-molecular amidation
urements revealed that both hD i and scattering light
h
intensities of PP2 LCV dispersion decreased upon PGA
digestion (Figure S22a,b). However, it was found that PGA
cannot completely digest PP2 LCVs into monomers even
upon extended incubation, and PGA-treated LCVs disper-
reactions, aided by suppressed amine pK owing to high local
a
sion exhibited an hD i of ꢀ 100 nm when diluted into DMF
density and the relatively hydrophobic microenvironment.
Intermolecular amidation reactions then lead to crosslinking
h
(
Figure S22c). The hD i measured in DMF is comparable to
h
1
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of nanoparticles. Enzymes may also directly digest into
aggregates and in situ generate primary amines, followed by
amidation-induced crosslinking (Figure S23). This crosslink-
ing chemistry is quite comparable to our recent work
concerning the fabrication cross-linked polymersomes under
[8d]
light irradiation.
Furthermore, PGA and Bla-triggered
transition from polymeric vesicles to CCL micelles for PP4
1
and PC1 were also confirmed by H NMR, DLS, and TEM
measurements (Figures 3e and S24,S25).
Microenvironmental polarity variation within polymeric
vesicles during enzymatic degradation was explored. For 3-
HF-labeled PP6 LCVs, continuous increase of ESICT
(
451 nm) and decrease of ESIPT (537 nm) band intensities
were observed when incubated with PGA (Figure S26),
revealing a hydrophobic-to-hydrophilic transition of bilayers.
This is reasonable considering that enzymatic uncapping and
self-immolative rearrangement will remove hydrophobic side
groups; newly generated amines will either be protonated or
participate in amidation, and all of these reactions will endow
CCL nanoparticle interiors with hydrophilicity.
Enzyme-triggered cargo release was then evaluated.
CLSM and fluorescence analysis confirmed sustained release
of calcein, NR, and tetramethylrhodamine-dextran from PP2
LCVs mediated by PGA (Figures S27–S29). Moreover, initial
enzyme concentrations considerably affect the rate of micro-
environmental polarity switching and cargo release profiles
Figure 4. a) GEN-equivalent concentration dependent P. aeruginosa
inhibition of GEN, GEN-loaded PP2 LCVs in the absence and presence
of PGA. b) Time-dependent MRSA growth inhibition of Synercid-loaded
PC1 vesicles. c) Growth inhibition effects of VAN and VAN-loaded PC1
vesicles against MRSA, B. longum, L. acidophilus, and E. faecalis.
d) Burn wound size analysis. ns, no statistically significant differences;
*p<0.05, student’s t-test.
À1
a drug concentration higher than 1.0 mgmL , VAN-loaded
PC1 vesicles significantly inhibited MRSA growth, compara-
ble to that of free VAN (Figure S32a). In contrast, Bla-inert
PS1 vesicles loaded with VAN cannot efficiently kill MRSA
even at much higher drug concentrations. In control experi-
ments, blank PC1 and PS1 vesicles exhibited negligible effect
on MRSA growth (Figure S31c,d). The crucial role of Bla in
regulating VAN release from PC1 vesicles was further
verified by the addition of metallo-Bla inhibitor, zinc ion
chelator EDTA. Although both free VAN and VAN-loaded
PC1 vesicles prominently inhibited MRSA growth, the co-
incubation with Bla inhibitor led to negligible inhibitory
effects (Figure S32b).
We further examined MRSA-selective antibiotic release
from PC1 vesicles (Figure 4c). Free VAN can completely
inhibit the growth of both probiotics (B. longum, L. acid-
ophilus, and E. faecalis) and MRSA, which should be avoided
under practical circumstances. Significantly, when co-incu-
bated with VAN-loaded PC1 vesicles at equivalent drug
concentrations, only the MRSA strain was significantly
inhibited. The selectivity should be ascribed to the fact that
common probiotics cannot produce Bla, thus PC1 vesicles
encapsulating VAN exhibit no bioactivities.
Bacterial strain-selective delivery of antibiotics is highly
advantageous for slowing the evolution of drug resistance and
avoiding antibiotic abuse/misuse. For example, the combina-
tion of conventional antibiotics with enzyme-responsive
vesicles loaded with new-generation drugs could be utilized,
so that resistant strains can be killed by potent drugs released
from vesicles by specific enzymes, whereas non-resistant
strains could be eliminated by conventional ones. Enzyme-
responsive polymeric vesicles loaded with antibiotics could
also directly serve as wound healing enhancers (Figure S33).
As a proof-of-concept, topical treatment of MRSA-infected
burn wound with VAN-loaded PC1 vesicles were conducted.
(
(
Figure S30). Next, PGA-triggered release of gentamicin
GEN), a hydrophilic aminoglycoside antibiotic, from PP2
LCVs was tested against the highly opportunistic Gram-
negative pathogen, P. aeruginosa. Neither PP2 LCVs nor
PGA exhibited appreciable growth inhibition of P. aeruginosa
(
Figure S31a,b). However, upon GEN loading and PGA
actuation, PP2 LCVs showed a prominent growth inhibition
effect comparable to that of free GEN when the equivalent
À1
GEN concentration was higher than 1.0 mgmL . In contrast,
largely compromised antibacterial activity was observed
without PGA, which can be ascribed to the poor permeability
of hydrophilic guest molecules through intact PP2 LCV
bilayers (Figure 4a).
Methicillin-resistant Staphylococcus aureus (MRSA)
poses a huge threat to public health, and one main cause of
MRSA resistance is the production of Bla. Considering
combination therapy is a clinically important treatment
option for MRSA, a pair of semisynthetic streptogramin
antibiotics, quinupristin/dalfopristin (Synercid), was concom-
itantly loaded into PC1 vesicles to evaluate anti-MRSA
activity (Figure 4b). Significantly, both free Synercid and PC1
vesicles encapsulating Synercid efficiently inhibited MRSA
growth. In contrast, MRSA strains continued to grow in the
presence of Bla-inert PS1 vesicles encapsulating Synercid at
comparable equivalent drug concentrations.
In addition to enhanced stability, reduced side effects, and
the feasibility of combination therapy, encapsulation of
antibiotics into enzyme-responsive polymeric vesicles could
also achieve resistant bacteria-selective drug delivery. To this
end, vancomycin (VAN), a glycopeptide antibiotic for effec-
tively treating MRSA infections, was encapsulated into PC1
vesicles. After studying in vitro VAN release activated by Bla
(
Figure S27d), the anti-MRSA activity was examined. At
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Note that blank PC1 vesicles exhibited negligible effects on
the growth of both mammalian and bacterial cells (Figure S34
and Table S4). In the in vivo murine infected burn wound
model, enhanced wound healing process was observed when
treated with VAN and VAN-loaded PC1 vesicles, as com-
pared to the control groups (Figures 4d; Supporting Infor-
mation, Figure S35).
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In summary, PGA and Bla-responsive polymeric vesicles
were fabricated and enzyme-mediated degradation processes
and microstructural evolution were studied. Enhanced struc-
tural stability, reduced side effects, feasibility of combination
therapy, bacterial strain-selective delivery of potent anti-
biotics, and enhanced burn wound healing were achieved on
the basis of antibiotic-loaded responsive vesicles. Overall, the
modular design of enzyme-responsive vesicles from amphi-
philic block copolymers, sharing the same backbone scaffold
attached with different types of enzymatic substrates in the
side chain, opens up new avenues to construct next-gener-
ation antimicrobial systems with improved efficacy for
combating against virulent resistant pathogens.
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This work was supported by the National Natural Scientific
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