A Polymer Solution To Prevent Nanoclustering and Improve the Selectivity of Metal Nanoparticles for Electrocatalytic CO2 Reduction

Article abstract of DOI:10.1002/anie.201909069

The stability of metal nanocatalysts for electrocatalytic CO₂ reduction is of key importance for practical application. We report the use of two polymeric N-heterocyclic carbenes (NHC) (polydentate and monodentate) to stabilize metal nanocatalysts (Au and Pd) for efficient CO₂ electroreduction. Compared with other conventional ligands including thiols and amines, metal–carbene bonds that are stable under reductive potentials prevent the nanoclustering of nanoparticles. Au nanocatalysts modified by polymeric NHC ligands show an activity retention of 86 % after CO₂ reduction at ?0.9 V for 11 h, while it is less than 10 % for unmodified Au. We demonstrate that the hydrophobicity of polymer ligands and the enriched surface electron density of metal NPs through σ-donation of NHCs substantially improve the selectivity for CO₂ reduction over proton.

Full text of DOI:10.1002/anie.201909069

Article
Cite This: ACS Appl. Bio Mater. 2019, 2, 1270−1277
www.acsabm.org
Antibiotic-Derived Lipid Nanoparticles to Treat Intracellular
Staphylococcus aureus
†
†
‡
†
§
§,∥
Chengxiang Zhang, Weiyu Zhao, Cong Bian, Xucheng Hou, Binbin Deng, David W. McComb,
,
†,‡
,†,⊥,#,¶,△,○
Xiaofang Chen,* and Yizhou Dong*
†Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio
3210, United States
4
‡
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050,
China
§
Center for Electron Microscopy and Analysis, The Ohio State University, Columbus, Ohio 43212, United States
Department of Materials Science and Engineering, The Ohio State University, Columbus, Ohio 43210, United States
∥
⊥
#
¶
Department of Biomedical Engineering, The Center for Clinical and Translational Science, The Comprehensive Cancer Center,
△
○
Dorothy M. Davis Heart & Lung Research Institute, and Department of Radiation Oncology, The Ohio State University,
Columbus, Ohio 43210, United States
*
S Supporting Information
ABSTRACT: Intracellular survival of pathogenic bacteria leads to high chances of
bacterial persistence and relapse in the bacteria-infected host. However, many
antibiotics fail to clear the intracellular bacteria due to their low internalization by
cells. In order to increase delivery of antibiotics in cells and eliminate intracellular
bacteria, we developed antibiotic-derived lipid nanoparticles. First, we synthesized
antibiotic-derived lipid conjugates using two widely used antibiotics including
penicillin G (PenG) and levofloxacin (Levo). Then, we formulated them into
antibiotic-derived lipid nanoparticles and evaluated their antibacterial effects. We
found that PenG-derived phospholipid nanoparticles (PenG-PL NPs) were able to
enhance cellular uptake of penicillin G as compared with free penicillin G and
8.5
eliminate up to 99.9998% of ∼10 intracellular methicillin-sensitive Staphylococcus
aureus (S. aureus) in infected A549 cells, a lung epithelial cell line. The PenG-PL NPs
showed the potential for inhibiting intracellular S. aureus and are promising to be
further studied for in vivo antibacterial applications.
KEYWORDS: intracellular bacteria, penicillin-G-derived phospholipid, antibiotic-derived lipid nanoparticles
INTRODUCTION
intracellular survival behavior of S. aureus, poor penetration
of antibiotics across the mammalian cell membrane also leads
■
The formidable prevalence of pathogenic bacteria remains a
severe threat to public health. Meanwhile, an increasing
concern has been raised upon intracellular survival of bacteria
due to the limitation of treatment options for approaching and
eliminating these intracellular pathogens. Two major types of
1
to the difficulty in the treatment and relapse of infectious
2
disease. β-Lactam antibiotics are the major treatment option
10
for the methicillin-sensitive S. aureus infection. However, the
cellular pharmacodynamics studies revealed that the β-lactam
antibiotics cannot effectively penetrate the mammalian cell
2
intracellular bacteria have been reported including facultative
intracellular bacteria that can reproduce both inside and
outside the host cell, e.g., Mycobacterium tuberculosis and S.
aureus, and obligate intracellular bacteria that can only
reproduce inside the host cell, e.g., Chlamydophila pneumo-
2
membrane due to their physicochemical characteristics. A few
strategies were studied to increase the accumulation of
antibiotics in cells, such as increasing the dose and prolonging
11
the infusion administration, using efflux transporter inhib-
2
3
itors, and developing antibiotic nanoparticle drug delivery
niae.
12−17
systems.
In this study, we aimed to develop antibiotic-
S. aureus is a Gram-positive, facultative intracellular
bacterium that can cause serious infection. Previous studies
demonstrated that intracellular S. aureus was related to several
4
derived lipid nanoparticles to improve the internalization of
antibiotics in infected cells and enable the clearance of
intracellular bacteria.
5
diseases, including S. aureus bacteremia, bone and joint tissue
6
7
8
infection, chronic rhinosinusitis, and pneumonia. These
studies also pointed out that the host cells, for example, lung
epithelial cells, may provide a protective compartment for local
Received: December 20, 2018
Accepted: February 13, 2019
Published: February 14, 2019
9
or metastatic infection of S. aureus. Apart from the
©
2019 American Chemical Society
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Figure 1. Chemical structures of the four antibiotic−lipid conjugates.
Figure 2. Characterization of antibiotic-derived lipid nanoparticles. (A) Size distribution of the selected formulation 6, PenG-L NPs. (B) Size
distribution of antibiotic-derived lipid nanoparticles. Mean ± SD (n = 3). (C) A TEM image of PenG-PL NPs. Scale bar = 100 nm.
Drug-conjugated liposomal antimicrobials were used as a
potential strategy to inhibit intracellular pathogens because of
[alkane-based lipid chain (L) and phospholipid chain (PL)]
through a multiple step synthetic route. Briefly, penicillin-G-
derived lipid, PenG-L was prepared by installing two stearoyl
chains to the extended hydroxyl groups on penicillin G (Figure
S1A). Similarly, levofloxacin-derived lipid, Levo-L, was made
by adding two stearoyl chains to the extended hydroxyl groups
on levofloxacin (Figure S1C). PenG-derived phospholipid,
PenG-PL, and levofloxacin-derived phospholipid, Levo-PL,
were synthesized by adding modified dicetyl phosphate to
18−22
their enhanced drug internalization by host cells.
Herein,
we synthesized a series of lipid conjugates using FDA-approved
antibiotics and formulated them to antibacterial nanoparticles
for treating intracellular bacteria. Using the methicillin-
sensitive S. aureus infected lung epithelial cells as an infection
model, we identified that optimized penicillin-G-derived
phospholipid nanoparticles (PenG-PL NPs) were able to
8.5
eliminate up to 99.9998% of ∼10 intracellular S. aureus,
which were significantly more efficacious than free penicillin G
under the same drug concentration. Furthermore, this
enhanced antibacterial effect was consistent with the high
internalization of penicillin G after treatment of PenG-PL NPs
in infected cells. Compared to other antibiotic drug delivery
systems, such as drug-encapsulated liposomal nanoparticles,
antibiotic-derived lipid nanoparticles may provide a more
penicillin G and levofloxacin, respectively (Figure S1B and
1
1
D). All products were confirmed by H NMR and high-
resolution mass spectrum.
Formulation and Characterization of Antibiotic-
Derived Lipid Nanoparticles. The resulting antibiotic−
lipid conjugates were formulated with 2-diphytanoyl-sn-
glycero-3-phosphocholine (DPhPC), cholesterol, and 1,2-
dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-
PEG2000) to prepare the antibacterial nanoparticles through a
18
efficient drug loading and reduce burst release. The results
showed that PenG-PL NPs may be promising to treat
intracellular bacteria in the future.
23
thin-film hydration method. In order to find an appropriate
formulation, we first used PenG-L to prepare the nanoparticles.
Sixteen different PenG-L-based formulations with diverse
molar ratio of four components were made according to the
RESULTS
23
■
formulation table (Table S1). Among these 16 formulations,
Synthesis of Antibiotic-Derived Lipid Conjugates. To
synthesize the antibiotic-derived lipids (Figure 1, Figure S1),
two FDA-approved antibiotics, penicillin G (PenG) and
levofloxacin (Levo), were conjugated with two types of lipids
most formulations displayed two or more particle peaks
(Figure S2A), indicating the particle heterogeneity. Formula-
tion 6 (Figure 2A) and formulation 14 showed a relatively
homogeneous peak. Previous studies reported that cholesterol
1
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2
4
may hinder the disassembly of nanoparticles. Due to the
higher antibiotic percentage and simpler formulation compo-
sition without cholesterol in formulation 6 over 14, the molar
ratio of formulation 6 was selected and further used for
characterization of other antibiotic-derived lipid nanoparticles.
The other three newly synthesized antibiotic−lipid con-
jugates with similar structure as PenG-L were included in the
formulation (Table S2) following the selected molar ratio of
nanoparticle components (phospholipid/antibiotic−lipid con-
jugates/PEG = 45/15/2). The size and distribution were
measured, and these nanoparticles displayed similar properties
as PenG-L NPs (Figure 2B). To study the morphology of the
PenG-PL NPs, a TEM imaging was conducted. As shown in
Figure 2C and Figure S2B, PenG-PL NPs were spherical with a
lipid membrane outside.
drug concentration. Importantly, the inhibition rate of PenG-
PL nanoparticles (PenG-PL NPs) at the lowest tested
concentration (0.17 μg/mL, equivalent penicillin G concen-
tration) was still high, 97.39 ± 3.65%, indicating that PenG-PL
NPs had the strongest antibacterial potential among these
antibiotic-derived lipid nanoparticles. Further statistical test
revealed that the antibacterial effect of PenG-PL NPs was
significantly higher than other formulations. In addition, the
MIC was 0.34−0.68 μg/mL for PenG-PL NPs.
To study the mechanism of antimicrobial activity of PenG-
PL NPs, drug release rate was first measured. PenG-PL NPs
displayed relatively faster drug release rate in the first 3 h when
compared to the other three nanoparticles (Figure S3). The
inhibition test also showed that PenG-PL had the strongest
antimicrobial activity among the four conjugates when directly
incubating with MSSA (Figure S4). These data may explain
the different potency of antimicrobial activities observed in
Figure 3.
Bacterial Growth Inhibition by the Antibiotic-Derived
Lipid Nanoparticles. After characterizing the antibiotic-
derived lipid nanoparticles, we evaluated their antibacterial
effects by incubating them with methicillin-sensitive S. aureus
Inhibition of Intracellular Bacteria by PenG-PL NPs.
Next, we evaluated PenG-PL NPs for their inhibitory effect on
intracellular bacteria in the MSSA-infected A549 cells, a lung
epithelial cancer cell line. The inhibitory effect was determined
(
MSSA) in 96-wells plates. As shown in Figure 3, penicillin G
25
by counting and analyzing the colony forming units (CFUs).
After eliminating the extracellular bacteria by gentamicin, the
number of intracellular bacteria in A549 was determined to be
8
.49±0.15
10
(n = 3) after 20 h of incubation. To compare the
inhibitory effect of free penicillin G and PenG-PL NPs to the
intracellular bacteria, different concentrations of free drug and
nanoparticles were added to the MSSA-infected A549 cells.
The CFU number of each treatment group was quantified
(
Figure S5A), and the percentage losses of log CFU in free
10
PenG-treated groups were less than 10% (Figure 4A).
However, in PenG-PL NP treated groups, the percentage
losses of log CFU were all significantly higher than that of the
Figure 3. Bacterial growth inhibition by the antibiotic-derived lipid
nanoparticles. Comparisons were made between the four nano-
particles at their highest concentration tested. **, PenG-PL vs PenG-
L; ****, PenG-PL vs Levo-L and PenG-PL vs Levo-PL. Mean ± SD
1
0
free drug-treated group, with the highest percentage loss at
7.4 ± 0.6% in the 20 μg/mL (equivalent PenG concen-
tration) group, which was equal to the elimination of
6
(n = 3). (One-way ANOVA with Bonferroni posthoc test.)
8
.5
9
9.9998% of ∼10
bacteria. Meanwhile, no significant
differences of cytotoxicity were found in free PenG and
PenG-PL NPs treated cells (Figure S6). The results suggested
that the enhanced anti-intracellular bacteria effect of PenG-PL
NPs was not relevant to the particle cytotoxicity. Moreover,
these cells were well tolerated with the PenG-PL NPs.
(
free PenG) and levofloxacin (free Levo) completely inhibited
bacterial growth under the tested concentrations. The
inhibition rates of PenG-L nanoparticles (PenG-L NPs),
Levo-L nanoparticles (Levo-L NPs), and Levo-PL nano-
particles (Levo-PL NPs) were all less than 80% at the highest
Figure 4. Growth inhibition of intracellular MSSA by PenG-PL NPs. (A) Inhibitory effect of free penicillin G (free PenG) and PenG-PL NPs on
intracellular bacteria. Mean ± SD (n = 3) (**, p < 0.01, ****, p < 0.0001, t-test, double-tailed). (B) Cellular internalization of free penicillin G
(free PenG) and PenG-PL NPs in A549 cells. Mean ± SD (n = 3) (***, p < 0.001, ****, p < 0.0001, t-test, double-tailed).
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Figure 5. Intracellular trafficking of PenG-PL NPs in MSSA-infected A549 cells. The PenG-PL NPs were labeled with FITC. Lysosomes were
labeled with lysotracker. MSSA was stained with Hoechst33342. Colocalization of PenG-PL NPs and lysosomes was observed. No colocalization of
MSSA and PenG-PL NPs was found. Scale bar = 10 μm.
Next, we quantified cellular uptake of PenG and PenG-PL
NPs (Figure 4B). After incubating cells with free PenG for 1 h,
the PenG uptake was less than 2% of total PenG treatment in
all groups. On the other hand, the percentage of intracellular
PenG uptake in the PenG-PL NPs treatment groups was
significantly higher than that in the free PenG treatment group,
with the highest percentage at 10.1 ± 0.3% in 2.5 μg/mL of the
equivalent PenG treatment group. Although the percentage of
uptake decreased from the more concentrated nanoparticles,
the total amount of internalized PenG was gradually increased
conjugates were formulated with phospholipids and PEG−
lipids to prepare antibiotic-derived lipid nanoparticles. Results
showed that PenG-PL NPs had significantly stronger
antibacterial effect than PenG-L NPs. Then, we quantified
the drug release rate from the nanoparticles. The data
indicated that PenG-PL NPs released the drug much faster
than PenG-L NPs in the early time point. Next, we performed
an inhibition assay using free antibiotic-derived lipid
conjugates. Interestingly, PenG-PL displayed antibacterial
activity, while the other conjugates did not. Previous studies
reported that phospholipids (PLs) helped anchor the antibiotic
(
Figure S5B).
26
To study the intracellular trafficking profile of the nano-
to the bacteria membrane through its lipid part, which may
lead to stronger antimicrobial activities of PenG-PL conjugates
in comparison to PenG-L conjugates. In order to test whether
PenG-PL NPs can be efficiently internalized into cells and kill
the intracellular bacteria, the MSSA-infected lung epithelial
cells were treated with either free penicillin G or PenG-PL NPs
after eliminating the extracellular bacteria. The results showed
that the PenG-PL NPs underwent an endocytic pathway when
uptaken by infected cells. More importantly, the PenG-PL NPs
were more potent than free penicillin G to inhibit intracellular
bacteria. This strong antibacterial effect was consistent to the
observation of increased cellular internalization of PenG-PL
NPs. Meanwhile, these nanoparticles induced little cytotoxicity
to the treated cells. The findings suggested that PenG-PL NPs
were able to efficiently eliminate the intracellular bacteria
MSSA by increasing cellular internalization.
particles, we imaged the MSSA-infected A549 cells after
treating with PenG-PL NPs. The MSSA was stained with
Hoechst33342; the nanoparticles were labeled with FITC; and
the lysosomes were labeled with lysotracker. As shown in
Figure 5, bacteria were found in the cytoplasm. No
colocalization between bacteria and PenG-PL NPs was
noticed. In addition, apparent colocalization was found
between FITC-labeled PenG-PL NPs and lysosomes. These
results indicated that PenG-PL NPs underwent an endocytic
pathway.
DISCUSSION
■
Previous studies reported that free penicillin G can hardly
enter cells to eliminate intracellular bacteria, which leads to
serious complications and makes several infectious diseases
2
hard to cure. In order to address this issue and construct
With this proof-of-concept study, these antibiotic-derived
lipid nanoparticles can be applied to a wide variety of infected
cells, such as macrophages or neutrophils, and in vivo studies in
antibacterial nanoparticles, antibiotic-derived lipid conjugates
were first synthesized, and their structures were confirmed via
NMR and mass spectra. In our experiments, the antibiotics
were conjugated with two forms of lipid chains forming
carbonate and phosphate linkers. These antibiotic−lipid
27−31
bacterial infection models.
Moreover, this conjugation
and formulation approach is applicable to other types of
antibiotics. Since there is an unmet and urgent need for
1
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treating drug-resistant bacteria, we anticipate developing
diverse antibiotics-conjugated nanoparticles for killing intra-
cellular drug-resistant bacteria with a new antimicrobial
reaction was vigorously stirred at 0 °C for 10 min, followed by the
addition of chloromethyl chlorosulfate (160 mg, 0.96 mmol) in DCM
(
4 mL) with continuous vigorous stirring. NaHCO (200 mg, 2.4
3
3
2
33
mmol) was added to the solution, and the reaction was stirred
overnight at room temperature. The organic layer was separated,
washed with brine, and dried with sodium sulfate. The residue was
purified by flash silica gel column chromatography using ethyl
hexane/acetate (85:15, v/v) to give the product (2) in a white solid
mechanism or a combinational strategy.
CONCLUSION
■
In this study, we designed and synthesized antibiotic-derived
lipid conjugates and then developed a series of antibiotic-
derived lipid nanoparticles. Among them, the PenG-PL NPs
incorporating a phosphate linker of antibiotic−lipid conjugates
formed relatively homogeneous particle distribution and
displayed strong antibacterial effects in cells. PenG-PL NPs
enhanced the cellular internalization of penicillin G and
efficiently inhibited the intracellular bacteria in the MSSA-
infected lung epithelial cells. Our study demonstrated the
potential of using antibiotic-derived lipid nanoparticles to treat
intracellular bacteria.
(
236 mg, yield: 49.6%). Penicillin G (100 g, 0.27 mmol) and 2 (80
mg, 0.13 mmol) were dissolved in 8 mL of dry DMF and stirred at
3
5−40 °C for 4 h. The mixture was dissolved in CH Cl and washed
2
2
with water, and then the organic layer was dried with sodium sulfate.
The crude product solution was concentrated in vacuum. The residue
was purified by column chromatography with EA/PE (30:70, v/v) to
give the phosphate (PE) product in a white solid (35 mg, yield
1
29.2%). H NMR (400 MHz, CDCl
) d 7.34 (m, 5H, Ph), 6.18 (d,
3
1
H, CHS), 5.61−5.75 (m, 3H, OCH O, CHNH), 5.51 (m, 1H,
2
CHN), 4.40 (b, 1H, NH), 4.05 (m, 4H, 2CH O), 3.64 (s, 2H,
2
CH Ph), 1.49 (m, 4H, 2CH ), 1.45 (d, 6H, 2CH ), 1.27 (m, 52H,
2
2
3
+
2
6CH ), 0.90 (m, 6H, 2CH ). MS (m/z): [M + H] calcd for
C H N O PS, 893.5842; found, [M + H] = 893.5839.
2
3
+
4
9
86
2
8
EXPERIMENTAL SECTION
Materials. Penicillin G sodium salt was purchased from Alfa Aesar
Tewksbury, MA). Levofloxacin was purchased from Ark Pharm
Arlington Heights, IL). 1,2-Diphytanoyl-sn-glycero-3-phosphocho-
Synthesis of Levo-L. Levofloxacin (300 mg, 0.83 mmol) was
dissolved in 15 mL of CH Cl . Then 2,2-dimethyl-1,3-dioxolane-4-
■
2
2
methanol (132 mg, 1 mmol), trimethylamine (303 mg, 3 mmol), and
HBTU (380 mg, 1 mmol) were added successively. The reaction
mixture was stirred at room temperature for 72 h. The mixture was
then concentrated in vacuum, and the residue was purified by flash
silica gel column chromatography to give the product (3) (210 mg,
yield: 44.21%). An amount of 120 mg of product 3 was dissolved in 4
mL of DMC. The solution was cooled to 0 °C, and 200 μL of
trifluoroacetic acid was added (solution turned yellow). The mixture
was stirred at 0 °C for 1 h and then stirred at room temperature for 3
(
(
line (DPhPC) was purchased from Avanti Polar Lipids. 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
purchased from Amresco (Solon, OH). Other chemicals and solvents
were purchased from Sigma-Aldrich (St. Louis, MO).
Bacteria and Cell Line. Methicillin-sensitive Staphylococcus aureus
subsp. Aureus Rosenbach strain (MSSA, ATCC 6538) was obtained
from the ATCC and cultured in tryptic soy broth/agar (BD) at 37 °C.
The lung epithelial cancer cell line (A549, ATCC CCL-185) was
cultured in Ham’s F-12K (Kaighn’s) (Thermo Fisher, Waltham, MA)
supplemented with 10% inactivated fetal bovine serum (FBS)
h. The NaHCO and 5 drops of water were added, and the solution
3
turned no color. The solution was dried by sodium sulfate to give the
product 4 (35 mg, yield: 32.11%). Product 4 (20 mg, 0.046 mmol)
was dissolved in 2 mL of CH Cl . Triethylamine (33 mg) and 4-
(
Thermo Fisher) at 37 °C in humidified atmosphere containing 5%
2
2
(
N,N-dimethylamino) pyridine (1 mg) were then added to the
CO2.
solution. The reaction mixture was cooled to 0 °C, and then stearoyl
chloride (38 mg) was added dropwise. The reaction was warmed to
room temperature and quenched after 4 h by pouring over saturated
Synthesis and Characterization of Antibacterial Drug−Lipid
Conjugates. In order to prepare the antibiotic-derived lipid
nanoparticles, four antibiotic−lipid conjugates were afforded. These
conjugates are amphiphilic materials, which can be formed with
phospholipid, cholesterol, and PEG to construct stable lipid
nanoparticles.
aqueous NaHCO
organic layer was washed successively with saturated aqueous
NaHCO , water, and brine and then dried with sodium sulfate. The
crude product was concentrated in vacuum and was purified by flash
column chromatography with CH Cl
(5 mL). The aqueous layer was separated, and the
3
3
Synthesis of PenG-L. Penicillin G sodium salt (100 mg, 0.3 mmol)
and 3-bromo-1,2-propanediol (156 g, 1 mmol) were dissolved in 2
mL of DMF. The mixture was stirred at 35 °C for 24 h. CH Cl and
/methanol (100:0−90:10, v/v)
1
2
2
to give the product (14 mg, yield: 31.4%). H NMR (400 MHz,
2
2
water were then added to the reaction mixture. The organic phase was
collected and dried with sodium sulfate. The crude product was
purified by column chromatography with CH Cl /CH OH (98:2, v/
CDCl ) d 8.31 (d, 1H, Ph), 7.70 (dd, 1H, Ar), 5.43 (m, 1H, CHO),
3
4.30−4.51 (m, 7H, 3CH O, CHN), 3.41 (m, 4H, 2CH N), 2.68 (m,
2
2
4H, 2CH N), 2.44 (s, 3H, CH N), 2.35 (m, 2CH C(O), 4H), 1.57−
2
2
3
2
3
2
v) to give the product (1) (30 mg, yield 26.3%). Triethylamine (22
mg, 0.22 mmol) and 4-(N,N-dimethylamino) pyridine (1 mg) were
added to the solution of 1 (40 mg, 0.1 mmol) in CH Cl (2 mL). The
1.63 (m, 7H, 2CH , CH ), 1.27 (m, 56H, 28CH ), 0.90 (t, 6H,
2
3
2
+
2CH ). MS (m/z): [M + H] calcd for C H FN O , 968.7103;
3
57 95
3
8
found, 968.7151.
2
2
reaction mixture was cooled to 0 °C, and then stearoyl chloride (75.5
mg, 0.25 mmol) was added dropwise. The reaction was warmed to
room temperature (RT) and quenched after 4 h by pouring over
saturated aqueous NaHCO3 (5 mL). The aqueous layer was
separated, and the organic layer was washed successively with
Synthesis of Levo-PL. Following the same procedures as PenG-PL,
the last reaction was performed at 90 °C for 2 h. The product is 14.4
1
mg, yield 15.7%. H NMR (400 MHz, CDCl ) d 8.46 (s, 1H, Ph),
3
7.76 (d, 1H, Ar), 5.87 (m, 1H, CHN), 4.40 (m, 3H, OCH , CHN),
2
4.08 (m, 4H, 2CH O), 3.41 (m, 4H, 2CH N), 2.58 (m, 4H, 2CH N),
2
2
2
saturated aqueous NaHCO , water, brine, and then dried with sodium
2.40 (s, 3H, NCH ), 1.58 (m, 4H, 2CH ), 1.28 (m, 52H, 26CH ),
3
3
2
2
+
sulfate. The crude product was purified by column chromatography to
0.90 (m, 6H, 2CH ). MS (m/z): [M + H] calcd for C H FN O P,
3
51 88
3
8
1
give the PenG-L in a white solid (100 mg, yield 33.4%). H NMR
920.6293; found, 920.6273.
(
1
(
400 MHz, CDCl ) d 7.38 (m, 5H, Ph), 6.06 (d, 1H, CHS), 5.69 (m,
Preparation and Characterization of Antibiotic-Derived
3
H, CHNH), 5.52 (m, 1H, CHN), 5.30 (s, 1H, CHN), 4.14−4.41
Lipid Nanoparticles. The antibiotic-derived lipid nanoparticles
23
m, 5H, 2CH O, CHO), 3.66 (s, 2H, CH Ph), 2.32 (t, 4H,
(NPs) were prepared by the thin-film dispersion method. To
prepare PenG-L NP formulations, DPhPC, cholesterol, antibiotic−
lipid conjugates, and 1,2-dimyristoyl-rac-glycero-3-methylpolyoxy-
ethylene (DMG-PEG2000)2 were dissolved in chloroform at 16
2
2
2
5
CH C(O)), 1.62 (m, 4H, 2CH ), 1.45 (d, 6H, 2CH ), 1.28 (m,
2 2 3
+
6H, 28CH ), 0.90 (t, 6H, 2CH ). MS (m/z): [M + Na] calcd for
2
3
C H N O SNa, 963.6472; found, 963.6469.
55
92
2
8
3
Synthesis of PenG-PL. Dicetyl phosphate (440 mg, 0.8 mmol),
different ratios (Table S1). The solvent was then removed at 50°
sodium bicarbonate (84 mg, 1 mmol), and tetra-n-butylammonium
hydrogen sulfate (272 mg, 0.8 mmol) were dissolved in 8 mL of
water. Dichloromethane (8 mL) was then added to the mixture. The
by a rotary vacuum evaporator (Hedolph G5, Schwabach, Germany).
The remaining lipid film was hydrated with H O using water bath
2
sonication for 1 min and further homogenized by ultrasonication at 50
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W, 20 kHz (QSonica, Newtown, CT) for 5 min. The theoretical
concentration of antibacterial drug−lipid conjugates was 160 μg/mL.
Particle size distribution of the NPs was determined using a NanoZS
Zetasizer (Malvern, Worcestershire, U.K.). FITC-labeled PenG-PL
NPs were prepared using the same method to prepare the PenG-PL
NPs with addition of fluorescein (fluorescein to total lipid, 1:200, w/
w) when dissolving the formulation components in chloroform.
For TEM imaging, samples were prepared by applying a small
aliquot (3 μL) of PenG-PL NPs on carbon-coated copper grids. After
blotting away excess liquid, the grids were air-dried. Electron
micrographs were collected under STEM mode on a Tecnai F20 S/
TEM (Thermo Fisher Scientific, Hillsboro) with a high-angle angular
dark-field (HAADF) detector. The microscope was operated at an
acceleration voltage of 200 kV.
h. Then the cells were washed with F12K containing 0.5% FBS and
treated with gentamicin (50 μg/mL) in F12K for 2 h to eliminate the
extracellular bacteria. A549 cells were then washed with F12K 0.5%
FBS three times and incubated with penicillin G or antibacterial NPs
at 37° for 1 h. After incubation, the cells were washed with F12K 0.5%
FBS again and incubated with full media at 37° for 18 h. The
intracellular bacteria were then fully released by adding Triton X-100
0.1% (PBS, v/v) at 37° for 10 min and then counted by serial dilution
and the pour plate technique. Percentage loss of log10(colony forming
units, CFU) = log10(CFU of untreated control/CFU of treated
group)/log10(CFU of untreated control) × 100%.
Comparison of Cytotoxicity between Antibiotic-Derived
Lipid Nanoparticles and Free Drug. To test if cells treated after
PenG-PL NPs or penicillin G can still be intact, the cytotoxicity of
these two formulations was evaluated. A549 cells were seeded at 5000
cells/well in a 96-well plate. After 24 h of incubation, the free PenG or
PenG-PL NPs were added. Following 18 h of further incubation, the
cytotoxicity of both treatment groups was analyzed by MTT assay.
Briefly, 20 μL of MTT was added to 200 μL of treated cells. After 4 h
of incubation at 37°, the media was removed, and 150 μL of DMSO
was added. The absorbance was then recorded at 570 nm. The cell
viability of A549 cells was calculated using the following formula: cell
The encapsulation efficiency (EE%) of drug−lipid conjugates was
calculated using the formula: EE% = (W /W ) × 100%, where
encap
total
the Wencap is the measured amount of conjugates in the antibiotic-
derived lipid nanoparticle precipitate after centrifuging the freshly
made nanoparticle solution at 10 000 rpm for 20 min, and the Wtotal is
the measured amount of conjugates in an equal amount of
nanoparticle solution. The NPs were disrupted by CHCl , and the
3
concentration of drug−lipid conjugates was quantified by UV
absorbance at 215 nm with the standard curve (Figure S7) (Molecular
Devices, Sunnyvale, CA). The encapsulation efficiency of PenG-PL in
PenG-PL NPs was 48.2 ± 3.9% (n = 3).
We then quantified drug release rate by measuring the remaining
amount of antibiotic−lipid conjugates after incubating the nano-
particles in water solution at 37°. The rationale is that PenG-derived
phospholipid, PenG-PL, will be hydrolyzed by esterase after cell
internalization. Thus, drug release was calculated by subtracting the
remaining PenG-PL conjugates from the total conjugates in the
solution. After 0, 3, 6, and 24 h, chloroform was added to extract the
remaining antibiotic lipid conjugates. The organic phase and the water
phase were then separated by centrifuge at 10 000g for 10 min. Next,
the chloroform phase was transferred and mixed with methanol
viability % = (A570 nm for treated cells − A
blank well)/ (A570 nm
570 nm
blank well) × 100%.
for nontreated cells − A
570 nm
Intracellular Uptake of PenG-PL NPs in A549 Cells. To
further study the internalization of PenG-PL NPs in A549 cells, the
intracellular PenG was quantified using the mass spectrum (MS, LTQ
5
Orbitrap XL, Thermo Scientific). The A549 cells were seeded at 10
cells/0.5 mL/well in a 24-well plate for 24 h. The cells were then
washed with the PBS, and free PenG or PenG-PL NPs in F12K 0.5%
FBS were added. After incubation for 1 h, the cells were washed with
PBS and then disrupted with 0.5 mL of ice cold water for 30 min. For
the PenG-PL NPs treated group, the cell solution was transferred to a
1
.5 mL tube, and 0.5 mL of CHCl was added to disrupt the NPs. The
3
mixture was then centrifuged to separate the water/CHCl layer, and
3
1
00 μL of the CHCl layer was diluted with 900 μL of methanol for
3
(
(
chloroform:methanol = 1:1, v/v) for MS quantification. Drug release
%) = (1 − (wtconjugates at different time points/wtconjugates at 0 h)) × 100%.
Antibacterial Effect of Antibiotic-Derived Lipid Nano-
MS measurement. For the free PenG-treated group, after the cell
disruption, 0.5 mL of methanol was added. The mixture was then
filtered with a 0.22 μm membrane and diluted with methanol for
further MS measurement.
particles. The antibacterial effect was analyzed by the turbidity
(
34
OD600 nm) after incubating the NPs with MSSA. The MSSA was
5
Intracellular Trafficking Profile of the Nanoparticles in
MSSA-Infected A549 Cells. The intracellular trafficking profile of
the nanoparticles in infected A549 cells was determined by the
confocal imaging. A549 cells were seeded on coverslips in a 6-well
first prepared at ∼10 bacterial cells/well in the 96-well plate. After 2-
fold serial dilution, each newly formulated antibacterial NP was added
to the bacteria suspension, and the final concentration of each drug
was determined by the formula: drug concentration (μg/mL) =
MWfree drug/MWdrug‑lipid conjugates × EE% × 160 μg/mL. The NP-treated
bacteria plates were then incubated at 37° with continuous shaking at
5
plate at a density of 3 × 10 cells/2 mL/well and were cultured for 24
h at 37°. Then, the cells were washed with F12K 0.5% FBS media.
6
MSSA suspension containing approximate 10 bacteria were added to
1
50 rpm for 24 h. After incubation, the inhibition effect of
antibacterial NPs was evaluated by a plate reader at OD600 nm
SpectraMax M5, Molecular Devices, LLC., Sunnyvale, CA). When
studying the bacterial growth inhibition by the antibiotic-derived lipid
conjugates, drug concentration (μg/mL) = MWfree drug
A549 cells at MOI = 10 (multiplicity of infection) and incubated with
the cells for 2 h. Next, the cells were washed with F12K containing
(
0.5% FBS and treated with FITC-labeled PenG-PL NPs for 2 h. A549
cells were then washed with F12K 0.5% FBS three times and
incubated with lysotracker and Hoechst33342 at 37° for 15 min. After
incubation, the cells were fixed with 4% paraformaldehyde and
washed with PBS for confocal imaging (Nikon A1R, Melville, NY).
Statistics. Data were presented as the mean ± standard deviation.
The GraphPad Prism 6 (La Jolla, CA) was used to analyze the data.
The double-tailed student t-test and one-way ANOVA were used in
different experiments to determine the significance among groups. A
value of p < 0.05 was considered to be statistically significant.
/
MWdrug‑lipid conjugates × Conc.drug‑lipid conjugates. The inhibition rates of
MSSA were calculated using the following formula: inhibition rate %
=
(1 − A6
for treated bacteria cells/A600 nm for nontreated control
00 nm
bacteria cells) × 100%. To measure the minimum inhibitory
concentration (MIC) of drug-loaded NPs and free drug, the pour
plate method was used. Briefly, the colony forming units of drug-
treated bacteria were determined by serial diluting and plating on
tryptic soy agar (BD) followed by incubation overnight at 37 °C. The
MIC was determined by the lowest concentration of drugs that
completely inhibited the growth of bacteria on the agar plate.
Inhibition of Intracellular Bacteria by Antibiotic-Derived
Lipid Nanoparticles. To quantify anti-intracellular bacteria effects,
MSSA-infected A549 cells were treated with antibiotic-derived lipid
nanoparticles, and then the intracellular bacteria were quantified by
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsabm.8b00821.
■
*
S
Figure S1. Synthetic routes to antibiotic-derived lipid
conjugates. Figure S2. Size distribution of 16 penicillin-
G-derived lipid nanoparticles (PenG-L NPs), and TEM
images of PenG-PL NPs. Figure S3. Drug release rate of
penicillin-G-derived phospholipid nanoparticles (PenG-
the pour plate method. A549 cells were seeded in a 24-well plate at
5
1
0 cells/0.5 mL/well and allowed to grow for 24 h at 37°. Then the
cells were washed with F12K 0.5% FBS media. MSSA suspension
6
containing approximate 10 bacteria cells were added to A549 cells at
MOI = 10 (multiplicity of infection) and incubated with the cells for 2
1
275
DOI: 10.1021/acsabm.8b00821
ACS Appl. Bio Mater. 2019, 2, 1270−1277

ACS Applied Bio Materials
Article
(
4) Lawes, T.; Lopez-Lozano, J. M.; Nebot, C. A.; Macartney, G.;
PL NPs), PenG-L NPs, levofloxacin-derived phospholi-
pid nanoparticles (Levo-PL NPs), and levofloxacin-
derived lipid nanoparticles (Levo-L NPs). Figure S4.
Bacterial growth inhibition by the antibiotic-derived
lipid conjugates. Figure S5. Inhibition of intracellular
bacteria by PenG-PL NPs. Figure S6. Cytotoxicity of
free penicillin G and PenG-PL NPs on A549 cell. Figure
S7. Standard curve of PenG-PL absorbance at 215 nm.
Table S1. Formulation components of 16 penicillin-G-
derived lipid nanoparticles. Table S2. Formulation
components of antibiotic-derived lipid nanoparticles
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Corresponding Authors
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013, 3, 261−266.
*Dr. Xiaofang Chen: chenchemistry@hotmail.com
Dr. Yizhou Dong: dong.525@osu.edu.
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ORCID
Weiyu Zhao: 0000-0003-3345-4925
Yizhou Dong: 0000-0001-5786-0659
Author Contributions
C.Z., X.C., and Y.D. conceived and designed the experiments;
C.Z., W.Z., C.B., X.H., B.D., D.M., and X.C. performed the
experiments; C.Z., X.C., and Y.D. analyzed the data; C.Z., X.C.,
X.H., and Y.D. wrote the paper with edits and comments from
all authors. All authors have given approval to the final version
of the manuscript.
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for Intracellular Persistence. Trends Microbiol. 2009, 17, 59−65.
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Funding
(13) Xiong, M. H.; Bao, Y.; Yang, X. Z.; Zhu, Y. H.; Wang, J.
This work was supported by CAMS Innovation Fund for
Medical Sciences (2017-I2M-1−012) and the Drug Innovation
Major Project of China (2018ZX09711001-007-002) for Dr.
Xiaofang Chen, and by the Maximizing Investigators’ Research
Award 1R35GM119679 from the National Institute of General
Medical Sciences as well as the start-up fund from the College
of Pharmacy at The Ohio State University for Dr. Yizhou
Dong.
Delivery of Antibiotics with Polymeric Particles. Adv. Drug Delivery
Rev. 2014, 78, 63−76.
(14) Liu, L.; Xu, K.; Wang, H.; Tan, P. K.; Fan, W.; Venkatraman, S.
S.; Li, L.; Yang, Y. Y. Self-Assembled Cationic Peptide Nanoparticles
as an Efficient Antimicrobial Agent. Nat. Nanotechnol. 2009, 4, 457−
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63.
(15) Lam, S. J.; O’Brien-Simpson, N. M.; Pantarat, N.; Sulistio, A.;
Wong, E. H. H.; Chen, Y.-Y.; Lenzo, J. C.; Holden, J. A.; Blencowe,
A.; Reynolds, E. C.; Qiao, G. G. Combating Multidrug-Resistant
Gram-Negative Bacteria with Structurally Nanoengineered Antimi-
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Notes
The authors declare no competing financial interest.
(
16) Onyeji, C. O.; Nightingale, C. H.; Marangos, M. N. Enhanced
ACKNOWLEDGMENTS
■
Killing of Methicillin-Resistant Staphylococcus aureus in Human
Macrophages by Liposome-Entrapped Vancomycin and Teicoplanin.
Infection 1994, 22, 338−342.
C.Z. acknowledges the support from the Professor Sylvan G.
Frank Graduate Fellowship. Electron microscopy was
performed at the Center for Electron Microscopy and Analysis
(17) Gao, W.; Chen, Y.; Zhang, Y.; Zhang, Q.; Zhang, L.
Nanoparticle-Based Local Antimicrobial Drug Delivery. Adv. Drug
Delivery Rev. 2018, 127, 46−57.
(
CEMAS) at The Ohio State University. Confocal images were
generated using the instruments and services at the Campus
Microscopy and Imaging Facility, The Ohio State University.
This facility is supported in part by grant P30 CA016058,
National Cancer Institute, Bethesda, MD.
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