Hyaluronic acid-based levofloxacin nanomicelles for nitric oxide-triggered drug delivery to treat bacterial infections

Article abstract of DOI:10.1016/j.carbpol.2019.115479

Antibiotics are powerful weapons to fight against bacterial infections, while most of them lack of selective targeting towards pathological site which could restrict their antibacterial efficacy. To overcome this challenge, an antimicrobial levofloxacin(L

Full text of DOI:10.1016/j.carbpol.2019.115479

Journal Pre-proof
Hyaluronic acid-based levofloxacin nanomicelles for nitric oxide-triggered
drug delivery to treat bacterial infections
Chunbo Lu, Yao Xiao, Yinyin Liu, Feifei Sun, Yuanhao Qiu, Haibo
Mu, Jinyou Duan
PII:
S0144-8617(19)31147-6
DOI:
https://doi.org/10.1016/j.carbpol.2019.115479
CARP 115479
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Carbohydrate Polymers
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Revised Date:
Accepted Date:
13 June 2019
14 October 2019
14 October 2019
Please cite this article as: Lu C, Xiao Y, Liu Y, Sun F, Qiu Y, Mu H, Duan J, Hyaluronic
acid-based levofloxacin nanomicelles for nitric oxide-triggered drug delivery to treat bacterial
infections, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115479
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Hyaluronic acid-based levofloxacin nanomicelles for nitric
oxide-triggered drug delivery to treat bacterial infections
Chunbo Lu,† Yao Xiao,† Yinyin Liu, Feifei Sun , Yuanhao Qiu, Haibo Mu*, Jinyou
Duan *
Author Affiliations:
College of Chemistry & Pharmacy, Shaanxi Key Laboratory of Natural Products &
Chemical Biology, Northwest A&F University, Yangling 712100, Shaanxi, China
† These authors contributed equally to this work.
*Corresponding author:
E-mail addresses: mhb1025@nwsuaf.edu.cn (H. Mu), jduan@nwsuaf.edu.cn (J.
Duan).
Tel.: +86 29 87092226; Fax: +86 29 87092226
Highlights:
 Levofloxacin conjugated hyaluronic acid was synthesized by
o-phenylenediamine group linkage.
 HA-NO-LF nanomicelles could enter macrophages via a CD44
mediated endocytosis.
 HA-NO-LF nanomicelles showed better bactericidal effects against S.
aureus than LF in vivo.
Abstract
Antibiotics are powerful weapons to fight against bacterial infections, while most
of them lack of selective targeting towards pathological site which could restrict their
antibacterial efficacy. To overcome this challenge, an antimicrobial levofloxacin（LF）
1

was conjugated onto hyaluronic acid (HA) moieties via an o-phenylenediamine linker
to prepare a NO-sensitive nanosystem (HA-NO-LF) in this study. The HA-NO-LF
nanomicelles could enter host cells via a CD44 mediated endocytosis and release drug
gradually upon exposure to endogenous NO. Furthermore, the more promising
therapeutic effect of the nanomicelles in ameliorating inflammatory levels was
observed in a mouse pneumonia model than that of LF. These results suggest that the
HA-NO-LF nanomicelles may exert potent curative effect in infectious diseases.
Keywords: Hyaluronic acid; NO-sensitivity; Nanomicelles; Drug release;
1. Introduction
Infectious diseases caused by pathogenic microorganisms have long been a
prevalent threat to human health (Gao, Thamphiwatana, Angsantikul, & Zhang, 2014).
The discovery and application of antibiotics has created a new era of human
resistance to infectious diseases. It is well-documented that antibiotics are powerful
weapons to fight against bacterial infections due to their high sterilization efficiency,
broad spectrum applications, and low cost. However, most of them are effective only
at a very high dose owing to the lack of selective targeting towards pathological sites,
which could not only induce multiple drug resistance in microbes but also cause side
effects to other tissues and organs (Chen et al., 2018). Accordingly, to combat
infectious diseases efficiently, there is an urgent need for antimicrobial agents to
achieve targeted delivery and controlled drug release towards the lesion sites, thus
improving therapeutic effects and avoiding tissue injury or organ dysfunction.
Hyaluronic acid (HA), known as a natural linear polysaccharide with outstanding
biodegradability, low toxicity and high water-affinity, has been extensively applied in
biomedical field. Besides, the presence of multiple acid and hydroxyl groups in the
HA molecules makes it an ideal candidate for chemical modification. Recently,
scientists have paid more attention to HA as a drug carrier since it can bind to CD44
on the surface of certain cells (Huang & Huang, 2018). Specifically, CD44 is a type I
transmembrane glycoprotein that is highly expressed on endothelial and mesenchymal
2

cells, especially in inflamed tissues (McDonald & Kubes, 2015). During the process
of pathogenic attack, inflammatory reactions occur as an adaptive defense response in
the organism. In this way, HA is expected to achieve targeted drug delivery as a
carrier of antibiotics, thus increasing the effective dose of drug at inflammatory site
and minimizing the side-effects due to overusing of antibiotics.
Recently, the functional polymer-based nanosystem has been elegantly designed
to enable responsiveness to biological signaling molecules including reactive oxygen
species (ROS) (Xu et al., 2017), hydrogen sulfide (H2S) (Ma, Zheng, Zhang, & Xing,
2018) and nitric oxide (NO) (Yeo et al., 2019). It has been scientifically proven that
macrophages secret a large amount of NO by activating an inducible nitric oxide
synthases (NOS) enzyme in inflammation process (Hoey, Grabowski, Ralston,
Forrester, & Liversidge, 1997). Appropriate levels of NO influences critically
important physiological activities including apoptosis, antisepsis and inflammatory
regulation, while excessive NO is thought to be involved in the pathogenesis of severe
diseases such as rheumatoid arthritis (RA), hypertension and cancer (Coussens &
Werb, 2002). In that sense, harnessing endogenous NO to cleave synthetic HA-based
nanosystem will open up great potential not only for triggered ‘‘on-demand” drug
release but also removal of redundant NO radicals to prevent tissue damage.
Herein, to realize targeted delivery and controlled release of antibiotics, the NO-
sensitive HA-based nanomicelles were developed via modification with NO-reactive
moieties. It has been reported that the o-phenylenediamine group has been frequently
utilized as a NO-sensitive unit due to its highly reactive nature toward NO (J. Hu et al.,
2014; Jinming Hu, Whittaker, Yu, Quinn, & Davis, 2015; Park, Pramanick, Kim, Lee,
& Kim, 2017). Levofloxacin (LF), a third-generation fluoroquinolone antibiotic
effective against both Gram-positive and Gram-negative bacteria was selected as a
model antibiotic in this study. The hypothesized NO-triggered nanomicelles
(HA-NO-LF) were prepared by linking a hydrophilic HA chain and a lipophilic LF
chain via a functionalized intermediate bearing o-phenylenediamine groups, followed
3

by self-assembly in aqueous condition. When encountered with NO, the
o-phenylenediamine moieties could be transformed into benzotriazole moieties in a
highly efficient manner, facilitating chain scission and propelling release of LF
gradually. The properties of the nanomicelles including morphology, biocompatibility,
NO-sensitive behavior as well as antibacterial effects were investigated in vitro. To
further evaluate the therapeutic efficacy in vivo, pneumonia mice infected by
Staphylococcus aureus were selected as the infectious model.
Fig.1 Schematic illustration of the proposed NO-triggered hyaluronic acid- levofloxacin
nanomicelles targeting infection site.
2. Experimental and methods
2.1. Materials
Sodium hyaluronate (10 kDa) was bought from QuFu GuangLong Biochem Co.,
Ltd. Tetrabutylammonium hydroxide (TBAH), 3-nitro-4-amino-phenol (NA)
1,3-dibromopropane (DP), levofloxacin (LF) were purchased from Aladdin Chemical
Reagent Company, China. Nω-Nitro-L-arginine methyl ester hydrochloride
4

(L-NAME), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT),
glutaraldehyde solution were obtained from Sigma Co. (St. Louis, MO, USA). All of
other chemical reagents and solvents were of analytical grade and were used without
further purification.
Staphylococcus aureus（ATCC 29213）and RAW 264.7 cells were generous gifts
received from Prof. Xia (College of Food Science and Engineering, Northwest A&F
University). Kunming mice were obtained from Experimental Animal Center of Basic
Medical College, Xi’an Jiaotong University, China. This study was performed with
the approval of the Experimental Animal Manage Committee (EAMC) of Northwest
A&F University. Animals were treated as the guidelines of EAMC.
2.2. Preparation of HA-NO-LF conjugates
2.2.1. Synthesis of NA-DP
NA-DP was synthesized according to a reported procedure with slight
modifications (Jinming Hu et al., 2015). NA (385 mg, 2.5 mmol) was previously
dissolved in 50 mL acetone in a conical flask equipped with a magnetic bar, then
K2CO3 (345 mg, 2.5 mmol) and DP (606 mg, 3 mmol) were added respectively. The
reaction continuously proceeded for 36 h at 80 °C with gentle agitation. Further, the
crude product was purified using silica gel column chromatography with ethyl
acetate/petroleum ether as eluent to yield a red solid (526 mg, 76.5%) named NA-DP.
1H NMR (500 MHz, d-DMSO): δ = 7.38 (d, J = 8.0 Hz, 1H), 7.24 - 7.16 (m, 2H),
7.00 (d, J = 8.5 Hz, 1H), 4.02 (t, J = 6.0 Hz, 2H), 3.65 (t, J = 6.0 Hz, 2H), 2.24 - 2.19
(m, 2H).
2.2.2. Synthesis of LF-NA-DP
LF-NA-DP conjugates were prepared by chemical grafting chlorinated
levofloxacin (LF-Cl) to NA-DP through amide formation as previously described with
minor modifications (Jinming Hu et al., 2015; Tomišić, Kujundžić, Krajačić,
Višnjevac, & Kojić-Prodić, 2002). In detail, to obtain LF-Cl, LF (722 mg, 2 mmol)
was dissolved in 50 mL anhydrous CH2Cl2, and an appropriate amount of SOCl2 was
5

added dropwise in an ice bath, followed by stirring overnight under N2 atmosphere.
Eventually, the mixture was concentrated under reduced pressure using a rotary
evaporator at 45 °C to remove unreacted CH2Cl2 in dispersions. The freshly prepared
LF-Cl (380 mg, 1 mmol) and moderate content of DIEA were then added to CH2Cl2
solution containing NA-DP (330 mg, 1.2 mmol) and stirred overnight at room
temperature to ensure complete reaction. And the resulting solution was passed
through a silica gel column chromatography (eluted with CH2Cl2/CH3OH) to give the
1
pure LF-NA-DP (422 mg, yield: 68.3%). H NMR (500 MHz, d-DMSO): δ = 12.94
(s, 1H), 8.94 (s, 1H), 8.38 (d, J = 8.5 Hz, 1H), 7.65 (s, 1H), 7.44 (d, J = 8.5 Hz, 1H),
4.93 (s, 1H), 4.60 (d, J = 10.5 Hz, 1H), 4.22 (d, J = 10.5 Hz, 1H), 4.22 (s, 2H), 3.72 (s,
2H), 3.00-2.86 (m, 4H), 2.61 (s, 2H), 2.53 (s, 5H), 2.32-2.94 (m, 2H), 1.48 (d, J = 4.5
Hz, 3H) .
2.2.3. Synthesis of HA-TBAH
Following a reported method (Sun et al., 2019), sodium hyaluronate was
dissolved in distilled water to meet designated concentrations (50 mg/mL) and treated
with a cationic-exchange resin (H+ form). Then the above solution was adjusted to the
desired pH 7 by TBAH, resulting in the ideal product HA-TBAH.
2.2.4. Synthesis of HA-NO-LF
The lyophilized HA-TBAH (200 mg) was dissolved in dry DMSO, followed by
dropping of LF-NA-DP (278 mg, 0.45 mmol) in the mixture. The reaction was
incubated for 48 h at room temperature under stirring, and the resulting solution was
dialyzed (MWCO 3500 Da) exhaustively against distilled water and then 0.1 M NaCl
to remove impurities. Finally, the polymer solutions were lyophilized to obtain a
sponge-like solid and stored at 4 °C for further use. Moreover, the solid above was
redissolved in 20 mL of distilled water, after sequential addition of methanol and
acetic acid at an appropriate amount, zinc powder was progressively added under
stirring vigorously. The mixture was stirred for a further 1 h after the solution turned
colorless to ensure the successful synthesis of NO-triggered conjugates. Then the final
6

product was purified and collected following the same procedure as conducted before,
which was designated as HA-NO-LF.
LF content in the nanomicelles was determined by UV-Vis measurements as
detailed described in supplementary information.
Fig.2 Synthetic routes of HA-NO-LF conjugates.
2.3. Characterization of HA-NA-LF conjugates
The UV−Vis spectra of HA, LF and HA-NO-LF were acquired on a Thermo
Evolution 300 spectrophotometer in the range of 250-450 nm, respectively.
13
1H NMR and C NMR spectra of the prepared conjugates were measured on a
Bruker AM 500 spectrometer with reference to the chemical shift of the solvent peak.
Chloroform (CDCl3), deuterium oxide (D2O) and d-DMSO were used as the solvents,
depending on the particular substance being analyzed. FT-IR spectrophotometer
(BRUKER TEMSOR 27, Germany) was employed to determine the intramolecular
interactions between different components in the prepared conjugates with a
resolution of 4 cm-1, scanning from 500 to 4000 cm-1 wavenumber.
2.4. Preparation and characterization of HA-NO-LF nanomicelles
2.4.1. Preparation of HA-NO-LF nanomicelles
7

In an attempt to prepare HA-NO-LF nanomicelles, 20 mg of HA-NO-LF
conjugates were re-dissolved in 15 mL of DMSO. Then the solution was sonicated for
5 min using an ultrasonic cleaner (SB 5200-D; Ningbo Scientz Biotechnology Co.,
Ltd, Nanjing, China) at 200 W to ensure the uniform dispersion of nanoparticles.
Finally, the solution was dialyzed against deionized water by using a dialysis bag with
3500 Da molecular cutoff for 3 days to obtain HA-NO-LF nanomicelles.
2.4.2. Characterization of HA-NO-LF nanomicelles
The particle size and zeta potential of freshly prepared nanomicelles were
measured by dynamic light scattering (DLS) using a Malvern Nano ZetaSizer ZS
model (Malvern Zetasizer NANOZS90, Malvern Instruments, Ltd., UK). Prior to each
measurement, the samples were diluted with pure water to meet optimal instrument
sensitivity. In addition, the stability of prepared nanomicelles stored in refrigerator at
4 °C were checked for particle size and zeta potential at predetermined intervals (0,
0.5, 1, 2, 3, 4, 5 and 6 days). The morphology of the HA-NO-LF micelles was
characterized by Hitachi S-4800 field emission scanning electron microscopy (SEM)
with an accelerating voltage of 10 kV (Hitachi, Japan).
2.5. In vitro drug release behavior
A dialysis membrane method was employed for quantitatively analyzing the
kinetic release profile of LF from nanomicelles. Briefly, PBS buffer saturated with
NO was prepared as release medium for LF(Liu, Hu, Whittaker, Davis, & Boyd,
2017). Then a dialysis bag (MWCO 3500 Da) containing 5 mL HA-NO-LF
nanomicelles was immersed in 30 mL PBS (10mM, pH 7.4) at 37 °C. Noticeable, the
PBS buffer containing saturated NO was continually applied after 6 h. Furthermore, 2
mL of release medium was collected at scheduled time intervals for measurement and
fresh release medium was replenished to maintain the constant volume. And the same
procedure was performed without NO as control. The absorbance of these samples at
293 nm was recorded using a UV-vis spectrophotometer to determine the percentage
of released drug.
8

2.6. In vitro NO-sensitive antibacterial assay
The NO-sensitive antibacterial assay of HA-NO-LF nanomicelles against S.
aureus was performed by following the method of Mu et al. (Mu, Liu, Niu, Sun, &
Duan, 2016) with slight modifications. In detail, S. aureus strain was first grown in
broth for 12 h, and then centrifuged at 5000 rpm and washed three times with PBS
buffer to adjust to an appropriate concentration for further use. Subsequently, bacteria
samples (OD600=0.4, 0.1 mL) were co-incubated with 3.9 mL tryptone soya broth
(TSB) containing HA-NO-LF nanomicelles which had been stimulated by NO in a
constant temperature shaker (37 °C, 120 rpm). In addition, the antimicrobial
properties of PBS, LF and HA-NO-LF nanomicelles without exposure to NO were
also assessed under the same procedure. The OD600 was monitored at intervals.
In addition, the four bacteria groups treated above were isolated and stained with
propidium iodide (PI,
1
μg mL-1) for 15 min, then stained with
4′-6-diamidino-2-phenylindole (DAPI, 5 μg/mL) for further 5 min in the dark. A
fluorescence microscope (Olympus BX53, Japan) was employed to visualize the
distribution of live and dead colonies.
2.7. Biocompatibility analysis
The cytotoxicity of nanomicelles was assessed against RAW 264.7 cells by MTT
assay. The RAW 264.7 cells were first seeded in 96-well plates at a density of 1×104
cells/well in 200 μL of RPMI 1640 medium and incubated at 37 °C in a humidified
atmosphere with 5% CO2 for 24 h. The cells were then treated with HA-NO-LF
nanomicelles (50, 100 and 200 μg/mL) respectively. PBS was used as blank control.
After incubation for another 24 h, the culture medium was removed and 20 μL of
MTT (5 mg/mL) was added into each well and incubated for 4 h at 37 °C.
Subsequently, 100 μL DMSO was added to dissolve the dark blue formazan crystals
generated by mitochondrial dehydrogenase in living cells. The optical density (OD) at
570 nm was monitored by a microplate reader (Perlong DNM-9062, Perlong New
technology Co., Ltd, Beijing, China).
9

In order to determine the compatability of HA-NO-LF nanomicelles with blood,
the hemolysis assay was performed as described in our recent work (Lu et al., 2019).
In brief, 0.2 mL of red blood cells (RBC) solution (2% w/w) was dispersed in 0.8 mL
of HA-NO-LF nanomicelles (50, 100, 200 µg/mL). Pure water and PBS were used as
positive controls (PC, 100% lysis) and negative controls (NC, 0% lysis), respectively.
After incubation for 3 h at 37 °C, the obtained mixtures were centrifuged at 6500 rpm
for 10 min to remove unruptured cells. Then 200 µL supernatant was collected and
measured by an automated microplate reader as mentioned above at 545 nm to record
respective OD value.
Hemolysis ratio (%) = (ODsample-ODNC)/(ODPC-ODNC) × 100
The in vivo biosafety test was similar with a previous report (Wang et al., 2016).
2.8. Intracellular uptake of nanomicelles
To evaluate the intracellular uptake capacities of nanomicelles in RAW 246.7 cell
lines, RAW 246.7 cells were first cultured in 6-well microplates in accordance with
the previous report (J. Li et al., 2012). Then the culture media were replaced with
media containing DIO-loaded HA-NO-LF nanomicelles (150 µg/mL). To investigate
whether nanomicelles were specifically taken up by RAW 246.7 cells through
HA-receptors mediated endocytosis, cells were preincubated with free-HA (4 mg/mL)
for 2 h before adding the fluorescently-labeled nanomicelles. After co-incubation for 3
h, cells were washed with PBS buffer for three times and stained by DAPI (5 μg/mL)
to label the cell nucleus. The cells were then imaged on a fluorescence microscopy
(Olympus BX53).
2.9. Intracellular bacteria experiment
According to our previous work (Qiu et al., 2017), RAW 264.7 cells were
co-incubated with S. aureus in a cell culture incubator for 1 h to establish an infection
model in vitro. Then 50 μg/mL of gentamicin was added to eradicate extracellular
bacteria. Additionally, the cells were washed three times with PBS and then incubated
in 1 mL fresh medium containing LF alone, HA plus LF, HA-NO-LF nanomicelles
10

and HA-NO-LF plus L-NAME (300 µmol/L) respectively. PBS was applied as blank
control. After incubation for further 12 h at 37 °C, the cells were lysed with Triton
X-100. And colony forming unit (CFU) of intracellular bacteria in the lysates was
recorded by counting live bacteria in tryptic soy agar (TSA) plates after serial
dilution.
2.10. In vivo pharmacodynamic assessment
Firstly, female Kunming mice (weighing approximately 25 g) between 6 and 8
weeks of age were anesthetized with sodium phenobarbital at a dose of 30 mg/kg,
then 10 µL of S. aureus (109 CFU/mL) was injected through weasand to establish the
pneumonia model for further use.
To assess the therapeutic efficacy of HA-NO-LF nanomicelles in vivo, the mice
were randomly assigned into four experimental groups: control, LF, HA plus LF and
HA-NO-LF nanomicelles (n=7). Subsequently, the infected mice were treated with
saline solution, LF, a simple mixture of HA and LF, HA-NO-LF nanomicelles (10
mg/kg) via tail vein injection respectively for three consecutive days. 24 h after the
last treatment, the mice were sacrificed. Then the lungs of each group were separated
and mashed into homogenate. The CFU of live bacteria in each organ was calculated
with the same system as operated above. Furthermore, tissue sections were prepared
according to conventional methods (Lou et al., 2018; C. Y. Zhang et al., 2019) to
observe the level of inflammation.
To investigate the extent of nanomedicine accumulation in pathological site,
DIO-loaded HA-NO-LF nanomicelles were injected to the infectious mice and lung
homogenates were prepared at scheduled time intervals. The same DIO dose was
administered for comparison. Then the fluorescence intensities of DIO were obtained
by using a BRUKER TEMSOR 27 fluorescence spectrophotometer with an excitation
wavelength of 480 nm.
2.11. Statistical analysis
Experimental data were expressed as mean ± standard deviation (SD) and plotted
11

and statistically analyzed using the software GraphPad Prism 5.01.
3. Results and discussion
3.1. Synthesis and characterization of HA-NO-LF
The synthesis of HA-NO-LF was performed by following the synthetic route
outlined in Fig.2. Formation of HA-NO-LF conjugate was confirmed by 1H NMR and
FTIR. As shown in Fig.3, the 1H NMR of hybrids LF-NA-DP, the characteristic peaks
of NH, aromatic hydrogens and vinyl hydrogen appear at δ = 12.94 ppm, δ =
8.94-7.43 ppm and 5.78 ppm, respectively. These signals are in full agreement with
the target products. After the condensation reaction, the chemical shifts of NH and
aromatic hydrogens of compound HA-NO2-LF are in accordance with the signals of
compound LF-NA-DP. Moreover, the signal of methyl group of the amide moiety
appears at δ = 2.24 ppm. After reduction, the chemical shifts of the antibiotic moiety
and the methyl group of the amide moiety are observed at δ = 8.91, 7.62, 7.41, 6.45
ppm and δ = 2.28 ppm, which indicates the formation of HA-NO-LF. In the 13C NMR
spectra (Fig. S1), the characteristic peaks of the carbonyls appeared at 176.2, 174.4
and 171.1 ppm. The chemical shifts of the aromatic carbons ranging from 155.7 ppm
to 102.2 ppm, showed the presence of the aromatic rings. Further, the carbons of the
hyaluronic acid and the aliphatic carbons showed multi-peaks ranging from 83.9 ppm
to 18.6 ppm. The above information certified the formation of the conjugates. FTIR
analysis was also used to verify the connection. As Shown in Fig. S2, the spectrum of
LF-NA-DP revealed typical characteristic peaks for amide bond, such as a strong peak
at 1571 cm-1 assigned to N-H bending vibrations，at 1293 cm−1 due to stretching of
amines (C-N), indicating the successful conjugation of LF into NA-DP moieties.
Similarly, The spectra of HA-NO2-LF and HA-NO-LF showed sharp peaks of N-H
moieties around 1571cm−1, which could also verify the connection as NMR claimed
before.(Yang et al., 2010). UV spectra of HA, LF, and HA-NO-LF are shown in
Fig.S3. There was no absorption peak of HA and an absorption peak of LF at 293 nm
from 250 to 450 nm. HA-NO-LF showed an absorption peak at 295 nm. Compared to
12

LF, the absorption peak of HA-NO-LF was slightly red shifted (2 nm) due to the
conjugation of amino in the NA-DP molecules with carboxide in the LF molecules.
The esterification degree of free carboxyl acid in HA-NO-LF was 21.9%, according to
the molar ratio of integration between N-acetyl and vinyl hydrogen. (Y. Zhang et al.,
2019). UV-Vis measurement at 293 nm showed a drug content of 18.07 wt %,
confirming successful synthesis of HA-NO-LF.
Fig. 3 1H NMR spectra of HA-NO-LF (A), HA-NO2-LF (B), LF-NA-DP (C).
3.2. Characterization of HA-NO-LF nanomicelles
As shown in Fig. 4A, the size of HA-NO-LF nanomicelles was investigated by
DLS to have an intensity-averaged hydrodynamic diameter of about 129 nm. It was
supposed that the small particle size of nanomicelles could be ascribed to the effect of
preformed hydrophobic unit acting as heterogeneous nucleation sites, which
prevented the formation of larger particles during dialysis process. Besides, the
nanomicells were found to partially carry negative charges according to Fig. 4B,
13

which might create favorable conditions for inducing electrostatic adsorption of the
drug vehicle onto the surface of inflammatory sites, as demonstrated in previous
studies (W. Li et al., 2018).
The micromorphology of HA-NO-LF nanomicelles was then performed using
SEM, and the images are shown in Fig. 4C. In a view of Fig. 4C, the micelles were
observed to exhibit well-separated individuals and a smooth surface. They were also
found to remain sphere-like structures with diameter sizes around 120 nm, which was
well corroborated with the results measured by DLS (Fig. 4A). The digital photo of
nanomicelles was inserted in Fig. 4C (a), it was apparent that the nanomicelles
showed a bright path once illuminated with a laser pointer, confirming the successful
formation of the nanostructures once again.
To investigate storage stability of nanomicelles, the change of size distribution as
well as zeta potential was monitored through 6 days using DLS measurement. As
shown in Fig. 4D, with the function of time, the mean particle size of HA-NO-LF
nanomicelles was essentially unchanged, similar to the result of zeta potential. The
results described above confirmed the successful self-assembly and good stability of
nanomicelles, avoiding abnormal release and inactivation of drug in normal storage
conditions, which provided a promising prospect for the product to be applied as
effective antibacterial agent.
14

Fig.4 Particle size distribution curve (A), zeta potential (B) and SEM images (C) attached
with an inset macroscopic image (a) of HA-NO-LF nanomicelles; Changes of particle size and
zeta potential of HA-NO-LF nanomicelles after storing up to 6 days (D).
3.3. NO-triggered drug release in vitro
In order to confirm whether HA-NO-LF nanomicelles could achieve controlled
release effect triggered by external NO, we performed NO-sensitive LF release
experiments in a time-dependent manner in the presence and absence of NO. The
release curve of LF showed in Fig. 5 exhibits a saltation once the NO was injected at
6 h (+ NO), indicating highly reactive nature of the nanomicelles. Upon extending the
incubation time, the drug was released continuously in a highly efficient matter, as
evident by a sharp release rate of LF between 6 to 15 h, achieving the total drug
release efficiency at nearly 70%. By sharp contrast, there was no significant release of
the drug in the group without NO stimulation with the function of time. It is
noticeable, however, that the LF could also be detected for slight release without NO
trigger (- NO), presumedly attributed to the partial cleavage of nanomicelles affected
by environment inevitably. Although only an initial proof of concept, the experiment
above has demonstrated the potential of the HA-NO-LF nanomicelles to act as a drug
delivery vehicle with sustained controlled release.
Fig.5 The time course of LF release profile of HA-NO-LF nanomicelles
15

in the presence and absence of NO.
3.4. In vitro antibacterial activity
A Gram-positive bacterium (S. aureus) was selected as the model bacteria to
qualitatively analyze the antibacterial property of NO-sensitive nanomicelles, and the
results were cheerfully presented in Fig. 6A, B. It is apparent that the HA-NO-LF
nanomicelles exhibited almost no inhibition of bacterial growth compared with the
blank control, which reveals the fact that LF was hardly cleaved from the
nanomicelles in the absence of NO stimulus. Nevertheless, an unaltered OD value
which tends to zero over time was observed (Fig. 6A) while treated by HA-NO-LF
nanomicelles in the presence of NO, consistent with the antibacterial effect of pure LF.
Besides, the macroscopic photos of colony growth shown in Fig. 6B revealed that the
number of colonies decreased significantly when incubated with HA-NO-LF
nanomicelles in the presence of NO, which provided powerful evidence supporting
the successful cleavage and remarkable antibacterial effects of LF-loaded nanomicells.
Meanwhile, the bactericidal effects of different mediations could also be clearly
observed from the fluorescence microscopy images in Fig. S4 since the dead bacteria
could by specificly dyed by PI. The discovery above showed conclusion that
HA-NO-LF nanomicells could only possess the antibiotic effect when it came to
contact with NO stimulus, which renders the NO-sensitive micelles suitable for
intracellular delivery of drugs.
16

Fig.6 Growth curves of S. aureus cultured by PBS, LF, HA-NO-LF nanomicelles with and
without exposure to NO, respectively (A); Macroscopic photos of colony growth above with
different treatments (B).
3.5. Study on biocompatibility
It has been universally acknowledged that biosafety as well as biocompatibility
are fundamental requirements for biomaterial. Then in vitro cytotoxicity of
HA-NO-LF nanomicelles were investigated by MTT assays in RAW 246.7 cells. As
depicted in Fig. 7A, the cell viability decreased slightly with the increase in the
concentration of HA-NO-LF nanomicelles from 50 to 200 µg/mL. However, no
significant toxicity (cell viability＞90%) was found in RAW 246.7 cells even at a
high concentration of HA-NO-LF nanomicelles at 200 µg/mL. Moreover, assessing
the interaction of intravenous preparations with blood components is also of great
concern just in case pathophysiological events. The percentage of hemolysis was
around 7% for the nanomicelles over a wide range of concentrations, revealing blood
compatibility of HA-NO-LF nanomicelles (Fig. 7B) (Figueiredo et al., 2017). The
potential acute toxicity of the nanomicelles was further assessed by analyzing tissue
functions. As shown in Fig. S5, no significant difference between nanomicelles
treated group and the control group treated with PBS, demonstrating negligible acute
17

toxicity of HA-NO-LF nanomicelles. In general, the multifunctional nanomicelles we
designed are of good biocompatibility, which laid a solid foundation for subsequent in
vivo experiments and practical applications in the future.
Fig.7 In vitro cytotoxicity of different concentrations of nanomicelles on RAW 246.7 cells
(A); Hemolysis reaction of different concentrations of nanomicelles on red blood cells (B).
3.6. Intracellular uptake of nanomicelles
Having validated the desired chemical responsiveness of HA-NO-LF
nanomicelles, we ulteriorly investigated cellular uptake of nanomicelles and
competitive inhibition of specific receptors on the surface macrophages. DIO is a
crucial stain which exhibits green fluorescence in cells, and DIO payload inside the
nanomicelles was used as fluorescent marker to qualitatively analyze the amount of
HA-NO-LF nanomicelles located in intracellular compartments.
As displayed obviously in Fig. 8A, strong green fluorescence was observed when
RAW 246.7 cells incubated with the nanomicelles for 3 h, implying high intracellular
uptake capabilities for the HA-NO-LF nanomicelles. When it comes to competition
inhibition assay, the fluorescence intensity (green) of HA-NO-LF nanomicelles
treated with free-HA was weaker to that of untreated one, while blue fluorescence
which referred to cell nucleus of those two test groups remained almost the same
intensity (Fig. 8A, B). It was suggested that free-HA could competitively interact with
specific HA-receptors such as CD44 receptors, hindering the endocytosis mediated by
HA-receptors of the HA-NO-LF nanomicelles and resulting in the decreased
18

fluorescent intensity (J. Li et al., 2012). This result appears to be consensus with our
hypothesis that HA-NO-LF nanomicelles could be specifically adsorbed onto the
surface of macrophages via receptor-mediated nature, facilitating nanomicelles
passage through cytomembrane.
Fig.8 Confocal microscopy images of RAW 246.7 cell line incubated with DIO-loaded
HA-NO-LF nanomicelles (A), and with free-HA polymer pretreated DIO-loaded HA-NO-LF
nanomicelles(B). Scale bars correspond to 50 µm in all the images.
3.7. Intracellular bacteria experiment
On the basis of above results, we attempted to further explore the NO-sensitive
performance under simulated biological inflammatory conditions. To investigate
whether the nanomicelles are capable of responding to endogenous NO secreted from
cells cultured in vitro, the RAW 264.7 cell was chosen as model cell since it is a type
of murine macrophage that produces NO when infected by bacteria(Park et al., 2017).
As shown in Fig. 9, the intracellular bacteria were eliminated efficiently by LF,
whereas the simple mixture of HA and LF didn’t contribute to further decrease of
CFU remained in infected cells. As such, it is conceivable that HA alone has no
contribution to the antibacterial activity of testing groups. In addition, the HA-NO-LF
nanomicelles group had a marked decrease in CFU value compared with single LF
with the same effective dose of antibiotic. We could thus make a bold speculation that
free LF can be transferred into cells by passive diffusion or dispersed in the liquid
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medium without specific targeting, while HA-NO-LF nanomicelles could be
internalized by endocytosis via HA receptors which were highly expressed by
activated macrophages and then controlled release of effective LF, resulting in better
therapeutic effect against intracellular bacteria.
To further confirm the NO-sensitive behavior of HA-NO-LF nanomicelles in
activated macrophages, L-NAME was added to the macrophages to inhibit the
production of NO(He & Frost, 2016). The result presented in Fig.8 is consistent with
our conjecture that the bactericidal activity of HA-NO-LF nanomicelles was
significantly decreased without stimulated by enough NO.
Fig. 9 RAW264.7 cells infected with S. aureus were treated with PBS, LF alone, simple mixture of
HA and equivalent LF, HA-NO-LF nanomicelles, HA-NO-LF nanomicelles added with L-NAME,
respectively. Values are given as the mean ± SD of triplicates. *p < 0.05, **p < 0.005, ***p <
0.001.
3.8. Therapeutic efficacy of HA-NO-LF nanomicelles in vivo
To evaluate in vivo antibacterial activities of the formulated drugs, the infected
mice were tail-intravenously injected with various agents as vividly depicted in Fig.
10A. After drugs administration, various degrees of inhibition to the propagation of
pathogenic bacteria compared with control group were observed in Fig. 10B. Notably,
20

the HA-NO-LF nanomicelles group showed more effective suppression of S. aureus
than LF group, while the germicidal ability of tested group treated with simple
mixture of HA and LF was equivalent to LF alone. Taken in this sense, the
HA-NO-LF nanomicelles exhibited better antibacterial efficacy under the same
content of LF compared with other groups. From histological analysis in Fig. 10C, the
lung in mice treated with saline displayed obvious signs of inflammatory response,
containing alveolar wall hyperemia and thickening. Specifically, the blood vessels
were significantly dilated and part of the alveolar tissue structure collapsed to form
abscesses in the alveolar cavity. Moreover, we found numerous inflammatory cells
infiltrating the alveolar septum and spaces with hemorrhage and congestion. However,
the group treated with HA-NO-LF nanomicelles decreased pathological tissue damage
and showed lower pulmonary inflammatory characteristics compared with other
groups. In addition, it is evident that the fluorescence intensity of DIO from the
nanomicelles was significantly higher than that of free DIO at all three time points
from 2 to 8 h (Fig. S6), which confirmed the accumulation of nanomicelles in
pathological lung tissues. Taken together, these results indicated that HA-NO-LF
nanomicelles could effectively reverse S.aureus infection in vivo.
21

Fig.10 The schematic illustration about establishment and treatment of pneumonia model (A);
Average CFU values recorded in lung tissues of four animal groups: control, LF, simple mixture of
HA and LF and HA-NO-LF nanomicelles, respectively (B); Analysis in lung tissues of the impact
of four experimental groups: control, LF, simple mixture of HA and LF, HA-NO-LF nanomicelles
(C). The black arrows indicate that alveolar tissue collapse. The green arrows depict alveolar wall
hyperemia. Values are given as the mean ± SD of triplicates. *p < 0.05, **p < 0.005, ***p <
0.001.
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4. Conclusions
In this paper, the novel HA-based nanomicelles with NO-sensitive antibacterial
properties was prepared by conjugating LF with HA using a functionalized
intermediate bearing o-phenylenediamine groups. It was found in vitro that the
nanomicelles were cleaved to release LF gradually and exerted great antibacterial
effect against S.aureus upon exposure to NO. Meanwhile, the NO-triggered
nanomicelles retained good biosafety as well as biocompatibility. The results of the
competition inhabitation assay proved that the nanomicelles could enter macrophages
via a CD44 mediated endocytosis. Moreover, in vivo pharmacodynamic assessment
acknowledged their ability to fight against bacteria and reduce inflammatory levels.
All these results suggested that NO-sensitive HA-based nanomicelles hold great
promise as a targeting vehicle for selective delivery of drugs to pathogenic sites,
thereby enhancing the therapeutic efficacy for infectious diseases.
5. Conflicts of interest
There are no conflicts of interest to declare.
6. Acknowledgements
This work was supported by the National Natural Science Foundation of China
(NSFC) (31870799 and 31700707) and the Fundamental Research Funds for the
Central Universities (2452017026). The authors are grateful to Life Science Research
Core Services (LSRCS), Northwest A&F University for providing SEM
measurement.
23

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