Carbohydrate Polymers 252 (2021) 117162

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Composite inclusion complexes containing hyaluronic acid/chitosan

nanosystems for dual responsive enrofloxacin release
Yuda Liu a, 1, Dongmei Chen a, b, 1, Aoxue Zhang a, Man Xiao c, Zhenxia Li b, Wanhe Luo b,
Yuanhu Pan b, Wei Qu b, Shuyu Xie a, b, *
a
National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, China
b
MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, 430070, China
c
School of Bioengineering and Food Science, Hubei University of Technology, Wuhan, 430068, China

A R T I C L E I N F O A B S T R A C T

Keywords: In order to overcome treatment difficulty of S. aureus infections, a pH/hyaluronidase dual responsive
Staphylococcus aureus enrofloxacin-cyclodextrin (β-CD) inclusion complexes (IC) containing hyaluronic acid/chitosan (HA/CS) self-
Hyaluronic acid/chitosan nanogels assemble composite nanosystems covered by poloxamer 188 (F68) was firstly explored for targeted “on-de­
pH responsiveness
mand” delivery. The FTIR, DSC and PXRD showed that enrofloxacin was embedded into IC and then distributed
Hyaluronidase responsiveness
into F68 coating nanogels formulated by electrostatic interaction between CS and HA. The optimal nanosystems
Inclusion complexes
of 118.8 ± 30.7 nm showed excellent stability and responsive release in the acid medium, hyaluronidase con­
taining medium, and LB broth medium where S. aureus present. The nanosystems displayed strong surface
adsorption on S. aureus and enhanced activity against S. aureus. It had stronger sustained release than the
polymeric nanoparticles formulated by entrapping of IC into F68 and the single HA/CS nanogels. This study
provides a promising multi-functionalized nanosystems to overcome the treatment challenge of S. aureus and
other bacterial infections.

1. Introduction
Staphylococcus aureus (S. aureus) seriously threaten human health
and result in huge economical losses for livestock over the world, which
has aroused extensive concerns for long time (Zhou et al., 2018).
Enrofloxacin is specially and widely used to treat various infections of
animals caused by S. aureus because of its strong antibacterial properties
(Serrano-Rodríguez et al., 2017; Viveros, Lopez-Ordaz, Gutiérrez,
Miranda-Calderón, & Sumano, 2018). However, the poor solubility,
rapid metabolism and excretion, and nonspecific distribution limit its
clinical application. The long period treatments with high frequency and
doses are required, which often lead to notable side effects and serious
resistance due to the huge fluctuation of drug concentrations. The design
of novel smart delivery systems for enrofloxacin is urgent to improve its
solubility and sustained release, and realize target delivery to the
infection site of S. aureus.
Cyclodextrins (CD) are the cyclic oligosaccharides which can form
inclusion complexes (IC) with drugs. The β-CD complexations could take
up drug molecule into the cavity and has been widely studied to improve
the solubility and sustained release of drug (Ding, Pang, Vara Prasad
Chamakura, & Wang, 2020). Due to the limited sustained release per­
formance, IC could be further incorporated into polymeric particles to
further modify the release pattern and obtain superior controlled release
(Li et al., 2016). However, the premature and non-target release of IC
and its polymeric based micro/nanoparticles remain challenging.
Nanogels, a nano-sized cross-linked polymeric networks, is used to
incorporate drugs. They hold excellent structural stability and favorable
responses to a wide variety of environmental stimuli (Hajebi et al.,
2019). Inspired by the special microenvironment of bacterial infection
sites (e.g., lower pH about 5.5, overexpressed enzymes including hyal­
uronidase and gelatinase, and exotoxins), the intelligent responsive
nanogels can enable on demand delivery by achieving a responding
responsiveness to specific signals of bacteria (Qi, Zhang, Liu, Qiao, &
Wang, 2017; Wu et al., 2018). For example, bacteria-responsive gelatin
nanoparticles could be degraded by gelatinase in infected areas and
therefore released the encapsulating Ru-complex-modified selenium

* Corresponding author at: National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Res­
idues, China.
E-mail address: snxsy1@126.com (S. Xie).
1
Yuda Liu and Dongmei Chen contributed equally.

https://doi.org/10.1016/j.carbpol.2020.117162
Received 2 July 2020; Received in revised form 20 September 2020; Accepted 24 September 2020
Available online 4 October 2020
0144-8617/© 2020 Elsevier Ltd. All rights reserved.
Y. Liu et al.
nanoparticles to destroy the MRSA cells (Lin et al., 2019). The nanogels
with environmental stimuli are mainly related with its components. The
negatively charged hyaluronic acid (HA), a naturally occurring linear
polysaccharide, is an excellent material for design of antibiotics nano­
gels due to its biocompatibility and favorable responses to hyaluroni­
dase secreted by bacteria (Shyam, Pratik, & Deepak, 2020). Many kinds
of bacteria including S. aureus generate hyaluronidase, therefore, HA
can be used as a capping material in responsive to hyaluronidase pre­
sented at bacteria infectious microenvironment. For instance, the
capping HA-dopamine conjugates of the ascorbic acid @
graphene-mesoporous silica nanosheet @ ferromagnetic nanoparticles
were degraded by hyaluronidase upon their arrival at the infection site
(Ji et al., 2016). Due to its lack of inherent amphiphilicity, HA cannot
spontaneously assemble into stable and segregated nanogels in aqueous
and thus it should be with other polymers or functional transformation
to render its amphiphilicity. Chitosan (CS), an abundant natural and
biodegradable carbohydrate polymer, is an effective material to prepare
the nanogels with exhibiting a proton sponge effect (Kurdtabar, Koute­
naee, & Bardajee, 2018). The proton sponge effect will lead to the
swelling of CS at acid condition, which will contribute CS particles to
rapid release of drugs at the low pH of the bacteria cloning site. For
example, 100 % of rifampicin was released from SA/CS microparticles
within 2 h at pH 6.8 (Lacerda, Parize, Fávere, Laranjeira, & Stulzer,
2014). These properties make it a promising carrier for increasing the
concentration gradient at bacterial infection site and controlling drug
release (Shuo et al., 2020). The HA/CS fibrous/hydrogel formed by the
electrostatic interactions has been used as surface self-healing agents
(Barroso et al., 2019), wound healing material (Bazmandeh, Mirzaei,
Fadaie, Shirian, & Ghasemi, 2020; Wang et al., 2019) and tissue engi­
neering (Deng et al., 2017). However, hydrogel including HA/CS
nanohydrogel have disadvantages of relatively rapid release profiles and
premature release.
In view of these, a novel targeted “on-demand” delivery composite
nanosystems was designed based on the triple controlled release of IC,
polymeric nanocapsulation, and nanogels as well as the responsiveness
of HA and CS. It was hypothesized that the designed composite nano­
systems can decrease premature release and realize bacteria targeted
responsive release by ingenious dispersing enrofloxacin IC into HA/CS
nanogels capsulating with poloxamer 188. The formation mechanism,
structural characteristics, bacterial responsiveness, and antibacterial
activity of the composite nanosystems were studied (Detail experiment
scheme is showed in Fig. S1).
2. Materials and methods
2.1. Materials
Hydroxypropyl methyl cellulose (HPMC, methoxy group: 28–30 %;
hydroxyl value: 7.0–12 %, 2 % viscosity: 6 mPa.s) was obtained from
Tianjin guangfu fine chemical research institute. HA (MW: 403.31,
content: 97 %) was purchased from Macklin (Shanghai, China). CS
(MW:161.16KDa, viscosity: 50–800 mPa.s, degree of acetylation 80–95
%), sodium tripolyphosphate (TPP, MW: 367.86), β-CD (MW: 1134.98;
reducing sugar≤2.0 %), sodium hydroxide, polyvinylpyrrolidonek30
(PVP k30., MW: 37,900), sodium alginate (SA, MV: 398.32; viscosity:
≥0.02 Pa.s in 10 g/L, 20℃), sodium carboxymethyl cellulose (CMC-Na,
MW: 263.198, viscosity: 800~1200 mPa.s), acetic acid, potassium
dihydrogen phosphate (KH2PO4, MW136.09), and potassium chloride
(KCl) were purchased from Sinopharm group chemical reagent co. Ltd.
Ethyl alcohol was bought from Wuhan xingheda trading co. Ltd. Poly­
vinyl alcohol (PVA, 87–90 % hydrolyzed, average MW: 30,000–70,000)
and poloxamer 188 (F68, MW: ~7680− 9510, oxyethylene: 79.9–83.7
%) were purchased from sigma-aldrich, USA. Sodium chloride (NaCl,
≥99.5 %) and disodium hydrogen phosphate (Na2HPO4⋅12H2O, ≥99 %)
were purchased from Wuxi jiani chemical co. Ltd. Ultrapure water was
prepared by a Microporous Purification® Purified Water System (Milli-
2
Carbohydrate Polymers 252 (2021) 117162
Q Ltd., France).
2.2. Preparation of polymeric nanoparticles containing IC
The formation of enrofloxacin-β-CD-IC was optimized and modified
with reference to previous reports (Ding et al., 2020; Li et al., 2016).
Briefly, 2 g enrofloxacin and a certain amount (6 or 12 g) of β-CD (in a
molar ratio of CD and enrofloxacin = 1:1 or 1:2) were weighed into a
250 mL beaker containing 30 mL solvent (absolute ethanol, or 0.1 mol/L
NaOH). The solution was heated to a fixed temperature (60℃, 70℃ or
80℃) on a hot plate magnetic stirrer (IT-07A3, Shanghai Yi Heng Co.,
Ltd, Shanghai, China) and then stirred at a rotational speed of 1700
r/min for 1, 2, or 3 h to form the IC. After stop heating, 50 mL water
solution containing different concentration (2 %, 4 % or 6 %) of poly­
mers (HPMC, SA, CMC-Na, PVPk30, PVA, or F68) was quickly added
under a rotational speed of 1000 r/min to form nanoparticles. The
stirring was continued to blend until the nanosuspension was milky
white and the final volume was maintained at 80 mL to ensure that the
drug content in the nanosuspension was 2.5 %.
2.3. Preparation of enrofloxacin-composite nanosystems
According to the optimized inclusion conditions, 2 g enrofloxacin
and 12 g β-CD were weighed into a 250 mL beaker containing 30 mL 0.1
mol/L NaOH. The solution was heated to 80℃ on a hot plate magnetic
stirrer and then stirred at a rotational speed of 1700 r/min for 2 h to
form IC. After stop heating, a mixture of 30 mL water containing 10 %
F68 and a certain (0.4 %, 1.2 %, or 1.6 %) HA was rapidly added under a
rotational speed of 1000 r/min. The stirring was continued to blend until
the liquid is milky white and then 20 mL 0.5 % acetic acid solution
containing a proper concentration (0.6 %, 1.8 %, 2.4 %, 4.8 %) of CS was
added by drops. After stirring for 15 min, 1 mL aqueous solution con­
taining 16 % TPP was added by drops under a constant stirring for 2 h to
form a stable composite nanosystems. Finally, the volume of composite
nanosystems was maintained at 80 mL to ensure that the drug content in
the nanosuspension was 2.5 %. The optimal CS and HA concentrations
were evaluated by single factor experiment. In order to confirm the
sustained release of our designed composite nanosystems, the simple
nanogels was also prepared as the above-mentioned process without the
preparation process of IC and addition of F68. The enrofloxacin was
directly weighed into 30 mL 0.1 mol/L NaOH.
2.4. Determination of inclusion rate
The inclusion rate was used to evaluate the inclusion effects. The
inclusion rate was calculated through the following for­
Enrofloxacin in IC(mg)
mulas:Inclusion rats(%) Enrofloxacin added(mg) × 100%. The enrofloxacin
content was quantitatively determined by UV-high performance liquid
chromatography (HPLC).
2.5. Characterization of the nanoparticles and composite nanosystems
2.5.1. Determination of sizes, polydispersity index (PDI), and zeta potential
(ZP)
The size, PDI and ZP of the nanoparticles and composite nanosystems
were determined by Zetasizer ZX3600 (Malvern Instruments, UK) at
25℃. Before determination, the nanoparticles and composite nano­
systems were diluted to 2.5 mg/mL to get the optimum kilo counts per
second for the measurements.
2.5.2. Morphology observation
The surface of IC and the mixture of enrofloxacin and β-CD were
coated with a thin gold overlay and analyzed with a scanning electron
microscope (SEM, Hitachi S-4800, HITACHI, Japan). Morphology of the
nanoparticles and composite nanosystems was observed by the light
Y. Liu et al.
Fig. 1. Optimization procedure of IC and its appearance. A: SEM image of mixture of β-CD and enrofloxacin; B: SEM image of IC; C: Appearance of IC; Effect of
temperature (D), molar ratio (E), time (F), and solvent (G) on the inclusion rate.
electron microscope (SDP TOP, CX 40, Ningbo Sunny Instruments Co.,
Ltd) and transmission electron microscope (TEM) after that 2 μL samples
were placed on the copper grids with films and dried at room after
negative staining with 2 % sodium phosphotungstate for 30 s.
2.5.3. Fourier transform infrared (FTIR)
The interactions of enrofloxacin with individual components were
analyzed by FTIR spectrophotometer (Nicolet iS50, Thermo Scientific
Inc.). The dried samples were ground into a fine powder and then placed
into a disc, respectively. Each disc was scanned for 32 times at 2 mm/
size at a resolution of 4 cm− 1 using carson apodization.
2.5.4. Differential scanning calorimeters (DSC)
The thermal analysis of composite nanosystems and individual
components was determined by using DSC (DSC200PC, NETZSCH, Selb,
Germany). Briefly, 15 mg dried samples were added into sample cell and
heated from 25 to 275℃ under the nitrogen purge with a heating rate of
10℃/min to record the endothermic peak.
2.5.5. Powder X-Ray diffraction (PXRD) study
The PXRD of composite nanosystems and individual components was
done with an accelerating voltage of 45 kV, a beam current of 40 mA, a
step size of 0.0170◦ , scanning range (2θ) from 5◦ to 49◦ angles and a data
acquisition time of 25.1954s per step.
2.5.6. Determination of loading capacity (LC) and encapsulation efficiency
(EE)
The nanoparticles and composite nanosystems were collected by
centrifugation at 14,000 rpm (Hitachi Centrifugation CR21 GIII; Hitachi
Koki Co., Ltd., Japan) for 60 min at 4 ℃. The enrofloxacin in the su­
pernatant after centrifugation was determined by HPLC to determine the
EE. The precipitation after re-suspending with distilled water was
lyophilized to test the LC (Meng et al., 2020).
2.5.7. Determination of sedimentation rate (F)
The original height (H0) of 50 mL nanosuspension and composite
nanosystems suspension in 100 mL graduated cylinder after shaking was
measured. The height (H) of the sediment in the cylinder after standing
for 3 h was recorded. F was calculated according to the equation: F= H/
H0.
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Carbohydrate Polymers 252 (2021) 117162
2.6. Stimuli-responsive release study
The composite nanosystems (equivalent to 10 mg enrofloxacin) was
placed into a dialysis bag (molecular weight: 3500), and then dialyzed
against 98 mL pH 5.5 and 7.4 release medium without and with 80 μg/
mL hyaluronidase or LB broth culture with and without logarithmic
growth phase of S. aureus (Hubei-01, ShangHai-H1908− 01, and HuNan-
25, respectively) at 106 CFU/mL (receiver solution) in a 500 mL disso­
lution cup at 37 ± 0.5 ◦ C using a dissolution tester stirring at 50 rpm. At
fixed time, 0.2 mL sample was collected from the receiver solution for
measurement and the same volume of fresh medium was added to
maintain a constant volume. The cumulative release curve vs time was
plotted.
2.7. Determination of anti-bacterial activity
2.7.1. Agar diffusion method
The S. aureus (Hubei-01, ShangHai-H1908-01, and HuNan-25) sus­
pensions in logarithmic growth phase were diluted to the concentration
of 107 CFU/mL, respectively, and then the diluted bacteria suspensions
of 100 μL were evenly covered on the surface of the agar medium. After
evenly coating, 3 holes were made in each plate and subsequently 200
μL solution, nanoparticles, and composite nanosystems of different
concentrations were added for the three strains of S. aureus, respectively.
The inhibition zone sizes of each formulation in agar plate were deter­
mined after incubation at 37℃ for 16− 18 h.
2.7.2. Broth dilution method
The antibacterial activity of the three different enrofloxacin formu­
lations was studied using the broth dilution technique as recommended
by CLSI. Briefly, serial dilutions of the nanosystems, polymeric nano­
particles or native enrofloxacin in Mueller-Hinton broth were prepared.
Then S. aureus (Hubei-01, ShangHai-H1908-01, and HuNan-25) were
added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The
minimal inhibitory concentration (MIC) of enrofloxacin was defined as
the lowest concentration inhibiting visible growth of bacteria after 24 h
incubation of the cultures at 37℃.
2.7.3. In vitro time-kill curve study
The in vitro kill curves of enrofloxacin solution and nanosystems
against three different strains of S. aureus were established by plotting
time versus log10 CFU/mL. The S. aureus from a mid-log phase culture
was added to 2 mL of MH broth to give a starting inoculum of 106 CFU/
Y. Liu et al.
Fig. 2. Appearance and optical microscope images of polymeric nanoparticles prepared with different stabilizers at different concentration.
mL. The drug was added to obtain the concentrations corresponding to 2
× MIC and 20 μg/mL. The tubes were placed in a 37 ◦ C shaker at 220
rpm for incubation and the bacterial count (CFU/mL) was determined
by the agar dilution method for each tube after incubation for fixed time.
Briefly, each culture sample was subjected to 10-fold serial dilutions
with sterile saline, then 100 μL of each dilution was spread onto the agar
plates. The plates were incubated at 37 ◦ C and the viable colonies were
counted after 24 h. Each concentration was tested in triplicate. The limit
of detection was 10 CFU/mL.
The liveness of the bacteria treatment by different formulations was
qualitatively evaluated using the Live/Dead backlight bacterial viability
kit (Shanghai Bestbio Biotechnology Co., Ltd, China). The three different
strains (1 × 105-1 × 106 CFU/mL) were treated with 20 μg/mL enro­
floxacin solution, nanoparticles and composite nanosystems for 2 and 4
h. At the fixed time, the samples were collected by centrifugation. After
washing with 0.9 % NaCl, the bacteria were stained with 100 μL green
fluorescent dye for 15 min. Subsequently, the bacterial suspension of 5
μL was dropped onto the slide and then observed by fluorescence
microscope.
2.7.4. Determination of adhesion of composite nanosystems to bacteria
The diluted composite nanosystems was put into the liquid medium
containing the S. aureus of logarithmic growth phase of 106 CFU/mL at
the final enrofloxacin concentrations of 20 μg/mL (Hubei-01, Shanghai-
01) or 40 μg/mL (HuNan-25), the liquid culture was drawn on the
copper mesh after incubation for 15 min and then observed whether the
composite nanosystems was adsorbed on the surface of bacteria by TEM.
2.8. Statistical analysis
Data were presented as mean ± standard deviation (SD) and
analyzed by the SPSS software (version 20, IBM, New York, USA). Sta­
tistical significance was defined as a p-value of 0.05 by the one-way
ANOVA.
3. Results and discussion
3.1. Optimization of enrofloxacin-β-CD-IC
The formation of enrofloxacin-β-CD-IC is affected by many factors, e.
g., temperature, time, and feed ratio (Li et al., 2016). In this study, the IC
were optimized using inclusion rate as selection index. As shown in
Fig. 1D, the inclusion rate was increased from 72.0 ± 3.1 % to 84.1 ± 0.6
% as the temperature was increased from 60 ℃ to 80 ℃. When the molar
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Carbohydrate Polymers 252 (2021) 117162
ratio of enrofloxacin and β-CD was fixed at 1:1 and 1:2, the inclusion rate
was 58.5 % and 83.6 %, respectively (Fig. 1E). The inclusion rate was
79.5 ± 2.8 %, 83.6 ± 1.6 %, and 84.1 ± 0.6 %, respectively, when the
inclusion time was 1, 2 and 3 h (Fig. 1F). A high inclusion rate of 79.5 %
was obtained at 1 h, which might be due to that the IC was quickly and
effectively formulated under the stirring speed of 1700 rpm at 80℃. It
was reported that the enrofloxacin-HP-β-CD IC could be formed during
2− 4 h under the stirring speeds of 400− 600 rpm at 55− 65℃ (Ding
et al., 2020). In this study, the high temperature (80℃) and stirring
speed (1700 rpm) might help to short the inclusion time. It is consistent
with previous report that magnetic mesoporous carbon could be effec­
tive embedd into β-CD at 80℃ for 1 h (Mashile, Dimpe, & Nomngongo,
2020). When the inclusion time was continued to increase, the inclusion
rate was not dramatically improved, which might be due to that it was
near the maximum inclusion capacity. When using absolute ethanol and
0.1 mol/L NaOH as solvents, the inclusion rates were 83.6 ± 1.6 % and
82.9 ± 0.3 %, respectively (Fig. 1G). Acetic acid was not considered due
to its unsuitable for large-scale production of the pungent odor although
it is reported to prepare the enrofloxacin-IC (Ding et al., 2020). The
inclusion rate of IC prepared by absolute ethanol was not significantly
higher compared to those using 0.1 mol/L NaOH. Due to the high price
and less environmentally friendly of absolute ethanol, 0.1 mol/L NaOH
was finally used. Based on the above results, the optimal temperature,
feed molar ratio, time, and solvent were set at 80℃, 1: 2, 2 h and 0.1
mol/L NaOH, respectively.
The dried IC were white or yellowish and loose texture powder
without graininess and crystallization (Fig. 1C). Under the observation
of SEM, β-CD was irregular and bulky with relatively smooth and flat
surface (Fig. 1A). Pure enrofloxacin was in the shape of smooth needles
or sticks. The IC displayed block-shaped, larger ball-like and cagelike
shape with uneven surface due to attaching a little of unincorporated
enrofloxacin (Fig. 1B). The morphological change directly confirmed the
formation of the enrofloxacin-β-CD IC (Ding et al., 2020). The FTIR
(Fig. 5C), DSC (Fig. 6), and PXRD (Fig. 7C) also showed that enro­
floxacin was effectively embedded into IC.
3.2. Optimization of IC containing composite nanosystems covered by
polymer
In order to avoid swiftly premature release, the IC were encapsulated
into nanogels coating with hydrophilic polymer emulsifier to prepare
the composite nanosystems with on-demand release.
The polymers were commonly used to form and stabilize the particles
(Li et al., 2016; Wei et al., 2018) and thus the suitable polymers were
Y. Liu et al. Carbohydrate Polymers 252 (2021) 117162

Fig. 3. In vitro release profiles of composite nanosystems, single nanogels, and polymeric nanoparticles. A: Effect of HA and CS concentration; B: Effect of HA and CS
mass ratio; C: In vitro release in pH 5.5 and 7.4 mediums with and without hyaluronidase; D: In vitro release in broth culture containing S. aureus of Hubei-01,
ShangHai-H1908-01, and HuNan-25, respectively.

5
Y. Liu et al.
firstly selected. The polymeric nanoparticle suspensions prepared by
entrapping of IC into 2.5 % of HPMC, SA and CMC-Na was very viscous
or even solidified with unencapsulated crystals of IC (Fig. 2), indicating
that these polymers could not completely encapsulate even disintegrate
the IC. When the nanoparticle suspension prepared by entrapping of IC
into 2.5 % of PVPk30, PVA, or F68 was a milky white suspension with
good fluidity. This might be due to the excellent function of PVPk30, PVA
and F68 to inhibit crystallization of guest molecules and their excellent
performance as coating agents (Li et al., 2016). The polymeric nano­
particle suspension prepared by PVPk30 and PVA had a few unencap­
sulated needle and bulk crystals of IC under the light microscope. When
the concentration was decreased to 1.25 %, the F of the nanosuspension
prepared with PVP k30 and PVA was below 0.3, while the value of the
nanoparticle suspension prepared by F68 was above 0.9. This is due to
its excellent surface adsorption and surfactant properties. It is also an
excellent material for preparing hydrogel (Collett & Popli, 2000). Thus,
F68 was used to encapsulate the IC to prepare the IC containing poly­
meric nanoparticles. The polymeric nanoparticles prepared by 3.75 %
F68 had the smallest size of 548.4 ± 6.7 nm and PDI of 0.225 ± 0.006
compared to those prepared by 1.25 % and 2.5 %, and thus the 3.75 %
was used. TEM images showed that the nanoparticles formulated by
entrapping of IC into F68 were irregular shape with uneven and
incomplete covering of F68 on the surface (Fig. 4A).
In order to avoid the premature release and implement responsive
release, HA mixed with F68 and IC was used to self-assemble nanogels
by crosslinking with CS under the addition of TPP and simultaneously be
covered by F68. Their formation depends on the molecular weight, and
concentration and mixing ratio of polyelectrolytes and thus the nanogels
was optimized. Firstly, they were prepared by varying concentrations of
HA and CS from 0.15 % to 0.60 % at the mass ratio of 1:1. When their
concentration was increased from 0.15 % to 0.60 %, the IC containing
composite nanosystems covered by F68 obtained the best sustained
release in pH 7.4 and swiftest release rate in pH 5.5 (Fig. 3A) and the size
of the nanosystems was decreased from 711.5 ± 41.1–118.8 ± 30.7 nm
with more uniform (Table 1). The increased HA content was help to form
a gel layer instead of burst release (Bazmandeh et al., 2020). The for­
mation of ionic complex between HA and CS hinders the undesirable
6
Carbohydrate Polymers 252 (2021) 117162
Fig. 4. Appearance and TEM image of different
enrofloxacin formulations. A: Appearance of
polymeric nanoparticle suspensions; B:
Appearance of simple nanogels; C: Appearance
of designed composite nanosystems; TEM image
of polymeric nanoparticles (D), simple nanogels
(E), and designed composite nanosystems (F);
Size distribution of polymeric nanoparticles
(G), simple nanogels (H), and designed com­
posite nanosystems (I). The intensity (Y axis) of
scattered light is related with the number of
nanoparticles. In the three different enro­
floxacin suspension, the number of nano­
particles was hardly to ensure complete
consistence, thus the Y axis of Fig. 4 F-I had
some difference.
solubility of HA and thus hindering the undesirable drug release from
the HA/CS nanogels (Bazmandeh et al., 2020). The release velocity of
the nanosystems was significantly slower than those of polymeric
nanoparticles formulated by entrapping IC into F68 and the simple
HA/CS nanogels without IC and F68 in both pH 7.4 and 5.5 (Fig. 3A).
This might be due to the best stabilization of the crosslinking layer to
disperse the IC by increasing of gel polymers and thus obtaining a
satisfactory pH responsiveness because of enough amount CS. While
continuing to increase the amount of HA and CS, the composite nano­
systems became aggregated due to increase in the viscosity. Then, the
concentration of HA was fixed to 0.60 %, the concentration of CS was
changed to select the best mass ratio of HA and CS. As it is showed in
Fig. 3B and Table 1, the release velocity of enrofloxacin from the
nanosystems in different mediums showed no difference when the CS
was decreased to 0.30 %. This might be that CS on the composite
nanosystems is too little to induce the pH-responsiveness. Simulta­
neously, it was found that the composite nanosystems with 0.30 % CS
was released faster than those with 0.60 % CS. This might be due to that
there is no enough CS to form the ionic complex between HA and CS to
hinder the solubility of HA (Bazmandeh et al., 2020). When CS was
increased to 1.2 %, the in vitro release velocity of composite nanosystems
was faster than those using 0.6 % CS although it displayed a certain pH
sensitiveness. The high concentration of CS was too viscous to prepare
the uniform and stable composite nanosystems verified by its larger size
and PDI of 808.4 ± 38.4 nm and 0.55 ± 0.03 (Table 1). The respective
ingredient usages of the optimum enrofloxacin formulations are showed
in Table S1.
3.3. Properties of composite nanosystems and single nanogels
The composite nanosystems was homogenous bright white uniform
suspension with a sedimentation rate of 1. Optical images showed that
the composite nanosystems containing IC was relative uniform. TEM
images displayed that the nanosystems were transparent spherical shape
with smooth surface and good particle size distributions. The mean EE,
LC, sizes, PDI, and ZP of the optimal composite nanosystems were 95.4
± 0.6 %, 10.2 ± 0.3 %, 118.8 ± 30.7 nm, 0.26 ± 0.02, and − 2.20 ± 0.24
Y. Liu et al.
Fig. 5. FTIR of nanogels and its individual components A: Enrofloxacin; B: β-CD; C: IC; D: F68; E: Polymeric nanoparticles; F: HA; G: CS; H: Single nanogels: I:
Designed composite nanosystems.
mv, respectively. The diameter of the nanosystems determined by TEM
was smaller than measured by DLS nm. The difference was probably
because the sizes detected by DLS were carried out in an aqueous state
and the sizes were hydrated diameters, which are usually larger than
their genuine sizes. In the case of TEM sample preparation, all the free
water and even some of hydrated water were evaporated. This implies
that the sizes of solid nanoparticles derived from TEM might be
considerably smaller than their real diameters. The phenomena were
also reported in our previous report (Algharib et al., 2020). LC and EE
are the important parameters as far as the delivery of any drugs is
concerned. The high drug loading is beneficial for reducing the number
7
Carbohydrate Polymers 252 (2021) 117162
of required doses, yet avoid side effects. Although the zeta potential of
the nanosystems was in near to neutral or low range, the certain thick­
ness of F68 layer was effective for its efficient and complete steric sta­
bilization (Xie, Wang, Zhao, Han, & Zhou, 2008). According to the
theory of Derjaguin, Landau, Verwey and Overbeek, the stability of
colloidal system was good after they were covered by a surfactant.
In order to verified the advantages of the designed nanosystems, the
simple nanogels consist of 0.6 % HA and 0.60 % CS without IC and F68
was also prepared. The simple nanogels was homogenous faint yellow
uniform suspension with a sedimentation rate of 1(Fig. 4 B). The mean
EE, LC, sizes, PDI, and ZP were 85.9 ± 3.1 %, 55.4 ± 1.3 %, 107.9 ± 17.7
Y. Liu et al.
Fig. 6. DSC of different enrofloxacin formualtions and its individual components.
nm, 0.18 ± 0.03, and − 12.3 ± 0.2.5 mv, respectively. TEM images
showed that the nanogels was transparent spherical shape (Fig. 4 E).
3.4. Structure features of different enrofloxacin formulations
3.4.1. FTIR
FTIR spectroscopy is an ideal tool to get the information about the
molecular interactions between drugs and ingredients because any
interaction occurred will cause frequency shifts or splitting of the ab­
sorption peaks. The FTIR spectra of enrofloxacin-HA/CS composite
nanosystems and its individual components is showed in Fig. 5.
The characteristic peaks in FTIR spectrum of enrofloxacin (Fig. 5A)
and β-CD (Fig. 5B) were similar with previous reports (Gao et al., 2019;
Kumar et al., 2014; Mashile et al., 2020). The characteristic peaks in
FTIR spectrum of enrofloxacin showed at 1250 cm− 1, 1340 cm− 1,
1500− 1510 cm− 1, 1629 cm− 1, 1730 and 1737 cm− 1, 2968, 2874, and
2823 cm− 1(Kumar et al., 2014). Through the comparison with the FTIR
spectra of β-CD and the IC (Fig. 5C), the FTIR spectrum of IC showed the
peaks at 1629 cm− 1 (C– – O stretching) moved to lower frequencies with
reduced intensities and the disappeared peaks at 1737 cm− 1 (CO2H).
The keto groups of enrofloxacin moved to higher energy frequencies and
disappeared in the IC indicated the complexes formation, since both keto
groups are entrenched in β-CD cavity. The aliphatic vibration absorption
of (CH–) at 2968, 2874, and 2823 cm− 1 of enrofloxacin was dis­
appeared (Fig. 5C), indicating that the piperazine ring was incorporated
into β-CD cavity by van der Waals forces and hydrophobic interactions
(Ding et al., 2020).
For the polymeric nanoparticles, Fig. 5E formulated by entrapping of
IC into F68, the two characteristic spectra of F68 Fig. 5D were dis­
appeared, which indicated that the hydroxy group of F68 might overlap
with the hydroxy group of the IC.
In the spectrum of HA/CS composite nanosystems (Fig. 5I), the
characteristic peak of 1586 cm− 1 for NH+ 3 on CS and the characteristic
peak of 1408 cm− 1, 1602 cm− 1 for COO− of HA was disappeared.
Furthermore, two other bands of 1331 and 1372 cm− 1 were identified,
which coincide with the symmetric stretching of HA carboxylate and CS
C–N, respectively (Barroso et al., 2019). A peak at 3302 cm− 1 was
broadened due to the interaction of phosphate group from TPP with
ammonium group from CS. The results suggested that the spontaneous
interactions between CS and HA generated an ionically cross-linked
network-structure. This formulation mechanism agrees with other
report (Hadidi, Pouramin, Adinepour, Haghani, & Mahdi, 2020). The
FTIR spectra of the single nanogels (Fig. 5H) was different from our
designed composite nanosystems. The characteristic FTIR spectrum of
enrofloxacin were showed in the single nanogels (Fig. 5H). This might be
that the enrofloxacin might be evenly dispersed in the network structure
of nanogels and the drug in single nanogels was not triple-wrapped as
8
Carbohydrate Polymers 252 (2021) 117162
our designed nanosystems.
3.4.2. DSC
The DSC of the nanosystems, intermediate products and its individ­
ual components were also performed to determine the structural inter­
ference amongst enrofloxacin and components and thus to judge
whether the formation of the composite nansystems.
As shown in Fig. 6, the DSC thermogram of enrofloxacin showed one
endothermic peak at 229℃ corresponding to its melting point (Kumar
et al., 2014). There is a broad endothermic peak for β-CD at 180.9℃,
which might be due to the liberation of crystal water from β-CD (Gao
et al., 2019). The melting peaks of enrofloxacin and β-CD disappeared
completely in the IC, indicating that the drug is amorphous or it is
present in the bulk of β-CD (Kumar et al., 2014). The new endothermic
melting peaks at 128℃ and 150.4℃ and small exothermic peak at
97.5℃ were present, which could be associated with the new supra­
molecular compound formation. The measured endothermic peak of F68
was observed at 57.2℃, which is similar with previous reports of 55.99
◦
C and 55.09 ◦ C. The melting peak of the F68 stabilized polymeric
nanoparticles containing IC as core was at 131.2℃, while the endo­
thermic peaks of F68 and IC were completely absent.
DSC curve of CS characterized by an endothermic wide band that
extends from 40℃ to 160℃ corresponding to polymeric dehydration
(Saeed, Dmour, & Taha, 2020). There was obvious endothermic peak for
HA, which is consistent with the report of Barroso et al. (2019). The
second exothermic peak of the HA occurred at approximately 239.9 ◦ C,
which might be attributed to the polysaccharide degradation of HA
(Wang, Tang, Huang, & Hui, 2020). The unique absorption peak of
composite nanosystems containing IC was at 118.1℃, which is different
with each components and intermediate products of the IC and poly­
meric nanoparticles. In addition, the endothermic peak of enrofloxacin
was completely absent, indicating the disordered-crystalline phase of
enrofloxacin was present in the composite nanosystems. It has been
reported that if the encapsulated bioactive material is not being
adequately incorporated into the encapsulating polymer, individual
peaks of each compound will appear (Hadidi et al., 2020). The quite
different of DSC curve of the composite nanosystems from free compo­
nents revealed the successful encapsulation of enrofloxacin by the β-CD
and then dispersed throughout the intertwined supra-molecules of CS
and HA complexation by the help of TPP and F68 in a pattern of stag­
gered arrangements (Barroso et al., 2019).
3.4.3. XRD
XRD was used to analyze the degree of crystallinity and the physical
state of enrofloxacin once incorporated into different carriers. Enro­
floxacin exhibited big diffraction peaks at 7.24◦ , 9.77◦ , and 14.89◦ (2θ)
(Fig. 7A), confirming the drug is crystalline. The main characteristic
Y. Liu et al.
Fig. 7. XRD of designed nanogels and individual components. A: Enrofloxacin; B: β-CD; C: IC; D: F68; E: Polymeric nanoparticles; F: HA; G: CS; H: Composite
nanosystems.
diffraction peaks of the IC were at 10.7◦ , 12.5◦ , and 22.7◦ (2θ) (Fig. 6C),
which were very similar to the latest report (Gao et al., 2019) and was
similar to the main characteristic diffraction peaks of β-CD. The char­
acteristic diffraction peaks of enrofloxacin had almost disappeared in IC
due to the formation of IC (Gao et al., 2019), and which was mutually
corroborated with the SEM image (Fig. 1B). The XRD patterns showed
strong evidence for the IC formulation of enrofloxacin with β-CD (Gao
et al., 2019) and more amorphous pattern when compared to the free
components. The characteristic diffraction peaks of the polymeric
nanoparticles remained the IC and F68 (Fig. 7D), which might be due to
the physical and incomplete encapsulation of IC by F68 confirmed by
TEM image (Fig. 4A). The intensity peak CS at 20◦ (2Ɵ) (Fig. 7F) (Hadidi
et al., 2020; Shetta, Kegere, & Mamdouh, 2018) was disappeared upon
9
Carbohydrate Polymers 252 (2021) 117162
interacting with HA and crosslinking with TPP, indicating a less crys­
talline structure related to the formulation of CS/HA nanogels. As re­
ported before, a decrease in the crystalline structure is due to the
intermolecular and intramolecular network structure of CS when
crosslinked by TPP counter ions, caused disarray in the chain alignment
and subsequent decrease in crystallinity (Milligan, Winstead, & Smith,
2018). The disappeared characteristic diffraction peaks of each
component in the composite nanosystems can be credited to the exis­
tence of strong interactions amongst different components. The dis­
appeared characteristic peaks of F68 in the nanosystems indicate that
the molecule of F68 might be partly inserted into the network structure
of nanogels.
Y. Liu et al. Carbohydrate Polymers 252 (2021) 117162

Table 1 3.7. Enhanced antibacterial activity

Effect of HA and CS on the properties of nanosystems.
Formulation Inclusion HA:CS Size PDI Zeta As showed in the Fig. S2, the zones of microbial inhibitions of the
(%) (%/%) (nm) potential three different enrofloxacin formulations at the same drug concentration
(mV) was the order: composite nanosystems > solution > nanoparticles. The
Nanoparticles 18.75 0.00:0.00 548.4 ± 0.23 ± − 1.3 ± composite nanosystems showed slightly larger zones compared to those
(0:0) 6.7 0.01 0.16 of the solution at the same concentration (Fig. S2). In broth method, the
Simple 0.00 0.60:0.60 107.9 ± 0.18 ± − 12.3 ± MIC values of composite nanosystems against HuBei-01, ShangHai-01,
nanogels (1:1) 17.7 0.03 2.5
0.15:0.15 711.5 ± 0.43 ±
and HuNan-25 were 28, 6, and 1 μg/mL, respectively, and the MIC
18.75 − 7.6 ± 0.7 values of the polymeric nanoparticles were 32, 6, and 1 μg/mL,
(1:1) 41.1 0.03
0.45:0.45 283.6 ± 0.33 ± respectively. These values were smaller than those of enrofloxacin so­
18.75 − 7.0 ± 0.4
(1:1) 38.1a 0.03a lution against the three clinical strains (32, 8, and 2 μg/mL) (Table S2).
Composite 0.60:0.60 118.8 ± 0.26 ± − 2.2 ±
18.75 The in vitro kill curves showed that the enrofloxacin composite nano­
nanosystems (1:1) 30.7a,b,f 0.02a,e 0.2a,b,e
0.60:0.30 615.0 ± 0.22 ± − 10.7 ± systems had stronger activity against the three different strains of
18.75 S. aureus at the same drug concentration (Fig. 8A–C). The number of
(2:1) 29.0c 0.02 0.4c

18.75
0.60:1.20 808.4 ± 0.55 ± − 8.3 ± living S. aureus of the three different clinical strains when incubated
(1:2) 38.4c,d 0.03c,d 0.4c,d with the composite nanosystems for 2 and 4 h was smaller than those
a
Statistical significances compared with HA:CS = 0.15 %:0.15 % are p < 0.05. treated with enrofloxacin solution (Figs. 8D, S3). The enhanced anti­
b
Statistical significances compared with HA:CS = 0.45 %:0.45 % are p < 0.05. bacterial activity by encapsulating drug into nanogels was also reported
c
Statistical significances compared with HA:CS = 0.60 %:0.60 % are p < 0.05. by others. In order to clarify the enhanced mechanism, the co-culture of
d
Statistical significances compared with HA:CS = 0.60 %:0.30 % are p < 0.05. bacteria and composite nanosystems for 30 min was observed by TEM.
e
Statistical significances of composite nanosystems with 0.60 %:0.60 % (1:1) The TEM images showed that the nanosystems were contacted with or
compared with nanoparticles are p < 0.05. absorbed on the bacteria (Fig. 8E–G), which might influence the integ­
f
Statistical significances of composite nanosystems prepared with 0.60
rity of bacterial cell membranes or increase the drugs entering bacteria.
%:0.60 % (1:1) compared with simple nanogels are p < 0.05.
The absorption might be due to that the cationic nature of CS in acid
medium interact with negative charge of bacteria as well as the adhesion
3.5. Hyaluronidase/pH dual responsive release of nanogels (Cheung, Ng, Wong, & Chan, 2015; Logithkumar et al.,
2016). It is reported the adhesion of nanofiber was improved by incor­
The release rate of enrofloxacin from our designed composite poration of HA (Bazmandeh et al., 2020). It is interesting that the
nanosystems containing IC was significantly slower than the single antimicrobial activity of polymeric nanoparticles in the LB agar diffu­
nanogels and polymeric nanoparticles in pH 7.4 and pH 5.5 (Fig. 3A and sion was smaller compared to the enrofloxacin solution while it was
B). This indicates our designed composite nanosystems could effectively stronger than solution by the broth method. This might be due to that
avoid the premature release before reaching the site of infection. the polymeric nanoparticles could not diffuse in agar and thus could not
The bacterial infection site usually presents at pH values of ≈5.5 due contact with bacteria, while it could be absorbed on the bacterial cells in
to the anaerobic fermentation and subsequent inflammation. Therefore, broth medium. For the nanosystems, it was highly hydrophilic and much
the release of the composite nanosystems in pH 5.5 and 7.4 were smaller, so it was easy to diffuse in agar and interact with bacteria and
investigated to evaluate the effects of the environmental pH changes on thus displayed enhanced activity in both methods.
the release. During 4 h, 100 % enrofloxacin was released in the pH 5.5
and 7.4 medium containing hyaluronidase. However, about 72.87 %
4. Conclusions
were released within 4 h and released completely up to 24 h in the pH
5.5 without hyaluronidase. In the pH 7.4 without hyaluronidase, only
In this study, a novel pH/hyaluronidase dual responsiveness IC
39.43 % and 61.83 % were released at 24 h and 36 h, respectively
containing self-assemble HA/CS composite nanosystems covered by F68
(Fig. 3C). The swift release might be induced by the enzymatic degra­
for targeted “on-demand” delivery was successfully explored to treat
dation of the capping HA and structural transformation of CS under the
S. aureus infections. The composite nanosystems formulated by elec­
acid condition. The protonation and surface charge reversal of CS at low
trostatic interaction between CS and HA containing IC and coating with
pH is owe to the effective release in the acidic medium. The CS has an
F68. The nanosystems displayed more excellent sustained release
amino group that can undergo protonation at low pH owing to increase
compared to the polymeric nanoparticles containing IC and the simple
its solubility in acidic solution. Moreover, the surface charge reversal of
nanogels without IC. The designed nanosystems showed quick release in
the nanogels occurs when protons or hydronium ions transfer from the
the acid medium with hyaluronidase and a liquid medium where
bulk solution to the particle surface at the acidic pH, leading to desta­
S. aureus grows. The composite nanosystems could be absorbed on the
bilization for effective release in the acidic release medium.
surface of S. aureus and thus displayed enhanced antibacterial activity.
This result confirmed that our designed composite nanosystems can
3.6. Bacterial environmental responsiveness
decrease the premature drug release and enhance the targeting release
at S. aureus by ingenious combining of IC with HA/CS nanogels stabi­
There was a significant burst release when S. aureus was existing,
lized by F68. Therefore, the present study may serve as a fruitful strategy
reaching a nearly 100 % cumulative release of total encapsulated
to overcome the treatment challenge of S. aureus as well as other bac­
enrofloxacin within 1− 4 h. The release rate of composite nanosystems
terial infections.
was swiftest in broth culture containing HuNan-25 (most resistance) and
slowest in culture containing HuBei-01 (most sensitivity). The differ­
CRediT authorship contribution statement
ences might be due to the different enzyme secretion level of different
strains. The hyaluronidase secreted by S. aureus can affect β-1, 4
Yuda Liu: Conceptualization, Methodology, Data curation, Writing -
glycosidic bonds. The release rate was significantly slowest in broth
original draft. Dongmei Chen: Supervision, Writing - original draft,
culture without S. aureus compared to those in the bacteria-containing
Writing - review & editing. Aoxue Zhang: Visualization, Validation.
medium. These results suggest that the triggered rapid drug release
Man Xiao: Investigation. Zhenxia Li: Investigation. Wanhe Luo:
will occur after its arrival at the S. aureus infectious sites.
Investigation. Yuanhu Pan: Validation. Wei Qu: Validation. Shuyu
Xie: Supervision, Conceptualization, Methodology, Data curation,

10
Y. Liu et al. Carbohydrate Polymers 252 (2021) 117162

Fig. 8. Antibacterial activity of different enrofloxacin formulations against S. aureus. Kill curves of solution, and composite nanosystems (A) against S. aureus Hubei-
01(A), ShangHai-H1908-01 (B), and HuNan-25 (C); D1-9: Number of living S. aureus incubation with different formulation for 2 h; E-F: TEM images of co-culture of
bacteria and nanogels for 30 min.

Writing - review & editing.
Acknowledgments
This work was supported by the National Natural Science Foundation
of China (grant no. 31772797) and the Fundamental research Funds for
the Central Universities (2662020DKPY008).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2020.117162.
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