Biomaterials 299 (2023) 122129

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Sustained release of levobupivacaine from temperature-sensitive injectable

hydrogel for long-term local anesthesia in postoperative pain management
YuJun Zhang a, c, 1, Kun Shi b, 1, Xi Yang b, Wen Chen b, TianHong Wang a, c, Yi Kang a, c,
DeYing Gong a, c, ZhiYong Qian b, **, WenSheng Zhang a, c, *
a
Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu, 610041, China
b
Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China
c
Laboratory of Anesthesia and Critical Care Medicine, National-Local Joint Engineering Research Centre of Translational Medicine of Anesthesiology, West China
Hospital, Sichuan University, 610041, China

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

Keywords: Postoperative pain is a major concern for most of the surgical patients, and an inadequate postoperative pain
Injectable thermo-sensitive hydrogel control may cause a series of complications. With an effective pain control and lesser side effects, local anes­
pH adjustment thetics are preferred for use in postoperative pain management. However, the action duration of current local
Long-acting local anesthetic
anesthetics is too short to meet the requirements of postoperative analgesia. In this study, an injectable levo­
Levobupivacaine
Postoperative analgesia
bupivacaine (LB)-loaded thermo-sensitive hydrogel system based on biodegradable poly(D,L-lactide)-poly
(ethylene glycol)-poly(D,L-lactide) (PLEL) was developed for long-acting local anesthetic, in which the soluble
charged cation form of LB (LB HCl) was partly alkalified to the poorly soluble base form (LB base). This hybrid LB
loaded PLEL system (hLB/PLEL) is a free flowable liquid at room temperature and changes into a semi-solid
hydrogel once injection in response to the physiological temperature. Then, the dissolved LB HCl could
release firstly from the hydrogel contributing to a quick work, and the insoluble LB base dissolved and released
gradually as the decrease of the pH during the biodegradation of PLEL hydrogel, resulting in a long-term LB
release in local. The drug release behavior, pharmacokinetic, and biocompatibility of the thermo-sensitive hLB/
PLEL were studied in vitro and in vivo. The anesthetic effects of hLB/PLEL system were evaluated in the rat models
of sciatic nerve block, subcutaneous infiltration anesthesia and postoperative pain as well. This hLB/PLEL system
generated a significantly prolonged analgesic effect in rat models, which produced approximately 7 times longer
duration than 0.75% LB HCl and effectively relieved the spontaneous pain for 3 days. In general, the presented
hLB/PLEL system can not only achieve a fast-acting but also sustainably release LB to block the nerve and
significantly extend the effect of local analgesia, which means a promising candidate for long-acting post­
operative pain management.

1. Introduction
More than 80% of applicable patients experience have postoperative
pain, and 75% of which has moderate to extreme pain scores [1].
Inadequate postoperative pain control may cause a series of complica­
tions, such as prolonged hospital stay, chronic pain, and drug addiction
[2]. The major drug categories for postoperative pain treatment are
opioid analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), and
local anesthetics [3]. In recent years, multimodal analgesic strategies for
enhanced recovery after surgery have focused on reducing the use of
opioid agents to avoid opioid-related adverse effects such as drug
addiction, gastrointestinal distress, sedation, ileus, pruritus, and respi­
ratory depression [3–5]. Furthermore, an important warning published
by the US Food and Drug Administration indicated that opioid abuse was
the most pressing public health crisis [6]. It is generally accepted that
NSAIDs are not necessary for postoperative pain control because of their

Abbreviations: LB, Levobupivacaine; PLEL, poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide); hLB/PLEL, hybrid LB loaded PLEL system.
* Corresponding author. Department of Anesthesiology, Laboratory of Anesthesia and Critical Care Medicine, National-Local Joint Engineering Research Centre of
Translational Medicine of Anesthesiology, West China Hospital, Sichuan University, China.
** Corresponding author. Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, China.
E-mail addresses: zhiyongqian@scu.edu.cn (Z. Qian), zhang_ws@scu.edu.cn (W. Zhang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.biomaterials.2023.122129
Received 18 November 2022; Received in revised form 10 April 2023; Accepted 20 April 2023
Available online 1 May 2023
0142-9612/© 2023 Elsevier Ltd. All rights reserved.
Y. Zhang et al.
analgesic ceiling effect as well as a series of severe side effects, including
gastrointestinal toxicities, cardiovascular risks, renal injuries and hep­
atotoxicity [7–11]. Thus, with effective pain control and fewer side ef­
fects, local anesthetics are preferred for postoperative pain
management. The most serious degree of postoperative pain was within
72 h after surgery, which did not exceed 7 days [1]. However, the effects
of currently available local anesthetics do not exceed 12 h after subcu­
taneous infiltration [12]. The development of a long-acting local anes­
thetic for postoperative pain management is urgently needed [13].
Local anesthetics-induced analgesic effect usually achieved through
reversible blockade of neuronal sodium channels to prevent the gener­
ation and the conduction of nerve impulses [14]. The investigation
strategies for a novel long-acting local anesthetic primarily includes the
invention of new chemical compounds and the development of
slow-release drug delivery systems. A few new chemical compounds,
such as QX-314 [15], QX-OH [16], Neosaxitoxin [17], and EN3427 [18]
have been shown to produce long-acting local anesthesia in animal
models. Unfortunately, one of the most promising new chemical com­
pounds with the longest duration of action, EN3427, have been proven
to produce analgesia for only 24 h after a single injection. Slow-release
drug delivery systems developed by encapsulating a local anesthetic via
a carrier hold the potential to provide a prolonged period of analgesia
effect and to overcome the short duration of local anesthetic effect [19].
Specifically, a variety of formulations, including microparticles [20],
nanoparticles [21], liposomes [22], cyclodextrins [23] hydrogels [24]
and composite systems [25], have all been investigated to develop a
long-acting local anesthetic drug products. Typically, Exparel™ (Pacira
Pharmaceuticals, USA) is a liposomal encapsulation of 13.3 mg/mL of
bupivacaine arranged in a microscopic honeycomb-like structure and is
delivered directly to the surgical site via injection, which provides
post-surgical local analgesia for up to 72 h [26]. However, patients who
underwent total knee arthroplasty and received Exparel™ treatment did
not report a clinically meaningful reduction in inpatient opioid pre­
scription, resource utilization, or opioid-related complications [27]. In
short, the action duration of current local anesthetic-based formulations
is still too short to meet the requirements of postoperative pain
management.
Scheme 1. Schematic illustration of levobupivacaine-loaded temperature-sensitive injectable hydrogel system (hLB/PLEL), which is used for sustained release of
anesthetic to achieve long-term local anesthesia in postoperative pain management.
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Biomaterials 299 (2023) 122129
For long-term local anesthesia in postoperative pain management,
how to effectively prolong the action duration of local anesthetic re­
mains a challenge currently. Several studies have reported that the
alkalization of local anesthetic benefited to extend the duration of action
[28,29]. However, unexpected soft tissue necrosis at the injection site
induced by the precipitate of the base form of local anesthetic was
observed, which limited the application of this strategy [30,31]. To
solve this problem, drug delivery systems were tried to encapsulate the
base form of local anesthetic. Although some of them have reported to
prolong the analgetic duration and avoid the risks of local toxicity to
some extent, the effects are still far from satisfactory [25,32,33]. Thus,
developing a novel local anesthetic-based formulation for long-acting
postoperative pain control should be urgently addressed.
Levobupivacaine (LB), the S(− )-enantiomer of bupivacaine, is a
common used long-acting local anesthetic for infiltration, nerve block,
ophthalmic, epidural and intrathecal anesthesia. What’s more, levobu­
pivacaine has been proven to produce a greater length of action and a
lower risk of cardiotoxicity comparison to bupivacaine, thus attracted
more and more attention [34]. Similar to the other local anesthetics,
levobupivacaine normally exist in two forms in solution, specifically
presents as cycles through a rapid equilibrium between a charged
cationic form (good solubility in water) and uncharged base form (base
form, poorly soluble in water). In this study, we proposed an injectable
hybrid LB-loaded hydrogel (hLB/PLEL) system based on an biodegrad­
able thermosensitive hydrogel, in which the soluble charged cation form
(LB HCl) and insoluble base form (LB base) co-existed through adjusting
the initial pH of the PLEL hydrogel system (Scheme 1). The thermo­
sensitive hydrogel was made of poly(D,L-lactide)-poly(ethylene glyco­
l)-poly(D,L-lactide) (PLEL) amphiphilic triblock copolymer, whose
biocompatibility and potential for local drug sustained delivery has been
demonstrated in our several previous studies already [35–37]. This
developed hLB/PLEL system is a free-flowable liquid at room tempera­
ture and changes into a semi-solid hydrogel in situ once injected in
response to the physiological temperature. This characteristic is not only
helpful for easier management but also conducive to avoid the local
precipitation of the LB base. More importantly, after administration, the
dissolved LB HCl could initially release from the hydrogel and
Y. Zhang et al.
contributed to a quick nerve block for a rapid analgesic effect, while the
undissolved LB base dissolved and released gradually as the pH
decreased during the biodegradation of the PLEL hydrogel, resulting in a
long-term local LB HCl release. In another words, this designed
hLB/PLEL system characterized by self-adjustable drug release due to its
changing pH probably achieve fast-acting, long-term anesthesia, and
less side effect at the same time. Here, the drug release behavior and
biocompatibility of the thermosensitive hLB/PLEL were studied in vitro
and in vivo detailedly. The anesthetic effects of the hLB/PLEL system
were evaluated in sciatic nerve block, subcutaneous infiltration anes­
thesia, and acute postoperative pain rat models systematically.
2. Materials and methods
2.1. Materials
Poly(ethylene glycol) (PEG, Mn = 1500) was purchased from Nanj­
ing Well Pharmaceutical co., Ltd. (China.). Stannous octoate (Sn(Oct)2),
acetonitrile, formic acid, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe­
nyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich
(USA). D,L-lactide (D,L-LA) was purchased from Jinan Daigang Bioma­
terial Co., Ltd. (China.). Levobupivacaine hydrochloride (LB HCl) was
purchased from Hubei Humanwell Pharmaceutical, Ltd. (China). Ropi­
vacaine (purity: 94.2%) was purchased from the China National In­
stitutes for Food and Drug Control. Dulbecco’s modified Eagle’s medium
(DMEM), penicillin-streptomycin, and fetal bovine serum (FBS) were
obtained from Gibco (USA). A live-dead cell staining kit was purchased
from Keygen Biotech Corp., Ltd. (China.). Cy5.5 was purchased from the
Beijing Fanbo Biochemicals Company (China). Isoflurane and normal
saline were purchased from Hebei Yipin Pharmaceutical Co., Ltd.
(China) and Chengdu Qingshanlikang Pharmaceutical Co., Ltd. (China),
respectively.
Sprague-Dawley rats (aged 7–9 weeks, weighing 240–280 g) were
purchased from Chengdu Dossy Biological Technology Co., Ltd. (China)
and maintained in a controlled environment with a 12 h light/dark
cycle. Food and water were provided ad libitum. Before experiments
were initiated, the rats underwent a minimum 5 d adaptation period. All
experiments were approved by the Committee of Animal Care of the
West China Hospital, Sichuan University, China (2015015A) and carried
out in accordance with the Guidance on the Care and Use of Laboratory
Animals (U.S. National Institutes of Health Publication No. 80-23,
revised in 1996).
2.2. Preparation and characterization of levobupivacaine-loaded
thermosensitive hydrogel
2.2.1. PLEL copolymer synthesis
The PLEL triblock copolymer was synthesized via ring-opening
copolymerization to prepare an injectable thermosensitive hydrogel,
as described in our previous study [35]. Briefly, the amounts of PEG and
D,L-LA were calculated, and Sn(Oct)2 was introduced into a nitrogen
atmosphere under a dry glass flask. The mixtures were kept at 140 ◦ C
under mild agitation and nitrogen protection. After 12 h, the resulting
crude copolymer was washed with water at approximately 80 ◦ C for
purification. The sediment was then lyophilized to a constant weight and
stored at − 20 ◦ C until needed. The molecular weight of the obtained
PLEL copolymer was characterized using nuclear magnetic resonance
spectroscopy (1H NMR, Varian 400 spectrometer, USA) and gel perme­
ation chromatography (GPC, Agilent 110 HPLC, USA).
2.2.2. Levobupivacaine loaded PLEL hydrogel preparation
The composites of LB/PLEL and hLB/PLEL were fabricated by dis­
solving LB HCl in a PLEL copolymer solution. Briefly, the prepared PLEL
copolymer (10 wt%, 15 wt%, 20 wt%) was fully dissolved in phosphate
buffered saline (PBS) with different pH by mild agitation for 10 h to get a
transparent micelle solution with a different pH (pH 5.3, pH 7.0, and pH
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Biomaterials 299 (2023) 122129
8.0). A predetermined amount of LB HCl was then added to the copol­
ymer solution and stirred at room temperature for 10 min to obtain a
homogeneous suspension.
2.2.3. Dynamic rheological study of hLB/PLEL
The thermosensitive solution-hydrogel (sol-gel) phase transition of
hLB/PLEL was investigated using a rheometer (HAAKE Rheostress 6000,
Thermo Scientific, USA) through a parallel plzate (diameter: 20 mm).
The storage modulus (G′ ) and loss modulus (Gʺ) of hLB/PLELs with
different PLEL concentrations and LB concentrations were measured as
functions of temperature from 20 ◦ C to 50 ◦ C. The heating rate was set at
2 ◦ C/min. The data were collected at frequency of 1.0 Hz and a
controlled stress of 1 Pa. Rheological analysis of the PLEL hydrogel (10
wt%, 15 wt%, 20 wt%) was conducted for comparison. The viscosity of
blank solution with different polymer concentrations were tested around
room temperature.
2.2.4. Undissolved drug analyses
Because of the initial neutral pH of the PLEL, dissolved LB HCl and
undissolved LB base co-existed in the hLB/PLEL system. The undissolved
LB base dissolved gradually with a decrease in pH during biodegradation
of the PLEL hydrogel. Therefore, the pH and undissolved LB base
amounts were detected at different time points. Briefly, hLB/PLEL
samples with different drug concentrations were maintained at 37 ◦ C to
form a stable hydrogel. At predetermined intervals, three samples from
each group were collected and kept at room temperature for 20 min to
obtain fluid samples. The pH of each sample was tested and centrifuged
for 10 min at 3000 rpm at 4 ◦ C. The sediment was completely dissolved
in acidic normal saline and quantitatively measured using HPLC with a
reversed-phase C18 column (4.6 mm × 5 mm, 5 μm) at a wavelength of
250 nm. A solution of 20 mM aqueous sodium bicarbonate and aceto­
nitrile (35:65, v/v) was used as the mobile phase for isocratic elution and
delivered at a flow rate of 1 mL/min. The pH change in the PLEL
hydrogel at 37 ◦ C was also detected.
2.3. In vitro cell experiments
2.3.1. MTT test
HT22 and C2C12 cells were used to examine the in vitro cytotoxicity
of the PLEL hydrogel on nerve and muscle cells using the MTT assay.
C2C12 and HT22 cells were purchased from the American Type Culture
Collection (ATCC, USA) and cultured in DMEM medium containing 10%
FBS, supplemented with 1% penicillin and streptomycin at 37 ◦ C in a
humidified 5% CO2 atmosphere. Briefly, different concentrations of
PLEL polymer solution were prepared in DMEM medium, and the
leachate of the PLEL hydrogel was prepared by extraction in DMEM
medium for 24 h and diluted sequentially. The cell suspensions were
seeded in 96-well plates at a density of 3500 cells/well and incubated for
24 h. The medium was then replaced with fresh medium with different
concentrations of the PLEL polymer or PLEL hydrogel leachates. After
culturing for another 48 h at 37 ◦ C, the cells were subjected to the MTT
assay, and the absorbance was measured using a microplate reader (Bio-
Rad) at 570 nm. The relative viability of the treated cells was calculated
as a percentage of the untreated control group.
2.3.2. Live/dead assay
A live/dead assay was conducted to assess the biocompatibility of the
PLEL hydrogel using C2C12 and HT22 cells. The cells were cultured in
24-well plates (1 × 104 cells/well) for 24 h, and the medium was
replaced with the prepared PLEL polymer solution (1 mg/mL) or PLEL
hydrogel leachate (100%). After 24 h or 48 h of culture, cells were
stained with 2 μM Calcein AM and 8 μM PI (Live/Dead cell staining kit)
for 30 min at room temperature. Fluorescent images were then observed
under a fluorescence microscope (Olympus, Tokyo, Japan) and images
were analyzed using ImageJ 7.0 software.
Y. Zhang et al.
2.4. In vivo histological analyses and immunohistochemistry analyses
2.4.1. Histological analyses
Male rats (n = 8) were anesthetized with 2% isoflurane in oxygen
delivered in an induction chamber and placed them in the right lateral
position, following the method published in our previous study [16] A
23G needle with a syringe was inserted into the center of the line, linking
the greater trochanter and the ischial tuberosity of the left leg. Once the
needle made bone contact, 0.2 mL of normal saline or the PLEL solution
was administered. The animals were euthanized with an overdose of
pentobarbital sodium after the 1, 3, 7, and 14 days of sciatic nerve block
(n = 2 per day). The sciatic nerves and adjacent muscles were collected
and soaked in 10% buffered formalin. Hematoxylin-eosin (H&E) stain­
ing was performed [16], and the degree of inflammation of the sciatic
nerve and muscle of each rat was scored by two blinded investigators in
accordance with an established scoring standard [38,39]: 0 = normal; 1
= 0%–25% of area involved; 2 = 25%–50% of area involved; 3 = 50%–
75% of area involved; and 4 = 75%–100% of area involved.
2.4.2. Immunohistochemistry analyses
The inflammatory factors, including interleukin-1 (IL-1), IL-6, and
tumor necrosis factor-α (TNF-α), was further carried out to evaluate the
inflammatory response caused by the PLEL [25]. The sciatic nerve and
surrounding muscles were dewaxed and rehydrated in gradual ethanol
series, followed by immersion in boiling citrate solution (pH = 6) for
antigen retrieval for 60 min. Then, the sections were blocked with
donkey serum for 30 min at 37 ◦ C. The antibody solutions of IL-1, IL-6,
and TNF-α (1:1000; Abcam Company, USA) were added and incubated
overnight at 4 ◦ C. Next day, the sections were rinsed with PBS and
incubated with secondary antibody solution (1:100; Life Technologies
Co., Ltd., USA) for 45 min at 37 ◦ C. Then, the cell nuclei were stained
with DAPI. The sections were visualized under CaseViewer (3DHISTECH
Co., Ltd., HU), and semi-quantitatively analyzed by ImageJ software
(National Institutes of Health, USA).
2.5. Drug release of hLB/PLEL
2.5.1. In vitro drug release
Dialysis method was used to study the in vitro drug release behavior
of LB/PLEL and hLB/PLEL. Briefly, 1 mL of the prepared 0.75% LB so­
lution or hLB/PLEL solutions with different LB concentrations (0.75%,
2%, and 4%) and 0.75% LB solution with different PLEL concentrations
(10 wt%, 15 wt%, and 20 wt%) were transferred to a dialysis bag
(molecular weight cut-off = 8000). The dialysis bags were placed in test
tubes and equilibrated at 37 ◦ C for 30 min to form a stable hydrogel.
Thereafter, 20 mL of pre-warmed phosphate-buffered saline (PBS, pH
7.4) was added to the test tubes, and the samples were incubated at 37
◦
C in a shaking incubator at 100 rpm. At predetermined intervals, the
release medium was collected and replaced with 20 mL of fresh pre-
warmed PBS. The in vitro release of the LB solution from the dialysis
bag was studied using the same method. The amount of LB released into
the medium was measured using HPLC, as described previously. The
studies were conducted three times, and all data were averaged over
three individual results.
2.5.2. In vivo drug release
To intuitively observe the in vivo drug release of the PLEL hydrogel,
water-soluble Cy5.5 was used as a substitute for LB. After the rats were
anesthetized with 2% isoflurane in oxygen, 0.2 mL of Cy5.5 solution or
the Cy5.5 loaded PLEL solution (Cy5.5/PLEL, 20 wt%) was dorsally and
injected around the sciatic nerve with a 23G needle. Rats were anes­
thetized at time intervals of 0.25, 8, 24, 48, and 72 h, and fluorescence
images were acquired using an IVIS Lumina III imaging system (Perki­
nElmer, Caliper Life Sciences, MA, USA; excitation = 673 nm, emission
= 692 nm). The images were analyzed using the onboard software.
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Biomaterials 299 (2023) 122129
2.6. Pharmacokinetic study
2.6.1. Sample preparation and liquid chromatography-mass spectrometry
analysis
After the sciatic nerve block, male rats (n = 6 in each group) were
used to investigate the pharmacokinetics of 0.75% LB, 4% LB, and 4%
hLB/PLEL. Blood samples (0.2 mL) were collected from the caudal vein
and transferred to heparinized polypropylene tubes. Subsequently,
plasma was collected after centrifugation at 800×g for 10 min. The
sampling time points were as follows: pre-dose; 0.17, 0.33, 0.5, 0.67,
0.83, 1, 2, 3, 4, 5, 6, and 8 h after dosing. According to the method
described in a previous study [40], plasma samples were deproteinized
using acetonitrile solution with an internal standard (ropivacaine;
50:150, v/v). The mixture was then fully vortexed and centrifuged for
10 min at 28,621×g in 4 ◦ C, and the resultant supernatant was diluted
with ultrapure water (50:50, v/v). The liquid chromatography-mass
spectrometry system consisted of an Agilent 6460 triple quadrupole
mass spectrometer and an electrospray ionization source (Agilent
Technologies, CA, USA). Chromatographic separation was performed
using an Agilent Extend C18 column (100 mm × 3 mm, 3.5 μm) at 35 ◦ C
with an isocratic elution containing 0.05% formic acid and acetonitrile
at a volume ratio of 78:22 and a flow velocity of 0.3 mL/min. The total
running time for each sample was 5 min. Mass spectrometry was per­
formed in the positive ionization mode under the following conditions:
sheath gas flow rate, 11.0 L/min; sheath gas heater temperature, 300 ◦ C;
nebulizer pressure, 45 psi; capillary voltage, 3500 V. MassHunter soft­
ware was used to analyze the data (B.04.00 Build 4.0.479.0, Agilent
Technologies).
2.6.2. Pharmacokinetic analysis
A non-compartmental pharmacokinetic model was used to calculate
the value of maximum concentration (Cmax), the time to acquire Cmax
(Tmax), half-life (t1/2), and area under curve (AUC) from zero to the last
time point (AUC0-last), and from zero to infinity (AUC0-∞) by using DAS
software (version 3.2.1, Drug and Statistics, China).
2.7. Anesthetic effects of hLB/PLEL system in rat models
2.7.1. Sciatic nerve blockade model
Male rats (n = 8 in each group) were randomly divided into eight
groups: (1) normal saline, (2) PLEL, (3) 0.75% LB, (4) 0.75% hLB/PLEL,
(5) 1% hLB/PLEL, (6) 2% hLB/PLEL, (7) 3% hLB/PLEL, and (8) 4% hLB/
PLEL groups. The method of sciatic nerve blockade was elaborated in the
2.4.1 section. In accordance with a previous study [16], thermal noci­
ception was used to evaluate sensory function, which was determined in
the same anatomic area by using a modified hotplate at 56 ◦ C (model
RB-200 Hotplate Analgesia Meter; Chengdu Technology & Market Co.,
Ltd., China), and the cutoff value was set to 6 s to prevent injury or
hyperalgesia. The time taken by the rats to withdraw the paw was
measured using a stopwatch. Motor function was determined by
measuring the extensor muscle force using a digital balance (model
HZT-B5000, Huazhi Scientific Instrument Co., Ltd., Fuzhou, China). The
rat was held upright over a digital balance, with the hind limbs extended
to the platform. When the force was less than 60 g, motor function was
considered to be blocked. The measurements were recorded at pre-dose,
10, 30, 60 min, then hourly until 12 h, then six hourly until 24 h, and
then once every 12 h until sensory and motor functions were fully
recovered. At each point, thermal nociception was measured only once;
three repeated measurements of extensor muscle force were obtained
and averaged.
The animals were euthanized with an overdose of pentobarbital so­
dium after the 1, 3, 7, and 14 days of sciatic nerve block (n = 2 per day).
The sciatic nerves and adjacent muscles were collected and soaked in
10% buffered formalin. Hematoxylin-eosin (H&E) staining was per­
formed [16], and the degree of inflammation of the sciatic nerve and
muscle of each rat was scored by two blinded investigators in
Y. Zhang et al.
accordance with an established scoring standard [38,39]: 0 = normal; 1
= 0%–25% of area involved; 2 = 25%–50% of area involved; 3 = 50%–
75% of area involved; and 4 = 75%–100% of area involved.
2.7.2. Subcutaneous infiltration anesthesia model
Male rats (n = 8 in per group) were randomly divided into two
groups: (1) 4% LB and (2) 4% hLB/PLEL. Cutaneous trunci muscle reflex
(CTMR) was used to measure the effect of subcutaneous infiltration
anesthesia effect [16]. After the rats were anesthetized with 2% isofl­
urane in oxygen, they were administered subcutaneously with 0.2 mL of
the test solution in the lateral thoracispinal site, which was marked with
a nontoxic pen for the pinprick responsiveness (PPR) measurement.
Then, the standardized stimulus to determine PPR was measured using
an 18G needle affixed to a 26 g von Frey filament, and six pinpricks were
tested at pre-dose, 10, 30 min, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 36, and 48
h until the CTMR fully recovered. For each stimulation, it was accepted
that PPR = 1 if the lateral thoracispinal muscles induced a skin move­
ment over the back and that PPR = 0 if reflex movement was absent.
2.7.3. Acute postoperative pain model
Male rats (n = 6 in per group) were randomly divided into four
groups: (1) saline, (2) PLEL, (3) 0.75% LB, and (4) 4% hLB/PLEL. The
commonly used incisional pain model was used as described in a pre­
vious study [41]. Briefly, rats were anesthetized with 2% isoflurane in
oxygen, and the left leg was prepared using an iodophor solution for
disinfection. Then, a 1 cm longitudinal incision, starting 0.5 cm from the
proximal edge of the heel and extending toward the toes, was made with
a number 11 blade through the skin and fascia. The plantaris muscle was
elevated and incised in all rats. Finally, the skin was sutured using a 5-0
nylon surgical sutures. After surgery, 25 μL of the drug was injected
adjacent to the wound, and the rats were placed in their cages for
recovery.
The mechanical withdrawal threshold and pain score by weight were
used to measure pain behavior. The mechanical withdrawal threshold
was determined by placing the rats on a plastic mesh floor (12 mm × 12
mm grid) and using von Frey filaments starting with 0.4 g and
continuing until a withdrawal response occurred or the cutoff value (26
g) was reached. The testing site was located in the same area adjacent to
the wound near the heel. The pain score by weight was determined by
placing the rats on a plastic mesh floor and allowing them to acclimate
for at least 15 min. Each rat was observed once every 5 min for 1 h. The
scoring standard depended on the foot position: 0 = the wound was
blanched or distorted; 2 = the foot was completely off the mesh;
otherwise, the score was 1. The total pain score at 1 h represents the
degree of spontaneous pain. The time points for pain behavior mea­
surements were baseline, 2 h after surgery, once daily in the first 3 days
after surgery, and 7 days after surgery.
2.8. Statistical analyses
Homogeneity-variance data were assessed using ANOVA followed by
Dunnett’s test to compare the treatment groups with the control.
Otherwise, the data were assessed using the Kruskal-Wallis test followed
by the Wilcoxon signed-rank test to compare with the control, and
Bonferroni correction was used to adjust the p values. The level of sta­
tistical significance was set at p < 0.05. Data were analyzed using SPSS
Statistics (version 23, Chicago, IL, USA).
3. Results and discussion
3.1. Preparation and characterization of levobupivacaine-loaded PLEL
hydrogel
The biodegradable PLEL copolymer was prepared via ring-opening
copolymerization of D,L-LA initiated by PEG, using Sn(Oct)2 as the
catalyst. According to the 1H NMR spectrum and GPC measurements, the
5
Biomaterials 299 (2023) 122129
average molecular weight of the prepared copolymer was 4520 (PEG/
PDLLA ratio: 1500: 3020), and the molecular weight was distributed in a
unimodal manner, indicating that the PLEL copolymer was successfully
synthesized.
LB is a clinic long-acting local anesthetic. With the similarly physi­
cochemical property of other local anesthetics, LB consist of two forms
in solution, and which exist in a rapid equilibrium between the basic
uncharged form (LB base, poorly soluble in water) and the charged
cationic form (LB HCl, good soluble in water). The amount of LB that
exists in solution as LB base is determined by its acid dissociation con­
stant (pKa) and the pH of solvent, and then it can change to soluble LB
HCl when the pH changes from alkalinity or neutral or to acidic [42]. In
clinical practice, LB commonly is used at the highest concentration of
0.75%. Thus, we used the 0.75% LB HCl solution as a positive control
when optimized the LB-loaded PLEL hydrogel systems in this study. As
shown in Fig. 1A, the thermo-sensitive behavior of three types of
LB-loaded PLEL hydrogel composites (0.75% LB) made by PLEL solu­
tions with different pH were tested firstly. The LB-loaded PLEL hydrogel
composites made by acidic was transparent and homogeneous, while the
ones made by neutral and alkaline PLEL solution were cloudy, suggested
a hybrid of LB HCl form and LB base form. Both the LB/PLEL made by
acidic or neutral PLEL solution were free flowable liquids at room
temperature and changed into a semi-solid gel rapidly around body
temperature, which were similar to the sol-gel of blank PLEL hydrogel.
However, in the case of LB/PLEL made by alkaline PLEL solution, a
longer time was needed to be hydrogel at 37 ◦ C, and the hydrogel is
unstable and weak. Therefore, we chose the acidic and neutral PLEL
solution to prepare LB-loaded hydrogel systems for further study, which
were denoted as LB/PLEL and hLB/PLEL, respectively. In addition, We
noticed that the pH of the PLEL hydrogel and hLB/PLEL were decreased
with time when the samples were kept at 37 ◦ C, which may be caused by
the generation of D,L-lactide and low-molecular-weight oligomers dur­
ing the biodegradation of the PLEL polymer (Fig. 1B). As a result of the
pH decrease of hydrogel, the hLB/PLEL samples became clearer with
time and almost transparent after 96 h, suggesting the gradual dissolu­
tion of the insoluble LB base (Fig. 1C). Simultaneously, the hLB/PLEL
samples presented stable and homogeneous hydrogels at 37 ◦ C without
precipitation. Generally, drug delivery systems made by physical
hydrogels exhibit an initial burst release owing to the quick diffusion of
small-molecule drugs. The dynamic dissolution of the LB base in
hLB/PLEL may relieve the initial burst release and extend the period of
drug release. Thus, the drug release behavior of LB/PLEL and hLB/PLEL
was studied in vitro. As expected, hLB/PLEL showed the slowest drug
release compared with the LB/PLEL and free LB solution groups, sug­
gesting the potential for long-term local anesthesia (Fig. 1D). Consid­
ering the possible effect of hydrogel concentration on drug release,
different concentrations of hydrogel (10 wt%, 15 wt%, and 20 wt%)
were used to prepare hLB/PLEL as well. With the increase of PLEL
concentration from 10 wt% to 20 wt%, the slow-release effect of
hLB/PLEL was improved gradually (Fig. 1E), implying that a higher
polymer concentration benefits to the sustained drug release. Although
we have tried to make hLB/PLEL using PLEL hydrogel in 25 wt% to get a
delaying drug release, it failed to carry out. Because the 25 wt% PLEL
was found to have a higher viscosity at room temperature (Fig. S1),
which is not only unfriendly to the dissolution and dispersion of LB, but
also difficult to inject in vivo and sterilize by filtration. Thus, hLB/PLEL
with a concentration of 15 wt% and 20 wt% seemed to be the superior
candidates for further formulation optimization.
Encouraged by the in vitro drug release results, we preliminarily
evaluated the local anesthetic effect of hLB/PLEL and LB/PLEL using a
rat sciatic nerve block model (Fig. 1F). The duration of sensory blockade
(5.88 ± 1.13 h) in 0.75% hLB/PLEL treated group showed a significant
difference with that of 0.75% LB/PLEL group (4.63 ± 0.74 h, p = 0.02)
and 0.75% LB (3.13 ± 0.35 h). For motor blockade, 0.75% hLB/PLEL
(6.38 ± 1.69 h) also produced a significantly longer duration than
0.75% LB/PLEL (4.63 ± 0.74 h, p = 0.018) and 0.75% LB (3.25 ± 0.46
Y. Zhang et al. Biomaterials 299 (2023) 122129

Fig. 1. The sol-gel phase transition behavior of levobupivacaine-loaded thermosensitive hydrogel system. (A) Thermosensitive sol-gel phase transition of blank PLEL
hydrogel and LB-loaded hydrogel systems. (B) Changes in pH of PLEL hydrogel and hLB/PLEL upon time. (C) Transparency changes of hLB/PLEL upon time. (D) In
vitro drug release of different LB-loaded hydrogel systems. (E) In vitro drug release of hLB/PLEL with different concentrations of PLEL hydrogel. (F) Schematic
representation of rat sciatic nerve block model. (G) Duration of sensory blockade and motor blockade on the rat sciatic nerve block model after treated with different
LB preparations. (H) Effect of local anesthesia of hLB/PLEL with different polymer concentrations on the rat sciatic nerve block model. Data are presented as mean
± SD.

h) (Fig. 1G). These results indicate that hLB/PLEL containing a hybrid of
LB HCl and LB base is superior in sustainably releasing LB and extending
the effect of local anesthesia. Furthermore, we compared the local
blockade effect on the sensory and motor blockade functions of hLB/
PLEL made with 15 wt% and 20 wt% PLEL hydrogels (Fig. 1H). Both the
sensory and motor blockade durations in rats treated with hLB/PLEL
(20 wt% PLEL) were longer than those in rats treated with hLB/PLEL
(15 wt% PLEL). Therefore, we chose the hLB/PLEL with 20 wt% PLEL
for further studies.
3.2. Sol-gel phase transition and dynamic drug composition of hLB/PLEL
Dynamic rheological measurements were performed to study the
mechanical properties of the PLEL hydrogel and hLB/PLEL during the
sol-gel phase transition. According to the results in Fig. 2A and Fig. S2,
both the blank PLEL and hLB/PLEL formulations showed an evident
change in storage modulus (G′ ) upon temperature, characterized as a
rapidly growing after the temperature increasing to the thermores­
ponsive temperature. It is worth mentioning that the gelation temper­
ature of hLB/PLEL is closely related to the loaded LB concentration and
hydrogel concentration. As shown in Fig. 2B, increase in LB encapsula­
tion resulted in an increase in the thermoresponsive temperature. Spe­
cifically, when the LB concentration reached to 3% and 4%, the
thermoresponsive temperature of hLB/PLEL samples at polymer con­
centrations of 20 wt% were around 34 ◦ C, while that of samples in 10 wt
% and 15 wt% hydrogels were above 37 ◦ C. As an injectable in-site
sustained drug delivery system, enough LB encapsulation and appro­
priate gelation temperature less than 37 ◦ C of the hLB/PLEL is expected.
hLB/PLEL made by 20 wt% PLEL showed the more potential to resist the
drug concentration limitation. However, when the LB concentration
reached to 5%, the gelation temperature of hLB/20 wt% PLEL was above
37 ◦ C and its G′ at 37 ◦ C was measured as less than 2 Pa, which was not
applicable for in vivo drug delivery any more (Fig. 2B and C). Thus, an
hLB/20 wt% PLEL with LB encapsulation of less than 4% was preferred
here.
The initial concentration of LB base in hLB/PLEL with 0.75%, 2%,
and 4% LB were 2.23 ± 0.43 mg/mL, 7.19 ± 0.11 mg/mL, and 9.34 ±
0.21 mg/mL, respectively (Fig. 2D). This difference could be ascribed to
the pH of the hLB/PLEL and the amount of the loaded LB HCl. We further
investigated the pH and LB base dynamic changes in hLB/PLEL samples

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Y. Zhang et al. Biomaterials 299 (2023) 122129

Fig. 2. Rheological properties and dynamic drug composition of hLB/PLEL. (A) Changes in G′ of hLB/PLEL (20 wt%) with different LB concentrations. (B) Ther­
moresponsive temperature of hLB/PLEL with different polymer concentration and LB concentrations. (C) G′ of hLB/20 wt% PLEL with different LB concentrations at
37 ◦ C. (D) The initial concentration of LB base in hLB/PLEL with different drug loading. (E) Changes in pH of hLB/PLEL systems upon time. (F) Concentration
changes in LB base of hLB/PLEL systems upon time. Data are presented as mean ± SD.

with different LB encapsulations at 37 ◦ C. The results are shown in
Fig. 2E and F. The initial pH of the hLB/PLEL decreased with the LB
encapsulation, and the pH of all the samples decreased with time.
Accordingly, the amount of LB base decreased when time went on,
suggesting the gradual dissolution of the LB base.
3.3. Drug release of hLB/PLEL
The in vitro drug release behavior of hLB/PLEL was evaluated under
conditions mimicking the physiological microenvironment. Fig. 3A
shows that LB was released from the hLB/PLEL formulation in a sus­
tained manner over an extended period, whereas the free LB solution
showed a quick release within the first 2 h. The release profiles of hLB/
PLEL presented a biphasic pattern, with an initial burst release in the

Fig. 3. Drug release from the PLEL hydrogel both in vivo and in vitro. (A) In vivo drug release behavior of hLB/PLEL. (B) Real-time intravital fluorescence images of
local retention of Cy5.5 after injected around the sciatic nerve of rats. (C) Quantitative analysis of the fluorescence intensity of local Cy5.5 at different time. (D)
Results of pharmacokinetic study of hLB/PLEL systems on rat sciatic nerve block model. Data are presented as mean ± SD.

7
Y. Zhang et al.
first 2 h, followed by a sustained release period, suggesting a swelling
diffusion-controlled process [35]. Logically, the initial burst of LB can
contribute to quick work after injection, and the subsequent sustained
drug release is preferred for a long-lasting action. Moreover, the increase
in LB encapsulation from 0.75% to 4% seemed to have benefited the
long-term drug release, which may have resulted from the dynamic
dissolution of the insoluble fraction during the pH decrease of the
hLB/PLEL system.
To intuitively observe the drug release capacity of the PLEL hydrogel
in vivo, the water-soluble fluorescence chromogenic agent Cy5.5, was
used as a substitute for LB, and noninvasive intravital fluorescence im­
aging was used to monitor the local retention of Cy5.5 in real-time, as
shown in Fig. 3B. After injected around the sciatic nerve of rats, the
fluorescence of Cy5.5, observed mainly at the injection site, showed a
persistent maintenance and slow decay for longer than 72 h in the
Cy5.5/PLEL treatment group. In contrast, the fluorescence intensity in
the Cy5.5 solution group decayed rapidly with time owing to its rapid
clearance in vivo. According to the quantitative analysis of the fluores­
cence intensity, the fluorescence of Cy5.5/PLEL was stronger than that
of the Cy5.5 solution during all observations, indicating that PLEL
hydrogel holds the potential to prolong the drug release and extend the
local drug retention in the model of sciatic nerve blockade (Fig. 3C).
3.4. Pharmacokinetic study of hLB/PLEL
Local anesthetics were administered and absorbed into the circula­
tion through blood flow. The systemic concentration of local anesthetics
is closely related to their concentration in local tissues. Therefore, we
determined the plasma concentration and concentration-time profiles
for 0.75% LB, 4% LB, and 4% hLB/PLEL application near the rat sciatic
nerve. The data are shown in Fig. 3D and the computed pharmacokinetic
parameters are listed in Table 1. As shown in the plasma concentration-
time curve, the maximum concentration of 4% LB (993.03 ± 55.88 ng/
mL) was significantly higher than that of 0.75% LB (377.77 ± 16.57 ng/
mL, p < 0.001). However, the Cmax of 4% hLB/PLEL was 614.28 ±
54.19 ng/mL, which was not significantly different from that of 0.75%
LB (p = 0.216). Meanwhile, the maximum concentrations were observed
at 1.00 ± 0.22 h for 0.75% LB, 1.00 ± 0.21 h for 4% LB, and 1.50 ± 0.47
h for 4% hLB/PLEL after dosing. The hydrogel was found to slowly
release LB. The calculated AUC0-8 of 4% LB–hydrogel was 2820.70 ±
243.63 ng/mL*h, which was reduced at 37.69% than 4% LB (4526.61 ±
182.48 ng/mL*h). Given the blood volume in the rats [43], we did not
continue to collect the blood samples to determine the time to reach the
minimal concentration in the 4% LB and 4% hLB/PLEL groups. How­
ever, we calculated the AUC0-∞ of 4% LB and 4% hLB/PLEL, which were
5877.52 ± 422.85 ng/mL*h and 5642.31 ± 313.34 ng/mL*h, respec­
tively. These results preliminarily indicate that the PLEL hydrogel sys­
tem could release LB slowly in vivo for long-lasting local drug delivery in
vivo.
3.5. In vitro cytotoxicity and in vivo biocompatibility of PLEL hydrogel
Considering the local delivery of anesthetics, the biocompatibility
Table 1
Systemic pharmacokinetic parameters in sciatic nerve block (Mean ± SEM).
Pharmacokinetic 0.75% LB 4% LB solution 4% hLB/PLEL
parameters solution
Cmax (ng/mL) 377.77 ± 993.03 ± 55.88 614.28 ± 54.19
16.57
Tmax (h) 1.00 ± 0.22 1.00 ± 0.21 1.50 ± 0.47
t1/2 (h) 1.13 ± 0.22 3.54 ± 0.35 7.58 ± 3.38
AUC0–8 (ng/mL*h) 949.59 ± 4526.61 ± 2820.70 ±
50.18 182.48 243.63
AUC0-∞ (ng/mL*h) 937.39 ± 5877.52 ± 5642.31 ±
52.11 422.85 313.34
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Biomaterials 299 (2023) 122129
and cytotoxicity of the PLEL hydrogel on the nerve and muscle cells were
assessed using HT22 and C2C12 cells in vitro. According to the results of
the MTT test in Fig. 4A, the PLEL polymer solution showed mild cyto­
toxicity to both HT22 and C2C12 cells, whose viability was more than
90%, even at a high polymer concentration of 1000 μg/mL. The viability
of HT22 and C2C12 cells was approximately 90% after co-culture with
the PLEL hydrogel leachate for 48 h (Fig. 4B). Similar results were
observed in the live/dead assay as shown in Fig. 4C. Compared with the
untreated group, the addition of the PLEL polymer or hydrogel leachate
did not affect the proliferation and morphology of HT22 and C2C12
cells. These results indicate that the PLEL hydrogel had little cytotoxicity
on the nerve and muscle cells, indicating that it is a safe biomaterial for
the local delivery of anesthetics.
Mild tissue compatibility is of considerable importance for the clin­
ical application of local anesthetic formulations. We evaluated the in
vivo local toxicity of the PLEL hydrogel on the sciatic nerve and adjacent
muscles using a sciatic nerve blockade model in rats. Rats were eutha­
nized 1,3, 7, and 14 d after PLEL hydrogel administration, and sciatic
nerves with adjacent tissues were collected for macroscopic examination
and H&E staining. As shown in Fig. 4D, on macroscopic examination, all
rats injected with PLEL exhibited gelatinous white deposits of hydrogel
surrounding the sciatic nerve. The PLEL hydrogel was biodegradable,
and most of it degraded within 7 d. Moreover, these findings show that
the degradation rate of the PLEL hydrogel was increased with time. This
phenomenon may be related to the degradation of the PLEL generated
acidic products (lactic acid), which led to a pH decrease in the local
microenvironment and expedited degradation of the PLEL hydrogel.
PLEL did not cause significant pathological and morphological changes
in the rats (Fig. 4D). The total score of inflammation in the sciatic nerve
and muscle demonstrated that PLEL had a mild tissue compatibility
(Fig. S3A). Then, the results of immunofluorescent staining and semi-
quantitative analyses of the inflammatory factors are shown in
Fig. 4E. The red fluorescence representing the inflammatory factors was
observed mainly in the muscle around the sciatic nerve. As shown in
Figs. S3B–D, the fluorescence intensities of the rats treated with PLEL
hydrogel were not significantly different from those treated with saline.
The observed inflammatory response was moderate, which was consis­
tent with the results of H&E staining. Additionally, no statistical dif­
ferences were found between these groups, indicating that the
inflammatory response to the PLEL hydrogel was mild. This quality
makes it highly desirable for drug delivery applications.
3.6. Anesthetic effects of hLB/PLEL in sciatic nerve blockade model
For local anesthetics, the duration of anesthesia was determined
using two principal factors: concentration and volume [44]. However, a
concentration that exceeds the limitation will induce local and even
systematic toxicity. Therefore, the duration and histopathological ex­
aminations of different concentrations of hLB/PLEL were measured in a
rat sciatic nerve blockade model to determine the efficacy and safety of
hLB/PLELs for local anesthesia. In pain research, the effect of sex on
nociceptive processing has received attention. Previous studies have
shown that the response to pain stimulation varies between sexes [45].
Therefore, to eliminate the effect of sex, all rats in the in vivo pharma­
cological studies were male.
The duration of effect in different concentrations of LB in the PLEL
hydrogel was measured. The sensory and motor functions of rats injec­
ted with saline and PLEL hydrogel did not differ at the baseline, which is
not presented. The onset time of rats injected with LB or hLB/PLEL was
determined in the first 10 min post-dosing, indicating that the sustained-
release formulation could achieve the minimum effective concentration
quickly (Fig. 5A). However, the duration of 0.75% and 1% hLB/PLEL-
induced sensory and motor blockades were not significantly different
from that of 0.75% LB (Fig. 5B). Then, rats treated with 2%, 3%, or 4%
hLB/PLEL presented sensory blockades lasting for 7.88 ± 1.54 h (p =
0.036), 15.17 ± 1.83 h (p = 0.001), and 17.17 ± 2.24 h (p = 0.001),
Y. Zhang et al.
respectively, which were significantly longer than that induced by
0.75% LB (2.63 ± 0.16 h, Fig. 5B). Prolonged sensory blockade had a
linear relationship with LB concentration. Similar to the sensory
blockade, the duration of motor blockade induced by hLB/PLEL dis­
played a concentration-dependent relationship (Fig. 5C and D). The
hLB/PLEL with LB at concentration of 2%–4% (8.42 ± 1.54 h at 2%, p =
0.047; 16.50 ± 2.16 h at 3%, p = 0.007; and 20.33 ± 2.84 h at 4%, p =
0.001, respectively) produced a significantly longer effect than that
induced by 0.75% LB (3.00 ± 0 h). Then, the 6% hLB/PLEL and 8% hLB/
PLEL were further measured to determine whether it is safe to increase
the concentration of LB in the PLEL hydrogel (n = 3). The duration of 6%
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Biomaterials 299 (2023) 122129
Fig. 4. In vitro Cytotoxicity and in vivo
biocompatibility of PLEL hydrogel. (A)
Cytotoxicity of PLEL copolymer against
HT22 cells and C2C12 cells after co-
culture for 48 h. Data are presented as
mean ± SD. (n = 4). (B) Cell viability of
HT22 cells and C2C12 cells after treated
with PLEL hydrogel leachate for 48 h.
Data are presented as mean ± SD. (n =
3). (C) Results of live/dead assay at
different treatment time. (Scale bar:
100 μm) (D) Macroscopic examination
(green arrow: hydrogel; red arrow:
sciatic nerve) and representative histo­
logical images of the sciatic nerves and
adjacent muscles (with H&E staining).
The scale bars in the H&E staining panel
represent 500 μm. (E) Immunohisto­
chemistry analyses of sciatic nerve and
surrounding muscles after treated with
PLEL hydrogel. Red fluorescence in­
dicates the inflammatory factors and
blue fluorescence represents the nuclei.
Scale bar = 200 μm. (For interpretation
of the references to colour in this figure
legend, the reader is referred to the Web
version of this article.)
hLB/PLEL was increased to 38.33 h. Then, the sensory (n = 1, 33%) and
motor (n = 2, 67%) function of rats in 8% hLB/PLEL group did not re­
turn to normal even after the 14 d sciatic nerve block. However,
macroscopic changes, including muscle stiffness of the hind limb and
abnormal gait, were observed in rats injected with 6% and 8% hLB/
PLEL, which may have been induced by neuropathological injury. Thus,
4% LB was regarded as the maximum safe concentration of hLB/PLEL.
Mild tissue compatibility is of considerable importance for the clin­
ical application of local anesthetic formulations. The rats were eutha­
nized for histological analysis 1, 3, 7, and 14 d after dosing (n = 2 in per
day). Macroscopic examination and representative histological images
Y. Zhang et al. Biomaterials 299 (2023) 122129

Fig. 5. Anesthetic effects and tissue toxicity of hLB/PLEL in sciatic nerve blockade model. (A) The actual measurement value of paw withdraw latency. (B) Time to
recovery from sciatic nerve block for sensory blockade. (C) The actual measurement value of extensor postural thrust force. (D) Time to recovery from sciatic nerve
block for motor blockade. (E) The representative histological images of the sciatic nerves and adjacent muscles of hLB/PLEL (with H&E staining). The scale bars in
the H&E staining panel represent 500 μm. (F) The sciatic nerve inflammation score of hLB/PLEL. (G) The muscle inflammation score of hLB/PLEL.

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Y. Zhang et al. Biomaterials 299 (2023) 122129

of the sciatic nerves and adjacent muscles are presented in Fig. 5E. We
compared the total score of inflammation in the sciatic nerve and muscle
within 14 days in the 0.75% LB and 0.75%–4% hLB/PLEL groups
(Fig. 5F and G). The degree of inflammation in the local tissue of hLB/
PLEL was not significant than that recorded for LB at the maximum
clinical concentration.
Exparel™ (Pacira Pharmaceuticals, USA) is a liposomal encapsula­
tion of 13.3 mg/mL of bupivacaine arranged in a microscopic
honeycomb-like structure, which is delivered directly to the surgical
site, provides post-surgical local analgesia for up to 72 h, and is
approved for postoperative pain [26]. In a previous study, the effect of
the clinically available Exparel™ lasted only for 4–5 h in a rat sciatic
blockade model. The effect 1% hLB/PLEL in the sciatic nerve block
model lasted for 5.29 ± 0.61 h, which was not significantly different
with that of Exparel™ (contains 1.31% bupivacaine). However, 4%
hLB/PLEL produced an anesthesia duration four times longer than that
of Exparel™. Meanwhile, we compared 0.75%–4% hLB/PLEL with
0.75% LB in terms of sciatic nerve and muscle inflammation. The

Fig. 6. Anesthetic effects of hLB/PLEL in subcutaneous infiltration blockade model and acute postoperative pain model. (A) Schematic representation of rat sub­
cutaneous infiltration blockade model. (B) The actual measurement value of cutaneous trunci muscle reflex (pinprick responsiveness, PPR). (C) Time to recovery from
subcutaneous infiltration blockade. *: P < 0.05. (D) Schematic representation of rat acute postoperative pain model. (E) The actual measurement value of withdrawal
threshold (mechanical pain). *: compared with saline (*: P < 0.05; **: P < 0.01; ***: P < 0.001); #: compared with PLEL (#: P < 0.05; ##: P < 0.01; ###: P <
0.001); &: compared with levobupivacaine (&: P < 0.05). (F) The actual measurement value of cumulative pain score (spontaneous pain). *: compared with saline (*:
P < 0.05; **: P < 0.01; ***: P < 0.001); #: compared with PLEL (#: P < 0.05; ##: P < 0.01; ###: P < 0.001); &: compared with levobupivacaine (&: P < 0.05).

11
Y. Zhang et al.
degrees of inflammation in the local tissue of 4% hLB/PLEL were
considerably not more than that recorded for LB at the clinically
maximal concentration. Based on these results, 4% hLB/PLEL may be a
possible method for the prolonged postoperative pain management in
clinical settings.
3.7. Anesthetic effects of hLB/PLEL in subcutaneous infiltration blockade
model
We used a sciatic nerve blockade model to consider the 4% hLB/PLEL
as the optimal concentration and to determine the longest duration with
mild tissue toxicity in rats. The subcutaneous anesthetic effect of 4%
hLB/PLEL and the reason for the extended duration were verified using
the well-established CTMR model, and the effects of 4% hLB/PLEL and
4% LB were explored (Fig. 6A). The measured values are shown in
Fig. 6B. The 4% hLB/PLEL produced a subcutaneous anesthetic effect
with a significantly longer duration than that recorded with 4% LB (34
± 2 h vs. 18 ± 3 h, p = 0.003, Fig. 6C). The concentration of local
anesthetic is a major factor affecting the duration of anesthesia [46].
Thus, we chose 4% LB as the control group to determine whether the
long-acting local anesthesia was generated by the slow-release effect and
not by the increased concentration. However, 4% hLB/PLEL produced a
nearly two-fold longer duration than that of 4% LB. Thus, the prolonged
duration of this drug delivery system was not the result of drug
concentration.
3.8. Efficiency evaluation of hLB/PLEL in acute postoperative pain model
Previously used animal models, including sciatic nerve and subcu­
taneous infiltration blocks, are standard models of neurobehavioral
assessment for drug development, but they are not used for studying
postoperative pain. Therefore, surgical incision of the hind paw in rats
was used to estimate the effect of hLB/PLEL (Fig. 6D) [41]. The baseline
of paw withdrawal threshold for von Frey hair stimulation was not
significantly different between the two groups (Fig. 6E). However, the
incised rats injected with 4% hLB/PLEL showed a higher paw with­
drawal threshold than the rats injected with 0.75% LB and control
groups. Skin incision and treatment with 4% hLB/PLEL resulted in a
mild spontaneous pain after incision (Fig. 6F). Postoperative pain is
characterized by constant spontaneous pain near the surgical site and
acute exacerbation due to movement or other forms of stimulation. The
pain scores measured in this study were related to either touch-evoked
pain or ongoing pain during the post-operative period. Therefore, our
findings that a single postoperative dose of 4% hLB/PLEL attenuates the
pain scores is valuable from a therapeutic perspective.
The common types of local anesthesia are topical anesthesia, infil­
tration anesthesia, and peripheral nerve blockade. In this study, the rat
sciatic nerve block is the standard animal model used to simulate the
method of peripheral nerve blockade in human for surgeries at or below
the knee. Then, the infiltration anesthesia is to anesthetize nerve ter­
minal in a finite area of tissue by the injection of local anesthetics
nearby. Contrast to nerve block, in which nerve axons are the target and
the injection may take place in an area removed from the surgical site.
Infiltration anesthesia alone can provide adequate analgesia for
superficial procedures (such as inguinal herniorrhaphy, breast and
anorectal surgery, shoulder and knee arthroscopy) but is vastly
underutilized in clinical practice. However, advantages of recom­
mended wound infiltration with local anesthetics are safety, simplicity,
and enhanced postoperative analgesia [47], and the effect of hLB/PLEL
was evaluated in acute postoperative pain rat model by wound infil­
tration. Thus, multiple animal models were used to demonstrate the
sufficient feasibility of the proposed approach and significantly
advanced pain management.
12
Biomaterials 299 (2023) 122129
4. Conclusions
In this study, a biodegradable thermosensitive hydrogel was fabri­
cated to release LB for postoperative analgesia. LB existed in the neutral
base form and the charged cation form due to the chemical construction
of LB and the initial pH of the PLEL hydrogel. After local injection, the
dissolved LB HCl was first released from the hydrogel, and the insoluble
LB base dissolved and was released gradually as the pH decreased during
biodegradation of the PLEL hydrogel, resulting in long-term LB release.
Furthermore, the PLEL hydrogel showed mild cytotoxicity to nerve and
muscle cells, and histological analyses demonstrated that the degree of
inflammation in local tissues of the hLB/PLEL was acceptable.
Furthermore, the hLB/PLEL system significantly extended the effect of
local analgesia in sciatic nerve block, subcutaneous infiltration anes­
thesia, and acute postoperative pain models in rats, suggesting that it is a
promising candidate for local anesthetic delivery and postoperative pain
management.
Credit author statement
YuJun Zhang, Conceptualization, Methodology, Investigation, Re­
sources, Data Curation, Writing - Original Draft; Kun Shi, Conceptuali­
zation, Methodology, Investigation, Resources, Data Curation, Writing -
Original Draft; Xi Yang, Investigation, Data Curation; Wen Chen,
Investigation, Data Curation; TianHong Wang, Investigation, Data
Curation; Yi Kang, Investigation, Data Curation; DeYing Gong, Investi­
gation, Data Curation; WenSheng Zhang, Conceptualization, Method­
ology, Resources, Supervision, Project administration, Writing - Review
& Editing Supervision; ZhiYong Qian, Conceptualization, Methodology,
Resources, Supervision, Project administration, Writing - Review &
Editing Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
This work was supported by the National Natural Science Foundation
of China (81973274, U21A20417, 31800797), the Sichuan Science and
Technology Program, China (2020YJ0053, 2022YFS0333,
2022YFS0308), the China Postdoctoral Science Foundation
(2020M673235), the Post-Doctor Research Project, West China Hospi­
tal, Sichuan University, China (2019HXBH012, 2018HXBH066), the
Science and Technology Department of Chengdu City, China (2021-
YF05-00855-SN), and the 1•3•5 project for disciplines of excellence,
West China Hospital, Sichuan University, China (ZYGD18002,
ZYJC21068).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biomaterials.2023.122129.
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