International Journal of Pharmaceutics 579 (2020) 119178

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Dimeric artesunate phospholipid-conjugated liposomes as promising anti- T

inflammatory therapy for rheumatoid arthritis
⁎
Yaru Zhanga, Wei Heb, Yawei Dub, Ying Dua, Chao Zhaoa, Ying Zhanga, Hu Zhanga, Lihong Yina, ,
⁎
Xinsong Lib,
a
School of Public Health, Southeast University, Nanjing 210009, PR China
b
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Objective: The dimeric artesunate phospholipid conjugate (Di-ART-GPC) is a novel amphipathic artemisinin
Artesunate derivative, which can be assembled into liposomes. Di-ART-GPC liposomes were prepared and evaluated as
Rheumatoid arthritis potential anti-inflammatory agents for rheumatic arthritis (RA).
Liposome Methods: Di-ART-GPC was assembled into liposomes utilizing thin film dispersion-high pressure homogeniza-
Anti-inflammatory
tion. Dynamic light scattering (DLS), transmission electron microscopy (TEM), and electron cryo microscopy
(cryo-EM) were employed to characterize the liposomal size and morphology. The in vitro cytotoxicity of the Di-
ART-GPC liposomes was assessed using Cell Counting Kit-8 (CCK8). The anti-inflammatory effects were studied
utilizing the inflammatory cell model. Finally, the in vivo efficacy of the Di-ART-GPC-conjugated liposomes was
investigated using the arthritis rat model.
Results: The particle size of the Di-ART-GPC liposomes decreased to a narrow range of approximately 70 nm
following high-pressure homogenization. The in vitro studies revealed low cytotoxicity and good anti-in-
flammatory effects of the Di-ART-GPC liposomes, which exhibited significantly higher inhibition of the cell
secretion of pro-inflammatory cytokines than ART. The in vivo evaluation confirmed that treatment with Di-ART-
GPC resulted in a decline in the ankle swelling rate and a low inflammatory response compared with the model
control and ART.
Conclusion: Di-ART-GPC liposomes demonstrate remarkable potential as novel ART-based anti-inflammatory
agents for RA.

1. Introduction
Rheumatoid arthritis (RA), an immune-mediated systemic disease,
is the most common form of chronic inflammatory arthritis (Uttra et al.,
2018). RA is a lifetime advancing disease affecting up to 1% of the
global adult population (Pal et al., 2015; Smolen et al., 2016). Un-
fortunately, the conventional therapeutics, including non-steroidal anti-
inflammatory drugs (NSAIDs) and disease-modifying anti-rheumatic
drugs (DMARDs) are not a cure for the disease. The available agents
merely temporarily delay its progress and cause various undesirable
side effects (Choudhary et al., 2014). Thus, it is necessary to develop
novel RA therapies exhibiting improved efficacy and safety.
Plant-derived medicines, such as ones obtained from herbal re-
medies, play important roles in the therapy of various diseases, in-
cluding arthritis, due to their favorable potency and low toxicity
(Prakash et al., 2016). For instance, in addition to its significant anti-
malarial activity, artemisinin isolated from the Artemisia annua plant
has been reported to display potential RA therapeutic effects (Vilar
et al., 2019). The compound was first isolated in 1972 by Youyou Tu,
who in 2015 was awarded half of the Nobel Prize in Physiology or
Medicine for this discovery. Subsequently, artemisinin derivatives
(ARTs), including dihydroartemisinin (DHA), artesunate (ART), and
artemether, were developed and have been widely employed as im-
portant anti-malarial drugs (Miller and Su, 2011). In recent years, the
anti-inflammatory, anti-tumor, immune regulation, and other pharma-
cological activities of ARTs have been extensively reported (Jiang et al.,
2016; Lai et al., 2015; Liu et al., 2019). It has been revealed that ARTs
affect the activation and regulation of immune cells by inhibiting sig-
naling pathways associated with inflammation. The compounds reduce
the corresponding symptoms of various inflammatory diseases, in-
cluding rheumatoid arthritis, gastritis, nerve inflammation, and pan-
creatitis (Cen et al., 2016; Jang et al., 2017; Li et al., 2015).

⁎
Corresponding authors.
E-mail addresses: lhyin@seu.edu.cn (L. Yin), lixs@seu.edu.cn (X. Li).

https://doi.org/10.1016/j.ijpharm.2020.119178
Received 5 December 2019; Received in revised form 4 February 2020; Accepted 23 February 2020
Available online 24 February 2020
0378-5173/ © 2020 Elsevier B.V. All rights reserved.
Y. Zhang, et al.
Furthermore, in the collagen II-induced RA rat model, ARTs have been
demonstrated to considerably improve symptoms by reducing the ex-
pression of pro-inflammatory cytokines in the serum and preventing the
destruction of the cartilage and bone (Li et al., 2013). Notably, several
new ARTs have been developed for the treatment of RA, and one of
them (SM934) has already been approved by the Chinese Food and
Drug Administration (CFDA) as a novel agent to enter clinical trials
(Costa et al., 2013). SM934 was shown to significantly reduce the se-
verity of collagen-induced arthritis, decreasing the degree of joint
swelling and bone destruction (Lin et al., 2016). Nevertheless, the tra-
ditional ARTs include fast-acting drugs with extremely short half-lives
in vivo (< 1 h) (Huang et al., 2019). Hence, the drug efficacy against RA
could be further improved by modifications resulting in a prolonged
circulation time in the body.
Efficient nanotechnology-based therapies for the treatment of RA
have also attracted considerable attention. For example, nano-
technology-mediated interventions have been proved to be more fea-
sible in reducing systemic toxicity and improving the therapeutic effi-
cacy for the treatment of RA and osteoporosis (co-morbidity of RA)
(Dong et al., 2018; Hussain, 2019). Nonetheless, the pharmacokinetic
and pharmacodynamic challenges following intravenous and intra-ar-
ticular administration still remain. Thus, multi-functionalization, such
as nanoencapsulation in liposomes, is an emerging strategy for the
optimization of the therapeutic efficacy (Gang et al., 2019). Liposomes
are widely employed as nanocarriers for targeted delivery of pharma-
ceuticals (Allen and Cullis, 2013). Overall, recent reports demonstrated
that nanoencapsulation of pharmaceuticals in liposomes enhanced the
anti-inflammatory, anti-RA as well as remission efficacy of osteo-
porosis, proving that liposomes are promising nanometer delivery sys-
tems (Gottschalk et al., 2015; Sultana et al., 2017).
We previously reported a novel artemisinin derivative, a dimeric
artesunate phospholipid conjugate (Di-ART-GPC), which exhibited re-
markable liposomal assembly ability (Ismail et al., 2018). The improved
chemical stability, prolonged in vivo half-life (t1/2), and enhanced anti-
malarial activity of the Di-ART-GPC liposomes have been confirmed in
preliminary experiments. In the previous study, the in vitro release of
the Di-ART-GPC liposomes was evaluated using a dialysis technique.
The obtained results suggested that the ART-conjugated heparin pro-
drug effectively released ART in a weakly acidic microenvironment
(Ismail et al., 2018). It is speculated that the improvement in the drug
activity of the Di-ART-GPC liposomes is attributed to the relatively
stable and long-acting effects in comparison to the traditional ARTs,
which typically include short-acting drugs with extremely short t1/2
(~1 h). As stable and relatively long acting artemisinin derivatives, Di-
ART-GPC liposomes are expected to display better efficacy against RA
than traditional ARTs, while sharing a similar anti-inflammatory me-
chanism of action.
In the current work, we focused on the anti-inflammatory effects of
the Di-ART-GPC liposomes against RA. In the first instance, we further
optimized the method for the preparation of the Di-ART-GPC liposomes
utilizing a modified thin film dispersion-high pressure homogenization
approach. Subsequently, the proliferation rate of the mouse leukemia
cells of monocyte macrophage (RAW264.7 cells) incubated with the Di-
ART-GPC liposomes was measured using the cell counting kit-8 (CCK8)
method. The influence of the Di-ART-GPC liposomes on the levels of
pro-inflammatory cytokines in the LPS-induced RAW264.7 cell model
was also evaluated. Finally, the in vivo drug efficacy of the Di-ART-GPC
liposomes was investigated employing an arthritis rat model.
2. Materials and methods
2.1. Materials
Artesunate (ART, purity ≥99%) and 1,8-diazabicyclo-[5.4.0]-
undec-7-ene (DBU, purity ≥99%) were purchased from Macklin Co.
(Shanghai, China). L-α-Glycerophosphorylcholine (GPC, purity ≥99%)
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International Journal of Pharmaceutics 579 (2020) 119178
was obtained from J&K Industries (Shanghai, China). N,N-
Carbonyldiimidazole (CDI, purity ≥97%) was purchased from Aladdin
Co. (Shanghai, China).
Lipopolysaccharides (LPS) and Freund’s complete adjuvant (FCA)
were purchased from Sigma-Aldrich Co. (Shanghai, China). Cell
Counting Kit-8 (CCK8) was obtained from 7 Sea Biotech Co. (Shanghai,
China). The Griess Reagent Kit was purchased from Beyotime Co.
(Nantong, China). Prednisolone acetate injection was supplied by
Huazhong Pharma Co. (Xiangyang, China). The ELISA kits for mouse
TNF-α, IL-1β, and IL-6 were purchased from Yfxbio Biotechnology Co.,
Ltd. (Nanjing, China). The ELISA kits for rat TNF-α, and IL-6 were
obtained from Senbeijia Biological Technology Co., Ltd. (Nanjing,
China). All of the chemical reagents were of analytical-grade quality
and were used without any further purification.
Mouse leukemia cells of monocyte macrophage (RAW264.7 cells)
were purchased from Conservation Genetics Kunming Cell Bank,
Chinese Academy of Sciences. The cells were cultured in Dulbecco's
Minimum Essential Medium (DMEM, Hyclone, Logan, UT) containing
10% (v/v) fetal bovine serum (FBS, Invitrogen, Grand Island, NY),
100 μg/mL streptomycin, and 100 U/mL penicillin. The cells were in-
cubated at 37 °C under a humidified atmosphere containing 5% CO2.
2.2. Synthesis of the Di-ART-GPC conjugate
The Di-ART-GPC conjugate was synthesized using a previously re-
ported modified esterification reaction (Ismail et al., 2018). Briefly,
ART (1 eq.) and CDI (1.5 eq.) were dissolved in dichloromethane (DCM)
and stirred for 4 h at 30 °C. The reaction was monitored by thin layer
chromatography (TLC). Subsequently, the organic solvent was evapo-
rated in vacuo and the remaining complex was re-dissolved in DMSO,
followed by the addition of 0.3 eq. of GPC and 1 eq. of DBU. The re-
action was continued at 40 °C for 24 h. The resulting product was
purified by Biotage flash column using elution solvent A (CH2Cl2) and
solvent B (CH3OH/H2O, 25/4, v/v). The desired fractions were eva-
porated in vacuo and vacuum dried to afford the desired product as a
white solid with a purity of 97.3%, as determined by high-performance
liquid chromatography (HPLC).
2.3. Preparation of the Di-ART-GPC liposomes
The Di-ART-GPC liposomes were prepared using a thin film dis-
persion-high pressure homogenization method. Briefly, 10 mg of the Di-
ART-GPC conjugate was dissolved in 20 mL of CH2Cl2 in a 100 mL
round-bottom flask. The organic solvent was evaporated at 40 °C under
reduced pressure using a rotary evaporator until a thin film of Di-ART-
GPC was visible on the walls of the flask. Subsequently, 5 mL of PBS
was added to hydrate the Di-ART-GPC thin film at 50 °C for 30 min and
the crude liposome solution was obtained. The liposome solution was
homogenized using a high-pressure homogenizer (Niro Soavi S.p.A.,
Parma, Italy).
The morphology of the liposomes was observed by transmission
electron microscopy (TEM, JEOL Inc., Tokyo, Japan) and electron cryo
microscopy (Cryo-EM, Tecnai G2 F20, FEI, Hillsboro, OR). The particle
size before and after homogenization was measured by dynamic light
scattering (DLS) using a particle size analyzer (NanoBrook Omni,
Brookhaven, Holtsville, NY).
2.4. In vitro cytotoxicity assay
The cytotoxicity of the drugs was evaluated utilizing the CCK8
method employing the RAW264.7 cells. Briefly, the cells were seeded in
96-well plates at a density of 1 × 104 cells/well. After 5 h, different
treatments were used: (a) blank complete medium; (b) complete
medium containing 0.1% dimethylsulfoxide (DMSO); (c) ART at con-
centrations of 0.625, 1.25, 2.5, 5, 10, and 20 μg/mL (dissolved in the
complete medium containing 0.1% DMSO); (d) Di-ART-GPC solution at
Y. Zhang, et al.
concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 μg/mL (dissolved in
the complete medium containing 0.1% DMSO); (e) Di-ART-GPC lipo-
somes at concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 μg/mL
(dissolved in the complete medium containing 0.1% DMSO).
Following incubation for 6, 12, and 24 h, the cell viability of each
group was assessed using the CCK8 method. The medium was removed
from each well and 200 μL of the DMEM medium containing 10 μL of
CCK8 was added. The plates were incubated for 2 h, after which the
optical density (OD) values were measured at 450 nm using a micro-
plate reader (ELX800, Bio Tek Instruments Inc., Winooski, VT). The
experiment was repeated three times. The cell viability was calculated
according to the following formula:
Cell viability (%) = (AX /A 0) × 100%
(Ax: OD of the experimental groups; A0: OD of the blank control group).
2.5. In vitro analysis of the released NO
Following treatment with different drugs, the NO released from the
RAW264.7 cells was analyzed utilizing the Griess reagent method.
Briefly, the RAW264.7 cells were seeded in 24-well plates at a density
of 1 × 105 cells/well, and treated with different conditions. The fol-
lowing four groups were used for the analyses: (a) blank control group
treated with the blank medium; (b) model control group treated with
1 μg/mL of LPS; (c) ART experiment group treated with 1.25 μg/mL of
ART following induction with 1 μg/mL of LPS; (d) Di-ART-GPC solution
group (AG group) treated with 1.25 μg/mL of the Di-ART-GPC solution
(ART equal concentration) following induction with 1 μg/mL of LPS
(n = 3); (e) Di-ART-GPC liposomes group (AGL group) treated with
1.25 μg/mL of the Di-ART-GPC liposomes (ART equal concentration)
following induction with 1 μg/mL of LPS (n = 3).
For ART, AG and AGL groups, the cells were pre-incubated under a
humidified atmosphere containing 5% CO2 at 37 °C. Subsequently, the
existing supernatants were removed and new supernatants containing
1 μg/mL of LPS (pre-dissolved in PBS) were added to the specified wells
and incubated for 24 h. After that, ART (1.25 μg/mL), Di-ART-GPC
solution (with ART equal concentration of 1.25 μg/mL), and Di-ART-
GPC liposomes (with ART equal concentration of 1.25 μg/mL) were
added in the specified wells. After incubation for 12 h, the cell culture
medium was taken into 1.5 mL enzyme-free EP tubes. After cen-
trifugation at 2000 rpm for 20 min, the supernatant was collected. The
NO concentration in each sample was measured with the Griess reagent
kit by measuring the OD values at 540 nm using a microplate reader
(ELX800, Bio Tek Instruments Inc., Winooski, VT).
2.6. In vitro analysis of the pro-inflammatory cytokines
Following treatment with different drugs, the secretion changes of
the pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 in the
RAW264.7 cells were analyzed utilizing the enzyme-linked im-
munosorbent assay (ELISA). Briefly, the RAW264.7 cells were seeded in
24-well plates at a density of 1 × 105 cells/well, and were divided into
different groups, analogously to the method described in Section 2.5.
The cells were incubated in the presence of LPS for 9 h, after which ART
(1.25 μg/mL), the Di-ART-GPC solution (1.25 μg/mL), and Di-ART-GPC
liposomes (1.25 μg/mL) were added in the specified wells of experi-
ment groups. After 12 h, the cell culture medium was collected and
centrifuged at 2000 rpm for 20 min. The levels of TNF-α, IL-1β, and IL-6
of each sample were determined using the ELISA kits (Yfxbio Bio-
technology Co., Ltd., Nanjing, China).
2.7. Animals
Sprague-Dawley (SD) rats (male, 180–200 g, 6–8 weeks of age) were
purchased from Sibeifu Laboratory Animal Co., Ltd. (Beijing, China).
All animals were fed in a specific pathogen free (SPF) grade
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International Journal of Pharmaceutics 579 (2020) 119178
environment. The temperature of the room was controlled at
23 ± 1 °C, with lighting for 12 h per day. All experiments were ap-
proved by the Ethics Review Committee for Animal Experimentation,
School of Public Health, Southeast University (Nanjing, China).
2.8. Evaluation of the in vivo anti-arthritic activity using the FCA-induced
RA rat model
2.8.1. Establishment of the RA rat model
The rat model of RA was established by injecting Freund’s complete
adjuvant into the sub-plantar surface of the right hind paw of the rats.
FCA-induced rat arthritis model is an extensively employed animal
model for demonstrating autoimmune inflammation and reliably pre-
sents characteristics similar to those observed in human RA patho-
physiology (Lee et al., 2019). The injection volume of FCA was 0.1 mL.
After 8 days, the ankle swelling rate was measured, and the statistical
difference from the control group confirmed the successful establish-
ment of the RA rat model. The in vivo anti-arthritic activity was in-
vestigated using the established FCA-induced model involving in-
travenous injection of different drugs every day for a 21-day period (8th
to 28th day). The RA model rats were randomly divided into six groups
(n = 5): (a) the control group (normal rats) injected with saline (3 mL/
kg); (b) model group injected with saline (3 mL/kg); (c) ART group
injected with ART (2.4 mg/kg); (d) low dose Di-ART-GPC liposomes
(AGL-low) group injected with 1.5 mg/kg (Di-ART-GPC concentration)
of the Di-ART-GPC liposomes; (e) medium dose Di-ART-GPC liposomes
(AGL-medium) group injected with 3.0 mg/kg (Di-ART-GPC con-
centration) of the Di-ART-GPC liposomes; (f) high dose Di-ART-GPC
liposomes (AGL-high) group injected with 6.0 mg/kg of the Di-ART-
GPC liposomes.
2.8.2. Ankle swelling rate evaluation
The anti-arthritic activity was evaluated by the comparison of the
thickness of the ankle utilizing a Vernier caliper. Initial thickness of the
ankle (T0) and body weight (W0) were determined prior to the FCA
injection. The thickness of the ankle (Tx) and body weight (Wx) of every
rat were measured twice, i.e., on the 8th and 28th day. The degree of
the ankle swelling and the growth rate were calculated using the fol-
lowing formulas:
Ankles welling (%) = (Tx − T0)/T0 × 100%
Growth rate (%) = (Wx − W0)/W0 × 100%
2.8.3. Inflammatory cytokine evaluation
On the 28th day, the rats were anaesthetized with ether, and a blood
sample of each animal was drawn via eyeball extirpation. Following
centrifugation at 5000 rpm for 10 min at 4 °C, the supernatant serum
was collected for the evaluation of the TNF-α and IL-6 expression levels
using the TNF-α and IL-6 ELISA kits (Senbeijia Biotechnology Co.,
Nanjing, China).
2.8.4. Spleen/thymus index evaluation
The rats were sacrificed following the blood collection, and the
spleen and thymus were separated and weighted (recorded as Ws and
Wt, respectively). The spleen and thymus indexes of the rats were cal-
culated according to the following formulas:
Spleen index (10 mg/g) = 10 × Ws/Wb
Thymus index (10 mg/g) = 10 × Wt /Wb
2.8.5. Histological studies
The ankle samples were separated after sacrificing the animals. All
specimens were fixed using 4% paraformaldehyde, decalcified, em-
bedded with paraffin, and cut into slices for histological examination
Y. Zhang, et al.
Fig. 1. (A) Preparation of Di-ART-GPC liposomes; TEM images of Di-ART-GPC liposomes before (B) and after (C) 5 times homogenizations; (D) size distribution
measured by DLS after 0-5 times of homogenizations; (E) Cryo-EM image of Di-ART-GPC liposomes after 5 times of homogenizations.
after hematoxylin-eosin (H&E) staining.
2.9. Statistical analysis
Data was expressed as the mean ± standard error (SE). All statis-
tical analyses were performed using the GraphPad Prism 6.0 software.
The statistical significance comparing multiple groups was evaluated by
one-way ANOVA. P-value of < 0.05 was considered to be statistically
significant.
3. Results
3.1. Preparation of the Di-ART-GPC liposomes
The Di-ART-GPC conjugate was synthesized according to a pre-
viously reported method, which involved an esterification reaction of
ART and GPC in the presence of the CDI/DBU catalytic system (Ismail
et al., 2018). Similar to natural phosphatidylcholines, Di-ART-GPC
could be assembled into liposomes utilizing a classical thin film dis-
persion method (Fig. 1A). Subsequently, high-pressure homogenization
was carried out to uniformize the size of the Di-ART-GPC liposomes. To
obtain a narrow size distribution, the homogenization was conducted
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International Journal of Pharmaceutics 579 (2020) 119178
multiple times. The morphology of the Di-ART-GPC liposomes was
observed by TEM before and after homogenization. Compared with the
images prior to homogenization (Fig. 1B), the liposomes after five cy-
cles of homogenization exhibited sphere structures with a narrow size
distribution (Fig. 1C). Following each cycle, the size distribution of the
liposomes was measured by DLS. The results are demonstrated in
Fig. 1D and Table 1. The obtained data clearly show that subsequent
homogenizing cycles generated smaller mean size of the liposomes,
which was consistent with the results found in the literature (Fan et al.,
2018; Pandey et al., 2019). Before the first homogenization, the particle
Table 1
Particle size of Di-ART-GPC liposomes measured by DLS after
homogenization for 0–5 times.
Homogenizing times Size (nm)
0 209.1 ± 5.8
1 172.7 ± 6.4
2 129.5 ± 2.3
3 84.3 ± 3.8
4 72.0 ± 4.5
5 76.6 ± 6.7
Y. Zhang, et al.
size of the Di-ART-GPC liposomes was 209.1 ± 5.8 nm. This value
decreased to 172.7 ± 6.4 and 129.5 ± 2.3 nm after the 2nd and 3rd
homogenization, respectively. Following the 4th cycle, the particle size
of the liposomes remained stable at approximately 70 nm. The mean
average of the particle size after four homogenization cycles was close
to the value following the fifth homogenization; however, the standard
deviation after five cycles was smaller. With a view to obtaining more
reliable and repeatable results, five homogenization cycles were em-
ployed as a standard method in the following experiments. The dia-
meter of the liposomes under TEM was consistent with the data mea-
sured by DLS. Furthermore, the liposomal structure was confirmed by
the cryo-EM analysis, as shown in Fig. 1E. Notably, the observed similar
particle size was in agreement with the obtained TEM images. More-
over, obvious liposomal structure of the Di-ART-GPC liposomes was
also determined.
3.2. In vitro cytotoxicity assay
In vitro cytotoxicity of the Di-ART-GPC liposomes was evaluated
utilizing the CCK8 method employing the RAW264.7 cells in the pre-
sence of ART and the Di-ART-GPC solution as controls. Cell viabilities
(% of control group) were detected after incubation for 6, 12, and 24 h,
as shown in Fig. 2A, B, and C, respectively. The cells treated with the
Di-ART-GPC liposomes demonstrated the highest viabilities under
nearly all of the evaluated conditions (P < 0.05). After incubation for
6 h, the remaining cell viability for the AGL group at the highest con-
centration was more than 60%. Conversely, in comparison to the AGL
group, the value for the AG group was less and the value for the ART
group was only half (50%). After incubation for 12 and 24 h, the AGL
group also resulted in much lower 50% inhibitory concentration (IC50)
values compared with the ART (approx. 2-fold higher) and AG groups.
The cell viability data provided an important reference for the assays
conducted in the subsequent investigations.
3.3. Analysis of the in vitro NO, TNF-α, IL-1β, and IL-6 release
Cells exhibited inflammatory responses by up-regulating NO and
increasing the levels of pro-inflammatory cytokines, such as TNF-α, IL-
1β, and IL-6, following the stimulation with LPS. To evaluate the in vitro
anti-inflammatory activity of ART, the Di-ART-GPC solution as well as
the Di-ART-GPC liposomes, the release levels of NO, TNF-α, IL-1β, and
IL-6 from the LPS-treated RAW264.7 cells (model cells) were in-
vestigated after treatment with the drugs for 12 h. The obtained results
are demonstrated in Fig. 3. As it can be seen, all of the drug-treated
groups showed significantly lower NO, TNF-α, IL-1β, and IL-6 levels
than the model cell group (P < 0.001). Moreover, the AGL and AG
groups (with ART equivalent concentration of 1.25 μg/mL) exhibited
Fig. 2. The cell viability of RAW264.7 cells after 6 h (A), 12 h (B) and 24 h (C) treatment with ART, Di-ART-GPC, Di-ART-GPC liposomes. (*P < 0.05, ** P < 0.01
and *** P < 0.001 compared with ART group at the same concentration, and #P < 0.05, ## P < 0.01 and ### P < 0.001 compared with AG group at the same
concentration).
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International Journal of Pharmaceutics 579 (2020) 119178
lower values than the ART group (1.25 μg/mL) (P < 0.05), whereas
the AGL group displayed enhanced inhibitory effects in comparison to
the AG group (P < 0.05).
3.4. Investigation of the in vivo anti-arthritic activity using the FCA-induced
RA rat model
3.4.1. Evaluation of the ankle swelling rate
The in vivo anti-arthritic activity of the Di-ART-GPC liposomes was
evaluated using the FCA-induced RA rat model. The ankle swelling rate
was measured on the 8th day after the FCA injection, and the statistical
differences between the control group and other groups confirmed the
successful establishment of the RA rat model. The RA model rats were
intravenously injected with the Di-ART-GPC liposomes and ART for
21 days. The ankle swelling rates, body weights, spleen/thymus in-
dexes, pro-inflammatory cytokine levels, and pathological sections of
each group were thoroughly investigated.
Photographs of the rat ankles subjected to different treatments are
illustrated in Fig. 4A. After the FAC-induction, all experimental groups
demonstrated significantly swollen ankles. In addition, the calculated
swelling rates shown in Fig. 4B also exhibited considerable differences
between the model groups and the control group (P < 0.001). These
data indicate that the ideal rat model of arthritis induced by FCA was
successfully established. As shown in Fig. 4B, the ankle swelling rates
for the rats in the ART and Di-ART-GPC liposomes groups were lower
than for the rats in the model group. Furthermore, both the Di-ART-GPC
liposomes medium dose (AGL-medium) and high dose (AGL-high)
groups displayed significantly decreased ankle swelling rates in com-
parison to the ART group (P < 0.05). Moreover, cardinal symptoms of
inflammation, such as redness and swelling, were considerably relieved
in rats treated with ART and the Di-ART-GPC liposomes compared with
the model group without any drug treatment, as shown in Fig. 4A.
3.4.2. Evaluation of the body weight
The changes in the body weight are demonstrated in Fig. 4C. The Di-
ART-GPC liposomes-treated groups exhibited significantly higher
growth rates than the model group. Moreover, the AGL-medium group
(treated with 3.0 mg/kg of the Di-ART-GPC liposomes) displayed the
highest body weight among all experimental groups and its mean value
was even slightly higher than that of the control group. Although the
values for the ART group were also higher than those for the model
group, the differences were not significant (P > 0.05).
3.4.3. Evaluation of the spleen/thymus indexes
The spleen/thymus index data are shown in Fig. 4A and B. The ART
and Di-ART-GPC liposomes groups exhibited a considerable reduction
in the spleen and thymus indexes compared with the model group.
Y. Zhang, et al.
Fig. 3. The level of NO (A), TNF-α (B), IL-1β (C) and IL-6 (D) in the supernatant after different treatments (n = 5). (*P < 0.05, ** P < 0.01 and *** P < 0.001
compared with model group, and #P < 0.05, ## P < 0.01 and ### P < 0.001 compared with ART group).
Furthermore, the differences between the AGL-medium and AGL-high
groups were significant (P < 0.05). The AGL-medium and AGL-high
groups also showed the closest values to the spleen/thymus index data
for the control group (see Fig. 5).
3.4.4. Evaluation of the pro-inflammatory cytokines
The levels of the pro-inflammatory cytokines, including TNF-α and
IL-6, are presented in Fig. 4C and D. The ART and Di-ART-GPC lipo-
somes groups exhibited significantly lower levels of the cytokines
compared with the model group (P < 0.05). Moreover, there was
obvious dose dependence in the Di-ART-GPC treated groups. Notably,
the AGL-high group showed the lowest TNF-α and IL-6 levels in com-
parison to all three Di-ART-GPC groups and significantly lower levels
than the ART group (P < 0.05).
3.4.5. Histological study
The paraffin sections of the ankle specimens were observed under
an optical microscope following H&E staining (Fig. 6). The analysis of
the ankle joints in the control group revealed smooth articular surface
with normal synovial tissue, as well as joint space with no inflamma-
tion. Additionally, there was no infiltration of inflammatory cells in the
articular cavity. Histological evaluation of the ankle joints of the ar-
thritic rats in the model group showed intense synovial proliferation
and inflammatory cell infiltration. However, the ART and Di-ART-GPC
liposomes treatment produced remarkable improvements in the in-
flammatory cell infiltration and joint destruction. Moreover, the de-
crease in the synovial inflammation resulting from the Di-ART-GPC li-
posome treatment was dependent on the dose used. Notably, the AGL-
high group (6 mg/kg) demonstrated the most optimal results in the
6
International Journal of Pharmaceutics 579 (2020) 119178
histopathological assessment among all groups.
4. Discussion
In our previous study, the Di-ART-GPC prodrug was successfully
synthesized, and the in vitro and in vivo experiments confirmed the
improved chemical stability, prolonged half-life (t1/2), and enhanced
anti-malarial activity of the Di-ART-GPC liposomes (Ismail et al., 2018).
Considering the reported anti-inflammatory potential of different short-
acting ARTs used for the treatment of RA (Shi et al., 2015; Wu et al.,
2016; Zhao et al., 2017), long-acting Di-ART-GPC liposomes are sus-
pected to display better efficacy than traditional ARTs. In the current
study, the Di-ART-GPC liposomes were characterized by the TEM and
DLS analyses and the liposomal structure was confirmed by cryo-EM.
The particle size of the Di-ART-GPC liposomes was slightly smaller than
the typical liposomes. Liposomes have been previously formulated as
novel drug delivery systems to achieve effective drug concentrations,
concurrently avoiding significant systemic side effects of high doses of
drugs (Vyas et al., 1995). Due to their biocompatibility, biodegrad-
ability, non-toxicity, and non-immunogenicity via various administra-
tion routes, liposomes have been demonstrated to provide an efficient
and convenient drug delivery system (Gangwar et al., 2012; Mufamadi
et al., 2011). Numerous liposomal formulations of NSAIDs and
DMARDs have been studied for the therapies of RA (Kapoor et al.,
2014); however, the safety and stability of such systems remain chal-
lenging. Nevertheless, considering the success of the liposomal anti-
cancer drug (Yeh et al., 2012) and the antifungal agent amphotericin B
(Allen and Cullis, 2013b) in the clinic, similar achievements are an-
ticipated in the area of liposomal delivery of anti-arthritic drugs.
Y. Zhang, et al.
Fig. 4. (A) Photos of rat ankle treated with different treatments (n = 5); Ankle swelling rate (B) and the body weight (C) of different groups. (*P < 0.05, **
P < 0.01 and *** P < 0.001 compared with model group, and #P < 0.05, ## P < 0.01 and ### P < 0.001 compared with ART group).
The conducted in vitro assessment confirmed low cytotoxicity and
good anti-inflammatory effects of the Di-ART-GPC liposomes. The via-
bility of the cells treated with the Di-ART-GPC liposomes was much
higher than the viability of cells incubated with ART and the Di-ART-
GPC solution, indicating lower cytotoxicity of the Di-ART-GPC lipo-
somes. As a widely used antimalarial agent, ART is undeniably a safe
and reliable drug (Wang et al., 2015). However, the side-effects of ART
cannot be ignored. Based on our study, we suspect that the Di-ART-GPC
liposomes displaying lower cytotoxicity may show improved bio-safety
in comparison to ART. In addition, the Di-ART-GPC liposomes group
revealed higher inhibition of the secretion of the signal molecule (NO),
as well as of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6),
compared to the ART and Di-ART-GPC solution groups. As a positive
control, ART exhibits good anti-inflammatory activity, effectively re-
ducing the levels of various pro-inflammatory cytokines.
The in vivo evaluation was carried out using the FCA-induced RA rat
model, which is generally accepted as a reliable immunosuppression
and inflammation model, presenting characteristics analogous to those
observed in human RA pathophysiology (Wang et al., 2015). The pedal
swelling rate is one of the typical indicators utilized to evaluate anti-
arthritic potential of drugs. It is typically associated with intensification
of vascular perviousness, fluid and proteins extravasation, and cellular
invasion at the site of inflammation (Bose et al., 2014). Pleasingly, the
Di-ART-GPC liposomes exhibited a decline in the ankle swelling rate as
well as low inflammatory response compared with the model control
and ART. Spleen and thymus are important organs of the immune
system; therefore, to some extent, the spleen and thymus indexes can
reflect the strength of the immune function of the body. The Di-ART-
7
International Journal of Pharmaceutics 579 (2020) 119178
GPC liposomes group exhibited a considerable reduction in the values
of the spleen and thymus indexes compared with the model group,
indicating that liposomes had a certain inhibitory effect on the en-
hanced immune system caused by inflammation. These outcomes ver-
ified that the FCA-induced RA symptoms notably decreased after
treatment with the Di-ART-GPC liposomes. Compared with the dose of
3.0 mg/kg/d, the dose of 6.0 mg/kg/d achieved more optimal results.
Pro-inflammatory cytokines correlate with the pathogenesis of RA
and are involved in numerous pathological and physiological processes
(Achi et al., 2019; Stevanin et al., 2017). It is noteworthy that the in-
hibitory effects of the Di-ART-GPC liposomes against the pro-in-
flammatory cytokines were found as a result of both in vitro and in vivo
evaluations. Because pro-inflammatory cytokines are highly correlated
with the inflammation severity (Shabbir et al., 2014), the obvious de-
crease in the levels of the pro-inflammatory cytokines following the
treatment with the Di-ART-GPC liposomes can be regarded as an im-
portant verification of the improved anti-inflammatory effect. More-
over, it is assumed that the decreased levels of cytokines may further
affect the apparent pedal swelling symptoms. The improved pedal
swelling was consistent with the detected levels of the pro-in-
flammatory cytokines. Histopathological studies further confirmed the
anti-arthritic effects of the Di-ART-GPC liposomes. In RA, the synovial
membrane is predominantly affected, while joints are the primary loci
of inflammation. Histopathology analysis is the most reliable method
for the determination of pathological changes at the locations of the
lesions (Pan et al., 2019). The Di-ART-GPC treatment revealed obvious
improvement in the overall inflammatory signs, which was comparable
to the reference standard drug ART.
Y. Zhang, et al.
Fig. 5. The levels of spleen index (A), thymus index (B), TNF-α (C) and IL-6 (D) in different groups. (*P < 0.05, ** P < 0.01 and *** P < 0.001 compared with
model group, and #P < 0.05, ## P < 0.01 and ### P < 0.001 compared with ART group).
In the treatment of RA, high systemic doses are typically necessary
to achieve effective drug concentrations in the affected joints, which is
accompanied by various systemic side effects. Reduction in the drug
doses may result in alleviation of the side effects; however, the ther-
apeutic efficacy can decrease at the same time. Therefore, drugs spe-
cifically targeted to the synovial capsule of the affected joint have been
popular, particularly in the treatment of RA with only a limited number
of affected joints (Gerwin et al., 2006). Liposomes have been reported
to retain drugs in the synovial cavity as a consequence of their size and
chemical composition. Such an effective delivery system avoids the fast
clearance of the intra-synovial administered drugs, increases the uptake
of drugs by the target synovial cells, reduces the exposure of the non-
target sites, and eliminates many side effects (Kapoor et al., 2014).
Consequently, the intra-synovial injection of the Di-ART-GPC liposomes
into the affected joints may exhibit improved curative effects for RA
compared to systemic administration. The results obtained in the pre-
sent study provide a valuable platform for further research in this area.
5. Conclusions
In conclusion, the Di-ART-GPC liposomes were successfully pre-
pared and utilized as a novel ART-based anti-inflammatory therapy for
RA. Following high-pressure homogenization, the particle size of the Di-
ART-GPC liposomes decreased to a narrow range of approximately
8
International Journal of Pharmaceutics 579 (2020) 119178
70 nm. Moreover, the liposomal structure was confirmed by the cryo-
EM analysis. Notably, the in vitro evaluations confirmed the low cyto-
toxicity and good anti-inflammatory effects of the Di-ART-GPC lipo-
somes. In addition, the Di-ART-GPC liposomes were demonstrated to
result in significantly higher inhibition of the cell secretion of the stu-
died signal molecule (NO) and pro-inflammatory cytokines (TNF-α, IL-
1β, and IL-6) than ART. The conducted in vivo assessment using the
FCA-induced RA rat model showed that Di-ART-GPC led to a decline in
the ankle swelling rate and low inflammatory response compared with
the model control and ART. All of the obtained results proved that the
Di-ART-GPC liposomes show remarkable potential for application as
novel ART-based anti-inflammatory drugs for the treatment of RA.
CRediT authorship contribution statement
Yaru Zhang: Methodology, Investigation, Data curation, Writing -
original draft. Wei He: Methodology, Investigation, Validation. Yawei
Du: Visualization, Writing - review & editing. Ying Du: Investigation,
Validation. Chao Zhao: Investigation, Formal analysis. Ying Zhang:
Investigation. Hu Zhang: Resources. Lihong Yin: Conceptualization,
Supervision, Project administration. Xinsong Li: Supervision, Funding
acquisition.
Y. Zhang, et al.
Fig. 6. H&E stained sections of ankle joints. The black arrow indicates hyperplasia of connective tissues, the yellow arrow indicates inflammatory cell infiltrate in
articular cartilage and the red arrow indicates inflammatory cell infiltrate of synovial connective tissue. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This research was supported by the Major National Science and
Technology Program for Innovative Drugs from the Ministry of Science
and Technology of China (2017ZX09101002-001-004).
References
Achi, S.C., Talahalli, R.R., Halami, P.M., 2019. Prophylactic effects of probiotic
Bifidobacterium spp. in the resolution of inflammation in arthritic rats. Appl.
Microbiol. Biotechnol. 103, 6287–6296.
Allen, T.M., Cullis, P.R., 2013. Liposomal drug delivery systems: From concept to clinical
applications. Adv. Drug Deliv. Rev. 65, 36–48.
Bose, M., Chakraborty, M., Bhattacharya, S., Bhattacharjee, P., Mandal, S., Kar, M.,
Mishra, R., 2014. Suppression of NF-kappaB p65 nuclear translocation and tumor
necrosis factor-alpha by Pongamia pinnata seed extract in adjuvant-induced arthritis.
J. Immunotoxicol. 11, 222–230.
Cen, Y., Liu, C., Li, X., Yan, Z., Kuang, M., Su, Y., Pan, X., Qin, R., Liu, X., Zheng, J., Zhou,
H., 2016. Artesunate ameliorates severe acute pancreatitis (SAP) in rats by inhibiting
expression of pro-inflammatory cytokines and Toll-like receptor 4. Int.
Immunopharmacol. 38, 252–260.
Choudhary, M., Kumar, V., Gupta, P., Singh, S., 2014. Investigation of antiarthritic po-
tential of Plumeria alba L. leaves in acute and chronic models of arthritis. Biomed.
Res. Int. 2014, 474616.
Costa, E.A., Lino, R.C., Gomes, M.N., Nascimento, M.V., Florentino, I.F., Galdino, P.M.,
Andrade, C.H., Rezende, K.R., Magalhaes, L.O., Menegatti, R., 2013. Anti-in-
flammatory and antinociceptive activities of LQFM002 - A 4-nerolidylcatechol deri-
vative. Life Sci. 92, 237–244.
Dong, J., Tao, L., Mas, A., Hussain, Z., 2018. Design and development of novel hyalur-
onate-modified nanoparticles for combo-delivery of curcumin and alendronate: fab-
rication, characterization, and cellular and molecular evidences of enhanced bone
regeneration. Int. J. Biol. Macromol. 116, 1268–1281.
Zhuo, F., Abourehab, M.A., Hussain, Z., 2018. Hyaluronic acid decorated tacrolimus-
loaded nanoparticles: efficient approach to maximize dermal targeting and anti-
dermatitis efficacy. Carbohydrate Polym. 197, 478–480.
Gang, F., Qinghuai, Z., Yuzhou, P., Hnin, E.T., Zahid, H., 2019. Nanomedicines for im-
proved targetability to inflamed synovium for treatment of rheumatoid arthritis:
9
International Journal of Pharmaceutics 579 (2020) 119178
Multi-functionalization as an emerging strategy to optimize therapeutic efficacy. J.
Control. Release 303, 181–208.
Gangwar, M., Singh, R., Goel, R.K., Nath, G., 2012. Recent advances in various emerging
vescicular systems: an overview. Asian Pacific J. Tropical Biomed. 2, S1176–S1188.
Gerwin, N., Hops, C., Lucke, A., 2006. Intraarticular drug delivery in osteoarthritis. Adv.
Drug Deliv. Rev. 58, 226–242.
Gottschalk, O., Metz, P., Dao Trong, M.L., Altenberger, S., Jansson, V., Mutschler, W.,
Schmitt-Sody, M., 2015. Therapeutic effect of methotrexate encapsulated in cationic
liposomes (EndoMTX) in comparison to free methotrexate in an antigen-induced
arthritis study in vivo. Scand. J. Rheumatol. 44, 456–463.
Huang, Z.Z., Xu, Y., Xu, M., Shi, Z.R., Mai, S.Z., Guo, Z.X., Tang, Z.Q., Luo, Y.J., Guo, Q.,
Xiong, H., 2019. Artesunate alleviates imiquimod-induced psoriasis-like dermatitis in
BALB/c mice. Int. Immunopharmacol. 75, 105817.
Hussain, Z., 2019. Nanotechnology guided newer intervention for treatment of osteo-
porosis: efficient bone regeneration by up-regulation of proliferation, differentiation
and mineralization of osteoblasts. Int. J. Polym. Mater. Polym. Biomater. 1–13.
Ismail, M., Ling, L., Du, Y., Yao, C., Li, X., 2018. Liposomes of dimeric artesunate phos-
pholipid: a combination of dimerization and self-assembly to combat malaria.
Biomaterials 163, 76–87.
Jang, S., Jeong, M.H., Lim, K.S., Bae, I.H., Park, J.K., Park, D.S., Shim, J.W., Kim, J.H.,
Kim, H.K., Sim, D.S., Hong, Y.J., Ahn, Y., Kang, J.C., 2017. Effect of stents coated
with artemisinin or dihydroartemisinin in a porcine coronary restenosis model.
Korean Circ. J. 47, 115–122.
Jiang, W., Luo, F., Lu, Q., Liu, J., Li, P., Wang, X., Fu, Y., Hao, K., Yan, T., Ding, X., 2016.
The protective effect of Trillin LPS-induced acute lung injury by the regulations of
inflammation and oxidative state. Chem. Biol. Interact. 243, 127–134.
Kapoor, B., Singh, S.K., Gulati, M., Gupta, R., Vaidya, Y., 2014. Application of liposomes
in treatment of rheumatoid arthritis: quo vadis. Sci. World J. 2014, 978351.
Lai, L., Chen, Y., Tian, X., Li, X., Zhang, X., Lei, J., Bi, Y., Fang, B., Song, X., 2015.
Artesunate alleviates hepatic fibrosis induced by multiple pathogenic factors and
inflammation through the inhibition of LPS/TLR4/NF-kappaB signaling pathway in
rats. Eur. J. Pharmacol. 765, 234–241.
Lee, H.H., Moon, Y., Shin, J.S., Lee, J.H., Kim, T.W., Jang, C., Park, C., Lee, J., Kim, Y.,
Kim, Y., Werz, O., Park, B.Y., Lee, J.Y., Lee, K.T., 2019. A novel mPGES-1 inhibitor
alleviates inflammatory responses by downregulating PGE2 in experimental models.
Prostaglandins Other Lipid Mediat. 144, 106347.
Li, Y., Wang, S., Wang, Y., Zhou, C., Chen, G., Shen, W., Li, C., Lin, W., 2013. Inhibitory
effect of the antimalarial agent artesunate on collagen-induced arthritis in rats
through nuclear factor kappa B and mitogen-activated protein kinase signaling
pathway. Transl. Res. 161, 89–98.
Li, Y.J., Guo, Y., Yang, Q., Weng, X.G., Yang, L., Wang, Y.J., Chen, Y., Zhang, D., Li, Q.,
Liu, X.C., Kan, X.X., Chen, X., Zhu, X.X., Kmoniekova, E., Zidek, Z., 2015. Flavonoids
casticin and chrysosplenol D from Artemisia annua L. inhibit inflammation in vitro
and in vivo. Toxicol. Appl. Pharmacol. 286, 151–158.
Lin ZM, Y.X., Zhu FH, et al., 2016. Artemisinin analogue SM934 attenuate collagen-in-
duced arthritis by suppressing T follicular helper cells and T helper 17 cells. 38115.
Liu, X., Lu, J., Liao, Y., Liu, S., Chen, Y., He, R., Men, L., Lu, C., Chen, Z., Li, S., Xiong, G.,
Yang, S., 2019. Dihydroartemisinin attenuates lipopolysaccharide-induced acute
Y. Zhang, et al.
kidney injury by inhibiting inflammation and oxidative stress. Biomed.
Pharmacother. 117, 109070.
Miller, L.H., Su, X., 2011. Artemisinin: discovery from the Chinese herbal garden. Cell
146, 855–858.
Mufamadi, M.S., Pillay, V., Choonara, Y.E., Du, T., Lisa, C., Modi, G., Naidoo, D.N.,
Valence, M.K., 2011. A review on composite liposomal technologies for specialized
drug delivery. J. Drug Deliv. 2–19.
Pal, R., Chaudhary, M.J., Tiwari, P.C., Babu, S., Pant, K.K., 2015. Protective role of
theophylline and their interaction with nitric oxide (NO) in adjuvant-induced rheu-
matoid arthritis in rats. Int. Immunopharmacol. 29, 854–862.
Pan, T.C., Tsai, Y.H., Chen, W.C., Hsieh, Y.L., 2019. The effects of laser acupuncture on
the modulation of cartilage extracellular matrix macromolecules in rats with ad-
juvant-induced arthritis. PLoS ONE 14, e0211341.
Pandey, M., Choudhury, H., Gunasegaran, T.A.P., Nathan, S.S., Md, S., Gorain, B.,
Tripathy, M., Hussain, Z., 2019. Hyaluronic acid-modified betamethasone en-
capsulated polymeric nanoparticles: fabrication, characterisation, in vitro release
kinetics, and dermal targeting. Drug Deliv. Transl. Res. 9, 520–533.
Prakash, D., Katiyar, N.S., Singh, A.P., Gangwar, A.K., 2016. Evaluation of anti-arthritic
potential of Zingiber officinale in experimental rats. Eur. J. Pharm. Med. Res. 3,
305–308.
Shabbir, A., Shahzad, M., Ali, A., Zia-ur-Rehman, M., 2014. Anti-arthritic activity of N'-
[(2,4-dihydroxyphenyl)methylidene]-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c]
[1,2]benzothiazin-1(4H)-yl)acetohydrazide. Eur. J. Pharmacol. 738, 263–272.
Shi, C., Li, H., Yang, Y., Hou, L., 2015. Anti-inflammatory and immunoregulatory func-
tions of artemisinin and its derivatives. Mediators Inflamm. 2015, 435713.
Smolen, J.S., Aletaha, D., McInnes, I.B., 2016. Rheumatoid arthritis. Lancet 388,
2023–2038.
Stevanin, M., Busso, N., Chobaz, V., Pigni, M., Ghassem-Zadeh, S., Zhang, L., Acha-Orbea,
10
International Journal of Pharmaceutics 579 (2020) 119178
H., Ehirchiou, D., 2017. CD11b regulates the Treg/Th17 balance in murine arthritis
via IL-6. Eur. J. Immunol. 47, 637–645.
Sultana, F., Neog, M.K., Rasool, M., 2017. Targeted delivery of morin, a dietary bio-
flavanol encapsulated mannosylated liposomes to the macrophages of adjuvant-in-
duced arthritis rats inhibits inflammatory immune response and osteoclastogenesis.
Eur. J. Pharm. Biopharm. 115, 229–242.
Uttra, A.M., Alamgeer, Shahzad, M., Shabbir, A., Jahan, S., 2018. Ephedra gerardiana
aqueous ethanolic extract and fractions attenuate Freund Complete Adjuvant induced
arthritis in Sprague Dawley rats by downregulating PGE2, COX2, IL-1beta, IL-6, TNF-
alpha, NF-kB and upregulating IL-4 and IL-10. J. Ethnopharmacol. 224, 482–496.
Vilar, K.M., Pereira, M.C., Dantas, A.T., Rego, M., Pitta, I.D.R., Marques, C.D.L.,
Goncalves, R.S.G., Junior, L., Duarte, A., Pitta, M., 2019. Galectin-1, -4, and -7 were
associated with high activity of disease in patients with rheumatoid arthritis.
Autoimmune Dis. 2019, 3081621.
Vyas, S.P., Singh, R., Asati, R.K., 1995. Liposomally encapsulated diclofenac for sono-
phoresis induced systemic delivery. J. Microencapsul. 12, 149–154.
Wang, D., Shi, J., Lv, S., Xu, W., Li, J., Ge, W., Xiao, C., Geng, D., Liu, Y., 2015. Artesunate
attenuates lipopolysaccharide-stimulated proinflammatory responses by suppressing
TLR4, MyD88 expression, and NF-kappaB activation in microglial cells. Inflammation
38, 1925–1932.
Wu, Y., Tang, W., Zuo, J., 2016. Development of artemisinin drugs in the treatment of
autoimmune diseases. Sci. Bull. 61, 37–41.
Yeh, M.K., Chang, H.I., Cheng, M.Y., 2012. Clinical development of liposome based drugs:
formulation, characterization, and therapeutic efficacy. Int. J. Nanomed. 7, 49–60.
Zhao, D., Zhang, J., Xu, G., Wang, Q., 2017. Artesunate Protects LPS-Induced Acute Lung
Injury by Inhibiting TLR4 Expression and Inducing Nrf2 Activation. Inflammation 40,
798–805.