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Liposomal glutathione as a promising candidate for immunological
rheumatoid arthritis therapy
Mai O. Kadry
*
National Research Center, Therapeutic Chemistry Department, Al Bouhooth Street, Dokki, Giza, Egypt
ARTICLE INFO
Keywords:
Biochemistry
Biotechnology
Cell biology
Molecular biology
Toxicology
ABSTRACT
Nano-medicine can passively accumulate in chronic inflammatory tissues via the enhanced permeability and
retention phenomenon, or by being conjugated with a ligand that can bind to receptors over expressed by cells
inside chronic inflammatory tissues, contributing to reduced systemic side-effects and increased efficacy. This
article highlights the utilization of nanomedicine for potential treatment of rheumatoid arthritis. Rheumatoid
arthritis was induced in rat model via 2 weeks intradermal injection of pristane at the base of the tail in a daily
dose of 150
μ
l. Susceptible rat strains developed severe arthritis with a sudden onset 3 weeks post pristane in-
jection. Three weeks post pristane administration; rats were treated intravenously with glutathione or liposomal-
glutathione in a dose of 5 mg/kg daily for 30 days. Concomitant supplementation with the aforementioned an-
tioxidants effect on proinflammatory marker C-reactive protein (CRP) was assessed. On the other hand, oxidative
stress biomarker malondialdehyde (MDA) and rheumatoid factor (RF) compared with pristane treated group was
also investigated. The results elucidated that glutathione and liposomal -glutathione significantly reduced rheu-
matoid factor, malondialdehyde and C-reactive protein levels with the superiority of liposomal -glutathione in this
side reflecting its pronounced effect as anti-rheumatoid agent.
1. Introduction
A widespread understanding in the pathophysiology of chronic in-
flammatory autoimmune diseases, such as RA, affirms that the diseased
tissue has high prevalence of macrophages, inflammatory mediators and
necrotic monocytes lacking clearance via lymphatic system, which can
become an inflammatory trigger in itself contributing to adaptive im-
mune response [1,2]. This can lead to boosting the delivery of
nano-medicine. Nano-medicine can passively accumulate into chronic
inflammatory tissues via the augmented permeability and retention
phenomenon, leading to decreased systemic adverse-effects [1].
RA affects women three-times more than men and affecting 10% of
the population in developing countries [3]. RA is characterized by sy-
novial inflammation, which can lead to deformation, bone erosion in
addition to loss of joint function. Other clinical manifestations include
elevation in rheumatoid factor (RF), inflammatory markers (CRP, TNF-
α
and IL-6) in the blood, muscle soreness and joint tenderness [4].
Vast majority of inflammatory autoimmune diseases can be
controlled, however not completely cured; NSAIDs and corticosteroids
are the current available therapies for RA [5,6]. NSAIDs are accom-
pained with some side-effects as fluid retention, gastrointestinal bleeding
and increased risk of heart disease. Corticosteroids in high-dose can
cause gastrointestinal complications, glucose intolerance, liver toxicity
and adrenal suppression [7,8].
Nano-medicines are designed nowadays to target certain receptors,
prevent the degradation of therapeutic agent, extend the time of reten-
tion in blood circulation and to be tailored for macrophage [9,10].
The use of nanoparticles such as liposomal -glutathione increase site-
specific drug targeting to inflamed tissues, by utilizing the disease state
including, enhanced permeability or changes in pH of inflamed tissues.;
thereby, allowing for maximum drug action with less adverse effect than
traditional drug therapy [11,12].
The focus of this study lies on the elucidation of the impact of lipo-
somal -glutathione in comparison with glutathione on rheumatoid
arthritis induced in rat model.
2. Material and methods
2.1. Chemicals
Pristane, glutathione and liposomal -glutathione were purchased
from Sigma-Aldrich Co (St. Louis, MO, USA). ELISA kits for C-reactive
* Corresponding author.
E-mail address: maiosman666@yahoo.com.
Contents lists available at ScienceDirect
Heliyon
journal homepage: www.heliyon.com
https://doi.org/10.1016/j.heliyon.2019.e02162
Received 24 March 2019; Received in revised form 6 June 2019; Accepted 24 July 2019
2405-8440/©2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Heliyon 5 (2019) e02162
protein and rheumatoid factor determination were provided from R &D
systems (MN, USA). Kits used for the determination of malondialdehyde
was obtained from Randox Company (UK). All other chemicals were of
the highest analytical grade.
2.2. Animals
Forty male Wister albino rats, weighing 190–200 gm, obtained from
the animal house of National Research Center were used in this study.
Animals were housed in cages kept at standardized conditions (22 5C,
55 5% humidity, and 12 h light/dark cycle). They were allowed free
access to water and pelleted standard chow diet.
All procedures relating to animal care and treatments strictly adhered
to the ethical procedures and policies approved by Animal Care and Use
Committee of National Research Center (AR110204), and complied with
the Guide for Care and Use of Laboratory published by the US National
Institute of Health.
2.3. Experimental design
After 1 week of acclimatization, animals were randomly divided into
four groups (10 rats each) and were divided according to the following
schedule:
Group 1: Animals were treated by saline (control group).
Group 2: Animals treated with pristane via 2 weeks intradermal in-
jection at the base of the tail in a daily dose of 150
μ
l (0.04028 gm) in a
total dose of 0.56gm which represent (2.8 gm/kg BW) and served as
rheumatoid arthritis model [13].
3 weeks post pristane administration; rats were treated with gluta-
thione or liposomal -glutathione [14,15].
Group 3: pristane - treated animals were treated intravenously with
glutathione in a dose of 5 mg/kg daily for 30 days [14].
Group 4: pristane - treated animals were treated intravenously with
liposomal-glutathione in a dose of 5 mg/kg daily for 30 days [15].
2.4. Blood sampling
Animals were followed for any sign of sickness. At the end of the
experimental period, rats were weighed, slightly anesthetized by ether
and blood samples were collected from the sublingual vein. Sera were
separated by centrifugation at 2555g for 10 min and were kept at 80 C
for subsequent estimation of biochemical parameters. Animals were then
sacrificed by cervical dislocation.
2.5. Measured parameters
2.5.1. Serum malondialdehyde (MDA) level
MDA expressed as thiobarbituric acid reactive substances was
measured using diagnostic kits provided from Randox Company as pre-
viously described by [16].
2.5.2. Serum C-reactive protein and rheumatoid factor activities
The activities of C-reactive protein and rheumatoid factor were
assayed using ELISA kits (R &D systems MN, USA) according to the
manufacturer's instructions. The assays estimated the quantitative
sandwich enzyme immunoassay technique. Specific antibodies were pre-
coated onto the microplate. The standards, and samples were pipetted
into the wells and C-reactive protein was bound by the immobilized
antibody. After washing away any unbound substances, an enzyme-
linked secondary antibody specific for C-reactive protein and rheuma-
toid factor were added to the wells. Following color development, the
assay was stopped, and the absorbance was read at 450 nm [17].
2.5.3. Statistical analysis
Data were expressed as means S.E.M. Statistical analysis was per-
formed using Instat-3 computer program (Graph pad software Inc, San
Diego, CA, USA). One way analysis of variance (ANOVA) by SPSS 12
program followed by Post hoc test was conducted. The level of signifi-
cance was set at p<0.05 using Tukey ҆s test.
3. Results
3.1. Body weight coefficient
Body weight revealed a significant decrease post pristane treatment
which was significantly increased post glutathione and liposomal gluta-
thione treatment as represented in (Table 1).
3.2. Evaluation of clinical arthritis
Inflamed toes were observed every day; each inflamed one was given
1 point and up to 5 points for the affected 5 toes or the ankle and data was
recorded in (Table 2).
3.3. Modulation of rheumatoid factor biomarker
A significant elevation in rheumatoid factor level was elucidated post
pristane treatment by a percentage of 400% as compared with the normal
value indicating a state of rheumatoid arthiritis (Fig. 1). Treatment with
glutathione and nano-glutathione evidenced a significant reduction in RF
level with a percentage of 200 and 140% respectively in comparison to
animals treated with pristane with liposomal -glutathione showing the
most significant effect.
3.4. Modulation of oxidative stress biomarker
Significant elevation in malondialdehyde level was elucidated post
pristane treatment by a percentage of 180% as compared with the normal
value indicating a state of severe oxidative stress (Fig. 2). Treatment with
glutathione and nano-glutathione evidenced a significant reduction in
MDA level with a percentage of 160 and 95% respectively in comparison
to animals treated with pristane with liposomal -glutathione showing the
most significant effect.
3.5. Modulation of inflammatory biomarker
Fig. 1 elucidated that, pristane treatment produced a significant
Table 1
Effect of glutathione and liposomal glutathione on body weight following pris-
tane administration.
Groups
Parameter Control Pristane Glutathione Liposomal
glutathione
Body
weight
197.5
2.5
a
182.3
1.8
b
189.9 1.7
c
192.5 1.65
d
Data are expressed as means S.E.M (n ¼10). P-value <0.05 is considered sig-
nificant. Groups having the same letter are not significantly different, while those
having different letters are significantly different from each other.
Table 2
Effect of glutathione and liposomal glutathione on clinical investigations
following pristane administration.
Groups
Parameter Control Pristane Glutathione Liposomal
glutathione
Score of toe
deformation
0
a
56.4
2.1
b
29.2 1.6
c
20.3 1.3
d
Data are expressed as means S.E.M (n ¼10). P-value <0.05 is considered sig-
nificant. Groups having the same letter are not significantly different, while those
having different letters are significantly different from each other.
M.O. Kadry Heliyon 5 (2019) e02162
2
elevation in serum C-reactive protein level by a value of 500% as
compared with the control value. On the other hand, there was a sig-
nificant reduction in its level post glutathione and liposomal -glutathione
treatment with a percentage of 112.5 and 102.5% respectively with
liposomal -glutathione showing the most significant effect as compared
with pristane treated group.
4. Discussion
A growing body of evidence contributes to nano-medicine application
in the field of diagnosis, treatment or prevention of disease. Nano-
medicines may include drug-loaded liposomes, nano-capsules and
nanoparticles [9]. Nano-medicines were performed to: prevent the
degradation of therapeutic agents, increase retention time and to be
tailored for macrophage uptake or target certain receptors [18].
Over expression of inflammatory mediators in RA, contributed to
increased tissue permeability. Moreover, inflamed tissues have more
activated macrophages or other monocytes and changes in pH that can be
utilized as targets for site-specific drug delivery systems as nano-carriers
[19,20].
In the current study, oxidative stress biomarker malondialdehyde
(MDA) and RA biomarker rheumatoid factor (RF) were significantly
elevated post pristane intoxication as compared with the control value.
The results elucidated that liposomal-glutathione and glutathione
significantly reduced rheumatoid factor and malondialdehyde levels
with liposomal -glutathione reflecting the most pronounced effect.
Fig. 1. Effect of glutathione and liposomal glutathione on serum rheumatoid factor and C-reactive protein following pristane administration. Data are expressed as
means S.E.M (n ¼10). P-value <0.05 is considered significant. Groups having the same letter are not significantly different, while those having different letters are
significantly different from each other.
Fig. 2. Effect of glutathione and liposomal glutathione on serum malondialdehyde following pristane administration. Data are expressed as means S.E.M (n ¼10). P-
value <0.05 is considered significant. Groups having the same letter are not significantly different, while those having different letters are significantly differentfrom
each other.
M.O. Kadry Heliyon 5 (2019) e02162
3
Exposure to pristane for 12 or 24 weeks promoted macrophage acti-
vation syndrome, characterized by hemophagocytosis in spleen and pe-
ripheral blood, as well as hypercytokinemia of IFN-γ, TNF-
α
, IL-4, and IL-
6. In addition to a profound elevation in MDA and reduction in SOD,
GSH, and catalase activities were also observed [21,22].
In harmony, GSH is a good supplement for RA which can regulate
immune cells preventing them from attacking the normal body cells. GSH
can neutralize free radicals which can destroy tissues and muscles of the
joints. It increases body energy and owns anti-inflammatory effect [23,
24].
Liposomal GSH is capable to bypass de novo glutathione synthesis
contributing to elevating glutathione level, improving redox homeostasis
and decreasing the effect of TGF-β[24,25].
A significant elevation in C-reactive protein activity was elucidated in
the present study meanwhile treatment with glutathione and liposomal
glutathione was observed with liposomal glutathione elucidating the
most significant effect.
Liposomal glucocorticoids elucidate local delivery and mounting in
inflammatory sites, contributing to increased therapeutic efficiency and
decreased systemic side-effects. Hofkens et al [26] showed that liposomal
prednisolone phosphate reduced the proinflammatory macrophages
activation in vivo [26,27]. After macrophage uptake, significant re-
ductions were seen in the expression of proinflammatory cytokines
including CRP, TNF-
α
and IL-8 [26]. Selective biodistribution in inflamed
tissues due to enhanced permeability and the resultant lower effective
dose and longer duration of drug action leading to decreased dose fre-
quency are a recurring theme with nano-medicines in RA models with
less systemic side-effects [28].
5. Conclusion
Nano-glutathione may be a promising supplement for rheumatoid
arthritis therapy and can improve oxidative stress as well as inflamma-
tory markers. Further studies may be required and further investigations.
Declarations
Author contribution statement
Mai O Kadry: Conceived and designed the experiments; Performed
the experiments; Analyzed and interpreted the data; Contributed re-
agents, materials, analysis tools or data; Wrote the paper.
Funding statement
This work was supported by grants from the National Research Center
internal projects (AR110204).
Competing interest statement
The authors declare no conflict of interest.
Additional information
No additional information is available for this paper.
Acknowledgements
Is directed to the National Research Center Egypt.
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