The Effects of Liposomale Methylprednisolone

Palmitate on the Production of TNFα in Mice

Aprilita Rina Yanti Eff

, F. D. Suyatna

and Erni H. Purwaningsih

Department of Pharmacy, Faculty of Health Sciences, Universitas Esa Unggul, Jl,Arjuna Utara, West Jakarta, Indonesia

Department of Pharmacology and Therapeutics, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Department of Pharmacy, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Keywords: Glucocorticoid, liposome, methylprednisolone palmitate, TNFα

Abstract: Introduction & Aim: Liposomes are used in this study as the carrier of an immunosuppressive drug, namely

methylprednisolone palmitate (MPLP), to reduce the drug side effects on various organ systems, such as

musculoskeletal, gastrointestinal, and cardiovascular. This study aimed to investigate the effects of low-dose

liposomal methylprednisolone palmitate (L-MPLP) on the in vitro and in vivo production of tumor necrosis

factor alpha (TNFα) derived from the culture of C3H mice spleen. Methods: For the in vitro culture, the

TNFα levels were identified using the splenic lymphocyte cultures. Meanwhile, for the in vivo culture, the

mice were divided into eight groups of five (5) mice randomly. Forty-eight hours after the drug

administration, these mice were sacrificed. The spleen was removed and used for lymphocyte culture. The

TNFα levels were measured with ELISA at a wavelength of 450 nm. Results: The in vitro and in vivo

assays showed that, when administered at the same dose, L-MPLP produced lower TNFα than

methylprednisolone (MPL). Conclusion: At small doses, L-MPLP can significantly inhibit the in vitro and

in vivo production of TNFα, as opposed to the control group MPL.

1 INTRODUCTION

Liposomes are phospholipid vesicles containing

polar and non-polar groups with a phospholipid

membrane structure similar to the cell membrane.

Liposomal vesicles are formed spontaneously when

lipids or phospholipids exposed to water. Liposomes

carry drug molecules in various ways, namely the

encapsulation of hydrophobic drug substances by

interacting with the lipophilic substance membrane

and the entrapment of hydrophilic substance inside

the vesicle (Jone, 2013).

As a drug carrier, liposomes have been tested in

animals and humans either by oral or parenteral

route. The oral administration of liposomes is

ineffective because they are enzymatically

hydrolyzed before entering the blood circulation.

They are also unstable in the intestines, and they can

interact with bile salts. Meanwhile, their parenteral

administration, especially intravenous, is more easily

monitored in terms of extravasation and uptake by

tissues. Several studies have used liposomes as a

drug carrier with the following reasons: liposomes

extend the half-life of drugs and increase the

distribution of drugs to the targeted organs, which

consequently reduce drug dose and minimize the

drug side effects (Anwekar et al., 2011; Ait-Oudhia

et al., 2014).

MPL is a lipophilic glucocorticoid containing a

hydroxyl (-OH) group at C

which makes it

amphoteric and able to form micelles. The formation

of micelles makes MPL easily detached from the

liposome membrane, resulting in unstable

liposomes. Benameur et al. (1993) incorporate

dexamethasone palmitate (DMP) into liposomes by

replacing the -OH group at C

with a palmitate

group, forming a more lipophilic drug and creating a

better DMP interaction with liposome membrane

than dexamethasone alone. Shaw et al. (1976) also

manage to incorporate cortisone in the form of

cortisone palmitate into liposomes. Following the

success of Benaumer et al. (1993) and Shaw et al.

(1976) and relying on the similarity of the basic

structures of dexamethasone- and cortisone-

incorporated methylprednisolone, this research

hypothesized that the addition of a palmitate group

to the C

of methylprednisolone would give similar

results. The initial experiment in our research proved

Eff, A., Suyatna, F. and Purwaningsih, E.

The Effects of Liposomale Methylprednisolone Palmitate on the Production of TNFÎ

s in Mice.

DOI: 10.5220/0008239700770083

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 77-83

ISBN: 978-989-758-349-0

that the preparation of liposomal methylprednisolone

palmitate (L-MPLP) using phosphatidylcholine from

egg yolk (Egg-Yolk Phosphatidyl Choline/EPC)

resulted in an incorporation of about 70% and that

the combination of EPC and 2.5 mol% tetraether

lipid (TEL) increased the incorporation of MPLP

into the liposome membrane to 95%.

In spite of having strong anti-inflammatory

effects, dexamethasone has a long half-life,

stimulates activity in bone demineralization, and

suppresses the growth factor better than

methylprednisolone; therefore, it is not suitable for

long-term therapy, for example in post-organ

transplantation (Becker, 2013). This condition is the

reason behind the exclusion of dexamethasone in our

study. Immunosuppressants play an important role in

preventing the recipient’s body from rejecting

transplanted organ or tissue and in treating several

autoimmune diseases. Immunosuppressive drugs

promote the success of organ transplants, such as

kidney, bone marrow, liver, heart, pancreas, and

lungs. Some autoimmune diseases and disorders,

such as hemolytic anemia, idiopathic

thrombocytopenia purpura, Hashimoto's thyroiditis,

systemic lupus erythematosus, acute

glomerulonephritis, and acquired hemophilia, have

improved with the use of this drug (Luisa and

Piedras, 2013).

Liposomes are used as drug carriers, which in

this case is methylprednisolone palmitate (MPLP),

to reduce the drug side effects and retain the

therapeutic function of the resultant liposomal drug

even when administered at a small dose. MPLP is a

novel compound created using the same synthesis

method as that of dexamethasone palmitate

developed by Benameur et al. (1993), and it has

been successfully incorporated into the liposome

membrane by Purwaningsih et al. (2007), forming

methylprednisolone palmitate (L-MPLP). However,

this new compound has never been evaluated for its

immunosuppressive effect. The initial step of the

biological activity test of L-MPLP is assessing its

immunosuppressive effects on lymphocyte

proliferation. This new compound is expected to

inhibit the proliferation of lymphocytes, which is

indicated by the decreased production of TNFα, as

the mitogen ‘concanavalin A’ stimulates the other

glucocorticoids in cultured lymphocytes.

TNFα is a cytokine playing a major role in the

activation of immune reactions, including the

specific and non-specific immune system. The other

roles of TNFα are to stimulate endothelial cells,

express adhesion molecules, activate inflammatory

cells, and stimulate other cells in expressing major

histocompatibility (MHC) class I molecules on the

cytotoxic T cells (Keystone and Ware, 2010).

Lymphocyte proliferation test mainly aims to

determine the proliferation ability of lymphocyte

cells after mitogen stimulation with or without

drugs. Some mitogens stimulate a specific

subpopulation of lymphocytes. For instance,

concanavalin A (con-A) stimulates T lymphocyte

cells (Abbas et al., 2014).

This study aimed to evaluate the biological

effects of L-MPLP by measuring the TNFα levels

in the lymphocyte cultures of C3H mice spleen after

the administration of L-MPLP with different

concentrations, namely 0.005 mM, 0.05 mM, and

0.5 mM, and 48 hours after the intravenous

administration of L-MPLP at different doses, i.e., 2,

8, and 16 mg/kg BW.

2 MATERIALS AND METHODS

2.1 Materials

The liposomes and liposomal methylprednisolone

palmitate (L-MPLP) were made fresh from Egg-

Yolk Phosphatidylcholine (EPC), the Tetraether

Lipid (TEL) was obtained from Purwaningsih et al.

(2007), and the MPLP was donated by Bernina

Biosystems GmBH. The methylprednisolone-Na

succinate (Solu-Medrol) was purchased from

Upjohn. This research also used ethyl acetate,

methanol, chloroform PA, NaOH, HCl, Tris buffer

solution (pH 7.4) from Merck (filtered through a

Millipore membrane before use), and Aquabidest

(sterilized water) from IKA Farma. The other

materials were RPMI 1640 (pH 7.2-7.4, Gibco), fetal

bovine serum/FBS (ICN Flow), gentamicin (Gibco),

fungizone (Gibco), sodium bicarbonate (ICN Flow),

concanavalin A (Sigma) as a mitogen, nylon wool

(Biotest), nylon mesh, 2M HCl, aqua Millipore,

aquabidest, 70% alcohol, sterile cottons, candles,

gas from Perum Aneka Gas, and TNFα ELISA

kits (Quantikine). The male C3H mice (12-16 weeks

old, 20-22 gr in weight) were obtained from the

Department of Anatomic Pathology, Faculty of

Medicine, Universitas Indonesia. Before the

experiment, the test animals were acclimatized in

captivity in the Laboratory of Pharmacology,

Faculty of Medicine. They were given ad libitum

access to food and drink. The study approval (No

556/PT02.FK/ETIK/2012) was obtained from the

Ethics Committee, Faculty of Medicine, Universitas

Indonesia before the study began.

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

2.2 Methods

2.2.1 In Vitro Measurement of TNFα Levels

The TNFα assay was performed in the splenic

lymphocyte cultures using the Tris buffer solution

and liposomes as controls. RPMI solution containing

1x10

-6

cells/ml and 2.5 g/ml concanavalin A were

added to each of the following groups: the liposome

solution (20 ml, 0.5 mM), MPL, and the three L-

MPLP concentrations (0.5 mM, 0.05 mM, and

0.005 mM). After 48-hour incubation, the cultures

were centrifuged at 2,000g for 10 min. The

supernatants were discharged and stored at -20

before being used for the measurement of TNFα

levels using ELISA at a wavelength of 450 nm.

2.2.2 In Vivo Measurement of TNFα Levels

Drug Administration

The drug was administered intravenously to each

mouse based on the group division, i.e., eight (8)

groups of 5 mice, via vena lateralis in the tail. The

group division was as follows: Group I was used as a

control (Tris buffer, 5ml/kg); Groups II, III, and IV

were administered with methylprednisolone sodium

succinate/MPL at different concentrations, i.e., 8, 16,

and 32 mg/kg BW, respectively; Groups V, VI, and-

VII were given liposomal methylprednisolone (L-

MPLP) at different concentrations, i.e., 2, 8, and 16

mg/kg BW, respectively; and Group VIII was treated

with 0.5 mM liposomes. Forty-eight hours after drug

administration, these mice were sacrificed by

exposing them to the aether. Their spleens were

removed using scissors, and the lymphocyte culture

was collected with tweezers.

The Lymphocyte Culture

The spleens were placed in a sterile 60mm Petri dish

containing 5 ml of RPMI medium. To get the

lymphocyte cell suspension, the cell was filtered

through a sterile nylon mesh, and the erythrocytes

were lysed with ammonium chloride buffer solution.

The amount of T lymphocytes was enriched by

flowing the cell suspension through a nylon wool

column, which had been soaked in 2M HCl

overnight and then washed with distilled water, for

5-6 times and aerating it to allow drying.

Nylon wool fibers were separated with tweezers

and inserted into a polyethylene plastic syringe. As

much as 0.3 gram of nylon wool was inserted into a

5ml syringe to form the column for the spleen cell

suspension. The insertion was conducted with

moderate pressure and followed by the slow release

of the piston. The column, piston, and needle syringe

were sterilized in an autoclave for 15 minutes at a

temperature of 110-115

C. The column was

preincubated with RPMI 1640 medium containing

5% fetal bovine serum (FBS). The cell suspension

was resuspended to achieve a population of 5x10

cells/ml. It was loaded onto the column and, then,

incubated at 37

C for 60 minutes. The unretained

lymphocytes were eluted with a medium (eluent) as

much as one volume of the syringe. The eluate (i.e.,

T lymphocytes) was washed two (2) times and

centrifuged at 2,000g for 5 minutes. Afterward, a

population of 1x10

cells/ml was prepared from the

cell suspension. Lymphocytes with a concentration

of 1x10

cells/ml were maintained in RPMI 1640

medium containing 5% FBS, fungizone, gentamicin,

and concanavalin A (10 µg/ml). The in vitro cultures

were created by adding MPL and L-MPLP with four

different concentrations (i.e., 0.005 mM, 0.05 mM,

0.5 mM, and 0.5 mM) to the mice spleen. After a 48-

hour incubation, the cultures were centrifuged at

2,000g for 10 min. The supernatants were

discharged and stored at -20

C before being used for

the measurement of TNFα levels using ELISA at a

wavelength of 450 nm (Wohler & Barnum, 2010;

Bhattacharjee & Das, 2008)

2.2.3 Data Analysis

The data obtained from in vitro and in vivo cultures

were analyzed in SPSS 20.0. The mean values of the

measurement results of the groups were compared

using One-way ANOVA and expressed as

mean±SD. The p-value of <0.05 meant that the

difference between the mean values was statistically

significant.

3 RESULTS AND DISCUSSION

The results of the TNFα level measurement from the

in-vitro culture are shown in Figure 1A, while the

percentage of TNFα level in each group to the

TNFα level in the control group (represented as %

levels of TNFα) is depicted in Figure 1B. The TNFα

levels and the % levels of TNFα from the in-vivo

culture can be seen in Figure 2A and Figure 2B,

respectively.

The statistical test results showed that percentages of

the TNFα levels in Groups MPL1, MPL2, MPL3,

L-MPLP1, and liposomes were not significantly

different from the control group (p>0.05).

Meanwhile, the percentages of the TNFα levels in

Groups L-MPLP2 and L-MPLP3 were significantly

The Effects of Liposomale Methylprednisolone Palmitate on the Production of TNFÎ

s in Mice

different (p <0.05) from the control group (without

the administration of drugs or MPL). The

administration of L-MPLP at a dose of 2 mg/kg BW

resulted in an equal TNFα level to MPL given at a

dose of 16 mg/kg BW, i.e., 94.85% (see Figure 2B).

Meanwhile, at the same dose of 8 mg/kg BW, the

treatment using L-MPLP produced lower TNFα than

MPL by 1.5 times. At a dose of 16 mg/kg BW, L-

MPLP yielded TNFα level 3 times lower than MPL

at the same dose.

Several theories suggest that liposomes as drug

carriers can extend the half-life of drugs and increase

the distribution of drugs into the organ selectively so

that the drug dose can be minimized (Sercombe et

al., 2015; Mishina et al., 1994; Binder et al., 1994).

Mishina et al. (1994) state that, when compared to

methylprednisolone (MPL) at a dose of 2 mg/kg

BW IV), the same dose of liposomal

methylprednisolone (L-MPL) in male Sprague-

Dawley rats can prolong the half-life from 0.48

hours to 30.13 hours and increase the distribution

volume from 2.1 L/kg to 21.87 L/kg. However, the

use of L-MPLP at a dose of 2 mg/kg BW in our

study did not exhibit any biological effects on mice.

This finding contradicts the research conducted by

Mishina et al. (1994) and Binder et al. (1994) where

the administration of L-MPL at this dose exhibits

biological effects. These two studies successfully

examine the immunosuppressive effects of the same

dose of L-MPL in male Sprague-Dawley rats, as

evident in the increase of survival to 30 days after a

heart transplant (instead of only 10-day survival in

the control group).

Although Mishina et al. (1994) and Binder et al.

(1994) identify the immunosuppressive effects of

MPL without measuring the TNFα level, these two

studies provide a conclusion that at a dose of 2

mg/kg BW, L-MPL gives a good

immunosuppressive effect. Meanwhile, in our study,

the use of L-MPLP at the same dose has no

biological effects on male C3H mice because of

several reasons. The first is the different strains of

Figure 1: (A) TNFα levels (mean ± SD) and (B) the

percentages of TNFα levels in each group compared to

the TNFα level in the control group (in vitro culture).

(Control group) Tris Buffer 5 ml/kg BW; (MPL1) 0.5 mM

methylprednisolone sodium succinate; (MPL2) 0.05 mM

methylprednisolone sodium succinate; (MPL3) 0.005 mM

methylprednisolone sodium succinate; (L-MPLP1) 0.5

mM liposomes methylprednisolone palmitate; (L-MPLP2)

0.05 mM liposome methylprednisolone palmitate; (L-

MPLP3) 0.005 mM liposome methylprednisolone

palmitate

Figure 2: (A) TNFα Level (mean ± SD) and (B) the

percentages of TNFα levels in each group compared to

the TNFα level in the control group (in vivo culture).

(Control group) Tris Buffer 5 ml/kg BW; (MPL1) 0.5 mM

methylprednisolone sodium succinate; (MPL2) 0.05 mM

methylprednisolone sodium succinate; (MPL3) 0.005 mM

methylprednisolone sodium succinate; (L-MPLP1) 0.5

mM liposomes methylprednisolone palmitate; (L-MPLP2)

0.05 mM liposome methylprednisolone palmitate; (L-

MPLP3) 0.005 mM liposome methylprednisolone

palmitate

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

the test animals. This study uses mice with C3H

strain, whereas Mishina et al. (1994) and Binder et

al. (1994) use the Sprague-Dawley strain. Such

difference in the test animal species leads to

dissimilar sensitivity to glucocorticoids. The second

reason is the type of the liposome used in the

experiments. These two studies use liposome made

from a combination of EPC and

phosphatidylglycerol. Meanwhile, the liposome in

our research is small-sized with a diameter of 73 nm,

and it is made from a combination of EPC and TEL.

The different types of phospholipids determine the

size and diameter of the liposome, which, in turn,

affect the speed of the drug uptake (Sercombe et al.,

2015; Shashi et al., 2012).

The small unilamellar liposome vesicles

(SUVs) are absorbed at a slower pace compared to

the large-sized ones (LUVs). Therefore, the

circulation time of liposomal SUVs is longer than

the liposomal LUVs. Liposomes made from the

combination of EPC and phosphatidylglycerol are

medium-sized (MUVs), i.e., about 100 nm

(Sercombe et al., 2015; Kumar et al., 2012), whereas

the liposomes in our research are small-sized

(SUVs), i.e., about 73 nm (Purwaningsih et al.,

2007). The third reason is that both Mishina et al.

(1994) and Binder et al. (1994 use MPL, which is

already widely used and known for its effects,

whereas our study chooses a novel compound

(MPLP) that is expected to be a pro-drug. Therefore,

even though administered during the same period,

MPLP has not shown its effect yet, as opposed to

MPL.

Methylprednisolone (MPL) sodium succinate is

a pro-drug (an MPL derivative) that is rapidly

hydrolyzed to methylprednisolone with a half-life of

2.5 hours in humans when administered at a dose of

1 mg/kg BW. At higher doses than 10 mg/kg BW,

the half-life becomes longer, i.e., up to 3.6 hours.

The half-life of MPL sodium succinate after the

intravenous administration to the C3H mice was 10-

30 minutes. MPL sodium succinate did not decrease

the TNFα levels due to its short half-life.

Accordingly, after 48 hours of administration, the

drug levels in the blood were very low. This finding

is similar to several references that mention that the

administration of a single dose of glucocorticoids

reduces lymphocytes, monocytes, basophils, and

eosinophils for 4-6 hours and returns them to their

normal levels after 48 hours.

Figure 2A shows that L-MPLP is still exhibiting

an effect on T lymphocytes and reducing the TNFα

levels after 48 hours of treatment. This condition

shows that the use of liposomes as drug carriers can

extend the half-life of MPLP, but the length of the

half-life of MPLP remains unclear. Using different

concentrations of dexamethasone (DEX), namely at

a concentration of 0.3, 0.8, and 10 μg/ml, against the

mitogen-induced proliferation of lymphocytes in

rats, Miller et al. (1991) explain that DEX influences

T cell proliferation and the in vitro mitogen-induced

proliferation of lymphocyte in cell cultures.

As seen in Figure 1A, the different

concentrations of MPL and L-MPLP (i.e., 0.5, 0.05,

and 0.005 mM) added to the cultures before being

incubated for 48 hours inhibit lymphocyte

proliferation, which is significantly different from

the control group (without drug administration,

p<0.05). The figure also shows that at a

concentration of 0.5 mM, L-MPLP inhibits the

lymphocyte proliferation by decreasing the TNFμ

levels, which are significantly different from the

control MPL at the same concentration (p<0.05).

Furthermore, the application of liposome without

any immunosuppressive drugs at a concentration of

0.5 mM does not decrease the TNFα levels (p>0.05).

This finding shows that liposomes do not affect

lymphocyte proliferation and TNFα level. It also

indicates that 0.5 mM liposomes comprised of a

combination of EPC and TEL are not toxic to C3H

mice. From the results of a toxicity study of 6 μg/ml

TEL in L5178Y murine lymphoma cells (EMAT

cells) and mutagenicity or antimutagenicity tests of

Salmonella typhimurium strain TA 100, Freisleben

et al. (1993) affirm that TEL is nontoxic and

nonmutagenic.

The additions of MPL and L-MPLP to the

cultures before 48 hours of incubation resulted in the

proliferation of lymphocyte that reduced the TNFα

levels significantly, as opposed to the controls (p

<0.05). This condition is explainable by the ability

of glucocorticoids to suppress lymphocyte

proliferation. According to Cidlowski (2013), the

sensitivity of glucocorticoid receptor varies

depending on the antigen or mitogen used.

Benameur et al. (1995) state that liposomes as

drug carriers are useful for reducing the

administration dose of dexamethasone palmitate

while retaining its therapeutic effects. As an initial

test to assess the biological activity of liposomal-

dexamethasone (DMP-SUVs), they employ

lymphocyte proliferation test and interferon gamma

level (IFNγ) measurement and compare the results to

the use of dexamethasone (DEX) without liposomes

at 1/6 times of the dose of DEX. Based on the

proliferation test and measurement results, they

conclude that DMP-SUVs inhibit lymphocyte

proliferation and reduce the levels of IFNγ six (6)

The Effects of Liposomale Methylprednisolone Palmitate on the Production of TNFÎ

s in Mice

times greater than dexamethasone alone. In other

words, the use of DMP-SUVs in therapy can be

reduced without altering their pharmacological

effects because liposomes carry the drug to the

targeted organs, especially the ones that are rich in

reticulum-endothelial systems like liver and spleen

(Anwekar et al., 2011). The sustained drug release

offered by liposomes prolongs the drug exposure in

the cells, allowing more drug cellular uptake (Jone,

2013).

In our study, the administration of L-MPLP at a

dose of 2 mg/kg to the C3H mice inhibited the

formation of TNFα. This result was equal to the

administration of MPL at a dose of 16 mg/kg.

Meanwhile, the administrations of L-MPLP at the

doses of 8 mg/kg and 16 mg/kg inhibited the

formation of TNFα, respectively, by 1.4 times and 3

times greater than MPL at the same doses.

The in vitro administrations of 0.005 mM L-

MPLP and MPL to the cell cultures inhibited the

formation of TNFα by nearly equal numbers.

Meanwhile, the applications of 0.05 mM and 0.5

mM L-MPLP inhibited the formation of TNFα,

respectively, by 1.4 and 6.6 times greater than MPL

at the same concentrations.

Tumor Necrosis Factor (TNF) plays an important

role in a broad range of immune and inflammatory

processes, including cellular activation, survival, and

proliferation, as well as cell death by necrosis and

apoptosis (Keystone, 2010). Glucocorticoids are

used as immunosuppressant drugs that inhibit or

prevent cellular and humoral immunity. They

suppress cellular immune responses that suppress

hinder T cell proliferation by inhibiting enzymes

with the following cytokines: IL-1, IL-2, IL-3, IL-4,

IL-5, IL-6, IL-8, and TNF. Glucocorticoids suppress

humoral immunity’s response and lead to the

expression of IL-2 and IL-2 receptors on B cells

(Rathee et al., 2012). Applying 10 µg/ml

lipopolysaccharide to male CBA/J mice in an in vivo

study, Nguyen et al. (1990) affirm that the 2-week

administration of cyclosporine at a dose of 75 mg/kg

decreases the levels of TNFα, which are

significantly different from the TNFα level in the

control group. They also affirm the inhibition of in

vitro TNFα formation by using murine macrophages

that are stimulated with lipopolysaccharide and

administered with cyclosporine at a dose of 0.001 to

1 g/ml. This inhibition is significantly different from

the one exhibited in the control groups (without

cyclosporine) (Nguyen et al., 1990). Our research

used a small dose of liposomal methylprednisolone

palmitate, which also inhibited the formation of

TNFα. The resulting TNFα level was significantly

different from the one in the control groups. In this

case, such administration is expected to reduce the

side effects and toxicity of methylprednisolone

palmitate.

4 CONCLUSIONS

A small dose of liposomal methylprednisolone

palmitate (L-MPLP) can significantly inhibit both in

vivo and in vitro productions of TNFα, as opposed

to the control group, i.e., methylprednisolone

(MPL).

ACKNOWLEDGMENTS

The authors would like to express their gratitude to

Prof. Dr. Ernie H. Purwaningsih and Prof. F.D.

Suyatna, Ph.D. from the Faculty of Medicine,

Universitas Indonesia, for their invaluable helps in

providing liposomes and liposomal

methylprednisolone palmitate (L-MPLP) for this

study.

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