The effect of addition NaCI 150 mOsmol pH 7 on liposomes Tetraether Lipid (EPC-TEL 2,5) with sonication

International Journal of Current Medical Science and Dental Research (IJCMSDR)
Volume 1 Issue 2 ǁ July-August 2019 ǁ PP 01-07
ISSN: 2581-866X || www.ijcmsdr.com
|Volume 1| Issue 2 | www.ijcmsdr.com | 1 |
The effect of addition NaCI 150 mOsmol pH 7 on liposomes
Tetraether Lipid (EPC-TEL 2,5) with sonication
1,
Yulhasri, 2,
Widya Safitri, 3,
Erni H Purwaningsih, 4,
Kusmardi Kusmardi
1,2,3,4, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
ABSTRACT:As a drugs carrier, liposome can alter the pharmacokinetics of the entrapped drugs. Thus, drugs
can act directly on the targeted cell while their systemic side effects are reduced. To become an effective drugs
carrier, liposome must reach its stability in chemical, physical, and biological conditions. Liposome stability
can be achieved by changing the lipid composition, such as EPC-TEL 2,5 which is made from the combination
of Egg Yolk Phosphatydyl Coline (EPC) and TEL 2,5 mol % that is extracted from Thermoplasma acidofilum.
The aim of this study is to test the chemical stability of liposome EPC-TEL 2,5 with sonication by addition of
NaCI 150 mOsmol pH 7 solution. The increase in number of liposome larger than 100 nm is the stability
parameter in this study. After observation at day 0, 7, 30, 60, 90, there was no significant increase in the
number of liposome larger than 100 nm after addition of NaCI 150 mOsmol pH 7 compared with control.
KEYWORDS:EPC-TEL 2,5, Liposome, Thermoplasma acidofilum, NaC1 150 mOsmol pH 7
I. INTRODUCTION
It is undeniable that the use of high-dose long-term medicines for certain diseases such as Systemic Lupus
Erythematosus, nephrotic syndrome, cancer or post-organ transplantation still causes various problems,
especially in terms of the side effects of their use. The drugs used for the treatment of these diseases are
generally very toxic such as corticosteroids, cyclosporine, methotrexate etc. These drugs can suppress the
immune system and cause a variety of adverse side effects such as the appearance of seconder infection. By
utilizing technological developments, especially in the field of pharmacology, various efforts have been found to
reduce the side effects of drugs, one of them is by incorporating drugs into drug carriers.1-5
One of the drug
carriers that is widely developed and proven to be able to reduce drug side effects to a minimum is liposomes.
Drugs that are incorporated into the liposome will be changed pharmacokinetics, so that the drugs can be
concentrated in the target cell or organ while the amount of the drug in another place that allows the occurrence
of side effects will decrease. 6,7
Liposomes are a nanotechnology product that was discovered about 4 decades ago. Until now liposomes have
been widely used as a multifunctional tool in various scientific fields, including one in the health and
pharmaceutical fields. Applications in the health sector use 80-200 nm liposomes and must meet the exact
requirements, including; lipid and drug concentration, liposome size distribution, pH, and osmolarity. 8,9
One of
the conditions is distribution, obtained by extrusion through a polycarbonate membrane 100-200 nm or
sonication using water probes or sonication.10-11
Another requirement is that liposomes can be used as drug
carriers, which must be proven to be chemically, physically and biologically stable. Liposomes that are
physically, chemically and biologically stable will bring the drug to the target better.
Stability is a major problem faced in efforts to develop liposomes. The liposomes that were first developed have
not been profitable because the stability is still low and the half-life is short even though it is stored in the
cooling phase. To get a stable liposome and fulfill the requirements as a drug carrier, several manipulations can
be carried out such as changing the shape, size and composition. 12
The main components that make up the
structure of liposomes are phospholipids and cholesterol which can come from nature or synthetically. One type
of substance that promises to produce stable liposomes is a tetraeter lipid derivative obtained from natural
sources. 13-14
Lipid tetraeter is the result of destruction of the Archaeabacteria membrane, including from
Sulfolobus acidocalcidarius or Thermoplasma acidophilum. Based on previous research by Freisleben et al., It
was proved that TEL from Thermoplasma acidophilum was not toxic, not mutagenic or antimutagenic in acute
toxicity tests 15-16
. One of the liposome formulations currently being developed is EPC-TEL 2.5 liposomes. 17
EPC-TEL 2.5 liposomes composed of egg yolk Phosphatidylcholine (EPC) and Lipid Tetraeter lipids (TEL)
derived from the bacterium Thermoplasma acidolilum. 18
The results of the study by Purwaningsih et al. EPC-
TEL 2.5 liposomes using TEL from Sulfolobus acidocalcidarius have been shown to bind the drug
methylprednisolone palmitate better, show therapeutic effects, and are well distributed in organs compared to
controls. 19-20
However, this study has not been equipped with stability tests which are the main requirements for

The effect of addition NaCI 150 mOsmol pH 7 on liposomes…
liposomes as drug carriers. To prove that EPC-TEL 2.5 liposomes remain stable or more stable as chemical
carriers compared to other liposomes, further research is needed on the stability test.
In this study, researchers chemically tested EPC-TEL 2.5 liposome stability. The study was carried out by
adding NaC1 150 mOsmol salt pH 7 to the liposomes that had been modified and stored at 4 ° C and incubated
at a certain time. If proven to be stable, it is hoped that this formulation liposome can be used as a drug carrier
for long-term therapy so that the drug can be more effective even with low doses and adverse side effects can be
reduced as low as possible.
II. METHODS
Making a solution of NaCl 150 mOsmol pH 7The NaC1 150 mOsmol solution was made by mixing 175.5 mg of
NaCI salt into the aquabidest liquid. The degree of acidity of the desired solution is obtained by adding a
solution of NaOH and HCl. At the pH of the solution> 7, the solution must be added to the HCI solution and
vice versa if the pH of the solution is <7, then the solution is added to the NaOH solution. After obtaining the
desired pH, the aquabidest liquid is added to reach a volume of 20 ml (according to calculations).
Making Liposome Preparations : The liposome solution needed is as much as 50 ml. In every 1 ml of
liposome solution, EPC is needed as much as 10 mMolar, so EPC required is 390 mg. TEL is needed as much as
2.5% of mMolar EPC, so that TEL is needed as much as 0.25 mMolar or as much as 18.605 mg. Buchi
Rotavapor is heated, then pour the water into the waterbath with the appropriate amount so that the pumpkin can
be submerged. EPC and TEL (2.5% TEL levels of EPC moles) were weighed according to calculations. EPC
and TEL were mixed then Cloroform and ethanol were added with a ratio of 1: 1 (5 ml each). Put a mixture of
EPC-TEL, Cloroform, Ethanol into a pumpkin containing beads. The mixture is dispersed with Buchi Rotavapor
for 2 hours. The water in the waterbath must be 40 ° C and the pressure on the vacuum is maintained at 200
barrels and after drying the pressure on the vacuum is maintained below 50 barr. After the mixture dries, add
Aquadest until the volume reaches 50 ml (according to the required volume), then rotate again until
homogeneous. The homogeneous mixture is divided into 3 parts (each containing 15 mI) then labeled into
sample I (control), II (sonication) and III (extrusion). Sample II was given treatment in the form of sonication
carried out in the Department of Pharmacology FMUI for 100 minutes. After being treated, Quinacrine was
added to the sonication sample according to the calculations. Liposomes that have been sonicated and given
Quinacrine are divided into 2 parts, each of which is 2 ml. Both parts are labeled with the label Control and pH
NaCl 7. Added a pH 7 NaCl solution with a ratio of 1: 1 to the liposome that matches the label, except the
control.
Measurement and Calculation of Liposomes : Calculation of the number of liposomes and the estimated size
of liposomes is done manually based on a predetermined scale. There were 2 categories of liposome sizes: <100
nm and> 100 nm. The measurement and calculation of the number of liposomes is carried out on days 0, 7, 30,
60 and 90 according to the following steps. Prepared glass is cleaned using 70% alcohol and the microscope is
prepared for data collection. The measuring line is determined to measure the size of the liposome. A total of 25
µL liposomes mixed with Quinacrine were dropped on the glass preparations. Prepared glass with liposomes is
closed with a cover glass, then observed under a microscope with 40x magnification (adjustable). Every
preparationare taken at 10 fields of view, then record with a duration of 15 seconds. Calculated the number of
liposomes <100 nm and> 100 nm.
Statistical analysis:Data analysis was performed by 1-way ANOVA statistical analysis method.
III. RESULT AND DISCUSSION
Liposome preparations made by mixing EPC as much as 390 mg and TEL 18.605 mg produce 50 ml EPC-TEL
2.5 liposomes. The liposomes obtained were still MLV, so to obtain a <100nm small unilamelar vesicle
liposome, sonication was performed using Branson type 1510 sonicator. The number and diameter of liposomes
were calculated. One of the photos is shown in Figure 1.

Figure 1. EPC-TEL 2.5 Liposomes on Day 90 (A) EPC-TEL 2.5 Liposomes with Addition of NaC1 150
mOsmol pH 7. (B) Liposomes EPC-TEL 2.5 Control.
The above liposome photo was taken on the 90th day of the study. From Figure A which is a liposome with the
addition of NaC1 150 mOsmol pH 7, liposome vesicles with a number that tend to be more than the control
liposomes seen in Figure B. From the calculation of the number and diameter of liposomes on the 90th day,
from 10 Liposome photos taken, obtained an average number of control liposomes with a size of > 100 nm as
many as 12 pieces and the average NaCI 150 mOsmol liposomes pH 7 sizes> 100 nm as many as 52.67. While
for the average <100 nm liposomes as much as 163.33 for control liposomes and 169.33 for liposomes with
NaCI 150 mOsmol pH 7. From these results, it can be seen that there are differences in the number of liposomes
between the two treatments, where the number of liposomes measuring> 100 nm tends to be more in liposomes
with NaC1 150 mOsmol pH 7 compared to controls, whereas for liposomes <100 nm in size, there is more in the
control liposomes compared to NaCl 150 mOsmol liposomes pH 7.
Photographs of liposomes taken on days 0, 7, 30 and 60 are not included. Based on the calculation of the
number and diameter of the liposomes for the photos, the number of liposomes was obtained in two categories,
those that were> 100 nm in size and <100nm in size. The data obtained can only be divided into two categories,
due to the unavailability of liposome particle gauges (particle sizers). From the data obtained starting from day 0
to day 90, there are fluctuations in the number of liposomes measuring> 100 nm and <100 nm in both treatments
(control and by the addition of NaC1 150 mOsmol pH 7 (Figures 2 and Figure 3).
Figure 2. Number of Liposomes with a Diameter> 100 nm

Figure 3. Number of liposomes <100 nm in diameter
From Figure 2, it can be seen that untreated liposomes (control) have the highest number of vesicles> 100 nm at
30 days, whereas for liposomes with NaCI 150 mOsmol pH 7 has the highest number of vesicles> 100 nm on 90
days. For liposomes with diameter 5 100 nm (Figure 3) in both treatments showed almost the same number on
days 0, 7, 30, and 90. However, on day 60 there was an increase in the number of significant vesicles on
liposomes with NaCI 150 mOsmol pH 7 Based on the data shown in this graph, it was found that the liposomes
that were treated or not were of a less stable nature. Therefore to ascertain whether these liposomes are
chemically stable or not, then data processing is carried out using Non-parametric statistical analysis.The size of
liposomes> 100 nm is an indicator of changes in liposome stability in this study. Liposome data measuring> 100
nm will be processed processed using the Non-Parametric statistical analysis method, namely Kruskall-Wallis 1-
way ANOVA, while for liposomes that are <100 nm in size not analyzed in this study. This analysis method was
used to compare the stability between control liposomes and NaCl 150 mOsmol liposomes pH 7.
From the results of the analysis using the Kruskall-Wallis method, there is a p value of 0.332, which states that
there is no significant difference in the stability of the liposomes in each treatment during the study.Over time,
various changes in liposomes can be found, such as the occurrence of chemical degradation (oxidation and
hydrolysis) which causes clumping and rupture of the contents of the liposome. 20
However, this change was not
found in EPC-TEL liposomes 2.5 to 90 days of storage. The TEL structure used in this formulation makes it
impossible for these changes not to occur.TEL which comes from Thermoplasma acidophilum has a structure in
the form of 2 polar head groups with a membrane thickness of about 4 nm (from one head to the other head), so
it is expected to be able to incorporate EPC liposome membranes that resemble pegs as shown in Figure 13. 21,
24-26
Figure 13. Schematic drawing of TEL incorporation of EPC28 '3 liposome membrane

The stability test of liposomes derived from TEL Thermoplasma acidophilum, as done by Freisleben HJ, is
known that liposomes composed of pure TEL or liposomes with a combination of egg lecithin and high
concentrations of TEL are proven to have high stability, can even be stored in infinite time and not can be
penetrated by protons if the TEL concentration reaches 100%.21
In addition, another Freisleben HJ studystated
that Iiposom with TEL Thermoplasma acidophilum proved to be quite stable at a low pH compared to neutral
and alkaline pH. The ability to add negative charges to the surface of the liposome membrane also makes the
liposome structure more stable. 22
The use of TEL as much as 2.5 mol% in this study, is more due to safety factors for long-term use. Although
TEL Thermoplasma acidophilum has been proven to be non-toxic in-vivo and in-vitro, until now, the
mechanism of breakdown and metabolism of TEL in the body has not been known in-vivo mechanism.23,24,27
In
addition to the TEL factor used in making liposomes, pH, temperature, and size also influence the stability that
occurs. 14
Neutral pH functions to slow down the hydrolysis process that occurs, consequently the structure of
liposomes can be more stable.
Storage of liposomes at a low temperature of around 4 °C allows the liposomes to be stored for an infinite
period of time without affecting their stability. However, this process of storing in low temperatures still causes
problems, namely causing damage to liposomes by osmotic dehydration. This dehydration occurs when an extra
liposomal fluid pulls out the fluid contained in the liposome core chamber. 7
Cooling also causes changes in
liposome transition temperature, this leads to aggregation of liposomes, formation of MLV from SLV and
rupture of liposome contents.7,14
. In general, the transition temperature is strongly influenced by the chain length
and saturation of the fat chain. Increased saturation and chain length increase the transition temperature. At
elevated temperatures, the fatty acid chain will change its conformation from the all-tran chain. Straight into the
gauche conformation as seen in Figure 12. This gauche structure reduces the length of the hydrocarbon chain
and decreases the thickness of the membrane. As a result the membrane becomes less dense, and its stability
decreases.14
Figure 12. All-trans conformation and Gauche 14
Sonication carried out on EPC-TEL 2, 5 liposomes produces liposomes of smaller size, which is around 10-50
nm (SUV). With smaller sizes, liposomes can be more stable. In liposomes of a larger size, it is likely that the
liposome membrane that is in contact with another membrane is larger, making it more likely for clumping. In
addition, large-sized liposomes can experience sedimentation due to the Van der Waals force.However, some
losses can still occur due to the sonication carried out on the liposome, as sonication still allows for oxidation of
lipids and small size of liposomes because sonication decreases the concentration of the drug that can be carried
by the liposome. For example, small liposomes, also known as small-unilarnellar vesicles, which are 25-30 nm
in size, can only capture 0.3 L of water per mole of lipid compared to multi-lamellar vesicles which can carry 3
L per mole. 27
Another way that can be used to minimize the oxidation process is by adding antioxidants to the
structure of liposomes (a-tocopherol and phosphatidic acid), avoiding high temperatures in the process of
making and storing them and using fresh liposome constituents.NaC1 150 mOsmol pH 7 salt used in this study
is useful as a liposome solvent, so it is possible for EPC-TEL 2.5 liposomes to be mixed into 0.9% NaCI
physiological solution. This salt is the main electrolyte component in the body and is often used in intravenous
fluid therapy.
IV. CONCLUSION
From the results of this study it can be concluded that EPC-TEL 2.5 liposomes containing tetraeter lipid
derivatives from small concentrations of Thermoplasma acidofilum bacteria proved to be chemically stable,
after exposure to NaC1 150 mOsmol salt solution pH 7 for a period of 3 months of storage.

Besides that there was no significant stability difference, between EPC-TEL 2,5 liposomes which added NaC1
150 mOsmol pH 7 with liposomes which were not given any treatment (control).
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