https://doi.org/10.52973/rcfcv-e34401
Received: 26/02/2024 Accepted: 01/04/2024 Published: 15/07/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34401
ABSTRACT
The aim of this study is to by converting albendazole and levamisole,
which are antiparasitic drugs used in both humans and animals, into
liposomal formulations under laboratory conditions. To ascertain
the circumstance in practice, characterization studies were
additionally conducted. The study was performed by modifying the
hydration of the thin lipid lm. Experiments were carried out with
egg phosphatidylcholine, cholesterol, chloroform and methanol
in different amounts. Albendazole and levamisole formulations
were made with the substances used in liposomes. Zeta potential,
polydispersity index, encapsulation efficiency, particle size
measurements and scanning electron microscopy were performed
as part of characterization studies. The results show that Lipo LVM
has the smallest particle size value at 380.87 ± 19.52 nm, whereas Lipo
LVM–PBS has the largest particle size value at 7236.67 ± 443.89 nm.
Values for the polydispersity index fall between 0.527 and 0.896. Zeta
potential levels, on the other hand, range from -7.6 mV to -46.8 mV.
While this value was determined as -8.2 ± 0.4 mV in LD Lipo ABZ and
-18.4 ± 0.6 mV in HD Lipo ABZ, respectively. Both HD Lipo ABZ and
LD Lipo ABZ have polydispersity indices for ABZ of 0.529 ± 0.066 and
0.896 ± 0.085, respectively. It was found that the particle size rose
as the desired amount of liposomal albendazole increased. It was
found that the liposomization of albendazole was higher than that
of levamisole. Albendazole and levamisole liposomal formulations
were successfully developed in the investigation. By carrying out
characterization studies, it was discovered that it may be employed in
clinical trials. In the upcoming years, it is anticipated that continuous
research in the eld of nanotechnology will improve human and animal
health and aid to more effectively control parasite infestations.
Key words: Albendazole, levamisole, liposome
RESUMEN
El objetivo de este estudio es convertir albendazol y levamisol,
fármacos antiparasitarios utilizados tanto en humanos como en
animales, en formulaciones liposomales en condiciones de laboratorio.
Para comprobar la circunstancia en la práctica, se realizaron además
estudios de caracterización. El estudio se realizó modicando la
hidratación de la na película lipídica. Se realizaron experimentos
con fosfatidilcolina de huevo, colesterol, cloroformo y metanol en
diferentes cantidades. Se realizaron formulaciones de albendazol
y levamisol con las sustancias utilizadas en los liposomas. Como
parte de los estudios de caracterización se realizaron mediciones
del potencial zeta, el índice de polidispersidad, la eficacia de
encapsulación, el tamaño de partícula y la microscopía electrónica
de barrido. Los resultados muestran que Lipo LVM tiene el valor de
tamaño de partícula más pequeño con 380,87 ± 19,52 nm, mientras
que Lipo LVM–PBS tiene el valor de tamaño de partícula más grande
con 7236,67 ± 443,89 nm. Los valores del índice de polidispersidad se
sitúan entre 0,527 y 0,896. Por otra parte, los niveles de potencial
zeta oscilan entre -7,6 mV y -46,8 mV. Mientras que este valor se
determinó como -8,2 ± 0,4 mV en LD Lipo ABZ y -18,4 ± 0,6 mV en
HD Lipo ABZ, respectivamente. Tanto HD Lipo ABZ como LD Lipo
ABZ tienen índices de polidispersidad para ABZ de 0,529 ± 0,066
y 0,896 ± 0,085, respectivamente. Se observó que el tamaño de
partícula aumentaba a medida que aumentaba la cantidad deseada
de albendazol liposomal. Se comprobó que la liposomización del
albendazol era mayor que la del levamisol. En la investigación se
desarrollaron con éxito formulaciones liposomales de albendazol y
levamisol. Al realizar estudios de caracterización, se descubrió que
pueden emplearse en ensayos clínicos. Se prevé que en los próximos
años la investigación continua en el campo de la nanotecnología
mejore la salud humana y animal y ayude a controlar más ecazmente
las infestaciones parasitarias.
Palabras clave: Albendazol, levamisol, liposoma
Preparation and Characterisation of Liposomal Formulations of Levamisole
and Albendazole Used in Veterinary Medicine
Preparación y caracterización de formulaciones liposomales de
levamisol y albendazol utilizadas en medicina veterinaria
Hasan Susar
1
* , Murat Çelebi
2
, Çağla Çelebi
1
, Özlem Çoban
3
, Hüseyin Şen
1
, İzzet Karahan
1
1
Balikesir University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology. Balıkesir, Türkiye.
2
Balikesir University, Savastepe Vocational School, Department of Laboratory and Veterinary Health. Balıkesir, Türkiye.
3
Karadeniz Technical University, Faculty of Pharmacy, Drug and Pharmaceutical Technology Application and Research Center,
Department of Pharmaceutical Technology. Trabzon, Türkiye.
*Corresponding author: hasan.susar@balikesir.edu.tr
Formulations of Levamisole and Albendazole used in Veterinary Medicine / Susar et al. ____________________________________________
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INTRODUCTION
Tetramisole (levamisole), an ingredient of the imidazothiazole
derivatives class of anthelmintic medications, is particularly effective
against nematodes in the respiratory tract and gastrointestinal tract.
Tetramizole’s L – isomer, levamisole, is a broad – spectrum medication.
The condence interval was enlarged by employing the L – isomer
alone because it was discovered that the drug’s anthelmintic action
emanated from the L – isomer. Levamisole is also known to have an
immunomodulatory. For example, in a study; It has been stated that
the vaccine has a strengthening effect on the immune system by
increasing the protective effect of the Brucella vaccine in mice [1, 2].
For the treatment of parasitic infestations brought on by helminths,
the drug albendazole, a benzimidazole derivative, is used in both
veterinary and human medicine. It exhibits great ecacy at low
doses and a broad spectrum of anthelmintic effects. Its chemical
formulation is C
12
H
15
N
3
O
2
S and its molecular weight is 265.33 g·mol
-1
.
Albendazole is a whitish powder with a melting point of 209°C [3].
It has been stated by researchers that albendazole shows specic
and high toxicity in parasites compared to mammals and forms the
mechanism of action by inhibiting the polymerization of tubulins into
microtubules. Since albendazole is less soluble in water and organic
solvents, increasing its solubility greatly increases its absorption
and therefore its effectiveness Biotransformation of albendazole
is primarily mediated by cytochrome P450 and microsomal avin
monooxygenase enzymes. Following absorption, it undergoes
rst–pass effects in the liver and intestines and is subsequently
metabolized to sulfoxide. The plasma half–life is 4 – 15 hours (h) and
its metabolites are excreted through urine, feces, and bile [4, 5].
Liposomes, dendrimers, nanoemulsions, polymeric, and metallic
nanoparticles are examples of nanomaterials used in medicine.
These materials are primarily used for purposes such as directing
drugs to the target tissue, reducing drug side effects, and reducing
labor and costs as a result of increasing the bioavailability of drugs.
Liposomes were rst described as a model for the cell membrane
by Alec Bangham in the 1960s. Liposomes, one of the drug delivery
systems are biocompatible spherical vesicles with a diameter of
about 2 – 3.5 µm, consisting of single or intertwined layers with
an aqueous phase between them. They can carry water–soluble
active components in the hydrophilic central part and hydrophobic
components that are insoluble in water in their membranes. Its
advantages are that it is resistant to environmental effects without
side effects, and that it is a carrier system that allows the delivery of
bioactive components into the cell and even to the compartments
within the cell, reducing the side and toxic effects of drugs. It also
has properties such as increasing the permeability and bioavailability
of drugs with a short half–life, ensuring the stability of drugs,
changing the pharmacokinetics of liposomal substances, and
bringing the size of drugs to the desired size [6, 7, 8]. The use of
nanotechnologically prepared drugs in the eld of medicine is limited.
The formation of resistance in parasites to commercially available
drugs is a major problem in the ght against parasitic infestations.
In this sense, new drug delivery systems are needed to increase the
effectiveness of drugs.
Some studies, it is aimed to develop a new drug delivery system
and to increase drug effectiveness against parasites thanks to this
carrier system. For this purpose, the use of liposomal formulations and
nanoparticles in drugs used in parasite control has become interesting
[9]. The development of drug resistance by parasites appears to
be the major obstacle for research in the ght against parasites.
This is where the serious benets of nanoparticles come into play.
These are very reasonable systems to reduce the resistance that can
develop against traditional drugs used against parasites, to prevent
the formation of some of the resistance development mechanisms,
and to improve the bioavailability of drugs. In contrast to other uses,
the world of medicine now has a new therapeutic option available
thanks to nanotechnology. Because the ability to produce and manage
nano–sized substances in this eld means new opportunities.
The aim of this study was to produce and characterise liposomal
formulations of albendazole and levamisole for medical use in the
treatment and prevention of parasite infestations. In the subsequent
study, the pharmacokinetics of the liposomes produced according to
the routes of administration and dosages in animals will be evaluated
and their effectiveness will be demonstrated.
MATERIALS AND METHODS
Different physicochemical features of the active ingredient were
identied, and pre–formulation trials were conducted to maximize
medication distribution. Naeem et al. [10] used chloroform as 2.5 ml
while preparing lecithin liposomes. Khoshneviszadeh et al. [11] used
15 ml chloroform and 5 ml methanol while preparing hydroquinone
liposomes. Ahmad et al. [12] preferred the ratio of chloroform : methanol
as 1 : 1 while preparing isotretinoin liposomes. The solvents and ratios
used in this study were decided by considering the chloroform :
methanol ratios and amounts used in the studies. During the preparation
of liposomal levamisole, chloroform–methanol 2.5 mL–2.5 mL, 5 mL–5
mL used as a solvent were tested. Chloroform–methanol, the solvent
used to generate liposomal albendazole, was tested as 2.5 mL–2.5 mL,
5 mL–5 mL, 7.5 mL–7.5 mL, and 12.5 mL–12.5 mL.
It is well established that the formulation and preparation
methodologies have a pivotal effect on controlling particle size,
shape, polydispersity, and nally capability of liposomes [13, 14].
The Bangham method, also called the thin lm hydration method,
has been preferred in the preparation of liposomal formulations.
This approach has a lot of benets, including simplicity of use and
a low time and labor requirement. This technique works by drying
lipids that have been dissolved in an organic solvent, creating and
acquiring liposomes in an aqueous medium, and then analyzing the
liposomes that have been produced [15].
Levamisole ((LVM), Santa Cruz Biotechnology – sc – 205730, USA),
albendazole ((ABZ) Cayman – 23705, USA), egg phosphatidylcholine
((PC) L – α – phosphatidylcholine, USA), and cholesterol ((CL) Acros
Organics (Belgium), active substances that are intended to be
liposomal, were weighed on a precise scale (Denver Instrument
SI–234, Germany), and administered in beakers. Methanol (Sigma –
Aldrich, USA) and chloroform (Sigma – Aldrich, USA) were added to
them. The beaker’s contents were entirely dissolved. To obtain the
lipid lm, evaporation was carried out for 15 min at a rotational speed
(Isolab Laborgerâte GmbH 605.01.001, Germany) of 200 G in a rotary
evaporator (Isolab Laborgerâte GmbH 605.01.001, Germany), set to
37°C. In order to take the resulting dry lipid lm, 10 mL of distilled water
(Lipo LVM–PBS, Oxoid – BR0014G UK) was added to the evaporation
bottle and rotated without vacuum. The mixture was then vortexed
(Vortex MS 3 basic ika 3617000, Germany), for 3 min. To reduce particle
size, it was placed in an ultrasonic bath (MEDISSON, Turkey) for 5 min.
The nal mixture was placed in tubes for centrifugation and stored
in the refrigerator (Arçelik 270530EB, Turkey) at 4°C with the mouths
TABLE I
Liposome–forming substances and their amounts
Formulation
Name
Lipid/Cholesterol
Active
Drug
Amount of
Solvent
Amount of
Dispersion
PC (mg) / CL (mg)
LVM
(mg)
ABZ
(mg)
Chloroform :
Methanol (ml)
Distilled
Water (ml)
Lipo LVM 2.5 (50) 1.0 (20) 37 - 1,0 (5) 1.0 (5) 10
HD Lipo LVM 2.0 (50) 1.0 (29) 49 - 1.0 (5) 1.0 (5) 10
LD Lipo LVM–1 3.0 (49) 1.0 (19) 25 - 1.0 (5) 1.0 (5) 10
LD Lipo LVM–2 2.0 (49) 1.0 (21) 25 - 1.0 (5) 1.0 (5) 10
Lipo LVM–PBS 2.0 (49) 1.0 (25) 37 - 1.0 (5) 1.0 (5) 10 (PBS)
HD Lipo ABZ 3.0 (50) 1.0 (19) - 50 1.0 (12.5) 1.0 (12.5) 10
LD Lipo ABZ 3.0 (50) 1.0 (19) - 25 1.0 (12.5) 1.0 (12.5) 10
Lipo LVM: Liposomal Levamisole; HD Lipo LVM: High Dose Liposomal Levamisole; LD
Lipo LVM–1: Low Dose Liposomal Levamisole–1; LD Lipo LVM–2: Low Dose Liposomal
Levamisole–2; Lipo LVM–PBS: Liposomal Levamisole–Phosphate Buer Solution; HD Lipo
ABZ: High Dose Liposomal Albendazole; LD Lipo ABZ: Low Dose Liposomal Albendazole
TABLE II
Characterization results of liposomal LVM and ABZ formulations
Formulation
Particle size (nm)
(mean±SD)
Polydispersity
index
(mean±SD)
Zeta potential
(mV)
(mean±SD)
Encapsulation
eciency
(%) (mean±SD)
Lipo LVM 353.4 ± 19.52 0.527 ± 0.037 -46.8 ± 0.3 25.82 ± 0.03
HD Lipo LVM 1397.67 ± 72.63 0.852 ± 0.158 -15.7 ± 0.7 34.19 ± 0.17
LD Lipo LVM–1 1169.67 ± 66.51 0.630 ± 0.130 -43.9 ± 0.8 35.90 ± 0.19
LD Lipo LVM–2 2000.00 ± 84.67 0.701 ± 0.191 -14.9 ± 0.4 32.31 ± 0.85
Lipo LVM–PBS 7236.67 ± 443.89 0.756 ± 0.081 -7.6 ± 0.2 43.14 ± 0.17
HD Lipo ABZ 4777.33 ± 1150.22 0.529 ± 0.066 -18.4 ± 0.6 99.33 ± 0.00
LD Lipo ABZ 2243.00 ± 288.31 0.896 ± 0.085 -8.2 ± 0.4 99.56 ± 0.00
nm: nanometer, mV: milivolt, SD: standard deviation, Lipo LVM: Liposomal Levamisole,
HD Lipo LVM: High Dose Liposomal Levamisole, LD Lipo LVM–1: Low Dose Liposomal
Levamisole–1, LD Lipo LVM–2: Low Dose Liposomal Levamisole–2, Lipo LVM–PBS: Liposomal
Levamisole–Phosphate Buer Solution, HD Lipo ABZ: High Dose Liposomal Albendazole,
LD Lipo ABZ: Low Dose Liposomal Albendazole
FIGURE 1. Calibration graphs of active substances
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34401
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tightly closed. Centrifugation (ALLEGRA–X64R, USA) was performed
at 27000 G for 40 min. The collapsing liposomal part and the aqueous
part were placed in separate test tubes and stored in the refrigerator
at 4°C for analysis. The amounts and lipid / cholesterol ratios of all
the ingredients in the formulations, and the amounts of active drug
molecules, solvents, and dispersion liquids are shown in TABLE I.
The encapsulation eciency (EE) of liposomes was calculated
using the following formula (Eq.1) [17].
EE(%)
Totaldrug
TotaldrugFreedruginsupernatant
100
#
=
-
Eq.1
For this purpose, rstly separate calibration samples were prepared
for LVM and ABZ. After a known amount of LVM was dissolved in some
distilled water, it was completed to a certain volume with distilled
water. Based on this stock solution of LVM, a series of dilutions were
made with distilled water and the absorbances of the calibration
samples prepared at 0.05, 0.5, 2.5, 5, and 10 µg·ml
-1
concentrations
were measured with a UV – Vis spectrophotometer (Shimadzu, Japan)
at a wavelength of 213 nm. The calibration equation is obtained as
y = 0.1344x + 0.0098 and y = 0.1438x + 0.0126, the R
2
value of
both equations are 0.9997 and 0.9996 for LVM and ABZ, respectively.
Calibration graphs were shown in FIG. 1.
Particle size, polydispersity index, and zeta potential of the
liposomes were measured using Malvern Zetasizer Nano – ZS
(ZEN3600, UK) equipped with dynamic light scattering (DLS) and
electrophoretic light scattering techniques [16]. For this purpose,
1 ml of distilled water was added to the precipitated liposomes as a
result of centrifugation and dispersed. 120 µl of these samples were
taken and placed in the measuring cuvette, and 1880 µl of distilled
water was added to it. All measurements were performed at 25 ± 0.1°C.
Obtained results are presented in TABLE II.
By using the calibration equation of the relevant drug (LVM or ABZ),
rst of all, the amount of free drug in the supernatant was determined.
Then, the amount of LVM and ABZ – loaded into liposomes with Eq.1
was calculated. After these procedures, a known amount of ABZ was
dissolved in some methanol and then completed to the desired volume
with methanol. A stock solution was prepared by taking 5 ml of this
solution and adding 5 ml of distilled water. Calibration samples at
the same concentrations as LVM were prepared by making a series
of dilutions with methanol : distilled water (1 : 1 v/v) mixture using
this stock solution of ABZ. The absorbances of the samples were
measured using a UV – Vis spectrophotometer (Shimadzu UV – 1900i,
Japanese) at a wavelength of 215 nm.
For encapsulation eciency analysis, measurement samples were
prepared by making various dilutions from the supernatant parts of
the prepared and centrifuged liposomes. Distilled water was used to
dilute the supernatant of LVM – loaded liposomes. In ABZ – loaded
a
b
FIGURE 2. a: Liposomal levamizole imaging under SEM, b: Liposomal albendazole
imaging under SEM
Formulations of Levamisole and Albendazole used in Veterinary Medicine / Susar et al. ____________________________________________
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liposomes, the supernatants were rst diluted with methanol at a ratio
of 1 : 1 (v/v supernatant : methanol), and then a 1 : 1 methanol:water
mixture was used for further dilution when necessary. The absorbance
measurements of these samples were performed at 213 and 215
wavelengths for LVM and ABZ, respectively at 25 ± 0.5°C.
Scanning Electron Microscopy (SEM) Analysis
About 0.02 g of liposomized levamisole and albendazole were weighed
out to obtain scanning electron microscopic images. The sample was
placed on a bidirectional carbon tape. Gold plating was rst carried
out by applying a vacuum of 8 × 10
-1
mbar·Pa
-1
and a voltage of 10 mA in
a quorum coating device (Quorum, Japanese). Liposomal levamisole
and albendazole was examined in SEM (JEOL Neoscope JCM – 5000,
Japanese) at 2400× magnication, respectively (FIGS. 2a and 2b).
RESULTS AND DISCUSSIONS
Particle Size, Zeta Potential, Polydispersity Index, Encapsulation
Eciency
In the preparation of liposomal levamisole, initially 2.5–2.5 mL
of chloroform–methanol was preferred as the solvent. However,
levamisole did not completely dissolve. Therefore, chloroform–
methanol in an amount of 5 mL–5 mL was taken. Here, solvent ratios
of 1 : 1 were preferred.
In the preparation of liposomal albendazole, initially 2.5–2.5 mL
of chloroform – methanol was preferred as the solvent. However,
albendazole did not completely dissolve. Therefore, chloroform–
methanol in an amount of 5 mL–5 mL was taken. Once more,
dissolved was not accomplished. Low–dose albendazole dissolved
when chloroform–methanol contained 7.5 mL–7.5 mL. High dose
albendazole dissolved in chloroform–methanol 12.5 mL–12.5 mL.
Therefore, 12.5mL–12.5 mL were preferred in both formulations to
ensure that the results would not be impacted by the solvent utilized.
Here, solvent ratios of 1 : 1 were preferred.
According to the results, the lowest particle size value belongs to Lipo
LVM with 380.87 ± 19.52 nm, and the highest particle size value belongs
to Lipo LVM – PBS with 7236.67 ± 443.89 nm. When phosphate buffer
was used instead of distilled water for liposomal levamisole, it was
found that the particle size increased while the encapsulation eciency
increased. Except for the lipo LVM–PBS formulation, it was thought
to be in sizes that could be used in animals. The polydispersity index
values for LVM are in the range of 0.527–0.756. The polydispersity index
of ABZ was found to be 0.529 ± 0.066 for HD Lipo ABZ and 0.896 ± 0.085
for LD Lipo ABZ. When looking at the table, it is seen that the lowest
and highest polydispersity indices are in Lipo LVM and LD Lipo ABZ,
respectively. Zeta potential values for liposomal levamisole were found
to be between -7.6 ± 0.2 and -46.8 mV ± 0.3. Here, the smallest zeta
potential value belongs to Lipo LVM–PBS with -7.6 ± 0.2 mV, and the
highest zeta potential value belongs to Lipo LVM with -46.8 ± 0.3 mV.
On the other hand, this value was measured as -18.4 ± 0.6 mV in HD Lipo
ABZ and -8.2 ± 0.4 mV in LD Lipo ABZ. Polydispersity indices for ABZ
were found 0.529 ± 0.066 in HD Lipo ABZ and 0.896 ± 0.085 in LD Lipo
ABZ. It was determined that the particle size increased as the amount of
albendazole desired to be liposomal increased. The liposoming rate of
albendazole was found to be higher than levamisol. According to Table
II, the encapsulation yields of HD Lipo ABZ and LD Lipo ABZ seem to
be quite good. It has been found that the particle size increases as the
amount of the drug that is required to be encapsulated in ABZ increases.
Although the particle size of albendazole liposomal formulations is high,
it is within the usable range in animals. Zeta potential values indicate
that it is monodisperse, but the result is better in LD Lipo ABZ where
the polydispersity index is close to 1. It was considered to reduce the
particle size by prolonging the sonication time. However, it was thought
that prolonged sonication may damage the active substance in the
liposome. The particle size was considered to be suitable for use in
animals. Accordingly, the lowest EE value was found to be 25.82 ± 0.03%
and the highest 43.14 ± 0.17% in LVM–loaded liposomes, while this value
was approximately 99% in ABZ – loaded liposomes (Table II).
When liposomes are classified according to size and number
of layers; Small Unilamellar Vesicles (SUV): 20 – 100 nm, Medium
Unilamellar Vesicles (MUV): 40 – 100 nm in size and 1 lipid bilayer, Large
Unilamellar Vesicules (LUV): Larger than 100 nm, Giant Unilameller
Vesicules (GUV): Larger than 1 μm Multilayer Vesicles (OLV): 100 – 1000
Statistical Analysis
Statistical analysis of experimental ndings was performed using
GraphPad Prism 5.0 by student t–test and all data were expressed
as mean ± standard deviation (SD). P–values less than 0.05 were
considered statistically signicant values.
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nm Multilayer vesicles (MLV): larger than 500 nm, Multiple Vesicles
(MVV): Larger than 5000 nm have been reported [18, 19]. When the
prepared liposomes were classied according to their size and layers;
Lipo LVM was determined as Large Unilamellar Vesicules (LUV), HD
Lipo LVM, LD Lipo LVM – 1, LD Lipo LVM – 2, HD Lipo ABZ, LD Lipo
ABZ were determined as Giant Unilameller Vesicules (GUV) and Lipo
LVM – PBS was determined as Multiple Vesicles (MVV).
Scanning Electron Microscopy (SEM)
Images of the liposomal formulations are shown in Figure 2. The
prepared liposomes were observed to be nano–sized and roughly
spherical in shape in suspension by SEM analyses. Liposomal levamisole
was visualised at 10 KV × 2400 magnication in 10 µm size and liposomal
albendazole was visualised at 10 KV × 2400 magnication in 5 µm size.
Nanotechnology opens up new perspectives for applications in the
elds of biology, biotechnology, medicine, and veterinary medicine.
New possibilities in animal and human health are made possible by the
use of nanotechnology to the development of ecient products and
applications for animals. The effectiveness of medications used in human
and veterinary medicine will soon change as a result of this research.
In a study by Liu et al. [20], engineered stem cell biomimetic
liposomes carrying levamisole were prepared. They reported that the
particle size of liposomal levamisole was 108.4 ± 1.2 nm, 138.3 ± 2.9nm,
116.1 ± 1.9 nm, respectively. The small particle size facilitates the entry
of the drug into the cells, but shortens the circulation time. Because
particle size affects the distribution of liposomes in the body and those
with very small size are eliminated from the body faster. This may
cause a decrease in the bioavailability of the drug. They found that the
polydispersity index was 0.108 ± 0.042, 0.280 ± 0.062, and 0.102 ± 0.026,
and the zeta potential was -16.85 ± 0.91mV, -6.72 ± 0.50mV,
-7.23 ± 0.91mV. Zeta potential values are similar with HD Lipo LVM,
LD Lipo LVM–2, Lipo LVM–PBS in this study. Zeta potential values are
one of the stability indicators of liposomal formulation. Therefore,
monodisperse or polydisperse values are important parameters.
When the stability of the liposomal formulation is good, bioresistance
in the body, mean residence time, area under the curve values are
higher than the free formulation. Although the active substance is
different, it is similar to HD Lipo ABZ and LD Lipo ABZ. Particle size
and polydispersity index gave better results than this study. The
researchers did not calculate the encapsulation rate. It was thought
that the different results may be due to the materials used in liposome
preparation and the preferred method. In addition, the charge of
the liposomal formulation obtained, surface conditions, number of
layers and electrostatic interactions between the substances added
to the formulation may have caused different results. Liposomal
albendazole was prepared by Fülöp et al. [21]. Here, although zeta
potential and polydispersity index values were better than this study,
encapsulation eciency values were found to be low. Zhang et al. [22]
conducted a study on the pharmacokinetics and tissue distribution
of liposomal albendazole. Particle size, polydispersity index, zeta
potential, encapsulation eciency and SEM analyses of liposomal
albendazole characterisation studies were not performed here.
According to TABLE II, although the encapsulation eciency is low, the
best results in terms of particle size and zeta potential were obtained with
Lipo – LVM for LVM – loaded liposomes. The low encapsulation eciency
of the liposomal formulation limits its usability. Since other results
are better, the usefulness of the drug can be increased by processes
that can increase the encapsulation eciency (such as increasing
the amount of lipid). When the use of PBS instead of distilled water as
the dispersion medium (Lipo LVM – PBS), the highest particle size and
lowest zeta potential were obtained with Lipo LVM – PBS. EPC is a neutral
phospholipid, on the other hand in some literature it is said that is a
zwitterionic lipid [23, 24, 25, 26]. The zeta potential of liposomes prepared
with these lipids is usually lower [27]. Increased negative values of the
zeta potential can lead to the system becoming more monodisperse.
However, this would be more favourable if the value is greater than -30
mV. However, in TABLE II, see that the zeta potentials of liposomes are
different from zero. This may be due to the adsorption of Cl
–
ions formed
in the aqueous environment as a result of the ionization of the active
substance (LVM HCl) to the liposome surface.
The system is considered monodisperse if the positive or negative
zeta potential is greater than 30 mV positive or -30 mV negative,
respectively. However, values that are around the neutral value that
is, less than 5 mV and greater than -5 mV can also lead to aggregation
[24]. In the study of the zeta potential values are consistent with the
literature. Lipo LVM and LD Lipo LVM–1 formulations are polydisperse,
others are monodisperse.
Since the Na
+
and K
+
ions in the buffer will also be adsorbed on the
surface of the liposome when PBS is used as the dispersion medium,
the decrease in the zeta potential can be explained by this situation
[26, 28]. Because the dispersion medium affects the electrostatic
interactions of the substances added to the liposomal formulation.
The zeta potential value varies according to electrostatic interactions.
In addition, by increasing the ionic strength of the medium, PBS
may have reduced the electrical double layer barrier around the
liposomes and caused the particles to aggregate, and then because of
aggregation particle size reduction may have occurred [29, 30]. When
PBS was used as a dispersion medium, the encapsulation eciency
of the liposomes signicantly increased (P<0.0001).
In another study, the opposite results were observed in
cinnamaldehyde–loaded liposomes, and the decrease in encapsulation
efficiency was attributed to the increase in ionic strength by
the researchers [31]. However, this difference may be because
cinnamaldehyde is water–insoluble [32]. This may be because water
– soluble LVM creates a common ion effect (Cl
-
) with salts from PBS,
decreasing the anity of the active substance to the dispersion medium
and consequently increasing both its adsorption to the liposome bilayer
and retention in the internal phase of the liposome [33]. Moreover,
sodium counter ions in the dispersion medium can reduce the charge of
phospholipid and cause a decrease in the interlamellar space of multilayer
vesicles formed during the hydration phase, thereby reducing the volume
of aqueous inner phase and encapsulation eciency [34]. LD Lipo LVM
– 2 has a lipid / cholesterol ratio of 2 / 1 and contains more cholesterol
for the same amount of lipid than LD Lipo LVM – 1. It was shown that the
zeta potential of liposomes decreased as the amount of cholesterol in
them increased [35]. In this investigation, LD Lipo LVM – 1 and LD Lipo
LVM – 2, this was proven. On the other hand, as the amount of cholesterol
increased there was a signicant decrease in encapsulation eciency
despite the increase in particle size. (P<0.0042).
Active substances with high water solubility, such as levamisole, are
located in the internal aqueous phase of the liposome [36]. According
to TABLE II, the EE value of levamisole was in the range of 25 – 43%.
These values are similar to Egg PC liposomes Katragadda et al. [36]
containing the hydrophilic active ingredient stavudine. In addition, as
shown in this study, it is seen in this results that the lipid : cholesterol
ratio does not affect the EE value.
Formulations of Levamisole and Albendazole used in Veterinary Medicine / Susar et al. ____________________________________________
6 of 8
The encapsulation eciency of ABZ – loaded liposomes was higher
than LVM – loaded liposomes. It has been thought that the reason
for this is due to the high content of ABZ in the bilayer structure of
the liposome instead of the aqueous dispersion medium due to its
lipophilic natüre. It was observed that the zeta potential of ABZ –
loaded liposomes was lower than that of LVM – loaded liposomes.
This may be because ABZ is not ionized in an aqueous medium such
as LVM. Although many factors can affect the zeta potential value,
this results for the ABZ–loaded liposome are considered acceptable.
Because liposomes prepared with egg phosphatidylcholine loaded
with cyclosporine Chen et al. [37], another lipophilic drug, have also
been observed to have low zeta potentials. However, since ABZ is
located in the bilayer of the liposome, the particle size and zeta
potential increased as the amount of ABZ in the formulation increased.
Pensel et al. [38] studied liposomal albendazole in experimentally
infected mice. However, characterisation studies are also lacking here.
Even if liposomes are used in animal experiments, characterisation
studies should be carried out rst. Ergin et al. [39] prepared and
evaluated cholesterol – free liposomes. Although the particle sizes
were smaller than this study, the results of polydispersity index, zeta
potential and encapsulation eciency were similar. Zhang et al.
[40] prepared liposomal ciprooxacin. They found the particle size
smaller than this study. Although encapsulation rates were similar
to levamisole, they were much lower than albendazole. Reigada etal.
[41] prepared liposomal isotretinoin and loratadine formulations.
They found the particle size smaller than this study. Although
encapsulation rates were similar to levamisole, they were much
lower than albendazole. This may be due to the preparation method.
CONCLUSIONS
With the help of this research tried to prepare liposomal formulations
of levamisole and albendazole from antiparasitic drugs that are often
used in medicine. Different formulation ratios were tried. The tests
applied in the characterization studies of liposomes were performed
within the laboratory facilities. In this respect, even if the highest
encapsulation eciency in LVM – loaded liposomes is obtained with
Lipo LVM – PBS, it will not be the best formulation because the zeta
potential is too low – it can negatively affect the colloidal stability –
and also the particle size is too high. In the remaining LVM – loaded
liposomes, it concluded that HD Lipo LVM is the optimal formulation,
which has particle size compatible with the literature data, the highest
encapsulation eciency, and high zeta potential. When viewed with
a similar approach, it can be said that HD Lipo ABZ with higher zeta
potential is the optimal formulation, since the particle size of both
liposomes is compatible with the literature data and the encapsulation
eciency is over 99% in ABZ–loaded liposomes. When the possible
mechanisms of action of liposomes on parasites are evaluated,
it is possible to benet from the fact that they provide more drug
permeability and increase the bioavailability of the drug, or even directly
inactivate the parasites by targeting and delay the re–occurrence of
resistance. In order to better understand these effects of liposomal
levamisole and albendazole, it was thought that they should be studied
in natural or experimentally induced parasitic infestations.
Funding
The study was nanced and supported as a project by the Balikesir
University Scientic Research Co–ordinatorship (BAP Project No:
2020 / 095).
ACKNOWLEDGEMENTS
This research was carried out with the support of the devices in
the laboratories of Karadeniz Technical University (KTU) Drug and
Pharmaceutical Technology Application and Research Center and
experienced instructors in the eld.
Since the research carried out is a laboratory study and does not
include animal experiments, it does not contain any ethical issues.
Conict of Interest
The authors declared that there is no conict of interest.
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