Key words
cyanidinum chloride - liposome -
Pseudomonas aeruginosa
- killing rates -
in vitro
-
in vivo
Introduction
Pseudomonas aeruginosa is an opportunistic pathogen that causes serious wound infections [1]. Since P. aeruginosa can quickly colonize and infect wound sites and rapidly disseminate from wounds into
the bloodstream, the clinical outcome in these patients can lead to sepsis, which
is often fatal [2]. One of the major problems associated with P. aeruginosa infection is resistance to most conventional antibiotics [1]. Therefore, there is a compelling need to develop novel agents, strategies, and
methods to overcome this resistance [3].
Cyanidinum ion ([Fig. 1]), a hydrolysis product from cyanidin salts and a flavonoid occurring in many red
berries, possesses a range of biological and medicinal properties, including antioxidant,
anticancer, antiobesity, and antiviral activities [4], [5]. Other physiological functions of cyanidinum ion include anti-inflammatory activity
and reduction in memory impairment effects [6], [7]. It has been shown that plant extracts that contain a wide range of polyphenolic
compounds such as cyanidinum ion have antimicrobial activity against drug-resistant
bacteria [8], [9]. Cyanidinum ion has a major antibacterial and antifungal activity effect on Bacillus subtilis and yeast, respectively [10]. Also, this compound has antituberculosis activity [11].
Fig. 1 Cyanidinum ion structure.
Liposomes are spherical and colloidal vesicles that range from a few nanometers to
several micrometers in diameter [12], [13]. These carriers are composed of natural phospholipids and other lipids, such as
cholesterol, and can be used as a vehicle for the administration of nutrients and
pharmaceutical drugs [14], [15], [16].
Previous studies showed that the encapsulation of plant-derived materials into liposomes
markedly alter their pharmacokinetics, increasing their half-lives and effectiveness
[17], [18]. While some of these compounds, such as usnic acid, have relative antibacterial
effectiveness in the liposomal form, others, such as oleic acid, have been more effective
in this formulation [19], [20]. The anti-P. aeruginosa effectiveness of cyanidinum chloride-loaded liposomes has not yet been studied. The
primary aim of this study was to prepare cyanidinum chloride-loaded liposomes and
evaluate their in vitro antibacterial activity against the resistant strain of P. aeruginosa ATCC 15692. A secondary aim was to investigate the therapeutic efficacy of prepared
liposomes using a mouse model of skin wounds infected with this bacterium.
Materials and Methods
Chemicals
Cyanidinum chloride (purity ≥ 95 %), cholesterol, egg lecithin, and amikacin hydrate
(an aminoglycoside antibiotic, as the positive control, purity ≥ 78 %) were purchased
from the Sigma Chemical Company. Chloroform, HPLC grade methanol, and Mueller-Hinton
broth were purchased from Merck.
Animals
Fifty male BALB/c mice (18–20 g) were obtained from the National Institute of Pasture,
Iran. The mice were handled according to the national guidelines for laboratory animals,
received food and water ad libitum and were housed in separate and pathogen-free cages [21]. Animal care and protocols were performed and approved by the Institutional Animals
Ethics Committee of Tarbiat Modares University (Number: 145, Tehran, Iran, 3/3/2011).
Microorganism
P. aeruginosa ATCC 15692 was purchased from the American Type Culture Collection. This strain was
inoculated onto blood agar plates and then incubated for 24 h at 37 °C and used for
experiments.
Preparation of liposomes
The cyanidinum chloride-loaded liposomes were prepared by the extrusion method, as
described previously [17]. Briefly, the egg lecithin and cholesterol in the molar ratio of 4 : 1 were dissolved
in chloroform and dried to a lipid film with a rotary evaporator (Brinkman) under
vacuum and N2 flow at 30 °C. The dried lipids were dispersed by agitation in 6 mL of an aqueous
solution of cyanidinum chloride (150 mg/mL in PBS, pH 7.4) and sonicated at 4 °C in
ultrasonic bath (Braun-sonic 2000). Finally, cyanidinum chloride-loaded liposomes
were obtained by extruding the respective suspension through a polycarbonate membrane
with 100 nm-sized pores 12 times, and separating the excess free drug and larger lipid
aggregation by ultracentrifugation (100 000 g, 30 min). The control liposomes were
prepared similarly, but PBS (pH 7.4) was used instead of the cyanidinum chloride solution.
Determination of encapsulation efficacy
The content of the cyanidinum chloride in the liposomes was determined by HPLC as
previously described [22], and the percentage of cyanidin loading was then calculated as: amount of cyanidinum
chloride in liposome × total volume tested × 100/total sample volume × initial amount
of cyanidinum chloride. This experiment was done in triplicate.
Particle size, zeta-potential, size distribution, and polydispersity index determination
The mean particle size, zeta-potential, size distribution, and polydispersity index
of the liposomes were evaluated using a Malvern zetasizer (Malvern instrument) apparatus,
as reported previously [23], [24]. Each experiment was done in triplicate.
Antimicrobial susceptibility testing
The minimum inhibitory concentrations (MICs) of the free and cyanidinum chloride-loaded
liposomes and amikacin for P. aeruginosa ATCC 15692 were determined by the broth dilution technique as recommended by CLSI
(formerly NCCLS) [25]. A bacterial suspension of ~ 5 × 105 cells/mL was diluted in the Mueller-Hinton broth and dispensed (100 µL) into a microtiter
tray containing serial twofold dilutions of cyanidinum chloride. The tray was then
incubated for 24 h at 37 °C. The MICs were recorded as the lowest concentrations of
cyanidinum chloride in the free and liposomal forms that prevented visible bacterial
growth and were expressed in µg/mL. All experiments were done in triplicate.
Time-kill studies
Time-kill studies were performed in triplicate in 10 mL tubes containing 2 mL of Mueller-Hinton
broth as previously described [26]. Briefly, 100 µL of the bacterial suspension were resuspended in 10 mL of the Mueller-Hinton
broth, incubated overnight at 37 °C and adjusted to a McFarland standard of 0.5. Then,
100 µL of this standardized inoculum were added to separate culture tubes containing
1 mL of Mueller-Hinton broth with 1 mL cyanidinum chloride solutions in the free and
liposomal forms at 1, 2, and 4 times the MIC and incubated at 37 °C. Subsequently,
colony counts were performed at 0, 2, 4, 6, and 24 h, and the results were expressed
as log colony forming unit per milliliter (CFU/mL). All experiments were performed
in triplicate.
Animal studies
The in vivo therapeutic efficacy of cyanidinum chloride-loaded liposomes in a mouse skin suture-wound
model was tested according to a described method [27], with some modifications. Briefly, sterile silk sutures were cut and threaded onto
sterile surgical needles and soaked for 45 min in undiluted broth cultures of the
P. aeruginosa ATCC 15692 that had been incubated at 35 °C for 8 h. The mice were anesthetized with
a ketamine-xylazine mixture (50 mg/kg each, given intramuscularly). Subsequently,
the fur on the back and flanks was clipped, and the skin was swabbed with 70 % ethanol.
A 1-cm length of inoculated suture was inserted under the skin of the mid-back and
secured by knotting the other side of the suture. Then the infected mice were divided
into 5 groups. Prior to the treatment starting, the free and liposomal forms of cyanidinum
chloride, empty liposomes, amikacin, and physiological saline solutions were prepared.
Subsequently, the gel forms of these treatments were prepared according to a previously
described method [28]. Then, all groups were treated topically as follows: group 1 received cyanidinum
chloride-loaded liposomes gel (contained 250 mg cyanidinum chloride/kg/24 h); group
2 received free cyanidinum chloride gel (250 mg/kg/24 h); group 3 received empty liposomes
gel (250 mg/kg/24 h); group 4 received physiological saline gel (1 mL/kg/24 h); and
group 5, as the positive control group, received amikacin gel (250 mg/kg/24 h); for
8 days starting from the 3rd day postinfection. Two days after the last dose, the
surviving animals were anesthetized and sacrificed by cervical dislocation, and the
skin, liver, and spleen of each animal was removed under sterile conditions and homogenized
for 5–10 min in PBS (2 mL/g). The homogenates were serially diluted and plated for
growth in the soybean-casein digest agar medium and incubated at 35 °C for 24 h, and
then the colony forming unit (CFU) was counted. The colony counts were performed in
triplicate.
Data analysis
The results are expressed as means ± standard errors for all of the experimental measurements.
The data from the killing rate study were statistically evaluated by paired Studentʼs
t-test, and a p value < 0.05 was considered significant. The results of the survival
rates of the control and treated animals were determined by using the chi-squared
test with Yates correction and by Fisherʼs exact test.
Supporting information
The particle size, zeta-potential, and polydispersity index of the empty and cyanidinum
chloride-loaded liposomes as well as the size distribution pattern of cyanidinum chloride-loaded
liposomes are shown in the Supporting Information.
Results and Discussion
The use of plant-derived compounds to eliminate harmful and resistant bacterial infections
has been widely investigated [8], [9]. However, the main problems associated with the application of these materials are
insufficient quantities in the target site and in vivo instability [9]. To overcome these problems, the investigators focused on the encapsulation of plant-derived
compounds in carriers such as liposomes [29], [30]. However, the preparation of plant-derived liposomes with high encapsulation efficacy
may not be easy because the variable interactions between these materials and bilayer
lipids can occur [31]. In this study, we evaluated the potential of incorporating cyanidinum chloride
into liposomes. The results showed that cyanidinum chloride can be encapsulated into
the prepared liposomes with high entrapment efficacy (85.00 % ± 0.15). It has been
shown that positive interaction between lipids and loaded components could enhance
entrapment efficacy [16], [17], and probably some of the various known types of weak links make the increase of
cyanidinum chloride encapsulation efficacy.
The homogeneity of the mean particle size of the empty and loaded liposomes suggested
that the cyanidinum chloride was entrapped in the lipid bilayer, according to previous
studies [17], [23]. The zeta-potential of the liposomes revealed that the prepared cyanidinum chloride-loaded
nanoparticles have appropriate stability in aqueous dispersion [31], [32]. The size distribution study showed a monomodal distribution with a mean diameter
of 92.5 ± 0.2 nm.
The MIC values of cyanidinum chloride in both of the free and liposomal forms and
amikacin for P. aeruginosa ATCC 15692 were 1.5 × 10−3, 7.7 × 10−4, and 6.8 × 10−6 M, respectively. The difference between the MICs of the free and liposomal cyanidinum
chloride was significant (p < 0.05). Our results suggest that the entrapped cyanidinum
chloride in the liposomes enhanced its antibacterial activity against P. aeruginosa when compared to the free cyanidinum chloride. The killing curves of the cyanidinum
chloride in the free and encapsulated forms at 1, 2, and 4 times the MICs are shown
in [Fig. 2]. In all of the conditions, the encapsulated cyanidinum chloride was more effective
on reducing the bacterial counts when compared to the free cyanidinum chloride ([Fig. 2]). At one time MIC only, the cyanidinum chloride-loaded liposomes could eliminate
P. aeruginosa ATCC 15692 after 24 h ([Fig. 2 A]). At two times MIC, both the free and encapsulated forms of cyanidinum chloride
eradicated the bacteria after 6 and 24 h, respectively ([Fig. 2 B]). At four times MIC, the cyanidinum chloride-loaded liposomes eliminated the bacteria
after 4 h ([Fig. 2 C]).
Fig. 2 Killing curves for P. aeruginosa ATCC 15692 when exposed to various concentrations (A = 1 × MIC, B = 2 × MIC, and C = 4 × MIC) of cyanidinum chloride in the free and liposomal forms. All experiments
were done in triplicate.* Significant difference between the killing rate of the empty
liposomes versus free and cyanidinum chloride-loaded liposomes (p < 0.01); ** significant
difference between the killing rate of the cyanidinum chloride-loaded liposomes versus
free cyanidinum chloride (p < 0.05); *** significant difference between the killing
rate of the cyanidinum chloride-loaded liposomes versus free cyanidinum chloride (p < 0.01);
+
significant difference between the killing rate of the free cyanidinum chloride and
the empty liposomes (p < 0.05);
++
significant difference between the killing rate of the free cyanidinum chloride and
the empty liposomes (p < 0.01).
It has been reported that encapsulating methyl-N-methylanthranilate and alcohol α-bisabolol (organic compounds from Zanthoxylum tingoassuiba) in liposomes could eliminate the resistant strain of P. aeruginosa as well [33]. Some hypotheses, including increasing the penetration of plant-derived materials
into bacteria cells and their non-sensitivity to degradation bacterial enzymes, may
explain the mechanism of the enhanced antimicrobial activities of these liposomal
formulations [8], [33]. The time-kill assays confirmed that the potency of cyanidinum chloride-loaded liposomes
was higher than free cyanidinum chloride. As reported previously [20], we hypothesized that the electrostatic interaction between the outer membrane lipopolysaccharides
of P. aeruginosa and the liposomes could enhance the mechanism of cyanidinum chloride entry into this
microorganismʼs cell. Wounds and other exposed tissues are particularly susceptible
to bacterial contamination and infections [34], [35]. Compared to the untreated animals, the treatment of the wound-infected mice with
cyanidinum chloride-loaded liposomes showed a significant reduction in CFU values
in the evaluated organs, especially in the skin and liver ([Table 1]). It was found that the mortality of the mice without the administration of cyanidinum
chloride was 100 % after 8 days, whereas the animals treated with cyanidinum chloride
in free and encapsulated liposomes showed an increase in the survival rate of 40 and
100 %, respectively. Also, the viability of the amikacin-treated mice (as positive
control) was 100 %.
Table 1 Survival rate of infected mice and colony-forming units (CFUs) of P. aeruginosa ATCC 15692 in different organs.
|
Treatment
|
Tissue/Organ
|
Log CFU/Gram tissue
|
Percentage of survival mice (n = 10)
|
|
The results are expressed as mean ± standard error of mean. The analysis of variance
of one-way classification between the treatment means was heterogeneous, and the t-test
values (two-tailed) were significant. * p < 0.001 and ** p < 0.05
|
|
Control without drug administration (received physiological saline, topically)
|
Skin
Spleen
Liver
|
3.213 ± 0.200
3.871 ± 0.400
3.182 ± 0.500
|
none survived
|
|
Empty liposomes (250 mg · kg−1, topically)
|
Skin
Spleen
Liver
|
3.316 ± 0.500
3.766 ± 1.100
3.210 ± 0.500
|
none survived
|
|
Free cyanidinum chloride (250 mg · kg−1, topically)
|
Skin
Spleen
Liver
|
2.122 ± 0.300
2.795 ± 1.100
2.547 ± 0.120
|
40
|
|
Cyanidinum chloride-loaded liposomes (250 mg · kg−1, topically)
|
Skin
Spleen
Liver
|
Nil*
0.623 ± 0.100**
Nil*
|
100
|
|
Amikacin (250 mg · kg−1, topically)
|
Skin
Spleen
Liver
|
0.120 ± 0.200**
0.843 ± 0.500**
0.355 ± 0.300**
|
100
|
It has been proven that the potential mortality from wound infections, even with aggressive
antibiotic therapy, may approach or exceed 50 % [36]. The treatment of mice with cyanidinum chloride-loaded liposomes resulted in a 100 %
survival rate and in an almost complete eradication of the bacteria from the skin
and liver of each of the infected animals. This data may be due to optimal antibacterial
delivery, which has been reported by several investigators [19], [37]. It has also been reported that oleic acid-loaded liposomes (LipoOA) could eliminate
methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo [20]. Also, the encapsulation of usnic acid (a dibenzofuran originally isolated from
lichens) improved the burn healing process in rats [19]. When liposomes containing compounds are applied topically, they can interact with
the cell membranes of exposed tissues, and therefore protect the wound tissues from
further bacterial infections [19], [20], [37].
In conclusion, the in vitro and in vivo testing showed that cyanidinum chloride-loaded liposomes have a strong protective
effect of wounds infected by P. aeruginosa ATCC 15692 and may be a good choice for the treatment of patients with such infections.
Acknowledgements
This study was supported by the Islamic Azad University, Borujerd Branch, Iran. The
authors would like to acknowledge staffs of the university.
