Medical

Communication

Biosci. Biotech. Res. Comm. 9(4): 633-642 (2016)

In vitro

evaluation of antifungal effects of nanoliposomal

 uconazole against  uconazole susceptible and resistant

Candida

species isolated from patients

Asadi Mehrdad

, Hashemi Seyed Jamal

*, Hamishehkar Hamed

, Kordbacheh Parivash

Ghasemi Zeinab

, Mojtaba Didehdar

, Nazari Maryam

and Sadeghi Sanam

Department of Medical Parasitology and Mycology, Faculty of Public Health, Tehran University of Medical

Sciences, Tehran-Iran

Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical

Sciences, Tehran, Iran

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical

Sciences, Tehran, Iran

Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical

Sciences, Tehran, Iran

Department of Medical Parasitology and Mycology, Arak University of Medical Sciences, Arak, Iran

Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

Department of Nanobiotechnology, Faculty of Biological Sciences, University of Tarbiat Modares, Tehran, Iran

ABSTRACT

The aim of this study was to produce  uconazole loaded liposomal nanoparticles, to analyze their physicochemical

properties and to compare their antifungal effects with the free  uconazole drug in vitro against the  uconazole

susceptibleand resistant Candida species isolated from patients.Six common candida species including C.albicans,

C.parapsilosis, C.tropicalis, C.glabrata, C.krusei and C.guilliermondii were tested. The Liposomal nanoparticles

were prepared using thin layer hydration method and soybean lecithin, cholesterol, and  uconazole at a ratio of

10: 1: 1. The nanoparticles were analyzedin terms of size, poly dispersity index, zeta potential, morphology,

entrapment ef ciency of drug and the amount of drug released. To investigate the antifungal effects of liposomal

633

ARTICLE INFORMATION:

*Corresponding Author: sjhashemi@tums.ac.ir

Received 13

Nov, 2016

Accepted after revision 19

Dec, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

634 IN VITRO EVALUATION OF ANTIFUNGAL EFFECTS OF NANOLIPOSOMAL FLUCONAZOLE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Asadi Mehrdad et al.

nanoparticles and compare them with the free form of  uconazole, we used Broth Microdilution as described in CLSI

M27-A3.The results were analyzed using Student’s T-test and indicated the greater antifungal effects of the liposomal

nanoparticles containing  uconazole than the normal form of the drug. It was shown that MIC of Fluconazole was put in

the range of sensitive species after exposure with the Fluconazole Nanoliposomal in most Fluconazole resistant Candida

species except for the krusei species. Therefore, it is likely that we can use the new system for drug delivery to prevent

drug release from the cell. In addition, this is the  rst research using  uconazole lipid nanoparticles against C.krusei.

KEY WORDS: NANOLIPOSOME, FLUCONAZOLE, ANTIFUNGAL ACTIVITY, CANDIDA

INTRODUCTION

Nowadays, the opportunistic pathogenic fungi are

among the life threatening infections in patients with

impaired immune systems. Yeasts, especially the can-

dida species are the most common fungi that are isolated

from human infections. Despite many advances in the

 eld of health care and methods of treatment, the inci-

dence of invasive systemic candidiasis is signi cantly

increasing. Although Candida albicans was and is the

most common agent responsible for infections in differ-

ent clinical forms of candidiasis, the other species belong

to the Candida types such as Candida tropicalis, Candida

glabrata, Candida krusei, Candida parapsilosis, Candida

guilliermondii and so on, are more or less isolated from

patients. The importance of the non-albicans species has

increased due to the relative resistance in some of these

species like Candida tropicalis and Candida glabrata to

the antifungal drugs in the recent years, (Price etal 1994,

Pfaller, 1995, Wingard, 1995 Neppelenbroek et al 2006).

The increase of the different and various reports from

all over the world about the drug resistance increase

among the fungi and Candida species, and on the other

hand, the new antifungal drugs production indicates the

need for testing the susceptibility to these drugs and

makes the researchers eager to determine susceptibility

pattern for the various antifungal drugs, (White et al.,

2002 Mohammadi et al 2016).

The side effects of anti-fungal drugs is one of the

main reasons of extensive research on new anti-fungal

compounds and their therapeutic effects, (Falkiewicz-

Dulik and Macura, 2008). Meanwhile nano particles are

of the particular importance, among the metal nano par-

ticles, silver nano particles has attracted many research-

ers due to their interesting physicochemical traits (S.

Schultz et al., 2000). Recently nano particles synthesis

by microbes has been considered as a suitable alterna-

tive for the mass production of nano particles, (Verma et

al 2009 and Omidi et al., 2014). Solid lipid nanoparticles

(SLNs) were introduced for the  rst time in 1991 as an

alternative to common colloidal carriers such as emul-

sions and polymeric nanoparticles. SLNs are colloidal

carriers with sizes ranging from 50 to 1000 nm, which

are formed from biolipids (Thassu et al., 2007, Yadav

et al., 2013).

Recently, these particles are used as antifungal drug

carriers such as Itraconazole, (Mohanty et al., 2015),

ketoconazole (Souto and Muller, 2005), Griseofulvin

(Aggarwal and Goindi, 2013) and Miconazole ( Mendes

et al., 2013). The small size of these lipid nanoparticles

increases their access to the tissues and the more in u-

ence of drugs( Jenning et al., 2000). These drug delivery

systems bring the controlled release of drugs, increas-

ing chemical stability of the trapped drugs. In addition,

these systems are among the safe and secure carriers

that can be easily produced on a large scale (Muhlen et

al., 1998 and Mehnert and Mader, 2001).

The treatment using azole antifungal agents, espe-

cially,  uconazole is introduced as an effective solution

for treating infections caused by Candida, but the drug

resistances have faced this treatment method with dif-

 culties ( Revankar et al., 1996, Silva et al., 2012).

Thus, for treating these types of cases, that are not

curable applying the conventional methods, the use of

new treatment strategies seems to be essential. In this

research, nanoliposome-containing  uconazole is pre-

pared, and its antifungal effects are studied in vitro

on the  uconazole susceptible and resistant strains of

C.albicans, C.parapsilosis, C.glabrata, C.tropicalis and

the resistant C.krusei species comparing to the conven-

tional form of  uconazole.

MATERIALS AND METHODS

87 positive cultures of yeast organisms isolated from

cutaneous, mucosal and systemic fungal lesions of

patients referring Razi hospital in Tehran, Sina Hospital

in Tabriz city and another private lab in Tabriz were col-

lected. Clinical specimens included skin, nail, mucous

discharges and the BAL samples.

Isolation of yeast: clinical samples were cultured on

Sabouraud dextrose agar medium with chloramphenicol,

and after the growth of yeast colonies were transferred

to sterile Eppendorf tubes containing distilled water

and 20% glycerol so were kept in the freezer with -20 °

C.CHROMagar Candida culture medium: Yeasts isolated

from patients were cultured applying linear method on

the CHROMagar Candida medium in HIMEDIA Company

and incubated for 48 hours at 35 ° C and then the plates

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Asadi Mehrdad et al.

FIGURE 1. Gel electrophoresis of ITS-PCR prod-

ucts of six candida species, L is 50 bp molecular

marker, well 1) C. guilliermondii, well 2) C. par-

apsilosis, well 3) C. glabrata, well 4) C. krusei, 5)

C. albicans, well 6) C. tropicalis.

FIGURE 2.PCR-RFLP electrophoresis pattern

with the HpaII enzyme on Agarose gel, L is 50

bp molecular marker, well 1) C. guilliermondii,

well 2) C. parapsilosis, well 3) C.glabrata, well 4)

C. krusei, well 5) C. albicans, well 6) C. tropicalis.

were examined macroscopically. For the green samples

testing of Candida albicans/dubliniensis, chlamydocan-

idia and mycelium forms production using corn meal

agar medium containing Tween 80 for 48 hours at 30 °

C was carried out.To differentiate albicans species from

dubliniensis the test of chlamydoconidia production by

dubliniensis species in Niger seed agar medium and the

inability of C.albicans was used in the production of

chlamydoconidia in the mentioned medium.

The identi cation of Non-Albicans candidaisolates:

all of the yeasts that their colony color was not exclu-

sive to identify them in CHROMagar medium and iso-

lates that their color was blue and purple in CHROMagar

Candida agar medium were identi ed using PCR-RFLP.

This method is based on ampli cation of ribosomal DNA

fragments in ITS1-5.8S-ITS2 PCR method using univer-

sal primers ITS1, ITS4 and then digestion the PCR prod-

ucts ampli ed by restriction enzyme HpaII. ( gure 1, 2)

PREPARATION OF LIPOSOME

Fluconazole and cholesterol were purchased from Sigma

company, and soybean lecithin was purchased from Lip-

oid Company. Liposomal formulation  uconazole was

prepared by the method of thin layer  lm hydration (H.

Ola et al., 2010).

For the preparation of liposomes, lecithin, choles-

terol and  uconazole were used with a ratio of 10: 1:

1. A thin layer was formed by solving these two sub-

stances in the organic solvent of chloroform-methanol

(1: 1) containing  uconazole at a rate of 5.12mg / ml,

then evaporation of the solvent in the rotary evaporator

under the temperature of 45 ° C. Then, it was hydrated

by 9% sucrose slowly at 65 ° C. The homogenization

action of samples was performed by a homogenizer at

20000 rpm and ata temperature above the liposomes

phase transition (70) for 10 minutes. The liposomal sam-

ple sonication operation was carried out in an ice bath.

The amount of the drug that was not loaded, by the

amicon  lter was removed by centrifugation at 4000 × g

for 4 minute and was passed for sterilization twice using

0/22 micron  lter needle. This new formulation trans-

ferred to the laboratory and used freshly to evaluate the

antifungal activity.

BLANK LIPOSOMAL PREPARATION

To prepare this formulation, it was treatedin accordance

with the formulation of  uconazole liposomal formula-

tions and the difference was that it was prepared with-

out  uconazole.Determining the size of nanoliposomes

containing  uconazole:Particle size, average diameter,

nanoliposome zeta potential distribution was measured

by zetasizer device based on laser light scattering. Par-

ticle size distribution in the device, was evaluated based

on PDI (Poly Dispersity Index).

NANOPARTICLE STRUCTURE

For this purpose, the scanning electron microscope

device was used. That a quantity of sample is placed on

a glass surface with dimensions of 1 x 1 cm. Then it was

placed inside an incubator at 37 ° C until the sample is

completely dry. After that the particles were coated with

gold and the images were taken with the magni cation

of 20,000 and 40,000.

636 IN VITRO EVALUATION OF ANTIFUNGAL EFFECTS OF NANOLIPOSOMAL FLUCONAZOLE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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DRUG ENTRAPMENT EFFICIENCY (DE)

To check the encapsulated drug in the prepared liposomal

formulations, the maximum absorption of  uconazole

in the UV, 255 nm was obtained. Then, the calibration

curve was drawn, and a speci c amount of formulation

is placed above the amicon  lter and centrifuged for

5 minutes at 4 ° C and 4000 × g. A certain amount of

free drug passes on the underside of Falcon removed by

pipette and unloaded  uconazole absorption was meas-

ured by spectrophotometer at a wavelength of 255 nm,

and the percent of  uconazole loading was obtained by

the following formula:

RESULTS AND DISCUSSION

Eighty-seven positive cultures of yeast isolated from

cutaneous, mucosal and systemic fungal lesions of

patients referring Razi hospital in Tehran, Sina Hospital

in Tabriz city and another private lab in Tabriz were

collected. Due to the fact that more than one type of

yeast wereisolated from some patient samples, the total

number of 91 yeast colonies were isolated and tested in

the next steps. The relevant results and methods of iden-

tifying can be seen in Table 1.

The particle size of liposomal nanoparticles: In the

present study, our goal was to obtain an ideal formula-

tion in terms of particle size and the  uconazole encap-

sulation amount for the evaluation of the antifungal

effects and its comparison with the normal form of

the drug, which is reached. As can be seen, the particle

size of liposome containing  uconazole is 88.9± 12.1

nm(Figure 3). Zeta potential for the  uconazole lipo-

some formulation is -12.20± 1.88mv (Figure 4).

The SEM results for the nanoliposomes contain-

ing  uconazole, to study the structure and form of the

nanoliposomes the scanning electron microscope was

used. The results are presented in Figure 5,6. Figure 5 is

captured with a lower magni cation and to show a uni-

form distribution of particle size and Figure 6 is captured

with a higher magni cation to better show the structure

and form of the nanoliposomes, which are spheral.

The amount of  uconazole loaded in the liposomal

formulation is obtained as 75.1% in accordance with the

aforementioned method.

ANTIFUNGAL SUSCEPTIBILITY TESTING

Table 2 represents the susceptibility of the examined

Candida species to the  uconazole and nanoliposomal

 uconazole medicines.In the present study, the produced

liposomal formulation for more loading of the  ucona-

zole drug as well as its antifungal effects on the  u-

conazole susceptible and resistant Candida species iso-

lated from patients in the current form of  uconazole

were created.Poly dispersity index or PDI of the prepared

nanoparticles:

The results of the measurements using malvernzeta-

sizer indicated that the formulation had a narrow par-

ticle distribution domain and the samples prepared to

apply this method had a proper PDI and particle dis-

tribution. About the examination of the results of poly

dispersity index or PDI of the prepared nanoparticles,it

can be said that the lower the PDI, the lower the distri-

bution of particle sizes is and the system will be more

homogeneous. Generally, if PDI is in the range of 0 to

0.5, the suspension is mono disperse, and if it is higher

than 0.5, it is poly disperse.

ANTIFUNGAL SUSCEPTIBILITY TESTING

In this study, the standard method of Broth Microdilu-

tion described in CLSI M27-A3-S4 is presented and the

standard quality control of C. parapsilosis ATCC 22019

was used to evaluate the MIC of  uconazole and  u-

conazole nanolipososmal formulation (J.H. Rex et al.,

2008).

To perform this test, 96-well microplateswere used. In

order to dilute the  uconazole stock solution, we used

the growth medium RPMI1640 (with glutamine, with-

out bicarbonate and buffered to pH 7.0). To prepare this

medium, we solved 10.43 grams of RPMI powder and

34.53 grams of MOPS buffer (N-MorpholinoPropane-

sulfoni Acid) in one liter of distilled water with gentle

shaking on the  ame. We used normal saline for dilu-

tion of the Nanoliposomes containing  uconazole. In

the case of the two drugs, after adding to each drug the

yeast cell suspension which had been prepared accord-

ing to CLSI M27-A3 protocol and contained 2.5×10

0.5 × 10

cfu/ml of yeast, we obtained the highest and

lowest  nal concentrations as 64μg / ml and 0.063μlg

/ml respectively. The well No. 12 was used as posi-

tive control. Each of the isolates was examined as dual

series.

After 48 hours of incubation, to determine the Mini-

mum Inhibitory Concentration (MIC) of  uconazole and

nanoliposomal  uconazole that the fungus growing

inside were not observed in the microplates and 80% of

its growth was blocked, examined and the results were

read.To ensure of the non-antifungal activity of nanoli-

posomes particles, blank nanoparticles liposome were

used in this test and no antifungal effect was observed.

We have used SPSS version 18 and Student’s T-test in

order to analyze and compare the antifungal effects of

the liposomal nanoparticles containing  uconazole and

the free  uconazole.

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Asadi Mehrdad et al.

Table 1:

Identifying method Percent Quantity Species Total (%) Candida Species

(quantity)

Clinical

species

Chrome Candida Agar,

the production of

chlamydocanidia

61.4 56 Candida albicans 29 (31.8) Albicans (16),

parapsilosis (5),

tropicalis (3), glabrata

(1), krusei (2),

guilliermondii (2)

Fingernail

PCR-RFLP 16.5 15 Candida parapsilosis 11 (12) Albicans (6), parapsilosis

(3), tropicalis(2)

Toenail

Chrome Candida Agar,

PCR-RFLP

8.8 8 Candida tropicalis 15 (16.5) Albicans (9), parapsilosis

(3), tropicalis (2), krusei

(1)

Hand

PCR-RFLP 6.6 6 Candida glabrata 6 (6.6) Albicans (4),

parapsilosis(2)

Foot

Chrome Candida

Agar,PCR-RFLP

4.4 4 Candida krusei 19 (20.9) Albicans (15), glabrata

(3), krusei (1)

Vagina

PCR-RFLP 2.2 2 Candida guilliermondii 6 (6.6) Albicans (4),

parapsilosis(2)

Groin

100 91 Total 5 (5.5) Albicans (2), tropicalis

(1), glabrata (2)

BAL

FIGURE 3. Curve of Particle size distribution of liposomes containing

 uconazole.

Zeta potential of the  uconazole liposome formula-

tion:

Zeta potential is an important factor in determin-

ing the stability of the colloidal system and is the best

indicator to determine the status of super cial electrical

dispersions. Usually, cholesterol is used to improve the

stability of liposomes. Adding cholesterol increases the

zeta potential of the colloidal system; as the zeta poten-

tial increases in the particles, the particles will be more

stable, and on the other hand, adding cholesterol will

increase the rigidity of bilayer phospholipid (Mcnaught

and Wilkinson, 1997, Panyam and V. Labhasetwar,

2003).

In liposomal formulations, in the lipid composition of

which there is cholesterol in addition to the lecithin, zeta

potential was negative. About the stability of stearic and

electrostatic combined, a minimum of ±20 ZP is desir-

able (Tang et al., 2014). In a research by El-Nesr et al.

in 2010 on multilayer liposomes containing  uconazole,

formulations with negative, neutral and positive zeta

Asadi Mehrdad et al.

638 IN VITRO EVALUATION OF ANTIFUNGAL EFFECTS OF NANOLIPOSOMAL FLUCONAZOLE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

the stearylamine particles in liposome and the slightly

negative charge of the  uconazole in comparison with

the liposome particles with negative and neutral charge,

(Ola et al., 2010).

The amount of drug loaded in the liposomal formula-

tion of  uconazole:

The amount of drug loaded in the liposomal formula-

tion was 75.11%. In fact, a signi cant amount of  u-

conazole was encapsulated in the nanoliposomes, which

is due to the hydrophilic nature of  uconazole that are

not willing to be present in the external phase and as a

result, a signi cant amount of  uconazole is encapsu-

lated during the formation of nanoparticles.

The SEM results of nanoliposomes containing  u-

conazole:

In the scanning electron microscope images, the

narrow range of particle distribution and the spherical

shape of the formed particles was clearly observable.

According to the results of SEM, the prepared particles

were in the same particle size range that was obtained

by nanozetasizer.

The comparison of the antifungal effects of  ucona-

zole and liposomal nanoparticles containing  ucona-

zole:

Table 2 shows the results of in vitro susceptibility of 6

susceptible and resistant Candida isolates to the  ucona-

zole comparing to the  uconazole containing nanolipo-

some. As can be seen in the table 2, the use of the lipo-

somal form of  uconazole signi cantly decreased the

MIC of C.albicans species (P <0.0001), C.parapsilosis (P

<0.05), C.tropicalis (P = 0.0005), C.glabrata (P = 0.002)

and C.guilliermondii (P <0.05), but no difference was

observed in the amount of MIC of the two forms of drug

in the case of C.krusei.

The MIC

amount for C.glabrata was obtained as

8μg/ml, which had the highest amount of MIC relative

to the nanoliposomes containing  uconazole among the

isolated Candida species of patients. The resistant strain

FIGURE 4. Curve of Zeta Potential of liposomes containing

 uconazole.

FIGURE 5. Image of nanoparticles with lower

magni cation.

FIGURE 6. Image of nanoparticles with greater

magni cation.

potential were evaluated. This examination indicated

that the nanoliposomes having positive, neutral and

negative electric charge had the highest drug loading

amount, respectively, and this can be possibly related

to the electrostatic force between the positive charge of

Asadi Mehrdad et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS IN VITRO EVALUATION OF ANTIFUNGAL EFFECTS OF NANOLIPOSOMAL FLUCONAZOLE 639

to the  uconazole of C.glabrata which has MIC equal to

64 μg/ml, had 4 times decrease in the MIC and placed in

the susceptible species after exposing to the  uconazole

liposomal form. The amount of MIC

for the  ucona-

zole susceptible strains of C.albicans, C.parapsilosis,

C.tropicalis, and C.guilliermondii against to the  ucona-

zole liposomal form, were 1μg/ml, 1μg/ml, 1.5μg/ml and

2μg/ml, respectively. The amount of MIC

for the resist-

ant strains of C.albicans, C.parapsilosis, C.krusei were

3μg/ml, 4μg/ml and 64 μg/ml, respectively.

There is a high possibility that the reason for the

low amount of MIC in the  uconazole resistant Can-

dida strains relative to the nanoliposomal  uconazole

is related to the identi ed resistance mechanisms of

pathogenic fungi to drugs; the mechanisms that make

fungi survive in the vicinity of a series of toxic sub-

strates as well as antifungal drugs( Mishra et al., 2007,

Morschhäuser, 2010). Various molecular mechanisms

have been identi ed in the development of strains resist-

ant to antifungal drugs (Franz et al., 1998), that these

mechanisms include reducing the transmission and dif-

fusion of the drug into the cell, changes in the enzymes

of the pathway of ergosterol Biosynthesis, changes in

the target enzyme (point mutations, expression increase

Table 2: In vitro antifungal susceptibility of all yeast isolates (n = 91)

Species/Antifungal

agent

MIC Range

MIC (μ/ml)

MIC50 MIC90 GM

C. albicans (30)

Fluconazole 0.5-4 1 4 1.33

LE-Fluconazole 0.063-4 0.125 2 0.21

C. albicans(26)*

Fluconazole 8-64 32 64 28.64

LE-Fluconazole 0.063-8 4 8 4.34

C. parapsilosis(13)

Fluconazole 0.25-4 1 2 0.89

LE-Fluconazole 0.063-0.5 0.125 0.45 0.13

C.parapsilosis(2)*

Fluconazole 8-32 20 -- 16

LE-Fluconazole 2-4 3 -- 2.82

C.tropicalis(8)

Fluconazole 0.5-2 1.5 -- 1.29

LE-Fluconazole 0.125-0.5 0.25 -- 0.27

C. glabrata (5)

Fluconazole 4-16 8 -- 6.96

LE-Fluconazole 1-8 2 -- 2

C.glabrata(1)*

Fluconazole 64 64 -- --

LE-Fluconazole 8 8 -- --

C.krusei (4)*

Fluconazole 64 64 -- 64

LE-Fluconazole 64 64 -- 64

C.guilliermondii(2)

Fluconazole 2 2 -- 2

LE-Fluconazole 0.125 0.125 -- 0.125

C.parapsilosis 22019

Fluconazole 0.5 0.5 -- --

LE-Fluconazole 0.125 0.125 -- --

*Percent resistance using interpretive breakpoint criteria of the CLSI(M27-A3s4); Fluconazole

resistance C. albicans, C.parapsilosis ≥ 8 μg/ml and C.glabrata≥ 64 μg/ml MIC(minimum

inhibitory concentration), GM (Geometricmean)

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640 IN VITRO EVALUATION OF ANTIFUNGAL EFFECTS OF NANOLIPOSOMAL FLUCONAZOLE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

and gene change) and the increase of drug release to

the outside by membrane diffusion pumps, activation of

the enzymes that break down drugs outside the cell wall

(Favre et al., 1999, Hitchcock et al., 1990, Li et al., 2004,

Löf er et al., 1997, Sanglard and Odds, 2002, White

et al., 1998).

From the mentioned cases, the most important reason

for the resistance of Candida to the azole drugs is related

to the expression of the high amount of transporter

membrane proteins that lead to the pumping of azoles to

the outside of the cell. The result is the reduction of drug

concentration in cell and in this case, the drug concen-

tration inside the cell is under the necessary level needed

for creating the inhibitory effect on Erg11p (Caira et al.,

2004). Two main types of drug release pumps from the

fungus cells causes their resistance to drugs: ABS and

MFS (Lamping et al., 2010). Cdr1 and Cdr2 are among

the important transporters of the drug to the outside of

the cell in C.albicans, C.glabrata and C.parapsilosis and

the increase in their expression leads to the release of

drug from the cell, which  nally leads to the resistance

of mentioned species to the  uconazole (White et al.,

2002, Bennett et al., 2004 and Souza et al., 2015).

In the examination conducted by us, the lipid nano-

particles are fused quickly with the cell membrane of

yeast due to the hydrophobic outer surface, and as they

are small in terms of size, they are capable of pene-

trating to the yeast cell and causes the drug protection

against enzymes and ef ux pumps. Also, the lipid drug

delivery systems due to the use biodegradable lipids are

low toxic, have the ability to get lyophilized (Garud et

al., 2012) and are capable of controlled release of the

drugs, (Bose et al., 2013).

In a recent study conducted by Moazeni et al. in 2016,

using lipid nanoparticles against  uconazole suscepti-

ble and resistant species in vitro, the results indicated

the satisfying effect of lipid nanoparticles contain-

ing  uconazole on the resistant strains of C.albicans,

C.parapsilosis and C.glabrata (M. Moazeni et al., 2016).

Similarly, Gupta et al. had observed that the use of

drug delivery system can lead to the increase of  u-

conazole penetration from the skin surface (Gupta et al.,

2013). As the  uconazole prevents the synthesis of ergos-

terol by affecting the -demethylase14 enzyme, in the

investigations of Venkateswarlu et al. it was determined

that the -demethylase14 existing in the C.krusei differs

the -demethylase14 of C.albicans (Venkateswarlu et al.,

1997, Venkateswarlu et al., 1996).

Studies by Orozco et al. indicated that the intra-

cellular accumulation of  uconazole of C.krusei and

C.albicans are similar (Orozco et al., 1998). This in turn

could explain the reason why C.krusei was resistant to

the  uconazole exposure as well as the form of  ucona-

zole nanoparticles.

CONCLUSION

In the current study, the function of a new drug delivery

system was evaluated on the  uconazole resistant and

susceptible Candida strains isolated from the patients.

Although different mechanisms of resistance to  ucona-

zole are expressed, it seems that the resistance is due to

the pumps releasing drug from the cell and it is one of the

main reasons for these resistances. Therefore, it is likely

that we can use the new system for drug delivery to pre-

vent drug release from the cell. In addition, this is the  rst

research using  uconazole lipid nanoparticles against

C.krusei.

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