بررسی تاثیر کورکومین انکپسوله در نانوذرات پلیمرزومی بر بیان ژن افلاکس پمپ MDR1 در جدایه های کاندیدا آلبیکنس مقاوم به فلوکونازول

الموضوعات : Biotechnological Journal of Environmental Microorganisms

سحر پوراصغر ¹ ( گروه زیست شناسی، واحد رشت، دانشگاه آزاد اسلامی، رشت، ایران. )
آیدا بجاری ² ( گروه زیست شناسی، واحد رشت، دانشگاه آزاد اسلامی، رشت، ایران. )
مهکامه هدایت صفا ³ ( university of parma, Italy )
مهدی شهریاری نور ⁴ ( گروه زیست شناسی، واحد رشت، دانشگاه آزاد اسلامی، رشت، ایران. )
نجمه رنجی ⁵ ( گروه زیست شناسی، واحد رشت، دانشگاه آزاد اسلامی، رشت، ایران. )

تاريخ الإرسال : 02 السبت , شوال, 1444 تاريخ التأكيد : 16 الإثنين , ذو القعدة, 1444 تاريخ الإصدار : 19 الخميس , ذو القعدة, 1444

الکلمات المفتاحية: : کاندیدا آلبیکنس, کورکومین, نانوذرات, MDR1, qRT-PCR,

ملخص المقالة :

کورکومین، یک ترکیب طبیعی از زردچوبه است که به عنوان یک عامل ضدباکتریایی و ضدقارچی شناخته می شود. کاندیدا آلبیکنس یکی از پاتوژن‌‌های قارچی مهم با میزان کشندگی بالا در بیماران با نقص ایمنی است. شایع ترین پاتوژن فرصت‌طلب قارچی است که مصرف پیوسته ضد قارچ‌ها در درمان آن منجر به ظهور و افزایش مقاومت چند‌دارویی شده است. در این مطالعه اثر کورکومین محبوس‌شده در نانوذرات پلیمرزومی و فلوکونازول بر‌ بیان ژن MDR1 در جدایه‌های کاندیدا آلبیکنس مقاوم به دارو ارزیابی شد. در این مطالعه مقطعی توصیفی، 50 نمونه‌ بالینی از زنان با عفونت ولووواژینیت از بیمارستان الزهرا (رشت) تهیه شد. بعد از تعیین هویت جدایه ها، مقاومت به فلوکونازول به روش انتشار از دیسک و براث دایلوشن بررسی شد. شش جدایه کاندیدا آلبیکنس مقاوم به فلوکونازول، تحت تیمار با فلوکونازول به تنهایی (1/2MIC) (نمونه شاهد) و در ترکیب با کورکومین محبوس‌شده در نانوذرات (نمونه تست) قرار گرفت. بعد از 24 ساعت، دو گروه سلولی جهت تخمین نرخ مرگ سلولی در محیط کشت سابوروددکستروز آگار کشت داده شد. بیان ژن MDR1 بطور کمی به روش Q-RT-PCR در سلول های تیمار‌شده و تیمار‌نشده با کورکومین مورد بررسی قرار‌گرفت. نتایج ما نشان داد که درمان ترکیبی فلوکونازول (1/2MIC) و کورکومین محبوس‌شده در نانوذرات(400µg/ml) طی 24 ساعت رشد قارچی را تا 50% کاهش داد. در سلولهای تیمار‌شده با کورکومین، آنالیز Q-RT-PCR کاهش بیان ژن MDR1 را در مقایسه با سلول‌های تیمار‌نشده با کورکومین نشان داد. به نظر می رسد کورکومین بتواند از طریق کاهش بیان ژن MDR1 در جدایه‌های مقاوم به فلوکونازول، اثربخشی قلوکونازول را افزایش دهد.

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نص كامل:



Antibacterial effect of Curcumin encapsulated in polymersome nanoparticles on the expression of efflux pump MDR1 gene in fluconazole resistant isolates of Candida albicans 

Sahar Pourasgar1, Aida Bejari1, Mahkameh Hedayatsafa2, Mahdi Shahriarinour1, Najmeh Ranji1*,

 

1 Department of Biology, Faculty of Sciences, Rasht branch, Islamic Azad University, Rasht, Iran.

2 Department of Natural Resources, Faculty of Sciences, University of Parma, Italy.

 

*Correspondence to: 

Najmeh Ranji, Department of Biology, Faculty of Sciences, Rasht Branch, Islamic Azad University, Rasht, Iran. P.O. Box:3516-41335.Tel: [phone]. Fax: [phone].

Email: [e-mail] 

 

ABSTRACT

Curcumin, a natural product of turmeric, is known for its antibacterial and antifungal properties. Candida albicans is a major fungal pathogen with high mortality rate, particularly in immunocompromised patients. This study aimed to evaluate the effect of curcumin encapsulated in nanoparticles (polymersomes) in combination with fluconazole on the expression of the MDRI gene in drug-resistant isolates of C. albicans. This descriptive cross-sectional study involved obtaining 50 clinical samples, from women with vulvovaginal infections at Al-Zahra hospital (Rasht, Iran). After identifying the strains, resistance to fluconazole was assessed using disc diffusion and broth dilution methods. Six fluconazole-resistant isolates of C. albicans were treated with ½ MIC fluconazole (control) alone and in combination with curcumin encapsulated in nanoparticles. After 24 hours, the two cell groups were cultured on Sabouraud dextrose agar (SDA) to estimate the cell death rate. The expression of the MDR1 gene was quantitatively investigated using the qRT-PCR method in treated and untreated isolates. Our finding indicated that combined therapy with ½ MIC fluconazole and curcumin encapsulated in nanoparticles (at a concentration of 400µg/ml) reduced fungal growth by up to 50% within during 24 hours. In treated cells, qRT-PCR analysis revealed a decrease in MDR1 gene expression compared to untreated cells. Curcumin appears to enhance the effectiveness of fluconazole in fluconazole-resistant isolates by reducing MDR1 gene expression.

Key words: Candida albicans; Curcumin; Nanoparticles; MDR1; qRT-PCR.

1. INTRODUCTION

Candida species, fungal infections, have become more prevalent worldwide over the past few decades. Consequently, C. albicans has emerged  as the primary cause of fungal infection in humans (Garcia-Gomes, Curvelo, Soares, & Ferreira-Pereira, 2012). Candidiasis,  a common fungal infection, poses significant health challenges for individuals with conditions such as HIV infection (Awanish Kumar et al., 2014), organ transplantation, and cancer chemotherapy (Garcia-Gomes et al., 2012). Despite the availability of numerous antifungal medications, the use  of azole and non-azole drugs  can lead to adverse  effects, including  liver damage, changes in oestrogen levels, allergic reactions, and the development of drug-resistant (Awanish Kumar et al., 2014).Azole resistance in C. albicans arises from various molecular mechanisms, such as alterations  in ergosterol biosynthesis pathways, overexpression of genes  involved in efflux pump systems like CDR1, CDR2, and MDR1, and overexpression or mutation of  ERG11 genes (Paul et al., 2020). Evidence suggests that reduced  intracellular drug accumulation,  associated with the upregulation of genes like MDR and CDR,  plays a crucial  role in azole resistance in C. albicans (Zhang et al., 2019). 

Due to the side effects of antifungal drugs and the increasing,  resistance to these medications, the use of natural herbal drugs with  inhibitory effect has generated the attention of researchers as the third generation of treatments (Awanish Kumar et al., 2014). Given that curcumin, the primary and active ingredient in turmeric, possesses anti-inflammatory, anti-tumor, anti-fungal, and antibacterial properties, it has emerged as a significant herbal components (Awanish Kumar et al., 2014; Rahbar Takrami, Ranji, & Sadeghizadeh, 2019). 

There is mounting evidence supporting the hypothesis that curcumin, when   combined with antibacterial agents,  can have a significant impact on microorganisms (Khalil et al., 2012). Although the role of curcumin in the diets of many  countries and its antifungal properties have been recognized since ancient times  (Ranji, 2014; Sharma et al., 2011), its poor solubility in aqueous environments results in  low bioavailability  in both culture environments and in vivo studies (Ranji, 2014). In recent years, there has been widespread interest in using carriers, such as liposome, micelle, chitosan, nano capsule and polymersome to deliver curcumin into cells. Studies have shown that curcumin loaded in polymersome nanoparticles exhibits greater release and efficacy on target cells compared to  curcumin alone (Pakizehkar, Ranji, Sohi, & Sadeghizadeh, 2020). Given the increasing resistance of C. albicans isolates to azoles, particularly fluconazole, it is imperative to explore approaches to mitigate this resistance. The objective of this research was to investigate the antifungal effects of polymersome nanoparticles containing curcumin in reducing MDR1 gene expression in fluconazole-resistant C. albicans isolates.

 

2. MATERIALS AND METHODS

2.1. Collection of C. albicans isolates

This study was conducted at Al-Zahra Hospital in Rasht, Iran.  Screening for the presence of C. albicans was performed on samples of vaginal and cervical secretions from 50 female patients. After  a 24-hour incubation period at 37°C, yeast-like colonies on Sabouraud Dextrose Agar (SDA) were evaluated  using colony morphology, germ-tube tests, and CHROMagar Candida (Charles, Kali, & Joseph, 2015).

2.2. Disk diffusion testing

Disk diffusion testing was conducted  strictly according to CLSI standard M44-A2 (Balouiri, Sadiki, & Ibnsouda, 2016). Paper disks containing 10 mcg of fluconazole were obtained from HiMedia, India. Following 24 hours incubation period at 37°C, the zone diameter was measured, and fluconazole-resistant isolates were identified and selected.

2.3. Determination of minimum inhibitory concentrations

In accordance with CLSI M27A2 guidelines, the MIC test for fluconazole was conducted using Sabouraud Dextrose Broth (SDB). C. albicans isolates were cultured in SDB in a 96-well microplate with fluconazole concentrations ranging from 2048 µg/ml to 4 µg/ml for a duration of 48 hours at 37°C.

2.4. Preparation of curcumin entrapped in polymersome nanoparticles

Polymersome nanoparticles, comprising a blend of oleic acid (OA) and polyethylene glycol (PEG400) were procured from Nano Daro Alborz Company (Tehran, Iran). Curcumin (E, E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)-1,6-heptadiene3,5-dione) (Chemical formula: C21H20O6) with a purity of ≥94% (MW: 368,38 g/mol) was obtained from Sigma-Aldrich (Germany). Triethylamine (Billerica, USA) and chloroform (Sigma Aldrich, America) served as solvents during the four-hour synthesis of oleoyl chloride (Sigma Aldrich, USA) and polyethylene glycol (Sigma Aldrich, USA), with the reaction conducted at 24°C. Subsequently, the organic phase underwent filtration to eliminate trimethylaniline hydrochloride, and a vacuum device was utilized to evaporate chloroform at 40°C. 

The chemical structure of the polymersome nanoparticles was analyzed using FTIR. Next, curcumin and polymersome nanoparticles (PEG400-OA) were mixed (Pakizehkar et al., 2020). The efficiency of curcumin entrapment in nanoparticles and the size of the curcumin entrapped in polymersome nanoparticles (Cur: PEG400-OA) were assessed through various physico-chemical analysis. The final product was stored in a light-protected environment at 4°C.

2.5 Treatment of fluconazole-resistant C. albicans isolates with Cur: PEG400-OA

To investigate the antifungal effect of Cur: PEG400-OA, 60 μl of microbial suspension containing C. albicans isolates (1.5 x 108 cfu/ml) was added to the wells, along with ½ MIC fluconazole and varying concentrations of Cur: PEG400-OA (0, 100, 200, and 400 μg/ml). The plates were then incubated at 37°C for 24 hours. After incubation, 20 microliters from each treated sample were cultured on Sabouraud Dextrose Agar (SDA) to determine the Minimum Fungicidal Concentration (MFC). Each test was carried out at least twice (Alalwan et al., 2017).

 

2.6. Quantitative analysis of MDR1 gene

The transcription levels of the MDR1 gene were assessed via qRT-PCR in C. albicans isolates treated with 400 μg/ml of Cur: PEG400-OA and ½ MIC fluconazole, with the control group receiving only ½ MIC fluconazole. Isolate RNA from both study and control groups was extracted using the RNX-PlusTM kit (Cinaclon, Tehran, Iran). Subsequently, cDNA was synthesized using the cDNA Synthesis Kit (Yekta Tajhiz Azma, Iran). Quantitative RT-PCR was performed with SYBR®Premix Ex Taq™ II (TaKaRa, Japan) on the Rotor-Gene Q instrument (QIAGEN). The PCR protocol involved an initial denaturation step of three minutes at 95°C, followed by forty cycles of denaturation for five seconds at 95°C, annealing for thirty seconds at 60°C, and extension for five seconds at 75°C.The ACT1 (Actin) gene served as an internal endogenous control. The primers were in the investigation are listed in Table 1. Each reaction was conducted in triplicate, and the ΔΔCT method was employed for outcome analysis. The fold change for each was computed using the formula 2−ΔΔCT.

 

Table. 1 Sequence of primers used for quantitative RT-PCR

Primer name	Primer sequence	Length of production	Reference
MDR1-F	5'-TCAGTCCGATGTCAGAAAATGC-3'	81 bp	(Alalwan et al., 2017)
MDR1-R	5'-GCAGTGGGAATTTGTAGTATGACAA-3'
ACT1-F	5'-GCTTTTGGTGTTTGACGAGTTTCT-3'	72 bp	(Alalwan et al., 2017)
ACT1-R	5'-GTGAGCCGGGAAATCTGTATAGTC-3'

 

2.7. Statistical analysis

The significance differences between the groups were examined using the student’s t-test, one-way, and two-way ANOVA. A 0.05 P-value was employed to ascertain statistical significance. The mean ± SD was used to express the results.

 

3. RESULTS

3.1. Physico-chemical properties of Cur: PEG400-OA

In our previous investigation, the ratio of curcumin in Cur: PEG400-OA  solvent (PBS) was reported to be 1 mg/ml (Pakizehkar et al., 2020). Furthermore, it was discovered that curcumin loaded into nanoparticles at a density of approximately 16% with an encapsulation efficiency in Cur: PEG400-OA reaching approximately 97%. The average size of Cur: PEG400-OA was determined to be nearly 259 nm based on dynamic light scattering (DLS) results, while  it measured  close to 300 nm in the scanning electron microscope (SEM) analysis (Pakizehkar et al., 2020).

 

 

3.2. Anti-fungal properties of Cur: PEG400-OA

The morphology and color of the colonies on CHROMagar Candida were utilized for the identification of C. albicans. The results indicated that among the 23 isolates in the disc diffusion studies, 20 were resistant to fluconazole, one was sensitive, and two were semi-sensitive (Fig.1. A). Moreover, 50% of the isolates exhibited the highest MIC (2048 µg/ml), indicating a significant level of fluconazole resistance among the isolates (Fig.1. B).

 

 

Figure.1. A. Resistance of C. albicans isolates to fluconazol via disc diffusion method. B. MIC of fluconazole in isolates

 

It was determined that the lethal dose to 50% (LD50) of Cur: PEG400-OA in combination with ½ MIC fluconazole was 400 µg/mL. The isolates were cultured on Sabouraud Dextrose Agar (SDA) to validate the MIC results. Based on the Minimum Fungicidal Concentration (MFC) results, 50% of the cells can be annihilated by 400 µg/ml of Cur: PEG400-OA (Fig.2).

 

 

Figure. 2. MFC colony figures in C. albicans isolate number 7 treated with ½ MIC fluconazole (F) and ½ MIC fluconazole combined with 400 µg/ml of Cur: PEG400-OA (F+N)

 

3.3. Reduction of MDR1 gene expression after treatment with Cur: PEG400-OA

The findings revealed that, compared to the control group (treated with ½ MIC fluconazole alone), the MDR1 gene expression level in the three isolates under study, which were treated with 400 µg/ml of Cur: PEG400-OA in combination with ½ MIC fluconazole, confirmed a reduction in MDR1 gene expression in all three isolates (Fig. 3).

 

Figure. 3. Quantitative expression of MDR1 gene in C. albicans isolate treated with 1400 µg/ml of Cur: PEG400-OA in combination with ½MIC fluconazole compared to the control (½MIC fluconazole). The results are represented as mean±SD. Significant difference between treat and control was calculated based on *P>0.05, **P<0.01, and ***P<0.001.

4. DISCUSSION 

Researchers often regard  curcumin as a potential  new adjunctive  medication for  treating  various  diseases due to  its  broad-spectrum qualities and safety profile even  at high doses (Rahbar Takrami et al., 2019). In this study, it was found that after 24 hours, Cur: PEG400-OA (400 µg/ml) combined with ½ MIC of fluconazole inhibited the growth of isolates. Moreover, the qRT-PCR method confirmed downregulation of the MDR1 gene compared to the control group. Approximately 90% of C. albicans cases are attributed to Candida vaginitis, which constitutes one-third of vulvovaginitis cases and is experienced by at least 70% of women at some point in their lives (Baldesi et al., 2017; Ishikane, Hayakawa, Kutsuna, Takeshita, & Ohmagari, 2016). The prevalence of recurrent Vulvovaginal Candidiasis (VVC) in women is approximately 8% (Jeanmonod, Jeanmonod, Christopherson, & Spivey, 2019). Various researchers are currently exploring different methods of treating infections, including the use of antimicrobial herbal compounds as supplements or alternatives to traditional treatments to expedite patient recovery and reduce hospital mortality rates (Hu, Zhang, Kong, Zhao, & Yang, 2017; Sadiq et al., 2016; Vinodkumar, Nakkeeran, & Renukadevi, 2017). Additionally, due to the increase in resistance to opportunistic infections and their high prevalence in hospitals over the past few decades, researchers are seeking alternative approaches to their treatment (Motahhary Tashi & Ranji, 2017; Rahbar Takrami et al., 2019; Ranji & Rahbar Takrami, 2017).

Several studies have shown the curcumin, as an antifungal compound, can downregulation genes have involved in the synthesis of the cell wall of C. albicans (Rajasekar et al., 2021; Zhou et al., 2024). Kali et al. identified  a clear relationship between curcumin and antibiotics in  several biofilm-producing bacterial isolates (Kali, Bhuvaneshwar, Charles, & Seetha, 2016). Numerous  studies (Bezerra, y Araújo, Alves-Júnior, Damasceno, & Oshiro-Junior, 2024; Cheraghipour et al., 2021; Garcia-Gomes et al., 2012; Hajifathali, Lesan, Lotfali, Salimi-Sabour, & Khatibi, 2023; Tsopmene et al., 2024) have demonstrated the antifungal effect of curcumin (at concentrations ranging from 10 to650 µg/ml) alone or in combination with antifungal agents such as MCD (α-methyl cinnamaldehyde) (Narayanan et al., 2020), caspofungina (Alalwan et al., 2017), and fluconazole (Lee et al., 2022) against  C. albicans. In a previous study by Soliman et al., extracts of Asparagus tenuifolius, toothbrush tree (Salvadora persica), henna leaf (Lawsonia inermis), and purslane (Portulaca oleracea) at concentrations between 25 and 100 µm/ml exhibited  anti-Candida effects after 24 hours using  the disc diffusion method (Soliman et al., 2017). Furthermore, Garcia-Gomes et al. discovered that curcumin had a remarkable ability to prevent a C. albicans isolate from developing resistance to fluconazole. At 11 μM, it was able to restore   sensitivity to this azole. Notably,  an efflux pump may be involved in  contributing to the  isolate's resistance to fluconazole (Garcia-Gomes et al., 2012). In the present study, the anti-fungal property of Cur: PEG400-OA was confirmed    at 400 µg/ml within 24 hours, a concentration lower than that required in the study by Garcia-Gomes et al.

The general mode of action of azole antifungals involves inhibiting  lanosterol 14-alpha-demethylase, which converts  lanosterol in fungal cell membranes into ergosterol (Monk & Keniya, 2021). MDR1 is typically expressed at low levels, but a significant proportion of fluconazole-resistant in C. albicans clinical isolates permanently overexpress MDR1. Additionally, it has been demonstrated that MDR1 overexpression plays a crucial role in the isolate's resistance; deletion of the MDR1 gene  from overexpressing C. albicans strains results in a decrease in the mutants' resistance to fluconazole (Bergin et al., 2023). In the study by  Khosravi et al., overexpression of MDR1 and CDR2 genes was observed in 75% of C. albicans isolates (Rad, Falahati, Roudbary, Farahyar, & Nami, 2016).Chen et al. observed  that all  fluconazole-resistant isolates in their study exhibited  elevated expression of CDR1 and CDR2, and some of these isolates also showed  increased expression of MDR1 and ERG11 (Chen et al., 2010). In the present study, a reduction in MDR1 gene expression was observed in several fluconazole-resistant C. albicans isolates. It appears that curcumin, with its inhibitory effect on the expression of this gene, reduces the presence of this transporter of the efflux pump system on the cell surface, leading to less fluconazole being pumped out of the cell. This reduction in the release of fluconazole results in an increase in the drug's cytoplasmic concentration and a decrease in the amount that affects the cell. Considering that various factors play a crucial role in the development of drug resistance, and resistance can vary among different fungal strains due to various mutations or the acquisition of new genes, the limitations of this research include the variability in the response of each strain to the drug at specific dose, which can affect the expression of genes involved in drug resistance.

 

5. CONCLUSION

According to this study, curcumin trapped in polymersome nanoparticles exhibits a synergistic effect with fluconazole on C. albicans isolates. Additionally, reducing the expression of the MDR1 gene allows more fluconazole to be taken up and retained in the cell, while decreasing the efflux of the drug from the cell. Consequently, this enhances the antifungal drug's efficacy in inducing cell death. 

 

ACKNOWLEDGEMENTS

The authors of this article would like to be grateful to Islamic Azad University, Rasht, for funding the research based on research grant number 19280/1/16/17.

 

CONFLICT OF INTEREST

The authors declare no conflict of interest.

 

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