Folate receptor alpha targeted delivery of artemether to breast cancer cells with folate-decorated human serum albumin nanoparticles

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Abstract

The pharmaceutical application of artemether (ARM) as an anticancer natural agent is hampered due to its poor solubility and bioavailability. In the present study, ARM was encapsulated in human serum albumin nanoparticles (HSA NPs) via desolvation method led to improvement of the water solubility by 50 folds. In further, folate-decorated ARM-HSA NPs (F-ARM-HSA NPs) were developed to enhance targeted delivery to folate receptor alpha (FRα)-overexpressing breast cancer cells. The hydrodynamic diameter and the zeta potential value of F-ARM-HSA NPs were 198 ± 11.22 nm and −23 ± 0.88 mV, respectively. Fluorescent microscopy demonstrated an enhanced cellular uptake of F-ARM-HSA NPs by high FRα-expressing MDA-MB-231 breast cancer cells compared to low FRα-expressing SK-BR-3 breast cancer cells. Cytotoxicity assay revealed a small significant difference between cytotoxicity effect of targeted and non-targeted NPs in SK-BR-3 cells. However, in MDA-MB-231 cells due to FRα-mediated endocytosis, the F-conjugated NPs had less inhibitory concentration (IC50) value (19.82 μg/mL) and higher cytotoxicity after 72 h compared to non-targeted ARM-HSA NPs. Flow cytometry analysis indicated a more potent drug-induced apoptosis rather than necrosis. The results suggest that our novel F-ARM-HSA NPs are likely to be recommended as a promising candidate for combination therapy of FRα-overexpressing breast cancer cells.

Introduction

Despite efforts in the field of cancer therapy, cancer still is the leading cause of deaths among non-communicable diseases and the single most important threat to life expectancy worldwide. According to the International Agency for Research on Cancer (IARC) report in 2018, 18.1 million new cases and 9.6 million cancer deaths are estimated annually. Among these, breast cancer is the first primary cause of death among women suffering from cancer [1]. Chemotherapy as a current treatment for cancer, due to its non-selective actions, requires higher doses and consequently develops severe toxicity to normal tissues and also leads to drug resistance [2]. Therefore, researchers have always sought treatment approaches with appropriate efficacy and specific function that induce less toxicity to the normal tissues. The drug with these specifications might be used along with chemical drugs and other treatments to reduce the possible complications; this approach nowadays is known as combination therapy [3].

Within the available alternative therapies, naturally occurring drugs such as herbal derivatives have always been of interest to the researchers because of their low toxicity [4]. Artemisinin and its semi-synthetic derivatives such as artemether (ARM) are anti-malarial compounds containing endoperoxidase moiety which has been shown to have anti-cancer cytotoxic effects in recent years. ARM is a methyl ether derivative of artemisinin a compound derived from Artemisa annua L. plant. Although artemisinin has been identified as a first-line treatment against Plasmodium falciparum malaria, but since 1990 several papers have reported the anti-cancer potency of artemisinin derivatives [4,5]. In vitro and in vivo studies have shown that ARM not only induces apoptosis as its main mechanism of action [[5], [6], [7]], but also inhibits proliferation, migration, and invasion of cancer cells and enhances chemotherapy or radiotherapy sensitization [7,8].

Despite the aforementioned advantages, ARM has some pharmacological drawbacks that have limited its uses in clinical application. Due to ARM properties such as low solubility in water and oil, low bioavailability, and short half-life in vivo, a drug delivery system formulation is needed to alleviate these obstacles [9,10]. Among different drug delivery systems, protein-based NPs have been extensively studied as a drug delivery system owing to their advantages such as low toxicity, high biodegradability, and high absorbability in comparison to the industrial polymers. Albumin is the most abundant plasma protein (MW = 66,500 Da) with a half-life of 19 days [11]. Due to its characteristics such as non-toxicity, non-immunogenicity, high availability, and degradability albumin has a potential to be used as a proper candidate for drug delivery. Accordingly, in this study human serum albumin (HSA) was used for encapsulation of ARM to enhance the solubility and obtaining other advantages of albumin nano-carrier. Furthermore, due to the structural specificities of the tumor microenvironment and high demand of tumor cells for albumin as a nutrition source, nano-carriers such as albumin NPs can accumulate at the tumor site via enhanced permeability and retention (EPR) effect leading to passive tumor targeting [12]. Some in vivo and in vitro studies have previously used HSA to encapsulate ARM by preparing nano-formulations via the desolvation method in which they have succeeded to achieve nanoparticles with enhanced solubility property and thereby increased drug efficacy [13,14].

Nevertheless, passive tumor targeting drug delivery is not strong enough to achieve an ideal delivery to the tumor site; therefore, an active targeting is needed to enhance the specificity and effectiveness of the drug delivery [15]. Consequently, conjugation of NPs for active targeting along with the intrinsic passive targeting capacity of the albumin can improve the specificity and efficacy of drug delivery to tumor tissues. The presence of amine (single bondNH2) and carboxyl (single bondCOOH) groups on the albumin enables the surface modification opportunity to manipulate the albumin NPs for advanced tumor targeting which leads to reduce drug side effects significantly [15].

Among the available ligands, folic acid (FA) (MW = 441 Da) is more desirable for selective targeting of folate receptor alpha (FRα)-expressing cancer cells [16]. FRα is a membrane bound protein abundantly expressed on the surface of many human tumor cells such as breast cancer cells attributing to the high folate requirement, whereas its expression is restricted in normal cells. Highly aggressive and metastatic triple negative breast cancer cells are particularly enriched with the FRα expression on their membrane surface to the extent that knockdown of FRα inhibits proliferation of triple negative cell lines [17]. Therefore, the overexpression of FRα on the surface of these cells such as MDA-MB-231 could be considered as an important target for inhibiting the growth of triple negative breast cancer cells [18].

In this article, we have taken advantage of the HSA NPs as a solid colloidal drug delivery system to enhance the efficiency of the anti-cancer properties of ARM and reduce the aforementioned problems. It should be noted that HSA maintains the ARM from outside stress, until it reaches the tumor target cells, as well as extends the ARM half-life span. Hence, ARM-HSA NPs were prepared by the desolvation technique and then in order to target the drug to the FRα-expressing tumor cells, FA was covalently attached to ARM-HSA NPs using NHS/EDC coupling agents. Our prepared NPs formulation was assessed by morphology, particle size, zeta potential, and drug release analyses. Furthermore, the cytotoxic activity and the anti-cancer efficacy of the developed NPs were evaluated. The cellular drug uptake by high FRα-expressing cancer cells such as MDA-MB-231 has been also compared to the low FRα-expressing cancer cells (SK-BR-3) [19]. We have succeeded to achieve ARM-HSA nanoparticles with 50 folds greater solubility and <200 nm in diameter. The effects of ARM and its newly designed HSA NPs formulation on human breast cancer cells especially on the drug resistant cell line have not been previously reported.

Section snippets

Materials

The artemether powder (CAS no: 71963-77-4, >98.0% pure) was kindly donated as a gift by Dr. Zuhair Hassan, Tarbiat Modares University (Sigma -Aldrich, USA). N–hydroxysulfosuccinimide (NHS, >98.0% pure), 1-ethyl3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, >98.0% pure), 3-(4,5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT, >98.0% pure), 2-mercaptoethanol solution (2-ME, >97.5% pure), Human Serum Albumin (HSA, >98.0% pure), Folic Acid (FA, >97.0% pure), and

Preparation and characterization of ARM-HSA and F-ARM HSA nanoparticles

In the present study, ARM-HSA NPs were successfully prepared using desolvation method. Ethanol was used as desolvating agent due to its ability to reduce dissolution of HSA and simplifies the formation of NPs [25]. In order to conjugate the FA to the HSA nanoparticles, NHS/EDC coupling agents were used to activate carboxylic groups of FA by creating reactive folate NHS ester (FA-NHS) [16,29]. Then, amide bond was formed between carboxylic group of FA and amine group of albumin in the

Conclusion

In this study to enhance the anti-cancer potency of ARM, HSA-based NPs were prepared by the desolvation method. In order to achieve active targeting, folate-decorated ARM-HSA NPs were created by chemical conjugation of FA on the surface of ARM-HSA NPs. The results show that the prepared NPs have a good biocompatibility and suitable physiochemical features. The mean particle size of ARM-HSA NPs and F-ARM-HSA NPs was not >200 nm (PDI < 0.182) and the average of DL and EE of the ARM-HSA NPs were

CRediT authorship contribution statement

Asiye Akbarian: Writing - original draft, Software, Validation, Investigation, Formal analysis, Software. Masoumeh Ebtekar: Conceptualization, Methodology, Writing - review & editing, Investigation, Project administration, Visualization. Nafiseh Pakravan: Methodology, Investigation, Writing - review & editing, Visualization. Zuhair Mohammad Hassan: Investigation, Visualization, Writing - review & editing, Resources, Visualization.

Acknowledgment

The authors would like to acknowledge Zahra Hasanpor for her kind collaboration in this project and also Zahra Matloobi and Ardeshir Abbasi for their excellent technical assistance. This project was financially supported by Tarbiat Modares University (TMU).

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