Research paper
Reversible Targeting and controlled release delivery of daunorubicin to cancer cells by aptamer-wrapped carbon nanotubes

https://doi.org/10.1016/j.ejpb.2010.12.005Get rights and content

Abstract

Aim

Single-walled carbon nanotubes (SWNTs) have been already used as drug carriers. In this study, we introduced sgc8c aptamer (this aptamer targets leukemia biomarker protein tyrosine kinase-7) to complex between Dau (daunorubicin) and SWNT to enhance targeted delivery of Dau to acute lymphoblastic leukemia T-cells (Molt-4).

Material and methods

Dau-aptamer-SWNTs tertiary complex formation was analyzed by visible spectroscopy and spectrofluorophotometric analysis. Dau release profiles from the complex were investigated in pH 7.4 and 5.5. For cytotoxic studies (MTT assay), Molt-4 (target) and U266 (B lymphocyte human myeloma, non-target) cells were treated with Dau, Dau-aptamer-SWNTs tertiary complex. Internalization was analyzed by flow cytometry. Targeted delivery of Dau was antagonized using antisense of aptamer.

Results

Dau was efficiently loaded onto SWNTs (efficiency ∼157%). Dau was released from Dau-aptamer-SWNTs tertiary complex in a pH-dependent manner (higher release rate at pH 5.5). Flow cytometric analysis showed that the tertiary complex was internalized effectively to Molt-4 cells, but not to U266 cells. Cytotoxicity of Dau-aptamer-SWNTs tertiary complex also confirmed internalization data. Dau-aptamer-SWNTs tertiary complex was less cytotoxic in U266 cells when compared to Dau alone. No significant change in viability between Dau- and complex-treated Molt-4 cells was observed. Cytotoxicity of Dau-aptamer-SWNTs complex was efficiently and quickly reversed using antisense in Molt-4 cells.

Conclusion

Dau-aptamer-SWNTs complex is able to selectively target Molt-4 cells. The other advantages of this system are reversibility and pH-dependant release of Dau from its complex.

Graphical abstract

Effects of Dau and Dau-aptamer-SWNTs tertiary complex on control and target cells viability (MTT assay). Cells were treated with aptamer-SWNTs, Dau, and Dau-aptamer-SWNTs tertiary complex for 3 h. After 72 h post-treatment viability of the cells was assessed using MTT technique. * Indicates there is a significant difference between viability of Molt-4 and U266 cells after treatment with Dau-aptamer-SWNTs tertiary complex (p < 0.005).

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Introduction

Leukemia is an uncontrolled proliferation of blood cells and bone marrow, leading to death in many cases [1]. Acute lymphoblastic leukemia (ALL) is ranked among the most diagnosed malignancy in children [2].

Daunorubicin (Dau) is classified as anthracycline antitumor antibiotics. Dau is one of the most widely used chemotherapy agents in treatment of leukemia [3], [4], [5]. Clinical administration of Dau is limited by cumulative cardiotoxicity and myelosuppression, aside from its conventional side effects such as nausea, vomiting, and alopecia [4], [5].

Tumor-specific delivery of anticancer drugs maximizes the efficacy of drugs and minimizes their off-target effects [6]. The aim of targeted delivery is to selectively accumulate drug in the target site [7], [8].

In recent years, aptamers have been used as molecular targeting agents for specific drug delivery [9], [10], [11]. Originally discovered in 1990 [12], aptamers are single-stranded DNA or RNA nanomaterials. Their size could vary from 20 to 80 nucleotides [13], [14]. Because of their three-dimensional structures, aptamers could selectively bind to a variety of targets ranging from small molecules to proteins and whole cells [12], [15], [16]. Aptamers are generated in an in vitro process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) [12]. Cell-SELEX has been recently developed to isolate cell-specific aptamers. So far, several aptamers have been generated by cell-SELEX for cancer cells such as liver cancer and small and non-small lung cancers [17]. No toxicity and immunogenicity has been reported for aptamers to date of preparation of this article. Beside selectivity and high affinity, aptamers are stable in heat and reduced condition. Moreover, aptamers can be chemically synthesized in large scale. Aptamers have potential applications in medicine such as therapeutic, diagnostic, or biosensor [15], [16], [18], [19]. Sgc8c aptamer (Fig. 1) targets leukemia biomarker protein tyrosine kinase-7 (PTK7) [10], [22], [23]. Sgc8c aptamer can recognize target leukemia cells (T-cell ALL) with high affinity (kd = 1 nM) among normal human bone marrow aspirates [10].

Discovered by Iijima in 1991, carbon nanotubes (CNTs) are molecular-scale tubes of graphitic carbon [24], [25], [26]. CNTs are classified structurally as single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) [24], [25]. Because of their unique physical and chemical properties, SWNTs have been the focus of interest in electronics, chemistry, biosensing, and drug delivery research projects [26], [27], [28], [29].

In this study, we investigated the possibility of targeted delivery of Dau to Molt-4 cells (T-cell line, ALL) using SWNTs and sgc8 aptamer.

Section snippets

Cell culture

Molt-4 (C149, T-cell line, human ALL) and U266 (C151, B lymphocyte, human myeloma) were purchased from Pasteur Institute of Iran and cultured in RPMI 1640 (Euroclone) supplemented with 10% fetal bovine serum (FBS, heat inactivated, Gibco) and 100 units/ml penicillin–streptomycin (Sigma). All experiments were performed in FBS-free media.

Functionalization of SWNTs with aptamer

Sgc8c aptamer, 5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3′, was synthesized using a Polygene DNA synthesizer (Polygene, Germany). Synthesized

Water soluble aptamer-SWNTs complex characterization

The SWNTs concentration in solution after functionalization with ssDNA was calculated to be 40 mg/l (≈11.5% of the starting nanotube suspension). The functionalized SWNTs are well dispersed. Both SWNTs and aptamer-SWNTs were characterized using TEM. The overall diameter of functionalized nanotubes are about 6 nm (Fig. 2b), which is greater than the diameter of SWNTs (about 4 nm, Fig. 2a). This increase is due to the wrapping of SWNT by aptamers, 1–2 nm [31], [32].

Dau loading onto functionalized SWNTs

Dau-aptamer-SWNTs tertiary complex

Discussion

Surgery, chemotherapy, radiotherapy, and immunotherapy are common methods of cancer treatment. Chemotherapy is still the treatment of choice when a cancer is metastasized. Anticancer agents are associated with severe side effects because of their poor specificity. Targeted drug delivery can effectively minimize the adverse effect of chemotherapy and enhance their therapeutic efficacy by specific delivery of anticancer drugs to tumor area [35], [36]. Targeted drug delivery system consists of a

Conclusion

Dau-aptamer-SWNTs tertiary complex delivery system is able to specifically deliver and internalize Dau to Molt-4 cells. Rate of Dau release in this system depends on pH of environment. In pH 5.5, rate of drug release increased about six folds. Moreover, application of antisense oligos against the aptamer can almost prevent delivery of Dau to Molt-4 cells. Therefore, this system can reduce cytotoxic effects of Dau by selective delivery and controllable release of this drug to tumor cells.

Conflict of interest

There is no conflict of interest about this article.

Acknowledgment

Financial support of this study was provided by Mashhad University of Medical Sciences.

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