Cancer Letters

Cancer Letters

Volume 400, 1 August 2017, Pages 1-8
Cancer Letters

Original Article
Chitosan-modified PLGA nanoparticles tagged with 5TR1 aptamer for in vivo tumor-targeted drug delivery

https://doi.org/10.1016/j.canlet.2017.04.008Get rights and content

Highlights

  • Targeted nanoparticle was synthesized by electrostatic attachment of 5TR1 aptamer to Epi-PLGA-CS.

  • In vitro study revealed a pH-depended release profile.

  • In vitro cytotoxicity demonstrated the MUC1 mediated endocytosis of Epi-PLGA-CS-Apt into MCF-7 cells (+MUC1).

  • In vivo experiment revealed the higher therapeutic index of Epi-PLGA-CS-Apt in comparison with free Epi and Epi-PLGA-CS.

Abstract

In this study, we reported epirubicin (Epi) encapsulated nanoparticles (NPs) formulated with biocompatible and biodegradable poly (lactic-co-glycolic acid) (PLGA) modified with chitosan (CS) through a physical adsorption method. Using chitosan, the solubility and surface charge of PLGA was modified to make efficient drug carriers for cancer cells. To improve the anti-tumor efficacy, we developed targeted therapy of tumor cells using a 5TR1 DNA aptamer (Apt) against the MUC1 receptor. To prove the MUC1 receptor-mediated uptake of Epi-PLGA-CS-Apt NPs in the cells, competition experiments were carried out.

In vitro experiments, cytotoxicity assay and fluorescence uptake assay demonstrated that fabricated NPs with or without aptamers showed significantly high therapeutic efficiency in MCF7 cells (breast cancer cell) compared with free Epi, while in BALB/c mice bearing C26 cells (murine colon carcinoma cell), targeted NP groups exhibited significant tumor growth inhibition and higher inclination to tumor compared with non-targeted NPs. Hence, our in vivo results revealed that non-targeted NPs may diffuse away from the tumor site and release Epi in the extracellular space and decrease concentration of the drug in the targeted tissue. This study indicated Epi-PLGA-CS-Apt has great potential as a promising nanoplatform for in vivo cancer therapy and could be of great value in medical use.

Introduction

Anthracyclines are the most effective anti-tumors among all classes of chemotherapeutic agents. Epirubicin (Epi) is a widely used anthracycline in breast cancer chemotherapy [1], [2]. Epi intercalates to DNA and inhibits topoisomerase II thereby leading to cell death [1], [3]. Even though chemotherapy is still the frontline treatment strategy for cancers, its side effects, including effects on normal cells and limited treatment duration or dosing, reduce clinical applications [4], [5]. Today, nanotechnology is paving the way to overcome the many barriers for efficient drug delivery [6]. A favorable approach to advance the selectivity and specificity of drugs for tumor cells is targeted drug delivery using nanoparticle functionalized with targeting ligands [4], [7], [8].

Choice of tumor marker and tumor-targeting ligand are important elements for targeted cancer therapy. Development of targeting ligands could reduce the off-target effects of drugs and cause them to be accumulated at the site of action [2]. The novel generation of targeting ligands is aptamers.

Aptamers are a class of nucleic acid-based ligands that fold into well-defined 3D structures to specifically bind to their targets [9], [10]. Low toxicity, slow degradation kinetics and notable stability in a wide range of pH, temperature and organic solvents without loss of activity make aptamers powerful candidates for pharmaceutical applications [11], [12], [13]. These ligands also play a critical role in the development of novel biosensors [14].

Overexpression of a specific maker on the cell surface is used to justify cell-specific targeted drug delivery [15]. Mucin 1 (MUC1) is a cell surface glycoprotein with extensive glycosylated extracellular domain [16]. Striking overexpression of the MUC1 receptor on the surface of tumor cells suggested that 5TR1 (Apt), anti-MUC1 aptamer would be suitable for tumor targeting [17], [18].

The development of targeted drug delivery systems with nanoparticles is expected to have a big impact on the clinical approaches in tumor chemotherapy [19], [20]. So far, a few aptamer-based targeted nanosystems have been introduced for treatment of breast and colon cancers. However, great attempts are being devoted to develop targeted delivery systems with better function and characteristic. One of these targeted delivery systems for treatment of breast and colon cancers has been introduced previously by our group. In this work, an aptamer-based dendrimer nanostructure was presented for targeted delivery of Epi to cancer cells [21]. The dendrimer structure was composed of several DNA building blocks. Although the developed delivery system showed good function in treatment of cancer cells in vitro and in vivo, the design of DNA origamis like this structure is complicated, and because of the presence of several DNA building blocks, they have a high cost. Thus here, we developed a targeted delivery system which did not have these shortcomings and has a simple design, low cost, more Epi loading and lower drug release in peripheral circulation.

Drug delivery systems using biodegradable and biocompatible nanoparticles, which have been approved by the Food and Drug Administration (FDA), like poly (lactic-co-glycolic acid) (PLGA), are of interest as they can be easily extended to clinical trials [5], [22], [23]. PLGA is a copolymer of poly lactic acid (PLA) and poly glycolic acid (PGA), and its breakdown products are hydrophilic, diffusible and rapidly metabolized in the human body [24]. Nevertheless, the short residence time of this nanosystem represents a major limitation of PLGA for achieving effective drug targeting to the site of action [6], [25]. Thus, to maximize the therapeutic effects of these carriers, they should bypass the phagocytic effects via surface coating of PLGA with hydrophilic polymers such as chitosan (CS) [6]. Chitosan is a biodegradable and biocompatible cationic polymer with relatively low immunogenicity. Because of its unique properties, chitosan has been widely used as a vehicle in drug delivery systems [26], [27], [28]. Also, the presence of primary amino groups on the chitosan polymer backbone could facilitate its attachment to other chemical reagents [24].

The present study is aimed at developing a targeted delivery system using modified PLGA nanoparticles with the chitosan and MUC1 aptamer. MCF7 and CHO cell lines were chosen to evaluate the effects of the designed delivery system in vitro. Consequently, using mice bearing C26 colon carcinoma, we assessed therapeutic efficacy of fabricated NPs with or without aptamers.

Section snippets

Materials

The MUC1 aptamer (5TR1) (GAAGTGAAAATGACAGAACACAACA) was obtained from Bioneer (South Korea). MCF7 (C135, breast cancer cell) and CHO (C111, Chinese hamster ovary cell) cell lines were purchased from Pasteur Institute of Iran, and C26 cells (murine colon carcinoma cell) were obtained from Cell Lines Service (Eppelheim, Germany) and cultured in RPMI 1640 and 10% fetal bovine serum (Gibco, Gaithersburg, USA). DNase RNase-free water was purchased from Sigma. Epirubicin (Epi), chitosan (CS, medium

Characterization of the nanoparticle

FTIR spectroscopy was used to demonstrate the presence of CS on the surface of PLGA. Fig. 1 shows the FTIR spectra of PLGA, CS and PLGA-CS. The spectra of PLGA (Fig. 1a) contained a strong peak at 1091 cm−1 for Csingle bondOsingle bondC stretching, at 1760 cm−1 for Cdouble bondO and around 3500 cm−1 for OH stretching. Also, the presence of peaks around the 1490 cm−1 could be assigned to the Csingle bondH stretching in methyl groups and between 2800 and 3000 cm−1 corresponding to CH, CH2 and CH3 stretching vibrations. A characteristic

Discussion

Even though chemotherapy is still the frontline treatment strategy for cancers, its side effects, including effects on normal cells and limited treatment duration or dosing, reduce clinical applications.

Previously, it was proved that in comparison with free Epi, liposomal formulation of Epi exhibited the lowest induction of body weight loss and also has a better survival rate output of mice [33].

However, while liposomal formulations of Epi are versatile, they cannot provide a sustained release

Conclusion

The present study developed a nanoparticulate drug delivery system functionalized with 5TR1 aptamer as the targeting ligand for anti-tumor drug delivery. The aptamer was electrostatically coupled to the surface of an Epi-PLGA-CS nanoparticle. The conjugation of aptamer was confirmed by increase in particle size and decrease in zeta potential of targeted NP.

As confirmed by a competition study in both cellular drug uptake and in vitro cytotoxicity, the 5TR1-MUC1 interaction facilitated the

Declaration of interest

All authors declare that they have no conflicts of interest. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Acknowledgements

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

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