Elsevier

European Journal of Pharmacology

Volume 784, 5 August 2016, Pages 121-128
European Journal of Pharmacology

Molecular and cellular pharmacology
Loading of Gemcitabine on chitosan magnetic nanoparticles increases the anti-cancer efficacy of the drug

https://doi.org/10.1016/j.ejphar.2016.05.016Get rights and content

Abstract

Targeted delivery of anti-cancer drugs increase the efficacy, while decreasing adverse effects. Among various delivery systems, chitosan coated iron oxide nanoparticles (CsMNPs) gained attention with their biocompatibility, biodegradability, low toxicity and targetability under magnetic field. This study aimed to increase the cellular uptake and efficacy of Gemcitabine.

CsMNPs were synthesized by in situ co-precipitation and Gemcitabine was loaded onto the nanoparticles. Nanoparticle characterization was performed by TEM, FTIR, XPS, and zeta potential. Gemcitabine release and stability was analyzed. The cellular uptake was shown. Cytotoxicity of free-Gemcitabine and Gem-CsMNPs were examined on SKBR and MCF-7 breast cancer cells by XTT assay.

Gemcitabine loading was optimized as 30 µM by spectrophotometric analyses. Drug release was highest (65%) at pH 4.2, while it was 8% at pH 7.2. This is a desired release characteristic since pH of tumor-tissue and endosomes are acidic, while the blood-stream and healthy-tissues are neutral. Peaks reflecting the presence of Gemcitabine were observed in FTIR and XPS. At neutral pH, zeta potential increased after Gemcitabine loading. TEM images displayed, Gem-CsMNPs were 4 nm with uniform size-distribution and have spherical shape. The cellular uptake and targetability of CsMNPs was studied on MCF-7 breast cancer cell lines. IC50 value of Gem-CsMNPs was 1.4 fold and 2.6 fold lower than free-Gem on SKBR-3 and MCF-7 cell lines respectively, indicating the increased efficacy of Gemcitabine when loaded onto nanoparticles.

Targetability by magnetic field, stability, size distribution, cellular uptake and toxicity characteristics of CsMNPs in this study provides a useful targeted delivery system for Gemcitabine in cancer therapy.

Introduction

Breast cancer is a prevalent cancer for women in the vast majority of countries worldwide (Bray et al., 2013). Despite the advances in treatments, the overall survival rate has not been improved substantially (Jemal et al., 2010). Conventional chemotherapeutic agents are unspecifically distributed all over the body where they affect both cancerous and normal cells. This treatment results in excessive toxicities. There is a need to develop novel approaches for therapies based on targeting of cancer cells. Recent advances in nanotechnology have explored new targeting strategies for enhancing intra-tumoral drug concentrations while limiting the systemic toxicity and side effects (Maeda, 2001).

Antitumor activity of Gemcitabine (2′,2′-difluorodeoxycytidine), a nucleoside analogue, has been reported in a variety of human tumors, including breast cancer in both experimental and clinical studies (Reddy and Couvreur, 2008). Gemcitabine inhibits DNA synthesis by incorporating into DNA. With its incorporation, DNA polymerase cannot add nucleotides leading to the termination of chain elongation, and induces apoptosis (Toschi et al., 2005). However, Gemcitabine could cause major systemic toxicities and drug resistance, which also restricts its therapeutic efficacy (Dasanu, 2008). The plasma level of this drug can quickly drop below the effective threshold level due to the short biological half-life (8–17 min) and its clinical benefit becomes limited. Thus, much larger doses are required to reach the effective plasma concentrations, increasing the risk of side effects. In addition, Gemcitabine is hydrophilic and it could not pass the plasma membrane by passive transportation (Lilly and Company, 1997) Therefore, it must be transported into the cells by nucleoside transporters, such as the human Equilibrative Nucleoside Transporters (hENT) (Bildstein et al., 2010, Chitkara et al., 2013). Cancer patients with tumors conveying lower hENT1 expression, have a considerably lower survival rate following Gemcitabine therapy as compared to patients with tumors that has a higher hENT1 expression. Moreover, many patients fail to benefit from the treatment due to the lack of the receptor (Farrell et al., 2009).

Few studies in the literature have been carried out for the development of Gemcitabine delivery system using nanoparticles which could reduce its side effects, increase internalization of the drug without receptor mediation and prolong its retention time (Arya et al., 2011, Hosseinzadeh et al., 2012, Garg et al., 2012, Arias et al., 2011). Chitosan has gained considerable attention due to its biocompatibility, biodegradability, and non-toxicity. Chitosan based delivery systems are widely used for the controlled delivery of drugs, proteins, and peptides (Braz et al., 2011, Rodrigues et al., 2012, Yongmei and Yumin, 2003). Chitosan coated magnetic nanoparticles were previously synthesized and characterized by our group (Unsoy et al., 2012, Unsoy et al., 2014a, Unsoy et al., 2014b). These nanocarriers, containing a magnetite (Fe3O4, iron oxide) core, could be actively targeted to the tumor site by an externally applied magnetic field after loaded with anti-cancer drugs. This is the main advantage of MNPs. Another important utility of MNPs is their role in tumor visualization as MRI agents, which is also approved by Food and Drug Administration in clinical use (Gao et al., 2009, Wilczewska et al., 2012).

Section snippets

Material and methods

In this study, chitosan coated magnetic nanoparticles (CsMNPs) were loaded with Gemcitabine for tumor targeting. Characterization of the drug loaded nanoparticles was performed by Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and zeta potential analyses. Drug loading and release characteristics of the nanoparticles were investigated. The cellular uptake and targetability characteristics of CsMNPs were studied on

Gemcitabine loading onto CsMNPs

Gemcitabine loading was achieved as 16, 22, 30 µM with different initial drug concentrations 7.5, 15, 22.5 µg/ml, respectively (Fig. 1). The highest loading efficiency was obtained as 39% (30 µM) with the highest Gemcitabine concentration (22.5 µg/ml) and all the experiments continued with this preparation.

Release of Gemcitabine from nanoparticles

Gemcitabine release studies showed that CsMNPs have a pH dependent release pattern. Chitosan is soluble in diluted acids with a pH lower than pKa (about 6.3) (Mucha, 1997). At low pH, the free

Discussion

Easy synthesis, chemical stability in physiological conditions, possibility of coating by polymeric shells and loading with various agents make iron oxide (Fe3O4, magnetite) nanoparticles favorable for biomedical use (Sun et al., 2008, Nune et al., 2009). Chitosan coated magnetic nanoparticles (CsMNPs) were synthesized for targeting drugs to the tumor cells in the presence of magnetic field. By this way the drug concentration may be increased in a specific target tissue compared with the rest

Conclusion

Targeted drug delivery prevents adverse off-target effects, drug toxicities, and unnecessary systemic immunosuppression, while providing increased therapeutic efficacy. Moreover, tissue specific delivery may also increase the benefit of some drugs whose their use have been eliminated due to low bioavailability in affected tissues or prohibitively high toxicities in the rest of the body. Therefore, targeted drug delivery may solve many concerns.

In this study, synthesized chitosan magnetic

Conflict of interest

The authors of this study declare that they have no conflict of interest.

References (38)

  • G. Unsoy et al.

    Chitosan magnetic nanoparticles for pH responsive Bortezomib release in cancer therapy

    Biomed. Pharm.

    (2014)
  • M. Vandana et al.

    Long circulation and cytotoxicity of PEGylated gemcitabine and its potential for the treatment of pancreatic cancer

    Biomaterials

    (2010)
  • A.Z. Wilczewska et al.

    Nanoparticles as drug delivery systems

    Pharmacol. Rep.

    (2012)
  • F. Andrade et al.

    Chitosan formulations as carriers for therapeutic proteins

    Curr. Drug Discov.

    (2011)
  • J.L. Arias et al.

    Superior preclinical efficacy of gemcitabine developed as chitosan nanoparticulate system

    Biomacromolecules

    (2011)
  • F. Bray et al.

    Global estimates of cancer prevalence for 27 sites in the adult population in 2008

    Int. J. Cancer

    (2013)
  • L. Braz et al.

    Mechanisms of chemical and enzymatic chitosan biodegradability and its application on drug delivery

  • C. Celia et al.

    Gemcitabine-loaded innovative nanocarriers vs GEMZAR: Biodistribution, pharmacokinetic features and in vivo antitumor activity

    Expert Opin. Drug Deliv.

    (2011)
  • D. Chitkara et al.

    Self-assembling, amphiphilic polymer-gemcitabine conjugate shows enhanced antitumor efficacy against human pancreatic adenocarcinoma

    Bioconjug. Chem.

    (2013)
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