Elsevier

Free Radical Biology and Medicine

Volume 77, December 2014, Pages 230-238
Free Radical Biology and Medicine

Original Contribution
PEGylated silver doped zinc oxide nanoparticles as novel photosensitizers for photodynamic therapy against Leishmania

https://doi.org/10.1016/j.freeradbiomed.2014.09.005Get rights and content

Highlights

  • Biocompatible newly synthesized daylight responsive PEGylated silver doped zinc oxide nanoparticles.

  • Production of measurable amount of reactive oxygen species causes the death of Leishmania tropica.

  • Singlet oxygen and hydroxyl radicals are the main ROS produced.

  • Nontoxic in dark and the daylight response make the current nanoparticles better therapeutic agents against Leishmania and also other infections.

Abstract

We describe daylight responsive silver (Ag) doped semiconductor nanoparticles of zinc oxide (DSNs) for photodynamic therapy (PDT) against Leishmania. The developed materials were characterized by X-ray diffraction analysis (XRD), Rutherford backscattering (RBS), diffused reflectance spectroscopy (DRS), and band-gap analysis. The Ag doped semiconductor nanoparticles of zinc oxide were PEGylated to enhance their biocompatibility. The DSNs demonstrated effective daylight response in the PDT of Leishmania protozoans, through the generation of reactive oxygen species (ROS) with a quantum yield of 0.13 by nondoped zinc oxide nanoparticles (NDSN) whereas 0.28 by DSNs. None of the nanoparticles have shown any antileishmanial activity in dark, confirming that only ROS produced in the daylight were involved in the killing of leishmanial cells. Furthermore, the synthesized nanoparticles were found biocompatible. Using reactive oxygen species scavengers, cell death was attributable mainly to 77–83% singlet oxygen and 18–27% hydroxyl radical. The nanoparticles caused permeability of the cell membrane, leading to the death of parasites. Further, the uptake of nanoparticles by Leishmania cells was confirmed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). We believe that these DSNs are widely applicable for the PDT of leishmaniasis, cancers, and other infections due to daylight response.

Introduction

Infectious agents can bring alterations in the biological system in the form of diseases. Some of these alterations can be life threatening and many others can cause disfiguring, disabling, and frequently stigmatizing disorders [1]. Leishmaniasis is one such disease that can either be fatal (visceral leishmaniasis) or may cause disfiguring of skin (cutaneous leishmaniasis) or face (mucocutaneous leishmaniasis). The World Health Organization has ranked this disease as the sixth most important infectious disease. Worldwide, new cases and deaths from leishmaniasis are 1.5–2 million and 70,000, respectively, and an estimated 0.35 billion people are at risk for this disease and infection [2]. The most common between these manifestations is cutaneous leishmaniasis, which is caused by many species of Leishmania including Leishmania tropica and Leishmania major. The patient’s skin develops single or multiple ulcers at the biting place, by sandflies [3]. The mucocutaneous form is the most severe and causes disfiguring of the face. Many of the drugs available for the treatment of leishmaniasis are toxic and Leishmania species are also becoming resistant to drugs [2].

Chemotherapy and chromotherapy are the only treatments used for the cure of leishmaniasis. But these therapies are expensive such as carbon dioxide laser therapy and the Leishmania parasite is developing resistance to some drugs while other causes nephrotoxicity. Some of the drugs (glucantime and antimonial) are not approved by the Food and Drug Administration (FDA) and there is no acceptable vaccine for this disease [4], [5]. Leishmaniasis is considered mainly a disease of the underdeveloped world and therefore commercial interest of pharmaceutical industries is to develop effective but cheaper antileishmanial agents. In this study we aim to develop novel but cheaper antileishmanial photoactive agents which might be helpful for a low-cost regimen for principally the cutaneous, subcutaneous, and mucocutaneous leishmaniasis. During leishmanial infection, the macrophage phagocytizes the parasites and releases ROS such as hydroxyl radicals/ions, superoxides, and hydrogen peroxide [6]. Leishmania parasites are sensitive to these ROS and can be easily killed [7]. To cope with these ROS, Leishmania contain enzymes such as peroxiredoxins and acid phosphatase as well as lipophosphoglycan, which are responsible for the scavenging of ROS produced by the macrophages. Helping the immune system by producing more ROS from the outside environment may therefore present a potential solution [8]. This increase in ROS production will easily kill the Leishmania parasite but it may also have an effect on the human cells. There is a need therefore to develop a system for controlled ROS production with a reasonable toxicity to Leishmania protozoans while being relatively safe to humans. Our approach was similar to photodynamic therapy (PDT), including light, oxygen, and a photoreactive material as the principal units. The polyethylene glycol (PEG)-functionalized Ag doped semiconductor nanoparticles of ZnO absorb light energy, particular to the physical aspects of Ag/ZnO, and ultimately transfer it to molecular oxygen and water molecules in the biological environment, leading to the production of ROS. Cutaneous and subcutaneous leishmanial lesions are well exposed and are easily accessible for the light exposure. Surface functionalization of DSNs may overcome the biological barriers resulting in an effective delivery of a therapeutic dose to the diseased area.

Section snippets

Reagents

Zinc acetate, silver nitrate, PEG, ethanol, Triton X-100, ficoll, percoll, trypan blue, sea salt, sodium azide, mannitol, and penicillin were purchased from Sigma (USA), Medium 199, RPMI, SYTOX green, diphenylisobenzofuran (DPBF) from Invitrogen (USA), fetal bovine serum from PAA (Austria), streptomycin from Bio Basic Inc., methylene blue, HCl, from Merck, Hepes from Roth Germany, and shrimp (nauplii) from Ocean Star International.

DSNs synthesis

Initially, we synthesized the ZnO-based DSNs with varying

Characterization of DSNs

All the synthesized particles were rounded in shape and were 20–50 nm in size, with an average size of 23 nm. The XRD graphs of developed DSNs confirmed the presence of Ag in crystalline materials when incorporated at relatively higher concentrations (7 and 9% in DSN5 and DSN6) (Fig. 1). The dominant peaks of ZnO at 2θ=31.4°, 34.2°, 35.9°, 47.2°, and 56.3° (marked with o) were found in all the DSNs and NDSN, and were indexed to wurzite ZnO (hexagonal) structure, whereas Ag peaks (marked with *)

Conclusions

We have studied the photobiology properties of our synthesized DSNs on the L. tropica KWH23 strain. The Leishmania cells were easily killed by the DSNs, when activated by daylight. Our focus was on the development of polychromatic daylight stimulated doped semiconductor nanoparticles (DSNs) of ZnO for the production of ROS, which were able to induce death of L. tropica KWH23. Furthermore there was no hyperthermia involved in the killing of Leishmania cells. Potent low concentrations of DSNs,

Acknowledgments

This work was financially supported by Higher Education Commission (HEC) of Pakistan and Pakistan Science Foundation (PSF) and their support is gratefully acknowledged. The authors thankfully acknowledge the kind support of the late Dr. Tajammul Hussain (Nanosciences and Catalysis Division, National Centre for Physics, Islamabad) and Dr. Ashraf Gondal (King Fahad University of Petroleum and Minerals, KFUPM, Saudi-Arabia) for their help and guidance in starting this project. The authors are

References (44)

  • X. Zhang et al.

    Identification of possible reactive oxygen species involved in ultraviolet radiation-induced oxidative DNA damage

    Free Radic. Biol. Med.

    (1997)
  • M. Ahamed et al.

    ZnO nanorod-induced apoptosis in human alveolar adenocarcinoma cells via p53, survivin and bax/bcl-2 pathways: role of oxidative stress

    Nanomed. Nanotechnol. Biol. Med

    (2011)
  • WHO Leishmaniasis;...
  • D. Molyneux et al.

    Morphology, ultrastructure and life cycles. The leishmaniases in biology and medicine, volume I. Biology and epidemiology

    (1987)
  • D.O. Santos et al.

    Leishmaniasis treatment—a challenge that remains: a review

    Parasitol. Res.

    (2008)
  • B.M. Babior et al.

    Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent

    J. Clin. Invest.

    (1973)
  • H.W. Murray

    Susceptibility of Leishmania to oxygen intermediates and killing by normal macrophages

    J. Exp. Med.

    (1981)
  • A.M. Allahverdiyev et al.

    Antileishmanial effect of silver nanoparticles and their enhanced antiparasitic activity under ultraviolet light

    Int. J. Nanomed.

    (2011)
  • R.S. Zeferino et al.

    Photoluminescence and Raman scattering in Ag-doped ZnO nanoparticles

    J. Appl. Phys.

    (2011)
  • Q. Fu

    Radiation (solar)

    (2003)
  • M.C. d. Almeida et al.

    A simple method for human peripheral blood monocyte isolation

    Mem. Inst. Oswaldo Cruz

    (2000)
  • I. Walev et al.

    Resealing of large transmembrane pores produced by streptolysin O in nucleated cells is accompanied by NF-κB activation and downstream events

    FASEB J.

    (2002)
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