Date Published: July 12, 2017
Publisher: Public Library of Science
Author(s): Lukas Nejdl, Jiri Kudr, Amitava Moulick, Dagmar Hegerova, Branislav Ruttkay-Nedecky, Jaromir Gumulec, Kristyna Cihalova, Kristyna Smerkova, Simona Dostalova, Sona Krizkova, Marie Novotna, Pavel Kopel, Vojtech Adam, Hélder A. Santos.
Sparsely tested group of platinum nanoparticles (PtNPs) may have a comparable effect as complex platinum compounds. The aim of this study was to observe the effect of PtNPs in in vitro amplification of DNA fragment of phage λ, on the bacterial cultures (Staphylococcus aureus), human foreskin fibroblasts and erythrocytes. In vitro synthesized PtNPs were characterized by dynamic light scattering (PtNPs size range 4.8–11.7 nm), zeta potential measurements (-15 mV at pH 7.4), X-ray fluorescence, UV/vis spectrophotometry and atomic absorption spectrometry. The PtNPs inhibited the DNA replication and affected the secondary structure of DNA at higher concentrations, which was confirmed by polymerase chain reaction, DNA sequencing and DNA denaturation experiments. Further, cisplatin (CisPt), as traditional chemotherapy agent, was used in all parallel experiments. Moreover, the encapsulation of PtNPs in liposomes (LipoPtNPs) caused an approximately 2.4x higher of DNA damage in comparison with CisPt, LipoCisPt and PtNPs. The encapsulation of PtNPs in liposomes also increased their antibacterial, cytostatic and cytotoxic effect, which was determined by the method of growth curves on S. aureus and HFF cells. In addition, both the bare and encapsulated PtNPs caused lower oxidative stress (determined by GSH/GSSG ratio) in the human erythrocytes compared to the bare and encapsulated CisPt. CisPt was used in all parallel experiments as traditional chemotherapy agent.
The first anticancer drug cisplatin (cis-diamminedichloroplatinum(II)) was discovered in 1965 by Rosenberg during his studies on the effects of an electric current on bacterial growth . The binding of cisplatin to DNA and the interactions with non-DNA targets with subsequent triggering of cell death through apoptosis, necrosis or both belong to the most important parameters of cisplatin cytotoxicity . Since then, numerous platinum complexes of second generation (carboplatin, oxaliplatin, nedaplatin) and third generation (lobaplatin, heptaplatin) have been developed  and evaluated as anticancer agents [4, 5]. The undesired side effects can be eliminated by specific molecular interactions of a drug with cancer cells and by selective targeting, which can be done by their encapsulation in the molecules suitable for selective transportation. The most common transporters belong to functionalized polymers, nanoparticles with conjugated platinum drugs, carbon nanotubes and micelles. Among them, liposomes, which consist of a phospholipid bilayer surrounding an aqueous core, are a very important class of compounds . Liposomes have interesting biological activities including effective drug loading capacity, biocompatibility and improved pharmacokinetics. The liposomal formulations of cisplatin and oxaliplatin (Lipoplatin™ and Lipoxal™) are now widely used to reduce drug toxicity and also improve drug targeting [7, 8]. Experimentally, many nanoparticles consisting of the polymers or copolymers and the Pt-based drugs have been tested. Among these, polyethylene glycol-functionalized poly-isobutylene-maleic acid copolymer can form complexes with cisplatin. The nanoparticles are internalized into the endolysosomal compartment of cancer cells releasing cisplatin in a pH-dependent manner. These nanoparticles have been developed for better antitumor efficacy, which was confirmed in a 4T1 breast cancer model in vivo with limited nephrotoxicity . Furthermore, the nanoparticles treatment resulted in reduced systemic and nephrotoxicity, which was confirmed by the decreased biodistribution of platinum in kidney [10, 11]. The nanoparticles of oxaliplatin and carboplatin with polyisobutylene maleic acid copolymer with glucosamine and albumin-bound paclitaxel, respectively, were also tested [12, 13].
In the present experiment, the effects of PtNPs, or CisPt alone and encapsulated in liposomes (LipoPtNPs or LipoCisPt) on bacterial cultures (Staphylococcus aureus), HFF, erythrocytes and DNA (PCR product of Xis gene of λ phage) were studied. The platinum complexes are the active substance used as effective cytostatics . Hundreds of Pt(II) and Pt(IV) complexes have been synthesized since the discovery the antibacterial effects of platinum in 1965 [42–44]. The effect of the platinum complexes (cisplatin, carboplatin and oxaliplatin) is most probably based on their covalent binding with DNA bases [45, 46] forming intra- and interstrand crosslinks, DNA—protein crosslinks, and monoadducts with DNA . DNA secondary structures can block transcription and replication with subsequent apoptosis . Pt drugs enter cells mainly via passive diffusion, although some evidence of its active transport by Ctr1 system involved in the maintaining of copper homeostasis was reported (Fig 5A) [49–52]. Another possibility is the intake of the nanoparticles via endocytosis . Cisplatin-induced DNA damage activates ATR kinase, which triggers the effector molecules. One of the ATR kinase targets is a tumour suppressor protein p53, which is phosphorylated by the kinase on serine 15. This phosphorylation causes the decrease of p53 affinity to its negative regulator Mdm-2, which leads to an increased p53 concentration. The protein p53 then initiates the transcription of p21 gene, which inhibits the cyclin-dependent kinases Cdk2 and Cdk4 and consequent arrests the cell cycle. p53 also induces the expression of pro-apoptotic members of Bcl-2 family such as Bax, Puma and Noxa in response of the activation of mitochondrial apoptotic pathway.
In this paper, we showed that PtNPs primarily inhibit the activity of Taq DNA polymerase and damage to the DNA structure. We tend to believe that this effect together with the transition of PtNPs to Pt2+ causes mutagenicity and increases DNA damage compared to CisPt. The cytotoxic effect of the PtNPs may be increased by their encapsulation in liposome. The results suggest that the activation of p53 in PtNPs treated cells was caused by the genotoxic stress with subsequent activation of p21 leading to a proliferating cell nuclear antigen-mediated growth arrest in S phase and following apoptosis . It is conceivable that the PtNPs with the effective antitumor activity may provide an alternative treatment for cancer.