Synergistic and enhanced anticancer effect of a facile surface modified non-cytotoxic silver nanoparticle conjugated with gemcitabine in metastatic breast cancer cells

https://doi.org/10.1016/j.mtcomm.2019.100884Get rights and content

Highlights

  • Polyvinyl pyrrolidone stabilized silver nanoparticles (AgNP) were developed.

  • Gemcitabine (GEM)-AgNP were prepared by electrostatic attraction for the first time.

  • Physicochemical characteristics of developed AgNP and GEM-AgNP were evaluated.

  • GEM and AgNP showed synergistic effect against breast cancer MDA-MB-453 cell line.

  • GEM-AgNP showed enhanced cytotoxicity against MDA-MB-453 cell line.

Abstract

The safety and efficacy of metallic nanoparticles was one of the major challenges that limit their use in the treatment of cancer. Nanotechnology is applied in the field of pharmaceutical sciences with focus on improving the therapeutic outcome in various diseases. Recently, many novel nano-formulations comprising two or more drugs were studied to improve their efficacy with better safety profile. In this study, we investigated the synergistic cytotoxic effect of gemcitabine (GEM) conjugated non-cytotoxic dose of silver nanoparticles (AgNP) in MDA-MB-453 human triple-negative metastatic breast cancer cells. Synthesized AgNP and electrostatic conjugates were characterized by UV-spectroscopy, dynamic light scattering (DLS), Transmission electron microscopy (TEM), Scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDAX). GEM-(non-cytotoxic AgNP) conjugated system (IC50 = 37.64 μM) showed better cytotoxic activity in MDA-MB-453 cells when compared to individual treatments of GEM (IC50 = 56.54 μM) or AgNP (IC50 = 71.45 μg/ml). The synergism between the GEM-(non-cytotoxic AgNP) for all the tested doses were evaluated using CompuSyn software. The combination index (CI) of ED50, ED75 and ED90 showed synergism for GEM-(non-cytotoxic AgNP) conjugation. According to the calculated dose reduction index (DRI), it requires 1.70-fold less GEM plus 42.55-fold less AgNP to achieve the same 50 % inhibition at 18.38 (GEM): 1 (AgNP) ratio.

Introduction

Nanotechnology is applied in various fields of pharmaceutical sciences to improve the physicochemical and ADME properties, efficacy and safety of the drugs [1]. One such approach is combining effective metallic nanoparticles with chemotherapeutic agents [2]. Metallic nanoparticles possess unique properties due to their size, shape, surface structure and aggregation characteristics [3]. Amongst, AgNP is the one which is extensively studied in pharmaceutical sciences because of its antimicrobial activity and cytotoxicity against various cancer cell lines [[4], [5], [6]]. Nano silver can be easily oxidized in the presence of oxygen leading to the release of silver ions (Ag+) which is the major source of toxicity. Thus, AgNP often acts as a source of Ag+ inside the cells. Ag+ ions induce oxidative stress through the generation of reactive oxygen species and causes damage to cellular components such as cell membrane, protein and DNA. This leads to apoptotic and necrotic cell deaths triggering the release of pro-inflammatory cytokines which cause further damage to nearby tissues or cells. AgNP can also deplete the antioxidant molecules like glutathione and aggravate the cytotoxic effects. AgNP are reported to cause genotoxicity, neurotoxicity, pulmonary toxicity, hepatotoxicity and immunotoxicity [7]. Necropsy analysis of rats orally administered with AgNP (5 mg/kg b.w. and 10 mg/kg b.w.) for 28 days showed no signs of toxicity in the organs such as kidney, brain, lungs, heart and testis. Liver cells had some anomaly in 10 mg/kg b.w. treatment and not in 5 mg/kg b.w. treated rats [8]. Hepatoma cells (HepG2) treated with non-cytotoxic dose (<0.5 mg/L) of AgNP exhibited protective effect. The toxic effects were observed only in the doses excess of 1 mg/l in HepG2 cells [9]. Moreover, the protective effect of AgNP at low doses was correlated with its ability to activate Nrf2 (nuclear factor erythroid 2(NFE2)-related factor 2) and P38 MAPK mediated transcriptional process. Hence, reducing the dose of AgNP is a prime prerequisite to minimize toxicity associated with AgNP usage to treat disease conditions [10].

Monotherapy for cancer treatment has many drawbacks such as drug resistance, excessive toxic effects and undesired side effects. The advantage of combination therapy includes the use of chemotherapeutic agents and NP at lower doses. This could result in the elimination/reduction of undesired toxic effects and increased efficacy. In practice, combination chemotherapy results in better response and improved survival compared with single-agent therapy [11]. Metastatic breast cancer is often treated with combinations such as cyclophosphamide / doxorubicin / fluorouracil, cyclophosphamide / doxorubicin, cyclophosphamide / methotrexate / fluorouracil, gemcitabine / paclitaxel and gemcitabine / carboplatin [12]. However, many chemotherapeutic drugs have systemic toxicity because they nonspecifically accumulate in normal cells [13]. Hence, improving the safety and synergy of chemotherpeutic agents in monotherapy is the need of the hour to treat cancer. The dose limitation of AgNP could be achieved by combining a proper cytotoxic drug to exploit the synergism between the two agents [14]. Hence, in this study, we controlled the ratio and tested the synergistic effect of non-cytotoxic dose of AgNP conjugated with a cytotoxic agent gemcitabine (GEM). Synthesis of AgNP generally involves reducing the precursor Ag+ by techniques such as biological, chemical, electrochemical reduction and radiolysis. Among them, size controllable chemical reduction is the most popular to get uniform AgNP using chemical reductants such as sodium citrate [15], tannic acid [16], sodium borohydride (NaBH4) [17], hydrazine [18] and hydroxylamine hydrochloride [19]. Generally, AgNP have stability issues such as aggregation, crystal growth and poor size distribution. After stabilization, chemically reduced AgNP are reported to be highly stable at room temperature (23 °C) over a period of one year when compared to bare AgNP [20].

AgNP with different surface charges can be synthesized using different types of reducing agents. To obtain AgNP with negative, neutral and positive surface charge, NaBH4, rice starch and 1-dodecyl-3-methylimidazolium chloride were used, respectively [[21], [22], [23]]. During the synthesis of nanoparticles, the ligand molecules bound to the surface prevent the growth and aggregation of nanoparticles. The ligand molecules bind to the surface of the particles by attractive interactions such as chemisorption, electrostatic attraction and hydrophobic interactions [[24], [25], [26]]. Electrostatic attraction of ligands on metallic nanoparticles has been widely studied. Metallic nanoparticle system such as folic acid-conjugated AgNP, polymyxin B-conjugated gold nanoparticles and pyrethroid-conjugated on nanosilver have been previously studied [2,[27], [28], [29]]. Surface functionalization of AgNP is important for their applicability, compatibility and safety. AgNP act as a drug carrier with enhanced stability of surface adsorbed drug. It has diagnostic potency, enhanced catalytic activity, reduced toxicity, and high-density surface for ligand attachment without additional use of any linkers and protects the surface attached drug from degradation. Surface functionalization defines how nanoparticles behave in physiological conditions [30,31]. In this study, we developed a system with AgNP as a core which is electrostatically attached to GEM. This system was evaluated in human triple-negative metastatic breast cancer cell line, MDA-MB-453.

Section snippets

Materials

GEM (as gemcitabine hydrochloride) was a gift sample from Dr. Reddy’s Laboratories, Hyderabad, India. Silver nitrate (AgNO3), NaBH4, polyvinyl pyrrolidone (PVP), sucrose and sodium chloride (NaCl) were purchased from Sigma Aldrich, India. MDA-MB-453 was purchased from National Centre for Cell Science (NCCS), Pune, India.

Synthesis of AgNP

AgNP were synthesized by chemical reduction method using NaBH4 as a reducing agent [21]. Briefly, 10 ml of 1.0 mM AgNO3 solution was added drop wise to 30 ml of 2.0 mM ice-cold

Results and discussion

AgNP were prepared from AgNO3 by chemical reduction method with the indication of nanoparticle formation by bright yellow color and the addition of GEM did not result in any color transition (Fig. 2A and B). Prepared AgNP were stabilized using 0.3 % PVP (w/v). PVP is a polymer which has been reported to be strongly adhere to the surface of AgNP, preventing aggregation and enhances the stability of AgNP for over a year [21,36]. The synthesized AgNP were added with sucrose as a cryoprotectant to

Conclusion

In this study, we investigated the synergism between GEM and AgNP in order to reduce the dose of AgNP and its associated toxicity. To improve the safety and efficacy of metallic nanoparticles, we used GEM as a “model” drug and prepared GEM conjugated with non-cytotoxic doses of AgNP. The stable AgNP were prepared using PVP. The prepared GEM-(non-cytotoxic AgNP) improved the cytotoxic effect of GEM in MDA-MB-453 cell line. The IC50 value of GEM-(non-cytotoxic AgNP) was significantly lower when

CRediT authorship contribution statement

Arjunan Karuppaiah: Conceptualization, Methodology, Data curation, Validation, Writing - original draft. Karthik Siram: Software. Divakar Selvaraj: Software, Writing - review & editing. Mohankandhasamy Ramasamy: Validation, Writing - review & editing. Dinesh Babu: Visualization, Investigation, Writing - review & editing. Veintramuthu Sankar: Supervision, Investigation, Validation, Writing - review & editing.

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors would like to thank Peelamedu Saamanaidu Govindaswamy (PSG) Sons & Charities for providing all the necessary research facilities. The authors also would like to thank Dr. Reddy’s Laboratories Ltd. (Hyderabad, Andhra Pradesh, India) for providing gift sample of gemcitabine hydrochloride to carry out this research study. Also, we would like to thank pharmacology team, Department of Pharmacology, JSS College of Pharmacy, Tamil Nadu, India, for helping to carry out combination index

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    Affiliated to TN Dr M.G.R Medical University, Guindy, Chennai 600032, Tamil Nadu, India.

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