Reducing the cytotoxicity while improving the anti-cancer activity of silver nanoparticles through α-tocopherol succinate modification

Abstract

By releasing Ag⁺ ions and generating reactive oxygen species (ROS), silver nanoparticles (Ag NPs) not only have good anti-tumor activity but also display cytotoxicity towards normal cells which limits their further application in the medical field. Up to now, there was still no appropriate method to reduce the cytotoxicity while improving the anti-cancer activity of Ag NPs. This paper focuses on counteracting the toxic side effect of the ROS from Ag NPs while simultaneously improving their anti-cancer effect. We used α-TOS to modify Ag NPs and investigated their bioactivity in vitro for the first time. The modified Ag NPs with a high α-TOS concentration not only show much higher anti-tumor activity than Ag NPs alone but also promote the survival of normal cell lines slightly, while the modified Ag NPs with a low α-TOS concentration display a lower cytotoxicity against normal cell lines without affecting their anti-cancer activity when compared to Ag NPs alone. Therefore, this work presents a higher potential for cancer treatment than using Ag NPs alone.

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Introduction

Breast cancer disease is a global disease and endangers human life and health as a result of high incidence and mortality. Chemotherapy, the main conventional treatment strategy, has several limitations including damaging healthy cells, nonspecificity, the toxicity of anticancer drugs and drug resistance.^1,2 Therefore, it is immediately necessary to establish an economically safer and more effective alternative treatment strategy for the development of breast cancer treatments rather than the existing orthodox approaches.

Nanomaterials have attracted the interest of researchers in cancer research, due to their unusual chemical and physical properties. The enhanced permeability and retention (EPR) effect could permit passive accumulation into cancer tissues to a certain degree, and the surface effect of nanomaterials could lead to the insufficiency of surface atomic coordination and high surface energy, which makes them easily modifiable with multiple functionalities such as targeting molecules, imaging agents, and drugs.^3,4 With unique properties and structures at the nanometer scale, nanomaterials could be applied in safe and efficient cancer treatments.

Ag NPs have also been explored as inhibitors to suppress angiogenesis and tumor growth, vectors for drug delivery, as well as nanoprobes for the detection and imaging of tumors.^5–7 Ag NPs have good anti-tumor activity and the suggested cause for death in Ag NP-treated cancer cells is the release of Ag⁺ and the generation of ROS, which is known to cause apoptosis and a sequence of irreversible damage.^8–11 However, the side effects of Ag⁺ and ROS to normal cells such as DNA damage and oxidative stress have also been reported in a number of in vitro studies, and limit further application of silver nanoparticles in the medical field.^12,13

Now, there are two main methods to reduce the cytotoxicity of Ag NPs in cancer treatment: one is to increase their targeting ability by grafting targeting molecules such as hyaluronic acid,¹⁴ aptamer AS1411,¹⁵ and epidermal growth factor receptor antibodies,¹⁶ and so on; the other is to avoid direct exposure to the bloodstream through capping agents such as mesoporous silica,¹⁷ protein corona,¹⁸ plant latex,¹⁹ and so forth. In essence, neither of the two methods alleviate the side effects of Ag⁺ and ROS from Ag NPs directly.

Here, we not only aim at alleviating the side effects of ROS but also at increasing the anti-cancer activity of Ag NPs simultaneously. α-TOS is known as a kind of vitamin E derivative, which can induce apoptosis of various types of cancer cells while not affecting the proliferation of most normal cells.^20,21 This is because α-TOS stimulates the production of ROS via interaction with the coenzyme Q binding site in complex II of the mitochondrial respiratory chain of a cancer cell which then causes the apoptosis of the cancer cell.^22,23 Lippard et al. reported that two platinum(IV) complexes conjugated with α-TOS had caused damage to the DNA and mitochondria of cancer cells simultaneously.²⁴ Reyes-Leyva et al. demonstrated that magnetite nanoparticles functionalized with α-TOS retained anticancer activity on cervical cancer cells selectively and were biocompatible with non-malignant fibroblasts.²⁵

Up to now, there has been no report about modifying Ag NPs with α-TOS. In this work, we prepared three kinds of Ag NPs with good anti-cancer activity and attempted to modify Ag NPs with α-TOS for the first time. Cell experiments were used to evaluate the cytotoxicity and anti-cancer effect of the modified Ag NPs.

Experimental

Materials

Silver nitrate (AgNO₃, 99.9%) was purchased from Alfa Aesar (Tianjin) Chemical Co. Ltd. Trisodium citrate dihydrate (Na₃C₆H₅O₇·2H₂O, 99.0%), cetyltrimethylammonium bromide (CTAB, 99.0%) and sodium borohydride (NaBH₄, 96.0%) were of analytical grade (AR) and purchased from China Medicine (Group) Shanghai Chemical Reagent Corp. All chemical reagents were used as received without further purification. Deionized water was used for all experiments.

Formation of Ag NPs

In previous articles, we have studied the factors that influence the controllable preparation of Ag NPs.^26,27 And Ag NPs were synthesized under conditions which are the same as those shown in ref. 28. For the formation of the Ag NPs, silver nitrate (AgNO₃, 99.8%) served as the precursor, ascorbic acid (Vc, 99.7%) served as the reductant and NaOH (96.0%) acted as the reaction initiator. CTAB was introduced as a structure-directing agent and silver nanocrystals were used as seeds. CTAB was dissolved in 10% aqueous alcohol to avoid CTAB precipitating out at a low temperature.

Modification of Ag NPs

The pre-prepared Ag NPs were centrifuged at 12 000 rpm for 10 min and the precipitate was collected in two flasks (A and B). Flask A contained 50 mg of α-TOS and 4 mL of ethyl alcohol, and flask B contained 12 mg of α-TOS and 2 mL of ethyl alcohol. The molar ratios of Ag NPs to α-TOS in these two flasks were 1 : 25 and 2 : 3, respectively. Some precipitate appeared and the color of the solution became clear in the flasks after stirring for hours. Some solutions from these two flasks were used in cell experiments as mother liquors. The precipitate was collected by centrifugation and then dried in a vacuum desiccator, followed by infrared analysis.

Characterization

An AU-3010 UV-vis spectrophotometer (Hitachi) was used to record the absorbance of the formed Ag NP colloidal sol. An FEI transmission electron microscope (Tecnai G2 20, an accelerating voltage of 200 kV) was used to observe the different morphologies of the pre-prepared Ag NPs. A Fourier transform infrared (FT-IR) spectrometer (Nicolet 6700 Series) was used to obtain the interactions of the modified Ag NPs with a high α-TOS concentration (50 mg) and the IR spectra were measured using the KBr method.

Cell culture

The human breast cancer cell line MCF-7, human leukemia cell line K562, human lung cancer cell line A549 and human liver cell line HL7702 were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. The cells were routinely grown and maintained in RPMI or DMEM medium with 10% FBS and 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO₂ water-jacketed incubator with 5% CO₂ in air at 37 °C.

Cell viability assay

The cytotoxicity and anti-tumor activity of the Ag NPs and modified Ag NPs were assessed using a CCK-8 assay. The modified Ag NP solutions were appropriately diluted based on the Ag NP concentration. The cells were plated in 96-well flat bottom plates at a density of 1 × 10⁵ cell per mL. After 24 h of incubation, the cells were exposed to various concentrations of Ag NPs, modified Ag NPs or drugs for 48 h, followed by CCK-8 assay. The absorbance was measured at 450 nm by an Envision 2104 multi-label reader (Perkin-Elmer, USA).

Results and discussion

Characteristics of Ag NPs

Because of the surface plasmon resonance of electrons at the surface of Ag NPs, silver nanospheres have an absorption peak at about 470 nm while silver nanorods have two absorption peaks: the longitudinal plasmon resonance peak (centered at about 500–600 nm) and the transversal plasmon resonance peak (centered at about 410 nm). Fig. 1(a) is the UV-vis spectra of Ag NPs formed by adding different volumes of seed solution. The longitudinal plasmon resonance peak was obviously red shifted from 512 nm to 606 nm with a decrease in the seed solution from 0.25 mL to 0.062 mL, which meant that the aspect ratio of the silver nanorods increased. Fig. 1(b)–(d) show the TEM images of Ag NPs corresponding to different volumes of the seed solution (1.0 mL–0.062 mL). In Fig. 1(b), when adding 1.0 mL of the seed solution, the main components of the Ag NPs were nanospheres formed in the range of 70 nm–100 nm. Short nanorods appeared in the presence of nanospheres when the seed solution was reduced to 0.25 mL (Fig. 1(c)). The aspect ratio of the silver nanorods sequentially increased after adding 0.062 mL of the seed solution, and long nanorods were obtained (Fig. 1(d)).

Fig. 1 (a) UV-vis spectra of Ag NPs showing the effect of the seed solution, (b–d) typical TEM images of Ag NPs prepared by adding (b) 1.0 mL, (c) 0.25 mL, and (d) 0.062 mL of the seed solution.

FTIR analysis of the modified Ag NPs

In this work, we attempted to explain how α-TOS interacted with Ag NPs by using IR spectroscopy (Fig. 2). After centrifugation, the supernatant containing the most CTAB in the Ag NP colloidal sol was removed and there still existed some CTAB molecules on the surface of the Ag NP precipitate. So there are some peaks of CTAB in the Ag NP precipitate IR spectrum and the peak intensity is weaker than in the CTAB spectrum. The peaks at 2850 and 2918 cm⁻¹ are due to the CH₂ symmetric and asymmetric stretching vibrations of CTAB, and the bandwidths of these peaks in the Ag NP precipitate spectrum are narrower than those in the CTAB spectrum, which may imply that the CTAB molecules are arranged in some order on the Ag NP precipitate surface.²⁹ The peaks at 1396 and 1479 cm⁻¹ are related to the ⁺N-CH₃ symmetric and asymmetric deformation vibrations of the CTAB headgroup.³⁰ Because CTAB molecules cap Ag NPs via their headgroups, the peak at 1396 cm⁻¹ becomes wider in the Ag NP precipitate spectrum compared to the CTAB spectrum. The peaks at 3462 and 1641 cm⁻¹ are related to the stretching and bending vibrations of adsorbed water. After mixing the Ag NP precipitate with α-TOS through physical grinding, we performed infrared analysis and found that the spectrum was similar to the α-TOS spectrum, which means that Ag NPs did not interact with α-TOS in the mixture.

Fig. 2 FTIR spectra of the α-TOS modified Ag NPs.

By comparing the modified Ag NP and α-TOS spectra, it appeared that the modifying agent (α-TOS) was linked to the Ag NP precipitate. In the α-TOS spectrum, the peaks at 1753 and 1714 cm⁻¹ are due to the stretching vibrations of the carbonyl groups of the ester and carboxylic acid, respectively,³¹ and the peaks around the region of 1450–1360 cm⁻¹ are due to the C–H bending vibrations of the –CH₂ and –CH₃ groups;²⁵ they can also be slightly observed in the α-TOS modified Ag NP spectrum. The peak at 1109 cm⁻¹ is assigned to CH₂–O–CH₂ of cyclic ether,^32,33 and it becomes wider and larger in the α-TOS modified Ag NP spectrum than in the α-TOS spectrum, which may imply that α-TOS interacted with Ag NPs through cyclic ether.

Ag NPs and electron-rich groups such as those groups containing oxygen/nitrogen could form coordination,³⁴ and the changes in the peaks around 1109 cm⁻¹ in our modified Ag NP spectrum may be attributed to the formation of coordination bonds between the Ag NP precipitate and the CH₂–O–CH₂ group of the cyclic ether in α-TOS.

Anti-cancer activity of Ag NPs

Lung cancer is the most common cancer in China, followed by female breast cancer.³⁵ Leukemia is a malignant disease of blood-forming cells in the bone marrow, also known as “cancer of the blood”. These cancer diseases are characterized by high incidence and mortality. In Fig. 3, we investigated whether Ag NPs have an effective anti-cancer efficacy against those malignant tumors in vitro.

Fig. 3 Anti-tumor activity of Ag NPs on (a) MCF-7, (b) A549 and (c) K562 cancer cells.

We took trichostatin A (TSA) and taxol as positive controls to evaluate the anti-cancer activity of Ag NPs against the MCF-7, K562 and A549 cell lines. Taxol, a mature drug, acts as an effective antimicrotubule drug for the treatment of a range of intractable cancers, especially breast and ovarian cancer. TSA is one of the representative examples for histone deacetylase inhibitors which are now emerging as a new class of anticancer agents. TSA has been used in several cancer treatment studies, such as breast cancer,³⁶ human leukemia,³⁷ and ovarian and lung cancer.³⁸

Many studies have shown that Ag NPs have satisfactory anti-cancer activity in a wide spectrum of tumors.^8–11 In Fig. 3, the results of the anti-tumor experiment show that Ag NPs with different morphologies have better anti-tumor activity on the three cancer cell lines compared to TSA, but exhibit weaker anti-cancer activity than taxol in general. One reason why Ag NPs have good anti-tumor activity is because of the release of Ag⁺ ions into cancer cells, which may cause subsequent cytotoxicity and induce cell death.³⁹ Some scholars thought that the cytotoxicity mechanism of Ag NPs was mostly related to the intracellular release of silver ions.^13,40 Yuan et al. suggested that the {111} crystal plane of Ag NPs could influence the release of Ag⁺ ions.⁴¹ So it may explain why silver nanorods with {111} basal planes display better anti-cancer activity than silver nanospheres that predominantly have {100} crystal facets. Silver nanorods may release Ag⁺ ions more easily than nanospheres in our experiment. In Fig. 3(a), Ag NPs nearly kill all MCF-7 cancer cells at the concentration of 4.12 × 10⁻² μM and the efficacy is even better than taxol at that concentration. Because of this potent anti-tumor activity, we selected the MCF-7 cancer cells for further study on reducing the cytotoxicity of Ag NPs.

Cell viability assay of the modified Ag NPs

Most Ag NPs are found to accumulate in the liver and the excessive deposition of Ag NPs would cause certain adverse effects.⁴² α-TOS can be associated with lipoproteins in the bloodstream so the Ag NPs could be not only transported into the tumor microvasculature but also to the liver where α-TOS is hydrolyzed by the competent esterases.⁴³ We would mainly investigate the side effect of Ag NPs or modified Ag NPs through the human liver cell line HL7702.

The in vitro study on HL7702 and MCF-7 cells was divided into the control group (Ag NPs), the experimental group (modified Ag NPs) as well as the blank group. The solutions of modified Ag NPs with a high α-TOS concentration (50 mg) were used in the experiment after appropriate dilution based on the Ag NP concentration. At the Ag NP concentration of 1.60 × 10⁻² μM, the three experimental groups kill all cancer cells while the survival of MCF-7 cells is at least 95% in each corresponding control group, which means that high α-TOS concentration modified Ag NPs have a synergistic potent anti-tumor effect. In addition, the experimental groups show less cytotoxicity on HL7702 cells than the corresponding control groups in general. In Fig. 4(b), the MCF-7 cell survival is similar between the control and experimental groups at the experimental concentration. In Fig. 4(a) and (c), the HL7702 cell survival in the control groups is about 50% at the Ag NP concentration of 4.00 × 10⁻¹ μM and it is 12% lower in Fig. 4(a) and 9% lower in Fig. 4(c) compared to their corresponding experimental groups. With the help of a high α-TOS concentration, the modified Ag NPs not only exhibit less cytotoxicity on HL7702 cells to a certain degree but also display much greater anti-tumor activity against MCF-7 cells than Ag NPs alone.

Fig. 4 Cytotoxicity and anti-cancer activity of three different Ag NPs with high α-TOS concentrations: (a) nanospheres, (b) short nanorods, and (c) long nanorods.

In Fig. 5, the solutions of modified Ag NPs with a low α-TOS concentration (12 mg) are taken as experimental groups after dilution. In Fig. 5(a), the survival of the HL7702 cells is almost the same between the control group and experimental group while the modified Ag NPs show less toxicity on HL7702 cells when compared to the control groups in Fig. 5(b) and (c). All three experimental groups and their corresponding control groups display similar anti-cancer activity on MCF-7 cells at Ag NP concentrations from 8.00 × 10⁻² μM to 2.00 μM.

Fig. 5 Cytotoxicity and anti-cancer activity of three different Ag NPs with a low α-TOS concentration: (a) nanospheres, (b) short nanorods, and (c) long nanorods.

α-TOS has a highly selective toxicity towards various cancer cells, without any adverse effects on organs or normal cells.^20,44 It may be explained by esterases which exist in normal cells but not in cancer cells, as esterases could hydrolyze α-TOS into tocopherol, which has antioxidant activity.⁴³ α-TOS induces apoptosis in cancer cells by targeting the ubiquinone binding sites of the mitochondrial complex II to cause an excessive production of ROS which activates the proteins that regulate mitochondrial permeabilization and triggers apoptosis.⁴⁵ Breast cancer cells are induced to apoptosis by a rapid formation of ROS generated from exogenously added α-TOS.⁴⁶ On the contrary, α-TOS is converted into tocopherol by esterase hydrolysis in normal cells.⁴⁷

In MCF-7 cells, the α-TOS modified Ag NPs could generate high levels of ROS not only from Ag NPs but also from α-TOS while α-TOS is cleaved into tocopherol to counterwork the side effects of the ROS from Ag NPs in the HL7702 cells (Scheme 1). So α-TOS modified Ag NPs show much greater anti-cancer activity against MCF-7 cancer cells but are safer for HL7702 cells compared to Ag NPs alone. The efficiency of α-TOS to produce ROS in MCF-7 cells or form tocopherol is unclear and this may contribute to the different bioactivities of the modified Ag NPs shown in Fig. 4 and 5. In our experiment, a more suitable α-TOS concentration for modified Ag NPs may be further studied.

Scheme 1 Schematic illustration for reducing the cytotoxicity while improving the anti-cancer activity of modified Ag NPs.

Conclusion

In this paper, we prepared Ag NPs with anti-cancer activity on cancer cell lines (MCF-7, A549, K562) and they had a better efficacy against MCF-7 cancer cells than taxol at the Ag NP concentration of 4.12 × 10⁻² μM. To reduce the cytotoxicity and improve the anti-cancer activity of Ag NPs simultaneously, we modified Ag NPs with α-TOS. The modified Ag NPs with a high α-TOS concentration kill all MCF-7 cancer cells at the experimental concentration and promote the HL7702 cell survival slightly. The modified Ag NPs with a low α-TOS concentration display less cytotoxicity on HL7702 than Ag NPs alone but their anti-cancer activity is not affected. From this study, modified Ag NPs display potential for cancer treatment and show less cytotoxicity to normal cells than Ag NPs alone.

Acknowledgements

The Project was supported by the National Natural Science Foundation of China (Grant no. 81071254) and the Natural Science Foundation of Guangdong Province, China (Grant no. 10451051501004706).

Notes and references

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