Mn‐doped ZnS quantum dots‐chlorin e6 shows potential as a treatment for chondrosarcoma: an in vitro study

Abstract

Chondrosarcoma is the second‐most malignant cancer of the bone and routine treatments such as chemotherapy and radiotherapy have not responded to the treatment of this cancer. Due to the resistance of chondrosarcoma to radiotherapy, the combination of therapeutic methods has been considered in recent years. In this study, a novel combination approach is used that allows photodynamic therapy to be activated by X‐rays. The synthesis of Mn‐doped zinc sulphide (ZnS) quantum dots was carried out and chlorin e6 photosensitiser attached by covalent and non‐covalent methods and their application as an intracellular light source for photodynamic activation was investigated. The toxicity of each nanoparticles was evaluated on chondrosarcoma cancer cells (SW1353) before and after radiation. Also, the effect nanoparticle‐photosensitiser conjugated type was investigated in the therapeutic efficacy. The characterisation test (SEM, TEM, EDS, TGA, XRD and ICP analyses) was shown successful synthesis of Mn‐doped ZnS quantum dots. Chondrosarcoma cancer cell viability was significantly reduced when cells were treated with MPA‐capped Mn‐doped ZnS quantum dots‐chlorin e6 with spermine linker and with covalent attachment (P  ≤ 0.001). These results indicate that X‐ray can activate the quantum dot complexes for cancer treatment, which can be a novel method for treatment of chondrosarcoma.

1 Introduction

Chondrosarcoma is a type of cancer that affects the bones. It is diagnosed in ∼700 patients each year in Iran [1, 2]. Historically, surgical removal of the tumour along with a wide margin of healthy tissue has been crucial in the treatment of chondrosarcoma. Most chondrosarcoma cancers do not respond to radiation therapy or chemotherapy. However, these treatments are not effective [3].

Nanoparticles, because of their small size and unique optical, biological, and chemical properties possess wide application in biotechnology and medicine [4, 5, 6]. Quantum dots (QDs) are an example of this type. QDs are nanocrystals that have many advantages such as broad absorption spectra and tunable emission wavelengths [7, 8, 9, 10, 11].

Photodynamic therapy is a non‐invasive method for treating malignancies. In this treatment method, a suitable photosensitiser is administered. The photosensitiser is activated by exposure to light for a specified period. The light dose supplies sufficient energy to stimulate the photosensitiser as the result of activation reactive oxygen is generated. The cell target reacts with oxygen and kills the cells. Photodynamic therapy is an effective treatment but due to the limited tissue penetration of light has rarely applied for deep cancer [12, 13, 14].

Radiation therapy is another commonly used treatment for cancer. One of the limitations of this method is the destructive effects of ionising radiation on healthy tissues around the tumour. Other limitations of this method is the response of different types of malignancies to radiation [15, 16].

Nowadays, the use of combination methods has been considered by many researchers. In other words, radiotherapy is used to increase the efficacy of photodynamic therapy [17, 18, 19, 20]. It means that, the light is generated by QDs upon exposure of X‐rays and activate the photosensitisers. The utilisation of QDs as an intracellular light source for photosensitiser activation is a novel method to enable photodynamic therapy of deep cancers [21, 22, 23]. However, there are few studies in this area.

In this study, the application of the MPA‐capped Mn‐dopped ZnS quantum dots for photosensitiser activation in chondrosarcoma cancer cells in vitro was investigated. The reason for using ZnS:Mn is that material is non‐toxic and easy‐made in water by simple wet chemistry. It is also to be excited by various sources, including UV light and X‐ray, which further allows its application in both fluorescence imaging and light source in photodynamic therapy for deep cancer treatment [24].

2 Materials and methods

2.1 Materials

ZnSO₄, Na₂ S, MnCl₂, 3‐Mercaptopropionic acid (MPA), ethylenediamine (ED), N‐hydroxysuccinimide (NHS), and Spermine (SP) were purchased from Merck (Germany). 1‐ethyl‐3‐[3‐dimethylaminopropyl] carbodieimide hydrochloride (EDC) were purchased from Sigma–Aldrich (Munich, Germany) and Chlorin e6 (Ce6) were purchased from Medkoo (USA). All solvents and chemicals were supplied by Sigma–Aldrich and have the highest grade available. Dialysis was carried out using Spectra/Por dialysis membranes (Spectrum Laboratories, Houston, USA).

The prepared nanoquantum dots were characterised by transmission electron microscopy (TEM) and scanning electron microscope (SEM) experiments that were conducted on a Leo 912AB microscope (Germany) operated at 120 kV and Leo 1450VP (Germany), respectively. X‐ray powder diffraction (XRD) patterns were recorded on a Bruker D4 X‐ray diffractometer with Ni‐filtered Cu KR radiation (40 kV, 30 mA). The weight loss of shell and cysteine percentage was studied using thermogravimetric analysis (TGA), (Mettler Toledo LF, Switzerland). Fourier‐transform infrared spectroscopy (FT‐IR) spectra were collected on a Nicolet Fourier spectrophotometer, using KBr pellets (USA). The elemental analysis was studied using by inductively coupled plasma optical emission spectrometry (ICP‐OES) (USA). Proton nuclear magnetic resonance (¹ HNMR) [300 MHz] spectra were obtained by using a Bruker Avance DRX‐300 Fourier transformer spectrometer. Chemical shifts are reported in parts per million (δ) downfield from tetramethylsilane (TMS).

2.2 MPA‐capped Mn‐dopped ZnS quantum dots synthesis

Mn‐doped ZnS QDs were synthesised in an aqueous solution in accordance with previously reported method [25]. Briefly, the aqueous solution of ZnSO₄ (0.1 M, 5 ml), MnCl₂ (0.01 M, 2 ml), and MPA (0.04 M, 50 ml) was added to a three‐neck flask. The pH of the mixed solution was adjusted to 11 with NaOH (1 M) and then stirred under N₂ at room temperature for 30 min. Subsequently, a solution of Na₂ S (0.1 M, 5 ml) was added immediately and continues stirring for 30 min. After a further 2 h of stirring at 50°C under open air, the formed MPA‐capped Mn‐doped ZnS QDs were purified by precipitation with ethanol and centrifuged. The obtained QDs washed with ethanol and dried.

2.3 QD‐ED and QD‐SP (V, VI) synthesis

The QD‐ED and QD‐SP are species of nanoquantum dots with amino terminal group that showed as Mn: ZnS‐CONH₂. The Mn: ZnS‐CONH₂ was synthesised by the reaction between carboxylic acid end groups of QDs with a series of diamnio groups (ED and SP) with different chain lengths using amide linkage formation. Briefly, the first flask contained 25 mg of QDs, 5 mmol of EDC, and 5 mg of NHS that dissolved in 10 ml of distilled water and stirred at dark for 1 h. The second flask contained the excess amounts of ethyelendiamine (15 mmol) or spermine (15 mmol) that dissolved in 5 ml of distilled water. Subsequently, the contents of the second flask were added dropwise to the first flask over a period of 15 min. The reaction mixture was stirred for 48 additional hours at room temperature. The reaction mixture was centrifuged for 15 min and the resulting products resolved in distilled water and then freeze‐dried.

2.4 Covalent and non‐covalent synthesis of QD‐ED‐Ce6 and QD‐SP‐Ce6 (VII–X)

The synthesis of the QD‐ED‐Ce6 and QD‐SP‐Ce6 was done in two pathways, covalent and non‐covalent.

For covalent synthesis, 25 mg of Chlorin e6 (Ce6), 16 mg of EDC, and 9.6 mg of NHS were added to 10 ml of distilled water and stirred at dark for 1 h. Then, another flask contains 5 mg of QD‐ED or QD‐SP that was dissolved in 5 ml distilled water added to the reaction mixture of Chlorin e6 to form the amide linkage, separately. The reaction mixtures were stirred in room temperature for 48 h at dark. Finally, the crude products of QD‐ED‐Ce6 and QD‐SP‐Ce6 were purified using 500 Da cut‐off Spectra/Por dialysis tubing by dialysing against three changes of water.

For non‐covalent synthesis, the aqueous solution of Ce6 (25 mg in 10 ml of D. H₂ O) was reacted with 5 mg of QD‐ED (in 5 ml of D. H₂ O) or 10 mg of QD‐SP (in 5 ml of D. H₂ O), separately. Two reaction mixtures were stirred in room temperature for 48 h at dark. Finally, the crude products of QD‐ED‐Ce6 and QD‐SP‐Ce6 were purified using 500 Da cut‐off Spectra/ Por dialysis tubing by dialysing against three changes of water.

2.5 Structural characterisation

The prepared QDs were characterised by TEM method Leo 912AB microscope (Germany) operated at 120 kV. Powder XRD patterns were recorded on a Bruker D4 X‐ray diffractometer with Ni‐filtered Cu KR radiation (40 kV, 30 mA). FT‐IR spectra were obtained by using a Thermo Nicolet (AVATAR 370 FT‐IR, USA). ¹ HNMR (300 MHz) spectra were obtained by using a Bruker Avance DRX‐300 Fourier transformer spectrometer. Chemical shifts are reported in parts per million (δ) downfield from TMS.

2.6 Effect of quantum dots synthesised on the cell line SW1353

2.6.1 Cellular uptake study

Chondrosarcoma cells (SW1353 line; American Type Culture Collection HTB‐94) were grown under standard cell culture procedure in Dulbecco's Modified Eagles medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin. The SW1353 cells (7 × 10³ cells/ml) were seeded on sterile 96‐well plates, and incubated to obtain nearly confluent cell layers after 24 h at 37°C in a humidified atmosphere with 5% Co₂. On the day of the experiment, culture medium was removed and aqueous solutions of free Ce6, free QD, and QD‐Ce6 complexes (QD‐ED, QD‐SP, QD‐ED‐Ce6 by covalent bond, QD‐ED‐Ce6 by non‐covalent bond, QD‐SP‐Ce6 by covalent bond, QD‐SP‐Ce6 by non‐covalent bond) with different concentrations (0.1, 0.25, 0.5, 1, 2, and 5 µmol l⁻¹) were added to each wells for 24 h. At the time points of 2, 4, and 24 h, the cells viewed under the florescent microscope to determine maximum uptake.

2.6.2 Irradiation of the cells

X‐ray irradiation (6 MV) was performed by linear Accelerator (Siemens, Germany) at a dose rate of 200 cG/min.

2.6.3 Cell viability study

To determine the effect of the Ce6, QD and QD‐Ce6 complexes on cell growth, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was performed. The medium of the wells was removed and 100 μl of medium without FBS and then 10 μl of the MTT solution was added. Plates were incubated for 4 h at 37°C. The medium containing unreacted dye was removed. Then, 200 μl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals formed by live cells. The optical absorbance was recorded by ELISA Reader at 570 nm (reference wavelengths 630 nm). Finally, survival rate was expressed as a percentage of viable treated cells relative to untreated control cells. All experiments were done in triplicates, and results were reported as mean ± standard deviation.

2.7 Statistical analysis

To analyse the results of the experiments, SPSS software was used. Statistical significance was determined using t test and a P ‐value of less than 0.05 was considered significant.

3 Results and discussion

3.1 Characterisation of QD‐Ce6 complexes

3.1.1 FTIR and NMR spectra

The FTIR spectra were used to determine the successful synthesis of QDs and incorporation of ED and SP to synthesis of QD‐ED and QD‐SP, respectively. The FTIR spectra of QD shown in Fig. 1 a. Since all the free ions and capping molecules were removed by solvent and centrifugal purification and only the purified precipitates of QDs were gathered, the observed peaks of MPA in the FTIR spectra of QDs could only be related to bond formation between QDs and MPA (QD‐bound MPA). The significant absorbance peaks at 3235, 2940, 1645, 1275, and 845 cm⁻¹ were associated with OH (H‐bonded), C‐H stretching vibrations, C = O stretching vibrations, and C‐S vibration, respectively [26]. In addition, the FTIR observed peak around 600 cm⁻¹ is assigned to the Zn‐S and Mn‐S bonds [27, 28]. These results indicated the successful incorporation of MPA in synthesis of Mn‐doped ZnS. The FTIR spectra of QD‐ED showed in Fig. 1 b. The significant absorbance peaks at 3287, 2930, 2860, 1645, and 1566 cm⁻¹ were associated with N‐H stretching vibrations, C‐H stretching vibrations, C = O stretching vibrations of amide, and N‐H bending vibrations, respectively. The broad adsorbed band at 3200–3400 cm⁻¹ is assigned to O‐H stretching mode (H bonded). Also, the results of FTIR confirmed the presentation of ethyelendimaine in QD‐ED structure. Fig. 1 c showed the FTIR spectra of QD‐ED‐Ce6. The same absorbance peaks with QD‐ED was observed but the C = O band was appeared in the 1680 cm⁻¹. Since there are two different C = O groups in final product, the C = O group of formed amide (resulted of reaction between QD and ED, and QD‐ED and Ce6) and remained carboxylic acid groups of Ce6, the C = O stretching vibrations peaks due to overlapping of different C = O groups at 1680 and 1691 cm⁻¹ were appeared. The FTIR spectra of QD‐SP shown in Fig. 1 d. The significant absorbance peaks at 3435, 3055, 2989, 1635, 1559, and 1263 cm⁻¹ were associated with OH (H‐bonded), N‐H stretching vibrations, C‐H stretching vibrations, C = O stretching vibrations of amide, N–H bending, and C‐N vibrations, respectively. The FTIR spectra of QD‐SP‐Ce6 (Fig. 1 e) shows significant absorbance peaks at 3431, 2923, 2875, 1675, and 1653 cm⁻¹ that related with OH (H‐bonded), N‐H stretching vibrations, C‐H stretching vibrations, C = O stretching vibrations of acid and amide bonds, respectively.

Fig. 1.

FT‐IR spectra of synthetised nanoparticles

(a) QD, (b) QD‐ED, (c) QD‐ED‐Ce6, (d) QD‐SP, (e) QD‐SP‐Ce6

¹ HNMR spectra of QDs in deuterium oxide is shown in Fig. 2. There are two triplet signals at 2.84 and 2.51 ppm that the integration of all these peaks shows two protons. Due to being formed the carboxylate salt (sodium salt) of QDs in alkaline medium, the hydrogen signal of carboxylic acid was disappeared. Also due to incorporation of MPA in synthesis of QDs, the hydrogen signal of S‐H group in MPA was missed.

Fig. 2.

HNMR spectra of QDs

3.1.2 TEM, SEM, XRD and ICP analysis

The TEM images of QDs, QD‐ED, and QD‐ED‐Ce6 are illustrated in Figs. 3 a –d. Fig. 3 a shows that QDs have spherical shape and the size of these particles, which are uniformly distributed, is about 5 nm. Fig. 3 b shows the TEM image of QD‐ED. The QDs have a core‐shell structure of ED, which is spherical shape and consistent with the results of NMR and FTIR analyses. The overall shape of QD‐ED particles is like to Aliens face. The TEM images of QD‐ED‐Ce6 are illustrated in Fig. 3 c. TEM image of QD‐SP is showed in Fig. 3 d. The QD‐SP nanoparticles have spherical shape like QD‐ED.

Fig. 3.

TEM images of synthetised nanoparticles

(a) QDs, (b) QD‐ED, (c) QD‐ED‐Ce6, (d) QD‐SP

The surface morphology and morphology changes of the QDs, QD‐ED, QD‐ED‐Ce6, and QD‐SP were studied using scanning electron microscopy (SEM) and shown in Figs. 4 a –d. The SEM image of prepared MPA‐capped Mn‐dopped ZnS QDs (Fig. 4 a) shows that the nanoparticles have spherical and regular shape. The previous research has shown that the capping agents play a very important role to control the shape and size of the growing nanoquantum dots [29]. The SEM images of the QD‐ED are shown in Fig. 4 b. The QD‐EDs have the spherical shape and uniformly distributed in the nanostructure. The SEM image of QD‐ED‐Ce6 (Fig. 4 c) shows a spherical nanostructure with uniform distribution. The QD‐SP nanoparticles have spherical and regular shape similar to QD‐ED image (Fig. 4 d).

Fig. 4.

SEM images of synthetised nanoparticles

(a) QDs, (b) QD‐ED, (c) QD‐ED‐Ce6, (d) QD‐SP

The X‐ray diffraction pattern of the MPA‐capped ZnS QDs is shown in Fig. 5. The average crystal size of the MPA‐capped ZnS QDs was obtained by calculation of Sherrer's equation, is about 3.9 nm. In XRD pattern, the diffraction of MPA‐capped ZnS QDs with cubic zinc blende crystal structure by comparing with the pattern of the bulk ZnS material (Ref code: 01‐080‐0020) from (111), (220), and (311) at peaks of 28.8, 47.8, and 57.1° is shown in Fig. 2 c. The small crystalline size of QDs caused the peaks in the pattern were broad and overlapped at the higher diffraction angle.

Fig. 5.

XRD patterns of MPA‐capped ZnS QDs

The inductively coupled plasma analysis (ICP‐OES) was shown that QD consists 6.00% Mn, 2.94% S, and 29.04% Zn.

3.1.3 EDS and TGA analysis

The energy dispersive X‐ray spectroscopy (EDS) analysis of QD‐ED, QD‐SP, and QD‐ED‐Ce6 is shown in Fig. 6 a –c. This analysis shows that the elemental compositions of the nanostructure are C, N, O, S, Mn, and Zn. The EDS analysis for QD‐ED‐Ce6 shows that amount of carbon and nitrogen is more than the QD‐ED and QD‐SP EDS analysis.

Fig. 6.

EDS result of synthetised nanoparticles

(a) QD‐ED, (b) QD‐ED‐Ce6, (c) QD‐SP

The thermal gravimetric analysis curves of QDs, QD‐ED, and QD‐ED‐Ce6 were carried out and presented in Fig. 7. The starting heating treatment was about 20°C and then increased up to 800°C. In the TGA synthesis, the landing of TGA of QDs until it becomes horizontal around 200°C showed the initial weight loss that related to removal of surface adsorbed water, while the second weight loss from 200 to 800°C is probably due to the decomposition of chemically bound groups. The TGA pattern of QDs shows about 9.76% weight loss in the region of 20–800°C. The TGA curves of QD‐ED show the primary weight loss around 200°C that due to the removal of surface adsorbed water and the next weight loss at 200–800°C could be attributed to decomposition of chemically bound groups and evaporation of the decomposed product. Due to adding the ethylenediamine to QD as an organic linker, the TGA pattern of QD‐ED shows more weight loss than QDs in the same region. The TGA pattern of QD‐ED shows about 58.70% weight loss in the region of 20–800°C. The TGA curves of QD‐ED‐Ce6 show the same manner with QD‐ED, but the more weight loss was observed that related to presented of Ce6 in final structure. The TGA pattern of QD‐ED‐Ce6 shows about 66.10% weight loss in the region of 20–800°C.

Fig. 7.

TGA analyses of the QDs, QD‐ED and QD‐ED‐Ce6

3.2 MTT assay

Cell viability of QDs and its conjugates was performed by MTT assay at different concentrations each in three replicates on SW1353 cells. The cytotoxic effects of MPA‐capped Mn‐dopped ZnS quantum dots‐chlorin e6 in SW1353 cells are tested by the exposure of X‐ray. Fig. 8 displays the percentage cell viabilities for free Ce6, free QD, and QD‐Ce6 complexes (QD‐ED, QD‐SP, QD‐ED‐Ce6 by covalent bond, QD‐ED‐Ce6 by non‐covalent bond, QD‐SP‐Ce6 by covalent bond, QD‐SP‐Ce6 by non‐covalent bond) with and without X‐ray treatment, respectively.

Fig. 8.

Cell viabilities for free Ce6, free QD and QD‐Ce6 complexes (0.5 µmol l⁻¹) before and after X‐ray treatment on SW1353 cells, respectively

In this study, after the X‐ray exposure, the metabolic activity of QD‐SP‐Ce6 by covalent bond‐treated cells dropped heavier to 61.1% that statistically was significant (P  ≤ 0.001). In explained mechanism of cell killing by X‐ray photodynamic therapy, the QDs are exposed to ionising radiation (X‐rays). The emitted light of QD activates the photosensitiser (PS). The photosensitiser is transmitted from the ground state to the singlet excited state by absorption of emitted light of quantum dot. The excited photosensitiser can be gone to a long‐lived triplet state. Then, photosensitiser transferred its energy into the molecular oxygen. These reactions led to generate free radicals and induce cell death. These reactive species caused cell necrosis or apoptosis with effects on mitochondria, plasma membrane, lysosomes, endoplasmic, and endosomal networks [30]. In this study, a possible explanation is that the photosensitiser Ce6 absorbed some energy from X‐ray radiation and killed a few cells consequently. As represented in Geoffrey D. Wang study, X‐ray‐induced photodynamic therapy (X‐PDT) is essentially a radiotherapy and photodynamic therapy (PDT) combination. This interactions attack both cell membrane and DNA, and finally leading to lethal damage that is beyond the repairs of cells [21]. However, in our study, the cell viability reduced to 84.7%, 78.5%, 75.4%, and 78.4% after the X‐ray exposure in the free QD, free Ce6, QD‐ED‐Ce6 by covalent bond and QD‐SP‐Ce6 by non‐covalent bond–treated cell samples, respectively. For QD‐ED‐Ce6 by non‐covalent bond–treated cells, there is no further cell killing after exposure of X‐ray. From the data shown, the efficiency of cell killing by QD‐SP‐Ce6 with covalent bond combination is around 40% compared to tumour cell line before radiation, and around 50% compared to tumour cell line without QD (control group). These results indicate that X‐ray can activate these conjugate for chondrosarcoma treatment.

4 Conclusion

Photodynamic therapy is an effective treatment but due to the limited tissue penetration of light has rarely applied for Chondrosarcoma. The utilisation of QDs as an intracellular light source for photosensitiser activation is a novel method to enable photodynamic therapy of deep cancers. In this study, the efficiency of cell killing by QD‐SP‐Ce6 by covalent bond combination is around 40%. The obtained results indicate that X‐ray can activate all of the mentioned QD complexes for Chondrosarcoma treatment.