Characterization and Application of Nanomaterials (2022) Volume 5 Issue 1 doi:10.24294/can.v5i1.1408 10 Original Research Article Synthesis and in Vitro anticancer properties of Cu2–xSe–AIPH nano- materials Juan Lu, Yini Mao, Jun Yang* School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China. E-mail: jyang@swu.edu.cn ABSTRACT The Cu2–xSe nanoparticles were synthesized by high temperature pyrolysis, modified with aminated polyethylene glycol in aqueous solution and loaded with compound 2,2′–azobis[2–(2–imidazolin–2–yl)propane] dihydrochloride (AIPH). The obtained nanomaterials can induce photothermal effect and use heat to promote the generation of toxic AIPH radicals under the irradiation of near-infrared laser (808 nm), which can effectively kill cancer cells. A series of in vitro experiments can preliminarily prove that Cu2–xSe–AIPH nanomaterials have strong photothermal conversion abil- ity, good biocompatibility and anticancer properties. Keywords: Cu2–xSe Nanoparticles; AIPH; Photothermal Effect; Anticancer Properties ARTICLE INFO Received: 26 September 2021 Accepted: 19 November 2021 Available online: 30 November 2021 COPYRIGHT Copyright © 2022 Juan Lu, et al. EnPress Publisher LLC. This work is li- censed under the Creative Commons Attribu- tion-NonCommercial 4.0 International Li- cense (CC BY-NC 4.0). https://creativecommons.org/licenses/by-nc/4 .0/ 1. Introduction Cancer has now become one of the major threats to people’s health all over the world, and countless people lose their lives due to cancer every year. However, there are problems lying in treatment methods of traditional chemotherapy, radiotherapy, and surgery, such as great side effects and low treatment efficiency. Therefore, it is ur- gent to develop some novel and effective cancer treatment methods[1–5]. In recent years, photothermal therapy (PTT) has attracted more and more scientists’ attention in the field of cancer treatment due to its low invasiveness[6–8]. The therapeutic principle is that photothermal materi- als can effectively convert light energy into heat energy after absorbing the external light energy, thereby triggering the withering or death of cancer cells. Based on this, batches of excellent photothermal materials have been created continuously, including binary chalcogenides with unique physical and chemical properties[9]. At the same time, near-infrared light is also closely connected to cancer treatment due to its deeper penetration depth in biological tissues compared with ultra- violet light[10–12]. Take advantage of the photothermal effect of selenide Cu2–xSe and combine it with other therapeutic agents. Cu2–xSe nanoparticles with a particle size of 20–40 nm were synthesized by a classical high-temperature pyrolysis method, and the compound 2,2′–azobis[2– (2–imidazoline–2–yl)propane] dihydrochloride (AIPH), an azo com- pound that rapidly decomposes during heating to produce toxic alkyl groups, was loaded after aminated polyethylene glycol modification. 11 Due to the large tumor cell gap, the tumor microen- vironment has high enhanced permeability and re- tention effect (EPR), and anti-tumor nanomaterials will passively be targeted to tumor sites to play a role of treatment; without extra light and heating, the body temperature about 37 ℃ will not cause the decomposition of AIPH to generate free radicals. Therefore, AIPH itself will not cause damage to the normal tissues of the organism. Due to the lack of copper atoms, Cu2–xSe nanoparticles have strong plasmon resonance absorption (LSPR) and excel- lent photothermal properties in the near-infrared region where the penetration of biological tissues is strong, and have been increasingly used in the field of photothermal therapy. The Cu2–xSe–AIPH nano- particles synthesized by this system can generate photothermal temperature up to 48 ℃ after absorb- ing 808 nm near-infrared light, which is sufficient for photothermal therapy. Under the action of near-infrared light, the photothermal effect of Cu2– xSe nanoparticles can play a synergistic effect with AIPH free radicals to kill tumor cells. The anti- cancer properties of Cu2–xSe–AIPH materials were preliminarily proved through a series of in vitro ex- periments. 2. Experiment 2.1 Experimental reagents The reagents used in this experiment including cuprous chloride (CuCl), aminated polyethylene glycol (NH2–PEG(2000)–NH2), 1–(3–dimethy- laminopropyl)–3–ethylcarbodiimide hydrochloride (EDC), N–hydroxysuccinimide (NHS), oleylamine, octadecene, and anhydrous ethanol (C2H5OH) were all at an analytical grade and were used directly in the experimental process without further purifica- tion. All chemicals were purchased from Sig- ma-Aldrich, except for the selenium powder (Se) which was purchased from Sinopharm Chemical Reagent Co., Ltd. 2.2 Preparation process Cu2–xSe nanoparticles were synthesized by a typical solvent injection method with slight modi- fications from the previous literature[13]. The prepa- ration process was mainly divided into two stages. First, the precursor of Se–OAm (seleni- um-oleylamine) was synthesized. Weigh 10 mmol of selenium powder and 10 mL of oleylamine into a three-necked flask, set up a pyrolysis device, add nitrogen, and heat it to 140 ℃ with a heating mantle for 10 min to remove moisture and some low-boiling impurities. Then the temperature was raised to 320 ℃ for a period of time, and the ob- tained brown solution was the precursor Se–OAm. At the same time of the constant temperature reac- tion, take another four-necked bottle, weigh 0.5 mmol of CuCl, 8 mL of octadecene (ODE) and 2 mL of oleyl amine in it, also keep it in a nitrogen environment for 10 min, and then heat it up to 200 ℃ and immediately inject the Se–OAm solution pre- pared in the previous step, and then continue to heat it up to 220 ℃ for a period of time. The ob- tained black-brown solution was washed three times alternately with ethanol, cyclohexane and ethanol to obtain Cu2–xSe nanoparticles, wherein the amount of each washing solvent was equal to the volume of the reaction solution, about 10 mL. The synthesized Cu2–xSe nanoparticles were then modi- fied with amino groups to improve their hydro- philicity for subsequent compound loading and testing applications. The prepared nanoparticles were mixed with 10 mg of EDS and 20 mg of NHS in methanol solvent and stirred for 1.5 h in the dark environment. Then 40 mg of NH2–PEG(2000)–NH2 was added to the above solution, and the obtained solution was washed with methanol and deionized water and centrifuged after the reaction was con- tinued for 12 h. Subsequently, 0.2 g of AIPH and the aminated product were kept in a water bath at 70 ℃ for 4 h. Finally, the reactant was washed several times with ethanol to remove excess AIPH, and the precipitate was collected[14]. 2.3 Characterization of properties Phase analysis was performed using a Rigaku D/max–TTR–III diffractometer (Cu–Kα radiation λ = 0.15405 nm), the scanning range 2θ = 10°–90°, 10°/min of the scanning speed; a FEI Tecnai G2S– Twin transmission electron microscope was used to observe the morphology (the sample was first dis- persed in cyclohexane or ethanol solvent, and then dropped on a carbon support film for observation 12 after the solvent was volatilized); confocal laser scanning microscope observation was performed on a Leica SP8 device (CLSM); X-ray electron spec- troscopy (XPS) measurements were performed us- ing a surface analysis system (Thermofisher Esca- lab Xi+) with AlKα radiation (hν = 1486.6 eV) from a monochromatic X-ray source and a spot size of 500 μm. The above characterizations were all done at room temperature. 3. Results and discussion 3.1 Morphology, phase and elemental analy- sis Figure 1(a) is the XRD pattern of the product synthesized by the high temperature pyrolysis method in the first step. It can be seen that the XRD pattern of the product shows the position and rela- tive intensity of the main characteristic peaks, which are consistent with the standard pattern of Cu2–xSe (JCPDS No.06–0680), proving the suc- cessful synthesis of Cu2–xSe crystals with good crystallinity. Figure 1. (a) XRD pattern of Cu2–xSe nanoparticles; (b) dynamic light scattering particle size distribution of Cu2–xSe and Cu2–xSe– AIPH nanoparticles. Figure 2. (a) TEM of Cu2–xSe nanoparticles; (b) TEM of Cu2–xSe–AIPH nanoparticles. Figure 2 shows the morphology of Cu2–xSe and Cu2–xSe–AIPH nanoparticles. It can be clearly seen from Figure 2(a) that the particle size of Cu2– xSe nanoparticles prepared by high temperature py- rolysis is 20–40 nm, and the morphology is mostly square or spherical with clearly visible outline. Af- ter transferring water and loading AIPH, it can be seen from Figure 2(b) that the particle size in- creases to about 80 nm, and the morphology changes to a single spherical shape. It can be con- cluded that the increase in the particle size of nano- particles may be due to the coagulation of the oily Cu2–xSe nanoparticles in the subsequent reaction process of the aqueous solution. The mapping im- ages of Cu, Se, C, N, and O elements in Figure 3 further verify the successful synthesis of the prod- uct. Figure 1(b) shows the dynamic light scattering particle size distribution of Cu2–xSe and Cu2–xSe– AIPH. It can be seen from the figure that the parti- cle size of the particles is basically the same as the 13 value in the previous transmission diagram. Figure 3. Elemental mapping images of Cu2–xSe–AIPH nanoparticles. Figure 4. XPS spectra of Cu 2p (a) and Se 3d (b) in Cu2–xSe–AIPH nanoparticles. Figure 4 is the XPS spectrum of Cu and Se elements in Cu2–xSe–AIPH nanoparticles. Specifi- cally, the binding energy of the characteristic peaks of Cu 2p in Figure 4(a) is 952.2 eV and 932.4 eV, proving the existence of monovalent copper in the material; the binding energy of the characteristic peaks of Se 3d in Figure 4(b) is 54.7 eV, which proves that selenium in the material is negative di- valent. The change of Zeta potential during the re- action also confirmed the progress of each step of the reaction. Figure 5(a) is the Zeta potential change diagram of each step of the reaction product aqueous solution (Cu2–xSe, NH2–Cu2–xSe, Cu2–xSe– AIPH). It can be seen that the potential of the initial Cu2–xSe particle solution was –10.35 mV, which increased significantly after the introduction of amino groups (–1.3 mV). The potential was further increased to 16 mV. The value of Zeta potential can reflect the stability of the system to some extent. The lower the Zeta potential value (positive or neg- ative) is, when the attractive force exceeds the re- pulsive force, the more likely the nanoparticles in the system are to agglomerate. Therefore, combined with the change of the Zeta potential value, the change of the particle size and morphology in the transmission image can be explained to a certain extent. At the beginning, the potential of the Cu2– xSe particle solution was –10.35 mV, and the parti- cles began to have a tendency to coagulate, but it was relatively stable; after the amino group was modified in the aqueous solution, the potential of the material became –1.3 mV, and the negative val- ue of Zeta potential became lower , the system be- gan to become unstable, and the particles will co- agulate to a certain extent; finally, the Zeta potential of the Cu2–xSe–AIPH material became 16 mV, the positive value of Zeta potential was higher, and the system gradually tended to be stable. After the square or spherical Cu2–xSe particles coagulated in the solution, the particle size of the final material increased and the morphology changed to a single spherical shape. 14 Figure 5. (a) Zeta potential change diagram of products of each step in the reaction process; (b) standard absorption curve of AIPH. Figure 5(b) is the standard curve of the sup- ported compound AIPH. From the figure, the linear relationship between the concentration (X) of AIPH and its UV-Vis absorbance (Y) can be obtained: Y = 0.01207X – 0.05899. Figure 6(a) is the UV-Vis absorption curve of the Cu2–xSe material before and after loaded with AIPH. It can be seen that, com- pared with the absorption curve of Cu2–xSe nano- particles, that of Cu2–xSe–AIPH appeared at 360 nm. The characteristic absorption peaks of AIPH proved the successful loading of AIPH. Combined with the linear relationship obtained from the AIPH standard curve, the loading of AIPH can be calculated to be 8.1%. To verify the generation of AIPH radicals, 1 mg/mL 2,2′–azobis(3–ethylbenzothiazoline–6– sulfonic acid) (ABTS) and Cu2–xSe–AIPH of the same concentration were mixed and reacted for 2 h in a water bath at 45 ℃, and the UV-Vis absorption spectrum of the reaction solution was measured. Figure 6(b) is the characteristic absorption curve of the reaction product ABTS, indicating that AIPH decomposes to generate free radicals under the heating condition of 45 ℃, which further react with ABTS to generate ABTS free radicals. Figure 6. (a) UV-Vis absorption curves of Cu2–xSe, Cu2–xSe–AIPH and AIPH; (b) the reaction curve of AIPH and ABTS when heat- ed at 45 ℃. 3.2 Photothermal conversion performance Since the photothermal effect of nanoparticles plays a key role in the subsequent treatment of tu- mor cells, the related photothermal properties of synthetic materials were first explored in aqueous solution[15–17]. The temperature curves of Cu2–xSe, Cu2–xSe–AIPH solution and solvent water record- ed by an infrared thermal imager under the irradia- tion of near-infrared light (808 nm) are shown in Figure 7(a). It can be clearly seen that the temper- ature of the solvent water basically fluctuates around room temperature, while the temperature of Cu2–xSe and Cu2–xSe–AIPH materials increases sig- nificantly under the irradiation of 808 nm laser, which preliminarily proves the photothermal con- version properties of the materials. Moreover, the temperature of the material can reach 48 ℃ or above in a short period of 5 min, meeting the tem- perature requirements for the treatment of cancer cells in vitro and in vivo. The subsequent connec- tion of amino groups and loading of AIPH may block the absorption of near-infrared light by the photothermal agent Cu2–xSe to a certain ex- tent, but happily, on the contrast, the temperature 15 rise rate of Cu2–xSe–AIPH nanoparticles every 50 s and the final stable temperature value only decrease slightly, which does not affect the performance and application of the material[18]. Figure 7. (a) Heating curves of Cu2–xSe, Cu2–xSe–AIPH solution and water under 808 nm laser irradiation; (b) temperature variation curves of Cu2–xSe–AIPH solution with different concentrations under 808 nm laser irradiation. All laser power is 1 W/cm2. Then, the temperature changes of Cu2–xSe– AIPH solutions with different concentrations (31.25, 62.5, 125, 250, 500 μg/mL) during the irradiation time of 500 s were further explored. It can be seen from Figure 7(b) that the temperature of each group of solutions increases regularly with the pro- longation of infrared light irradiation time and the greater the concentration, the faster the temperature rises. When the material’s concentration is 31.25 μg/mL, the heating rate is the smallest, and the final stable temperature is only about 33 ℃, which is lower than the normal temperature of the hu- man body; when the material’s concentration is 500 μg/mL, the heating rate is the largest, and the final stable temperature can reach around 48 ℃, which is enough to induce apoptosis and death of cancer cells[19,20]. Figure 8. Infrared thermal images of the Cu2–xSe–AIPH material (500 μg/mL). In order to observe the photothermal effect of the Cu2–xSe–AIPH material more intuitively, we used an infrared thermal imager to record the eve- ry-minute infrared thermal imaging pictures of 500 μg/mL material when the material was irradiated by 808 nm laser for 5 min. It can be seen from Figure 8 that with the extension of time, the color of the thermal imaging picture of the material gradually changes from light yellow to dark red to white-hot. It can be determined that the material can generate a large amount of heat after absorbing near-infrared light, causing a significant increase of the solution temperature. Compared with the standard tempera- ture bar, it can be roughly judged that the final photothermal temperature of the material can reach about 48 ℃. The above tests show that Cu2–xSe– AIPH nanomaterials can effectively convert near-infrared light into thermal energy, and thus have the potential for photothermal therapy. 3.3 Anticancer properties in vitro Before carrying out biological experiments, the biocompatibility of the materials must be effec- tively evaluated to avoid unnecessary side effects to the organism[21]. After co-culturing Cu2–xSe–AIPH materials of different concentrations (15.6, 31.3, 62.5, 125, 250, 500 μg/mL) with L929 fibroblasts for 24 h, the cell biocompatibility was evaluated by MTT analysis. Figure 9(a) shows the state of cells after cultured with different concentrations of Cu2– xSe–AIPH nanomaterials. It can be seen that even 16 when the material’s concentration is as high as 500 μg/mL, the cell viability is still as high as 85%, which preliminarily proves Cu2–xSe–AIPH nano- materials have good biocompatibility. Figure 9. (a) Biocompatibility of Cu2–xSe–AIPH nanomaterials with different concentrations; (b) cytotoxicity of Cu2–xSe and Cu2– xSe–AIPH nanoparticles with different concentrations to HeLa cells. Next, the cytotoxicity of nanoparticles to HeLa cells was analyzed by a similar MTT method. Fig- ure 9(b) is a histogram of the viability of HeLa cells cultured for the same time under different conditions. As shown in the figure, the cells were divided into four groups, namely: AIPH, NIR, Cu2– xSe+NIR, Cu2–xSe–AIPH+NIR. It can be seen from the figure that the survival rate of cells in all AIPH and NIR groups can reach more than 95%, which proves that AIPH culture alone or infrared light ir- radiation has basically no effect on cells. The cell viability of the Cu2–xSe+NIR group dropped to about 50% (with a concentration of 500 μg/mL), which was attributed to the good photothermal properties of Cu2–xSe nanoparticles under the action of near-infrared light, while the cell viability of Cu2–xSe–AIPH+NIR group can be reduced to 20% (with a concentration of 500 μg/mL). It can be seen from the analysis that this is because compared with Cu2–xSe+NIR, AIPH in the Cu2–xSe–AIPH material can also be decomposed into free radicals under the photothermal action of Cu2–xSe, and the synergistic effect of the two further enhances the ability of the system to kill cancer cells[22,23]. In order to illustrate the above cytotoxic results more intuitively, a staining experiment was per- formed using propidium iodide (PI) (this staining method can only mark dead cells with red)[24,25]. Figure 10 shows the CLSM images of HeLa cells under different incubation conditions. After similar incubation in the cytotoxicity experiment, similar results were obtained in the staining experiment, namely: in the AIPH and NIR groups, the number of dead red cells was the least; the number of dead red cells in Cu2–xSe–AIPH+NIR was the highest, i.e., the treatment effect was the best, followed by Cu2–xSe+NIR. In conclusion, the above cell experi- ments can preliminarily prove that the Cu2–xSe– AIPH material has good biocompatibility and anti- cancer properties[26]. Figure 10. CLSM images of HeLa cells incubated under different conditions (the scale bar of all images is 100 μm). 4. Conclusion In summary, a series of experiments have proved that the photothermal conversion tempera- ture of the constructed Cu2–xSe–AIPH system is as high as 48 ℃, which can be used for photothermal therapy; in vitro cell experiments prove that the bi- ocompatibility of the material is better, and the cell 17 survival rate is as high as 85%, when the material concentration is 500 μg/mL; the survival rate of cancer cells in the Cu2–xSe–AIPH+NIR group could be reduced to 20%, when the material con- centration was 500 μg/mL. The above work shows that the Cu2–xSe–AIPH material has the potential for tumor therapy and can be further developed for bi- ological applications. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements National Natural Science Foundation of China (51302229). References 1. Sun Y, Zhou Q, Su S. Research progress of Chinese medicine compatibility in cancer treatment (in Chi- nese). 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