Facile Synthesis of Sulfobetaine-Stabilized Cu2O Nanoparticles and

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Facile synthesis of sulfobetaine-stabilized CuO nanoparticles and their biomedical potential. Marta Joanna Wo#niak-Budych, #ucja Przysiecka, Barbara Malgorzata Maciejewska, Daria Wieczorek, Katarzyna Staszak, Marcin Jarek, Teofil Jesionowski, and Stefan Jurga ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00465 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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ACS Biomaterials Science & Engineering

Facile synthesis of sulfobetaine-stabilized Cu2O nanoparticles and their biomedical potential

Marta J. Woźniak-Budycha, Łucja Przysieckaa, Barbara M. Maciejewskaa, Daria Wieczorekb, Katarzyna Staszakc, Marcin Jareka, Teofil Jesionowskic and Stefan Jurgaa

a

NanoBioMedical Centre, Adam Mickiewicz University in Poznan, Umultowska 85, Poznan, Poland, corresponding author: [email protected], b

Department of Technology and Instrumental Analysis, Faculty of Commodity Science,

Poznan University of Economics and Business, al. Niepodległości 10, Poznan, Poland, c

Institute of Technology and Chemical Engineering, Poznan University of Technology, ul. Berdychowo 4, Poznan, Poland.

a

Ł. Przysiecka: [email protected], B.M. Maciejewska: [email protected] ,

M. Jarek: [email protected], S. Jurga: [email protected] b

D. Wieczorek: [email protected]

c

K. Staszak: [email protected], T. Jesionowski: [email protected].

Highlights Spherical Cu2O nanoparticles were synthesized using zwitterionic surfactant as a stabilizing agent. Sulfobetaine-stabilized Cu2O nanoparticles have potential to be applied in cancer therapy. The nanotoxicity of Cu2O nanoparticles is related to the intercellular localization and ROS formation.

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Abstract A novel approach using a zwitterionic sulfobetaine-based surfactant for the synthesis of spherical copper oxide nanoparticles (Cu2O NPs) has been applied. For the first time, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate has been used as stabilizer to control the size and morphology of Cu2O NPs. Several techniques, such as transmission electron microscopy (TEM), X-ray diffraction (XRD) and fluorescence spectroscopy are used to investigate the size, structure and optical properties of synthesized Cu2O nanocrystals. The results indicate that copper(I) oxide nanoparticles with size in the range of 2 to 45 nm and crystalline structure, exhibit intense yellow fluorescence (λem = 575 nm). Furthermore, the cytotoxicity studies show that sulfobetaine-stabilized copper oxide nanoparticles prompt inhibition of cancer cell proliferation in a concentration-dependent manner, however, the adverse effect on the normal cells has been also observed. The results indicate that the sulfobetaine-stabilized Cu2O, due to their unique properties, have a potential to be applied in medical fields, such as cancer therapy and bioimaging.

Keywords: copper oxide nanoparticles, sulfobetaine, zwitterionic surfactant, flurorescence properties


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Introduction Despite the general concern related to toxicity of copper nanoparticles and copper-based materials, there are still number of their prospective applications in medicine.1 According to the common pharmaceutical concept, it cannot be stated unambiguously, whether the particles are harmful or therapeutic because the adverse effect is dose-dependent.2,3 The toxicity of copper-based nanomaterials results mainly from the generation of reactive oxygen species (ROS) which leads to damage of various proteins, lipids, and DNA.4 However, it should be clearly indicated that this effect is observed only at a relatively high concentration of nanoparticles.5 The toxicity of copper nanoparticles depends also on their size. Prabhu et al. have demonstrated that the oral lethal dose (LD50) of nano-sized copper is around 20% lower than LD50 assigned to micrometric ones (in vivo studies).6 It is worth noting that, in contrast to the other metallic nanomaterials (such as silver, gold or platinum nanoparticles), copper is classified as a trace metal, which is required for the regulation of metabolic processes.7 For example, copper is essential for healthy muscles tone and their function. It is crucial for the brain and the nervous system development. It takes a part in the transformation of melanin in the skin pigmentation, helps to collagen and elastin cross-link and plays a crucial role in the distribution of iron throughout the body. Furthermore, the copper deficiency can cause the development of cardiovascular diseases.8 According to the WHO recommendations, the daily mean intake of copper should not exceed 1.3-1.6 mg for adults and 0.7-1.1 mg for children aged 1 to 10 years.9 Moreover, the cost of the synthesis of noble metal nanoparticles is relatively high in comparison with the synthesis of copper and copper-based nanomaterials. For example, the cost of 7.25⋅1017 (1 mole of gold atoms) of Au nanoparticles production is 52.200$US and the cost of 1.55⋅1020 (1 mole of CuS molecules) of CuS nanoparticles is around 330$US.10



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Copper and copper-based nanomaterials have efficient biocidal properties, thus they can be used as an antimicrobial and antifungal agent or even antibiotic.11 They can also serve as a vehicle for drug delivery and as a photothermal agent for bioimaging, including positron emission tomography (PET), photothermal therapy (PTT) and photodynamic therapy (PDT).12 Zha et al. have investigated ultrasmall gelatin-stabilized CuS nanoparticles as multifunctional doxorubicin delivery system for combined photoacoustic imaging, tumor-selective chemotherapy, and PTT.12 In addition, the last four decades have exposed several applications of copper-based nanoparticles, as a catalyst, conductive inks, sensors/biosensors and component of the transparent solar cell.13,14 Goel and co-workers have reported that Cu2O hollow nanoparticles can be used for methylene blue-based DNA biosensing of Hepatitis B virus.15 Yuan et al. have indicated that Cu2O nanospheres reveal excellent adsorption ability for organic dyes, such as methyl orange, methyl blue or fuchsin acid, thus can be applied for photon catalytic degradation of organic pollutants under visible light.16 The favorable characteristics of cuprous oxide (Cu2O) nanoparticles, like low toxicity and good environmental acceptability, may facilitate a wide range of biomedical applications, especially as antibacterial and antifouling agent, but also as theranostic platform for imageguided therapy.17-19 Recently developed synthesis methods for copper and copper oxide nanoparticles production include chemical or hydrothermal reduction, laser ablation, vapour deposition, microwave radiation, electrochemical techniques etc.20-22 Among these methods, the chemical reduction is usually preferred, because it is quite fast, simple and cost-efficient. The properties of copper nanoparticles, including biodistribution and biosafety, depend upon surrounding environment of nanoparticles, as well as their surface modification. Several polymers (natural and/or synthetic), such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), dextran or chitosan, as

well

as

various

surfactants

like

cetyltrimethylammonium


bromide

(CTAB),

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dodecylbenzenesulphonic acid (DBSA) or N-Dodecylphosphonic acid, can be applied to reduce the toxicity of nanoparticles and/or improve their stability.23-25 One of the most interesting classes of surfactants are zwitterionic (amphoteric) surfactants, like sulfobetaines and their derivatives.26 Zwitterionic surfactants have anionic and cationic head both attached to the same molecule. The anionic part is based on sulfonates, phosphateor carboxylate moieties, and the cationic part generally consists of quaternary ammonium group. Due to the unique structure, zwitterionic surfactants exhibit excellent properties such as biodegradability, pH- and temperature-insensitivity and good solubility in water.27 Moreover, zwitterionic surfactants have lower critical micelle concentration (CMC) than anionic, cationic or nonionic ones. For example, the CMCs of CTAB, DBSA, and N-Dodecylphosphonic acid, determined in water at room temperature are 0.90, 0.55 and 0.54 mM/L, respectively.28-30 The CMC of sulfobetaine surfactant SB3C16 is 0.02 mM/L.31 It is very important parameter, because nanoparticles are formed at a concentration of surfactant above its critical value. The minimalization of the surfactant content during the nanoparticles synthesis is preferred from economical point of view. The application of zwitterionic surfactants as a capping agent in the nanoparticles synthesis is a relatively novel concept of the nanomaterials production. Suoza et al. have shown that imidazolium-based zwitterionic surfactant, i.e 3-(1-dodecyl-3imidazolio)propanesulfonate (ImS3-12), can be used as a stabilizer for the synthesis of highly dispersible in water and chloroform, palladium nanoparticles.32 The same group have presented the possibility of using 3-(1-butadecyl-3-imidazolio)propanesulfonate (IMS3-14) in palladium nanoparticle synthesis.33 An interesting approach towards the synthesis of metallic nanoparticles in the presence of zwitterionic surfactant has been reported by Gao et al. The authors have demonstrated that zwitterionic wormlike micelles consisting of 3-(N,N-dimethylpalmitylammonio)-propanesulfonate (PAPS) with AuCl4− counter-ions can be applied as a soft template to formulate gold nanoparticles and their alloys.34



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This report is devoted to the synthesis of the sulfobetaine-stabilized copper oxide nanoparticles (Cu2O NPs) and characterization of their properties, such as fluorescence, longterm stability, and cytotoxicity. The effect of the sulfobetaine surfactant, i.e. N-hexadecylN,N-dimethyl-3-ammonio-1-propanesulfonate (SB3C16), as well as the reducer and salt precursor concentration on the final properties of the copper oxide nanoparticles is also investigated and summarized. Materials and methods Materials Copper (II) chloride anhydrous (purity >99.995%), hydrazine monohydrate (purity 98%), were purchased from Sigma-Aldrich. All reagents were used without further purification. The sulfobetaine: N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB3C16) was prepared by Wieczorek D. et al. according to the procedure presented in the manuscript.31 Synthesis of copper oxide nanoparticles Copper oxide nanoparticles (Cu2O NPs) were synthesized using copper(II) chloride as a precursor, hydrazine as a reducing agent and sulfobetaine SB3C16 as a capping agent. The chemical reduction reactions were performed above critical micelle concentration of SB3C16, i.e. 0.02 mM/L. The Cu2O nanoparticles were obtained by mixing an equal volume of sulfobetaine solutions (10-40 mM/L) containing hydrazine (10-80 mM/L) and copper(II) chloride (1-10 mM/L). The reaction mixture was stirred for 3h at 25°C. When the colour of mixture turned from green to dark brown, the copper oxide nanoparticles were formed. In order to purify the final product, the copper oxide nanoparticles were centrifuged (24000 rpm, 30 min) and washed with water and absolute ethanol. For further experiments, the Cu2O NPs suspension was stored in the fridge (5°C). The synthesis of copper oxide nanoparticles was performed under different operating conditions in terms of surfactant, 6 ACS Paragon Plus Environment

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reducing agent and precursor content, as listed in Table 1. In order to examine the effect of the individual parameter, each experiment was performed on the condition that only one processing parameter was changed while other two parameters remained constant. Table 1

Characterization of nanoparticles The size, shape and crystalline structure of nanoparticles were determined using High Resolution Transmission Electron Microscope (HRTEM) and X-ray diffraction (XRD) techniques. HRTEM micrographs were obtained using HRTEM Jeol ARM 200F. The accelerating voltage used during the experiment was 200 kV. The XRD spectra were obtained using X-ray diffraction technique on Empyrean (PANalytical) diffractometer. The zeta-potential measurements for copper nanoparticles suspension were made at room temperature on the ZetaPlus Analyzer (Malvern Instruments). Size distribution of Cu2O NPs was measured by DLS measurments. The luminescence emission spectra of the nanoparticles suspended in water (25oC, 60 µg/mL) were measured under excitation at 470 nm. The fluorescence emission maximum was detected at λ = 580 nm. The emission and the excitation spectra were measured using FluoroSENS Gilden Photonics fluorescence spectrometer. Cytotoxicity test The apoptotic potential of the copper oxide nanoparticles was evaluated by WST-1 assay (Roche). Briefly, human fibroblasts (MSU 1.1) and human epithelial cervical cancer (HeLa) cells were seeded on 96-well plate at equal density 1×103 cell/well and cultured overnight at 37°C under a 5% CO2 atmosphere in a complete medium DMEM (Dulbecco Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 µg/ml streptomycin. Then, cells were exposed to different concentrations of the



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nanoparticles in the range of 0.5 µg/mL to 250 µg/mL for 24 h. The cells without any treatment were used as a negative control. 10 µl of WST-1 solution was added to each well (previously rinsed with PBS) and the plates were further incubated for 2 h. The absorbance was measured with a microplate reader (Anthos Zenyth 340rt) at 450 nm and 650 nm as reference. All experiments were performed in triplicate, and the relative cell viability (%) was expressed as a percentage relative to the negative control. The IC50 values were obtained by nonlinear regression using GraphPad Prism 7.0. Confocal laser scanning microscope (CLSM) To investigate the intracellular distribution of internalized Cu2O nanoparticles, confocal microscopy was performed on human cervical cancer cell line (HeLa). Briefly, cells were placed on a chambered labtek dish (1×104 cells/well), grown overnight, and then incubated with 100 µg/ml of sulfobetaine-stabilized Cu2O NPs samples (P1, R40, SB40) for 24 h. The cells were then rinsed 3 times with phosphate buffered saline (PBS, pH 7.4) and stained with Hoechst 33342 dye. The distribution of Cu2O NPs was analyzed using a confocal scanning laser microscope (FV1000, Olympus). Hoechst dye was excited with an argon laser 405 nm, while Cu2O NPs samples were excited by 488 nm laser light and the emission was recorded between 505-600 nm. Intracellular ROS generation The intracellular reactive oxygen species (ROS) level was determined using CellROX Green Reagent (Life Technologies) according to the manufacturer’s instructions. Briefly, HeLa cells were incubated with 100 µg/ml of Cu2O NP samples for 3 h. Afterwards, cells were loaded with Cell ROX (final concentration of 5 µM for 30 minutes at 37°C), washed 3 times with PBS buffer, stained with nuclear fluorescent dye Hoechst 33342, and then observed under CLSM (FV1000, Olympus). 8 ACS Paragon Plus Environment

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Results and discussion In the synthesis of metal and metal oxide nanoparticles, the choice of relevant capping agent and reducer is crucial. In particular, the nucleation and particles growth, as well as their size distribution and stability may be affected by the nature of reducing agent.35 The smallest and stable nanoparticles with a narrow size distribution are usually obtained using a strong reducing agents, such as sodium borohydride or lithium aluminium hydride.36, 37 In the case of metal oxide nanoparticles synthesis, the capping agent should contain oxygen and nitrogen atoms, capable of coordinating the metal ions and preventing the nanoparticles aggregation or agglomeration due to the steric hindrance.38 Although there are number of scientific reports concerning the synthesis and various applications of metal and metal oxide nanoparticles, their potential is not fully utilized.39-41 Moreover, the studies involving the application of zwitterionic surfactants in the nanoparticles synthesis are relatively new. Most of the scientific reports concern on the use of cationic, anionic or sometimes non-ionic surfactant to produce nanomaterials. The novelty of this research is the synthesis of copper oxide nanoparticles (Cu2O NPs) in the presence of newly synthesized zwitterionic surfactant SB3C16 and its influence on the final size, shape, stability, and properties of Cu2O nanoparticles. The influence of sulfobetaine concentration A facile one-step approach was employed to obtain the sulfobetaine-stabilized copper oxide nanoparticles (Figure 1).The sulfobetaine applied in this investigation, i.e. N-hexadecyl-N,Ndimethyl-3-ammonio-1-propanesulfonate (SB3C16), was a zwitterionic surfactant that contains two ionic centers with different charges: -N+ and -SO3-. Zwitterionic surfactants are neutral, however, in aqueous salt solutions, their micelles incorporate ions depend on electrostatic interactions and ion-specific effect.42 Mafi et al. have reported that in water, the positively charged head of sulfobetaine surfactants was more capable of orienting interfacial,



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than negatively charged one.43 The density of positively charged quaternary ammonium group was slightly higher than that of the sulfonate anions. Thus, sulfobetaine surfactants interacted preferentially with anions and became negatively charged.44 In turns, the higher Zeta-potential inducted differential binding of the counter-ions to the sulfobetaine surface. Taking into consideration all the above-mentioned arguments, the mechanism of sulfobetaine-stabilized Cu2O NPs synthesis was proposed (Figure 1). The results presented in Table 2 clearly show that the sulfobetaine micelles exhibited negative Zeta-potential. The Zeta-potential decreased after addition of copper salt, as a result of electrostatic interaction between positively charged quaternary ammonium head of surfactants and negatively charged chloride anions. The results are consistent with literature reports.43, 44

Table 2

The negative charge of modified sulfobetaine micelles caused the effective copper cations Cu2+(counter-ions) binding to the micelle surface. The attracted ions directly in contact with the charged surface of zwitterions and the other mobile ions in the solution formed two layers of charge, i.e. the electrical double layer. This effect is also electrostatic by nature and is strongly dependent on the cation size and valence. According to the Priebe et al. report, these interactions were stronger for divalent and trivalent cations, such as Be2+, Mg2+, Al3+, La3+, in comparison with monovalent cations, i.e. Li+, Na+ or K+.44 Due to the preferential interaction between copper ions and anionic head group on the outer surface of the zwitterions, the formation of zwitterionic amphiphiles with the entrapped counter-ions (Cu2+) was favoured. When the strong reducing agent (i.e. hydrazine) was injected into the SB3C16-Cu2+solution, the colour of reaction mixture changed from green to light orange, indicating the beginning of the reduction reaction. Firstly, during the reaction between hydrazine and water, the hydroxyl 10 ACS Paragon Plus Environment

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anions were generated. These OH- ions reacted with the entrapped copper ions, forming copper hydroxide on the outer surface of that double layer. It is also likely, that OH- anions could compete with Cl- anions for micelle modification.45 The copper hydroxide reacted with NH2NH3+ giving copper oxide nanoparticles (Cu2O NPs) and volatile by-products (nitrogen and hydrogen). Presumably, the presence of sulfobetaine in the Cu2O NPs synthesis implied the interaction of copper ions with sulfobetaine surface, and might significantly affect the nucleation and nanoparticles growth.46 Surfactant may form different kind of multimolecular assemblies from simple micelles to organized structures, like fibers or tubes.47 As shown in Figure 2a-c, during the one-step reaction, spherical copper nanoparticles with size varied from 2-7 nm were synthesized. The smallest particles size was obtained at the lowest surfactant concentration. A further increase of surfactant content caused the increase of particles size up to 6.5 nm. Based on HRTEM measurements it can be also observed that surfactant concentration affected both, the dispersion and the sizes of the copper oxide nanoparticles. It was found that variation of surfactant concentration caused changes in the particles morphology. Adjustment of various surfactant concentration might change the surface energies of nanoparticles, thus their final size and specific surface area can be different.48 In order to investigate the effect of sulfobetaine on the final size, shape and stability of copper oxide nanoparticles, the synthesis without the addition of zwitterionic surfactant SB3C16 was also carried out. These results are summarized in Supporting Information. The diffraction rings of SAED pattern shown in the inset of Figure 2a-c, confirmed the polycrystalline character of as-prepared copper oxide nanoparticles. The crystal planes of all samples were determined according to X-Ray diffraction patterns (Figure 3a). It can be observed that the positions of the characteristic reflection peaks were consistent with the standard diffraction reflections of copper(I) oxide (Cu2O). All peaks from Cu2O could be indexed to the cubic phase. The X-Ray diffraction patterns of Cu2O NPs exhibited additional



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crystalline phases, which can be attributed to copper, copper(II) oxide (CuO) and to the remains of the unreacted chloride-based precursor. However, the highest dose of impurities was observed for nanoparticles obtained at the lowest surfactant content. The intensities of the reflection peaks for all samples were generally smaller. This effect is typical for the small metallic nanomaterials with a particle size less than 10 nm, such as quantum dots. The size and morphology of the nanoparticles play an important role in changing the entire properties of nanomaterials, including optical properties. The emission spectra of Cu2O nanoparticles obtained at the various content of sulfobetaine (Sample SB10, SB30, SB40) and recorded at the same concentration of nanoparticles (60 µg/mL) were presented in Figure 4a. It can be observed that the fluorescence emission for all samples was located in the range of 565-585 nm with the maximum emission at 575 nm. Moreover, the FWHM (full width at half maximum) of emission peaks for all samples was narrow (around 10 nm). When the surface of Cu2O NPs was changed, due to the presence of various content of surfactant, the fluorescence emission peak was not shifted, while the fluorescence intensity was significantly changed. Similar results were observed and described by Zhang et al. for gold nanoparticles with different surface structures.49 The influence of precursor concentration The size and shape of nanomaterials determine their physical and chemical properties. One of the important factor affecting the structure and morphologies of nanoparticles is the precursor concentration.50 Moloto and co-workers have investigated that an increase of precursor concentration changed the shape of the CdS nanoparticles from spherical to rod-like.51 In turn, Zhu et al. have demonstrated that a decrease in the copper ions concentration, resulting in a decrease in the number of copper nuclei and an increase in the nanoparticle size.52 The effect of copper salt concentration on the size and shape of nanoparticles was studied according to HRTEM micrographs. As shown in Figure 2d-f, the increase in precursor 12 ACS Paragon Plus Environment

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concentration caused the increase in nanoparticles size. This effect is well-known and extensively investigated in the literature. It might be explained due to the fact that at higher precursor concentration, more nanoseeds turned to deposition on the existing particle surface rather than homogeneously nucleated.53 Similar results have been reported by Zielinska et al.54 and Young et al.55 The low concentration of copper salt (1 mM/L) resulted in the formation of single spherical nanoparticles with the diameter varying between 1.5-3 nm and an average diameter of 2.2 nm (Figure 2d). It is worth mentioning that in some regions, the presence of bigger clusters (around 120 nm) surrounded by the smaller nanoparticles was also observed. The further increase in precursor concentration led to the production of polycrystalline, oblate and elongated nanoparticles with the size varied from 5 nm to 11 nm (5 mM/L) and even larger than 40 nm (10 mM/L) (Figure 2e-f). Figure 2 Figure 3 In order to identify the crystallinity and crystal phases of nanoparticles, the XRD measurements were carried out (Figure 3b, sample P1, P5, and P10). Three peaks at 2Ѳ: 36.4, 42.3 and 61.3deg, corresponding to the (111), (200) and (220) planes of Cu2O were observed and compared with JCPDS01-080-7711. All three peaks could be indexed to the cubic phase with lattice constants a = b = c = 4.2712 Å and with the space group of Pn-3m. The XRD patterns confirmed that the final product of the synthesis was pure and crystalline (Figure 3b, sample P1 and P10). The HRTEM micrographs revealed that the copper oxide nanoparticles obtained for the molar ratio of copper salt to the sulfobetaine equal to 1:4 (sample P5) and 1:2 (sample P10) exhibit polycrystalline character (Figure 2e-f). It suggests, that in case of structural characterization of nanoparticles, especially smaller than 10 nm, the XRD analysis should be supported by HRTEM measurements. The additional diffraction peaks arising from 13 ACS Paragon Plus Environment


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impurities and CuO phase were detected for sample P5. Their presence might be responsible for the micrometric-sized clusters formation (Figure 2e). Figure 4 The photoluminescence (PL) emission spectra of Cu2O NPs were shown in Figure 4b (sample P1, P5, P10). The spectra consist of a single sharp peak centered at 575 nm in the yellow region, arising from the band edge emission from Γ 1 + to the new sub-levels.56 The sub-levels could be developed as a result of the imperfections due to the interaction of two excitons or the 3D—1D splitting in Cu+ (3d94s2).57 The FWHM was less than 10 nm. It means that the size distribution of Cu2O NPs was remarkably narrow. Small humps were also observed at 560 and 620 nm and may be associated with defects from singly ionized oxygen vacancy of Cu2O. Das et al. have indicated that the peak around 620-650 nm might correspond to the conversion of some upper shell of the Cu2O into more thermodynamically stable CuO layers.58 The fluorescence intensity was found to be size-dependent. The significant decrease in fluorescence intensity with the Cu2O NPs size increase was clearly observed (Figure 4b). The loss of fluorescence can be explained by the occurrence of selfquenching phenomena due to short-range interactions between polycrystalline nanoparticles and distortions in the local environment.59,

60

The PL results are consistent with HRTEM

measurements. The low emission intensity was detected only for nanoparticles that revealed polycrystalline character (Sample P5 and P10, Figure 2e-f). The influence of reducing agent concentration Chemical reduction method is one of the common approaches for metal and metal oxide nanoparticles synthesis. The nature and concentration of reducing agent have a significant influence on the physical and chemical properties of the final products, including size, shape, specific surface area, crystallinity etc.61 For example, the use of strong reducing agent lead to 14 ACS Paragon Plus Environment

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the formation of smaller nanoparticles, however, due to the fact that the rate of reduction reaction is high, there is no opportunity to cap the individual nanoparticles by the surfactant. The application of milder or weak reducing agents may provide a slow reduction of metal ions and lead to the formation of bigger nanoparticles or large aggregates.62 In this investigation, copper oxide nanoparticles were obtained using hydrazine (usually classified as strong reducing agent). The size, shape, surface morphology and crystallinity of copper oxide nanoparticles at a various concentration of hydrazine were determined according to the HRTEM micrographs. As shown in Figure 2g-i, the increase in reducer concentration caused the increase in the nanoparticle sizes. The HRTEM image indicated that Cu2O NPs obtained at the lowest concentration of hydrazine were spherical in shape with the size varied from 4 to 12 nm (Figure 2g). The further increase of the hydrazine content caused an increase of nanoparticle size from around 40 nm (at 40 mM/L of hydrazine) to more than 45 nm (at 80 mM/L of hydrazine). Moreover, the HRTEM micrographs confirmed that copper oxide nanoparticles obtained at a concentration 40 mM/L and 80 mM/L of hydrazine were elongated and rougher, in comparison with the Cu2O NPs obtained at the lowest hydrazine concentration (Figure 2h-i). Similar relationships were described by Zielinska et al.54 This effect was probably associated with the pH of the reaction mixture, which influenced the reducing power of hydrazine. The redox potential of hydrazine decreases with increasing pH.63 The reduction reaction was carried out at pH around 6-7. It means that in these conditions, the hydrazine action might be weakened. The crystalline character of all presented nanoparticles was also confirmed by XRD measurements (Figure 3c, sample R20, R40, and R80). According to the JCPDS Card No. 01080-7711, the pattern can be indexed as a cubic structure. The characteristic peaks were observed at 2ϴ of 29.3, 36.4, 42.3, 61.4 and 77.6 deg and assigned to the crystal planes of (110), (111), (200), (220) and (311) Bragg reflection of Cu2O, respectively.



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The size and morphology of nanoparticles usually induce significant changes in their optical properties. Figure 4c exhibits the PL spectra of as-prepared Cu2O NPs (Sample R20, R40, R80). The fluorescence emission maximum was detected at 580 nm (excitation wavelength 460 nm, Figure 4d). The presence of some small peaks in the wavelength region 545-565 nm and 600-640 nm can be associated with the defects in the Cu2O nanostructures. According to the PL spectra presented in Figure 4c, it can be concluded that the size of copper oxide nanoparticles significantly affected the fluorescence intensity. The intensity of the emission peak at 580 nm increased together with increasing size of Cu2O nanoparticles. The differences in the intensities could be related to the different surface imperfections and specific surface area of copper oxide nanoparticles.64 Based on the HRTEM micrographs, it can be noticed that the copper oxide nanoparticles obtained at the highest content of hydrazine were more irregular with clearly visible particles thresholds (Figure 2i). Stability of nanoparticles The stability of the nanoparticles suspended in water or biological media is crucial to prevent their aggregation, which usually leads to undesirable biological response. In order to investigate the long-term stability of Cu2O NPs, the Zeta-potential measurements were carried out (Table 3). Table 3 Zeta-potential is one of the fundamental parameters to investigate the nature of electrostatic interactions between nanoparticles suspended in the solvent. According to the well-known theory, the balance of the attractive versus repulsive forces can be used to determine the stability of the nanoparticle solutions. If the repulsive forces are greater than the attractive forces, the suspension remains stable.65


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Based on the results presented in Table 3, it can be concluded that all prepared Cu2O NPs revealed negative Zeta-potential. The magnitude of the electrokinetic potential of the copper nanoparticles suspended in water at 25°C decreased with time, resulting in their aggregation. However, the Cu2O NPs in sample P1, P5, R40, and SB40 were inherently more stable than the other nanoparticles. The highest stability of the Cu2O NPs was observed during the first 48h. The stabilization effect can be related to the formation of a double layer of surfactant molecules around the copper oxide surface. However, the template effect, resulted from the heterogeneous structure of zwitterionic surfactant, could also affect the Cu2O NPs stability.32,34 After 1 months the stability of nanoparticles decreased sharply, especially for the Cu2O NPs prepared at the lower molar ratio of sulfobetaine to the copper precursor (Sample P10 and SB10). The low Zeta-potential resulted in agglomeration of the nanoparticles, causing a decrease in their physical stability in water. The state of nanoparticles agglomeration was monitored 24 h, 48 h and 1 month after Cu2O NPs synthesis, based on HRTEM (Figure 5a-c) and DLS measurements (Figure 5d). The small Cu2O agglomerates were found after 48 (Figure 5b-c, sample P1). The results confirmed that the long-term stability of the nanoparticles can be associated with the concentration of the sulfobetaine. According to the Zeta-potential measurements, the highest stability was observed for the sulfobetaine-stabilized Cu2O NPs in samples: P1, R40, and SB40, thus only these nanoparticles were used for further cytotoxicity studies.

Figure 5

Cytotoxicity of nanoparticles The rapid growth of interest in nanoparticles research causes that the toxicity of nanomaterials becomes an important approach in nanotechnology studies. A number of scientific reports have investigated the toxic effect of nanoparticles, depending on their chemical composition, 17 ACS Paragon Plus Environment


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size, shape and surface activity.66 Nowadays, the in vitro toxicity studies are more desirable due to direct estimation of the nanoparticles effect on the viability of cells. In this report, the cytotoxicity of the Cu2O NPs was investigated using human fibroblast (MSU 1.1) and cervical cancer (HeLa)cell line, based on the WST-1 assay. The concentration-dependent toxicity profile in both cell lines after incubation with the Cu2O NPs for 24 h was presented in Figure 6a-b. The results exhibited the increase of the copper oxide nanoparticles toxicity with their concentration increase. A significant decrease in HeLa cell viability was observed at a concentration from 25 to 250 µg/mL of copper oxide nanoparticles (Figure 6a, sample P1, SB40, and R40). The IC50 values of Cu2O NPs confirmed also that toxicity of sample SB40 is similar to the toxicity of sample R40 (Table 4). At concentration lower than 10 µg/mL no effect on mitochondrial activity and any cell mortality were found. However, at concentration 10 µg/mL of the Cu2O NPs, a slight toxicity for sample SB40 and R40 was observed. As was expected, the HeLa cells appeared to be more resistant to the toxic action of the Cu2O NPs than human fibroblasts. Cancer cells are usually more resilient towards metallic nanoparticle toxicity than normal cells due to an increased rate of proliferation and metabolic activity.67 Figure 6 Table 4 For fibroblast cells (Figure 6b), the viability decreased from 5 µg/mL of Cu2O NPs. However, the significant toxic effect was detected for sample SB40 and R40 at concentration 50 µg/mL. From the range 50-250 µg/mL of Cu2O, both, the SB40 and R40 samples demonstrated an almost equal degree of toxicity (Figure 6).However, the IC50 values presented in Table 4 indicated that the sample SB40 was less toxic than the sample R40. According to the results obtained it can be concluded that the toxicity of the copper oxide nanoparticles, in both cell lines, increased in the order: P1< SB40 ≤R40. The lowest observed


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toxic effect on human fibroblasts and HeLa cells was detected for sample P1 at concentration below 100 µg/mL. Recently, a number of scientific reports have demonstrated that the toxicity of nanoparticles-surfactant conjugates can be also related to the toxicity of surfactant molecules. According to the results presented by Dong et al. it can be concluded that commonly applied surfactants, such as sodium dodecyl sulfate (SDS) and sodium dodecylbenzene sulfonate (SDBS) are toxic to the cells.68 The sulfobetaines are generally regarded as non-toxic and environmentally friendly surfactants,31 however it cannot be stated unambiguously that sulfobetaine SB3C16 had no effect on cell proliferation. Intracellular distribution of Cu2O NPs in cells

Figure 7

To investigate the intracellular distribution of Cu2O NPs and their influence on the cytotoxicity, the in vitro imaging experiments were conducted (using human cervical cancer cell line- HeLa). The cells were incubated in DMEM medium for 24 h with 100 µg/ml of sulfobetaine-stabilized Cu2O nanoparticles (sample P1, SB40, and R40). All of the samples exhibited bright yellow fluorescence emission. To visualize the nuclei, the additional staining with Hoechst (blue) was carried out. The copper nanoparticles were internalized efficiently into HeLa cells (Figure 7), however, some differences were observed between the samples. In the case of sample R40 (composed of particles from 5 nm up to 40 nm, Figure 7a), the fluorescent signal was observed mainly in the cytoplasm, which origins from aggregates, and suggests the endosome vesicle formation. Moreover, these nanoparticles were also visible as monodispersed signal in the nucleus. Smaller nanoparticles (SB40 ~ 6.5 nm, Figure 7c) were found to be monodispersed within the cells. Interestingly, the smallest nanoparticles (sample P1 ~ 2.2 nm, Figure 7b) were located only in vesicles within the cytoplasm. None of the nanoparticles entered the cell nucleus. Macara has reported that nuclear pore complexes allow 19 ACS Paragon Plus Environment


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to the transport of cargos smaller than 9 nm into the nucleus.69 In contrast, the obtained results have suggested that sulfobetaine-stabilized Cu2O NPs cell distribution was affected not only by the particle size but also by their stability in the culture medium. Furthermore, the cytotoxicity level of the obtained nanoparticles is highly influenced by their intracellular localization.71 ROS generation of Cu2O NPs in cancer cells Figure 8 One of the most important nanotoxicity mechanisms is the generation of reactive oxygen species (ROS). The overproduction of ROS results in the formation of the oxidative stress in the biological system, may cause the damage of cell functions, DNA-strand breaks, modulation of gene expression or even cell death.71 Due to determine the ROS level in the living cells, we used a confocal laser scanning microscopy. It will allow to understand whether the interaction of cells with Cu2O NPs causes the cells death. The 100 µg/ml of nanoparticles, i.e. P1, R40 and SB40 were added to the cells placed in the separated wells and then incubated with CellROX Green Oxidative Stress Reagent. Cells labelled only with fluorescent dye represent non-treated cells (control). The results presented in Figure 8, indicated that the Cu2O NPs significantly increases intracellular ROS production. The obtained results are consistent with the literature reports.72,73 After 3 h of Cu2O NPs incubation with HeLa cells, the highest ROS level was observed for R40 and the lowest one for P1. Generally, due to their small size and large surface area, nanoparticles are very reactive leading to ROS generation. Thus oxidative stress plays an important role in their nanotoxic mechanism.74, 75 We conclude that the ROS formation can be the main reason of copper oxide nanotoxicity.73, 76 Conclusion 20 ACS Paragon Plus Environment

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This report indicates that copper oxide nanoparticles (Cu2O NPs) can be prepared by a simple, one-step reaction using sulfobetaine (exemplary of zwitterionic surfactant) as a stabilizing agent. The class of zwitterionic sulfobetaine-based surfactants is very promising for metallic and metal oxide nanomaterials synthesis because it provides both control of the final properties of nanoparticles and their stabilization. The results confirm that content of sulfobetaine affects the size, shape, and crystallinity of the copper oxide nanoparticles. It was also found that at the constant content of sulfobetaine, the size of nanoparticles can be controlled by changing both, the copper precursor and the reducing agent concentration. Moreover, it was proven that all as-prepared copper oxide nanoparticles exhibit fluorescence in the yellow region. The cytotoxicity tests show that the copper oxide nanoparticles cause inhibition of cell proliferation and viability of cancer cells in a concentration-depended manner, however, it should be pointed out that they also impair the growth and division of normal cells. The nanotoxicity is associated with the nanoparticles intercellular localization and ROS formation. The results indicate that the sulfobetaine-stabilized copper oxide nanoparticles have potential to be applied in cancer therapy. Nevertheless, in order to decrease the side effect on healthy cells the additional biofunctionalization of their surface by tumor targeting agent should be performed. Supporting Information Available The following file is available free of charge at the website http://pubs.acs.org. Synthesis of copper oxide nanoparticles without surfactant, HRTEM micrographs of the copper oxide nanoparticles obtained without sulfobetaine SB3C16. Acknowledgments



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Table 1. The composition of the reaction mixtures. Sample P1 P5 P10 R20 R40 R80 SB10 SB30 SB40

Precursor concentration, mM/L 1 5 10

Reducer concentration, mM/L

Sulfobetaine concentration, mM/L

10

20

20 40 80

20

10

10 30 40

10

10


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Table 2. Zeta-potential of sulfobetaine aqueous solution and its mixture with the copper precursor. Zeta-potential, mV 1.01±0.15 1.23±0.21 1.17±0.27 -2.93±0.31 -1.80±0.34 -2.58±0.29

SB3C16 [10mM/L] SB3C16 [30mM/L] SB3C16 [40mM/L] SB3C16 [10mM/L]+CuCl2 [10mM/L] SB3C16 [30mM/L]+CuCl2 [10mM/L] SB3C16 [40mM/L]+CuCl2 [10mM/L]



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Table 3. Zeta-potential of copper oxide nanoparticles suspended in water as a function of time (25°C).

Sample P1 P5 P10 R20 R40 R80 SB10 SB30 SB40

1h -23.4±1.8 -16.4±1.3 -16.7±1.9 -17.9±1.1 -14.0±0.8 -22.9±2.1 -14.4±1.5 -19.7±1.3 -26.2±1.1

Zeta-potential [mV] 24h -22.0±1.7 -15.0±1.2 -10.7±0.8 -16.5±2.4 -14.5±1.5 -20.8±2.3 -14.5±0.5 -13.9±1.8 -26.5±1.8

48h -22.9±1.8 -16.5±1.3 -9.8±0.9 -16.0±1.8 -14.6±1.2 -16.0±1.5 -9.8±0.4 -12.8±2.6 -25.9±2.6


1 month -17.4±1.6 -15.1±1.5 -9.2±0.5 -6.3±1.2 -15.0±0.2 -7.8±0.3 -3.1±0.3 -8.40±0.26 -16.0±1.2

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Table 4. The cytotoxicity effects of copper oxide nanoparticles on MSU1.1 and HeLa cell lines. The concentration of compounds causing a 50% reduction in cell growth after 24 h of incubation (IC50). IC50 [µg/mL] Sample P1 R40 SB40

HeLa 62.5 13.7 13.5

MSU1.1 75.0 8.7 4.0



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Graphical abstract


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Figure 1a) Chemical reducing reaction of sulfobetaine-stabilized copper oxide nanoparticles; b) Proposed mechanism of the sulfobetaine-stabilized copper oxide nanoparticles formation.



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Figure 2.HRTEM micrographs of copper oxide nanoparticles; a) sample SB10; b) sample SB30; c) sample SB40; d) sample P1; e) sample P5; f) sample P10; g) sample R20; h) sample R40; i) sample SB40.


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Figure 3.XRD spectra of sulfobetaine-stabilized copper oxide nanoparticles; a) samples 20R-80R; b) P1-P10; c) SB10-SB30.



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Figure 4. Photoluminescence (PL) spectra of sulfobetaine-stabilized copper oxide nanoparticles; a) SB10-SB30; b) P1-P10; c) R20-R40; d) excitation spectra of sulfobetaine-stabilized copper oxide nanoparticles.


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Figure 5.The time-dependent aggregation studies of sulfobetaine-stabilized copper oxide nanoparticles (sample P1) after a) 24h; b) 48h and c) 1 month; based on HRTEM and d) DLS measurements.



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Figure 6.Cytotoxicity profile of copper oxide nanoparticles in a) HeLa cells and b) fibroblasts; sample P1, R40, SB40).


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Figure 7.The confocal laser scanning microscopy (CLSM) images of HeLa cancer cells after 24h incubation with sulfobetaine-stabilized copper oxide nanoparticles a) sample R40; b) sample P1; c) sample SB40; scale 5µm.



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Figure 8.Cellular ROS generation in HeLa cells analyzed via CLSM after Cu2O NPs treatment; a) sample P1, b) sample SB40, c) sample R40. Cells were stained with CellROX Green dye (green), nucleus were stained with Hoechst (blue). Scale 10 µm.


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Figure 1. a) Chemical reducing reaction of sulfobetaine-stabilized copper oxide nanoparticles; b) Proposed mechanism of the sulfobetaine-stabilized copper oxide nanoparticles formation. 191x250mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Figure 2. HRTEM micrographs of copper oxide nanoparticles; a) sample SB10; b) sample SB30; c) sample SB40; d) sample P1; e) sample P5; f) sample P10; g) sample R20; h) sample R40; i) sample R80. 299x299mm (300 x 300 DPI)


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Figure 3. XRD spectra of sulfobetaine-stabilized copper oxide nanoparticles; a) samples 20R-80R; b) P1P10; c) SB10-SB30. 297x237mm (300 x 300 DPI)



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Figure 4. Photoluminescence (PL) spectra of sulfobetaine-stabilized copper oxide nanoparticles; a) SB10SB30; b) P1-P10; c) R20-R40; d) excitation spectra of sulfobetaine-stabilized copper oxide nanoparticles. 345x264mm (300 x 300 DPI)


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Figure 5. The time-dependent aggregation studies of sulfobetaine-stabilized copper oxide nanoparticles (sample P1) after a) 24h; b) 48h and c) 1 month; based on HRTEM and d) DLS measurements. 199x199mm (300 x 300 DPI)



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Figure 6. Cytotoxicity profile of copper oxide nanoparticles in a) HeLa cells and b) fibroblasts; sample P1, R40, SB40). 172x86mm (300 x 300 DPI)


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Figure 7. The confocal laser scanning microscopy (CLSM) images of HeLa cancer cells after 24h incubation with sulfobetaine-stabilized copper oxide nanoparticles a) sample R40; b) sample P1; c) sample SB40; scale 5µm. 38x12mm (300 x 300 DPI)



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Figure 8. Cellular ROS generation in HeLa cells analyzed via CLSM after Cu2O NPs treatment; a) sample P1, b) sample SB40, c) sample R40. Cells were stained with CellROX Green dye (green), nucleus were stained with Hoechst (blue). Scale 10 µm. 139x130mm (300 x 300 DPI)


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Facile Synthesis of Sulfobetaine-Stabilized Cu2O Nanoparticles and

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