Evaluation of cytotoxicity, photoluminescence, bio-imaging, and sonosensitizing properties of silicon nanoparticles (SiNPs) prepared by ultrasound grinding of porous silicon nanowires (SiNWs) have been investigated. SiNWs were formed by metal (silver)-assisted wet chemical etching of heavily boron-doped (100)-oriented single crystalline silicon wafers. The prepared SiNWs and aqueous suspensions of SiNPs exhibit efficient room temperature photoluminescence (PL) in the spectral region of 600 to 1,000 nm that is explained by the radiative recombination of excitons confined in small silicon nanocrystals, from which SiNWs and SiNPs consist of. On the one hand, in vitro studies have demonstrated low cytotoxicity of SiNPs and possibilities of their bio-imaging applications. On the other hand, it has been found that SiNPs can act as efficient sensitizers of ultrasound-induced suppression of the viability of Hep-2 cancer cells.
Recently, a lot of bio-applications of different silicon nanostructures were reported. The most popular material in this field of interest is porous silicon (PSi). PSi consists of a network of intersecting silicon nanocrystals (nc-Si) separated by nanometer-sized pores . Usually, PSi films are formed by a method of electrochemical etching of bulk crystalline silicon (c-Si) in hydrofluoric acid (HF), which was firstly showed in 1956 by Uhlir . The size of nc-Si and pores in PSi depends on the formation parameters, such as HF concentration, current density, and substrate doping density [1, 3]. In [4–6], it has been shown that PSi, which consists of nc-Si with sizes of 2 to 5 nm, demonstrates efficient photoluminescence (PL) in the visible spectral range at room temperatures, originating from quantum confinement in nc-Si . In 1995, Canham  discussed bio-friendly properties of PSi and suggested its applications in bio-medicine. Such properties of PSi and Si-based nanoparticles (SiNPs) as bio-compatibility and bio-degradability have been previously investigated [9, 10]. SiNPs can be used as luminescent labels [10–12], nanocontainer for drug delivery [13, 14], sensitizers of photo- [15–18] and ultrasound irradiation (USI) therapy [19, 20]. Furthermore, other bio-medical applications of SiNPs were also proposed [21–26].
It is known a cheap and efficient method of the formation of PSi and silicon nanowires (SiNWs) based on metal-assisted chemical etching (MACE) of c-Si [27–31]. MACE-prepared SiNWs consists of an almost non-intersecting nanowires with diameters from several to hundreds nm . The morphology of the as-synthesized SiNWs was highly dependent on the doping level of original silicon wafers and on the concentrations of etching solutions . SiNWs show unique physical properties such as room temperature PL [33, 34], enhanced Raman scattering , low reflectance in the visible spectral range, and a strong broadband optical absorption [31, 36] and gained much attention due to their possible applications in the fields of photovoltaics, photocatalysis, gas sensors, lithium-ion battery, and drug delivery carriers [33, 37]. Also, a long-term antiseptic effect of SiNWs decorated with silver nanoparticles was shown in .
In this paper, we propose a simple method for the preparation of SiNP suspensions, which is based on grinding of SiNWs in water by ultrasound irradiation. The prepared SiNPs are used as PL probes for bio-imaging and sensitizers for sonodynamic suppression of the cancer cells proliferation in vitro.
SiNWs were prepared by MACE of heavily boron-doped (doping level 1020 cm-3; conductivity < 0.005 Ω cm) (100) single crystalline wafers. Prior to the MACE procedure, the Si substrates were rinsed in 5% HF aqua solution for 1 min to remove the native oxide. Then, in the first step of MACE, thin (approximately 100 nm) layers of Ag nanoparticles of different morphology were deposited on the substrates by immersing them in aqueous solution of 0.02 M of silver nitrate (AgNO3) and 5 M of HF in the volume ratio of 1:1 for 30 s. In the second step, the Si substrates covered with Ag nanoparticles were immersed in the solution containing 5 M of HF and 30% H2O2 in the volume ratio of 10:1 in a teflon vessel for 20 min. The etching was performed at room temperature. Then, SiNW arrays were rinsed several times in deionized water and additionally immersed in concentrated (65%) nitric acid (HNO3) for 15 min to remove the residual Ag nanoparticles from the SiNWs. Finally, the samples were rinsed several times in deionized water and dried at room temperature. Aqueous suspensions of SiNPs were prepared by 3 h ultrasound grinding (37 kHz, 90 W) of SiNWs. Afterward, the suspensions were centrifuged for 3 min at 2,000 rpm, and the resulting supernatant was used in the experiments. The in vitro experiments were done with freshly prepared SiNP suspensions to avoid an influence of the dissolution of SiNPs in water.
Structural investigations of SiNW-based samples were carried out by using a field emission scanning electron microscope (Carl Zeiss ULTRA 55 FE-SEM, Oberkochen, Germany) and a transmission electron microscope (LEO 912 AB OMEGA, Oberkochen, Germany). A Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., UK) instrument was used to determine the size and zeta potential (ZP) of SiNPs from the dynamic light scattering (DLS) data. The PL was excited by the radiation of an Ar+ -ion laser at 364 nm (power 10 mW, spot diameter 1 mm). The PL signal was detected using a grating monochromator (MS750, SOLAR TII, Moscow, Russia) equipped with a CCD array. The external quantum yield (QY) of PL was measured by using a calibrated detector as it was described in . In vitro cytotoxicity experiments were performed with Hep-2 human lung cancer cells. The cells were incubated with SiNPs and cultural medium (Dulbecco’s modified Eagle medium, Biolot, St. Petersburg, Russia) for 24 h. The cancer cells were directly counted in a hemocytometer by using the standard method when dead cells were separated from living ones by preliminary coloring with trypan blue (0.4%, 1:1) (PanEco Ltd., Moscow, Russia). The results were statistically processed.
In vitro SiNPs visualization experiments were carried out with CF2Th (dog thymus) cells infected with a green fluorescent protein (GFP) gene. Thirty hours before the fluorescence analysis, RSL-1 inducer was added to the CF2Th culture, which caused the synthesis of GFP. The luminescence wavelength of GFP is near 515 nm (green light). SiNPs were introduced into CF2Th 5 h after the inducer injection and 24 h before the measurements. Cell nuclei were imbued with 5 μg Hoechst (PanEco Ltd., Moscow, Russia) 30 min before the experiment. The luminescence wavelength of Hoechst is near 460 nm (blue light). The cells with incorporated SiNPs were studied using a Leica TCS SP5 confocal microscope (Wetzlar, Germany).
To evaluate the effect of ultrasound and SiNPs on the Hep-2 cell viability, SiNPs in the cultural medium were added to the cells to achieve the nanoparticles concentration 0.1 mg/ml and left for 14 h prior to the experiment. Afterward, the cells were washed three times by Hanks’ balanced salt solution (PanEco Ltd., Moscow, Russia) and removed from the substrate by trypsinization. The obtained suspension of cells was exposed to USI (0.88 MHz, 1 W/cm2, pulse mode, modulation 2/20), with the UST-1.3.01 F ‘MeDTeKo’ equipment. Degassed distilled water (at 37°C) was used as a contact medium between flat emitters with a radius of 2 cm and cuvette filled with the sample. In the control group, the cells without SiNPs were investigated.
Results and discussion
Figure 1a shows typical cross-sectional scanning electron microscopy (SEM) micrographs of heavily doped SiNW arrays on c-Si. Inset in Figure 1a shows a porous structure of SiNW. Quasi-ordered similarities in SiNW arrays with preferential orientation along the (100) crystallographic direction have been observed. The length of SiNWs was observed to be about 5 μm after 20 min etching time. As you can see from SEM (inset in Figure 1a), SiNWs have a highly porous structure, which is also confirmed by the transmission electron microscopy (TEM) image of single SiNW as presented in Figure 1b. Such porous microstructure of nanowires is typical for SiNWs obtained from MACE etching of highly doped c-Si . The diameter of SiNW is about 200 nm, and it is nearly constant through the whole SiNW length.Inset in Figure 2b shows the corresponding electron diffraction pattern obtained in the ‘transmission’ geometry for SiNWs. The presence of rings in the pattern indicates the preservation of misoriented nanocrystals in the porous structure of SiNWs.
A TEM image of SiNPs, fabricated by ultrasound grinding of SiNWs in water, is presented in Figure 1c. Inset shows the electron diffraction pattern for SiNPs, which indicates the preservation of misoriented nanocrystals in SiNPs. Figure 1d shows a size distribution function of SiNPs, obtained by DLS, which was characterized by a maximum of 135 nm. ZP of SiNPs in the initially obtained aqueous suspensions was -20 ± 2 mV. The ZP value is typical for porous silicon nanoparticles and related to the negative charge of hydroxyl groups on SiNPs surfaces . Such negative ZP determines the existence of stable suspensions of nanoparticles. A typical view of SiNP aqueous suspension with the concentration of 1 mg/ml is presented in the inset of Figure 1d. Note that the Raman spectra (not shown) of as-prepared SiNWs and dried SiNP suspensions showed that both samples consisted of Si nanocrystals with mean size of 3 to 4 nm. A negligible fraction of the amorphous silicon was also found. The preservation of nanocrystalline structure of the samples after the preparation of SiNW suspension can be explained by the effect of surface oxide formed during the last stage of MACE and nitric acid treatment.
The PL spectra of SiNWs consist of a broad band in the visible range with the maximum in the photon energy at 850 nm (1.7 eV) as shown in Figure 2a. The external QY of PL was estimated to be about 2%, and the PL emission could be easily observed with a naked eye (see the inset in Figure 2a). The PL spectra of the samples are well explained by the radiative recombination of excitons confined in small silicon nanocrystals with an average size of 2 to 6 nm . Such nanocrystals were found by electron microscopy in porous volume of SiNWs as shown in Figure 1a,b.The intensity and character of the PL spectra of SiNPs suspensions is similar to the corresponding SiNW layers, and their PL could be easily observed with a naked eye (see the inset in Figure 2b). As mentioned above, the 2 to 6 nm nanocrystals are responsible for this PL. Thus, it can be argued that the obtained SiNPs have a porous structure Therefore, the SiNWs milling procedure does not significantly quenching their PL properties, and obtained PL SiNPs can be used in different bio-medical application, in particular, for cells labeling.
Figure 3 shows in vitro cytotoxicity of SiNPs towards living Hep-2 cells. As it is clearly visible, SiNPs are relatively non-toxic to Hep-2 cells in the concentration range of 2 to 125 μg/ml. However, at larger concentrations of SiNPs, of 250 to 1,000 μg/ml, the cell viability was dropped to 5% as shown in Figure 3.
The in vitro fluorescence photographs of living CF2Th cells with and without (the control) introduced 0.1 mg/ml SiNPs are presented in Figure 4a,b, respectively. Green, blue, and red colors correspond to the luminescence of cell membranes, cell nuclei and SiNPs, respectively.
A significant luminescence of SiNPs was observed in the cells 24 h after their incubation. It is evident that the majority of SiNPs could penetrate into the cells and locate in their cytoplasm. We assume that nanoparticles penetrate into the cells due to the mechanism of endocytosis . The localization of SiNPs in the cell cytoplasm was confirmed by z-scan imaging [see Additional file 1: Movie S1].
Figure 5 represents the living cell viability dependence on USI duration. Setup of in vitro experiments is given in the inset of Figure 5. It was found out that USI essentially no affect on the cell viability within the time interval from 0 to 10 min. At the same time, the combined action of ultrasound and SiNPs led to 50% drop in the number of living cells as compared to the control.
The effect of ultrasound and SiNPs on the cell viability can be explained by the appearance of local increasing of temperature near SiNPs, so-called hyperthermia, caused by the adsorption of the ultrasound energy . Also, the possibility of appearance of cavitation near the nanoparticles under ultrasound irradiation cannot be excluded.
The obtained results open a new perspective for the application of bio-compatible SiNPs, produced from ultrasound milling of porous SiNWs, in SDT.
In summary, low toxicity, photoluminescence and sonosensitizing properties of SiNPs, prepared by ultrasound milling of porous SiNW arrays, open new possibility of their theranostic (therapy and diagnostic) applications.
We have shown that aqueous suspensions of SiNPs can be obtained by a simple and cheap way, i.e., by ultrasound milling of porous SiNW arrays in water. After 2 h of grinding, SiNPs represent a mixture of 200 nm porous agglomerates composed of 2 to 6 nm nc-Si. The prepared PL SiNPs are characterized by low cytotoxicity in vitro even at concentrations of 125 μg/ml and can be used for bio-imaging of cancer cells. The prepared SiNPs also exhibit the properties of efficient sonosensitizer of therapeutic ultrasound. It was found out that the combined action of ultrasound and SiNPs with concentration of 100 μg/mL led to 50% decrease in the number of cancer cells. The obtained results open a new perspective for the usage of bio-compatible porous SiNPs prepared from SiNWs in the sonodynamic therapy of cancer.
Cullis AG, Canham LT, Calcott PD: The structural and luminescence properties of porous silicon. J Appl Phys 1997, 82: 909–965. 10.1063/1.366536
Uhlir A: Electrolytic shaping of germanium and silicon. Bell Syst Tech 1956, 35(2):333–347. 10.1002/j.1538-7305.1956.tb02385.x
Lehmann V, Stengl R, Luigart A: On the morphology and the electrochemical formation mechanism of mesoporous silicon. Mater Sci Eng B 2000, 69–70: 11–22.
Canham LT: Silicon quantum wire array fabrication by electrochemical and chemical dissolution of wafers. Appl Phys Lett 1990, 57(10):1046–1048. 10.1063/1.103561
Delerue C, Allan G, Lannoo M: Theoretical aspects of the luminescence of porous silicon. Phys Rev B 1993, 48: 11024. 10.1103/PhysRevB.48.11024
Ledoux G, Guillois O, Porterat D, Reynaud C, Huisken F, Kohn B, Pillard V: Photoluminescence properties of silicon nanocrystals as a function of their size. Phys Rev B 2000, 62: 15942. 10.1103/PhysRevB.62.15942
Lehmann V, Gösele U: Porous silicon formation: a quantum wire effect. Appl Phys Lett 1991, 58: 856–858. 10.1063/1.104512
Canham LT: Bioactive silicon structure fabrication through nanoetching techniques. Adv Mater 1995, 7(12):1033–1037. 10.1002/adma.19950071215
Canham LT: Nanoscale semiconducting silicon as a nutritional food additive. Nanotechnology 2007, 18: 185704. 10.1088/0957-4484/18/18/185704
Park JH, Gu L, von Maltzahn G, Ruoslahti E, Bhatia SN, Sailor MJ: Biodegradable luminescent porous silicon nanoparticles for in vivo applications. Nat Mater 2009, 8: 331–336. 10.1038/nmat2398
Erogbogbo F, Yong KT, Roy I, Xu GX, Prasad P, Swihart MT: Biocompatible luminescent silicon quantum dots for imaging of cancer cells. ACS Nano 2008, 5: 873–878.
Santos HA, Bimbo LM, Herranz B, Shahbazi M, Hirvonen J, Salonen J: Nanostructured porous silicon in preclinical imaging: moving from bench to bedside. J Mater Res 2013, 28(2):152–164. 10.1557/jmr.2012.271
Anglin EJ, Cheng L, Freeman WR, Sailor MJ: Porous silicon in drug delivery devices and materials. Adv Drug Deliv Rev 2008, 60: 1266–1277. 10.1016/j.addr.2008.03.017
Santos HA, Bimbo LM, Lehto VP, Airaksinen AJ, Salonen J, Hirvonen J: Multifunctional porous silicon for therapeutic drug delivery and imaging. Curr Drug Discov Technol 2011, 8(3):228–249. 10.2174/157016311796799053
Timoshenko VY, Kudryavtsev AA, Osminkina LA, Vorontsov AS, Ryabchikov YV, Belogorokhov IA, Kovalev D, Kashkarov PK: Silicon nanocrystals as photosensitizers of active oxygen for biomedical applications. JETP Lett 2006, 83(9):423–426. 10.1134/S0021364006090128
Xiao L, Gu L, Howell SB, Sailor MJ: Porous silicon nanoparticle photosensitizers for singlet oxygen and their phototoxicity against cancer cells. ACS Nano 2011, 5(5):3651–3659. 10.1021/nn1035262
Osminkina LA, Tamarov KP, Sviridov AP, Galkin RA, Gongalsky MB, Solovyev VV, Kudryavtsev AA, Timoshenko VY: Photoluminescent biocompatible silicon nanoparticles for cancer theranostic applications. J Biophotonics 2012, 5(7):529–535. 10.1002/jbio.201100112
Low SP, Williams KA, Canham LT, Voelcker NH: Generation of reactive oxygen species from porous silicon microparticles in cell culture medium. J Biomed Mater Res Part A 2010, 93A(3):1124–1131.
Osminkina LA, Luckyanova EN, Gongalsky MB, Kudryavtsev AA, Gaydarova AK, Poltavtseva RA, Kashkarov PK, Timoshenko VY, Sukhikh GT: Effects of nanostructurized silicon on proliferation of stem and cancer cell. Bull Exper Biol Med 2011, 151(1):79–83. 10.1007/s10517-011-1264-5
Osminkina LA, Gongalsky MB, Motuzuk AV, Timoshenko VY, Kudryavtsev AA: Silicon nanocrystals as photo- and sono-sensitizers for biomedical applications. Appl Phys B 2011, 105: 665–668. 10.1007/s00340-011-4562-8
O’Farrell N, Houlton A, Horrocks BR: Silicon nanoparticles: applications in cell biology and medicine. Int J Nanomedicine 2006, 1(4):451–472. 10.2147/nano.2006.1.4.451
Salonen J, Lehto VP: Fabrication and chemical surface modification of mesoporous silicon for biomedical applications. Chem Eng J 2008, 137: 162–172. 10.1016/j.cej.2007.09.001
Sweetman MJ, Ronci M, Ghaemi SR, Craig JE, Voelcker NH: Porous silicon films micropatterned with bioelements as supports for mammalian cells. Adv Funct Mater 2012, 22: 1158–1166. 10.1002/adfm.201102000
Granitzer P, Rumpf K: Porous silicon—a versatile host material. Materials 2010, 3(2):943–998. 10.3390/ma3020943
Godin B, Liu TE, Serda REX, Ferrari M: Multistage nanovectors: from concept to novel imaging contrast agents and therapeutics. Acc Chem Res 2011, 44(10):979–989. 10.1021/ar200077p
Haidary SM, Corcoles EP, Ali NK: Nanoporous silicon as drug delivery systems for cancer therapies. J Nanomaterials 2012, 2012: 1–15.
Peng KQ, Yan YJ, Gao SP, Zhu J: Synthesis of large-area silicon nanowire arrays via self-assembling nanoelectrochemistry. Adv Mater 2002, 14(16):1164–1167. 10.1002/1521-4095(20020816)14:16<1164::AID-ADMA1164>3.0.CO;2-E
Peng K, Huang Z, Zhu J: Fabrication of large‒area silicon nanowire p–n junction diode arrays. Adv Mater 2004, 16(1):73–76. 10.1002/adma.200306185
Huang Z, Geyer N, Werner P, de Boor J, Gösele U: Metal-assisted chemical etching of silicon: a review. Adv Mater 2011, 23(2):285–308. 10.1002/adma.201001784
Nassiopoulou AG, Gianneta V, Katsogridakis C: Si nanowires by a single-step metal-assisted chemical etching process on lithographically defined areas: formation kinetics. Nano Res Let 2011, 6: 597. 10.1186/1556-276X-6-597
Sivakov VA, Brönstrup G, Pecz B, Berger A, Radnoczi GZ, Krause M, Christiansen SH: Realization of vertical and zigzag single crystalline silicon nanowire architectures. J Phys Chem C 2010, 114: 3798–3803. 10.1021/jp909946x
Qu Y, Zhoua H, Duan X: Porous silicon nanowires. Nanoscale 2011, 3: 4060–4068. 10.1039/c1nr10668f
Qu Y, Liao L, Li Y, Zhang H, Huang Y, Duan X: Electrically conductive and optically active porous silicon nanowires. Nano Lett 2009, 9(12):4539–4543. 10.1021/nl903030h
Sivakov VA, Voigt F, Berger A, Bauer G, Christiansen SH: Roughness of silicon nanowire sidewalls and room temperature photoluminescence. Phys Rev B 2010, 82: 125446–125452.
Timoshenko VY, Gonchar KA, Golovan LA, Efimova AI, Sivakov VA, Dellith A, Christiansen SH: Photoluminescence and Raman scattering in arrays of silicon nanowires. J Nanoelectr Optoelectr 2011, 6(4):519–524. 10.1166/jno.2011.1205
Bronstrup G, Jahr N, Leiterer C, Csaki A, Fritzsche W, Christiansen S: Optical properties of individual silicon nanowires for photonic devices. ACS Nano 2010, 4(12):7113–7122. 10.1021/nn101076t
Sivakov V, Andrä G, Gawlik A, Berger A, Plentz J, Falk F, Christiansen SH: Silicon nanowire-based solar cells on glass: synthesis, optical properties, and cell parameters. Nano Lett 2009, 9: 1549–1554. 10.1021/nl803641f
Lv M, Su S, He Y, Huang Q, Hu W, Li D, Fan C, Lee ST: Long-term antimicrobial effect of silicon nanowires decorated with silver nanoparticles. Adv Matt 2010, 22(48):5463–5465. 10.1002/adma.201001934
Timoshenko VY, Lisachenko MG, Kamenev BV, Shalygina OA, Kashkarov PK, Heitmann J, Schmidt M, Zacharias M: Highly efficient sensitizing of erbium ion luminescence in size-controlled nanocrystalline Si/SiO2 superlattice structures. Appl Phys Lett 2004, 84: 2512–2514. 10.1063/1.1690465
Kovalainen M, Mönkäre J, Kaasalainen M, Riikonen J, Lehto VP, Salonen J, Herzig KH, Järvinen K: Development of porous silicon nanocarriers for parenteral peptide delivery. Mol Pharmaceutics 2013, 10: 353–359. 10.1021/mp300494p
Slowing I, Trewyn BG, Lin VS-Y: Effect of surface functionalization of MCM-41-type mesoporous silica nanoparticles on the endocytosis by human cancer cells. J Am Chem Soc 2006, 128: 14792–14793. 10.1021/ja0645943
Sviridov AP, Andreev VG, Ivanova EM, Osminkina LA, Tamarov KP, Timoshenko VY: Porous silicon nanoparticles as sensitizers for ultrasonic hyperthermia. Appl Phys Lett 2013, 103: 193110. 10.1063/1.4829148
This work was supported by the German Federal Ministry of Education and Research (BMBF) under Grant No. 01DJ13010 in frame of German-Russian ‘Nanoscaled Semiconductors as a Key for Future Industry’ project. LAO greatly acknowledge the financial support by the joint program of MSU-DAAD ‘Vladimir Vernadsky’ (A/12/89292).
The authors declare that they have no competing interests.
LAO performed the optical measurements and data analysis. VAS contributed in the development of the preparation method and analysis of the results. GAM and VAG performed the measurements of the PL spectra of the samples. UAN performed the structural measurements and the DLS spectra of the samples. FT performed the SiNWs fabrication. VVS and AAK performed in vitro measurements. VYT performed the general data analysis and discussion of the obtained data. All authors participated in writing the manuscript and approved its final version.
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Additional file 1: (‘Z-scan movie of living CF2Th cells with added SiNPs’) shows a z-scan movie of living CF2Th cells with added SiNPs. Green, blue, and red colors correspond to the luminescence of cell cytoplasm (colored with GFP), cell nuclei (colored with Hoechst) and SiNPs, respectively. (ZIP 377 KB)
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Osminkina, L.A., Sivakov, V.A., Mysov, G.A. et al. Nanoparticles prepared from porous silicon nanowires for bio-imaging and sonodynamic therapy. Nanoscale Res Lett 9, 463 (2014). https://doi.org/10.1186/1556-276X-9-463
- Silicon nanowires
- Silicon nanoparticles
- Sonodynamic therapy