The Anti-Proliferative Activity of Coordination Compound-Based ZnO Nanoparticles as a Promising Agent Against Triple Negative Breast Cancer Cells

ORIGINAL RESEARCH The Anti-Proliferative Activity of Coordination Compound-Based ZnO Nanoparticles as a Promising Agent Against Triple Negative Breast Cancer Cells Hana Stepankova 1,2 Marcin Swiatkowski 3 Rafal Kruszynski 3 Pavel Svec 1 Hana Michalkova 1 Vendula Smolikova 1,2 Andrea Ridoskova 1,4 Zbynek Splichal 1,2 Petr Michalek 1,2 Lukas Richtera 1,2 Pavel Kopel 5 Vojtech Adam 1,2 Zbynek Heger 1,2 Simona Rex 1,2 1 Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czechia; 2 Central European Institute of Technology, Brno University of Technology, Brno, Czechia; 3 Institute of General and Ecological Chemistry, Lodz University of Technology, Lodz, Poland; 4 Central European Institute of Technology, Mendel University in Brno, Brno, Czechia; 5 Department of Inorganic Chemistry, Faculty of Science, Palacky University, Olomouc, Czechia Purpose: The present study deals with the in vitro evaluation of the potential use of coordination compound-based zinc oxide (ZnO) nanoparticles (NPs) for the treatment of triple negative breast cancer cells (TNBrCa). As BrCa is one of the most prevalent cancer types and TNBrCa treatment is difficult due to poor prognosis and a high metastasis rate, finding a more reliable treatment option should be of the utmost interest. Methods: Prepared by reacting zinc carboxylates (formate, acetate, propionate, butyrate, isobutyrate, valerate) and hexamethylenetetramine, 4 distinct coordination compounds were further subjected to two modes of conversion into ZnO NPs – ultrasonication with oleic acid or heating of pure precursors in an air atmosphere. After detailed characterization, the resulting ZnO NPs were subjected to in vitro testing of cytotoxicity toward TNBrCa and normal breast epithelial cells. Further, their biocompatibility was evaluated. Results: The resulting ZnO NPs provide distinct morphological features, size, biocompatibility, and selective cytotoxicity toward TNBrCa cells. They internalize into two types of TNBrCa cells and imbalance their redox homeostasis, influencing their metabolism, morphology, and ultimately leading to their death via apoptosis or necrosis. Conclusion: The crucial properties of ZnO NPs seem to be their morphology, size, and zinc content. The ZnO NPs with the most preferential values of all three properties show great promise for a future potential use in the therapy of TNBrCa. Keywords: carboxylate, hexamethylenetetramine, nanoparticles, redox homeostasis imbalance, triple negative breast cancer, zinc oxide coordination compounds Introduction As breast cancer is the cancer type with the second highest prevalency, a huge attention is paid toward the development of novel drug modalities for its therapy. 1 Based on its molecular subtypes, it is usually categorized into human epidermal growth factor receptor 2 (HER2) positive, estrogen receptor (ER) positive, progesterone receptor (PR) positive, luminal A, or luminal B, 2 where ER and PR positive subtypes form nearly 70% of all breast cancer cases and 20% lack HER2, ER, and PR. Called triple negative breast cancer, 3 these often possess an aggressive phenotype with an increased metastasis rate, poor prognosis, 1 and unreliable treatability with numerous side effects due to conventional chemotherapy inhibiting both healthy and cancer cells. 4 These side effects could be overcome by the employment of nano-scaled particles (NPs) with a specific affinity toward cancer cells. 1 NPs have received Correspondence: Simona Rex Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czechia Tel +420-5-4513-3350 Fax +425-5-4521-2044 Email [email protected] International Journal of Nanomedicine 2021:16 4431–4449 4431 © 2021 Stepankova et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/ terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). International Journal of Nanomedicine Dovepress open access to scientific and medical research Open Access Full Text Article Received: 5 February 2021 Accepted: 27 May 2021 Published: 1 July 2021 International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) increased attention thanks to the enhanced permeability and retention (EPR) effect, causing suitably sized particles to intrinsically accumulate in many types of solid tumors due to the presence of large pores in rapidly formed tumor neovasculature together with a lack of lymph vessels. 5 However, the EPR effect is not present in all types of solid tumors and even where it is, there is only 2× increased delivery of NPs to tumors compared to critical off-target organs. 6 For this reason, engineering of NPs to selectively bind to tumor cells rather than normal cells can greatly improve their therapeutic efficacy. 7–9 Recently, zinc oxide (ZnO) NPs, generally known for their antibacterial properties, 10 attracted much attention as a suitable platform for the therapy of various types of cancer. They provide different photo-oxidizing and photocatalytic effects, owning to their ultraviolet light absorption, 11 semiconducting, and magnetic properties. 12 They can induce selective inhibition of cancer cells, 13 with additive or synergistic effects together with anti-cancer compounds. 14 The induction of apoptosis by ZnO NPs was observed in breast cancer cells, 15,16 including triple negative breast cancer. 17,18 On the other hand, certain ZnO NPs can accumulate in various off-target organs, leading to toxicity for heart, brain, thymus, pancreas, liver, kidney, and others. 19–22 This work is focused on the development of ZnO NPs converted from distinct coordination compounds providing selective cytotoxicity toward triple negative breast cancer cells of human origin, while maintaining biocompatibility, and avoiding the normal cells of breast epithelium. Materials and Methods Chemicals Most of the listed chemicals were purchased from SigmaAldrich (St. Louis, MO, USA) in an ACS purity. The MilliQ ® Water Purification System (Merck Millipore, Burlington, MA, USA) was employed to produce deionized water. Synthesis of Coordination Compounds Coordination compounds were synthesized according to literature procedure in direct reactions featuring Zn carboxylate and hexamethylenetetramine (HMTA) in aqueous medium 23 (synthesis and analytical 24 details are given in Supplementary Materials Paragraph S1.1). The reactions between Zn carboxylates (formate, acetate, propionate, butyrate, isobutyrate, valerate) and HMTA were carried out with metal to ligand (M:L) molar ratio of 2:1 (complementary to the previous research, 23 in which stoichiometries of 1:1 and 1:2 were applied) and led to the formation of 4 coordination compounds (Supplementary Table S1). Similar to the previously reported data, in the synthesis with Zn valerate, both substrates crystallized separately and there was no evidence of a formation of coordination compounds (despite the used M:L molar ratio). The separate crystallization of salt and HMTA also occurred in the synthesis involving Zn butyrate with M:L molar ratio of 2:1. Synthesis of ZnO NPs Two strategies of a single precursor method were applied to the synthesis of ZnO NPs. In the first one (S1), samples of coordination precursors (containing 0.01 mol of Zn) were suspended in oleic acid (10 mL) with support of ultrasonic irradiation generated by the ultrasonic processor Hielscher 100UP (Hielscher Ultrasonics, Teltow, Germany) equipped with a sonotrode of diameter 10 mm (power 100 W, working frequency 30 kHz, maximum amplitude 70 µm) followed by heating at 220 °C for 2 h. After that the temperature was raised to 800 °C with a maximum heating range (~30 °C/min) in order to purify ZnO from oleic acid. In this approach, NPs A, B, C, and D were produced from precursors 1, 2, 3, and 5, respectively. In the second strategy (S2), precursor samples (containing 0.01 mol of Zn) were heated in an air atmosphere up to 500 °C (5 °C/min heating rate), and then left at this temperature for 1 h. In this approach, NPs E, F, G, and H were produced from precursors 1, 2, 3, and 5, respectively. Surface morphologies of NPs were studied using a scanning electron microscope (SEM, Hitachi S-4700 [Hitachi, Tokyo, Japan]) with an energy-dispersive X-ray spectrometer (EDS Thermo NORAN [Thermo Fisher Scientific, Waltham, MA, USA]). Determination of Crystal Structure The colorless prism crystals of compounds 3 and 5 were mounted on the Rigaku Synergy Dualflex automatic diffractometer equipped with Pilatus 300K detector (Rigaku, Tokyo, Japan). X-ray intensity data were collected using the monochromated CuK α mirror (λ=1.54184 Å, microfocus sealed PhotonJet X-ray tube), radiation at 100.0(1) K, ω scan and the shuterless mode. The reflections inside an Ewald sphere were collected up to ϴ=79°. 61102 and 12308 strongest reflections were used for 3 and 5, respectively, to determine the unit cell parameters. The details are provided in Supplementary Table S2. No loss of intensity during the measurement occurred. Polarization, https://doi.org/10.2147/IJN.S304902 DovePress International Journal of Nanomedicine 2021:16 4432 Stepankova et al Dovepress Powered by TCPDF (www.tcpdf.org) International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) Lorentz, and numerical absorption corrections (using a Gaussian integration over a multifaceted crystal model) were used. A dual-space algorithm was used to solve the structure. The full-matrix, least-squares technique on F2 was used to anisotropically refine the non-hydrogen atoms, whereas the hydrogen atoms were found after four cycles of anisotropic refinement from difference Fourier synthesis, and refined as “riding” on the adjacent atom with geometric idealization after each cycle of the refinement. The individual isotropic displacement factors were 1.5× of parent methyl carbon and oxygen atoms and 1.2× of parent non-methyl carbon atoms. Rotation about local three-fold axes was allowed for the methyl groups. Programs SHELXT, 25 SHELXL, 26 and SHELXTL 27 were used for all the calculations. International Tables for Crystallography were used to obtain atomic scattering factors. 28 Supplementary Table S3, shows the interatomic bond distances and Supplementary Table S4, shows the intramolecular interactions. Further details are in the Supplementary Paragraph S1.2. The supplementary crystallographic data are in CCDC 1980739−1980740 and they can be obtained free of charge via www.ccdc.cam.ac.uk/ data_request/cif, [email protected], or The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK. Other Physical Measurements The infrared (IR) spectra were recorded on the Jasco Fourier transform (FT)/IR 6200 spectrophotometer (Jasco Deutschland GmbH, Pfungstadt, Germany), in a form of KBr pellets. The spectral range was 4000−400 cm −1 . The thermal analyses were performed using the TG/DTASETSYS−16/18 thermoanalyser coupled with the ThermoStar mass spectrometer (Balzers, Liechtenstein). Platinum crucibles were used to heat the samples up to 1000 °C (5 °C/min) in 1 L/h flow of synthetic air (composed of N 2 :O 2 79:21). X-ray powder diffraction (XRPD) using the Powder Diffraction File and XPert PRO XRPD system (CENIMAT|i3N, Caparica, Portugal) equipped with CuKα1 radiation, Bragg-Brentano PW 3050/65 highresolution goniometer, and PW 3011/20 proportional point detector were used to confirm the final products of decomposition. 29 Zn Content Analysis The stock ZnO NPs suspensions (1 mg/mL) were prepared by suspending the NPs in the appropriate solution (water in the case of Zn content determination), followed by ultrasonication for 2 h. To evaluate the Zn content, a standard solution of Zn (1 mg/mL; Merck Millipore) was used. Calibration solutions were acidified by HNO 3 suprapure (1%, Penta Chemicals Unlimited, Prague, Czech Republic), obtained by sub-boiling distillation using the BSB-939IR (Berghof, Eningen unter Achalm, Germany). Suspensions of ZnO NPs were diluted with HNO 3 (5%) for the selected calibration range (0.0–10.0 µg/mL). The atomic absorption spectrometer 240FS AA (Agilent Technologies, Santa Clara, CA, USA), with a deuterium background correction and flame atomization was used for Zn content analysis. The instrument operated under the oxygen flow of 13.5 L/min and acetylene flow of 2.0 L/min. The radiation source was the ultrasensitive hollow cathode lamp (Agilent Technologies) with the operational wavelength for Zn detection of 213.9 nm. Hydrodynamic Diameter and ζ-Potential Measurement The ZnO NPs were suspended in water (1 mg/mL) and diluted 200× for hydrodynamic diameter (HDD) measurement and 50× for ζ-potential determination. Quasielastic dynamic light scattering and Doppler microelectrophoresis were used, respectively, both employing the Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The refractive index of 2.000 was used for the dispersive phase and 1.333 for the dispersive environment. Equilibration lasted for 120 s. To determine the HDD and polydispersive index (PdI), the cuvettes ZEN0040 (Brand GmbH, Wertheim, Germany) were used with the measurement of each suspension in hexaplicates. For ζ-potential determination, the cells DTS1070 (Brand GmbH, Wertheim, Germany) were used with the measurement of each suspension in triplicates. The measurements were performed for three separate suspensions of each ZnO NPs. The calculations were performed as in. 30 Stability of ZnO NPs in Water ZnO NPs were suspended in deionized water and imaged using a SEM Tescan MAIA 3 (Tescan Ltd., Brno, Czech Republic) with a field-emission gun. To obtain 768×858 pixel images, the In-lens SE detector was used (working distance of 2.41–3.06 mm, acceleration voltage 5 kV, magnification 100,000×, sample area 2.08 µm). UH resolution mode was employed for full frame capture with the enabled image shift correction for image accumulation (~0.32 µs/pixel dwell time, 2.4 nm spot size). International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S304902 DovePress 4433 Dovepress Stepankova et al Powered by TCPDF (www.tcpdf.org) International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) Stability of ZnO NPs Size in Ringer’s Solution ZnO NPs suspended in Ringer’s solution pH 7.4 (containing 6.50 g NaCl, 0.42 g KCl, 0.25 g CaCl 2 , and 0.20 g NaHCO 3 in 1 L of deionized water) were kept at 37 °C with the evaluation of HDD at different time points. Cell Lines Normal breast cell line HBL-100 and triple negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were purchased from American Type Culture Collection (Manassas, VA, USA). HBL-100 were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) and both cancer cell lines were cultured in RPMI (Roswell Park Memorial institute) 1640 medium, both supplemented with penicillin (100 U/mL), streptomycin (0.1 mg/mL), and fetal bovine serum (FBS, 10% v/v). Humidified incubator (Galaxy 170 R, Eppendorf, Hamburg, Germany) with 5% CO 2 was employed to maintain the cells. 24hIC 50 Determination The cell viability was assayed by measuring mitochondrial activity using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as in. 31 Incubation in 1:1 ratio with the varying concentrations of ZnO NPs in medium (maximum of 1 mg/mL) lasted for 24 h. The 24hIC 50 concentration was determined from the viability data. Wound Healing Assay An artificial wound was formed in a cell monolayer as in. 31 The influence of the treatment with ZnO NPs (24hIC 50 ) in medium was captured at the beginning and at each time point (0; 2; 4, 8; 12; 24; 48; and 72 h [only 24, 48 and 72 h are shown in this work]). Hard Protein Corona Formation ZnO NPs were exposed to human plasma or FBS. Human plasma was obtained as in 31 and kept at –80 °C until use. Plasma pooled from multiple donors and FBS were defrosted and spun at 21,000 rcf and 4 °C for 30 min to remove aggregates formed during the defrosting. Next, the supernatant was incubated at 37 °C and 350 rpm for 35 min and again spun to remove aggregates. ZnO NPs in water (1 mg/mL) were mixed in 1:1 ratio (v/v) with FBS or plasma and the mixture was incubated at 37 °C and 350 rpm for 35 min. As a control of the follow-up cleaning process from unbound proteins, phosphate buffered saline (PBS) mixed with FBS or plasma was used. The rest of the assay was performed as in 31 with some notable changes. The pellets were washed 5× with PBS by centrifugation at 15,000 rcf and 4 °C for 15 min. Finally, the pellets were resuspended in deionized water (100 µL) and mixed (10 µL) with loading buffer (5 µL; 50 mM Tris/HCl, 20% glycerol, 0.1% bromophenol blue, and 2% sodium dodecyl sulphate [SDS]). NEB protein ladder 10–250 kDa (5 µL, New England Biolabs, Ipswich, MA, USA) was used as a molar mass marker. The protein bands were imaged using near infrared irradiation at 700 nm on Azure c600 (Azure Biosystems, Dublin, CA, USA). Opsonization with Complement C3b Element ZnO NPs in water were mixed with human serum from male AB clotted whole blood (H6914, cleaned from aggregates in the same way as plasma and FBS) in 1:1 ratio and incubated at 37 °C and 350 rpm for 35 min. After the incubation, ZnO NPs with bound proteins were 3× centrifuged at 15,000 rcf and 4 °C for 15 min with the washing of the pellets containing NPs and bound proteins by PBS. Finally, pellets were resuspended in deionized water (100 µL), mixed in 2:1 ratio with loading buffer and resolved on 6% SDS PAGE at 200 V and 4 °C for 30 min. After the electrotransfer of the proteins onto the Immobilon ® -FL PVDF membrane (Merck Millipore), the membrane was blocked with skim milk for 1 h at 20 °C. Primary complement C3 monoclonal antibody (LF-MA0132, Thermo Fisher Scientific, 1:1000) was used, with incubation at 20 °C for 1 h. Secondary antibody labelled with horseradish peroxidase (HRP, p0260, Dako, Santa Clara, CA, USA, 1:5000) was incubated with the membrane at 20 °C for 1 h. Chemiluminescent signal was developed with Clarity Western ECL Blotting Substrate (Bio-Rad, Hercules, CA, USA) and membranes were imaged using Azure c600 (Azure Biosystems). Hemocompatibility To determine the hemocompatibility of ZnO NPs in water, fresh human red blood cells (RBCs) were used as in 31 with the ZnO NPs diluted in PBS (pH 7.4; 500, 250, 125, and 62.5 µg/mL). Ethidium Bromide (EtBr) Displacement The ability of ZnO NPs to displace EtBr was studied as in 31 with some notable changes. Plasmid MP px459 was used and ZnO NPs in water (1000; 500; 250; 125 µg/mL, diluted in PBS with 10 mM NaCl) were added (final volume 25 µL). As https://doi.org/10.2147/IJN.S304902 DovePress International Journal of Nanomedicine 2021:16 4434 Stepankova et al Dovepress Powered by TCPDF (www.tcpdf.org) International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) a control, plasmid (1 µg) with EtBr (6 µM), and ZnO NPsfree PBS with NaCl (10 mM) was used. The rate of displacement was calculated as a percentage decrease of EtBr fluorescence. Plasmid Cleaving Assay ZnO NPs in water were diluted (to 500 µg/mL) with Tris-HCl buffer (50 mM) containing NaCl (50 mM, pH 7.2). After that, plasmid MP px330 (50 µg/mL) was incubated with the ZnO NPs (final volume 10 µL), according to. 31 Loading buffer (5 µL, 30% glycerol with 0.1% bromophenol blue) was used, with 2-Log DNA ladder as a molar mass marker. Reactive Oxygen Species (ROS) Formation 10,000 cells seeded in a 24-well plate were incubated with ZnO F-H in medium (55 µg/mL) for 6 h, stained with CellROX ® Deep Red (Thermo Fisher Scientific, 1:500 in PBS), detached by accutase, and analyzed on flow cytometer BD Accuri C6 Plus (BD Biosciences, Franklin Lakes, NJ, USA), with the 35 µL/min flow rate and at least 10,000 cells analyzed in each group. Lipid Peroxidation The analysis was performed as in 32 with the cells treated with ZnO F-H in medium (55 µg/mL) for 12 h. Cumene hydroperoxide (CH, 150 µM) was administered as a positive control. Fiji ImageJ software (National Institute of Health, Bethesda, MD, USA) was used to calculate the fluorescence intensity. F-Actin Disorganization, Nuclei-toCytoplasm (N/C) Ratio, and Cell Morphology ~40,000 cells/coverslip seeded were treated with ZnO F-H in medium (55 µg/mL) for 24 h, followed by fixation, labeling of F-actin filaments, imaging, and analysis as in 32 with the following exceptions. To visualize ZnO NPs, confocal reflectance microscopy was employed, with irradiation by a solid state 633 nm laser and detection window at 631–636 nm. To evaluate morphometric parameters, a minimum of 20 cells in each group were analyzed. Orientation J plugin with a cubic-spline gradient and 7 ROI in each picture was used to analyze coherency. 33 Cell morphology upon treatment with ZnO F-H in medium (55 µg/mL) for 6 h was evaluated by Cryo-SEM as in. 32 Apoptosis Induction ~600,000 cells seeded into each well of a 6-well plate were treated with ZnO F-H in medium (55 µg/mL) for 6 or 24 h. Cells detached by accutase, as well as spontaneously detached cells were dual stained for translocated phosphatidylserine and cell permeability using the PE Annexin V Apoptosis Detection Kit I (BD Biosciences). The analysis was performed using flow cytometry as in 22 with at least 100,000 cells analyzed in each group. Further, cytopathological May-Grünwald-Giemsa staining was performed upon treatment with ZnO F-H (55 µg/mL) for 24 h according to. 30 Descriptive Statistics The main tendency was measured using the mean and the dispersion using the standard deviation (SD). To analyze the differences between groups, an unpaired t-test in software GraphPad (GraphPad Software, San Diego, CA, USA) was used. Figures were processed using Microsoft Office PowerPoint software (Redmond, WA, USA) and images using Fiji Image J (National Institute of Health), unless otherwise mentioned. Results and Discussion Synthesis Stoichiometry Affects the Composition of the Coordination Compounds Formed by Zn Propionate and Isobutyrate but Not Formate or Acetate The reactions between Zn carboxylates and HMTA led to the formation of two previously described (1 and 2) 23 and two new (3 and 5) coordination compounds (Supplementary Table S3). The stoichiometry used in the synthesis (metal: HMTA equal to 2:1 [this work], 1:1, and 1:2) 23 does not affect the composition of formed compounds 1 and 2, i. e. M:L is always 1:2 in 1 and 1:1 in 2. Additionally, both these compounds (in opposition to the other discussed) are mononuclear molecular systems. The synthesis stoichiometries used previously (1:2 and 1:1) 23 were not reflected in compounds containing propionate and butyrate ions (3A and 4A with M:L of 2:1). Repetition of these syntheses with resulting product M:L ratio (i. e. 2:1) led to the formation of compound 3 (M:L of 3:1) and separate crystallization of pure Zn butyrate [polymeric solventless Zn(n-C 3 H 7 COO) 2 ] form 34 and pure HMTA. It proves that excess of a neutral ligand is International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S304902 DovePress 4435 Dovepress Stepankova et al Powered by TCPDF (www.tcpdf.org) International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) necessary for the formation of the 3, 3A, and 4A because in these cases the final compound possesses a smaller amount of ligand than was used in the synthesis. Hypothetical coordination compound of Zn butyrate with M:L of 3:1 was not formed in the solid state probably because of a lower dissociation constant (and higher stability) of polymeric Zn butyrate 34 than Zn propionate 35,36 (Zn butyrate crystallized as a first, and HMTA later, after precipitation of the whole salt). The synthesis stoichiometry affects the composition of the coordination compounds formed by Zn isobutyrate but it does not affect the M:L ratio in the product. In both cases, it is 1:1, but the compound formed in reactions with 1:2 and 1:1 stoichiometries of substrates is zig-zag one-dimensional polymer (5A), 23 while a 2:1 reaction stoichiometry leads to the formation of a dinuclear molecular compound (5). In this reaction, the competition between anions and HMTA in an inner coordination sphere leads to the formation of four carboxylate bridges between two neighboring Zn cations in case of HMTA deficiency (compound 5) and one carboxylate bridge between two subsequent atoms in chains in case of a stoichiometric amount of HMTA (compound 5A). In general, a lengthening of anions with non-branched aliphatic chains leads to a lower number of neutral molecules present in the resulting compounds. This effect is not observed for anions with branched aliphatic chains. The Four Compounds Have Distinct Crystal and Molecular Structure The appliance of carboxylate anions differing slightly in the length of the aliphatic chain allows constructing diverse coordination moieties containing the same neutral ligand (HMTA). Coordination numbers of Zn cations, as well as coordination modes of ligands also vary in the studied compounds. All studied compounds are dissimilar, the 1 and 2 are mononuclear compounds with HMTA molecules located in outer and inner coordination sphere, respectively, the 3 (Figure 1Aa) is a two-dimensional coordination polymer with a hexagonal tiling (Figure 1B), and 5 is a dinuclear molecular compound (Figure 1Ab). The structures of 1 and 2 were described in, 23 thus the respective data are not discussed in the current work. All atoms of 3 (Figure 1Aa) are located at general positions but the presence of the glide planes going through the asymmetric unit moiety expands it into a polymeric net. Each asymmetric unit contains six structurally different Zn cations divided into two distinguishable coordination systems composed of Zn 3 (C 2 H 5 COO) 5 (OH) heteroionic units interlinked by the neutral HMTA molecules. Each unit possesses cations in two different coordination environments. Two Zn cations of each unit (Zn1/Zn2 and Zn4/Zn5) adopt a slightly distorted trigonal bipyramidal geometry 37 with apexes occupied by HMTA N atom and O atom of OH − ion, and three base corners created by the O atoms of bridging carboxylate ions. One Zn cation of each unit (Zn3 and Zn6) exists in an almost ideal tetragonal bipyramidal geometry 38 with apexes inhabited also by HMTA N atom and O atom of OH − ion, and four O atoms of bridging carboxylate ions creating polyhedron base. Consequently, all carboxylate anions bridge two Zn 2+ ions; however, one pair of cations is bridged by one carboxylate anion, while two other pairs of cations are bridged by two carboxylate anions. The central OH − ion links all three cations forming one heteroionic unit. The Zn 3 (C 2 H 5 COO) 5 (OH) moieties are connected to three tridentate HMTA molecules via Zn–N coordination bonds (Supplementary Table S2). The Zn···Zn distances within Zn 3 (C 2 H 5 COO) 5 (OH) units vary in the range of 3.32–3.73 Å. Compound 3 is a very rare example of a polymer composed of trinuclear Zncarboxylate moieties assembled further by bridging N ligands. The two-dimensional polymeric net of 3 extends along the crystallographic (0 1 0) plane and is composed of rings with nodes occupied by Zn cations and HMTA molecules. The whole polymeric system creates a 3,3,4-c 3-nodal net with stoichiometry (3-c)(3-c)3(4-c)3 and is described by the {3.12 2 }3{3 3 .12 2 .13}3{3 3 } Schlafli symbol. Considering the simplified net with nodes located only at Zn cations, the polymer adopts a kagome pattern (kgm topological type; Shubnikov plane net (3.6.3.6)), i. e. 4-c unimodal net described by the {3 2 .6 2 .7 2 } Schlafli symbol (Supplementary Figure S9). 39 This is distinctly different from the simple 3-c unimodal net ({4.8 2 } Schlafli symbol) existing in compounds 3A and 4A. The asymmetric unit of 5 (Figure 1Ab) contains one fourth of the molecular compound. Consequently, one molecule occupies four asymmetric units and the unit cell contains two complex molecules. The Zn1, N1, N2, C1, and C4 atoms occupy a special position i of C2/m space group with site symmetry m and multiplicity 4, thus half of each of them resides in the asymmetric unit. The central atom in 5 is surrounded by four O atoms (each belonging to one bridging carboxylate ion) creating the tetragonal pyramid 37 base and one N atom of HMTA molecule located at polyhedron apex. Therefore, two Zn https://doi.org/10.2147/IJN.S304902 DovePress International Journal of Nanomedicine 2021:16 4436 Stepankova et al Dovepress Powered by TCPDF (www.tcpdf.org) International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) atoms existing in the molecule are bridged by four carboxylate ions and HMTA molecules acting as monodentate ligands. The Zn cations within one molecule are separated by about 2.892(1) Å which is larger than the doubled Van der Waals radius of Zn (2.78 Å). Carboxylate anions bridging five-coordinated central atoms of 3 exhibit a very slight asymmetricity of formed coordination bonds, and all the other carboxylate anions (of 3 and 4) display a strong asymmetricity of Zn-O bonds (Supplementary Table S3). The computed bond valences 40,41 where R ij is the bond-valence parameter for i-j bond 42–44 equal to 1.7588 Å for R Zn-N and 1.6950 Å for R Zn-O , 45 b is the constant equal to 0.37 Å, 46,47 show that in the compound 3 the Zn(pentacoordinated)–O(carboxylate) bonds are about 2× stronger than Zn(pentacoordinated)–N bonds and the strength of Zn(pentacoordinated)–O(OH − ) is in the middle of the previous ones (Supplementary Table S3). The coordination bonds formed by hexacoordinated Zn ions possess similar strength, which agrees with the considerable regularity of coordination polyhedra. Compound 5 possesses the reverse strength of coordination bonds, i. e. the Zn–N bonds are stronger than Zn–O ones. This originates from constraints imposed by a rigid polymeric net existing in 3, which prevents the formation of coordination Zn–O of the most privileged strength (with bond valence equal to 0.4 v.u. for pentacoordinated divalent cations and 0.33 v.u. for hexacoordinated divalent cations). The computed total valences of Zn cations are close to the expected value +2 (Supplementary Table S3), which proves attainment of an optimal coordination sphere geometry for used building block ligands and lack of significant strains in the coordination moieties. The absence of classical hydrogen bond donors (except one) leads to the presence of only weak hydrogen bonds in the studied compounds. The OH – ion (present in compound 3) is formally a classical hydrogen bond donor; however, it shares its electron density with three Zn 2+ cations (via three Zn–O coordination bonds); thus, a partial negative charge on the O atom is diminished, which subsequently reduces a polarization of O–H bond and a partial positive charge of H atom. This results in a decreasing propensity of possible O–H···A interactions. ZnO A ZnO B ZnO C ZnO D ZnO E ZnO F ZnO G ZnO H NPs c*d (mg/L) RSD % ZnO A 733.1 0.7 ZnO B 294.0 0.7 ZnO C 207.6 1.0 ZnO D 512.3 0.6 ZnO E 728.4 0.8 ZnO F 798.9 0.5 ZnO G 712.1 0.6 ZnO H 918.5 0.4 Aa B C Ab D Figure 1 Distinct morphology of the produced nanoparticles originates in the molecular and crystal structure of the coordination compounds and their conversion method. (A) The molecular structure of the compounds 3 (a) and 5 (b), with atom numbering scheme, plotted with 50% probability of displacement ellipsoids of non-hydrogen atoms. The hydrogen atoms are omitted to improve clarity in 3, and they are plotted as spheres of arbitrary radii in 5. The symmetry generated atoms, indicated by the d letters, were generated via the following symmetry transformations: 3 (a) x+1, y, z; (b) x, -y+1.5, z-0.5; (c) x, -y+1.5, z+0.5; (d) x-1, y, z; 5 (a) x, -y+1, z; (b) -x+1, -y+1, -z; (c) -x+1, y, -z. (B) The part of a polymeric net of compound 3. The kagome pattern (left) and the general hexagonal motif (right). (C) The SEM micrographs showing morphology of the produced NPs. Scale bar, 3 µm for ZnO F and 10 µm for the rest. (D) Zinc content in the NPs determined by atomic absorption spectroscopy. Abbreviations: NPs, nanoparticles; RSD, relative standard deviation; SEM, scanning electron microscope; ZnO, zinc oxide. International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S304902 DovePress 4437 Dovepress Stepankova et al Powered by TCPDF (www.tcpdf.org) International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) The polymeric net of compound 3 contains multiple intermolecular weak hydrogen bonds connecting the C-H(HMTA) donors and O(carboxylate) acceptors (Supplementary Table S4). The subsequent nets are relatively well separated, and only H···H distances close to the doubled Van der Waals radius exist. In compound 5 (beside intramolecular C–H···O), one intermolecular C– H···N hydrogen bond exists, and it links the molecules to the piles extending along the crystallographic [0 0 1] axis (Supplementary Table S4). IR Spectra Show Multiplication of Most of HMTA Bands in Compounds 3 and 5 The IR spectra of compounds 1 and 2 were described in 23 thus respective data are not discussed in the current work. Most of HMTA bands present in the spectra of compounds 3 and 5 (Supplementary Figure S1, Supplementary Table S5) are multiplicated (in comparison to the spectrum of pure HMTA) 48 due to the formation of the coordination bonds by some (but not all) nitrogen atoms. 49 The bands corresponding to ρCH 2 (1240 cm −1 ) and νCN (1007 cm −1 ) vibrations split up into quartet and doublet, respectively, in the spectrum of 3, and into doublet and doublet, respectively, in the spectrum of 5. The multiplication to a larger number of bands in the case of 3 in comparison to 5 is a consequence of the tridentate function of HMTA in 3 in contrast to monodentate HMTA in 5. The separation parameter Δν, concerning bands of stretching vibrations of the carboxylate group, is 179 cm −1 (3) and 197 cm −1 (5). These values are in the range characteristic for bridging anions 23 which agrees with the structural data. The absence of the considerable shifts of bands involving non-coordination N atoms of HMTA (e. g. bending NCN vibrations) is caused by a lack of strong or medium strength D–H···N hydrogen bonds. 50,51 Blue (3) and red (5) shifts of bands assigned to stretching symmetric and asymmetric vibrations of carboxylate anions methyl groups originates from a different environment of each anion in a crystal net and consequently different vibration freedom of these groups. The hydroxide anion is represented in a spectrum of 3 by a typical broadened band with a maximum at about 3447 cm −1 , caused by the stretching OH vibrations. Thermogravimetric Analysis Shows Decomposition of Compounds 3 and 5 at Similar Temperatures The thermal decompositions of compounds 1 and 2 were described in 23 thus respective data are not discussed in the current work. The thermal decompositions of 3 and 5 are a one stage processes composed of multiple (7 and 6, respectively) overwrapping substages (Supplementary Figure S2). Due to the absence of the water molecules in the structures of these compounds, both are stable above 100 °C. The presence of some analogy between coordination moieties (occurrence of the bridging carboxylate ions) causes the start of decomposition of 3 and 5 at similar temperatures (142 °C and 155 °C, respectively). The first substage is connected to the decomposition of coordination moieties accompanied by sublimation of the HMTA molecules (endothermic process, Supplementary Figure S2), which, upon elevation of the temperature, is changed to exothermic combustion of HMTA. 52 Upon further increase of the temperature, the simultaneous oxidation of the anions occurs, accompanied by the formation of carbon deposit, which is oxidized much slower in comparison to ligands (it is visible on the TG curves as a mild weight loss at the end of the decomposition process, Supplementary Figure S2). The temperature needed for the formation of pure ZnO slightly differs for the studied compounds, and it is equal to 428 °C and 450 °C for compounds 3 and 5, respectively. Total losses (found/calculated: 67.3/66.0% for 3 and 78.1/78.6% for 5) correspond to the registered (by XRPD) final product, i. e. ZnO. Morphology of ZnO NPs is Bound to the Structure of Coordination Compounds and the Conversion Method The structure of the coordination compounds and the conversion conditions are crucial for controlling the morphology of the resulting ZnO NPs (Figure 1C). In all cases, a hexagonal form of ZnO was formed (wurtzite structure, a = b = 3.249 Å, c = 5.203 Å, Z = 2, space group = P6 3 mc). The NPs synthesized with the usage of oleic acid (S1) are straight nanopoles, while those produced from pure precursors (S2) create complex spherical and polyhedral agglomerates. The mononuclear precursors 1 and 2 allow the production of cylindrical nanowires, polymeric 3 lead to production of hexagonal rods, and dinuclear 5 to https://doi.org/10.2147/IJN.S304902 DovePress International Journal of Nanomedicine 2021:16 4438 Stepankova et al Dovepress Powered by TCPDF (www.tcpdf.org) International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) a mixture of hexagonal rods and spherical/polyhedral particles. It must be outlined that the currently used alteration of the precursor conversion procedure described previously 23 allows change of the shape of the particles (obtained from 1 and 2) from nanoplates to nanopoles and nanospheres. Furthermore, the used conversion method also influences the Zn content in the produced NPs (Figure 1D). In general, NPs synthesized from pure precursors contain significantly more Zn than NPs synthesized with the usage of oleic acid. No correlation between the structure of the coordination compounds and the Zn content in the NPs was found. ZnO NPs Show Favorable Stability for Future in vivo Use The HDD is one of the most crucial parameters when considering NPs for therapy. NPs need to be >5.5 nm to avoid renal clearance and >10 nm to avoid rapid clearance to healthy tissues through the pores in normal blood vessels. 53 At the same time, size <200 nm is required to exploit the EPR effect and escape tumor vasculature 54 and NPs >100 nm can be cleared in the liver, kidneys, and spleen by phagocyting cells of the reticuloendothelial system (RES). 55 Therefore, the size impairment of the ZnO NPs synthesized in this work needs to be addressed before commencing in vivo testing. The differences in dimensions of solid NPs result mainly from precursor structures (Figure 2A top). A comparison of NPs prepared from mononuclear precursors via both strategies demonstrate that the larger NPs are created from 1 (375 and 385 nm for ZnO A and E vs 280 and 195 nm for ZnO B and F, respectively), due to its larger outer coordination sphere and presence of the easily degradable [Zn(H 2 O) 6 ] 2+ coordination moieties. 23 The dimensions of NPs produced from polynuclear precursors are larger for 3 (450 and 325 nm for ZnO C and G, respectively) because the polymeric net of 3 is more stable and cations are packed more closely than in the supramolecular net of dinuclear 5 (375 and 275 nm for ZnO D and H, respectively). The usage of additional dispersing agent and ultrasonication, beside the shape of NPs, also changes their size and uniformity at some degree. For compounds without an outer coordination sphere (2, 3, and 5), the application of S2 (without additional dispersing factors) leads to NPs more uniform in size. Such effect is not observed for 1 as the coordination units are “dispersed” (well separated) by the outer coordination sphere species also in S2. A similar trend was obtained for the HDD of the produced ZnO NPs suspended in water (Figure 2A bottom). Interestingly, NPs with larger HDD were prepared using the synthesis with the usage of oleic acid and ultrasonication (ZnO A = 825 nm; B = 712 nm; C = 825 nm; D = 531 nm), rather than those prepared from pure precursors (ZnO E = 459 nm; F = 531 nm; G = 712 nm; H = 342 nm). The HDD in water was also quite heterogeneous, with the PdI ranging from 0.35 for ZnO F to 1.00 for ZnO H. Except for ZnO F, the NPs were more homogeneous when synthesized with the usage of oleic acid and ultrasonication. The HDD did not significantly change upon suspension of the NPs in Ringer’s solution at 37 °C (Supplementary Figure S3), mimicking human plasma environment 56 and it changed only slightly during further incubation. All ZnO NPs showed slightly negative ζ-potential upon suspension in water (Figure 2A bottom, inset). For ZnO A and D, the ζ-potential was close to the neutral value (−1.14 and −0.61 mV, respectively). The other NPs possessed ζ-potential in the range of −7.5 through −14.8 mV without dependence on the synthesis method. Further, the ζ-potential was stable for up to 72 h. For potential in vivo use, the ζ-potential needs to be large enough to prevent aggregation of the NPs (at least |3−8| mV) 53 but at the same time small enough not to provoke rapid clearance into neighboring tissues and RES through electrostatic means. 57 Also, slightly negative ζ-potential prevents interactions between the NPs and negatively charged membranes of cells forming the endothelial layer of blood vessels and hinders clearance from the organism. 54 It has been shown that the ζ-potential for larger NPs (above 250 nm) does not differ between blood plasma, interstitial fluid, and intracellular fluid, 53 so measuring in one can serve as a good approximation of the other environments. Taken together, the ζ-potential of most of these ZnO NPs is favorable for future in vivo use. Further, the stability of the ZnO NPs morphology upon suspending in a liquid was tested (Figure 2B). It was shown that suspension of ZnO NPs leading to a sustained release of Zn 2+ ions can facilitate their pronounced internalization into cells and enable the anticancer activity 58 by causing mitochondrial damage and disrupting cellular Zn homeostasis, leading to a disequilibrium in protein activity. 59 Most of the ZnO NPs did not show any morphological changes even after 72 h incubation in water, suggesting that the potential International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S304902 DovePress 4439 Dovepress Stepankova et al Powered by TCPDF (www.tcpdf.org) International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) toxicity for normal cells. Further fine-tuning of these properties can therefore lead to synthesis of NPs with distinct mode of cytotoxicity. To the best of our knowledge, this is the first study taking a complex look at the intrinsic triple negative breast cancer cell targeting ability of coordination compound-based ZnO NPs. It is clear that this description offers great opportunities for future synthesis of cancer nanomedicines naturally exploiting the responses of triple negative breast cancer cells to ZnO NPs. Acknowledgments The authors wish to express their thanks to Lucie Markova for perfect technical assistance. 80 85 90 95 100 Negative control ZnO F ZnO G ZnO H Negative control ZnO F ZnO G ZnO H 6h 24h )%(noitubirtsiD Live Early apoptosis Late apoptosis Necrosis 80 85 90 95 100 Negative control ZnO F ZnO G ZnO H Negative control ZnO F ZnO G ZnO H 6h 24h noitubirtsiD (%) 60 70 80 90 100 Negative control ZnO F ZnO G ZnO H Negative control ZnO F ZnO G ZnO H 6h 24h noitubirtsiD (%) HBL-100 MDA-MB-231 MDA-MB-468 ZnO H ZnO G ZnO F ZnO H ZnO G ZnO F ZnO H ZnO G ZnO F 6 h 24 h 6 h 24 h 6 h 24 h Figure 7 ZnO NPs lead to distinct modes of cell death in the exposed cells. Induction of early and late apoptosis and necrosis in cells dual-stained for translocated phosphatidylserine and cell permeability. Insets show representative micrographs of cells stained for cytopathological features according to May-Grünwald-Giemsa. Abbreviations: NPs, nanoparticles; ZnO, zinc oxide. https://doi.org/10.2147/IJN.S304902 DovePress International Journal of Nanomedicine 2021:16 4446 Stepankova et al Dovepress Powered by TCPDF (www.tcpdf.org) International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 78.128.147.172 on 12-Jul-2021 For personal use only. Powered by TCPDF (www.tcpdf.org) Funding The synthesis and characterization of coordination compounds and ZnO nanoparticles, as well as crystallographic part of this work, was financed by funds allocated by the Ministry of Science and Higher Education (Poland) to the Institute of General and Ecological Chemistry, Lodz University of Technology. For testing of the in vitro effects of these nanoparticles, the financial support from ERDF “Multidisciplinary research to increase application potential of nanomaterials in agricultural practice” (No. CZ.02.1.01/0.0/0.0/16_025/0007314), ERC Starting Grant 759585 and the Czech Science Foundation (project no. 1913766J) are gratefully acknowledged. Disclosure The authors report no conflicts of interest in this work. References 1. Jain V, Kumar H, Anod HV, et al. A review of nanotechnology-based approaches for breast cancer and triple-negative breast cancer. J Control Release. 2020;326:628–647. doi:10.1016/j. jconrel.2020.07.003 2. Greish K, Mathur A, Al Zahrani R, et al. Synthetic cannabinoids nano-micelles for the management of triple negative breast cancer. J Control Release. 2018;291:184–195. doi:10.1016/j. jconrel.2018.10.030 3. Saraiva DP, Cabral MG, Jacinto A, Braga S. How many diseases is triple negative breast cancer: the protagonism of the immune microenvironment. 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