IntroductionDespite the widespread use of modern strategies, cancer is still one of the most threatening factors of human’s life1. Clinically, established metal-based chemotherapy drugs such as Cisplatin and its derivatives are widely used2,3. Researchers are seeking alternative metal˗based drugs to maximize the anticancer properties and minimize systemic toxicity and drug resistance. In this regard, there have been remarkable advances in the introduction of the Cu(II) complexes as alternative agents due to the Cu(II) ion is an essential element in the human body as well as being widely present in many biological pathways and enzyme structures as cofactor4,5. Additionally, the Cu(II)˗based drugs have been screened by the National Cancer Institute’s (NCI) Developmental Therapeutics Program (DTP) and have been indicated to possess unique activity in sixty cancerous cell lines6.One of the noticeable challenges and limitations encountered in both the approval and clinical application of a large number of new synthetic metal˗based drugs is their poor water solubility, which leads to poor bioavailability and ultimately low bioactivity7. To accomplish this, approaches have been made as novel drug delivery systems (DDSs) and become a foremost objective in cancer therapy8. Liposomes8,9,10,11 polymeric micelles8,12,13,14 proteins15,16,17,18 metal˗based nanoparticles19,20,21 solid lipid nanoparticles8,22,23 graphene oxide and carbon nanotubes8,24,25,26 mesoporous silica nanoparticles27,28 and metal˗organic frameworks (MOFs)8,29,30 are renowned instances. Despite extensive studies and promising results of these DDSs, their bio-applications are often restricted by non-controlled drug release, low drug loading, high cost, toxicity, and non-biocompatibility of some carriers31,32,33.Among different DDSs, a facile, efficient, and low-cost approach based on nanosuspension (NS) formulation has been developed as a versatile option for the drug delivery purposes. NSs are defined as biphasic colloidal systems containing a solid drug with a particle size less than 1000 nm. In these systems, the drug core is surrounded by a layer of a stabilizing agent (surfactants and/or polymers)34,35. Pharmaceutically, benign compounds like the non-ionic polysorbates (Polyoxyethylene sorbitan fatty acid esters), also known as Tweens (TWs), are suitable candidates in this regard36. The NS architecture not only improves the solubility, stability, and bioavailability of drugs but also increases drug loading of poorly soluble drugs nearing 100%. Additionally, NSs have a simple structure and their formation is typically one-pot35,37. The nanosuspension technique has been reported in several studies as an outstanding DDS for cancer therapy with better anticancer activity and lower toxicity38,39,40,41,42,43,44.Accordingly, the development of the novel nanosuspension˗based delivery systems for insoluble metal complexes can overcome the drawbacks of other DDSs. We found few studies addressing biological activities of surfactant-NS formulations of metal complexes. In 2014, Kowol’s group prepared a formulation of KP1019 (a Ru(III) complex) to overcome its instability and insolubility in aqueous solution. They incorporated KP1019 into poly lactic acid (PLA) nanoparticles using two surfactants (Pluronic F68 and Tween 80). Tween 80 as a stabilizer led to very high stability and loading efficiency, ranging from 92 to 95%. Their KP1019 formulation exhibited much higher cytotoxicity on hepatocellular and colon carcinoma cell lines (Hep3B and SW480) than the free KP1019, an effect they assigned to the interaction between KP1019 and Tween 8045. In 2020, Zheng and co-workers prepared NS formulation of a Zn(II) complex (ZnPc-NS) with different surfactants. Then, they investigated the ZnPc-NS and Zn(II) complex dissolved in DMF as a photosensitizer in photodynamic therapy. Their ZnPc-NS with excellent stability and biocompatibility showed an optimal photocytotoxicity in HepG2 cells both in vitro and in vivo evaluations as compared to its low cytotoxicity in HELFX normal cells46.Glioblastoma (GB) is formed in the central nervous system and includes all tumors that origin from the brain glial cells and is one the most aggressive and deadliest brain tumors. Nowadays, the common treatment for GB includes surgery and followed by chemo˗radiotherapy which can improve the survival of GB patients to some extent. The aggressive and invasive characteristics of GB, extensive drug resistance, and the decrease of the bioavailability and efficacy of therapeutic drugs by blood–brain barrier (BBB) and blood brain˗tumor˗barrier (BBTB) restrict the GB treatment. Recent studies indicate that the use of nanomedicine and DDSs can overcome the factors limiting GB patient’s treatment38,47,48,49. Therefore, we choose the U˗87 cell line as the most frequently studied cells representing GB cancer for in vitro studies to validate the antitumor efficacy of the nanosuspension˗based delivery system for our previously reported insoluble Cu(II) complex. Herein, we embarked on the development, characterization, and anti˗GB activity of a TW˗assisted nanosuspension formulation of a Cu(II) complex, [Cu(2MeObpy)2](PF6)2. First, the Cu(II) complex was synthesized, purified, and characterized according to our previously published method50. Then, the nanosuspension formulation was prepared in aqueous solution with the non˗ionic polysorbate 60 (TW60) as the stabilizer agent by using the antisolvent precipitation method, namely hereafter CuTW60-NS. Elucidation of CuTW60-NS was performed by FT˗IR, UV˗Vis, dynamic light scattering (DLS), X˗ray diffraction powder (XRD) analysis, thermal analyses, and field emission scanning electron microscopy (FE˗SEM) in comparison with Cu-S (water suspension). The following in vitro biological studies of CuTW60-NS in parallel with its Cu-DMSO counterpart (DMSO˗dissolved complex) and Cisplatin were carried out: (i) cytotoxic effect by using the MTT assay, (ii) cellular morphological changes, (iii) inhibition of cell migration and colony formation, (iv) induction of apoptosis using flow cytometry, (v) the expression of apoptosis˗related genes by using Real˗Time PCR, and (vi) plasmid DNA cleavage.Results and discussionPreparation of CuTW60-NSThe insoluble Cu(II) complex with formula [Cu(2MeObpy)2](PF6)2, where 2MeObpy is 4,4′-dimethoxy-2,2′-bipyridine, was prepared from the reaction of CuCl2∙H2O and 2MeObpy in ethanol in a stoichiometric ratio of 1:2 under stirring condition. The resultant product was purified and characterized according to our previously published method50. The synthesized complex was soluble in organic solvents such as CH3CN, DMF, and DMSO and insoluble in water. In line with the former X˗ray crystallography data, the complex has a square˗planar structure and consists of a [Cu(2MeObpy)2]2+ cation and two PF6− anions (Fig. 1). The Cu(II) ion is coordinated by four N atoms from two 2MeObpy ligands forming CuN4 chromophore.Initially, a series of trials was carried out to reach optimal conditions suitable for preparation of CuTW60-NS formulation with minimum particle size, maximum solubility, and long˗term stability. The parameters expected to impact the NS formulation, included surfactant type (sodium dodecyl sulfate (SDS), TW80, TW60, Polyethylene glycol (PEG), Sorbitan monostearate (Span 60)), surfactant concentration, complex concentration, solvent (DMSO, DMF, and CH3CN), antisolvent (H2O, normal saline, and phosphate-buffered saline (PBS)), solvent/antisolvent ratio, and temperature were examined. Details of three trial formulations (namely F1-F3) are reported in Table S1 (in supplementary file).Afterwards, the optimal formulation, namely CuTW60-NS, was prepared with TW60 as a stabilizer by using antisolvent precipitation method according to conditions in the experimental section. Figure 1 indicates the structures of the Cu(II) complex and TW60 used in this study as well as a schematic illustration of CuTW60-NS formulation as a result of the combination between the Cu(II) complex and TW60. Where, TW60 is a non˗ionic surfactant with a hydrophilic head group (non˗ionic head) and a hydrophobic tail. TW60 can be adsorbed on the nanoparticle surface and form a layer in which the hydrophobic tail has the affinity for the nanoparticle surface and the hydrophilic head possesses the affinity for the aqueous dispersion medium36. As shown in Fig. 1, formation of a layer onto the Cu(II) complex by TW60 in CuTW60-NS formulation leads to an increased distance between particles and provision of a steric barrier36,51.Fig. 1The structures of the Cu(II) complex and TW60 used in this study as well as a proposed schematic illustration of CuTW60-NS. In the complex structure, the counter anions (PF6) and hydrogen atoms have been omitted for clarity. In TW60 structure, black color represents the alkyl tail and the pink circle denotes the hydrophilic head group.Full size imageCharacterization of CuTW60-NSSize, polydispersity index, and zeta potentialFigure 2 displays the DLS particle size distribution of the three experimental formulations (F1-F3). The size distribution of the optimal formulation, F3 = CuTW60-NS, falls within the range of 400–700 nm, where a majority of particles (36%) have the size of 600 nm. The PDI value for CuTW60-NS is 0.5, indicative of a moderate size distribution. The DLS results indicate that F1 and F2 formulations have the particle size more than 1 µm. On the other hand, the particle size of Cu-S has been found to exceed 6 µm (data not shown). The zeta potential of CuTW60-NS is -3.0 mV, higher than that of Cu-S (0.0 mV). This zeta potential value is similar to those of other non˗ionic surfactant˗stabilized nanosuspensions46. Consequently, the optimal formulation, CuTW60-NS, with lower size was opted to undergo further characterization and biological experiments.FT-IR spectraFigure 3A demonstrates FT-IR spectra of Cu-S, TW60, and CuTW60-NS denoting possible interactions between the Cu(II) complex and TW60 in CuTW60-NS formulation. In the FT-IR spectrum of Cu-S, two bands are observed at 1663 and 1613 cm− 1 which are assigned to C = N and C = C stretching vibrations, respectively. Cu-S also exhibits a strong band at 837 cm− 1 which is assigned to the stretching frequency for P–F groups50. In the FT-IR spectrum of TW60, the broad and strong bands are observed at 3423 cm− 1 (O–H stretching vibrations), 2922 and 2856 cm− 1 (C–H symmetric and asymmetric stretching vibrations), and 1735 cm− 1 (C = O stretching vibrations)52. Most importantly, the characteristic bands observed for the Cu (II) complex and TW60 alone are also found in the FT-IR spectrum of CuTW60-NS, confirming that the chemical structure of the Cu(II) complex and TW60 has been preserved in CuTW60-NS. On the other hand, the wavenumber of these characteristic bands is slightly shifted in the FT-IR spectrum of CuTW60-NS which can be attributed to weak interactions between the Cu(II) complex and TW60. For example, the band of O–H has shifted from 3423 to 3445 cm− 1 and bands of C–H shifted from 2922 to 2856 cm− 1 to 2918 and 2850, respectively. Notably, the band of the C = O group at 1735 cm− 1 has been distinctly weakened in the spectrum of CuTW60-NS. A similar observation in the FT-IR spectrum of NS formulations has been also reported in other papers51,52.Fig. 2The DLS particle size distribution of F1, F2, and the optimal formulation of F3 = CuTW60-NS.Full size imageUV–Vis spectroscopyTo achieve further insights into the structural alterations in the architecture of the Cu(II) complex by TW60 at molecular level, the UV-Vis spectra of Cu-DMSO, CuTW60-NS, and Cu-S at 20 µg/ml concentration were recorded and compared. As seen in Fig. 3B, the absorption spectra of Cu-S and Cu-DMSO at 20 µg/ml concentration exhibit the characteristic absorption bands at 228, 277, 286, and 299 nm, which are assigned to intraligand electronic transitions of the metal˗bound MeObpy ligands50. TW60 itself has no characteristic absorption bands at the used concentration in the CuTW60-NS formulation at the range of 200–350 nm. The UV spectrum of CuTW60-NS is consistent with Cu-S and Cu-DMSO and no change in wavelengths is observed. This assured that TW60 has no influence on the molecular structure of the complex. Interestingly, the absorbance intensity of the bands in CuTW60-NS are higher than Cu-DMSO and Cu-S at the same concentrations, suggesting its higher solubility in aqueous solution53. This increase in the UV˗absorbance intensity of the corresponding peak, could also support a complete uptake of the Cu(II) complex into the final CuTW60-NS form to merely a 100% yield, as acclaimed in the similar literature35,37,45.Fig. 3(A) The FT˗IR spectra of Cu-S, TW60, and CuTW60-NS. (B) The UV˗Vis spectra of Cu-DMSO, CuTW60-NS, and Cu-s at 20 µg/ml concentration.Full size imageX-ray diffraction (XRD) analysisThe XRD patterns of the Cu(II) complex and CuTW60-NS were achieved to explore whether the crystalline state of the complex changes during the preparation process of NS. Figure 4A reveals the XRD patterns of the Cu(II) complex and CuTW60-NS. No XRD analysis was exercised on TW60 due to its being liquid at room temperature (RT)40. The Cu(II) complex has a crystalline structure and presents sharp diffraction peaks between 2θ values of 10° and 30°. Thus, the crystalline nature of the complex is evident from its powder XRD spectrum. Obviously, the characteristic diffraction peaks of the Cu(II) complex are still retained in the XRD spectrum of CuTW60-NS, indicating that TW60 has no influence on the crystal lattice of the complex. While the diffraction peaks intensity of CuTW60-NS are lower than the Cu(II) complex, indicating that the preparation process of NS may disorder or cleave the intrinsic crystal lattice of the complex and lead to the reduction in its crystallinity40,51. The XRD results confirm the preparation of a stable formulation because the crystalline structures are physically more stable compared to amorphous structures.Thermal analysesTemperature˗related Cu(II) complex and CuTW60-NS properties were measured by thermo˗gravimetric analysis and differential thermal analysis (TGA-DTA). Figure 4B displays TGA-DTA curves of the Cu(II) complex and CuTW60-NS, where dominant weight loss of the Cu(II) complex and CuTW60-NS occur in the temperature between 250 and 400 °C. There is almost no weight loss below 250 °C. The major weight loss of the Cu(II) complex is initiated at about 290 °C and completed at about 400 °C while for CuTW60-NS is initiated at about 270 °C and completed at about 400 °C. In the DTA curve of the Cu(II) complex and CuTW60-NS two endothermic peaks are observable. For the Cu(II) complex, a narrow sharp endothermic peak at 290 °C which can be attributed to its melting point is observed. While a small endothermic peak is observed at 280 °C for CuTW60-NS formulation. These findings indicate that the crystallinity and consequent stability of the complex in CuTW60-NS formulation has changed and are greatly consistent with the XRD results.Fig. 4(A) XRD patterns of the Cu(II) complex and CuTW60-NS. (B) TGA-DTA curves of the Cu(II) complex and CuTW60-NS.Full size imageSurface morphology by FE-SEMFigure 5 indicates the surface morphology of Cu-S and CuTW60-NS by FE˗SEM images in two magnifications each. The particles of Cu-S show a broad size distribution in the micron range and are mostly agglomerated. On the other hand, nanoparticles of CuTW60-NS are observed at a narrower size variation and more dispersed than Cu-S. Moreover, TW60 seems to have settled as an adsorbed layer on the particlesʹ surface. Nevertheless, the cylindrical morphology of CuTW60-NS looks very much similar to that of Cu-S. The DLS and FE˗SEM results are entirely consistent with each other and indicate that the size distribution of CuTW60-NS is in the range of 300–700 nm.Fig. 5The surface morphology of Cu-S and CuTW60-NS by using FE-SEM images in 10.0 and 20.0 KX magnifications. Error bars are 1 µm (left) and 200 nm (right).Full size imageStability and saturation solubility studiesFigure 6A indicates the physical appearance of Cu-DMSO (dissolved in DMSO), CuTW60-NS (suspended in H2O), Cu-S (suspended in H2O), and TW60 (dissolved in H2O). Both CuTW60-NS and Cu-S display sky-blue suspensions, but Cu-S rapidly forms sedimentation while CuTW60-NS remains consistent. This may indicate that CuTW60-NS forms stable nanoparticle formation devoid of apparent agglomeration owing to the interference of TW60. The stabilization of NSs by the non˗ionic surfactants is due to steric repulsion, as previously shown in Fig. 1. Besides, no obvious change in the appearance and color of CuTW60-NS was observed at 4 °C over 6 months.The part B of Fig. 6 displays the particle size distribution of CuTW60-NS at 4 °C after 6 months. The DLS results indicate that the particle size of CuTW60-NS is in the range of 800–1200 nm, indicating that CuTW60-NS is stable after 6 months of storage at 4 °C. Such increase in the particle size of NS formulations has been also reported elsewhere53,54. Figure 6C demonstrates time˗dependent stability of CuTW60-NS (20 µg/ml concentration) in PBS by UV˗Vis studies at different times over 6 months. The overall pattern of the UV spectrum of CuTW60-NS remains constant and no new absorption bands appear over 6 months. The results display only slight reduction in absorption intensity, which probably is due to the slight change in particle size. These observations also confirm the long˗term stability of CuTW60-NS in aqueous medium at 4 °C.The saturation solubility of the Cu(II) complex in two Cu-S and CuTW60-NS forms were examined in deionized water by UV˗Vis technique. As seen in Fig. 6D, the supernatant of CuTW60-NS indicates the higher absorbance intensity at 280 nm than Cu-S. The observed solubility for CuTW60-NS and Cu-S was found to be 65 and 25 µg/ml, respectively. The 2.6˗fold increase of solubility of CuTW60-NS than Cu-S may be attributed to surfactant behaviour of TW60, but also reduction in particle size of CuTW60-NS which lead to larger surface area and ultimately higher solubility53.Fig. 6(A) The physical appearance of Cu-DMSO, CuTW60-NS, Cu-S, and TW60. (B) The particle size distribution of CuTW60-NS at 4 °C after 6 months. (C) Time-dependent stability of CuTW60-NS (20 µg/ml concentration) in PBS by UV˗Vis studies at different times over 6 months. (D) The saturation solubility of CuTW60-NS and Cu-S by UV˗Vis spectra.Full size imageIn vitro cellular and molecular studiesInhibition of cancer cells growthIn vitro cytotoxic activity of CuTW60-NS was investigated after 24 and 48 h against U˗87 by MTT method. The U˗87 cells were also treated with Cu-DMSO, Cisplatin, and equalvolume amounts of DMSO and TW60. Results are expressed as half maximal inhibitory concentrations (IC50 values). Figure S1 (in supplementary file) and Fig. 7 illustrate the plots of cell viability (%) vs. different concentrations of Cu-DMSO, CuTW60-NS, and Cisplatin after 24 and 48 h, respectively as well as the corresponding IC50 values. The substantial reduction in cell viability with increasing concentrations of Cu-DMSO, CuTW60-NS, and Cisplatin plus the high cytotoxic effect with extension the exposure time from 24 to 48 h are evident. Accordingly, concentration˗ and time˗dependent cytotoxic effects are observed after cells˗exposure to Cu-DMSO, CuTW60-NS, and Cisplatin. CuTW60-NS exerts higher inhibitory effects on the U˗87 cells growth in comparison with Cu-DMSO, specifically at 3.7 µg/ml concentration after 48 h (P ≤ 0.0001). Cu-DMSO and CuTW60-NS indicate the IC50 values of 17.5 ± 2.6 and 7.8 ± 0.7 µg/ml, respectively against U˗87 cells after 48 h (Fig. 7). Therefore, the IC50 value of CuTW60-NS is twice as much as Cu-DMSO. This may support the possible role of TW60 in the cell-uptake of the complex which can in˗turn result in a higher cytotoxic activity45. The enhanced antitumor efficacy of CuTW60-NS could be explained by the fact that the non˗ionic surfactant Tween can facilitate cell membrane-nanosuspension attachment, which lead to enhance the complex entry into cancer cells. While, the complex alone enters the cells through passive diffusion55. Additionally, Cisplatin displays a higher cytotoxic effect on the U˗87 cells in comparison to both Cu-DMSO and CuTW60-NS with IC50 value of 2.5 ± 0.2 µg/ml. Meanwhile, the cytotoxicity of equivolume amounts of DMSO and TW60 under the same conditions was found insignificant and negligible at the used concentrations in Cu-DMSO and CuTW60-NS, respectively (data not shown).According to the above findings, the treatment time of 48 h was chosen for further evaluations.Cells morphology alterationsAlterations in the shape and size of U˗87 cells treated with different concentrations of Cu-DMSO, CuTW60-NS, and Cisplatin were examined under an inverted microscope after 48 h without staining. Figure S2 (in supplementary file) compares the microscopic images of the untreated cells with Cu-DMSO-, CuTW60-NS-, and Cisplatin-treated cells after 48 h. Here, untreated U˗87 cells demonstrate their original spindle˗shaped epithelial morphology. Whereas Cu-DMSO-, CuTW60-NS-, and Cisplatin-treated cells are noticeably observed as round and small entities. Besides, a sharp decline in the number of viable cells and a significant increase in the dead cells is observable. These observations are in solid support of cytotoxicity results as well as suggesting apoptotic cell death.Fig. 7Plots of cell viability (%) vs. concentrations of Cu-DMSO, CuTW60-NS, and Cisplatin on U˗87 cells after 48 h (n = 3). The significant difference of cytotoxicity at each concentration was statistically calculated by one-way ANOVA with Dunnett’s multiple comparisons test, * represents *p