Micelle encapsulation zinc‐doped copper oxide nanocomposites reverse Olaparib resistance in ovarian cancer by disrupting homologous recombination repair

Abstract Micelle Encapsulation Zinc‐doped copper oxide nanocomposites (MEnZn‐CuO NPs) is a novel doped metal nanomaterial prepared by our group based on Zinc doped copper oxide nanocomposites (Zn‐CuO NPs) using non‐micellar beam. Compared with Zn‐CuO NPs, MEnZn‐CuO NPs have uniform nanoproperties and high stability. In this study, we explored the anticancer effects of MEnZn‐CuO NPs on human ovarian cancer cells. In addition to affecting cell proliferation, migration, apoptosis and autophagy, MEnZn‐CuO NPs have a greater potential for clinical application by inducing HR repair defects in ovarian cancer cells in combination with poly (ADP‐ribose) polymerase inhibitors for lethal effects.

NPs are fundamentally different from their corresponding bulk materials because the quantum mechanical nature of atomic interactions is influenced by their size. 21 Nanometal oxides (nMeOs) are among the most promising NPs due to their potential physicochemical properties such as high affinity, low molecular weight, and large specific surface area. 22 nMeOs such as ZnO, CuO, and Fe3O4 NPs have been reported to cause genotoxicity, mitochondrial dysfunction and induction of apoptosis and autophagy in many cancer cell lines. 21,23,24 The dope NPs is a kind of mechanism that metal ions are doped in the unit cell of the monoclinic lattice replacing some of the metal ions by sono-chemical method by our collaborator. 25 Enhanced activity of doped nMeOs has been reported due to their increased structural defects and increased ROS production. [26][27][28] A zinc-doped CuO nanocomposite (Zn-CuO NPs) with efficient antibacterial activity was synthesized by the acoustic chemistry method in our collaborator group previously. 29 Previous studies have shown that Zn-CuO NPs can induce apoptosis through ROS-mediated pathway, thus inhibiting cancer cell proliferation. 30, 31 We also found that Zn-CuO NPs can have antiglioma effects both in vitro and in vivo. 32 However, due to their special structure, Zn-CuO NPs suffer from low solubility. Polymeric micelles as a novel drug delivery system have the advantages of prolonging drug circulation time, enhancing drug accumulation, improving drug dissolution range, and reducing side effects. 33 In this study, we used surfactants and polyether polyol as stabilizers to prepare stabilized nanoparticles by strong ultrasound technique encapsulation to enhance the durability of metal nanoparticles, reduce their accumulation, enhance their distribution, and form a nanosystem of 50-nm metal nanomicelles. We continued the characterization using transmission electron microscopy (TEM) and found that the metal nanoparticles were dispersed in the interior of the micellar system and the micellar encapsulation improved the homogeneity of the metal nanoparticles. We named them as Micelle Encapsulation Zinc-doped copper oxide nanocomposites (MEnZn-CuO NPs). The results suggest that MEnZn-CuO NPs increase the range of organic solvent selection, reduce the adverse effects of surfactants, and are more stable in nature. However, it is not clear whether MEnZn-CuO NPs have greater antitumor potential than Zn-CuO NPs.
In this study, we examined the therapeutic effects of MEnZn-CuO NPs in ovarian cancer in vitro and in vivo, and explored the underlying molecular mechanisms. We found that MEnZn-CuO NPs exerted strong antitumor effects by causing cellular genomic damage. More importantly, MEnZn-CuO NPs significantly increased the therapeutic sensitivity of ovarian cancer cells to the PARP inhibitor Olaparib by inhibiting the cellular HR repair ability. These findings provide strong evidence that MEnZn-CuO NPs may be applied as a novel antitumor nanodrug for ovarian cancer treatment, especially for Olaparib-resistant patients.

| Compared with Zn-CuO NPs, MEnZn-CuO NPs have stronger homogeneity and stability
Previously, our team reported the anti-tumor activity of metal nanoparticles Zn-CuO NPs, 31 but due to the properties of metal nanoparticles are prone to aggregation as well as liquid settling. 25,30,31 Therefore, this study proposes to reduce the aggregation between metal nanoparticles and increase the stability of metal nanoparticles in solution by colloidal technique. The particle size distribution of Zn-CuO NPs was wider, ranging from 100 to 500 nm (Figure 1a), and its particle diameter is mainly distributed at 144 and 453 nm, in addition, it is more distributed at 453 nm. We prepared MEnZn-CuO NPs by using high-intensity ultrasound, and the particle size distribution of MEnZn-CuO was more homogeneous than that of Zn-CuO NPs, with the particle size around 200 nm (Figure 1b). The TEM results also suggested that Zn-CuO NPs tend to aggregate into larger metal nanoparticle aggregates compared to MEnZn-CuO NPs, causing the accumulation of nanoparticles resulting in weakened nanoparticle properties (Figure 1c,d). NPs in a group of ovarian cancer cell lines. After 72 h of drug action, cytotoxic effects were determined by the CCK-8 assay, followed by a medianmedian IC50s analysis. All six ovarian cancer cell lines were more susceptible to MEnZn-CuO NPs compared to IOSE80, with A2780 and OVCAR8 being the most prominent ( Figure 2b). Because the ZnCuO could inhibited the cell growth by the autophagy enhancing, the autophagy phenotype was test by the double label autophagy. As shown in the Figure S1C, the cell lines A2780 and OVCAR8 was treatment with the Zn-CuO NPs (4 μg/mL) and MEnZn-CuO NPs (4 μg/mL), the MEnZn-CuO NPs shown more fluorescent intensity of red than the control group and Zn-CuO NPs treatment group. MEnZn-CuO NPs have more autophagy transmission capacity than the Zn-CuO NPs. So, the MEnZn-CuO NPs could be an autophagy enhancer to inhibited the cells growth. We further examined the drug responses of A2780 and OVCAR8 to MEnZn-CuO NPs over a long period, and the results showed that the metal nanoparticles produced greater inhibition of proliferation of both cell types (Figure 2c   NPs could also induce autophagy in cells. As shown in Figure S1B, there were no differences in retroviral transfection efficiency with 80% positive cells before the nanoparticle treatment. As shown in Figure 3c

| MEnZn-CuO NPs and PARP inhibitor Olaparib synergistically inhibit the growth of ovarian cancer cell lines in vitro
To test the conjecture, we first determined the IC 50        which are all involved in maintaining genomic stability. 52 Another research has also shown that activation of autophagy leads to recruitment of BRCA1/Rad51, which are involved in the HR pathway. 53 And the ligand YM155 regulates BIRC5, leading to the induction of autophagy that affects genome stability through the downregulation of RAD54L and RAD51, resulting in autophagy-dependent ROS-mediated DNA damage. 54 Our result has shown that MEnZn-CuO NPs may be an autophagy enhancer to disrupting the autophagy and HR repair to inhibit the ovarian cancer cells by the synergy effect in the Figure S4. The HR and autophagy related HR might involve in synergy effect to inhibit the ovarian cancer. As nano-sized agents with long circulation times leak into tumor tissue preferentially due to permeable tumor vasculature, then they retained in the tumor bed by their reduced lymphatic drainage. 55

| The synthesis and characterization of MEnZn-CuO NPs
The synthesis and the characterization of the Zn-CuO was described the previous studies. 57 Briefly, MEnZn-CuO NPs were prepared from a mixed solution of copper acetate and zinc acetate at a molar ratio of  NPs is a novel doped metal nanomaterial synthesized by our group using the sonochemical method, stock concentration is 10 mg/mL in 4 C. Olaparib (AZD2281) was purchased from Aladdin (USA).

| Cell viability assay and determination of drug synergy
Cell viability was assayed using the cell counting kit-8 assay according to the manufacturer's protocol (Dojindo Molecular Technologies, Japan). Synergistic effects were determined by the Chou-Talalay method to calculate the combination index (CI). 59

| Wound-healing assay
Wound-healing assay was used to evaluate cell migration as described previously. 60,61 Briefly, cells were seeded in 24-well plates and grown until confluent state and then cells were scratched using sterile tips.
Then the cell monolayer was rinsed twice with PBS to remove debris.
Fresh culture medium was added with indicated drugs. The mean width of each scratch was measured using Image Pro Plus (Media Cybernetics) and ImageJ. 62

| Immunofluorescence staining analysis
Cells were fixed with 4% formaldehyde in PBS after drug treatment, blocked using 5% BSA, and permeabilized with 0.2% Triton X-100.
The primary antibodies were diluted in 1% BSA and incubated at 4 C overnight. Then, secondary antibodies were added to the samples and

| Comet assay
A comet assay was performed as previously described. 64 100 randomly selected cells were analyzed using Casplab software. The level of DNA damage was presented as percentage of DNA in tail.
Images were captured with a fluorescence microscope (Olympus, Japan).

| Metaphase chromosome spread assay
Cells were treated with colchicine (0.5 μg/mL) (Beyotime, China) for 12 h prior to harvest. Metaphase spreads were prepared as described previously. 65 Images were captured with oil lens of microscope (Olympus, Japan).

| Autophagy detection by fluorescence microscope
Autophagy flow was analyzed as described previously. 66

| Statistical analyses
Differences between two independent groups were calculated using Unpaired Student's t test and one-way ANOVA with Tukey's multiplecomparisons tests as indicated in the figure legends. p values less than 0.05 were considered statistically significant and are denoted as follows: *< 0.05, **< 0.01, and ***< 0.001. All data were analyzed with GraphPad Prism 8 software.