# Preparation and Synergistic Anti-Tumor Effect of Iridium Oxide Nanocomposites under Microscope

Authors: Xudong He; Feng Xiang; Zhangyi Xu
Journal: International Journal of Analytical Chemistry
 (2022)

---

## Abstract

In order to solve the great difficulties in cancer prevention, diagnosis, and treatment, a preparation method of iridium oxide nanocomposites under the microscope was proposed in this paper. Through a retrospective analysis of an experiment, IrOx nanoparticles were prepared by direct hydrothermal hydrolysis and loaded with chemotherapy drug adriamycin to construct nanodrug-loaded complex IrOx@DOX. At the same time, IrOx, as a sound-sensitive agent, can produce ROS under US irradiation, amplify intracellular oxidative stress, accelerate tumor cell death, and finally achieve the effect of SDT chemotherapy synergistic therapy. The experimental results show that IrOx@DOX has the dual response of pH and US, and the inhibition rates are 27%, 57%, and 76%, respectively. At the same time, ultrasound not only can enhance the uptake of nanoparticles by cells but also can promote the release of DOX in cells, which provides a basis for subsequent SDT chemotherapy synergistic therapy.Conclusion. Iridium oxide nanocomposite DOX combined with SDT can obtain a good therapeutic effect, which has positive feedback on the efficacy of chemotherapy and the therapeutic effect of cancer surgery.


---

## Body

## 1. Introduction

Cancer is expected to become a major problem that seriously threatens human health in the world in the twenty-first century, and it is also the most important obstacle to prolonging human life [1]. According to the data released by the World Health Organization (WHO) in 2015, among the 172 countries counted, 91 countries listed cancer as the first or second major cause of death before the age of 70, while in the other 22 countries, it was the third or fourth cause. The reasons for the rapid growth of global cancer incidence rate and mortality are complex, but it reflects that the aging of the population and the rapid growth of the population are the main reasons, as well as the changes in the main risk factors and distribution of cancer prevalence, which are closely related to social and economic development [2]. More seriously, cancer affects almost all types of human organs, and more than 17.2 million new cases are diagnosed every year. Therefore, developing effective cancer prevention, diagnosis, and treatment methods is the top priority of research teams and clinicians around the world. At the same time, if cancer can be detected or diagnosed early, especially before its metastasis, it can prolong life and improve quality of life. However, the early diagnosis of cancer is still a huge challenge. In order to conquer the huge fortress of cancer, scientific research and clinical workers around the world have made great efforts to develop effective methods to diagnose and treat cancer. So far, many treatment methods including chemotherapy, surgical resection, molecular targeted therapy, gene therapy, radiotherapy, and so on have been widely used in the clinical treatment of cancer [3]. However, these therapies have unavoidable defects, such as poor specificity, large side effects, and certain damage to the immune system. In this context, the rapid development of nanotechnology makes nanomaterials show great potential in tumor diagnosis and treatment. The development of nanomedicine provides a new strategy for improving clinical efficacy and reducing side effects [4] as shown in Figure 1.Figure 1
Iridium oxide nanoscale drug-carrying complex.


## 2. Literature Review

Targeted preparation is a carrier preparation that can selectively transport drugs to lesion sites and improve drug utilization. It can reduce the frequency of administration, improve the adaptability of patients, and reduce medical cost [5]. At present, many drug carriers have been used in the targeted delivery system of chemotherapy drugs. The drug delivery system can be modified to avoid being engulfed by macrophages, or it can be combined with the target cell receptor to reach the target site and change the distribution of drug carriers in the body [6]. Starch/β cyclodextrin composite microspheres were prepared from starch and β cyclodextrin. The microspheres were activated by epichlorohydrin and cross-linked with EGFR antibodies to prepare immune composite microspheres. After the EGFR antibody modified on the surface of the composite microspheres spontaneously binds with EGFR in thyroid cancer and breast cancer cells, EGFR is highly expressed in cancer cells, making the microspheres located at the cancerous site [7]. Using sodium alginate as carrier material, Konkoová prepared epirubicin-loaded nanodrug carriers with anti-VEGFR2 monoclonal antibodies by polyelectrolyte method. VEGFR2 mainly exists on the surface of tumor cells and is expressed at a low level in normal cells and tissues. It is the main receptor of vascular endothelial growth factor. The expression rate of VEGFR2 in liver cancer tissues is as high as 70%. The combination of drug carrier and anti-VEGFR2 monoclonal antibody can locate the drug in liver cancer cells, improve the anti-tumor effect of the drug on liver cancer cells, and reduce the side effects of epirubicin [8]. After intravenous administration, most of the passive targeted particles with particle sizes ranging from 2.5 to 10 μm were gathered in macrophages. When the particle size was less than 7 um, they could be phagocytized by macrophages in the liver and spleen. In addition, the properties of the carrier materials on the surface of particles also play an important role in their distribution in the body. Talaván used lecithin and stearic acid as carrier materials to prepare ashitali and lipid nanoparticles by low-temperature curing emulsification evaporation method. The average particle size was determined to be 120 nm by a dynamic light scattering instrument, which provided a certain experimental basis for the development of passive targeting formulation of actalide [9]. Barbosa used a hydrazone bond to connect doxorubicin and chitosan derivatives, prepared a pH-sensitive drug carrier, and studied its in vitro release performance [10]. The drug carrier can exist stably in the physiological environment with a pH value of 7.4, and the drug is basically not released. In an acid environment, the drug carrier can release the drug rapidly. Kumar coated titanium substrate with L-polylactic acid/eicosane to prepare a thermosensitive drug carrier containing adenosine triphosphate, which automatically releases the drug only at body temperature. Muzzarelli prepared the drug carrier with a strong magnetic response by emulsion method, with superparamagnetic ferric oxide as the magnetic core and chitosan as the carrier material, which provided a theoretical basis for the study of magnetic targeted drug carrier [11], as shown in Table 1.Table 1
Targeted delivery system of chemotherapy drugs.
Drug delivery systemDrugs containedPolylactic acid microspheresAraubicinAlbumin microspheresCisplatinStarch microsphereDoxorubicinChitosan gelatin composite microspheres5-FluorouracilPoly (butyl cyanoacrylate)AdriamycinEthyl cellulose ballMitoxantroneThermosensitive liposomeMethotrexateBased on the above research background, this study focused on the application of IrOx in SDT to carry out systematic research. IrOx nanoparticles were prepared by direct hydrothermal hydrolysis and loaded with chemotherapy drug doxorubicin (DOX) to construct the nanodrug-loaded complex IrOx@DOX. Because the tumor cells are slightly acidic, IrOx@DOX can realize the dual stimulation of pH and US in response to drug release to improve the effect of chemotherapy. At the same time, IrOx, as a sound-sensitive agent, can produce ROS under US irradiation, amplify intracellular oxidative stress, accelerate tumor cell death, and finally achieve the effect of SDT chemotherapy synergistic therapy. This study provides a new idea for the development of IrOx-based nanodrugs and will also provide a universal scheme for the formulation of SDT combined chemotherapy strategy [12].


## 3. Research Methods

### 3.1. Research Materials and Laboratory Animals

Through retrospective analysis of an experiment, the research materials include iridium chloride, adriamycin, 4′, 6-diamidino-2-phenylindole, 2,2,6,6-tetramethylpiperidine, 3-diphenylisobenzofuran, reactive oxygen species detection kit, CCK-8 kit, calcein PI kit, Annexin V-FITC cell apoptosis detection kit, human umbilical vein endothelial cells, and mouse breast cancer 4T1 cells. Fifteen female nude mice with BABL/c at 4 to 6 weeks weighed about 18g.


### 3.2. Research Instruments

The transmission electron microscope, Fourier infrared spectrometer, x-ray photoelectron spectrometer, x-ray crystal powder diffractometer, paramagnetic reson nce spectrometer, particle size and zeta potential analyzer, UV visible spectrophotometer, laser confocal microscope, fluorescence microscope, flow cytometer, multifunctional enzyme marker, and ultrasonic therapeutic instrument [13] are used.Dissolve iridium chloride (bIrCl3, 0.1493 g) in 50 ml deionized water, adjust the pH value to 12 with sodium hydroxide solution (1 mol/L), and stir at room temperature for 2.5 h. Then, the solution was transferred to an oil bath (80°C) and stirred for 1 h to obtain the product dispersion IrOX. Transfer the above solution to an ultrafiltration tube (MWCO = 14 kDa) for centrifugation (660 × g) for 15 min, resuspend it with deionized water, and repeat 3 times to remove excess IrOX and by-products. The obtained B dispersion was mixed with DOX in different proportions (b IrCl3 ∶DOX = 1∶2, 1∶1, 2∶1, 4∶1, 6∶1). Stir at room temperature for 24 hours without light. Centrifuge the product (20,000 × g) for 15 min, collect the supernatant, and repeat 3 times to remove excess DOX. Finally, it was resuspended with deionized water to obtain IrOx@DOX dispersion. Collect all supernatants and washing solutions, determine the absorbance of DOX by UV Vis, and calculate the loading rate under different conditions according to the formula [14]. Loading rate = (initial mass of DOX – mass of DOX in supernatant)/IrOx@ mass of DOX × 100%. The shape of IrOx was observed by TEM; the samples were characterized by XRD; the different elements and valence states in IrOx were characterized by XPS; the functional groups on the surface of nanoparticles were investigated by FTIR; and UV-Vis was used to measure the UV absorption spectra of different samples: zeta potential analyzer was used to measure the potential of products in each stage of the material synthesis process.


#### 3.2.1. Investigation of Drug Release Behavior In Vitro

The drug release behavior of IrOx@DOX was investigated by dialysis. Specifically, a certain quality of DOX was accurately weighed and dissolved in PBS buffer solution (200 mL) with pH values of 7.4 and 5.5, and its absorbance was measured to obtain the standard curve of DOX under different pH values. Then put 2 ml of IrOx@DOX dispersion into a dialysis bag (MWCO = 3 kDa) and put it into a buffer solution with different pH values (pH value is 5.5 or 7.4) and stir at room temperature without light. US group was given ultrasound irradiation (1 W/cm2) for 30 s at a certain time. Take 1 ml of release solution from the beaker at different time points and add the same volume of fresh buffer. UV-Vis was used to determine the absorbance of the diluent at 500 nm. The cumulative release of DOX under different conditions is calculated through the standard curve, and the release curve of DOX is obtained [15]. Drug release rate = (mass of DOX released/total mass of DOX in the complex) × 100%.


#### 3.2.2. In Vitro SDT Effect Evaluation

Mix 200μL IrOx@DOX dispersion (100 ug/ml) with 200 μL temp solution (100 mmol/L), irradiate with US (1.0 MHz, 1.0 W/) for 2 min, and immediately detect the generation of oxygen by ESR spectrometer. At the same time, no ultrasonic treatment was used as control. In order to explore the effect of different ultrasonic power on oxygen generation, we irradiated IrOx@DOX dispersion with the same concentration with US of different power (0.5, 1.0, 1.5, 2.0, and 2.5 W/cm2) and detected oxygen generation by ESR. In addition, mix 2.96 ml of the iroxdox solution (100 μg/ml) and 40 μL of DBPF (2 mg/ml) away from light, conduct US radiation (1.0 MHz, 1 W/cm2, 2 min) every 2 min, and detect the generation of ROS by UV-Vis recording the absorption curve of the solution at the wavelength of 200∼650 nm [16].


#### 3.2.3. In Vitro Cellular Uptake

CISM was used to observe the uptake of IRO and @DOX by 4TI cells. 4Ti cells (1 × 105/well) were inoculated into confocal Petri dishes. After the cells adhered to the wall, the blank control group was added with PBS containing medium for 6 hours; In the experimental group, the medium containing IrOx@DOX was added and treated with or without ultrasonic irradiation (1 MHz, 1.0 W/cm2, 1 min), and then continued to culture for 6 hours. The cells were washed with PBS 3 times to remove the nonphagocytized nanoparticles. The cells were fixed with 4% paraformaldehyde for 15 min and stained with DAPI for 5 min. Finally, CISM was used for observation.


#### 3.2.4. Cell SDT Effect in Vitro

4T1 cells were inoculated into 6-well plates (1 × 10³/well) and cultured for 24 hours. The cells were divided into the following groups: ① PBS group, ② PBS + US group, ③ IrOx group, ④ IrOx + US group, ⑤ IrOx@DOX group, and ⑥ IrOx@DOX + US group. Add 2 mL of fresh medium containing the above nanoparticles and continue to incubate for 12 h. PBS was washed 3 times, and then ROS probe (DCFH-DA) was added to incubate for 30 min. After that, PBS was washed 3 times. At the same time, groups ②, ④, and ⑥ were incubated for 2 h after ultrasonic irradiation (1.0 MHz, 1.0 W/cm2, 1 min). Finally, the cells were observed by a fluorescence microscope. Furthermore, ROS production was detected by flow cytometry. Cell treatment was as described above.


#### 3.2.5. In Vitro Chemotherapy SDT Synergistic Treatment Effect

Firstly, the cytocompatibility of IrOx was investigated. Specifically, HUVEC cells were inoculated into 96 well plates (1 × 104/well) and cultured in an incubator at 37°C and 5% carbon dioxide for 24 h. Then add 100 μ·L of fresh medium containing different concentrations of IRO that were incubated for 12 h. Remove the medium, wash it with PBS 3 times, and then add 100 μ·L of medium containing 10 μL CCK-3 reagent to continue incubation for 1 h [17]. Finally, the absorbance value at 450 nm was read with a microplate reader, and the cell survival rate was calculated according to the instructions. Each group of samples is set with 5 multiple holes, and each sample is measured in parallel 3 times. CCK-8 method was used to evaluate the synergistic effect of IrOx@DOX-induced chemotherapy SDT. 4T1 cells were inoculated into 96 well plates (1 × 104/well) and cultured for 24 hours.


### 3.3. Experimental Grouping

In the US irradiation group, the cells were incubated with nanoparticles for 6 hours and then exposed to US irradiation (1.0 MHz, 1 min) and continued to incubate for 12 hours. Then the medium was removed and washed with PBS 3 times. The cell survival rate was calculated by the CCK-8 method. In addition, after different treatments, the cells were stained with Calcein AM/PI (Calcein AM 2μmol/L, PI4.5 μmol/L, 100 μL) for 15 min. Then the culture medium was discarded and washed with PBS. Finally, the survival state of the cells was observed under the fluorescence microscope.Apoptosis was detected by flow cytometry: 4T1 cells (1 × 105/well) were inoculated and incubated in 6-well plates for 24 hours. The cells were treated according to the above experimental steps of CCK-8. After incubation, the cells were digested with trypsin, washed with PBS, centrifuged, and collected, and the number of early apoptosis, late apoptosis, and necrotic cells were analyzed with flow cytometry apoptosis staining kit (Annexin VFITC/PI staining solution) [18].


### 3.4. Establishment and Treatment of the Tumor Model

One hundred microliter HEPA 1–6 cell suspension containing 2 × 106 cells was subcutaneously injected into BALB/c female nude mice to establish a subcutaneous transplanted tumor model. When the tumor volume reached 120 mm, they were randomly divided into five groups (n = 3): ① normal saline group, ② IrOx group, ③ Irox@DOX group, ④IrOx + US group, and ⑤ IrOx@DOX + US group. Nude mice were injected with normal saline, IrOx, and IrOx@DOX (8 mg/mL, 100 μL). The ultrasonic irradiation group received ultrasonic irradiation (1.5 W/cm2, 1.5 MHz, 50% duty cycle, 5 min) 12 hours after drug injection. The treatment process was repeated every 3 days. The body mass and tumor volume of the mice were recorded every 3 days for 14 days. The tumor volume calculation formula is V = L × W2/2, where V, L, and W represent the tumor volume, length, and width, respectively. After 14 days of treatment, a contrast agent was injected intravenously to observe the growth of the tumor. The nude mice were euthanized. The corresponding tumor bodies were photographed and observed by HE staining. Calculate the tumor volume inhibition rate = (V control group – V test group)/V control group × 100%.


### 3.5. Statistical Analysis

Origin and SPSS 26.0 statistical software were used for mapping and statistical analysis. The independent samplet-test was used for data pairwise comparison, and a one-way analysis of variance was used for the comparison of the multivariate mean. P<0.05 indicates that the difference is statistically significant.


## 3.1. Research Materials and Laboratory Animals

Through retrospective analysis of an experiment, the research materials include iridium chloride, adriamycin, 4′, 6-diamidino-2-phenylindole, 2,2,6,6-tetramethylpiperidine, 3-diphenylisobenzofuran, reactive oxygen species detection kit, CCK-8 kit, calcein PI kit, Annexin V-FITC cell apoptosis detection kit, human umbilical vein endothelial cells, and mouse breast cancer 4T1 cells. Fifteen female nude mice with BABL/c at 4 to 6 weeks weighed about 18g.


## 3.2. Research Instruments

The transmission electron microscope, Fourier infrared spectrometer, x-ray photoelectron spectrometer, x-ray crystal powder diffractometer, paramagnetic reson nce spectrometer, particle size and zeta potential analyzer, UV visible spectrophotometer, laser confocal microscope, fluorescence microscope, flow cytometer, multifunctional enzyme marker, and ultrasonic therapeutic instrument [13] are used.Dissolve iridium chloride (bIrCl3, 0.1493 g) in 50 ml deionized water, adjust the pH value to 12 with sodium hydroxide solution (1 mol/L), and stir at room temperature for 2.5 h. Then, the solution was transferred to an oil bath (80°C) and stirred for 1 h to obtain the product dispersion IrOX. Transfer the above solution to an ultrafiltration tube (MWCO = 14 kDa) for centrifugation (660 × g) for 15 min, resuspend it with deionized water, and repeat 3 times to remove excess IrOX and by-products. The obtained B dispersion was mixed with DOX in different proportions (b IrCl3 ∶DOX = 1∶2, 1∶1, 2∶1, 4∶1, 6∶1). Stir at room temperature for 24 hours without light. Centrifuge the product (20,000 × g) for 15 min, collect the supernatant, and repeat 3 times to remove excess DOX. Finally, it was resuspended with deionized water to obtain IrOx@DOX dispersion. Collect all supernatants and washing solutions, determine the absorbance of DOX by UV Vis, and calculate the loading rate under different conditions according to the formula [14]. Loading rate = (initial mass of DOX – mass of DOX in supernatant)/IrOx@ mass of DOX × 100%. The shape of IrOx was observed by TEM; the samples were characterized by XRD; the different elements and valence states in IrOx were characterized by XPS; the functional groups on the surface of nanoparticles were investigated by FTIR; and UV-Vis was used to measure the UV absorption spectra of different samples: zeta potential analyzer was used to measure the potential of products in each stage of the material synthesis process.


### 3.2.1. Investigation of Drug Release Behavior In Vitro

The drug release behavior of IrOx@DOX was investigated by dialysis. Specifically, a certain quality of DOX was accurately weighed and dissolved in PBS buffer solution (200 mL) with pH values of 7.4 and 5.5, and its absorbance was measured to obtain the standard curve of DOX under different pH values. Then put 2 ml of IrOx@DOX dispersion into a dialysis bag (MWCO = 3 kDa) and put it into a buffer solution with different pH values (pH value is 5.5 or 7.4) and stir at room temperature without light. US group was given ultrasound irradiation (1 W/cm2) for 30 s at a certain time. Take 1 ml of release solution from the beaker at different time points and add the same volume of fresh buffer. UV-Vis was used to determine the absorbance of the diluent at 500 nm. The cumulative release of DOX under different conditions is calculated through the standard curve, and the release curve of DOX is obtained [15]. Drug release rate = (mass of DOX released/total mass of DOX in the complex) × 100%.


### 3.2.2. In Vitro SDT Effect Evaluation

Mix 200μL IrOx@DOX dispersion (100 ug/ml) with 200 μL temp solution (100 mmol/L), irradiate with US (1.0 MHz, 1.0 W/) for 2 min, and immediately detect the generation of oxygen by ESR spectrometer. At the same time, no ultrasonic treatment was used as control. In order to explore the effect of different ultrasonic power on oxygen generation, we irradiated IrOx@DOX dispersion with the same concentration with US of different power (0.5, 1.0, 1.5, 2.0, and 2.5 W/cm2) and detected oxygen generation by ESR. In addition, mix 2.96 ml of the iroxdox solution (100 μg/ml) and 40 μL of DBPF (2 mg/ml) away from light, conduct US radiation (1.0 MHz, 1 W/cm2, 2 min) every 2 min, and detect the generation of ROS by UV-Vis recording the absorption curve of the solution at the wavelength of 200∼650 nm [16].


### 3.2.3. In Vitro Cellular Uptake

CISM was used to observe the uptake of IRO and @DOX by 4TI cells. 4Ti cells (1 × 105/well) were inoculated into confocal Petri dishes. After the cells adhered to the wall, the blank control group was added with PBS containing medium for 6 hours; In the experimental group, the medium containing IrOx@DOX was added and treated with or without ultrasonic irradiation (1 MHz, 1.0 W/cm2, 1 min), and then continued to culture for 6 hours. The cells were washed with PBS 3 times to remove the nonphagocytized nanoparticles. The cells were fixed with 4% paraformaldehyde for 15 min and stained with DAPI for 5 min. Finally, CISM was used for observation.


### 3.2.4. Cell SDT Effect in Vitro

4T1 cells were inoculated into 6-well plates (1 × 10³/well) and cultured for 24 hours. The cells were divided into the following groups: ① PBS group, ② PBS + US group, ③ IrOx group, ④ IrOx + US group, ⑤ IrOx@DOX group, and ⑥ IrOx@DOX + US group. Add 2 mL of fresh medium containing the above nanoparticles and continue to incubate for 12 h. PBS was washed 3 times, and then ROS probe (DCFH-DA) was added to incubate for 30 min. After that, PBS was washed 3 times. At the same time, groups ②, ④, and ⑥ were incubated for 2 h after ultrasonic irradiation (1.0 MHz, 1.0 W/cm2, 1 min). Finally, the cells were observed by a fluorescence microscope. Furthermore, ROS production was detected by flow cytometry. Cell treatment was as described above.


### 3.2.5. In Vitro Chemotherapy SDT Synergistic Treatment Effect

Firstly, the cytocompatibility of IrOx was investigated. Specifically, HUVEC cells were inoculated into 96 well plates (1 × 104/well) and cultured in an incubator at 37°C and 5% carbon dioxide for 24 h. Then add 100 μ·L of fresh medium containing different concentrations of IRO that were incubated for 12 h. Remove the medium, wash it with PBS 3 times, and then add 100 μ·L of medium containing 10 μL CCK-3 reagent to continue incubation for 1 h [17]. Finally, the absorbance value at 450 nm was read with a microplate reader, and the cell survival rate was calculated according to the instructions. Each group of samples is set with 5 multiple holes, and each sample is measured in parallel 3 times. CCK-8 method was used to evaluate the synergistic effect of IrOx@DOX-induced chemotherapy SDT. 4T1 cells were inoculated into 96 well plates (1 × 104/well) and cultured for 24 hours.


## 3.2.1. Investigation of Drug Release Behavior In Vitro

The drug release behavior of IrOx@DOX was investigated by dialysis. Specifically, a certain quality of DOX was accurately weighed and dissolved in PBS buffer solution (200 mL) with pH values of 7.4 and 5.5, and its absorbance was measured to obtain the standard curve of DOX under different pH values. Then put 2 ml of IrOx@DOX dispersion into a dialysis bag (MWCO = 3 kDa) and put it into a buffer solution with different pH values (pH value is 5.5 or 7.4) and stir at room temperature without light. US group was given ultrasound irradiation (1 W/cm2) for 30 s at a certain time. Take 1 ml of release solution from the beaker at different time points and add the same volume of fresh buffer. UV-Vis was used to determine the absorbance of the diluent at 500 nm. The cumulative release of DOX under different conditions is calculated through the standard curve, and the release curve of DOX is obtained [15]. Drug release rate = (mass of DOX released/total mass of DOX in the complex) × 100%.


## 3.2.2. In Vitro SDT Effect Evaluation

Mix 200μL IrOx@DOX dispersion (100 ug/ml) with 200 μL temp solution (100 mmol/L), irradiate with US (1.0 MHz, 1.0 W/) for 2 min, and immediately detect the generation of oxygen by ESR spectrometer. At the same time, no ultrasonic treatment was used as control. In order to explore the effect of different ultrasonic power on oxygen generation, we irradiated IrOx@DOX dispersion with the same concentration with US of different power (0.5, 1.0, 1.5, 2.0, and 2.5 W/cm2) and detected oxygen generation by ESR. In addition, mix 2.96 ml of the iroxdox solution (100 μg/ml) and 40 μL of DBPF (2 mg/ml) away from light, conduct US radiation (1.0 MHz, 1 W/cm2, 2 min) every 2 min, and detect the generation of ROS by UV-Vis recording the absorption curve of the solution at the wavelength of 200∼650 nm [16].


## 3.2.3. In Vitro Cellular Uptake

CISM was used to observe the uptake of IRO and @DOX by 4TI cells. 4Ti cells (1 × 105/well) were inoculated into confocal Petri dishes. After the cells adhered to the wall, the blank control group was added with PBS containing medium for 6 hours; In the experimental group, the medium containing IrOx@DOX was added and treated with or without ultrasonic irradiation (1 MHz, 1.0 W/cm2, 1 min), and then continued to culture for 6 hours. The cells were washed with PBS 3 times to remove the nonphagocytized nanoparticles. The cells were fixed with 4% paraformaldehyde for 15 min and stained with DAPI for 5 min. Finally, CISM was used for observation.


## 3.2.4. Cell SDT Effect in Vitro

4T1 cells were inoculated into 6-well plates (1 × 10³/well) and cultured for 24 hours. The cells were divided into the following groups: ① PBS group, ② PBS + US group, ③ IrOx group, ④ IrOx + US group, ⑤ IrOx@DOX group, and ⑥ IrOx@DOX + US group. Add 2 mL of fresh medium containing the above nanoparticles and continue to incubate for 12 h. PBS was washed 3 times, and then ROS probe (DCFH-DA) was added to incubate for 30 min. After that, PBS was washed 3 times. At the same time, groups ②, ④, and ⑥ were incubated for 2 h after ultrasonic irradiation (1.0 MHz, 1.0 W/cm2, 1 min). Finally, the cells were observed by a fluorescence microscope. Furthermore, ROS production was detected by flow cytometry. Cell treatment was as described above.


## 3.2.5. In Vitro Chemotherapy SDT Synergistic Treatment Effect

Firstly, the cytocompatibility of IrOx was investigated. Specifically, HUVEC cells were inoculated into 96 well plates (1 × 104/well) and cultured in an incubator at 37°C and 5% carbon dioxide for 24 h. Then add 100 μ·L of fresh medium containing different concentrations of IRO that were incubated for 12 h. Remove the medium, wash it with PBS 3 times, and then add 100 μ·L of medium containing 10 μL CCK-3 reagent to continue incubation for 1 h [17]. Finally, the absorbance value at 450 nm was read with a microplate reader, and the cell survival rate was calculated according to the instructions. Each group of samples is set with 5 multiple holes, and each sample is measured in parallel 3 times. CCK-8 method was used to evaluate the synergistic effect of IrOx@DOX-induced chemotherapy SDT. 4T1 cells were inoculated into 96 well plates (1 × 104/well) and cultured for 24 hours.


## 3.3. Experimental Grouping

In the US irradiation group, the cells were incubated with nanoparticles for 6 hours and then exposed to US irradiation (1.0 MHz, 1 min) and continued to incubate for 12 hours. Then the medium was removed and washed with PBS 3 times. The cell survival rate was calculated by the CCK-8 method. In addition, after different treatments, the cells were stained with Calcein AM/PI (Calcein AM 2μmol/L, PI4.5 μmol/L, 100 μL) for 15 min. Then the culture medium was discarded and washed with PBS. Finally, the survival state of the cells was observed under the fluorescence microscope.Apoptosis was detected by flow cytometry: 4T1 cells (1 × 105/well) were inoculated and incubated in 6-well plates for 24 hours. The cells were treated according to the above experimental steps of CCK-8. After incubation, the cells were digested with trypsin, washed with PBS, centrifuged, and collected, and the number of early apoptosis, late apoptosis, and necrotic cells were analyzed with flow cytometry apoptosis staining kit (Annexin VFITC/PI staining solution) [18].


## 3.4. Establishment and Treatment of the Tumor Model

One hundred microliter HEPA 1–6 cell suspension containing 2 × 106 cells was subcutaneously injected into BALB/c female nude mice to establish a subcutaneous transplanted tumor model. When the tumor volume reached 120 mm, they were randomly divided into five groups (n = 3): ① normal saline group, ② IrOx group, ③ Irox@DOX group, ④IrOx + US group, and ⑤ IrOx@DOX + US group. Nude mice were injected with normal saline, IrOx, and IrOx@DOX (8 mg/mL, 100 μL). The ultrasonic irradiation group received ultrasonic irradiation (1.5 W/cm2, 1.5 MHz, 50% duty cycle, 5 min) 12 hours after drug injection. The treatment process was repeated every 3 days. The body mass and tumor volume of the mice were recorded every 3 days for 14 days. The tumor volume calculation formula is V = L × W2/2, where V, L, and W represent the tumor volume, length, and width, respectively. After 14 days of treatment, a contrast agent was injected intravenously to observe the growth of the tumor. The nude mice were euthanized. The corresponding tumor bodies were photographed and observed by HE staining. Calculate the tumor volume inhibition rate = (V control group – V test group)/V control group × 100%.


## 3.5. Statistical Analysis

Origin and SPSS 26.0 statistical software were used for mapping and statistical analysis. The independent samplet-test was used for data pairwise comparison, and a one-way analysis of variance was used for the comparison of the multivariate mean. P<0.05 indicates that the difference is statistically significant.


## 4. Results and Discussion

### 4.1. Preparation, Characterization, and Physical and Chemical Property Evaluation of IrOx@DOX

IrOx nanoparticles were prepared by hydrothermal method. TEM shows that the prepared IrOx has an irregular nanocluster structure, and the particle size is about 5 nm [19]. At the same time, the lattice fringes of IrOx can be clearly observed. Comparing the color of the solution before and after the reaction, it can be seen that the synthesized IrOx is a blue-purple dispersion, which is different from the iridium chloride solution. It is proved that the oxidation reaction Ir3+ is initiated by high temperatures. In the XRD spectrum, IrOx shows an obvious wide peak, indicating that it tends to the aggregation and growth of small particles. The valence states of elements in IrOx were confirmed by XPS. The characteristic peaks related to Ir4f, O1s, and C1s can be seen from the full spectrum of XPS, which preliminarily indicates the successful preparation of IrOx [20].The XPS spectra of ir4f show two characteristic peaks at 64.7 eV and 61.8 eV, which can be attributed toIr4f5/2 and Ir4f7/2. FT-IR spectrum shows that IrOx has strong and wide absorption near 3,300 cm−1, which is attributed to some hydroxides on the surface of IrOx, which may be caused by the oxidation of Ir3+ [21]. It can be seen from the UV-Vis spectrum that IrOx has good absorption at 200–650 nm. The characteristic absorption peak (485 nm) of DOX appeared in the UV-Vis spectrum of IrOx@DOX, which proved the successful loading of DOX [22]. In addition, zeta potential measurement showed that the potential of IrOx was (–22.30 ± 0.47) mV, while that of DOX was (23.60 ± 0.62) mV. After DOX is loaded, the potential of IrOx@DOX becomes (1.40 ± 0.39) mV, indicating that IrOx and DOX can be combined by electrostatic attraction [23, 24].


### 4.2. Drug Loading and In Vitro Drug Release

The drug loading was investigated by UV-Vis, and the standard curve of DOX was established, as shown in Figure2. The loading amount of IrOx to DOX increases with the increase of DOX concentration within a certain drug concentration (as shown in Figure 3). When the ratio of IrOx to DOX was 4:1, the drug loading rate reached (18.7 ± 0.2)%. IrOx@DOX drug release in vitro; as shown in Figure 4, when pH = 7.4, the DOX release rate is (9.7 ± 1.2)%; and when pH = 5.5, the DOX release rate is (40.0 ± 1.7)%. This is due to the protonation of DOX due to the acidic pH value. In addition, US stimulation can further accelerate DOX release. Under pH = 5.5 and US stimulation, the release rate of DOX increased to (61.1 ± 1.3)%. It may be that the cavitation effect stimulated by US weakens the force between DOX and IrOx and releases the drug. In conclusion, IrOx@DOX has the dual response of pH and US to drug release, which is expected to improve the chemotherapy effect of DOX.Figure 2
Standard curve of DOX.Figure 3
Drug loading concentration.Figure 4
DOX release rate.


### 4.3. Investigation of In Vitro ROS Production Performance of IrOx

ROS production performance of IrOx in vitro: compared with the group without US irradiation, the generation of oxygen can be significantly detected in the US irradiation group (as shown in Figure5). Meanwhile, with the increase of US irradiation power, the peak value of the TEMP/oxygen spectrum gradually increases, indicating that the oxygen production caused by IrOx is power-dependent. In addition, DPBF was used as the active oxygen probe, and the absorption peak intensity at different time points was recorded by UV-Vis. It can be seen from Figure 6 that at the wavelength of 398 nm, the absorption peak of DPBF decreases with time, indicating that under US irradiation, IrOx can continuously produce oxygen with time.Figure 5
ROS performance.Figure 6
Singlet oxygen generation performance.


### 4.4. In Vitro Cellular Uptake

The uptake of IrOx@DOX by 4T1 cells was observed by CLSM. It was found that the nuclei were stained with DAPI and showed blue fluorescence, while DOX showed red fluorescence under excitation light. There was no red fluorescence in the blank control group, but obvious red fluorescence could be observed in the cells treated with IrOx@DOX, indicating that 4Ti cells could ingest IrOx@DOX. In contrast, IrOx@DOX + US group showed a stronger red fluorescence signal, indicating that ultrasound can not only enhance the uptake of nanoparticles by cells but also promote the release of DOX in cells, which provides a basis for subsequent SDT chemotherapy synergistic therapy [25].


### 4.5. SDT Performance Evaluation at Cell Level

DCFH-DA has no fluorescence and can pass through the cell membrane. After entering the cell, it is hydrolyzed by esterase in the cell to produce DCFH. However, DCFH cannot penetrate the cell membrane, so the probe is loaded into the cell. Intracellular reactive oxygen species can oxidize nonfluorescent DCFH to produce fluorescent DCF, so as to detect the level of intracellular reactive oxygen species. Under the fluorescence microscope, it is observed that the control group has almost no green fluorescence, while the green fluorescence of the IrOx + US group is significantly stronger than that of the IrOx group alone or the PBS + US group (see Figure7). Furthermore, prove the SDT effect of IrOx. In addition, the production of reactive oxygen species in the IrOx@DOX + US group was significantly higher than that in other groups. At the same time, quantitative detection by flow cytometry also confirmed the above conclusions. The production of reactive oxygen species in the IrOx@DOX + US group was the highest.Figure 7
Evaluation of ROS production performance of IrOX@DOX at the cell level.


### 4.6. In Vivo Anti-Tumor Effect

The subcutaneous transplanted tumor was established to verify the therapeutic effect. There was no significant difference in the body mass of mice in each group during the treatment, indicating that the injected nanoparticles in each group had no significant effect on the body mass of mice. Compared with the control group, the tumor volume of the IrOx + US group, the IrOx@DOX group, and the IrOx@DOX + US group decreased, and the difference was statistically significant (P<0.05). The inhibition rates were 27%, 57%, and 76%, respectively, which was consistent with the digital image, indicating that IrOx@DOX combined with US radiography could produce an obvious synergistic therapeutic effect. In addition, the contrast-enhanced ultrasound results showed that compared with the control group, the IrOx@DOX + US group had a small tumor volume and necrosis of the internal tissues of the tumor, and the treatment effect was the best, which was consistent with the results of the experiment. The above experimental results show that a good therapeutic effect can be obtained when DOX and SDT are combined.


## 4.1. Preparation, Characterization, and Physical and Chemical Property Evaluation of IrOx@DOX

IrOx nanoparticles were prepared by hydrothermal method. TEM shows that the prepared IrOx has an irregular nanocluster structure, and the particle size is about 5 nm [19]. At the same time, the lattice fringes of IrOx can be clearly observed. Comparing the color of the solution before and after the reaction, it can be seen that the synthesized IrOx is a blue-purple dispersion, which is different from the iridium chloride solution. It is proved that the oxidation reaction Ir3+ is initiated by high temperatures. In the XRD spectrum, IrOx shows an obvious wide peak, indicating that it tends to the aggregation and growth of small particles. The valence states of elements in IrOx were confirmed by XPS. The characteristic peaks related to Ir4f, O1s, and C1s can be seen from the full spectrum of XPS, which preliminarily indicates the successful preparation of IrOx [20].The XPS spectra of ir4f show two characteristic peaks at 64.7 eV and 61.8 eV, which can be attributed toIr4f5/2 and Ir4f7/2. FT-IR spectrum shows that IrOx has strong and wide absorption near 3,300 cm−1, which is attributed to some hydroxides on the surface of IrOx, which may be caused by the oxidation of Ir3+ [21]. It can be seen from the UV-Vis spectrum that IrOx has good absorption at 200–650 nm. The characteristic absorption peak (485 nm) of DOX appeared in the UV-Vis spectrum of IrOx@DOX, which proved the successful loading of DOX [22]. In addition, zeta potential measurement showed that the potential of IrOx was (–22.30 ± 0.47) mV, while that of DOX was (23.60 ± 0.62) mV. After DOX is loaded, the potential of IrOx@DOX becomes (1.40 ± 0.39) mV, indicating that IrOx and DOX can be combined by electrostatic attraction [23, 24].


## 4.2. Drug Loading and In Vitro Drug Release

The drug loading was investigated by UV-Vis, and the standard curve of DOX was established, as shown in Figure2. The loading amount of IrOx to DOX increases with the increase of DOX concentration within a certain drug concentration (as shown in Figure 3). When the ratio of IrOx to DOX was 4:1, the drug loading rate reached (18.7 ± 0.2)%. IrOx@DOX drug release in vitro; as shown in Figure 4, when pH = 7.4, the DOX release rate is (9.7 ± 1.2)%; and when pH = 5.5, the DOX release rate is (40.0 ± 1.7)%. This is due to the protonation of DOX due to the acidic pH value. In addition, US stimulation can further accelerate DOX release. Under pH = 5.5 and US stimulation, the release rate of DOX increased to (61.1 ± 1.3)%. It may be that the cavitation effect stimulated by US weakens the force between DOX and IrOx and releases the drug. In conclusion, IrOx@DOX has the dual response of pH and US to drug release, which is expected to improve the chemotherapy effect of DOX.Figure 2
Standard curve of DOX.Figure 3
Drug loading concentration.Figure 4
DOX release rate.


## 4.3. Investigation of In Vitro ROS Production Performance of IrOx

ROS production performance of IrOx in vitro: compared with the group without US irradiation, the generation of oxygen can be significantly detected in the US irradiation group (as shown in Figure5). Meanwhile, with the increase of US irradiation power, the peak value of the TEMP/oxygen spectrum gradually increases, indicating that the oxygen production caused by IrOx is power-dependent. In addition, DPBF was used as the active oxygen probe, and the absorption peak intensity at different time points was recorded by UV-Vis. It can be seen from Figure 6 that at the wavelength of 398 nm, the absorption peak of DPBF decreases with time, indicating that under US irradiation, IrOx can continuously produce oxygen with time.Figure 5
ROS performance.Figure 6
Singlet oxygen generation performance.


## 4.4. In Vitro Cellular Uptake

The uptake of IrOx@DOX by 4T1 cells was observed by CLSM. It was found that the nuclei were stained with DAPI and showed blue fluorescence, while DOX showed red fluorescence under excitation light. There was no red fluorescence in the blank control group, but obvious red fluorescence could be observed in the cells treated with IrOx@DOX, indicating that 4Ti cells could ingest IrOx@DOX. In contrast, IrOx@DOX + US group showed a stronger red fluorescence signal, indicating that ultrasound can not only enhance the uptake of nanoparticles by cells but also promote the release of DOX in cells, which provides a basis for subsequent SDT chemotherapy synergistic therapy [25].


## 4.5. SDT Performance Evaluation at Cell Level

DCFH-DA has no fluorescence and can pass through the cell membrane. After entering the cell, it is hydrolyzed by esterase in the cell to produce DCFH. However, DCFH cannot penetrate the cell membrane, so the probe is loaded into the cell. Intracellular reactive oxygen species can oxidize nonfluorescent DCFH to produce fluorescent DCF, so as to detect the level of intracellular reactive oxygen species. Under the fluorescence microscope, it is observed that the control group has almost no green fluorescence, while the green fluorescence of the IrOx + US group is significantly stronger than that of the IrOx group alone or the PBS + US group (see Figure7). Furthermore, prove the SDT effect of IrOx. In addition, the production of reactive oxygen species in the IrOx@DOX + US group was significantly higher than that in other groups. At the same time, quantitative detection by flow cytometry also confirmed the above conclusions. The production of reactive oxygen species in the IrOx@DOX + US group was the highest.Figure 7
Evaluation of ROS production performance of IrOX@DOX at the cell level.


## 4.6. In Vivo Anti-Tumor Effect

The subcutaneous transplanted tumor was established to verify the therapeutic effect. There was no significant difference in the body mass of mice in each group during the treatment, indicating that the injected nanoparticles in each group had no significant effect on the body mass of mice. Compared with the control group, the tumor volume of the IrOx + US group, the IrOx@DOX group, and the IrOx@DOX + US group decreased, and the difference was statistically significant (P<0.05). The inhibition rates were 27%, 57%, and 76%, respectively, which was consistent with the digital image, indicating that IrOx@DOX combined with US radiography could produce an obvious synergistic therapeutic effect. In addition, the contrast-enhanced ultrasound results showed that compared with the control group, the IrOx@DOX + US group had a small tumor volume and necrosis of the internal tissues of the tumor, and the treatment effect was the best, which was consistent with the results of the experiment. The above experimental results show that a good therapeutic effect can be obtained when DOX and SDT are combined.


## 5. Conclusion

In this paper, a preparation method of iridium oxide nanocomposites under the microscope is proposed, focusing on the application of IrOx in SDT, and the shape of IrOx is observed by TEM. The samples were characterized by XRD. The different elements and valence states in IrOx were characterized by XPS. The functional groups on the surface of nanoparticles were investigated by FTIR. UV-Vis was used to measure the UV absorption spectra of different samples: zeta potential analyzer was used to measure the potential of products in each stage of the material synthesis process. It is proved that iridium oxide nanocomposite has a good anti-tumor effect, which is reflected in the good therapeutic effect when the chemotherapy drug DOX and SDT are combined.



---

*Source: 9694425-2022-07-13.xml*