CA4 (purity > 98%) and MIT (purity > 98%) were purchased from Shanghai Aladdin Biochemical Co., Ltd. CuCl₂∙2H₂O and triethylamine (TEA) were purchased from Shanghai Macklin Bio-Technology Co., Ltd. All chemicals were of analytical grade and used as received without further purification. Ultrapure water (18.2 MΩ/cm) was used throughout the work.

A precursor ethanol solution consisting of CA4, MIT, and CuCl₂∙2H₂O with a particular molar ratio (1:1:2.5) was prepared. Then, TEA was added to the precursor ethanol solution to reach pH 7.4, and the mixture was stirred for 24 h. After ultrafiltration centrifugation and washing with DI water three times, CMMOPs were finally formed. Finally, CMMOPs were redisposed in water to form CMMOPs suspension (1 mg/ml) for later use.

The morphology, energy dispersive spectroscopy (EDS) spectrum, and EDS elemental mapping of CMMOPs were examined by TEM (Tecnai G2 F20 S-TWIN) at 10 kV. The size and zeta-potential values were determined by a Malvern Zetasizer Nano-ZS machine (Malvern Instruments, Malvern). Three parallel measurements were carried out to determine the average values.

The drug loading content of CA4 and MIT in CMMOPs was determined by high-performance liquid chromatography (HPLC) at 305 and 620 nm, respectively. The drug loading content was calculated as follows: Drug loading content of CA 4   ( % ) = weight of CA 4  in CMMOPs weight of CMMOPs × 100 % , (1) Drug loading content of MIT  ( % ) = weight of MIT in CMMOPs weight of CMMOPs × 100 % . (2)

The CMMOP suspension (0.1 mg/ml) was placed in a dialysis bag (MWCO 3500 Da). The dialysis bag was then immersed in PBS (150 ml, 0.15 M) at pH 5.0, 5.5, 6.0, 6.5, or 7.4 and oscillated continuously in a shaker incubator (160 rpm) at 37°C. Then, 1.0 ml of the release medium was withdrawn at predetermined time intervals and replaced with 1.0 ml of fresh release medium. All samples were assayed by HPLC.

BALB/C mice (5–6 weeks, 18–22 g) were purchased from Hunan STA Laboratory Animal Co., Ltd. The tumor models were set up by injecting H22 cells subcutaneously in the selected position of the mice.

When the tumor volume of the H22 tumor-bearing mice was approximately 50 mm³, mice were randomly divided into five groups (12 mice per group) and treated by intravenous injection of 0.9% NaCl, the free CA4, the free MIT, the mixture of CA4 and MIT, and the CMMOPs [(CA4) = 7.3 mg/kg, (MIT) = 10.0 mg/kg] every 3 days three times. The survival rate was recorded every day. The tumor volume and body weight were monitored every 3 days. The tumor volume was calculated by the following formula: tumor volume = 0.5 × length × width². The highest and lowest data were discarded at last.

CMMOPs were simply assembled from the hydrophobic CA4 and MIT by coordination between C-O-Cu and C=O-Cu bonds (Figure 2A). Because both drugs possessed large amounts of hydroxyl groups, they could easily coordinate to Cu(II) and form the [-CA4-Cu(II)-MIT-Cu(II)-]_n complex, which was dispersed in water to form CMMOPs (1 mg/ml). Then, the morphology of CMMOPs was characterized by transmission electron microscopy. The TEM image showed that CMMOPs were spherical and possessed a size of about 90 nm (Figure 2B). The energy dispersive spectroscopy (EDS) spectrum (Figure 2F) of the CMMOPs indicated the existence of Cu, C, O, and N elements in the nanoparticles. Moreover, the EDS elemental mapping (Figures 2C–E) of CMMOPs indicated the uniform distribution of Cu, C, and N elements in the nanoparticles. Results suggested that CMMOPs were assembled from the coordination of CA4, MIT, and Cu(II).

Then, the size distribution and zeta potential of CMMOPs were obtained via Dynamic Light Scattering analysis. As shown in Figure 3A, CMMOPs possessed a hydrodynamic size of 133.6 ± 12.4 nm. With a zeta potential of 27.6 ± 3.7 mV (Figure 3B), the size of CMMOPs could be kept within an acceptable range over 4 weeks at 4 °C, suggesting their good stability as a nanodrug (Figure 3C). The drug loading of CA4 and MIT in the CMMOPs was 37.6% and 51.4%, respectively. Data indicated that 89.0% of CMMOPs were composed of chemotherapeutic agents, much higher than the NDDS prepared via traditional strategies.

As the coordination bonds between Cu(II) and the drugs were sensitive to acid conditions, the drug release profiles of CA4 and MIT from CMMOPs in PBS under different pH conditions (pH 5.0, 5.5, 6.0, 6.5, and 7.4) were investigated. The amounts of released CA4 and MIT were determined by HPLC at pre-decided time points. As shown in Figures 4A,B, only 6.4% of the CA4 and 10.2% of the MIT were released from the CMMOPs after 48 h (pH 7.4, 37°C), indicating that CMMOPs were very stable at the pH value of the physiological condition. When reducing the pH value, both drugs were released more quickly. More strikingly, the cumulative released percentages of CA4 and MIT increased up to 90.2% and 93.2% at pH 5.0, and the burst release of the dual drugs was very sharp. Results indicated that CMMOPs would possess a slight drug release in the blood circulation but a rapid release at the tumor site. This feature could serve efficient targeting property for CMMOPs.

Moreover, the in vivo anticancer effects of the CMMOPs were investigated by evaluating their tumor inhibition efficacy after injection. The individual drug and the physical mixture of the dual drugs were used as control. As shown in Figure 5A, only 16.7% of mice in the control group could survive for 21 days after the first drug injection. The individual administration of CA4 or MIT could improve the survival rate to 41.7% or 33.3%, indicating a certain anticancer effect. The combination of the dual drugs could largely increase the survival rate to 66.7%, suggesting the synergistic effect of the dual drugs. MIT is a cell cycle nonspecific agent, which could act directly on the tumor cells and kill them. However, CA4 is an anti-angiogenic drug that suppresses tumor growth by inhibiting angiogenesis. Dual drugs could synergistically kill the cancer cells through different routes. Furthermore, CMMOPs possessed the most pronounced inhibition effect. The survival rate of the mice injected with CMMOPs was up to 83.3%, and only two mice died from tumors in this group. The tumor volume was also recorded (Figure 5B). Tumors in the mice of the CA4 group were significantly smaller than those of the MIT group. The mice administrated with CA4 showed gradually increased body weight, while those injected with MIT possessed severe body weight loss, as well as listlessness and laziness (Figure 5C). This might be owing to the targeting and slight side effects of CA4. Although tumors in the mice administrated with the physical mixture of CA4 and MIT were much smaller than those administrated with the individual drug, the side effect of MIT was not improved. In comparison, the mice of the CMMOPs group possessed the minimum tumor volume, indicating their efficient anticancer effect. The gradually increased body weight suggested their better health conditions than those administrated with MIT formulations. More strikingly, the tumors of this group decreased with the injection of the formulation, indicating its outstanding anticancer efficacy. Overall, results forcefully indicated that CMMOPs with significant antitumor and slight side effects would greatly improve the efficacy of cancer therapy.