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In vivo formation of Cu(DDC)2 complex induced by nanomedicine for mesothelioma chemotherapy
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Yixin Zhanga, Shunjie Dingb, Junhua Lia, Xinyu Penga, Jing Lia, Jing Changc, Wenxia Gaod, *, Bin Hea, *
Chinese Chemical Letters | 2020, 31(12) : 3168 - 3172
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Chinese Chemical Letters | 2020, 31(12): 3168-3172
• Communication •
In vivo formation of Cu(DDC)2 complex induced by nanomedicine for mesothelioma chemotherapy
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Yixin Zhanga, Shunjie Dingb, Junhua Lia, Xinyu Penga, Jing Lia, Jing Changc, Wenxia Gaod, *, Bin Hea, *
Affiliations
  • a National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
  • b Army Logistics University of PLA, Chongqing 401331, China
  • c College of Marine Life Science, Ocean University of China, Qingdao 266003, China
  • d College of Chemistry and Materials Engineering, Wenzhou University, Wenzhou 325027, China
Published: 2020-12-15 doi: 10.1016/j.cclet.2020.04.051
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The copper(II) diethyldithiocarbamate (Cu(DDC)2) complex exhibited excellent inhibition to cancer cells. The usual administration is intravenous injection for disulfram and oral for copper. A new strategy was reported to improve the administration efficiency of the Cu(DDC)2 drug. Poly(lactide-co-glycolide) (PLGA) nanoparticles were used to trap disulfram and copper gluconate separately, the two types of drug loaded nanoparticles were injected in mesothelioma-bearing nude mice via intraperitoneal injection. The in vivo formation of Cu(DDC)2 complex was induced by disulfiram and Cu2+ released from PLGA nanoparticles. This strategy avoided many obstacles in the use of Cu(DDC)2 complex as chemotherapeutic and exhibited excellent anticancer activity to mesothelioma.

Mesothelioma  /  Nanoparticles  /  In vivo formation  /  PLGA  /  Cu(DDC)2 complex
Yixin Zhang, Shunjie Ding, Junhua Li, Xinyu Peng, Jing Li, Jing Chang, Wenxia Gao, Bin He. In vivo formation of Cu(DDC)2 complex induced by nanomedicine for mesothelioma chemotherapy[J]. Chinese Chemical Letters, 2020 , 31 (12) : 3168 -3172 . DOI: 10.1016/j.cclet.2020.04.051
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Mesothelioma is a malignant tumor occurred at mesothelial lining of the pleura, peritoneum, pericardium and tunica vaginalis. It is rapidly progressing with high morality and gets constantly growing attention as its peak is expected to reach in the next few years. The pathopoiesia of mesothelioma is overexposure to asbestos which was consumed excessively around the world, especially in Asia, in the past couple of decades [1-3]. Recent research reveals that carbon nanotubes would lead to mesothelioma [4]. As mesothelioma has a very long latency, even over 40 years, and the patients with mesothelioma have only approximately 10 months once diagnosed, if the treatment cannot be implemented in the early stage [5]. Current treatment of mesothelioma is inefficient and local recurrence is common. Antifolate combined with platinum is the only established chemotherapy for mesothelioma but benefits are usually modest at best and prognosis is poor. Some other clinical schemes were reported, however, no optimistic results were received at present [6].
The drawbacks of chemotherapy such as the toxicity of drug, multidrug resistance (MDR) and huge R&D cost of a new drug are particularly obvious [7-10]. "Drug repurposing" is an effective strategy that could greatly reduce time and cash consumption. Disulfiram (DS) as one example of "drug repurposing" is attracted more and more interests in cancer treatment. Studies have explored that DS is highly specific to cancer cells and could inhibit drug resistance [11]. The anticancer mechanism of DS is that DS could decompose into diethyldithiocarbamate (DDC), which easily chelates with copper(Ⅱ) to form Cu(DDC)2 complex to generate reactive oxygen species (ROS) and kill cancer cells [12]. The Cu(DDC)2 complex is easily precipitated if DS and Cu2+ are complexed directly, the Cu(DDC)2 precipitations could not be dispersed into nano-scale particles for encapsulation. In applications, DS and Cu2+ were administrated in different routes. DS was injected via intravenous injection, copper-containing preparations were administrated orally to increase the content of copper ions in tumor cells to generate more ROS intracellularly [13, 14]. However, the utilization of oral copper ions was low, taking large doses of copper ions would cause serious poisoning [15].
Free DS is very unstable in the blood transportation [16], encapsulation of DS in nanoparticles could elongate the metabolic time, liposomes and polymeric micelles were reported for DS delivery [17, 18]. In order to avoid the precipitation of Cu(DDC)2 complex, an in situ complexation was carried out to encapsulate DS and Cu2+ in a same nanoparticle [19]. A new poly(ethylene glycol)-b-poly(ester-carbonate) block copolymer with carboxyl pendant groups was synthesized to chelate Cu2+, DS was encapsulated in the self-assembly polymeric micelles, as the chelating ability between Cu2+ and DS was much stronger than that between Cu2+ and carboxyl groups, the Cu(DDC)2 complex was formed in the micelles and the drug loaded micelles were intravenously injected for non-small cell lung cancer (NSCLC) treatment. As the formation of Cu(DDC)2 was too fast, it was hard to control the formation process. Other than encapsulation, a ROS responsive DDC prodrug with an aryl boronic ester modification was presented to overcome the rapid metabolism of DS in blood. The bond between DDC and aryl boronic ester in the prodrug was cleaved to return free DDC in the presence of high concentrated H2O2 in tumors, the complexation of DDC and oral administrated Cu2+ exerted efficient anticancer activity in breast cancer bearing mice model [20].
The mesotheliomas are composed of many nodules, which is very different from NSCLC and/or breast cancer with a solid tumor tissue in subcutaneous cancer bearing mice models. Nanomedicine is convenient for sustaining or controlled release and drug protection with syringe ability [21-23]. The in situ injection of nanomedicine facilitates local drug depot to maintain effective drug concentration over a long period of time to broaden the therapeutic window. In this study, a new strategy was used to be aimed at dispersive mesothelioma. Poly(lactide-co-glycolide) (PLGA75/25) was synthesized and fabricated nanoparticles (NPs) to load both hydrophobic DS and hydrophilic copper gluconate separately. The two kinds of nanoparticles were injected to the mesothelioma-bearing nude mice via intraperitoneal injection. The released DS and Cu2+ from the nanoparticles formed Cu(DDC)2 in situ to kill mesothelioma cells. This in situ formation strategy of Cu(DDC)2 complex could not only increase the Cu2+ utilization, but also avoid the precipitation and reduce systemic toxicity (Scheme 1).
The experimental section was presented in Supporting information. The 1H NMR spectrum of PLGA75/25 was exhibited in Fig. S1 (Supporting information), and the molar ratio between lactide and glycolide was 77:23 according to the integrals calculation. The intrinsic viscosity of the copolymer was 1.10. The morphology of PLGA75/25 nanoparticles loaded with DS and copper gluconate was shown in Figs. 1A and B. It demonstrated that most of the nanoparticles were spherical with the size ranged from 90 nm to 240 nm. The drug loading contents of the two nanoparticles were presented in Fig. 1C. They were 3.70% and 0.40% for DS and copper gluconate loaded nanoparticles. The drug loading content of DS was much higher than that of copper gluconate due to the hydrophobicity of DS. As a water soluble molecule, copper gluconate was hard to be trapped in PLGA75/25 nanoparticles via double emulsion method. In order to promote the loading capacity of hydrophilic copper salt, copper gluconate was added to external phase to enhance the osmotic concentration [24]. Although the drug loading content of Cu2+ in PLGA75/25 nanoparticles was only 0.40%, it was high enough comparing to the dose of Cu2+ absorbed from oral administration. The drug release profiles of DS and Cu2+ in Fig. 1D revealed the release rate of Cu2+ was much faster than that of DS due to its hydrophilic essence, whose diffusion rate from nanoparticles was much higher than that of DS. Both accumulative release rates of DS and Cu2+ were around 60%, however, the reaching time for DS and Cu2+ were about 26 h and 13 h, respectively. The fast release of Cu2+ could also make up for deficiency of low drug loading content.
The cytotoxicity of blank PLGA75/25 nanoparticles was investigated and the results were demonstrated in Fig. 2A. The blank nanoparticles in different concentrations were incubated with mesothelioma cells for three days, the cell viabilities were tested for 24, 48 and 72 h incubation, interestingly, and the cell viabilities for 48 h incubation were the lowest no matter what the concentration was, they were in the range of 80%–90%. The cell viabilities in other conditions were all higher than 90%. It was probably because that the intracellular concentration of blank NPs reached the maximum when the incubation time was 48 h. The cell viabilities of all nanoparticles samples were higher than 80%, which could be considered as non-toxic according to the evaluation standard of biomaterials. Other than cancer cells, normal L929 cells were also incubated with blank NPs for 48 h to evaluate the cytotoxicity, the NPs were non-toxic as all the cell viabilities were higher than 80% (Fig. S2 in Supporting information).
Four formulations of free DS, free DS with free copper gluconate (DS + Cu2+), DS loaded NPs with free copper gluconate (DS/NP + Cu2+) and DS loaded NPs with copper gluconate loaded NPs (DS/NP + Cu2+/NP) were used to study the in vitro anticancer activity (Fig. 2B). The formulations with different concentrations were incubated with mesothelioma cells to explore the values of half maximal inhibitory concentration (IC50), which were 16.0, 0.0436, 0.0217 and 0.00915 μg/mL calculated by Graph Pad Prism 5 (Fig. 2C). The results clearly demonstrated that only DS without Cu2+ would not kill mesothelioma cells efficiently (IC50 = 16.0 μg/mL) while the in situ formation of Cu(DDC)2 complex with DS and Cu2+ released from PLGA75/25 NPs exhibited absolutely the best in vitro anticancer activity as the IC50 was as low as 0.00915 μg/mL. These results exhibited the superiority of nanomedicine [25-27].
Different from the classic application of DS nanomedicine with oral Cu2+ administration [28], two nanomedicine groups of DS loaded NPs plus oral copper gluconate (DS/NP + Cu2+) and DS loaded NPs plus copper gluconate loaded NPs (DS/NP + Cu2+/NP) were utilized to investigate the in vivo anticancer activity. Two groups of saline and blank NPs were used as controls. The nanomedicines were administrated via intraperitoneal injection under regulations approved by the Laboratory Animal Center of Sichuan University. As mesothelioma was composed of nodules, the nodules volumes and numbers were used to evaluate the in vivo anticancer activity of the nanomedicine in mesotheliomabearing nude mice. The in vivo therapeutic effects of nanomedicines were showed in Fig. 3. As shown in Figs. 3A and B, the values of average volumes and numbers of DS/NP + Cu2+/NP group were 0.41 mm3 and 0.33, however, those average volumes and numbers of the other three groups were from 16 mm3 to 69 mm3 and 5.67–8.33, respectively. Both numbers and volumes of DS/NP + Cu2+/NP group were much lower than those of the other three groups. The mesothelioma-bearing nude mice were dissected to observe the tumors in the mice (Fig. 3C) and their amplified images were shown in Fig. S3 (Supporting information). There were many nodules observed in the peritoneum of saline and blank NP groups mice. Several nodules were found in the peritoneum of DS/NP + Cu2+ group, nearly no nodule was found in the peritoneum of DS/NP + Cu2+/NP group. All these results revealed that the therapeutic efficacy of DS/NP + Cu2+/NP formulation was excellent.
To most nanomedicines administrated by intravenous injection, the side effects of chemotherapeutics could be reduced greatly due to the utilization of carriers as well as the enhanced permeation and retention (EPR) effect. Body weight variation is an important parameter to show the toxicity of nanomedicine. The body weight variations of the nude mice administrated with the four formulations were presented in Fig. S4 (Supporting information). Different from other nanomedicines, the body weight variation of DS/NP + Cu2+/NP formulation was undulated around the initial weight, however, the weight variations of the other three formulations were directly increased with time elongated.
After the mice were sacrificed, the organs and tumor nodules were stained. The H&E staining images were showed in Fig. 4. For all groups, there was no obvious damage in the H&E staining images of heart, lung, liver, spleen and kidney, indicating the safety of this strategy for Cu(DDC)2 administration. Themesenteryimage of saline displayed that the mesothelioma cells formed papillary nodules which attached on the surface of mesentery. And the mesentery images of blank NPs and DS/NP + Cu2+ formulations showed the normal tissue architecture of mesentery while the mesentery in DS/NP + Cu2+/NP group showed papillary lesion. To the nodules images of saline, blank NPs and DS/NP + Cu2+ formulations, papillomatous growth of the mesothelioma cells were clearly observed, and partial cells were destroyed in the image of DS/NP + Cu2+ formulation. As the nodules in DS/NP + Cu2+/NP group were too rare and little to be separated, thus, no H&E staining image was received.The above H&E staining results revealed the anticancer efficacy of DS/NP + Cu2+/NP formulation was the best.
In other subcutaneous tumor models, the excellent therapeutic effect of nanomedicine would be consistent with good results in both body weight increase and none or weak toxicity observed in H&E staining images. To our research, it was very different. The relationship between body weight variation and therapeutic effects on nodule volumes and numbers as well as H&E staining images seemed contradictory. In fact, these results were not conflict. The mesothelioma tumor models were implanted in the peritoneum of mice, abundant abdominal dropsy was produced. We measured the abdominal dropsy in the four groups and the volumes were 3, 1.98, 6.38 and 0.2 mL to saline, blank NP, DS/NP + Cu2+ and DS/NP + Cu2+/NP groups. The abdominal dropsy gave great contribution to the body weight increase, thus, resulted in the contradictory appearance. The smallest volume of 0.2 mL abdominal dropsy to DS/NP + Cu2+/NP formulation was perfectly consistent with the results of nodules and H&E staining images.
In summary, a new strategy of nanomedicine application with in situ formation of Cu(DDC)2 complex as the chemotherapeutic was introduced. Two kinds of PLGA75/25 nanoparticles trapped with disulfiram and copper gluconate were administrated via intraperitoneal injection to the mesothelioma-bearing nude mice. The released disulfiram and Cu2+ were complexed as Cu(DDC)2 in peritoneum to resolve the encapsulation problem of Cu(DDC)2 precipitation and exert excellent chemotherapeutic function. The side effect of Cu(DDC)2 was greatly reduced and efficient in vivo anticancer activity was received. Therefore, this strategy could be potential for the application of disulfiram in chemotherapy.
Declaration of competing interest
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
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This work was supported by the National Natural Science Foundation of China (No. 51773130), Natural Science Foundation of Shandong Province (No. ZR2017MC072). The authors thank Prof. Weiguang Wang for providing mesothelioma cells.
Appendix A. Supplementary data
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Supplementary material related to this article canbefound, in the online version, at doi: https://doi.org/10.1016/j.cclet.2020.04.051.
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Year 2020 volume 31 Issue 12
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doi: 10.1016/j.cclet.2020.04.051
  • Receive Date：2020-03-24
  • Online Date：2026-01-31
  • Published：2020-12-15
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  • Received：2020-03-24
  • Revised：2020-04-24
  • Accepted：2020-04-27
Affiliations
    a National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
    b Army Logistics University of PLA, Chongqing 401331, China
    c College of Marine Life Science, Ocean University of China, Qingdao 266003, China
    d College of Chemistry and Materials Engineering, Wenzhou University, Wenzhou 325027, China
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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