Self-assembled metal-polyphenol nanomedicines for tumor chemotherapy/chemokinetic synergistic therapy

Chemodynamic therapy (CDT) converts endogenous H2O2 into highly toxic reactive oxygen species through the Fenton reaction or Fenton-like reaction. With the “one-pot” approach, the nanodrug EGCG-Cu-cisplatin NPs were created. The nanodrug has a particle size of approximately 150 nm and a homogeneous spherical shape. In vitro, results showed that the nanodrug degraded in a weakly acidic environment and released Cu2+ and cisplatin. Cisplatin raises intracellular H2O2 through a cascade reaction, Cu2+ consumes GSH while generating Cu+. Cu+ catalyzes H2O2 to generate •OH, which breaks the intracellular redox balance, induces ferroptosis, reduces tumor resistance, and promotes chemotherapy. CDT uses the Fenton /Fenton-like reaction to transform endogenous H2O2 into extremely hazardous reactive oxygen species.


Introduction
Cancer is a well-known main barrier to raising life expectancy in nations all over the world [1].Traditional tumor treatments, such as radiotherapy and chemotherapy, have a low quality of life due to severe side effects and are prone to drug resistance, resulting in tumor treatment failure.As a result, investigating simpler and more efficient treatment strategies has significant research and therapeutic significance [2].Recent years have seen the emergence of innovative cancer treatments, such as immunotherapy, chemodynamic therapy, and others [3][4] [5] [6].
The main characteristics of the TME are weak acidity [7] and high levels of hydrogen peroxide and GSH [8].As a result, creating smart nanomaterials with TME responsiveness is likely to benefit tumorspecific treatment.CDT is a Fenton/Fenton-like reaction that activates a tumor-specific microenvironment and has a logical response related to endogenous therapeutic method.It avoids the toxicity of typical drug carriers that persist in the body for extended periods of time and has therapeutic translation potential [9].The pH of the TME is about 6.5~7.0, while the Fenton-like reaction mediated by Cu + is more pH compatible.Copper ions combine with GSH to create cuprous ions, which accelerate the formation of hydroxyl radicals from H2O2.
NADPH oxidase, which encourages the oxidation of NADPH to NADP + , is particularly activated by cisplatin.The O2 molecule receives electrons at the same moment to create O 2•− , which is later decomposed by superoxide dismutase.CDT is improved by increased H2O2 production [10].
Polyphenols are increasingly being employed in the fabrication of nanoparticles [11][12].The tea polyphenol epigallocatechin gallate (EGCG) contains a vicinal di-or trihydroxy structure, which allows for metal ion chelation [13].Furthermore, the metal polyphenol network is pH-responsive and antitumor [14].In this study, EGCG was used to coordinate with Cu 2+ and cisplatin to form a network of metal polyphenols for chemotherapy/ chemodynamic therapy.

Synthesis and Characterization of EGCG-Cu NPs and EGCG-Cu-Pt NPs.
A previous paper, with some alterations, alluded to the synthesis process of EGCG-Cu nanoparticles [15].Micron-scale particles were created in the article.The nanoparticles were created after testing the experimental circumstances.In summary, the CuCl2 (1 mM) solutions were gently added to the EGCG (1 mM) solution.Cu 2+ to EGCG has a molar ratio of 1:2, and the pH was adjusted to 7 using NaHCO3 solutions (1 M), stirring overnight at room temperature.After that, the EGCG-Cu nanoparticles were centrifuged at 4000 rpm for 4 minutes and rinsed four times with ultrapure water.
The cisplatin solution (1 mg/mL) and the CuCl2 solution (1 mM) were added to the EGCG solutions while stirring in order to create the EGCG-Cu-Pt NPs.After that, they were disturbed in the dark for 24 hours [16].
The EGCG-Cu NPs had a diameter of about 70~100 nm, as illustrated in Figures 1 and 2, while the EGCG-Cu-Pt NPs had a diameter of around 50~120 nm.The sphere forms of EGCG-Cu and EGCG-Cu-Pt nanoparticles were visible in TEM images (Figures 3 and 4, respectively).According to Figure 5, EGCG joined forces with copper and cisplatin to form a complex.The maximum absorbance changed from 275nm to 320 nm, which indicated that metal ions had bound to EGCG.

GSH depletion of EGCG-Cu-Pt NPs.
GSH has high reducibility and high ROS scavenging ability.Glutathione can combine with DTNB to form a yellow product.At a wavelength of 412 nm, it possesses the greatest optical absorption.We IOP Publishing doi:10.1088/1742-6596/2671/1/0120194 2.2.1.GSH depletion at different time.500 μL system comprising 100 μL GSH, 390 μL PBS (pH=5.4), and 10 μL EGCG-Cu-Pt NPs (final concentration: 50 μg/mL) in a 1.5mL EP tube.Samples were incubated on a rotating plate at 37°C for 5, 10, 15, 20, 30, 40, 50, 60, 90, and 120 minutes before being centrifuged.Pour 20 μL of supernatant into each well.Add 140 μL of Assay Buffer to each well, followed by 40 μL of Chromogen to each well.Incubate at ambient temperature for 2 minutes, shielded from light.Measure the optical density of each well with a microplate reader at 412nm.. Figure 6 shows that when the concentration of EGCG-Cu-Pt NPs was 50 μg/mL, the consumption of GSH increased with time.At 1.5 hours, the GSH in the solution was exhausted.Figure 6.GSH consumption at different time points.

GSH depletion after 1.5 h reaction with different concentrations of EGCG-Cu-Pt NPs.EGCG-Cu-
Pt NPs at concentrations of 1.25, 2.25, 5, 10, 25, and 50 μg/mL, respectively.The samples were centrifuged after 1.5 hours on a rotating plate at 37°C.According to Figure 7, during the same reaction period, the consumption of GSH rose as the concentration of EGCG-Cu-Pt NPs increased.

•OH generation of EGCG-Cu-Pt NPs.
To begin, 10 μL of EGCG-Cu-Pt NPs (copper ion final concentration: 30 μg/mL) were distributed in 50 μL of GSH solution at various concentrations (GSH concentrations of 1 mM, 2 mM, 5 mM, 10 mM, and 20 mM).For 2 hours, incubated gently on a shaker at 37°C.After centrifuging the supernatant, 50 μL of it was mixed with 500 μL of TMB Color liquid and incubated for 1 hour.UV-visible spectroscopy was used to monitor the •OH-induced oxidation of TMB by measuring the change in absorbance between 500 and 800 nm.The EGCG-Cu-Cisplatin NPs were treated with 10 mM GSH for 3 hours before being examined under an electron microscope.Figure 11 depicts the results: After 3 hours of incubation with 10 mM GSH, EGCG-Cu-Cisplatin NPs showed deconjugation.Because of the conflict for coordination between -NH2 and -COOH and Cu 2+ in the GSH structure, the nanoparticles disassembled.

Conclusion
This thesis created EGCG-Cu-cisplatin NPs, a copper-based nanocomplex loaded with cisplatin.It was suggested that a combined chemotherapy/chemokinetic treatment plan be used.The nanodrug can deplete GSH while producing ROS, enhancing anti-tumor activity.The nanodrug offers a lot of potential in the realm of tumor therapy. .References

Figure 7 .
Figure 7.The ability of different concentrations of EGCG-Cu-Pt NPs to consume GSH.

Figure 8
indicated that when the concentration of GSH increased, the quantity of •OH increased first and subsequently decreased.

2. 4 .
In Vitro Cytotoxicity Test.Cell Counting Kit-8 was used to assess the capacity of EGCG-Cu-Pt NPs to destroy cancer cells.A549 and MCF-7 cells (5×10 4 cells) were planted into 96-well plates and cultivated for 36 hours.100 μL of DMEM solution (0, 10, 20, 50, and 100 μg/mL) containing cisplatin, EGCG-Cu NPs, and EGCG-Cu-Pt NPs was added and incubated for 12 hours.All experiments were done three times.Each well received 10 μL of cck-8 solution, which was incubated for 2 hours.A microplate reader was used to read the absorbance values at 450 nm.when illustrated in Figures9 and 10, there is a clear trend of decreased cell viability rate when concentration of EGCG-Cu-Pt NPs increases.