Synthesis and characterization of EGCG&Cys(DOX) novel chemotherapeutic drug

As shown in Fig. 1a and b, EGCG, L-cysteine (Cys), and formaldehyde (HCHO) were rapidly polymerized in an aqueous phase reaction system to form highly active phenolic EGCG&Cys nanoformulation. Previous literature reports have demonstrated that the formation of nanoparticles through classical phenolic condensation is challenging. Therefore, the Mannich reaction is dominant in forming EGCG&Cys nanoformulation [26]. To further synthesize EGCG&Cys nanoformulation with controllable particle size and stable physical and chemical properties, we investigated the effect mechanisms of the synthesis parameters (concentration of reactants, EGCG/Cys mass ratios, formaldehyde content, and ethanol content) on the reaction process and the particle size distribution of the products. In the presence of Cys, the reaction system gradually changed from a transparent solution to a milky-white suspension with a reaction time increase for different reaction conditions (Fig. S1). One-way experiments showed significant correlations between the four synthesis parameters and the average particle size, dispersibility, and stability of EGCG&Cys nanoformulation (Table S1, Fig. S2).

Fig. 1

Synthesis and characterization of EGCG&Cys and EGCG&Cys(DOX) nanoparticles. a Schematic illustration of the preparation process of EGCG&Cys; b Schematic illustration of the preparation process of EGCG&Cys(DOX), and the photos of the EGCG solution, EGCG&Cys, and EGCG&Cys(DOX) dispersion; c Reaction kinetics curves of EGCG&Cys nanoformulation synthesized under different reactant mass concentrations; d, e TEM image and size distribution of EGCG&Cys nanoformulation; f Ultraviolet–visible-infrared absorption spectra of EGCG, Cys, and EGCG&Cys nanoformulation obtained under different reactant mass concentrations; g, h DLS and zeta potential of EGCG&Cys and EGCG&Cys(DOX); i The DOX loading efficiency and capacity of EGCG&Cys(DOX) obtained under different DOX/EGCG mass ratio reaction conditions; j Fluorescence spectra of EGCG&Cys and EGCG&Cys(DOX) of various DOX loading capacity; k FT-IR spectra of EGCG, Cys, EGCG&Cys, DOX, and EGCG&Cys(DOX); l ABTS and (m) DPPH radical scavenging activity of EGCG&Cys and EGCG&Cys(DOX)

Taking the reaction system with different reactants (EGCG and Cys) concentrations as an example, Fig. 1c shows the relationship curve between the turbidity of the reaction solution and the reaction time during the reaction process, used to analyze the reaction kinetics characteristics of the reaction solution for the generation of ECGC&Cys nanoparticles with different particle sizes. The kinetic curve measurements showed that the higher the concentration of reactants in the reaction system, the larger the average particle size and dynamic light scattering DLS of the synthesized nanoparticles (Fig. S5 a-i). To further investigate the relationship between the formation process of nanoparticles and the reaction parameters, we used a mathematical model of chemical reactions, the Hill function, to fit the reaction kinetic curves to obtain the Hill coefficients, the reaction onset time, the maximum reaction rate, the time for the reaction to reach half of the equilibrium state, and the absorbance values of the resulting nanoparticle suspensions when the reaction reached the equilibrium state (Fig. 1c). Wherein, the Hill coefficient (n) has the following three situations: n = 1, reactant concentration, proportion, ethanol addition amount, formaldehyde addition amount, and reaction start time have no cooperation; n > 1, have a positive, cooperative relationship; n < 1, indicating a negative partnership. The reaction onset time was 14 min when the reactant concentration was CEGCG = 0.02 M and CCys = 0.008 M. In contrast, after reducing the concentration of the reactants by half (CEGCG = 0.01 M and CCys = 0.004 M), the reaction onset time was extended to 34 min. The correlation results showed that the concentration of the reactants was negatively correlated with the time required for the formation of nanoparticles (Supplementary Table S2). The reason is that when the concentration of the reactants is higher, there are more reactant molecules per unit volume, resulting in a higher probability of reactant molecule collisions, thereby increasing the reaction rate and shortening the time for the reaction to reach equilibrium.

Furthermore, it was also found that in addition to varying the concentration, different choices of EGCG/Cys mass ratio, formaldehyde, and ethanol content were effective ways to regulate the reaction rate and the particle size of polyphenol nanoparticles (Fig. S9). Reducing the feed ratio of Cys and the amount of formaldehyde added in the reaction system respectively played a role in reducing the number of nucleophilic and electrophilic groups in the reaction process, thereby reducing the particle size of the generated EGCG&Cys nanoformulation. In addition, adding an appropriate ratio of ethanol in the reaction system also reduces the particle size of EGCG&Cys nanoformulation (Fig. S6, S7). When the ratio of ethanol is too high, EGCG&Cys nanoformulation is not easily generated. The main reasons include two aspects: (1) ethanol's polarity is lower than water’s. The polarity of the solvent has a more significant impact on the Mannich reaction; the lower the polarity, the lower the reaction recovery rate. (2) The solubility of EGCG in water is more excellent than in ethanol. Increasing the proportion of ethanol will reduce the concentration of EGCG in the whole reaction system, thereby reducing the mass transfer of the reaction and affecting the generation rate and recovery rate of EGCG nanoparticles (Fig. S8). More importantly, the fitting results of reaction kinetics curves under different reaction conditions demonstrated that the Hill coefficients are all greater than 1, indicating a positive synergistic relationship between the formation process of synthesized EGCG&Cys nanoformulation and the four synthesis parameters (Supplementary Table S2).

The zeta potential of the synthesized EGCG&Cys nanoformulation decreased from −25.8 mV to −45.3 mV as the concentration of the reactants decreased, indicating that the nanoformulation would be better dispersed in pure water, PBS, or culture solution (Fig. S5 j). In addition, the EGCG&Cys nanoformulation exhibited excellent ABTS and DPPH radical scavenging ability, and Fig. S5 k shows that there is no difference in the ABTS radical clearing performance of the synthesized in EGCG&Cys with different particle sizes at different reactant concentrations. Fig. S5 l shows that EGCG&Cys synthesized at a high reactant concentration with a particle size of 584.2 nm has a weak DPPH radical scavenging ability. In contrast, the other three small-sized nanoparticles have nearly the same DPPH radical scavenging ability.

In drug delivery research, it is generally clear that using small-sized nanoparticles as carriers for intravenous drug delivery is a promising approach [27, 28]. Small particle-size drug carriers are more conducive to the enrichment of drugs in tumor cells and improve treatment through the enhanced permeation and retention (EPR) effect [29]. Therefore, we selected the synthesis parameters of EGCG&Cys nanoformulation with small particle size, low polydispersity index (PDI) value, and potent antioxidant properties as the optimal reaction conditions, which are named A-2 and B-3 in Table S1, for synthesizing nanoparticles to facilitate subsequent antitumor therapeutic experiments at the level of the tumor cells and tumor-bearing mice and achieve excellent good antitumor efficacy.

Transmission electron microscopy images show that the synthesized EGCG&Cys nanoformulation is spherical and has a smooth surface. By measuring 50 NPs, the average particle size was calculated to be 101.7 ± 22.9 nm (Fig. 1d, e). The characteristic absorbance peaks of Cys (215 nm) and EGCG (273 nm) can be observed from the UV–visible absorption spectra in Fig. 1f, indicating the successful preparation of EGCG&Cys nanoformulation. The slight red shift of the EGCG characteristic absorption peak may be due to the aggregation of π-π bonds of the nanoformulation during polyphenol assembly [30]. A schematic diagram of EGCG&Cys(DOX) nanoformulation synthesis process is shown in Fig. 1b. The average DLS diameters of EGCG&Cys and EGCG&Cys(DOX) (CDOX: CEGCG&Cys = 1:8, 1:4, 1:2) are approximately 220 ± 8.76 nm, 255 ± 15.9 nm, 327 ± 3.75 nm, 295 ± 4.74 nm respectively (Fig. 1g). The PDI coefficients are about 0.092, 0.096, 0.113, and 0.136. The zeta potentials are around -27.6 ± 0.5 mV, −20.6 ± 1.2 mV, −27.8 ± 0.57 mV, and −22.4 ± 0.64 mV (Fig. 1h). The characterization results of DLS and zeta potentials indicate that the EGCG&Cys nanoformulation still exhibit good stability after loading DOX on the surface. Figure 1i shows that the highest DOX load efficiency is 100%, and the load capacity increases from 11.98% to 33.12% under different CDOX / CEGCG&Cys mass concentration ratios. In addition, Fig. 1j shows that the DOX fluorescence intensity of EGCG&Cys(DOX) produced by 488 nm laser excitation is precisely consistent with the upward trend of DOX loading capacity in the sample. As shown in Fig. 1k, in the FTIR spectra of EGCG, the C-H out-of-plane bending vibration absorption peak appears between 739 and 623 cm−1. After EGCG&Cys nanoformulation was formed, all these bands disappeared due to the EGCG molecule's self-assembly. The 1690 cm−1 band is a unique peak of EGCG carbon-based stretching, which is commonly used for qualitative analysis but is also relatively weak in polycondensation polyphenols. The absorption of DOX at 1730 cm−1 is attributed to the stretching of the carboxyl group. Additionally, this characteristic absorption peak of DOX still appears in the FTIR spectrum of EGCG&Cys(DOX). These results indicate the successful preparation of EGCG&Cys(DOX) nanoformulation.

In vitro drug release and stability

We detected the DOX release curves in EGCG&Cys(DOX) under pH values of 7.4, 6.5, and 5.0. The experimental results showed that in a buffer solution with a pH value of 7.4, the release rate of DOX was less than 14.20% within 48 h. Under acidic conditions with a pH of 5.0, the release rate of DOX is as high as 65.39% (Fig. S10). DOX is released much more rapidly within tumor or endolysosomes (pH = 5) than under physiological conditions (pH = 7.4). This experimental result indicates that EGCG&Cys(DOX) has a low DOX leakage rate under physiological pH conditions, which is beneficial for alleviating the toxic side effects of DOX. Additionally, according to the experimental results in Fig. S11, it can be seen that the dispersion and morphology of the drug carrier EGCG&Cys nanoparticles did not change after being dispersed under neutral and acidic conditions for different times, indicating that EGCG&Cys has good stability in environments with various pH values.

In vitro multi-catalytic activity

The phenolic hydroxyl groups on the surface of EGCG&Cys nanoformulation can contribute to hydrogen protons reacting with free radicals and playing a role in scavenging them. To verify this, we further evaluated the scavenging capacity of EGCG-based nanoparticles on 1,1-diphenyl-2-pyridazine (DPPH) and 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic Acid Ammonium Salt) (ABTS) radical. It was found that with the increase of EGCG&Cys nanoformulation concentration, its DPPH and ABTS free radical scavenging ability was significantly enhanced. When the concentration of EGCG&Cys was 6.7 μg/mL, the ABTS clearance rate could be as high as 98.3%, and when the concentration of EGCG&Cys was 16 μg/mL, the DPPH clearance rate reached 72.5%, indicating that EGCG&Cys was an excellent antioxidant (Fig. 1l, m). In addition, loading DOX onto EGCG&Cys nanoformulation will consume a portion of the phenolic hydroxyl groups on the surface, which leads to a decrease in antioxidant performance. At the same concentration, the scavenging rate of DPPH and ABTS free radicals of EGCG&Cys(DOX) was 41.5% and 43.9%, respectively (Fig. 1l, m). These experimental results indicate that EGCG&Cys(DOX) still has good free radical scavenging performance. EGCG&Cys, EGCG&Cys(DOX), and DOX are determined by a standard curve of their mass concentrations versus the corresponding absorbance values at 488 nm (Fig. S3, S4).

In vitro antitumor efficacy assessment

The anthracycline chemotherapy drug DOX has an excellent killing effect on tumor cells, but it has also been shown to damage myocardial cells, leading to fatal cardiotoxicity in chemotherapy patients. To investigate the anti-tumor efficacy and myocardial cell toxicity of EGCG&Cys(DOX) nanoformulation, we used 4T1 murine mammary cancer cells and HL-1 mouse cardiomyocytes for in vitro efficacy evaluation and myocardial toxicity assessment, respectively. The EGCG&Cys(DOX), the equivalent concentration of DOX, and the EGCG&Cys groups were incubated with 4T1 cells for 24 h, within the concentration range of 0 ~ 27.6 μg/mL, the lowest cell viability of the EGCG&Cys group remained above 93%, indicating that low-concentration EGCG&Cys nanoformulation had almost no inhibitory effect on 4T1 cells. In contrast, the viability of 4T1 cells was 51.3 ± 1.16% and 45.4 ± 2.33% in the EGCG&Cys(DOX) and DOX groups at DOX concentrations of 6.2 μg/mL (Fig. 2a). The measured IC50 value of EGCG&Cys(DOX) was 5.1 μg/mL. EGCG &Cys (DOX) has a similar inhibitory effect on 4T1 breast cancer cells compared to DOX (IC50 = 3.56 μg/mL). On top of that, flow cytometry results showed that when the concentration of DOX was 1.55 μg/mL, the apoptosis rates of 4T1 cells induced by EGCG&Cys(DOX) and DOX were 74.03% and 71.3%, respectively (Fig. 2b, S13a). The results of 4T1 cell survival rates and apoptosis rates showed that at the same DOX concentration, EGCG&Cys(DOX) had the same damage effect on breast cancer cells as DOX, which indicates that modifying the chemotherapy drug formulation by loading DOX onto EGCG&Cys nanoformulation surface did not weaken the efficacy of chemotherapy.

Fig. 2

Cytotoxicity and in vitro antitumor efficacy. a, c Cytotoxicity of EGCG&Cys, EGCG&Cys(DOX), and DOX to 4T1 cell and HL-1 cell at different equivalent concentrations; b, d Annexin V-FITC/7-AAD dual dye staining flow cytometry was used to analyze the apoptosis rate of 4T1 and HL-1 cells after co-incubation with DOX or EGCG&Cys(DOX) for 24 h (EL: EGCG&Cys(DOX) 2.5 μg/mL, EH: EGCG&Cys(DOX) 5.0 μg/mL, DL: DOX 0.775 μg/mL, DH: DOX 1.55 μg/mL); e, f Cellular uptake by confocal microscopy images analysis of 4T1 and HL-1 cells coincubated with different concentrations of DOX and EGCG&Cys(DOX) for 4 h; g The protein expression levels of Bax, Bcl-2 and caspase 3 in 4T1 cells was determined by Western blot (DL: DOX 1.96 μg/mL, EDL: EGCG&Cys(DOX) 5.92 μg/mL, EGCG&Cys 7.92 μg/mL, DH: DOX 3.92 μg/mL); h Protein expression of caspase3 compared to the control group; i Protein expression of Bax/Bcl2

Excitingly, we incubated HL-1 cells with three drugs at the same concentration for 24 h. The results in Fig. 2c showed that when the DOX concentration reached 6.2 μg/mL, the cell viability of HL-1 cells was only 25.5 ± 1.96%, while in the EGCG&Cys(DOX) group could be as high as 78 ± 1.79%. Furthermore, the IC50 of EGCG&Cys(DOX) is 16.23 μg/mL. EGCG&Cys(DOX) nanoformulation has superior protection against HL-1 cells compared to DOX (IC50 = 3.55 μg/mL). What’s more, flow cytometry results showed that the EGCG&Cys(DOX) group (EL: 26.28%, EH: 29.57%) reduced the rate of chemotherapy-induced apoptosis of HL-1 cells compared to the DOX group (DL: 33.93%, DH: 34.4%) (Fig. 2d, S13b). Therefore, these experimental results significantly indicate that EGCG&Cys(DOX) can effectively protect HL-1 myocardial cells from damage induced by DOX.

The cell uptake results showed that the amount of drug phagocytosis by 4T1 and HL-1 cells towards EGCG&Cys(DOX) and DOX increased with the prolongation of co-incubation time (Fig. 2e, f; Fig. S12 a, b). A very advantageous point is that within the same co-incubation time, 4T1 cells have a similar uptake of EGCG&Cys(DOX) and DOX, while HL-1 has a significantly lower EGCG&Cys(DOX) uptake than DOX. In addition, the red fluorescence intensity of DOX in the confocal images representing cell phagocytosis was statistically analyzed, as shown in Fig. S13 c-f, which also well illustrated the difference in phagocytosis of the two drugs by 4T1 and HL-1 cells. We can be inferred that the large amount of 4T1 cells engulfing EGCG&Cys(DOX) is due to the 67 kDa laminin receptor (67LR) overexpression level on the membrane of 4T1 cells is much higher than that of HL-1 cells. Meanwhile, EGCG is the target ligand for the 67LR, and there is a robust affinity between the two kinds of cells [31,32,33]. These results sufficiently demonstrate that EGCG&Cys(DOX) has excellent targeting ability towards 4T1 cells and further affects the phagocytic behavior of different cells. Meanwhile, Fig. S13 g and h show that both EGCG&Cys(DOX) and DOX exhibit excellent nuclear-targeting performance, as EGCG&Cys(DOX) drug-loaded nanoformulation enters tumor cells through endocytosis and then release DOX to achieve nuclear targeting. This process can avoid p-glycoprotein efflux [34] while ensuring the nuclear-targeting properties of DOX. These results indicate that EGCG&Cys can significantly reduce the DOX-induced damage to HL-1 cells without weakening the killing effect of DOX on 4T1 cells when EGCG&Cys is used to deliver the DOX.

Moreover, after co-incubation of different concentrations of EGCG&Cys with 4T1 cells for 24 h, the viability of 4T1 cells was only 29.78% when the EGCG&Cys concentration was 400 μg/mL, indicating that EGCG&Cys has specific tumor cells suppressive effect (Fig. S14). To clarify the mechanism by which EGCG&Cys inhibits tumor cells, we further explored the regulatory impact of EGCG&Cys on the expression of proteins related to the apoptosis signaling pathway, such as Bax, Bcl-2, and caspase3 proteins. The Western blot results showed that the expressions of Bax and caspase3 in the DOX group were higher than those in the control group. Like the DOX group, the Bax/Bcl2 ratio and caspase3 protein expression levels also increased in the EGCG and EGCG&Cys(DOX) groups (Fig. 2 g-i). Therefore, the protein expression results demonstrate that EGCG&Cys induces apoptosis in 4T1 cells by upregulating caspase 3 and Bax proteins and downregulating Bcl-2 protein expression, followed by Bax/Bcl2 and caspase pathways.

In vivo antitumor efficacy assessment

Balb/c mice bearing breast cancer were injected with chemotherapy drugs 7 times in 14 days to investigate the anti-tumor efficacy of EGCG&Cys(DOX) nanoformulation in vivo (Fig. 3a). The results showed that the low-dose DOX (2.5 mg/kg), EGCG&Cys (10.16 mg/kg), EGCG&Cys(DOX) (7.58 mg/kg, Loading capacity of DOX is 33.1%) and high-dose DOX (5 mg/kg) groups had inhibitory effects on subcutaneous tumors in mice (Fig. 3b). In addition, under the same DOX concentration, the EGCG&Cys(DOX) group showed better anti-tumor effects compared to the low-dose DOX concentration group. On the 14th day of treatment, the tumor growth inhibition index (TGI) of these two treatment groups of mice was 64.21% and 51.65%, respectively. We consider that there are two main reasons why EGCG&Cys(DOX) has a better chemotherapy effect: firstly, EGCG&Cys(DOX) has a DOX slow and sustained-release effect, which can maintain a higher DOX concentration in mouse blood for an extended time, resulting in more DOX enriched in the tumor site. Secondly, EGCG&Cys nanoformulation as a drug carrier also kills tumor cells and has a synergistic antitumor effect with DOX. The tumor growth curve, extracted tumor photos, tumor weight, and TGI (24.05%) can all effectively demonstrate the antitumor efficacy of EGCG&Cys (10.16 mg/kg) (Fig. 3b, e, g, Fig. S15 a). It is worth noting that the TGI of the high-dose DOX group was as high as 88.34%, which was significantly higher than that of the other three experimental groups. Moreover, the tumor weight, TGI curves, photographs of ex vivo tumors, and 4T1 tumor-bearing mouse images (days 0, 8, and 14) after different treatment outcomes all showed different therapeutic efficacy, which was consistent with tumor growth curves of each group of mice (Fig. 3b, c, e, g, Fig. S15 a).

Fig. 3

In vivo chemotherapy efficacy of EGCG&Cys(DOX). a Schematic illustration of in vivo anti-tumor chemotherapy experiment process; b Tumor growth curves within 14 days for different treatment groups; c Photos of 4T1 tumor-bearing mice in different treatment groups on days 0, 8, and 14; d Photos of the main organs dissected from 4T1 tumor-bearing mice after euthanasia on the 14th day; e, g Photographs and weights of tumors excised from mice after sacrifice; f, h Body weight change curves of mice during the 14-day chemotherapy process and organ coefficients of the principal organs of mice after chemotherapy; i-k H&E, DAPI, TUNEL, and Ki67 staining images of mice tumor tissue sections at 14 days of chemotherapy with different drugs

Besides, while achieving excellent antitumor efficacy with high-dose DOX chemotherapy, the systemic toxic side effects it produces are also an unavoidable and vital issue. Previous studies have shown that splenic atrophy is caused by DOX-induced damage to the CD169+ macrophage population in the splenic marginal zone [35]. After receiving DOX chemotherapy, the organ coefficient of the spleen in tumor-bearing mice significantly decreased. The spleen coefficient decreased by 59.5% and 85.5% in the 2.5 mg/kg and 5 mg/kg DOX chemotherapy groups, respectively (Fig. 3d, h). However, the spleen coefficient of mice in the EGCG&Cys(DOX) treatment group decreased by only 24.0%, significantly lower than that of the DOX chemotherapy group. Besides, during the continuous chemotherapy process, the body weight of the EGCG&Cys(DOX) group mice was consistent with that of the control group. However, the body weight of mice in the low-dose and high-dose DOX groups decreased by 14.5% and 32.4%, respectively, indicating that DOX has severe systemic toxicity to mice (Fig. 3f).

Each experimental group's hematological and serum biochemical indexes showed significantly reduced white blood cells (WBC) and platelets (PLT). The reason is that DOX cannot specifically kill tumor cells, resulting in severe damage to normal cells, especially the proliferating bone marrow hematopoietic cells, during chemotherapy, decreasing white blood cells. Furthermore, DOX inhibits bone marrow megakaryocyte cells, resulting in insufficient platelet production and excessive destruction, and platelet counts are below average in the peripheral blood. Additionally, the AST index of mice in the low and high-concentration DOX chemotherapy groups increased dramatically, which were 1.23 and 1.10 times higher than those in the EGCG&Cys(DOX) group, respectively, indicating that DOX has significant toxicity to the liver. All the hematological and serum biochemistry parameters of the EGCG&Cys(DOX) group were consistent with those of the control group (Fig. S15 b, c), fully demonstrating that EGCG&Cys nanoformulation can effectively alleviate the systemic toxicity of DOX.

At the histopathological level, the therapeutic effect of the EGCG&Cys(DOX) nanoformulation on breast cancer was further evaluated by H&E, TUNEL, and Ki67 staining tumor tissue sections. As shown in Fig. 3i, the H&E staining results showed that the tumor cells in the regular saline group were morphologically intact, with no nuclear destruction and cell necrosis. However, the 4T1 cell morphology of the four treatment groups all changed, such as severe nuclei atrophies, cell destruction, and necrosis in the EGCG&Cys(DOX) and high-dose DOX groups.

Moreover, no significant differences were observed in H&E tissue sections of other vital organs (liver, lung, and kidney) among the treatment groups of mice (Fig. S16). The Fig. S17 indicated that the spleen structure was relatively intact. In the saline, EGCG&Cys, and low-dose DOX groups, the medullary areas of the spleens were enlarged, while the cortical areas were significantly dilated. Conversely, in these groups, the cortical area of the spleen was reduced, and the structure of the lymph nodules was unclear. In contrast, the spleens of the EGCG&Cys(DOX) group showed improvements compared to the groups mentioned above. Specifically, the medullary area was slightly reduced, the cortical area was somewhat increased, and the lymph nodes were more distinct. In the high-dose DOX group, there was a significant improvement in the spleen's medullary and cortical areas, and the lymph nodes were clearly delineated (Fig. S17).

In addition, to assess the apoptosis ability of EGCG&Cys(DOX) on 4T1 cells, we performed Tunel staining on 4T1 cells, and the results showed that the EGCG&Cys(DOX) group had a significantly higher number of positive cells (green fluorescent cells) compared to the equivalent concentration of DOX group (Fig. 3j). In addition, the results of ki67 showed that the antiproliferative activity of the EGCG&Cys(DOX) group was significantly higher than that of the equivalent concentration DOX group (Fig. 3k, S18). The results of TUNEL staining and Ki67 further verified that the EGCG&Cys carrier has the advantages of promoting tumor cell apoptosis and inhibiting tumor cell proliferation. The above experimental results demonstrated that EGCG&Cys carrier significantly enhances the chemotherapy efficacy of DOX and reduces the DOX-induced organ damage and systemic toxicity, which has considerable clinical application prospects in developing breast cancer treatment.

Alleviate DOX-induced myocardial toxicity

To evaluate the role of EGCG&Cys nanoformulation in alleviating DOX-induced myocardial injury, we first investigated the scavenging ability of EGCG&Cys(DOX) to ROS in cardiomyocytes by DCFH-DA fluorescent dyes. Figure 4a shows that as the concentration of DOX increases, the green fluorescence intensity also increases. When the DOX concentration is 0.31 μg/mL, DCFH exhibited intense green fluorescence, indicating that DOX-induced HL-1 cells generated a large amount of ROS. In contrast, EGCG&Cys(DOX) nanoformulations with concentrations of 0.5 and 1.0 μg/mL showed weak green fluorescence. These results demonstrated that DOX can induce severe oxidative stress imbalance in HL-1 cells and produce much ROS, causing severe damage to cardiomyocytes. However, EGCG&Cys plays a crucial role in alleviating oxidative stress damage in myocardial cells by eliminating ROS induced by DOX.

Fig. 4

Evaluation of EGCG&Cys nanoformulation in vitro for alleviating DOX-induced myocardial toxicity. a DCF fluorescence images for detecting ROS produced by HL-1 cells after 4 h incubation with DOX or EGCG&Cys(DOX); b JC-1 fluorescence images for detecting mitochondrial membrane potential of HL-1 cells after 24 h incubation with DOX or EGCG&Cys (DOX); c The fluorescence intensity of JC-1 aggregates/JC-1 monomers ratio of various chemotherapy groups (EL: EGCG&Cys(DOX) 0.5 μg/mL, EH: EGCG&Cys(DOX) 1.0 μg/mL, DL: DOX 0.155 μg/mL, DH: DOX 0.31 μg/mL

Mitochondria are the prominent organelles for intracellular ROS production and the main target organs for ROS attack and damage. Therefore, the imbalance of cellular oxidative stress caused by DOX directly affects the function of mitochondria. Changes in mitochondrial membrane potential are critical indicators to evaluate cells' health and functional status [36]. The red fluorescence intensity of JC-1 aggregates and the green fluorescence intensity of JC-1 monomers were used to judge changes in mitochondrial membrane potential. The transition from JC-1 aggregates to monomers indicates that the mitochondrial membrane potential of the cell decreases, and the cell is in an early apoptotic state. It is evident in Fig. 4b that the intensity of green fluorescence gradually increases as the concentration of DOX increases, and when the DOX is 0.31 μg/mL, the intensity of green fluorescence is consistent with that of the positive control group. By comparison, 1.0 μg/mL of EGCG&Cys(DOX) had stronger red fluorescence, and the intensity of green fluorescence was also significantly lower than that of the DOX group. In addition, the red/green fluorescence intensity ratio in the DOX group was substantially lower than that in the negative control group, indicating that the mitochondria in the DOX group underwent a depolarization process. In contrast, the mitochondrial membrane potential and degree of depolarization after treatment in the EGCG&Cys(DOX) group were consistent with those of the negative control cells (Fig. 4c).

The efficacy of EGCG&Cys(DOX) on alleviating DOX damage cardiac function of tumor-bearing mice before and after chemotherapy was further evaluated by ultrasound echocardiography (UCG) and electrocardiogram (ECG) (days 0 and 14). The results of pre-treatment UCG (Fig. S19 a-e) and post-treatment UCG (Fig. 5a-e) showed a significant decrease of 54.4% and 63.4% in the mean LVEF and LEFS values in the 5 mg/kg DOX treatment group compared to the non-chemotherapy group. These two crucial indicators suggest that DOX chemotherapy caused severe dilated cardiomyopathy, leading to a decrease in myocardial contractility and cardiac blood pumping function. ECG results further elucidated that the mean heart rate of the non-chemotherapy group mice was 434 BPMI, and the QT interval was 137 ms. However, the two indicators of the 5 mg/kg DOX treatment group mice were 261 BPMI and 166 ms, respectively. Significantly, we found that the LVEF of 80.26%, LVFS of 47.3%, mean heart rate of 432 BPMI, and QT interval of 149 ms in the EGCG&Cys (DOX) treated group was essentially the same as that of the untreated group (LVEF: 84.5%, LVFS: 49.7%, mean heart rate 434 BPMI, QT interval 137 ms) was essentially the same and there were no abnormalities (Fig. 5f-j, S19f-j).

Fig. 5

In vivo echocardiography and electrocardiogram evaluation. a Echocardiographic images of mice in different groups the day before the end of chemotherapy; b-e The values of LVEF, LVFS, LVIDd, and LVIDs were detected by echocardiography in various groups of mice; f Electrocardiogram of mice in different groups; g-j The results of heart rate, QRS, QT, and ST intervals of different chemotherapy groups were calculated based on electrocardiogram data. k H&E and Masson trichrome staining images of mouse heart tissue after 14 days of chemotherapy with various drugs

At the end of 14 days of chemotherapy, mice were euthanized, and cardiac tissue from each group was collected for further pathological analysis. As shown in Fig. 5k, the H&E staining results showed that some cardiomyocytes in the DOX group had lost nuclei and had vacuolar changes. In contrast, the EGCG&Cys(DOX) group had a neat myocardial arrangement, rich and uniform cytoplasm, normal interstitium, and consistent cell morphology with the control group. By Masson staining, we observed that two DOX-treated groups with different concentrations were stained blue due to the disappearance of cardiomyocytes and their replacement by fibro-collagen deposition. In particular, large areas of myocardial fibrosis appeared in the 5 mg/kg DOX group. However, in the EGCG&Cys(DOX) group of mice, only a tiny amount of heart tissue was stained blue, showing mild tissue fibrosis.

Additionally, we assayed four typical plasma cardiac function biomarkers (AST, CK, CKMB, and LDH). We observed that EGCG&Cys can effectively reduce the increase of AST, CK, CKMB, and LDH indicators in the blood caused by DOX. For example, the CK value was 825 U/L in the control group and 1218 U/L in the DOX 2.5 mg/kg group, while the CK value was 770 U/L in the EGCG&Cys (DOX) group at the equivalent concentration (Fig. S15 c). In conclusion, EGCG&Cys(DOX) can effectively alleviate DOX-induced myocardial injury both in vitro and in vivo. As a result, using EGCG&Cys as a carrier to load DOX can effectively mitigate the severe toxic side effects of DOX on normal tissues while enhancing the chemotherapy efficacy of DOX, providing a safer and more effective novel chemotherapy drug for oncotherapy.

Alleviate myocardial injury mechanisms of EGCG&Cys nanoformulation

To further elucidate the mechanism of EGCG&Cys(DOX) nanoformulation in attenuating myocardial injury, we evaluated mitochondrial function by analyzing mitochondrial morphology in bio-electron microscopy images of HL-1 cardiomyocytes and detecting the activity of mitochondrial complex I/NADH-CoQ and the release concentration of ATP in HL-1 cardiomyocytes. Transmission electron microscopy (TEM) results showed that the mitochondria of the untreated group were oval, containing double-membrane-bound vesicles with an internal crest. After the addition of DOX, the mitochondria contract and become rounded, the average length decreases, the inner crest is missing, and the mitochondria are damaged. However, the EGCG&Cys (DOX) group supplemented with equivalent concentrations showed improvements in mitochondrial morphology and average length compared to the DOX group (Fig. 6a). The results of Fig. 6b show that the activity of the mitochondrial complex I/NADH-CoQ in the EGCG&Cys(DOX) group is 9.4 U per 106 cells. However, the activity of the equivalent concentration of DOX is 4.4 U per 106 cells, which is significantly lower than that in the EGCG&Cys(DOX) group. In addition, DOX treatment can decrease intracellular ATP synthesis, whereas EGCG&Cys(DOX) treatment can block DOX-induced reduction in intracellular ATP content (Fig. 6c, S20). The Keap1-Nrf2/HO-1 signaling pathway is considered one of the most critical endogenous antioxidant stress pathways and an important therapeutic target for oxidative stress-related disorders. In Fig. 6d, the results of Western blot experiments showed that the expression of Keap1 in the DOX group decreased compared with the control group. In contrast, the expression of Keap1 in the EGCG and EGCG&Cys(DOX) groups increased, and the expressions of Nrf2 and HO-1 were also increased compared with the DOX group (Fig. 6e-g). The results demonstrated that EGCG&Cys nanoformulation inhibited DOX-induced oxidative stress imbalance by activating the Nrf2/HO-1 signaling pathway, thereby reducing the damaging effect of DOX on HL-1 cells.

Fig. 6

Exploration of the mechanisms of EGCG&Cys nanoformulation in cardioprotective effect in vitro. a Characterization images of mitochondrial morphology under electron transmission microscopy after co-incubation of HL-1 cells with DOX or EGCG&Cys(DOX) for 24 h; b Mitochondrial complex I/NADH-CoQ enzyme activity of HL-1 cells after 12 h of incubation with DOX or EGCG&Cys(DOX). (DL: DOX 1.96 μg/mL, EDL: EGCG&Cys(DOX) 5.92 μg/mL, EGCG&Cys 7.92 μg/mL, DH: DOX 3.92 μg/mL); c ATP concentrations released into the culture medium by HL-1 cells after 12 h incubation with DOX or EGCG&Cys(DOX); d The protein expression levels of HO-1, Keap1, and Nrf2 in HL-1 cells were determined by Western blot. e–g Keap1, Nrf2, and HO-1 protein expressions were compared with those in the control group

Acute toxicity of EGCG&Cys(DOX)

Figure 7a shows that the body weight of mice treated with different doses of EGCG&Cys(DOX) did not decrease but showed a steady increase trend. Moreover, during the acute toxicity evaluation period, the mice had no behavioral abnormalities, reduced food intake, or deaths observed. There was no difference in the organ coefficients and the images of main organs between the different groups compared with the control group (Fig. 7b, c). Similarly, the blood routine and serum biochemical parameters of the three groups injected with varying EGCG&Cys(DOX) doses were also within the normal range (Fig. 7d, e). Moreover, the H&E staining results (Fig. 7f) showed no abnormal changes or pathological damage in major organs and tissues. According to literature reports, the liver and spleen are part of the mononuclear phagocytic cell system, and intravenous delivery of nanomedicines is prone to accumulate in the liver or spleen, thereby sharply reducing the proportion of drugs entering the lesion. However, no significant aggregation of nanomedicines, cell edema, inflammatory cell infiltration, or necrosis was observed in the liver and spleen sections. These results confirm the outstanding biosafety of EGCG&Cys(DOX) nanoformulation, which can achieve effective chemotherapy through intravenous injection at 100 mg/kg and below doses.

Fig. 7

In vivo acute toxicity of EGCG&Cys(DOX) nanoformulation after administered intravenously in mice. a Body weight of mice was measured every other day within 35 days of EGCG&Cys(DOX) exposure; b, c Organ coefficients and photos of significant ex vivo organs of mice on the 35th day after EGCG&Cys(DOX) exposure; d, e Serum biochemical indicators and routine blood test results of mice euthanized on the 35th day; f H&E staining images of primary organ slices in mice

Pharmacokinetic and tissue distribution analysis

The relationship between the average plasma concentration and administration time of two groups of mice injected with DOX and EGCG&Cys(DOX) is shown in Figure S21a. The results showed that the plasma concentration of the DOX group rapidly decreased after 5 min of administration and gradually cleared. In contrast, the plasma concentration of the EGCG&Cys(DOX) group also showed a decreasing trend after administration but an increasing trend during the 15–20 min and 45–60 min periods. We believe this results from the combined action of DOX sustained release from EGCG&Cys(DOX) and clearance of DOX in plasma. The slight decrease in EGCG&Cys(DOX) blood drug concentration indicates that the nanoformulation has more persistent drug release characteristics.

In addition, DOX rapidly and widely distributes in various tissues after entering the body. At 1 h after injection, the highest concentration of DOX in the DOX group was 1161.7 ng/g in renal tissue, indicating that DOX was rapidly metabolized out of the body through the kidneys. The concentration of DOX released by EGCG&Cys(DOX) in lung tissue was 8678 ng/g, followed by 501.3 ng/g in liver tissue. The DOX concentration in the heart of the DOX group was 169.8 ng/g, significantly higher than the 69.1 ng/g in the EGCG&Cys(DOX) group (Figure S21 b). After 24 h, the DOX concentration in the DOX group's heart tissue increased by 1.8%, while the concentration of DOX in the EGCG&Cys(DOX) group decreased by 10.9%. These results indicate that EGCG&Cys loaded with DOX alters DOX’s metabolic pathways and tissue distribution characteristics, reducing its accumulation in the heart and facilitating the reduction of DOX-induced cardiac toxicity. In addition, compared to 1 h, the concentration of DOX in tumor tissue of the EGCG&Cys(DOX) group (88.7 ng/g) increased by 54.5% in 24 h, significantly higher than that of the DOX group (29.9 ng/g). the distribution results indicate that EGCG&Cys(DOX) exhibits slow release of DOX, which will prolong the maintenance time of higher DOX concentrations in mouse blood and increase the chance of DOX entering the tumor from the blood. In addition, EGCG&Cys targeting the 67LR protein on the surface of tumor cells can significantly enhance the enrichment of EGCG&Cys(DOX) at the tumor site, thereby releasing DOX in the acidic tumor microenvironment and achieving better chemotherapy efficacy (Figure S21c).