Introduction

Cancer remains a significant public health issue worldwide.1–4 Cancer treatment strategy involves surgery, chemotherapy, radiotherapy (RT), and immunotherapy.5,6 RT is an important multidisciplinary management of many types of cancer. It exerts high energy beams to target tumor tissue, causing oxidative stress and DNA double-strand breaks (DSBs), which in turn trigger cell apoptosis. This treatment can serve as a curative strategy for early-stage cancer, a method to control local recurrence, and palliative care for advanced cancer.7,8 However, the tumors are much less sensitive than late-responding healthy tissues, due to tumor radioresistance resulting from specific tumor microenvironment (TME).9,10 Thus, the use of RT should be carefully weighed to overcome radioresistance and reduce side effects. Therefore, it is urgent to develop a novel method for improving the efficacy of RT.

Radiosensitizers have been applied for the improvement of RT during clinic, such as cisplatin, antibody-drug conjugates (ADC) and epoxygenase-2 inhibitors.11–15 But these sensitizers have serious side effects.16–18 Recently, nanotechnology can improve RT treatment effects and overcome the above limitations.19,20 Nanoplatforms containing high Z elements can accumulate large dosages by increasing the amounts of photoelectrons and Auger electrons. Ertas’s team developed an alginate coated Pt nanoradiosensitizer for breast cancer.21 Han et al found that Au infinite coordination complex causes dose enhancement after the metal-organic nanocomplex entry to cells.22 Cao reported that platinum nano-assemblies can not only act as radiosensitizers enhance radiodynamic therapy and activate immunotherapy against tumors but also contrast agent for computed tomography imaging.23 Meanwhile, the exogenous stimuli-responsive therapy strategy by merging photothermal therapy, photodynamic therapy, ultrasonic therapy and other treatments with RT showed potential in removal of tumors.24–26 For instance, nanoagents for dual enhancement of photothermal therapy and RT lead to synergistic effects. This was due to the hypoxia in TME can be alleviated by the accelerated blood flow induced by photothermal therapy, which results in enhancement of RT.27,28 However, it is necessary to consider the TME triggered burst release of nanoradiosensitizer to reduce toxicity to normal tissues. It is well known, the TME is characterized by high levels of glutathione (GSH), and H2O2 with hypoxia status under low pH.29,30 Responsive to these endogenous stimulations, nanoplatforms are widely developed in biomedical applications to enhance radiosensitization. An acid-triggered aggregation gold nanosystem realizes controllable size monitoring by changing the surface charge after the nanosystem accumulates in tumor regions by enhanced permeability and retention (EPR) effects.31 Janus nanoparticles were fabricated which dissolved responsive to GSH to improve RT.32 These smart designs of stimuli-nanoplatform enhance radio sensitization efficacy. However, positive tumor-targeting properties are necessary when considering the circulation time and side effects.

Cuproptosis, which was discovered recently, has provided novel ideas for cancer treatment.33,34 Cuproptosis is caused by DLAT aggression and Fe-S cluster protein loss, which causes proteotoxic stress and cell death. In addition, immunogenic cell death (ICD) can be triggered by cuproptosis.35–37 However, the cuproptosis was hindered due to the lack of transporter proteins and high level of GSH content in TME which eliminate copper ions.38 Based on this background, here this work constructed a copper-doped polydopamine nanoparticles (PDA@Cu) and camouflaged them with platelet cell membrane (PCM), as shown in Scheme 1. This PCM coating PDA@Cu was termed PC NPs. Because platelet cells tend to accumulate in the inflammatory tumor sites, PC with PCM coating is capable of highly accumulating in tumor sites. Meanwhile, the disguise of PCM enables escape of PC from immune system clearance, prolonging circulation time and enhancing the delivery efficiency. After intracellular entry, PC NPs cracked and dissolved to release PDA@Cu, the latter of which dissembled under acidic TME and releases Cu2+. This process enabled an influx of copper ions in tumor cells. Cu2+ next was reduced to more toxic Cu+ in the existence of GSH. Cu+ leads to aggregation of DLAT to further enhance the cuproptosis. RT also led to generations of reactive oxygen species (ROS) causing oxidative stress, which decreases the amount of GSH and hence strengthens the cuproptosis. Copper overloading also promoted the enhancement of RT by exhausting the intracellular GSH to inhibit tumor cell self-repair. Hereby, this cuproptosis involved PC NPs with improved tumor targeting ability, and showed potential in radiosensitization.

Scheme 1 Schematic illustration of synthesize procedure and functional pattern of PC NPs.

Result and DiscussionCharacterization of Acidic pH Responsive PC NPs

PDA@Cu was prepared by using CuCl2 and PDA as precursors in dispersion and stable solution (Supporting information, Experimental section). Figure 1A shows the transmission electron microscopy (TEM) images of sphere structures of PDA@Cu. After the coating of PCM, a thin film boundary appeared on the edge of PC NPs (Figure 1B). Then, the dynamic light scattering (DLS) confirmed that the average diameter of PCM NPs remained stable in both PBS and FBS over 7 days observation (Figure 1C). To further verify the characterization of PCM on PDA@Cu, the diameter and zeta potential of PDA@Cu, PC, and PCM were measured, as shown in Figure 1D. Before camouflaging of PCM whose diameter is about 188.6 nm, the diameter of PDA@Cu was increased from 123.1 nm to 139.2 nm. Meanwhile, the zeta potential of PC shifted from −13.2 mV (PDA@Cu) to −17.6 mV (PC) after coating of PCM (−17.9 mV). These results verified the successful coating of PCM on PC NPs. Elemental mapping patterns and Energy-dispersive X-ray spectroscopy (EDX) were used to confirm the composition of PC NPs, as shown in Figure 1E and F. The results confirmed the existence of Cu, N and C elements, and these elements were distributed in PC NPs uniformly.

Figure 1 Characterization of PC. (A) TEM images of PDA@Cu NPs. (B) TEM images of PC NPs. (C) Time-dependent observation of PC diameter in PBS and FBS respectively. (D) Average diameter and zeta potential of PDA@Cu, PC and PCM respectively. (E) Elemental mapping of PC. (F) XPS survey of PC. (G) Release profile of Cu in buffer solution of different pH values. (H) Representative fluorescence imaging of cellular uptake in 4T1 after FITC labeled RC and PC. Data are presented as mean ± SD. Student t test. *** p< 0.001.

The release behavior of Cu at different pH buffers was further investigated, as shown in Figure 1G. Almost no significant ion Cu release was detected in a buffer with pH 7.4. While significant amount of Cu content was observed in the buffer of the group with a pH value of 6.0, reaching as high as 79.2% amount of total Cu.

In this work, 4T1, CT26 and MCF-10A cells were obtained from purchased from the Pricella Biotechnology (China). To evaluate and compare the cellular uptake efficacy of PC NPs, erythrocyte membrane camouflaged PDA@Cu (termed RC) was also synthesized. 4T1 cells were co-incubated with either FITC-labeled RC or PC NPs, respectively. Next, the cells were fixed for measurements of the number of NPs cellular uptake by CLSM imaging. A significant amount of green fluorescence representing NPs was observed in the PC NPs treatment group (Figure 1H) than that of RC NPs group, indicating higher intracellular entry and accumulation of PC NPs.

Evaluation of Anti-Tumor Effect of PC NPs Enhanced RT

The cytotoxicity of both RC and PC NPs on MCF-10A was verified through CCK8 method based on the studies in the previous section. As shown in Figure S1, cell viability of MCF-10A decreased with RC or PC NPs concentrations increase, which exceeded 90% survival rate under 30 μg/mL Cu concentration, suggesting satisfied biocompatibility at the concentration of 30 μg/mL. The antitumor efficacy in vitro was further evaluated. As highly reactive molecules, ROS with strong oxidation property can lead to cell death. The ROS production after various treatments was assessed using DCFH-DA probe. As shown in Figure 2A, the intensity of green fluorescence was strongest in PC+RT group among all groups, while weaker intensity was observed in RC+RT group. This result indicated that significant amount of ROS was generated by PC+RT under GSH conditions at acidic buffer due to the PCM coating. Because ionization irradiation can induce cell death by direct DNA damage, γ-H2AX staining was used for DNA DSBs staining. Slight red fluorescence was observed in 4T1 cells under RT. In addition, RC combined with RT exerted apparent DNA damage on 4T1 cells, however remarkable amount of red fluorescence intensity appeared in PC+RT group. The fluorescence intensity evaluation further confirmed the result, as shown in Figure S2. Next, cell viability evaluation was performed using CCK8 kit on CT26. As shown in Figure 2B, PC group showed modestly reduced cell viability while RC+RT group obviously destroy tumor cells. By striking contrast, PC+RT significantly killed tumor cells. Next, GSH consumption in different groups was qualified in Figure 2C. It showed that the GSH level decreased in RT group and PC group because RT exhausted GSH content and cuproptosis process of PC in cells. Compared with RC+RT group, PC+RT group showed more robust GSH elimination efficacy. The cancer cell proliferation in all groups was assessed by colony formation assay, as shown in Figure 2D. The surviving fraction of 4T1 cancer cells and CT26 cancer cells reduced to 10% in PC+RT group. It shows that this therapy is effective for breast cancer and colon cancer.

Figure 2 PC NPs could radiosensitize by inducing cuproptosis. (A) ROS production and DNA damage in 4T1 cells after different treatments (RT: radiotherapy). (B) Cell viability in all treatment groups on CT26 cells. (C) Measurement of GSH level. (D) Surviving fraction in each group. (E) Immunofluorescence staining of DLAT. (F) WB analysis and gray analysis. Data are presented as mean ± SD. Student t test. *** p< 0.001.

Subsequently, the cuproptosis induced by PC+RT was assessed. As an important intracellular signaling related to cuproptosis, DLAT aggression was investigated by fluorescence staining (Figure 2E). A large amount of DLAT was observed in 4T1 cells treated with PC NP, indicating that PC can induce cuproptosis. PC+RT showed the most significant DLAT aggression. Ferredoxin 1 (FDX1) showed an important role in copper metabolism, since it involved in the Fe-S proteins and electron transfer processes.37 From the result of Western blot analysis and protein quantification, the FDX1 downregulated after the PC treatment (Figure 2F). This confirmed that the cuproptosis was induced by PC. Surprisingly, PC combined RT exhibited the most obvious downregulation of FDX1 protein. These above results verified that both RT and PC can consume intracellular GSH, PC showed higher cellular uptake of RC which increased the antitumor efficacy in vitro. The PC showed potential in inducing cuproptosis, while the cuproptosis was more robust in combination therapy of PC and RT.

Promotion of ICD by PC NPs Enhanced RT via Cuproptosis

ICD can activate immune response, which is characterized by damage-associated molecular patterns (DAMPs) including releasing of ATP, surface-exposed calreticulin (CRT) and DNA bound high-mobility histone 1 (HMGB1). An experiment in vitro was designed for the study of in vitro ICD induction and dendritic cell (DC) maturation assays of 4T1 cells (Figure 3A). The ICD-related DAMPs by fluorescence imaging under various treatment groups were tested. The “eat me” signal of CRT was observed using CLSM imaging (Figure 3B). Negligible amounts of green fluorescence were observed in RT or PC or RC+RT treatment groups. However, PC+RT treatment group exhibited significant amount of CRT exposure among all groups. Besides, significant amount of HMGB1 was released into medium after treatment of PC+RT group (Figure 3C and D), which was 1.50 times higher than that of RC+RT group. Meanwhile, PC+RT group had the highest relative concentration of ATP, which was 1.54 times higher than that of RC+RT group (Figure 3E). Maturated mouse bone marrow-derived DCs were incubated with pretreated 4T1 cells before using flow cytometry to assess the DC maturation, as shown in Figure 3F. PC+RT showed the highest population of mature DC proportion, reaching as high as 54.1% CD80+CD86+ DC, which was significantly higher than that of RT group. These results indicated a more robust ICD effect after the addition of PC, suggesting the radiosensitization by PC.

Figure 3 ICD effect assessment in vitro. CLSM for (A) Schematic illustration for PC+RT induced ICD (B)CRT efflux detection and (C) HMGB1 release and (D) HMGB1 release in medium. (E) Released ATP detection. (F) DC maturation. Data are presented as mean ± SD. Student t test. *** p< 0.001.

Antitumor Efficacy Evaluation on Subcutaneous Tumor Model

Tumor-specific targeting in vivo was studied on subcutaneous 4T1 tumor bearing mice. Animal experiments were conducted following the guidelines of the National Institutes of Health and approved by the Animal Ethics Committee of Guangxi Medical University (Approval number: 2023-KY (0932)). 105 4T1 cells were subcutaneously injected on the right lap of 6-week-old female BALB/c mice to form tumor models. After 24 h injection of RC or PC NPs via tail vein, Cu concentration biodistribution in main organs and tumors was quantified using ICP-AES, as shown in Figure 4A. The results confirmed that PC had higher tissue accumulation than that of RC, implying PC NPs had significant active tumor targeting ability than RC. Besides, the subcutaneous 4T1 tumor bearing model on BALB/c mice was established to examine the antitumor efficacy of this integral method. The day 1 was set as the next day of treatment and all the mice were executed at day 16, as shown in Figure 4B. Mild tumor inhibition was observed under treatment of RT alone, while obvious tumor suppressing was observed in RC+RT group. Surprisingly, integral treatment of PC+RT was proven to be the most efficient strategy among all, significantly higher than that of other groups, which was consistent with the results in Figure 4C.

Figure 4 Identification of PC enhanced RT in tumor growth inhibition in vivo. (A) Biodistribution in main organs and tumors after administration of RC or PC NPs respectively. (B) Relative tumor volume curve. (C) Immunofluorescence staining of LIAS and FDX1, HE staining. (D) Tumor weight measurement. (E) Body weight fluctuation. Data are presented as mean ± SD. Student t test. *** p< 0.001.

Immunofluorescence and HE staining were further performed. Lipoic acid synthetase (LIAS) and FDX1 play important roles in influencing cuproptosis. The tumor tissue of PC+RT treatment exhibited decreasing expression of LIAS with minimal red fluorescence intensity, as confirmed in Figure 4D. PC+RT treatment also effectively promoted FDX1 reduction. And most substantial cell death was observed in PC+RT group. These in vivo results demonstrated that the integral treatment of PC and RT showed potential in tumor growth inhibition.

Moreover, body weight had no significant change after these treatment strategies (Figure 4E). It is necessary to evaluate the biosafety of nano-radiosensitizers for cancer treatment. Therefore, the biosafety of PC NPs in vivo was then further evaluated. Ten healthy mice received administration of PBS buffer or PC NPs solution and were executed after 10 days. Histological analysis of main organs including heart, liver, spleen, lung and kidney confirmed positive compatibility of PC NPs, as shown in Figure 5A. In addition, blood biochemical analyses of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine (CRE) were evaluated, and found that there was no significant difference between PBS and PC group (Figure 5B–D).

Figure 5 Observation of biosafety. (A) Identification of damage to main organs with HE staining analysis. (B) The ALT, AST and ALP levels in mice received injection of PBS and PC. (C) BUN and (D) CRE concentration in PBS and PC treatment groups.

Conclusion

This work developed a biomimicking TME responsive cuproptosis induced nanoplatform PC for radiosensitization. PC NPs can efficiently target and accumulate tumor tissues through PCM camouflage. Since the high cellular uptake property of PC NPs, PDA@Cu is successful transport into cells. Under acidic TME, PDA@Cu is dissembled in cell matrix, releasing a burst of Cu2+ ions which consume reducible GSH to generate Cu+. Next, Cu+ induces DLAT aggression occurred under RT irradiation. Meanwhile, ROS generated from RT also lead to decreasing GSH content. Both Cu+ and RT lead to severe cell cuproptosis, enhance ICD effect in vitro, and promote DC maturation. The antitumor efficacy of integral treatment of PC and RT is significant as proven both in vitro and in vivo. This work hereby provided a potential strategy by radiosensitization induced by PC to enhance cuproptosis, holding great promise in the treatment of cancer.

Acknowledgments

We previously published an article on cuproptosis (reference 37). The published article used Cu doped polypyrrole nanoparticles loaded with BPTES, which can release copper ions upon hydrogen peroxide stimulation and exhibit POD like enzyme activity. Combined with BPTES mediated glutamine metabolism, it enhances cuproptosis treatment. The innovation of this work is reflected in the BPTES sensitization of cuproptosis mediated by nanoparticles.

In this work, we used polydopamine nanoparticles that adsorb copper ions, which are significantly different from previously published nanomedicine. These nanoparticles release copper ions under acidic conditions, causing cuproptosis in tumor cells and enhancing the sensitivity of radiation immunotherapy. This work focuses on the application of cuproptosis in RT immune-sensitization, and there is currently limited research on this topic.

Funding

We are grateful for the financial support from the Shenzhen Science and Technology Program (JCYJ20220530153816038).

Disclosure

The authors report no conflicts of interest in this work.

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