Engineered charge adaptive nanoplatform overcomes the drug penetration barriers to potentiate the efficacy of chemotherapy

Abstract

Breast cancer continues to present a major clinical hurdle, largely attributable to its aggressive metastatic behavior and the suboptimal efficacy of standard chemotherapeutic regimens. Cisplatin (CDDP) is a representative platinum drug in the treatment of breast cancer, however, its therapeutic application is often constrained by systemic toxicity and the frequent onset of chemoresistance. Here, we introduce a novel charge-adaptive nanoprodrug system, referred to as PP@, engineered to respond to tumor-specific conditions. This platform was constructed by conjugating ibuprofen and polyethylene glycol (PEG) to the hydrophobic and hydrophilic termini of an amphiphilic dendrimer, respectively, enabling the formation of uniform and stable nanostructures through spontaneous self-assembly. Importantly, PP@ undergoes charge reversal in response to acidic pH and elevated glutathione levels (GSH), facilitating deeper tumor penetration. Cisplatin was subsequently encapsulated within the nanoprodrug to yield the PP@-based CDDP nanoformulation (PP@CDDP). The physicochemical properties and therapeutic performance of PP@CDDP were systematically evaluated. The results demonstrated that PP@CDDP significantly improves cellular uptake, suppresses drug efflux, and reduces intracellular GSH levels, collectively contributing to prolonged drug retention at the tumor site. In vivo studies further confirmed that PP@CDDP significantly improved the antitumor efficacy of cisplatin, as evidenced by marked inhibition of tumor growth and metastasis, along with a favorable safety profile. These results underscore the potential of this charge-adaptive nanoprodrug platform to address key limitations of traditional cisplatin chemotherapy. The rational integration of smart material design with pharmacological strategies offers a promising pathway for improving therapeutic outcomes in cancer treatment.

Graphical abstract

Highlights

• •Constructed a novel nanoprodrug with charge-adaptive properties, allowing enhanced tumor penetration.
• •The nanoprodrug enables tailored chemotherapeutic loading and pH/GSH-responsive release.
• •Antitumor efficacy was enhanced through improved uptake, efflux inhibition, GSH depletion, and tumor accumulation.
• •This synergy-driven design offers a new strategy for breast cancer chemotherapy.

1. Introduction

Breast cancer is one of the most common malignancies worldwide, accounting for nearly 30 % of all cancer diagnoses among women [1]. Despite advancements in early detection and multimodal therapies, metastatic breast cancer remains a significant clinical challenge, the primary cause of related mortality due to tumor spread to distant organs, such as the lungs [2,3]. Current treatment regimens—including surgery, radiation, and chemotherapy—often fail to eliminate metastases, highlighting the urgent need for therapies that effectively target both primary tumors and metastatic progression.

Cisplatin, a cornerstone chemotherapeutic for breast cancer, mediates antitumor activity via DNA crosslinking and apoptosis induction [4,5]. However, its clinical application is limited by two major factors: drug resistance, which reduces tumor cell sensitivity, and systemic toxicity [[6], [7], [8]]. Most malignant tumors, including breast cancer, develop resistance to cisplatin over time. While the precise mechanisms underlying this resistance remain incompletely understood, it is generally attributed to three key factors: reduced intracellular accumulation [9], increased cisplatin inactivation [10,11], and enhanced DNA damage repair [8,12]. Additionally, cisplatin-based therapies are frequently associated with various adverse effects, particularly nephrotoxicity, approximately one-third of patients experience nephrotoxicity, primarily due to renal excretion of the drug [7,13]. Notably, patients receiving high-dose cisplatin (>100 mg/m²) are at increased risk for severe and irreversible nephrotoxicity [14]. Dose-limiting toxicity further complicates the management of acquired resistance, as increasing the dose is not a viable solution. Therefore, novel therapeutic strategies are urgently required to enhance the therapeutic index by improving tumor-selective cisplatin sensitization and mitigating its associated toxicity.

Nanotechnology offers a transformative approach to these issues. By encapsulating cisplatin in nanocarriers, researchers aim to leverage the nanoscale effect for selective tumor accumulation [14,15]. For instance, liposomal formulations of cisplatin have demonstrated significantly enhanced intratumoral drug accumulation compared to free cisplatin in preclinical models [14,16,17]. However, the clinical translation of nanomedicines is often hindered by inadequate tumor penetration [18,19], primarily due to the complex tumor microenvironment, which impedes nanoparticle diffusion [20,21]. Recent efforts have focused on engineering "smart" nanocarriers capable of dynamically modulating their physicochemical properties—such as size and surface charge—in response to the tumor microenvironment [22,23].

In this study, we present a tumor-microenvironment-responsive nanoformulation designed to combat multiple biological barriers in breast cancer therapy (Fig. 1). Our system utilizes a novel amphiphilic prodrug carrier (PSSP), which incorporates four key innovations: (i) Stealth-to-sticky surface transformation: Polyethylene glycol (PEG) chains anchored via reducible disulfide bonds provide stability in circulation while exposing positively charged of low-generation polyamidoamine (PAMAM) dendrons, enhancing cellular uptake and deep tumor penetration. (ii) Dual-function hydrophobic core: Ibuprofen (IBU) acts as both a structural stabilizer for nanoparticle assembly and a sensitizer that augments the anticancer effects of cisplatin in 4T1 cancer cells. (iii) Hierarchical drug release: The release kinetics of cisplatin are finely regulated, with less than 7 % leakage during circulation and over a fivefold increase in release within 48 h under simulated tumor conditions. (iv) GSH depletion-mediated chemosensitization: Simultaneous reduction of intracellular GSH levels and inhibition of cisplatin efflux boost drug utilization efficiency.

Fig. 1.

Schematic of the PSSP-based nanoprodrug with adaptive charge switching for sensitizing cisplatin against breast cancer.

In 4T1 breast cancer models, this newly constructed dual drugs codelivery nanoformulation PP@CDDP demonstrates more efficient accumulation in tumor tissue compared to free cisplatin, resulting in significantly greater inhibition of tumor growth while minimizing kidney damage. The charge-variable nanodrug exhibits enhanced tumor-penetrating capacity, which, in combination with the synergistic action between ibuprofen and cisplatin, results in notably reduces lung metastasis in treated mice compared to those administered free cisplatin. These findings indicate that this innovative cisplatin nanoformulation, which integrates smart material design with pharmacological synergy, represents a promising strategy for combating breast cancer.

2. Results and discussion

2.1. Synthesis and characterization of an amphiphilic dendritic prodrug with self-assembly properties and charge-adaptive nanostructures for deep tumor penetration

The amphiphilic dendritic prodrug was synthesized through a three-step modular strategy (Scheme 1). First, a polyethylene glycol-functionalized polyamidoamine dendrimer (I) and an azido-functionalized ibuprofen derivative (II) were synthesized separately. These two precursors underwent an efficient click reaction (azide-alkyne cycloaddition) to yield the disulfide-containing amphiphilic prodrug, PSSP, with structural confirmation by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (Figs. S1–S3).

Scheme 1.

Synthetic route of pH/GSH-responsive amphiphilic dendritic prodrug (PSSP). (A–C) Polyethylene glycol-functionalized polyamidoamine dendron I and an azido-terminated ibuprofen derivative II were synthesized as the hydrophilic and hydrophobic units of PSSP, respectively. These components were then conjugated via click chemistry to form the amphiphilic prodrug. The synthetic routes for the hydrophilic segment (A), the hydrophobic segment (B), and the overall PSSP (C) are depicted.

The self-assembly behavior of PSSP was systematically characterized (Fig. 2A). The pyrene fluorescence method was employed to determine the critical micelle concentration (CMC) of PSSP, which was found to be 1.0 μM, indicative of its propensity to self-assemble into stable structures (Fig. 2B). Dynamic light scattering (DLS) analysis (Fig. 2C) revealed that PSSP could spontaneously form nanoparticles (defined as PP@) with an average hydrodynamic diameter of 170.3 ± 0.6 nm in aqueous solution and displayed good dispersity. Transmission electron microscopy (TEM) corroborated that PSSP could assemble into uniform spherical nanostructures (Fig. 2C). The particle sizes measured by DLS were marginally larger than those observed by TEM, likely due to the shrinkage of nanoparticles upon solvent evaporation during TEM sample preparation. Notably, the nanoprodrug PP@ displayed stable particle size distribution when stored at room temperature for 5 days and in PBS containing 10 % FBS at 37 °C for 24 h (Fig. 2D and E). This stability underscores its potential for clinical applications. These data suggest PSSP as a robust self-assembling prodrug system with precisely engineered architecture, establishing its potential as a versatile nanoplatform for therapeutic delivery.

Fig. 2.

Preparation and physicochemical properties of the self-assembled nanoprodrug. (A) Schematic illustration of PSSP self-assembly into nanoparticles and its potential charge variation in response to tumor acidity and the intracellular reductive environment. (B) Critical micelle concentration of PSSP. (C) Size distribution and morphological characteristics of the PSSP-based nanoprodrug (PP@). (D) Stability of PP@ in PBS (pH 7.4). (E) Stability of PP@ in PBS (pH 7.4) supplemented with 10 % fetal bovine serum (FBS). Size was measured by dynamic light scattering (mean ± SD, n = 3). (F) Zeta potential variation of PP@ over time in PBS under different pH conditions and glutathione (GSH) levels. Zeta was measured by dynamic light scattering (mean ± SD, n = 3). (G) Deep penetration of PP@ in 3D spheroids formed by 4T1 cells, visualized through DOX fluorescence (red) using fluorescence microscopy, compared to free DOX as a control.

To validate the stimuli-responsive behavior of amphiphilic prodrug PSSP, we monitored zeta potential variations of PP@ nanoprodrugs under controlled GSH/pH conditions (Fig. 2F). At physiological conditions (10 μM GSH, pH 7.4), PP@ maintained a negative surface charge (−4 mV), whereas significant charge reversal occurred in tumor-mimicking conditions: reaching +4 mV at extracellular tumor (10 μM GSH, pH 6.5) and +15 mV under intracellular tumor settings (10 mM GSH, pH 6.5). This pH/GSH-coactivated charge reversal originates from acid-induced protonation of dendritic secondary amines synergized with GSH-triggered disulfide cleavage [[24], [25], [26]], progressive PEG detachment, and exposure of latent amines that amplify protonation efficiency.

To directly validate the generation of transformation products under tumor-relevant conditions, we performed mass spectrometric analysis of PP@ following incubation at 37 °C for 4 h under three distinct conditions: (i) pH 7.4 with 10 μM GSH, (ii) pH 6.5 with 10 μM GSH, and (iii) pH 6.5 with 10 mM GSH. Under both physiological and mildly acidic conditions with low GSH levels (i and ii), the dominant species remained the intact amphiphilic prodrug PSSP (Fig. S4). In contrast, incubation under the intracellular tumor-mimicking condition (pH 6.5 with 10 mM GSH) yielded a major product corresponding to a disulfide-cleaved, PEG-detached derivative (designated as compound III, Fig. S4). These results provide direct evidence that high GSH concentrations can induce PEG shedding, thereby unmasking polyamide moieties on the nanoparticle surface, which in turn facilitates the pH/GSH-triggered surface charge reversal.

The smart charge switching confers dual functionality: (i) Blood-compatible negative surface minimizes nonspecific adsorption during circulation, while (ii) tumor-specific positive charge promotes electrostatically enhanced cellular uptake through membrane interaction [27]. Such context-adaptive surface engineering balances circulation stability with tumor penetration efficiency, providing a rational strategy for tumor-precision drug delivery.

Subsequent experiments utilizing three-dimensional tumor spheroids further supported these conclusions. We incorporated the hydrophobic drug doxorubicin (DOX) into the PP@ nanoformulation via a thin-film dispersion method, thus constructing a fluorescently labeled nanosystem PP@DOX. Confocal microscopy revealed that, as shown in Fig. 2G, PP@DOX demonstrated superior accumulation in the deeper regions of tumor spheroids compared to free DOX. This indicates that the PP@ nanoprodrug significantly enhances the permeability of the loaded drug within tumors, potentially augmenting its anticancer efficacy.

2.2. Amphiphilic dendrimer based nanoprodrugs PP@ quantitatively encapsulate cisplatin and enable tumor microenvironment-responsive drug release

Building on the exceptional stability, tumor microenvironment-responsive charge-switching properties, and enhanced penetration capabilities of PP@ nanoparticles, we explored its potential to augment the antitumor efficacy of cisplatin, a widely utilized chemotherapeutic agent, using PP@ as a drug carrier. To optimize the synergistic effects between cisplatin and ibuprofen, we first evaluated the cytotoxicity of various ibuprofen-to-cisplatin ratios (1:1, 1:2, 1:4, and 1:6) in 4T1 breast cancer cells (Table S1). This evaluation involved calculating the combination index (CI) based on IC₅₀ values to determine synergy, where a CI < 1 signifies synergy, CI > 1 indicates antagonism, and CI = 1 denotes an additive effect [28]. As detailed in Table S1, the CI was lowest (0.237) at an ibuprofen-to-cisplatin mass ratio of 1:4, highlighting the most pronounced synergistic antitumor effect at this ratio. Consequently, we used this ratio to develop the cisplatin-loaded nanoparticle formulation PP@CDDP based on the PP@ nanoprodrug. Characterization by DLS and TEM confirmed that constructed PP@CDDP nanoparticles were spherical with an average diameter of 186.4 ± 0.7 nm (Fig. 3A).

Fig. 3.

Characterization and biological evaluation of the dual drug co-delivery nanosystem (PP@CDDP). (A) Size distribution and morphological characteristics of PP@CDDP. (B) Cisplatin release from PP@CDDP under different conditions: pH 7.4 PBS with 10 μM GSH, pH 7.4 PBS with 10 mM GSH, pH 5.0 PBS with 10 μM GSH, and pH 5.0 PBS with 10 mM GSH. (C) Ibuprofen release from PP@CDDP at pH 7.4 and pH 5.0, with or without the enzyme papain. (D) Antiproliferative activity of PP@CDDP against 4T1 breast cancer cells, assessed using the CCK-8 assay and compared to untreated cells, cisplatin alone (CDDP), ibuprofen alone (IBU), and the physical mixture of cisplatin and ibuprofen (CDDP + IBU).

A primary challenge in chemotherapeutic applications is the nonspecific release of drugs, which can adversely affect normal tissues [29]. To examine the drug release characteristics of PP@CDDP nanoparticles under physiological and tumor-mimic conditions [20,26], we studied the microenvironment-responsive release kinetics under varying GSH concentrations and pH levels. Under physiological conditions (pH 7.4, 10 μM GSH), cisplatin release was restricted to less than 7 % within 48 h (Fig. 3B), indicating substantial circulatory stability. In contrast, conditions simulating the tumor endosomal environment (pH 5.0, 10 mM GSH) facilitated significant cisplatin release, exceeding that observed under neutral pH/10 μM GSH controls by 5.2-fold within 48 h, thereby demonstrating selective activation in the tumor microenvironment. Moreover, the release of ibuprofen from PP@CDDP was investigated under acidic, enzyme-rich conditions (pH 5.0, in the presence of papain), simulating the endosomal environment [20]. Papain, serving as a model enzyme for cathepsin B, enabled the exploration of enzyme-triggered drug release [30,31]. As illustrated in Fig. 3C, ibuprofen release substantially increased under these conditions relative to neutral pH (7.4) and enzyme-free settings, with over 60 % released in 48 h. These findings highlight a multiphasic activation mechanism—combining pH, GSH and enzyme triggering—enables efficiently control over drug release, potentially widening the therapeutic window through tumor-selective activation.

2.3. PP@CDDP nanoparticles exhibit strong antiproliferative, antimigratory, and anti-invasive effects on breast cancer cells

The in vitro antiproliferative efficacy of the dual-drug delivery system PP@CDDP nanoparticles against 4T1 breast cancer cells was assessed using the CCK-8 assay. Control groups included free CDDP, free IBU, a physical mixture of IBU and CDDP (IBU + CDDP), the nanoprodrug PP@, and untreated cells. As illustrated in Fig. 3D, both PP@CDDP nanoparticles and the physical mixture IBU + CDDP demonstrated dose-dependent antiproliferative activity alongside free CDDP in 4T1 cells. Notably, PP@CDDP nanoparticles exhibited more significant cell proliferation inhibition at multiple concentrations compared with free CDDP and the physical mixture of IBU and CDDP. Interestingly, while free IBU alone did not significantly inhibit 4T1 cell proliferation, it did enhance the antiproliferative effect of CDDP, particularly at lower concentrations. This finding aligns with previous studies that IBU could sensitize cancer cells to chemotherapeutic agents [32,33].

A significant clinical challenge in breast cancer management is tumor metastasis, characterized by the migration and invasion of tumor cells into surrounding tissues, which are critical steps in disease progression [34]. Nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, have demonstrated effectiveness in inhibiting tumor metastasis by reducing cancer cell migration [35]. To evaluate the antimetastatic potential of the dual-drug delivery system PP@CDDP, we performed wound healing and transwell invasion assays. As shown in Fig. 4A, the wound healing ability of 4T1 cells was significantly reduced after 24 h of treatment with free CDDP, free IBU, the physical mixture of CDDP and IBU, the prodrug PP@, and the dual-drug delivery system PP@CDDP, compared to the negative control group. Free IBU reduced the wound healing rate by about 6 % compared to the control group, while free CDDP caused a 23 % decrease in wound healing rate (Fig. 4C). The physical combination of IBU and CDDP significantly reduced the wound healing rate by 40 %, indirectly demonstrating the sensitization effect of IBU on cisplatin. Importantly, the inhibitory effect of the nanosystem PP@CDDP on wound healing was significantly better than that of the other groups, with a wound healing rate of less than 4 % after 24 h of co-culture with 4T1 cells, underscoring the PP@-based nanostructure can also enhance the inhibitory effect on tumor cell migration.

Fig. 4.

PP@CDDP enhances the synergistic effect of cisplatin and ibuprofen in inhibiting 4T1 cell motility. (A) Representative wound healing images at 0 and 24 h after treatment with different agents. (B) Images of 4T1 cells that migrated through the membrane in invasion assays. (C) Relative migration rate after 24 h of treatment. (D) Statistical analysis of invasion assays. Data are presented as mean ± SD (n ≥ 3), ∗ p < 0.05; ∗∗∗∗P < 0.0001.

Consistent with the wound healing assay, the results from the transwell invasion assay supported our findings. As shown in Fig. 4B, untreated 4T1 cells nearly fully covered the bottom surface of the transwell chamber after 24 h, indicating pronounced invasive potential, which is consistent with previous reports. Treatment with various formulations significantly reduced the number of cells that traversed the matrigel-coated transwell (Fig. 4D), reflecting the inhibitory effects of both IBU and CDDP on 4T1 cell invasion. Notably, the physical mixture of IBU and CDDP resulted in a considerable decrease in the number of invasive cells, suggesting a synergistic antimetastatic effect. Remarkably, PP@CDDP nanoparticles demonstrated the most robust inhibitory effect on cell invasion, with nearly no cells crossing the matrigel-coated transwell after 24 h of treatment, indicating superior efficacy compared to both free drugs and their physical mixture. Overall, these results suggest that the PP@CDDP nanodrug delivery system holds considerable promise for enhancing the efficacy of anticancer therapeutics in both tumor suppression and metastasis prevention.

2.4. PP@-based nanosystem enhances cellular drug uptake via endocytosis and inhibits drug efflux

To investigate the mechanisms underlying the significantly improved antiproliferative and antimetastatic effects of the PP@CDDP dual-drug delivery nanoplatform, we analyzed the cellular uptake and retention of the PP@ based nanosystem in 4T1 cells. Given that cisplatin lacks intrinsic fluorescence and is not suitable for direct visualization, DOX, a fluorescent agent, was used as a surrogate to assess cellular uptake efficiency [36,37]. Flow cytometry analysis demonstrated that the intracellular uptake of both PP@DOX and free DOX increased over time (Fig. 5A). Notably, at all assessed time points, the average uptake percentage and fluorescence intensity in PP@DOX-treated cells exceeded those observed in free DOX-treated cells significantly. After 4 h of incubation, the fluorescence intensity in 4T1 cells treated with PP@DOX was approximately five times greater than in those treated with free DOX (Fig. 5B), indicating that PP@DOX nanoparticles are internalized more efficiently and rapidly by tumor cells.

Fig. 5.

PP@-based nanosystem exhibited rapid cellular uptake and suppressed drug efflux. (A, B) Cellular uptake of DOX and PP@DOX in 4T1 cells, quantified by flow cytometry. (C) Inhibition of DOX efflux by PP@ based nanosystem. (D) Flow cytometric analysis of PP@DOX uptake in 4T1 breast cancer cells after treatment with the endocytosis inhibitors genistein and β-cyclodextrin, or under energy-depleting conditions (4 °C). Data are presented as mean ± SD (n ≥ 3). Statistical significance: ns, no significance; ∗∗P < 0.01; ∗∗∗∗P < 0.0001.

A critical challenge in chemotherapy pertains to the efflux of intracellular drugs mediated by P-glycoprotein (P-gp), a transmembrane glycoprotein that actively transports drugs out of cancer cells, leading to decreased intracellular drug concentrations and diminished therapeutic efficacy [33,38,39]. To assess drug retention, breast cancer cells were incubated with PP@DOX or free DOX for 4 h, followed by replacing the drug-containing medium with fresh medium and subsequent monitoring of intracellular fluorescence. Free DOX exhibited rapid efflux, with ∼70 % expelled within 1 h and continued depletion over time (Fig. 5C). In contrast, PP@DOX nanoparticles displayed significantly lower efflux rate of DOX at all time points compared to free DOX, suggesting that the PP@ based nanoplatform can mitigates P-gp-mediated efflux, thereby enhancing intracellular drug retention and therapeutic potency.

The rapid and substantial accumulation of PP@ based nanosystem in cancer cells is likely attributed to its cell uptake pathway. To gain further mechanistic insights into the cellular uptake pathway, we employed a panel of endocytic inhibitors and monitored the intracellular accumulation of PP@DOX in 4T1 cells by flow cytometry. As shown in Fig. 5D, pretreatment with the endocytosis inhibitors genistein and β-cyclodextrin did not result in a significant reduction in intracellular fluorescence intensity, indicating that these pathways are not primarily involved in PP@ uptake. In contrast, incubating cells with PP@DOX at 4 °C, a condition that broadly suppresses energy-dependent endocytic processes [26], led to a marked decrease in fluorescence signal, suggesting that internalization of PP@ proceeds via an energy-dependent mechanism. These findings were consistent with the observed threefold increase in IC₅₀ of PP@CDDP under 4 °C conditions (Fig. S5), further supporting the reliance on energy-mediated uptake. These results indicate that energy-dependent endocytosis is a crucial pathway for PP@CDDP internalization. Therefore, the remarkable in vitro antitumor cell proliferation and anti-metastasis efficacy demonstrated by PP@CDDP nanoparticles may be attributed to their rapid cellular uptake and decreased cellular elimination, leading to sustained intracellular drug accumulation.

2.5. PP@CDDP nanoparticles suppress tumor progression and metastasis while reducing cisplatin-associated toxicity

Given the robust in vitro antiproliferative activity of PP@CDDP nanosystem, we further evaluated its in vivo antitumor efficacy and biocompatibility. Prior to efficacy studies, hemocompatibility—a critical parameter for clinical translation of nanocarriers—was assessed by incubating PP@ nanoprodrugs (1.56–100 μM) with murine red blood cells (RBCs). Even at 100 μM, which far exceeds the dosage required for animal experiments, no visible hemolysis occurred, with hemolysis rates remaining <2 % across all concentrations (Fig. S6). This confirms the excellent blood compatibility of the PP@ nanoprodrug, a prerequisite for systemic administration.

Subsequently, we established a 4T1 breast cancer-bearing mouse model to evaluate the in vivo antitumor efficacy and safety of PP@CDDP nanoparticles. The administered doses of IBU and CDDP were 1.1 mg/kg and 4.4 mg/kg, respectively. Throughout the study, mouse body weight and tumor volume were recorded. As indicated in the tumor growth curves (Fig. 6A), there were varying degrees of tumor growth inhibition in the groups treated with free CDDP, the physical mixture of IBU and CDDP, and PP@CDDP nanoparticles compared to the PBS control group. Consistent with our in vitro findings, PP@CDDP nanoparticles achieved nearly complete tumor inhibition (96.3 % inhibition rate; Fig. 6B), which was superior to free CDDP and physical co-administered IBU and CDDP. Tumor weight analysis further confirmed this result, and the average tumor weight of the PP@CDDP treated group was the smallest among all treatment groups (Fig. 6C and D). This enhanced efficacy likely stems from the tumor microenvironment-responsive PP@ nanosystem synergizing IBU to enhance cisplatin cytotoxicity while improving tumor penetration.

Fig. 6.

PP@CDDP enhances anticancer activity in tumor-bearing mice. The therapeutic efficacy of different treatments in 4T1 tumor-bearing mice was evaluated. Mice received intravenous (i.v.) administration of CDDP (4.4 mg/kg) and IBU (1.1 mg/kg) twice per week. (A) Growth curves represent individual tumor progression for each mouse within the respective treatment groups, (B) tumor inhibition rate, (C) tumor images, and (D) tumor mass. (E) Representative photomicrographs of tumor sections stained with H&E and Ki67 immunohistochemical staining to analyze tumor proliferation, along with Caspase-3 for apoptosis assessment. Data are presented as mean ± SD (n ≥ 3), ∗ p < 0.05; ∗∗P < 0.01.

To validate the notable antitumor activity of the PP@CDDP nanosystem, we performed immunohistochemical analyses on tumor sections, including hematoxylin and eosin (H&E) staining, as well as Caspase-3 and Ki67 immunolabeling. The findings indicated that the PP@CDDP-treated group exhibited more substantial tumor tissue damage (Fig. 6E), characterized by significantly increased expression of the apoptosis marker Caspase-3 and markedly decreased expression of the proliferation marker Ki67. This suggests that the PP@CDDP nanoplatform more effectively inhibits tumor growth.

During the tumor growth inhibition experiments, we also monitored the body weight of the mice. As presented in Fig. 7A, the body weights of mice in the free IBU, PP@, and PP@CDDP groups did not significantly differ from those in the control group, suggesting acceptable biocompatibility. Conversely, mice treated with free CDDP and the IBU + CDDP combination experienced notable weight loss, likely attributable to known toxicity of cisplatin [40]. These results indicate that both the PP@CDDP nanocarrier and the PP@ self-assembled nanoprodrug exhibit good biocompatibility.

Fig. 7.

PP@CDDP nanosystem significantly reduces toxicity and suppress tumor metastasis in tumor-bearing mice. (A) Changes in body weight during the treatment period. (B) Survival curves of mice under different treatments. (C) Representative lung images from BALB/c mice intravenously injected with 4T1 cells, showing metastatic nodules. (D) Histological analysis of kidney tissue to assess toxicity. (E) Representative lung tissue sections H&E-stained lung sections from mice in the following treatment groups, from left to right: PP@CDDP, CDDP + IBU, CDDP, PP@, IBU, and PBS. (F) GSH concentrations in tumor tissue following various treatments. Data are presented as mean ± SD (n ≥ 3),∗∗∗∗P < 0.0001. (G) Fluorescent imaging of PP@ nanoprodrugs loaded with the near-infrared dye DIR, captured 4h, 24h and 48h post-intravenous injection in 4T1 tumor-bearing mice. (H) Fluorescence images of tumor tissues and major organs (heart, liver, spleen, lung, and kidney) collected 4h, 24h and 48h post-injection.

A survival study provided additional evidence of the superior safety of PP@CDDP compared to the free drugs (Fig. 7B). After 40 days of treatment, the PP@CDDP group maintained a survival rate of 100 %, whereas the survival rates for the IBU + CDDP and free CDDP groups were only 33.3 %. Notably, all mice in the PBS control group died during this period (Fig. 7B). These findings underscore that the PP@CDDP nanoplatform not only significantly enhances the antitumor efficacy of cisplatin but also effectively minimizes its systemic toxicity.

Cisplatin is widely known for its severe side effects, with nephrotoxicity being among the most concerning. To assess this, we conducted histological analyses of kidney morphology across various treatment groups using H&E staining. As illustrated in Fig. 7D, the kidney cells of mice treated with PBS, free IBU, the PP@ nanoprodrug, and the PP@CDDP nanoplatform maintained regular morphology, exhibiting no apparent pathological changes. In contrast, mice administered free CDDP or the physical mixture of IBU and CDDP displayed significant kidney congestion and tubular dilation. These findings underscore the ability of the PP@ nanoprodrug to encapsulate cisplatin, thereby effectively mitigating its nephrotoxic effects. The safety profile of PP@CDDP was further evaluated in healthy mice. After 17 days of treatment, mice receiving free CDDP or the CDDP + IBU mixture showed significant elevations in ALT, BUN, and CRE levels, indicating hepatic and renal toxicity. In contrast, PP@CDDP-treated mice exhibited no significant changes in these parameters relative to the control group (Fig. S7), demonstrating that the CDDP nanoformulation based on charge-adaptive PP@ nanoprodrug substantially alleviates the systemic toxicity typically associated with cisplatin treatment.

A particularly promising discovery was the PP@CDDP nanoplatform's ability to markedly inhibit tumor metastasis in vivo. As demonstrated in Fig. 7C and E, 16 days post-treatment, the PBS-treated mice exhibited numerous white metastatic nodules in lung tissues (Fig. 7C). Remarkably, the lungs of mice treated with PP@CDDP exhibited almost no clearly visible metastatic nodules. Supporting this observation, H&E staining of lung tissue sections revealed significantly fewer metastatic lesions in the PP@CDDP group compared to other groups (Fig. 7E). These findings highlight the potent potential of the PP@CDDP nanoplatform in effectively suppressing tumor metastasis in vivo.

2.6. PP@-based nanosystem effectively reduces glutathione levels and enhances tumor accumulation

To elucidate the mechanisms underlying the enhanced antitumor efficacy and reduced systemic toxicity of PP@CDDP, we investigated two critical aspects of the nanoplatform's function. First, the high disulfide bond content within the PP@ nanoprodrug is hypothesized to play a pivotal role. Disulfide bonds can effectively consume the elevated intracellular glutathione levels commonly found in tumor cells, thus preventing the deactivation of cisplatin [41]. Prior studies have shown that resistance to cisplatin in tumor cells is frequently due to high intracellular GSH concentrations, which can form complexes with cisplatin and diminish its antitumor activity [[41], [42], [43]]. The ibuprofen-based nanoprodrug PP@ possesses disulfide bonds capable of consuming GSH, making PP@CDDP likely to reduce GSH levels and enhance the antitumor efficacy of cisplatin.

To validate this hypothesis, we quantified the GSH content in tumor tissues. As shown in Fig. 7F, GSH levels in the free CDDP and free IBU groups showed no significant changes compared to the control group, with only a slight reduction noted in the IBU + CDDP group. In contrast, GSH levels in the PP@CDDP and PP@ nanoprodrug groups were significantly reduced to 24 % and 59 %, respectively. This pronounced reduction is likely attributed to thiol–disulfide exchange between intracellular GSH and the disulfide bonds within the PP@ scaffold, thereby disrupting the redox balance and mitigating cisplatin deactivation. While these findings are consistent with the hypothesized mode of action, they represent indirect evidence. Future studies will aim to directly quantify tumor redox shifts—such as through GSH/GSSG ratio analysis—to further substantiate the mechanism.

In addition, we evaluated the tumor-targeting capability of the nanoplatform using the near-infrared fluorescent dye DIR. DIR was encapsulated into the nanoprodrug, resulting in the fluorescently labeled nanoplatform PP@DIR. Using 4T1 tumor-bearing BALB/c mice, we administered either free DIR or PP@DIR via tail vein injection and monitored biodistribution with an in vivo small animal imaging system. As shown in Fig. 7G, the PP@DIR-treated group exhibited consistently stronger fluorescence signals at tumor sites than the free DIR group across all observed time points. Notably, at 48 h post-injection, fluorescence remained clearly detectable in the tumors of mice treated with PP@DIR, whereas the signal in the free DIR group was not obvious. These results suggest that the PP@ nanoprodrug significantly improves tumor accumulation and retention of the payload.

This trend was further corroborated by ex vivo fluorescence imaging of excised tumors and major organs. As presented in Fig. 7H, tumor tissues from the PP@DIR group exhibited substantially higher fluorescence intensity than those from the free DIR group at each examined time point, reinforcing the enhanced tumor-targeting performance of the charge-adaptive nanoplatform. This pronounced tumor accumulation is likely due to the structural and physicochemical properties of the PP@ nanoplatform, which facilitates enhanced retention and prolonged presence of the encapsulated drug within the tumor microenvironment.

3. Conclusions

In this work, we developed an innovative strategy to address the limitations of traditional chemotherapeutics like cisplatin by integrating a non-steroidal anti-inflammatory drug into a delivery system featuring tumor microenvironment-responsive chemical bonds. The novel nanoplatform PP@ capable of charge-adaptive switching at tumor sites, achieving enhanced tumor penetration. The PP@-based cisplatin nanoformation PP@CDDP is engineered to optimize the synergistic index, thereby maximizing therapeutic synergy. This system facilitates controlled drug release in response to intracellular acidic pH and elevated GSH concentrations, ensuring efficient payload delivery within tumor cells. By enhancing tumor tissue penetration and cell uptake, minimizing drug efflux, and lowering intracellular GSH levels to prevent CDDP deactivation, our nanosystem significantly improves the antitumor activity of cisplatin. Supporting in vitro studies and in vivo mouse tumor models provide robust cellular and biochemical evidence for the unique therapeutic advantages of the PP@CDDP nanoparticles. Importantly, this system effectively inhibits tumor growth and substantially suppresses breast cancer metastasis while exhibiting high biosafety in vivo. These findings underscore the potential of PP@CDDP as a promising candidate for breast cancer treatment. The multifunctional nanoprodrug PP@ not only provides a powerful solution to the application challenges faced by traditional cisplatin-based chemotherapy drugs, but also provides an effective strategy for the design of advanced nanomedicines for cancer care.

4. Experimental section

4.1. Materials and instruments

All chemical reagents were purchased from Alfa Aesar, TCL, Macklin, or Aladdin. Methyl acrylate, ethylenediamine, dichloromethane, and methanol were distilled before use, while other reagents were used as received. Column chromatography was performed on silica gel (Merck, 200–300 mesh). The 4T1 breast cancer cell line was kindly provided by Dr. Ting Zhuang (Henan Medical University) and approved by the university's Ethics Committee (XYLL-20240097, dated 2024/03/06). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and maintained at 37 °C in a humidified incubator with 5 % CO₂.

4.2. Synthetic procedure and characterization of PSSP

4.2.1. Synthesis of compound I

The PAMAM dendron (2A) was synthesized following established laboratory protocols [44]. Subsequently, the synthesis of I was performed according to the method described by Hu [45]. Briefly, SPDP (43.7 mg) was added to compound 2A (19.8 mg) in 2.0 mL of methanol containing 10.0 μL of triethylamine, and the mixture was stirred at room temperature for 2 h. PEG-SH (49.9 mg) was then added, and the reaction continued at room temperature. The reaction mixture was subsequently evaporated to obtain a crude product, which was purified using a CH₂Cl₂:CH₃OH system, yielding the pure product I, 42.1 mg (51 %). ¹H NMR (400 MHz, CDCl₃) δ7.68–7.62 (m, 2H), 7.31–7.26 (m, 2H), 3.73–3.57 (m, 47H), 3.37 (s, 13H), 3.14–3.02 (m, 2H), 2.97–2.86 (m, 10H), 2.72 (s, 1H), 2.63 (t, J = 6.0 Hz, 4H), 2.50–2.39 (m, 4H), 2.32 (s, 1H), 1.43–1.24 (m, 7H). ¹³C NMR (101 MHz, MeOD) δ 174.58, 72.70, 71.33, 71.26, 71.23, 71.19, 71.11, 71.06, 70.27, 59.22, 54.93, 39.55, 39.22, 36.29, 34.77. HRMS: calcd. for C₄₉H₉₃N₅O₁₈S₄,[M+H]⁺ 1168.5477, found 1168.5412.

4.2.2. Synthesis of compound II

EDCI (76.7 mg) and HOBt (54.0 mg) were added to the reaction flask in an ice bath, followed by DCM addition at room temperature. A solution of 3PEG-2A (44.6 mg) which was synthesized following the reported protocols [26], and ibuprofen (45.4 mg) was then introduced and reacted for 5 h in darkness under a nitrogen atmosphere. After evaporating the solvent and washing with distilled water and a saturated sodium bicarbonate solution, the mixture was dried over Na₂SO₄. The resulting crude product was purified using a CH₂Cl₂:CH₃OH system to obtain a pure white solid product II, 59.7 mg (73 %). ¹H NMR (400 MHz, CDCl₃), 7.73 (br, 2H), 7.18–7.17 (m, 4H), 7.04–7.02 (m, 4H), 6.68 (br, 2H), 3.60–3.45 (m, 14H), 3.33–3.20 (m, 10H), 2.72–2.59 (m, 6H), 2.38 (d, J = 4.0 Hz, 4H), 2.18 (t, J = 6.0 Hz, 4H), 1.74–1.84 (m, 2H), 1.44–1.41 (m, 6H), 1.22 (s, 1H), 0.86–0.84 (d, J = 8.0 Hz, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 175.4, 173.2, 140.5, 138.7, 129.4, 127.2, 70.6, 70.6, 70.5, 70.3, 70.0, 68.5, 53.3, 50.6, 50.6, 46.5, 45.0, 40.2, 38.8, 33.5, 30.2, 22.4, 18.6. HRMS: calcd. for C₄₄H₇₀N₈O₇,[M+H]⁺ 823.5446, found 823.5436.

4.2.3. Synthesis of PSSP

Finally, Tris (3-hydroxypropyl triazolyl methyl) amine (5.0 mg) was added to a Shecks tube. Compound I (135.5 mg) and Ⅱ (115.2 mg) were each dissolved in 1.6 mL of THF. Additionally, CuSO₄·5H₂O (3.0 mg) and sodium ascorbate (2.5 mg) were dissolved in 0.8 mL of water. The resulting reaction mixture was heated at 60 °C for 5 h, monitored by TLC until completion. The organic solvent was then evaporated, and the product was lyophilized to yield a crude product, which was further purified using a CH₂Cl₂:CH₃OH system to obtain the pure product PSSP, 126.7 mg (55 %). ¹H NMR (400 MHz, CDCl₃) δ7.84–7.70 (m, 4H), 7.51 (br, 2H), 7.23–7.16 (m, 4H), 7.07–7.06 (m, 4H), 6.89 (br, 2H), 5.35–5.30 (m, 1H), 4.55–4.50 (m, 2H), 3.85–3.23 (m, 72H), 2.94–2.62 (m, 17H), 1.42–1.63 (m, 27H), 1.47–1.25 (m, 16H), 0.90–0.86 (m, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 175.4, 172.8, 171.7, 142.6, 140.1, 138.5, 129.1, 128.8, 127.0, 124.5, 77.4, 71.6, 70.2, 70.2, 70.0, 69.9, 69.2, 69.0, 67.8, 58.7, 52.7, 50.1, 50.0, 49.2, 47.2, 46.0, 44.7, 39.5, 39.2, 38.9, 38.6, 37.9, 35.3, 33.9, 33.1, 32.7, 29.9, 22.2, 18.4, 13.5. HRMS: calcd. for C₉₃H₁₆₃N₁₃O₂₅S₄,[M+H]⁺ 1991.0844, found 1992.0918.

4.3. Critical micelle concentration measure of PSSP

The critical micelle concentration of PSSP was determined using a pyrene fluorescence probe method [26]. PSSP aqueous solutions (3.9 × 10⁻⁶ to 1.0 × 10⁻⁴ M, 2.0 mL each) were prepared and mixed with 20.0 μL of 1.0 mg/mL pyrene solution. After sonication for 30 min, samples were left to evaporate at room temperature for 2 h. Fluorescence spectra were recorded at 335 nm excitation using an RF-5301PC spectrophotometer (Shimadzu, Japan). The I₃₇₃/I₃₈₄ ratio was plotted against PSSP concentration to determine the CMC via linear fitting.

4.4. Preparation of PSSP-based nanoprodrugs (PP@)

The PP@ nanoprodrugs were prepared via the thin-film hydration method. Briefly, 3.0 mg of PSSP was dissolved in 3.0 mL of chloroform, followed by rotary evaporation at 32 °C to remove the solvent. The resulting film was further dried under oil-pump vacuum, then hydrated with PBS (pH 7.4) and sonicated for 30 min to obtain the nanoprodrug solution (PP@).

4.5. Stability analysis of PP@

To assess serum stability, 1.0 mL of freshly prepared PP@ nanoprodrug was mixed with 1.0 mL of complete medium containing 10 % FBS. The mixture was incubated at 37 °C with gentle shaking (110 rpm). Particle size was recorded at designated time points (0, 2, 4, 6, 8, 10, 12, and 24 h).

4.6. GSH/pH-responsiveness of PP@ nanoprodrugs

To evaluate the pH and GSH responsiveness of PP@, two complementary methods were employed. First, 900 μL of the filtered blank PP@ solution was mixed with 100 μL of GSH solution (100 mM or 100 μM) to achieve final GSH concentrations of 10.0 mM or 10.0 μM, respectively. The mixture was incubated at 37 °C in a shaker (110 rpm), and particle zeta potential were measured at 0, 4, 8, 12, and 24 h. Experiments at pH 6.5 were conducted using the same protocol.

In parallel, the same PP@–GSH mixtures were incubated under identical conditions for 4 h, after which aliquots were collected for mass spectrometric analysis to identify the major degradation products. This approach allowed for direct structural characterization of GSH- and pH-responsive cleavage events.

4.7. Permeability assessment of PP@ nanoprodrugs in 3D tumor spheroids

4T1 cells were seeded in U-5 S plates at 3500 cells per well, centrifuged at 500 g for 5 min, and incubated at 37 °C for 2–3 days. Tumor sphere formation was monitored daily. Once spheroids reached the desired size, the medium was replaced with DOX-labeled PP@ (PP@DOX); free DOX served as the control. After 4 h of incubation, spheroids were washed twice with PBS, fixed in 4 % paraformaldehyde for 15 min, rinsed with PBS, and transferred to confocal dishes. Images were captured using confocal laser scanning microscopy (CLSM).

4.8. Determination of the combination index (CI₅₀)

The cytotoxicity of free CDDP, IBU, and their physical mixture (IBU + CDDP) against breast cancer cells was assessed using a Cell Counting Kit-8 (CCK-8) assay. Cells were seeded in 96-well plates at 4 × 10³ cells/well and incubated at 37 °C for 24 h. Afterward, the medium was replaced with fresh drug-containing medium, and cells were cultured for an additional 72 h. Cell viability was determined via CCK-8. All experiments were performed in triplicate and repeated independently three times.

The synergistic effect of CDDP and IBU was analyzed using the combination index (CI) method. CI₅₀ was calculated as [28].

CI₅₀ =IC _CDDP in _{IBU + CDDP mixture}/IC _{single CDDP} + IC _{IBU in IBU + CDDP mixture/}IC _{single IBU}

Here, IC₅₀ values represent drug concentrations required to inhibit 50 % of cells. IC_{50 single IBU} was defined as 100 μg/mL. CI₅₀ values indicate: synergy (CI₅₀ < 1), additivity (CI₅₀ = 1), or antagonism (CI₅₀ > 1).

4.9. Preparation of PP@-based cisplatin nanoformation (PP@CDDP)

The cisplatin-loaded nanomicelles (PP@CDDP) were prepared via a thin-film hydration method. Briefly, PSSP and CDDP were dissolved in 3.0 mL of chloroform/methanol (3:2, v/v), followed by solvent removal through rotary evaporation at 32 °C and further vacuum drying. The resulting thin film was rehydrated with PBS or water and sonicated for 30 min to yield the PP@CDDP nanomicelle solution. Particle size and morphology were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively.

4.10. Cumulative release of CDDP and IBU from PP@CDDP

The cumulative release profiles of cisplatin (CDDP) and ibuprofen (IBU) from PP@CDDP nanomicelles were evaluated using a dialysis method under various conditions: (i) CDDP release was assessed in PBS (0.01 M) at pH 7.4 and pH 5.0, containing either 10 μM or 10 mM GSH. (ii) IBU release was tested in PBS (0.01 M) at pH 7.4 and pH 5.0, with or without papain (25.0 μg/mL).

For each condition, 450 μL of PP@CDDP solution was mixed with 50 μL of GSH solution (100 μM or 100 mM) and transferred into dialysis bags (MWCO: 500 Da), which were then immersed in 10.0 mL of release medium at 37 °C with shaking. At predetermined intervals (1–48 h), 2.0 mL of external medium was withdrawn and replaced with fresh PBS. Drug concentrations were measured by UV–Vis at 308 nm (CDDP) and 272 nm (IBU). Cumulative release percentages were plotted over time. All experiments were performed in triplicate, with results expressed as mean ± SD.

4.11. CCK8 assay for antiproliferation activity

The antiproliferative effects of free CDDP, IBU, their physical mixture (IBU and CDDP), PP@, and PP@CDDP on 4T1 cells were evaluated using the CCK-8 assay. 4T1 cells were seeded in 96-well plates at 3500 cells per well (100 μL/well) and incubated at 37 °C overnight. The next day, the medium was replaced with 190 μL of fresh medium containing various concentrations of the test formulations. Each concentration was tested in triplicate. A blank control (no cells, no drugs, no CCK-8) and a negative control (cells without drug treatment) were included. After 72 h incubation at 37 °C, the medium was removed and cells were gently washed with cold PBS. Then, 100 μL of complete medium containing 10 % CCK-8 reagent was added to each well and incubated for 1 h at 37 °C. Absorbance at 450 nm was measured using a microplate reader.

4.12. Wound healing assay

4T1 cells were seeded into 6-well plates at a density of 3 × 10⁵ cells/well and incubated overnight at 37 °C. A sterile 200 μL pipette tip was used to create a scratch in the cell monolayer, followed by PBS washing to remove detached cells. The medium was then replaced with fresh medium containing 2 % FBS and supplemented with free IBU, free CDDP, IBU + CDDP mixture, PP@, or PP@CDDP nanoparticles (final concentrations: CDDP 5.0 μM, IBU 1.25 μM). After 24 h incubation at 37 °C, wound closure was imaged at 0 and 24 h. Cell migration was quantified by measuring the wound area using ImageJ software.

4.13. Cell invasion

4T1 cells (3 × 10⁵ cells/well) were seeded into 6-well plates and cultured until adherence. Cells were then treated with 1.5 mL/well of PP@CDDP, IBU + CDDP, CDDP, PSSP, IBU, or DMEM complete medium (all at 5 μM cisplatin equivalent) for 24 h. After treatment, cells were harvested by trypsinization and centrifugation.

Matrigel was diluted 1:8 in serum-free medium, and 60 μL was added to the upper chamber of each Transwell insert using a chilled pipette tip. Following a 3 h incubation at 37 °C, excess Matrigel was removed, and 100 μL of serum-free medium was added to ensure no leakage before seeding cells. The inserts were pre-equilibrated in the incubator for 30 min.

Treated cells (1 × 10⁴ cells/well) were seeded into the upper chambers with 600 μL of complete medium added to the lower chambers. After 24 h incubation at 37 °C, non-invading cells and Matrigel were removed from the upper surface. Cells that had invaded to the lower surface were fixed with 4 % paraformaldehyde for 15 min, washed with PBS, stained with 0.5 % crystal violet for 10 min, and air-dried. Invasive cells were imaged under a light microscope.

4.14. Cell uptake

The internalization of PP@DOX and free DOX by 4T1 cells was quantified using flow cytometry. Cells were seeded in 6-well plates (3 × 10⁵ cells/well) and cultured overnight at 37 °C. The medium was then replaced with fresh medium containing either PP@DOX or free DOX (both at 11.0 μM DOX), followed by incubation for 5, 20, 60, 120, 180, or 240 min. After incubation, cells were harvested, washed with PBS ( × 3), and analyzed by flow cytometry (FACS Calibur, BD). Each assay was tested in triplicate.

4.15. Cell uptake mechanism

To explore the internalization pathway of PP@-based nanocarriers, 4T1 cells were pre-incubated with genistein or β-cyclodextrin at 37 °C for 1 h to selectively inhibit endocytosis. Subsequently, PP@DOX (equivalent to 10 μM DOX) was added and cells were exposed for an additional 2 h under the same conditions. To assess the involvement of energy-dependent uptake, parallel experiments were conducted at 4 °C and 37 °C. After incubation, cells were rinsed with ice-cold PBS and subjected to flow cytometric analysis. All assays were performed in triplicate to ensure data reliability.

4.16. Hemolysis experiment

Fresh blood (1.0 mL) was collected from healthy female mice, and red blood cells (RBCs) were isolated by centrifugation at 3500 rpm for 5 min. After washing with PBS until the supernatant was clear, RBCs were resuspended in 15.0 mL PBS. Then, 0.5 mL of RBC suspension was incubated with 0.5 mL of PP@ solutions at varying concentrations (final PP@: 1.56–100 μM). For controls, RBCs were treated with 0.5 mL distilled water (positive control) or PBS (negative control). All samples were gently mixed and kept at room temperature for 2 h, followed by centrifugation at 3500 rpm for 5 min. Supernatant (100 μL) was transferred to a 96-well plate for absorbance measurement at 540 nm. Hemolysis was calculated as [26].

Hemolysis%= (sample absorbance negative control)/(positive control negative control) × 100 %.

4.17. In vivo anticancer activity assay

BALB/c mice were subcutaneously injected with 100 μL of 4T1 cells (6 × 10⁶ cells/mL). Once tumors reached approximately 100 mm³, mice were randomly divided into six groups: PBS (control), CDDP, IBU, PP@, IBU + CDDP, and PP@CDDP. Treatments were administered via tail vein every three days (CDDP: 4.4 mg/kg; IBU: 1.1 mg/kg). Tumor dimensions were recorded every other day over a 16-day period. Four days after the final treatment, mice were euthanized for the collection of tumors, major organs, and blood samples.

A parallel survival study followed the same treatment regimen but extended observation beyond day 16, with continuous monitoring of body weight, tumor progression, and general condition.

4.18. Pathological analysis and detection of GSH content

Tumor tissues were paraffin-embedded and stained for Ki-67 to assess proliferation. Apoptosis was evaluated using a TMR-labeled Caspase-3 assay kit (Servicebio, G1502). H&E staining was conducted using a commercial kit (Servicebio, G1003) with minor modifications. Intratumoral GSH levels were quantified using a GSH Assay Kit (Servicebio, G1503).

4.19. In vivo biodistribution

A near-infrared fluorescent probe, DIR, was used to label nanoprodrugs (PP@DIR) for in vivo imaging. PSSP (1.0 mg) in 100.0 μL THF was mixed with 20.0 μL DIR ethanol solution (1.0 mg/mL), followed by dropwise addition into 1.0 mL PBS to allow nanoparticle self-assembly. Free DIR solution, prepared by the same method without PSSP addition, served as a control.

Female BALB/c mice bearing 4T1 tumors were intravenously injected with either free DIR or PP@DIR (DIR dose: 60.0 μg/kg). Biodistribution was imaged at 4h, 24h and 48h post-injection using an in vivo imaging system (IVIS Lumina III). Afterward, mice were sacrificed, and tumors and major organs were harvested for ex vivo fluorescence imaging.

4.20. Biosafety assessment

Biosafety assessment was performed based on established protocols. Kunming mice were randomly divided into six groups (n = 3–6 per group) and intravenously administered various formulations, each containing cisplatin (CDDP) at 4.4 mg/kg and ibuprofen (IBU) at 1.1 mg/kg, following the same dosing schedule used in the therapeutic study on the 4T1 tumor model. On day 17 after the first injection, blood samples were collected for serum biochemical analysis. Liver function markers (ALT and AST) and kidney function markers (BUN and CRE) were quantified using commercially available ELISA kits, with validation performed according to the manufacturer's instructions.

4.21. Statistics

Statistical analyses were performed using GraphPad Prism. Data are presented as mean ± standard deviation (SD). Unpaired two-tailed Student's t-test was used for pairwise comparisons. For multiple comparisons, two-way ANOVA with Tukey's post hoc test was applied. A p-value <0.05 was considered statistically significant.

CRediT authorship contribution statement

Guangxing Xie: Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. Weidong Zhao: Writing – review & editing, Methodology, Funding acquisition, Formal analysis, Conceptualization. Qian Liu: Methodology. Bingjie Wang: Methodology. Rongrong Feng: Methodology. Hao Sun: Formal analysis. Mei Cong: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

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.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.