Abstract

   Currently, given variations in the abnormal tumor microenvironment
   (TME), significant progress has been made in the treatment of breast
   cancer (BC) based on TME-responsive nanosystems-mediated ferroptosis.
   Due to inherent deficiencies such as the limited efficacy of Fe-induced
   catalysis, the lack of endogenous hydrogen peroxide (H[2]O[2]), and the
   overexpression of glutathione (GSH) in tumor cells, the treatment of
   ferroptosis is restricted. Here, this document highlighted polymeric
   nanoparticles with GSH resection (ssP-tHB@Fe/DOX), demonstrating
   significant improvements in TME responses, biocompatibility, and
   anti-tumor effects. Our study offers a platform for combined BC
   amplification therapy involving ferroptosis and DOX-induced
   chemotherapy, and it highlights the use of 3,4,5-trihydroxybenzaldehyde
   (tHB) in a Fe(III)-Fe(II) cyclic reaction to deplete GSH and enhance
   ferroptosis. Notably, ssP-tHB@Fe/DOX could effectively disrupt
   mitochondrial structure and reduce membrane potential in tumor cells,
   leading to decreased ATP production, depletion of GSH via multi-channel
   approach, inhibition of GPX4 expression, and accumulation of lipid
   peroxidation, thereby inducing ferroptosis enhancement to achieve BC
   therapy. Meanwhile, the release of the anticancer drug DOX has dual
   effects: it interferes with NADPH to enhance the Fe-mediated Fenton
   reaction and induces apoptosis in tumor cells. Moreover, RNA sequencing
   (RNA-seq) analysis robustly supported the anti-tumor mechanism of
   ssP-tHB@Fe/DOX, confirming involvement in the amino acid metabolism
   ferroptosis signaling pathway and the p53 signaling pathway, etc.
   Therefore, in this study, we thoroughly introduced the design of
   intelligent nanosystems, proposed methods to enhance DOX-induced
   apoptosis and ferroptosis, offering a novel approach for cancer
   treatment.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12951-025-03569-7.

   Keywords: Ferroptosis amplification, GSH response, Chemotherapy, Breast
   Cancer, Polymeric nanoparticles

Introduction

   Breast cancer (BC), one of the most prevalent malignancies, is also the
   leading cause of cancer-related deaths in women worldwide [[48]1,
   [49]2]. Despite numerous standard treatment options for BC, including
   chemotherapy, radiotherapy, endocrine therapy, and surgery, these
   treatments often have undesirable side effects, in which chemotherapy
   remains central to BC treatment, whether preoperative or postoperative
   [[50]3, [51]4]. Generally, chemotherapy often yields significant
   therapeutic benefits initially, but its effectiveness diminishes over
   time [[52]5, [53]6]. Doxorubicin (DOX), a primary anti-tumor
   anthracycline drug known for its ability to induce tumor cell death
   through apoptosis, also causes significant cytotoxicity by inhibiting
   DNA replication via topoisomerase II or triggering
   mitochondria-mediated apoptosis through the formation of semiquinone
   radicals [[54]7, [55]8]. However, like other similar chemotherapeutic
   agents, DOX has inherent issues that cannot be overlooked, including
   multidrug resistance, poor targeted delivery, and damage to normal
   organs or tissues, such as cardiotoxicity and skin inflammation [[56]6,
   [57]9, [58]10], leading to often severely restricted in their
   applications. Therefore, a new strategy is urgently needed to enhance
   the effectiveness of BC therapy and reduce the adverse effects of
   chemotherapy.

   Recently, addressing these defects has become crucial for the
   successful implementation of traditional chemotherapy drugs in clinical
   treatments. Stimuli-responsive nanoparticles, as intelligent
   nano-carriers, offer numerous advantages, including high encapsulation
   of hydrophobic drugs, tumor-specific treatment, and low cytotoxicity,
   making them a focal point in precision nanomedicine therapy [[59]8,
   [60]11–[61]13]. Polymer micelles are formed spontaneously when
   two-parent block copolymers dissolve in water, creating a nuclear shell
   structure to facilitate the encapsulation of drugs, making them
   suitable for precise drug delivery in cancer treatment [[62]14]. Given
   the peculiarity of the tumor microenvironment (TME), polymer-based
   nanomicelles often incorporate mechanisms that control drug release in
   specific spatial and temporal contexts, such as acidic pH values
   [[63]15, [64]16], strong levels of reactive oxygen species (ROS)
   [[65]17, [66]18], and endogenous glutathione (GSH) [[67]19, [68]20]
   etc., to achieve significant improvements in the chemo drug’s drawbacks
   as well as improve the effect of tumor therapy. In addition, responsive
   nanomicelles that can be activated externally through light,
   ultrasound, X-rays [[69]21, [70]22], and other stimuli have garnered
   significant interest, but it is difficult to obtain large-scale
   expansion due to the limitations of the expensive instrument,
   complexity of stimulation conditions, etc. Therefore, among these,
   researchers seek endogenous GSH-triggered responsive polymers because
   the GSH content in tumor tissue is significantly higher than in normal
   cells, ranging from 2 to 20 µM, which is 100 to 1,000 times greater
   [[71]17, [72]23]. Moreover, GSH imbalance aids in inducing apoptosis in
   tumor cells by increasing ROS levels, thereby enhancing the
   effectiveness of chemotherapy drugs. For example, platinum (II) readily
   reacts with intracellular GSH, forming water-soluble Pt-GSH compounds
   that are excreted from tumor cells, thereby reducing the cross-linking
   between platinum and DNA [[73]24]. Furthermore, research findings
   indicate that GSH plays a role in the process of cell detoxification
   through catalytic reaction via glutathione-s-transferases (GSTs),
   resulting in reduced efficacy of chemotherapy drugs (DOX and
   carmustine) and tumor cell resistance [[74]25]. Although GSH-responsive
   polymeric nanomicelles-loaded chemotherapy drugs have been used in a
   variety of anti-tumor treatments, how to accurately regulate GSH level
   in tumors to maximize synergistic antitumor therapy remains a
   challenge.

   Ferroptosis, as a new type of regulatory cell death recently
   discovered, is primarily driven by iron overload, ROS accumulation and
   lipid peroxidation (LPO), differs from other forms of cell death such
   as necrosis, apoptosis, and autophagy [[75]26, [76]27]. In simple
   terms, there are two primary mechanisms that trigger ferroptosis: the
   iron-mediated ROS pathway and the glutathione-dependent peroxidase 4
   (GPX4) pathway [[77]28]. Over the past decade, the strategies of
   ferroptosis-dependent tumor therapy have evolved rapidly and yielded
   good results, especially in multimodal anti-tumor therapy. Because of
   their complexity and variability, it is well known that treating tumors
   with a single chemotherapy drug can easily lead to resistance in tumor
   cells, resulting in chemotherapy failure and potentially worsening the
   patient’s condition. Therefore, it is a trend to combine multiple death
   patterns to overcome the tumor’s ability to self-adjust [[78]29]. At
   present, iron-based nanocarriers induce ferroptosis in tumor cells,
   such as iron oxide nanoparticles [[79]30, [80]31], iron-monatomic
   nanoparticles [[81]32, [82]33], and iron-organic frameworks [[83]34],
   through increasing intracellular iron dosage and ROS levels to achieve
   tumor ablation. Despite significant advancements in tumor treatment
   using inorganic or hybrid nanoparticles rich in iron ions, their
   anticancer efficacy is somewhat limited due to the complex and diverse
   tumor microenvironment, along with issues related to poor stability and
   uncontrolled degradation. Therefore, developing new iron-rich
   nanocarriers and effectively inducing ferroptosis for tumor treatment
   remains a significant challenge.

   Herein, we fabricated a GSH-activated and polyphenol-modified
   amphiphilic three-block polymers (named ssP-tHB), and effectively
   loaded the FDA-approved anti-cancer drug DOX into hydrophobic regions
   based on the self-assembly strategy, while the introduction of Fe^3+
   induced cross-linking again through the iron ion-phenolic network to
   form a stable nanoplatform (named ssP-tHB/Fe@DOX) for the treatment of
   BC (Scheme [84]1). Key challenges in chemotherapy involving
   nanocarriers carrying chemotherapeutic drugs include ensuring
   controlled drug release at the tumor site and preventing drug leakage
   during blood circulation. Therefore, we proposed to further secure the
   DOX in the hydrophobic layer through cross-linking with
   Fe^3+-polyphenol coordination, enhancing intracellular iron storage and
   the release of tHB small molecules to synergistically induce
   ferroptosis. Additionally, extensive studies show that DOX drug can
   exacerbate ferroptosis by activating the expression of NADPH oxidase 4
   (NOX4), a family of membrane-bound enzyme complexes that enhance
   H[2]O[2] production [[85]35]. After arriving to the over-expressed of
   GSH and acidic conditions in tumor cells, ssP-tHB/Fe@DOX undergoes
   GSH-triggered in situ decomposition, and releasing Fe^2+, tHB molecule,
   and DOX drug. Consequently, intracellular GSH levels are viciously
   consumed, leading to a disruption in the balance of GSH/ROS, which
   further results in GSH-driven chemotherapy and ferroptosis
   synergistically reinforcing therapy to mediate BC tumor cell death.

Scheme 1.

   [86]Scheme 1
   [87]Open in a new tab

   Schematic illustration of the fabrication processes of polymeric
   ssP-tHB@Fe/DOX and its application as a ferroptosis amplifier and
   DOX-induced apoptosis for synergistic antitumor therapy

Results and discussion

Synthesis and characterization of block copolymers and NPs

   The synthetic routes were minutely depicted in Fig. [88]S1. Initially,
   employing a classical one-pot method [[89]36], the diblock
   three-component copolymer with tunable stability,
   poly(cyclobutane−1,2,3,4-tetracarboxylic dianhydride-co-hydroxyethyl
   disulfide)-polyethylene glycol (mPEG[5K]-p(CBDA-co-BiBi)-mPEG[5K],
   abbreviated as ssP-COOH), was synthesized, using 2-hydroxyethyl
   disulfide (BiBi) monomer and cyclobutane−1,2,3,4-tetracarboxylic
   dianhydride (CBDA) molecular synthesis as functional skeletons, as well
   as the introduction of mPEG[5k]-NH[2] chain as hydrophilic fragment
   (Fig. [90]S1). Based on the nucleophilic reactions, thus, the
   as-prepared ssP-COOH copolymer displayed numerous disulfide bonds
   (-S-S-) and pendent pairwise carboxylic acid (-COOH) groups, providing
   a foundation for creating diverse copolymers. Subsequently, an standard
   EDC/HOBT-catalyzed amidation reaction was performed to couple the side
   carboxylic acid group with ethylenediamine-monoboc (EDA-Boc), and then
   the modified copolymer was named as ssP-EDA-Boc copolymer. The next
   work was a simple in-situ Boc-deprotection reaction using
   trifluoroacetic acid (TFA) solution to form an amino-modified polymer
   (ssP-EDA copolymer). The chemical structures of these block copolymers
   were characterized through ^1H nuclear magnetic resonance (^1H NMR)
   spectroscopy (Fig. [91]S2) and fourier transform infrared (FTIR)
   spectroscopy (Fig. [92]S3) and UV-vis spectra (Fig. [93]S4), confirming
   their successful synthesis.

   Furthermore, it is well known that, in aqueous solution, amphiphilic
   block copolymers can spontaneously drive the formation of diverse
   nanoparticles (such as spheres, cylinders and lamellaes) through
   self-assembly approach [[94]36, [95]37]. Therefore, the distinct NPs,
   including ssP-COOH, ssP-EDA-Boc, and ssP-EDA, were fabricated in mild
   solution. The diameter of these NPs decreased progressively from
   197.6 nm to 146.1 nm and then to 108.1 nm, as analyzed by DLS, due to
   the gradual modification of copolymer (Fig. [96]S5a, [97]S5c), mainly
   probably due to the increased hydrophobic capacity and electrostatic
   adsorption effect. Moreover, variations in zeta potential further
   proved the success of grooming (Fig. [98]S5b).

   To enhance the strong active metal (e.g., Fe^3+) carrying capacity of
   block copolymers, we incorporated a polyphenol-like structure with
   acid-responsive Schiff-base bonding, utilizing the
   3,4,5-trihydroxybenzaldehyde (tHB) monomer. A new peak appeared around
   7.9–6.7 ppm in the ^1H NMR spectrum was attributed to the benzene ring
   structure, indicating the successful preparation of tHB-decorated block
   copolymer (ssP-tHB) (Fig. [99]S2). Besides, the presence of v (C = N)
   azomethine stretching vibrations was confirmed by the detection of
   absorption peaks at 1720 cm ^− 1 for tHB, which indicates the
   establishment of Schiff base linkage [[100]19].

   Next, the FDA-approved doxorubicin (DOX), as a hydrophobic broad
   spectrum anti-cancer drug, often lacks bio-targeting, leading to
   cellular resistance and limiting its effectiveness in cancer treatment
   [[101]19, [102]38]. To that end, using nano-precipitation, the ssP-tHB
   block copolymer created square nanoparticles that encapsulated DOX
   through hydrophobic interactions. These were then enhanced with
   polyphenol-iron coordination to form ssP-tHB@Fe/DOX (Fig. [103]1a),
   which boasts strong stability, minimal drug leakage, and high iron
   ion-carrying capacity.

Fig. 1.

   [104]Fig. 1
   [105]Open in a new tab

   Preparation and characterization of ssP-tHB@Fe/DOX. (a) Synthesis
   routes of different nanoparticles. (b, c) Hydrodynamic sizes and TEM
   images of ssP-tHB (b), and ssP-tHB@Fe/DOX (c). (d) Zeta potential
   changes of nanoparticles (n = 5). (e) EDS elemental mapping images of
   ssP-tHB@Fe/DOX. (f) EDS elements of ssP-tHB@Fe/DOX. (g) Powder XRD
   patterns of free DOX, ssP-tHB, and ssP-tHB@Fe/DOX. (h) UV-vis
   absorption spectra of free DOX, ssP-tHB, and ssP-tHB@Fe/DOX. Inset: The
   corresponding photos of samples. (i) XPS spectra of ssP-tHB and
   ssP-tHB@Fe/DOX. (j) High-resolution S 2p XPS spectra of ssP-tHB and
   ssP-tHB@Fe/DOX

   After that, the composition and structure of ssP-tHB and
   Fe/DOX-coloaded ssP-tHB were minutely characterized. By DLS testing, in
   aqueous solution, the self-assembled ssP-tHB block copolymer
   prominently formed spherical nanoparticles with a hydrodynamic particle
   size of 147 nm and a polydispersity index (PDI) of 0.134
   (Fig. [106]1b). Fe/DOX-loaded NPs have a larger hydrodynamic diameter
   of approximately 220 nm and a PDI of 0.164 compared to blank NPs,
   demonstrating successful payload incorporation (Fig. [107]1c).

   In TEM images, ssP-tHB@Fe/DOX in rough tablet-like morphology with
   about 350 nm in length and 200 nm in width was showed, whereas blank
   NPs were uniformly spherical, measuring 50–60 nm (Fig. [108]1b),
   indicting that DOX loading and iron ions coordination can influence the
   morphology of the nanoassemblies. Furthermore, the self-assembled
   ssP-tHB solution shrunk in its dry state due to the dehydration effect
   [[109]19, [110]20]. However, this similar phenomenon did not occur with
   ssP-tHB@Fe/DOX, primarily because iron-phenol crosslinking stabilized
   the nanoparticle structure.

   The zeta potential of ssP-tHB decreased from 13.42 mV (ssP-EDA) to 0.46
   mV, attributed to the negative charges of tHB pendant groups [[111]39].
   Meanwhile, a positively charged ssP-tHB@Fe/DOX (9.88 mV) was observed
   compared to blank NPs (ssP-tHB) (Fig. [112]1d), mainly due to the
   successful incorporation of the positively charged DOX drug and iron
   ions. As showed in UV-vis absorption spectra (Fig. [113]1h), ssP-tHB
   illustrated no obvious characteristic absorption peak. Conversely, the
   absorption peak at 410–550 nm, characteristic of ssP-tHB@Fe/DOX or DOX,
   indicates the successful incorporation of DOX into the nanoplatform.

   To investigate the loading of DOX and iron ions, we subsequently
   conducted a series of experiments. In the study, through solution
   self-assembly, the anticancer drug DOX was encapsulated, followed by
   the introduction of trivalent iron ions for metal coordination to
   further prevent leakage of DOX and enhance the stability of NPs.
   Therefore, the prepared nanoparticles (ssP-tHB@DOX) had an average
   particle size of approximately 165 nm, with a PDI of 0.121 and a zeta
   potential of 4.56 mV, via DLS measurement (Fig. [114]S6a), indicating
   that the hydrophobic chemotherapeutic drug DOX was primarily
   encapsulated within the hydrophobic region of block copolymers through
   self-assembly method. Furthermore, the UV-vis spectra of different
   samples were analyzed. As shown in Fig. [115]S6b, after mixing free DOX
   with Fe^3 + solution, the characteristic peak of DOX at about 480 nm
   did not fluctuate, indicating that the chelation between iron ions and
   DOX did not interfere with the characteristic absorption peak of DOX.
   Subsequently, when ssP-tHB@Fe/DOX was added in DMSO solution, it was
   clearly observed through digital photography that the red color of the
   DMSO solution became deeper compared to in aqueous solution, and the
   characteristic peaks of DOX also enhanced by UV-vis spectra (Fig.
   [116]S6c), indicating that the DMSO solution effectively disrupted the
   polymer-based nanoparticles, promoting the release of DOX from the
   hydrophobic cavities of the copolymer. Furthermore, the DMSO-treated
   ssP-tHB@Fe/DOX was divided into two segments, with sizes ranging from
   40 to 68 nm and 531–955 nm (Fig. [117]S6d), further demonstrating that
   DMSO solution could effectively disrupt NPs stability. As a result, the
   weakening of DOX’s characteristic peaks after drug loading could be
   attributed to: (a) effective incorporation of the hydrophobic
   chemotherapeutic anticancer drug DOX into the polymer’s hydrophobic
   layer during the solution self-assembly process; (b) interference with
   DOX’s characteristic absorption peak due to π-π stacking interactions
   involving its large benzene rings [[118]20, [119]39]. Simultaneously,
   Fe^3 + mainly utilized the coordination between phenol group and iron
   ions to effectively carry iron ions on the copolymer surface.

   According to the DOX concentration-dependent absorption standard curve,
   the content of DOX in ssP-tHB@Fe/DOX estimated to be 21.2% (Fig.
   [120]S7). Moreover, the mass percentage of Fe in ssP-tHB@Fe/DOX about
   was 17.3% through ICP technology. The Fig. [121]1e showed the
   distribution of elements for ssP-tHB@Fe/DOX, and related elements such
   as C, N, O, S and Fe were evenly dispersed on its the surface,
   indicating the successful preparation of nanoparticles. In addition,
   the energy-dispersive X-ray (EDC) spectrum further corroborated this
   finding (Fig. [122]1f). A consequent X-ray photoelectron spectroscopy
   (XPS) characterization further confirmed the construction of the
   ssP-tHB@Fe/DOX (Fig. [123]1i). Meanwhile, the typical peak about at
   163.8 eV indexed as S-S or S-C bonds [[124]40], was observed in S
   sophisticated XPS spectra of ssP-tHB and ssP-tHB@Fe/DOX, effectively
   confirming the presence of nanoparticles responsive to GSH
   (Fig. [125]1j). Following this, we performed an XRD analysis on the
   crystal structures of ssP-tHB, ssP-tHB@Fe/DOX, and free DOX powder. The
   results indicated that both the solution self-assembled nanoparticles
   and the DOX drug exhibited an amorphous structure [[126]39]. During
   self-assembly, the characteristic diffraction peaks at 10º−40º of DOX
   vanished compared to ssP-tHB or ssP-tHB@Fe/DOX (Fig. [127]1g), likely
   due to π-π stacking and hydrophobic interactions between them
   [[128]39].

GSH-responsive degradation and catalytic performance

   The favorable colloidal stability and appropriate size of nanoparticles
   are crucial factors for the accumulation in tumor regions enhanced by
   the permeability and retention (EPR) effect [[129]19, [130]41]. We
   further investigated the stability of ssP-tHB@Fe/DOX in different
   solution (PBS, 10% FBS medium, and dH[2]O) (Fig. [131]2a). There were
   no significant changes in particle distribution across different media.
   Additionally, the hydrodynamic diameters of ssP-tHB@Fe/DOX remained
   constant over the seven-day tracking experiment (Fig. [132]2b). This
   establishes a foundation for using ssP-tHB@Fe/DOX in tumor therapy
   through caudal intravenous injection.

Fig. 2.

   [133]Fig. 2
   [134]Open in a new tab

   GSH responsive performance and ·OH generation assessments. (a)
   Hydrodynamic size of ssP-tHB@Fe/DOX in different media (water, PBS, and
   10% FBS) (n = 5). (b) Changes of size and zeta potential of
   ssP-tHB@Fe/DOX in PBS for 7 days (n = 5). (c) The diagram of
   H^+/GSH-emitted degradation and release of ssP-tHB@Fe/DOX. (d) Changes
   in hydrodynamic size of ssP-tHB@Fe/DOX in pH 5.0 + 10 mM GSH at 12 h.
   Inset: The corresponding TEM photo of samples. (e) UV-vis spectra for
   GSH level detection using DTNB as a probe in the presence of
   ssP-tHB@Fe/DOX. (f) Schematic illustration for Fe^2+ detection using
   1,10-Phenanthroline (Phen) probe. (g) UV-vis spectra for Fe^2+
   detection using 1,10-phenanthroline as a probe in different mixtures.
   Inset: The corresponding photographs of samples (from left to right).
   (h) High-resolution XPS spectrum of Fe 2p in ssP-tHB@Fe/DOX. (i) The
   release profile of DOX and Fe ions from ssP-tHB@Fe/DOX under different
   stimulation conditions (n = 5). (j) EPR spectra of DMPO-OH in aqueous
   solutions in different conditions. (k) UV-vis absorption spectra of
   regarding ssP-tHB@Fe/DOX-mediated MB degradation under different
   conditions. (l) Digital photos showing interactions between iron ions
   and tHB molecules under different conditions

   Current research indicates that tumor regions display significant
   differences from normal tissues, such as elevated levels of GSH, an
   acidic environment, and various enzymes that promote tumor growth
   [[135]16, [136]19]. Therefore, these tumor-specific nurturing
   microenvironments will enhance the effectiveness of intelligent
   nanotechnology delivery systems, particularly tumor-responsive drug
   release. Based on this, the nanoparticles containing S-S bonds and
   Fe^3 + ions were assessed under GSH conditions (Fig. [137]2c). As
   depicted in Fig. [138]2d, ssP-tHB@Fe/DOX undergone severe
   disintegration at pH 5.0 with 10 mM GSH over 12 h, as observed through
   TEM imaging. DLS analysis revealed three distinct particle size signals
   for acid/GSH-treated ssP-tHB@Fe/DOX: small fragments (5–10 nm), larger
   particles (300–400 nm), and micron-sized particles. This suggests that
   acid and GSH conditions can cause the degradation of ssP-tHB@Fe/DOX. We
   then explored the capacity of GSH consumption of ssP-tHB@Fe/DOX or
   ssP-tHB through the 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB)
   chromogenic method (Fig. [139]2e, S8). Nanoparticles containing
   plentiful S-S bonds showed a robust GSH-depleting capability, relying
   mainly on the presence of nanoparticle-rich disulfide bonds and
   high-valence iron ions. So, these findings indicate that ssP-tHB@Fe/DOX
   has GSH-triggered ferroptosis-inducing potential for tumor treatment.

   Consequently, the crucial role of iron in the induction of ferroptosis
   was examined. Therefore, the 1,10-phenanthrolline (Phen) probe was
   selected for detecting bivalent iron ions in ssP-tHB@Fe/DOX using
   UV-vis spectroscopy (Fig. [140]2f). The red flocculation was clearly
   showed when GSH treated ssP-tHB@Fe/DOX, indicating the production of
   Fe^2 + by GSH reduction (Fig. [141]2g). The high-resolution XPS
   spectrum of Fe 2p revealed that Fe in ssP-tHB@Fe/DOX exists as both
   Fe(II) and Fe(III). Specifically, Fe 2p[3/2] and Fe 2p[1/2] peaks were
   observed at 711.4 eV and 727.5 eV [[142]42], respectively
   (Fig. [143]2h), indicating tHB-induced generation of Fe^2 + from
   ssP-tHB@Fe/DOX. Thus, we also investigated the chemical transformations
   of small molecule tHB and iron ions in various oxidation states across
   different media (Fig. [144]2l). The conversion of Fe^3+/Fe^2 + was
   tested by a 1,10-phenanthroline probe (Fig. [145]2f). Under acidic
   conditions (pH 5.5), tHB and trivalent iron ions exhibited a pale brown
   color, unlike the Fe(II) solution. At a pH of 5.5, the solution of
   Fe(II) exhibited a red color change upon the introduction of tHB,
   indicating the reduction of Fe(II) by tHB. Interestingly, the presence
   of H[2]O[2] resulted in both Fe (II) and tHB small molecules displaying
   similar red colors. Thus, we can anticipate that the tHB-mediated
   Fe^3+-to-Fe^2 + conversion following ssP-tHB@Fe/DOX internalization by
   tumor cells will ensure continuous Fe^2 + production, facilitating
   highly efficient Fenton reactions [[146]43].

   Next, given the high GSH expression in tumors that mediates the
   response to ssP-tHB@Fe/DOX, the drug release curves of the loaded DOX
   and iron ions were investigated (Fig. [147]2c). As shown in
   Fig. [148]2i, in pH 5.0 and 10 mM GSH (simulating tumor
   microenvironments), DOX from ssP-tHB@Fe/DOX displayed a continuous
   release action, achieving a cumulative release rate of 64.2% in 36 h,
   whereas only 11.3% of DOX was released during incubation in pH 7.4 and
   10 µM GSH (simulating normal conditions) over the same time period. In
   addition, for iron ions in ssP-tHB@Fe/DOX, the analogous release
   profiles were collected by ICP testing, and approximately 68.9% of iron
   ions were released under simulated tumor conditions, while only 11.1%
   was released in the simulated normal conditions (Fig. [149]2i). Some
   factors contribute to these behaviors: (a) ssP-tHB@Fe/DOX contains
   -S-S- bonds that create the hydrophobic region leading to disassembly
   and the release of DOX and iron ions; (b) Fe (III) is reduced to Fe
   (II), weakening its coordination with polyphenol groups and
   accelerating release; (c) the Schiff base bonds attached to the tHB
   monomer detach in the presence of weak acid (pH 5.0), accelerating the
   disintegration of ssP-tHB@Fe/DOX by destroying the hydrophobic layer
   (Fig. [150]2c).

   The release of iron ions from ssP-tHB@Fe/DOX induced the generation of
   highly toxic hydroxyl radicals (·OH) via the Fe-mediated Fenton
   reaction. Methylene blue (MB), used as a colorimetric substrate, was
   chosen for detection. As depicted in Fig. [151]2j, no obvious·OH
   formation was observed in a pH 7.4 environment with 10 mM H[2]O[2]
   after 8 h, indicating the non-toxic nature of ssP-tHB@Fe/DOX prior to
   reaching the tumor site. However, under similar tumor conditions (pH
   5.0 + 10 mM H[2]O[2]), ssP-tHB@Fe/DOX produced a considerable abundance
   of ·OH, resulting in the oxidation of MB. Amusingly, rich ·OH was
   detected when 10 mM GSH was present (resembling the tumor intracellular
   environment), indicating that GSH-treated Fe (III) produced active Fe
   (II) to generate ·OH, further demonstrating that ssP-tHB@Fe/DOX has
   tumor-activated therapeutic property. Electron spin resonance (ESR)
   spectroscopy, utilizing 5.5-dimethylpyrroline N-oxide (DMPO), was
   employed to determine ·OH levels (Fig. [152]2k). We found that pH 7.4
   environment was not responsible for the production of ·OH, whereas
   iron-containing nanoparticles in pH 5.0 condition could generate
   abundant ·OH through the iron-based Fenton reaction in a manually
   simulated tumor microenvironments [[153]44], indicating that its
   potential to induce ferroptosis in tumors.

Apoptosis and ferroptosis mechanism analysis in vitro

   At present, treatment model of chemotherapy alone is prone to allow
   poor tumor efficacy and chemoresistance of tumor cells, thereby
   resulting in the failure of tumor treatment or tumor progression.
   Therefore, non-apoptotic death property, namely ferroptosis, was
   successfully combined to coordinate tumor therapy, in this study
   (Fig. [154]3h). It is well known that iron accumulation and subsequent
   lipid peroxidation (LPO) play crucial roles in triggering and
   accelerating ferroptosis. Therefore, the molecular pathways and
   signaling mechanisms involved in iron homeostasis and LPO play crucial
   regulatory roles in this process [[155]26, [156]32, [157]40]. To this
   end, we evaluated the iron uptake in Fe-containing nanoparticles
   (ssP-tHB@Fe/DOX) to indirectly understand the accumulation of iron
   levels in 4T1 cells and evaluate the ability of Fe-induced ferroptosis.
   As presented in Fig. [158]3b, the time-dependent uptake of iron ions in
   4T1 cells showed that ssP-tHB@Fe/DOX could be internalized by cells and
   resided inside cells, laying the foundation for inducing ferroptosis.
   Furthermore, iron ions alone also showed an increase in
   time-dependence, possibly due to the presence of tumor cells with
   ferrophilic effect.

Fig. 3.

   [159]Fig. 3
   [160]Open in a new tab

   Antitumor mechanisms of ssP-tHB@Fe/DOX. (a) ROS levels (indicated by
   DCFH-DA) in 4T1 cells after various treatments using CLSM. (b) Analysis
   of intracellular Fe^2+ content in 4T1 cells at different time points
   (0, 4, 8, and 12 h) after incubation with PBS, FeCl[3], and
   ssP-tHB@Fe/DOX. (c) Confocal microscopic images of 4T1 cells stained
   with JC-1 probes to evaluate intracellular mitochondrial membrane
   potential. (d) CLSM images of C11-BODIPY-stained 4T1 cells after
   different treatments. Scale bar = 20 μm. (e) Analysis of the GSH/GSSG
   ratio in cells. (f) Western blot analysis of GPX4 expression. (g)
   Representative bio-TEM images of 4T1 cells. (h) The proposed schematic
   diagram of ssP-tHB@Fe/DOX-induced ferroptosis/apoptosis. (i, j, and k)
   Intracellular MDA levels (i), ATP content (j), and H[2]O[2]
   concentration (k) of 4T1 cells after different treatments. (l) Western
   blot analysis of the expressions of Bax, Bcl-2, and NOX4. Note: PBS
   (G1), ssP-tHB (G2), FeCl[3] (G3), DOX (G4), and ssP-tHB@Fe/DOX (G5)

   Subsequently, treatments by different samples of 4T1 intracellular GSH
   levels were examined through GSH/GSSG assay kit. Figure [161]3e
   indicated that GSH levels significantly decreased compared to the PBS
   group following treatment with various samples, including DOX, FeCl[3],
   ssP-tHB, and ssP-tHB@Fe/DOX. As anticipated, the consumption of
   endogenous GSH levels was optimal during treatment with ssP-tHB@Fe/DOX
   compared to other groups. This phenomenon occurred due to two main
   reasons: (i) the involvement of reduction reactions in groups
   containing Fe^3+, and (ii) the consumption of nanoparticles through a
   sulfhydryl-dissulfhydryl exchange reaction with GSH.

   Additionally, according to earlier reports, the consumption of GSH
   disrupts the equilibrium of intracellular redox system, leading to the
   emergence of cell oxidative stress and LPO [[162]45]. We primarily
   monitored the generation of intracellular ROS utilizing
   2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. As shown
   in Fig. [163]3a, the strong intensity of green fluorescence from DCF
   was displayed after incubation of ssP-tHB@Fe/DOX. Besides, other
   treated groups (FeCl[3] and DOX) also showed low intensity of green
   signal compared to control group or ssP-tHB, demonstrating that the
   generation of ROS. To investigate the production of •OH via
   iron-mediated Fenton reaction within tumor cells, the fluorescence
   probe of HPF (indicating intracellular •OH levels) was employed
   [[164]46]. As shown in Fig. [165]S9, a notable increase in HPF green
   fluorescence was observed in the ssP-tHB@Fe/DOX group compared to PBS,
   ssP-tHB, FeCl[3], and DOX groups. This analysis further confirmed that
   ssP-tHB@Fe/DOX-treated 4T1 cells induced the generation of abundant •OH
   through iron-mediated Fenton reaction, leading to oxidative damage of
   cell membranes and triggering ferroptosis.

   Owing to the strong oxidative capacity of ROS in 4T1 cells,
   intracellular polyunsaturated fatty acids (PUFA) readily undergo
   oxidation, forming lipid peroxides (LPO), which leads to cell
   ferroptosis [[166]45]. Therefore, we selected C11-BODIPY^581/591 probe
   to detect LPO utilizing changes in the excitation emission wavelength
   in cells, to assess its ability to induce ferroptosis. Figure [167]3d
   demonstrated that red fluorescence decreased (reduction state) and
   green fluorescence increased (oxidized form) following ssP-tHB@Fe/DOX
   treatment, compared to other treatment groups, indicating the strongest
   LPO activity. Moreover, DOX-treated 4T1 cells demonstrated a similar
   level of LPO activity, aligning with the observed production of ROS. As
   expected, by malondialdehyde (MDA) concentration in 4T1 cells testing,
   similar results were observed (Fig. [168]3i). Since the Fe-mediated
   Fenton reaction depends on high levels of H[2]O[2], we investigated the
   production of H[2]O[2] in 4T1 cells by various samples. As already
   known, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4),
   primarily derived from mitochondria, efficiently generates H[2]O[2] by
   catalyzing the transfer of electrons from NADPH to oxygen [[169]47,
   [170]48]. In early experiments, DOX not only induces cell apoptosis but
   also triggers oxidative stress by increasing the level of H[2]O[2]. A
   significant amount of H[2]O[2] was produced during DOX treatment,
   contributing to the ROS generation in tumor cells induced by DOX
   [[171]49]. In addition, DOX-containing nanoparticles also produced
   abundant H[2]O[2], compared to blank carriers (ssP-tHB) (Fig. [172]3k).
   Therefore, in the tumor microenvironments, the circulatory
   amplification therapeutic strategies were formed, on the one hand, DOX
   induced chemotherapy, and on the other, DOX-assisted H[2]O[2]
   production also aided in the amplification of ferroptosis.

   The morphology of mitochondria in 4T1 cells treated with ssP-tHB@Fe/DOX
   was examined using bio-TEM. Compared to the PBS group, the
   ssP-tHB@Fe/DOX-treated mitochondria exhibited typical ferroptosis
   characteristics, including smaller size, increased membrane density,
   reduced or absent mitochondrial ridges, and disrupted outer
   mitochondrial membranes [[173]50] (Fig. [174]3g). Figure [175]3c showed
   CLSM images of 4T1 cells after treatments of different samples and
   stained with
   5,5′,6,6′-tetrachloro−1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine
   iodide (JC−1) to monitor the intracellular mitochondrial membrane
   potential (MMP). The JC−1 showed red fluorescence after treatment with
   PBS or ssP-tHB, indicating a state of aggregation within mitochondria.
   However, the JC−1 showed increased green fluorescence after treatment
   with free FeCl[3] or DOX alone, and the most intense green fluorescence
   when treated with ssP-tHB@Fe/DOX, suggesting the presence of monomers
   within mitochondria in 4T1 cells and indicating the strong •OH
   generation capability of ssP-tHB@Fe/DOX. This occurs because the •OH
   can harm the mitochondria, resulting in decreased MMP.

   Extensive studies have identified glutathione peroxidase 4 (GPX4) as a
   classic indicator of ferroptosis [[176]23, [177]24]. Abnormalities in
   intracellular mitochondrial activity, particularly in energy
   metabolism, were observed due to ssP-tHB@Fe/DOX-induced ferroptosis.
   The laboratory results indicated a significant decrease in ATP levels
   in 4T1 cells during ssP-tHB@Fe/DOX cultivation, compared to the control
   group. Other treatment groups (ssP-tHB, free FeCl[3], and DOX alone)
   also showed the reduction of ATP levels (Fig. [178]3j). To better
   understand the mechanisms of ssP-tHB@Fe/DOX-induced cell death,
   specifically apoptosis and ferroptosis, we investigated the related
   pathways in pre-treated 4T1 cells. The results of the western blot (WB)
   assays visually demonstrated that ssP-tHB@Fe/DOX inhibits GPX4
   expression in vitro (Fig. [179]3f). In the free FeCl[3] treatment
   group, there was a lower reduction in GPX4 levels compared to the
   normal group, which mirrored the results of GSH consumption due to
   their positive correlation [[180]39]. Additionally, the decrease in
   GXP4 expression in blank ssP-tHB also could be attributed to the
   consumption of GSH. Hence, the expression of levels of NADPH oxidase 4
   (NOX4) in cells was investigated. As illustrated in Fig. [181]3l, S10c,
   DOX (G4) distinctly raised an enhancement in NOX4 level in comparison
   to the control group. Additionally, Additionally, treatment with
   ssP-tHB (G2) and iron ions (G3) showed minimal change. However, during
   the conversion of low-toxicity H[2]O[2] into high-toxicity ·OH,
   ssP-tHB@Fe/DOX-treated mitochondria also experienced a certain degree
   of mitochondrial dysfunction, such as mitochondrial shrinkage and
   dissipation of membrane potential. Therefore, through WB analysis (Fig.
   [182]S10c), we intriguingly observed that free DOX-induced NOX4 level
   was slightly higher than those induced by ssP-tHB@Fe/DOX. Subsequently,
   the impact of ssP-tHB@Fe/DOX on apoptotic pathways in 4T1 cells was
   further investigated. As illustrated in Fig. [183]3l, S10a, and S10b,
   G5 significantly mediated the increase in pro-apoptotic protein Bax and
   the decrease in anti-apoptotic protein Bcl−2. In other therapeutic
   groups, such as G2, G3, and G4, there was a decrease in anti-apoptotic
   Bcl−2 proteins and an increase in pro-apoptotic Bax proteins compared
   to the control group, indicating their ability to induce apoptosis to
   some extent.

Cellular internalization and cytotoxicity assays in vitro

   Next, relevant cytotoxicity experiments in vitro were evaluated through
   CCK−8 assays (Fig. [184]4a). Blank ssP-tHB and tHB-treated 4T1 cells
   showed significant biocompatibility, even at a concentration of
   640 µg/mL, with only a minor decrease in cell viability (Fig.
   [185]S11). Subsequently, the normal L929 cells were used to investigate
   the tumor-specific characteristics of ssP-tHB@Fe/DOX and free DOX
   (Fig. [186]4b, S12). After 24 h of incubation, the CCK−8 measurement
   results showed that increasing concentrations of ssP-tHB@Fe/DOX showed
   minimal cytotoxicity, demonstrating significant biosafety for normal
   cells. However, due to the spectral anti-cancer effect of free DOX and
   its potent cytotoxicity, healthy L929 cells exhibited
   concentration-dependent cytotoxic effects (Fig. [187]S12).
   Additionally, a dose-dependent cytotoxicity effect on 4T1 cells was
   exhibited in the presence of ssP-tHB@Fe/DOX, further indicating its
   tumor-specific therapeutic potential (Fig. [188]4b). As shown in
   Fig. [189]4c, trivalent iron ions (G3) also exhibited a degree of
   anti-tumor effect, with a cell survival rate of 72.7%, which may be
   attributed to iron-induced cell death through the Fenton reaction. In
   DOX-treated experiments (G4) at equal doses, ssP-tHB@Fe/DOX (G5,
   125 µg/mL) demonstrated significantly greater cytotoxicity compared to
   free DOX.

Fig. 4.

   [190]Fig. 4
   [191]Open in a new tab

   In vitro cytotoxicity related assessment. (a) The viability of 4T1
   cells after different ssP-tHB concentrations treatment. (b) Cell
   viability of 4T1 cells and L919 cells treated with ssP-tHB@Fe/DOX for
   24 h. (c) Relative viability of 4T1 cells in different samples. (d)
   Cellular uptake detected by CLSM. (e) CLSM images of Calcein
   AM/PI-stained 4T1 cells after different treatments. (f) Quantification
   of live and dead cells after treatment. (g) 4T1 cell apoptosis assessed
   through annexin V-FITC and PI staining, analyzed by FCM. (h) Histogram
   showing quantification of different apoptotic forms. Note: PBS (G1),
   ssP-tHB (G2), FeCl[3] (G3), DOX (G4), and ssP-tHB@Fe/DOX (G5)

   Cellular uptake behavior is considered a crucial indicator for
   assessing the anti-tumor efficacy of “nano-vehicles” [[192]51].
   Therefore, as free DOX or ssP-tHB@Fe/DOX-cultured time increased, the
   intensity of red fluorescence signals became stronger by CLSM images
   (Fig. [193]4a and d). Simultaneously, for free DOX, 4T1 cells showed a
   time-dependent increase in fluorescence. Importantly, as the culture
   time increases, red fluorescence (DOX) overlapped with blue
   fluorescence (nucleus), indicating that DOX released from
   ssP-tHB@Fe/DOX had moved from the cytoplasm to the nucleus for
   chemotherapy. The effect of phagocytosis of ssP-tHB@Fe/DOX was more
   pronounced compared to free DOX, likely due to the enhanced cellular
   uptake facilitated by nanoparticles.

   Calcein-AM (green fluorescence for live cells) and propidium iodide
   (PI, red fluorescence for dead cells) were used together to
   differentiate between live and dead 4T1 cancer cells. As shown in
   Fig. [194]4e and f, after processing with blank ssP-tHB (G2), nearly
   all green fluorescence signals were observed, indicating its non-toxic
   nature. Free FeCl[3] (G3) and DOX (G4) were found to kill cells to some
   extent, but their therapeutic effect was limited compared to the
   ssP-tHB@Fe/DOX. The results aligned with the conclusions of the CCK−8
   assay.

   Given that the chemotherapy drug DOX effectively induces apoptosis in
   tumor cells [[195]42, [196]47], we also examined the apoptosis of
   different samples on 4T1 cells using flow cytometry (Fig. [197]4g and
   h). Compared to the control group (G1) or blank NPs (G2), the
   proportion of apoptotic cells significantly increased in the
   FeCl[3]-treated group (G3). Furthermore, free DOX (G4) showed strong
   cell apoptosis, with the apoptosis rate of 50.5%. Of note, the cell
   apoptosis rate rose to 77.9% after treatment with ssP-tHB@Fe/DOX (G5).

Hemolysis and acute toxicity test in vivo

   Inspired by the brilliant performance of ssP-tHB@Fe/DOX against 4T1
   cells in vitro, its anti-tumor efficacy was further assessed in vivo.
   To investigate the biosafety of ssP-tHB@Fe/DOX, we performed hemolysis
   tests and acute toxicity measurements in vivo. The hemolysis rates were
   low, at less than 10%, compared to the control group. At a
   concentration of 200 µg/mL, the ssP-tHB@Fe/DOX caused a hemolysis rate
   of only 8.78%, indicating that the erythrocyte structure remained
   normal (Fig. [198]S13). As a result, ssP-tHB@Fe/DOX, with its
   tumor-specific therapy, demonstrated significant blood compatibility
   and can be further utilized in in vivo bio-related assessments.
   Subsequently, female Balb/c mice were randomly divided into two groups
   (n = 6), and then injected intravenously with PBS (control group),
   ssP-tHB@Fe/DOX (DOX: 6 mg/kg, 100 µL), respectively. After 8 and 14
   days of treatment, all mice were euthanized, and their major organs
   (including the heart, liver, spleen, lung, and kidney) were removed for
   hematoxylin and eosin (H&E) staining (Fig. [199]S14), and blood was
   also collected from their eyeballs for biochemical analysis (Tables
   [200]S1, [201]S2) [[202]52]. The H&E analysis revealed no significant
   organ damage or inflammation in the ssP-tHB@Fe/DOX-treated mice.
   Furthermore, ssP-tHB@Fe/DOX injection had no effect on liver and kidney
   functions or blood parameters. In summary, the GSH/pH dual-responsive
   ssP-tHB@Fe/DOX showed significant potential for biological
   applications.

RNA-seq analysis of ssP-tHB@Fe/DOX

   RNA sequencing (RNA-seq) analysis was conducted to evaluate the
   therapeutic mechanism of ssP-tHB@Fe/DOX in 4T1 cells. As shown in
   Fig. [203]5a, the heat map of mRNA reveals changes in the cell
   transcriptome induced by treatment with ssP-tHB@Fe/DOX. Moreover,
   principal component analysis (PCA) revealed a significant clustering
   relationship between the ssP-tHB@Fe/DOX treatment group and the PBS
   control group (Fig. [204]5b). We then conducted bioinformatics analyses
   to compare the differences (p-value < 0.05 and|log[2] FC| > 1)
   (Fig. [205]5c). The results of the collection found that the threshold
   of differentially expressed genes (DEGs) under ssP-tHB@Fe/DOX-mediated
   4T1 cells was 3373, of which 1873 gene up-regulated, 2101 gene
   down-regulated. Then, the key pathways involved in the therapeutic
   effect of ssP-tHB@Fe/DOX were analyzed through enrichment analysis.
   Gene Ontology (GO) enrichment analysis (Fig. [206]S15) and pathway
   enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes
   (KEGG) database (Fig. [207]5g) revealed significant enrichment in p53
   signaling pathways, apoptosis-related processes, and amino acid
   metabolism. Therefore, the obtained results showed that treatment with
   ssP-tHB@Fe/DOX in 4T1 cells could partially exert its anti-cancer
   effect by activating p53 signaling to induce cellular apoptosis and by
   modulating amino acid metabolism to induce ferroptosis.

Fig. 5.

   [208]Fig. 5
   [209]Open in a new tab

   (a) Heat map of mRNAs related to treatment progression. (b) PCA based
   on bulk RNA-seq results of the PBS and ssP-tHB@Fe/DOX. (c) Volcano map
   of the transcriptomic profiles. (d, c, and f) GSEA of apoptosis
   (WP1254) (d), p53 signaling (WP2902) (e), and amino acid metabolism
   (WP662) (f) signaling pathway after ssP-tHB@Fe/DOX therapy. (g) Bubble
   diagram of differentially expressed genes enriched in KEGG

   In addition, to better illustrate the transcriptome changes induced by
   ssP-tHB@Fe/DOX, we conducted gene set enrichment analysis (GSEA) on the
   RNA-seq data. As expected, the three significantly enriched signature
   pathways, such as apoptosis (WP1254) (Fig. [210]5d), p53 signaling
   (WP2902) (Fig. [211]5e), and amino acid metabolism (WP662)
   (Fig. [212]5f), were displayed. More encouragingly, gene regulators
   involved in cell apoptosis and ferroptosis mediated by ssP-tHB@Fe/DOX,
   such as drivers, suppressors, and markers, were identified (Fig.
   [213]S16, [214]S17, and [215]S18). Ultimately, we could speculate that
   breast cancer could be effectively treated through ssP-tHB@Fe/DOX by
   inducing both apoptosis and ferroptosis.

Therapeutic efficiency against 4T1 tumors and biosafety in vivo

   To evaluate the therapeutic efficacy of the cyclic magnification
   ferroptosis and DOX-induced chemotherapy in ssP-tHB@Fe/DOX, the
   treatment’s effect on a subcutaneous 4T1 tumor model was assessed in
   vivo. Firstly, 4T1 tumor-bearing mice were successfully established
   (approximately tumor volume: 80 mm^3) and were randomly assigned five
   groups, PBS group (G1), ssP-tHB (G2), FeCl[3] (G3), DOX (G4), and
   ssP-tHB@Fe/DOX (G5, DOX: 6 mg/kg), respectively. Afterwards, we
   administered the diverse samples with an equal volume via intravenous
   injections every two days (five times in total) for a total of 17 days
   and recorded the tumor volume changes and body weight fluctuations, as
   visually depicted in Fig. [216]6a. Throughout the treatment period, 4T1
   tumor-bearing mice in all groups showed venial upward trend in weight
   (Fig. [217]6b), suggesting that the therapeutic samples did not
   interfere with normal growth in mice. The tumor growth curves showed
   that, unlike the PBS (G1) and ssP-tHB (G2) groups, which exhibited
   rapid tumor growth, the other therapeutic groups (G3, FeCl[3]; G4, DOX;
   and G5, ssP-tHB@Fe/DOX) demonstrated significant tumor volume
   reduction. Among these, G5 (ssP-tHB@Fe/DOX) was the most effective,
   aligning with results from in vitro cell experiments (Fig. [218]6f and
   g). Actually, this supports the results that
   ferroptosis-chemotherapeutic combination therapy is superior to
   mono-therapy. This observation was further supported by digital
   photographs of the tumors taken 17 days after treatment with different
   samples, as shown in Fig. [219]6c. At 14 days post-treatment, as shown
   in Fig. [220]6d and e, G5 demonstrated the most significant tumor
   inhibition due to the combined effects of ssP-tHB@Fe/DOX-induced
   efficient ferroptosis and DOX-mediated apoptosis.

Fig. 6.

   [221]Fig. 6
   [222]Open in a new tab

   Anti-tumor treatments with ssP-tHB@Fe/DOX in 4T1 tumor model. (a)
   Schematic illustration of the in vivo antitumor treatment experiment.
   (b) Body-weight changes of mice (n = 5). (c) Representative tumor
   images of 4T1 tumor-bearing mice receiving various treatments. (d)
   Weights of tumors harvested from 4T1 tumor-bearing mice in different
   groups. (e) Tumor growth inhibition rate for each treatment group after
   17 days of treatment. (f) Spaghetti curves showing tumor growth in mice
   treated differently. (j) Tumor growth curves (n = 5) for tumor-bearing
   mice injected intravenously with G1, G2, G3, G4, and G5, respectively.
   (h) MDA content in serum of mice from each treatment group
   post-treatment. (i) Histological images of H&E, and TUNEL, antigen
   Ki-67, GPX4 immunofluorescence-stained tumor slices from the 4T1
   tumor-bearing mice at day 17. PBS (G1), ssP-tHB (G2), FeCl[3] (G3), DOX
   (G4), and ssP-tHB@Fe/DOX (G5), **p < 0.01, ***p < 0.001, and
   ****p < 0.0001

   Subsequently, for specific analysis of pathology, the collected tumors
   were stained via hematoxylin and eosin (H&E), TdT-mediated dUTP
   Nick-End Labeling (TUNEL), Ki−67, and GPX4 (Fig. [223]6i). As
   anticipated, H&E and TUNEL imaging revealed that the ssP-tHB@Fe/DOX
   group (G5) exhibited a higher level of apoptosis compared to the other
   groups. The cell proliferation ability of ssP-tHB@Fe/DOX exhibited a
   significant decrease, as indicated by Ki−67 protein staining, compared
   to other treatment groups. These results showed that ssP-tHB@Fe/DOX
   with multi-mode combination exhibits significant anti-tumor effects.
   Next, we assessed the expression of GPX4 in different samples-treated
   tumors. Comparatively, the expression of GPX4 in tumors was receded by
   ssP-tHB@Fe/DOX (G5) treatment, proving its ability to enhance
   ferroptosis (Fig. [224]6i). MDA is natural byproducts of LPO. We
   measured the MDA expression levels in the serum of each group after
   treatment. As presented in Fig. [225]6h, compared to the PBS group, the
   expression of MDA was the highest in the G5, which again indicates that
   ssP-tHB@Fe/DOX could enhance LPO and lead to ferroptosis.

   The biosafety of mice with 4T1 tumors was also a crucial factor in the
   treatment process. Therefore, the functions of liver (indicators:
   alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate
   transaminase (AST)) and kidney (indicators: creatinine (CREA), urea
   (UREA)) were analyzed, as well as routine bloods (indicators: platelet
   hematocrit (PCT), white blood cell (WBC) and red blood cell (RBC)) of
   mice were detected [[226]52]. As shown in Fig. [227]S19, the levels of
   ALP, ALT, and AST were significantly elevated in DOX alone-treated mice
   (G4), indicating the induction of hepatotoxicity. No significant
   changes were observed in the levels of ALT, ALP, AST, UREA, CREA, PCT,
   WBC, and RBC in the G2, G3, and G5 treatment groups. These data
   indicated no marked toxic effects at the current dose of
   ssP-tHB@Fe/DOX. Correspondingly, H&E staining results indicated
   significant liver damage exclusively in the DOX treatment group,
   whereas no notable injuries were observed in the livers, kidneys, or
   hearts of mice treated with ssP-tHB@Fe/DOX (Fig. [228]S20). These
   results indicated that ssP-tHB@Fe/DOX had an excellent
   biocompatibility.

Conclusions

   Currently, single-agent chemotherapy, like DOX, has several drawbacks
   in treating breast cancer, often resulting in chemotherapy treatment
   failure. Furthermore, combining different treatment methods with
   intelligent nanosystems has become a trend to enhance therapeutic
   results. Therefore, in this study, we developed GSH consumption
   polymeric nanoparticles (ssP-tHB@Fe/DOX) that effectively co-load the
   chemotherapeutic drug DOX and store iron ions to enhance both
   ferroptosis and apoptosis in breast cancer treatment. The as-prepared
   ssP-tHB@Fe/DOX displayed GSH-triggered degradation and sustained
   release of TME-activated drugs, as well as remarkable biocompatibility
   and tumor-targeted cytotoxicity. Meanwhile, the treatment platform that
   circularly enhanced ferroptosis and DOX-assisted apoptosis was
   proposed. The study focused on demonstrating that the tHB compound
   could consistently increase Fe(II) production, thereby enhancing the
   Fe-mediated Fenton reaction and significantly depleting GSH levels. The
   presence of ssP-tHB@Fe/DOX led to the effective destruction of
   mitochondrial structure and a reduction in mitochondrial membrane
   potential, meanwhile, cellular energy ATP and GPX4 protein levels
   significantly decreased, indicating ferroptosis. More importantly, the
   continuous release of DOX not only induced apoptosis in tumor cells but
   also generated significant amounts of H[2]O[2], which aids in
   amplifying ferroptosis via mitochondrial activity. RNA sequencing
   (RNA-seq) analysis robustly supported the anti-tumor mechanism of
   ssP-tHB@Fe/DOX, confirming involvement in the amino acid metabolism
   ferroptosis signaling pathway and the p53 signaling pathway. Therefore,
   in this study, we developed an intelligent nanosystem featuring GSH
   cutting, validated the mechanism of DOX-induced cell apoptosis and
   ferroptosis enhancement, and introduced a novel approach for breast
   cancer treatment.

Experimental procedures

Chemicals, cells, and animals

   Cyclobutane-1,2,3,4-tetracarboxylic dianhydride (CBDA, 95%),
   N-boc-ethylenediamine (EDA-Boc, 98%), hydrogen peroxide (H[2]O[2],
   30%), iron (III) chloride hexahydrate (FeCl[3]•6H[2]O, 99.5%), N,
   N-dimethylformamide (DMF, AR), trifluoroacetic acid (TFA, AR), and
   1,10-phenanthroline (Phen, ≥ 99%) were provided by Sinopharm Chemical
   Reagent Co. Ltd. 3,4,5-trihydroxybenzaldehyde (tHB, 98%),
   2,2’-dithiodiethanol (BiBi, ≥ 90%), doxorubicin hydrochloride (DOX·HCl,
   98%), methylene blue (MB), and reduced glutathione (GSH, 98%) were
   purchased from Aladdin (Shanghai, China). N-(3-dimethyl
   aminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC, 95%),
   1-hydroxybenzotriazole (HOBT, 99%) were supplied by Beijing J&K
   Technology Co. Ltd. Methanol and dimethyl sulfoxide (DMSO) were
   supplied by Tianjin Fuyu Fine Chemical Co., Ltd. C11-BODIPY^581/591 and
   Trizol were purchased from Invitrogen. Cell-counting kit-8 (CCK-8) and
   5,5-dimethyl-1-pyrroline N-oxide (DMPO) were obtained from Dojindo
   Molecular Technologies. 2′,7′-dichlorofluorescin diacetate (DCFH-DA),
   4% paraformaldehyde, 4′,6-diamidino-2-phenylindole (DAPI), GSH/GSSG
   assay kit, and Annexin V-FITC & PI dual staining kit were all bought
   from Beyotime Biotechnology. Hydroxyl radical (·OH) assay kit (HR8841)
   was obtained from Beijing Bio-Labor Biotechnology Co., Ltd. All
   chemical reagents were directly used without further purification.

   4T1 breast cancer cell line (4T1 cells) and mouse fibroblast cell line
   (L929 cells) were purchased from ATCC. All cells were cultured in
   Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) containing 10%
   FBS (Gibco, USA) and 1% Penicillin-Streptomyc, and humid incubator
   (37℃, 5% CO[2]).

   All animals, 6–8 weeks, SPF environments, were fed in standardized
   experimental animal center. All animal experiments were approved by the
   institutional ethics committee of Shanghai Chest Hospital and all
   procedures were performed according to the rules of the Institutional
   Animal Care and Use Committee of Shanghai Chest Hospital (approval
   number: KS22004). All BALB/c mice were provided by Sibeifu
   Biotechnology Co., Ltd (Beijing, China).

Preparation of range of block copolymers

   5 mL of DMF was used to dissolve 162 mg of 2-hydroxyethyl disulfide
   (BiBi, 1.0 mmol) and 224 mg of cyclobutane-1,2,3,4-tetracarboxylic
   dianhydride (CBDA, 1.1 mmol). The reaction proceeded for 24 h under
   nitrogen protection. Afterwards, 300 mg of PEG[5K]-NH[2] was added to
   the system and continued to react overnight at 50 °C. Finally, 10 mL of
   distilled H[2]O (dH[2]O) was added to the reacted system. The product
   was then dialyzed for three days and lyophilized, resulting in the
   mPEG-poly(CBDA-co-BiBi) block copolymer, abbreviated as ssP-COOH
   copolymer [[229]36].

   Subsequently, 150 mg of ssP-COOH copolymer was dissolved in DMF (5 mL),
   and added into HOBT (170 mg) and EDC (150 mg) continuously stirred for
   30 min. Then, N-boc-ethylenediamine (EDA-Boc, 80 mg) was added, and
   continued to react for 24 h. Finally, the Boc group-grafted polymer was
   obtained by dialysis, and freezing, and was named ssP-EDA-Boc
   copolymer. Additionally, to obtain amino-modified copolymer, we
   conducted an amino deprotection experiment using TFA reagent. After
   24 h of reaction, the purified polymer (labelled as ssP-EDA copolymer)
   was obtained by dialysis and freeze-drying. The ssP-EDA block-copolymer
   (125 mg) was dissolved into methanol (4.5 mL), and then added into
   3,4,5-trihydroxybenzaldehyde (tHB, 125 mg). The mixture was allowed to
   react under nitrogen conditions for 24 h at room temperature. The
   purified polymer, ssP-tHB copolymer, was then obtained via dialysis and
   freeze-drying.

Preparation and characterization of ssP-tHB@DOX and ssP-tHB@Fe/DOX

   To prepare ssP-tHB@DOX, 40 mg of ssP-tHB copolymer was dissolved in 2
   mL of methanol. Separately, 10 mg of DOX was dissolved in another 2 mL
   of methanol. After thoroughly mixing the two solutions, the mixture was
   added to 15 mL of distilled water with stirring at room temperature.
   The solution was then stirred for 12 h, and the final product was
   obtained through dialysis and freeze-drying.

   ssP-tHB copolymer (40 mg) was dissolved in 2 mL of methanol. 2 mL of
   methanol was used to dissolve DOX (10 mg). After evenly mixing the two
   solutions, the mixing solution dropped into 15 mL of dH[2]O with
   stirring at room temperature, followed by stirring for 12 h to obtain
   well-mixed solution. Afterwards, 0.3 mL of FeCl[3] solution (10 mg/mL)
   was added, and stirred for 12 h. Finally, the mixture was transferred
   to the dialysis bag (MWCO = 14, 000). The ssP-tHB@Fe/DOX was obtained
   through freeze drying. Meanwhile, the ssP-tHB and other nanoparticles
   also were prepared according to the above operations.

Characterization

   The structure of the synthetic samples was characterized using a
   Fourier transform infrared spectrometer (FTIR, Thermo Fisher Scientific
   Nicolet iS50), a nuclear magnetic resonance spectrometer (^1H NMR,
   Bruker ARX 400 MHz, Germany), X-ray photoelectron spectroscopy (XPS,
   ESCALAB 250 X-ray photoelectron spectrometer) and X-ray diffraction
   (XRD, PANalytical X-ray diffractometer). The DOX related experiments
   were measured using an ultraviolet spectrophotometer (UV-3101PC
   Shimadzu). The hydrodynamic size and zeta potential distribution of the
   NPs were characterized by Zetasizer Nanoseries (Nano ZS90, Malvern
   Instrument Ltd.). The morphology of ssP-tHB, ssP-tHB@Fe/DOX, and
   GSH-treated NPs were observed by transmission electron microscopy (TEM,
   (JEOL JEM 2100 F). Confocal laser microscopy (Carl Zeiss, Zeiss 710)
   was used to observe the uptake of DOX formulations by 4T1 cells. Flow
   cytometry experiments were conducted using a BD Accuri C6 flow
   cytometer (Becton, Dickinson and Company). Cytotoxicity was determined
   using a multifunctional microplate reader (Tecan infinite M200, Tecan).

Release of DOX and Fe ions from ssP-tHB@Fe/DOX

   The release curves of DOX and Fe ions from ssP-tHB@Fe/DOX were
   characterized in vitro. In short, ssP-tHB@Fe/DOX (5 mg) was dispersed
   in pH 7.4 PBS with 10 µM GSH (simulating normal cell microenvironment)
   and pH 5.0 ABS with 10 mM GSH (simulating tumor microenvironment),
   respectively [[230]19]. After cultivation at different time points (0,
   0.5, 1, 2, 5, 10, 24, 36 h), the supernatant was collected by
   centrifugation (rpm = 13, 000, 20 min) and detected by UV-vis
   spectrophotometer and ICP technology, respectively, and then the curves
   of release of DOX and Fe ions were calculated.

Detection of Fe (II) by 1,10-phenanthroline color reaction

   1,10-phenanthroline is a sensitive reagent for Fe (II), which could
   react with Fe (II) to form the orange red complex [[231]47]. Therefore,
   the generation of Fe (II) could be judged by the changes of color. 100
   µL of 1,10-phenanthroline solution (1 mg/mL) was mixed with 2 mL
   ssP-tHB@Fe/DOX (200 µg/mL) containing GSH (100 µL, 1 mg/mL), the color
   change and absorbance of the mixture solution were recorded.

GSH-related experiments

   ssP-tHB@Fe/DOX (100 µL) and GSH solution (3 mM, 50 µL) were mixed in
   PBS (pH 7.4, 1.85 mL). After 2 h of incubation, the reacted solution
   was centrifuged to collect the supernatant. Then,
   5,5’-dithiobis-(2-nitrobenzoic acid) solution (DTNB, 10 mg/mL, 50 µL)
   was added and reacted for 30 min. The corresponding absorbance at
   412 nm was tested using a UV-vis spectrophotometer.

   The as-prepared ssP-tHB was con-incubated in PBS (pH 7.4) with
   different concentrations of GSH (0, 0.625, 1.25, 2.5, 5, and 10 mM).
   After 20 min of reaction, 1 mL of the mixture was transferred and added
   into 2 mM DTNB ethanol solution. The residual GSH level was detected
   via UV-vis spectrophotometer.

Measurement of •OH generation

   ssP-tHB or ssP-tHB@Fe/DOX were mixed with an aqueous solution
   containing with H[2]O[2] (10 mM) and 10 µL DMPO. The resulting mixture
   was analyzed using an ESR spectrometer to detect characteristic peak
   signals.

   The Fenton reaction-based generation of •OH using iron ions was
   assessed with methylene blue (MB) as a probe. MB was incubated with
   different solutions and the corresponding absorbance was measured using
   a UV-vis spectrophotometer.

Hemolysis test in vitro

   Simply put, ssP-tHB@Fe/DOX solution at different concentrations was
   added to an equal volume of 2% red blood cell suspension. After 1 h of
   incubation at 37 °C, the supernatant was collected via centrifugation
   (3500 rpm, 10 min) and then measured by UV-vis spectrophotometer. The
   haemolysis rate was calculated according to the following formula:
   haemolysis rate (%) = (A[sample] - A[negative]) / (A[positive] -
   A[nagatibe]) × 100% [[232]52].

Cytotoxicity assay in vitro

   4T1 cells and L929 cells were seeded in 96-well plates (10, 000 cells
   per well) and cultured for 12 h. Then, the cells were co-incubated with
   different samples (n = 5) for 24 h. After that, the cells were washed 3
   times with PBS, and the cell viability was measured using a CCK-8
   assay.

Intracellular ROS detection

   4T1 cells were seeded in 12-well plates (5 × 10^5 cells per well) and
   cultured for 24 h. After 24 h of co-incubation with the different
   samples, the cells were washed 3 times with PBS. Then, the cells were
   stained using DCFH-DA (ROS probe) and DAPI (nucleus probe) for 30 min.
   The fluorescence images were captured via a confocal laser scanning
   microscope (CLSM 710, Carl Zeiss, Germany).

GSH depletion, H[2]O[2] level and MDA level in 4T1 cells

   Glutathione (GSH) assay kit was used to determine the levels of 4T1
   intracellular GSH after different samples treatments for 24 h. Then the
   total glutathione assay kit (Beyotime, S0052) was used to measure GSH
   contents in treated cells.

   The intracellular ATP levels were tested using the enhanced ATP assay
   kit (Beyotime, S0026), and the different samples-induced H[2]O[2]
   concentration in 4T1 cells were measured using the H[2]O[2] assay kit
   (Beyotime, S0038). To assess LPO in 4T1 cells, the intracellular
   malondialdehyde (MDA) levels were measured using the MDA assay kit
   (Beyotime, S0131S) [[233]47].

Detection of intracellular ·OH

   To measure the intracellular ·OH generation, 4T1 cells were seeded into
   6-well plates and incubated for 24 h. After that, the cells were
   incubated with HPF solution for 30 min. Then, the cells were treated
   with different samples for 4 h. The fluorescence signals were measured
   by fluorescence images.

Cellular uptake

   The cellular uptake of ssP-tHB@Fe/DOX was evaluated using CLSM. Due to
   its fluorescence properties, the fluorescence intensity of DOX in 4T1
   cells (1 × 10^4/well) was observed after co-incubating them with
   various samples containing equal amounts of DOX.

Apoptosis experiment

   In this experiment, the Annexin V-FITC/PI double-staining method was
   employed to assess the percentage of 4T1 cells (5 × 10^5/well)
   undergoing early and late apoptosis after a 24-hour incubation with
   various formulations.

Western blotting

   The protein samples were separated via SDS-polyacrylamide gel
   electrophoresis and then transferred to PVDF membranes in an ice bath.
   Following sealing and washing, the membrane was incubated first with
   the primary antibody, followed by the secondary antibody, and the
   signal was then detected using the ChemiDoc™ Touch Imaging System
   (Catalog No. 1708370, Bio-Rad).

RNA sequencing and Bioinformatic analysis

   4T1 cells treated with control group and ssP-tHB@Fe/DOX group were
   collected for RNA extraction. After 6 h of incubation, the process of
   RNA extraction process was implemented. Total RNA was extracted in
   triplicate using the mirVana miRNA Isolation Kit (Ambion) following the
   manufacturer’s instructions. RNA quality control was performed using
   the Agilent 2100 Bioanalyzer from Agilent Technologies, Santa Clara,
   CA, USA. The TruSeq Stranded mRNA LTSample Prep Kit from Illumina (San
   Diego, CA, USA) was used for library construction. The libraries were
   then sequenced on the Illumina HiSeqTM 2500 platform, generating
   125 bp/150 bp paired-end reads. Subsequently, transcriptome sequencing
   and analysis were performed by OE Biotech Co., Ltd. ( Shanghai, China).
   PCA analysis was conducted using R (version 3.2.0) to assess the
   biological replication of samples. Using DEseq2 method to identify
   differentially expressed genes between the treatment group and the
   control group (parameters: cQ value < 0.05, P value < 0.05 and fold
   Change > 2 or < 0.5). KEGG pathway enrichment analysis of DEGs was
   conducted using R based on the hypergeometric distribution.

Bio-security in vivo

   Six-week-old female BALB/c mice, classified as SPF, were used for in
   vivo biosecurity studies. The mice received intravenous injections of
   PBS and ssP-tHB@Fe/DOX (5 mg/kg, 100 µL). The body weight of the mice
   was measured every other day. At the conclusion of the treatment (8
   days and 14 days), blood samples were collected to conduct a blood
   routine examination (WBC, HGB, PCT, and RBC) and kidney/liver
   biofunctions (ALT, ALT, AST, CREA, and UREA indicators). The major
   organs (heart, liver, spleen, lung, and kidney) were sliced and stained
   with hematoxylin and eosin (H&E) staining to evaluate toxicity of
   ssP-tHB@Fe/DOX.

In vivo antitumor efficacy

   Female BALB/c mice (n = 10) at 7 weeks were depilated over the right
   abdominal femur, and then each mouse was injected with 1 × 10^6 4T1
   cells. When tumors reached approximately 50 mm³, various formulations
   (PBS (G1), free FeCl[3] (G2), DOX alone (G3), ssP-tHB (G4), and
   ssP-tHB@Fe/DOX (G5, containing 2.5 mg/kg DOX)) were administered via
   the tail vein at 2-day intervals (days 0, 2, 4, 6, and 8). The first
   administration was marked as day 0, and the tumor’s vertical diameter
   was measured every other day. Body weights and tumor volumes were
   recorded. The condition of the mice was monitored 17 days after
   discontinuing the drug treatment. On the 17th day, five randomly
   selected mice were euthanized, and their tumors were removed and
   weighed. The heart, liver, spleen, lung, kidney, and tumor were
   collected and stained using H&E and immunohistochemical methods for
   Ki-67, GPX4, and TUNEL.

Statistical analysis

   Data were presented as mean ± standard deviation (SD) through at least
   three experiments. Statistical analysis was performed using Student’s t
   test for two-group differences. A p-value < 0.05 was considered
   statistically significant: *p < 0.05 (significant), **p < 0.01
   (moderately significant), and ***p < 0.001 (highly significant).

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [234]Supplementary Material 1^ (7.5MB, docx)

Acknowledgements