Abstract

   Near‐infrared‐II (NIR‐II) ferroptosis activators offer promising
   potentials in in vivo theranostics of deep tumors, such as glioma.
   However, most cases are nonvisual iron‐based systems that are blind for
   in vivo precise theranostic study. Additionally, the iron species and
   their associated nonspecific activations might trigger undesired
   detrimental effects on normal cells. Considering gold (Au) is an
   essential cofactor for life and it can specifically bind to tumor
   cells, Au(I)‐based NIR‐II ferroptosis nanoparticles (TBTP‐Au NPs) for
   brain‐targeted orthotopic glioblastoma theranostics are innovatively
   constructed. It achieves the real‐time visual monitoring of both the
   BBB penetration and the glioblastoma targeting processes. Moreover, it
   is first validated that the released TBTP‐Au specifically activates the
   effective heme oxygenase‐1‐regulated ferroptosis of glioma cells to
   greatly extend the survival time of glioma‐bearing mice. This new
   ferroptosis mechanism based on Au(I) may open a new way for the
   fabrication of advanced and high‐specificity visual anticancer drugs
   for clinical trials.

   Keywords: aggregation‐induced emission, brain‐targeting, ferroptosis,
   glioma, near‐infrared imaging
     __________________________________________________________________

   A near‐infrared (NIR) aggregation‐induced emission ferroptosis molecule
   (TBTP‐Au) is first designed by introducing an Au(I) active center. The
   self‐assembled TBTP‐Au nanoparticles with targeted modification display
   superior NIR imaging performance to monitor the blood–brain barrier
   penetration of nanoparticles. The controlled release of TBTP‐Au
   specifically activates the effective heme oxygenase‐1‐regulated
   ferroptosis to greatly prolong the survival of glioma‐bearing mice.

   graphic file with name ADVS-10-2206333-g002.jpg

1. Introduction

   Glioblastoma multiforme (GBM) is the most common and fatal malignant
   brain tumor,^[ [52]^1 , [53]^2 , [54]^3 ^] which faces the challenges
   of blood–brain barrier (BBB) and the lack of effective drugs,^[ [55]^4
   , [56]^5 , [57]^6 ^] so there has been little progress in the
   treatment. Ferroptosis has emerged as a newly discovered iron‐dependent
   programmed cell death paradigm,^[ [58]^7 , [59]^8 , [60]^9 ^] and it is
   different from the common programmed cell death modalities including
   apoptosis, autophagy, necroptosis, and pyroptosis.^[ [61]^10 ^] Given
   neoplastic cells show higher sensitivity toward iron than those of the
   nonmalignant cells,^[ [62]^11 , [63]^12 ^] ferroptosis possesses great
   potential to kill malignancies. As documented, lipid peroxidation‐based
   ferroptosis generally can be classified into two types, i.e., canonical
   and noncanonical ferroptosis.^[ [64]^13 ^] Canonical ferroptosis
   involves the inactivation of glutathione peroxidase 4 (GPX4) which
   leads to uncontrolled and toxic lipid peroxidation.^[ [65]^14 , [66]^15
   ^] Noncanonical ferroptosis includes excessive activation of heme
   oxygenase‐1 (HMOX1), where the increased labile iron pool could
   directly catalyze free radical formation via Fenton reaction and induce
   lipid peroxidation.^[ [67]^16 , [68]^17 , [69]^18 ^] Considering the
   short in vivo half‐lives and the incapability of penetrating BBB for
   current small‐molecule ferroptosis activators, such as sorafenib,
   sulfasalazine, and artemisinin,^[ [70]^19 ^] the iron‐based ferroptosis
   nanosystems^[ [71]^20 , [72]^21 , [73]^22 ^] and nonferrous (Mo and
   Cu)‐based nanomaterials^[ [74]^23 , [75]^24 , [76]^25 ^] were developed
   in recent years. However, these iron species and the associated
   nonspecific ferroptosis activation may trigger undesired detrimental
   effects,^[ [77]^25 ^] such as anaphylactic reactions in normal
   tissues.^[ [78]^26 , [79]^27 , [80]^28 ^] Moreover, these blind
   ferroptosis systems lack imaging capability and were unable to afford
   visual and precise theranostics in vivo. Therefore, constructing new
   nonferrous ferroptosis activators with visual imaging function in vivo
   is highly in demand.

   Near‐infrared‐II (NIR‐II) fluorescence imaging has acquired
   considerable attention due to the advantages of deep tissue penetration
   and high imaging resolution,^[ [81]^29 , [82]^30 , [83]^31 , [84]^32 ^]
   which enables the real‐time observation of BBB penetration for
   nanoparticles.^[ [85]^33 ^] However, the existing inorganic NIR‐II
   probes have high risks of toxicity,^[ [86]^34 ^] such as upconverting
   nanoparticles (UCNP) and lead sulfide (PbS) quantum dots.^[ [87]^35 ,
   [88]^36 , [89]^37 ^] In another aspect, organic dyes usually face
   severe aggregation‐caused quenching (ACQ) effects that may induce low
   imaging sensitivity.^[ [90]^38 ^] Aggregation‐induced emission
   luminogens (AIEgens), a type of special fluorophores that can overcome
   the above ACQ limitation,^[ [91]^39 , [92]^40 , [93]^41 ^] feature high
   biocompatibility and flexibly tailored photophysical performance and
   may be a promising option for the desired NIR‐II fluorophores. However,
   the short excitation/emission wavelength and lack of ferroptosis‐active
   centers make them still not been established for orthotopic glioma
   until now, which inevitably limit their applications. This reflects the
   huge challenge in molecule design and synthesis of NIR‐II ferroptosis
   activators including AIE systems.

   Based on the above points, we thus innovatively selected the
   biocompatible Au(I), an essential cofactor in humans, as the nonferrous
   active center to design a NIR‐II ferroptosis activator (TBTP‐Au) by
   incorporating an AIE‐active molecule backbone. The pertinent results
   turned out that real‐time NIR‐II imaging could be realized, which
   efficiently monitored the BBB penetration and targeting process of
   apolipoprotein E (ApoE) peptide^[ [94]^42 ^] modified TBTP‐Au
   nanoparticles (ApoE‐TBTP‐Au NPs). Additionally, triggered by the tumor
   microenvironment,^[ [95]^43 ^] the controlled release of TBTP‐Au from
   the reactive oxygen species (ROS)‐responsive nanosystem could initiate
   effective death of glioma cells through selectively binding to the
   overexpressed thioredoxin reductase (TrxR),^[ [96]^44 , [97]^45 ^] and
   specifically activate HMOX1‐regulated ferroptosis pathway (Figure
   [98]1A). Impressively, this finding is completely distinct from the
   current reports on the mechanisms of nonspecific noncanonical
   ferroptosis and the known Au‐induced autophagy and apoptosis.^[ [99]^46
   , [100]^47 ^] This study demonstrates the first example of a
   high‐specificity NIR‐II ferroptosis activator against glioma. Moreover,
   the present strategy may inspire more discoveries and be helpful for
   the development of advanced visual anticancer drugs.

Figure 1.

   Figure 1
   [101]Open in a new tab

   Structure and photophysical performance of TBTP‐Au. A) Schematic
   illustration of brain‐targeting ApoE‐TBTP‐Au NPs for ferroptosis in
   orthotopic glioma via the TrxR‐HMOX1 axis. B) Chemical structure and
   optimized S[0] geometry of TBTP‐Au. C) Illustration of the frontier
   molecular orbitals (LUMO and HOMO) determined at the
   M062X‐D3/6‐31G(d,p) Lanl2DZ (Au) level of theory for TBTP‐Au. D)
   Absorption spectra of TBTP and TBTP‐Au in tetrahydrofuran (THF). E)
   Photoluminescence (PL) spectra (excitation: 808 nm) of TBTP‐Au in
   THF/n‐hexane mixtures with different fractions (f) of n‐hexane. F) Plot
   of PL peak intensity at 1015 nm of TBTP and 1030 nm of TBTP‐Au at
   various fraction values.

2. Results and Discussion

2.1. Molecular Design, Synthesis, and Photophysical Properties of TBTP‐Au

   Regarding the design of AIE core, we selected a unique donor
   (D)‐π‐acceptor (A)‐π‐A type of twisted structure (TBTP), where
   benzo[1,2‐c:4,5‐c’]bis([1,2,5]thiadiazole) (BBTD) was a common core
   acted as the electron acceptor for NIR‐II molecule, thiophene ring was
   π‐conjugation part and triphenylamine (TPA) played as the electron
   donor (Figure [102]1B). As we mentioned above, Au(I) is a biocompatible
   active center of clinically approved antirheumatic drug (auranofin),^[
   [103]^45 ^] which has the potential to trigger ferroptosis via
   specifically inhibiting the highly expressed TrxR in tumor cells. So we
   further engaged the Au(I) unit into the AIE‐active TBTP core to obtain
   the target molecule, TBTP‐Au, and further evaluated its potential both
   in NIR‐II imaging in vivo and ferroptosis activation. To this end, the
   target molecule TBTP‐Au was synthesized via the well‐established
   synthetic route (Figure [104]S1, Supporting Information). To validate
   the contribution of Au(I), the control molecule, TBTP, was
   simultaneously studied. The structures of all intermediates were
   confirmed by nuclear magnetic resonance (NMR) and high‐resolution mass
   spectrum (HRMS) (Figures [105]S2–S11, Supporting Information).

   Next, the photophysical performance of TBTP‐Au was evaluated. Firstly,
   the preliminary theoretical calculation was performed. The electron
   cloud of the lowest unoccupied molecular orbital (LUMO) was dominantly
   distributed on the central BBTD unit, and the highest occupied
   molecular orbital (HOMO) was delocalized across the whole conjugated
   backbone of TPA and two thiophene rings, which generated a bandgap of
   2.79 eV (Figure [106]1C). Thus, this typical charge separation
   character in the donor–acceptor structure was favorable for the
   redshift of both the absorption and emission spectra compared with the
   electron‐withdrawing BBTD core.^[ [107]^33 ^] Therefore, both TBTP and
   TBTP‐Au exhibited a long absorption to ≈1000 nm (Figure [108]1D).
   Further photoluminescence (PL) experiments demonstrated the typical AIE
   properties of both TBTP and TBTP‐Au. As depicted in Figure [109]1E,F, a
   gradually intensified PL profile with the maximum peak at 1030 nm for
   TBTP‐Au was observed when increasing the fraction of insoluble solvent
   (n‐hexane). Comparatively, TBTP showed a blue‐shifted peak at 1015 nm
   in the aggregate state (Figure [110]S12, Supporting Information). Given
   the long NIR‐II luminescent property, the developed TBTP‐Au has the
   potential to act as a visual platform for in vivo deep‐penetration
   bioimaging.

2.2. Physical, Optical Performance, and Tumor Selective Inhibition

   Furthermore, the TBTP‐Au molecule was self‐assembled into 126.5 nm of
   sphere‐like smart nanoparticles (NPs) (Figure [111]2A). The results of
   element mapping demonstrated the homogeneous distribution of Au in
   nanoparticles, and the two characteristic peaks at 84.5 eV (Au4f[7/2])
   and 88.2 eV (Au4f[5/2]) in X‐ray photoelectron spectroscopy (XPS)
   spectra (Figure [112]2B,C) further indicated Au(I) not Au(III) or zero
   states.^[ [113]^48 ^] The NPs also showed superior stability over a
   5‐day incubation in various media (Figure [114]S13, Supporting
   Information). The maximum peaks of the absorption and PL spectra for
   TBTP‐Au NPs red‐shifted to 820 and 1150 nm, respectively
   (Figure [115]2D), which was well consistent with the results of strong
   concentration‐dependent NIR‐II brightness under 1350 nm (image inset;
   other wavelengths are shown in Figure [116]S14, Supporting
   Information). Furthermore, bright cerebrovascular and blood vessel
   frames under a 1350 nm filter were observed (Figure [117]2E) and
   preliminarily confirmed the prominent NIR‐II imaging ability of TBTP‐Au
   NPs.

Figure 2.

   Figure 2
   [118]Open in a new tab

   Photophysical performance and in vitro anticancer activity of TBTP‐Au
   NPs. A) Size distribution graph and corresponding TEM image of TBTP‐Au
   NPs. B) Element mapping images of Au, F, N, and S in TBTP‐Au NPs by
   scanning transmission electron microscopy (STEM). C) XPS spectra of Au
   in TBTP‐Au NPs. D) Absorption and fluorescence spectra (excitation:
   808 nm) of TBTP‐Au NPs. The inset shows NIR imaging at various
   concentrations of TBTP‐Au NPs aqueous solution using a 1350 nm filter.
   E) In vivo NIR imaging at 1350 nm of cerebrovascular and body blood
   vessels. F) Changes in diameter of TBTP‐Au NPs in PBS and H[2]O[2]
   solution during a 24 h of incubation. The inserted TEM image shows
   degraded TBTP‐Au NPs incubated with H[2]O[2] for 6 h. G) Confocal
   images of U87 cells treated with two NPs for 2 h. Scale bars: 10 µm. H)
   Cell toxicity of two NPs with various concentrations to U87 cells
   (n = 3). I) Toxicity assessment of two NPs to various cell lines
   (n = 3). Data are presented as mean ± standard deviation (SD).
   *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Unless
   otherwise stated, all concentrations of nanoparticles were 100 µg mL^−1
   and U87‐luciferase cells (U87‐Luc) were used in whole‐cell assays.

   Prior to in vitro tumor selective inhibition assays, we first verified
   the ROS‐responsive release triggered by hydrogen peroxide (H[2]O[2])
   and the tumor‐targeting capability of NPs. As shown in Figure [119]2F
   (inset image), the increased diameter and transmission electron
   microscopy (TEM) image confirmed the successful release. The results of
   Fourier‐transform infrared spectroscopy (FTIR) further proved the
   successful modification of ApoE (Figure [120]S15, Supporting
   Information). Assisted by the green fluorescence ranging from 500 to
   600 nm of NPs (Figure [121]S16, Supporting Information), the confocal
   images of U87 cells incubated with target NPs showed higher uptake in
   the cytoplasm than that of nontarget NPs (Figure [122]2G). The optical
   value at 820 nm also verified it and revealed the maximum at 8 h
   (Figure [123]S17, Supporting Information). The result of 3D tumor
   spheroids indicated ApoE‐TBTP‐Au NPs have superior tumor‐penetrating
   and targeting capability (Figure [124]S18, Supporting Information).
   Additionally, in sharp contrast to the neglectable toxicity of Au‐free
   ApoE‐TBTP NPs, the released TBTP‐Au molecules from ApoE‐TBTP‐Au NPs
   triggered dramatic concentration‐dependent toxicity in U87 cells
   (Figure [125]2H). Furthermore, it exhibited significant selective
   damage to tumor cells (HeLa and U251 cells) compared with 293T and
   HA1800 cells (Figure [126]2I). The results of flow cytometry also
   presented a higher death rate of U87 cells (70.47%) caused by
   ApoE‐TBTP‐Au NPs than that of PBS (8.78%) and Au‐free NPs (7.77%,
   Figure [127]S19, Supporting Information). These results indicated that
   ApoE‐TBTP‐Au NPs could play as specific and effective NIR‐II
   nanotheranostic for glioma cells, where Au(I) acted as the active
   center.

2.3. Unveiling of TrxR/HMOX1 Ferroptosis Axis

   To unveil the underlying molecular mechanism of ApoE‐TBTP‐Au NPs
   against glioma cells, we conducted RNA sequencing to investigate the
   gene expression profile of U87 cells after various treatments. Firstly,
   all gene expressed levels were performed (Figure [128]S20, Supporting
   Information), and we identified 3426 differentially expressed genes
   (DEGs) for the ApoE‐TBTP‐Au NPs post‐treated group, including 1452
   down‐regulated DEGs and 1974 up‐regulated DEGs (Figure [129]3A). To
   pull out the potential anticancer pathways from these DEGs, Kyoto
   Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were
   performed. The term plot results presented in Figure [130]3B and Figure
   [131]S21 (Supporting Information) clearly showed that the
   ferroptosis‐related pathway was significantly enriched, implying the
   critical role in inducing cell death by NPs treatment. Further
   clustering enrichment analysis and Gene Set Enrichment Analysis (GSEA,
   Figure [132]S22A, Supporting Information) corroborated that the
   ferroptosis response was coupled with positive regulation of external
   stimulus and cell death (Figure [133]3C). To identify the hub genes in
   modulating ferroptosis pathways during NPs treatment, we performed the
   protein–protein interaction (PPI) network analysis, and we discovered
   some HMOX1‐centered markers that had strong regulatory effects on
   ferroptosis (Figure [134]S22B, Supporting Information). Similar to
   TrxR, HMOX1 was also a redox regulator. Therefore, we speculated a TrxR
   inhibition‐induced HMOX‐1 activation as the Au(I) could specifically
   inhibit the activity of TrxR.^[ [135]^44 ^] To verify this hypothesis,
   the level of cellular TrxR and HMOX1 were determined using the TrxR
   assay kit and the quantitative polymerase chain reaction (qRT‐PCR),
   respectively. We observed an obvious decrease of TrxR (Figure [136]3D)
   and substantial up‐regulation (89.1‐fold) of HMOX1 than the other DEGs
   (Figure [137]3E and Figure [138]S23, Supporting Information). Likewise,
   the result of immunoblots confirmed the same conclusion
   (Figure [139]3F). Taken together, the above results convincingly
   pointed out that TBTP‐Au selectively activated the HMOX1‐regulated
   noncanonical ferroptosis but not autophagy and apoptosis.

Figure 3.

   Figure 3
   [140]Open in a new tab

   TrxR/HMOX1 ferroptosis and cell cycle arrest induced by ApoE‐TBTP‐Au
   NPs. A) Volcano plot of all identified genes in U87 cells treated with
   ApoE‐TBTP‐Au NPs. Threshold value to consider as significance (FDR q
   value < 0.05 and log[2]|Fold Change| ≥ 1.0). B) Bubble diagram of KEGG
   enrichment analysis of the up‐regulated DEGs after treatment with
   ApoE‐TBTP‐Au NPs. FDR and gene count are represented by bubble color
   and size, respectively. FDR q value of all presented terms is less than
   0.05. C) Pathway enrichment analysis of the up‐regulated DEGs using
   Metascape. Enrichment significance of gene or gene clusters was
   presented as bubble color, which is less than 0.05. Dots occurred by
   red circle were attributed to same pathway. D) In vitro TrxR levels in
   U87 cells treated with various NPs for 24 h (n = 3). E) Relative mRNA
   expression of HMOX1 in U87 cells treated with NPs and PBS (n = 3). F)
   Western blot of TrxR, Trx, HMOX1, and P53 proteins in U87 cells after
   treatments for 24 h. G) Cell cycle analysis and quantification of U87
   cells with various treatments for 24 h by flow cytometry (n = 3). H)
   Representative crystal violet staining of U87 cells after various
   treatments for 24 h. I) Schematic illustration of ApoE‐TBTP‐Au NPs
   induced ferroptosis following cell cycle arrest after binding with
   TrxR. ApoE‐TBTP NPs and ApoE‐TBTP‐Au NPs were respectively termed “−Au”
   and “+Au”. Data are presented as mean ± standard deviation (SD).
   ***p < 0.001, ****p < 0.0001.

   Furthermore, we verified the detailed process of HMOX1‐mediated
   ferroptosis characterized by lipid peroxidation to cell death. Firstly,
   the cell cycles that reflected cell activity were assessed on the U87
   cells. The ApoE‐TBTP‐Au NPs group indicated an S phase cell‐cycle
   arrest (6.57%) compared with the PBS (11.86%) or Au‐free NPs (11.88%)
   group, revealing a DNA replication inhibition, which might be induced
   by the lipid peroxidation stress as shown in Figure [141]3G. The
   crystal violet staining was used to assess the therapeutic efficiency,
   and the result indicated an obvious cell proliferation inhibition after
   the treatment of ApoE‐TBTP‐Au NPs for 24 h (Figure [142]3H). In
   addition, as shown in Figure [143]3I, the ApoE‐TBTP‐Au NPs efficiently
   induced the death of U87 cells through ferroptosis, accompanied by
   cycle arrest and suppression of the growth. Besides the cellular
   proliferation inhibition, it simultaneously damaged the cell membranes
   and organelles, which might further induce cell death. As depicted in
   Figure [144]4A and Figure [145]S24 (Supporting Information), drastic
   morphological damage of the cell membranes, decreased volume of the
   mitochondrial cristae and obvious shrinkage of the mitochondrial
   profile as well as condensation of the mitochondrial membranes were
   observed in Bio‐TEM images, indicating typical ferroptosis‐involved
   cell death. Moreover, the lipid peroxidation was triggered by HMOX1
   that catalyzed the degradation of heme into labile iron and increased
   the ROS level via Fenton reaction. In addition to the above
   morphological changes, these three intermediates were also tested using
   three specific probes, respectively, i.e., FerroOrange for labile Fe^2+
   detection, 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) for ROS
   detection, and commercial BODIPY probe for lipid peroxidation. As
   clearly depicted in Figure [146]4B, bright fluorescence for the
   ApoE‐TBTP‐Au NPs treated groups were all observed under the signaling
   of these three different probes, strongly suggesting the lipid
   peroxidation‐associated cell death pathway. Moreover, both the results
   of electron paramagnetic resonance (EPR) and
   3,3′,5,5′‐tetramethylbenzidine (TMB) screening indicated that
   ApoE‐TBTP‐Au NPs were unable to generate radical (Figure [147]S25,
   Supporting Information), and the radical in the ferroptosis was
   generated from the HMOX1‐regulated endogenous Fe^2+, but not from
   Au(I).

Figure 4.

   Figure 4
   [148]Open in a new tab

   In vitro analyses of ferroptosis phenotype. A) Ferroptosis‐induced
   morphological change by Bio‐TEM observation in mitochondria from U87
   cells after the treatment for 24 h. B) Confocal images of labile iron
   (Fe^2+), ROS and lipid peroxidation in U87 cells stained with
   FerroOrange, DCFH‐DA, and BODIPY probes, respectively. Scale bars:
   10 µm. C–F) Cell viability of U87 cells co‐incubated with ApoE‐TBTP‐Au
   NPs (100 µg mL^−1) and different concentrations of ferroptosis
   inhibitors deferoxamine (DFO (C)), Zinc protoporphyrin (ZnPP (D)),
   Ferrostatin‐1 (Fer‐1 (E)) and other specific inhibitors (F). The
   control group was ApoE‐TBTP‐Au NPs treatment without any inhibitor in
   (F), which was the same as the 0 groups in (C), (D), and (E). G) TrxR
   expression by qRT‐PCR and immunoblot in TrxR over‐expressing U87 cells
   (n = 3). H) Corresponding reversal of cell viability in TrxR
   over‐expressing U87 cells after treatment with ApoE‐TBTP‐Au NPs for
   24 h (n = 3). I) HMOX1 expression by qRT‐PCR and immunoblot in HMOX1
   down‐regulating U87 cells. J) The corresponding cell viability treated
   with ApoE‐TBTP‐Au NPs for 24 h (n = 3). K) Schematic illustration of
   the ferroptosis process in U87 cells. Unless stated, all concentrations
   of two NPs were 100 µg mL^−1. ApoE‐TBTP NPs and ApoE‐TBTP‐Au NPs were
   respectively termed “−Au” and “+Au.” Data are presented as mean ± SD,
   **p < 0.01, ***p < 0.001, and ****p < 0.0001, NS, no significance.

   Apart from the above validation, a series of reverse cellular
   investigations of various inhibitors on cell viability were also
   assessed to confirm the ferroptosis regulation axis. As presented in
   Figure [149]4C,D, the viability of U87 cells treated by a labile iron
   chelator (DFO)^[ [150]^49 ^] or the HMOX1 inhibitor (ZnPP)^[ [151]^50
   ^] exhibited an obvious increase. Whereas the treatment of ferroptosis
   inhibitor (Fer‐1) failed to improve the cell viability (Figure [152]4E)
   because of Fer‐1 was a common inhibitor for GPX4‐mediated
   ferroptosis.^[ [153]^51 ^] In addition, the necrosis inhibitor
   (Necrostatin‐1, Nec‐1), autophagy inhibitor (3‐Methyladenine, 3‐MA),
   and apoptosis inhibitor (Z‐DEVD‐FMK, termed as DEVD) were also utilized
   to further assess the cell death modality.^[ [154]^52 ^] However, these
   all failed to improve the cell viability (Figure [155]4F), suggesting
   that the inhibition pathway of tumor cells was independent of the above
   three modalities. Subsequently, the effects of the ferroptosis targets
   (TrxR and HMOX1) on cell viability were assessed using
   TrxR‐overexpressed and HMOX1 knocking down U87 cells, respectively. In
   the TrxR‐overexpressed cells verified by the mRNA and protein levels
   (Figure [156]4G), the cell viability of the ApoE‐TBTP‐Au NPs treated
   group showed more obvious improvement than that of the control one
   (Figure [157]4H). Likewise, in the HMOX1 knocking down cells
   (Figure [158]4I), a significantly improved cell viability was also
   observed as shown in Figure [159]4J. These results consistently
   demonstrated that ApoE‐TBTP‐Au NPs could act as an efficient
   ferroptosis activator and effectively exert antitumor activity via the
   TrxR‐HMOX1 axis, which caused lipid peroxidation and mitochondrial
   dysfunction and further resulted in cell death (Figure [160]4K).

2.4. In Vivo Theranostic Effects of ApoE‐TBTP‐Au NPs

   Considering the superior anticancer property of ApoE‐TBTP‐Au NPs at the
   cellular level, we then evaluated the in vivo therapeutic efficacy in
   the orthotopic GBM mouse model according to the predesigned protocol
   (Figure [161]5A). Prior to the therapy, the biocompatibility of the NPs
   was first determined by assessing the effects on hemolysis and
   morphology of red blood cells (Figure [162]S26, Supporting
   Information), as well as routine blood parameters and blood
   biochemistry (Figure [163]S27, Supporting Information) in healthy
   BALB/c mice. The low hemolysis ratio (<5%), intact morphology of red
   blood cells as well as normal blood parameters and biochemistry
   together showed the excellent biocompatibility of ApoE‐TBTP‐Au NPs. The
   in vivo pharmacokinetic study of mice after i.v. injection of
   ApoE‐TBTP‐Au NPs (dosage: 10 mg kg^−1) showed a similar circulation
   time (t [1/2,] [β ] = 3.3 h) to that of the treatment with nontargeting
   TBTP‐Au NPs (t [1/2,] [β ] = 3.2 h) (Figure [164]S28, Supporting
   Information). The results of NIR‐II imaging applied to monitor the
   tumor accumulation of NPs (Figure [165]5B) together with the
   fluorescence intensity quantification (Figure [166]S29, Supporting
   Information) indicated that ApoE‐TBTP‐Au NPs showed much higher
   BBB‐penetrating and tumor‐accumulating abilities compared with that of
   TBTP‐Au NPs group. Simultaneously, the ApoE‐TBTP‐Au NPs mainly
   accumulated in the liver and kidney as determined by the absorption of
   tissue homogenates at 835 nm (Figure [167]S30, Supporting Information).
   After a therapeutic process performed by i.v. injection of ApoE‐TBTP‐Au
   NPs every three days for four cycles, the expression of TrxR, Trx,
   HMOX1, and P53 proteins for the harvested brain tumor tissues showed
   evident changes, which were well consistent with the results observed
   at the cellular level (Figure [168]5C). The tumor growth was also
   monitored by bioluminescence images (Figure [169]5D) and evaluated by
   the quantified luminescent intensity (Figure [170]5E). The group
   treated with ApoE‐TBTP‐Au NPs showed a strong tumor inhibition effect
   with negligible change in body weight (Figure [171]5F). More
   impressively, the treatment of ApoE‐TBTP‐Au NPs could significantly
   improve the survival period to more than 60 days (Figure [172]5G), in
   sharp contrast to the 28 days of the PBS control group and the 31 days
   of the non‐Au group. Furthermore, biosafety assessment by
   hematoxylin‐eosin (H&E) staining indicated no obvious physiological
   abnormalities (Figure [173]S31, Supporting Information). Therefore, the
   fabricated biocompatible ApoE‐TBTP‐Au NPs as a powerful NIR‐II
   ferroptosis activator could be applied in NIR‐II imaging‐guided
   theranostics of glioma.

Figure 5.

   Figure 5
   [174]Open in a new tab

   Theranostic effects of ApoE‐TBTP‐Au NPs on orthotopic glioma model. A)
   Timeline of theranostic treatment in mice bearing orthotopic U87‐Luc
   tumor. B) Real‐time NIR imaging of orthotopic glioma‐bearing mice
   before and at 4 h post injection of ApoE‐TBTP‐Au NPs (+ApoE) and
   TBTP‐Au NPs (−ApoE) (dosage: 10 mg kg^−1). C) Expression of
   ferroptosis‐related proteins by Western blot in ex vivo tumors after
   finishing treatment. D) In vivo bioluminescence imaging of mice at
   various days post i.v. injection with PBS, ApoE‐TBTP NPs, and
   ApoE‐TBTP‐Au NPs, respectively (n = 5). The images were captured on the
   next day after injection except that on day 9. E) Quantified
   luminescence values in tumors (n = 5). Data are presented as mean with
   standard error of the mean (SEM). F) The corresponding weight changes
   of mice (n = 6) undergoing treatment. G) Survival graph of the
   tumor‐bearing mice (n = 6). NS, no significance, *p < 0.05. ApoE‐TBTP
   NPs and ApoE‐TBTP‐Au NPs were respectively termed “−Au” and “+Au.”

3. Conclusion

   The high‐specificity anticancer drugs can minimize the toxicity to
   normal cells and maximize therapeutic efficiency. To address the
   challenges of iron toxicity, nonspecific activation and
   non‐visualization of the current iron‐based systems, we first
   synthesized an Au(I)‐based NIR‐II ferroptosis activator (TBTP‐Au) with
   AIE property by integrating Au(I) unit and AIE backbone. Then, TBTP‐Au
   was self‐assembled into the brain‐targeting nanodrug via ROS‐responsive
   template and ApoE modification. As a result, the fabricated nanodrug
   achieved real‐time NIR‐II imaging to monitor the BBB penetration and
   the targeting to glioma cells. It was well demonstrated that the higher
   ROS gradient in the glioma microenvironment stimulated the release of
   TBTP‐Au from the NPs, and Au(I) selectively bound with the
   over‐expressed TrxR and triggered the specific HMOX1‐regulated
   noncanonical ferroptosis activation of glioma cells, which induced
   notable tumoral suppression and the extension of the survival time. The
   proposed fabrication and modulation rules in this work would give
   favorable impetus for the development of new NIR‐II ferroptosis
   activators. Moreover, the Au‐based nanoparticles also have great
   potential as powerful photothermal agents or radiosensitizers to boost
   anticancer efficacy, which may provide a toolbox for developing more
   exciting metal‐engaged drugs with visualization capacity and high
   therapeutic specificity, and further inspire researchers to explore
   more unknown mechanisms.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [175]Click here for additional data file.^ (2.9MB, pdf)

Acknowledgements