Abstract

   Anti‐PD(L)1 immunotherapy recently arises as an effective treatment
   against triple‐negative breast cancer (TNBC) but is only applicable to
   a small portion of TNBC patients due to the low PD‐L1 expression and
   the immunosuppressive tumor microenvironment (TME). To address these
   challenges, a multifunctional “drug‐like” copolymer that possesses the
   auto‐changeable upper critical solution temperature and the capacity of
   scavenging reduced nicotinamide adenine dinucleotide phosphate (NADPH)
   inside tumor cells is synthesized and employed to develop a
   hypoxia‐targeted and BMS202 (small molecule antagonist of PD‐1/PD‐L1
   interactions)‐loaded nanomedicine (BMS202@HZP NPs), combining the
   anti‐PD‐L1 therapy and the low‐dose radiotherapy (LDRT) against TNBC.
   In addition to the controlled release of BMS202 in the hypoxic TNBC,
   BMS202@HZP NPs benefit the LDRT by upregulating the pentose phosphate
   pathway (PPP, the primary cellular source for NADPH) of TME whereas
   scavenging the NADPH inside tumor cells. As a result, the BMS202@HZP
   NPs‐mediated LDRT upregulate the PD‐L1 expression of tumor to promote
   anti‐PD‐L1 therapy response while reprogramming the immunometabolism of
   TME to alleviate its immunosuppression. This innovative
   nanomedicine‐mediated radio‐immunometabolism regulation provides a
   promising strategy to reinforce the anti‐PD‐L1 therapy against TNBC.

   Keywords: drug‐like copolymer, immunometabolism regulation,
   immunosuppression of tumor microenvironment, low‐dose radiotherapy,
   selective depletion of NADPH
     __________________________________________________________________

   A multifunctional “drug‐like” copolymer with the auto‐changeable upper
   critical solution temperature and selective depletion of reduced
   nicotinamide adenine dinucleotide phosphate (NADPH) inside tumor cells
   is synthesized and employed to develop a multifunctional nanomedicine
   (BMS202@HZP NPs), which sensitizes the low‐dose radiotherapy and
   achieves the radio‐immunometabolism regulation against triple‐negative
   breast cancer (TNBC), providing a promising strategy to reinforce
   anti‐PD‐L1 against TNBC.

   graphic file with name ADVS-10-2203788-g002.jpg

1. Introduction

   According to the International Agency for Research on Cancer and the
   American Cancer Society, breast cancer makes the most common cancer
   diagnosed in women.^[ [44]^1 ^] Among different types of breast cancer,
   triple‐negative breast cancer (TNBC) possesses more aggressive
   invasiveness, higher metastasis rate, and more frequent recurrence,
   which leads to a consequence that the 5‐year relative survival rate for
   TNBC patients at late stage is only 14%. Additionally, the immune
   checkpoint blockade (ICB) anti‐PD‐(L)1 treatment only showed moderate
   efficacy in TNBC even in combination with chemotherapeutic drugs in the
   latest clinical trial,^[ [45]^2 ^] and its therapeutic efficacy is
   highly determined by the expression of PD‐L1 in TNBC.^[ [46]^3 ^] Given
   the fact that only 20% of the tumors overexpressed PD‐L1 due to the
   individual difference among TNBC patients,^[ [47]^4 ^] the treatment
   effectiveness of anti‐PD‐L1 therapy in clinical practice has been
   hindered. Moreover, the immune evasion caused by the tumor
   microenvironment (TME) also causes a reduction in the therapeutic
   effect of ICB.^[ [48]^5 ^] Therefore, a strategy that can promote the
   anti‐PD‐L1 therapy response while simultaneously alleviating the
   immunosuppression of TME would be valuable for improving the
   therapeutic efficacy of PD‐(L)1 blockade against TNBC.

   Due to the significant impacts of radiotherapy (RT) on TME and systemic
   immunity, the combination of RT and anti‐PD‐L1 therapy provides a
   promising strategy to treat TNBC. On the one hand, high‐dose RT would
   pose damages to the DNA of cancer cells, which can not only induce
   immunogenic cell death (ICD), but also activate the ATM/ATR/Chk1
   signaling pathway to upregulate PD‐L1 expression, eventually reversing
   the “cold tumor” to “hot tumor” and increasing the anti‐PD‐L1 therapy
   response.^[ [49]^6 ^] On the other hand, low‐dose RT (LDRT, 0.5–2 Gy)
   would remodel the TME and alleviate its immunosuppression through
   regulating the immunometabolism of TME (Figure [50] 1 ).^[ [51]^7 ^]
   The radiation dosage employed during RT is closely related to its
   immunomodulatory effectiveness,^[ [52]^8 ^] where it is challenging to
   simultaneously increase the expression of PD‐L1 in tumor cells and
   relieve the immunosuppression of TME through RT alone with a
   fixed‐dosage. One of the key factors for this dilemma is the existence
   of the antioxidant compounds in the tumor,^[ [53]^9 ^] such as the
   reduced nicotinamide adenine dinucleotide phosphate (NADPH). Responding
   to the enhanced oxidative stress induced by RT, the pentose phosphate
   pathways (PPP) of the whole tumor tissue would be upregulated, followed
   by enhanced production of NADPH.^[ [54]^10 ^] When NADPH is
   over‐generated in immune cells, such as the tumor‐associated
   macrophages (TAMs), it would facilitate the polarization of immune
   cells toward M1‐like phenotype.^[ [55]^11 ^] Beyond, NADPH that drives
   many metabolic alterations would also participate in the synthesis of
   fatty acid and plasma membrane in newly activated CD8+ T cells and M1
   macrophages,^[ [56]^12 ^] which can contribute to the modulation of
   immunometabolism and the relief of immunosuppression of TME. However,
   when the level of NADPH inside tumor cells is elevated, it can turn
   into a building block of plasma membrane in rapidly growing tumor
   cells, which would assist the repairment of the damaged DNA in tumor
   cells and inhibit their immunogenic death and PD‐L1 upregulation.^[
   [57]^13 ^] Therefore, selective depletion of NADPH in tumor cells is
   the key to solve the puzzle of RT‐based treatments of TNBC.

Figure 1.

   Figure 1
   [58]Open in a new tab

   Production and application of the BMS202@HZP NPs. a) Schematic
   illustration of design and preparation of BMS202@HZP NPs. b)
   Dose‐dependent RT immunoregulation in clinic. c) Mechanism of
   BMS202@HZP NPs‐mediated NADPH selective depletion and
   radio‐immunometabolism regulation for synergistically anti‐PDL1 therapy
   against TNBC.

   In order to reduce the intratumoral antioxidant compounds for RT
   sensitization, in addition to PPP inhibitors, a few functional
   nanomaterials have been developed to not only function as vehicles for
   drug delivery and controlled release, but to possess the capacity of
   scavenging the antioxidant compounds.^[ [59]^9 , [60]^10 ^] Therefore,
   it is urgent to develop an effective “drug‐like” adjuvant nanomaterial
   to selectively deplete the tumor cellular NADPH and immobilize the DNA
   damage during LDRT to increase the expression of PD‐L1, while
   simultaneously alleviating the immunosuppression of TME by maintaining
   NADPH at a high level in immune cells, eventually improving the immune
   response of anti‐PD‐L1 therapy plus LDRT against TNBC. Herein, a
   multifunctional “drug‐like” copolymer with the auto‐changeable upper
   critical solution temperature (UCST), named
   poly(acrylonitrile‐co‐acrylamide‐co‐N‐(2‐(2‐nitro‐1H‐imidazol‐1‐yl)ethy
   l)acrylamide (PAAN), was synthesized and exploited to prepare
   nanoparticles (NPs) for delivering BMS202 (an antagonist of PD‐1/PD‐L1
   interaction) and facilitate the depletion of NADPH in cancer cells,
   which was beneficial for LDRT to regulate the immunometabolism of TME
   and reinforce the anti‐PD‐L1 therapy against TNBC (Figure [61]1).
   Briefly, this type of nanomedicine, BMS202@HZP NPs, could be prone to
   accumulate in TNBC cells by virtue of its dual‐targeting ability to
   both CD44 receptors and carbonic anhydrase IX (CA IX) on cell membrane.
   Meanwhile, with the assistance of nitroreductase (NTR) over‐expressed
   in cancer cells under hypoxia, the NADPH inside cancer cells could be
   scavenged by the nitroimidazole groups in PAAN copolymer to sensitize
   LDRT, immobilize DNA damage of cancer cells, and facilitate their PD‐L1
   expression and immunogenic death. Contrastly, due to lack of NTR in
   TAMs, the elevated NADPH in TAMs would be not depleted, which could
   facilitate their polarizations toward M1‐like phenotypes and relieve
   the immunosuppression of TME. Additionally, once the nitroimidazole
   groups in this nanomedicine were reduced to the hydrophilic
   aminoimidazole groups by NADPH in tumor cells, the UCST of PAAN
   copolymer could be automatically decreased to the point below the
   physiological temperature by alternating the hydrophilicity to
   hydrophobicity ratio, causing the rapid disintegration of this
   nanomedicine and on‐demand release of the PD‐L1 inhibitor BMS202. In
   comparison with the preclinical protocol (24 Gy in 3 fractions) and
   previous studies,^[ [62]^6 , [63]^10 , [64]^14 ^] the significantly
   lower dose RT (6 Gy in 3 fractions) in our study simultaneously
   achieved the reconstruction of the tumor immuno‐microenvironment and
   the reinforcement of anti‐PD‐L1 therapy while further avoiding the
   adverse effects, because of the above advantages of the BMS202@HZP NPs
   (Figure [65]1a,c). Generally, if traditional nanomedicines are regarded
   as flowering plants, the encapsulated drugs would be like “flowers” to
   initiate the therapeutic efficacy, while the carrier materials would
   act as “green leaves” to augment their treatment effectiveness. In
   sharp contrast, our novel nanocarriers could not only serve as the
   “green leaves” for drug delivery and controlled release, but also play
   a unique role as the “flowers” for specific clearance of NADPH in
   boosting the therapy against TNBC via metabolic immunoregulation, which
   provides an alternative strategy for combining RT with anti‐PDL1
   therapy against TNBC.

2. Results and Discussion

2.1. Preparation and Characterization of BMS202@HZP NPs

   The auto‐changeable UCST polymer PAAN was synthesized through the
   radical polymerization of acrylonitrile (AN), acrylamide (AAm), and
   N‐(2‐(2‐nitro‐1H‐imidazol‐1‐yl)ethyl)acrylamide (NIEAAm)
   (Figure [66]S1a, Supporting Information), as the carrier materials for
   encapsulating BMS202 and its subsequent controlled release. Meanwhile,
   a lipoid (i.e., hyaluronic acid‐hexadecylamine‐ acetazolamide,
   HA‐HDA‐Z) was obtained by introducing acetazolamide (Z) and
   hexadecylamine (HDA) groups into hyaluronic acid (HA) in succession
   (Figure [67]S1b, Supporting Information), as the emulsifier for
   formulating the PAAN NPs with a shell of lipoid via hexadecyl anchors.
   The chemical structures of PAAN and HA‐HDA‐Z were confirmed by ^1H NMR
   (Figure [68]S2a,b, Supporting Information). The content of
   nitroimidazole groups in PAAN polymer was 261 µg per mg of polymer
   according to UV‐vis spectrum analysis of NIEAAm and PAAN polymer in
   dimethyl sulfoxide (Figure [69]S3, Supporting Information). The
   electrochemical test results of cyclic voltammogram revealed that PAAN
   polymer possessed similar electron affinity with half‐wave potential E
   [1/2] = −0.660 V to NIEAAm with E [1/2] = −1.049 V (Figure [70]S4,
   Supporting Information), suggesting the nitroimidazole groups in PAAN
   polymer still showed the efficient electron affinity. Due to the
   electron affinity, the nitroimidazole groups in PAAN polymer could be
   reduced by NADPH under hypoxic condition containing NTR, which was
   verified by the reduced absorbance intensity of nitroimidazole group at
   330 nm after incubating PAAN in the presence of NADPH and NTR under
   hypoxia for 24 h. However, addition of a single reductant (e.g., GSH or
   NADPH) into the solution of PAAN induced a negligible change or a
   slight decrease (Figure [71]S5, Supporting Information). These results
   confirmed that the PAAN polymer could react with NADPH and deplete it
   with assistance of NTR, impairing the DNA damage repair to sensitize RT
   even under hypoxia.

   The blank NPs without BMS202 loading, denoted as HZP NPs, were prepared
   using HA‐HDA‐Z and PAAN polymers, which exhibited spherical shapes with
   an average diameter of 210 nm and ζ‐potential of −31.2 mV
   (Figure [72]S6a–c, Supporting Information). Anionic HA, present on the
   surface of HZP NPs, played a critical role in promoting the stability
   of HZP NPs in a simulated physiological condition, reflected as the
   negligible change of NPs’ hydrodynamic diameters during 48 h of
   incubation (Figure [73]S6d, Supporting Information). The targeting
   capacity of HZP NPs on 4T1 cells under normoxia (21% O[2]) and hypoxia
   (1% O[2]) was evaluated using flow cytometry and confocal laser
   scanning microscopy (CLSM) with coumarin 6 (C6) as a fluorescent probe
   and CD44 single‐targeting HP NPs as control. It has been reported that
   CA IX receptors are often over‐expressed in cancer cells under hypoxia
   and CD44 is a cell surface receptor indicating malignancy.^[ [74]^15 ^]
   Thus, we hypothesized that the HZP NPs could efficiently interact with
   4T1 cells under hypoxia that benefited from the specific binding of HA
   and Z to CD44 and CA IX receptors, respectively. Compared to the HP
   NPs/C6 lacking Z modification, more HZP NPs/C6 accumulated in 4T1 cells
   and highlighted its advantages in targeting cancer cells under hypoxia
   (Figure [75] 2a–c). Moreover, in the competitive inhibition
   experiments, the amount of HZP NPs/C6 existing in 4T1 cells was
   significantly reduced because of the preblockade of CD44 and CA IX by
   free HA and Z, further confirming their targeting ability to 4T1 cells
   under hypoxia. Furthermore, to further demonstrate the high selectivity
   of HZP NPs to 4T1 cells under hypoxia, we incubated HZP NPs/C6 with 4T1
   cells, RAW264.7 macrophages, and T cells under normoxia and hypoxia for
   4 h, respectively. As shown in Figure [76]S7a–d in the Supporting
   Information, compared to T cells and RAW264.7 macrophages, more HZP
   NPs/C6 accumulated in 4T1 cells. Especially under hypoxia, the
   intracellular fluorescence intensity of 4T1 cells was 2.4‐ and 2.3‐fold
   higher than that of T cells and RAW264.7 macrophages, indicating that
   HZP NPs were prone to target TNBC cells.

Figure 2.

   Figure 2
   [77]Open in a new tab

   Targeting capacity, structure, payload release profiles, and blocking
   PD‐L1 receptor efficacy of NPs. a) CLSM images and b,c) flow cytometry
   analysis of 4T1 cells incubated with HP NPs/C6 and HZP NPs/C6 with or
   without pretreatment of HA ([HA]  =  10 mg mL^−1) and Z ([Z]   = 0.2 mg
   mL^−1). The blue color represents nucleus and green color represents
   C6‐loaded NPs. d) Morphology of BMS202@HZP NPs. e–g) Characterization
   of different NPs, including e) diameter, f) zeta potential, and g)
   encapsulation efficiency. h) Cumulative release of BMS202 from
   BMS202@HZP NPs incubated in PBS (pH 7.4, 0.01 m) containing NADPH and
   NTR under normoxia and hypoxia at 37 °C, respectively. i,j) Turbidity
   heating profiles of i) the PAA polymer and j) PAAN polymer after
   incubation with NADPH and NTR at 37 °C under argon protection for 0,
   12, and 24 h. k) Schematic diagram of the BMS202 released from
   BMS202@HZP NPs to inhibit the interaction between PE‐anti‐PD‐L1
   antibody and PD‐L1 receptor under hypoxia. l) PE fluorescence
   intensities of anti‐PD‐L1 antibodies in 4T1 cells treated with or
   without different drug formulations under normoxia and hypoxia. m)
   Blocking PD‐L1 efficiency of BMS202 and BMS202@HZP NPs in hypoxia (mean
   ± SD, n = 3, *p < 0.05, **p < 0.01).

   After loading the antagonist of PD‐1/PD‐L1 interactions (BMS202), the
   as‐obtained spherical BMS202@HZP NPs presented the similar
   morphological characteristics (222.2 nm, −35.4 mV) to the blank HZP NPs
   (Figure [78]2d–f), and the encapsulation efficiency of BMS202 for
   BMS202@HZP NPs was 54.5% (Figure [79]2g). The release profiles of
   BMS202 from BMS202@HZP NPs under various conditions were measured to
   evaluate the responsiveness of PAAN core with auto‐changeable UCST
   property under hypoxia in presentence of NADPH and NTR. Under normoxia,
   only 20.1% of BMS202 was released after 144 h incubation of BMS202@HZP
   NPs at pH 7.4 (Figure [80]2h). In contrast, the release of up to 61.6%
   and 80.3% BMS202 was observed for BMS202@HZP NPs incubated under
   hypoxia in presentence of NADPH and NTR for 6 and 144 h, respectively,
   revealing that the HZP NPs achieved the effectively controlled drug
   release. As reported in our previous studies, the hydrophobicity acted
   an important role in thermo‐responsiveness of UCST polymers.^[ [81]^16
   ^] When the ratio of hydrophilicity versus hydrophobicity of UCST
   polymers increased, the intermolecular forces among polymers would be
   weakened, leading to a decreased UCST. As above demonstrated, the
   hydrophobic nitroimidazole groups in the PAAN chains could transform
   into hydrophilic aminoimidazole groups under hypoxia in the presence of
   NADPH and NTR,^[ [82]^17 ^] which should be the main reason for
   realizing its auto‐changeable UCST. Thus, compared to PAA polymer
   without nitroimidazole group (Figure [83]2i), the UCST of PAAN polymer
   significantly decreased from 79.9 to 22.4 and 4.5 °C after 12 and 24 h
   of incubation under hypoxic condition containing NADPH and NTR
   (Figure [84]2j), respectively, demonstrating that the UCST of PAAN
   polymer could be auto‐changed under hypoxia in the presence of NADPH
   and NTR. By virtue of this advantage of PAAN as carrier, HZP NPs would
   avoid several adverse side effects caused by the undesired leakage of
   BMS202 during systemic circulation. Once entering the tumor under
   hypoxia, BMS202@HZP NPs would collapse rapidly to release BMS202. More
   importantly, when 4T1 cells were treated with BMS202@HZP NPs under
   hypoxia, the released BMS202 from BMS202@HZP NPs would specifically
   bind to PD‐L1 receptors, potentially inhibiting the PD‐1/PD‐L1
   interaction to realize ICB therapy. The high binding capacity of BMS202
   to PD‐L1 was observed, as shown by a reduced binding efficiency of
   PE‐anti‐PD‐L1 antibody to PD‐L1 receptors for the cells pretreated with
   BMS202@HZP NPs, revealing a good potentiality of anti‐PD‐L1 ICB therapy
   of BMS202@HZP NPs (Figure [85]2k–m).

   To further demonstrate that the imidazole groups in PAAN were prone to
   selectively scavenge the NADPH inside cancer cells, the RAW264.7
   macrophages and 4T1 cells were radiated with 2 Gy of X‐ray, and then
   treated with 0.9% NaCl, blank HZP NPs, and BMS202@HZP NPs,
   respectively. To adapt to the enhanced oxidative stress induced by RT,
   the intracellular NADPH/NADP^+ ratios of both macrophages and 4T1 cells
   were obviously increased (Figure [86]S8, Supporting Information).
   Moreover, when the RAW264.7 macrophages were treated with the blank HZP
   NPs and BMS202@HZP NPs, the NADPH/NADP^+ ratio still stayed at the high
   level compared to the macrophages without any treatments
   (Figure [87]S8a, Supporting Information). However, the NADPH/NADP^+
   ratio of 4T1 cells would obviously decrease under both normoxia
   (Figure [88]S8b, Supporting Information) and hypoxia (Figure [89]S8c,
   Supporting Information) once co‐culturing the blank HZP NPs and
   BMS202@HZP NPs with 4T1 cells together. Thus, it was demonstrated that
   the BMS202@HZP NPs could scavenge the NADPH inside 4T1 cells, which was
   attributable to the targeting capacity of HZP NPs to 4T1 cells as well
   as the overexpression of NTR in cancer cells.^[ [90]^18 ^]

2.2. In Vitro Antitumor Efficacy of BMS202@HZP NPs

   The nitroimidazole groups in PAAN polymer could deplete NADPH and
   immobilize DNA damage during RT due to their higher electron affinity,
   contributing to an increased in vitro antitumor efficacy. To prove
   this, we evaluated the cytotoxicity of BMS202@HZP NPs against 4T1 cells
   upon the low‐dose (2 Gy) X‐ray radiation using 3‐(4,
   5‐dimethylthiazolyl‐2)‐2, 5‐diphenyltetrazolium bromide assay. Indeed,
   the viability of 4T1 cells gradually decreased with an increase of
   nitroimidazole concentration after the blank HZP NPs‐mediated LDRT,
   regardless of the oxygen level (Figure [91] 3a,b). Meanwhile, since
   BMS202 can not only inhibit the formation of PD‐1/PD‐L1 complex but
   also induce cytotoxicity to cancer cells, BMS202@HZP NPs showed the
   enhanced inhibitory on 4T1 cells growth (Figure [92]3c,d).
   Additionally, the combination indexes of BMS202 and
   nitroimidazole‐sensitized LDRT at IC[50] values (Table [93]S1,
   Supporting Information) under normoxia (0.37) and hypoxia (0.42) were
   much lower than 1 for the BMS202@HZP NPs‐mediated LDRT, indicating a
   good synergism mediated by BMS202@HZP NPs. Then, the long‐term
   radiation‐sensitization effect of BMS202@HZP NPs was also examined on
   4T1 cells using clone formation assay. Once 4T1 cells were treated with
   BMS202@HZP NPs plus LDRT, there were only around 30.8% and 57.1% clones
   formed under normoxia (Figure [94]3e,f) and hypoxia (Figure [95]3g,h),
   respectively, which were much less for the 4T1 cells undergoing the
   treatments of free BMS202 and blank HZP NPs‐mediated LDRT.
   Collectively, BMS202@HZP NPs‐mediated LDRT could suppress the
   proliferation of 4T1 cells, potentially implying their capacity of
   scavenging the tumor intracellular NADPH to sensitize LDRT.

Figure 3.

   Figure 3
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   In vitro therapeutic and immunoregulatory effects of BMS202@HZP NPs.
   a–d) Cytotoxicity evaluation of blank a,b) HZP NPs, c,d) BMS202 and
   BMS202@HZP NPs with or without LDRT (2 Gy) against 4T1 cells at various
   NI‐equivalent concentrations under hypoxia and normoxia (n = 5). e–l)
   Representative images of e,g) colony formation assay, f,h) clone
   formation efficacy, i,k) CRT expression, and j,l) western blot analysis
   of 4T1 cells treated with free BMS202, blank HZP NPs, and BMS202@HZP
   NPs‐mediated 2 Gy X‐ray radiation (nitroimidazole [NI]  =  238 × 10^−6
   m, [BMS202] = 2.57 µg mL^−1) under hypoxia or normoxia (n = 3). m)
   Schematic diagram of the experimental methodology for distant tumor
   cell treatment in vitro using a co‐culture model in the transwell
   system. n,o) The levels of n) IFN‐γ and o) TNF‐α in cell supernatants
   collected from the lower chamber (n = 3). p) Cell survival rate and q)
   image of distant 4T1 cells stained with calcein‐AM in the lower chamber
   (n = 3) (mean ± SD, *p < 0.05, **p < 0.01).

   In addition to enhancing the LDRT efficiency, the purpose of
   incorporating nitroimidazole groups into BMS202@HZP NPs is to realize
   the efficient ICD and activate the immune response against TNBC, by
   virtue of depleting NADPH inside tumor cells to impair the DNA repair
   during LDRT. Besides, the cytotoxic BMS202 could also make
   contributions to inducing ICD. Calreticulin (CRT) is one of major
   hallmarks of ICD, which is a well‐known “eat‐me” signal for dendritic
   cells (DCs) uptake and activating immune system.^[ [97]^19 ^] Thus, the
   expressions of CRT on 4T1 cells with different treatments were first
   measured using western blot. It was found that the CRT expressions of
   4T1 cells after the treatments of HZP NPs or BMS202@HZP NPs‐mediated
   LDRT were upregulated (Figure [98]3i–l), especially for the treatment
   of BMS202@HZP NPs plus LDRT, which increased by 4.4‐fold and 1.2‐fold
   compared to the untreated 4T1 cells under normoxia and hypoxia,
   respectively. As such, BMS202@HZP NPs‐mediated LDRT could effectively
   induce TNBC immunogenic death.

   To further evaluate activities of immune cells against metastatic cells
   after BMS202@HZP NPs‐mediated LDRT, a double‐layered cell model was
   established using a transwell system as illustrated in Figure [99]3m.
   Briefly, the peripheral blood mononuclear cells (PBMCs) and 4T1 cells
   were co‐cultured in upper wells under hypoxia and performed with
   different treatments, then the level of several cytokines that promoted
   immune responses in lower wells (preseeded with 4T1 cells) was
   measured. Meanwhile, the viability of tumor cells in lower wells was
   determined via calcein‐AM staining. Due to the hypoxia, LDRT alone
   hardly generated radicals to induce the ICD and facilitated PBMCs to
   secrete IFN‐γ and TNF‐α, causing the negligible inhibitory effect on
   the proliferation of metastatic 4T1 cells (Figure [100]3n,o). In sharp
   contrast, the enhanced content of IFN‐γ and TNF‐α were minored in the
   groups of BMS202@HZP NPs with or without LDRT, further inducing the 4T1
   cells death in lower wells. Especially, BMS202@HZP NPs‐mediated LDRT
   presented the most significant killing effect against 4T1 cells and
   reflected as only 25.4% of viable cells stained by calcein‐AM in lower
   cells, which were 3.6‐fold and 2.3‐fold lower than the 4T1 cells that
   only received LDRT or BMS202@HZP NPs treatments (Figure [101]3p,q).
   This phenomenon could be explained by the following factors: for one
   thing, BMS202 could relieve PD‐1/PD‐L1 immunosuppressive pathway to
   boost T cells response and facilitate cytokines production; for another
   thing, due to the existence of nitroimidazole in HZP NPs, BMS202@HZP
   NPs were prone to scavenge the NADPH inside the 4T1 cells to impair the
   DNA damage repair and induce immunogenic death of 4T1 cells under
   hypoxia, which would also activate PBMCs to release more cytotoxic
   cytokines against metastatic 4T1 cells.

2.3. In Vivo Biodistribution of BMS202@HZP NPs

   The effective delivery of nanomedicine to tumor is the prerequisite to
   achieving better therapeutic efficacy in vivo. However, there are some
   factors, such as the dense blood vessels and elevated interstitial
   fluid pressure, limiting the intratumoral delivery of nanomedicine. The
   preclinical data implied that only 0.7% of injected dose of NPs
   accumulated in human tumors.^[ [102]^20 ^] To address this challenge,
   besides modifying the surface of NPs with targeted ligands, several
   exogenous stimuli (e.g., radiation and heat) are also effective in
   promoting accumulation of NPs in tumors.^[ [103]^21 ^] According to the
   previous studies, X‐ray radiation could alter the fluid dynamics of
   tumor blood vessels and enhance the accumulation of NPs in tumors.^[
   [104]^21 ^] In this study, DiR (a near‐infrared fluorescence
   probe)‐loaded HZP NPs could indeed better accumulate in tumors
   pretreated with X‐ray radiation (Figure [105]S9, Supporting
   Information), presenting 2.5‐fold, 1.5‐fold, and 1.1‐fold enhancement
   in tumor fluorescence intensity as compared to the group of free DiR,
   DiR@HP NPs, and DiR@HZP NPs without the pretreatment of X‐ray
   radiation, respectively. To further confirm whether the increased
   vascular permeability was caused by X‐ray radiation, the mice
   pretreated with or without X‐ray radiation were i.v. injected the Evans
   blue dye at 24 h post‐injection of 0.9% NaCl, BMS202@HP NPs, or
   BMS202@HZP NPs. After measuring the content of extravasated Evans blue
   in tumors, we found that there was no significant difference in
   intratumoral Evans blue level among the groups of 0.9% NaCl, BMS202@HP
   NPs, and BMS202@HZP NPs. Excitingly, there was 1.6–1.7‐fold enhancement
   on Evans blue dye extravasation for the group of BMS202@HZP NPs plus
   LDRT in comparison to the other groups (Figure [106]S10, Supporting
   Information). These results further demonstrated that the X‐ray
   radiation could serve as an enhancer for augmenting enhanced
   permeability and retention effect. Besides, CD44/CA IX dual‐targeted
   DiR@HZP NPs presented higher fluorescence intensity than the
   single‐targeted DiR@HZP NPs, verifying that the specific interaction
   between HA/CD44 receptor and Z/CA IX receptor could promote HZP NPs to
   target hypoxic 4T1 tumor. Finally, both X‐ray radiation and HA/Z
   modification jointly improved the accumulation of therapeutics at tumor
   sites, which would lay the foundation for effective TNBC therapy.

2.4. In Vivo Therapeutic Efficacy of BMS202@HZP NPs

   RT was reported to facilitate the PD‐L1 upregulation,^[ [107]^22 ^]
   which also has been verified in our study with results shown in
   Figure [108]S11 in the Supporting Information. Compared to the LDRT (2
   Gy) alone, the BMS202@HZP NPs‐mediated LDRT resulted in the enhanced
   PD‐L1 expression in 4T1 tumor (Figure [109]S11, Supporting
   Information). As mentioned above, the low or negative expression of
   PD‐L1 was one of the major reasons for the low response rate of
   anti‐PD‐L1 therapy among TNBC patients. Thus, promoting the PD‐L1
   expression by BMS202@HZP NPs‐mediated LDRT has the potential to
   increase the response rate and efficiency of anti‐PD‐L1 therapy against
   TNBC, facilitate the CD8+ T cells recruitment, and relieve the
   immunosuppression of TME. To prove this, the mice bearing a single 4T1
   subcutaneous tumor were subjected to 0.9% NaCl, BMS202@HP NPs, and
   BMS@HZP NPs with or without LDRT (2 Gy) when the tumor volume reached
   around 150 mm^3, and part of tumors and plasma were harvested to
   evaluate the immune responses on 10 days as illustrated in Figure [110]
   4a. Additionally, the transcriptome sequencing and analysis of total
   RNA were performed. BMS202@HZP NPs‐mediated LDRT induced the
   significant changes in 731 genes in tumor in comparison to 0.9% NaCl
   treatment (Figure [111]4b). Thereinto, there were 361 genes upregulated
   and 370 downregulated. Moreover, through gene ontology analysis, it is
   shown that among the differentially expressed genes, 61 genes were
   associated with immune system. As shown in Figure [112]4b,c, BMS202@HZP
   NPs‐mediated LDRT caused the upregulation of the genes correlated with
   the proliferation and activation of T cells (e.g., Igf2, Zbtb32,
   Il12rb1, and Tacr1), the interferon response genes (e.g., Oas2 and
   Oas3) for effector T‐cell recognition of tumor cells and the genes
   polarized TAMs into immunostimulatory M1 phenotype (e.g., Nos2, Acod1,
   Isg15, and Fcer1a). Meanwhile, the expression of genes relative to the
   process of polarizing TAMs into an immunostimulatory M2 phenotype
   (e.g., Epx, Prg2, Alox15, Fbn2) reduced obviously after the treatment
   of BMS202@HZP NPs plus LDRT. Accordingly, it was indicated that
   BMS202@HZP NPs‐mediated LDRT possessed the potential to increase
   antitumor immune responses and relieve the immunosuppression of TME.

Figure 4.

   Figure 4
   [113]Open in a new tab

   BMS202@HZP NPs plus LDRT relieving the immunosuppression of TME to
   achieve the enhanced antitumor efficacy. a) Treatment schedule for
   BMS202@HZP NPs‐mediated LDRT against subcutaneous 4T1 tumor on Balb/c
   mice. b) Volcano plot for displaying genes expression in tumor. Red
   dots represent upregulated genes, gray dots represent not
   differentially expressed genes, and blue dots represent downregulated
   genes in BMS202@HZP NPs‐mediated LDRT group versus 0.9% NaCl group (cut
   off: fold change ≥ 2, FDR‐adjusted p‐value < 0.05). c) Heatmap of
   differentially expressed genes associated with immune response of 4T1
   tumors following the indicated treatments on day 10 (fold change ≥ 2,
   FDR‐adjusted p‐value < 0.05). d) DCs, e) CD8+ T cells, f) CD8+ T
   cells/T[regs] ratio, and g) M1/M2 ratio in 4T1 tumors following the
   indicated treatments on day 10 (n = 5). h) IHC staining for CD8 and
   Gran B, and immunofluorescence staining for CD86 (hallmark of M1
   phenotype, red), F4/80 (hallmark of TAM, green), CD206 (hallmark of M2
   phenotype, red), and nucleuses (blue) of tumor tissue in 4T1 tumors
   following the indicated treatments on day 10. i) Primary tumor growth
   and j) their spaghetti curves during experiment (n = 5). k) Survival
   curves of mice‐bearing 4T1 tumors after tumor inoculation (n = 5,
   log‐rank test) (mean ± SD, *p < 0.05, **p < 0.01).

   On account of LDRT capacity to regulate immune response, there was a
   slight increase in the amount of CD3+CD8+ T cells and matured DCs in
   tumors received 2 Gy of radiation in comparison to 0.9% NaCl‐treated
   group (Figure [114]4d,e), and the ratios of CD8+ T cells versus
   regulator T cells (T[reg]) and M1 versus M2 also increased
   (Figure [115]4f,g). However, the ratios of various immune cells were
   not obviously altered for the mice administrated with BMS202@HP NPs or
   BMS202@HZP NPs in the absence of low‐dose X‐ray radiation (2 Gy).
   Moreover, BMS202@HZP NPs‐mediated LDRT could further strengthen the
   immune activities in vitro, and thus this treatment achieved the
   maximal elevation of the CD8+ T cells infiltration (Figure [116]4e,h),
   the DCs maturation (Figure [117]4d), and the Granzyme B (Gran B)
   production (Figure [118]4h) in tumor among various groups. Meanwhile,
   there were more M1‐like phenotype macrophages and less M2‐like
   phenotype macrophages detected in tumor after BMS202@HZP NPs‐mediated
   LDRT. The ratio of M1/M2 in this group was 3.3‐, 1.3‐, 3.1‐, and
   3.1‐fold higher than that in groups of 0.9% NaCl, 2 Gy radiation,
   BMS202@HP NPs, and BMS202@HZP NPs, respectively (Figure [119]4g).
   Immunofluorescence staining for CD86 (hallmark of M1) and CD206
   (hallmark of M2) also presented the consistent results
   (Figure [120]4h). Considering TAMs account for a portion even up to 50%
   in tumor mass and represent the major population of immune cells in
   TME,^[ [121]^23 ^] BMS202@HZP NPs‐mediated LDRT could polarize the TAMs
   into M1‐phenotype and relieve the immunosuppression of TME.

   The above results confirmed that BMS202@HZP NPs‐mediated LDRT could
   activate immune response and reprogram TME toward immunostimulatory
   phenotype, so as to enhance the capacity for inhibiting tumor growth.
   As expected, there was an obvious improvement in suppressing tumor
   growth (Figure [122]4i,j and Figure [123]S12, Supporting Information)
   and prolonging survival rate (Figure [124]4k) for the mice received
   BMS202@HZP NPs‐mediated LDRT, and its tumor inhibitory efficiency was
   76.2% at the end of experiment (Figure [125]S13, Supporting
   Information). In contrast, the treatment of 2 Gy radiation alone and
   nanomedicines (BMS202@HP NPs or BMS202@HZP NPs) in absence of LDRT just
   moderately suppressed the growth of 4T1 tumors. Overall, BMS202@HZP
   NPs‐mediated LDRT could achieve an excellent capacity of eliminating
   tumor tissue.

2.5. Mechanisms of Immune Regulation Analysis based on Metabolomics

   Metabolomics is a state‐of‐the‐art tool to identify and quantify the
   downstream small molecules present in biological systems, aiming to
   gain insight into the system's metabolic state.^[ [126]^24 ^] More
   importantly, the metabolism of both cancer cells and immune cells is
   believed to determine their fate and relevant TME.^[ [127]^12a,d ^]
   Therefore, we measured and analyzed the metabolisms of the tumors
   treated by BMS202@HZP NPs‐mediated LDRT using gas chromatography‐mass
   spectrometry, with the purpose of elucidating the synergism mechanisms
   behind anti‐PD‐L1 therapy and LDRT. In this study, a total of 52
   metabolites were identified in tumors for the groups of BMS202@HZP
   NPs‐mediated LDRT and 0.9% NaCl, which are shown in Table [128]S2 in
   the Supporting Information. The principal component analysis (PCA) was
   then performed to produce an unbiased overview of the metabolic
   datasets, which exhibited a clear and separated clustering trend of
   metabolic datasets between these two groups (Figure [129] 5a).
   Furthermore, the supervised orthogonal partial least‐squares
   discrimination analysis (OPLS‐DA) was employed to optimize the group
   discrimination model. As shown in Figure [130]5b, the score plots of
   BMS202@HZP NPs‐mediated LDRT group were far from those of the 0.9% NaCl
   group, which revealed that the metabolic profiles in tumor
   significantly changed after the synergistic therapy. It is worth
   mentioning that the cumulative R2 and Q2 values of OPLS‐DA were 0.860
   and 0.800, respectively, verifying the good qualities of this model. As
   exhibited in S‐plot of OPLS‐DA (Figure [131]5c), the variable
   importance for the projection (VIP) values of labeled metabolites (14
   compounds) was greater than 1, suggesting these compounds might be
   altered metabolites between these two groups. Besides, the heatmap
   clustering analysis of the tumor differential metabolites between 0.9%
   NaCl and BMS202@HZP NPs‐mediated LDRT groups was also performed, and a
   total of 16 differential metabolites (false discovery rate
   (FDR)‐adjusted p‐value <0.05) were screened out and are shown in
   Figure [132]5d. Based on these results, the significantly differential
   metabolites, matching the condition of both VIP > 1 and FDR‐adjusted
   p‐value < 0.05, were identified, which revealed that BMS202@HZP
   NPs‐mediated LDRT induced the significant elevation of adenosine,
   D‐ribose‐5‐phosphate, glycerol 1‐phosphate, ribulose‐5‐phosphate, etc.,
   and decline of D‐glucose‐6‐phosphate, glyceric acid, etc.
   (Table [133]S3, Supporting Information).

Figure 5.

   Figure 5
   [134]Open in a new tab

   Analysis of metabolites in tumor tissue treated with BMS202@HZP NPs
   plus LDRT. a) PCA score plot and b) OPLS‐DA score plot for 0.9% NaCl
   (red dots) and BMS202@HZP NPs‐mediated LDRT groups (black dots). c)
   S‐plot of OPLS‐DA model. d) Heatmap of differential metabolites of
   tumor in BMS202@HZP NPs‐mediated LDRT compared to 0.9% NaCl group. e)
   Metabolic pathway enrichment analysis based on differential
   metabolites. f–i) Enzyme activity of f) G6PD and g) 6PGD, h) level of
   NADPH, and i) ratio of NADPH/NADP^+ in tumor tissue after different
   treatments (mean ± SD, n = 5, *p < 0.05, **p < 0.01). j) Schematic
   illustration of the proposed mechanism for BMS202@HZP NPs‐mediated LDRT
   to reverse relieve suppression of TME by upregulating PPP.

   The pathway enrichment analysis based on significantly differential
   metabolites was then conducted, and seven metabolic pathways were
   significantly altered in BMS202@HZP NPs‐mediated LDRT group. Among
   them, the most significant difference occurred in PPP (Figure [135]5e);
   thus, the activity and level of enzymes and compounds associated with
   PPP were measured. The activities of glucose‐6‐phosphate dehydrogenase
   (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) for BMS202@HZP
   NPs‐mediated LDRT were increased by 2.4‐ and 2.3‐fold as compared with
   0.9% NaCl group, respectively (Figure [136]5f,g). In agreement with
   enzymatic activity levels of G6PD and 6PGD, there were also 1.5‐fold
   and 1.8‐fold enhancements on the level of NADPH and the ratio of
   NADPH/NADP^+ for the synergistically therapeutic group, respectively
   (Figure [137]5h,i). Since BMS202@HZP NPs could selectively target 4T1
   cells and scavenge their intracellular NADPH by nitroimidazole group,
   these results suggested the PPP of TME was further elevated to produce
   more NADPH after this therapy. As discussed above, the PPP, as one of
   the important specific glycolytic mediators, influences the division
   and activation of immune cells.^[ [138]^12a,b ^] NADPH generated by PPP
   is a molecule with essential roles in the synthesis of biomolecules,
   like fatty acid synthesis, which is the primary source for fatty acid
   and plasma membrane synthesis in newly activated CD8+ T cells.^[
   [139]^12b,d ^] Furthermore, NADPH is the crucial substrate of the NADPH
   oxidase (NOX) pathway, which is exploited by activated macrophages to
   kill cancer cells.^[ [140]^25 ^] Thus, the engagement of the PPP is a
   peculiar feature of activated CD8+ T cells and M1. Besides, since NADPH
   also plays the key role in maintaining cellular redox, the upregulated
   NADPH could facilitate the polarization of TAMs from M2‐like phenotype
   toward M1‐like phenotype upon scavenging reactive oxygen species.^[
   [141]^11 ^] Therefore, the elevated PPP of TME during BMS202@HZP
   NPs‐mediated LDRT should be responsible for relieving the
   immunosuppression of TME, serving as an important proinflammatory
   programmer of antitumor response (Figure [142]5j).

2.6. Abscopal Effect and Lung Metastasis Treatment

   To evaluate abscopal effects of BMS202@HZP NPs‐mediated LDRT, the 4T1
   bilateral tumor model was established and various treatments were
   performed, as illustrated in Figure [143] 6a. It was found that the
   growth profiles of primary tumors on bilateral tumor model were
   consistent with those on unilateral tumor model, where the optimal
   tumor‐suppressive effect was achieved during the therapeutic process of
   combining BMS202@HZP NPs and LDRT (Figure [144]6b). Similarly, it was
   found that BMS202@HZP NPs‐mediated LDRT could greatly suppress the
   growth of distant tumors; however, other treatments (X‐ray radiation
   alone, BMS202@202HP NPs and BMS202@HZP NPs without X‐ray radiation) had
   only a slight influence on the distant tumors’ growth (Figure [145]6c,d
   and Figure [146]S14, Supporting Information). We hypothesized that the
   enhanced abscopal effect induced by BMS202@HZP NPs‐mediated LDRT
   potentially inhibited the proliferation of distant tumor tissues. As
   our hypothesis, the flow cytometry analysis of the distant tumors
   revealed a significant increase in the number of lymphocytes (CD3+ T
   cells and CD3+CD8+ T cells) after the BMS202@HZP NPs‐mediated LDRT
   (Figure [147]6e,f). We also observed that the expression of Gran B and
   the number of CD8+ T cells was significantly increased in distant
   tumors via immunohistochemistry (IHC) staining (Figure [148]6g),
   indicating that more activated CD8+ T cells infiltrated into distant
   tumor and attacked the target tumor cells. In addition, after
   BMS202@HZP NPs‐mediated LDRT, there were much more CD8+ T cells and
   CD20+ B cells accumulating in spleen (Figure [149]6g). Since spleen was
   one of major peripheral lymphoid organs for lymphocyte activation by
   antigens, these lymphocytes would be activated and mature in spleen and
   then recirculate between blood until they encounter their specific
   antigen.^[ [150]^26 ^] To further confirm the abscopal effect, the
   level of IFN‐γ and TNF‐α in plasma was measured, which was believed to
   promote the proliferation and activation of CD8+ T cells. As shown in
   Figure [151]6h,i, the single treatment, including 2 Gy X‐ray radiation,
   BMS202@HP NPs and BMS202@HZP NPs, facilitated TNF‐α and IFN‐γ
   expression to some extent due to the immune responses induced by LDRT
   or BMS202‐induced PD‐1/PD‐L1 blockade, both of which could boost immune
   cells to produce cytokines. Thus, when combining anti‐PD‐L1 and LDRT
   through HZP NPs, their synergism would further enhance the level of
   IFN‐γ and TNF‐α.

Figure 6.

   Figure 6
   [152]Open in a new tab

   Abscopal effects and systemic antitumor immunity induced by BMS202@HZP
   NPs‐mediated LDRT. a) Treatment schedule for BMS202@HZP NPs‐mediated
   LDRT against primary and distant 4T1 tumors. b) Primary tumor growth,
   c) distant tumor growth, and d) spaghetti curves of distant tumors
   during experiments. e–i) The level of e) CD3+ T cells and f) CD8+ T
   cells in distant tumor tissue, and the level of h) TNF‐α and i) IFN‐γ
   in plasma (n = 3) following the indicated treatments on day 21. g) IHC
   staining of CD8+ T cells and Gran B of distant tumor, and CD20 and CD8
   of spleen following the indicated treatments on day 21. j) Morphology,
   k) number, and l) H&E staining images of lung metastatic nodules. m)
   Schematic diagram of BMS202@HZP NPs‐mediated LDRT inducing strong
   antitumor immunological responses to reinforce the abscopal effect
   against distant tumor growth and tumor metastasis (mean ± SD, n = 5, *p
   < 0.05, **p < 0.01).

   On the other hand, to further evaluate this therapeutic potency on
   inhibiting lung metastasis of 4T1 cells, the lungs after different
   treatments were harvested and stained with Bouin's solution and
   hematoxylin and eosin (H&E). Figure [153]6j,k reveals that there were
   37.2 nodules detected at the lung site in 0.9% NaCl group, while the
   number of metastatic nodules remarkably decreased to 6.8 after
   BMS202@HZP NPs‐mediated LDRT, which were also less than those of 2 Gy
   X‐ray radiation (27.6), BMS202@HP NPs (16.4), and BMS202@HZP NPs (12.2)
   treatments. These results were further validated by the images of
   H&E‐stained lung tissues, where both the number and area of lung
   metastatic nodules in the group of BMS202@HZP NPs‐mediated LDRT were
   obviously reduced in comparison to other groups (Figure [154]6l),
   indicating this synergistically therapeutic strategy could also inhibit
   the lung metastasis of TNBC. To sum up, BMS202@HZP NPs‐mediated LDRT
   could induce strong antitumor immunological responses to reinforce the
   abscopal effect against distant tumor growth and tumor metastasis
   (Figure [155]6m).

2.7. Biosafety

   Biosafety of nanomedicine is an important evaluation criterion for its
   further clinical application. Thus, we not only evaluated the
   hemocompatibility of blank HZP NPs but also collected plasma and major
   organs of mice after different treatments for histopathologic,
   bodyweight, biochemical, blood routine, and metabolomics analysis. When
   the blank HZP NPs were incubated with erythrocytes, there only appeared
   the low hemolysis (<3%) at different concentrations (50–500 µg mL^−1)
   (Figure [156]S15, Supporting Information), demonstrating that blank HZP
   NPs possessed good hemocompatibility. The results of mice body weight,
   H&E staining images, and biochemical analysis (AST, ALT, CRE, BUN) did
   not show obvious differences between the BMS202@HZP NPs group and 0.9%
   NaCl group, indicating that BMS202@HZP NPs could not trigger severe
   tissue damage and dysfunction in major organs (Figures [157]S16–S18,
   Supporting Information). However, free BMS202 treatment caused more
   infiltration of inflammatory cells in kidney. In routine blood test,
   free BMS202 induced the significant enhancement of WBC and lymph
   compared to normal group, suggesting it might cause an inflammatory
   reaction in mice body (Figure [158]S19, Supporting Information). In
   contrast, the similar symptom was not observed in BMS202@HZP NPs group.
   Meanwhile, through analyzing the metabolites of serum, there existed
   five metabolites with statistically significant difference (p value <
   0.05) between free BMS202 group and normal group (Figure [159]S20 in
   the Supporting Information), such as the decrease of glycerol and the
   increase of gluconic acid, myo‐inositol, glycine, and L‐lysine.
   However, there was only one metabolite (succinic acid) in BMS202@HZP
   NPs from normal group. Taken together, it is believed that HZP NPs with
   the controllable UCST would be a suitable carrier for delivering
   BMS202, which could avoid adverse side effects caused by nonspecific
   drug leakage and thus achieve the preferable biosafety for further
   medical application.

3. Conclusions

   In summary, a multifunctional “drug‐like” copolymer with the
   auto‐changeable UCST and selective depletion of NADPH inside tumor
   cells was successfully synthesized and exploited to fabricate a safe
   and effective nanocarrier for sensitizing radiotherapy and controlled
   release of antagonist (BMS202) of PD‐1/PD‐L1 interactions. Due to the
   modification of CD44 receptor HA and hypoxia‐dependent factor CA IX
   inhibitor Z on the surface of nanocarrier, it achieved the enhanced
   strike on hypoxic TNBC and mediated LDRT to induce ICD of TNBC,
   converting the immune “cold” tumor into the “hot” tumor. More
   importantly, BMS202@HZP NPs‐mediated LDRT significantly promoted the
   PPP in TME and generated more NADPH to facilitate the polarization of
   TAMs toward M1‐like phenotype and activation of CD8+ T cells,
   reprogramming the immunometabolism of TME to alleviate its
   immunosuppression. Meanwhile, BMS202@HZP NPs were prone to scavenge the
   NADPH inside tumor cells to impair DNA repair, resulting in the
   upregulated PD‐L1 expression of tumor cells to reinforce the anti‐PD‐L1
   therapy. Based on these advantages, BMS202@HZP NPs‐mediated LDRT
   achieved the significantly improved synergism effect against TNBC.
   Thus, it is believed that this nanomedicine‐mediated
   radio‐immunometabolism regulation would be a promising strategy to
   reinforce anti‐PD‐L1 against TNBC.

4. Experimental Section

   The Experimental Section is available in the Supporting Information.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [160]Click here for additional data file.^ (2.7MB, pdf)

Acknowledgements