Abstract

   Low persistence, metabolic dysfunction in microenvironment, and
   tumor‐derived immunosuppression of Natural killer (NK) cells in
   patients are greatly limited the successful clinical application of NK
   cell‐based cancer immunotherapy. Interestingly, herein that human serum
   albumin‐encapsulated black phosphorus quantum dots (BPQDs@HSA) can
   effectively augment antitumor efficacy of clinical patients‐derived NK
   cell immunotherapy is found. As the donor of phosphate group, BPQDs@HSA
   binds with the protein of phosphatidylinositol 4‐phosphate 5‐kinase
   type‐1 gamma (PIP5K1A) and activates the downstream PI3K‐Akt and mTOR
   signaling pathways to reprogram cell metabolism of glycolysis and
   further promote the oxidative phosphorylation, sequentially maintains
   the cell viability and immunity of NK cells. And multiomics analysis is
   therefore conducted to reveal the underlying immunoregulation
   mechanisms, and that BPQDs@HSA can interact with the Toll‐like receptor
   (TLR) on the NK cell surface and increase the expression level of mTOR,
   and thus activate downstream NF‐κB signalling pathways to regulate
   cytokine secretion and enhance immune tumoricidal is found. BPQDs@HSA
   can also enhance immune surveillance, relieve immune suppression, and
   inhibit tumor immune escape. Collectively, this study not only
   demonstrates a successful strategy for nanomedicine‐potentiated
   immune‐cancer therapy, but also sheds light on the understanding of
   interface between nanomedicine and immune cells activation.

   Keywords: black phosphorus, cell metabolism, immunological enhancement,
   immunotherapy, natural killer cell, relieving immunosuppression
     __________________________________________________________________

   Herein a successful strategy for cancer treatment by
   nanomedicine‐potentiated NK cell immunotherapy is presented. BPQDs@HSA
   activates PI3K‐Akt‐mTOR signaling pathways to reprogram cell metabolism
   of glycolysis and further promote the oxidative phosphorylation,
   sequentially maintains the cell viability and immunity of NK cells.
   BPQDs@HSA can also enhance immune tumoricidal and immune surveillance,
   relieve immune suppression, and inhibit tumor immune escape.

   graphic file with name ADVS-10-2202519-g006.jpg

1. Introduction

   Currently, natural killer (NK) cell therapy, a new type of cancer
   immunotherapy, has a unique advantage of discriminating the malignant
   from healthy cells, thus achieving encouraging output in pre‐clinic and
   clinic state research.^[ [40]^1 ^] According to statistics, totally,
   there are over two hundred of clinical trials investigating variety of
   NK cell immunotherapies to treat hematologic and solid malignancies in
   2021.^[ [41]^2 ^] NK cells exert the anti‐cancer activity mainly
   through: 1) secreting perforin and granzymes to kill cancer cell; or 2)
   directly induce cell apoptosis mediated by death receptor. Another
   important hallmark of NK cells is the major histocompatibility complex
   (MHC)‐unrestricted anti‐tumor activity, which provided higher
   cells‐mediated immunity than other cells in cancer therapy.^[ [42]^3 ^]
   The MHC non‐restrictive characteristic makes NK cells play an important
   role in tumor immune surveillance, and show a significant advantage in
   immunotherapy to kill cancer cell more directly and rapidly.^[ [43]^4
   ^] However, although NK cells exhibit impressive anticancer activity,
   the severe dysfunction, diminished activity and tumor‐derived
   immunosuppression of NK cells in tumor microenvironment (TME) limit the
   successful of cancer treatments.^[ [44]^5 ^] The process of tumor
   establishment, progression and metastasis forms an immunosuppressive
   microenvironment, including the highly secreted immunosuppressive
   factors such as TGF‐β and IL‐10, and the activation of PD‐1/PD‐L1
   signaling pathways, to affect the immune anergy, exhaustion and cell
   death of NK cells or even leads to the transmission of an
   anti‐apoptotic signal to tumor cells. The damage of NK cells can
   promote the tumor establishment, progression and metastasis, and also
   show correlative with the prognosis of many cancer patients.^[ [45]^6
   ^] The fundamental mechanisms of NK cell immunosuppression in TME are
   still poorly understood. Therefore, it is of great significance to
   utilize high‐efficiency and low‐toxicity sensitization agents to
   re‐activate and amplify NK cells activity in TME, thus enhancing their
   sustained anticancer activity, and further explore the potential action
   mechanisms.

   The complexity of malignant tumors and their microenvironment makes it
   impossible for successful clinical treatment to be accomplished by a
   single technique or method, which requires scientists to continuously
   explore new problems that arise in the treatment process and
   accordingly to propose new strategies based on multimodal and
   integrated approaches.^[ [46]^7 ^] Cancer immunotherapy by utilizing
   nanotechnology to stimulate the immunological systems of patients to
   enhance anticancer effects has sparked great interest in scientists,
   and tremendous progress has been made in this next‐generation cancer
   therapeutic strategy.^[ [47]^8 ^] Activation of immunological systems
   can effectively clear residual, scattered, and dormant cancer cells in
   blood and lymph; stop the regeneration of cancer cells; and prevent the
   recurrence and metastasis of tumors. Recent studies have reported that
   nanomedicine‐based immunotherapy in combination with immune checkpoint
   inhibitors, laser and/or X‐ray can effectively enhance antitumor
   efficacy by stimulating the host immune system^[ [48]^9 ^] and
   regulating T cells.^[ [49]^10 ^] Consistently, studies also found that,
   targeting delivery of active molecules/cytokine by nanoparticles were
   able to overcome the challenges of cancer immunotherapy.^[ [50]^11 ^]
   Based on these findings, the evolving and emerging nanotechnology could
   provide a greater opportunity to augment immunotherapy of malignant
   cancers. However, the current nano‐immunotherapy only stimulates the
   host immune system by activating limited amounts of cytotoxic T
   lymphocytes (CTLs) and the tumoricidal immune cells. CTLs express the
   unique T cell receptor (TCR) in the surface to recognize and combine
   with the specific target antigen of tumor cells, which appear as the
   MHC‐restricted tumor‐killing effect. For example, the CD^4+ and CD^8+ T
   cells attack tumor cells through recognizing and binding with the
   MHC‐II and MHC‐I class molecules on the surface of targeted cells,
   respectively. While, tumor cells can evade immune system recognition
   and attack through various mechanisms, such as downregulating the
   expression of MHC class molecules. While, the NK cells exhibit the MHC
   non‐restrictive characteristic, and kill cancer cell more directly and
   rapidly, which providing higher cells‐mediated immunity than CTLs in
   cancer therapy. Furthermore, the immune cells can also be negatively
   regulated by the PD‐1/PD‐L1 pathway, and easily inactivated by
   immunosuppressive effects in tumor environment.^[ [51]^12 ^] To
   re‐activate and amplify the effect of immune cell therapy, we recently
   reported that cytokine‐induced killer cells expanded using IL‐15 could
   be boosted by selenium‐based nanoemulsions, and were capable of
   eliminating established tumors in mice bearing human breast cancer
   through regulation of NKG2D/NKG2DL activation and modulating
   TGF‐β/TGF‐β RI/Smad2/3 signaling.^[ [52]^13 ^] These findings suggest
   that, strategies based on stimulating the NK cell immune response could
   be further improved to achieve successful cancer therapy, and the
   potentiation mechanisms of NK cells also need more in‐depth
   investigation.

   Herein, we designed and synthesized biocompatible black phosphorus
   quantum dots encapsulated with human serum albumin (BPQDs@HSA) as
   immunosensitizers to enhance NK cell activity against tumor cells, thus
   achieving synergistic tumor inhibition. Black phosphorus (BP) has
   attracted great interest owing to its sensitization for X‐ray‐induced
   photodynamic therapy and excellent near‐infrared (NIR) photothermal
   performance.^[ [53]^14 ^] Furthermore, BP also exhibits high
   biocompatibility and biodegradability, and its metabolites are nontoxic
   phosphate and phosphonate.^[ [54]^14b ^] In this study, we have found
   that BPQDs@HSA can interact with Toll‐like receptor (TLR) on the
   surface of NK cells and thus activate downstream NF‐κB signalling
   pathways to regulate the secretion of cytokines by NK cells and enhance
   the Th1 immune response. And as the donor of phosphate group, BPQDs@HSA
   binds with the protein of phosphatidylinositol 4‐phosphate 5‐kinase
   type‐1 gamma (PIP5K1A) and activates the downstream PI3K‐Akt‐mTOR
   signaling pathways to reprogram cell metabolism of glycolysis and
   further promote the oxidative phosphorylation, sequentially maintaining
   the cell viability and immunity of NK cells. Examination of the
   mechanisms underlying the action of BPQDs@HSA as an immunosensitizer
   revealed that BPQDs@HSA enhances immune effects, relieves tumor‐derived
   immunosuppression of NK cells and inhibits the immune escape of tumor
   cells, leading to amplified anticancer efficacy. Multiomics analysis
   further revealed that, BPQDs@HSA regulated diverse effects at the
   individual gene and protein levels but converged to common responses at
   the pathway level, finally resulting in enhancement of immunocidal
   effects of NK cells (Figure [55]1A). Collectively, our data contribute
   to a successful and translational strategy for cancer treatment by
   nanomedicine‐potentiated NK cell immunotherapy, and elucidate the
   fundamental mechanisms for regulation of cell metabolism and antitumor
   immunity by a BP‐based nanosystem.

Figure 1.

   Figure 1
   [56]Open in a new tab

   BPQDs@HSA enhances cancer‐killing activity of NK cells derived from
   patients (A). B) TEM image of BPQDs@HSA. XPS (C) and P 2p spectra (D)
   of BPQDs and BPQDs@HSA. E) ITC data for the adsorption process of HSA
   on BPQDs. The upper graphs exhibit the raw data obtained from the
   titrations, and lower graphs show the integrated energy of each peak
   with the corresponding fitting curve. F) BPQDs@HSA enhances
   cancer‐killing activity of NK cells derived from patients. G) The
   purity of NK cells after isolation and culture from clinical patients.
   H) Cell viability of HepG‐2 cells after co‐treatment with BPQDs@HSA and
   NK cells obtained from different patients. The ratio of NK cells to
   HepG‐2 cells was approximately 5:1 (means ± S.D., n = 3). I) Fold
   enhancement in anticancer activity of NK cells obtained from different
   patients enhanced by BPQDs@HSA. PBMC, peripheral blood mononuclear
   cells; NKs, NK cells.

2. Results and Discussion

2.1. BPQDs@HSA Reprogrammes NK Cells to Potentiate its Anticancer Action

   NK cell inhibition in the TME remains a major limit to hinder NK cell
   immunotherapy for malignant tumors. Therefore, in this study, we
   constructed this BPQDs@HSA nanosystem to overcome the drawbacks of NK
   cells (severe dysfunction and diminished activity) to achieve
   synergistic cancer immunotherapy. First, ultrasmall black phosphorus
   quantum dots (BPQDs) were prepared by the ultra‐sonication liquid
   exfoliation technique as previously described.^[ [57]^15 ^] Here, we
   have developed a simple method to synthesize biocompatible
   nanoparticles by employing human serum albumin (HSA) to decorate
   isolated BPQDs and reduce the degradation of BPQDs under physiological
   conditions, thus increasing the stability of BPQDs. As shown in
   Figure [58]1B, BPQDs@HSA appears as highly monodisperse particles with
   a diameter of ≈5 ± 2 nm. The peaks at 2930 and 1650 cm^−1 in the Raman
   spectra were assigned to the stretching vibration of CH3 and the α
   helical structure corresponding to amide band I of protein, suggesting
   successful encapsulation of BPQDs by HSA (Figure [59]S1, Supporting
   Information). The presence of N in the BPQDs@HSA and the change in
   binding energy of P 2p in X‐ray photoelectron spectroscopy (XPS)
   analysis also demonstrated the successful coating of HSA on the BPQDs
   surface (Figure [60]1C,D). The results of isothermal titration
   calorimetry (ITC) analysis (ΔG < 0, ΔH < 0, and ΔS < 0) suggest that
   the interaction of HSA and BPQDs is mainly due to van der Waals’ forces
   (Figure [61]1E). We also explored the BPQDs stability after HSA
   encapsulation. As shown in Figure [62]S2A–C, Supporting Information,
   when dispersed in water and exposed to air for 4 days, the color of the
   BPQDs solution became lighter over time, while the BPQDs@HSA solution
   retained color well. The corresponding UV–vis absorption spectra also
   demonstrate the degradation of BPQDs in aqueous solution and the high
   stability of BPQDs@HSA. This stable property is important to support
   the application of P‐based nanomaterials in NK cells potentiation under
   physiological conditions. Studies have found that BP is biodegradable
   and its degradation products are nontoxic phosphate and phosphonate.^[
   [63]^16 ^] We then also examined the metabolites of BPQDs by ^31P NMR
   spectra at different times in aqueous solution. As shown in Figure
   [64]S2D, Supporting Information, after being exposed in the aqueous
   solution for 4 days, the characteristic absorption peaks of phosphate
   group appeared in supernatant of BPQDs, which can act as the donor of
   phosphate group to participate in phosphorylation in vivo.

   To better simulate the real conditions of NK cells in patients, we
   isolated and expanded NK cells from ten clinically identified cancer
   patients (Figure [65]1F), and confirmed the purity of NK cells (the
   ratio of CD56^+/CD3^− NK cells at about 96.7%, Figure [66]1G). By using
   these purified NK cells, we further confirmed the high safety of
   BPQDs@HSA, even at a high concentration of 10 µg mL^−1 (Figure [67]S3,
   Supporting Information). We also found that different modes of
   BPQDs@HSA pretreatment significantly enhanced NK cell‐induced HepG‐2
   cell growth inhibition (Figure [68]S4, Supporting Information), while
   free BPQDs@HSA did not inhibit HepG‐2 cell proliferation, and NK cells
   alone slightly inhibited HepG‐2 cell growth when applied at a 2.5:1
   ratio of NK cells to HepG‐2 cells. In addition, pretreatment of NK
   cells with BPQDs@HSA (2 µg mL^−1, group 7) led to a higher anticancer
   effect than pretreatment of HepG‐2 cells with the nanosystem (groups 4,
   5, and 6), indicating a better anticancer effect from pretreatment with
   NK cells than from pretreatment with cancer cells. We then examined the
   cancer‐killing activity of NK cells from 10 patients against HepG‐2
   hepatocellular carcinoma cells with BPQDs@HSA. The information of these
   patients and the ratio of NK cells from these patients are shown in
   Table [69]S1, Supporting Information. Unexpectedly, upon stimulation by
   BPQDs@HSA, the NK cells from seven of ten patients demonstrated twofold
   higher cancer‐killing effects, and the maximum of a 6.7‐fold higher
   effect (Figure [70]1H,I and Figures [71]S5 and [72]S6, Supporting
   Information), indicating that BPQDs@HSA enhances the anticancer
   efficacy of NK cell immunotherapy.

2.2. Enhancement of Cancer Recognition Ability of NK Cells to Inhibit Immune
Escape

   NK cells can express natural killer group 2D (NKG2D) to recognize and
   activate its ligands, which are expressed in cancer cell membranes,
   including the UL16 binding protein family (ULBP 1–6) and MHC class I
   chain‐related molecules A and B (MICA, and MICB),^[ [73]^17 ^] thus
   enhancing the immune surveillance against tumor cells (Figure
   [74]2A).^[ [75]^18 ^] Therefore, we examined the expression level of
   NKG2D on the NK cell surface after being pre‐treated with BPQDs@HSA for
   6 h and co‐incubated with HepG‐2 cells for another 24 h. As shown in
   Figure [76]2B, although BPQDs@HSA didn't increase the NKG2D‐positive
   cell proportion in the absence of cancer cells, the mean fluorescence
   intensity (the value of MFI) of NKG2D expression was obviously enhanced
   after being treated by BPQDs@HSA. In the presence of cancer cells,
   NKG2D‐positive cell population and NKG2D expression level in NK cells
   were significantly increased, which exhibit the high cancer killing
   activity of these NK cells. We then also examined the NKG2D expression
   on the NK cell surface after incubating with BPQDs@HSA for a longer
   time (24 h). The results showed that the addition of BPQDs@HSA also can
   effectively increase the population of NKG2D‐positive cells and NKG2D
   expression on the NK cell surface (Figure [77]S7, Supporting
   Information), suggesting the potentiation of NK cell immunotherapy by
   BPQDs@HSA. With Western blotting, we also examined the expression level
   of NKG2D in NK cells after incubation with BPQDs@HSA, and found that
   the NKG2D expression was also significantly increased (Figure [78]2C).
   The expression of NKG2D ligands on HepG‐2 cells was also examined after
   treatment with NK cells with or without BPQDs@HSA. Consistently, the
   expression level of MICA and ULBP‐2 ligands in HepG‐2 cells
   significantly increased with the presence of NK cells and BPQDs@HSA.
   Furthermore, as examined by fluorescence flow cytometry, BPQDs@HSA and
   NK cells increased the expression level of MICA/B and ULBP 1–4 on the
   HepG‐2 surface to varying degrees (Figure [79]2D). Taken together, the
   results indicate that upregulation of NKG2D in NK cells and its
   activating ligands of the MICA/B and ULBP family in cancer cells by the
   nanosystem could enhance the immune surveillance of tumor growth and
   thus prevent tumor escape from NK cell‐mediated cytotoxicity. Next, we
   also performed experiments to evaluate the interaction of NK cells with
   HepG2 cells and the ability of NK cells‐penetrated in HepG‐2 tumor
   spheroids, as spheroids with diameters up to 400 µm can exhibit the
   pathophysiological characteristics of solid tumors. As shown in
   Figure [80]2E and Video [81]S1, Supporting Information, NK cells (red
   fluorescence) accumulated around HepG‐2 tumor spheroids (blue) after
   2 h of treatment and then began to penetrate the tumor spheroids. After
   12 h of treatment, NK cells penetrated and became dispersed in the
   whole tumor spheroid. We then further evaluate penetration depth of NK
   cells in tumor spheroids by confocal laser scanning. The fluorescence
   images at different layers of the spheroids from the top to the middle
   at −80 nm showed that BPQDs@HSA effectively potentiated the penetration
   ability of NK cells into HepG‐2 tumor spheroids and increased the
   penetration depth (Figure [82]2F), with a time‐dependent reduction in
   tumor spheroid size (Video [83]S2, Supporting Information). Studies
   have found that the chemokine secreted by tumor cells can interact with
   chemokine receptors on the surface of NK cells, and promote NK cells
   infiltration into solid tumors. Therefore, we examined the expression
   of chemokine receptor of CXCR3 on the NK cell surface. As shown in
   Figure [84]S8, Supporting Information, BPQDs@HSA can effectively
   increase the CXCR3 expression on the NK cell surface. Furthermore, we
   also examined two important chemokines secretion of CXCL9 and CXCL10
   (the ligands of CXCR3) in the cultured medium of HepG‐2 cells after
   being incubated with BPQDs@HSA and NK cells. The results showed that
   the secretion of CXCL9 has no obvious change, while CXCL10 secretion in
   the cultured medium of HepG‐2 cells after being incubated with
   BPQDs@HSA and NK cells was significantly enhanced compared with the
   control group (Figure [85]S9, Supporting Information). Furthermore, we
   also examined the CXCL10 level in the supernatant of HepG‐2 tumor
   spheroids, and found that BPQDs@HSA combined with NK cells effectively
   increased the CXCL10 secretion of tumor spheroids in a dose‐dependent
   manner (Figure [86]S10, Supporting Information), which exhibited the
   positive correlation between the CXCL10 secretion and penetration depth
   of NK cells in tumor spheroids. These results indicate that the
   treatment of BPQDs@HSA can promote the CXCR3 expression of NK cells and
   interact with the enhancement secretion of CXCL10 by HepG‐2 cells to
   enhance the penetration of NK cells into HepG‐2 tumor spheroids. Taken
   together, the upregulation of NKG2D induced by BPQDs@HSA can enhance
   the tumor‐targeting and killing effects of NK cells, which can
   effectively interact with HepG‐2 cells and further enhance the
   penetration depth in HepG‐2 tumor spheroids. The higher cancer
   recognition and cancer‐penetrating ability of NK cells may increase the
   overall anticancer efficacy.

Figure 2.

   Figure 2
   [87]Open in a new tab

   BPQDs@HSA enhances the targeting and penetration ability of NK cells
   against HepG‐2 cells. A) Schematic diagram of NK cells targeting tumor
   cells to enhance immune surveillance against tumor cells. B) Expression
   level of NKG2D on the NK cell surface after pre‐treated with BPQDs@HSA
   for 6 h and co‐incubated with HepG‐2 cells for another 24 h. NK:HepG‐2
   = 5:1. C) BPQDs@HSA increases the expression of NKG2D in NK cells and
   the expression of MICA and ULBP‐2 in HepG‐2 cells. D) Expression level
   of MICA/B and ULBP 1–4 on HepG‐2 cells surface treated with BPQDs@HSA
   (2.5 and 5 µg mL^−1) and NK cells (NK:HepG‐2 = 5:1). E,F) Penetration
   ability of NK cells into HepG‐2 tumor spheroids. NK cells were
   pretreated with BPQDs@HSA (5 µg mL^−1), then labeled with CellTracker
   Red CMTPX Dye and visualized with red fluorescence. Scale bar, 500 µm.

2.3. Multiomics Analysis Reveals the Mechanisms for NK Cells‐Regulation of
BPQDs@HSA

   High throughput multiomics approaches provide an unprecedented
   opportunity and technique for dissecting the molecular mechanisms
   accounting for immune‐regulation effects of anticancer drugs.^[ [88]^19
   ^] Therefore, in this study, multiomics analysis was employed to
   explore the mechanism of action of BPQDs@HSA‐enhanced NK cell‐mediated
   cytotoxicity, and to discover potential therapeutic targets accounting
   for this effect. From the results of the transcriptomic volcano plot,
   389 genes were upregulated and 302 genes were downregulated after 12‐h
   incubation of NK cells with BPQDs@HSA (Figure [89]S11A, Supporting
   Information). In contrast, when NK cells were incubated with BPQDs@HSA
   for 12 h in the tumor microenvironment, the change in intracellular
   gene expression was significantly different, with 835 genes upregulated
   and 90 genes downregulated (Figure [90]S11B, Supporting Information).
   Especially, the genes involved in PI3K‐AKT, mTOR, and cGMP‐PKG
   signaling pathway genes were significantly up‐regulated. In addition,
   the KEGG signaling pathway enrichment analysis reveals that, the number
   of genes enriched in these signaling pathways was also significantly
   increased (Figure [91]S12, Supporting Information), including PI3K‐AKT,
   mTOR, MAPK, AMPK, TGF‐β and Toll‐like receptor signaling pathways, et
   al, which indicates that, under the tumor microenvironment, BPQDs@HSA
   could cause more pronounced changes in gene expression in NK cells.

   To understand the underlying mechanisms and potential relationship
   between the changes that were induced at transcriptome level and
   protein executors of cellular functions, we used three tumor
   patients‐donated NK cells to further explore the effect of BPQDs@HSA by
   proteomics (Figure [92]3A). As shown in the heatmap and volcano plot of
   the differentially expressed proteins in NK cells (Figure [93]3B,C), a
   large number of intracellular proteins were upregulated in all the
   three tumor patients‐donated NK cells after treatment by BPQDs@HSA,
   with 246 ones upregulated and 39 ones downregulated. Among them,
   proteins related to NK cell immune function were significantly
   up‐regulated by BPQDs@HSA treatment, such as Granzyme A and K, TNF
   receptor‐associated factor 6 (TRAF6), tumor necrosis factor ligand
   superfamily member (TNLG6A, TNFRSF1B) and stimulator of interferon
   protein (STING). Furthermore, in these 285 changed proteins, a large
   number of proteins involved in NK cell viability and immune‐mediated
   regulation were significantly upregulated after NK cells after
   incubation with BPQDs@HSA for 12 h (Figure [94]3D). Through KEGG
   signaling pathway enrichment analysis (Figure [95]3E), these proteins
   participated in cytokine–cytokine receptor interaction, Th1 and Th2
   cell differentiation, NK cell‐mediated cytotoxicity, and other
   immunoregulatory pathways. Moreover, consistent with the transcriptome,
   the changed proteins in NK cells after BPQDs@HSA treatment were
   similarly enriched to signaling pathways such as cGMP‐PKG signaling
   pathway, Jak‐STAT signaling pathway, and RIG‐I‐like receptor signaling
   pathway. Protein functional gene ontology (GO) enrichment analysis
   further reveals (Figure [96]3F) that the differential proteins after
   BPQDs@HSA treatment were significantly involved in tumor necrosis
   factor‐activated receptor activity, tumor necrosis factor receptor
   binding, activation of innate immune response, and cytokine receptor
   binding in enhancing the immunological effects of NK cells.

Figure 3.

   Figure 3
   [97]Open in a new tab

   Multiomics analysis reveals the mechanisms for NK cells‐regulation
   effects of BPQDs@HSA. A) Schematic of multiomics analysis of NK cells
   derived from patients. Heatmap (B) and volcano plot (C) of the
   differentially expressed proteins in NK cells from three tumor patients
   with BPQDs@HSA incubation. D) Heatmap of immunoregulatory proteins in
   the three tumor patients‐donated NK cells treated by BPQDs@HSA (red
   indicates the increased expression and blue indicates the decreased
   expression). E) KEGG bubble chart of the differential proteins
   enrichment signaling pathway. F) Protein functional GO enrichment
   analysis of the differential proteins in NK cells with BPQDs@HSA
   treatment. The NK cells incubated with BPQDs@HSA at 5 µg mL^−1 for 12 h
   were then subjected to multiomics analysis. G) Fluorescence imaging of
   HepG‐2 cells after treatment with NK cells for 24 h. NK cells were
   pretreated with FITC‐labelled BPQDs@HSA for 6 h, and the BPQDs@HSA were
   visualized by the green fluorescence (Scale bar, 50 µm). The white
   arrows indicated the NK cells. H) Quantitative analysis of the cellular
   uptake efficiency of BPQDs@HSA in NK cells and HepG‐2 cells (means ±
   S.D., n = 3). I) Process of BPQDs@HSA capturing the target proteins of
   NK cells. J) GO analysis on biological process and functions of bound
   proteins. K) Heatmap of the captured target proteins associated with
   the immune system by BPQDs@HSA. L) Scatterplots display the relative
   quantity of the target proteins captured by BPQDs@HSA. M) The
   expression levels of TLR‐2, TLR‐5, TLR‐9, and NF‐κB in NK cells treated
   with BPQDs@HSA. N) Cytokines secretion from NK cells induced by 2 µg
   mL^−1 BPQDs@HSA (means ± S.D., n = 3). *p < 0.05, **p < 0.01.

   The treatment strategy in this study used BPQDs@HSA was as
   immunosensitizer to enhance NK cell immunocompetence against tumor
   cells. Therefore, the regulation of immune response and activation of
   signaling pathways in NK cells treated by BPQDs@HSA may be due to its
   interaction with proteins in NK cells. To further evaluate the main
   action of BPQDs@HSA in tumor and NK cells, we then examined the
   biodistribution and translocation of BPQDs@HSA in NK and HepG‐2 cells.
   As shown in Figure [98]3G,H, NK cells were pretreated with BPQDs@HSA
   for 12 h and then co‐incubated with HepG‐2 cells for another 24 h. It
   was found that most of the BPQDs@HSA remained in NK cells, and only a
   few BPQDs@HSA were internalized into HepG‐2 cells. These results
   suggest that BPQDs@HSA mainly accumulate in NK cells to regulate their
   immunologic function, thus enhancing anticancer activity, while the
   metabolites of BPQDs@HSA are phosphate and phosphonate, which are
   endogenous substances in vivo. Therefore, it is possible that the
   immune regulation mechanism is caused by the direct interaction between
   the complete nanoparticle of BPQDs@HSA and the proteins in NK cells.
   Based on these hypotheses, we employed surface plasmon resonance (SPR)
   technology and LC‐MS analysis to identify the protein in NK cells that
   was interacted by the nanosystem of BPQDs@HSA (Figure [99]3I). Totally,
   91 proteins have been captured by BPQDs@HSA (Figure [100]S13,
   Supporting Information). These bound proteins were further classified
   according to their biological process and functions by GO analysis.
   Among them, 21 proteins bound to BPQDs@HSA are involved in the immune
   system process, while 14 of them contribute to the regulation of immune
   response. 71 bound proteins are involved in the cell metabolic process
   and 30 ones contribute to intracellular protein phosphorylation,
   indicating the important roles of P element in the cell metabolism
   events (Figure [101]3J). Among the 21 proteins of the immune system
   process, most of them can interact strongly with BPQDs@HSA
   (Figure [102]3K). Furthermore, the scatterplot displays that the 11
   bound proteins with relatively high quantity are related to the
   immunologic function of NK cells, especially the Toll‐like receptor,
   mTOR and GSK3 (Figure [103]3L). These results demonstrate the
   regulatory effects of BPQDs@HSA on the immune response of NK cells.

2.4. Enhancement of Immunologic Function of NK Cells by Activating TLR
Pathway

   Toll‐like receptor (TLR) family plays an important role in tumor immune
   surveillance and response by activating downstream NF‐κB signalling
   pathways to promote pro‐inflammatory cytokine secretion and the
   inflammatory response. Herein, the results of target identification
   indicated that the Toll‐like receptor family exhibited high interaction
   with BPQDs@HSA, including TLR‐2, TLR‐5 and TLR‐9, so we examined the
   expression levels of TLR‐2, TLR‐5, TLR‐9 and NF‐κB in NK cells after
   BPQDs@HSA treatment. As shown in Figure [104]3M, BPQDs@HSA obviously
   increased the expression levels of these proteins in NK cells,
   especially TLR‐5, TLR‐9 and NF‐κB. This activated signalling pathway
   plays an important role in regulating cytokine secretion by NK cells to
   kill cancer cells by changing the tumor immune environment and
   attacking cancer cells. Therefore, we examined the levels of cytokines
   released from NK cells induced by BPQDs@HSA. In the supernatant of NK
   cells, TGF‐β (an important immune suppressive factor), was
   significantly decreased after BPQDs@HSA treatment, which could
   effectively relieve the immune suppression of NK cells suffered from
   the TME. Moreover, the expression of IFN‐γ and IL‐10 after being
   treated with BPQDs@HSA also showed an upward and downward trend,
   respectively (Figure [105]3N). Taken together, BPQDs@HSA potentiated NK
   cell immunotherapy mainly through relieving immune suppression, but
   also regulating NK cells trends to Th1 immune response, which is
   consistent with the result of Th1 and Th2 cell differentiation by KEGG
   pathway enrichment analysis of genomics and proteomics.

2.5. BPQDs@HSA Relieves Immune Suppression of NK Cells in TME

   The process of tumor establishment, progression and metastasis forms an
   immunosuppressive microenvironment, including the highly secreted
   immunosuppressive factors such as TGF‐β and IL‐10, and the activation
   of PD‐1/PD‐L1 signalling pathways, which affects the immune anergy,
   exhaustion and cell death of NK cells or even leads to the transmission
   of an anti‐apoptotic signal to tumor cells (Figure [106]4A).^[ [107]^20
   ^] Interestingly, we have found that BPQDs@HSA obviously captured the
   proteins related to NK cell growth, proliferation and differentiation,
   including EGFR, mTOR and GSK‐3β. Although these proteins also play an
   important role in tumorigenesis, the treatment strategy of BPQDs@HSA
   was pre‐incubated with NK cells and then co‐treated with tumor cells.
   And from the localization of BPQDs@HSA in the co‐treatment of NK cells
   and HepG‐2 cells, most of the BPQDs@HSA remained in NK cells, and only
   a few BPQDs@HSA were internalized into HepG‐2 cells (Figure [108]3G,H).
   Therefore, we consider that BPQDs@HSA mainly accumulate in NK cells to
   regulate their immunologic function, thus enhancing anticancer
   activity. Then, we examined the expression levels of these proteins in
   NK cells incubated with BPQDs@HSA under TME. As shown in Figure
   [109]S14, Supporting Information, the expression level of these
   proteins increased significantly after BPQDs@HSA treatment, indicating
   that BPQDs@HSA could maintain the vitality of NK cells and activate the
   immunologic function of NK cells. To further explore the negative
   effects of tumor cells against NK cells, we also examined the
   expression levels of EGFR, mTOR, and GSK‐3β in the supernatant of NK
   cells after co‐incubation with HepG‐2 cells by Western blotting. As
   shown in Figure [110]4B, BPQDs@HSA effectively reversed the
   downregulation of EGFR, mTOR and GSK‐3β induced by tumor cells,
   indicating that cancer cells may inhibit the differentiation and immune
   response of NK cells, but BPQDs@HSA could effectively block this
   negative influence. To further verify the importance of TLR pathway and
   mTOR pathway in the potentiation of immune response of NK cells by
   BPQDs@HSA, we used rapamycin (mTOR inhibitor) and MyD88 inhibitory
   peptide (TLR signalling inhibitor) to examine the effects of BPQDs@HSA
   on the cytokine secretion and anticancer activity of NK cells. As shown
   in Figure [111]4C,D, both inhibitors effectively suppressed the
   pro‐inflammatory cytokine (IL‐2 and IFN‐γ) secretion from NK cells
   alone, but could not affect the enhancement triggered by BPQDs@HSA. The
   inhibitors could also suppress the inhibitory effects of BPQDs@HSA on
   immune suppressive factor TGF‐β. Furthermore, we also examined the
   effects of these inhibitors on the anticancer activity of NK cells. As
   shown in Figure [112]4E, rapamycin and MyD88 inhibitory peptide at
   non‐toxic dose effectively suppressed the NK cells‐induced HepG‐2 cell
   growth inhibition, indicating that mTOR and TLR pathway play important
   roles in the anticancer action of NK cells. However, interestingly, the
   presence of BPQDs@HSA significantly stopped this effect. Furthermore,
   the results of polymerase chain reaction (PCR) analysis also revealed
   that, BPQDs@HSA treatment increased the expression of TLR‐1, ‐2, ‐3, ‐6
   and ‐9, mTOR and GSK3β at mRNA level, which is consistent with those
   change induced by the nanosystem at protein level (Figure [113]4F).
   Furthermore, mRNA sequencing analysis was conducted to examine the
   change of NK cells. As shown in Figure [114]4G, treatment with
   BPQDs@HSA up‐regulated the gene expression levels of GSK3β, mTOR, TLR1,
   TLR6, TLR9, and NFκB1. In addition, PPI was constructed by String for
   genes of interest, unspecific catalysis interaction was found between
   mTOR and NFκB1, TNF and NFκB1. In the TLR family, TLR1 and TLR6 are
   tied together in an unspecified manner. Additionally, TGF‐β could
   inhibit IFN‐γ expression at the transcriptional regulatory level,
   leading to activation of TNF secretion from NK cells. Taking the
   results of mRNA sequencing, captured target protein analysis and
   Western blotting analysis together, we can conclude that, BPQDs@HSA
   interact with TLR on the NK cell surface to activate downstream
   GSK3B‐mTOR‐NF‐κB signaling pathways to regulate cytokine secretion,
   finally resulting in enhanced cancer therapeutic efficacy
   (Figure [115]4H).

Figure 4.

   Figure 4
   [116]Open in a new tab

   BPQDs@HSA relieves immune suppression of NK cells in TME. A) The
   immunosuppressive microenvironment affects the immune anergy,
   exhaustion, and cell death of NK cells or even leads to the
   transmission of an anti‐apoptotic signal to tumor cells. B) Effects of
   BPQDs@HSA on the expression levels of EGFR, mTOR and GSK‐3β in NK cells
   associated with cancer cell growth, proliferation and differentiation.
   Effects of BPQDs@HSA on the cytokine secretion from NK cells with
   pre‐incubation of 100 nm rapamycin (C) and 10 µm MyD88 inhibitory
   peptide (D) (means ± S.D., n = 3). E) Effects of rapamycin (Ra, 100 nm)
   and MyD88 inhibitory peptide (My, 10 µm) on anticancer effect of
   BPQDs@HSA (2 µg mL^−1) and NK cells (NK:HepG‐2 = 5:1) (means ± S.D., n
   = 3). NK cells were pre‐treated with different concentration of these
   two inhibitors for 2 h. F) Effects of BPQDs@HSA (5 µg mL^−1) on mRNA
   expression level of NK cells (means ± S.D., n = 3). *p < 0.05, **p <
   0.01. G) Effects of BPQDs@HSA on related gene sequencing of NK cells.
   H) BPQDs@HSA enhanced the immunologic function of NK cells by
   activating TLR pathway. I) The expression level of PD‐1 and PD‐L1 in NK
   cells and HepG‐2 cells with different treatments. J) Inhibitory effects
   of BPQDs@HSA on the expression of PD‐1 on the surface of NK cells.
   NK:HepG‐2 = 5:1. K) Effects of BPQDs@HSA on the cytokine secretion from
   NK cells after co‐incubation with HepG‐2 cells for 24 h (means ± S.D.,
   n = 3).

   Studies have found that the activation of PD‐1/PD‐L1 signalling
   pathways of NK cells in tumor immunosuppressive microenvironment is the
   main reason of NK cells immune dysfunction. Furthermore, the results of
   KEGG enrichment signalling pathway analysis showed that some of the
   captured target proteins by BPQDs@HSA were also enriched in the
   PD‐1/PD‐L1 checkpoint pathway. Therefore, we then evaluated the
   expression levels of PD‐1 in NK cells after treatment with BPQDs@HSA.
   As expected, we found that the expression levels of PD‐1 in NK cells
   decreased significantly after BPQDs@HSA treatment (Figure [117]4I).
   Interestingly, free NK cell treatment induced an increase in PD‐L1
   expression in HepG‐2 cells. However, BPQDs@HSA‐potentiated NK cells
   significantly inhibited the expression of PD‐L1 in HepG‐2 cells. We
   then examined the expression of PD‐1 on the NK cell surface by flow
   cytometry. As expected, treatment with BPQDs@HSA significantly
   decreased the PD‐1‐positive cell population and PD‐1 expression level
   on the surface of NK cells in a dose‐dependent manner (Figure [118]4J).
   Interestingly, under the cancer cells‐simulated microenvironment,
   coincubation with HepG‐2 cells significantly enhanced the PD‐1‐positive
   cell population from 0.8% (control) to 35.1% to achieve immune escape,
   but this enhancement was also effectively inhibited by treatment of
   BPQDs@HSA. In addition, under the co‐incubation with HepG‐2 cell, the
   BPQDs@HSA‐triggered enhanced secretion of pro‐inflammatory cytokine
   (IL‐2 and IFN‐γ) from NK cells was inhibited, while still exhibiting
   the inhibitory effects on the secretion of immune suppressive factor
   IL‐10 and TGF‐β (Figure [119]4K), which further verified the remission
   of immune suppression effect on NK cells in a tumor microenvironment by
   BPQDs@HSA. These results indicate that cancer cells could activate
   PD‐1/PD‐L1 recognition with NK cells to cause immune anergy of NK
   cells, leading to the failure of immunotherapy. In contrast, BPQDs@HSA
   could effectively inhibit PD‐1 expression in NK cells and prevent the
   activation of the PD‐1/PD‐L1 pathway to relieve immune suppression of
   NK cells.

2.6. Metabolic Reprogramming of NK Cells by BPQDs@HSA

   The immunosuppressive microenvironment in solid tumors also reduces the
   viability of immune cells by affecting their metabolic activity,
   thereby inhibiting their tumor‐killing immune effects.^[ [120]^21 ^]
   Studies have found that, suppression and inhibition of NK cell
   anticancer action by the developed TME plays a critical role in tumor
   development and progression.^[ [121]^5 , [122]^22 ^] In this study, the
   results of proteomic analysis reveal that, BPQDs@HSA treatment
   upregulated most of the metabolism‐related proteins in NK cells from
   the three selected cancer patients (Figure [123]5A). These differential
   proteins clearly participated in oxidative phosphorylation as well as
   lipid metabolism (Figure [124]5B). The results of GO enrichment
   analysis further reveals that a large number of proteins in NK cells
   after BPQDs@HSA treatment were involved in mitochondrial functions as
   well as ATP synthesis processes, including mitochondrial pyruvate
   transport, mitochondrial respiratory chain and proton‐transporting ATP
   synthase complex assembly, ATP synthesis coupled electron transport and
   NADH dehydrogenase activity (Figure [125]5C). These results suggest
   that BPQDs@HSA significantly affects the oxidative phosphorylation and
   glycolytic processes in NK cells. Moreover, the target‐capturing
   experiments also revealed that more than 70% of the intracellular
   proteins captured by BPQDs@HSA were involved in cellular metabolic
   processes, which also suggest the importance of the metabolic
   regulation in NK cell immunotherapy (Figure [126]5D). GO enrichment
   analysis reveals that these proteins were mainly involved in the
   intracellular organic substance metabolic process (66 proteins),
   phosphorus metabolic process (42 proteins), lipid metabolic process (21
   proteins), monosaccharide metabolic process (8 proteins), and et al.
   Among them, 23 proteins participate in the positive regulation of
   metabolic process (Figure [127]5E). Further investigation by KEGG
   signaling pathway enrichment analysis of these proteins reveals that,
   28% of these target proteins are involved in cellular metabolic
   pathways, and several other signaling pathways related to lipid
   metabolism and gluconeogenesis, such as glycerophospholipid metabolism,
   inositol phosphate metabolism, sphingolipid metabolism, pentose
   phosphate pathway, and fructose and mannose metabolism (Figure
   [128]S15, Supporting Information). In addition, the relevant metabolic
   signaling pathways also point to the PI3K‐Akt signaling pathway, mTOR
   signaling pathways, and MAPK signaling pathway (Figure [129]5F).
   Therefore, we then examined the signaling pathway of PI3K‐Akt/mTOR in
   NK cells activated by BPQDs@HSA. As shown in Figure [130]S16,
   Supporting Information, the expression levels of phosphoinositide 3
   kinase (PI3K), AKT, and mTOR in NK cells were upregulated after treated
   with BPQDs@HSA. Especially, the protein of PIP5K1A was well captured by
   BPQDs@HSA. And as the donor of phosphate group, BPQDs@HSA binding can
   promote the phosphorylation‐catalyzing effect on phosphatidylinositol
   4‐phosphate (PI4P) to form phosphatidylinositol 4,5‐bisphosphate
   (PIP2). PIP2 act as substrate to form phosphatidylinositol
   3,4,5‐trisphosphate (PIP3) under the phospholipid turnover of
   phosphoinositide 3 kinase (PI3K), subsequently activating the
   downstream PI3K‐Akt‐mTOR signaling pathways to enhance the cell
   metabolism, especially the glycolysis (Figure [131]5G).^[ [132]^23 ^]
   To further evaluate the effects of BPQDs@HSA on the metabolism of NK
   cells, we measured the oxygen consumption rate (OCR) and extracellular
   acidification rate (ECAR) of NK cells incubated with BPQDs@HSA to
   detect their oxidative phosphorylation and glycolysis. As shown in
   Figure [133]5H, BPQDs@HSA significantly enhanced oxidative
   phosphorylation processes in NK cells with a dose‐dependent manner.
   Furthermore, we also found that the NK cells had a low basal
   glycolysis, after incubation with BPQDs@HSA for 24 h, the glycolysis of
   NK cells were significantly upregulated, which is owing to the
   activation of PI3K‐Akt signaling pathway by BPQDs@HSA (Figure [134]5I).
   Furthermore, the metabolomics analysis of NK cells was performed to
   further explore the effects of BPQDs@HSA on NK cell metabolism. From
   the results of the differential metabolite in NK cells (Figure
   [135]S17, Supporting Information) and the KEGG bubble map of
   differential metabolite‐enriched signaling pathways, after BPQDs@HSA
   treatment, the intracellular differential metabolites could be enriched
   to pathways such as PI3K‐Akt and mTOR signaling pathways
   (Figure [136]5J). These results indicate that BPQDs@HSA effectively
   reprogram cell metabolism of glycolysis and further promote the
   oxidative phosphorylation of NK cells to improve cell viability and
   activates through PI3K‐Akt and mTOR axis to enhance the immunocidal
   function of NK cells.

Figure 5.

   Figure 5
   [137]Open in a new tab

   Metabolic reprogramming of NK cells by BPQDs@HSA. A) Heatmap of the
   proteins associated with cell metabolism in the three tumor
   patients‐donated NK cells treated by BPQDs@HSA (red color indicates the
   increased expression and blue color indicates the decreased
   expression). B) KEGG bubble map of differential protein enrichment in
   pathways related to cell metabolism. C) Protein functional GO
   enrichment analysis of the differential proteins associated with
   cellular energy metabolism. D) Heatmap of the captured target proteins
   associated with the cell metabolism by BPQDs@HSA. E) GO analysis on
   metabolic process of bound proteins (1: organic substance metabolic
   process, 2: organonitrogen compound metabolic process, 3: phosphorus
   metabolic process, 4: organophosphate metabolic process, 5: small
   molecule metabolic process, 6: lipid metabolic process, 7: positive
   regulation of macromolecule metabolic process, 8: phospholipid
   metabolic process, 9: glycerophospholipid metabolic process, 10:
   phosphatidylinositol metabolic process, 11: monosaccharide metabolic
   process, 12: cellular carbohydrate metabolic process, 13: pentose
   metabolic process, 14: D‐ribose metabolic process, 15: positive
   regulation of metabolic process,16: positive regulation of cellular
   metabolic process). F) KEGG enrichment signaling pathway of the
   captured target proteins by BPQDs@HSA. G) BPQDs@HSA promotes NK cell
   metabolism through activating PI3K‐Akt and mTOR pathway. H) OCR of NK
   cells treated by BPQDs@HSA for 24 h to sequential addition of Oligo,
   FCCP, and rotenone and antimycin (Rot/Ant). I) ECAR of NK cells treated
   by BPQDs@HSA for 24 h to sequential addition of glucose (Gluc),
   oligomycin (Oligo), and 2‐deoxy‐D‐glucose (2DG) and quantified basal
   glycolysis and glycolytic capacity (*p < 0.05, **p < 0.01). J) KEGG
   bubble map of differential metabolite‐enriched signaling pathways by
   metabolomics analysis.

2.7. X‐Ray‐Assisted Enhancement of Infiltration and Cancer‐Killing Activity
of NK Cells

   The dysfunction of NK cells in tumor microenvironment and the
   difficulty to penetrate into solid tumors has been identified as
   barriers for NK cell immunotherapy.^[ [138]^5a ^] Recent studies have
   showed that, low‐dose X‐ray can assist nanoscale metal–organic
   frameworks to elicit anticancer immune‐response, through increasing
   immune cell infiltration and enhancing the requirement of functional B
   and T cells.^[ [139]^10b ^] Therefore, we may ask whether X‐ray could
   assist NK cell to penetrate into solid tumor and enhance the in vivo
   anticancer efficacy of BPQDs@HSA (Figure [140]6A). To confirm this
   hypothesis, first, we examined the toxicity of BPQDs@HSA against NK
   cells with X‐ray, and further investigate the responsiveness of the
   nanoparticles to X‐rays on NK cell activity. As expected, NK cells
   exposed on the lower X‐ray radiation (2 Gy) didn't exhibit obvious side
   effects, even combined with BPQDs@HSA treatment at 10 µg mL^−1 (Figure
   [141]S18, Supporting Information). Moreover, it is interestingly found
   that the combined treatment with NK cells and nanoparticles triggered
   singlet oxygen (^1O[2]) generation in HepG‐2 hepatocellular carcinoma
   cells to ≈135.9% higher than that of the BPQDs@HSA treatment alone
   (Figure [142]6B), which may be due to the release of granzyme B and
   perforin to promote tumor cell lysis, while pretreatment of NK cells
   with BPQDs@HSA only slightly increased ^1O[2] generation in HepG‐2
   cells. However, with X‐ray assistance, ^1O[2] generation significantly
   increased by 157.3% and 159.4% after incubation with
   BPQDs@HSA‐potentiated NK cells. In addition, as assisted by X‐ray,
   BPQDs@HSA‐potentiated NK cell demonstrated much higher anticancer
   activity (Figure [143]6C). The result of Annexin V and PI co‐staining
   assay indicate that BPQDs@HSA‐potentiated NK cell significantly
   enhanced HepG‐2 cell apoptosis, which was further increased with X‐ray
   assistance (Figure [144]6D).

Figure 6.

   Figure 6
   [145]Open in a new tab

   X‐ray assists BPQDs@HSA‐potentiated NK cell to penetrate into solid
   tumor and enhance in vivo anticancer efficacy (A). B)
   BPQDs@HSA‐potentiated NK cell therapy increases X‐ray‐induced ^1O[2]
   overproduction in HepG‐2 cells (means ± S.D., n = 3). C) X‐ray enhanced
   anticancer activity of BPQDs@HSA and NK cells (means ± S.D., n = 3).
   The cells were treated with 2 µg mL^−1 of BPQDs@HSA or X‐ray (2 Gy) and
   NK cells (NK:HepG‐2 = 2.5:1) (*p < 0.05, **p < 0.01). D) Annexin
   V‐FITC/PI double staining to evaluate the HepG‐2 cell apoptosis and
   necrosis induced by BPQDs@HSA (2 µg mL^−1) combined with NK cells
   (NK:HepG‐2 = 2.5:1) and X‐ray assistance (2 Gy). Changes in tumor
   volume (E), tumor weight (F), tumor inhibition (G), and body weight (H)
   of HepG‐2 xenografts in nude mice after intravenous injection of NK
   cells pre‐incubated with BPQDs@HSA (50 µg kg^−1) and/or subjected to
   X‐ray radiation (2 Gy) for 7 times within 21 days (means ± S.D., n = 10
   mice). I) Immunostaining to evaluate CD56 expression in NK cells
   penetrating into solid tumor tissues. Scale bar, 100 µm. J) Pseudocolor
   signals of MRI, H&E staining and TUNEL‐Hoechst co‐staining assays of
   tumor tissues from different treatment groups. Scale bar, 100 µm. K)
   Quantitative analysis of IVIM‐DWI parameters of tumor tissues in
   different treatment groups by MRI analysis (means ± S.D., n = 4 mice).
   L) Quantitative analysis of TUNEL expression of tumor tissues in
   different treatment groups by ImageJ analysis (means ± S.D., n = 4). M)
   Hematological analysis of liver, kidney, and heart function in the
   different mouse treatment groups (means ± S.D., n = 4 mice). Bars with
   different characters are statistically different at p < 0.05 level.

   We then further examined the X‐ray assisted enhancement of
   BPQDs@HSA‐potentiated NK cell immunotherapy in vivo. As shown in
   Figure [146]6E–G, the treatment of BPQDs@HSA, X‐ray radiation and NK
   cells alone inhibit HepG‐2 tumor growth in vivo at 17.9%, 31.4% and
   29.4%, while the combination of BPQDs@HSA and NK cells slightly
   increased tumor inhibition to 41.3%, demonstrating the enhancement of
   antitumor activity of NK cells by BPQDs@HSA alone in the solid tumors.
   However, X‐ray‐assisted BPQDs@HSA effectively potentiated NK
   cells‐induced tumor inhibition rate to 81.1%, with no obvious change
   observed in the body weight of each group (Figure [147]6H). This
   enhancement may be due to the improvement of deep penetration of NK
   cells into solid tumors assisted by X‐ray radiation. We then examined
   the ability of NK cells to penetrate tumor tissues by CD56 (an
   important marker of NK cell) immunostaining. As expected, with X‐ray
   assistance, BPQDs@HSA‐pretreated NK cells can penetrate more into tumor
   tissue, as indicated by the higher expression of CD56 marker
   (Figure [148]6I and Figure [149]S19, Supporting Information). We also
   employed magnetic resonance (MR) to examine tumor necrosis in living
   tissue and tumor growth inhibition. As shown in Figure [150]6J, under
   X‐ray assistance, BPQDs@HSA‐potentiated NK cells significantly enhanced
   the tumor necrosis, as evidenced by the highest values of standard ADC
   and slow ADC (Figure [151]6K). Moreover, the results of H&E staining
   indicated that this combined strategy significantly decreased the
   nuclear abnormalities, abnormal mitoses and microvascular formation,
   and increased the necrosis of tumor cells. This strategy also induced
   typical apoptotic DNA fragmentation in cancer cells in vivo, as
   reflected by the strong green fluorescence in the TUNEL expression of
   tumor tissues (Figure [152]6L). Taken together, these results confirm
   that X‐ray could assist BPQDs@HSA‐potentiated NK cell to penetrate into
   solid tumor and enhance the in vivo anticancer efficacy via triggering
   tumor cell apoptosis.

   In addition, we also examined the possible toxicity of the combined
   strategy in vivo. As shown in Figure [153]S20, Supporting Information,
   no obvious pathological changes could be observed in the major organs
   (including the heart, liver, spleen, lung, and kidney) after the
   combined treatment. Furthermore, from the images of the H&E staining,
   there was no inflammatory cell infiltration in the main organs with
   different treatment groups, which indicate the high safety of the
   BPQDs@HSA‐potentiated NK cell immunotherapy. Hematological analysis was
   also employed to examine the effects of each treatment on the functions
   of the liver, kidney, and heart of nude mice. As shown in
   Figure [154]6M, the tumor‐bearing nude mice exhibited a loss of balance
   in liver function, as revealed by changes in various blood indexes,
   indicating the obvious acute toxicity of tumor formation affecting the
   health of nude mice. However, the combined strategy significantly
   alleviated these damaging effects caused by tumor formation and even
   restored the indexes to the levels of the healthy group. Taken
   together, these results further confirm the safety of X‐ray‐assisted
   BPQDs@HSA‐potentiated NK cell immunotherapy.

3. Conclusion

   NK cell therapy exhibits impressive anticancer activity for high
   cells‐mediated immunity and low tumor immune escape, but the low
   persistence, metabolic dysfunction in microenvironment and
   tumor‐derived immunosuppression of NK cells in patients limit the
   successful clinical application. Therefore, it is of great significance
   to utilize high‐efficiency and low‐toxicity sensitization agents to
   activate and amplify NK cells to enhance their sustained anticancer
   activity. Inspired by this, herein we have synthesized biocompatible
   BPQDs@HSA to achieve synergistic tumor inhibition by combining
   BPQDs‐potentiated clinical patients‐derived NK cell immunotherapy with
   X‐ray radiotherapy. And as the donor of phosphate group, BPQDs@HSA
   binds with the protein of PIP5K1A and activates the downstream PI3K‐Akt
   and mTOR signaling pathways to reprogram cell metabolism of glycolysis
   and further promote the oxidative phosphorylation, sequentially
   maintains the cell viability and immunity of NK cells. Multiomics
   analysis was therefore conducted to reveal the underlying
   immunoregulation mechanisms, and we found that BPQDs@HSA could interact
   with the TLR on the NK cell surface and increase the expression level
   of mTOR, and thus activate downstream NF‐κB signalling pathways to
   regulate cytokine secretion. Interestingly, we also found that this
   nanosystem could downregulate PD‐1 expression in NK cells to block
   PD‐1/PD‐L1 recognition, thus relieving immune suppression by tumor
   cells and the microenvironment. In addition, this system also
   upregulates NKG2D to enhance immune surveillance and inhibit tumor
   immune escape. Furthermore, X‐ray radiation could assist
   BPQDs@HSA‐potentiated NK cells to penetrate into deep solid tumors,
   which effectively triggered tumor cell apoptosis and enhanced the in
   vivo antitumor activity of BPQDs@HSA‐potentiated NK cells.
   Collectively, our data contribute to a successful and translational
   strategy for cancer treatment by nanomedicine‐potentiated NK cell
   immunotherapy, and elucidate the fundamental mechanisms for regulation
   of cell metabolism and antitumor immunity by BP‐based nanosystem. This
   study also presents an important scientific basis for the development
   of high‐efficiency and low‐toxicity nanosystem based on stimulating the
   NK immune response and thus to achieve successful clinical cancer
   therapy.

4. Experimental Section

Materials and Cell Lines

   The BP crystals were purchased from a commercial supplier
   (Smart‐Elements). N‐methyl‐2‐pyrrolidone (NMP) (99.5%, anhydrous) and
   HSA were purchased from Aladdin Chemistry Co., Ltd. Thiazolyl Blue
   tetrazolium bromide (MTT), propidium iodide (PI), Hoechst,
   2″,7″‐dichlorodihydrofluorescein diacetate (DCF‐DA) and a bicinchoninic
   acid (BCA) kit were purchased from Sigma‐Aldrich. Rapamycin (Sirolimus;
   AY 22 989) was purchased from Med Chem Express. MyD88 inhibitory
   peptide (RQIKIWFQNRRMKWKK‐RDVLPGTCVNS‐NH[2]) was purchased from
   InvivoGen. Fluorescein isothiocyanate (FITC)‐CD3 and phycoerythrin
   (APC)‐CD56 were purchased from BD Biosciences. Interferon‐γ (IFN‐γ),
   recombinant human interleukin‐2 (rhIL‐2), and monoclonal antibody CD3
   (Mab‐CD3) were purchased from Cyagen. ELISA kits, including IL‐2,
   IL‐10, TGF‐β and IFN‐γ kits, were obtained from Bioss Antibodies.
   Hepatocellular carcinoma cell line (HepG‐2) was purchased from American
   Type Culture Collection (Manassas, VA). The cells were cultured in DMEM
   with 10% fetal bovine serum, 100 units/mL penicillin, and 50 units/mL
   streptomycin at 37 °C in a CO[2] incubator (95% relative humidity, 5%
   CO[2]).

NK Cell Culture and Expansion

   The NK cells were obtained, cultured, and expanded in the liver
   transplantation center of 3rd Affiliated Hospital of Sun Yat‐Sen
   University. Briefly, peripheral blood mononuclear cells (PBMCs) were
   obtained from 50 mL of a volunteer's peripheral blood by density
   gradient centrifugation with Ficoll. The cells were suspended in fresh
   NK cell medium (ALYS505NK‐AC medium, Cell Science & Technology
   Institute, Japanese) and adjusted to a density of 2 × 10^6 cells/mL,
   then incubated for 1 day at 37 °C in CO[2] incubator (95% relative
   humidity, 5% CO[2]). After that, 1000 U/mL of rhIL‐2 and 10% autologous
   plasma were added into the cell culture medium, and continue to
   incubate the NK cells for another 1 day. At the third day, 30 mL of
   fresh ALYS505NK‐EX medium with 1000 U/mL of rhIL‐2 and rhIL‐15 were
   added into the cells to incubate for two days. At the fifth day, 60 mL
   of fresh ALYS505NK‐EX medium (Cell Science & Technology Institute,
   Japanese) with 1000 U/mL of rhIL‐2 were added to the cells to incubate
   for another two days. During the seventh to fourteen days, double fresh
   serum‐free ALYS505NK‐EX medium with 1000 U/mL of rhIL‐2 was added into
   the cells according to the medium color change. On the 14th day, the
   purity of NK cells was assessed by fluorescence‐activated cell sorting
   (CytoFLEX S, Beckman Coulter) with anti‐CD3‐FITC (Biolegend, Catalog:
   300306) and anti‐CD56‐APC (Biolegend, Catalog: 362504), and the
   qualified cells were harvested for later use.^[ [155]^24 ^]

Synthesis of BPQDs@HSA Nanoparticles

   The BPQDs were synthesized according to previously described
   procedures.^[ [156]^15b ^] For BPQDs@HSA preparation, 200 µg of BP
   quantum dots (in NMP) were centrifuged to remove the NMP and washed
   with ethanol and ultrapure water. Then, 1 mL of an aqueous HSA solution
   (1 mg mL^−1) was added to the aqueous BPQD solution (200 µg mL^−1) and
   stirred for 24 h. In addition, a BPQDs@HSA aqueous solution was freshly
   synthesized.

Characterization of BPQDs@HSA Nanoparticles

   The synthesized BPQDs and BPQDs@HSA were characterized by different
   microscopic and spectroscopic methods. Briefly, TEM images were taken
   on a FEI Tecnai G2 F30 transmission electron microscope at an
   acceleration voltage of 200 kV. Raman scattering was performed on
   Horiba LabRAM HR Evolution. XPS measurement was analyzed on Thermo
   Fisher ESCALAB 250Xi XPS. UV–vis absorption spectra were recorded on a
   Lambda 25 spectrophotometer (PerkinElmer) using QS‐grade quartz
   cuvettes at room temperature. Isothermal titration calorimetry (ITC)
   (Affinity ITC, TA) was conducted at room temperature and used to
   examine the interaction of BPQDs and HSA.

Cell Viability Analysis

   Briefly, HepG‐2 cells (2 × 10^3 cells/well) were seeded in 96‐well
   micro‐plates and attached for 24 h. The treatment modes included
   pre‐treatment of BPQDs@HSA with NK cells for 12 h and co‐incubation
   with HepG‐2 cells, administration of BPQDs@HSA and NK cells to HepG‐2
   cells at the same time, and pre‐treatment of BPQDs@HSA with HepG‐2
   cells for various times (2, 6, or 12 h) before administration to NK
   cells for another 24 h. In addition, the free BPQDs@HSA was applied to
   HepG‐2 cells under dark conditions for 24 h. The concentration of
   BPQDs@HSA in this experiment was 2 µg mL^−1, and the NK cells were
   added to HepG‐2 cells at a ratio of 2.5:1 or 5:1. After incubation, the
   NK cells were removed and washed three times with PBS. Then, HepG‐2
   cell viability was evaluated by the MTT assay as previously reported.

Interaction of BPQDs@HSA with NK Cells and Identification Captured Proteins

   To further explore the interaction between BPQDs@HSA and NK cells,
   surface plasmon resonance (SPR) technology using a bScreen LB 991
   Label‐free Microarray System (BERTHOLD TECHNOLOGIES, Germany) and LC‐MS
   analysis to identify the protein in NK cells that was targeted by the
   nanosystem were employed. Briefly, BPQDs were suspended with ddH[2]O at
   1 µg mL^−1, and then immobilized in a sensor chip (Betterways Inc.,
   China) with a label‐free reactive amino group modification through
   coordination bond. NK cell total cellular proteins were extracted by
   incubating cells in a lysis buffer. BPQDs were flowed through the chip
   surface and immobilized to the surface through coordination bond in a
   pH = 5.2 environment. Then NK cell lysate was flowed through chip
   surface. Finally, the chip was washed to remove non‐specifically
   attached proteins on the chip surface. The captured proteins or
   peptides by BPQDs were digested in situ by trypsin, and then identified
   with HPLC‐MS/MS. MS data were collected by Xcalibur (Thermo Scientific,
   version 2.2.0), and MS experiments were performed triply for each
   sample. The MS data were analyzed using MaxQuant software (COX LAB,
   version 1.3.0.5). Peptides were identified by database searching and
   the MS[2] results for selected proteins that changed quantity between
   sample types were annotated via BLASTP. The GO analysis was carried out
   with Blast2GO.

Determination of Biomarker on Surface of NK Cells

   The NK cells were treated with different concentration (2.5 or 5 µg/mL)
   of BPQDs@HSA for 6 h. After that the cells were collected and
   co‐stained with anti‐CD3‐FITC (Biolegend, Catalog: 300306) and
   anti‐CD56‐APC (Biolegend, Catalog: 362504) according to the
   manufacturer's method. BPQDs@HSA treated NK cells also incubated with
   anti‐PD‐1 (Cell Signaling Technology) or anti‐NKG2D (Abcam) for 2 h,
   and then incubated with FITC‐Rabbit IgG[1] isotype for another 0.5 h,
   according to the manufacturer's protocols. These biomarkers on the
   surface of NK cells were determined on Beckman CytoFLEX S flow
   cytometer.

   The effects of BPQDs@HSA changing the expression of biomarker on the
   surface of NK cells in the tumor microenvironment were also examined
   with flow cytometer. NK cells were pretreated with or without BPQDs@HSA
   for 6 h, and then added into HepG‐2 cells at ratio of 5:1 for another
   24 h. The supernatant NK cells were collected and used to determine the
   expression of anti‐PD‐1 and anti‐NKG2D according the methods as above.
   Furthermore, according to the manufacturer's method, the HepG‐2 cells
   were collected and respectively incubated with anti‐MICA/B (Abcam,
   Catalog: ab203679), anti‐ULBP1 (Abcam, Catalog: ab176566), anti‐ULBP2
   (Abcam, Catalog: ab88645), anti‐ULBP3 (Abcam, Catalog: ab89931),
   anti‐ULBP4 (Abcam, Catalog: ab95202) for 2 h, and then respectively
   incubated with FITC‐Rabbit IgG1 for another 0.5 h. These biomarkers on
   the surface of NK cells and HepG‐2 cells were determined on Beckman
   CytoFLEX S flow cytometer.

OCR and ECAR Analyses

   Measurement of OCR and ECAR of NK cells were conducted by using XFp
   Cell Mito Stress Test Kit and XFp Glycolysis Stress Test kit on a
   Seahorse XF96 Extracellular Flux Analyzer (Agilent). The NK cells (2 ×
   10^4/well) were seeded in metabolic 96‐wells plate, and incubated with
   different concentrations of BPQDs@HSA for 24 h. The reagents used in
   this assay were oligomycin (1.0 mm), FCCP (1.0 mm), rotenone/antimycin
   A (0.5 mm), glucose (10 mm), and 2‐DG (50 mm).

Penetrating Ability of NK Cells to Tumor Spheroids

   The HepG‐2 tumor spheroids were formed and grown in 6‐well plates with
   ultra‐low attachment surface (Corning Incorporate) according to
   previous procedures.^[ [157]^25 ^] Briefly, 1 mL of HepG‐2 cells was
   seeded into each well at a density of 400 000 cells/well. After tumor
   spheroids with diameters up to 400 µm, the tumor spheroids were removed
   to 35‐mm confocal dishes. Then the NK cells were labeled with Cell
   Tracker Red CMTPX Dye and were visualized with red fluorescence. The NK
   cells (1 × 10^6 cells) were pretreated with or without BPQDs@HSA (5 µg
   mL^−1) for 6 h, and then added into HepG‐2 tumor spheroids. The process
   of NK cells penetrated in tumor spheroids was determined by a cell
   imaging multi‐mode reader (Cytation 5, BioTek Instruments, Inc.).
   Furthermore, after NK cells were incubated with tumor spheroids for
   12 h, the tumor spheroids were rinsed with PBS and scanned at the
   different layers from the top of the tumor spheroids to the middle
   using a confocal laser scanning fluorescent microscope. The video of
   HepG‐2 tumor spheroids attacked by BPQDs@HSA‐potentiated NK cells were
   determined by a cell imaging multi‐mode reader (Cytation 5, BioTek
   Instruments, Inc.).

Determination of ROS Generation

   The effects of BPQDs@HSA under X‐ray radiation on intracellular
   reactive oxygen species (ROS) generation in HepG‐2 cells (5 × 10^4
   cells/well, 96‐well micro‐plates) were examined using a DCF‐DA
   fluorescence probe on a micro‐plate reader (SpectraMax M5, MD, USA). In
   addition, BPQDs@HSA (2.5 µg mL^−1) were applied to HepG‐2 cells for 4 h
   and then subjected to X‐ray radiation at 2 Gy.

   The effects of drug treatment on ^1O[2] overproduction in HepG‐2 cells
   were examined by DPBF assay. The HepG‐2 cells (2 × 10^4 cells/well,
   0.1 mL) were seeded on 96‐well micro‐plates and attached for 24 h.
   BPQDs@HSA at 8 and 16 µg mL^−1 incubated with NK cells for 6 h and then
   added in HepG‐2 cells at a ratio of 5:1, then the cells treated with or
   without X‐ray radiation at 2 Gy. After co‐incubation for another 12 h,
   the NK cells were removed and added with fresh medium, then the HepG‐2
   cells were stained with the probe of DPBF (20 µm) for 1 h. The 96‐well
   plates were analyzed for ^1O[2] generation in the HepG‐2 cells using a
   cell imaging multi‐mode reader (Cytation 5, BioTek Instruments, Inc.)
   at excitation and emission wavelengths of 410 and 484 nm, respectively.

Biodistribution and Translocation of BPQDs@HSA

   The biodistribution and translocation of nanoparticles between NK and
   HepG‐2 cells were measured by the fluorescence intensity of
   FITC‐labelled BPQDs@HSA by fluorescence microscope and fluorescence
   microplate reader. The HepG‐2 cells were seeded into 6‐well plates at 8
   × 10^4 cells/mL (2 mL) and attached for 24 h. NK cells were pretreated
   with FITC‐labeled BPQDs@HSA for 6 h, and then co‐incubated with HepG‐2
   cells at a ratio of 5:1 for another 24 h. The cells were then examined
   by fluorescence microscope, and the BPQDs@HSA was visualized by green
   fluorescence. Then the NK cells were removed to another blank well of
   the same plate and fresh medium was added, the quantitative analysis of
   cellular uptake efficiency of BPQDs@HSA in NK cells and HepG‐2 cells
   were examined by a cell imaging multi‐mode reader (Cytation 5, BioTek
   Instruments, Inc.).

Flow Cytometric Analysis

   Flow cytometry was employed to examine the effects of BPQDs@HSA and NK
   cells under X‐ray radiation on HepG‐2 cell cycle progression. Briefly,
   BPQDs@HSA (2 µg mL^−1) were incubated with NK cells for 12 h and then
   added to HepG‐2 cells at a ratio of 2.5:1. After 24‐h incubation, the
   NK cells were removed and washed three times with PBS. Then, the HepG‐2
   cells were collected, fixed, and stained with PI in the dark and then
   analyzed on a Beckman Cytomics FC500 flow cytometer. The collected
   cells were also stained with Annexin V‐FITC and PI according to the
   method in manufacturer's instructions of assay kit (Solarbio), and then
   analyzed on Beckman CytoFLEX S flow cytometer.

Detection Cytokine Levels Secreted by NK Cells

   One milliliter of NK cells (1 × 10^6 cells/mL) was incubated with 2 µg
   mL^−1 BPQDs@HSA for 12 h. Then, the cells were centrifuged to obtain
   the medium. The levels of the cytokines IL‐2, IL‐10, TGF‐β, and IFN‐γ
   secreted by NK cells were examined by ELISA kits (obtained from Bioss
   Antibodies) according to the instructions.

Detection Chemokine Levels Secreted by HepG‐2 Cells

   The HepG‐2 cells (10 × 10^4 cells/well, 1 mL) were seeded on 24‐well
   micro‐plates and attached for 24 h. BPQDs@HSA at 4 µg mL^−1 incubated
   with NK cells for 6 h and then added in HepG‐2 cells at ratio of 5:1.
   After co‐incubation for another 24 h, the supernatant medium was
   collected and examined the secretion of CXCL9 and CXCL10 by ELISA kits
   (obtained from Bioss Antibodies) according to the instructions.
   Furthermore, the NK cells were treated with BPQDs@HSA for 24 h, and
   then stained with anti‐CXCR3‐Alexa Fluor 488 (Biolegend, Catalog:
   353709) according to the manufacturer's protocols.

Western Blot Analysis

   Total cellular proteins (1 × 10^5 cells/mL, 10 mL/vessel) in the groups
   treated with different concentration of free BPQDs@HSA (2.5, 5.0, and
   10 µg mL^−1, in the dark) or NK cells (NK:HepG‐2 = 5:1) or co‐treated
   with BPQDs@HSA and NK cells were extracted by cell lysis buffer from
   Cell Signaling Technology. The protein concentrations were examined by
   the BCA protein assay. The effects of BPQDs@HSA and NK cells on the
   expression levels of related proteins were determined by Western
   blotting.

Real‐Time PCR Analysis

   Total RNA of NK cells after different treatment was extracted using
   TRIzol and subjected to reverse transcription with a ReverTra Ace‐a‐kit
   (Takara) according to the manufacturer's protocol. Real‐time PCR
   analysis was performed using a SYBR Green PCR Master Mix kit (Bimake)
   on an Opticon 2 Real Time Cycler instrument (Bio‐red, CFX Connect). The
   primer sequences of the related genes were listed in the Table [158]S2,
   and the ΔΔCt method was used to quantify the mRNA expression level.

mRNA Sequencing Analysis of NK Cells after Treatment

   After treatment with BPQDs@HSA (5 µg mL^−1) and X‐ray (2 Gy), RNA from
   the NK cells was extracted using TRIzol reagent. RNA was precipitated
   at −20 °C overnight. Then the mRNA library was constructed using VAHTS
   mRNA‐seq V3 Library Prep Kit following the manufacturer's instructions.
   Libraries were sequenced on an Illumina NovaSeq 6000 sequencer for 318
   cycles. Adapters were trimmed from the reads, and the reads shorter
   than 17 nt were discarded. The reads were mapped to the human mRNA
   reference database on Chi‐Cloud NGS Analysis Platform (Chi‐Biotech Co.
   Ltd., Shenzhen, China). PPI analysis of differentially expressed genes
   was based on the STRING database, which known and predicted
   protein–protein interactions.

In Vivo Antitumor Activity

   HepG‐2 cell xenografts were established with ≈1 × 10^6 cells in 100 µL
   of serum‐free medium injected into the right oxter of male BALB/c nude
   mice. After the tumor volume increased to ≈70 mm^3, the mice were
   randomly divided into six treatment groups (n = 10). The radiotherapy
   treatment mice were conducted the tumor sites under X‐ray radiation for
   2 Gy. After 24 h, the mice received caudal vein injection of the NK
   cells with pre‐incubation of BPQDs@HSA (50 µg kg^−1) for 12 h. In
   addition, each mouse was injected with 5 × 10^5 NK cells in 100 µL of
   X‐vivo medium via the tail vein twice at intervals of one week.
   Throughout the 3 weeks, changes in the tumor volume and body weight
   were monitored. On the 21st day, the tumor tissues of each treatment
   group were harvested and weighed. The tumor sections were subjected to
   H&E staining and TUNEL‐Hoechst co‐staining. The expression levels of
   PD‐1 and PD‐L1 protein in 2‐µm tumor sections were examined by
   immunohistochemical and immunofluorescence methods. Venous blood was
   collected from the nude mouse eyes at the end of the experiments, and
   hematological analysis was conducted to examine the kidney, liver and
   heart functions of the nude mice. Intravoxel incoherent motion
   diffusion‐weighted imaging (IVIM‐DWI) was performed on a 3.0‐T MR
   scanner (General Electric, Milwaukee, WI, USA) in living nude mice on
   the 21st day. The mice with tumor volume larger than 2.5 cm^3 were
   regarded as dead. All of the animal studies were approved by the Animal
   Experimentation Ethics Committee of Jinan University (GB/T 35892‐2018).

Statistical Analysis

   The experiments were carried out at least three times and all the data
   were expressed as mean ± standard deviation. Statistical analysis was
   performed with the SPSS statistical package (SPSS 13.0 for Windows;
   SPSS, Inc. Chicago, IL) and differences of p < 0.05 (*) or p < 0.01
   (**) were considered statistically significant.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [159]Click here for additional data file.^ (1.7MB, pdf)

   Supplemental Video 1
   [160]Click here for additional data file.^ (1.9MB, wmv)

   Supplemental Video 2
   [161]Click here for additional data file.^ (2.5MB, wmv)

Acknowledgements