Abstract

   Photosynthesis, essential for life on earth, sustains diverse processes
   by providing nutrition in plants and microorganisms. Especially,
   photosynthesis is increasingly applied in disease treatments, but its
   efficacy is substantially limited by the well‐known low penetration
   depth of external light. Here, ultrasound‐mediated photosynthesis is
   reported for enhanced sonodynamic tumor therapy using organic
   sonoafterglow (ultrasound‐induced afterglow) nanoparticles combined
   with cyanobacteria, demonstrating the proof‐of‐concept sonosynthesis
   (sonoafterglow‐induced photosynthesis) in cancer therapy. Chlorin e6, a
   typical small‐molecule chlorine, is formulated into nanoparticles to
   stimulate cyanobacteria for sonosynthesis, which serves three roles,
   i.e., overcoming the tissue‐penetration limitations of external light
   sources, reducing hypoxia, and acting as a sonosensitizer for in vivo
   tumor suppression. Furthermore, sonosynthetic oxygenation suppresses
   the expression of hypoxia‐inducible factor 1α, leading to reduced
   stability of downstream SLC7A11 mRNA, which results in glutathione
   depletion and inactivation of glutathione peroxidase 4, thereby
   inducing ferroptosis of cancer cells. This study not only broadens the
   scope of microbial nanomedicine but also offers a distinct direction
   for sonosynthesis.

   Keywords: cyanobacteria, sonoafterglow, sonodynamic therapy
   ferroptosis, sonosynthesis
     __________________________________________________________________

   Ultrasound‐mediated photosynthesis for enhanced sonodynamic tumor
   therapy using organic sonoafterglow (ultrasound‐induced afterglow)
   nanoparticles combine with cyanobacteria, demonstrating the
   proof‐of‐concept sonosynthesis (sonoafterglow‐induced photosynthesis)
   in cancer therapy (Created with BioRender.com).

   graphic file with name ADVS-11-2400251-g002.jpg

1. Introduction

   Photosynthesis, the fundamental biological process on earth, harnesses
   solar energy to convert carbon dioxide and water into chemical energy
   and oxygen.^[ [42]^1 ^] It is crucial in addressing contemporary
   challenges in food, energy, and environmental resources.^[ [43]^2 ^]
   Various artificial photosynthetic systems have been constructed and
   employed for green energy.^[ [44]^3 ^] Even a plant‐derived
   photosynthetic system was transferred to mammalian cells to cure
   osteoarthritis.^[ [45]^4 ^] Beyond plants, microbial photosynthesis is
   vital in nature and has been innovatively integrated into biomedical
   applications,^[ [46]^5 ^] particularly for oxygen‐dependent disease
   treatments.^[ [47]^6 ^] The photocatalytic oxygen production capacity
   and biocompatibility make photosynthetic microorganisms suitable for
   treating hypoxia‐related diseases, including tumor therapy,^[ [48]^7 ^]
   wound healing,^[ [49]^8 ^] tissue engineering,^[ [50]^9 ^] and ischemic
   myocardial injury treatment.^[ [51]^10 ^] However, the effectiveness of
   photosynthesis in medical applications is limited by the poor tissue
   penetration of light sources and interference from endogenous tissue
   substances, hindering activation in deep tissues and reducing the
   therapeutic effect.^[ [52]^11 ^] Moreover, prolonged light irradiation
   or increased power densities pose potential issues, challenging the
   clinical translation of photostimulated microbial therapy.^[ [53]^12 ^]
   Therefore, optimizing the activation source is highly crucial for
   effective photosynthetic oxygen therapy.

   The development of nanotechnology, particularly light conversion
   nanomaterials such as upconversion nanoparticles (UCNPs),^[ [54]^13 ^]
   nanoscintillators,^[ [55]^14 ^] and persistent luminescence
   nanoparticles (PLNPs),^[ [56]^15 ^] has significantly advanced therapy
   based on light. UCNPs and nanoscintillators efficiently convert
   near‐infrared (NIR) light and X‐ray into ultraviolet (UV) or visible
   light, offering deeper tissue penetration.^[ [57]^11 ^] PLNPs,
   stimulated by external light sources such as UV light and X‐rays
   maintain prolonged afterglow luminescence, potentially shorting
   irradiation time and minimizing tissue photodamage.^[ [58]^16 ^]
   Additionally, surgically implanted photonic patches address light
   source limitations through direct irradiation.^[ [59]^17 ^] Despite
   these advances, challenges such as poor biodegradability, low light
   conversion efficiency, and inefficient targeting limit their potential.
   UCNPs suffer from non‐degradability and relatively low light conversion
   efficiencies.^[ [60]^18 ^] Nanoscintillators require constant X‐ray
   irradiation, posing side effects on patients. PLNPs still need frequent
   charging from external energy sources.^[ [61]^19 ^] Photonic patches
   restrict patient mobility and pose biosafety concerns for long‐term
   implantation.^[ [62]^20 ^] Consequently, there is a critical need for a
   treatment strategy with high penetration capabilities to enhance
   microbial therapy efficacy. Ultrasound (US) emerges as a promising
   alternative, offering superior penetration and targetability than
   light, especially in vivo.^[ [63]^21 ^] However, the sonostimulated
   microbial photosynthesis in disease treatment remains underexplored.

   In this study, we develop a sonoafterglow‐induced cyanobacterial
   photosynthesis‐based sonodynamic therapy (SDT) platform and demonstrate
   a proof‐of‐concept sonosynthesis (sonoafterglow‐induced photosynthesis)
   by combining Synechococcus elongatus PCC 7942 (PCC) with sonoafterglow
   Ce6 nanoparticles (NPs‐Ce6) (Figure [64]1 ). This platform mitigates
   tumor hypoxia using the sustained afterglow of NPs‐Ce6 to stimulate PCC
   oxygen production while enhancing SDT by singlet oxygen (^1O[2])
   generation under US irradiation. Tumor hypoxia contributes
   significantly to resistance against ferroptosis,^[ [65]^22 ^] due to
   the stabilization of solute carrier family 7 member 11 (SLC7A11) mRNA,
   a gene vital for cystine transport and glutathione (GSH) synthesis,^[
   [66]^23 ^] facilitated by hypoxia‐inducible factor 1α (HIF‐1α).^[
   [67]^24 ^] Under US irradiation, NPs‐Ce6 emits continuous red light,
   inducing prolonged PCC photosynthesis, alleviating tumor hypoxia, and
   improving SDT. The reduced hypoxia leads to lower HIF‐1α levels,
   decreasing SLC7A11 subunit activity, impeding GSH synthesis, and
   reducing tumor cell resistance to ferroptosis. Additionally, the
   SDT‐produced ^1O[2] disrupts ferritin, releasing Fe^3+ ions, further
   depleting GSH and intensifying tumor cell ferroptosis. This
   sonosynthesis‐based therapeutic strategy has the potential to overcome
   the deep hypoxic disease environment by enhancing oxygenation, thereby
   improving the effectiveness of treatments such as SDT.

Figure 1.

   Figure 1
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   Schematic illustration of sonoafterglow‐enhanced photosynthetic
   oxygenation to boost SDT. a) Diagram of the fabrication process of
   NPs‐Ce6. b) The antitumor mechanisms of sonosynthesis‐enhanced SDT lead
   to ferroptosis and apoptosis (Created with
   [69]https://www.BioRender.com).

2. Results and Discussion

2.1. Synthesis and Characterization of NPs‐Ce6

   A bottom‐up nanoprecipitation technique was employed to synthesize the
   NPs‐Ce6 from the homogeneously dissolved tetrahydrofuran (THF) solution
   of Ce6. Briefly, PEG‐b‐PPG‐b‐PEG and Ce6 were dissolved into a THF
   solution. The mixture was then injected into ultrapure water (45 mL)
   under magnetic stirring. Then the THF was evaporated with a rotary
   evaporator. Finally, the resulting solution of NPs‐Ce6 was received by
   ultracentrifugation with 30 K centrifugal filter units and the
   concentrated solution of NPs‐Ce6 was diluted with 1 × PBS buffer. The
   obtained nanoparticle dispersion was stored in the dark at 4 °C (Figure
   [70]2a). NPs‐Ce6 showed spherical morphology with a hydrodynamic size
   of 24.6 ± 3.6 nm (Figure [71]2b,c). The ultraviolet‐visible (UV‐vis)
   absorbance spectrum of the Ce6 exhibited a prominent absorption peak at
   ≈400 nm, and this distinctive absorption peak was likewise observed in
   NPs‐Ce6 (Figure [72]2d). The emission of NPs‐Ce6 excited at 400 nm was
   tracked between 610 and 750 nm (Figure [73]2e), in which the
   fluorescence intensity was dependent on the concentration
   (Figure [74]2f). Notably, the examination of the afterglow spectrum of
   NPs‐Ce6 following US irradiation revealed the presence of a prominent
   peak at 680 nm (Figure [75]2g). Importantly, it was observed that
   NPs‐Ce6 could effectively produce afterglow luminescence under US
   stimulation (Figure [76]2h), confirming the sonoafterglow ability of
   NPs‐Ce6. Moreover, after co‐incubation PCC, the afterglow luminescence
   intensity of NPs‐Ce6 after US activation further increased compared
   with only NPs‐Ce6. Furthermore, the sonoafterglow intensity immediately
   recovered upon re‐exposure to the US (three rounds) after a continuous
   decline (Figure [77]2k; Figure [78]S1a, Supporting Information).
   Additionally, the sonoafterglow intensity closely correlated with the
   duration and power of US irradiation (Figure [79]2i; Figure [80]S1b,
   Supporting Information), in which the sonoafterglow intensity increased
   with the incremental US time and power density. To investigate the
   advantage of US penetration depth, NPs‐Ce6 dispersion was covered with
   chicken breast tissues, followed by US or laser irradiation (Figure
   [81]S3, Supporting Information). Due to the superior penetration
   ability of US, the intensity of sonoafterglow was notably higher than
   that of laser‐mediated photoafterglow when penetrating chicken breast
   tissues at the thicknesses of 3 and 5 mm (Figure [82]2j), demonstrating
   the effective activation of sonoafterglow in deep tissues for the
   enhanced therapeutic potential.

Figure 2.

   Figure 2
   [83]Open in a new tab

   Synthesis and characterization of NPs‐Ce6. a) Schematic illustration
   showing the synthesis of NPs‐Ce6 (Created with BioRender.com). b) TEM
   images of NPs‐Ce6. c) Size distribution of NPs‐Ce6 in PBS. d) UV‐vis
   absorbance spectra of Ce6 in THF and NPs‐Ce6 in PBS. e) Normalized
   fluorescence spectrum of NPs‐Ce6 in PBS under excitation at 400 nm. f)
   Concentration‐dependent fluorescence intensity of NPs‐Ce6 (n = 3). g)
   Normalized afterglow luminescence spectrum of NPs‐Ce6 after US
   irradiation. h) Sonoafterglow intensity of NPs‐Ce6 after different
   treatment (n = 3). i) US irradiation power‐dependent sonoafterglow
   intensity of NPs‐Ce6 (n = 4). j) Afterglow intensity of NPs‐Ce6 after
   US or light irradiation at different chicken breast tissue thicknesses.
   (Ultrasound parameters: 1.0 MHz, 50% duty, 1.5 W cm^−2, 60 s. Laser
   parameters: 660 nm, 1.5 W cm^−2, 60 s. n = 3). NPs‐Ce6 concentration:
   500 µg mL^−1, PCC concentration: 1 × 10^6 cell mL^−1. Statistical
   differences of p values were determined by One‐way ANOVA. k)
   Sonoafterglow luminescence images of NPs‐Ce6 after US irradiation for
   2 min and re‐activation for another 2 min. Data are presented as mean
   values ± SD. ^** p < 0.01.

   To elucidate the molecular mechanism underlying the sonoafterglow
   phenomenon induced by Ce6 after US irradiation, the structural
   alterations of Ce6 were analyzed. The absorption spectra detailed in
   Figure [84]S2a (Supporting Information) reveal a significant decrease
   in the primary absorption peak of Ce6 ≈ 400 nm following US
   irradiation, suggesting substantial Ce6 decomposition. Further
   examination through mass spectrometry to assess the Ce6 changes before
   and after US treatment indicated the emergence of a new compound,
   exhibiting a molecular ion peak at m/z = 363.90 ([M + H]+). Correlating
   with existing literature, the entity was identified as Ce6‐F (Figure
   [85]S2c, Supporting Information).^[ [86]^25 ^] These findings lead to
   the proposition of sonoafterglow mechanism for Ce6: energy absorbed by
   Ce6 is transferred to oxygen, facilitating the ^1O[2] production.
   Subsequently, ^1O[2] engages in a [π2 + π2] cycloaddition with the
   vinylene linkage (C═C) of Ce6, generating a Ce6‐dioxetane intermediate.
   The eventual breakdown of this intermediate is responsible for the
   sonoafterglow luminescence. Given the similarity between the
   sonoafterglow and Ce6 fluorescence, it is inferred that the spontaneous
   disintegration of the Ce6‐dioxetane intermediate channels the requisite
   energy for elevating Ce6 to an excited state (Ce6*), which emits
   sonoafterglow luminescence upon returning to the ground state.
   Meanwhile, Ce6‐F formation occurs (Figure [87]S2d, Supporting
   Information).^[ [88]^26 ^]

2.2. Characterization of Cyanobacteria

   PCC displayed a distinctive rod‐shaped morphology with micrometer size
   (Figure [89]3a,b). In addition, the surface of PCC exhibited a negative
   charge attribute (Figure [90]S4, Supporting Information). During their
   mid‐exponential phase, PCC was cultured in BG11 medium and continuously
   supplemented with fresh medium to promote healthy growth, in which the
   characteristic green coloration of the culture medium was attributed to
   the optical density at 730 nm (OD730) in the ranges of 0.4 to 0.7
   (Figure [91]3c, inset). The red autofluorescence, attributed to
   phycocyanin fluorescence in PCC, was confirmed using a confocal laser
   scanning microscope (CLSM) (Figure [92]3d; Figure [93]S5, Supporting
   Information). The broad absorption spectrum, with prominent peaks at
   440, 630, and 681 nm, was attributed to the presence of chlorophyll a.
   It was important to note that the absorption intensity showed a
   positive correlation with the concentration of PCC (Figure [94]3e,f).
   To determine the amount of PCC, a standard curve fcor PCC concentration
   was established by measuring OD730 (Figure [95]S6, Supporting
   Information). The overlap between the emission spectrum of NPs‐Ce6 and
   the absorption spectrum of PCC is a crucial factor in photonic
   activation. The absorption peak at 680 nm, attributed to chlorophyll a,
   as shown in Figure [96]3g, confirmed the effective activation of
   photosynthesis under red light irradiation, which guaranteed the
   oxygenation by the afterglow of NPs‐Ce6 (Figure [97]3h). Subsequently,
   to assess the oxygen evolution capacity of PCC, a portable dissolved
   oxygen electrode was utilized. At first, we excluded the impact of
   varying NPs‐Ce6 concentrations on dissolved oxygen levels after US
   irradiation (Figure [98]S7, Supporting Information). After that, the
   effect of NPs‐Ce6 on the oxygen production of PCC was examined
   (Figure [99]3i). It was found that NPs‐Ce6, after US irradiation,
   effectively enhanced the oxygen production of PCC, as indicated by the
   increased dissolved oxygen levels. In contrast, NPs‐Ce6 without US
   irradiation not only failed to promote dissolved oxygen production but
   also reduced it due to the respiration of PCC. This confirmed that
   sonoafterglow could effectively enhance the oxygen‐producing capacity
   of PCC. Moreover, both NPs‐Ce6 and PCC exhibited a
   concentration‐dependent relationship with the generation of dissolved
   oxygen (Figure [100]3j,k). To verify the viability of PCC, it was
   cultured with PBS (pH 6.4), 1640 medium, and different concentrations
   of NPs‐Ce6. Remarkably, even in the presence of a high concentration of
   NPs‐Ce6 at 500 µg mL^−1, PCC maintained its proliferative ability after
   US stimulation during a 7‐day observation period (Figure [101]S8,
   Supporting Information), suggesting that NPs‐Ce6 and ^1O[2] have no
   obvious toxicity on PCC growth. Furthermore, the oxygen‐generating
   capacity of PCC was evaluated after 7 days (Figure [102]S9, Supporting
   Information). The photosynthetic oxygen production of PCC remained
   largely consistent with its performance in BG11 medium, establishing a
   solid foundation for the following in vitro and in vivo experiments.

Figure 3.

   Figure 3
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   Sonoafterglow‐mediated photosynthetic oxygenation. a) TEM image of PCC.
   b) Scanning electron micrograph (SEM) image of PCC. c) Optical
   microscope image of PCC (inset, digital photo of PCC during
   preparation). d) Fluorescence microscope image of PCC. e) UV‐vis
   absorbance spectra and f) the corresponding optical images of PCC at
   different concentrations. g) UV‐vis absorbance spectrum of PCC and
   sonoafterglow luminescence spectrum of NPs‐Ce6. h) Schematic
   illustration showing the photosynthetic mechanism of PCC (Created with
   BioRender.com). i) Dissolved oxygen levels of PCC with NPs‐Ce6 after
   different treatments. j) Concentration‐dependent oxygenation of PCC
   treated with NPs‐Ce6 under US irradiation (NPs‐Ce6 500 µg mL^−1, 2 W
   cm^−2, 3 min). k) Concentration‐dependent oxygenation of NPs‐Ce6 under
   US irradiation with PCC (PCC 5 × 10^7 cell mL^−1, 2 W cm^−2, 3 min).

2.3. Sonosynthetic Oxygenation for Self‐Enhanced SDT

   Benefiting from the ability of NPs‐Ce6 to promote oxygen production in
   PCC through photosynthesis via sonoafterglow, we hypothesized that
   sonosynthesis could increase the ROS production of NPs‐Ce6.
   1,3‐diphenylisobenofuran (DPBF), a yellow indicator that fades after
   reacting with ^1O[2], was used to examine the enhancement of SDT
   through sonoafterglow‐induced oxygen production (Figure [104]4a).
   First, the potential adsorption effects of NPs‐Ce6 and PCC on
   characteristic peak reduction were eliminated (Figure [105]S10,
   Supporting Information). Under US irradiation, a significant reduction
   in characteristic peaks was observed over time in both the NPs‐Ce6 and
   NPs‐Ce6 + PCC groups compared to that in PBS and PCC groups
   (Figure [106]4b–e), indicating that the PCC group does not generate
   ^1O[2], while both NPs‐Ce6 and NPs‐Ce6 + PCC groups exhibited a
   time‐dependent ^1O[2] production. Furthermore, under the same US
   conditions, the NPs‐Ce6 + PCC group exhibited a higher ^1O[2] yield
   than the NPs‐Ce6 group alone, suggesting that sonoafterglow‐enhanced
   oxygen production led to increased ROS production (Figure [107]4f).
   When subjected to varying US power density levels, the characteristic
   peaks in the NP‐Ce6 + PCC group were diminished with the power
   densities increased (Figure [108]4g). These results were further
   confirmed by the 9,10‐anthracenediyl‐bis‐(methylene)dimalonic acid
   (ABDA) indicator, which showed similar outcomes to DPBF and provided
   additional evidence for sonoafterglow‐augmented oxygen production in
   PCC, resulting in enhanced ^1O[2] production (Figure [109]4h; Figure
   [110]S11, Supporting Information). Additionally, electron spin
   resonance (ESR) spectroscopy was utilized to detect and identify the
   ^1O[2] generation, using 2,2,6,6‐tetramethyl‐4‐piperidone (TEMP) as a
   trapping agent. The pronounced ^1O[2]‐induced ESR signals were observed
   in the ESR spectrum following NPs‐Ce6 treatment under US irradiation
   (Figure [111]4i), evidenced by the appearance of a characteristic 1:1:1
   signal. Compared to the NPs‐Ce6 + US group, the NPs‐Ce6 + PCC + US
   group exhibited a stronger ESR signal, indicating higher ^1O[2] yielded
   due to photosynthetic oxygenation stimulated by sonoafterglow, thereby
   effectively enhancing the sonodynamic effect.

Figure 4.

   Figure 4
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   Sonodynamic performance of NPs‐Ce6 + PCC. a) DPBF structure and color
   change before and after interacting with ^1O[2]. Time‐dependent ^1O[2]
   generation of b) PBS, c) PCC, d) NPs‐Ce6, and e) NPs‐Ce6 + PCC under US
   irradiation (1.5 W cm^−2) for 4 min detected with the DPBF probe. f)
   Quantitative analysis of DPBF oxidation after different treatments. g)
   Power‐dependent ^1O[2] generation of NPs‐Ce6 + PCC under US treatment
   with the DPBF probe (0, 0.5, 1, 1.5, and 2 W cm^−2, 2 min). h)
   Quantitative analysis of ABDA oxidation after different treatments
   (1.5 W cm^−2). i) ESR spectra using TEMP as trapper after different
   treatments. (1.5 W cm^−2, 2 min). NPs‐Ce6 concentration: 500 µg mL^−1,
   PCC concentration: 1 × 10^6 cell mL^−1.

2.4. In Vitro Therapeutic Effect

   Inspired by the excellent sonoafterglow‐mediated oxygen generation and
   sonodynamic capabilities, the in vitro SDT performance was further
   evaluated at the cellular level. Initially, the cytocompatibility of
   PCC and NPs‐Ce6 was evaluated independently. The results of the Cell
   Counting Kit‐8 (CCK‐8) assay demonstrate that neither PCC nor NPs‐Ce6
   display significant cytotoxicity after co‐incubation for 24 and 48 h,
   confirming their benign biocompatibility (Figure [113]5a; Figure
   [114]S12, Supporting Information). Furthermore, the cytotoxicity of
   NPs‐Ce6 remained negligible even up to 500 µg mL^−1 when tested on
   murine mammary carcinoma 4T1 cells and murine embryonic fibroblast 3T3
   cells (Figure [115]5b), reinforcing its high biosafety. Therefore, in
   subsequent in vitro experiments, 400 µg mL^−1 NPs‐Ce6 and 5 × 10^7 cell
   mL‐1PCC were applied. The cellular uptake was examined through CLSM
   (Figure [116]S13, Supporting Information), which revealed the NPs‐Ce6
   uptake in 4T1 cells after co‐incubation for 2 h with an increase over
   time. The sonodynamic effect on 4T1 cancer cells was subsequently
   evaluated (Figure [117]5c), which induced a notable decrease in cell
   viability under normoxia in the NPs‐Ce6 + US group, highlighting the
   critical role of oxygen in efficient SDT. Significantly, the cell
   viability in the NPs‐Ce6 + PCC group decreased substantially post‐US
   irradiation under hypoxic conditions, demonstrating that sonoafterglow
   could promote oxygen generation in PCC, thereby enhancing SDT efficacy.
   In addition, the prolonged US irradiation duration further decreased
   the cell survival rates (Figure [118]S14, Supporting Information).

Figure 5.

   Figure 5
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   In vitro therapeutic effect. a) Relative viabilities of 4T1 cancer
   cells after treatment with cyanobacteria at different concentrations
   for 24 and 48 h (n = 3). b) Relative viabilities of 4T1 cells and 3T3
   cells after treatment with NPs‐Ce6 at different concentrations for 24 h
   (n = 3). c) Relative viabilities of 4T1 cancer cells after different
   treatments (n = 3). d) Fluorescence images of Calcein‐AM/PI co‐staining
   4T1 cells. e) flow cytometry analysis of 4T1 cells staining with
   DCFH‐DA. f) CLSM of 4T1 cells staining with JC‐1 probe, and g) flow
   cytometry analysis of 4T1 cells staining with fluorescein‐annexin
   V‐FITC and PI after indicated treatments under hypoxic conditions.
   NPs‐Ce6 concentration: 400 µg mL^−1, PCC concentration: 5 × 10^7 cell
   mL^−1 1.2 W cm^−2, 5 min. Data are presented as mean values ± SD.
   Statistical differences in p values were determined by One‐way ANOVA.
   ^**** p < 0.0001.

   Calcein acetoxymethyl ester (Calcein‐AM) and propidium iodide (PI)
   co‐staining assay were utilized for evaluating the live and death cells
   of SDT effect (Figure [120]5d; Figure [121]S15, Supporting
   Information). As expected, the NPs‐Ce6 + US group showed partial red
   fluorescence, whereas the NPs‐Ce6 + PCC + US group exhibited strong red
   fluorescence, aligning with the CCK‐8 assay results. Additionally,
   dichlorofluorescein diacetate (DCFH‐DA) was utilized to assess ROS
   production. Flow cytometry analysis and CLSM images of 4T1 tumor cells
   stained with DCFH‐DA revealed significant green fluorescence
   (Figure [122]5e; Figures [123]S16,  [124]S17, Supporting Information),
   indicating that the sonosynthetic oxygenation boosted ROS generation.
   ROS production is known to damage mitochondria, as evidenced by the
   decrease in mitochondrial membrane potential observed through the
   transition of potential‐sensitive dye JC‐1 from red to green
   fluorescence. Finally, apoptosis in 4T1 cancer cells was investigated
   using flow cytometry analysis with fluorescein isothiocyanate
   (FITC)‐labeled Annexin V and PI staining method (Figure [125]5f).
   Compared to the NPs‐Ce6 + US group showing a significant late apoptosis
   ratio at 18.6%, the addition of PCC led to a higher rate of apoptosis
   (late apoptosis ratio at 41.3%), confirming the enhanced SDT
   therapeutic performance.

2.5. In Vitro Therapeutic Mechanism

   NPs‐Ce6 in synergy with PCC had demonstrated remarkable cell‐killing
   efficiency under US radiation. We delved further into the mechanism
   underlying the cell‐killing effect. NPs‐Ce6, leveraging its
   sonoafterglow and sonodynamic properties, could enhance photosynthesis
   in PCC and alleviate tumor hypoxia, thereby augmenting SDT. Under
   hypoxic conditions, the NPs‐Ce6 + PCC + US group exhibited a
   significant decrease in HIF‐1α levels, demonstrating the effectiveness
   of sonosynthetic oxygen production in vitro (Figure [126]6a). On the
   contrary, the NPs‐Ce6 + US group showed a marked increase in HIF‐1α
   levels, likely due to increased intracellular oxygen consumption during
   SDT. To further visualize the oxygen production, the same effect was
   observed using the intracellular hypoxia indicator [Ru(dpp)[3]]Cl[2],
   which operates on an oxygen‐dependent fluorescence‐quenching mechanism.
   The weakest red fluorescence in the NPs‐Ce6 + PCC + US group indicated
   a reduction in hypoxia (Figure [127]6e). Moreover, western blot
   analysis indicated that, compared to other groups, the NPs‐Ce6 + PCC +
   US group led to the down‐regulation of HIF‐1α expression, inhibition of
   amino acid transporter SLC7A11 expression, and the destruction of
   ferritin, ultimately resulting in GPX4 inactivation in cancer cells
   (Figure [128]6b; Figure [129]S18, Supporting Information).

Figure 6.

   Figure 6
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   In vitro therapeutic mechanism. a) The concentration of HIF‐1α after
   different treatments in hypoxic conditions (n = 3). b) Western blot
   assay of HIF‐1α, SLC7A11, Ferritin, and GPX4 level in 4T1 cells treated
   after indicated treatments. c) Relative Cys content (n = 3) and d)
   relative GSH content in 4T1 cells after indicated treatments (n = 3).
   e) CLSM images of 4T1 cells staining with Ru(dpp)[3]Cl[2] after
   indicated treatments. f) Relative MDA content in 4T1 cells after
   indicated treatments (n = 3). g) CLSM images of 4T1 cells staining with
   C11‐BODIPY^581/591 dye after indicated treatments. h) Cell viability of
   4T1 treated with NPs‐Ce6 + PCC+US after addition of different
   concentrations of Fer‐1 (n = 4). i) Schematic illustration showing that
   oxygen generation promotes SDT and ferroptosis (Created with
   BioRender.com, NPs‐Ce6 concentration: 400 µg mL^−1, PCC concentration:
   5 × 10^7 cell mL^−1, 1.2 W cm^−2, 5 min). Data are presented as mean
   values ± SD. Statistical differences of p values were determined by
   One‐way ANOVA. ^*** p < 0.001, ^**** p < 0.0001.

   Additionally, ROS facilitated the destruction of ferritin in cells,
   releasing endogenous iron ions and further depleting GSH, which
   indirectly decreased GPX4 expression. The cytoplasmic iron levels, a
   critical indicator of ferroptosis, were assessed using Calcein (CAL).
   Since iron chelates with CAL to quench its fluorescence, the
   fluorescence intensity inversely correlates with iron content. As
   expected, the signal was the weakest in the NPs‐Ce6 + PCC + US group,
   signifying the most extensive damage to ferritin and the highest iron
   ions release (Figure [131]S19, Supporting Information). Consequently,
   following the decrease in SLC7A11 expression, cellular levels of
   cysteine (Cys) and GSH were further measured (Figure [132]6c,d). Cys
   and GSH levels were significantly lower in the NPs‐Ce6 + PCC + US
   group, confirming that hypoxia relief inhibited SLC7A11 expression,
   thereby reducing Cys transport and GSH synthesis in cells.
   Additionally, malondialdehyde (MDA), a key marker of lipid peroxidation
   (LPO), was quantified to assess LPO levels (Figure [133]6f).
   C11‐BODIPY^581/591, a probe specific to LPO, showed marked red
   fluorescence decay and green fluorescence intensification in the
   NPs‐Ce6 + PCC + US group, indicating increased LPO due to
   oxygenation‐enhanced ROS generation (Figure [134]6g). To validate the
   role of ferroptosis in cancer cell killing, inhibitors like
   deferoxamine (DFO) and ferrostatin‐1 (Fer‐1) were added to the NPs‐Ce6
   + PCC + US group, resulting in a concentration‐dependent increase in
   cell viability (Figure [135]6h; Figure [136]S20, Supporting
   Information). Moreover, Fer‐1 and DFO did not induce significant
   toxicity to the cells, thereby excluding their potential cytotoxic
   effects (Figure [137]S21, Supporting Information). NPs‐Ce6 + PCC + US
   effectively elevated O[2] levels to alleviate the hypoxic environment
   and led to decreased SLC7A11 expression, reduced GSH biosynthesis,
   diminished GPX4 activity, increased intracellular LPO, and ultimately,
   inducing ferroptosis. In summary, hypoxia induced the expression of
   HIF‐1α, which consequently increased SLC7A11 protein expression.
   SLC7A11, a specific amino acid transporter, is crucial in regulating
   ferroptosis. Reducing hypoxia could downregulate SLC7A11 expression,
   thus impeding Cys transport into cells. This reduction led to lower
   intracellular Cys levels and decreased GSH biosynthesis, which
   indirectly inhibited GPX4 activity, resulting in LPO accumulation and
   ultimately inducing ferroptosis. Furthermore, SDT‐mediated ^1O[2]
   generation disrupted intracellular ferritin, releasing ferric iron and
   further depleting GSH (Figure [138]6i). Based on the aforementioned
   data and analysis, it can be concluded that mitigating tumor hypoxia
   not only bolsters the therapeutic efficacy of SDT but also augments
   ferroptosis.

   After confirming the anti‐cancer effect in vitro, RNA sequencing was
   conducted to further elucidate the potential therapeutic mechanism. As
   shown in Figure [139]7a, there were 56981 differentially expressed
   genes (DEGs), with 46328 upregulated (red dots) and 10653 downregulated
   genes (blue dots) in the volcano. These DEGs underwent Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway analysis for deeper
   insights into NPs‐Ce6 + PCC + US on 4T1 tumor cell therapy. This
   treatment led to significant gene enrichment changes related to the
   HIF‐1α signaling pathway, apoptosis, ferroptosis, GSH metabolism, and
   glycolysis (Figure [140]7b). Additionally, gene set enrichment analysis
   (GSEA) of the DEGs, using the Molecular Signature Database (MSigDB),
   revealed enrichment in pathways associated with disrupted amino acid
   biosynthesis and the HIF‐1α signaling pathway, suggesting that the
   treatment resulted in tumor cell death and hypoxia remission,
   potentially via photosynthesis enhanced by sonoafterglow
   (Figure [141]7c). To further explore this mechanism, heat maps of genes
   related to the HIF‐1α signaling pathway, apoptosis, and ferroptosis
   were generated (Figure [142]7d–f), confirming the activation of these
   pathways. Moreover, protein‐protein interaction network analysis of
   differentially expressed genes linked to the HIF‐1α signaling pathway,
   apoptosis, and ferroptosis were conducted (Figure [143]7g).
   Downregulation of HIF‐1α and GPX4, as observed in the differential
   expression analysis, contributed to hypoxia relief and ferroptosis
   induction (Figures [144]S22 and [145]S23, Supporting Information).
   Last, crossover analysis based on KEGG highlighted gene function
   enrichment (Figure [146]7h), confirming changes in the HIF‐1α signaling
   pathway, apoptosis, and ferroptosis, which suggested a vital role of
   NPs‐Ce6 + PCC + US in tumor therapy.

Figure 7.

   Figure 7
   [147]Open in a new tab

   Underlying therapeutic mechanism. a) Volcano plot of identified DEGs
   between the control group and NPs‐Ce6 + PCC + US group, in which red
   dots represent upregulated genes and blue dots represent downregulated
   genes with a fold change ≥ 1.5 (or ≤ −1.5), p < 0.05. b) KEGG pathway
   enrichment analysis of identified DEGs. c) GSEA analysis of identified
   DEGs. Heat map of differential genes associated with d) HIF‐1α, e)
   apoptosis, and f) ferroptosis in 4T1 cells after different treatments.
   g) Protein–protein interaction network of differential genes associated
   with HIF‐1α, ferroptosis, and apoptosis. h) KEGG enrichment chord
   diagram of identified DEGs. NPs‐Ce6 concentration: 400 µg mL^−1, PCC
   concentration: 5 × 10^7 cell mL^−1.

2.6. In Vivo Antitumor Performance

   Based on the substantial induction of ferroptosis and the amelioration
   of hypoxia achieved in vitro with NPs‐Ce6 + PCC‐mediated SDT (500 µg
   mL^−1 NPs‐Ce6 and 1 × 10^8 cells mL^−1 PCC, 100 µL), we investigated
   the antitumor effect in vivo. 4T1 tumor‐bearing mice were randomly
   divided into six groups: G1) PBS control; G2) US only; G3) NPs‐Ce6 +
   PCC (simultaneously injecting NPs‐Ce6 and PCC; i.t. injection of the
   mixture of NPs‐Ce6 (500 µg mL^−1) and PCC (1 × 10^8 cell mL^−1),
   100 µL); G4) NPs‐Ce6 + US; G5) NPs‐Ce6 + PCC + US and G6) NPs‐Ce6 + PCC
   + US 3 mm (coating with 3 mm chicken breast to mimic deep tumor
   therapy) (Figure [148]8a). Notably, Group G2 (US) and Group G3 (NPs‐Ce6
   + PCC) exhibited a rapid tumor growth, similar to the Group G1
   (control). In contrast, Group G4 (NPs‐Ce6 + US) showed a significant
   reduction in tumor progression, with a 41.9% inhibition rate (Figure
   [149]S24, Supporting Information). Remarkably, Group G5 (NPs‐Ce6 + PCC
   + US) and Group G6 (NPs‐Ce6 + PCC + US, 3 mm) demonstrated
   significantly enhanced tumor suppression, with suppression rates of
   92.2% and 86.0%, respectively, which was attributed to ferroptosis and
   hypoxia alleviation‐improved SDT (Figure [150]8b,c; Figure [151]S25,
   Supporting Information). The efficacy in Group G6 highlights the high
   potential of this therapeutic strategy for treating deep‐seated tumors
   based on the high tissue‐penetrating capability of the US. Ex vivo
   analysis of representative tumors confirmed this, with tumor weights
   measured at the end of therapy (Figure [152]8d,e). Biosafety
   assessments indicated no significant body weight changes (Figure
   [153]S26, Supporting Information) in all experimental groups during the
   treatment period. Furthermore, comprehensive assessments of hematology
   parameters and blood biochemical indexes were conducted (Figure
   [154]S27, Supporting Information). The results demonstrate no
   significant differences across the different groups, indicating
   remarkable biocompatibility without noticeable hepatic and renal
   toxicity. Additionally, the histopathological examinations through H&E
   staining of the major organs such as the heart, liver, spleen, lung,
   and kidney suggest neglectable toxicity associated with the treatment
   (Figure [155]S28, Supporting Information).

Figure 8.

   Figure 8
   [156]Open in a new tab

   In vivo evaluation for antitumor therapy. a) Schematic illustration
   showing the process of sonosynthesis‐mediated SDT (Created with
   BioRender.com). b) Individual tumor growth of tumor‐bearing mouse in
   each group G1) PBS control; G2) US only; G3) NPs‐Ce6 + PCC; G4) NPs‐Ce6
   + US; G5) NPs‐Ce6 + PCC + US and G6) NPs‐Ce6 + PCC + US 3 mm. c) Tumor
   growth curve of each group (n = 5). d) Weight and e) digital photo of
   harvested tumors from the 4T1 tumor‐bearing mice after different
   treatments (n = 5). f) H&E staining, TUNEL staining, HIF‐1α
   immunohistochemical staining, and GPX4 immunohistochemical staining
   images of 4T1‐tumor sections from the tumor‐bearing mice different
   treatments. (NPs‐Ce6 concentration: 500 µg mL^−1, PCC concentration: 1
   × 10^8 cell mL^−1, 1.5 W cm^−2, 5 min) Data are presented as mean
   values ± SD. Statistical differences of p values were determined by
   One‐way ANOVA. ^**** p < 0.0001.

   The antitumor effects were further corroborated by hematoxylin and
   eosin (H&E), Ki‐67, TdT‐mediated dUTP‐biotin nick end labeling (TUNEL),
   and cleaved caspase‐3 staining of tumor sections. These analyses
   revealed the highest levels of apoptosis and necrosis, coupled with the
   lowest proliferation, in the tumor tissue from Group G5 (Figure [157]8;
   Figure [158]S25, Supporting Information). Considering hypoxia typically
   triggers HIF‐1α, which transactivates various signaling molecules
   including vascular endothelial growth factor (VEGF) and CD31 to promote
   angiogenesis and tumor growth, immunohistochemical staining was used to
   assess HIF‐1α, VEGF, and CD31 levels. Notably, Groups G5 and G6
   exhibited significantly reduced expression of HIF‐1α, CD31, and VEGF,
   highlighting the reduction of hypoxia in the tumor region
   (Figure [159]8f; Figures [160]S29,  [161]S30, Supporting Information).
   Given that GPX4 reduction is a hallmark event in the ferroptosis
   process, we conducted a thorough investigation of GPX4 expression in
   tumor tissues post‐therapy. Immunohistochemical analysis displayed a
   significant decrease of GPX4 expression in Groups G5 and G6 compared to
   the other groups, further supporting the occurrence of ferroptosis
   within the tumors. These results validated the function of the
   sonosynthesis‐based SDT platform in effectively remodeling the tumor
   hypoxic microenvironment, which could not only enhance the therapeutic
   efficacy of SDT but also trigger ferroptosis in cancer cells.

3. Conclusion

   In summary, this work has provided a proof‐of‐concept on sonosynthesis
   and established a therapeutic strategy that augments SDT by
   sonosynthetic cyanobacteria oxygenation. The engineered NPs‐Ce6,
   featuring inherent sonosensitizer and sonoafterglow properties, enables
   the generation of ^1O[2] and activation of cyanobacteria for
   US‐activated photosynthetic oxygenation, thus augmenting the
   therapeutic effects and outcomes of SDT. Stimulating cyanobacterial
   photosynthesis via US‐activated sonoafterglow overcomes the limitations
   of external light penetration and avoids potential side effects of
   prolonged light exposure. Our findings, both in vivo and in vitro,
   confirm the capability of sonoafterglow to trigger photosynthetic
   oxygenation in cyanobacteria, effectively remodeling the tumor hypoxic
   microenvironment. This desirable effect not only enhances the
   therapeutic efficacy of SDT but also triggers ferroptosis in cancer
   cells. The developed sonosynthesis‐based therapeutic platform
   effectively addresses the twin challenges of hypoxia and light
   penetration by ultrasound‐photo conversion, heralding a new era in
   aerobic precision medicine, particularly for in vivo applications.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [162]ADVS-11-2400251-s001.pdf^ (2.5MB, pdf)

Acknowledgements