X-ray preactivated reversible persistent luminescence enables photodynamic immunotherapy of deep tumors

Domain: biology
Published: 2026-03-21
Editorial outcome: major_revision

Abstract
Persistent luminescence is a promising approach for photodynamic therapy (PDT) in deep-seated tumors,
as it provides sustained light within tissues, eliminating the need for continuous external illumination.
However, the uncontrollability of light within the body complicates precise spatiotemporal regulation. In
this study, we report X-ray preactivated elimusertib-loaded tumor-targeted photodynamic nanoparticles
(ETPNs), featuring reversible “on-off” afterglow properties. The excellent afterglow properties of X-ray-
activated porous NaYF :Er@NaGdF persistent luminescence nanoparticles enable the continuous
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activation of chlorin e6 (Ce6) to generate reactive oxygen species (ROS), leading to DNA damage. The
integration of elimusertib potentiates ROS-induced DNA damage and activates the cGAS-STING
pathway, thereby enhancing immuno-photodynamic therapeutic efficacy. All in vivo experiments were
conducted using female mice. Our findings highlight the potential of ETPNs to advance the therapeutic
landscape for deep-seated tumors, offering a robust and controllable platform for combined immuno-
photodynamic therapy.

Introduction
Persistent luminescence has emerged as a promising approach to overcome the challenges associated with
photodynamic therapy (PDT) for the treatment of deep tumors1-3. Unlike conventional PDT, which
requires a direct excitation light source to activate the photosensitizers4,5, persistent luminescence
materials can store the excitation energy in energy traps and gradually release it over time after the
removal of the external light source6. This grants the availability of light within deep tissues, eliminating
the necessity for continuous direct light delivery to inaccessible tumors7. Although this promising
therapeutic approach for deep tumors has been shown by our previous study8, several challenges still need
to be solved. For instance, the inherent uncontrollability of light within the body9,10, characterized by its
inevitable decay and limited penetration, is a major challenge. Additionally, it is difficult to precisely
control the location and timing of light delivery, which can lead to off-target reactive oxygen species
(ROS) generation and damage the adjacent healthy tissues11. Thus, it is crucial to develop a safe and
biocompatible approach that can improve light stability and regulate light emissions for an effective PDT
in deep tumors.

Pancreatic ductal adenocarcinoma (PDAC) is a deep-seated and notoriously lethal malignancy due to its
late diagnosis, rapid progression, and poor response to conventional treatments such as chemotherapy and
radiation12,13. It is classified as a “cold” tumor due to its immunosuppressive microenvironment, which
makes immune activation especially challenging14-16. Elimusertib, an ataxia telangiectasia and Rad3-
related (ATR) kinase inhibitor, offers a promising therapeutic alternative for PDAC due to its ability to
disrupt the DNA damage response (DDR)17-19, thereby increasing DNA damage and activating the cyclic
GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway to promote immune
activation20. Nevertheless, its application in PDAC encounters several obstacles. For instance, the dense
stroma of PDAC limits elimusertib delivery21, and resistance may arise as cancer cells adapt by activating
alternative DNA repair mechanisms or modifying cell cycle regulation22. Additionally, conventional
treatments such as chemotherapy and radiotherapy often fail to achieve durable responses in pancreatic
cancer. An integrated approach combining PDT with immunotherapy could offer a more effective strategy.
PDT provides localized cytotoxicity that targets tumor cells, while immunotherapy enhances the
antitumor immune response, potentially overcoming the resistance caused by the immunosuppressive
tumor microenvironment (TME).

To address these challenges, we propose persistent luminescence nanoparticles (PLNPs) with “on-off”
afterglow properties to enhance control over light emission for applications such as bioimaging,
environmental sensing, and targeted drug delivery. Achieving effective "on-off" afterglow in PLNPs is
challenging due to the nature of persistent luminescence, where emission typically continues for extended
periods after excitation, necessitating precise control over energy transfer mechanisms to facilitate
reversible transitions23-25. Of note, we specifically focus on X-ray-excited PLNPs, which exhibit long-
lasting afterglow26,27, providing significantly higher excitation efficiency compared to ultraviolet (UV) or
near-infrared (NIR) excitation28. In addition, X-ray excitation offers distinct advantages for large-scale
commercialization: the prevalent accessibility of X-ray sources facilitates effective, large-area irradiation,
unlike infrared lasers, which typically possess limited coverage29,30. This makes X-ray excitation
particularly well-suited for large-scale applications and future drug commercialization.

In this work, we develop elimusertib-loaded tumor-targeted photodynamic nanoparticles (ETPNs) with
reversible “on-off” afterglow properties for the treatment of deep tumors (Fig. 1). ETPNs consist of X-
ray preactivated porous NaYF :Er@NaGdF persistent luminescence nanoparticles (pPLNPs) that serve
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as both a drug delivery carrier for elimusertib and nanotransducers for PDT through chlorin e6 (Ce6)
activation. These pPLNPs were then self-assembled with estrone-targeted pH-responsive photodynamic
polymeric ligands (EPPLs), which provide tumor-targeting via estrone and photodynamic functionality
via Ce6, to form ETPNs. The afterglow properties of X-ray-activated pPLNPs allow for the continuous
activation of Ce6 for ROS generation, leading to DNA damage. Notably, ETPNs can be turned “on” and
“off” for several cycles by decreasing or increasing the pH, respectively. This presents a precise and
minimally invasive tool for surgeons to differentiate tumor tissue from healthy tissue. Furthermore, the
integration of elimusertib with PDT offers a synergistic advantage, as PDT generates ROS to induce
additional DNA damage, further enhancing the efficacy of elimusertib. Elimusertib plays a crucial role in
activating the cGAS-STING pathway via ATR inhibition, which increases DNA damage, resulting in the
release of cytosolic DNA fragments that trigger the cGAS-STING pathway to recruit immune cells and
enhance antitumor immunotherapy. Overall, the excellent reversible pH-dependent assembly and
disassembly process of ETPNs ensures that they can target tumors safely and effectively, enhancing
specificity and surgical accuracy in deep tumor therapy.

Results
Synthesis and characterization of NaYF :Er@NaGdF pPLNPs
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Herein, we developed a class of porous shell structure NaYF :Er@NaGdF as a multipurpose theranostic
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intermediary. These pPLNPs were utilized as drug delivery carriers and as nanotransducers for PDT via
Ce6 activation. The NaYF :Er core was synthesized using a co-precipitation method8,31, followed by the
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formation of a porous NaGdF shell to enhance the persistent luminescence performance and serve as an
4
effective drug carrier. As depicted by high-angle annular dark-field scanning transmission electron
microscopy (HAADF-STEM) and transmission electron microscopy (TEM) images, the
NaYF :Er@NaGdF pPLNPs exhibit excellent dispersion and a uniform shape, with an average diameter
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of ~60 nm in dynamic light scattering (DLS) measurement (Fig. 2a and Supplementary Fig. 1-2). The
core and shell layers of pPLNPs can be distinguished by the Z contrast discrepancy between Y (Z = 39)
and Gd (Z = 64), and the pore on the shell can be observed in the high-resolution STEM image (Fig. 2b).
The porous shell is well-crystallized and adopts the hexagonal phase structure, as evidenced by the atom
configuration of Gd (Fig. 2c). Likewise, X-ray diffraction (XRD) analysis confirms that both the core and
shell possess a hexagonal phase structure (Supplementary Fig. 3). The successful construction of the core-
shell structure is further validated by the high concentration of Y and Gd elements in the core and shell
areas, respectively, as demonstrated by energy dispersive spectrometry (EDS) line scan analysis (Fig. 2d).
The uniform distribution of all elements in the pPLNPs is also indicated by the EDS mapping analysis
(Supplementary Fig. 4).

As shown in Fig. 2e, small-angle X-ray scattering (SAXS) spectra of the pPLNPs show a strong scattering
peak at ~0.15 nm-1 and several small scattering peaks between 0.25 and 0.7 nm-1 stemming from the
nanoparticles and the multilevel ordered substructure of the shell, thereby validating the porous nature of
the pPLNPs. To further explore the porous properties and the specific surface area of the
NaYF :Er@NaGdF pPLNPs, the Brunauer-Emmett-Teller (BET) analysis was employed
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(Supplementary Fig. 5). The BET surface area of the pPLNPs is approximately 59.11 m2/g, and the pore
size distribution obtained using the Barrett-Joyner-Halenda (BJH) model has a narrow peak with an
average value of 7 nm. Cyclohexane was instrumental in the development of the porous shell due to its
low boiling point, with the formation of the porous shell being attributed to the abundance of cyclohexane
bubbles encasing the core. Multiple studies have suggested that gas bubbles may serve as the aggregation
centers32,33. Cyclohexane was primarily employed for dispersing NaYF :Er core PLNPs and
4
was subsequently eliminated from the reaction system prior to the thermal deposition process for shell
production. Initially, the NaGdF monomers form at 100 ºC through initial nucleation, while cyclohexane
4
microbubbles emerge around the OA-capped cores. Next, interfacial energy minimization allows these
nuclei to aggregate around the bubbles and develop into ultra-small nanocrystals34. Finally, the
evaporation of cyclohexane under increased temperature in vacuum leads to the elimination of the bubbles,
leading to the formation of porous NaGdF shells.
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Notably, the porous shell formation on the PLNP core can significantly improve X-ray activated emission
intensity34,35. As shown in Fig. 2g, the emission intensity for NaYF :Er@NaGdF core@shell pPLNPs
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under X-ray irradiation is dramatically higher than NaYF :Er core PLNPs. After the X-ray induces
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photoelectric ionization and energy transfer process, the Er3+ ions doped in the crystals can be excited,
resulting in distinctive emissions at 540 nm and 655 nm27. Furthermore, the NaYF :Er@NaGdF pPLNPs
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also exhibit remarkable X-ray-induced afterglow properties that can last for several hours (Fig. 2h and
Supplementary Movie 1). The system can continuously emit light after the excitation source is removed
due to the stored energy, which is gradually released from traps in the crystal lattice after the initial
excitation by X-rays36. In addition, the pPLNPs exhibited excellent photostability and recyclability under
X-ray irradiation for 30 cycles (Supplementary Fig. 6). Overall, the core is responsible for the persistent
luminescence, while the shell augments emission intensity and provides additional functionalities,
including drug delivery and long-lasting afterglow properties.

Synthesis and characterization of ETPNs
The synthesis of elimusertib-loaded tumor-targeted photodynamic nanoparticles (ETPNs) was modified
from previous work8,37. To synthesize estrone-targeted pH-responsive photodynamic polymeric ligands
(EPPLs), we prepared a template consisting of carboxyl-poly(ethylene glycol)-poly(β-benzyl- -aspartate)
(COOH-PEG-PBLA), in which the primary amines can react with the flanking carboxyl groups through
amidation (Supplementary Fig. 7). The carboxyl group of COOH-PEG-PBLA was then conjugated with
estrone and grafted with Ce6 to obtain ES-PEG-PBLA-Ce6. Finally, the ionizable group of 1-(3-
aminopropyl) imidazole (API) was incorporated for pH sensitivity to the acidic TME, while 3-phenyl-1-
propylamine (PPA) was added as high-affinity anchors for the pPLNPs to promote self-assembly. The
successful synthesis of EPPLs was confirmed with 1H-NMR, 13C-NMR, and MALDI-TOF MS
(Supplementary Fig. 8-13). Moreover, to investigate the effect of PPA:API ratio on polymer structure and
functionality, several batches with varying ratios were synthesized (Supplementary Fig. 14 and
Supplementary Table 1). According to the results, the optimal PPA:API ratio of 0.625 provided the best
balance of stability at pH 7.4 and pH-responsive release. To obtain ETPNs, the porous structure of the
pPLNPs was incorporated with elimusertib, and Pluronic F127, an FDA-approved surfactant, was added
to ensure colloidal stability. The elimusertib-loaded photodynamic nanoparticles (EPNs), tumor-targeted
photodynamic nanoparticles (TPNs), and elimusertib-loaded pH-insensitive tumor-targeted
photodynamic nanoparticles (EITPNs) were fabricated with the same procedure but without estrone
conjugation, elimusertib loading, or imidazole-containing ligands, respectively.

As shown in Fig. 3a and Supplementary Fig. 15, the TEM images revealed that multiple pPLNPs
aggregate to form ETPNs at neutral pH and disaggregate into isolated pPLNPs in an acidic environment.
At pH 7.4, ETPNs are negatively charged (-27 mV) and have a hydrodynamic size of ~180 nm (Fig. 3a,
b). Upon exposure to acidic conditions, which increases imidazole ionization, the surface charge rapidly
shifted from negative to positive (+10 mV and +19 mV for pH 6.5 and 5.5, respectively). This charge
reversal results in an increased electrostatic repulsive force between the pPLNPs inside the ETPNs and
enhances the cellular uptake of the positively charged ETPNs by tumor cells38,39. Ultimately, as the pH
falls to 5.5, the hydrophobic interactions inside the ETPNs weaken in strength compared to the repulsive
forces among ionized unimers, resulting in complete ETPNs dissociation. The disassembly mechanism
significantly increased the photoactivity of Ce6, which breaks down the self-quenched clustered
photosensitizers into dequenched free molecules (Supplementary Fig. 16)40,41. This enables the emitted
light from the pPLNPs upon X-ray irradiation to continuously activate Ce6 photoactivity within the acidic
TME. Of note, the size and Ce6 fluorescence intensity of EITPNs are not influenced by pH variations
(Supplementary Fig. 17). Moreover, ETPNs have no distinct size changes in both PBS and cell culture
medium at 37 ºC for 5 days (Supplementary Fig. 18), which suggests the excellent stability of ETPNs
under physiological conditions. Next, we examined whether the pPLNPs emissions can activate Ce6 under
the acidic TME. As demonstrated in Fig. 3c, the red emission from the pPLNPs at wavelengths ~650 nm
matches with one of the Ce6 absorbance peaks, indicating that pPLNPs can be employed as transducers
for Ce6 activation.

To further investigate the potential biomedical applications of ETPNs, the drug release profile,
photodynamic activity, and afterglow properties were analyzed. According to the HPLC results, ETPNs
have elimusertib and Ce6 loading efficiency of 88% and 86.4%, respectively (Supplementary Fig. 19).
ETPNs are comprised of a pH-responsive moiety that will partially release the encapsulated drugs in
response to the acidic TME and completely release the remaining drugs upon X-ray irradiation (Fig. 3d).
Only 9% of elimusertib was released at pH 7.4 after 72 h, while 35% and 48% of elimusertib were released
at pH 6.5 and 5.5, respectively. This results from ETPNs response to acidic pH, wherein the ionic
interactions between pPLNPs and EPPLs weakened its structure, hence facilitating the release of
elimusertib. However, the structure of the EPPLs on ETPNs is still intact before X-ray irradiation, which
prevents the complete release of elimusertib. Additionally, just 22% of elimusertib was released after X-
ray irradiation at pH 7.4, illustrating the remarkable stability of ETPNs that limit the release of elimusertib
during X-ray irradiation. Ultimately, the X-ray irradiation of ETPNs will yield 75% and 88% of released
elimusertib at pH 6.5 and 5.5, respectively. These results indicate that ETPNs have exceptional pH-
responsive drug release properties and stability, ensuring biosafety in normal tissue.

Subsequently, we used 1,3-diphenylisobenzofuran (DPBF) as the ROS indicator to examine the pH-
dependent ROS generation elicited by Ce6 during X-ray irradiation42. The absorption intensity of DPBF
exhibited a minor reduction during X-ray irradiation at neutral pH and a substantial decrease under acidic
pH conditions (Supplementary Fig. 20). Furthermore, we used DPBF to evaluate ROS production upon
the termination of X-ray irradiation. Likewise, ROS production at pH 7.4 is negligible, whereas pH 6.5
and 5.5 can sustainably produce ROS for over 1.5 h after the cessation of X-ray irradiation (Fig. 3e).
Notably, the plateau observed in DPBF degradation after ~50 minutes at pH 5.5 is due to the rapid initial
release of ROS following X-ray preactivation, driven by fast disassembly of ETPNs and strong Ce6
activation in the highly acidic environment. In contrast, at pH 6.5, the disassembly process is more gradual,
resulting in a sustained yet slower ROS release over time. To further validate this observation, we
employed 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) as an additional 1O probe43. The
2
decrease in ABDA absorbance confirmed effective pH-dependent 1O generation (Supplementary Fig.
2
21), in agreement with the DPBF results. Moreover, we performed electron spin resonance (ESR)
spectroscopy using 2,2,6,6-tetramethylpiperidine (TEMP) as a singlet oxygen (1O ) trapping agent and
2
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a superoxide (·O -) trapping agent to confirm the
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generation and identity of ROS by ETPNs (Supplementary Fig. 22)44. Upon X-ray preactivation of ETPNs
under acidic conditions, a characteristic three-line ESR signal corresponding to the TEMP-1O product
2
(TEMPO) was clearly detected, indicating efficient singlet oxygen generation. In contrast, the ESR signal
for the DMPO-·O - adduct was negligible, suggesting that 1O is the dominant ROS produced by ETPNs.
2 2
Meanwhile, the pH-dependent afterglow emission intensity of ETPNs also demonstrates the lowest
emission intensity at pH 7.4 and the highest emission intensity at pH 5.5 (Fig. 3f and Supplementary Fig.
23). Finally, we evaluated the afterglow emission and ROS generation of ETPNs under different X-ray
doses (Supplementary Fig. 24). Although luminescence intensity increased with X-ray dose, 5 Gy caused
an initial ROS burst followed by rapid depletion, whereas 1 Gy produced minimal ROS. Therefore, we
selected 3 Gy as the optimal X-ray preactivation dose for ETPNs, providing strong persistent
luminescence and stable ROS generation over time.

Switchable “on-off-on” persistent luminescence of ETPNs for precise PDT activation
In this study, NaYF :Er@NaGdF pPLNPs were self-assembled with ionizable EPPLs for pH sensitivity
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to form ETPNs. The excellent persistent luminescence of X-ray-activated pPLNPs can be used as long-
term nanotransducers to continuously activate Ce6 for ROS generation. Notably, the “on-off” afterglow
properties of ETPNs are attributed to their pH-responsive ability (Fig. 4a). The persistent luminescence
lifetime of the nanoparticles is primarily determined by the intrinsic properties of NaYF :Er@NaGdF
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pPLNPs, resulting in an afterglow lifetime of approximately 6 hours, which is largely independent of pH
(Supplementary Fig. 25 and Supplementary Movie 1). However, when incorporated into ETPNs, the
afterglow can vary due to the pH-induced changes in the ETPNs assembly state. At pH 7.4, the ETPNs
are in the quenched (off) state, in which the pPLNPs and Ce6 are in close proximity to enable efficient
resonance energy transfer. Upon X-ray irradiation, the pPLNPs transfer most of their energy directly to
Ce6 instead of emitting visible fluorescence, resulting in limited emission (shutter “off”, “dim” emission)
and ROS generation45. Subsequently, when the hydrophobic interactions within ETPNs diminish below
the repulsive force between ionized unimers, ETPNs will begin to dissociate at pH 6.5 and will further
disintegrate into isolated pPLNPs at pH 5.5. This separation reduces resonance energy transfer due to the
increased distance between the donor and acceptor. Consequently, this dequenched (on) state allows the
restoration of pPLNPs to emit light (shutter “on”, “bright” emission) and Ce6 to efficiently produce ROS,
as energy is no longer being transferred to Ce6 non-radiatively25. The disassembly process significantly
increased the emission intensity of ETPNs, with the pH-dependent afterglow showing the highest intensity
in acidic pH (Fig. 3f). Notably, the intensity difference between the “on” and “off” states was
approximately 10-fold, demonstrating the significant enhancement in emission intensity upon dissociation.
Additionally, DPBF was employed to demonstrate the on-off activity of pH-activated PDT in ETPNs
when the pH fluctuates between 7.4 and 5.5 (Fig. 4b and Supplementary Fig. 26). Initially, the DPBF
absorption intensity exhibited a minor reduction during X-ray irradiation at pH 7.4, while the following
pH adjustment to 5.5 drastically decreased the DPBF absorption intensity. When the pH was returned to
7.4, the DPBF absorption reverted to its minimally reduced state, highlighting the biosafety of pre-
activated ETPNs under physiological conditions.

Moreover, the disassembly mechanism of ETPNs has been further examined through the observation of
spectral alterations. As depicted in Fig. 4c and Supplementary Fig. 27, the absorbance of ETPNs gradually
drops at acidic pH as a result of the rise in transmittance (%T), which enhances the emission from ETPNs
due to reduced luminescence quenching46. Subsequently, the acid-base titration exhibited a hysteretic %T
recovery when the pH was increased once again, thus revealing the reversible pH-dependent assembly
and disassembly process of ETPNs. Next, we evaluated the reversible “on-off” afterglow properties of
ETPNs under acidic and neutral pH fluctuations (Fig. 4d). Owing to ETPNs reversible assembly and
disassembly properties, ETPNs can be turned “on” and “off” for several cycles by decreasing or increasing
the pH, respectively. Due to the electrostatic repulsion that leads to the recovery of pPLNPs photoactivity
at pH 5.5, the emission intensity of ETPNs is significantly higher at acidic pH (3 x 105 p/sec/cm2/sr) than
at neutral pH (4 x 104 p/sec/cm2/sr), with a gradual decrease in radio-afterglow intensity over time as the
afterglow from the pPLNPs slowly decays.

To further validate the “on-off” afterglow properties of ETPNs, we administered X-ray preactivated
EPTNs (120 mg kg-1) on orthotopic PDAC mice models (2 weeks after tumor implantation) and monitored
the radio-afterglow of ETPNs around the tumor under acidic and neutral pH fluctuations via the IVIS
imaging system (Fig. 4e, Supplementary Fig. 28, and Supplementary Movie 2). Likewise, the reversible
radio-afterglow intensity of ETPNs increases with acidic PBS solution injection and decreases with basic
PBS solution injection. ETPNs reversible “on-off” afterglow properties provide a controlled imaging
mechanism, especially in deep-seated tumors, by leveraging the acidic TME to activate luminescence.
This targeted illumination helps surgeons distinguish tumor tissue from healthy tissue, while the “off”
state in normal cells can minimize exposure to the surrounding areas. Furthermore, the pre-activation of
ETPNs with X-ray irradiation allows for clear visualization, which improves surgical precision in
complex tumor environments.

In vitro antitumor properties of ETPNs
Previous studies have reported that estrogen receptor (ER) is overexpressed in over 30% of PDAC and
correlates with tumor aggressiveness and poor prognosis47. Estrone, with its high affinity for ER and good
biocompatibility, is an effective targeting ligand. While ER-positive breast cancer is commonly used in
targeting studies, PDAC was selected due to its greater clinical challenge and acidic microenvironment,
where the pH-responsiveness of ETPNs provides a distinct therapeutic advantage. Therefore, KPC and
BxPC-3 cell lines were selected for subsequent studies because they exhibited higher levels of ER
expression than other PDAC cell lines (Supplementary Fig. 29)48. Confocal laser scanning microscopy
(CLSM) revealed that a significant amount of ETPNs entered the cells and accumulated around the
nucleus 1 h after administration (Fig. 5a). Notably, Ce6 fluorescence from ETPNs was stronger at pH 6.5
after 0.5 h of administration, suggesting that cellular uptake was improved in acidic conditions. This pH-
responsive phenomenon is ascribed to the recovered photoactivity of Ce6, which dissociated from ETPNs
in the presence of an acidic environment. Nevertheless, the fluorescence intensity in acidic and neutral
conditions became similar after 4 h as a result of ETPN degradation. Likewise, the cellular uptake process
of ETPNs was further confirmed with Cryo-TEM imaging (Fig. 5b), which depicts the breakdown of
ETPNs and their aggregation around the cell nucleus upon entering the acidic TME. To better quantify
cellular uptake, we also performed flow cytometry analysis (Supplementary Fig. 30). The results were
consistent with the CLSM observations, showing that the acidic environment significantly enhanced
cellular uptake of ETPNs. However, the difference in uptake attributed to acidity became less pronounced
over extended incubation periods, which also aligns with the trends observed in confocal imaging.

Moreover, to investigate the role of ER receptors in cellular uptake, we compared the uptake efficiency
of ETPNs across PDAC cell lines with varying ER expression levels using flow cytometry
(Supplementary Fig. 31). We observed that cell lines with high ER expression (KPC and BxPC-3)
exhibited greater uptake of ETPNs than those with low ER expression (Pan02 and PANC-1). To further
validate the involvement of ER, we established ER-α/β double-knockdown models in high-ER-expressing
PDAC cells (KPC and BxPC-3) via shRNA and quantified their uptake under acidic conditions. The
results confirmed a significant reduction in cellular uptake of ETPNs following ER knockdown
(Supplementary Fig. 32). This further validates CLSM results, indicating that ETPNs can bind to ER and
undergo endocytosis to interact with tumor cells.

Next, we assessed the cytotoxicity of X-ray preactivated ETPNs (3 Gy). To determine the ideal ETPN
concentration and treatment duration, KPC and BxPC-3 were subjected to different concentrations of
elimusertib or ETPNs over 0-72 h (Supplementary Fig. 33). As a result, a relatively low concentration of
20 nM (based on elimusertib dosage) was chosen to evaluate the antitumor effects of ETPNs after 48 h.
X-ray preactivated ETPNs (20 nM, elimusertib concentration in ETPNs) induce significant anti-
proliferative response in both PDAC cell lines compared to free elimusertib after 48h of treatment (Fig.
5c). This observation was further supported by flow cytometric analysis of apoptosis, which showed that
the X-ray preactivated ETPNs had the highest proportion of late apoptotic cells (Supplementary Fig. 34).
Notably, the half-maximal inhibitory concentration (IC50) of X-ray-preactivated ETPNs on KPC cells
was 16.7 nM (based on elimusertib dosage), which is significantly lower than elimusertib alone (142.4
nM) (Supplementary Fig. 35), with comparable results observed in BxPC-3 cells. Additionally, in the
absence of X-ray irradiation, ETPNs did not exhibit increased cytotoxicity relative to elimusertib,
indicating that the elimusertib released from ETPNs is not the sole mechanism behind the antitumor effect
of X-ray-preactivated ETPNs (Fig. 5c, d). Meanwhile, there was a slight increase in cytotoxicity of X-
ray-preactivated TPNs compared to inactivated Ce6, suggesting that Ce6 may not fully account for the
antitumor efficacy of ETPNs (Fig. 5c, e). These results suggest that the remarkable anti-proliferative
ability of X-ray-preactivated ETPNs arises from the synergistic interaction between elimusertib and Ce6.
Interestingly, X-ray-preactivated ETPNs exhibited pan-cancer antitumor effects across various cell lines,
including colorectal adenocarcinoma cells (MC38), breast adenocarcinoma cells (E0771), cervical
carcinoma cells (HeLa), and gallbladder carcinoma cells (NOZ), indicating potential for broad clinical
application (Fig. 5f and Supplementary Fig. 36).

ETPNs induce damaged DNA accumulation and trigger the cGAS-STING pathway
The synergistic antitumor mechanism between elimusertib and Ce6 in ETPNs was further investigated
(Fig. 6a). Theoretically, elimusertib released by ETPNs could disrupt the DDR by abrogating the G2/M
checkpoint, resulting in the accumulation of unrepaired DNA49,50. However, the functional redundancies
among DDR pathways may mitigate the inhibition of ATR, decreasing the efficacy of elimusertib.
Meanwhile, ROS generated by ETPNs following X-ray irradiation near the nucleus may damage the DNA,
thereby disrupting the compensation for elimusertib-induced DNA damage. Consequently, a reduced
elimusertib concentration, in conjunction with an appropriate amount of ROS to provoke DNA damage,
will yield a potent synergic antitumor effect. As depicted in Fig. 6b and Supplementary Fig. 37, TPNs and
ETPNs generated two-fold ROS under the presence of X-ray irradiation compared to those without.
Consequently, X-ray-preactivated TPNs and ETPNs resulted in a greater accumulation of γ-H2AX, a
marker of DNA breaks, compared to TPNs and ETPNs without X-ray pre-irradiation, respectively (Fig.
6c). In addition, cell cycle assays demonstrated that X-ray-preactivated ETPNs significantly abrogated
G2/M arrest, which is similar to elimusertib monotherapy (Fig. 6d). However, X-ray-preactivated ETPNs
resulted in the highest γ-H2AX expression compared to other treatment, indicating the synergistic effect
of ATR inhibition and ROS (Fig. 6c). Notably, ETPNs without X-ray irradiation also elicit minor G2/M
arrest and γ-H2AX expression, potentially due to the dissociation of elimusertib from ETPNs in the acid
TME (Fig. 6c).

Since ATR inhibitors have been reported to activate cGAS-STING, we further investigated the role of X-
ray-preactivated ETPNs on cGAS-STING activation51. As expected, immunofluorescence analysis
confirmed that X-ray-preactivated ETPNs induced a higher frequency of micronuclei in both KPC and
BxPC-3 cell lines compared to other treatments. Given that micronuclei are known to activate cGAS, a
key cytosolic dsDNA sensor, these results suggest that the enhanced micronucleation resulting from X-
ray-preactivated ETPNs treatment promotes cGAS pathway activation (Fig. 6e and Supplementary Fig.
38). Subsequently, activated cGAS phosphorylated STING by producing cyclic GMP-AMP (cGAMP),
as demonstrated by immunoblotting, which showed that X-ray-preactivated ETPNs significantly induced
the expression of phosphorylated STING (Fig. 6c). Additionally, to illustrate that STING activation
prompts the mobilization of downstream signaling cascades and transcriptional activities, we examined
the activation of the TBK1-IRF3 axis, which regulates the transcription of proinflammatory genes52.
Immunoblot analysis revealed that X-ray-preactivated ETPNs enhance the expression of phosphorylated
TBK1 relative to other treatments (Fig. 6c). NF-κB, another major downstream component of the STING
pathway, was also activated by X-ray-preactivated ETPNs treatment, as evidenced by the highest
expression of phosphorylated p65 in PDAC cells that received X-ray-preactivated ETPNs rather than
other treatments (Fig. 6c). Overall, X-ray-preactivated ETPNs exhibited excellent performance in the
accumulation of DNA damage and the activation of the cGAS-STING pathway in vitro, which indicates
their potential for immune regulation.

ETPNs shape the immune microenvironment and eliminate tumors in vivo
Next, we established an orthotopic PDAC mouse model to verify the antitumor effect of X-ray-
preactivated ETPNs in vivo (Fig. 7a). 6 days after KPC inoculation, mice were randomly assigned to 8
experimental groups receiving the following treatments: (1) PBS control; (2) X-ray-preactivated PBS (3
Gy); (3) elimusertib monotherapy (8 mg kg-1); (4) X-ray-preactivated elimusertib (3 Gy + 8 mg kg-1); (5)
TPNs nanoparticles (112 mg kg-1); (6) X-ray-preactivated TPNs (3 Gy + 112 mg kg-1); (7) ETPNs
composite (120 mg kg-1, equivalent to elimusertib dose); (8) X-ray-preactivated ETPNs (3 Gy + 120 mg
kg-1). Luc-KPC tumor-bearing mice received biweekly treatments via tail vein injection, and tumor
progression was monitored through bioluminescence imaging at days 6, 12, 18, and 24 post-implantation.
As shown in Fig. 7b and Supplementary Fig. 39-40, mice treated with X-ray-preactivated ETPNs
exhibited a significant reduction in tumor-associated bioluminescence signal intensity compared to other
treatment cohorts, indicating robust tumor suppression. In contrast, PBS- and TPNs-treated groups
showed minimal inhibitory effects on tumor proliferation. Notably, non-irradiated ETPNs demonstrated
inferior antitumor activity compared to free elimusertib, likely due to limited drug release from
nanoparticles without X-ray preactivation. Control experiments confirmed that X-ray preradiation alone
(applied to PBS or free elimusertib) had no additional therapeutic impact. These findings were
corroborated by terminal tumor weight measurements and quantitative bioluminescence intensity analysis
(Fig. 7c, d). Histopathological evaluation of excised pancreatic tumors further validated the promising
antitumor effect of X-ray-preactivated ETPNs. H&E staining of tumors from X-ray-preactivated ETPNs-
treated mice displayed marked reductions in tumor burden compared to other treatments, which correlated
with decreased proliferative activity as evidenced by Ki-67 immunohistochemistry (Supplementary Fig.
41). Kaplan-Meier survival analysis revealed that X-ray-preactivated ETPNs significantly prolonged
median survival to 44 days, compared to 30 days for PBS, 31 days for X-ray-preactivated PBS, 37 days
for free elimusertib, 39 days for X-ray-preactivated elimusertib, 28 days for TPNs, 32 days for X-ray-
preactivated TPNs, and 35 days for non-irradiated ETPNs (Supplementary Fig. 42). Notably, elimusertib
works through a synthetic lethality mechanism, selectively inducing cell death in TP53-mutant tumor cells
while sparing normal cells without these mutations. This targeted action results in fewer side effects,
which was reflected in the absence of significant body weight loss in the elimusertib-treated mice. While
PBS-treated mice experienced significant weight loss due to cancer cachexia (Supplementary Fig. 43), no
such loss was observed in the X-ray-preactivated ETPNs cohort, suggesting that the nanoparticles may
improve quality of life in pancreatic cancer patients.

X-ray-preactivated ETPNs demonstrated excellent ability to activate the cGAS-STING pathway in vitro.
Next, we further validated the activation status of the cGAS–STING pathway following ETPNs treatment
in vivo using immunohistochemistry. Although elimusertib monotherapy or X-ray preactivated TPNs
induced DNA damage and partially activated the cGAS-STING pathway, X-ray-preactivated ETPNs
significantly enhanced the downstream phosphorylation of key proteins, including γ-H2AX, p-P65, and
p-IRF3. These findings are consistent with the strongest apoptotic signal indicated by TUNEL staining in
tissues treated with X-ray preactivated ETPNs (Fig. 7e and Supplementary Fig. 44). Given the established
role of the cGAS-STING pathway as a key mediator of antitumor immune responses53, we next
investigated the impact of X-ray-preactivated ETPNs on the tumor immune microenvironment.
Immunofluorescence analysis of excised pancreatic tumors revealed a marked increase in CD3+CD8+ T
cell infiltration in mice treated with X-ray-preactivated ETPNs (Fig. 7f and Supplementary Fig. 45),
indicating reversal of the immunosuppressive microenvironment typically observed in PDAC. To further
characterize the immunomodulatory effects of X-ray-preactivated ETPNs, tumors and tumor-draining
lymph nodes (TDLNs) were harvested for flow cytometry following 3 weeks of treatment. Flow
cytometry confirmed a significant improvement in the immune microenvironment, with CD8+ T cell
levels significantly increasing from 21.3% in the PBS control group to 30.56% in mice treated with non-
irradiated ETPNs. Remarkably, X-ray-preactivated ETPNs further elevated CD8+ T cell infiltration to
42.56%, consistent with immunofluorescence findings (Fig. 7g and Supplementary Fig. 46-47). This
robust enhancement is attributed to the synergistic action of Ce6 and elimusertib in amplifying DNA
damage and augmenting cGAS-STING pathway activation. Furthermore, we also assessed the impact of
treatment on immunosuppressive myeloid-derived suppressor cells (MDSCs, CD11b+Gr1+), which are
negatively regulated by cGAS-STING activation54. The proportion of MDSCs decreased substantially
from 65.86% in the PBS group to 42.13% in the X-ray-preactivated ETPNs group (Supplementary Fig.
48). Additionally, analysis of TDLNs revealed a pronounced expansion of mature dendritic cell
populations, increasing from 9.26% in controls to 28.08% following treatment with X-ray-preactivated
ETPNs (Fig. 7h and Supplementary Fig. 49). Collectively, these results demonstrate that X-ray-
preactivated ETPNs not only stimulate robust CD8+ T cell infiltration but also mitigate
immunosuppressive cell populations and enhance dendritic cell maturation, effectively reprogramming
the tumor immune microenvironment in PDAC.

To further investigate the immunomodulatory effects of ETPNs, we examined IFN-β secreted by tumor
cells in vitro. ELISA results confirmed that ETPNs treatment markedly induced IFN-β production
(Supplementary Fig. 50). Moreover, when co-cultured with tumor cells, bone marrow-derived dendritic
cells (BMDCs) exhibited enhanced maturation, a finding consistent with in vivo flow cytometry results
(Supplementary Fig. 51-52), strongly supporting the role of ETPNs in immune activation. Furthermore,
cGAS knockout abolished both IFN-β induction and BMDC maturation, indicating that the
immunostimulatory effects of ETPNs are primarily mediated through the cGAS pathway (Supplementary
Fig. 53).

In vivo biodistribution and biosafety of ETPNs
To evaluate biodistribution, fluorescence imaging was performed following intravenous injection of
ETPNs and EPNs. ETPNs exhibited extensive distribution throughout the body 0.5 h post-injection,
similar to EPNs (Fig. 7i). However, after 4 h post-injection, ETPNs demonstrated substantial
accumulation in tumors, exhibiting stronger fluorescence intensity than EPNs. After 48 h, the fluorescence
intensity throughout the entire body markedly diminished, indicating that ETPNs were eliminated.
Notably, the observed uniform fluorescence intensity across the body of mice, even without drug
administration, can be attributed to porphyrin-like metabolic products in the blood (e.g. pheophorbide)55,56,
which have absorption properties near 660 nm and overlap with Ce6’s fluorescence. Furthermore, tumors
and vital organs (heart, lung, liver, kidney, and spleen) were removed from mice at 0.5, 1, 4, 12, 24, and
48 h following ETPNs injection for ex-vivo imaging to confirm the biodistribution of ETPNs. The tumors
demonstrated the highest fluorescence intensity 4 h after intravenous injection, consistent with the
findings from the in vivo imaging. ETPNs within the tumors were completely degraded after 24 h, whereas
ETPNs in other sites exhibited a slower degradation rate, persisting over 48 h post-administration
(Supplementary Fig. 54). This extended retention in major organs is typically attributed to organ-specific
uptake and extended circulation times of nanoparticles57, aligning with the behavior observed in the
reticuloendothelial system, as reported by previous studies58.

Finally, we examined the toxicity of X-ray-preactivated ETPNs following 4 weeks of treatment to
determine their biosafety (Supplementary Fig. 55). There were no significant differences in hematological
indicators, liver function, and renal function between the treatment and the control group. Although ER
receptors are expressed in some vital organs (Supplementary Fig. 56), H&E staining of sections from
these organs showed no discernible histological differences between the treated and untreated groups
(Supplementary Fig. 55). Consequently, these findings indicate that X-ray-preactivated ETPNs exhibit
exceptional biosafety, demonstrating no systemic toxicity or detrimental effects on key organs, thereby
highlighting their potential for clinical applications.

Methods
Ethical regulations
All animal handling protocols and experiments were approved by the Guidelines for Care and Use of
Laboratory Animals of Zhejiang University (Protocol No. 29761). According to the Laboratory Animal
Welfare and Ethics Committee of Zhejiang University, the subcutaneous and body tumors of mice must
not exceed 2000 mm3, with a diameter of no more than 20 mm in any dimension. Euthanasia is mandatory
upon attainment of this size. The mouse's maximum tumor size/burden was never exceeded in any of the
animal experiments described in this article.

Materials
All precursors and solvents were procured commercially and utilized without additional purification.
Gd O (99.99%), trifluoroacetic acid (TFA), sodium trifluoroacetate (99.0%), cyclohexane, β-benzyl- -
2 3 L
aspartate (BLA), bis-(trichloromethyl)-carbonate (triphosgene), tetrahydrofuran (THF), hexane, amine-
poly(ethylene glycol)-carboxyl (NH -PEG -COOH), N,N-dimethylformamide (DMF), dichloromethane
2 2K
(DCM), dimethyl sulfoxide (DMSO), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride
(EDC), 4-Dimethylaminopyridine (DMAP), estrone, Chlorin e6 (Ce6), N-hydroxysuccinimide (NHS), 3-
phenyl-1-propylamine (PPA), 1-(3-aminopropyl) imidazole (API), 1,3-diphenylisobenzofuran (DPBF),
and acetonitrile were purchased from Aladdin Co. (China). Pluronic F-127 was purchased from Macklin
Co. (China). Ethanol, diethyl ether, and hydrochloric acid (HCl) were purchased from Sinopharm Co.
(China). YCl ∙6H O (99.99%), ErCl ∙6H O (99.99%), sodium hydroxide, ammonium fluoride, oleic acid
3 2 3 2
(OA), 1-octadecene (ODE), methanol, and 9,10-anthracenediyl-bis(methylene)-dimalonic acid (ABDA)
were purchased from Sigma-Aldrich Co. (USA). Elimusertib (BAY1895344) was purchased from MCE
Co. (USA).

Gd(CF COO) precursor synthesis
3 3
The synthesis of Gd(CF COO) was modified from previous work37. Gd O (3.625 g, 10.0 mmol) was
3 3 2 3
dissolved in 50% aqueous trifluoroacetic acid (TFA) (30 mL) at 70 ºC and stirred overnight into a
homogenous solution. The excess water and TFA were slowly evaporated at 70 ºC under vacuum and
stored in a desiccator.

NaYF :Er core PLNPs synthesis
4
The synthesis of NaYF :Er core PLNPs was modified from previous work8,31. YCl ∙6H O (297.3 mg, 0.98
4 3 2
mmol) and ErCl ∙6H O (7.6 mg, 0.02 mmol) were added into a three-neck flask containing oleic acid (OA)
3 2
(6 mL) and 1-octadecene (ODE) (15 mL). The mixture was then heated to 160 ºC for 30 min under argon
gas to remove water and oxygen. Once a clear solution formed, reduce the temperature to 50 ºC and
gradually add 10 mL of a methanol solution with sodium hydroxide (100.0 mg, 2.50 mmol) and
ammonium fluoride (148.2 mg, 4.00 mmol) to the reaction mixture and stir for 30 min under argon gas.
Next, heat the solution to 100 ºC for 30 min, alternating between cycles of argon aeration and vacuum to
remove the methanol, then to 300 ºC for 60 min under argon gas. Finally, the mixture was cooled to room
temperature, precipitated and washed with ethanol three times, and dispersed in cyclohexane.

NaYF :Er@NaGdF pPLNPs synthesis
4 4
The synthesis of NaYF :Er@NaGdF pPLNPs was modified from previous work34. Gd(CF COO) (248.2
4 4 3 3
mg, 0.5 mmol) and sodium trifluoroacetate (136.0 mg, 1.0 mmol) were added into a three-neck flask
containing oleic acid (OA) (6 mL) and 1-octadecene (ODE) (15 mL). The mixture was stirred at room
temperature for 10 min before NaYF :Er (0.5 mmol) in cyclohexane dispersion was added. Next, the
4
solution was heated to 100 ºC for 30 min with alternate cycles of argon aeration and vacuum to remove
cyclohexane, then to 280 ºC for 60 min under argon gas. Finally, the mixture was cooled to room
temperature, precipitated and washed with ethanol three times, and dispersed in cyclohexane.

β-Benzyl-ʟ-aspartate N-carboxy anhydride synthesis
The synthesis of β-Benzyl-ʟ-aspartate N-carboxy anhydride was carried out following our previous
procedure41,70,71. β-benzyl- -aspartate (BLA) (10.0g, 44.8 mmol) and triphosgene (10.0g, 33.7 mmol) were
dissolved in anhydrous tetrahydrofuran (THF) (200mL) and stirred at 60 ºC for 3 h under argon gas.
The mixture was then allowed to cool to room temperature and filtered. The product was purified by
precipitation with cold hexane (500 mL) three times, followed by filtration and drying under vacuum at
room temperature.

EPPL synthesis
Estrone-targeted pH-responsive photodynamic polymeric ligand (EPPL) was synthesized using carboxyl-
poly(ethylene glycol)-poly(β-benzyl- -aspartate) (COOH-PEG-PBLA) as a template (Supplementary Fig.
7). COOH-PEG-PBLA was prepared by polymerizing BLA-NCA (3.0 g, 12.0 mmol) in a mixture of N,N-
dimethylformamide (DMF) (20 mL) and dichloromethane (DCM) (50 mL) with the primary amino group
of amine-poly(ethylene glycol)-carboxyl (NH -PEG -COOH) (molecular weight = 2000 Da, 0.24 g, 0.12
2 2K
mmol) at 40 ºC for 48 h under argon gas. Next, COOH-PEG-PBLA was purified through precipitation
with excess diethyl ether three times. ES-PEG-PBLA was prepared by dissolving COOH-PEG-PBLA
(1.0 g, 57.21 µmol), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) (13.63 mg,
71.10 µmol), and 4-Dimethylaminopyridine (DMAP) (8.18 mg, 66.96 µmol) in dimethyl sulfoxide
(DMSO) (10 mL) and stirred for 3 h at room temperature. Estrone (19.22 mg, 71.09 mmol) was then
dissolved in DMSO (2 mL), added dropwise to the solution, and stirred for 72 h at room temperature.
Finally, the solution was dialyzed against deionized water (MWCSO: 1000 Da) and lyophilized to obtain
ES-PEG-PBLA. ES-PEG-PBLA-Ce6 was prepared by attaching Chlorin e6 (Ce6) to the amine groups of
ES-PEG-PBLA via the carbodiimide reaction. ES-PEG-PBLA (0.8 g, 72.91 µmol) and a mixture of Ce6
(47.85 mg, 80.20 µmol), EDC (33.96 mg, 218.72 µmol), and N-hydroxysuccinimide (NHS) (41.95 mg,
364.54 µmol) were separately dissolved in dimethyl sulfoxide (DMSO) (10 mL) and stirred for 3 h at
room temperature before the condensation reaction. Next, both solutions were mixed and stirred for 24 h
at room temperature under argon gas. The solution was filtered and dialyzed against deionized water for
48 h (MWCO: 1000 Da) to remove insoluble by-products and lyophilized to obtain ES-PEG-PBLA-Ce6.
Finally, platform ligand aminolysis was employed to prepare ES-PEG-p(API/PPA)-Ce6 using 1-(3-
aminopropyl) imidazole (API) and 3-phenyl-1-propylamine (PPA). ES-PEG-PBLA-Ce6 (0.8 g, 74.99
µmol) was dissolved in DMSO (10 mL) and PPA (190.3 mg, 1.41 mmol) was added and stirred at room
temperature under argon gas. After 2 h, API (704.7 mg, 5.63 mmol) was added and continued stirring at
room temperature for 12 h under argon gas. Finally, the reaction mixture was then added dropwise to
cool hydrochloric acid (HCl) solution (0.1 M, 20 mL), dialyzed against 0.01 M HCl aqueous solution
(MWCO: 1000 Da), and lyophilized to obtain ES-PEG-p(API/PPA)-Ce6.

ETPNs synthesis
ES-PEG-p(API/PPA)-Ce6 (20 mg, 1.6 µmol) and elimusertib (4 mg) were dissolved in deionized water
(10 mL and 2 mL, respectively). Next, NaYF :Er@NaGdF pPLNPs (200 µL, 10 mg/mL) was slowly
4 4
added into the solution under an ultrasonic condition to induce PLNPs encapsulation and micelle
formation. The reaction mixture was then subjected to a probe sonicator for 10 min before stirring for 1 h
at room temperature and cyclohexane removal via rotary evaporation at 60 ºC. Pluronic F-127 (8 mg) was
added, followed by a pH adjustment to 7.4 using sodium hydroxide, and continued stirring at room
temperature for 1 h. The excess ligands were removed with a spin filter (Millipore; MWCO: 10 kDa,
10,000 g, 10 min), washed with deionized water three times, and re-dispersed in PBS. The synthesis of
TPNs and EPNs were prepared using the same process, but without the addition of elimusertib or estrone
conjugation, respectively.

Characterization
Transmission electron microscopy (TEM) was taken with Talos L120C (Thermo Fisher Scientific, USA)
at 120kV for morphology characterization. High-angle annular dark-field scanning transmission electron
microscopy (HADFF-STEM) imaging and mapping were conducted on Tecnai G2 F20 S-Twin (FEI,
USA). X-ray diffraction (XRD) patterns were conducted on D8 ADVANCE (Bruker, Swiss) with Cu Kα
irradiation (λ = 1.54056 Å) in the range of 10-70º (2θ). Small-angle X-ray scattering (SAXS) was
conducted on Xeuss Pro (Xenocs, France) (Supplementary Table 3). Nitrogen adsorption-desorption
isotherms were measured using Micromeritics ASAP 2020 Plus HD88 (Micromeritics Instruments, USA).
The Brunauer-Emmett-Teller (BET) method was employed to determine the specific surface area using
adsorption data within the relative pressure (P/P ) range of 0.05 to 0.3. The pore size distribution was
0
obtained utilizing the Barrett-Joyner-Halenda (BJH) method. Zeta potentials and dynamic light scattering
(DLS) were conducted on Zetasizer Nano ZS90 (Malvern Instruments, UK). 1H-NMR analysis was
conducted on Ascend 600 (Bruker, Swiss) using DMSO-d as the solvent. MALDI-TOF MS analysis was
6
conducted on UltrafleXtreme MALDI−TOF/TOF MS (Bruker Daltonic, Germany). High-performance
liquid chromatography (HPLC) analysis was conducted on Agilent 1200 (Marshall Scientific, USA).
Absorption spectrum was conducted on Shimadzu UV-1900i (Shimadzu, Japan). A fiber-coupled
fluorescence spectrometer (Ocean Optics QE PRO), an X-ray tube (Mini-X2, Amptek Inc, USA), and a
quartz mold were constructed for the measurement of radioluminescence spectrum and intensity. A color
complementary metal oxide semiconductor (CMOS) ASI224MC camera (ZWO, China) was used to
capture color images and videos.

ETPNs transmittance analysis at different pH
The light-transmittance of ETPNs solutions (2 mg/mL) was measured using a UV-vis spectrophotometer
at 500 nm. The pH of the solution was adjusted from 7.5 to 4.5 by adding HCl (0.1 M).

Fluorescence intensity analysis at different pH
The fluorescence intensity of ETPNs or EITPNs solutions (2 mg/mL) was measured using a fluorescence
spectrophotometer with 400 nm excitation. The pH was adjusted from 7.4 to 5.5 by adding HCl (0.1 M).

HPLC analysis
HPLC analysis was conducted on Agilent 1200 (Marshall Scientific, USA). The separation was performed
using Zorbax SB-C18 5 µm column (250 mm x 4.6 mm) under the following gradient. Solvent A:
acetonitrile. Solvent B: 0.1% trifluoroacetic acid. For elimusertib, the gradient was mixed from solvent A
and B: 0-25 min, 10% A; 25-31 min, 95% A; 31-40 min, 10% A. For Ce6, the gradient was mixed from
solvent A and B: 0-25 min, 60% A. The volume injected was 20.0 µL at a flow rate of 1.0 mL/min.

In vitro drug release
3 mL of fresh PBS was placed into an MWCO 1 kDa dialysis tube (Spectra/Por, USA). The dialysis tube
was then immersed into ETPNs PBS solutions (1 mg, 10 mL) at different pH values, and stirred at 37 ºC,
100 rpm. At designated time intervals, 3 mL of PBS was removed from the dialysis tube and replaced
with fresh PBS. For X-ray irradiated samples, the samples were exposed to a 3 Gy dose of X-ray. All
experiments were conducted in triplicate. The drug loading efficiency (DLE, %) was obtained according
to the following equation:
𝐷𝑟𝑢𝑔𝑠 𝑖𝑛 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 (𝑤𝑡)
Drug Loading Efficiency (DLE) % = ( )𝑥100%
𝐷𝑟𝑢𝑔𝑠 𝑎𝑑𝑑𝑒𝑑 (𝑤𝑡)

Singlet oxygen generation analysis via DPBF
For pH-dependent DPBF absorbance, DPBF (20 μL, 8 mM) was mixed with acetonitrile and added to
ETPNs solutions (2 mg/mL, 3 mL) at various pH levels. The solution was irradiated with X-ray (3 Gy) at
specified time intervals. For time course 1O generation, DPBF (20 μL, 8 mM) was mixed with acetonitrile
2
and added to ETPNs solutions (2 mg/mL, 3 mL), followed by X-ray irradiation (3 Gy). Absorption spectra
were measured using a UV-vis spectrophotometer. To ensure data reliability, freshly opened DPBF
reagent was used for each experiment. All experiments were performed in triplicate.

Singlet oxygen generation analysis via ABDA
For pH-dependent ABDA absorbance, ABDA (50 μL, 2.5 mM) was dissolved in deionized water and
added to ETPNs solutions (1 mg/mL, 2 mL) at various pH levels. The solution was irradiated with X-ray
(3 Gy) at specified time intervals. Absorption spectra were measured using a UV-vis spectrophotometer.
To ensure data reliability, freshly opened ABDA reagent was used for each experiment. All experiments
were performed in triplicate.

Singlet oxygen detection via ESR
ESR spectra were used to detect ROS generation from ETPNs. Briefly, ETPNs solutions (150 μg/mL, 200
μL) were mixed with 2,2,6,6-tetramethylpiperidine (TEMP) or 5,5-dimethyl-1-pyrroline-N-oxide
(DMPO), followed by X-ray irradiation (3 Gy). The reaction mixture was then injected into a quartz
capillary for ESR analysis.

Cell culture
Human pancreatic ductal adenocarcinoma (PDAC) cell line BxPC-3 (Cat. #SCSP-529), murine colon
adenocarcinoma cell line MC38 (Cat. #SCSP-5431), and human cervical carcinoma cell line HeLa (Cat.
#SCSP-504) were originally obtained from the Shanghai Institute for Biological Sciences, Chinese
Academy of Sciences (China). Murine PDAC cell line KPC was generated from a KPC [C57BL/6Smoc-
Trp53em4(R172H)Krasem4(LSL-G12D)Tg(Pdx1-cre)Smoc] mouse (Shanghai Model Organisms Center, Cat.
#NM-KI-210096). Murine mammary cancer cell line E0771 and human gallbladder carcinoma cell line
NOZ were provided by Dr. Jin HC (Zhejiang University). All cell lines used in this study were
authenticated by STR profiling. KPC, NOZ, and HeLa were cultured in Dulbecco's Modified Eagle
Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at
37°C in 5% CO . BxPC-3, MC38, and E0771 were cultured in Roswell Park Memorial Institute (RPMI)
2
1640 supplemented with 10% FBS and 1% penicillin/streptomycin under the same conditions.

Cellular uptake
KPC (5 x 104/mL) and BxPC-3 (5 x 104/mL) cells were seeded in 500 µL of DMEM or RPMI 1640
medium in 24-well cell culture plates and incubated overnight. Next, the ETPNs (20 nM, elimusertib
concentration in ETPNs) were added to the plates, and the culture medium was replaced with fresh
medium adjusted to the corresponding pH. After incubation for 0.5 h, 1 h, and 4 h, the cells were washed
three times with PBS. The samples were fixed with 4% paraformaldehyde for 10 min and subsequently
incubated with DAPI (0.01%) for another 10 min. The images were collected using a confocal laser
scanning microscope (Zeiss LSM 880, Germany). BV650 was selected for the quantitative assessment of
nanomaterial uptake via flow cytometry.

Cryo-TEM cell imaging
KPC and BxPC-3 cells were seeded in 6 cm Petri dishes at 1.5 x 106/mL per well until they adhered,
exposed to ETPNs (8.2 μg/mL), and incubated for 0.5 h and 4 h. The cells were then fixed with 2.5%
glutaraldehyde in PBS (0.1 mol/L, pH 7.0) for 12 h, washed three times with PBS, and post-fixed with 1%
OsO in PBS for 1 h, followed by three washes with PBS. The samples were dehydrated in ethanol at
4
concentrations of 30%, 50%, 70%, and 80%, followed by acetone at 90%, 95%, and 100%, with each step
lasting 15 min. Next, the samples were immersed in a 1:1 acetone-Spurr resin mixture for 1 h, then in a
1:3 mixture for 3 h. The specimens were then transferred to a final Spurr resin mixture and incubated
overnight. The samples were polymerized in Spurr resin at 70°C for 9 h. Finally, the specimens were
sectioned into ultrathin slices using a LEICA EM UC7 and stained with uranyl acetate and alkaline lead
citrate for 10 min. The morphology of the cells was observed using a TEM Talos L120C (Thermo Fisher
Scientific) at 120 kV.

Cell counting kit-8 (CCK-8) assay
The in vitro cytotoxicity of the nanoparticles was evaluated using the CCK-8 assay (Yeasen, China),
following the manufacturer’s instructions. KPC and BxPC-3 cells were seeded into 96-well plates at a
density of 0.6 x 104 per well until adherence, and then treated with elimusertib (2.00 – 632.46 nmol/L) or
ETPNs (2.00 – 632.46 nmol/L, elimusertib concentration in ETPNs) at varying concentrations for 12 h.
After an additional 36 h, the absorbance was measured with a microplate reader (Thermo Fisher Scientific)
at 450 nm. All experiments were performed in triplicate.

Cell damage caused by reactive oxygen species (ROS) production from Ce6 in nanoparticles under X-ray
irradiation was assessed similarly. KPC and BxPC-3 cells were seeded in 96-well plates at 0.6 x 104 per
well until adherence and then treated with different concentrations (0.36 – 113.84 µg/mL, Ce6
concentration in TPNs) of TPNs or X-ray-preactivated TPNs (3 Gy). After 12 h, the cells were transferred
to fresh media. After incubating for an additional 36 h, viability was measured with a microplate reader
at 450 nm, comparing treated cells to control unirradiated cells. All experiments were conducted in
triplicate.

The impact of nanoparticles on PDAC cell proliferation was assessed using the CCK-8 assay. KPC and
BxPC-3 cells were seeded in 96-well plates at a density of 0.6 x 104 cells per well and allowed to adhere.
The cells were then treated with different media for 12 h. Each cell line was divided into eight groups: (1)
NC (normal media), (2) NC with X-ray irradiation, (3) elimusertib (20 nM), (4) elimusertib with X-ray
irradiation, (5) TPNs (same nanoparticle concentration as group 7), (6) TPNs with X-ray irradiation, (7)
ETPNs (20 nM, elimusertib concentration in ETPNs), and (8) ETPNs with X-ray irradiation. The
treatment in groups 2, 4, 6, and 8 was irradiated with X-ray (3 Gy) before administration. After 12 h, the
cells were transferred to fresh media and then incubated at 37°C for an additional 36 h before measuring
viability at 450 nm relative to the cells in the control group. All experiments were performed in triplicate.

The effects of nanoparticles on the proliferation of other cancer cell lines were assessed using the same
methods as described above. Each cancer cell line was divided into eight groups: (1) NC (normal media),
(2) NC with X-ray irradiation, (3) elimusertib (20 nM), (4) elimusertib with X-ray irradiation, (5) TPNs
(same nanoparticle concentration as group 7), (6) TPNs with X-ray irradiation, (7) ETPNs (20 nM,
elimusertib concentration in ETPNs), and (8) ETPNs with X-ray irradiation. The treatment in groups 2,
4, 6, and 8 was irradiated by X-ray (3 Gy) before administration.

Immunofluorescence
To detect the activation of the cGAS-STING pathway by ETPNs, we measured the cGAS expression.
KPC and BxPC-3 cells were cultured in confocal dishes, divided into eight groups, and treated as
described previously. 4 h after treatment, the cells were stained with anti-dsDNA (Santa Cruz
Biotechnology, sc-58749) primary antibody and FITC secondary antibody (Proteintech, SA00013-2),
and visualized using a confocal laser scanning microscope (Zeiss LSM 880, Germany).

Western blotting
To validate the activation of the cGAS-STING signaling pathway, KPC and BxPC-3 cells were seeded in
6-well plates at a density of 4 x 105 per well until adherence and treated with different media. Each cell
line was divided into eight groups and treated as described previously. After 48 h, proteins were extracted
from each group, and western blotting was performed according to standard methods. Anti-pSTING (Cat.
#72971S and Cat. #19781T), anti-P65 (Cat. #8242T), anti-pP65 (Cat. #3033T), γ-H2AX (Cat. #2577S),
and anti-beta actin (Cat. #4970T) were obtained from Cell Signaling Technology (USA). Anti-TBK1 (Cat.
#DF7026), anti-pTBK1 (Cat. #AF8190), and anti-STING (Cat. #DF12090) were obtained from
Affinity Biosciences (China). Anti-Vinculin (Cat. #26520-1-AP) were obtained from Proteintech (China).

Cell cycle assays
Cell cycle distribution was analyzed using a cell cycle staining kit (MultiSciences, China) following the
manufacturer’s instructions. KPC and BxPC-3 cells were seeded in 24-well plates at a density of 1 x 105
cells per well and allowed to adhere. The cells were then replaced with various media and incubated for
12 h. Each cell line was divided into eight groups and treated as described previously for 48 h. All cells
in each group were digested with EDTA-free trypsin and resuspended in 1 mL of DNA staining solution.
After incubation in the dark with 10 μL of permeabilization solution for 30 min at room temperature, data
acquisition was performed using a flow cytometer (BD LSR FortessaTM, USA).

ROS assays
KPC and BxPC-3 cells were seeded in 24-well plates at a density of 1 x 105 per well and allowed to adhere.
Afterward, the cells were replaced with various media and incubated for 12 h. Each cell line was divided
into eight groups and treated as described previously. For flow cytometry analysis, adherent cells were
harvested and washed three times with PBS, 4 h after administration. For fluorescence cell imaging, the
cells were also washed three times with PBS, 4 h after administration. Each group was stained in the dark
with 500 μL (25 μmol/L) 2,7-dichlorodihydrofluorescein diacetate (Sigma-Aldrich, USA) for 30 min at
37°C, then washed three times with PBS. Finally, the cells were analyzed using flow cytometry (BD LSR
FortessaTM, USA) and observed via a fluorescence cell imager (Bio-Rad, USA). All experiments were
performed in triplicate.

Animals
All mice were bred and housed under specific pathogen-free conditions in IVC cages at the central animal
facility of the Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University. All mice had ad
libitum access to food and water, under 50% humidity at 22 ºC with normal light/dark cycles. Only female
C57BL/6J mice were used in the experiments, as sex and gender were not considered as variables in the
study design. All animal handling protocols and experiments were approved by the Guidelines for Care
and Use of Laboratory Animals of Zhejiang University (Protocol No. 29761).

Subcutaneous tumor model
C57BL/6J mice (female, 6 weeks old) were anesthetized by isoflurane before receiving a subcutaneous
injection of KPC cells (2 × 105) into the right flank. Experiment was initiated when palpable tumors
formed (average volume ~100 mm3), at which point the mice were randomized into groups (n = 5 mice
per group). Tumor volume was monitored every three days using the formula (L × W2)/2. Mice were
euthanized if the tumor volume exceeded 2000 mm3 or body weight loss was >20%.

Orthotopic tumor model
Luc-KPC cells were dissociated from culture using 0.25% trypsin (Gibco), washed, and resuspended in
fresh culture medium. The left abdominal side of the animal (C57BL/6J mice, female, 6 weeks old) was
shaved to expose the incision site. Animals were anesthetized by isoflurane. A total of 1 x 105 cells was
injected into the tail of the pancreas as a 25 μL mixture of Matrigel (2 mg/mL). Animals were monitored
for 3 days after surgery for recovery, and skin clips were removed 7 – 10 days after surgery (n = 5 mice
per group).

In vivo antitumor treatment
To evaluate the in vivo antitumor efficacy of ETPNs, a bioluminescent orthotopic PDAC model was
established in C57BL/6J mice (female, 6 weeks old). On day 6 post-implantation, tumor engraftment was
confirmed by in vivo bioluminescence imaging. Mice with established tumors were then randomly
assigned to eight treatment groups (n = 5 mice) as follows: (1) PBS; (2) PBS + X-ray (3 Gy); (3)
Elimusertib (8 mg kg-1); (4) Elimusertib (8 mg kg-1) + X-ray (3 Gy); (5) TPNs (112 mg kg-1); (6) TPNs
(112 mg kg-1) + X-ray (3 Gy); (7) ETPNs (120 mg kg-1); (8) ETPNs (120 mg kg-1) + X-ray (3 Gy). The
dosage of ETPNs is within the range of clinically attainable levels, and injections in groups 2, 4, 6, and 8
were irradiated by X-ray (3 Gy) before administration. All formulations were intravenously administered
(200 μL) three times per week, for a total of 6 times. Tumor dimensions and body weights were recorded
every three days. At the end of treatment, mice from the endpoint cohort (n = 5 mice per group) were
euthanized. Tumors and lymph nodes were harvested for subsequent analysis, including: H&E, IHC for
Ki-67/p-IRF3/p-P65/γ-H2AX, TUNEL, IF for CD8+ T cells, and flow cytometry for dendritic cells,
BMDCs and CD8+ T cells.

Afterglow and fluorescence luminescence imaging
Afterglow and fluorescence imaging were conducted utilizing an IVIS Spectrum imaging system
(PerkinElmer IVIS® Lumina LT, USA) in bioluminescence and fluorescence modalities, respectively.
For afterglow imaging, 50 μL ETPNs (120 mg kg-1, the same nanoparticle content as the treatment group)
in a tube were subjected to X-ray irradiation (3 Gy). In vitro afterglow signals were recorded every 120 s
for a duration of 2 h using an open filter. The afterglow spectra were acquired for 60 s using designated
emission filters. The afterglow intensities were quantified by assessing the signal intensity of the region
of interest (ROI) using Living Imaging 4.7.3 Software. For in vivo “on-off-on” afterglow imaging, the
tumor-bearing mice (C57BL/6J mice, female, 6 weeks old, n = 3 mice per group) were anesthetized with
isoflurane and received an intratumoral injection of 150 μL ETPNs pre-irradiated with X-rays (3 Gy).
Subsequently, basic PBS (pH 8.0) and acidic PBS (pH 5.0) was intratumorally injected to induce local
pH changes. In vivo afterglow signals were immediately acquired after each injection for 60 s using an
open filter. The afterglow intensities were quantified by assessing the signal intensity of the ROI using
Living Imaging 4.7.3 Software. For fluorescence imaging, the tumor-bearing mice were administered
distinct intravenous injections of EPNs and ETPNs (10 days after KPC implantation). Fluorescence
images (λ = 430 nm; λ = 650 nm) of the mice were obtained using the NIR imaging system at pre-
ex em
injection and 0.5, 1, 4, 12, 24, and 48 h after injection. Likewise, ETPNs were administered via
intravenous injections individually for tumor-bearing model mice. The mice (C57BL/6J mice, female, 6
weeks old, n = 3 mice per group) were subsequently sacrificed at different time intervals (0.5, 1, 4, 12,
24, 48 h) following intravenous injection, and the heart, liver, spleen, lung, kidney, and tumor were
excised to observe the biodistribution of nanoparticles through imaging.

ER-α and ER-β shRNA knockdown
Lentiviral particles expressing small hairpin RNAs (shRNAs) targeting ER-α or ER-β were obtained from
Santa Cruz Biotechnology. The following constructs were used: two distinct shRNAs against ERα (sc-
29305-V and sc-29306-V) and two against ERβ (sc-35325-V and sc-35326-V). A non-targeting scramble
shRNA (shRNA-NC) was used as a negative control. Pancreatic ductal adenocarcinoma (PDAC) cells
were transfected in accordance with the manufacturer’s protocol.

cGAS CRISPR/Cas9 knockout
cGAS CRISPR/Cas9 plasmid (sc-437363, Santa Cruz Biotechnology) was used to generate knockout
KPC cell lines. Lentiviruses carrying the sgRNA and Cas9 were generated in HEK293T with the
packaging plasmid psPAX2 and the envelope plasmid pMD2.G. Following transduction, cells were
selected using antibiotics for several days. Single-cell clones were subsequently isolated and validated to
establish the CRISPR-Cas9-mediated knockout cell lines.

Biosafety assessment
Normal C57BL/6J mice (female, 6 weeks old) were randomly divided into 2 groups (n = 3 mice per group).
Healthy mice in the experimental group were intravenously injected 3 times a week, for a total of 12
injections, with ETPNs (120 mg kg-1, irradiated with X-ray (3 Gy) before injection). Healthy mice in the
control group were intravenously injected with PBS. After four weeks of treatment, the mice were
sacrificed, and major organs (heart, liver, spleen, lung, and kidney) were collected for H&E staining.
For blood analysis, blood samples (600 μL) were collected from the mice to perform serum biochemistry
assays and a complete blood count. White blood cells (WBC), red blood cells (RBC), mean corpuscular
volume (MCV), platelets (PLT), mean corpuscular hemoglobin (MCH), and hemoglobin concentration
(MCHC) were measured. Blood biochemical examination parameters, including alanine aminotransferase
(ALT), aspartate aminotransferase (AST), albumin (ALB), and total bilirubin (TBIL), were analyzed.
Kidney function was assessed by measuring creatinine (CR), uric acid (UA), and blood urea nitrogen
(BUN).

Statistical analysis
Statistical analyses were conducted utilizing GraphPad Prism 9, ImageJ v1.54f, Python 3.12.11, and
OriginPro 9.5.1. Data are expressed as means ± standard deviation (SD). The Welch's t-test was employed
to analyze the disparities between the two groups. P < 0.05 is considered statistically significant. All P-
values were calculated using a two-tailed approach.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this work, the authors developed a nanoplatform, which could be preactivated by X-ray to show pH-dependent persistent luminescence for continuous activation of Ce6 to generate ROS for photodynamic therapy without light irradiation. This work solved the penetration depth issue in traditional photodynamic therapy, and therefore is valuable to be published. Following are some special suggestions.\n1. The alkaline of imidazole groups in the side chains of ES-PEG-p(API/PPA)-Ce6 is the key for pH response. Therefore, the ratio between API and PPA should be the key. Has the feed ratio been optimized? Any differences between feed ratio and actual ratio? Here needs further investigation.\n2. What is the persistent luminescence lifetime of the nanoparticles? Is it different under different pH values?\n3. In Figure 3e, it seems that the degradation of DPBF was inhibited after 50 min at the pH of 5.5. Why? Does this mean the amount ROS generated by such method is limited? Would it affect treatment efficacy? In addition, from Supplementary Figure 18, the peak shape of DPBF changes over time. Why?\n4. Will the intensity of X-ray irradiation affect the persistent luminescence, and subsequent ROS generation? Since the nanoparticles is preactivated, to some extent, should higher power density of X-ray be better?\n5. In Figure 5a, there are some red fluorescence from ETPNs were not surround the blue one from DAPI. It is very strange, please give some explanation.\n6. Figure 7b is too small. It was suggested to show only one example in main text, and put the other ones into supporting information. A quantitative intensity is also suggested.", "reviewer_2": "In this manuscript, the authors developed an X-ray preactivated elimusertib-loaded photodynamic nanoparticles (ETPNs) with reversible \"on-off\" persistent luminescence for precise photodynamic-immunotherapy of deep tumors. ETPNs integrate porous NaYF4:Er@NaGdF4 nanoparticles (PLNPs) with pH-responsive polymeric ligands, enabling pH-dependent assembly-disassembly to regulate afterglow emission and ROS generation. X-ray pre-activation triggers sustained luminescence, activating chlorin e6 (Ce6) for ROS-induced DNA damage, while elimusertib inhibits ATR kinase to amplify DNA damage and activate the cGAS-STING pathway. In orthotopic pancreatic cancer models, ETPNs demonstrated pH-sensitive tumor targeting, suppressed tumor growth, and reprogrammed the immunosuppressive microenvironment via enhanced CD8+ T cell infiltration. However, some issues remain unexplored in terms of material design and related characterizations, I would like to suggest the authors to address the following concerns before considering publication in Nature Communications.\n1. In the Introduction section, the authors mentioned that “in the treatment of PDAC, where conventional treatments frequently fail to penetrate the dense tumor microenvironment (TME), an integrated PDT and immunotherapy approach could provide an effective strategy to overcome treatment resistance.”. The relationship between cause and effect in this sentence is not clear, and the authors are suggested to enhance the logic of the presentation.\n2. Reported measures to improve the penetration efficiency of PDAC solid tumors include modulation of the extracellular matrix (ECM), and induction of cancer-associated fibroblast (CAF) quiescence, etc. Obviously, ETPNs do not possess the relevant functions, and I am curious about the mechanism of their penetration into the dense stroma. It is necessary to explain this issue in detail.\n3. As shown in Fig. 3a, the authors proposed that ETPNs disaggregate into isolated pPLNPs in acidic TME, leading to a decrease in particle size. However, as shown in Fig. 1, after co-assembly with F127, the ETPNs did not show a clear tendency to disaggregate, and I wonder if this charge repulsion-mediated disaggregation behavior is feasible? The authors are suggested to resubmit the TEM images of ETPNs under different pH values, the current TEM images are too few samples to be informative.\n4. In order to make the drug release experiments more convincing, it is necessary to provide the loading efficiencies of elimusertib as well as Ce6, and the corresponding calculation formulas.\n5. To demonstrate the in vitro 1O2 generation capacity of ETPNs more intuitively, electron spin resonance experiments need to be complemented.\n6. As shown in Fig. 5a, the difference in fluorescence intensity of Ce6 at various pH values was not significant when ETPNs were taken up by cells for 1 h. The authors need to quantitatively analyze the cellular uptake experiments, which is feasible by flow cytometry.\n7. The authors systematically evaluated the in vitro cell killing performance of ETPNs for a wide range of tumor cells, but the data from CCK-8 experiments were not intuitive. It is necessary to supplement the more convincing cell killing experiments with flow cytometry or live-dead cell staining for CLSM as feasible.\n8. As shown in Fig. 6b, the contrast of the ROS-stained background was too low to influence the judgment of readers, and the authors are suggested to resubmit the ROS-stained images.\n9. The authors mentioned that ETPNs have an immune-activating effect, but did not validate this at the in vitro level. Therefore, the authors are suggested to supplement the effect of ETPNs on the maturation of bone marrow-derived dendritic cells or other ways.\n10. As shown in Fig. 7b, the authors showed images of tumor-bearing mice without annotation of the monitoring time, which could be confusing to the readers. Meanwhile, the authors indicated in the figure caption that the number of mice used to assess tumor suppression efficiency was five/group, but the authors only showed 4 of them, which could be confusing for the readers as well. In addition, in the PBS-treated group, I am curious as to why the fluorescence intensity of the tumors appeared to increase and then decrease over time? The authors are invited to explain this phenomenon.\n11. Intravenous administration of chemotherapeutic agents is known to cause systemic toxicity in the body. However, as shown in Supplementary Figure 32, neither the tumor inhibition rate nor the body weight of the mice after administration of elimusertib showed a significant downward trend, and the authors are suggested to explain this.\n12. In order to make the investigation of therapeutic mechanisms more complete, the authors are suggested to supplement the immunofluorescence staining of tumor sections with alterations in markers validated at the cellular level, including but not limited to TUNEL, ROS, cGAS, γ-H2AX, and others.\n13. As shown in Fig. 7h, why did the mice exhibit a uniform distribution of fluorescence intensity throughout the body when the drug was not administered? The authors are invited to explain this phenomenon.\n14. As shown in Supplementary Figure 37, why did the lungs of the mice demonstrate fluorescence intensity even when they were untreated? Please explain this phenomenon. In addition, ETPNs demonstrated liver, lung, and kidney accumulation up to 24 hours after intravenous administration, is there any relevant reported work to support this phenomenon?", "reviewer_3": "In this study, the authors reported the development of a novel photodynamic therapy (PDT) using X-ray preactivated elimusertib (ATR inhibitor)-loaded tumor-targeted photodynamic nanoparticles (ETPNs). By combining reactive oxygen species (ROS) induced by luminescence nanoparticles and inhibition of ROS-induced DNA damage response by ATR inhibitor, this new therapeutic agent exhibits a strong inhibitory effect on pancreatic cancer cells with estrogen receptor expression in vitro and in vivo. Furthermore, this new therapeutic agent triggers activation of DNA sensor cGAS-STING-TBK1/IRF3 pathway and promotes anti-tumor immune responses in vitro and also in vivo. Collectively, these data show that ETPNs may open a new avenue for developing anti-tumor agents by combining immune-photodynamic therapy.\nThe study utilized a multi-disciplinary approach including chemistry, cell biology, and molecular biology to develop and validate this new therapeutic agent. This is a major strength of collaborative teamwork testing hypotheses. The in vivo animal model showed a significant reduction in tumor growth in mice treated by ETPHs. The therapeutic phenotype is convincing. Overall, the hypothesis is supported by the in vitro and in vivo data. The finding of ETPNs as a novel and promising photodynamic therapy approach is significant. Results from this study will have a clear impact on developing new anti-cancer drugs using X-ray preactivated elimusertib (ATR inhibitor)-loaded tumor-targeted photodynamic nanoparticles.\nThere are several major and minor concerns regarding the data interpretation and study design.\n1. ETPN is using ER as a tumor target. ER is present in normal tissues. It is not a tumor-specific receptor. It is not clear why ER is selected, and the pancreatic cancer model is selected, for example, not ER-positive breast cancer is selected. Clear justifications for selecting ER and pancreatic cancer can be helpful.\n2. ETPN is intended to target ER-positive tumor cells. As shown in Supplementary Figure 24, KPC and BxPC-3 cancer cells showed ERα expression. Can ETPN be uptake by ER-negative cancer cells such as Pan-02 and PANC-1? Or can loss of ER (ER knockdown) block ETPN uptake by KPC or BxPC-3? To what extent does the cellular uptake of ETPN rely on ER expression?\n3. In Figure 4e, ETPNs luminescence intensity was analyzed under acidic and neutral pH fluctuations. IVIS imaging was used in orthotopic PDAC after receiving ETPNs, followed by the injection of basic and acidic PBS solution to induce pH changes. The presence of ETPNs was only observed in injected PDAC tumor cells. In Supplementary Figure 37 and Figure 7h, the ETPC fluorescence can be detected in normal tissues and other organs. It is not clear why these signals were not observed in Figure 4e. In addition, in Figure 4e, the highest signals were concentrated in the center of the tumor. As discussed in the introduction, the penetration of nanoparticles in general is challenging. Why is the signal in the center of the tumor stronger than at the peripheral regions of the tumor? ETPNs are intended to target ER-receptor positive cells. Why is the signal not detected in mammary glands or other ER-positive tissues?\n4. ETPNs are intended to work under acidic environments. It is not clear why the strong signals were detected in liver, kidney, heart, and lung in Supplementary Figure 37. What is the PH range in vivo to activate ETPNs? Further analyses showed no obvious toxicity in these normal organs. It is unclear why the strong enrichment of ETPNs in these organs does not cause damage while tumors can be targeted.\n5. In Figure 6b, gamma-H2AX staining looks like pan-nuclear staining instead of foci staining. This staining pattern is normally associated with apoptosis/cell death. In Figure 6d, ETPNs treatment did not induce apoptosis in KPC or BxPC-3 cells. Does ETPN induce cell death? The overall phenotype in Figure 6d does not show a remarkable difference as observed in tumor suppression (Figure 7b, c, and d).\n6. In Figure 6e, cGAS staining was used to detect micronuclei. The quality of the images is poor. For this purpose, PicoGreen staining might be easier to detect micronuclei.\n7. Similar to Figure 6c, the phenotype of activation of cGAS-STING-TBK1 and p-P65 induced by ETPNs compared to ATR inhibitor alone is at relatively same level. It is not clear whether the phenotype can explain the phenotype presented in Figure 7b, c, and d. The downstream type-I interferon signaling can be detected to further show whether the phenotype of ETPNs is stronger than ATR inhibitor alone. Additionally, cGAS or STING knockdown can be used to genetically prove the involvement of this pathway in the therapeutic efficacy of ETPNs.\n8. The figure legends can be further improved by adding key details, e.g. Figure 7, what cells were injected to form tumors; 7b, time points should be labeled; the scale bar of fluorescence for Figure 4e."}, "author_response": "Comments: In this work, the authors developed a nanoplatform, which could be preactivated by\nX-ray to show pH-dependent persistent luminescence for continuous activation of Ce6 to generate\nROS for photodynamic therapy without light irradiation. This work solved the penetration depth\nissue in traditional photodynamic therapy, and therefore is valuable to be published. Following\nare some special suggestions.\nResponse: Thank you very much for your kind comments. We have revised our manuscript based\non your suggestions. We believe that this revision has significantly improved the quality of our\nmanuscript.\nQ1: The alkaline of imidazole groups in the side chains of ES-PEG-p(API/PPA)-Ce6 is the key for\npH response. Therefore, the ratio between API and PPA should be the key. Has the feed ratio been\noptimized? Any differences between feed ratio and actual ratio? Here needs further investigation.\nResponse: Thank you for this insightful question. The reproducibility of polymer synthesis\ndepends on multiple factors, such as raw materials, reaction time, sample addition order,\ntemperature, and so on. To address this, we systematically investigated five feed ratios of PPA (3-\nphenyl-1-propylamine) to API (1-(3-aminopropyl)imidazole) while keeping all other synthesis\nvariables constant, as shown in Table A.\nTable A: Feed ratio and actual mass of ES-PEG-p(API/PPA)-Ce6 with varying PPA and API ratios.\nFeed ratio Actual mass*\nGroup\nPPA API PPA API\nA 0.15 1.35 10.44 86.98\nB 0.3 1.2 20.88 77.32\nC 0.5 1 34.8 64.43\nD 0.75 0.75 52.2 48.32\nE 1 0.5 69.6 32.22\n* per 100 mg PEG-PBLA\nNext, we conducted 1H-NMR analysis on all groups to determine the actual composition\n(Supplementary Fig. 13). By normalizing PEG peaks and integrating characteristic peaks of PPA\n(7.13, 7.21 ppm) and API (9.17 ppm), we confirmed that the actual PPA:API ratio closely\ncorrelates with the feed ratio, validating the robustness of our synthetic strategy (Table B). Notably,\nat higher API feed ratios (groups A, B, C), not all PBLA sites were substituted. This is likely due\nto API’s relatively weaker substitution efficiency within a fixed reaction time.\nSupplementary Fig. 13: 1H-NMR analysis of ES-PEG-p(API/PPA)-Ce6 with varying PPA and\nAPI ratios.\nTable B: Feed ratio and actual ratio of ES-PEG-p(API/PPA)-Ce6 with varying PPA and API ratios.\nFeed ratio Actual degree of polymerization Actual Ratio\nGroup\n(PPA:API) PPA API (PPA:API)\nA 0.11 14 47 0.297\nB 0.25 25 40 0.625\nC 0.5 33 34 0.97\nD 1 70 30 2.333\nE 2 88 18 4.888\nFinally, functional performance testing under different pH conditions showed that Group B (actual\nratio PPA:API ~0.625) achieved the optimal balance, offering excellent stability at pH 7.4 and\nefficient nanoparticle release at acidic tumor pH (Supplementary Table 1). The polymer used in\nthis study had a feed ratio of PPA:API = 0.246, which yielded a zeta potential behavior closely\nmatching that of Group B, thus confirming its suitability for pH-responsive release.\nSupplementary Table 1: Zeta potential of ES-PEG-p(API/PPA)-Ce6 with varying PPA and\nAPI ratios.\nActual ratio Zeta potential (± SD)\nGroup\n(PPA:API) pH 7.4 pH 6.5 pH 5.5\nA 0.297 -5.278 ± 1.465 12.17 ± 1.33 25.07 ± 1.12\nB 0.625 -27.09 ± 1.202 10.25 ± 1.508 19.09 ± 1.04\nC 0.97 -23.06 ± 1.01 -20.7 ± 1.702 -17.8 ± 1.4\nD 2.333 -23.4 ± 1.611 -23.1 ± 1.2 -21.0 ± 1.01\nE 4.888 -29.3 ± 1.115 -29.5 ± 1.5 -28.2 ± 1.306\nThese results demonstrate that the feed ratio has been systematically optimized, and the actual\nmonomer incorporation ratios are consistent with the feed ratios, within expected reactivity\nconstraints. In addition, we have included this investigation in the results section of the revised\nmanuscript (Page 10, Lines 177-181).\nQ2: What is the persistent luminescence lifetime of the nanoparticles? Is it different under different\npH values?\nResponse: The NaYF :Er@NaGdF nanoparticles exhibit an afterglow lasting up to\n4 4\napproximately 6 hours (Supplementary Video 1). This intrinsic lifetime of the nanoparticles, before\nany surface modification, remains largely unaffected by pH (Supplementary Fig. 23), as the\nNaYF :Er emission core is well protected by the NaGdF shell. Notably, when incorporated into\n4 4\nETPNs (elimusertib-loaded tumor-targeted photodynamic nanoparticles) with EPPLs (estrone-\ntargeted pH-responsive photodynamic polymeric ligands), the polymeric component of ETPNs\ndoes not alter the luminescence lifetime but mainly influences the luminescence intensity. Under\ndifferent pH conditions, particularly in acidic environments, the pH-responsive disassembly of\nETPNs results in the release and better dispersion of individual pPLNPs (Fig. 3f). This reduces the\nself-quenching effects and enhances the observed brightness of the afterglow, although the actual\nlifetime remains constant. This clarification has been included in the results section of the revised\nmanuscript (Page 14, Lines 263-267).\nSupplementary Fig. 23: Afterglow luminescence decay of NaYF :Er@NaGdF pPLNPs at room\n4 4\ntemperature under different pH after X-ray irradiation (3 Gy).\nQ3: In Figure 3e, it seems that the degradation of DPBF was inhibited after 50 min at the pH of\n5.5. Why? Does this mean the amount ROS generated by such method is limited? Would it affect\ntreatment efficacy? In addition, from Supplementary Figure 18, the peak shape of DPBF changes\nover time. Why?\nResponse: The plateau observed in DPBF degradation after ~50 minutes at pH 5.5 (Fig. 3e) is due\nto the rapid initial release of ROS following X-ray preactivation, driven by fast disassembly of\nETPNs and strong Ce6 activation in the highly acidic environment. In contrast, at pH 6.5, the\ndisassembly process is more gradual, resulting in a sustained yet slower ROS release over time.\nImportantly, ROS levels at pH 6.5 remain sufficient to induce DNA damage and activate the cGAS-\nSTING pathway, as confirmed by our in vitro and in vivo therapeutic outcomes. It is also worth\nnoting that solid tumors, particularly PDAC, commonly exhibit an extracellular pH of ~6.5 due to\nlactic acid accumulation, hypoxia, and metabolic reprogramming1,2. Therefore, the observed\nplateau does not imply inadequate ROS generation under physiologically relevant conditions.\nMoreover, the changes in DPBF peak shape in Supplementary Fig. 18 are characteristic of singlet\noxygen reactions, caused by photochemical degradation products and microenvironmental\nchanges such as polarity, oxidation state, and nanoparticle aggregation3,4.\nWe have now elaborated these points in the results section of the revised manuscript (Page 12,\nLines 225-228 and 231-235).\nQ4: Will the intensity of X-ray irradiation affect the persistent luminescence, and subsequent ROS\ngeneration? Since the nanoparticles is preactivated, to some extent, should higher power density\nof X-ray be better?\nResponse: Thank you for your insightful comment. The intensity of X-ray irradiation significantly\ninfluences both the persistent luminescence and the subsequent generation of ROS in ETPNs. We\nevaluated the persistent luminescence and ROS generation of ETPNs under different X-ray doses\n(Supplementary Fig. 22). As anticipated, afterglow luminescence intensity increased with higher\nX-ray doses (1 Gy < 3 Gy < 5 Gy). However, when assessing ROS generation via DPBF\nabsorbance, we observed that the 5 Gy dose resulted in an initial burst of ROS, followed by a\ndepletion phase as indicated by a plateau after about 20 minutes. In contrast, the 1 Gy dose\ngenerated minimal ROS, while the 3 Gy dose produced a steady and consistent amount of ROS\nover time.\nThese patterns suggest that while a high dose (5 Gy) triggers excessive ROS generation initially,\nit eventually leads to saturation and depletion of reactive sites. On the contrary, a low dose (1 Gy)\ngenerates insufficient ROS for therapeutic efficacy. Based on these findings, 3 Gy was selected as\nthe optimal X-ray dose for preactivating ETPNs. It provides a sufficient afterglow luminescence\nintensity for consistent ROS generation without leading to the rapid depletion of ROS that occurs\nat higher doses. Therefore, 3 Gy was selected as the optimal dose for preactivating ETPNs,\nproviding a balanced, sustained ROS production suitable for therapeutic use. This has been\nclarified in the results section of the revised manuscript (Page 13, Lines 244-249).\nSupplementary Fig. 22: a) Afterglow luminescence decay and b) relative DPBF absorbance of\nETPNs under different X-ray doses.\nQ5: In Figure 5a, there are some red fluorescence from ETPNs were not surround the blue one\nfrom DAPI. It is very strange, please give some explanation.\nResponse: We apologize for the confusion caused by the previous image and have now uploaded\na more representative image of cellular uptake (Fig. 5a). The uptake of ETPNs is mediated by\nestrogen receptors (ER), which are present both on the cell membrane and nuclear membrane.\nTherefore, internalized ETPNs may appear either surrounding the DAPI-stained nuclei or slightly\nfarther from the nuclear membrane. In addition, since the confocal images were captured in a two-\ndimensional plane, ETPNs located directly above or below the nucleus may appear to overlap with\nthe DAPI signal, giving the impression that fluorescence does not surround the nucleus.\nFig. 5a: CLSM image of ETPNs cellular uptake by KPC or BxPC-3 cells at different time points.\nDAPI-stained cells are shown in blue; ETPNs fluorescence signals are shown in red (scale bar =\n10 μm).\nQ6: Figure 7b is too small. It was suggested to show only one example in main text, and put the\nother ones into supporting information. A quantitative intensity is also suggested.\nResponse: Thank you for your suggestion. We have rearranged Fig. 7b and included its\nquantitative intensity in Fig. 7d.\nFig. 7: X-ray-preactivated ETPNs augment antitumor immune response and eliminate\nestablished tumors in vivo. a Schematic illustration of experimental procedure for in vivo\ntherapeutic efficacy evaluation; KPC pancreatic cancer cells were injected into C57BL/6 mice for\ntumor implantation, divided into eight treatment groups, and monitored via bioluminescence\nimaging every 6 days. b In vivo orthotopic PDAC bioluminescence images of mice receiving PBS,\nelimusertib, TPNs, and ETPNs with or without X-ray preactivation. c Terminal tumor weights of\ndifferent treatment groups (n = 5 biologically independent samples, the data are represented as\nmean ± SD). d Quantified tumor bioluminescence levels for analysis of orthotopic tumor growth\nin each group (n = 5 biologically independent samples, the data are represented as mean ± SD). e\nRepresentative immunohistochemical staining of γ-H2AX, P-IRF3, P-P65, and TUNEL in PDAC\ntissues from different groups. Tissues treated with X-ray-preactivated EPTNs showed more\npronounced activation of the cGAS–STING pathway compared to the other groups. f\nImmunofluorescence image of CD8+ T cells in different groups. CD3 fluorescence signals are\nshown in green; CD8 fluorescence signals are shown in red; DAPI-stained cells are shown in blue.\ng Quantification of CD8+ T cells within the primary tumor after various treatments. The data are\nrepresented as mean ± SD (n = 3). h Quantification of mature dendritic cells within TDLNs after\nvarious treatments. The data are represented as mean ± SD (n = 3). i Fluorescence imaging of\nETPNs and EPNs administration at different time points post-intravenous injection. *P < 0.05,\n**P < 0.01, ***P < 0.001, ****P < 0.0001.\nReviewer Comments to Author\nReviewer #2\nComments: In this manuscript, the authors developed an X-ray preactivated elimusertib-loaded\nphotodynamic nanoparticles (ETPNs) with reversible \"on-off\" persistent luminescence for precise\nphotodynamic-immunotherapy of deep tumors. ETPNs integrate porous NaYF :Er@NaGdF\n4 4\nnanoparticles (PLNPs) with pH-responsive polymeric ligands, enabling pH-dependent\nassembly-disassembly to regulate afterglow emission and ROS generation. X-ray pre-activation triggers\nsustained luminescence, activating chlorin e6 (Ce6) for ROS-induced DNA damage, while\nelimusertib inhibits ATR kinase to amplify DNA damage and activate the cGAS-STING pathway.\nIn orthotopic pancreatic cancer models, ETPNs demonstrated pH-sensitive tumor targeting,\nsuppressed tumor growth, and reprogrammed the immunosuppressive microenvironment via\nenhanced CD8+ T cell infiltration. However, some issues remain unexplored in terms of material\ndesign and related characterizations, I would like to suggest the authors to address the following\nconcerns before considering publication in Nature Communications.\nResponse: Thank you very much for your suggestions. The manuscript was revised and elaborated\nas your comment suggested. We believe that this revision has made our manuscript much more\ncoherent and comprehensible.\nQ1: In the Introduction section, the authors mentioned that “in the treatment of PDAC, where\nconventional treatments frequently fail to penetrate the dense tumor microenvironment (TME),\nan integrated PDT and immunotherapy approach could provide an effective strategy to overcome\ntreatment resistance.”. The relationship between cause and effect in this sentence is not clear, and\nthe authors are suggested to enhance the logic of the presentation.\nResponse: We apologize for the lack of clarity. The original sentence in the introduction section\ncould benefit from a clearer cause-and-effect relationship. We have revised the statement to better\nexplain why PDAC treatment is challenging and how PDT combined with immunotherapy\naddresses these challenges (Page 4, Lines 65-70).\nQ2: Reported measures to improve the penetration efficiency of PDAC solid tumors include\nmodulation of the extracellular matrix (ECM), and induction of cancer-associated fibroblast (CAF)\nquiescence, etc. Obviously, ETPNs do not possess the relevant functions, and I am curious about\nthe mechanism of their penetration into the dense stroma. It is necessary to explain this issue in\ndetail.\nResponse: Thank you for raising this important point regarding the penetration mechanism of\nETPNs in PDAC. While ETPNs does not directly modify the ECM or induce CAF quiescence,\nthey utilize a distinct mechanism for tumor penetration, including (1) estrone functionalization that\ntargets estrogen receptor (ER)-positive tumor cells for specific binding and receptor-mediated\nendocytosis; (2) pH-responsive disassembly at acidic tumor microenvironment that enhances\ninterstitial diffusion and facilitates deeper nanoparticle distribution; and (3) ROS generation,\nwhich further promotes intratumoral penetration and improves therapeutic efficacy5,6. Together,\nthese mechanisms enable ETPNs to overcome some of the limitations that conventional\nnanocarriers face in PDAC. We have clarified this issue in the discussion section of the revised\nmanuscript (Pages 29-30, Lines 589-592).\nQ3: As shown in Fig. 3a, the authors proposed that ETPNs disaggregate into isolated pPLNPs in\nacidic TME, leading to a decrease in particle size. However, as shown in Fig. 1, after co-assembly\nwith F127, the ETPNs did not show a clear tendency to disaggregate, and I wonder if this charge\nrepulsion-mediated disaggregation behavior is feasible? The authors are suggested to resubmit\nthe TEM images of ETPNs under different pH values, the current TEM images are too few samples\nto be informative.\nResponse: We agree that direct morphological evidence of ETPNs disaggregation under acidic\nconditions is essential to support our proposed pH-responsive mechanism. To provide further\nevidence for pH-responsive disassembly, we provided additional TEM images of ETPNs under\ndifferent values (Supplementary Fig. 14). As shown in Fig. 3a and Supplementary Fig. 14, multiple\npPLNPs aggregate to form ETPNs at pH 7.4 (~180 nm), whereas at pH 5.5, individual pPLNPs\n(~80 nm) become predominant, confirming charge-repulsion-mediated dissociation. These results\nare consistent with zeta potential reversal (Fig. 3b) and enhanced Ce6 fluorescence\n(Supplementary Fig. 15) under acidic conditions.\nSupplementary Fig. 14: TEM image of ETPNs at different pH (scale bar = 200 nm).\nQ4: In order to make the drug release experiments more convincing, it is necessary to provide the\nloading efficiencies of elimusertib as well as Ce6, and the corresponding calculation formulas.\nResponse: We have included the drug loading efficiencies of both elimusertib and Ce6, as well as\nthe corresponding calculation formula in the revised manuscript (Page 11, Lines 208-210; Page 36,\nLines 743-745).\n𝐷𝑟𝑢𝑔𝑠 𝑖𝑛 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 (𝑤𝑡)\nDrug Loading Efficiency (DLE) % = ( )𝑥100\n𝐷𝑟𝑢𝑔𝑠 𝑎𝑑𝑑𝑒𝑑 (𝑤𝑡)\nElimusertib DLE = 88%\nCe6 DLE = 86.4%\nQ5: To demonstrate the in vitro 1O generation capacity of ETPNs more intuitively, electron spin\n2\nresonance experiments need to be complemented.\nResponse: To further confirm the generation and identity of ROS by ETPNs, we performed ESR\nspectroscopy using 2,2,6,6-tetramethylpiperidine (TEMP) as a 1O trapping agent and 5,5-\n2\ndimethyl-1-pyrroline-N-oxide (DMPO) as a ·O - trapping agent (Supplementary Fig. 20). Upon\ncorresponding to the TEMP-1O product (TEMPO) was clearly detected, indicating efficient\n2\nsinglet oxygen generation. In contrast, the ESR signal for the DMPO-·O - adduct was negligible,\n2\nsuggesting that 1O is the dominant ROS produced by ETPNs. We have included this information\n2\nin the results section of the revised manuscript (Page 12, Lines 235-242).\nSupplementary Fig. 20: ESR spectra of ETPNs for a) 1O detection by TEMP and b) ·O -\n2 2\ndetection by DMPO after X-ray irradiation (3 Gy).\nQ6: As shown in Fig. 5a, the difference in fluorescence intensity of Ce6 at various pH values was\nnot significant when ETPNs were taken up by cells for 1 h. The authors need to quantitatively\nanalyze the cellular uptake experiments, which is feasible by flow cytometry.\nResponse: We sincerely apologize for any confusion caused by the initial image selection, which\nmay have been misleading. To ensure clarity and prevent misinterpretation, we have carefully\nreselected and provided more representative CLSM images (Fig. 5a) and performed quantitative\nanalysis of cellular uptake by flow cytometry (Supplementary Fig. 29). Using the control group as\nbaseline, we quantified the relative fluorescence intensity to represent ETPNs uptake. The results\nshowed that at 0.5 h and 1 h, acidic pH conditions promoted greater cellular uptake of ETPNs\ncompared with neutral pH. However, by 4 h, uptake levels became comparable between the two\nconditions, which we attribute to the gradual self-disassembly of the nanoparticles (Page 17, Lines\n338-343).\nSupplementary Fig. 29: Quantitative analysis of cellular uptake in PDAC cells under different\nconditions, as measured by flow cytometry. Data are presented relative to the control group (set as\nbaseline). Results are from n = 3 independent experiments.\nQ7: The authors systematically evaluated the in vitro cell killing performance of ETPNs for a wide\nrange of tumor cells, but the data from CCK-8 experiments were not intuitive. It is necessary to\nsupplement the more convincing cell killing experiments with flow cytometry or live-dead cell\nstaining for CLSM as feasible.\nResponse: While the CCK-8 assay provides an initial measure of cell viability, we understand the\nneed for more intuitive and direct evidence of ETPNs cell killing performance. In response, we\nperformed flow cytometry to assess the in vitro cytotoxicity (Supplementary Fig. 33) and\nperformed TUNEL staining of animal tissue sections (Fig. 7e) to evaluate the in vivo cell-killing\nefficacy of ETPNs (Page 18, Lines 359-361; Page 24, Lines 473-479).\nSupplementary Fig. 33: Flow cytometric analysis of apoptosis in PDAC cells under different\ntreatment conditions: normal media, elimusertib, TPNs, or ETPNs, with or without X-ray\nirradiation. Data are presented as mean ± SD from three independent experiments. **P < 0.01,\n***P < 0.001, ****P < 0.0001.\nFig. 7e: Representative immunohistochemical staining of γ-H2AX, P-IRF3, P-P65, and TUNEL\nin PDAC tissues from different groups. Tissues treated with X-ray-preactivated EPTNs showed\nmore pronounced activation of the cGAS–STING pathway compared to the other groups.\nQ8: As shown in Fig. 6b, the contrast of the ROS-stained background was too low to influence the\njudgment of readers, and the authors are suggested to resubmit the ROS-stained images.\nResponse: We apologize for the poor image quality. We have adjusted the contrast of the ROS-\nstained background in the revised manuscript.\nFig. 6b: Intracellular ROS observation of KPC or BxPC-3 cells treated as indicated (scale bar =\n100 μm).\nQ9: The authors mentioned that ETPNs have an immune-activating effect, but did not validate this\nat the in vitro level. Therefore, the authors are suggested to supplement the effect of ETPNs on the\nmaturation of bone marrow-derived dendritic cells or other ways.\nResponse: We apologize for the oversight in not including the in vitro immune activation\nexperiments. In response, the immune-activating effects of ETPNs have been further validated by\nevaluating their impact on the maturation of bone marrow-derived dendritic cells (BMDC)\n(Supplementary Fig. 49) (Pages 25-26, Lines 503-510).\nSupplementary Fig. 49: Flow cytometry plots and quantitative analysis of BMDC maturation\nfollowing co-culture with tumor cells receiving various treatments: normal media, elimusertib,\nTPNs, or ETPNs, with or without X-ray irradiation. Data are expressed as mean ± SD; n = 3\nindependent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001.\nQ10: As shown in Fig. 7b, the authors showed images of tumor-bearing mice without annotation\nof the monitoring time, which could be confusing to the readers. Meanwhile, the authors indicated\nin the figure caption that the number of mice used to assess tumor suppression efficiency was\nfive/group, but the authors only showed 4 of them, which could be confusing for the readers as\nwell. In addition, in the PBS-treated group, I am curious as to why the fluorescence intensity of the\ntumors appeared to increase and then decrease over time? The authors are invited to explain this\nphenomenon.\nResponse: We sincerely apologize for the confusion. In the previous version, all five mice across\nfour time points (days 6, 12, 18, and 24) were shown for each treatment group, which made the\nfigure overly large and unclear. To improve clarity, we have updated Fig. 7b by adding monitoring\ntime point annotations (previously only indicated in Fig. 7a) and included the quantitative intensity\ndata in Fig. 7d.\nFig. 7: X-ray-preactivated ETPNs augment antitumor immune response and eliminate\nestablished tumors in vivo. a Schematic illustration of experimental procedure for in vivo\ntherapeutic efficacy evaluation; KPC pancreatic cancer cells were injected into C57BL/6 mice for\ntumor implantation, divided into eight treatment groups, and monitored via bioluminescence\nimaging every 6 days. b In vivo orthotopic PDAC bioluminescence images of mice receiving PBS,\nelimusertib, TPNs, and ETPNs with or without X-ray preactivation. c Terminal tumor weights of\ndifferent treatment groups (n = 5 biologically independent samples, the data are represented as\nmean ± SD). d Quantified tumor bioluminescence levels for analysis of orthotopic tumor growth\nin each group (n = 5 biologically independent samples, the data are represented as mean ± SD). e\nRepresentative immunohistochemical staining of γ-H2AX, P-IRF3, P-P65, and TUNEL in PDAC\ntissues from different groups. Tissues treated with X-ray-preactivated EPTNs showed more\npronounced activation of the cGAS–STING pathway compared to the other groups. f\nImmunofluorescence image of CD8+ T cells in different groups. CD3 fluorescence signals are\nshown in green; CD8 fluorescence signals are shown in red; DAPI-stained cells are shown in blue.\ng Quantification of CD8+ T cells within the primary tumor after various treatments. The data are\nrepresented as mean ± SD (n = 3). h Quantification of mature dendritic cells within TDLNs after\nvarious treatments. The data are represented as mean ± SD (n = 3). i Fluorescence imaging of\nETPNs and EPNs administration at different time points post-intravenous injection. *P < 0.05,\n**P < 0.01, ***P < 0.001, ****P < 0.0001.\nQ11: Intravenous administration of chemotherapeutic agents is known to cause systemic toxicity\nin the body. However, as shown in Supplementary Figure 32, neither the tumor inhibition rate nor\nthe body weight of the mice after administration of elimusertib showed a significant downward\ntrend, and the authors are suggested to explain this.\nResponse: We apologize for the confusion. Elimusertib functions through a synthetic lethality\nmechanism, which differs from conventional chemotherapeutic agents. It primarily induces cell\ndeath in tumor cells with TP53 mutations, while normal cells without these mutations are able to\nresist this effect. As a result, elimusertib generally exhibits fewer side effects than traditional\nchemotherapy, which is why the body weight of the mice did not show significant loss.\nFurthermore, after elimusertib treatment, the survival curve of the mice significantly improved,\nand the tumors were considerably smaller compared to the control group (Supplementary Fig. 42).\nWe have clarified this in the results section of the revised manuscript (Page 24, Lines 465-468).\nQ12: In order to make the investigation of therapeutic mechanisms more complete, the authors\nare suggested to supplement the immunofluorescence staining of tumor sections with alterations\nin markers validated at the cellular level, including but not limited to TUNEL, ROS, cGAS, γ-\nH2AX, and others.\nResponse: To enhance the investigation of therapeutic mechanisms, we have supplemented the\nimmunofluorescence staining of tumor sections with additional markers, including TUNEL,\nPIRF3, p65, and γ-H2AX (Fig. 7e and Supplementary Fig. 43). We believe that these additional\nassays provide a more complete understanding of the therapeutic effects at the cellular level and\nfurther validate ETPNs mechanisms of action (Page 24, Lines 473-479).\nFig. 7e: Representative immunohistochemical staining of γ-H2AX, P-IRF3, P-P65, and TUNEL\nin PDAC tissues from different groups. Tissues treated with X-ray-preactivated EPTNs showed\nmore pronounced activation of the cGAS–STING pathway compared to the other groups.\nSupplementary Fig. 43: Representative immunohistochemical staining of γ-H2AX, P-IRF3, P-\nP65 and TUNEL in PDAC tissues from different group. Tissues treated with X-ray-preactivated\nEPTNs showed more pronounced activation of the cGAS–STING pathway compared to the other\ngroups.\nQ13: As shown in Fig. 7h, why did the mice exhibit a uniform distribution of fluorescence intensity\nthroughout the body when the drug was not administered? The authors are invited to explain this\nphenomenon.\nResponse: We apologize for the confusion. The fluorescence observed in Fig. 7h (now Fig. 7i)\nrepresents Ce6 fluorescence imaging, not persistent luminescence, and thus reflects the overall\nbiodistribution of the nanoparticles rather than functional activation. The uniform fluorescence\nintensity seen throughout the body, even in the absence of drug administration, may be due to\nmetabolic products of certain porphyrin-like compounds in the blood7,8. These metabolites, which\ncan have overlapping excitation wavelengths with Ce6, may include chlorophyll derivatives such\nas pheophorbide, which can remain in the blood and absorb light near 660 nm. This results in the\nobserved fluorescence across the body (Page 27, Lines 536-539).\nQ14: As shown in Supplementary Figure 37, why did the lungs of the mice demonstrate\nfluorescence intensity even when they were untreated? Please explain this phenomenon. In\naddition, ETPNs demonstrated liver, lung, and kidney accumulation up to 24 hours after\nintravenous administration, is there any relevant reported work to support this phenomenon?\nResponse: We apologize for the lack of clarity. Regarding the observed fluorescence in the lungs\nobserved in Supplementary Fig. 37 (now Supplementary Fig. 52), this is due to the presence of\nchlorophyll-like metabolites, such as pheophorbide, which have absorption properties near 660 nm\nand overlap with Ce6 fluorescence. We apologize for not appropriately adjusting the background\nin the original image and have corrected this mistake in the revised manuscript (Supplementary\nFig. 52).\nSupplementary Fig. 52: Ex vivo fluorescence (FL) images of tumors and other organs (heart,\nlung, liver, kidney, and spleen) from mice treated with X-ray-preactivated ETPNs after intravenous\ninjection at different time points (0.5, 1, 4, 12, 24, 48 h).\nAs for the biodistribution of ETPNs in the liver, lung, and kidney up to 24 hours post-\nadministration, it is consistent with reported studies on nanoparticle metabolism, where\nnanoparticles can remain in major organs for extended periods due to reticuloendothelial system\n(RES) uptake and organ-specific accumulation9,10. Studies have shown that certain nanoparticles,\ndepending on their size and surface properties, can exhibit prolonged circulation times and\naccumulation in these organs for more than 24 hours. We have clarified this matter in the results\nsection of the revised manuscript (Pages 27-28, Lines 545-547).\nReviewer Comments to Author\nReviewer #3\nComments: In this study, the authors reported the development of a novel photodynamic therapy\n(PDT) using X-ray preactivated elimusertib (ATR inhibitor)-loaded tumor-targeted photodynamic\nnanoparticles (ETPNs). By combining reactive oxygen species (ROS) induced by luminescence\nnanoparticles and inhibition of ROS-induced DNA damage response by ATR inhibitor, this new\ntherapeutic agent exhibits a strong inhibitory effect on pancreatic cancer cells with estrogen\nreceptor expression in vitro and in vivo. Furthermore, this new therapeutic agent triggers\nactivation of DNA sensor cGAS-STING-TBK1/IRF3 pathway and promotes anti-tumor immune\nresponses in vitro and also in vivo. Collectively, these data show that ETPNs may open a new\navenue for developing anti-tumor agents by combining immune-photodynamic therapy.\nThe study utilized a multi-disciplinary approach including chemistry, cell biology, and molecular\nbiology to develop and validate this new therapeutic agent. This is a major strength of\ncollaborative teamwork testing hypotheses. The in vivo animal model showed a significant\nreduction in tumor growth in mice treated by ETPNs. The therapeutic phenotype is convincing.\nOverall, the hypothesis is supported by the in vitro and in vivo data. The finding of ETPNs as a\nnovel and promising photodynamic therapy approach is significant. Results from this study will\nhave a clear impact on developing new anti-cancer drugs using X-ray preactivated elimusertib\n(ATR inhibitor)-loaded tumor-targeted photodynamic nanoparticles. There are several major and\nminor concerns regarding the data interpretation and study design.\nResponse: Thank you very much for your encouraging remarks. We have modified our manuscript\nbased on your suggestions. We believe that the quality of our manuscript has been significantly\nimproved by your comments.\nQ1: ETPN is using ER as a tumor target. ER is present in normal tissues. It is not a tumor-specific\nreceptor. It is not clear why ER is selected, and the pancreatic cancer model is selected, for\nexample, not ER-positive breast cancer is selected. Clear justifications for selecting ER and\npancreatic cancer can be helpful.\nResponse: Thank you for your comment. Although ER is not tumor-specific, it is overexpressed\nin a subset of aggressive cancers, including ~30% of PDAC, where its expression correlates with\ntumor invasiveness and poor prognosis11. Estrone, as a ligand for ER, offers advantages such as\ngood biocompatibility, small molecular size, and high affinity for ER, making it an attractive\ntargeting group. While ER-positive breast cancer is a commonly studied model, we chose PDAC\ndue to its greater clinical challenge. Although ER expression in breast cancer is typically stronger,\nPDAC presents a more aggressive and clinically relevant target with an acidic and highly fibrotic\nmicroenvironment. The unique ability of ETPNs to penetrate PDAC’s dense stroma and respond\nto the acidic TME gives them a distinct advantage in tumor targeting and therapy. Therefore,\ntargeting ER in PDAC provides a promising strategy to overcome treatment resistance in this\nchallenging cancer (Page 17, Lines 323-328).\nQ2: ETPN is intended to target ER-positive tumor cells. As shown in Supplementary Figure 24,\nKPC and BxPC-3 cancer cells showed ERα expression. Can ETPN be uptake by ER-negative\ncancer cells such as Pan-02 and PANC-1? Or can loss of ER (ER knockdown) block ETPN uptake\nby KPC or BxPC-3? To what extent does the cellular uptake of ETPN rely on ER expression?\nResponse: We performed flow cytometry analysis to evaluate ETPNs uptake under different ER\nexpression conditions (Supplementary Fig. 31). As shown in Supplementary Figure 24 (now\nSupplementary Fig. 28), both KPC and BxPC-3 cells exhibit detectable ERα expression, and these\ncells show high ETPN uptake, consistent with receptor-mediated internalization. In ER\nknockdown KPC and BxPC-3 cells, the loss of ER expression significantly decreases the cellular\nuptake of ETPNs. Consistently, cell lines with intrinsically low ER expression, such as Pan-02 and\nPANC-1, also exhibited relatively lower uptake of ETPNs compared with ER-positive cells\n(Supplementary Fig. 30). These results suggest that cellular uptake of ETPNs is correlated with\nER expression and is at least partially mediated by ER receptors (Pages 18, Lines 344-351).\nSupplementary Fig. 31: Quantitative analysis of cellular uptake in wild-type and ER-knockdown\nPDAC cell lines, as measured by flow cytometry. Data were normalized to the 0 h control group\n(set as baseline) and are presented as mean ± SD from three independent experiments. *P < 0.05,\n**P < 0.01, ***P < 0.001.\nSupplementary Fig. 30: Quantitative analysis of cellular uptake across PDAC cell lines with\nvarying ER-α/β expression levels, as assessed by flow cytometry. Data are normalized to the 0h\ncontrol group (set as baseline) and represent mean ± SD from n = 3 independent experiments.\nQ3: In Figure 4e, ETPNs luminescence intensity was analyzed under acidic and neutral pH fluctuations. IVIS imaging was used in orthotopic PDAC after receiving ETPNs, followed by the injection of basic and acidic PBS solution to induce pH changes. The presence of ETPNs was only observed in injected PDAC tumor cells. In Supplementary Figure 37 and Figure 7h, the ETPN fluorescence can be detected in normal tissues and other organs. It is not clear why these signals were not observed in Figure 4e. In addition, in Figure 4e, the highest signals were concentrated in the center of the tumor. As discussed in the introduction, the penetration of nanoparticles in general is challenging. Why is the signal in the center of the tumor stronger than at the peripheral regions of the tumor? ETPNs are intended to target ER-receptor positive cells. Why is the signal not detected in mammary glands or other ER-positive tissues?\nResponse: In response to the reviewer’s concerns, we would like to clarify the differences\nobserved in the results presented in Fig. 4e, Fig. 7h (now Fig. 7i), and Supplementary Fig. 37 (now\nSupplementary Fig. 52). Fig. 4e shows bioluminescence signals related to persistent luminescence,\nwhich is sensitive to pH fluctuations and activated by X-ray pre-irradiation. This afterglow\nemission is triggered by pH changes in the tumor microenvironment, providing a visual signal of\nETPN activity. In contrast, Fig. 7h (now Fig. 7i) and Supplementary Fig. 37 (now Supplementary\nFig. 52) display Ce6 fluorescence imaging, which is not affected by pH changes and provides\ndifferent information, detecting the presence of Ce6 within the ETPNs but not involving the same\nafterglow properties as in Fig. 4e. Notably, the uniform fluorescence intensity seen throughout the\nbody in Supplementary Fig. 37 (now Supplementary Fig. 52), even in the absence of drug\nadministration, may be due to metabolic products of certain porphyrin-like compounds in the\nblood7,8. These metabolites, which can have overlapping excitation wavelengths with Ce6, may\ninclude chlorophyll derivatives such as pheophorbide, which can remain in the blood and absorb\nlight near 660 nm. This results in the observed fluorescence across the body (Page 27, Lines 536-\n539).\nThe stronger signal observed in the center of the tumor in Fig. 4e can be attributed to several factors.\nAlthough nanoparticle penetration is generally challenging, X-ray preactivation of ETPNs before\ninjection enhances their activity. The enhanced signal in the center of the tumor likely results from\nthe better penetration of the acidic PBS solution into the tumor core12, influenced by vascular\nperfusion dynamics and interstitial fluid pressure gradients. These factors promote more effective\ndissociation of the ETPNs and ROS activation in the tumor core13. The pH-driven disassembly and\nROS generation may be more thoroughly activated in the center compared to the peripheral regions,\nwhere the environment may be less acidic and therefore hinder the full dissociation of the\nnanoparticles.\nRegarding the absence of signal in ER-positive non-tumor tissues, such as the mammary glands,\nthe lack of signal can be explained by the fact that systemic ER expression levels in normal tissues\nare lower and more heterogeneous than in ER-overexpressing PDAC tumor cells. The persistent\nluminescence observed in the tumors is primarily driven by the pH-triggered disassembly process,\nwhich is more pronounced in the acidic tumor microenvironment. In normal tissues, the lower ER\nexpression and the absence of a strong acidic environment for nanoparticle dissociation likely\nresult in no detectable signal. Therefore, the relatively low and variable ER expression in normal\ntissues reduces the specific targeting of ETPNs, which are more efficiently targeted to the tumor\ntissues where ER expression is higher.\nTo further assess the potential on-target activity in other ER-positive tissues, we evaluated the\nexpression of ER across major organs in PDAC tumor-bearing mice (Supplementary Fig. 54). The\nresults demonstrated that PDAC tissues exhibited the highest expression levels of both ER-α and\nER-β among all organs examined. To ensure the biosafety of ETPNs, histological analysis was\nperformed using H&E staining on these organs (Supplementary Fig. 53). No significant\nhistological differences were observed between the treated and untreated groups, suggesting\nminimal treatment-associated toxicity, even in organs with elevated ER expression. Furthermore,\nhematological and biochemical analyses were conducted in healthy mice to provide a\ncomprehensive assessment of the systemic safety of X-ray preactivated ETPNs (Supplementary\nFig. 53) (Page 28, Lines 551-553).\nSupplementary Fig. 54: Relative mRNA expression of ER-α and ER-β in PDAC tissues and other\nmajor functional organs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.\nQ4: ETPNs are intended to work under acidic environments. It is not clear why the strong signals\nwere detected in liver, kidney, heart, and lung in Supplementary Figure 37. What is the pH range\nin vivo to activate ETPNs? Further analyses showed no obvious toxicity in these normal organs.\nIt is unclear why the strong enrichment of ETPNs in these organs does not cause damage while\ntumors can be targeted.\nResponse: Thank you for your comment. ETPNs were meticulously designed to only be activated\nin the acidic tumor microenvironment. As previously mentioned, the detected fluorescence signal\nin Supplementary Fig. 37 (now Supplementary Fig. 52) was captured using Ce6 fluorescence\nimaging, not bioluminescence. This distinction is critical because Ce6 fluorescence measures the\noverall biodistribution of the nanoparticles, not their functional activation. In contrast, the\npersistent luminescence of ETPNs only occurs under acidic conditions, meaning that the emission\ndetected here does not indicate the active state of ETPNs.\nETPNs are designed to disassemble and activate in acidic environments. They remain in the “off”\nstate at physiological pH 7.4. Upon exposure to more acidic conditions (pH ~6.5), as typically\nfound in tumor tissues due to factors such as lactic acid accumulation, hypoxia, and metabolic\nreprogramming, ETPNs disassemble, switch to the \"on\" state, and begin emitting light while\ngenerating ROS for targeted PDT. This pH-sensitive activation ensures that ETPNs are primarily\nactive within tumor sites, particularly in cancers like PDAC, which have a naturally acidic\nmicroenvironment1,2.\nIn normal tissues, such as the liver, kidney, heart, and lungs, the pH remains neutral (~7.4), so\nETPNs stay in the \"off\" state, preventing activation and potential harm. Moreover, ETPNs are\nrapidly cleared from these organs, as shown in Supplementary Fig. 37 (now Supplementary Fig.\n52), further minimizing any prolonged exposure and ensuring minimal off-target effects. The\nbiosafety data in Supplementary Fig. 38 (now Supplementary Fig. 53) also support the absence of\ntoxicity in these organs.\nQ5: In Figure 6b, gamma-H2AX staining looks like pan-nuclear staining instead of foci staining.\nThis staining pattern is normally associated with apoptosis/cell death. In Figure 6d, ETPNs\ntreatment did not induce apoptosis in KPC or BxPC-3 cells. Does ETPN induce cell death? The\noverall phenotype in Figure 6d does not show a remarkable difference as observed in tumor\nsuppression (Figure 7b, c, and d).\nResponse: We apologize for the confusion regarding Fig. 6b. The staining in this panel is not for\nγ-H2AX, but rather for ROS generation, which plays a critical role in the therapeutic mechanism\nof ETPNs. This distinction is crucial, as γ-H2AX is a well-established marker of DNA double-\nstrand breaks, while the signal in Fig. 6b reflects ROS generation, consistent with the\nphotodynamic effect mediated by ETPNs. Additional γ-H2AX data, which provide a more accurate\nreflection of DNA damage, are presented in Fig. 7e.\nWe also regret the misunderstanding related to Fig. 6d. This figure does not depict apoptosis, but\ninstead demonstrates the antitumor effect resulting from the release of elimusertib from ETPNs,\nspecifically through abrogation of the G2/M checkpoint.\nETPNs consist of two major cytotoxic components: elimusertib and TPNs. In Fig. 6b, we detected\nROS production upon TPNs activation. Fig. 6d illustrates cell cycle arrest following elimusertib\nrelease, showing substantial accumulation of cells in the G2 phase for repair, which is\ncharacteristic of the DNA damage response. Meanwhile, elimusertib effectively abrogates the\nG2/M checkpoint, leading to an impaired repair mechanism and further contributing to tumor cell\ndeath. The combination of these two mechanisms (ROS generation and G2/M checkpoint\ninhibition) in ETPNs results in significantly enhanced tumor cell killing.\nTo further evaluate the cytotoxicity of ETPNs, in addition to the CCK-8 assay, we have also\nperformed flow cytometry-based apoptosis detection, as well as in vivo TUNEL and Ki67 staining.\nAll results consistently demonstrate that X-ray preactivated ETPNs induce significantly stronger\nkilling effects compared to the control groups.\nQ6: In Figure 6e, cGAS staining was used to detect micronuclei. The quality of the images is poor.\nFor this purpose, PicoGreen staining might be easier to detect micronuclei.\nResponse: We apologize for the poor image quality in the original figure. To address this, we have\nreplaced Fig. 6e and included Supplementary Fig. 37, which uses dsDNA staining for improved\nvisualization of micronuclei. The results indicate that cells treated with X-ray-preactivated ETPNs\nexhibited a significantly higher frequency of micronuclei (Page 21, Lines 408-413). Due to local\nregulatory restrictions, we were unable to obtain the PicoGreen dye. Therefore, we used a\ncommercially available dsDNA marker (Santa Cruz Biotechnology) for micronuclei detection.\nThis approach provided sufficient resolution to confirm that X-ray preactivated ETPNs markedly\nincreased micronuclei formation.\nFig. 6e: Immunofluorescence image of cytosolic dsDNA in KPC and BxPC-3 cells treated with\nnormal media and X-ray preactivated ETPNs. dsDNA fluorescence signals are shown in green;\nDAPI-stained cells are shown in blue; arrow indicates micronuclei (scale bar = 10 μm).\nSupplementary Fig. 37: Immunofluorescence image of cytosolic dsDNA in KPC and BxPC-3\ncells treated under various conditions. dsDNA fluorescence signals are shown in green;\nDAPI-stained cells are shown in blue; arrow indicates micronuclei (scale bar = 10 μm).\nQ7: Similar to Figure 6c, the phenotype of activation of cGAS-STING-TBK1 and p-P65 induced\nby ETPNs compared to ATR inhibitor alone is at relatively same level. It is not clear whether the\nphenotype can explain the phenotype presented in Figure 7b, c, and d. The downstream type-I\ninterferon signaling can be detected to further show whether the phenotype of ETPNs is stronger\nthan ATR inhibitor alone. Additionally, cGAS or STING knockdown can be used to genetically\nprove the involvement of this pathway in the therapeutic efficacy of ETPNs.\nResponse: Thank you for your insightful question. ETPNs are composed of two primary cytotoxic\ncomponents, elimusertib and TPNs. In the absence of X-ray activation, ETPNs gradually release\nthe elimusertib payload due to the acidic tumor environment, while TPNs remain inactive and do\nnot contribute to tumor killing. As a result, the overall therapeutic effect observed in this condition\nis similar to the effect of elimusertib monotherapy. This explains why the phenotype presented in\nFig. 6c, showing the activation of the cGAS-STING-TBK1 and P-P65 pathways, appears similar\nbetween ETPNs and elimusertib monotherapy, as TPNs’ contribution to tumor suppression is not\nactivated in the absence of X-ray irradiation.\nTo further validate the involvement of the cGAS-STING signaling and its downstream effects,\nELISA analysis was performed to quantify IFN-β levels in tumors under different treatment\nconditions (Supplementary Fig. 48). X-ray preactivated ETPNs induced a significantly higher IFN-\nβ production compared with all other groups, whereas elimusertib monotherapy TPNs elicited only\na modest increase. Notably, ETPNs without X-ray preactivation generated IFN-β levels\ncomparable to elimusertib monotherapy, consistent with the lack of Ce6 activation in the absence\nof irradiation.\nSupplementary Fig. 48: ELISA analysis of IFN-β expression in KPC cells treated under various\nconditions: normal media, elimusertib, TPNs, or ETPNs, with or without X-ray irradiation. Data\nare expressed as mean ± SD; n = 3 independent experiments. *P < 0.05.\nAdditionally, we performed cGAS knockdown experiments to confirm the role of the cGAS-\nSTING pathway in the therapeutic efficacy of ETPNs (Supplementary Fig. 51). Silencing cGAS\nmarkedly reduced IFN-β secretion and significantly decreased the proportion of mature dendritic\ncells in BMDC co-culture assays. These results demonstrate that the therapeutic activity of ETPNs\ndepends in part on the activation of the cGAS–STING pathway (Pages 25-26, Lines 503-510).\nSupplementary Fig. 51: a, b) Flow cytometry profiles and quantitative analysis of BMDC\nmaturation following co-culture with either wild-type KPC cells or cGAS-knockdown KPC cells,\nboth treated with X-ray-preactivated ETPNs. c) ELISA analysis of IFN-β expression in KPC cells\ntreated with X-ray-preactivated ETPNs or subjected to cGAS knockdown. Data are expressed as\nmean ± SD; n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.\nQ8: The figure legends can be further improved by adding key details, e.g. Figure 7, what cells\nwere injected to form tumors; 7b, time points should be labeled; the scale bar of fluorescence for\nFigure 4e.\nResponse: We apologize for the lack of clarity. We have added additional details in the figure\nlegends of Fig. 7, updated Fig. 7b by adding monitoring time point annotations, and included the\nfluorescence scale bar in Fig. 4e.\nFig. 7: X-ray-preactivated ETPNs augment antitumor immune response and eliminate\nestablished tumors in vivo. a Schematic illustration of experimental procedure for in vivo\ntherapeutic efficacy evaluation; KPC pancreatic cancer cells were injected into C57BL/6 mice for\ntumor implantation, divided into eight treatment groups, and monitored via bioluminescence\nimaging every 6 days. b In vivo orthotopic PDAC bioluminescence images of mice receiving PBS,\nelimusertib, TPNs, and ETPNs with or without X-ray preactivation. c Terminal tumor weights of\ndifferent treatment groups (n = 5 biologically independent samples, the data are represented as\nmean ± SD). d Quantified tumor bioluminescence levels for analysis of orthotopic tumor growth\nin each group (n = 5 biologically independent samples, the data are represented as mean ± SD). e\nRepresentative immunohistochemical staining of γ-H2AX, P-IRF3, P-P65, and TUNEL in PDAC\ntissues from different groups. Tissues treated with X-ray-preactivated EPTNs showed more\npronounced activation of the cGAS–STING pathway compared to the other groups. f\nImmunofluorescence image of CD8+ T cells in different groups. CD3 fluorescence signals are\nshown in green; CD8 fluorescence signals are shown in red; DAPI-stained cells are shown in blue.\ng Quantification of CD8+ T cells within the primary tumor after various treatments. The data are\nrepresented as mean ± SD (n = 3). h Quantification of mature dendritic cells within TDLNs after\nvarious treatments. The data are represented as mean ± SD (n = 3). i Fluorescence imaging of\nETPNs and EPNs administration at different time points post-intravenous injection. *P < 0.05,\n**P < 0.01, ***P < 0.001, ****P < 0.0001.\nFig. 4e: IVIS images of orthotopic PDAC mice depicting tumor bioluminescence (X-ray 3 Gy)\nafter receiving ETPNs, followed by the injection of basic and acidic PBS solution to induce pH\nchanges.\nReferences\norganoid growth and drug resistance. Cell Rep 43, doi:10.1016/j.celrep.2024.114409\n(2024).\nAnnu Rev Physiol 82, 103-126, doi:10.1146/annurev-physiol-021119-034627 (2020).\nNear-Infrared-Triggered Photodynamic Therapy of Deep Tumors. Adv Mater 30, e1802808,\ndoi:10.1002/adma.201802808 (2018).\noxygen probes in aqueous environments. Journal of Photochemistry and Photobiology B:\nBiology 204, 111787, doi:https://doi.org/10.1016/j.jphotobiol.2020.111787 (2020).\nmethod to pave the way for subsequent therapies. Biomaterials 289, 121813,\ndoi:https://doi.org/10.1016/j.biomaterials.2022.121813 (2022).\nthe intrinsic/extrinsic apoptotic pathway of anoikis for triple-negative breast cancer\ntreatment. Biomaterials Advances 160, 213859,\ndoi:https://doi.org/10.1016/j.bioadv.2024.213859 (2024).\nfor Practical Applications and Everyday Use. Anal Chem 92, 9429-9440,\ndoi:10.1021/acs.analchem.0c00415 (2020).\nDeliv Rev 182, 114134, doi:https://doi.org/10.1016/j.addr.2022.114134 (2022).\nNanomaterials. Adv Mater 30, e1704307, doi:10.1002/adma.201704307 (2018).\nAdenocarcinoma. Adv Sci (Weinh) 8, 2002545, doi:10.1002/advs.202002545 (2021).\nin resected pancreatic adenocarcinoma. BMC Cancer 18, 1049, doi:10.1186/s12885-018-\n4973-6 (2018).\nand Nucleic Acids with pH-Dependent Nanoparticles. Pharmaceutics 15,\ndoi:10.3390/pharmaceutics15051482 (2023).\nHypoxia-Induced Chemotherapy Resistance: Combination of Optimizing Size and Self-\nInducing High Level of Reactive Oxygen Species. ACS Appl Mater Interfaces 11, 31743-\n31754, doi:10.1021/acsami.9b12129 (2019)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I do not agree with the authors’ reply on my previous comment 3.\nFirstly, the DPBF could be degraded by singlet oxygen very fast, when used for measuring singlet oxygen generation efficiency, it could be usually completely degraded within several minutes. From Supplementary Figure 19 (Supplementary Figure 19 in the previous version), the singlet oxygen generation efficiency is very poor, which could be in fact affect the PDT efficacy.\nIn addition, the degradation of DPBF could only result in the decrease of its absorbance, but hardly affect its peak shape.\nThis problem is directly related to the accuracy of the key performance. Therefore, this issue must be addressed before publication.", "reviewer_2": "It seems that the authors have addressed all the concerned issues, and I would like to recommend its publication without any other changes.", "reviewer_3": "The manuscript and the responses to the reviewer are accepted after the revision."}, "author_response": "Response: We sincerely thank the reviewer for the careful and insightful evaluation, especially for\nidentifying the issue in our previous DPBF measurements. Upon re-examination, we found that\nthe DPBF reagent used earlier had partially oxidized after prolonged exposure to air, which led to\nthe atypical spectral change observed. We truly appreciate the reviewer’s correction on this point.\nTo prevent any misunderstanding, we have since used freshly opened DPBF for each test, and the\nnew results show the expected decrease in absorbance without peak distortion, consistent with\ncanonical DPBF quenching by singlet oxygen (1O ) (Fig. 4b, Supplementary Fig. 19, and\nTo ensure data reliability, we repeated the 1O detection experiments under rigorously controlled\n2\nconditions and further validated the results using another singlet-oxygen probe, 9,10-\nanthracenediyl-bis(methylene)dimalonic acid (ABDA)1, which confirmed efficient 1O generation\n2\nunder the same activation conditions (Supplementary Fig. 20) (Page 12, Lines 232-235; Page 37,\nLines 755-760).\nFrom our measurements, the ROS generation efficiency under our excitation conditions is indeed\nmoderate. Unlike traditional photoactivation methods that produce high ROS bursts for short-term\ntumor killing, our approach utilizes persistent luminescence to achieve continuous ROS production\nfor prolonged PDT treatment. Such sustained ROS generation is particularly advantageous for\ntreating deep-seated tumors and, when combined with elimusertib, results in excellent therapeutic\nefficacy. This was intentionally designed to ensure effective synergy while minimizing excessive\ncytotoxicity. Consistent with this, both our in vitro and in vivo results show that TPNs (tumor-\ntargeted photodynamic nanoparticles, no elimusertib) alone, which generate similar ROS levels,\nfail to induce significant tumor cell death, whereas ETPNs (elimusertib-loaded tumor-targeted\nphotodynamic nanoparticles) achieve strong therapeutic outcomes.\nSupplementary Fig. 19: Changes in DPBF-absorbance spectra of ETPNs at a) pH 7.4, b) 6.5, and\nc) 5.5 under X-ray irradiation. d) pH-dependent DPBF absorbance of ETPNs under X-ray\nirradiation at different pH.\nFig. 4b: Changes in the DPBF-absorbance of ETPNs when the pH values fluctuate under X-ray\nirradiation, exhibiting a negligible decrease at pH 7.4 and a significant decrease at pH 5.5.\nSupplementary Fig. 25: Changes in the DPBF-absorbance spectra of ETPNs when the pH values\nfluctuate under X-ray irradiation, exhibiting a negligible decrease at pH 7.4 and a significant\ndecrease at pH 5.5.\nSupplementary Fig. 20: Changes in the ABDA-absorbance spectra of ETPNs at a) pH 7.4, b) 6.5,\nand c) 5.5 under X-ray irradiation. d) pH-dependent DPBF absorbance of ETPNs under X-ray\nirradiation at different pH.\nReferences\nOxygen Generation of Nano-Photosensitizers Toward Advanced Phototherapy. Nano-\nmicro letters 14, 123, doi:10.1007/s40820-022-00856-y (2022)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed my comments well, and I think this work can be published now."}, "author_response": ""}]

Decision letter