D-π-A sensitized carbon dots as long-lived type-I/Ⅱ photosensitizers for NIR-excited hypoxia-regulated photodynamic therapy

Domain: biology
Published: 2026-04-02
Editorial outcome: major_revision

Abstract
In the development of clinically translatable triplet photosensitizers for hypoxia regulated
photodynamic therapy (PDT), there is an unmet need for engineering sensitizers as near-infrared
(NIR)-responsive, type I/type Ⅱ dual photosensitizers and mild photothermal agents. Herein, we
develop a binary precursor-engineering strategy for precise regulation of the D-π-A configuration
of carbon dots (CDs) as ultralong-lived triplet, type I/Ⅱ dual photosensitizers by utilizing phenolic
hydroxyl as an electron-rich donor and pyridinic N as an electron-withdrawing acceptor. The
photodynamic performance of CDs is enhanced by intramolecular charge transfer and mild
photothermal conversion. We further design M1-like macrophage-derived cell
membrane‑camouflaged CDs to realize preferential tumor accumulation while guaranteeing rapid
systemic clearance. D-π-A sensitized CD-mediated PDT induces anti-tumor activity against
primary and distant tumors. Our work highlights the crucial roles of D-π-A sensitization of CDs
in boosting PDT by triplet state tuning, surface charge transfer, and mild photothermal relief of
hypoxia.

Introduction
Phototherapy including photodynamic and photothermal therapy (PDT/PTT) utilizes light of a
specific wavelength to activate photosensitizers (PSs) or photothermal conversion reagents
enriched in the tumor site, and selectively kills tumors by generating cytotoxic reactive oxygen
species (ROS) or local hyperthermia with spatiotemporal precision.1-5 Recently, both PDT and
PTT have demonstrated their ability to elicit immunogenic immune responses for
photoimmunotherapy against both primary and abscopal/metastatic tumors, and thus hold potential
for establishing long-term cancer-free health.6-10 While PTT also induces immunogenic cell death
(ICD) to activate innate immunity, significant ICD of irradiated tumor cells was induced by PTT
only in a high photothermal window (50−60 °C).11-13 This means that high doses of irradiation at
a high laser power density is required to effectively trigger ICD for photothermal immunotherapy,
which instead may raise severe concerns about thermal damage to the surrounding anatomical
structures, including blood vessels, ECM scaffolds and nerves.14-17 In contrast, immunogenic PDT
is superior to immunogenic PTT in photoimmunotherapy since irradiation at rather lower laser
power density can trigger desirable ICD mode of tumor cells.18-20 However, the effectiveness of
PDT for photoimmunotherapy is subject to the hypoxic tumor microenvironment (TME), since
conventional PDT generates ROS typically in the single product of only 1O through the energy-
2
transfer type-Ⅱ mechanism that is highly dependent on oxygen level.21-23 Albeit respective
drawbacks for PTT or PDT as a single treatment mode, type-Ⅱ PDT and mild PTT may synergize
to enhance phototherapy efficacy owing to partial alleviation of hypoxia by mild hyperthermia.
With rapid development in multimodal nanoplatforms composed of photodynamically and
photothermally active agents, PDT/PTT combinations have recently gained momentum in tumor
phototherapy.24-27 Nevertheless, currently available PDT/PTT combined systems suffer from two
intrinsic shortcomings. On the one hand, owing to a lack of a common optimal excitation
wavelength for PDT and PTT agents, the utilization of two different lasers for sequential excitation
complicates treatment and prolongs treatment duration.28-31 On the other hand, although the dual
modes can be triggered by a single near-infrared (NIR) laser, relatively high-power
photoirradiation or utilization of highly efficient photothermal agents is required, leading to an
interrupted tumor perfusion and oxygenation owing to hyperthermia-induced vascular damage and
collapse.32-34

Very recently, an emerging class of PSs with type-I and type-Ⅱ dual modes have been developed
as alternative avenues for treating normoxic/hypoxic tumors by switching between oxygen-
dependent type-Ⅱ and oxygen-independent type-I mechanisms based on local oxygen
concentration.25,35,36 Different from the energy-transfer type-Ⅱ mechanism, the type-I mechanism
works by photoinduced single electrons transferring to molecular oxygen in a low O -dependence
2
and reduction manner to produce superoxide radical (O •ˉ) or/and hydroxyl radical (•OH).24,37-39
2
To date, most type-I/Ⅱ PSs are constructed from inorganic semiconducting nanomaterials, whose
electron-hole separation and charge transfer can be enhanced by surface defects or/and
heterojunctions.18,37,40,41 Despite they show excellent photostability compared to less-stable
organic PSs, their PDT applications are limited by short absorption/excitation wavelengths,
ultrashort excited-state lifetimes (~ ns), and poor biocompatibility owing to metal toxicity and less
degradability.42,43 It is still a terrible challenge to develop single-component, metal-free, NIR-
responsive, type-I/Ⅱ photosensitizers with matching photothermal tunability, high photostability
and high biocompatibility to meet the above standard for robust photoimmunotherapy.

As a metal-free zero-dimensional semiconductor, carbon dots (CDs) have garnered increasing
attention for a wide range of applications, since their electronic, optical and catalytic properties
can be facilely tailored through molecular engineering, by which functional groups containing
heteroatoms of O, N, S with lone pairs of electrons can be in situ incorporated at core and surface
sites during hydrothermal/solvothermal processes.44-49 Given their superior properties, such as
high photostability, high biocompatibility, excellent water-solubility, and tunable fluorescent or
phosphorescent properties, small-sized CDs have been extensively employed as highly stable and
kidney-excretable theranostic agents for bioimaging and imaging guided tumor multimodal
treatments, including sonodynamic therapy (SDT), PTT, and type-I/Ⅱ PDT.50,51 As type-I/Ⅱ PSs,
however, all CDs reported so far exhibit ultrashort excited state lifetimes (~ ns), leading to
unsatisfactory PDT efficacy owing to inefficient triplet population through intersystem
crossing.52,53 Moreover, most CDs exhibited a wide bandgap, with absorption/excitation
wavelengths in the UV-vis range.54,55 Although there also were a few reports on NIR-responsive
type-I/Ⅱ CDs excited at 808 nm, light intensity of 0.8-1.0 W cm−2 was still required, which far
exceeds the maximum permissible exposure (MPE) (808 nm, 0.33 W cm−2) to skin and tissue.2,56

In this work, we report the rational design of D-π-A sensitized photodynamic CDs as NIR-
excited type-Ⅰ/Ⅱ sensitizers for hypoxia regulated photodynamic immunotherapy, which inspires
by the design principle of D-A sensitized molecular photosensitizers. Generally, D-A sensitized
photosensitizers are organic molecules designed to enhance the efficiency of photovoltaic or
photodynamic processes in dye-sensitized solar cells and photodynamic therapy by harnessing the
properties of donor (D) and acceptor (A) components.57-59 Especially, the D-π-A structure
facilitates electron transfer between the donor and acceptor groups through an extended π bridge
for enhancing type-I/Ⅱ photodynamic performances.60-62 As an extended π-conjugated nano-
system of CDs in the D-π-A configuration, electron-rich oxygen atoms from -OH modifications at
edge sites significantly improve the electron-donating capability, which combine with electron-
withdrawing moieties (largely pyridinic N) to form the D-π-A configuration, resulting in an
elongated triplet state lifetime (tens of ms), an enhanced intramolecular charge transfer, and a
reduced bandgap (1.49 eV). To demonstrate the CD-based design of the D-π-A sensitized type-Ⅰ/Ⅱ
PSs and the precise regulation of PDT/PTT performances of CDs, a molecular-engineering
combination strategy is employed to synthesize three types of CDs with different regional
modifications from two molecular precursors: O-CDs from OPD + ICG, M-CDs from MPD + ICG,
and P-CDs from PPD + ICG (Fig. 1). Among them, D-π-A type CDs (P-CDs) with an average
carrier lifetime of 36.32 ms demonstrate the highest efficacy in generation of both 1O and O •ˉ but
2 2
the lowest photothermal conversion efficiency (32.2%), revealing the D-π-A enhanced type-I/Ⅱ
photodynamic mechanisms. In contrast, donor N-rich (pyrrolic N) yet acceptor-deficient M-CDs
as D-π-D type CDs show the highest photothermal conversion efficiency of 69.6% but the poor
PDT efficacy. Mechanically, an intramolecular charge transfer (CT) state with a ultralong lifetime
mediated by the D-π-A structure enables the enhanced PDT performance. Importantly, the hypoxia
condition is substantially relieved by mild PTT + PDT, as evidenced by immunofluorescence
staining of HIF-1α in tumor. We further design and synthesize M1-like macrophage-derived cell
membrane (MM)‑camouflaged P-CDs (P-CD@MM) to realize preferential tumor accumulation
while guarantee rapid systemic clearance. On the one hand, the encapsulation of P-CDs in MM
can prevent the rapid clearance of CDs, actively targeting the tumor tissue to improve the tumor
accumulation level of P-CDs. On the other hand, MM-encapsulated P-CD can enhance reversion
of the immunosuppressive TME and realize the cascade amplification of antitumor immune
response through triggering ICD and reprogramming tumor-associated macrophages (TAMs) into
antitumoral M1-like phenotype. Collectively, satisfactory therapeutic outcomes for primary and
distant tumors are achieved by P-CD@MM-mediated precise PDT-enhanced immunotherapy at
low laser power density of 0.30 W/cm2 (808 nm). Our work highlights the crucial roles of D-π-A
sensitization of CDs in boosting NIR-photoimmunotherapy by triplet state stabilization, surface
charge transfer, and mild photothermal relief of hypoxia.

Results
Molecular engineering of D-π-A sensitized CDs. To screen D-π-A sensitized nanosensitizers
from surface-functionalized CDs, we developed a one-step combination strategy for controllable
solvothermal synthesis of a series of O, N, S co-doped NIR-CDs with narrow bandgaps by
combining one of the three isomers of o-, m-, and p-phenylenediamines (OPD, MPD, PPD) with
indocyanine green (ICG) as key precursors, as schemed in Fig. 2a. Benefited from the combination
synthetic strategy, three kinds of NIR-CDs with an decreasing graphitization level and significantly
reduce bandgaps (1.40-1.56 eV), denoted as O-CDs, M-CDs, and P-CDs, were successfully
synthesized from OPD/ICG, MPD/ICG, PPD/ICG, respectively. In contrast, without the
combination with ICG, N-doped CDs with wide bandgaps (1.93-2.28 eV) were derived from one
of the phenylenediamine isomers (Supplementary Fig. 1), which was similar to the previous
report.63 On the other hand, without addition of any phenylenediamine isomers, the solvothermal
treatment of only ICG precursor, O, S co-doped CDs (ICG-CDs) with slightly reduced bandgap
(1.92 eV) were synthesized (Fig. 2j), but exhibited a high oxygen content (19.27 at. %) and low
carbonization level, as revealed by XPS and Raman characterization (Supplementary Fig. 2, 3a).
As verified by evaluation of the potential in the following phototherapy for NIR-CDs, P-CDs were
eventually screened out as superior a type-Ⅰ photodynamic agent, while M-CDs were found to
serve as a superior PTT agent, suggesting that the combination synthetic strategy was effective in
the regulation of the PDT/PTT performances.

We then characterized the structural features of three distinct types of CDs and attempted to
unveil the structure-activity relationship between the N doping types of CDs with the enhanced
photothermal or photodynamic performances. TEM images presented in Fig. 2b-d demonstrated
the successful preparation of these CDs, revealing their dot-like morphology. We also found that
M-CD and P-CD exhibited comparable average diameters, measuring 1.69 ± 0.45 nm for M-CDs
and 1.38 ± 0.40 nm for P-CDs, while O-CD displayed a significantly larger average size of 5.30 ±
0.75 nm. The high crystallinity of these CDs was confirmed by high-resolution TEM images (Fig.
2e-g), which revealed well-defined lattice fringes with a spacing of 0.21 nm, corresponding to the
(100) crystallographic plane characteristic of graphitic carbon. Raman spectroscopy further
validated the graphitic structures of all four types of CDs (Supplementary Fig. 3). The D/G
intensity ratios for ICG-CD, O-CD, M-CD, and P-CD were determined to be 0.817, 0.784, 0.854,
and 0.867, respectively, indicating the lowest degree of graphitization in P-CD, which could be
ascribed to the presence of abundant surface functional groups. In addition, the para-configuration
of PPD could impede the generation of ordered graphitic domains. The symmetrical structure of
PPD lacks sufficient spatial degrees of freedom during the carbonization process, thereby
hindering effective structural rearrangement and aromatization. Therefore, many disordered
structures are formed within the carbon core, leading to the low graphitization level of P-CDs.
XPS analysis revealed that the content of N element in P-CD (10.31 at. %) was significantly higher
than that of ICG-CD (3.06 at. %), O-CD (5.82 at. %), and M-CD (8.33 at. %) (Supplementary Fig.
2, 4-6). The predominant form of N in P-CD was pyridinic N, which exhibited peaks at 398.6 eV.
In contrast, no peak of pyridinic N can be detected in the high-resolution N 1s spectrum of the
other three CDs. However, M-CD contained graphitic N with a content of 3.19 at. %, suggesting
that M-CD possessed graphitic N-doped features. For O-CD, no peaks of graphitic N and pyridinic
N can be detected, illustrating that O-CD possessed pyrrolic N-doped characteristics (Fig. 2k).
Collectively, we can confirm that M-CD had the characteristic of graphitic-N-doping, while P-CD
possessed the features of pyridinic-N-doping. We also performed 1H NMR and 13C NMR analysis
to clarify the specific structures and molecular arrangements of these CDs. As depicted in
Supplementary Fig. 7a, the 1H NMR exhibited that the peaks of P-CD and ICG monomers partially
overlapped at δ 7.5-8.5 ppm, indicating that the conjugated motif of ICG composed of several -
CH= units is likely retained in the extended π conjugated aromatic section in the P-CD, which can
be beneficial to extend its absorption in the NIR region. However, the additional strong signal of
hydroxyl (-OH) was observed at δ 6.35 ppm in the P-CD 1H NMR spectrum, further confirming
the structural characteristics of P-CD containing the -OH donor. Additionally, the 13C NMR
analysis of P-CD revealed the existence of conjugated carbon skeletons at δ 110-150 ppm, which
were the basis of π bridges in the D-π-A structure, but such extended conjugated carbon skeletons
cannot be observed in the ICG 13C NMR spectrum (Supplementary Fig. 7b).

We further investigated the characteristics of surface functional groups of these CDs using FT-
IR spectroscopy. As depicted in Supplementary Fig. 8, the broad peak at approximately 3430 cm-
1 was detected in the three kinds of CDs, which indicated the presence of O-H or N-H on the edge
of these CDs. Because O-CDs, M-CDs, and P-CDs all contain hydroxyl groups, we believe that
the hydroxyl groups may originate from ethanol solvents during the solvothermal synthesis of O-
CDs, M-CDs, and P-CDs. These surface functional groups could endow these CDs with good
colloidal stability and dispersibility. Additionally, stretching vibration bands associated with -SO ,
3
C-N, C=N, and C-S were evident in the FT-IR spectrum, indicating the presence of SO ˉ and N-
3
containing heterocycles in these CDs. Subsequently, we performed Zeta potential characterization
to determine the surface charge characteristics of O-CD, M-CD, and P-CD. The measured values
of zeta potential were found to be -21.63 ± 0.8 mV, -31.4 ± 0.8 mV, and -21.83 ± 0.4 mV,
respectively (Supplementary Fig. 9). The characteristics of negative charges of these CDs could
be ascribed to the presence of SO ˉ and O-H. The bandgap values of these CDs were determined
3
by measuring their absorption spectra. As depicted in Fig. 2j, O-CD, M-CD, and P-CD exhibit
broad UV to NIR absorption, with significant absorption bands observed between 600-850 nm,
demonstrating that the doping of N could increase the NIR absorption of CDs. Based on the (αE)2-
E diagram, the bandgap for O-CD, M-CD, and P-CD were calculated to be 1.56, 1.40, and 1.49
eV, respectively. In contrast, the absorption spectrum of ICG-CD was restricted to the UV-visible
light region, displaying a band gap of 1.92 eV.

Photothermal properties of CDs. We performed NIR-responsive photothermal performance
measurements on O-CDs, M-CDs and P-CDs to investigate the influence of N doping on the
photothermal conversion properties. To avoid damage to skin tissue caused by laser irradiation, we
measured the PTT performances at low power density of 0.3 W/cm2 with an 808 nm laser. We
found that the temperature of the M-CD aqueous solution increased rapidly from 26 °C to 54.7 °C
during 5 min irradiation (Fig. 3a and Supplementary Fig. 10), achieving a remarkable photothermal
conversion efficiency of 69.6%. In contrast, P-CD and O-CD solutions exhibited slow rise in
solution temperature and significantly lower conversion efficiencies at only 32.2% and 42.9%,
respectively (Fig. 3b, c). We also performed photothermal stability evaluation of M-CD. As shown
in Supplementary Fig. 11, after 9 cycles of heating and cooling, the temperature of M-CD still
reached 53.7 ℃ under 808 nm laser irradiation (0.3 W/cm2), indicating the excellent photothermal
stability of M-CD. We monitored solution temperature changes with reduced laser-power (0.3-0.1
W/cm2) and reduced concentration (0.1-0.3 mg/mL) under consistent irradiation of 808 nm laser.
With decreasing laser-power or CD concentration, the photothermal effect of all the three CDs was
markedly reduced (Fig. 3d-i). Notably, for P-CD solutions at low concentration (0.1 mg/mL) with
808 nm laser at low power density (0.3-0.1 W/cm2), the rise in temperature was rather low (~10 °C),
suggesting that P-CDs exhibited a rather mild NIR PTT effect compared with M-CDs. Previous
reports have demonstrated that the doping of graphitic N and pyrrolic N with electron donating
capability can trigger the red-shift of absorption spectrum of CDs, thereby improving their
photothermal conversion properties. Indeed, M-CDs contained the highest N doping level (8.33
at. %) in forms of graphitic and pyrrolic N compared with other two CD samples, which endowed
them with the highest photothermal conversion efficiency. For PTT-poor P-CDs, almost all N
doping was in the pyridinic N form (an electron-withdrawing group) with absence of graphitic and
pyrrolic N, despite the highest N doping level (10.31 at. %). These results indicate that molecular
engineering can be harnessed to regulate photothermal performances of NIR-responsive CDs as a
photothermal conversion reagent by varying N doping level and N-contained species.

Enhanced photodynamic properties of P-CD. Having demonstrated the molecular regulation
of the NIR photothermal effect of CDs, the NIR photodynamic property of the three types of CDs
as type II and type I photosensitizers was then investigated first using DPBF as a chemical capture
agent for ROS including singlet oxygen (1O ) and superoxide radicals (O •ˉ) after photoreactions
2 2
with an 808 nm laser (0.3 W/cm2). As the irradiation time increased from 0 to 10 min, the
characteristic absorption peak of DPBF at 418 nm diminished gradually (Fig. 4a). Among these
CDs, P-CDs exhibited the strongest ROS generation efficiency. The ROS generation efficiency of
P-CDs was measured to be 0.038 min-1, which was approximately 2.9 times greater than that of
O-CD and about 4.75 times greater than that of M-CD, respectively (Supplementary Fig. 12a). To
differentiate the generated ROS type, we then utilized DHR123 as a specific fluorescent probe for
detecting O •ˉ. In the presence of O •ˉ, DHR123 exhibited a distinct fluorescence emission peak at
2 2
525 nm. Following irradiation with an 808 nm laser, the fluorescence intensity induced by P-CDs
was significantly enhanced, thereby confirming the generation of O •ˉ by the type I photodyanmic
2
mechanism (Fig. 4c). Moreover, the O •ˉ generation efficiency in P-CD group was higher than that
2
of M-CDs and O-CDs (Supplementary Fig. 12b), further demonstrating the enhanced type-I
photodynamic activity of P-CDs in generation of O •ˉ. To confirm the type I photodyanmic
2
mechanism, we utilized the ESR spectrum to measure O •ˉ signals by using the DMPO as the
2 2
scavenger for O •ˉ. The characteristic ESR signal of O •ˉ in P-CDs was stronger than that in M-
2 2
CDs and O-CDs (Fig. 4e). Furthermore, the generation of another ROS type (1O ) was tested by
2
ESR spectra using TEMP as the capture agent of 1O . Following irradiation for a duration of 5
2
minutes, three characteristic peaks exhibiting an intensity ratio of 1:1:1 were observed (Fig. 4f).
The ESR signal intensity of TEMPO progressively increased from M-CDs to O-CDs and
subsequently to P-CDs, verifying that P-CDs exhibited highest 1O production efficiency. We then
2
evaluated the photostability of P-CD through measuring photothermal conversion efficiency and
ROS generation efficiency under multiple laser irradiations. As presented in Supplementary Fig.
13a, no significant change of the photothermal conversion efficiency of P-CD was detected during
the six consecutive laser irradiation cycles. In addition, the O •ˉ generation efficiency of P-CD
2
remained unchanged after five cycles (Supplementary Fig. 13b). These results demonstrated the
good photostability of P-CD under multiple laser irradiations.

We then attempted to unveil the ambiguous structure-activity relationship of CD-based PSs
owing to their complex components and understand why type II/I P-CDs possessed the strongest
photodynamic property in greatly enhanced generation of 1O and O •ˉ compared with the other
2 2
CDs. For organic triplet PS molecules, the type II PDT mechanism has been widely accepted,
which involves relatively stable population of long-lived triplet states (T ) through photoexcitation
1
from S to S state, and then to T by intersystem crossing (ISC). Excited PSs return from T to S
0 n 1 1 0
state by energy transfer to triplet oxygen (3O ) to generate 1O . Accordingly, prolonging carrier
2 2
lifetime in stable T states is a crucial factor in augmenting photodynamic activity of triplet PSs.
1
We determined their photoexcited carrier lifetime by measuring time-resolved PL spectra. For O-
CDs and M-CDs, time-resolved PL spectra exhibited relatively rapid decay, with average lifetimes
of 2.02 ns and 8.88 ns determined, respectively (Fig. 4g, h), suggesting a characteristic for common
fluorescent CDs with an ultrashort lifetime (~ ns). In contrast, P-CDs demonstrated slow
phosphorescence decay with ultralong carrier lifetime of 36.32 ms, which was six orders of
magnitude longer than those observed for O-CDs and M-CDs (Fig. 4i). These results suggest that
P-CDs can emit phosphorescence rather than fluorescence. Given the enhanced catalytic potential
of long-lived triplet excited states, we propose an enhanced mechanism for both 1O and O •ˉ
2 2
generation mediated by long-lived triplet excited states.

Spin-orbit coupling (SOC) serves as the primary driving force for ISC, a process that can be
significantly enhanced by modifying the features of the participating triplet states. In accordance
with the El-Sayed rule, heteroatom groups such as oxygen, nitrogen, and sulfur—characterized by
their abundant lone pairs of electrons—are incorporated into aromatic compounds to augment SOC
and thus ISC. There is a negligible difference in heteroatom-induced SOC effect on electronic
configurations since the total heteroatom content of O, N, S in O-CDs, M-CDs and P-CDs are
24.89%, 20.72%, 21.90%, respectively (Fig. 2i). However, notably, there is a large difference in
contents of donor and acceptor groups between donor-rich, acceptor-rich CDs (P-CDs) and donor-
rich yet acceptor-deficient CDs (M-CDs and O-CDs). Since D-A sensitized photosensitizers
commonly possess stable triplet states with lifetime of ~ μs to ms, we further proposed that
phosphorescent P-CDs have a D-π-A configuration with a long-lived intramolecular charge
transfer (CT) state, which may facilitate electron transfer between the donor and acceptor groups
through an extended π bridge for enhancing type-I/Ⅱ photodynamic performances (Fig. 2h, 4i). In
contrast, donor-rich yet acceptor-deficient O-CDs and M-CDs with a D-π-D configuration
exhibited low-efficacy type-I photodynamic performances likely owing to absence of effective
intramolecular CT between the donor and acceptor groups. Intramolecular CT excited states in D-
A sensitized photosensitizers generally exhibit solvent polarity-dependent optical properties such
as red shift in emitting wavelength with increasing solvent polarity. To test this, we measured PL
spectra of O-CDs, M-CDs and P-CDs in solvents (EA, acetone, EtOH, and H O) with increased
2
polarity ordering. It was found that there was a pronounced PL red shift effect consistently induced
by solvent polarity in P-CDs, while in M-CDs and O-CDs, the solvent polarity effect on PL red-
shift or blue-shift was irregular (Fig. 4j). Combining the long-lived T and solvent polarity effect
1
clearly revealed the evidence for D-π-A sensitized type II/I photodynamic mechanism of molecule-
engineered CDs with enhanced charge-transfer dynamics.

To highlight the advantages of the D-π-A configured P-CDs, we compared the excited-state
lifetime, ROS generation efficiency, and hypoxic adaptability between D-π-A structured CDs (P-
CDs), traditional CDs (O-CD), and commercial photosensitizers (ICG, TCPP). First, P-CDs
exhibited ultralong triplet state lifetime of 36.32 ms, which was six orders of magnitude longer
than those observed for O-CDs and M-CDs (Fig. 4i). The triplet state lifetime of P-CDs was also
significantly higher than that of commercial photosensitizers (0.8 ns for ICG and 8.48 ns for TCPP)
(Supplementary Table 1). Second, this ultralong triplet state lifetime is a crucial factor in
augmenting photodynamic activity of P-CDs. The calculated 1O quantum yield of P-CD was
2
measured to be 1.49%, which was much higher than that of O-CD (0.58%), ICG (0.20%), and
TCPP (0.10%). Third, under hypoxic conditions, the P-CD with type I/II photodyanmic
mechanism can still effectively produce O •ˉ (Fig. 4b, d). In contrast, no ROS generation of ICG
2
and TCPP can be detected under hypoxic conditions (Supplementary Fig. 14), which could be
attributed to their O -dependent type II photodyanmic mechanism. These results demonstrate that
2
the D-π-A configured P-CDs possess the largest excited-state lifetime, highest ROS generation
efficiency, and optimal hypoxic adaptability compared with the traditional CDs and commercial
photosensitizers.

Theoretical calculation of CDs. We then performed the density functional theory (DFT)
calculations to visually illustrate molecular orbital distributions, electron localization, and charge
transfer pathways. We establish three kinds of sp2 hybrid carbon molecules as simple CD models
according to the theoretical models presented in Fig. 2h. Although the simplified CD model
established in the theoretical calculations did not represent the true structure of these CDs, the
relative comparison among the model enabled identification of trends and suggested explanations
for the observed findings. Fig. 5a exhibits the spatial distributions of the HOMO (highest occupied
molecular orbital) and LUMO (lowest unoccupied molecular orbital) for O-CD, M-CD, and P-CD.
In the P-CD model, the HOMO is predominantly localized on the hydroxyl-rich region ("donor",
D), whereas the LUMO is strongly concentrated on the pyridinic nitrogen-containing domain
("acceptor", A). This clear spatial separation of frontier orbitals directly demonstrates that, in the
ground state of P-CD, the highest-energy electrons reside primarily in the donor moiety, while the
lowest-energy vacant orbitals are located in the acceptor moiety, with both regions bridged by a
conjugated π-system (carbon core). This configuration constitutes definitive evidence for a well-
defined D-π-A architecture. Fig. 5b presents the calculated energy levels of the first singlet excited
state (S ) and the first triplet state (T ), along with their respective singlet-triplet energy gaps
1 1
(ΔE ). Notably, P-CD exhibits a significantly smaller ΔE (0.39 eV) compared to O-CD (0.82
ST ST
eV) and M-CD (0.96 eV). Such a reduced energy gap facilitates efficient intersystem crossing (ISC)
from the singlet to the long-lived triplet state. The theoretical calculation results consistent with
the experiment results of ultralong phosphorescence lifetime of P-CD (36.32 ms), offering a
mechanistic explanation for its excellent performance as a photosensitizer.

We then performed the surface electrostatic potential (ESP) analysis of O-CD, M-CD, and P-
CD. As depicted in Fig. 5c, red regions represent the negative potential (electron-rich zones) and
blue regions indicate positive potential (electron-deficient zones). In the P-CD model, a
pronounced negative potential is observed in the hydroxyl-enriched region, contrasting sharply
with the positive potential in the pyridinic nitrogen zone. This phenomenon confirms that hydroxyl
groups act as the primary electron-donating center (D), while the pyridinic nitrogen serves as the
electron-accepting center (A). These findings are further demonstrated by Fukui-DD function
analysis (Fig. 5d), which reveals electrophilic features at the pyridinic nitrogen site with a high
propensity for electron acceptance. In addition, the nucleophilic features over the hydroxyl and π-
bridge regions was consistent with electron donation. These two complementary analyses confirm
that the hydroxyl groups function as the strongest electron donor within the P-CD framework,
serving as the driving force for intramolecular charge transfer. In summary, the DFT calculations
provide comprehensive evidence from orbital distribution, energy-level alignment, and
quantitative reactivity descriptors for the proposed D-π-A model. Orbital visualization confirms
the spatial segregation of donor and acceptor units linked via a π-conjugated bridge. Energy-level
calculations rationalize the experimentally observed long-lived triplet state. Electrostatic potential
and Fukui function analyses jointly establish the functional roles of the hydroxyl group as the
dominant donor and pyridinic nitrogen as the key acceptor.

In vitro antitumor effectiveness evaluation of P-CD@MM. Having demonstrated the mild
photothermal and D-π-A enhanced photodynamic effects of ultrasmall-sized P-CDs as superior
type-I and type-Ⅱ photosensitizers, we then prepared size-suitable and biomimetic P-CD@MM
nanoparticles by harnessing tumor-associated MM as the nanocarrier to encapsulate P-CDs for
enhancing both active and passive tumor-targeting capabilities, as previously reported in
biomimetic nanomedicines.64-66 It is noted that the encapsulation of M1-like MM can not only
avoid immune clearance of free CDs for extended circulation time, but also promote the
polarization of TAMs from protumoral M2- like phenotype to antitumoral M1-like phenotype. The
loading efficiency and Zeta potential of P-CD@MM were determined to be about 20% and -30.8
± 1.4 mV, respectively (Supplementary Fig. 15). The TEM images showed a size of ~ 150 nm
suitable for affording the passive tumor-targeting capability via the EPR effect (Fig. 6a, b). As WB
analysis revealed, there was the high expression level of M1-like macrophage specific proteins
CD86 in P-CD@MM, while there was no significant band of CD206 as the M2-like macrophage
specific protein, confirming the successful preparation of P-CD@MM with tumor-associated M1-
like phenotype (Fig. 6c). The quantitative result of the expression levels of CD86 was added in
Supplementary Fig. 16a. We then investigated the stability evaluation of P-CD@MM during
storage. As depicted in Supplementary Fig. 16b, c, no significant change of size and Zeta potential
of P-CD@MM were observed within 9 days, demonstrating the good colloidal stability of P-
CD@MM. We then investigated the cellular uptake behaviors of P-CD@MM based on the intrinsic
fluorescence of P-CD. Confocal imaging of NIH-3T3 normal cells and 4T1 tumor cells exposed
to P-CD@MM exhibited a much higher level of cell internalization in cancer cells, further
clarifying the augmentation in tumor targeting levels of P-CDs through M1-like MM encapsulation
(Fig. 6d). We then investigated the biocompatibility of P-CD@MM exposed to NIH-3T3 normal
cells without photoirradiation through the MTT assay across a concentration range of 0-100 μg/mL.
Even at the concentration up to 100 μg/mL, P-CD and P-CD@MM exhibited insignificant
cytotoxicity to normal cells (Fig. 6e and Supplementary Fig. 17a), indicating their favorable
biosafety profiles for potential applications in PDT.

To investigate the in vitro PDT efficacy of P-CD@MM, MTT assays were conducted under 808
nm laser irradiation (0.3 W/cm2) for 5 minutes. As depicted in Fig. 6f, almost all 4T1 cells were
killed by P-CD@MM +laser, suggesting the high in vitro antitumor efficacy of P-CD@MM under
5-min photoirradiation. The in vitro antitumor efficacy of MM-free P-CDs was also evaluated for
comparison. Although a concentration-dependent cytotoxic profile of P-CD@MM similar to that
of MM-free P-CDs, the real photoactive concentration of P-CDs in P-CD@MM was merely one-
fifth of MM-free P-CDs owing to the only 20% loading efficiency. In other words, real antitumor
efficacy of P-CD@MM + laser was five times higher than MM-free P-CD + laser owing to
increased tumor targeting capability through MM coating, as confirmed by the cell imaging result
(Supplementary Fig. 17b). We also performed the MTT assay to evaluate the relative cell viability
of NIH-3T3 cells after being treated with P-CD@MM + L. As depicted in Supplementary Fig. 18,
no significant decrease of the cell viability of NIH-3T3 cells was detected in the P-CD@MM + L
group, which could be ascribed to the low cellular internalization efficiency of P-CD@MM in
normal cells.

To further investigate the therapeutic effects of P-CD@MM-mediated PDT, we conducted
live/dead cell staining using Calcein-AM and PI as the probes of live and dead cells, respectively.
The experimental groups were as follows: Group 1: PBS; Group 2: laser; Group 3: P-CD@MM;
Group 4: P-CD@MM + laser. No dead cells can be detected in the control, laser alone, and P-
CD@MM alone (Fig. 6g). In contrast, when the NIR laser was applied, the images presented in P-
CD@MM + laser group exhibited no green fluorescence signal, verifying the excellent antitumor
effectiveness of P-CD@MM-mediated PDT.

Notably, the photodynamic property of P-CDs was excellent (Supplementary Fig. 19, 20), while
their photothermal property was of low-efficiency (only 32.2%). At the mild PTT conditions (0.3
W/cm2) and a low P-CD concentration (<100 μg/mL), the rise in temperature was rather low
(~10 °C) (Fig. 3i). Therefore, the excellent phototherapy effectiveness of P-CDs and P-CD@MM
was not attributive to the mild PTT effect. To confirm the PDT effect as the main phototherapy
mechanism, intracellular ROS levels under different treatment conditions were tested by ROS
detection assay using DCFH-DA as the probe of ROS. This ROS-sensitive probe is oxidized by
intracellular ROS to generate green fluorescence in cells. No significant ROS fluorescence signal
was detected in groups 1-3 (Fig. 6h), implying no ROS generation in the absence of laser treatment
or P-CD@MM. In contrast, strong green fluorescence was observed in the cytoplasm of cells in
the P-CD@MM + laser group, confirming that ROS-mediated cell death was effectively induced
by P-CD@MM based PDT. To further investigate the mechanisms of cell death, we analyzed
apoptosis levels using Annexin V-FITC and PI staining combined with flow cytometry. As shown
in Supplementary Fig. 21, significant apoptosis of 4T1 cells was detected in the P-CD + laser and
P-CD@MM + laser groups. Extensive studies have demonstrated that PDT can induce tumor cell
apoptosis while PTT can induce tumor cell necrosis.2 Collectively, these results indicated that P-
CD@MM exhibits excellent antitumor efficacy in vitro by PDT induced cell apoptosis.

P-CD@MM-mediated PDT induces ICD and M1-like polarization. After confirming that P-
CD@MM induced cancer cell apoptosis through PDT, we then investigated the ROS-triggered
ICD effect to evaluate the tumor-specific immune responses. It is well known that the generation
of ROS in tumors could trigger the exposure of CRT, secretion of HMGB1, and leakage of ATP.12,67
On this basis, we initially evaluated CRT exposure level on 4T1 cells through immunofluorescence
staining. Notable CRT signals were observed in both the P-CD + laser group and the P-CD@MM
+ laser group, indicating a translocation of CRT from the endoplasmic reticulum to the cell surface
(Fig. 7a). Subsequently, ELISTA assay and ATP detection kit were utilized to assess the HMGB1
and APT levels, respectively. The concentration of HMGB1 released from 4T1 cells treated with
P-CD@MM + laser was measured at 30.7 ± 1.6 μg/L (Fig. 7b), significantly surpassing that
observed in other groups. This finding suggests that the PDT effects of P-CD and P-CD@MM
enhance HMGB1 release, which may trigger pro-inflammatory cytokine secretion and
subsequently stimulate DC maturation. In addition, ATP levels were markedly diminished in both
the P-CD + laser group and the P-CD@MM + laser group (Fig. 7c). Collectively, these results
indicate that P-CD@MM can induce an enhanced ICD effect via PDT.

Having demonstrated that P-CD@MM can induce ICD and enhance the secretion of DAMPs,
we proceeded to investigate DAMP-activated DC maturation and the subsequent immune
responses. BMDCs were isolated from the tibias and femurs of BALB/c mice. 4T1 cells were
pretreated with P-CD@MM + laser, after which the supernatant containing DAMPs was collected
to stimulate BMDCs (Fig. 7d). The expression ratios of mature DC markers CD80+ and CD86+ in
the P-CD@MM + laser group were measured at 15.7  0.8%, significantly higher than those
observed in the other groups (Fig. 7e, f). These results indicate that P-CD@MM-mediated ICD
effectively triggers BMDC maturation, potentially activating a systemic immune response capable
of targeting tumor cells for destruction.

It is well known that macrophages can switch between their main phenotypes, M1-like and M2-
like, depending on cytokines or pathogenic signals.68 M1-like macrophages are known for their
pro-inflammatory, immunogenic, and anti- tumoral features, whereas M2-like macrophages are
recognized for their anti-inflammatory, tolerogenic, and pro-tumoral traits.68 Within the TME,
most macrophages showcase the M2-like phenotype, prompting the exploration of reprogramming
M2-like to M1-like macrophages as a strategy for cancer therapy. Increased ROS levels have been
shown to significantly induce TAMs to switch from the pro-tumoral M2-like to the anti-tumoral
M1-like phenotype, thus reversing the immunosuppressive TME. Based on this situation, we
utilized commercialized RAW 264.7 cells and BMDMs as model systems to evaluate the ability
of P-CD@MM + laser to reprogram macrophages. Similar to the evaluation of DC maturation,
4T1 cells were pre-treated with P-CD@MM + laser, and the supernatant was collected to stimulate
RAW 264.7 cells (M0-like) (Supplementary Fig. 22a). A significant increase of CD86+CD206ˉ
TAMs (M1-like) was observed in P-CD@MM + laser group, suggesting that the treatment of P-
CD@MM plus laser irradiation can promote the polarization of TAMs from M2-like to M1-like
phenotype (Supplementary Fig. 22b, c). For BMDMs, we first pre-polarized TAMs with M0-like
phenotype to M2-like phenotype. After treatment with P-CD@MM + laser, a pronounced shift
from the M2-like to M1-like phenotype was observed compared to the other groups (Fig. 7g).
Specifically, CD86+CD206ˉ TAMs (M1-like) increased from 6.3 ± 0.6% to 36.0 ± 1.0%, while
CD86ˉCD206+ TAMs (M2-like) decreased from 41.7 ± 0.8% to 8.4 ± 0.8% (Fig. 7h, i). A similar
M2-like to M1-like polarization was also observed in RAW 264.7 cells (M2-like) after being
treated with P-CD@MM + laser (Supplementary Fig. 23). We also noted that the M1-like
macrophage membrane can inherently promote M1-like polarization. On this basis, we explored
the interaction mechanism between M1-like macrophage membrane and TAMs. As shown in
Supplementary Fig. 24, compared with the Control, Laser, and P-CD groups without M1-like
macrophage membrane, P-CD@MM treatment alone significantly increased the expression levels
of phosphorylated NF-κB p65 (p-NF-κB p65) and phosphorylated STAT1 (p-STAT1) in TAMs.
This enhancement effect was further amplified after adding laser irradiation (P-CD@MM + Laser).
The results provide direct molecular evidence that the M1-like macrophage membrane is not an
inert carrier, but interacts with TAMs through its surface retained ligands, actively activating the
core signaling pathways (NF-κB and STAT1) that drive M1-like polarization, thereby
reprogramming the pro-tumoral M2-like phenotype to the anti-tumoral M1-like phenotype. These
findings confirm that P-CD@MM-triggered ICD can reprogram TAMs from the M2-like to M1-
like phenotype, effectively reversing the immunosuppressive TME.

In vivo antitumor effectiveness evaluation. We established a unilateral tumor model to
determine the time-dependent accumulation level of intravenously injected P-CD@MM in tumor
sites and the optimal time window for PDT by in vivo imaging (Fig. 8a). For comparison, free P-
CDs without MM encapsulation was also evaluated by the in vivo imaging. As presented in the
Fig. 8b, c, MM-free P-CDs exhibited a rather low accumulation level in the tumor sites at 24 h
post-injection owing to rapid metabolization out of the body, while P-CD@MM showed a high
level of accumulation in the tumor sites at 12-48 h (Supplementary Fig. 25), clearly confirming
the crucial role of MM encapsulation in enhancing tumor targeting capability and elongating blood
circulation time. The optimal time for laser irradiation was thus determined to be at 24 h post-
injection since at this time point the strongest fluorescent signal of P-CDs was observed. This
tumor targeting distribution of P-CD@MM was also revealed by the ex vivo imaging (Fig. 8d and
Supplementary Fig. 26). For MM-free P-CDs, following intravenous administration, P-CD
predominantly accumulated in the liver of mice (Supplementary Fig. 27), which was consistent
with the in vivo NIR imaging results (Fig. 8b), suggesting the low accumulation level of P-CDs in
tumors. We also investigated the in vivo pharmacokinetics of MM-free P-CDs and MM-
encapsulated P-CDs by measuring plasma concentration-time profiles. As depicted in
Supplementary Fig. 28a, a typical double-compartment pharmacokinetic behavior of P-CD and P-
CD@MM can be detected. The elimination half-life (t ) of P-CD and P-CD@MM was
1/2
determined to be 0.9 ± 0.1 h and 10.6 ± 1.1 h, respectively (Supplementary Fig. 28b). The
prolonged half-life of P-CD after coating of MM illustrates that P-CD@MM possesses longer
circulation time compared with the pristine P-CD, enabling the accumulation of P-CD@MM in
tumors at high levels.

We also monitored a PTT induced moderate rise in local temperature at the tumor location
exposed to 808 nm NIR laser (0.3 W/cm2) using an IR thermal camera. In Fig. 8e, the tumor
temperature treated with P-CD@MM under 10 min NIR irradiation slowly reached 49.1 °C, which
was insufficient for tumor thermal ablation but could relieve tumor hypoxia. Generally, PDT
efficacy is compromised due to its oxygen dependent nature as the continuous oxygen
consumption during the treatment prohibits further ROS generation and induces a hypoxic
environment in tumor to trigger the hypoxia-inducing factor-1α (HIF-1α)-mediated signaling
cascade that could desensitize tumors to PDT and even provoke tumor invasion and metastasis.
Therefore, the exploration of the moderate PTT effect on alleviation of hypoxia in TME is of great
importance for improving PDT efficiency and safety. To confirm this effect, we conducted the
immunofluorescence hypoxia staining assay to verify the alleviation of hypoxia in the tumor tissue
induced by mild NIR PTT. As expected, the hypoxia signal of tumor slices in P-CD@MM plus
NIR laser group was less than that of the control group, only NIR laser group, and only P-
CD@MM group (Fig. 8f), illustrating that the severe tumor hypoxia was alleviated by the mild
PTT.

According to these pre-examinations, we finally designed the treatment protocol for PTT-
assisted PDT, by which single dosing and single irradiation under 808 nm at low power density
(0.3 W/cm2) for 10 min irradiation were administrated (Fig. 7a). To evaluate the in vivo antitumor
efficacy of P-CD@MM by photoimmunotherapy, we established a bilateral tumor model and
examined the inhibitory rates of local tumors and distant metastases by measuring the volumes of
primary and distant tumors. As shown in Fig. 8g, h, P-CD and P-CD@MM alone showed limited
antitumor effects in vivo. However, when combined with 808 nm laser irradiation, tumor growth
was significantly inhibited. Notably, the P-CD@MM + laser treatment achieved complete
eradication of the primary tumor, suggesting that P-CD@MM-mediated PDT can achieve optimal
therapeutic outcomes. Furthermore, the volume of distant tumors also exhibited a significant
inhibiting effect after treatment of P-CD@MM + laser, indicating that P-CD@MM-mediated PDT
also possessed antitumor immune response. Mice in the P-CD@MM + laser group survived for
over 50 days, whereas mice in the control group survived only up to 26 days (Supplementary Fig.
29a). This phenomenon indicates that P-CD@MM-mediated PDT can induce a strong immune
response for synergistic therapy, thereby prolonging the survival of mice. We also investigated the
influence of laser irradiation parameters on the therapeutic effects of P-CD@MM-mediated
combination therapy. To evaluate the impact of irradiation spatial coverage and duration, we
evaluated tumor growth under different light spot sizes (d = 0.4 cm, insufficient to fully cover the
tumor; d = 0.8 cm, slightly larger than the tumor; d = 1.2 cm, substantially exceeding tumor
dimensions) at a fixed power density of 0.3 W/cm2 and irradiation time of 10 min. Additionally,
under the same power density and a spot size of 0.8 cm, we examined the effects of varied
irradiation durations (0, 5, 10, and 15 min) on therapeutic outcomes. As shown in Supplementary
Fig. 30, both insufficient irradiation area and suboptimal exposure time failed to achieve complete
tumor growth inhibition. The optimal therapeutic efficacy was observed at a power density of 0.3
W/cm2, a spot diameter of 0.8 cm, and an irradiation time of 10 min. While longer irradiation times
or larger spot sizes also yielded strong antitumor effects, they may increase the risk of collateral
damage to surrounding healthy tissues due to excessive energy deposition. ROS staining of tumor
tissues showed the highest ROS signals in the P-CD@MM + laser group (Fig. 8i). H&E and
TUNEL staining images indicated apoptosis or necrosis in both primary and distant tumors in the
P-CD@MM + laser group (Supplementary Fig. 31, 32). These findings suggest that P-CD@MM-
mediated PDT induces a strong immune response, leading to severe tumor cell apoptosis/necrosis.

In vivo photoimmunotherapy antitumor mechanism. As indicated by in vitro cellular
experiments, P-CD@MM-mediated PDT can effectively trigger ICD, promote DC maturation, and
reverse the immunosuppressive TME. To further elucidate the synergistic therapy of PDT and
immunotherapy, we investigated the immune responses induced by ICD at the solid tumor level.
We detected the levels of pro-inflammatory cytokines in mice including interleukin-6 (IL-6),
interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) using ELISA assay. As shown in
Supplementary Fig. 33, the levels of IL-6, IFN-γ, and TNF-α in P-CD@MM + laser group were
significantly higher than those in other groups. These results suggest that P-CD@MM-mediated
PDT can activate the immune response in vivo by releasing tumor-associated antigen, thus
inducing ICD and M1-like polarization. Then, immunofluorescence staining for CRT expression
in tumor tissues showed the most evident green fluorescence in the P-CD@MM + laser group (Fig.
9a). ELISA and an ATP detection kit were used to measure the other two ICD markers, revealing
significantly higher HMGB1 and ATP levels in tumor tissues of the P-CD@MM + laser group than
in other groups (Supplementary Fig. 34), further confirming marked ICD induction by P-
CD@MM-mediated PDT.

Subsequently, tumor-draining lymph nodes, spleens, and tumor tissues were collected from mice
in each group for flow cytometric analysis of immune cell populations. Analysis of DCs in tumor-
draining lymph nodes revealed a significantly higher proportion of CD80+CD86+ DCs in the P-
CD@MM + laser group (Supplementary Fig. 35). This confirmed that P-CD@MM + laser
effectively promoted DC maturation, attributed to ROS induced ICD and M1-like polarization of
TAMs. We also noted that the treatment of P-CD@MM + laser induced the higher DC maturation
level compared with the treatment of P-CD + laser, which could be ascribed to the stronger tumor
targeting and enrichment capabilities of P-CD after encapsulating by MM. The higher local tumor
concentration means that a stronger photodynamic effect can be generated under NIR laser
irradiation, thereby triggering stronger ICD to activate antitumor immune response. After P-
CD@MM + laser treatment, the proportions of CD8+CD3+ T cells and CD4+CD3+ T cells in mouse
spleens increased significantly from 1.3 ± 0.3% to 16.0 ± 0.7% and from 1.6 ± 0.3% to 18.0 ±
0.9%, respectively (Supplementary Fig. 36). Further assessment of T cell activation in tumor
tissues showed that the proportions of CD8+CD3+ T cells and CD4+CD3+ T cells in both primary
and distant tumors were significantly higher in the P-CD@MM + Laser group compared with the
other groups (Fig. 9b and Supplementary Fig. 37). These results clearly indicate that P-CD@MM
effectively triggered immune responses through PDT. As the TME is enriched with M2-like TAMs
that promote tumor progression, we used flow cytometry to analyze macrophage polarization in
primary and distant tumors following various treatments. As shown in Fig. 9c and Supplementary
Fig. 38, in primary tumors treated with P-CD@MM + laser, M2-like TAMs (CD206+F4/80+) were
markedly reduced, while M1-like TAMs (CD86+F4/80+), which have anti-tumor properties, were
significantly increased. Similar results were observed in distant tumors (Supplementary Fig. 39).
These findings forcefully indicate that the photosensitizer P-CD@MM triggered tumor-specific
ICD, reprogramed TAMs from M2-like to M1-like phenotype, promoted DC maturation, and
activated T cells.

To demonstrate the pivotal role of P-CD-mediated phototherapy in activation of antitumor
immune response, we employed the M0-like phenotype RAW264.7 macrophage membrane as an
empty cell membrane to encapsulate P-CD (P-CD@EM). As depicted in Supplementary Fig. 40a,
b, no significant antitumor effectiveness of P-CD@EM alone can be detected, suggesting the
encapsulation of P-CD in EM did not trigger the antitumor immune response. For comparison, a
slight inhibition effect on the growth of primary and distant tumors was observed in the P-
CD@MM, demonstrating that cell membrane derived from M1-like phenotype macrophage can
activate a certain antitumor immune response. We also noted that the treatment of P-CD@EM + L
can inhibit the tumor growth, which could be attributed to the P-CD-induced PDT. However, the
stronger antitumor effectiveness can be detected in the P-CD@MM + L group, which was superior
to that in the P-CD@EM + L group, illustrating that the antitumor immune response of P-CD-
mediated PDT could be further enhanced by the encapsulation of P-CD in MM rather than in EM.
Immunological analyses revealed that P-CD@EM alone did not elicit antitumor immune responses.
P-CD@EM failed to promote DC maturation, induce T cell infiltration, or drive polarization of
macrophages from M2-like phenotype to M1-like phenotype (Supplementary Fig. 40c-n). In
contrast, P-CD@MM exhibited a certain immunostimulatory activity, implying that the
encapsulation of P-CD in MM can endow immunoregulation functions to P-CD. We also noted
that P-CD@EM combined with laser irradiation triggered strong immunological activation,
demonstrating that P-CD-mediated PDT served as the essential core component for the activation
of antitumor immune responses.

Long-term immunological memory evaluation. It is well known that the immune-memory
response represents an essential aspect of adaptive immunity, enabling the immune system to
remember previous pathogen invasions and provide long-lasting immunity. On this basis, we
constructed a re-challenging tumor model to evaluate the long-term immune memory effect
induced by P-CD@MM-mediated combination therapy (Fig. 10a). After the first tumor was cured
by P-CD@MM + L, we inoculated 4T1 cells on the opposite axilla of mice to fabricate the re-
challenging tumor model. No injection of P-CD@MM and no irradiation of 808 nm laser were
performed. For comparison, five healthy mice were also inoculated with 4T1 cells as the control
group. We then monitored the change of tumor volume and performed the immunological analysis.
As depicted in Fig. 10b, c, the significantly inhibited tumor growth was detected in the P-CD@MM
+ L group, while rapid tumor growth was observed in the control group, suggesting that the
treatment of P-CD@MM + L can induce an effective immune memory response for the long-term
protection against tumor recurrence of mice. During the treatment period, no loss of weight can be
found in the control and P-CD@MM + L groups (Fig. 10d), demonstrating that the treatment of P-
CD@MM + L possessed good biosafety. Subsequently, we investigated the T cell activation level
in each group to clarify the immune memory response. As presented in Fig. 10e, the higher
expression levels of CD4 and CD8 were detected in the P-CD@MM + L group compared with the
control group. Specifically, the proportion of CD3+CD8+ and CD3+CD4+ T cells in the P-CD@MM
+ L group were measured to be approximately 24.5 ± 1.4% and 22.2 ± 1.3%, respectively (Fig.
10f, g), which was much higher than those in the control group (3.9 ± 0.5% for CD3+CD8+ and
2.9 ± 0.7% for CD3+CD4+). Additionally, the proportion of effector memory T cells
(CD3+CD8+CD44+CD62Lˉ) in the spleens of both groups was analyzed. As depicted in Fig. 10h,
i, in the P-CD@MM + L group, a significantly higher proportion of effector memory T cells (34.3
± 1.4%) was observed, which was four times greater than that in the control group (8.6 ± 0.9%).
Collectively, these results indicate that the administration of P-CD@MM + L induces a long-
lasting immune memory response, which can enable the immune system to remember previous
pathogen invasions and provide long-lasting immunity to prevent the reappearance of tumors.

Finally, we assessed the biological safety of the P-CD@MM + laser treatment modality. During
the treatment period, no significant weight loss was observed in any group compared to normal
mice (Supplementary Fig. 29b). We then explored the in vivo biodistribution and excretion
pathways of P-CD@MM. Supplementary Fig. 41a showed that P-CD@MM mainly accumulated
in the liver at 24 h post-injection, which was attributed to the capture of the reticuloendothelial
system. Importantly, no obvious P-CD signal was observed in these organs after 14 days, indicating
that P-CD@MM has been almost completely cleared. To study the excretion pathways, the
concentrations of P-CD in urine and feces were also measured. The results indicated that P-
CD@MM was mainly excreted from the mice through the liver and kidney. We also noted that no
P-CD signal can be detected after 14 days (Supplementary Fig. 41b), suggesting the complete
clearance of P-CD@MM. The major organs of mice subjected to different treatments were
analyzed via H&E staining. The results reveal no significant differences compared to the control
group, with no evident damage observed (Supplementary Fig. 42). Furthermore, blood
biochemical analysis (Supplementary Fig. 43a) and hematological parameters (Supplementary Fig.
43b) demonstrated that all blood indices in treated mice remained within the normal physiological
range. We also performed the hemolysis test of P-CD@MM. As shown in Supplementary Fig. 44,
hemolysis assays confirmed the good blood compatibility of P-CD@MM, with a hemolysis rate
below 5% at concentration of 100 μg/mL. Collectively, these results substantiate the good
biocompatibility and biosafety profile of P-CD@MM-mediated PDT, thereby supporting its
potential utility in antitumor therapy.

Methods
Ethical statement
All animal experiments were approved by the Animal Protection and Use Committee of Shanghai
University (SYXK2019-0020). All mice were housed in an animal facility under constant
environmental conditions (room temperature, 22 ± 1 °C, relative humidity, 40-70% and a 12 h
light-dark cycle). The experiment was designed without considering the sex of the animals. Both
male and female mice were used for the in vivo experiments. Maximal tumor sizes were within
permission limits (≤1200 mm3). Tumor volume was calculated using the formula: Volume =
(Length × Width × Width)/2.

Synthesis of P-CDs, M-CDs and O-CDs
For the preparation of P-CDs, ICG (5 mg) (Aladdin, China) and p-phenylenediamine (10 mg)
(Aladdin, China) were dissolved in ethanol (10 mL) (Sinopharm Chemical Reagent Co., Ltd.,
China). The solution was transferred into a 25 mL Teflon-lined autoclave and reacted at 150 oC for
4 h. The reactor is then cooled to room temperature. Large particles were removed by 0.22 μm
membrane, followed by dialysis for 36 h using a dialysis bag. The preparation methods for M-CDs
and O-CDs were analogous to that of P-CDs, with the exception that meta-phenylenediamine
(Aladdin, China) was used as the precursor for M-CDs and ortho-phenylenediamine (Aladdin,
China) for O-CDs.

Synthesis of M1-like macrophage cell membrane
RAW264.7 cells (Haixing Biosciences, TCM-C766-UD, China) were co-cultured with LPS (1
μg/mL) (Beyotime, China) for 24 h to obtain M1-like macrophages. The cells were resuspended
in lysis buffer (Beyotime, China) and sonicated on ice (80 W, 20 min). The solution was
centrifuged at 4 °C for 30 min, and the supernatant was collected to obtain macrophage membranes
(MM). The M0-like macrophage membranes were obtained from RAW 264.7 cells using the same
method.

Synthesis of P-CD@MM
The P-CD aqueous solution (1 mL, 0.5 mg/mL) and the M1-like macrophage cell membrane (1
mL, 1 mg/mL) were separately transferred to syringes and successively extruded through a 100
nm water-phase filter using an extruder to form P-CD@MM. Finally, the P-CD@MM solution
was centrifuged to remove unbound P-CD. We also employed the M0-like phenotype RAW264.7
macrophage membrane as an empty cell membrane (EM) to encapsulate P-CD (P-CD@EM) using
the same method.

Characterization
TEM images of O-CD, M-CD, and P-CD were acquired using a JEOL JEM-2100F microscope.
XPS, FT-IR, and Raman spectra of CDs were obtained with a Kratos Axis Ultra DLD spectrometer,
Nicolet AVATAR 370 spectrophotometer, and Renishaw in plus laser Raman spectrometer,
respectively. NMR of these CDs were obtained by Bruker Avance NEO 500MHz.
Phosphorescence lifetimes were determined via an Edinburgh Instruments FLS 1000
spectrofluorometer. UV-vis-NIR absorption and fluorescence spectra were detected by Agilent
Cary 5000 and Hitachi 7000 spectrophotometer, respectively.

Photothermal performance measurements
The photothermal properties of P-CD were studied by irradiating centrifuge tubes containing P-
CD solutions (0-0.3 mg/mL, 1 mL) for 5 min. The temperature of the P-CD solution was measured
every 30 s by Fluke infrared thermal imager. The photothermal conversion efficiency of P-CD was
calculated according to the following four formula.
𝜂 = hA(Tmax−Tsurr )−Q
Dis (1)
I(1−10−Aλ)
θ = T−Tsurr
(2)
Tmax−Tsurr
τ = mDCD
(3)
s
hA
t = −τ(ln θ) (4)

Photodynamic performance measurements
The generation efficiency of ROS was recorded by measuring the absorbance of DPBF (Aladdin,
China) at 418 nm after irradiating the mixture of 30 μg DPBF and P-CD (0.1 mg/mL) with an 808
nm laser (0.3 W/cm2) for 10 min.

The generation efficiency of O •ˉ was determined by recording the green fluorescence of
2
DHR123 (Aladdin, China) at 525 nm after irradiating the mixture of 10 μM DHR123 and P-CD
(0.1 mg/mL) under the same laser and time conditions. To detect O
2
•ˉ under hypoxia, a similar
method was performed except that the mixed solution was bubbled with N for 15 min before
2
exposure to 808 nm laser.

For the ESR measurements of 1O and O •ˉ, TEMP (20 μL) (Sigma) and DMPO (10 μL) (Sigma)
2 2
were respectively added to the P-CD solution (0.1 mg/mL). The ESR spectrometer is used to detect
1O and O •ˉ produced by P-CD for 5 min under 808 nm laser irradiation (0.3 W/cm2). The 1O and
2 2 2
O •ˉ generation efficiencies of O-CD and M-CD were also obtained under the same conditions.
2
We then utilized an indirect method to determine the quantum yield (QY) of 1O produced by
2
P-CD. Free ICG solution was used as a comparison standard with a 1O QY of 0.2%. The 1O QY
2 2
of the P-CD solution was calculated using the following Eq. (1)
Φ = Φ ( K sample)( A ICG ) (1)
sample ICG
KICG A
sample
Where K and K represent the decomposition rate constants of DPBF by P-CD and free
sample ICG
ICG solution, respectively. A and A are the integral areas of the optical absorption bands
ICG sample
between 808 and 810 nm by P-CD and free ICG solution, respectively.

DFT details
All quantum chemical calculations were performed using the Gaussian 16 program package.
The geometries of all molecules were optimized within the framework of DFT using the B3LYP
functional in combination with the def2-SVP basis set, without imposing any symmetry constraints.
Vibrational frequency analyses were carried out at the same level of theory to confirm that all
optimized structures correspond to true minimum on the potential energy surface, with no
imaginary frequencies. Based on the optimized ground-state geometries, the energy and spatial
distributions of the frontier molecular orbitals, including HOMO and LUMO, were calculated to
analyze the intramolecular charge transfer characteristics. Excited-state properties were
investigated using time-dependent DFT (TD-DFT) to obtain the low-lying singlet and triplet
excited-state energy levels. The resulting data were used to construct excited-state energy level
diagrams, providing insight into the fluorescence and phosphorescence transition processes.
Molecular electrostatic potential (ESP) surface analyses were performed on the optimized ground-
state structures. The electrostatic potential distributions were calculated and visualized using the
Multiwfn program to elucidate the surface charge distribution characteristics of the molecules. In
addition, Fukui function analyses were carried out to identify potential reactive sites and regions
with high electronic responsiveness, thereby assisting in understanding charge redistribution
behaviors in the excited states. All molecular orbital isosurfaces and electron density-related
images were visualized using VMD and Gauss View.

MTT assay
To evaluate the cytotoxicity of M-CD, P-CD, and P-CD@MM on NIH-3T3 (Haixing
Biosciences, TCM-C752, China) and 4T1 cells (Haixing Biosciences, TCM-C705, China), NIH-
3T3 or 4T1 cells were first inoculated and cultured in 96-well plates, and then incubated with
different concentrations of M-CD, P-CD, and P-CD@MM for 24 h. The survival rate was detected
by standard MTT assay. After incubating with M-CD, P-CD and P-CD@MM for 4 h, NIH-3T3 or
4T1 cells were irradiated with 808 nm laser (0.3 W/cm2) for 5 min, and then incubated for another
24 h.

Live/dead cell staining, ROS detection, O •ˉ detection, and apoptosis assay
2
4T1 cells were seeded in 6-well plates at 2  105 cells per well. They were treated with PBS,
808 nm laser (0.3 W/cm2), P-CD, P-CD@MM, P-CD + laser, or P-CD@MM + laser. After 24 h
of incubation, cells were stained with Calcein-AM (488 nm) and PI (561 nm) (Beyotime, China),
and live/dead cell fluorescence images were captured via fluorescence microscopy (Olympus,
Japan). For ROS staining, the cells were stained with DCFH-DA and DAPI (Beyotime, China).
Confocal images were captured using a confocal microscope (Olympus, Japan) to assess
intracellular ROS levels. For O •ˉ staining, replace the culture medium with DHE staining solution
2
(Beyotime, China) and incubate for 30 min. Then, the cells were exposed to an 808 nm (0.3 W/cm2)
laser for 5 min and imaged under a confocal microscope. For apoptosis analysis, cells were
collected by centrifugation (300 × g, 5 min) and apoptosis was detected using an Annexin V-
FITC/PI kit (Dojindo, Japan) via flow cytometry (Beckman, CytoFLEX LX).

In vitro ICD marker detection
4T1 cells were seeded in a 6-well plate at 2  105 cells per well and treated with control, laser
(808 nm, 0.3 W/cm2), P-CD, P-CD@MM, P-CD + laser, or P-CD@MM + laser. The intracellular
ATP and HMGB1 levels were measured using an ATP kit (Beyotime, China) and an HMGB1 kit
(Beyotime, China), respectively. CRT was detected using confocal microscope after 4T1 cells were
incubated with calreticulin rabbit mAb (Beyotime, AF1666, 1:500) and FITC-conjugated goat-
anti-rabbit IgG (Beyotime, A0562, 1:500).

In vitro evaluation of DC maturation
BMDCs were extracted from the femurs and tibias of 5-week-old Balb/c mice and cultured for
7 days in 1640 medium with GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) (Beyotime, China). Then,
BMDCs were co-incubated for 24 h with 4T1 cell suspensions that had been treated with control,
laser (808 nm, 0.3 W/cm2), P-CD, P-CD@MM, P-CD + laser, or P-CD@MM + laser. BMDCs
were collected and stained with anti-CD11c-FITC (Biolegend, 117306, 1:200), anti-CD80-PE
(Biolegend, 104707, 1:200), and anti-CD86-APC (Biolegend, 105011, 1:200), then analyzed by
flow cytometry.

In vitro macrophage polarization evaluation
RAW264.7 macrophages (M0-like phenotype) were first cultured in a 6-well plate for 24 h and
then treated with IL-4 (20 ng/mL) to differentiate into M2-like phenotype. These cells were treated
with PBS, laser (808 nm, 0.3 W/cm2), P-CD, P-CD@MM, P-CD + laser, or P-CD@MM + laser.
Finally, these cells were collected and analyzed by flow cytometry after staining by anti-F4/80-
APC (Biolegend, 123116, 1:200), anti-CD86-PE (Biolegend, 159204, 1:200), and anti-CD206-
FITC (Biolegend, 141704, 1:200). We also utilized BMDMs to evaluate the polarization effect of
P-CD@MM-mediated combination therapy. BMDMs were extracted from the tibiae and femurs
of 5-week-old Balb/c mice and then treated with M-CSF and IL-4 to obtain M2-like phenotype.

Western blot analysis
Proteins in macrophage lysates, macrophage membranes, and P-CD@MM were characterized
by Western blot analysis. Cells were lysed in RIPA buffer containing 1% PMSF. Protein
concentrations were measured using the BCA method. Twenty micrograms of protein were
separated by SDS-PAGE and transferred to a PVDF membrane, which was blocked with 5% skim
milk at room temperature for 1 h and washed with TBST. The membrane was incubated overnight
at 4 oC with primary antibodies: anti-β-actin Rabbit mAb (ABclonal, AC026, 1:5000), anti-CD206
Rabbit mAb (ABclonal, A26948, 1:5000), and anti-CD86 Rabbit mAb (ABclonal, A21198,
1:10000). STAT1 monoclonal antibody (Proteintech, 66545-1-Ig, 1:5000), p-STAT1 monoclonal
antibody (Proteintech, 82674-10-RR, 1:2000), NF-κB p65 monoclonal antibody (Proteintech,
80979-1-RR, 1:5000), phospho-NF-κB p65 monoclonal antibody (Proteintech, 82335-1-RR,
1:2000), After washing with TBST, the membrane was incubated with the secondary antibody
HRP-labeled Goat Anti-Rabbit IgG (H+L) (Beyotime, A0208, 1:8000) or secondary antibody
HRP-labeled Goat Anti-Mouse IgG (H+L) (Beyotime, A0216, 1:8000) at room temperature for 2
h. Finally, the membrane was washed with TBST and the images were captured using a digital gel
imaging system.

In vivo antitumor effectiveness evaluation in bilateral tumor model
Balb/c mice, 5 weeks old, were purchased from SLAC Laboratory Animal Co. in Shanghai,
China. On day -8, 1 × 106 4T1 cells were implanted into the right axilla of the mice to establish a
primary tumor. On day -1, 1 × 106 4T1 cells were implanted into the left axilla to create a distant
tumor. Mice were divided into six groups for different treatments (PBS, laser (808 nm, 0.3 W/cm2),
P-CD, P-CD@MM, P-CD + laser, P-CD@MM + laser). On day 0, mice were treated with PBS,
P-CD, or P-CD@MM via intravenous injection. On day 1, the primary tumor was irradiated with
a laser for 10 min. On day 7, tumors were collected for H&E and TUNEL staining. Primary tumors,
distant tumors, spleens, and lymph nodes were collected to assess ICD markers, DC maturation,
and T cell activation. We measured the HMGB1 and ATP level in the supernatant of tumor tissue
homogenate. Tumor tissues of equal mass were processed identically by adding 500 μL of RIPA
lysis buffer and homogenizing on ice. Following homogenization, samples were centrifuged at
12,000 rpm for 15 min at 4 °C, and the resulting supernatants were collected. ATP levels were
quantified using an ATP assay kit, while HMGB1 concentrations were determined by ELISA. The
evaluation of ICD markers and DC maturation level was similar to the cellular experiments. For
the evaluation of T cells, single cell suspensions of tumor tissues and spleen were obtained after
mixed collagenase digestion and filtration using cell strainers. Then, anti-FITC-CD3 (Biolegend,
100203, 1:200), anti-APC-CD4 (Biolegend, 100412, 1:200) and anti-PE-CD8a (Biolegend,
162304, 1:200) were utilized to stain cells, which were then analyzed by flow cytometer.

In vivo antitumor effectiveness evaluation in rechallenge tumor model
To assess the long-term immune memory response induced by P-CD@MM + L, a tumor re-
challenge model was established. 4T1 tumor-bearing Balb/c mice in each group were
intravenously injected with PBS or P-CD@MM on days 0, 2, and 4. After 24 h, tumors were
irradiated with an 808 nm laser (0.3 W/cm2) for 10 min. After tumors were eradicated by P-
CD@MM + L, on day 30, 4T1 cells were injected into the opposite axilla of mice to fabricate the
rechallenge tumor model. Another 6 healthy mice were utilized as the control, which were also
inoculated by 4T1 cells. On day 50, spleens were harvested to analyze the proportion of TEM
(CD3+) cells, particularly CD8+ T cells exhibiting high CD44 and low CD62L expression. The
single cell suspensions of spleen were stained by anti-CD3-FITC (Biolegend, 102023, 1:200), anti-
CD8a-PE (Biolegend, 162304, 1:200), anti -CD44-PE-Cy7 (Biolegend, 103030, 1:200), and anti-
CD62L-APC (Biolegend, 161218, 1:200) and then analyzed by flow cytometer.

Pharmacokinetic and metabolism evaluation
Balb/c mice aged 5 weeks (n = 3) were intravenously administered P-CD or P-CD@MM at a
dose of 2.0 mg/kg of P-CD. For pharmacokinetic study, blood samples (10 μL) were collected
from the tail vein at predetermined time points. Then, blood samples were transferred into
heparinized microcentrifuge tubes, followed by dilution with 990 μL of PBS. The samples were
centrifuged at 1500 × g for 10 min to isolate plasma. Both P-CDs and P-CD@MM exhibited
plasma concentration-time profiles characteristic of a two-compartment pharmacokinetic model,
indicating biphasic distribution and elimination kinetics in vivo. For metabolism evaluation, the
major organs were collected on the 1st, 7th, and 14th day after administration. In the excretion
pathway study, Balb/c mice were intravenously injected with the same dose of P-CD@MM, and
then immediately placed in metabolic cages to collect fecal and urine samples. The collected
samples (organs, feces, or urine) were digested with aqua regia and the absorbance at 720 nm was
measured to determine the metabolism and excretion pathway of P-CD@MM.

Statistical analysis
Data are representative results from at least three independent experiments and are presented as
mean ± standard deviation. All statistical analyses were performed using Origin 2024 and Excel
software. Statistical comparisons were performed using one-way ANOVA for multiple
comparisons or two-tailed Student’s t-test for comparisons between two groups.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study reports the molecular engineering of NIR-responsive CDs as low-efficacy photothermal but high-efficacy type-I/Ⅱ photodynamic agents for hypoxia-regulated photodynamic immunotherapy. The ultralong carrier lifetime (36.32 ms) of P-CDs was six orders of magnitude longer than singlet excited states observed in previously reported CDs (several ns). The authors demonstrated that the phosphorescent P-CDs have a D-π-A configuration with a long-lived intramolecular charge transfer (CT) state, which may facilitate electron transfer between the donor and acceptor groups through an extended π bridge for enhancing type-I/Ⅱ photodynamic performances. Therefore, whether from the exploration of experimental results or mechanisms, this work exhibits high innovation. In my opinion, this work is important for the application of CDs for NIR-photoimmunotherapy through D-π-A sensitization, triplet state stabilization, surface charge transfer, and mild photothermal relief of hypoxia. Therefore, this manuscript should be considered for publication in Nature Communications after a minor revision.\n1.The cyclic test of photothermal stability is crucial for evaluating photothermal performance. It is suggested that the authors assess the performance attenuation of M-CD under multiple laser irradiations.\n2.For in vivo immune activation, the higher immune response induced by P-CD@MM + Laser compared with P-CD + Laser was observed. Please explain the reason.\n3.More biocompatibility evaluation should be performed, such as hemolysis test.\n4.The metabolic pathway of P-CD@MM should be investigated.\n5.The authors should provide the source of the materials during the preparation of CDs. In addition, the experimental details should be supplemented.\n6.The statistical significance should be added in the MTT results.", "reviewer_2": "The authors successfully constructed near-infrared-excited carbon dots (P-CDs) with a D–π–A configuration and employed them as novel photosensitizers possessing both type I and type II activities. Furthermore, by encapsulating the P-CDs with M1 macrophage membranes (MM), they developed a biomimetic nanoplatform termed P-CD@MM for precise photodynamic immunotherapy. Addressing key limitations of conventional photodynamic therapy—such as tumor hypoxia, limited tissue penetration, and metal-induced toxicity—this work proposes an all-metal-free, carbon dot-based photosensitizing system that can be activated by near-infrared light and exhibits dual type I/II photodynamic activities. The study demonstrates significant theoretical innovation and considerable potential for clinical translation. However, this study is subject to certain limitations that need to be addressed. A more comprehensive evaluation of the manuscript would be warranted once these concerns are resolved.\n1. In this study, the authors prepared four types of carbon dots using OPD, MPD, and PPD, respectively, in combination with ICG. However, the formation mechanism remains unclear: First, in the co-precursor system comprising ICG and the three phenylenediamine isomers, which component serves as the primary precursor for the carbon core? Second, by what specific chemical bonds (e.g., covalent bonds) or intermolecular interactions are these molecules effectively integrated?\n2. The manuscript lacks direct evidence defining ICG's role in the D-π-A system. Its specific function—as an electron donor or acceptor—and the quantitative impact of its inclusion on the material's photodynamic performance are unclear. These aspects are fundamental to assessing the design's validity and innovation.\n3. The assertion that hydroxyl groups serve as electron donors in the proposed D–π–A model currently lacks sufficient experimental and theoretical support. Given that the synthetic precursors contain no hydroxyl groups, the origin of such functional groups and their specific role as electron donors in charge separation require a thorough explanation. The current inference of the D–π–A structure relies primarily on XPS spectra and optical properties, which provide only indirect evidence. We recommend supplementing the study with theoretical calculations, such as density functional theory (DFT), to visually illustrate molecular orbital distributions, electron localization, and charge transfer pathways, thereby furnishing more convincing electronic structure-level evidence for the proposed model.\n4. The authors' interpretation of the highest ID/IG ratio observed in P-CDs remains superficial, merely attributing it to the introduction of surface functional groups. To gain deeper insight into the fundamental nature of the structural defects, further clarification is needed: during material formation, does the specific structure of the precursor molecule (e.g., the para-configuration of PPD) impede the generation of ordered graphitic domains, or does the functionalization process itself disrupt the continuous growth of sp²-hybridized carbon domains?\n5. The use of ICG-labeled P-CD@MM, instead of the carbon dots' intrinsic fluorescence, undermines the interpretation of cellular uptake. This approach cannot confirm whether the signal comes from the nanoparticles or free/leaked ICG, and requires justification.\n6. The experimental design that uses the red channel to represent CRT exposure is questionable. Since the P-CD@MM nanomaterials themselves may produce background fluorescence in the red light region, this would cause the red fluorescence signal of CRT to be mixed with the background fluorescence, thereby making it impossible to draw reliable conclusions about the localization and expression of CRT. To clearly prove the membrane translocation of CRT, a channel (such as the green channel) that does not overlap with the emission spectrum of carbon dots must be used for labeling.\n7. The citations of figure numbers (e.g., Fig. 5a, Fig. 5e and Fig.5f) in parentheses throughout the manuscript appear in a disordered sequence and do not follow the order in which they are first mentioned in the text. This inconsistency disrupts the flow and professionalism of the narrative. It is recommended that the authors systematically review the entire manuscript and reorganize all figure citations according to the logical sequence of result presentation.", "reviewer_3": "This manuscript pioneers the molecular engineering of carbon dots (CDs) with a D-π-A configuration, achieving the regulation of ultralong triplet excited states (36.32 ms) and breaking the bottleneck of short excited-state lifetimes (nanosecond scale) in traditional CDs. The research proposes precise regulation of the photothermal/photodynamic properties of CDs by modulating nitrogen doping types (pyridinic N/graphitic N) and combines M1 macrophage membrane camouflage technology to synergistically improve tumor targeting and immune microenvironment remodeling. This design provides a novel paradigm for photodynamic immunotherapy of hypoxic tumors, demonstrating prominent innovation. However, there are some issues need to be addressed before acceptance for publishment.\n1. Direct comparative data on key indicators (e.g., excited-state lifetime, ROS generation efficiency, hypoxic adaptability) between D-π-A configured CDs, traditional CDs, and commercial photosensitizers (such as ICG and porphyrins) should be provided to further highlight the advantages of the configuration design.\n2. Theoretical calculations (e.g., DFT) should be conducted to explain the effects of different nitrogen doping types (pyridinic N, graphitic N, pyrrolic N) on the electronic structure and charge transfer efficiency of CDs, to strengthen the mechanistic interpretation of the \"configuration-property\" correlation.\n3. The photostability of CDs under multiple laser irradiations (e.g., attenuation of ROS generation efficiency, changes in photothermal conversion efficiency) needs to be clarified to support the feasibility of clinical applications.\n4. Tumor recurrence experiments: after primary tumor treatment, perform secondary tumor inoculation in mice, detect the proportion of CD8+ T cell memory subsets (e.g., CD8+CD44+CD62L+) and tumor growth needs to be conducted to further verify the immune memory effect.\n5. Pharmacokinetic experiments of P-CD@MM in vivo (e.g., blood clearance curve, organ distribution and metabolism patterns) should be performed to clarify its biosafety and in vivo retention characteristics.\n6. Please increase the number of biological replicates (n≥6 is recommended) for key experiments (e.g., ROS generation, DC maturation rate, T cell infiltration) and supplement detailed parameters of statistical analysis (e.g., results of homogeneity of variance test).\n7. Pleas add characterization data of M1 macrophage membranes. For example, quantitatively detect the purity of membrane surface markers (iNOS, CD86) by Western blot, and supplement stability tests of the cell membranes during storage (e.g., changes in Zeta potential, duration of integrity maintenance).\n8. Please optimize the in vivo laser irradiation protocol: clarify the matching relationship between laser spot size, irradiation time, and tumor volume, and supplement comparative data on therapeutic effects under different irradiation parameters to standardize the protocol.\n9. Please supplement negative control experiments: set up a \"P-CD camouflaged with empty cell membranes\" group to exclude the interference of membrane components themselves on the immune response, and further confirm the core role of P-CD in photodynamic therapy.", "reviewer_4": "In this study, the authors construct three types of NIR-responsive carbon dots (CDs) by using a type-Ⅱ organic photosensitizer/NIR probe (ICG) as the active ingredient and three isomers of o-, m-, and p-phenylenediamines (OPD, MPD, PPD) as regulators, to obtain phototherapy agents with low-efficacy photothermal but high-efficacy type-I/Ⅱ photodynamic activity. After evaluating their photothermal and photodynamic properties, P-CDs was selected as the superior type-I/Ⅱ photosensitizers, and subsequently coated with M1-macrophage cell membranes to form P-CD@MM nanoparticles for potent photo-immunotherapy. Although the study presents many data, there are lots of major issues that must be addressed prior to any further consideration.\n1. Given the extensive prior work on M1-macrophage membrane-coated nanoparticles and PDT-immunotherapy, the latter part of this study lacks novelty. Moreover, the authors have previously reported similar NIR phosphorescent CDs constructed using ICG as a precursor, which were also coated with cancer cell membranes for precision SDT (Nature Communications (2022) 13:5735). Compared with this previous work, the improvements in this study are minimal.\n2. The advantages of this study and the key problems it aims to address remain unclear. Importantly, many critical data are lacking in the current manuscript.\n1) Direct characterization about structure of O-CDs, M-CDs, and P-CDs should be provided, instead of only presenting TEM, XPS, and FT-IR data.\n2) The authors described in the Introduction that “type-Ⅱ PDT and mild PTT may synergize to enhance phototherapy efficacy owing to partial alleviation of hypoxia by mild hyperthermia, which can increase tumor blood flow and improves hemoglobin oxygen saturation within the tumor vasculature”, however, the current manuscript only provides HIF-α immunofluorescence staining and does not include other experiments demonstrating hypoxia alleviation by mild hyperthermia, such as tumor blood flow, or hemoglobin oxygen saturation.\n3) The authors claimed to develop superior type-I/II photosensitizers with matching photothermal effects for robust photoimmunotherapy; however, the study lacks comparative experiments between O-CDs/M-CDs/P-CDs and widely used photosensitizers or photothermal agents, making it impossible to determine the actual advantages of the proposed system or to assess its practical therapeutic potential.\n3. Please provide more data about the D-π-A sensitized type II/I photodynamic mechanism of CDs with enhanced charge-transfer dynamics,\n4. In Fig. 4c, it is unclear why iNOS appears in the M1 membrane, as it is typically a cytoplasmic protein.\n5. In Fig. 4f, only the relative cell viability of 4T1 cells after laser irradiation is presented; please include corresponding experiments using NIH/3T3 cells. Moreover, the number of live cells appears to decrease in P-CD@MM group compared with the control group in Fig. 4g, which shows an inconsistent trend with Fig. 4e.\n6. In Fig. 5f and Supplementary Fig. 22, whether the measured ATP levels in the intracellular or supernatant? Please add more details about this issue, especially the in vivo experiments.\n7. In Supplementary Fig. 23, gating on CD11b+ is not appropriate for identifying DCs.\n8. For better comparison, please provide ex vivo NIR imaging and quantification of major tissues and tumors for P-CD, similar to the data shown for P-CD@MM in Fig. 6d and Supplementary Fig. 18."}, "author_response": "Itemized Responses to Reviewers’ Comments\nReviewer #1\nThis study reports the molecular engineering of NIR-responsive CDs as low-efficacy photothermal but high-efficacy type-I/Ⅱ photodynamic agents for hypoxia-regulated photodynamic immunotherapy. The ultralong carrier lifetime (36.32 ms) of P-CDs was six orders of magnitude longer than singlet excited states observed in previously reported CDs (several ns). The authors demonstrated that the phosphorescent P-CDs have a D-π-A configuration with a long-lived intramolecular charge transfer (CT) state, which may facilitate electron transfer between the donor and acceptor groups through an extended π bridge for enhancing type-I/Ⅱ photodynamic performances. Therefore, whether from the exploration of experimental results or mechanisms, this work exhibits high innovation. In my opinion, this work is important for the application of CDs for NIR-photoimmunotherapy through D-π-A sensitization, triplet state stabilization, surface charge transfer, and mild photothermal relief of hypoxia. Therefore, this manuscript should be considered for publication in Nature Communications after a minor revision.\nReply: We thank the reviewer for the positive evaluation and kind recommendation. We have addressed all of Reviewer 1’s comments and concerns in detail, and the corresponding revisions have been made in the revised manuscript and supporting information. Our responses to these issues are attached as follows.\n1. The cyclic test of photothermal stability is crucial for evaluating photothermal performance. It is suggested that the authors assess the performance attenuation of M-CD under multiple laser irradiations.\nReply: Thanks for your suggestion. We have performed photothermal stability evaluation of M-CD. As shown in Supplementary Fig. 10, after 9 cycles of heating and cooling, the temperature of M-CD still reached 53.7 ℃ under 808 nm laser irradiation (0.3 W/cm2), indicating the excellent photothermal stability of M-CD. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 10. Photothermal stability evaluation of M-CD. Source data are provided as a Source Data file.\n2. For in vivo immune activation, the higher immune response induced by P-CD@MM + Laser compared with P-CD + Laser was observed. Please explain the reason.\nReply: Thanks for your suggestion. Firstly, at the cellular level, the M1-type macrophage membrane can inherently promote maturation of dendritic cells (DCs) and M1 polarization (Biomaterials, 2024, 305, 122456), enabling the P-CD@MM + laser treatment to elicit a significantly enhanced immune response. Secondly, M1-type macrophage membrane (MM) encapsulation significantly enhances the tumor targeting and enrichment capabilities. As shown in Fig. 7b-c and SupplementaryFig. 22, 23, the unencapsulated P-CDs accumulated only minimally at the tumor site due to rapid metabolism in the body. In contrast, after encapsulating the P-CDs with MM, P-CD@MM effectively avoided immune clearance by its biomimetic membrane structure, prolonging the circulation time in the blood and achieving active targeting through the M1-type macrophage-specific proteins retained on the membrane surface (such as CD86). This phenomenon enabled more P-CDs to accumulate in the tumor area (Fig. 5c, d). The higher local tumor concentration means that a stronger photodynamic effect can be generated under NIR laser irradiation, thereby triggering stronger ICD to activate antitumor immune response. We have added the corresponding discussion in the revised manuscript.\n3. More biocompatibility evaluation should be performed, such as hemolysis test.\nReply: Thanks for your suggestion. We have performed the hemolysis test of P-CD@MM. As shown in SupplementaryFig. 41, hemolysis assays confirmed outstanding blood compatibility of P-CD@MM, with a hemolysis rate below 5% at concentration of 100 μg/mL. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 41. The hemolysis test of P-CD@MM (n = 3 independent samples). Data are presented as mean ± SD. Source data are provided as a Source Data file.\n4. The metabolic pathway of P-CD@MM should be investigated.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we explored the in vivo biodistribution and excretion pathways of P-CD@MM. Supplementary Fig. 38a showed that P-CD@MM mainly accumulated in the liver at 24 h post-injection, which was attributed to the capture of the reticuloendothelial system. Importantly, no obvious P-CD signal was observed in these organs after 14 days, indicating that P-CD@MM has been almost completely cleared. To study the excretion pathways, the concentrations of P-CD in urine and feces were also measured. The results indicated that P-CD@MM was mainly excreted from the mice through the liver and kidney. We also noted that no P-CD signal can be detected after 14 days (Supplementary Fig. 38b), suggesting the complete clearance of P-CD@MM. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 38. (a) Biodistribution of P-CD@MM post i.v. injection in mice on different days (n = 6 independent samples). (b) The detected P-CD mass in urine and feces at different time points post i.v. injection of P-CD@MM (n = 6 independent samples). Data are presented as mean ± SD. Source data are provided as a Source Data file.\n5. The authors should provide the source of the materials during the preparation of CDs. In addition, the experimental details should be supplemented.\nReply: Thanks for your suggestion. We have provided the source of the materials and supplemented the experimental details in the revised manuscript.\nFor example, during the synthesis of P-CDs, ICG (5 mg) (Aladdin, China) and p-phenylenediamine (10 mg) (Aladdin, China) were dissolved in ethanol (10 mL) (Sinopharm Chemical Reagent Co., Ltd., China). The solution was transferred into a 25 mL Teflon-lined autoclave and reacted at 150 °C for 4 h. The reactor is then cooled to room temperature. Large particles were removed by 0.22 μm membrane, followed by dialysis for 36 h using a dialysis bag. The preparation methods for M-CDs and O-CDs were analogous to that of P-CDs, with the exception that meta-phenylenediamine (Aladdin, China) was used as the precursor for M-CDs and ortho-phenylenediamine (Aladdin, China) for O-CDs.\n6. The statistical significance should be added in the MTT results.\nReply: Thanks for your suggestion. We have calculated and added statistical significance in the MTT results.\nFig. 5 (e, f) Relative cell viability of NIH-3T3 and 4T1 cells after different treatments (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. P value: ***P < 0.001. Source data are provided as a Source Data file.\nSupplementary Fig. 16. Relative cell viability of NIH-3T3 and 4T1 cells after different treatments (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. P value: ***P < 0.001. Source data are provided as a Source Data file.\nSupplementary Fig. 17. Relative cell viability of NIH-3T3 cells after being treated with P-CD@MM in the presence of NIR laser irradiation (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. Source data are provided as a Source Data file.\nReviewer #2\nThe authors successfully constructed near-infrared-excited carbon dots (P-CDs) with a D–π–A configuration and employed them as novel photosensitizers possessing both type I and type II activities. Furthermore, by encapsulating the P-CDs with M1 macrophage membranes (MM), they developed a biomimetic nanoplatform termed P-CD@MM for precise photodynamic immunotherapy. Addressing key limitations of conventional photodynamic therapy—such as tumor hypoxia, limited tissue penetration, and metal-induced toxicity—this work proposes an all-metal-free, carbon dot-based photosensitizing system that can be activated by near-infrared light and exhibits dual type I/II photodynamic activities. The study demonstrates significant theoretical innovation and considerable potential for clinical translation. However, this study is subject to certain limitations that need to be addressed. A more comprehensive evaluation of the manuscript would be warranted once these concerns are resolved.\nReply: We thank the reviewer for the positive evaluation and kind recommendation. We have addressed all of Reviewer 2’s comments and concerns in detail, and the corresponding revisions have been made in the revised manuscript and supporting information. Our responses to these issues are attached as follows.\n1. In this study, the authors prepared four types of carbon dots using OPD, MPD, and PPD, respectively, in combination with ICG. However, the formation mechanism remains unclear: First, in the co-precursor system comprising ICG and the three phenylenediamine isomers, which component serves as the primary precursor for the carbon core? Second, by what specific chemical bonds (e.g., covalent bonds) or intermolecular interactions are these molecules effectively integrated?\nReply: Thanks for your questions about the formation mechanism of CDs synthesized from the co-precursor systems.\nTo date, the exact formation mechanism of all reported CDs under hydrothermal or solvothermal conditions has not been elucidated clearly owing to a lack of in situ, real-time tools for direct observation of evolution processes of organic molecules as precursors. In our cases, for the synthesis of O-CDs, M-CDs, and P-CDs from the relatively complex binary co-precursor systems, the CD formation may involve multiple steps, such as initial fusion/polymerization, partial decomposition, and final graphitization under the low-temperature (150 °C), short-time (4 h) solvothermal synthesis conditions. Besides the binary precursors, the alcohol solvent also participated in these reaction processes because phenolic hydroxyl at high content was incorporated into the CDs. Therefore, we speculated that these molecules could be initially integrated through a hydrogen bond network formed among molecules of ICG, phenylenediamine isomers, and alcohol for driving subsequent solvothermal reactions.\nIn response to another question “which component serves as the primary precursor for the carbon core”, we proposed that the core of CDs should be more graphitized that the heteroatom functional edge of CDs. Considering that M-CDs and P-CDs from the binary combination of ICG + MPD, ICG + PPD have a much higher graphitization level than CDs from ICG only, the phenylenediamine component may serve as the primary precursor for construction of the carbon core as well as N-heteroatom modification at the edge.\nWe have added the corresponding discussion in the revised manuscript.\n2. The manuscript lacks direct evidence defining ICG's role in the D-π-A system. Its specific function—as an electron donor or acceptor—and the quantitative impact of its inclusion on the material's photodynamic performance are unclear. These aspects are fundamental to assessing the design's validity and innovation.\nReply: Thanks for your suggestion on defining of ICG's role in the construction of the D-π-A system and further understanding the quantitative impact of its inclusion on the D-π-A system's photodynamic performance.\nFirst, the ICG precursor indirectly provided D-π-A sensitized CDs with the D-type modification, majorly because ICG as electrophilic centers at positively charged N sites can react with nucleophilic alcohol, leading to the electron-rich functionalization of P-CDs with rich -OH groups as donors (7.92 at.%). Although the ICG precursor also provided P-CDs with electron-deficient modification by inclusion of an acceptor -SO H/-SO ˉ, but there was a much lower doping level for the S-based acceptor (1.80 at.%) than the N-based acceptor (pyridinic N 8.68at.%) from another precursor (PPD). In other words, the A-type modification in P-CDs was largely ascribed to PPD rather than ICG.\nSecond, the new D-π-A system in the type-I/Ⅱ dual photosensitizer (P-CD) was different from the previously reported D-π-A system in the type-Ⅱ photosensitizer (termed p-n-CDs) with an intermolecular p-n junction (Nat. Commun. 2022, 13, 5735). The p-n-CDs were synthesized by microwave-assisted co-treatment of ICG and BPEI (branched polyethylenimine) as dual precursors. In the p-n-CDs, the D-π-A configuration was composed of -SO H/-SO ˉ groups as the electron acceptor (A) and pyrrolic N primarily as the electron donor (D). Notably, the p-n-CDs exhibited no activity in O •ˉ generation under ultrasonic or photo-irradiation (Supplementary Fig. 42), suggesting that the type-I mechanism was deactivated in the pyrrolic N/-SO H configuration with a high S doping level (8.1 at.%). We speculated that -SO H/-SO ˉ groups enriched in p-n-CDs with the strong electron-withdrawing capability may block photoinduced electron transfer from CDs to molecular O , leading to ineffective generation of O •ˉ. In the new D-π-A system with high photosensitization activity in both type-I and type-Ⅱ, however, the electron withdrawing effect by the strong S-based acceptor was greatly weakened owing to the low S doping level (1.80 at.%), and the high-content pyridinic N (8.68 at.%) as a weak N-based acceptor plays a crucial role in the D-π-A system for optimization of desirable photodynamic types, activation wavelength, and ultra-stable triplets.\nCollectively, to overcome such limitations as short excited-state lifetime (ns) and absence of NIR-photodynamic activity of common CDs, and deactivated type-I mechanism in the type-Ⅱ D-π-A photo-system, we designed the novel type-I/Ⅱ D-π-A NIR-photo-system by changing the chemical components of the D-π-A configuration, i.e. using electron-rich phenolic hydroxyl groups as donors and pyridine N with weak electron withdrawing ability as acceptors. this work demonstrates that it is possible to improve the electron donating and withdrawing ability of long-lived excited states within the D-π-A framework to activate the type-I photodynamic mechanism under NIR irradiation.\nSupplementary Fig. 42. (a-d) Measurements of the Type I and II photodynamic activity of p-n-CDs under 660 or 808 nm laser irradiation. (e, f) Measurements of the Type I and II sonodynamic activity of p-n-CDs under US irradiation.Source data are provided as a Source Data file.\n3. The assertion that hydroxyl groups serve as electron donors in the proposed D-π-A model currently lacks sufficient experimental and theoretical support. Given that the synthetic precursors contain no hydroxyl groups, the origin of such functional groups and their specific role as electron donors in charge separation require a thorough explanation. The current inference of the D-π-A structure relies primarily on XPS spectra and optical properties, which provide only indirect evidence. We recommend supplementing the study with theoretical calculations, such as density functional theory (DFT), to visually illustrate molecular orbital distributions, electron localization, and charge transfer pathways, thereby furnishing more convincing electronic structure-level evidence for the proposed model.\nReply: Thanks for your suggestion. As shown in Fig. 1i and Supplementary Fig. 4-6, O-CDs, M-CDs, and P-CDs exhibited strong oxygen signals. High-resolution O 1s spectrum revealed the peak of O-H, providing direct evidence for the presence of hydroxyl groups. Furthermore, FT-IR analysis showed a broad absorption band at ~3430 cm-1 for O-CDs, M-CDs, and P-CDs, characteristic of O–H stretching vibrations, which further confirmed the existence of hydroxyl groups (Supplementary Fig. 7). Because O-CDs, M-CDs, and P-CDs all contain hydroxyl groups, we believe that the hydroxyl groups may originate from ethanol solvents during the solvothermal synthesis of O-CDs, M-CDs, and P-CDs.\nAccording to the reviewer’s suggestion, we performed the density functional theory (DFT) calculations to visually illustrate molecular orbital distributions, electron localization, and charge transfer pathways. We establish three kinds of sp2 hybrid carbon molecules as simple CD models according to the theoretical models presented in Fig. 1h. Fig. 4a exhibits the spatial distributions of the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) for O-CD, M-CD, and P-CD. In the P-CD model, the HOMO is predominantly localized on the hydroxyl-rich region (\"donor\", D), whereas the LUMO is strongly concentrated on the pyridinic nitrogen-containing domain (\"acceptor\", A). This clear spatial separation of frontier orbitals directly demonstrates that, in the ground state of P-CD, the highest-energy electrons reside primarily in the donor moiety, while the lowest-energy vacant orbitals are located in the acceptor moiety, with both regions bridged by a conjugated π-system (carbon core). This configuration constitutes definitive evidence for a well-defined D-π-A architecture. Fig. 4b presents the calculated energy levels of the first singlet excited state (S ) and the first triplet state (T ), along with their respective singlet-triplet energy gaps (ΔE ). Notably, P-CD exhibits a significantly smaller ΔE (0.39 eV) compared to O-CD (0.82 eV) and M-CD (0.96 eV). Such a reduced energy gap facilitates efficient intersystem crossing (ISC) from the singlet to the long-lived triplet state. The theoretical calculation results consistent with the experiment results of ultralong phosphorescence lifetime of P-CD (36.32 ms), offering a mechanistic explanation for its excellent performance as a photosensitizer.\nWe then performed the surface electrostatic potential (ESP) analysis of O-CD, M-CD, and P-CD. As depicted in Fig. 4c, red regions represent the negative potential (electron-rich zones) and blue regions indicate positive potential (electron-deficient zones). In the P-CD model, a pronounced negative potential is observed in the hydroxyl-enriched region, contrasting sharply with the positive potential in the pyridinic nitrogen zone. This phenomenon confirms that hydroxyl groups act as the primary electron-donating center (D), while the pyridinic nitrogen serves as the electron-accepting center (A). These findings are further demonstrated by Fukui-DD function analysis (Fig. 4d), which reveals electrophilic features at the pyridinic nitrogen site with a high propensity for electron acceptance. In addition, the nucleophilic features over the hydroxyl and π-bridge regions were consistent with electron donation. These two complementary analyses confirm that the hydroxyl groups function as the strongest electron donor within the P-CD framework, serving as the driving force for intramolecular charge transfer. In summary, the DFT calculations provide comprehensive evidence from orbital distribution, energy-level alignment, and quantitative reactivity descriptors for the proposed D-π-A model. Orbital visualization confirms the spatial segregation of donor and acceptor units linked via a π-conjugated bridge. Energy-level calculations rationalize the experimentally observed long-lived triplet state. Electrostatic potential and Fukui function analyses jointly establish the functional roles of the hydroxyl group as the dominant donor and pyridinic nitrogen as the key acceptor.\nWe have added these results and corresponding discussion in the revised manuscript.\nFig. 4. Theoretical calculation of CDs. (a) Illustration of the frontier molecular orbitals (LUMOs and HOMOs) of O-CD, M-CD and P-CD. (b-d) Singlet and triplet energy levels and ΔE (b), Surface electrostatic potential (c), Fukui-DD function analysis (d) of O-CD, M-CD and P-CD.\n4. The authors' interpretation of the highest I /I ratio observed in P-CDs remains superficial, merely attributing it to the introduction of surface functional groups. To gain deeper insight into the fundamental nature of the structural defects, further clarification is needed: during material formation, does the specific structure of the precursor molecule (e.g., the para-configuration of PPD) impede the generation of ordered graphitic domains, or does the functionalization process itself disrupt the continuous growth of sp2-hybridized carbon domains?\nReply: Thanks for your suggestion. As the reviewer’s said, the para-configuration of PPD could impede the generation of ordered graphitic domains. The symmetrical structure of PPD lacks sufficient spatial degrees of freedom during the carbonization process, thereby hindering effective structural rearrangement and aromatization. Therefore, many disordered structures are formed within the carbon core, leading to the low graphitization level of P-CDs. We have added the corresponding discussion in the revised manuscript.\n5. The use of ICG-labeled P-CD@MM, instead of the carbon dots' intrinsic fluorescence, undermines the interpretation of cellular uptake. This approach cannot confirm whether the signal comes from the nanoparticles or free/leaked ICG, and requires justification.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we investigated the cellular uptake behaviors of P-CD@MM based on the intrinsic fluorescence of P-CD. As depicted in Fig. 5d, confocal imaging of NIH-3T3 normal cells and 4T1 tumor cells exposed to P-CD@MM exhibited a much higher level of cell internalization in cancer cells, further clarifying the augmentation in tumor targeting levels of P-CDs through M1 MM encapsulation (Fig. 5d). We have added these results and corresponding discussion in the revised manuscript.\nFig. 5. (d) Representative fluorescence images of cellular uptake of P-CD@MM (red fluorescence) in NIH-3T3 or 4T1 cells.\n6. The experimental design that uses the red channel to represent CRT exposure is questionable. Since the P-CD@MM nanomaterials themselves may produce background fluorescence in the red light region, this would cause the red fluorescence signal of CRT to be mixed with the background fluorescence, thereby making it impossible to draw reliable conclusions about the localization and expression of CRT. To clearly prove the membrane translocation of CRT, a channel (such as the green channel) that does not overlap with the emission spectrum of carbon dots must be used for labeling.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we performed the CRT immunofluorescence staining experiment using FITC-labeled secondary antibodies. Notable CRT signals were observed in both the P-CD + laser group and the P-CD@MM + laser group, indicating a translocation of CRT from the endoplasmic reticulum to the cell surface (Fig. 6a). We have added these results and corresponding discussion in the revised manuscript.\nFig. 6. (a) Confocal images of CRT exposure in 4T1 cells after different treatments.\n7. The citations of figure numbers (e.g., Fig. 5a, Fig. 5e and Fig. 5f) in parentheses throughout the manuscript appear in a disordered sequence and do not follow the order in which they are first mentioned in the text. This inconsistency disrupts the flow and professionalism of the narrative. It is recommended that the authors systematically review the entire manuscript and reorganize all figure citations according to the logical sequence of result presentation.\nReply: Thanks for your suggestions. We have reorganized Fig. 6 according to the logical sequence of result presentation.\n[Editorial note: icons in figure 6d Created in BioRender. zhenlin, Z. (2026) https:// BioRender.com/9u519d6]\nFig. 6. P-CD@MM-mediated PDT induces ICD and M1 polarization in vitro. (a) Confocal images of CRT exposure in 4T1 cells after different treatments. (b, c) Evaluation of HMGB1 and ATP levels in 4T1 cells after different treatments (n = 6 independent samples). (d) A scheme to show the experimental process of the evaluation of DC maturation. (e, f) Evaluation of DC maturation level in each group (n = 6 independent samples). (g-i) Flow cytometry analysis and the corresponding quantitative results of CD86+ and CD206+ expression in TAMs of BMDM after different treatments (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. P value: **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.\nReviewer #3\nThis manuscript pioneers the molecular engineering of carbon dots (CDs) with a D-π-A configuration, achieving the regulation of ultralong triplet excited states (36.32 ms) and breaking the bottleneck of short excited-state lifetimes (nanosecond scale) in traditional CDs. The research proposes precise regulation of the photothermal/photodynamic properties of CDs by modulating nitrogen doping types (pyridinic N/graphitic N) and combines M1 macrophage membrane camouflage technology to synergistically improve tumor targeting and immune microenvironment remodeling. This design provides a novel paradigm for photodynamic immunotherapy of hypoxic tumors, demonstrating prominent innovation. However, there are some issues need to be addressed before acceptance for publishment.\nReply: We thank the reviewer for the positive evaluation and kind recommendation. We have addressed all of Reviewer 3’s comments and concerns in detail, and the corresponding revisions have been made in the revised manuscript and supporting information. Our responses to these issues are attached as follows.\n1. Direct comparative data on key indicators (e.g., excited-state lifetime, ROS generation efficiency, hypoxic adaptability) between D-π-A configured CDs, traditional CDs, and commercial photosensitizers (such as ICG and porphyrins) should be provided to further highlight the advantages of the configuration design.\nReply: Thanks for your suggestion. To highlight the advantages of the D-π-A configured P-CDs, we compared the excited-state lifetime, ROS generation efficiency, and hypoxic adaptability between D-π-A structured carbon dots (P-CDs), traditional CDs (O-CD), and commercial photosensitizers (ICG, TCPP). First, P-CDs exhibited ultralong triplet state lifetime of 36.32 ms, which was six orders of magnitude longer than those observed for O-CDs and M-CDs (Fig. 3i). The triplet state lifetime of P-CDs was also significantly higher than that of commercial photosensitizers (0.8 ns for ICG and 8.48 ns for TCPP) (Supplementary Table 1). Second, this ultralong triplet state lifetime is a crucial factor in augmenting photodynamic activity of P-CDs. The calculated 1O quantum yield of P-CD was measured to be 1.49%, which was much higher than that of O-CD (0.58%), ICG (0.20%), and TCPP (0.10%). Third, under hypoxic conditions, the P-CD with type I/II photodyanmic mechanism can still effectively produce O •ˉ (Fig. 3b, d). In contrast, no ROS generation of ICG and TCPP can be detected under hypoxic conditions (Supplementary Fig. 13), which could be attributed to their O -depecdent type II photodyanmic mechanism. These results demonstrate that the D-π-A configured P-CDs possess the largest excited-state lifetime, highest ROS generation efficiency, and optimal hypoxic adaptability compared with the traditional CDs and commercial photosensitizers. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 13. Photodynamic activity evaluation of ICG, TCPP, and O-CD at the hypoxia conditions using DPBF (a) or DHR123 (b) as the probe. Source data are provided as a Source Data file.\nSupplementary Table 1. Comparison of D-π-A sensitized CDs with commercial photosensitizers in the excited-state lifetime and 1O generation efficiency.\nType II ICG 0.80 ns 0.20 3,4\nType II TCPP 8.48 ns 0.10 5,6\nReferences\n3. Swamy MMM, Murai Y, Monde K, Tsuboi S, Swamy AK, Jin T. Biocompatible and Water-Soluble Shortwave-Infrared (SWIR)-Emitting Cyanine-Based Fluorescent Probes for In Vivo Multiplexed Molecular Imaging. ACS Appl. Mater. Interfaces16, 17253-17266 (2024).\n4. Cui M, Tang D, Wang B, Zhang H, Liang G, Xiao H. Bioorthogonal Guided Activation of cGAS-STING by AIE Photosensitizer Nanoparticles for Targeted Tumor Therapy and Imaging. Adv. Mater.35, 2305668 (2023).\n5. Huang ZW, et al. Thermally Induced Orderly Alignment of Porphyrin Photoactive Motifs in Metal-Organic Frameworks for Boosting Photocatalytic CO Reduction. J. Am. Chem. Soc.145, 18148-18159 (2023).\n2\n6. Liu S, Hu Y, Hu C, Xiong Y, Duan M. Quantum yields of singlet oxygen of tetrakis (4-carboxyphenyl) porphyrin in different solvents. J. Porphyr. Phthalocya.23, 1084-1091 (2019).\n2. Theoretical calculations (e.g., DFT) should be conducted to explain the effects of different nitrogen doping types (pyridinic N, graphitic N, pyrrolic N) on the electronic structure and charge transfer efficiency of CDs, to strengthen the mechanistic interpretation of the \"configuration-property\" correlation.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we performed the density functional theory (DFT) calculations to visually illustrate molecular orbital distributions, electron localization, and charge transfer pathways. We establish three kinds of sp2 hybrid carbon molecules as simple CD models according to the theoretical models presented in Fig. 1h. Fig. 4a exhibits the spatial distributions of the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) for O-CD, M-CD, and P-CD. In the P-CD model, the HOMO is predominantly localized on the hydroxyl-rich region (\"donor\", D), whereas the LUMO is strongly concentrated on the pyridinic nitrogen-containing domain (\"acceptor\", A). This clear spatial separation of frontier orbitals directly demonstrates that, in the ground state of P-CD, the highest-energy electrons reside primarily in the donor moiety, while the lowest-energy vacant orbitals are located in the acceptor moiety, with both regions bridged by a conjugated π-system (carbon core). This configuration constitutes definitive evidence for a well-defined D-π-A architecture. Fig. 4b presents the calculated energy levels of the first singlet excited state (S ) and the first triplet state (T ), along with their respective singlet-triplet energy gaps (ΔE ). Notably, P-CD exhibits a significantly smaller ΔE (0.39 eV) compared to O-CD (0.82 eV) and M-CD (0.96 eV). Such a reduced energy gap facilitates efficient intersystem crossing (ISC) from the singlet to the long-lived triplet state. The theoretical calculation results consistent with the experiment results of ultralong phosphorescence lifetime of P-CD (36.32 ms), offering a mechanistic explanation for its excellent performance as a photosensitizer.\nWe then performed the surface electrostatic potential (ESP) analysis of O-CD, M-CD, and P-CD. As depicted in Fig. 4c, red regions represent the negative potential (electron-rich zones) and blue regions indicate positive potential (electron-deficient zones). In the P-CD model, a pronounced negative potential is observed in the hydroxyl-enriched region, contrasting sharply with the positive potential in the pyridinic nitrogen zone. This phenomenon confirms that hydroxyl groups act as the primary electron-donating center (D), while the pyridinic nitrogen serves as the electron-accepting center (A). These findings are further demonstrated by Fukui-DD function analysis (Fig. 4d), which reveals electrophilic features at the pyridinic nitrogen site with a high propensity for electron acceptance. In addition, the nucleophilic features over the hydroxyl and π-bridge regions were consistent with electron donation. These two complementary analyses confirm that the hydroxyl groups function as the strongest electron donor within the P-CD framework, serving as the driving force for intramolecular charge transfer. In summary, the DFT calculations provide comprehensive evidence from orbital distribution, energy-level alignment, and quantitative reactivity descriptors for the proposed D-π-A model. Orbital visualization confirms the spatial segregation of donor and acceptor units linked via a π-conjugated bridge. Energy-level calculations rationalize the experimentally observed long-lived triplet state. Electrostatic potential and Fukui function analyses jointly establish the functional roles of the hydroxyl group as the dominant donor and pyridinic nitrogen as the key acceptor.\nWe have added these results and corresponding discussion in the revised manuscript.\nFig. 4. Theoretical calculation of CDs. (a) Illustration of the frontier molecular orbitals (LUMOs and HOMOs) of O-CD, M-CD and P-CD. (b-d) Singlet and triplet energy levels and ΔE (b), Surface electrostatic potential (c), Fukui-DD function analysis (d) of O-CD, M-CD and P-CD.\n3. The photostability of CDs under multiple laser irradiations (e.g., attenuation of ROS generation efficiency, changes in photothermal conversion efficiency) needs to be clarified to support the feasibility of clinical applications.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we evaluated the photostability of P-CD through measuring photothermal conversion efficiency and ROS generation efficiency under multiple laser irradiations. As presented in Supplementary Fig. 12a, no significant change of the photothermal conversion efficiency of P-CD was detected during the six consecutive laser irradiation cycles. In addition, the O •ˉ generation efficiency of P-CD remained unchanged after five cycles (Supplementary Fig. 12b). These results demonstrated the good photostability of P-CD under multiple laser irradiations. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 12. Photothermal conversion efficiency (a) and ROS generation efficiency (b) of P-CD under multiple laser irradiations. Source data are provided as a Source Data file.\n4. Tumor recurrence experiments: after primary tumor treatment, perform secondary tumor inoculation in mice, detect the proportion of CD8+ T cell memory subsets (e.g., CD8+CD44+CD62L+) and tumor growth needs to be conducted to further verify the immune memory effect.\nReply: Thanks for your suggestion. It is well known that the immune-memory response represents an essential aspect of adaptive immunity, enabling the immune system to remember previous pathogen invasions and provide long-lasting immunity. On this basis, we constructed a re-challenging tumor model to evaluate the long-term immune memory effect induced by P-CD@MM-mediated combination therapy (Fig. 9a). After the first tumor was cured by P-CD@MM + L, we inoculated 4T1 cells on the opposite axilla of mice to fabricate the re-challenging tumor model. No injection of P-CD@MM and no irradiation of 808 nm laser were performed. For comparison, five healthy mice were also inoculated with 4T1 cells as the control group. We then monitored the change of tumor volume and performed the immunological analysis. As depicted in Fig. 9b, c, the significantly inhibited tumor growth was detected in the P-CD@MM + L group, while rapid tumor growth was observed in the control group, suggesting that the treatment of P-CD@MM + L can induce an effective immune memory response for the long-term protection against tumor recurrence of mice. During the treatment period, no loss of weight can be found in the control and P-CD@MM + L groups (Fig. 9d), demonstrating that the treatment of P-CD@MM + L possessed good biosafety. Subsequently, we investigated the T cell activation level in each group to clarify the immune memory response. As presented in Fig. 9e, the higher expression levels of CD4 and CD8 were detected in the P-CD@MM + L group compared with the control group. Specifically, the proportion of CD3+CD8+ and CD3+CD4+ T cells in the P-CD@MM + L group were measured to be approximately 24.5 ± 1.4% and 22.2 ± 1.3%, respectively (Fig. 9f, g), which was much higher than those in the control group (3.9 ± 0.5% for CD3+CD8+ and 2.9 ± 0.7% for CD3+CD4+). Additionally, the proportion of effector memory T cells (CD3+CD8+CD44+CD62Lˉ) in the spleens of both groups was analyzed. As depicted in Fig. 9h, i, in the P-CD@MM + L group, a significantly higher proportion of effector memory T cells (34.3 ± 1.4%) was observed, which was four times greater than that in the control group (8.6 ± 0.9%). Collectively, these results indicate that the administration of P-CD@MM + L induces a long-lasting immune memory response, which can enable the immune system to remember previous pathogeninvasionsand provide long-lasting immunity to prevent the reappearance of tumors. We have added these results and corresponding discussion in the revised manuscript.\n[Editorial note: icons in figure 9a Created in BioRender. zhenlin, Z. (2026) https:// BioRender.com/9u519d6]\nFig. 9. Long-Term Immunological Memory Evaluation. (a) Schematic illustration of the experimental timeline to assess the antitumor immunological memory response triggered by P-CD@MM-mediated combination therapy. (b-d) Tumor volume and body weight of mice after different treatments (n = 6 independent samples). (e-g) The expression of CD4+CD8+ T cells in the rechallenged tumors (n = 6 independent samples). (h, i) The expression of CD8+ memory T cells (CD3+CD8+CD44+CD62Lˉ) in spleen of mice with different treatments (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. P value: ***P < 0.001. Source data are provided as a Source Data file.\n5. Pharmacokinetic experiments of P-CD@MM in vivo (e.g., blood clearance curve, organ distribution and metabolism patterns) should be performed to clarify its biosafety and in vivo retention characteristics.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we investigated the in vivo pharmacokinetics of MM-free P-CDs and MM-encapsulated P-CD by measuring plasma concentration-time profiles. Female Balb/c mice (n = 3 biologically independent samples) were i.v. injected with P-CD and P-CD@MM, respectively, at a P-CD dose of 2.0 mg/kg. We then collected 10 μL of blood from the tail vein at different times after intravenous injection. The concentration of P-CD was determined by measuring the absorbance of supernatants at 720 nm. As depicted in Supplementary Fig. 26a, a typical double-compartment pharmacokinetic behavior of P-CD and P-CD@MM can be detected. The elimination half-life (t ) of P-CD and P-CD@MM was determined to be 0.9 ± 0.1 h and 10.6 ± 1.1 h, respectively (Supplementary Fig. 26b). The prolonged half-life of P-CD after coating of MM illustrates that P-CD@MM possesses longer circulation time compared with the pristine P-CD, enabling the accumulation of P-CD@MM in tumors at high levels.\nThe organ distribution and metabolism pathways of P-CD@MM were then investigated to clarify its biosafety and in vivo retention characteristics. Supplementary Fig. 38a showed that P-CD@MM mainly accumulated in the liver at 24 h post-injection, which was attributed to the capture of the reticuloendothelial system. Importantly, no obvious P-CD signal was observed in these organs after 14 days, indicating that P-CD@MM has been almost completely cleared. To study the excretion pathways, the concentrations of P-CD in urine and feces were also measured. The results indicated that P-CD@MM was mainly excreted from the mice through the liver and kidney. We also noted that no P-CD signal can be detected after 14 days (Supplementary Fig. 38b), suggesting the complete clearance of P-CD@MM. We have added these results and corresponding discussion in the revised manuscript.\nWe have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 26. Plasma concentration-time profiles (a) and pharmacokinetic parameters (b) of P-CD and P-CD@MM after intravenous administration at the P-CD dose of 2.0 mg/kg (n = 3 independent samples). Data are presented as mean ± SD. Source data are provided as a Source Data file.\nSupplementary Fig. 38. (a) Biodistribution of P-CD@MM post i.v. injection in mice on different days (n = 6 independent samples). (b) The detected P-CD mass in urine and feces at different time points post i.v. injection of P-CD@MM (n = 6 independent samples). Data are presented as mean ± SD. Source data are provided as a Source Data file.\n6. Please increase the number of biological replicates (n≥6 is recommended) for key experiments (e.g., ROS generation, DC maturation rate, T cell infiltration) and supplement detailed parameters of statistical analysis (e.g., results of homogeneity of variance test).\nReply: Thanks for your suggestion. We have increased the number of biological replicates (n≥6) for key experiments and supplement detailed parameters of statistical analysis.\n7. Please add characterization data of M1 macrophage membranes. For example, quantitatively detect the purity of membrane surface markers (iNOS, CD86) by Western blot, and supplement stability tests of the cell membranes during storage (e.g., changes in Zeta potential, duration of integrity maintenance).\nReply: Thanks for your suggestion. The quantitative results of the expression levels of CD86 was added in Supplementary Fig. 15a. We then investigated the stability evaluation of P-CD@MM during storage. As depicted in Supplementary Fig. 15b, c, no significant change of size and Zeta potential of P-CD@MM were observed within 9 days, demonstrating the good colloidal stability of P-CD@MM. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 15. (a) The quantitative results of the expression levels of CD86 (n = 3 independent samples). (b, c) The Zeta potential and DLS measurements of P-CD@MM during the long-term storage (n = 3 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. P value: ***P < 0.001. Source data are provided as a Source Data file.\n8. Please optimize the in vivo laser irradiation protocol: clarify the matching relationship between laser spot size, irradiation time, and tumor volume, and supplement comparative data on therapeutic effects under different irradiation parameters to standardize the protocol.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we investigated the influence of laser irradiation parameters on the therapeutic effects of P-CD@MM-mediated combination therapy. To evaluate the impact of irradiation spatial coverage and duration, we investigated tumor growth under different light spot sizes (d = 0.4 cm, insufficient to fully cover the tumor; d = 0.8 cm, slightly larger than the tumor; d = 1.2 cm, substantially exceeding tumor dimensions) at a fixed power density of 0.3 W/cm2 and irradiation time of 10 min. Additionally, under the same power density and a spot size of 0.8 cm, we examined the effects of varied irradiation durations (0, 5, 10, and 15 min) on therapeutic outcomes. As shown in Supplementary Fig. 28, both insufficient irradiation area and suboptimal exposure time failed to achieve complete tumor growth inhibition. The optimal therapeutic efficacy was observed at a power density of 0.3 W/cm2, a spot diameter of 0.8 cm, and an irradiation time of 10 min. While longer irradiation times or larger spot sizes also yielded strong antitumor effects, they may increase the risk of collateral damage to surrounding healthy tissues due to excessive energy deposition. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 28. The influence of laser irradiation parameters on the therapeutic effects of P-CD@MM-mediated combination therapy, including laser spot sizes (a) and irradiation durations (b) (n = 6 independent samples). Data are presented as mean ± SD. Source data are provided as a Source Data file.\n9. Please supplement negative control experiments: set up a \"P-CD camouflaged with empty cell membranes\" group to exclude the interference of membrane components themselves on the immune response, and further confirm the core role of P-CD in photodynamic therapy.\nReply: Thanks for your suggestion. We employed the M0-phenotype RAW264.7 macrophage membrane as an empty cell membrane to encapsulate P-CD (P-CD@EM), thereby confirming the core role of P-CD in antitumor immune response. As depicted in Supplementary Fig. 37a, b, no significant antitumor effectiveness of P-CD@EM alone can be detected, suggesting the encapsulation of P-CD in EM did not trigger the antitumor immune response. For comparison, a slight inhibition effect on the growth of primary and distant tumors was observed in the P-CD@MM, demonstrating that cell membrane derived from M1 phenotype macrophage can activate a certain antitumor immune response. We also noted that the treatment of P-CD@EM + L can inhibit the tumor growth, which could be attributed to the P-CD-induced PDT. However, the stronger antitumor effectiveness can be detected in the P-CD@MM + L group, which was superior to that in the P-CD@EM + L group, illustrating that the antitumor immune response of P-CD-mediated PDT could be further enhanced by the encapsulation of P-CD in MM rather than in EM.\nImmunological analyses revealed that P-CD@EM alone did not elicit antitumor immune responses. P-CD@EM failed to promote DC maturation, induce T cell infiltration, or drive polarization of macrophages from M2 phenotype to M1 phenotype (Supplementary Fig. 37c-n). In contrast, P-CD@MM exhibited a certain immunostimulatory activity, implying that the encapsulation of P-CD in MM can endow immunoregulation functions to P-CD. We also noted that P-CD@EM combined with laser irradiation triggered strong immunological activation, demonstrating that P-CD-mediated PDT served as the essential core component for the activation of antitumor immune responses.\nWe have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 37. (a, b) Primary and distant tumor volume of mice after different treatments (n = 6 independent samples). (c-f) Expression levels of CD8+ T cells, CD4+ T cells, CD86+, CD206+ in TAMs in the distant tumors after different treatments (n = 6 independent samples). (g, h) Flow cytometry analysis of CD80+ and CD86+ expression in lymph nodes after different treatments (n = 6 independent samples). (i-k) Expression levels of CD4+ T cells and CD8+ T cells in the primary tumors after different treatments (n = 6 independent samples). (l-n) Expression levels of CD86+ and CD206+ in TAMs in the primary tumors after different treatments (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. P value: ***P < 0.001. Source data are provided as a Source Data file.\nReviewer #4\nIn this study, the authors construct three types of NIR-responsive carbon dots (CDs) by using a type-Ⅱ organic photosensitizer/NIR probe (ICG) as the active ingredient and three isomers of o-, m-, and p-phenylenediamines (OPD, MPD, PPD) as regulators, to obtain phototherapy agents with low-efficacy photothermal but high-efficacy type-I/Ⅱ photodynamic activity. After evaluating their photothermal and photodynamic properties, P-CDs was selected as the superior type-I/Ⅱ photosensitizers, and subsequently coated with M1-macrophage cell membranes to form P-CD@MM nanoparticles for potent photo-immunotherapy. Although the study presents many data, there are lots of major issues that must be addressed prior to any further consideration.\nReply: We thank the reviewer for the positive evaluation and kind recommendation. We have addressed all of Reviewer 4’s comments and concerns in detail, and the corresponding revisions have been made in the revised manuscript and supporting information. Our responses to these issues are attached as follows.\n1. Given the extensive prior work on M1-macrophage membrane-coated nanoparticles and PDT-immunotherapy, the latter part of this study lacks novelty. Moreover, the authors have previously reported similar NIR phosphorescent CDs constructed using ICG as a precursor, which were also coated with cancer cell membranes for precision SDT (Nature Communications (2022) 13:5735). Compared with this previous work, the improvements in this study are minimal.\nReply: Thanks for your suggestion. Compared with our previous report (Nat. Commun. 2022, 13, 5735), P-CDs and P-CD@MM prepared in this work possessed many innovations. We have summarized these innovations as follows.\nFirst, the previously reported NIR phosphorescent CDs (p-n-CDs) exhibited excellent sonodynamic activity owing to their long-lived triplet states. However, p-n-CDs showed insignificant photodynamic activity at 808 nm (Supplementary Fig. 42c, d), which could be ascribed to the poor absorption of p-n-CDs at 808 nm. For comparison, P-CDs prepared in this work exhibited excellent photodynamic activity at 808 nm. In addition, the triplet lifetime of P-CDs is limited to the microsecond level (11.4 μs). For comparison, P-CDs exhibited millisecond-level ultralong carrier lifetime (36.32 ms), three orders of magnitude longer than p-n-CDs (11.4 μs). The excellent NIR absorption and ultralong triplet lifetime of P-CDs was achieved by the rational design of D-π-A configuration in P-CDs. As an extended π-conjugated nano-system of CDs in the D-π-A configuration, electron-rich oxygen atoms from -OH modifications at edge sites significantly improve the electron-donating capability, which combine with electron-withdrawing moieties (largely pyridinic N) to form the D-π-A configuration, resulting in an elongated triplet state lifetime (tens of ms), an enhanced intramolecular charge transfer, and a reduced bandgap (1.49 eV). Therefore, from the perspective of red-shifting absorption and enhancing triplet lifetime, this work has significant advantages compared to the previous report.\nSecond, the p-n-CDs exhibited no activity in O •ˉ generation under ultrasonic or photo-irradiation (Supplementary Fig. 42), suggesting that the type-I mechanism was deactivated in the pyrrolic N/-SO H configuration with a high S doping level (8.1 at.%). We speculated that -SO ˉ/-SO H groups 3 3 3 enriched in p-n-CDs with the strong electron-withdrawing capability may block photoinduced electron transfer from CDs to molecular O , leading to ineffective generation of O •ˉ. In the new D-π-A system with high photosensitization activity in both type-I and type-Ⅱ, however, the electron withdrawing effect by the strong S-based acceptor was greatly weakened owing to the low S doping level (1.80 at.%), and the high-content pyridinic N (8.68at.%) as a weak N-based acceptor plays a crucial role in the D-π-A system for optimization of desirable photodynamic types, activation wavelength, and ultra-stable triplets. Therefore, from the perspective of elucidating the mechanism of structure-activity relationship, this mechanism has not been reported in previous study.\nThird, we reported for the first time the single materials exhibited both mild photothermal effects and excellent photodynamic effects, thereby achieving mild PTT-enhanced PDT and amplifying the therapeutic effect of PDT through alleviating hypoxia. Therefore, from the perspective of regulating photothermal and photodynamic properties and therapeutic applications, P-CDs reported in this work possess both excellent photodynamic activity and mild photothermal effects, which have not been reported in the previous work.\nFourth, from the perspective of cell membrane carriers, this work chooses macrophage membranes with immunoregulatory functions to encapsulate P-CD, while the previous work (Nat. Commun. 2022, 13, 5735) have chosen to encapsulate p-n-CD with only homologous targeting ability of the cancer cell membrane. The encapsulation of P-CDs in M1-type macrophage membranes (MM) not only provides tumor-targeting function, but also reprogram tumor-associated macrophages from the pro-tumoral M2 phenotype to anti-tumoral M1 phenotype, thereby reversing the immunosuppressive microenvironment. We then investigated the immune-related pathway induced by P-CD@MM-mediated PDT, including ICD, maturation of DCs, activation of T cells, and reprogramming of macrophages. However, the previous report only focused on SDT and did not involve research on immune-related pathways.\nTherefore, the innovations of this work compared to our previous report (Nat. Commun. 2022, 13, 5735) have been elucidated as the above four aspects. We have added these innovative explanations in the revised manuscript.\nSupplementary Fig. 42. (a-d) Measurements of the Type I and II photodynamic activity of p-n-CDs under 660 or 808 nm laser irradiation. (e, f) Measurements of the Type I and II sonodynamic activity of p-n-CDs under US irradiation.Source data are provided as a Source Data file.\n2. The advantages of this study and the key problems it aims to address remain unclear.\n1) Direct characterization about structure of O-CDs, M-CDs, and P-CDs should be provided, instead of only presenting TEM, XPS, and FT-IR data.\nReply: Thanks for your suggestion. In addition to TEM, XPS, and FT-IR characterizations, we further analyzed the UV-Vis absorption spectrum (Fig. 1j), Raman spectrum (Supplementary Fig. 3), and Zeta potential (Supplementary Fig. 8) of O-CD, M-CD, and P-CD. These characterization experiments demonstrate that these CDs were successfully prepared.\nFig. 1. (j) Absorption spectrum of O-CD, M-CD and P-CD. Source data are provided as a Source Data file.\nSupplementary Fig. 3. Raman spectrum of ICG-CD (a), O-CD (b), M-CD (c), and P-CD (d). Source data are provided as a Source Data file.\nSupplementary Fig. 8. Zeta potential measurements of O-CD, M-CD, and P-CD (n = 3 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. Source data are provided as a Source Data file.\n2) The authors described in the Introduction that “type-Ⅱ PDT and mild PTT may synergize to enhance phototherapy efficacy owing to partial alleviation of hypoxia by mild hyperthermia, which can increase tumor blood flow and improves hemoglobin oxygen saturation within the tumor vasculature”, however, the current manuscript only provides HIF-α immunofluorescence staining and does not include other experiments demonstrating hypoxia alleviation by mild hyperthermia, such as tumor blood flow, or hemoglobin oxygen saturation.\nReply: Thanks for your suggestion. Many previous reports have claimed that mild PTT can alleviate hypoxia through increasing tumor blood flow and improves hemoglobin oxygen saturation within the tumor vasculature (Bioact. Mater. 2022, 8, 409-419, International Journal of Nanomedicine 2024, 19, 6499-6513, International Journal of Nanomedicine 2024, 19, 3367-3386, Nano Res. 2023, 16, 9859-9872). However, due to limitations in equipment and experimental conditions, we are unable to measure tumor blood flow and hemoglobin oxygen saturation. We will investigate the tumor blood flow and hemoglobin oxygen saturation in the future.\nTo avoid misunderstandings and make our statement more rigorous, we removed the description in the Introduction section that mild PTT can increase tumor blood flow and improve hemoglobin oxygen saturation within the tumor vasculature. We only emphasized that mild PTT could alleviate hypoxia.\n3) The authors claimed to develop superior type-I/II photosensitizers with matching photothermal effects for robust photoimmunotherapy; however, the study lacks comparative experiments between O-CDs/M-CDs/P-CDs and widely used photosensitizers or photothermal agents, making it impossible to determine the actual advantages of the proposed system or to assess its practical therapeutic potential.\nReply: Thanks for your suggestion. To highlight the advantages of the D-π-A configured P-CDs, we compared the excited-state lifetime, ROS generation efficiency, and hypoxic adaptability between D-π-A structured carbon dots (P-CDs), traditional CDs (O-CD), and commercial photosensitizers (ICG, TCPP). First, P-CDs exhibited ultralong triplet state lifetime of 36.32 ms, which was six orders of magnitude longer than those observed for O-CDs and M-CDs (Fig. 3i). The triplet state lifetime of P-CDs was also significantly higher than that of commercial photosensitizers (0.8 ns for ICG and 8.48 ns for TCPP) (Supplementary Table 1). Second, this ultralong triplet state lifetime is a crucial factor in augmenting photodynamic activity of P-CDs. The calculated 1O quantum yield of P-CD was measured to be 1.49%, which was much higher than that of O-CD (0.58%), ICG (0.20%), and TCPP (0.10%). Third, under hypoxic conditions, the P-CD with type I/II photodyanmic mechanism can still effectively produce O •ˉ (Fig. 3b, d). In contrast, no ROS generation of ICG and TCPP can be detected under hypoxic conditions (Supplementary Fig. 13), which could be attributed to their O -depecdent type II photodyanmic mechanism. These results demonstrate that the D-π-A configured P-CDs possess the largest excited-state lifetime, highest ROS generation efficiency, and optimal hypoxic adaptability compared with the traditional CDs and commercial photosensitizers. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 13. Photodynamic activity evaluation of ICG, TCPP, and O-CD at the hypoxia conditions using DPBF (a) or DHR123 (b) as the probe. Source data are provided as a Source Data file.\nSupplementary Table 1. Comparison of D-π-A sensitized CDs with commercial photosensitizers in the excited-state lifetime and 1O generation efficiency.\nType II ICG 0.80 ns 0.20 3,4\nType II TCPP 8.48 ns 0.10 5,6\nReferences\n3. Swamy MMM, Murai Y, Monde K, Tsuboi S, Swamy AK, Jin T. Biocompatible and Water-Soluble Shortwave-Infrared (SWIR)-Emitting Cyanine-Based Fluorescent Probes for In Vivo Multiplexed Molecular Imaging. ACS Appl. Mater. Interfaces16, 17253-17266 (2024).\n4. Cui M, Tang D, Wang B, Zhang H, Liang G, Xiao H. Bioorthogonal Guided Activation of cGAS-STING by AIE Photosensitizer Nanoparticles for Targeted Tumor Therapy and Imaging. Adv. Mater.35, 2305668 (2023).\n5. Huang ZW, et al. Thermally Induced Orderly Alignment of Porphyrin Photoactive Motifs in Metal-Organic Frameworks for Boosting Photocatalytic CO Reduction. J. Am. Chem. Soc.145, 18148-18159 (2023).\n2\n6. Liu S, Hu Y, Hu C, Xiong Y, Duan M. Quantum yields of singlet oxygen of tetrakis (4-carboxyphenyl) porphyrin in different solvents. J. Porphyr. Phthalocya.23, 1084-1091 (2019).\n3. Please provide more data about the D-π-A sensitized type II/I photodynamic mechanism of CDs with enhanced charge-transfer dynamics.\nReply: Thanks for your suggestion. To further demonstrate the D-π-A sensitized type II/I photodynamic mechanism of CDs with enhanced charge-transfer dynamics, we performed the density functional theory (DFT) calculations to visually illustrate molecular orbital distributions, electron localization, and charge transfer pathways. We establish three kinds of sp2 hybrid carbon molecules as simple CD models according to the theoretical models presented in Fig. 1h. Fig. 4a exhibits the spatial distributions of the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) for O-CD, M-CD, and P-CD. In the P-CD model, the HOMO is predominantly localized on the hydroxyl-rich region (\"donor\", D), whereas the LUMO is strongly concentrated on the pyridinic nitrogen-containing domain (\"acceptor\", A). This clear spatial separation of frontier orbitals directly demonstrates that, in the ground state of P-CD, the highest-energy electrons reside primarily in the donor moiety, while the lowest-energy vacant orbitals are located in the acceptor moiety, with both regions bridged by a conjugated π-system (carbon core). This configuration constitutes definitive evidence for a well-defined D-π-A architecture. Fig. 4b presents the calculated energy levels of the first singlet excited state (S ) and the first triplet state (T ), along with their respective singlet-triplet energy gaps (ΔE ). Notably, P-CD exhibits a significantly smaller ΔE (0.39 eV) compared to O-CD (0.82 eV) and M-CD (0.96 eV). Such a reduced energy gap facilitates efficient intersystem crossing (ISC) from the singlet to the long-lived triplet state. The theoretical calculation results consistent with the experiment results of ultralong phosphorescence lifetime of P-CD (36.32 ms), offering a mechanistic explanation for its excellent performance as a photosensitizer.\nWe then performed the surface electrostatic potential (ESP) analysis of O-CD, M-CD, and P-CD. As depicted in Fig. 4c, red regions represent the negative potential (electron-rich zones) and blue regions indicate positive potential (electron-deficient zones). In the P-CD model, a pronounced negative potential is observed in the hydroxyl-enriched region, contrasting sharply with the positive potential in the pyridinic nitrogen zone. This phenomenon confirms that hydroxyl groups act as the primary electron-donating center (D), while the pyridinic nitrogen serves as the electron-accepting center (A). These findings are further demonstrated by Fukui-DD function analysis (Fig. 4d), which reveals electrophilic features at the pyridinic nitrogen site with a high propensity for electron acceptance. In addition, the nucleophilic features over the hydroxyl and π-bridge regions was consistent with electron donation. These two complementary analyses confirm that the hydroxyl groups function as the strongest electron donor within the P-CD framework, serving as the driving force for intramolecular charge transfer. In summary, the DFT calculations provide comprehensive evidence from orbital distribution, energy-level alignment, and quantitative reactivity descriptors for the proposed D-π-A model. Orbital visualization confirms the spatial segregation of donor and acceptor units linked via a π-conjugated bridge. Energy-level calculations rationalize the experimentally observed long-lived triplet state. Electrostatic potential and Fukui function analyses jointly establish the functional roles of the hydroxyl group as the dominant donor and pyridinic nitrogen as the key acceptor.\nWe have added these results and corresponding discussion in the revised manuscript.\nFig. 4. Theoretical calculation of CDs. (a) Illustration of the frontier molecular orbitals (LUMOs and HOMOs) of O-CD, M-CD and P-CD. (b-d) Singlet and triplet energy levels and ΔE (b), Surface electrostatic potential (c), Fukui-DD function analysis (d) of O-CD, M-CD and P-CD.\n4. In Fig. 4c, it is unclear why iNOS appears in the M1 membrane, as it is typically a cytoplasmic protein.\nReply: Thanks for your suggestion. As the reviewer’s said, iNOS was classically described as a predominantly cytoplasmic protein. However, the previous report (J. Control. Release 2023, 360, 482-495) have claimed that a fraction of iNOS can transiently associate with the inner leaflet of the plasma membrane through interactions with membrane-associated proteins or lipid rafts. Furthermore, our membrane isolation protocol was based on mechanical disruption followed by differential centrifugation which yields a membrane-enriched fraction rather than a pure plasma membrane preparation. This fraction typically contains intact plasma membranes, partial intracellular membranes, and tightly bound peripheral proteins. In M1-phenotype macrophages with high iNOS expression, the previous report (J. Control. Release 2023, 360, 482-495) demonstrated that iNOS can be co-isolated in such membrane-enriched preparations. To avoid misunderstandings, we have removed the WB assay of iNOS. The expression of CD86 in M1 membrane and P-CD@MM can confirmed the successful preparation of MM-encapsulated P-CDs. The quantitative results can be found in Supplementary Fig. 15a. We also investigated the stability evaluation of P-CD@MM during storage. As depicted in Supplementary Fig. 15b, c, no significant change of size and Zeta potential of P-CD@MM were observed within 9 days, demonstrating the good colloidal stability of P-CD@MM. We have added these results and corresponding discussion in the revised manuscript.\nFig. 5. (c) Measurements of the expression levels of CD86 and CD206 in each group.\nSupplementary Fig. 15. (a) The quantitative results of the expression levels of CD86 (n = 3 independent samples). (b, c) The Zeta potential and DLS measurements of P-CD@MM during the long-term storage (n = 3 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. P value: ***P < 0.001. Source data are provided as a Source Data file.\n5. In Fig. 4f, only the relative cell viability of 4T1 cells after laser irradiation is presented; please include corresponding experiments using NIH/3T3 cells. Moreover, the number of live cells appears to decrease in P-CD@MM group compared with the control group in Fig. 4g, which shows an inconsistent trend with Fig. 4e.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we performed the MTT assay to evaluate the relative cell viability of NIH-3T3 cells after being treated with P-CD@MM + L. As depicted in Supplementary Fig. 17, no significant decrease of the cell viability of NIH-3T3 cells was detected in the P-CD@MM + L group, which could be ascribed to the low cellular internalization efficiency of P-CD@MM in normal cells.\nFor the live/dead cell staining, the reason why the number of live cells in the P-CD@MM group was smaller than that in the control group might be because we did not maintain a consistent number of cells in each group. Therefore, we re-performed the live/dead cell staining of 4T1 cells after different treatments. As presented in Fig. 5g, the number of live cells in the P-CD@MM group was similar to that in the control group.\nWe have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 17. Relative cell viability of NIH-3T3 cells after being treated with P-CD@MM in the presence of NIR laser irradiation (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. Source data are provided as a Source Data file.\nFig. 5. (g) Live/dead cell staining of 4T1 cells after different treatments.\n6. In Fig. 5f and Supplementary Fig. 22, whether the measured ATP levels in the intracellular or supernatant? Please add more details about this issue, especially the in vivo experiments.\nReply: Thanks for your suggestion. For the evaluation of ATP level at the cellular level, we detected the intracellular ATP level in 4T1 cells after different treatments. As depicted in Fig. 6c, the intracellular ATP level in 4T1 cells was markedly diminished in both the P-CD + laser group and the P-CD@MM + laser group.\nFor the in vivo experiments, we measured the ATP level in the supernatant of tumor tissue homogenate. On day 7 post-treatment, primary tumors were harvested from mice in each group. Tumor tissues of equal mass were processed identically by adding 500 μL of RIPA lysis buffer and homogenizing on ice. Following homogenization, samples were centrifuged at 12,000 rpm for 15 minutes at 4 °C, and the resulting supernatants were collected. ATP levels were quantified using an ATP assay kit, while HMGB1 concentrations were determined by ELISA.\nWe have added these details in the revised manuscript.\n7. In Supplementary Fig. 23, gating on CD11b+ is not appropriate for identifying DCs.\nReply: Thanks for your suggestion.Due to a typing error, we mistakenly wrote \"CD11c+\" as \"CD11b+\". We have modified this mistake in the revised manuscript.\nSupplementary Fig. 31. Flow cytometry analysis of CD80+ and CD86+ expression in lymph nodes after different treatments (a) and corresponding quantitative results (b) (gated on CD11c+ cells) (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via two-tailed Student’s t-test. P value: ***P < 0.001. Source data are provided as a Source Data file.\n8. For better comparison, please provide ex vivo NIR imaging and quantification of major tissues and tumors for P-CD, similar to the data shown for P-CD@MM in Fig. 6d and Supplementary Fig. 18.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we performed the ex vivo NIR imaging and quantification of major tissues and tumors for P-CD. Consistent with the in vivo NIR imaging results (Fig. 7b), following intravenous administration, P-CD predominantly accumulated in the liver of mice (Supplementary Fig. 25), suggesting the low accumulation level of P-CDs in tumors. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 25. Ex vivo NIR imaging and quantification of major tissues and tumors for P-CD (n = 3 independent samples). Data are presented as mean ± SD. Source data are provided as a Source Data file.\nItemized Responses to Reviewers’ Comments\nReviewer #1\nThe revised manuscript can be accepted after the thorough revision.\nReply: We thank the reviewer for the positive evaluation\nReviewer #2\nThe authors answered the reviewers' concerns properly.\nReply: We thank the reviewer for the positive evaluation\nReviewer #3\nThe issues arosed by the Reviewers have been addressed well, and I am pleased to recommend acceptance for published in this journal.\nReply: We thank the reviewer for the positive evaluation\nReviewer #4\nAlthough the authors have added some additional experiments, many major issues remain insufficiently addressed. The special issues are as follows:\nReply: We thank the reviewer for the positive evaluation and kind recommendation. We have addressed all of Reviewer 4’s comments and concerns in detail, and the corresponding revisions have been made in the revised manuscript and supporting information. Our responses to these issues are attached as follows.\n1. The revised manuscript still lacks direct evidence supporting the specific structures and molecular arrangements of O-CDs, M-CDs, and P-CDs shown in Scheme 1 and Figure 1h. Direct experimental evidence, such as NMR or other appropriate techniques, is required to verify the proposed molecular connectivity. Since the D-π-A structure is critical to the photodynamic performance of CDs, these conclusions are not convincing without such direct data. Most importantly, are the specific structural motifs of ICG preserved in the final CDs? If not, what is the rationale for selecting ICG instead of other conventional N/S-containing molecules as precursors?\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we conducted 1H NMR and 13C NMR analysis. As depicted in Supplementary Fig. 7a, the 1H NMR exhibited that the peaks of P-CD and ICG monomers partially overlapped at δ 7.5-8.5 ppm, indicating that the conjugated motif of ICG composed of several -CH=units is likely retained in the extended π conjugated aromatic section in the P-CD, which can be beneficial to extend its absorption in the NIR region. However, the additional strong signal of hydroxyl (-OH) was observed at δ 6.35 ppm in the P-CD 1H NMR spectrum, further confirming the structural characteristics of P-CD containing the -OH donor. Additionally, the 13C NMR analysis of P-CD revealed the existence of conjugated carbon skeletons at δ 110-150 ppm, which were the basis of π bridges in the D-π-A structure, but such extended conjugated carbon skeletons cannot be observed in the ICG 13C NMR spectrum (Supplementary Fig. 7b). In carbon nanomaterials such as CDs, the carbon signals related to pyridine/pyrrole N completely overlap at the δ 100-150 ppm and cannot be distinguished by conventional NMR. At present, the standard method for determining the type of N doping in CDs is XPS analysis. XPS results clearly showed that P-CDs were mainly composed of pyridine N, M-CDs contained graphite N and pyrrole N, and O-CDs were mainly composed of pyrrole N (Fig. 1k). Therefore, the specific structures and molecular arrangements of O-CDs, M-CDs, and P-CDs can be determined by the FTIR, XPS, and NMR characterization. NMR confirmed the existence of aromatic frameworks (π bridges) and hydroxyl groups (donors), FTIR further verified the existence of hydroxyl groups, and XPS clarified the nitrogen doping type (receptor type). We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 7.1H NMR (a) and 13C NMR (b) of P-CD and ICG. Source data are provided as a Source Data file.\n2. The CD model established in the theoretical calculations is an idealized planar sp2 hybrid carbon molecules. However, carbon dots (CDs) synthesized from ICG and phenylenediamine possess highly complex structures, typically containing many amorphous domains and randomly distributed functional groups. Therefore, this assumption appears to be unrealistic.\nReply: As the reviewer’s said, CDs synthesized from ICG and phenylenediamine possess highly complex structures, typically containing many amorphous domains and randomly distributed functional groups. The highly complex structures make it difficult to perform accurate all atom modeling and perform first principles calculations. Although the simplified CD model established in the theoretical calculations did not represent the true structure of these CDs, the relative comparison among the model enabled identification of trends and suggested explanations for the observed findings. We can clarify the core physical mechanism and key structural factors that determine its photoelectric properties (such as the D-π-A configuration, energy band gap, and long-lived triplet state). In addition, many previous reports utilized the simplified CD model to perform the theoretical calculations (Nat. Commun. 2023, 14, 160; ACS Nano 2017, 11, 12, 12402-12410; Adv. Funct. Mater. 2025, e13584; Biomaterials, 2024, 309, 122622; Biomaterials, 2026, 324, 123496). We have added the corresponding discussion in the revised manuscript.\n3. The immunomodulatory effect of the M1 macrophage membrane carrier is only indirectly inferred from CD86 expression and tumor growth inhibition. Is there any direct evidence for the interaction mechanism between this membrane and tumor-associated macrophages (TAMs)?\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we provided direct evidence for the interaction mechanism between M1 macrophage membrane and TAMs. As shown in Supplementary Fig. 24, compared with the Control, Laser, and P-CD groups without M1 macrophage membrane, P-CD@MM treatment alone significantly increased the expression levels of phosphorylated NF-κB p65 (p-NF-κB p65) and phosphorylated STAT1 (p-STAT1) in TAMs. This enhancement effect was further amplified after adding laser irradiation (P-CD@MM + Laser). The results provide direct molecular evidence that the M1 macrophage membrane is not an inert carrier, but interacts with TAMs through its surface retained ligands, actively activating the core signaling pathways (NF-κB and STAT1) that drive M1 polarization, thereby reprogramming the pro-tumoral M2 phenotype to the anti-tumoral M1 phenotype. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 24. Measurements of the expression levels of p-STAT, STAT, p-NF-κB p65, NF-κB p65 in each group.\n4. The pro-inflammatory cytokines, including IL-6, TNF-α, and IFN-γ, as markers of systemic immunity activation should be analyzed.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we detected the levels of pro-inflammatory cytokines in mice including interleukin-6 (IL-6), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) using ELISA assay. As shown in Supplementary Fig. 33, the levels of IL-6, IFN-γ, and TNF-α in P-CD@MM + laser group were significantly higher than those in other groups. These results suggest that P-CD@MM-mediated PDT can activate the immune response in vivo by releasing tumor-associated antigen, thus inducing ICD and M1 polarization. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 33. The expression levels of IL-6 (a), TNF-α (b), and IFN-γ (c) in blood serum of mice after different treatments (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA. P value: *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.\n5. All statistical comparisons involving multiple groups in the manuscript are still analyzed using two-tailed Student’s t-test, which is statistically inappropriate. Appropriate statistical methods, such as one-way ANOVA followed by suitable post hoc tests, should be applied instead. This should be corrected throughout the entire manuscript.\nReply: Thanks for your suggestion. We have analyzed the multiple groups’ data in the manuscript using the one-way ANOVA method."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript can be accepted after the thorough revision.", "reviewer_2": "The authors answered the reviewers' concerns properly.", "reviewer_3": "The issues arosed by the Reviewers have been addressed well, and I am pleased to recommend acceptance for published in this journal.", "reviewer_4": "Although the authors have added some additional experiments, many major issues remain insufficiently addressed.\nThe special issues are as follows:\n1. The revised manuscript still lacks direct evidence supporting the specific structures and molecular arrangements of O-CDs, M-CDs, and P-CDs shown in Scheme 1 and Figure 1h. Direct experimental evidence, such as NMR or other appropriate techniques, is required to verify the proposed molecular connectivity. Since the D-π-A structure is critical to the photodynamic performance of CDs, these conclusions are not convincing without such direct data. Most importantly, are the specific structural motifs of ICG preserved in the final CDs? If not, what is the rationale for selecting ICG instead of other conventional N/S-containing molecules as precursors?\n2. The CD model established in the theoretical calculations is an idealized planar sp2 hybrid carbon molecules. However, carbon dots (CDs) synthesized from ICG and phenylenediamine possess highly complex structures, typically containing many amorphous domains and randomly distributed functional groups. Therefore, this assumption appears to be unrealistic.\n3. The immunomodulatory effect of the M1 macrophage membrane carrier is only indirectly inferred from CD86 expression and tumor growth inhibition. Is there any direct evidence for the interaction mechanism between this membrane and tumor-associated macrophages (TAMs)?\n4. The pro-inflammatory cytokines, including IL-6, TNF-α, and IFN-γ, as markers of systemic immunity activation should be analyzed.\n5. All statistical comparisons involving multiple groups in the manuscript are still analyzed using two-tailed Student’s t-test, which is statistically inappropriate. Appropriate statistical methods, such as one-way ANOVA followed by suitable post hoc tests, should be applied instead. This should be corrected throughout the entire manuscript."}, "author_response": "Itemized Responses to Reviewers’ Comments\nReviewer #1\nThe revised manuscript can be accepted after the thorough revision.\nReply: We thank the reviewer for the positive evaluation\nReviewer #2\nThe authors answered the reviewers' concerns properly.\nReply: We thank the reviewer for the positive evaluation\nReviewer #3\nThe issues arosed by the Reviewers have been addressed well, and I am pleased to recommend acceptance for published in this journal.\nReply: We thank the reviewer for the positive evaluation\nReviewer #4\nAlthough the authors have added some additional experiments, many major issues remain insufficiently addressed. The special issues are as follows:\nReply: We thank the reviewer for the positive evaluation and kind recommendation. We have addressed all of Reviewer 4’s comments and concerns in detail, and the corresponding revisions have been made in the revised manuscript and supporting information. Our responses to these issues are attached as follows.\n1. The revised manuscript still lacks direct evidence supporting the specific structures and molecular arrangements of O-CDs, M-CDs, and P-CDs shown in Scheme 1 and Figure 1h. Direct experimental evidence, such as NMR or other appropriate techniques, is required to verify the proposed molecular connectivity. Since the D-π-A structure is critical to the photodynamic performance of CDs, these conclusions are not convincing without such direct data. Most importantly, are the specific structural motifs of ICG preserved in the final CDs? If not, what is the rationale for selecting ICG instead of other conventional N/S-containing molecules as precursors?\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we conducted 1H NMR and 13C NMR analysis. As depicted in Supplementary Fig. 7a, the 1H NMR exhibited that the peaks of P-CD and ICG monomers partially overlapped at δ 7.5-8.5 ppm, indicating that the conjugated motif of ICG composed of several -CH=units is likely retained in the extended π conjugated aromatic section in the P-CD, which can be beneficial to extend its absorption in the NIR region. However, the additional strong signal of hydroxyl (-OH) was observed at δ 6.35 ppm in the P-CD 1H NMR spectrum, further confirming the structural characteristics of P-CD containing the -OH donor. Additionally, the 13C NMR analysis of P-CD revealed the existence of conjugated carbon skeletons at δ 110-150 ppm, which were the basis of π bridges in the D-π-A structure, but such extended conjugated carbon skeletons cannot be observed in the ICG 13C NMR spectrum (Supplementary Fig. 7b). In carbon nanomaterials such as CDs, the carbon signals related to pyridine/pyrrole N completely overlap at the δ 100-150 ppm and cannot be distinguished by conventional NMR. At present, the standard method for determining the type of N doping in CDs is XPS analysis. XPS results clearly showed that P-CDs were mainly composed of pyridine N, M-CDs contained graphite N and pyrrole N, and O-CDs were mainly composed of pyrrole N (Fig. 1k). Therefore, the specific structures and molecular arrangements of O-CDs, M-CDs, and P-CDs can be determined by the FTIR, XPS, and NMR characterization. NMR confirmed the existence of aromatic frameworks (π bridges) and hydroxyl groups (donors), FTIR further verified the existence of hydroxyl groups, and XPS clarified the nitrogen doping type (receptor type). We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 7.1H NMR (a) and 13C NMR (b) of P-CD and ICG. Source data are provided as a Source Data file.\n2. The CD model established in the theoretical calculations is an idealized planar sp2 hybrid carbon molecules. However, carbon dots (CDs) synthesized from ICG and phenylenediamine possess highly complex structures, typically containing many amorphous domains and randomly distributed functional groups. Therefore, this assumption appears to be unrealistic.\nReply: As the reviewer’s said, CDs synthesized from ICG and phenylenediamine possess highly complex structures, typically containing many amorphous domains and randomly distributed functional groups. The highly complex structures make it difficult to perform accurate all atom modeling and perform first principles calculations. Although the simplified CD model established in the theoretical calculations did not represent the true structure of these CDs, the relative comparison among the model enabled identification of trends and suggested explanations for the observed findings. We can clarify the core physical mechanism and key structural factors that determine its photoelectric properties (such as the D-π-A configuration, energy band gap, and long-lived triplet state). In addition, many previous reports utilized the simplified CD model to perform the theoretical calculations (Nat. Commun. 2023, 14, 160; ACS Nano 2017, 11, 12, 12402-12410; Adv. Funct. Mater. 2025, e13584; Biomaterials, 2024, 309, 122622; Biomaterials, 2026, 324, 123496). We have added the corresponding discussion in the revised manuscript.\n3. The immunomodulatory effect of the M1 macrophage membrane carrier is only indirectly inferred from CD86 expression and tumor growth inhibition. Is there any direct evidence for the interaction mechanism between this membrane and tumor-associated macrophages (TAMs)?\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we provided direct evidence for the interaction mechanism between M1 macrophage membrane and TAMs. As shown in Supplementary Fig. 24, compared with the Control, Laser, and P-CD groups without M1 macrophage membrane, P-CD@MM treatment alone significantly increased the expression levels of phosphorylated NF-κB p65 (p-NF-κB p65) and phosphorylated STAT1 (p-STAT1) in TAMs. This enhancement effect was further amplified after adding laser irradiation (P-CD@MM + Laser). The results provide direct molecular evidence that the M1 macrophage membrane is not an inert carrier, but interacts with TAMs through its surface retained ligands, actively activating the core signaling pathways (NF-κB and STAT1) that drive M1 polarization, thereby reprogramming the pro-tumoral M2 phenotype to the anti-tumoral M1 phenotype. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 24. Measurements of the expression levels of p-STAT, STAT, p-NF-κB p65, NF-κB p65 in each group.\n4. The pro-inflammatory cytokines, including IL-6, TNF-α, and IFN-γ, as markers of systemic immunity activation should be analyzed.\nReply: Thanks for your suggestion. According to the reviewer’s suggestion, we detected the levels of pro-inflammatory cytokines in mice including interleukin-6 (IL-6), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) using ELISA assay. As shown in Supplementary Fig. 33, the levels of IL-6, IFN-γ, and TNF-α in P-CD@MM + laser group were significantly higher than those in other groups. These results suggest that P-CD@MM-mediated PDT can activate the immune response in vivo by releasing tumor-associated antigen, thus inducing ICD and M1 polarization. We have added these results and corresponding discussion in the revised manuscript.\nSupplementary Fig. 33. The expression levels of IL-6 (a), TNF-α (b), and IFN-γ (c) in blood serum of mice after different treatments (n = 6 independent samples). Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA. P value: *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.\n5. All statistical comparisons involving multiple groups in the manuscript are still analyzed using two-tailed Student’s t-test, which is statistically inappropriate. Appropriate statistical methods, such as one-way ANOVA followed by suitable post hoc tests, should be applied instead. This should be corrected throughout the entire manuscript.\nReply: Thanks for your suggestion. We have analyzed the multiple groups’ data in the manuscript using the one-way ANOVA method."}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors have carefully addressed the comments in the revised manuscript. Therefore, I recommend it for publication."}, "author_response": ""}]

Decision letter