Engineering NIR-II carbon dots through aniline extension with graphene and nitrogen enrichment for hepatobiliary theranostics

Abstract

Near-infrared-II carbon dots offer exceptional deep-tissue penetration for biomedical imaging, but challenges remain in their synthesis and photoluminescence mechanisms. Here, we report three carbon dots (CDs-1, CDs-2, CDs-3) with tunable emission from the visible to the Near-infrared-II (480–1265 nm), synthesized by constructing extended aniline-based frameworks from p-phenylenediamine. Combined structural and density functional theory analyses reveal that the Near-infrared-II redshift arises from the enhanced molecular dipole moments and electron-acceptor ability of the precursor, as well as the accumulation of graphene domains and pyrrolic nitrogen doping during carbonization polymerization, which collectively drive the narrowing of the energy gap. CDs-3 shows 15 mm penetration depth in gallbladder Near-infrared-II imaging (vs. clinically used indocyanine green 2 mm). With 1.44 signal-to-noise ratio and 334.5 μm resolution, it enables precise monitoring of biliary strictures/leakage. Selenium-doping-derived functionalized composite nanomaterials (CDs-3@pPB) exhibit potent reactive oxygen species scavenging and theranostic efficacy in liver fibrosis. This work elucidates the mechanism underlying the redshift of carbon dots emission into the Near-infrared-II and establishes a nanoplatform for hepatobiliary theranostics, demonstrating substantial clinical potential.

Near-infrared-II carbon dots offer exceptional deep-tissue penetration for biomedical imaging, but challenges remain in their synthesis and photoluminescence mechanisms. In this work, the authors designed and synthesized a series of carbon dots (CDs-1 to CDs-3) through extension of aniline-based frameworks, achieving tunable emission extending into the NIR-II region, and applied these CDs in deeptissue NIR-II imaging of the hepatobiliary system.

Introduction

Compared with luminescent small molecules, carbon nanotubes, lanthanide-doped nanoparticles and inorganic quantum dots, carbon dots (CDs) have better photostability, anti-photobleaching properties, nano-sized structural features, biocompatibility and easy modifiability^1–3, which give them significant advantages in the field of bio-diagnosis and therapy^4,5. Currently, many CDs with excellent optical characteristics have been successfully synthesized, with their emission wavelengths ranging from visible to near-infrared (NIR) regions (400–900 nm). However, short-wavelength emitting CDs suffer from limitations due to strong tissue autofluorescence and photon scattering, facing drawbacks such as low signal-to-noise ratios, increased photodamage and application in vivo disease detection and image-guided surgery spatial resolution. These issues represent major obstacles to their application in in vivo disease detection and image-guided surgery^6,7. NIR-II (1000-1700 nm) CDs, benefiting from the significant reduction of tissue autofluorescence and the suppression of photon scattering, exhibit high tissue penetration ability and non-invasive high-precision diagnosis and thus have more advantages in bio-diagnosis and therapy^8,9. Therefore, the development of CDs with intense NIR-II fluorescence is expected to pave the way for a paradigm in disease detection and real-time, image-guided surgical procedures.

Since 2004, when the CDs were found¹⁰, a great deal of effort has been made to explore the intrinsic mechanism of redshifted emission wavelengths¹¹. Currently, there are numerous reports on the preparation of red-emitting (Em <700 nm) CDs based on aniline derivatives. For instance, Zhang et al.¹² successfully prepared CDs that simultaneously exhibit single-photon (630 nm) and two-photon (680 nm) red light emission through the protonation of o-phenylenediamine (OPD)-derived CDs, providing a approach for regulating and predicting high-purity red light-emitting CDs¹³. In contrast, some CDs exhibit only NIR-II absorption and are primarily applied in photothermal therapy¹⁴, which cannot emit in NIR-II. To date, only a limited number of CDs exhibiting NIR-II emission have been successfully developed, as summarized in Table 1. The diversity of available probes as well as their emission wavelengths remains highly restricted and require further optimization. In these studies, the design of NIR-II CDs mainly relied on the incorporation of NIR-active small-molecule chromophores onto the CD surface¹⁵. However, as a rising star among CDs materials, CDs exhibit distinct luminescence mechanisms from small molecules. These mechanisms involve multiple cooperative fluorescence-regulation pathways, such as surface-controlled luminescence, cross-linking enhanced emission effect, quantum size effect, and carbon core-controlled luminescence^16–20. The complexity of the carbonization process of CDs makes it difficult to characterize the details of the entire reaction pathways¹³. Notably, intrinsic NIR-II emission originating from CDs with graphene-like carbonized structures has not yet been reported. Key challenges remain unresolved, such as how to precisely control the carbonization process to regulate the evolution of various nitrogen components and make the CDs emit in NIR-II²¹. In recent years, with the rapid development of artificial intelligence-assisted computation²², density functional theory (DFT) calculations and molecular simulation have achieved significant breakthroughs in the field of material property research^23,24. Marzari et al.²⁵ have emphasized the critical role of electronic structure information in the identification, characterization, and optimization, which may suggesting a powerful theoretical strategy to solve the above challenges. Therefore, utilizing DFT to analyze the electronic structure of CDs at the atomic scale provides a theoretical framework for understanding their fluorescence mechanism²⁶. Combined with the analysis of experimental characterization data, it is possible to predict and calculate the evolution process of CDs, thereby elucidating the derivation process and the influence of individual factors on optical properties. This approach is of great significance for investigating the research on the redshift of CDs toward NIR-II.

Table 1.

Reported the NIR-II emission wavelengths, luminescence mechanisms and applications of CDs

Object	Max emission	NIR-II luminescence mechanism	Property and application	Year	Ref.
CDs	1085 nm	Small molecule dye: IR1061	Radiometric pH sensing	2022	15
CQDs	1053 nm	Diverse defects in CQDs	NIR-II imaging and photothermal therapy	2024	11
Fe-CD	1000 nm	-	PH response and NIR-II bioimaging	2023	75
CyCDs	1150 nm	Small molecule dye: Cy7	Identification of drug-resistant bacteria	2023	76
OPDG	1091 nm	-	NIR-II imaging for the diagnostic of inflammatory bowel disease	2024	77
CDs-3	1080/1265 nm	Extended aniline precursors and carbonization-mediated graphene/pyrrolic nitrogen enrichment	NIR-II imaging, fibrosis theranostics	2025	This work

Biliary injury remains the most severe complication in laparoscopic cholecystectomy^27,28, intraoperative cholangiography can help prevent misidentification of the biliary tract, thereby reducing the risk of bile duct injury²⁹. Fluorescence surgical navigation technology not only facilitates the visualization of gallbladder resection and the diagnosis of biliary diseases³⁰, but also significantly enhances the visualization of bile leakage through its NIR-II fluorescence imaging, which features a high tissue penetration depth. However, traditional clinical contrast agents (e.g., indocyanine green, ICG) have limitations in NIR-I imaging, including insufficient penetration depth (<2 mm)³¹ and low spatial resolution, which hinder the further development of fluorescence navigation in cholecystectomy and the detection of bile leakage³². Notably, biliary diseases such as bile leakage, cholecystitis, and cholestasis can directly trigger local liver inflammation^33–35, In the liver microenvironment, overactivated reactive oxygen species (ROS) and abnormally activated hepatic stellate cells (HSCs) can promote the excessive deposition of extracellular matrix (ECM), thus accelerating the progression of liver fibrosis and even leading to liver cirrhosis³⁶. Considering the high mortality rate of advanced liver cirrhosis or hepatocellular carcinoma (HCC), early intervention in hepatitis and liver fibrosis has become a crucial clinical treatment goal³⁷. CDs have been proven to have liver and kidney targeting abilities³⁸. Their unique optical properties, small size, high stability, and excellent biocompatibility show great potential in prolonging blood circulation time, promoting specific uptake by hepatocytes, controlling drug release, and integrating optical diagnosis and treatment^4,5,39. Therefore, the development of multifunctional CDs with both NIR-II biliary imaging/bile leakage detection capabilities and the diagnostic and therapeutic effects for hepatitis/liver fibrosis holds significant clinical implications.

In this work, we design and synthesize a series of carbon dots (CDs-1 to CDs-3) through extension of aniline-based frameworks, achieving tunable emission extending into the NIR-II region. We reveal that the redshift mechanism is governed by enhanced molecular dipole moments, increased graphitization, and enrichment of pyrrolic nitrogen, which collectively narrow the energy gap. Furthermore, we demonstrate the application of these CDs in deep-tissue NIR-II imaging of the hepatobiliary system and establish a theranostic platform for liver fibrosis by constructing a functionalized composite (CDs-3@pPB) capable of reactive oxygen species scavenging. Specifically, we design a series of carbon dots (CDs-1, CDs-2, and CDs-3) by sequentially introducing aniline moieties to the amino groups of p-phenylenediamine derivatives, including 1,4-diaminobenzene, N1-(4-aminophenyl)benzene-1,4-diamine and tris(4-aminophenyl)amine. This structural modification enables fine-tuning of the electron-withdrawing capability of the benzene ring and modulates the charge state of the key nitrogen atoms. CDs-3 was successfully synthesized, which exhibit intense and ultralong near-infrared second window (NIR-II) photoluminescence with maximum emission peaks at 1080 nm and 1265 nm. Through comprehensive physical characterization and density functional theory (DFT)-based electronic structure calculations, we systematically investigated the influence of graphitization degree and nitrogen configurations—specifically amino, pyrrolic, and pyridinic nitrogen—on the electronic properties of the carbon dots during their formation and evolution. Special emphasis was placed on elucidating how these factors govern the modulation of the energy bandgap. Furthermore, we demonstrate the utility of these CDs in deep-tissue NIR-II imaging of the hepatobiliary system and establish a theranostic platform for liver fibrosis through the development of a functional composite (CDs-3@pPB) with reactive oxygen species (ROS)-scavenging capabilities ((Fig. 1).

Fig. 1. Design, mechanism and application of CDs and application in NIR-II imaging of the biliary system and theranostics in liver fibrosis.

a Preparation of CDs. b Redshift mechanism of CDs. c Preparation of CDs-3@pPB. d Treatment of liver fibrosis. e NIR-II imaging of biliary system.

Results and discussion

Preparation and characterization of CDs-1, CDs-2, and CDs-3

Aniline, as an important aromatic amine compound, can supply an abundant carbon source for the synthesis of CDs due to its benzene ring structure and facilitate the formation of sp²-hybridized graphene-like carbon frameworks. During the preparation of CDs, the amino group of aniline has dual functions: (1) as a nitrogen source to participate in the doping of the carbon core, introducing structural defects to modulate the electronic structure of CDs and optimize their optical properties; (2) the retained surface amino groups provide active sites for subsequent functionalization of CDs, making them ideal precursors for constructing multifunctional carbon-based nanoplatforms⁴⁰. Based on these considerations, in this study, p-phenylenediamine was chosen as the core structure. By means of an aniline-group extension strategy at the amino termini, monomer 1, monomer 2, and monomer 3 were sequentially selected as precursors. Using a one-step bottom-up solvothermal method, CDs were successfully prepared (Fig. 2a).

Fig. 2. Preparation and characterization of CDs.

a The carbonization reaction process of CDs (CDs-1, CDs-2, CDs-3). b–d TEM images and corresponding size distribution of CDs-1 b CDs-2 c and CDs-3 (d). e–g HRTEM images of CDs-1 e CDs-2 f and CDs-3 (g). h–j XRD pattern of CDs-1 h, CDs-2 i, and CDs-3 j. k–m Raman spectrum of CDs-1 k CDs-2 l and CDs-3 (m). For Fig. 2e–m, a.u. stands for arbitrary units. Source data are provided as a Source Data file.

Transmission electron microscopy (TEM) characterization results (Fig. 2b–d) show that CDs-1, CDs-2, and CDs-3 exhibit excellent monodispersity and uniform spherical morphology, with their particle size distributions showing good uniformity. Statistical analysis yielded average particle dimensions of 3.5 nm, 9.1 nm, and 13.8 nm, respectively (Supplementary Fig. 1), respectively. As shown in HRTEM images (Fig. 2e–g), all three CD variants exhibit distinct lattice fringes, with corresponding interplanar distances of 0.21 nm, 0.28 nm, and 0.33 nm. These values align well with the lattice spacing of the (100, 020, 002) planes in graphene^41–43, indicating the formation of partially graphitized crystalline domains. To deeply investigate the crystal characteristics and graphitization degree of carbon dots, we conducted X-ray diffraction (XRD) and Raman spectroscopy characterizations. The XRD patterns (Fig. 2h–j) show that all three samples have a distinct diffraction peak at 2θ ≈ 21°, which is significantly different from the diffraction peaks of the precursors and the unpurified samples (Supplementary Fig. 2), This peak corresponds to the (002) plane of graphite^44,45, which confirms the presence of sp²-hybridized graphite domains in the carbon cores. Raman spectroscopy analysis (Fig. 2k–m) further reveals the structural features of the CDs. All samples display characteristic peaks at 1360 cm⁻¹ (D band, attributed to disordered sp³-hybridized carbon) and 1580 cm⁻¹ (G band, attributed to sp²-hybridized graphite carbon). Quantitative analysis reveals that the I_D/I_G ratios decrease from 2.73 (CDs-1) to 2.42 (CDs-2) and 2.27 (CDs-3), suggesting a progressive enhancement in the graphitization degree with increasing particle size^46,47. In summary, CDs-1, CDs-2, and CDs-3 are all spherical nanoparticles with partial crystalline properties, and their structures consist of both ordered graphite domains and amorphous carbon domains. Notably, the graphitization degree increases monotonically from CDs-1 to CDs-3, providing a critical structural basis for subsequent analysis of the fluorescence redshift toward the NIR-II region.

Analysis of chemical constituents and speculation on the reaction process

The surface chemical compositions of the three CD types (CDs-1, CDs-2, and CDs-3) were thoroughly analyzed using Fourier-transform infrared spectroscopy (FT-IR) (Fig. 3b and Supplementary Fig. 3). The results revealed that all samples displayed six distinct absorption peaks in their FT-IR spectra, corresponding to the following functional groups: 3100 cm⁻¹ (O–H/N–H stretching vibrations), 2820 cm⁻¹ (C–H stretching vibrations), 1630 cm⁻¹ and 1500 cm⁻¹ (C = C/C = N/C = O skeletal vibrations), 1320 cm⁻¹ (C–N stretching vibrations), 1135 cm⁻¹ (C–N/C–O stretching vibrations), and 590 cm⁻¹ (C–Cl vibrations)^46,47. The presence of these characteristic peaks not only confirmed that the aniline-based precursors successfully constructed the CDs framework (reaction progress shown in Fig. 3a) but also indicated that selenourea and hydrochloric acid enabled the co-doping of Se and Cl during the solvothermal process. Notably, the absorption bands near 1500 cm⁻¹ in the three CDs exhibit a systematic increase in intensity, indicating an enhanced abundance or more ordered arrangement of aromatic rings and conjugated structural units within their cores. This trend is further supported by the gradual decrease in the I_D/I_G ratio observed in Raman spectra and the increasing particle size revealed by TEM imaging. Collectively, these findings reflect the progressive expansion of sp²-hybridized carbon domains and improved structural ordering. The strong correlation among FT-IR, Raman, and TEM results supports the conclusion that the carbonization degree and graphitic order are progressively enhanced from CDs-1 to CDs-3, establishing a structural foundation for NIR-II fluorescence redshift.

Fig. 3. Analysis of chemical constituents and speculation on the reaction process of CDs.

a Chemical structure diagram of the precursor extending the aniline structure on the amino group of aniline and the process of preparing CDs. b-e The FT-IR spectra of CDs. c-e XPS spectra of CDs-1 c, CDs-2 d, and CDs-3 e. f Detailed element content analysis after XPS sub-peak fitting of three kinds of CDs. g-i Shows the C 1 s, N 1 s spectrum of the CDs-1 g, CDs-2 h, the CDs-3 i. j Possible chemical reaction processes and chemical structures of intermediate products. k-m ¹H-NMR spectra k, ¹³C-NMR spectra l and HR-mass spectra m of CDs-3. Source data are provided as a Source Data file.

To deeply examine the elemental composition and chemical state distribution of CDs, comprehensive X-ray photoelectron spectroscopy (XPS) characterization was carried out^48,49. The full-spectrum analysis results (Fig. 3c–e) revealed that characteristic peaks of C 1 s (284.8 eV), N 1 s (399 eV), O 1 s (532 eV), Se 3 d (55 eV), and Cl 2p (198 eV) were detected in all samples, confirming the successful doping of Se and Cl elements in the three types of CDs. Quantitative analysis indicated that the contents of C 1 s and N 1 s in CDs-1 to CDs-3 showed a regular increasing trend (Fig. 3f and Supplementary Fig. 4). The high-resolution C 1 s spectra (Fig. 3g–i) could be deconvoluted into five components at 284.5 eV (C-C/C = C), 285.3 eV (C-N), 286.2 eV (C-O), 287.3 eV (C-Cl), and 284.5 eV (C = N/C = O). Notably, the relative contributions of C-C/C = C and C-N bonds increased from 35.95% to 38.41% and from 20.92% to 23.62%, respectively, from CDs-1 to CDs-3 (Fig. 2f and Supplementary Fig. 5). The N 1 s spectra could be decomposed into four characteristic peaks: 401.6 eV (graphitic N), 400.3 eV (amino N), 399.4 eV (pyrrolic N), and 398.5 eV (pyridinic N)⁴⁹. Notably, the N 1 s and pyrrolic N content increased from 6.95% and 2.61% in CDs-1 to 9.98% and 7.10% in CDs-3, whereas other nitrogen species showed minimal variation. These results, together with O 1 s and Se 3 d analyses (Supplementary Figs. 6, 7), are consistent with FT-IR data and provide quantitative evidence for graphitic domain expansion and nitrogen-enriched structural evolution.

Based on reported condensation mechanisms of urea and o-phenylenediamine⁵⁰ and considering the similar electronegativity of selenium and oxygen, we propose that selenourea undergoes an analogous nucleophilic substitution and dehydration process to form guanidine-type intermediates (compound 1). Subsequent polycondensation reactions generate nitrogen-rich structures containing amino, pyrrolic, and pyridinic nitrogen species (Fig. 3j), consistent with FT-IR and XPS results. Further ¹H–NMR, ¹³C–NMR, and HR-MS analyses (Fig. 3k–m and Supplementary Figs. 8–10) confirm the formation of these intermediates and their protonated forms during CDs-3 synthesis.

Overall, these results demonstrate that precise regulation of aniline group number in p-phenylenediamine derivatives, combined with selenourea-assisted Carbonization polymerization, enables controlled formation of nitrogen-rich, graphitized CD structures. The evolution of these intermediates and nitrogen configurations plays a pivotal role in modulating graphitization degree, surface states, and ultimately the optical properties of CDs, which are further discussed below.

Study of optical properties and redshift mechanisms

Firstly, the UV-Vis absorption spectra of CDs were systematically characterized (Fig. 4a). All three samples exhibit a distinct absorption band at around 360 nm, which can be primarily attributed to π-π* electronic transitions originating from both the conjugated carbon skeleton of the carbon dots and functional groups such as C=C and C=O³⁹. In addition, absorption peaks at 420 nm (CDs-1), 530 nm (CDs-2), and 860 nm (CDs-3) were observed, with corresponding emission maxima at 480 nm, 590 nm, and 1080/1265 nm, respectively (Supplementary Fig. 11), demonstrating a pronounced redshift trend. This represents the report of NIR-II emission from CDs derived from aniline-based precursors, highlighting their potential as next-generation NIR-II probes²⁹. To elucidate the underlying redshift mechanism, combined DFT calculations and experimental analyses were performed, focusing on precursor molecular engineering, nitrogen doping, and the carbonization–polymerization process.

Fig. 4. Study of optical properties and redshift mechanisms of CDs.

a Normalized absorption spectra and emission spectra of CDs, the emission spectra were obtained under excitation at 420 nm, 530 nm, and 860 nm, respectively. b The measurement of the distance between adjacent nitrogen atoms, charge calculation and LUMO-HOMO calculation after the DFT+ optimization of monomers. c Comparison of the HOMO and LUMO electron spatial distribution, energy levels for Amino Ns, Pyrrolic Ns, Pyridinic Ns, Pyrrolic Ns-graphene, and Pyridinic Ns-graphene based on DFT calculations. d The HOMO and LUMO electron spatial distribution, energy levels change after continuous graphitization and pyrrole nitrogen accumulation during CDS-3 formation. e Schematic diagram of the mechanism of CDs redshift. f Schematic diagram of energy level changes during fluorescence excitation and emission of CDs. Source data are provided as a Source Data file.

The structures of three precursors with different numbers of aniline units were geometry-optimized. The electrostatic potential analysis (Supplementary Fig. 12) showed that the amino groups were in regions of negative electrostatic potential (blue), while the benzene rings and imines were in regions of positive electrostatic potential (red), thereby forming a well-defined electron donor–acceptor system. Quantitative analysis (Fig. 4b and Supplementary Fig. 13) of precursors indicated that as the number of aniline units increased, the partial charge on the central nitrogen atom decreased significantly (from −0.65e to −0.38e) (Fig. 4b), while the charge on the peripheral amino groups changed little, suggesting an enhanced electron acceptor ability of the benzene rings. Meanwhile, the spatial separation between the peripheral amino groups and the central nitrogen atom increased slightly (5.663 Å → 5.686 Å), accompanied by a continuous increase in the molecular dipole moment, thereby promoting the intramolecular charge transfer (ICT) effect. Frontier molecular orbital analysis of the monomers (Fig. 4b) demonstrated that the highest occupied molecular orbital (HOMO) was delocalized over the entire molecular framework, whereas the lowest unoccupied molecular orbital (LUMO) was predominantly localized on the benzene ring moieties, further substantiating the electron transfer pathway from the amino donors to the aromatic acceptors. Notably, both the HOMO and LUMO energy levels progressively decreased (HOMO: −4.64 → −4.03 eV, LUMO: 0.29 → −0.02 eV), leading to a gradual narrowing of the band gap (Eg: 4.94 → 4.01 eV). The band gaps of the three monomers, extracted from Tauc plots (Supplementary Fig. 14) from UV absorption spectra (Supplementary Fig. 15), corroborated this trend, thereby confirming the reliability of the theoretical calculations. Collectively, these results demonstrate that increasing the number of aniline units in the precursor molecules enhances the electron-accepting ability of the benzene rings⁵¹, while simultaneously extending the donor–acceptor distance and strengthening the ICT effect^52,53, ultimately resulting in a reduced LUMO energy level and narrowed electronic band gap.

Drawing from the FT-IR characterization findings and XPS results, this study further integrated density functional theory (DFT) calculations to systematically investigate the mechanistic roles of different nitrogen doping configurations—namely amino N, pyrrolic N, and pyridinic N—as well as graphene structure extension in governing the fluorescence redshift of CDs. Focusing on CDs-3 as the representative system, the partial charge of the key nitrogen atom was found to no longer decrease further (Supplementary Fig. 16). Based on frontier molecular orbital analysis, it was demonstrated that compound 1 and the amino-nitrogen-doped structure generated from the reaction between monomer 3 and selenourea not only increase the nitrogen doping content but also significantly reduce the electronic band gap (compound 1: 3.96 eV; amino nitrogen: 3.97 eV) (Fig. 4c and Supplementary Fig. 17). Further analysis revealed that introducing a triphenylamine group at the pyrrolic N site effectively extends the conjugated sp² framework, leading to an enhanced spatial separation between the HOMO and LUMO, thereby strengthening the intramolecular electron transfer process. This structural modification resulted in a pronounced band gap narrowing (pyrrolic N: 4.03 eV → pyrrolic N–graphene system: 3.74 eV)¹⁸. Collectively, these results demonstrate that the increased pyrrolic N content in CDs, together with the incorporation of a strong electron-donating triphenylamine moiety, synergistically enhances the electron-donating capability of the system, concomitantly increasing the molecular dipole moment and ultimately achieving an effective narrowing of the band gap.

The fluorescence excitation and emission maxima for the three CD types were observed at 420/480 nm, 530/590 nm, and 860/1080 nm, respectively. According to the energy equation Eg = hν, their corresponding fluorescence excitation and emission band gaps were 2.63/2.58 eV (420/480 nm), 2.12/2.10 eV (420/480 nm), 1.38/1.15 eV (420/480 nm), respectively^12,54. This observation suggests that traditional organic molecular systems constructed solely from aniline and selenourea are insufficient to generate NIR-II emission in the 1080–1250 nm range. To elucidate the origin of this redshift, we constructed a series of progressively enlarged pyrrolic N-modified graphene models (pyrrolic N-graphene) to simulate the structural evolution during CD formation (Fig. 4d). Theoretical calculations revealed that, with increasing system size, the band gap continuously decreased (3.74 → 3.72 → 3.54 eV). This trend is primarily attributed to enhanced spatial separation between the electron-donating HOMO and electron-accepting LUMO orbitals within the extended π-conjugated framework, as well as a concomitant increase in the molecular dipole moment^52,53. These structural factors collectively result in a pronounced redshift of the fluorescence spectrum. These results clearly demonstrate that the unique formation process of CDs enables optical property modulation beyond the limits of conventional molecular systems. During the transformation from small-molecule precursors to carbonized nanostructures, ultranarrow band gaps (1.38/1.15 eV) that are unattainable in traditional organic fluorescent systems were successfully realized (Fig. 4e). This structure-evolution-driven luminescence mechanism not only overcomes the intrinsic performance limitations of small-molecule fluorophores but also establishes a design paradigm for next-generation NIR-II fluorescent imaging agents.

Based on the aforementioned studies, the electronic structure evolution of the three CDs was further analyzed through a combination of ultraviolet photoelectron spectroscopy (UPS) and optical bandgap measurements, revealing a strong intrinsic correlation with the observed fluorescence redshift. As shown in Supplementary Fig. 18, the UPS spectra displayed the typical features of the CDs near the Fermi level and in the secondary electron cutoff region. Based on the method reported in the literature (see Experimental “Methods” section)⁵⁵, we precisely calculated the changes in the HOMO energy levels of these three types of CDs using an excitation source with an incident photon energy of 21.2 eV from the UPS data, which were 7.79 eV, 7.87 eV and 8.43 eV, respectively (Fig. 4f). Their corresponding fluorescence excitation and emission band gaps were 2.63/2.58 eV (420/480 nm), 2.12/2.10 eV (530/590 nm), 1.38/1.15 eV (860/1080-1265 nm). On this basis, we derived their LUMO energy levels as −5.16 eV, −5.75 eV and −7.05 eV, respectively. As illustrated in Fig. 4f, the intramolecular energy level arrangements and electron transition processes of the three types of CDs exhibited a clear and progressive evolutionary trend. With the evolution of the CDs structure, the HOMO energy level shifted downward from −7.79 eV to −8.43 eV, whereas the HOMO–LUMO energy gap narrowed from 2.63 eV to 1.38 eV, with the associated fluorescence emission band gap progressively decreasing from 2.58 eV to 1.15 eV, thereby accounting for the continuous redshift of the emission wavelength into the NIR-II region.

NIR-II imaging of the biliary system

Based on the demonstrated excellent optical performance of CDs-3, this study systematically evaluated its practical applicability in gallbladder NIR-II imaging. With approval from the institutional clinical ethics committee, five surgically resected human gallbladder specimens were employed for NIR-II imaging (Fig. 5a). To mimic the physiological process whereby CDs-3 is secreted from the liver into the gallbladder, a biomimetic gallbladder-CDs-3 model was constructed, in which a PCR tube containing 0.2 mL of CDs-3 bile solution was wrapped with human gallbladder tissue, and its potential for clinical imaging was evaluated. We systematically assessed the imaging performance of this model under dual excitation sources (808 nm and 980 nm lasers) and across multiple detection bands (long-pass (LP) filters: 1000–1400 nm) (Fig. 5b). Under 808 nm excitation, the 1000–1300 nm LP filters clearly captured bright NIR-II fluorescence from CDs-3, accurately delineating the contour of the simulated gallbladder (PCR tube), whereas no detectable signal was observed at 1400 nm. Under 980 nm excitation, high-contrast images were also obtained using the same filters, and weak but discernible fluorescence appeared at 1400 nm. These results demonstrate that CDs-3 is compatible with dual-wavelength excitation and supports broadband NIR-II multichannel imaging.

Fig. 5. NIR-II imaging of the biliary system.

a Schematic diagram of the experimental operation for NIR-II imaging of human isolated gallbladder tissue with CDs-3(created with BioRender.com). b Gallbladder-CDs-3 at different emission bands (FEL: 1000 nm; 1100 nm; 1200 nm; 1300 nm; 1400 nm) c Schematic diagram of the NIR-II imaging experiment operation of gallbladder-CDs-3 covered with chicken tissues of different thicknesses (0 mm, 5 mm, 8 mm, 10 mm, 15 mm) (created with BioRender.com). d NIR-II imaging images of gallbladder-CDs-3 covered with chicken tissues of different thicknesses (0 mm, 5 mm, 8 mm, 10 mm, 15 mm). e Bright-field images of the normal rat extrahepatic bile duct anatomy and its NIR-II images at different emission wavelengths. f Bright-field images of the extrahepatic bile duct anatomy of rat models with biliary stricture and biliary leakage and their NIR-II images in the abdominal cavity. Scale bar = 500 μm. Source data are provided as a Source Data file.

To further evaluate its performance under conditions that simulate clinically relevant surgical scenarios, in which the gallbladder may be obscured by surrounding tissues, imaging capability was assessed under increasing tissue coverage. After placing chicken breast tissue of varying thicknesses (0–15 mm) over the model (Fig. 5c and Supplementary Fig. 19), NIR-II imaging revealed that CDs-3 maintained strong fluorescence signals even beneath 15 mm of tissue, clearly delineating the underlying simulated gallbladder structure with high signal-to-noise ratio and spatial resolution (Fig. 5d). Notably, its penetration depth exceeded that of the clinically used contrast agent indocyanine green (ICG) by ~2 mm³¹, and was superior to most reported NIR-II contrast agents (Supplementary Table 1).

Further, multiband NIR-II imaging studies were performed in the normal biliary system. According to the literature method³², CDs-3 was injected into the biliary tract, and the imaging characteristics of different LP filters (1000–1400 nm) were systematically investigated. The results showed (Fig. 5e) that the 1000–1300 nm LP filters enabled clear visualization of the biliary architecture in rat. To comprehensively evaluate the imaging quality, cross-sectional intensity distribution diagrams were drawn along the selected biliary tract (white line) to compare the signal-to-background ratio (SBR) and spatial resolution: the SBR values corresponding to 1000, 1100, 1200, and 1300 nm LP filters were 1.31, 1.39, 1.44, and 1.26, respectively (Supplementary Fig. 20), respectively. The full width at half maximum (FWHM) diameters obtained by Gaussian fitting after normalization of the fluorescence intensity were 326.261 μm, 321.273 μm, 266.507 μm, and 334.549 μm, respectively. The 1200 nm LP filter provided the highest SBR and spatial resolution, confirming the benefit of employing CDs-3 in in vivo imaging applications in the 1200 nm and above band (Fig. 5f). Subsequently, the biliary obstruction or stricture model was established through bile duct ligation, and it was clearly observed that the probe was uniformly distributed at the upper end of the stricture and there was no NIR-II fluorescence signal in the biliary tract distal to the ligation point. The biliary leakage model was constructed by transecting the bile duct, showing that the probe leaked at the transection point and there was a local fluorescence signal enhancement, confirming the ability of CDs-3 to visualize biliary stricture and leakage³⁰. Time-series NIR-II fluorescence imaging of intragastrically administered rats revealed a time-dependent decrease in gastric tissue fluorescence (Supplementary Fig. 21). Liver and kidney showed an initial increase followed by a decline in fluorescence, while no significant signals were observed in spleen or lung. These findings confirm the stomach-to-liver-kidney metabolic pathway of CDs-3, and its organ-selective distribution supports a favorable biosafety profile.

Collectively, the high fluorescence brightness, dual-excitation and multichannel compatibility, deep tissue penetration, and strong anti-interference capability of CDs-3 provide robust technical support for its application in complex laparoscopic procedures, such as cholecystectomy, enabling precise real-time biliary navigation as well as the diagnosis and monitoring of biliary leakage, obstruction, and stenosis, thereby highlighting its strong potential for clinical translation.

Preparation, characterization and performance study of CDs-3@pPB

Excessive production of ROS has been shown to be intricately linked to the progression of liver fibrosis^56,57. Specifically, Hepatic oxidative stress induced by ROS can lead to the activation of HSCs. This activation subsequently drives the proliferation and accumulation of ECM, which is a key pathological hallmark of liver fibrosis⁵⁸. The low electronegativity and weak Se–H bond in selenium urea make it an efficient hydrogen atom donor, enabling free radical elimination via single-electron transfer. Monomer 3’s trianiline structure acts as a strong electron donor, forming stable radical cations through its conjugated system after electron loss. During carbonization, these two molecules were integrated into nano-carbon dots, creating multimodal synergistic antioxidant sites that give CDs-3 ROS scavenging ability. Therefore, targeting the excessive ROS and remodeling the liver microenvironment could represent a potential therapeutic strategy for alleviating hepatic fibrosis. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging test showed that selenourea exhibited stronger antioxidant activity than thiourea and urea. Monomer 3 also efficiently quenched DPPH radicals, as evidenced by the characteristic fading of its purple color (Supplementary Fig. 22). Based on these findings, selenourea and monomer 3 were used to synthesize CDs-3. CDs-3 demonstrated excellent antioxidant activity (Supplementary Fig. 23), with an EC50 value of 0.051 μg/mL against 0.1 mM DPPH radicals and a marked reduction in the absorption peak at 517 nm of DPPH (Supplementary Fig. 24), confirming its high radical scavenging efficiency. The antioxidant performance demonstrated by CDs-3 has laid a core functional foundation for the subsequent treatment of hepatic fibrosis.

The enrichment of targeted probes at lesion sites can not only reduce the side effects of drugs in non-target tissues but also enhance therapeutic efficacy and improve diagnostic accuracy. Studies have shown that nanoparticles with a particle size of less than 6 nm are usually excreted through the kidneys⁵⁹, while neutral lipophilic particles (<100 nm) can pass through the sinusoidal endothelial space of the liver and target liver parenchymal cells and interstitial cells, thus having the potential for liver-targeted delivery and anti-fibrotic treatment^17,59. Traditional unmodified CDs often exhibit passive dual targeting to the liver and kidneys. To further develop an integrated diagnostic and therapeutic probe for liver fibrosis, this study systematically optimized CDs-3: (1) regulating particle size to enhance passive liver targeting; (2) introducing HSC-specific targeting peptide modification; (3) optimizing hydrophilicity to improve biocompatibility and circulation stability. PLGA (poly(lactic-co-glycolic acid)) is extensively utilized in drug delivery systems owing to its remarkable biodegradability and biocompatibility^60,61, and can achieve sustained and controlled drug release. Polyethylene glycol (PEG) can endow nanoparticles with good hydrophilicity⁶², while thioether (TK) bonds can achieve intelligent drug release through ROS-responsive cleavage⁶³. Research has demonstrated that HSCs strongly express platelet-derived growth factor receptor beta (PDGFR-β). Additionally, the cyclic peptide pPB, derived from the PDGF-B chain and containing arginine (R) and isoleucine (I) residues, is capable of selectively binding to this receptor, making it highly significant for HSC-targeted therapeutic strategies⁶⁴. Based on the above mechanism, this study designed the PLGA-TK-PEG-pPB polymer chain and encapsulated it on the surface of CDs-3 (Supplementary Figs. 25–27) by the nanoprecipitation method to construct the composite probe CDs-3@pPB.

By regulating the ratio of CDs-3 to polymer (20:1, 10:1, 6.66:1, 5:1), the particle size of the probe gradually increased from the original 12.5 nm to 45 nm, 80 nm, 100 nm and above (forming aggregates) (Fig. 6a–d). Finally, 100 nm CDs-3@pPB was selected for subsequent studies. FT-IR analysis (Fig. 6e) showed that compared with the original CDs-3, CDs-3@pPB exhibited peaks at 2577 cm⁻¹ (-SH vibration), and the C = O vibration (2577 cm⁻¹) was significantly enhanced, confirming the successful loading of PLGA-TK-PEG-pPB onto CDs-3. In addition, compared to CDs-3@pPB, the CDs-3 aqueous solution showed visible precipitation after standing for 10 min, indicating that the modified probe not only has better dispersibility but also good stability (Supplementary Fig. 28). Meanwhile, NIR-II imaging (Supplementary Fig. 29) of both the CDs-3 and CDs-3@pPB aqueous systems, which aggregated in the aqueous solutions, was significantly lower than that of the CDs-3 ethanol system⁶⁵. Meanwhile, compared with CD-3, the characteristic UV absorption peak at 860 nm in ethanol solution of CD-3@pPB was significantly weaker Supplementary Fig. 30) This reduction is attributed to the formation of ~100 nm aggregates after CDs-3 modification, which causes aggregation-induced fluorescence quenching (ACQ). After incubation at 37 °C for 12 h, the 860 nm UV absorption recovered in both aqueous and ethanol solutions of CD-3@pPB (Fig. 6f and Supplementary Fig. 31). In 0.01 M H₂O₂ solution, UV absorption was rapidly restored (Fig. 6g and Supplementary Fig. 32), and ethanol-based fluorescence intensity increased significantly (Supplementary Fig. 33), confirming that CD-3@pPB responds to ROS by dissociating aggregates and enhancing fluorescence, this lays the foundation for fluorescence enhancement based on the high ROS microenvironment CD-3@pPB of liver fibrosis, thus having the potential to diagnose liver fibrosis. The preparation and ROS-responsive release mechanism of CD-3@pPB are illustrated in Fig. 6h.

Fig. 6. Preparation, characterization and performance study of CDs-3@pPB.

a–d TEM images of CDs-3@pPB prepared by PLGA-TK-PEG-pPB polymer chain and CDs-3 in different proportions, 20:1 (a) 10:1 (b) 6.66:1 (c) and 5:1 (d). Inset is corresponding lateral size distribution of CDs-3@pPB in different proportions. e The FT-IR spectra of CDs-3 and CDs-3@pPB. f The UV-vis absorption of CDs-3@pPB released for different times in a 37°C constant temperature shaker. g The UV of CDs-3@pPB released in different concentrations of H₂O₂. h Schematic diagram of CDs-3@pPB preparation and ROS response release. i, j Ex vivo NIR-II imaging(Created with BioRender.com) i and analysis of fluorescence intensity (j) of major organs in different times after times after intragastric injection by CDs-3@pPB (100 µL, 2 mg/mL) (n = 3, biologically independent mice, mean ± SD). k Schematic diagram of in vivo modeling and imaging of CCl₄ in C57BL/6 J mice(created with BioRender.com). l, m NIR-II images of in vivo (l) and ex vivo livers (m). n TUNEL staining of liver tissue. Scale bar = 100 μm. o Analysis of the fluorescence signal ratio from NIR-II imaging (in vivo and ex vivo; n = 3) and TUNEL staining (n = 5). Data are derived from biologically independent mice and shown as mean ± SD. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test (two-sided). For Fig. 6f, g, j, a.u. stands for arbitrary units. Source data are provided as a Source Data file.

Imaging performance validation and hepatic fibrosis diagnosis

NIR-II optical imaging technology has demonstrated significant application value in biomedical imaging and diagnosis owing to its tissue penetration capability and superior spatial resolution. Next the imaging performance of the prepared CDs-3@pPB probe was evaluated through simulation experiments. Different CDs solutions (CD-3@pPB, CD-s3, CDs-2, CDs-1) were loaded into capillary glass tubes (diameter 0.3 mm) to simulate vascular distribution, and 3 mm thick chicken (Supplementary Figs. 34, 35) breast tissue was used to simulate the biological environment^31,66. The NIR-II imaging results indicated that CDs-3@pPB and CDs-3 maintained excellent imaging contrast under the condition of 3 mm chicken breast tissue coverage, and CDs-3@pPB exhibited superior spatial resolution. Further dynamic imaging of rabbit ear vessels confirmed the real-time dynamic imaging capability of CDs-3@pPB. As illustrated in the figure, the imaging clearly recorded the entire dynamic process of CDs-3@pPB entering, circulating, and disappearing in the vessels (Supplementary Fig. 36 and Supplementary Movie 1), and even micro vessels with diameters less than 100 μm were clearly visible, demonstrating the excellent high-resolution and real-time imaging capabilities of CDs-3@pPB. This lays the necessary imaging foundation for the subsequent diagnosis of liver fibrosis.

To systematically evaluate the in vivo targeted distribution characteristics of CDs-3@pPB, we studied its biodistribution by tail vein injection. NIR-II imaging demonstrated that the hepatic fluorescence enhanced within 0 to 24 h after administration, and then began to weaken (Supplementary Fig. 37). Meanwhile, fluorescence signals of CDs-3@pPB were observed in the gastrointestinal tract, confirming the existence of the haematogenetic circulation metabolic pathway³⁸. Notably, the whole-body fluorescence signal returned to the baseline level after 120 h (Fig. 6i, j), which conformed to the first-order elimination kinetic model, indicating that the probe had ideal biodegradability and safety⁷.vMeanwhile, compared with the CDs-3 tail vein administration group, CDs-3@pPB exhibited significantly enhanced NIR-II fluorescence in the liver region, while the fluorescence intensity in the kidney region was notably weakened. Quantitative analysis revealed that the liver/kidney signal ratio of CDs-3@pPB was 1.5 times that of the unmodified group (Supplementary Fig. 38), which fully confirmed the effectiveness of pPB peptide modification in enhancing liver-targeting efficiency. This laid an important foundation for the subsequent diagnosis and treatment application of liver fibrosis.

In the context of liver fibrosis diagnosis, we developed a CCl₄-induced murine model of liver fibrosis and utilized NIR-II imaging technology for non-invasive disease monitoring (Fig. 6k). When compared to the control group, a notable enhancement in NIR-II fluorescence intensity was observed both in the livers of CCl₄-treated mice in vivo (Fig. 6l) and in the excised livers (Fig. 6m). Quantitative analysis revealed that the fluorescence intensity in both the in vivo liver tissue and the dissected liver samples from the fibrotic group increased by ~1.2-fold and 1.5-fold, respectively (Fig. 6o), relative to the control group. The increased ROS environment promoted the release of the CDs-3 component in CDs-3@pPB, thereby generating a fluorescence enhancement effect in the hydrophobic microenvironment. To verify the reliability of the diagnostic results, we used the TUNEL staining method to assess the apoptosis status of liver tissue cells (Fig. 6n). The results showed a significant positive correlation between fluorescence signal intensity and apoptosis index, proving that CDs-3@pPB can diagnose liver fibrosis in mouse liver in vivo and dissected liver. This also laid the foundation for further clinical applications.

Antioxidant properties of CDs-3@pPB intracellular

Given the high reactivity of ROS and their potential to damage DNA and proteins⁶⁷, we systematically evaluated the in vitro scavenging performance of CDs-3@pPB. EPR analysis demonstrated that both CDs-3 and CDs-3@pPB exhibited significant scavenging effects on ·OH, ¹O₂, and O₂·⁻ (Supplementary Fig. 39). In concentration-dependent experiments, the scavenging rates of CDs-3@pPB for H₂O₂ and O₂·⁻ reached ~80% at a concentration of 100 μg/mL (Fig. 7a, b). To further verify its broad-spectrum radical scavenging ability, we conducted tests in two classic model systems, ABTS·⁺ and DPPH·. The results indicated that CDs-3@pPB could effectively scavenge these two radicals in a concentration-dependent manner, with scavenging rates of ~70% at a concentration of 100 μg/mL (Fig. 7c, d). In conclusion, CDs-3@pPB demonstrated robust ROS scavenging ability and has the potential to be an efficient antioxidant.

Fig. 7. Intracellular Antioxidant Activity and Therapeutic Potential of CDs-3@pPB in the Treatment of Hepatic Fibrosis.

a–d O₂·⁻ (n = 6, biologically independent samples, mean ± SD) a H₂O₂ (n = 4, biologically independent samples, mean ± SD) b ABTS·⁺ (n = 4, biologically independent samples, mean ± SD) c and DPPH· (n = 3, biologically independent samples, mean ± SD) d scavenging ability of CDs-3@pPB. e Schematic diagram of intracellular treatment of liver fibrosis with CDs-3@pPB. f Representative fluorescence and corresponding brightfield images of ROS staining in LX2 cells under different treatment conditions. Scale bar = 50 μm. g, h The quantitative analysis of western blot and western blot of α-SMA and COL1A1 expression in LX2 cells. All bands shown were derived from the same membrane and cropped for presentation; Uncropped and unprocessed scans of the corresponding blots are provided in the Source Data file (n = 3, biologically independent experiments, mean ± SD). i Ex vivo liver images of mice in different treatment groups. j, k The concentration of blood ALT (j) and AST (k) after different treatments (n = 3, biologically independent mice, mean ± SD). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test (two-sided). l Hepatic histology images with Sirius red, Masson staining, and immunohistochemical staining. m-o Analysis of Sirius Red (% area) of Sirius red m Collagen Volume Fraction (%) of Masson staining n a-SMA Positive area (%) of immunohistochemical staining (o). n = 5, biologically independent mice, mean ± SD. For g, j–k and m–o, statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparisons test (two-sided). Exact P values are indicated in the figure. Exact P values are indicated in the figure. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.

Leveraging the superior ROS scavenging properties of CDs-3@pPB, we explored its potential protective effects against ROS-induced oxidative stress in HSCs. Initially, we assessed the biocompatibility of CDs-3 and CDs-3@pPB by incubating them with LX2 and AML12 cells. CCK-8 assays revealed no significant cytotoxicity, indicating excellent biocompatibility (Supplementary Fig. 40). Subsequently, we incubated CDs-3 and CDs-3@pPB with transforming growth factor-beta 1 (TGF-β1)⁶⁸-activated LX2 cells to evaluate their cellular uptake profiles. Confocal laser scanning microscopy (CLSM) images (Supplementary Fig. 41) revealed that the cellular uptake of both CDs-3 and CDs-3@pPB gradually improved as the incubation period extended. Notably, the uptake efficiency of CDs-3@pPB was markedly greater than that of CDs-3 without modification. Flow cytometry further confirmed that activated LX2 cells internalized CDs-3@pPB at a higher rate than CDs-3 (Supplementary Fig. 42). This observation implies that the presence of pPB in CDs-3@pPB may facilitate more efficient cellular uptake by aHSCs.

Intracellular treatment of hepatic fibrosis

HSC activation represents a key process in the development of liver fibrosis. To evaluate the anti-fibrotic effect of CDs-3@pPB, a class of experiments were conducted (Fig. 7e). The intracellular ROS clearance ability of CDs-3@pPB was evaluated (Fig. 7f and Supplementary Fig. 43). CDs-3@pPB had a better ROS clearance effect than CDs-3 and was comparable to SLB. Subsequently, the expression of activation markers, including α-SMA and COL1a1, was evaluated in activated hepatic stellate cells (aHSCs). Western blotting and immunofluorescence assays demonstrated that TGF-β stimulation led to a marked increase in the levels of α-SMA and COL1a1 in LX2 cells (Fig. 7g, h). In contrast, treatment with silymarin and CDs-3 resulted in a reduction of α-SMA and COL1a1 signals. Notably, in LX2 cells treated with CDs-3@pPB, the induction of α-SMA and COL1a1 signals by TGF-β1 was almost completely abolished Supplementary Figs. 44, 45). Additionally, Ki67 staining demonstrated that CDs-3@pPB could more effectively inhibit the proliferation of activated LX2 cells (Supplementary Fig. 46). These findings indicate that CDs-3@pPB not only effectively inhibits the activation of HSCs but also significantly suppresses the proliferation of already activated HSCs, thereby showing potential therapeutic value in the prevention and treatment of liver fibrosis.

In vivo biosafety evaluation of CDs-3 and CDs-3@pPB

The biocompatibility of nanomedicines plays a vital role in their effectiveness for biomedical imaging and therapeutic applications. Despite extensive research have confirmed the excellent biocompatibility of CDs⁶⁹, this study still conducted a systematic safety assessment of CDs-3 and CDs-3@pPB before their clinical application. The hemolysis test results showed that at a concentration of 100 μg/mL, CDs-3 and CDs-3@pPB (Supplementary Fig. 47) displayed low hemolysis rates is 5.86 % and near to 0 % respectively. Notably, the surface-modified CDs-3@pPB showed a markedly reduced hemolytic effect, with no significant difference compared to the PBS control group, indicating excellent hemocompatibility. To comprehensively evaluate the in vivo toxicity of CDs-3@pPB, we injected 400 μg/mL CDs-3@pPB solution (100 μL) via the tail vein into healthy C57BL/6 J mice and conducted hematological analysis after continuous observation for 7 days. Blood routine tests (Supplementary Fig. 48) and serum biochemical indicators (Supplementary Fig. 49) showed that compared with the control group, the counts of red blood cells, white blood cells, platelets, and key parameters such as renal and cardiac function indicators (creatinine (CREA), urea, lactate dehydrogenase (LDH), and creatine kinase (CK)) in the experimental group mice remained within the normal physiological range, showing no statistically significant variation compared to the PBS group. Histopathological analysis of major organs, including the heart, liver, spleen, lungs, and kidneys, using hematoxylin-eosin (H&E) staining (Supplementary Fig. 50) showed no obvious inflammatory infiltration, tissue necrosis, or other pathological changes in either the treatment group or the control group. These results collectively indicate that CDs-3@pPB has good in vivo safety, providing an important safety basis for its subsequent clinical application.

Evaluating the therapeutic efficacy in CCl₄-induced hepatic fibrosis in vivo

To evaluate CDs-3@pPB against liver fibrosis, a CCl₄-induced mouse model was established (Supplementary Fig. 51). Groups included: control (Olive oil + PBS), model (20% CCl₄ + PBS), and treatments (CDs-3@pPB 11.5 mg/kg, CDs-3 10 mg/kg, SLB 10 mg/kg)⁶⁸. Initial body weights were similar (Supplementary Fig. 52), but by day 30, model mice lost weight, indicating metabolic disruption from fibrosis. Liver anatomy revealed increased fibrosis severity (CCl₄-2w: granular surface; CCl₄-4w: enlarged volume) (Fig. 7i), while treated groups showed moderate improvement⁷⁰. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important clinical indicators for evaluating liver injury (Fig. 7j, k). ALT/AST levels surged in CCl₄ groups (2w: ALT 206.7 U/L, AST 315.9 U/L; 4w: further elevated), but treatments significantly reduced transaminases, with CDs-3@pPB being most effective (CDs-3@pPB: ALT 101.2 U/L, AST 29.5 U/L vs. SLB: 147.7 U/L, 39.5 U/L) (Fig. 6j, k), highlighting CDs-3@pPB’s superior therapeutic potential⁷¹.

H&E staining (Supplementary Fig. 53) revealed severe fibrosis features (inflammation, collagen hyperplasia) in the model group vs. normal control. CDs-3@pPB treatment markedly improved histopathology, reducing inflammation and fibrosis. TUNEL staining (Supplementary Fig. 54) confirmed higher hepatocyte apoptosis in CCl₄ groups (2w: 8.0%; 4w: 9.6%) vs. control (2.7%), with CDs-3@pPB (4.7%) showing near-normal recovery, suggesting ROS-scavenging protection. To further evaluate the anti-fibrotic effects of each treatment regimen, we conducted quantitative analysis of collagen deposition using Sirius Red and Masson’s trichrome staining (Fig. 7l–n). Sirius Red/Masson staining quantified collagen deposition (CCl₄-2w: 3.758%/6.1358%), which was reduced by all treatments, especially CDs-3@pPB (0.149%/2.047%). α-SMA immunohistochemistry (Fig. 7l, o) confirmed HSC activation in CCl₄ groups (2w: 5.9%; 4w: 7.4%) vs. control (0.27%), with CDs-3@pPB (0.16%) outperforming SLB (0.98%) in suppression. These results demonstrate superior anti-fibrotic efficacy of CDs-3@pPB, rivalling or exceeding SLB clinically.

To investigate the long-term therapeutic effect of the drug, we established a 30-day CCl₄-induced liver fibrosis model and treated the groups with PBS (model control group) and CDs-3@pPB (11.5 mg/kg) for 30 days (Supplementary Fig. 55). The findings indicated that, in comparison to the model group, the CDs-3@pPB treatment group exhibited significant efficacy. The smoothness of the liver surface was significantly improved (Supplementary Fig. 56), the levels of serum ALT (42.37 vs 58 U/L) and AST (130.67 vs 205.07 U/L) were significantly reduced (Supplementary Fig. 57). Besides, H&E staining showed reduced inflammatory infiltration (Supplementary Fig. 58). TUNEL staining indicated a decrease in apoptotic cells (4.60% vs 10.61%). The expression of fibrosis markers was decreased (Supplementary Fig. 59) (Sirius Red: 0.46% vs 3.11%, Masson: 0.40% vs 1.89%, α-SMA: 0.76% vs 1.59%).

Histological analysis of major organs (Supplementary Figs. 60, 61) confirmed CDs-3@pPB’s safety with no obvious toxicity. Notably, compared to the CCl₄-4W group, fibrosis markers partially improved after drug withdrawal, suggesting liver self-repair potential. These results demonstrate CD3@pPB’s excellent safety profile and superior anti-fibrotic effects—both short-term prevention and long-term therapy—comparable to or even exceeding SLB clinically.

Discussion

This research introduces a strategy for the synthesis of CDs with emission spanning from the visible to the NIR-II region by extending the aniline structure from the amino group of p-phenylenediamine as the precursor. The NIR-II emissive CDs were successfully prepared from aniline-derived molecular structures. Through a combination of systematic characterization and theoretical calculations, the unique mechanism of fluorescence redshift of these CDs to the NIR-II region, that is fundamentally distinct from that of conventional small-molecule dyes, was elucidated. The enhanced molecular dipole moments and electron-acceptor ability of the extended aniline structure of the precursor and the accumulation of graphene structure/pyrrolic nitrogen doped during the carbonization process jointly drive the narrowing of the energy gap. This finding offers a theoretical foundation for the development of NIR-II fluorescent probes but also offers a potential type of nanomaterial for hepatobiliary theranostics.

The three types of CDs (CDs-1, CDs-2, and CDs-3) with emission covering the visible-to-NIR-II range were successfully prepared. Characterization studies indicated that the carbonization degree of CDs-1/2/3 deepened, the proportion of graphene structure increased, and the pyrrolic nitrogen content rose, whereas the amino/pyridine nitrogen levels fell. DFT calculations clarified the triple redshift mechanism: (1) the change of the precursor led to an synergistic enhancement electron-accepting capability of the benzene ring and an increase in the molecular dipole moment; (2) High pyrrolic nitrogen doping and the graphene structure together reduce the energy gap; (3) the degree of redshift showed a dynamic cumulative effect with the progress of the carbonization process. CDs-3 offers deep tissue penetration and high SNR and spatial resolution in NIR-II biliary imaging, making it effective for diagnosing conditions like bile duct strictures and leaks, including gallbladder leakage in humans. This highlights its strong potential for clinical use in biliary diseases. Modified with PLGA-TK-PEG-pPB (CDs-3@pPB), the probe gains enhanced liver targeting, biocompatibility, and hydrophilicity, allowing specific binding to HSC. By reducing ROS and inhibiting HSC activation, it significantly lowers collagen COL1 and α-SMA expression, with anti-fibrosis effects similar to silybin. Furthermore, the CDs-3@pPB probe enables dynamic imaging in the NIR-II window, supporting ROS-responsive drug release and fluorescence activation, combining diagnosis and therapy into one system.

In summary, through the combination of systematic characterization and theoretical calculations, analyzed the structure-activity relationship of NIR-II emission. The enhanced molecular dipole moments and electron-acceptor ability of the extended aniline structure of the precursor and the accumulation of graphene domains and pyrrolic nitrogen during the carbonization process jointly drive the narrowing of the electronic band gap, ultimately achieving redshift to the NIR-II band. CDs-3 is effective in diagnosing gallbladder leakage and biliary tract diseases. Engineered CDs-3@pPB nanocomposites also show integrated diagnostic and therapeutic potential in liver fibrosis models. Overall, this study provides a synthetic paradigm for synthesizing NIR-II CDs and presents an innovative mechanism for developing NIR-II probes, paving the way for future advances in hepatobiliary theranostics.

Methods

Materials

1,4-Diaminobenzene dihydrochloride, N1-(4-aminophenyl)benzene-1,4-diamine,Tris(4 aminophenyl)amine (TAPA)and selenourea were obtained from Shanghai Acmec Biochemical Co., Ltd (Shanghai, China). Propanoicacid,3,3’-[(1-methylethylidene)bis(thio)]bis-,1,1’-bis(2,5-dioxo-1-pyrrolidinyl)ester (PLGA-TK-PEG-pPB) were purchased from RuixiBiotech Co., Ltd (Jilin, China). DPPH· and potassium persulfate (K₂S₂O₈) were purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). ABTS was obtained from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai,China). Dimethyl sulfoxide (DMSO) was obtained from Chengdu Kelong Chemical Co., Ltd (Chengdu, China). DCFH-DA (2’,7’-dichlorodihydrofluorescein diacetate) was bought from Yeasen Biotechnology Co., Ltd (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM)/F12, Dulbecco’s modified Eagle’s (DMEM) medium, Fetal bovine serum (FBS) and penicillin/streptomycin were provided by Thermo Fisher Bio-chemical Product (Beijing) Co., Ltd (Beijing, China). Phosphate Buffered Saline (PBS, 0.01 M, pH 7.2-7.4) was bought from was obtained from Wuhan Servicebio Technology Co., Ltd (Wuhan, China). 4% paraformaldehyde and Phosphate buffer saline (PBS) was acquired from Wuhan Servicebio Technology Co., Ltd (Wuhan, China). Recombinant Human TGFβ1 was bought from Dongkang Biological Technology Co., LTD (Suzhou, China).

Synthesis of CDs-1, CDs-2, CDs-3

The synthesis of CDs-1, CDs-2, CDs-3 was conducted following established protocols. Initially, 50 mg of 1,4-Diaminobenzene dihydrochloride (Mnnomer1, M = 181.06, 0.276 mmol), 26.2 mg of N1-(4-Aminophenyl)benzene-1,4-diamine (Mnnomer1, M = 190.28, 0.138 mmol), 26.7 mg of Tris(4 aminophenyl) amine (Mnnomer1, M = 290.36, 0.092 mmol) and 6 mg of selenourea were dissolved in 15 mL of 50 mM hydrochloric acid (HCl), which the ratio of aniline to selenium-urea is equal to 0.276: 0.048. This solution underwent sonication for 10 min before being transferred to a 20 mL high-pressure reactor. The reactor was then heated at 200 °C for 6 h, resulting in a brownish black of CDs. Following purification, CDs-1, CDs-2, and CDs-3 were collected by centrifugation at 11180 × g for 10 min, taking the supernatant, and repeating this process three times, and then filtered through a 0.22 μm membrane filter, and dialyzed using a 1000 D dialysis bag. Finally, the purified CDs-1, CDs-2, CDs-3 were lyophilized to obtain a solid product for subsequent applications.

Synthesis of PLGA2K-TK-PEG2K-pPB

Weigh and dissolve 100 mg PLGA2K-TK-PEG2K-NHS in 3 mL DMF, add pPB peptide (1.1 eq.) and triethylamine (3.0 eq), and react at room temperature for 12 h. The reaction solution was transferred to a dialysis bag (molecular weight 2000 Da), dialysis in pure water for 24 h, and the product was collected after freezing and drying.

Synthesis of CDs-3@pPB

Weigh and dissolve 100 mg PLGA2K-TK-PEG2K-NHS in 3 mL DMF, add pPB peptide (1.1 eq) and triethylamine (3.0 eq), and react at room temperature for 12 h. The reaction solution was transferred to a dialysis bag (molecular weight 2000 Da), dialysis in pure water for 24 h, and the product was collected after freezing and drying. PLGA2K-TK-PEG2K-pPB was successfully prepared.

2 mg/mL PLGA₂K-TK-PEG₂K-pPB DMSO solution (0.2, 0.4, 0.6, and 0.8 mL, respectively) was added to 0.8 mL of CDs-3 (10 mg/mL in DMSO). The resulting mixture was stirred at 300 r/min for 4 h at room temperature, then transferred to 2–8 °C and allowed to react for 24 h. Subsequently, the solution was slowly introduced into 10 mL of aqueous phase under continuous stirring (300 r/min, 8 h). Finally, the product was lyophilized (freeze-dried) and stored for future use, yielding CDs-3@pPB as the target conjugate.

DFT+ calculation

All geometry optimizations and frequency analyses were performed using Gaussian 16⁷². The B3LYP functional and the 6-31 G(d) basis set were used for geometry optimization^73,74. Frequency analyses were conducted at the same level of theory to confirm that the stationary points correspond to minima or transition states. The isovalue for all molecular orbitals was set to 0.01.

Fluorescence emission spectroscopy measurements

CDs-1 and CDs-2: The tests were conducted using a Cary Eclipse fluorescence spectrometer from Varian, USA. The samples were dissolved in ethanol to prepare solutions with a concentration of 10 µg/mL and placed in a 4 mL quartz cuvette with four transparent sides for measurement. Both the excitation and emission slit widths were set to 5 nm. To determine the optimal emission, excitation wavelength scans were performed: for CDs-1, the excitation wavelength range was 380-470 nm, and the corresponding emission wavelength collection range was (excitation wavelength + 20 nm) to 650 nm; for CDs-2, the excitation wavelength range was 480-580 nm, and the corresponding emission wavelength collection range was (excitation wavelength + 20 nm) to 750 nm. The scanning speed was set to medium.

CDs-3: The tests were conducted using a FLS1000 steady-state/transient fluorescence spectrometer from Edinburgh Instruments, UK. The samples were prepared as 10 µg/mL ethanol solutions and placed in a 4 mL quartz cuvette. The excitation slit width was set to 5 nm, and the emission slit width was set to 9 nm. The excitation wavelength scan range was 800-1000 nm, and the corresponding emission wavelength collection range was (excitation wavelength + 20 nm) to 1500 nm. The measurement used an MCS laser as the excitation source and a NIR-PMT as the detector, with a data point interval (step) of 1 nm. All raw data were processed and analyzed using Origin software.

Methods for Raman I_D/I_G ratio calculation and XPS peak deconvolution

Methods for Raman I_D/I_G ratio calculation

For the data analysis of Raman spectra, the specific steps are as follows: Firstly, the original spectra are preprocessed using the baseline correction tool in Origin software to eliminate interference such as fluorescence background. Then, the D peak at ~1360 cm⁻¹ and the G peak at ~1580 cm⁻¹ are fitted separately using the Lorentz function, which is more in line with the physical line shape of Raman scattering of carbon materials. Through iterative optimization, the peak position, half-width at half-maximum, and peak area of each peak are obtained, and the goodness of fit (R² > 0.98) is recorded. Finally, the intensity ratio (I_D/I_G) is calculated based on the integral area of the D peak and the G peak to quantitatively evaluate the structural order of carbon dots.

XPS peak deconvolution

X-ray photoelectron spectroscopy (XPS) data analysis was conducted using XPSPEAK 4.1 software. All spectra (including C 1 s, N 1 s, O 1 s and Se 3 d) were first corrected for Shirley background to eliminate inelastic scattering background. Subsequently, the peaks were deconvoluted and fitted using a Gaussian-Lorentzian product function. To ensure the reliability and consistency of quantitative comparison of chemical states among different samples, strict constraints were imposed on the peaks attributed to the same chemical functional groups during the fitting process: that is, for the three samples CDs-1, CDs-2 and CDs-3, the binding energy positions, full width at half maximum (FWHM) and Gaussian-Lorentzian mixing ratio (%GL) of the peaks corresponding to the same chemical state were kept consistent. For example, the %GL of the sp² hybridized graphitic carbon (C = C) component in the C 1 s spectrum was uniformly constrained to 81%, and its FWHM was limited to 1.38–1.39 eV. The corresponding parameters of other carbon, nitrogen, oxygen and selenium species were also set to fixed values. The peak areas obtained through this constrained fitting were further converted to atomic percentages (at%) for subsequent quantitative comparison and analysis. The fitted spectra and the corresponding residual curves are shown in Fig. 2 of the main text, indicating that this model can well reproduce the experimental data.

ROS Scavenging capacity assessment of CDs-3 and CDs-3@pPB

·OH scavenging capacity assessment of CDs-3 and CDs-3@pPB via EPR spectroscopy

A Fenton reaction system was employed to generate hydroxyl radicals (·OH): 100 μM ferrous sulfate (FeSO₄) and 500 μM hydrogen peroxide (H₂O₂) were mixed in phosphate-buffered saline (PBS, pH 7.4), followed by the immediate addition of 50 mM DMPO as a spin trapping agent. The resulting reaction mixture was then combined with CDs-3 or CDs-3@pPB solution and vortexed for 5 s. Subsequently, 50 μL of the mixture was rapidly transferred into a quartz capillary and analyzed using a Bruker E500 electron paramagnetic resonance (EPR) spectrometer within 60 s post-mixing. Instrument parameters were as follows: center field, 3510 G; scan width, 100 G; microwave power, 20 mW; modulation frequency, 100 kHz; and modulation amplitude, 1.0 G. In this system, DMPO specifically traps ·OH to form DMPO-OH adducts, yielding a characteristic EPR signal. In the presence of effective ·OH scavengers such as CDs-3 and CDs-3@pPB, competitive consumption of ·OH occurs, leading to reduced formation of DMPO-OH adducts and a concomitant decrease in EPR signal intensity.

¹O₂ Scavenging Capacity Assessment of CDs-3 and CDs-3@pPB via EPR Spectroscopy

¹O₂ generation system was established using PBS containing 50 μM indocyanine green (ICG), with 50 mM TEMPO employed as the spin trapping agent. The system was irradiated for 60 s using an 808 nm laser at a power density of 0.5 W/cm² and a fixed distance of 1 cm from the sample to induce ¹O₂ production. During irradiation, CDs and CDs@pPB samples were introduced into the reaction mixture. Immediately following irradiation, the reaction solutions were transferred to an EPR spectrometer for analysis. Instrument parameters were configured identically to those used for ·OH detection. TEMPO reacts specifically with ¹O₂; therefore, in the presence of effective ¹O₂ scavengers such as CDs-3 and CDs-3@pPB, the concentration of reactive ¹O₂ decreases, leading to reduced consumption of TEMPO and a consequent increase in EPR signal intensity. Thus, compared to the blank control group—where ICG was irradiated to generate ¹O₂ in the absence of any scavenger—an enhanced EPR signal indicates the ¹O₂-scavenging activity of CDs-3 and CDs-3@pPB.

O₂·⁻ Scavenging Capacity Assessment of CDs-3 and CDs-3@pPB via EPR Spectroscopy

A photoexcitation system was established to generate O₂·⁻. A reaction solution containing 50 μM riboflavin and 50 mM DMPO was prepared in PBS (pH 7.4) and irradiated under a white light source (3000 lux) for 120 s to initiate O₂·⁻ production. The CDs-3 material was added simultaneously with the onset of illumination. Immediately after irradiation, samples were transferred to a quartz capillary and analyzed by EPR spectroscopy. In this system, DMPO specifically traps O₂·⁻ to form the DMPO-OOH spin adduct, yielding a characteristic EPR signal. In the presence of effective O₂·⁻ scavengers such as CDs-3 and CDs-3@pPB, competitive consumption of O₂·⁻ occurs, leading to reduced formation of DMPO-OOH and a concomitant decrease in EPR signal intensity. Therefore, compared to the blank control group (ROS generation system without CDs materials), the addition of CDs materials exhibiting scavenging activity results in a significant reduction in EPR signal intensity.

The blank control group was a pure ROS generation system without any CDs and CDs@pPB materials.

H₂O₂ scavenging activity of CDs-3@pPB

H₂O₂ scavenging activity was evaluated using a Hydrogen Peroxide assay kit (Nanjing Jiancheng, A064-1-1, China) following the manufacturer’s protocol. In this assay, H₂O₂ reacts with ammonium molybdate to produce a stable yellow complex that absorbs strongly at 405 nm. For the experiment, various concentrations of CDs-3@pPB (0-200 μg/mL) were incubated with 2 mM H₂O₂ at 37 °C for 2 h. Following incubation, the residual H₂O₂ concentration was determined according to the kit instructions.

O₂·⁻ scavenging activity of CDs-3@pPB

The ability of CDs-3@pPB to O2·⁻ was evaluated using a Superoxide Anion Assay Kit (Nanjing Jiancheng, A052-1-1, China) in strict accordance with the provided protocol.

Radical scavenging assays

To assess the antioxidant properties of CDs-3@pPB, ABTS·⁺ and DPPH· radical scavenging assays were performed.

ABTS·⁺ scavenging assay

To produce ABTS radicals, a 7.4 mM ABTS solution was combined with a 2.6 mM potassium persulfate solution in equal volumes. The mixture was then incubated in the dark for 12 h. After incubation, the ABTS·⁺ solution was diluted to an optimal concentration using acetate buffer (pH 4.5). Next, CDs-3@pPB was introduced into the ABTS·+ solution at different concentrations ranging from 0 to 200 μg/mL. The mixture was incubated at room temperature in the dark for 30 min. The absorbance of the reaction mixture was measured at 734 nm.

DPPH· scavenging assay

The free radical scavenging ability of CDs-3 was determined by 2,2-Diphenyl-1-picric hydrazide (DPPH) scavenging method. DPPH is a stable nitrogen-centered free radical with two lone pairs of electrons surrounded by three benzene rings. Its alcohol solution is purple and has the highest absorption at 517 nm. In the presence of antioxidants, DPPH free radicals are scavenged, the free radical reaction from purple to yellow can take place, and the absorbance at the highest absorption wavelength decreases, therefore, it can be used to evaluate the scavenging activity of free radicals and explore the oxidation resistance of materials. 0.1 mL ethanol solution with different concentrations of L-CDs (0.02-1 mg/mL) was added to 0.9 mL 0.1 mM DPPH ethanol solution. After keeping the reagent solution in the dark for 30 min, the absorbance of the solution at 517 nm was measured and recorded.

Initially, DPPH was dissolved in ethanol to create a 0.1 mM solution, which was then stirred in the dark for 2 h to ensure proper dissolution. This DPPH· solution was subsequently mixed with equal volumes of CDs-3@pPB solutions at various concentrations (0–200 μg/mL). The mixtures were allowed to react at room temperature in the dark for 30 min. The absorbance of the resulting mixtures was measured at 517 nm. The scavenging efficiency was calculated using the formula:

$$\mathrm{scavenging}\left(\%\right)=\left[\left(A_0-A\right)/A_0\right]\times 100\%$$

where A₀ represents the absorbance of the control group (without CDs-3@pPB) and A is the absorbance in the presence of CDs-3@pPB.

Cell line source

The LX2, AML12 and RAW264.7 cell lines used in this study were obtained from our laboratory stocks. The original sources were ATCC (LX2: ATCC® SCRC-1046™; AML12: ATCC® CRL-2254™; RAW264.7: ATCC® TIB-71™). LX2 and RAW264.7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin, and AML12 cells were cultured in DMEM/F-12 supplemented with 10% FBS and 1% penicillin–streptomycin.

In vitro biocompatibility evaluation of CDs-3@pPB

The cytotoxic effects of CDs-3@pPB were evaluated using the CCK-8 assay in vitro. First, LX2 cells and AML12 cells were plated in 96-well plates at a density of 10⁴ cells per well and incubated at 37 °C with 5% CO₂ for 24 h to allow cell attachment. The culture medium was then removed, and fresh medium containing different concentrations of CDs-3@pPB (0-80 μg/mL) was added to the wells. After 24 h of incubation, the cells were washed twice with sterile PBS and treated with 100 μL of fresh culture medium and 10 μL of CCK-8 solution (TargetMol, C0005, USA) for an additional 2 h at 37 °C. Cell viability was assessed by measuring the absorbance at 450 nm using a microplate reader (Thermo Varioskan Flash, USA).

Cellular uptake of CDs-3 and CDs-3@pPB

Flow cytometry assay

LX2 cells were seeded into a 24-well plate at a density of 3×10⁴ cells per well and cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics for 24 h. Subsequently, CDs-3 and CDs-3@pPB (at a concentration of 1 μg/mL) were introduced into the wells by mixing with fresh medium. The cells were incubated with these compounds for various time intervals. After incubation, the cells were rinsed with PBS, detached by trypsinization, and collected via centrifugation. The fluorescence intensity of the drug within the cells was then measured using flow cytometry. Gating strategy used to determine cellular uptake in the Y675 channel, as presented in Supplementary Fig. 62

CLSM Imaging

For confocal laser scanning microscopy (CLSM) studies, LX2 cells were plated in glass-bottom confocal dishes (with a 20 mm diameter) at a density of 1 × 10⁵ cells per dish. Prior to imaging, the cells were stained with DAPI nuclear dye for 5 min to label the nuclei, which appeared blue under CLSM. The fluorescence of CDs-3 and CDs-3@pPB was observed as red signals, allowing for the visualization of their intracellular distribution relative to the nuclei.

In vitro ROS scavenging ability

To assess the ROS-scavenging ability of CDs-3@pPB, LX2 cells were plated in 12-well plates at a density of 5 × 10⁴ cells per well. After incubating for 24 h, the culture medium was replaced with fresh medium containing silymarin, CDs-3, and CDs-3@pPB (each at 50 μg/mL), and the cells were incubated for an additional 24 h. Following treatment, the cells were exposed to 250 μM H₂O₂ for 4 h to induce oxidative stress.

Flow cytometry assay

For ROS detection, cells were harvested using trypsin-EDTA and collected by centrifugation. They were then washed twice with cold PBS and incubated with 10 μM DCFH-DA (2’,7’-dichlorodihydrofluorescein diacetate) in serum-free medium for 30 min at 37 °C in the dark. Following incubation, cells were washed twice with PBS and analyzed immediately by flow cytometry to measure the fluorescence intensity of the oxidized product (DCF) at 525 nm (excitation wavelength 488 nm). Gating strategy used to determine intracellular reactive oxygen species (ROS) levels, as presented in Supplementary Fig. 62

Fluorescence staining

For fluorescence microscopy, the cells were first washed three times with PBS. They were then incubated with a 10 μM solution of DCFH-DA at 37 °C for 30 min. Following incubation, the cells were rinsed three times with serum-free medium to remove any residual dye. The intracellular ROS levels were subsequently visualized by examining the green fluorescence of DCF using an inverted fluorescence microscope (Leica DMi8).

Immunohistochemistry

For immunofluorescence staining of cultured cells, start by washing the cells twice with PBS to remove any residual culture medium. Fix the cells with 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature. After fixation, rinse the cells three times with PBS to eliminate excess fixative. Permeabilization is performed by treating the cells with 0.1% Triton X-100 in PBS for 15 min at room temperature. To block non-specific binding, incubate the cells in 5% bovine serum albumin (BSA) in PBS for 60 min at room temperature. Next, apply the primary antibodies (α-SMA, rabbit polyclonal, Proteintech, 14395-1-AP, 1:500; COL1a1, mouse monoclonal IgG1, Santa Cruz Biotechnology, sc-59772, clone COL-1, 1:500) diluted in the blocking solution and incubate for 3 h at room temperature or overnight at 4 °C. Afterward, wash the cells three times with PBS to remove any unbound primary antibodies. Then, incubate the cells with a fluorophore-conjugated secondary antibodies (Alexa Fluor™ 594 goat anti-rabbit IgG (H + L), Thermo Fisher Scientific, A-11012, 1:500; Alexa Fluor™ 488 goat anti-mouse IgG (H + L), Thermo Fisher Scientific, A-11001, 1:500) in the blocking solution for 45 min at room temperature, ensuring protection from light. Following three additional PBS washes to remove excess secondary antibodies, mount the cells using a DAPI-containing mounting medium for nuclear staining. Finally, observe the samples under a fluorescence microscope (Zeiss LSM980).

Western blot

Cells and tissues were lysed via sonication in RIPA buffer containing PMSF. After lysis, the lysates were then centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was collected. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, P0009, China). For protein analysis, 20 μg of each sample was loaded onto an SDS-PAGE gel for electrophoresis. The separated proteins were subsequently transferred onto PVDF membranes, which were then blocked with 5% non-fat dry milk in TBST at room temperature for 1 h. The membranes were incubated overnight at 4 °C with primary antibodies (α-SMA, rabbit polyclonal, Proteintech, 14395-1-AP, 1:1000; COL1a1, mouse monoclonal IgG1, Santa Cruz Biotechnology, sc-59772, clone COL-1, 1:1000; Tubulin, rabbit mAb, ABclonal, A12289, 1:5000). After three washes with TBST (5 minutes each), the membrane was incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG, Proteintech, SA00001-2, 1:5000; goat anti-mouse IgG, Proteintech, SA00001-1, 1:5000) for 1 h at room temperature. Protein bands were visualized using ECL reagents (Bio-Rad) and detected with a Bio-Rad ChemiDoc chemiluminescent imaging system.

Animal model and ethics statement

7–8-week-old male C57BL/6 J mice (n = 5 per group) weighing ~20 g were sourced from the Laboratory Animal Center of Xiamen University were raised in SPF environment under constant temperature (23–25 °C) and humidity (50%) with 12-h light/12-h dark circadian cycle. This choice was based on established literature and method indicating that this sex yield more reproducible and robust fibrosis models. This study exclusively used male mice to ensure faster disease progression and more accurate evaluation. This study results were independent of sex or gender. To establish a liver fibrosis mouse model, C57BL/6 J mice were treated with a 20% CCl4 olive oil solution (intraperitoneal injection). The animal experiments adhered to the guidelines of the Xiamen University Animal Care and Use Committee.

5–6-week-old female Sprague-Dawley (SD) rats (n = 5 per group) weighing ~120 g were sourced from the Laboratory Animal Center of Xiamen University and were raised in an SPF environment under constant temperature (23–25 °C) and humidity (50%) with a 12-h light/12-h dark circadian cycle. This choice was based on established literature and methods indicating that this sex yields more consistent and clearer biliary tract imaging outcomes. This study exclusively used female rats to ensure anatomical stability and optimal imaging conditions. The results of this study were independent of sex or gender. The animals were used for biliary tract imaging studies. All animal experiments were approved by the Animal Ethics Committee of Xiamen University and conducted in accordance with its guidelines.

In vivo biosafety evaluation

Blood (total 1.0 mL) was collected from 5 healthy 7–8-week-old male C57BL/6 J mice and centrifuged for a hemolysis assay to assess the biosafety of CDs-3 and CDs-3@pPB. Blood was washed five times with saline until the supernatant was colorless and clear. Then, the erythrocytes were collected and dispersed into saline to make a 4% erythrocyte suspension. Different concentrations of CDs-3, CDs-3@pPB, saline, and deionized water were added. After a 3 h incubation at 37 °C, the mixture was photographed and recorded by centrifugation (250 × g, 3 min). Its absorption of supernatant at 570 nm was measured. On the first and seventh day of dosing, mouse blood was collected for blood routine analysis and chemical analysis.

In vitro liver-targeting evaluation of CDs-3@pPB

Three groups of C57BL/6 J mice (n = 3 in each group) were set up. The mice in each group were injected via the tail vein with the following probes: the first group received unmodified CDs-3 (10 mg/kg), the second group received CDs-3@pPB modified with pPB peptide (11.5 mg/kg), and the third group was given PBS as control. The injection volume for all groups was uniformly 100 µL, and the dose was adjusted to ensure that the actual delivery dose of the CDs-3 core was the same in the first and second groups. At 12 and 24 h after administration, the mice were sacrificed by cervical dislocation, and the heart, liver and kidney were collected for NIR-II imaging (specific parameters: 808 nm laser excitation, 1000 nm long-pass filter, exposure time 0.5 s).

In vivo imaging

SD rats were orally administered CDs-3 (10 mg/kg) via gavage. At designated time points (1, 3, and 5 h), a subset of animals was euthanized, and major organs were excised for ex vivo imaging. Bile duct obstruction models were established via ligation, while bile duct leakage models were created by transection. Probe-based drug administration followed Reference³², involving injection of 100 μL CDs-3 solution (1 mg/mL) directly into the bile duct of model rats. Fluorescence signals were systematically recorded using an NIR-II imaging system (Teledyne Asia DB-1)

The C57BL/6 J mice were administered by the tail vein with CDs-3 (10 mg/kg) and CDs-3@pPB (11.5 mg/kg). Fluorescence signals emitted were systematically recorded using NIR-II imaging system (Teltec. asia DB-1) at various time points (0 h, 1 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h). Meanwhile, A part of mice was euthanized, and major organs were excised for ex vivo imaging.

In vivo prevention and alleviation of liver fibrosis

The treatment options are the same as those that have been reported for SLB. Four groups of mice were injected with CDs-3@pPB, SLB, CDs-3, and PBS with a 20% CCl₄ olive oil solution (100 μL intraperitoneal injection), while mice injected with 100 μL olive oil solution were used as a control group, and relevant indexes were detected 15 days after injection. Orbital blood was taken from mice, and the mice were euthanized. The heart, liver, spleen, lung, and kidney of each group were fixed with 4 % paraformaldehyde to make sections for H&E staining histological analysis. The liver is further used for TUNEL immunofluorescent staining and Sirius Red, Masson, and A-SMA staining. Finally, ALT and AST of Serum were measured by automatic biochemical analyzer, H&E staining, TUNEL, Sirius Red, Masson, A-SMA staining images were collected by slice scanning system.

Human tissue and ethics statement

Tissue samples from 5 patients with acute cholecystitis were collected for ex vivo CDs-3 NIR-II imaging, patients’ clinical data are presented in Supplementary Table 2. All samples are discarded surgical specimens, we obtained written informed consent from each patient or their legally authorised representative(s) prior to collection. This study was approved by the Ethics Committee of Dazhou Central Hospital (No. 2025096). The study was performed in accordance with the Declaration of Helsinki.”

Statistics and reproducibility

All experiments were independently repeated at least three times. Data are presented as the mean ± standard deviation (SD). Microscopy and staining images shown are representative of at least three independent experiments. Reported results were consistently replicated across multiple experiments with all replicates generating similar results: cell experiments, n = 3 biologically independent experiments; animal experiments, typically n = 3–5 biologically independent animals per group. Statistical analyses were performed using GraphPad Prism 8.0 software. Differences between groups were assessed using ordinary one-way ANOVA or two-tailed Student’s t-test, as appropriate. For multiple comparisons following ANOVA, appropriate post-hoc tests (e.g., Tukey’s or Dunnett’s) were applied with adjustment. Significance levels are denoted as follows: ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

L.Y.: Writing original draft, Project administration, Methodology, Investigation, Conceptualization. M.Li.: Writing original draft, Methodology, Investigation, Conceptualization. Y.P.: Writing–original draft, Project administration, Methodology, Data curation. Y.Z.: Resources, Methodology, Formal analysis. J.Z.: Resources, Methodology, Formal analysis. H.L. and W.Z.: Supervision, Methodology, Formal analysis. J.L. (Jie Liu), P.H., F.D., J.Z. and J.L. (Jing Lin): Resources, Investigation. G.L., Z.L., S.Q.: Writing–review & editing, Writing–original draft, Funding acquisition, Data curation.

Peer review

Peer review information

Nature Communications thanks Ding-Kun Ji and the other anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

Data availability

All data are available in the main text or the supplementary materials. The atomic coordinates of the optimized computational models reported in this paper have been deposited in the figshare database under the (10.6084/m9.figshare.30889529). Any additional data supporting the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-026-70150-7.