All chemical reagents necessary for the synthesis of IMPP-c NPs were procured from commercial sources and were utilized without any further purification. Manganese (II) chloride (MnCl₂), Dopamine-hydrocholoride (DA-HCl), and ICG were supplied by Sigma-Aldrich (Shanghai, China). D-Luciferin and PEG-SH (5 kDa) were procured from Aladdin (Shanghai, China). Cell culture medium, PBS, trypsin, and the cell counting kit-8 (CCK-8) were obtained from Thermo Fisher Scientific (Shanghai, China). The anti-CXCR4 antibody (ab208128) was acquired from Abcam (Shanghai, China). A detailed account of the synthesis process for IMPP-c NPs is provided in the Supporting information.

The morphology was analyzed using a transmission electron microscope (TEM) (TF20, JEOL, Japan). The Zeta potential and hydrodynamic particle size were measured with a Zetasizer Nano ZS (SZ-100, Horiba, Japan). The UV-Vis-NIR absorption spectrum of IMPP-c NPs was recorded utilizing a Shimadzu spectrophotometer (Sintec Optronics Technology, Singapore). Fluorescent spectra were obtained using a spectrofluorometer (Hitachi, Japan). Temperature variations were monitored with a thermal camera (FLIR One Pro, USA). Serum stability was assessed by dissolving the sample (0.2 mg·mL⁻¹) in serum to simulate physiological conditions at 37°C for durations of 6, 12, 24, and 48 hours via the Zetasizer Nano ZS.

Different concentrations of IMPP-c aqueous solutions (0.025, 0.05, 0.1, 0.2, and 0.4 mg·mL^-1) were sealed in distinct rubber capillaries. Subsequently, the PA signals of each sample at a wavelength of 808 nm were detected and quantified using the PA imaging system (Vevo LAZR-X, Visual Sonics, Fujifilm, Japan), which is equipped with an LZ-550 linear sensor operating at a center frequency of 40 MHz. Concurrently, the PA stability of IMPP-c NPs was assessed utilizing the PA imaging system following laser irradiation for durations ranging from 0 to 30 minutes. MRI was conducted using a 9.4 T BioSpec 94/30 (Bruker, Germany) equipped with an animal abdominal coil. Various concentrations of IMPP-c aqueous solutions (0.025, 0.05, 0.1, 0.2, and 0.4 mg·mL^-1) were prepared in EP tubes and positioned on a wax holder to assess the T₁-weighted signals. The T₁-weighted signals were acquired by manually delineating the region of interest (ROI) on the images, and the relaxation rate (r₁) of IMPP-c aqueous solutions was determined as the reciprocal of T₁ (i.e., r₁ = 1/T₁).

Different concentrations of IMPP-c aqueous solutions, ranging from 0 to 0.4 mg·mL⁻¹, were injected into quartz cuvettes. Temperature changes in these samples were recorded and photographed every two minutes using a FLIR camera, under an 808-nm laser emitting at a power output of 0.8 W·cm². The laser was cycled on and off five times to allow the temperature to equilibrate during heating and subsequently cool naturally to room temperature, thereby assessing the photothermal stability of IMPP-c NPs. To verify the photo-switchable PDT potential of IMPP-c NPs, we utilized a single-line oxygen sensor green (SOSG) as a probe for detecting singlet oxygen (¹O₂). The variation in SOSG fluorescence intensity, assessed both under laser irradiation and in its absence, indicates the levels of ¹O₂ present across each experimental group. Additionally, ethanol was combined with IMPP-c, ICG, and PBS followed by the introduction of 1,3-diphenylisobenzofuran (DPBF). The depletion of DPBF after approximately five minutes of exposure to an 808-nm laser serves as an indicator for reactive oxygen species (ROS) generation and reflects the efficacy of PDT performance.

The human HCC cell lines HepG2 and HCCLM3, and the normal human hepatocyte LO2 cells (American Type Culture Collection, ATCC) were cultured in 89% Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cultures were maintained at a temperature of 37°C in a humidified atmosphere containing 5% CO₂. For confocal imaging, HepG2 and LO2 cells were seeded into different laser confocal dish and allowed to incubate for 24 hours. Following this incubation period, the culture medium containing IMPP-c NPs (0.4 mg·mL^-1) was added to each dish and further incubated for an additional 4 hours. Subsequent to these treatments, the NPs-containing culture medium was removed, and the cells were fixed using 4% paraformaldehyde (500 µL for 10 minutes). The cell nuclei were then stained with DAPI at a concentration of 1.5 µg·mL^-1 for 12 minutes before rinsing the dishes three times with PBS (1 mL each time, over a period of 10 minutes). Finally, fluorescence images of the cells were acquired utilizing a laser confocal microscope (Leica, Germany). For TEM analysis of HepG2 cells, the cells underwent incubation with IMPP-c NPs for a duration of 4 hours before being prepared on copper grids suitable for TEM observation.

To evaluate the toxicity of IMPP-c NPs under dark conditions, a CCK8 assay was performed. HepG2, HCCLM3 and LO2 cells were seeded at appropriate densities into separate 96-well plates and incubated for 24 hours. Subsequently, the growth medium was replaced with a mixture containing varying concentrations of NPs (0, 0.05, 0.1, 0.2, and 0.4 mg·mL^-1) and incubated in darkness for an additional 24 hours. Following this incubation period, the medium-NPs mixture was removed; CCK solution was then added to each well for further incubation lasting another 4 hours. The absorbance at a wavelength of 450 nm in each well was measured using a microplate reader. To assess the cellular PTT and PDT efficacy of IMPP-c NPs, HepG2 and HCCLM3 cells were also plated in individual wells within a separate 96-well plate and cultured for an initial duration of 24 hours. Afterward, the culture medium was exchanged with one containing various concentrations of IMPP-c NPs (0, 0.05, 0.1, 0.2, and 0.4 mg·mL^-1), followed by incubation for an additional period of 4 hours prior to exposure to an infrared laser at a wavelength of 808-nm for 10 minutes; subsequently allowing another incubation phase in darkness that lasted an additional 24 hours thereafter. The cell survival rates across all samples were determined through the aforementioned CCK8 assay methodology; final results represent averages obtained from three independent experimental repetitions. To accurately reflect cell survival status, HepG2 and HCCLM3 cells were independently cultured in separate dishes for 24 hours before replacing the culture medium with one containing IMPP-c NPs while continuing incubation for an additional 4 hours period post-replacement. Following this treatment step; samples from both Laser + IMPP-c/PBS group underwent exposure to an infrared laser at wavelengths of 808-nm over 10 minutes while those designated as IMPP-c/PBS remained unirradiated during this interval. Subsequently these samples underwent staining utilizing Calcein-AM as well as propidium iodide (PI) over 15 minutes’ time span after which cell fluorescence images were acquired via fluorescence microscopy.

In vivo PA imaging was conducted using subcutaneous/orthotopic tumor-bearing BALB/c nude mice. Following anesthesia, IMPP-c NPs (30 mg·Kg^-1)/ICG were administered to the tumor-bearing mice via the tail vein. To assess the accumulation time of IMPP-c NPs in both subcutaneous and orthotopic tumor regions, real-time monitoring of the PA signal within the tumor area was performed utilizing a PA imaging system over a subsequent 24-hour period. Furthermore, to further verify the accumulation of IMPP-c NPs in the orthotopic tumors, surgical procedures were carried out on the mice at designated time points. The tumors along with major organs—including heart, liver, spleen, lung, and kidney—were excised for analysis. Subsequently, these excised tumors and organs were imaged using the PA imaging system.

The BALB/c nude mice bearing subcutaneous/orthotopic tumors were utilized for in vivo MR imaging studies. To investigate the distribution of the NPs at the tumor site, IMPP-c NPs (administered at a dose of 30 mg·Kg^-1)/ICG were injected via the tail vein into mice with subcutaneous/orthotopic tumors. The mice were anesthetized using 2% isoflurane and connected to an ECG device for continuous monitoring of electrocardiogram, respiration, and body temperature. Tumors located in both subcutaneous and orthotopic mice were imaged utilizing the Turbo RARE T₁ imaging sequence (field of view: 3 cm; slice thickness: 0.5 mm).

The in vivo PTT and PDT experiment was performed using HepG2 tumors implanted in BALB/c nude mice, encompassing both subcutaneous and orthotopic tumor models. Prior to the initiation of the experiment, subcutaneous tumors exhibited an approximate volume of 80 mm³, while all orthotopic SHCC formations were confirmed via an in vivo bioluminescence imaging system. Subsequently, the mice were categorized into four groups: treatment groups (laser + IMPP-c/PBS group) and a control group (IMPP-c/PBS group). In the laser + IMPP-c/IMPP-c group, following intravenous injection at a dosage of 30 mg·kg⁻¹, IMPP-c NPs were administered; conversely, an equivalent volume of PBS was injected for the laser + PBS/PBS control group. Tumor locations were verified by gross examination for subcutaneous tumors and through optical imaging guidance employing bioluminescence imaging for orthotopic tumors. At a predetermined accumulation time point for IMPP-c NPs, an 808-nm laser with an intensity of 0.8 W·cm⁻² was utilized to irradiate the tumor sites in mice assigned to the laser treatment group over a duration of 12 minutes—this method being minimally invasive without requiring laparotomy—while no irradiation occurred in either control group. During PTT/PDT treatment sessions, temperature changes within the tumor area were monitored using infrared thermal imaging. Following this procedure, tumor volumes within the subcutaneous treatment cohort were recorded every other day; meanwhile, measurements for orthotopic tumors were taken on days 7 and 14 post-treatment. Bioluminescence imaging as well as MR imaging assessments were performed on each mouse cohort using an in vivo bioluminescence/MR imaging system to quantify results accurately. All mice underwent evaluations regarding their general condition and weight every other day throughout this period. On day 15 post-treatment completion, euthanasia was executed through cervical dislocation across all experimental groups. Subsequently, the treated tumors and major organs were surgically excised for comprehensive histological analysis utilizing hematoxylin and eosin (H&E) staining techniques.

The data are presented as mean ± standard deviation (M ± SD), and statistical comparisons were conducted using the Analysis of ANOVA with post-hoc corrections as necessary. Statistical analysis and figure plotting were performed utilizing MATLAB software (MathWorks, USA) and Origin software (OriginLab, USA). P value of less than 0.05 was considered statistically significant.

As depicted in Figure 1 , the reaction process can be optimized through the incorporation of ICG/Mn precursors and anti-CXCR4 on the surface. This approach facilitates the development of multifunctional targeted PDA NPs, designated as ICG/Mn@PDA-PEG-CXCR4 (abbreviated as IMPP-c), which are intended for PA/MR imaging and targeted phototherapy applications. TEM imaging reveals that the particle size of IMPP-c is approximately 100-120 nm (as shown in Figure 2A ), which aligns closely with the particle size measured via DLS testing (120 ± 14.5 nm) (as presented in Figure 2B ). The zeta potentials of both IMPP and IMPP-c were recorded at -36.6 ± 5.6 mV and -34.2 ± 5.2 mV, respectively. This variation in zeta potential indicates that anti-CXCR4 has been electrostatically adsorbed onto the surface of IMPP NPs, as illustrated in Figure 2C . Furthermore, discrepancies observed in fluorescence spectra between IMPP and IMPP-c provide additional evidence supporting successful attachment of anti-CXCR4 through electrostatic adsorption, with an estimated antibody loading amount calculated to be approximately 1.6% based on fluorescence peak analysis (as indicated in Figure 2D ). Meanwhile, the study also confirmed the quenching of ICG fluorescence after the synthesis of IMPP NPs. The UV-NIR spectra for both IMPP-c and PDA NPs reveal distinct NIR absorption peaks at wavelengths of 690 nm and 830 nm for IMPP-c ( Figure 2E ), while a significant decrease in absorption is noted for PDA with increasing wavelength. As illustrated in Figures 2F and 2G , an increase in concentration correlates with a gradual rise in NIR absorption for IMPP-c, demonstrating a linear relationship at an 808-nm wavelength characterized by R² =0.9996. As illustrated in Figure 2H , IMPP-c NPs exhibit excellent dispersion in deionized (DI) water, and their particle size remains relatively stable after being stored at 4°C for a duration of 14 days. Furthermore, as demonstrated in Figure 2I , IMPP-c NPs maintain good dispersion across various liquids—including phosphate-buffered saline (PBS), fetal bovine serum (FBS), and Dulbecco’s Modified Eagle Medium (DMEM)—with no significant changes in particle size observed following storage at 37°C for 48 hours, thereby simulating physiological conditions. In summary, these findings indicate that IMPP-c possesses an appropriate particle size along with favorable NIR absorption characteristics while exhibiting commendable stability within biological fluids; thus, positioning it well for potential biomedical applications.

IMPP-c NPs exhibit strong NIR absorption capabilities, rendering them suitable for NIR PA imaging. The PA imaging system was employed to visualize IMPP-c NPs in vitro. As illustrated in Figures 3A and 3B , at a wavelength of 808-nm and a light intensity of 5.0 mJ·cm⁻², the PA intensity demonstrated a positive correlation with the concentration of IMPP-c NPs, which ranged from 0 to 0.4 mg·mL⁻¹ (R² = 0.97844). Subsequent stability tests indicated that the IMPP-c NPs maintained reliable optical stability after irradiation with an 808-nm laser for a duration of 30 minutes (20 pulses/second at a light intensity of 5 mJ·cm⁻²), as shown in Figure 3E ; no significant changes were detected in the PA signal by the PA imaging system. Furthermore, Mn²⁺ incorporated within the IMPP-c NPs contributes to a robust magnetic resonance (MR) imaging signal. Figures 3C and 3D present MR images along with quantitative analyses of IMPP-c dispersions at varying concentrations. The relaxation rates 1/T1 (s^-1) is directly proportional to Mn concentration (mM) (R² = 0.99359). The strong PA/MR signals combined with excellent optical stability indicate that IMPP-c serves as an outstanding contrast agent for in vivo biological imaging.

Moreover, we evaluated the NIR PTT and PDT performance of IMPP-c NPs prior to in vivo application. The efficacy of PDT with IMPP-c NPs was validated using the SOSG ¹O2 detection probe, which exhibited significant generation of reactive oxygen species (ROS), thereby indicating remarkable effectiveness in PDT (see Figure 3F ). As depicted in Figures 3G and 3H , the temperature of the IMPP-c NPs solution at varying concentrations (ranging from 0 to 0.4 mg·mL⁻¹) increased to differing extents under laser irradiation. After 10 minutes of exposure to an 808-nm laser, the temperature for the group at a concentration of 0.4 mg·mL⁻¹ elevated from 23°C to 49.2°C when subjected to an energy density of 0.8 W·cm⁻²; in contrast, deionized water exposed to similar laser conditions only experienced a temperature rise of 5.8°C. Furthermore, we assessed the PTT stability of IMPP-c NPs through 5 cycles of on/off laser testing (refer to Figure 3I ), complemented by morphological examinations via TEM as shown in Supplementary Figure S1 in Supporting Information ; no significant changes in temperature or morphology were observed, confirming exceptional photothermal stability. As shown in Figure 3G , the photothermal conversion efficiency (PCE) for IMPP-c NPs was calculated through linear regression analysis against both IMPP-c and DI water values, yielding a PCE value of 67.9%—a result derived from methodologies detailed within the Supporting information—which ranks among the highest for both organic and inorganic materials as indicated in Table 1 in the Supporting Information The pronounced photothermal conversion characteristics exhibited by IMPP-c may be attributed to non-radiative processes associated with melanin-derived materials (30–39). Nanoparticles exhibiting high optical absorption capabilities—such as those doped with ICG/Mn²⁺—also facilitate efficient electron/charge transfer processes (40–42); their photothermal performance is comparable to that observed with CoF nanoparticles and polyoxyethylene ether nanoparticles (43–45). With reliable PA/MR signals, commendable light stability, and elevated PCE values, IMPP-c presents promising potential for further exploration in both vitro and vivo studies.

In a recent series of research advancements, rigorous and comprehensive experiments have scientifically demonstrated that the CXCR4 protein is highly expressed on the cell membrane of HCC cells, including the HepG2 and HCCLM3 cell lines (46–48). Consequently, in vitro studies demonstrated that the overexpression of CXCR4 in HepG2 cells significantly enhanced their ability to actively internalize IMPP-c NPs. As illustrated in Figures 4A and 4B , CLSM and TEM imaging clearly reveal substantial accumulation of CXCR4-modified (Alexa Fluro@488) IMPP-c NPs at both the cell membrane and within the cytoplasm of HepG2 cells after incubation with IMPP-c NPs for up to 4 hours. In contrast, LO2 cell lines incubated with IMPP-c NPs for an equivalent duration exhibited nearly undetectable levels of targeted uptake for this substance, suggesting the safety of normal human hepatocyte cells of the nanomaterials. This observation suggests that HCC cells possess specific uptake capabilities, underscoring the significant advantages offered by IMPP-c in this context. Therefore, it emerges as a promising candidate for targeted tumor diagnosis and treatment facilitated by in vivo biomedical imaging.

The cellular cytotoxicity of IMPP-c NPs was evaluated to assess their biocompatibility. As shown in Figures 4C and Supplementary Figure S2 in the Supporting Information , after co-culturing IMPP-c NPs (0.4 mg·mL^-1) with HepG2, HCCLM3 and LO2 cells in the absence of light for 24 hours, the survival rates for both cell types were found to exceed 91.2%, 92.5% and 96.8%, respectively, indicating that their cytotoxicity is negligible. Following this, a cellular phototoxicity test was conducted to confirm the efficacy of PTT and PDT using IMPP-c NPs under irradiation from an 808-nm laser for a duration of 10 minutes at an energy density of 0.8 W·cm^-². Supplementary Figure S3 in the Supporting Information illustrates the ROS generation characteristics of both IMPP-c NPs and ICG upon exposure to the 808-nm laser as detected by the DCFH-DA probe, further validating the PDT action of IMPP-c NPs. Figure 4D demonstrates that an increase in NP concentration corresponds with a decline in survival rates among HepG2 and HCCLM3 cells; specifically, following laser irradiation at a concentration of NPs-0.4 mg·mL^-1, HepG2 cell viability decreased to approximately 13.2%, while HCCLM3 cell viability fell to around 11.6%. These results indicate significant phototoxic effects associated with increased NP concentrations. The photothermal effect on HepG2 and HCCLM3 cells was directly observed through Calcein-AM and PI dual staining experiments ( Figure 4E ). In both the HepG2 and HCCLM3 groups treated with PBS or subjected to either laser treatment or IMPP-c treatment alone, strong green fluorescence signals indicative of viable cells were observed. However, following the combined treatment with laser and ICG, a robust green fluorescence signal was noted alongside minimal red fluorescence signals representing dead cells. In contrast, significant red fluorescence signals were observed following treatment with laser and IMPP-c NPs. These results indicate that the targeted delivery utilizing IMPP-c NPs can effectively engage HCC cells through PTT and PDT modalities, while enabling appropriate adjustments in laser intensity based on its targeting capabilities combined with low toxicity profiles in vitro. Consequently, due to its selective targeting ability alongside its advantageous PTT/PDT characteristics, IMPP-c is considered a reliable and specific therapeutic agent for the treatment of HCC.

Because IMPP-c has demonstrated effective targeting capabilities in vitro, a PA and MR imaging study was conducted on a subcutaneous HepG2 tumor model to validate the tumor-specific accumulation characteristics of IMPP-c in vivo. A dose of 30 mg·kg^-1 of IMPP-c NPs combined with ICG, at equal dosage, was administered via the tail vein. The PA signal within the tumor region was monitored in real-time using a PA imaging system over the course of 24 hours post-administration. As illustrated in Figure 5A , NPs effectively targeted and accumulated within the tumor region at 12 hours following injection in the IMPP-c group; conversely, no significant aggregation was observed in the ICG group. Quantitative analysis of the PA signal from the tumor region, as depicted in Figure 5B , revealed that both at 6 and 12 hours after injection, the PA signal intensity for the IMPP-c group was significantly greater than that for the ICG group (P<0.001). Furthermore, at 24 hours post-injection, there was a notable enhancement of PA signal intensity within the tumor region for the IMPP-c group compared to that of ICG. IMPP-c possesses an appropriate size along with established biocompatibility and targeted homing capabilities toward tumors, enabling effective accumulation at tumor sites. Additionally, these accumulation characteristics were corroborated through MR imaging techniques. The subcutaneous mouse tumors were imaged utilizing Turbo RARE T₁ imaging sequences (field of view: 3 cm; slice thickness: 0.5 mm). As depicted in Figures 5C and 5D , average values within the tumor region gradually increased up to 12 hours following injection before diminishing by 24 hours—results consistent with those obtained from PA imaging. In conclusion, we recommend employing IMPP-c for efficient PA imaging as well as MR imaging-guided precise theranostic applications in vivo.

Under the precise guidance of PA and MR imaging, targeted PTT/PDT for a subcutaneous HepG2 tumor model were scheduled to be conducted 12 hours post-injection of IMPP-c NPs. As illustrated in Supplementary Figure S4 in the supporting information, details regarding the NIR PTT/PDT device and its configuration are provided. The PTT/PDT treatment utilizing IMPP-c NPs is depicted in Figures 5E and 5F . Under real-time accurate guidance from both an 808-nm laser (energy density: 0.8 W·cm^-²) and optical imaging within the Laser + IMPP-c group, tumor region temperatures surged to approximately 53.8°C within just 5 minutes while being maintained at elevated levels for 8 minutes thereafter. In contrast, temperatures within the tumor region treated with Laser + PBS only reached about 39.5°C. Consequently, owing to the PTT/PDT effects induced by IMPP-c NPs—exceeding thresholds above 53.8°C—tumor tissues can be effectively ablated without significantly affecting surrounding healthy tissues containing minimal amounts of IMPP-c NPs that remained below approximately 39.5°C; thus minimizing collateral damage to normal tissue. The tumor growth curves and corresponding digital images of the mice are presented in Figures 5G and Supplementary Figure S5 in the Supporting Information . As anticipated, throughout the entire study period of 15 days, tumors in the laser + IMPP-c group were completely eradicated, while no significant inhibition of tumor growth was observed in the other groups (laser + PBS, PBS, and IMPP-c). The body weight across all groups remained stable during the treatment period ( Figure 5H ). Furthermore, pronounced necrosis of tumor tissue was evident in H&E-stained sections from mice treated with laser + IMPP-c; conversely, there were no notable signs of apoptosis or tissue necrosis in tumor cells from mice treated with laser + PBS, PBS alone, or IMPP-c ( Figure 5I ). The successful therapeutic outcomes observed in subcutaneous tumor models may serve as a catalyst for further investigations into treatments and diagnostics related to orthotopic SHCC models facilitated by assistance from IMPP-c.

In prior in vitro and in vivo studies, the targeting efficacy of IMPP-c NPs has been rigorously validated. To evaluate the performance of PA imaging utilizing these NPs in abdominal tumors, an orthotopic SHCC mouse model was employed for PA imaging assessment. Following a 10-day period post-cell implantation, the establishment of SHCC in nude mice was confirmed via biological fluorescence monitoring. Subsequently, IMPP-c NPs conjugated with ICG at a dosage of 30 mg·Kg^-1 were administered through intravenous injection into the nude mice; optimal tumor imaging regions were then assessed using a NIR PA imaging system within 24 hours post-injection. As depicted in Figure 6A , within the IMPP-c group, NPs exhibited effective targeting and significant accumulation within the tumor region as early as 6 hours following injection; conversely, no substantial accumulation was observed in the ICG group. Furthermore, quantitative analysis depicted in Figure 6C indicated that PA signal intensity within the tumor region for the IMPP-c group was considerably greater than that observed for the ICG group (P<0.001) at the 6-hour mark post-injection. Ex vivo PA imaging conducted at this time point further verified NPs accumulation across various major organs and tumors, as shown in Figure 6E . The PA signal corresponding to orthotopic tumors exhibited substantially higher intensity within the IMPP-c group compared to that within the ICG group (P<0.001), confirming targeted aggregation of IMPP-c NPs specifically at orthotopic SHCCs.

In previous studies, orthotopic SHCC has been imaged using a PA imaging system to monitor cross-sections of the tumor. However, in vivo MR imaging can also visualize the distribution throughout the body following the injection of IMPP-c NPs/ICG. Specifically, IMPP-c NPs conjugated with ICG were administered to nude mice via intravenous injection at a dosage of 30 mg·kg⁻¹. The optimal region for tumor imaging was assessed using an MR imaging system within 24 hours post-injection. As demonstrated in Figure 6B , within the IMPP-c group, NPs exhibited targeted accumulation in the tumor region as early as 6 hours after administration; in contrast, no significant accumulation was observed in the ICG group. Furthermore, as demonstrated in Figure 6D , average signal values within the tumor area gradually increased over a period extending up to 6 hours post-injection and were significantly greater than those observed in the ICG group (P<0.001), which aligns with findings from PA imaging. These results—both in vivo and ex vivo—indicate that the targeted nanocarrier IMPP-c is actively taken up by CXCR4-overexpressing SHCC tumor cells and accumulates within tumoral tissues; whereas ICG is rapidly metabolized and fails to be absorbed by tumor cells. Therefore, we recommend the utilization of IMPP-c for effective PA/MR imaging, in conjunction with imaging-guided precision theranostics for SHCC in vivo.

Under the guidance of PA/MR imaging, the optimal time point for treating orthotopic SHCC using PTT and PDT has been determined to be 6 hours post-injection. Previous studies have indicated that the depth of SHCC within the abdominal cavity is approximately 4 mm (49), which can be effectively penetrated by an 808-nm laser with minimal attenuation (50–52) (refer to Supplementary Figure S6 in the Supporting Information ). Prior to treatment, a PTT experiment was conducted on IMPP-c, whole blood, PBS, and normal liver tissue obtained via laparotomy to evaluate the safety profile of PTT (see Supplementary Figure S7 in the Supporting Information ). As anticipated, following irradiation with an 808-nm laser at an energy density of 0.8 W·cm⁻², the temperature of IMPP-c exhibited a rapid increase of 26.3°C within just 10 minutes. In contrast, temperatures for whole blood, liver tissue, and PBS only increased by 3.4°C, 3.0°C, and 2.8°C respectively—demonstrating reliable biocompatibility. Figures 7A and 7B depict PTT/PDT treatments for SHCC utilizing IMPP-c NPs; here it is evident that real-time accurate optical imaging guided under irradiation from an 808-nm laser resulted in a swift elevation of tumor region temperatures to around 50°C while maintaining this elevated temperature for a duration of 8 minutes. Such high temperatures are effective for tumor ablation without inflicting secondary damage upon surrounding healthy tissues. Conversely, temperatures in the tumor region within both laser + PBS groups reached only approximately up to 40°C. As demonstrated through both in vivo and in vitro PA/MR imaging investigations highlighting their active and passive targeting capabilities, IMPP-c NPs achieve significantly high concentrations within tumorous lesions while exhibiting low concentrations in normal liver tissues. Consequently, the noninvasive PTT/PDT results in effective elimination of tumor cells due to heat application (exceeding temperatures above 50°C) localized at areas rich with IMPP-c NPs; meanwhile normal liver tissue containing sparse amounts yields negligible phototherapy effects (remaining below or around capacities like under ~40°C), thereby minimizing phototherapeutic damage inflicted upon healthy tissues.

Figures 7C and 7D illustrate the findings from evaluating the effectiveness of noninvasive PTT/PDT in orthotopic tumor models, utilizing MR and biofluorescence imaging. The MR/biofluorescence imaging results demonstrate a significant reduction in signal within the tumor area of the laser + IMPP-c group, whereas no notable change is observed in the tumor area of the laser + PBS group. As depicted in Figure 7E , there is a gradual decrease in the average signal within the tumor area for the laser + IMPP-c group, while no significant alteration occurs in the laser + PBS group. Additionally, as illustrated in Supplementary Figure S8 ( Supporting Information ), quantitative data obtained from biofluorescence imaging indicate a significant reduction in biofluorescence signals within the tumor region for the laser + IMPP-c group. In contrast, an increase in biofluorescence signals is observed in tumors treated with laser + PBS. Given that cell survival rates directly correlate with orthotopic SHCC outcomes, these promising MR/biofluorescence imaging results support light-based therapies as effective treatments for SHCC. Throughout the treatment period, weight measurements across all groups remained stable (refer to Figure 7F ). Figure 7G presents H&E staining results and measurements of tumor diameter across all groups 15 days post-treatment. The laser + IMPP-c cohort exhibited marked tumor necrosis and suppression following phototherapy, while notable proliferation without necrosis was evident in tumors from the laser + PBS group after treatment. These findings further validate the efficacy of utilizing IMPP-c NPs to specifically target SHCCs for noninvasive PTT/PDT. According to existing literature reports, comparable studies conducted on mice have demonstrated successful outcomes with phototherapy targeted at SHCCs; however, non-targeted or imprecise treatments are less than optimal. Therefore, targeted approaches utilizing noninvasive methods combined with accurate optical imaging guidance provide an innovative strategy for PTT/PDT interventions applied to orthotopic SHCCs in mice—a method that holds considerable promise for clinical translation potential.

To further explore the metabolic clearance of IMPP-c NPs in vivo, we examined the distribution of these particles within major organs—including the heart, liver, spleen, lungs, and kidneys—at 24, 36, and 48 hours following intravenous injection. As a control measure, an equivalent volume of PBS was administered to a separate cohort of mice to establish baseline PA signals in each organ. Supplementary Figure S9 in the Supporting Information demonstrates that relative PA signals in major organs diminished progressively during the first 24 hours post-injection and eventually disappeared within 48 hours when compared to the control group. Considering their effective tumor-targeting capabilities and favorable metabolic clearance properties, we conclude that IMPP-c NPs presents itself as a promising contrast agent for in vivo tumor diagnosis and therapy employing PA imaging techniques.

For successful clinical translation, it is crucial to conduct a comprehensive evaluation of the safety profile associated with phototherapy. The potential for tissue damage or harm to vital organs arising from PTT and PDT can be systematically assessed utilizing various methodologies: monitoring NPs metabolism, recording body weight and vital signs, evaluating biochemical indicators, and performing histological examinations of organs post-surgery. Importantly, the PA signal in liver, spleen, heart, lung, and kidney tissues returned to baseline levels within 48 hours following NPs injection. This observation indicates that IMPP-c NPs are effectively metabolized in vivo. We continuously monitored the weight and vital signs across all groups for 14 days after treatment. No significant abnormal behaviors or weight loss—such as unusual movements, aggression, or restlessness—were observed in any group. Furthermore, H&E staining results for major organs at day 15 post-treatment are presented in Supplementary Figure S10 ( Supporting Information ). There was no evident tissue or organ damage detected in any group during this period; thus confirming the safety throughout the entire treatment course involving NIR laser irradiation and NPs injection. Additionally, we evaluated the biocompatibility of IMPP-c NPs and phototherapy by measuring routine blood parameters and biochemical indicators in nude mice across all groups. No significant abnormalities were identified in these assessments ( Supplementary Figure S11 in Supporting Information ), suggesting that IMPP-c NPs exhibit excellent biocompatibility in vivo. The targeted nanocarrier IMPP-c has demonstrated commendable stability and biocompatibility along with superior PA/MR imaging capabilities and reliable noninvasive PTT/PDT performance. This positions it favorably for advancing nanomedicine applications related to photothermal therapy across various tumor models. In conclusion, our findings suggest that this nanoplatform, when integrated with imaging-guided phototherapy, has significant potential for precise diagnosis and effective treatment of HCC.